Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Diabetes-induced changes in cardiac sarcoplasmic reticulum function Lopaschuk, Gary David 1983

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1983_A1 L66.pdf [ 7.9MB ]
Metadata
JSON: 831-1.0095881.json
JSON-LD: 831-1.0095881-ld.json
RDF/XML (Pretty): 831-1.0095881-rdf.xml
RDF/JSON: 831-1.0095881-rdf.json
Turtle: 831-1.0095881-turtle.txt
N-Triples: 831-1.0095881-rdf-ntriples.txt
Original Record: 831-1.0095881-source.json
Full Text
831-1.0095881-fulltext.txt
Citation
831-1.0095881.ris

Full Text

DIABETES-INDUCED CHANGES IN CARDIAC SARCOPLASMIC RETICULUM FUNCTION by GARY DAVID LOPASCHUK M S c , The U n i v e r s i t y of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES i n THE FACULTY OF PHARMACEUTICAL SCIENCES DIVISION OF PHARMACOLOGY AND TOXICOLOGY We accept t h i s t h e s i s as conforming to the re q u i r e d standards THE © UNIVERSITY OF BRITISH COLUMBIA A p r i l 1983 GARY DAVID LOPASCHUK, 1983 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f Pharmacology and Toxicology The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date March 22, 1983. DE-6 (3/81) - i -ABSTRACT A prominent f i n d i n g i n the d i a b e t i c r a t heart i s a decrease i n the rate at which the v e n t r i c u l a r muscle can c o n t r a c t and r e l a x . Since c a r d i a c sarcoplasmic r e t i c u l u m i s thought to be i n t i m a t e l y i n v o l v e d i n muscle c o n t r a c t i o n and r e l a x a t i o n we s t u d i e d the a b i l i t y of d i a b e t i c r a t c a r d i a c 2+ sarcop l a s m i c r e t i c u l u m to t r a n s p o r t Ca . Hearts were obtained from female Wistar r a t s 7, 30, 42, and 120 days a f t e r the i n d u c t i o n of di a b e t e s by a s i n g l e i . v . i n j e c t i o n of e i t h e r a l l o x a n (65 mg/kg) or s t r e p t o z o t o c i n (60 mg/kg). At a l l 2+ 2+ Ca c o n c e n t r a t i o n s t e s t e d (0.2 uM-5.0 uM f r e e Ca ) c a r d i a c sarcoplasmic r e t i c u l u m obtained from 42 and 120 day d i a b e t i c r a t s showed a s i g n i f i c a n t decrease i n the rate of 2+ ATP-dependent t n s - o x a l a t e f a c i l i t a t e d Ca t r a n s p o r t . T h i s 2+ was accompanied by a decrease i n Ca -ATPase a c t i v i t y . The l e v e l s of long chain a c y l c a r n i t i n e s a s s o c i a t e d with the microsomal sarcoplasmic r e t i c u l u m p r e p a r a t i o n from 42 and 120 day d i a b e t i c r a t s were s i g n i f i c a n t l y higher than those present i n s arcoplasmic r e t i c u l u m from c o n t r o l r a t s . P a l m i t y l c a r n i t i n e , the most abundant of the long c h a i n a c y l c a r n i t i n e s , i n c o n c e n t r a t i o n s < 7 uM was found to be a 2+ potent time-dependent i n h i b i t o r of both Ca t r a n s p o r t and 2+ Ca -ATPase i n both c o n t r o l and d i a b e t i c r a t sarcoplasmic 2+ r e t i c u l u m p r e p a r a t i o n s ; i n h i b i t i o n of Ca t r a n s p o r t was found to be more marked i n the c o n t r o l p r e p a r a t i o n s . T h i s would i n d i c a t e that a degree of i n h i b i t i o n produced by the high endogenous l e v e l s of p a l m i t y l c a r n i t i n e may al r e a d y be - i i -present i n the d i a b e t i c r a t heart p r e p a r a t i o n s . Cardiac sarcoplasmic r e t i c u l u m prepared from a c u t e l y d i a b e t i c r a t s (7 2+ days) d i d not show any decrease i n Ca t r a n s p o r t a b i l i t y . L e v e l s of long c h a i n a c y l c a r n i t i n e s a s s o c i a t e d with the microsomal p r e p a r a t i o n enriched i n sarcoplasmic r e t i c u l u m were a l s o unchanged. I n s u l i n treatment of d i a b e t i c r a t s could s i g n i f i c a n t l y i n c r e a s e the a b i l i t y of c a r d i a c sarcoplasmic 2+ r e t i c u l u m to t r a n s p o r t Ca , although at the time p e r i o d ob-t e s t e d (30 days) the SR Ca t r a n s p o r t a c t i v i t y was only s l i g h t l y depressed as compared to c o n t r o l . I n s u l i n treatment a l s o r e s u l t e d i n a s l i g h t , but n o n - s i g n i f i c a n t , lowering of the l e v e l s of long chain a c y l c a r n i t i n e s a s s o c i a t e d with the sarcoplasmic r e t i c u l u m microsomal p r e p a r a t i o n s . These f i n d i n g s suggest that the a l t e r a t i o n i n sarcoplasmic r e t i c u l u m f u n c t i o n 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 may be due to the b u i l d u p of c e l l u l a r long chain a c y l c a r n i t i n e s which i n h i b i t 2+ sarcoplasmic r e t i c u l u m Ca t r a n s p o r t . The absence of any 2+ s i g n i f i c a n t change i n Ca t r a n s p o r t a c t i v i t y or l e v e l s of long chain a c y l c a r n i t i n e s at 7 and 30 days suggests that the a l t e r a t i o n s i n 42 and 120 day d i a b e t i c r a t s must be of gradual onset. C a r d i a c sarcoplasmic r e t i c u l u m i s known to be r e g u l a t e d by a number of f a c t o r s , among them calmodulin, cAMP-dependent + 2+ p r o t e i n k i n a s e , and K . Since Ca t r a n s p o r t a c t i v i t y i n c a r d i a c sarcoplasmic r e t i c u l u m from 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 depressed, the r o l e t h at these r e g u l a t o r s play was i n v e s t i g a t e d . Calmodulin ( 0.61 uM), cAMP (10 uM) plus cAMP-dependent p r o t e i n kinase (0.2 mg/0.5 ml), and K + (0-110 — i i i — mM) a l l s t i m u l a t e d Ca t r a n s p o r t i n both c o n t r o l and s t r e p t o z o t o c i n - t r e a t e d d i a b e t i c r a t s to the same degree. T h i s suggests that the d e p r e s s i o n observed i n sarcoplasmic r e t i c u l u m f u n c t i o n from d i a b e t i c r a t s i s not due to a l t e r e d 2+ r e g u l a t i o n by these p u t a t i v e mediators of Ca uptake. A number of s t u d i e s suggest that c a r n i t i n e a d m i n i s t r a t i o n may lower myocardial l e v e l s of long chain a c y l c a r n i t i n e s i n the d i a b e t i c r a t . T h e r e f o r e , D , L - c a r n i t i n e (1 g/kg/day, o r a l l y ) was administered to 120 day d i a b e t i c r a t s f o r a 30 day p e r i o d . The e l e v a t e d l e v e l s of long chain a c y l c a r n i t i n e s normally seen i n d i a b e t i c r a t s were s i g n i f i c a n t l y reduced i n the d i a b e t i c r a t s administered c a r n i t i n e . C a r n i t i n e a d m i n i s t r a t i o n , however, could not reverse the p r e v i o u s l y noted d e p r e s s i o n i n d i a b e t i c r a t heart f u n c t i o n , as measured on an i s o l a t e d working heart apparatus. In an e f f o r t to prevent the onset of the d i a b e t i c cardiomyopathy D , L - c a r n i t i n e was administered (3 g/kg/day, o r a l l y ) 3 days a f t e r the i n d u c t i o n of d i a b e t e s f o r a 42 day p e r i o d . As p r e v i o u s l y 2+ mentioned, sarcoplasmic r e t i c u l u m Ca t r a n s p o r t a c t i v i t y was depressed i n d i a b e t i c r a t s , as compared to c o n t r o l r a t s , 2+ at a l l f r e e Ca c o n c e n t r a t i o n s t e s t e d (0.1 uM-3.5 uM). S i m i l a r l y , sarcoplasmic r e t i c u l u m l e v e l s of long c h a i n a c y l c a r n i t i n e s were s i g n i f i c a n t l y e l e v a t e d i n these d i a b e t i c r a t s . The d i a b e t i c r a t s t r e a t e d with c a r n i t i n e d i d not show 2+ any d e p ression i n Ca t r a n s p o r t a c t i v i t y ; long chain a c y l c a r n i t i n e l e v e l s were a l s o s i m i l a r to c o n t r o l . The c a r n i t i n e - t r e a t e d d i a b e t i c r a t s , however, showed no improvement i n heart f u n c t i o n compared to u n t r e a t e d - d i a b e t i c - i v -r a t s . These data suggest that although the long chain a c y l c a r n i t i n e s are i n h i b i t i n g c a r d i a c sarcoplasmic r e t i c u l u m f u n c t i o n 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 other f a c t o r s must a l s o be c o n t r i b u t i n g to the depression i n heart f u n c t i o n . John H. M c N e i l l , PhD. A s s o c i a t e Dean,Fac. Pharm. S c i . Sidney Katz, PhD. A s s o c i a t e P r o f e s s o r , Fac. Pharm. S c i . -V-ACKNOWLEDGEMENTS I am deeply g r a t e f u l to my s u p e r v i s o r s , Dr. J.H. M c N e i l l and Dr. S. Katz, f o r t h e i r guidance and support throughout t h i s study. I would l i k e to thank my committee members (Drs. B.D. R o u f o g a l i s , Dr. J . Diamond, Dr. R.A. Pederson, and Dr. D. Godin) f o r t h e i r c o n s t r u c t i v e c r i t i c i s m and suggestions during t h i s study. I would a l s o l i k e to thank Dr. J.T. Penniston, Dept. of Biochemistry, Mayo C l i n i c , f o r a l l o w i n g me to spend 4 months working i n h i s l a b o r a t o r y . I would l i k e to acknowledge the generous g i f t of L - p a l m i t y l c a r n i t i n e from Dr. P. Hahn. I am indebted to the Canadian Heart Foundation f o r f i n a n c i a l support throughout t h i s study period.. I would l i k e to thank Mr. R. Vadlamudi, Dr. M. Bri d g e s , Mr. B. E i b s c h u t z , and the r e s t of my l a b o r a t o r y c o l l e a g u e s f o r t h e i r t e c h n i c a l a s s i s t a n c e and moral support. F i n a l l y , I a l s o wish to thank a l l members of the f a c u l t y , s t a f f , and graduate student body i n the F a c u l t y of Pharmaceutical S c i e n c e s , U.B.C., f o r making t h i s d o c t o r a l program e n j o y a b l e . - v i -DEDICATION To my parents, f o r a l l they have done - v i i -TABLE OF CONTENTS Page ABSTRACT i ACKNOWLEDGEMENTS v DEDICATION v i TABLE OF CONTENTS v i i LIST OF FIGURES X LIST OF TABLES x i i LIST OF ABBREVIATIONS x i v INTRODUCTION 1 LITERATURE REVIEW 3 A) DIABETES AND CARDIOVASCULAR DISEASE 3 1) Angiopathies 6 2) Autonomic neuropathies 8 3) Metabo l i c changes 10 B) DIABETIC CARDIOMYOPATHY 13 C) ALLOXAN AND STREPTOZOTOCIN DIABETES 19 D) CARDIAC SARCOPLASMIC RETICULUM CALCIUM UPTAKE ACTIVITY 21 E) OBJECTIVES OF THE STUDY 24 MATERIALS AND METHODS 26 A) MATERIALS 26 1) Radioisotopes 26 2) Enzymes 26 3) Chemicals 26 4) Apparatus 28 METHODS 29 A) ANIMALS 29 1) I n s u l i n treatment u t i l i z i n g osmotic minipumps 29 2) D , L - c a r n i t i n e treatment 31 - v i i i -PREPARATION OF CARDIAC MICROMES ENRICHED WITH SARCOPLASMIC RETICULUM 32 1) Method 1 32 2) Method 2 33 MEASUREMENT OF CALCIUM UPTAKE BY CARDIAC MICROSOMES ENRICHED IN SARCOPLASMIC RETICULUM VESICLES 34 2+ 1) Measurement of c a r d i a c SR Ca t r a n s p o r t a c t i v i t y 34 2) C a l c u l a t i o n of calcium uptake a c t i v i t y by c a r d i a c microsomes enriched i n sarcoplasmic r e t i c u l u m 35 MEASUREMENT OF (CA 2 +-MG 2 +)-ATPASE ACTIVITY IN CARDIAC MICROSOMES ENRICHED IN SARCOPLASMIC RETICULUM 36 MEASUREMENT OF CALCIUM EFFLUX FROM C A 2 + LOADED CARDIAC SARCOPLASMIC RETICULUM 37 WORKING HEART PERFUSION ' 38 DETERMINATION OF CARNITINE AND LONG CHAIN ACYL-CARNITINE LEVELS 40 A) SAMPLE PREPARATION 40 1) Microsomal SR 40 2) V e n t r i c u l a r t i s s u e 40 3) Serum 41 B) ASSAY TO DETERMINE CARNITINE LEVELS 41 GLYCOSYLATED HEMOGLOBIN ASSAY 43 CYTOCHROME C OXIDASE 44 ELECTRON MICROSCOPY OF CARDIAC SARCOPLASMIC RETICULUM VESICLES 45 PROTEIN ASSAY 45 STATISTICAL ANALYSIS 46 RESULTS 47 A) THE CHEMICALLY-INDUCED DIABETIC RAT MODEL 47 B) CHARACTERIZATION OF CARDIAC SARCOPLASMIC RETICULUM FROM CONTROL AND DIABETIC RATS 51 C) EFFECT OF CHRONIC DIABETES ON CARDIAC MICROSOMAL SARCOPLASMIC RETICULUM FUNCTION 63 - i x -1) Calcium uptake and ca l c i u m ATPase a c t i v i t y 63 i) C a r n i t i n e and long c h a i n a c y l c a r n i t i n e s i n c a r d i a c microsomal sarcoplasmic r e t i c u l u m 63 i i ) Chronic d i a b e t e s and the p e r m e a b i l i t y of microsomal sarcoplasmic r e t i c u l u m to ca l c i u m 76 i i i ) E f f e c t of calm o d u l i n , cAMP-dep_endent p r o t e i n k i nase, and K on Ca -uptake a c t i v i t y i n c a r d i a c SR from c h r o n i c a l l y d i a b e t i c r a t s . 76 iv) E f f e c t of acute d i a b e t e s giji microsomal sarcoplasmic r e t i c u l u m Ca , and s a r c o -plasmic r e t i c u l u m l e v e l s of c a r n i t i n e and long chain a c y l c a r n i t i n e s . 80 v) E f f e c t of i n s u l i n treatment on c a r d i a c sarcoplasmic r e t i c u l u m f u n c t i o n i n one month s t r e p t o z o t o c i n d i a b e t i c r a t s . 85 D) EFFECT OF ORAL CARNITINE TREATMENT ON DIABETIC RAT HEART FUNCTION; PREVENTION AND REVERSAL STUDIES 102 1) Reversal study 104 2) Prevention study 104 DISCUSSION 127 A) EFFECTS OF DIABETES ON CARDIAC SR FUNCTION 130 B) MECHANISMS BY WHICH DIABETES MAY ALTER CARDIAC SR FUNCTION 131 1) Diabetes and the r e g u l a t i o n of c a r d i a c SR f u n c t i o n 132 2) Diabetes-induced metabolic changes and c a r d i a c SR f u n c t i o n 135 C) EFFECT OF INSULIN AND CARNITINE TREATMENT ON DIABETIC RAT CARDIAC SR FUNCTION 143 1) I n s u l i n treatment 143 2) C a r n i t i n e treatment 144 CONCLUSIONS 157 BIBLIOGRAPHY 159 - X -LIST OF FIGURES  FIGURE 1. Serum l e v e l s of i n s u l i n and glucose from a c u t e l y (7 days) and c h r o n i c a l l y (120 days) d i a b e t i c r a t s 48 2+ 2. Time course of Ca -uptake i n c a r d i a c s arcoplasmic r e t i c u l u m p r e p a r a t i o n s from c o n t r o l and c h r o n i c a l l y d i a b e t i c r a t s (120 days) 54 3. E l e c t r o n micrograph of c a r d i a c s a r c o -plasmic r e t i c u l u m obtained from c o n t r o l r a t s 57 4. E l e c t r o n micrograph of c a r d i a c s a r c o -plasmic r e t i c u l u m obtained from s i x week d i a b e t i c r a t s 59 5. E f f e c t of c h r o n i c d i a b e t e s (120 days) on c a r d i a c s a r c o p l a s m i c 2 ^ e t i c u l u m Ca -uptake at v a r i o u s Ca c o n c e n t r a t i o n s 64 2+ 6. E f f e c t of L - p a l m i t y l c a r n i t i n e on Ca -uptake i n sarcoplasmic r e t i c u l u m prep-a r a t i o n s from c h r o n i c a l l y d i a b e t i c r a t s (120 days) 69 2+ 7. E f f e c t of L - p a l m i t y l c a r n i t i n e on (Ca -Mg )-ATPase a c t i v i t y i n sarcoplasmic r e t i c u l u m p r e p a r a t i o n s from c h r o n i c a l l y d i a b e t i c r a t s (120 days) 72 2+ 8. E f f e c t of D , L - c a r n i t i n e on Ca -uptake i n sarcoplasmic r e t i c u l u m p r e p a r a t i o n s from c h r o n i c a l l y d i a b e t i c r a t (120 days) 74 9. Effec£ +of c h r o n i c d i a b e t e s (120 days) on Ca e f f l u x from c a r d i a c s a r c o -plasmic r e t i c u l u m . 77 10. E f f e c t of v a r i o u s KC1 c o n c e n t r a t i o n s on c a l c i u m uptake i n sarcoplasmic r e t i c u l u m from c o n t r o l and s t r e p t o z o t o c i n - t r e a t e d d i a b e t i c r a t s 81 11. An Eadie-Hofstee p l o t of the e f f e c t s of v a r i o u s KC1 c o n c e n t r a t i o n s on c a l c i u m uptake i n sarcoplasmic r e t i c u l u m prep-a r a t i o n s from c o n t r o l and s t r e p t o z o t o c i n -t r e a t e d d i a b e t i c r a t s 83 - X I -12. E f f e c t of acute d i a b e t e s (7 days) on Ca -uptake i n c a r d i a c sarcoplasmic 9 r e t i c u l u m p r e p a r a t i o n s at v a r i o u s Ca c o n c e n t r a t i o n s 86 2+ 13. E f f e c t of L - p a l m i t y l c a r n i t i n e on Ca uptake i n c a r d i a c sarcoplasmic r e t i c u l u m p r e p a r a t i o n s from a c u t e l y d i a b e t i c r a t s (7 days) 89 2+ 14. E f f e c t of D f L - c a r n i t i n e on Ca -uptake i n c a r d i a c sarcoplasmic r e t i c u l u m p r e p a r a t i o n s from the a c u t e l y d i a b e t i c r a t (7 days) 91 15. Serum glucose l e v e l s of c o n t r o l , d i a b e t i c and i n s u l i n - t r e a t e d d i a b e t i c r a t s 94 16. E f f e c t of di a b e t e s (31 days) on c a r d i a c sarcoplasmic r e t i c u l u m Ca -uptake at v a r i o u s Ca c o n c e n t r a t i o n s 98 17. E f f e c t of c h r o n i c d i a b e t e s and c a r n i t i n e treatment on l e f t v e n t r i c u l a r developed pressure of i s o l a t e d perfused working hearts at v a r i o u s f i l l i n g p ressures 106 18. E f f e c t of c h r o n i c d i a b e t e s and c a r n i t i n e treatment on p o s i t i v e dP/dT of i s o l a t e d perfused working hearts at v a r i o u s f i l l i n g p r e ssures 108 19. E f f e c t of c h r o n i c d i a b e t e s and c a r n i t i n e treatment on negative dP/dT of i s o l a t e d working hearts at v a r i o u s f i l l i n g p r e s s u r e s . . . . 110 20. E f f e c t of s i x week diabe t e s and c a r n i t i n e treatment on l e f t v e n t r i c u l a r developed pressure of i s o l a t e d perfused working hearts at v a r i o u s f i l l i n g p r essures 115 21. E f f e c t of s i x week di a b e t e s and c a r n i t i n e treatment on p o s i t i v e dP/dT of i s o l a t e d working hearts at v a r i o u s f i l l i n g p r e s s u r e s . . . . 117 22. E f f e c t of s i x week diabe t e s and c a r n i t i n e treatment on negative dP/dT of i s o l a t e d working hearts at v a r i o u s f i l l i n g p r e s s u r e s . . . . 119 23. E f f e c t of s i x we,^ k dia b e t e s and c a r n i t i n e treatment on Ca -uptake i n c a r d i a c sarcoplasmic r e t i c u l u m 124 24. Sequence of f a t t y a c i d metabolism i n the myocardial c e l l 136 - x i i -LIST OF TABLES TABLE 1. Body weights of c o n t r o l , s t r e p t o z o t o c i n , 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 at 7 days and 120 days a f t e r the i n d u c t i o n of d i a b e t e s 50 2. Measurement of g l y c o s y l a t i o n of hemoglobin i n r a t s at v a r i o u s time p e r i o d s a f t e r the i n d u c t i o n of d i a b e t e s with s t r e p t o z o t o c i n 52 3. Y i e l d of c a r d i a c sarcoplasmic r e t i c u l u m from c o n t r o l and c h r o n i c a l l y d i a b e t i c r a t s (120 days) 53 4. Marker enzyme assays on c a r d i a c sarcoplasmic r e t i c u l u m from c o n t r o l and 42 day d i a b e t i c r a t s 62 2+ 5. Measurement of Ca -ATPase a c t i v i t y i n c a r d i a c sarcoplasmic r e t i c u l u m prepared from c h r o n i c a l l y d i a b e t i c r a t s (120 days) 66 6. Measured l e v e l s of c a r n i t i n e and long c h a i n a c y l c a r n i t i n e s i n c a r d i a c microsomal prep-a r a t i o n s enriched i n sarcoplasmic r e t i c u l u m from c h r o n i c a l l y d i a b e t i c r a t s (120 days) 67 7. E f f e c t of K , c almodulin, and cAMP-depen-dent p r o t e i n kinase on c a l c i u m uptake a c t i v i t y i n c a r d i a c s a r c o p l s m i c r e t i c u l u m p r e p a r a t i o n s from c h r o n i c a l l y d i a b e t i c r a t s (120 days) 79 8. Measured l e v e l s of c a r n i t i n e and long chain a c y l c a r n i t i n e s i n c a r d i a c microsomal prep-a r a t i o n s enriched i n sarcoplasmic r e t i c u l u m from a c u t e l y d i a b e t i c r a t s (7 days) 88 9. Body weights of c o n t r o l , d i a b e t i c , and i n s u l i n - t r e a t e d d i a b e t i c r a t s 97 10. L e v e l s of c a r n i t i n e and long chain a c y l -c a r n i t i n e s i n c a r d i a c microsomal prepar-a t i o n s enriched i n sarcoplasmic r e t i c u l u m from 31 day d i a b e t i c , and i n s u l i n - t r e a t e d d i a b e t i c r a t s 101 11. Measurement of body weight, serum glucose l e v e l s , g l y c o s y l a t i o n of hemoglobin, and y i e l d of SR i n c o n t r o l and d i a b e t i c r a t s administered D, L - c a r n i t i n e 103 12. L e v e l s of f r e e c a r n i t i n e i n serum obtained from c h r o n i c a l l y d i a b e t i c and c o n t r o l r a t s t r e a t e d with c a r n i t i n e 105 - x i i i -13. L e v e l s of c a r n i t i n e and long c h a i n a c y l -c a r n i t i n e s from v e n t r i c l e of i s l o a t e d per-fused working h e a r t s from c o n t r o l and d i a b e t i c c a r n i t i n e - t r e a t e d r a t s 112 14. L e v e l s of f r e e c a r n i t i n e i n serum obtained from s i x week d i a b e t i c and c o n t r o l r a t s t r e a t e d with c a r n i t i n e 114 15. L e v e l s of c a r n i t i n e and long chain a c y l -c a r n i t i n e s from v e n t r i c l e of i s l o a t e d per-fused working hearts from c o n t r o l and d i a b e t i c c a r n i t i n e - t r e a t e d r a t s 121 16. C a r d i a c sarcoplasmic r e t i c u l u m l e v e l s of c a r n i t i n e and long chain a c y l c a r n i t i n e s from s i x week d i a b e t i c and c o n t r o l c a r n i t i n e -t r e a t e d r a t s 126 17. L e v e l s of c a r n i t i n e and long c h a i n a c y l -c a r n i t i n e s from v e n t r i c l e of perfused and non-perfused hearts from c o n t r o l and d i a b e t i c r a t s 123 L I S T OF A B B R E V I A T I O N S ADP a d e n o s i n e 5 ' - d i p h o s p h a t e ATP a d e n o s i n e 5 1 - t r i p h o s p h a t e A T P a s e a d e n o s i n e t r i p h o s p h a t a s e C c e n t i g r a d e 2+ 2+ 2+ (Ca - M g ) A T P a s e Mg - d e p e n d e n t c a l c i u m - s t i m u l a t e d A T P a s e cAMP c y c l i c a d e n o s i n e 5 ' - m o n o p h o s p h a t e CHD c o r o n a r y h e a r t d i s e a s e C i C u r i e cpm c o u n t s p e r m i n u t e E enzyme EDTA e t h y l e n e d i a m i n e t e t r a a c e t a t e EGTA e t h y l e n e g l y c o l - b i s - ( y S - a m i n o e t h y 1 e t h e r ) N , N ' - t e t r a - a c e t i c a c i d EP p h o s p h o r y l a t e d enzyme i n t e r m e d i a t e e t a l a n d o t h e r s FADH f l a v i n e a d e n i n e d i n u c l e o t i d e ( r e d u c e d f o r m ) HEPES N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N - 2 -e t h a n e s u l f o n i c a c i d g g r a m g a c c e l e r a t i o n o f g r a v i t y HMF h y d r o x y m e t h y l f u r f u r a l d e h y d e K C a a s s o c i a t i o n c o n s t a n t o f t h e enzyme f o r Ca2+ K , . d i s s o c i a t i o n c o n s t a n t d i s s Km M i c h a e l i s - M e n t e n c o n s t a n t LC l o n g c h a i n -XV-m m i l l i u micro M mo 1 a r mg s m i l l i g r a m min minute ml m i l l i l i t e r u l m i c r o l i t e r MW molecular weight NAD + nicotinamide adenine d i n u c l e o t i d e NADH nicotinamide adenine d i n u c l e o t i d e (reduced form) nmole nanomoles PEP phosphoenolpyruvate Pi i n o r g a n i c phosphate pmoles picomoles PMSF p a r a - m e t h y l s u l f o n y l f l u o r i d e RBC red blood c e l l S.E.M. standard e r r o r of the mean S.D. standard d e v i a t i o n SR sarcoplasmic r e t i c u l u m TCA t r i c h l o r o a c e t i c a c i d T r i s t r i s (hydroxymethyl aminomethane) VCa2+ maximum v e l o c i t y of Ca2+-transport Vmax maximum v e l o c i t y of enzyme r e a c t i o n % percent / per -1-INTRODUCTION Diabetes m e l l i t u s i s a l i f e - t h r e a t e n i n g d i s o r d e r which has been known to mankind f o r thousands of ye a r s . In 1921, two Canadian s c i e n t i s t s , F r e d r i c k Banting and Ch a r l e s Best, s u c c e s s f u l l y t r e a t e d the acute symptoms of di a b e t e s with an i n s u l i n - c o n t a i n i n g p a n c r e a t i c e x t r a c t from dogs. Despite t h i s major medical achievement the d i a b e t i c p a t i e n t s t i l l runs the r i s k of developing a number of s e r i o u s c h r o n i c c o m p l i c a t i o n s . These i n c l u d e r e n a l d i s o r d e r s , autonomic and p e r i p h e r a l neuropathies, r e t i n o p a t h i e s , and c a r d i a c and c e r e b r o v a s c u l a r problems. I t i s w e l l e s t a b l i s h e d that c a r d i o v a s c u l a r d i s e a s e and i t s c o m p l i c a t i o n s can be 4 to 20 times more p r e v a l e n t i n the d i a b e t i c than i n the gen e r a l p o p u l a t i o n (Goldenberg et a l , 1958; Marks, 1965; G a r c i a et a l , 1974). Although the d i a b e t i c p a t i e n t runs the r i s k of developing any one of the aforementioned c o m p l i c a t i o n s , m o r t a l i t y i s o f t e n the r e s u l t of c a r d i o v a s c u l a r c o m p l i c a t i o n s of the d i s o r d e r ( E p s t e i n et a l , 1965; Bennett, 1981) . The i n c i d e n c e of coronary heart d i s e a s e , c o n g e s t i v e heart f a i l u r e , and autonomic neuropathies of the heart are a l l i n c r e a s e d i n d i a b e t i c p a t i e n t s (Kannel et a l , 1974; Knowles, 1981; Dyrberg et a l , 1981). Recent c l i n i c a l and experimental, evidence suggests that the long term d i a b e t i c i s a l s o s u s c e p t i b l e to an obscure cardiomyopathy which cannot be a t t r i b u t e d to coronary a r t e r y disease or microangiopathies (Regan et a l , 1974; Kannel, 1978; Ledet, 1979). To date, the u n d e r l y i n g causes of the cardiomyopathy -2-are not understood. F u n c t i o n a l changes i n the heart o c c u r r i n g as a r e s u l t of the d i a b e t i c cardiomyopathies have been c h a r a c t e r i z e d u t i l i z i n g animal models of d i a b e t e s (Ingebretson et a l , 1980; Penpargkul et a l , 1980; Vadlamudi et a l , 1982). On the other hand s u b c e l l u l a r biochemical s t u d i e s performed on myopathic d i a b e t i c h e arts are few and do not adequately e x p l a i n the observed f u n c t i o n a l changes. The aim of t h i s research p r o j e c t , t h e r e f o r e , was to attempt to c o r r e l a t e f u n c t i o n a l changes r e s u l t i n g from d i a b e t i c cardiomyopathy with a l t e r a t i o n s i n s u b c e l l u l a r biochemical parameters. The biochemical s t u d i e s performed focused on sarcoplasmic r e t i c u l a r f u n c t i o n as w e l l as the intermediary metabolism of f a t t y a c i d s . -3-LITERATURE REVIEW Diabetes m e l l i t u s i s c h a r a c t e r i z e d by the presence of prolonged hyperglycemia. Often e x c e s s i v e l i p o l y s i s and ketogenesis are p r e s e n t . These metabolic e f f e c t s can be a t t r i b u t e d to a r e l a t i v e i n s u l i n d e f i c i e n c y , as a r e s u l t of e i t h e r an abnormality i n i n s u l i n s y n t h e s i s and r e l e a s e , or a decreased s e n s i t i v i t y to i n s u l i n at i t s s i t e s of a c t i o n . The e f f e c t s of i n s u l i n d e f i c i e n c y are widespread and i n v o l v e a number of organ systems such as l i v e r , muscle and adipose t i s s u e . In t h i s review the d i s c u s s i o n of the metabolic consequences of i n s u l i n d e f i c i e n c y w i l l be c o n f i n e d to those aspects known to d i r e c t l y or i n d i r e c t l y a f f e c t the heart muscle. A) DIABETES AND CARDIOVASCULAR DISEASE An a s s o c i a t i o n between d i a b e t e s and c a r d i o v a s c u l a r disease was f i r s t suggested by Levine (1922) who reported that angina p e c t o r i s occurred more f r e q u e n t l y i n p a t i e n t s with d i a b e t e s than the general p o p u l a t i o n . M o r b i d i t y and m o r t a l i t y s t a t i s t i c s have s i n c e confirmed that d i a b e t i c s run an i n c r e a s e d r i s k of developing a number of c a r d i o v a s c u l a r c o m p l i c a t i o n s . Indeed, coronary heart d i s e a s e (CHD), the most common c o m p l i c a t i o n i n d i a b e t e s , accounts f o r over h a l f the m o r t a l i t y i n the t o t a l d i a b e t i c p o p u l a t i o n (Bradley, 1971). T h i s compares to the general p o p u l a t i o n i n which 35% of deaths -4-are a t t r i b u t a b l e to CHD (Heart F a c t s , 1976). A number of s t u d i e s have been done on other p o p u l a t i o n groups, and although the numbers vary, the trend c o n t i n u e s . Palumbo ej; a l (1976) reported a 42% i n c i d e n c e of deaths due to CHD i n the d i a b e t i c p o p u l a t i o n , as compared to 22.5% i n the general p o p u l a t i o n . P e l l and D'Alonzo (1970) a t t r i b u t e d 49% of d i a b e t i c deaths to CHD, while 44% of deaths i n the normal p o p u l a t i o n were a l s o a t t r i b u t e d to CHD. The o v e r a l l i n c i d e n c e of CHD i n t h e i r d i a b e t i c group was however 2.8 times as p r e v a l e n t as i n the c o n t r o l group. T h i s o b s e r v a t i o n was confirmed i n the Framingham Heart Study ( G a r c i a , 1974) i n which the i n c i d e n c e of CHD was 3 times as p r e v a l e n t i n d i a b e t i c p a t i e n t s as compared to c o n t r o l p a t i e n t s . S t a t i s t i c a l s t u d i e s have a l s o revealed that female d i a b e t i c s do not e x h i b i t the decreased r i s k of developing CHD r e l a t i v e to males seen i n the n o n - d i a b e t i c p o p u l a t i o n . Autopsy s t u d i e s by Goldenberg et a l (1958) revealed a 2 f o l d i n c r e a s e d i n c i d e n c e of myocardial i n f a r c t i o n s i n female d i a b e t i c s compared to male d i a b e t i c s . S i m i l a r l y , B e l l (1960) noted that f a t a l CHD, normally twice as common i n n o n - d i a b e t i c males as females, was e q u a l l y p r e v a l e n t i n d i a b e t i c females. A number of s t u d i e s have a l s o demonstrated that the prognosis i s more s e r i o u s f o r d i a b e t i c s diagnosed as having CHD, than f o r n o n - d i a b e t i c s . The probab i l i t y of dying from an i n i t i a l myocardial i n f a r c t i o n i s twice as great i n the d i a b e t i c p a t i e n t as f o r n o n - d i a b e t i c s (Partamian and Bradley, 1965; S o l e r et a l , 1974) . The long term s u r v i v a l rate of -5-p a t i e n t s with angina i s a l s o lower i n the d i a b e t i c (Root and Graybel, 1931) . S u r p r i s i n g l y , the d i a b e t i c p a t i e n t encounters l e s s pain during a myocardial i n f a r c t i o n or angina attack (Bradley and Schonfeld, 1962) . The g r e a t e r i n c i d e n c e of neuropathies i n the d i a b e t i c may account f o r t h i s o b s e r v a t i o n . Other forms of c a r d i o v a s c u l a r d i s e a s e , besides CHD, are a l s o more common i n d i a b e t i c s . Congestive heart f a i l u r e i s two to f i v e times as p r e v a l e n t i n the d i a b e t i c p a t i e n t (Kannel et a l , 1974) . Autonomic neuropathies, which c o n t r i b u t e to 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 , occur with g r e a t e r frequency i n d i a b e t i c s (Dryberg et a l , 1981). The Framingham study (Kannel et a l , 1974) has revealed that the i n c i d e n c e of c e r e b r o v a s c u l a r a c c i d e n t s , such as s t r o k e s , is e l e v a t e d as w e l l . I t i s u n l i k e l y that any s i n g l e f a c t o r could account f o r the i n c r e a s e d i n c i d e n c e of c a r d i o v a s c u l a r c o m p l i c a t i o n s noted i n d i a b e t i c s . The r e l a t i v e c o n t r i b u t i o n of i n d i v i d u a l p a t h o l o g i c a l changes seen i n d i a b e t e s i s not c l e a r . In t h i s t h e s i s , t h e r e f o r e , each of the c o n t r i b u t i n g f a c t o r s w i l l be d i s c u s s e d s e p a r a t e l y . -6-1) Angiopathies Diabetes i s a w e l l known r i s k f a c t o r f o r the development of coronary s c l e r o s i s (Root and Sharkey, 1936; K e s s l e r , 1971). Diabetes-induced a n g i o p a t h i e s i n v o l v e both l a r g e coronary v e s s e l s (macroangiopathies) and smaller a r t e r i o l e s and c a p i l l a r i e s ( m i c r o a n g i o p a t h i e s ) . The l a r g e extramural coronary a r t e r i e s are a p p r e c i a b l y t h i c k e r i n d i a b e t i c hearts ( L e f k o v i t s , 1937); Goodale et a l , 1962) perhaps due to increased d e p o s i t i o n of calcium, loose connective t i s s u e , c o l l a g e n , l i p i d , e l a s t i c t i s s u e , and g l y c o p r o t e i n (Ledet, 1968; B e r t e l s e n , 1961). Data by C r a l l and Roberts (1978) a l s o c o n f i r m the presence of n e c r o s i s and d e p o s i t i o n of c h o l e s t e r o l c r y s t a l s at s i t e s of t h i c k e n i n g . Diabetes-induced metabolic a l e r a t i o n s such as h y p e r l i p i d e m i a , h y p e r t r i g l y c e r i d e m i a , hyperglycemia, and abnormal g l y c o p r o t e i n metabolism have been i m p l i c a t e d as the e t i o l o g i c a l f a c t o r s producing a r t e r i a l d i sease (Renolds, 1972) . Another r i s k f a c t o r i n the development of angiopathies i s hype r t e n s i o n , which i s q u i t e common i n d i a b e t i c p a t i e n t s . S t u d i e s by Factor et a l (1981) suggest that hypertension can a l s o c o n t r i b u t e to c a r d i a c d y s f u n c t i o n i n the absence of coronary a r t e r y d i s e a s e . D i e t a r y f a c t o r s such as high l i p i d intake have been suggested to p o t e n t i a t e d i a b e t i c macroangiopathies (Kalant et a l , 1964, Lehner et a l , 1971) , although recent work by Haider et a l (1981) does not s u b s t a n t i a t e t h i s . M i c r o a n g i o p a t h i e s have a l s o been i m p l i c a t e d as ca u s a l f a c t o r s i n c a r d i o v a s c u l a r disease induced by d i a b e t e s . -7-Microaneurysms, which may be important i n the pathogenesis of heart d i s e a s e , have been found i n the human d i a b e t i c heart by Factor et a l (1980) . The microangiopathies occur as a r e s u l t of g l y c o p r o t e i n and c o l l a g e n accumulation i n the small i n t r a m u r a l a r t e r i e s and c a p i l l a r i e s . In d i a b e t e s t h i s occurs with the same frequency as i n the l a r g e coronary a r t e r i e s (Blumenthal et a l , 1960; C r a l l and Roberts, 1978; F i s h e r et a l , 1979). Recent i n v i t r o s t u d i e s have shown that serum f a c t o r s i n the d i a b e t i c promote g l y c o p r o t e i n and p r o c o l l a g e n s y n t h e s i s by a r t e r i a l medial c e l l s ( Ledet et a l , 1976; Baandrup et a l , 1982). Although s c l e r o s i s i n the a r t e r i e s of the heart c o n t r i b u t e s to 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 i n d i a b e t i c s , other f a c t o r s appear to be i n v o l v e d . T h i s i s evident from c l i n i c a l s t u d i e s which demonstrate c a r d i a c d y s f u n c t i o n i n the absence of coronary a r t e r y d i s e a s e . For i n s t a n c e , Ledet (1976) could not r e l a t e small v e s s e l l e s i o n s to c a r d i a c pathology. S i m i l a r l y , Regan et a l (1978) demonstrated abnormal 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 p a t i e n t s o c c u r r i n g i n the absence of coronary s c l e r o s i s . Another study by V i h e r t et a l (1969) suggested that the s e v e r i t y of d i a b e t i c c a r d i o v a s c u l a r c o m p l i c a t i o n s could not be q u a n t i t a t i v e l y c o r r e l a t e d with the degree o f a t h e r e o s c l e r o s i s . Experimental animal s t u d i e s have a l s o shown a b n o r m a l i t i e s i n myocardial f u n c t i o n independent of a t h e r o s c l e r o s i s ( G i o c o m e l l i and Wiener, 1979; Regan et a l , 1974) . -8-2) Autonomic neuropathies Autonomic neuropathies that can lead to p e r s i s t e n t t a c h y c a r d i a are a common f i n d i n g i n diab e t e s ( C h r i s t e n s e n , 1972; Ewing et a l , 1974). In severe cases, t h i s may c o n t r i b u t e to a form of CHD r e f e r r e d to as sudden c a r d i a c death, or c a r d i o r e s p i r a t o r y a r r e s t (Page and Watkins, 1977; Watkins and McKay, 1980) . In the m a j o r i t y of d i a b e t i c s , however, autonomic neuropathy simply decreases the beat-to-beat v a r i a t i o n of the heart rate (Wheeler and Watkins, 1975; Bennett et a l , 1975). Beat-to-beat v a r i a t i o n s , i n normal h e a r t s , are a r e f l e x response to changes i n a c t i v i t y such as standing up or a l t e r i n g the r e s p i r a t o r y r a t e . Denervation of the parasympathetic nervous system (with a decrease i n vagal tone) appears to be the main f a c t o r c o n t r i b u t i n g to the abnormal c a r d i o v a s c u l a r r e f l e x e s i n d i a b e t i c s (Lloyd-Mostyn and Watkins, 1975). Recent work by P f e i f e r et a l (1982) suggests that parasympathetic nervous a c t i v i t y may be diminished i n d i a b e t i c s even before c l i n i c a l symptoms of autonomic neuropathy are pres e n t . Sympathetic neuropathies have a l s o been reported i n d i a b e t e s . In d i a b e t i c p a t i e n t s the presence of an autonomic neuropathy a l t e r s the c a r d i o v a s c u l a r response to catecholamine a d m i n i s t r a t i o n i n that i n f u s i o n of catecholamines leads to a s u p e r s e n s i t i v e response (Moorehouse et a l , 1966). C i r c u l a t i n g and t i s s u e l e v e l s of catecholamines are a l s o low i n d i a b e t i c p a t i e n t s with autonomic neuropathies (Neubauer and -9-C h r i s t e n s e n , 1976). The presence of autonomic neuropathies i n animal models of d i a b e t e s has not been w e l l documented. Foy and Lucus (1976) reported a depressed parasympathetic response i n a l l o x a n - d i a b e t i c r a t s . Stuesse et a l (1982), on the other hand, have demonstrated an accentuated vagal response i n these animals. T h i s d i s c r e p a n c y may be e x p l a i n e d by a recent study done by Vadlamudi and M c N e i l l (1983) . Using both a l l o x a n - and s t r e p t o z o t o c i n - d i a b e t i c r a t s , they have shown that the s e n s i t i v i t y of hearts to c h o l i n e r g i c agents v a r i e d during the course of diabetes.As the d u r a t i o n of d i a b e t e s increased hearts were f i r s t s u b s e n s i t i v e , then s u p e r s e n s i t i v e to c a r b a c h o l . P a r a l l e l i n g t h i s study, Vadlamudiand McNeill (1980) have a l s o demonstrated that i s o l a t e d d i a b e t i c h e arts are l e s s responsive to the c a r d i a c r e l a x a n t e f f e c t of exogenously administered a d r e n e r g i c agents ( i s o p r o t e r e n o l ) . How t h i s may r e l a t e to a sympathetic neuropathy i s s t i l l u n c l e a r . In d i a b e t e s , the development of neuropathy i s most l i k e l y secondary to a b n o r m a l i t i e s i n l i p i d or carbohydrate metabolism. For i n s t a n c e , accumulation of membranous l i p i d - r i c h bodies and g e n e r a l i z e d c e l l u l a r d i l a t a t i o n have been observed i n autonomic neuronal bodies (Duchen et a l , 1980). T h i s may i n p a r t be a consequence of s o r b i t o l and f r u c t o s e accumulation i n the c e l l (Anderson, 1980) . Demyelination of the vagus nerve and p r e g a n g l i o n i c f i b r e s are a l s o a common o b s e r v a t i o n ( K r i s t e n s s o n et a l , 1971; Olsson and Sourande, 1968). Duchen et a l (1980) have suggested -10-that inflammatory changes may be o c c u r r i n g at these s i t e s of d e m y e l i n a t i o n . 