c THE ACTIONS OF CALCIUM ANTAGONISTS ON ARRHYTHMIAS AND OTHER RESPONSES TO MYOCARDIAL ISCHAEMIA IN THE RAT By MICHAEL JOHN CURTIS B.Sc. (Hon.), Univers i ty of London (Chelsea College) 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Pharmacology and Therapeutics, Faculty of Medicine We accept th is thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Ju ly 1986 ®Michael John Cu r t i s , 1986 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 0 DE-6 (2/81) - i i -ABSTRACT Studies were carr ied out in order to examine the actions of calcium antagonists in acute myocardial ischaemia and the mechanism(s) responsible for these act ions; the' s c i e n t i f i c hypothesis under test was that calcium antagonism in the vent r ic les i s antiarrhythmic in acute myocardial i s c h -aemia. In addi t ion, experiments were carr ied out to invest igate the ro le of the sympathetic nervous system in arrhythmogenesis. The actions of seven calcium antagonists on responses to myocardial ischaemia were investigated in vivo using the conscious rat preparat ion. I t was found that the drugs with i den t i f i ab le actions in the heart a t t r ibutab le to calcium antagonism possessed antiarrhythmic a c t i v i t y , whereas the drugs producing only systemic vasodi la tat ion were without tangible antiarrhythmic a c t i v i t y . The resu l ts were taken as evidence in support of the main hypoth-esis (above). In no instance did any of the drugs produce consistent dose-dependent - infarct-reducing act ions. It was establ ished from the comparison of the opt ica l enantiomers of verapamil that antiarrhythmic potency corresponded with calcium antagonist potency. I t was also shown that these drugs appeared to have no ef fect on g N a in the heart in vivo (as predicted from work by others, in v i t r o ) . Evidence that arrhythmias were reduced as a resu l t of ef fects on i . si in the ischaemic ven t r i c le was accrued from several s tud ies. In iso la ted Langendorff-perfused rat vent r ic les the calcium antagonist a c t i v i t y of the verapamil enantiomers was great ly potentiated by ra i s ing K + concentration to levels seen during acute myocardial ischaemia, whereas n i fed ip ine , which showed l i t t l e i f any antiarrhythmic a c t i v i t y in v-ivo did not show marked + K -dependent calcium antagonist a c t i v i t y . In a separate ser ies of experiments i t was demonstrated that se r i a l ablat ions in the CNS had profound ef fects on occlusion-induced arrhythmias, but that these ef fects occurred independently of the level of adrenoceptor ac t i va t i on . It was hypothesised that surgery reduced ischaemia-induced arrhythmias, by v i r tue of e i ther i t s ef fects on serum K + concentration or i t s ef fects on the number of c i r cu la t i ng thrombocytes. - iv -TABLE OF CONTENTS CHAPTER Page TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES x LIST OF FIGURES xi LIST OF ABBREVIATIONS xiv ACKNOWLEDGEMENTS XV AUTHORISATION FROM ANIMAL CARE UNIT, U. B. C. xvi 1 INTRODUCTION 1 1.1 C l i n i c a l myocardial ischaemia and in fa rc t ion 1 1.1.1 Overview 1 1.1.2 Infarct ion 3 1.1.3 Arrhythmias 4 1.1.4 Therapeutic approaches 14 1.2 Experimental myocardial ischaemia and in fa rc t ion 17 1.2.1 Overview 17 1.2.2 Methods for producing ischaemia 18 1.2.3 Differences between species 24 1.2.3.1 Infarct ion 24 1.2.3.2 Arrhythmias 26 1.2.3.3 Choice of species 28 1.2.4 A h is tory of coronary occlusion in rats 36 1.3 Ventr icu lar arrhythmias in acute myocardial ischaemia 46 1.3.1 The electrophysiology of exc i tab le t issue 47 1.3.2 The electrophysiology of the normal ven t r i c le 54 1.3.3 Elect rophysio log ica l changes caused by myocardial ischaemia 57 1.3.4 Models Of Arrhythmogenesis 60 1.3.4.1 Reentry 60 1.3.4.2 Abnormal Automaticity 62 1.3.4.3 Triggered Automaticity 66 1.3.5 Epicard ia l Act ivat ion Mapping 67 1.3.6 The slow inward current and arrhythmogenesis in acute ischaemia 70 1.3.6.1 Introduction 70 1.3.6.2 How might i s l - contr ibute to arrhythmogenesis? 70 1.4 The pharmacology of calcium antagonists 73 1.4.1 Def in i t ion 73 1.4.2 Pharmacology of phenethylalkylamines and 1,4-dihydropyridines 74 - V -1.5 Aims of studies 77 1.5.1 The action of calcium antagonists in acute myocardial ischaemia 77 1.5.2 Arrhythmogenesis in acute myocardial ischaemia 78 2 METHODS 80 2.1 Coronary occlusion in rats 80 2.1.1 Overview 80 2.1.2 Preparation 81 2.1.2.1 Occluders 82 2.1.2.2 Leads and l ines 87 2.1.3 Coronary occlusion 91 2.1.3.1 Sequence of events 92 2.1.3.2 Monitoring of responses to occlusion 96 2.1.3.3 Occluded zone (OZ) 97 2.1.3.4 Infarct zone (IZ) 98 2.1.4 Def in i t ion of occlusion-induced arrhythmias 99 2.1.4.1 Introduction 99 2.1.4.2 Premature vent r icu lar contractions (PVC) 102 2.1.4.3 Ventr icu lar tachycardia (VT) 103 2.1.4.4 Ventr icular f i b r i l l a t i o n (VF) 103 2.1.4.5 Other arrhythmias 104 2.1.4.6 Rationale for d e f i b r i l l a t i o n 105 2.1.4.7 Arrhythmia scores 107 2.1.5 ECG changes produced by occlusion 110 2.1.5.1 ' S - T ' segment elevat ion 110 2.1.5.2 Pathological R-waves 114 2.1.5.3 Pathological Q waves 114 2.1.6 ECG changes produced by drugs 115 2.1.6.1 P-R interval 115 2.1.6.2 QRS interval 115 2.1.7 Measurement of serum K + concentration 116 2.1.8 Exclusion c r i t e r i a 117 2.1.9 S t a t i s t i c s 122 2.1.9.1 Normalisation procedures 122 2.1.9.2 Censoring 123 2.1.9.3 S t a t i s t i c a l tests 123 2.2 Calcium antagonist studies in coronary-occluded rats 124 2.2.1 General experimental design 124 2.2.2 Phenethylalkylamines 125 2.2.2.1 Anipamil and Ronipamil 125 2.2.2.2 (+)- And (-)-verapami1 126 2.2.3 1,4-Dihydropyridines 127 2.2.3.1 Felodipine 127 2.2.3.2 Nifedipine and DHM9 , 128 2.3 Arrhythmogenesis and the ro le of the CNS 129 2.3.1 Introduction 129 2.3.2 Preparation 130 2.3.2.1 P i th ing 130 2.3.2.2 Sp ina l i sa t ion 131 2.3.2.3 Decerebration 131 - v i -2.3.3 Other manipulations 132 2.3.4 S t a t i s t i c s 135 2.4 Prel iminary Screen for drug a c t i v i t y in acute ischaemia 135 2.4.1 Introduction 135 2.4.2 Preparation 135 2.4.3 Experimental endpoints 136 2.4.3.1 Def in i t ions 136 2.4.3.2 Val idat ion of behaviour endpoints 137 2.4.4 Comprison of ( + )-, ( - ) - and (ij-verapami1 137 2.4.5 S t a t i s t i c s 139 2.5 E lec t r i ca l l y - induced arrhythmias in conscious rats 139 2.5.1 Introduction 139 2.5.2 Preparation 140 2.5.3 Experimental endpoints 140 2.5.3.1 Maximum fol lowing frequency 140 2.5.3.2 Threshold voltage 141 2.5.3.3 Threshold pulse width 141 2.5.4 Comparison of (+)- and (-)-verapamil 141 2.6 Haemodynamic ef fects of calcium antagonists in pithed rats 142 2.6.1 Introduction 142 2.6.2 Preparation 143 2.6.3 Variables measured 143 2.6.4 Comparison of (+)- and (-)-verapami1 143 2.6.5 S t a t i s t i c s 144 2.7 Actions of calcium antagonists in perfused ra t ven t r i c les 144 2.7.1 Introduction 144 2.7.2 Perfusion apparatus 145 2.7.3 Spec i f i ca t ions for Langendorff perfusion 146 2.7.4 Variables measured 146 2.7.4.1 Isochoric l e f t vent r icu lar developed pressure 146 2.7.4.2 Coronary blood flow 147 2.7.4.3 Ventr icu lar e x c i t a b i l i t y 147 2.7.5 Comparison of (+)- and (-)-verapami1 147 2.7.6 Comparison of n i fed ip ine and DHM9 148 2.7.7 S t a t i s t i c s 148 2.8 Metabolism of (^O-verapamil in acute myocardial ischaemia 149 2.8.1 Introduction 149 2.8.2 Animals 149 2.8.3 Drug administrat ion and t issue samples 149 2.8.4 Extract ion 150 2.8.5 HPLC 152 2.8.6 Calcu la t ion of (^J-verapami1 concentration 152 3 RESULTS 154 3.1 Metabolism of (^-verapami l in acute myocardial ischaemia 154 3.1.1 Concentration of (^-verapami l in blood matrices 154 3.1.2 Concentration of (±)-verapami1 in the vent r icu lar myocardium 155 - v i i -3.2 Arrhythmogenesis and the ro le of the CNS 158 3.2.1 Overview 158 3.2.2 Occluded zones (OZ) 158 3.2.3 Arrhythmias 160 3.2.3.1 Arrhythmia scores 160 3.2.3.2 VT and VF 160 3.2.3.3 PVC 160 3.2.4 Haemodynamic var iables 165 3.2.5 ECG changes 165 3.2.6 Thrombocytes, leukocytes and serum K + 168 3.2.7 Summary 168 3.3 Actions of anipamil and ronipamil in acute myocardial ischaemia 170 3.3.1 Overview 170 3.3.2 OZ and in fa rc t zone (IZ) 170 3.3.3 Arrhythmias 171 3.3.4 Mor ta l i t y 175 3.3.5 Haemodynamic var iables 175 3.3.6 ECG changes 177 3.3.7 Plasma concentrations of anipamil 177 3.3.8 Summary 180 3.4 Actions of fe lod ip ine in acute myocardial ischaemia 180 3.4.1 Overview 180 3.4.2 OZ, IZ and morta l i ty 180 3.4.3 Arrhythmias 181 3.4.4 Haemodynamic var iables 186 3.4.5 ECG changes 186 3.4.6 Summary 186 3.5 Actions of verapamil enantiomers in acute myocardial ischaemia 189 3.5.1 Overview 189 3.5.2 Effects of enantiomers before occlusion 190 3.5.2.1 P-R and QRS in terva ls 190 3.5.2.2 Blood pressure and heart rate 190 3.5.3 OZ, IZ and morta l i ty 190 3.5.4 Arrhythmias 195 3.5.4.1 VT and VF 195 3.5.4.2 PVC 195 3.5.4.3 Arrhythmia scores 195 3.5.4.4 Arrhythmias at 24 h 203 3.5.5 Haemodynamic var iables 203 3.5.6 ECG changes 203 3.5.7 Summary 208 3.6 Actions of n i fed ip ine and DHM9 in acute myocardial ischaemia 208 3.6.1 Overview 208 3.6.2 Ef fects of enantiomers before occlusion 208 3.6.2.1 P-R and QRS in terva ls 208 3.6.2.2 Blood pressure and heart rate 210 3.6.3 OZ, IZ and morta l i ty 210 3.6.4 Arrhythmias 210 3.6.5 Haemodynamic var iables 216 - v i i i -3.6.6 ECG changes 216 3.6.7 Serum K + concentration 218 3.6.8 Summary 218 3.7 Prel iminary Screen for drug a c t i v i t y in acute ischaemia 218 3.7.1 Overview 218 3.7.2 Val idat ion of behaviour endpoints 218 3.7.3 OZ and IZ 219 3.7.4 Mor ta l i t y and morbidity 219 3.7.5 Summary 223 3.8 E lec t r i ca l l y - i nduced arrhythmias in conscious rats 223 3.8.1 Overview and summary 223 3.9 Ef fects of verapamil enantiomers in pithed rats 225 3.9.1 Overview 225 3.9.2 S t a b i l i t y of the preparation 225 3.9.3 Ef fects of verapamil enantiomers 227 3.9.3.1 Blood pressure and heart rate 227 3.9.3.2 P-R and QT in terva ls 227 3.9.4 Summary 227 3.10 Actions of verapamil enantiomers in perfused rat ven t r i c les 232 3.10.1 Overview 232 3.10.2 Isochoric l e f t vent r icu lar developed pressure 233 3.10.3 Ventr icu lar e x c i t a b i l i t y 233 3.10.4 Addit ional comments 237 3.10.5 Summary 237 3.11 Actions of n i fed ip ine and DHM9 in iso la ted perfused vent r i c les 237 3.11.1 Overview 237 3.11.2 Isochoric l e f t vent r icu lar developed pressure 238 3.11.3 Ventr icular e x c i t a b i l i t y 240 3.11.4 Summary 240 4 DISCUSSION 243 4.1 The conscious rat preparation for myocardial ischaemia studies 243 4.1.1 Overview 243 4.1.2 Precis ion of var iables 243 4.1.2.1 Occluded zone and in fa rc t zone 243 4.1.2.2 Arrhythmias 245 4.1.2.3 ECG changes 245 4.1.3 Responses to drugs 246 4.1.3.1 Overview 246 4.1.3.2 Class 1 antiarrhythmics 247 4.1.3.3 Class 2 antiarrhythmics 248 4.1.3.4 Class 3 antiarrhythmics 250 4.1.3 .5 Class 4 antiarrhythmics 250 4.1.3.6 Arachidonic acid metabolites and an t iph log is t i cs 252 4.1.3.7 Other en t i t i es 253 4.1.4 The conscious rat versus other preparations 254 4.1.4.1 Advantages and disadvantages of rats 255 - ix -4.2 Important determinants of arrhythmogenesis in acute ischaemia 256 4.2.1 Role of OZ 256 4.2.2 Role of the autonomic nervous system 258 4.2.3 Role of K + 261 4.2.4 Role of thrombocytes and leucocytes 263 4.2.5 Role of arachidonic acid metabolites 264 4.2.6 Role of l i p i d metabolites 264 4.2.7 Role of heart rate and blood pressure 265 4.2.8 Role of the fast and slow inward currents 265 4.3 Mechanism of action of calcium antagonists in acute ischaemia 268 4.3.1 Role of calcium antagonism 268 4.3.1 .1 Overview 268 4.3.1 .2 Anipamil versus ronipamil 269 4.3.1 .3 (+)- Versus (-)-verapamil 272 4.3.1 .4 Felod ip ine, n i fed ip ine and DHM9 278 4.3.2 Role of calcium antagonism in the myocardium 282 4.3.3 Role of calcium antagonism in the ischaemic ven t r i c le 289 4.3.4 Role of other pharmacological properties 293 4.3.4 .1 Inh ib i t ion of the fast inward current 293 4.3.4 .2 Blockade of a-adrenoceptors 294 4.3.4 .3 Indirect actions 296 4.4 General conclusions 297 4.4.1 Arrhythmogenesis in acute myocardial ischaemia 297 4.4.2 Action of calcium antagonists in acute myocardial ischaemia 298 5 REFERENCES 301 LIST OF TABLES TABLE 1 Summary of groups in the CNS ablat ion study. 2 Concentration of (^-verapami l in blood matr ices. 3 D is t r ibu t ion of (^J-verapami1 in the ven t r i c l es . 4 Haemodynamic ef fects of anipamil and ronipamil before and af ter occ lus ion. 5 Extent of ischaemia, i n f a r c t i on , and ECG changes after occ lus ion: ef fects of verapamil enantiomers. 6 Arrhythmias and morta l i ty fo l lowing coronary occ lus ion: ef fects of verapamil enantiomers. 7 Extent of ischaemia, i n f a r c t i on , and ECG changes after occ lus ion: ef fects of n i fed ip ine and DHM9. 8 Arrhythmias and morta l i ty fo l lowing coronary occ lus ion: ef fects of n i fed ip ine and DHM9. 9 Pre-drug values for haemodynamic and ECG var iables in pithed ra t s . 10 Incidence of PVC in iso la ted ven t r i c l es : ef fects of verapamil enantiomers. - XI -LIST OF FIGURES FIGURE Page 1 Sequence of events on the day of occlusion fo l lowing connection 93 of leads and l i n e s . 2 Anecdote of ECG changes caused by occlusion in a conscious ra t . I l l 3 Effects of ablat ions in the CNS on occluded zone. 159 4 Ef fects of ablat ions in the CNS on arrhythmia score. 161 5 Ef fects of ablat ions in the CNS on the incidence of vent r icu lar 162 f i b r i l l a t i o n . 6 Ef fects of ablat ions in the CNS on the incidence of vent r icu lar 163 tachycardia. 7 Ef fects of ablat ions in the CNS on the log^o number of PVC. 164 8 Ef fects of ablat ions in the CNS on heart rate and blood pressure 166 before and af ter coronary occ lus ion. 9 Ef fects of ablat ions in the CNS on ECG changes fo l lowing coronary 167 occ lus ion. 10 Changes in serum K + concentrat ion, leukocytes and thrombocytes 169 in pithed rats before and af ter coronary occ lus ion. 11 Ef fect of ronipamil and anipamil on arrhythmia score. 172 12 Ef fect of ronipamil and anipamil on the incidence and duration 173 of various arrhythmias during the f i r s t 4 h fo l lowing coronary occlusion 13 Ef fect of ronipamil and anipamil on occlusion-induced ECG changes 179 (R-wave and 'S -T ' segment e levat ion) . 14 Ef fects of fe lod ip ine on 0Z and IZ, and to ta l and arrhythmia- 182 induced morta l i ty during the 0 - 4 h period fo l lowing occ lus ion. 15 Ef fects of fe lod ip ine on the incidence of vent r icu lar f i b r i l l a t i o n 183 and tachycardia during the 0 - 3 0 min and 0 - 4 h periods fo l lowing occ lus ion . 16 Ef fects of fe lod ip ine on arrhythmia score for the 0 - 3 0 min and 184 0 - 4 h periods fo l lowing occ lus ion. 17 Ef fects of fe lod ip ine on log^o n u m D e r of PVC during the 0 - 3 0 185 min and 0 - 4 h periods fo l lowing occ lus ion. - X I 1 -18 Ef fects of fe lod ip ine on blood-pressure and heart rate 1 min 187 before and 1 h after occ lus ion. 19 Ef fects of fe lod ip ine on maximum S-T elevat ion and the time at 188 which maximum S-T elevat ion occurred. 20 Changes in P-R and QRS in response to {*)- and (-)-verapami1. 191 21 The ef fects of ( ± ) - and (-)-verapamil on blood pressure and 193 heart rate at 1 min before occ lus ion. 22 The ef fects of (+)- and (-)-verapamil on the incidence of PVC 197 in re la t ion to the time after occ lus ion. 23 The ef fects of (+)- and (-)-verapami1 on the incidence of 199 vent r icu lar tachycardia in re la t ion to the time after occ lus ion. 24 The ef fects of (+)- and (-)-verapamil on the incidence of 200 vent r icu lar f i b r i l l a t i o n in re la t ion to the time after occ lus ion. 25 The ef fects of (+)- and (-)-verapami1 on arrhythmia score. 201 26 Mean percent changes in blood pressure and heart rate from pre- 205 drug values in response to (+)- and (-)-verapami1, before and af ter occ lus ion. 27 The ef fects of (± ) - and (-)-verapami1 on S-T segment elevat ion 206 at various times before and af ter occ lus ion. 28 The ef fects of (± ) - and (-)-verapamil on R-wave amplitude 207 at various times before and af ter occ lus ion. 29 The ef fects of n i fed ip ine , DHM9 and vehic le on P-R and QRS 211 in terva l before occ lus ion. 30 Mean percent changes in blood pressure and heart rate from pre- 212 drug values in response to DHM9 and n i fed ip ine , before and af ter occ lus ion. 31 Ef fects of n i fed ip ine and DHM9 on arrhythmia score. 213 32 Effects of n i fed ip ine and DHM9 on serum K + concentrat ion. 217 33 Ef fects of (+)-, (*)- and (-)-verapami1 on OZ and IZ in the 220 'prel iminary sc reen ' . 34 Ef fects of (+)-, (*)- and (-)-verapami1 on tota l morta l i ty 221 and morta l i ty associated with sudden convulsive-type behaviour during the 0 - 4 h period after occ lus ion, and morbidity during the 5 min period before occ lus ion . - x i i i -35 Ef fects of (+)- and (-)-verapami1 on mean aor t ic blood pressure 222 heart rate and P-R in terva l in experiments to supplement the 'prel iminary sc reen ' . 36 Ef fects of (+)- and (-)-verapamil on threshold voltage and 224 threshold pulse width for induction of f i b r i l l o - f l u t t e r , and maximum fol lowing frequency in conscious rats subjected to e l e c t r i c a l st imulat ion of the l e f t ven t r i c l e . 37 Time-course of changes in mean aor t ic blood pressure and heart 226 rate in rats fo l lowing p i th ing . 38 Effects of (+)- and (-)-verapamil on mean aor t ic blood pressure 228 heart rate in pithed ra t s . 39 Effects of (+)- and (-)-verapami1 on Q-T and P-R in te rva ls in 229 pithed ra t s . 40 Dose-response curves for the ef fects of (-)- and (+)-verapami1 234 on developed pressure in Langendorff-perfused rat ven t r i c les paced at 300/min. 41 The re la t ionsh ip between negative inotropic potency of (-)- and 235 (+)-verapamil and K + concentration in iso lated Langendorff-perfused ra t ven t r i c l es . 42 The re la t ionsh ip between negative inotropic potency of n i fed ip ine 235 and K + concentration in iso la ted Langendorff-perfused rat ven t r i c l es . 43 The ef fect of d i f ferent buffer K + concentrations on the 241 threshold voltage and pulse width for capture of the l e f t ven t r i c le in the Langendorff-perfused rat ven t r i c le preparat ion. - X I V -LIST OF ABBREVIATIONS action potent ial AP act ion potent ial duration APD arrhythmia score AS calcium Ca conductance g conduction ve loc i ty e current i e f fec t i ve ref ractory period ERP hour(s) h in fa rc t zone IZ length constant x maximum d ias to l i c potent ial MDP membrane potent ial E m minute(s) min occluded zone OZ potassium K premature vent r icu lar contract ion PVC resistance r second(s) sec slow inward current i $ ^ sodium Na sodium current n*Na standard error of the mean s.e.mean time constant tau vent r icu la r f i b r i l l a t i o n VF vent r icu lar tachycardia VT - XV -ACKNOWLEDGEMENTS In order to ensure blindness in experimentation, the fo l lowing people k ind ly assisted with drug d i l u t i on and (occasional ly) with drug administra-t i on : Kathleen Saint , Tony Au and Michael Walker. Experiments c i ted in the text as having been carr ied out by others in the laboratory (measurement of c i r cu la t i ng p la te le ts and leukocytes) were carr ied out by Kathleen Saint . Dr R Wall i s thanked for his guidance with the HPLC experiments. The calcium antagonists used in the experiments were requested from and supplied by the fo l lowing: Dr B. Ljung of Haessle ( fe lod ip ine) ; Drs M. Raschack and R Kretzschmar of Knoll (opt ical enantiomers of verapamil, anipamil and ron ipami l ) ; Dr R. Whiting of Syntex (DHM9); Dr Kazda of Bayer (n i fed ip ine) . I am indebted to the Department of Pharmacology and Therapeutics, The Univers i ty of B r i t i sh Columbia and the B. C. Heart Foundation, a l l of whom provided me with f i nanc ia l support. I would l i k e to thank the fo l lowing people for the i r support and encouragement concerning the experiments which were carr ied out, and for the i r kindness and advice during my time in the department; Dr Bernard MacLeod, Dr Catherine Pang, Dr Morley Sut ter , Carol ine Bruce and Hari Nai r . I must also thank Tannis for putt ing up with my work being scattered across the kitchen tab le , and my bad temper. F i n a l l y , I am indebted to my supervisor, Dr Michael Walker, not only for his encouragement, for thr ightness, and for his example as a person searching for the truth (even in the face of advers i ty ) , but also for presenting me with a problem to so lve , a hypothesis to t es t , an establ ished preparation with which to begin my studies and an essen t ia l l y f ree hand to chose my own d i rec t i on . One could ask for nothing more. - xvi -AUTHORISATION FROM ANIMAL CARE UNIT, U. B. C. The experiments carr ied out in pa r t ia l fu l f i lment of the requirements for the degree of doctor of philosophy, and described in th is t hes i s , were approved as e th ica l by the Animal Care Unit of U. B. C. (reference number 511/W8). - 1 -1 INTRODUCTION 1.1 C l i n i c a l myocardial-ischaemia-and in fa rc t ion 1.1.1 Overview Myocardial ischaemia is def ined, simply, as an impairment of coronary blood f low, and myocardial in fa rc t ion is defined as the necrot ic changes resu l t ing from myocardial ischaemia ( e . g . , Steadman's Medical Dict ionary, 1972). However, myocardial ischaemia and in farc t ion i s a heterogenous disease, varying from patient to patient in cause, l oca t ion , sever i t y , t ime-course and sequelae (Henderson, 1984; O l i ve r , 1982; Poole-Wilson, 1983; H i l l is and Braunwald, 1977; Maseri et a l 1 9 7 8 ) . I t i s general ly accepted that coronary blood flow insu f f i c iency i s the common event which produces the s igns, symptoms and sequelae of myocardial ischaemia and i n fa rc t i on . Ischaemia may involve par t ia l r e s t r i c t i o n or complete loss of blood f low, and may be permanent or temporary. Temporary ischaemia is associated with reper fus ion, which may occur gradually or abruptly (Fol ts et a l ; , 1982; Maseri et a l ; , 1978). Symptoms of acute myocardial ischaemia include angina pector is (chest pain) and d i zz iness , signs include syncope, vent r icu lar arrhythmias, hypotension, S-T segment a l te ra t ions , a reduction in cardiac output and leakage into the systemic c i r cu la t i on of creat ine phosphokinase, and sequelae include vent r icu lar arrhythmias, i n fa r c t i on , a prominant Q wave in chest lead ECGs, heart f a i l u r e , pulmonary oedema, and hypertension (Henderson, 1984; O l i ve r , 1982). The most common presentation of myocardial ischaemia is angina pecto-r i s . Angina of e f fo r t i s associated with rad iat ing chest pain and S-T seg-ment a l te ra t ions resu l t ing from an increase in myocardial oxygen demand in the set t ing of an inadequate oxygen supply, and r e l i e f may be obtained by reducing sympathetic dr ive to the heart with B-adrenoceptor antagonists, or by d i l a t i ng coronary vessels with organic n i t r i t e s (see Poole-Wilson, - 2 -1983). Pr inzmetal 's variant form of angina (Prinzmetal e t - a l ; , 1959) may occur at rest in the absence of any apparent sympathetic nervous system-med-iated increase in myocardial oxygen demand. Angina of e f fo r t i s general ly associated with coronary a r te r i osc le ros i s^ whereas Pr inzmetal 's var iant i s not. The l a t t e r i s believed to be associated with coronary vasospasm (Hel lstrom, 1973; 1977), although th is i s not yet f i rm ly estab l ished. Ischaemic heart disease can often be at t r ibuted to coronary a r t e r i o -s c l e r o s i s . Stenosis (narrowing of vessels) leads to a loss of physio logical reserve, resu l t ing in i nsu f f i c i en t oxygen supply during periods of increased demand (angina pec to r i s ) . Sustained stenosis of su f f i c i en t sever i ty may lead to i n fa r c t i on . Coronary a r te r iosc le ros i s is pr imar i ly a large vessel disease (Gensini et a l , 1971), and morta l i ty resu l t ing from acute coronary occlusion is associated with s ingle vessel disease in 84% of cases (Liberthson et al 1982). Thus, the l e f t anter ior descending (LAD), l e f t circumflex and r ight coronary ar ter ies are the most common s i tes of coronary a r t e r i o s c l e r o s i s - i n -duced ischaemia and mor ta l i ty . In patients with occlusive stenosis in one or more coronary ar tery , the most important determinant of ischaemia is coronary artery anatomy, which var ies with age. Functional co l l a te ra l anastamoses are rare in young humans, but co l l a t e ra l s become larger and more numerous with age, perhaps in conjunction with (and as a consequence of) the development of a r te r i osc le ro -s i s (Baroldi and Scomazzoni, 1967; Fu l ton, 1965; Harr is et a l ; , 1969; Gensini and Bruto da Costa, 1972; Newman, 1981). It i s probable that slowly developing coronary s tenos is , from whatever cause, leads to a compensatory development of coronary co l l a te ra l anastamoses, as has been demonstrated in pigs (Schaper, 1971). - 3 -1.1.2 Infarct ion Most of our knowledge of the in fa rc t ion process and i t s re la t ion to morbidity and surv ival has come from animal experimentation (see below). For th is reason, an exhaustive review of the in fa rc t ion process and i t s prognostic s ign i f icance w i l l not be given here. It i s c l ea r l y undesirable for working muscle t issue to d ie . If s u f f i -c ient myocardial muscle becomes necrot ic and f ibrous then the a b i l i t y of the heart to pump blood w i l l be impaired. Experimental evidence has shown that in fa rc t ion leads to myocyte hypertrophy in surv iv ing t i s sue , but that large in fa rc ts preclude complete funct ional recovery, owing to the surv ival of an i nsu f f i c i en t number of c e l l s ; hyperplasia does not occur (Anversa . e t - a l . , 1984; 1985a; 1985b; 1986). There are two important points which must be examined when in farc t s ize i s considered. F i r s t l y , a c lear d i s t i nc t i on must be drawn between preven-t ion and delay of necros is . It i s conceivable that necrosis could be delay-ed by a drug which causes vasodi la tat ion of co l l a te ra l anastamoses or by a drug which slows myocardial metabolism (reducing the rate of formation of cytotoxic products of anaerobic metabolism). Both types of drug would pre-vent in fa rc t ion provided that the stenosis were resolved (by thrombolysis or bypass surgery). I t i s unfortunate, therefore, that in most c l i n i c a l stud-ies aimed at reducing in fa rc t s ize no del ineat ion of the patient population into those with and those without co l l a te ra l anastamoses i s made, and l i t t l e attempt i s made to ra t i ona l i se the aims of the study in terms of the known pharmacology of the drug under inves t iga t ion . Some drugs have been invest igated for the i r a b i l i t y to reduce in fa rc t s ize in humans on the basis of the i r a b i l i t y to reduce myocardial oxygen consumption or oxygen demand (heart ra te , force of contract ion, e t c . ) . However, i f occlusion is complete, and co l l a te ra l c i r cu la t i on is minimal or - 4 -absent, then no amount of o f f - loading w i l l salvage the ischaemic myocar-dium. Attempting to prevent in fa rc t ion by such a strategy is analogous to attempting to survive in a vacuum by holding one's breath. I t should also be noted that most of the reports in the l i t e ra tu re con-cerning c l i n i c a l in farc t s ize refer to enzymatical ly determined in fa rc t s i z e . There are a var ie ty of techniques for measuring in fa rc t s ize by det-ermining the concentration in the blood of enzymes which are normally only found i n t r a c e l l u l a r ^ , for example creat ine phosphokinase (CPK). I t has been shown that in experimental animals the peak leve ls of serum CPK c o r r e l -ate with in fa rc t s ize determined h i s t o l o g i c a l l y fo l lowing s a c r i f i c e (Shel l et a l ; , 1971). However, i t i s not c lear whether th is re la t ionsh ip s t i l l holds under the inf luence of drugs. It is quite possible that a drug may inf luence CPK without a l te r ing in fa rc t s i z e . The problem c l i n i c a l l y , of course, is that one cannot examine a heart h i s t o l o g i c a l l y unless the patient d ies . Therefore one is dependent in most cases on ind i rec t measures of in fa rc t s i z e . There are angiographic techniques for measuring coronary 133 blood flow in v ivo , e .g . using radioact ive contrast imaging with xenon ( P i t t et a l . , 1969), and ultrasound techniques for imaging an in fa rc t (e .g . Mattrey and Mi t ten, 1984), but these techniques are not normally used in large scale mult icentre c l i n i c a l t r i a l s . 1.1.3 Arrhythmias C l i n i c a l l y , the major type of death in associat ion with myocardial ischaemia i s 'sudden' . Sudden death has been defined as death occurring within 1 h of the patient l as t being seen a l i v e , and has been suggested to occur in approximately 30% of the to ta l patient population with coronary artery disease (Armstrong et a l ; , 1972). Sudden death i s the major cause of death in pat ients with myocardial ischaemia. I t i s estimated that approxi-mately 400,000 such deaths occur per year in the USA alone (Lown, 1982). - 5 -The cause of sudden death is usual ly uncerta in. I t has been shown that approximately 43%of sudden deaths occurring during the 4 weeks fo l lowing the onset of chest pain occur during the f i r s t , hour (Armstrong e t -a l . - , 1972). In add i t ion , i t has been estimated that less than 10% of pat ients dying during the f i r s t hour af ter the onset of chest pain are seen by a physician (Ol iver , 1982). In other words, the majority of patients who die as a resu l t of myocardial ischaemia do so without a de f in i te establishment of the cause of death. In the fol low-up reports of the Framingham study of the epidemiology of sudden death, the fo l lowing comments were made: 'The assignment of sudden deaths to coronary aet iology i s la rge ly by inference, since few other diseases can k i l l in a matter of minutes. Coronary ae t i o lo -gy i s assumed when aor t ic d i ssec t ion , ruptured aneurysm, and pulmonary embo-l ism are excluded c l i n i c a l l y or on postmortem examination. Thus, death within minutes in persons not i l l at the time with a po ten t ia l l y le thal i l l n e s s permits c l a s s i f i c a t i o n as coronary sudden deaths with reasonable cer ta in ty ' (Kannel e t - a l ; , 1984). The cause of sudden death is general ly at t r ibuted to VF (Ol iver , 1972; Campbell, 1983; 1984; e t c . ) . Many c l i n i c i a n s use the terms sudden death and fa ta l VF interchangeably, ( e . g . , O l i ve r , 1982). There are several persuasive reasons in support of t h i s , despite the absence of d i rec t evidence in most cases. F i r s t l y , evidence from work using experimental animals (see exper i -mental sect ion of Introduction) c lea r l y demonstrates that VF occurs during the f i r s t hour af ter coronary occ lus ion, and that th is is the major cause of death at th is time (cardiogenic shock and pulmonary oedema being r e l a t i v e l y rare during the f i r s t hour af ter occ lus ion) . Secondly, i f VF i s the major cause of sudden death then a reduction in ear ly VF should lead to a corres-ponding reduction in sudden death. With regard to th is point , i t was found, in 1956, (Zol l e t - a l . ) that VF in humans could be reverted by applying a DC - 6 -shock to the chest. This procedure was r igourously applied in Seatt le by paramedics in order to d e f i b r i l l ate patients before admission to hosp i ta l , and the resu l t was a reduction in out -of -hospi ta l sudden death of approxim-ate ly 55% (Cobb et a l ; , 1980), confirming that VF and sudden death were re la ted . In add i t ion , an invest igat ion of the ECGs of patients co l laps ing outside of hospital and receiv ing paramedic care within 15 min of col lapse has provided a strong suggestion that sudden death and VF are one and the same; of 426 pat ients , 72 % were in VF when the ECG was f i r s t recorded, and of the remainder, only 1 % were not experiencing vent r icu la r arrhythmias (Liberthson et a l ; , 1982). However, i t i s important to consider that although sudden death is probably caused by VF, there i s no establ ished c r i t e r i o n , in the absence of ECG evidence, for categor is ing a death as hav-ing resulted from VF, and that most sudden death occurs out of hospital (Campbell, 1984) in the absence of ECG monitoring. I r respect ive of the exact incidence of fa ta l VF in c l i n i c a l myocardial ischaemia and i t s contr ibut ion to the body of 'sudden death ' , i t neverthe-less remains that VF can occur during myocardial ischaemia in humans, and that th is event is often fa ta l ( e . g . , Ju l ian e t - a l 1 9 6 4 ) . The natural h is tory of vent r icu lar arrhythmias during myocardial ischaemia and in farc t ion is not well establ ished in humans. This is under-standable since many patients die before admission to hosp i ta l . There is very l i t t l e information concerning the very ear ly phase ( f i r s t few minutes) of acute myocardial ischaemia in humans. Campbell e t - a l . (1981) evaluated 38 previously unmedicated patients admitted to hospital with 'acute myocar-d ia l i n f a r c t i o n ' , and expressed arrhythmia incidence in re la t ion to the onset of the symptoms (not def ined, presumably chest pa in) . I t was found that primary VF (defined as VF occurring in the absence of ' shock ' , heart f a i l u r e or heart block) occurred p r i nc i pa l l y within the f i r s t 4 h af ter the - 7 -onset of symptoms. VF was almost always associated with an i n i t i a l 'R on T' premature vent r icu lar contraction (defined as a QRS complex f a l l i n g within 85% of the prevai l ing QT interval of a normal sinus QRS complex), whereas vent r icu lar tachycardia (VT) was almost never associated with such an occur-rence. VF and R on T premature vent r icu lar contract ions (PVCs) were almost absent af ter 4 h. In contrast , non R on T PVCs and VT (defined as 3 or more consecutive PVCs at a rate of 120/min or more) increased in frequency bet-ween 4 and 12 h af ter the onset of symptoms. It i s of in terest that despite the uncertainty concerning the exact onset of occ lus ion , the degree of s ten-o s i s , the volume of ischaemia, e t c . , in th is study (Campbell et a l ; , 1981), the time d is t r ibu t ion of arrhythmias was not grossly d i f ferent from that reported for dogs (Harr is , 1950), and rats (Johnston et a l . , 1983a). Adgey et a l ; (1971) also examined the natural h is tory of arrhythmias occurring during the f i r s t few hours af ter the onset of symptoms. They examined 284 patients with ECG evidence of 'acute myocardial i n f a r c t i on ' (S-T segment changes or bundle branch block) and found a 31 % incidence of bradyarrhythmia, 25% incidence of PVCs, 10% incidence of VF, 4% incidence of a t r i a l f i b r i l l a t i o n and 0.4% incidence of supraventr icular tachycardia dur-ing the f i r s t hour af ter the onset of symptoms ( i t must be noted that the above does not include the incidence of fa ta l vent r icu lar arrhythmias, which were not analysed). Corresponding incidences during the 3rd and 4th hour were 2, 6, 0.7, 2, 0 and 0%, respect ive ly . The decl ine with time in the incidence of VF corresponds with that reported by Campbell et a l ; (1981). Adgey e t - a l ; (1971) also reported that the incidence of VF 'a f te r 4 h' was low (4%). Since the p o s s i b i l i t y of a second episode of myocardial ischaemia was not explored, i t i s possible that the true incidence of VF occurring af ter 4 h as a resu l t of the i n i t i a l episode of myocardial ischaemia was less than 4%. It is a problem of most c l i n i c a l studies that there i s gener-- 8 -a l l y l i t t l e examination of the time course of ischaemia in re la t ion to the p o s s i b i l i t y of further c r i t i c a l stenosis and occlusion higher up the a r t e r i -osc le ro t i c t ree . Recent c l i n i c a l studies of arrhythmias associated with myocardial ischaemia and in fa rc t ion have concentrated to a cer ta in extent on the desire to predict sudden death/ fatal VF. This is par t ly a resu l t of the hesitance in prescr ib ing antiarrhythmic agents to a l l patients at r i sk of developing myocardial ischaemia and i n fa r c t i on , owing to the occurrence of serious side ef fects with long-term use ( e . g . , Kosowsky et a l ; , 1973; Jel ineck e t a l ; , 1974; Campbell, 1983). In addi t ion, there are no drugs which have been shown to unequivocally reduce c l i n i c a l l i fe - th rea ten ing arrhythmias asso-ciated with myocardial ischaemia and in farc t ion (Campbell, 1983; 1984). Therefore there is not su f f i c i en t j u s t i f i c a t i o n , in terms of cost versus benef i t , for whole-sale prescr ip t ion of a par t i cu la r drug in pat ients at r i sk of developing myocardial ischaemia and i n fa rc t i on . As a resu l t of these considerat ions, corre la t ions between benign arrhythmias and l i fe - th rea ten ing arrhythmias have been sought, under the assumption that a spec i f i c patient population 'at high r i s k 1 of sudden death can be del ineated. In 1967, Lown et a l . proposed that cer ta in arrhythmias (for example, R on T PVCs) const i tuted 'warning arrhythmias' . A r e l a t i o n -ship between R on T PVCs and sudden death had been speculated e a r l i e r (Smirk and Palmer, 1960). For many years, therapeutic decisions were based on the detection of these warning arrhythmias. However, there were contradictory reports ( e . g . , E l -Sher i f e t a l . , 1976; Rabkin et a l ; , 1982) which suggested that 'warning arrhythmias' were just as common in patients not subsequently developing VF as in those who d i d . The la t te r suggestion has been supported by a recent study in which i t was found that a high incidence of complex PVCs ( including R on T) and VT in otherwise healthy subjects was not assoc i -- 9 -ated with sudden death during a 10 year period (Kennedy et a l . - , 1985). The natural h is tory study of Campbell (Campbell et_al±, 1981) showed that whi ls t R on T PVCs were extremely common in patients during the 10 min preceeding VF, they were almost as common in patients not developing VF. Campbell concluded that 'warning arrhythmias' were of no pred ic t ive value for th is reason. In addi t ion, i t i s worth considering that any 'warning' which does not appear un t i l 10 min before a l i fe - th reaten ing event i s of l i t t l e use as a guide to therapy, espec ia l l y out of hosp i ta l . Part of the confusion concerning 'warning arrhythmias' stems from a fundamental phi losphical dichotomy. This concerns the model of arrhythmo-genesis in myocardial ischaemia and in fa rc t ion to which an invest igator subscribes. S p e c i f i c a l l y , i s ischaemia more or less important than i n fa rc -t ion in arrhythmogenesis? In experimental preparations, VF occurs before t issue has become in fa rc -ted (see experimental sect ion in Introduct ion). Therefore, when considering myocardial ischaemia and i n fa r c t i on , the pr inc ipa l prerequis i te for VF would appear to be myocardial ischaemia. In support of th is premise derived from experimental animal studies are the fo l lowing pieces of c l i n i c a l evidence. F i r s t l y , i t has been suggested that most patients who die from sudden death do not have myocardial i n fa rc -t ion when examined postmortem (Lovegrove and Thompson, 1978), ind icat ing that sudden death may have resulted from myocardial ischaemia, but not myo-card ia l i n fa r c t i on . Secondly, i t has been reported that less than 20% of patients resusci tated from VF exhib i t ECG signs of in fa rc t ion such as Q waves in V leads (Cobb e t - a l ; , 1980). This has lead some c l i n i c i a n s (e .g . O l i ver , 1982) to voice the opinion that c l i n i c a l l y , VF i s associated with ischaemia, whereas e l e c t r i c a l i n s t a b i l i t y i s not a feature of in farcted t i ssue , since i t is not v iab le . - 10 -Proponents of the 'warning arrhythmia 1 hypothesis take a d i f ferent view. The ra t iona le for 'warning arrhythmias' i s based on the premise that ' a d i s t i n c t i v e myocardial pathophysiologic derangement long preexists in vict ims of sudden death ' , and that 'because such deaths are the resu l t of VF, i t i s not i l l o g i c a l to assume the abnormality to be e lectrophysio logic and the r i sk indicator to re la te to al tered cardiac rhythm' (Lown, 1982). This philosophy is not supported by the evidence described above. Lown did not include in his scheme any recognit ion of the duration of symptoms (whether the patient is experiencing ischaemia or i n fa r c t i on ) , nor did he examine the re la t i ve importance of re - in fa rc t i on versus re- ischaemia. Lown dismisses the evidence which suggests that 'warning arrhythmias' are of no value in predict ing VF (see Campbell, 1983), and comments that 'the pre-va i l i ng pessimism concerning the p o s s i b i l i t y of protect ing a patient who has experienced VF against recurrence i s unwarranted' (Lown, 1982). Lown may be overstat ing his case, because whereas Campbell considers the ent i re popu-la t ion of myocardial ischaemia and in fa rc t ion pat ients , Lown's studies were car r ied out on a selected subset of myocardial ischaemia/ infarct ion pat ients , namely those patients 'who have experienced malignant vent r icu lar arrhythmias' , and the prognostic c r i t e r i a which were developed therefore only apply to these pat ients . As discussed in deta i l above, th is subset of patients (those who have experienced complex VT) are the very patients who are least l i k e l y to subsequently experience VF (Campbell e t - a l ; , 1981). These are the patients who have e i ther survived the VF occurring during the f i r s t 3 - 4 h of myocardial ischaemia, or passed through th is danger period without experiencing VF. This group, therefore, does not include any of the t ru l y a t - r i sk pat ients . Indeed, pat ients who die of sudden death before receiv ing medical attent ion were not considered in th is a r t i c l e (Lown, 1982). Therefore the statement that ' abo l i t i on of advanced grades of ven-- 11 -t r i c u l a r premature beats prevents the recurrence of po ten t ia l l y le thal arrhythmias' (Lown, 1982) does not apply to myocardial ischaemia/ infarct ion patients as a whole. Campbell, re fer r ing to a l l ischaemia/ infarct ion pat ients , stated that whi ls t many studies have demonstrated that cer ta in drugs reduce R on T PVCs and complex vent r icu lar contract ions, 'no tangible benef i t of suppressing these arrhythmias has been detec ted ' , (Campbell, 1984). Campbell also stated that 'no current strategy for vent r icu lar arrhythmia prophylaxis or treatment in acute myocardial in fa rc t ion i s s a t i s -factory ' (Campbell, 1983). The f a i l u r e to d is t inguish between the various stages and condit ions of myocardial ischaemia and i n fa r c t i on , pa r t i cu la r l y between the i n i t i a l occlusion and the subsequent course of the disease(s) may be responsib le, in part , for the confusion over both prognosis and the assessment of a n t i -arrhythmics. It has been suggested that improvements must be made in th is regard in re la t ion to the design of c l i n i c a l t r i a l s , or 'the treatment of these patients w i l l continue to be made from a posi t ion of abject ignorance' (Bigger, 1984). In summary, there i s no evidence that 'warning arrhythmias' usefu l l y predict sudden death/VF, pa r t i cu la r l y in pat ients who have passed through the f i r s t few hours of myocardial ischaemia. The 'warning arrhythmia' concept is contingent upon d i f ferent arrhyth-mias being part of a continuum. There i s abundant evidence from studies with experimental animals (see experimental sections) which suggests that PVCs, VT and VF do const i tute a continuum, e i ther in non-ischaemic prepa-rat ions (Dresel and Sutter , 1961) or in experimental myocardial ischaemia. Our laboratory has shown that some antiarrhythmics can reduce the incidence of VF without af fect ing PVCs in rats subjected to coronary artery occlusion (Johnston et a l ; , 1983a). With regard to var ia t ions in the natural h is tory of the disease, in par t i cu la r , the incidence of re-ischaemia and r e - i n f a r c t i o n , there is no reason to bel ieve that the arrhythmias resu l t ing from a f i r s t episode of myocardial ischaemia predict in any way the outcome of a subsequent episode of ischaemia. Therefore, in terms of therapy, i t would be f o l l y to withold an e f fec t ive prophylaxis against VF ( i f such a drug were shown to ex is t ) on the basis of an absence of VF (or R on T PVCs) during a f i r s t episode of myocardial ischaemia in one pat ient , while administering that drug to a second patient with a h is tory of VF (or R on T PVCs) during myocardial ischaemia. There are strategies which have been used c l i n i c a l l y to determine which drug should be prescribed in order to prevent subsequent sudden death in patients who have experienced myocardial i n fa rc t i on , based on responses to drugs. An example is programmed e l e c t r i c a l st imulat ion of the r igh t ven-t r i c l e (Fisher et a l ; , 1977). The technique i s based on the p r inc ip le that i f a premature stimulus is del ivered during the terminal phase of r e p o l a r i -sat ion (the 'vulnerable per iod ' ) then VT or f i b r i l l a t i o n may be e l i c i t e d (de Boer, 1921; Wiggers and Wegria, 1940). Essen t i a l l y , the threshold (current or pulse width) for induction of VT or VF is determined before and af ter the administrat ion of various drugs, with the aim of f ind ing a drug which com-p le te ly suppresses the induction of the arrhythmia upon s t imula t ion. I t has been claimed that a pos i t ive response to a drug predicts that treatment with that drug w i l l prevent spontaneous arrhythmias in 90 - 95 % of patients dur-ing the fo l lowing 1 - 2 years (Ruskin et a l • , 1983). Nevertheless, i t should be recognised that 1 5 - 6 0 % of patients in the studies reviewed by Ruskin who did not respond to any drug during e l e c t r i c a l st imulat ion went on to experience no VT or VF during the fol lowing 1 - 2 years, while receiv ing apparently ' i n e f f e c t i v e ' therapy according to the resu l ts of the st imulat ion tes ts . Therefore the 90-95% 'success' rate (Ruskin et a l . , 1983) is perhaps - 13 -misleading. The problem with th is method i s that although i t may be reason-ably e f fec t i ve in demonstrating that a par t icu lar drug may prevent ven t r i cu -la r arrhythmias occurring in the f i r s t 2 years fo l lowing myocardial ischae-mia and i n fa rc t i on , i t does not benef i t the vast majority of pat ients who suf fer sudden death without warning and in whom no programmed st imulat ion has been undertaken. A lso , despite the claimed success of such methods, i t remains that there are no e f fec t i ve agents for preventing sudden death (Campbell, 1983; 1984), and that enormous numbers of people s t i l l die from sudden death/VF. I f one accepts the premise that the arrhythmias occurring during the f i r s t few hours of myocardial ischaemia (Campbell e t - a l ; , 1981; Adgey e t - a l ; , 1971) are the major cause of death in patients with myocardial ischaemia and in farc t ion (Ol iver , 1982), what are the charac te r i s t i cs and importance of the arrhythmias which begin approximately 4 h af ter the onset of symptoms (Campbell et a l . - , 1981)? Essen t i a l l y , la te arrhythmias have only been of in terest c l i n i c a l l y in terms of whether they predict subsequent sudden death (see foregoing paragraphs) or pose a s ign i f i can t health r isk in themselves. There are no c l i n i c a l studies which have attempted to estab l ish the re la t ionsh ip between late vent r icu lar arrhythmias and the onset of ischaemia, the extent of ischaemic muscle mass, the degree of stenosis (residual blood f low) , the frequency and extent of re-ischaemia or the r e l a -t i ve importance of ischaemia versus i n fa rc t i on . The possible ro le of arrhythmias occurring more than 24 h af ter the onset of symptoms in the genesis of sudden death has been discussed, and i t appears that there i s no prognostic value in the analysis of such arrhythmias (Campbell, 1984). In add i t ion , the poor corre la t ion between the i n i t i a l episode of ischaemia and la te arrhythmias is exemplif ied by the observation that although the i n c i -dence of PVCs and VT occurring during the f i r s t 10 h after the onset of - 14 -chest pain correlated with in fa rc t s ize (determined by measuring serum CPK), the rate of PVC measured 1 - 1 0 months af ter the onset of chest pain bore no re la t ion to in farc t s ize in the same patients (Roberts, et a l ; , 1975). This implies that i t i s possible that the so-ca l led la te arrhythmias in humans may be a mixture of arrhythmias associated with an old in farc t (generated by reentry around the i n fa rc t , perhaps; see section on arrhythmogenesis in Introduct ion), arrhythmias associated with pers is t ing par t ia l ischaemia and arrhythmias associated with new bouts of ischaemia. In summary, c l i n i c a l information concerning the aet iology of sudden death and i t s re la t ionsh ip with VF and other vent r icu lar arrhythmias is incomplete, owing to the fact that the majority of patients dying suddenly do so out of hosp i ta l . C l i n i c a l information concerning the natural h is tory of arrhythmias and the i r underlying causes in the min, h, days, weeks and months fo l lowing the onset of symptoms is confusing, perhaps as a resu l t of the desire to estab l ish an ideal protocol for treatment without having to tediously order and c l a s s i f y each condit ion and i t s cha rac te r i s t i cs . How-ever, such an approach i s necessary in order to remove the confusion concer-ning mechanisms of arrhythmogenesis and the contentious aspects of progno-s i s . It i s c l ea r , however, that the current c l i n i c a l approach has not pro-vided any e f fec t i ve prophylaxis against sudden death (Campbell, 1983; 1984; Furberg, 1983b). 1.1.4 Therapeutic approaches Theore t ica l ly the primary therapeutic aim would be to prevent myocardial ischaemia from occurr ing. The Framingham study (Kannel e t a l . - , 1984) suggested that in previously healthy humans, the incidence of sudden death increases with increasing age, t r i p l i n g in males from 2 to 6 per 1000 per-sons between the age groups 45 - 54 and 65 - 74 years. In addi t ion, the frequency in males i s approximately 2 - 3 times that in females. Age and - 15 -sex are not t reatable condit ions at present. However, the Framingham study also iden t i f i ed hypertension, diabetes, c igaret te smoking, obesity and the ra t io of low versus high density l ipoprote in serum cholesterol as important indicators of coronary artery disease and sudden death. While cor re la t ion does not prove cause-and-effect, i t i s general ly considered that a healthy l i f e s t y l e , a balanced d ie t and exercise are associated with a low incidence of coronary artery disease and sudden death. A safe e f fec t i ve prophylaxis against coronary occlusion would be des i r -able. In th i s regard, the resu l ts of a long term t r i a l of asp i r in in appa-rent ly healthy volunteers is expected to be published reasonably soon. In theory, thrombolysis w i l l only be of benefi t in pat ients with e f fec t i ve co l l a te ra l vascu lar isat ion or par t ia l occ lus ion, in whom ischaemia is not complete ( in whom the delay between the onset of symptoms and the i n i t i a t i o n of thrombolysis i s not as c r i t i c a l as i t would be expected to be in patients with complete occlusion in the absence of funct ioning co l l a te ra l anastamo-ses) . Since th is has not been thoroughly invest igated in the many c l i n i c a l studies of thrombolysis (Yusuf et a l ; , 1985), then i t is not possible to comment further in th is regard. Since coronary artery disease remains a s ign i f i can t health r i s k , then i t is expedient to l im i t the sequelae, namely arrhythmias and i n fa rc t i on . There are at least two ways in which attempts can be made to reduce VF and other arrhythmias. F i r s t l y , automatic d e f i b r i l l a t o r s may be implanted. This has been found to be a successful approach (Echt et a l . - , 1985), but since surgery i s required, i t i s desirable to ident i fy patients most at r i sk of sudden death; the implantation of d e f i b r i l l a t o r s is not a pract ice which can be carr ied out on a very large scale at present. In add i t ion , i t has recent ly been suggested that automatic d e f i b r i l l a t o r s can reduce the qua l i ty of l i f e by v i r tue of the anxiety and fear associated with the i r use (Cooper - 16 -et a l ; , 1985). A l te rna t i ve l y , pharmacological prophylaxis could be admini-s tered. Such a strategy would be expected to protect the pat ient without undue inconvenience in terms of side e f f ec t s , inconvenient dosing regimen and frequent fo l low-up. However, a drug su i tab le for th is purpose does not e x i s t , owing to a lack of proven benefi t on one hand, or unacceptable s ide-e f fec ts on the other ( e . g . , Campbell et a l ; , 1984). Along with the search for a cure for cancers, the search for a safe e f fec t i ve agent which may be used in a large number of subjects who might be categorised as at r i sk of coronary artery disease would appear to be the branch of drug research with the greatest potent ial c l i n i c a l impact in terms of prolonging l i f e . In addit ion to arrhythmia prevention, attempts should be made to l im i t i n fa r c t i on . While i t would c l ea r l y be benef ic ia l to prevent in fa rc t ion resu l t ing from coronary occ lus ion, th is is not a r e a l i s t i c goal at present, par t ly because therapy i s usual ly not i n i t i a t e d un t i l hours af ter the onset of symptoms. Experimental studies in animals without e f fec t i ve co l l a te ra l anastomoses have i l l u s t r a t ed that 15 min of ischaemia w i l l lead to i r reve r -s i b l e myocardial c e l l death, even i f complete reperfusion is achieved (Hort and Da Canal i s , 1965b). While unequivocal c l i n i c a l information i s lack ing , i t i s reasonable to suppose that in the absence of extensive co l l a te ra l vascu la r i sa t ion , much the same w i l l occur in humans. Theore t i ca l l y , any measure which reduces in fa rc t s ize i s desirable in order to reduce the l i ke l ihood of "cardiac output f a i l u r e (Maroko e t - a l ; , 1971). However, unless an agent i s given as prophylaxis, i t i s d i f f i c u l t to imagine how i t might prevent i n f a r c t i on , unless i t possesses the capab i l i t y of e i ther converting f ibrous t issue into muscle, st imulat ing myocardial m i tos is , or converting dead c e l l s into l i v i n g c e l l s . At present, no such drugs e x i s t , and prevention of myocardial in farc t ion with a view to l im i t i ng - 17 -pump f a i l u r e and cardiogenic shock (Agress e t a l ; , 1952) has not been demon-strated e i ther c l i n i c a l l y or experimentally (Reimer and Jennings, 1985). However, i t might be hoped that a fas t -ac t ing myocardioplastic growth factor might one day be developed. 1.2 Experimental-myocardial ischaemia and in fa rc t ion 1.2.1 Overview For an animal model of any human disease to be i d e a l , i t should exhib i t the fo l lowing cha rac te r i s t i cs . I t should: a . completely mimic at least one aspect of the disease b. respond in the same manner as humans to drugs c. have the precis ion and accuracy of a good bioassay d. allow various responses to be measured e. be simple and cost l i t t l e in terms of apparatus, t ime, and exper-t i s e . There is no model of myocardial ischaemia and in fa rc t ion which dup l i -cates the human condi t ion. This was the conclusion of the Coronary Heart Disease Task Group Panel Report of 1973 (see Winbury, 1975). In 1978, the Workshop on More Uniform Animal Models and Protocols for Assessment of Interventions to Protect Ischemic Myocardium held at the NIH recommended the study of 3 coronary occlusion preparations, the rat for ' r e l a t i v e l y inexpen-sive and rapid screening of po ten t ia l l y useful t reatments' , the anaesthe-t ised dog ' fo r v e r i f i c a t i o n of ef fect iveness in a more physio logical s i t u -a t ion ' and the conscious dog ' fo r test ing the most promising agents under the most physio logical condi t ions ' (see Reimer et a l ; , 1985). The l a t t e r recommendations may perhaps appear pecul iar when factors other than 'physio-log ica l relevance' are considered (see sect ion dealing with choice of spec ies) . In addi t ion, i t has been suggested that 'no animal heart i s t ru l y comparable to that of man' (Sasyniuk and Na t te l , 1982) implying that r e l a -- 18 -t i ve rather than absolute physio logical relevance i s the issue in question, and as such should not be the primary consideration in the choice of exper i -mental species. 