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The actions of calcium antagonists on arrhythmias and other responses to myocardial ischaemia in the.. Curtis, Michael John 1986-7-31

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c THE ACTIONS OF CALCIUM ANTAGONISTS ON ARRHYTHMIAS AND OTHER RESPONSES TO MYOCARDIAL ISCHAEMIA IN THE RAT By MICHAEL JOHN CURTIS B.Sc. (Hon.), U n i v e r s i t y 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 t h i s t h e s i s as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1986 ® M i c h a e l John C u r t i s ,  1986  In p r e s e n t i n g requirements  this thesis f o r an  of  British  it  freely available  agree t h a t for  that  Library  s h a l l make  for reference  and  study.  I  f o r extensive copying of  h i s or  be  her  g r a n t e d by  s h a l l not  be  0  DE-6 (2/81)  of  further this  Columbia  thesis  head o f  this  my  It is thesis  a l l o w e d w i t h o u t my  permission.  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3  the  representatives.  copying or p u b l i c a t i o n  f i n a n c i a l gain  University  the  s c h o l a r l y p u r p o s e s may  understood  the  the  I agree that  permission by  f u l f i l m e n t of  advanced degree at  Columbia,  department or for  in partial  written  - ii  -  ABSTRACT Studies were c a r r i e d out antagonists for  in acute myocardial  these a c t i o n s ;  antagonism in aemia.  in  order  examine the  actions  of  ischaemia and the mechanism(s)  the' s c i e n t i f i c  the v e n t r i c l e s  to  is  hypothesis  under  antiarrhythmic  in  test  calcium  responsible  was that  calcium  acute myocardial  isch-  In a d d i t i o n , experiments were c a r r i e d out to investigate the r o l e 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 preparation. was found that the drugs with i d e n t i f i a b l e  actions in the heart a t t r i b u t a b l e  to calcium antagonism possessed antiarrhythmic  activity,  whereas the  producing only systemic v a s o d i l a t a t i o n were without tangible activity.  It  drugs  antiarrhythmic  The r e s u l t s were taken as evidence in support of the main hypoth-  e s i s (above).  In no instance did any of the drugs produce consistent dose-  dependent -infarct-reducing a c t i o n s . It  was established from the  comparison of  the  optical  enantiomers  verapamil  that antiarrhythmic  potency.  It was also shown that these drugs appeared to have no e f f e c t  g  N a  potency corresponded with calcium  of  antagonist on  in the heart in vivo (as predicted from work by others, in v i t r o ) . Evidence that arrhythmias  were reduced as a r e s u l t  of  effects  on i .  si in the  ischaemic v e n t r i c l e  was accrued from several s t u d i e s .  Langendorff-perfused rat v e n t r i c l e s verapamil  enantiomers was greatly  the calcium antagonist potentiated  In  isolated  activity  by r a i s i n g K  +  the  concentration  to l e v e l s seen during acute myocardial ischaemia, whereas n i f e d i p i n e , showed l i t t l e i f any antiarrhythmic a c t i v i t y + K -dependent calcium antagonist a c t i v i t y . In a separate series of experiments i t  of  which  in v-ivo did not  show marked  was demonstrated  that  serial  ablations in the CNS had profound e f f e c t s on occlusion-induced arrhythmias, but that these e f f e c t s occurred independently of the level of adrenoceptor activation.  It  was  hypothesised  arrhythmias, by v i r t u e of e i t h e r  that its  surgery  effects  reduced  on serum K  i t s e f f e c t s on the number of c i r c u l a t i n g thrombocytes.  ischaemia-induced +  concentration  or  - iv TABLE OF CONTENTS CHAPTER  Page TITLE PAGE  i  ABSTRACT  ii  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 1.1.1 1.1.2 1.1.3 1.1.4  C l i n i c a l myocardial ischaemia and i n f a r c t i o n Overview Infarction Arrhythmias Therapeutic approaches  1 1 3 4 14  1.2 1.2.1 1.2.2 1.2.3 1.2.3.1 1.2.3.2 1.2.3.3 1.2.4  Experimental myocardial ischaemia and i n f a r c t i o n Overview Methods for producing ischaemia Differences between species Infarction Arrhythmias Choice of species A h i s t o r y of coronary occlusion in rats  17 17 18 24 24 26 28 36  1.3 1.3.1 1.3.2 1.3.3 1.3.4 1.3.4.1 1.3.4.2 1.3.4.3 1.3.5 1.3.6 1.3.6.1 1.3.6.2  V e n t r i c u l a r arrhythmias in acute myocardial ischaemia 46 The electrophysiology of e x c i t a b l e t i s s u e 47 The electrophysiology of the normal v e n t r i c l e 54 E l e c t r o p h y s i o l o g i c a l changes caused by myocardial ischaemia 57 Models Of Arrhythmogenesis 60 Reentry 60 Abnormal Automaticity 62 Triggered Automaticity 66 E p i c a r d i a l A c t i v a t i o n Mapping 67 The slow inward current and arrhythmogenesis in acute ischaemia 70 Introduction 70 How might i - contribute to arrhythmogenesis? 70  1.4 1.4.1 1.4.2  The pharmacology of calcium antagonists Definition Pharmacology of phenethylalkylamines and 1,4-dihydropyridines  s l  73 73 74  -  V  -  1.5 1.5.1 1.5.2  Aims of studies The action of calcium antagonists in acute myocardial ischaemia Arrhythmogenesis in acute myocardial ischaemia  77 77 78  2  METHODS  80  2.1 2.1.1 2.1.2 2.1.2.1 2.1.2.2 2.1.3 2.1.3.1 2.1.3.2 2.1.3.3 2.1.3.4 2.1.4 2.1.4.1 2.1.4.2 2.1.4.3 2.1.4.4 2.1.4.5 2.1.4.6 2.1.4.7 2.1.5 2.1.5.1 2.1.5.2 2.1.5.3 2.1.6 2.1.6.1 2.1.6.2 2.1.7 2.1.8 2.1.9 2.1.9.1 2.1.9.2 2.1.9.3  Coronary occlusion in rats Overview Preparation Occluders Leads and l i n e s Coronary occlusion Sequence of events Monitoring of responses to occlusion Occluded zone (OZ) I n f a r c t zone (IZ) D e f i n i t i o n of occlusion-induced arrhythmias Introduction Premature v e n t r i c u l a r contractions (PVC) V e n t r i c u l a r tachycardia (VT) V e n t r i c u l a r f i b r i l l a t i o n (VF) Other arrhythmias Rationale for d e f i b r i l l a t i o n Arrhythmia scores ECG changes produced by occlusion ' S - T ' segment elevation Pathological R-waves Pathological Q waves ECG changes produced by drugs P-R i n t e r v a l QRS i n t e r v a l Measurement of serum K concentration Exclusion c r i t e r i a Statistics Normalisation procedures Censoring S t a t i s t i c a l tests  80 80 81 82 87 91 92 96 97 98 99 99 102 103 103 104 105 107 110 110 114 114 115 115 115 116 117 122 122 123 123  2.2 2.2.1 2.2.2 2.2.2.1 2.2.2.2 2.2.3 2.2.3.1 2.2.3.2  Calcium antagonist studies in coronary-occluded rats General experimental design Phenethylalkylamines Anipamil and Ronipamil (+)- And (-)-verapami1 1,4-Dihydropyridines Felodipine Nifedipine and DHM9 ,  124 124 125 125 126 127 127 128  2.3 2.3.1 2.3.2 2.3.2.1 2.3.2.2 2.3.2.3  Arrhythmogenesis and the r o l e of the CNS Introduction Preparation Pithing Spinalisation Decerebration  129 129 130 130 131 131  +  - vi 2.3.3 2.3.4  Other manipulations Statistics  132 135  2.4 2.4.1 2.4.2 2.4.3 2.4.3.1 2.4.3.2 2.4.4 2.4.5  Preliminary Screen for drug a c t i v i t y in acute ischaemia Introduction Preparation Experimental endpoints Definitions V a l i d a t i o n of behaviour endpoints Comprison of ( + )-, (-)- and (ij-verapami1 Statistics  135 135 135 136 136 137 137 139  2.5 2.5.1 2.5.2 2.5.3 2.5.3.1 2.5.3.2 2.5.3.3 2.5.4  E l e c t r i c a l l y - i n d u c e d arrhythmias in conscious rats Introduction Preparation Experimental endpoints Maximum following frequency Threshold voltage Threshold pulse width Comparison of (+)- and (-)-verapamil  139 139 140 140 140 141 141 141  2.6 2.6.1 2.6.2 2.6.3 2.6.4 2.6.5  Haemodynamic e f f e c t s of calcium antagonists in pithed rats Introduction Preparation Variables measured Comparison of (+)- and (-)-verapami1 Statistics  142 142 143 143 143 144  2.7 2.7.1 2.7.2 2.7.3 2.7.4 2.7.4.1 2.7.4.2 2.7.4.3 2.7.5 2.7.6 2.7.7  Actions of calcium antagonists in perfused r a t v e n t r i c l e s Introduction Perfusion apparatus S p e c i f i c a t i o n s for Langendorff perfusion Variables measured Isochoric l e f t v e n t r i c u l a r developed pressure Coronary blood flow Ventricular e x c i t a b i l i t y Comparison of (+)- and (-)-verapami1 Comparison of n i f e d i p i n e and DHM9 Statistics  144 144 145 146 146 146 147 147 147 148 148  2.8 2.8.1 2.8.2 2.8.3 2.8.4 2.8.5 2.8.6  Metabolism of (^O-verapamil in acute myocardial ischaemia Introduction Animals Drug administration and t i s s u e samples Extraction HPLC C a l c u l a t i o n of (^J-verapami1 concentration  149 149 149 149 150 152 152  3 RESULTS 3.1 3.1.1 3.1.2  154 Metabolism of ( ^ - v e r a p a m i l in acute myocardial ischaemia Concentration of ( ^ - v e r a p a m i l in blood matrices Concentration of (±)-verapami1 in the v e n t r i c u l a r myocardium  154 154 155  - vii  -  3.2 3.2.1 3.2.2 3.2.3 3.2.3.1 3.2.3.2 3.2.3.3 3.2.4 3.2.5 3.2.6 3.2.7  Arrhythmogenesis and the r o l e of the CNS Overview Occluded zones (OZ) Arrhythmias Arrhythmia scores VT and VF PVC Haemodynamic v a r i a b l e s ECG changes Thrombocytes, leukocytes and serum K Summary  3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.6 3.3.7 3.3.8  Actions of anipamil and ronipamil in acute myocardial ischaemia 170 Overview 170 OZ and i n f a r c t zone (IZ) 170 Arrhythmias 171 Mortality 175 Haemodynamic variables 175 ECG changes 177 Plasma concentrations of anipamil 177 Summary 180  3.4 3.4.1 3.4.2 3.4.3 3.4.4 3.4.5 3.4.6  Actions of f e l o d i p i n e in acute myocardial ischaemia Overview OZ, IZ and m o r t a l i t y Arrhythmias Haemodynamic variables ECG changes Summary  180 180 180 181 186 186 186  3.5 3.5.1 3.5.2 3.5.2.1 3.5.2.2 3.5.3 3.5.4 3.5.4.1 3.5.4.2 3.5.4.3 3.5.4.4 3.5.5 3.5.6 3.5.7  Actions of verapamil enantiomers in acute myocardial ischaemia Overview Effects of enantiomers before occlusion P-R and QRS i n t e r v a l s Blood pressure and heart rate OZ, IZ and m o r t a l i t y Arrhythmias VT and VF PVC Arrhythmia scores Arrhythmias at 24 h Haemodynamic variables ECG changes Summary  189 189 190 190 190 190 195 195 195 195 203 203 203 208  3.6 3.6.1 3.6.2 3.6.2.1 3.6.2.2 3.6.3 3.6.4 3.6.5  Actions of n i f e d i p i n e and DHM9 in acute myocardial ischaemia Overview Effects of enantiomers before occlusion P-R and QRS i n t e r v a l s Blood pressure and heart rate OZ, IZ and m o r t a l i t y Arrhythmias Haemodynamic v a r i a b l e s  208 208 208 208 210 210 210 216  +  158 158 158 160 160 160 160 165 165 168 168  - viii  -  3.6.6 3.6.7 3.6.8  ECG changes Serum K concentration Summary  216 218 218  3.7 3.7.1 3.7.2 3.7.3 3.7.4 3.7.5  Preliminary Screen for drug a c t i v i t y in acute ischaemia Overview V a l i d a t i o n of behaviour endpoints OZ and IZ M o r t a l i t y and morbidity Summary  218 218 218 219 219 223  3.8 3.8.1  E l e c t r i c a l l y - i n d u c e d arrhythmias in conscious rats Overview and summary  223 223  3.9 3.9.1 3.9.2 3.9.3 3.9.3.1 3.9.3.2 3.9.4  E f f e c t s of verapamil enantiomers in pithed rats Overview S t a b i l i t y of the preparation Effects of verapamil enantiomers Blood pressure and heart rate P-R and QT i n t e r v a l s Summary  225 225 225 227 227 227 227  3.10 3.10.1 3.10.2 3.10.3 3.10.4 3.10.5  Actions of verapamil enantiomers in perfused r a t v e n t r i c l e s Overview Isochoric l e f t v e n t r i c u l a r developed pressure Ventricular e x c i t a b i l i t y Additional comments Summary  232 232 233 233 237 237  3.11 3.11.1 3.11.2 3.11.3 3.11.4  Actions of n i f e d i p i n e and DHM9 in i s o l a t e d perfused v e n t r i c l e s Overview Isochoric l e f t v e n t r i c u l a r developed pressure Ventricular e x c i t a b i l i t y Summary  237 237 238 240 240  4  DISCUSSION  243  4.1 4.1.1 4.1.2 4.1.2.1 4.1.2.2 4.1.2.3 4.1.3 4.1.3.1 4.1.3.2 4.1.3.3 4.1.3.4 4.1.3.5 4.1.3.6 4.1.3.7 4.1.4 4.1.4.1  The conscious rat preparation for myocardial ischaemia studies Overview P r e c i s i o n of variables Occluded zone and i n f a r c t zone Arrhythmias ECG changes Responses to drugs Overview Class 1 antiarrhythmics Class 2 antiarrhythmics Class 3 antiarrhythmics Class 4 antiarrhythmics Arachidonic acid metabolites and a n t i p h l o g i s t i c s Other e n t i t i e s The conscious r a t versus other preparations Advantages and disadvantages of rats  243 243 243 243 245 245 246 246 247 248 250 250 252 253 254 255  +  - ix 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 4.2.7 4.2.8  Important determinants of arrhythmogenesis in acute ischaemia Role of OZ Role of the autonomic nervous system Role of K Role of thrombocytes and leucocytes Role of arachidonic acid metabolites Role of l i p i d metabolites Role of heart rate and blood pressure Role of the f a s t and slow inward currents  256 256 258 261 263 264 264 265 265  4.3 4.3.1 4.3.1 .1 4.3.1 .2 4.3.1 .3 4.3.1 .4 4.3.2 4.3.3 4.3.4 4.3.4 .1 4.3.4 .2 4.3.4 .3  Mechanism of action of calcium antagonists in acute ischaemia Role of calcium antagonism Overview Anipamil versus ronipamil (+)- Versus (-)-verapamil F e l o d i p i n e , n i f e d i p i n e and DHM9 Role of calcium antagonism in the myocardium Role of calcium antagonism in the ischaemic v e n t r i c l e Role of other pharmacological properties I n h i b i t i o n of the f a s t inward current Blockade of a-adrenoceptors I n d i r e c t actions  268 268 268 269 272 278 282 289 293 293 294 296  4.4 4.4.1 4.4.2  General conclusions Arrhythmogenesis in acute myocardial ischaemia Action of calcium antagonists in acute myocardial ischaemia  297 297 298  5  REFERENCES  301  +  LIST OF TABLES TABLE 1  Summary of groups in the CNS ablation study.  2  Concentration of ( ^ - v e r a p a m i l  3  D i s t r i b u t i o n of (^J-verapami1 in the v e n t r i c l e s .  4  Haemodynamic e f f e c t s of anipamil and ronipamil before and a f t e r occlusion.  5  Extent of ischaemia, i n f a r c t i o n , and ECG changes after o c c l u s i o n : e f f e c t s of verapamil enantiomers.  6  Arrhythmias and m o r t a l i t y following coronary o c c l u s i o n : e f f e c t s of verapamil enantiomers.  7  Extent of ischaemia, i n f a r c t i o n , and ECG changes after o c c l u s i o n : e f f e c t s of n i f e d i p i n e and DHM9.  8  Arrhythmias and m o r t a l i t y following coronary o c c l u s i o n : e f f e c t s of n i f e d i p i n e and DHM9.  9  Pre-drug values for haemodynamic and ECG variables in pithed r a t s .  10  Incidence of PVC in i s o l a t e d v e n t r i c l e s : e f f e c t s of verapamil enantiomers.  in blood matrices.  -  XI  -  LIST OF FIGURES FIGURE  Page  1  Sequence of events on the day of occlusion following connection of leads and l i n e s .  93  2  Anecdote of ECG changes caused by occlusion in a conscious r a t .  Ill  3  Effects of ablations in the CNS on occluded zone.  159  4  Effects of ablations in the CNS on arrhythmia score.  161  5  E f f e c t s of ablations in the CNS on the incidence of v e n t r i c u l a r fibrillation.  162  6  E f f e c t s of ablations in the CNS on the incidence of v e n t r i c u l a r tachycardia.  163  7  Effects of ablations in the CNS on the log^o number of PVC.  164  8  Effects of ablations in the CNS on heart rate and blood pressure before and after coronary o c c l u s i o n .  166  9  Effects of ablations in the CNS on ECG changes following coronary occlusion.  167  10  Changes in serum K concentration, leukocytes and thrombocytes in pithed rats before and a f t e r coronary o c c l u s i o n .  169  11  E f f e c t of ronipamil and anipamil on arrhythmia score.  172  12  E f f e c t of ronipamil and anipamil on the incidence and duration of various arrhythmias during the f i r s t 4 h following coronary occlusion  173  13  E f f e c t of ronipamil and anipamil on occlusion-induced ECG changes (R-wave and ' S - T ' segment e l e v a t i o n ) .  179  14  E f f e c t s of f e l o d i p i n e on 0Z and IZ, and t o t a l and arrhythmiainduced m o r t a l i t y during the 0 - 4 h period following o c c l u s i o n .  182  15  E f f e c t s of f e l o d i p i n e on the incidence of v e n t r i c u l a r 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 following occlusion.  16  E f f e c t s of f e l o d i p i n e on arrhythmia score for the 0 - 3 0 min and 0 - 4 h periods following o c c l u s i o n .  184  17  E f f e c t s of f e l o d i p i n e on log^o of PVC during the 0 - 3 0 min and 0 - 4 h periods following o c c l u s i o n .  185  +  n u m D e r  -  XI 1 -  18  E f f e c t s of f e l o d i p i n e on blood-pressure and heart rate 1 min before and 1 h after o c c l u s i o n .  187  19  E f f e c t s of f e l o d i p i n e on maximum S-T elevation and the time at which maximum S-T elevation occurred.  188  20  Changes in P-R and QRS in response to {*)-  191  21  The e f f e c t s of ( ± ) - and (-)-verapamil on blood pressure and heart rate at 1 min before o c c l u s i o n .  193  22  The e f f e c t s of (+)- and (-)-verapamil on the incidence of PVC in r e l a t i o n to the time after o c c l u s i o n .  197  23  The e f f e c t s of (+)- and (-)-verapami1 on the incidence of v e n t r i c u l a r tachycardia in r e l a t i o n to the time after o c c l u s i o n .  199  24  The e f f e c t s of (+)- and (-)-verapamil on the incidence of v e n t r i c u l a r f i b r i l l a t i o n in r e l a t i o n to the time after o c c l u s i o n .  200  25  The e f f e c t s of (+)- and (-)-verapami1 on arrhythmia score.  201  26  Mean percent changes in blood pressure and heart rate from predrug values in response to (+)- and (-)-verapami1, before and after occlusion.  205  27  The e f f e c t s of ( ± ) - and (-)-verapami1 on S-T segment elevation at various times before and a f t e r o c c l u s i o n .  206  28  The e f f e c t s of ( ± ) - and (-)-verapamil on R-wave amplitude at various times before and a f t e r o c c l u s i o n .  207  29  The e f f e c t s of n i f e d i p i n e , DHM9 and v e h i c l e on P-R and QRS i n t e r v a l before o c c l u s i o n .  211  30  Mean percent changes in blood pressure and heart rate from predrug values in response to DHM9 and n i f e d i p i n e , before and  212  and (-)-verapami1.  after occlusion. 31  E f f e c t s of n i f e d i p i n e and DHM9 on arrhythmia score.  213  32 33  E f f e c t s of n i f e d i p i n e and DHM9 on serum K concentration. E f f e c t s of (+)-, (*)- and (-)-verapami1 on OZ and IZ in the 'preliminary screen'.  217 220  34  E f f e c t s of (+)-, (*)- and (-)-verapami1 on t o t a l m o r t a l i t y and m o r t a l i t y associated with sudden convulsive-type behaviour during the 0 - 4 h period after o c c l u s i o n , and morbidity during the 5 min period before o c c l u s i o n .  221  +  - xiii  -  35  E f f e c t s of (+)- and (-)-verapami1 on mean a o r t i c blood pressure heart rate and P-R i n t e r v a l in experiments to supplement the 'preliminary s c r e e n ' .  222  36  E f f e c t s of (+)- and (-)-verapamil on threshold voltage and threshold pulse width for induction of f i b r i l l o - f l u t t e r , and maximum following frequency in conscious rats subjected to e l e c t r i c a l stimulation of the l e f t v e n t r i c l e .  224  37  Time-course of changes in mean a o r t i c blood pressure and heart rate in rats following p i t h i n g .  226  38  E f f e c t s of (+)- and (-)-verapamil heart rate in pithed r a t s .  228  39  E f f e c t s of (+)- and (-)-verapami1 on Q-T and P-R i n t e r v a l s in pithed r a t s .  229  40  Dose-response curves for the e f f e c t s of (-)- and (+)-verapami1 on developed pressure in Langendorff-perfused r a t v e n t r i c l e s paced at 300/min.  234  41  The r e l a t i o n s h i p between negative i n o t r o p i c potency of (-)- and (+)-verapamil and K concentration in i s o l a t e d Langendorffperfused r a t v e n t r i c l e s .  235  The r e l a t i o n s h i p between negative i n o t r o p i c potency of n i f e d i p i n e and K concentration in i s o l a t e d Langendorff-perfused rat ventricles.  235  The e f f e c t of d i f f e r e n t buffer K concentrations on the threshold voltage and pulse width for capture of the l e f t v e n t r i c l e in the Langendorff-perfused r a t v e n t r i c l e preparation.  241  on mean a o r t i c blood pressure  +  42  +  43  +  -  XIV  -  LIST OF ABBREVIATIONS action potential  AP  action potential duration  APD  arrhythmia score  AS  calcium  Ca  conductance  g  conduction v e l o c i t y  e  current  i  e f f e c t i v e r e f r a c t o r y period  ERP  hour(s)  h  i n f a r c t zone  IZ  length constant  x  maximum d i a s t o l i c potential  MDP  membrane potential  E m  minute(s)  min  occluded zone  OZ  potassium  K  premature v e n t r i c u l a r contraction  PVC  resistance  r  second(s)  sec  slow inward current  i ^ $  sodium  Na  sodium current  n*  standard error of the mean  s.e.mean  time constant  tau  ventricular f i b r i l l a t i o n  VF  v e n t r i c u l a r tachycardia  VT  Na  -  -  XV  ACKNOWLEDGEMENTS In order to ensure blindness in experimentation, k i n d l y a s s i s t e d with drug d i l u t i o n  the following  and ( o c c a s i o n a l l y ) with drug administra-  t i o n : Kathleen S a i n t , Tony Au and Michael Walker.  Experiments c i t e d in the  text as having been c a r r i e d out by others in the laboratory circulating  platelets  Dr R Wall  is  and leukocytes)  thanked  for  his  people  were c a r r i e d  guidance  with  out  the  (measurement of  by Kathleen  Saint.  HPLC experiments.  The  calcium antagonists used in the experiments were requested from and supplied by the f o l l o w i n g : Dr B. Ljung of Haessle ( f e l o d i p i n e ) ; Kretzschmar  of  Knoll  (optical  enantiomers  of  Drs M. Raschack and R  verapamil,  r o n i p a m i l ) ; Dr R. Whiting of Syntex (DHM9); Dr Kazda of Bayer I  anipamil  (nifedipine).  am indebted to the Department of Pharmacology and Therapeutics, The  U n i v e r s i t y of B r i t i s h Columbia and the B. C. Heart Foundation, a l l provided me with f i n a n c i a l I  would  like  to  kindness  of whom  support.  thank  encouragement concerning the their  and  the  following  experiments  and advice during  people  for  which were  my time  in  the  their  carried  support out,  department;  Dr  and  and for  Bernard  MacLeod, Dr Catherine Pang, Dr Morley S u t t e r , Caroline Bruce and Hari N a i r . I must also thank Tannis for  putting up with my work being  scattered  across the kitchen t a b l e , and my bad temper. Finally, for  his  I am indebted to my supervisor,  encouragement,  searching f o r  the  truth  forthrightness, (even  in  the  Dr Michael Walker, not  and f o r  his  face  adversity),  of  example  presenting me with a problem to s o l v e , a hypothesis to t e s t ,  as but  only  a person also  for  an established  preparation with which to begin my studies and an e s s e n t i a l l y free hand to chose my own d i r e c t i o n .  One could ask f o r nothing more.  - xvi -  AUTHORISATION FROM ANIMAL CARE UNIT, U. B. C. The experiments c a r r i e d out in p a r t i a l  f u l f i l m e n t of  the  requirements  for the degree of doctor of philosophy, and described in t h i s t h e s i s , were approved as e t h i c a 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 i n f a r c t i o n 1.1.1 Overview Myocardial  blood flow, resulting 1972).  ischaemia i s  defined, simply,  and myocardial  from myocardial However,  infarction  is  defined  ischaemia ( e . g . ,  myocardial  ischaemia  as an impairment as the  of  necrotic  Steadman's Medical  and  infarction  is  a  coronary changes  Dictionary, heterogenous  disease, varying from patient to patient  in cause, l o c a t i o n , s e v e r i t y ,  course  Oliver,  and sequelae  (Henderson,  1984;  1982;  H i l l i s and Braunwald, 1977; Maseri et a l 1 9 7 8 ) . that coronary blood flow i n s u f f i c i e n c y  It  time-  Poole-Wilson, 1983; i s generally accepted  i s the common event which produces  the s i g n s , symptoms and sequelae of myocardial ischaemia and i n f a r c t i o n . Ischaemia may involve flow,  partial  restriction  and may be permanent or temporary.  or  complete  loss  of  blood  Temporary ischaemia i s associated  with r e p e r f u s i o n , which may occur gradually or abruptly ( F o l t s et a l ; , 1982; Maseri et a l ; , 1978). pectoris  (chest  arrhythmias,  pain)  Symptoms of acute myocardial ischaemia include angina and d i z z i n e s s ,  hypotension,  signs  include  S-T segment a l t e r a t i o n s ,  syncope,  a reduction  ventricular in  cardiac  output and leakage into the systemic c i r c u l a t i o n of creatine phosphokinase, and sequelae include v e n t r i c u l a r arrhythmias, i n f a r c t i o n , in  chest  lead  ECGs,  heart  failure,  pulmonary  a prominant Q wave  oedema,  and  hypertension  (Henderson, 1984; O l i v e r , 1982). The most common presentation ris.  of myocardial  ischaemia i s  angina pecto-  Angina of e f f o r t i s associated with r a d i a t i n g chest pain and S-T seg-  ment a l t e r a t i o n s  r e s u l t i n g from an increase in myocardial oxygen demand in  the s e t t i n g of an inadequate oxygen supply, and r e l i e f  may be obtained by  reducing sympathetic drive to the heart with B-adrenoceptor antagonists, by  dilating  coronary  vessels  with  organic  nitrites  or  (see Poole-Wilson,  - 2 1983).  P r i n z m e t a l ' s variant  form of  angina  (Prinzmetal  et-al;,  1959) may  occur at r e s t in the absence of any apparent sympathetic nervous system-mediated increase in myocardial oxygen demand.  Angina of e f f o r t  is  generally  associated with coronary a r t e r i o s c l e r o s i s ^ whereas P r i n z m e t a l ' s variant not.  The  latter  is  believed  to  be  associated with  is  coronary vasospasm  (Hellstrom, 1973; 1977), although t h i s i s not yet f i r m l y e s t a b l i s h e d . Ischaemic heart sclerosis.  disease can often  demand (angina  al,  pectoris).  Sustained stenosis  of  arterio-  sufficient  severity  may  infarction.  1971),  and m o r t a l i t y  resulting  a large vessel disease (Gensini  from acute  associated with s i n g l e vessel disease in 84% of 1982).  coronary  oxygen supply during periods of increased  Coronary a r t e r i o s c l e r o s i s i s p r i m a r i l y et  to  Stenosis (narrowing of vessels) leads to a loss of p h y s i o l o g i c a l  r e s e r v e , r e s u l t i n g in i n s u f f i c i e n t  lead to  be a t t r i b u t e d  coronary  cases  occlusion  (Liberthson  is  et al  Thus, the l e f t anterior descending (LAD), l e f t circumflex and r i g h t  coronary a r t e r i e s are the most common s i t e s of coronary a r t e r i o s c l e r o s i s - i n duced ischaemia and m o r t a l i t y . In patients with o c c l u s i v e stenosis in one or more coronary a r t e r y , most important determinant varies  with  age.  of  Functional  ischaemia i s  coronary  artery  collateral  anastamoses  are  the  anatomy,  which  rare  young  in  humans, but c o l l a t e r a l s become larger and more numerous with age, perhaps in conjunction with (and as a consequence of) the development of a r t e r i o s c l e r o sis  (Baroldi  and  Scomazzoni,  1967;  Fulton,  Gensini and Bruto da Costa, 1972; Newman, 1981).  1965; It  Harris  et a l ; ,  1969;  i s probable that slowly  developing coronary s t e n o s i s , from whatever cause, leads to a compensatory development of coronary c o l l a t e r a l pigs (Schaper, 1971).  anastamoses, as has been demonstrated  in  -  1.1.2  morbidity this  -  Infarction  Most of  For  3  our knowledge of  and s u r v i v a l  the  infarction  has come from  process and i t s  animal  reason, an exhaustive review of  experimentation  the  infarction  relation (see  to  below).  process and  its  prognostic s i g n i f i c a n c e w i l l not be given here. It  i s c l e a r l y undesirable for working muscle t i s s u e to d i e .  If  suffi-  c i e n t myocardial muscle becomes necrotic and fibrous then the a b i l i t y of the heart to pump blood w i l l infarction  be impaired.  Experimental evidence has shown that  leads to myocyte hypertrophy in s u r v i v i n g t i s s u e , but that large  i n f a r c t s preclude complete functional insufficient  number of c e l l s ;  recovery, owing to the s u r v i v a l of an  hyperplasia does not occur  (Anversa . e t - a l . ,  1984; 1985a; 1985b; 1986). There are two important i s considered.  Firstly,  points which must be examined when i n f a r c t  size  a c l e a r d i s t i n c t i o n must be drawn between preven-  t i o n and delay of n e c r o s i s .  It  i s conceivable that necrosis could be delay-  ed by a drug which causes v a s o d i l a t a t i o n of c o l l a t e r a l  anastamoses or by a  drug which slows myocardial metabolism (reducing the rate of formation c y t o t o x i c products of anaerobic metabolism).  of  Both types of drug would pre-  vent i n f a r c t i o n provided that the stenosis were resolved (by thrombolysis or bypass surgery).  It  i s unfortunate,  ies aimed at reducing i n f a r c t  t h e r e f o r e , that in most c l i n i c a l stud-  s i z e no delineation of the patient  population  into those with and those without c o l l a t e r a l anastamoses i s made, and l i t t l e attempt i s made to r a t i o n a l i s e the aims of the study in terms of the known pharmacology of the drug under i n v e s t i g a t i o n . Some drugs have been investigated for  their  ability  to reduce  infarct  s i z e in humans on the basis of t h e i r  ability  consumption  or  rate,  However, i f  occlusion i s complete, and c o l l a t e r a l c i r c u l a t i o n i s minimal or  oxygen  demand  (heart  to reduce myocardial oxygen  force  of  contraction,  etc.).  - 4 absent,  then  dium.  no amount of  off-loading  Attempting to prevent  will  infarction  salvage the  ischaemic myocar-  by such a strategy  i s analogous to  attempting to survive in a vacuum by holding one's breath. It  should also be noted that most of the reports  cerning  clinical  size.  infarct  size  refer  to  in the l i t e r a t u r e  enzymatically  determined  There are a v a r i e t y of techniques for measuring i n f a r c t  ermining the concentration found  intracellular^,  con-  infarct  s i z e by det-  in the blood of enzymes which are normally  for  example creatine  phosphokinase  (CPK).  only  It  has  been shown that in experimental animals the peak l e v e l s of serum CPK c o r r e l ate with i n f a r c t et a l ; , 1971).  s i z e determined h i s t o l o g i c a l l y However, i t  is  not  holds under the influence of drugs. influence CPK without a l t e r i n g  following  c l e a r whether It  infarct  this  sacrifice  (Shell  relationship  i s quite possible that a drug may size.  The problem c l i n i c a l l y ,  course, i s that one cannot examine a heart h i s t o l o g i c a l l y unless the dies.  Therefore  infarct  size.  one i s There  dependent  are  in most cases  angiographic  still  on i n d i r e c t  techniques  for  patient  measures  measuring  of  of  coronary 133  blood flow (Pitt  in v i v o , e . g . using r a d i o a c t i v e  et a l . , 1969), and ultrasound techniques for  Mattrey  and M i t t e n ,  1984), but these techniques  large scale multicentre c l i n i c a l 1.1.3  ischaemia  is  the  major  'sudden'.  imaging an i n f a r c t are not  normally  (e.g.  used  in  trials.  approximately  type  of  death  in  association  Sudden death has been defined  within 1 h of the patient in  imaging with xenon  Arrhythmias  Clinically,  occur  contrast  with  myocardial  as death  occurring  l a s t being seen a l i v e , and has been suggested to  30% of  the  total  artery disease (Armstrong et a l ; , 1972).  patient  population  with  coronary  Sudden death i s the major cause of  death in patients with myocardial ischaemia.  It  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  usually u n c e r t a i n .  It  approximately 43%of  sudden deaths occurring during  the  pain occur during  onset  1972).  of  chest  In a d d i t i o n , i t  dying during the f i r s t  the  has been shown that the 4 weeks  f i r s t , hour  following  (Armstrong  has been estimated that l e s s than 10% of hour a f t e r  et-al.-, patients  the onset of chest pain are seen by a  physician ( O l i v e r , 1982).  In other words, the majority of patients who die  as a r e s u l t of myocardial  ischaemia do so without a d e f i n i t e  of the cause of death. the epidemiology of  establishment  In the follow-up reports of the Framingham study of  sudden death, the following  comments were made:  'The  assignment of sudden deaths to coronary aetiology i s l a r g e l y by inference, since few other diseases can k i l l  in a matter of minutes.  Coronary a e t i o l o -  gy i s assumed when a o r t i c d i s s e c t i o n , ruptured aneurysm, and pulmonary embolism  are excluded c l i n i c a l l y  within minutes illness  permits  certainty'  in  or  persons not  ill  classification  on postmortem at  examination.  Thus,  the time with a p o t e n t i a l l y  as coronary  sudden deaths with  death lethal  reasonable  (Kannel e t - a l ; , 1984).  The cause of sudden death i s generally a t t r i b u t e d to VF ( O l i v e r , 1972; Campbell, 1983; 1984; e t c . ) .  Many c l i n i c i a n s use the terms sudden death and  f a t a l VF interchangeably, ( e . g . , O l i v e r , 1982).  There are several persuasive  reasons in support of t h i s , despite the absence of d i r e c t evidence in most cases.  F i r s t l y , evidence from work using experimental animals (see e x p e r i -  mental section of  Introduction) c l e a r l y demonstrates that VF occurs during  the f i r s t hour a f t e r coronary o c c l u s i o n , and that t h i s i s the major cause of death at t h i s 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 a f t e r o c c l u s i o n ) .  Secondly, i f  VF i s the major  cause of sudden death then a reduction in e a r l y VF should lead to a c o r r e s ponding reduction in sudden death. in 1956, ( Z o l l e t - a l . )  With regard to t h i s p o i n t , i t was found,  that VF in humans could be reverted by applying a DC  - 6 shock to the chest.  This procedure was rigourously applied in S e a t t l e by  paramedics in order to d e f i b r i l l ate patients before admission to h o s p i t a l , and the r e s u l t was a reduction in o u t - o f - h o s p i t a l ately  55% (Cobb et a l ; , 1980), confirming  related.  that  sudden death of approximVF and sudden death were  In a d d i t i o n , an i n v e s t i g a t i o n of the ECGs of patients c o l l a p s i n g  outside of hospital and r e c e i v i n g paramedic care within 15 min of collapse has provided a strong suggestion that sudden death and VF are one and the same; of 426 p a t i e n t s , 72 % were in VF when the ECG was f i r s t recorded, and of  the  remainder,  (Liberthson although  only  et a l ; ,  1 % were  1982).  sudden death  is  not  However,  experiencing v e n t r i c u l a r it  is  important  to  probably caused by VF, there  is  arrhythmias  consider  that  no established  c r i t e r i o n , in the absence of ECG evidence, for c a t e g o r i s i n g a death as having resulted  from VF, and that most sudden death occurs out  of  hospital  (Campbell, 1984) in the absence of ECG monitoring. I r r e s p e c t i v e of the exact incidence of f a t a l ischaemia and i t s contribution  to the body of  'sudden d e a t h ' , i t  less remains that VF can occur during myocardial that t h i s event i s often f a t a l The  natural  history  ischaemia and i n f a r c t i o n  of  VF in c l i n i c a l myocardial  ischaemia in humans, and  (e.g., Julian e t - a l 1 9 6 4 ) . ventricular  arrhythmias  during  i s not well established in humans.  standable since many patients  neverthe-  myocardial  This i s under-  die before admission to h o s p i t a l .  There  is  very l i t t l e information concerning the very e a r l y phase ( f i r s t few minutes) of acute myocardial ischaemia in humans.  Campbell e t - a l .  (1981)  38 previously unmedicated patients admitted to hospital with dial  infarction',  and expressed arrhythmia  onset of the symptoms (not  incidence  in  evaluated  'acute myocar-  relation  defined, presumably chest p a i n ) .  that primary VF (defined as VF occurring in the absence of  It  to  the  was found  'shock',  f a i l u r e or heart block) occurred p r i n c i p a l l y within the f i r s t 4 h a f t e r  heart the  - 7 onset of symptoms.  VF was almost always associated with an i n i t i a l  premature v e n t r i c u l a r  contraction  85% of the p r e v a i l i n g QT i n t e r v a l  'R on T'  (defined as a QRS complex f a l l i n g  within  of a normal sinus QRS complex), whereas  v e n t r i c u l a r tachycardia (VT) was almost never associated with such an occurrence.  VF and R on T premature v e n t r i c u l a r contractions (PVCs) were almost  absent after 4 h.  In c o n t r a s t , 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 between 4 and 12 h a f t e r the onset of symptoms.  It  i s of i n t e r e s t that despite  the uncertainty concerning the exact onset of o c c l u s i o n , the degree of s t e n o s i s , the volume of ischaemia, e t c . , i n t h i s study (Campbell et a l ; , 1981), the  time  distribution  of  arrhythmias  was not  grossly d i f f e r e n t  from  that  reported for dogs ( H a r r i s , 1950), and rats (Johnston et a l . , 1983a). Adgey et a l ; occurring during  (1971)  the f i r s t  examined 284 patients (S-T  also examined the few hours a f t e r  with ECG evidence of  natural the  history  onset of  of  arrhythmias  symptoms.  'acute myocardial  They  infarction'  segment changes or bundle branch block) and found a 31 % incidence of  bradyarrhythmia, 25% incidence of PVCs, 10% incidence of VF, 4% incidence of atrial  fibrillation  ing the f i r s t  and 0.4% incidence of supraventricular tachycardia dur-  hour a f t e r  the onset of symptoms ( i t  must be noted that the  above does not include the incidence of f a t a l v e n t r i c u l a r arrhythmias, which were not  analysed).  Corresponding incidences during the 3rd and 4th hour  were 2 , 6, 0 . 7 , 2, 0 and 0%, r e s p e c t i v e l y .  The decline with time  incidence of VF corresponds with that reported by Campbell et a l ; Adgey e t - a l ; (1971) also reported that the incidence of VF ' a f t e r low (4%).  in  the  (1981). 4 h' was  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 a f t e r 4 h as a r e s u l t less than 4%.  i s possible that the true of the  initial  incidence of  episode of myocardial  VF occurring ischaemia was  It i s 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 r e l a t i o n to p o s s i b i l i t y of further c r i t i c a l  the  stenosis and occlusion higher up the a r t e r i -  osclerotic tree. Recent  clinical  studies  of  arrhythmias  associated  with  myocardial  ischaemia and i n f a r c t i o n have concentrated to a c e r t a i n extent on the desire to predict sudden death/fatal VF. in p r e s c r i b i n g antiarrhythmic  This i s p a r t l y a r e s u l t of the hesitance  agents to a l l  patients  at r i s k of  developing  myocardial ischaemia and i n f a r c t i o n , owing to the occurrence of serious side effects  with long-term use ( e . g . , Kosowsky et a l ; , 1973; Jelineck  1974; Campbell, shown to ciated  1983).  In  addition,  there  unequivocally reduce c l i n i c a l  with  myocardial  Therefore there benefit,  for  is  not  ischaemia and sufficient  are no drugs  life-threatening infarction  justification,  whole-sale p r e s c r i p t i o n  of  which  have been  arrhythmias  (Campbell, 1983; in terms  a particular  etal;,  of  asso1984).  cost versus  drug in  patients  at  r i s k of developing myocardial ischaemia and i n f a r c t i o n . As  a  arrhythmias  result  of  these  considerations,  and l i f e - t h r e a t e n i n g  arrhythmias  correlations  have been sought,  assumption that a s p e c i f i c patient population 'at high r i s k can be d e l i n e a t e d . (for  between  1  under  the  of sudden death  In 1967, Lown et a l . proposed that c e r t a i n  example, R on T PVCs) constituted  benign  'warning arrhythmias'.  arrhythmias A relation-  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 y e a r s , therapeutic decisions were based on the  detection of these warning arrhythmias. reports  However, there were  contradictory  ( e . g . , E l - S h e r i f e t a l . , 1976; Rabkin et a l ; , 1982) which suggested  that 'warning arrhythmias' were j u s t as common in patients not subsequently developing VF as in those who d i d . by a recent study in which i t  The l a t t e r suggestion has been supported  was found that a high incidence of complex  PVCs (including R on T) and VT in otherwise healthy subjects was not a s s o c i -  - 9 ated with sudden death during a 10 year period (Kennedy et a l . - , 1985).  The  natural h i s t o r y study of Campbell (Campbell e t _ a l ± , 1981) showed that w h i l s t R on T PVCs were extremely common in patients during the 10 min preceeding VF, they were almost as common in concluded that reason.  patients  'warning arrhythmias'  In a d d i t i o n , i t  not  developing VF.  were of no p r e d i c t i v e  Campbell  value for  this  i s worth considering that any 'warning' which does  not appear u n t i l 10 min before a l i f e - t h r e a t e n i n g  event i s of l i t t l e use as  a guide to therapy, e s p e c i a l l y out of h o s p i t a l . Part  of  the  confusion  concerning  fundamental  p h i l o s p h i c a l dichotomy.  genesis  myocardial  in  subscribes.  'warning  arrhythmias'  stems from  This concerns the model of  ischaemia and  infarction  to  which  an  a  arrhythmoinvestigator  S p e c i f i c a l l y , i s ischaemia more or less important  than  infarc-  t i o n in arrhythmogenesis? In experimental preparations, VF occurs before t i s s u e has become i n f a r c ted (see experimental section in Introduction).  Therefore, when considering  myocardial ischaemia and i n f a r c t i o n , the p r i n c i p a l p r e r e q u i s i t e for VF would appear to be myocardial ischaemia. In support of t h i s premise derived from experimental animal studies are the following  pieces of c l i n i c a l evidence.  Firstly,  it  has been suggested  that most patients who die from sudden death do not have myocardial tion  when examined postmortem  (Lovegrove  and Thompson, 1978),  infarc-  indicating  that sudden death may have resulted from myocardial ischaemia, but not myocardial patients  infarction.  Secondly, i t  has been reported that  r e s u s c i t a t e d from VF e x h i b i t  waves in V leads (Cobb e t - a l ; , 1980).  ECG signs of  infarction  whereas e l e c t r i c a l  t i s s u e , since i t i s not v i a b l e .  such as Q  This has lead some c l i n i c i a n s  O l i v e r , 1982) to voice the opinion that c l i n i c a l l y , VF i s ischaemia,  less than 20% of  instability  is  not  (e.g.  associated with  a feature  of  infarcted  - 10 Proponents of the 'warning arrhythmia The r a t i o n a l e distinctive  for  'warning  myocardial  arrhythmias'  i s not i l l o g i c a l  and the r i s k  is  pathophysiologic  victims of sudden d e a t h ' , and that VF, i t  hypothesis take a d i f f e r e n t view.  1  indicator  based on the  derangement  long  that  preexists  'because such deaths are the r e s u l t  to assume the abnormality to r e l a t e  premise  to  'a in of  to be e l e c t r o p h y s i o l o g i c  altered cardiac rhythm'  (Lown, 1982).  This philosophy i s not supported by the evidence described above. Lown did not include in his scheme any recognition symptoms (whether the patient  of the duration  i s experiencing ischaemia or i n f a r c t i o n ) ,  did he examine the r e l a t i v e importance of r e - i n f a r c t i o n  of nor  versus re-ischaemia.  