3) Metabolic changes The metabolic e f f e c t s of i n s u l i n are d i v e r s e and widespread. Consequently numerous metabolic a b n o r m a l i t i e s occur i n d i a b e t i c s , i n c l u d i n g a l t e r a t i o n s i n carbohydrate and l i p i d s y n t h e s i s and metabolism, as w e l l as abnormal p r o t e i n s y n t h e s i s and t r a n s p o r t . As would be expected, these changes have marked u l t r a s t r u c t u r a l and i n t r a c e l l u l a r e f f e c t s on the h e a r t . As mentioned e a r l i e r , u l t r a s t r u c t u r a l changes have been observed i n both the l a r g e extramural coronary a r t e r i e s and smaller i n t r a m u r a l a r t e r i o l e s of d i a b e t i c s . S i m i l a r but more d i f f u s e changes have a l s o been observed across the myocardium. H i s t o p a t h o l o g i c a l s t u d i e s on d i a b e t i c hearts have revealed d i f f u s e f i b r o t i c strands extending between muscle f i b e r s , as w e l l as m y o f i b r i l l a r hypertrophy (Rubier et a l , 1972). Factor et a l (1981), using d i a b e t i c r a t s , have r e c e n t l y demonstrated that the degree of t h i s f i b r o s i s i n c r e a s e s to an even g r e a t e r extent i n the presence of h y p e r t e n s i o n . Regen et a l (1975,1978) have a l s o i d e n t i f i e d i n t e r s t i t i a l c o l l a g e n o s i s throughout the muscle area of both d i a b e t i c human and d i a b e t i c dog h e a r t s . In a d d i t i o n , they reported what appears to be i n c r e a s e d amounts of g l y c o p r o t e i n a s s o c i a t e d with the myocardial t i s s u e of d i a b e t i c p a t i e n t s . T h i s d e p o s i t i o n of c o l l a g e n (and g l y c o p r o t e i n ) may p a r t i a l l y account f o r the abnormally s t i f f h e a rts observed i n d i a b e t i c s -11-(Regen, 1969). M u l t i p l e i n t r a c e l l u l a r a b n o r m a l i t i e s have been noted i n d i a b e t i c h e a r t s . I n s u l i n d e f i c i e n c y r e s u l t s i n a marked impairment of glucose t r a n s p o r t and u t i l i z a t i o n (Morgan et a l , 1961), as w e l l as increased metabolism of f a t t y a c i d s (Randle et a l , 1964). For i n s t a n c e , i n the normal heart approximately 60% of the t o t a l energy of metabolism i s s u p p l i e d by o x i d a t i o n of f a t t y a c i d s , and the other 40% by glucose and l a c t a t e u t i l i z a t i o n . In the d i a b e t i c heart o x i d a t i o n of f a t t y a c i d s may account f o r over 90% of the ATP p r o d u c t i o n (Randle, 1978). Consequent to t h i s are m u l t i p l e changes i n the biochemical pathways l e a d i n g to the p r o d u c t i o n of ATP. The number of metabolic a b n o r m a l i t i e s that can occur i n d i a b e t i c s have been summarized by Opie et a l (1979). These i n c l u d e d e f e c t s i n glucose uptake, g l y c o l y s i s , glycogen metabolism, g l y c e r i d e s y n t h e s i s , and p r o t e i n s y n t h e s i s . In a d d i t i o n , i n c r e a s e d uptake of f r e e f a t t y a c i d s , t r i g l y c e r i d e s , and ketone bodies o c c u r s . P a r a l l e l i n g the i n c r e a s e d u t i l i z a t i o n of f a t t y a c i d s as an energy s u b s t r a t e i s an expected i n c r e a s e i n the m e t a b o l i t e s a s s o c i a t e d with f a t t y a c i d metabolism (Denton and Randle, 1967). E l e v a t e d l e v e l s of these i n t e r m e d i a t e s may r e s u l t i n adverse e f f e c t s on other c e l l u l a r b i o c h e m i c a l f u n c t i o n s (Opie, 1979; Katz and Messineo, 1981). I t i s p o s s i b l e that such a l t e r a t i o n s could c o n c e i v a b l y c o n t r i b u t e to abnormal heart f u n c t i o n i n d i a b e t i c s . The s i g n i f i c a n c e of these i n t e r m e d i a t e s on o v e r a l l heart f u n c t i o n , -12-however, remains u n c l e a r . These metabolic changes (and t h e i r c o n t r i b u t i o n to d i a b e t i c cardiomyopathies) w i l l be f u r t h e r d i s c u s s e d l a t e r . -13-B) DIABETIC CARDIOMYOPATHY The term "cardiomyopathy" i s used to d e s c r i b e myocardial d y s f u n c t i o n which cannot be accounted f o r by the usual causes of heart d i s e a s e . I t i s d i s t i n c t from c a r d i o p a t h i e s i n that i t i n f e r s the lack of involvement of atherogenic mechanisms. Cardiomyopathies can be hemodynamically c h a r a c t e r i z e d as 1) co n g e s t i v e , 2) h y p e r t r o p h i c , 3) r e s t r i c t i v e , or 4) o b l i t e r a t i v e . The cardiomyopathy found i n d i a b e t i c s , however, does not f a l l i n t o any s i n g l e one of these c a t e g o r i e s , and i s , t h e r e f o r e , c l a s s i f i e d e t i o l o g i c a l l y as a " d i a b e t i c cardiomyopathy". As mentioned e a r l i e r , c a r d i a c d y s f u n c t i o n i n diabetes can occur i n the absence of l a r g e coronary a r t e r y d i s e a s e . The Framingham study (Garcia et a l , 1974) has concluded that "some form of cardiomyopathy was a s s o c i a t e d with diabetes as a r e s u l t of small v e s s e l d i s e a s e or metabolic d i s o r d e r " . Since then, Ledet (1976), Regen et a l (1978), and Shi r e y et a l (1980) have shown that microangiopathies have l i t t l e or no r e l a t i o n to the onset of d i a b e t i c cardiomyopathies. T h i s s t r o n g l y suggests that metabolic d i s o r d e r s c o n t r i b u t e to the cardiomyopathies noted i n d i a b e t i c s . I t i s t h e r e f o r e of i n t e r e s t , not onl y to f u r t h e r e l u c i d a t e what metabolic d i s o r d e r s are o c c u r r i n g i n the myocardium of d i a b e t i c s , but al s o to determine how these d i s o r d e r s c o n t r i b u t e to myopathies of the h e a r t s . Before t h i s can be done the f u n c t i o n a l changes that occur i n the heart as a r e s u l t of the cardiomyopathy must be determined. Studi e s of t h i s nature have r e c e n t l y been -14-performed i n humans and experimental d i a b e t i c animal models. In man the most prominent f e a t u r e of d i a b e t i c cardiomyopathy i s a depression of l e f t v e n t r i c u l a r f u n c t i o n (Regen et a l , 1978; Seneviratne, 1977; Shapiro et a l , 1981) as measured by the r a t i o of t h e ' p r e - e j e c t i o n p e r i o d to the l e f t v e n t r i c u l a r e j e c t i o n time (PEP/LVET). Regen et a l (1978) observed a s i g n i f i c a n t l y e l e v a t e d PEP/LVET r a t i o i n d i a b e t i c p a t i e n t s , suggesting a r e d u c t i o n i n the v e n t r i c u l a r e j e c t i o n f r a c t i o n , p o s s i b l y due to a decrease i n the l e f t v e n t r i c u l a r d i a s t o l i c compliance. Shapiro et a l (1981), using a l a r g e r sample s i z e , have a l s o observed an increased PEP/LVET r a t i o i n hearts from d i a b e t i c p a t i e n t s . In a d d i t i o n , d i a b e t i c s with a p p a r e n t l y normal heart f u n c t i o n have a s i g n i f i c a n t l y lengthened isovolumic r e l a x a t i o n time. The a b n o r m a l i t i e s i n l e f t v e n t r i c u l a r f u n c t i o n were a l s o more pronounced i n insulin-dependent d i a b e t i c s , as compared to d i a b e t i c s not r e q u i r i n g i n s u l i n . I t has been suggested by Seneviratne (1977) that l e f t v e n t r i c u l a r f u n c t i o n a l changes can occur even i n the p r e - c l i n i c a l phase of a d i a b e t i c cardiomyopathy. Hemodynamic s t u d i e s could t h e r e f o r e be u s e f u l i n d e t e c t i n g p a t i e n t s at r i s k of developing cardiomyopathies. Recent s t u d i e s on d i a b e t i c animal models have supported human s t u d i e s . In dogs with a l l o x a n - i n d u c e d d i a b e t e s , Regen et a l (1974) demonstrated an i n c r e a s e i n l e f t v e n t r i c u l a r w a l l s t i f f n e s s , r e s u l t i n g i n a decreased d i a s t o l i c compliance. In other s t u d i e s , i s o l a t e d working hearts fromchemically-induced d i a b e t i c r a t s have shown a decreased a b i l i t y to respond to -15-i n c r e a s i n g f i l l i n g p r essures (Penpargkul et a l , 1980; Vadlamudi and M c N e i l l , 1982) and a f t e r l o a d (Ingebretson et a l , 1980). A prominent o b s e r v a t i o n noted i n these d i a b e t i c hearts was a depressed r a t e of c o n t r a c t i o n and r e l a x a t i o n at higher workloads. The depressed r e l a x a t i o n rate c o r r e l a t e s with the longer isovolumic r e l a x a t i o n time noted i n human d i a b e t i c s (Shapiro et a l , 1981). S i m i l a r l y , the depressed rate of c o n t r a c t i o n noted i n animals may a l s o correspond to the lengthened p r e - e j e c t i o n p e r i o d found i n d i a b e t i c humans. Evidence that these changes are o c c u r r i n g at the l e v e l of the myocardium were provided by F e i n et a l (1980) . They demonstrate that p a p i l l a r y muscle i s o l a t e d from d i a b e t i c r a t s had a depressed v e l o c i t y of s h o r t e n i n g , and a delayed onset of r e l a x a t i o n . The e f f e c t s of ischemia on heart f u n c t i o n i n d i a b e t i c r a t s a l s o appears to c o r r e l a t e with the f i n d i n g s i n humans. Feuvray et a l (1979) have shown that when ischemia i s produced, hearts from a l l o x a n d i a b e t i c r a t s f a i l e d at a f a s t e r rate than c o n t r o l h e a r t s . T h i s p a r a l l e l s what has been observed i n humans. A d i a b e t i c i s l e s s l i k e l y to s u r v i v e an acute'myocardial i n f a r c t i o n than a n o n d i a b e t i c (Bennett, 1981). Although human and animal s t u d i e s of d i a b e t i c cardiomyopathies show s i m i l a r i t i e s , d i f f e r e n c e s have a l s o been noted. Penpargkul et a l (1980) demonstrated that d i a b e t i c r a t s have a lower r a t i o of developed l e f t v e n t r i c u l a r end d i a s t o l i c pressure to l e f t v e n t r i c u l a r end d i a s t o l i c volume -16-(LVEDP/LVEDV). The s t i f f myocardium noted i n humans (and dogs) would be expected to i n c r e a s e the LVEDP, as a r e s u l t of the decreased d i a s t o l i c compliance of the v e n t r i c l e . Conforming with t h i s , Regen et a l (1981) have r e c e n t l y shown that i n d i a b e t i c dogs, the r a t i o of LVEDP/LVEDV i s e l e v a t e d . The reason f o r the d i f f e r e n c e s i n the LVEDP/LVEDV r e l a t i o n s h i p seen i n d i a b e t i c dogs as compared to r a t s i s s t i l l u n c l e a r . Recent s t u d i e s with r a t s have shown that the development of a d i a b e t i c cardiomyopathy i s time dependent. In a l l o x a n - d i a b e t i c r a t s , depressed heart f u n c t i o n i s evident by 30 days a f t e r i n j e c t i o n (Vadlamudi et a l , 1982) . S t r e p t o z o t o c i n - t r e a t e d r a t s have s i g n i f i c a n t l y depressed heart f u n c t i o n by s i x weeks p o s t - i n j e c t i o n ( T a h i l i a n i et a l , 1983). M i l l e r (1979) has reported d i f f e r e n c e s i n c a r d i a c performance as e a r l y as 3 days a f t e r i n d u c t i o n of a l l o x a n d i a b e t e s . The decrease i n c a r d i a c f u n c t i o n was, however, reversed i f the hearts were perfused with 10 mM g l u c o s e . Since depressed heart f u n c t i o n i n d i a b e t i c animals i s u l t i m a t e l y a r e s u l t of i n s u l i n d e f i c i e n c y there has been i n t e r e s t i n determining whether heart f u n c t i o n can be r e s t o r e d with i n s u l i n treatment. In d i a b e t i c r a t s with demonstrable myopathic changes, i n s u l i n treatment reversed depressed p a p i l l a r y muscle f u n c t i o n (Fein et a l , 1981) . Complete r e v e r s a l of the d i a b e t i c cardiomyopathy was g r a d u a l , o c c u r r i n g between 10 and 28 days a f t e r i n s u l i n treatment was i n i t i a t e d . I s o l a t e d working heart s t u d i e s by T a h i l i a n i et a l (1983) revealed that i n s u l i n treatment can a l s o prevent the onset of -17-the cardiomyopathy. Furthermore, these workers a l s o demonstrated that diabetes-induced depression of l e f t v e n t r i c u l a r f u n c t i o n 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 was reversed by i n s u l i n treatment. Non-invasive s t u d i e s by Pfaffman (1980) have a l s o demonstrated the r e v e r s a l by i n s u l i n of c a r d i o v a s c u l a r changes. The decreased heart r a t e and blood pressure i n 7 week s t r e p t o z o t o c i n d i a b e t i c r a t s was reversed w i t h i n 4 days a f t e r i n s t i t u t i o n of i n s u l i n therapy. I n s u l i n treatment cannot, however, reverse c a r d i o v a s c u l a r a b n o r m a l i t i e s i n a l l animal models of d i a b e t e s . In long term a l l o x a n - i n d u c e d d i a b e t i c dogs the el e v a t e d LVEDP/LVEDV r a t i o was not a l t e r e d by i n s u l i n treatment (Regen et a l , 1981). These i n v e s t i g a t o r s , however, d i d not dis c o u n t a p o t e n t i a l r o l e f o r i n s u l i n replacement i n decreasing d i a b e t i c - i n d u c e d c a r d i o v a s c u l a r a b n o r m a l i t i e s . O v e r a l l , the i n s u l i n p r e v e n t i o n and r e v e r s a l s t u d i e s performed on animals suggest that the development of cardiomyopathy i n d i a b e t i c humans probably r e s u l t s from inadequate i n s u l i n therapy. Poor management of insulin-dependent d i a b e t i c s may, t h e r e f o r e , hasten the development of c a r d i o v a s c u l a r a b n o r m a l i t i e s . The s t u d i e s c i t e d above suggest that a l t e r e d l e f t v e n t r i c u l a r f u n c t i o n , seen i n d i a b e t i c s with cardiomyopathy, can be d u p l i c a t e d i n the ch e m i c a l l y - i n d u c e d d i a b e t i c r a t . T h i s animal model can t h e r e f o r e be u s e f u l i n attempting to e l u c i d a t e the biochemical changes o c c u r r i n g i n the myocardium. One may r e c a l l the c o n s i s t e n t o b s e r v a t i o n i n both d i a b e t i c humans and r a t s of an impaired v e l o c i t y of c o n t r a c t i o n and -18-r e l a x a t i o n i n heart muscle. T h i s i m p l i e s a d e f e c t at one or more of the s e q u e n t i a l biochemical events l e a d i n g to muscle c o n t r a c t i o n . I f sarcoplasmic r e t i c u l a r f u n c t i o n was impaired 2+ one would expect the ra t e of Ca removal from the c o n t r a c t i l e u n i t to be decreased, r e s u l t i n g i n a decreased rate of r e l a x a t i o n . The r a t e of subsequent c o n t r a c t i o n may 2+ a l s o be decreased s i n c e l e s s Ca would be a v a i l a b l e f o r r e l e a s e from the sarcoplasmic r e t i c u l u m . We, t h e r e f o r e , decided to i n v e s t i g a t e whether d i a b e t i c cardiomyopathy may i n p a r t be due to an a l t e r a t i o n i n sarcoplasmic r e t i c u l a r f u n c t i o n . -19-C) ALLOXAN AND STREPTOZOTOCIN-INDUCED DIABETES A number of drugs can produce the symptoms of diabe t e s i n experimental animals. These i n c l u d e agents that act as i r r e v e r s i b l e c e l l - s p e c i f i c t o x i n s , agents which r e v e r s i b l y decrease i n s u l i n r e l e a s e , and agents which i n c r e a s e endogenous i n s u l i n requirements (Mordes and R o s s i n i , 1981). The c e l l - s p e c i f i c t o x i n s , a l l o x a n and s t r e p t o z o t o c i n , are widely used d i a b e t o g e n i c agents because they produce an i r r e v e r s i b l e d i a b e t i c s t a t e . The mechanism of a c t i o n i n v o l v e s a r e l a t i v e l y s e l e c t i v e n e c r o s i s of p a n c r e a t i c - i s l e t beta c e l l s (Rerup and Tarding , 1970; Veleminsky et a l , 1970). The a c t i o n s of a l l o x a n as a di a b e t o g e n i c agent have been known f o r a considerable p e r i o d of time (Kennedy, 1944). The mechanism by which a l l o x a n induces dia b e t e s i s , however, s t i l l unknown. U n t i l r e c e n t l y , i t was thought that a l l o x a n i n t e r a c t e d with the s u r f a c e of beta c e l l s , a l t e r i n g membrane p e r m e a b i l i t y (Mordes and R o s s i n i , 1981). Boquist (1980), however, suggests that a l l o x a n passes through the plasma membrane of beta c e l l s v i a a glucose t r a n s p o r t channel. Once i n s i d e i t may impair m i t o c h o n d r i a l a c t i v i t y by i n h i b i t i n g phosphate t r a n s p o r t . Evidence i n favour of t h i s hypothesis includes the f i n d i n g that glucose and l e u c i n e can p r o t e c t p a n c r e a t i c c e l l s from the adverse e f f e c t s of a l l o x a n a d m i n i s t r a t i o n . Whether t h i s i s a r e s u l t of competition f o r a l l o x a n at plasma membrane bi n d i n g s i t e s or an i n t r a c e l l u l a r m e t a b o l i c a l l y induced antagonism of phosphate accumulation, i s -20-unknown. The o v e r a l l e f f e c t of a l l o x a n i s to decrease the i s l e t c e l l volume,as a r e s u l t of a decrease i n e i t h e r the s i z e or the number of i s l e t c e l l s ( H o f t i e z e r and Carpenter, 1973). The c l i n i c a l symptoms of diabe t e s i n the r a t occur w i t h i n 24 hours of a s i n g l e i . v . i n j e c t i o n of a l l o x a n (65 mg/kg). These symptoms i n c l u d e hyperglycemia, weight l o s s , p o l y d y p s i a , hyperphagia, p o l y u r i a , g l y c o s u r i a , hyper1ipidemia, and h y p e r t r i g l y c e r i d e m i a . K e t o s i s has been reported (Mansford and Opie, 1968) although more recent data do not support t h i s o b s e r v a t i o n (Opie et a l , 1979). Non-diabetogenic a c t i o n s of a l l o x a n i n c l u d e n e p h r o t o x i c i t y and r e d u c t i o n i n alpha c e l l number (although c o n f l i c t i n g evidence e x i s t s ) ( W r i g h t , 1968). The mechanism by which s t r e p t o z o t o c i n produces diabetes i s a l s o unknown. I t i s thought that the glucose moiety of s t r e p t o z o t o c i n allows i n t r a c e l l u l a r entry of the drug v i a s p e c i f i c t r a n s p o r t mechanisms. The N-nitrosomethylurea moiety of s t r e p t o z o t o c i n may then a l k y l a t e c e l l u l a r c o n s t i t u e n t s i n v o l v e d i n i n s u l i n s y n t h e s i s and r e l e a s e ( S r i v a s t a v a et a l , 1982). S t e r e o s p e c i f i c carbohydrates such as 3-O-methylglucose can p r o t e c t beta c e l l s from the adverse e f f e c t s of s t r e p t o z o t o c i n , p o s s i b l y by competing f o r c e l l u l a r uptake. The c l i n i c a l symptoms of s t r e p t o z o t o c i n - i n d u c e d d i a b e t e s are s i m i l a r to those produced by a l l o x a n . Non-diabetogenic a c t i o n s i n c l u d e n e p h r o t o x i c i t y , as w e l l as tumourogenic and anti-tumourogenic a c t i v i t y . Both a l l o x a n and s t r e p t o z o t o c i n appear to be u s e f u l agents i n producing an experimental d i a b e t i c s t a t e which -21-p a r a l l e l s the c l i n i c a l j u v e n i l e onset d i a b e t e s . Although a l l o x a n i s the c l a s s i c a l d i a b e t o g e n i c agent, s t r e p t o z o t o c i n i s becoming i n c r e a s i n g l y favoured i n experimental s t u d i e s . T h i s i s due t o : 1) the lower m o r t a l i t y r a t e seen i n s t r e p t o z o t o c i n - i n d u c e d d i a b e t i c animals ( H o f t i e z e r and Carpenter, 1973), 2) the gr e a t e r s e l e c t i v i t y of s t r e p t o z o t o c i n f o r beta c e l l s (Junoud et a l , 1969), and 3) the longer h a l f l i f e of s t r e p t o z o t o c i n i n the body (Agarawal, 1980). N e v e r t h e l e s s , both agents were used to produce dia b e t e s i n our s t u d i e s . The e f f e c t of both types of diab e t e s on f u n c t i o n a l and biochemical a l t e r a t i o n s i n hearts c o u l d , t h e r e f o r e , be compared. Furthermore, d i r e c t t o x i c e f f e c t s of the i n d i v i d u a l d i a b e t o g e n i c agents on the heart could more e a s i l y be r u l e d out. D) CARDIAC SARCOPLASMIC RETICULUM CALCIUM UPTAKE ACTIVITY 2+ The c e n t r a l r o l e of Ca i n the c o n t r a c t i l e response of muscle was suggested by Heibrunn and W i e r c i n s k i (1947). T h i s was l a t e r confirmed by other re s e a r c h e r s who demonstrated 2+ the a b i l i t y of Ca to c o n t r o l myocardial c o n t r a c t i l i t y (Weber and Herz, 1961; B o z l e r , 1954). In a d d i t i o n , a membranous system, termed the sarcoplasmic r e t i c u l u m (SR), 2+ which i s capable of removing Ca from the c y t o s o l (Marsh, 1951; Ebashi and Lipmann, 1962) has been i d e n t i f i e d . The SR 2+ was found to a c t i v e l y accumulate Ca a g a i n s t a 2+ c o n c e n t r a t i o n g r a d i e n t and a Mg -dependent ATPase enzyme -22-has been i m p l i c a t e d i n t h i s process (Hasselbach and Makinose, 1962). I t was thus suggested that the SR a l t e r s myocardial 2+ c o n t r a c t i l i t y by changing cytoplasmic Ca l e v e l s , e i t h e r by 2+ a c t i v e l y accumulating cytoplasmic Ca , or by r e l e a s i n g 2+ accumulated Ca i n t o the cytoplasm (Ebashi, 1969; Reuter, 1974) . I t has s i n c e been shown that c a r d i a c SR f u n c t i o n may be r e g u l a t e d by a v a r i e t y of f a c t o r s . C y c l i c AMP-dependent p r o t e i n k i n a s e , c a l m o d u l i n , and monovalent c a t i o n s a l l a f f e c t the a b i l i t y of c a r d i a c SR to accumulate Ca (Kirchberger et a l , 1972; Lopaschuk et a l , 1980; Shigekawa and P e a r l , 1976). Such r e g u l a t o r y e f f e c t s conform to what would be expected i f SR was indeed i n t i m a t e l y i n v o l v e d i n c a r d i a c muscle c o n t r a c t i o n . S i m i l a r l y , using the r a t , a number of s t u d i e s have c o r r e l a t e d the i n v i t r o r a t e of c a r d i a c SR 2+ Ca -uptake a c t i v i t y to p a t h o l o g i c a l l y - i n d u c e d changes i n myocardial f u n c t i o n (Limas, 1978; F r o e l i c h et a l , 1978; Limas et a l , 1980; A l t o and D h a l l a , 1981; Penpargkul et a l , 1981; Limas et a l , 1980). As mentioned e a r l i e r , a number of groups, i n c l u d i n g our own, have i d e n t i f i e d a cardiomyopathy i n d i a b e t i c r a t s . A prominent o b s e r v a t i o n i s a depressed r a t e of c o n t r a c t i o n and r e l a x a t i o n of the myocardium. We, t h e r e f o r e , decided to use the c h e m i c a l l y - i n d u c e d d i a b e t i c r a t model to i n v e s t i g a t e whether these rate changes i n v o l v e an a l t e r a t i o n i n c a r d i a c SR f u n c t i o n . Experiments were designed to determine i f the onset of myocardial d y s f u n c t i o n p a r a l l e l e d any a l t e r a t i o n s i n -23-c a r d i a c SR f u n c t i o n . Furthermore, a p o s s i b l e mechanism accounting f o r a l t e r a t i o n s i n SR f u n c t i o n was i n v e s t i g a t e d . The premise f o r t h i s p o s s i b l e mechanism was that " a l t e r e d l i p i d metabolism can a l t e r c a r d i a c f u n c t i o n by changing the p r o p e r t i e s of c a r d i a c c e l l membranes" such as the sarcoplasmic r e t i c u l u m (Katz and Messineo, 1981). As mentioned e a r l i e r , l i p i d metabolism i s d r a m a t i c a l l y a c c e l e r a t e d i n the heart muscle of d i a b e t i c s . One c l a s s of intermediate i n v o l v e d i n f a t t y a c i d metabolism i n the heart are the long c h a i n a c y l c a r n i t i n e s . These a m p i p h i l i c compounds, which are el e v a t e d i n v e n t r i c u l a r t i s s u e from d i a b e t i c r a t s (Feuvray et a l , 1979), are potent i n h i b i t o r s of c a r d i a c SR f u n c t i o n (Adams et a l , 1979; P i t t s et a l , 1978). We t h e r e f o r e p o s t u l a t e d that changes i n c a r d i a c SR f u n c t i o n i n d i a b e t i c r a t s may r e s u l t from changes i n v e n t r i c u l a r t i s s u e l e v e l s of long c h a i n a c y l c a r n i t i n e s . T h i s w i l l be d i s c u s s e d i n d e t a i l l a t e r . -24-E) OBJECTIVES OF THE STUDY 1) To determine i f the a b i l i t y of c a r d i a c sarcoplasmic r e t i c u l u m to accumulate c a l c i u m i s depressed i n a l l o x a n - and s t r e p t o z o t o c i n - d i a b e t i c r a t s . 2) To determine i f the development of c a r d i a c SR a b n o r m a l i t i e s i n d i a b e t i c r a t s i s time-dependent, and c o r r e l a t e s with the development of the p r e v i o u s l y noted depression i n the r a t e of c o n t r a c t i o n and r e l a x a t i o n of v e n t r i c u l a r muscle. 3) To c h a r a c t e r i z e the d e p r e s s i o n i n SR f u n c t i o n by measuring not o n l y the c a l c i u m t r a n s p o r t a c t i v i t y , but a l s o c a l c i u m - s e n s i t i v e ATPase a c t i v i t y , and c a l c i u m e f f l u x from SR v e s i c l e s i s o l a t e d from the d i a b e t i c r a t v e n t r i c l e . A l s o , t o determine i f r e g u l a t i o n of c a r d i a c SR c a l c i u m t r a n s p o r t d i f f e r s i n microsomes i s o l a t e d from d i a b e t i c r a t s . 4) To determine i f i n s u l i n treatment can prevent the d i a b e t e s - i n d u c e d d e p r e s s i o n i n c a r d i a c SR f u n c t i o n . 5) To i n v e s t i g a t e the mechanism by which d i a b e t e s depresses c a r d i a c SR f u n c t i o n . T h i s problem w i l l be approached by attempting to r e l a t e d i a b e t e s - i n d u c e d i n t r a c e l l u l a r metabolic changes to a l t e r a t i o n s i n SR f u n c t i o n ; more s p e c i f i c a l l y to determine how long chain a c y l c a r n i t i n e s may e f f e c t SR f u n c t i o n . -25-6) By bioch e m i c a l i n t e r v e n t i o n to attempt to prevent or reverse the di a b e t e s - i n d u c e d a l t e r a t i o n i n SR f u n c t i o n . A l s o , to determine the e f f e c t of a d m i n i s t r a t i o n of c a r n i t i n e , a s u b s t r a t e i n v o l v e d i n the metabolism and t r a n s p o r t of the long c h a i n a c y l c a r n i t i n e s , on o v e r a l l heart performance i n d i a b e t i c r a t s . -26-MATERIALS A N D METHODS A) MATERIALS 2) R a d i o i s o t o p e s - ( 4 5 C a ) C l 2 (10 Ci/mmmole) , © < - 3 2 N - A T P (16.4 Ci/mmole) , 14 r and (1- C) a c e t y l CoA were purchased from Amersham rad i o c h e m i c a l s (Toronto, O n t a r i o ) . 2) Enzymes - 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 , l a c t i c dehydrogenase, pyruvate kinase, and cAMP-dependent kinase (Type II) were obtained from the Sigma r Chemical Co. 3) Chemicals The f o l l o w i n g chemicals were purchased from the Sigma r Chemical Co. Trizma adenosine t r i p h o s p h a t e (equine muscle) ( T r i s ATP) Nicotinamide adenine d i n u c l e o t i d e (NADH) Phosphoenolpyruvate (PEP) Adenosine 3 ' , 5 ' - c y c l i c n u c l e o t i d e monophosphate ( T r i s s a l t ) Ferrocytochrome c (type I I I ) 1X8-400 Dowex-1 Anion Exchange Resin Bovine Serum Albumin L - h i s t i d i n e f r e e base, EGTA, EDTA, Imidazole, Magnesium C h l o r i d e , Potassium C h l o r i d e , Sodium C h l o r i d e , Sodium A z i d e , -27-Sucrose, Trizma Base, Trizma HC1, T r i t o n X-100, Trizma maleate, D i t h i o t h r e i t o l , D , L - c a r n i t i n e , L - c a r n i t i n e , L - p a l m i t y l c a r n i t i n e , S t r e p t o z o t o c i n , A l l o x a n , A c e t y l Coenzyme A, 2 - t h i o b a r b i t u r i c a c i d , 5-hydroxymethyIfurfuraldehyde, T r i c h l o r o a c e t i c a c i d , P a l m i t i c a cid,/^-glycerophosphate, HEPES, Tetrasodium pyrophosphate, Magnesium Ace t a t e , Sodium f l u o r i d e , M e t h y l i s o b u t y l x a n t h i n e , Potassium Hydroxide, Copper s u l f a t e , Deoxycholate, Potassium f e r r o c y a n a t e , Potassium phosphate, G l u t a r a l d e h y d e , c a c o d y l a t e CaCl2 d i h y d r a t e , L-ascorbate, Trizma o x a l a t e , Sodium carbonate, D-glucose, Sodium b i c a r b o n a t e , and C i t r i c a c i d were purchased from A n a l a r r chemicals. Sodium t e t r a t h i o n a t e was purchased from the P i e r c e r chemical co. L - p a l m i t y l c a r n i t i n e was a generous g i f t of Dr. P. Hahn, Center fo r Developmental Medicine, F a c u l t y of Medicine, U n i v e r s i t y of B. C., Vancouver, Canada. Calmodulin was obtained from Calbiochem r or prepared from bovine b r a i n according to the method of Watterson et a l , (1976) . P u r i f i e d s y m - c o l l i d i n e and other chemicals used i n the e l e c t r o n microscopy study were obtained from P o l y s c i e n c e r Inc. 2-methylbutane was purchased from MCBr manufacturing chemists Aquasol s c i n t i l l a t i o n f l u i d was purchased from New England N u c l e a r r A l l s o l v e n t s were reagent grade S t r e p t o z o t o c i n was a generous g i f t of the Upjohn r -28-pharmaceutical co. -U-500 I l l e t i n II pork i n s u l i n (500 U/cc) was a generous g i f t of E l i L i l l y r and Co. 4) Apparatus A l z e t r Miniosmotic pumps (Model 2002) with 0.228ml c a p a c i t y and a pumping r a t e of 0.458ul/hr were purchased from A l z a r corp. -29-METHODS A) Animals Female Wistar r a t s (Charles River Canada r, Montreal, Canada) between 150-200g were used. Diabetes 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 of s t r e p t o z o t o c i n (60 or 50mg/kg), or a l l o x a n (65 or 50mg/kg), d i s s o l v e d i n c i t r a t e b u f f e r (pH 4.0). C o n t r o l r a t s were i n j e c t e d with c i t r a t e b u f f e r alone. Food and water were provided ad l i b i t u m throughout the study p e r i o d (7, 30, 42, 120, and 150 days). In the i n i t i a l study, r a t s were f a s t e d 16 hours p r i o r to s a c r i f i c e . In some s t u d i e s r a t s were t r e a t e d with i n s u l i n or D , L - c a r n i t i n e as d e s c r i b e d below. At the end of the study p e r i o d the animals were stunned by a blow to the head and the hearts were removed and i) used to prepare c a r d i a c sarcoplasmic r e t i c u l u m , or i i ) perfused f o r working heart s t u d i e s . Blood was c o l l e c t e d i n hepartiized tubes and c e n t r i f u g e d at 3000 times g f o r 5 minutes. Serum was assayed f o r i n s u l i n by a radioimmunoassay method (B e c t o n - D i c k i n s o n r I n s u l i n Radioimmunoassay K i t 125 ( I ) ) ; and f o r glucose by the glucose oxidase method r r (Ames Reagent K i t f o r blood g l u c o s e , or Sigma Glucose K i t ) . C e l l s were used to determine g l y c o s y l a t i o n of hemoglobin, as d e s c r i b e d l a t e r . 