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Effect of free fatty acids and dichloroacetic acid on the diabetic isolated working rat heart Nicholl, Tessa Anne 1990

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EFFECT OF FREE FATTY ACIDS AND DICHLOROACETIC ACID ON THE DIABETIC ISOLATED WORKING RAT HEART By TESSA ANNE NICHOLL B . S c , The University of Br i t ish Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Division of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1990 © Tessa Anne Nichol1, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada Department DE-6 (2/88) i i ABSTRACT: I t i s we l l e s t a b l i s h e d t ha t a cardiomyopathy independent o f a t h e r o s c l e r o s i s develops i n both humans and animals w i t h d iabe tes m e l l i t u s . The e t i o l o g y o f d i a b e t i c cardiomyopathy i s ve ry complex i n v o l v i n g many d i f f e r e n t p rocesses , one o f which may be the i nc reased f a t t y a c i d u t i l i z a t i o n , and/or the concomitant decrease i n g lucose u t i l i z a t i o n , by the d i a b e t i c h e a r t . We compared c o n t r o l and 6-week s t r e p t o z o t o c i n ( S T Z ) - i n d u c e d d i a b e t i c i s o l a t e d working r a t hear ts and were ab le to demonstrate c a r d i a c dys func t i on i n the d i a b e t i c as assessed by depressed hear t r a t e (HR), hear t r a t e peak s y s t o l i c p ressure product (HR X P S P ) , l e f t v e n t r i c u l a r developed pressure (LVDP), r a t e o f p ressure r i s e (+dP/dt) and r a t e o f p ressure d e c l i n e ( - d P / d t ) . P a r a l l e l i n g depressed c a r d i a c f u n c t i o n i n the d i a b e t i c were hyperg lycemia , h y p e r l i p i d e m i a and decreased body weight ga in as compared to age-matched c o n t r o l s . The a d d i t i o n o f f r ee f a t t y a c i d s , i n the form of 1.2 mM p a l m i t a t e , t o the i s o l a t e d working hear t pe r fusa te had no e f f e c t on e i t h e r c o n t r o l or d i a b e t i c hear t f u n c t i o n , w i th the excep t ion o f a dep ress i ve e f f e c t on +dP/dt o f d i a b e t i c hear ts and - d P / d t o f con t ro l h e a r t s . Bu t , d i a b e t i c hear ts per fused w i th p a l m i t a t e - c o n t a i n i n g pe r f usa te p lus the g lucose o x i d a t i o n s t i m u l a t o r d i c h l o r o a c e t a t e (DCA) showed a marked improvement i n f u n c t i o n . Heart r a t e , HR X PSP, LVDP and + / -dP /d t were a l l r es to red to c o n t r o l hear t va lues i n d i a b e t i c hear ts per fused i n the presence of DCA. C r e a t i n e phosphate and adenosine 5'-t r i p h o s p h a t e (ATP) l e v e l s were s i m i l a r under a l l p e r f u s i o n c o n d i t i o n s , t h e r e f o r e e l i m i n a t i n g energy s t o r e s as the l i m i t i n g f a c t o r i n hear t f u n c t i o n . R e s u l t s i n d i c a t e t ha t DCA-induced s t i m u l a t i o n o f g lucose o x i d a t i o n a c u t e l y reversed d i a b e t i c c a r d i a c f u n c t i o n d e p r e s s i o n . T h e r e f o r e , dep ress ion o f g lucose o x i d a t i o n i n the d i a b e t i c hear t may be c o n t r i b u t i n g to d i a b e t i c card iomyopathy. i v TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGEMENTS v DEDICATION v i LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS x i i INTRODUCTION 1 MAJOR OBJECTIVES OF THIS STUDY 16 MATERIALS AND METHODS 17 I. M a t e r i a l s 17 I I . Methods 19 Animals 19 Working Heart Apparatus 19 Heart Pe r fus i ons 23 E x t r a c t i o n o f ATP, C rea t i ne Phosphate, and Long Chain C a r n i t i n e from V e n t r i c u l a r T i ssue 25 P e r c h l o r i c Ac i d E x t r a c t i o n 25 ATP /C rea t i ne Phosphate Assay 26 Long Chain C a r n i t i n e Assay 27 E x t r a c t i o n o f T r i g l y c e r i d e s from V e n t r i c u l a r T i ssue 28 T r i g l y c e r i d e Assay 28 Serum Ana lyses 30 S t a t i s t i c a l A n a l y s i s 31 RESULTS 32 DISCUSSION 94 CONCLUSIONS 106 REFERENCES 107 V ACKNOWLEDGEMENTS: I would l ike to express my thanks to my supervisor, Dr. John H. McNeill , for his knowledgeable guidance, encouragement and patience which enabled me to complete this work. I wish to express my deepest gratitude to Dr. Gary D. Lopaschuk for valuable suggestions, continuous interest, support and encouragment throughout this study. The valuable suggestions and constructive cr i t ic isms of a l l the members of my supervisory committee are gratefully acknowledged. I am indebted to the Br i t ish Columbia Heart Foundation for the financial support they provided me throughout this study. v i DEDICATION: To Murray, f o r h i s l o v e , p a t i e n c e , moral support and encouragment whenever I needed i t most To my p a r e n t s , f o r always being there f o r me LIST OF TABLES Pressure produced by increased flow rate through a resistance-type afterload system LIST OF FIGURES The sequence of events thought to ultimately cause heart fa i lure and death in the diabetic population Regulation of the entry and use of glucose residues in glycolysis Mechanism by which dichloroacetate activates the pyruvate dehydrogenase complex Schemata of the working heart apparatus Serum glucose levels of 6 week diabetic rats and age-matched controls Serum free fatty acid levels of 6 week diabetic rats and age-matched controls Body weight change of diabetic and control rats six weeks following injection of STZ or i ts vehicle Effect of metabolic substrates on heart rate in control and diabetic isolated working hearts at a medium workload Effect of metabolic substrates on HR X PSP in control and diabetic isolated working rat hearts at a medium workload Effect of metabolic substrates on LVDP in control and diabetic working rat hearts at a medium workload i x 11. E f f e c t o f me tabo l i c subs t ra tes on maximal LVDP 52 i n c o n t r o l and d i a b e t i c i s o l a t e d working r a t hea r t s 12. E f f e c t o f me tabo l i c subs t ra tes on LVDP, 54 measured a t f i v e d i f f e r e n t a f t e r l o a d p r e s s u r e s , i n c o n t r o l and d i a b e t i c i s o l a t e d working r a t hea r t s 13. E f f e c t o f me tabo l i c subs t ra tes on +dP/dt , 56 measured a t f i v e d i f f e r e n t a f t e r l o a d p r e s s u r e s , i n c o n t r o l and d i a b e t i c i s o l a t e d working r a t hea r t s 14. E f f e c t o f me tabo l i c subs t ra tes on - d P / d t , 58 i n c o n t r o l and d i a b e t i c i s o l a t e d working r a t hea r t s 15. E f f e c t o f DCA on hear t r a te i n FFA per fused 60 c o n t r o l and d i a b e t i c i s o l a t e d working r a t hear ts a t a medium work load 16. E f f e c t o f DCA on HR X PSP i n FFA per fused 62 c o n t r o l and d i a b e t i c i s o l a t e d working r a t hear ts a t a medium work load 17. E f f e c t o f DCA on LVDP i n FFA per fused 64 c o n t r o l and d i a b e t i c i s o l a t e d working r a t hea r t s a t a medium workload 18. E f f e c t o f DCA on maximal LVDP i n FFA per fused 66 c o n t r o l and d i a b e t i c r a t hear ts 19. E f f e c t o f DCA on LVDP, measured a t f i v e 68 d i f f e r e n t a f t e r l o a d p r e s s u r e s , i n FFA per fused c o n t r o l and d i a b e t i c i s o l a t e d work ing r a t hear ts 20 . E f f e c t o f DCA on +dP/dt , measured a t f i v e 70 d i f f e r e n t a f t e r l o a d p r e s s u r e s , i n FFA per fused c o n t r o l and d i a b e t i c i s o l a t e d work ing r a t hear ts 2 1 . E f f e c t o f DCA on - d P / d t , measured at f i v e 72 d i f f e r e n t a f t e r l o a d p r e s s u r e s , i n FFA per fused c o n t r o l and d i a b e t i c i s o l a t e d work ing r a t hear ts 22 . E f f e c t o f me tabo l i c subs t ra tes on t i s s u e ATP 74 i n c o n t r o l and d i a b e t i c i s o l a t e d work ing hear ts a t a maximal workload 23 . E f f e c t o f me tabo l i c subs t ra tes on t i s s u e 76 c r e a t i n e phosphate i n con t ro l and d i a b e t i c i s o l a t e d work ing r a t hear ts a t maximal work load 24. E f f e c t o f me tabo l i c subs t ra tes on t i s s u e 78 LCC 's i n c o n t r o l and d i a b e t i c i s o l a t e d working hear t s a t a maximal workload 25 . E f f e c t o f me tabo l i c subs t ra tes on t i s s u e 80 t r i g l y c e r i d e s i n c o n t r o l and d i a b e t i c i s o l a t e d work ing hear t s at a maximal work load 26. E f f e c t o f DCA on t i s s u e ATP i n FFA per fused 82 c o n t r o l and d i a b e t i c i s o l a t e d working r a t hea r t s at a maximal workload 27. E f f e c t o f DCA on t i s s u e c r e a t i n e phosphate 84 i n FFA per fused con t ro l and d i a b e t i c i s o l a t e d work ing r a t hear ts at a maximal workload x i 28. E f f e c t o f DCA on t i s s u e LCC's i n FFA per fused 86 c o n t r o l and d i a b e t i c i s o l a t e d working r a t hear ts a t a maximal workload 29. E f f e c t o f DCA on t r i g l y c e r i d e s i n FFA per fused 88 c o n t r o l , and d i a b e t i c i s o l a t e d working r a t hear ts a t a maximal workload 30. P ressu re produced by inc reased f low r a t e 90 through a r e s i s t a n c e - t y p e a f t e r l o a d system x i i L i s t o f A b b r e v i a t i o n s : Ace t y l COA Ace ty l Coenzyme A ADP adenosine 5 ' -d iphospha te ATP adenosine 5 ' - t r i p h o s p h a t e C cen t i g rade CAT c a r n i t i n e a c e t y l t r a n s f e r a s e Ci C u r i e CoA Coenzyme A CP c r e a t i n e phosphate CPK c r e a t i n e phosphokinase CPT c a r n i t i n e pa lm i toy l t r a n s f e r a s e DCA d i c h l o r o a c e t i c a c i d DTT d i t h i o t h r e i t o l - d P / d t r a t e o f l e f t v e n t r i c u l a r p ressure d e c l i n e +dP/dt r a t o f l e f t v e n t r i c u l a r p ressure development EDTA e thy lene diamine t e t r a a c e t a t e et al and o thers Etomoxi r ( 2 - [ 6 - ( 4 - ch l o rophenoxy )hexy l ] - ox i r a n e - 2 - c a r b o x y l a t e g gram G-6-P g lucose-6-phosphate G-6-PDH g lucose-6-phosphate dehydrogenase GPO g l y c e r o l - 3 - p h o s p h a t e ox idase -p -ch lo ropheno l Hepes N - 2 -hyd roxye thy l p i pe raz i ne -N - 2 -e thanesu l f on i c a c i d HK hexokinase HR hear t r a t e IDDM insu l i n -dependen t d iabe tes n i e l l i t u s x i i i i . p . i n t r a p e r i t o n e a l i . v . in t ravenous Kg k i l og ram KH Krebs H e n s e l e i t LCC long c h a i n a c y l c a r n i t i n e LVDP l e f t v e n t r i c u l a r developed p ressure u l m i c r o l i t e r mg mi 11i gram ml m i l l i l i t e r mM m i l l i m o l a r min minute mw mo lecu la r weight N Normal N sample number NADP n i co t i nam ide adenine d i n u c l e o t i d e phosphate NIDDM non insu l in -dependen t d iabe tes m e l l i t u s nm nanometer nmol nanomoles PCA p e r c h l o r i c a c i d PDH pyruvate dehydrogenase complex POCA 2 - [ 5 - ( 4 - c h l o r o p h e n y l ) p e n t y l ] - o x i r a n e - 2 - c a r b o x y l a t e PSP peak s y s t o l i c p ressure / Per % percent rpm r e v o l u t i o n s per minute S . E . M . s tandard e r r o r o f the mean SR sa rcop lasm ic r e t i c u l u m x i v STZ s t r e p t o z o t o c i n TG t r i g l y c e r i d e U u n i t s vo l volume wt weight 1 INTRODUCTION: D iabetes n i e l l i t u s i s the name g iven to a group o f d i s o r d e r s c h a r a c t e r i z e d by f a s t i n g hyperg lycemia and a l t e r e d l i p i d metabo l ism, the u n d e r l y i n g cause o f which i s a l a c k o f i n s u l i n or a l a c k o f i n s u l i n a c t i v i t y . Th i s i s a d i s e a s e which has been recogn ized s i n c e a n t i q u i t y . An Egypt ian papyrus o f 1500 BC con ta ins a d e s c r i p t i o n o f the symptoms. In the second cen tu ry AD Aretaeus o f Cappadocia named the d i s e a s e d i a b e t e s , a Greek word meaning " to f l ow through a syphon" . Th i s g raph ic d e s c r i p t i o n imp l i ed t ha t the re was a was t ing o f the whole body i n a cop ious f l u x o f u r i n e . Hindu manuscr ip ts o f the s i x t h cen tu ry recogn ized t ha t the u r i ne was sweet. I t was not u n t i l the e igh teen th cen tu ry t ha t t h i s sweet substance was e s t a b l i s h e d to be g l u c o s e , and the word m e l l i t u s (honeyed) was added. D iabetes m e l l i t u s i s now, s t i l l , a c l i n i c a l d i s o r d e r o f major medical s i g n i f i c a n c e and has been repor ted to be the t h i r d l e a d i n g cause o f death i n the Un i ted S t a t e s , behind c a r d i o v a s c u l a r d i s e a s e and c a n c e r ; the i n c i d e n c e i n Canada i s p r o p o r t i o n a l . In the e a r l y 1980's there were es t imated to be 10 m i l l i o n Americans showing ove r t symptoms o f d iabe tes m e l l i t u s ( N o t k i n s , 1979). However, i t has been es t imated t ha t so many cases o f d iabe tes go unreported or undiagnosed t ha t as many as twenty m i l l i o n people i n the Un i ted S ta tes may be d i a b e t i c (Dav idson, 1981). T h i s number i s es t imated to double by the year 2000. 