Lown dismisses the evidence which suggests that 'warning arrhythmias' no value in p r e d i c t i n g VF (see Campbell, 1983), and comments that  are of  'the pre-  v a i l i n g pessimism concerning the p o s s i b i l i t y of protecting a patient who has experienced VF against recurrence i s unwarranted' overstating  out  on  a  selected  subset  p a t i e n t s , namely those patients arrhythmias',  and the  are  of  patients,  myocardial  criteria  which were  As discussed in d e t a i l  popu-  Lown's studies were ischaemia/infarction  'who have experienced malignant  prognostic  only apply to these p a t i e n t s . patients  Lown may be  his case, because whereas Campbell considers the e n t i r e  l a t i o n of myocardial ischaemia and i n f a r c t i o n carried  (Lown, 1982).  developed  ventricular therefore  above, t h i s subset of  (those who have experienced complex VT) are the very patients who  least  likely  to  subsequently  experience  VF (Campbell  et-al;,  1981).  These are the patients who have e i t h e r survived the VF occurring during the f i r s t 3 - 4 h of myocardial ischaemia, or passed through t h i s danger period without experiencing VF.  This group, therefore, does not include any of the  truly  Indeed,  at-risk  receiving 1982).  patients.  medical  attention  were  patients not  Therefore the statement that  who die of  considered 'abolition  in  sudden death  before  this  (Lown,  article  of advanced grades of ven-  - 11 tricular  premature  arrhythmias'  beats  prevents  the  recurrence  of  (Lown, 1982) does not apply to myocardial  patients  as  a  whole.  Campbell,  patients,  stated that w h i l s t  referring  many studies  to  all  potentially  ischaemia/infarction ischaemia/infarction  have demonstrated  that  drugs reduce R on T PVCs and complex v e n t r i c u l a r c o n t r a c t i o n s , benefit  of  suppressing these  1984).  Campbell  also  arrhythmias  stated  that  'no  has  strategy  for  certain  'no tangible  been d e t e c t e d ' ,  current  lethal  (Campbell, ventricular  arrhythmia prophylaxis or treatment in acute myocardial i n f a r c t i o n i s s a t i s factory'  (Campbell, 1983).  The f a i l u r e to d i s t i n g u i s h between the various stages and conditions of myocardial  ischaemia  and  infarction,  particularly  between  the  initial  occlusion and the subsequent course of the disease(s) may be r e s p o n s i b l e , in part,  for  the  arrhythmics.  confusion over both It  prognosis  and the  assessment of  anti-  has been suggested that improvements must be made in  regard i n r e l a t i o n to the design of c l i n i c a l t r i a l s ,  or  'the treatment  this of  these patients w i l l continue to be made from a p o s i t i o n of abject ignorance' (Bigger, 1984).  In summary, there i s no evidence that 'warning arrhythmias'  u s e f u l l y predict sudden death/VF, p a r t i c u l a r l y  in patients who have passed  through the f i r s t few hours of myocardial ischaemia. The  'warning arrhythmia'  mias being part of with experimental  concept i s contingent  a continuum.  abundant  animals (see experimental  PVCs, VT and VF do c o n s t i t u t e rations  There i s  (Dresel and Sutter,  upon d i f f e r e n t evidence from  arrhythstudies  sections) which suggests that  a continuum, e i t h e r  in non-ischaemic prepa-  1961) or in experimental  myocardial ischaemia.  Our laboratory has shown that some antiarrhythmics can reduce the incidence of VF without a f f e c t i n g PVCs in rats subjected to coronary artery occlusion (Johnston et a l ; , 1983a). With regard to  variations  in  the natural  history  of  the  disease,  in  particular,  the  incidence of  re-ischaemia and r e - i n f a r c t i o n ,  reason to believe that the arrhythmias  resulting  there  from a f i r s t  is  no  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 f e c t i v e prophylaxis against VF ( i f  such a drug were shown to e x i s t ) on  the basis of an absence of VF (or R on T PVCs) during a f i r s t  episode of  myocardial  drug  ischaemia in  second patient  one p a t i e n t ,  with a h i s t o r y  of  while  VF (or  administering that  R on T PVCs)  to a  during myocardial  ischaemia. There are s t r a t e g i e s 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 f a r c t i o n , based on responses to drugs.  An example i s programmed e l e c t r i c a l stimulation of the r i g h t  t r i c l e (Fisher et a l ; , 1977).  ven-  The technique i s based on the p r i n c i p l e that  i f a premature stimulus i s delivered during the terminal phase of r e p o l a r i sation (the 'vulnerable p e r i o d ' ) 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).  E s s e n t i a l l y , the threshold  (current  or pulse width) for induction of VT or VF i s determined before and a f t e r  the  administration of various drugs, with the aim of f i n d i n g a drug which comp l e t e l y suppresses the induction of the arrhythmia upon s t i m u l a t i o n .  It has  been claimed that a p o s i t i v e response to a drug predicts that treatment with that drug w i l l ing  the  prevent spontaneous arrhythmias in 90 - 95 % of patients  following  1-2  years  (Ruskin et a l • ,  should be recognised that 1 5 - 6 0 % of patients  1983).  Nevertheless,  durit  in the studies reviewed by  Ruskin who did not respond to any drug during e l e c t r i c a l s t i m u l a t i o n went on to experience no VT or VF during the following 1 - 2  years, while r e c e i v i n g  apparently ' i n e f f e c t i v e ' therapy according to the r e s u l t s of the stimulation tests.  Therefore the 90-95% 'success' rate (Ruskin et a l . , 1983) i s perhaps  - 13 misleading.  The problem with t h i s method i s that although i t may be reason-  ably e f f e c t i v e in demonstrating that a p a r t i c u l a r drug may prevent v e n t r i c u l a r arrhythmias occurring in the f i r s t 2 years f o l l o w i n g myocardial mia and i n f a r c t i o n , suffer  it  does not benefit  the vast majority  of patients who  sudden death without warning and in whom no programmed  has been undertaken. remains  that  there  ischae-  stimulation  A l s o , despite the claimed success of such methods, are no e f f e c t i v e  agents  for  preventing  sudden  (Campbell, 1983; 1984), and that enormous numbers of people s t i l l  it  death  die from  sudden death/VF. If  one accepts the  first  few  et-al;,  hours  1971)  of  premise that  myocardial  are the major  ischaemia and i n f a r c t i o n  the  arrhythmias  ischaemia  cause of  (Oliver,  occurring during  (Campbell  death  in  1982), what  et-al;,  patients  the  1981; Adgey  with  myocardial  are the c h a r a c t e r i s t i c s and  importance of the arrhythmias which begin approximately 4 h after the onset of  symptoms  (Campbell et a l . - , 1981)?  Essentially, late  arrhythmias  have  only been of i n t e r e s t 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 i g n i f i c a n t health r i s k themselves. the  There are no c l i n i c a l studies which have attempted to e s t a b l i s h  relationship  ischaemia,  in  the  between extent  of  late  ventricular  arrhythmias  ischaemic muscle mass,  the  and the degree of  onset  of  stenosis  (residual blood f l o w ) , the frequency and extent of re-ischaemia or the r e l a tive  importance  arrhythmias  of  ischaemia  versus  infarction.  occurring more than 24 h a f t e r  The  the onset of  genesis of sudden death has been discussed, and i t  l a t e arrhythmias dence of  role  of  symptoms in  the  appears that there i s no  prognostic value in the analysis of such arrhythmias a d d i t i o n , the poor c o r r e l a t i o n between the i n i t i a l  possible  (Campbell, 1984).  episode of ischaemia and  i s exemplified by the observation that although the  PVCs and VT occurring during the f i r s t  In  10 h after  inci-  the onset  of  - 14 chest pain correlated with i n f a r c t s i z e (determined by measuring serum CPK), the rate of PVC measured 1 - 1 0  months after the onset of chest pain bore no  r e l a t i o n to i n f a r c t s i z e in the same patients (Roberts, et a l ; , 1975). implies that i t  i s possible that the s o - c a l l e d l a t e  arrhythmias  may be a mixture of arrhythmias associated with an old i n f a r c t reentry  around  Introduction),  the  infarct,  arrhythmias  perhaps;  see section  This  in humans  (generated by  on arrhythmogenesis  associated with p e r s i s t i n g p a r t i a l  in  ischaemia and  arrhythmias associated with new bouts of ischaemia. In death  summary, and i t s  clinical  information  relationship  with VF and other  incomplete, owing to the fact do so out of h o s p i t a l . of arrhythmias  and t h e i r  concerning  that the majority  C l i n i c a l information  the  aetiology  of  sudden  ventricular  arrhythmias  of patients  dying suddenly  concerning the natural  is  history  underlying causes in the min, h, days, weeks and  months f o l l o w i n g the onset of symptoms i s confusing, perhaps as a r e s u l t  of  the desire to e s t a b l i s h an ideal  to  protocol  for  tediously order and c l a s s i f y each condition  treatment  and i t s  without having  characteristics.  How-  ever, such an approach i s necessary in order to remove the confusion concerning mechanisms of arrhythmogenesis and the contentious sis.  It  aspects of  progno-  i s c l e a r , however, that the current c l i n i c a l approach has not pro-  vided any e f f e c t i v e prophylaxis against sudden death (Campbell, 1983; 1984; Furberg, 1983b). 1.1.4 Therapeutic approaches T h e o r e t i c a l l y the primary therapeutic aim would be to prevent myocardial ischaemia  from  occurring.  The  Framingham  study  (Kannel  etal.-,  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 persons between the age groups 45 - 54 and 65 - 74 years. frequency in males i s approximately  2-3  In  addition,  times that in females.  the  Age and  - 15 sex are not treatable conditions at present. also i d e n t i f i e d  However, the Framingham study  hypertension, diabetes, c i g a r e t t e  smoking, obesity and the  r a t i o of low versus high density l i p o p r o t e i n serum c h o l e s t e r o l as important i n d i c a t o r s of coronary artery disease and sudden death. does not prove cause-and-effect, i t  While  correlation  i s generally considered that a healthy  l i f e s t y l e , a balanced d i e t and exercise are associated with a low incidence of coronary artery disease and sudden death. A safe e f f e c t i v e prophylaxis against coronary occlusion would be d e s i r able.  In t h i s regard, the r e s u l t s of a long term t r i a l of a s p i r i n in appa-  rently  healthy volunteers i s expected to be published reasonably soon.  theory,  thrombolysis  collateral  will  only  be of  v a s c u l a r i s a t i o n or p a r t i a l  benefit  in  patients  with  effective  o c c l u s i o n , in whom ischaemia i s  complete ( i n whom the delay between the onset of symptoms and the  In  not  initiation  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 functioning ses).  collateral  anastamo-  Since t h i s has not been thoroughly investigated in the many c l i n i c a l  studies of thrombolysis  (Yusuf et a l ; , 1985), then  it  is  not  possible  to  Since coronary artery disease remains a s i g n i f i c a n t health r i s k , then  it  comment f u r t h e r in t h i s regard.  i s expedient to l i m i t the sequelae, namely arrhythmias and i n f a r c t i o n . There are at l e a s t 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), since surgery i s r e q u i r e d , i t  but  i s desirable to i d e n t i f y patients most at r i s k  of sudden death; the implantation of d e f i b r i l l a t o r s  i s not a p r a c t i c e which  can be c a r r i e d out on a very large scale at present. r e c e n t l y been suggested that automatic d e f i b r i l l a t o r s  In a d d i t i o n , i t can reduce the  of l i f e by v i r t u e of the anxiety and fear associated with t h e i r  has  quality  use (Cooper  - 16 et a l ; , 1985). stered.  Alternatively,  pharmacological prophylaxis could be admini-  Such a strategy would be expected to protect  undue inconvenience in terms of and frequent follow-up. exist,  owing to  side-effects  side e f f e c t s ,  the patient  inconvenient  dosing  a lack  of  on the other  proven (e.g.,  benefit  on one hand,  Campbell et a l ; ,  may be used in a large number of of  coronary  regimen  However, a drug s u i t a b l e for t h i s purpose does not  1984).  or  unacceptable  Along with  search for a cure for cancers, the search for a safe e f f e c t i v e  risk  without  artery  subjects who might  disease would  research with the greatest potential  appear  clinical  to  the  agent which  be categorised as at  be the  branch  impact in terms of  of  drug  prolonging  life. In addition infarction.  to arrhythmia prevention,  While  it  would  clearly  attempts  be b e n e f i c i a l  r e s u l t i n g from coronary o c c l u s i o n , t h i s  Experimental  anastomoses have i l l u s t r a t e d  studies  it  is  reasonable to  prevent  that 15 min of ischaemia w i l l  in  collateral  lead to  irrever-  complete reperfusion i s achieved (Hort  While unequivocal c l i n i c a l  suppose that  infarction  u n t i l hours a f t e r the onset  in animals without e f f e c t i v e  s i b l e myocardial c e l l death, even i f and Da Canal i s , 1965b).  to  limit  i s not a r e a l i s t i c goal at present,  p a r t l y because therapy i s usually not i n i t i a t e d of symptoms.  should be made to  the  information  absence of  is lacking,  extensive  collateral  v a s c u l a r i s a t i o n , much the same w i l l occur in humans. Theoretically, order 1971).  to  reduce the  likelihood  size  of "cardiac output f a i l u r e  However, unless an agent i s given as prophylaxis, i t  imagine how i t of  any measure which reduces i n f a r c t  either  might prevent i n f a r c t i o n ,  converting  fibrous  tissue  m i t o s i s , or converting dead c e l l s  unless i t  into  desirable  (Maroko  in  et-al;,  is d i f f i c u l t  to  possesses the  capability  stimulating  myocardial  muscle,  into l i v i n g  is  cells.  At present, no such  drugs e x i s t , and prevention of myocardial i n f a r c t i o n with a view to  limiting  - 17 pump f a i l u r e and cardiogenic shock (Agress e t a l ; , 1952) has not been demonstrated  either  clinically  or  experimentally  (Reimer  and Jennings,  1985).  However, i t might be hoped that a f a s t - a c t i n g myocardioplastic growth factor might one day be developed. 1.2  Experimental-myocardial ischaemia and i n f a r c t i o n 1.2.1 Overview For an animal model of any human disease to be i d e a l , i t  the following c h a r a c t e r i s t i c s .  should  exhibit  It should:  a.  completely mimic at l e a s t one aspect of the disease  b.  respond in the same manner as humans to drugs  c.  have the precision 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, time, and exper-  tise. There i s no model of myocardial cates the human c o n d i t i o n .  ischaemia and i n f a r c t i o n which  This was the conclusion of  the Coronary Heart  Disease Task Group Panel Report of 1973 (see Winbury, 1975). Workshop on More Uniform  Animal  Models and Protocols  dupli-  for  In 1978, the Assessment of  Interventions to Protect Ischemic Myocardium held at the NIH recommended the study of 3 coronary occlusion preparations, the rat for sive and rapid screening of t i s e d dog ' f o r ation'  verification  potentially  of effectiveness  and the conscious dog ' f o r  the most p h y s i o l o g i c a l  useful  conditions'  testing  'relatively  treatments',  inexpen-  the anaesthe-  in a more p h y s i o l o g i c a l  situ-  the most promising agents under  (see Reimer et a l ; , 1985).  The  latter  recommendations may perhaps appear peculiar when factors other than ' p h y s i o logical species).  relevance'  are  considered  (see  section  dealing  with  choice  of  In a d d i t i o n , i t has been suggested that 'no animal heart i s t r u l y  comparable to that of man' (Sasyniuk and N a t t e l , 1982) implying that r e l a -  - 18 t i v e rather than absolute p h y s i o l o g i c a l relevance i s the issue in question, and as such should not be the primary consideration in the choice of e x p e r i mental species. 1.2.2 Methods for producing ischaemia A r t e r i o s c l e r o s i s may be induced with a high cholesterol with  the  possible  exceptions  et a l ; , 1983), mini-pig  of  the  diet.  hypercholesterolaemic  (Jacobsson, 1984), and quail  However,  hare  (Pearson  (Cheung et - a l ; , 1983)  t h i s method i s not considered to be s p e c i f i c for  the coronary v e s s e l s .  most  produced,  investigations,  approach  is  rarely  generalised used  stenosis  (Winbury,  is  1964).  therefore  In this  Hypercholesterolaemia-induced  coronary a r t e r i o s c l e r o s i s models are also compromised owing to the necessity that  the  within  endpoint  a  (development  prescribed  inconvenient.  It  time-frame,  of  infarction or  or  arrhythmias)  experimentation  may be possible to circumvent t h i s  becomes  must  occur  unacceptably  problem by increasing  oxygen demand by a t r i a l  pacing in the s e t t i n g of a compromised oxygen supply  induced by cholesterol  feeding, but t h i s  method produces only  reversible,  pacing-dependent ischaemia (demonstrated by S-T segment e l e v a t i o n ) ;  arrhyth-  mias and i n f a r c t i o n do not occur (Lee and Baky, 1973). There are several techniques for diffuse  myocardial  ischaemia.  producing small  By i n j e c t i n g  starch  vessel  occlusion and  suspensions  (Roos and  Smith, 1948), p l a s t i c microspheres (Weber et a l . , 1972) or lycopodium spores (Guzman et a l . , 1962), i t ing  to  generalised  i s possible to produce a r t e r i o l a r  and d i f f u s e  myocardial  infarcts.  occlusion l e a d -  This  technique  was  f i r s t t r i e d using powder, wax, o i l and ink, more than 100 years ago (Panum, 1862, see Tillmanns e t a l ; , techniques. it  Firstly,  1975).  these  in order to preclude generalised systemic o c c l u s i o n ,  i s necessary to i n j e c t the material  itates either  There are many disadvantages of  into a coronary a r t e r y .  open-chest experimentation,  This necess-  or t e c h n i c a l l y demanding coronary  c a t h e t e r i s a t i o n techniques.  Secondly, most animals die from cardiac output  f a i l u r e (Weber et a l ; , 1972), which i s not the major cause of death in c l i n i c a l myocardial ischaemia (Campbell, 1983; 1984) or in other models of myoc a r d i a l ischaemia.  Thirdly, arteriolar  ischaemia and i n f a r c t i o n d i f f e r s  (Gensini et a l ; , 1971).  from  arteriosclerosis  While t h i s ,  of  in i t s e l f ,  expected to be d i f f i c u l t to quantify,  thereby making i t  mine the e f f e c t  size.  of a drug on i n f a r c t  large  coronary  i s not n e c e s s a r i l y  a disadvantage, the d i f f u s e character of the experimental  importantly,  diffuse  extensively from c l i n i c a l occlusion-induced  ischaemia which generally r e s u l t s arteries  occlusion and the r e s u l t a n t  infarct  would be  d i f f i c u l t to  In a d d i t i o n ,  deter-  and perhaps more  models of arrhythmogenesis (see elsewhere) are contingent  the presence of  a well  defined focus of  ischaemia which must be of  upon suff-  i c i e n t volume to serve as a substrate for conduction delays and the generat i o n of  i n j u r y currents  (Gettes,  etc.).  These models of  arrhythmogenesis  ischaemia preparations.  1974; Janse,  1982; Kleber e t - a l . ,  would not  Indeed, the d i f f u s e  appear to be associated with v e n t r i c u l a r  apply  to  1978;  the  diffuse  ischaemia preparations  arrhythmias  at  all  do not  (see Winbury,  1975). There are a v a r i e t y  of  techniques  coronary artery by embolisation. t i o n of Direct  may be  producing occlusion of  The 2 major approaches are d i r e c t  an o c c l u s i v e embolus and the embolisation  for  induced  1969), placing a s t a i n l e s s steel  induction by  of  injecting  cylinder  an occlusive mercury  et a l . , 1974).  In a d d i t i o n ,  or i n f l a t i n g it  producthrombus.  (Lluch  et-al;,  (Nakhjavan et a l ; , 1968),  b a l l bearing ( R i b i e l i m a , 1964) or detachable catheter t i p 1962) into a coronary a r t e r y ,  a major  steel  (Hammer and P i s a ,  an intracoronary balloon (Corday  has been demonstrated that i f  a cylindrical  magnet i s placed around a coronary a r t e r y ,  and small  particles  be captured by the magnet and  administered,  the  particles  will  (4 um diameter)  iron  - 20 occlude the vessel (Elzinga et - a l , 1969).  These techniques are a l l capable  of producing complete o c c l u s i o n , although t h i s must be v e r i f i e d by measuring coronary  blood  flow.  Partial  embolisation by i n s e r t i n g  occlusion may be brought  a cylinder  of  lead f o i l  about  via  direct  into a coronary  artery;  the c y l i n d e r w i l l pass down the artery for a distance governed by i t s  outer  circumference, while the degree of stenosis i s governed by the inner circumference (Johnsrude and Goodrich, 1969). known degree of  s t e n o s i s , but  the  This technique i n i t i a l l y  device  is  thrombogenic,  produces a  and serves  to  induce secondary progressive s t e n o s i s . Deliberate thrombogenic techniques include e l e c t r i c a l l y - i n d u c e d genesis, and placement of a thrombogenic foreign object ery.  Placement of an electrode in a coronary artery  the chest wall can produce thrombi i f z a r , 1961). electrode.  current  thrombo-  in a coronary  art-  lumen and another on  i s passed between them ( S a l a -  This study reported d i f f u s e thrombi d i s t a l to the  intraluminal  However, Weiss (1971) managed to produce complete occlusion of  the circumflex or LAD artery using s i m i l a r techniques, and used the preparation to evaluate antiarrhythmic  drugs.  The e l e c t r i c a l production of  bi i s used today by Lucchesi for evaluating antiarrhythmic agents in dogs ( e . g . , thrombi vessels,  Patterson et a l • , 1981; 1983).  may be produced for  by  inserting  thrombogenic  thrombus  anti-infarct  Foreign-body-induced objects  example, magnesium a l l o y or copper h e l i c e s  1972); the r e s u l t a n t  and  throm-  into  (Kordenat  is generally located at the s i t e  coronary et-aU,  of the  in-  sertion. The most common means of producing myocardial ischaemia in use today i s occlusion of a major coronary artery by c o n s t r i c t i o n . of techniques for producing t h i s end (see below).  There are a v a r i e t y  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 rings of hygroscopic material which swell and progressively reduce the lumen of the artery which they have been placed around.  Rings of  formation  of  gelatin  impregnated with  granulomatous  diacetyl  t i s s u e which w i l l  phosphate  gradually  induce  constrict  the  c i r c l e d vessel (Asada e t a l . - , 1962), in a manner analogous with aneroids. similar  result  can be produced by the use of pneumatic cuffs  Gregg, 1967; Bond et a l . - , 1973) Hood et a l ; , 1970).  or  hydraulic  cuffs  (Khouri  (Khouri  the enA and  et a l . , 1968;  A major l i m i t a t i o n of these techniques i s the r e q u i r e -  ment for the placement of a coronary flow-probe  in order to e s t a b l i s h the  extent of occlusion and the moment of complete o c c l u s i o n .  A l s o , the prob-  lems associated with the a r t e r i o s c l e r o s i s models (in addition to the problem of generalised systemic a r t e r i o s c l e r o s i s ) also apply to the gradual coronary occlusion  models,  namely  that  production  of  ischaemia,  arrhythmias  i n f a r c t i o n does not take place within a convenient time period for  and  experi-  mentation. Abrupt, complete coronary artery occlusion i s perhaps the simplest and most r a t i o n a l because two still  technique variables  for  investigating  which  are d i f f i c u l t  myocardial to  ischaemia.  measure and more  to regulate are removed from the experimental arena.  are the  time course of  is  difficult  These v a r i a b l e s  occlusion and the absolute blood flow.  course of occlusion i s known only for  This  The time  abrupt occlusion techniques,  whilst  bloodflow in an occluded vessel i s only known i f occlusion i s 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 i s therefore the extent collateral  blood  flow.  Abrupt  of  occlusion may be produced by clamping an  artery with screw clamps (Gregg e t a l -., 1939), bulldog clamps or Goldblatt clamps (Jennings et a l ; , 1960). coronary a r t e r i e s  it  is  In small animals (such as r a t s ) with small  possible to cauterise a major vessel  electrically  - 22 (Staab et a l . - , 1977;  Prum e t - a l . - ,  1984).  More simply,  a vessel  may be  1igated. Coronary l i g a t i o n was f i r s t undertaken by Chirac in 1698, who observed a loss  of  heart  Interest  in  movement  as  a consequence  the dependence of  heart  (see  activity  Tillmanns  et-al.,  on the coronary  1975).  circulation  resumed in the 19th century, when Erichsen (1842) determined the duration of occlusion necessary for quently,  Cohnheim (1881)  producing v e n t r i c u l a r ligated  standstill  dog coronary  arteries  in dogs.  Subse-  and developed the  hypothesis that the coronary c i r c u l a t i o n i s comprised mainly of end a r t e r i e s (see Tillmanns e t - a l . - , 1975 for  translation  and d i s c u s s i o n ) .  In the  20th  century, l i g a t i o n of a coronary artery together with ECG recording was f i r s t c a r r i e d out  in 1918 (Smith),  (Johnston e t - a l : ) . dial  The reason for the apparent lack of i n t e r e s t  ischaemia in the e a r l y  belief  but was r a r e l y c a r r i e d out again u n t i l  part  of  this  century  at that time that coronary occlusion i s  1935  in myocar-  stems from the  general  a universally fatal  event  (see Fye, 1985 f o r review). In most l i g a t i o n  techniques a simple s i l k  ligature  is  used.  To gain  access to the designated artery and tighten the l i g a t i o n , experiments were initially  carried  However, for  out  using  open-chest  dogs  (Townshend  long term experiments on m o r t a l i t y ,  Porter,  1894).  chests were subsequently  closed and animals allowed to regain consciousness ( e . g . , Smith, 1918; Le Roy et a l ; , 1942). tion.  Most e a r l y experiments involved 1-stage coronary l i g a -  However, an adaptation of a technique designed for  of a coronary artery was used by Harris to stages  (Harris,  1950).  Harris  ligate  had observed that  partial  ligation  coronary a r t e r i e s abrupt  occlusion of  in 2 the  l e f t anterior descending coronary artery produced VF in approximately 50 % of dogs ( H a r r i s , 1948) within the f i r s t 10 min after  ligation.  Animals s u r v i -  ving t h i s i n s u l t experienced few arrhythmias u n t i l approximately 4.5 h a f t e r  l i g a t i o n , whereupon PVCs ensued, increasing in i n t e n s i t y with time; by 8 h, frequent VT developed, p e r s i s t i n g 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 o c c l u sion  could be circumvented  creased,  allowing  for  the  frequency of  a more d e t a i l e d  second phase (4.5 - 8 h) Therefore, an i n i t i a l  then  and t h i r d  partial  survival  and s p e c i f i c  would  be  in-  investigation  of  the  phase (8 h to 4 days)  of  arrhythmias.  l i g a t i o n was undertaken by including a needle  in the loop of the l i g a t u r e , then removing the needle once the l i g a t u r e had been tightened; the lumen of the a r t e r y was therefore stenosed to the diameter of the needle. After 30 min or 1 h, a second l i g a t u r e was tightened the p o s i t i o n of the f i r s t to f u l l y occlude the v e s s e l . e l i m i n a t i o n of phase-1 arrhythmias after  phase-2 and phase-3  arrhyth-  It i s of i n t e r e s t to note that t h i s much-quoted work of Harris (1950)  was in f a c t pre-empted, to a c e r t a i n extent, lis,  The r e s u l t was the  (those occurring during the f i r s t 10 min  complete occlusion) without change in  mias.  at  1894),  in  which  it  was demonstrated  branches were t i e d before the  larger  by much e a r l i e r work (Michaethat  if  small  coronary  artery  a r t e r i e s then cardiac s t a n d s t i l l  was  prevented. A s i g n i f i c a n t advance was made by the development of a noose-type device for  'atraumatic'  occlusion.  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 r a c t i o n  the v e s s e l .  between the tubes would occlude  Rushmer e t - a l ; , (1963) used the same p r i n c i p l e , s u b s t i t u t i n g a  nylon suture  for  occlusion  conscious dogs.  in  the  small  polythene This  tube,  in  technique  order  allowed  to  produce  arrhythmias  coronary and  in-  f a r c t i o n to be investigated in the absence of anaesthetic and acute s u r g i c a l preparation for the f i r s t time. technique was made for  Unfortunately,  almost 20 y e a r s .  perhaps, l i t t l e use of  this  Coronary occlusion in conscious  animals was not  investigated e x t e n s i v e l y u n t i l  our  laboratory  developed a  method for coronary occlusion in conscious rats (Au et a l ; , 1979a), using an occluder  similar  to  that  used by Rushmer.  The use of  a  'pre-prepared  1  animal with a loose occluder implanted around a coronary artery  and e x t e r -  iorised  absence  through  the  skin  for  induction  of  occlusion  in  the  anaesthetic and recent surgery may be an extremely important recent work has suggested that much of the information gations  into  ischaemia-induced arrhythmogenesis  in  of  advance, since  provided by i n v e s t i -  anaesthetised,  acutely  prepared animals i s misleading (see section dealing with arrhythmogenesis  in  Discussion). 1.2.3 Differences between species 1.2.3.1 I n f a r c t i o n .  I n f a r c t s i z e reduction has been  investigated in dogs, and therefore  the majority  traditionally  of the l i t e r a t u r e  ning myocardial i n f a r c t i o n r e f e r s to t h i s species.  However, the  concer-  variability  of i n f a r c t s i z e in dogs has been suggested to make t h i s species e s s e n t i a l l y useless for This fact  quantitative  assessment of  infarct  size  and i t s  modification.  was recognised as long ago as 1918, and was a t t r i b u t e d to  presence of large and varied c o l l a t e r a l anastamoses (Smith, 1918). reason, Johns and Olson (1954) developed the rat infarction,  and  concluded  that  any  species  the  For t h i s  preparation for assessing  with  undeveloped  collateral  v a s c u l a r i s a t i o n (rats and mice) would be s u i t a b l e for evaluating  infarction,  whereas species with v a r i a b l e coronary vasculature (hamsters and dogs) and species with extensive c o l l a t e r a l anastomoses (guinea-pigs) would be unsuitable for study.  Perhaps s u r p r i s i n g l y , t h i s work was completely ignored (see  section dealing with coronary occlusion in r a t s ) ,  and studies of myocardial  ischaemia and i n f a r c t i o n  continued to be c a r r i e d out  using dogs.  was work  Indeed,  it  using  dogs which  (almost led  exclusively)  Braunwald's  (Maroko e t - a l . , 1971) to propose the concept of myocardial salvage.  group It was  - 25 suggested that  it  should be possible to  reduce the  ultimate  size  of  an  i n f a r c t by measures 'designed for reduction of myocardial oxygen demands and improvement of coronary p e r f u s i o n .  It  1  investigators  (Maroko et a l • , 1971)  manipulation  (ouabain,  isoproterenol)  glucagon,  is  did  perhaps s i g n i f i c a n t  not  in  propranolol,  fact  demonstrate  haemorrhage,  reduced i n f a r c t s i z e as such.  that  The index of  these  that  any  methoxamine infarction  or used  was S-T segment e l e v a t i o n , measured from DC electrograms recorded from the surface of the heart.  In only 2 groups was i n f a r c t s i z e (at 24 h) recorded,  and evidence that i n f a r c t s i z e was a l t e r e d was not convincing. The many experimental  and c l i n i c a l  ing years have not confirmed the infarct drug  s i z e can be reduced in  treatment,  Although i t  is  under  the  hypothesis  of  a potentially  condition  of  Maroko et - al.-  clinically  irreversible  of  that  manner by  coronary  occlusion.  ischaemia and the  (see Reimer and Jennings, 1984).  C e r t a i n l y , no treatment has yet been claimed to have prevented nature  (1971)  the evidence does not suggest that any treatment  death in non-perfused t i s s u e  The v a r i a b l e  follow-  useful  possible to delay both the ECG signs of  development of i n f a r c t i o n , can prevent  studies c a r r i e d out over the  the outcome of  coronary  infarction.  occlusion  in  dogs  is  exemplified by a study c a r r i e d out by Sobel and associates (Shell et a l ; , 1971).  These authors developed the method of q u a n t i f i c a t i o n  based on serum l e v e l s of CPK.  of i n f a r c t s i z e  They found that peak serum CPK correlated  l i n e a r l y with i n f a r c t s i z e determined at 24 h by measuring the myocardial content of CPK.  However, the actual  24 h was highly  variable,  the  range of  that the c o e f f i c i e n t grel  dogs and 73% in  infarct size  21.5 ± 18 (mean ± s . d . )  values being 1-55%.  i n f a r c t s i z e in dogs.  value of  These values  in 22 dogs at  as % v e n t r i c u l a r are  typical  of  For example, Burmeister and Reynolds (1983) of v a r i a t i o n  ( s . d . as a%of  beagles, while  weight,  published reported  the mean) was 23% in mon-  Miyazaki et-al.-  (1984)  reported  that  - 26 i n f a r c t s i z e varied from 0.6 to 46 %of t o t a l v e n t r i c u l a r weight, with a 60% c o e f f i c i e n t of v a r i a t i o n in mongrel dogs. With v a r i a b i l i t y reports  of  generated  such as t h i s ,  'myocardial (e.g.,  salvage'  Jugdutt,  it  is d i f f i c u l t  have been generated  imagine how so many and continue  to  1985; Bednar et a l . , 1985; Tumas et a l . ,  Perhaps the many reports of i n f a r c t s i z e reductions of sampling e r r o r ,  to  or measurement of  'ultimate'  be  1985).  in dogs are the  result  i n f a r c t s i z e too soon after  occlusion. 1.2.3.2 arrhythmias  first  Arrhythmias. occur  In  in the  second major phase appears after  conscious  period 4 - 2 0  rats,  severe  min a f t e r  1 h and l a s t s for  ventricular  o c c l u s i o n , and a  2 - 6 h (Clark  et-al;,  1980; Johnston e t - a l ; , 1983a).  In dogs, ischaemia-induced arrhythmias were  originally  occurring during  peaking  at  described as e a r l y , approximately  24  h  after  coronary  the f i r s t  hour,  and l a t e ,  occlusion  (Harris,  1950).  However, recently the e a r l y (phase-1) arrhythmias have been subdivided into p h a s e - l a , occurring during the f i r s t 1 - 3 occurring  5-20  Meesman, 1982).  min after  coronary  min of ischaemia, and phase-lb,  occlusion  (Haase and S c h i l l e r ,  1969;  Phase-la arrhythmias, which u s u a l l y comprise only of PVC,  have only been reported in anaesthetised dogs; information occurrence in conscious dogs i s not a v a i l a b l e at present.  concerning  their  However, the type  of anaesthetic used appears to influence the occurrence of phase-la arrhythmias, in that they are f a r more frequent barbitone  than  dogs  anaesthetised  nitrous oxide (Meesman, 1982).  in dogs anaesthetised with pento-  with  morphine-chloralose-urethane  In a d d i t i o n , phase-la arrhythmias  reported to occur in pentobarbitone anaesthetised rats not conscious rats (Johnston et a l . , 1983a).  or  have been  (Fagbemi, 1984),  but  Phase-la arrhythmias have also  been reported in pentobarbitone anaesthetised pigs (Bergey e t - a l . , 1982); i s not established whether such arrhythmias occur in conscious p i g s .  it  - 27 In  dogs 24 h a f t e r  o c c l u s i o n , episodes of  commonly observed (Smith, 1918; H a r r i s , 1950).  VT and m u l t i f o c a l  In our laboratory, we have  r a r e l y observed VT and never observed VF in rats a l l 24 h survivors e x h i b i t m u l t i f o c a l between rats  and dogs has not  PVCs.  PVC are  at 24 h, although  The reason for t h i s  been examined, however  it  is  almost  difference possible  to  speculate that the presence or absence of c o l l a t e r a l  anastamoses may govern  the s e v e r i t y of 24 h arrhythmias.  it  that abnormal automaticity by c o l l a t e r a l perfusion) man e t a l ; ,  nature  of  functional  collateral  regard,  has been suggested  in p a r t i a l l y ishaemic Purkinje f i b r e s  (maintained  i s responsible f o r 24 h arrhythmias in dogs ( F r i e d -  1973; Lazzara e t a l ; ,  multifocal  In t h i s  these  1973).  This idea i s consistent with the  arrhythmias.  In  rats,  the  relative  lack  of  anastamoses compared with dogs (Maxwell e t - a l ; , 1984;  Winkler e t a l ; , 1984) may not permit s u r v i v a l of s u f f i c i e n t  Purkinje t i s s u e  to t r i g g e r VT and VF 24 h after o c c l u s i o n . In conscious r a t s , arrhythmias occurring during the f i r s t 4 hours occlusion have been described in d e t a i l our  laboratory  after  on the basis of many experiments  (Johnston et a l . , 1983a).  Ventricular  tremely common and include PVC, VT and VF. v e n t r i c u l a r blocks are much less common.  arrhythmias  in  are ex-  Sinus bradycardia and  Very o c c a s i o n a l l y , a t r i a l  atrioarrhyth-  mias ( f i b r i l l o f l u t t e r ) and supraventricular tachycardia are seen. Spontaneous reversion of VT during acute myocardial ischaemia i s common, both experimentally reported  and c l i n i c a l l y .  Spontaneous reversion  evidence to substantiate t h i s b e l i e f . (approximately  90 %), and the  However, there  sustained VF i s even higher  is l i t t l e  but  objective  In conscious rats the incidence of VF incidence of  ation i s correspondingly high (approximately of  VF has been  in humans (Robinson and Bredeck, 1917; Maseri et - a l . - , 1982),  the incidence i s considered to be low.  i s high  of  60 %).  (approximately  spontaneous  defibrill-  However, the incidence  90 %), according to  experi-  - 28 merits  from our  laboratory  been occasional reports Gibson e t - a l . , 1986).  (Johnston e t - a l . ,  ular  In  dogs, there  In p i g s , spontaneous reversion of VF has been observlaboratory),  although  this  event  In the absence of any evidence that spontaneously reverting arrhythmias  differ  fundamentally  from sustained v e n t r i c u l a r  in  arrhythmias,  origin  there  maintenance  or  disadvantage.  ventricmechanism  On the  t r a r y , our laboratory has shown that spontaneous d e f i b r i l l a t i o n animal  is  i s no reason to believe that  spontaneous reversion of VF i s an experimental  experimental  have  of spontaneous reversion of VF ( e . g . , Smith, 1918;  ed (unpublished observations from our rare.  1983a).  to s u r v i v e , allowing for  an increase in  con-  permits  the  the y i e l d  of  information from each preparation (Johnston e t - a l . - , 1983a). 1.2.3.3 myocardial  Choice- of -species.  ischaemia and i n f a r c t i o n  The  section  dealing  with  clinical  suggested that most deaths in  patients  with myocardial ischaemia occur from VF, and that the incidence of VF declines  exponentially  (Campbell  et-al;,  treatments  for  with  time  1981).  following  Therefore  arrhythmias  it  the is  initial  of  great  onset  of  importance  associated with acute myocardial  chest to  pain  develop  ischaemia.  It  has been suggested that a drug which can be taken as prophylaxis by patients at high r i s k of myocardial ischaemia to prevent VF would have far more impact  on  mortality  (Campbell, 1984). few  hours,  than  therapy  after  admission  to  hospital  This implies that arrhythmias occurring during the  and p a r t i c u l a r l y  during  ischaemia are the  arrhythmias  Clinically,  acute  these  initiated  the  first  few  minutes  which must be investigated  arrhythmias  are  generally  of  myocardial  in t h i s  missed;  first  regard.  a patient  has  e i t h e r died from them, been resuscitated by paramedics, o r , perhaps, recovered spontaneously by the time they are admitted to h o s p i t a l . a dilemma.  One may e i t h e r  investigate  early  arrhythmias  animals (phase-1 according to H a r r i s , 1950) or late (phase-3)  This leads to in  experimental  arrhythmias.  - 29 -  Any experimental  preparation  cannot be evaluated for  its  in which phase-1 arrhythmias  are measured  ' c l i n i c a l r e l e v a n c e ' , because there i s  i c i e n t information concerning these arrhythmias and t h e i r drugs in humans to provide a useful template.  insuff-  susceptibility  Therefore i t  to  i s desirable to  use several animal species for such s t u d i e s . Alternatively, assumption that  one may concentrate  drugs which  on phase-3 arrhythmias,  reduce phase-3 arrhythmias  will  phase-1 arrhythmias (in other words that common mechanisms of  under  the  also reduce arrhythmogen-  e s i s operate for phase-1 and phase-3 arrhythmias). Essentially, criteria  therefore,  one must  establish  (depending on which experimental  one of  strategy  the  two  following  i s 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  it  i s decided to investigate phase-1 arrhythmias  the only concern should be the l o g i s t i c  then  c h a r a c t e r i s t i c s of the model.  It  must be established 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 i s redundant). Alternatively,  if  phase-3 arrhythmias are to be studied as a model of  phase-1 arrhythmias then i t  follows that a drug which i s found to  phase-3 arrhythmias should also i n h i b i t  phase-1 arrhythmias.  inhibit  Therefore,  it  i s of absolute necessity to e s t a b l i s h that a l l drugs which are e f f e c t i v e or ineffective  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 preparation.  The majority  of published reports  been c a r r i e d out do s a t i s f y t h i s c r i t e r i o n .  in which such comparisons have It f o l l o w s , t h e r e f o r e , that the  evaluation of drugs for potential c l i n i c a l use as prophylaxis against phase1 arrhythmias must not be based upon an extrapolation from a c t i v i t y phase-3 arrhythmias. ventions f o r  Such a strategy  preventing f i r e s  i s analogous to  investigating  in the home by measuring the e f f e c t  against interof  the  - 30 experimental intervention on room temperature. It own  i s , however, permissible to investigate phase-3 arrhythmias for  sake.  In  this  regard,  clinical  relevance  may be  tested  their  directly,  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  important c l i n i c a l r a m i f i c a t i o n s .  it  in the home, i f  acute  out-of-hospital  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) enormous consequences in  may have  With further reference to the analogy of  the study of the prevention of f i r e s VF i s analogous to a f i r e ,  are studied  terms of  lives  saved,  the  prevention  will  have  of phase-3  arrhythmias may have l i t t l e therapeutic impact. All  the techniques for  producing myocardial ischaemia, with the excep-  t i o n of e l e c t r o c a u t e r y , were developed using dogs.  Until  1985, dogs were  e a s i l y the most common species used in studies of myocardial ischaemia and infarction.  