1) I n s u l i n treatment u t i l i z i n g osmotic minipumps A l z e t r Miniosmotic pumps (Model 2002) with 0.228 + 0.26 (S.D.) ml c a p a c i t y and a pumping rate of 0.458 u l / h r were obtained from A l z a r C o r p o r a t i o n , Palo A l t o , C a l i f o r n i a . U-500 I l l e t i n II pork i n s u l i n (500 U/cc) was a generous g i f t of E l i L i l l y and Company, I n d i a n a p o l i s , Indiana. -30-Female Wistar r a t s were a n e s t h e t i z e d with ether and i n j e c t e d v i a the t a i l v e i n with 50 mg/kg s t r e p t o z o t o c i n d i s s o l v e d i n c i t r a t e b u f f e r (pH 4.0). C o n t r o l r a t s were i n j e c t e d with c i t r a t e b u f f e r alone. Three days a f t e r i n j e c t i o n r a t s d i s p l a y i n g g l y c o s u r i a and e l e v a t e d serum glucose l e v e l s were randomly d i v i d e d i n t o two groups. The f i r s t group r e c e i v e d no a d d i t i o n a l treatment. The second group had A l z a r Miniosmotic pumps (Model 2002) s u r g i c a l l y implanted subcutaneously as" described by A l z e t (1981). Fur was shaved from the r o s t r a l h a l f of the back of ether a n e s t h e t i z e d r a t s , and a 1/2" h o r i z o n t a l i n c i s i o n made through the s k i n . A hemostat was i n s e r t e d i n t o the wound p a r a l l e l to the s p i n e , then separated s l i g h t l y . The osmotic minipump was p o s i t i o n e d i n t h i s c a v i t y ( p o r t a l end f i r s t ) and the wound c l o s e d with three s t i t c h e s . Each r a t was caged s e p e r a t e l y throughout the study to prevent chewing of the s t i t c h e s by other r a t s . Each minipump was f i l l e d with 273 U/ml of U-500 I l l e t i n pork i n s u l i n ( L i l l y r ) . At a pumping r a t e of 0.458 u l / h r a dose of 3 U of i n s u l i n was d e l i v e r e d subcutaneously during every 24 hour p e r i o d . Minipumps were r e p l a c e d every 10 to 14 days depending on the r e s u l t s of serum glucose assays. Blood samples were c o l l e c t e d every 3 days by t a i l v e i n puncture of a n e s t h e t i z e d r a t s . Serum was separated from c e l l s by s p i n n i n g i n 50 u l c a p i l l a r y tubes (non-heparinized) at 3000 times g f o r 10 minutes. Serum glucose was determined by the glucose oxidase method (Sigma r glucose k i t s ) . I f serum glucose exceeded 150 mg % i n d i a b e t i c r a t s implanted with minipumps the a p p r o p r i a t e amounts of protamine z i n c i n s u l i n -31-(Connaught L a b o r a t o r i e s , Toronto, Canada) were i n j e c t e d subcutaneously u n t i l the pump was r e p l a c e d . 2) D , L - c a r n i t i n e treatment In the attempted r e v e r s a l study D , L - c a r n i t i n e was administered to d i a b e t i c and c o n t r o l r a t s , v i a t h e i r d r i n k i n g water, at a dose of 1 g/kg/day. C a r n i t i n e treatment commenced 120 days a f t e r the i n d u c t i o n of diab e t e s and continued f o r 30 days, a f t e r which time the hearts were removed and perfused using a working heart apparatus. In the attempted p r e v e n t i o n study d i a b e t i c and c o n t r o l r a t s were administered D , L - c a r n i t i n e (3 g/kg/day) 3 days a f t e r the i n d u c t i o n of diab e t e s f o r a 42 day p e r i o d . The r a t s were then randomly d i v i d e d i n t o two experimental groups. Hearts from one group were used to prepare c a r d i a c sarcoplasmic r e t i c u l u m , while the second group were perfused using a working heart apparatus. -32-PREPARATION OF CARDIAC MICROSOMES ENRICHED WITH SARCOPLASMIC RETICULUM 1) Method 1 In i n i t i a l s t u d i e s sarcoplasmic r e t i c u l u m - e n r i c h e d v e s i c l e s were prepared using the methodology of Harigaya and Schwartz (1969) with s l i g h t m o d i f i c a t i o n s . The e n t i r e procedure was performed at 4 degrees c e n t i g r a d e . Rat v e n t r i c l e (0.5-1.0g) was minced and placed i n 15ml of b u f f e r 1 (10mM NaHC0 3, 5mM NaN 3, and 0.2mM ascorbate pH 6.8). T h i s was homogenized continuously i n a g l a s s homogenizer with a t e f l o n p e s t l e , at 1500 r.p.m. f o r 15 seconds. The suspension was t r a n s f e r r e d to a Co r e x r c e n t r i f u g e tube and c e n t r i f u g e d at 10,000 times g f o r 20 minutes i n a Beckman r Model J2-21 c e n t r i f u g e . The supernatant was then c e n t r i f u g e d at 40,000 times g f o r 60 minutes. The r e s u l t a n t supernatant was di s c a r d e d and the p e l l e t resuspended by g e n t l e homogenization i n 15ml of b u f f e r 2 (0.6M KC1, 2.0mM T r i s - C l pH 7.3, 1.0mM M g C l 2 ) . The suspension was r e c e n t r i f u g e d at 40,000 times g fo r 60 minutes, the supernatant d i s c a r d e d , and the p e l l e t suspended i n 10 ml b u f f e r 3 (10mM T r i s - C l pH 7.3). A f t e r r e c e n t r i f u g a t i o n at 40,000 times g f o r 60 minutes, the supernatant was d i s c a r d e d and the p e l l e t suspended i n 1.5ml of b u f f e r 4 (40% sucrose 10mM T r i s - C l pH 7.4, 5mM d i t h i o t h r e i t o l ) . The sucrose s o l u t i o n had p r e v i o u s l y been p u r i f i e d by passage through a Bio-Rad r AG 50W-X8 c a t i o n exchange r e s i n column i n the Na + form. The concentrated microsomal suspension (1.2-1.5mg/ml) was then d i v i d e d e q u a l l y i n t o 4 t e s t tubes. These were used e i t h e r immediately, or -33-quick f r o z e n , by immersing the a l i q u o t i n 2-methyl butane on dry i c e , and s t o r e d at -80 degrees c e n t i g r a d e . P r i o r to use the concentrated a l i q u o t s were d i l u t e d with b u f f e r 4 such that the f i n a l p r o t e i n content ranged from 0.4-0.6 mg/ml. A l l microsomal suspensions were used w i t h i n one month of p r e p a r a t i o n . The a c t i v i t y of the suspension, as measured by 2+ the a b i l i t y to t r a n s p o r t Ca i n t o the SR v e s i c l e s , was s i m i l a r i n f r e s h and p r e v i o u s l y f r o z e n a l i q u o t s . 2) Method 2 Rats were d e c a p i t a t e d and t h e i r hearts e x c i s e d , trimmed of a t r i a , and the v e n t r i c l e s weighed and placed i n i c e - c o l d 10 mM T r i s maleate b u f f e r , pH 6.8. C a rdiac microsomes enriched i n SR were then prepared by a m o d i f i c a t i o n of the method of Sumida et a l (1978). V e n t r i c l e s were homogenized i n 15ml of 10 mM T r i s maleate pH 6.8 with a t e f l o n p e s t l e f o r 15 seconds at 1500 r.p.m.. The homogenate was then c e n t r i f u g e d at 4000 times g f o r 10 minutes, and the supernatant passed through c h e e s e c l o t h . T h i s supernatant was then c e n t r i f u g e d at 40,000 times g f o r 80 minutes. The r e s u l t i n g p e l l e t was then re-suspended i n 10 mM T r i s maleate pH 6.8 c o n t a i n i n g 0.6 M KCl and c e n t r i f u g e d at 40,000 times g f o r 80 minutes. In order to minimize K + contamination, the p e l l e t was re-suspended, washed i n 10 mM T r i s maleate pH 6.8, and r e - c e n t r i f u g e d at 40,000 times g f o r 100 minutes. The f i n a l p e l l e t was suspended i n 10mM T r i s maleate c o n t a i n i n g 40% sucrose, q u i c k - f r o z e n i n 2-methylbutane on dry i c e , and s t o r e d at -70 degrees c e n t i g r a d e u n t i l use. -34-MEASUREMENT OF CALCIUM UPTAKE BY CARDIAC MICROSOMES ENRICHED IN SARCOPLASMIC RETICULUM VESICLES 2+ 1) Measurement of c a r d i a c SR Ca t r a n s p o r t a c t i v i t y ATP-dependent calcium uptake was measured by the method of Tada et a l (1974) with a few m o d i f i c a t i o n s . O x a l a t e - f a c i l i t a t e d c a l c i u m uptake was determined i n an i n c u b a t i o n medium c o n t a i n i n g 10-50 ug of microsomal sarcoplasmic r e t i c u l u m p r o t e i n , 40 mM h i s t i d i n e h y d r o c h l o r i d e , pH 6.8, 5 mM MgCl 2, 110 mM KCl, 5 mM Tris-ATP, 2.5 mM T r i s - o x a l a t e , and C a C l 2 c o n t a i n i n g 4 5 C a C l 2 . The d e s i r e d f r e e c a l c i u m c o n c e n t r a t i o n was maintained by the a d d i t i o n of ethylene g l y c o l b i s (^-aminoethyl e t h e r ) - N , N 1 - t e t r a a c e t a t e (EGTA), and the f r e e c a l c i u m c o n c e n t r a t i o n s present were determined by the equations of Katz et a l (1970). Samples were pre-incubated f o r 11 minutes at 30 degrees c e n t i g r a d e , and the r e a c t i o n s t a r t e d by the a d d i t i o n of 4 5 C a C l 2 . When L - p a l m i t y l c a r n i t i n e or D , L - c a r n i t i n e was present, samples were 45 incubated 20 minutes p r i o r to C a C l 2 a d d i t i o n to allow fo r complete e q u i l i b r a t i o n of the sarcoplasmic r e t i c u l u m membrane with the L - p a l m i t y l c a r n i t i n e or c a r n i t i n e i n the i n c u b a t i o n medium ( P i t t s et a l , 1978). A f t e r 5 minutes, the r e a c t i o n was terminated by f i l t e r i n g an a l i q u o t of the r e a c t i o n mixture through a M i l l i p o r e f i l t e r (HA 45, M i l l i p o r e r Co.). The f i l t e r was then washed twice with 10 ml of 40 mM T r i s - C l , pH 7.2, d r i e d and counted f o r -35-r a d i o a c t i v i t y i n A q u a s o l r (New England N u c l e a r r Co.) by using standard l i q u i d s c i n t i l l a t i o n counting techniques. 2) C a l c u l a t i o n of calcium uptake a c t i v i t y by c a r d i a c microsomes enriched i n sarcoplasmic r e t i c u l u m . 2+ The r a t e of Ca -uptake by the microsomal p r e p a r a t i o n 2+ i s expressed as nmoles Ca taken up per mg p r o t e i n per minute. T h i s i s determined by the f o l l o w i n g formula: 2+ / sample counts -blank d i l u t i o n t o t a l Ca / mg p r o t e i n * >< / t o t a l counts f a c t o r / i n c u b a t i o n time where sample counts= 4 ^ C a 2 + counts obtained per i n d i v i d u a l samples. t o t a l counts= t o t a l 4 5 C a 2 + counts present i n the i n c u b a t i o n medium blank counts= 4 5 C a 2 + counts obtained i n the absence of microsomal p r o t e i n 2+ t o t a l Ca = t o t a l c a l c i u m c o n c e n t r a t i o n i n the i n c u b a t i o n medium d i l u t i o n f a c t o r = c o r r e c t i o n f o r i n c u b a t i o n volume sampled mg protein= weight of microsomal p r o t e i n present i n the i n c u b a t i o n medium i n c u b a t i o n time= length of time the microsomal p r o t e i n was incubated i n the presence of C a C ^ . -36-MEASUREMENT OF (CA 2 +-MG 2 +)-ATPASE ACTIVITY IN CARDIAC MICROSOMES ENRICHED IN SARCOPLASMIC RETICULUM 2+ 2+ (Ca -Mg )ATPase a c t i v i t y was measured s p e c t r o p h o t o m e t r i c a l l y u t i l i z i n g a coupled enzyme assay (Watterson et a l , 1976). The i n c u b a t i o n medium was s i m i l a r 2+ to that used i n the Ca t r a n s p o r t s t u d i e s except 0.22 mg phosphoenolpyruvate, 0.28 mg NADH, 7 u n i t s l a c t a t e dehydrogenase, and 42 u n i t s pyruvate kinase were present OJ. i n s t e a d of t r l s - o x a l a t e . In order to o b t a i n Ca s e n s i t i v i t y 4 uM ATP and 20 uM Mg 2 + were used. T h i s d i d not 2+ 2 + a l t e r the t o t a l (Ca -Mg )ATPase a c t i v i t y obtained i n the 2+ presence of 5 mM ATP and 5 mM Mg . The i n c u b a t i o n medium ( f i n a l volume 1 ml) was e q u i l i b r a t e d at 30 degrees c e n t i g r a d e i n a Beckman r-24 spectrophotometer. Sarcoplasmic r e t i c u l u m (2-10 ug) was added to the cuvette and pre-incubated f o r 5 minutes. When L - p a l m i t y l c a r n i t i n e or D , L - c a r n i t i n e was present, samples were incubated 20 minutes p r i o r to C a C l 2 a d d i t i o n to allow f o r complete e q u i l i b r a t i o n of the SR membrane with the L - p a l m i t y l c a r n i t i n e or c a r n i t i n e i n the in c u b a t i o n medium ( P i t t s et a l , 1978). The r e a c t i o n was s t a r t e d by the a d d i t i o n of v a r i o u s c o n c e n t r a t i o n s of C a C l 2 c o n t a i n i n g 0.1 mM EGTA, the f r e e c a l c i u m c o n c e n t r a t i o n present being determined by the equations of Katz et a l (1970). The decrease i n absorbance at 340 nm was monitored. As shown below, one ATP hydrolyzed i s e q u i v a l e n t to one NADH converted to NAD +. One nmole of NADH converted i s e q u i v a l e n t to an -37-absorbance change of 0.00622. ATP h y d r o l y s i s i s determined by measuring the absorbance change per mg SR p r o t e i n per minute. P r i n c i p l e of the coupled enzyme assay pyruvate kinase l a c t a t e dehydrogenase phosphoenolpyruvate' * pyruvate ^  l a c t a t e 1 ADP SR ATPase 1 ATP NADH *NAD~ (measure decrease i n absorbance at 340nm) MEASUREMENT OF CALCIUM EFFLUX FROM C A 2 + LOADED CARDIAC SARCOPLASMIC RETICULUM VESICLES Calcium e f f l u x from c a r d i a c microsomes was measured using a m o d i f i c a t i o n of the procedure of Weller and Laing (1979). Heart microsomes enriched i n SR (0.5 mg/ml) were suspended i n 2 + the same medium used to measure Ca -uptake ( i n the absence of t r i s - o x a l a t e ) and incubated i n 2 uM fr e e 4 5 C a 2 + . A f t e r 45 minutes, the i n i t i a l l e v e l of 4 5 C a 2 + bound to c a r d i a c microsomes was determined by tak i n g a 0.1 ml sample and f i l t e r i n g as d e s c r i b e d above. The suspension (2 ml) was then c e n t r i f u g e d at 40,000 times g f o r 25 minutes at 4 degrees c e n t i g r a d e , and the p e l l e t suspended i n i c e - c o l d 40% sucrose, 10 mM T r i s - C l , pH 7.4, and 5 mM D i t h i o t h r e i t o l . E x a c t l y 2 minutes a f t e r resuspension, a 0.1 ml a l i q u o t was f i l t e r e d to determine the amount of 4 5 C a 2 + bound to the microsomes. Sampling was then repeated at v a r i o u s time i n t e r v a l s . -38-WORKING HEART PERFUSION Hearts were perfused on a working heart apparatus f i r s t d e s c r i b e d by Neely and Rovetto (1971) ,and modified by Rod.gers et a l (1981) and Vadlamudi et a l (1982). Hearts were removed from r a t s and d i s s e c t e d f r e e of extraneous t i s s u e i n warm aerated Chenoweth-Koelle s o l u t i o n , and t i e d to a 15 gauge s t a i n l e s s s t e e l cannula i n s e r t e d i n the a o r t a . The heart was then perfused i n the Langendorff mode with Chenoweth-Koelle b u f f e r (37 degrees centigrade) c o n t a i n i n g ( i n m i l l i m o l a r ) NaCl,120; KC1,5.6; CaCl 2,2.18; MgCl 2,2.1; NaHC0 3,19; glucose,10; and EDTA,0.03;aerated with 95% 0 2~5% C0 2 to give a pH of 7.4. A 16 gauge s t a i n l e s s s t e e l cannula, connected to a t r i a l f i l l i n g r e s e r v o i r s , was then i n s e r t e d i n t o , and t i e d t o , the pulmonary v e i n . L e f t v e n t r i c u l a r developed pressure was measured by means,of a Statham P23AA transducer (Gould Statham Instruments Inc.) attached to a 3 cm piece of PE90 t u b i n g . A 20 gauge needle attached onto the tubing was i n s e r t e d i n t o the l e f t v e n t r i c l e through the apex of the h e a r t . I n t r a o r t i c pressure was a l s o measured with a statham P23AA transducer attached to a s i d e arm on the a o r t i c outflow system. The heart was switched to the working heart mode by p e r f u s i n g the l e f t atrium with Chenoweth-Koelle b u f f e r . A o r t i c outflow passed through a s e r i e s of p i p e t t e s which produced an a f t e r l o a d r e s i s t a n c e of 5.6 mm Hg/ml per minute (340 p e r i p h e r a l r e s i s t a n c e u n i t s or 453,200 dynes sec/cm ). Hearts were s t i m u l a t e d with a platinum e l e c t r o d e -39-placed on the l e f t atrium; a Grass Model SD9D s t i m u l a t o r d e l i v e r e d a 1.5 mV square pulse of 5 msec d u r a t i o n at a rate of 280 beats/minute. L e f t v e n t r i c u l a r f u n c t i o n data were recorded and analyzed on an Apple II microcomputer, as de s c r i b e d by H a r r i s et a l (1982). The l e f t v e n t r i c u l a r pressure pulse (sampled every 1.5 msec f o r 1024 sample points) was obtained from the polygraph with the a i d of an Analog to D i g i t a l (A/D) c o n v e r t e r . A n a l y s i s of the pressure pulse was made using an A p p l e s o f t program which u t i l i z e d v a r i o u s curve f i t t i n g techniques ( M a r r i o t t , 1982). Hearts were e q u i l i b r a t e d at 10 cm H 2 O f i l l i n g pressure f o r 10 minutes before f u n c t i o n curves were measured. L e f t a t r i a l f i l l i n g pressure was a l t e r e d by v a r y i n g the height of the l e f t a t r i a l f i l l i n g r e s e r v o i r between 5 and 22.5 cm H 2 o , i n 2.5 cm increments. Function curves were obtained i n d u p l i c a t e , a f t e r which time hearts were q u i c k l y f r o z e n with Wollenberger clamps precooled i n l i q u i d n i t r o g e n . -40-DETERMINATION OF CARNITINE AND LONG CHAIN ACYLCARNITINE LEVELS C a r n i t i n e and long c h a i n a c y l c a r n i t i n e l e v e l s were measured i n microsomal SR samples, v e n t r i c u l a r t i s s u e from perfused working h e a r t s , and serum samples. A) Sample P r e p a r a t i o n 1 ) Microsomal SR In order to measure l e v e l s of f r e e and e s t e r i f i e d c a r n i t i n e , an a l i q u o t of microsomal sarcoplasmic r e t i c u l u m ( c o n t a i n i n g 0 . 5 mg p r o t e i n ) was c e n t r i f u g e d at 4 0 , 0 0 0 times g f o r 45 minutes at 4 degrees c e n t i g r a d e , and the p e l l e t resuspended i n c o l d 6 % (w/v) p e r c h l o r i c a c i d ( 0 . 6 ml). A 1 0 0 u l a l i q u o t was n e u t r a l i z e d with 75 u l of 2 M t r i s base and used to determine the l e v e l s of t o t a l c a r n i t i n e p r e s e n t . The remaining sample was then c e n t r i f u g e d at 1 2 , 0 0 0 times g f o r 1 0 minutes. A 2 0 0 u l a l i q u o t of supernatant was n e u t r a l i z e d with 150 u l 2 M t r i s base and used to determine l e v e l s of a c i d s o l u b l e f r e e c a r n i t i n e . The p e l l e t was washed with 6 % p e r c h l o r i c a c i d and suspended i n 1 0 0 u l H 2 O and used to determine l e v e l s of long c h a i n a c y l c a r n i t i n e s (acid i n s o l u b l e ) . T o t a l c a r n i t i n e and long c h a i n a c y l c a r n i t i n e s were assayed as f r e e c a r n i t i n e f o l l o w i n g a l k a l i n e h y d r o l y s i s of the samples by i n c u b a t i o n at 70 degrees c e n t i g r a d e f o r 1 hour i n 1 0 0 u l of 1 . 0 M t r i s base and 50 u l of 0.4 N KOH (pH approximately 13). F o l l o w i n g i n c u b a t i o n , samples were n e u t r a l i z e d with 2 0 0 u l of 0.575 N H C 1 . 2 ) V e n t r i c u l a r t i s s u e -41-Approximately 150 mg of f r o z e n v e n t r i c u l a r t i s s u e , obtained from i s o l a t e d perfused h e a r t s , was thoroughly homogenized i n 2 ml of c o l d 6% p e r c h l o r i c a c i d . A 200 u l a l i q u o t was n e u t r a l i z e d with 150 u l 2 M t r i s base and used to determine the l e v e l s of t o t a l c a r n i t i n e p resent. The remaining sample was c e n t r i f u g e d at 12,000 times g f o r 10 minutes at 4 degrees c e n t i g r a d e . A 400 u l a l i q u o t of supernatant was n e u t r a l i z e d with 300 u l 2 M t r i s base and used to determine l e v e l s of a c i d s o l u b l e f r e e c a r n i t i n e . The p e l l e t was washed with 6% p e r c h l o r i c a c i d and resuspended i n 1 ml d i s t i l l e d H 2 o . A 200 u l a l i q u o t was used to determine l e v e l s of long c h a i n a c y l c a r n i t i n e s . T o t a l c a r n i t i n e and long c h a i n a c y l c a r n i t i n e s were assayed as f r e e c a r n i t i n e f o l l o w i n g a l k a l i n e h y d r o l y s i s of the samples by i n c u b a t i o n at 70 degrees c e n t i g r a d e f o r 1 hour i n 200 u l of 1.0 M t r i s base and 100 u l 0.4 N KOH (pH approximately 13). Samples were then n e u t r a l i z e d with 400 u l 0.575 N HC1. 3) serum Serum c a r n i t i n e was determined by adding 0.5 ml c o l d 12% p e r c h l o r i c a c i d to 0.5 ml of serum and homogenizing thoroughly. The suspension was c e n t i f u g e d at 12,000 time g fo r 10 minutes. A 200 u l a l i q u o t of the supernatant was n e u t r a l i z e d with 150 u l 2 M t r i s base and used f o r f r e e c a r n i t i n e d e t e r m i n a t i o n . B) Assay to determine c a r n i t i n e l e v e l s Free c a r n i t i n e was measured using a r a d i o i s o t o p i c procedure developed by McGarry and F o s t e r (1976) which -42-u t i l i z e s (1- C ) - A c e t y l CoA and the enzyme c a r n i t i n e a c e t y l t r a n s e r a s e . To a 150 u l sample c o n t a i n i n g c a r n i t i n e was added a 1.05 ml volume of 120 umole t r i s - C l pH 7.3, 2 umole sodium t e t r a t h i o n a t e , and 25 nmoles (0.025 uCi) of 14 (1- C ) - A c e t y l CoA. The r e a c t i o n , as d e s c r i b e d below was then i n i t i a t e d by the a d d i t i o n of 10 u l (lu) 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 suspension. 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 c a r n i t i n e (sample) ^ ( 1 - 1 4 C ) - a c e t y l c a r n i t i n e 14, + (1- C ) - A c e t y l CoA) + CoASH Na t e t r a t h i o n a t e (prevents reverse r e a c t i o n ) A f t e r i n c u b a t i o n f o r 30 minutes at room temperature a 0.3 ml a l i q u o t of Dowex 1X8-400 anion exchange r e s i n (200-400 mesh i n c h l o r i d e form, a 0.3 ml suspension c o n t a i n i n g 0.22 ml H 20) was added and the sample vortexed and placed on i c e . The sample was vortexed twice at 10 minute i n t e r v a l s , and c e n t r i f u g e d at 3000 times g f o r 5 minutes. A 0.7 ml a l i q u o t of the supernatant was added to 5 ml of A q u a s o l r and assayed fo r r a d i o a c t i v i t y using standard l i q u i d s c i n t i l l a t i o n t echniques. The q u a n t i t y of ( 1 - 1 4 C ) - a c e t y l c a r n i t i n e formed (supernatant f r a c t i o n ) i s s t o i c h i o m e t r i c a l l y r e l a t e d to the amount of c a r n i t i n e present i n the sample. L e v e l s of f r e e c a r n i t i n e and long c h a i n a c y l c a r n i t i n e s were expressed as nmoles/mg sarcoplasmic r e t i c u l u m , nmoles/g wet v e n t r i c l e weight, or nmol/ml serum. -43-GLYCOSYLATED HEMOGLOBIN ASSAY G l y c o s y l a t i o n of hemoglobin i n blood from c o n t r o l and d i a b e t i c r a t s was determined s p e c t r o p h o t o m e t r i c a l l y by the method of Subramanian et a l (1980). Approximately 2 ml of blood was c o l l e c t e d i n h e p a r i n i z e d tubes. C e l l s were seperated from serum by c e n t r i f u g a t i o n (3000 times g f o r 5 minutes) and washed three times with normal s a l i n e (4 degrees c e n t i g r a d e ) . Packed c e l l s were then l y s e d with 2 ml H 20 and 0.5 ml carbon t e t r a c h l o r i d e . The suspension was c e n t r i f u g e d at 1500 times g f o r 15 minutes, and the p e l l e t d i s c a r d e d . A c i d i f i e d acetone (15 ml,4 degrees c e n t r i g r a d e ) was added to 1 ml of c l e a r hemolysate to seperate heme and i r o n from the g l o b u l i n . The suspension was c e n t r i f u g e d at 3600 times g f o r 5 minutes and the supernatant d i s c a r d e d . The p e l l e t (globin) was washed once with a c i d i f i e d acetone, and twice with acetone. The f i n a l p e l l e t was suspended i n d i e t h y l ether and d r i e d under n i t r o g e n . A 75 mg sample of the g l o b i n was di g e s t e d with 4.0 ml of 10 M a c e t i c a c i d f o r 16 hours at 80 degrees c e n t i g r a d e i n tubes capped with marbles. F o l l o w i n g i n c u b a t i o n , 2 ml of 2% t r i c h l o r o a c e t i c a c i d was added to 1 ml of d i g e s t and the mixture allowed to s i t f o r 5 minutes. To measure the formation of hydroxymethyIfurfuraldehyde (HMF) 2 ml of t h i o b a r b i t u r a t e reagent (0.35 g TBA i n 100 ml 0.25 M sodium s u l f a t e ) was added. The r e a c t i o n mixture (now c o n t a i n i n g a f i n a l c o n c e n t r a t i o n of 2 M a c e t i c acid) was incubated f o r 50 minutes at 40 degrees c e n t r i g r a d e , and the absorbance measured at 443 nm. Standard s o l u t i o n s of v a r y i n g c o n c e n t r a t i o n s of hydroxymethylfurfuraldehyde (20 to 150 uM -44-HMF) were .prepared i n 2 M a c e t i c a c i d and a l s o read at 443 nm. The degree of g l y c o s y l a t i o n was c a l c u l a t e d as the HMF Index. A HMF Index of one i s e q u i v a l e n t to one umol of HMF present per g of globin p r o t e i n . CYTOCHROME C OXIDASE Cytochrome C oxidase was assayed s p e c t r o p h o t o m e t r i c a l l y by the method of Wharton and T z a g a l o f f (1967). The decrease i n absorbance at 550 nm was used as an index of the rate of o x i d a t i o n of ferrocytochrome C. To each of 2 cuvettes was added 100 u l of 0.01 mM potassium phosphate b u f f e r pH 7.0, 70 u l 1% ferrocytochrome C, and 0.83 ml H 2 o . Ferrocytochrome C had p r e v i o u s l y been reduced with ascorbate and d i a l y z e d e x t e n s i v e l y . To the blank cuvette was added 10 u l 0.1 M potassium f e r r o c y a n a t e to o x i d i z e the ferrocytochrome p r e s e n t . Foll o w i n g e q u i l i b r a t i o n at 37 degrees c e n t r i g r a d e the r e a c t i o n was i n i t i a t e d by adding 10 u l of c a r d i a c SR ( approximately 5 ug p r o t e i n ) to the r e a c t i o n c u v e t t e . The decrease i n absorbance was measured continuously at 550 nm f o r 3 minutes. Cytochrome C oxidase a c t i v i t y of the SR was determined using the f o l l o w i n g f i r s t order k i n e t i c equations. k = 2.3 log (absorbance at time 0) min""1, (Absorbance at one minute) S p e c i f i c A c t i v i t y = k ( c o n c e n t r a t i o n of cytochrome c) ( c o n c e n t r a t i o n of SR) -45-Th e s p e c i f i c a c t i v i t y was expressed as nmol of cytochrome c o x i d i z e d per mg SR p r o t e i n per min. ELECTRON MICROSCOPY OF CARDIAC SARCOPLASMIC RETICULUM VESICLES A 0.5 ml suspension of c o n t r o l and d i a b e t i c c a r d i a c SR c o n t a i n i n g approximately 100 ug of p r o t e i n was c e n t r i f u g e d at 40,000 times g f o r 40 minutes. The r e s u l t a n t p e l l e t was f i x e d i n 5 ml 2% g l u t a r a l d e h y d e , 0.06 M c a c o d y l a t e b u f f e r pH 7.4. The SR p e l l e t was then s t o r e d i n 7% sucrose s o l u t i o n at 4 degrees cent igrade u n t i l f u r t h e r processed. The f i x e d p e l l e t was p o s t - f i x e d f o r 2 hours i n 1 % OsO^ contained i n a c o l l i d i n e b u f f e r pH 7.4. The c o l l i d i n e b u f f e r was prepared by adding 5.34 ml of p u r i f i e d s y m - c o l l i d i n e and 18 ml of 1 N HC1 to 200 ml d i s t i l l e d H 20. F o l l o w i n g f i x a t i o n the p e l l e t was dehydrated by a number of s e q u e n t i a l ethanol and propylene oxide washes. Small p o r t i o n s of t i s s u e were then embedded i n an Epon 813 r e s i n u t i l i z i n g an EM k i t produced by P o l y s c i e n c e r Inc., Warrington PA). The embedded t i s s u e was then processed using standard e l e c t r o n microscopy procedures. PROTEIN ASSAY Microsomal SR (between 10-150 ug p r o t e i n ) was suspended i n d i s t i l l e d H 20 to a f i n a l volume of 1.5 ml. To t h i s was added 12.5 u l of a 2% deoxycholate s o l u t i o n . F o l l o w i n g a 10 minute i n c u b a t i o n at room temperature, 0.5 ml of c o l d 24% t r i c h l o r o a c e t i c a c i d was added to p r e c i p i t a t e any p r o t e i n . - 4 6 -Th e suspension was c e n t r i f u g e d at 3000 times g r a v i t y f o r 30 minutes. The r e s u l t a n t p e l l e t was assayed f o r p r o t e i n using the standard Lowry (1951) p r o t e i n assay. Varying c o n c e n t r a t i o n s of serum albumin were used as standards. STATISTICAL ANALYSIS When two samples were compared, s t a t i s t i c a l a n a l y s i s was performed using the unpaired Student's t - t e s t . For m u l t i p l e comparison, One-way a n a l y s i s of v a r i a n c e , followed by the Newman-Keuls t e s t was used. A p r o b a b i l i t y of p<0.05 was used as the l e v e l of s i g n i f i c a n c e . S t a t i s t i c a l a n a l y s i s on slopes of curves was performed using r e g r e s s i o n a n a l y s i s f o r each set of data from the treatment groups, followed by one-way a n a l y s i s of v a r i a n c e and the Newman-Keuls t e s t on the r e s u l t a n t slopes from the i n d i v i d u a l data s e t s . -47-RESULTS A) The c h e m i c a l l y - i n d u c e d d i a b e t i c r a t model In i n i t i a l s t u d i e s d i a b e t e s was induced by a s i n g l e i . v . i n j e c t i o n of 60 mg/kg s t r e p t o z o t o c i n or 65 mg/kg a l l o x a n . In subsequent s t u d i e s the dose of s t r e p t o z o t o c i n was lowered to 55 mg/kg, and f i n a l l y to 50 mg/kg i . v . . T h i s was done because the animals appeared to become more s e n s i t i v e to the drug as the study progressed. During the f i r s t three days f o l l o w i n g i n j e c t i o n , approximately 10 % of the s t r e p t o z o t o c i n - i n j e c t e d , and 30 % of the a l l o x a n - i n j e c t e d r a t s d i e d ; probably as a r e s u l t of hypoglycemic coma due to massive i n s u l i n r e l e a s e . The c o n d i t i o n of the r a t s s t a b i l i z e d a f t e r 3 days. Animals which became d i a b e t i c (90 % of s t r e p t o z o t o c i n - i n j e c t e d , 60 % of a l l o x a n - i n j e c t e d ) had an e l e v a t e d u r i n e glucose ( g r e a t e r than 2 %) throughout the study p e r i o d . F i g u r e 1 i n d i c a t e s the serum glucose and i n s u l i n l e v e l s measured i n c h e m i c a l l y - i n d u c e d d i a b e t i c r a t s 7 days and 120 days a f t e r the i n d u c t i o n of d i a b e t e s . Serum i n s u l i n l e v e l s of f a s t e d r a t s , measured at the time of s a c r i f i c e , w e r e s i g n i f i c a n t l y lower i n both c h r o n i c a l l y (120 days) and a c u t e l y (7 days) d i a b e t i c r a t s . Serum glucose l e v e l s of f a s t e d r a t s , a l s o measured at the time of s a c r i f i c e , were markedly increased i n both the c h r o n i c a l l y and a c u t e l y d i a b e t i c r a t s . Table 1 shows that the weight g a i n i n both 7 day and 120 day d i a b e t i c r a t s was s i g n i f i c a n t l y lower than i n the c o n t r o l r a t s . During the study p e r i o d the i n j e c t e d r a t s d i s p l a y e d a number of symptoms a s s o c i a t e d with d i a b e t e s . These i n c l u d e d polyphagia, p o l y d y p s i a , p o l y u r i a , -48-FIGURE 1: Serum l e v e l s of i n s u l i n and glucose from a c u t e l y (7 days) and c h r o n i c a l l y (120 days) d i a b e t i c r a t s . Serum samples were c o l l e c t e d at time of s a c r i f i c e i n c o n t r o l , s t r e p t o z o t o c i n - and a l l o x a n - t r e a t e d r a t s f a s t e d f o r 16 hours. Measurement of i n s u l i n (open bars) and glucose (closed bars) are as d e s c r i b e d i n Methods. Numbers i n bars i n d i c a t e sample s i z e . R e s u l t shown is the mean + S.D. (*p<0.05, Student's unpaired t - t e s t , as compared to c o n t r o l ) . Serum Insulin (jull/ml) o CJ O O Cn O n O 3 :' »o ".-x-'.