1.2.2 Methods for producing ischaemia Ar te r iosc le ros i s may be induced with a high cholesterol d ie t . However, with the possible exceptions of the hypercholesterolaemic hare (Pearson et a l ; , 1983), mini-p ig (Jacobsson, 1984), and quai l (Cheung et - a l ; , 1983) th is method is not considered to be spec i f i c for the coronary vesse ls . In most inves t iga t ions , generalised stenosis is produced, therefore th is approach i s ra re ly used (Winbury, 1964). Hypercholesterolaemia-induced coronary a r te r iosc le ros i s models are also compromised owing to the necessity that the endpoint (development of in fa rc t ion or arrhythmias) must occur wi thin a prescribed time-frame, or experimentation becomes unacceptably inconvenient. It may be possible to circumvent th is problem by increasing oxygen demand by a t r i a l pacing in the set t ing of a compromised oxygen supply induced by cholesterol feeding, but th is method produces only reve rs ib le , pacing-dependent ischaemia (demonstrated by S-T segment e levat ion) ; arrhyth-mias and in fa rc t ion do not occur (Lee and Baky, 1973). There are several techniques for producing small vessel occlusion and d i f fuse myocardial ischaemia. By in jec t ing starch suspensions (Roos and Smith, 1948), p l as t i c microspheres (Weber et a l . , 1972) or lycopodium spores (Guzman et a l . , 1962), i t i s possible to produce a r te r i o l a r occlusion lead-ing to generalised and d i f fuse myocardial i n f a r c t s . This technique was f i r s t t r i ed using powder, wax, o i l and ink, more than 100 years ago (Panum, 1862, see Tillmanns e t a l ; , 1975). There are many disadvantages of these techniques. F i r s t l y , in order to preclude generalised systemic occ lus ion, i t i s necessary to in jec t the material into a coronary ar tery. This necess-i ta tes e i ther open-chest experimentation, or techn ica l l y demanding coronary catheter isat ion techniques. Secondly, most animals die from cardiac output f a i l u r e (Weber et a l ; , 1972), which is not the major cause of death in c l i n -i ca l myocardial ischaemia (Campbell, 1983; 1984) or in other models of myo-card ia l ischaemia. Th i rd ly , a r t e r i o l a r occlusion and the resul tant d i f fuse ischaemia and in fa rc t ion d i f fe rs extensively from c l i n i c a l occlusion-induced ischaemia which general ly resu l ts from a r te r iosc le ros i s of large coronary ar ter ies (Gensini et a l ; , 1971). While t h i s , in i t s e l f , i s not necessar i ly a disadvantage, the d i f fuse character of the experimental in fa rc t would be expected to be d i f f i c u l t to quant i fy , thereby making i t d i f f i c u l t to deter-mine the ef fect of a drug on in farc t s i z e . In add i t ion , and perhaps more importantly, models of arrhythmogenesis (see elsewhere) are contingent upon the presence of a well defined focus of ischaemia which must be of suf f -i c ien t volume to serve as a substrate for conduction delays and the genera-t ion of in jury currents (Gettes, 1974; Janse, 1982; Kleber e t - a l . , 1978; e t c . ) . These models of arrhythmogenesis would not apply to the d i f fuse ischaemia preparations. Indeed, the d i f fuse ischaemia preparations do not appear to be associated with vent r icu lar arrhythmias at a l l (see Winbury, 1975). There are a var ie ty of techniques for producing occlusion of a major coronary artery by embol isat ion. The 2 major approaches are d i rect produc-t ion of an occlusive embolus and the induction of an occlusive thrombus. Direct embolisation may be induced by in jec t ing mercury (Lluch e t - a l ; , 1969), placing a s ta in less steel cy l inder (Nakhjavan et a l ; , 1968), steel ba l l bearing (R ib ie l ima, 1964) or detachable catheter t i p (Hammer and P i s a , 1962) into a coronary ar tery , or i n f l a t i ng an intracoronary balloon (Corday et a l . , 1974). In add i t ion , i t has been demonstrated that i f a cy l i nd r i ca l magnet i s placed around a coronary ar tery , and small (4 um diameter) iron par t i c les administered, the par t i c les w i l l be captured by the magnet and - 20 -occlude the vessel (Elz inga et - a l , 1969). These techniques are a l l capable of producing complete occ lus ion, although th is must be ve r i f i ed by measuring coronary blood f low. Par t i a l occlusion may be brought about v ia d i rect embolisation by inser t ing a cyl inder of lead f o i l into a coronary ar tery; the cy l inder w i l l pass down the artery for a distance governed by i t s outer circumference, while the degree of stenosis is governed by the inner circum-ference (Johnsrude and Goodrich, 1969). This technique i n i t i a l l y produces a known degree of s tenos is , but the device is thrombogenic, and serves to induce secondary progressive s tenos is . Deliberate thrombogenic techniques include e lec t r i ca l l y - i nduced thrombo-genesis, and placement of a thrombogenic foreign object in a coronary ar t -ery. Placement of an electrode in a coronary artery lumen and another on the chest wall can produce thrombi i f current i s passed between them (Sala-zar , 1961). This study reported d i f fuse thrombi d i s ta l to the intraluminal e lectrode. However, Weiss (1971) managed to produce complete occlusion of the circumflex or LAD artery using s im i la r techniques, and used the prepar-ation to evaluate antiarrhythmic drugs. The e l e c t r i c a l production of throm-bi is used today by Lucchesi for evaluating antiarrhythmic and an t i - i n f a r c t agents in dogs ( e . g . , Patterson et a l • , 1981; 1983). Foreign-body-induced thrombi may be produced by inser t ing thrombogenic objects into coronary vesse ls , for example, magnesium a l l oy or copper hel ices (Kordenat e t - a U , 1972); the resul tant thrombus is general ly located at the s i t e of the i n -se r t i on . The most common means of producing myocardial ischaemia in use today is occlusion of a major coronary artery by cons t r i c t i on . There are a var ie ty of techniques for producing th is end (see below). Occlusion may be gradual or abrupt. Gradual coronary occlusion may be brought about by the use of aneroids - 21 -(Berman et a l ; , 1956), which are r ings of hygroscopic material which swell and progressively reduce the lumen of the artery which they have been placed around. Rings of ge lat in impregnated with diacety l phosphate induce the formation of granulomatous t issue which w i l l gradual ly cons t r i c t the en-c i r c l e d vessel (Asada e t a l . - , 1962), in a manner analogous with aneroids. A s im i la r resu l t can be produced by the use of pneumatic cuffs (Khouri and Gregg, 1967; Bond et a l . - , 1973) or hydraul ic cuffs (Khouri et a l . , 1968; Hood et a l ; , 1970). A major l im i ta t ion of these techniques is the requi re-ment for the placement of a coronary flow-probe in order to estab l ish the extent of occlusion and the moment of complete occ lus ion. A lso , the prob-lems associated with the a r te r iosc le ros i s models ( in addit ion to the problem of generalised systemic a r te r iosc le ros is ) also apply to the gradual coronary occlusion models, namely that production of ischaemia, arrhythmias and in fa rc t ion does not take place within a convenient time period for exper i -mentation. Abrupt, complete coronary artery occlusion is perhaps the simplest and most ra t iona l technique for invest igat ing myocardial ischaemia. This is because two var iables which are d i f f i c u l t to measure and more d i f f i c u l t s t i l l to regulate are removed from the experimental arena. These var iables are the time course of occlusion and the absolute blood f low. The time course of occlusion i s known only for abrupt occlusion techniques, whi ls t bloodflow in an occluded vessel is only known i f occlusion is complete (flow in p a r t i a l l y occluded vessels must be measured d i r e c t l y ) . The major v a r i -able in abrupt, complete occlusion preparations is therefore the extent of co l l a te ra l blood f low. Abrupt occlusion may be produced by clamping an artery with screw clamps (Gregg e t a l -., 1939), bulldog clamps or Goldblat t clamps (Jennings et a l ; , 1960). In small animals (such as rats) with small coronary ar te r ies i t i s possible to cauter ise a major vessel e l e c t r i c a l l y - 22 -(Staab et a l . - , 1977; Prum e t -a l . - , 1984). More simply, a vessel may be 1igated. Coronary l i ga t ion was f i r s t undertaken by Chirac in 1698, who observed a loss of heart movement as a consequence (see Tillmanns e t - a l . , 1975). Interest in the dependence of heart a c t i v i t y on the coronary c i r cu la t i on resumed in the 19th century, when Erichsen (1842) determined the duration of occlusion necessary for producing vent r icu lar s t a n d s t i l l in dogs. Subse-quently, Cohnheim (1881) l igated dog coronary ar ter ies and developed the hypothesis that the coronary c i r cu la t i on i s comprised mainly of end ar ter ies (see Tillmanns e t -a l . - , 1975 for t rans la t ion and d iscuss ion) . In the 20th century, l i ga t ion of a coronary artery together with ECG recording was f i r s t carr ied out in 1918 (Smith), but was rare ly carr ied out again un t i l 1935 (Johnston e t - a l : ) . The reason for the apparent lack of in terest in myocar-d ia l ischaemia in the ear ly part of th is century stems from the general be l ie f at that time that coronary occlusion is a un iversa l ly fa ta l event (see Fye, 1985 for review). In most l i ga t ion techniques a simple s i l k l igature i s used. To gain access to the designated artery and t ighten the l i g a t i o n , experiments were i n i t i a l l y carr ied out using open-chest dogs (Townshend Por ter , 1894). However, for long term experiments on mor ta l i ty , chests were subsequently closed and animals allowed to regain consciousness ( e . g . , Smith, 1918; Le Roy et a l ; , 1942). Most ear ly experiments involved 1-stage coronary l i g a -t i o n . However, an adaptation of a technique designed for par t ia l l i ga t ion of a coronary artery was used by Harris to l iga te coronary ar ter ies in 2 stages (Harr is , 1950). Harr is had observed that abrupt occlusion of the l e f t anter ior descending coronary artery produced VF in approximately 50 % of dogs (Harr is , 1948) within the f i r s t 10 min after l i g a t i o n . Animals s u r v i -ving th is i nsu l t experienced few arrhythmias un t i l approximately 4.5 h af ter l i g a t i o n , whereupon PVCs ensued, increasing in in tens i ty with time; by 8 h, frequent VT developed, pers is t ing for a further 2 - 4 days. However, i t was reasoned that i f the arrhythmias occurring during the f i r s t 10 min of occ lu -sion could be circumvented then the frequency of surv ival would be i n -creased, al lowing for a more deta i led and spec i f i c invest igat ion of the second phase (4.5 - 8 h) and th i rd phase (8 h to 4 days) of arrhythmias. Therefore, an i n i t i a l par t ia l l i ga t i on was undertaken by including a needle in the loop of the l i ga tu re , then removing the needle once the l igature had been t ightened; the lumen of the ar tery was therefore stenosed to the diame-ter of the needle. After 30 min or 1 h, a second l iga ture was tightened at the posi t ion of the f i r s t to f u l l y occlude the vesse l . The resu l t was the el iminat ion of phase-1 arrhythmias (those occurring during the f i r s t 10 min af ter complete occlusion) without change in phase-2 and phase-3 arrhyth-mias. I t i s of in terest to note that th is much-quoted work of Harr is (1950) was in fact pre-empted, to a cer ta in extent, by much e a r l i e r work (Michae-l i s , 1894), in which i t was demonstrated that i f small coronary artery branches were t ied before the larger ar ter ies then cardiac s t ands t i l l was prevented. A s ign i f i can t advance was made by the development of a noose-type device for 'atraumatic ' occ lus ion. Fischer and Edwards (1963) threaded a small polythene tube under a coronary artery and then passed both ends through a larger polythene tube, such that t rac t ion between the tubes would occlude the vesse l . Rushmer e t - a l ; , (1963) used the same p r i n c i p l e , subst i tu t ing a nylon suture for the small polythene tube, in order to produce coronary occlusion in conscious dogs. This technique allowed arrhythmias and i n -fa rc t ion to be invest igated in the absence of anaesthetic and acute surg ica l preparation for the f i r s t t ime. Unfortunately, perhaps, l i t t l e use of th is technique was made for almost 20 years. Coronary occlusion in conscious animals was not invest igated extensively un t i l our laboratory developed a method for coronary occlusion in conscious rats (Au et a l ; , 1979a), using an occluder s im i la r to that used by Rushmer. The use of a 'pre-prepared 1 animal with a loose occluder implanted around a coronary artery and exter-io r ised through the skin for induction of occlusion in the absence of anaesthetic and recent surgery may be an extremely important advance, since recent work has suggested that much of the information provided by i n v e s t i -gations into ischaemia-induced arrhythmogenesis in anaesthetised, acutely prepared animals is misleading (see sect ion dealing with arrhythmogenesis in Discussion). 1.2.3 Differences between species 1.2.3.1 In fa rc t ion . Infarct s ize reduction has been t r a d i t i o n a l l y invest igated in dogs, and therefore the majority of the l i t e ra tu re concer-ning myocardial in fa rc t ion re fers to th is species. However, the v a r i a b i l i t y of in fa rc t s ize in dogs has been suggested to make th is species essen t ia l l y useless for quant i tat ive assessment of in fa rc t s ize and i t s modi f icat ion. This fact was recognised as long ago as 1918, and was at t r ibuted to the presence of large and varied co l l a te ra l anastamoses (Smith, 1918). For th is reason, Johns and Olson (1954) developed the rat preparation for assessing i n fa r c t i on , and concluded that any species with undeveloped co l l a t e ra l vascu lar isat ion (rats and mice) would be sui tab le for evaluating i n f a r c t i on , whereas species with var iable coronary vasculature (hamsters and dogs) and species with extensive co l l a te ra l anastomoses (guinea-pigs) would be unsui t -able for study. Perhaps su rp r i s ing l y , th is work was completely ignored (see sect ion deal ing with coronary occlusion in r a t s ) , and studies of myocardial ischaemia and in farc t ion continued to be carr ied out (almost exc lus ive ly ) using dogs. Indeed, i t was work using dogs which led Braunwald's group (Maroko e t - a l . , 1971) to propose the concept of myocardial salvage. I t was - 25 -suggested that i t should be possible to reduce the ult imate s ize of an in fa rc t by measures 'designed for reduction of myocardial oxygen demands and improvement of coronary per fus ion 1 . It i s perhaps s i gn i f i can t that these invest igators (Maroko et a l • , 1971) did not in fact demonstrate that any manipulation (ouabain, glucagon, propranolol , haemorrhage, methoxamine or isoproterenol) reduced in fa rc t s ize as such. The index of in fa rc t ion used was S-T segment e leva t ion , measured from DC electrograms recorded from the surface of the heart. In only 2 groups was in fa rc t s ize (at 24 h) recorded, and evidence that in fa rc t s i ze was al tered was not convincing. The many experimental and c l i n i c a l studies carr ied out over the fo l low-ing years have not confirmed the hypothesis of Maroko et - al.- (1971) that in fa rc t s ize can be reduced in a po ten t ia l l y c l i n i c a l l y useful manner by drug treatment, under the condit ion of i r reve rs ib le coronary occ lus ion. Although i t is possible to delay both the ECG signs of ischaemia and the development of i n fa r c t i on , the evidence does not suggest that any treatment can prevent death in non-perfused t issue (see Reimer and Jennings, 1984). Cer ta in ly , no treatment has yet been claimed to have prevented i n fa rc t i on . The var iab le nature of the outcome of coronary occlusion in dogs is exemplif ied by a study carr ied out by Sobel and associates (Shell et a l ; , 1971). These authors developed the method of quant i f i ca t ion of in fa rc t s ize based on serum levels of CPK. They found that peak serum CPK correlated l i nea r l y with in fa rc t s ize determined at 24 h by measuring the myocardial content of CPK. However, the actual value of in fa rc t s ize in 22 dogs at 24 h was highly va r iab le , 21.5 ± 18 (mean ± s .d . ) as % vent r icu lar weight, the range of values being 1-55%. These values are typ ica l of published in fa rc t s ize in dogs. For example, Burmeister and Reynolds (1983) reported that the coe f f i c ien t of var ia t ion ( s . d . as a%of the mean) was 23% in mon-grel dogs and 73% in beagles, while Miyazaki et-al . - (1984) reported that - 26 -in fa rc t s i ze varied from 0.6 to 46 %of to ta l vent r icu lar weight, with a 60% coe f f i c ien t of var ia t ion in mongrel dogs. With v a r i a b i l i t y such as t h i s , i t i s d i f f i c u l t to imagine how so many reports of 'myocardial salvage' have been generated and continue to be generated ( e . g . , Jugdutt, 1985; Bednar et a l . , 1985; Tumas et a l . , 1985). Perhaps the many reports of in fa rc t s ize reductions in dogs are the resu l t of sampling er ror , or measurement of 'u l t imate ' i n fa rc t s ize too soon af ter occ lus ion. 1.2.3.2 Arrhythmias. In conscious ra t s , severe vent r icu lar arrhythmias f i r s t occur in the period 4 - 2 0 min af ter occ lus ion , and a second major phase appears af ter 1 h and las ts for 2 - 6 h (Clark e t - a l ; , 1980; Johnston e t - a l ; , 1983a). In dogs, ischaemia-induced arrhythmias were o r i g i n a l l y described as ear ly , occurring during the f i r s t hour, and l a t e , peaking at approximately 24 h af ter coronary occlusion (Harr is , 1950). However, recent ly the ear ly (phase-1) arrhythmias have been subdivided into phase- la , occurring during the f i r s t 1 - 3 min of ischaemia, and phase-lb, occurr ing 5 - 2 0 min after coronary occlusion (Haase and S c h i l l e r , 1969; Meesman, 1982). Phase-la arrhythmias, which usual ly comprise only of PVC, have only been reported in anaesthetised dogs; information concerning the i r occurrence in conscious dogs i s not avai lab le at present. However, the type of anaesthetic used appears to inf luence the occurrence of phase-la arrhyth-mias, in that they are far more frequent in dogs anaesthetised with pento-barbitone than dogs anaesthetised with morphine-chloralose-urethane or ni trous oxide (Meesman, 1982). In add i t ion , phase-la arrhythmias have been reported to occur in pentobarbitone anaesthetised rats (Fagbemi, 1984), but not conscious rats (Johnston et a l . , 1983a). Phase-la arrhythmias have also been reported in pentobarbitone anaesthetised pigs (Bergey e t - a l . , 1982); i t i s not establ ished whether such arrhythmias occur in conscious p igs. - 27 -In dogs 24 h af ter occ lus ion, episodes of VT and mul t i focal PVC are commonly observed (Smith, 1918; Har r i s , 1950). In our laboratory, we have ra re ly observed VT and never observed VF in rats at 24 h, although almost a l l 24 h survivors exhib i t mul t i focal PVCs. The reason for th is di f ference between rats and dogs has not been examined, however i t i s possible to speculate that the presence or absence of co l l a te ra l anastamoses may govern the sever i ty of 24 h arrhythmias. In th is regard, i t has been suggested that abnormal automaticity in p a r t i a l l y ishaemic Purkinje f ib res (maintained by co l l a te ra l perfusion) i s responsible for 24 h arrhythmias in dogs (F r ied -man e t a l ; , 1973; Lazzara e t a l ; , 1973). This idea i s consistent with the mul t i focal nature of these arrhythmias. In r a t s , the re la t i ve lack of funct ional co l l a te ra l anastamoses compared with dogs (Maxwell e t - a l ; , 1984; Winkler e t a l ; , 1984) may not permit surv ival of su f f i c i en t Purkinje t issue to t r igger VT and VF 24 h after occ lus ion. In conscious ra t s , arrhythmias occurr ing during the f i r s t 4 hours af ter occlusion have been described in deta i l on the basis of many experiments in our laboratory (Johnston et a l . , 1983a). Ventr icular arrhythmias are ex-tremely common and include PVC, VT and VF. Sinus bradycardia and a t r i o -vent r icu lar blocks are much less common. Very occas iona l l y , a t r i a l arrhyth-mias ( f i b r i l l o f l u t t e r ) and supraventr icular tachycardia are seen. Spontaneous reversion of VT during acute myocardial ischaemia i s common, both experimentally and c l i n i c a l l y . Spontaneous reversion of VF has been reported in humans (Robinson and Bredeck, 1917; Maseri et - a l . - , 1982), but the incidence i s considered to be low. However, there is l i t t l e object ive evidence to substantiate th i s be l i e f . In conscious rats the incidence of VF i s high (approximately 90 %), and the incidence of spontaneous d e f i b r i l l -ation i s correspondingly high (approximately 60 %). However, the incidence of sustained VF is even higher (approximately 90 %), according to exper i -- 28 -merits from our laboratory (Johnston e t - a l . , 1983a). In dogs, there have been occasional reports of spontaneous reversion of VF ( e . g . , Smith, 1918; Gibson e t - a l . , 1986). In p igs , spontaneous reversion of VF has been observ-ed (unpublished observations from our laboratory) , although th is event i s ra re . In the absence of any evidence that spontaneously revert ing ven t r i c -ular arrhythmias d i f f e r fundamentally in o r ig in maintenance or mechanism from sustained vent r icu lar arrhythmias, there is no reason to bel ieve that spontaneous reversion of VF i s an experimental disadvantage. On the con-t ra ry , our laboratory has shown that spontaneous d e f i b r i l l a t i o n permits the experimental animal to surv ive, al lowing for an increase in the y i e l d of information from each preparation (Johnston e t -a l . - , 1983a). 1.2.3.3 Choice- of -species. The sect ion dealing with c l i n i c a l myocardial ischaemia and in fa rc t ion suggested that most deaths in patients with myocardial ischaemia occur from VF, and that the incidence of VF dec-l ines exponent ial ly with time fo l lowing the i n i t i a l onset of chest pain (Campbell e t - a l ; , 1981). Therefore i t i s of great importance to develop treatments for arrhythmias associated with acute myocardial ischaemia. It has been suggested that a drug which can be taken as prophylaxis by pat ients at high r i sk of myocardial ischaemia to prevent VF would have far more im-pact on morta l i ty than therapy i n i t i a t e d after admission to hospi ta l (Campbell, 1984). This implies that arrhythmias occurr ing during the f i r s t few hours, and pa r t i cu la r l y during the f i r s t few minutes of myocardial ischaemia are the arrhythmias which must be invest igated in th i s regard. C l i n i c a l l y , these acute arrhythmias are general ly missed; a patient has e i ther died from them, been resusci tated by paramedics, or , perhaps, recov-ered spontaneously by the time they are admitted to hosp i ta l . This leads to a dilemma. One may ei ther invest igate ear ly arrhythmias in experimental animals (phase-1 according to Har r i s , 1950) or late (phase-3) arrhythmias. - 29 -Any experimental preparation in which phase-1 arrhythmias are measured cannot be evaluated for i t s ' c l i n i c a l re levance ' , because there is insuf f -i c i en t information concerning these arrhythmias and the i r suscep t i b i l i t y to drugs in humans to provide a useful template. Therefore i t i s desirable to use several animal species for such s tud ies . A l te rna t i ve l y , one may concentrate on phase-3 arrhythmias, under the assumption that drugs which reduce phase-3 arrhythmias w i l l also reduce phase-1 arrhythmias ( in other words that common mechanisms of arrhythmogen-esis operate for phase-1 and phase-3 arrhythmias). Essen t i a l l y , therefore, one must estab l ish one of the two fol lowing c r i t e r i a (depending on which experimental strategy is chosen) in order to j u s t i f y the choice of model and species. F i r s t of a l l , i f i t i s decided to invest igate phase-1 arrhythmias then the only concern should be the l o g i s t i c charac te r i s t i cs of the model. I t must be establ ished that the model provides accurate and precise information (any debate concerning the c l i n i c a l relevance of such a model is redundant). A l t e rna t i ve l y , i f phase-3 arrhythmias are to be studied as a model of phase-1 arrhythmias then i t fol lows that a drug which i s found to i nh ib i t phase-3 arrhythmias should also i nh ib i t phase-1 arrhythmias. Therefore, i t is of absolute necessity to estab l ish that a l l drugs which are e f fec t i ve or ine f fec t ive against phase-3 arrhythmias in the chosen preparation possess the same p r o f i l e of a c t i v i t y against phase-1 arrhythmias in that same prepa-ra t i on . The majority of published reports in which such comparisons have been carr ied out do sa t i s f y th is c r i t e r i o n . It fo l lows, therefore, that the evaluation of drugs for potent ial c l i n i c a l use as prophylaxis against phase-1 arrhythmias must not be based upon an extrapolat ion from a c t i v i t y against phase-3 arrhythmias. Such a strategy i s analogous to invest igat ing i n te r -ventions for preventing f i r e s in the home by measuring the ef fect of the - 30 -experimental intervention on room temperature. I t i s , however, permissible to invest igate phase-3 arrhythmias for the i r own sake. In th is regard, c l i n i c a l relevance may be tested d i r e c t l y , because phase-3 arrhythmias are easy to study c l i n i c a l l y . However, whether phase-1 or phase-3 arrhythmias are studied may have important c l i n i c a l rami f ica t ions . With further reference to the analogy of the study of the prevention of f i r e s in the home, i f acute out-of -hospi ta l VF is analogous to a f i r e , i t may also be that c l i n i c a l phase-3 arrhythmias are merely analogous to an increase in room temperature. In other words, while the prevention of out of hospital VF (phase-1 arrhythmias) w i l l have enormous consequences in terms of l i ves saved, the prevention of phase-3 arrhythmias may have l i t t l e therapeutic impact. A l l the techniques for producing myocardial ischaemia, with the excep-t ion of e lectrocautery, were developed using dogs. Unt i l 1985, dogs were eas i l y the most common species used in studies of myocardial ischaemia and i n fa rc t i on . Recently, the rat has become popular. Popular i ty , however, does not necessar i ly imply s u i t a b i l i t y . There is no concensus at present concerning the ideal choice of species for invest igat ing myocardial ischae-mia and i n fa r c t i on . I t has been argued that there is no ideal species for such s tud ies, and that the choice depends upon the objects and goals of an invest igat ion (Fozzard, 1975; Harken et a l . , 1981). In contrast with animal models of other diseases, such as bacter ia l i n fec t i on , in which a high degree of s i m i l a r i t y between the animal t issue and human t issue is des i rab le , i t i s possible to argue that animals with hearts s im i la r to the human heart are paradoxical ly unsuitable for studies of myocardial ischaemia and i n fa rc t i on . There are several unrelated reasons for th is viewpoint. F i r s t l y , an animal with a heart which resembles the healthy, young human heart in terms of co l l a te ra l vascu lar isat ion is by de f in i t i on a poor substrate for invest igat ions concerned with the behaviour of the chron ica l l y s ick human heart with extensive atheromae and co l l a te ra l development. Such an animal ought, however, to provide a good model of the young human heart . It has been suggested that young humans are more suscep-t i b l e to fa ta l VF than older humans (Morgan Jones, 1969), and i t has been argued that th is is a resu l t of the re la t i ve lack of co l l a te ra l anastamoses in the younger human (Ol iver , 1982). Pig hearts lack co l l a te ra l anastamoses and have consequently been suggested to resemble young human hearts (Fedor et a l . , 1978; Verdouw et a l . , 1983). The problem in th is instance i s that while such an animal may in theory provide a good model of myocardial ischaemia in otherwise healthy young humans, there i s very l i t t l e in fo r -mation concerning the charac ter is t i cs of th is c l i n i c a l s i t ua t i on . Moreover, there is essen t i a l l y no information concerning the effect iveness of drug therapy in such pat ients . Furthermore, un t i l the improbable eventual i ty of a large scale c l i n i c a l study of the effect iveness of a range of drugs in preventing the signs and sequelae of acute myocardial ischaemia in young healthy humans, then such information w i l l remain unavai lable. In such a circumstance, any argument that an animal with few e f fec t i ve co l l a te ra l anastamoses provides a good model of acute myocardial ischaemia in healthy young humans i s merely hypothet ica l . At the other end of the spectrum, i t has been suggested that a species with extensive or varied co l l a te ra l vascu lar isat ion resembles the mature or e lder ly human heart . The mature human heart may develop co l l a t e ra l anasta-moses in response to the slow developmemt of a r te r i osc le ros i s (Schaper, 1971). In dogs the development of co l l a te ra l anastamoses over a period of weeks fo l lowing coronary artery l i ga t i on may represent such a process (Eckstein e t - a l ; , 1941). Amongst common experimental species, dog hearts usual ly have we l l -deve l -- 32 -oped co l l a te ra l anastamoses and may resemble the mature or e lder ly human heart (Schaper, 1971; 1979). C lea r l y , i f we assume that a l l other var iables are equal , then a species with an extensive coronary co l l a te ra l c i r cu la t i on is preferred when myocardial ischaemia in the mature human ( in whom myocar-d ia l ischaemia occurs more frequently than younger humans) is to be modeled. However, there are other considerat ions. One important considerat ion in any s c i e n t i f i c invest igat ion is the pr ior knowledge of the power of the experiment. I f one is interested in the ef fect of a treatment on a Gaussian d is t r ibu ted var iab le then the sample s i z e , n, i s dependent on two indepen-dent va r iab les , the ef fect iveness of the treatment and the var iance. If two species d i f f e r in terms of the variance of a par t i cu la r va r iab le , then provided there are no other confounding fac to rs , the preferred species w i l l be the one with the lower variance. Consideration of which of the 2 species is the most c lose ly re lated to humans becomes of minor importance. There i s a tendency, nevertheless, for aesthet ic rather than l o g i s t i c considerations to d ic tate the choice of species in myocardial ischaemia and in fa rc t ion s tud ies. The dog, un t i l recent ly , has been the most popular species for i n v e s t i -gating myocardial ischaemia and i n fa rc t i on . For example, a large general review of models of coronary artery disease published in 1975 is almost exc lus ive ly devoted to dog preparations (Winbury, 1975). However, there is an h is tory of evidence which warns that var ia t ion in the coronary anatomy of dogs jeopardises myocardial ischaemia studies by const i tu t ing a large source of variance ( e . g . , Smith, 1918). Paradoxica l ly , the dog has been favoured for th i s very reason; the var iable nature of the coronary anatomy of the dog i s q u a l i t a t i v e l y s im i la r to that of the human (see above). However, the question a r i ses : i s i t desirable to use an animal myocardial ischaemia preparation in which the coronary anatomy pa ra l l e l s the human coronary - 33 -anatomy? In th is regard i t is important to consider the sample s ize necess-ary for a c l i n i c a l study of the effect iveness of a drug in reducing mortal-i t y occurring during acute myocardial ischaemia. The problem in th is instance is that the required information is not ava i l ab le , since the acute phase of myocardial ischaemia occurs out of hospital in most cases, and has resolved or has lead to death (see preceeding sec t ions) . Nevertheless, with regard to the la te r ' pos t - i n fa r c t i on ' per iod, in order to show a s t a t i s t i c -a l l y s i gn i f i can t reduction of morta l i ty of 10 - 20% (from approximately 5 - 15% during the f i r s t year) one requires a study sample of several thou-sands (May, 1983a; 1983b). It i s therefore undesirable to use a preparation which c lose ly mimics the human s i t ua t i on . As Meesman has c l ea r l y demonstra-ted, the major source of variance in dogs with regard to the outcome of myocardial ischaemia is the extent of co l l a te ra l vascu lar iza t ion (Meesman, 1982). Co l la te ra l flow correlates not only with arrhythmias (Meesman, 1982) but also with in fa rc t s i z e . I t has recent ly been shown that in fa rc t s ize in dogs can be predicted on the basis of the extent of co l l a te ra l vascu la r i -sation independently of the s ize of the occluded bed ( ' r i sk zone') (Yel lon e t - a l ; , 1986b). In addi t ion, a recent study showed that whereas simple occlusion of the LAD produced only minor vent r icu lar arrhythmias, LAD occ lu -sion coupled with the obstruct ion of retrograde co l l a te ra l blood flow to the occluded bed resul ted in severe vent r icu lar arrhythmias in 75% dogs (Shoji e t - a l ; , 1986). Therefore, a species with l i t t l e e f fec t i ve co l l a te ra l perfu-s ion , productive of consistent and predictable regions of ischaemia upon coronary occ lus ion, would be more appropriate than the dog, in terms of the precis ion and accuracy for a given to sample s i z e . The two species in which a well defined and reproducible area of ischae-mia fo l lowing coronary occlusion has been documented are the pig and the ra t . Coronary co l l a te ra l anastamoses connecting to the l e f t anter ior - 34 -descending coronary artery in the pig are almost absent (Schaper, 1971), leading to a sharp demarcation between the normal and the ischaemic zones fo l lowing occlusion of th is artery (Kleber e t -a l . - , 1978; Janse e t - a l ; , 1979). Several reports suggest that co l l a te ra l s are neg l ig ib le in the rat ven t r i c le (Johns and Olson, 1954; Selye e t -a l . - , 1960; Maxwell e t -a l . - , 1984; Winkler e t - a l ; , 1984; Schaper et a l . , 1986). This suggestion is substant i -ated by the consistent extent of ischaemia and in fa rc t ion in rat hearts fol lowing coronary occlusion (Johnston e t - a l . , 1983a; Bernauer, 1982; Lepran e t -a l . - , 1983; e t c . ) , which compares favourably with the consistency seen in pig studies (Verdouw e t - a l ; , 1983c; Sjoquist e t - a l ; , 1983; e t c . ) . There is one report , however, which suggests that there is a di f ference between rats and pigs in that gradual stenosis induced over a period of 3 months w i l l induce co l l a te ra l growth in p igs , su f f i c ien t to prevent in fa rc t formation upon complete occ lus ion, whereas rats have end a r te r i es , and are unable to develop co l l a te ra l anastamoses, even fo l lowing slow occlusion (Schaper e t - a l ; , 1986). In summary, various hypotheses concerning the nature of arrhythmias occurring c l i n i c a l l y during and af ter myocardial ischaemia have t ranslated into a template for determining the ' c l i n i c a l relevance' of experimental studies (see Reimer et a l . , 1985). This template may be ser ious ly mislead-ing. It must be remembered that far more i s known about the nature of ischaemia-induced arrhythmias and in fa rc t ion in experimental animals than in humans, pa r t i cu la r l y during the c r i t i c a l f i r s t few hours af ter occ lus ion. C l i n i c a l l y , i t has been suggested that a lack of s c i e n t i f i c basis for the management of vent r icu lar arrhythmias is founded in poorly contro l led c l i n i -cal studies (Campbell, 1983). C lea r l y , the confusion concerning the nature of r i s k , cause and the strategy of management of arrhythmias occurring during acute myocardial ischaemia dictates that the c l i n i c a l concepts of - 35 -arrhythmogenesis should fol low from studies with experimental animal prepar-at ions, rather than govern the acceptab i l i t y of an experimental approach. I r respect ive of the arguments concerning c l i n i c a l relevance of d i f ferent species, un t i l a ser ies of drugs have been character ised for the i r a c t i v i t y against arrhythmias and in fa rc t ion in a range of experimental preparations with establ ished precis ion and accuracy, and un t i l these resu l ts have been compared with the analogous condit ion in humans with c lear d i s t i nc t i on between complete and par t ia l occ lus ion, abrupt and gradual occ lus ion, the time of onset of occ lus ion , large and small r i sk zones, the extent of c o l l a -tera l blood supply, the duration of ischaemia in the case of reperfusion, the degree of reper fus ion, the extent of pre-existant in fa rc t ion and the locat ion of pre-existant i n f a r c t i on , then any discussion of c l i n i c a l r e l e -vance is redundant. At present, most experimental preparations have not been characterised s a t i s f a c t o r i l y , and sources of error variance have not been recognised and el iminated. The foregoing chapters have alluded to one fact which ought to play the key ro le in the choice of experimental preparat ion. That i s , without ischaemia there is no i n fa r c t i on , and no ischaemia/ infarct ion re lated arrhythmias. Therefore, the primary consideration should be the reprodu-c ib le production of ischaemia of known sever i ty . The most ra t iona l means of producing ischaemia of known sever i ty is to abrupt ly, completely and perman-ent ly occlude a coronary artery in a species with minimal co l l a te ra l vascu-l a r i sa t i on and l i t t l e intraspecies v a r i a b i l i t y . In add i t ion , i t would be an advantage i f the species were small (not requir ing techn ica l l y demanding preparative equipment or inconvenient housing), inexpensive (to allow large sample s i z e s ) , robust (to allow rapid recovery from preparative surgery) and 'good natured' (to permit conscious animal experiments). There is a species which f i t s a l l these requirements, the common laboratory ra t . - 36 -1.2.4 A h is tory of coronary occlusion in rats Coronary occlusion in rats was f i r s t undertaken in 1946 by Heimburger. His experiments were prompted by the desire to invest igate whether the production of adhesion of the pericardium to the epicardium was capable of preventing in farc t ion (by generation of co l l a te ra l anastamoses of ext racar-diac o r i g i n ) . His experiments were a f a i l u r e , in as much as in ject ions into the per icard ia l sac of cod l i v e r o i l , a lcoho l , sodium s a l i c y l a t e , urea, sodium stearate, soap and sodium morrhuate f a i l e d to prevent i n fa r c t i on . Nevertheless, Heimburger establ ished that l i ga t i on of the l e f t coronary artery in rats produced death within 24 h of occlusion in approximately 25 % of animals, and that extensive in fa rc t ion could be produced. The technique used for occlusion was l i ga t ion using a s i l k suture under pos i t i ve pressure anaesthesia. The study was essen t i a l l y qua l i t a t i ve , and no attempt was made to ve r i f y occ lus ion. Before continuing with the discussion of myocardial ischaemia in r a t s , the question of the v e r i f i c a t i o n of occlusion should be considered. V e r i -f i ca t i on of occlusion is an aspect of experimentation which has been neglec-ted throughout the h is tory of myocardial ischaemia and in fa rc t ion s tud ies . Without such ve r i f i ca t i on i t becomes impossible to d is t ingu ish between animals (or humans) in which a treatment has prevented or delayed in fa rc t ion and animals (or humans) in which occlusion was par t ia l or absent, or present but o f fset by co l l a te ra l vascu la r i sa t ion . Although the nature of co l l a te ra l vascu lar izat ion has been extensively documented in pigs and dogs (e .g . Schaper 1971; Meesman, 1982), very l i t t l e work has been undertaken in r a t s . Ear ly work (Johns and Olson, 1954; Selye et a l ; , 1960) showed that the coronary c i r cu la t i on in the ra t heart is very uniform, and that the coronary ar te r ies are end ar te r ies ( in contrast with guinea pigs in which extensive co l l a te ra l vascu lar izat ion is present) . - 37 -Recent radiolabeled microsphere studies in rats have shown that the extent of co l l a te ra l perfusion in the occluded zone (OZ) is neg l i g i b l e , flow being less than 0.01 ml/min/g according to Winkler e t a l ; (1984) and 6.1 ± 0.7% of normal flow according to Maxwell e t a l ; (1984). It should be mentioned that Kannengiesser et a l • (1975) found that residual flow in the OZ in iso la ted rat hearts was 18% of pre-occlusion f low, as assessed by microsphere techniques. This value i s somewhat higher than the values measured in -v ivo by Winkler e t - a l ; (1984) and Maxwell e t - a l . (1984). The higher estimate of flow i s l i k e l y to be the more accurate, owing to the fact that flow measurements are not dependent simply on the a b i l i t y of a bed to trap microspheres, as is general ly assumed; microspheres tend to d is t r ibu te p re fe ren t ia l l y into beds rec iv ing high f low. The resu l t is an under-estimation of flow in beds receiv ing lower f low. I t has been reported that rats do not develop co l l a t e ra l anastamoses during prolonged and gradual s tenos is , in contrast with p igs , and that in farc t ion is inev i tab le upon complete occlusion in r a t s , in contrast with guinea pigs in which in fa rc t ion is prevented by the extensive pre-existant co l l a te ra l vascu lar isat ion (Schaper e t - a l ; , 1986). Bloor et a l ; (1967) have also invest igated the rat coronary c i r c u l a t i o n . Although they did not look for co l l a te ra l anastamoses, they reported that the l e f t ven t r i c le i s supp-l i ed by a s ing le large coronary artery in more than 90 % of rats in an ex-tremely reproducible and consistent manner. Of the remaining 10 % of r a t s , almost a l l received l e f t vent r icu la r perfusion from 2 main ar ter ies a r is ing from separate o s t i a , and that the second artery was usual ly an independent septal ar tery , not supplying the l e f t vent r icu lar f ree w a l l . The work of Heimburger generated no in terest for many years. However, in 1954, Johns and Olson decided to undertake a detai led comparison of several small animals for the i r a p p l i c a b i l i t y to the study of myocardial - 38 -ischaemia and i n fa rc t i on . The experiments were prompted by the view that the species which had been exc lus ive ly used up to that t ime, the dog, was in essence useless for the study of myocardial ischaemia and i n fa r c t i on ; 'as a test procedure, coronary occlusion in the dog produces resu l ts so var iab le as to have only l imi ted s t a t i s t i c a l usefu lness ' . In th is regard, Johns and Olson were among the f i r s t to recognise that l o g i s t i c s play at least as an important part in the choice of a species for study as physio logical iden-t i t y with the human species. Johns and Olson examined the outcome of coro-nary occlusion in over 500 mice, r a t s , hamsters and guinea p igs. The exper-iments were carr ied out in a manner ident ica l with that used by Heimburger, (1946) except that hearts were exposed v ia an in tercosta l i nc i s ion rather than by thoracotomy. In prel iminary experiments (Johns and Olson, 1954), the coronary c i r c u -la t ion was invest igated by in jec t ing lead oxide (as a suspension in melted gelat in) into the ascending aorta ( b r i e f l y clamped to r e s t r i c t flow to the coronary a r t e r i es ) . The coronary c i r cu la t i on of the rat and mouse were described as s im i l a r . Vessels were c l ea r l y v i s i b l e fo l lowing in jec t ion of contrast medium and d issec t ion . The coronary ar ter ies lay jus t beneath the ep icard ia l surface, and were described as predominantly end ar ter ies ( c o l -la te ra l i n te ra r te r i a l anastomoses being infrequent) . In add i t ion , the l e f t anter ior descending coronary artery (LAD) was described as the dominant ar tery , whereas the circumflex artery was described as merely a small branch of the LAD. For th is reason, i t i s appropriate to describe the artery of occlusion in rats (and mice), that artery which emerges from under the a t r i a l appendage, as the l e f t main coronary ar tery , although most i n v e s t i -gators commonly refer to i t as the LAD. Johns and Olson's studies of the guinea pig (1954) are perhaps worth mentioning, since a comparison of the responses of the guinea pig and rat to - 39 -coronary occlusion has been used to invest igate the ro le of co l l a te ra l c i r cu la t i on in l im i t i ng in fa rc t ion (Winkler e t - a l , 1984). Johns and Olson described the guinea pig as having prominent coronary ar ter ies c l ea r l y v i s i b l e on the ep icard ia l surface. The circumflex was well developed, and both th is vessel and the r ight coronary artery were described as the source of la rge, prominent and profuse co l l a te ra l anastomoses feeding into the region supplied by the LAD. In keeping with the extent and the reproduc ib i l i t y of the co l l a te ra l vascu lar isat ion in rats and mice compared with hamsters and guinea p igs , the respect ive incidences of in fa rc t ion were 83, 64, 31 and 25% (Johns and Olson, 1954). Despite the extent of the i r study, Johns and Olson (1954) generated cer ta in gross pieces of misinformation. In pa r t i cu la r , the authors stated that VF was absent in r a t s . However, there is a simple reason for th is misleading statement. The authors did not record the ECG, and VF was d iag-nosed by observing ' s t r i k i n g changes in rate and rhythm' of the heart while the chest was open. Since the chest was closed immediately af ter occ lus ion , and since la ter studies ( e . g . , Johnston e t - a l . , 1983a) have c lea r l y shown that vent r icu lar arrhythmias do not occur unt i l several min af ter occ lus ion , i t i s no wonder that the authors did not ' see ' VF. Nevertheless, the s ta te -ment that ' i n contrast to dogs, mice and rats are able to survive occlusion of the l e f t main coronary ar tery; VF does not occur' (Johns and Olson, 1954) undoubtedly served to dissuade researchers from using rats in the study of myocardial ischaemia and i n fa r c t i on , despite the conclusion that ' t h i s method of coronary occlusion produces a test in fa rc t which is more nearly standard than any current ly a v a i l a b l e ' . Before discussing further developments in the h is tory of coronary occ lu -sion in the ra t , a comment should be made concerning the smallness of the - 40 -coronary ar ter ies of the ra t , as described by Johns and Olson (1954). Neither Johns and Olson, nor Heimburger (1946) mentioned that the i r occ lu -sions must (by v i r tue of the imposs ib i l i t y of d issect ing free almost i n v i s -ib le artery imbedded in the myocardium) have included some myocardium with the l igated ar tery. It i s possible that i f the artery i s missed and only muscle l igated then spurious 'small i n f a r c t s ' may form, confounding analy-s i s . However, according to Heimburger (1946), l i ga t i on of myocardium alone, or the l e f t coronary vein alone produced no s ign i f i can t sequelae ( in fa rc -t i on ) . In other words, unless the LAD is included in the l i g a t i o n , no in fa rc t ion occurs. From 1954 to 1973 very l i t t l e work was done using rat occlusion prepara-t i ons . Bryant e t - a l ; (1958) used Johns and Olson's technique (1954) to invest igate the in fa rc t process by electron microscopy. They found that gross s t ructura l changes in the myocardium did not occur un t i l at least 5 h af ter occ lus ion, by l i gh t microscopy. However, electron microscopy revealed swel l ing of the sarcoplasmic ret iculum and mitochondrial enlargement af ter only 1 h of occ lus ion. By 2 h, deposit ion of l i p i d bodies, uniform enlarge-ment of mitochondria and par t ia l d isrupt ion of myof ibr i ls were present. Animals sac r i f i ced at la te r times showed increasingly severe i n t r a c e l l u l a r d is rup t ion , such as l i p i d body formation in c e l l nuclei (4 h) , loss of de f in i t i on of myofilaments (5 h) general d isarray of i n t r a c e l l u l a r order (24 h) and f i n a l l y extensive i n f i l t r a t i o n of neutrophi ls and macrophages, with associated phagocytosis (48 h) . The authors made no comments on the i r reasons for using r a t s , and gave no ind icat ion of the reproduc ib i l i t y of the extent of i n f a r c t i on . A year l a te r , Kaufman et a l ; (1959) embarked on a ser ies of studies on in fa rc t ion in rats (a ser ies which appears to have terminated af ter 1 pub l i -ca t ion) . Rats were chosen because they were considered to provide ' large - 41 -numbers' and 'uniform s tock ' . Johns and Olson were c i ted as the source of the technique of occ lus ion. These invest igators (Kaufman e t -a l . - , 1959) showed that occlusion caused depletion of succinate dehydrogenase which began 4 h after occlusion and slowly progressed over the fo l lowing 12 h. In confirmation of the resu l ts of Bryant et a l ; , Kaufman e t - a l ; found that no apparent a l terat ions occurred in gross mycardial structure unt i l at least 6 h af ter occ lus ion . However, these authors observed i n f i l t r a t i o n by mononu-c lear leukocytes beginning at 18 h af ter occ lus ion, reaching 'moderate' proportions by 24 h, at a time when Bryant e t - a l ; (1958) detected no such i n f i l t r a t i o n . The scarr ing process began 36 h af ter occlusion with the i n f i l t r a t i o n of f i b rob las t s . Kaufman e t - a l ; (1959) reported that the dec-reases in succinate dehydrogenase and other enzymes with time para l le led those reported fo l lowing occlusion in dogs according to Jennings e t - a l . (1957), and concluded that the rat was a ' sa t i s fac to ry animal for the study of sequential histochemical and morphologic changes in the myocardium'. The fo l lowing year, the much-cited work of Selye et-al .- (1960) was publ ished. These authors re i tera ted the view of Johns and Olson (1954) that the dog, by v i r tue of the var iab le outcome of occlusion in th is species, i s unsuitable for the study of myocardial ischaemia and i n fa rc t i on . However, Selye e t - a l ; (1960) c r i t i c i s e d Heimburger (1946) and Johns and Olson (1954) for the i r 'complicated' procedure, the high post-operative morta l i ty and the f a i l u r e to produce an in fa rc t with every occ lus ion. To be f a i r to Heimburg-er (1946) and Johns and Olson (1954), the f i r s t 2 c r i t i c i sms are completely i n v a l i d . F i r s t l y , i t appears that a l l 3 groups prepared rats using essen t i -a l l y the same technique. The e a r l i e r 2 studies are superior in as much as they both employed a r t i f i c i a l r esp i ra t i on . Selye e t - a l . (1960) used no a r t i f i c i a l r esp i ra t i on , claiming that they could occlude an artery so quick-l y that none was required. However, the speed was dependent on the rather - 42 -crude expedient of ' e x t e r i o r i s i n g ' the heart, i . e . , temporari ly 'popping' i t through the in tercosta l i n c i s i o n . Secondly, although i t i s d i f f i c u l t to make d i rect comparisons owing to the imprecise declarat ions of time of death, i t appears that per i -operat ive morta l i ty was 25% in Heimburger's study (1946), 21% in Johns and Olson's (1954), and 10% in Selye 's (1960), di f ferences which are not pa r t i cu la r l y s t r i k i n g . In addi t ion, no d i s t i n c -t ion was made between death resu l t ing from occlusion and death resu l t ing from bad surgery and anaesthetic overdosage. The myth that rats do not develop VF fo l lowing occlusion (Johns and Olson, 1954) presumably confounded Selye e t - a l i ' s perception of the super io r i ty of the i r technique, g iv ing r i s e to the assumption that any per i -operat ive death occurs as a resu l t of poor technique. Although Selye e t - a l ; (1960) did not record the ECG, they appeared to acknowledge that the extent of occlusion inf luences s u r v i v a l , since they found that the production of 'smal ler ' in fa rc ts by de l ibera te ly occluding the LAD d i s ta l to i t s o r ig in was associated with a marked reduction in mor ta l i ty , whereas l i ga t i on of the both l e f t and r ight coronary ar ter ies in the same rat resul ted in immediate death in 80% of animals. These authors (Selye e t - a l . , 1960) also confirmed the report of Heimburger (1946) that occlusion of the coronary veins alone produced no sequelae (unless the ent i re coronary sinus was occluded, in which case pe r i ca rd i t i s and super f ic -i a l ep icard ia l c a l c i f i c a t i o n developed). The only other point of in terest in Se lye 's work concerns long term su rv i va l . It was found that animals surv iv ing the f i r s t 24 h af ter occlusion general ly a l l survived the fo l low-ing month, death general ly occurring only as a resu l t of 'accidental i n fec -t i ons ' (Selye e t - a l . , 1960). Several pecul iar publ icat ions appeared in the fo l lowing years. The f i r s t report of ECG information fo l lowing occlusion in rats was given in - 43 -1961, when Normann e t - a l ; , using the technique of Johns and Olson (1954) recorded 5 leads (3 limb and 2 chest) at 2 h af ter occlusion (presumably during br ie f ether anaesthesia). Remarkably, no arrhythmias were observed, although ST segment elevat ion in the anter ior chest lead was apparent, and deep Q waves were seen in the same lead at 24 h and 7 days af ter occ lus ion. A s im i la r study was performed by Zsoter and Bajusz in 1962, and the only arrhythmias seen were a few PVCs in 1 ra t . However in th is study (Zsoter and Bajusz, 1962) the ECG was not recorded un t i l 2 days af ter occ lus ion. No information concerning surv ival af ter occlusion was given. Bajusz, who had previously worked with Selye and Zsoter, re i tera ted the be l i e f that coronary occlusion causes no arrhythmias in r a t s , in his book on in fa rc t ion (Bajusz, 1963). In 1964 and 1965, 4 deta i led and extensive, but completely neglected works were published by Hort 's group (Hort et a l . , 1964; Hort and Da Canal-i s , 1965a; 1965b; Hort 1965). These authors carr ied out coronary occlusion in over 1000 rats by 1 stage and 2 stage l i g a t i o n , with and without reperfu-s ion . According to the English language abstracts fo l lowing the German tex t , the c r i t i c a l duration of ischaemia necessary for production of i r r ev -e rs ib le damage was 13 - 15 min. These authors also stated that in fa rc t ion could only be delineated s u f f i c i e n t l y c l ea r l y for ana lys is , according to the t r iphenyl tet razol ium sta in ing method (Jestadt and Sandr i t ter , 1959), when measured 10 h or more af ter occ lus ion , whereas that t issue destined to become infarcted could be d i f fe rent ia ted from normal vent r icu lar t issue at only 15 min af ter occlusion by l i gh t microscopy, using the c r i t e r i on of 's t reched' versus 'non-streched' muscle f ib res ( ' s t re tched ' being i r r eve rs -ib l y damaged but not yet dead). This c r i t e r i on was suggested to be potent i -a l l y useful in the diagnosis of acute myocardial ischaemia in pat ients who have died from unknown causes (sudden death) without evidence of coronary - 44 -thrombosis or i n fa rc t i on ; th is recommendation has been ignored. The authors concluded that since the standard error of mean in fa rc t s ize was always less than 10% of the mean, then the coronary-1igated rat prepartion was sui tab le for measuring the ef fect of drug treatment on in fa rc t s i z e . In th is regard, these authors (Hort and Da Canal i s , 1965a;1965b) provided sound quant i tat ive evidence of rep roduc ib i l i t y to support the e a r l i e r semiquantitat ive evidence of Heimburger (1946) and Johns and Olson (1954). Despite the c lear exposit ion of ear ly vent r icu lar arrhythmias in dogs fo l lowing coronary occ lus ion, which had been known for decades ( e . g . , Town-shend Por ter , 1894; Smith 1918), i t was not un t i l 1973 that the myth that rats do not experience arrhythmias fo l lowing occlusion was d ispe l led by the simple expedient of monitoring the ECG during and immediately af ter occ lu -s ion . This work was not carr ied out by any of the invest igators who had ea r l i e r proclaimed the rat to be superior to the dog for studying myocardial ischaemia and in fa rc t ion ( e . g . , Heimburger, 1946; Selye e t -a l . - , 1960) but by 2 members of the Hungarian Army Medical Corps (Kenedi and Losconci , 1973a). Kenedi and Losconci recorded the ECG during the 10 min period fo l lowing occlusion (while the ether anaesthesia used in preparation wore o f f ) . Before discussing the i r r esu l t s , i t i s worth mentioning that the technique of Selye e t - a l . (1960) was used in preparat ion, and that in every case the act of ex te r i o r i s i ng the heart through the in tercosta l i nc is ion was reported to produce t ransient i r regu lar VT, a t r ioven t r i cu la r block and sinus brady-card ia ; the technique of Heimburger (1946) as used by Johns and Olson (1954) would not be expected to lead to arrhythmias in th is way, since heart exter-io r i s a t i on was not employed. Kenedi and Losconci (1973a) found that during the f i r s t 10 min af ter occ lus ion , a high incidence of vent r icu lar arrhythmias occurred. General ly, a short run of PVC occurred immediately upon occ lus ion, but these i n i t i a l - 45 -arrhythmias ( la ter ca l led phase-la by Fagbemi, 1985) resolved in a l l cases. However, i t was the opinion of these authors that the run of PVC occurring upon occlusion was a resu l t of the mechanical trauma of preparation rather than ischaemia, because these arrhythmias were s im i la r to the pre-occlusion arrhythmias induced by ex te r io r i s ing the heart through the in tercosta l i n c i s i o n , rather than the arrhythmias which occurred a few minutes after occ lus ion. After an unspecif ied i n t e r v a l , a high incidence of PVC, VT and VF occurred. Unfortunately, the exact incidence and time course of the arrhythmias was not given, although of the 5/20 rats which experienced VF or ' f l u t t e r ' , 2/5 d ied. In addit ion to th is f i r s t exposit ion of arrhythmias associated with acute myocardial ischaemia in r a t s , Kenedi and Losconsci (1973a) also noted that a l l rats surv iv ing 24 h af ter occlusion exhibi ted a deep Q wave, and ST segment elevat ion in lead 1 of the ECG. Arrhythmias induced by coronary occlusion in rats were not invest igated again un t i l 1979, when Szekeres' group (Lepran e t -a l . - , 1979; Siegmund e t - a l ; , 1979a; 1979b) and our laboratory (Au et a l . , 1979a; 1979b) began the i r ser ies of experiments, which have continued throughout the 1980s, along with work by Pa r ra t t ' s group (Clark e t - a l ; , 1980; e t c . ) , Winslow (Kane and Winslow, 1980; e t c . ) , Bernauer (1980; e tc . ) and others. In general , the above Investigators have concentrated on arrhythmias, although our labora-tory and Bernauer always measure the OZ and the extent of i n f a r c t i on . Other researchers have concentrated on in fa rc t s ize reductions in ra t s , and have completely ignored arrhythmias, for example, the Harvard contingent (MacLean e t - a l ; , 1976; Kloner e t - a l . , 1977; Pfef fer e t - a l . , 1979; 1982; 1985), Chiar-i e l l o ' s group (Ch ia r ie l l o et a l . , 1980; 1983; 1984; 1985), Flaim and Ze l i s (1981) and others. In doing so, these authors ignore censoring associated with VF-induced morta l i ty ; in most cases, the numbers of ear ly deaths and the i r causes are not given. - 46 -Independently of whether in fa rc t ion or arrhythmias are the main subject of study, the h is tory and past l i t e ra tu re concerning the use of the rat have been almost un iversa l ly ignored. For example, the o r ig ina l work of Kenedi and Losconci (1973a) has only been appropriately c i ted twice (Clark e t a l , 1980; Abrahamsson and Almgren, 1980). In add i t ion , c red i t is general ly given to Selye (1960) for inventing coronary occlusion in ra t s , whereas in r e a l i t y the technique was developed by Heimburger, 14 years e a r l i e r . 1.3 Ventri eu1ar-arrhythmi as -i n - acute myocardi al i schaemi a This chapter is concerned with the genesis of ischaemia-induced ven t r i -cular arrhythmias. Many of the concepts associated with arrhythmogenesis, such as reentry (Mines, 1913) have been under invest igat ion for many years. An understanding of what l i e s behind the gross pathologic expressions of heart disease such as VF would be expected to ass is t in the predict ion of which type of drug might be of benef i t . However, i t i s also worth consider-ing that the cart often comes before the horse, in that many c l i n i c a l l y useful drugs have been developed on the basis of a purely empirical app-roach. In essence, a mult i layered approach to the problem of ischaemia- in-duced arrhythmias i s ind icated, and i t would be foo l i sh to dismiss an empir-i ca l approach on the grounds that i t does not take su f f i c i en t considerat ion of the underlying pathologic mechanisms. For example, the an t i -u lcer H,, receptor histamine antagonists were developed from the premise that h i s t a -mine was somehow involved in ulcerogenesis v ia a mepyramine-insensitive mechanism. What that mechanism i s , in terms of the biochemical basis for the generation of a gast r ic les ion and i t s precise re la t ionsh ip with stomach pH, remains uncertain. Nevertheless, cimetidine and ran i t i d ine have proven to be e f fec t i ve an t i -u lcer agents. This chapter attempts to i l l u s t r a t e what i s known and what remains to be ascertained with regard to the e lect rophys io log ica l basis of arrhythmo-- 47 -genesis in acute myocardial ischaemia. In add i t ion , an attempt has been made to examine why and how calcium antagonists might be of benefi t in such circumstances. As background information, a br ie f summary of some of the major features of the electrophysiology of exc i table t issue i s also given. 