Recently, the  rat  has become popular.  does not n e c e s s a r i l y imply s u i t a b i l i t y . concerning the ideal choice of species for mia and i n f a r c t i o n .  It  Popularity,  however,  There i s no concensus at  present  i n v e s t i g a t i n g myocardial  ischae-  has been argued that there i s no ideal species for  such s t u d i e s , and that the choice depends upon the objects and goals of an i n v e s t i g a t i o n (Fozzard, 1975; Harken et a l . , 1981). In  contrast  infection,  with  animal  models of  other  diseases,  in which a high degree of s i m i l a r i t y  and human t i s s u e i s d e s i r a b l e , i t  such as  bacterial  between the animal  i s possible to argue that  animals with  hearts s i m i l a r to the human heart are paradoxically unsuitable for of myocardial ischaemia and i n f a r c t i o n . for  this  healthy,  viewpoint.  Firstly,  young human heart  in  studies  There are several unrelated reasons  an animal with terms  tissue  of  a heart  collateral  which resembles the  vascularisation  is  by  definition  a poor substrate for  i n v e s t i g a t i o n s concerned with the behaviour  of the c h r o n i c a l l y sick human heart with extensive atheromae and c o l l a t e r a l development.  Such an animal ought, however, to provide a good model of the  young human heart. t i b l e to f a t a l  It has been suggested that young humans are more suscep-  VF than older humans (Morgan Jones, 1969), and i t  argued that t h i s i s a r e s u l t of the r e l a t i v e in the younger human ( O l i v e r , 1982).  has been  lack of c o l l a t e r a l anastamoses  Pig hearts lack c o l l a t e r a l anastamoses  and have consequently been suggested to resemble young human hearts et a l . , 1978; Verdouw et a l . , 1983).  The problem in t h i s  while  provide  such an animal  may in  ischaemia in otherwise  theory  is  mation concerning the c h a r a c t e r i s t i c s of t h i s c l i n i c a l there  is  e s s e n t i a l l y no information  therapy in such p a t i e n t s . a large scale c l i n i c a l preventing healthy  the  concerning the  study of the effectiveness  signs and sequelae of  any argument  of  very  myocardial  little  situation.  infor-  Moreover,  effectiveness  of  drug  Furthermore, u n t i l the improbable eventuality  humans, then such information  circumstance,  instance i s that  a good model  healthy young humans, there  (Fedor  that  of  acute myocardial will  an animal  a range of drugs ischaemia in  of in  young  remain u n a v a i l a b l e .  In such a  with  collateral  few  anastamoses provides a good model of acute myocardial  effective  ischaemia i n  healthy  young humans i s merely h y p o t h e t i c a l . At the other end of the spectrum, i t  has been suggested that a species  with extensive or varied c o l l a t e r a l v a s c u l a r i s a t i o n resembles the mature or e l d e r l y human heart. moses 1971). weeks  in  response to  The mature human heart may develop c o l l a t e r a l anastathe  slow  developmemt  of  In dogs the development of c o l l a t e r a l following  coronary  artery  ligation  arteriosclerosis  (Schaper,  anastamoses over a period of  may  represent  such  a process  (Eckstein e t - a l ; , 1941). Amongst common experimental species, dog hearts u s u a l l y have w e l l - d e v e l -  - 32 oped c o l l a t e r a l  anastamoses and may resemble the mature or  heart (Schaper, 1971; 1979).  e l d e r l y human  C l e a r l y , i f we assume that a l l other variables  are equal, then a species with an extensive coronary c o l l a t e r a l  circulation  is preferred when myocardial ischaemia in the mature human ( i n whom myocard i a l ischaemia occurs more frequently than younger humans) i s to be modeled. However, there are other c o n s i d e r a t i o n s . any s c i e n t i f i c experiment.  investigation  is  the  prior  One important consideration in knowledge of  the  power of  the  If one i s interested in the e f f e c t of a treatment on a Gaussian  d i s t r i b u t e d v a r i a b l e then the sample s i z e , n, i s dependent on two independent v a r i a b l e s , the effectiveness of the treatment and the v a r i a n c e . species  differ  in  terms  of  the  variance of  a particular  If  variable,  two then  provided there are no other confounding f a c t o r s , the preferred species w i l l be the one with the lower variance.  Consideration of which of the 2 species  i s the most c l o s e l y r e l a t e d to humans becomes of minor importance.  There i s  a tendency, nevertheless, for aesthetic rather than l o g i s t i c considerations to  dictate  the  choice of  species in  myocardial  ischaemia and  infarction  studies. The dog, u n t i l r e c e n t l y , has been the most popular species for gating myocardial  ischaemia and i n f a r c t i o n .  review of models of  coronary artery  investi-  For example, a large  disease published  e x c l u s i v e l y devoted to dog preparations (Winbury, 1975).  in  1975 i s  general almost  However, there  is  an h i s t o r y of evidence which warns that v a r i a t i o n in the coronary anatomy of dogs jeopardises myocardial ischaemia studies by c o n s t i t u t i n g a large source of variance ( e . g . , Smith, 1918).  P a r a d o x i c a l l y , the dog has been favoured  for t h i s very reason; the v a r i a b l e nature of the coronary anatomy of the dog is qualitatively question  arises:  preparation  in  similar is which  it  to that of desirable  the  coronary  the human (see above). to  use an animal  anatomy  parallels  However,  myocardial the  human  the  ischaemia coronary  - 33 anatomy?  In t h i s regard i t  i s important to consider the sample s i z e necess-  ary for a c l i n i c a l study of the effectiveness of a drug in reducing mortality  occurring  during  acute  myocardial  instance i s that the required information  ischaemia.  The  ally  significant  reduction  of  this  in most cases, and has  resolved or has lead to death (see preceeding s e c t i o n s ) . 'post-infarction'  in  i s not a v a i l a b l e , since the acute  phase of myocardial ischaemia occurs out of hospital  regard to the l a t e r  problem  Nevertheless, with  p e r i o d , in order to show a s t a t i s t i c -  mortality  of  10 - 20% (from  approximately  5 - 15% during the f i r s t year) one requires a study sample of several thousands (May, 1983a; 1983b).  It  i s therefore undesirable to use a preparation  which c l o s e l y mimics the human s i t u a t i o n . ted,  the major  source of variance in  myocardial ischaemia i s the extent 1982).  As Meesman has c l e a r l y demonstra-  dogs with regard to  of c o l l a t e r a l  the outcome of  v a s c u l a r i z a t i o n (Meesman,  C o l l a t e r a l flow c o r r e l a t e s not only with arrhythmias (Meesman, 1982)  but also with i n f a r c t s i z e .  It has r e c e n t l y been shown that i n f a r c t s i z e in  dogs can be predicted on the basis of the extent  of c o l l a t e r a l  vasculari-  sation independently of the s i z e of the occluded bed ( ' r i s k zone') et-al;,  1986b).  In  addition,  a recent  study  (Yellon  showed that whereas simple  occlusion of the LAD produced only minor v e n t r i c u l a r arrhythmias, LAD o c c l u sion coupled with the obstruction of retrograde c o l l a t e r a l blood flow to the occluded bed resulted in severe v e n t r i c u l a r e t - a l ; , 1986). sion,  arrhythmias  in 75% dogs (Shoji  Therefore, a species with l i t t l e e f f e c t i v e c o l l a t e r a l  productive  of  consistent  and predictable  regions  of  perfu-  ischaemia upon  coronary o c c l u s i o n , would be more appropriate than the dog, in terms of the p r e c i s i o n and accuracy for a given to sample s i z e . The two species in which a well defined and reproducible area of ischaemia following rat.  coronary occlusion has been documented are the pig and the  Coronary  collateral  anastamoses  connecting  to  the  left  anterior  - 34 descending coronary  artery  in  the  pig  are almost  absent  leading to a sharp demarcation between the normal following 1979).  occlusion  of  Several reports  ventricle  this  artery  (Kleber  (Schaper, 1971),  and the ischaemic zones  et-al.-,  1978;  Janse  suggest that c o l l a t e r a l s are n e g l i g i b l e  et-al;,  in the  rat  (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). ated by the  consistent  extent  of  This suggestion is  ischaemia and i n f a r c t i o n  substanti-  in  rat  hearts  following 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 . ) . one r e p o r t ,  however, which suggests that there i s a difference between rats  and pigs in that gradual induce c o l l a t e r a l  stenosis induced over a period of 3 months  growth in p i g s , s u f f i c i e n t  to  prevent  infarct  upon complete o c c l u s i o n , whereas rats have end a r t e r i e s , develop  There i s  collateral  anastamoses,  even  following  slow  will  formation  and are unable to occlusion  (Schaper  e t - a l ; , 1986). In  summary,  various  hypotheses  concerning  occurring c l i n i c a l l y during and a f t e r  myocardial  into  'clinical  a template  for  determining  studies (see Reimer et a l . , 1985). ing.  It  must be remembered that  the  Clinically,  it  nature  of  arrhythmias  ischaemia have relevance'  of  translated  experimental  This template may be s e r i o u s l y misleadfar  more i s  ischaemia-induced arrhythmias and i n f a r c t i o n humans, p a r t i c u l a r l y  the  during the c r i t i c a l  nature  of  in experimental animals than  in  first  known about  few hours  has been suggested that a lack of  the  after  scientific  occlusion.  basis for  the  management of v e n t r i c u l a r arrhythmias i s founded in poorly c o n t r o l l e d  clini-  cal studies (Campbell, 1983).  nature  of  risk,  during  cause and the  acute myocardial  C l e a r l y , the confusion concerning the  strategy  of  management  ischaemia d i c t a t e s  that  of the  arrhythmias clinical  occurring  concepts  of  - 35 -  arrhythmogenesis should follow from studies with experimental animal prepara t i o n s , rather than govern the a c c e p t a b i l i t y of an experimental approach. I r r e s p e c t i v e of the arguments concerning c l i n i c a l relevance of  different  species, u n t i l a s e r i e s of drugs have been characterised for t h e i r against arrhythmias  and i n f a r c t i o n  in a range of experimental  with established p r e c i s i o n and accuracy, and u n t i l compared with  the  analogous  between complete and p a r t i a l  condition  in  preparations  these r e s u l t s have been  humans with  o c c l u s i o n , abrupt  activity  clear  and gradual  distinction  o c c l u s i o n , the  time of onset of o c c l u s i o n , large and small r i s k zones, the extent of c o l l a teral the  blood supply, the duration degree of  r e p e r f u s i o n , the  l o c a t i o n of p r e - e x i s t a n t vance i s redundant.  of  ischaemia in the case of  extent  infarction,  At present,  been characterised s a t i s f a c t o r i l y ,  of  pre-existant  reperfusion,  infarction  and the  then any discussion of c l i n i c a l  most experimental and sources of  preparations  error  rele-  have  not  variance have not  been recognised and e l i m i n a t e d . The foregoing chapters have alluded to one f a c t which ought to play the key  role  ischaemia  in  the  there  arrhythmias.  choice is  no  Therefore,  of  experimental  is,  without  ischaemia/infarction  related  primary consideration should be the  reprodu-  infarction, the  and  preparation. no  c i b l e production of ischaemia of known s e v e r i t y .  That  The most r a t i o n a l means of  producing ischaemia of known s e v e r i t y i s to abruptly, completely and permane n t l y occlude a coronary artery in a species with minimal c o l l a t e r a l l a r i s a t i o n and l i t t l e i n t r a s p e c i e s v a r i a b i l i t y . advantage i f  the  species were small  (not  vascu-  In a d d i t i o n , i t would be an  requiring  technically  preparative equipment or inconvenient housing), inexpensive (to  demanding  allow large  sample s i z e s ) , robust (to allow rapid recovery from preparative surgery) and 'good natured'  (to permit conscious animal experiments).  which f i t s a l l these requirements, the common laboratory  There i s a species rat.  - 36 1.2.4 A h i s t o r y of coronary occlusion in rats Coronary occlusion in rats was f i r s t His  experiments  were  prompted  by the  undertaken in 1946 by Heimburger. desire  to  investigate  whether  the  production of adhesion of the pericardium to the epicardium was capable of preventing i n f a r c t i o n diac o r i g i n ) . the  anastamoses of  extracar-  His experiments were a f a i l u r e , in as much as i n j e c t i o n s  pericardial  sodium s t e a r a t e , Nevertheless,  (by generation of c o l l a t e r a l  sac of  cod l i v e r  oil,  a l c o h o l , sodium s a l i c y l a t e ,  soap and sodium morrhuate  Heimburger  established that  failed ligation  to  prevent  of  the  into urea,  infarction.  left  coronary  artery in rats produced death within 24 h of occlusion in approximately 25 % of animals, and that extensive i n f a r c t i o n could be produced.  The technique  used for occlusion was l i g a t i o n using a s i l k suture under p o s i t i v e pressure anaesthesia.  The study was e s s e n t i a l l y q u a l i t a t i v e , and no attempt was made  to v e r i f y o c c l u s i o n . Before continuing with the discussion of myocardial the question of the v e r i f i c a t i o n  ischaemia in  of occlusion should be considered.  rats, Veri-  f i c a t i o n of occlusion i s an aspect of experimentation which has been neglected throughout the h i s t o r y of myocardial ischaemia and i n f a r c t i o n Without  such  verification  it  becomes  impossible  to  studies.  distinguish  between  animals (or humans) in which a treatment has prevented or delayed i n f a r c t i o n and animals (or humans) in which occlusion was p a r t i a l or absent, or present but o f f s e t by c o l l a t e r a l v a s c u l a r i s a t i o n . Although the nature of c o l l a t e r a l  v a s c u l a r i z a t i o n has been extensively  documented in pigs and dogs ( e . g . Schaper 1971; work has been undertaken in r a t s .  Meesman, 1982), very  little  E a r l y work (Johns and Olson, 1954; Selye  et a l ; , 1960) showed that the coronary c i r c u l a t i o n in the r a t heart i s very uniform, and that the coronary a r t e r i e s are end a r t e r i e s guinea  pigs  in  which extensive  collateral  (in contrast with  vascularization  is  present).  - 37 Recent radiolabeled microsphere studies in rats have shown that the  extent  of c o l l a t e r a l perfusion in the occluded zone (OZ) i s n e g l i g i b l e , flow being less than 0.01 ml/min/g according to Winkler e t a l ; normal flow according to Maxwell e t a l ; It  should  residual flow,  flow  (1984).  be mentioned  that  in  i s o l a t e d rat  the OZ in  (1984) and 6.1 ± 0.7% of  Kannengiesser et a l • hearts  as assessed by microsphere techniques.  (1975)  was 18% of  found  that  pre-occlusion  This value i s somewhat higher  than the values measured i n - v i v o by Winkler e t - a l ; (1984) and Maxwell e t - a l . (1984).  The higher  owing to  the f a c t  estimate of  flow  is  likely  to  be the more accurate,  that flow measurements are not dependent simply on  the  a b i l i t y of a bed to trap microspheres, as i s generally assumed; microspheres tend to d i s t r i b u t e  preferentially  into beds r e c i v i n g high flow.  The r e s u l t  i s an under-estimation of flow in beds r e c e i v i n g lower flow. It during  has been reported prolonged  infarction  that rats  and gradual  is inevitable  do not  stenosis,  in  develop c o l l a t e r a l contrast  upon complete occlusion in r a t s ,  guinea pigs in which i n f a r c t i o n  also investigated the rat coronary c i r c u l a t i o n . collateral  pigs,  and  that  in contrast  with  is prevented by the extensive  c o l l a t e r a l v a s c u l a r i s a t i o n (Schaper e t - a l ; , 1986).  for  with  anastamoses  pre-existant  Bloor et a l ; (1967) have Although they did not  anastamoses, they reported that the l e f t v e n t r i c l e  l i e d by a s i n g l e large coronary artery  almost a l l received l e f t v e n t r i c u l a r  i s supp-  in more than 90 % of rats  tremely reproducible and consistent manner.  look  in an ex-  Of the remaining 10 % of  perfusion from 2 main a r t e r i e s  rats,  arising  from separate o s t i a , and that the second artery was u s u a l l y an independent septal a r t e r y , not supplying the l e f t v e n t r i c u l a r free w a l l .  in  The work of Heimburger generated no i n t e r e s t  for many years.  1954,  a detailed  several  Johns and Olson decided to small  animals for  their  undertake  applicability  to  However,  comparison  the study of  of  myocardial  - 38 -  ischaemia and i n f a r c t i o n .  The experiments were prompted by the view that  the species which had been e x c l u s i v e l y used up to that time, the dog, was in essence useless for the study of myocardial ischaemia and i n f a r c t i o n ;  'as a  test procedure, coronary occlusion in the dog produces r e s u l t s so v a r i a b l e as to have only l i m i t e d s t a t i s t i c a l  usefulness'.  In t h i s 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 l e a s t as an important part in the choice of a species f o r study as p h y s i o l o g i c a l ident 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 i g s .  The exper-  iments were c a r r i e d out in a manner i d e n t i c a l with that used by Heimburger, (1946)  except that hearts were exposed v i a an i n t e r c o s t a l  incision  rather  than by thoracotomy. In preliminary experiments (Johns and Olson, 1954), the coronary c i r c u l a t i o n was investigated by i n j e c t i n g  lead oxide (as a suspension in melted  gelatin)  into the ascending aorta ( b r i e f l y clamped to r e s t r i c t  coronary  arteries).  The coronary c i r c u l a t i o n  described as s i m i l a r .  lateral  rat  injection  of  The coronary a r t e r i e s lay j u s t beneath the  anastomoses being infrequent).  descending coronary  the  and mouse were  surface, and were described as predominantly end a r t e r i e s  interarterial  anterior  the  Vessels were c l e a r l y v i s i b l e f o l l o w i n g  contrast medium and d i s s e c t i o n . epicardial  of  flow to  artery  (LAD)  (col-  In a d d i t i o n , the  was described as the  left  dominant  a r t e r y , whereas the circumflex artery was described as merely a small branch of the LAD.  For t h i s reason, i t  occlusion  rats  atrial  in  (and mice),  i s appropriate to describe the artery  that  artery  which  emerges from under  appendage, as the l e f t main coronary a r t e r y ,  of the  although most i n v e s t i -  gators commonly r e f e r 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 r a t  to  - 39 coronary  occlusion has been used to  c i r c u l a t i o n in l i m i t i n g i n f a r c t i o n described  the  guinea  pig  as  investigate  the  role  (Winkler e t - a l , 1984).  having  v i s i b l e on the e p i c a r d i a l surface.  prominent  coronary  of  collateral  Johns and Olson arteries  clearly  The circumflex was well developed, and  both t h i s vessel and the r i g h t coronary artery were described as the source of  large,  prominent  and profuse  collateral  anastomoses feeding  into  the  region supplied by the LAD. In  keeping with the extent  and the r e p r o d u c i b i l i t y  of  the  collateral  v a s c u l a r i s a t i o n in rats and mice compared with hamsters and guinea p i g s , the respective  incidences of  infarction  were 83,  64,  31 and 25% (Johns  and  Olson, 1954). Despite  the  extent  of  their  study,  c e r t a i n gross pieces of misinformation. that VF was absent in r a t s . misleading statement.  Johns and Olson (1954) In p a r t i c u l a r ,  However, there  is  generated  the authors  stated  a simple reason for  this  The authors did not record the ECG, and VF was d i a g -  nosed by observing ' s t r i k i n g changes in rate and rhythm' of the heart while the chest was open. and since l a t e r  Since the chest was closed immediately a f t e r o c c l u s i o n ,  studies  ( e . g . , Johnston e t - a l . , 1983a) have c l e a r l y shown  that v e n t r i c u l a r arrhythmias do not occur u n t i l several min a f t e r o c c l u s i o n , i t i s no wonder that the authors did not ' s e e ' VF.  Nevertheless, the s t a t e -  ment that ' i n contrast to dogs, mice and r a t s are able to survive occlusion of  the  1954)  left  main coronary a r t e r y ;  undoubtedly  served to  study of myocardial 'this  VF does not  occur'  (Johns and Olson,  dissuade researchers from using rats  ischaemia and i n f a r c t i o n ,  in  the  despite the conclusion that  method of coronary occlusion produces a t e s t  infarct  which  i s more  nearly standard than any c u r r e n t l y a v a i l a b l e ' . Before discussing further developments in the h i s t o r y of coronary o c c l u sion in the r a t ,  a comment should be made concerning the smallness of  the  - 40 coronary  arteries  of  the  rat,  as  described by  Johns  and Olson  (1954).  Neither Johns and Olson, nor Heimburger (1946) mentioned that t h e i r o c c l u sions must (by v i r t u e of the i m p o s s i b i l i t y of d i s s e c t i n g free almost i n v i s i b l e artery  imbedded in the myocardium) have included some myocardium with  the l i g a t e d a r t e r y .  It  i s possible that i f  muscle l i g a t e d then spurious sis.  the artery  'small i n f a r c t s '  i s missed and only  may form, confounding analy-  However, according to Heimburger (1946), l i g a t i o n of myocardium alone,  or the l e f t tion).  coronary vein alone produced no s i g n i f i c a n t  In other  words,  unless the  LAD i s  included  in  sequelae the  (infarc-  ligation,  no  i n f a r c t i o n occurs. From 1954 to 1973 very l i t t l e work was done using rat occlusion preparations.  Bryant  investigate  et-al;  the  (1958)  infarct  used Johns and Olson's technique  process by electron  microscopy.  (1954)  They found  to that  gross s t r u c t u r a l changes in the myocardium did not occur u n t i l at l e a s t 5 h a f t e r o c c l u s i o n , by l i g h t microscopy.  However, electron microscopy revealed  swelling of the sarcoplasmic reticulum and mitochondrial only 1 h of o c c l u s i o n . ment of  mitochondria  Animals s a c r i f i c e d at disruption,  enlargement  after  By 2 h, deposition of l i p i d bodies, uniform enlargeand p a r t i a l later  such as l i p i d  disruption  of  myofibrils  were  times showed i n c r e a s i n g l y severe body formation  in  cell  nuclei  (4  present.  intracellular h),  loss  of  d e f i n i t i o n of myofilaments (5 h) general d i s a r r a y 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 associated  extensive i n f i l t r a t i o n of neutrophils  phagocytosis  (48  h).  The authors  and macrophages, with  made no comments on  their  reasons for using r a t s , and gave no i n d i c a t i o n of the r e p r o d u c i b i l i t y of the extent of  infarction.  A year l a t e r ,  Kaufman et a l ; (1959) embarked on a s e r i e s of studies on  i n f a r c t i o n in rats (a s e r i e s which appears to have terminated a f t e r 1 p u b l i cation).  Rats were chosen because they were considered to provide  'large  - 41 numbers' and 'uniform s t o c k ' .  Johns and Olson were c i t e d as the source of  the  These  technique  showed that  of  occlusion.  occlusion  investigators  caused depletion  of  (Kaufman  succinate  et-al.-,  1959)  dehydrogenase  which  began 4 h after occlusion and slowly progressed over the following 12 h.  In  confirmation of the r e s u l t s of Bryant et a l ; , Kaufman e t - a l ; found that no apparent a l t e r a t i o n s h after occlusion. clear  leukocytes  proportions  occurred in gross mycardial structure  However, these authors observed i n f i l t r a t i o n by mononu-  beginning  at  The s c a r r i n g  in  occlusion,  Kaufman e t - a l ;  succinate dehydrogenase and other  those reported  following  occlusion  in  dogs  reaching  'moderate'  (1958) detected no such  process began 36 h after  i n f i l t r a t i o n of f i b r o b l a s t s . reases  18 h after  by 24 h, at a time when Bryant e t - a l ;  infiltration.  u n t i l at l e a s t 6  occlusion with  the  (1959) reported that the decenzymes with  time  according  Jennings  to  paralleled et-al.  (1957), and concluded that the rat was a ' s a t i s f a c t o r y animal for the study of sequential histochemical and morphologic changes in the myocardium'. The following published.  year,  the  much-cited  work  of  Selye et-al.-  (1960)  was  These authors r e i t e r a t e d the view of Johns and Olson (1954) that  the dog, by v i r t u e of the v a r i a b l e outcome of occlusion in t h i s species, unsuitable for  the study of myocardial  ischaemia and i n f a r c t i o n .  is  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 t h e i r  'complicated' procedure, the high post-operative m o r t a l i t y  f a i l u r e to produce an i n f a r c t with every o c c l u s i o n .  and the  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 s m s are completely invalid.  Firstly, it  appears that a l l 3 groups prepared rats using e s s e n t i -  a l l y the same technique. they  both  artificial  The e a r l i e r 2 studies are superior  employed a r t i f i c i a l  respiration.  Selye e t - a l .  in as much as (1960)  used no  r e s p i r a t i o n , 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 ' through  the  make d i r e c t death, study  it  the heart, i . e . ,  intercostal  incision.  comparisons  owing  appears that  temporarily  Secondly, although  to  it  is  'popping'  it  difficult  to  the  imprecise  declarations  of  time  peri-operative  mortality  was 25% in  Heimburger's  (1946), 21% in Johns and Olson's (1954), and 10% in S e l y e ' s  differences which are not p a r t i c u l a r l y  striking.  t i o n was made between death r e s u l t i n g  from occlusion and death  from bad surgery and anaesthetic overdosage.  In  addition,  The myth  of  (1960),  no d i s t i n c -  that  resulting  rats  do  not  develop VF f o l l o w i n g occlusion (Johns and Olson, 1954) presumably confounded Selye e t - a l i ' s perception of the s u p e r i o r i t y of t h e i r  technique, g i v i n g r i s e  to the assumption that any p e r i - o p e r a t i v e death occurs as a r e s u l t of poor technique. Although Selye e t - a l ; acknowledge that  the extent  found that the production the  LAD d i s t a l  mortality,  (1960)  to  its  of  of  did not record the ECG, they appeared to occlusion  'smaller'  origin  influences  infarcts  survival,  by d e l i b e r a t e l y  was associated with  they  occluding  a marked reduction  in  whereas l i g a t i o n of the both l e f t and r i g h t coronary a r t e r i e s  in  the same r a t resulted in immediate death in 80% of animals. (Selye e t - a l . , occlusion  since  of  1960) the  also confirmed  coronary  veins  the report  alone  of  produced  These authors  Heimburger  no sequelae  (1946)  that  (unless  the  e n t i r e coronary sinus was occluded, in which case p e r i c a r d i t i s and s u p e r f i c ial  of  interest  that  animals  s u r v i v i n g the f i r s t 24 h a f t e r occlusion generally a l l survived the  follow-  in  epicardial Selye's  work  c a l c i f i c a t i o n developed).  The only other  concerns long term s u r v i v a l .  It  was found  ing month, death generally occurring only as a r e s u l t of tions'  'accidental  infec-  (Selye e t - a l . , 1960).  Several first  point  report  peculiar  publications  of ECG information  appeared following  in  the  following  occlusion in r a t s  years. was given  The in  - 43 1961, when Normann e t - a l ; , using the  technique of  recorded 5 leads (3 limb and 2 chest) during b r i e f ether anaesthesia).  Johns and Olson  at 2 h a f t e r  occlusion  (1954)  (presumably  Remarkably, no arrhythmias were observed,  although ST segment elevation in the anterior  chest lead was apparent, and  deep Q waves were seen in the same lead at 24 h and 7 days a f t e r o c c l u s i o n . A similar  study was performed by Zsoter and Bajusz in 1962, and the  arrhythmias seen were a few PVCs in 1 r a t .  However in t h i s  study  only  (Zsoter  and Bajusz, 1962) the ECG was not recorded u n t i l 2 days a f t e r o c c l u s i o n . information concerning s u r v i v a l a f t e r occlusion was g i v e n .  No  Bajusz, who had  previously worked with Selye and Zsoter, r e i t e r a t e d the b e l i e f that coronary occlusion causes no arrhythmias in r a t s , in his book on i n f a r c t i o n  (Bajusz,  1963). In  1964 and 1965, 4 d e t a i l e d  and extensive, but  completely neglected  works were published by Hort's group (Hort et a l . , 1964; Hort and Da Canali s , 1965a; 1965b; Hort 1965).  These authors c a r r i e d out coronary occlusion  in over 1000 rats by 1 stage and 2 stage l i g a t i o n , with and without r e p e r f u sion.  According to  t e x t , the c r i t i c a l  the  English  language  abstracts  following  the German  duration of ischaemia necessary f o r production of  e r s i b l e damage was 13 - 15 min.  These authors also stated that  irrev-  infarction  could only be delineated s u f f i c i e n t l y c l e a r l y f o r a n a l y s i s , according to the triphenyltetrazolium measured 10 h or  s t a i n i n g method  more a f t e r  (Jestadt  o c c l u s i o n , whereas that  become i n f a r c t e d could be d i f f e r e n t i a t e d only 15 min a f t e r  occlusion by l i g h t  tissue  1959), when destined  from normal v e n t r i c u l a r microscopy,  ' s t r e c h e d ' versus 'non-streched' muscle f i b r e s i b l y damaged but not yet dead).  and S a n d r i t t e r ,  using the  ('stretched'  to  tissue  at  criterion  of  being i r r e v e r s -  This c r i t e r i o n was suggested to be p o t e n t i -  a l l y useful i n the diagnosis of acute myocardial ischaemia in patients who have died from unknown causes (sudden death) without evidence of  coronary  - 44 thrombosis or i n f a r c t i o n ; t h i s recommendation has been ignored.  The authors  concluded that since the standard error of mean i n f a r c t s i z e was always less than 10% of the mean, then the coronary-1igated rat prepartion was s u i t a b l e for measuring the e f f e c t of drug treatment on i n f a r c t s i z e .  In t h i s regard,  these authors (Hort and Da Canal i s , 1965a;1965b) provided sound q u a n t i t a t i v e evidence of r e p r o d u c i b i l i t y to support the e a r l i e r semiquantitative evidence of Heimburger (1946) and Johns and Olson (1954). Despite the c l e a r exposition of e a r l y v e n t r i c u l a r  arrhythmias  in dogs  f o l l o w i n g coronary o c c l u s i o n , which had been known f o r decades ( e . g . , Townshend P o r t e r , 1894; Smith 1918), i t  was not u n t i l 1973 that the myth that  rats do not experience arrhythmias f o l l o w i n g occlusion was d i s p e l l e d by the simple expedient of monitoring the ECG during and immediately a f t e r sion.  occlu-  This work was not c a r r i e d out by any of the i n v e s t i g a t o r s who had  e a r l i e r proclaimed the r a t to be superior to the dog f o r studying myocardial ischaemia and i n f a r c t i o n ( 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 f o l l o w i n g occlusion  (while  the  ether  anaesthesia used  Before discussing t h e i r r e s u l t s , i t  in  preparation  wore  off).  i s worth mentioning that the technique  of Selye e t - a l . (1960) was used in preparation, and that in every case the act of e x t e r i o r i s i n g the heart through the i n t e r c o s t a l i n c i s i o n was reported to produce t r a n s i e n t  i r r e g u l a r VT, a t r i o v e n t r i c u l a r  block and sinus brady-  c a r d i a ; the technique of Heimburger (1946) as used by Johns and Olson (1954) would not be expected to lead to arrhythmias in t h i s way, since heart e x t e r i o 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  o c c l u s i o n , a high incidence of v e n t r i c u l a r arrhythmias occurred.  after  Generally,  a short run of PVC occurred immediately upon o c c l u s i o n , but these  initial  - 45 arrhythmias  ( l a t e r c a l l e d 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 r e s u l t of the mechanical trauma of preparation  rather  than ischaemia, because these arrhythmias were s i m i l a r to the pre-occlusion arrhythmias incision,  induced  rather  occlusion.  by  exteriorising  the  than the arrhythmias  Unfortunately,  the  exact  through  the  intercostal  which occurred a few minutes  After an unspecified i n t e r v a l ,  VF occurred.  heart  after  a high incidence of PVC, VT and  incidence and time course of  the  arrhythmias was not given, although of the 5/20 rats which experienced VF or 'flutter',  2/5 d i e d .  In  addition  associated with acute myocardial  to t h i s  first  ischaemia in  exposition of  arrhythmias  rats,  Kenedi and Losconsci  (1973a) also noted that a l l rats s u r v i v i n g 24 h a f t e r  occlusion exhibited a  deep Q wave, and ST segment elevation in lead 1 of the ECG. Arrhythmias induced by coronary occlusion in r a t s were not again et-al;, their  until  1979,  when  1979a; 1979b) s e r i e s of  Szekeres'  and our  experiments,  group  laboratory which  (Lepran  et-al.-,  investigated  1979;  Siegmund  (Au et a l . , 1979a; 1979b) began  have continued  throughout  the  1980s,  along with work by P a r r a t t ' s group (Clark e t - a l ; , 1980; e t c . ) , Winslow (Kane and Winslow, 1980; e t c . ) , Bernauer (1980; e t c . ) and others. above Investigators  have concentrated on arrhythmias,  In general, the  although our l a b o r a -  tory and Bernauer always measure the OZ and the extent of i n f a r c t i o n .  Other  researchers have concentrated on i n f a r c t s i z e reductions in r a t s , and have completely ignored arrhythmias, for example, the Harvard contingent (MacLean e t - a l ; , 1976; Kloner e t - a l . , 1977; P f e f f e r e t - a l . , 1979; 1982; 1985), C h i a r iello's  group ( C h i a r i e l l o et a l . , 1980; 1983; 1984; 1985), Flaim and Z e l i s  (1981) and others.  In doing s o , these authors ignore censoring associated  with VF-induced m o r t a l i t y ; t h e i r causes are not given.  in most cases, the numbers of e a r l y deaths and  - 46 Independently of whether i n f a r c t i o n  or arrhythmias are the main subject  of study, the h i s t o r y and past l i t e r a t u r e concerning the use of the r a t have been almost u n i v e r s a l l y ignored.  For example, the o r i g i n a l  work of Kenedi  and Losconci (1973a) has only been appropriately c i t e d twice (Clark 1980;  Abrahamsson and Almgren,  given to Selye (1960) f o r  1980).  inventing  In  addition,  credit  is  etal,  generally  coronary occlusion in r a 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 i s concerned with the genesis of ischaemia-induced v e n t r i -  cular arrhythmias. such as reentry  Many of the concepts associated with  arrhythmogenesis,  (Mines, 1913) have been under i n v e s t i g a t i o n 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 a s s i s t in the prediction which type of drug might be of b e n e f i t . ing that the useful roach.  cart  often comes before  However, i t the  i s also worth consider-  horse, in  drugs have been developed on the basis of  of  that many  a purely  clinically  empirical  app-  In essence, a multilayered approach to the problem of i s c h a e m i a - i n -  duced arrhythmias i s i n d i c a t e d , and i t would be f o o l i s h to dismiss an empiri c a l approach on the grounds that i t of  the  underlying  pathologic  does not take s u f f i c i e n t  mechanisms.  For example,  the  consideration anti-ulcer  receptor histamine antagonists were developed from the premise that mine was mechanism.  somehow  involved  in  ulcerogenesis  What that mechanism i s ,  via  a  H,,  hista-  mepyramine-insensitive  in terms of the biochemical basis  for  the generation of a g a s t r i c l e s i o n and i t s precise r e l a t i o n s h i p with stomach pH, remains uncertain.  Nevertheless, cimetidine  and r a n i t i d i n e  have proven  to be e f f e c t i v e a n t i - u l c e r 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  electrophysiological  basis  of  arrhythmo-  - 47 -  genesis  in  acute myocardial  ischaemia.  In  addition,  an attempt has been  made to examine why and how calcium antagonists might be of benefit circumstances.  As background information,  a brief  in such  summary of some of  the  major features of the electrophysiology of e x c i t a b l e t i s s u e i s also given. 1.3.1 The electrophysiology of e x c i t a b l e t i s s u e Arrhythmogenesis  is  an e l e c t r o p h y s i o l o g i c a l  phenomenon.  understand the electrophysiology of myocardial ischaemia i t understand normal  heart  electrophysiology.  in the case of conduction in nerves, by i o n s . are governed in part by the  order  to  i s necessary to  Normal v e n t r i c u l a r  dependent upon the propagation of d e p o l a r i s a t i o n .  duction  In  function  is  The charge i s c a r r i e d , as  The c h a r a c t e r i s t i c s of  passive e l e c t r i c a l  properties  con-  of  the  properties may be approximated according to  the  t i s s u e , and in part by the a c t i v e or dynamic properties of the t i s s u e . The passive e l e c t r i c a l  cable equations developed by Kelvin (1855). cell  membrane  is  high  compared with  the  If  intracellular  r e s i s t a n c e , then any transmembrane potential considered as functions  of longitudinal  the resistance across the and  extracellular  differences and currents can be  distance and time, only.  an ' i d e a l ' c a b l e , the change in membrane potential  For such  r e s u l t i n g from the focal  a p p l i c a t i o n of current w i l l decay in distance and time according t o : d E /dx .l/r 2  2  m  2  = C .dE /dt m  m  i.  +  where x i s distance along the cable from the current J  rane  potential,  capacitance,  t  r is  is  2  intracellular  time  and i .  is  the  fluid  source, E i s membm m  resistance,  component  of  the  C  m  is  membrane  membrane current  c a r r i e d by ions (Hodgkin and Huxley, 1952a). Rearrangement of the cable equation gives r i s e to two membrane constants which length  are  often  constant,  useful x  =J  in r  / 2 r  m  the  understanding  describes  the  of  electrophysiology.  distance  from a current source at which the r e s u l t a n t potential  along  the  The  membrane  change has f a l l e n  to  - 48 1/e  (approximately  37%) of that at the point of the current  time required for this  V (E )  to  m  value gives  the  the membrane ( t a u ) .  decay from i t s  product  R C , m  initial  value  commonly c a l l e d  m  the  source.  (V )  to  Q  63% of  time constant  This arises because the membrane potential  m  The  of  at time t  a f t e r a p p l i c a t i o n of a current across the membrane i s given by:  When t i s equal to R C , then V. = V m  m  Ill  ill  U  The a c t i v e e l e c t r i c a l rical  nature  ATPase,  of  the  (l-e ),  and 1 - e "  - 1  m  = 0.63.  1  ITlaX  properties of the t i s s u e depend upon the asymmet-  excitable tissue. The oriented enzyme, Na -K -dependent + ++ ++ Na -Ca exchanger, intracellular Ca sequestrating +  +  proteins such as calsequestrin and other energy-dependent processes serve to generate a chemical  potential  gradient  for  several  charged e n t i t i e s .  a d d i t i o n , Donnan e q u i l i b r i u m , the presence of a r e l a t i v e l y body of i n t r a c e l l u l a r resting  K  the establishment below, i . (g.)  of  versus  that  of  other  a membrane p o t e n t i a l .  does not obey Ohm's Law.  constitutes  impermeant  anionic p r o t e i n , and the presence of a r e l a t i v e l y  permeability  +  fixed  In  the operational  charged e n t i t i e s ,  Finally,  This v a r i a b i l i t y  mechanism of  the  as w i l l in  leads  to  be discussed  ionic  active  high  conductance  electrical  prop-  e r t i e s of e x c i t a b l e t i s s u e which leads to propagation. At r e s t , t i s s u e in the heart ate  a resistance-capacitance  (excluding nodal) can be said to approxim-  circuit  r e s u l t i n g r e s t i n g membrane potential E +  where  [K ]  and  +  [K ] .  m  =  with  a K  +  battery,  such  that  the  i s given by the Nernst equation:  RT/F.ln([K ] /[K ].) +  +  o  are  the  local  concentrations  +  of  K  on  for  the  the  immediate outside and inside of the membrane, r e s p e c t i v e l y . The Nernst  equation  for  K  +  gives  the  reversal  potential  current, which i s approximately -90 mV under normal p h y s i o l o g i c a l ([K ] +  Q  approximately  2.5 - 4.5  meq/1  and  [K ] +  i  120 - 140  K  +  conditions  meq/1),  and  - 49 t h i s corresponds reasonably well with the r e s t i n g membrane potential of most non-nodal heart t i s s u e . the K  +  This model i s based on the assumption that at r e s t ,  permeability of the membrane f a r  rying s p e c i e s , such as Na  and Ca  exceeds that of other  charge-car-  .  The a c t i v e dynamic properties of e x c i t a b l e t i s s u e may be approximated by variations  in  the  equations  which  were  developed by  (1952a; 1952b; 1952c; 1952d) to explain the r e c t i f y i n g able t i s s u e . rent-voltage dependent  In t h i s  instance, r e c t i f i c a t i o n  relationship  changes  in  from  ionic  linear  and Huxley  properties of e x c i t -  i s the deviation of the cur-  (ohmic)  conductance.  Hodgkin  as  a  result  Rectification  is  of  explained  invoking the concepts of separate i o n i c currents associated with i o n - s e l e c t i v e channels which may vary with membrane potential ( s o - c a l l e d voltage and time dependence).  voltageby  relatively and/or  time  The equations describe the beha-  viour of these conductances in r e l a t i o n to membrane potential and time. R e c t i f i c a t i o n has been demonstrated in Purkinje f i b r e s wein, 1964).  (Deck and Traut-  At membrane p o t e n t i a l s p o s i t i v e to approximately -30 mV, there  i s an increase in a conductance which i s r e l a t i v e l y s e l e c t i v e for K .  The  +  properties of t h i s conductance resemble those of a conductance described by Hodgkin and Huxley in the squid axon which activates slowly f o l l o w i n g changes in holding p o t e n t i a l , rectifier carried  (see below). mainly  by  K , +  and i s c a l l e d ,  The current is  as a consequence, the delayed  associated with  outward-going,  step  causes  this  conductance  repolarization  and  is is  activated by d e p o l a r i s a t i o n . There i s with a K  +  a second type of r e c t i f i c a t i o n  conductance.  This conductance i s  in Purkinje t i s s u e associated activated by changing holding  potential  in voltage clamped (see below) t i s s u e from -30 mV to more negative  values.  Since  (as opposed to  depolarisation  reduces the  the behaviour of  the  conductance for  delayed r e c t i f i e r ) ,  this this  K current +  current  is  - 50 known as the anomalous r e c t i f i e r c u r r e n t , with 'inward going' It  i s perhaps important,  clamping.  (i ).  at t h i s stage, to comment on the use of voltage  When i n v e s t i g a t i n g  membrane  potential,  it  is  current  flowing  necessary  to  as a r e s u l t  eliminate  (the  holding  dE /dt  is  m  potential),  zero  if  E  m  because is  i  held  =  at  C .dE /dt. m  m  (clamped)  of  a change  capacitance  This i s done by maintaining membrane potential  c  rectification.  current  a constant Since  constant,  the  then  in  value value  the  term  C . d E / d t disappears from the cable equation, l e a v i n g : m  m  d E /dx .l/r 2  = i.  2  m  2  In other words, the transmembrane current simple function of membrane p o t e n t i a l ,  c a r r i e d by ions becomes a  under clamped c o n d i t i o n s .  fairly  The p r i n -  c i p l e 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 matically  by invoking  conductance channel.  voltage-dependent  parameters  which govern a  In the case of the delayed r e c t i f i e r ,  conductance i s  governed by the parameter, n.  fitting  may be described mathe-  For a population of delayed r e c t i f i e r  conduc-  tance channels, the t o t a l current i s dependent on the proportion of channels in the open s t a t e .  The v a r i a b l e n behaves according to the equation: dn/dt = a ( l - n ) - 8 ( n ) n  where c*  n  fraction  and & of  are  n  voltage  n  dependent  rate  channels in the open state  constants,  and 1-n  is  n  is  the molar  the  molar  fraction  of  channels in the closed s t a t e . Hodgkin and Huxley found that t h e i r conductance  (gK)  was proportional  to  r e s u l t s best f i t n^.  The simplest  a model where K physical  concep-  t u a l i s a t i o n of the above r e l a t i o n i s that n i s a p a r t i c l e which functions ' g a t e ' the conductance channel and e x i s t s e i t h e r  in an open state  closed state  to the rate of  (1-n).  The rate constant a  n  refers  +  (n)  to  or a  conversion  of  a channel  state,  from the  and v i c e - v e r s a .  resting  (closed)  state  to  the  depolarised  (open)  The voltage-dependence  of  the  channel, which  ac-  counts for i t s r e c t i f y i n g p r o p e r t i e s , i s explained by the voltage-dependence of B  n  c*  n  and  e ;  the  n  value  of  ct  increases  n  upon  depolarisation,  while  decreases upon d e p o l a r i s a t i o n . Although t h i s model was developed for nerve, manipulation of the values  of  a  n  and e  n  can provide  theoretical  conductance-voltage  relations  correspond well with experimentally-derived data in Purkinje t i s s u e  which (Noble,  1960; Noble 1962). It  i s suspected that there are many d i f f e r e n t conductances in the heart  which show a range of p r o p e r t i e s . activation  by changes in  These properties include ion  membrane  potential,  inactivation  selectivity,  by changes  in  membrane p o t e n t i a l , i n a c t i v a t i o n with time (see below) and r e a c t i v a t i o n . a d d i t i o n , under pathological  conditions  such as myocardial  In  ischaemia, some  of these properties may be influenced by changes in pH, c y c l i c AMP, temperat u r e , e t c . (see Hauswirth and Singh, 1978). The delayed r e c t i f i e r  is  voltage-dependent,  s i z e with membrane p o t e n t i a l .  However, the  Na  squid axon (and the s i m i l a r ' f a s t inward current' additional  property,  the increase in g ^ ing  off  g  time dependence.  since a +  with  time.  p  (g^ ) a  in the v e n t r i c l e )  This r e f e r s to  This  and B  conductance  which occurs upon depolarisation  (inactivating)  n  vary  in  in  the  shows an  the observation is transient,  phenomenon was explained  that  switchby  the  invocation of two gating variables ( i n contrast with the s i n g l e species for the delayed r e c t i f i e r ) , rectifier,  one of  which,  m, behaves l i k e  n of  the  delayed  s h i f t i n g to the open channel state upon d e p o l a r i s a t i o n , while the  second, h, behaves in the opposite manner, s h i f t i n g to the closed state upon depolarisation.  If  either n or h i s in the closed s t a t e , then that  partic-  ular channel i s closed (conductance f o r that channel i s minimal), s i n c e ,  in  - 52 -  analogy with g e n e t i c s , the open state i s recessive while the closed state  is  dominant. In order for it  is  g^  to f i r s t  a  necessary that  the  increase upon d e p o l a r i s a t i o n , then decrease,  rate  constant,  a , which  governs  m  the  rate  of  conversion of m from the r e s t i n g (closed) state to the open s t a t e , be larger than a^, the rate constant for the conversion of h from the r e s t i n g  (open)  state to the closed s t a t e , both of which reactions occur upon d e p o l a r i s a t i o n . The i n t e r a c t i o n rectifier  in  via  a  inward current  in time governs the a b i l i t y  propagate, g^i  between the f a s t  many e x c i t a b l e  effects  on  m  However, d e p o l a r i s a t i o n  Depolarisation  effect  leads  to  also serves to reduce g ^  also serves to increase gK v i a e f f e c t s  on a .  to  since the  repolarise  much larger enough  (at  steady-state feedback  of  the  membrane.  than that of a or  above  However,  n  of  depolarisation  a  n  a  serves more  to  These l a t t e r  increase  on c^, and  effects  maximum value  will  to  (so  as  'inactivation')  occur before  of  a  is  m  is  large  to  preclude  then  positive  the r e p o l a r i z a t i o n  processes  and a c t i v a t i o n of the delayed r e c t i f i e r . leads to the p o s s i b i l i t y  of  t i o n , since K i r c h o f f ' s law i s s a t i s f i e d by the induction of outward some distance away from the inward current, (intracellular  turn by the longitudinal tion.  serve  The AP i s terminated by a combination of  The assymetry of e x c i t a b l e c e l l s  longitudinal  to  depolarisation.  via effects  and occurs q u i c k l y  leading  are a c t i v a t e d , producing the AP. i n a c t i v a t i o n of g ^  and the delayed  or a^, then provided d e p o l a r i s a t i o n  'threshold')  elevation  Na  of an AP to occur, and in space,  tissues. This  a .  (i' )  current,  which i s induced in  gradient r e s u l t i n g from focal d e p o l a r i s a -  A m p l i f i c a t i o n may occur in t h i s c i r c u i t  cesses which lead to the AP, as f o l l o w s . depolarisation is s u f f i c i e n t l y  current  linked to the inward current by  and e x t r a c e l l u l a r )  potential  propaga-  If  as a r e s u l t  of s i m i l a r  pro-  within a region of membrane  l a r g e , and occurs s u f f i c i e n t l y  quickly,  then  will  swamp r e p o l a r i s i n g  K  current,  +  leading . to  further  t i o n at points further and f u r t h e r away from the i n i t i a l ation in a manner described by the cable equation. of d e p o l a r i s a t i o n  (the  'propagated A P ' ) .  propagated AP depends upon x  m  This manifests as a wave  and t a u . m  e x c i t a b l e t i s s u e , leading  depolarisation-1inked events,  such as neurotransmitter  case  coordinated  ventricular  properties  of  ventricular  the  tissue,  muscle  conductance channels confer  tissue.  This  is  because the  of  g^  a  is  a consequence of  This i n e x c i t a b i l i t y riness  under  repolarisation  serves to  for  of  is  on  tissue  as is  gating  The i n a c t i v a in a ^ .  increases  refracto-  repolarisation, to  since  a  shift  With the passing of time, more  and more  said to  in  excitation  The duration of  dependent  polarisation, the  wave of  to the  conduction  and reduce a ^ , leading  increase  opening,  to  depolarisation-induced  circumstances  re-establishment  re-available  direction  in  The  is inactivated.  a  in the e q u i l i b r i u m of h to the open-state. the  contraction.  i s known as ' r e f r a c t o r i n e s s ' .  normal  r e l e a s e , or  propagating  leaves behind a band of t i s s u e in which g ^ tion  point of d e p o l a r i s -  The threshold for generation of a  A propagated AP passes as a wave through  of  depolarisa-  channels  pass from  the  and  become  stage  absolute r e f r a c t o r i n e s s to r e l a t i v e r e f r a c t o r i n e s s , and f i n a l l y to the  of  fully  excitable state. The conduction v e l o c i t y (e = x / t )  of a propagated AP can be derived  in  terms of the cable equation: e  2  = (x .d V/dt )/(tau .dV/dt 2  2  + V)  2  m  This d e r i v a t i o n predicts that e i s d i r e c t l y proportional proportional  to  the square root of  tau . m  In  to the square root of the maximum r i s e rate  to x, and i n v e r s e l y  addition, of  e is  proportional  the AP (dV/dt  ).  Under  max normal  circumstances,  almost  linearly  dV/dt  related  to  m 3 v  the  is  the  square  major root  determinant of  dV/dt  m a x  of .  ©,  with ©  Manipulations  - 54 -  which e i t h e r  prevent channels from opening  i n a c t i v a t e d s t a t e , preventing a c t i v a t i o n  (by f i x i n g  m  and E  to  m  the  right  (to  more p o s i t i v e  In most nerve t i s s u e and normal  channel  in  the  or p h y s i c a l l y blocking the chann-  e l s ) , or slow the k i n e t i c s of opening by s h i f t i n g a  the  the r e l a t i o n s h i p between  values of  ventricular  E )  will  m  t i s s u e , dV/dt  reduce is  e.  governed  Hid A  by  i^ .  In  is  dependent on currents  a  ischaemic v e n t r i c u l a r other  tissue,  than the  it  is  normal  possible that  fast  inward  dV/dt  current  m a x  (see  below). 1.3.2 The electrophysiology of the normal v e n t r i c l e Variations  in the a c t i v e and passive e l e c t r i c a l  properties  t i s s u e between d i f f e r e n t regions of the v e n t r i c l e propagating AP. dimensions  of  These v a r i a t i o n s  the  cells  in  are brought  specific  of  ventricular  govern the shape of  about by v a r i a t i o n s  regions  and also  variations  the  in  the  in  the  conductance channels present in the d i f f e r e n t regions. By manipulation of e x t r a c e l l u l a r ion composition (for ment of  Na with choline or L i ) , +  +  addition  example, r e p l a c e -  of substances shown to  selec-  t i v e l y i n h i b i t s p e c i f i c conductances, such as tetrodotoxin which s e l e c t i v e l y blocks g  (Moore et a l . , 1967), and by a p p l i c a t i o n of various techniques  N a  for clamping voltage or current such as the recently-introduced patch-clamp technique (Lee e t - a l ; , 1980), i t conductance While i t  has been possible to describe some of  systems which contribute  is  possible to  study  to  isolated  the  propagated AP of  heart  s i z e ) by patch clamping (Lee e t - a l ; , 1979), i t cells  in vivo  cellular  because of  movement  (the  and i n t e r c e l l u l a r  geometry  (Attwell  cells  (despite  is d i f f i c u l t  organ  beats'.),  et-al.,  the  heart.  their  small  to study heart  and  1979).  unfavourable Nevertheless,  although some conductances have yet to be f u l l y c h a r a c t e r i s e d , i t a l l y accepted that the shape of the v e n t r i c u l a r three  current  systems,  i  N  ,  the  outward  the  i s gener-  AP i s mainly governed by  repolarising  currents  (carried  - 55 mainly  by  K )  and  +  the  slow  inward  current  (i -»  carried  sl  mainly  by  Ca ). + +  In v e n t r i c u l a r  tissue, i^  with i t s rapid opening and c l o s i n g k i n e t i c s  a  dominates the i n i t i a l r i s e of the AP, and governs the d V / d t , of max m  isation  (Beeler and Reuter, 1970a).  velocity. the  The proportion  excitability  of  of  the  i*  Na  tissue.  This  in  channels  turn governs the  available  Ventricular  i^  for  is  a  depolar-  v  conduction  opening  similar  governs  to  the  Na  +  current described by Hodgkin and Huxley for the squid giant axon, with minor differences  (such  as the  voltage-dependence  of  the  inhibitory  action  of  tetrodotoxin in the v e n t r i c l e ) . Repolarization  is  associated  with  current s i m i l a r to the delayed r e c t i f i e r i 2 K  repolarization,  for  (  N o b l e  \2  a n d  duration,  since  t a i l of the AP. for is  K  current  in the squid axon, c a l l e d  +  increased.  example, mixed ion currents a n d  Tsien,  inhibition  of  1969). gK delays  (predominantly  The gK systems  K)  the AP  by prolonging  the  If the t o t a l duration of the AP (often measured as the time  This  negative for  restored.  to  called  +  influence  repolarization  a  variable  Several other outward currents may also contribute  is  because the membrane potential  a time s u f f i c i e n t  for  In t h i s regard, i f  must remain  conversion of  from t h e i r closed to t h e i r open state ( r e a c t i v a t i o n )  N a  including  90 % r e p o l a r i s a t i o n , or APD90) i s increased, then the r e f r a c t o r y  ciently  g  currents  ( M c A l l i s t e r and Noble, 1966), which i s governed by the gating  s (analogous to n ) .  \l  several  the  period suffi-  h variables  for e x c i t a b i l i t y  to be  Purkinje t i s s u e i s clamped at -70 mV then the  system i s 50 ^ i n a c t i v a t e d (Weidmann, 1955a). The t h i r d major current in v e n t r i c u l a r t i s s u e i s the slow inward current ++  (i .j). s  This current  is  c a r r i e d mainly by Ca  minant of the plateau of the AP in v e n t r i c u l a r  ,  and i s  tissue  the major  deter-  (Mascher and Peper,  1969; Beeler and Reuter, 1970a; 1970b; New and Trautwein, 1972).  As such,  - 56 serves to govern the AP d u r a t i o n , outward K partly  current  +  governed  intracellular conductances normal well  systems described above.  by  Ca  i^,  a  (Isenberg,  i ..  may themselves be  i ^ causes a t r a n s i e n t  1977b; 1977c).  after  the  In  rise  a large extent,  Inhibition  of  i'  will  s i  Since the duration  shorten  that  +  of  i .. as  of the AP i s  is  AP duration  unbound  restoration  an AP requires  then r e f r a c t o r i n e s s  in  enhance some K  the v e n t r i c l e s ,  propagation of  recovers from i n a c t i v a t i o n .  governed by i .. to on  since  The l a t t e r  concentration which has been shown to  + +  excitability  as i ^  in conjunction with the r e p o l a r i s i n g  also dependent  at  25% r e p o l a r i -  sation (APD25), but APD90 may e i t h e r increase or decrease, presumably according to  the  dependence of  intracellular  Ca  the  tail  of  the  repolarising  ( t h i s may vary from one type of  + +  current  ventricular  another, and may also vary from species to s p e c i e s ) .  It  during  role  myocardial  ischaemia,  i  .  plays  an  aspects of conduction in the v e n t r i c u l a r  important  on  free  tissue  to  i s possible that  t i s s u e , as w i l l  in  additional  be discussed in  d e t a i l below. Just as i ^  possesses two gating v a r i a b l e s , i . has been shown to  a  governed by two v a r i a b l e s , denoted as d and f, tively  (Reuter,  threshold that for (Bf)  far  for  1973). i  .  .  is  For f ,  rate  of  a  constant  for  is  such that  the  more  positive  than  d  -50 mV, s l i g h t l y  the rate constant for  exceeds the  analogous to m and h, respec-  The voltage-dependence approximately  conversion to the open state  conversion  to  the  closed  (df) at membrane p o t e n t i a l s more negative than -60 mV.  The rate  for  larger  activation  slower k i n e t i c s )  (a^) and  inactivation  (a^) are  much  than corresponding rate constants for  species v a r i a t i o n , a  £  be  i^ .  state  constants (indicating  There i s some  being 80-200 msec in cat and dog v e n t r i c u l a r  tissue  (McDonald and Trautwein, 1978; Reuter and Scholz, 1977), and 10-30 msec in rat  ventricular  tissue  (Isenberg  and  Klockner,  1980).  In  the  latter  - 57 species,  the  fast  kinetics  of  i ^  the  abbreviated  plateau of the AP (compared with the c a t , dog, pig and human  ventricular  $  probably  account  for  AP), which i s associated with a shorter APD90 of 100 instead of 300 msec (Langer,  1978),  and accords with the high  resting  heart  rate  of  350-450  beats/min. Since v e n t r i c u l a r giant  c e l l s are small compared w i t h , f o r example, the squid  axon, and are part  investigate their information  of  a functional  syncytium,  it  is  difficult  to  electrophysiology in a manner which provides unequivocal  (Beeler  and  McGuigan,  beating c e l l s are d i f f i c u l t  to  1978;  McDonald,  1982).  Furthermore,  impale with microelectrodes.  Patch clamp  studies are favoured at present for t h e i r power to reveal the properties of ventricular  conductances.  from t h e i r  p h y s i o l o g i c a l environment one may introduce  found i n v e s t i g a t i o n .  Nevertheless, as one removes c e l l s or membranes  For example, i • i s  factors  of  inactivation  1984).  of  phosphorylation of the f i .  and  an  ventricular suggest  properties  t i s s u e remain to a c e r t a i n  many explanations  for  'particle',  enhancement  Therefore, since the basic  the  con-  enhanced by pharmacological and  p h y s i o l o g i c a l manipulation which increases c y c l i c AMP (cAMP). that cAMP t r i g g e r s  which  extent  of of  peak  It  i s thought  leading to slowing i .  (Bean  et-al.,  conductance channels  unclear,  mechanism by which  it  is  a drug  possible  in to  influences  v e n t r i c u l a r e l e c t r o p h y s i o l o g y , whether t i s s u e i s normal or abnormal (ischaemic, f o r example). 1.3.3 E l e c t r o p h y s i o l o g i c a l changes caused by myocardial ischaemia The study of the electrophysiology of myocardial ischaemia i s confounded by the dynamic unstable c h a r a c t e r i s t i c s of myocardial ischaemia.  The e l e c t -  r o p h y s i o l o g i c a l properties of the ischaemic v e n t r i c l e vary from one region to another, and also from time to time, as i n t r a - and i n t e r c e l l u l a r biochemistry  varies from one region to another,  and changes from time to  time.  - 58 Furthermore,  the  electrophysiological  changes  vary  blood supply i s completely or p a r t i a l l y reduced.  according  to  whether  In s h o r t , these considera-  tions d i c t a t e that 'the ischaemic myocardium' i s not a s i n g l e e n t i t y . The electrophysiology of the squid giant axon i s considered to be well understood, that of the normal heart  less s o , and that of the heart  the influence of regional ischaemia even l e s s . scopic changes in electrophysiology the macroscopic consequences of provide a consensus.  Attempts to c o r r e l a t e micro-  during acute myocardial  ischaemia with  such changes - arrhythmias  This i s p a r t l y  under  - have yet  a r e s u l t of the d i f f i c u l t i e s  to  in study-  ing the electrophysiology of the heart and p a r t l y a r e s u l t of the v a r i a b i l ity  in experimental preparations and approach.  of convincing c o r r e l a t i o n myocardial  Nevertheless, despite a lack  between s p e c i f i c e l e c t r o p h y s i o l o g i c a l  ischaemia and arrhythmias,  changes  in  much has been speculated concerning  mechanisms of arrhythmogenesis. Recently, i t was demonstrated, using anaesthetised p i g s , that the maximum d i a s t o l i c potential precipitously  within  et a l 1 9 7 8 ) . '  (MDP) in subepicardial muscle c e l l s begins to  seconds of  Simultaneously,  occlusion  (Downar  the AP amplitude,  et-al;, dV/dt  1977b;  fall  Kleber  and APD90  m a x  all  decrease. The decrease in dV/dt the f a l l  v  and APD90 are considered to  in MDP, s i n c e , as discussed in  diastolic  depolarisation  leads  to  detail  inactivation  be secondary  previously, of  g » Na  However,  possible that ischaemia d i r e c t l y reduces g ^  a  although  such a mechanism.  it  i s not  necessary to  quence of  the  decreases,  since r e a c t i v a t i o n  situation  reduction  in  i s not maintained.  postulate  APD90, of  g^  the a  state it  is  independently of MDP changes,  effective  refractory  occurs more q u i c k l y .  As MDP continues to f a l l  p i t e a maintained narrow APD90.  steady  to  Since ERP continues  As a conseperiod (ERP) However,  this  ERP lengthens,  des-  beyond the  point  of  - 59 f u l l r e p o l a r i s a t i o n , t h i s phenomenon i s known as toriness  (Lazzara e t - a l ; ,  1978).  Since  post-repolarisation-refrac-  post-repolarisation-refractoriness  can be induced by d e p o l a r i s a t i o n alone in i s o l a t e d v e n t r i c u l a r t i s s u e (Inoue e t - a l ; , 1984), i t follows that i t may be possible to explain t h i s phenomenon in terms of the normal v e n t r i c u l a r conductance channels (see below). It  i s important to note that the e l e c t r o p h y s i o l o g i c a l  are not  uniform  in time or space.  similar resting E  m  changes described  For example, adjacent  c e l l s with  very  may vary in ERP from 180 to 500 msec in pig ischaemic  subepicardial muscle (Downar e t - a l . , 1977b). As a r e s u l t of the f a l l slow conduction  in the  in d V / d t  ischaemic  between -55 and -60 mV is  m a x  ,  e falls,  tissue.  In  associated with  producing  addition,  complete  characteristic  depolarisation  inactivation  of  to g^,  and under these circumstances complete conduction block may occur. The phenomena described above were recorded in vivo from subepicardial t i s s u e (Downar et a l . , 1977b; Kleber et a l . , 1978).  It  i s not possible to  record from deeper layers in -vivo, owing to the l i m i t a t i o n s  of  intracellular  recording electrodes (electrodes are too f r a g i l e to be plunged deep into the myocardium and penetrate  cells).  It  is  possible to  s t r i p s of subendocardial t i s s u e in v i t r o ,  but  it  record  from  isolated  i s unclear to what extent  the experimental technique influences the v a r i a b l e s under i n v e s t i g a t i o n l e a s t because the t i s s u e i s generally superfused, in which case i t icult the  to regulate the experimental isolated  ischaemia).  Langendorff-perfusion  rat  heart  is  (not diff-  A compromise preparation in  which  careful  is  incision  exposes the sub-endocardial t i s s u e s , while coronary occlusion may be undertaken to produce regional atory  using t h i s  cribed above for tion,  it  ischaemia.  preparation subepicardial  Experiments c a r r i e d out in our  have revealed s i m i l a r pig t i s s u e  changes to  (Inoue e t - a l . - , 1984).  was shown that the changes were more severe and less  those  labordes-  In a d d i reversible  - 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 e a r l y demonstrated in these experiments (Inoue et a l . , 1984). Qualitatively,  it  i s c l e a r that the active and passive e l e c t r i c a l  e r t i e s of v e n t r i c u l a r t i s s u e may change with time in a f a i r l y manner during myocardial this  information  to  ischaemia, and attempts  theoretical  mechanisms of  reentry, automaticity and triggered  prop-  reproducible  have been made to  arrhythmogenesis,  relate  including  automaticity.  1.3.4 Models Of Arrhythmogenesis 1.3.4.1 Reentry. genesis f o l l o w s .  A b r i e f d e s c r i p t i o n of some models of  Reentry was o r i g i n a l l y  arrhythmo-  described as r e e x c i t a t i o n  myocardium v i a a c i r c u l a r route (Mines, 1913).  The p r e r e q u i s i t e for  i s one-way block of propagation in one limb of the reentrant  of  the  reentry  circuit.  The  nature and mechanism of reentry, and the roles of r e f r a c t o r i n e s s , conduction v e l o c i t y and duration of the 'wave of e x c i t a t i o n ' were e x p l i c i t l y described by Mines (1913) Ehrlanger  using the t o r t o i s e  and r e i t e r a t e d  (1928) using a t u r t l e heart preparation.  that the normal wave of e x c i t a t i o n circuit  heart,  c l o s e r to threshold for  brings  E  m  in  by Schmitt  and  The mechanism requires the blocked limb  of  the  conduction, such that the normal wave can  then travel retrogradely up t h i s limb and reenter i t s previous pathway.  For  t h i s to happen, the t i s s u e of the anterograde pathway must be e x c i t a b l e at the time the wave f r o n t reenters. mechanisms operate.  Firstly,  if  This c r i t e r i o n w i l l the c i r c u i t  is  be met i f  long enough i t  one of two will  allow  s u f f i c i e n t time to elapse for the anterograde pathway to recover e x c i t a b i l ity.  This type of reentry i s c a l l e d macroreentry. The pathway w i l l  be f u n c t i o n a l l y long i f conduction v e l o c i t y i s reduced,  e i t h e r in the anterograde or retrograde limb. tory  period  in  the  anterograde  pathway i s  A l t e r n a t i v e l y , i f the r e f r a c very short  (excitability  being  - 61 restored  quickly),  excitation  then the requirement  and r e - e x c i t a t i o n  for  i s reduced.  a prolonged  In t h i s  interval  between  case the reentry  circuit  may be extremely s h o r t , perhaps only a few mm (Sasyniuk and Mendez, 1971). Indeed, transmural  ( e p i - to endocardial) reentry has recently been reported  (Kramer et a l ; , 1985).  This type of reentry i s c a l l e d microreentry.  Acute myocardial ischaemia provides a l l the t h e o r e t i c a l reentry:  slow conduction, dispersion of r e f r a c t o r i n e s s  conduction block around which a reentrant  requirements  and areas of  for  2-way  impulse may e n c 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  investigation  in open-chest animals have not been s u c c e s s f u l l y applied  to c l i n i c a l or closed-chest animal s t u d i e s .  In open-chest animals an array  of DC electrodes may be placed on the epicardium for mapping the  activation  pathways.  paragraphs  By t h i s  method,  dealing with automaticity, prepare experimental  arrhythmias below).  It  may be v i s u a l i s e d  (see  may be p o s s i b l e , in the f u t u r e ,  animals with i n - d w e l l i n g  DC electrode  arrays.  In  to the  c l i n i c a l s i t u a t i o n , or in closed-chest animals, standard 12 lead ECGs do not permit one to d i s t i n g u i s h between reentry and automaticity Reentry, t h e o r e t i c a l l y ,  may give r i s e to PVCs or VT.  (see below). 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 lost  to  analysis,  since VF i s  activity  (Moe e t - a l ; ,  electrical  usually  defined  as uncoordinated  be  chaotic  1964), without recognisable QRS complexes  (Bigger, 1980). In closed-chest experimental animals undergoing repeated episodes of VT, it  has been suggested that the d e l i v e r y of a premature e l e c t r i c a l  to the v e n t r i c l e can terminate or i n i t i a t e the arrhythmia, is reentry  (Bigger and Goldreyer, 1970).  if  stimulus  the mechanism  The premature stimulus  presumably  e i t h e r conducts through the one-way blocked t i s s u e to i n i t i a t e VT, or depol-  - 62 arises  the one-way blocked t i s s u e  normal impulse to terminate VT.  allowing  anterograde  conduction  The use of premature stimulation  of  the  does not  permit the recognition of reentrant PVCs, only r e l a t i v e l y sustained arrhythmias, such as VT. Trains of high frequency v e n t r i c u l a r  stimulation w i l l  often  initiate  terminate reentrant VT by the same mechanism as premature s t i m u l a t i o n . stimulation w i l l is  ventricle  terminated, sinus rhythm w i l l is  not  this  response i s  with  automaticity  changed, the not  will  fail  resume.  the  (see below).  follow.  i b l e to prove reentry  will  shortly  post-overdrive  In  Therefore,  open-chest  techniques, the s i t u a t i o n i s d i f f e r e n t 1.3.4.2 automaticity  in  of normal automaticity.  AP.  greatest  (for  also show t h i s  in the sinus node.  The  for  i s imposs-  VF, according to  using  is  epicardial  the  mapping  understanding  of  abnormal  understanding in normal  However, in the sinus node a slow  precedes and t r i g g e r s  the  propagated  example the a t r i o v e n t r i c u l a r slow d e p o l a r i s a t i o n , but  As a r e s u l t ,  propagated APs i s highest in the sinus node. originating  Furthermore, i t  During d i a s t o l e , the membrane potential  Other tissues in the heart  the Purkinje f i b r e s )  Unfortunately,  arrhythmogenesis requires the  depolarisation  is  (see below).  v e n t r i c u l a r muscle i s almost constant. spontaneous d i a s t o l i c  resume.  closed chest animals i t  animals,  Abnormal - Automaticity.  as a mechanism of  stimulus  suppression response seen  as the underlying t r i g g e r  considerations.  Once the  However, since the underlying condi-  arrhythmia  unlike  to  d i f f i c u l t to prove reentry as the cause of VT.  above  Such  i n i t i a l l y capture the reentrant c i r c u i t , then, as frequency  increased, the  tion  or  the frequency of  node and  dV/dt  m a x  is  triggering  of  Consequently the propagated AP  from the sinus node overdrives the spontaneous depolarisations  in other regions of the heart, and d i c t a t e s the heart r a t e . utes the pacemaker property of the sinus node.  This  constit-  The current responsible for  the spontaneous d i a s t o l i c d e p o l a r i s a t i o n i s known as the pacemaker current. In the sinus node the pacemaker current has r e c e n t l y been shown to  result  from the i n t e r a c t i o n of two currents (Shibata and G i l e s , 1985), the decay of a  delayed  rectifier  K  +  current  called  i ,  and  K  the  activation  of  an  ++  inward current  carried  voltage-dependent  over  by Ca the  ,  range  called -80  i  to  .  c  The decay of  -55 mV.  i  K  is  The a c t i v a t i o n  highly of  i^  a  occurs at between -60 and -55 mV with peak current at 0 mV. Abnormal  automaticity  simply means that  heart  tissue  other  than  the  sinus node has taken over the pacemaker r o l e of the sinus node (Hoffman and Dangman, 1982; Sasyniuk, 1984).  U s u a l l y , in order for  to occur,  the  rate  pacemaker region must exceed that of  the  of  firing  of  the  abnormal  this  sinus node and overdrive the heart (but see comments concerning p a r a s y s t o l e , below).  The anatomical source of  ectopic focus.  this  pacemaker a c t i v i t y  Abnormal pacemaker a c t i v i t y  of  an  increase in the steepness of the slope of the d i a s t o l i c d e p o l a r i s a t i o n  in  l a t e n t pacemaker t i s s u e , or a reduction in the threshold for  of  the (abnormal) propagated AP.  may occur  i s known as the  to  i n the maximum d i a s t o l i c  (MDP) to a more p o s i t i v e value.  The d i f f i c u l t i e s apply  generation  The l a t t e r may r e s u l t from e i t h e r a reduction  in the absolute threshold in mV, or from a s h i f t potential  as a r e s u l t  the  in  recognising reentry  recognition  of  abnormal  in  closed-chest animals  automaticity.  Automaticity  also  may be  associated with i n t e r m i t t a n t e x i t block whereby the ectopic impulse f a i l s propagate (Fisch et a l . - , 1971). charges j u s t after  This w i l l  occur i f  to  the ectopic focus d i s -  the normal wave of e x c i t a t i o n has passed by (leaving a  band of r e f r a c t o r y t i s s u e ) .  Under such circumstances one w i l l see, perhaps,  only PVC in the ECG. A l t e r n a t i v e l y , the ectopic focus may be protected, by entrance block, from overdrive suppression by the normal wave of  excitation  (Wennemark and Bandura, 1974), in which case automatic arrhythmias may occur  - 64 -  even when the  cycle length  sinus cycle length. of  t i s s u e which  abnormalities.  of  the  abnormal  pacemaker i s  longer  than  T h e o r e t i c a l l y , entrance block may be caused by a band  possesses  one-way blocking  properties,  or  by  anatomical  Conceivably, entrance block may also r e s u l t from the induc-  t i o n of a band of r e f r a c t o r y t i s s u e by the previous ectopic impulse. normal are  sinus frequency  similar,  the  the  and the  normal  entrance-block-protected  and abnormal  ectopic  If  the  frequency  pacemakers may alternate  in  their  dominance, producing p a r a s y s t o l e . It  has been suggested that  differentiated  abnormal  automaticity  and reentry  may be  by studying the e f f e c t s of overdrive pacing (see Bigger and  Goldreyer, 1970; V a s a l l e , 1977). by pacing, then following  If  an automatic arrhythmia  termination  of  overdrive  one w i l l  is  overdriven  see a delay  (post-overdrive suppression), then a resumption of the automatic arrhythmia. Reentrant arrhythmias do not show t y p i c a l post-overdrive suppression. The mechanism of Purkinje f i b r e s ing,  post-overdrive  ( V a s a l l e , 1977).  MDP f a l l s .  Providing  suppression has  been studied  in  dog  E a r l y during the onset of overdrive pac-  that  stimulation  does not  exceed a  critical  frequency, maximum d i a s t o l i c d e p o l a r i s a t i o n then r i s e s and the slope of the pacemaker potential  is  reduced.  Immediately  the ectopic frequency i s lower than i t a r e s u l t of potential.  the e f f e c t  upon cessation of  had been before overdrive pacing, as  of overdrive pacing on the slope of  Over a short  priod  of  overdrive,  time the  original  the pacemaker  electrophysiological  c h a r a c t e r i s t i c s of the ectopic focus r e t u r n , and the arrhythmia resumes. the case of reentry  there  i s generally no post-overdrive suppression, and  the reentrant arrhythmia resumes after an unpredictable i n t e r v a l . less, it drive,  i s possible that reentrant after  an  interval  In  similar  Neverthe-  arrhythmias may resume, following overto  suppression of abnormal automaticity.  that  seen f o l l o w i n g  post-overdrive  Therefore a reentrant  arrhythmia may  - 65 be  incorrectly  city,  classed as automatic.  In  addition,  parasystolic  protected from overdrive by entry block, w i l l  automati-  not e x h i b i t  post-over-  drive suppression, and may therefore be i n c o r r e c t l y classed as reentrant. As discussed above, overdrive techniques are only useful for relatively  sustained arrhythmias.  However, i t  assessing  i s possible to gain  insight  into arrhythmogenic mechanisms (including mechanisms of PVC induction)  from  ECG r e c o r d i n g ,  this  coupled with  simple p h y s i o l o g i c a l  regard, reentrant PVCs are triggered i n i t i a l l y gation. rate,  manipulations.  In  by the normal wave of propa-  Therefore, by analysing the frequency of PVC in r e l a t i o n  it  may be possible to e s t a b l i s h a r e l a t i o n s h i p .  to heart  Furthermore,  heart  rate may be slowed or stopped by s t i m u l a t i n g the vagal e f f e r e n t s , or a c c e l erated  by administering  atropine  measured over a wide range of  (etc.),  heart  such that  rate.  PVC incidence may be  Theoretically,  PVCs  resulting  from automaticity should not vary in frequency with changes in heart whereas the frequency of reentrant and f a l l  with bradycardia.  rate,  PVCs should increase with t a c h y c a r d i a ,  However, the study of the e f f e c t  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 i a g n o s i s , owing to the p o s s i b i l i t y of entry- and e x i t - b l o c k .  In a d d i t i o n , i t  i s necessary  to ensure that the manipulation designed to a l t e r heart rate does not ence other  variables, directly  PVC frequency. It  It  or  indirectly,  leading to  i s almost impossible to control for t h i s  influ-  an a l t e r a t i o n  in  possibility.  i s possible to make c e r t a i n speculations concerning the mechanism of  arrhythmogenesis of PVCs by simply observing the ECG without other manipulation.  In t h i s regard, sustained f i x e d coupling of PVCs (of consistent QRS  configuration)  with  reentry  than automaticity  rather  the  normal  sinus beats (Langendorf  is  regarded as i n d i c a t i v e  and P i c k ,  1967).  of  The reason  for t h i s i s that the frequency of an ectopic arrhythmia would not be expected to be the same as the sinus frequency, whereas a reentrant  arrhythmia  - 66 should be linked to the sinus cycle in a f i x e d manner. It  i s questionable whether  any of  the tests  and experiments described  are useful for d i s t i n g u i s h i n g between reentry and automaticity  during acute  myocardial ischaemia, despite t h e i r possible usefulness in other circumstances. ical time,  During the development of myocardial ischaemia, the e l e c t r o p h y s i o l o g c h a r a c t e r i s t i c s of  a hypothetical  causing changes in coupling  myocardial criterion  reentrant  interval.  ischaemia may be i n c o r r e c t l y for  possible that  circuit  Therefore,  wave of normal e x c i t a t i o n  change with  reentry  in  described as automaticity  diagnosis i s a f i x e d coupling i n t e r v a l . parasystolic  will  (entry-blocked) via electrotonic  foci  an area of entry block ( J a l i f e and Moe, 1976).  if  In a d d i t i o n ,  may be influenced  intercourse  acute the it by  (reflection)  is the  across  F i n a l l y , in t h i s regard, the  functional d i s t i n c t i o n between reentry and automaticity becomes questionable when the phenomenon of triggered automaticity i s considered. 1.3.4.3  Triggered Automat-i c i t y .  been demonstrated in v i t r o .  The reason for t h i s  to demonstrate triggered automaticity i t t i s s u e , and i t  Triggered  automaticity  i s quite simple.  has  only  In order  i s necessary to stimulate quiescent  i s not possible to have quiescent v e n t r i c l e s i n - v i v o .  Since  triggered automaticity i s , by d e f i n i t i o n , automaticity which i s i n i t i a t e d by an exogenous source, and the  exogenous source t h e o r e t i c a l l y  normal wave of d e p o l a r i s a t i o n , then i t  includes  the  i s possible that abnormal automatic-  i t y in vivo may be triggered by the sinus beat (Spear and Moore, 1982).  In  such a case the frequency of automatic PVC would c o r r e l a t e with heart r a t e . Therefore, i t  i s not possible to d i s t i n g u i s h between automaticity  and r e -  entry on the basis of the r e l a t i o n s h i p between heart rate and PVC frequency unless triggered automaticity  i s ruled out.  However, i t would be necessary  to induce asystole in order to test for triggered automaticity t h i s i s not f e a s i b l e .  in v i v o , and  - 67 -  1.3.5 E p i c a r d i a l A c t i v a t i o n Mapping Although i t or reentrant  i s not possible to prove whether an arrhythmia i s  in closed-chest animals, the  technique  of  automatic  epicardial  mapping  allows for v i s u a l i s a t i o n of conduction pathways in anaesthetised open-chest animals and i n - v i t r o .  E p i c a r d i a l mapping has been c a r r i e d out in conjunc-  t i o n with simultaneous  intracellular  recording  in acute myocardial  mia, and attempts have been made to r e l a t e the pattern of abnormal  ischaeelectro-  genesis and conduction with the underlying e l e c t r o p h y s i o l o g y . How do the  electrophysiological  changes in  acute myocardial  ischaemia  c o r r e l a t e with arrhythmogenic mechanisms v i s u a l i s e d by e p i c a r d i a l mapping ? In coronary-occluded blood-perfused dog and pig h e a r t s , records of but not  automaticity,  1981; Janse, 1982).  were observed (Janse e t - a l ; ,  reentry,  1980; Janse and Kleber  Most of these arrhythmias arose from the normal  tissue  close to the occluded bed within the f i r s t few minutes of o c c l u s i o n .  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). Interestingly,  the i n i t i a l  trigger  which arose from the normal t i s s u e . this  phenomenon was as f o l l o w s .  that of ischaemic c e l l s i t  for  the reentrant  VT was an impulse  The mechanism proposed to account  By comparing the AP of normal  for  c e l l s with  was found that many ischaemic c e l l s were in the  plateau phase of the c y c l e when normal c e l l s were at the r e s t i n g r e p o l a r i s e d phase.  This s i t u a t i o n occurred as a r e s u l t of the heterogeneity of conduc-  tion velocity  in the ischaemic t i s s u e versus the non-ischaemic t i s s u e ,  association with slow conduction in the ischaemic t i s s u e . of t h i s  situation  included a large potential  and the  ischaemic t i s s u e .  between  the  depolarised  It  The consequences  difference between the normal  was proposed that a flow of  ischaemic t i s s u e  in  and the  polarised  injury  current  non-ischaemic  - 68 tissue  may have  Alternatively,  been  sufficient  to  re-excite  the  non-ischaemic  tissue.  since the non-ischaemic t i s s u e could be in the plateau phase  of the AP when the ischaemic t i s s u e was r e p o l a r i s e d , i n j u r y current may have flowed in the opposite d i r e c t i o n to r e - e x c i t e the ischaemic t i s s u e . For t h i s exists  mechanism to operate,  (separating  least temporarily,  the current  it  i s necessary that a layer of  source and sink)  and capable of  which  is  cells  inexcitable,  permitting e l e c t r o t o n i c  at  coupling between  the source and sink (Janse and Kleber, 1981). Whatever the mechanism of i n i t i a t i o n ,  a c t i v a t i o n maps revealed fragmen-  t a t i o n and fusion of wavefronts, one- and two-way block and reentry. ry was associated with VT. ing slowly along tortuous which  changed  position  VF was associated with m u l t i p l e wavelets t r a v e l routes among m u l t i p l e  and magnitude  i s l e t s of conduction  from moment  terminated as a r e s u l t of major s i n g l e wavefronts  to  moment.  arriving  VF did not terminate  sec allowed before d e f i b r i l l a t i o n  either  at a large area  spontaneously  was applied in t h i s  block  VT  of i n e x c i t a b l e t i s s u e , or degenerated to VF as major wavefronts subsidiary wavefronts.  Reent-  split  into  (within the 10  study).  It  was sug-  gested that t h i s was because VF was associated with more wavefronts than VT, therefore  if  arrhythmia.  some wavefronts This  is  terminated, others remained to perpetuate  a possible explanation  for  the  higher  the  frequency  of  spontaneously reverting VF in small animals such as r a t s (Kenedi and Losconci,  1973a; Clark et a l . , 1980) compared with  (Townshend P o r t e r ,  1894; Smith, 1918).  larger  animals such as dogs  Presumably the  larger  hearts  have  more wavefronts in VF than smaller hearts, and consequently express a lower p r o b a b i l i t y of spontaneous It  was found that  conduction VF.  It  defibrillation.  stimulation  of  the  left  stellate  in the ischaemic t i s s u e , and increased the  ganglion  incidence of  i s possible that t h i s enhanced sympathetic a c t i v i t y  improved VT and  increased e x c i -  - 69 tability  in the  ischaemic t i s s u e , f a c i l i t a t i n g  the conduction of PVCs and  t h e i r degeneration into VT and VF (Janse and Kleber, 1981). tions  of  l i d o c a i n e increased the proportion  of the  High concentra-  ischaemic t i s s u e which  was i n e x c i t a b l e at any given moment, thereby causing termination  of VT and  prevention of progression to VF. To summarise, of only  reentry  the major  theoretical  has been demonstrated  in  mechanisms of  acute  arrhythmogenesis,  myocardial  ischaemia.  evidence was provided from e p i c a r d i a l mapping experiments.  However, reentry  appeared to be triggered in many cases by an ectopic i n j u r y current from normal Purkinje f i b r e s .  The p r e r e q u i s i t e for  This  arising  induction of an ectopic  impulse appeared to be a dispersion of r e f r a c t o r i n e s s between the normal and the ischaemic t i s s u e .  