-x CO a a a O X a 3 n o 3 h CO O O a O X Q 3 1 i 1 1 r o CO Cn O o o O o o o O o o Serum Glucose (mg/IOOml) -50-Table 1: Body weights of control, streptozotocin-treated, and alloxan-treated diabetic rats at 7 days and 120 days after the induction of diabetes. a) Acute diabetes Day 0 Body Weight (g) Control n=29 177 ± 8 Day 7 Control n=8 192 ± 8 Streptozotocin-treated n=5 163 ± 6* Alloxan-treated n=4 170+4* b) Chronic diabetes Day 0 Control n=40 190+7 Day 120 Control n=8 270 ± 24 Streptozotocin-treated n=7 226 ± 24* Alloxan-treated n=4 232 + 22* Diabetes was i n i t i a t e d at day 0 by an i . v . i n j e c t i o n of 60 mg/kg streptozotocin, or 65 mg/kg alloxan. Results are expressed as the mean + S.D. • s i g n i f i c a n t l y d i f f e r e n t from control (p <0.05, analysis of variance, followed by Newman-Keuls test) -51-and c a t a r a c t formation i n the eye. As d i s c u s s e d l a t e r , i n s u l i n treatment of d i a b e t i c r a t s not on l y prevented these symptoms of d i a b e t e s , but a l s o reduced serum glucose l e v e l s to c o n t r o l l e v e l s . As a f u r t h e r index of d i a b e t e s , g l y c o s y l a t i o n of blood hemoglobin was measured. E l e v a t i o n of g l y c o s y l a t e d hemoglobins i n d i c a t e s that blood glucose l e v e l s have been el e v a t e d f o r extended p e r i o d s of time. Table 2 i n d i c a t e s the l e v e l s of g l y c o s y l a t e d hemoglobin obtained from 30 day, 42 day, and 150 day s t r e p t o z o t o c i n - t r e a t e d d i a b e t i c r a t s . The l e v e l of g l y c o s y l a t i o n was e l e v a t e d i n d i a b e t i c r a t s , as compared to c o n t r o l , at a l l 3 time p e r i o d s . In the 30 day d i a b e t i c r a t s t r e a t e d with i n s u l i n there was no s i g n i f i c a n t i n c r e a s e i n g l y c o s y l a t i o n , as compared to c o n t r o l . However,there was a l s o no s i g n i f i c a n t d i f f e r e n c e i n g l y c o s y l a t i o n between the 30 day d i a b e t i c and i n s u l i n - t r e a t e d d i a b e t i c r a t s . The reason f o r t h i s i s unclear s i n c e , as i n d i c a t e d l a t e r (Figure 15), the serum glucose l e v e l s measured i n the i n s u l i n - t r e a t e d d i a b e t i c r a t s were reduced to c o n t r o l l e v e l s . B) C h a r a c t e r i z a t i o n of c a r d i a c sarcoplasmic r e t i c u l u m from c o n t r o l and d i a b e t i c r a t s . In most s t u d i e s c a r d i a c SR was prepared by the method of Sumida et a l (1978). The y i e l d of SR obtained from c o n t r o l , s t r e p t o z o t o c i n - t r e a t e d , and a l l o x a n - t r e a t e d r a t s i s i n d i c a t e d -52-Table 2: Measurement of glycosylation of hemoglobin i n rats at various time periods after the induction of diabetes with streptozotocin. Condition HMF Index (umol/g globin p r o t e i n ) 9 a) 30 day diabetic rats control n=5 1.69 + 0.05 diabetic n=6 . 2.08 + 0.08* diabetic plus i n s u l i n n=5 1.94+0.08 b) 42 day diabetic rats control n=16 1.34 ± 0.09 diabetic n=12 2.60 + 0.17* c) 150 day diabetic rats control n=12 1.02 ± 0.04 diabetic n=6 1.79 + 0.13* a) HMF index i s equivalent to 1 umol 5-hydroxymethylfurfuraldehyde per g of globin protein b) i n s u l i n (3U/day) was administered via osmotic minipumps, as described in Methods, throughout the study period, commencing 3 days a f t e r the induction of diabetes. Results are expressed as the mean ± S.E. * s i g n i f i c a n t l y d i f f e r e n t from control (p<0.05, analysis of variance, followed by Newman-Keuls test) -53-Table 3: Yield of cardiac sarcoplasmic reticulum from control and chronically diabetic rats (120 day). rvmHi+inn Yield of Sarcoplasmic Wet Weight mg Sarcoplasmic uoncntion Reticulum (mg) Ventricle (g) Reticulum/g tissue Control (n=9) 1.05 ± 0 . 3 2 ^ 1.09 ± 0.06 0.96 S t r L P t e d ° ( " 5 ) " 0.89 * 0.15 0.91 * 0.07 0.97 A t £ £ ( n . 6 > ° - 9 5 t ° - " " < b ) a) Results are expressed as the mean + S.E. b) Data not available. -54-FIGURE 2 2+ Time course of Ca -uptake i n c a r d i a c sarcoplasmic r e t i c u l u m p r e p a r a t i o n s from c o n t r o l and c h r o n i c a l l y d i a b e t i c r a t s (120 2+ days). Ca -uptake was measured i n the presence of 2 uM 2+ f r e e Ca as d e s c r i b e d i n Methods, i n sarcoplasmic r e t i c u l u m p r e p a r a t i o n s from c o n t r o l (• •) or s t r e p t o z o t o c i n - t r e a t e d ( A • ) r a t s . 250-, Time (minutes) -56-i n Table 3. Although the v e n t r i c l e weights were lower i n the d i a b e t i c r a t s , the y i e l d of SR per g of wet v e n t r i c l e was s i m i l a r . A l s o , as shown i n F i g u r e 3 and 4, the degree of v e s i c u l a t i o n was s i m i l a r i n both c o n t r o l and d i a b e t i c r a t SR 2+ p r e p a r a t i o n s . In these p r e p a r a t i o n s , at 2 uM f r e e Ca , the 2+ rate of Ca t r a n s p o r t a c t i v i t y i n c o n t r o l and d i a b e t i c r a t SR was 31.4 ± 3.7 (S.D.) nmol/mg/min, and 20.9 ± 2.4 r e s p e c t i v e l y . In i n i t i a l s t u d i e s , c a r d i a c SR was prepared by the method of Harigaya and Schwartz (1969). At 2 uM f r e e 2+ 2+ Ca , the r a t e of Ca t r a n s p o r t a c t i v i t y i n c o n t r o l and d i a b e t i c r a t SR was 12.2 1.1 and 9.0 ± 1.4 r e s p e c t i v e l y . Although the a c t i v i t y of the i n i t i a l p r e p a r a t i o n was lower, the d e p r e s s i o n i n C a 2 + t r a n s p o r t , seen i n the d i a b e t i c r a t SR, was p r o p o r t i o n a t e l y s i m i l a r to that seen i n the SR p r e p a r a t i o n s of Sumida et a l . These r a t e s of C a 2 uptake, i n the c o n t r o l p r e p a r a t i o n s , are comparable to those reported by other authors ( F r o e l i c h et a l , 1978; Ho l l i n g w o r t h and England, 1978). 2+ When measuring Ca -uptake a c t i v i t y i n c a r d i a c SR va r i o u s i n c u b a t i o n c o n d i t i o n s were determined. The length of the p r e - i n c u b a t i o n time was found not to a f f e c t the rate of 2+ Ca -uptake. S i m i l a r l y i t was found t h a t , up to 20 minutes, the length of the i n c u b a t i o n time d i d not a f f e c t the rate of 2+ Ca -uptake. T h i s i s i n d i c a t e d i n Fig u r e 2 which shows that 2+ between 2 and 10 minutes Ca -uptake was l i n e a r i n c o n t r o l and d i a b e t i c r a t s . In t h i s experiment the amount of SR p r o t e i n used (36 ug/0.5 ml i n c u b a t i o n volume) was s i m i l a r to that used throughout the study. Using the ATP h y d r o l y s i s -57-FIGURE 3: E l e c t r o n micrograph of c a r d i a c sarcoplasmic r e t i c u l u m obtained from c o n t r o l r a t s . Sarcoplasmic r e t i c u l u m microsomes were prepared as d e s c r i b e d i n Methods. T i s s u e p r e p a r a t i o n s are shown magnified 19,000 times. C o n t r o l r a t m i c r o s o m a l p r e p a r a t i o n 2 -59-FIGURE 4: E l e c t r o n micrograph of c a r d i a c sarcoplasmic r e t i c u l u m obtained from s i x week d i a b e t i c r a t s . Diabetes was induced by a s i n g l e i . v . i n j e c t i o n of 50 mg/kg s t r e p t o z o t o c i n . Sarcoplasmic r e t i c u l u m microsomes were prepared as d e s c r i b e d i n the Methods. T i s s u e p r e p a r a t i o n are shown magnified 19,000 times. Diabetic rat microsomal preparation 2 - 6 1 -values shown i n Table 5 i t was determined that i n no i n c u b a t i o n sample was more than 7% of the ATP u t i l i z e d , and end product i n h i t i o n was t h e r e f o r e of no consequence. 2+ Ca -ATPase a c t i v i t y was a l s o measured i n the SR p r e p a r a t i o n s prepared by the method of Sumida et a l . Using the ATP r e g e n e r a t i n g system, d e s c r i b e d i n the Methods, i t was found that ATP h y d r o l y s i s was l i n e a r with time. Continuous spectrophotometric readings were l i n e a r up to and beyond 25 minutes when 5 ug SR was used i n the i n c u b a t i o n v i a l . To f u r t h e r c h a r a c t e r i z e the c a r d i a c SR p r e p a r a t i o n s v a r i o u s marker enzyme a c t i v i t i e s were determined (Table 4). The degree of m i t o c h o n d r i a l contamination, as i n d i c a t e d by cytochrome c oxidase a c t i v i t y , was the same i n both c o n t r o l and d i a b e t i c r a t c a r d i a c SR. T h i s m i t o c h o n d r i a l contamination 2 + d i d not c o n t r i b u t e to SR Ca accumulation, as determined by 2+ the absence of any a z i d e - s e n s i t i v e Ca t r a n s p o r t a c t i v i t y . A l s o , m i t o c h o n d r i a l and sarcolemmal contamination d i d not appear to c o n t r i b u t e to ATP h y d r o l y s i s measurements, as determined by the absence of a z i d e - s e n s i t i v e and 2+ 2+ ouabain-sensitive (Ca -Mg )-ATPase a c t i v i t y . -62-Table 4: Marker enzyme assays on cardiac sarcoplasmic reticulum from control and 42 day diabetic rats. Assay 3 Control Diabetic cytochrome c oxidase a c t i v i t y b 53.0 + 5.9 49.4 + 3.7 azid_e-sensitive Ca -transport a c t i v i t y N.D. N.D. azide-sensitive (Ca -Mg^ )-ATPase acti v t y N.D. N.D. oua bain-sensitive (Ca -Mg^ )-ATPase N.D. N.D. a c t i v i t y a) cytochrome c oxidase a c t i v i t y i s expressed as +the nmol of cytochrome c oxidized per mg SR per min_ Ca -transport a c t i v i t y i s expressed as the nmol of Ca tranported per mg SR per min. ATPase a c t i v i t y i s expressed as the nmol of ATP hydrolyzed per mg SR per min. b) Result i s the mean + S.E. of 5 control and 5 diabetic SR preparations N.D.-not detectable Diabetes was induced by a single i . v . injection of 50 mg/kg streptozotocin -63-C) E f f e c t of c h r o n i c d i a b e t e s on c a r d i a c microsomal sarcoplasmic r e t i c u l u m f u n c t i o n : 1) Calcium uptake and Calcium ATPase a c t i v i t y ATP-dependent t r i s - o x a l a t e - f a c i 1 itated Ca -uptake was measured i n c a r d i a c microsomal p r e p a r a t i o n s enriched i n s a rcoplasmic r e t i c u l u m from c o n t r o l and c h r o n i c a l l y d i a b e t i c 2+ r a t s (Figure 5). At a l l f r e e Ca c o n c e n t r a t i o n s t e s t e d 2+ (0.2-5.0 uM) the r a t e of Ca -uptake was s i g n i f i c a n t l y lower (p<0.05) i n the p r e p a r a t i o n s obtained from s t r e p t o z o t o c i n and a l l o x a n d i a b e t i c r a t s , compared to c o n t r o l s . R e c i p r o c a l p l o t s 2+ of these data revealed that the lower l e v e l s of Ca -uptake 2 + were accompanied by a decrease i n the V f o r Ca , with max 2+ no s i g n i f i c a n t change i n the apparent K ^ g g f o r Ca 2+ Ca -ATPase a c t i v i t y was determined i n c a r d i a c s arcoplasmic r e t i c u l u m from c o n t r o l and 120 day s t r e p t o z o t o c i n 2+ d i a b e t i c r a t s . As shown i n Table 5, both b a s a l Mg -ATPase 2+ a c t i v i t y and Ca -dependent ATPase a c t i v i t y were s i g n i f i c a n t l y (p<0.05) decreased i n sarcoplasmic r e t i c u l u m from d i a b e t i c r a t s . i) C a r n i t i n e and long c h a i n a c y l c a r n i t i n e s i n c a r d i a c microsomal sarcoplasmic r e t i c u l u m . L e v e l s of c a r n i t i n e and long chain a c y l c a r n i t i n e were measured i n microsomal p r e p a r a t i o n s e n r i c h e d i n SR prepared from c o n t r o l and c h r o n i c a l l y d i a b e t i c r a t s (Table 6). P r e p a r a t i o n s from the d i a b e t i c r a t s contained s i g n i f i c a n t l y (p <0.05) higher l e v e l s of f a t t y a c y l c a r n i t i n e s . L e v e l s of -64-FIGURE 5 E f f e c t of c h r o n i c d i a b e t e s (120 days) on c a r d i a c sarcoplasmic 2+ 2+ r e t i c u l u m Ca -uptake at v a r i o u s Ca c o n c e n t r a t i o n s . 2+ 2+ Ca -uptake and f r e e Ca c o n c e n t r a t i o n s were determined as d e s c r i b e d i n Methods i n sarcoplasmic r e t i c u l u m p r e p a r a t i o n s d e r i v e d from c o n t r o l (• • ) , s t r e p t o z o t o c i n - t r e a t e d ( O - O ) , or a l l o x a n - t r e a t e d r a t s (A A ) . The r e s u l t shown i s the mean + S.D. of ob s e r v a t i o n s from 7 c o n t r o l , 6 s t r e p t o z o t o c i n - t r e a t e d , and 5 a l l o x a n - t r e a t e d r a t s . The i n s e r t i s a double r e c i p r o c a l p l o t of the same data. A s t e r i s k i n d i c a t e s s i g n i f i c a n t l y d i f f e r e n t than c o n t r o l , p<0.05. [Ca*+] (juM) -66-Table 5: 2+ Measurement of Ca -ATPase activity in cardiac sarcoplasmic reticulum prepared from chronically diabetic rats (120 days) Conditions Control (n=8) Streptozotocin-treated (n=8) Mg2+-ATPase Activity (nmol/mg/min) 833.9 ± 66.4 518.5 ± 43.9 (Ca2+-Mg2+)-ATPase Activity (nmol/mg/min) 1270.9 ± 102.8 712.6 ± 184.3* * Significantly less than control p < 0.05 : a 2 + + M in Methods Ca2+-ATPase Activity (nmol/mg/min) 437.0 ± 48.8 194.1 ± 40.8* 2+ 2+ 2+ (Ca  Mg )-ATPase was measured at 2 yM free Ca as described -67-Table 6: Measured levels of carnitine and long chain acylcarnitines in cardiac microsomal preparations enriched in sarcoplasmic reticulum from chronically diabetic rats (120 days). CONDITIONS METABOLITE TISSUE LEVELS (nmol/mg S.R.) Control Total carnitine 8.47 + 1.66 (n = 11) Acid-soluble carnitine 6.93 + 1.33 Fatty acylcarnitine 0.96 + 0.18 Streptozotocin-Treated Total carnitine 12.49 + 1.68* (n = 6) Acid-soluble carnitine 9.75 + 1.91* Fatty acylcarnitine 1.51 + 0.28* Alloxan-Treated Total carnitine 13.65 + 2.84* (n = 5) Acid-soluble carnitine 10.53 + 1.38* Fatty acylcarnitine 1.27 + 0.19* Preparation of metabolites and measurement of carnitine levels are as described in Methods. Data are expressed as nmoles per mg of cardiac microsomal S.R. Results are expressed as the mean + S.D. * S i g n i f i c a n t l y different from control p < 0.05. -68-a c i d - s o l u b l e c a r n i t i n e were a l s o s i g n i f i c a n t l y higher i n these p r e p a r a t i o n s . L e v e l s of t o t a l c a r n i t i n e , a measure of both a c i d - s o l u b l e and f a t t y a c y l c a r n i t i n e , were a l s o s i g n i f i c a n t l y i n c reased i n p r e p a r a t i o n s from both a l l o x a n - and s t r e p t o z o t o c i n - t r e a t e d r a t s compared to c o n t r o l s . 2+ The r a t e of microsomal SR Ca - t r a n s p o r t was determined i n the presence of L - p a l m i t y l c a r n i t i n e i n c o n t r o l and c h r o n i c a l l y d i a b e t i c r a t s (Figure 6). L - p a l m i t y l c a r n i t i n e was chosen f o r study s i n c e i t i s the most abundant of the long chain a c y l c a r n i t i n e s i n the h e a r t . Since L - p a l m i t y l c a r n i t i n e 2+ i s a time-dependent i n h i b i t o r of Ca - t r a n s p o r t , microsomes were incubated i n i t s presence f o r 20 minutes p r i o r to the 2+ measurement of Ca -uptake. The length of the p r e - i n c u b a t i o n time was found to have no subsequent e f f e c t on 2+ the r a t e of Ca -uptake. A n a l y s i s of v a r i a n c e revealed that the c a l c i u m uptake l e v e l s i n the c o n t r o l r a t SR was s i g n i f i c a n t l y higher than that observed i n the a l l o x a n - or s t r e p t o z o t o c i n - t r e a t e d r a t p r e p a r a t i o n s up to 7 uM p a l m i t y l c a r n i t i n e . In p r e p a r a t i o n s from both c o n t r o l and 2+ d i a b e t i c r a t s , Ca t r a n s p o r t was completely i n h i b i t e d i n the presence of low c o n c e n t r a t i o n s of L - p a l m i t y l c a r n i t i n e (15-20 uM). At sub-maximal i n h i b i t o r y c o n c e n t r a t i o n s of 2+ L - p a l m i t y l c a r n i t i n e , Ca t r a n s p o r t was i n h i b i t e d to a g r e a t e r degree i n p r e p a r a t i o n s from c o n t r o l s ; i n the presence 2+ of 7 uM L - p a l m i t y l c a r n i t i n e Ca t r a n s p o r t was reduced to 55% of i t s o r i g i n a l r a t e , whereas at t h i s c o n c e n t r a t i o n SR p r e p a r a t i o n s from a l l o x a n - and s t r e p t o z o t o c i n - t r e a t e d r a t s 7 + r e t a i n e d 75% of the o r i g i n a l Ca t r a n s p o r t a c t i v i t y . At -69-FIGURE 6 E f f e c t of L - p a l n u t y l c a r n i t i n e on Ca"" -uptake i n sarcoplasmic r e t i c u l u m p r e p a r a t i o n s from c h r o n i c a l l y d i a b e t i c r a t s (120 2+ day s ) . Ca -uptake was measured i n the presence of 2 uM 2+ fr e e Ca , as d e s c r i b e d i n Methods, i n sarcoplasmic r e t i c u l u m p r e p a r a t i o n s from c o n t r o l (• •) , s t r e p t o z o t o c i n - t r e a t e d (O O) , or a l l o x a n - t r e a t e d (A A ) r a t s . The r e s u l t shown i s the mean + S.D. of obs e r v a t i o n s from 6 c o n t r o l s , 5 s t r e p t o z o t o c i n - , and 4 a l l o x a n - t r e a t e d r a t s . The i n s e r t i n d i c a t e s the percent of maximal uptake i n the c o n t r o l s and the t r e a t e d p r e p a r a t i o n s at each p a l m i t y l c a r n i t i n e c o n c e n t r a t i o n . A s t e r i s k i n d i c a t e s 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 p<0.05. -71-c o n c e n t r a t i o n s above 7 uM the r a t e of Ca t r a n s p o r t i n p r e p a r a t i o n s from c o n t r o l and d i a b e t i c r a t s was s i m i l a r . S t a t i s t i c a l a n a l y s i s of the s l o p e s of the curves revealed that between 2 and 10 uM L - p a l m i t y l c a r n i t i n e the slope of the c o n t r o l group was s i g n i f i c a n t l y d i f f e r e n t from that of the t r e a t e d groups (eg the c o n t r o l slope was -11.10 + 0.71, compared to slope of,-3.48 +_ 0.62, and -4.62 + 0.55, i n the a l l o x a n - and s t r e p t o z o t o c i n - t r e a t e d animals r e s p e c t i v e l y ) . A s t a t i s t i c a l a n a l y s i s was performed using r e g r e s s i o n a n a l y s i s on each s e t of data from the three treatment groups, followed by one-way a n a l y s i s of v a r i a n c e on the r e s u l t i n g slopes from the i n d i v i d u a l data s e t s . Although markedly i n h i b i t e d , the 2+ r a t e of Ca -uptake remained l i n e a r with time i n SR t r e a t e d with 7 uM p a l m i t y l c a r n i t i n e . F i g u r e 7 i n d i c a t e s that SR 2+ 2+ (Ca +Mg )-ATPase a c t i v i t y i s a l s o i n h i b i t e d i n c o n t r o l and d i a b e t i c r a t s by s i m i l a r c o n c e n t r a t i o n s of L - p a l m i t y l c a r n i t i n e . In c o n t r o l p r e p a r a t i o n s the c o n c e n t r a t i o n of L - p a l m i t y l c a r n i t i n e necessary to h a l f - m a x i m a l l y i n h i b i t (Ca 2 ++Mg 2 +)-ATPase was 4.5 uM. T h i s c o r r e l a t e s with the c o n c e n t r a t i o n of L - p a l m i t y l c a r n i t i n e 2+ necessary to h a l f-maximally i n h i b i t Ca t r a n s p o r t a c t i v i t y . 7 + The e f f e c t of D , L - c a r n i t i n e on SR Ca -uptake a c t i v i t y was a l s o i n v e s t i g a t e d (Figure 8). In microsomal p r e p a r a t i o n s from both c o n t r o l and d i a b e t i c r a t s t o t a l i n h i b i t i o n of 2+ Ca -uptake occurred i n the presence of 50 mM D , L - c a r n i t i n e . T h i s c o n c e n t r a t i o n i s 1500 f o l d g r e a t e r than the c o n c e n t r a t i o n 2+ of L - p a l m i t y l c a r n i t i n e necessary to i n h i b i t Ca - t r a n s p o r t . -72-FIGURE 7 2+ 2+ E f f e c t of L - p a l m i t y l c a r n i t i n e on (Ca -Mg )-ATPase a c t i v i t y i n sarcoplasmic r e t i c u l u m p r e p a r a t i o n s from c h r o n i c a l l y d i a b e t i c r a t s (120 days). (Ca 2 +-Mg 2 +)-ATPase 2+ was measured i n the presence of 2 uM f r e e Ca i n SR pr e p a r a t i o n s from c o n t r o l (• •) and s t r e p t o z o t o c i n - t r e a t e d ( A - - " - A ) r a t s . The r e s u l t i s the mean + SE of 7 c o n t r o l and 7 s t r e p t o z o t o c i n - t r e a t e d r a t s . A s t e r i s k i n d i c a t e s s i g n i f i c a n t l y d i f f e r e n t than c o n t r o l , p<0.05. 7 J -74-FIGURE 8 2+ E f f e c t of D , L - c a r n i t i n e on Ca -uptake i n sarcoplasmic r e t i c u l u m p r e p a r a t i o n s from c h r o n i c a l l y d i a b e t i c r a t s (120 2+ days). Ca -uptake, was measured i n the presence of 2 uM 2 + f r e e Ca as d e s c r i b e d i n Methods, i n sarcoplasmic r e t i c u l u m p r e p a r a t i o n s d e r i v e d from c o n t r o l (• • ) , s t r e p t o z o t o c i n - t r e a t e d (O O) / or a l l o x a n - t r e a t e d (A- A ) r a t s . The r e s u l t shown i s the mean + S.D. of o b s e r v a t i o n s from 6 c o n t r o l s , 4 s t r e p t o z o t o c i n - , and 3 a l l o x a n - t r e a t e d r a t s . A s t e r s i k i n d i c a t e s s i g n i f i c a n t l y d i f f e r e n t than c o n t r o l p < 0.05. D,L-Carnitine (mM) -76-i i ) Chronic d i a b e t e s and the p e r m e a b i l i t y of the microsomal sarcoplasmic r e t i c u l u m to calcium 2+ The r a t e of Ca - e f f l u x from c a r d i a c microsomal SR was determined 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 (Figure 9). 45 2+ Microsomes were preloaded with Ca as d e s c r i b e d i n the 2+ Methods. The r a t e of Ca - e f f l u x from these p r e p a r a t i o n s was s i m i l a r to that of the c o n t r o l p r e p a r a t i o n s . T h i s 2+ suggests that the i n h i b i t i o n of Ca -uptake seen 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 not due to an i n c r e a s e i n the 2+ p e r m e a b i l i t y of the SR to Ca . i i i ) E f f e c t of c a l m o d u l i n , cAMP-dependent p r o t e i n k i n a s e , and + 2+ K on Ca -uptake a c t i v i t y i n c a r d i a c SR from c h r o n i c a l l y d i a b e t i c r a t s . The e f f e c t of three documented r e g u l a t o r s of c a r d i a c SR f u n c t i o n were determined i n SR from 120 day s t r e p t o z o t o c i n - i n d u c e d (55mg/kg i.v.) d i a b e t i c r a t s . Table 7a and 7b shows that both i n the absence (Table 7a) and presence (Table 7b) of 110 mM KC1, c a r d i a c SR from d i a b e t i c r a t s showed 2+ s i g n i f i c a n t l y l e s s Ca -uptake a c t i v i t y compared to c o n t r o l s . When calmodulin (0.61 uM) was added to the i n c u b a t i o n medium i n the absence of KCl (Table 7a), the degree of s t i m u l a t i o n observed was s i m i l a r i n both c o n t r o l and d i a b e t i c animals. KCl was excluded when examining the e f f e c t of calmodulin s i n c e previous work (Lopaschuk et a l , 1980) had shown calmodulin s t i m u l a t i o n to be decreased i n the presence of t h i s c a t i o n . Table 7b shows that i n the presence -77-F I G U R E 9 2+ E f f e c t o f c h r o n i c d i a b e t e s ( 1 2 0 d a y s ) on Ca e f f l u x f r o m c a r d i a c s a r c o p l a s m i c r e t i c u l u m . M i c r o s o m e s e n r i c h e d i n S . R . w e r e p r e l o a d e d w i t h 4 5 C a 2 + b y i n c u b a t i o n i n 2 uM f r e e 2+ 2+ Ca f o r 45 m i n u t e s . Ca e f f l u x was m e a s u r e d a s d e s c r i b e d ' i n M e t h o d s , i n S . R . p r e p a r a t i o n s d e r i v e d f r o m c o n t r o l ( • # ) , s t r e p t o z o t o c i n - t r e a t e d (O O) , o r a l l o x a n - t r e a t e d r a t s (A A ) . The r e s u l t i s t h e mean + S . D . o f o b s e r v a t i o n s f r o m 5 c o n t r o l s , 4 s t r e p t o z o t o c i n - , and 4 a l l o x a n - t r e a t e d r a t s . -78 -79-Table 7: Effect of K +, calmodulin, and cAMP-dependent protein kinase on calcium uptake a c t i v i t y in cardiac sarcoplasmic reticulum prep-arations from chronically diabetic rats (120 days). Condition Calcium Uptake A c t i v i t y % change (nmol/mg/min) + S.E.) a) 0 KC1 control n=8 7.29 + 0.38 control plus calmodulin (0.61 uM) 10.22 ± 0.47 +40.1% streptozotocin-treated n=7 4.92 + 0.46 streptozotocin-treated plus calmodulin (0.61 uM) 7.18 + 0.71 +45.9% b) 110 mM KC1 control n=7 18.98 + 1.07 control plus cAMP-dependent protein kinase (0.2 mg/0.5 ml) 24.86 + 1.66 +30.9% streptozotocin-treated n=7 14.79 +1.77 streptozotoci n-treated plus cAMP-dependent protein kinase (0.2mg/0.5ml) 18.28 + 2.06 +23.6% a) 10 uM cAMP was also present Diabetes was induced by a single i . v . in j e c t i o n of 60 mg/kg streptozotocin Calcium uptake was measured as described in Methods at 2 uM free Ca Results are expressed as the mean + S.E. 2+ -80-of cAMP (10 uM) and cAMP-dependent p r o t e i n kinase (0.2 mg/0.5 ml i n c u b a t i o n volume), the degree of s t i m u l a t i o n of 2+ . . Ca -uptake a c t i v i t y i n c o n t r o l r a t s i s not s i g n i f i c a n t l y d i f f e r e n t from that observed i n d i a b e t i c animals. From Table 2+ 7 i t was c a l c u l a t e d that 110 mM KCl s t i m u l a t e d Ca -uptake i n d i a b e t i c animals to a g r e a t e r extent than i n c o n t r o l r a t s (200 % compared to 160 % ) . We t h e r e f o r e undertook an i n v e s t i g a t i o n of the e f f e c t s of v a r y i n g c o n c e n t r a t i o n s of KCl 2+ on Ca -uptake a c t i v i t y i n c o n t r o l versus d i a b e t i c animals. As shown i n F i g u r e 10 c a r d i a c microsomes from s t r e p t o z o t o c i n - t r e a t e d animals accumulated s i g n i f i c a n t l y l e s s 2+ Ca than microsomes from c o n t r o l r a t s . An examination of the Km f o r K + of the enzyme uptake a c t i v i t y i n c o n t r o l and d i a b e t i c r a t s , as determined by an Eadie-Hofstee p l o t (Figure 11), showed the slopes of the two l i n e s to be p a r a l l e l , suggesting that the a f f i n i t y of the enzyme f o r K + remained unchanged. iv) E f f e c t of acute dia b e t e s on microsomal sarcoplasmic 2+ r e t i c u l u m Ca t r a n s p o r t , and sarcoplasmic r e t i c u l u m l e v e l s of c a r n i t i n e and long c h a i n a c y l c a r n i t i n e s . 2+ Ca -uptake a c t i v i t y i n microsomal p r e p a r a t i o n s enriched i n SR was measured 7 days a f t e r the i n d u c t i o n of d i a b e t e s by a l l o x a n or s t r e p t o z o t o c i n (Figure 12). SR p r e p a r a t i o n s from the a c u t e l y d i a b e t i c r a t s showed no decrease 2+ 2+ i n the r a t e of Ca -uptake at any of the Ca c o n c e n t r a t i o n s t e s t e d . L e v e l s of a c i d s o l u b l e c a r n i t i n e and -81-FIGURE 10 E f f e c t of v a r i o u s KC1 c o n c e n t r a t i o n s on calcium-uptake i n sarcoplasmic r e t i c u l u m p r e p a r a t i o n s from c o n t r o l (• •) and s t r e p t o z o t o c i n - t r e a t e d c h r o n i c a l l y d i a b e t i c ( A • ) r a t s . 2+ Calcium-uptake and f r e e Ca c o n c e n t r a t i o n s were determined as d e s c r i b e d i n Methods. R e s u l t s shown are the mean + S.E. of ob s e r v a t i o n s from 8 c o n t r o l and 7 s t r e p t o z o t o c i n - t r e a t e d r a t s . A s t e r i s k i n d i c a t e s s i g n i f i c a n t l y d i f f e r e n t than c o n t r o l , p < 0.05. 0-1 , 1 1 1 1 1 1 0 10 30 50 70 90 110 KCl concentration (mM) -83-FIGURE 1 1 An Eadie-Hofstee p l o t of the e f f e c t s of v a r i o u s K C 1 c o n c e n t r a t i o n s on calcium-uptake i n sarcoplasmic r e t i c u l u m p r e p a r a t i o n s from c o n t r o l (• •) and s t r e p t o z o t o c i n - t r e a t e d 2+ d i a b e t i c ( A - - * - A ) r a t s . Calcium uptake and f r e e Ca c o n c e n t r a t i o n s were determined as d e s c r i b e d i n Methods. R e s u l t s shown are the mean of ob s e r v a t i o n s from 8 c o n t r o l and 7 s t r e p t o z o t o c i n - t r e a t e d r a t s . 2jc V/(S) (nmol/mg/min)/mM KCl -85-f a t t y a c y l c a r n i t i n e were a l s o compared i n microsomal p r e p a r a t i o n s from 7 day d i a b e t i c r a t s and c o n t r o l s ( t a b l e 8). At t h i s time p e r i o d no d i s c e r n a b l e d i f f e r e n c e i n the l e v e l s of t o t a l c a r n i t i n e was observed. The c o n t r o l l e v e l s of t o t a l c a r n i t i n e are higher i n the 7 day c o n t r o l samples than i n the 120 day samples (see Table 6). There i s no e x p l a n a t i o n f o r t h i s except the d i f f e r e n c e i n age and weight of the c o n t r o l s used i n the 7 day and 120 day s t u d i e s , or experimental v a r i a t i o n between the two study p e r i o d s . 2+ Microsomal SR Ca t r a n s p o r t was a l s o measured i n a c u t e l y d i a b e t i c r a t s i n the presence of i n c r e a s i n g c o n c e n t r a t i o n s of L - p a l m i t y l c a r n i t i n e (Figure 13) or D , L - c a r n i t i n e (Figure 14). At a l l c o n c e n t r a t i o n s of L - p a l m i t y l c a r n i t i n e or D , L - c a r n i t i n e Ca -uptake a c t i v i t y was i d e n t i c a l i n c o n t r o l , a l l o x a n - , or s t r e p t o z o t o c i n - t r e a t e d r a t s . v) E f f e c t of i n s u l i n treatment on c a r d i a c sarcoplasmic r e t i c u l u m f u n c t i o n i n one month s t r e p t o z o t o c i n d i a b e t i c r a t s . Approximately 80% of the r a t s i n j e c t e d with a s i n g l e i . v . dose of 50 mg/kg s t r e p t o z o t o c i n were found to have el e v a t e d serum glucose l e v e l s w i t h i n 3 days of i n j e c t i o n . Only d i a b e t i c r a t s which had serum glucose l e v e l s g r e a t e r than 500 mg% were used i n the study. F i g u r e 15 shows the average serum glucose l e v e l s determined throughout the study p e r i o d f o r c o n t r o l , d i a b e t i c , and i n s u l i n - t r e a t e d d i a b e t i c r a t s . Implantation of A l z e t osmotic minipumps r e s u l t e d i n an -86-FIGURE 12 2+ E f f e c t of acute d i a b e t e s (7 days) on Ca -uptake i n c a r d i a c 2+ sarcoplasmic r e t i c u l u m p r e p a r a t i o n s at v a r i o u s Ca 2+ 2+ c o n c e n t r a t i o n s . Ca -uptake and f r e e Ca c o n c e n t r a t i o n s were determined as d e s c r i b e d i n Methods i n sarcoplasmic r e t i c u l u m p r e p a r a t i o n s d e r i v e d from c o n t r o l (• • ) , s t r e p t o z o t o c i n - t r e a t e d (O O) , or a l l o x a n - t r e a t e d (A A) r a t s . The r e s u l t i s the mean +, S.D. f o r o b s e r v a t i o n s from 6 c o n t r o l s , 5 s t r e p t o z o t o c i n - , and 4 a l l o x a n - t r e a t e d r a t s . C a 2 + Uptake : nmoles/mg/5min XI -88-Table 8: Measured levels of carnitine and long chain acylcarnitines in cardiac microsomal preparations enriched in sarcoplasmic reticulum from acutely diabetic rats (7 days). CONDITIONS Control (n = 7) Streptozotoci n-Treated (n = 5) Alloxan-Treated (n = 4) METABOLITE Total carnitine Acid soluble carnitine Fatty acylcarnitine Total carnitine Acid-soluble carnitine Fatty acylcarnitine Total carnitine Acid soluble carnitine Fatty acylcarnitine TISSUE LEVELS  (nmol/mg S.R.) 10.22 ± 2.48 7.78 ± 2.41 1.11 ± 0.28 10.96 ± 0.77 8.73 ± 1.38 1.09 ± 0.12 10.63 ± 2.25 7.55 ± 1.93 1.02 ± 0.19 Preparation of metabolites and measurement of carnitine levels are as described in Methods.Data are; expressed as nmoles per mg of cardiac microsomal S.R. Results are expressed as the mean + S.D. - 8 9 -F I G U R E 13 2+ E f f e c t o f L - p a l m i t y l c a r m t i n e on Ca - u p t a k e i n c a r d i a c s a r c o p l a s m i c r e t i c u l u m p r e p a r a t i o n s f r o m a c u t e l y d i a b e t i c r a t s 2+ (7 d a y s ) . Ca - u p t a k e was m e a s u r e d i n t h e p r e s e n c e o f 2 uM 2+ f r e e Ca as d e s c r i b e d i n M e t h o d s , i n s a r c o p l a s m i c r e t i c u l u m p r e p a r a t i o n s d e r i v e d f r o m c o n t r o l ( • • ) , s t r e p t o z o t o c i n - t r e a t e d (O O ) , o r a l l o x a n - t r e a t e d (A ' • • • A ) r a t s . The r e s u l t i s t h e mean +. S . D . o f o b s e r v a t i o n s f r o m 6 c o n t r o l , 4 s t r e p t o z o t o c i n , and 4 a l l o x a n - t r e a t e d r a t s . 9o -91-FIGURE 14 2+ E f f e c t of D , L - c a r n i t i n e on Ca -uptake i n c a r d i a c sarcoplasmic r e t i c u l u m p r e p a r a t i o n s from the a c u t e l y d i a b e t i c 2+ r a t (7days). Ca -uptake was measured i n the presence of 2 2+ uM f r e e Ca as d e s c r i b e d i n Methods, i n sarcoplasmic r e t i c u l u m p r e p a r a t i o n s d e r i v e d from c o n t r o l (• • ) , s t r e p t o z o t o c i n - t r e a t e d (O O) , or a l l o x a n - t r e a t e d ( A A ) r a t s . The r e s u l t shown i s the mean ± S.D. of o b s e r v a t i o n s from 6 c o n t r o l s , 5 s t r e p t o z o t o c i n , and 4 a l l o x a n - t r e a t e d r a t s . 100 D,L-Carnitine (mM) - 9 3 -immediate drop i n serum glucose l e v e l s . These minipumps d e l i v e r e d 3U of i n s u l i n d a i l y at a constant r a t e . Serum glucose determinations were made every 3 days to check the e f f i c i e n c y of the minipumps. Of the 20 d i a b e t i c r a t s s u r g i c a l l y implanted with i n s u l i n minipumps 4 died w i t h i n the f i r s t 3 days, probably as a r e s u l t of hypoglycemic coma. Serum glucose l e v e l s were w e l l maintained i n the remaining animals at 7 days p o s t - i m p l a n t a t i o n . By 10 days, 9 of the remaining 16 r a t s r e q u i r e d new i n s u l i n minipumps. In the remaining 7 r a t s mini pumps l a s t e d f o r the two week expected d u r a t i o n of a c t i o n . When new minipumps were implanted i n these 7 r a t s normal serum glucose l e v e l s and osmotic pump f u n c t i o n were w e l l maintained over a subsequent two week t e s t p e r i o d . In the remaining 9 r a t s , minipumps had to be again r e p l a c e d w i t h i n 6 to 10 days of i m p l a n t a t i o n . Within t h i s group, 4 r a t s demonstrated u n c o n t r o l l e d serum glucose l e v e l s . In these r a t s we found membranous t i s s u e e n c a p s u l a t i n g the minipump each time the minipump was r e p l a c e d . I t was necessary to i n j e c t 1-2 U of protamine-zinc i n s u l i n (Connaught r l a b o r a t o r i e s , Toronto, Ontario) s.c. i n these r a t s d a i l y to maintain normal serum glucose l e v e l s . The shortened d u r a t i o n of a c t i o n of the minipumps i n the remaining 5 r a t s of t h i s group of 9 could not r e a d i l y be e x p l a i n e d . D i s s e c t i o n of the minipumps upon removal revealed that i n s u l i n s o l u t i o n was s t i l l present w i t h i n the r e s e r v o i r . At the end of the 31 day study p e r i o d , body weights of c o n t r o l , d i a b e t i c and i n s u l i n - t r e a t e d d i a b e t i c r a t s were compared. As shown i n Table 9, weight gain i n i n s u l i n t r e a t e d - 9 4 -FIGURE 15 Serum glucose l e v e l s of c o n t r o l , d i a b e t i c and i n s u l i n - t r e a t e d d i a b e t i c r a t s . Serum glucose was determined i n 11 c o n t r o l (• •) , 19 d i a b e t i c ( A A ) , and 16 i n s u l i n - t r e a t e d r a t s (• • ) . I n s u l i n treatment was i n i t i a t e d at 3 days by s u r g i c a l i m p l a n t a t i o n of A l z e t r osmotic mini-pumps c o n t a i n i n g 0.228 +. 