1.3.1 The electrophysiology of exc i table t issue Arrhythmogenesis is an e lect rophysio log ica l phenomenon. In order to understand the electrophysiology of myocardial ischaemia i t i s necessary to understand normal heart e lectrophysiology. Normal vent r icu la r function is dependent upon the propagation of depolar isat ion. The charge i s ca r r i ed , as in the case of conduction in nerves, by ions. The charac te r i s t i cs of con-duction are governed in part by the passive e l e c t r i c a l propert ies of the t i s sue , and in part by the act ive or dynamic propert ies of the t i s sue . The passive e l e c t r i c a l propert ies may be approximated according to the cable equations developed by Kelv in (1855). If the resistance across the c e l l membrane i s high compared with the i n t r a c e l l u l a r and ex t race l lu la r res is tance, then any transmembrane potent ial di f ferences and currents can be considered as functions of longi tudinal distance and time, only. For such an ' i d e a l ' cab le , the change in membrane potent ia l resu l t ing from the focal appl icat ion of current w i l l decay in distance and time according to : d 2 E m / d x 2 . l / r 2 = C m .dE m /d t + i . where x is distance along the cable from the current source, E m i s memb-J m rane po ten t ia l , r 2 i s i n t r ace l l u l a r f l u i d res is tance, C m i s membrane capacitance, t i s time and i . is the component of the membrane current carr ied by ions (Hodgkin and Huxley, 1952a). Rearrangement of the cable equation gives r i se to two membrane constants which are often useful in the understanding of e lectrophysiology. The length constant, x = J r m / r 2 describes the distance along the membrane from a current source at which the resul tant potent ia l change has f a l l en to - 48 -1/e (approximately 37%) of that at the point of the current source. The time required for V (Em) to decay from i t s i n i t i a l value (VQ) to 63% of th is value gives the product R m C m , commonly ca l led the time constant of the membrane ( tau m ) . This ar ises because the membrane potent ia l at time t a f ter appl icat ion of a current across the membrane i s given by: When t is equal to R m C m , then V. = V m ( l - e - 1 ) , and 1-e" 1 = 0.63. Ill i l l U IT laX The act ive e l e c t r i c a l propert ies of the t issue depend upon the asymmet-r i c a l nature of exc i tab le t i ssue . The oriented enzyme, Na +-K +-dependent + ++ ++ ATPase, the Na -Ca exchanger, i n t r a c e l l u l a r Ca sequestrating proteins such as calsequestr in and other energy-dependent processes serve to generate a chemical potent ial gradient for several charged e n t i t i e s . In addi t ion, Donnan equi l ib r ium, the presence of a r e l a t i v e l y f i xed impermeant body of i n t r a c e l l u l a r anionic pro te in , and the presence of a r e l a t i v e l y high rest ing K + permeabil i ty versus that of other charged e n t i t i e s , leads to the establishment of a membrane po ten t ia l . F i n a l l y , as w i l l be discussed below, i . does not obey Ohm's Law. This v a r i a b i l i t y in ion ic conductance (g.) const i tutes the operational mechanism of the act ive e l e c t r i c a l prop-e r t ies of exc i tab le t issue which leads to propagation. At r es t , t issue in the heart (excluding nodal) can be said to approxim-ate a resistance-capacitance c i r c u i t with a K + bat tery, such that the resu l t ing rest ing membrane potent ial i s given by the Nernst equation: E m = R T / F . l n ( [ K + ] o / [ K + ] . ) + + + where [K ] and [K ] . are the local concentrations of K on the immediate outside and inside of the membrane, respec t ive ly . The Nernst equation for K + gives the reversal potent ial for the K + current , which is approximately -90 mV under normal physio logical condit ions ( [ K + ] Q approximately 2.5 - 4.5 meq/1 and [ K + ] i 120 - 140 meq/1), and - 49 -th is corresponds reasonably well with the rest ing membrane potent ial of most non-nodal heart t i ssue . This model is based on the assumption that at r es t , the K + permeabil i ty of the membrane far exceeds that of other charge-car-rying species, such as Na and Ca . The act ive dynamic propert ies of exc i tab le t issue may be approximated by var ia t ions in the equations which were developed by Hodgkin and Huxley (1952a; 1952b; 1952c; 1952d) to explain the rec t i f y i ng propert ies of e x c i t -able t i ssue . In th is instance, r e c t i f i c a t i o n is the deviat ion of the cur-rent-voltage re la t ionsh ip from l inear (ohmic) as a resu l t of vol tage-dependent changes in ion ic conductance. Rec t i f i ca t i on i s explained by invoking the concepts of separate ion ic currents associated with r e l a t i v e l y ion-se lec t ive channels which may vary with membrane potent ial and/or time (so-ca l led voltage and time dependence). The equations describe the beha-viour of these conductances in re la t ion to membrane potent ia l and time. Rec t i f i ca t ion has been demonstrated in Purkinje f ib res (Deck and Traut-wein, 1964). At membrane potent ia ls pos i t ive to approximately -30 mV, there is an increase in a conductance which i s r e l a t i v e l y se lec t ive for K + . The propert ies of th is conductance resemble those of a conductance described by Hodgkin and Huxley in the squid axon which act ivates slowly fo l lowing step changes in holding po ten t ia l , and is c a l l e d , as a consequence, the delayed r e c t i f i e r (see below). The current associated with th is conductance is car r ied mainly by K + , i s outward-going, causes repo lar iza t ion and i s act ivated by depolar isat ion. There i s a second type of r e c t i f i c a t i o n in Purkinje t issue associated with a K + conductance. This conductance is act ivated by changing holding potent ial in voltage clamped (see below) t issue from -30 mV to more negative values. Since depolar isat ion reduces the conductance for th is K +current (as opposed to the behaviour of the delayed r e c t i f i e r ) , th i s current is - 50 -known as the anomalous r e c t i f i e r current , with 'inward going' r e c t i f i c a t i o n . I t i s perhaps important, at th is stage, to comment on the use of voltage clamping. When invest igat ing current f lowing as a resu l t of a change in membrane po ten t ia l , i t i s necessary to el iminate capacitance current ( i c ) . This is done by maintaining membrane potent ial at a constant value (the holding po ten t ia l ) , because i = C m . d E m / d t . Since the value dE m /dt is zero i f E m i s held (clamped) constant, then the term C m . dE m / d t disappears from the cable equation, leav ing: d 2 E m / d x 2 . l / r 2 = i . In other words, the transmembrane current carr ied by ions becomes a f a i r l y simple function of membrane po ten t ia l , under clamped condi t ions. The p r i n -c ip le of voltage clamping was u t i l i s e d by Hodgkin and Huxley (1952a; 1952b; 1952c; 1952d), in order to measure i - in the squid giant axon. According to Hodgkin and Huxley, r e c t i f i c a t i o n may be described mathe-mat ica l ly by invoking voltage-dependent f i t t i n g parameters which govern a conductance channel. In the case of the delayed r e c t i f i e r , conductance i s governed by the parameter, n. For a population of delayed r e c t i f i e r conduc-tance channels, the to ta l current i s dependent on the proportion of channels in the open s ta te . The var iable n behaves according to the equation: dn/dt = a n ( l - n ) - 8 n ( n ) where c*n and &n are voltage dependent rate constants, n i s the molar f rac t ion of channels in the open state and 1-n is the molar f rac t ion of channels in the closed s ta te . Hodgkin and Huxley found that the i r resu l ts best f i t a model where K + conductance (gK) was proportional to n^. The simplest physical concep-tua l i sa t i on of the above re la t ion is that n is a pa r t i c l e which functions to 'gate ' the conductance channel and ex is ts e i ther in an open state (n) or a closed state (1-n). The rate constant a n refers to the rate of conversion of a channel from the rest ing (closed) state to the depolarised (open) s ta te , and v ice-versa . The voltage-dependence of the channel, which ac-counts for i t s rec t i f y i ng proper t ies, is explained by the voltage-dependence of c*n and e n ; the value of ct n increases upon depolar isat ion, while Bn decreases upon depolar isat ion. Although th is model was developed for nerve, manipulation of the values of a n and e n can provide theoret ica l conductance-voltage re la t ions which correspond well with experimental ly-derived data in Purkinje t issue (Noble, 1960; Noble 1962). I t i s suspected that there are many d i f fe rent conductances in the heart which show a range of propert ies. These propert ies include ion s e l e c t i v i t y , ac t iva t ion by changes in membrane po ten t ia l , inac t iva t ion by changes in membrane po ten t ia l , inact iva t ion with time (see below) and reac t i va t ion . In add i t ion , under pathological condit ions such as myocardial ischaemia, some of these propert ies may be influenced by changes in pH, c y c l i c AMP, tempera-ture, e tc . (see Hauswirth and Singh, 1978). The delayed r e c t i f i e r i s voltage-dependent, since a n and Bp vary in s ize with membrane po ten t ia l . However, the Na + conductance (g^ a) in the squid axon (and the s im i la r ' f as t inward current ' in the ven t r i c le ) shows an addit ional property, time dependence. This refers to the observation that the increase in g ^ g which occurs upon depolar isat ion i s t rans ien t , swi tch-ing off ( inact ivat ing) with time. This phenomenon was explained by the invocation of two gating var iables ( in contrast with the s ing le species for the delayed r e c t i f i e r ) , one of which, m, behaves l i k e n of the delayed r e c t i f i e r , sh i f t i ng to the open channel state upon depolar isa t ion, while the second, h, behaves in the opposite manner, sh i f t i ng to the closed state upon depo lar isa t ion . If e i ther n or h is in the closed s ta te , then that pa r t i c -ular channel i s closed (conductance for that channel is minimal), s ince , in - 52 -analogy with genet ics, the open state is recessive while the closed state i s dominant. In order for g^ a to f i r s t increase upon depolar isat ion, then decrease, i t i s necessary that the rate constant, a m , which governs the rate of conversion of m from the rest ing (closed) state to the open s ta te , be larger than a^, the rate constant for the conversion of h from the rest ing (open) state to the closed s ta te , both of which reactions occur upon depolar isat ion. The in teract ion between the fas t inward current ( i ' N a ) and the delayed r e c t i f i e r in time governs the a b i l i t y of an AP to occur, and in space, to propagate, in many exci table t i ssues . Depolar isat ion serves to increase g^ia v i a ef fects on a m . This e f fect leads to more depolar isa t ion. However, depolar isat ion also serves to reduce g^ a v ia ef fects on c^, and also serves to increase gK v ia ef fects on a n . These l a t t e r ef fects serve to repolar ise the membrane. However, since the maximum value of a m i s much larger than that of a n or a^, then provided depolar isat ion is large enough (at or above ' threshold ' ) and occurs qu ick ly (so as to preclude steady-state elevat ion of leading to ' i nac t i va t i on ' ) then pos i t ive feedback of depolar isat ion w i l l occur before the repo lar iza t ion processes are act ivated, producing the AP. The AP is terminated by a combination of inact iva t ion of g^ a and act ivat ion of the delayed r e c t i f i e r . The assymetry of exc i tab le c e l l s leads to the p o s s i b i l i t y of propaga-t i o n , since K i r cho f f ' s law is sa t i s f i ed by the induction of outward current some distance away from the inward current , l inked to the inward current by longi tudinal ( i n t r ace l l u l a r and ex t race l lu la r ) current , which i s induced in turn by the longi tudinal potent ial gradient resu l t ing from focal depolar isa-t i o n . Ampl i f ica t ion may occur in th is c i r c u i t as a resu l t of s im i la r pro-cesses which lead to the AP, as fo l lows. If wi thin a region of membrane depolar isat ion is s u f f i c i e n t l y la rge, and occurs s u f f i c i e n t l y qu ick ly , then w i l l swamp repolar is ing K + current , leading . to further depolar isa-t ion at points further and further away from the i n i t i a l point of depo lar is -ation in a manner described by the cable equation. This manifests as a wave of depolar isat ion (the 'propagated A P ' ) . The threshold for generation of a propagated AP depends upon x m and t a u m . A propagated AP passes as a wave through exc i tab le t i s sue , leading to depolarisation-1 inked events, such as neurotransmitter re lease, or in the case of vent r icu lar t i ssue , coordinated muscle contract ion. The gating propert ies of the conductance channels confer d i rec t ion to conduction in vent r icu lar t i ssue . This is because the propagating wave of exc i ta t ion leaves behind a band of t issue in which g ^ a i s inac t iva ted. The i nac t i va -t ion of g ^ a i s a consequence of depolar isat ion-induced increases in a ^ . This i n e x c i t a b i l i t y i s known as ' r e f r a c t o r i n e s s ' . The duration of re f rac to -r iness under normal circumstances i s dependent on repo la r i sa t ion , since repo lar isa t ion serves to increase and reduce a ^ , leading to a sh i f t in the equi l ibr ium of h to the open-state. With the passing of t ime, and the re-establishment of po la r i sa t i on , as more and more channels become re -ava i lab le for opening, the t issue i s said to pass from the stage of absolute re f ractor iness to re la t i ve re f rac to r iness , and f i n a l l y to the f u l l y exc i tab le s ta te . The conduction ve loc i t y (e = x / t ) of a propagated AP can be derived in terms of the cable equation: e 2 = ( x 2 . d 2 V / d t 2 ) / ( t a u m . d V / d t + V) This der ivat ion predicts that e is d i r ec t l y proportional to x, and inversely proport ional to the square root of t au m . In addi t ion, e i s proportional to the square root of the maximum r i se rate of the AP (dV/dt ). Under max normal circumstances, d V / d t m 3 v i s the major determinant of ©, with © almost l i n e a r l y re lated to the square root of d V / d t m a x . Manipulations - 54 -which ei ther prevent channels from opening (by f i x i n g the channel in the inact ivated s ta te , preventing act ivat ion or phys ica l l y blocking the chann-e l s ) , or slow the k ine t ics of opening by sh i f t i ng the re la t ionsh ip between am and E m to the r ight (to more pos i t i ve values of Em) w i l l reduce e. In most nerve t issue and normal vent r icu lar t i s sue , dV/dt i s governed Hid A by i ^ a . In ischaemic vent r icu lar t i s sue , i t i s possible that d V / d t m a x i s dependent on currents other than the normal fas t inward current (see below). 1.3.2 The electrophysiology of the normal ven t r i c le Variat ions in the act ive and passive e l e c t r i c a l propert ies of vent r icu la r t issue between d i f ferent regions of the ven t r i c le govern the shape of the propagating AP. These var ia t ions are brought about by var ia t ions in the dimensions of the c e l l s in spec i f i c regions and also var ia t ions in the conductance channels present in the d i f fe rent regions. By manipulation of ex t race l lu la r ion composition (for example, replace-ment of Na + with choline or L i + ) , addit ion of substances shown to se lec -t i v e l y i nh i b i t spec i f i c conductances, such as tetrodotoxin which se lec t i ve l y blocks g N a (Moore et a l . , 1967), and by appl icat ion of various techniques for clamping voltage or current such as the recent ly- introduced patch-clamp technique (Lee e t - a l ; , 1980), i t has been possible to describe some of the conductance systems which contr ibute to the propagated AP of the heart . While i t i s possible to study iso la ted heart c e l l s (despite the i r small s ize) by patch clamping (Lee e t - a l ; , 1979), i t i s d i f f i c u l t to study heart c e l l s in vivo because of movement (the organ beats'.) , and unfavourable c e l l u l a r and i n t e r ce l l u l a r geometry (Attwell e t - a l . , 1979). Nevertheless, although some conductances have yet to be f u l l y character ised, i t i s gener-a l l y accepted that the shape of the vent r icu lar AP is mainly governed by three current systems, i N , the outward repo lar is ing currents (carr ied - 55 -mainly by K + ) and the slow inward current ( i s l -» car r ied mainly by C a + + ) . In vent r icu lar t i s s u e , i ^ a with i t s rapid opening and c los ing k ine t i cs dominates the i n i t i a l r i s e of the AP, and governs the d V / d t m , v of depolar-max isa t ion (Beeler and Reuter, 1970a). This in turn governs the conduction ve loc i t y . The proportion of i* N a channels ava i lab le for opening governs the e x c i t a b i l i t y of the t i ssue . Ventr icular i ^ a i s s im i la r to the Na + current described by Hodgkin and Huxley for the squid giant axon, with minor di f ferences (such as the voltage-dependence of the inh ib i to ry action of tetrodotoxin in the v e n t r i c l e ) . Repolar izat ion is associated with several K + currents including a current s im i la r to the delayed r e c t i f i e r current in the squid axon, ca l led i K 2 (McAl l i s ter and Noble, 1966), which is governed by the gating var iable s (analogous to n ) . Several other outward currents may also contribute to repo la r i za t i on , for example, mixed ion currents (predominantly K + ) ca l led \l a n d \2 ( N o b l e a n d Ts ien, 1969). The gK systems inf luence the AP durat ion, since inh ib i t i on of gK delays repo lar iza t ion by prolonging the t a i l of the AP. I f the to ta l duration of the AP (often measured as the time for 90 % repo la r i sa t ion , or APD90) is increased, then the ref ractory period is increased. This i s because the membrane potent ial must remain s u f f i -c i en t l y negative for a time su f f i c i en t for conversion of the h var iables from the i r closed to the i r open state ( react ivat ion) for e x c i t a b i l i t y to be restored. In th is regard, i f Purkinje t issue i s clamped at -70 mV then the g N a system is 50 ^ inact ivated (Weidmann, 1955a). The th i rd major current in vent r icu lar t issue i s the slow inward current ++ ( i s . j ) . This current is carr ied mainly by Ca , and is the major deter-minant of the plateau of the AP in vent r icu lar t issue (Mascher and Peper, 1969; Beeler and Reuter, 1970a; 1970b; New and Trautwein, 1972). As such, - 56 -serves to govern the AP durat ion, in conjunction with the repo lar is ing outward K + current systems described above. The l a t t e r may themselves be par t ly governed by i ^ , since i ^ causes a t ransient r i se in unbound i n t r ace l l u l a r C a + + concentration which has been shown to enhance some K + conductances (Isenberg, 1977b; 1977c). In the ven t r i c l es , restorat ion of normal e x c i t a b i l i t y af ter the propagation of an AP requires that i .. as well as i ^ a recovers from inac t i va t ion . Since the duration of the AP is governed by i .. to a large extent, then re f ractor iness i s also dependent on i . . Inh ib i t ion of i ' s i w i l l shorten AP duration at 25% r e p o l a r i -sat ion (APD25), but APD90 may ei ther increase or decrease, presumably accor-ding to the dependence of the t a i l of the repo lar is ing current on free i n t r a c e l l u l a r C a + + ( th is may vary from one type of vent r icu lar t issue to another, and may also vary from species to spec ies) . It i s possible that during myocardial ischaemia, i . plays an important ro le in addit ional aspects of conduction in the vent r icu lar t i s sue , as w i l l be discussed in deta i l below. Just as i ^ a possesses two gating var iab les , i . has been shown to be governed by two var iab les , denoted as d and f , analogous to m and h, respec-t i v e l y (Reuter, 1973). The voltage-dependence of a d i s such that the threshold for i . i s approximately -50 mV, s l i g h t l y more pos i t ive than that for . For f , the rate constant for conversion to the open state (Bf) fa r exceeds the rate constant for conversion to the closed state (df) at membrane potent ia ls more negative than -60 mV. The rate constants for ac t iva t ion (a^) and inact iva t ion (a^) are much larger ( ind icat ing slower k ine t ics ) than corresponding rate constants for i^ . There is some species va r i a t i on , a£ being 80-200 msec in cat and dog ven t r i cu la r t issue (McDonald and Trautwein, 1978; Reuter and Scholz, 1977), and 10-30 msec in rat vent r icu lar t issue (Isenberg and Klockner, 1980). In the la t te r - 57 -species, the fast k ine t ics of i $ ^ probably account for the abbreviated plateau of the AP (compared with the cat , dog, pig and human vent r icu lar AP), which i s associated with a shorter APD90 of 100 instead of 300 msec (Langer, 1978), and accords with the high rest ing heart rate of 350-450 beats/min. Since vent r icu lar c e l l s are small compared wi th , for example, the squid giant axon, and are part of a funct ional syncytium, i t i s d i f f i c u l t to invest igate the i r electrophysiology in a manner which provides unequivocal information (Beeler and McGuigan, 1978; McDonald, 1982). Furthermore, beating c e l l s are d i f f i c u l t to impale with microelectrodes. Patch clamp studies are favoured at present for the i r power to reveal the propert ies of vent r icu lar conductances. Nevertheless, as one removes c e l l s or membranes from the i r physio logical environment one may introduce factors which con-found inves t iga t ion . For example, i • i s enhanced by pharmacological and physio logical manipulation which increases c y c l i c AMP (cAMP). It i s thought that cAMP t r iggers phosphorylation of the f ' p a r t i c l e ' , leading to slowing of inac t iva t ion of i . and an enhancement of peak i . (Bean e t - a l . , 1984). Therefore, since the basic propert ies of conductance channels in vent r icu lar t issue remain to a cer ta in extent unclear, i t i s possible to suggest many explanations for the mechanism by which a drug inf luences vent r icu lar e lectrophysiology, whether t issue i s normal or abnormal ( ischae-mic, for example). 1.3.3 E lect rophys io log ica l changes caused by myocardial ischaemia The study of the electrophysiology of myocardial ischaemia is confounded by the dynamic unstable charac te r is t i cs of myocardial ischaemia. The e l ec t -rophysio logical propert ies of the ischaemic ven t r i c le vary from one region to another, and also from time to t ime, as i n t r a - and i n t e r c e l l u l a r biochem-i s t r y var ies from one region to another, and changes from time to time. - 58 -Furthermore, the e lect rophysio log ica l changes vary according to whether blood supply i s completely or p a r t i a l l y reduced. In short , these considera-t ions d ic tate that 'the ischaemic myocardium' i s not a s ing le en t i t y . The electrophysiology of the squid giant axon is considered to be well understood, that of the normal heart less so, and that of the heart under the inf luence of regional ischaemia even l ess . Attempts to corre late micro-scopic changes in electrophysiology during acute myocardial ischaemia with the macroscopic consequences of such changes - arrhythmias - have yet to provide a consensus. This is par t ly a resu l t of the d i f f i c u l t i e s in study-ing the electrophysiology of the heart and par t ly a resu l t of the v a r i a b i l -i t y in experimental preparations and approach. Nevertheless, despite a lack of convincing cor re la t ion between spec i f i c e lect rophys io log ica l changes in myocardial ischaemia and arrhythmias, much has been speculated concerning mechanisms of arrhythmogenesis. Recently, i t was demonstrated, using anaesthetised p igs , that the maxi-mum d i a s t o l i c potent ial (MDP) in subepicardial muscle c e l l s begins to f a l l p rec ip i tous ly within seconds of occlusion (Downar e t - a l ; , 1977b; Kleber et a l 1 9 7 8 ) . ' Simultaneously, the AP amplitude, d V / d t m a x and APD90 a l l decrease. The decrease in dV/dt v and APD90 are considered to be secondary to the f a l l in MDP, s ince , as discussed in deta i l prev iously , steady state d i a s t o l i c depolar isat ion leads to inact ivat ion of g N a » However, i t i s possible that ischaemia d i r ec t l y reduces g ^ a independently of MDP changes, although i t is not necessary to postulate such a mechanism. As a conse-quence of the reduction in APD90, the e f fec t i ve ref ractory period (ERP) decreases, since react ivat ion of g ^ a occurs more qu ick ly . However, th is s i tua t ion is not maintained. As MDP continues to f a l l ERP lengthens, des-p i te a maintained narrow APD90. Since ERP continues beyond the point of - 59 -f u l l r epo la r i sa t i on , th is phenomenon i s known as pos t - repo la r i sa t ion- re f rac -tor iness (Lazzara e t - a l ; , 1978). Since post - repo lar isa t ion- re f rac tor iness can be induced by depolar isat ion alone in iso la ted vent r icu la r t issue (Inoue e t - a l ; , 1984), i t fol lows that i t may be possible to explain th is phenomenon in terms of the normal vent r icu lar conductance channels (see below). I t i s important to note that the e lect rophys io log ica l changes described are not uniform in time or space. For example, adjacent c e l l s with very s imi la r res t ing E m may vary in ERP from 180 to 500 msec in pig ischaemic subepicardial muscle (Downar e t - a l . , 1977b). As a resu l t of the f a l l in d V / d t m a x , e f a l l s , producing charac te r i s t i c slow conduction in the ischaemic t i ssue . In add i t ion , depolar isat ion to between -55 and -60 mV is associated with complete inac t iva t ion of g ^ , and under these circumstances complete conduction block may occur. The phenomena described above were recorded in vivo from subepicardial t issue (Downar et a l . , 1977b; Kleber et a l . , 1978). I t i s not possible to record from deeper layers in -vivo, owing to the l im i ta t ions of i n t r a c e l l u l a r recording electrodes (electrodes are too f r a g i l e to be plunged deep into the myocardium and penetrate c e l l s ) . I t i s possible to record from iso la ted s t r ips of subendocardial t issue in v i t r o , but i t i s unclear to what extent the experimental technique inf luences the var iables under invest igat ion (not least because the t issue is general ly superfused, in which case i t i s d i f f -i c u l t to regulate the experimental ischaemia). A compromise preparation is the iso la ted Langendorff-perfusion rat heart in which careful i nc is ion exposes the sub-endocardial t i ssues , while coronary occlusion may be under-taken to produce regional ischaemia. Experiments carr ied out in our labor-atory using th is preparation have revealed s im i la r changes to those des-cr ibed above for subepicardial pig t issue (Inoue e t -a l . - , 1984). In addi -t i on , i t was shown that the changes were more severe and less revers ib le - 60 -with time in the deeper layer c e l l s versus surface c e l l s . The heterogeneity of ischaemic electrophysiology in time and space was c l ea r l y demonstrated in these experiments (Inoue et a l . , 1984). Qua l i t a t i ve l y , i t i s c lear that the act ive and passive e l e c t r i c a l prop-er t ies of vent r icu lar t issue may change with time in a f a i r l y reproducible manner during myocardial ischaemia, and attempts have been made to re la te th i s information to theoret ica l mechanisms of arrhythmogenesis, including reentry, automaticity and tr iggered automatic i ty. 1.3.4 Models Of Arrhythmogenesis 1.3.4.1 Reentry. A b r ie f descr ipt ion of some models of arrhythmo-genesis fo l lows. Reentry was o r i g i n a l l y described as reexc i ta t ion of the myocardium v ia a c i r cu la r route (Mines, 1913). The prerequis i te for reentry is one-way block of propagation in one limb of the reentrant c i r c u i t . The nature and mechanism of reentry, and the roles of re f rac to r iness , conduction ve loc i t y and duration of the 'wave of exc i t a t i on ' were e x p l i c i t l y described by Mines (1913) using the to r to ise heart , and re i tera ted by Schmitt and Ehrlanger (1928) using a t u r t l e heart preparat ion. The mechanism requires that the normal wave of exc i ta t ion brings Em in the blocked limb of the c i r c u i t c loser to threshold for conduction, such that the normal wave can then travel retrogradely up th is limb and reenter i t s previous pathway. For th is to happen, the t issue of the anterograde pathway must be exc i tab le at the time the wave front reenters. This c r i t e r i on w i l l be met i f one of two mechanisms operate. F i r s t l y , i f the c i r c u i t is long enough i t w i l l allow su f f i c i en t time to elapse for the anterograde pathway to recover e x c i t a b i l -i t y . This type of reentry is ca l led macroreentry. The pathway w i l l be func t iona l l y long i f conduction ve loc i t y is reduced, e i ther in the anterograde or retrograde l imb. A l t e rna t i ve l y , i f the re f rac -tory period in the anterograde pathway i s very short ( e x c i t a b i l i t y being - 61 -restored qu ick l y ) , then the requirement for a prolonged in terva l between exc i ta t ion and re-exc i ta t ion is reduced. In th is case the reentry c i r c u i t may be extremely short , perhaps only a few mm (Sasyniuk and Mendez, 1971). Indeed, transmural (ep i - to endocardial) reentry has recent ly been reported (Kramer et a l ; , 1985). This type of reentry i s ca l led microreentry. Acute myocardial ischaemia provides a l l the theoret ica l requirements for reentry: slow conduction, dispersion of re f rac tor iness and areas of 2-way conduction block around which a reentrant impulse may enc i r c l e , and of 1-way block through which retrograde conduction may occur ( e . g . , Janse, 1982). How does one recognise reentry experimentally ? The techniques employed for invest igat ion in open-chest animals have not been successfu l ly applied to c l i n i c a l or closed-chest animal s tudies. In open-chest animals an array of DC electrodes may be placed on the epicardium for mapping the act ivat ion pathways. By th is method, arrhythmias may be v isua l i sed (see paragraphs dealing with automatici ty, below). I t may be poss ib le , in the fu ture, to prepare experimental animals with in-dwel l ing DC electrode arrays. In the c l i n i c a l s i t ua t i on , or in closed-chest animals, standard 12 lead ECGs do not permit one to d is t inguish between reentry and automaticity (see below). Reentry, t heo re t i ca l l y , may give r i se to PVCs or VT. VT may degenerate to VF, in which case the mechanism of i n i t i a t i o n of the arrhythmia w i l l be los t to ana lys is , since VF is usual ly defined as uncoordinated chaotic e l e c t r i c a l a c t i v i t y (Moe e t - a l ; , 1964), without recognisable QRS complexes (Bigger, 1980). In closed-chest experimental animals undergoing repeated episodes of VT, i t has been suggested that the de l ivery of a premature e l e c t r i c a l stimulus to the ven t r i c le can terminate or i n i t i a t e the arrhythmia, i f the mechanism is reentry (Bigger and Goldreyer, 1970). The premature stimulus presumably e i ther conducts through the one-way blocked t issue to i n i t i a t e VT, or depol-- 62 -ar ises the one-way blocked t issue al lowing anterograde conduction of the normal impulse to terminate VT. The use of premature st imulat ion does not permit the recognit ion of reentrant PVCs, only r e l a t i v e l y sustained arrhyth-mias, such as VT. Trains of high frequency vent r icu lar st imulat ion w i l l often i n i t i a t e or terminate reentrant VT by the same mechanism as premature s t imula t ion. Such st imulat ion w i l l i n i t i a l l y capture the reentrant c i r c u i t , then, as frequency is increased, the ven t r i c le w i l l f a i l to fo l low. Once the stimulus is terminated, sinus rhythm w i l l resume. However, since the underlying condi-t ion is not changed, the arrhythmia w i l l shor t ly resume. Unfortunately, th is response is not unl ike the post-overdrive suppression response seen with automaticity (see below). Therefore, in closed chest animals i t i s d i f f i c u l t to prove reentry as the cause of VT. Furthermore, i t i s imposs-ib le to prove reentry as the underlying t r igger for VF, according to the above considerat ions. In open-chest animals, using ep icard ia l mapping techniques, the s i tua t ion is d i f ferent (see below). 1.3.4.2 Abnormal - Automatici ty. The understanding of abnormal automaticity as a mechanism of arrhythmogenesis requires the understanding of normal automatic i ty. During d ias to le , the membrane potent ial in normal vent r icu la r muscle is almost constant. However, in the sinus node a slow spontaneous d i a s t o l i c depolar isat ion precedes and t r iggers the propagated AP. Other t issues in the heart ( for example the a t r ioven t r i cu la r node and the Purkinje f ib res) also show th is slow depolar isat ion, but d V / d t m a x i s greatest in the sinus node. As a resu l t , the frequency of t r igger ing of propagated APs is highest in the sinus node. Consequently the propagated AP or ig inat ing from the sinus node overdrives the spontaneous depolar isat ions in other regions of the heart , and dictates the heart ra te . This cons t i t -utes the pacemaker property of the sinus node. The current responsible for the spontaneous d i a s t o l i c depolar isat ion i s known as the pacemaker current. In the sinus node the pacemaker current has recent ly been shown to resu l t from the in teract ion of two currents (Shibata and G i l e s , 1985), the decay of a delayed r e c t i f i e r K + current ca l led i K , and the act ivat ion of an ++ inward current carr ied by Ca , ca l led i c . The decay of i K i s h ighly voltage-dependent over the range -80 to -55 mV. The act ivat ion of i ^ a occurs at between -60 and -55 mV with peak current at 0 mV. Abnormal automaticity simply means that heart t issue other than the sinus node has taken over the pacemaker ro le of the sinus node (Hoffman and Dangman, 1982; Sasyniuk, 1984). Usual ly , in order for th is to occur, the rate of f i r i n g of the abnormal pacemaker region must exceed that of the sinus node and overdrive the heart (but see comments concerning parasystole, below). The anatomical source of th is pacemaker a c t i v i t y is known as the ectopic focus. Abnormal pacemaker a c t i v i t y may occur as a resu l t of an increase in the steepness of the slope of the d i a s t o l i c depolar isat ion in latent pacemaker t i s sue , or a reduction in the threshold for generation of the (abnormal) propagated AP. The l a t t e r may resu l t from ei ther a reduction in the absolute threshold in mV, or from a sh i f t in the maximum d i a s t o l i c potent ial (MDP) to a more pos i t ive value. The d i f f i c u l t i e s in recognising reentry in closed-chest animals also apply to the recognit ion of abnormal automatici ty. Automaticity may be associated with intermit tant ex i t block whereby the ectopic impulse f a i l s to propagate (Fisch et a l . - , 1971). This w i l l occur i f the ectopic focus d i s -charges jus t af ter the normal wave of exc i ta t ion has passed by (leaving a band of re f rac tory t i s sue ) . Under such circumstances one w i l l see, perhaps, only PVC in the ECG. A l te rna t i ve l y , the ectopic focus may be protected, by entrance block, from overdrive suppression by the normal wave of exc i ta t ion (Wennemark and Bandura, 1974), in which case automatic arrhythmias may occur - 64 -even when the cycle length of the abnormal pacemaker is longer than the sinus cycle length. Theore t ica l l y , entrance block may be caused by a band of t issue which possesses one-way blocking proper t ies, or by anatomical abnormal i t ies. Conceivably, entrance block may also resu l t from the induc-t ion of a band of ref ractory t issue by the previous ectopic impulse. If the normal sinus frequency and the entrance-block-protected ectopic frequency are s im i l a r , the normal and abnormal pacemakers may al ternate in the i r dominance, producing parasystole. I t has been suggested that abnormal automaticity and reentry may be d i f fe rent ia ted by studying the ef fects of overdrive pacing (see Bigger and Goldreyer, 1970; Vasa l l e , 1977). If an automatic arrhythmia is overdriven by pacing, then fo l lowing termination of overdrive one w i l l see a delay (post-overdrive suppression), then a resumption of the automatic arrhythmia. Reentrant arrhythmias do not show typ ica l post-overdrive suppression. The mechanism of post-overdrive suppression has been studied in dog Purkinje f ib res (Vasal le , 1977). Ear ly during the onset of overdrive pac-ing , MDP f a l l s . Providing that st imulat ion does not exceed a c r i t i c a l frequency, maximum d i a s t o l i c depolar isat ion then r i ses and the slope of the pacemaker potent ia l is reduced. Immediately upon cessation of overdr ive, the ectopic frequency i s lower than i t had been before overdrive pacing, as a resu l t of the ef fect of overdrive pacing on the slope of the pacemaker po ten t ia l . Over a short priod of time the or ig ina l e lect rophys io log ica l charac te r i s t i cs of the ectopic focus re turn, and the arrhythmia resumes. In the case of reentry there i s general ly no post-overdrive suppression, and the reentrant arrhythmia resumes after an unpredictable i n t e r v a l . Neverthe-l e s s , i t i s possible that reentrant arrhythmias may resume, fo l lowing over-d r i ve , af ter an in terval s im i la r to that seen fo l lowing post-overdrive suppression of abnormal automatici ty. Therefore a reentrant arrhythmia may - 65 -be incor rec t ly classed as automatic. In add i t ion , parasysto l ic automati-c i t y , protected from overdrive by entry block, w i l l not exh ib i t post-over-dr ive suppression, and may therefore be incor rec t l y classed as reentrant. As discussed above, overdrive techniques are only useful for assessing r e l a t i v e l y sustained arrhythmias. However, i t i s possible to gain ins ight into arrhythmogenic mechanisms ( including mechanisms of PVC induction) from ECG recording, coupled with simple physio logical manipulations. In th is regard, reentrant PVCs are tr iggered i n i t i a l l y by the normal wave of propa-gat ion. Therefore, by analysing the frequency of PVC in re la t ion to heart ra te , i t may be possible to estab l ish a re la t i onsh ip . Furthermore, heart rate may be slowed or stopped by st imulat ing the vagal e f fe rents , or acce l -erated by administering atropine ( e t c . ) , such that PVC incidence may be measured over a wide range of heart ra te . Theore t ica l l y , PVCs resu l t ing from automaticity should not vary in frequency with changes in heart ra te , whereas the frequency of reentrant PVCs should increase with tachycardia, and f a l l with bradycardia. However, the study of the ef fect of changes in heart rate on PVC incidence i s not an i n f a l l i b l e means of d iagnosis, owing to the p o s s i b i l i t y of entry- and ex i t -b lock . In add i t ion , i t i s necessary to ensure that the manipulation designed to a l te r heart rate does not i n f l u -ence other var iab les , d i r ec t l y or i n d i r e c t l y , leading to an a l te ra t ion in PVC frequency. It i s almost impossible to control for th is p o s s i b i l i t y . I t i s possible to make cer ta in speculations concerning the mechanism of arrhythmogenesis of PVCs by simply observing the ECG without other manipula-t i o n . In th is regard, sustained f ixed coupling of PVCs (of consistent QRS conf igurat ion) with the normal sinus beats is regarded as ind ica t ive of reentry rather than automaticity (Langendorf and P ick , 1967). The reason for th is i s that the frequency of an ectopic arrhythmia would not be expec-ted to be the same as the sinus frequency, whereas a reentrant arrhythmia - 66 -should be l inked to the sinus cycle in a f ixed manner. I t i s questionable whether any of the tests and experiments described are useful for d is t inguish ing between reentry and automaticity during acute myocardial ischaemia, despite the i r possible usefulness in other circumstan-ces. During the development of myocardial ischaemia, the e lect rophysio log-i ca l charac te r i s t i cs of a hypothetical reentrant c i r c u i t w i l l change with time, causing changes in coupling i n t e r v a l . Therefore, reentry in acute myocardial ischaemia may be incor rec t l y described as automaticity i f the c r i t e r i on for diagnosis i s a f i xed coupling i n t e r v a l . In add i t ion , i t i s possible that parasysto l ic (entry-blocked) foc i may be influenced by the wave of normal exc i ta t ion v ia e lec t ro ton ic intercourse ( re f lec t ion) across an area of entry block ( J a l i f e and Moe, 1976). F i n a l l y , in th is regard, the funct ional d i s t i nc t i on between reentry and automatici ty becomes questionable when the phenomenon of tr iggered automaticity i s considered. 1.3.4.3 Triggered Automat-i c i t y . Triggered automaticity has only been demonstrated in v i t r o . The reason for th is is quite simple. In order to demonstrate tr iggered automaticity i t i s necessary to stimulate quiescent t i s sue , and i t is not possible to have quiescent ven t r i c les i n -v i vo . Since tr iggered automaticity i s , by d e f i n i t i o n , automaticity which is i n i t i a t e d by an exogenous source, and the exogenous source theore t i ca l l y includes the normal wave of depo lar isa t ion, then i t is possible that abnormal automatic-i t y in vivo may be tr iggered by the sinus beat (Spear and Moore, 1982). In such a case the frequency of automatic PVC would corre la te with heart ra te . Therefore, i t i s not possible to d is t inguish between automaticity and re -entry on the basis of the re la t ionsh ip between heart rate and PVC frequency unless tr iggered automaticity i s ruled out. However, i t would be necessary to induce asystole in order to test for t r iggered automaticity in v i vo , and th is i s not f eas ib l e . - 67 -1.3.5 Epicard ia l Act ivat ion Mapping Although i t i s not possible to prove whether an arrhythmia is automatic or reentrant in closed-chest animals, the technique of ep icard ia l mapping allows for v i sua l i sa t i on of conduction pathways in anaesthetised open-chest animals and i n - v i t r o . Epicardia l mapping has been car r ied out in conjunc-t ion with simultaneous i n t r ace l l u l a r recording in acute myocardial ischae-mia, and attempts have been made to re la te the pattern of abnormal e lec t ro -genesis and conduction with the underlying e lectrophysio logy. How do the e lect rophysio log ica l changes in acute myocardial ischaemia corre la te with arrhythmogenic mechanisms v isua l i sed by ep icard ia l mapping ? In coronary-occluded blood-perfused dog and pig hearts, records of reentry, but not automatic i ty, were observed (Janse e t - a l ; , 1980; Janse and Kleber 1981; Janse, 1982). Most of these arrhythmias arose from the normal t issue close to the occluded bed within the f i r s t few minutes of occ lus ion. The time course of arrhythmias corresponded reasonably well with the time course of ischaemia-induced arrhythmias in - vivo in dogs and pigs ( e . g . , Bergey et a l . , 1982; 1984). In teres t ing ly , the i n i t i a l t r igger for the reentrant VT was an impulse which arose from the normal t i ssue . The mechanism proposed to account for th is phenomenon was as fo l lows. By comparing the AP of normal c e l l s with that of ischaemic c e l l s i t was found that many ischaemic c e l l s were in the plateau phase of the cycle when normal c e l l s were at the rest ing repolar ised phase. This s i tua t ion occurred as a resu l t of the heterogeneity of conduc-t ion ve loc i t y in the ischaemic t issue versus the non-ischaemic t i s sue , in associat ion with slow conduction in the ischaemic t i s sue . The consequences of th is s i tua t ion included a large potent ial d i f ference between the normal and the ischaemic t i ssue . It was proposed that a flow of in jury current between the depolarised ischaemic t issue and the polar ised non-ischaemic - 68 -t issue may have been su f f i c i en t to re-exc i te the non-ischaemic t i ssue . A l te rna t i ve l y , since the non-ischaemic t issue could be in the plateau phase of the AP when the ischaemic t issue was repo la r ised , in jury current may have flowed in the opposite d i rec t ion to re -exc i te the ischaemic t i s sue . For th is mechanism to operate, i t i s necessary that a layer of c e l l s ex is ts (separating the current source and sink) which i s inexc i tab le , at least temporari ly, and capable of permitt ing e lec t ro ton ic coupling between the source and sink (Janse and Kleber, 1981). Whatever the mechanism of i n i t i a t i o n , ac t iva t ion maps revealed fragmen-tat ion and fusion of wavefronts, one- and two-way block and reentry. Reent-ry was associated with VT. VF was associated with mul t ip le wavelets t r a v e l -ing slowly along tortuous routes among mul t ip le i s l e t s of conduction block which changed posi t ion and magnitude from moment to moment. VT ei ther terminated as a resu l t of major s ingle wavefronts a r r i v ing at a large area of inexc i tab le t i s sue , or degenerated to VF as major wavefronts s p l i t into subsidiary wavefronts. VF did not terminate spontaneously (within the 10 sec allowed before d e f i b r i l l a t i o n was applied in th i s study). I t was sug-gested that th is was because VF was associated with more wavefronts than VT, therefore i f some wavefronts terminated, others remained to perpetuate the arrhythmia. This is a possible explanation for the higher frequency of spontaneously revert ing VF in small animals such as rats (Kenedi and Loscon-c i , 1973a; Clark et a l . , 1980) compared with larger animals such as dogs (Townshend Por ter , 1894; Smith, 1918). Presumably the larger hearts have more wavefronts in VF than smaller hearts, and consequently express a lower p robab i l i t y of spontaneous d e f i b r i l l a t i o n . I t was found that st imulat ion of the l e f t s t e l l a t e ganglion improved conduction in the ischaemic t i s sue , and increased the incidence of VT and VF. I t i s possible that th is enhanced sympathetic a c t i v i t y increased e x c i -- 69 -t a b i l i t y in the ischaemic t i ssue , f a c i l i t a t i n g the conduction of PVCs and thei r degeneration into VT and VF (Janse and Kleber, 1981). High concentra-t ions of l idocaine increased the proportion of the ischaemic t issue which was inexc i tab le at any given moment, thereby causing termination of VT and prevention of progression to VF. To summarise, of the major theoret ica l mechanisms of arrhythmogenesis, only reentry has been demonstrated in acute myocardial ischaemia. This evidence was provided from epicard ia l mapping experiments. However, reentry appeared to be tr iggered in many cases by an ectopic in jury current a r is ing from normal Purkinje f i b r e s . The prerequis i te for induction of an ectopic impulse appeared to be a dispersion of re f ractor iness between the normal and the ischaemic t i ssue . This heterogeneity was produced by the slow conduc-t ion in the ischaemic t i ssue . Abnormal slow conduction and var iab le conduc-t ion block in the ischaemic t issue appeared to be responsible for the con t i -nuation of the arrhythmias and the i r progression into VT and VF. Conduction block was made worse by l i doca ine , such that arrhythmias terminated soon af ter i n i t i a t i o n , while l e f t s t e l l a t e ganglion st imulat ion improved conduc-t ion (lessened block) and worsened the arrhythmias. Do these phenomena occur in rats fol lowing coronary occlusion ? Our laboratory has demonstrated ef fects of occlusion in -v i t ro on i n t r a c e l l u l a r potent ia ls (Inoue et a l . , 1984) s im i la r to those demonstrated in other species. In add i t ion , ind i rec t evidence in v ivo such as a reduction in ref ractory period (Northover, 1986) and a lack of e f fec t of vagal s t imula-t ion-induced bradycardia on arrhythmia incidence (Mertz and Kaplan, 1982) suggests that the arrhythmias occurring during the 30 min period af ter occlusion are more l i k e l y to be reentrant than automatic. A question which ar ises i s , to what extent does i ' N a contribute to arrhythmogenesis compared with i $ i ? The ef fects of l idocaine and l e f t - 70 -s t e l l a t e st imulat ion (Janse, 1982) suggest that both may be involved. 1.3.6 The slow inward current and arrhythmogenesis in acute ischaemia 1.3.6.1 Introduction As discussed previously , in normal vent r icu lar t issue the i n i t i a l up-stroke of the AP (phase-0) is governed by i N a , and the plateau by i .. The changes in phase-0 and rest ing E m produced by myocardial ischaemia, as described above, may represent one of four e lect rophys io log ica l phenomena. F i r s t l y , the reduction in dV/dt may represent a severely depressed Hid A ^Na' 1 S because t n e depolar isat ion associated with acute myocardial ischaemia w i l l cause inac t iva t ion of i N a . Secondly, the upstroke may represent an enhanced i $ ^ . This i s because depolar isat ion to -60 to -65 mV w i l l bring E m c loser to the threshold for i . without inducing J m s i steady-state inac t iva t ion of i s - (o^ should be s im i la r to that at an MDP of -90 mV), whereas i ^ a w i l l be almost completely inact ivated at th is vol tage. Th i rd ly , the upstroke may involve both depressed i ^ a and en-hanced i $ . j . F i n a l l y , the upstroke may be associated with an abnormal current system which possesses elements of i ^ a and/or i s l - , but perturbed by factors of ischaemia other than depolar isa t ion, such as increases in pH ( I i j ima e t - a l . , 1986) and the products of anaerobic metabolism (Corr et a l ; , 1984). 1.3.6.2 How - might- - j ,. - contr ibute to -arrhythmogenesis ? The e lec -t rophys io log ica l changes produced by acute myocardial ischaemia (Downar et a l . , 1977a; 1977b; Kleber et a l ; , 1978), and the attendant hypothetical arrhythmogenic mechanisms (reentry and automatic i ty, e tc . ) reveal that i $ ^ may play an important ro le in arrhythmogenesis (Hauswirth and Singh, 1978). The pr inc ipa l reasons for such a suggestion are as fo l lows. a . Acute myocardial ischaemia is associated with depolar isat ion (Downar e t - a l . , 1977b; Kleber e t - a l . , 1978; Inoue et a l . , 1984) to E m values close to those producing complete inact iva t ion of i ^ a (Beeler and Reuter, 1970a). However, from the known voltage-dependence of i . (Reuter, 1973), i t would be expected that th is depolar isat ion should not reduce i . , but rather enhance i t by v i r tue of a reduction in threshold for ac t i va t i on . b. Acute myocardial ischaemia is characterised by slow conduction in the ischaemic t issue (Downar et a l ; , 1977b; Kleber e t -a l . - , 1978). This slow conduction can be accounted fo r , almost completely, by the f a l l in dV/dt of the upstroke of the ischaemic AP (Dodge and Cranef ie ld , 1982); max r 3 the increase in longi tudinal i n t e r ce l l u l a r resistance associated with increases in intercalated disc resistance may also contr ibute (Weidmann, 1982). Slow conduction in depolarised Purkinje t issue has been shown to be dependent on C a + + (Cranef ield e t a l . , 1972), From considerations of res t ing E m , as discussed above, the current responsible for the upstroke of the slow conduction AP may be i •. In th is regard, slow conduction in Purkinje t issue in -v i t ro has been shown to be inh ib i ted by (^-verapami l at concentrations below those which i nh ib i t i ^ a (Cranef ield e t a l ; , 1974). c . Acute myocardial ischaemia i s associated with a large increase in ex t race l l u la r K + concentration ( [K + ] Q ) (H i l l and Gettes, 1980; Hirche et a l . , 1980). While th is phenomenon may contr ibute to the depolar isat ion occurr ing in acute myocardial ischaemia and i t s attendant consequences (see above), i t may also serve to release noradrenaline from sympathetic nerve endings (Hirche e t - a l ; , 1985). By act iva t ing adrenoceptors, noradren-a l ine i nh ib i t s inact ivat ion of i s i (Bean et a l . , 1984). This arrhythmo-genic mechanism is highly specula t ive, however, and recent evidence from our laboratory (Botting et a l . , 1983) and elsewhere (Daugherty e t -a l . - , 1986) does not support a major ro le for catecholamines in arrhythmogenesis in acute myocardial ischaemia. Therefore, i f i' s^ is involved in arrhythmo-genesis, th is involvement is essen t ia l l y independent of the modulating inf luence of adrenoceptor act ivat ion and antagonism on i . . d. Although current evidence does not support a ro le for automatici ty in arrhythmogenesis in acute myocardial ischaemia (Janse, 1982; Janse and Kleber 1981; Mertz and Kaplan, 1982; Northover, 1986), th is p o s s i b i l i t y i s not precluded. If abnormal automaticity i s the expression of enhanced latent pacemaker currents in Purkinje t issue (Spear and Moore, 1982), then i t is possible that i . , or a s imi la r current plays an important ro le in arrhythmogenesis. This is because the pacemaker current in the sinus node ++ comprises, in part , of i*£a, a Ca current s im i la r to i $ ^ (Shibata and G i l e s , 1985), and phase-0 depolar isat ion is carr ied almost exc lus ive ly by i s i (Yangihara e t - a l . , 1980; Brown, 1982). e. Act ivat ion mapping experiments have shown that a d ispar i t y in AP phase between the normal and ischaemic t issue generates in jury currents which t r igger reentry (Janse, 1982; Janse and Kleber 1981). The pr inc ipa l determinant of such t r igger ing i s the occurrence of an AP plateau adjacent to exc i table t i ssue . The plateau of the AP in normal (and probably also in ischaemic) vent r icu lar t issue is governed by i • (Reuter, 1973). The AP plateau in non-ischaemic t issue i s much longer than the plateau in ischaemic t issue (Downar et a l ; , 1977b; Kleber et a l . , 1978; Inoue e t - a l ; , 1984). I t fo l lows that the longer the plateau in non-ischaemic t i s s u e , the higher the probab i l i t y of t r igger ing an e lect ro ton ic re -exc i ta t ion of the ischaemic t i s sue . Therefore, i • may be involved in arrhythmogenesis by v i r tue of i t s ro le in creating a dispersion of AP plateau (and consequently a d isper-sion in re f rac tor iness) between the ischaemic and non-ischaemic t i ssue . f . I t i s possible to hypothesize other mechanisms by which i .. i s instrumental in i n i t i a t i n g or maintaining arrhythmias in acute myocardial ischaemia. Only one further p o s s i b i l i t y w i l l be discussed here, and th is concerns the hypothetical mechanism of arrhythmogenesis known as tr iggered automatic i ty. From i n - v i t r o s tud ies , tr iggered automaticity has been l inked to the occurrence of o s c i l l a t o r y a f terpotent ia ls (Cranef ie ld , 1977), also known as ear ly or delayed af ter -depolar isat ions (DADs), depending on whether they occur before or af ter f u l l r epo la r i za t i on , respect ive ly . While there is no evidence to support a ro le for tr iggered automaticity in arrhythmogen-es is in acute myocardial ischaemia, i t has been suggested that DADs and tr iggered a c t i v i t y occur in surv iv ing Purkinje t issue excised from the occluded vent r icu lar regions of dog hearts fo l lowing the development of in fa rc t ion (El Sher i f e t - a l ; , 1982). The involvement of i g i in DADs has been suggested by the observations that DADs are enhanced by ra i s ing ex t ra-c e l l u l a r C a + + , occur in depolarised t issue (espec ia l l y t issue depolarised by cardiac g lycos ides) , and that DADs are inh ib i ted by compounds which i nh ib i t i . (Ferr ier and Moe, 1973). However, other evidence suggests that DADs are generated by the t ransient inward current (Lederer and Ts ien, 1976), a current which appears to be d i s t i nc t from i $ ^ . Recently i t was shown that simulated ischaemia (16.2 meq/1 K + , low pH, lactate and hypoxia) abolished DADs induced by high frequency pacing in Purkinje t issue in v i t r o (Opie e t - a l ; , 1986; Coetzee e t a l . , 1986), sugges-t ing that th is mechanism of arrhythmogenesis in acute myocardial ischaemia may not be important. 1.4- The-pharmaeology-of caleium-antagonists 1.4.1 Def in i t ion Calcium antagonists were o r i g i n a l l y described as drugs which produce the i r pharmacological e f fects predominantly by i nh ib i t i ng vol tage-act ivated 2+ 2 + entry of Ca in a manner which can be inh ib i ted by Ca (Fleckenstein et a l ; , 1969). This de f in i t i on has been extended to include drugs which 2+ 2+ i nh ib i t Ca entry resu l t ing from the opening of Ca channels coupled - 74 -with drug receptors (see Jam's and Tr igg le , 1983), and drugs which i nh ib i t 2+ i n t r a c e l l u l a r Ca -dependent processes such as binding with calmodulin (Rahwan, 1983; Lynch and Rahwan, 1982). The expansion of the de f in i t i on has occurred in conjunction with the introduct ion of new terms such as 'slow channel b lockers ' and 'calcium entry b lockers ' (see Nayler, 1983). The term calcium antagonist i s used here in accordance with F leckenste in 's o r ig ina l de f in i t i on (see above and F leckenste in , 1983 for review). 1.4.2 Pharmacology of phenethylalkylamines and 1,4-dihydropyridines Phenethylalkylamine calcium antagonists include verapamil, ga l lopami l , b e p r i d i l , t i apami l , anipamil and a var ie ty of t r i a l preparations such as D888. 1,4-dihydropyridines include n i fed ip ine , fe lod ip ine , nimodipine, and n i t rend ip ine. Verapamil, gallopamil and n i fed ip ine have been studied in the most deta i l (see Henry, 1979; 1980; Tr igg le , 1981; F leckenste in , 1983; Nayler and Horowitz, 1983). Phenethylalkylamines have been suggested to possess a higher a f f i n i t y for the channel associated with i • when the channel is in the open or inact ivated state (Kohlhardt and Mnich, 1978; Pelzer e t - a l . , 1982; Lee and Ts ien, 1983), whereas 1,4-dihydropyridines are equipotent in rested and open channels (Lee and Tsien, 1983). This di f ference may account for the w e l l -known frequency-dependence of phenethylalkylamines (Sanguinetti and West, 1982) compared with 1,4-dihydropyr idines, which possess l i t t l e or no f r e -quency-dependence, but considerably more rest ing block (Woods and West, 1983; 1985; Hachisu and Pappano, 1983). Both 1,4-dihydropyridine and phen-ethylalkylamine calcium antagonists have been suggested to act by slowing the recovery from inact ivat ion of the i • channel (Lee and Tsien, 1983). Studies with permanently charged analogues of gallopamil (Hescheler et a l ; , 1982), and studies carr ied out using 'sk inned' (plasmalemma-free) smooth-muscle and cardiac t issue (Fleckenste in, 1977; Kreye e t - a l ; , 1983; - 75 -Itoh e t a l ; , 1984) have indicated that both phenethylalkylamine and 1 ,4-d i -hydropyridine calcium antagonists produce the i r pharmacological ef fects v ia an action on the inner surface of the plasmalemma, and that i n t r ace l l u l a r ?+ actions such as inh ib i t ion of Ca binding with calmodulin (S i lver at a l , 1984) do not contr ibute to the ef fect of these drugs on exc i tab le t i s sue . Both phenethylalkylamines and 1,4-dihydropyridines possess a wide spec-trum of pharmacological proper t ies, such as i nh ib i t i on of i ^ a (Bayer et a l . , 1975a; Nawrath e t - a l . , 1981; Yatani and Brown, 1985), i nh ib i t i on of co r t i cos te r iod release from the adrenal cortex (Costa et a l ; , 1983), i n h i b i -t ion of myosin l i gh t chain kinase a c t i v i t y (Movsesian e t - a l . , 1984), e tc . However, consideration of the concentrations required to produce these —6 ef fects (general ly 10 M or more) has lead most invest igators to regard these ef fects as r e l a t i v e l y unimportant compared with ef fects on vascular smooth-muscle and myocardial t issue resu l t ing from inh ib i t i on of vol tage-operated calcium entry (see Nayler and Horowitz, 1983; F leckenste in, 1977; 1983; Henry, 1979; T r igg le , 1981; 1982). It is of in terest to determine the mechanism of any action of a drug. In order to ascribe an ef fect of a drug to calcium antagonism a var ie ty of approaches may be taken, since i t is often impossible to d i r ec t l y measure calcium entry through voltage-operated channels at the same time as other var iab les , espec ia l l y in -v ivo. Of fundamental importance is information concerning the concentration range over which calcium antagonism is produ-ced; i dea l l y one would l i k e to know EC^Q values. For example, i f one wished to know whether the antiarrhythmic actions of verapamil and n i f ed ip -ine were produced by calcium antagonism in the ven t r i c l es , one might app-roach the problem by comparing E D 5 Q values for reductions in arrhythmias with EDgQ (or EC^Q ) values for calcium antagonism in ven t r i c l es . In order to support th is information i t might be considered of in terest to - 76 -invest igate the re la t i ve calcium antagonist potency of these drugs in a var ie ty of t issues under a var ie ty of condi t ions. There are a number of studies which have compared several calcium antagonists for the i r re la t i ve potencies in vascular and cardiac muscle, in vivo and in v i t r o . In order to i l l u s t r a t e some of the problems inherent in determining whether the ef fect of a drug may be at t r ibuted to calcium antagonism, some studies in which n i fed ip ine and (^-verapami l were compared are summarised. EC^Q or ED^Q values were e i ther taken d i r ec t l y from the text of the publ icat ions or approximated by in terpolat ion (or extrapolat ion) of the data presented. 2+ ECgg values for i nh ib i t i on of depolar isat ion/Ca -dependent contrac-t ion in vascular smooth muscle range from 3 x 10 - ^ M (Kenakin and Beek, 1985) to 2.4 x IO" 8 M (Mi l la rd e t - a l . , 1983) for (^)-verapamil, and from 1.5 x 10" 8 M (Mi l la rd e t - a l ; , 1983) to 6.3 x I O - 9 M (Kenakin and Beek, 1985) for n i fed ip ine . In a l l of the studies considered (Fleckenstein-Grun et a l . , 1976; Lee e t - a l ; , 1983; Kenakin and Beek, 1985; M i l l a rd e t - a l ; , 1983; Nakayama et a l ; , 1985) n i fed ip ine was more potent than (±)-verapami1. However, the di f ference in potency varied considerably, from 1.7:1 (Lee et a l ; , 1983) to 350:1 (Fleckenstein-Grun e t -a l . - , 1976). This may re la te to the var ie ty of preparations, test condit ions and methods used. In heart preparations (vent r ic les and a t r ia ) a s im i la r survey reveals an even greater va r ia t i on . EC^Q values for (^)-verapamil have been reported _5 to range from 10 M (Nabata, 1976; Raschack, 1976a; Briscoe and Smith, 1982) to 1.5 x 10~ 8 M (Clarke et a l . , 1984a; 1985). Corresponding E C 5 Q for n i fed ip ine range from 7.2 x 10~ 6 (Briscoe and Smith, 1982) to _ Q 5.8 x 10 M (Nabata, 1976). In a t r i a and vent r i c les n i fed ip ine has been reported to be as much as 62.5 times as potent as (±)-verapamil (Raschack, 1976a), but other invest igators have found (^)-verapami1 to be up to 5 times as potent as n i fedip ine (Clarke e t - a l ; , 1985). Ni fedipine was found to be - 77 -more potent than (±)-verapamil in 4 studies (Clarke e t - a l ; , 1984; 1985; Lee et a l ; , 1983; M i l l a rd e t -a l . - , 1983), whereas (±)-verapami 1 was found to be more potent than n i fedip ine in 7 studies (Raschack, 1976a; Nabata, 1976; Briscoe and Smith, 1982; Winslow e t - a l . , 1983; Kenakin and Beek, 1985; Nakayama et a l . , 1985). The data d iscussed above i l l u s t r a t e s that i t i s dangerous to re l y on published reports of the potency and re la t i ve potency of calcium antagonists with regard to the invest igat ion of the mechanism of action of calcium antagonists (as ant iarrhythmics, for example). 1.5 Aims of-studies 1.5.1 The action of calcium antagonists in acute myocardial ischaemia The pharmacologist functions to examine and describe the propert ies of drugs, and then account for the mechanism(s) underlying these e f fec ts . In acute myocardial ischaemia, the ef fects of calcium antagonists (Flecken-s t e i n , 1969) are not well character ised. The f i r s t report of the actions of a calcium antagonist in acute myocar-d ia l ischaemia appeared in 1968, when Kaumann and Aramendia demonstrated that 0.79 mg/kg (±)-verapamil abolished arrhythmias and prevented death when administered 10 min before coronary occlusion in anaesthetised dogs. Since th is time there have been a number of reports confirming the antiarrhythmic a c t i v i t y of (^-verapami 1 and d i l t iazem in dogs, although n i fedip ine has general ly not been found to be antiarrhythmic ( e . g . , Guelker et a l ; , 1983; Kobayashi et a l . , 1983; Clusin e t - a l . , 1984). Most of these studies used low doses of calcium antagonists (0.1 - 0.5 mg/kg (±)-verapamil , 0.04 - 0.08 mg/kg n i fed ip ine ) . The resu l ts were highly var iab le between studies, and a c lear exposi t ion of the antiarrhythmic actions of calcium antagonists, and possible mechanism(s) of action remained e lus i ve . In the development and character isat ion of the conscious rat preparation - 78 -by our laboratory, i t was found that (^-verapami 1 and quinidine appeared to have antiarrhythmic a c t i v i t y (Johnston e t - a l ; , 1983a). I t was decided, therefore, to invest igate in more deta i l the classes of antiarrhythmics of which these drugs represent, beginning with c lass-4 (calcium antagonist) antiarrhythmics such as (^-verapami 1 (Singh and Vaughan Wi l l iams, 1972), using the conscious rat preparat ion. The experiments with calcium antagonists described in the Methods sec-t ion were designed to answer the fo l lowing questions: a. Do calcium antagonists reduce arrhythmias induced by acute myocar-d ia l ischaemia in conscious ra ts? b. Do calcium antagonists reduce in fa rc t s ize fo l lowing permanent coronary occlusion? c. Do these ef fects ( i f any) occur as a resu l t of calcium antagonism? d. What factors determine these e f fec ts? It was expected that by carrying out experiments b l ind and assembling dose-response curves for a range of va r iab les , using several preparations ( in vivo and in -v i t ro ) i t would be possible to provide unequivocal answers to questions a and b. In add i t ion , by considering the dose-dependence of the ef fects of d i f ferent types of calcium antagonists on the same var iab les , i t was hoped that c ircumstant ial evidence in support of the hypothesis described in question c, or unequivocal disproof of th is hypothesis would be provided. By considering the overal l resu l ts of the experiments, specula-t i ve hypotheses concerning question d were expected to be generated. 