This heterogeneity was produced by the slow conduc-  t i o n in the ischaemic t i s s u e .  Abnormal slow conduction and v a r i a b l e conduc-  t i o n block in the ischaemic t i s s u e appeared to be responsible for the c o n t i nuation of the arrhythmias and t h e i r progression into VT and VF. block was made worse by l i d o c a i n e , such that after  initiation,  arrhythmias  while l e f t s t e l l a t e ganglion s t i m u l a t i o n  Conduction  terminated  soon  improved conduc-  t i o n (lessened block) and worsened the arrhythmias. Do these phenomena occur  in  rats  laboratory has demonstrated e f f e c t s potentials species. refractory  (Inoue In  addition,  1984)  to  those  evidence in vivo  bradycardia on arrhythmia  that  the  arrhythmias  occlusion ?  of occlusion in - v i t r o on  similar  indirect  coronary  during  in  other  such as a reduction  incidence (Mertz  occurring  Our  intracellular  demonstrated  period (Northover, 1986) and a lack of e f f e c t  tion-induced suggests  et a l . ,  following  the  in  of vagal s t i m u l a and Kaplan,  30 min  period  1982) after  occlusion are more l i k e l y to be reentrant than automatic. A question arrhythmogenesis  which  arises  is,  compared with  to  i ? $ i  what  extent  The e f f e c t s  does of  i'  N a  contribute  lidocaine  and  to left  - 70 s t e l l a t e stimulation  (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 p r e v i o u s l y , stroke  of  the AP (phase-0)  in normal is  ventricular  governed by i  The changes in phase-0 and r e s t i n g E  the  reduction  in  the  and the  dV/dt  initial  plateau  produced by myocardial  m  described above, may represent one of four Firstly,  ,  N a  tissue  represent  a  ..  ischaemia, as  electrophysiological  may  by i  up-  phenomena.  severely  depressed  Hid A  ^Na'  because  1 S  depolarisation  t n e  will  represent  an enhanced i ^ .  This i s  because d e p o l a r i s a t i o n  mV w i l l  bring  to  threshold  $  J  steady-state  E closer m m  inactivation  -90 mV), whereas  voltage. hanced current  inactivation  Thirdly, i .j. $  i^  of  the  Finally,  the  i -  be  upstroke the  i  N a  .  Secondly,  for  almost  completely  may involve  upstroke  both  may be  system which possesses elements of  the  i . si  (o^ should be s i m i l a r  s  will  a  of  acute  ischaemia  of  cause  associated with  upstroke  may  to -60 to  -65  without to  a  inducing  that at  an MDP  inactivated  depressed i ^  associated with i^  myocardial  and/or  i -,  an  this  and en-  a  but  s l  at  abnormal perturbed  by factors of ischaemia other than d e p o l a r i s a t i o n , such as increases in pH ( I i j i m a e t - a l . , 1986) and the products of anaerobic metabolism (Corr et a l ; , 1984). 1.3.6.2 How - might- - j ,. - contribute trophysiological  changes  produced  by  to -arrhythmogenesis ? The e l e c -  acute  myocardial  ischaemia  et a l . , 1977a; 1977b; Kleber et a l ; , 1978), and the attendant arrhythmogenic mechanisms (reentry may play an important  and automaticity,  r o l e in arrhythmogenesis  etc.)  (Downar  hypothetical  reveal  that  i ^ $  (Hauswirth and Singh, 1978).  The p r i n c i p a l reasons for such a suggestion are as f o l l o w s . a. (Downar  Acute et-al.,  myocardial 1977b;  ischaemia  Kleber  et-al.,  is  associated 1978;  Inoue  with et a l . ,  depolarisation 1984)  to  E  m  values  close to  Reuter,  1970a).  (Reuter,  1973),  reduce i . , for  those producing complete However, it  but  from  the  inactivation  known  enhance i t  i^  (Beeler and  a  voltage-dependence  would be expected that t h i s  rather  of  by v i r t u e  depolarisation  of  a reduction  of  i .  should  in  not  threshold  activation. b.  Acute myocardial  ischaemia i s characterised by slow conduction  the ischaemic t i s s u e (Downar et a l ; , 1977b; Kleber e t - a l . - , 1978). conduction dV/dt the  can  be  for,  almost  completely,  by  This slow  the  fall  in  of the upstroke of the ischaemic AP (Dodge and C r a n e f i e l d , 1982);  max  r  increase  increases 1982).  accounted  in  in  3  in  longitudinal  intercalated  intercellular  resistance  disc resistance may also  associated  contribute  with  (Weidmann,  Slow conduction in depolarised Purkinje t i s s u e has been shown to be  dependent resting  on  Ca  (Cranefield  + +  etal.,  E , as discussed above, the  1972),  current  m  of the slow conduction AP may be i •.  From  considerations  responsible for  the  of  upstroke  In t h i s regard, slow conduction  in  Purkinje t i s s u e in - v i t r o has been shown to be i n h i b i t e d by ( ^ - v e r a p a m i l  at  concentrations below those which i n h i b i t i ^ c.  Acute myocardial  extracellular  K  et a l . , 1980).  +  a  (Cranefield e t a l ; , 1974).  ischaemia i s associated with a large increase  concentration  While t h i s  ([K ] )  and  Gettes,  phenomenon may contribute  to the  +  Q  (Hill  occurring in acute myocardial ischaemia and i t s attendant above),  it  endings  (Hirche  aline  1980;  inhibits  1985).  inactivation  of  By a c t i v a t i n g i  s i  consequences (see  adrenoceptors,  (Bean et a l . ,  Hirche  depolarisation  may also serve to release noradrenaline from sympathetic et-al;,  in  1984).  This  nerve  noradrenarrhythmo-  genic mechanism i s highly s p e c u l a t i v e , however, and recent evidence from our laboratory does not  (Botting support  acute myocardial  et a l . ,  a major ischaemia.  1983) role  for  and elsewhere  (Daugherty  catecholamines  Therefore,  if  i' ^ s  is  in  et-al.-,  1986)  arrhythmogenesis  involved  in  in  arrhythmo-  genesis,  this  involvement  is  essentially  independent  of  influence of adrenoceptor a c t i v a t i o n and antagonism on i d.  the  ..  Although current evidence does not support a r o l e for  in arrhythmogenesis  in acute myocardial  precluded.  If  abnormal  automaticity  ischaemia (Janse, 1982; Janse and  Kleber 1981; Mertz and Kaplan, 1982; Northover, 1986), t h i s not  modulating  automaticity  is  the  possibility  expression  of  enhanced  latent pacemaker currents  in Purkinje t i s s u e (Spear and Moore, 1982),  it  or  i s possible that  arrhythmogenesis.  i .,  a similar  current  then  plays an important r o l e  This i s because the pacemaker current  is  in  in the sinus node  ++  comprises, Giles, i  s i  in  part,  1985),  of  i*£ ,  a Ca  a  current  and phase-0 d e p o l a r i s a t i o n  is  similar carried  to  i ^  (Shibata and  $  almost e x c l u s i v e l y by  (Yangihara e t - a l . , 1980; Brown, 1982). e.  Activation  phase between the which t r i g g e r  mapping experiments have shown that normal  reentry  and  generates  injury  (Janse, 1982; Janse and Kleber 1981).  determinant of such t r i g g e r i n g to e x c i t a b l e t i s s u e . ischaemic)  ischaemic t i s s u e  a disparity  in AP  currents  The p r i n c i p a l  i s the occurrence of an AP plateau  adjacent  The plateau of the AP in normal (and probably also in  ventricular  tissue  is  governed by i  • (Reuter,  1973).  The AP  plateau in non-ischaemic t i s s u e i s much longer than the plateau in ischaemic t i s s u e (Downar et a l ; , 1977b; Kleber et a l . , 1978; Inoue e t - a l ; , 1984).  It  follows that the longer the plateau in non-ischaemic t i s s u e , the higher  the  probability tissue.  of  triggering  Therefore,  i  an e l e c t r o t o n i c  • may be involved  re-excitation in  of  arrhythmogenesis  the  ischaemic  by v i r t u e  of  i t s r o l e in creating a dispersion of AP plateau (and consequently a d i s p e r sion in r e f r a c t o r i n e s s ) between the ischaemic and non-ischaemic t i s s u e . f.  It  instrumental ischaemia.  is in  possible to initiating  hypothesize  other  or maintaining  Only one f u r t h e r  mechanisms by which  arrhythmias  possibility will  in  acute  i ..  is  myocardial  be discussed here, and  this  concerns the hypothetical automaticity. to  the  mechanism of  arrhythmogenesis  known as  From i n - v i t r o s t u d i e s , triggered automaticity has been linked  occurrence of  oscillatory  afterpotentials  known as e a r l y or delayed a f t e r - d e p o l a r i s a t i o n s they occur before or after  full  ( C r a n e f i e l d , 1977),  in  acute myocardial  triggered  activity  occur  occluded v e n t r i c u l a r infarction  also  (DADs), depending on whether  repolarization,  respectively.  is no evidence to support a r o l e for triggered automaticity esis  triggered  While  there  in arrhythmogen-  ischaemia, i t  has been suggested that  DADs and  in  Purkinje  from  regions  (El Sherif e t - a l ; ,  surviving of  dog hearts  1982).  tissue  following  excised the  The involvement  of  the  development i  in  g i  of  DADs has  been suggested by the observations that DADs are enhanced by r a i s i n g e x t r a cellular by  Ca , + +  cardiac  inhibit  occur  in  glycosides),  i .  (Ferrier  depolarised t i s s u e and that  and Moe,  DADs are  1973).  that DADs are generated by the t r a n s i e n t  (especially tissue inhibited  However,  by compounds  other  inward current  depolarised  evidence  which  suggests  (Lederer and T s i e n ,  1976), a current which appears to be d i s t i n c t from i ^ . $  Recently i t lactate  was shown that simulated ischaemia (16.2 meq/1 K ,  low pH,  +  and hypoxia)  abolished DADs induced by high  frequency  Purkinje t i s s u e in v i t r o (Opie e t - a l ; , 1986; Coetzee e t a l . , t i n g that t h i s mechanism of arrhythmogenesis  pacing  in  1986), sugges-  in acute myocardial  ischaemia  may not be important. 1.4-  The-pharmaeology-of caleium-antagonists 1.4.1  Definition  Calcium antagonists their  were  pharmacological e f f e c t s  entry of  2+ Ca  et a l ; ,  1969).  inhibit  2+ Ca  in  originally  predominantly by i n h i b i t i n g  a manner which  This d e f i n i t i o n entry  resulting  described as drugs  can be i n h i b i t e d  from the  opening  of  produce  voltage-activated  2 by Ca  has been extended to  which  +  include  (Fleckenstein drugs  2+ Ca channels  which  coupled  - 74 with drug receptors (see Jam's and T r i g g l e , 1983), and drugs which 2+ intracellular  Ca -dependent  processes  (Rahwan, 1983; Lynch and Rahwan, 1982). occurred in conjunction  such  as  binding  with  inhibit  calmodulin  The expansion of the d e f i n i t i o n has  with the introduction  of  new terms  such as  'slow  channel b l o c k e r s ' and 'calcium entry b l o c k e r s ' (see Nayler, 1983).  The term  calcium antagonist  original  definition  i s used here in accordance with F l e c k e n s t e i n ' s  (see above and F l e c k e n s t e i n , 1983 for review).  1.4.2 Pharmacology of phenethylalkylamines and 1,4-dihydropyridines Phenethylalkylamine bepridil, D888.  tiapamil,  anipamil  1,4-dihydropyridines  nitrendipine. most  calcium antagonists  detail  include  and a v a r i e t y  of  verapamil,  trial  include n i f e d i p i n e ,  gallopamil,  preparations  felodipine,  such as  nimodipine,  and  Verapamil, gallopamil and n i f e d i p i n e have been studied in the (see  Henry,  1979;  1980;  Triggle,  1981;  F l e c k e n s t e i n , 1983;  Nayler and Horowitz, 1983). Phenethylalkylamines have been suggested to for  the  channel  associated with  i n a c t i v a t e d state  (Kohlhardt  i  possess a higher  • when the channel  channels (Lee and Tsien, 1983). known frequency-dependence of  the  open  or  are equipotent in rested and open  This difference may account for phenethylalkylamines  (Sanguinetti  1982) compared with 1,4-dihydropyridines,  which possess  quency-dependence,  resting  considerably  more  1983; 1985; Hachisu and Pappano, 1983). ethylalkylamine  in  and Mnich, 1978; Pelzer e t - a l . , 1982; Lee and  T s i e n , 1983), whereas 1,4-dihydropyridines  but  is  affinity  calcium antagonists  block  little  the w e l l and West, or  no  fre-  (Woods  and West,  Both 1,4-dihydropyridine  and phen-  have been suggested to  act  by slowing  the recovery from i n a c t i v a t i o n of the i • channel (Lee and Tsien, 1983). Studies et a l ; ,  with  1982),  permanently  and studies  charged  carried  smooth-muscle and cardiac t i s s u e  out  analogues using  of  gallopamil  'skinned'  (Hescheler  (plasmalemma-free)  ( F l e c k e n s t e i n , 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 t h e i r  pharmacological e f f e c t s  an action on the inner surface of the plasmalemma, and that  via  intracellular  ?+  actions such as i n h i b i t i o n  of Ca  binding with calmodulin  (Silver  at  al,  1984) do not contribute to the e f f e c t of these drugs on e x c i t a b l e t i s s u e . Both phenethylalkylamines and 1,4-dihydropyridines trum  of  pharmacological  properties,  such  as  possess a wide spec-  inhibition  of  i^  a  (Bayer  et a l . , 1975a; Nawrath e t - a l . , 1981; Yatani and Brown, 1985), i n h i b i t i o n  of  c o r t i c o s t e r i o d release from the adrenal cortex (Costa et a l ; , 1983), i n h i b i t i o n of myosin l i g h t chain kinase a c t i v i t y However,  consideration  of  the  (Movsesian e t - a l . , 1984),  concentrations  required  to  produce  etc. these  —6  effects  (generally 10  these e f f e c t s  M or more)  as r e l a t i v e l y  has lead most i n v e s t i g a t o r s  unimportant  compared with e f f e c t s  smooth-muscle and myocardial t i s s u e r e s u l t i n g from i n h i b i t i o n operated calcium entry  to  regard  on vascular of  voltage-  (see Nayler and Horowitz, 1983; F l e c k e n s t e i n , 1977;  1983; Henry, 1979; T r i g g l e , 1981; 1982). It  i s of  interest  to determine the mechanism of any action of a drug.  In order to ascribe an e f f e c t of a drug to calcium antagonism a v a r i e t y approaches may be taken, since i t calcium entry variables,  through  especially  i s often  impossible to d i r e c t l y measure  voltage-operated channels at the same time as other in - v i v o .  Of  fundamental  importance  is  information  concerning the concentration range over which calcium antagonism i s ced;  ideally  of  one would  like  to  know E C ^ Q values.  wished to know whether the antiarrhythmic  For example,  produif  one  actions of verapamil and n i f e d i p -  ine were produced by calcium antagonism in the v e n t r i c l e s , one might approach the with order  EDgQ to  problem by comparing E D (or support  E C ^ Q ) values this  for  information  5Q  values for  calcium it  might  reductions  antagonism  in  in  arrhythmias  ventricles.  be considered of  interest  In to  - 76 investigate  the  relative  calcium antagonist  v a r i e t y of tissues under a v a r i e t y  potency of  of c o n d i t i o n s .  these drugs  There are a number  studies which have compared several calcium antagonists for  their  potencies in vascular and cardiac muscle, in vivo and in v i t r o . illustrate of  some of the problems inherent  values  and ( ^ - v e r a p a m i l  In order to  in determining whether the  were e i t h e r  taken  were compared are  directly  from the  summarised.  text  of  the  of  relative  a drug may be a t t r i b u t e d to calcium antagonism, some studies  nifedipine  in a  effect  in which  E C ^ Q or E D ^ Q publications  or  approximated by i n t e r p o l a t i o n (or extrapolation) of the data presented. 2+ ECgg values tion  for  inhibition  of  d e p o l a r i s a t i o n / C a -dependent  in vascular smooth muscle range from  1985)  to  2.4 x I O "  1.5 x 1 0 "  8  1985) for et a l . ,  M (Millard  8  M (Millard nifedipine.  1976;  et-al;, In a l l  Lee e t - a l ; ,  et-al., 1983)  3 x 1 0 ^ M (Kenakin and Beek, -  1983)  to  contrac-  for  6.3 x I O  (^)-verapamil, - 9  M (Kenakin  and from and Beek,  of the studies considered (Fleckenstein-Grun  1983; Kenakin and Beek, 1985; M i l l a r d  et-al;,  1983; Nakayama et a l ; , 1985) n i f e d i p i n e was more potent than (±)-verapami1. However,  the  difference  in  potency  varied  considerably,  et a l ; , 1983) to 350:1 (Fleckenstein-Grun e t - a l . - , 1976).  from  1.7:1  (Lee  This may r e l a t e to  the v a r i e t y of preparations, t e s t conditions and methods used. In heart preparations ( v e n t r i c l e s and a t r i a ) even greater to  range from 10  1982) for  variation. _5  to  (^)-verapamil  have been reported  M (Nabata, 1976; Raschack, 1976a; Briscoe and Smith,  1.5 x 10~  nifedipine  E C ^ Q values for  a s i m i l a r survey reveals an  8  M (Clarke  range  from  et a l . ,  7.2 x 10~  1984a; 1985). 6  (Briscoe  and  Corresponding Smith,  1982)  EC  5Q  to  _Q  5.8 x 10  M (Nabata, 1976).  In a t r i a  and v e n t r i c l e s  reported to be as much as 62.5 times as potent  nifedipine  has been  as (±)-verapamil (Raschack,  1976a), but other i n v e s t i g a t o r s have found (^)-verapami1 to be up to 5 times as potent as n i f e d i p i n e  (Clarke e t - a l ; , 1985).  Nifedipine 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 r d e t - a l . - , 1983), whereas (±)-verapami 1 was found to be more potent than Briscoe  nifedipine  and Smith,  1982;  in  7 studies  Winslow  (Raschack, 1976a; Nabata, 1976;  et-al.,  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  it  is  dangerous to  rely  on  published reports of the potency and r e l a t i v e potency of calcium antagonists with  regard  to  the  investigation  of  the  mechanism of  action  of  calcium  antagonists (as antiarrhythmics, f o r example). 1.5  Aims of-studies 1.5.1 The action of calcium antagonists in acute myocardial ischaemia The pharmacologist functions  drugs, and then account for acute myocardial  to examine and describe the properties  the mechanism(s) underlying these e f f e c t s .  ischaemia, the  effects  of  calcium  antagonists  of In  (Flecken-  s t e i n , 1969) are not well c h a r a c t e r i s e d . The f i r s t report of the actions of a calcium antagonist in acute myocardial  ischaemia appeared in  that 0.79 mg/kg (±)-verapamil  1968, when Kaumann and Aramendia  demonstrated  abolished arrhythmias and prevented death when  administered 10 min before coronary occlusion in anaesthetised dogs. t h i s time there have been a number of reports confirming the activity  of  (^-verapami 1 and d i l t i a z e m  in  dogs,  generally not been found to be antiarrhythmic Kobayashi et a l . , 1983; Clusin e t - a l . ,  although  Since  antiarrhythmic nifedipine  has  ( e . g . , Guelker et a l ; , 1983;  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 f e d i p i n e ) .  The r e s u l t s were highly v a r i a b l e between s t u d i e s , and a  c l e a r exposition of the antiarrhythmic  actions of  calcium antagonists, and  possible mechanism(s) of action remained e l u s i v e . In the development and c h a r a c t e r i s a t i o n of the conscious rat  preparation  - 78 by our laboratory, have  i t was found that (^-verapami 1 and quinidine appeared to  antiarrhythmic  therefore, which  activity  to investigate  these  drugs  antiarrhythmics  (Johnston  et-al;,  in more d e t a i l  represent,  beginning  1983a).  It  was decided,  the classes of antiarrhythmics with  class-4  (calcium  antagonist)  such as (^-verapami 1 (Singh and Vaughan W i l l i a m s ,  using the conscious r a t  of  1972),  preparation.  The experiments with calcium antagonists described in the Methods sect i o n were designed to answer the f o l l o w i n g questions: a.  Do calcium antagonists reduce arrhythmias  induced by acute myocar-  d i a l ischaemia in conscious r a t s ? b.  Do calcium  antagonists  reduce  infarct  size  following  permanent  coronary occlusion? c.  Do these e f f e c t s ( i f any) occur as a r e s u l t of calcium antagonism?  d.  What f a c t o r s determine these e f f e c t s ?  It  was expected that by carrying out experiments  dose-response curves for (in vivo and i n - v i t r o ) to questions a and b.  a range of  it  b l i n d and assembling  v a r i a b l e s , using several  preparations  would be possible to provide unequivocal answers  In a d d i t i o n ,  by considering the dose-dependence of  the e f f e c t s of d i f f e r e n t types of calcium antagonists on the same v a r i a b l e s , it  was hoped that  circumstantial  evidence  in  support  of  the  hypothesis  described in question c , or unequivocal disproof of t h i s hypothesis would be provided.  By considering the o v e r a l l r e s u l t s of the experiments,  specula-  t i v e hypotheses concerning question d were expected to be generated. 1.5.2 Arrhythmogenesis in acute myocardial ischaemia The introduction myocardial ischaemia.  has broached the subject of  arrhythmogenesis  An understanding of the mechanism(s) of  in  acute  arrhythmoge-  nesis i s a necessary adjunct to the understanding of the mechanism of action of  beneficial  drugs.  The experiments  c a r r i e d out  as part of  this  thesis  - 79 were not designed to provide a d e f i n i t i v e  statement concerning the determin-  ants of arrhythmogenesis in acute myocardial ischaemia.  However, in view of  the c o n f l i c t i n g evidence concerning the r o l e of the autonomic nervous system in  arrhythmogenesis,  and the  relationship  between  i ^  and  adrenoceptors  (Bean et a l ; , 1984), an attempt was made to resolve t h i s p a r t i c u l a r  question  by c a r r y i n g out a series of graded ablations in the CNS. In r a t s , evidence concerning the r o l e of the sympathetic nervous system in  arrhythmogenesis  is  contradictory.  Campbell  and P a r r a t t  (1983)  that a v a r i e t y of s-adrenoceptor antagonists reduced arrhythmias occlusion in anaesthetised r a t s . naline infusion  noradrenaline reduced the s e v e r i t y .  influence  arrhythmias  while adrenaline and  However, in conscious r a t s , our labora-  labetalol  (Botting  induced by  Marshall e t - a l ; (1981a) found that i s o p r e -  increased the s e v e r i t y of arrhythmias  tory found that propranolol,  found  and chemical  et a l ; ,  1983).  sympathectomy  Therefore  did  experiments  not were  c a r r i e d out in order to answer the following questions: a.  Does  the  autonomic  nervous  system play  an  independent  role  in  arrhythmogenesis following coronary occlusion in rats? b.  Is the r o l e of the autonomic nervous system of s u f f i c i e n t  magnitude  for i t to c o n s t i t u t e a major determinant of arrhythmogenesis? The use of s e r i a l ablation in the CNS coupled with s e l e c t i v e replacement of catecholamines was chosen as a unique method for answering the questions o u t l i n e d without r e s o r t i n g to the use of drugs as t o o l s , often involves u n j u s t i f i e d t i v i t y of  drug a c t i o n .  a practice  which  assumptions concerning the s p e c i f i c i t y and s e l e c If  the autonomic nervous system ( p a r t i c u l a r l y  sympathetic nervous system) plays a n e g l i g i b l e r o l e i s important to e s t a b l i s h t h i s f a c t .  in arrhythmogenesis  the it  Such a r e s u l t has important consequen-  ces, since i t would be d i f f i c u l t , in t h i s 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 brief  duction.  history.of  coronary occlusion in rats was given in the  A conscious animal preparation was developed by our laboratory  order to remove the p o t e n t i a l l y  confounding influences  recent major surgery from the experimental It  of  in  anaesthesia and  arena (Johnston et a l ; , 1983a).  i s our opinion that when i n v e s t i g a t i n g the actions of drugs or the nature  of a disease process, the  initial  experiments should be c a r r i e d out  conscious unrestrained preparations, free from ' a r t i f i c i a l ' as  Intro-  anaesthesia.  ischaemia, t h i s  In  the  case  of  the  conscious r a t  approach i s consistent with the f a c t  using  constraints such  model that  of  myocardial  the human con-  d i t i o n s of myocardial ischaemia generally 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 d e t a i l  in  the  Introduction.  stressed that c l i n i c a l l y , information concerning the c r i t i c a l the onset of symptoms (when sudden death and v e n t r i c u l a r a premium), and information of  ischaemia, i t s  vance  are  essentially  was  f i r s t h after  arrhythmias are at  concerning the r e l a t i o n s h i p between the  time-course and i t s  are both incomplete at best.  It  sequelae ( p a r t i c u l a r l y  extent  arrhythmias)  Therefore, arguments concerning c l i n i c a l r e l e -  pointless,  since  no  human  template  of  myocardial  ischaemia and i n f a r c t i o n 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 p r i n c i p a l aim of a myocardial ischaemia model should be the reproducible and unequivocal  production  of  myocardial  ischaemia of known s e v e r i t y and reproducible sequelae, in a manner which can be manipulated simply, whereby the preparation  serves as a bioassay.  The  - 81 r a t may be the only species which meets a l l  these requirements  (dogs, ham-  sters and guinea-pigs are too v a r i a b l e , pigs are too l a r g e , primates are too expensive and mice are too s m a l l ) . The following been described, et-al;,  chapters in  part,  attempt to in various  collate  all  publications  the methods which have from our  laboratory  (Au  1979a; 1979b; Johnston et a l ; , 1983a; 1983b; Botting et a l ; , 1983;  C u r t i s et a l . , 1984; 1985a; 1985b; 1986a; 1986b; C u r t i s 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 a s s i s t a n c e . 2.1.2 Preparation Before preparation,  all  instruments,  placed in a bath of 70% ethanol In  addition  ethanol.  leads,  lines  and occluders  were  in d i s t i l l e d water for a n t i s e p t i c purposes.  hands were c a r e f u l l y washed in soapy water  and rinsed  As a f u r t h e r precaution against i n f e c t i o n , f i n g e r  in 70%  n a i l s were cut  as short as p o s s i b l e . All rats,  experiments  were c a r r i e d  230 - 350 g.  out  A glass gas j a r was e q u i l i b r a t e d  oxygen delivered v i a a vapourisor a r a t i o n s , a humidifier prised through  simply  of  (Fluotec).  flask  anaesthetic  containing  had shown that i f  a saturated  gas was bubbled.  halothane, or i f rats  was placed in  the j a r  the e x c i t a t o r y  plane of  solution  The s a l i n e prevented  before  a lower concentration of halothane ( 1 - 3  passed through  This com-  saline  the  Each rat was  min a f t e r halothane had been introduced.  a rat  in  For the l a s t 300 - 400 prep-  growth of algae and yeasts for periods of at l e a s t 9 months. placed in the j a r 2 - 3  Wistar  with 5 % halothane  was included in the anaesthetic c i r c u i t .  a conical  which the  using male Sprague Dawley or  Experience  equilibration  with  %) was used, then  anaesthesia more s l o w l y ,  and  exhibited coprophagic behaviour, which compromised the subsequent stage of preparation  (intubation).  - 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 e d by slow, shallow r e s p i r a t i o n ) . bated as f o l l o w s .  Following i n d u c t i o n , each r a t was i n t u -  The mouth was f i x e d  open by temporarily  attaching  upper jaw to the bench, and a 14 gauge human intravenous catheter was inserted into the trachea with the aid of The sharp point blunted  of  the metal  and smoothed for  insert  atraumatic  of  the  a paediatric  catheter  location  of  the  (Jelco)  laryngoscope.  had been previously  the catheter.  The vocal  cords, v i s i b l e as two white bands on the l e f t and r i g h t side of the entrance to  the  trachea, were used as landmarks.  The p a e d i a t r i c  laryngoscope had  been f i l e d on one side in order to permit atraumatic entry into the small buccal c a v i t y of the r a t s . Intubation was v e r i f i e d 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 t a b l e , maintained on 1% h a l o thane, and prepared with an occluder, i n t r a v a s c u l a r l i n e s and ECG l e a d s , as follows.  Anaesthesia was adjusted during preparation such that movement was  j u s t prevented. 2 . 1 . 2 . 1 Occluders.  In contrast with e a 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 .  Essentially,  a guide was made from an 11 cm length of PE10 polythene tubing.  One end was  f l a r e d by b r i e f exposure to heat from a soldering i r o n , w h i l s t 1 cm from the other end, a flange was made by b r i e f l y melting the tubing by r o t a t i n g i t f r o n t of a j e t of hot a i r .  in  The hot a i r j e t was also used in the manufacture  of blood pressure and intravenous l i n e s (see below), and was created simply  - 83 -  by passing pressurised a i r through a t h i n 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 l a r e d end of the guide. The occluder was implanted as f o l l o w s . over the 4th to 6th r i b s on the l e f t dissection  (Spencer Wells f o r c e p s ) .  A 1 cm skin i n c i s i o n was made  thorax.  This was enlarged by blunt  The forceps were then  inserted  under  the Pectoral i s muscle, which was gently separated from the underlying Rectus Abdominus, exposing the Intercostal muscles beneath. was immediately i n i t i a t e d .  Artificial  respiration  Using a Palmer pump, or equivalent, at a stroke  volume of 4 ml per r a t and a stroke rate of 54/min, the anaesthetic regimen described above was d e l i v e r e d , and c o n t r o l l e d using the same c r i t e r i a cribed above.  Voluntary r e s p i r a t i o n was switched to a r t i f i c i a l  by the simple  r e d i r e c t i o n of gas flow through the plumbing of the anaesthesia s e t - u p . 5th or 6th forceps.  intercostal  space was then  punctured  using the  This i n c i s i o n was enlarged by blunt d i s s e c t i o n .  versus 6th i n t e r c o s t a l space was a r b i t r a r y ,  des-  The  Spencer Wells  The choice of 5th  and generally not noted.  If  the  heart was exposed unfavourably for placement of the occluder, then a second intercostal  i n c i s i o n was made.  arations.  The i n t e r c o s t a l  retractors  attached  tubing.  to  This was necessary in less than 5 % of prep-  i n c i s i o n was widened by i n s e r t i n g  braided  silk  The aluminium r e t r a c t o r s  thread  or  lengths  were placed c a r e f u l l y  of  4  aluminium  PE90  polythene  in order to  avoid  damaging the l e f t lung, and were f i x e d by attachment to the small s t a i n l e s s steel operating table (via s l i t s e a s i l y moved and r e p o s i t i o n e d .  in i t s In  later  corners).  The r e t r a c t o r s  experiments,  all  could be  retractors  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 i g h t thorax,  and replacing 1 of  - 84 the r e t r a c t o r s  under the thymus (which i s large and o v e r l i e s the heart  in  most r a t s ) ,  it  was a simple matter to expose the l e f t v e n t r i c l e and a t r i a l  appendage.  Using a pair of small blunt f o r c e p s , the t h i n p e r i c a r d i a l mem-  brane was retracted at a p o s i t i o n overlying the junction  between the  a t r i a l appendage and the l e f t v e n 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 f o r c e p s , a small tear  the pericardium was created. one after  left  another,  in  The aluminium r i b r e t r a c t o r s were then moved,  by i n s e r t i n g  their  tips  through  the small  pericardial  t e a r , such that the pericardium was now included with the retracted t i s s u e . As a r e s u l t ,  the heart was l i f t e d toward the i n t e r c o s t a l  i n c i s i o n by t h i s  p e r i c a r d i a l c r a d l e , f a c i l i t a t i n g subsequent surgery. Without d i s t u r b i n g the heart by gripping i t finger  between the thumb and f o r e -  (Johns and Olson, 1954), or e x t e r i o r i s i n g i t  incision  through the  (Selye e t - a l . , 1960), the nylon suture of  the  intercostal  occluder was sewn  under the l e f t coronary artery (generally referred to as the LAD, see I n t r o duction) by i n s e r t i n g the needle into the l e f t v e n t r i c l e under the overhanging l e f t a t r i a l appendage, and bringing i t The a t r i a l  out high on the pulmonary conus.  appendage was displaced s l i g h t l y  manipulation with a small pair of blunt  for  forceps.  this  purpose by  gentle  The LAD i s 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  coronary veins as landmarks.  LAD using the highly  visible  In our experiments, although the veins were  found to be v i s i b l e , i t was usually possible to see the artery quite c l e a r l y as a t h i n pink l i n e emerging from under the a t r i a l  appendage.  The s t i t c h of  the suture was made wide, such that the needle entered and l e f t the myocardium approximately 2 mm e i t h e r side of the a r t e r y .  As Heimberger (1946) and  Selye et al.- (1960) have pointed out, unless the artery ligation  there are no sequelae of consequence.  i s included in the  This whole process usually  - 85 produced no blood l o s s . Occasionally (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  this  occurred, 5 min was allowed for c l o t t i n g , then the t h o r a c i c c a v i t y 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 a r t e r y , suture was sewn through the f l a r e d end of the guide tubing.  the  Therefore, the  suture emerged from the guide t u b i n g , passed into the myocardium, under the LAD, out of the myocardium and back through making a loose loop or snare. and the remaining  length  of  the t i p  of the guide  tubing,  The needle of the suture was then cut nylon c a r e f u l l y melted down to  form a small  b a l l , which prevented the suture from being pulled back through the t i p of the guide tubing. guide tubing.  The nylon suture extended the f u l l  The s i z e of the loop at the end proximal  adjusted by p u l l i n g on the suture at the end d i s t a l  off,  flared  length of  the  to the heart was  to the heart.  It was  found in some e a r l y experiments that occlusion sometimes resulted in part of the thymus being caught in the occluder.  This could be completely prevented  by making the loop s m a l l , such that the f l a r e d t i p of the guide tubing was positioned adjacent t o , or j u s t underneath the a t r i a l s i z e of the loop had been adjusted, the d i s t a l down to a small b a l l adjacent to the d i s t a l manner s i m i l a r  to that by which the  Once the  length of suture was melted  tip  proximal  appendage.  of the guide tubing, in a  end of  the  suture  had been  affixed. The Pectoral i s muscle was sutured l i g h t l y to the Rectus Abdominus muscle with s i l k . to f i x  it  The loose ends of the s i l k suture were then t i e d to the occluder in p o s i t i o n in the thorax.  In a l l but the l a s t 50 - 60 prepara-  t i o n s , the pneumothorax was evacuated at the time the chest was closed by i n s e r t i n g a length of PE90 polythene tubing through the i n t e r c o s t a l i n c i s i o n , and applying negative pressure to the thorax as the chest was c l o s e d .  This  - 86 precaution was r e c e n t l y s u c c e s s f u l l y replaced by h y p e r i n f l a t i o n by t r a n s i e n t l y closed.  increasing stroke volume to 6 ml per r a t  of the lungs  as the chest was  This modification reduced the time taken to prepare the r a t s .  As soon as the chest was c l o s e d , the halothane anaesthetic was replaced with 100 %oxygen.  The occluder was then fed subcutaneously to the subscap-  ular region using a s t a i n l e s s steel t r o c a r .  The occluder was e x t e r i o r i s e d  between the shoulder blades, and a s i l k suture was attached to the occluder j u s t below the flange at the d i s t a l end ( r e l a t i v e  to the h e a r t ) .  This piece  of s i l k was designed to serve as a ' f l a g ' to a s s i s t in the location of the occluder on the day of o c c l u s i o n .  The length to which the occluder projected  from the subscapular region was l i m i t e d  to no more than 1 cm, in order  reduce the l i k e l i h o o d of the occluder being chewed or tugged at by the  to rat  during the week of recovery from surgery.  This was arranged by p u l l i n g on  the occluder with a pair of small forceps.  In l a t e r experiments the occluder  was allowed to s l i p j u s t beneath the s k 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 i c a t r i n ( b a c i t r a c i n ,  neomycin and streptomycin)  powder,  and Marcaine  (1% bupivacaine). The skin over the Pectoral i s muscle was closed with a p u r s e - s t r i n g suture of s i l k . breath  The r a t was disconnected from the r e s p i r a t o r y pump, and allowed to spontaneously.  and c l o s i n g the  skin  The i n t e r v a l  between c l o s i n g the  i n c i s i o n was generally  thoracic  approximately  since the rat was r e c e i v i n g only oxygen during t h i s  2-3  incision min, and  time, s u f f i c i e n t  thane was expired to permit the r e s p i r a t o r y centre to override the  halo-  artificial  r e s p i r a t o r y rhythm, such that once the rat was disconnected from the r e s p i ratory pump, i t generally  immediately began to breath spontaneously.  remained  immobile  for  a further  C i c a t r i n and Marcain were i n f i l t r a t e d  2-5  min.  However, the rat During  this  time,  into the subscapular wound, and the  - 87 other e x t e r i o r i s e d leads and l i n e s (see below) were t i d i e d up. The l o c a t i o n of the occluder was the l a s t stage in preparation, but was described f i r s t since i t i s the focus of the experiment.  A f t e r the induction  of anaesthesia, the f i r s t stage of preparation was the l o c a t i o n of the blood pressure and intravenous l i n e s . 2.1.2.2 Leads -and - 1 i n e s .  Blood pressure and intravenous l i n e s were  made from PE polythene t u b i n g , 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 r o t a t i n g and melting t h e i r pressing them together.  This process was c a r r i e d out a f t e r  tips,  then  the lengths  of  tubing had been threaded onto a length of t h i n w i r e , in order to prevent the welding process from occluding the lumen.  The seal between the tubings was  tested by c l o s i n g 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 , o s t e n s i b l y closed system, while  it  was submerged in  distilled  water.  seal was revealed by bubbles emerging from the junction curl was put into the PE10 end of the l i n e , by looping i t and submerging i t  in b o i l i n g water for 3 s e c .  An incomplete  of the tubings.  A  around a glass rod  The loop was f i x e d by sub-  merging i t in i c e cold water. The l i n e s were implanted using a modification of the method developed by Weeks (Weeks and Jones,1960; Weeks, 1981).  A midline  laparotomy was per-  formed, extending from approximately 1 cm posterior to the xiphoid process to the region overlying the b i f u r c a t i o n of the common i l i a c v e s s e l s .  Using  f i n g e r s o n l y , the connective t i s s u e overlying the abdominal aorta and vena cava was c l e a r e d .  A s i l k thread was then placed round these v e s s e l s , using  a p a i r of small forceps, in the standard manner.  The thread was u s u a l l y  positioned in the region of the b i f u r c a t i o n s of the renal veins and a r t e r i e s , at which point the small forceps passed under the aorta and vena cava most  - 88 -  easily.  A more posterior approach o c c a s i o n a l l y produced haemorrhaging  the vena cava.  Whilst t h i s was r a r e l y f a t a l ,  and u s u a l l y resolved without  sequelae by the time of o c c l u s i o n ' and s a c r i f i c e , i t (5 min was allowed for naturally, rations  clotting  delayed surgery).  the  if  from  was deemed inconvenient  ever haemorrhaging occurred, and t h i s ,  Therefore in most of the l a s t 500 - 600 prepa-  aorta and vena cava were  accessed from  the  former,  anterior  approach. Once the aorta and vena cava had been l o c a t e d , these vessels were separated  using small  forceps.  Again, t h i s  plished when the more anterior  procedure was most e a s i l y accom-  approach was made.  The thread was divided  such that tension on one or the other of the r e s u l t a n t in either the aorta or the vena cava. in the rat used for  as f o l l o w s . locating  the  The l i n e s themselves were then located  Using the same trocar occluder,  muscle and subcutaneously to  the  a channel  leaving the  lines  in  which would subsequently be  was created  subscapular r e g i o n .  p l a c e , two l i n e s were threaded into t h i s removed,  t i e s would stop flow  place.  through With the  the  trocar  channel, and the trocar  A syringe  heparin) was attached to each of the l i n e s at t h e i r  full exit  of  the  occluder).  (using two r e t r a c t o r s  The abdomen was then  retracted  in  was then  saline  (without  point between the  shoulder blades (note that t h i s e x i t point was used subsequently for orising  Psoas  on the  exteri-  left  side  in the same manner as for the placement of the o c c l u -  der). Both the l i n e s 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 facilitate  the  subsequent i n s e r t i o n  caval l i n e was located f i r s t . and the l i n e was l i g h t l y  into  the  vessels.  Usually,  the  to  vena  The a i r in the l i n e was displaced with s a l i n e ,  held with a pair of small forceps.  The vena cava  was temporarily occluded with the s i l k thread, and a t i n y hole was created  - 89 -  d i s t a l to the occlusion (proximal veins.  to the renal  The l i n e was then inserted into the h o l e , and the occlusion removed.  The process was repeated  for  exception  distal  heart.  to the h e a r t ) , j u s t anterior  that  the h o l e ,  the  placement of to  the  the occluder,  aortic  line,  with  the  to  the  line,  the  towards  the  was also d i s t a l  In both the case of the vena caval and also the a o r t i c  PE10 tubing  was inserted 2 - 3 cm into  the v e s s e l s ,  pointing  heart. The l i n e s , and t h e i r placement, were designed in order that blood pressure recording and drug administration (non-occluded) v e s s e l s .  could be undertaken v i a  functioning  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 t u b i n g , such that the l i n e s could be moved f r e e l y without blood leakage.  In a d d i t i o n , the small diameter  of the PE tubing did not occlude the aorta or vena cava; t h i s 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 l a r g e , e a s i l y a c c e s s i b l e v e s s e l s , s u i t a b l e for  cannulation  with chronic indwelling non-occluding l i n e s . The abdomen was dusted with C i c a t r i n , and the body wall running s t i t c h .  closed with a  The skin was then c l o s e d , also with a running s t i t c h ,  the wound was i n f i l t r a t e d with C i c a t r i n and Marcaine.  Following placement  of the occluder by the method described above, the e x t e r i o r i s e d ends of cannulae were treated as f o l l o w s .  and  the  S i l k f l a g s were attached to each cannula  in the same way that a f l a g was attached to the occluder.  Approximately 0.3  ml of s a l i n e was then injected into the vena caval l i n e , which was abruptly clamped with a pair of  Spencer Wells f o r c e p s , the t i p s  made atraumatic by encasement in soft polythene tubing. exposed PE50 portion  of which had been The open end of the  of the cannula was then sealed by m e l t i n g ,  ordinary c i g a r e t t e l i g h t e r .  using an  The same process was c a r r i e d out for the a o r t i c  - 90 line.  