0.26 (S.D.) ml of 273 U/ml i n s u l i n ( L i l l y r , U-500 I l l e t i n I I ) . At a pumping r a t e of 0.458 u l / h r , 3U of i n s u l i n were r e l e a s e d subcutaneously every 24 hours. Minipumps were changed i f serum glucose exceeded 150 mg %. d a y s -96-r a t s was s i g n i f i c a n t l y g r e a t e r than d i a b e t i c r a t s . As would be expected, weight g a i n i n the i n s u l i n - t r e a t e d d i a b e t i c animals approximated that of c o n t r o l r a t s . C a r d i a c SR was prepared from these r a t s and Ca t r a n s p o r t measured. Fi g u r e 16 i n d i c a t e s the r a t e of 2+ • 2+ Ca -uptake by SR at v a r i o u s f r e e Ca c o n c e n t r a t i o n s 2+ between 0.2 and 5 uM. The a b i l i t y of SR to t r a n s p o r t Ca was s l i g h t l y depressed i n the d i a b e t i c r a t as compared to c o n t r o l , although t h i s was s i g n i f i c a n t only at 2 uM f r e e 2+ 2+ Ca . However, Ca t r a n s p o r t a c t i v i t y i n SR from i n s u l i n - t r e a t e d d i a b e t i c r a t s was s i g n i f i c a n t l y g r e a t e r at a l l 2+ Ca c o n c e n t r a t i o n s t e s t e d . In t h i s study HEPES b u f f e r was used i n s t e a d of h i s t i d i n e - H C l i n the assay medium. T h i s may 2+ 2+ e x p l a i n the g r e a t e r Ca -uptake r a t e s at lower f r e e Ca c o n c e n t r a t i o n s noted here. In membranes from red blood c e l l s 2+ HEPES has been shown to i n c r e a s e the Ca a f f i n i t y of the ATPase enzyme. When c a r d i a c microsomal SR l e v e l s of c a r n i t i n e and long chain a c y l c a r n i t i n e s were measured i n these p r e p a r a t i o n s (Table 10) no d i f f e r e n c e between c o n t r o l and d i a b e t i c r a t s was seen. C a r d i a c SR from i n s u l i n - t r e a t e d d i a b e t i c r a t s contained s l i g h t l y lower l e v e l s of long c h a i n a c y l c a r n i t i n e s , although t h i s was not s i g n i f i c a n t l y d i f f e r e n t than c o n t r o l . S i m i l a r s t u d i e s performed by Arun T a h l l i a n i (unpublished observations) on r a t s 10 weeks a f t e r the i n d u c t i o n of d i a b e t e s showed s i g n i f i c a n t l y decreased C a 2 + transport a c t i v i t y at a l l C a 2 + c o n c e n t r a t i o n s t e s t e d . Furthermore, i n s u l i n treatment prevented any d e p r e s s i o n of SR C a 2 + t r a n p o r t from o c c u r r i n g . -97-Table 9: Body weights of control, rats. Day 0 control Day 31 control (n=8) diabetic (n=8) insulin-treated (n=8) diabetic and insulin-treated diabetic Body Weight (g) 166±8 g 199±4 172±15* 213±11 a) diabetes initiated at day 0 by I .V. injection of 50 mg/kg streptozotocin. b) insulin treatment started at day 3 by surgical implantation of p insulin-f i l led Alza osmotic minipumps. •Significantly different from insulin-treated diabetic (p<0.05 analysis of variance, followed by Newman-Keuls test). -98-FIGURE 16 E f f e c t of d i a b e t e s (31 days) on c a r d i a c sarcoplasmic r e t i c u l u m 2+ 2+ 2+ Ca -uptake at v a r i o u s Ca c o n c e n t r a t i o n s . Ca -uptake 2+ and f r e e Ca c o n c e n t r a t i o n s were determined as d e s c r i b e d i n Methods, i n SR p r e p a r a t i o n s from c o n t r o l (• • ) , d i a b e t i c ( • A ) , and i n s u l i n - t r e a t e d d i a b e t i c r a t s (• • ) . Administration of i n s u l i n was as d e s c r i b e d i n Methods. The r e s u l t i s the mean ± S.E. of 8 c o n t r o l , 8 u n t r e a t e d - d i a b e t i c , and 8 i n s u l i n - t r e a t e d d i a b e t i c r a t s . A s t e r i s k s i n d i c 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 than c o n t r o l , p<0.05. 9? 1 1 1 02 [ C o ' i (MM) -100 -Cardiac SR l e v e l s of LC a c y l c a r n i t i n e s were not measured i n t h i s study. -101-Table 1 0: Levels of carnitine and long chain acylcarnitines in cardiac microsomal preparations enriched in sarcoplasmic reticulum from 31 day diabetic, and insulin-treated diabetic rats. Condition control n=8 diabetic n=8 diabetic plus i n s u l i n n=8 Metabolite acid-soluble carnitine f a t t y acylcarnitine acid-soluble carnitine f a t t y acylcarnitine acid-soluble carnitine f a t t y acylcarnitine Tissue levels (nmol carnitine/mq SR) 5.66 + 0.05 0.82 + 0.06 5.65 + 0.24 0.85 + 0.06 4.91 + 0.30 0.65 + 0.07 Preparation of metabolites and measurement of carnitine levels are as described in Methods. Data are expressed as nmoles of carnitine per mg of cardiac microsomal SR protein. Results are expressed as the mean + S.D. Diabetes was induced by a single i . v . injection of 50 mg/kg streptozotocin -102-D) E f f e c t of o r a l c a r n i t i n e treatment on d i a b e t i c r a t heart f u n c t i o n ; p r e v e n t i o n and r e v e r s a l s t u d i e s . The e f f e c t of o r a l l y administered D f L - c a r n i t i n e on f u n c t i o n a l and b i o c h e m i c a l changes seen i n d i a b e t i c h e arts was i n v e s t i g a t e d . In the i n i t i a l study ( r e v e r s a l s t u d y ) , D , L - c a r n i t i n e was administered to d i a b e t i c and c o n t r o l r a t s , v i a t h e i r d r i n k i n g water, at a dose of 1 g/kg/day. C a r n i t i n e treatment commenced 120 days a f t e r the i n d u c t i o n of d i a b e t e s and continued f o r 30 days, a f t e r which time the hearts were removed and perfused on a working heart apparatus. A f t e r monitoring heart f u n c t i o n at v a r i o u s p r e l o a d s , hearts were q u i c k l y f r o z e n and v e n t r i c u l a r t i s s u e l e v e l s of c a r n i t i n e and long c h a i n a c y l c a r n i t i n e s measured. In the second study (prevention study) d i a b e t i c and c o n t r o l r a t s were administered 3 g/kg/day D , L - c a r n i t i n e 3 days a f t e r the i n d u c t i o n of d i a b e t e s f o r a 42 day p e r i o d . The r a t s were then randomly d i v i d e d i n t o two experimental groups. Hearts from one group were used to prepare SR, while hearts from the second group were perfused on a working heart apparatus. Table 11 i n d i c a t e s v a r i o u s parameters measured i n the d i a b e t i c r a t s . Body weights were s i g n i f i c a n t l y decreased i n a l l groups of d i a b e t i c r a t s . The degree of g l y c o s y l a t i o n of hemoglobin i n the d i a b e t i c r a t was s i g n i f i c a n t l y g r e a t e r than that found i n the c o n t r o l r a t s . In the r e v e r s a l study, g l y c o s y a t e d hemoglobin was 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 the c a r n i t i n e - t r e a t e d c o n t r o l r a t s . The reason f o r t h i s i s -1 03-Table 11: Measurement of body weight, serum glucose l e v e l s , glycosylation of hemoglobin, and y i e l d of SR in control and diabetic rats administered D,L-carnitine. Reversal study Prevention Study (120 day diabetes, (3 days diabetes, then then 30 days 1g/kg/day 42 days 3 g/kg/day D,L-D,L-carnitine treatment) carnitine treatment 1) Body weight (g) (at time of s a c r i f i c e ) control 229 + 36 (10) 278 + 13 (19) control plus carnitine 249 + 6 (9) 282 ± 11 (12) diabetic 164 + 14*(17) 208 ± 21*(15) diabetic plus carnitine 161 + 19*(7) 193 + 21*(14) 2) Serum glucose (mg %) control 162 + 25 (10) 131 ± 9 (20) control plus carnitine 159 ± 26 (9) 144 + 12 (12) diabetic 463 + 16*(5) 425 ± 22*(13) diabetic plus carnitine 485 ± 22*(7) 430 ± 5 *(14) 3) Glycosylated hemoglobin (HMF Index) 3 control 1.02+0.04(12) 1.18 ± 0.09 (16) control plus carnitine 1.31 + 0.06*(5) 1.34 + 0.13 (11) diabetic 1.79 + 0.13*(6) 2.60 + 0.17*(12) diabetic plus carnitine 2.37 + 0.07*(7) 2.43 ± 0.14*(13) 4) Yield of SR (mg/g wet ventricle) control - 1.02 control plus carnitine - 0.94 diabetic - 1.05 diabetic plus carnitine , - 0.86 Numbers in parentheses indicate the number of animals a) umol HMF/g globin protein Results are expressed as the mean + S.E. •S i g n i f i c a n t l y d i f f e r e n t from control (p<0.05, analysis of variance, followed by Newman-Keuls test) -104-unknown. The y i e l d of SR per g of v e n t r i c u l a r t i s s u e was s i m i l a r i n both c o n t r o l and d i a b e t i c r a t s . 1) R eversal study When 1 g/kg/day of D , L - c a r n i t i n e was administered to d i a b e t i c r a t s there was a small i n c r e a s e i n serum c a r n i t i n e l e v e l s (Table 12). The s i g n i f i c a n t l y lower (p<0.05) l e v e l s of serum c a r n i t i n e i n d i a b e t i c r a t s was reversed by c a r n i t i n e a d m i n i s t r a t i o n . Since serum c a r n i t i n e l e v e l s are normally low , due to r a p i d uptake of c i r c u l a t i n g c a r n i t i n e by the kidney, l i v e r , and h e a r t , l a r g e changes i n serum c a r n i t i n e l e v e l s due to c a r n i t i n e a d m i n i s t r a t i o n would not n e c e s s a r i l y be expected. F i g u r e s 17,18, and 19 i n d i c a t e the e f f e c t of v a r y i n g the f i l l i n g pressure on three parameters of heart f u n c t i o n ( l e f t v e n t r i c u l a r developed p r e s s u r e , +dP/dT, and -dP/dT) i n d i a b e t i c and c o n t r o l c a r n i t i n e - f e d r a t s . As shown, heart f u n c t i o n i s depressed i n d i a b e t i c r a t s at higher p r e l o a d s . The depressed f u n c t i o n i s not reversed by 30 day c a r n i t i n e treatment. T i s s u e l e v e l s of c a r n i t i n e e s t e r s were then measured i n these h e a r t s . The l e v e l s of f r e e c a r n i t i n e were s i m i l a r i n a l l groups of r a t s (Table 13). In d i a b e t i c r a t s the t i s s u e l e v e l s of long c h a i n a c y l c a r n i t i n e s were s i g n i f i c a n t l y e l e v a t e d (Table 13). Administration of c a r n i t i n e to d i a b e t i c r a t s r e s u l t e d i n a lowering of long chain a c y l c a r n i t i n e l e v e l s . 2) P revention study In the r e v e r s a l study i t was shown that c a r n i t i n e -105-Table 12: Levels of free carnitine i n serum obtained from chronically diabetic and control rats treated with carnitine. Carnitine (lg/kg/day) was administered for 30 days to rats 120 days after the induction of diabetes with 50mg/kg i . v . streptozotocin. Conditions Serum Levels (nmol/ml) control 17.82 + 0.62 n=9 control plus carnitine 20.74 + 0.63* n=8 diabetic 15.36 + 0.43* n=9 diabetic plus carnitine 17.58 + 0.51 n=7 Measurement of serum carnitine was as described in the Methods, Data are expressed as nmol of carnitine per ml of serum. Results are expressed as the mean + S.E. ••Significantly di fferent from control (p<0.05, analysis of variance, followed by the Newman-Keuls t e s t ) . -106-FIGURE 17 E f f e c t of c h r o n i c d i a b e t e s and c a r n i t i n e treatment on l e f t v e n t r i c u l a r developed pressure of i s o l a t e d perfused working h e a r t s at v a r i o u s f i l l i n g p r e s s u r e s . Hearts were i s o l a t e d from c o n t r o l (• #) , c a r n i t i n e - t r e a t e d c o n t r o l (• •) , d i a b e t i c ( • A ) , and c a r n i t i n e - t r e a t e d d i a b e t i c (• •) r a t s 150 days a f t e r the i n d u c t i o n of diab e t e s with 50mg/kg i . v . s t r e p t o z o t o c i n . C a r n i t i n e - t r e a t e d c o n t r o l and d i a b e t i c r a t s r e c i e v e d lg/kg/day D , L - c a r n i t i n e o r a l l y f o r 30 days p r i o r to s a c r i f i c e . The r e s u l t i s the mean £. S.E. of 6 c o n t r o l , 5 c a r n i t i n e - t r e a t e d c o n t r o l , 5 d i a b e t i c , and 5 c a r n i t i n e - t r e a t e d d i a b e t i c r a t s . A s t e r i s k i n d i c a t e s s i g n i f i c a n t l y d i f f e r e n t than c o n t r o l , p<0.05. /oi -108-FIGURE 18 E f f e c t of c h r o n i c d i a b e t e s and c a r n i t i n e treatment on p o s i t i v e dP/dT of i s o l a t e d perfused working hearts at v a r i o u s f i l l i n g p r e s s u r e s . Hearts were i s o l a t e d from c o n t r o l (•< • ) , c a r n i t i n e - t r e a t e d c o n t r o l (• •) , d i a b e t i c ( A A ) , and c a r n i t i n e - t r e a t e d d i a b e t i c + ) r a t s 150 days a f t e r the i n d u c t i o n of di a b e t e s with 50 mg/kg i . v . s t r e p t o z o t o c i n . C a r n i t i n e - t r e a t e d c o n t r o l and d i a b e t i c r a t s recei ved lg/kg/day D , L - c a r n i t i n e o r a l l y f o r 30 days p r i o r to s a c r i f i c e . The r e s u l t i s the mean + S.E. of 6 c o n t r o l , 5 c a r n i t i n e - t r e a t e d c o n t r o l , 5 d i a b e t i c , and 5 c a r n i t i n e - t r e a t e d d i a b e t i c r a t s . A s t e r i s k i n d i c a t e s s i g n i f i c a n t l y d i f f e r e n t than c o n t r o l , p< 0.05. - 1 1 0 -FIGURE 19 E f f e c t of c h r o n i c d i a b e t e s and c a r n i t i n e treatment on negative dP/dT of i s o l a t e d perfused working hearts at v a r i o u s f i l l i n g p r e s s u r e s . Hearts were i s o l a t e d from c o n t r o l (• • ) , c a r n i t i n e - t r e a t e d c o n t r o l ( • — — • ) , d i a b e t i c (• A), and c a r n i t i n e - t r e a t e d d i a b e t i c (4 •) r a t s 150 days a f t e r the i n d u c t i o n of diabe t e s with 50 mg/kg i . v . s t r e p t o z o t o c i n . C a r n i t i n e - t r e a t e d c o n t r o l and d i a b e t i c r a t s recei ved lg/kg/day D , L - c a r n i t i n e o r a l l y f o r 30 days p r i o r to s a c r i f i c e . The r e s u l t i s the mean ±. S.E. of 6 c o n t r o l , 5 c a r n i t i n e - t r e a t e d c o n t r o l , 5 d i a b e t i c , and 5 c a r n i t i n e - t r e a t e d d i a b e t i c r a t s . A s t e r i s k i n d i c a t e s s i g n i f i c a n t l y d i f f e r e n t than c o n t r o l , p< 0.05. / / / fining pressure (cm H 2 0 ) -112-Table 13: Levels of carnitine and long chain acylcarnitines from ventricle of isolated perfused working hearts from control and diabetic carnitine-treated rats. Hearts were isolated from control and diabetic rats 150 days after the induction of diabetes. Carnitine, treated rats received 1g/kg/day D,L-carnitine o r a l l y for 30 days prior to s a c r i f i c e . Condition Metabolite Tissue levels (nmol/g ventricle)' control n=6 acid-soluble carnitine fatty acylcarnitine 2675 + 148 295.3 + 28.9 control plus carnitine n=6 di abeti c n=4 diabetic plus carnitine n=4 acid-soluble carnitine fatty acylcarnitine acid-soluble carnitine fatty acylcarnitine acid-soluble carnitine fatty acylcarnitine 2722 + 89 268.1 + 13.3 2833 ± 265 538.8 +13.3* 2727 + 248 340.5 ± 48.0 Preparation of metabolites and measurement of carnitine levels are as described in the Methods. Data are expressed as nmol of carnitine per g frozen v e n t r i c l e . Results are expressed as the mean + S.E. a) indicates wet ventricle weight • S i g n i f i c a n t l y different from control (p<0.05, analysis of variance, followed by the Newman-Keuls t e s t ) . -113-treatment r e s u l t e d i n a lowering of v e n t r i c u l a r t i s s u e long chain a c y l c a r n i t i n e l e v e l s i n d i a b e t i c r a t s , but d i d not reverse the depressed heart f u n c t i o n . C a r n i t i n e was t h e r e f o r e administered from the onset of d i a b e t e s i n an attempt to prevent the d i a betes-induced heart d y s f u n c t i o n . When serum c a r n i t i n e l e v e l s were measured i n these r a t s , no d e p r e s s i o n was found i n the d i a b e t i c r a t s (Table 14). There was, however, a s i g n i f i c a n t e l e v a t i o n i n serum c a r n i t i n e l e v e l s i n c a r n i t i n e - f e d d i a b e t i c r a t s as compared to both c o n t r o l and d i a b e t i c r a t s . F i g u r e s 20,21, and 22 i n d i c a t e the e f f e c t of v a r y i n g f i l l i n g p r essures on v a r i o u s parameters of heart f u n c t i o n ( l e f t v e n t r i c u l a r developed p r e s s u r e , +dP/dT, and -dP/dT) i n d i a b e t i c and c o n t r o l c a r n i t i n e - f e d r a t s . In s i x week d i a b e t i c r a t s there was a s i g n i f i c a n t d e p ression i n heart function at higher f i l l i n g p r e s s u r e s . Treatment of d i a b e t i c r a t s with c a r n i t i n e d i d not prevent t h i s heart d y s f u n c t i o n from o c c u r r i n g . T i s s u e l e v e l s of c a r n i t i n e e s t e r s were then measured i n these h e a r t s . Again the l e v e l s of f r e e c a r n i t i n e were s i m i l a r i n a l l groups of r a t s (Table 15). L e v e l s of long chain a c y l c a r n i t i n e s were a l s o not s i g n i f i c a n t l y d i f f e r e n t (Table 15). I t has p r e v i o u s l y been shown by Feuvray et a l (1979) that t i s s u e l e v e l s of long c h a i n a c y l c a r n i t i n e s are e l e v a t e d 300-400% i n d i a b e t i c r a t h e a r t s . Vary and Neely (1982) however have r e c e n t l y shown that p e r f u s i o n of hearts with glucose w i l l lower t i s s u e long c h a i n . a c y l c a r n i t i n e l e v e l s . T h i s c o u l d e x p l a i n the absence of any s i g n i f i c a n t i n c r e a s e i n -114-Table 14: Levels of free carnitine in serum obtained from s i x week diabetic and control rats treated with carnitine. Carnitine (3g/kg/day o r a l l y ) was administered throughout the study period, commencing 3 days after the induction of diabetes with:50 mg/kg i . v . strep-tozotocin. Conditions Serum Levels (nmol/ml) control 20.07 + 0.71 n=18 control plus carnitine 23.06 ± 0.98 n=14 diabetic 22.00 ± 0.44 n=12 diabetic plus carnitine 25.13 + 0.49* n=14 Measurement of serum carnitine was as described in the Methods. Data are expressed as nmol of carnitine per ml of serum. Results are expressed as the mean + S.E. •S i g n i f i c a n t l y different from control (p<0.05, analysis of variance, followed by the Newman-Keuls t e s t ) . -115-FIGURE 20 E f f e c t of s i x week diabe t e s and c a r n i t i n e treatment on l e f t v e n t r i c u l a r developed pressure of i s o l a t e d perfused working h e a r t s at v a r i o u s f i l l i n g p r e s s u r e s . Hearts were i s o l a t e d from c o n t r o l (• •) , c a r n i t i n e - t r e a t e d c o n t r o l (•....•), d i a b e t i c ( A A ) , and c a r n i t i n e - t r e a t e d d i a b e t i c •) r a t s 42 days a f t e r the i n d u c t i o n of diabetes with 50 mg/kg i . v . s t r e p t o z o t o c i n . C a r n i t i n e - t r e a t e d c o n t r o l and d i a b e t i c r a t s r e c e i v e d 3 g/kg/day D f L - c a r n i t i n e o r a l l y throughout the study p e r i o d , commencing 3 days a f t e r the i n d u c t i o n of d i a b e t e s . The r e s u l t i s the mean + S.E. of 8 c o n t r o l , 7 c a r n i t i n e - t r e a t e d c o n t r o l , 6 d i a b e t i c , and 7 c a r n i t i n e - t r e a t e d d i a b e t i c r a t s . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , p < 0.05. 150-, X E E <D k-3 (A 0) d) 100 H "D a a o o v. £ 50-r 5 I 10 15 fining pressure (cm H 2 0 ) -117-FIGURE 21 E f f e c t of s i x week diabe t e s and c a r n i t i n e treatment on p o s i t i v e dP/dT of i s o l a t e d perfused working hearts at v a r i o u s f i l l i n g p r e s s u r e s . Hearts were i s o l a t e d from c o n t r o l ( • — • ) , c a r n i t i n e - t r e a t e d c o n t r o l (• •) , d i a b e t i c (• •) , and c a r n i t i n e - t r e a t e d d i a b e t i c 4) r a t s 42 days a f t e r the i n d u c t i o n of di a b e t e s with 50 mg/kg i . v . s t r e p t o z o t o c i n . C a r n i t i n e - t r e a t e d c o n t r o l and d i a b e t i c r a t s r e c e i v e d 3 g/kg/day D , L - c a r n i t i n e o r a l l y throughout the study p e r i o d , commencing 3 days a f t e r the i n d u c t i o n of d i a b e t e s . The r e s u l t i s the mean + S.E. of 8 c o n t r o l , 7 c a r n i t i n e - t r e a t e d c o n t r o l , 6 d i a b e t i c , and 7 c a r n i t i n e - t r e a t e d d i a b e t i c r a t s . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , p<0.05. "8 5000 O J , r — 1 1 -I 5 10 15 20 25 filing pressure (cm H20) -119-FIGURE 22 E f f e c t of s i x week diabe t e s and c a r n i t i n e treatment on negative dP/dT of i s o l a t e d perfused working h e a r t s at v a r i o u s f i l l i n g p r e s s u r e s . Hearts were i s o l a t e d from c o n t r o l (•• • ) , c a r n i t i n e - t r e a t e d c o n t r o l (• • ) , d i a b e t i c (• •) , and c a r n i t i n e - t r e a t e d d i a b e t i c (• •) r a t s 42 days a f t e r the i n d u c t i o n of di a b e t e s with 50 mg/kg i . v . s t r e p t o z o t o c i n . C a r n i t i n e - t r e a t e d c o n t r o l and d i a b e t i c r a t s recei ved 3 g/kg/day D , L - c a r n i t i n e o r a l l y throughout the study p e r i o d , commencing 3 days a f t e r the i n d u c t i o n of d i a b e t e s . The r e s u l t i s the mean + S.E. of 8 c o n t r o l , 7 c a r n i t i n e - t r e a t e d c o n t r o l , 6 d i a b e t i c , and 7 c a r n i t i n e - t r e a t e d d i a b e t i c r a t s . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , p<0.05. A 2 C 5000-, fining pressure (cm H20) -121-Table 15 Levels of carnitine and long chain acylcarnitines from ventricle of isolated perfused working hearts from control and diabetic carnitine-treated rats. Hearts were isolated from control and diabetic rats 42 days after the induction of diabetes with 50 mg/kg i.v . streptozotocin. Carnitine-treated rats recieved 3 g/kg/day D,L-carnitine o r a l l y throughout the study period, commencing three days after the induction of diabetes. Condition Metabolite Tissue Levels (nmol/g ven t r i c l e ) ' control n=8 acid-soluble carnitine fatty acylcarnitine 2184 +.90 259.9 + 26.9 control plus carnitine n=7 acid-soluble carnitine fatty acylcarnitine 2440 +216 273.7 + 32.9 diabetic n=8 acid-soluble carnitine fatty acylcarnitine 2061 + 181 354.5 + 36.9 diabetic plus carnitine n=7 acid-soluble carnitine 2459 ± 180 fatty acylcarnitine 339.4 ± 50 preparation of metabolites and measurement of carnitine levels are as described in the Methods. Data are expressed as nmol of carnitine per g frozen v e n t r i c l e . Result i s expressed as the mean + S.E. a-indicates wet ventricle weight -122-t i s s u e long c h a i n a c y l c a r n i t i n e s i n d i a b e t i c r a t s from t h i s study. Table 17 confirmed t h i s o b s e r v a t i o n i n that non-perfused h e a r t s from s i x week d i a b e t i c r a t s were found to have s i g n i f i c a n t l y e l e v a t e d l e v e l s of myocardial LC a c y l c a r n i t i n e s . The intermediate 100% i n c r e a s e i n t i s s u e long c h a i n a c y l c a r n i t i n e l e v e l s i n d i a b e t i c r a t s from the r e v e r s a l study may r e f l e c t the s h o r t e r p e r f u s i o n time used. To examine t h i s hypothesis hearts from s i x week d i a b e t i c r a t s were perfused f o r v a r y i n g lengths of time on an i s o l a t e d working heart apparatus. Hearts perfused f o r 5 minutes contained s i m i l a r t i s s u e l e v e l s of LC a c y l c a r n i t i n e s as non-perfused h e a r t s . A f t e r 10 minutes of p e r f u s i o n t i s s u e l e v e l s of LC a c y l c a r n i t i n e s were decreased by 50% (Table 17). 2+ Ca -uptake a c t i v i t y was measured i n c a r d i a c SR , from c o n t r o l and d i a b e t i c r a t s fed c a r n i t i n e , at v a r i o u s f r e e 2+ Ca c o n c e n t r a t i o n s (Figure 23). Six weeks a f t e r the i n d u c t i o n of d i a b e t e s c a r d i a c SR f u n c t i o n was s i g n i f i c a n t l y 2+ depressed i n d i a b e t i c r a t s . SR Ca -uptake a c t i v i t y i n c a r n i t i n e t r e a t e d d i a b e t i c r a t s , however, remained s i m i l a r to c o n t r o l r a t s . L e v e l s of c a r n i t i n e were then measured i n these SR p r e p a r a t i o n s . No d i f f e r e n c e was observed i n l e v e l s of f r e e c a r n i t i n e a s s o c i a t e d with the SR (Table 16). In c a r d i a c SR from d i a b e t i c r a t s , however, a s i g n i f i c a n t i n c r e a s e i n l e v e l s of long c h a i n a c y l c a r n i t i n e s was observed (Table 16). In SR from c a r n i t i n e - t r e a t e d d i a b e t i c r a t s there was no e l e v a t i o n i n the l e v e l s of long c h a i n a c y l c a r n i t i n e s . -123-Table 17: Levels of carnitine and long chain acylcarnitines from ventricle of perfused and non-perfused hearts from control and diabetic rats. Hearts were isolated from control and diabetic rats 42 days after the induction of diabetes with 50 mg/kg i.v. streptozotocin. a) Non-perfused hearts Condition Metabolite Tissue Levels (nmol/g ventricle) control acid-soluble carnitine 2483 + 90 n=8 fatty acylcarnitine 540.2 + 28.2 diabetic acid-soluble carnitine 2924 ± 239 * n=7 fatty acylcarnitine 891.4+57.4 b) Perfused hearts Condition diabetic i) Langendorff mode fatty acylcarnitine 658 i i ) working heart mode 5 minutes fatty acylcarnitine 618 10 minutes fatty acylcarnitine 263 Preparation of metabolites and measurement of carnitine levels are as described in the Methods. Data are expressed as nmol of carnitine per g frozen ventricle. Results are expressed as the mean + S.E. •Significantly different from control (p <0.05) -124-FIGURE 23 E f f e c t of s i x week diabe t e s and c a r n i t i n e treatment on 2+ Ca -uptake i n c a r d i a c s arcoplasmic r e t i c u l u m . 2+ 2+ Ca -uptake was measured at v a r i o u s f r e e Ca c o n c e n t r a t i o n s between 0.1 and 3.5 uM as d e s c r i b e d i n Methods. Hearts were i s o l a t e d from c o n t r o l ( • — • ), c a r n i t i n e - t r e a t e d c o n t r o l (• •) , d i a b e t i c ( A A), and c a r n i t i n e - t r e a t e d d i a b e t i c (• •) r a t s 42 days a f t e r the i n d u c t i o n of d i a b e t e s with 50 mg/kg i . v . s t r e p t o z o t o c i n . C a r n i t i n e - t r e a t e d r a t s recei ved 3 g/kg/day D , L - c a r n i t i n e o r a l l y throughout the study p e r i o d , commencing three days a f t e r the i n d u c t i o n of d i a b e t e s . The r e s u l t i s the mean + S.E. of 8 c o n t r o l , 5 c a r n i t i n e - t r e a t e d c o n t r o l , 5 d i a b e t i c , and 6 c a r n i t i n e - t r e a t e d d i a b e t i c r a t s . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s from c o n t r o l , p<0.05. — r -0.1 i i i i i i i ) 1 [Co'*] ( M M ) "as \ -126-Table 16: Cardiac sarcoplasmic reticulum levels of carnitine and long chain acylcarnitines from s i x week diabetic and control carnitine-treated rats. Hearts were isolated from control and diabetic rats 42 days after the induction of diabetes with 50 mg/kg i . v . streptozotocin. Carnitine-treated rats recieved 3 g/kg/day D,L-carnitine o r a l l y throughout the study period, commencing three days after the induction of diabetes. Condition Metabolites Tissue Levels (nmol carnitine/mg SR) control n=8 acid-soluble carnitine fatty acylcarnitine 7.01 + 0.63 0.74 + 0.09 control plus carnitine n=5 di abeti c n=5 diabetic plus carnitine n=6 acid^soluble carnitine fa t t y acylcarnitine acid-soluble carnitine fatty acylcarnitine acid-soluble carnitine fatty acylcarnitine 6.78 + 0.56 0.85 ± 0.09 7.33 ± 0.41 1 .82 + 0.40* 7.67 ± 0.76 0.86 + 0.14 Preparation of metabolites and measurement,of carnitine levels are as described in the Methods. Data are expressed as nmol carnitine per mg of S.R. protein. Results are expressed as the mean + S.E. • S i g n i f i c a n t l y different from control (p<0.05, analysis of variance, followed by the Newman-Keuls test) -127-DISCUSSION Before i n f e r e n c e s can be drawn concerning the e f f e c t s of d i a b e t e s on c a r d i a c SR f u n c t i o n i t i s necessary to confirm that 1) the d i a b e t e s induced c h e m i c a l l y i n our l a b o r a t o r y p a r a l l e l s s i m i l a r p r e p a r a t i o n s documented by other i n v e s t i g a t o r s (which as d i s c u s s e d i n the I n t r o d u c t i o n , have been shown to be good models of human d i a b e t e s ) , and that 2) c a r d i a c SR p r e p a r a t i o n s d e r i v e d from c o n t r o l and d i a b e t i c animals do not d i f f e r i n c e r t a i n c r i t i c a l parameters. In t h i s study, d i a b e t e s was induced by i n j e c t i o n of e i t h e r a l l o x a n or s t r e p t o z o t o c i n , both of which destroy p a n c r e a t i c beta c e l l s . In our hands these agents produced a c o n s i s t e n t and r e p r o d u c i b l e d i a b e t i c c o n d i t i o n i n the r a t . The parameters used to assess d i a b e t e s confirms what has been p r e v i o u s l y documented ( H o f t i e z e r and Carpenter, 1973; Junod et a l , 1969). Blood samples taken at v a r i o u s time i n t e r v a l s , or at the time of s a c r i f i c e , r evealed that d i a b e t i c r a t s c o n s i s t e n t l y had serum glucose l e v e l s g r e a t e r than 400 mg%. The e l e v a t e d l e v e l s of n o n - s p e c i f i c a l l y g l y c o s y l a t e d hemoglobin found i n these d i a b e t i c r a t s a l s o suggested that hyperglycemia was present i n these r a t s f o r long p e r i o d s of time. Serum i n s u l i n l e v e l s measured i n d i a b e t i c r a t s were approximately 60-75% of that seen i n c o n t r o l animals. In a d d i t i o n , the d i a b e t i c r a t s d i s p l a y e d a number of symptoms normally a s s o c i a t e d with the d i s e a s e s t a t e , i n c l u d i n g weight l o s s , p o l y u r i a , p o l y d y p s i a , hyperphagia, and c a t a r a c t formation. K e t o s i s could not be detected i n these animals. -128-The p r e v e n t i o n of a l l of the above symptoms by i n s u l i n therapy was a l s o c o n v i n c i n g evidence that we do indeed have an a p p r o p r i a t e animal model of d i a b e t e s . The methodology used to prepare microsomal c a r d i a c SR has been e x t e n s i v e l y employed by many l a b o r a t o r i e s , i n c l u d i n g our own. The microsomal p r e p a r a t i o n has a l s o been thoroughly c h a r a c t e r i z e d by other i n v e s t i g a t o r s (for a review see Jones and Besch, 1979). For our study, however, i t was necessary to i d e n t i f y any q u a l i t a t i v e d i f f e r e n c e s i n SR p r e p a r a t i o n s from c o n t r o l and d i a b e t i c r a t s . S p e c i f i c a l l y , we were i n t e r e s t e d i n determining i f the degree of m i t o c h o n d r i a l membrane contamination was a l t e r e d i n the SR prepared from d i a b e t i c r a t s . A l s o i n v e s t i g a t e d were p o s s i b l e u l t r a s t r u c t u r a l d i f f e r e n c e s , namely the degree of microsomal SR v e s i c u l a t i o n . M i t o c h o n d r i a l contamination of our SR p r e p a r a t i o n s was determined by measuring the marker enzyme cytochrome C oxidase. As shown i n Table 4, c a r d i a c SR from both c o n t r o l and d i a b e t i c r a t s o x i d i z e d approximately 50 nmoles of cytochrome C per mg SR per minute. T h i s i n d i c a t e s that the degree of m i t o c h o n d r i a l membrane contamination (within the l i m i t s normally found i n c a r d i a c SR prepared by our procedure (Jones and Besch, 1979)) i s not a l t e r e d i n c a r d i a c SR obtained from d i a b e t i c r a t s . In support of our data, a recent r e p o r t by Ganguly et a l (1983) has a l s o determined that the degree of m i t o c h o n d r i a l contamination i s s i m i l a r i n SR d e r i v e d from e i t h e r c o n t r o l or d i a b e t i c r a t h e a r t s . The m i t o c h o n d r i a l membrane contamination present i n our SR p r e p a r a t i o n s does not 2+ appear to c o n t r i b u t e to microsomal Ca uptake. T h i s i s -129-evident by the o b s e r v a t i o n that Na a z i d e , a metabolic 2+ i n h i b i t o r of m i t o c h o n d r i a l Ca uptake, had no e f f e c t on the 2+ a b i l i t y of our microsomal p r e p a r a t i o n to accumulate Ca E l e c t r o n micrographs were made of c a r d i a c SR from c o n t r o l and d i a b e t i c r a t s to determine the degree of v e s i c u l a t i o n . An 2+ apparent d e p r e s s i o n i n c a r d i a c SR Ca uptake may simply r e f l e c t a s m a l l e r number of i n t a c t v e s i c l e s , compared with unsealed membranes. As shown i n F i g u r e s 3 and 4, however, the degree of v e s i c u l a t i o n was s i m i l a r i n both c o n t r o l and d i a b e t i c SR p r e p a r a t i o n s . These data are supported by the f i n d i n g t h a t any change i n the a b i l i t y of the SR to accumulate 2+ 2+ Ca was accompanied by changes i n Ca s e n s i t i v e ATPase 2+ a c t i v i t y . I f an apparent depression i n SR Ca uptake was due to a decrease i n the r a t i o of sealed to unsealed v e s i c l e s 2+ or of i n s i d e - o u t to r l g h t s i d e - o u t v e s i c l e s , then Ca -ATPase a c t i v i t y would not be c o n c o m i t t a n t l y depressed. Another parameter that was used to determine i f the i s o l a t i o n procedure a l t e r e d the c h a r a c t e r i s t i c s of the SR d e r i v e d from d i a b e t i c r a t h e a r t s , was to measure the y i e l d of SR. Since d i a b e t i c h e arts were sma l l e r , the y i e l d of SR per v e n t r i c l e was lower i n d i a b e t i c r a t s as compared to c o n t r o l r a t s . However, the y i e l d of SR per gram weight of v e n t r i c l e was found to be the same i n d i a b e t i c and c o n t r o l r a t s (Tables 3 and 11) . 