2 Diabetes m e l l i t u s can be subd iv ided i n t o two major c a t e g o r i e s : i n s u l i n - d e p e n d e n t (IDDM) and non insu l in -dependent (NIDDM). A l a c k o f panc rea t i c i n s u l i n c h a r a c t e r i z e s IDDM and r e s u l t s i n the dependence upon d a i l y i n s u l i n i n j e c t i o n s to c o n t r o l b lood g lucose l e v e l s , avo id k e t o t i c coma, and remain a l i v e . IDDM appears most commonly i n youth and the e t i o l o g y i s complex w i t h g e n e t i c de te rm inan ts , env i ronmenta l f a c t o r s and immune system a b n o r m a l i t i e s i n c l u d e d as p o s s i b l e f a c t o r s i n the genes is o f the d i s e a s e . Non insu l in -dependent d i a b e t i c s do not u s u a l l y r e q u i r e i n s u l i n t o ma in ta i n good con t ro l o f blood g lucose l e v e l s and they seldom k e t o t i c . U s u a l l y NIDDM appears i n obese adu l t s and they are f r e q u e n t l y h y p e r i n s u l i n e m i c and e x h i b i t c h a r a c t e r i s i t i c s o f i n s u l i n r e s i s t a n c e . Gene t i c and environmental f a c t o r s p lay a prominent r o l e i n the e t i o l o g y o f NIDDM. Non insu l in -dependent d iabe tes i s the most p r e v a l e n t type o f d i abe tes m e l l i t u s , w i th most o f the d i a b e t i c p a t i e n t s i n America be ing o f the NIDDM t ype . With the famous d i scove ry o f i n s u l i n i n 1921 by Ban t ing and B e s t , d i abe tes m e l l i t u s was thought to be " c u r e d " . A l though the average l i f e expectancy o f a d i a b e t i c p a t i e n t inc reased d r a m a t i c a l l y i n the pos t -i n s u l i n e r a , i t remains an inescapab le f a c t t ha t t h i s d i s e a s e i s a t best t r e a t a b l e but c e r t a i n l y not c u r a b l e . I n s u l i n a d m i n i s t r a t i o n has v i r t u a l l y e l i m i n a t e d coma as a cause o f death i n the d i a b e t i c p o p u l a t i o n , but secondary d i s o r d e r s remain p r e v a l e n t . Some o f the comp l i ca t i ons of d iabe tes i n c l u d e be ing t w e n t y - f i v e t imes more prone to b l i n d n e s s , due to r e t i n o p a t h y , than the normal 3 p o p u l a t i o n . In f a c t , r e t i nopa thy i s the l e a d i n g cause o f new cases o f b l i n d n e s s . Macroangiopathy, i n c l u d i n g coronary hear t d i s e a s e and p e r i p h e r a l v a s c u l a r d i s e a s e , occurs premature ly and a c c e l e r a t e s r a p i d l y , k i l l i n g s e v e n t y - f i v e percent o f n o n - i n s u l i n dependent d i a b e t i c s . As w e l l , n i n e t y - t h r e e percent o f d i a b e t i c s o f g rea te r than twenty years d u r a t i o n have abnormal basement membrane t h i c k e n i n g . D i a b e t i c s are a l s o seventeen t imes more prone to k idney d i sease w i t h approx imate ly f i f t y percent o f i n s u l i n dependent d i a b e t i c s dy ing o f c h r o n i c rena l f a i l u r e and twenty f i v e percent o f new rena l t r a n s p l a n t r e c i p i e n t s being d i a b e t i c . I t i s a l s o noted tha t f i f t y percent o f men w i t h long d u r a t i o n o f d i abe tes are impotent and d i a b e t i c s are f i v e t imes more prone to gangrene, i n par t due to pe r i phe ra l neuropathy (Podo l sky , 1981) . One o f the more s i g n i f i c a n t secondary d i s o r d e r s a s s o c i a t e d w i t h d iabe tes i s t ha t o f hear t d i s e a s e , d i a b e t i c s e x h i b i t tw i ce the i nc i dence o f hear t a t t a c k s and s t rokes as the normal p o p u l a t i o n . T h i s i nc reased r i s k i s present i n both i nsu l i n -dependen t and non insu l i n -dependen t d i a b e t i c s (S ieve rs et al, 1963). A number o f c l i n i c a l s t u d i e s have shown t ha t hear t d i sease accounts f o r approx imate ly e i g h t y percent o f deaths among d i a b e t i c s (Kannel et al, 1974; Kannel and McGee, 1979). M o r t a l i t y due to c a r d i o v a s c u l a r causes i n d i a b e t i c women i s f i v e t imes g rea te r than i n nond iabe t i cs (Kannel et al, 1974) . S i m i l a r i l y , i n the Rochester p r o j e c t , the observed cases o f coronary hear t d i s e a s e (angina p e c t o r i s , myocard ia l i n f a r c t i o n or sudden unexpected death) and o f conges t i ve hear t f a i l u r e wi thout antecedent hear t d i s e a s e i n p a t i e n t s w i t h NIDDM exceeded those expected from the general p o p u l a t i o n (Palumbo et al, 1981) . 4 The pathogenes is o f hear t f a i l u r e i n d iabe tes i s u n c e r t a i n . Four major f a c t o r s , namely a t h e r o s c l e r o s i s , m i c r o v a s c u l a r a l t e r a t i o n s , autonomic neuropathy and pr imary cardiomyopathy, may be i m p l i c a t e d i n v a r y i n g degrees , i n conce r t o r a c t i n g independent l y , i n c o n t r i b u t i n g t o c a r d i o v a s c u l a r d y s f u n c t i o n dur ing d iabe tes (F igu re 1 ) . The p o s s i b i l i t y t ha t impai red c a r d i a c performance du r i ng d iabe tes may be due to a l e s i o n i n the c a r d i a c muscle i t s e l f , independent o f a t h e r o s c l e r o t i c a r t e r y d i s e a s e , was r a i s e d and suppor ted by both c l i n i c a l and exper imenta l ev idence . P a t i e n t s i n the Framingham s tudy who had d i abe tes had an exaggerated r i s k o f c l i n i c a l conges t i ve hear t f a i l u r e deve lop ing compared w i th t h e i r nond iabe t i c coun te rpa r t s (Kannel and McGee, 1979) . Th i s excess r i s k p e r s i s t e d even a f t e r the i n f l u e n c e o f o ther a the rogen ic r i s k f a c t o r s and p r i o r coronary d i s e a s e s t a t u s was e l i m i n a t e d , sugges t ing t ha t f a c t o r s o ther than coronary a r t e r y d i s e a s e may be impor tan t . Exper imenta l ev idence showed tha t both d i a b e t i c dogs and r a t s a l s o had c h a r a c t e r i s t i c a b n o r m a l i t i e s i n the c o n t r a c t i l e f u n c t i o n o f the h e a r t . Regan and co-workers (1974) showed pr imary myocard ia l abnorma l i t y i n a l l o x a n - i n d u c e d d i a b e t i c dogs. F o l l o w i n g one year o f exper imenta l d i a b e t e s , dogs showed v e n t r i c u l a r s t i f f e n i n g a s s o c i a t e d w i t h s h o r t e n i n g o f l e f t - v e n t r i c u l a r e j e c t i o n t ime . As w e l l , i s o l a t e d work ing hear t s from s t r e p t o z o t o c i n - (STZ) and a l l o x a n - i n d u c e d d i a b e t i c r a t s showed a decreased a b i l i t y to respond t o i nc reased l e f t - a t r i a l f i l l i n g p ressures 5 Increased Mortality Rate Defective Cardiovascular System Increased Incidence of; Myocardial Infarcta Silent Infarcts Stroke Thrombosis Hypertension Primary Cardiomyopathy Autonomic Neuropathy Figure 1: The sequence of events thought to ul t i m a t e l y cause heart f a i l u r e and death i n the dia b e t i c population (Pierce et a l , 1989). 6 (Penparagkul et al, 1980; Vadlamudi et al, 1980). I nd i ces o f c a r d i a c f u n c t i o n such as l e f t - v e n t r i c u l a r developed p r e s s u r e , r a t es o f c o n t r a c t i o n and r e l a x a t i o n , c a r d i a c output and a o r t i c output were a l l dep ressed . Ca rd i ac f u n c t i o n depress ion was a l s o shown i n d i a b e t i c r a t hea r t s i n the presence o f i n c r e a s i n g a f t e r l o a d p r e s s u r e s , r e s u l t i n g i n decreased peak l e f t - v e n t r i c u l a r p ressure and r a t e o f c o n t r a c t i o n ( Ingebre tsen et a7 , 1980). F i n a l l y , i s o l a t e d p a p i l l a r y muscles from STZ- induced d i a b e t i c r a t s had a depressed v e l o c i t y o f sho r ten ing and de layed onset o f r e l a x a t i o n (Fe in et al, 1980). The e t i o l o g y o f t h i s myocard ia l abnorma l i t y a s s o c i a t e d w i th d i abe tes i s u n c e r t a i n . One exp lana t i on comes from the obse rva t i on tha t hea r t s from d i a b e t i c dogs appear to accumulate c o l l a g e n which may r e s u l t i n v e n t r i c u l a r s t i f f n e s s and thus decreased compl iance (Regan et al, 1981). However, i n c r e a s i n g data suggest t ha t the d i a b e t i c cardiomyopathy may be due, i n p a r t , t o a combinat ion o f changes i n myocard ia l enzyme systems and changes i n s u b c e l l u l a r o r g a n e l l e s such sarcolemma, m i t o c h o n d r i a , sa rcop lasmic r e t i c u l u m (SR) , as we l l as c o n t r a c t i l e p r o t e i n s ( D h a l l a et al, 1985). Myosin and actomyosin ATPase a c t i v i t i e s have been s t u d i e d du r ing d i abe tes and have been found to d i s p l a y a d i abe tes - i nduced depress ion i n r a t hea r t s ( D i l l m a n , 1980; Malhot ra et a / , 1981; Garber et al, 1983). S i m i l a r i l y , m y o f i b r i l l a r ATPase from d i a b e t i c r a t hear ts d i s p l a y e d depressed a c t i v i t y ( P i e r c e and D h a l l a , 1981). Th i s depress ion was suggested to be the r e s u l t o f a change i n myosin isoenzyme d i s t r i b u t i o n from the most a c t i v e v j form, found to predominate i n c o n t r o l r a t s , t o ma in l y the l e s s a c t i v e V3 form i n d i a b e t i c r a t s ( D i l l m a n , 1980). Hence, 7 i t can be suggested tha t d iabe tes - i nduced dep ress ion o f myosin ATPase i n the hear t may lead to a l t e r e d c o n t r a c t i l i t y . Depress ion o f ca l c i um uptake i n t o the SR has a l s o been shown to occur i n d i a b e t i c hear ts (Lopaschuk et a7 , 1983a, 1983b). Th i s same group proposed t ha t a p o s s i b l e mechanism f o r t h i s dep ress ion cou ld be an accumula t ion o f a m p h i p h i l i c i n te rmed ia tes o f f a t t y a c i d metabol ism i n t e r f e r i n g w i t h SR membrane f u n c t i o n (Lopaschuk et al, 1 9 8 3 1 , 2 ) . De fec ts i n the sarcolemmal enzymat ic a c t i v i t i e s and compos i t ion have a l s o been observed i n the d i a b e t i c r a t myocardium. A l t e r e d p h o s p h o l i p i d compos i t ion o f the sarcolemma has been found and cou ld p o t e n t i a l l y a f f e c t i t s f u n c t i o n ( P i e r c e et al, 1983). As w e l l , a de fec t a t the l e v e l o f the basement membrane ( g l y c o c a l y x ) has been found i n the form o f a l t e r e d s i a l i c a c i d syn thes i s i n sarcolemma o f d i a b e t i c hear ts ( P i e r c e et al, 1983). The N a + - K + - A T P a s e enzyme r e s p o n s i b l e f o r m a i n t a i n i n g the r e s t i n g membrane p o t e n t i a l o f the sarcolemma has a l s o been shown to be depressed ( P i e r c e and D h a l l a , 1981) . Depress ion o f t h i s i on pump w i l l o b v i o u s l y a l t e r the r e s t i n g membrane p o t e n t i a l and subsequent c a r d i a c c o n t r a c t i o n . S i m i l a r i l y , adeny la te c y c l a s e a c t i v i t y has been repor ted to be depressed i n the d i a b e t i c h e a r t , and t h e r e f o r e may be p a r t l y r e s p o n s i b l e f o r the reduced s e n s i t i v i t y t o ca techo lamines seen i n these hear ts (Smith et al, 1984; Miche l et al, 1985). A long w i t h the repor ted decrease i n a lpha and beta ad rene rg i c recep to r numbers i n d i a b e t i c hear ts (Hey l i ge r et al, 1982; L a t i f p o u r and M c N e i l l , 1984), a dep ress ion i n adeny la te c y c l a s e may c o n t r i b u t e to the l o s s o f ad rene rg i c respons iveness o f the d i a b e t i c myocardium and, hence, the normal f u n c t i o n i n g of h e a r t . F i n a l l y , both the N a + - C a 2 + exchanger and 8 Ca -pump o f the sarcolemma have been repor ted to be d e f e c t i v e i n the d i a b e t i c myocardium (Makino et a7 , 1987) and may, t h e r e f o r e a l s o c o n t r i b u t e t o a l t e r e d c o n t r a c t i l i t y o f the d i a b e t i c h e a r t . M i t ochond r i a l a c t i v i t y i s a l s o a f f e c t e d by the d i a b e t i c s t a t e , w i t h c a l c i u m t r a n s p o r t dep ress ion and an a l t e r a t i o n i n r e s p i r a t o r y a c t i v i t y repo r ted to occur i n the m i tochondr ia o f d i a b e t i c hear ts (Haugaard and Haugaard, 1964; P i e r c e and D h a l l a , 1983). A depress ion i n m i tochond r i a l a c t i v i t y may e v e n t u a l l y l ead to a r e d u c t i o n i n ATP l e v e l s , as repor ted p r e v i o u s l y , (P iepe r et a7 , 1984; P iepe r and Murray, 1987), which may r e s u l t i n an impairment o f normal c e l l u l a r f u n c t i o n and hence, the development o f d i a b e t i c card iomyopathy. What i s apparent from the above d i s c u s s i o n i s t ha t many de t r imen ta l and c h r o n i c changes occur i n d i a b e t i c hear t t i s s u e which u l t i m a t e l y r e s u l t i n depressed c a r d i a c f u n c t i o n . The exac t mechanism o f these changes i s not known but are suggested to be due to me tabo l i c a l t e r a t i o n s a t the l e v e l o f the hear t c e l l . I t i s known t ha t a prominent me tabo l i c a l t e r a t i o n o f d iabe tes i s an i nc reased c o n c e n t r a t i o n o f c i r c u l a t i n g f a t t y a c i d s and concomitant i nc reased r e l i a n c e o f the hear t on f a t t y ac i ds as an energy s u b s t r a t e (Morgan et a7 , 1961; Denton et a7 , 1967) . I n s u l i n l a c k may be the major cause o f l i p i d d i s o r d e r s o f d iabe tes because i t p lays a r o l e i n both p roduc t ion and removal o f t r i g l y c e r i d e -r i c h l i p o p r o t e i n . Adipose l i p o p r o t e i n l i p a s e , r e s p o n s i b l e f o r the removal o f the t r i g l y c e r i d e - r i c h l i p o p r o t e i n s , has been demonstrated to have decreased a c t i v i t y a f t e r i n s u l i n wi thdrawl i n i nsu l i n -dependen t 9 d i a b e t i c an imals and man and to have low a c t i v i t y i n poo r l y c o n t r o l l e d d i a b e t i c sub jec t s ( H a v e l , 1976). An e l e v a t i o n o f serum l i p o p r o t e i n s was a s s o c i a t e d w i t h the decreased l i p o p r o t e i n l i p a s e a c t i v i t y . I n s u l i n d e f i c i e n c y a l s o r e s u l t s i n a marked i nc rease i n ad ipose t i s s u e l i p o l y s i s , accompanied by enhanced r e l e a s e o f f r e e f a t t y a c i d s ( F F A ' s ) (McGarry and F o s t e r , 1977) . Th i s occurs du r ing d iabe tes because t r i g l y c e r i d e l i p a s e , the r a t e - d e t e r m i n i n g enzyme i n ad ipose t i s s u e h y d r o l y s i s , i s no rma l l y i n h i b i t e d by the presence o f i n s u l i n . The c l i n i c a l s i g n i f i c a n c e o f an i n c r e a s e i n c i r c u l a t i n g serum FFA l e v e l s i s t ha t these l e v e l s can i nc rease the s e v e r i t y o f a myocard ia l i n f a r c t i o n ( D e L e i r i s and Op ie , 1978; L i e d t k e et al, 1978; V ik-Mo and Mjos , 1981) and promote and a c c e l e r a t e l i f e - t h r e a t e n i n g ar rhy thmias (Corr et al, 1984). Exper imenta l ev idence suggests t ha t n o r m a l i z a t i o n o f v a r i o u s aspec ts o f f a t t y a c i d metabol ism i s a s s o c i a t e d w i th b e n e f i c i a l e f f e c t s on the mechanical f u n c t i o n o f the d i a b e t i c h e a r t . D i a b e t i c r a t s t r e a t e d w i th 1 i p i d - a l t e r i n g agents such as c a r n i t i n e (Rodr igues et al, 1988), methyl pa lmox i ra te ( T a h i l i a n i and M c N e i l l , 1985), myo inos i t o l (X iang et al, 1988), and meth ion ine and c h o l i n e (Hey l i ge r et a7 , 1986) d i s p l a y e d an improvement i n c a r d i a c performance a s s o c i a t e d w i th a decrease i n v a r i o u s l i p i d l e v e l s . Treatment w i t h h y d r a l a z i n e (Rodr igues et a7 , 1986) a l s o r e s u l t e d i n a b e n e f i c i a l e f f e c t on the d i a b e t i c r a t h e a r t , aga in accompanied by n o r m a l i z a t i o n o f the serum l i p i d p r o f i l e s . These data s t r o n g l y i m p l i c a t e a l t e r e d l i p i d metabol ism as a c o n t r i b u t o r to d i a b e t i c card iomyopathy. 