The melted seals were f l a r e d such that t h e i r  imately double that of the PE50 tubing.  diameters were approx-  The l i n e s were allowed to  from between the r a t s ' shoulder blades by about 1 cm.  extend  As in the case of the  occluder, the l i n e s sometimes slipped beneath the skin during the  interval  between preparation and o c c l u s i o n ; the s i l k f l a g s permitted easy l o c a t i o n of the l i n e s , and t h e i r p o s i t i o n under the skin was considered acceptable, since the cannulae were therefore out of the reach of the r a t s .  In general, how-  ever, the f l a r e d , melted ends of the cannulae prevented them from s l i p p i n g completely under the s k i n . The rats appeared to be unconcerned by the presence of the e x t e r i o r i s e d cannulae and the occluder. around the e x i t  site  There was an extremely low incidence of  (fewer  than  1 % of  rats  infection  had pus exudation  from  the  wound), presumably as a r e s u l t of the prophylaxis with C i c a t r i n . ECG  leads  were  prepared  from  teflon-coated  stainless  steel  wire.  Approximately 1 cm of one end of each lead was de-insulated using an ordinary c i g a r e t t e l i g h t e r flame.  The lead was approximately V3.  implanted with the occluder, as f o l l o w s . of the lead by wrapping i t was then  A t i g h t b a l l was made in one end  around a 21 gauge hypodermic needle.  positioned underneath the  needle as a t r o c a r .  The chest lead was  This end  Pectoral i s muscle, using the 21 gauge  The d i s t a l end of the chest lead was e x t e r i o r i s e d with  the occluder between the shoulder blades.  The large trocar  purpose was directed through the hole made e a r l i e r for  the  used for  this  exteriorization  of the i n t r a v a s c u l a r cannulae. The limb leads were implanted on the day of o c c l u s i o n . They were made of the  same material  as the  chest  lead,  but  were  positioned  in  a  slightly  d i f f e r e n t manner (see below). After preparation, the animals were placed in i n d i v i d u a l cages and given tap water and Purina rat chow ad l i b i t u m .  The body weight of each rat was  - 91 recorded before preparation.  The procedures described usually took  approx-  imately 25 - 40 min to complete, from induction of anaesthesia to recovery of consciousness. 2.1.3 Coronary occlusion This  section deals with the experiment  proper,  which was c a r r i e d  approximately 7 days after preparation (range 4 - 1 5 days). events i s described below.  out  The sequence of  The actual process of occlusion was as f o l l o w s .  The occluder was gripped by the Spencer Wells forceps with the  atraumatic  t i p s (see above) j u s t above, and adjacent to the flange located approximately 1 cm from the end d i s t a l provide a stock for occluder  to the heart.  The purpose of  the flange was to  the forceps which would permit the outer guide of  to be held f i r m without excessive clamping, such that  the  snare of the occluder remained free to move w i t h i n the outer guide.  the inner  Using  f i n g e r s , the small bobble on the d i s t a l end of the inner snare of the o c c l u der was pulled from the adjacent outer guide tubing to the extent of approximately 2 mm.  A second pair of forceps was then used to f i r m l y grip  exposed inner  snare of  between the  the occluder.  inner snare and the outer  Traction was then applied guide tubing  Successful occlusion occurred when the outer and i t  was no longer possible to move the  outer guide.  to  smoothly  produce o c c l u s i o n .  guide tubing  became c r i n k l e d  inner snare in r e l a t i o n  This moment was designated time z e r o .  the  to  the  The atraumatic forceps  were clamped down f i r m l y on the occluder at t h i s 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 t a l end of the outer  guide tubing,  fixing  i t in p l a c e . Time zero was accompanied in most cases by a sudden change in the ECG, but t h i s was not taken as a c r i t e r i o n for o c c l u s i o n , since i t  was possible  that drug treatment could delay the ECG changes caused by o c c l u s i o n .  Never-  - 92 theless,  in  the  present experiments  it  was found that  an absence of ECG  changes w i t h i n 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), that occlusion had been unsuccessful.  indicating  This event occurred in less than 5%  of r a t s . 2.1.3.1 Sequence of events. occlusion i s outlined in Figure 1.  The sequence of events on the day of 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 f o l l o w s .  Each wire was threaded through a  23 gauge  the  hypodermic  needle,  such  that  non-insulated  approximately 0.5 cm from the sharp end of the needle. bent back c l e a n l y , forming a barb.  tip  projected  The wire was then  The needle, together with the electrode  was passed subcutaneously, e i t h e r to the elbow region of the forelimb aspect)  for  the  2 forelimb  l e a d s , or  region for the r i g h t hind limb l e a d .  to  the  right  to the e x i t  flank  point of  all the  The ECG leads were disconnected from the  hypodermic needle by pinching the skin around the needle t i p withdrawing the needle.  dorsal  The entry point was the same for  three leads, namely approximately 0.5 cm anterior occluder and i n t r a v a s c u l a r l i n e s .  anterior  (dorsal  and c a r e f u l l y  As the needle was withdrawn, the electrode remained  in p l a c e , owing to the action of the barb. In approximately the f i r s t 50 preparations, the limb leads were implanted on the day of preparation.  Although they did not appear to cause d i s -  comfort, they were sometimes removed by the r a t during the i n t e r v a l  between  preparation and o c c l u s i o n .  implant  Therefore, i t was considered sensible to  limb leads on the day of occlusion in every case.  In t h i s way an element of  consistency was introduced, which was absent  some r a t s  if  fresh electrodes placed on the day of o c c l u s i o n , and some not.  were used with  CORONARY OCCLUSION IN CONSCIOUS RATS Sequence of events on the day of study (time scale in min) -60  0 •15 -5,+5  +15  +30  +60  +120  +180  1 day l a t e r +240  0  30  stabi1isation  drug admi ni s t r a t i on r o CD  OCCLUSION  DEATH?  SACRIFICE  measure occlusion Figure 1.  measure i n fa rc t  Sequence of events on the day of occlusion following connection of leads and l i n e s  - 94 The 3 limb leads were connected to one another by simply t w i s t i n g  their  exposed ends together, forming a composite whole body l e a d , which was trimmed to  approximately  2 cm length  using a p a i r  of  scissors.  The t e f l o n was  removed from the end of the composite lead as described above.  It should be  noted that the anaesthetic mask was temporarily moved well away from the r a t during  this  procedure.  The un-insulated  leads were connected to  1 of 2  lengths of enamel-coated s t a i n l e s s steel wire by t w i s t i n g the wires together. The t i p s of the wires had been scraped with a scalpel blade to remove the i n s u l a t i n g enamel.  The connections were i s o l a t e d by applying a small piece  of s u r g i c a l tape to each.  The tape was attached in such a way that i t  e a s i l y be removed at a l a t e r time.  Additionally  in t h i s  could  regard, when the  leads and wires were connected, the t w i s t was always clockwise, f a c i l i t a t i n g subsequent disconnection. Whilst the r a t was s t i l l  anaesthetised with halothane, the blood pressure  l i n e was connected to a pressure transducer v i a a length of PE tubing and a blunted 23 gauge needle t i p . full  The intravenous l i n e was connected to a syringe  of s a l i n e in a s i m i l a r manner.  imately 0.3 ml s a l i n e . arterial  Both l i n e s were flushed with approx-  The intravenous l i n e was i n d i s t i n g u i s h a b l e from the  l i n e by v i s u a l i n s p e c t i o n , but once the melted t i p of the l i n e had  been snipped o f f ,  the l i n e s were e a s i l y d i s t i n g u i s h a b l e in most cases by the  f a c t that blood immediately f i l l e d the a o r t i c line.  l i n e , but not the vena caval  In the infrequent event that blood did not flow back through  either  l i n e , both l i n e s were flushed with s a l i n e u n t i l a d i s t i n c t i o n could be made. The a o r t i c l i n e was kept open by attaching a leak pump in s e r i e s with the line,  according to the method of Weeks (1981).  In at  least 75% of  cases,  negative pressure on the vena caval l i n e caused dark venous blood to appear in the l i n e .  In the remaining 25% of cases, the venous l i n e was tested by  the i n j e c t i o n of a small amount of adrenaline which e l i c i t e d a small pressor  - 95 response when the l i n e was patent. The placement of the ECG leads and the connection of the blood pressure and intravenous l i n e s to the transducer and syringe of s a l i n e , r e s p e c t i v e l y , took no more than 10 min. anaesthetic imately 2 - 5  Rats regained consciousness upon termination  generally within 30 s e c , and were f u l l y min.  alert  within  of  approx-  Blood pressure and the ECG were displayed on a Grass  Polygraph (4 channel, model 7 ) , and ECG was also displayed on a delayed loop o s c i l l o s c o p e with a 4 sec delay and a 4 sec real Type E for  M).  The l a t t e r  arrhythmias  (see below).  was p a r t i c u l a r l y  The ECG channel  l i t t l e adjustment was ever r e q u i r e d .  time d i s p l a y  useful  for  the  was c a l i b r a t e d  (Honeywell  diagnosis  once per  of  week;  The blood pressure channel was c a l i -  brated every time i t was used. After approximately 1 h, the administration of drugs, i f  any, and coro-  nary occlusion were c a r r i e d out while the r a t was f u l l y conscious and not r e s t r a i n e d in any way.  The procedure was as f o l l o w s .  Intravenously admin-  i s t e r e d drugs were injected slowly over a 10 min period.  A f a s t (100 mm/sec  chart speed) record of ECG and blood pressure was taken before drug admini s t r a t i o n and 4 min a f t e r completion of i n j e c t i o n .  One min a f t e r the second  f a s t recording of blood pressure, the occluder was tightened, as described above.  Occlusion did not appear to cause the rats any d i s t r e s s .  in behaviour ever occurred during the f i r s t few min after blood pressure f e l l  precipitously  following  occlusion  (in  No changes  o c c l u s i o n , unless which case  the  animal became subdued), or unless acute pulmonary oedema, (characterised by laboured r e s p i r a t i o n ) developed. to  In general, the f i r s t behavioural response  occlusion was sudden convulsive-type behaviour  which occurred in  con-  junction with VF (see below). The rats were monitored for at l e a s t 4 h following o c c l u s i o n . polygraph recordings were made every min f o r the f i r s t  Fast speed  15 min, every 5 min  - 96 -  for the following 15 min, then every 15 min. it  was disconnected from the ECG wires  reversing the processes used for devices.  the r a t  survived for 4 h,  and blood pressure transducer  by  connecting the animal to these recording  The animal was conscious and not r e s t r a i n e d during t h i s process.  Usually the rat occlusion  If  (see  was subdued at t h i s below)  time as a r e s u l t  of  and was submissive and compliant  the sequelae of enough for  this  disconnection to be completed without any d i f f i c u l t y . After 24 h, i f the r a t was s t i l l pressure and ECG recorders whereupon  it  a l i v e , i t was reconnected to the blood-  and c a r e f u l l y monitored  was s a c r i f i c e d .  Reconnection to  the  for  a further  recording  30 min,  devices was  c a r r i e d 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. the  bench and wrapped in  exposed.  a lab  coat,  with  only  Instead, i t was placed on the  subscapular  region  Rats, l i k e many rodents, do not struggle when they cannot see.  t h i s manner the l i n e s and leads were reconnected without i n c i d e n t .  It  In is  worth mentioning that in e a r l y experiments, when the limb leads were implanted 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 t h i s manner ( i . e . , when the r a t was conscious) on the day of o c c l u s i o n . Upon death or s a c r i f i c e , the heart was excised and the occluded zone and i n f a r c t s i z e were determined (see below), and a general postmortem examination was performed  (the  appearance of the lungs,  liver,  kidneys, spleen,  bladder and snout were noted, and any gross abnormalities were recorded). Exclusion c r i t e r i a  (see below) were considered at each stage of experimen-  tation. 2.1.3.2 Monitoring of - responses -t-o o c c l u s i o n . the  ECG and blood  pressure were recorded continuously  As described above, for  4 h  following  - 97 o c c l u s i o n , and again at 24 h a f t e r o c c l u s i o n .  Behavioural changes were noted  in a general way, and any p e c u l i a r i t i e s were recorded. niques described below have been described in b r i e f  Most of the t e c h -  previously (Au e t - a l ; ,  1979; Johnston et a l . , 1983a; C u r t i s et a l . , 1984; 1985b).  All  information  concerning each r a t was recorded permanently on i n d i v i d u a l a n a l y s i s sheets. 2 . 1 . 3 . 3 Occluded-zone (OZ).  The OZ was recorded for every r a t .  In  24 h s u r v i v o r s , s a c r i f i c e was undertaken by d e l i v e r i n g a blow to the head with a metal cosh. fused v i a the  No anaesthetic was used.  aorta according to  The heart was excised and per-  Langendorff  (1895)  with s a l i n e  (0.9 %).  Once blood was no longer present in the perfusate, the s a l i n e was replaced with s a l i n e containing Indocyanine (Fast Green dye, BDH) 0.5 g / 1 .  Approx-  imately 20 - 50 ml of t h i s s o l u t i o n was allowed to pass through the coronary circulation.  The d e l i v e r y of the 2 s o l u t i o n s , s a l i n e and Fast Green dye was  c o n t r o l l e d using a T-tube device connected to 2 large r e s e r v o i r s containing each s o l u t i o n , 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  stasis  thrombi  present  in  the  left  ventricle.  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 r e s h l y dead animals.  However, generalised c l o t t i n g  in the coronary c i r c u l a t i o n  had probably  not  occurred, because the hearts from rats dying overnight provided OZs as well defined and of a s i m i l a r extent to those from f r e s h l y dead animals. more l i k e l y that perfusion rate was reduced as a r e s u l t of the  It  is  contracture  which was a common feature of hearts removed from rats dying overnight s i n c e , with  reference  to  the  effect  of  systole,  coronary  perfusion  is  highly  s e n s i t i v e to the c o n t r a c t i l e state of the myocardium. A f t e r p e r f u s i o n , 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 v e n t r i c u l a r myocardium into normal and occluded t i s s u e was made by  visual  inspection.  The  two  zones were  almost  invariably  well  d i f f e r e n t i a t e d with a c l e a r - c u t border, as might be expected on the basis of the  reported  lack  of  collaterals  in  rat  hearts  Maxwell et a l . , 1984; Winkler et a l . , 1984).  (Johns  and Olson,  1954;  Using a small p a i r of s c i s s o r s  the perfused (green) and non-perfused (pink) t i s s u e s were separated,  lightly  blotted and weighed. 2.1.3.4  I n f a r c t -zone - ( I Z ) .  The i n f a r c t  for animals which survived f o r 24 h. an i n t e r v a l size  of  at  s i z e was only  determined  In t h i s regard, i t has been shown that  l e a s t 10 h must be allowed a f t e r  occlusion for  (measured in the manner described below) to be s u f f i c i e n t l y  for q u a n t i f i c a t i o n  in rats  (Hort and Da C a n a l i s , 1965a).  infarct developed  I n f a r c t s i z e was  determined by a modification of the method of Jestadt and S a n d r i t t e r  (1959),  based on the production of formazan red by the reduction of 2 , 3 , 5 - t r i p h e n y l tetrazolium ases.  (TTZ)  The normal  by  H  emanating  +  from  NAD- or  and occluded t i s s u e s were cut  NADP-1inked into s t r i p s  dehydrogenapproximately  2 mm wide and incubated with TTZ (Fischer S c i e n t i f i c ) 0.1 g in 10 ml phosphate  buffer  at  37 °C.  and 0.5 g NaHgPO^ in 0.1 M NaOH).  The buffer  2 1 distilled  was made by water  dissolving  (pH adjusted  Incubation was c a r r i e d out for  to  25 g Na^HPO^  8.5 - 8.6  with  approximately 10 min (incuba-  t i o n was terminated when the s t r i p s of non-occluded t i s s u e had turned deep purple from the formazan red r e a c t i o n , rather than after a s p e c i f i c time). In l a t e r  experiments, the OZ t i s s u e was cut not  2 discs by s l i c i n g through the subendocardium. more than 2 mm thick this  into s t r i p s ,  investigate  whether  surfaces were salvaged to any extent Canalis (1965b) and others.  the  into  The r e s u l t a n t discs were no  (by v i s u a l inspection) at any p o i n t .  approach was to  but  The r a t i o n a l e for  endocardial  and e p i c a r d i a l  as has been suggested by Hort and Da  Although no q u a n t i t a t i v e data was assembled, i t  - 99 -  appeared that the endocardial surface was usually not infarcted  (white)  at  24 h, and o c c a s i o n a l l y parts of the e p i c a r d i a l surface were also not white, although a gentle scrape with a pair of sharp s c i s s o r s was always s u f f i c i e n t to remove t h i s  'salvaged' t i s s u e .  In a d d i t i o n , by separate weighing of  t i s s u e i t was found that i t s presence changed the i n f a r c t  this  s i z e by only 2 to  3 %. Infarct s i z e was q u a n t i f i e d by separating the white and purple t i s s u e s and weighing them.  However, the t i s s u e was f i r s t f i x e d f o r 2-3 days in 10  ml formal s a l i n e (made by d i s s o l v i n g 3.56 g NaCl and 125 ml 40% formaldehyde in 375 ml d i s t i l l e d water). by  this  method,  It  corresponds  has been shown that the i n f a r c t , with  the  histologically  as detected  identified  infarct  (Fishbein e t a ! . , 1981). 2.1.4 D e f i n i t i o n of occlusion-induced arrhythmias 2.1.4.1 treatments  Introduction.  on a v a r i a b l e ,  necessary.  In  order  to  an unequivocal  definition  of  In the case of occlusion-induced arrhythmias,  the case that v a r i a t i o n s e x i s t in d e f i n i t i o n s . not  investigate  n e c e s s a r i l y be important  between studies  difficult  to  in  in  effects  of  that  variable  is  it  is  generally  While these differences may  themselves, they  interpret  the  make d i r e c t  some cases.  comparison  Although there  is  v a r i a t i o n in the d i s t i n c t i o n between a run of PVC and VT, and differences in the q u a n t i f i c a t i o n tinction  of PVC, the main source of contention  between VT ( p a r t i c u l a r l y  lies  in the  that of the torsade de pointes  dis-  variety)  and VF. VF was f i r s t described by Erichsen (1842)  in terms of the  contractile  behaviour of the myocardium ( t h i s was more than 60 years before the development of the ECG).  VF has been defined in various ways.  Moe e t - a l ;  defined VF as c h a o t i c , asynchronous f r a c t i o n a t e d e l e c t r i c a l a c t i v i t y . (1980)  gave an operational  definition,  based on  the  (1964) Bigger  c h a r a c t e 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 u n d u l a t i o n s ' . quantitative differential  VT.  are not amenable to  diagnosis because they are subjective  and they do not d i f f e r e n t i a t e of  These d e f i n i t i o n s  This i s not r e a l l y  definitions,  between abrupt VF and VF occurring after a run a serious problem c l i n i c a l l y ;  the  distinction  between i r r e g u l a r torsade de pointes, v e n t r i c u l a r f l u t t e r and VF i s , perhaps academic, since a l l  3 arrhythmias  are associated with cardiac output  that necessary for the maintenance of l i f e , prevention of m o r t a l i t y .  below  and the primary concern i s  However, more rigorous d e f i n i t i o n s  the  are necessary  experimentally. Abrahamsson's group who induce myocardial ischaemia in rats define VF as asynchronous disorganised e l e c t r i c a l (Abrahamsson e t - a l ; , 1985).  activity  While t h i s  of  at  definition  least  5 sec  duration  introduces an  important  caveat, namely that some i n d i c a t i o n concerning the duration of the phenomenon may be useful definition,  in  diagnosis, i t  nevertheless f a i l s  since how does one define  Opie's group have defined VF as  'total  repetitive  at  ectopic complexes for  as a t r u l y  'asynchronous' and irregularity  least 6 c y c l e s '  of  an a r b i t r a r y  but  s p e c i f i e d duration.  element in the diagnosis and d e f i n i t i o n  morphology of  disorder i s  C l e a r l y there  of VF.  'disorganised' ? the  (Lubbe et a l ; , 1978),  introducing once more the notion that VF i s only VF i f for  objective  is  present  a subjective  This i s an important  point  and attempts to deal with t h i s problem are discussed below. It  i s unfortunate  set of c r i t e r i a for  that many researchers do not appear to have a r i g i d defining arrhythmias.  A glance through the  literature  will  reveal that most researchers do not define what they mean by VT and  VF.  In p a r t i c u l a r ,  it  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 r e n t i a t e d , VT.  If  and how many consecutive PVCs c o n s t i t u t e a run of  PVC, VT, torsade de pointes, f l u t t e r  and VF represent a continuum,  - 101 as was once believed ( e . g . , H a r r i s , 1950), then the d i s t i n c t i o n between these arrhythmias would not be s e r i o u s l y important; be expected to reduce VT and VF as w e l l . from  studies  with  investigations  in  animals healthy  (Dresel  a drug which reduces PVC would  However, there i s some i n d i c a t i o n  and S u t t e r ,  humans with  a high  1961)  and from  frequency of  follow-up  PVC (Kennedy  e t - a l . , 1985), that PVC, VT and VF are not n e c e s s a r i l y part of a continuum. Therefore the p o s s i b i l i t y e x i s t s that a drug may influence s e l e c t i v e l y one type of v e n t r i c u l a r arrhythmia but not another.  The l a t t e r  issue i s 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 'classical  antiarrhythmic  agents'  a g e n t s ' , with s e l e c t i v i t y f o r  and  'antiarrhythmic/antifibrillatory  VF versus PVCs (Anderson, 1984)  i s somewhat  premature. I r r e s p e c t i v e of whether ischaemia-induced v e n t r i c u l a r arrhythmias represent a continuum or not, i t define endpoints.  i s nevertheless important  Since arrhythmia d e f i n i t i o n s  to another, a l l one can aim for  in any experiment to  vary from one i n v e s t i g a t o r  i s internal consistency at present.  describing the c r i t e r i a which were used for defining v e n t r i c u l a r  Before  arrhythmias  produced by coronary occlusion in r a t s , the subjective nature of c l a s s i f i c ation  of  arrhythmias  must  be r e i t e r a t e d .  No matter  what  definition  chosen for VF and VT, there remains an element of s u b j e c t i v i t y ,  is  particularly  with regard to d i f f e r e n t i a t i o n between torsade de pointes and VF. A simple way around the problem of s u b j e c t i v i t y i s to carry  out studies  using a b l i n d and random p r o t o c o l .  In t h i s manner, i n c o n s i s t e n c i e s in d e f i -  nition  and evenly  should  be  spread randomly  between  conscious and unconscious bias should be e l i m i n a t e d .  study It  groups,  and  must be stressed  that attempts were made to carry out a l l the experiments described in a b l i n d and random manner, where p o s s i b l e .  - 102 The  following  definitions  were  used in  the  diagnosis of  arrhythmias  f o l l o w i n g coronary occlusion in r a t s . 2.1.4.2 Premature v e n t r i c u l a r - c o n t r a c t i o n s - ( P V G ) .  PVC were defined  as premature QRS complexes occurring independently of the P wave. generally accompanied by a transient  PVC were  drop in a o r t i c 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  intervals  of  coupling  i n t e r v a l s i s believed to give an i n d i c a t i o n of whether the PVC i s  reentrant  or automatic (see I n t r o d u c t i o n ) . ment  available  intervals, consuming. PVCs.  was not  was  undertaken.  Measurement  However, i t was considered that the equip-  amenable to  the  routine  measurement  of  coupling  since measurement of such by hand would be e x t r a o r d i n a r i l y Only s i n g l e t s , doublets (bigemini)  time  and t r i p l e t s were counted as  Longer runs were recorded as VT (see below).  It  i s noteworthy  that  other workers include every d e f l e c t i o n in a run of VT as a PVC (Kane and Winslow, 1980; Clark et a l . , 1980).  This manoeuvre i s based on the assump-  t i o n that PVC and VT are i d e n t i c a l in mechanism and drug s e n s i t i v i t y . our d e f i n i t i o n  implies that s i n g l e t s , doublets  and t r i p l e t s  'same' arrhythmia, we only associate these arrhythmias  for  While  represent analytical  the con-  venience; since the incidence of doublets and t r i p l e t s i s v a r i a b l e and lower than the incidence of s i n g l e t s , a useful drug on these i n d i v i d u a l the study group s i z e .  It  i n v e s t i g a t i o n of the e f f e c t s of a  types of arrhythmia would require an increment  in  was considered that the c u t - o f 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 n e c e s s a r i l y the product of a common e l e c t r o p h y s i o l o g i c a l mechanism (namely short runs of VT and long runs of PVCs).  In other words,  in the absence of clear evidence concerning the point at which a run of PVCs ceases to be PVCs and becomes VT, i t PVCs and VT.  was decided to a r t i f i c i a l l y  segregate  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,  it  i s sensible to separate them  for analysis by generating an a r b i t r a r y c u t - o f f p o i n t . 2.1.4.3 Ventricular-tachycardia-(VT). or  more consecutive PVC.  No r e s t r i c t i o n  VT was defined as a run of 4  was made on the associated r a t e .  This d e f i n i t i o n d i f f e r s from that of other workers who measure ischaemiainduced arrhythmias in r a t s . rate of  VT has been defined as 7 or more PVCs at a  > 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 i m i t a t i o n  on rate (Fagbemi and  P a r r a 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 t h i s  definition  work  has been adopted by Verdouw's group  in  their  with  pigs  (Verdouw et a l . , 1978). 2.1.4.4 V e n t r i c u l a r - f i b r i l l a t i o n - (VF).  VF was defined as disorder  in the ECG accompanied by a p r e c i p i t o u s f a l l in blood pressure.  In a d d i t i o n ,  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 f e r e n t i a t e between 'primary' VF and VF 'secondary' to VT, namely that disorder had to have begun w i t h i n 10 sec of the l a s t sinus beat for the arrhythmia to be classed as VF, otherwise 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 -  u l a r c y c l e length with no i d e n t i f i a b l e QRS complex. word  'disorder'  lends an element of s u b j e c t i v i t y  As discussed above, the to the d e f i n i t i o n .  Bias  was eliminated as f a r as possible by b l i n d and random experimental design. In the introduction to t h i s subsection, a v a r i e t y of operational d e f i n i tions of VF were given, and i t was suggested that the timing and duration.of ' d i s o r d e r ' needs to be defined, as well as the actual nature of  'disorder'.  Some Authors have avoided the question of the d e f i n i t i o n of VF altogether. Winslow i n i t i a l l y  reported the incidence of VF in r a t s f o l l o w i n g occlusion  - 104 (Kane and Winslow,  1980),  but  in  later  studies  (Marshall  etal.-,  1981a;  1981b; 1981c; Marshall and Winslow, 1981; e t c . ) VT and VF were combined, and the arrhythmia was c a l l e d ' f i b r i 1 l o f l u t t e r ' . s t r a t e g y , avoiding ' a r b i t r a r y  Northover has adopted a s i m i l a r  and subjective d i s t i n c t i o n s '  between VT and VF  by simply recording the time spent in the 'combined forms' (Northover, 1985). Other groups have taken a completely absolute terms.  different  For example, Harron et a l .  approach, defining  (1985)  VF in  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 d e f i n i t i o n simply a d e f i n i t i o n  of VF.  The method used in the current experiments is  of VF which was f e l t  clinical definitions,  to be reasonably consistent with  and more importantly,  amenable to routine  use,  n a l l y c o n s i s t e n t , but not obsessively unequivocal to the point of  inter-  absurdity  (see the d e f i n i t i o n used by Harron e t - a l ; , 1985, above). Despite various  the  groups  extremely  different with  Na  consistent,  all  classifications +  channel studies  used,  blocking  the  drugs  demonstrating  results (see  obtained  Discussion)  antifibrillatory  are  activity  for quinidine and Org-6001, for example, suggesting that the v a r i a b i l i t y arrhythmia c l a s s i f i c a t i o n appears not to compromise i n v e s t i g a t i o n s , of the recognition  of a n t i f i b r i l l a t o r y  drugs.  by  in  in terms  However, the v a r i a b i l i t y  in  control incidence of VF w i t h i n research groups suggests that there may be an element of i n t e r n a l  inconsistency.  This may r e l a t e  from o u r s e l v e s , research groups do not  appear to  to the f a c t carry out  This may give r i s e  to  that  their  using b l i n d  and random p r o t o c o l s .  information,  p a r t i c u l a r l y concerning drugs with weak antiarrhythmic  apart  studies  some misleading actions,  as a r e s u l t of the loss of p r e c i s i o n of endpoints. 2.1.4.5 Other-arrhythmias. arrhythmias  of  major  interest  and  Although VF, VT and PVC are the importance,  all  arrhythmias  following  - 105 occlusion  are recorded.  arrhythmias  is,  however,  considered worthwhile ventricular  The incidence of extremely  low  investigating  in  non-ventricular control  rats,  them in a q u a n t i t a t i v e  (and non-nodal) and  it  manner.  is  not  Atrio-  (AV) blocks occur from time to time, p a r t i c u l a r l y in association  with pulmonary oedema-induced gasping.  Anecdotal observations have strongly  suggested that gasps in rats with severe pulmonary oedema are coupled, with Moebitz Type-2 AV block.  1:1  Presumably the AV block i s the r e s u l t of a  vagal r e f l e x . A l l types of AV block and d i s s o c i a t i o n have been observed. AV block  i s usually only seen in  rats  Third degree  experiencing p e r s i s t e n t  and severe  r e s p i r a t o r y d i s t r e s s (exudation of sputum), and according to the exclusion c r i t e r i a (see below) are not usually included in s t u d i e s . Sinus bradycardia and tachycardia do not occur very often  during  the  f i r s t 4 h a f t e r o c c l u s i o n , but both have been observed in 24 h survivor r a t s . Atrial  arrhythmias are extremely r a r e .  between a t r i a l atrial  f l u t t e r and f i b r i l l a t i o n  'fibrilloflutter  1  It  is d i f f i c u l t  distinguish  using the V3 l e a d , but some sort of  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 . which i s l i f e - t h r e a t e n i n g  to  if  it  VF i s a serious  does not spontaneously r e v e r t .  arrhythmia It  i s well  established that r a t s 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 r a t s which experience VF die from VF (values being dependent on whether  conscious or  anaesthetised animals are  used, and on how VF i s defined).  Therefore,  i s expected in any study  it  that the sample s i z e w i l l vary with time, as animals d i e , and w i l l also vary between groups.  This means f i r s t l y  that  ultimate  group s i z e s w i l l  vary,  compromising the value of s t a t i s t i c a l t e s t s , secondly that group s i z e 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  interrelationships  of v a r i a b l e s i s l o s t as animals die during the course of the study p e r i o d . This censoring can only be eliminated i f  the animals are a l l  kept a l i v e 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 order  to  preclude the generation  'severity' f o r VF.  of  arbitrary  was considered that  definitions  concerning  in the  of VT, a l l VT would be reverted a f t e r 10 sec in the manner used  The procedure was as f o l l o w s .  After 10 sec of continuous VF or VT the r a t was l i f t e d out of the home cage by  the  tail.  Within  a few  seconds convulsive-type  behaviour  (see  section concerning preliminary screen) ceased and syncope ensued, whereupon the chest was f l i c k e d with the were required to revert  index f i n g e r .  the arrhythmia  Usually only 1 or 2 f l i c k s  to sinus rhythm.  not occur immediately then the f l i c k s were continued. nosed by the  combination  of  a sudden return  to  If  reversion  did  Reversion was d i a g -  consciousness, a sudden  increase in a o r t i c blood pressure and the termination of the arrhythmia (as seen on the ECG r e c o r d ) . Because 10 sec was the maximum time allowed before initiated,  differentiation  longer than 10 s e c , i t  was  between VT and VF was only attempted during the  10 sec period of the arrhythmia. for  defibrillation  Of course, i f  VT were allowed to continue  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  induced m o r t a l i t y ,  version  greatly  reduces  censoring  due  to  arrhythmia-  i t introduces a new form of censoring, namely that h i t t i n g  the chest may 'change t h i n g s ' compared with r a t s which have not experienced VF.  This p o s s i b i l i t y was examined by our  vations) mortality  and  it  was found  from VF, but did not  other words, d e f i b r i l l a t i o n VF.  that  Therefore  it  laboratory  defibrillation  (unpublished  reduced the  obser-  incidence  influence the incidence of VF i t s e l f .  of In  does not influence the l i k e l i h o o d of subsequent  was considered that  the benefits  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 r e l e v a n t in terms of a n a l y s i s ; 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 influence SVF also  influence  NVF, and drugs  which  influence  SVT also  influence  NVT.  Therefore i t i s reasonable to assume that SVT and NVT are i d e n t i c a l in terms of mechanism of generation; the same can be said for SVF and NVF. an important  point because i t  sinus rhythm in rats  This i s  implies that spontaneous reversion of VF to  i s not a disadvantage of the preparation.  Moreover,  the occurrence of SVF, and the r e a d i l y revertable nature of NVF (see r e s u l t s ) means that most animals w i l l  survive the period of  continuous  monitoring  such that censoring produced by e a r l y m o r t a l i t y i s reduced. 2 . 1 . 4 . 7 Arrhythmia scores. record t h e i r  In quantifying arrhythmias, many workers  incidence, number, type  and duration.  Such  l i m i t e d value since the number of episodes of VT and VF are  information  log^-normally  d i s t r i b u t e d variables (Johnston et a l ; , 1983a); t h i s f a c t i s not taken consideration by other workers in the f i e l d .  has  into  In a d d i t i o n , thump-version may  - 108 influence the duration of VT and VF compared with the values found by other investigators.  In the hands of workers who do not revert  mean duration of VF i s dominated by the terminal measures of duration reason.  of VF should be log^g normally  I n t u i t i v e l y , such  distributed  for  this  However, correction of data i s never undertaken (Clark et a l . - , 1980;  Kane and Winslow, 1980; e t c . ) . with thump-version,  the  In our laboratory,  durations  t r i b u t e d (Johnston et a l • , 1983a).  of  upon such variables  for  value for  was found that even log-^Q normally  dis-  Nevertheless, since thump-version c l e a r l y i s not reasonable to completely r e l y  antiarrhythmic  reason for t h i s c o n c l u s i o n ; i f  it  VT and VF were  censors the durations of VT and VF, i t  matical  event.  VT and VF, the  quantification.  There  is  another  a drug abolishes VF then there i s no mathe-  log^g VF duration.  If  an a r b i t r a r y  value of  1 sec i s  given to rats not experiencing VF then the r e s u l t i s a standard deviation of zero in the group in which VF i s absent, creating a variance inhomogeneity which i n v a l i d a t e s the use of standard parametric s t a t i s t i c a l tests the non-parametric Mann-Whitney U t e s t may be used).  (although  In summary, measuring  arrhythmia duration alone i s not an acceptable means of gauging the s e v e r i t y of arrhythmias. An a l t e r n a t i v e approach i s to generate a number scale which can be used to summarise and grade a l l incidence and s e v e r i t y .  the arrhythmias f o l l o w i n g occlusion in terms of  This scale (arrhythmia score) should accomodate and  summarise complex arrhythmia data s e t s .  Arrhythmia scores should be normally  d i s t r i b u t e d (to permit parametric s t a t i s t i c a l t e s t i n g ) and l i n e a r l y a d d i t i v e . The  arrhythmia  score which  has been used for  occlusion  studies  in  conscious r a t in our laboratory has been shown to be Gaussian d i s t r i b u t e d control  rats  (Johnston et a l ; , 1983a)  and amenable to modified t  the in  tests.  - 109 This score is as f o l l o w s : 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 t o t a l  duration,  4 = VT and/or VF of 60 - 119 sec t o t a l duration, 5 = VT and/or VF of > 119 sec t o t a l  duration,  6 = Fatal VF occuring 15 min - 4 h after o c c l u s i o n , 7 = Fatal VF occurring 4 min - 14 min 59 sec a f t e r o c c l u s i o n , 8 = Fatal VF occurring 1 min - 3 min 59 sec after o c c l u s i o n , 9 = Fatal VF occurring before 1 min a f t e r o c c l u s i o n . 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. (1981)  have used a score based on the product  of  Martinez and Crampton  arrhythmia  duration  and  arrhythmia type (the l a t t e r being graded from 1 to 5 according to s e v e r i t y ) . However, t h i s score was found not to be amenable to parametric t e s t i n g , since drug treatments  altered the v a r i a n c e .  statistical  Mueller e t - a l ;  (1984)  have used a simple 3 point score which they sum for each group and submit to the chi  t e s t for  analysis.  Woodward's group have used a group  arrhythmia  score, whereby arrhythmias for a whole group are summed, and a score assigned to the group arrhythmia  (Daugherty e t - a l ; ,  score seems to  1986).  However, the object  have been forgotten  by t h i s  of  group,  p a r t i c u l a r score does not seem to be amenable to any s t a t i s t i c a l  (analysis  of  variance  followed  comparisons, such as Tukey's t e s t ) . plotted  against  log  dose  in  order  by  modified  t  tests  since  this  test.  The ideal arrhythmia score should be amenable to parametric tests  having an  statistical  for  multiple  In t h i s manner, arrhythmia score may be to  estimate  ED™ values  for  overall  - 110 antiarrhythmic actions (see section concerned with s t a t i s t i c s ) . At l e a s t 6 other  arrhythmia  scores were investigated during the course  of experimentation, since i t was considered worthwhile to investigate whether more c l o s e l y Gaussian-distributed scores could be invented. 2.1.5 ECG changes produced by occlusion Coronary occlusion produces c h a r a c t e r i s t i c S-T segment elevation (Pardee, 1920)  and changes  occlusion,  in  R wave s i z e .  Since  and are presumably the r e s u l t  considered of  interest  has attempted to measure and quantify The optimum method for  therefore not w e i 1 ' e s t a b l i s h e d . as f o l l o w s .  of myocardial  are  produced by  ischaemia, i t  to measure and record these v a r i a b l e s .  ourselves (Johnston e t - a l . , 1983a; e t c . ) ,  in r a t s .  these e f f e c t s  only Bernauer (1982;  was  Apart from 1983; 1985)  the ECG changes produced by occlusion  measuring and expressing such changes is  A v a r i e t y of techniques have been considered  Figure 2 i l l u s t r a t e s the changes in ECG configuration with time  following occlusion. 2 . 1 . 5 . 1 ' S - T ' segment e l e v a t i o n . ECG in rats  i s not as c l e a r - c u t as i t  The p o s i t i o n of the T wave of the  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 beginning  portion  before  of the QRS complex, i n d i c a t i n g that r e p o l a r i z a t i o n  depolarisation  is  complete  measure S-T segment e l e v a t i o n , an a r b i t r a r y , the T wave was determined. ECGs recorded  at  fast  (Cooper,  1969).  In  order  but standardised p o s i t i o n  is to for  P r i o r to o c c l u s i o n , a sample of approximately 50  chart  speed (100 mm/sec)  p o s i t i o n of the S wave in r e l a t i o n  were  inspected,  to the Q wave was measured.  found to be approximately 30 msec (3 mm on the f a s t speed r e c o r d ) . f o r e , as an index of S-T segment e l e v a t i o n , the height of t h i s  and  the  This was There-  S wave p o s i -  t i o n above i s o e l e c t r i c was measured before (S ) and a f t e r occlusion (S ).  - Ill  KEY:  _  ECG  lmv  '200 msec 1  'I  U A A  -  HR Time PVC no. After VT no. Occlusion VF no. BP  l\  !  -  456 beats/mtn -5 m1n  444 beats/mln -1 m1n  123 itmHg  453 beats/mln 1 min  123 iraiHg  444 beats/mtn  444 beats/mln 3 m1n  2 m1n 97 irniHg  103 mmHg  95 umHj  h  522 beats/mln 485 beats/mtn 474 beats/m1n 468 beats/mtn 434 beats/mln 512 beats/mtn 19 PVC 2 PVC 22 PVC 20 mtn 25 min 1 VT 5 m1n 10 min 15 m1n 1 VF 98 mmHg 95 mmHg 83 mmHg 88 mmHg 93 mmHg 105 mmHg  4 m1n  I  -I V J V  "v-A 474 beats/m1n 30 m1n  500 beats/mtn 45 min  105 mmHg  105 nroHg  I. '  536 beats/m1n 69 PVC  2.25 h  103 irniHg  525 beats/mln. 2 PVC  1.25 h  108 «Hg  I 525 beats/m1n 16 PVC  519 beats/mtn . 1h  95 mmHg  2 h  A  98 irniHg  '  540 beats/mtn 24 PVC  2.5 h  90 itmHg  496 beats/mtn 2 PVC 52 nmHg  Figure 2.  524 beats/mtn. 2 PVC  1.5 h  J  \  525 beats/mtn 50 PVC  1.75 h  103 mmHg  105 irniHg  p.  525 beats/mln 544 beats/mtn 503 beats/mtn 63 PVC 435 PVC 36 PVC 2 VT 3.25 h 29 VT 1 VT 3 h 3 VF 75 nuiHg 85 irniHg 83 ItmHg  2.75 h  522 beats/mtn 517 beats/mtn 512 beats/mln 514 beats/mtn 24 PVC 84 PVC 376 PVC 1 PVC 12 VT 4.25 h 3.5 h 3.75 h 4 h 65 mmHg 65 mmHg 70 mmHg 70 limHg  A., J  v  A A  511 beats/mtn 1 PVC 60 mmHg  A A  505 beats/mtn 1 PVC  4.75 h  60 imHg  - A - A  491 beats/mtn 475 beats/mtn 482 beats/mtn 475 beats/mtn 365 beats/mtn 4 PVC 10 PVC 8 PVC 12 PVC/mtn 3 PVC 5 VT 5.5 h 5.75 h 6 h 24 h 4 VF 55 mmHg 55 irniHg 60 mmHg 55 nmHg 75 flmHg  5.25 h  Anecdote of ECG changes caused by occlusion i n a conscious r a t  - 112 I s o e l e c t r i c was defined as the voltage at the foot of the P wave of preceding beat.  Before o c c l u s i o n , values are negative,  the  since the S wave  p o s i t i o n i s negative to i s o e l e c t r i c .  ' S - T ' segment elevation begins immedi-  ately  to  upon o c c l u s i o n , and in  order  simplify  subsequent a n a l y s i s ,  all  negative (pre-occlusion) values are assigned the value z e r o . ' S - T ' segment elevation was expressed in a v a r i e t y of ways. originally  used in  our  laboratory  was the  dimensionless dSTR.  determined by measuring the R wave amplitude (R )  and  Q  at  times  after  In  other  (S -S ).