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Effects of insulin or thyroid treatment on diabetes-induced myocardial abnormalities Tahiliani, Arunkumar Govindram 1983

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EFFECTS Of INSULIN OR THYROID TREATMENT ON DIABETES-INDUCED MYOCARDIAL ABNORMALITIES by Arunkumar Govindram T a h i l i a n i B. Pharm., Bombay U n i v e r s i t y , I n d i a , 1979; M. Pharm., Gujarat U n i v e r s i t y , I n d i a , 1981 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES v i s i o n of Pharmacology and Toxicology i n the Faculty of Pharmaceutical Sciences) We accept t h i s t h e s i s as conforming to the required standards THE UNIVERSITY OF BRITISH COLUMBIA May 1983 (TWunkumar Govindram T a h i l i a n i , 1983 I n 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 o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e 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 s t u d y . 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 c o p y i n g o f 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 g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) i i ABSTRACT Diabetes induced i n female Wistar r a t s by an intravenous i n j e c t i o n o f s t r e p t o z o t o c i n r e s u l t e d i n myocardial changes s i x weeks a f t e r i n j e c t i o n o f the drug. The l e f t v e n t r i c u l a r developed pressure (LVDP), the rate of pressure r i s e ( p o s i t i v e dP/dt) and the rate of pressure d e c l i n e (negative dP/dt) were depressed i n d i a b e t i c r a t hearts as compared to c o n t r o l s when measured using the working heart technique. The a b i l i t y o f myocardial SR to t r a n s p o r t calcium was a l s o depressed i n d i a b e t i c animals and the l e v e l of long chain a c y l c a r n i t i n e s was elevated i n these animals. Treatment of the d i a b e t i c animals w i t h i n s u l i n (0.9 U/lOOg/day) immediately a f t e r the disease was detected prevented the d e t e r i o r a t i o n of the p h y s i c a l c o n d i t i o n of d i a b e t i c animals. The t r e a t e d animals did not lose weight, had normal plasma glucose and i n s u l i n values and the degree of g l y c o s y l a t i o n of hemoglobin d i d not d i f f e r s i g n i f i c a n t l y from c o n t r o l s . Cardiac f u n c t i o n as r e f l e c t e d by LVDP and p o s i t i v e and negative dP/dt was not depressed i n 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 . In p a r a l l e l to these r e s u l t s , i t was found that the l e v e l of long chain a c y l c a r n i t i n e s i n SR of tr e a t e d animals was not e l e v a t e d . Calcium uptake a c t i v i t y of SR was a l s o normal i n d i a b e t i c animals t r e a t e d with i n s u l i n . The data suggest t h a t i n s u l i n treatment i s capable of preventing changes i n the myocardium of d i a b e t i c r a t s . In the next set of experiments, d i a b e t i c animals were t r e a t e d with i n s u l i n s i x weeks a f t e r the disease was induced. Four weeks o f treatment normalized the ph y s i c a l features i . e . body weight, plasma . i i i glucose and plasma i n s u l i n values. However, g l y c o s y l a t e d hemoglobin values were not completely reversed to normal. Studying the heart f u n c t i o n , i t was found that such treatment was e f f e c t i v e i n r e v e r s i n g the depressed c a r d i a c f u n c t i o n to normal. Calcium uptake a c t i v i t y and long chain a c y l c a r n i t i n e l e v e l s i n SR were a l s o reversed to normal. These experiments suggest that i n s u l i n treatment i s capable of reversing as well as preventing diabetes-induced myocardial a l t e r a t i o n s . The e f f e c t of i n s u l i n treatment on hearts of r a t s from f i v e month d i a b e t i c animals was then examined. General features o f the f i v e