10 However, the exac t mechanism o f these e f f e c t s and the degree to which they occur i s not we l l unders tood. P o s s i b l y , the de t r imen ta l e f f e c t s o f f a t t y a c i d s are due to an inc reased requirement f o r oxygen f o r c a t a b o l i s m , or t o an accumulat ion o f p o t e n t i a l l y t o x i c i n t r a c e l l u l a r i n t e r m e d i a t e s . Long cha in acy l CoA and long c h a i n a c y l c a r n i t i n e have been shown to accumulate i n d i a b e t i c and i schemic myocardium ( I d e l l -Wenger et a7 , 1978; Lopaschuk et a7 , 1983a, 1983b) and, as mentioned p r e v i o u s l y , have subsequent ly been shown t o a l t e r the f u n c t i o n o f a number o f c r i t i c a l membrane p r o t e i n s (Lopaschuk et a7 , 1983b; P i t t s and Okhuysen, 1984; Shug et a7 , 1975; Adams et a7 , 1979) . In a d d i t i o n to the above d i scussed de t r imen ta l e f f e c t s o f F F A ' s , F F A ' s may have an i n h i b i t o r y e f f e c t on g lucose use . Under normal p h y s i o l o g i c a l c o n d i t i o n s the hear t u t i l i z e s about 70% f a t t y a c i d s and about 30% g lucose f o r energy p roduc t ion (Neely and Morgan, 1974). In the d i a b e t i c s t a t e g lucose u t i l i z a t i o n i s e s s e n t i a l l y abo l i shed and f a t t y a c i d s can account f o r up to 99% o f the o x i d a t i v e fue l o f c a r d i a c muscle (Rand le , 1986). R e c e n t l y , i t was shown tha t g lucose o x i d a t i o n r a t e s i n i s o l a t e d work ing hear ts from d i a b e t i c r a t s were markedly depressed i n the presence o f p h y s i o l o g i c a l l e v e l s o f f a t t y ac i ds (Wall and Lopaschuk, 1989). An i n s u l i n l a c k r e s u l t i n g i n a depress ion o f g lucose uptake i s a c o n t r i b u t o r t o the marked decrease i n g lucose u t i l i z a t i o n , but another major f a c t o r i s the i n c r e a s e i n c i r c u l a t i n g F F A ' s (Randle et a7 , 1964, 1984). Accord ing to the concept o f the g l u c o s e - f a t t y a c i d c y c l e , e l eva ted serum and t i s s u e f a t t y a c i d l e v e l s w i l l decrease g lucose o x i d a t i o n . F i r s t l y , i nc reased c i t r a t e concen t ra t i ons w i l l i n h i b i t 11 phospho f ruc tok inase 1, w i th g l y c o g e n o l y s i s and g lucose uptake being subsequent l y decreased due to an accumulat ion o f g lucose-6 -phospha te (Newsholme et al, 1962; Kerbey et al, 1985). As w e l l , g lucose o x i d a t i o n i s decreased i n d iabe tes due to an i n h i b i t i o n o f pyruvate dehydrogenase (PDH), the f i r s t i r r e v e r s i b l e r e a c t i o n i n the m i tochond r i a l o x i d a t i v e pathway (Gar land et al, 1962) . F a t t y ac ids i n h i b i t the PDH complex by i n c r e a s i n g the p r o d u c t s / s u b s t r a t e s r a t i o (ace t y l CoA/CoA) o f the r e a c t i o n . Th i s r e s u l t s i n a s t i m u l a t i o n o f PDH k inase which phosphory la tes PDH and decreases the p ropo r t i on o f the complex i n the the a c t i v e dephosphory la ted form (Randle et a7 , 1984) . F igu re 2 shows the d i s c u s s e d mechanisms by which f a t t y ac ids i n h i b i t g lucose use . In d i a b e t e s , the PDH complex i s i n h i b i t e d even f u r t h e r by enhanced i n t r i n s i c PDH k inase a c t i v i t y and inc reased a c t i v i t y o f an as ye t u n i d e n t i f i e d " k i n a s e / a c t i v a t o r " f a c t o r which i s e x t r i n s i c t o the complex and produces a more s t a b l e a c t i v a t i o n o f PDH k inase (Kerbey et al, 1985; Randle et al, 1984). Even though the re i s an inc reased r e l i a n c e o f the d i a b e t i c hear t on f a t t y a c i d s , i t has been shown tha t o v e r a l l r a t es o f exogenous f a t t y a c i d o x i d a t i o n are not changed (Lopaschuk and Tsang, 1987; K r e i s b e r g , 1966; Chen et al, 1983). The dep ress ion o f g lucose o x i d a t i o n , i n d i a b e t e s , by f a t t y a c i d metabol ism has l e d many l a b o r a t o r i e s to t r y t o i n c r e a s e g lucose u t i l i z a t i o n i n the d i a b e t i c h e a r t . Th i s has i n i t i a t e d the development o f a number o f hypoglycemic drugs which b lock c a r n i t i n e pa lm i toy l t r a n s f e r a s e 1 (CPT 1 ) , the enzyme which i s r e s p o n s i b l e f o r c a t a l y z i n g the key r e g u l a t o r y s tep i n f a t t y a c i d o x i d a t i o n . Th i s CPT1 c a t a l y z e d . phosphorylase Glucose h c x o k i a a s c G i P Glycogen phaspharylase b ATP 12 V G 6 P w 1 F6P p h o s p h o f r u c t o k m a $ « I n-FDP ! TP ATP' P h o s p h o r y l a s e a Phosphory lase & Fatty acids Figure 2 : A M P citrate ATP \ NAD- - ADP NADH * 3PG I 2 P G I PEP p y r u v a t e ATP — ' ^ f ^ A D P Q trie Citrate y C O . + H.O Regulation of the entry and use of glucose residues i n g l y c o l y s i s . Regulatory i n h i b i t i o n i s shown by dashed feedback arrows leading to bars across the reaction arrows. Regulatory stimulation i s shown by a wide arrow p a r a l l e l to the reaction arrow (adapted from Lehninger, A.L. In: P r i n c i p l e s of Biochemistry, 1982). 13 r e a c t i o n produces long cha in a c y l c a r n i t i n e from c y t o s o l i c c a r n i t i n e and long cha in acy l CoA (Cook, 1987; Stephens et a7 , 1985; T u t w i l e r et a7 , 1978; Wolf et al, 1982). S tud ies us ing a CPT1 i n h i b i t o r (POCA) i n i s o l a t e d per fused STZ d i a b e t i c r a t s demonstrated an i n h i b i t i o n o f l i p o l y s i s and a r e s t o r a t i o n o f i n s u l i n s e n s i t i v i t y w i t h respec t t o g lucose o x i d a t i o n (Rosen and Re inauer , 1984) . With the use o f another CPT 1 i n h i b i t o r , the s y l f o n y l u r e a t o l bu tam ide , Tan et al (1984) a l s o demonstrated i nc reased g lucose o x i d a t i o n r a t e s i n i s o l a t e d work ing hear ts from c h r o n i c a l l y STZ-d i a b e t i c r a t s . However, these s t u d i e s were c a r r i e d out i n the absence o f exogenous f a t t y a c i d s , a procedure which w i l l i n i t s e l f s t i m u l a t e g lucose o x i d a t i o n . Th i s i s e s p e c i a l l y r e l e v a n t i n the c h r o n i c d i a b e t i c s t a t e which i s c h a r a c t e r i z e d by both e leva ted serum g lucose and FFA l e v e l s (Denton and Rand le , 1967). R e c e n t l y , Wal l and Lopaschuk (1989) , us ing the CPT 1 i n h i b i t o r E tomox i r , demonstrated a s i g n i f i c a n t i n c r e a s e i n g lucose o x i d a t i o n r a t e s i n both the presence and absence o f p a l m i t a t e . They a l s o found Etomoxir t o s i g n i f i c a n t l y i n c r e a s e hear t f u n c t i o n i n pa lm i t a te -pe r f used hear ts from both c o n t r o l and d i a b e t i c r a t s . S tud ies have a l s o demonstrated t h a t Etomoxir can i n c r e a s e g lucose u t i l i z a t i o n i n t i s s u e s o ther than the hear t (Wolf and E n g e l , 1985). Etomoxir was a l s o demonstrated to p ro tec t the d i a b e t i c hear t from the adverse e f f e c t s o f i schemia independent o f a lower ing o f long cha in acy l c a r n i t i n e or long cha in acy l CoA l e v e l s (Lopaschuk and S p a f f o r d , 1989). These da ta suggest t ha t accumulat ion o f f a t t y a c i d o x i d a t i o n 14 i n t e rmed ia tes i n the myocardium cannot account f o r the de t r imen ta l e f f e c t s o f f a t t y ac i ds and d iabe tes i n i schemic hea r t s nor the b e n e f i c i a l e f f e c t o f Etomoxi r . The re fo re , i t can be argued t ha t impa i red g lucose use i n d i a b e t i c r a t hear ts and i n f a t t y a c i d per fused hea r t s c o n t r i b u t e s t o i nc reased s u s c e p t i b i l i t y t o i schemic i n j u r y . D i c h l o r o a c e t a t e (DCA) i s another pharmaco log ica l agent which i s e f f i c a c i o u s i n i n c r e a s i n g myocard ia l g lucose o x i d a t i o n . In 1962 i t was repo r ted t h a t DCA lowered blood and u r i n e g lucose l e v e l s and i nc reased the r e s p i r a t o r y quo t i en t i n d i a b e t i c r a t s ( L o r i n i and Ciman, 1962) . In t h a t same year V a i l a t i and Rabass in i admin is te red DCA to d i a b e t i c p a t i e n t s and found decreases i n b lood g lucose l e v e l s . Because o f t h i s d i s c o v e r e d p o t e n t i a l as a hypoglycemic agent , DCA has s i n c e become the s u b j e c t o f many i n v e s t i g a t i o n s . I t i s now known tha t DCA i s an i n h i b i t o r o f the p r o t e i n k i nase i n v o l v e d i n the r e g u l a t i o n o f the PDH complex. In 1973 Whitehouse and Randle f i r s t demonstrated tha t DCA a c t i v a t e s PDH i n r a t h e a r t s . The f o l l o w i n g year they found t h a t DCA i n h i b i t s the ATP-dependent phospho ry l a t i on and i n a c t i v a t i o n o f p u r i f i e d p i g hear t PDH k i nase (Whitehouse and Rand le , 1974). F igu re 3 shows the mechanism by which DCA a c t i v a t e s the PDH complex. M c A l l i s t e r et al (1973) demonstrated t h a t a d d i t i o n o f m i l l i m o l a r c o n c e n t r a t i o n s o f DCA to the pe r fusa te can i n c r e a s e g lucose o x i d a t i o n r a t e s i n normal and d i a b e t i c r a t hear ts per fused w i t h g lucose and i n s u l i n . Th i s was a l s o shown to occur i n hear ts from normal or s ta rved r a t s per fused i n the presence o f added a c e t a t e , ketone bod ies , or 15 Pyruvate Dichloroacetate Kinase Pyruvate Dehydrogenase a form (active) Pyruvate Dehydrogenase b form (inactive) Phosphatase Figure 3 Mechanism by which dichloroacetate a c t i v a t e s the pyruvate dehydrogenase complex (Crabb et a l , 1981). 16 pa lm i t a te (H igg ins et al, 1978). D i r e c t a d d i t i o n o f t h i s agent t o i s o l a t e d work ing hear t s from d i a b e t i c r a t s a l s o markedly s t i m u l a t e s g lucose o x i d a t i o n ; DCA was shown to i nc rease g lucose o x i d a t i o n by 5000% i n d i a b e t i c r a t hea r t s per fused i n the presence o f F F A ' s (Lopaschuk and McVeigh, personal communicat ion) . Thus , i t i s apparent t ha t DCA would be a use fu l t o o l i n i n v e s t i g a t i n g whether a depress ion o f g lucose o x i d a t i o n i s a c o n t r i b u t o r t o d i a b e t i c cardiomyopathy by examining any acute e f f e c t s t h i s agent may have on i s o l a t e d work ing d i a b e t i c hear t f u n c t i o n . The purpose o f t h i s t h e s i s was to determine i f f a t t y ac i ds and /or an i n h i b i t i o n o f g lucose o x i d a t i o n have any de t r imen ta l e f f e c t s on d i a b e t i c hear t f u n c t i o n . The f o l l o w i n g are the major o b j e c t i v e s o f t h i s s tudy : 1. To attempt to more c l o s e l y s imu la te in vivo c o n d i t i o n s o f energy s u b s t r a t e a v a i l a b i l i t y i n the i s o l a t e d work ing d i a b e t i c r a t h e a r t . 2 . To i n v e s t i g a t e the e f f e c t s o f f r e e f a t t y a c i d s on d i a b e t i c r a t hear t f u n c t i o n . 3 . To i n v e s t i g a t e the e f f e c t s o f g lucose o x i d a t i o n s t i m u l a t i o n , through the use o f d i c h l o r o a c e t a t e , on a f r e e f a t t y a c i d / g l u c o s e per fused d i a b e t i c r a t h e a r t . 17 MATERIALS AND METHODS MATERIALS: Below i s a l i s t o f the chem ica l s , assay k i t s , r a d i o i s o t o p e s , and enzymes used i n t h i s s tudy and the companies from which they were s u p p l i e d : A v e r s t . M o n t r e a l . Canada ha lo thane BDH. Vancouver The f o l l o w i n g chemica ls were reagent grade: sodium c h l o r i d e ( N a C l ) , potass ium c h l o r i d e (KC1) , potassium phosphate (KH2P04), magnesium su lpha te (MgS0/i.-7H20), ca l c ium c h l o r i d e (CaCl2-2H20) , potass ium hydrox ide (KOH), sodium hydrox ide (NaOH), p e r c h l o r i c a c i d (PCA) , D-g l u c o s e , sodium hydrogen carbonate (NaHC03), Hepes, d i e t h y l e t h e r , c h l o r o f o r m , methano l , Spect rapor membrane t u b i n g , Whatman GF/C f i l t e r s Boehr inger Mannheim. Montreal g lucose assay k i t , ace t y l coenzyme A, n i co t i namide adenine d i n u c l e o t i d e phosphate (NADP), hexokinase (HK), c r e a t i n e phosphokinase (CPK) , g lucose-6 -phospha te dehydrogenase (G-6-PDH), adenosine 5 ' - d i p h o s p h a t e (ADP), e t h y l e n e d i a m i n e - t e t r a a c e t i c a c i d (EDTA) Canada Packers Chemica l . O n t a r i o . Canada Euthanyl - sodium pen toba rb i t a l 18 New England Nuc lear A c e t y l Coenzyme A , Aquasol l i q u i d s c i n t i l l a n t Sigma Chemical C o . . S t . L o u i s . MO s t r e p t o z o t o c i n (STZ) , d i c h l o r o a c e t i c a c i d (DCA), p a l m i t a t e - f r e e a c i d , bov ine serum albumin (BSA) , sodium carbonate (Na2C03), c a r n i t i n e a c e t y l t r a n s f e r a s e (CAT) , sodium t e t r a t h i o n a t e , Dowex 1-xlO an ion exchange r e s i n (7 x 12 mm) Wako Pure Chemical I n d u s t r i e s . Osaka. Japan serum FFA assay k i t , t r i g l y c e r i d e assay k i t 19 METHODS;  A n i m a l s : Male Wis ta r r a t s , weighing between 225-275 grams, were t r a n s i e n t l y a n e s t h e t i z e d w i th d i e t h y l e ther or ha lothane to a l l o w i n j e c t i o n o f e i t h e r s t r e p t o z o t o c i n (STZ) or i t s v e h i c l e i n t o the t a i l v e i n . D iabetes was induced by a s i n g l e t a i l v e i n i n j e c t i o n o f 60 mg/kg STZ d i s s o l v e d i n 0.9% s a l i n e . STZ was f r e s h l y prepared before i n j e c t i o n and was kept on i c e . Con t ro l animals were i n j e c t e d w i th app rop r i a t e volumes o f s a l i n e a l o n e . Animals were prov ided food and water ad libitum f o r 6 weeks. P roduc t i on o f the d i a b e t i c s t a t e was diagnosed 48 hours a f t e r i n j e c t i o n by a p o s i t i v e t e s t f o r g l y c o s u r i a w i th L i l l y T e s - t a p e . B lood samples were c o l l e c t e d a t the t ime of k i l l i n g , a l lowed to c l o t , c e n t r i f u g e d and the serum was ana lyzed f o r serum g lucose and t r i g l y c e r i d e s (TG). STZ an imals hav ing serum g lucose above 16 mmoles/L were used i n t h i s s t udy . Working Heart Appara tus : The work ing hear t pe r fus ion apparatus used i n t h i s s tudy was a r e c i r c u l a t i n g t y p e , a mod i f ied v e r s i o n o f N e e l y ' s o r i g i n a l work ing hear t apparatus (Neely et al, 1967). The appara tus , as shown i n F i gu re 4 , c o n s i s t e d o f a wa te r - j acke ted pr imary r e s e r v o i r connected to a 12 gauge s t e e l a o r t i c c a n n u l a . The r e s e r v o i r was 60 cms above the a o r t i c cannu la and con ta ined Krebs H e n s e l e i t (KH) bu f fe r aera ted w i th 95% 02/5% C02- A second j a c k e t e d r e s e r v o i r / o x y g e n a t o r was connected to a 20 gauge s t e e l a t r i a l cannu la at a he igh t o f 15 cms above the c a n n u l a . The bu f f e r l e v e l was main ta ined at a constant l e v e l by means of an ' o v e r - f l o w ' tube connected to the t h i r d j acke ted r e s e r v o i r which was s i t u a t e d underneath 20 the h e a r t . The t h i r d r e s e r v o i r a l s o served to ma in ta in the hear t a t a cons tan t temperature as i t sea led around the cork i n which the cannulas were l o c a t e d , thereby c o n t a i n i n g the hear t i n an enc losed chamber. From the t h i r d r e s e r v o i r the pe r fusa te passed through a s c i n t e r e d - g l a s s f i l t e r and was then r e c i r c u l a t e d to the secondary o x y g e n a t o r / r e s e r v o i r by means o f a M a s t e r f l e x R pump. The a o r t i c cannu la was a l s o connected to a wa te r - j acke ted compl iance chamber wh ich , i n t u r n , was connected to a l eng th o f t ub ing p o s i t i o n e d at va r i ous he igh ts above the a o r t a , i n o rder t o p rov ide an ex te rna l pe r i phe ra l r e s i s t a n c e , or h y d r o s t a t i c a f t e r l o a d . Th i s l eng th o f tub ing was connected to the t h i r d r e s e r v o i r where the pe r fusa te was, as a forement ioned, r e c i r c u l a t e d . The r e c i r c u l a t i n g pe r fusa te c o n s i s t e d o f KH bu f fe r i n the presence o f 3% bovine serum albumin (BSA) , w i th p a l m i t a t e , when used, be ing pre-bound to the a lbumin . The pe r fusa te was mainta ined a t a cons tan t temperature o f 37°C by means o f a Hakke R cons tan t - tempera ture water r e c i r c u l a t i n g pump which prov ided the water f o r the j acke ted r e s e r v o i r s and compl iance chamber. The KH s o l u t i o n conta ined the f o l l o w i n g i n m i l l i m o l a r (mM) q u a n t i t i e s : NaCl 118 .0 , KC1 4 . 