1.5.2 Arrhythmogenesis in acute myocardial ischaemia The introduct ion has broached the subject of arrhythmogenesis in acute myocardial ischaemia. An understanding of the mechanism(s) of arrhythmoge-nesis i s a necessary adjunct to the understanding of the mechanism of action of benef ic ia l drugs. The experiments carr ied out as part of th is thesis - 79 -were not designed to provide a de f i n i t i ve statement concerning the determin-ants of arrhythmogenesis in acute myocardial ischaemia. However, in view of the con f l i c t i ng evidence concerning the ro le of the autonomic nervous system in arrhythmogenesis, and the re la t ionsh ip between i ^ and adrenoceptors (Bean et a l ; , 1984), an attempt was made to resolve th i s par t i cu la r question by carry ing out a series of graded ablat ions in the CNS. In r a t s , evidence concerning the ro le of the sympathetic nervous system in arrhythmogenesis is contradictory. Campbell and Parrat t (1983) found that a var ie ty of s-adrenoceptor antagonists reduced arrhythmias induced by occlusion in anaesthetised ra t s . Marshall e t - a l ; (1981a) found that isopre-nal ine infusion increased the sever i ty of arrhythmias while adrenaline and noradrenaline reduced the sever i t y . However, in conscious ra t s , our labora-tory found that propranolol , labeta lo l and chemical sympathectomy did not inf luence arrhythmias (Botting et a l ; , 1983). Therefore experiments were carr ied out in order to answer the fo l lowing questions: a. Does the autonomic nervous system play an independent ro le in arrhythmogenesis fol lowing coronary occlusion in rats? b. Is the ro le of the autonomic nervous system of su f f i c i en t magnitude for i t to const i tute a major determinant of arrhythmogenesis? The use of se r i a l ablat ion in the CNS coupled with se lec t ive replacement of catecholamines was chosen as a unique method for answering the questions out l ined without resor t ing to the use of drugs as too l s , a pract ice which often involves un jus t i f ied assumptions concerning the s p e c i f i c i t y and se lec -t i v i t y of drug ac t ion. If the autonomic nervous system (pa r t i cu la r l y the sympathetic nervous system) plays a neg l ig ib le ro le in arrhythmogenesis i t is important to estab l ish th is f ac t . Such a resu l t has important consequen-ces, since i t would be d i f f i c u l t , in th is event, to j u s t i f y the use of B - a d -renoceptor antagonists as antiarrhythmics in the prophylaxis of sudden death. - 80 -2 METHODS 2.1 Goronary-occlusion-in rats 2.1.1 Overview A br ie f h i s t o r y . o f coronary occlusion in rats was given in the Intro-duct ion. A conscious animal preparation was developed by our laboratory in order to remove the po ten t ia l l y confounding inf luences of anaesthesia and recent major surgery from the experimental arena (Johnston et a l ; , 1983a). I t i s our opinion that when invest igat ing the actions of drugs or the nature of a disease process, the i n i t i a l experiments should be car r ied out using conscious unrestrained preparations, f ree from ' a r t i f i c i a l ' constraints such as anaesthesia. In the case of the conscious rat model of myocardial ischaemia, th is approach is consistent with the fact that the human con-d i t ions of myocardial ischaemia general ly occur in the absence of anaesthetic and concurrent surgery. The choice of animal species in ischaemia studies and the human spectrum of disease were discussed in some deta i l in the Introduct ion. It was stressed that c l i n i c a l l y , information concerning the c r i t i c a l f i r s t h af ter the onset of symptoms (when sudden death and vent r icu la r arrhythmias are at a premium), and information concerning the re la t ionsh ip between the extent of ischaemia, i t s time-course and i t s sequelae ( p a r t i c u l a r l y arrhythmias) are both incomplete at best. Therefore, arguments concerning c l i n i c a l r e l e -vance are essen t ia l l y po in t less , since no human template of myocardial ischaemia and in fa rc t ion e x i s t s . The philosophy behind the use of the conscious rat preparation i s based on a simple premise, namely that the pr inc ipa l aim of a myocardial ischaemia model should be the reproducible and unequivocal production of myocardial ischaemia of known sever i ty and reproducible sequelae, in a manner which can be manipulated simply, whereby the preparation serves as a bioassay. The - 81 -ra t may be the only species which meets a l l these requirements (dogs, ham-sters and guinea-pigs are too va r iab le , pigs are too la rge, primates are too expensive and mice are too smal l ) . The fo l lowing chapters attempt to co l la te a l l the methods which have been described, in part , in various publ icat ions from our laboratory (Au e t - a l ; , 1979a; 1979b; Johnston et a l ; , 1983a; 1983b; Bott ing et a l ; , 1983; Cur t is et a l . , 1984; 1985a; 1985b; 1986a; 1986b; Cur t is and Walker, 1986a; 1986b), in an e x p l i c i t manner which would permit the reader to reproduce the techniques without further reading or ass is tance. 2.1.2 Preparation Before preparat ion, a l l instruments, leads, l ines and occluders were placed in a bath of 70% ethanol in d i s t i l l e d water for ant isept ic purposes. In addit ion hands were ca re fu l l y washed in soapy water and r insed in 70% ethanol. As a further precaution against i n fec t i on , f inger na i l s were cut as short as poss ib le . A l l experiments were carr ied out using male Sprague Dawley or Wistar r a t s , 230 - 350 g. A glass gas ja r was equi l ibrated with 5 % halothane in oxygen del ivered v ia a vapourisor (F luotec) . For the l as t 300 - 400 prep-arat ions, a humidi f ier was included in the anaesthetic c i r c u i t . This com-prised simply of a conical f lask containing a saturated sa l ine solut ion through which the anaesthetic gas was bubbled. The sa l ine prevented the growth of algae and yeasts for periods of at least 9 months. Each rat was placed in the j a r 2 - 3 min af ter halothane had been introduced. Experience had shown that i f a rat was placed in the ja r before equ i l ib ra t ion with halothane, or i f a lower concentration of halothane ( 1 - 3 %) was used, then rats passed through the exc i ta tory plane of anaesthesia more s lowly, and exhibi ted coprophagic behaviour, which compromised the subsequent stage of preparation ( in tubat ion) . - 82 -Induction of anaesthesia took approximately 1 min, but rats were kept in the gas chamber for a further 1 - 2 min to induce deep anaesthesia (exempli-f i ed by slow, shallow resp i ra t i on ) . Following induct ion, each rat was in tu -bated as fo l lows. The mouth was f ixed open by temporari ly attaching the upper jaw to the bench, and a 14 gauge human intravenous catheter (Jelco) was inserted into the trachea with the aid of a paediat r ic laryngoscope. The sharp point of the metal insert of the catheter had been previously blunted and smoothed for atraumatic locat ion of the catheter. The vocal cords, v i s i b l e as two white bands on the l e f t and r igh t side of the entrance to the trachea, were used as landmarks. The paediat r ic laryngoscope had been f i l e d on one side in order to permit atraumatic entry into the small buccal cav i ty of the ra t s . Intubation was ve r i f i ed by observing the condensation produced when the exhaled a i r was directed onto the surface of the bench. The rats were transferred to a small square metal operating tab le , maintained on 1% halo-thane, and prepared with an occluder, int ravascular l ines and ECG leads, as fo l lows. Anaesthesia was adjusted during preparation such that movement was just prevented. 2.1.2.1 Occluders. In contrast with ea r l i e r work in rats in which coronary occlusion was brought about using s i l k thread (Heimburger, 1946; Johns and Olson, 1954; e t c . ) , our laboratory uses a snare device made from nylon and polythene (Au et a l ; , 1979a; 1979b; Johnston et a l ; , 1983a). The manufacture of the occluder has never been described in d e t a i l . Essen t i a l l y , a guide was made from an 11 cm length of PE10 polythene tubing. One end was f la red by b r ie f exposure to heat from a soldering i r on , wh i ls t 1 cm from the other end, a f lange was made by b r i e f l y melting the tubing by ro ta t ing i t in f ront of a je t of hot a i r . The hot a i r j e t was also used in the manufacture of blood pressure and intravenous l ines (see below), and was created simply - 83 -by passing pressurised a i r through a th in copper tube over a Bunsen burner. The occluder was made complete by threading a 5.0 gauge atraumatic nylon suture (Ethicon) through the polythene guide such that the needle end of the suture appeared at the f la red end of the guide. The occluder was implanted as fo l lows. A 1 cm skin i nc i s ion was made over the 4th to 6th r ibs on the l e f t thorax. This was enlarged by blunt d issect ion (Spencer Wells forceps) . The forceps were then inserted under the Pectoral is muscle, which was gently separated from the underlying Rectus Abdominus, exposing the Intercostal muscles beneath. A r t i f i c i a l resp i ra t ion was immediately i n i t i a t e d . Using a Palmer pump, or equivalent, at a stroke volume of 4 ml per ra t and a stroke rate of 54/min, the anaesthetic regimen described above was de l ivered, and contro l led using the same c r i t e r i a des-cr ibed above. Voluntary resp i ra t ion was switched to a r t i f i c i a l by the simple red i rec t ion of gas flow through the plumbing of the anaesthesia set-up. The 5th or 6th in tercosta l space was then punctured using the Spencer Wells forceps. This inc is ion was enlarged by blunt d i ssec t ion . The choice of 5th versus 6th in tercosta l space was a rb i t ra ry , and general ly not noted. If the heart was exposed unfavourably for placement of the occluder, then a second in tercosta l i nc is ion was made. This was necessary in less than 5 % of prep-arat ions. The in tercosta l inc is ion was widened by inser t ing 4 aluminium ret ractors attached to braided s i l k thread or lengths of PE90 polythene tubing. The aluminium ret ractors were placed ca re fu l l y in order to avoid damaging the l e f t lung, and were f ixed by attachment to the small s ta in less steel operating table (v ia s l i t s in i t s corners). The ret ractors could be eas i l y moved and reposi t ioned. In la ter experiments, a l l re t ractors were made with polythene attachments, since these were easier to replace ( i f they broke during surgery) than the braided s i l k attachments. By applying gentle pressure under the r igh t thorax, and replacing 1 of - 84 -the ret ractors under the thymus (which i s large and over l ies the heart in most r a t s ) , i t was a simple matter to expose the l e f t ven t r i c le and a t r i a l appendage. Using a pair of small blunt forceps, the th in per icard ia l mem-brane was retracted at a posi t ion overlying the junct ion between the l e f t a t r i a l appendage and the l e f t ven t r i c l e , at which point i t could be manipu-lated e a s i l y . Using a second pair of small blunt forceps, a small tear in the pericardium was created. The aluminium r i b ret ractors were then moved, one after another, by inser t ing the i r t ips through the small per icard ia l tear , such that the pericardium was now included with the retracted t i ssue . As a r e s u l t , the heart was l i f t e d toward the in tercosta l i nc is ion by th is per icard ia l c rad le , f a c i l i t a t i n g subsequent surgery. Without d isturbing the heart by gripping i t between the thumb and fo re-f inger (Johns and Olson, 1954), or ex te r io r i s ing i t through the in tercosta l i nc is ion (Selye e t - a l . , 1960), the nylon suture of the occluder was sewn under the l e f t coronary artery (general ly referred to as the LAD, see Intro-duction) by inser t ing the needle into the l e f t ven t r i c le under the overhang-ing l e f t a t r i a l appendage, and bringing i t out high on the pulmonary conus. The a t r i a l appendage was displaced s l i g h t l y for th is purpose by gentle manipulation with a small pair of blunt forceps. The LAD is described as i n v i s i b l e to the naked eye in rats by some Authors (Heimburger 1946; Johns and Olson 1954). These Authors located the LAD using the highly v i s i b l e coronary veins as landmarks. In our experiments, although the veins were found to be v i s i b l e , i t was usual ly possible to see the artery quite c l ea r l y as a thin pink l i ne emerging from under the a t r i a l appendage. The s t i t ch of the suture was made wide, such that the needle entered and l e f t the myocar-dium approximately 2 mm ei ther side of the ar tery . As Heimberger (1946) and Selye et al.- (1960) have pointed out, unless the artery i s included in the l i ga t i on there are no sequelae of consequence. This whole process usual ly - 85 -produced no blood l oss . Occasional ly ( in less than 10% of preparations) there was a l i t t l e bleeding, amounting to less than 1 ml of blood. If th is occurred, 5 min was allowed for c l o t t i n g , then the thoracic cav i ty was gently cleared with gauze in order to restore a blood-less f i e l d . Following the sewing of the suture under the l e f t coronary ar tery , the suture was sewn through the f la red end of the guide tubing. Therefore, the suture emerged from the guide tubing, passed into the myocardium, under the LAD, out of the myocardium and back through the t i p of the guide tubing, making a loose loop or snare. The needle of the suture was then cut o f f , and the remaining length of nylon ca re fu l l y melted down to form a small b a l l , which prevented the suture from being pul led back through the f la red t i p of the guide tubing. The nylon suture extended the f u l l length of the guide tubing. The s ize of the loop at the end proximal to the heart was adjusted by pu l l ing on the suture at the end d i s ta l to the heart . I t was found in some ear ly experiments that occlusion sometimes resul ted in part of the thymus being caught in the occluder. This could be completely prevented by making the loop smal l , such that the f la red t i p of the guide tubing was posit ioned adjacent to , or jus t underneath the a t r i a l appendage. Once the s ize of the loop had been adjusted, the d i s ta l length of suture was melted down to a small ba l l adjacent to the d i s ta l t i p of the guide tubing, in a manner s im i la r to that by which the proximal end of the suture had been a f f i xed . The Pectoral is muscle was sutured l i g h t l y to the Rectus Abdominus muscle with s i l k . The loose ends of the s i l k suture were then t ied to the occluder to f i x i t in posi t ion in the thorax. In a l l but the l as t 50 - 60 prepara-t i ons , the pneumothorax was evacuated at the time the chest was closed by inser t ing a length of PE90 polythene tubing through the in tercosta l i n c i s i o n , and applying negative pressure to the thorax as the chest was c losed. This - 86 -precaution was recent ly successfu l ly replaced by hyper inf la t ion of the lungs by t rans ien t ly increasing stroke volume to 6 ml per rat as the chest was c losed. This modif icat ion reduced the time taken to prepare the r a t s . As soon as the chest was c losed, the halothane anaesthetic was replaced with 100 %oxygen. The occluder was then fed subcutaneously to the subscap-ular region using a s ta in less steel t rocar . The occluder was ex ter io r ised between the shoulder blades, and a s i l k suture was attached to the occluder jus t below the flange at the d i s ta l end ( re la t i ve to the heart ) . This piece of s i l k was designed to serve as a ' f l a g ' to ass is t in the locat ion of the occluder on the day of occ lus ion. The length to which the occluder projected from the subscapular region was l imi ted to no more than 1 cm, in order to reduce the l i ke l ihood of the occluder being chewed or tugged at by the rat during the week of recovery from surgery. This was arranged by pu l l ing on the occluder with a pair of small forceps. In la te r experiments the occluder was allowed to s l i p jus t beneath the sk i n , with only the s i l k ' f l a g ' exposed. The rat was then turned onto i t s back, and the chest wound was i n f i l t r a t e d with C ica t r i n (bac i t rac in , neomycin and streptomycin) powder, and Marcaine (1% bupivacaine). The skin over the Pectoral is muscle was closed with a purse-str ing suture of s i l k . The rat was disconnected from the respi ratory pump, and allowed to breath spontaneously. The in terva l between c los ing the thoracic i nc i s ion and c los ing the skin i nc i s ion was general ly approximately 2 - 3 min, and since the rat was receiv ing only oxygen during th is t ime, su f f i c i en t halo-thane was expired to permit the respi ratory centre to override the a r t i f i c i a l respi ratory rhythm, such that once the rat was disconnected from the r e s p i -ratory pump, i t immediately began to breath spontaneously. However, the rat general ly remained immobile for a further 2 - 5 min. During th is t ime, C ica t r i n and Marcain were i n f i l t r a t e d into the subscapular wound, and the - 87 -other ex ter io r ised leads and l ines (see below) were t i d i ed up. The locat ion of the occluder was the las t stage in preparat ion, but was described f i r s t since i t is the focus of the experiment. Af ter the induction of anaesthesia, the f i r s t stage of preparation was the locat ion of the blood pressure and intravenous l i n e s . 2.1.2.2 Leads -and -1ines. Blood pressure and intravenous l ines were made from PE polythene tubing, using the hot a i r welding technique described for manufacture of the occluder. A 14 cm length of PE50 tubing was welded to a 10 cm length of PE10 tubing by ro tat ing and melting the i r t i p s , then pressing them together. This process was carr ied out af ter the lengths of tubing had been threaded onto a length of th in wire, in order to prevent the welding process from occluding the lumen. The seal between the tubings was tested by c los ing the open end of the PE10 tubing, attaching a needle and a i r - f i l l e d syringe to the open end and applying pressure to t h i s , ostens ib ly closed system, while i t was submerged in d i s t i l l e d water. An incomplete seal was revealed by bubbles emerging from the junct ion of the tubings. A cur l was put into the PE10 end of the l i n e , by looping i t around a glass rod and submerging i t in bo i l ing water for 3 sec. The loop was f ixed by sub-merging i t in ice cold water. The l ines were implanted using a modif icat ion of the method developed by Weeks (Weeks and Jones,1960; Weeks, 1981). A midl ine laparotomy was per-formed, extending from approximately 1 cm poster ior to the xiphoid process to the region overly ing the b i furcat ion of the common i l i a c vesse ls . Using f ingers only, the connective t issue overly ing the abdominal aorta and vena cava was c leared. A s i l k thread was then placed round these vesse ls , using a pair of small forceps, in the standard manner. The thread was usual ly posit ioned in the region of the b i furcat ions of the renal veins and a r te r i es , at which point the small forceps passed under the aorta and vena cava most - 88 -e a s i l y . A more poster ior approach occasional ly produced haemorrhaging from the vena cava. Whilst th is was rare ly f a t a l , and usual ly resolved without sequelae by the time of occ lus ion ' and s a c r i f i c e , i t was deemed inconvenient (5 min was allowed for c lo t t i ng i f ever haemorrhaging occurred, and t h i s , na tu ra l l y , delayed surgery). Therefore in most of the las t 500 - 600 prepa-rat ions the aorta and vena cava were accessed from the former, anter ior approach. Once the aorta and vena cava had been located, these vessels were sepa-rated using small forceps. Again, th is procedure was most eas i l y accom-pl ished when the more anter ior approach was made. The thread was divided such that tension on one or the other of the resul tant t i es would stop flow in ei ther the aorta or the vena cava. The l ines themselves were then located in the rat as fo l lows. Using the same trocar which would subsequently be used for locat ing the occluder, a channel was created through the Psoas muscle and subcutaneously to the subscapular region. With the trocar in p lace, two l ines were threaded into th is channel, and the trocar was then removed, leaving the l ines in p lace. A syringe f u l l of sal ine (without heparin) was attached to each of the l ines at the i r ex i t point between the shoulder blades (note that th is ex i t point was used subsequently for e x t e r i -o r i s ing the occluder) . The abdomen was then retracted on the l e f t side (using two ret ractors in the same manner as for the placement of the occ lu -der) . Both the l ines at the end proximal to the blood vessels (the PE10 ends) were severed, using a pair of sharp s c i s s o r s , to produce a pointed bevel to f a c i l i t a t e the subsequent inser t ion into the vesse ls . Usual ly , the vena caval l i ne was located f i r s t . The a i r in the l i ne was displaced with s a l i n e , and the l i ne was l i g h t l y held with a pair of small forceps. The vena cava was temporari ly occluded with the s i l k thread, and a t iny hole was created - 89 -d is ta l to the occlusion (proximal to the hear t ) , just anter ior to the renal ve ins. The l i ne was then inserted into the hole, and the occlusion removed. The process was repeated for the placement of the aor t ic l i n e , with the exception that the hole, d i s ta l to the occluder, was also d i s ta l to the heart . In both the case of the vena caval and also the aor t ic l i n e , the PE10 tubing was inserted 2 - 3 cm into the vesse ls , point ing towards the heart . The l i n e s , and the i r placement, were designed in order that blood press-ure recording and drug administrat ion could be undertaken v ia funct ioning (non-occluded) vesse ls . The holes in the vessels were made using a 27 gauge hypodermic needle which had been bent to an angle of approximately 120 ° . The hole was of a smaller diameter than the PE10 tubing, such that the l ines could be moved f ree ly without blood leakage. In add i t ion , the small diameter of the PE tubing did not occlude the aorta or vena cava; th is was obviously c r u c i a l , since these were recovery r a t s . The aorta and vena cava were used because they are large, eas i l y accessib le vesse ls , su i tab le for cannulation with chronic indwell ing non-occluding l i n e s . The abdomen was dusted with C i c a t r i n , and the body wall closed with a running s t i t c h . The skin was then c losed, also with a running s t i t c h , and the wound was i n f i l t r a t e d with C ica t r i n and Marcaine. Following placement of the occluder by the method described above, the ex ter io r ised ends of the cannulae were treated as fo l lows. S i l k f lags were attached to each cannula in the same way that a f l ag was attached to the occluder. Approximately 0.3 ml of sa l ine was then injected into the vena caval l i n e , which was abruptly clamped with a pair of Spencer Wells forceps, the t ips of which had been made atraumatic by encasement in soft polythene tubing. The open end of the exposed PE50 portion of the cannula was then sealed by mel t ing, using an ordinary c igaret te l i gh te r . The same process was car r ied out for the aor t ic - 90 -l i n e . The melted seals were f la red such that the i r diameters were approx-imately double that of the PE50 tubing. The l ines were allowed to extend from between the ra t s ' shoulder blades by about 1 cm. As in the case of the occluder, the l ines sometimes sl ipped beneath the skin during the in terva l between preparation and occ lus ion; the s i l k f lags permitted easy locat ion of the l i n e s , and the i r posi t ion under the skin was considered acceptable, since the cannulae were therefore out of the reach of the ra t s . In general, how-ever, the f l a r e d , melted ends of the cannulae prevented them from s l ipp ing completely under the sk in . The rats appeared to be unconcerned by the presence of the ex ter io r ised cannulae and the occluder. There was an extremely low incidence of in fect ion around the ex i t s i t e (fewer than 1 % of rats had pus exudation from the wound), presumably as a resu l t of the prophylaxis with C i c a t r i n . ECG leads were prepared from tef lon-coated s ta in less steel wi re. Approximately 1 cm of one end of each lead was de-insulated using an ordinary c igaret te l igh te r f lame. The lead was approximately V3. The chest lead was implanted with the occluder, as fo l lows. A t ight ba l l was made in one end of the lead by wrapping i t around a 21 gauge hypodermic needle. This end was then posit ioned underneath the Pectoral is muscle, using the 21 gauge needle as a t rocar . The d i s ta l end of the chest lead was ex ter io r ised with the occluder between the shoulder blades. The large trocar used for th is purpose was directed through the hole made e a r l i e r for the ex te r io r i za t ion of the int ravascular cannulae. The limb leads were implanted on the day of occ lus ion. They were made of the same material as the chest lead, but were posit ioned in a s l i g h t l y d i f fe rent manner (see below). After preparat ion, the animals were placed in indiv idual cages and given tap water and Purina rat chow ad l ib i tum. The body weight of each rat was - 91 -recorded before preparat ion. The procedures described usual ly took approx-imately 25 - 40 min to complete, from induction of anaesthesia to recovery of consciousness. 2.1.3 Coronary occlusion This sect ion deals with the experiment proper, which was car r ied out approximately 7 days after preparation (range 4 - 1 5 days). The sequence of events is described below. The actual process of occlusion was as fo l lows. The occluder was gripped by the Spencer Wells forceps with the atraumatic t i ps (see above) jus t above, and adjacent to the f lange located approximately 1 cm from the end d i s ta l to the heart . The purpose of the flange was to provide a stock for the forceps which would permit the outer guide of the occluder to be held f i rm without excessive clamping, such that the inner snare of the occluder remained free to move within the outer guide. Using f i nge rs , the small bobble on the d i s ta l end of the inner snare of the occ lu -der was pul led from the adjacent outer guide tubing to the extent of approx-imately 2 mm. A second pair of forceps was then used to f i rmly gr ip the exposed inner snare of the occluder. Traction was then applied smoothly between the inner snare and the outer guide tubing to produce occ lus ion. Successful occlusion occurred when the outer guide tubing became cr ink led and i t was no longer possible to move the inner snare in re la t ion to the outer guide. This moment was designated time zero. The atraumatic forceps were clamped down f i rmly on the occluder at th is moment, and the length of exposed inner snare of the occluder was melted down with a soldering iron to form a bobble adjacent to the d i s ta l end of the outer guide tubing, f i x i ng i t in p lace. Time zero was accompanied in most cases by a sudden change in the ECG, but th i s was not taken as a c r i t e r i on for occ lus ion , since i t was possible that drug treatment could delay the ECG changes caused by occ lus ion. Never-- 92 -the less , in the present experiments i t was found that an absence of ECG changes within 15 min of occlusion was associated in every instance with an absence of an occluded zone (measured upon s a c r i f i c e , see below), ind icat ing that occlusion had been unsuccessful. This event occurred in less than 5% of r a t s . 2.1.3.1 Sequence of events. The sequence of events on the day of occlusion is out l ined in Figure 1. Rats were anaesthetised with halothane in the manner described above, and allowed to breathe 1.5% halothane through a small face mask. The limb leads of the ECG (made in the same way as the chest lead) were then placed as fo l lows. Each wire was threaded through a 23 gauge hypodermic needle, such that the non-insulated t ip projected approximately 0.5 cm from the sharp end of the needle. The wire was then bent back c lean ly , forming a barb. The needle, together with the electrode was passed subcutaneously, e i ther to the elbow region of the forelimb (dorsal aspect) for the 2 forelimb leads, or to the r igh t anter ior dorsal f lank region for the r ight hind limb lead. The entry point was the same for a l l three leads, namely approximately 0.5 cm anter ior to the ex i t point of the occluder and intravascular l i n e s . The ECG leads were disconnected from the hypodermic needle by pinching the skin around the needle t i p and ca re fu l l y withdrawing the needle. As the needle was withdrawn, the electrode remained in p lace, owing to the action of the barb. In approximately the f i r s t 50 preparat ions, the limb leads were implan-ted on the day of preparat ion. Although they did not appear to cause d i s -comfort, they were sometimes removed by the rat during the in terva l between preparation and occ lus ion. Therefore, i t was considered sensible to implant limb leads on the day of occlusion in every case. In th is way an element of consistency was introduced, which was absent i f some rats were used with fresh electrodes placed on the day of occ lus ion, and some not. CORONARY OCCLUSION IN CONSCIOUS RATS r o C D -60 Sequence of events on the day of study (time scale in min) 1 day la te r 0 •15 -5,+5 +15 +30 +60 +120 +180 +240 0 30 stab i1 i sa t ion drug admi ni s t r a t i on OCCLUSION DEATH? SACRIFICE measure occlusion measure i n f a rc t Figure 1. Sequence of events on the day of occlusion fol lowing connection of leads and l ines - 94 -The 3 limb leads were connected to one another by simply twist ing the i r exposed ends together, forming a composite whole body lead, which was trimmed to approximately 2 cm length using a pair of s c i s so rs . The te f lon was removed from the end of the composite lead as described above. It should be noted that the anaesthetic mask was temporari ly moved well away from the ra t during th is procedure. The un-insulated leads were connected to 1 of 2 lengths of enamel-coated s ta in less steel wire by twis t ing the wires together. The t ips of the wires had been scraped with a scalpel blade to remove the insu la t ing enamel. The connections were iso la ted by applying a small piece of surg ical tape to each. The tape was attached in such a way that i t could eas i l y be removed at a la ter time. Add i t iona l l y in th i s regard, when the leads and wires were connected, the twist was always c lockwise, f a c i l i t a t i n g subsequent disconnect ion. Whilst the rat was s t i l l anaesthetised with halothane, the blood pressure l i ne was connected to a pressure transducer v ia a length of PE tubing and a blunted 23 gauge needle t i p . The intravenous l i ne was connected to a syringe f u l l of sa l ine in a s im i la r manner. Both l ines were flushed with approx-imately 0.3 ml sa l i ne . The intravenous l i ne was ind is t inguishable from the a r te r i a l l i ne by v isual inspect ion, but once the melted t i p of the l i ne had been snipped o f f , the l ines were eas i l y d is t inguishable in most cases by the fact that blood immediately f i l l e d the aor t ic l i n e , but not the vena caval l i n e . In the infrequent event that blood did not flow back through ei ther l i n e , both l ines were flushed with sa l ine un t i l a d i s t i nc t i on could be made. The aor t i c l i ne was kept open by attaching a leak pump in ser ies with the l i n e , according to the method of Weeks (1981). In at least 75% of cases, negative pressure on the vena caval l i ne caused dark venous blood to appear in the l i n e . In the remaining 25% of cases, the venous l i ne was tested by the in jec t ion of a small amount of adrenaline which e l i c i t e d a small pressor - 95 -response when the l ine was patent. The placement of the ECG leads and the connection of the blood pressure and intravenous l ines to the transducer and syringe of s a l i n e , respect ive ly , took no more than 10 min. Rats regained consciousness upon termination of anaesthetic general ly within 30 sec, and were f u l l y a le r t within approx-imately 2 - 5 min. Blood pressure and the ECG were displayed on a Grass Polygraph (4 channel, model 7 ) , and ECG was also displayed on a delayed loop osci l loscope with a 4 sec delay and a 4 sec real time display (Honeywell Type E for M). The la t te r was pa r t i cu la r l y useful for the diagnosis of arrhythmias (see below). The ECG channel was ca l ib ra ted once per week; l i t t l e adjustment was ever required. The blood pressure channel was c a l i -brated every time i t was used. After approximately 1 h, the administrat ion of drugs, i f any, and coro-nary occlusion were carr ied out while the ra t was f u l l y conscious and not restra ined in any way. The procedure was as fo l lows. Intravenously admin-is tered drugs were injected slowly over a 10 min per iod. A fast (100 mm/sec chart speed) record of ECG and blood pressure was taken before drug admin-i s t ra t i on and 4 min af ter completion of i n j ec t i on . One min af ter the second fas t recording of blood pressure, the occluder was t ightened, as described above. Occlusion did not appear to cause the rats any d i s t ress . No changes in behaviour ever occurred during the f i r s t few min after occ lus ion, unless blood pressure f e l l prec ip i tous ly fo l lowing occlusion ( in which case the animal became subdued), or unless acute pulmonary oedema, (characterised by laboured resp i ra t ion) developed. In general , the f i r s t behavioural response to occlusion was sudden convulsive-type behaviour which occurred in con-junct ion with VF (see below). The rats were monitored for at least 4 h fo l lowing occ lus ion. Fast speed polygraph recordings were made every min for the f i r s t 15 min, every 5 min - 96 -for the fol lowing 15 min, then every 15 min. If the rat survived for 4 h, i t was disconnected from the ECG wires and blood pressure transducer by reversing the processes used for connecting the animal to these recording devices. The animal was conscious and not restra ined during th is process. Usual ly the rat was subdued at th is time as a resu l t of the sequelae of occlusion (see below) and was submissive and compliant enough for th is disconnection to be completed without any d i f f i c u l t y . After 24 h, i f the rat was s t i l l a l i v e , i t was reconnected to the blood-pressure and ECG recorders and care fu l l y monitored for a fur ther 30 min, whereupon i t was s a c r i f i c e d . Reconnection to the recording devices was carr ied out in the same way as the i n i t i a l connection the previous day, with the exception that the rat was not anaesthetised. Instead, i t was placed on the bench and wrapped in a lab coat, with only the subscapular region exposed. Rats, l i k e many rodents, do not struggle when they cannot see. In th is manner the l ines and leads were reconnected without inc ident . I t i s worth mentioning that in ear ly experiments, when the limb leads were implan-ted on the day of preparation along with the occluder and blood pressure l i n e , e t c . , i t was routine to connect a l l rats to the blood pressure and ECG recording devices in th is manner ( i . e . , when the ra t was conscious) on the day of occ lus ion. Upon death or s a c r i f i c e , the heart was excised and the occluded zone and in fa rc t s i ze were determined (see below), and a general postmortem examin-ation was performed (the appearance of the lungs, l i v e r , kidneys, spleen, bladder and snout were noted, and any gross abnormalit ies were recorded). Exclusion c r i t e r i a (see below) were considered at each stage of experimen-ta t i on . 2.1.3.2 Monitoring of - responses -t-o occ lus ion. As described above, the ECG and blood pressure were recorded continuously for 4 h fo l lowing - 97 -occ lus ion , and again at 24 h af ter occ lus ion. Behavioural changes were noted in a general way, and any pecu l i a r i t i es were recorded. Most of the tech-niques described below have been described in br ie f previously (Au e t - a l ; , 1979; Johnston et a l . , 1983a; Cur t is et a l . , 1984; 1985b). A l l information concerning each rat was recorded permanently on ind iv idual analysis sheets. 2.1.3.3 Occluded-zone (OZ). The OZ was recorded for every ra t . In 24 h surv ivors , s a c r i f i c e was undertaken by de l iver ing a blow to the head with a metal cosh. No anaesthetic was used. The heart was excised and per-fused v ia the aorta according to Langendorff (1895) with sa l ine (0.9 %). Once blood was no longer present in the perfusate, the sa l ine was replaced with sa l ine containing Indocyanine (Fast Green dye, BDH) 0.5 g/1. Approx-imately 20 - 50 ml of th is solut ion was allowed to pass through the coronary c i r c u l a t i o n . The del ivery of the 2 so lu t ions , sa l ine and Fast Green dye was contro l led using a T-tube device connected to 2 large reservoi rs containing each so lu t i on , and was regulated with a simple glass 2 by 2-way stopcock. In the case of animals which died overnight, care was taken not to d i s -lodge the s tas is thrombi present in the l e f t ven t r i c l e . Perfusion with approximately 20 ml Fast Green dye sometimes took 5 - 1 0 min in such hearts, compared with the 2 - 3 min in the case of hearts from f resh ly dead animals. However, general ised c lo t t i ng in the coronary c i r cu la t i on had probably not occurred, because the hearts from rats dying overnight provided OZs as well defined and of a s imi la r extent to those from f resh ly dead animals. It i s more l i k e l y that perfusion rate was reduced as a resu l t of the contracture which was a common feature of hearts removed from rats dying overnight s ince , with reference to the ef fect of sys to le , coronary perfusion is highly sens i t i ve to the con t rac t i l e state of the myocardium. Af ter perfusion, the heart was removed and processed. F i r s t of a l l , the a t r i a , aorta and pulmonary vessels were removed and discarded. Delineation - 98 -of the remaining vent r icu lar myocardium into normal and occluded t issue was made by v isual inspect ion. The two zones were almost invar iab ly well d i f fe rent ia ted with a c lear-cut border, as might be expected on the basis of the reported lack of co l l a t e ra l s in rat hearts (Johns and Olson, 1954; Maxwell et a l . , 1984; Winkler et a l . , 1984). Using a small pai r of sc issors the perfused (green) and non-perfused (pink) t issues were separated, l i g h t l y blot ted and weighed. 2.1.3.4 Infarct -zone - ( IZ) . The in fa rc t s ize was only determined for animals which survived for 24 h. In th is regard, i t has been shown that an in terva l of at least 10 h must be allowed af ter occlusion for in farc t s ize (measured in the manner described below) to be s u f f i c i e n t l y developed for quant i f i ca t ion in rats (Hort and Da Cana l is , 1965a). Infarct s ize was determined by a modif icat ion of the method of Jestadt and Sandr i t ter (1959), based on the production of formazan red by the reduction of 2 ,3 ,5 - t r i pheny l -tetrazol ium (TTZ) by H + emanating from NAD- or NADP-1inked dehydrogen-ases. The normal and occluded t issues were cut into s t r i ps approximately 2 mm wide and incubated with TTZ (Fischer S c i e n t i f i c ) 0.1 g in 10 ml phos-phate buffer at 37 °C. The buffer was made by d isso lv ing 25 g Na^HPO^ and 0.5 g NaHgPO^ in 2 1 d i s t i l l e d water (pH adjusted to 8.5 - 8.6 with 0.1 M NaOH). Incubation was carr ied out for approximately 10 min ( incuba-t ion was terminated when the s t r ips of non-occluded t issue had turned deep purple from the formazan red reac t ion , rather than after a spec i f i c t ime). In la ter experiments, the OZ t issue was cut not into s t r i p s , but into 2 discs by s l i c i n g through the subendocardium. The resul tant discs were no more than 2 mm thick (by v isual inspection) at any point . The ra t iona le for th is approach was to invest igate whether the endocardial and ep icard ia l surfaces were salvaged to any extent as has been suggested by Hort and Da Canalis (1965b) and others. Although no quant i ta t ive data was assembled, i t - 99 -appeared that the endocardial surface was usual ly not infarcted (white) at 24 h, and occasional ly parts of the epicard ia l surface were also not white, although a gentle scrape with a pair of sharp sc issors was always su f f i c i en t to remove th is 'salvaged' t i s sue . In addi t ion, by separate weighing of th is t issue i t was found that i t s presence changed the in fa rc t s i ze by only 2 to 3 %. Infarct s ize was quant i f ied by separating the white and purple t issues and weighing them. However, the t issue was f i r s t f i xed for 2-3 days in 10 ml formal sa l ine (made by d isso lv ing 3.56 g NaCl and 125 ml 40% formaldehyde in 375 ml d i s t i l l e d water). I t has been shown that the i n f a r c t , as detected by th is method, corresponds with the h i s t o l o g i c a l l y i den t i f i ed in fa rc t (Fishbein e t a ! . , 1981). 2.1.4 Def in i t ion of occlusion-induced arrhythmias 2.1.4.1 Introduct ion. In order to invest igate the ef fects of treatments on a va r iab le , an unequivocal de f in i t i on of that var iab le i s necessary. In the case of occlusion-induced arrhythmias, i t i s general ly the case that var ia t ions ex is t in de f i n i t i ons . While these dif ferences may not necessar i ly be important in themselves, they make d i rect comparison between studies d i f f i c u l t to in terpret in some cases. Although there is var ia t ion in the d i s t i nc t i on between a run of PVC and VT, and di f ferences in the quant i f i ca t ion of PVC, the main source of contention l i e s in the d i s -t i nc t ion between VT (pa r t i cu la r l y that of the torsade de pointes var iety) and VF. VF was f i r s t described by Erichsen (1842) in terms of the con t rac t i l e behaviour of the myocardium ( th is was more than 60 years before the develop-ment of the ECG). VF has been defined in various ways. Moe e t - a l ; (1964) defined VF as chaot ic , asynchronous f ract ionated e l e c t r i c a l a c t i v i t y . Bigger (1980) gave an operational d e f i n i t i o n , based on the charac te r i s t i c ECG - 100 -pattern of 'the absence of QRS complexes and T waves and the presence of low-amplitude baseline undulat ions ' . These de f in i t ions are not amenable to quant i tat ive d i f f e ren t i a l diagnosis because they are subject ive de f i n i t i ons , and they do not d i f fe ren t ia te between abrupt VF and VF occurr ing after a run of VT. This i s not r e a l l y a serious problem c l i n i c a l l y ; the d i s t i nc t i on between i r regu lar torsade de pointes, vent r icu lar f l u t t e r and VF i s , perhaps academic, since a l l 3 arrhythmias are associated with cardiac output below that necessary for the maintenance of l i f e , and the primary concern i s the prevention of mor ta l i ty . However, more rigorous de f in i t i ons are necessary experimental ly. Abrahamsson's group who induce myocardial ischaemia in rats define VF as asynchronous disorganised e l e c t r i c a l a c t i v i t y of at least 5 sec duration (Abrahamsson e t - a l ; , 1985). While th is de f in i t i on introduces an important caveat, namely that some indicat ion concerning the duration of the phenomenon may be useful in d iagnosis, i t nevertheless f a i l s as a t ru l y object ive d e f i n i t i o n , since how does one define 'asynchronous' and 'd isorganised' ? Opie's group have defined VF as ' to ta l i r r egu la r i t y of morphology of the repe t i t i ve ectopic complexes for at least 6 cyc les ' (Lubbe et a l ; , 1978), introducing once more the notion that VF is only VF i f disorder is present for an arb i t ra ry but spec i f ied durat ion. C lear ly there i s a subject ive element in the diagnosis and de f in i t i on of VF. This is an important point and attempts to deal with th is problem are discussed below. I t i s unfortunate that many researchers do not appear to have a r i g i d set of c r i t e r i a for def in ing arrhythmias. A glance through the l i t e ra tu re w i l l reveal that most researchers do not define what they mean by VT and VF. In pa r t i cu la r , i t i s often unclear by what c r i t e r i a torsade de pointes and VF are d i f f e ren t ia ted , and how many consecutive PVCs const i tute a run of VT. If PVC, VT, torsade de pointes, f l u t t e r and VF represent a continuum, - 101 -as was once believed ( e . g . , Har r i s , 1950), then the d i s t i nc t i on between these arrhythmias would not be ser ious ly important; a drug which reduces PVC would be expected to reduce VT and VF as w e l l . However, there is some indicat ion from studies with animals (Dresel and Sut ter , 1961) and from fol low-up invest igat ions in healthy humans with a high frequency of PVC (Kennedy e t - a l . , 1985), that PVC, VT and VF are not necessar i ly part of a continuum. Therefore the p o s s i b i l i t y ex is ts that a drug may inf luence se lec t i ve l y one type of vent r icu lar arrhythmia but not another. The la t te r issue is neither proven nor disproven at present, and wholesale r e - c l a s s i f i c a t i o n of drugs as ' c l a s s i c a l antiarrhythmic agents' and ' an t i a r rhy thm ic /an t i f i b r i l l a to ry agents ' , with s e l e c t i v i t y for VF versus PVCs (Anderson, 1984) is somewhat premature. I r respect ive of whether ischaemia-induced vent r icu lar arrhythmias repre-sent a continuum or not, i t i s nevertheless important in any experiment to define endpoints. Since arrhythmia de f in i t ions vary from one invest igator to another, a l l one can aim for is internal consistency at present. Before describing the c r i t e r i a which were used for def ining vent r icu lar arrhythmias produced by coronary occlusion in r a t s , the subject ive nature of c l a s s i f i c -at ion of arrhythmias must be re i te ra ted . No matter what de f in i t i on is chosen for VF and VT, there remains an element of sub jec t i v i t y , pa r t i cu la r l y with regard to d i f fe ren t ia t ion between torsade de pointes and VF. A simple way around the problem of sub jec t i v i t y i s to carry out studies using a b l ind and random protocol . In th is manner, inconsistencies in d e f i -n i t ion should be spread randomly and evenly between study groups, and conscious and unconscious bias should be e l iminated. I t must be stressed that attempts were made to carry out a l l the experiments described in a b l ind and random manner, where poss ib le . - 102 -The fo l lowing def in i t ions were used in the diagnosis of arrhythmias fo l lowing coronary occlusion in r a t s . 2.1.4.2 Premature vent r icu lar -cont ract ions-(PVG). PVC were defined as premature QRS complexes occurr ing independently of the P wave. PVC were general ly accompanied by a t ransient drop in aor t ic blood pressure. Owing to the high frequency f i l t e r i n g of the ECG by the Grass polygraph, no attempt to measure coupling in terva ls was undertaken. Measurement of coupling in terva ls i s believed to give an ind icat ion of whether the PVC is reentrant or automatic (see Introduct ion). However, i t was considered that the equip-ment avai lab le was not amenable to the routine measurement of coupling in te rva ls , since measurement of such by hand would be ex t raord inar i l y time consuming. Only s i ng le t s , doublets (bigemini) and t r i p l e t s were counted as PVCs. Longer runs were recorded as VT (see below). I t i s noteworthy that other workers include every def lect ion in a run of VT as a PVC (Kane and Winslow, 1980; Clark et a l . , 1980). This manoeuvre is based on the assump-t ion that PVC and VT are ident ica l in mechanism and drug s e n s i t i v i t y . While our de f in i t i on implies that s i ng le t s , doublets and t r i p l e t s represent the 'same' arrhythmia, we only associate these arrhythmias for ana ly t ica l con-venience; since the incidence of doublets and t r i p l e t s is var iab le and lower than the incidence of s i ng l e t s , a useful invest igat ion of the ef fects of a drug on these ind iv idual types of arrhythmia would require an increment in the study group s i z e . I t was considered that the cut -of f value of 4 PVCs, which was low, removed the p o s s i b i l i t y of spurious amalgamation of arrhyth-mias which are not necessar i ly the product of a common e lect rophys io log ica l mechanism (namely short runs of VT and long runs of PVCs). In other words, in the absence of c lear evidence concerning the point at which a run of PVCs ceases to be PVCs and becomes VT, i t was decided to a r t i f i c i a l l y segregate PVCs and VT. E s s e n t i a l l y , since one cannot be sure whether VT and PVCs are - 103 -expressions of the same arrhythmia or not, i t i s sensible to separate them for analysis by generating an arb i t ra ry cut -o f f po int . 2.1.4.3 Ventr icu lar- tachycard ia-(VT). VT was defined as a run of 4 or more consecutive PVC. No res t r i c t i on was made on the associated ra te . This de f in i t i on d i f f e rs from that of other workers who measure ischaemia-induced arrhythmias in r a t s . VT has been defined as 7 or more PVCs at a rate of > 600/min (Kane e t - a l ; , 1980), heart rate exceeding 500 beats/min (Lepran et a l ; , 1981b), 7 or more PVCs, no l im i ta t ion on rate (Fagbemi and Par ra t t , 1981b; McLennan et a l . , 1985) and 5 or more PVCs (Mertz and Kaplan, 1982; Manning e t - a l ; , 1984; Daugherty e t - a l . , 1986). C l i n i c a l l y , VT has been defined as 3 or more consecutive PVCs (Lown et - a l ; , 1973), and th is de f in i t i on has been adopted by Verdouw's group in the i r work with pigs (Verdouw et a l . , 1978). 2.1.4.4 Ventr icular - f i b r i l l a t i o n - (VF). VF was defined as disorder in the ECG accompanied by a precip i tous f a l l in blood pressure. In add i t ion , since d e f i b r i n a t i o n was undertaken in a l l rats experiencing 10 sec of VT or VF, a time constraint was imposed in order to d i f fe ren t ia te between 'primary' VF and VF 'secondary' to VT, namely that disorder had to have begun within 10 sec of the las t sinus beat for the arrhythmia to be classed as VF, other-wise the arrhythmia was c l a s s i f i e d as VT. Disorder was defined as an i r r e g -ular cycle length with no i den t i f i ab le QRS complex. As discussed above, the word 'd isorder ' lends an element of sub jec t i v i t y to the d e f i n i t i o n . Bias was eliminated as far as possible by b l ind and random experimental design. In the introduction to th is subsect ion, a var ie ty of operational d e f i n i -t ions of VF were given, and i t was suggested that the timing and durat ion.of 'd isorder ' needs to be def ined, as well as the actual nature of ' d i so rde r ' . Some Authors have avoided the question of the de f in i t i on of VF al together. Winslow i n i t i a l l y reported the incidence of VF in rats fo l lowing occlusion - 104 -(Kane and Winslow, 1980), but in la te r studies (Marshall e t a l . - , 1981a; 1981b; 1981c; Marshall and Winslow, 1981; e tc . ) VT and VF were combined, and the arrhythmia was ca l led ' f i b r i 1 l o f l u t t e r ' . Northover has adopted a s imi la r s t rategy, avoiding 'a rb i t ra ry and subject ive d i s t i n c t i ons ' between VT and VF by simply recording the time spent in the 'combined forms' (Northover, 1985). Other groups have taken a completely d i f ferent approach, def in ing VF in absolute terms. For example, Harron et a l . (1985) defined VF as a run of ectopic beats with a rate of 720/min or more. It is evident that there i s a lack of consistency between research groups in terms of de f in i t i on of VF. The method used in the current experiments is simply a de f in i t i on of VF which was f e l t to be reasonably consistent with c l i n i c a l de f i n i t i ons , and more importantly, amenable to routine use, i n te r -na l l y consis tent , but not obsessively unequivocal to the point of absurdity (see the de f in i t i on used by Harron e t - a l ; , 1985, above). Despite the d i f ferent c l a s s i f i c a t i o n s used, the resu l ts obtained by various groups with Na + channel blocking drugs (see Discussion) are extremely consis tent , a l l studies demonstrating a n t i f i b r i l l a t o r y a c t i v i t y for quinidine and Org-6001, for example, suggesting that the v a r i a b i l i t y in arrhythmia c l a s s i f i c a t i o n appears not to compromise inves t iga t ions , in terms of the recognit ion of a n t i f i b r i l l a t o r y drugs. However, the v a r i a b i l i t y in control incidence of VF within research groups suggests that there may be an element of internal inconsistency. This may re la te to the fact that apart from ourselves, research groups do not appear to carry out the i r studies using b l ind and random protocols. This may give r i s e to some misleading information, pa r t i cu la r l y concerning drugs with weak antiarrhythmic act ions, as a resu l t of the loss of precis ion of endpoints. 2.1.4.5 Other-arrhythmias. Although VF, VT and PVC are the arrhythmias of major in terest and importance, a l l arrhythmias fol lowing - 105 -occlusion are recorded. The incidence of non-ventr icular (and non-nodal) arrhythmias i s , however, extremely low in control r a t s , and i t is not considered worthwhile invest igat ing them in a quant i tat ive manner. A t r i o -vent r icu la r (AV) blocks occur from time to t ime, pa r t i cu l a r l y in associat ion with pulmonary oedema-induced gasping. Anecdotal observations have strongly suggested that gasps in rats with severe pulmonary oedema are coupled, 1:1 with Moebitz Type-2 AV block. Presumably the AV block i s the resu l t of a vagal r e f l ex . A l l types of AV block and d issoc ia t ion have been observed. Third degree AV block is usual ly only seen in rats experiencing pers istent and severe respiratory d is t ress (exudation of sputum), and according to the exclusion c r i t e r i a (see below) are not usual ly included in s tud ies . Sinus bradycardia and tachycardia do not occur very often during the f i r s t 4 h af ter occ lus ion, but both have been observed in 24 h survivor r a t s . A t r i a l arrhythmias are extremely ra re . It i s d i f f i c u l t to d is t inguish between a t r i a l f l u t t e r and f i b r i l l a t i o n using the V3 lead, but some sort of a t r i a l ' f i b r i l l o f l u t t e r 1 has been observed ( in fewer than 1% of r a t s ) . 2.1.4.6 Rationale for- - d e f i b r i l l a t i o n . VF is a serious arrhythmia which is l i fe - th reaten ing i f i t does not spontaneously rever t . It is well establ ished that rats can spontaneously d e f i b r i l l ate ( e . g . , Johnston e t - a l . , 1983a). However, between approximately 25% (Kenedi and Losconci , 1973a) and 95% (Siegmund et a l . , 1979b) of control rats which experience VF die from VF (values being dependent on whether conscious or anaesthetised animals are used, and on how VF is def ined). Therefore, i t i s expected in any study that the sample s ize w i l l vary with t ime, as animals d ie , and w i l l a lso vary between groups. This means f i r s t l y that ult imate group s izes w i l l vary, compromising the value of s t a t i s t i c a l t es ts , secondly that group s ize may be so small in controls that meaningful comparisons cannot be made, and t h i r d l y - 106 -that much information concerning the time course and the in ter re la t ionsh ips of var iables is los t as animals die during the course of the study per iod. This censoring can only be eliminated i f the animals are a l l kept a l i ve by d e f i b r i l l a t i n g VF as i t occurs. In a l l rats an attempt was made to revert a l l episode of VT and VF l a s t -ing longer than 10 sec by thump-version. VT was reverted as well as VF because many (but not a l l ) episodes of VT reduce blood pressure to close to zero in a manner analogous to that seen with VF; i t was considered that in order to preclude the generation of a rb i t ra ry de f in i t i ons concerning the ' seve r i t y ' of VT, a l l VT would be reverted af ter 10 sec in the manner used for VF. The procedure was as fo l lows. After 10 sec of continuous VF or VT the rat was l i f t e d out of the home cage by the t a i l . Within a few seconds convulsive-type behaviour (see sect ion concerning prel iminary screen) ceased and syncope ensued, whereupon the chest was f l i c ked with the index f inger . Usual ly only 1 or 2 f l i c k s were required to revert the arrhythmia to sinus rhythm. If reversion did not occur immediately then the f l i c k s were continued. Reversion was d iag-nosed by the combination of a sudden return to consciousness, a sudden increase in aor t i c blood pressure and the termination of the arrhythmia (as seen on the ECG record) . Because 10 sec was the maximum time allowed before d e f i b r i l l a t i o n was i n i t i a t e d , d i f fe ren t ia t ion between VT and VF was only attempted during the 10 sec period of the arrhythmia. Of course, i f VT were allowed to continue for longer than 10 sec, i t may possibly degenerate to VF. Therefore our method of diagnosing VF may lead to an underestimation of VF compared with other workers who do not use d e f i b r i l l a t i o n . To our knowledge, only 1 other group attempts thump-version, Charnock's group (McLennan e t - a l . , 1985), using our technique. - 107 -Although thump version great ly reduces censoring due to arrhythmia-induced mor ta l i ty , i t introduces a new form of censoring, namely that h i t t i ng the chest may 'change th ings ' compared with rats which have not experienced VF. This p o s s i b i l i t y was examined by our laboratory (unpublished obser-vations) and i t was found that d e f i b r i l l a t i o n reduced the incidence of mor ta l i ty from VF, but did not inf luence the incidence of VF i t s e l f . In other words, d e f i b r i l l a t i o n does not inf luence the l i ke l ihood of subsequent VF. Therefore i t was considered that the benef i ts of thump-version out-weighed the possible disadvantages. Thump-version creates 2 classes of VT and VF, that which spontaneously reverts to sinus rhythm within 10 sec of onset (SVT and SVF) and that which does not spontaneously revert before thump-version (NVT and NVF). The generation of subclasses of VT and VF may be i r re levant in terms of ana lys is ; while we continue to note the incidence and log^Q number of episodes of VT, SVT, NVT, VF, SVF and NVF, we have found that drugs which inf luence SVF also inf luence NVF, and drugs which inf luence SVT also inf luence NVT. Therefore i t i s reasonable to assume that SVT and NVT are ident ica l in terms of mechanism of generation; the same can be said for SVF and NVF. This is an important point because i t implies that spontaneous reversion of VF to sinus rhythm in rats is not a disadvantage of the preparat ion. Moreover, the occurrence of SVF, and the read i l y revertable nature of NVF (see resu l ts ) means that most animals w i l l survive the period of continuous monitoring such that censoring produced by ear ly morta l i ty i s reduced. 