2+ F i n a l l y , i n c u b a t i o n c o n d i t i o n s f o r the c a r d i a c SR Ca uptake assay were optimized; SR p r o t e i n and i n c u b a t i o n times were adjusted so that no more than 10% of the ATP present i n 2+ the i n c u a t i o n medium was h y d r o l y z e d . T h i s ensured that Ca -130-uptake was l i n e a r throughout the i n c u b a t i o n p e r i o d (Figure 2) i n both c o n t r o l and d i a b e t i c r a t c a r d i a c SR p r e p a r a t i o n s . A) EFFECTS OF DIABETES ON CARDIAC SR FUNCTION Th i s study demonstrates that c a r d i a c microsomal p r e p a r a t i o n s enriched i n sarcoplasmic r e t i c u l u m obtained from c h r o n i c a l l y (120 day) d i a b e t i c r a t s have a s i g n i f i c a n t l y 2+ 2+ depressed rate of Ca t r a n s p o r t . The depression i n Ca uptake appears to be a s s o c i a t e d with a decrease i n 2+ Ca -ATPase a c t i v i t y (Table 5), rather than any change i n 2+ the p e r m e a b i l i t y of the membrane to Ca (Figure 9). A recent r e p o r t by Penpargkul et a l (1981) suggests that 2+ . . Ca t r a n s p o r t a c t i v i t y can be depressed as e a r l y as f i v e weeks a f t e r the i n d u c t i o n of d i a b e t e s . From our study i t i s evident that by 31 days there i s a s l i g h t d e p ression i n SR 2+ Ca -uptake a c t i v i t y i n d i a b e t i c r a t s (Figure 16). By 42 days t h i s d e p r e s s i o n i s maximal (Figure 23, d i a b e t i c r a t 2+ c a r d i a c SR Ca -uptake i s 65% of that found i n c o n t r o l r a t SR) and remains the same f o r up to 120 days a f t e r the i n d u c t i o n of d i a b e t e s (Figure 5). Our data are supported by the recent r e s u l t s of Ganguly et a l (1983) who demonstrated that the d e f e c t i n the a b i l i t y of the microsomal v e s i c l e s to 2+ . t r a n s p o r t Ca i s not evident u n t i l 28 days a f t e r s t r e p t o z o t o c i n i n d u c t i o n of d i a b e t e s . The above o b s e r v a t i o n s provide a p o s s i b l e e x p l a n a t i o n f o r the decreased c a r d i a c 2+ performance seen i n d i a b e t i c r a t s . Removal of Ca from the c y t o s o l and i t s subsequent r e l e a s e by c a r d i a c SR appears to -131-play a major r o l e i n beat to beat r e g u l a t i o n of myocardial c o n t r a c t i o n (Reuter, 1974). Var i o u s i n v e s t i g a t o r s have p r e v i o u s l y reported that i s o l a t e d d i a b e t i c working hearts have a depressed r a t e of c o n t r a c t i o n (+LVdP/dT) and r e l a x a t i o n (-LVdP/dT) (Ingebretson et a l , 1980; Penpargkul et a l , 1980; Vadlamudi and M c N e i l l , 1980). I s o l a t e d p a p i l l a r y muscle from d i a b e t i c r a t s a l s o had a decreased v e l o c i t y of shortening and r a t e of r e l a x a t i o n (Fein et a l , 1980). The impaired v e l o c i t y of c o n t r a c t i o n and r e l a x a t i o n i n d i a b e t i c heart 2+ muscle may be due to the decreased rate of Ca -uptake observed i n SR d e r i v e d from the v e n t r i c u l a r muscle of d i a b e t i c r a t s . I f SR f u n c t i o n i s impaired i t would be expected 2+ that the rate at which Ca can be removed from the c o n t r a c t i l e u n i t would be decreased, r e s u l t i n g i n a decreased r a t e of r e l a x a t i o n . The r a t e of subsequent c o n t r a c t i o n could 2+ a l s o be decreased s i n c e l e s s Ca would be a v a i l a b l e f o r r e l e a s e from the SR. MECHANISMS BY WHICH DIABETES MAY ALTER CARDIAC SR FUNCTION I t i s evident that i s o l a t e d c a r d i a c SR microsomes from c h r o n i c a l l y d i a b e t i c r a t s have a depressed a b i l i t y to 2+ accumulate Ca . In t h i s study we were a l s o i n t e r e s t e d i n determining the mechanism by which d i a b e t e s a l t e r s SR f u n c t i o n . One approach was to i n v e s t i g a t e p o s s i b l e a l t e r a t i o n s i n the r e g u l a t i o n of c a r d i a c SR. The other approach taken was to attempt to r e l a t e changes i n SR f u n c t i o n to some diabetes-induced metabolic change. 1) Diabetes and the r e g u l a t i o n of c a r d i a c SR f u n c t i o n -132-As d i s c u s s e d e a r l i e r , a c t i v e uptake of Ca in t o the SR + 2+ i s achieved by a K - s e n s i t i v e Ca -dependent ATPase enzyme. T h i s enzyme appears to be re g u l a t e d e i t h e r d i r e c t l y v i a i o n i c c o n t r o l at a l l o s t e r i c s i t e s or i n d i r e c t l y through p h o s p h o r y l a t i o n of phospholamban, the 22,000 MW p r o t e i n c l o s e l y a s s o c i a t e d with the ATPase p r o t e i n i n the membrane (Kranias et a l , 1980) . Changes i n the c o n c e n t r a t i o n of r e g u l a t o r y i o n s , the degree to which the enzyme recognizes 2+ these i o n s , or the extent to which the Ca -ATPase i n t e r a c t s with phospholamban could a l l be expected to a l t e r the rate at 2+ which the enzyme t r a n s p o r t s Ca . C l e a r l y , then, r e g u l a t o r y changes i n the SR pump may be important i n attempting to e l u c i d a t e the mechanism whereby a p a t h o l o g i c a l s t a t e , such as 2+ d i a b e t e s , depresses Ca t r a n s p o r t a c t i v i t y . I t was, t h e r e f o r e , of i n t e r e s t to examine the v a r i o u s documented 2+ r e g u l a t o r s of the Ca -ATPase enzyme (calmodulin, cAMP-dependent p r o t e i n k i n a s e , and potassium) i n the d i a b e t i c model. The data presented suggest that none of the three 2+ r e g u l a t o r s of the SR Ca pump examined p l a y a r o l e i n the 2+ depression of c a r d i a c SR Ca t r a n s p o r t observed i n s t r e p t o z o t o c i n - i n d u c e d d i a b e t e s i n the r a t . The e f f e c t s of calmodulin on the SR pump have been r e c e n t l y examined i n d e t a i l (Lopaschuk et a l , 1980). 2+ S t i m u l a t i o n by calmodulin of Ca t r a n s p o r t i n c a r d i a c microsomes enriched i n SR was reported by Katz and Remtulla i n 2+ 1978. The Ca -ATPase enzyme i s a l s o s t i m u l a t e d by calmodulin (Lopaschuk et a l , 1980). I t has been suggested -133-2+ that a Ca -calmodulin complex, a c t i n g through a s p e c i f i c p r o t e i n kinase which phosphorylates phospholamban, s t i m u l a t e s 2+ Ca t r a n s p o r t a c t i v i t y (LePeuch et a l , 1979) although a d i r e c t a c t i o n of calmodulin on the ATPase enzyme i t s e l f can not be discounted (Katz, 1980). Regardless of the mechanism, i t does not appear that a change i n the r e g u l a t i o n of SR 2+ f u n c t i o n by calmodulin i s r e s p o n s i b l e f o r the depressed Ca t r a n s p o r t observed i n d i a b e t i c r a t heart SR. In dog heart, the e f f e c t s of cAMP-dependent p r o t e i n kinase have been w e l l documented (Kirchberger et a l , 1972; Kranias et a l , 1980) . P h o s p h o r y l a t i o n of phospholamban r e s u l t s i n a conformational change i n the 22,000 MW 2+ g l y c o p r o t e i n r e s u l t i n g i n d e - r e g u l a t i o n of the Ca -ATPase p r o t e i n . The consequent i n c r e a s e d turnover r a t e of the pump 2+ r e s u l t s i n an i n c r e a s e i n both Ca t r a n s p o r t and 2+ Ca -ATPase a c t i v i t y (Kranias et a l , 1980) . In the only study we are aware of i n v o l v i n g r a t h e a r t , H o l l i n g w o r t h and 2+ England (1978) have noted a 150% s t i m u l a t i o n of Ca -uptake a c t i v i t y by c y c l i c AMP-dependent p r o t e i n k i n a s e , a much l a r g e r i n c r e a s e than we were able to o b t a i n i n the present study. The d i s c r e p a n c y may l i e i n the more f a v o r a b l e i n c u b a t i o n c o n d i t i o n s f o r s t i m u l a t i o n of cAMP-dependent p r o t e i n kinase used by those workers. In p a r t i c u l a r they used 20 mM ATP, 20 2+ mM MgCl 2, and 150 uM Ca unbuffered with EGTA, c o n d i t i o n s 2+ which are not normally u t i l i z e d i n Ca t r a n s p o r t assays. N e v e r t h e l e s s , l i k e c a lmodulin, a l t e r a t i o n i n cAMP-dependent p r o t e i n kinase r e g u l a t i o n of c a r d i a c SR does not appear to be 2+ the mechanism whereby Ca t r a n s p o r t i s depressed i n -134-d i a b e t i c r a t s . Monovalent c a t i o n s have a number of e f f e c t s on the 2 + intermediate events a s s o c i a t e d with SR Ca t r a n s p o r t . K 2+ appears to s t i m u l a t e Ca -uptake a c t i v i t y by i n c r e a s i n g both the r a t e s of dephosphorylation and ph o s p h o r y l a t i o n of the 2+ Ca -ATPase (Shikeg,awa and Akowitz, 1979) r e s u l t i n g i n an increased turnover of the pump. An inc r e a s e i n turnover means 2+ that more Ca i s able to be t r a n s l o c a t e d . Since the i n t e r i o r of the c a r d i a c c e l l i s normally bathed i n high K + c o n c e n t r a t i o n s (100-150 mM), the p h y s i o l o g i c a l s i g n i f i c a n c e of vary i n g the K + c o n c e n t r a t i o n s i s unknown. However, by + 2+ examining the e f f e c t of K on SR Ca t r a n s p o r t on i n v i t r o p r e p a r a t i o n s , i t might be p o s s i b l e to determine what d i r e c t changes t h a t p a t h o l o g i c a l s t a t e s such as diabe t e s may 2+ have on the Ca pump. Changes i n a l l o s t e r i c r e g u l a t i o n of the ATPase p r o t e i n by ion s , f o r example, could be r e f l e c t e d i n + 2+ the degree to which K s t i m u l a t e s Ca t r a n s p o r t a c t i v i t y . In t h i s study, however, K + exerted an e q u i v a l e n t s t i m u l a t i o n i n both c o n t r o l and d i a b e t i c p r e p a r a t i o n s and appeared to have 2+ an e q u i v a l e n t a f f i n i t y f o r the Ca t r a n s p o r t system as r e f l e c t e d by a comparable Km under both c o n d i t i o n s . As p r e v i o u s l y mentioned, diab e t e s does not a l t e r the a f f i n i t y of 2+ the enzyme f o r Ca . These data suggest, t h e r e f o r e , that 2+ i o n i c c o n t r o l of the Ca -dependent ATPase i s not a l t e r e d i n c a r d i a c SR from d i a b e t i c r a t s . T h i s i m p l i e s that the 2+ diabetes-induced depression of SR Ca t r a n s p o r t i s not r e a d i l y e x p l a i n a b l e by s p e c i f i c a l t e r a t i o n s i n the response to r e g u l a t o r y i o n s . The same holds true f o r calmodulin and -135-cAMP-dependent p r o t e i n k i n a s e . In both cases, these two r e g u l a t o r s appear to act i n d i a b e t i c c a r d i a c SR as they would normally f u n c t i o n i n c o n t r o l SR. 2) Diabetes induced metabolic changes and c a r d i a c SR f u n c t i o n . I t i s w e l l recognized that a l t e r a t i o n s i n l i p i d metabolism, a common f i n d i n g i n d i a b e t i c p a t i e n t s , can a l t e r heart f u n c t i o n by modifying the s t r u c t u r e of c a r d i a c membranes (Katz and Messineo, 1981) . We, t h e r e f o r e , hypothesized that i n t r a c e l l u l a r accumulation of l i p i d s and of intermediates i n v o l v e d i n l i p i d metabolism may be depressing SR f u n c t i o n i n the d i a b e t i c r a t h e a r t . T h i s may be due to an a s s o c i a t i o n of am p h i p h i l i c l i p i d substances with the membrane of the SR which then d i s r u p t the f u n c t i o n of membrane-dependent t r a n s p o r t p r o t e i n s . The intermediate steps i n v o l v e d i n the u t i l i z a t i o n of f a t t y a c i d s as an energy source i n the heart are summarized i n Figu r e 24. Diabetes, which a c c e l e r a t e s the rate of f a t t y a c i d metabolism i n the heart, r e s u l t s i n an e l e v a t i o n of a number of these in t e r m e d i a t e s i n c l u d i n g c y t o s o l i c l e v e l s of t r i g l y c e r i d e s and f r e e f a t t y a c i d s (Denton and Randle, 1967). C e l l u l a r uptake and i n c o r p o r a t i o n of pantothenic a c i d i n t o Coenzyme A (CoA) i s a c c e l e r a t e d i n d i a b e t e s , which r e s u l t s i n inc r e a s e d t i s s u e l e v e l s of CoA (Riebel et a l , 1981a,b). A consequence of the e l e v a t e d l e v e l s of f r e e f a t t y a c i d and CoA are i n c r e a s e s i n a c y l CoA and LC a c y l c a r n i t i n e s y n t h e s i s (Feuvray et a l , 1979). I f t i s s u e l e v e l s of l i p i d i n t e rmediates are r a i s e d above c e r t a i n c o n c e n t r a t i o n s , Sarcolemma : Mitochondria outer inner membrane membrane Figure 24: Sequence of fatt y acid metabolism in the myocardial c e l l 1) cytosolic f f a i s obtained either by metabolism of triglycerides or uptake of ffa by the c e l l . 2) f f a i s converted to acyl CoA by acyl CoA synthetase. 3) acyl CoA which cannot cross the mitochondrial membrane is converted to acylcarnitine by an acylcarnitine transferase located on the outer mitochondrial membrane. 4) acylcarnitine crosses the membrane coupled to carnitine transport via an acylcarnitine translocase enzyme. 5) acylcarnitine i s converted back to acyl CoA by an acylcarnitine transferase located on the inner mitochondrial membrane. 6 ) acyl CoA i s metabolized to acetyl CoA, and f i n a l l y c i t r a t e . The NADH and FADH produced enter the respiratory chain producing ATP. -137-d e t r i m e n t a l e f f e c t s on c e l l u l a r membranes can occur. These intermediates can modify membrane f u n c t i o n by d i s r u p t i n g important i n t e r a c t i o n s that e x i s t between hydrophobic regions of membrane p r o t e i n s and the l i p i d b i l a y e r (Gordon et a l , 1980). For i n s t a n c e , high c o n c e n t r a t i o n s of f r e e f a t t y a c i d s 2+ w i l l uncouple o x i d a t i v e p h o s p h o r y l a t i o n and i n h i b i t SR Ca 2+ t r a n s p o r t and Ca ATPase a c t i v i t y (Pressman and Landy, 1956; Cheah, 1981). A c y l CoA d e r i v a t i v e s , such as p a l m i t y l CoA, can not o n l y i n h i b i t m i t o c h o n d r i a l r e s p i r a t i o n and adenine n u c l e o t i d e t r a n s l o c a s e (Pande and Blanchaer, 1971; Shug et a l , 1975), but a l s o i n h i b i t sarcolemmal + + 2+ Na -K -ATPase and SR Ca t r a n s p o r t (Adams et a l , 1979, P i t t s et a l , 1979). Of these metabolic intermediates the LC a c y l c a r n i t i n e s 2+ appear to be the most potent i n h i b i t o r s of SR Ca t r a n s p o r t . T h e i r potency r e s u l t s from the s t r o n g l y charged c a r n i t i n e moeity of the molecule, which makes i t an e x c e l l e n t d etergent. As a consequence LC a c y l c a r n i t i n e s are c o n s i d e r a b l y more potent than a c y l CoA or f r e e f a t t y a c i d s i n i n h i b i t i n g membrane t r a n s p o r t p r o t e i n s (Woods et a l , 1977; Adams et a l , 1979; Opie et a l , 1979). Since LC a c y l c a r n i t i n e s are markedly e l e v a t e d i n d i a b e t i c r a t hearts we wanted to determine i f they might be c o n t r i b u t i n g to the 2+ depression i n SR Ca t r a n s p o r t noted i n d i a b e t i c r a t s . We c o n f i n e d our s t u d i e s to the LC a c y l c a r n i t i n e s , not only because of t h e i r potency, but a l s o because t i s s u e c o n c e n t r a t i o n s i n d i a b e t i c hearts are 2 to 4 times that of a c y l CoA d e r i v a t i v e s . -138-In t h i s study, l e v e l s of c a r n i t i n e and long chain a c y l c a r n i t i n e s were e l e v a t e d i n the microsomal SR p r e p a r a t i o n s d e r i v e d from c h r o n i c a l l y d i a b e t i c r a t s (Table 6,16). The i n c r e a s e i n LC a c y l c a r n i t i n e a s s o c i a t e d with the SR p a r a l l e l s the i n c r e a s e i n t o t a l t i s s u e l e v e l s of LC a c y l c a r n i t i n e s observed p r e v i o u s l y i n d i a b e t i c r a t h e arts (Feuvray et a l , 1979). The high l e v e l s of LC a c y l c a r n i t i n e s a s s o c i a t e d with 2+ the microsomal SR could account f o r the i n h i b i t o n of Ca t r a n s p o r t noted. As shown i n F i g u r e 7, and p r e v i o u s l y reported by other i n v e s t i g a t o r s (Adams et a l , 1979, P i t t s et a l , 1979), L - p a l m i t y l c a r n i t i n e , the most abundant of the LC a c y l c a r n i t i n e s , i s a potent i n h i b i t o r of c a r d i a c SR 2+ Ca t r a n s p o r t . I t has been estimated that under ischemic c o n d i t i o n s the c o n c e n t r a t i o n of p a l m i t y l c a r n i t i n e i n the c y t o s o l of heart c e l l s can i n c r e a s e to such a l e v e l that 2+ i n h i b i t i o n of SR Ca t r a n s p o r t may occur (Idell-Wenger et. a l , 1979; P i t t s et a l , 1979). Since t i s s u e l e v e l s of p a l m i t y l c a r n i t i n e are e l e v a t e d i n d i a b e t i c h e arts i t i s 2+ p o s s i b l e that some i n h i b i t i o n of SR Ca may occur, a f f e c t i n g o v e r a l l c a r d i a c performance. A d d i t i o n of low c o n c e n t r a t i o n s of exogenous L - p a l m i t y l c a r n i t i n e (3-7 uM) to SR obtained from c o n t r o l r a t s 2+ r e s u l t e d i n a s u b s t a n t i a l i n h i b i t o n i n the r a t e of Ca t r a n s p o r t (Figure 7). However, a d d i t i o n of s i m i l a r c o n c e n t r a t i o n s of L - p a l m i t y l c a r n i t i n e to SR obtained from c h r o n i c a l l y d i a b e t i c r a t s produced a l e s s marked i n h i b i t i o n . 2+ T h i s would i n d i c a t e that a degree of i n h i b i t i o n of Ca t r a n s p o r t produced by the high endogenous l e v e l s of -139-p a l m i t y l c a r n i t i n e may al r e a d y be present i n the d i a b e t i c r a t p r e p a r a t i o n s . I t i s p o s s i b l e that the SR membrane reaches e q u i l i b r i u m with the exogenous L - p a l m i t y l c a r n i t i n e . The e q u i l i b r i u m c o n c e n t r a t i o n i n the membrane would not n e c e s s a r i l y have to equal the c o n c e n t r a t i o n i n the i n c u b a t i o n medium. Endogenous l e v e l s of LC a c y l c a r n i t i n e s i n the d i a b e t i c SR membrane may a l r e a d y exceed the e q u i l i b r i u m c o n c e n t r a t i o n obtained by adding low c o n c e n t r a t i o n s of L - p a l m i t y l c a r n i t i n e s and t h e r e f o r e f u r t h e r i n h i b i t i o n of 2+ Ca t r a n s p o r t would not be observed. At higher exogenous L - p a l m i t y l c a r n i t i n e c o n c e n t r a t i o n s the e q u i l i b r a t e d membrane c o n c e n t r a t i o n would be g r e a t e r than the i n i t i a l membrane 2+ c o n c e n t r a t i o n , r e s u l t i n g i n f u r t h e r i n h i b i t i o n of Ca uptake. T h i s i s f u r t h e r i n d i c a t e d by the o b s e r v a t i o n that at c o n c e n t r a t i o n s of p a l m i t y l c a r n i t i n e above 7 uM the rat e s of SR 2+ Ca t r a n s p o r t i n both d i a b e t i c and c o n t r o l r a t s are s i m i l a r . Since these s t u d i e s i n d i c a t e that higher l e v e l s of c a r n i t i n e may be a s s o c i a t e d with SR p r e p a r a t i o n s from d i a b e t i c r a t s i t was necessary to determine what e f f e c t c a r n i t i n e had 2+ on SR Ca t r a n s p o r t . Although c a r n i t i n e can t o t a l l y 2+ i n h i b i t Ca t r a n s p o r t (Figure 8 ), the c o n c e n t r a t i o n necessary to do so (30 mM) was 1500 f o l d g r e a t e r than the c o n c e n t r a t i o n of L - p a l m i t y l c a r n i t i n e causing t o t a l i n h i b i t i o n . The high c o n c e n t r a t i o n s of c a r n i t i n e necessary to i n h i b i t 2+ Ca t r a n s p o r t suggest that i n the i n t a c t c e l l c a r n i t i n e does not a f f e c t c a r d i a c SR f u n c t i o n . The inc r e a s e d l e v e l s of c a r n i t i n e a s s o c i a t e d with c a r d i a c SR from d i a b e t i c r a t s i s -140-s t i l l i n s u f f i c i e n t to a l t e r Ca t r a n s p o r t . I t has p r e v i o u s l y been shown that t i s s u e l e v e l s of f r e e c a r n i t i n e are decreased i n d i a b e t e s (Feuvray et a l , 1979). The i n c r e a s e d l e v e l s of c a r n i t i n e a s s o c i a t e d with our SR p r e p a r a t i o n are t h e r e f o r e not r e a d i l y e x p l a i n e d . The technique used to measure c a r n i t i n e l e v e l s does not d i f f e r e n t i a t e between f r e e c a r n i t i n e , a c e t y l c a r n i t i n e , or short c h a i n a c y l c a r n i t i n e s . I t i s p o s s i b l e that i n d i a b e t e s a g r e a t e r p r o p o r t i o n of the a c i d - s o l u b l e c a r n i t i n e s may be i n the form of short c h a i n a c y l c a r n i t i n e s . I f short chain a c y l c a r n i t i n e s bind to c y t o s o l i c membranes more r e a d i l y than f r e e c a r n i t i n e , the e l e v a t e d l e v e l s of a c i d s o l u b l e c a r n i t i n e a s s o c i a t e d with our p r e p a r a t i o n could be e x p l a i n e d . In d i a b e t i c h e a r t s , accumulation of LC a c y l c a r n i t i n e s , 2+ i n h i b i t i o n of SR Ca t r a n p o r t , and depression of c a r d i a c performance must occur c o n c u r r e n t l y i f the three are c a u s a l l y 2+ r e l a t e d . To i n v e s t i g a t e t h i s r e l a t i o n s h i p SR Ca t r a n s p o r t and l e v e l s of SR a c y l c a r n i t i n e s were measured i n a c u t e l y d i a b e t i c r a t s (7 days). T h i s time p e r i o d was chosen because there i s no d i s c e r n a b l e decrease i n +LVdP/dT or -LVdP/dT i n i s o l a t e d working hearts 7 days a f t e r the i n d u c t i o n of d i a b e t e s (Vadlamudi et a l , 1981) . At t h i s time we observed no 2+ s i g n i f i c a n t decrease i n SR Ca t r a n s p o r t (Figure 12) and no s i g n i f i c a n t i n c r e a s e i n LC a c y l c a r n i t i n e s a s s o c i a t e d with the microsomal SR (Table 8 ) . The e f f e c t of exogenously added L - p a l m i t y l c a r n i t i n e or D , L - c a r n i t i n e was a l s o i d e n t i c a l to c o n t r o l s i n these a c u t e l y d i a b e t i c r a t s (Figures 13,14). The la c k of i n h i b i t o n of SR 7 days a f t e r the i n d u c t i o n of d i a b e t e s 141-c o r r e l a t e s with the normal c a r d i a c performance seen i n a c u t e l y 2+ d i a b e t i c h e a r t s . The a l t e r a t i o n i n Ca t r a n s p o r t a c t i v i t y and i n c r e a s e d l e v e l s of f r e e and LC a c y l c a r n i t i n e s (Figure 7, Table 6,16) observed 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 p r e p a r a t i o n s must t h e r e f o r e be of gradual onset. In support of t h i s we observed only a s l i g h t n o n - s i g n i f i c a n t i n c r e a s e i n microsomal SR l e v e l s of LC a c y l c a r n i t i n e s i n 30 day s t r e p t o z o t o c i n d i a b e t i c r a t s (Table 10). By 42 days p o s t - i n d u c t i o n SR l e v e l s of LC a c y l c a r n i t i n e s were s i g n i f i c a n t l y e l e v a t e d (Figure 16). I t was at t h i s time that 2+ the d e p r e s s i o n i n SR Ca t r a n s p o r t was f i r s t observed (Figure 23). The absence of any change at 7 days a l s o suggests t h a t the a l t e r a t i o n s seen at longer time p e r i o d s are not due to a d i r e c t t o x i c e f f e c t of a l l o x a n or s t r e p t o z o t o c i n . M i l l e r (1980) has reported d i f f e r e n c e s i n c a r d i a c performance as soon as 3 days a f t e r the i n d u c t i o n of a l l o x a n d i a b e t e s , although i n t h i s time p e r i o d t o x i c e f f e c t s of the drug cannot be r u l e d out. Furthermore, t h i s decrease i n c a r d i a c f u n c t i o n was reversed i n the presence of 10 mM gl u c o s e . 2+ The e f f e c t of L - p a l m i t y l c a r n i t i n e on SR Ca t r a n s p o r t may be due to i t s detergent a c t i o n . As an i n d i c a t i o n of p o s s i b l e a l t e r a t i o n s , experiments were performed to determine i f the c h r o n i c a l l y d i a b e t i c r a t s showed any a l t e r a t i o n i n SR 2+ Ca p e r m e a b i l i t y (Figure 9 ) . The r e s u l t s suggest that the SR p r e p a r a t i o n s from the d i a b e t i c r a t s , which had higher l e v e l s of LC a c y l c a r n i t i n e s a s s o c i a t e d with the SR, d i d not 2+ show any change i n Ca p e r m e a b i l i t y . Another p o s s i b l e e f f e c t of LC a c y l c a r n i t i n e i n c o r p o r a t i o n i n t o the SR membrane -142-i s an a l t e r a t i o n i n p r o t e i n f u n c t i o n . SR Ca -ATPase a c t i v i t y i s h i g h l y dependent on i t s p h o s p h o l i p i d environment (Warren et a l , 1974; Hidalgo et a l , 1978). In SR from c h r o n i c a l l y d i a b e t i c r a t s the LC a c y l c a r n i t i n e s may a l t e r the 2+ p h o s p h o l i p i d environment, thereby decreasing Ca -ATPase a c t i v i t y , r e s u l t i n g i n a decreased a b i l i t y of the SR to 2+ t r a n s p o r t Ca . T h i s i s supported by our o b s e r v a t i o n s that L - p a l m i t y l c a r n i t i n e had the same e f f e c t on SR (Ca 2 +-Mg 2 +)-ATPase a c t i v i t y as i t d i d on C a 2 + t r a n s p o r t (Table 10). Our data c o n t r a d i c t the data of P i t t s et a l (1979) who suggest that a g r e a t e r c o n c e n t r a t i o n of 2+ L - p a l m i t y l c a r n i t i n e i s necessary to i n h i b i t Ca -ATPase 2+ a c t i v i t y than Ca t r a n s p o r t . However, the work of Adams et a l (1979) conforms with our r e s u l t s which suggest a s i m i l a r i n h i b i t o r y potency of L - p a l m i t y l c a r n i t i n e on both pro c e s s e s . -143-C) EFFECT OF INSULIN AND CARNITINE TREATMENT ON DIABETIC RAT CARDIAC SR FUNCTION Re s u l t s to date suggest that c a r d i a c SR f u n c t i o n 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 depressed, p o s s i b l y as a r e s u l t of a bu i l d u p of t i s s u e LC a c y l c a r n i t i n e s . We were i n t e r e s t e d 2+ i n determining i f the dep r e s s i o n i n SR Ca t r a n s p o r t could be reversed by lowering t i s s u e l e v e l s of LC a c y l c a r n i t i n e s . 1) I n s u l i n treatment One c o n c e i v a b l e way to prevent the accumulation of LC a c y l c a r n i t i n e s i n the myocardium of d i a b e t i c r a t s i s to reverse the abnormally high u t i l i z a t i o n of f a t t y a c i d s as an energy source. T h e o r e t i c a l l y , by lowering t i s s u e l e v e l s of LC a c y l c a r n i t i n e s the degree by which SR f u n c t i o n i s depressed would be less e n e d . To decrease f a t t y a c i d u t i l i z a t i o n i n s u l i n was administered to d i a b e t i c r a t s f o r a four week pe r i o d commencing 3 days a f t e r the i n d u c t i o n of d i a b e t e s . U n f o r t u n a t e l y , as determined l a t e r , at t h i s time p e r i o d changes i n c a r d i a c SR f u n c t i o n were b a r e l y d e t e c t a b l e . 2+ However, SR Ca t r a n s p o r t a c t i v i t y from i n s u l i n - t r e a t e d d i a b e t i c r a t s was s i g n i f i c a n t l y increased as compared to untreated d i a b e t i c r a t s (Figure 16). In support of our hypot h e s i s , SR i s o l a t e d from these i n s u l i n - t r e a t e d r a t s d i d co n t a i n s l i g h t l y lower l e v e l s of LC a c y l c a r n i t i n e s (Table 10). Since these experiments were performed other s t u d i e s have i n d i c a t e d that i n s u l i n treatment can prevent the depression i n SR f u n c t i o n . In our l a b o r a t o r y A. T a h i l i a n i (Lopaschuk et -144-a l , 1983) has shown that s i x weeks a f t e r the i n d u c t i o n of d i a b e t e s the s i g n i f i c a n t l y depressed SR f u n c t i o n seen i n d i a b e t i c r a t s can be t o t a l l y prevented by i n s u l i n treatment. S i m i l a r l y , Ganguly et a l (1983) has been able to reverse the 2+ depression i n c a r d i a c SR Ca t r a n s p o r t i n 8 week d i a b e t i c r a t s by a d m i n i s t e r i n g i n s u l i n f o r a 2 week p e r i o d . 2) C a r n i t i n e treatment Although i n s u l i n treatment w i l l prevent the depression i n c a r d i a c SR from o c c u r r i n g , few c o n c l u s i o n s can be made concerning the mechanism i n v o l v e d . The widespread metabolic a l t e r a t i o n s t hat occur as a r e s u l t of i n s u l i n treatment preclude us from making c o n c l u s i o n s which i n v o l v e s p e c i f i c metabolic changes. T h e r e f o r e , we were i n t e r e s t e d i n using an agent that was more s p e c i f i c i n a l t e r i n g myocardial LC a c y l c a r n i t i n e l e v e l s . A number of o b s e r v a t i o n s suggested to us that c a r n i t i n e a d m i n i s t r a t i o n might lower c e l l u l a r l e v e l s of LC a c y l c a r n i t i n e s i n the d i a b e t i c r a t h e a r t . These were: a) p a t h o l o g i c a l c o n d i t i o n s such as d i a b e t e s which e l e v a t e myocardial LC a c y l c a r n i t i n e s may be a s s o c i a t e d with decreased serum c a r n i t i n e l e v e l s (McNab et a l , 1978; Genuth and Hoppel, 1979). b) some of the adverse e f f e c t s that accompany myocardial ischemia have been a t t r i b u t e d to the r a p i d accumulation of LC a c y l c a r n i t i n e s (Idell-Wenger et a l , 1978; Shug et a l , 1978). Although c o n t r o v e r s i a l , a number of i n v e s t i g a t o r s have suggested that c a r n i t i n e a d m i n i s t r a t i o n can reduce the d e t r i m e n t a l e f f e c t s of c l i n i c a l or experimentally-produced -145-ischemia (Shug, 1979;. Thomsen et a l , 1979; L i e d t k e et a l , 1981) . c) members of a f a m i l y with a serum c a r n i t i n e d e f i c i e n c y syndrome developed severe cardiomyopathies (Regitz et a l , 1982) . O r a l c a r n i t i n e treatment r e s u l t e d i n dramatic c l i n i c a l improvement i n the only s u r v i v i n g c h i l d . Other systemic m a n i f e s t a t i o n s of c a r n i t i n e d e f i c i e n c y such as s k e l e t a l myopathies can a l s o be reversed by o r a l c a r n i t i n e a d m i n i s t r a t i o n (Karpati et a l , 1975, Engel et a l , 1980). d) C a r n i t i n e a d m i n i s t r a t i o n to h e arts perfused with high f a t t y a c i d s (a c o n d i t i o n that i s seen i n diabetes) has been shown to prevent the i n t r a c e l l u l a r accumulation of v a r i o u s l i p i d s (namely a c y l g l y c e r o l , f r e e f a t t y a c i d s , and LC a c y l CoA d e r i v a t i v e s ) (Liedtke and N e l l i s , 1979; L i e d t k e et a l , 1981; Hulsmann et a l , 1982). In these s t u d i e s i n t r a c e l l u l a r l e v e l s of LC a c y l c a r n i t i n e s were not measured. e) i f r a t s are f a s t e d ( i . e . the d i e t a r y intake of c a r n i t i n e and i t s p r e c u r s o r s are e liminated) the myocardial l e v e l s of LC a c y l c a r n i t i n e s become e l e v a t e d (Choi et a l , 1979). T h i s may r e s u l t from a decrease i n serum c a r n i t i n e , although the s h i f t from glucose to f a t t y a c i d metabolism i n h e a r t s during s t a r v a t i o n must be considered as a p o s s i b l e c a u s a l f a c t o r . These o b s e r v a t i o n s suggested to us that a l t e r a t i o n s i n serum c a r n i t i n e l e v e l s can a f f e c t myocardial l i p i d metabolism. We t h e r e f o r e decided to determine i f r a i s i n g serum c a r n i t i n e 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 could lower the e l e v a t e d t i s s u e l e v e l s of LC a c y l c a r n i t i n e s . -146-C a r n i t i n e (3 hydroxy-4-N trimethylammoniumbutyrate) i s an amino a c i d r e q u i r e d f o r normal f a t t y a c i d o x i d a t i o n , and i s t h e r e f o r e p a r t i c u l a r l y abundant i n muscle t i s s u e . I t i s not a true v i t a m i n i n that the l i v e r and kidney can s y n t h e s i z e the compound from l y s i n e , methionine, and ascorbate (Rebouche, 1980). A c a r n i t i n e d e f i c i e n c y s t a t e can a r i s e i n v a r i o u s p a t h o l o g i c a l c o n d i t i o n s (Rudman et a l , 1977) or i n the absence of an adequate intake of c a r n i t i n e p r e c u r s o r s (Penn et a l , 1980). Supplementing the d i e t with c a r n i t i n e or i t s p r e c u r s o r s has proven very s u c c e s s f u l i n r e v e r s i n g the d e f i c i e n c y (Regitz et a l , 1982; K a r p a t i et a l , 1975; B i z z i et a l , 1978). We t h e r e f o r e chose to administer c a r n i t i n e o r a l l y , v i a d r i n k i n g water, i n an e f f o r t to reverse the dep r e s s i o n i n serum c a r n i t i n e l e v e l s noted i n these animals (McNab et a l , 1978; Genuth and Hoppel, 1979). Although the L form of c a r n i t i n e i s the n a t u r a l l y o c c u r r i n g isomer we were o b l i g e d to use the D,L form of c a r n i t i n e because of the p r o h i b i t i v e c o s t of L - c a r n i t i n e . As shown i n Table 12 serum c a r n i t i n e l e v e l s returned to normal 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 fed 1 g of D,L-carnitine/kg/day f o r 30 days. A high dose was chosen s i n c e plasma c a r n i t i n e i s exchanged by l i v e r , kidney, and spleen at a f a s t e r r a t e than by s k e l e t a l or c a r d i a c muscle (Brooks and Mcintosh, 1975). A f t e r 30 days of c a r n i t i n e treatment to c h r o n i c a l l y d i a b e t i c r a t s , v e n t r i c u l a r l e v e l s of LC a c y l c a r n i t i n e s were measured. Table 13 i n d i c a t e s that c a r n i t i n e a d m i n i s t r a t i o n d i d indeed lower myocardial l e v e l s of LC a c y l c a r n i t i n e s . The mechanism by which c a r n i t i n e does t h i s i s as yet unknown. I t -147-has been suggested by L i e d t k e et a l (1981) that c a r n i t i n e may d i r e c t l y i n t e r f e r e with the p a s s i v e uptake of f r e e f a t t y a c i d s at the plasma membrane. T h i s would e x p l a i n not o n l y our r e s u l t s , but a l s o the reduced i n t r a c e l l u l a r i n c o r p o r a t i o n of f r e e f