7 , K H 2 P 0 4 1 .2, MgS0 4 -7H 2 0 1.2, C a C l 2 - 2 H 2 0 3 . 0 , g lucose 1 1 . 0 , NaHC03 2 5 . 0 , and EDTA 5 . 0 . When aera ted w i th 95% C 0 2 / 5 % 0 2 a t 37°C, the bu f f e r had a pH o f 7 . 4 . 21 F igu re 4 : Schemata o f The Working Heart Apparatus The work ing hear t apparatus c o n s i s t e d o f t h ree j a c k e t e d r e s e r v o i r s ( 1 , 2 , 3 ) . The pr imary r e s e r v o i r (1) was connected to a 12 gauge s t e e l a o r t i c cannu la (4) a t a he igh t o f 60 cms above the c a n n u l a . A second j a c k e t e d r e s e r v o i r (2) was connected to a 12 gauge s t e e l a t r i a l cannu la (5) a t a he igh t o f 15 cms above the cannu la . A t h i r d j a c k e t e d r e s e r v o i r (3) was s i t u a t e d below, and sea led around, the hear t ( 6 ) . An ' o v e r -f l o w ' tube (7) ma in ta ined the pe r fusa te a t a cons tan t l e v e l and was fed i n t o the t h i r d r e s e r v o i r . The a o r t i c cannula was a l s o connected t o a w a t e r - j a c k e t e d compl iance chamber (8) wh ich , i n t u r n , was connected to a l eng th o f t ub ing (9) p o s i t i o n e d at v a r i o u s he igh ts above the a o r t a i n o rder t o p rov ide a h y d r o s t a t i c a f t e r l o a d . The h y d r o s t a t i c a f t e r l o a d fed i n t o the t h i r d r e s e r v o i r where pe r fusa te then passed through a s c i n t e r e d - g l a s s f i l t e r (10) and was pumped back up to the second r e s e r v o i r by means o f a M a s t e r f l e x pump (11 ) . The pe r fusa te i n the pr imary and secondary r e s e r v o i r s was c o n s t a n t l y aera ted w i t h 95% C02/5% O2 (12) and a l l r e s e r v o i r s and the compl iance chamber were main ta ined a t 37°C by means o f a cons tan t - tempera tu re water r e c i r c u l a t i n g pump (not shown h e r e ) . The hear t (6) was a t t a c h e d , v i a the a o r t a , t o the a o r t i c cannu la and i n i t i a l l y per fused i n a re t rograde manner. The l e f t a t r i a was then cannu la ted and the p e r f u s i o n then swi tched to the work ing hear t mode. In the work ing hear t mode, pe r fusa te e n t e r i n g the l e f t a t r i a , from the secondary r e s e r v o i r , was pumped out through the a o r t i c cannula i n t o the compl iance chamber and then up the h y d r o s t a t i c a f t e r l o a d tub ing and i n t o the t h i r d r e s e r v o i r where i t was r e c i r c u l a t e d . A o r t i c p ressure was measured v i a a Statham P23 AA t ransducer (13) i n l i n e w i t h the a o r t i c c a n n u l a . L e f t v e n t r i c u l a r pressure was measured v i a a t ransducer (14) i n l i n e w i t h PE-90 t ub ing (15) p ie rced i n t o the apex o f the h e a r t . The hear t s were paced a t 240 beats /min by e l e c t r i c a l s t imu lus a p p l i e d to both cannulas v i a a Grass model SD9D s t i m u l a t o r (not shown h e r e ) . 23 Heart P e r f u s i o n s : Animals were weighed and then anes the t i zed by i n j e c t i o n ( i . p . ) w i t h sodium pen toba rb i t a l (240 mg/kg) . Hear ts were e x c i s e d from the a n e s t h e t i z e d animals and cannula ted as working h e a r t s , as desc r i bed p r e v i o u s l y (Lopaschuk et al, 1986). The hear ts were i n i t i a l l y per fused r e t r o g r a d e l y , a t 44 mm Hg p ressu re , through the a o r t a f o r 10 minutes w i t h KH b u f f e r , pH 7 . 4 , gassed w i th 95% 02/5% CO2, c o n t a i n i n g 3.0 mM c a l c i u m and 11 mM g l u c o s e . Dur ing t h i s t ime , the opening t o the l e f t a t r i um was cannu la ted . Hearts were then swi tched t o the work ing hear t model o f p e r f u s i o n w i th KH bu f fe r c o n t a i n i n g g lucose (11 mM), or g lucose (11 mM) p lus pa lm i ta te (1.2 mM). A l l pe r fus ions were done i n the presence o f 3% BSA, w i th p a l m i t a t e , when used, being pre-bound to the a lbumin . B ind ing pa lm i ta te to BSA invo l ved d i s s o l v i n g pa lm i t a te w i th 1.4 mM sodium carbonate i n a smal l amount o f 2:1 wa te r :e thano l and b o i l i n g u n t i l the ethanol had evapora ted . Th is was q u i c k l y poured i n t o warm KH, w i thout g lucose and w i th 3% BSA, fo l l owed by ove rn igh t d i a l y s i s u s i n g Spec t rapor membrane tub ing ( vo l / cm:5 m l , mw c u t o f f 6 , 000 -8 ,000 ) . A f t e r d i a l y s i s the bu f fe r was brought t o volume w i th KH and the a p p r o p r i a t e amount o f g lucose and was f i l t e r e d through a Whatman GF/C f i l t e r . Hear ts were per fused dur ing i n i t i a l work pe r i od at a l e f t a t r i a l f i l l i n g p ressure o f 15 cm H2O, and a h y d r o s t a t i c a f t e r l o a d o f 90 cm H 2 0 . Spontaneous ly bea t ing hear ts were ana lyzed f o r hear t r a t e (HR) and f o r f u n c t i o n at the medium work load. Func t ion was desc r i bed i n terms of HR - peak s y s t o l i c pressure product (HR X PSP) , measured through an a o r t i c t r ansduce r connected to a Grass model 79D po lyg raph . Hear ts were then 24 paced a t 240 beats /min and f u n c t i o n was assessed a t va r i ous a f t e r l o a d p r e s s u r e s . When hear ts were paced f u n c t i o n was based on l e f t v e n t r i c u l a r developed pressure (LVDP) and c o n t r a c t i l i t y ( + / - d P / d t ) , measured by means o f a t ransducer w i t h a t tached PE-90 tub ing p i e r c e d i n t o the apex o f the hear t and connected to a Grass po l yg raph . R e s u l t s were ana lyzed by an Apple II computer connected to the Grass po lygraph through an A/D+D/A conve r te r c a r d . The same procedure was c a r r i e d out on con t ro l and d i a b e t i c hear ts per fused i n the presence o f pa lm i t a te w i th and w i thout 0 .5 mM DCA. DCA was prepared as a 50 mM s o l u t i o n i n H2O and was ad jus ted to pH 7.4 w i t h NaOH. At the end o f the work pe r iod and a t a maximal work load , where the a f t e r l o a d was made i n f i n i t e by c lamping o f f the t u b i n g , hear ts were f r ozen us ing Wol lenberger clamps coo led to the temperature o f l i q u i d n i t r o g e n . Hear ts were then s to red i n a -80°C f r e e z e r . 2 5 E x t r a c t i o n o f ATP. C r e a t i n e Phosphate, and Long Chain C a r n i t i n e from  V e n t r i c u l a r T i s s u e : Frozen v e n t r i c u l a r t i s s u e was powdered i n a l i q u i d n i t r o g e n - c o o l e d p o r c e l a i n mortar and p e s t l e . Approx imate ly 200 mg o f the powdered t i s s u e was weighed, d r i e d overn igh t i n a 100°C oven and reweighed f o r de te rm ina t ion o f d ry - to -we t weight r a t i o . Us ing t h i s r a t i o , as w e l l as the t o t a l f r ozen v e n t r i c u l a r weight and the weight o f the d r i e d a t r i a l t i s s u e , t o t a l d ry weight o f the hear t was determined. P e r c h l o r i c A c i d E x t r a c t i o n : E x t r a c t i o n o f ATP, c r e a t i n e phosphate ( C P ) , and long c h a i n a c y l c a r n i t i n e (LCC) were done as desc r i bed p r e v i o u s l y ( Ide l l -Wenger et al, 1978). These me tabo l i t es were e x t r a c t e d w i t h the a i d o f mortar and p e s t l e , from approx imate ly 500 mg o f f r o z e n , powdered t i s s u e i n t o 2 ml o f i c e - c o l d p e r c h l o r i c a c i d (PCA) (6% w t / v o l ) c o n t a i n i n g 15 mM d i t h i o t h r e i t o l (DTT). The t i s s u e - P C A mix tu re was c e n t r i f u g e d (3500 rpm) a t 4°C, and a p o r t i o n (0 .5 ml) o f the supernatant was n e u t r a l i z e d w i t h 10 N KOH and used f o r de te rmina t ion o f t i s s u e l e v e l s o f ATP and CP. The PCA p r e c i p i t a t e was washed w i th 1.2% PCA and LCC was hydro lyzed at pH 13 (w i th 10 N KOH) f o r 60 min at 70°C i n the presence o f 15 mM DTT. The pH o f the hyd ro l yza te was decreased to 1 w i th 70% PCA to p r e c i p i t a t e any r e s i d u a l p r o t e i n . A f t e r c e n t r i f u g a t i o n a t 4°C, the supernatant was n e u t r a l i z e d w i th 10 N KOH and assayed fo r f r e e c a r n i t i n e r e l e a s e d from i t s acy l e s t e r s . 2 6 A T P / C r e a t i n e Phosphate Assay : Measurement o f ATP and CP l e v e l s from the PCA e x t r a c t was performed us ing a s tandard enzymat ic assay as desc r i bed p r e v i o u s l y (Bergmeyer, 1963) The f o l l o w i n g se t o f r e a c t i o n s forms the bas i s o f the assay : M g 2 + CP + ADP c r e a t i n e + ATP CPK M g 2 + g lucose + ATP G-6-P + ADP HK G-6-P + NADP 6-phosphogluconate + NADPH G-6-PDH the i n c r e a s e i n o p t i c a l d e n s i t y due to the format ion o f NADPH i s measured. Assay Procedure : To 100 u l o f sample, 100 ml o f Hepes (50 mM), MgCl2 (10 mM), EDTA (5 mM), 25 mg o f g l u c o s e , 200 u l o f g lucose-6-phosphate dehydrogenase (G-6-PDH) (300 U/ml) and 3.8 mg o f ADP was added. From t h i s m ix tu re a b a s e l i n e read ing was ob ta ined on a Hewlet t Packard 8452A spectrophotometer a t 340 nm. Th is was fo l l owed by the a d d i t i o n o f 10 u l o f hexok inase (HK) (3 U/10 u l ) . Once a c o n s i s t e n t read ing f o r the a d d i t i o n o f HK was o b t a i n e d , c r e a t i n e phosphokinase (CPK) 10 u l (10 U/assay) was added to the same r e a c t i o n media. 27 The va lues f o r ATP and CP are expressed as nmoles/g d ry weight o f hear t t i s s u e . The p e r f u s i o n medium conta ined no ATP or C P , t h e r e f o r e the va lues represen t ac tua l t i s s u e v a l u e s . Long Chain C a r n i t i n e Assay : Ex t rac ted c a r n i t i n e re leased from i t s acy l e s t e r s was measured r a d i o m e t r i c a l l y as desc r i bed by McGarry and Fos te r (1976) . B a s i c a l l y , the PCA e x t r a c t o f t i s s u e was incubated w i t h 3 H A c e t y l COA o f known s p e c i f i c a c t i v i t y and c a r n i t i n e a c e t y l t r a n s f e r a s e (CAT) . The l a b e l l e d a c e t y l c a r n i t i n e formed acco rd ing t o the f o l l o w i n g r e a c t i o n : _ CAT L - c a r n i t i n e + Ace ty l 3 H COA Ace ty l 3 H L - c a r n i t i n e + C0ASH + and was separated from the unreacted 3 H Ace t y l COA by pass ing the mix tu re through a column o f anion exchange r e s i n and de te rmin ing the i so tope content o f the e f f l u e n t f l u i d . Sodium t e t r a t h i o n a t e , a reduc ing agent , was added i n o rder to prevent the reve rse r e a c t i o n from o c c u r r i n g . Assay Procedure : The b a s i c r e a c t i o n mix ture c o n t a i n e d , i n a volume o f 1.2 m l , 120 nmoles o f Hepes b u f f e r , pH 7 . 3 , 2 umol o f sodium t e t r a t h i o n a t e , 1 u n i t o f c a r n i t i n e ace t y l t r a n s f e r a s e , 25 nmol (0.07 u C i ) o f 3 H Ace ty l COA, and 200 ul o f sample. The r e a c t i o n was i n i t i a t e d by a d d i t i o n o f 1.0 ml o f r e a c t i o n mix ture to an ependorf tube c o n t a i n i n g the sample. A f t e r 2 8 s tand ing a t room temperature f o r 30 min , 0 .5 ml o f the r e a c t i o n m ix tu re was t r a n s f e r r e d to a column of Dowex 1-xlO anion exchange r e s i n (7 x 12 mm) and washed th ree t imes w i th 0 .5 ml o f wa te r . The combined e f f l u e n t c o n t a i n i n g a c e t y l c a r n i t i n e was c o l l e c t e d i n s c i n t i l l a t i o n v i a l s , mixed w i th 5 ml o f aqueous count ing s c i n t i l l a n t and assayed f o r r a d i o a c t i v i t y i n a l i q u i d s c i n t i l l a t i o n counter ( T r i - C a r b 4530, Un i ted Techno log ies Packard) E x t r a c t i o n o f T r i g l y c e r i d e s from V e n t r i c u l a r T i ssue T i s s u e t r i g l y c e r i d e s were ex t rac ted as desc r i bed by Bowyer and K ing (1977) . A known weight o f f r o z e n , powdered t i s s u e (approx imate ly 200 mg) was e x t r a c t e d i n 18 ml o f i c e - c o l d ch lo ro fo rm and methanol (2 :1 v o l / v o l ) us ing a P o l y t r o n homogenizer. A f t e r the a d d i t i o n o f 3 ml o f methano l , the mix tu re was s t i r r e d and c e n t r i f u g e d at 3444 g f o r 15 min a t 4°C. The supernatant was evaporated to dryness under n i t r o g e n , i n a water bath se t a t 55°C. T r i g l y c e r i d e Assay : The method used to measure t r i g l y c e r i d e s was a g l y c e r o l - 3 -phosphate ox idase -p -ch lo ropheno l (GP0) c o l o u r i n g method. Th i s was accompl ished us ing a t e s t k i t purchased from Wako Pure Chemical I n d u s t r i e s , Japan . 29 T r i g l y c e r i d e s are decomposed to g l y c e r o l and f r e e f a t t y ac i ds by l i p o p r o t e i n l i p a s e . The g l y c e r o l thus produced i s conver ted to g l y c e r o l - 3 - p h o s p h a t e by g l y c e r o l k inase i n the presence o f ATP. Th i s g l y c e r o l - 3 - p h o s p h a t e i s then o x i d i z e d by GPO to y i e l d hydrogen p e r o x i d e . The hydrogen perox ide thus produced y i e l d s a red c o l o u r compound upon o x i d a t i v e condensat ion w i th p-ch lorophenol and 4-aminoant ipyrene i n the presence o f pe rox i dase . The amount o f t r i g l y c e r i d e i n the o r i g i n a l e x t r a c t sample i s then determined by measuring the absorbance o f the developed c o l o u r , a t a wavelength o f 492 nm, as compared w i t h absorbance of the s tandard s o l u t i o n . Assay P rocedure : The t i s s u e t r i g l y c e r i d e e x t r a c t s were d i s s o l v e d i n one mL o f ch lo ro fo rm and mixed w e l l . Two 10 uL samples were p laced i n two 12x75 mL t e s t tubes and the ch lo ro fo rm was evaporated o f f . Then, 0 .3 mL o f c o l o u r reagent was added to each tube and they were incubated a t 37°C f o r 5-10 minutes i n a water ba th . Fo l l ow ing i n c u b a t i o n , 200 uL o f each m ix tu re was p laced on a M i c r o t i t e r ^ p l a t e . The M i c r o t i t e r R p l a t e was read a t 492 nm on a SLT-Lab Instruments Easy Reader EAR340AT e l i z a reader w i t h a K i n e t i c Sof tware package. 