2.1.4.7 Arrhythmia scores. In quanti fying arrhythmias, many workers record the i r incidence, number, type and durat ion. Such information has l imi ted value since the number of episodes of VT and VF are log^ -norma l l y d is t r ibu ted var iables (Johnston et a l ; , 1983a); th is fac t i s not taken into considerat ion by other workers in the f i e l d . In addi t ion, thump-version may - 108 -inf luence the duration of VT and VF compared with the values found by other invest iga tors . In the hands of workers who do not revert VT and VF, the mean duration of VF is dominated by the terminal event. I n tu i t i ve l y , such measures of duration of VF should be log^g normally d is t r ibuted for th is reason. However, correct ion of data i s never undertaken (Clark et a l . - , 1980; Kane and Winslow, 1980; e t c . ) . In our laboratory, i t was found that even with thump-version, the durations of VT and VF were log-^Q normally d i s -t r ibuted (Johnston et a l • , 1983a). Nevertheless, since thump-version c lea r l y censors the durations of VT and VF, i t i s not reasonable to completely re l y upon such var iables for antiarrhythmic quan t i f i ca t ion . There is another reason for th is conclusion; i f a drug abolishes VF then there i s no mathe-matical value for log^g VF durat ion. If an arb i t ra ry value of 1 sec i s given to rats not experiencing VF then the resu l t i s a standard deviat ion of zero in the group in which VF is absent, creat ing a variance inhomogeneity which inval idates the use of standard parametric s t a t i s t i c a l tests (although the non-parametric Mann-Whitney U test may be used). In summary, measuring arrhythmia duration alone is not an acceptable means of gauging the sever i ty of arrhythmias. An a l te rnat ive approach is to generate a number scale which can be used to summarise and grade a l l the arrhythmias fo l lowing occlusion in terms of incidence and sever i t y . This scale (arrhythmia score) should accomodate and summarise complex arrhythmia data se ts . Arrhythmia scores should be normally d is t r ibu ted (to permit parametric s t a t i s t i c a l test ing) and l i n e a r l y add i t i ve . The arrhythmia score which has been used for occlusion studies in the conscious rat in our laboratory has been shown to be Gaussian d is t r ibuted in control rats (Johnston et a l ; , 1983a) and amenable to modified t t es t s . - 109 -This score is as fo l lows: 0 = No more than 49 PVCs, 1 = 50 - 499 PVC, 2 = No more than 1 episode of SVT or SVF and/or > 499 PVC, 3 = More than 1 episode of VT and/or VF < 60 sec to ta l durat ion, 4 = VT and/or VF of 60 - 119 sec to ta l durat ion, 5 = VT and/or VF of > 119 sec to ta l durat ion, 6 = Fatal VF occuring 15 min - 4 h after occ lus ion, 7 = Fatal VF occurring 4 min - 14 min 59 sec af ter occ lus ion, 8 = Fatal VF occurring 1 min - 3 min 59 sec af ter occ lus ion, 9 = Fatal VF occurr ing before 1 min af ter occ lus ion . This score has been adopted by Charnock's group for studies in anaesthetised rats (McLennan and Charnock, 1984; McLennan et a l . , 1985; McLennan, 1986). Other arrhythmia scores have also been developed. Martinez and Crampton (1981) have used a score based on the product of arrhythmia duration and arrhythmia type (the la t te r being graded from 1 to 5 according to seve r i t y ) . However, th is score was found not to be amenable to parametric s t a t i s t i c a l t es t i ng , since drug treatments al tered the var iance. Mueller e t - a l ; (1984) have used a simple 3 point score which they sum for each group and submit to the chi test for ana lys is . Woodward's group have used a group arrhythmia score, whereby arrhythmias for a whole group are summed, and a score assigned to the group (Daugherty e t - a l ; , 1986). However, the object of having an arrhythmia score seems to have been forgotten by th is group, since th is par t i cu la r score does not seem to be amenable to any s t a t i s t i c a l t es t . The ideal arrhythmia score should be amenable to parametric s t a t i s t i c a l tests (analysis of variance followed by modified t tests for mul t ip le comparisons, such as Tukey's t e s t ) . In th is manner, arrhythmia score may be plotted against log dose in order to estimate ED™ values for overal l - 110 -antiarrhythmic actions (see sect ion concerned with s t a t i s t i c s ) . At least 6 other arrhythmia scores were invest igated during the course of experimentation, since i t was considered worthwhile to invest igate whether more c lose ly Gaussian-distr ibuted scores could be invented. 2.1.5 ECG changes produced by occlusion Coronary occlusion produces charac te r i s t i c S-T segment elevat ion (Pardee, 1920) and changes in R wave s i z e . Since these e f fec ts are produced by occ lus ion, and are presumably the resu l t of myocardial ischaemia, i t was considered of in terest to measure and record these var iab les . Apart from ourselves (Johnston e t - a l . , 1983a; e t c . ) , only Bernauer (1982; 1983; 1985) has attempted to measure and quantify the ECG changes produced by occlusion in r a t s . The optimum method for measuring and expressing such changes is therefore not wei1 'establ ished. A var ie ty of techniques have been considered as fo l lows. Figure 2 i l l u s t r a t e s the changes in ECG conf igurat ion with time fo l lowing occ lus ion . 2.1.5.1 'S -T ' segment e leva t ion . The posi t ion of the T wave of the ECG in rats i s not as c lear-cut as i t i s in other species. In most chest leads ( including the V3 lead which we use), the T wave i s superimposed upon the terminal portion of the QRS complex, ind icat ing that repo lar iza t ion is beginning before depolar isat ion is complete (Cooper, 1969). In order to measure S-T segment e levat ion , an a rb i t ra ry , but standardised posi t ion for the T wave was determined. Pr io r to occ lus ion, a sample of approximately 50 ECGs recorded at fast chart speed (100 mm/sec) were inspected, and the pos i t ion of the S wave in re la t ion to the Q wave was measured. This was found to be approximately 30 msec (3 mm on the fast speed record) . There-fo re , as an index of S-T segment e levat ion , the height of th is S wave pos i -t ion above i s o e l e c t r i c was measured before (S ) and af ter occlusion (S ). - Ill -KEY: _ ECG lmv '200 msec 1 -HR Time PVC no. After VT no. Occlusion VF no. BP ! 'I U A A 456 beats/mtn 444 beats/mln 453 beats/mln 444 beats/mtn 444 beats/mln -5 m1n -1 m1n 1 min 123 itmHg 123 iraiHg 103 mmHg 2 m1n 3 m1n 97 irniHg 95 umHj l\ h 434 beats/mln 512 beats/mtn 22 PVC 4 m1n 1 VT 5 m1n 1 VF 93 mmHg 105 mmHg "v-A 522 beats/mln 485 beats/mtn 474 beats/m1n 468 beats/mtn 19 PVC 2 PVC 20 mtn 25 min 10 min 15 m1n 83 mmHg 88 mmHg 95 mmHg -I I A VJV 98 mmHg v J \ 474 beats/m1n 500 beats/mtn 519 beats/mtn . 525 beats/mln. 524 beats/mtn. 525 beats/mtn 2 PVC 2 PVC 50 PVC 30 m1n 45 min 1 h 1.25 h 1.5 h 1.75 h 95 mmHg 108 «Hg 105 mmHg 105 nroHg 103 mmHg 105 irniHg I I. ' ' p. 525 beats/m1n 536 beats/m1n 540 beats/mtn 525 beats/mln 544 beats/mtn 503 beats/mtn 16 PVC 69 PVC 24 PVC 36 PVC 2 h 2.25 h 2.5 h 2.75 h 1 VT 3 h 3 VF 103 irniHg 98 irniHg 90 itmHg 85 irniHg 63 PVC 435 PVC 2 VT 3.25 h 29 VT 83 ItmHg 75 nuiHg 522 beats/mtn 517 beats/mtn 512 beats/mln 514 beats/mtn 511 beats/mtn 3.5 h 84 PVC 376 PVC 3.75 h 4 h 70 mmHg 70 limHg 24 PVC 12 VT 4.25 h 65 mmHg 1 PVC 65 mmHg 1 PVC 60 mmHg 505 beats/mtn 1 PVC 4.75 h 60 imHg A., J A A A A - A - A 496 beats/mtn 491 beats/mtn 475 beats/mtn 482 beats/mtn 475 beats/mtn 365 beats/mtn 2 PVC 4 PVC 10 PVC 5.25 h 5 VT 5.5 h 4 VF 52 nmHg 55 mmHg 55 irniHg 5.75 h 3 PVC 6 h 60 mmHg 8 PVC 55 nmHg 24 h 12 PVC/mtn 75 flmHg Figure 2. Anecdote of ECG changes caused by occlusion in a conscious rat - 112 -I soe lec t r i c was defined as the voltage at the foot of the P wave of the preceding beat. Before occ lus ion, values are negative, since the S wave posi t ion is negative to i s o e l e c t r i c . 'S -T ' segment elevat ion begins immedi-a te ly upon occ lus ion, and in order to s impl i fy subsequent ana lys is , a l l negative (pre-occlusion) values are assigned the value zero. 'S -T ' segment elevat ion was expressed in a var ie ty of ways. The var iab le o r i g i n a l l y used in our laboratory was the dimensionless dSTR. This was determined by measuring the R wave amplitude (see below) before occlusion (RQ) and at times af ter occlusion (R t)» and was calculated as ( S t - S Q ) . ( R t / R Q ) . In other words, 'S -T ' segment was corrected for the change in R wave amplitude fo l lowing occlusion as a funct ion of pre-occlusion R wave. Other measures of 'S -T ' segment elevat ion have been subsequently evaluated, as fo l lows; Uncorrected e leva t ion , referred to as ST, calculated as S t mV. Elevat ion corrected for amplidude as a % of R wave amplitude, referred to as ST 2 /R , calculated as ( S t ) 2 / R t mV. Elevat ion as a % of R wave amplitude, referred to as ST%, calculated as (100) (S t ) /R t (dimensionless). The c r i t e r i on for determining which measure was most useful was simply to determine which var iab le produced the largest coe f f i c ien t of var ia t ion (defined as mean divided by standard dev ia t ion) . I t was found during the course of experimentation that ST% was consis tent ly the most precise v a r i -ab le. The coe f f i c ien ts of var ia t ion for ST% were approximately double those found for dSTR. The var iab le dSTR has been shown to be Poisson d is t r ibuted (Johnston e t - a l ; , 1983a). The problem with a l l measures of S-T segment elevat ion is that the var iab le regresses with time in a non-l inear manner. An attempt was made to account for th is by measuring the maximum value of S-T segment - 113 -elevat ion and the time at which th is occurred. Since the development of S-T segment elevat ion is not s t r i c t l y a mono-exponential process (see resu l ts ) i t was decided to measure the maximum value of S-T segment elevat ion (rather than the half-maximum value) . This was carr ied out according to the fo l low-ing c r i t e r i a . S-T segment elevat ion was measured at regular in terva ls af ter occlusion from 100 mm/sec chart speed records. The maximum value of S-T segment e l e -vation was determined by considering each time point consecutively and applying an arb i t rary '5% r u l e ' in the case of ST%, or a '0.05 mV ru le in the case of dSTR. This ru le operates as fol lows (using the der ivat ive ST% as the example). The f i r s t value i s i n i t i a l l y c l a s s i f i e d as the maximum. This is superceded by the f i r s t subsequent value which exceeds i t by 5% or more. In the example below, each successive apparent maximum has been underl ined. The f i na l underlined value, 85% was the value taken as the actual maximum according to the c r i t e r i a out l ined. The time at which the maximum occurred, 120 min, was taken as the 'time of maximum ST%' Time (min) 1 2 5 10 15 30 60 120 180 ST% 6 15_ 17 66 68 80 82 85 88 Of course, i f the re la t ionsh ip between S-T segment elevat ion and time af ter occlusion were a simple saturat ing monoexponential funct ion of the form: 'S -T ' e levat ion = 1/(1 + tau/ t ) where t is time af ter occlusion and tau is the time constant (defined here as the time at which 'S -T ' segment elevat ion is hal f maximal), then tau would be the ideal var iable to record. However, when 'S -T ' e leva-t ion i s plot ted against time ( e . g . , Johnston e t - a l . , 1983a) then i t i s c lear - 114 -that a simple monoexponential equation w i l l not f i t the re la t i onsh ip . Indeed, the re la t ionsh ip does not f i t any simple model. Since the action of drugs on 'S -T ' segment elevat ion was not the primary concern, i t was not considered worthwhile elaborat ing upon the analysis of the var iab le beyond using the semi-empirical approach out l ined above. 2.1.5.2 Pathological- -R-waves. Coronary occlusion in rats produces an i n i t i a l increase in the amplitude of the R wave, followed by a gradual decl ine in amplitude to values smaller than those seen before occ lus ion. An attempt was made to quantify R wave changes using a s imi la r approach to that used in assessing S-T segment e levat ion . R wave amplitude was easier to measure than S-T segment e leva t ion , since the peak of the R wave is c l ea r l y v i s i b l e on fas t chart speed recordings (100 mm/sec). R wave amplitude (mv) was taken as the def lect ion of the peak of the R wave above the i s o e l e c t r i c point . As in the case of S-T segment e levat ion, R wave does not corre la te with time af ter occlusion in a simple manner, and maximum R wave amplitude (using a 0.05 mV rule) and the time at which th is occurred were determined in a manner analogous to the determin-at ion of maximum S-T segment e leva t ion . 2.1.5.3 Pathological - Q waves. The chest lead ECGs of normal rats exh ib i t no Q wave, presumably because the rat heart f ronta l plane axis is perpendicular to the horizontal plane, according to ECG vector analysis (Cooper, 1969). However, coronary occlusion produces a deep Q wave in the chest leads which appears at approximately 2 h af ter occ lus ion, and pers is ts for at least 11 days (Normann et a l ; , 1961; Zsoter and Bajusz, 1962). If lead-I i s recorded, a Q wave appears wi thin 10 min of occlusion in approxim-ate ly 30% of r a t s , long before i t i s present in the chest leads (Kenedi and Losconci , 1973a). Using a V3 lead we ra re ly see a Q wave before 1 h af ter occ lus ion, but i t s presence is almost universal in rats surv iv ing 24 h. - 115 -Recently, attempts have been made to quanti fy Q wave development by measuring the time at which a s ign i f i can t Q wave is present in the ECG. A s ign i f i can t Q wave was defined as a downward def lect ion from i s o e l e c t r i c equal to approximately 10% of the R wave amplitude. 2.1.6 ECG changes produced by drugs Since the ECG i s always recorded before drug administrat ion and again 1 min before occ lus ion , there is an opportunity for assessing the e lec t ro -physio logical e f fects of drugs in the same rats in which ant iar rhythmic /ant i -in fa rc t a c t i v i t y is assessed. This i s achieved by measuring P-R interval and QRS i n t e r v a l . 2.1.6.1 P-R i n t e r v a l . P-R in terva l re f l ec t s the duration of conduc-t ion from high in the atrium through to the AV junct ion and bundle branches (see Horan and Flowers, 1980). Since conduction ve loc i t y in the AV node is much slower than conduction ve loc i t y in the atr ium, in accordance with the lower dV/dt „ of the upstroke of the action potent ia l and the lower e x c i -max t a b i l i t y (Merideth e t - a l . , 1968, and see Introduct ion), then P-R in terva l is essen t ia l l y a re f lec t ion of conduction through the AV node. As such, drugs inf luencing P-R in terva l therefore inf luence AV conduction. The P-R in terva l is f a i r l y easy to record in r a t s , although the foot of the P wave can be somewhat i nd i s t i nc t on occasions, pa r t i cu la r l y when recorded on a Grass Polygraph. P-R interval was measured according to the standard de f in i t i on (from the foot of the upstroke of the P wave to the s ta r t of the QRS). The ef fects of drug treatment on P-R interval were determined by comparing values 15 min before occlusion (pre-drug) with those 1 min before occlusion (4 min af ter drug adminis t rat ion) . 2.1.6.2 QRS i n t e r v a l . The QRS in terva l re f l ec t s vent r icu lar depol-a r isa t ion (see Horan and Flowers, 1980). If the QRS is widened, th is re f l ec ts a d ispers ion, or delay in vent r icu lar conduction, and reduction - 116 -in conduction ve loc i t y . The QRS interval was not measured in the conven-t ional manner (from the beginning of the Q wave to the end of the S) because i t was considered that the posi t ion of the downward going peak of the S was more amenable to measurement than the less wel l -def ined terminal end of the S wave. The ef fects of drug treatment on QRS interval were determined by comparing values 15 min before occlusion (pre-drug) with those 1 min before occlusion (4 min af ter drug adminis t rat ion) . In some experiments, an attempt was made to measure QT i n t e r v a l . The problems involved in measuring QRS were even more of a confounding factor here, since the T wave is the least wel l -def ined of the ECG waves in the ra t . The T wave re f l ec t s vent r icu lar repo la r i sa t i on , and i s therefore dependent on action potent ial durat ion. Therefore, QT in terva l re f l ec ts a combination of vent r icu lar conduction ve loc i t y (governed almost exc lus ive ly by dV/dt ), vent r icu lar action potent ial plateau (governed by i .) and H i d A S i vent r icu lar repo lar isa t ion (governed by inact iva t ion of i $ ^ and act iva t ion of repo lar is ing K + currents, see Introduct ion). It can be seen that QT interval is therefore a rather non-speci f ic va r iab le . In add i t ion , in rats the T wave is superimposed on the QRS (Cooper, 1969; D r i s c o l l , 1980). This makes measurement of QT interval pa r t i cu la r l y d i f f i c u l t . 2.1.7 Measurement of serum K + concentration Serum K + was measured in recent experiments as a consequence of the resu l ts of invest igat ions into mechanisms of arrhythymogenesis (see below). Since i t has been shown that ex t race l l u la r K + r i ses in the ischaemic t issue with a time course corresponding with that of ear ly occlusion-induced arrhythmias (Hirch e t - a l ; , 1980), and since the incidence of arrhythmias fo l lowing myocardial ischaemia is inversely proportional to serum K + c l i n i c a l l y (Nordrehaug and Von der L ippe, 1983; 1985) and experimentally in coronary-1igated rat hearts in v i t ro (Lubbe e t a l . , 1978; Daugherty e t a l ; , - 117 -1981), i t was decided to monitor serum K + in order to determine whether the antiarrhythmic actions of drugs were re lated to a l tera t ions in serum K + . A blood sample (0.5 - 0.8 ml) was withdrawn from the aor t i c blood press-ure l i ne at approximately 2 h af ter occ lus ion. The sample was spun at 10000 x g for 2 min, using an Eppendorf centr i fuge (Model 3200). The plasma was removed using a small pipette with a rubber bulb attached, and allowed to c l o t . The serum was then pipetted into a 1 ml stoppered vessel (occasion-a l l y a second spin was required to separate the serum from the c l o t ) . The K + concentration was determined in the ana ly t ica l laboratory of the Acute Care Hospital (by the i r techn ic ians) , using a K + - s e l e c t i v e electrode (Kodak Ektachem). 2.1.8 Exclusion c r i t e r i a I t is of paramount importance, when invest igat ing myocardial ischaemia, to ensure that animals are included into the study only when the coronary artery has been occluded and ischaemia has been produced. This is ensured by measuring the 0Z, ex v i vo , according to the technique described above. However, there are other less obvious sources of variance which may jeopar-dise the precis ion and accuracy of an experiment. Over the years, a set of exclusion c r i t e r i a has been developed which is designed to reduce the v a r i -ance not a t t r ibutab le to treatment. It must be stressed that the exclusion c r i t e r i a must be treated in the same manner as a l l other aspects of exper i -mentation and ana lys is , namely that i t must be applied b l i nd l y . The exclusion c r i t e r i a are t ie red in as much as rats must be excluded before, during or af ter occlusion according to a de f in i te chronological sequence. In other words, pre- or post-occlusion exclusion c r i t e r i a must not be applied post-hoc (except when postmortem v e r i f i c a t i o n i s requi red) . - 118 -A. The fo l lowing action is taken in re la t ion to the fo l lowing pre-occlusion abnormal i t ies, with the object of excluding rats in which ei ther the occluder has been acc identa l ly tightened during the in terval between preparation and experimentation, or in which in fect ions are present: a. The presence of a Q wave, as defined above, d ictates obl igatory exc lus ion, on the grounds that a Q wave i s only seen in chest leads in rats fol lowing coronary occlusion (Normann et a l . - , 1961; Zsoter and Bajusz, 1962), and that a Q wave i s associated in our experience only with an in fa rc t or scar t i s sue . b. If more than 5 PVC occur during the 15 min period pr ior to drug administrat ion then the rat should be excluded on the grounds that there is a lesion of some sort in the myocardium which may inf luence the outcome of occ lus ion. c . If there has been > 25% weight loss between preparation and occ lu -s ion , rats are only excluded i f there are other signs of i l l n e s s , such as diarrhoea and/or inflammation associated with surg ical wounds, and/or pre-drug mean a r te r i a l blood pressures of 85 mmHg or less (> 2 s . d . from mean pre-occlusion va lues) . d. I f there are signs of lung i n fec t i on , such as exudate around the snout and/or noisy resp i ra t i on , the rat is excluded. In th is regard, the s e n s i t i v i t y of rats to lung in fect ions i s wel l known. Evans et a l . (1985) reported that male Sprague Dawley rats have often been received from the suppl ier with 'a pulmonary in fect ion characterised by wheezing', and that the morta l i ty in these rats fol lowing coronary occlusion was increased. In ear ly experiments in our laboratory i t was probable that some rats were included despite having an underlying pulmonary i n fec t i on . During the las t 2 years the problem of respi ratory in fec t ion became so severe on occasions that rats died even before preparative surgery had been carr ied out. Mor ta l i ty associated with pneumopathy after occlusion is characterised by extensive exudation of sputum, often bloody, during the f i r s t 5 - 1 5 min af ter occ lus ion, ind ica t ive of severe pulmonary oedema. Postmortem examination reveals mott l ing and occasional ly haemorrhaging of the lungs, and severe pulmonary oedema. Often the thorax is f u l l of serum, and the lungs have a s im i la r appearance to the l i v e r (uniformly red) . Lung swabs and sputum swabs have been examined (by Charles Ford in Medical Micro-bio logy, UBC) and found to cons is tent ly grow monocultures of Psuedo-monas aeruginosa. Current ly , attempts have been made to remove th is problem by s l i g h t l y ac id i f y ing the dr inking water, and al lowing an in terval of at least a week between a r r i va l of the rats from the suppl ier and surg ical preparat ion. These manoeuvres, in conjunction with regular d is in fec t ion of the laboratory with bleach appear to have reduced the incidence of l i fe - th rea ten ing pneumopathy, e. We have had no reason to exclude rats from study on the grounds of low blood pressure. Pa r ra t t ' s group require such exclusion c r i t e r i a since they use acutely prepared anaesthetised animals (Clark et a l . , 1980), which can be obtunded by preparative surgery. The fo l lowing action is taken in the event of the fo l lowing post -occ lu-sion abnormal i t ies, with the object of excluding rats with incomplete or non-existant occlusions and rats which experience reperfusion as a resu l t of the loosening of a defect ive occluder: a . I f there is no increase in R wave, and/or no ST elevat ion fo l lowing occ lus ion, the rat i s excluded: i . i f postmortem examination reveals an unacceptably small OZ or IZ (see below), - 120 -i i . i f the rat dies within 4 hr of occlusion and is found to have in f lamination or scarr ing in the heart , i i i . i f postmortem examination reveals that the occluder i s loose, b. I f , at some time after occ lus ion, the ECG returns to the pre-occ lu-sion conf igurat ion, the rat is excluded: i . i f the OZ and/or IZ are unacceptably small (see below), i i . i f the l i ga to r is found to be loose. c . I f the rat dies within the f i r s t 10 min after occlusion in associat ion with immediate calamatous hypotension, the rat is excluded i f the thorax i s found to contain blood. I t is possible that overtightening of the occluder can cause haemorrhaging from the coronary vesse ls , perhaps as a resu l t of roughness in the region of the f la red outer guide tubing. This par t i cu la r problem is seen in less than 2% of r a t s . d. Animals experiencing fa ta l or non-fatal cardiogenic shock are not excluded from stud ies . Cardiogenic shock has been defined as a f a l l in blood pressure of at least 30%, maintained for at least 30 min, associated with ECG signs of ischaemia, but not caused by arrhythmias (Agress et a l . , 1952). This de f in i t i on adequately describes the syndrome which we recognise as cardiogenic shock in rats subjected to coronary artery occ lus ion . C. The fo l lowing action is taken in the event of the fol lowing postmortem abnormal i t ies, in order to exclude animals with inadequate or abnormal occ lus ions: a. Rats are excluded i f they are found to have an inappropr iately small OZ, defined as < 25 %ventr icular weight (more than 2 s . d . from the mean). - 121 -b. Rats having inappropriately small IZ, defined as < 50% of the OZ are excluded. This c r i t e r i on is somewhate a rb i t ra ry , and i s based on the assumption that since the ra t has no funct ional co l l a te ra l s capable of providing 'myocardial salvage 1 (Johns and Olson, 1954; Selye e t -a l . - , 1960; Maxwell et a l . , 1984; Winkler e t - a l . , 1984; Schaper et a l . , 1986), then the IZ at 24 h should be a reasonably f ixed % of the OZ. This appears to be the case, since to date, our laboratory has not found any drugs which cons is tent ly reduce IZ, which may mean that 'myocardial salvage' is impossible in rats (see Discussion). The arb i t rary nature of th is exclusion c r i t e r i on is not r e a l l y a problem, since no rats have ever been excluded from a study so le l y on the basis of th is c r i t e r i o n . c . I f in farcted t issue or scarr ing in the heart is found in a ra t which dies before 4 h after occ lus ion, the rat i s excluded, on the grounds that in farc t ion is not detectable using TTZ un t i l at least 6 h af ter occ lus ion, and is not well demarcated un t i l at least 10 h af ter occlusion (Hort and Da Cana l is , 1965a). Ear ly scarr ing is i nd i ca -t ive of in farc t ion produced during or shor t ly af ter preparat ion. Less than 1% of rats have been excluded on th i s bas is . d. If pus is found at the occlusion s i t e , the ra t i s excluded. Occasional ly , acutely prepared animals are used for experimentation. The exclusion c r i t e r i a out l ined above are used except where they are obvious-l y inappropriate. In accordance with Par ra t t ' s c r i t e r i a (Clark e t - a l ; , 1980), rats with pre-drug pre-occlusion mean a r te r i a l blood pressures of < 70 mmHg are excluded ( in the present experiments no rats ac tua l ly f a i l ed th is tes t ; pre-occlusion blood pressure was always > 80 mmHg). - 122 -2.1.9 S t a t i s t i c s In most experiments, a control group of rats was compared with at least two treated groups. The control group was defined as a group of animals treated with drug veh ic le . The var iables compared were e i ther Gaussian (normally) d is t r ibuted or binomial ly d is t r ibuted (Johnston e t a l ; , 1983a). In a l l comparisons, the l im i t of ' s t a t i s t i c a l s ign i f i cance ' was defined as p < 0.05. In accordance with the requirements for undertaking the types of s t a t i s t i c a l tests described below, randomisation to treatment and b l ind analysis of records were carr ied out. Whenever poss ib le , treatment was also given b l i n d . Obviously a procedure such as decerebration or pi th ing (see below) could not be carr ied out b l i n d . The group s i ze was kept to a minimum, and was based loosely on the minimum sample s ize required to reveal a 50 % reduction in VT and VF. The control incidence of VT and VT during the 0 - 4 h period after occlusion in conscious rats i s approximately 90 - 100 % 2 (Johnston et a l . - , 1983a; e t c . ) . In a 1- ta i led chi t es t , the minimum group s ize to reveal a 50 %reduction in VT and VF i s 9 (Mainland et a l ; , 1956). 2.1.9.1 Normalisation procedures. In order to carry out parametric t es t s , such as Duncan's mult ip le range tes t , i t was necessary to ensure that the var iab le was Gaussian d is t r ibu ted . I t has been shown by our laboratory (Johnston et a l ; , 1983a) that many of the var iables measured are log^g-Gaussian d i s t r i bu ted . Therefore, in order to compare means, the log^g values were ca lcu la ted. This manoeuvre was carr ied out for the fol lowing var iab les : PVC, number and duration of VT and VF and time of maximum R wave and S-T segment e levat ion . Correct ions for co-variance by the use of normalization procedures were carr ied out, in cer ta in instances, to improve p rec is ion . For example, i t has been shown that the arrhythmia score (AS) corre lates l i nea r l y with JbT (Johnston e t a l . , 1983a). Therefore AS can be expressed as a function of - 123 -OZ. There i s a major problem assoc i a ted w i th the r e l a t i o n s h i p between AS and OZ, namely tha t AS/JoT becomes meaningless i f AS i s z e r o . Th is i s never the case in c o n t r o l s , but i n d rug - t rea ted groups t h i s i s o f ten the case . An attempt was made to overcome t h i s problem by deve lop ing an ischaemia score (ASMC), which f unc t i ons in the same way as the AS , but i nc ludes scores f o r s i gns o f ischaemia (R wave s i z e , e t c . ) . The f a c t tha t AS c o r r e l a t e s w i th J"5z, but cannot e a s i l y be co r rec ted f o r \J0Z~ was a l s o approached by adding 1 to each AS v a l u e , then c o r r e c t i n g t h i s va lue f o r ^JOZ, g i v i n g the v a r i a b l e (AS + 1)1^01. However, the p r e c i s i o n of OZ i s h i g h , such tha t i n p r a c t i c e and AS are not l i n e a r l y c o r r e l a t e d but are i ns tead s c a t t e r va lues around a s i n g l e p o i n t . Only when smal l OZs are d e l i b e r a t e l y produced ( C u r t i s et a l ; , 1984) i s the r e l a t i o n s h i p between \[0Z~ and AS apparent . When la rge OZs on l y are produced there i s no need to c o r r e c t AS f o r \JoZ. 2 . 1 . 9 . 2 Censo r i ng . Censor ing as a r e s u l t o f death was d i scussed in the s e c t i o n concerned w i th thump-vers ion d e f i b r i l l a t i o n . Censor ing cannot be avoided and i t s e f f e c t can on ly be m in im ised . The type of censor ing which j e o p a r d i s e s an experiment must be i d e n t i f i e d and app rop r ia te a c t i o n taken . The censor ing in t roduced by thump-vers ion was cons idered to j e o p a r d i s e the experiment l e s s than the censor ing assoc i a t ed w i th a l l ow ing animals to d i e . 2 . 1 . 9 . 3 S t a t i s-t-i c a l - t e s t s . For b i n o m i a l l y d i s t r i b u t e d v a r i a b l e s 2 ch i t e s t s were used. Ma in land ' s cont ingency t a b l e s of minimum con t ras t s 2 were used f o r ch i t e s t i n g (Main land e t - a l ; , 1956) . For G a u s s i a n -d i s t r i b u t e d v a r i a b l e s , a n a l y s i s of va r i ance (ANOVA) was c a r r i e d o u t , us ing a U . B . C . s t a t i s t i c s program (Gregg and O s t e r l i n , 1977) . Only i f t reatment c o n s t i t u t e d a s i g n i f i c a n t source of va r i ance accord ing to an F t e s t were means compared (us ing Duncan's m u l t i p l e range t e s t ) . Th is t e s t was app rop r i a t e s i n c e the number of groups exceeded two in every s tudy . In no - 124 -instance was a simple t test used for comparing means, since the t test i s only appropriate when two groups are compared; i f a 1 in 20 probab i l i t y i s set as the l im i t of chance, and 20 groups are compared with a control group, then the probab i l i t y approaches 100% that at least one group w i l l be found to be d i f ferent from cont ro ls , according to the t t es t . Modified t t e s t s , such as Duncan's mult ip le range tes t , avoid th is p i t - f a l l , because the number and type of comparisons are accounted f o r . 2.2 Calcium-antagonist-studies in coronary-^occluded - rats 2.2.1 General experimental design Randomisation to treatment was carr ied out by assembling a table of l i n e s . Each l i ne was assembled by drawing playing cards numbered according to the number of groups in each study. Drug stocks were prepared, and appropriate d i lu t ions made by various members of the laboratory according to spec i f i c ins t ruc t ions . The stock solut ions were coded. Therefore the person preparing a syringe of drug for a par t i cu la r rat was also b l ind to treatment. Lines were chosen in random order by the person preparing the i n j ec t i on . This person prepared a second table in which the ra t code (assigned on the day of preparation) and the coded treatment were noted. In the case of rats treated with unstable drugs ( fe lod ip ine , n i fed ip ine and DHM9), the person making up the drugs was instructed to weigh out a spec i f ied amount of drug for each ra t , and was therefore not b l ind to the treatment. However, the person administering the drug, occluding the coron-ary ar tery , monitoring responses and analysing the records remained b l ind to the treatment. A l l other aspects of study were as out l ined in previous chapters. Up to 4 rats were occluded on each day of experimentation, with a 15 min in terva l between each occ lus ion. In th is manner, rat-1 had passed through the phase of ear ly arrhythmias (Clarke e t a l 1 9 8 0 ; Johnston et a l . , 1983a) - 125 -by the time rat-2 was due for occ lus ion; rat-2 received i t s drug treatment while rat-1 was being monitored for arrhythmias, and so on. For each rat a complete h is tory was kept, both in the laboratory day book and also on indiv idual analysis sheets. The analysis sheets contained a l l information concerning haemodynamic, ECG and arrhythmia data, as wel l as OZ, IZ, serum K + , body weight, date of preparation and occ lus ion, general comments concerning preparat ion, behavioural responses to drug treatment and occlusion and postmortem f ind ings . When the study was complete, the codes were broken and the resu l ts analysed. According to the p r i nc ip le of random-isat ion to l ines i t i s s t a t i s t i c a l l y acceptable to break the code upon the completion of each l i n e . However, th is pract ice was avoided. 2.2.2 Phenethylalkylamines 2.2.2.1 Anipamil -and-Ronipamil. The ef fects of anipamil and ronip-amil on responses to coronary occlusion were invest igated because these anal -ogues of verapamil d i f f e r in respect to the i r calcium antagonist a c t i v i t y , and were therefore considered to be valuble tools for tes t ing the hypothesis that calcium antagonism in the ven t r i c le is antiarrhythmic during acute myocardial ischaemia. Anipamil and ronipamil are analogues of verapamil. In the case of anip-amil the main chain of verapamil has been extended by 10 C-atoms and the methoxy substi tuents removed from the 4 posi t ion on the phenyl r i ngs , leaving l,7-bis-(3-methoxyphenyl)-3-methylaza-7-cyano-nonadecane. Ronipami1 res -embles anipamil except for a lack of methoxy substi tuents on the phenyl r i ngs , i .e i t i s l,7-bisphenyl-3-methylaza-7-cyano-nonadecane. Both drugs have been demonstrated to have anti- ischaemic a c t i v i t y in a var ie ty of preparations (Kovach, 1984; Kretzschmar and Raschack, 1984; Raschack 1984; Urbanics and Kovach 1984). Both anipamil and ronipamil were administered by the p.o. route. The - 126 -doses chosen were 50 and 150 mg/kg, based on prel iminary t o x i c i t y studies in which i t was found that administrat ion of up to 300 mg/kg was non-toxic (higher doses produced death in approximately 50 % of rats given anipamil ; the cause of death was unclear, since i t occurred overnight in every i ns -tance, but was considered to be the resu l t of cardiovascular depression, since the drug caused a progressive decrease in blood pressure with time in these r a t s ) . Both drugs were dissolved in d i s t i l l e d water and administered at a volume of 0.25 ml/kg body weight. Suspensions of 20 or 60 mg/ml of e i ther drug were prepared and gently heated to 60 °C to f a c i l i t a t e d isso-l u t i on , then allowed to cool s l i g h t l y before adminis t rat ion. Control animals received an equivalent volume of d i s t i l l e d water at the same temperature as the drug suspensions. Oral administrat ion was car r ied out 4 h before occlusion during a period of extremely br ie f anaesthesia (1 min) with halo-thane. An in t ragas t r i c tube for drug administrat ion was created by simply f i x i n g a 12 cm length of PE90 tubing to a 3 ml syr inge. The treatments were administered slowly in order to preclude accidental administrat ion into the trachea. The p o s s i b i l i t y of aspirat ion pneumonitis was discounted since rats lack a well-developed vomit re f lex (Briggs and Oehme, 1980). Drugs were made up fresh approximately once per week, re f r igerated and stored in l igh t -proof containers. 2.2.2.2 (-+-)- And (-)-verapami 1. The opt ica l enantiomers of verap-amil were compared for the i r actions on the responses to coronary occlusion in order to test the hypothesis that calcium antagonism in the vent r i c les is antiarrhythmic during acute myocardial ischaemia, and also to examine the complementary hypothesis that the antiarrhythmic act ion of (^J-verapamil in conscious rats (Curt is e t - a l . , 1984) occurred by v i r tue of calcium antagonism in the vent r ic les (see Introduct ion). Both hypotheses predict an a n t i -arrhythmic potency ra t i o equal to the calcium antagonist potency ra t i o in - 127 -the vent r icu la r myocardium, based on the reported potency di f ference between the enantiomers for calcium antagonism ( e . g . , Bayer et a l ; , 1975b; 1975c; Nawrath e t -a l . - , 1981; Ferry e t - a l ; , 1985). Drugs were dissolved in sa l ine and administered at a volume of 0.25 ml/kg by slow i . v . in jec t ion over 10 min, beginning 15 min before coronary occ lu -s ion , according to the protocol out l ined in the general methods sect ion (see Figure 1) . Ten groups of rats (n = 9 per group) were used. Two groups received sa l ine (cont ro ls ) . The remaining groups received ei ther (-)-verap-amil (0.2, 0.6, 2 or 6 mg/kg) or (+)-verapamil (0.4, 4 , 8 or 12 mg/kg). The doses studied were chosen on the basis of the e a r l i e r experiment with (^J-verapamil (Curt is e t - a l . , 1984), in which 6 mg/kg was found to be the EDgQ for reducing arrhythmia score (AS). Drug stocks were made up in advance of the study by d i l u t i o n , and were re f r igerated and stored in l ight -proof vesse ls , coded to ensure double-bl indness. 2.2.3 1,4-Dihydropyridines 2.2.3.1 Felodip ine. Felodipine was evaluated on the basis of i t s s e l e c t i v i t y as a calcium antagonist for the vasculature versus the myocardium ( e . g . , Au and Sutter , 1984). the hypothesis that calcium antagonists reduce ischaemia-induced arrhythmias v ia an action in the ven t r i c les predicts that fe lod ip ine (and other 1.4-dihydropyridine calcium antagonists) w i l l only reduce arrhythmias at high doses. In add i t ion , a calcium antagonist which shows marked s e l e c t i v i t y for the vasculature may prec ip i ta te 1i fe- threatening reductions in blood pressure at doses below those producing blood leve ls su f f i c i en t to reduce i .. in the vent r ic les and reduce the incidence and sever i ty of ischaemia-induced arrhythmias. Therefore, i t was predicted that fe lod ip ine would have l i t t l e i f any antiarrhythmic a c t i v i t y at doses which produced large reductions in blood pressure. - 128 -Eight groups of rats were used (n = 9 per group). In addit ion to the standard large OZ (LOZ) rats (5 groups), 3 groups of small OZ (SOZ) were prepared and subjected to coronary occlusion by Kathy Johnston. The LOZ rats received 1 of 3 doses, 0 .2 , 2.6 or 12.2 ymol/kg, according to the schedule out l ined in the general methods. These doses were 0.08, 1 and 4.68 mg/kg, respec t ive ly , and have been coded as L, I and H (low, intermediate and h igh, respect ive ly) for the purpose of label ing the f igures in the Results sec t ion . Only the lower and higher doses were given to the SOZ ra t s . Felodipine was dissolved in 20 %ethanol in sa l ine and administered at a volume of 0.25 ml/lOOg body weight. Care was taken to protect a l l syringes from d i rec t sunl ight / f luorescent room l i gh t ing before administ rat ion. Twenty seven control rats were used (18 LOZ rats and 9 SOZ r a t s ) . Control rats received 0.25 ml/lOOg body weight of 20%ethanol in sa l i ne . The LOZ and SOZ rats were analysed separately, since treatment was the only known source of variance within the LOZ and SOZ se r i es , but operators were a source of variance between the LOZ and SOZ se r i es . Drugs stocks were made up in advance of the study and were re f r igerated and stored in l ight -proof vesse ls . 2.2.3.2 Ni fedipine and DHM9. These 1,4-dihydropyridines were stud-ied in order to supplement the work with fe lod ip ine . DHM9 has been reported to possess a s e l e c t i v i t y of action for vent r icu lar t issue versus vascular smooth muscle, in contrast with a l l other 1,4-dihydropyridine calcium antag-onists (Clarke e t - a l ; , 1984b). Therefore, according to the hypothesis that calcium antagonism in the vent r i c les is antiarrhythmic during acute myocar-d ia l ischaemia, i t was expected that DHM9 should be more e f fec t i ve in reducing ischaemia-induced arrhythmias than n i fed ip ine at doses producing a s imi la r degree of blood pressure lowering. Five groups of rats were studied (n = 9 per group). Drugs were dissolved in 20%ethanol in sa l ine and administered at 0.25 ml/lOOg body - 129 -weight in accordance with the schedule out l ined in the general methods sect ion. Controls received the ethanol vehic le alone. The doses were 0.5 and 2 mg/kg n i fed ip ine and 5 and 20 mg/kg DHM9. These doses appear to be la rge, but they were based on a prel iminary t o x i c i t y study in which i t was found that conscious rats could to lerate at least 16 mg/kg n i fed ip ine . Although not invest igated in any d e t a i l , i t was considered that the dose-l im i t i ng factor was in fac t the ethanol vehic le rather than the drug i t s e l f . Prel iminary invest igat ions with DHM9 showed that no haemodynamic ef fects occurred at a l l at cumulative doses well in excess of 30 mg/kg. In accor-dance with the general method, stock solut ions were prepared in advance of the study, coded, re f r igerated and stored in l ight -proof containers. Care was taken to prevent exposure of the drug-containing syringes to l igh t before administ rat ion. True blindness could not be achieved in th is study, since the yellow colourat ion of the drugs could be seen in the thin PE tubing entering the subscapular region of the rats at the highest doses (both DHM9 and n i fed ip ine are v i v i d yellow in co lour ) . However, a l l records were analysed b l i n d . 2.3 Arrhythmogenesis and the- ro leof - the-GNS 2.3.1 Introduction The experiments out l ined here were not designed to answer a l l questions concerning arrhythmogenesis in r a t s , but were intended to examine the ro le of the CNS and the sympathetic nervous system. Our laboratory has car r ied out prel iminary experiments for th is purpose in the past (Bott ing et a l ; , 1983). The strategy was to remove, by surgery, the autonomic nervous system in a graded manner in some groups of animals, and replace i t by infusion of catecholamines in others. In addi t ion, the extent of surgery was considered as a possible source of var iance. In conjunction with th is l a t t e r consider-- 130 -a t ion , the ef fects of p i th ing (the most extensive surg ical ablat ion) on possible mediators and modulators of arrhythmias (serum K + , leukocytes and thrombocytes) were determined in a separate ser ies of experiments. A l l groups in th is study consis ted, as usual , of 9 ra t s . Occlusion was car r ied out as described above, and blood pressure was recorded v ia the aor ta , except when stated otherwise. Since there were many groups, and preparation was complicated, a summary table has been prepared which l i s t s a l l the groups (Table 1 ) . 2.3.2 Preparation 2.3.2.1 P i th ing . Male Sprague Dawley rats were l i g h t l y anaesthe-t ised with halothane (4% in oxygen) and intubated. The rats were prepared for occlusion in the normal manner, with an aor t ic blood pressure l i n e , jug-ular intravenous l i ne and Vg ECG leads. These rats were allowed to recover from preparative surgery for approximately 7 days. This group was subse-quently p i thed, as described below, and const i tuted the chron ica l l y prepared (c) pithed (P) group ( i . e . , cP ) . A second group was prepared with occluders, Vg ECG leads, carot id blood pressure l ines and femoral intravenous l ines (by standard ca the te r i -sation techniques using PE tubing). This group const i tuted the acutely prepared (a) pithed (P) group ( i . e . , aP), since they were pithed immediately af ter preparat ion. P i th ing was carr ied out as fo l lows. A s ta in less steel rod (3 mm d i a -meter) was passed through the orb i t and down the spinal cord during l i gh t anaesthesia (1% halothane in oxygen). Immediately afterwards, a r t i f i c i a l resp i ra t ion with 100% oxygen was inst igated (stroke volume 4 ml per 300 g body weight, 54 strokes/min). This a r t i f i c i a l resp i ra t ion regimen has been shown to produce blood gas and blood pH leve ls wi thin the normal range in pithed rats (Milmer and Clough, 1985). The rats were then mounted v e r t i c a l l y - 131 -with the head point ing downward, by securing the t a i l and the p i th ing rod in 2 clamps attached to a re tor t stand. The blood pressure transducer was elevated at th is time in order that i t should remain in the same horizontal plane as the heart . A recta l thermocouple was connected v ia an Indicat ing Contro l ler unit (YSI Model 73ATA) to a 100 W l i gh t bulb, placed 20 cm from the ra t , and a sheet of polythene was placed over the ent i re preparat ion, to maintain body temperatute at 37 - 38 °C. Sal ine (1 ml per 100 g body weight) was then in jected i . v . in an attempt to compensate for the i n i t i a l blood-pressure lowering ef fect of p i th ing . Coronary occlusion was carr ied out 30 min af ter p i th ing . Rats were monitored for 4 h, then sac r i f i ced (exsang-u inat ion) . A l l var iables were measured in the usual way (see general Methods sec t ion) . Infarct s ize (IZ) was not measured, since at least 10 h is required for quant i f iab le in fa rc t development according to the TTZ sta in ing technique (Hort and Da Cana l is , 1965b). 2.3.2.2 Sp ina l i sa t i on . Rats were prepared and occluded in a manner ident ica l with that described for acutely prepared pithed rats (the aP group) with the exception that instead of p i th ing , the rats were sp ina l i sed . This was achieved by inser t ing a steel rod into the sku l l through the foramen magnum at the level of C I , advancing i t r o s t r a l l y and rotat ing i t l a t e r a l l y to mascerate the b ra in . This group const i tuted the acutely prepared (a) sp ina l ised (S) group ( i . e . , aS). As in the case of the aP group, 30 min was allowed between ablat ion in the CNS and occ lus ion. At the time of occ lus ion, spinal ref lexes (foot withdrawal to pinching) were return ing. This group received s im i la r cardiovascular and respi ratory support to the pithed rat groups. This group was also monitored for 4 h then s a c r i f i c e d . 2.3.2.3 Decerebration. Rats were prepared and occluded in a manner ident ica l with the acutely prepared pithed and sp ina l ised groups (aP and aS respect ive ly) with the exception that the ablat ion carr ied out was decere-- 132 -brat ion. Decerebration was achieved by removing the brain ros t ra l to the midcol1icular level using the blunt end of a spatula fo l lowing craniotomy. The empty space was packed with gel foam. A 30 min in terva l was allowed between ablat ion and occ lus ion. In contrast with the previous groups, no cardiovascular or resp i ra tory support was required. After preparation the rats in th is group breathed spontaneously and maintained a high blood press-ure while hor i zon ta l . This group const i tuted the acutely prepared (a) decerebrate (D) group ( i . e . , aD). 2.3.3 Other manipulations The groups described above received ablat ions in the CNS at d i f ferent l eve l s . The pithed groups received complete CNS ab la t ion , the sp ina l ised rats were in tact d i s ta l to C l , and the decerebrate group possessed in tact resp i ra tory and vasomotor centres as well as spinal re f l exes . In a supplementary group of acutely prepared pithed ra t s , an attempt was made to restore the ef fects of the sympathetic nervous system by infusing a mixture of noradrenaline and adrenal ine. This infusion was begun 15 min before occlusion and was designed to elevate blood pressure to leve ls seen in conscious rats (mean of approximately 100 - 110 mmHg; Johnston e t - a l ; , 1983a). The mixture was 4:1 noradrenaline:adrenaline (on a weight b a s i s ) , and the infusion rate was var ied from 0.2 to 5 ug /kg/min noradrenal ine. Infusion volume was kept below 10 ml/kg/h. This group const i tuted the acutely prepared (a) , pithed (P) noradrenaline/adrenaline treated (N) group ( i . e . , aPN). Respiratory and cardiovascular support and a l l other aspects of experimentation were ident ica l with those used for the aP and aS groups. Four groups of ' con t ro l ' rats were used. A standard group of conscious chronical ly-prepared rats were prepared and occluded in the manner described in the general methods sec t ion . This group const i tuted the chron ica l l y prepared (c) conscious (C) group ( i . e . , cC) and served as a control group - 133 -free from recent surgery and ablat ions in the CNS. A second, s imi la r group received an infusion of noradrenaline/adrenaline mixture ident ica l with that administered to the aPN pithed group. This group of conscious rats const i tuted the chron ica l l y prepared (c) conscious (C) noradrenaline/adrenaline (N) treated group (cCN), and served as control for the ef fects of the catecholamine infusion in the absence of ablat ions in the CNS. A th i rd group of rats was prepared in the usual way, but instead of 7 days, these rats were allowed only 1 h to recover from preparative surgery before occ lus ion. This group const i tuted the acutely prepared (a) conscious (C) group ( i . e . , aC), and served as a control for recent surgery in the absence of surgical ablat ion in the CNS. F i n a l l y , a group of rats was prepared for occlusion according to Clark e t - a l ; (1980) using pentobarbitone (60 mg/kg i .p . ) anaesthesia, with the minor exception that our standard occluder (not the s i l k type of Clark e t - a l . ) and our V3 ECG leads (not lead 2) were used. Blood pressure was recorded from the l e f t carot id ar tery , and a cut-down tracheotomy was per-formed (for del ivery of a r t i f i c i a l resp i ra t ion according to the regimen used for the pithed and sp ina l ised groups). Occlusion was carr ied out 30 min af ter preparat ion, and rats were sac r i f i ced 4 h after occ lus ion. This group const i tuted the acutely prepared (a) barbi turate anaesthetised (B) group ( i . e . , aB), and served as a control for recent minor surgery in the absence of surgical ablat ion in the CNS but in the presence of chemical ablat ion in the CNS (anaesthesia). - 134 -Table 1. Summary of Groups in the CNS Ablations Study Acutely Prepared-Groups Code Conscious aC Anaesthetised aB Pithed aP Pithed plus catecholamine infusion aPN Spinal ized aS Decerebrate aD Chronical ly-Prepared Groups Code Conscious cC Conscious plus catecholamine infusion cCN Pithed CP Others Code Isolated perfused hearts I Detai ls of the surg ica l preparation are given in the tex t . - 135 -An addit ional group was included in th is study, a group of iso lated hearts perfused v ia the aorta at 37 °C with a modified Krebs Henseleit buffer containing 5.3 meq/1 K + . This group was prepared and occluded by Reza Tabrizchi according to the method of Kannengeisser et a l . (1975). This group const i tuted the iso la ted heart (I) group, and served as a general reference group. 2.3.4 S t a t i s t i c s Variables were measured and analysed as described for the standard occlusion preparat ion. The f u l l data set for th is study is extremely complicated, and i t is possible to discuss at great length the possible impl icat ions of the r e s u l t s . In order to s impl i fy matters somewhat, s t a t i -s t i c a l s ign i f icance has only been noted between the chron ica l l y prepared conscious group (cC) and the other groups. 2.4 Prel iminary-Screen for drug a c t i v i t y in acute-ischaemia 2.4.1 Introduction Although the conscious ra t preparation for invest igat ion of myocardial ischaemia and in farc t ion is capable of providing up to 10 data points per var iab le per week, i t was decided to develop a prel iminary screen for assessing new drugs with which 4 times as much information could be generated in the same time. This preparation was designed simply to provide i n fo r -mation concerning morta l i ty and in fa rc t s i z e . However, assessment of animal behaviour was also used, in order to attempt to del ineate between morta l i ty resu l t ing from vent r icu lar arrhythmias (VF) and other causes. 2.4.2 Preparation Male Sprague Dawley rats were prepared for occlusion in the manner described in deta i l in preceding chapters. No intravascular catheters and ECG leads were implanted, only the occluder. By th is technique, 16 rats could be prepared d a i l y , with ease. The rats were housed ind i v idua l l y and - 136 -allowed approximately 7 days before being subjected to coronary occlusion in the usual manner. 2.4.3 Experimental endpoints Following occ lus ion, rats were ca re fu l l y monitored by observation alone. The behaviour of each rat was recorded on ind iv idual analysis sheets. A l l observation and analysis was car r ied out b l i n d . 2.4.3.1 Def in i t ions . Certain behaviours were ca re fu l l y categorised for each time in terva l fo l lowing occlusion (the time in te rva ls corresponded with those used for the standard occlusion preparat ion). The time of death was noted, and OZ was measured in the usual manner. In rats surviv ing 24 h, the IZ was also measured. Behaviour was c l a s s i f i e d and assessed according to the fol lowing sub-jec t i ve c r i t e r i a . Morbidity was graded according to the presence or absence of the fo l lowing 3 behaviours, respi ratory d is t ress (which was defined as laboured breathing), head-down posture, and prone posture. These 3 end-points were considered to be signs of cardiogenic shock (Agress e t - a l . , 1952). If nothing more serious than minor panting was present, the rat was classed as normal. Severe vent r icu lar arrhythmias were diagnosed on the basis of sudden 'convuls ive- type ' behaviour. This was defined as sudden f renzied attempts to climb out of the home cage accompanied by convulsive-type limb movements and a sudden blanching of the ears and eyes. This behaviour i s en t i re l y cha rac te r i s t i c of VF (or severe torsade de pointes) las t ing longer than 10 sec in the standard instrumented rat preparat ion, and has never been observed in associat ion with other sequelae of coronary occlusion such as AV block or cardiogenic shock. Occasional ly , rats develop fa ta l pulmonary oedema fol lowing occ lus ion. The associated behaviour is d i f ferent from that caused by VF. Fatal pulmon-- 137 -ary oedema in conscious rats is associated with f renzied behaviour, but i s character ised by the absence of pal ing of the ears and eyes and the presence of expectoration of copious sputum (often bloody). Furthermore, convuls ive-type behaviour resu l t ing from VF las ts for less than 10 sec, since syncope rap id ly fol lows the loss of cardiac output, whereas the f renzied behaviour associated with acute pulmonary oedema general ly las ts for more than 30 sec, since cardiac output is maintained. Acute pulmonary oedema is always f a t a l ; the death throes are associated with serious expectoration and an unpleasant gagging sound. In contrast , sudden convulsive-type behaviour resu l t ing from vent r icu lar arrhythmias is never associated with sudden expectorat ion, and i s not invar iab ly f a t a l ; often a rat w i l l loose consciousness, then suddenly the ears and eyes w i l l turn pink and the rat w i l l regain consciousness without apparent i l l e f fec t . F i n a l l y , acute pulmonary oedema is general ly associated with a highly pathognomic prodroma, involv ing the signs of morbidity out l ined above. Sudden convulsive-type behaviour i s not associated with any prodroma. 2.4.3.2 Val idat ion of behaviour- end-points. The behaviours des-cr ibed above were tested in a b l ind study in which 12 standard f u l l y - i n s t r u -mented rats were prepared and occluded in the usual manner, while an observer recorded behaviour in the manner described. These par t i cu la r ra ts were not d e f i b r i l l a t e d i f VF developed, but were allowed to die (or spontaneously d e f i b r i l l a t e ) and were therefore ident ica l to rats subsequently used for comparing the verapamil enantiomers. 2.4.4 Comprison of (+)-, ( - ) - and (±)-verapamil As part of the invest igat ions of the actions of verapamil in myocardial ischaemia and i n fa r c t i on , a study was carr ied out with the opt ica l enantio-mers and the racema'te, using the above-described technique. Since the prec is ion of th is method was unknown, and since up to 16 rats could be - 138 -occluded and observed per day, i t was decided to use large group s izes (n = 25 per group). Rats were prepared and allowed to recover from surgery as descr ibed. On the day of study, up to 16 rats were stat ioned on a s ing le laboratory bench. Drugs were administered v ia a super f i c ia l t a i l vein using a bu t te r f l y hypo-dermic needle while each rat was temporari ly restra ined in a perspex res t -r a i n e d Drugs were in jected over a 10 min per iod, and 5 min was then allowed before occ lus ion , in a manner analogous to the method used for studies in standard ful ly- instrumented r a t s . The cycle time ( in terva l between success-ive occlusions) was therefore 15 min. This in terva l was designed such that close attent ion could be given to the behaviour of each rat during the c ruc ia l f i r s t 5 - 1 0 min af ter occlusion (when the highest incidence of VF and VT occurs) . Rats were continuously monitored for at least 4 h af ter occ lus ion . The fo l lowing treatments were administered: sa l ine (0.25 ml/100 g body weight), (+)-verapamil (6 mg/kg), (±)-verapamil (6 mg/kg) and (-)-verapamil (6 mg/kg). The dose of 6 mg/kg was chosen in accordance with the previously determined E D ^ Q for (±)-verapamil in ful ly- instrumented conscious rats (Curt is et a l . , 1984). The object of the study was essen t i a l l y ident ica l with the object of the study with the enantiomers in fu l ly- instrumented rats described previously , namely to test the hypothesis that the antiarrhythmic actions of ( ^ - ve rap -amil occur by v i r tue of calcium antagonism. The hypothesis predicts that the incidence of convulsive-type behaviour should be reduced more by (-)-verapamil than by (+)-verapami1, with (±)-verapami1 having intermediate a c t i v i t y . In addi t ion, the experiments were also carr ied out in order to examine and character ise th is new method of assessing drug a c t i v i t y in acute myocardial ischaemia and in fa rc t ion on the basis of a combination of object-- 139 -ive (OZ, IZ and mortal i ty) and subject ive (behavioural) end-points. 2.4.5 S t a t i s t i c s The incidence of morta l i ty and VF are binomial ly d is t r ibu ted in conscious rats fo l lowing occlusion (Johnston e t -a l . - , 1983a). Therefore, the incidence of sudden convulsive-type behaviour, morta l i ty and morbidity were analysed 2 using chi , in the manner described previously . The resu l ts were categor-ised in terms of the f i r s t 0.5 h and the 0.5 - 4 h periods af ter occ lus ion, in accordance with the bimodal d i s t r i bu t ion of ischaemia-induced arrhythmias with time in conscious rats (Johnston e t - a l . , 1983a). 2.5 Electr ical ly-^ induced-arrhythmias- in conscious rats 2.5.1 Introduction The opt ica l enantiomers of verapamil are known to block i N a in ven-t r i c u l a r muscles at high concentrations (Nawrath et a l . - , 1981). These concentrations are 50 - 150 times in excess of those necessary to abol ish i' s l- and c o n t r a c t i l i t y in the normal ven t r i c le (Nawrath et a l . - , 1981). Therefore, the p o s s i b i l i t y that blockade of i ' N a (sodium channel blockade) contr ibutes to the pharmacological actions of (^ -verapami l and i t s enant io-mers in vivo seems highly un l i ke l y . However, th is p o s s i b i l i t y was neverthe-less examined by comparing the actions of the enantiomers for the i r a b i l i t y to inf luence arrhythmias induced by e l e c t r i c a l st imulat ion of the l e f t ven t r i c le in conscious ra t s . I t was previously shown that quinidine i n f l u -enced e l e c t r i c a l l y induced arrhythmias in conscious rats (Curt is e t a l ; , 1984) at the same dose which reduced occlusion-induced arrhythmias (arrhyth-mia score) by 50% (Johnston et a l ; , 1983a), whereas (^J-verapami1 had no such actions at the dose reducing arrhythmia score by 50% (Curt is et a l ; , 1984). Therefore the assessment of e lec t r i ca l l y - i nduced arrhythmias was considered to be useful for d i f f e ren t ia t i ng between Na + channel blockers and calcium antagonists. - 140 -2.5.2 Preparation Male Sprague Dawley rats were prepared in a manner ident ica l with that for rats used for coronary occlusion s tud ies , with 1 exception. Instead of a coronary occluder, 2 tef lon-coated s ta in less steel wire electrodes were implanted approximately 3 mm apart into the l e f t v e n t r i c l e , in approximately the centre of the l e f t vent r icu lar w a l l . This was achieved by plunging the e thano l - s te r i l i sed leads into the v e n t r i c l e , using a 23 gauge hypodermic needle as a locator . A l l leads and l ines were ex ter io r ised in the subscap-ular reg ion, and approximately 7 days was allowed for recovery from th is preparative surgery. On the day of study, the rats were connected to the standard devices for intravenous drug adminis t rat ion, and blood pressure and ECG recording. 2.5.3 Experimental end-points The var iables described below were each measured 3 times every 5 min. Their measurement has been described previously , but not in great deta i l (Curt is et a l ; , 1984). Stimulation of the l e f t ven t r i c le with square wave pulses was undertaken using a Grass st imulator (Model SD9), which was c a l i -brated using a standard voltmeter (Beckman Model 3020B). A permanent record of the ECG and blood pressure was made using the standard Grass polygraph, while the ECG was also continuously monitored using a delayed loop o s c i l l o -scope (Honeywell Type E for M). Discr iminat ion of end-points was carr ied out using the osc i l l oscope . 