a t t y a c i d s and LC A c y l CoA d e r i v a t i v e s that i s observed by those workers i n ischemic swine hearts perfused with c a r n i t i n e . I t should be noted that both the L and D,L isomers of c a r n i t i n e were found to be b e n e f i c i a l i n improving mechanical f u n c t i o n i n these ischemic h e a r t s . A second p o s s i b l e mechanism by which c a r n i t i n e may lower i n t r a c e l l u l a r LC a c y l c a r n i t i n e s i s by a c a r n i t i n e - L C a c y l c a r n i t i n e t r a n s l o c a s e enzyme i n the sarcolemma, s i m i l a r to that found i n the m i t o c h o n d r i a l membrane. Although such a system has yet to be i d e n t i f i e d i t i s reasonable to assume that the myocardial c e l l must have some mechanism to lower i n t r a c e l l u l a r l e v e l s of amphiphiles, such as the LC a c y l c a r n i t i n e s . I f the c o n c e n t r a t i o n of these amphiphiles i n c r e a s e s to a c e r t a i n l e v e l the p o t e n t i a l e x i s t s f o r s e r i o u s d i s r u p t i o n of membrane-related f u n c t i o n s . Recently, Vary and Neely (1981) have i d e n t i f i e d a c a r r i e r - m e d i a t e d c a r n i t i n e t r a n s p o r t system i n r a t heart sarcolemma, although t h i s study d i d not i n v e s t i g a t e the p o s s i b i l i t y of a c o u n t e r - t r a n s p o r t of LC a c y l c a r n i t i n e s from the c y t o s o l to the blood. They d i d however, r e p o r t that the Km f o r c a r n i t i n e uptake was very s i m i l a r to the c o n c e n t r a t i o n of c a r n i t i n e found i n the blood under normal p h y s i o l o g i c a l c o n c e n t r a t i o n s . T h e r e f o r e , i f a c o - t r a n s p o r t system does e x i s t , a r e d u c t i o n i n blood c a r n i t i n e , such as that seen i n d i a b e t e s , could t h e o r e t i c a l l y -148-d e a c t i v a t e the enzyme r e s u l t i n g i n an accumulation of myocardial LC a c y l c a r n i t i n e s . Conversely, by r a i s i n g serum c a r n i t i n e , as we have done, the t r a n s l o c a s e system would be a c t i v a t e d r e s u l t i n g i n a lowering of myocardial LC a c y l c a r n i t i n e s . I t i s a l s o p o s s i b l e that p e r f u s i o n of i s o l a t e d h e a r t s with c a r n i t i n e can reduce t i s s u e l e v e l s of LC a c y l c a r n i t i n e s . However, the slow rate of myocardial c a r n i t i n e uptake suggests that i n such a s h o r t p e r i o d of time l i t t l e e f f e c t on l i p i d metabolism occurs (Dr. J.R. Neely, personal communication). A t h i r d mechanism by which c a r n i t i n e a d m i n i s t r a t i o n has been suggested to produce a b e n e f i c i a l e f f e c t i s by t r a p p i n g f r e e f a t t y a c i d s and LC a c y l CoA compounds as a c y l c a r n i t i n e s ( S i l i p r a n d i and Ramacci, 1980). T h e o r e t i c a l l y , u n l i k e f r e e f a t t y a c i d s and LC a c y l CoA d e r i v a t i v e s , t h e a c y l c a r n i t i n e s are more r e a d i l y r e l e a s e d from i n j u r e d t i s s u e , thereby reducing the damaging a c t i o n of these compounds on membrane s t r u c t u r e . T h i s h y p o t h e s i s , however, does not e x p l a i n why t i s s u e l e v e l s of LC a c y l c a r n i t i n e s are i n i t i a l l y e l e v a t e d i n d i a b e t i c or ischemic myocardium. As mentioned e a r l i e r , c a r n i t i n e a d m i n i s t r a t i o n has been shown to have a b e n e f i c i a l e f f e c t on heart f u n c t i o n i n e x p e r i m e n t a l l y - i n d u c e d ischemia p o s s i b l y by lowering the i n t r a c e l l u l a r accumulation of f a t t y a c i d i n t e r m e d i a t e s . As w e l l as showing that 30 day c a r n i t i n e treatment lowered myocardial l e v e l s of LC a c y l c a r n i t i n e 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 (Table 13) we determined the e f f e c t of c a r n i t i n e on heart f u n c t i o n . V a r i o u s parameters of heart f u n c t i o n were measured i n the c a r n i t i n e - t r e a t e d d i a b e t i c r a t s on an i s o l a t e d working -149-heart apparatus (Figures 17, 18 and 19). U n f o r t u n a t e l y we found no improvement i n the LVDP or the rate of myocardial c o n t r a c t i o n and r e l a x a t i o n i n d i a b e t i c r a t s fed c a r n i t i n e f o r a 30 day p e r i o d . T h i s could mean that the el e v a t e d l e v e l s of LC a c y l c a r n i t i n e s seen i n the d i a b e t i c r a t myocardium do not c o n t r i b u t e to the observed depression i n heart f u n c t i o n , or that the membrane damage that may r e s u l t from e l e v a t e d c e l l u l a r LC a c y l c a r n i t i n e s cannot be reversed by lowering t i s s u e l e v e l s of LC a c y l c a r n i t i n e s . To determine which mechanism was i n v o l v e d c a r n i t i n e was administered to r a t s from the onset of di a b e t e s to see i f the depression i n heart f u n c t i o n c o u l d be prevented. In these s t u d i e s the o r a l dose of c a r n i t i n e was r a i s e d to 3 g/kg/day on the advice of Dr. D. Paulson of Dr. A. Shug's l a b o r a t o r y ( V e t e r a n ' s A d m i n i s t r a t i o n H o s p i t a l , Madison, W i s c o n s i n ) . As shown i n Table 14 we could s i g n i f i c a n t l y e l e v a t e the serum l e v e l s of c a r n i t i n e i n both c o n t r o l and d i a b e t i c r a t s . The e f f e c t on heart f u n c t i o n of r a i s i n g serum c a r n i t i n e l e v e l s p a r a l l e l e d what was seen i n the o r i g i n a l study. That i s , c a r n i t i n e treatment d i d not prevent the onset of heart d y s f u n c t i o n i n these d i a b e t i c r a t s (Figures 20,21,22). I t d i d , however, prevent the depression i n c a r d i a c 2+ SR Ca t r a n s p o r t a c t i v i t y (Figure 23). Furthermore,there was an e l e v a t i o n i n the microsomal LC a c y l c a r n i t i n e l e v e l s i n the SR of d i a b e t i c r a t s , which was prevented by c a r n i t i n e treatment (Table 16). In t h i s study i t could not be a s c e r t a i n e d whether or not c a r n i t i n e treatment could lower myocardial LC a c y l c a r n i t i n e l e v e l s s i n c e the untreated d i a b e t i c r a t s d i d not have s i g n i f i c a n t l y e l e v a t e d l e v e l s of LC -150-a c y l c a r n i t i n e s (Table 15). T h i s study r a i s e d a number of i n t e r e s t i n g questions which we have attempted to answer. 1) What could account f o r the d i f f e r e n c e i n myocardial l e v e l s of LC a c y l c a r n i t i n e s i n the 42 day d i a b e t i c r a t s as compared to the 150 day d i a b e t i c r a t s ? Previous s t u d i e s by Feuvray et a l (1979) have shown an e l e v a t i o n of LC a c y l c a r n i t i n e s i n the myocardium of short -term d i a b e t i c r a t s . In our 150 day d i a b e t i c r a t s we a l s o found e l e v a t e d l e v e l s of LC a c y l c a r n i t i n e s . Yet i n our 42 day d i a b e t i c r a t s we d i d not see a s i g n i f i c a n t e l e v a t i o n i n LC a c y l c a r n i t i n e s . T h i s d i s c r e p a n c y i s not a r e s u l t of d i f f e r e n c e s i n the s e v e r i t y of d i a b e t e s , as i n d i c a t e d i n Table 11. Rather, i t probably r e f l e c t s the longer time p e r i o d that h e a rts from 42 day r a t s were perfused on the i s o l a t e d working heart apparatus. Hearts from 42 day r a t s were perfused f o r approximately 25 minutes, compared to 10 minutes f o r the 150 day d i a b e t i c r a t h e a r t s . Vary and Neely (1982) have r e c e n t l y reported that t i s s u e l e v e l s of LC a c y l c a r n i t i n e s w i l l decrease i f h earts are perfused with glucose as the s o l e energy s u b s t r a t e , which were the c o n d i t i o n s used i n our study. S i m i l a r l y , Shug et a l (1978) demonstrated that r a t myocardial l e v e l s of LC a c y l c a r n i t i n e s are 2.5 times as great i n non-perfused versus perfused h e a r t s . We have a l s o r e c e n t l y found that non-perfused c o n t r o l and d i a b e t i c h e arts have 3 times the t i s s u e l e v e l s of LC a c y l c a r n i t i n e s of perfused c o n t r o l and d i a b e t i c h e arts (experiments done i n c o n j u n c t i o n with Dr. D. Godin). The non-perfused d i a b e t i c hearts (42 -151-d a y s ) d i d h a v e e l e v a t e d l e v e l s o f LC a c y l c a r n i t i n e s as c o m p a r e d t o c o n t r o l h e a r t s ( T a b l e 1 7 ) . F u r t h e r m o r e , t h e t i s s u e l e v e l s o f LC a c y l c a r n i t i n e s p r o g r e s s i v e l y d e c r e a s e d w i t h i n c r e a s i n g p e r f u s i o n t i m e . The f a c t t h a t c a r d i a c SR p r e p a r e d f r o m d i a b e t i c r a t s i n t h e same 42 day e x p e r i m e n t a l g r o u p h a d e l e v a t e d l e v e l s o f LC a c y l c a r n i t i n e s s u p p o r t s t h e s e o b s e r v a t i o n s s i n c e t h e c a r d i a c SR was p r e p a r e d f r o m n o n - p e r f u s e d h e a r t s . Of c o u r s e one m us t c o n s i d e r t h a t t h e LC a c y l c a r n i t i n e s e q u i l i b r a t e d w i t h t h e SR membrane w o u l d n o t be p a r t o f t h e r e a d i l y e x c h a n g e a b l e p o o l o f c y t o s o l i c LC a c y l c a r n i t i n e s . I n f u t u r e s t u d i e s i t w o u l d be o f i n t e r e s t t o d e t e r m i n e t h e e f f e c t o f c a r n i t i n e a d m i n i s t r a t i o n on d i a b e t i c r a t h e a r t s p e r f u s e d w i t h a h i g h c o n c e n t r a t i o n o f f r e e f a t t y a c i d s . U n d e r t h e s e c o n d i t i o n s t h e m y o c a r d i a l l e v e l s o f LC a c y l c a r n i t i n e s w o u l d n o t be e x p e c t e d t o d e c r e a s e d u r i n g p e r f u s i o n o f t h e h e a r t . B e f o r e t h i s c a n be d o n e , h o w e v e r , t h e e f f e c t o f d i a b e t e s on i s o l a t e d w o r k i n g h e a r t f u n c t i o n m us t f i r s t be c h a r a c t e r i z e d u s i n g f a t t y a c i d s as t h e e n e r g y s u b s t r a t e . 2) What e f f e c t do t h e LC a c y l c a r n i t i n e s h a v e on c a r d i a c SR f u n c t i o n i n d i a b e t i c m y o c a r d i u m ? A l l o f o u r s t u d i e s t o d a t e s u p p o r t t h e h y p o t h e s i s t h a t t h e e l e v a t e d c y t o s o l i c l e v e l s o f LC a c y l c a r n i t i n e s i n d i a b e t i c r a t m y o c a r d i u m r e s u l t s i n an i n c r e a s e d a s s o c i a t i o n o f t h e LC a c y l c a r n i t i n e s w i t h t h e SR m e m b r a n e . By a l t e r i n g t h e i n t e g r i t y o f t h e membrane LC a c y l c a r n i t i n e s d e p r e s s t h e 2+ a b i l i t y o f t h e SR t o t r a n s p o r t Ca . The l a s t s t u d y -152-suggests that c a r n i t i n e treatment can lower SR l e v e l s of LC 2+ a c y l c a r n i t i n e s and thereby prevent the i n h i b i t i o n of Ca t r a n s p o r t . However, the c a r n i t i n e treatment d i d not r e s u l t i n an improvement i n heart f u n c t i o n i n these d i a b e t i c r a t s . An improvement i n heart f u n c t i o n would be expected i f SR i s p r i m a r i l y i n v o l v e d i n c o n t r o l l i n g the rate of r e l a x a t i o n , a n d subsequent c o n t r a c t i o n of c a r d i a c m y o f i b r i l s (Reuter, 1974). T h i s d i s c r e p a n c y can be i n t e r p r e t e d a number of ways. One ex p l a n a t i o n i s t h a t an i n v i t r o c a r d i a c SR p r e p a r a t i o n i s not t r u l y r e p r e s e n t a t i v e of c a r d i a c SR f u n c t i o n i n v i v o . With the techniques a v a i l a b l e today the v a l i d i t y of t h i s cannot be r e a d i l y determined. Numerous i n v e s t i g a t o r s , however, have attempted to c o r r e l a t e r e s u l t s from i n v i t r o SR experiments with changes i n heart f u n c t i o n , both p h y s i o l o g i c a l and p a t h o l o g i c a l . A second i n t e r p r e t a t i o n of the data i s that c a r d i a c SR i s not p r i m a r i l y r e s p o n s i b l e f o r changes i n the r a t e of c o n t r a c t i o n and r e l a x a t i o n of myocardium. A number of i n v e s t i g a t o r s would suggest a more important r o l e f o r the sarcolemmal membrane (Langer et al , 1982). An i n t e r p r e t a t i o n t h a t I favour i s that SR does not have to be 2+ maximally t r a n s p o r t i n g Ca i n v i v o to adequately maintain 2+ c y t o s o l i c Ca . I t may a l s o be p o s s i b l e that the degree to which SR i s a c t i v a t e d by c y t o s o l i c r e g u l a t o r s i s increased i n d i a b e t i c h e a r t s , thereby overcoming the LC a c y l c a r n i t i n e induced d e p r e s s i o n of SR f u n c t i o n . Our r e s u l t s support t h i s h y p o t h e s i s , suggesting that c a r d i a c SR from d i a b e t i c r a t s can 2+ + s t i l l be a c t i v a t e d by v a r i o u s r e g u l a t o r s such as Ca , K , calmo d u l i n , and cAMP-dependent p r o t e i n k i n a s e . In other -153; words, even though the rate at which the SR can t r a n s p o r t 2+ Ca i n d i a b e t i c myocardium i s compromised due to a l t e r a t i o n s i n the i n t e g r i t y of the membrane, other back-up mechanisms may e x i s t to maintain adequate f u n c t i o n . How does SR f u n c t i o n c o n t r i b u t e to depressed heart f u n c t i o n i n d i a b e t i c r a t s ? The i n i t i a l s t u d i e s performed suggest that d i a b e t e s -induced heart d y s f u n c t i o n was, i n p a r t , consequent to a depr e s s i o n i n SR f u n c t i o n . A decreased rate of myocardial c o n t r a c t i o n and r e l a x a t i o n i s a frequent f i n d i n g i n d i a b e t i c r a t s which suggests the involvement of the SR. The time p e r i o d r e q u i r e d f o r a dep r e s s i o n of SR f u n c t i o n to manifest i t s e l f a l s o c o r r e l a t e s with the onset of the d i a b e t i c cardiomyopathy (Vadlamudi et a l , 1982). An o b s e r v a t i o n that does not f i t with the hypothesis i s that c a r n i t i n e treatment of d i a b e t i c r a t s prevented the depression i n SR f u n c t i o n from o c c u r r i n g , but not the decrease i n the rate of c o n t r a c t i o n or r e l a x a t i o n . T h i s suggests that a depression i n SR f u n c t i o n i s not the main c o n t r i b u t i n g f a c t o r to d i a b e t i c cardiomyopathies. What other f a c t o r s could account f o r the d i a b e t i c - i n d u c e d c a r d i a c d y s f u n c t i o n ? I t i s obvious that a dep r e s s i o n i n SR f u n c t i o n i s not the only biochemical change that occurs i n the heart of d i a b e t i c r a t s . Numerous biochemical a l t e r a t i o n s occur that can d i r e c t l y or i n d i r e c t l y i n f l u e n c e myocardial c o n t r a c t i o n . -154-C h a n g e s i n t h e c e l l u l a r l e v e l s o f l i p i d i n t e r m e d i a t e s w h i c h may be a l t e r i n g SR f u n c t i o n may a l s o be a l t e r i n g o t h e r membranous s y s t e m s . F o r i n s t a n c e , LC A c y l CoA d e r i v a t i v e s h a v e b e e n shown t o be p o t e n t i n h i b i t o r s o f m i t o c h o n d r i a l o x i d a t i v e p h o s p h o r y l a t i o n a n d a d e n i n e n u c l e o t i d e t r a n s l o c a s e ( S h u g e t a l , 1 9 7 5 ) . A c o n s e q u e n c e o f t h i s may be a f a l l i n t i s s u e l e v e l s o f h i g h e n e r g y p h o s p h a t e s w h i c h h a v e , i n f a c t , b e e n r e p o r t e d i n 2 d a y and 7 d a y d i a b e t i c r a t s ( A l l i s o n e_t a l , 1 9 7 6 ; Opie e t a l ,1 979). L i p i d i n t e r m e d i a t e s may a l s o be e x e r t i n g an e f f e c t on s a r c o l e m m a l f u n c t i o n . B o t h LC a c y l CoA d e r i v a t i v e s and LC a c y l c a r n i t i n e s a r e p o t e n t i n h i b i t o r s o f s a r c o l e m m a l N a + - K + - A T P a s e a c t i v i t y (Woods e t a l , 1 9 7 7 ; Adams e t a l , 1 9 7 9 ) . An i n h i b i t o n o f N a + - K + - A T P a s e a c t i v i t y h a s b e e n r e p o r t e d i n d i a b e t i c r a t s (Ku and S e l l e r s , 1 9 8 2 ) . O n j i and L i u (1980) s u g g e s t t h a t t h e m a i n e f f e c t o f d i a b e t e s i n d o g h e a r t i s on t h e Na - K pump p r o d u c i n g a d e c r e a s e i n t h e K + a f f i n i t y . T h e s e c h a n g e s i n t h e N a + - K + pump may be h a v e some e f f e c t on m y o c a r d i a l c o n t r a c t i l i t y i n t h e d i a b e t i c h e a r t s . R e c e n t s t u d i e s h a v e f o c u s e d d i r e c t l y on m y o f i b r i l f u n c t i o n a s a p o s s i b l e e x p l a n a t i o n f o r d i a b e t i c i n d u c e d h e a r t d y s f u n c t i o n . P i e r c e a n d D h a l l a (1981) h a v e d e m o n s t r a t e d a d e c r e a s e i n A T P a s e a c t i v i t y i n m y o f i b r i l s f r o m d i a b e t i c r a t 2+ h e a r t s . S u p p o r t i n g t h i s , a d e c r e a s e i n Ca - A T P a s e a c t i v i t y w h i c h c a n be r e v e r s e d by i n s u l i n t r e a t m e n t h a s b e e n f o u n d i n t h e a c t o m y o s i n and m y o s i n c o m p o n e n t o f t h e d i a b e t i c r a t m y o f i b r i l s ( M a l h o l t r a e t a l , 1 9 8 1 ; D i l l m a n , 1 9 8 2 ) . A p o s s i b l e e x p l a n a t i o n f o r t h e d e p r e s s i o n i n m y o f i b r i l -155-Ca^ T-ATPase a c t i v i t y may be the presence of hypothyroidism i n d i a b e t i c r a t s (Pittman et al., 1979 Dillman (1982) has been able to overcome the de p r e s s i o n i n myosin ATPase a c t i v i t y with t h y r o i d hormone (T3 fT4) treatment. As with the SR s t u d i e s , however, biochemical events do not always c o r r e l a t e with p h y s i o l o g i c a l changes. T a h i l i a n i et a l (1983b) have r e c e n t l y demonstrated that t h y r o i d hormone treatment does not reverse the depression i n heart f u n c t i o n noted i n d i a b e t i c r a t s . Changes i n i n t r a c e l l u l a r and e x t r a c e l l u l a r p r o t e i n content may a l s o c o n t r i b u t e to c a r d i a c d y s f u n c t i o n i n d i a b e t i c h e a r t s . Further s t u d i e s must be done, however, s i n c e the evidence a v a i l a b l e i s c o n t r a d i c t o r y . For i n s t a n c e , although myocardial hypertrophy and cardiomegaly are common obs e r v a t i o n s i n d i a b e t i c s , p r o t e i n s y n t h e s i s i s reduced (Pain and G a r l i c k , 1974; W i l l i a m s et a l , 1980) and p r o t e i n degradation i s increased (Williams et a l , 1980). A l s o , Regan et a l (1981) have a t t r i b u t e d f u n c t i o n a l changes i n d i a b e t i c dog myocardium to c o l l a g e n d e p o s i t i o n , whereas Modrak (1980) f a i l e d to see any change i n myocardial c o l l a g e n content or s y n t h e s i s i n d i a b e t i c r a t s . In a d d i t i o n , Factor et a l (1981) d i d not see much i n t e r s t i t i a l f i b r o s i s i n d i a b e t i c r a t hearts unless the disease was accompanied by hy p e r t e n s i o n . In summary i t appears that a number of f a c t o r s c o n t r i b u t e to the de p r e s s i o n of myocardial f u n c t i o n i n d i a b e t e s . A l t e r a t i o n s i n i n t r a c e l l u l a r systems i n v o l v e d i n muscle c o n t r a c t i o n (eg SL, SR, mitochondrion, and m y o f i b r i l s ) as w e l l as e x t r a c e l l u l a r u l t r a s t r u c t u r a l changes may a l l c o n t r i b u t e to -156-the myopathy. In support of t h i s , a l t e r a t i o n s i n both heart f u n c t i o n and metabolism o c c u r r i n g as a r e s u l t of i n s u l i n d e f i c i e n c y , can be reversed or prevented by i n s u l i n replacement However, more s p e c i f i c c o r r e c t i o n s i n metabolic a l t e r a t i o n s , such as c a r n i t i n e or t h y r o i d replacement therapy, have f a i l e d to overcome the depression i n myocardial f u n c t i o n . These f i n d i n g s support the suggestion that no one, s p e c i f i c metabolic change o c c u r r i n g i n dia b e t e s can account f o r the depression i n heart f u n c t i o n . -157-CONCLUSIONS 1) Cardiac sarcoplasmic r e t i c u l u m i s o l a t e d from r a t s which have been d i a b e t i c f o r a p e r i o d of 42 days or longer have a 2+ s i g n i f i c a n t l y depressed c a p a c i t y to t r a n s p o r t Ca . The decrease i n SR f u n c t i o n i s a s s o c i a t e d with a d i r e c t depression 2+ of Ca -ATPase a c t i v i t y . At 7 days and 31 days a f t e r the 2+ i n d u c t i o n of diabe t e s c a r d i a c SR Ca t r a n s p o r t a c t i v i t y i n d i a b e t i c r a t s i s not 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 r a t s . 2) SR microsomes i s o l a t e d from d i a b e t i c r a t hearts have the same degree of m i t o c h o n d r i a l membrane contamination, and are u l t r a s t r u c t u r a l l y s i m i l a r , to c o n t r o l r a t SR microsomes. 2+ 3) S t i m u l a t i o n of c a r d i a c SR Ca t r a n s p o r t a c t i v i t y i n v i t r o by s p e c i f i c r e g u l a t o r s i s u n a l t e r e d i n d i a b e t i c r a t s . 2+ + S i m i l a r l y , the K m f o r Ca or K i s un a l t e r e d i n these SR p r e p a r a t i o n s . 4) L e v e l s of LC a c y l c a r n i t i n e s are el e v a t e d i n c h r o n i c a l l y d i a b e t i c r a t c a r d i a c SR p r e p a r a t i o n s . T h i s may r e s u l t i n a n o n - s p e c i f i c i n h i b i t i o n of SR f u n c t i o n by a l t e r i n g the 2+ membrane l i p i d environment surrounding the Ca -ATPase enzyme. T h i s e l e v a t i o n i n l e v e l s of LC a c y l c a r n i t i n e does not 2+ a l t e r the p e r m e a b i l i t y of the SR membrane to Ca 5) I n s u l i n treatment s i g n i f i c a n t l y improves c a r d i a c SR -158-f u n c t i o n i n d i a b e t i c r a t s at a time p e r i o d when changes i n SR 2+ Ca t r a n s p o r t a c t i v i t y are beginning to occur (31 days a f t e r the i n d u c t i o n of d i a b e t e s ) . 6) Treatment of c h r o n i c a l l y (120 day) d i a b e t i c r a t s with D , L - c a r n i t i n e (1 g/kg/day, o r a l l y ) f o r a 30 day p e r i o d can s i g n i f i c a n t l y lower myocardial l e v e l s of LC a c y l c a r n i t i n e s . I s o l a t e d working heart f u n c t i o n i n these c a r n i t i n e - t r e a t e d d i a b e t i c r a t s , however, remained depressed. 7) Treatment of r a t s with D , L - c a r n i t i n e (3 g/kg/day, o r a l l y ) f o r 42 days can prevent the accumulation of LC a c y l c a r n i t i n e s i n d i a b e t i c r a t c a r d i a c SR membranes. T h i s treatment a l s o 2+ prevents the dep r e s s i o n i n c a r d i a c SR Ca t r a n s p o r t a c t i v i t y from o c c u r r i n g i n these d i a b e t i c r a t s . T h i s supports the hypothesis that the noted depression i n SR f u n c t i o n i s a r e s u l t of an accumulation of LC a c y l c a r n i t i n e s i n the SR membrane. 8) I s o l a t e d working heart f u n c t i o n i n d i a b e t i c r a t s i s s i g n i f i c a n t l y depressed i n 42 day d i a b e t i c r a t s as compared to c o n t r o l . Treatment of these r a t s with D , L - c a r n i t i n e (3 g/kg/day) throughout the study p e r i o d d i d not improve heart f u n c t i o n . T h i s suggests that a depression i n SR f u n c t i o n i s not the only f a c t o r c o n t r i b u t i n g to the dep r e s s i o n i n c a r d i a c f u n c t i o n . -159-BIBLIOGRAPHY Adams RJ, Cohen DW, Gupte J , Johnson D, W a l l i c k ET, Wang T, Schwartz A (1979) In v i t r o e f f e c t s of 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 Na,K-ATPase, and sarcoplasmic r e t i c u l u m Ca -ATPase and Ca - t r a n s p o r t . J o u r n a l of B i o l o g i c a l Chemistry 254:12404-12410. Agarwal MK (1980) S t r e p t o z o t o c i n mechanisms of a c t i o n . FEBS L e t t e r s 120:1-3. A l l i s o n TB, B r u t i g SP, Crass I I I MF, E l i o t RS, Shipp JC (1976) Reduced high-energy phosphate l e v e l s i n r a t h e a r t s I. E f f e c t s of a l l o x a n d i a b e t e s . American J o u r n a l of P h y s i o l o g y 230:1744-1750. A l t o LE, D h a l l a NS (1981) Role of changes i n microsomal c a l c i u m uptake i n the e f f e c t s of r e p e r f u s i o n of Ca -de p r i v e d ra t h e a r t s . C i r c u l a t i o n Research 48:17-24. A l z e t r t e c h n i c a l b u l l e t i n (1981) A l z a r Corp., Palo A l t o , C a l i f o r n i a . Anderson JW (1975) Metabolic a b n o r m a l i t i e s c o n t r i b u t i n g to d i a b e t i c c o m p l i c a t i o n s . I. Glucose metabolism i n i n s u l i n - i n s e n s i t i v e pathways. American J o u r n a l of C l i n i c a l N u t r i t i o n 28:273-280. Baandrup U, Ledet T, Rasch R (1982) D i a b e t i c cardiopathy; Q u a n t i t a t i v e h i s t o l o g i c a l s t u d i e s of d i a b e t i c r a t s i n poor and good c o n t r o l (submitted f o r p u b l i c a t i o n ) . Bennett PH (1981) Diabetes and heart d i s e a s e - t h e magnitude of the problem. In: C l i n i c a l C a r d i o l o g y and Diabetes. V o l . 1 Part 1 Fundamental C o n s i d e r a t i o n s i n C a r d i o l o g y and Diabetes, S c o t t RC (Ed) Mount K i s c o , New York, Futura Pub. Co. 3-12. Bennett T, Hoskins DJ, Hampton JR (1975) C a r d i o v a s c u l a r c o n t r o l i n di a b e t e s m e l l i t u s . B r i t i s h Medical J o u r n a l 2:585-587. B e r t e l s e n S (1981) H i s t o c h e m i c a l i n v e s t i g a t i o n s on plagues i n human a t h e r o s c l e r o t i c a o r t a e . Acta Pathology and M i c r o b i o l o g y S c a n d i n a v i a 51:229-240. B i z z i , A, Mingardi G, Codegoni AM, Mecca G, C a r a t t i n i S (1978) A c c e l e r a t e d recovery of p o s t - d i a l y s i s plasma c a r n i t i n e f a l l by o r a l c a r n i t i n e . Biomedicine 29:183-184. Blumenthal HT, Alex M, Goldenberg S (1960) A study of l e s i o n s of the in t r a m u r a l coronary a r t e r y branches i n di a b e t e s m e l l i t u s . A r c h i v e s of Pathology 70:13-28. Boquist L (1980) A new hy p o t h e s i s f o r a l l o x a n d i a b e t e s . Acta Pathology and M i c r o b i o l o g y Scandinavia 88:201-209. Boz l e r E (1954) R e l a x a t i o n i n e x t r a c t e d muscle f i b e r s . J o u r n a l of General P h y s i o l o g y 38:149-159. -160-B r a d l e y R F , S c h o n f e l d A (1962 ) D i m i n i s h e d p a i n i n d i a b e t i c p a t i e n t s w i t h a c u t e m y o c a r d i a l i n f a r c t i o n . G e r i a t r i c s 1 7 : 3 2 2 . B r a d l e y RF ( i 9 7 1 ) C o r o n a r y a r t e r y d i s e a s e . I n : D i a b e t e s M e l l i t u s : D i a g n o s i s and T r e a t m e n t , F a j a n s S S , S u s s m a n KE ( e d s ) , V o l I I I , New Y o r k : A m e r i c a n D i a b e t i c A s s o c i a t i o n , C h 4 6 . B r o o k s D E , M c i n t o s h J E A (1975 ) T u r n o v e r o f c a r n i t i n e by r a t t i s s u e s . B i o c h e m i c a l J o u r n a l 1 4 8 : 4 3 9 - 4 4 5 . C h r i s t e n s e n N J ( 1 9 7 2 ) P l a s m a c a t e c h o l a m i n e s i n l o n g - t e r m d i a b e t i c s w i t h a n d w i t h o u t n e u r o p a t h y and i n h y p o p h y s e c t o m i z e d s u b j e c t s . J o u r n a l o f C l i n i c a l I n v e s t i g a t i o n 5 1 : 7 7 9 - 7 8 7 . C h e a h AM ( 1 9 8 1 ) E f f e c t s o f l o n g c h a i n u n s a t u r a t e d f a t t y a c i d s on t h e c a l c i u m t r a n s p o r t o f s a r c o p l a s m i c r e t i c u l u m . B i o c h i m i c a anD B i o p h y s i c a A c t a 6 4 8 : 1 1 3 - 1 1 9 . C h o i YR, F o g l e P J , B i e b e r L L (1979 ) The e f f e c t o f l o n g t e r m f a s t i n g on t h e b r a n c h e d - c h a i n a c y l c a r n i t i n e s a n d b r a n c h e d - c h a i n c a r n i t i n e a c y I t r a n s f e r a s e s . J o u r n a l o f N u t r i t i o n 1 0 9 : 1 5 5 - 1 6 1 . C r a l l FV, R o b e r t s WC ( 1 9 7 8 ) T h e e x t r a m u r a l a n d i n t r a m u r a l c o r o n a r y a r t e r i e s i n j u v e n i l e d i a b e t e s m e l l i t u s . A m e r i c a n J o u r n a l o f M e d i c i n e 6 4 : 2 2 1 . D e n t o n R M , R a n d l e R J ( 1 9 6 7 ) C o n c e n t r a t i o n o f g l y c e r i d e s and p h o s p h o l i p i d s i n r a t h e a r t g a s t r o c n e m i u s m u s c l e s . E f f e c t s o f a l l o x a n - d i a b e t e s and p e r f u s i o n . B i o c h e m i c a l J o u r n a l 1 0 4 : 4 1 6 - 4 2 2 . D i l l m a n WH ( 1 9 8 0 ) D i a b e t e s m e l l i t u s i n d u c e s c h a n g e s i n c a r d i a c m y o s i n o f t h e r a t . D i a b e t e s 2 9 : 5 7 9 - 5 8 2 . D u c h e n LW, A n j o r i n A , W a t k i n s P J (1980 ) P a t h o l o g y o f a u t o n o m i c n e u r o p a t h y i n d i a b e t e s m e l l i t u s . A n n a l s o f I n t e r n a l M e d i c i n e 9 2 : 3 0 1 - 3 0 3 . D y r b e r g T , B e n n J , S a n d a h l C , H i l s t e d J , N e r u p J ( 1981 ) P r e v a l e n c e o f d i a b e t i c a u t o n o m i c n e u r o p a t h y . D i a b e t o l o g i a 2 0 : 1 9 0 - 1 9 4 . E b a s h i S , Endo M (1969.) Ca i o n a n d m u s c l e c o n t r a c t i o n . P r o g r e s s i n B i o p h y s i c a l a n d M o l e c u l a r B i o l o g y 1 8 : 1 2 3 - 1 8 3 . E b a s h i S , L i p m a n n F (1962 ) A d e n o s i n e t r i p h o s p h a t a s e - 1 i n k e d c o n c e n t r a t i o n o f c a l c i u m i o n s i n a p a r t i c u l a t e f r a c t i o n o f r a b b i t m u s c l e . J o u r n a l o f B i o l o g i c a l C h e m i s t r y 1 4 : 3 8 7 - 4 0 0 . E p s t e i n F H , O s t r a n d e r L D J , J o h n s o n B C , P a y n e MW, H a y n e r N S , K e l l e r J B , F r a n c i s T J r . ( 1 9 6 5 ) E p i d e m i o l o g i c a l s t u d i e s o n c a r d i o v a s c u l a r d i s e a s e i n a t o t a l c o m m u n i t y - T e c h u m s e h , M i c h i g a n . A n n a l s o f I n t e r n a l M e d i c i n e 6 2 : 1 1 7 0 - 1 1 8 7 . -161-Ewing DJ, Irving JB, Kerr F, Wildsmith JAW, Clark BF (1974) Cardiovascular response to sustained hand g r i p in normal subjects and in patients with diabetes mellitus: A test of autonomic function. C l i n i c a l Science of Molecular Medicine 46:295-306. Factor SM, Bahn R, Minase T, Wolinsky H, Sonnenblick EH (1981) Hypertensive-diabetic cardiomyopathy in the rat. American Journal of Pathology 102:219-228. Factor SM, Okum EM, Minase T (1980) Ca p i l l a r y microaneurysms in the human diabetic heart. New England Journal of Medicine 30:384-388. Fein FS, Kornstein LB, Strobeck JE, Capasso JM, Sonnenblick EH (1980) Altered myocardial mechanics in diabetic rats. C i r c u l a t i o n Research 47:922-933. Feuvray D, Idell-Wenger JA, Neely JR (1979) Effects of ischemia on rat myocardial function and metabolism in diabetes. C i r c u l a t i o n Research 44:322-329. Fischer VW, Barner HB, Leskiu ML (1979) Ca p i l l a r y basal laminar thickness in diabetic human myocardium. Diabetes 28:713-719. Foy JM, Lucus PD (1976) Eff e c t of experimental diabetes , food deprivation and genetic obesity on the s e n s i t i v i t y of pithed rats to autonomic agents. B r i t i s h Journal of Pharmacology 57:229-234. F r i t z IB (1959) Action of carnitine on long chain fatty acid oxidation by l i v e r . American Journal of Physiology 197:297. Froelich JP, Lakatta EG, Beard E, Spurgeon HA, Weisfeldt, Gerstenblith G (1978) Studies on sarcoplasmic reticulum function and contraction duration in young and aged rat myocardium. Journal of Molecular and C e l l u l a r Cardiology 10:427-438. Ganguly PK, Pierce GN, Dhalla KS, Dhalla NS (1982) Insulin-induced r e v e r s i b i l i t y of the defect in sarcoplasmic r e t i c u l a r calcium transport in diabetic cardiomyopathy (submitted for pu b l i c a t i o n ) . Garcia MJ, McNamara PM, Gordon T, Kannell WB (1974) Morbidity and mortality in diabetics in the Framingham population: sixteen year followup study. Diabetes 23:105-111. Garland PB, Randle PJ (1964) Regulation of glucose uptake by muscle. Biochemical Journal 93:678-687. Genuth SM, Hoppel CL (1979) Plasma and urine carnitine in diabetic ketosis. Diabetes 28:1083-1087. -162-G i a c o m e l l i F, Wiener J (1979) Primary myocardial d i s e a s e i n the d i a b e t i c mouse. An u l t r a s t r u c t u r a l study. Laboratory I n v e s t i g a t i o n 40:460-473. Goldenberg S, Alex Mf Blumenthal HT (1958) Sequela of a r t e r i o s c l e r o s i s of a o r t i c and coronary a r t e r i e s . Diabetes 7:98. Goodale F, Dauod AS, F l o r e n t i n R, Lee KT, G i t t e l s o h n A (1962) Chemicoanatomic s t u d i e s of a r t e r i o s c l e r o s i s and thrombosis i n d i a b e t i c s . I: Coronary a r t e r i a l w a l l t h i c k n e s s , thrombosis, and myocardial i n f a r c t s i n autopsied North Americans. Experimental and Molecular Pathology 1:353-363. Gordon LM, Saverheber RD, Esgate JA, D i p p l e I, Marchmont RJ, Houslay MD (1980) An i n c r e a s e i n b i l a y e r f l u i d i t y of r a t l i v e r plasma membranes achieved by the l o c a l a n a e s t h e t i c benzyl a l c o h o l a f f e c t s the a c t i v i t y of i n t r i n s i c membrane enzymes. J o u r n a l of B i o l o g i c a l Chemistry 255:4519-4527. Haider B, Yeh CK, Thomas G, Oldewurtel HA, Lyons MM, Regan TJ (1981) In f l u e n c e of di a b e t e s on the myocardium and coronary a r t e r i e s of rhesus monkey fed an atherogenic d i e t . C i r c u l a t i o n Research 49:1278-1288. Harigaya S, Schwartz A (1969) Rate of c a l c i u m b i n d i n g and uptake i n normal animals and f a i l i n g c a r d i a c muscle. C i r c u l a t i o n Research 25:781-794. H a r r i s DP, M a r r i o t t ML, M c N e i l l JH (1983) Microcomputer a c q u i s i t i o