3 0 Serum A n a l y s e s : Serum c o l l e c t e d a t the t ime o f s a c r i f i c e was s t o r e d at -20°C u n t i l assayed . Serum g lucose was determined us ing a Boehr inger Mannheim d i a g n o s t i c k i t . The method i s an enzymat ic c o l o r i m e t r i c one where g lucose i s conver ted to g luconate i n the presence o f g lucose o x i d a s e . Hydrogen p e r o x i d e , i n the presence o f the enzyme p e r o x i d a s e , o x i d i z e d the c o l o u r l e s s reagen t , 4-aminophenazene, t o a dye whose c o l o u r development was read a t 510 nm w i th a Brinkmann model PC800 c o l o r i m e t e r . Serum g lucose va lues are expressed as mmoles/L. STZ an imals hav ing a serum g lucose above 16 mmoles/L were used i n t h i s s tudy . Serum f r e e f a t t y ac i ds were determined us ing a Wako Pure Chemical I n d u s t r i e s NEFA t e s t k i t which u t i l i z e s an in vitro enzymat ic c o l o r i m e t r i c method. The Wako enzymat ic method r e l i e s upon the a c y l a t i o n o f Coenzyme A (CoA) by the f a t t y ac i ds i n the presence o f added Acy l CoA Syn the tase . The Acy l CoA thus produced i s o x i d i z e d by added Acy l CoA Oxidase w i t h gene ra t i on o f hydrogen pe rox ide . Hydrogen p e r o x i d e , i n the presence o f p e r o x i d a s e , permi ts the o x i d a t i v e condensat ion o f 3 -methy l -N-e thy l -N - /J -h y d r o x y e t h y l - a n i l i n e w i th 4 -aminoan t ipy r ine to form a pu rp le co lou red adduct which can be measured co l o u r i m e t r i c a l l y a t 550 nm us ing a SLT-Lab Instruments Easy Reader EAR340AT e l i z a reader w i t h a K i n e t i c Sof tware package. 31 S t a t i s t i c a l A n a l y s i s : R e s u l t s are presented as mean ± S . E . M . (s tandard e r r o r o f mean). S t a t i s t i c a l a n a l y s i s was performed us ing a one-way ANOVA fo l l owed by a compar ison o f group means w i th a Newman-Keuls t e s t . For hear t f u n c t i o n c u r v e s , repeated measures o f a n a l y s i s o f v a r i a n c e was used , f o l l owed by a Newman-Keuls t e s t f o r comparison o f group means a t i n d i v i d u a l p o i n t s on the c u r v e . Where o n l y two group means were compared s t a t i s t i c a l a n a l y s i s was performed us ing an unpai red S t u d e n t ' s t - t e s t . A p r o b a b i l i t y o f p<0.05 was taken as the l e v e l o f s t a t i s t i c a l s i g n i f i c a n c e . 32 RESULTS The S t r e p t o z o t o c i n D i a b e t i c Rat Model D iabetes was induced by a s i n g l e i . v . i n j e c t i o n o f 60 mg/kg STZ. U r ine g lucose was q u a l i t a t i v e l y assessed 48 hours a f t e r STZ i n j e c t i o n us ing a L i l l y T e s - t a p e . On a s c a l e o f 1+ (0.1%) to 4+ (2.0%), i t was found to be between 3+ and 4+ f o r S T Z - i n j e c t e d r a t s . No d e t e c t a b l e g lucose was present i n the u r i ne o f con t ro l r a t s . In s i x weeks t i m e , a t the t ime o f s a c r i f i c e , b lood was c o l l e c t e d and serum g lucose l e v e l s were measured. Serum g lucose l e v e l s o f d i a b e t i c an imals were found to be s i g n i f i c a n t l y h igher than those o f age-matched c o n t r o l s (F igu re 5 ) . Serum t r i g l y c e r i d e l e v e l s were a l s o s i g n i f i c a n t l y e l e v a t e d i n d i a b e t i c r a t s compared w i th c o n t r o l s (F igu re 6 ) . Rats t r e a t e d w i th STZ e x h i b i t e d symptoms c h a r a c t e r i s t i c o f the d i a b e t i c s t a t e . D i a b e t i c r a t s gained l e s s weight than age-matched c o n t r o l s and, t h e r e f o r e , had a s i g n i f i c a n t l y lower body weight a t the t ime o f s a c r i f i c e (F igu re 7 ) . D i a b e t i c s a l s o had a n o t i c e a b l e i n c r e a s e i n u r i n e and f e c a l output which was not measured q u a n t i t a t i v e l y . E f f e c t o f Free F a t t y Ac ids on Cont ro l and D i a b e t i c I s o l a t e d Working Rat  Hear ts To i n v e s t i g a t e the e f f e c t s o f F F A ' s on d i a b e t i c hear t f u n c t i o n , i s o l a t e d work ing r a t hear ts were per fused i n the presence o f g lucose (11 mM) a lone or g lucose (11 mM) p lus pa lm i ta te (1 .2 mM). In spontaneous ly 33 bea t ing h e a r t s , a t a medium workload (15 cm H2O f i l l i n g p ressu re , 90 cm H2O a f t e r l o a d p r e s s u r e ) , hear t r a te was found to be s i g n i f i c a n t l y lower i n d i a b e t i c hear ts compared w i th c o n t r o l s (F igu re 8 ) . F igu re 8 a l s o shows t ha t F F A ' s had no s i g n i f i c a n t e f f e c t on hear t r a t e . HR X PSP was measured at a medium workload and was found to be depressed i n d i a b e t i c s , r ega rd l ess o f a v a i l a b l e subs t ra tes (F igu re 9 ) . Th i s f i g u r e a l s o shows t h a t , i n both c o n t r o l s and d i a b e t i c s , the presence o f F F A ' s had no s i g n i f i c a n t e f f e c t on HR X PSP. F i gu re 10 shows LVDP, measured a t a medium work load , to be s i g n f i c a n t l y depressed i n d i a b e t i c hear ts compared w i th con t ro l h e a r t s . The presence o f F F A ' s had no s i g n i f i c a n t e f f e c t on LVDP i n e i t h e r group o f h e a r t s . S i m i l a r i l y , maximal LVDP, measured at an i n f i n i t e a f t e r l o a d p r e s s u r e , was depressed i n d i a b e t i c hear ts compared w i th c o n t r o l s (F igu re 11) . The presence o f FFA ' s had no s i g n i f i c a n t e f f e c t on maximal LVDP i n e i t h e r c o n t r o l or d i a b e t i c h e a r t s . LVDP was a l s o measured at f i v e d i f f e r e n t a f t e r l o a d pressures (60, 70 , 90 , 140 cm H2O, and maximal) and was found to be s i g n i f i c a n t l y dep ressed , a t each a f t e r l o a d p ressu re , i n both d i a b e t i c groups w i t h respec t t o c o n t r o l s (F igu re 12) . A g a i n , the presence o f FFA 's had no s i g n i f i c a n t e f f e c t . P o s i t i v e dP /d t was measured a t f i v e d i f f e r e n t a f t e r l o a d pressures and was found t o be depressed , at each a f t e r l o a d p r e s s u r e , i n d i a b e t i c s per fused i n the presence o f FFA ' s compared w i th c o n t r o l s per fused i n e i t h e r c o n d i t i o n (F igu re 13) . P o s i t i v e dP /d t was a l s o found to be depressed i n d i a b e t i c s per fused i n the presence of FFA 's compared w i t h 34 d i a b e t i c s per fused i n the presence o f g lucose a l o n e . Th is depress ion was found at each a f t e r l o a d pressure w i th the excep t i on o f 140 cm H2O. Only a t an i n f i n i t e a f t e r l o a d p ressure d i d d i a b e t i c hear ts per fused i n the presence o f g lucose a lone have a +dP/dt s i g n i f i c a n t l y depressed from c o n t r o l s per fused under e i t h e r c o n d i t i o n . F F A ' s had no de t r imen ta l e f f e c t on +dP/dt i n c o n t r o l h e a r t s . F igu re 14 demonstrates - d P / d t measured a t f i v e d i f f e r e n t a f t e r l o a d p r e s s u r e s . Both d i a b e t i c groups had s i g n i f i c a n t l y depressed - d P / d t ' s , a t a l l a f t e r l o a d p r e s s u r e s , w i th respec t t o both c o n t r o l g roups. There was an e x c e p t i o n , where at a 70 cm H2O a f t e r l o a d , d i a b e t i c s per fused i n the presence o f g lucose a lone were on l y s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s per fused i n the presence o f g lucose a l o n e . FFA ' s had no de t r imen ta l e f f e c t on d i a b e t i c - d P / d t but had an e f f e c t on c o n t r o l h e a r t s . At a f t e r l o a d pressures o f 60, 70 , 90 and 140 cm H2O c o n t r o l s , per fused i n the presence o f F F A ' s , had s i g n i f i c a n t l y depressed - d P / d t ' s compared w i th c o n t r o l s per fused i n the presence o f g lucose a l o n e . E f f e c t o f DCA on FFA Per fused Cont ro l and D i a b e t i c I s o l a t e d Working Rat  Hear ts As desc r i bed i n the i n t r o d u c t i o n , DCA i s a PDH a c t i v a t o r which r e s u l t s i n i nc reased g lucose o x i d a t i o n r a t e s when added to i s o l a t e d work ing hear t p e r f u s a t e . F igu re 15 demonstrates t h a t , a t a medium work load , the a d d i t i o n o f 0 .5 mM DCA to the FFA per fusa te r e s u l t e d i n a s i g n i f i c a n t l y h igher HR i n 3 5 d i a b e t i c hear ts than i n d i a b e t i c hear ts per fused i n the presence o f F F A ' s a l o n e . The d i a b e t i c hear ts per fused i n the presence o f DCA showed s i m i l a r HR's to those o f c o n t r o l s . HR X PSP, measured a t a medium work load , was a l s o found to be s i g n i f i c a n t l y depressed i n d i a b e t i c s compared w i th c o n t r o l s and w i t h d i a b e t i c s per fused i n the presence o f 0 .5 mM DCA (F igu re 16 ) . F i gu re 17 shows LVDP, measured a t a medium work load , t o be s i g n i f i c a n t l y decreased i n d i a b e t i c s w i th respec t t o d i a b e t i c s per fused i n the presence o f 0 .5 mM DCA and w i th respec t t o c o n t r o l s . L i k e w i s e , maximal LVDP, demonstrated i n F igu re 18, was found to be s i g n i f i c a n t l y lower i n d i a b e t i c hea r t s compared w i th c o n t r o l s or w i t h d i a b e t i c hea r t s per fused i n the presence o f 0 .5 mM DCA. Measured a t f i v e d i f f e r e n t a f t e r l o a d p r e s s u r e s , LVDP was found t o be s i g n i f i c a n t l y lower i n d i a b e t i c h e a r t s , at 70, 90 cm H2O and maximal a f t e r l o a d p r e s s u r e s , w i t h respec t t o c o n t r o l s and w i th respec t t o d i a b e t i c s per fused i n the presence o f 0 .5 mM DCA (F igu re 19 ) . At a 60 cm H2O a f t e r l o a d d i a b e t i c hear ts per fused i n the absence o f DCA had a s i g n i f i c a n t l y lower LVDP than con t ro l hear ts o n l y . P o s i t i v e dP /d t was measured a t f i v e d i f f e r e n t a f t e r l o a d p ressures and was found to be s i g n i f i c a n t l y depressed at each a f t e r l o a d p ressure compared w i t h c o n t r o l s and w i th d i a b e t i c s per fused i n the presence o f 0 .5 mM DCA (F igu re 2 0 ) . F i gu re 21 demonstrates - d P / d t , measured a t f i v e d i f f e r e n t a f t e r l o a d p r e s s u r e s , t o be s i g n i f i c a n t l y lower i n d i a b e t i c h e a r t s , at 36 90, 140 cm H2O and maximal a f t e r l o a d s , compared w i t h c o n t r o l hear ts and compared w i th d i a b e t i c hear ts per fused i n the presence o f 0 .5 mM DCA. At 60 and 70 cm H2O a f t e r l o a d s , d i a b e t i c hea r t s per fused i n the absence o f DCA had a s i g n i f i c a n t l y depressed - d P / d t compared w i t h c o n t r o l hear ts o n l y . E f f e c t o f Free F a t t y Ac ids on V e n t r i c u l a r T i s s u e M e t a b o l i t e s i n I s o l a t e d  Working Cont ro l and D i a b e t i c Hearts Fo l l ow ing the aforement ioned i s o l a t e d work ing hear t p e r f u s i o n p r o t o c o l , and a t an i n f i n i t e a f t e r l o a d , hea r t s were immediate ly f r ozen w i t h Wol lenberger clamps coo led to the temperature o f l i q u i d n i t r o g e n . T i s s u e me tabo l i t es were ex t rac ted and assayed from the v e n t r i c u l a r t i s s u e , as desc r i bed i n the methods s e c t i o n . F igu re 22 demonstrates tha t ATP (umoles/g d ry wt) was not s i g n i f i c a n t l y d i f f e r e n t i n d i a b e t i c hear ts compared w i t h c o n t r o l h e a r t s . Th i s f i g u r e a l s o shows tha t the presence o f FFA ' s i n the work ing hear t pe r fusa te had no e f f e c t on the l e v e l s o f ATP found i n e i t h e r c o n t r o l or d i a b e t i c h e a r t s . I t was found t ha t c r e a t i n e phosphate l e v e l s were not s i g n i f i c a n t l y d i f f e r e n t i n d i a b e t i c hear ts compared w i t h c o n t r o l hear ts (F igu re 2 3 ) . I t was a l s o shown t ha t the presence o f F F A ' s i n the work ing hear t pe r fusa te had no e f f e c t on c r e a t i n e phosphate l e v e l s i n e i t h e r c o n t r o l or d i a b e t i c h e a r t s . 3 7 F igu re 24 demonstrates tha t v e n t r i c u l a r t i s s u e LCC l e v e l s were not s i g n i f i c a n t l y d i f f e r e n t i n d i a b e t i c s w i th respec t t o c o n t r o l s . Th i s f i g u r e a l s o shows t ha t the presence o f F F A ' s i n the work ing hear t pe r f usa te had no e f f e c t on LCC l e v e l s i n e i t h e r con t ro l o r d i a b e t i c h e a r t s . F i g u r e 25 shows tha t t i s s u e t r i g l y c e r i d e l e v e l s were not changed by e i t h e r the presence of d iabe tes or by the use o f FFA ' s i n the work ing hear t p e r f u s a t e . E f f e c t o f DCA on V e n t r i c u l a r T i ssue M e t a b o l i t e Leve ls i n Free F a t t y A c i d  Per fused Con t ro l and D i a b e t i c Hearts F i gu re 26 demonstrates tha t l e v e l s o f ATP i n v e n t r i c u l a r t i s s u e were not s i g n i f i c a n t l y a f f e c t e d by the presence o f d iabe tes or by the presence o f 0 .5 mM DCA i n the FFA work ing hear t p e r f u s a t e . No s i g n i f i c a n t d i f f e r e n c e was found f o r c r e a t i n e phosphate l e v e l s o f v e n t r i c u l a r t i s s u e i n d i a b e t i c s compared w i th c o n t r o l s , or i n d i a b e t i c s or c o n t r o l s per fused i n the presence o f 0 .5 mM DCA (F igu re 2 7 ) . F i g u r e 28 shows v e n t r i c u l a r LCC l e v e l s t o be s i m i l a r i n c o n t r o l and d i a b e t i c v e n t r i c u l a r t i s s u e r ega rd l ess o f the presence o f 0 .5 mM DCA i n the work ing hear t p e r f u s a t e . V e n t r i c u l a r t i s s u e t r i g l y c e r i d e s l e v e l s are demonstrated i n F igu re 29 . There was a t rend toward l e v e l s being inc reased i n d i a b e t i c hear ts 3 8 per fused i n the presence o f 0 .5 mM DCA a l though the i n c r e a s e was not s t a t i s t i c a l l y s i g n i f i c a n t . R a t i o n a l e f o r the Use o f A f t e r l o a d Curves Common throughout the l i t e r a t u r e are examinat ions o f c a r d i a c f u n c t i o n through comparison o f performances o f i s o l a t e d work ing hear t s i n response to changes i n f i l l i n g p ressure (Rodr igues et a l , 1988; T a h i l i a n i et al, 1985; Vadlamudi et al, 1982; X iang et a l , 1988) . I t has been we l l e s t a b l i s h e d tha t LVDP and + / -dP/d t i n c r e a s e , a t d i f f e r e n t r a t e s i n c o n t r o l s and d i a b e t i c s , i n response t o i n c r e a s e s i n f i l l i n g p ressu re (Vadlamudi et a l , 1982). However, we found ve ry l i t t l e , or no response , by way o f LVDP or +/-dP/dt, to changes i n f i l l i n g p r e s s u r e . Ra the r , we found a marked response to changes i n h y d r o s t a t i c a f t e r l o a d p ressu re and t h e r e f o r e , used a f t e r l o a d curves throughout our s tudy as opposed t o p r e l o a d , or f i l l i n g pressure c u r v e s . Upon f u r t h e r i n v e s t i g a t i o n i t became apparent t h a t the major f u n c t i o n a l d i f f e r e n c e between the methodology used by us and t ha t used by p rev ious workers was a d i f f e r e n c e i n the k ind o f a f t e r l o a d system used . T h i s was found to be the bas i s f o r the d i f f e r e n c e i n response to changes i n f i l l i n g p r e s s s u r e . The p rev ious researchers had u t i l i z e d a f i x e d r e s i s t a n c e - t y p e a f t e r l o a d whereas our s t u d i e s u t i l i z e d a h y d r o s t a t i c type a f t e r l o a d . I t i s obv ious t ha t any i nc rease i n f i l l i n g p ressure equa ls an i n c r e a s e i n f l ow r a t e through the a f t e r l o a d d e v i c e . An i n c r e a s e i n f l ow through a 39 hydrostatic afterload w i l l have no effect on the afterload pressure the heart experiences. However, as shown in Figure 30 and Table 1, an increase in flow rate through a resistance-type afterload results in an increase in pressure, or an increase in effective afterload the heart experiences. Therefore, i t can be assumed that previous workers showing changes in LVDP and +/-6P/dt in response to changes in f i l l i n g pressure were actually demonstrating a combined effect of both preload and afterload. For this reason, we chose to compare control and diabetic isolated working rat heart function through the comparison of responses to changes in afterload pressure. 