2.5.3.1 Maximum- following- frequency. Maximum fol lowing frequency was defined as the frequency at which the ven t r i c le f a i l e d to fo l low the stimulus on a 1:1 bas is . Fa i lu re to fol low was accompanied by a character-i s t i c blood pressure change; the dropped-beat produced a drop in blood pressure, and the subsequent beat produced a large pulse as a resu l t of the increase in f i l l i n g time and end-dioasto l ic pressure. The blood pressure - 141 -record could be used for d iscr iminat ion of the end-point, but in th is ser ies of experiments the osc i l loscope alone was used. Maximum fol lowing frequency was determined at 0.8 msec pulse width and twice threshold vol tage, by slowly increasing the st imulat ion frequency from a baseline of 5 Hz. 2.5.3.2 Threshold vol tage. The threshold voltage for inducing VT was determined at 50 Hz and 0.8 msec by the same p r inc ip le used for deter-mining maximum fol lowing frequency. The high frequency was used in order to maximise the probab i l i t y of de l iver ing a pulse during the vulnerable per iod, the terminal portion of the QT (de Boer, 1921; Wiggers and Wegria, 1940). In addit ion to the appearance in the ECG of VT, the threshold voltage for VT was character ised by a drop in blood pressure. VT was almost always non-sustained. 2.5.3.3 Threshold pulse- width. The threshold pulse width for inducing VT was determined from the osc i l loscope according to the same c r i t e r i a as those used for measuring the threshold vol tage. This var iab le was measured at 50 Hz and twice threshold vol tage. 2.5.4 Comparison of ( + )- and (-)-verapamil In the e a r l i e r study with (±)-verapamil , i t was found that 6 mg/kg had no ef fect on the var iables in question in conscious rats (Curt is et a l . , 1984). Therefore, i t was decided to evaluate only the equivalent of the highest doses of the enantiomers administered to coronary-occluded ra t s , since i t was not expected that the lower doses would inf luence the var iab les . Therefore e i ther 8 or 12 mg/kg ( + )-verapami 1 or 2 or 6 mg/kg (-)-verapami1 (n = 6 per group) were administered v ia the vena caval i . v . l i n e , over a 10 min per iod. The st imulat ion var iables were recorded 1 min before the s ta r t of drug administrat ion and 5 min af ter completion of drug administra-t i o n . The %changes in the var iables were recorded and analysed. The values for the var iables were noted on the ECG t races , and were analysed b l i n d . - 142 -Mean %changes in the var iables were compared by ANOVA and Duncan's mult ip le range tes t . 2.6 Haemodynami c and EGG - ef fects of -ca lc i um- antagon i s t s - i n pi thed-rats 2.6.1 Introduction In previous sect ions, the procedures for assessing the antiarrhythmic act ion of the enantiomers of verapamil were described. The premise was that (-)-verapamil is more potent as a calcium antagonist than (+)-verapamil. It was decided that rather than re ly on l i t e ra tu re values for the re la t i ve potency of the enantiomers of verapamil (Bayer e t - a l ; , 1975b; 1975c; Ferry e t - a l . , 1985; Nawrath et a l . - , 1981; Raschack 1976b; Echizen e t -a l . - , 1985), which give disparate values ranging from 4 to over 100 in favour of ( - ) -verapami l , various attempts should be made to gauge the re la t i ve potency of the enantiomers ourselves. The f i r s t method was to examine the ef fects of the enantiomers on blood pressure, heart ra te , P-R in terva l and QRS i n -terval in conscious ra t s . This par t i cu la r experiment const i tuted a portion of the occlusion study with the enantiomers, since a comparison of values before and af ter drug administrat ion provided th is information. However, i t was decided to supplement th is information with other s tud ies . Ea r l i e r work with pithed rats (see sect ion concerned with arrhythmo-genesis in myocardial ischaemia) had shown that i t was possible to produce a pithed ra t preparation with a high mean blood pressure of at least 60 mmHg, which remained v iable for well over 4 h. I t was decided to use th is prepa-rat ion to invest igate the actions of the verapamil enantiomers in the absence of autonomic tone and re f lexes . This information was considered to be of i n te res t , not least because there appears to be a large d ispar i t y between the potency of (^-verapami l in anaesthetised versus conscious rats (Curt is et a l . , 1984). - 143 -2.6.2 Preparation The pithed rat preparation was essen t ia l l y the same as that described prev ious ly , with the exception that in every instance blood pressure was recorded from the aor ta, and drugs were administered into the vena cava. Each preparation was allowed to s t a b i l i z e for at least 1.5 h before study, and mean aor t ic blood-pressure and the ECG were pe r iod i ca l l y recorded during th is t ime. 2.6.3 Variables measured Mean blood pressure, heart ra te , P-R in terva l and QT in terva l were recorded. The ECG var iables were measured at a chart speed of 100 mm/sec, using standard ECG c r i t e r i a (Horan and Flowers, 1980) as modified by Dr i sco l l (1980) for the ra t . Reductions in blood-pressure and prolongation of P-R in terva l are ef fects cha rac te r i s t i ca l l y produced by calcium antagonists, and are considered to be the resu l t of calcium antagonism (see Nayler and Horowitz, 1983). 2.6.4 Comparison of (+)- and (-)-verapamil Once mean blood pressure had reached a stable level (no more than a 5 mmHg var ia t ion over a 10 min per iod) , increasing doses of e i ther (+)-verapami1 (0.4, 0.8, 4 and 8 mg/kg) or (-)-verapami 1 (0.02, 0.06, 0.2 and 0.6 mg/kg) were administered (n = 6 per drug). Each successive dose was administered as a slow i . v . in jec t ion over a 10 min per iod. I t was observed in p re l im in-ary experiments that the peak blood-pressure lowering e f fec t of the enantio-mers occurred during the f i r s t 30 sec af ter f i n i sh ing an i n j ec t i on . In the present experiments, a l l var iables were recorded 10 - 15 sec af ter administ-ering each dose, in order to measure the peak e f fec t . An in terva l of 15 min was allowed between completing the f i r s t in jec t ion and s ta r t ing the second, and so on. - 144 -2.6.5 S t a t i s t i c s The 25 min cycle time used in the current experiments permitted recovery of mean blood pressure to pre-drug values by the time the subsequent dose was administered, j us t i f y i ng the expression of the data as s ing le point dose response curves, rather than cumulative dose response curves. Variables (ED^g or ED25 values) were compared, where appropriate, using ANOVA and Duncan's mul t ip le range tes t . 2.7 Act ions-of -ca lc ium antagonists i n - i so la ted per fused-rat -vent r ic les 2.7.1 Introduction The fo l lowing experiments were carr ied out in order to character ise further the calcium antagonist potency ra t i o of the opt ica l enantiomers of verapamil, and to invest igate the actions of n i fed ip ine and DHM9. I t was considered worthwhile to have a measure of calcium antagonist potency in rat vent r icu lar t i s sue , since th is was the t issue in which the antiarrhythmic actions of the calcium antagonists were suspected of being mediated. In addi t ion, verapamil was recent ly reported to exh ib i t s terose lec t ive plasma protein binding and hepatic metabolism in humans (Echizen et a l ; , 1985; Eichelbaum et a l . , 1984; Vogelgesang et a l ; , 1984). If the same phenomena occur in rats there i s a p o s s i b i l i t y that calcium antagonist potency ra t ios in vivo and in v i t r o may be d i f fe ren t . Therefore the potency ra t ios of the enantiomers in vivo (P-R interval prolongation and reductions in blood pressure in conscious and pithed rats) were compared with those obtained in v i t ro (negative inotropic act ions) in iso la ted vent r i c les per-fused v ia the aorta by the method of Langendorff (1895). The experiments were also designed to invest igate the p o s s i b i l i t y that ex t race l l u la r K + plays a ro le in governing calcium antagonist potency and the locus of antiarrhythmic a c t i v i t y of verapamil and other calcium antago-n i s t s . Since ex t race l l u la r K + concentration has been shown to r i s e rap id ly - 145 -fol lowing coronary occlusion (Hirche e t - a l ; , 1980; H i l l and Gettes, 1980), experiments were carr ied out over a range of buffer K + concentrat ion. 2.7.2 Perfusion apparatus The experiments were carr ied out using a perfusion apparatus which has recent ly been designed in the laboratory. The apparatus was designed with a small dead-space for rapid switching between d i f ferent solut ions in order to permit the generation of dose-response data. In b r i e f , 9 cy l inders of 70 ml capacity were machined into a block of p lex ig lass which contained channels for c i r cu la t i ng warm water (37 °C). Drug solut ions flowed from each cy l inder v ia separate s i l a s t i c tubes (2 mm outer diameter, 1 mm inner diameter) to meet in a common manifold with a low volume ( less than 0.1 ml) dead-space. The iso la ted ven t r i c le preparation (see below) was attached to the apparatus here. The flow from each cyl inder to the appropriate s i l a s t i c feed l i ne passed through machined Teflon taps such that flow could be switched between c y l i n -ders by simply opening 1 tap and c los ing another. The contents wi thin any cy l inder could be changed v ia a second Teflon tap at the opposite side of the cy l inder . The perfusion apparatus was pressurised to dr ive the drug solut ions though the coronary c i r c u l a t i o n , oxygenate each solut ion and regulate pH. The gas used was 5% CO2 in oxygen. A s ing le gas inf low branched at an exter ior manifold on the perspex box to give 9 separate s i l a s t i c tubes which served to del iver gas to each cy l inder . A l l cy l inders were sealed with a common perspex l i d . The l i d was tightened by brass screws c los ing on a rubber gasket. The atmosphere above each cyl inder was common, and was bled v ia a common ex i t through a regulat ing ' pop -o f f p ressure- re l ie f va lve. The pressures in the apparatus and at the aor t ic root were monitored by a manometer (anaeroid). - 146 -Oxygen del ivery in th is system was improved because of the design which reduces loss of oxygen to the dead space between the reservo i r and the heart , and because the apparatus oxygenates preheated buf fer , rather than heating pre-oxygenated buffer. A thermocouple inserted in the pulmonary artery was used to monitor temperature. 2.7.3 Spec i f ica t ions for Langendorff perfusion Hearts were excised from male Sprague Dawley rats (250 - 320 g) fo l lowing cerv ica l d is locat ion and exsanguination. The perfusion pressure was set at 95 mmHg. The a t r i a were removed and the vent r i c les were paced (300 st imul i /min) with square wave pulses at 4 V and 1 msec (supramaximal threshold voltage and pulse width) v i a tef lon-coated s ta in less steel plunge electrodes, using a Grass st imulator (Model SD9). The perfusing solut ion was a modified Krebs-Henseleit buffer comprising ( in mM) CaCl 2 0.7, NaCl 118, KCI 1.8 - 8 .8, MgCl 2 1.2, KH 2 P0 4 1.2, NaHC03 25 and dextrose 11, at 37 °C and pH 7.4. We used 0.7 mM CaCl 2 because in previous studies the E C ^ Q for pos i t i ve inotropism in perfused rat vent r ic les was found to be 0.5 ± 0.1 mM (Curt is e t - a l . , 1984). KCI was varied at the expense of NaCl to produce K concentrations from 3 to 10 meq/1 in order to simulate K + e levat ion seen in the ex t race l lu la r f l u i d during the ear ly period of myocardial ischaemia (Hirche et a l ; , 1980; H i l l and Gettes, 1980). 2.7.4 Variables measured 2.7.4.1 Isochori c 1 eft -ventr i cular-developed - pressure. A small com-p l i an t , but non-e las t i c , balloon was made from p l as t i c wrapping f i lm (Stretch n' Sea l ) . The balloon was connected to a transducer v ia a 30 cm length of PE50 tubing. The balloon was attached by ty ing a su i tab le sect ion of f i l m to the end of the PE tubing with s i l k thread, and was then f i l l e d with water and stretched by excess pressure to t r i p l e i t s o r ig ina l volume. - 147 -The balloon so-formed was non-e last ic and yet compliant enough to f i l l the cav i ty of the l e f t ven t r i c le without d is to r t ing i t s shape. Left in t raven-t r i c u l a r pressure was recorded af ter inser t ing the balloon into the l e f t ven t r i c le v ia a l e f t a t r i a l i n c i s i o n . The pressure within the balloon was adjusted to give an end d i a s t o l i c pressure of 15 mmHg. 2.7.4.2 Coronary blood f low. The perfusion apparatus was graduated to indicate the volume of f l u i d in each chamber. Coronary flow was estimated by measuring the volume of perfusion f l u i d passing from the graduated chambers, under the assumption that i f end d i a s t o l i c pressure remained constant when perfusion pressure was varied from 80 to 140 mmHg, then the aor t i c valve was competent. This re la t i on was tested during the s t a b i l i s a -t ion per iod. 2.7.4.3 Ventr icu lar e x c i t a b i l i t y . Prel iminary experiments showed that the threshold voltage and pulse width for capture of ven t r i c les were approximately 0.6 V and 0.1 msec at 5.9 meq/1 K + . The experiments were carr ied out at well above threshold (4 V and 1 msec), but at well below the threshold for inducing unwanted ef fects such as noradrenaline release and heating of the myocardium (as judged from the responses of hearts to large var ia t ions in st imulat ion var iables in prel iminary experiments). It was considered inappropriate to examine the strength-duration re la t ionsh ip in these par t i cu la r ven t r i c l es , since i t was possible that such an experiment may jeopardise the subsequent v i a b i l i t y of the preparat ions. Nevertheless, the threshold voltage (at 1 msec) and the threshold pulse width (at 4 V) for capture were determined in most experiments as an index of e x c i t a b i l i t y . 2.7.5 Comparison of (+)- and (-)-verapami1 Following 1 0 - 20 min s t a b i l i s a t i o n , the vent r ic les were perfused with (+)- or (-)-verapamil ( in half log^g increments) and steady state developed pressures were recorded. The enantiomers were loaded into the chambers of - 148 -the perfusion apparatus before each experiment from stock solut ions (prepared by se r i a l d i lu t ion) designed such that 0.2 ml of stock per 70 ml buffer gave the desired concentration wi th in each cy l inder . The stock solut ions were re f r igerated and stored in l ight -proof containers. Each preparation was used for a s ingle drug at a s ing le K buffer concentration (3, 5.9, 8 or 10 meq/1); therefore 8 groups of preparations (n = 6 per group) were used. Records were analysed b l i n d . Concentration-response curves were constructed and ECgQ and slope values were estimated for ind iv idual preparations. Potency ra t ios at each K + concentration were determined by contrast ing every E C ^ Q for (+)-verapamil with every E C ^ Q for ( - ) -verapami l . 2.7.6 Comparison of n i fed ip ine and DHM9 Nifedipine and DHM9 were compared in exact ly the same manner as (+)- and (-) -verapami l . However, special precautions were taken to protect these drugs from u l t r av i o l e t l i g h t . The experiments were carr ied out in a darkened room, i l luminated only by a 40 W bulb. The lamp was wrapped in red p l as t i c to f i l t e r out short wave-lengths. In addi t ion, the f l u i d c i r cu la t i ng and warming the perfusion apparatus was stained with red dye for the same purpose. The effect iveness of these procedures was exemplif ied by the rep roduc ib i l i t y of the resu l ts with time (see Resu l ts ) . The stock solut ions for both drugs used 50% ethanol in sa l ine as the solvent . When the drugs were added to the reservo i r chambers of the perfusion apparatus, d i l u t i on reduced the f i n a l ethanol concentration to 0.25%. 2.7.7 S t a t i s t i c s Slopes, E C ^ Q values and potency ra t ios were compared by ANOVA and Duncan's mul t ip le range tes t . - 149 -2.8 Metabolism-of racemie-verapamil - in-acute-myocardial- ischaemia 2.8.1 Introduction The fol lowing experiment was the f i r s t which was car r ied out as part of th is thes i s . This experiment was part of the i n i t i a l invest igat ion of (±)-verapamil (Curt is e t - a l ; , 1984). It had been shown by others in the laboratory that (^-verapami l reduced arrhythmias induced by coronary occlusion in conscious ra t s . As part of the attempt to invest igate the mechanism of action of (^-verapami 1, i t was decided to measure the amount of (±)-verapami 1 in the blood, the normal vent r icu lar t issue and the OZ of rats subjected to coronary occ lus ion . 2.8.2 Animals Male Sprague Dawley rats were prepared for coronary occlusion as described previously . The occluder was placed 3 mm below the a t r i a l appen-dage in order to produce small OZs. Approximately 7 days were allowed for the animals to recover from preparative surgery. 2.8.3 Drug administrat ion and t issue samples On the day of study, 6 mg/kg (^O-verapamil was administered, e i ther before occ lus ion, according to the usual pro toco l , or over a 10 min period s ta r t ing immediately af ter occ lus ion. I t had been shown that (±)-verapamil was approximately equal ly e f fec t i ve as an antiarrhythmic when given af ter occlusion as when given before occ lus ion, and the E D ^ Q for reducing arrhythmia score (AS) was approximately 6 mg/kg in both cases. Therefore i t was of in terest to determine whether the drug was capable of d i s t r i bu t ing into the OZ when given af ter occ lus ion , in re la t i on to the possible locus of action within the myocardium. Rats were monitored for 30 min after occ lus ion. Episodes of VF were thump-verted as required, in the usual manner. Af ter 30 min the rats were qu ick ly anaesthetised with halothane (5 %), using a face mask, and the aorta - 150 -was exposed by midl ine laparotomy for blood sampling. At least 10 ml of blood was taken and stored temporari ly in an iced heparinised test tube (Kymax). The heart was then removed. In several experiments, other t issues (ske le ta l muscle and l i ve r ) were also sampled. 2.8.4 Extract ion The fo l lowing procedures were carr ied out in order to prepare samples for in jec t ion onto an HPLC separation system. A l l the test tubes and syringes used were made of g lass . a. Blood was divided into 3 samples. One ml of whole blood was immediately pipetted into a Dreyer test tube and spiked with 16 pi of 1 ug/ul gallopamil ( internal standard for the HPLC and detector) . Extract ion of (^-verapami l was carr ied out using a modif icat ion of the method of Cole e t - a l . (1981). The sample was made basic (approximately pH 9) with approxi-mately 250 pi of 4 M NaOH for extract ion into organic so lvent , since the pKa of (^J-verapamil is approximately 8.5 (Dorrscheidt-Kafer, 1977). The organic solvent was 1 - 2 ml methyl- ter t-butylether (MTBE). In prel iminary exper i -ments i t was found that increasing MTBE volume beyond the recommended 1 ml did not inf luence y i e l d . The test tube was vortex-mixed for 30 sec and centri fuged at 9950 x g for 2 min in order to separate the organic and aqueous phases. Using a 1 ml syr inge, most of the upper organic phase was removed (the exact amount was immaterial, see below for explanat ion). The MTBE was then evaporated by blowing a je t of N 2 into the Dreyer tube, and the residue was reconst i tuted in 75 %aceton i t r i le in d i s t i l l e d water. This const i tuted the in jec t ion sample. A sample of plasma was prepared from the remainder of the blood, and 1 ml was processed in the same way as the whole blood sample, above. The remainder of the plasma was removed, using a glass syringe (Hamilton Rheo-dyne), and centri fuged at 900 x g for 40 - 60 min through a re-usable Amicon - 151 -centr i f low f i l t e r (type CF50A), in order to produce a sample of plasma water free of o^ -ac id g lycoprotein, with which (^-verapami l has been reported to be approximately 90% bound (Schomerus e t - a l . , 1976; McAl l i s te r e t - a l ; , 1983). The Amicon f i l t e r reta ins more than 95% of molecules > 50 kD, and more than 97% of serum prote in . The f i l t e r was prepared by soaking in d i s t i l l e d water for 3 h, followed by 10 min centr i fugat ion at 900 x g in order to remove excess water. The f i l t e r was cleansed af ter use by soaking in 0.1 M NaOH. The volume of the u l t r a f i l t r a t e was measured, and th is sample of f ree plasma water was then processed in the same way as the plasma and whole blood samples. b. Ventr icular t issue was processed according to a modif icat ion of the technique of McAl l i s te r and Howell (1976). Samples of normal and occluded vent r icu lar t issue were taken af ter b lo t t ing the t issue dry. Using small surg ical s c i s so r s , the t issue was subdivided on the basis of the known locat ion of the 0Z in r a t s , by v isua l inspect ion. A transmural sample of the centre of the 0Z was taken, as well as the ent i re 0Z and a large sample of normal v e n t r i c l e . A l l 3 samples were weighed, then processed as fo l lows . The t issue was minced using small surg ical sc issors and t issue forceps, and mixed in a glass homogenistation tube with 3 ml 0.1 M HCI and 16 nl of 1 yg/pl gallopamil ( internal standard). This a c i d i f i c a t i o n was carr ied out to encourage the (^-verapami l to par t i t i on out of the t issue into the aqueous melieu by promoting ion isat ion of th is weakly basic drug. The t issue was then homogenised using a pest le which f i t t i gh t l y in the homogenisation tube. The tube was kept cool by constant immersion in a s lu r ry of i c e . The contents of the tube were washed into a test tube, which was vortex-mixed for 2 min and then centri fuged at 10,000 x g for 30 min. The aquum was decanted into a Kymax test tube, and the pe l le t was resuspended in 1 ml 0.1 M HCI and reprocessed. The f i n a l extract was t i t r a ted to pH 8.5 with - 152 -20 %Na2C03 (approximately 400 p i ) in order to encourage subsequent par t i t i on into MTBE. The cautious adjustment of pH was carr ied out in order to preclude the development of a soapy scum, which was associated with the use of NaOH in prel iminary experiments. Subsequent processing steps were ident ica l to those used for blood matrix samples. 2.8.5 HPLC Separation of (^O-verapami1 and gallopamil was carr ied out by reverse-phase high pressure l i qu id chromatography (HPLC, SP8000), using MOS Hypersi l as the packing material and an Altex in jec t ion device. The running solvent was a 50:50 mixture of d i s t i l l e d water and ace ton i t r i l e containing 500 y l of sodium octylsulphate and 0.355 g of Na 2HP0 4 per l i t r e . The pH of the aqueous component was adjusted to 4.6 with 0.1 M phosphoric acid before mixing with a c e t o n i t r i l e . Flow rate was set at 1 ml/min using a Micrometrics pump. Inject ion of samples into the column was carr ied out using a 25 u l Hamilton Rheodyne glass syr inge. 2.8.6 Calculat ion of verapamil concentration (±)-Verapamil and gallopamil were detected using a f luorimeter (Schoef-fe l FS970). The optimum exc i ta t ion wavelength was found in prel iminary experiments to be 203 nm, and an ordinary glass detection f i l t e r was used. A permanent record of peaks was generated using a Rikadenki B-107 chart recorder. (^J-Verapami1 concentration was calculated under the assumption that the extract ion processes extracted (±)-verapamil and gallopamil equal ly . This assumption was tested and found to be the case in prel iminary exper i -ments. The column was ca l ibrated da i l y by in jec t ing 20 ng (±)-verapami 1 and 20 ng gallopamil simultaneously onto the column, and measuring the ra t io of peak heights and the retent ion times. The retent ion times were used to locate (^-verapami l and gallopamil on the traces from blood and t issue samples. The ra t io of heights was used to ca lcu la te the concentration of - 153 -(±)-verapamil in the samples, according to the fo l lowing formula: Concentration (mol/1) = (Vh s/Dh $)(16/491080)(R/Y) Where Vh g and Dh$ are the peak heights for (±)-verapami 1 and ga l lop-amil from the blood matrix samples, respec t ive ly , R is the ra t io of the peak heights of the (^-verapami l :gal lopamil standards, Y is the volume of blood matrix in ml, and 16 and 491080 are constants to account for the weight of gallopamil used to spike each sample (16 u l ) and the molecular weight of (^J-verapami1 (491.08). In the case of t issue samples, Y refers to g weight, and the units of (±)-verapamil concen-t ra t ion are mol/kg. The above ca lcu la t ion was based on prel iminary experiments which showed that the y ie lds of (^-verapami l and gallopamil from a sample spiked with known amounts of the drugs were s im i la r ( ra t io of recovery of (^J-verapami1:gal 1-opamil 1.003 ± 0.059 according to peak height, mean ± s.e.mean, n = 14), and experiments which showed that the ra t i o of peak heights, R, was constant over at least 3 orders of magnitude of drug concentrat ion. - 154 -3 RESULTS 3.1 Metabolism of -(*)-verapami1- in acute myocardial -ischaemia This study was carr ied out in conjunction with an invest igat ion into the ef fects of (^-verapami l on the responses to coronary occlusion in conscious r a t s . I t had been demonstrated that (±)-verapamil possessed dose-dependent antiarrhythmic ef fects when administered before occlusion and when adminis-tered af ter occ lus ion. Therefore, i t was of in terest to examine the concen-t ra t ions of ( i j -verapami l in the blood matrices af ter coronary occ lus ion, and also to examine whether the drug penetrated into the occluded zone. The l a t t e r considerat ion was expected to shed l i gh t on the question concerning the s i t e of antiarrhythmic action of the drug, since an absence of ( ^ - v e r a -pamil in the occluded zone would suggest that the antiarrhythmic ef fects were produced v ia an action elsewhere. The resu l ts of th is study have been published (Curt is et a l ; , 1984). 3.1.1 Concentration of (^-verapami 1 in blood matrices The concentration of (^O -verapamil in whole blood, plasma and plasma u l t r a f i l t r a t e (PU) i s shown in Table 2 . I t can be seen that concentrations were s l i g h t l y higher fo l lowing post-occlusion administrat ion compared with pre-occlusion administrat ion, although the di f ferences were s t a t i s t i c a l l y s i gn i f i can t only for the PU. Since drug administrat ion took approximately 10 min, whereas a l l rats were sac r i f i ced at 30 min af ter occ lus ion, then the di f ference between pre- and post-occlusion administrat ion in terms of blood matrix (^J-verapami 1 concentration re f l ec t s the short d i s t r i bu t ion hal f l i f e and the rapid hepatic metabolism of the drug (Schomerus et• - a l ; , 1976; McA l l i s t e r , 1982). The concentration of (^J-verapamil in the PU was in the same range as the concentration reported to i nh ib i t i^. but not i^ in vent r icu la r t issue (Kohlhardt et a l . , 1972; Nawrath e t - a l ; , 1981). The extent of plasma-protein binding was 82 ± 4%in rats given (*) -vera-- 155 -pamil af ter occ lus ion , and 84 ± 4% in rats given the drug before occ lus ion. These values were calculated by contrast ing the plasma concentrations with the PU concentrat ions. They compare favourably with published values for humans (Schomerus e t - a l . , 1976; Eichelbaum e t a l . , 1984). 3.1.2 Concentration of (^-verapami l in the vent r i cu la r myocardium. The amounts of (^-verapami l in the normal non-occluded vent r icu lar t issue (NZ), the occluded zone (OZ) and the centre of the OZ (0Z c)are shown in table 3. Values from rats in which the drug was administered before occlusion indicate that (±)-verapamil appeared to accumulate in the ischaemic t i ssue . The 0Z C , which was presumably the most ischaemic t issue (or the port ion of the OZ least contaminated with NZ) contained the most amount of drug (p < 0.05). The amount of (±)-verapami 1 in the NZ in rats given the drug af ter occlusion was approximately 3 times the value found in rats administered the drug before occlusion (although the di f ference was not s t a t i s t i c a l l y s i g n i f i c a n t ) . In th is regard, NZ values varied according to the time of administrat ion in a qua l i t a t i ve l y s im i la r manner to leve ls in the blood matr ices. I t was of considerable in terest to f ind that (^J-verapami1 was present in the OZ (and 0Z C) of rats administered the drug af ter occ lus ion. How-ever, unl ike the case in which (^)-verapami1 was given before occ lus ion, the leve ls in the OZ and 0Z C were not d isproport ionately h igh, compared with the NZ. Indeed, there were no s ign i f i can t di f ferences between the leve ls in the NZ, OZ and 0Z c fo l lowing post-occlusion adminis t rat ion. Although the amount of (^-verapami 1 in the NZ was greater fo l lowing post-occlusion adminis t rat ion, the amount in the 0Z c was l e s s , ind icat ing that although the drug did par t i t i on into the ischaemic t issue even when given af ter l i g a -t i o n , the a b i l i t y of the ischaemic t issue to accumulate the drug was dimin-ished. This presumably re f lec ted the fact that whereas (^O-verapami 1 had - 156 -Table 2. Concentration of (±)-verapamil in blood matrices Administrat ion n Whole Blood Plasma U l t r a f i U r a t e Pre-occlusion 6 2.4 ± 0.8 uM 2.7 ± 1, .1 vM 0.24 ± 0.04 yM Post-occlusion 6 3.2 ± 0.5 uM 3.6 i 0 .8 uM 0.57 ± 0.10 yM* The dose of (± )-verapamil was 6 mg/kg. Values are mean * s.e.mean. * Ind i -cates p < 0.05 versus the value in the pre-occlusion-administrat ion group (unpaired 2 - t a i l ed t t e s t ) . - 157 -Table 3. D is t r ibut ion of (^-verapami l in the ven t r i c les Administrat ion n Non--occluded Occluded Occluded centre Pre-occlusion 6 5 .7 ± 0.7 8.4 ± 0.8 22.1 ± 3.1 Post-occlusion 6 15 .8 ± 5.6 11.0 ± 2.3 13.9 ± 1.4 The data i s expressed exact ly in the same way as the data in Table 2, with the exception that values are in umol/kg. There were no s t a t i s t i c a l l y s i g -n i f i can t di f ferences between the pre- and post-occlusion administrat ion groups (unpaired 2 - ta i l ed t t e s t ) . - 158 -free access to the ischaemic t issue when given before occ lus ion , and indeed was probably trapped there in high concentrations (occlusion took place immediately af ter drug administrat ion in these r a t s ) , i t only had access to the ischaemic t issue v ia the veins which drain over the OZ in the rat heart (Taira et a l ; , 1985) when given af ter occ lus ion. 3.2 Arrhythmogenesis-and the ro le of the CNS 3.2.1 Overview In view of the contradictory evidence regarding the importance of the autonomic nervous system in arrhythmogenesis in the rat (Bot t ing, et a l . - , 1984; Siegmund, et a l . , 1979; Szekeres, 1979; Au, et a l . , 1983; Kenedi and Losconci , 1973a; 1983b; Campbell and Par ra t t , 1983; Marsha l l , et a l . - , 1981), and contradictory evidence regarding the ro le of the sympathetic nervous system in arrhythmogenesis in general ( e . g . , Har r i s , et - al-.-, 1951; Schaal et a l . , 1969; G i l l i s , 1971; Myers e t a l . , 1974; Hope, et a l . , 1974; Fowl iss , e t - a l . , 1974; Pantr idge, 1978; Johnston e t - a l ; , 1983a), a systematic i n v e s t i -gation of the importance of adrenoceptor act ivat ion and the central nervous system was car r ied out, using a ser ies of ablat ions in the CNS combined, in some cases, with catecholamine in fus ions. The groups in th is study are summarised in Table 1. The resu l ts of th is study have been published (Curt is et a l . , 1985b). 3.2.2 Occluded zone (OZ) OZ s ize (Figure 3) ranged from 33 ± 3 (%ventr icu lar weight) in aS rats to 45 ± 2 in cP ra t s . Differences in mean OZ s ize between groups were not s t a t i s t i c a l l y s i gn i f i can t (except for the aS group) and therefore did not account for var ia t ions in blood pressure, heart rate or arrhythmias (des-cribed below). - 159 -CD O Q LU Q CJ o 50 40 -5 30 20 -10 -1 X X 1 X 1 I a . re a . o Q_ ea re Q (TJ ca C J ro CJ CJ C J O F igu re 3 . Each h is togram represents mean ± s.e.mean 0Z s i z e as % v e n t r i c u l a r we igh t . The groups are i n d i c a t e d by symbols de f ined in the t e x t and in Tab le 1. * Ind ica tes p < 0.05 versus cC. - 160 -3.2.3 Arrhythmias 3.2.3.1 Arrhythmia scores. There were considerable dif ferences bet-ween the groups with regard to arrhythmias, which are summarized (Figure 4) as mean arrhythmia scores for the ear ly (0 - 0.5 h) and la te (0.5 - 4 h) per-iods. The most important f indings were in the 0.5 - 4 h post-occlusion per-iod . A dramatic f a l l in arrhythmia score was seen in a l l acutely prepared animals, in iso la ted hearts, and also in chron ica l l y prepared, but acutely p i thed, r a t s . In most pithed and sp ina l ized rats not even a s ing le PVC was seen in the la te (0.5 - 4 h) per iod. Only one iso la ted heart had VF (at 3.75 h af ter occ lus ion) . 3.2.3.2 VT- and VF. VF in the 0 . 5 - 4 h period was absent in a l l pithed rats including those receiv ing catecholamine infusions (Figure 5b). VF was also absent or of low incidence in the aS, aD, aB, and aC groups. The di f ferences in VF incidence between the groups during the ear ly period (Figure 5a) were qua l i t a t i ve l y s im i la r to the di f ferences during the late per iod, although less pronounced. Changes in the incidences of VT (Figure 6) resembled the changes seen in the incidences of VF. The most marked changes were seen in the la te period during which the incidence was low in a l l acutely prepared rats and absent in a l l pithed groups (aP, cP and aPN) including the group receiv ing the catecholamine infusion (aPN). 3.2.3.3 PVC. L o g ^ PVC number in the ear ly period (Figure 7) was s im i la r in a l l groups. Values were highest in the two acutely prepared non-ablated groups (aB and aC). In the late period (0.5 - 4 h) l o g l Q PVC was reduced by a l l types of surg ical ablat ion in the CNS. PVC were in fact absent in the aP group. Log-^ Q PVC number was also s l i g h t l y lower in the aB group versus conscious controls (cC). - 161 -a 0-h h after occlusion 5 1. cc o CO ac h— >-:n D C : < 4 — 3 -1 — _T_ ro ro IO . rO o o o o b %-4 h after occlusion O O co >-cc: 2 — 1 — * * D -X Gi-ro CO O, CO rO X C_5 Z O C_) o X Figure 4. Arrhythmia scores (mean ± s.e.mean). The groups in figure 3. * Indicates p < 0.05 versus cC. are the same - 162 -100 80 2 20 CO 0 •—* a: <: CJ I—I oi U J Q i—t o a 0-*s h a f ter occlusion 60 -40 -Q_ O- ZZ. fO O CL. ro CO Q ro ro b *s-4 h a f te r occlusion 100 80 -60 — 40 — 20 Cx. ro O ro CO ro Q ro co ro O rO CJ O CJ O CO ro CJ rO C J ST O O CJ Figure 5. Incidence of VF. The groups are indicated by symbols defined in the text and in Table 1. * Indicates p < 0.05 versus cC, - 163 -a 0-h h a f te r occlusion 100 Q CC o •>-zc <_J cc 80 60 -40 20 -Q-O ro OO (O Q ro CO ro C_> ro o o o i—i or: b %-4 h a f te r occlusion 100-o UJ r_> z: LU Q o 80 — 60 — 40 — 20 * * o_ ro (J o. ro 00 ro Q rO CO (O CJ (O CJ o CJ o Figure 6. Incidence of VT. The groups are indicated by symbols defined in the text and in Table 1. * Indicates p < 0.05 versus cC. - 164 -a 0-% h a f te r occlusion 1 _ X X X X X > Q-U-o UJ o CL. CJ a . co n3 a CO b %-4 h a f te r occlusion 2 -1 -X X CL. 2: C J Q . CO o •* cCN H * IH IH' Figure 8. Heart rates (a) and blood pressure (b) before (-1 min) and a f te r (30 min) coronary occ lus ion. Each value i s mean ± s.e.mean. The groups are indicated by symbols defined in the text and in Table 1. * Indicates p < 0.05 versus cC. - 167 -0.4-I I X x I — 0.3 CC I— co T 3 ^ 0 . 2 X X I X X $ 0 . 1 X 10-9 8 7 6 5 4 3 2 1 o-200-—150 H :100 L U 50 —| o Q Ek aii Figure 9. ECG changes fol lowing coronary occ lus ion. In each part of the' f i gu re , the groups (from l e f t to r ight) are aP, cP, aPN, aS, aD, aB, aC, cC and cCN (symbols defined in Table 1) . * Indicates p < 0.05 versus cC. - 168 -3.2.6 Thrombocytes, leukocytes and serum K + These var iables were measured in an anc i l l a r y group of acutely prepared pithed rats only. The most s t r i k i ng f ind ing was an elevat ion in serum K + fo l lowing p i th ing (Figure 10). At the same time periods the thrombocyte and leukocyte counts f e l l . The leukocyte count returned toward the pre-pi th ing value by 4 h af ter occ lus ion, whereas the reduction in thrombocyte count was maintained throughout the observation period (Figure 10). Arrhythmias in th is anc i l l a r y group (not shown) were almost ident ica l with those seen in the or ig ina l aP group. 3.2.7 Summary The ro le of the CNS in arrhythmogenesis fo l lowing coronary occlusion was invest igated in rats by use of CNS ablat ions and noradrenal ine/adrenal ine in fus ions. A l l procedures involv ing acute surg ica l preparation reduced the incidence and sever i ty of the arrhythmias induced by occlusion (Figures 4 - 7 ) . Such reductions were most marked in the la te (0.5 - 4 h period) af ter occ lus ion. The observed reductions in arrhythmias could not be explained in terms of involvement of the CNS or adrenoceptor ac t i va t i on . When c i r cu la t i ng leukocytes, thrombocytes and serum K + were measured in a group of pithed rats before and af ter occ lus ion, reduced leve ls (20-50 %) of both leukocytes and thrombocytes occurred while serum K + leve ls rose by 50-100 % . - 169 -cr E cc UJ CO 10 — 9 • 8 • 7 -6 -5 —I 4 -3 -2 -1 -0 I T ro i CO f= >-O —N O O CJ o o ro i >-CJ o CO s: o cc o o o * o o 14 12 10 8 6 4 2 0 10-8 6 4 2 0-I X * X I II +h +4 * * X I II +% +4 I II +4 Figure 10. Changes in serum K + concentration (part a ) , leukocytes (part b) and thrombocytes (part c) in pithed rats before and af ter coronary occ lu -s ion. The var iables were measured immediately fo l lowing preparative surgery but before p i th ing ( I ) , af ter p i th ing but 1 min before occlusion ( I I ) , 1/2 h af ter occlusion (+1/2) and 4 h af ter occlusion (+4). Each value i s mean ± s.e.mean (n = 9) . i n d i c a t e s p < 0.05 compared with values before pi th ing ( I ) . - 170 -3.3 Actions of anipamil and-ronipamil in acute myocardial ischaemia 3.3.1 Overview The hypothesis that the antiarrhythmic a c t i v i t y of (^O-verapami1 (demon-strated previously in our laboratory; Curt is et a l . - , 1984) occurred by v i r tue of calcium antagonism in the vent r icu lar myocardium predicts that a l l c a l -cium antagonists with such an action should be antiarrhythmic. Prel iminary experiments (Kretzschmar, personal communication) have suggested that anipa-mil is a calcium antagonist with re l a t i ve s e l e c t i v i t y for the myocardium, whereas ronipami l , a close s t ructura l analogue i s r e l a t i v e l y i nac t i ve . Therefore, i t was of in terest to determine whether these verapamil analogues could reduce ischaemia-induced arrhythmias at doses below those producing vasodi latat ion- induced hypotension, in a manner consistent with the i r c a l -cium antagonist p r o f i l e . Based on the l imi ted information concerning the pharmacological propert ies of these drugs, i t was predicted that anipamil should reduce ischaemia-induced arrhythmias, whereas ronipamil should be r e l a t i v e l y inac t i ve . In the f igu res , C refers to cont ro ls , R = ron ipami l , A = anipamil , L = low dose (50 mg/kg, p.o.) and H = high dose (150 mg/kg, p .o . ) . Therefore the 5 groups are C, RL, RH, AL and AH. This study has been published (Curt is e t - a l ; , 1986b). 3.3.2 OZ and in fa rc t zone (IZ) The s ize of the OZ was not found to be s t a t i s t i c a l l y s i g n i f i c a n t l y al tered by treatment. The mean ± s.e.mean OZ s ize (expressed as% ven t r i c -ular weight) was 42 ± 4 , 41 ± 4 , 40 ± 2, 39 ± 4 and 36 ± 3 % in the con t ro l , RL, RH, AL, and AH groups, respec t ive ly . The mean IZ s ize in the AH group was 29 ± 4%of to ta l vent r icu lar weight which was comparable with values in previous control groups (Curt is e t - a l ; , 1984). We were unable to obtain enough estimates of IZ s ize in the other groups to permit useful comparisons. - 171 -3.3.3 Arrhythmias Arrhythmias fol lowing occlusion in control rats have been shown by our laboratory to be bi-modally d is t r ibu ted with time (Johnston e t - a l ; , 1983a). Peaks occur at approximately 10 min and 2 h af ter occ lus ion, with a quiescent in terval las t ing from approximately 20 min to 1.5 h af ter occ lu -s ion . As arrhythmia incidence within groups was s im i la r during both the 0 - 0.5 h, and 0.5 - 4 h periods af ter occ lus ion , arrhythmia data i s presen-ted for the overal l 0 - 4 h per iod. The arrhythmia scores for the d i f fe rent groups are shown in Figure 11. Both doses of anipamil s t a t i s t i c a l l y s i g n i f -i can t l y reduced arrhythmia score, whereas ronipamil had no s ign i f i can t e f fec t . With regard to ind iv idual types of arrhythmias, the incidence of VT was high in a l l groups. In those rats having VT, the log^Q duration of such events (Figure 12a) was not s t a t i s t i c a l l y s i g n i f i c a n t l y reduced by e i ther drug. The log -^ number of episodes of VT in those rats having VT was 1.5 ± 0.2 (mean ± s.e.mean) in cont ro ls . This value was reduced to 1.4 ± 0.2, 1.1 ± 0.2, 1.1 ± 0.2 and 1.0 ± 0.2 by RL, RH, AL, and AH t rea t -ment, respec t ive ly . More importantly, anipamil dose-dependently reduced both the incidence (Figure 12b) and duration (Figure 12c) of VF. Log^Q number of PVC, how-ever, was not reduced to a s t a t i s t i c a l l y s ign i f i can t degree by e i ther anip-amil or ronipamil (Figure 12d). In summary, although anipamil was an e f fec t ive ant iarrhythmic, par t i cu -l a r l y against VF, ronipamil was far less e f fec t i ve and f a i l ed to produce any s t a t i s t i c a l l y s i gn i f i can t antiarrhythmic e f fec ts . - 172 -cc S 3 CO cc cc 1 — Figure 11. Ef fect of ronipamil and anipamil on arrhythmia score. The mean (± s.e.mean) arrhythmia scores are shown for the 4 h period fol lowing occ lu -s ion . The groups are: controls (C), 50 mg/kg ronipamil (RL), 150 mg/kg ron-ipamil (RH), 50 mg/kg anipamil (AL) and 150 mg/kg anipamil (AH); n = 9 for each group. * Indicates p < 0.05 versus cont ro ls . - 173 -Figure 12. Ef fect of ronipamil and anipamil on the incidence and duration of various arrhythmias during the f i r s t 4 h fo l lowing coronary occ lus ion. The groups are: controls (C), 50 mg/kg ronipamil (RL), 150 mg/kg ronipamil (RH), 50 mg/kg anipamil (AL) and 150 mg/kg anipamil (AH); n = 9 for each group. Part a i l l u s t r a t e s duration (log-^Q sec) of VT (mean ± s.e.mean) in those animals having th is arrhythmia. The number of rats per group which had VF is given in Part b. Part c i l l u s t r a t e s duration ( l o g 1 0 sec) of VF (mean ± s.e.mean) in those animals having th is arrhythmia. The s.e.mean data is omitted where n was less than 5. Part d shows the mean (* s .e . ) of log-^Q PVC. * Indicates p < 0.05 versus cont ro ls . DURATION OF VF ( l o g 1 0 s) o PVC ( l o g , 0 number) tn C RL RH AL AH DURATION OF VT ( l o g 1 Q s) tn c RL RH AL AH to INCIDENCE OF VF (per n = 9) o ' ro w * ui 0 1 M oo vo 4^ c RL RH AL AH - 175 -3.3.4 Morta l i ty Despite a high incidence of VF, pa r t i cu la r l y in control r a t s , a l l e p i -sodes of VF las t ing longer than 10 s were successfu l ly d e f i b r i l l a t e d by thump-version, thus a l l deaths occurring during the 4 h period fo l lowing occlusion were associated with hypotension (cardiogenic shock) or severe pulmonary oedema. In an e a r l i e r study in our laboratory (Curt is et a l . , 1984) i t was found that 20 mg/kg (±)-verapami1 exacerbated the morta l i ty associated with cardiogenic shock during the 4 h period fo l lowing occ lus ion. In the present study however, drug treatment did not increase the number of such deaths. The number of deaths (out of n = 9) which had occurred during the 4 h period af ter occlusion was 1, 5, 1, 1, 1 in the con t ro l , RL, RH, AL and AH groups, respec t ive ly . The high value seen with the 50 mg/kg dose of ronipamil (RL) was not s t a t i s t i c a l l y s i g n i f i c a n t l y d i f ferent from that for the other groups. By 24 h post -occ lus ion, morta l i ty (out of n = 9) was 5, 6, 5, 5, and 3 respect ive ly . 3.3.5 Haemodynamic var iables Mean a r te r i a l blood pressure and heart rate changes at various times pre- and post-occlusion are shown in Table 4. Before drug administ rat ion, the mean ± s.e.mean heart rates ranged from 376 ± 19 beats/min in the control group to 432 ± 20 beats/min in the AH group. The corresponding mean a r t e r i a l blood pressures ranged from 103 ± 9 to 117 ± 4 mmHg. There were no s t a t i s t i c a l l y s ign i f i can t di f ferences between these means. Immediately before occ lus ion, and for 4 h af ter drug administrat ion, neither drug, at e i ther 50 or 150 mg/kg, had marked actions on heart rate or blood pressure when compared with the untreated animals. The small reduc-t ions in blood pressure seen pr ior to occlusion (expressed in Table 4 as % changes from pretreatment values) were not s t a t i s t i c a l l y s i g n i f i c a n t l y d i f fe ren t . In the animals treated with anipamil , heart rate and blood Table 4. Haemodynamic Ef fects of Anipamil an Ronipamil Before and After Occlusion Percentage change in MAP Percentage change in HR Group i -30min - lmin +lmin +30min +lh +4h -30min - lmin +lmin +30min +lh +4h c 0±3 3±3 -5±7 -10±5 -8±5 -17±6 _1±2 2±3 g±4 -4±4 -5±4 -1±6 RL 4±4 5±4 -3±5 -4±6 -7±6 -9±8 0±6 2±7 3±5 0±6 -4±5 -15±9 RH 2±6 4±4 -15±7 -15±4 -16±4 -24±4 -5±6 -5±5 1±6 -13±5 -16±5 -14±5 AL -5±4 -7±3 -22±6 -22±6 -22±5 _41±4* -5±5 1±4 8±4 -1±5 -7±6 -18±4 AH -6±3 -5±2 -21±7 -22±4 -21±5 -36±4* -5±4 -4±4 2±6 -12±7 -15±9 -19±10 The ef fects of treatment on mean a r te r i a l pressure (MAP) and heart rate (HR) are shown. Control v a l -ues were recorded at the time of drug administration (4 h before coronary occ lus ion) , and the percen-tage changes in these values (mean ± s.e.mean) are shown for various time points before (-30 and -1 min) and af ter (+1 min to +4 h) occ lus ion. The symbol * indicates p < 0.05 versus the control group (C), at a par t i cu la r time point , by ANOVA and Duncan's range tes t . The groups are C = con t ro l , R = Ronipamil, A = Anipami l , L = low dose (50 mg/kg) and H = high dose (150 mg/kg. - 177 -pressure were reduced by occlusion to a greater degree than occurred in con-t ro l r a t s , although the only s t a t i s t i c a l l y s i gn i f i can t di f ferences were for mean blood pressure at 4 h af ter occlusion (owing to the large variance in each group). Thus ronipamil had no marked actions on blood pressure and heart rate while anipamil had l imi ted actions to lower heart rate and blood pressure. 3.3.6 ECG changes There were some ef fects of treatment on the ECG changes produced by cor-onary occlusion (S-T segment elevat ion and R-wave amplitude increases) (Figure 13). I t appeared that anipamil reduced the rate (Figure 13c and 13d) at which both changes occurred while both anipamil and ronipamil redu-ced maximum S-T segment elevat ion (Figure 13c). However, the maximum R-wave amplitude (Figure 13a) was the s im i la r in a l l groups. In Figure 13, S-T segment elevat ion i s expressed as dSTR. The elevat ion of the S-T segment was also reduced by anipamil and ronipamil i f changes in were expressed as % R-wave amplitude (ST% ) in the manner used by Bernauer (1982). 3.3.7 Plasma concentrations of anipamil To ensure that the drugs were being absorbed fo l lowing administ rat ion, plasma anipamil leve ls were measured (by Dr. Brode, Knol l A . G . , Ludwigshafen, using a technique developed in h is laboratory) in separate groups of rats (n = 5 per dose). Pooled plasma concentrations in rats given 50 mg/kg anip-amil p.o. were 3.2, 4 and 2.4 ug/ml at 1, 3 and 5 h af ter administrat ion, respec t ive ly . Concentrations at 1, 3 and 5 h af ter administrat ion of 150 mg/kg were 5, 8.5 and 3.4 ug/ml, respec t ive ly . - 178 -Figure 13. Ef fect of ronipamil and anipamil on occlusion-induced ECG changes (R-wave and "S-T" segment e levat ion) . Part a shows maximum R-wave amplitudes (mean ± s . e . ) . Part c shows the times at which maximum R-wave amplitudes were reached. Parts b and d show corresponding values for dSTR. Values in parts c and d were calculated as log-^g min, but are i l l u s t r a t e d as a n t i -logs ± 1 s.e.mean. The groups are as indicated in f igure 12. * Indicates p < 0.05 versus cont ro ls . TIME TO MAXIMUM R WAVE (min) tn ro tn c RL RH AL AH TIME TO MAXIMUM dSTR (min) ro tn tn ro tn _ L c RL RH AL AH o C RL RH AL AH MAXIMUM R WAVE (mV) o I— • i—> tn tn MAXIMUM dSTR (mV) o o o . ro to o c RL RH AL AH — 1 — 1 — 1 — H — 1 - 180 -3.3.8 Summary Ronipamil and anipamil (2 analogues of verapamil), were administered p.o. to conscious rats which were subjected to coronary occ lus ion. Only anipamil (50 and 150 mg/kg) s t a t i s t i c a l l y s i g n i f i c a n t l y reduced arrhythmias (Figures 11 and 12); i t was more e f fec t i ve against VF than VT or PVCs. Ron-ipamil at the same doses had l i t t l e antiarrhythmic a c t i v i t y . Only anipamil delayed the development of ECG signs of ischaemia, whereas both drugs redu-ced the extent of such changes (Figure 13). The apparent delays in the development of ischaemia produced by both drugs were not associated with delays in the onset of arrhythmias. The resu l ts appeared to conform with the hypothesis that ischaemia-induced arrhythmias are reduced by calcium antagonism in the ven t r i c l es . 3.4 Actions of fe lod ip ine in-acute-myocardial ischaemia 3.4.1 Overview Fe lod ip ine, a calcium antagonist with s e l e c t i v i t y for vascular smooth muscle versus the myocardium (Au and Sut ter , 1984), was evaluated for ac t i v -i t y against responses to coronary occlusion under the assumption that com-pared with ( ^ -ve rapami l , l i t t l e antiarrhythmic a c t i v i t y would be seen, as predicted from the hypothesis that calcium antagonism in the vent r icu lar myocardium is the pr inc ipa l determinant of the antiarrhythmic a c t i v i t y of calcium antagonists during acute myocardial ischaemia. In the f igu res , C re fers to con t ro ls , L = low dose (0.2 umol/kg), I = intermediate dose (2.6 umol/kg) and H = high dose (12.2 umol/kg). The resu l ts of th is study have been published (Curt is et a l . • , 1985a). 3.4.2 OZ, IZ and morta l i ty There were no s t a t i s t i c a l l y s i gn i f i can t di f ferences between control groups and fe lod ip ine- t reated groups with regard to OZ or IZ s i z e , within the subgroups of rats with del iberately-produced large (LOZ) or small (SOZ) - 181 -OZs (Figure 14a). Owing to the high morta l i ty rate during the f i r s t 4 h fo l lowing coronary occlusion in LOZ rats (Figure 14b), too few IZ measure-ments were taken to warrant useful comparison, thus IZ values have been omitted for LOZ r a t s . With regard to SOZ r a t s , when IZ was expressed asX OZ weight, the extent of in farc t ion was 88 ± 13, 71 ± 14 and 79 ^ 10% in con-t r o l , low dose and high dose ra t s , respec t ive ly , ind icat ing s l i gh t myocar-d ia l salvage (P < 0.05 versus control for the low dose on ly ) . Mor ta l i ty during the f i r s t 4 h fol lowing occlusion was low (0 - 11% ) in a l l SOZ groups, and higher (33 - 67 %) in a l l LOZ groups (Figure 14b). There were no obvious dose-related ef fects of fe lod ip ine on mor ta l i ty , whether resu l t ing from VF or cardiogenic shock. 3.4.3 Arrhythmias In SOZ ra t s , fe lod ip ine produced a small reduction in the incidence of VF and VT (Figure 15). This e f fect was re f lec ted in the arrhythmia score (Figure 16), but only the reduction in VT incidence produced by the high dose during the f i r s t 30 min fo l lowing occlusion was s t a t i s t i c a l l y s i g n i f i -cant. The log^Q number of PVC was reduced (P < 0.05) by high and low doses of fe lod ip ine only during the f i r s t 30 min fo l lowing occ lus ion, the ef fect being los t when the ent i re 4 h period was evaluated (Figure 17). In LOZ ra t s , fe lod ip ine had no e f fec t on the incidence of VT or VF (F ig -ure 15), log-^Q number of PVC (Figure 17) or arrhythmia score (Figure 16). The weak antiarrhythmic a c t i v i t y of fe lod ip ine is exemplif ied by the arrhythmia scores (Figure 16) and by the incidence of serious arrhythmias (VT and VF). In SOZ r a t s , 100% of controls experienced ei ther VT or VF during the 4 h fo l lowing occ lus ion. This incidence was reduced to 67% by both high and low doses of fe lod ip ine (not s t a t i s t i c a l l y s i g n i f i c a n t ) . In LOZ r a t s , a l l rats in a l l groups experienced e i ther VT or VF. -.. 182 -a C L H C L I H b SOZ LOZ _ 80 >> £ 60 4-> 1 40 •a-- 20 -0 — C L H C L I H Figure 14. Ef fects of fe lod ip ine on OZ (unhatched) and IZ (hatched) (part a ) , and to ta l (unhatched) and arrhythmia-induced (hatched) morta l i ty during the 0 - 4 h period fol lowing occlusion (part b) . The groups are indicated by: C = cont ro ls , L = 0 .2 , I = 2.6 and H = 12.2 umol/kg fe lod ip ine . * Indicates p < 0.05 versus the appropriate small (S) or large (L) OZ control group. - 183 -Figure 15. Ef fects of fe lod ip ine on the incidence of VF and VT during the 0 - 3 0 min (hatched) and 0 -• 4 h (unhatched) periods fo l lowing occ lus ion. Groups and s t a t i s t i c a l s ign i f i cance symbols are as indicated in f igure 14. - 184. -Figure 16. Ef fects of fe lod ip ine on arrhythmia score for the 0 - 3 0 min (hatched) and 0-4 h (unhatched) periods fol lowing occ lus ion. Values are mean ± s.e.mean. Groups and s t a t i s t i c a l s ign i f i cance symbols are as i nd i c -ated in f igure 14. - 185 -Figure 17. Ef fects of fe lod ip ine on l o g 1 Q number of PVC during the 0 - 30 min (hatched) and 0 - 4 h (unhatched) periods fo l lowing occ lus ion . Values are mean ± s.e.mean. Groups and s t a t i s t i c a l s ign i f icance symbols are as indicated in f igure 14. - 186 -3.4.4 Haemodynamic var iables Felodipine produced a large dose-dependent reduction of blood pressure before and af ter coronary occlusion (Figure 18). Heart rate changes produced by fe lod ip ine were neg l ig ib le (Figure 18). Heart rates 1 min before occ lu -sion were 447 ± 13 and 430 ± 10 beats/min in SOZ and LOZ control r a t s , res -pec t i ve ly . These values were both s l i g h t l y higher than h i s t o r i ca l control values of 392 ± 10 beats/minute (Johnston et a l ; , 1983). Coronary occlusion reduced blood pressure, pa r t i cu la r l y in LOZ ra t s . The ef fect of occlusion on blood pressure was neither enhanced nor attenuated by fe lod ip ine (Figure 18). In th is respect, fe lod ip ine does not resemble (^)-verapami 1 (Curt is et a l . - , 1984) which enhanced the f a l l in blood-pressure produced by coronary occlusion in previous studies in our laboratory. 3.4.5 ECG changes There were no obvious drug ef fects on ei ther maximum R wave amplitude nor the time at which th is occurred (not shown). However, fe lod ip ine delay-ed the time at which maximum S-T segment elevat ion occurred, whether expres-sed as maximum dSTR or ST %, in both SOZ and LOZ r a t s , pa r t i cu la r l y at the high dose (P < 0.05). In Figure 19, maximum ST% and the time at which th is occurred are shown. I t can be seen that the maximum ST% values were higher in a l l LOZ rats versus SOZ ra t s , and that fe lod ip ine did not inf luence th is var iable in any predictable manner. 3.4.6 Summary Felodipine was not a cons is tent ly e f fec t i ve antiarrhythmic agent (Figures 15 - 17) at doses which reduced blood pressure (Figure 18). Although the development of ECG signs of ischaemia was delayed (Figure 19), IZ s ize was not correspondingly reduced (Figure 14). - 187 -110 cn zc E E L U cc ZD OO oo LU CC Q. O o _1 ca cu J 3 cc cc LU 50 -0 — 1 500 400-300 200 100 0 1 min before occlusion SOZ * * — * LOZ C L H C L I H X 1 h af ter occlusion SOZ I I X LOZ JL C L H C L I H Figure 18. Ef fects of fe lod ip ine on blood-pressure (upper parts) and heart rate (lower parts) 1 min before and 1 h af ter occ lus ion. Values are mean ± s.e.mean. Groups and s t a t i s t i c a l s ign i f icance symbols are as i nd i c -ated in f igure 14. - 188 -i X 100 " 80 o -o §• 60 CU ^ I 40 o 20 -SOZ X C L H LOZ' X I C L I H i o o 50 5 ~ 40 111 o _ i •>-I i I l/l r. 30 o o « 20 ca S i CC 0 J 100 -> c CU o S-cu 50 -(+)-Verapami1 0 J 5 10 15 30 1 Occlusion mi n Time Af ter Occlusion 24 Figure 27. The ef fects of (-)-verapamil (upper f igure) and (+)-verapami1 (lower f igure) on S-T segment elevat ion (expressed as %R wave amplitude) at various times before and af ter occ lus ion. Each histogram represents the mean ± s.e.mean value at the time ind icated. In each f igure the histograms in the back row are control values. In the f ront row are the values for the highest dose (12 mg/kg (+)- or 6 mg/kg (-)-verapamil) , and successive rows represent decreasing doses of (+)- (8, 4 and 0.4 mg/kg) or (-)-verapamil (2, 0.6 and 0.2 mg/kg) in the lower and upper f i gu re , respect ive ly . - 207 -R-WAVE SIZE ( in mV) (-)-Verapamil 1.0-0 .5 -0 —' 1.0-0.5_ 0 -I Occlusion Time After Occlusion Figure 28. The ef fects of (-)-verapami1 (upper f igure) and (+)-verapami1 (lower f igure) on R-wave amplitude ( in mV). The format for th is f igure i s ident ica l with that used for f igure 27, with the exception that the values at 15 min before occlusion are included in th is f igure and not in f igure 27. - 208 -3.5.7 Summary Both (+)- and (-)-verapamil dose-dependently reduced arrhythmias during acute myocardial ischaemia (Figure 25, Table 6) without reducing IZ s ize (Table 5 ) . (-)-Verapami1 was approximately 4 times as potent as (-^-verapa-mil in i t s a b i l i t y to reduce arrhythmia score. The development of ECG signs of ischaemia appeared to be delayed by both enantiomers (Table 5) . Both enantiomers prolonged P-R interval at high doses, but QRS interval was not affected (Figure 20). Although both enantiomers slowed heart rate th is e f fect was lost af ter occ lus ion, and reductions in blood pressure produced by the enantiomers were also not maintained throughout the ent i re period over which antiarrhythmic ef fects were seen (Figure 26). 3.6 Ac t ions-o f -n i fed ip ine and-DHM9-in acute-myocardial-ischaemia 3.6.1 Overview The ef fects of n i fed ip ine and DHM9 were examined in conscious rats as part of the studies to invest igate the a c t i v i t y of calcium antagonists in acute myocardial ischaemia, and the mechanism by which the i r actions are mediated. Ni fedipine was examined because i t is the prototype 1,4-dihydro-pyr idine calcium antagonist (Fleckenstein e t - a l . , 1972), and DHM9 was studied because prel iminary reports suggest that i t i s a card iose lec t ive calcium antagonist, a lbe i t with low potency (Clarke et a l . , 1984b). 3.6.2 Ef fects of n i fed ip ine and DHM9 before occlusion 3.6.2.1 P-R and- QRS i n te r va l s . There were no dif ferences in P-R interval (43 ± 1 msec in contro ls) and QRS in terva l (29 ± 1 msec in controls) between the 5 groups before drug administ rat ion. P-R in terva l was not s i g n i f i c a n t l y affected by the drug solvent or by DHM9, but P-R in terva l was shortened by 0.5 mg/kg n i fed ip ine (by 6 ± 2 % ) and by 2 mg/kg n i fed ip ine (by 4 ± 1 %) (Figure 29a). QRS in terva l was not inf luenced by any treatment (Figure 29b). - 209.-cs zz> cc Q I LU cc o cc CD 20 V) under these condi t ions. 3.10.5 Summary (-)-Verapami 1 was more potent than ( + )-verapami1 in reducing the vent-r i c u l a r developed pressure in paced ven t r i c l es . The potency of both enant i -omers varied considerably with the concentration of K + to which the prepa-rat ion was exposed (Figure 41). In addi t ion, (-)-verapami1 was more sens i t -ive to the potent iat ing ef fect of K than (+)-verapami1. 3.11 Actions of n i fed ip ine and-DHM9 in - i so la ted perfused-rat ven t r i c les 3.11.1 Overview The actions of these 1,4-dihydropyridines in iso la ted vent r ic les were invest igated for the same reason that the verapamil enantiomers were studied (see above), namely to provide an independent estimate of calcium antagonist a c t i v i t y in vent r icu lar t issue in order to support explanations for the actions (or lack of actions) in the ischaemic heart i n - v i vo . - 238 -3.11.2 Isochoric l e f t vent r icu la r developed pressure _5 DHM9 at up to 3 x 10 M had no ef fect on l e f t vent r icu lar developed pressure, i r respect ive of the K + concentration in the perfusing buf fer . Figure 42 shows the re la t ionsh ip between the negative inotropic a c t i v i t y + + of n i fed ip ine and the concentration of K present. Increasing K caused a small increase in - l o g - ^ E C ^ Q , and the re la t ionsh ip appeared to be l inear over the range 3 - 1 0 meq/1 K + . Despite the fact that increasing K + concentration from 3 - 1 0 meq/1 only increased the potency of n i fed ip ine approximately 4 f o l d , each - log^g EC ^ Q value was s t a t i s t i c a l l y s i g n i f i -cant ly d i f ferent from each other value, i l l u s t r a t i n g that variance was low. In contrast with the enantiomers of verapamil (see above), the slope for the negative inotropic actions of n i fed ip ine was s i g n i f i c a n t l y greater than 1 at 3, 5.9 and 8 meq/1 K + (values being 1.46 ± 0.14, 1.7 ± 0.11 and 1.39 ± 0.12, respec t ive ly . However, at 10 meq/1 K + , the slope was not d i f ferent from 1 ( i t was 1.01 ± 0.03). Therefore i t i s not s t r i c t l y appro-pr iate to extrapolate from the -log-^Q EC ^ Q data and state that K + had changed 'potency 1 . Nevertheless, the di f ferences in slope were not p a r t i c -u la r l y large (slope values were always less than 2 ) , and variance values for -log-^Q ECgQ were always smal l . Ni fedipine caused increases in coronary flow which were not c l e a r l y con-centrat ion-re lated nor related to the K + concentrat ion. Since n i fed ip ine i s r e l a t i v e l y se lec t ive in i t s actions on vascular smooth muscle compared with the heart (see Discussion) , then i t may be argued that the lowest con-centrat ions applied probably produced the maximum coronary vasodi lat ion possible with th is drug, and that subsequent var ia t ions were a re f lec t ion of the inf luence of other var iables such as in t ravent r i cu la r pressure. Pre-drug flows were 9.3 ± 0.6, 11.3 * 1.2, 8.9 ± 0.9 and 11.2 ± 0.7 ml/min in the 3, 5.9, 8 and 10 meq/1 K + groups respec t ive ly . - 239 -Figure 42. The re la t ionsh ip between negative inotropic potency of n i fed ip -ine and K + concentration in iso la ted Langendorff-perfused rat vent r ic les i s shown. The abscissa is K + concentration (meq/1) in the perfusion, while the ordinate is mean EC ^ Q for negative inotropism (calculated as l o g ^ mol/1 but shown as an t i l og ) . Bars indicate ± s.e.mean (n = 6 preparat ions). - 240 -3.11.3 Ventr icular e x c i t a b i l i t y Prel iminary evidence from the previous study with the enantiomers of verapamil (see above) suggested that increasing the K + concentration redu-ced the e x c i t a b i l i t y of the vent r ic les (as determined by sampling the strength/durat ion curve at 4 V and 1 msec). This e f fect was confirmed quite e x p l i c i t l y in the present experiment (Figure 43). Raising K + concentration caused a large increase in the threshold voltage for capture at 1 msec and the threshold pulse-width for capture at 4 V. This e f fect appeared to be l inear over the K + range studied, but there was a suggestion that th is e f fec t was beginning to saturate; with only 4 points i t was impossible to d is t inguish between the 2 models, therefore the points have not been connec-ted in the f i gu re . Experiments at higher K + concentrations were not carr ied out because previous work with the enantiomers of verapamil had suggested that i t was not possible to pace the vent r i c les e f f i c i e n t l y when K + concentration was more than 10 meq/1. 