(R /R ). t  Q  t  Q  occlusion words,  R wave.  Other  measures of  This was  before occlusion  and  was  calculated  as  segment  was  corrected  for  t  the change in R wave amplitude following occlusion  (see below)  (R )»  'S-T'  The v a r i a b l e  occlusion as a function  'S-T'  segment  elevation  of  pre-  have been  subsequently evaluated, as f o l l o w s ; Uncorrected e l e v a t i o n , referred to as ST, c a l c u l a t e d as S mV. t  Elevation corrected for amplidude as a % of R wave amplitude,  referred  to as S T / R , calculated as ( S ) / R mV. 2  2  t  t  Elevation as a % of R wave amplitude, referred to as ST%, c a l c u l a t e d as (100)(S )/R t  The c r i t e r i o n determine  t  for  which  (dimensionless). determining which measure was most useful was simply variable  produced  the  largest  (defined as mean divided by standard d e v i a t i o n ) .  coefficient It  of  to  variation  was found during  the  course of experimentation that ST% was c o n s i s t e n t l y the most precise v a r i able.  The c o e f f i c i e n t s of v a r i a t i o n for ST% were approximately double those  found for dSTR. The v a r i a b l e dSTR has been shown to e t - a l ; , 1983a).  be Poisson d i s t r i b u t e d  (Johnston  The problem with a l l measures of S-T segment elevation  that the v a r i a b l e regresses with time in a non-linear manner.  is  An attempt  was made to account for t h i s by measuring the maximum value of S-T segment  - 113 elevation and the time at which t h i s occurred. segment elevation  is not s t r i c t l y  Since the development of S-T  a mono-exponential  process (see  results)  i t was decided to measure the maximum value of S-T segment elevation  (rather  than the half-maximum v a l u e ) .  follow-  This was c a r r i e d out according to the  ing c r i t e r i a . S-T segment elevation was measured at regular i n t e r v a l s from 100 mm/sec chart speed records. vation  was determined  applying an a r b i t r a r y the case of dSTR. as the example).  by  each  time  point  consecutively  value i s  initially  In  the  underlined.  example  The f i n a l  below,  each successive  c l a s s i f i e d as the maximum.  apparent  underlined v a l u e , 85% was the  actual maximum according to the c r i t e r i a  in  (using the d e r i v a t i v e ST%  This i s superceded by the f i r s t subsequent value which exceeds i t more.  and  in the case of ST%, or a '0.05 mV r u l e  This r u l e operates as follows The f i r s t  occlusion  The maximum value of S-T segment e l e -  considering  '5% r u l e '  after  outlined.  by 5% or  maximum has been  value  taken  as  the  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 after  the r e l a t i o n s h i p  occlusion were a simple  between S-T segment elevation  saturating  monoexponential  function  and time of  the  form: ' S - T ' elevation = 1/(1 + t a u / t ) where t  i s time a f t e r  occlusion and tau i s the time constant  (defined  here as the time at which ' S - T ' segment elevation i s half maximal), then tau would be the ideal v a r i a b l e to record.  However, when ' S - T ' e l e v a -  t i o n i s plotted against time ( e . g . , Johnston e t - a l . , 1983a) then i t  is clear  - 114 that  a  simple  monoexponential  equation  Indeed, the r e l a t i o n s h i p does not f i t  will  not  fit  the  relationship.  any simple model.  Since the action of drugs on ' S - T ' segment elevation was not the primary concern, i t  was not considered worthwhile  elaborating upon the analysis of  the v a r i a b l e beyond using the semi-empirical approach o u t l i n e d above. 2.1.5.2 Pathological- -R-waves. an i n i t i a l  Coronary occlusion in rats  produces  increase in the amplitude of the R wave, followed by a gradual  decline in amplitude to values smaller than those seen before o c c l u s i o n .  An  attempt was made to quantify R wave changes using a s i m i l a r approach to that used in assessing S-T segment e l e v a t i o n . R wave amplitude was easier to measure than S-T segment e l e v a t i o n , since the peak of  the R wave i s c l e a r l y v i s i b l e on f a s t  (100 mm/sec).  chart  speed recordings  R wave amplitude (mv) was taken as the d e f l e c t i o n of the peak  of the R wave above the i s o e l e c t r i c p o i n t .  As in the case of S-T segment  e l e v a t i o n , R wave does not c o r r e l a t e with time a f t e r  occlusion in a simple  manner, and maximum R wave amplitude (using a 0.05 mV rule)  and the time at  which t h i s occurred were determined in a manner analogous to the  determin-  ation of maximum S-T segment e l e v a t i o n . 2.1.5.3 Pathological - Q waves. exhibit  The chest lead ECGs of normal  no Q wave, presumably because the rat  perpendicular  to  (Cooper, 1969).  the  horizontal  plane,  heart  according  frontal to  rats  plane axis  ECG vector  is  analysis  However, coronary occlusion produces a deep Q wave in  the  chest leads which appears at approximately 2 h a f t e r o c c l u s i o n , and p e r s i s t s for  at l e a s t 11 days (Normann et a l ; , 1961; Zsoter and Bajusz,  1962).  If  lead-I i s recorded, a Q wave appears w i t h i n 10 min of occlusion in approxima t e l y 30% of r a t s , long before i t Losconci, 1973a). occlusion,  but  its  i s present in the chest leads (Kenedi and  Using a V3 lead we r a r e l y see a Q wave before 1 h after presence i s  almost universal  in rats  surviving  24 h.  - 115 Recently, attempts have been made to quantify Q wave development by measuring the time at which a s i g n i f i c a n t Q wave i s present in the ECG. A s i g n i f i c a n t Q wave was  defined  as  a downward  deflection  from  isoelectric  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 administration and again 1 min before o c c l u s i o n , there  is  an opportunity  for  assessing the  electro-  p h y s i o l o g i c a l e f f e c t s of drugs in the same rats in which a n t i a r r h y t h m i c / a n t i infarct  activity  i s assessed.  This i s achieved by measuring P-R i n t e r v a l  2.1.6.1 P-R i n t e r v a l .  P-R i n t e r v a l r e f l e c t s the duration of conduc-  and QRS i n t e r v a l .  t i o n from high in the atrium through to the AV junction (see Horan and Flowers, 1980).  Since conduction v e l o c i t y in the AV node i s  much slower than conduction v e l o c i t y lower dV/dt  and bundle branches  in the atrium,  in accordance with the  „ of the upstroke of the action potential  max  t a b i l i t y (Merideth e t - a l . , 1968, and see Introduction),  and the lower  then P-R i n t e r v a l  e s s e n t i a l l y a r e f l e c t i o n of conduction through the AV node. influencing P-R i n t e r v a l therefore influence AV conduction. is f a i r l y  easy to record in r a t s ,  somewhat i n d i s t i n c t Polygraph.  is  As such, drugs The P-R i n t e r v a l  although the foot of the P wave can be  on occasions,  P-R i n t e r v a l  exci-  particularly  when recorded on  was measured according to the standard  a Grass definition  (from the foot of the upstroke of the P wave to the s t a r t of the QRS).  The  e f f e c t s of drug treatment on P-R i n t e r v a l were determined by comparing values 15 min before occlusion (pre-drug)  with those 1 min before occlusion (4 min  a f t e r drug a d m i n i s t r a t i o n ) . 2 . 1 . 6 . 2 QRS i n t e r v a l . arisation reflects  (see  Horan  The QRS i n t e r v a l r e f l e c t s v e n t r i c u l a r depol-  and Flowers, 1980).  a d i s p e r s i o n , or  delay  in  If  ventricular  the  QRS i s  conduction,  widened,  this  and reduction  - 116 in conduction v e l o c i t y .  The QRS i n t e r v a l  was not measured in the conven-  t i o n a l manner (from the beginning of the Q wave to the end of the S) because i t was considered that the p o s i t i o n of the downward going peak of the S was more amenable to measurement than the less w e l l - d e f i n e d terminal S wave.  The e f f e c t s of drug treatment on QRS i n t e r v a l  end of  the  were determined by  comparing values 15 min before occlusion (pre-drug) with those 1 min before occlusion (4 min a f t e r drug a d m i n i s t r a t i o n ) . In some experiments, an attempt was made to measure QT i n t e r v a l . problems involved in measuring QRS were even more of a confounding  The factor  here, since the T wave i s the l e a s t w e l l - d e f i n e d of the ECG waves in rat.  The T wave r e f l e c t s  ventricular  dependent on action potential  duration.  repolarisation,  conduction v e l o c i t y  by dV/dt  action  ventricular  is  Therefore, QT i n t e r v a l  combination of v e n t r i c u l a r ),  and  potential  reflects a  plateau  (governed by i  .)  and  Si  ventricular repolarisation repolarising K  interval  therefore  (governed almost e x c l u s i v e l y  Hid A  of  the  +  (governed by i n a c t i v a t i o n  currents,  see I n t r o d u c t i o n ) .  It  of  i ^ $  and a c t i v a t i o n  can be seen that QT  i s therefore a rather n o n - s p e c i f i c v a r i a b l e .  In a d d i t i o n , in rats  the T wave i s superimposed on the QRS (Cooper, 1969; D r i s c o l l , 1980). makes measurement of QT i n t e r v a l p a r t i c u l a r l y 2.1.7 Measurement of serum K Serum K  difficult.  concentration  +  was measured in recent experiments  +  This  as a consequence of  the  r e s u l t s of i n v e s t i g a t i o n s into mechanisms of arrhythymogenesis (see below). Since  it  has  been  shown  that  extracellular  K  +  rises  in  the  ischaemic  t i s s u e with a time course corresponding with that of e a r l y occlusion-induced arrhythmias following  (Hirch e t - a l ; , myocardial  1980),  ischaemia  and since the is  inversely  incidence of  proportional  to  arrhythmias serum  K  c l i n i c a l l y (Nordrehaug and Von der L i p p e , 1983; 1985) and experimentally  in  +  coronary-1igated rat hearts in v i t r o (Lubbe e t a l . , 1978; Daugherty e t a l ; ,  - 117 1981), the  it  was decided to  antiarrhythmic  monitor  actions  of  serum K  +  in order  drugs were r e l a t e d  to  to  determine  alterations  whether in  serum  K . +  A blood sample (0.5 - 0.8 ml) was withdrawn from the a o r t i c blood p r e s s ure  line  at  approximately 2 h a f t e r  occlusion.  10000 x g for 2 min, using an Eppendorf centrifuge  The sample was spun (Model 3200).  at  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 ) . K  +  concentration was determined  in the a n a l y t i c a l  laboratory  of  The  the Acute  Care Hospital (by t h e i r t e c h n i c i a n s ) , 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 It  i s of paramount importance, when i n v e s t i g a t i n g myocardial ischaemia,  to ensure that animals are included into the study only when the artery has been occluded and ischaemia has been produced. by measuring the 0Z, ex v i v o ,  coronary  This i s ensured  according to the technique described above.  However, there are other less obvious sources of variance which may jeopardise the p r e c i s i o n and accuracy of an experiment.  Over the y e a r s , 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 i b u t a b l e 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 e x p e r i mentation and a n a l y s i s , namely that i t must be applied b l i n d l y . The exclusion c r i t e r i a before,  during  sequence.  In  or other  after  are t i e r e d occlusion  words,  pre- or  in as much as rats  according  to  must be excluded  a definite  chronological  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 r e q u i r e d ) .  - 118 A.  The f o l l o w i n g action i s taken in r e l a t i o n to the following pre-occlusion abnormalities, occluder  with the object  has been a c c i d e n t a l l y  of  excluding rats  tightened  in which either  during  the  interval  the  between  preparation and experimentation, or in which i n f e c t i o n s are present: a.  The presence of  a Q wave, as defined above, d i c t a t e s  obligatory  e x c l u s i o n , on the grounds that a Q wave i s only seen in chest leads in rats following 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 i n f a r c t or scar t i s s u e . b.  If  more than 5 PVC occur during the 15 min period p r i o r  to  drug  administration then the r a t should be excluded on the grounds that there i s a lesion of some sort in the myocardium which may influence the outcome of o c c l u s i o n . c.  If  there has been > 25% weight loss between preparation and o c c l u -  s i o n , rats are only excluded i f such  as  diarrhoea  and/or  there are other signs of  inflammation  associated with  illness, surgical  wounds, and/or pre-drug mean a r t e r i a l blood pressures of 85 mmHg or less (> 2 s . d . from mean pre-occlusion v a l u e s ) . d.  If  there are signs of  snout  and/or  noisy  lung i n f e c t i o n ,  respiration,  the  such as exudate around the rat  is  excluded.  regard, the s e n s i t i v i t y of r a t s to lung i n f e c t i o n s Evans et a l .  (1985)  reported  that  ' a pulmonary  characterised by wheezing', and that the m o r t a l i t y  in  our  coronary occlusion was increased.  laboratory  it  was probable  that  In  despite having an underlying pulmonary i n f e c t i o n . 2 years the problem of r e s p i r a t o r y  infection  in  early  some rats  this  i s well known.  male Sprague Dawley rats  often been received from the supplier with  following  In  have  infection these  rats  experiments  were  included  During the  last  became so severe on  occasions that rats died even before preparative surgery had been c a r r i e d out. is  M o r t a l i t y associated with pneumopathy after occlusion  characterised by extensive exudation of  during the f i r s t 5 - 1 5 pulmonary  oedema.  occasionally oedema. similar  min a f t e r  Postmortem  haemorrhaging  Often the thorax  of  sputum, often  o c c l u s i o n , i n d i c a t i v e of severe  examination  reveals  the  and  is f u l l  appearance to the l i v e r  bloody,  lungs,  mottling  severe  of serum, and the (uniformly r e d ) .  and  pulmonary  lungs have a  Lung swabs and  sputum swabs have been examined (by Charles Ford in Medical Microb i o l o g y , UBC) and found to c o n s i s t e n t l y grow monocultures of Psuedomonas aeruginosa.  Currently,  attempts  have been made to  remove  t h i s problem by s l i g h t l y a c i d i f y i n g the drinking water, and allowing an i n t e r v a l of at least a week between a r r i v a l of the rats from the supplier and s u r g i c a l preparation.  These manoeuvres, in conjunction  with regular d i s i n f e c t i o n of the laboratory with bleach appear to have reduced the incidence of l i f e - t h r e a t e n i n g pneumopathy, e.  We have had no reason to exclude rats from study on the grounds of low blood pressure.  P a r r a 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 following action i s taken in the event of the following p o s t - o c c l u sion abnormalities, with the object of excluding rats with incomplete or non-existant occlusions and r a t s which experience reperfusion as a r e s u l t of the loosening of a defective occluder: a.  If there i s no increase in R wave, and/or no ST elevation f o l l o w i n g o c c l u s i o n , the rat i s excluded: i.  if  postmortem examination reveals an unacceptably small OZ or  IZ (see below),  - 120 ii.  i f the rat dies within 4 hr of occlusion and i s found to have i n f lamination or scarring in the heart,  iii. b.  If,  i f postmortem examination reveals that the occluder i s l o o s e , at some time after o c c l u s i o n , the ECG returns to the p r e - o c c l u -  sion c o n f i g u r a t i o n , the r a t i s excluded:  c.  i.  i f the OZ and/or IZ are unacceptably small (see below),  ii.  i f the l i g a t o r  If  the  rat  dies  i s found to be loose. within  association  with  excluded i f  the thorax  that the  immediate  overtightening coronary  the  of  vessels,  first  10  calamatous  min  after  hypotension,  i s found to contain blood. the  occluder  perhaps  region of the f l a r e d outer  as  occlusion the  It  is  rat  guide tubing.  of  is  possible  can cause haemorrhaging  a result  in  roughness  This p a r t i c u l a r  from  in  the  problem  is seen in less than 2% of r a t s . d.  Animals experiencing f a t a l excluded from s t u d i e s .  or non-fatal  cardiogenic  Cardiogenic shock  shock are  has been defined  not as a  fall  in blood pressure of at l e a s t 30%, maintained for at l e a s t 30  min,  associated with  arrhythmias  (Agress  ECG signs et a l . ,  of  ischaemia, but  1952).  This  not  definition  caused by adequately  describes the syndrome which we recognise as cardiogenic shock  in  rats subjected to coronary artery o c c l u s i o n . C.  The following abnormalities,  action i s taken in the event of the following in order to exclude animals with inadequate or  postmortem abnormal  occlusions: a.  Rats are excluded i f they are found to have an inappropriately OZ, defined as < 25 %ventricular mean).  weight  small  (more than 2 s . d . from the  - 121 b.  Rats having inappropriately are excluded.  small  This c r i t e r i o n  IZ,  defined  as < 50% of  i s somewhate a r b i t r a r y ,  and i s based  on the assumption that since the r a t has no functional capable of providing Selye  et-al.-,  'myocardial  1960; Maxwell  salvage  et a l . ,  the OZ  collaterals  (Johns and Olson, 1954;  1  1984;  Winkler  et-al.,  1984;  Schaper et a l . , 1986), then the IZ at 24 h should be a reasonably f i x e d % of the OZ. laboratory  This appears to be the case, since to date, our  has not found any drugs which c o n s i s t e n t l y  reduce  IZ,  which may mean that 'myocardial salvage' i s impossible in rats (see Discussion).  The a r b i t r a r y  nature of t h i s  exclusion c r i t e r i o n  is  not r e a l l y a problem, since no rats have ever been excluded from a study s o l e l y on the basis of t h i s c.  If  criterion.  i n f a r c t e d t i s s u e or s c a r r i n g in the heart i s found in a r a t which  dies before 4 h after o c c l u s i o n , the r a t i s excluded, on the grounds that i n f a r c t i o n i s not detectable using TTZ u n t i l at l e a s t 6 h a f t e r occlusion,  and i s not well  demarcated u n t i l  occlusion (Hort and Da C a n a l i s , 1965a). t i v e of  infarction  produced during  or  at  least  10 h  after  Early scarring is indicashortly  after  preparation.  Less than 1% of rats have been excluded on t h i s b a s i s . d.  If pus is found at the occlusion s i t e , the r a t i s excluded.  Occasionally,  acutely  prepared  animals  are  used for  experimentation.  The exclusion c r i t e r i a outlined above are used except where they are obviously  inappropriate.  1980),  rats  In  accordance with  Parratt's  criteria  with pre-drug pre-occlusion mean a r t e r i a l  (Clark  blood  pressures  < 70 mmHg are excluded (in the present experiments no r a t s a c t u a l l y t h i s t e s t ; pre-occlusion blood pressure was always > 80 mmHg).  et-al;, of  failed  - 122 2.1.9 S t a t i s t i c s In most experiments, a control two treated treated  with  (normally) In a l l  groups.  The control  drug v e h i c l e .  distributed  group was defined as a group of  The v a r i a b l e s  compared were  or binomially d i s t r i b u t e d  comparisons, the l i m i t of  p < 0.05.  group of rats was compared with at l e a s t  'statistical  either  animals Gaussian  (Johnston e t a l ; ,  1983a).  s i g n i f i c a n c e ' was defined as  In accordance with the requirements f o r undertaking the types of  statistical  tests  described below,  randomisation  analysis of records were c a r r i e d out. given b l i n d .  to  treatment  and  blind  Whenever p o s s i b l e , treatment was also  Obviously a procedure such as decerebration or pithing  below) could not be c a r r i e d out b l i n d .  (see  The group s i z e was kept to a minimum,  and was based l o o s e l y on the minimum sample s i z e required to reveal a 50 % reduction in VT and VF. h period  after  The control  occlusion  in  incidence of VT and VT during the  conscious r a t s  is  approximately  0-4  90 - 100 %  2  (Johnston et a l . - , 1983a; e t c . ) .  In a 1 - t a i l e d chi  t e s t , the minimum group  s i z e 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 e s t s , such as Duncan's multiple range t e s t , i t was necessary to ensure that the v a r i a b l e was Gaussian d i s t r i b u t e d . (Johnston Gaussian  et a l ; ,  1983a)  distributed.  that  Therefore,  values were c a l c u l a t e d . variables:  many of in  It  has been shown by our  the order  variables to  laboratory  measured are  compare means, the  This manoeuvre was c a r r i e d out for  the  log^glog^g  following  PVC, number and duration of VT and VF and time of maximum R wave  and S-T segment e l e v a t i o n . Corrections for co-variance by the use of normalization procedures were c a r r i e d out,  in c e r t a i n  instances, to  improve p r e c i s i o n .  For example,  has been shown that the arrhythmia score (AS) c o r r e l a t e s l i n e a r l y with (Johnston e t a l . ,  1983a).  Therefore AS can be expressed as a function  it JbT of  - 123 OZ.  There  is  a major  problem a s s o c i a t e d w i t h  the case i n c o n t r o l s , but i n d r u g - t r e a t e d attempt was made to  overcome t h i s  (ASMC), which f u n c t i o n s  J"5z, to  of  T h i s i s never  i s o f t e n the c a s e .  i n the same way as the A S , but etc.).  but cannot e a s i l y be c o r r e c t e d f o r each AS v a l u e , then  groups t h i s  between AS  An  problem by d e v e l o p i n g an i s c h a e m i a s c o r e  i s c h a e m i a (R wave s i z e ,  (AS + 1)1^01.  relationship  AS/JoT becomes meaningless i f AS i s z e r o .  and OZ, namely t h a t  signs  the  correcting  The f a c t  includes scores  for  AS c o r r e l a t e s  with  that  \J0Z~ was a l s o approached by adding 1  this  v a l u e f o r ^JOZ, g i v i n g  However, the p r e c i s i o n of  OZ i s  high,  the  variable  in  practice  such t h a t  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 n s t e a d s c a t t e r v a l u e s around a  single  point.  e t a l ; , 1984)  Only  is  the  when  small  relationship  OZs  are  deliberately  between \[0Z~ and AS a p p a r e n t .  the be  section avoided  which  concerned w i t h and  its  jeopardises  taken.  The  effect  j e o p a r d i s e the  thump-version can o n l y  less  defibrillation.  be m i n i m i s e d .  must  introduced  experiment  When l a r g e  C e n s o r i n g as a r e s u l t o f death was d i s c u s s e d i n  an experiment  censoring  (Curtis  \JoZ.  OZs o n l y are produced t h e r e i s no need t o c o r r e c t AS f o r 2.1.9.2 Censoring.  produced  be  The type  identified  by  of  cannot  censoring  and a p p r o p r i a t e  thump-version  than the  Censoring  was  action  considered  censoring associated with  to  allowing  a n i m a l s 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  binomially  distributed  variables  2 chi  tests  were  used  distributed U.B.C.  for  testing  program  a significant  compared  appropriate  chi  Mainland's contingency tables (Mainland  et-al;,  of  minimum  1956).  For  contrasts Gaussian-  v a r i a b l e s , a n a l y s i s of v a r i a n c e (ANOVA) was c a r r i e d o u t ,  statistics  constituted means  were u s e d . 2  (using  (Gregg and s o u r c e of  Duncan's  s i n c e the number of  Osterlin, variance  multiple  1977).  Only  according  range  groups exceeded two  to  test). in  if  using a  treatment  an F t e s t This  were  test  every study.  was In  no  - 124 instance was a simple t test used for comparing means, since the t t e s t  is  only appropriate when two groups are compared; i f  is  a 1 in 20 p r o b a b i l i t y  set as the l i m i t of chance, and 20 groups are compared with a control then the p r o b a b i l i t y approaches 100% that at l e a s t one group w i l l to be d i f f e r e n t such  as  from c o n t r o l s , according to the t t e s t .  Duncan's m u l t i p l e  range  test,  avoid  this  be found  Modified t  pit-fall,  group,  tests,  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  lines. to  to  treatment  was c a r r i e d out  by assembling a table  of  Each l i n e was assembled by drawing playing cards numbered according  the number of  groups  in  each study.  Drug stocks were prepared, and  appropriate d i l u t i o n s made by various members of the laboratory according to specific instructions.  The stock solutions were coded.  Therefore the person  preparing a syringe of drug for a p a r t i c u l a r r a t was also b l i n d to treatment. Lines were chosen in random order  by the  person preparing the  This person prepared a second table in which the r a t  injection.  code (assigned on the  day of preparation) and the coded treatment were noted. In the case of rats treated with unstable drugs ( f e l o d i p i n e , and DHM9), the person making up the drugs was instructed s p e c i f i e d amount of drug for each r a t , treatment.  nifedipine  to weigh out a  and was therefore not b l i n d to  the  However, the person administering the drug, occluding the coron-  ary a r t e r y , monitoring responses and analysing the records remained b l i n d to the treatment.  All  other  aspects of  study were as outlined  in  previous  chapters. Up to 4 rats were occluded on each day of experimentation, with a 15 min interval  between each o c c l u s i o n .  In t h i s manner, rat-1  the phase of e a r l y arrhythmias (Clarke e t a l 1 9 8 0 ;  had passed through  Johnston et a l . , 1983a)  - 125 by the time r a t - 2 was due for o c c l u s i o n ; r a t - 2 received i t s  drug treatment  while rat-1 was being monitored for arrhythmias, and so on. For each r a t  a complete h i s t o r y  book and also on i n d i v i d u a l all  was kept,  analysis sheets.  both in the  laboratory  day  The analysis sheets contained  information concerning haemodynamic, ECG and arrhythmia data, as well as  OZ, IZ,  serum K , body weight, +  date of preparation  and o c c l u s i o n , general  comments concerning preparation, behavioural responses to drug treatment and occlusion and postmortem f i n d i n g s .  When the study was complete, the codes  were broken and the r e s u l t s analysed. i s a t i o n to l i n e s i t  is s t a t i s t i c a l l y  completion of each l i n e .  According to the p r i n c i p l e of randomacceptable to break the code upon the  However, t h i s p r a c t i c e was avoided.  2.2.2 Phenethylalkylamines 2 . 2 . 2 . 1 Anipamil -and-Ronipamil.  The e f f e c t s of anipamil and r o n i p -  amil on responses to coronary occlusion were investigated because these a n a l ogues of verapamil d i f f e r  in respect to t h e i r  calcium antagonist  activity,  and were therefore considered to be valuble tools for t e s t i n g the hypothesis that  calcium antagonism  in  the  ventricle  is  antiarrhythmic  during  acute  myocardial ischaemia. Anipamil and ronipamil  are analogues of verapamil.  amil the main chain of verapamil  In the case of anip-  has been extended by 10 C-atoms and the  methoxy substituents removed from the 4 p o s i t i o n on the phenyl r i n g s , leaving l,7-bis-(3-methoxyphenyl)-3-methylaza-7-cyano-nonadecane. embles anipamil rings, have  i .e i t  except for is  of  methoxy  substituents  on the  l,7-bisphenyl-3-methylaza-7-cyano-nonadecane.  been demonstrated  preparations  a lack  Ronipami1  to  have  anti-ischaemic  activity  in  resphenyl  Both drugs  a variety  of  (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 preliminary t o x i c i t y studies in which i t (higher  was found that administration  of  300 mg/kg was non-toxic  doses produced death in approximately 50 % of r a t s  the cause of death was unclear, since i t tance,  up to  but  was considered to  given anipamil;  occurred overnight  be the r e s u l t  of  in every i n s -  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. either  Suspensions of 20 or 60 mg/ml  drug were prepared and gently heated to 60 °C to f a c i l i t a t e  l u t i o n , then allowed to cool s l i g h t l y before a d m i n i s t r a t i o n .  of  disso-  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  administration  was  carried  out  4 h  before  occlusion during a period of extremely b r i e f anaesthesia (1 min) with h a l o thane.  An i n t r a g a s t r i c  tube for  drug administration was created by simply  f i x i n g a 12 cm length of PE90 tubing to a 3 ml s y r i n g e .  The treatments were  administered slowly in order to preclude accidental administration trachea.  The p o s s i b i l i t y  rats  a well-developed vomit r e f l e x  lack  of  aspiration  pneumonitis (Briggs  into  was discounted  and Oehme, 1980).  were made up fresh approximately once per week, r e f r i g e r a t e d  the  since Drugs  and stored  in  light-proof containers. 2.2.2.2 (-+-)- And (-)-verapami 1.  The o p t i c a l  enantiomers of verap-  amil were compared for t h e i r actions on the responses to coronary occlusion in order to test the hypothesis that calcium antagonism in the v e n t r i c l e s i s antiarrhythmic  during acute myocardial  ischaemia, and also to  complementary hypothesis that the antiarrhythmic  action of  examine the  (^J-verapamil  in  conscious rats (Curtis e t - a l . , 1984) occurred by v i r t u e of calcium antagonism in  the  ventricles  arrhythmic  (see  Introduction).  Both  hypotheses  potency r a t i o equal to the calcium antagonist  predict  an  anti-  potency r a t i o  in  - 127 the v e n t r i c u l a r myocardium, based on the reported potency difference 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 s a l i n e and administered at a volume of 0.25 ml/kg by slow i . v .  i n j e c t i o n over 10 min, beginning 15 min before coronary o c c l u -  s i o n , according to the protocol outlined in the general methods section (see Figure 1 ) .  Ten groups of  rats  received s a l i n e ( c o n t r o l s ) .  (n = 9 per group)  were used.  Two groups  The remaining groups received e i t h e r  (-)-verap-  amil ( 0 . 2 , 0.6, 2 or 6 mg/kg) or (+)-verapamil ( 0 . 4 , 4 , 8 or 12 mg/kg). doses  studied  (^J-verapamil  were  chosen on the  basis  of  the  earlier  experiment  The with  (Curtis 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 , refrigerated  and stored  in  light-proof  vessels,  coded to  and were  ensure  double-  blindness. 2.2.3 1,4-Dihydropyridines 2.2.3.1 Felodipine.  Felodipine was evaluated on the basis of  its  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 i a an action in the v e n t r i c l e s predicts  that  felodipine  only  (and  other  1.4-dihydropyridine  reduce arrhythmias at high doses.  calcium antagonists)  In a d d i t i o n , a calcium antagonist which  shows marked s e l e c t i v i t y for the vasculature may p r e c i p i t a t e reductions  in  blood pressure at  sufficient  to  reduce i .. in  the v e n t r i c l e s  if  1ife-threatening  doses below those producing blood  s e v e r i t y of ischaemia-induced arrhythmias. f e l o d i p i n e would have l i t t l e  will  and reduce the  incidence and  Therefore, i t was predicted that  any antiarrhythmic  produced large reductions in blood pressure.  levels  activity  at doses which  - 128 Eight groups of rats were used (n = 9 per group). standard large OZ (LOZ) rats prepared and subjected to rats  received 1 of  (5 groups), 3 groups of  In addition to  small OZ (SOZ) were  coronary occlusion by Kathy Johnston.  3 doses, 0 . 2 ,  2.6 or  12.2 ymol/kg,  schedule outlined in the general methods.  respectively)  Results s e c t i o n .  for  the  The LOZ  according to  purpose of  labeling  the  intermediate  figures  in  the  Only the lower and higher doses were given to the SOZ r a t s .  Felodipine was dissolved in  20 %ethanol  volume of 0.25 ml/lOOg body weight.  in  saline  and administered  Care was taken to protect a l l  rats were used (18 LOZ rats  and 9 SOZ r a t s ) .  received 0.25 ml/lOOg body weight of 20%ethanol in s a l i n e .  at a  syringes  from d i r e c t s u n l i g h t / f l u o r e s c e n t room l i g h t i n g before a d m i n i s t r a t i o n . seven control  the  These doses were 0.08, 1 and 4.68  mg/kg, r e s p e c t i v e l y , and have been coded as L, I and H (low, and h i g h ,  the  Twenty  Control  rats  The LOZ and SOZ  rats were analysed separately, since treatment was the only known source of variance within the variance between the  LOZ and SOZ s e r i e s , but LOZ and SOZ s e r i e s .  operators  were  a source  of  Drugs stocks were made up  in  advance of the study and were r e f r i g e r a t e d and stored in l i g h t - p r o o f v e s s e l s . 2 . 2 . 3 . 2 N i f e d i p i n e and DHM9.  These 1,4-dihydropyridines  were s t u d -  ied in order to supplement the work with f e l o d i p i n e .  DHM9 has been reported  to possess a s e l e c t i v i t y of action for  t i s s u e versus vascular  ventricular  smooth muscle, in contrast with a l l other 1,4-dihydropyridine onists (Clarke e t - a l ; , 1984b).  Therefore, according to the hypothesis that  calcium antagonism in the v e n t r i c l e s i s antiarrhythmic dial  ischaemia, i t  calcium antag-  was expected that  during acute myocar-  DHM9 should be more  effective  in  reducing ischaemia-induced arrhythmias than n i f e d i p i n e at doses producing a s i m i l a r degree of blood pressure lowering. Five dissolved  groups  of  rats  in 20%ethanol  were in  studied  (n = 9  per  group).  s a l i n e and administered at  Drugs  were  0.25 ml/lOOg body  - 129 weight  in  section.  accordance with  the  schedule outlined  in  the  Controls received the ethanol v e h i c l e alone.  and 2 mg/kg n i f e d i p i n e and 5 and 20 mg/kg DHM9.  general  methods  The doses were 0.5  These doses appear to be  l a r g e , but they were based on a preliminary t o x i c i t y study in which i t was found  that  conscious rats  could  tolerate  Although not investigated in any d e t a i l ,  at it  least  16 mg/kg  nifedipine.  was considered that the dose-  l i m i t i n g f a c t o r was in f a c t the ethanol v e h i c l e rather than the drug i t s e l f . Preliminary  investigations  with  DHM9 showed that  occurred at a l l at cumulative doses well  no haemodynamic  in excess of 30 mg/kg.  effects  In accor-  dance with the general method, stock solutions were prepared in advance of the study, coded, r e f r i g e r a t e d  and stored in l i g h t - p r o o f  containers.  Care  was taken to prevent exposure of the drug-containing syringes to l i g h t before administration.  True blindness could not be achieved in t h i s  the yellow colouration of the drugs could be seen in  the  study, since  t h i n PE tubing  entering the subscapular region of the rats at the highest doses (both DHM9 and n i f e d i p i n e  are  vivid  yellow  in  colour).  However,  all  records  were  analysed b l i n d . 2.3  Arrhythmogenesis and t h e - r o l e o f - t h e - G N S 2.3.1 Introduction The experiments outlined here were not designed to answer a l l questions  concerning arrhythmogenesis in r a t s , but were intended to examine the of the CNS and the sympathetic nervous system. out  preliminary  experiments for  this  role  Our laboratory has c a r r i e d  purpose in the past  (Botting  et a l ; ,  1983). The strategy was to remove, by surgery, the autonomic nervous system in a graded manner in some groups of catecholamines in others.  animals, and replace i t  by infusion  of  In a d d i t i o n , the extent of surgery was considered  as a possible source of variance.  In conjunction with t h i s l a t t e r consider-  - 130 ation,  the  effects  of  pithing  (the  most extensive  possible mediators and modulators of arrhythmias  surgical  (serum K , +  ablation)  on  leukocytes and  thrombocytes) were determined in a separate s e r i e s of experiments. A l l groups in t h i s study c o n s i s t e d , as u s u a l , of 9 r a t s . c a r r i e d out aorta,  Occlusion was  as described above, and blood pressure was recorded v i a  except when stated  otherwise.  Since  there  the  were many groups,  preparation was complicated, a summary table has been prepared which  and lists  a l l the groups (Table 1 ) . 2.3.2 Preparation 2.3.2.1 Pithing.  Male Sprague Dawley rats were l i g h t l y anaesthe-  t i s e d with halothane (4% in oxygen) and intubated.  The r a t s were prepared  for occlusion in the normal manner, with an a o r t i c blood pressure l i n e , j u g u l a r intravenous l i n e and Vg ECG leads. from preparative surgery for  These r a t s were allowed to recover  approximately 7 days.  This group was subse-  quently p i t h e d , as described below, and constituted the c h r o n i c a l l y prepared (c) pithed (P) group ( i . e . , c P ) . A second group  was  prepared  blood pressure l i n e s and femoral sation  techniques  with  occluders,  intravenous l i n e s  using PE t u b i n g ) .  This  group  Vg ECG leads, (by standard constituted  carotid  catheteri-  the  acutely  prepared (a) pithed (P) group ( i . e . , a P ) , since they were pithed immediately a f t e r preparation. P i t h i n g was c a r r i e d out as f o l l o w s .  A s t a i n l e s s steel rod (3 mm d i a -  meter) was passed through the o r b i t and down the spinal cord during anaesthesia  (1% halothane  in  oxygen).  Immediately  r e s p i r a t i o n with 100% oxygen was i n s t i g a t e d body weight, 54 strokes/min).  This a r t i f i c i a l  afterwards,  light  artificial  (stroke volume 4 ml per 300 g r e s p i r a t i o n regimen has been  shown to produce blood gas and blood pH l e v e l s w i t h i n the normal range pithed rats (Milmer and Clough, 1985).  in  The rats were then mounted v e r t i c a l l y  - 131 with the head pointing downward, by securing the t a i l 2 clamps attached to  a retort  stand.  elevated at t h i s time in order that i t plane as the heart.  A rectal  and the p i t h i n g rod in  The blood pressure transducer was should remain in the same horizontal  thermocouple was connected v i a an  Indicating  C o n t r o l l e r unit (YSI Model 73ATA) to a 100 W l i g h t bulb, placed 20 cm from the r a t , and a sheet of polythene was placed over the e n t i r e preparation, to maintain body temperatute at 37 - 38 °C. was then injected i . v .  in an attempt to compensate for the i n i t i a l  pressure lowering e f f e c t 30 min a f t e r p i t h i n g .  Saline (1 ml per 100 g body weight)  of  pithing.  blood-  Coronary occlusion was c a r r i e d  out  Rats were monitored for 4 h, then s a c r i f i c e d (exsang-  uination).  A l l v a r i a b l e s were measured in the usual way (see general Methods  section).  Infarct  size  required for q u a n t i f i a b l e  (IZ)  was not  infarct  measured, since  at  least  10 h  is  development according to the TTZ s t a i n i n g  technique (Hort and Da C a n a l i s , 1965b). 2.3.2.2 S p i n a l i s a t i o n .  Rats were prepared and occluded i n a manner  i d e n t i c a l with that described for acutely prepared pithed rats (the aP group) with the exception that instead of p i t h i n g , the rats were s p i n a l i s e d . was achieved by i n s e r t i n g  a steel rod into  magnum at the l e v e l of C I , advancing i t to  mascerate the b r a i n .  the s k u l l  r o s t r a l l y and r o t a t i n g  This group constituted  s p i n a l i s e d (S) group ( i . e . , aS).  reflexes  received s i m i l a r groups.  (foot withdrawal  the  the it  foramen laterally  acutely prepared  (a)  As in the case of the aP group, 30 min was  allowed between ablation in the CNS and o c c l u s i o n . spinal  through  This  to pinching)  cardiovascular and r e s p i r a t o r y  At the time of o c c l u s i o n ,  were r e t u r n i n g . support  to  the  This  group  pithed  rat  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  i d e n t i c a l with the acutely prepared pithed and s p i n a l i s e d groups (aP and aS r e s p e c t i v e l y ) with the exception that the ablation c a r r i e d out was decere-  - 132 bration.  Decerebration was achieved by removing the brain r o s t r a l  midcol1icular  level using the blunt end of a spatula f o l l o w i n g  The empty space was packed with gel foam. between ablation  and o c c l u s i o n .  cardiovascular or r e s p i r a t o r y  A 30 min i n t e r v a l  was r e q u i r e d .  After  the  craniotomy. was allowed  In contrast with the previous  support  to  groups, no  preparation  the  r a t s in t h i s group breathed spontaneously and maintained a high blood pressure  while  horizontal.  This  group  constituted  the  acutely  prepared  (a)  decerebrate (D) group ( i . e . , aD). 2.3.3 Other manipulations The groups described above received ablations levels.  The pithed  r a t s were i n t a c t  in the CNS at  groups received complete CNS a b l a t i o n ,  distal  the  different spinalised  to C l , and the decerebrate group possessed  intact  r e s p i r a t o r y and vasomotor centres as well as spinal r e f l e x e s . In a supplementary group of acutely prepared pithed r a t s , an attempt was made to restore the e f f e c t s of the sympathetic nervous system by i n f u s i n g a mixture  of  noradrenaline  and adrenaline.  This  infusion  was begun 15 min  before occlusion and was designed to elevate blood pressure to l e v e l s seen in  conscious r a t s  (mean of  approximately  100 - 110 mmHg; Johnston  1983a).  The mixture was 4:1 noradrenaline:adrenaline  and the  infusion  Infusion  volume  rate was  was varied  kept  below  from 0.2 10 m l / k g / h .  to  (on a weight  5 ug/kg/min This  group  aPN).  basis),  noradrenaline. constituted  acutely prepared ( a ) , pithed (P) noradrenaline/adrenaline treated (i.e.,  et-al;,  Respiratory and cardiovascular support and a l l  (N)  other  the group  aspects  of experimentation were i d e n t i c a l with those used for the aP and aS groups. Four groups of  'control'  r a t s were used.  A standard group of conscious  c h r o n i c a l l y - p r e p a r e d rats were prepared and occluded in the manner described in  the  general  prepared (c)  methods  section.  This  conscious (C) group ( i . e . ,  group  constituted  the  chronically  cC) and served as a control  group  - 133 free from recent surgery and ablations in the CNS. A second, s i m i l a r group received an infusion of noradrenaline/adrenaline mixture i d e n t i c a l with that administered to the aPN pithed group. of  conscious rats  constituted  the  chronically  prepared  (c)  This group  conscious (C)  noradrenaline/adrenaline (N) treated group (cCN), and served as control  for  the e f f e c t s of the catecholamine i n f u s i o n in the absence of ablations in the CNS. A t h i r d 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 before o c c l u s i o n . (C) group  (i.e.,  surgery  This group c o n s t i t u t e d the acutely prepared (a) conscious aC), and served as a control  for  recent  surgery  in  the  absence of s u r g i c a l ablation in the CNS. F i n a l l y , a group of r a t s was prepared for occlusion according to Clark et-al; minor et-al.)  (1980)  using pentobarbitone  exception  that  our  (60 mg/kg  standard occluder  and our V3 ECG leads (not  for  (not  anaesthesia, with the  lead 2) were used.  recorded from the l e f t c a r o t i d a r t e r y , formed (for d e l i v e r y of a r t i f i c i a l  i.p.)  silk  type  Blood pressure was  and a cut-down tracheotomy was per-  Occlusion was c a r r i e d out  a f t e r preparation, and rats were s a c r i f i c e d 4 h after o c c l u s i o n . the  acutely  prepared  Clark  r e s p i r a t i o n according to the regimen used  the pithed and s p i n a l i s e d groups).  