40 F igu re 5 : Serum g lucose l e v e l s o f d i a b e t i c r a t s 6 weeks a f t e r i n d u c t i o n o f d iabe tes w i th STZ and o f age-matched c o n t r o l s . Resu l t s are expressed as mean ± S . E . M . (* denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s a t p<0.05). 41 S e r u m G lucose Concen t ra t ion Of Six Week Cont ro l And Diabet ic Rats Diabet ic 30 r SS3 Cont ro l N = 18 20 10 0 N = 23 * - significantly different from control 4 2 F igu re 6: Serum f r e e f a t t y a c i d l e v e l s o f d i a b e t i c r a t s 6 weeks a f t e r i n d u c t i o n o f d iabe tes w i t h STZ and o f age-matched c o n t r o l s . R e s u l t s are expressed as mean ± S . E . M . (* denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s at p<0.05) A 3 S e r u m Free Fat ty Ac id Concen t ra t i on Of Six Week Cont ro l And Diabet ic Ra ts 1.000 c^O.750 o E E ^ 0-50.0 < LL. | 0.250 CD 0.000 I I D iabet ic K \N Cont ro l N = 1 6 * - I N = 23 -T * — s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l 44 F igu re 7: Body weight change o f d i a b e t i c and con t ro l r a t s s i x weeks f o l l o w i n g i n j e c t i o n o f STZ or i t s v e h i c l e . R e s u l t s are expressed as mean ± S . E . M . (* denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s at p<0.05). 45 Body Weight C h a n g e Over A Six Week Pe r i od Of Con t ro l And Diabet ic Ra ts I I D iabet ic N = 23 * - s ign i f i can t ly d i f ferent f rom con t ro l 4 6 F igu re 8 : E f f e c t o f me tabo l i c subs t ra tes on hear t r a t e i n c o n t r o l and d i a b e t i c i s o l a t e d working r a t hear ts at a medium workload (90 cm H2O a f t e r l o a d ) . Resu l t s are expressed as mean ± S . E . M . (* denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from both con t ro l groups at p<0.05). 4 7 EFFECT OF METABOLIC SUBSTRATES ON HEART RATE IN CONTROL AND DIABETIC ISOLATED WORKING RAT HEARTS AT A MEDIUM WORKLOAD (90 c m H 2 0 AFTERLOAD) 2 5 0 2 0 0 150 100 -I I Cont ro l G lucose f \S^ Cont ro l Pa lm i ta te g X 3 D iabet ic G l u c o s e F T H D iabet ic P a l m i t a t e * - s i g n i f i c a n t l y d i f f e r e n t f r o m b o t h c o n t r o l g r o u p s 48 F igu re 9 : E f f e c t o f me tabo l i c s u b s t r a t e s on HR X PSP i n c o n t r o l and d i a b e t i c i s o l a t e d work ing r a t hear ts at a medium workload (90 cm H2O a f t e r l o a d ) . R e s u l t s are expressed as mean ± S . E . M . (* denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from both con t ro l groups a t p<0.05). 4 9 EFFECT OF METABOLIC SUBSTRATES ON HR X P S P IN CONTROL AND DIABETIC ISOLATED WORKING RAT HEARTS AT A MEDIUM WORKLOAD (90 c m H 2 0 AFTERLOAD) 3E4 2E4 1 E4 0 ESS Cont ro l G lucose Cont ro l Pa lm i t a te g22S Diabet ic G l u c o s e \~7~] Diabet ic P a l m i t a t e * - s i g n i f i c a n t l y d i f f e r e n t f r o m b o t h c o n t r o l g r o u p s 50 F igu re 10: E f f e c t o f me tabo l i c s u b s t r a t e s on LVDP i n con t ro l and d i a b e t i c work ing r a t hea r t s a t a medium work load ( 90 cm H2O a f t e r l o a d ) . R e s u l t s are expressed as mean ± S . E . M . (* denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from both con t ro l groups at p<0.05). 51 E F F E C T OF METABOLIC SUBSTRATES ON LVDP IN CONTROL AND DIABETIC ISOLATED WORKING RAT HEARTS AT A MEDIUM WORKLOAD (90 c m H 2 0 AFTERLOAD) 1 5 0 -5 0 -N = 9 N = 7 N = 7 N=8 T _ Cont ro l G l u c o s e Con t ro l Pa lm i t a t e Diabet ic G l u c o s e Diabet ic Pa lm i t o t e t- — s i g n i f i c a n t l y d i f f e r e n t f r o m b o t h c o n t r o l g r o u p s 52 F igu re 11 : E f f e c t o f me tabo l i c subs t ra tes on maximal LVDP i n c o n t r o l and d i a b e t i c i s o l a t e d work ing r a t h e a r t s . R e s u l t s are expressed as mean ± S . E . M . (* denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from both con t ro l groups at p<0.05). 5 3 EFFECT OF METABOLIC SUBSTRATES ON MAXIMUM LVDP IN CONTROL AND DIABETIC ISOLATED WORKING RAT HEARTS 250 n I I Con t ro l G lucose g^S Diabet ic G l u c o s e RSN- Cont ro l Pa lm i ta te ^ D iabet ic Pa lm i t a t e * — s ign i f i cant ly di f ferent f r o m both con t ro l g roups 5 4 F igu re 12: E f f e c t o f me tabo l i c subs t ra tes on LVDP, measured a t f i v e d i f f e r e n t a f t e r l o a d p r e s s u r e s , i n con t ro l and d i a b e t i c i s o l a t e d work ing r a t h e a r t s . R e s u l t s expressed as mean ± S . E . M . (* denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from both con t ro l groups a t p<0.05) . E F F E C T OF METABOLIC SUBSTRATES ON LVDP IN CONTROL AND DIABETIC ISOLATED WORKING RAT HEARTS Af ter load P r e s s u r e c m H 2 0 — signi f icont ly di f ferent f rom both con t ro l g roups 5 6 Figure 13: Effect of metabolic substrates on +dP/dt, measured at f ive different afterload pressures, in control and diabetic isolated working rat hearts. Results expressed as mean ± S.E.M. •(* denotes values which are s ign i f icant ly different from al l other groups, + denotes values which are s ign i f icant ly different from Diabetic Palmitate, £ denotes values which are s ign i f icant ly different from Control Glucose, t denotes values which are s igni f icant ly different from Control Palmitate at p<0.05). EFFECT OF METABOLIC SUBSTRATES ON + D P / D T IN C O N T R O L AND DIABETIC ISOLATED WORKING RAT HEARTS o o • « A A X. X Cont ro l G lucose Cont ro l Pa lm i ta te Diabet ic G lucose Diabet ic Pa lm i ta te -O— Q-J L o-T A + A ->}; I 1 1 50 80 1 10 1 40 N = 7-+ £ 1-After lpad P ressu re c m H 2 0 s i g n i f i c a n t l y d i f f e r e n t f r o m a l l o t h e r g r o u p s s i g n i f i c a n t l y d i f f e r e n t f r o m D i a b e t i c P a l m i t a t e s i g n i f i c a n t l y d i f f e r e n t f r o m C o n t r o l G l u c o s e s i g n i f i c a n t l y d i f f e ren t f r o m C o n t r o l P a l m i t a t e 7~ MAX 58 Figure 14: Effect of metabolic substrates on -dP/dt in control and diabetic isolated working rat hearts. Results are expressed as mean ± S . E . M . (* denotes values which are significantly different from both control groups, + denotes values which are significantly different from Control Palmitate, £ denotes values which are significantly different from Control Glucose at p<0.05). E F F E C T OF METABOLIC SUBSTRATES ON - O P / D T IN C O N T R O L AND DIABETIC ISOLATED WORKING RAT HEARTS 5 0 0 0 -o o • « A A A. A. + O" + Cont ro l G lucose Cont ro l Pa lm i ta te Diabet ic G lucose Diabet ic Pa lm i ta te T — o -N = 7 - 9 JL o—-— X O 1 * * A ' " -" 1 ' A • 1 JL ^ ; 50 £ 8 0 1 1 0 ^ 4 0 iI f /Af ter load P r e s s u r e c m H 2 0 s i g n i f i c a n t l y d i f f e r e n t f r o m b o t h c o n t r o l g r o u p s s i g n i f i c a n t l y d i f f e r e n t f r o m C o n t r o l P a l m i t a t e s i g n i f i c a n t l y d i f f e r e n t f r o m C o n t r o l G l u c o s e 6 0 Figure 15: Effect of DCA on heart rate in FFA perfused control and diabetic isolated working rat hearts at a medium workload (90 cmh^ O afterload). Results are expressed as mean ± S.E.M. (* denotes values which are s ign i f icant ly different from al l other groups at p<0.05). 61 EFFECT of DICHLOROACETIC ACID on HEART RATE IN FATTY ACID P E R F U S E D CONTROL AND DIABETIC RAT HEARTS at a MEDIUM WORKLOAD (90 c m H 2 0 AFTERLOAD) 2 5 0 Cont ro l (553 Cont ro l + .5mM DCA r7n * — s i g n i f i c a n t l y d i f f e r e n t f r o m al l o t h e r g r o u p s Diabet ic Diabet ic + . 5mM DCA 62 F igu re 16: E f f e c t o f DCA on HR X PSP i n FFA per fused c o n t r o l and d i a b e t i c i s o l a t e d work ing r a t hear ts at a medium work load (90 cm H2O a f t e r l o a d ) . R e s u l t s are expressed as mean ± S . E . M . (* denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from a l l o ther groups at p<0.05). 63 EFFECT of DICHLOROACETIC ACID on HR X P S P IN FATTY ACID P E R F U S E D CONTROL AND DIABETIC RAT HEARTS at a MEDIUM WORKLOAD (90 c m H 2 0 AFTERLOAD) N = 6 EZTJ Cont ro l 883 Diabet ic ( 5 3 Cont ro l + .5mM DCA Diabet ic + . 5 m M DCA * — s i g n i f i c a n t l y d i f f e r e n t f r o m all o t h e r g r o u p s 64 F igu re 17: E f f e c t o f DCA on LVDP i n FFA per fused c o n t r o l and d i a b e t i c i s o l a t e d work ing r a t hea r t s a t a medium workload (90 cmH20 a f t e r l o a d ) . R e s u l t s are expressed as mean ± S . E . M . (* denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from a l l o ther groups at p<0.05). 6 5 cn X CL Q > 150 100 EFFECT of DICHLOROACETIC ACID on LVDP in FATTY ACID P E R F U S E D CONTROL AND DIABETIC RAT HEARTS at a MEDIUM WORKLOAD (90 c m H 2 0 AFTERLOAD) I I Cont ro l fx^S Cont ro l 4- .5mM DCA Diabet ic D iabet ic + . 5 m M DCA * — s i g n i f i c a n t l y d i f f e ren t f r o m al l o t h e r g r o u p s 66 F igu re 18: E f f e c t o f DCA on maximal LVDP i n FFA per fused c o n t r o l and d i a b e t i c r a t h e a r t s . R e s u l t s are expressed as mean ± S . E . M . (* denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from a l l o ther groups a t p<0.05). 6 7 EFFECT of DICHLOROACETIC ACID on MAXIMAL LVDP in F R E E FATTY ACID P E R F U S E D CONTROL AND DIABETIC RAT HEARTS 250 Cont ro l fv^q Con t ro l + .5mM DCA Diabet ic Diabet ic + . 5 m M DCA * — s ign i f icant ly di f ferent f rom all o the r g roups 68 F igu re 19: E f f e c t o f DCA on LVDP, measured a t f i v e d i f f e r e n t a f t e r l o a d p r e s s u r e s , i n FFA per fused con t ro l and d i a b e t i c i s o l a t e d work ing r a t h e a r t s . Resu l t s are expressed as mean ± S . E . M . (* denotes va lues which a re s i g n i f i c a n t l y d i f f e r e n t from a l l o ther groups, + denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from both con t ro l groups a t p<0.05) . 69 EFFECT OF DICHLOROACETIC ACID ON LVDP IN FATTY ACID P E R F U S E D CONTROL AND DIABETIC ISOLATED WORKING RAT HEARTS E E Q_ Q > 1 8 5 -155 125 9 5 50 o o A A A A Cont ro l Cont ro l + . 5 m M DCA Diabet ic Diabet ic + .5mM DCA N = 6 - 8 8 0 110 140 Af ter load P r e s s u r e c m H 2 0 s i g n i f i c a n t l y d i f f e r e n t f r o m al l o t h e r g r o u p s s i g n i f i c a n t l y d i f f e r e n t f r o m b o t h c o n t r o l g r o u p s MAX 70 F igu re 20 : E f f e c t o f DCA on +dP/dt , measured a t f i v e d i f f e r e n t a f t e r l o a d p r e s s u r e s , i n FFA per fused c o n t r o l and d i a b e t i c i s o l a t e d work ing r a t h e a r t s . R e s u l t s are expressed as mean ± S . E . M . (* denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from a l l o ther gorups a t p<0.05) . 7 1 EFFECT OF DICHLOROACETIC ACID ON + D P / D T IN FATTY ACID P E R F U S E C CONTROL AND DIABETIC ISOLATED WORKING RAT HEARTS 80 110 140 Af ter load P r e s s u r e c m H 2 0 s i g n i f i c a n t l y d i f f e r e n t f r o m al l o t h e r g r o u p s 7 2 F i g u r e 2 1 : E f f e c t o f OCA on - d P / d t , measured at f i v e d i f f e r e n t a f t e r l o a d p r e s s u r e s , i n FFA pe r fused c o n t r o l and d i a b e t i c i s o l a t e d working r a t h e a r t s . R e s u l t s a re expressed as mean ± S . E . M . (* denotes va lues which a re s i g n i f i c a n t l y d i f f e r e n t from a l l o ther groups, t denotes va lues which are s i g n i f i c a n t l y d i f f e r e n t from both con t ro l groups a t p<0.05) . 73 EFFECT OF DICHLOROACETIC ACID ON - D P / D T IN FATTY ACID P E R F U S E C CONTROL AND DIABETIC ISOLATED WORKING RAT HEARTS ' t 80 110 140 Af ter load P r e s s u r e c m H 2 0 s i g n i f i c a n t l y d i f f e r e n t f r o m a l l o t h e r g r o u p s s i g n i f i c a n t l y d i f f e r e n t f r o m b o t h c o n t r o l g r o u p s 74 F i g u r e 22: E f f e c t o f me tabo l i c s u b s t r a t e s on t i s s u e ATP i n c o n t r o l and d i a b e t i c i s o l a t e d work ing hear ts a t a maximal work load . R e s u l t s are expressed as mean ± S . E . M . 75 EFFECT OF METABOLIC SUBSTRATES ON TISSUE ATP IN CONTROL AND DIABETIC ISOLATED WORKING HEARTS AT A MAXIMAL WORKLOAD 2 5 7 6 F igu re 23 : E f f e c t o f me tabo l i c s u b s t r a t e s on t i s s u e c r e a t i n e phosphate i n c o n t r o l and d i a b e t i c i s o l a t e d work ing hear t s a t a maximal wo rk load . R e s u l t s are expressed as mean ± S . E . M . 77 EFFECT OF METABOLIC SUBSTRATES ON TISSUE CREATINE PHOSPHATE IN CONTROL AND DIABETIC ISOLATED WORKING HEARTS AT A MAXIMAL WORKLOAD 1 5 -1 0 -5 -0 I I Cont ro l G lucose ggg Diobet ic G l u c o s e ESS Cont ro l Pa lm i ta te Diabet ic Pa lm i t a t e 78 F igu re 24: E f f e c t o f me tabo l i c s u b s t r a t e s on t i s s u e LCC 's i n c o n t r o l and d i a b e t i c i s o l a t e d work ing hear t s a t a maximal work load . R e s u l t s are expressed as mean ± S . E . M . 79 EFFECT OF METABOLIC S U B S T R A T E S ON TISSUE L C C ' S IN CONTROL AND DIABETIC ISOLATED WORKING HEARTS AT A MAXIMAL WORKLOAD 3 0 0 0 Cont ro l G lucose Cont ro l Pa lm i ta te gg3 Diabet ic G l u c o s e (TH Diabet ic P a l m i t a t e 80 F igu re 25 : E f f e c t o f me tabo l i c subs t ra tes on t i s s u e t r i g l y c e r i d e s i n c o n t r o l and d i a b e t i c i s o l a t e d working hear ts at a maximal wo rk load . R e s u l t s are expressed as mean ± S . E . M . 81 EFFECT OF METABOLIC SUBSTRATES ON TISSUE TRIGLYCERIDES IN CONTROL AND DIABETIC ISOLATED WORKING HEARTS AT A MAXIMAL WORKLOAD I I Cont ro l G lucose f\\\] Cont ro l Pa lm i ta te gX3 Diabet ic G l u c o s e r / H D iabet ic P a l m i t a t e 82 F igu re 26 : E f f e c t o f DCA on t i s s u e ATP i n FFA per fused c o n t r o l and d i a b e t i c i s o l a t e d work ing r a t hear ts a t a maximal work load. R e s u l t s are expressed as mean ± S . E . M . 