3.11.4 Summary DHM9 was without e f fec t on isochor ic l e f t vent r icu lar developed pressure, even at high concentrat ions. Ni fedipine was an e f fec t i ve negative inotropic agent (Figure 42). Increasing buffer K + concentration caused a small but s t a t i s t i c a l l y s i gn i f i can t increase in the potency of n i fed ip ine , and a large reduction in e x c i t a b i l i t y (Figure 43) . As a general comment (which also applies to the in v i t r o work with the enantiomers of verapamil) i t w i l l be of in terest to examine the potency of n i fed ip ine in the perfused ven t r i c le preparation using another approach, by keeping the calcium antagonist concentration constant and construct ing dose-2+ response curves for Ca . Such curves would give an estimate of drug a f f i n i t y for i t s ' receptor ' at d i f ferent K + concentrat ions. - 241 -Figure 43. The ef fect of d i f ferent buffer K concentrations on the threshold voltage (at 1 msec pulse width, part a) and threshold pulse width (at 4 V, part b) for capture of the l e f t ven t r i c le in the Langendorff-perfu-sion rat ven t r i c le preparation (st imulat ion frequency 300/min). Values are mean ± s.e.mean and were recorded af ter 10 - 15 min s t a b i l i s a t i o n . Each preparation was subsequently used to measure the negative inotropic a c t i v i t y of n i fed ip ine (see f igure 42). The values for both var iables at 3 and 5.9 meq/1 K + were s i g n i f i c a n t l y d i f fe rent from each other and from the values at 8 and 10 meq/1 K + (p < 0.05). 1 . 5 ' - . a THRESHOLD VOLTAGE (V) T 0 . 3 - , b THRESHOLD PULSE WIDTH (ms) 1 H 0.5-J C M ^ -( X I 0 .2 i o . H o - 1 0 3 5.9 8 10 0 3 5.9 8 10 K + CONCENTRATION IN BUFFER - 243 -4 DISCUSSION 4.1 The conscious rat preparat ion-for myocardial-ischaemia studies 4.1.1 Overview The rat has been used sporad ica l ly since 1946, and in tens ive ly since 1979 for invest igat ing myocardial ischaemia and i n fa r c t i on . The rat i s used because i t s lack of coronary co l l a te ra l anastamoses (Johns and Olson, 1954; Selye, et a l . , 1960; Maxwell et a l . , 1984; Winkler et a l ; , 1984; Schaper et a l . , 1986) allows for reproducible and uniform in farc ts (Johns and Olson, 1954; Selye e t - a l . , 1960; Bajusz, 1963; Hort and Da Canal i s , 1965a; 1965b; MacLean et a l ; , 1976; Bernauer, 1980; Johnston et a l . , 1983a), and reprodu-c ib le vent r icu la r arrhythmias (Clark et a l . , 1980; Kane and Winslow, 1980; Coker and Par ra t t , 1981; Mertz and Kaplan, 1982; Lepran et a l . , 1983; Johnston et a l . , 1983a; 1983b; MacLeod et a l . , 1983). The preparation i s used to invest igate arrhythmias (Kenedi and Losconci , 1973a; 1973b; Szekeres et a l . , 1980; Clark et a l . , 1980; Kane and Winslow, 1980; Bernauer 1980; Clark et a l . , 1980; Mueller and Wilsmann, 1981; Martinez and Crampton, 1981; Bergey et a l . , 1982; Au et a l . , 1983; 1986; Johnston et a l . 1983a; 1983b; e tc . ) and in fa rc t ion (Bryant et a l ; , 1958; Zsoter and Bajusz, 1962; Hort and Da Canal i s , 1965a; 1965b; MacLean et a l . , 1976; 1978; Pfef fer et a l . , 1979; 1985a; 1985b; Bernauer 1980; 1982; Innes and Weisman, 1981; Flaim and Z e l i s , 1981; de Jong-Koster and van Zwieten, 1981; Byrne e t - a l . , 1982; Edoute et a l . , 1983; Au e t - a l . , 1983; 1986; Johnston e t - a l • 1983a; 1983b; Manning et a l . , 1983a; Godfraind and Sal eh, 1984; e t c . ) . 4.1.2 Prec is ion of var iables 4.1.2.1 Occluded - zone (OZ)- and - infarct- - zone ( IZ) . Drugs which reduce the extent or degree of ischaemia can be expected to be ant iar rhyth-mic. Therefore such actions must be considered when assessing ant iar rhyth-mic ac t ions . The quant i f ica t ion of ischaemic and infarcted t issue s ize as - 244 -well as arrhythmias in the same animals provides a ser ies of checks and balances by which i t can be ascertained that reductions in arrhythmias occurred as a resu l t of treatment rather than as a resu l t of 'missed' occ lu -s ion . In addi t ion, ischaemia and in fa rc t ion can be studied in the i r own r i gh t . The resu l ts of studies of in fa rc t s ize in rats (see below) should be considered within the wider context of whether in fa rc t s ize reduction is ac tua l ly possible in any species (Hearse and Ye l l on , 1984). It i s believed by Hearse's group that animals with l i t t l e coronary co l l a t e ra l anastamosis development such as the rabbi t (Dennis et a l . , 1986) and rat (Hearse et a l ; , 1986) are unsuitable for assessing potent ia l i n fa rc t -1 im i t ing drugs s ince, in the absence of ear ly reperfus ion, a l l the non-perfused t issue i s destined to become necro t ic . Even in dogs which often possess extensive co l l a te ra l vascu lar isa t ion (Schaper et a l ; , 1967; Schaper 1971; Meesman, 1982), i t has recent ly been suggested that although drug treatment may reduce in fa rc t s ize when assessed 24 h af ter occ lus ion , the ef fect i s los t i f in farc t s ize is measured 48 h after occlusion (Yellon et a l . , 1986a), i l l u s t r a t i n g that care must be taken in d is t inguish ing between prevention of necrosis and delays in the development of necros is . With regard to the re la t ionsh ip between co l l a te ra l anastamoses and i n fa r c t i on , Hearse's group recent ly reviewed the i r in fa rc t s i ze data and found that for control animals in fa rc t s ize could be predicted accurately from the extent of co l l a te ra l f low, independent of the ' r i s k zone' s ize (Yel lon et a l . , 1986b). It i s poss ib le , therefore, that fo l lowing permanent coronary occlusion the ult imate in fa rc t s ize i s a f i xed function of the extent of co l l a te ra l anastamoses. The present resu l ts suggest that th is appears to be the case for r a t s . For species such as dogs with var iab le co l l a te ra l development (Meesman, 1982) the p o s s i b i l i t y ex is ts that a combination of in fa rc t development - 245 -delays and sampling error (an excess of animals with wel l developed c o l l a t -era ls in the drug-treated group) has given r i se to the many reports of ' reductions in in farc t s i z e ' in th is species. Unless an experiment is carr ied out b l i nd l y and randomly with control for var ia t ions in c o l l a t e r a l s , and unless at least 3 doses of a drug are invest igated, then the resu l ts of i n fa rc t l im i ta t i on studies in dogs should be treated with scept ic ism. In species (such as the rat) with poorly developed co l l a t e ra l s i t i s d i f f i c u l t to imagine how IZ s ize (expressed as % of OZ size) can be reduced by t rea t -ment under any experimental circumstance. Nevertheless, many reports of i n fa rc t s ize reduction in rats have been published (see below). 4.1.2.2 Arrhythmias. Direct comparisons of arrhythmia incidence between various rat models is not s t r i c t l y possible owing to the lack of a common method for diagnosis and quant i f i ca t ion of arrhythmias. Despite t h i s , there appears to be a good concordance within and between groups of invest igators and for d i f ferent models. For example, the mean ± s . d . i n c i -dences of VT and VF for conscious rats calculated from the experiments of Szekeres' laboratory (82 ± 7, and 89 ± 10 respect ive ly) correspond well with the incidence found in the present experiments during the 0 - 0.5 h period af ter occ lus ion. 4 .1 .2 .3 ECG changes. In rats there is very l i t t l e information concerning the pattern of ECG changes fo l lowing coronary occ lus ion, since most invest igators use the ECG merely to diagnose arrhythmias. The ECG was f i r s t recorded in rats fol lowing coronary artery occlusion by Normann e t - a l ; (1961). S-T segment e levat ion, giant R waves and pathological Q waves were shown to be present by 2 h af ter occ lus ion. By 24 h, deep Q waves and abnormally small R waves were present. These f indings are charac te r i s t i c of the changes described in the present s tud ies . Of some in terest i s a set of experiments car r ied out in dogs in which - 246 -the ECG ef fects of coronary occlusion were compared with the ef fects of haemorrhage (Prinzmetal e t - a l . , 1961). I t was shown that whereas occlusion induced changes in the ECG of s im i la r nature to those described in rats fo l lowing coronary occ lus ion, haemorrhage c h a r a c t e r i s t i c a l l y resul ted in immediate S-T segment depression and a reduction in R wave s i z e . This e f fec t of haemorrhage in dogs corresponds with s imi la r f indings in rats (Cooper, 1969), and also corresponds with f indings in the present ser ies of experiments. In th is regard, i t was found that in rats in which occlusion caused haemorrhaging in the thorax (demonstrated by post-mortem examination) the ECG was characterised by S-T segment depression rather than e levat ion, and t iny R waves were present at the time that the R wave i s usual ly abnor-mally large. 4.1.3 Responses to drugs 4.1.3.1 Overview. Many drugs have been invest igated for actions against ischaemia-induced arrhythmias using rat preparat ions, both in vivo and in v i t r o , and the pattern of outcome is not consistent for a l l drugs. In in vivo s tud ies , some of the var ia t ions in outcome may be a resu l t of the low doses used in some studies ( e . g . , Fagbemi et a l . , 1984; Mueller and Wilsmann, 1982) s ince , i f c l i n i c a l dose regimens are given to r a t s , i ne f fec -t i v e l y low blood concentrations may resu l t owing to the rapid metabolic rate of r a t s . Unfortunately, blood concentrations have only been determined in conjunction with the assessment of antiarrhythmic a c t i v i t y in rats on rare occasions (Kane e t - a l . , 1982; Curt is et a l . , 1984; Hashimoto et a l . - , 1986). It i s important to examine the dose-dependence of the ef fects of a drug. In many studies only one or two doses of a drug have been adminis-tered. Mul t ip le dose studies generate dose-response curves which are one of pharmacology's more powerful tools for data ana lys is . Therapeutic ra t ios can be ca lcu la ted , and the curves may of fer ins ight into mechanisms of drug f/ - 247 -ac t ion . C l i n i c a l l y i t i s well establ ished that a therapeutic window ex is ts for many drugs, whereby i nsu f f i c i en t amounts of drug w i l l be i ne f fec t i ve , whereas high concentrations w i l l be t ox i c . Antiarrhythmic agents, by the i r very nature, have s ign i f i can t inf luence on cardiac electrophysiology in most instances. Therefore i t i s not surpr is ing to f ind that antiarrhythmic agents can prec ip i ta te or exacerbate arrhythmias during therapy. Since arrhythmogenic propert ies have been reported for most types of ant iar rhyth-mic drugs including l i doca ine , qu in id ine, procainamide, bethanidine, l o r -ca in ide , f l e ca i n i de , verapamil, bepr id i l and ethmozin both c l i n i c a l l y and experimentally (see Torres et a l ; , 1985), considerat ion of dose-response re la t ionsh ips in the study of antiarrhythmic agents cannot be over-empha-s i zed . Since, in general , i nsu f f i c i en t considerat ion has been given to the above factors and to other important aspects concerning use of rat models (see l a t e r ) , i t i s c lear why contradictory reports may be found in the l i t e ra tu re concerning the effect iveness of drugs as antiarrhythmics and in fa rc t l im i t i ng agents ( th is applies equally well to resu l ts from studies using other spec ies) . 4 .1.3.2 Class 1 ant iarrhythmics. With the exception of l idocaine (see below) a l l the Class I antiarrhythmics which have been tested (qu in i -d ine, disopyramide, procainamide, Org 6001, e tc . ) are e f fec t i ve in a l l models, and in a l l studies (e.g. Johnston et a l . , 1983a; Marshall et a l ; , 1981b; 1982; Marshall and Winslow, 1982; Winslow, 1980; Winslow e t a l . , 1983; Woodward, 1981). At e f fec t i ve antiarrhythmic doses most of these drugs reduce blood pressure, except for disopyramide which ra ises i t (Mar-sha l l e t - a l ; , 1982; Johnston e t a l ; , 1983a). E f fec t ive doses of Class I antiarrhythmics for in tact rats are general ly higher than those for dogs and p igs , as a consequence of di f ferences in the rate of metabolism of drugs - 248 -between these species. The mechanism of action of Class 1 drugs, in general terms, is most l i k e l y related to i nh ib i t i on of g ^ a , since in iso lated perfused hearts the concentrations of Class I drugs which are antiarrhythmic are s im i la r to the concentrations which i nh ib i t vent r icu la r g N a in v i t ro (Grant et a l . , 1984; Weidmann, 1955a). There are reports that l idocaine is antiarrhythmic in anaesthetised rats (Clark et a l . , 1980; Bergey et a l . , 1982; Lepran et a l . - , 1983), however there are contradictory reports which suggest that the antiarrhythmic e f fec -tiveness of l i d i c a i n e i s lower than that of Class Ia drugs such as quinidine (Mertz and Kaplan, 1982; Johnston et a l . , 1983a). Lidocaine has been used c l i n i c a l l y for many years, pa r t i cu la r l y as a consequence of a highly encour-aging report in the ear ly 1970s (Lie et a l . , 1974). However, as with a l l current ly avai lab le drugs (Campbell, 1984), the c l i n i c a l ef fect iveness of l idocaine i s not establ ished (Pentecost e t - a l . , 1981; May et a l ; , 1983). Recently i t was shown that l idocaine dose-dependently increased the i n c i -dence of VF fo l lowing coronary occlusion in pigs in associat ion with a slowing of conduction in the ischaemic and normal myocardium (Carson et a l . - , 1986). By slowing conduction without prolonging re f rac to r iness , l idocaine would be expected to f a c i l i t a t e reentry (Mines, 1913). High concentrations of l idocaine in v i t r o , however are capable of converting un id i rec t ion l block to b i -d i rec t i ona l block (Cardinal et a l . , 1981; Janse 1982) The example of l idocaine i l l u s t r a t e s the importance of consideration of dosage, since in iso la ted hearts in which s ide-ef fec ts of* drugs (such as 1 idocaine-induced convulsions) are not a considerat ion, i t may be possible to demonstrate an antiarrhythmic action of a drug which would be impossible to produce in v i vo . 4 .1.3.3 Glass 2 ant iarrhythmics. This sect ion should be considered in conjunction with the sect ion concerned with the ro le of the sympathetic nervous system in arrhythmogenesis. Results obtained with s-adrenoceptor - 249 -antagonists have to be considered within the context of dose (or concentra-t ion) and the associated pharmacological p r o f i l e , since the pos i t ive a n t i -arrhythmic actions reported for some e-adrenoceptor antagonists only occur in v ivo at concentrations above those necessary for e f fec t i ve (dose ra t io for isoprenal ine of > 10) e-adrenoceptor antagonism (Campbell and Par ra t t , 1983). Pa r ra t t ' s group (Campbell and Par ra t t , 1983; Campbell et a l . , 1984; Hughes e t a l . , 1984) have found that many e-adrenoceptor antagonists possess antiarrhythmic propert ies in anaesthetised ra t s . On the other hand, others ( inc luding our laboratory) have found only marginal antiarrhythmic actions of e-adrenoceptor antagonists in anaesthetised rats (Kenedi and Losconci , 1973b; Au et a l . , 1979a; 1979b; 1983), and an absence of antiarrhythmic a c t i v i t y of e-adrenoceptor antagonism and sympathectomy in conscious rats (Botting et a l . , 1983), and non-stereoselect ive antiarrhythmic actions of e-adrenoceptor antagonists in perfused rat hearts (Daugherty et a l . , 1986). e-Adrenoceptor antagonists are e f fec t ive against reperfusion-induced arrhythmias in -vi tro only at concentrations which i nh ib i t (Rochette e t a l . , 1984). An explanation for the contradictory reports of the effect iveness of e-adrenoceptor antagonism in vivo i s given in the sect ion dealing with the ro le of the autonomic nervous system. B r i e f l y , e-adrenoceptor antagonism in vivo only seems to be e f fec t ive in reducing arrhythmias in acutely pre-+ + pared animals. Since surgery elevates serum K , and elevated serum K reduces the incidence of arrhythmias during acute myocardial ischaemia (Lubbe et a l . , 1978; Daugherty e t a l ; , 1981; Nordrehaug and von der Lippe, 1983; 1985; Solomon, 1984), and since catecholamines lower serum K + where-as e-adrenoceptor antagonists i nh ib i t the ef fect of catecholamines to lower serum K + (Brown e t - a l . , 1983), then e-adrenoceptor antagonists may i nh i b i t ischaemia-induced arrhythmias in acutely-prepared rats by simply re in forc ing - 250 -the e f fec t of surgery to ra ise serum K + (by i nh ib i t i ng the K + - lower ing e f fec t of the sympathetic nervous system and c i r cu la t i ng catecholamines). 4.1.3.4 Glass - 3 ant iarrhythmics. This c lass of drugs has not been studied extensively in ra t s . Mertz and Kaplan (1982) reported that 5 mg/kg bretyl ium had no s ign i f i can t action on VF in anaesthetised r a t s . Sotalol (50 mg/kg) and melperone (5 and 10 mg/kg) have been shown to prevent coro-nary occlusion-induced f a l l s in the threshold current required for induction of VF in anaesthetised rats (Marshall et a l . , 1983). Amiodarone (2.5 - 20 mg/kg i . v . , or pretreatment with 25 or 50 mg/kg/day, p.o. for 2 weeks) has been found to dose-dependently reduce and abolish VF in anaesthetised rats (Schoenfeld et a l . , 1984). However, using the same preparat ion, Winslow et a l . (1984) found that 21 days of pretreatment with 20 mg/kg amiodarone p.o. had no ef fect on occlusion-induced arrhythmias. Doses of amiodarone which f a i l e d to inf luence arrhythmias were found to prolong act ion potent ia l duration in iso la ted non-ischaemic vent r icu lar t i s sue , but were found to have no inf luence on th is var iab le in ischaemic t issue (Winslow et a l . , 1984). However, meobentine and bethanidine, at doses which prolonged the e f fec t i ve ref ractory period in the normal vent r icu lar myocardium were found to be e f fec t i ve in reducing occlusion-induced arrhythmias in anaesthetised rats (Northover, 1985). In summary, i t i s not establ ished whether Class 3 antiarrhythmic drugs are e f fec t i ve antiarrhythmics in acute myocardial ischaemia in r a t s . In studies in which these drugs have been shown to be antiarrhythmic, the mechanism of action is unclear. There have been no invest igat ions into the ef fect of Class 3 antiarrhythmics on in fa rc t s ize in r a t s . 4 .1 .3 .5 Glass- 4 ant iarrhythmics. Drugs which reduce the duration of the plateau of the cardiac action potent ial (calcium antagonists) are c l a s s i f i e d as Class-4 antiarrhythmics (Singh and Vaughan Wi l l iams, 1972). - 251 -The antiarrhythmic ac t i v i t y of a var ie ty of calcium antagonists has been invest igated in acute myocardial ischaemia in r a t s . The resu l ts are d i s -cussed in deta i l in subsequent sec t ions , with par t i cu la r reference to the current inves t iga t ions . As an overview, i t was found in the present ser ies of experiments that phenethylalkylamine calcium antagonists (verapamil and anipamil) were very e f fec t i ve antiarrhythmics in conscious ra t s , whereas 1,4-dihydropyridines ( fe lod ip ine and n i fed ip ine) had l i t t l e a c t i v i t y . In contrast , in anaesthetised acutely prepared animals ( in which much lower doses of drugs were given) other invest igators found that (±)-verapamil was only marginal ly antiarrhythmic (Fagbemi et a l . , 1984), whereas 1,4-dihydro-pyridines were very e f fec t ive (Fagbemi and Par ra t t , 1981a; 1981b; 1981c). Differences in the resu l ts may r e f l e c t di f ferences in drug dosage in r e l a -t ion to the cardiovascular and neuronal status of the animals used. Dosage res t r i c t i ons depend on whether conscious or anaesthetized preparations are used, since much higher doses of calcium antagonists have been found to be to lerated in conscious versus anaesthetised animals (Curt is e t - a l . , 1984). With regard to studies in v i t r o , i t must be remembered that calcium antagonists possess a myriad of ' non-spec i f i c ' actions at high concentra-t ions (see Henry, 1980; Nayler and Horowitz, 1983) which may confound the in terpretat ion of r esu l t s . In pa r t i cu la r , many ' i n t e res t i ng ' e f fects of -4 (±)-verapamil have been reported at concentrations of 10 M, such as st imulat ion of noradrenaline release from sympathetic nerves (Karaki et a l . , 1984), i nh ib i t i on of Ca 2 +-dependent ATPase a c t i v i t y (Davis e t - a l . , 1984), i nh ib i t i on of plasminogen act ivator secret ion by aor t ic endothel ial c e l l s (Kant et a l . , 1985) e tc . Such ef fects cannot possib ly contr ibute to the antiarrhythmic ef fects of (^-verapami l in v i vo , since the associated e f f -ect ive concentrations are at least 100 times greater than those which abo l -ish contract ion in the heart (see the present resu l ts of studies with i s o l a -- 252 -ted v e n t r i c l e s ) . Ni fedipine i s antiarrhythmic in v i t r o only at concentrations producing calcium antagonism in the myocardium (Thandroyen, 1982; Opie and Thandroyen, 1983). In in tact animals the evidence suggests that even fo l lowing high doses, there are no d i rec t e f fects of 1,4-dihydropyridines on the myocardium ( e . g . , Raschack, 1976a). Therefore, i t i s d i f f i c u l t to imagine how 1 ,4 -d i -hydropyridines can reduce ischaemia-induced arrhythmias i n -v i vo . If 1,4-dihydropyridine calcium antagonists elevate serum K + , t h i s action would account for the antiarrhythmic ef fects observed in acutely prepared animals (Fagbemi and Par ra t t , 1981a; 1981b; 1981c). However, i t has been shown that n i fed ip ine has no ef fect on adrenaline-induced hypokal-aemia (Struthers and Reid, 1983). In add i t ion , the present experiments showed that n i fed ip ine did not inf luence serum K + fo l lowing coronary occlusion in conscious ra t s . Calcium antagonists have been extensively tested for in farct - reducing actions in the ra t . The general comments concerning in fa rc t s ize out l ined previously apply to these r e s u l t s . Experiments car r ied out by ourselves and others have revealed no in fa rc t s i ze reductions with (±)-verapamil (Johnston et a l . , 1983a; Cur t is et a l . , 1984; Baur et a l . , 1984; Evans et a l . , 1985) and n i fed ip ine (Leinot e t - a l . , 1983). Others have reported in fa rc t s ize reductions with d i l t iazem (Flaim and Z e l i s , 1981; Richard et a l . , 1984; Leinot et a l . , 1983), bepr id i l (Richard and de L e i r i s , 1983; Leinot et a l . , 1983) , n i fed ip ine (Richard et a l . , 1984), (±)-verapami 1 (Richard et a l . , 1984) and l i do f l az i ne (Leinot et a l . , 1983). 4.1.3.6 Arachidonic acid metabolites- and an t i ph log i s t i c s . I t has been suggested that eicosanoids inf luence ischaemia-induced arrhythmias in the rat (Coker, 1982; 1983; Martinez and Crampton, 1981), although studies in our laboratory have shown that that eicosanoids have l i t t l e e f fect on - 253 -ischaemia-induced arrhythmias in conscious rats (Au et_a_Li» 1979a; 1980; 1981; 1983). A l terat ions in prostaglandin production by non-steroidal antiinflammatory agents have also been reported to reduce arrhythmias (Coker and Par ra t t , 1981a; Fagbemi, 1984; 1985; Lepran et a l . , 1981a; 1985; F ied -l e r , 1983) in anaesthetised ra t s . However, in conscious r a t s , 2 d i f ferent asp i r in regimens were found to possess l i t t l e antiarrhythmic actions in our laboratory (Johnston et a l . , 1983b). Various invest igators (Kol ta i et a l . , 1982; 1983; Lepran et a l . , 1982b) have suggested that leukocytes may, perhaps, be involved in arrhythmogenesis v ia production of arrhythmogenic arachidonic acid metabolites (see above). Provis ional resu l ts from experiments from our laboratory suggest that deple-t ion of thrombocytes and leukocytes with ant isera do not reduce arrhythmias occurring during the 0 - 0.5 h period af ter occ lus ion , however, complete data for the 0.5 - 4 h period is not yet ava i l ab le . The present experiments with pithed rats suggested that reductions in c i r cu la t i ng thrombocyte leve ls may be associated with a lower incidence of occlusion-induced arrhythmias. Drugs af fect ing the eicosanoid system have been reported to reduce in fa rc t s ize (F ied le r , 1983; Lepran, e t - a l ; , 1985), although studies from our labor-atory do not support these claims (Johnston et a l . , 1983b, Au et a l ; , 1983). 4 .1.3.7 Other - e n t i t i e s . a-Adrenoceptor antagonists have been reported to be antiarrhythmic in cats (Sheridan et a l . , 1980). There is much disagreement concerning antiarrhythmic effect iveness of a-adrenoceptor antagonists, however. Questionable s e l e c t i v i t y of action of many a-adreno-ceptor antagonists complicates the in terpretat ion of most s tud ies. If large doses are required for antiarrhythmic e f fec ts , pharmacological actions other than a-adrenoceptor antagonism may be involved. Both in vivo (Daugherty et a l ; , 1986) and in v i t ro (Bralet et a l . , 1985) studies have shown that whereas some a-adrenoceptor antagonists reduce occlusion-induced arrhythmias - 254 -( e . g . , phentolamine and prazosin) others (phenoxybenzamine and tr imazosin) are without e f fec t . Therefore the antiarrhythmic ef fects of a-adrenoceptor antagonists appear to bear no re la t ion to the i r a c t i v i t y as a-adrenoceptor antagonists. a-Adrenoceptor antagonists are discussed again in re la t ion to the antiarrhythmic a c t i v i t y of verapamil, below. Most anaesthetics have l i t t l e e f fect on ischaemia-induced arrhythmias in r a t s . Halothane has cons is tent ly been reported to have a marked ant ia r -rhythmic a c t i v i t y , whereas other halogenated hydrocarbons have no such a c t i v i t y (Au et a l . , 1983; MacLeod et a l . , 1983). Chloroform and enflurane may have weak antiarrhythmic actions (Jang et a l . , 1983), whereas pentobarb-i tone, fentanyl and nitrous oxide have no inf luence on arrhythmias (Au et a l . , 1983). Anaesthetics do not appear to have in farct - reducing actions in rats (Au et a l . , 1983; Macleod et a l . , 1983; Jang et a l . , 1983), although there i s one pos i t i ve report for halothane (K iss in et a l . , 1981), and one report suggesting that morphine increases in fa rc t s i ze (Markiewicz et a l . , 1982) . 4.1.4 The conscious ra t versus other preparations The ra t iona le for the use of the conscious rat has been discussed in de ta i l in the Introduct ion. There are a few addi t ional points to be made, however. F i r s t l y , the conscious, chronical ly-prepared preparation (Johnston et a l . , 1983a; 1983b; Curt is et a l . , 1984) has an advantage over acutely-prepared preparations (Clark et a l . , 1980; Kane et a l . , 1980; Lepran e t - a l ; , 1983) as a resu l t of the higher degree of precis ion of var iab les . Secondly, and more importantly, some drugs (notably e-adrenoceptor antagonists and calcium antagonists) appear to possess quite d i f ferent e f fects on arrhythmias in acutely-prepared versus chronical ly-prepared animals, as discussed above. - 255 -The reasons for the di f ferences between acutely prepared and chron ica l l y prepared rats have not been e luc idated. Various p o s s i b i l i t i e s may be specu-la ted , for example, di f ferences in serum K + may a l te r both the incidence of arrhythmias and responses to drugs (see elsewhere). However, since the doses of drugs given to anaesthetised rats are general ly much lower than those given to conscious r a t s , i t i s perhaps premature to propose a hypothe-s i s to account for di f ferences between acutely prepared and chron ica l ly prepared animals which is testable by merely surveying the l i t e r a t u r e . Coronary occlusion in iso la ted ra t hearts has been car r ied out for many years (Kannengiesser et a l . , 1975). There are, unfortunately, no de f i n i t i ve descr ipt ions of th is preparat ion, in terms of prec is ion and rep roduc ib i l i t y . I t has recent ly been shown that PVCs peak at 14 - 16 min after occlusion in the iso la ted ra t heart preparation (Daugherty et a l . , 1986). However, the time-course of VF in th is preparation has not been adequately described. The incidence of arrhythmias in iso la ted ra t hearts has been show to depend absolutely on the concentration of K + in the perfusion f l u i d (Daugherty e t - a l . , 1981). 4.1.4.1 Advantages and disadvantages of r a t s . The re l a t i ve merit of ischaemia preparations using rats compared with other species has been considered in the Introduct ion. The rat is s im i la r to other species in terms of s e n s i t i v i t y to e l e c t r i c a l st imulat ion-induced and drug-induced arrhythmias (Winslow, 1984), including those induced by ouabain (Tepper et a l . , 1985; Wilson and Hewick, 1985). In summary, the advantages l i e i n : a. the reproduc ib i l i t y and ease of production of ischaemia b. the small s i z e , low cost and ready a v a i l a b i l i t y c. the large, wel l -def ined data-base (Johnston et a l . , 1983a) d. the large physiological data-base (Far r is and G r i f f i t h , 1967; Pet ty, 1982). - 256 -Disadvantages of the preparation are equivocal . a. The ra t has a high rest ing heart ra te . However, in conscious ra t s , heart rate does not corre late with arrhythmias (Johnston et a l . , 1983a) unl ike the s i tua t ion with dogs (Kaplinsky et a l . , 1981). b. Ventr icu lar action potent ia ls in the ra t myocardium are d i f ferent from those found in many laboratory species in that they are of short dura-t ion with a narrow plateau phase (Langer, 1978; Inoue et a l . , 1984). However, the plateau phase of the vent r icu lar action potent ia l is gov-erned in rats in a s imi la r manner to that of other mammals (Reuter, 1979) by the slow inward current (Payet et a l . , 1980a; 1980b). The narrow action potent ial of the rat ven t r i c le may re la te to the fas t heart ra te , in that one may argue tael i o log ica l l y that whereas high heart rate in small animals compensates for the low pumping e f f i c iency of small hearts, a narrow action potent ial (and i t s associated low absolute re f ractory period) reduces the probab i l i t y of the occurrence of un id i rect iona l block and reentry in normal circumstances. How (or whether) such dif ferences inf luence ischaemia-induced arrhythmias i s unknown. c . The rat has a higher rate of drug metabolism than larger animals. This i s a disadvantage only in that dose regimens for humans cannot be deter-mined d i r ec t l y from studies using ra t s . 4.2 Important- determinants of arrhythmogenesis - in acute -myocardial ischaemia 4.2.1 Role of OZ s ize According to previous studies in our laboratory, arrhythmias (incidence and duration) depend on the s ize of the ischaemic (occluded) zone such that arrhythmia score (AS) i s l i nea r l y correlated with the square root of the OZ (Johnston et a l . , 1983a). The in terpretat ion of th is f ind ing i s that there - 257 -is a l inear re la t ionsh ip between arrhythmias and the inter face area between the ischaemic and normal t i s sue . It has been suggested from studies with pigs and dogs that th is border zone i s the s i t e of arrhythmogenesis (Brofman et a l . , 1956; Beck, 1958; Janse e t - a l . t 1979; 1980). Indeed, global ischae-mia, which is characterised by the absence of a border zone, i s associated with comparatively few arrhythmias (Beck, 1958). This suggests that i t i s not ischaemia per se, rather the presence of both normal and ischaemic t issue which is necessary for arrhythmogenesis. Regardless of the impl icat ions of the above re la t ionsh ip , OZ s ize data can be used to normalise arrhythmia scores and thereby reduce var iance. The usefulness of such normalization has been tested in our laboratory by d e l i b -erate ly producing e i ther large or small OZs and observing the associated arrhythmias ( e . g . , Curt is et a l . , 1984). In the present study with f e l od i p -ine , arrhythmia scores for the large and small OZ control groups were almost ident ica l af ter normalization for OZ (10.9 ± 2.5 and 9.1 ± 0.6, respect ive-ly) according to the (AS + l)/Joz method (see Methods sect ion) . Cor re la -t ions between incidences of major arrhythmias and OZ s ize are non- l inear (Johnston et a l . , 1983a), therefore normalization i s more complex. Sigmoid-shaped corre la t ions have been shown for dogs (Aust in , 1982). I r respect ive of the exact re la t ionsh ip between the amount of ischaemic t issue and the consequences of myocardial ischaemia, i t nevertheless remains that some re la t ionsh ip must e x i s t , therefore the OZ should be measured in a l l rats in order to ve r i f y that occlusion has taken place. The necessity for th is has been emphasized by Bernauer (1982) who showed that post -occ lu-sion surv ival depends on OZ s ize in r a t s . Bernauer (1980; 1982; 1983a; 1983b; 1985) corrects surv iva l and ECG changes fo l lowing occlusion for OZ s ize assuming a d i rec t co r re la t i on . This may not be appropriate (Johnston e t a l . , 1983a). However, i r respect ive of whether the sequelae of acute - 258 -myocardial ischaemia are proportional to the OZ or the square root of the OZ, i t may not be necessary to correct var iables for OZ s i z e , provided the variance for OZ s ize is smal l . The resu l ts of extensive studies with rats in our laboratory suggest that th is is the case (Johnston et a l . , 1983a; 1983b; Au et a l . , 1983; MacLeod et a l . , 1983; Jang et a l . , 1983; Cur t is et a l . , 1984; e t c . ) . 4.2.2 Role of the autonomic nervous system Arrhythmogenesis fo l lowing the onset of myocardial ischaemia i s believed to resu l t from disordered electrophysiology which in turn depends pr imar i ly on ischaemia. Secondary factors which may or may not act independently, namely the a c t i v i t y of the sympathetic system, heart rate and blood pressure are also suspected to be involved in arrhythmogenesis in acute myocardial ischaemia by some invest igators (Harr is , e t - a l . , 1951; Scherlag et a l . , 1970; Hope, e t - a l . , 1974; Fowl iss , et a l . , 1974 G i l l i s et a l . , 1976; Hirche et a l . , 1980; Kaplinsky et a l . , 1981;). Some of these factors have been invest igated in rats subjected to coro-nary artery occlusion in our laboratory. Analysis of covariance in over 250 conscious rats showed no cor re la t ion between arrhythmias and blood pressure and/or heart rate (Johnston et a l . , 1983a). In add i t ion , in conscious ra t s , propranolol (acute or chronic treatment), l a b e t a l o l , and 6-OHDA plus adrena-lectomy did not inf luence arrhythmias produced by coronary occlusion (Bot-t i ng , et a l . , 1984). However, others, using conscious r a t s , found that chronic B-adrenoceptor antagonist treatment did reduce arrhythmias (Sieg-mund, et a l . , 1979). In acutely prepared anaesthetised rats propranolol (Au, et a l . , 1983) practo lo l and pindolol (Kenedi and Losconci , 1973b) were found to have very weak antiarrhythmic a c t i v i t y . However, Campbell and Parrat t (1983) demon-strated more powerful antiarrhythmic ef fects with e-adrenoceptor antagonists - 259 -in anaesthetised ra t s . In add i t ion , e-receptor ac t iva t ion has been found to exacerbate arrhythmias in anaesthetised rats (Kenedi and Losconci , 1973a; Marsha l l , e t - a l . , 1981a). Therefore the arrhythmogenic ro le of B-adrenocep-tor ac t iva t ion remains contentious, although i t appears to be more important in anaesthetised rather than conscious r a t s . In view of the contradictory evidence regarding the importance of the autonomic nervous system in arrhythmogenesis in the r a t , a systematic inves-t iga t ion was carr ied out, using a ser ies of ablat ions in the CNS combined, in some cases, with catecholamine in fus ions. The resu l ts of th is study have been out l ined and published (Curt is et a l . , 1985b). It was found that ablat ions in the CNS great ly reduced arrhythmias fo l lowing coronary occlusion in ra t s , almost abol ishing the la te arrhythmias (those occurr ing between 0.5 h and 4 h af ter occ lus ion) . However, a simple re la t ionsh ip between the funct ional status of the CNS and the arrhythmia incidence was not demonstrated. When CNS i n teg r i t y , and/or peripheral adrenoceptor a c t i v i t y was systemat ical ly a l tered in a graded manner the arrhythmias induced by occlusion were not s i m i l a r l y graded. According to blood pressure and heart rate data, sympathetic a c t i v i t y was r e l a t i v e l y normal in the aS group (see Table 1), above normal in the aD group, and absent in aP and cP ra ts , whereas arrhythmias were reduced in a l l 4 groups. An infusion of noradrenaline and adrenaline su f f i c i en t to restore blood pressure and heart rate to normal in a group of pithed rats (aPN) did not restore arrhythmias. Likewise, catecholamine infusions in in tact conscious chron ica l l y prepared rats (cCN) did not increase arrhythmias. When the CNS was obtunded by pentobarbitone anaesthesia (aB) there was no reduction in arrhythmias compared with conscious animals (aC). In contrast with the lack of cor re la t ion between CNS ablations and arrhythmias and between adrenoceptor act ivat ion and arrhythmias, there - 260 -appeared to be an inverse re la t ionsh ip between the extent of acute surgery and arrhythmias. Possible mechanisms to account for th is re la t ionsh ip are discussed in the sections concerned with the ro le of serum K + , and the ro le of leukocytes and thrombocytes in arrhythmogenesis. Regardless of the mechanisms by which acute surgery lowered the i n c i d -ence of arrhythmias fo l lowing coronary occ lus ion, the resu l ts of th is study may have important impl icat ions regarding the s u i t a b i l i t y of acute myocardi-al ischaemia models in which acute surg ical preparation is used. In th is regard, even acutely prepared conscious (aC) and pentobarbitone anaesthet-ised (aB) rats had fewer episodes of VF and VT than conscious chron ica l ly prepared (cC) animals. Therefore larger group s izes would be required i f acutely prepared animals were used for drug s tud ies . In addit ion (as d i s -cussed previously) some antiarrhythmic drugs such as e-adrenoceptor antagon-i s t s and calcium antagonists produce d i f fe rent ef fects in acutely prepared versus chron ica l l y prepared animals. Why might e-adrenoceptor antagonists reduce arrhythmias only in acutely prepared animals ? I t i s known that catecholamines reduce serum K + (Brown e t - a l . , 1983; Vincent et a l ; , 1985) and that th is e f fect is inh ib i ted and reversed by e-adrenoceptor antagonists (Vincent et a l • , 1985). Therefore, e-adrenoceptor antagonists may reduce arrhythmias in acutely prepared rats by simply exacerbating the ef fect of acute surgery to ra ise serum K + . This is because the incidence of a r -rhythmias during acute myocardial ischaemia appears to be c r i t i c a l l y depen-dent on serum K + (see sect ion discussing the ro le of K + in arrhythmogen-e s i s , below). In summary, acute surgery appears to reduce arrhythmias (pa r t i cu la r l y those occurr ing between 0.5 h and 4 h) af ter coronary occlusion in a manner graded with the degree of surgery, and th is e f fect is independent of , and l i t t l e affected by, the extent of adrenoceptor ac t i va t i on . In contrast , the - 261 -funct ional status of the autonomic nervous system does not appear to i n f l u -ence ischaemia-induced arrhythmias in the ra t . 4 .2.3 Role of K + In contrast with the lack of cor re la t ion between CNS abla t ions, adreno-ceptor ac t iva t ion and arrhythmias in the present s tud ies , there appeared to be an inverse cor re la t ion between the extent of acute surgery and the ar -rhythmias produced by occ lus ion. If such a cor re la t ion e x i s t s , what might be the mechanism? Elevat ions in serum K + are known to occur fo l lowing acute surgery, and were observed in the CNS ablat ion study fo l lowing p i th ing . Serum K + elevat ion may contr ibute to the resu l ts of th is study. Elevat ion of serum K + i s associated with a f a l l in the incidence of VF in patients with acute myocardial in fa rc t ion (Nordrehaug and von der L ippe, 1983; Solomon, 1984). In iso lated perfused hearts, elevat ion of the K + concentration of the perfusate d r a s t i c a l l y reduces arrhythmias induced by coronary occlusion (Lubbe et a l . , 1978; Daugherty et a l . , 1981). In the iso la ted heart group of the present CNS ablat ion study, a low incidence of arrhythmias was seen. This was not unexpected in view of the above, since the hearts were exposed to 5.3 mM K + in the perfusing so lu t i on . In add i t ion , prel iminary resu l ts of experiments (carr ied out by others in our laboratory) in which KCI i n fu -sion and other treatments were used to a l te r serum K + , large changes in VF incidence were produced. The incidence of VF was 100%, 77%, 44% and 11% in groups of rats whose serum K were in the ranges 3.0 - 3.9, 4 . 0 - 4 .9 , 5 . 0 - 5.9 and > 5.9 meq/1, respec t ive ly . Therefore, serum K + appears to govern the probab i l i t y of the manifestation of arrhythmias during acute myocardial ischaemia, such that mild hyperkalaemia can almost abol ish vent r i cu la r arrhythmias. If acute surgery elevates serum K + , as was demonstrated in pithed ra t s , then the low incidence of arrhythmias is ex-- 262 -p la ined. What might be the mechanism by which hyperkalemia protects against ischaemia-induced arrhythmias ? Occlusion in perfused ra t hearts induces i n t r a c e l l u l a r potent ial changes which include par t ia l depolar izat ion and action potent ia l narrowing (Inoue e t - a l . , 1984). These ischaemia-induced ef fects may resu l t in part from elevations in ex t race l l u la r K + in ischae-mic t i s sue , since the l a t te r have been shown to occur at the same time as the e lect rophys io log ica l changes which coincide with the onset of vent r icu-lar arrhythmias (Hirche e t - a l . , 1980; 1981; 1982; H i l l and Gettes, 1980). In add i t ion , many of the e lect rophys io log ica l and electrocardiographic changes associated with acute myocardial ischaemia can be simulated by e levat ing ex t race l l u la r K + in ven t r i c l es ; such changes include slow con-duct ion, un i -d i rec t iona l block and reentry (Schmitt and Erlanger, 1928), depolar isat ion and S-T segment elevat ion (Prinzmetal et a l . , 1961), slowly developing reductions in i n t r a c e l l u l a r Na + concentration (Wild and Kleber, 1986) and slowly developing i n e x c i t a b i l i t y (Kleber, 1986). The e lect rophys io log ica l d ispar i t y between ischaemic and normal t issue observed in several species (Cinca et a l ; , 1980; Downar, 1977a; 1977b; Janse et a l . , 1980; Janse and Kleber, 1981; Janse, 1982; Kleber et a l . , 1978) including rats (Inoue et a l . , 1984) might then be expected to be reduced by elevat ions in serum K + concentration (which elevate ex t race l l u la r K + concentration in the normal myocardium) since such an increase would be expected to cause the non-ischaemic myocardium to resemble the ischaemic t i s sue , e lec t rophys io log ica l l y . A l t e rna t i ve l y , or in add i t ion , i f the threshold for e l e c t r i c a l exc i ta t ion i s ra ised by elevated serum K + , the normal myocardium would be protected from invasion by aberrant (arrhythmo-genic) impulses emanating from the ischaemic t i ssue . Prel iminary resu l ts of studies with iso la ted vent r ic les showed that the strength-duration r e l a t i o n -- 263 -ship for exc i ta t ion was shi f ted in the d i rec t ion of reduced e x c i t a b i l i t y by increased perfusate K + concentrations (see Figure 43). 4.2.4 Role of thrombocytes and leukocytes An a l ternat ive explanation for the ef fects of surgery on arrhythmias produced by acute myocardial ischaemia re lates to possible involvement of leukocytes and thrombocytes in arrhythmogenesis. Poss ib ly , acute surg ical preparation and CNS destruct ion releases an antiarrhythmic substance into the c i r c u l a t i o n , or removes an arrhythmogenic substance from the c i r c u l a t i o n . In th is regard, p i th ing was found to dep-lete c i r cu la t i ng thrombocytes and leukocytes in the present s tud ies. A f a l l in c i r cu la t i ng thrombocytes fo l lowing pi th ing has also been observed by others (Bot t ing, personal communication to our laboratory) . There have been many reports suggesting that interference with the propert ies of thrombo-cytes and leukocytes may reduce both ischaemia-induced myocardial t issue damage and also arrhythmias (Romson, et a l . , 1983; Mullane, e t - a l . , 1984; F ied le r , 1983; Fagbemi, 1984; 1985; Cahn and Borzeix, 1983; Coker and Par ra t t , 1981; 1983a; 1983b; Coker et a l . , 1981; J o l l y and Lucchesi , 1983; Lucchesi , et a l . , 1983; Lepran, et a l . , 1981b; 1985). Therefore, leuko-cytes, and/or thrombocytes, may play an important ro le in governing arrhyth-mias and other responses of the myocardium to ischaemia. I f thrombocytes, and/or leukocytes, trapped within (Leinberger et a l . , 1979) or migrating to the ischaemic zone (Mullane et a l . , 1984) are par t ly responsible for arrhythmogenesis in acute myocardial ischaemia, pa r t i cu la r l y in the la te (0.5 - 4 h) per iod, then i t is possible that acute surgery produces t issue damage which acts as a trap for leukocytes, and/or thrombo-cytes, thereby reducing the i r a v a i l a b i l i t y for par t i c ipa t ing in ischaemia-induced arrhythmogenesis. Adjuvant a r t h r i t i s , which may cause s im i la r t rapping, has been shown to reduce arrhythmias in conscious rats (Ko l t a i , - 264 -et a l . , 1982). It i s not yet c lear by what mechanism leukocytes and/or thrombocytes inf luence arrhythmogenesis. However, i t may be speculated that some component of the inflammatory process may be involved, arachidonic acid metabolites for example. 4.2.5 Role of arachidonic acid metabolites The ro le of thromboxanes, prostaglandins and leukotr ienes in arrhythmo-genesis was not invest igated in the current ser ies of experiments, and w i l l be discussed only in b r i e f . Experiments car r ied out ( in a var ie ty of ex-perimental preparations) with non-steroidal anti- inflammatory agents (Cahn and Borziex, 1983; Fagbemi, 1984; Coker and Par ra t t , 1981a; 1983a; 1983b; Coker e t - a l . , 1981; Coker, 1982; 1983; Lepran e t - a l . , 1981c; 1985; F ied le r , 1983) and arachidonic acid metabolites (Coker, 1983; Coker and Par ra t t , 1981; 1983a; 1983b; Martinez and Crampton, 1981) support the hypothesis that thromboxane is arrhythmogenic during acute myocardial ischaemia and that other prostaglandins may also play a r o l e . However, our laboratory (Au et a l . , 1979a; 1980; 1983; Johnston e t - a l . , 1983b) has found that asp i r in and a var ie ty of prostaglandins have l i t t l e inf luence on occlusion-induced arrhythmias. Once again, most of the pos i t i ve reports or ig inate from stud-ies using acutely prepared anaesthetised animals, whereas the negative reports were generated from studies using conscious animals which had reco-vered from surgery. 4.2.6 Role of l i p i d metabolites As in the case of arachidonic acid metaboli tes, the ro le of l i p i d metab-o l i t e s in arrhythmogenesis was not invest igated in the current s tud ies. Long-chain acyl carn i t ines (for example pa lm i ty l ca rn i t i ne ) , and lysophospho-l i p i d s (for example lysophosphatidylchol ine) are amphipathic metabolites of unester i f ied free fa t t y acids (FFA) which may increase in concentration in myocytes during ischaemia, owing to l im i ta t ions in aerobic oxidat ion of FFA - 265 -brought about by the lack of oxygen. Some of these compounds, by v i r tue of the i r a b i l i t y to par t i t i on into the sarcolemma, are capable of producing e lect rophys io log ica l changes, and even arrhythmias under some experimental condi t ions. In pa r t i cu la r , pa lmi ty lcarn i t ine at high concentrations (3 x l O - ^ M) can depolarise vent r icu lar muscle, abbreviate re f ractor iness and act ion potent ial duration (Matsui et a l • , 1985), e f fects which a l l occur fo l lowing coronary cocclusion during the period of ear ly arrhythmias ( e . g . , Inoue et a l . , 1984). However, i t is not establ ished whether any of these compounds play a ro le in arrhythmogenesis (see Corr e t -a l . - , 1984 for review). 4.2.7 Role of heart rate and blood pressure In conscious ra t s , the frequency and incidence of occlusion-induced arrhythmias are not related to blood pressure or heart rate according to corre la t ion matrix analysis (Johnston et a l . , 1983a). Correlat ions between heart rate or blood pressure and arrhythmias have been reported in larger species ( e . g . , Scherlag et al • , 1970). With regard to the ro le of heart ra te , i t has been shown that vagal st imulat ion in anaesthetised rats does not unmask PVCs during the f i r s t 30 min after coronary occ lus ion, suggesting that i t i s more l i k e l y that ear ly arrhythmias fo l lowing occlusion in rats are reentrant rather than due to automaticity (Mertz and Kaplan, 1982). This data is consistent with the f indings of studies using ep icard ia l map-ping techniques in iso la ted perfused pig hearts (Janse, 1982, e t c . ) , and explains why heart rate would be expected to have l i t t l e inf luence on the incidence of arrhythmias during acute myocardial ischaemia. 4.2.8 Role of the fast and slow inward currents The possible mechanisms by which the slow inward current ( i s ^ ) may be involved in arrhythmogenesis in acute myocardial ischaemia were out l ined in the Introduct ion. Both normal and abnormal cardiac rhythms are dependent on the charac te r i s t i cs of propagation of the myocardial act ion po ten t ia l . This - 266 -is dependent on the behaviour of ion ic conductances. I f conduction in the ischaemic t issue could be abolished by se lec t ive i nh ib i t i on of the currents responsible for such conduction then arrhythmias might be abol ished. In th is regard i t has been suggested that chemical destruct ion of the ischaemic endocardium by local in jec t ion of phenol can abol ish arrhythmias in dogs fo l lowing coronary occlusion (Chilson e t a l . , 1983). As discussed in the in t roduct ion, myocardial ischaemia is character ised by depolar isat ion and a depression in the r i se rate of the upstroke of the action po ten t ia l . The consequence of th is change i s a slowing of conduction through the ischaemic t i s sue . Studies in v i t ro have shown that elevated K + concentrations in combination with adrenaline are capable of generating action potent ia ls of which the depolar is ing current i s i .. (Carmeleit and Vereecke, 1972), but i t i s not c lear how relevant th is observation is to acute myocardial ischaemia. Theore t i ca l l y , consideration of the r e l a t i o n -ship between the res t ing membrane potent ial and percentage of openable channels does not r e a l l y help in determining which conductance is the most important in arrhythmogenesis, since rest ing membrane potent ial does not remain constant during acute myocardial ischaemia (Janse, 1982), and since depolar isat ion is not the only consequence of coronary occlusion which produces e lect rophys io log ica l changes. For example, the f a l l in ex t race l l u -la r pH (Hirch et a l . , 1980) may a l te r both the k ine t ics of i . ( I i j ima et a l . , 1986) and the responses to drugs which in ter fere with i s ^ in vent r icu lar t issue (Briscoe and Smith, 1982). With regard to arrhythmogen-esis i n -v i vo , i t i s unknown whether the depolar is ing inward current in ischaemic t issue is carr ied by a depressed i ' N a , by i .. or a combination of the two. I t i s not techn ica l l y possible at present to determine which conduct-ances are responsible for arrhythmogenesis in acute myocardial ischaemia in - 267 -conscious animals (or humans) by e lect rophys io log ica l methods. Neverthe-l e s s , there i s abundant evidence that drugs which i nh ib i t i.. (see above) and i . (see below) can reduce arrhythmias induced by coronary artery occ lus ion. Although th is evidence i s not de f i n i t i ve proof of an involvement of e i ther of these conductances in arrhythmogenesis, i t i s h ighly sugges-t i v e . Pharmacological studies designed to ru le out the involvement of sub-s id ia ry propert ies of these drugs w i l l ass is t in removing confusion con-cerning mechanisms of arrhythmogenesis. As a general comment, i t i s un l ike ly that arrhythmogenesis in acute myocardial ischaemia is governed by a myriad of biochemical, physio logical and e lect rophysio log ica l mechanisms. It is possible to hypothesise that abnormal conduction in acute myocardial ischaemia is dependent pr imar i ly on the fast and slow inward currents, and that many drugs which have been reported to be antiarrhythmic are so by v i r tue of i nh ib i t i on of one or the other of these currents. If rats alone are considered, antiarrhythmic actions have been reported for d and 1 propranolol (Daugherty et a l ; , 1986), antihistamines (Dai , 1984), meptazinol (Fagbemi e t - a l , 1983) indomethacin (Fagbemi 1984), halothane (MacLeod e t - a l ; , 1983) and other drugs, a l l of which can i nh ib i t e i ther the fast or slow inward current in addit ion to the i r 'major' act ions. . For example, d- and 1-propranolol i nh ib i t i ^ a in vent r icu lar muscle (Pol len _et a l ; , 1969), SKF-93479 (an ant ih istamine), and meptazinol produced ef fects consistent with inh ib i t i on of i ^ g (Dai , 1984; Fagbemi et a l . , 1983), and indomethacin (Northover, 1977) and halothane (Lynch et a l . , 1981) have been reported possess calcium antagonist a c t i v -i t y . In add i t ion , qu in id ine, which is an e f fec t i ve antiarrhythmic in acute myocardial ischaemia ( e . g . , Johnston et a l . , 1983a) possesses calcium antagonist a c t i v i t y (Spedding, 1983) in addit ion to i t s well known a b i l i t y to i nh i b i t i., . - 268 -With regard to calcium antagonists and the ro le of i • in arrhythmo-genesis, many of the current experiments were designed to examine the mecha-nism of antiarrhythmic action of calcium antagonists. The resu l ts support the hypothesis that i • is important in arrhythmogenesis (see below). 4.3 Mechanism of action- of calcium antagonists in acute myocardial ischaemia 4.3.1 Role of calcium antagonism 4.3.1.1 Overview. (±)-Verapamil has been shown to be highly e f fec -t i ve in reducing occlusion-induced arrhythmias in dogs (Kaumann and Aramen-d i a , 1967; K ro l l and Knight, 1984), rats (Johnston et a l . , 1983; Mertz and Kaplan, 1982; Bernauer 1982; Fagbemi et a l . , 1984) and pigs (Bergey et a l . , 1984). The mechanism of action of (±)-verapami1 in these studies has not been proven. In the introduct ion i t was explained why i t i s d i f f i c u l t to prove a mechanism of antiarrhythmic act ion in closed-chest experimental animals, or in the c l i n i c . By studying act iva t ion maps in open-chest an i -mals, i t should be possible to test whether (^)-verapamil i nh ib i t s arrhyth-mia generation or maintainance. As ye t , such studies have not been carr ied out. Nevertheless, even i f (±)-verapami 1 were to be shown to i n h i b i t , for example, reentry in the ischaemic t issue in associat ion with the conversion of slow conduction to complete i n e x c i t a b i l i t y , th is would not prove that inh ib i t i on of i . was the mechanism of action unless i t could be estab-l ished that the abnormal conduction was dependent on i •. I t is d i f f i c u l t (perhaps impossible, depending on one's ph i losphica l approach) to estab l ish a mechanism of action by experiment. It has been argued that one can only disprove a hypothesis by experiment, and that f a i l u r e to disprove a hypothesis does not inf luence the probab i l i t y of that hypothesis being correct . At present there is no convincing evidence to disprove that (±)-verapamil i nh ib i t s ischaemia-induced arrhythmias by inh ib -- 269 -i t i ng This consideration appl ies to other calcium antagonists such as anipamil . Proponents of the hypothesis that calcium antagonists possess an t ia r -rhythmic actions have developed a hypothetical concept to account for the reported antiarrhythmic actions of calcium antagonists, such as ( ^ - ve rapa -m i l , ca l led the modulated receptor hypothesis (see Hondeghem and Katzung, 1984) ( th is concept applies equal ly well to i ' N a b lockers) . This hypothe-s i s essen t ia l l y attempts to incorporate the voltage-dependence of the inh ib -i to ry actions of drugs such as quinidine on i ^ a (Weidmann, 1955b) and (^-verapami l on i g ^ (Ehara and Kaufmann, 1968) into a mass-action model by proposing that the a f f i n i t y of a drug for a receptor associated with a conductance channel is dependent on whether the channel is res ted, act ivated or inac t iva ted. This model p red ic ts , on the basis of r e l a t i ve voltage-dep-endence, that (±)-verapamil should se lec t i ve l y reduce high frequency ar-rhythmias (VT and VF) compared with PVC, and select between t issues on the basis of E m and APD90. The resu l ts of the present experiments are cons is -tent with the former p red ic t ion , while the predicted s e l e c t i v i t y of ( ^ - v e r -apamil for t issue with a low ( re l a t i ve l y pos i t ive) rest ing E m may account for the a b i l i t y of (^-verapami l to i nh ib i t a t r ioven t r i cu la r conduction at doses below those inf luencing cardiac output ( s e l e c t i v i t y for the 'depolar-i sed ' a t r ioven t r i cu la r node versus the ' f u l l y po la r i sed ' vent r i c les) (Singh 1975; Singh et a l . , .1978;. Nayler and Horowitz, 1983; e t c ) . The sections below discuss the resu l ts of present studies with ( + ) - , ( ± ) - and (-)-verapamil , anipami l , ronipami l , f e lod ip ine , n i fed ip ine and DHM9, and attempt to explain the mechanism of action of calcium antagonists in acute myocardial ischaemia. 4.3.1.2 Anipamil versus- ron ipami l . Anipamil and ronipamil are analogues of verapamil. At present, l i t t l e i s known about the pharmacology - 270 -of these drugs. Prel iminary evidence suggests that anipamil appears s im i la r to verapamil in having calcium antagonist a c t i v i t y in the myocardium, where-as ronipamil has l i t t l e ac t i v i t y (Raschack, 1984; Kretzschmar and Raschack, 1984; Kretzschmar, personal communication to the laboratory) , in accordance with the known st ructura l requirements of verapamil analogues for calcium antagonism (Mannhold et a l . , 1978; 1981). The evidence suggests that anipa-mil i s a calcium antagonist with s e l e c t i v i t y for the myocardium versus the vasculature, whereas ronipamil appears to be r e l a t i v e l y inac t i ve . Most calcium antagonists including (±)-verapamil have s e l e c t i v i t y for vascular smooth-muscle versus the myocardium and produce profound hypotension at doses below those af fect ing i^.