n and a n a l y s i s system f o r the i s o l a t e d working heart p r e p a r a t i o n (submitted to J o u r n a l of Pharmacological Methods). Hasselbach W, Makinose M (1962) ATP and a c t i v e t r a n s p o r t . Biochemical and B i o p h y s i c a l Research Communications 7:132-136. Heart F a c t s (1976) American Heart A s s o c i a t i o n . H e i l b r u n n LV, W i e r c i n s k i FJ (1947) The a c t i o n of v a r i o u s c a t i o n s on muscle protoplasma. J o u r n a l of C e l l u l a r and Comparative P h y s i o l o g y 29:15-32. Hidalgo C, Thomas DD, Ikemoto N (1978) E f f e c t of the l i p i d environment on p r o t e i n motion and enzymatic a c t i v i t y of the sar c o p l a s m i c r e t i c u l u m ATPase. J o u r n a l of B i o l o g i c a l Chemistry 253:6879-6887. H o f t i e z e r V, Carpenter AM (1971) Comparison of s t r e p t o z o t o c i n and a l l o x a n - i n d u c e d d i a b e t e s i n the r a t , i n c l u d i n g v o l u m e t r i c q u a n t i t a t i o n of the p a n c r e a t i c i s l e t s . D i a b e t e o l o g i a 9:178-184. H o l l i n g w o r t h DN, England PJ (1978) The s t i m u l a t i o n of c a l c i u m uptake i n t o s a r c o p l a s m i c r e t i c u l u m v e s i c l e s from r a t heart by adenosine 3*,5'-phosphate-dependent p r o t e i n k i n a s e . B i o c h e m i c a l S o c i e t y T r a n s a c t i o n s 6:573-576. -163-Hulsmann WC, Stam H, Maccari F (1982) The e f f e c t of excess ( a c y l ) c a r n i t i n e on l i p i d metabolism i n r a t h e a r t . B i o c h i m i c a and B i o p h y s i c a Acta 713:39-45. Idell-Wenger JA, Grotyohann LW, N e e l y JR (1978) Coenzyme A and c a r n i t i n e d i s t r i b u t i o n i n normal and ischemic h e a r t s . J o u r n a l of B i o l o g i c a l Chemistry 253:4310-4318. Ingebretsen CG, Moreau P, Hawelu-Johnson C, Ingebretsen W (1980) Performance of d i a b e t i c r a t h e a r t s : e f f e c t s of anoxia and i n c r e a s e d work. American J o u r n a l of Physiology 239:H614-H620. Jones LR, Besch HR (1979) Calcium handling by c a r d i a c s a r c o p l a s m i c r e t i c u l u m . Texas Reports on B i o l o g y and Medicine 39:19-35. Junod A , Lambert AE, S t a u f f a c h e r W, Renold AE (1969) Diabetogenic a c t i o n of s t r e p t o z o t o c i n : R e l a t i o n s h i p of dose to metabolic response. J o u r n a l of C l i n i c a l I n v e s t i g a t i o n 48:2129-2139. Kalant N , Teitlbaum J I , Cooperberg AA, Harland WA (1964) D i e t a r y atherogenesis i n a l l o x a n d i a b e t e s . J o u r n a l of Laboratory C l i n i c a l Medicine 63:147-157. Kannel WB, H j o r t l a n d M, C a s t e l l i WP (1974) Role of d i a b e t e s i n c o n g e s t i v e heart f a i l u r e : The Framingham study. American J o u r n a l of C a r d i o l o g y 34:29-34. Kannel WB (1978) Role of d i a b e t e s i n c a r d i a c d i s e a s e . In: Diabetes and the Heart, Zoneraich S (Ed) Chase C Thomas (Publ) 97. K a r p a t i G, Carpenter S, Engel AG, watters G, A l l e n J , Rothman S, Klassen G, Mamer OA (1975) The syndrome of systemic c a r n i t i n e d e f i c i e n c y . Neurology 25:16-24. Katz AM (1979) Role of the c o n t r a c t i l e p r o t e i n s and s a r c o p l a s m i c r e t i c u l u m i n the response of the heart the catecholamines: A h i s t o r i c a l review. Advances i n C y c l i c N u c l e o t i d e Research 11:303-343. Katz AM, Repke DI, Upshaw JE, P o l a s c i k MA (1970) C h a r a c t e r i z a t i o n of dog c a r d i a c microsomes: Use of zonal c e n | r i ^ u g a t i o n to f r a c t i o n a t e s a r c o p l a s m i c r e t i c u l u m , (Na -K ) a c t i v a t e d ATPase and m i t o c h o n d r i a l fragments. Biochemical B i o p h y s i c a l Acta 205:473-490. Katz AM, Messineo FC (1981) Lipid-membrane i n t e r a c t i o n s and the pathogenesis of ischemic damage i n the myocardium. C i r c u l a t i o n Research 48:1-24. Katz S (1980) Mechanism of s t i m u l a t i o n of c a l c i u m t r a n s p o r t i n c a r d i a c s a r c o p l a s m i c r e t i c u l u m p r e p a r a t i o n s by c a l m o d u l i n . Annals of the New York Academy of Science 356:267-278. -165-K a t z S , R e m t u l l a MA ( 1 9 7 8 ) P h o s p h o d i e s t e r a s e p r o t e i n a c t i v a t o r s t i m u l a t e s c a l c i u m t r a n s p o r t i n c a r d i a c m i c r o s o m a l p r e p a r a t i o n s e n r i c h e d i n s a r c o p l a s m i c r e t i c u l u m . B i o c h e m i c a l B i o p h y s i c a l R e s e a r c h C o m m u n i c a t i o n s 8 3 : 1 3 7 3 - 1 3 7 9 . K e s s l e r H (1971) M o r t a l i t y e x p e r i e n c e i n d i a b e t i c p a t i e n t s . A t w e n t y - s i x y e a r f o l o w - u p s t u d y . A m e r i c a n J o u r n a l o f M e d i c i n e 5 1 : 7 1 5 - 7 2 4 . K i r c h b e r g e r M A , T a d a M , R e p k e D I , K a t z AM ( 1 9 7 2 ) C y c l i c a d e n o s i n e 3 ' , 5 1 - m o n o p h o s p h a t e - d e p e n d e n t p r o t e i n k i n a s e s t i m u l a t i o n o f c a l c i u m u p t a k e b y c a n i n e c a r d i a c m i c r o s o m e s . J o u r n a l o f M o l e c u l a r and C e l l u l a r C a r d i o l o g y 4 : 6 7 3 - 6 8 0 . K n o w l e s HC (1981 ) The n a t u r a l h i s t o r y o f c o r o n a r y h e a r t d i s e a s e and d i a b e t e s m e l l i t u s . I n : C l i n i c a l C a r d i o l o g y and D i a b e t e s . V o l 1 P a r t 1. F u n d a m e n t a l C o n s i d e r a t i o n s i n C a r d i o l o g y and D i a b e t e s , C o t t RC (Ed) M o u n t K i s c o , New Y o r k , F u t u r a P u b Co 2 9 - 3 9 . K r a n i a s EG, M a n d e l F , Wang T , S c h w a r t z A (1980) M e c h a n i s m o f t h e s t i m u l a t i o n o f c a l c i u m i o n d e p e n d e n t a d e n o s i n e t r i p h o s p h a t a s e o f c a r d i a c s a r c o p l a s m i c r e t i c u l u m by a d e n o s i n e 3 ' , 5 ' - m o n o p h o s p h a t e d e p e n d e n t p r o t e i n k i n a s e . B i o c h e m i s t r y 1 9 : 5 4 3 4 - 5 4 3 9 . K r i s t e n s s o n Y , N o r d b o r g C , O l s s o n Y , S o u r a n d e r P (1971 ) C h a n g e s i n t h e v a g u s n e r v e i n d i a b e t e s m e l l i t u s ^ A c t a P a t h o l o g y and M i c r o b i o l o g y S c a n d i n a v i a 7 9 : 6 8 4 - 6 8 5 . L a n g e r GA, F r a n k J S , P h i l i p s o n KD (1982) U l t r a s t r u c t u r a l and c a l c i u m e x c h a n g e o f t h e s a r c o l e m m a , s a r c o p l a s m i c r e t i c u l u m and m i t o c h o n d r i a o f t h e m y o c a r d i u m . P h a r m a c o l i c a l T h e r a p e u t i c s 1 6 : 3 3 1 - 3 3 6 . Ku DD, S e l l e r s BM (1982) E f f e c t s of s t r e p t o z o t o c i n d i a b e t e s and i n s u l i n t r e a t m e n t on m y o c a r d i a l sodium pump and c o n t r a c t i l i t y of the r a t h e a r t . J o u r n a l of Pharmacology and E x p e r i m e n t a l T h e r a p e u t i c s 222:395-400. L e d e t T ( 1 9 6 8 ) H i s t o l o g i c a l a n d h i s t o c h e m i c a l c h a n g e s i n t h e c o r o n a r y a r t e r i e s o f o l d d i a b e t i c p a t i e n t s . D i a b e t o l o g i a 4 : 2 6 8 - 2 7 2 . L e d e t T (1976 ) D i a b e t i c c a r d i o m y o p a t h y : Q u a n t i t a t i v e h i s t o l o g i c a l s t u d i e s o f t h e h e a r t f r o m y o u n g j u v e n i l e d i a b e t i c s . A c t a P a t h o l o g y M i c r o b i o l o g y S c a n d i n a v i a (A) 8 4 : 4 2 1 - 4 2 8 . L e d e t T , F i s c h e r - D z o g a K , W i s s l e r RW (1976 ) G r o w t h o f r a b b i t a o r t i c s m o o t h m u s c l e c e l l s c u l t u r e d i n m e d i a c o n t a i n i n g d i a b e t i c a n d h y p e r l i p e m i c s e r u m . D i a b e t e s 2 5 : 2 0 7 - 2 1 5 . L e d e t T , N e u b a u e r B , C h r i s t e n s e n N J , L u n d b a c k K ( 1 9 7 9 ) D i a b e t i c c a r d i o p a t h y . D i a b e t o l o g i a 1 6 : 2 0 7 - 2 0 9 . -166-L e f k o v i t s AM (1937) Coronary d i s e a s e i n d i a b e t e s m e l l i t u s . J o u r n a l of Laboratory and C l i n i c a l Medicine 23:354-357. Lehner NDM, C l a r k s o n TB, L o f l a n d HB (1971) The e f f e c t of i n s u l i n d e f i c i e n c y , hypothyroidism, and h y p e r t e n s i o n on a t h e r s c l e r o s i s i n the s q u i r r e l monkey. Experimental Molecular Pathology 15:230-244. LePeuch B, Haiech J , Demaille JC (1979) Concerted r e g u l a t i o n of c a r d i a c s a r c o p l a s m i c r e t i c u l u m c a l c i u m t r a n s p o r t by c y l i c adenosine monophosphate dependent and ca l c i u m - c a l m o d u l i n dependent p h o s p h o r y l a t i o n s . Biochemistry 18:5150-5157. Levine SA (1922) Angina p e c t o r i s ; some c l i n i c a l c o n s i d e r a t i o n s . J o u r n a l of the American Medical A s s o c i a t i o n 79:928-933. L i e d t k e AJ, N e l l i s SH (1979) E f f e c t s of c a r n i t i n e i n ischemic and f a t t y a c i d supplemented swine h e a r t s . J o u r n a l of C l i n i c a l I n v e s t i g a t i o n 64:440-447. L i e d t k e AJ, N e l l i s SH, W h i t e s e l l LF (1981) E f f e c t s of c a r n i t i n e isomers on f a t t y a c i d metabolism i n ischemic swine h e a r t s . C i r c u l a t i o n Research 48:859-866. Limas CJ (1978) Calcium t r a n s p o r t ATPase of c a r d i a c s a r c o p l a s m i c r e t i c u l u m i n experimental h y p e r t h y r o i d i s m . American J o u r n a l of Physiology 4:H745-H751. Limas CJ, S p i e r SS, Kahlon J (1980) Enhanced c a l c i u m t r a n s p o r t by s a r c o p l a s m i c r e t i c u l u m i n mild c a r d i a c hypertrophy. J o u r n a l of Molecular and C e l l u l a r C a r d i o l o g y 12:1103-1116. Lloyd-Mostyn RH, Watkins PJ (1975) D e f e c t i v e i n n e r v a t i o n of the heart i n d i a b e t i c autonomic neuropathy. B r i t i s h Medical J o u r n a l 3:15-17. Lopaschuk G, R i c h t e r B, Katz S (1980) C h a r a c t e r i z a t i o n of calmodulin e f f e c t s on c a l c i u m t r a n s p o r t i n c a r d i a c microsomes enriched i n s a r c o p l a s m i c r e t i c u l u m . Biochemistry 19:5603-5607. Lopaschuk GD, T a h i l i a n i A, Vadlamudi RSV, Katz S, M c N e i l l JH (1983) Long ch a i n a c y l c a r n i t i n e d e p r e s s i o n of c a r d i a c s a r c o p l a s m i c r e t i c u l u m f u n c t i o n i n d i a b e t i c r a t heart; E f f e c t s of i n s u l i n and c a r n i t i n e treatment. American J o u r n a l of P h y s i o l o g y (submitted). Lowry OH, Rosebrough NJ, F a r r AL, Randall RJ (1957) P r o t e i n measurment with the F o l i n phenol reagent. J o u r n a l of B i o l g i c a l Chemistry 193:265. -167-McGarry JD, F o s t e r DW (1976) An improved and s i m p l i f i e d r a d i o i s o t o p i c assay f o r the determination of f r e e and e s t e r i f i e d c a r n i t i n e . J o u r n a l of L i p i d Research 17:277-281. MacNab M, Goldberg RB, J o f f e B l , Botha A, S e f t e l HC (1978) C a r n i t i n e and experimental k e t o t i c and n o n - k e t o t i c d i a b e t i c stupor. Hormonal and M e t a b o l i c Research 11:78-79. M a l h o l t r a A, Penpargkul S, Fein FS, Sonnenblick E H , Scheuer J (1981) The e f f e c t of s t r e p t o z o t o c i n - i n d u c e d diabetes on c a r d i a c c o n t r a c t i l e p r o t e i n s . C i r c u l a t i o n Research 49:1243-1250. Mansford KRL, Opie L (1968) Comparison of metabolic a b n o r m a l i t i e s i n d i a b e t i c m e l l i t u s induced by s t r e p t o z o t o c i n or by a l l o x a n . Lancet 1:670. Marks HH (1965) Longevity and m o r t a l i t y of d i a b e t e s . American J o u r n a l of P u b l i c Health 55:416-423. M a r r i o t t ML (1982) Myocardial f u n c t i o n c h a r a c t e r i s t i c s , response to i s o p r o t e r e n o l and c a l c i u m uptake a c t i v i t y i n r a t s p r e t r e a t e d with t h y r o i d hormone. (Masters T h e s i s ) . Marsh BB (1951) A f a c t o r modifying muscle f i b e r s y n e r e s i s . Nature 167:1065-1066. M i l l e r TB (1979) C a r d i a c performance of i s o l a t e d perfused h e a r t s from a l l o x a n d i a b e t i c r a t s . American J o u r n a l of P h y s i o l o g y 236:H808-H812. Moorehouse JA, C a r t e r SA, Doupe S (1966) V a s c u l a r responses i n d i a b e t i c p e r i p h e r a l neuropathy. B r i t i s h Medical J o u r n a l 1:883-888. Mordes J P , R o s s i n i AA (1981) Animal models of d i a b e t e s . American J o u r n a l of Medicine 70:353-360. Morgan HE, Cadenas E, Regen DM, Park CR (1961) R e g u l a t i o n of glucose uptake i n muscle. I I I . Rate l i m i t i n g step and e f f e c t s of i n s u l i n and anoxia i n heart muscle from d i a b e t i c r a t s . J o u r n a l of B i o l o g i c a l Chemistry 236:262-268. Neely JR, Rovetto MJ (1975) Techniques f o r p e r f u s i n g i s o l a t e d r a t h e a r t s . Methods i n Enzymology 39:43-60. Neubauer B, C h r i s t e n s e n NJ (1976) Norepinephrine, e p i n e p h r i n e , and dopamine contents of the c a r d i o v a s c u l a r system i n long-term d i a b e t i c s . Diabetes 25:6-10. Olsson Y, Sourander P (1968) Changes i n the sympathetic nervous system i n d i a b e t e s m e l l i t u s : a p r e l i m i n a r y r e p o r t . J o u r n a l of N e u r o - V i s c e r a l R e l a t i o n s h i p s 31:86:95. Cn j i T, Liu M ( 1 980) E f f e c t s of a 11 oxan-dia betes on the sodium-potassium adenosine t r i p h o s p h a t a s e enzyme system i n dog h e a r t s . Biochemical and B i o p h y s i c a l Research Communications 96:799-804. -168-Opie LH (1979) Role of c a r n i t i n e i n f a t t y a c i d metabolism of normal and ischemic myocardium. American Heart J o u r n a l 97:375-388. Opie LH, Tansey MJ, K e n n e l l y BM (1979) The heart i n d i a b e t e s m e l l i t u s . P a r t i . Biochemical b a s i s f o r myocardial d y s f u n c t i o n . South A f r i c a n M e d i c a l J o u r n a l 56:207-211. Page MMcB, Watkins PJ (1977) The heart i n d i a b e t e s : autonomic neuropathy and cardiomyopathy. C l i n i c a l Endocrinology and Metabolism 6:377-388. Pain VM, G a r l i c k PJ (1974) E f f e c t of s t r e p t o z o t o c i n d i a b e t e s and i n s u l i n t r e a t m e n t on the r a t e of p r o t e i n s y n t h e s i s i n t i s s u e of the r a t i n v i v o . J o u r n a l o f B i o l o g i c a l C h e m i s t r y 249 : 451 0-451 4. Palumbo PJ, Elvesback LR, Chu C-P, Connolly DC, Kurland LT (1976) Diabetes m e l l i t u s : Incidence, prevalence, s u r v i v o r s h i p and causes of death i n Rochester, Minnesota, 1945-1970. Diabetes 25:566-573. Pande SV, Blanchaer MC (1971) R e v e r s i b l e i n h i b i t i o n of m i t o c h o n d r i a l adenosine diphosphate p h o s p h o r y l a t i o n by long c h a i n a c y l coenzyme A e s t e r s . J o u r n a l of B i o l o g i c a l Chemistry 243:6180-6185. Partamian JO, Bradley RF (1965) Acute myocardial i n f a r c t i o n in 258 cases of d i a b e t e s : Immediate m o r t a l i t y and f i v e - y e a r s u r v i v a l . New England J o u r n a l of Medicine 273:455. P e l l S, D'Alonzo CA (1970) F a c t o r s a s s o c i a t e d with long-term s u r v i v a l of d i a b e t i c s . J o u r n a l of the American Medical A s s o c i a t i o n 214:1833-1840. Penn D, Schmidt-Sommerfeld E, Wolf H (1980) C a r n i t i n e d e f i c i e n c y i n premature i n f a n t s r e c e i v i n g t o t a l p a r e n t e r a l n u t r i t i o n . E a r l y Human Development 4:23-34. Penpargkul S, F e i n F, Sonnenblick EH, Scheuer J (1981) Depressed c a r d i a c s a r c o p l a s m i c r e t i c u l u m f u n c t i o n from d i a b e t i c r a t s . J o u r n a l of Molecular and C e l l u l a r C a r d i o l o g y 13:303-309. Penpargkul S, S c h a i b l e T,, Y i p i n t s o i T, Scheuer J (1980) The e f f e c t of d i a b e t e s on performance and metabolism of r a t h e a r t s . C i r c u l a t i o n Research 47:911-921. Pfaffman MA (1980) The e f f e c t s of s t r e p t o z o t o c i n - i n d u c e d d i a b e t e s and i n s u l i n - t r e a t m e n t on the c a r d i o v a s c u l a r system o the r a t . Research Communications i n Chemical And P a t h o l o g i c a l Pharmacology 28:27-41. -169-P f e i f e r MA, Cook D, Brodsky J , T i c e D, Reenan A, Swedine S, H a l t e r TB (1982) Q u a n t i t a t i v e e v a l u a t i o n of c a r d i a c parasympathetic a c t i v i t y i n normal and d i a b e t i c man. Diabetes 31:339-345. P i e r c e GN, DhallaNS (1981) C a r d i a c m y o f i b r i l l a r ATPase a c t i v i t y i n d i a b e t i c r a t s . J o u r n a l of M o l e c u l a r and C e l l u l a r C a r d i o l o g y 13:1063-1069. Pi t t m a n CS, Suda AK, Chambers JB, Ray GY (1979) (T3) p r o d u c t i o n i n d i a b e t i c p a t i e n t s . Metabolism 28:333-338. P i t t s B J r., Tate CA, Van Winkle B, Wood JM, Entman ML (1978) P a l m i t y l c a r n i t i n e i n h i b i t i o n of the c a l c i u m pump in c a r d i a c s a r c o p l a s m i c r e t i c u l u m : A p o s s i b l e r o l e i n myocardial ischemia. L i f e S ciences 23:391-402. Pressman BC, Lardy H (1956) E f f e c t of s u r f a c e a c t i v e agents on the l a t e n t ATPase of m i t o c h o n d r i a . B i o c h i m i c a and B i o p h y s i c a Acta 21:458-466. Randle PJ, Newsholme EA, Garland PB (1964) R e g u l a t i o n of glucose uptake by muscle. 8. E f f e c t s of f a t t y a c i d s , ketone bodies and pyruvate, and of a l l o x a n d i a b e t e s on uptake and f a t e of glucose i n r a t heart diaphragm muscle. Biochemical J o u r n a l 93:652-656. Randle PJ (1978) I n t e r a c t i o n of metabolism and the p h y s i o l o g i c a l r o l e of i n s u l i n . Recent Progree i n Hormone Research 22:1-44. Rebouche CJ, Engel AG (1981) Primary systemic c a r n i t i n e d e f i c i e n c y : I. C a r n i t i n e b i o s y n t h e s i s . Neurology 31:813-818. Regen TJ, E t t i n g e r PO, Khan MI, J e s r a n i MU, Lyons MM, Oldewurtel HA, Wber M (1974) A l t e r e d myocardial f u n c t i o n a n d # metabolism i n c h r o n i c d i a b e t e s m e l l i t u s without ischemia i n dogs. C i r c u l a t i o n Research 35:222-237. Regen TJ, Haider B, Lyons MM (1978) A l t e r e d v e n t r i c u l a r f u n c t i o n and metabolism i n d i a b e t e s m e l l i t u s . In: Diabetes and the Heart, Zoneraich S (Ed) Chas. C. Thomas (Publ) 123. Regen TJ, Wu CF, Yeh CK, Oldewurtel HA, Haider B (1981) Myocar d i a l composition and f u n c t i o n i n d i a b e t e s : The e f f e c t s of c h r o n i c i n s u l i n use. C i r c u l a t i o n Research 49:1268-1277. Regi t z V, Hadach RJ, Shug AJ (1982) C a r n i t i n e d e f i c i e n c y . A t r e a t a b l e cause of cardiomyopathy i n c h i l d r e n . K l i n i s c h e Wochenschrift 60:393-400. R e i b e l DK, Wyse BW, B e r k i c h DA, Palko WM, Neely JR (1981a) E f f e c t s of d i a b e t e s and f a s t i n g on pantothenic a c i d metabolism i n r a t s . American J o u r n a l of P h y s i o l o g y 240:E597-E601. - 1 7 0 -R e i b e l DK, Wyse BW, B e r k i c h DA, Neely JR (1981b) Regula t i o n of coenzyme A s y n t h e s i s i n heart muscle: e f f e c t s of d i a b e t e s and f a s t i n g . American J o u r n a l of Physiology 240:h606-h611. Renold A (1972) WHO symposium on p r e v e n t i o n of ischemic h e a r t d i s e a s e : M e t a b o l i c a s p e c t s . Madrid 1972. Rerup CC, T a r d i n g F (1969) S t r e p t o z o t o c i n - and a l l o x a n -d i a b e t e s i n mice. D i a b e t o l o g i a 4:313. Reuter H (1974) Exchange of c a l c i u m ions i n the mammalian myocardium. Mechanics and p h y s i o l o g i c a l s i g n i f i c a n c e . C i r c u l a t i o n Research 34:599-605. Rodgers RL, MacLeod KM, M c N e i l l JH (1981) Responses of r a t and quinea p i g h e a r t s to glucagon. Lack of evidence f o r a d i s s o c i a t i o n between changes i n myocardial c y c l i c 3 1,5'-adenosine monophosphate and c o n t r a c t i l i t y . C i r c u l a t i o n Research 49:216-225. Root HF, G r a y b r e l A (1931) Angina p e c t o r i s and d i a b e t e s m e l l i t u s . J o u r n a l of the American Medical A s s o c i a t i o n 96:925. Root HF, Sharkey TP (1936) Coronary a r t e r i o s c l e r o s i s i n d i a b e t e s m e l l i t u s , New England J o u r n a l of Medicine 215:605. Root HF, Bradley RF (1959) C a r d i o v a s c u l a r - r e n a l d i s e a s e . In: Treatment of Diabetes M e l l i t u s , J o s l i n EP, Root HF, White P, Marble A (Ed) Lea and Febiger (Publ) 408. Rudman D, Sewell CW, Ansely JD (1977) D e f i c i e n c y of c a r n i t i n e i n c a c h e t i c c i r r i h o t i c p a t i e n t s . J o u r n a l of C l i n i c a l I n v e s t i g a t i o n 60:716-723. Seneviratne BIB (1977) D i a b e t i c cardiomyopathy: the p r e c l i n i c a l phase. B r i t i s h Medical J o u r n a l 1:1444-1446. Shapiro LM, Howat AP, C a l t e r MM (1981) L e f t v e n t r i c u l a r f u n c t i o n i n d i a b e t e s m e l l i t u s . I: Methodology, and prevalence and spectrum of a b n o r m a l i t i e s . B r i t i s h Heart J o u r n a l 45:122-128. Shigekawa M, P e a r l LJ (1976) A c t i v a t i o n of c a l c i u m t r a n s p o r t i n s k e l e t a l muscle sar c o p l a s m i c r e t i c u l u m by monovalent c a t i o n s . J o u r n a l of B i o l o g i c a l Chemistry 251:6947-6952. Shigekawa M, Akowitz Aa (1979) On the mechanism of Ca -dependent adenosine t r i p h o s p h a t a s e of sarcoplasmic r e t i c u l u m . J o u r n a l of B i o l o g i c a l Chemistry 254:4726-4729. Sh i r e y EK, P r o u d f i t WL, Hawk WA (1980) Primary myocardial d i s e a s e . C o o r e l a t i o n with c l i n i c a l f i n d i n g s , angiographic and biopsy d i a g n o s i s . Follow-up of 139 p a t i e n t s . American Heart J o u r n a l 99:198-207. -171-Shug AL, Thomsen JH, F o l t s JD, B i t t a r N, K l e i n MI, Kohe JR, Huth PJ (1978) Changes i n t i s s u e l e v e l s of c a r n i t i n e and other m e t a b o l i t e s during myocardial ischemia and anoxia. A r c h i v e s of Bio c h e m i s t r y and B i o p h y s i c s 187:25-33. Shug AL, Shargo E, B i t t a r N, F o l t s JD, Oe JR (1975) Acyl-CoA i n h i b i t i o n of adenine n u c l e o t i d e t r a n s l o c a t i o n i n ischemic myocardium. American J o u r n a l of Phy s i o l o g y 228:689-692. S h u r t l e f f D (1974) Some c h a r a c t e r i s t i c s r e l a t e d to i n c i d e n c e of C-V d i s e a s e and death: Framingham study 18 year f o l l o w up. Washington, U.S. Government P r i n t i n g O f f i c e , DHEW Publ. No (NIH) 74-599. S i l i p r a n d i N, Ramacci MA (1980) C a r n i t i n e as a "drug" a f f e c t i n g l i p i d metabolism. In: Drugs a f f e c t i n g l i p i d metabolism. Fumagallia R, Krisheusky D, P a s l e t t i R (eds) E l s e i v e r / N o r t h H o l l and Biomedical Press 381-392. S o l e r SL, Bennett MA, Lamb P', Pentecost B, F i t z G e r a l d MG, Malins JM (1974) Coronary care f o r myocardial i n f a r c t i o n i n d i a b e t i c s . Lancet 1:475-477. S r i v a s t a v a LM, Bora PS, Bhatt SD (1982) Diabetogenic a c t i o n of s t r e p t o z o t o c i n . Trends i n Pharmacological S c i e n c e s 376-378. Stearns SB (1980) C a r n i t i n e content of s k e l e t a l muscle from d i a b e t i c and i n s u l i n - t r e a t e d d i a b e t i c r a t s . Biochemical Medicine 24:33-38. Stuesse SL, W a l l i c k DW, Mace S (1982) Vagal c o n t r o l of heart p e r i o d i n a l l o x a n d i a b e t i c r a t s . L i f e S ciences 31:393-398. Subramanian CV, Radkishnamurthy B, Berenson GS (1980) Photometric d e t e r m i n a t i o n of g l y c o s y l a t i o n of hemoglobin i n di a b e t e s m e l l i t u s . C l i n i c a l Chemistry 26:1683-1687. Sumida M, Wang T, Mandel F, F r o e l i c h JP, Schwartz A (1978) T r a n s i e n t k i n e t i c s of Ca t r a n s p o r t of sarcoplasmic r e t i c u l u m ; a comparison of c a r d i a c and s k e l e t a l muscle. J o u r n a l of B i o l o g i c a l Chemistry 253:8772-8777. Tada M, K i r c h b e r g e r MA, Repke DI, Katz AM (1974) The s t i m u l a t i o n of calcium t r a n s p o r t i n c a r d i a c s a r c o p l a s m i c r e t i c u l u m by adenosine 3',5 * monophosphate dependent p r o t e i n k i n a s e . J o u r n a l of B i o l o g i c a l Chemistry 249:6174-6180. T a h i l i a n i AG, Vadlamudi RVSV, M c N e i l l JH (1983a) Pr e v e n t i o n and r e v e r s a l of a l t e r e d myocardial f u n c t i o n i n d i a b e t i c r a t s by i n s u l i n treatment (submitted f o r p u b l i c a t i o n ) . T a h i l i a n i AG, M c N e i l l JH (1983b) E f f e c t of t h y r o i d treatment on d i a b e t i c r a t c a r d i a c s a r c o p l a s m i c r e t i c u l u m f u n c t i o n and i s o l a t e d working heart f u n c t i o n (manuscript i n p r e p a r a t i o n ) . -172-Thomsen J H , S h u g A L , Y a p W , P a t t e l A K , K a r r a s T J , D e F e l i c e S L ( 1 9 7 9 ) I m p r o v e d p a c i n g t o l e r a n c e o f t h e i s c h e m i c human m y o c a r d i u m a f t e r a d m i n i s t r a t i o n o f c a r n i t i n e . A m e r i c a n J o u r n a l o f C a r d i o l o g y 4 3 : 3 0 0 - 3 0 6 . V a d l a m u d i R , M c N e i l l J H (1980 ) C a r d i a c f u n c t i o n i n n o r m a l a n d d i a b e t i c r a t s . P r o c e e d i n g s o f t h e W e s t e r n P h a r m a c o l o g y S o c i e t y 2 3 : 2 9 - 3 1 . V a d l a m u d i R V S V , R o d g e r s R L , M c N e i l l J H (1982 ) The e f f e c t o f c h r o n i c a l l o x a n - and s t r e p t o z o t o c i n - i n d u c e d d i a b e t e s on i s o l a t e d r a t h e a r t p e r f o r m a n c e . C a n a d i a n J o u r n a l o f P h y s i o l o g y and P h a r m a c o l o g y 6 0 : 9 0 2 - 9 1 1 . V a d l a m u d i R V S V , M c N e i l l J H (1983) E f f e c t o f a l l o x a n a n d s t r e p t o z o t o c i n - i n d u c e d d i a b e t e s on i s o l a t e d r a t h e a r t r e s p o n s i v e n e s s t o c a r b a c h o l ( s u b m i t t e d f o r p u b l i c a t i o n ) . V a r y T C , N e e l y J R (1982) C h a r a c t e r i z a t i o n o f c a r n i t i n e t r a n s p o r t i n i s o l a t e d p e f u s e d a d u l t r a t h e a r t s . A m e r i c a n J o u r n a l o f P h y s i o l o g y 2 4 2 : H 5 8 5 - H 5 9 2 . V e l e m i n s k y J , B u r r I M , S t a u f f a c h e r W (1970 ) C o m p a r a t i v e s t u d y o f e a r l y m e t a b o l i c e v e n t s r e s u l t i n g f r o m t h e a d m i n i s t r a t i o n o f t h e d i a b e t o g e n i c a g e n t s a l l o x a n a n d s t r e p t o z o t o c i n . E u r o p e a n J o u r n a l o f C l i n i c a l I n v e s t i g a t i o n 1 : 1 0 4 - 1 0 8 . V i h e r t A M , Z h a n d o v V S , M a t o v a EE ( 1 9 6 9 ) A t h e r o s c l e r o s i s o f t h e a o r t a a n d c o r o n a r y v e s s e l s o f t h e h e a r t i n c a s e s o f v a r i o u s d i s e a s e . J o u r n a l o f A t h e r s c l e r o s i s R e s e a r c h 9 : 1 7 9 - 1 9 2 . W a r r e n G B , T o o n P A , B i r d s a l l N J M , L e e A G , M e t c a l f e J C (1974) R e v e r s i b l e l i p i d t i t r a t i o n s o f t h e a c t i v i t y o f p u r e a d e n o s i n e t r i p h o s p h a t a s e - l i p i d c o m p l e x e s . B i o c h e m i s t r y 1 3 : 5 5 0 1 - 5 5 0 7 . W a t k i n s P J , M a c K a y J D (1980 ) C a r d i a c d e n e r v a t i o n i n d i a b e t i c n e u r o p a t h y . A n n a l s o f I n t e r n a l M e d i c i n e 9 2 : 3 0 4 - 3 0 7 . W a t t e r s o n D M , H a r r e l s o n WG, K e l l e r P M , S h a r i e f F , Vanaman TC (1976 ) S t r u c t u r a l s i m i l a r i t i e s b e t w e e n Ca - d e p e n d e n t r e g u l a t o r y p r o t e i n s o f 3 ' , 5 ' - c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e and a c t o m y o s i n A T P a s e . J o u r n a l o f B i o l o g i c a l C h e m i s t r y 2 5 1 : 4 5 0 1 - 4 5 1 3 . Weber A , H e r z R (1961) R e q u i r e m e n t f o r c a l c i u m i n . the s y n a e r e s i s o f m y o f i b r i l s . B i o c h e m i c a l B i o p h y s i c a l R e s e a r c h C o m m u n i c a t i o n s 6 : 1 0 6 5 - 1 0 6 6 . W e l l e r M , L a i n g W ( 1 9 7 9 ) The e f f e c t o f c y c l i c n u c l e o t i d e s a n d p r o t e i n p h o s p h o r y l a t i o n on c a l c i u m p e r m e a b i l i t y a n d b i n d i n g i n t h e s a r c o p l a s m i c r e t i c u l u m . B i o c h i m i c a B i o p h y s i c a A c t a 5 5 1 : 4 0 6 - 4 1 9 . W h a r t o n D C , T z a g o l o f f A ( 1 9 6 7 ) C y t o c h r o m e o x i d a s e f r o m b e e f h e a r t m i t o c h o n d r i a . M e t h o d s i n E n z y m o l g y 4 5 : 2 4 5 - 2 5 0 . -173-W h e e l e r T , W a t k i n s P J ( 1 9 7 3 ) C a r d i a c d e n e r v a t i o n i n d i a b e t e s . B r i t i s h M e d i c a l J o u r n a l 4 : 5 8 4 - 5 8 6 . W i l l i a m s IH, Chua BHL, Sahms RH, S i e h l D, Morgan HE (1980) E f f e c t s of d i a b e t e s on p r o t e i n t u r n o v e r i n c a r d i a c muscle. American J o u r n a l of P h y s i o l o g y 239:El78-E185 . Wood J M , B u s h B , P i t t s B J R , S c h w a r t z A ( 1 9 7 7 ) I n h i b i t i o n o f b o v i n e h e a r t Na , K - A T P a s e b y p a l m i t y l c a r n i t i n e a n d p a l m i t y l - C o A . B i o c h e m i c a l B i o p h y s i c a l R e s e a r c h C o m m u n i c a t i o n s 7 4 : 6 7 7 - 6 8 4 . Publications: Bellward GD, Li M, Lopaschuk GD, Warren P (1977) The Effect of Alpha-1-acetylmethadol Hydrochloride on Rat Hepatic Aryl Hydrocarbon Hydroxylase and Epoxide Hydrase. Res Commun Chem Path Pharmacol, 18(3): 415-422. Lopaschuk G, Katz S (1980) Calmodulin, a Possible Regulator of Calcium Transport in Cardiac Sarcoplasmic Reticulum. Proc West Pharmacol Soc, 23: 25-28. Lopaschuk G, Richter B, Katz S (1980) Characterization of Calmodulin Effects on Calcium Transport in Cardiac Microsomes Enriched in Sarcoplasmic Reticulum. Biochemistry, 19: 5603-5607. Lopaschuk GD, Katz S, McNeill JH (1982) Studies on the Mechanism by which Diabetes Alters Cardiac Sarcoplasmic Reticulum Function. Proc West Pharmacol Soc, 25: 47-50. McNeill JH, Lopaschuk GD (1982) Reserpine Treatment Affects Guinea Pig Heart S.R. Calcium Uptake. Proc West Pharmacol Soc, 25: 273-276 Graf E, Verma AK, Gorski JP, Lopaschuk G, Niggli V, Zurini M, Carafoli E, Penniston JT (1982) Molecular Properties of the C a ? + ATPase from Human Erythro-cytes, Biochemistry, 21:4511 -4516. Lopaschuk G, Katz S, McNeill JH (1982) The Effect of Alloxan and Streptozotocin-induced Diabetes on Calcium Transport in Rat Cardiac Sarcoplasmic Reticulum. The Possible Involvement of Long Chain Acylcarnitines. Canadian J. PhysHol. and Pharmacol, (in Press) Lopaschuk GD, Tahil i a n i A, McNeill, J.H. (1982) Continuous Long Term Insulin Delivery in Diabetic Rats U t i l i z i n g Implanted Osmotic Minipumps. J. Pharmacological Methods (in press) Rodgers RL, Lopaschuk GD, McNeill JH (1982) Effect of Reserpine Pretreatment on Calcium Transport and Ca2+-Mg2+ ATPase A c t i v i t y of Guinea Pig Cardiac Microsomes. Submitted to J Pharmacology and Experimental Therapeutics. Lopaschuk GD, Eibschutz B, Katz S, McNeill JH (1982) Depression of Calcium Transport in Sarcoplasmic Reticulum from Diabetic Rats; Lack of Involvement by Specific Regulatory Mediators. Submitted to Canadian J Physiol and Pharmacol. Lopaschuk GD, Katz S, McNeill JH (1983) Effect of oral carnitine treatment on cardiac sarcoplasmic reticulum function in streptozotocin-induced diabetic r a t s . Proc. Western Pharmacol.Soc. (in press) Eibschutz B, Lopaschuk GD, Katz S (1983) Calmodulin regulation of a purif i e d cardiac sarcoplasmic reticulum preparation. Proc. Western Pharmacol. Soc. (in press). Lopaschuk GD, Tah i l i a n i AG, Vadlamudi RVSV, Katz S, McNeill JH (1983) Insulin and carnitine effects on diabetic rat cardiac sarcoplasmic reticulum function. Am. J. Physiol, (manuscript in preparation). Lopaschuk GD, Katz S, McNeill JH (1983) Characterization of cardiac sarcoplasmic reticulum preparations from control and diabetic rats J. Pharmacological Methods (manuscript in preparation). Eibschutz B, Wong APG., Lopaschuk GD, Katz S (1983) Role of calmodulin in skeletal muscle sarcoplasmic reticulum function (manuscript in prep r a t i on). Abstracts: Lopaschuk G, Katz S (1980) Calmodulin Regulation of Calcium Transport in Cardiac Sarcoplasmic Reticulum. Calmodulin and Cell Functions: New York Academy of Sci 35: 404-405. 2 + Lopaschuk G, Katz S, McNeill JH (1981) Effects of Diabetes on Ca +-transport in Cardiac Sarcoplasmic Reticulum. Abst 447, The Pharmacologist 23: 198. American Society of Pharmacology and Experimental Therapeutics. Lopaschuk GD, Katz S, McNeill JH (1982> Involvement of Long Chain Acylcarnitines in the Depression of Cardiac Sarcoplasmic Reticulum Function in Diabetic Rats. J Molecular Ce l l u l a r Cardiol, 14(Supplement 1): 46. McNeill JH, Wenkstern BM, Lopaschuk GD, Rodgers RL (1982) Reserpine-induced Cardiac Supersensitivity to Calcium and Isoproterenol. J Molecular Cellular Cardiol, 14(Supplement 1): 50. Lopaschuk GD, Katz S, McNeill JH (1982) The Mechanism by which Diabetes Alters Cardiac Sarcoplasmic Reticulum Function. Proceedings Canadian Fed Biol Soc, 25: 1. McNeill JH, Rodgers RL, Riedel BE, Lopaschuk GD (1982) Alteration of Cardiac SR Ca?+ Transport May Account for Reserpine Supersensitivity. Proceedings Canadian Fed Biol Soc, 25: 88. Eibschutz B, Lopaschuk GD, Katz S (1982) Studies to Determine i f Calmodulin is Associated with Rabbit Skeletal Muscle Sarcoplasmic Reticulum. Proceedings Canadian Fed Biol Soc, 25: 161. McNeill JH, Tahi l i a n i AG, Lopaschuk GD (1983) Effect of various treatments on diabetes-induced alterations in myocardial function and biochemistry Proc. Canadian Fed. B i o l . Soc. (submitted) 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0095881/manifest

Comment

Related Items