83 EFFECT of DICHLOROACETIC ACID on TISSUE ATP IN FATTY ACID PERFUSED CONTROL AND DIABETIC ISOLATED WORKING RAT HEARTS AT A MAXIMAL WORKLOAD 2 0 -Con t ro l Con t ro l 4- .5mM DCA Diobet ic Diobet ic + . 5mM DCA 84 F igu re 27 : E f f e c t o f DCA on t i s s u e c r e a t i n e phosphate i n FFA per fused c o n t r o l and d i a b e t i c i s o l a t e d work ing r a t hea r t s at a maximal work load . R e s u l t s are expressed as mean ± S . E . M . 85 EFFECT of DICHLOROACETIC ACID on TISSUE CREATINE P H O S P H A T E IN FATTY ACID P E R F U S E D CONTROL AND DIABETIC ISOLATED WORKING RAT HEARTS AT A MAXIMAL WORKLOAD I I ESS Con t ro l Con t ro l + .5mM DCA Diabet ic D iabet ic + , 5 m M DCA 86 F igu re 28 : E f f e c t o f DCA on t i s s u e LCC's i n FFA per fused c o n t r o l and d i a b e t i c i s o l a t e d work ing r a t hear ts a t a maximal wo rk load . R e s u l t s are expressed as mean ± S . E . M . 87 EFFECT OF DICHLOROACETIC ACID ON TISSUE L C C ' S IN FATTY ACID P E R F U S E D CONTROL AND DIABETIC ISOLATED WORKING RAT HEARTS AT A MAXIMAL WORKLOAD I I Cont ro l Cont ro l 4- . 5 m M DCA g£2 Diabet ic j-71 Diabet ic + . 5 m M DCA 88 F igu re 29 : E f f e c t o f DCA on t i s s u e t r i g l y c e r i d e s i n FFA per fused c o n t r o l and d i a b e t i c i s o l a t e d work ing r a t hear ts at a maximal wo rk l oad . R e s u l t s are expressed as mean ± S . E . M . 89 EFFECT of DICHLOROACETIC ACID on TISSUE TRIGLYCERIDES IN FATTY ACID •• P E R F U S E D CONTROL AND DIABETIC ISOLATED WORKING RAT HEARTS AT A MAXIMAL WORKLOAD Cont ro l Cont ro l + .5mM DCA Diabet ic D iabet ic + . 5 m M DCA 90 Figure 30: Pressure produced by increased flow rate through a resistance-type afterload system. 91 P R E S S U R E P R O D U C E D BY INCREASED FLOW RATE THROUGH A R E S I S T A N C E - T U B I N G TYPE AFTERLOAD SYSTEM 10 20 30 FLOW RATE ( m L / m i n ) 4 0 92 Table 1: Pressure produced by i nc reased f l ow r a t e through a r e s i s t a n c e - t y p e a f t e r l o a d sys tem. Pressure Produced by Increased f l ow Rate Through a R e s i s t a n c e - t u b i Type A f t e r l o a d System Flow rate (mL/min) 3.2 7.8 10.8 14.5 22.8 34.0 Pressure (mm Hg) 30 68 100 140 212 228 94 DISCUSSION; The purpose o f t h i s i n v e s t i g a t i o n was to examine the e f f e c t s o f f r e e f a t t y a c i d s and the e f f e c t s o f DCA on S T Z - d i a b e t i c i s o l a t e d work ing hear t f u n c t i o n . S t r e p t o z o t o c i n (60 mg/Kg) was i n j e c t e d i n t o the t a i l v e i n o f r a t s i n o rder t o des t roy p a n c r e a t i c beta c e l l s , the reby i nduc ing a d i a b e t i c s t a t e . The d i a b e t i c s t a t e was c h a r a c t e r i z e d by hyperg lycemia , h y p e r l i p i d e m i a and a decrease i n body weight ga in over a s i x week pe r i od (F i gu res 5 - 7 ) . I n s u l i n d e f i c i e n c y , due t o beta c e l l l o s s , r e s u l t s i n e l e v a t e d b lood g lucose because o f a marked decrease i n c e l l u l a r r a t e o f g lucose up take . E leva ted blood l i p i d l e v e l s a l s o occur because o f an i n h i b i t i o n o f l i p o p r o t e i n l i p a s e and a s t i m u l a t i o n o f t r i g l y c e r i d e l i p a s e , as d i scussed i n the i n t r o d u c t i o n s e c t i o n o f t h i s t h e s i s . D imin ished body weight ga in occur red d e s p i t e no decrease i n food and f l u i d i n t a k e , p robab ly due to dehydra t ion accompanied by p r o t e i n and f a t c a t a b o l i s m (Oak ley , 1968). A t ime course s tudy on i s o l a t e d work ing hea r t s revea led t ha t c a r d i a c dep ress ion occur red between 30 days and 3 months a f t e r the onset o f d i abe tes (Penpargkul et al, 1980). L a t e r , i t was shown t h a t i s o l a t e d g l ucose -pe r f used hear ts from 6-week d i a b e t i c r a t s demonstrated myocard ia l dep ress ion at h igher f i l l i n g p r e s s u r e s , c h a r a c t e r i z e d by decreased LVDP and decreased p o s i t i v e and nega t i ve d P / d t compared w i t h age-matched c o n t r o l s ( T a h i l i a n i et al, 1983) . Th i s dep ress ion was found 95 not t o worsen w i th the p rog ress ion o f d iabe tes (Vadlamudi et a / , 1982). Based on the above f i n d i n g s we chose to examine c a r d i a c performance 6 weeks f o l l o w i n g STZ i n j e c t i o n . We were a l s o ab le t o demonstrate depressed c a r d i a c f u n c t i o n i n 6-week S T Z - d i a b e t i c r a t s compared w i th age-matched c o n t r o l s . Spontaneous ly bea t ing d i a b e t i c hear ts d i s p l a y e d depressed hear t r a t e and HR x PSP when at a medium a o r t i c h y d r o s t a t i c a f t e r l o a d , o therw ise d e s c r i b e d as a medium work load . S i m i l a r i l y , LVDP was found to be depressed i n paced d i a b e t i c hear ts a t a medium work load . A l though Ingebretsen et al (1980) observed o n l y minor d i f f e r e n c e s between the a b i l i t y o f d i a b e t i c and normal myocardium t o perform work aga ins t a low a o r t i c h y d r o s t a t i c a f t e r l o a d , they demonstrated t h a t i n c r e a s i n g the a f t e r l o a d by 70% r e s u l t e d i n the i n a b i l i t y o f the i s o l a t e d hear ts from d i a b e t i c r a t s t o ma in ta in v e n t r i c u l a r p ressure development. L i k e w i s e , we were ab le to demonstrate a l a r g e r depress ion i n LVDP i n d i a b e t i c hea r t s compared w i th c o n t r o l s as the a f t e r l o a d p ressure was i nc reased to a maximum (F igu res 11, 12 ) . A s i m i l a r r e l a t i o n s h i p was found when +/-d P / d t were measured i n con t ro l and d i a b e t i c i s o l a t e d working hear ts exposed t o i nc reased a f t e r l o a d pressures (F igu res 13, 14 ) . These r e s u l t s suppor t the we l l accepted proposal t ha t a cardiomyopathy e x i s t s i n the d i a b e t i c r a t hear t and t ha t 6 weeks o f STZ- induced d iabe tes i s s u f f i c i e n t t ime to cause an a l t e r e d myocard ia l f u n c t i o n . The pathogenesis o f d i a b e t i c cardiomyopathy i s complex and not we l l unders tood . I t has been suggested t ha t i t may be due, i n p a r t , t o a combinat ion o f changes i n myosin and actomyosin ATPase a c t i v i t i e s (Ma lho t ra et al, 1981), SR C a 2 + - M g 2 + - A T P a s e a c t i v i t y (Lopaschuk et a / , 96 1983a), sarcolemmal C a ^ + pump a c t i v i t y (Hey l i ge r et a7, 1987) , and m i t ochond r i a l a c t i v i t y (Haugaard and Haugaard, 1964; P i e r c e and D h a l l a , 1987) . How these changes occur i s u n c e r t a i n but they have been suggested to be due to metabo l i c a l t e r a t i o n s a t the l e v e l o f the hear t ee l 1. As d i s c u s s e d e a r l i e r , a major me tabo l i c a l t e r a t i o n o f d i abe tes i s an i nc reased concen t ra t i on o f c i r c u l a t i n g f a t t y a c i d s and concomitant i nc reased r e l i a n c e o f hear t c e l l s on f a t t y a c i d s f o r o x i d a t i v e phospho ry la t i on (Denton et a / , 1967; Morgan et a7, 1961) . Some workers b e l i e v e t h a t t h i s a l t e r a t i o n i n l i p i d metabol ism may be r e s p o n s i b l e f o r some o f the b iochemica l changes t ha t take p l ace i n the d i a b e t i c myocardium ( T a h i l i a n i and M c N e i l l , 1986; D h a l l a et a7, 1985; Lopaschuk et a7, 1983a; P i e r c e et a7, 1983). C l i n i c a l l y , i nc reased serum FFA l e v e l s have been shown t o aggravate ar rhy thmias (Corr et a7, 1984) and i n c r e a s e the s e v e r i t y o f a myocard ia l i n f a r c t i o n ( D e L e i r i s and Op ie , 1978; L i e d t k e et a7, 1978; Vik-Mo and Mjos , 1981). Expe r imen ta l l y , a l t e r e d l i p i d metabol ism has a l s o been i m p l i c a t e d as a c o n t r i b u t o r to d e f e c t i v e hear t f u n c t i o n . In d i a b e t i c r a t s t reatment w i th 1 i p i d - a l t e r i n g drugs such as c a r n i t i n e (Rodr igues et a7, 1988), methyl pa lmox i ra te ( T a h i l i a n i and M c N e i l l , 1985) , m y o i n o s i t o l (X iang et a7, 1988), and meth ion ine and c h o l i n e ( H e y l i g e r et a7, 1986) r e s u l t e d i n an improvement i n c a r d i a c f u n c t i o n a s s o c i a t e d w i t h a decrease i n v a r i o u s l i p i d l e v e l s . Th i s ev idence s t r o n g l y suggests tha t l i p i d s may be c o n t r i b u t i n g to d i a b e t i c card iomyopathy. 97 On an acute b a s i s , i t has been shown tha t i . v . i n f u s i o n o f F F A ' s i n t o anes the t i zed i n t a c t dogs had no e f f e c t on the mechanical a c t i v i t y o f the hear t (Mjos , 1971) , whereas excess F F A ' s were shown t o impa i r c a r d i a c f u n c t i o n i n both normal and ischemic i s o l a t e d work ing p i g and r a t hea r t s ( D e L e i r i s et a / , 1978; Henderson et a7 , 1970; L i e d t k e et al, 1978; Paulson et a / , 1988). We found tha t the a d d i t i o n o f F F A ' s , i n the form o f p a l m i t a t e , t o i s o l a t e d working r a t hear t pe r f usa te had some de t r imen ta l e f f e c t s on normal and d i a b e t i c hear t f u n c t i o n . Heart r a t e and HR X PSP were not a f f e c t e d by the presence o f F F A ' s i n the pe r fusa te o f spontaneous ly bea t ing con t ro l and d i a b e t i c hea r t s (F igu res 8 , 9 ) . S i m i l a r i l y , LVDP measured i n paced c o n t r o l and d i a b e t i c hear ts a t i n c r e a s i n g work loads was una f fec ted by the presence o f F F A ' s (F igu res 10 -12) . However, i n d i a b e t i c hear ts +dP/dt was found to be depressed by FFA p e r f u s i o n , at four out o f f i v e a f t e r l o a d p r e s s u r e s , but was found to be unchanged i n con t ro l hear ts (F igu re 13 ) . C o n v e r s e l y , the presence o f FFA ' s had no e f f e c t on - d P / d t o f d i a b e t i c hea r t s whereas i n c o n t r o l hear ts - d P / d t was depressed by FFA 's at four out o f f i v e a f t e r l o a d p ressures (F igu re 14) . In summary, FFA p e r f u s i o n had no de t r imen ta l e f f e c t s on c o n t r o l o r d i a b e t i c i s o l a t e d work ing h e a r t s , except f o r a dep ress i ve a c t i o n on +dP/dt o f d i a b e t i c hear ts and - d P / d t o f c o n t r o l h e a r t s . I t i s we l l documented tha t i n the myocardium o f the d i a b e t i c t he re i s an i nc reased amount o f t r i g l y c e r i d e s (Shipp et al, 1973; Denton and Rand le , 1967; H e y l i g e r et al, 1986). Th is bu i l dup o f t r i g l y c e r i d e s i s suggested to occur from inc reased l e v e l s o f c i r c u l a t i n g FFA 's r e s u l t i n g i n an i nc reased s y n t h e s i s o f , and i n h i b i t e d h y d r o l y s i s o f 98 t r i a c y l g l y c e r o l s (Denton and Rand le , 1967; Paulson and C r a s s , 1982). In a d d i t i o n , t o t a l CoA l e v e l s o f d i a b e t i c myocardium are e leva ted (Re ibe l et al, 1981) , which favours s to rage o f long cha in f a t t y ac i ds as t r i a c y l g l y c e r o l s r a t h e r than t r a n s p o r t across the m i tochond r i a l membrane and subsequent o x i d a t i o n (Lopaschuk et al, 1986). Myocard ia l t r i g l y c e r i d e l e v e l s were measured i n c o n t r o l and d i a b e t i c r a t s f o l l o w i n g p e r f u s i o n w i t h e i t h e r g lucose or g lucose and p a l m i t a t e as a v a i l a b l e me tabo l i c subs t ra tes but no s i g n i f i c a n t d i f f e r e n c e between any o f the groups was found (F igu re 2 5 ) . Knowing t ha t d i a b e t i c p r e - p e r f u s i o n t r i g l y c e r i d e l e v e l s i n the hear t are h igh suggests t h a t d i a b e t i c hear ts are r e l y i n g on endogenous t r i g l y c e r i d e s t o r e s f o r energy p r o d u c t i o n . Th i s p r o p o s i t i o n i s f u r t h e r suppor ted by work done by Wal l and Lopaschuk (1989) who demonstrated a decrease i n g lucose o x i d a t i o n r a t e s i n d i a b e t i c hear ts per fused i n the absence o f added F F A ' s compared w i t h con t ro l hear ts per fused under the same c o n d i t i o n s . They suggest t ha t t h i s occurs due to an i nc reased metabol ism o f f a t t y a c i d s from endogenous t r i g l y c e r i d e s t o r e s . T h i s same group found t h a t the a d d i t i o n o f pa lm i ta te to the pe r fusa te o f d i a b e t i c hea r t s r e s u l t e d i n an almost complete suppress ion o f g lucose o x i d a t i o n . However, a concomitant i nc rease i n o x i d a t i o n o f f a t t y a c i d s was not found . As w e l l , i n the presence o f p a l m i t a t e , endogenous f a t t y a c i d o x i d a t i o n from t r i g l y c e r i d e s to res has not been shown t o be e l eva ted i n d i a b e t i c r a t hear ts (Paulson and C r a s s , 1982) . Together , t h i s i n f o rma t i on suggests t ha t d i a b e t i c hear ts are r e l y i n g ma in ly on endogenous s t o r e s o f t r i g l y c e r i d e s f o r energy p roduc t ion w i th on l y a smal l c o n t r i b u t i o n from g lucose o x i d a t i o n . 