-dependent processes in the heart (Briscoe and Smith, 1982; M i l l a rd et a l . , 1983; Lee et a l . , 1983; Van Zwieten and Timmermans, 1983; Kenakin and Beek, 1985; e t c . ) . However, anipamil may be d i f fe rent in that i t appears to reduce cardiac output at doses below those inf luencing systemic blood-f low. Anipamil and ronipamil were developed as verapamil analogues with a long duration of ac t ion . The l i p o p h i l i c i t y conferred by the s t ra ight 10 C-chain extension on C-9 may be responsible for t h i s . Both compounds have been demonstrated to delay the depletion of myocardial enzymes in rat hearts induced by a period of hypoxia (Kretzschmar and Raschack, 1984; Raschack, 1984). In other tests s im i la r ant i- ischaemic or ant i-hypoxic actions have been observed (Kovach, 1984; Ferrar i e t - a l . , 1984; Urbanics and Kovach, 1984). In agreement with these previously reported act ions, i t was found in the present studies that both anipamil and ronipamil moderated the ischaemia induced by occlusion in terms of reductions in the maximum values of S-T segment e levat ion . Anipamil also delayed the development of S-T segment elevat ion and R-wave changes. However, there were no dif ferences in OZ s ize - 271 -between the groups. Therefore di f ferences in the patterns of ECG changes associated with drug treatment appeared to re f l ec t actions in the ischaemic t i s sue , rather than actions to l i m i t the s ize of the zone of ischaemia. Regardless of possible ant i- ischaemic act ions, i t was apparent that only anipamil possessed s ign i f i can t antiarrhythmic a c t i v i t y . Anipamil appeared to be a se lec t ive antiarrhythmic against VF compared with both VT and PVC. This resu l t does not support the concept of an arrhythmia continuum whereby PVCs 'cause' VT, and VT causes or 'degenerates to ' VF ( e . g . , Harr is et a l . , 1950). Anipamil 150 mg/kg p.o. reduced the incidence of VF from 9/9 in controls to 2 /9 . With respect to such a spec i f i c a n t i f i b r i l l a t o r y ac t ion , i t has been suggested by our laboratory (Curt is et a l . , 1984) that a se lec -t i ve a n t i f i b r i l l a t o r y drug would have frequency-dependence leading to e lec -t rophysio logic actions only at high frequencies ( f i b r i l l a t i o n ) . This prop-erty can also be described in terms of a s e l e c t i v i t y for the inact ivated state of the i • channel according to the modulated receptor hypothesis (Hondeghem and Katzung, 1984). A descr ipt ion of the frequency-dependence of the action of anipamil on vent r icu lar t issue is not, at present, ava i lab le . Neither ronipamil nor anipamil increased the incidence of cardiogenic shock in the present study. However, 20 mg/kg i . v . ( i j-verapami 1, in an e a r l i e r study from pur laboratory (Curt is et a l . , 1984) caused cardiogenic shock. I t was of in te res t , therefore, to compare anipamil with ( ^ - v e r a p a -mil for antiarrhythmic versus cardiovascular a c t i v i t y . In th is regard, anipamil reduced VF by about 50% and 80 % at 50 and 150 mg/kg, respec t ive ly , doses which reduced blood pressure jus t before occlusion by only 7% and 5%, respec t ive ly . In contrast , (±)-verapami 1, at a dose reducing the incidence of VF by 50%, reduced blood pressure by 23%. Thus, when one considers the haemodynamic and antiarrhythmic ef fects in tandem, i t fol lows that the therapeutic ra t i o of anipamil is higher than that of ( ^ -ve rapami l . - 272 -With regard to the antiarrhythmic actions of anipamil versus ronipamil in re la t i on to the i r pharmacological proper t ies, the major known dif ference between them appears to be in the i r a b i l i t y to reduce cardiac output (anipa-mil being much more potent in th is respect than ronipami l ) . Kretzschmar (personal communication to the laboratory) has suggested that the ef fect of anipamil to lower cardiac output in dogs can be reversed by the in jec t ion of 2+ bolus doses of Ca . Therefore, i t i s ten ta t i ve ly concluded from th is study that the antiarrhythmic actions of anipamil may be dependent on c a l -cium antagonist a c t i v i t y . A more f i rm conclusion can be made with regard to the re la t ionsh ip between the ECG signs of ischaemia and arrhythmias. There does not appear to be a l inear cor re la t ion between these var iab les , since anipamil reduced maximum S-T segment elevat ion to the same degree as ronipa-m i l , but was much more e f fec t i ve than ronipamil as an antiarrhythmic. Therefore the mechanisms responsible for the ECG signs of ischaemia and those responsible for arrhythmogenesis may d i f fe ren t . 4 .3.1.3 (+)- Versus (-)-verapami1. Before considering the actions of (+)- and (-)-verapamil fo l lowing coronary occlusion in conscious ra t s , i t i s of in terest to consider the i r calcium antagonist potencies in terms of e f fects on heart rate and blood pressure in vivo in conscious rats (from the occlusion study) and pithed ra t s , and on l e f t vent r icu lar developed pressure in v i t r o . These ef fects probably depend on inh ib i t i on of the transmembrane f lux of C a 2 + (Nayler and Horowitz, 1983). Without exception, (-)-verapamil was the more potent enantiomer. How-ever, both the absolute and re l a t i ve potencies of the enantiomers varied from one test s i tua t ion to another. In conscious rats the (-):( + ) potency ra t ios were the lowest. In pithed ra t s , higher potency ra t ios were obs-erved, while absolute potencies were also increased. In iso la ted vent r ic les both the absolute potencies and the (-):(+) potency ra t ios were highly - 273 -+ dependent on K concentrat ion. The ' t rue ' potency ra t i o i s best estimated from studies in v i t r o . Therefore, deviat ion in vivo from values measured in v i t ro probably resu l t from the a b i l i t y of the body to ' f i l t e r ' the drug. The present resu l ts suggest that the calcium antagonist potency of the verapamil enantiomers is governed, in v ivo , by at least 3 fac to rs , namely s tereoselect ive pharmaco-+ k i n e t i c s , sympathetic tone and ambient K concentrat ion. In conscious humans the potency of (-)-verapami1 is reduced compared with that of (+)-verapamil as a resu l t of s tereoselect ive hepatic metabolism (Vogelgesang et a l . , 1984; Eichelbaum et a l . , 1984). The low potency ra t io of ( - ) - to (+)-verapamil in conscious rats i s consistent with s tereoselect -ive metabolism. In pithed rats i t i s possible that the greater di f ference in potency between the enantiomers (compared with that found in conscious rats) resu l ts from a reduction in hepatic blood flow in pithed ra t s , serving to l i m i t the inf luence of s tereoselect ive metabolism. There is evidence of a ' f unc t iona l ' antagonism ( i . e . , not competit ive) between (^-verapami l and the sympathetic nervous system (Tung e t a l . , 1985). The enhanced absolute potencies of both enantiomers in pithed versus conscious rats may therefore resu l t from the lack of sympathetic ref lexes in pithed compared with conscious ra t s . In iso la ted ven t r i c l es , where potent ia l inf luences of pharmacokinetic factors and the sympathetic nervous system are removed, an important i n f l u -ence of extracel lu lar_ K + on potency was revealed. The mechanism by which elevat ion of K + increased the calcium antagonist potency of the enantio-mers while reducing the di f ference in potency between them is uncertain (see below). The resu l ts have impl icat ions regarding the hypothetical ro le of calcium antagonism in governing the antiarrhythmic action of (±)-verapamil during - 274 -acute myocardial ischaemia. F i r s t l y , ex t race l l u la r K + i s known to r i se (within min) to 8 - 15 meq/1 in the ischaemic t issue (Hirche et a l . , 1980; H i l l and Gettes, 1980). Therefore, according to the hypothesis that calcium antagonists reduce ischaemia-induced arrhythmias as a resu l t of calcium antagonism in the ven t r i c l es , (-)-verapamil should be no more than 8 times as antiarrhythmic as (+)-verapami1 i f , as the present experiments suggest, ex t race l l u la r K + concentration governs the calcium antagonist potency of phenethylalkylamines at the c e l l u l a r level in the ischaemic vent r icu lar t i s sue . Secondly, s tereoselect ive hepatic metabolism may occur in conscious rats as i t does in humans (Vogelgesang et a l . , 1984; Eichelbaum et a l . , 1984), in which case the antiarrhythmic potency ra t i o would be expected to be less than 8. The actions of the enantiomers of verapamil during acute myocardial ischaemia were f i r s t examined using a simple new conscious rat preparat ion. The end-points used were morbidi ty, death, OZ and IZ s i z e . Death was cate-gorized according to the behaviour pattern which accompanied i t . In separ-ate experiments i t was establ ished that convulsive-type behaviour always and only occurred af ter a minimum of 10 sec of VF or VT, and that morbidity was associated with hypotension and cardiogenic shock. The preparation was devised on the basis of considerable experience with coronary-occluded conscious r a t s , and was designed to provide information concerning the a n t i f i b r i l l a t o r y and in farct - reducing actions of drugs rap id ly and simply. The resu l ts of th is study were in agreement with those reported p rev i -ously by the laboratory for (±)-verapami 1 (Curt is et a l . , 1984). The i n c i d -ence of convulsive-type behaviour ( ind ica t ive of VF) during the f i r s t 4 h af ter occlusion in the present study was reduced from 84 to 43%by 6 mg/kg ( i j -verapami1. In the previous study the incidence of VF was reduced from 89 to 45% by the same dose (Curt is et a l . , 1984). Mor ta l i t y not associated - 275 -with convulsive-type behaviour was increased from 8 to 12% in the present study. This increase compares favourably with the increase from 11 to 22% found in the previous study (Curt is et a l . , 1984). The order of ef fect iveness of the treatments ( i . e . , - > ± > +) in redu-cing behaviour ind icat ive of VF suggests that calcium antagonism, rather than sodium channel blockade, accounted for the antiarrhythmic ac t ion . This i s because (-)-verapamil is more potent than (+)-verapamil as a calcium antagonist (Bayer et a l . , 1975b; 1975c; Raschach, 1976; Nawrath et a l . , 1981; Gloor and Ur tha ler , 1983; Ferry et a l . , 1985; Echizen et a l . , 1985) whereas the isomers are equipotent as sodium channel blockers (Nawrath et a l . , 1981). The order of ef fect iveness (- > ± > + > sal ine) of the treatments in producing morbidity and cardiovascular depression also cor res-ponded with the calcium antagonist potency order. However, since th is i n i t i a l study used only s ingle doses i t would be premature to ru le out other possible mechanisms of action in the absence of f u l l dose-response data. We found that ( + ), (± ) - and (-)-verapami 1 did not reduce in fa rc t s i z e . This agrees with previous f indings in rats for (±)-verapamil (Baur e t - a l . , 1984; Evans et a l . , 1985), including those from our laboratory (Curt is et a l . , 1984). Since rats have few e f fec t ive c o l l a t e r a l s , drug-induced reductions in in fa rc t s ize may not be possible in th is species (Schaper 1984; Hearse et a l . , 1986), as discussed prev iously . In summary, (A)-verapami1 was more e f fec t i ve than (+)-verapami1 and less e f fec t i ve than (-)-verapamil in reducing behaviour consistent with VF f o l l -owing permanent coronary artery occlusion in conscious ra t s . The order of potency corresponded qua l i t a t i ve l y with the calcium antagonist potency order. Infarct s ize was not reduced. Following th is prel iminary invest igat ion with the enantiomers of verap-ami l , an extensive dose-response study was carr ied out, in which blood - 276 -pressure and the ECG were monitored in the usual manner. In these ' f u l l y -instrumented' conscious ra t s , both (+)- and (-)-verapami1 possessed dose-dependent antiarrhythmic a c t i v i t y against coronary occlusion-induced ar-rhythmias. VT and VF appeared to be reduced more e f f ec t i ve l y than PVC. (-)-Verapamil was consis tent ly 4 times more potent in th is respect than (+)-verapamil. The antiarrhythmic potency ra t i o corresponded with that for ef fects on heart rate and blood pressure seen immediately before (but not af ter) coronary occ lus ion. The potency ra t i o for the antiarrhythmic actions corresponded reasonably well with recent estimates of the calcium antagonist potency ra t i o for the enantiomers in vent r icu lar muscle, based on pharmacol-ogical and radio l igand binding studies (Ferry et a l . , 1985). In order to supplement data from the l i t e ra tu re concerning the re la t i ve potencies of the opt ica l enantiomers of verapamil as calcium antagonists, experiments were carr ied out using iso la ted rat ven t r i c les and pithed rats (as discussed above). In iso la ted rat vent r ic les (-)-verapami 1 was more potent than the (+)-verapamil, and the di f ference in potency was dependent on the K + concentration in the perfusing buf fer . At high K + concentra-t i ons , the re l a t i ve potency of the enantiomers resembled the antiarrhythmic potency fo l lowing coronary occ lus ion. The antiarrhythmic potency ra t i o also corresponded with the potency ra t io for ef fects on blood pressure and heart rate in v i vo , reductions in which presumably occurred as a resu l t of calcium antagonism (Nayler and Horowitz, 1983). We consider the above to support the hypothesis that the antiarrhythmic actions of both enantiomers during acute myocardial ischaemia occurred by v i r tue of calcium antagonism, rather than v ia quin id ine-1ike Na + channel blockade, a property which is shared equal ly by both enantiomers and which is only manifest at concentrations in excess of those required to abol ish i$.j (Nawrath et a l . , 1981). The possible ro le of Na + channel blockade - 277 -in determining the antiarrhythmic a c t i v i t y of the enantiomers i s discussed fur ther , below. In support of the present conclusion is the experiment of Kaumann and Serur (1975) in which i t was found that both enantiomers of ver-apamil reduced arrhythmias induced by acute coronary occlusion in dogs, and that (-)-verapamil appeared to be the more potent enantiomer (the group s ize was described by the Authors as being too small for a meaningful assessment of potency di f ferences between the enantiomers, however). The experiments carr ied out in pithed rats were in te res t ing , but they did not ass i s t in supporting or disproving the hypothesis that (^-verapami l reduces ischaemia-induced arrhythmias by v i r tue of calcium antagonism. This was because the s e n s i t i v i t y of the pithed rats to the ef fects of the enant i -omers was great ly enhanced compared with conscious ra t s , making in terpre t -at ion of the resu l ts d i f f i c u l t . The increased s e n s i t i v i t y of pithed rats to the enantiomers compared with conscious rats suggests that the actions of the enantiomers are of fset in conscious animals by the sympathetic nervous system. However, since the potency ra t io of (-)- to (+)-verapamil in pithed rats for ef fects on blood pressure was not very d i f ferent from the cor res-ponding potency ra t io in conscious ra t s , then i t appears that i f the sympa-the t i c nervous system does of fset the ef fects of the enantiomers in con-scious rats then th is ef fect i s not se lec t ive for one enantiomer versus the other. With regard to the ef fects of the sympathetic nervous system on responses to calcium antagonists, i t has recent ly been suggested that a s im i la r e f fect occurs to l im i t the ef fects of some 1,4-dihydropyridine calcium antagonists in conscious animals (Che!ly et a l ; , 1985). There were 2 di f ferences between the present occlusion study with the opt ica l enantiomers of verapamil and the previous study with (^-verapami l from our laboratory (Curt is et a l . , 1984). F i r s t l y , in the present study arrhythmia-induced morta l i ty was abolished by improving the d e f i b r i l l a t i o n - 278 -technique, al lowing for improved precis ion of var iables (the previous con-t ro l incidence of VF-induced death was 30% ). Secondly, the incidence of mor ta l i ty associated with cardiogenic shock was lower in the present study. In the previous study such deaths mainly occurred with 20 mg/kg ( ^ - ve rapa -m i l , and the doses used in the present study were lower. These small d i f f -erences appear to be unimportant s ince , i f the E D ^ Q for (^-verapami l (determined from the previous study) during the 0-30 min period (6 mg/kg) i s compared with that for (-)-verapamil (2 mg/kg) and (+)-verapami1 (10 mg/kg), i t i s apparent that the order of potency ( - > * > + ) corresponds with that + expected i f calcium antagonism alone, rather than Na channel blockade alone, or a combination of the 2 propert ies was responsible for the a n t i -arrhythmic actions observed. In summary, the antiarrhythmic actions of the opt ica l enantiomers of verapamil during myocardial ischaemia corresponded with the i r calcium antagonist potency ra t ios in iso la ted vent r ic les under condit ions of ra ised K + . In addi t ion, (^-verapami l was found to d is t r ibu te into the ischaemic t i ssue , and appeared to accumulate there. It i s not possible to estimate the proportion of drug ava i lab le at th is s i t e for ef fects (much may have been bound 1 non -spec i f i ca l l y ' to the t i s sue ) . However, the current exper i -ments did es tab l ish the presence of (+)-verapami1 in the ischaemic t i s sue . Therefore, calcium antagonism in the ischaemic vent r icu la r myocardium appears to be the most l i k e l y explanation for the antiarrhythmic actions of (ij -verapami l in acute myocardial ischaemia. However, a l te rnat ive p o s s i b i l -i t i e s e x i s t , and these are discussed in subsequent chapters. 4.3.1.4 Felodip ine, Ni fedipine and DHM9. Ni fedipine is the proto-type 1,4-dihydropyridine calcium antagonist (Fleckenstein et a l ; , 1972; Kohlhardt and F leckenste in , 1977). Nifedipine inh ib i t s i . in vent r icu la r muscle (Kohlhardt and Fleckenste in, 1977) in a manner which is essen t i a l l y - 279 -independent of st imulat ion frequency according to most (Bayer et a l . , 1977; Hachisu and Pappano, 1983) but not a l l (Woods and West, 1983) s tud ies. In th is regard, n i fedip ine d i f fe rs from ( ^ -ve rapami l , which shows marked frequency-dependence (Sanguinetti and West, 1982; Hachisu and Pappano, 1983) . In add i t ion , n i fed ip ine does not appear to a f fect the inward sodium current in vent r icu lar t issue (Bayer et a l . , 1977), unl ike (^-verapami 1 which can block th is current at high concentrations in v i t r o (Bayer et a l ; , 1975a; Nawrath et a l . , 1981). DHM9 is a metabolite of an analogue of n i fed ip ine (n icardip ine) which, in contrast with both phenethylalkylamines such as verapamil and 1,4-dihy-dropyridines such as n i fed ip ine , appears to i nh i b i t i ^-dependent events se lec t i ve l y in vent r icu lar muscle versus vascular smooth muscle (Clark et a l ; , 1984b), although i t is in fact less potent than both n i fed ip ine and verapamil in vent r icu lar t i s sue . Felodipine i s a 1,4-dihydropyridine vasodi lator with st ructura l s i m i l a r -i t y to n i fed ip ine . Felodipine interacts with calmodulin (Bostrom et a l . , 1984) , and has been reported to increase calcium uptake by the sarcoplasmic ret iculum (Movsesian et a l . , 1984). However, even at high concentrations (0.8 x 10~^M), fe lod ip ine does not i nh ib i t calmodulin-dependent calcium-stimulated contract ion in chemically skinned rabbi t renal ar ter ies (Kreye et a l . , 1983). It appears from the above that fe lod ip ine d i la tes smooth muscle v ia an action on the plasma membrane. Owing to the close s t ructura l s i m i l a r i t i e s between fe lod ip ine and 1,4-dihydropyridines such as n i fed ip ine , n i so ld ip ine , n i lud ip ine and PY-108-068, i t seems l i k e l y that the major pharmacological actions of fe lod ip ine , which are shared with other 1 ,4 -d i -hydropyridines, occur as a resu l t of calcium antagonism. In th is regard, fe lod ip ine has been shown to possess calcium antagonist a c t i v i t y in rat aor t i c s t r i p s , portal ve ins , a t r i a and vent r ic les (Au and Sut ter , 1984). In - 280 -ven t r i c l es , and K + depolarized vesse ls , fe lod ip ine resembled n i fed ip ine more than (^-verapami l (Au and Sut ter , 1984). In view of the above, the ef fects of f e lod ip ine , n i fed ip ine and DHM9 were evaluated in conscious rats subjected to coronary artery occ lus ion , in order to test the hypothesis that calcium antagonism in vent r icu la r muscle i s responsible for the antiarrhythmic actions of calcium antagonists. None of these drugs would be expected to i nh ib i t i . in vent r icu lar t issue in vivo at doses producing moderate reductions in blood pressure, owing to the i r vascular s e l e c t i v i t y (n i fedip ine and fe lodip ine) or low potency (DHM9). In the e a r l i e r study (Curt is et a l . , 1984) with (±)-verapami1, i t was found that by de l ibera te ly producing a smaller area of ischaemia, the a n t i -arrhythmic actions of high doses of (±)-verapami 1 were more c lea r l y demon-s t ra ted, since fewer animals died from cardiogenic shock. Since 1,4-dihy-dropyridines are even more se lec t i ve systemic vasodi lators than ( ^ - ve rapa -mil (Van Zwieten and Timmermans, 1983; Briscoe and Smith, 1982), the tech-nique of production of large and small OZs was used again in order to of fset possible cardiogenic shock of fe lod ip ine . Since cardiogenic shock was not, in fac t , found to be a problem with fe lod ip ine , the subsequent studies with n i fed ip ine and DHM9 were car r ied out using large OZ rats only. Fe lod ip ine, at doses which produced profound hypotension in conscious ra t s , possessed only weak antiarrhythmic actions fo l lowing coronary occ lu -s ion . These actions were r ea l l y only apparent in rats with small OZs (SOZ). Our resu l ts are not en t i re l y in agreement with those of Verdouw and Wolffenbuttel (1983), who showed that 10 nmol/kg fe lod ip ine abolished VF during the 10 min period immediately fol lowing coronary occlusion in p igs. However, these Authors found that af ter a 20 min period of reper fus ion, a second 10 min occlusion produced fa ta l VF in 5/9 p igs , which was not d i f f e r -ent from the control incidence. The s ize of the 0Z was not determined in - 281 -the above study (Verdouw and Wolf fenbuttel , 1983); since we demonstrated in the present study a small a n t i f i b r i l l a t o r y action of fe lod ip ine in rats with small OZs, the di f ference between t h i s , and the present study may re la te to the s ize of the ischaemic area produced. An a l ternat ive explanat ion, also re la t ing to the extent of ischaemia, is provided by the observation that fe lod ip ine markedly elevates blood flow in the border of the ischaemic zone in pigs (Sjoquist et a l . , 1983). In the l a t t e r study (Sjoquist et a l . , 1983) the extent of subsequent in fa rc t ion was unfortunately not determined; an increase in blood flow to the border of the ischaemic t issue implies a potent ial degree of salvage. In the present study we were able to demons-t rate a small degree of myocardial salvage with fe lopine (reduction in IZ as a percentage of OZ). However, th is action was not dose-related and only occurred in one group of small OZ r a t s . In add i t ion , since r a t s , l i k e p igs , do not possess a well developed co l l a te ra l c i r cu la t i on (Johns and Olson, 1984; Selye et a l . , 1960; Schaper 1983; Hearse 1983; Verdouw et a l ; , 1983c; Maxwell e t - a l . , 1984; Winkler e t - a l . , 1984) i t seems un l i ke ly that an i n -crease in blood flow to the ischaemic t issue could produce a su f f i c i en t reduction in the s ize of the ischaemic area to great ly reduce arrhythmias. The present work has secondary impl ica t ions, in that secondary proper-t i es of fe lod ip ine such as i t s a b i l i t y to reduce af ter load (Verdouw et a l . , 1983a), to bind with calmodulin (Bostrum et a l . , 1981), to i nh ib i t calmodu-1 in -sens i t i ve phosphodiesterase a c t i v i t y (Norman et a l . , 1983), to in ter fere with calcium uptake by the sarcoplasmic ret iculum (Movsesian e t - a l . , 1984) and to inf luence calcium-ATPase (Wang et a l . , 1984), which may or may not occur over the dose range studied here, do not appear to confer ant iar rhyth-mic a c t i v i t y fo l lowing occlusion-induced myocardial ischaemia in conscious r a t s . Ni fedipine and DHM9 were both without antiarrhythmic a c t i v i t y . In the - 282 -case of DHM9, th is was not unexpected, since th is drug was without pharmac-o log ica l a c t i v i t y of any k ind, both in conscious rats and in iso la ted per-_5 fused rat vent r ic les at up to 3 x 10 M. Nifedipine produced dose-depen-dent reductions in blood pressure (as did anipamil , (+)- and (-)-verapamil in the previously-descr ibed s tud ies ) , but increased heart rate and shortened P-R i n t e r v a l , ef fects presumably mediated by sympathetic re f l exes . These resul ts show that the 1,4-dihydropyridines did not appear to have any d i rec t ef fects on the electrophysiology of the heart. In summary, the 1,4-dihydropyridine calcium antagonists did not reduce occlusion-induced arrhythmias, nor did they prolong P-R i n t e r v a l , cause a t r ioven t r i cu la r block or slow heart ra te . These actions contrast with the actions of (+)- and (-)-verapami1 and anipamil (the s ign i f icance of which is discussed below). 4.3.2 Role of calcium antagonism in the myocardium By integrat ing the resu l ts of the antiarrhythmic studies with the pharm-acological p r o f i l e of the 1,4-dihydropyridine and phenethylalkylamine c a l -cium antagonists, i t i s possible to suggest explanat ions, both for the di f ference in antiarrhythmic a c t i v i t y of the two classes of drugs, and also for the s i t e of action of the phenethylalkylamines. Phenethylalkylamine calcium antagonists (anipamil and the opt ica l enantiomers of verapamil) were found to possess antiarrhythmic a c t i v i t y in the present s tud ies. These resu l ts are consistent with previous f indings from our laboratory concerning the antiarrhythmic a c t i v i t y of (±)-verapami1, (Johnston et a l . , 1983a; Curt is e t - a l ; , 1984). Ronipamil (a close s t ructura l analogue of verapamil and anipamil without calcium antagonist ac t i v i t y ) had no antiarrhythmic a c t i v i t y . (^-Verapamil may have antiarrhythmic a c t i v i t y in anaesthetised rats (Fagbemi et a l . , 1984), and has been shown to possess marked a n t i f i b -r i l l a t o r y a c t i v i t y in dogs (Kaumann and Aramendia, 1968; Bren e t - a l . , 1982) - 283 -and pigs (Kro l l and Knight, 1984; Bergey et a l . , 1984) subjected to coronary artery occ lus ion. It has also been suggested that (-)-verapami 1 is more potent than (+)-verapamil as an antiarrhythmic in anaesthetised dogs (Kaumann and Serur, 1975). With regard to 1,4-dihydropyridine calcium antagonists, n i fed ip ine , n iso ld ip ine and n i lud ip ine have been reported to abol ish VF in rats (Fagbemi and Par ra t t , 1981) but not pigs (Bergey et a l . , 1984) fo l lowing coronary artery occ lus ion. Dihydropyridine calcium antagonists, un t i l now, have not been evaluated in conscious rats fo l lowing coronary occ lus ion, and fe lod ip -ine has not been tested in conscious or anaesthetised ra t s . Felodipine and n i fed ip ine were evaluated for antiarrhythmic a c t i v i t y in order to assess whether r e l a t i v e l y vascular se lec t ive calcium antagonists possess ant ia r -rhythmic a c t i v i t y in acute myocardial ischaemia in conscious ra t s . DHM9 was studied on the basis of i t s unusual property of myocardial s e l e c t i v i t y compared with other 1,4-dihydropyridines; however, the resu l ts suggest that DHM9 is in fac t completely inact ive as a calcium antagonist, at concentra-t ions < 30 pM. The lack of antiarrhythmic action of fe lod ip ine , n i fed ip ine , ronipamil and DHM9 compared with anipamil and the verapamil enantiomers was predicted from the hypothesis that calcium antagonists reduce ischaemia-induced a r -rhythmias v ia an action in the ven t r i c l es . If the actions of the 1,4-dihy-dropyridine calcium antagonists are compared with those of the phenethyl-alkylamines, some notable d i f ferences, which exemplify the above statement, may be seen. F i r s t l y , the antiarrhythmic phenethylalkylamines enhanced the f a l l in blood pressure produced by occ lus ion , whereas the 1,4-dihydropyridines did not. Secondly, the 1,4-dihydropyridines, unl ike high doses of (^J-verapami1 (Curt is et a l . , 1984) did not increase the number of deaths at t r ibutab le to - 284 -cardiogenic shock. Th i rd ly , a l l the antiarrhythmic phenethylalkylamines reduced pre-occlusion heart rate at doses which subsequently reduced ar -rhythmias, whereas the 1,4-dihydropyridines a l l increased heart ra te . F i n a l l y , (+)- and (-)-verapamil caused dose-dependent P-R prolongation at doses which reduced arrhythmias, whereas n i fedip ine caused dose-dependent shortening of P-R i n t e r v a l . Therefore, anipamil and the verapamil enantio-mers produced de f in i te actions a t t r ibutab le to d i rec t ef fects in the heart , and reduced arrhythmias, whereas the 1,4-dihydropyridines appeared to have no d i rec t actions in the heart , and did not reduce arrhythmias. I t is c lear that 1,4-dihydropyridine calcium antagonists possess a greater s e l e c t i v i t y for the systemic vasculature versus the vent r icu la r myocardium than phenethylalkylamines. Several invest igators have d i r ec t l y compared the potencies of n i fed ip ine and (±)-verapamil in vascular smooth muscle and cardiac t issue (Fleckenstein-Grun, et al • , 1976; Raschach, 1976a; Nabata, 1976; Briscoe and Smith, 1982; Winslow et a l . , 1983; M i l l a rd et a l . , 1983; Lee e t - a l . , 1983; Clarke e t - a l . ; 1984a; 1985; Kenakin and Beek, 1985; Nakayama et a l . , 1985). By comparing these studies i t i s possible to c a l -culate mean potency dif ferences in vascular and cardiac t issue for n i f ed ip -ine and (±)-verapami 1. I t was calculated that n i fed ip ine is approximately 43 times as potent as (^-verapami l as a calcium antagonist in vascular smooth muscle, whereas n i fed ip ine is only 10 times as potent as ( ^ - ve rapa -mil in cardiac t i ssue . According to the hypothesis that calcium antagonists reduce ischaemia-induced arrhythmias v ia calcium antagonism in the vent-r i c l e s , the fo l lowing predict ions are made on the basis of the calculated potency r a t i o s . F i r s t l y , i t was found previously that the antiarrhythmic E D 5 Q for (^)-verapamil was 6 mg/kg (Curt is et a l ; , 1984). Therefore, the antiarrhythmic E D ^ Q for n i fed ip ine should be 0.1 times th is dose: 0.6 mg/kg. However, in terms of actions in vascular smooth muscle, 0.6 mg/kg - 285 -n i fed ip ine is equivalent to 0.6 x 43 = 26 mg/kg (±)-verapami 1. This dose, 26 mg/kg of (±)-verapami1 cannot be to lerated in conscious rats fol lowing coronary occ lus ion. Indeed, in the e a r l i e r study (Curt is et a l . , 1984) 20 mg/kg of (^J-verapami 1 was found to cause cardiogenic shock in 6/9 rats wi th in 30 min of occ lus ion. Therefore these ca lcu la t ions predict that n i fed ip ine should not be capable of reducing arrhythmias i n -v i vo , owing to i t s s e l e c t i v i t y for vascular smooth muscle. These ca lcu la t ions were based on the assumption that n i fed ip ine and (^-verapami l are pharmacologically equivalent. This i s probably not the case; di f ferences e x i s t , for example, in frequency-dependence (see below). Differences in pharmacokinetics are also apparent. The higher degree of plasma-protein binding with n i fed ip ine (Hermann and M o r s e l l i , 1985) compared with (±)-verapamil would be expected to reduce the apparent potency of n i fed ip ine re l a t i ve to (^-verapami l in -v ivo. These factors may account for the fact that doses exceeding 0.6 mg/kg n i fed ip ine could be given to con-scious rats without adverse (or antiarrhythmic) e f f ec t . If fe lod ip ine resembles n i fed ip ine in terms of calcium antagonism and re la t i ve t issue s e l e c t i v i t y , then i t i s c lear why fe lod ip ine , a l so , had l i t t l e antiarrhythmic a c t i v i t y ; su f f i c i en t concentrations of fe lod ip ine to af fect calcium currents in the vent r ic les were probably not achieved. In common with n i fed ip ine , doses of fe lod ip ine su f f i c i en t to af fect the vent-r i c u l a r myocardium probably great ly exceed those given in the present s tud ies . There are addit ional factors to consider with regard to the di f ference between 1,4-dihydropyridines and phenethylalkylamines as ant iarrhythmics. Of par t i cu la r in terest i s frequency-dependence. Nifedipine has been repor-ted to possess l i t t l e (Woods and West, 1983; 1985) i f any (Kohlhardt and Fleckenste in, 1977; Hachisu and Pappano, 1983) frequency-dependence in - 286 -cardiac t i ssues . However, phenethylalkylamine calcium antagonists possess marked frequency-dependence (Ehara and Kaufmann, 1978; McDonald et a l . , 1980; Sanguinetti and West, 1982; Woods and West, 1985). If phenethyla lky l -amines possesses pronounced frequency-dependence such that calcium antago-n is t a c t i v i t y i s potentiated at fas t heart ra tes , but 1,4-dihydropyridines do not, th is may par t ly account for the re l a t i ve lack of a n t i f i b r i l l a t o r y a c t i v i t y of 1,4-dihydropyridines versus phenethylalkylamines, as has been suggested previously (Woods and West, 1985). With regard to the re la t ionsh ip between frequency-dependence and a n t i -arrhythmic a c t i v i t y , i t i s worthwhile to consider the possible mechanisms by which frequency-dependence operates. Frequency-dependence in heart t issues can refer to actions on i . and actions on force of contract ion. I t i s not c lear to what extent frequency-dependent reductions in i .. (McDonald e t - a l . , 1980) corre la te with f r e -quency-dependent inh ib i t ions of inotropism (Mannhold et a l . , 1981). How-ever, i t i s c lear that frequency-dependent negative inotropic actions of 2+ phenethylalkylamines are not due to depletion of i n t r ace l l u l a r Ca or non-speci f ic myocyte fat igue (Bayer and Ehara, 1979) since the ef fects of a l tera t ions in st imulat ion frequency are rap id ly revers ib le on resumption of control st imulat ion frequency. In attempting to understand the mechanism of frequency-dependence, i t i s important to consider the phenomenon of voltage-dependence (Weidmann, 1955a). It i s possible that frequency-dependence and voltage-dependence are manifestations of the same phenomenon, namely an ef fect of depolar isat ion to a l te r the apparant a f f i n i t y of a drug for i t s receptor. This may occur v ia an increase in the probab i l i t y of the i' s i- (or i N ) channel being in the open or inact ivated state (Lee and Ts ien , 1983), whereby the a f f i n i t y of cer ta in drugs for the channel increases (Hondeghem and Katzung, 1984). This - 287 -i s the 'modulated receptor hypothesis' which predicts that the a f f i n i t y of a drug for the receptor v ia which i t in ter feres with channel function is voltage-dependent. However, the modulated receptor hypothesis does not adequately account for the observation that a drug which exhib i ts vol tage-dependent behaviour may not necessar i ly exhib i t frequency-dependent beha-v iour . Although most studies suggest that 1,4-dihydropyridines do not possess marked frequency-dependent behaviour, (Hachisu and Pappano, 1983; Kohlhardt and Fleckenste in, 1977), i t has recent ly been shown that n i fed ip ine shows marked voltage-dependent behaviour, i nh ib i t i ng i's1- with an I C ^ Q of 20 nM at -40 mV holding po ten t ia l , and an I C 5 Q of 700 nM at -50 mV, and producing negative inotropism in vent r icu la r t issue with an I C ^ g of 480 nM at 5.9 meq/1 K + and an I C 5 0 of 0.63 nM at 37 meq/1 K + (Hoick and Osterr ieder, 1985). An a l ternat ive hypothesis, ' the guarded receptor hypothesis' of Starmer et a l . (1984) suggests that the a f f i n i t y of a channel-modulating drug for i t s receptor is constant, whereas access to the receptor is voltage-depen-dent and determined by the gating mechanism of the channel. The l a t t e r hypothesis appears to be f lawed, however, because i t predicts that the rate of change of apparant a f f i n i t y of a drug for i t s receptor is independent of the drug and the receptor, and is constant. The fact that (^O-verapami 1 exhib i ts frequency- and voltage-dependence but n i fedip ine only exh ib i ts voltage-dependence appears to disprove the guarded receptor hypothesis. The di f ference between the resu l ts of frequency-dependence and vol tage-dependence studies with n i fed ip ine may be explained by invoking a modulating inf luence which r e s t r i c t s the access of n i fed ip ine to i t s receptor in or adjacent to the i . channel, but which i s independent of the gating of the channel. I f sustained depolar isat ion removes th is modulating in f luence, whereas br ie f depolar isat ion does not, then the di f ference between the - 288 -ef fects of changing frequency compared with changing res t ing membrane poten-t i a l on responses to n i fed ip ine can be explained. This explanation i s contingent on the presence of separate receptors for 1,4-dihydropyridines and phenethylalkylamines. It i s possible to speculate that the modulating inf luence may simply represent a voltage-dependent in teract ion between the drug and i t s receptor, whereby depolar isat ion causes the a f f i n i t y of the receptor for the l igand to increase, but s lowly. This hypothesis side-steps the modulated receptor hypothesis in that voltage d i r ec t l y modulates the drug receptor, independently of whether the channel i s res ted, open or inac t iva ted. If verapamil and n i fed ip ine in teract with d i f ferent receptors within or in associat ion with the i . channel, then the most simple expla-si nation for the dif ference between n i fed ip ine and verapamil in the i r f r e -quency- and voltage-dependent propert ies is that the a f f i n i t y of the verapa-mil receptor for verapamil changes rap id ly (time constant of msec) with depo lar isa t ion , whereas the a f f i n i t y of n i fed ip ine for i t s receptor changes slowly (time constant of sec) with depolar isa t ion. The presence of d i f f e r -ent receptors for verapamil and n i fed ip ine is supported by extensive rad io -l igand binding studies (Glossman et a l . , 1985) and some pharmacological evidence (Spedding and Berg, 1984). There is a l imi ted amount of evidence which suggests that the frequency-dependence of verapamil is s te reose lec t ive . Bayer and Ehara (1979) found that whereas the negative inotropic ef fects of (-)-verapamil were enhanced by increasing st imulat ion frequency from 6 to 60 per min, the negative inotropic e f fects of (+)-verapami 1 were i nh ib i t ed . Although i t is d i f f i c u l t to suggest an explanation for th is which is consistent with a l l that is known concerning the pharmacology of the enantiomers, th is observation may par t ly explain the resul ts of the present studies with the enantiomers in + i so la ted ven t r i c l es , since ra i s ing K did not increase the negative ino-- 289 -t rop ic potency of (+)-verapamil to the same extent as i t did for (-)-verapa-m i l . However, ra is ing K + concentration in the present experiments did not ac tua l l y reverse the ef fects of (+)-verapami1, therefore i t would be prema-ture to suggest that the resu l ts of the present experiments are en t i re l y consistent with stereoselect ive frequency- or voltage-dependence, although the p o s s i b i l i t y cannot be ruled out. With regard to the hypothetical voltage-dependent modulating in f luence, the existence of such an ent i ty may explain the di f ference between the K a for dihydropyridines determined by measuring a physio logica l response such as vent r icu la r muscle contraction at d i f fe rent st imulat ion frequencies -7 -9 (general ly between 10 and 10~ M, see Nayler and Horowitz, 1983) and the KQ according to radiol igand binding studies (general ly no more than —9 10 M, see Glossman et a l . , 1982), since binding studies are often carr ied out using t issue depolarised in high K + . At present the mechanism of voltage-dependent in teract ions between drugs and ion channels i s uncertain. This discussion i s somewhat speculat ive, however i t may bear some re la t ion to the discussion of the ef fects of K + changes on the actions of calcium antagonists (see below). In summary, 1,4-dihydropyridine calcium antagonists were ine f fec t i ve as antiarrhythmic agents, probably because concentrations su f f i c i en t to produce calcium antagonism in the heart were not reached, despite evidence of sys -temic vasod i la ta t ion . In contrast , phenethylalkylamines were e f fec t i ve antiarrhythmic agents at doses which produced ef fects consistent with c a l -cium antagonism in the heart. 4 .3.3 Role of calcium antagonism in the ischaemic ven t r i c le The studies with the verapamil enantiomers, n i fed ip ine and DHM9 in iso la ted vent r ic les under condit ions of varied K + concentration were designed to examine the p o s s i b i l i t y that some calcium antagonists may - 290 -possess a s e l e c t i v i t y of action for ischaemic versus non-ischaemic t i ssue , by v i r tue of the di f ferences in ex t race l lu la r K + between ischaemic and non-ischaemic t issue (H i l l and Gettes, 1980; Hirche e t - a l . , 1980). Such a s i t e - s e l e c t i v i t y of action may account in part for di f ferences in an t ia r -rhythmic a c t i v i t y of d i f ferent calcium antagonists. The potency ra t io of ( - ) - to (+)-verapamil determined in iso la ted ven-t r i c l e s was highly dependent on the concentration of K + in the perfusing buf fer . The mechanism for th is e f fect is unclear. Elevat ion of K + from 5.4 to 8 meq/1 has been previously shown to produce only a small depo lar i -sation of res t ing membrane potent ial in vent r icu lar t issue (Buchanan et a l . , 1985) of i nsu f f i c i en t magnitude to af fect steady state act ivat ion and inac t -ivat ion of i .. (Reuter, 1979). Therefore the K + ion appeared to a l t e r the potency of the (^-verapami 1 enantiomers independently of gat ing. However, i t may be the case that the small depolar isat ions which are pred ic -ted to have occurred with raised K + were responsible for increases in the a f f i n i t y of the verapamil receptor for the enantiomers, in accordance with the proposed mechanism of frequency- and voltage-dependence discussed above. Experiments are current ly being car r ied out in the laboratory to examine whether the potency changes associated with K + concentration changes corre la te with changes in rest ing membrane po ten t ia l , by construc-t ing isobolograms. Prel iminary information suggests that th is may be the case, although the re la t ionsh ip between vent r icu la r e x c i t a b i l i t y and K + concentration in the present experiments did not appear to be ident ica l with the re la t ionsh ip between K + concentration and negative inotropic potency of the enantiomers of verapamil, suggesting that the re la t ionsh ip between rest ing membrane potent ial and the a f f i n i t y of verapamil for i t s receptor may be complex. The ef fect of elevat ions in K + to reduce the ra t i o of potency between - 291 -( - ) - and (+)-verapamil (from 21 to 8) may have important impl icat ions con-cerning the s i t e of action of antiarrhythmic phenethylalkylamines, since in ra ised K + , the potency ra t io was c loser to the antiarrhythmic potency ra t i o (4) during myocardial ischaemia. In th i s regard, i t i s important to consider that wi thin 5 - 1 0 min of coronary occ lus ion , ex t race l lu la r K + r i ses to 8 - 15 meq/1 in the ischaemic t issue ( H i l l and Gettes, 1980; Hirche et a l . , 1980). The present in v i t r o resu l ts pred ic t , therefore, that in ischaemic vent r icu lar t i ssue , the calcium antagonist potency ra t i o of (-)- to ( + )-verapami 1 should be 8 or l e s s . It fo l lows from th is that the antiarrhythmic potency ra t io of (-)- to ( + )-verapami 1 should be no more than 8, i f these phenethylamines reduce ischaemia-induced arrhythmias v ia calcium antagonism. In addi t ion, the calcium antagonist potency ra t i o in vivo may be even less than 8 in the ischaemic myocardium, in terms of dose, i f phar-macokinetic factors are considered (see above). In human t issue the potency ra t io of the enantiomers in vivo for prolonging P-R i n t e r v a l , in terms of unbound plasma concentration (Echizen et a l . , 1985), and the negative ino-t rop ic potency ra t io in v i t ro (Ferry et a l . , 1985) have been reported to be 4.5 and 8 in favour of (-)-verapami1, respec t ive ly . These values correspond almost exact ly with the antiarrhythmic and in v i t r o negative inotropic potency ra t ios in ra ised K + , respec t ive ly , reported here. Consideration of the antiarrhythmic potency ra t io of the verapamil enantiomers, and the i r ef fects on iso la ted rat ven t r i c les in the presence of ra ised K + concentrations suggests an explanation for the s i t e of action of the enantiomers, and explains how i . might be abolished by the enantio-mers in the ischaemic t issue while i • in the non-ischaemic t issue remains r e l a t i v e l y unaffected. Since the enantiomers of verapamil c lea r l y did not abol ish conduction in the non-ischaemic vent r icu lar t issue in v ivo , a se lec -t i ve act ion in the ischaemic t issue leading to a reduction in arrhythmias i s - 292 -an obvious hypothesis. The high ex t race l lu la r K + concentration seen in acutely ischaemic t issue is therefore a candidate for mediation of such s e l e c t i v i t y of ac t ion , provided that the actions of ( i j -verapami l on ar-rhythmogenesis during ischaemia depend on K + in a s im i la r manner to the actions of (±)-verapami1 on the force of contract ion in iso la ted ven t r i c l es . It i s of in terest to note that the ef fect of K + on the a c t i v i t y of n i fed ip ine was quite d i f ferent from that of (-)- and (+)-verapami1. In-creasing K + from 3 to 10 meq/1 increased the potency of n i fed ip ine only 4 f o l d . Therefore, K + did not confer s i t e - s e l e c t i v i t y of action on n i fed ip -ine to the same extent that was seen in the case of the verapamil enantio-mers. Qua l i t a t i ve l y , therefore, calcium antagonists which show great poten-cy changes with small changes in K + concentration (verapamil enantiomers) possess potent antiarrhythmic a c t i v i t y in acute myocardial ischaemia, where-as calcium antagonists without such a property (n i fedip ine) have l i t t l e or no antiarrhythmic a c t i v i t y . I t w i l l be of in terest to examine whether th is pattern is also a feature of other calcium antagonists. The spontaneous occurrence of PVC, and inh ib i t i on of such by the verapa-mil enantiomers in iso lated vent r ic les were 2 phenomena which were In-dependent. To our knowledge, such PVC have not been previously inves t iga-ted. (-)-Verapami1 was more potent than (+)-verapamil in reducing these PVC, in concordance with reductions of ischaemia-induced arrhythmias. I t is poss ib le , therefore, that these PVC share a common mechanism with ischaemia-induced PVC. However i t i s premature to reach such a conclusion at pre-sent. In the study of n i fedip ine and DHM9 in iso la ted vent r i c les the con-t ro l incidence of such PVC was much lower than that in the verapamil enant i -omer study, and therefore i t was not possible to state whether n i fed ip ine reduced these arrhythmias. - 293 -4.3.4 Role of other pharmacological propert ies Although the present resu l ts support the hypothesis that the an t ia r -rhythmic action of calcium antagonists occurs as a resu l t of calcium antago-nism, other explanations should be considered. Phenethylalkylamine and 1,4-dihydropyridine calcium antagonists possesses, in addit ion to the i r i blocking propert ies (Fleckenstein et a l . , 1969; Kohlhardt and Flecken-s t e i n , 1977; e t c . ) , numerous other actions (see Nayler and Horowitz, 1983). I t may be possible to hypothesize that some or a l l of these actions c o n t r i -bute to the antiarrhythmic actions described in the present experiments. However, close examination of the data reveals that most of these non-spec i f i c actions occur at concentrations greater than those required for i nh ib i t i on of i • (see below, and Nayler and Horowitz, 1983). 4 .3.4.1 Inh ib i t ion of the fas t inward current . (+)-, (*)- And (-)-verapami1 i nh ib i t i ^ a in vent r icu lar t issue i n - v i t r o (Bayer et a l . , 1975a; 1975b; 1975c), but only at 100-150 times the concentration required to i nh ib i t i • (Nawrath et a l . , 1981). Therefore, i t would seem un l ike ly that such actions contribute to the antiarrhythmic actions of (^J-verapamil , since doses e f fec t i ve in i nh ib i t i ng i^ would produce complete i nh ib i t i on of i $ 1 - , leading to a t r ioven t r i cu la r d issoc ia t ion and almost complete uncoupling of exc i ta t ion and contract ion in the heart and vascular smooth muscle. In the present s tud ies , and in antiarrhythmic studies car r ied out by other invest igators (Kaumann and Aramendia, 1968; Kaumann and Serur, 1975; K ro l l and Knight, 1984; e tc . ) th is was c l ea r l y not the case. Although i t i s general ly bel ieved that 1,4-dihydropyridines do not a f fect i N a ( e . g . , Kohlhardt and Fleckenste in, 1977), i t was recent ly found that n i t rendip ine blocked i ^ a in neonatal rat heart c e l l s (Yatani and Brown, 1985). However, as in the case of (^-verapami l (Nawrath et a l . , 1981), th is e f fect only occurred with concentrations great ly in excess of - 294 -those required to abolish i . . Therefore, i f i t were possible to admini-s ter massive doses of 1,4-dihydropyridines su f f i c i en t to i nh i b i t ischaemia-induced arrhythmias, inh ib i t i on of i ^ a could not account for such an ac t ion . Our laboratory previously found that quinidine at doses reducing ischae-mia-induced arrhythmias by 50 % (20 mg/kg) protected against e l e c t r i c a l l y induced arrhythmias, and increased maximum fol lowing frequency and QRS interval (Curt is et a l . , 1984). In the present s tud ies , neither ( + )- nor (-)-verapamil affected these var iables at doses which protected against ischaemia-induced arrhythmias. In add i t ion , Raschack (1976) showed that in anaesthetised ra t s , (+)-verapamil reduced aconit ine-induced arrhythmias, whereas high doses of (-)-verapamil (which caused second degree a t r i o -vent r icu lar block) were i ne f fec t i ve . This suggests that plasma concentra-t ions of (-)-verapamil which are e f fec t i ve in reducing the i ^ a (and asso-ciated aconit ine-induced arrhythmias) cannot be reached in v i vo . In summary, i t i s extremely un l i ke l y , i f not impossible, for n'N a blockade to be a contr ibutary factor in the antiarrhythmic a c t i v i t y of 1,4-dihydropyridine and phenethylalkylamine calcium antagonists. 4.3.4.2 Blockade of g -adrenoceptors. There have been suggestions that many calcium antagonists i nh ib i t responses to a-adrenoceptor agonists. However, current evidence does not support the suggestion (Motulsky et a l . , 1983) that a-adrenoceptor antagonism contributes to the antiarrhythmic a c t i v i t y of calcium antagonists, for the fo l lowing reasons. F i r s t l y , with regard to the antiarrhythmic a c t i v i t y of ( + )- and ( - ^ v e r -apamil , i t i s unclear whether (^-verapami l t ru l y inf luences a d r e n o c e p -to rs . While i t has been claimed that (±)-verapamil is a se lec t i ve antagon-i s t of a 2 -adrenoceptors, having l i t t l e e f fect on a-^ responses (Timmer-mans et a l . - , 1983; Cavero et a l . , 1983; Haeusler, 1985), i t has also been - 295 -claimed that the opposite i s the case (Vanhoutte 1982; Hoick and Gerrold, 1986). Proponents of the o^ - se lec t i ve hypothesis suggest that the antago-n i s t act ion of (^-verapami l is non-competit ive, and report that ( ^ - ve rapa-mil has almost neg l ig ib le a f f i n i t y for the receptors themselves (Van Meel et a l . , 1981). It was also suggested that while (^-verapami l i n h i b i t -ed a-adrenoceptor agonist-induced inotropic responses in a t r i a , the ef fect was not mediated by a-adrenoceptor antagonism (Tung et a l . , 1985). Secondly, i t i s doubtful whether a-adrenoceptor ac t iva t ion is of patho l -ogical importance in arrhythmogenesis during acute myocardial ischaemia. In non-ischaemic heart t i s sue , pharmacological and e lect rophys io log ica l studies revealed that a-adrenoceptor agonism is mediated essen t i a l l y by the a^ subtype and is expressed as a prolongation of act ion potent ia l duration (Dukes and Vaughan Will iams 1984; Black et a l • , 1985). I t was argued that , with regard to reentry, th is e lect rophys io log ica l e f fec t should be an t ia r -rhythmic rather than arrhythmogenic (Dukes and Vaughan Wil l iams 1984) owing to the dependence of reentry on the presence of a r e l a t i v e l y narrow act ion potent ia l and abbreviated ref ractor iness (Mines 1913). In add i t ion , in vent r icu lar muscle the ef fects of a-adrenoceptor agonism have been shown to be extremely sma l l ; maximum pos i t ive inotropic e f fec ts of phenylephrine were only 15% of the maximum ef fects of isoprenal ine, and the changes in act ion potent ia l conf igurat ion caused by a-adrenoceptor agonists were not s i g n i f i c -ant (Nawrath and Rupp, 1986). Studies with perfused rat hearts have shown that (-)-verapami1 and n i fed ip ine are equipotent in the i r a b i l i t y to i n h i b i t adrenaline-induced f a l l s in VF threshold (Higginson e t - a l , 1983). I t i s d i f f i c u l t to reconc i le data such as th is with the resu l ts of the present studies and, at the same time, hypothesize an involvement of cardiac a -adre-noceptors in arrhythmogenesis. Other studies in v i t r o have shown that ( - ) - but not (+)-verapamil i nh ib i t s release of noradrenaline from the heart - 296 -caused by global ischaemia (Nayler and Sturrock, 1983). However, t h i s e f fec t of (-)-verapamil was associated with a bel l-shaped dose-response curve, whereas the antiarrhythmic dose-response curve generated by the present studies (Figure 25) appeared to be a saturat ing sigmoidal funct ion. Th i rd ly , studies of the ef fect of a-adrenoceptor agonism and antagonism during myocardial ischaemia have produced con f l i c t i ng r esu l t s . a-Adrenocep-tor antagonism was reported to reduce arrhythmias in cats (Sheridan et al 1980), but ident ica l treatment was ine f fec t ive in dogs (Bo l l i et a l . , 1984). In add i t ion , the reported antiarrhythmic actions of a-adrenoceptor antagonists in iso la ted ischaemic rat hearts is not a t t r ibu tab le to a -adren-oceptor antagonism (Daugherty and Woodward, 1982; Daugherty et a l ; , 1986), rather to 'membrane s t a b i l i s i n g ' e f fects (Bralet et a l . , 1985). Our labora-tory has shown that neither combined a- and B-adrenoceptor blockade nor sympathectomy reduce arrhythmias in rats (Botting et a l . , 1983). In addi -t i o n , the present studies showed that graded ablat ions in the CNS and cate-cholamine infusions did not inf luence arrhythmias in a manner consistent with a ro le for a- (or B-) adrenoceptors in arrhythmogenesis during acute myocardial ischaemia. In summary, i t appears to be highly improbable that (+)- or (-)-verapa-mil reduced ischaemia-induced arrhythmias in conscious rats v ia myocardial a-adrenoceptor antagonism. 4.3.4.3 Indirect- ac t ions . It may be suggested that the verapamil enantiomers exerted the i r antiarrhythmic actions i nd i r ec t l y by reducing af ter load or heart ra te . However, th is may be ruled out for the fo l lowing reasons. F i r s t l y , our laboratory has shown that there i s no cor re la t ion between e i ther blood pressure or heart rate and arrhythmias in conscious rats (John-ston e t - a l . , 1983a). - 297 -Secondly, the antiarrhythmic ef fects of the calcium antagonists studied in the current ser ies of experiments did not corre la te with ef fects on blood pressure or heart ra te . Felodipine and n i fed ip ine produced large reductions in blood pressure without concomittant reductions in arrhythmias. Anipamil , which appears to be a calcium antagonist with s e l e c t i v i t y for the myocardium versus vascular smooth muscle, reduced arrhythmias without causing s i g n i f i c -ant reductions in blood pressure. The enantiomers of verapamil reduced arrhythmias both at doses with and without ef fects on blood pressure. Although heart rate was reduced by antiarrhythmic doses of the verapamil enantiomers and increased by the dihydropyridines at the time of occ lus ion , these ef fects were not sustained, and during the periods of arrhythmias the ef fects of treatment on heart rate did not corre la te with arrhythmias. I t may be suggested that the antiarrhythmic actions of the enantiomers of verapamil and anipamil occurred as a resu l t of the i r apparent a n t i -ischaemic ac t ions , since these drugs delayed the development of ECG indices of ischaemic such as S-T segment e leva t ion . However, these ef fects did not appear to corre la te with arrhythmias. The onset of arrhythmias was not delayed by the verapamil enantiomers or anipamil . In add i t ion , fe lod ip ine delayed S-T segment elevat ion in but did not reduce arrhythmias during myocardial ischaemia. It is perhaps worth commenting that delays in ECG signs of ischaemia did not corre late with in fa rc t s i z e , e i the r . Infarct s ize was not reduced by any treatment. 4.4 General conclusions 4.4.1 Arrhythmogenesis in acute myocardial ischaemia The experiments with calcium antagonists described here do not contra-d i c t the hypothesis that i . i s involved in arrhythmogenesis in acute myocardial ischaemia. On the contrary, they support the hypothesis. There are many theoret ica l reason why i ^ may be involved in arrhythmogenesis. - 298 -Slow conduction in the ischaemic t i s sue , which occurs during acute myocar-d ia l ischaemia and i s predicted to predispose to reentry (Mines, 1913) may depend on i ^ , since may be completely inact ivated by depolar isa-t i o n . Of the hypothetical mechanistic models of arrhythmogenesis, only reentry has been shown by act iva t ion mapping to occur during acute myocar-d ia l ischaemia. The resu l ts of the current experiments are consistent with a ro le for i .. in the ischaemic t issue in arrhythmogenesis in acute myo-card ia l ischaemia. Experimental evidence from the present studies and other published work has not yet disproven a ro le for i .. in arrhythmogenesis. However, un t i l a de f i n i t i ve l ink i s made between i $ - (or other currents) and reentry (or other mechanistic models of arrhythmogenesis) in myocardial ischaemia, i t w i l l not be possible to state with cer ta in ty by what mechanism an arrhythmia ar ises or is maintained in acute myocardial ischaemia, in terms of conduct-ances. The current experiments involv ing ablat ions in the CNS showed that the autonomic nervous system, heart rate and blood pressure are not s i gn i f i can t determinants of arrhythmogenesis in acute myocardial ischaemia. However, acute surgery, or a factor produced by acute surgery, i s capable of abo l -ishing arrhythmias resu l t ing from acute myocardial ischaemia. The iden t i t y of t h i s var iab le i s at present unknown. However prel iminary resu l ts suggest that th is factor may be e i ther hyperkalemia or leukopaenia. 4.4.2 Action of calcium antagonists in acute myocardial ischaemia The study of the ef fects of drugs on ischaemia-induced arrhythmias has lead to a cer ta in amount of tautology. Some researchers use drugs as too ls to probe for mechanisms of arrhythmogenesis, whereas others consider the underlying electrophysiology of ischaemia while invest igat ing the pharmaco-logy of these same drugs in an attempt to explain the i r mechanism of a n t i -- 299 -arrhythmic ac t ion . Neither approach has yet y ie lded a safe prophylact ic agent for prevention of arrhythmias in acute myocardial ischaemia. The current studies showed that some, but not a l l , calcium antagonists are capable of reducing ischaemia-induced arrhythmias. The di f ferences between (+)- and (-)-verapamil on one hand, and anipamil and ronipamil on the other suggest that the mechanism of antiarrhythmic action of calcium antagonists is calcium antagonism ( inh ib i t i on of i in e lect rophysio-log ica l terms). The dif ferences between phenethylalkylamines and 1,4-dihy-dropyridines suggest that the s i t e of action of antiarrhythmic calcium antagonists is the vent r icu la r t i s sue . Consideration of the re la t ionsh ip between calcium antagonist a c t i v i t y and ex t race l l u la r K + concentration suggests that the s i t e of antiarrhythmic action of calcium antagonists is the ischaemic vent r icu lar t i ssue . The resu l ts of the present studies with (±)-verapami1 suggest that phenethylalkylamine calcium antagonists can cer ta in ly penetrate into the ischaemic t i s sue , even when administered af ter occ lus ion. I t i s not possible to account for the antiarrhythmic actions of calcium antagonists, as catalogued in the present experiments, by invoking a l te rna-t i ve known actions of the drugs in quest ion. It is quite poss ib le , of course, to account for the resu l ts by invoking some as yet unknown ac t ion . With regard to possible therapeutic appl icat ions of the current crop of calcium antagonists, i t appears that most, i f not a l l , do not possess the necessary pharmacological p ro f i l e for prophylaxis of arrhythmias associated with acute myocardial ischaemia. According to the resu l ts of the present experiments, the ideal antiarrhythmic calcium antagonist, should: a. Have no act ion on vascular smooth muscle b. Have no action in non-ischaemic cardiac t issue at normal heart rates c. Abolish conduction in ischaemic t i s sue . - 300 -Current ly, the drug which appears to best f i t th i s p ro f i l e i s anipamil . Recent experiments in our laboratory suggest that anipamil is r e l a t i v e l y card iose lect ive compared with other calcium antagonists but, unl ike verapa-m i l , does not cause profound a t r ioven t r i cu la r block. Consideration of the pharmacological p ro f i l e of calcium antagonists suggests that there is much to be learned with regard to the i r mechanism of action and the processes which govern the i r apparent t issue s e l e c t i v i t y , frequency-dependence, voltage-dependence and subsidiary proper t ies. A f u l l e r understanding of these caveats may f a c i l i t a t e the design of therapeu-t i c a l l y useful prophylaxis against ischaemia-induced arrhythmias. - 301 -5 REFERENCES ABRAHAMSSON, T . , ALMGREN 0. Ventr icu lar f i b r i l l a t i o n fo l lowing coronary artery l i ga t i on in the ra t . In: Budden, R., Detwei ler, D. K. , Zbinden G. 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