constituted  of  the  (a)  barbiturate  anaesthetised  30 min  This group (B)  group  ( i . e . , aB), and served as a control for recent minor surgery in the absence of s u r g i c a l ablation in the CNS but in the presence of chemical ablation the CNS (anaesthesia).  in  - 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  Spinalized  aS  Decerebrate  aD  Chronically-Prepared Groups  Code  Conscious  cC  Conscious plus catecholamine  infusion  cCN  Pithed  CP  Others  Code  Isolated perfused hearts  I  D e t a i l s of the s u r g i c a l preparation are given in the t e x t .  - 135 An additional  group was included in  this  study,  a group of  isolated  hearts perfused v i a 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). c o n s t i t u t e d the i s o l a t e d heart (I)  This group  group, and served as a general reference  group. 2.3.4 S t a t i s t i c s Variables occlusion  were measured and  preparation.  complicated,  and i t  is  The f u l l possible to  i m p l i c a t i o n s of the r e s u l t s . stical  analysed as data  set  described for  discuss at  for  this  study  great  length  the  standard  is  extremely  the  possible  In order to s i m p l i f y matters somewhat, s t a t i -  s i g n i f i c a n c e has only been noted between  the  chronically  prepared  conscious group (cC) and the other groups. 2.4  Preliminary-Screen for drug a c t i v i t y in acute-ischaemia 2.4.1 Introduction Although the conscious r a t  ischaemia and i n f a r c t i o n variable  per  week,  it  preparation for  investigation  of myocardial  i s capable of providing up to 10 data points was decided to  develop  a preliminary  screen  per 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  mation concerning m o r t a l i t y and i n f a r c t s i z e .  infor-  However, assessment of animal  behaviour was also used, in order to attempt to delineate between m o r t a l i t y r e s u l t i n g from v e n t r i c u l a r arrhythmias (VF) and other causes. 2.4.2 Preparation Male  Sprague Dawley rats  described in d e t a i l  were  prepared for  in preceding chapters.  ECG leads were implanted, only the could be prepared d a i l y , with ease.  occlusion  No intravascular  occluder.  By t h i s  in  the  manner  catheters and  technique,  16 rats  The rats were housed i n d i v i d u a 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 o c c l u s i o n , rats were c a r e f u l l y monitored by observation alone. The behaviour of each rat was recorded on i n d i v i d u a l  analysis sheets.  All  observation and analysis was c a r r i e d out b l i n d . 2.4.3.1 D e f i n i t i o n s . for each time i n t e r v a l  Certain behaviours were c a r e f u l l y  f o l l o w i n g occlusion (the time i n t e r v a l s  with those used for the standard occlusion preparation). was noted, and OZ was measured in the usual manner.  categorised corresponded  The time of death  In rats surviving 24 h,  the IZ was also measured. Behaviour was c l a s s i f i e d and assessed according to jective c r i t e r i a . of the f o l l o w i n g  the following  Morbidity was graded according to the presence or absence 3 behaviours, r e s p i r a t o r y  distress  (which was defined as  laboured breathing), head-down posture, and prone posture. points 1952).  were  sub-  considered to  be signs  of  cardiogenic  These 3 end-  shock  (Agress  et-al.,  If nothing more serious than minor panting was present, the r a t was  classed as normal. Severe v e n t r i c u l a r 'convulsive-type'  arrhythmias  behaviour.  were diagnosed on the  basis of  This was defined as sudden f r e n z i e d  sudden attempts  to climb out of the home cage accompanied by convulsive-type limb movements and a sudden blanching of c h a r a c t e r i s t i c of VF (or  the ears and eyes.  This behaviour  severe torsade de pointes)  lasting  is  entirely  longer than 10  sec in the standard instrumented rat preparation, and has never been observed in association with other sequelae of coronary occlusion such as AV block or cardiogenic shock. Occasionally,  rats  develop f a t a l  pulmonary oedema following  The associated behaviour is d i f f e r e n t from that caused by VF.  occlusion.  Fatal pulmon-  - 137 ary oedema in conscious rats  i s associated with f r e n z i e d behaviour, but  is  characterised by the absence of paling of the ears and eyes and the presence of expectoration of copious sputum (often bloody). type behaviour r e s u l t i n g from VF l a s t s for  Furthermore, c o n v u l s i v e -  less than 10 s e c , since syncope  r a p i d l y follows the loss of cardiac output, whereas the f r e n z i e d behaviour associated with acute pulmonary oedema generally l a s t s for more than 30 sec, since cardiac output i s maintained.  Acute pulmonary oedema i s always f a t a l ;  the death throes are associated with serious expectoration and an unpleasant gagging sound. ventricular  In c o n t r a s t , sudden convulsive-type behaviour r e s u l t i n g from  arrhythmias  i s never associated with sudden expectoration, and  i s not i n v a r i a b l y f a t a l ; often a r a t w i l l loose consciousness, then suddenly the ears and eyes w i l l without apparent i l l associated  with  turn  effect.  a highly  morbidity outlined above.  pink  and the  rat  will  regain consciousness  F i n a l l y , acute pulmonary oedema i s pathognomic  prodroma,  involving  generally  the  signs  of  Sudden convulsive-type behaviour i s not associated  with any prodroma. 2.4.3.2  V a l i d a t i o n of  behaviour- end-points.  The behaviours  cribed above were tested in a b l i n d study in which 12 standard  des-  fully-instru-  mented rats were prepared and occluded in the usual manner, while an observer recorded behaviour in the manner described. defibrillated  if  defibrillate)  and were therefore  These p a r t i c u l a r r a t s were not  VF developed, but were allowed to identical  to  rats  die  (or  spontaneously  subsequently used  for  comparing the verapamil enantiomers. 2.4.4 Comprison of (+)-,  (-)-  and (±)-verapamil  As part of the i n v e s t i g a t i o n s of the actions of verapamil in myocardial ischaemia and i n f a r c t i o n , a study was c a r r i e d out with the o p t i c a l mers  and the  p r e c i s i o n of  racema'te, this  using  the  above-described technique.  method was unknown, and since up to  16 r a t s  enantio-  Since  the  could be  - 138 occluded and observed per  day,  it  was decided to  use large  group  sizes  (n = 25 per group). Rats were prepared and allowed to recover from surgery as described. On the day of study, up to 16 rats were stationed on a s i n g l e laboratory bench. Drugs were administered v i a a s u p e r f i c i a l t a i l  vein using a b u t t e r f l y hypo-  dermic needle while each rat was temporarily r e s t r a i n e d in a perspex r e s t rained  Drugs were injected over a 10 min p e r i o d , and 5 min was then allowed  before o c c l u s i o n , in a manner analogous to the method used for standard fully-instrumented r a t s .  The cycle time ( i n t e r v a l  ive occlusions) was therefore 15 min. close attention  could be given  crucial f i r s t 5 - 1 0 and VT o c c u r s ) .  min a f t e r  to  studies  in  between success-  This i n t e r v a l was designed such that  the  behaviour  of  each rat  during  the  occlusion (when the highest incidence of VF  Rats were continuously monitored for  at  least 4 h  after  occlusion. The f o l l o w i n g treatments were administered: s a l i n e (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  ED^Q  for  (±)-verapamil  in  fully-instrumented  conscious  rats  (Curtis et a l . , 1984). The object of the study was e s s e n t i a l l y i d e n t i c a l with the object of the study with the enantiomers in fully-instrumented  rats described p r e v i o u s l y ,  namely to test the hypothesis that the antiarrhythmic amil occur by v i r t u e the  incidence  (-)-verapamil activity.  of  of calcium antagonism.  convulsive-type  than by (+)-verapami1,  behaviour  actions of  (^-verap-  The hypothesis predicts should  be  reduced  with (±)-verapami1 having  In a d d i t i o n , the experiments were also c a r r i e d out  that  more  by  intermediate in order  to  examine and c h a r a c t e r i s e t h i s new method of assessing drug a c t i v i t y in acute myocardial ischaemia and i n f a r c t i o n on the basis of a combination of object-  - 139 ive (OZ, IZ and m o r t a l i t y ) and subjective (behavioural)  end-points.  2.4.5 S t a t i s t i c s The incidence of m o r t a l i t y and VF are binomially d i s t r i b u t e d in conscious rats f o l l o w i n g occlusion (Johnston e t - a l . - , 1983a). of sudden convulsive-type behaviour,  Therefore, the incidence  m o r t a l i t y and morbidity  were analysed  2  using chi , in the manner described p r e v i o u s l y .  The r e s u l t s were categor-  ised in terms of the f i r s t 0.5 h and the 0.5 - 4 h periods a f t e r o c c l u s i o n , in accordance with the bimodal d i s t r i b u t i o n  of ischaemia-induced arrhythmias  with time in conscious rats (Johnston e t - a l . , 1983a). 2.5  Electrically-^induced-arrhythmias-in conscious rats 2.5.1 Introduction The o p t i c a l  tricular  muscles  concentrations i' sl  and  enantiomers at  of verapamil  high  concentrations  are 50 - 150 times  contractility  are  in  the  Therefore, the p o s s i b i l i t y  known to  in  N a  in excess of those necessary to  abolish  that blockade of  i'  Na  (Nawrath  mers in vivo seems highly u n l i k e l y .  et a l . - ,  (sodium channel  contributes to the pharmacological actions of ( ^ - v e r a p a m i l  1981).  blockade)  and i t s  enantio-  However, t h i s p o s s i b i l i t y was neverthe-  less examined by comparing the actions of the enantiomers f o r t h e i r to  influence  ventricle  arrhythmias  induced  in conscious r a t s .  enced e l e c t r i c a l l y  It  by  electrical  stimulation  of  ability the  was previously shown that quinidine  induced arrhythmias  in  conscious rats  (Curtis  1984) at the same dose which reduced occlusion-induced arrhythmias mia score)  by 50% (Johnston  et a l ; ,  1983a),  such actions at the dose reducing arrhythmia 1984).  Therefore  considered to  the  be useful  and calcium antagonists.  assessment of for  venThese  ventricle  et a l . - ,  i  1981).  normal  (Nawrath  block  whereas  influetal;,  (arrhyth-  (^J-verapami1  had no  score by 50% (Curtis  et a l ; ,  electrically-induced  differentiating  left  between  Na  +  arrhythmias channel  was  blockers  - 140 2.5.2 Preparation Male Sprague Dawley rats were prepared in a manner i d e n t i c a l with that f o r rats used for coronary occlusion s t u d i e s , with 1 exception. a coronary occluder, 2 teflon-coated  s t a i n l e s s steel  wire  Instead of  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 v e n t r i c u l a r w a l l . ethanol-sterilised  leads  needle as a l o c a t o r . ular  region,  into  the  This was achieved by plunging the  ventricle,  using  a 23 gauge hypodermic  A l l leads and l i n e s were e x t e r i o r i s e d in the subscap-  and approximately  preparative surgery.  7 days was allowed for  recovery from  this  On the day of study, the rats were connected to  the  standard devices for intravenous drug a d m i n i s t r a t i o n , and blood pressure and ECG recording. 2.5.3 Experimental end-points The v a r i a b l e s described below were each measured 3 times every 5 min. Their measurement has been described p r e v i o u s l y , ( C u r t i s et a l ; , 1984).  but  not  in great  detail  Stimulation of the l e f t v e n t r i c l e with square wave  pulses was undertaken using a Grass stimulator brated using a standard voltmeter  (Model SD9), which was c a l i -  (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).  Discrimination of end-points was c a r r i e d  out using the o s c i l l o s c o p e . 2 . 5 . 3 . 1 Maximum- following- frequency.  Maximum following  was defined as the frequency at which the v e n t r i c l e stimulus on a 1:1 b a s i s . istic  blood  failed  frequency  to f o l l o w  the  F a i l u r e to follow was accompanied by a character-  pressure change;  the  dropped-beat  produced a drop  in  blood  pressure, and the subsequent beat produced a large pulse as a r e s u l t of the increase in f i l l i n g  time and e n d - d i o a s t o l i c  pressure.  The blood pressure  - 141 record could be used for d i s c r i m i n a t i o n of the end-point, but in t h i s s e r i e s of experiments the o s c i l l o s c o p e alone was used.  Maximum f o l l o w i n g  frequency  was determined at 0.8 msec pulse width and twice threshold v o l t a g e , by slowly increasing the stimulation frequency from a baseline of 5 Hz. 2.5.3.2 Threshold voltage.  The threshold voltage for  was determined at 50 Hz and 0.8 msec by the same p r i n c i p l e mining maximum f o l l o w i n g frequency.  inducing VT  used for  deter-  The high frequency was used in order to  maximise the p r o b a b i l i t y of d e l i v e r i n g a pulse during the vulnerable p e r i o d , the terminal  portion of the QT (de Boer, 1921; Wiggers and Wegria, 1940).  In addition to the appearance in the ECG of VT, the threshold voltage for VT was characterised by a drop in blood pressure.  VT was almost always non-  sustained. 2.5.3.3  Threshold  pulse- width. from the  The  threshold  pulse  inducing  VT was determined  o s c i l l o s c o p e according  criteria  as those used for measuring the threshold voltage.  width  to  the  This  for same  variable  was measured at 50 Hz and twice threshold voltage. 2.5.4 Comparison of ( + )- and  (-)-verapamil  In the e a r l i e r study with (±)-verapamil, no e f f e c t  on the variables  1984).  Therefore,  it  highest  doses of  the  in question  was decided to enantiomers  in  it  was found that 6 mg/kg had  conscious rats  evaluate only the  administered  to  (Curtis  et a l . ,  equivalent  of  coronary-occluded  the rats,  since i t was not expected that the lower doses would influence the v a r i a b l e s . Therefore e i t h e r  8 or 12 mg/kg ( + )-verapami 1 or 2 or 6 mg/kg (-)-verapami1  (n = 6 per group) 10 min p e r i o d .  were administered v i a the vena caval  The stimulation  s t a r t of drug administration tion. for  variables  and 5 min a f t e r  i.v.  line,  were recorded 1 min  over a  before  the  completion of drug administra-  The %changes in the v a r i a b l e s were recorded and analysed.  The values  the v a r i a b l e s were noted on the ECG t r a c e s , and were analysed b l i n d .  - 142 Mean %changes in the variables were compared by ANOVA and Duncan's multiple range t e s t . 2.6  Haemodynami c and EGG - e f f e c t s of - c a l c i um- antagon i s t s - i n pithed-rats 2.6.1 Introduction In  previous  s e c t i o n s , the  procedures for  assessing the  action of the enantiomers of verapamil were described. (-)-verapamil  than r e l y  on l i t e r a t u r e  potency of the enantiomers of verapamil  which  The premise was that  i s more potent as a calcium antagonist than (+)-verapamil.  was decided that rather  et-al.,  antiarrhythmic  values f o r  the  It  relative  (Bayer e t - a l ; , 1975b; 1975c; Ferry  1985; Nawrath et a l . - , 1981; Raschack 1976b; Echizen e t - a l . - , 1985), give  disparate  values  ranging  from  4  to  over  100  in  favour  of  ( - ) - v e r a p a m i l , various attempts should be made to gauge the r e l a t i v e potency of the enantiomers ourselves.  The f i r s t method was to examine the  of the enantiomers on blood pressure, heart r a t e , P-R i n t e r v a l terval  in conscious r a t s .  effects  and QRS i n -  This p a r t i c u l a r experiment constituted a portion  of the occlusion study with the enantiomers, since a comparison of values before and a f t e r drug administration provided t h i s information.  However,  it  was decided to supplement t h i s information with other s t u d i e s . Earlier  work  with pithed rats  (see section  concerned with  arrhythmo-  genesis in myocardial ischaemia) had shown that i t was possible to produce a pithed r a t preparation with a high mean blood pressure of at l e a s t 60 mmHg, which remained v i a b l e f o r well over 4 h.  It  was decided to use t h i s prepa-  r a t i o n to i n v e s t i g a t e the actions of the verapamil enantiomers in the absence of autonomic tone and r e f l e x e s . interest,  This information  was considered to be of  not l e a s t because there appears to be a large d i s p a r i t y  the potency of ( ^ - v e r a p a m i l et a l . , 1984).  in anaesthetised versus conscious r a t s  between (Curtis  - 143 2.6.2 Preparation The pithed r a t previously,  preparation was e s s e n t i a l l y the same as that described  with the  exception  that  in  every instance blood pressure was  recorded from the a o r t a , and drugs were administered Each preparation was allowed to s t a b i l i z e for  into  the vena cava.  at l e a s t 1.5 h before  study,  and mean a o r t i c blood-pressure and the ECG were p e r i o d i c a l l y recorded during this  time. 2.6.3 Variables measured Mean blood  recorded.  pressure,  heart  rate,  P-R i n t e r v a l  and QT i n t e r v a l  were  The ECG variables 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 D r i s c o l l (1980)  for  interval are  the r a t .  Reductions in blood-pressure and prolongation  of P-R  are e f f e c t s c h a r a c t e r i s t i c a l l y produced by calcium antagonists, and  considered  to  be the  result  of  calcium antagonism  (see  Nayler  and  Horowitz, 1983). 2.6.4 Comparison of (+)-  and  (-)-verapamil  Once mean blood pressure had reached a stable l e v e l variation  over a 10 min p e r i o d ) ,  (no more than a 5 mmHg  increasing doses of  either  (+)-verapami1  (0.4, 0 . 8 , 4 and 8 mg/kg) or (-)-verapami 1 (0.02, 0.06, 0.2 and 0.6 were administered (n = 6 per drug). as a slow i . v .  mg/kg)  Each successive dose was administered  i n j e c t i o n over a 10 min p e r i o d .  It was observed in p r e l i m i n -  ary experiments that the peak blood-pressure lowering e f f e c t of the enantiomers occurred during the f i r s t 30 sec a f t e r  f i n i s h i n g an i n j e c t i o n .  present experiments, a l l v a r i a b l e s were recorded 10 - 15 sec a f t e r ering each dose, in order to measure the peak e f f e c t . was allowed between completing the f i r s t i n j e c t i o n and so on.  In the administ-  An i n t e r v a l of 15 min  and s t a r t i n g  the second,  - 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 u s t i f y i n g the expression of the data as s i n g l e point dose response  curves, rather  (ED^g or  values)  ED25  than  cumulative  dose response curves.  were compared, where appropriate,  Variables  using ANOVA and  Duncan's m u l t i p l e range t e s t . 2.7  A c t i o n s - o f - c a l c i u m antagonists i n - i s o l a t e d p e r f u s e d - r a t - v e n t r i c l e s 2.7.1 Introduction The f o l l o w i n g  experiments  were  carried  out  in  order  to  f u r t h e r the calcium antagonist potency r a t i o of the o p t i c a l verapamil, and to i n v e s t i g a t e the actions of n i f e d i p i n e  characterise  enantiomers of  and DHM9.  It  was  considered worthwhile to have a measure of calcium antagonist potency in r a t ventricular  t i s s u e , since t h i s  was the t i s s u e in which the  antiarrhythmic  actions of the calcium antagonists were suspected of being mediated. In  a d d i t i o n , verapamil was r e c e n t l y reported to  plasma protein 1985;  binding  and hepatic  Eichelbaum et a l . ,  phenomena occur  in  rats  metabolism in  exhibit humans  1984;  Vogelgesang et a l ; ,  there  is  a possibility  potency r a t i o s in vivo and in v i t r o may be d i f f e r e n t . r a t i o s of the enantiomers in vivo (P-R i n t e r v a l  (negative i n o t r o p i c  actions)  If  calcium  the  same  antagonist  Therefore the potency  prolongation and reductions  in blood pressure in conscious and pithed r a t s ) obtained in v i t r o  (Echizen et a l ; ,  1984).  that  steroselective  in  were compared with isolated ventricles  those per-  fused v i a the aorta by the method of Langendorff (1895). The experiments were also designed to investigate the p o s s i b i l i t y extracellular  K  +  plays a r o l e in governing calcium antagonist potency and  the locus of antiarrhythmic nists.  that  a c t i v i t y of verapamil and other calcium antago-  Since e x t r a c e l l u l a r K concentration has been shown to r i s e r a p i d l y +  - 145 following coronary occlusion (Hirche e t - a l ; , 1980; H i l l experiments were c a r r i e d out over a range of buffer K  +  and Gettes, 1980),  concentration.  2.7.2 Perfusion apparatus The experiments were c a r r i e d out using a perfusion apparatus which has r e c e n t l y been designed in the laboratory.  The apparatus was designed with a  small dead-space for rapid switching between d i f f e r e n t solutions in order to permit the generation of dose-response data.  In b r i e f , 9 c y l i n d e r s of 70 ml  capacity were machined into a block of p l e x i g l a s s which contained channels for c i r c u l a t i n g warm water (37 °C). v i a separate s i l a s t i c tubes  Drug solutions flowed from each c y l i n d e r  (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 i s o l a t e d v e n t r i c l e preparation (see below) was attached to the apparatus here. The flow from each c y l i n d e r to the appropriate s i l a s t i c feed l i n e 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 l o s i n g another.  The contents w i t h i n any  c y l i n d e r could be changed v i a a second Teflon tap at the opposite side of the c y l i n d e r . solutions regulate  The perfusion  though pH.  branched at  the  apparatus was pressurised to  coronary  circulation,  The gas used was 5% CO2 in an e x t e r i o r  manifold  on the  oxygenate oxygen.  drive  each  c l o s i n g on a rubber gasket.  perspex box to  solution  and  give  gas  inflow  9 separate  All  cylinders  The l i d was tightened by brass screws  The atmosphere above each c y l i n d e r was common,  and was bled v i a a common e x i t through a regulating ' p o p - o f f valve.  drug  A single  s i l a s t i c tubes which served to d e l i v e r gas to each c y l i n d e r . were sealed with a common perspex l i d .  the  pressure-relief  The pressures in the apparatus and at the a o r t i c root were monitored  by a manometer (anaeroid).  - 146 Oxygen d e l i v e r y in t h i s system was improved because of the design which reduces loss of oxygen to the dead space between the r e s e r v o i r and the heart, and because the apparatus oxygenates preheated b u f f e r , pre-oxygenated buffer.  rather  than  heating  A thermocouple inserted in the pulmonary artery was  used to monitor temperature. 2.7.3 S p e c i f i c a t i o n s for Langendorff perfusion Hearts were excised from male Sprague Dawley rats (250 - 320 g) f o l l o w i n g c e r v i c a l d i s l o c a t i o n and exsanguination. 95 mmHg.  The  atria  (300 stimuli/min)  were  removed  The perfusion pressure was set at and  the  ventricles  were  paced  with square wave pulses at 4 V and 1 msec (supramaximal  threshold voltage and pulse width) v i a teflon-coated s t a i n l e s s s t e e l  plunge  e l e c t r o d e s , using a Grass stimulator (Model SD9). The perfusing s o l u t i o n was a modified Krebs-Henseleit buffer (in  mM)  NaHC0  3  CaCl  2  0.7,  NaCl  118,  25 and dextrose 11, at  because in  previous  studies  KCI  1.8 - 8 . 8 ,  MgCl  37 °C and pH 7.4. EC^Q  2  4  1.2,  We used 0.7 mM C a C l  KCI was  meq/1  in  order  to  simulate K  +  produce K  elevation  inotropism  2  r a t v e n t r i c l e s was found to be 0.5 ± 0.1 mM (Curtis e t - a l . , 1984). to  positive  KH P0  perfused  NaCl  for  1.2,  in  varied at the expense of  the  2  comprising  concentrations  seen in  the  from 3 to 10  extracellular  fluid  during the e a r l y 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 -ventri cular-developed - pressure. pliant,  but  (Stretch n'  non-elastic, Seal).  balloon  was  made  from  plastic  A small com-  wrapping  film  The balloon was connected to a transducer v i a a 30 cm  length of PE50 tubing.  The balloon was attached by t y i n g a s u i t a b l e section  of f i l m to the end of the PE tubing with s i l k  thread, and was then  filled  with water and stretched by excess pressure to t r i p l e i t s o r i g i n a l volume.  - 147 The balloon so-formed was n o n - e l a s t i c and yet compliant enough to f i l l c a v i t y of the l e f t v e n t r i c l e without d i s t o r t i n g  its  tricular  the balloon  pressure was recorded a f t e r  ventricle via a left atrial  inserting  incision.  shape.  Left  the  intraven-  into the  left  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 l o w .  The perfusion apparatus was graduated  to indicate the volume of f l u i d in each chamber. by  measuring  chambers,  the  under  volume the  of  perfusion  assumption  that  fluid if  Coronary flow was estimated passing from  end d i a s t o l i c  the  pressure  graduated remained  constant when perfusion pressure was varied from 80 to 140 mmHg, then a o r t i c valve was competent.  the  This r e l a t i o n was tested during the s t a b i l i s a -  tion period. 2.7.4.3  Ventricular  excitability.  Preliminary experiments  that the threshold voltage and pulse width for approximately  0.6 V and 0.1  msec at  showed  capture of v e n t r i c l e s were  5.9 meq/1 K . +  The experiments were  c a r r i e d out at well above threshold (4 V and 1 msec), but at well below the threshold for  inducing unwanted e f f e c t s  such as noradrenaline release and  heating of the myocardium (as judged from the responses of hearts to variations  in  stimulation  considered inappropriate  variables to  in  examine the  these p a r t i c u l a r v e n t r i c l e s , since i t may jeopardise the subsequent v i a b i l i t y  preliminary  experiments).  strength-duration  large It  was  relationship  in  was possible that such an experiment of the preparations.  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  excitability.  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 v e n t r i c l e s 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 solutions (prepared by s e r i a l d i l u t i o n ) designed such that 0.2 ml of stock per 70 ml buffer gave the desired concentration w i t h i n each c y l i n d e r . refrigerated used for  and stored  in  light-proof  a s i n g l e drug at a s i n g l e K  The stock s o l u t i o n s were  containers. buffer  Each  preparation  concentration  10 meq/1); therefore 8 groups of preparations  was  (3, 5.9, 8 or  (n = 6 per group) were used.  Records were analysed b l i n d .  Concentration-response curves were constructed  and  were  ECgQ  Potency  and  ratios  slope at  values  each K  +  estimated  concentration  for  were  every E C ^ Q for (+)-verapamil with every E C ^ Q for  individual determined  preparations. by  contrasting  (-)-verapamil.  2.7.6 Comparison of n i f e d i p i n e and DHM9 Nifedipine and DHM9 were compared in e x a c t l y the same manner as (+)- and (-)-verapamil.  However, s p e c i a l  drugs from u l t r a v i o l e t l i g h t .  precautions were taken  warming  out short wave-lengths.  the  purpose.  perfusion  apparatus  The effectiveness  these  of  The lamp was wrapped in red p l a s t i c  In a d d i t i o n ,  the f l u i d c i r c u l a t i n g and  was stained with these  procedures  red  both drugs used 50% ethanol  dye  for  the  was exemplified  r e p r o d u c i b i l i t y of the r e s u l t s with time (see R e s u l t s ) . for  protect  The experiments were c a r r i e d out in a darkened  room, illuminated only by a 40 W bulb. to f i l t e r  to  by  same the  The stock solutions  in s a l i n e as the s o l v e n t .  When the drugs  were added to the r e s e r v o i r chambers of the perfusion apparatus,  dilution  reduced the f i n a l ethanol concentration to 0 . 2 5 % . 2.7.7 S t a t i s t i c s Slopes,  EC^Q  values  and  Duncan's m u l t i p l e range t e s t .  potency  ratios  were  compared by  ANOVA and  - 149 2.8  Metabolism-of racemie-verapamil - in-acute-myocardial-ischaemia 2.8.1 Introduction The following experiment was the f i r s t which was c a r r i e d out as part of  this  thesis.  (±)-verapamil laboratory  This  in  was part  (Curtis e t - a l ; , 1984).  that  occlusion  experiment  (^-verapamil  conscious r a t s .  mechanism of action of  of  It  reduced  initial  investigation  had been shown by others arrhythmias  As part of  (^-verapami 1, i t  the  the  induced  attempt  to  by  in  of the  coronary  investigate  the  was decided to measure the amount  of (±)-verapami 1 in the blood, the normal v e n t r i c u l a r  t i s s u e and the OZ of  r a t s subjected to coronary o c c l u s i o n . 2.8.2 Animals Male  Sprague  Dawley  described p r e v i o u s l y .  rats  were  prepared  for  coronary  occlusion  as  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 administration and t i s s u e samples On the  day of  study,  6 mg/kg  (^O-verapamil  was administered,  either  before o c c l u s i o n , according to the usual p r o t o c o l , or over a 10 min period s t a r t i n g immediately a f t e r o c c l u s i o n . was approximately equally e f f e c t i v e occlusion  as  when  given  before  It  had been shown that  as an antiarrhythmic occlusion,  and  the  when given ED^Q  arrhythmia score (AS) was approximately 6 mg/kg in both cases. was of i n t e r e s t  to determine whether  (±)-verapamil  the drug was capable of  for  after  reducing  Therefore  it  distributing  into the OZ when given a f t e r o c c l u s i o n , in r e l a t i o n to the possible locus of action within the myocardium. Rats were monitored for  30 min after  occlusion.  thump-verted as r e q u i r e d , in the usual manner.  Episodes of  VF were  A f t e r 30 min the r a t s were  q u i c k l y anaesthetised with halothane (5 %), using a face mask, and the aorta  - 150 was exposed by midline blood was taken (Kymax).  laparotomy f o r  and stored  blood sampling.  temporarily  The heart was then removed.  in  At l e a s t  an iced heparinised  10 ml test  of  tube  In several experiments, other t i s s u e s  ( s k e l e t a l muscle and l i v e r ) were also sampled. 2.8.4 Extraction The f o l l o w i n g for  injection  procedures were c a r r i e d out  onto  an HPLC separation  in order  system.  All  to the  prepare samples test  tubes  and  whole  blood  was  syringes used were made of g l a s s . a.  Blood was  immediately  divided  pipetted  into  into  a Dreyer  1 ug/ul gallopamil ( i n t e r n a l of ( ^ - v e r a p a m i l et-al.  (1981).  3 samples. test  One ml  tube  of  and spiked with  standard f o r the HPLC and d e t e c t o r ) .  16 pi  of  Extraction  was c a r r i e d out using a modification of the method of Cole The sample was made basic (approximately pH 9) with approxi-  mately 250 pi of 4 M NaOH for e x t r a c t i o n i n t o organic s o l v e n t , since the pKa of (^J-verapamil i s approximately 8.5 (Dorrscheidt-Kafer, 1977). solvent was 1 - 2 ml methyl-tert-butylether  (MTBE).  The organic  In preliminary  experi-  ments i t  was found that increasing MTBE volume beyond the recommended 1 ml  did not  influence  centrifuged  at  aqueous phases.  yield.  The t e s t  9950 x g f o r  2 min  tube in  was vortex-mixed  order  to  separate  for the  30 sec and organic  and  Using a 1 ml s y r i n g e , most of the upper organic phase was  removed (the exact amount was immaterial,  see below f o r  MTBE was then evaporated by blowing a j e t  of  the residue was reconstituted  N  2  in 75 % a c e t o n i t r i l e  explanation).  The  into the Dreyer tube, and in d i s t i l l e d water.  This  c o n s t i t u t e d the i n j e c t i o n 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. remainder of the plasma was removed, using a glass syringe  (Hamilton  The Rheo-  dyne), and centrifuged at 900 x g f o r 40 - 60 min through a re-usable Amicon  - 151 c e n t r i f l o w f i l t e r (type CF50A), in order to produce a sample of plasma water free to  of  o ^ - a c i d g l y c o p r o t e i n , with  (^-verapamil  be approximately 90% bound (Schomerus e t - a l . ,  1983). more  The Amicon f i l t e r than  distilled  97% of water  serum p r o t e i n . for  3 h,  0.1 M NaOH.  has been  reported  1976; M c A l l i s t e r  et-al;,  r e t a i n s more than 95% of molecules > 50 kD, and was prepared  by  followed by 10 min c e n t r i f u g a t i o n  at  order to remove excess water. in  which  The f i l t e r  The f i l t e r was cleansed a f t e r  The volume of  the  ultrafiltrate  soaking  in  900 x g in  use by soaking  was measured, and  this  sample of free plasma water was then processed in the same way as the plasma and whole blood samples. b.  V e n t r i c u l a r t i s s u e was processed according to a modification of the  technique of M c A l l i s t e r and Howell ventricular surgical  (1976).  t i s s u e were taken a f t e r  scissors,  the  Samples of normal and occluded  blotting  the t i s s u e dry.  t i s s u e was subdivided on the  l o c a t i o n of the 0Z in r a t s , by v i s u a l  inspection.  basis  Using small of  A transmural  the  known  sample of  the centre of the 0Z was taken, as well as the e n t i r e 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 f o l l o w s .  The t i s s u e was minced using small s u r g i c a l s c i s s o r s and t i s s u e f o r c e p s , and mixed in a glass homogenistation tube with 3 ml 0.1 M HCI and 16 nl 1 yg/pl to  gallopamil  encourage the  (internal  standard).  (^-verapamil  to  This a c i d i f i c a t i o n was c a r r i e d out  partition  out  of  the  tissue  aqueous melieu by promoting i o n i s a t i o n of t h i s weakly basic drug. was then homogenised using a pestle which f i t tube.  into  2 min and then centrifuged  decanted i n t o  a Kymax t e s t  0.1 M HCI and reprocessed.  at  tube,  t i g h t l y in the homogenisation  The f i n a l  The  tube, which was vortex-mixed  10,000 x g f o r and the  the  The t i s s u e  The tube was kept cool by constant immersion in a s l u r r y of i c e .  contents of the tube were washed i n t o a t e s t for  of  pellet  30 min.  The aquum was  was resuspended in 1 ml  extract was t i t r a t e d to pH 8.5 with  - 152 20 %Na C03  (approximately  2  p a r t i t i o n into MTBE.  400 p i )  in  order  to  encourage  subsequent  The cautious adjustment of pH was c a r r i e d out in order  to preclude the development of a soapy scum, which was associated with the use of  NaOH in preliminary experiments.  Subsequent processing steps were  i d e n t i c a l to those used for blood matrix samples. 2.8.5 HPLC Separation of (^O-verapami1 and gallopamil was c a r r i e d out by reversephase high pressure l i q u i d chromatography (HPLC, SP8000), using MOS Hypersil as the packing material and an Altex i n j e c t i o n device.  The running solvent  was a 50:50 mixture of d i s t i l l e d water and a c e t o n i t r i l e containing 500 y l of sodium octylsulphate  and 0.355 g of  aqueous component was adjusted mixing with a c e t o n i t r i l e . pump.  to  Na HP0 2  4.6  with  per  4  litre.  The  pH of  0.1 M phosphoric acid  the  before  Flow rate was set at 1 ml/min using a Micrometrics  Injection of samples into the column was c a r r i e d out using a 25 u l  Hamilton Rheodyne glass s y r i n g e . 2.8.6 C a l c u l a t i o n of verapamil concentration (±)-Verapamil and gallopamil were detected using a fluorimeter fel  FS970).  The optimum  excitation  wavelength  was found  in  (Schoef-  preliminary  experiments to be 203 nm, and an ordinary glass detection f i l t e r was used. A permanent record of recorder.  peaks was generated using a Rikadenki  B-107  chart  (^J-Verapami1 concentration was c a l c u l a t e d under the assumption  that the e x t r a c t i o n processes extracted (±)-verapamil and gallopamil e q u a l l y . This assumption was tested and found to be the case in preliminary e x p e r i ments.  The column was c a l i b r a t e d d a i l y by i n j e c t i n g 20 ng (±)-verapami 1 and  20 ng gallopamil simultaneously onto the column, and measuring the r a t i o of peak heights locate samples.  and the retention  (^-verapamil  times.  and gallopamil  The retention  on the  traces  times  were used  to  from blood and t i s s u e  The r a t i o of heights was used to c a l c u l a t e the concentration  of  - 153 (±)-verapamil in the samples, according to the f o l l o w i n g formula: Concentration (mol/1) = (Vh /Dh )(16/491080)(R/Y) s  Where Vh  g  and Dh  are the  $  $  peak heights  for  (±)-verapami 1 and g a l l o p -  amil from the blood matrix samples, r e s p e c t i v e l y , R i s the r a t i o of the peak heights of the ( ^ - v e r a p a m i l :gallopamil standards, Y i s the volume of blood matrix the weight molecular  of  in ml, and 16 and 491080 are constants to account gallopamil  weight  of  used to  spike each sample  (^J-verapami1  samples, Y refers to g weight,  (491.08).  and the units  In of  the  (16  u l ) and  case  of  (±)-verapamil  for the  tissue concen-  t r a t i o n are mol/kg. The above c a l c u l a t i o n was based on preliminary experiments which showed that the y i e l d s of ( ^ - v e r a p a m i l  and gallopamil  amounts of the drugs were s i m i l a r  from a sample spiked with known  ( r a t i o 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 r a t i o  of  peak heights,  over at l e a s t 3 orders of magnitude of drug concentration.  R, was  constant  - 154 3  RESULTS  3.1  Metabolism of -(*)-verapami1- in acute myocardial -ischaemia This study was c a r r i e d out in conjunction with an i n v e s t i g a t i o n into the  e f f e c t s of ( ^ - v e r a p a m i l rats.  It  had been demonstrated that (±)-verapamil  antiarrhythmic  of  possessed dose-dependent  e f f e c t s when administered before occlusion and when adminis-  tered a f t e r o c c l u s i o n . trations  on the responses to coronary occlusion in conscious  Therefore, i t was of i n t e r e s t to examine the concen-  (ij-verapamil  in the blood matrices a f t e r  coronary o c c l u s i o n ,  and also to examine whether the drug penetrated into the occluded zone.  The  l a t t e r consideration was expected to shed l i g h t on the question concerning the s i t e of antiarrhythmic action of the drug, since an absence of pamil  in  the occluded zone would suggest that  were produced v i a an action elsewhere.  the  (^-vera-  antiarrhythmic  effects  The r e s u l t s of t h i s study have been  published ( C u r t i s et a l ; , 1984). 3.1.1 Concentration of (^-verapami 1 in blood matrices The  concentration  of  (^O-verapamil  u l t r a f i l t r a t e (PU) i s shown in Table 2 . were s l i g h t l y  higher following  pre-occlusion  administration,  s i g n i f i c a n t only for the PU.  in It  whole blood,  plasma and plasma  can be seen that concentrations  post-occlusion administration although  the  compared with  differences  were  statistically  Since drug administration  took  approximately  10 min, whereas a l l rats were s a c r i f i c e d at 30 min a f t e r o c c l u s i o n , then the difference between pre- and post-occlusion administration  in terms of blood  matrix (^J-verapami 1 concentration r e f l e c t s the short d i s t r i b u t i o n and  the  rapid  hepatic  metabolism of  the  drug  The concentration of (^J-verapamil  same range  concentration  reported  to  life  (Schomerus et• - a l ; , 1976;  M c A l l i s t e r , 1982). as the  half  inhibit  in the PU was in the i^.  but  not  i^  in  v e n t r i c u l a r t i s s u e (Kohlhardt et a l . , 1972; Nawrath e t - a l ; , 1981). The extent of plasma-protein binding was 82 ± 4 % i n rats given  (*)-vera-  - 155 pamil a f t e r o c c l u s i o n , and 84 ± 4% in rats given the drug before o c c l u s i o n . These values were calculated by contrasting the plasma concentrations with the PU concentrations.  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 ( ^ - v e r a p a m i l The tissue  amounts (NZ),  shown in  of  the  table  (^-verapamil  occluded zone  3.  in the v e n t r i c u l a r myocardium.  in  the  (OZ)  normal  and the  Values from rats  in  ischaemic t i s s u e . (or  the portion  centre  which  before occlusion i n d i c a t e that (±)-verapamil  non-occluded  the  of  the  ventricular OZ  (0Z )are c  drug was administered  appeared to  accumulate in  the  The 0 Z , which was presumably the most ischaemic t i s s u e C  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 a f t e r occlusion was approximately 3 times the value found in rats administered the drug before occlusion (although the difference was not statistically significant).  In t h i s  the time of administration  in  regard, NZ values varied according to  a qualitatively  s i m i l a r manner to l e v e l s  in  the blood matrices. It  was of considerable i n t e r e s t  in the OZ (and 0Z ) of r a t s C  to f i n d that (^J-verapami1 was present  administered the drug a f t e r  occlusion.  How-  ever, u n l i k e the case in which (^)-verapami1 was given before o c c l u s i o n , the levels the NZ.  in the OZ and 0Z  were not  disproportionately  h i g h , compared with  Indeed, there were no s i g n i f i c a n t differences between the l e v e l s in  the NZ, OZ and 0 Z amount  C  of  c  following  (^-verapami 1 in  administration,  the amount  post-occlusion a d m i n i s t r a t i o n . the  in the  NZ was 0Z  c  greater  was l e s s ,  Although  the  following  post-occlusion  indicating  that  although  the drug did p a r t i t i o n into the ischaemic t i s s u e even when given a f t e r  liga-  t i o n , the a b i l i t y of the ischaemic t i s s u e to accumulate the drug was diminished.  This presumably r e f l e c t e d the f a c t  that whereas (^O-verapami 1 had  - 156 -  Table 2.  Concentration of (±)-verapamil in blood matrices  Administration  n  Whole Blood  Pre-occlusion  6  2.4 ± 0.8  uM  2.7 ± 1,.1 vM  0.24 ± 0.04  Post-occlusion  6  3.2 ± 0.5  uM  3.6 i 0 .8 uM  0.57 ± 0.10  Plasma  The dose of (± )-verapamil was 6 mg/kg. cates  p < 0.05 versus the value  (unpaired 2 - t a i l e d t  test).  in  Ultrafi Urate  Values are mean * s.e.mean.  y  M yM*  * Indi-  the p r e - o c c l u s i o n - a d m i n i s t r a t i o n  group  - 157 -  Table 3.  D i s t r i b u t i o n of ( ^ - v e r a p a m i l  in the v e n t r i c l e s  Administration  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 exactly in the same way as the data in Table 2 , with the exception that values are in umol/kg. nificant  differences  between  groups (unpaired 2 - t a i l e d t  the  test).  There were no s t a t i s t i c a l l y  pre- and  post-occlusion  sig-  administration  - 158 free access to the ischaemic t i s s u e when given before o c c l u s i o n , and indeed was probably  trapped  there  in  high  immediately a f t e r drug administration  concentrations  (occlusion  in these r a t s ) ,  it  took  place  only had access to  the ischaemic t i s s u e v i a the veins which drain over the OZ in the r a t  heart  (Taira et a l ; , 1985) when given after o c c l u s i o n . 3.2  Arrhythmogenesis-and the r o l e of the CNS 3.2.1 Overview In view of  the contradictory  evidence rega