99 The f i n d i n g o f a dep ress i ve e f f e c t on +dP/dt i n d i a b e t i c hear ts pe r fused i n the presence o f FFA 's compared w i th d i a b e t i c hea r t s per fused i n the absence o f F F A ' s may be a t t r i b u t e d to f u r t h e r dep ress ion o f g lucose o x i d a t i o n from tha t which i s repor ted to a l r e a d y e x i s t i n the d i a b e t i c (Wal l and Lopaschuk, 1989). However, the presence o f F F A ' s d i d not a f f e c t HR, HR X PSP, LVDP or - d P / d t o f d i a b e t i c h e a r t s , sugges t ing t h a t energy from endogenous l i p i d s t o r e s was s u f f i c i e n t t o ma in ta in most aspec ts o f f u n c t i o n a t the l e v e l o f a d i a b e t i c hear t per fused i n the absence o f F F A ' s . Bu t , - d P / d t was a l ready very depressed i n the d i a b e t i c per fused i n the absence o f F F A ' s , t h e r e f o r e the a d d i t i o n o f F F A ' s would not show a f u r t h e r dep ress i ve e f f e c t . I n h i b i t i o n o f myocard ia l g lucose o x i d a t i o n i n the d i a b e t i c r e s u l t s from dep ress ion o f g lucose uptake as we l l as from dep ress ion by i n te rmed ia tes o f f a t t y a c i d metabo l ism. C i t r a t e accumula t ion i n h i b i t s phospho f ruc tok inase 1, r e s u l t i n g i n subsequent dep ress ion o f g l y c o g e n o l y s i s and g lucose uptake due to accumulat ion o f g l u c o s e - 6 -phosphate (Newsholme et a7 , 1962; Kerbey et a7, 1985). In a d d i t i o n , an i n c r e a s e i n the p r o d u c t s / s u b s t r a t e s r a t i o (ace ty l CoA/CoA) o f PDH, the f i r s t i r r e v e r s i b l e r e a c t i o n i n the m i tochondr ia l o x i d a t i v e pathway, r e s u l t s i n dep ress ion o f t h i s enzyme and subsequent dep ress ion o f g l ucose o x i d a t i o n (Gar land et a7 , 1962). The dep ress ion o f g lucose o x i d a t i o n by f a t t y a c i d metabol ism has l e d t o much resea rch i n t e r e s t regard ing the p o s s i b i l i t y o f i n c r e a s i n g g lucose o x i d a t i o n i n the d i a b e t i c h e a r t . The consequence o f t h i s i n t e r e s t has been the development o f a number o f hypoglycemic drugs which b l ock CPT 1, the enzyme r e s p o n s i b l e f o r c a t a l y z i n g the key 100 r e g u l a t o r y s tep i n f a t t y a c i d o x i d a t i o n . R e c e n t l y , drugs i n t h i s c l a s s , i n c l u d i n g POCA, the s u l f o n y l u r e a to lbu tamide and Etomox i r , have been demonstrated t o i n c r e a s e g lucose o x i d a t i o n r a tes i n i s o l a t e d work ing hear t s from c h r o n i c a l l y S T Z - d i a b e t i c r a t s (Rosen and Re inaue r , 1984; Tan et a7 , 1984; Wal l and Lopaschuk, 1989). In a d d i t i o n t o s t i m u l a t i n g g lucose o x i d a t i o n i n the presence and absence o f added p a l m i t a t e , Etomoxir was shown t o s i g n i f i c a n t l y improve hear t f u n c t i o n i n p a l m i t a t e -per fused d i a b e t i c r a t s (Wall and Lopaschuk, 1989). DCA i s another pharmacolog ica l agent which has proven capab le o f i n c r e a s i n g myocard ia l g lucose o x i d a t i o n . The f i r s t rev iew o f the pharmacolog ic and t h e r a p e u t i c e f f e c t s o f DCA was pub l i shed by S tacpoo le i n 1969. I t s repor ted p o t e n t i a l as a hypoglycemic agent made DCA the sub jec t o f seve ra l i n v e s t i g a t i o n s i n f o l l o w i n g y e a r s . I t i s now we l l e s t a b l i s h e d t ha t DCA i s an i n h i b i t o r o f the p r o t e i n k i nase i n v o l v e d i n the r e g u l a t i o n o f the PDH complex, a process which r e s u l t s i n PDH a c t i v a t i o n . DCA was o r i g i n a l l y demonstrated t o a c t i v a t e PDH i n r a t hear t (Whitehouse and Rand le , 1973) and was l a t e r found t o i n h i b i t the ATP-dependent phosphory la t ion and i n a c t i v a t i o n o f p u r i f i e d p i g hear t PDH by PDH k inase (Whitehouse and Rand le , 1974). I t became apparent t ha t a c t i v a t i o n o f PDH, by DCA, r e s u l t e d i n an i n c r e a s e o f g lucose o x i d a t i o n i n r a t hear ts ( M c A l l i s t e r e t a7, 1973; Higg ins e t a7 , 1978). R e c e n t l y , i t was shown tha t d i r e c t a d d i t i o n o f t h i s agent t o i s o l a t e d work ing d i a b e t i c r a t h e a r t s , per fused i n the presence o f F F A ' s , s t i m u l a t e s g lucose o x i d a t i o n by 5000% (Lopaschuk and McVeigh, personal communicat ion) . Thus, i t i s apparent t h a t DCA would 101 be use fu l i n i n v e s t i g a t i n g whether a dep ress ion o f g lucose o x i d a t i o n i s c o n t r i b u t i n g to depressed c a r d i a c f u n c t i o n i n the d i a b e t i c . We i n v e s t i g a t e d the e f f e c t s o f enhanced g lucose o x i d a t i o n on d i a b e t i c c a r d i a c f u n c t i o n by d i r e c t l y adding 0 .5 mM DCA to p a l m i t a t e -c o n t a i n i n g i s o l a t e d work ing hear t p e r f u s a t e . We were ab le t o demonstrate r e s t o r a t i o n t o c o n t r o l hear t va lues o f HR and HR X PSP i n spontaneous ly bea t ing d i a b e t i c hear ts per fused i n the presence o f DCA (F igu res 15, 16 ) . In paced h e a r t s , DCA was ab le t o r e s t o r e LVDP and -dP /d t i n d i a b e t i c hear ts t o c o n t r o l hear t l e v e l s a t t h ree out o f f i v e a f t e r l o a d pressures (F igu res 17-19, 2 1 ) . As w e l l , when per fused i n the presence o f DCA, +dP/dt va lues i n d i a b e t i c hear ts were s i m i l a r t o c o n t r o l hear t v a l u e s , at a l l a f t e r l o a d p ressures measured (F igu re 2 0 ) . In summary, r e s u l t s i n d i c a t e t ha t DCA-induced s t i m u l a t i o n o f g lucose o x i d a t i o n a c u t e l y reve rsed the depress ion o f d i a b e t i c c a r d i a c f u n c t i o n . Why g lucose o x i d a t i o n i s important i n the hear t o f the d i a b e t i c i s not we l l unders tood , but a few p o s s i b i l i t i e s have been proposed. A debatab le e x p l a n a t i o n i s t h a t g l y c o l y t i c a l l y produced ATP i n the c y t o s o l i s impor tant f o r membrane p r o t e i n s , such as the sarcolemmal ATPases ( B r i c k n e l l and Op ie , 1978). Bunger and co-workers (1986) o f f e r another theory t ha t i nc reased a c t i v i t y o f the PDH complex w i l l prevent the accumula t ion o f g l y c o l y t i c products such as l a c t a t e , which may accumulate under i schemic c o n d i t i o n s . Another e x p l a n a t i o n i s t ha t l e s s oxygen i s r e q u i r e d i n the p roduc t ion o f ATP when g lucose i s the s u b s t r a t e . I t i s c a l c u l a t e d tha t complete o x i d a t i o n o f g lucose produces 3.17 moles ATP/mole O2 whereas pa lm i ta te produces 2.80 moles ATP/mole O2 (Lehn inger , 1982). I t i s c l e a r t ha t more work needs to be done to 102 e l u c i d a t e the mechanism by which depressed g lucose o x i d a t i o n has a de t r imen ta l e f f e c t on d i a b e t i c hear t f u n c t i o n . The reason why DCA a c u t e l y reve rses the f u n c t i o n a l changes t h a t occur i n the c h r o n i c a l l y d i a b e t i c r a t hear t i s not c l e a r . P rev ious s t u d i e s have demonstrated tha t card iomyopath ic changes i n the d i a b e t i c r a t hear t p robab ly occur as a r e s u l t o f a l t e r a t i o n s i n enzymes i n v o l v e d i n e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g , i n c l u d i n g myosin and actomyosin ATPases a c t i v i t i e s (Malhot ra et al, 1981), SR Ca-ATPase and C a - t r a n s p o r t a c t i v i t y (Lopaschuk et al, 1983a; Penpargkul et a / , 1981) , sarcolemmal Ca-pump a c t i v i t y (Hey l i ge r et a / , 1987) and m i t ochond r i a l a c t i v i t y (Haugaard and Haugaard, 1964; P i e r c e and D h a l l a , 1987) . I f t h i s i s the c a s e , then these s t u d i e s demonstrate t ha t the e f f e c t s o f these changes i n enzymat ic a c t i v i t y on myocardia l f u n c t i o n can be masked by acu te s t i m u l a t i o n o f g lucose o x i d a t i o n . These s u b c e l l u l a r changes are s t i l l p robab ly present but not being mani fested as a dep ress ion i n hear t f u n c t i o n . The reason why a s t i m u l a t i o n o f g lucose o x i d a t i o n would overcome the card iomyopath ic changes i s not c l e a r . The f a c t t ha t hear t f u n c t i o n can be s i g n i f i c a n t l y i nc reased i n c h r o n i c a l l y d i a b e t i c r a t hea r t s by the acute a d m i n i s t r a t i o n o f E tomox i r , however, suppor ts the hypo thes i s t ha t acute metabo l i c changes i n d iabe tes are i n v o l v e d i n d i a b e t e s - i n d u c e d card iomyopath ies (Wall and Lopaschuk, 1989) . To d a t e , ve ry l i t t l e i s known about the r e l a t i o n s h i p between acu te changes i n myocard ia l energy subs t ra te u t i l i z a t i o n and the a c t i v i t i e s o f the enzymes i n v o l v e d i n e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g . I t i s c l e a r , however, t ha t c h r o n i c changes i n myocard ia l metabol ism a l t e r the a c t i v i t y o f these enzymes. Evidence f o r t h i s i s p rov ided by the 103 o b s e r v a t i o n s t ha t n o r m a l i z a t i o n o f va r i ous aspec ts o f f a t t y a c i d metabol ism (and the probable n o r m a l i z a t i o n o f g lucose o x i d a t i o n secondary t o these changes) reverse and prevent the changes i n hear t f u n c t i o n a s s o c i a t e d w i th d i a b e t e s , as we l l as the a c t i v i t i e s o f many o f the enzymes i n v o l v e d i n e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g (Rodr igues et al, 1986; Rodr igues et al, 1988; T a h i l i a n i and M c N e i l l , 1985; Lopaschuk et al, 1983b; X iang et al, 1988; Hey l i ge r et a / , 1986) . F o l l o w i n g p e r f u s i o n , we measured v e n t r i c u l a r t i s s u e t r i g l y c e r i d e l e v e l s i n d i a b e t i c and con t ro l hear ts and found a t rend towards l e v e l s be ing i n c r e a s e d i n d i a b e t i c hear ts per fused i n the presence o f DCA. However, the i n c r e a s e was not s t a t i s t i c a l l y s i g n i f i c a n t (F i gu re 2 9 ) . We had reasoned t h a t an i n c r e a s e i n t r i g l y c e r i d e l e v e l s i n d i a b e t i c hea r t s f o l l o w i n g p e r f u s i o n i n the presence o f DCA would i n d i c a t e t ha t the d i a b e t i c hear t was us ing g lucose as an energy source r a t h e r than r e l y i n g on endogenous s t o r e s o f t r i g l y c e r i d e s , as i s suggested to occur i n the absence o f DCA (Wall and Lopaschuk, 1989). However, we were unable t o suppor t our h y p o t h e s i s . One o f the me tabo l i c in te rmed ia tes o f f a t t y a c i d metabo l ism, LCC, has been shown to be e leva ted i n d i a b e t i c myocardium (Lopaschuk et al, 1983a) and has been shown to i n t e r f e r e w i th a number o f c r i t i c a l membrane p r o t e i n s (Lopaschuk et al, 1983 1 ; P i t t s and Okhuysen, 1984; Shug et a / , 1975; Adams et al, 1979). Hence, accumula t ion o f LCC has been i m p l i c a t e d as a c o n t r i b u t o r to depressed c a r d i a c f u n c t i o n du r i ng d i a b e t e s . We measured p o s t - p e r f u s i o n LCC l e v e l s o f the myocardium o f c o n t r o l s and d i a b e t i c s per fused i n the presence and absence o f pa lm i t a t e and DCA and found no s i g n i f i c a n t changes between any o f the groups 104 (F igu res 24, 2 8 ) . Th i s ev idence suggests t ha t t h i s i n te rmed ia te o f f a t t y a c i d metabol ism i s not the major c o n t r i b u t o r t o depressed d i a b e t i c hear t f u n c t i o n . An impor tant ques t i on i s whether a decrease i n myocard ia l ATP l e v e l s i n c h r o n i c a l l y d i a b e t i c r a t s i s c o n t r i b u t i n g t o a dep ress ion o f f u n c t i o n . P rev ious s t u d i e s have suggested t h a t reduced myocard ia l f u n c t i o n i n d i abe tes i s assoc i a t ed w i t h a l o s s o f ATP and adenine n u c l e o t i d e s (P iepe r et al, 1984; Rosen et al, 1986) . However, i n one o f these s t u d i e s , a l l measured l e v e l s o f ATP were ex t reme ly low (P iepe r et al, 1984). In c o n t r a s t , Lopaschuk and Spa f fo rd ( i n p ress ) found ATP l e v e l s were not depressed i n hear ts from c h r o n i c a l l y d i a b e t i c r a t s per fused w i t h p a l m i t a t e . L i k e w i s e , we demonstated t ha t p o s t - p e r f u s i o n l e v e l s o f both ATP and c r e a t i n e phosphate were s i m i l a r i n the myocardium o f c o n t r o l and d i a b e t i c r a t s r ega rd less o f a v a i l a b l e s u b s t r a t e s o r the presence o f DCA (F igu res 22 , 2 6 ) . Th i s ev idence suggests t h a t energy a v a i l a b i l i t y i s not the l i m i t i n g f a c t o r i n d i a b e t i c hear t f u n c t i o n . However, the query as to the r e l a t i o n s h i p between ATP l e v e l s and myocard ia l f u n c t i o n i n the c h r o n i c d i a b e t i c r e q u i r e s f u r t h e r i n v e s t i g a t i o n . In summary, we demonstrated tha t F F A ' s had no de t r imen ta l e f f e c t s on c o n t r o l o r 6-week d i a b e t i c i s o l a t e d work ing r a t h e a r t s , w i t h the excep t i on o f a dep ress i ve a c t i o n on +dP/dt o f d i a b e t i c hea r t s and - d P / d t o f c o n t r o l h e a r t s . We a l s o revea led tha t a d d i t i o n o f the g lucose o x i d a t i o n s t i m u l a t o r , DCA, to p a l m i t a t e - p e r f u s e d d i a b e t i c i s o l a t e d work ing hear t s r e s u l t e d i n a marked enhancement o f c a r d i a c f u n c t i o n . Th i s acute r e v e r s a l o f c h r o n i c c a r d i a c dep ress ion i n the d i a b e t i c , by 105 DCA, suggests t ha t i n h i b i t i o n o f g lucose o x i d a t i o n , p o s s i b l y due to h igh l e v e l s o f c i r c u l a t i n g and endogenous f a t t y a c i d s , p o t e n t i a t e s c a r d i a c f u n c t i o n depress ion i n the c h r o n i c d i a b e t i c . I t i s apparent , from t h i s s tudy , t ha t good m e t a b o l i c c o n t r o l o f the d i a b e t i c , r e s u l t i n g i n n o r m a l i z a t i o n o f myocard ia l f a t t y a c i d metabol ism and, t h e r e f o r e , n o r m a l i z a t i o n o f g lucose metabo l i sm, may be b e n e f i c i a l to d i a b e t i c hear t f u n c t i o n . Ch ron i c c o n t r o l o f f a t t y a c i d metabol ism has been proven to l e ssen the s e v e r i t y o f card iomyopathy i n d i a b e t i c animals and, as we have shown, r e v e r s a l o f g lucose o x i d a t i o n dep ress ion r e s u l t s i n enhancement o f c a r d i a c f u n c t i o n i n d i a b e t i c r a t s . Toge ther , t h i s i n fo rma t ion suggests t ha t l ong - te rm pharmaco log ica l the rapy aimed a t c o n t r o l l i n g and n o r m a l i z i n g f a t t y a c i d metabol ism i s an impor tant c l i n i c a l g o a l . 106 CONCLUSIONS: 1. S i x weeks f o l l o w i n g STZ t a i l v e i n i n j e c t i o n , r a t s d i s p l a y e d c h a r a c t e r i s t i c s o f d iabe tes i n c l u d i n g hyperg lycemia , h y p e r 1 i p i d e m i a , and decreased body weight ga in compared w i th age-matched c o n t r o l r a t s . 2 . D i a b e t i c an imals a l s o e x h i b i t e d c a r d i a c d y s f u n c t i o n as assessed by a dep ress ion i n HR, HR X PSP, LVDP, +dP/dt and - d P / d t i n the i s o l a t e d work ing h e a r t . 3 . The presence o f 1.2 mM pa lm i ta te i n the i s o l a t e d work ing hear t pe r f usa te had no e f f e c t on HR, HR X PSP, or LVDP o f c o n t r o l o r d i a b e t i c r a t h e a r t s , but had a dep ress i ve a c t i o n on +dP/dt o f d i a b e t i c hear ts and - d P / d t o f c o n t r o l h e a r t s . 4 . A d d i t i o n o f 0 .5 mM DCA to p a l m i t a t e - c o n t a i n i n g i s o l a t e d work ing hear t p e r f u s a t e a c u t e l y reversed d i a b e t i c c a r d i a c f u n c t i o n dep ress ion as assessed by i nc reased HR, HR X PSP, LVDP, +dP/dt and - d P / d t . 5 . P o s t - p e r f u s i o n v e n t r i c u l a r t i s s u e ATP, c r e a t i n e phosphate, LCC and t r i g l y c e r i d e l e v e l s were s i m i l a r r ega rd l ess o f the presence o f d i abe tes or o f the presence o f pa lm i ta te or DCA i n the i s o l a t e d work ing hear t p e r f u s a t e . 107 REFERENCES Adams, R . J . , Cohen, D.W., Gupte, J . , Johnson, D . , W a l l i c k , E . T . , Wang, T. and Schwar tz , A . 1979. In v i t r o e f f e c t s o f p a l m i t y l c a r n i t i n e on c a r d i a c plasma membrane N a - , K-ATPase, and sa rcop lasm ic r e t i c u l u m Ca - t r a n s p o r t . J . B i o l . Chem. 254:12404-12410. Bergmeyer, H e i n s - U l r i c h . 1963. 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