month animals were s i m i l a r to those described i n the ten week r e v e r s a l study. Heart f u n c t i o n a n a l y s i s revealed t h a t while the LVDP and p o s i t i v e dP/dt were only p a r t i a l l y reversed by i n s u l i n treatment, negative dP/dt was completely normalized. These s t u d i e s suggest t h a t the c a r d i a c a l t e r a t i o n s induced by diabetes can be normalized by i n s u l i n i f treatment i s i n i t i a t e d soon a f t e r the onset of diabetes. As diabetes r e s u l t s i n hypothyroidism, we studied the e f f e c t of t h y r o i d replacement therapy on diabetes-induced a l t e r a t i o n s . Treatment of d i a b e t i c animals with T3 (30 yg/kg/day) normalized the t h y r o i d s t a tus of d i a b e t i c animals. However, ca r d i a c f u n c t i o n remained depressed i n the T3 t r e a t e d animals as d i d the calcium uptake i n SR. Thus diabetes-induced myocardial a l t e r a t i o n s do not seem to be a r e s u l t of the hypothyroidism. Our study a l s o shows a c o r r e l a t i o n between the calcium uptake a b i l i t y of SR and i v l e v e l s o f long chain a c y l c a r n i t i n e s i n SR with myocardial f u n c t i o n . The c o r r e l a t i o n s h i p , however, does not n e c e s s a r i l y imply c a u s a l i t y . John H. M c N e i l l , Ph.D. Thesis Supervisor V CONTENTS PAGE ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i INTRODUCTION: A. The disease diabetes m e l l i t u s 1 B. Regulation of myocardial c o n t r a c t i l i t y 7 C. Diabetes and the heart 9 D. Thyroid hormones: Their a s s o c i a t i o n with diabetes m e l l i t u s and heart f u n c t i o n 18 SPECIFIC GOALS OF THE INVESTIGATION 23 MATERIALS AND METHODS: A. M a t e r i a l s 24 B. Methods: I. Preparation of animals 25 I I . I s o l a t e d working perfused heart preparation 27 I I I . Preparation of c a r d i a c microsomes enriched with sarcoplasmic r e t i c u l u m 28 IV. Measurement of calcium uptake by c a r d i a c microsomes enriched i n sarcoplasmic r e t i c u l u m v e s i c l e s 29 V. Determination of long chain a c y l c a r n i t i n e s i n sarcoplasmic r e t i c u l u m 30 VI. P r o t e i n assay 31 VI I . Blood a n a l y s i s 31 V I I I . S t a t i s t i c a l analyses 33 v i PAGE RESULTS 34 DISCUSSION 83 SUMMARY AND CONCLUSIONS 96 REFERENCES 98 v i i LIST OF TABLES PAGE I. E t i o l o g i c a l f a c t o r s involved i n diabetes m e l l i t u s 3 I I . E f f e c t s of i n s u l i n on major t a r g e t t i s s u e s 4 I I I . General features of animals used f o r studying heart f u n c t i o n ( s i x week i n s u l i n prevention study) 41 IV. General features of animals used f o r studying c a r d i a c sarcoplasmic r e t i c u l u m calcium uptake and 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 SR ( s i x week i n s u l i n prevention study) 41 V. General features of animals used f o r studying myocardial f u n c t i o n (ten week i n s u l i n r e v e r s a l study) 54 VI. General features of animals used f o r studying calcium uptake and long chain a c y l c a r n i t i n e s i n SR (ten week i n s u l i n r e v e r s a l study) 54 VII . General features of animals used f o r studying c a r d i a c f u n c t i o n ( s i x month i n s u l i n r e v e r s a l study) 67 V I I I . General c h a r a c t e r i s t i c s of animals used f o r studying heart f u n c t i o n ( s i x week t h y r o i d prevention study) 74 IX. General c h a r a c t e r i s t i c s of animals used f o r measurement of calcium uptake a c t i v i t y i n c a r d i a c SR ( s i x week t h y r o i d prevention study) 74 v i i i LIST OF FIGURES PAGE 1. E f f e c t o f diabetes on free f a t t y a c i d metabolism 6 2. Calcium f l u x e s involved i n myocardial c o n t r a c t i o n s 10 3. E f f e c t of i n s u l i n treatment on diabetes-induced depression of l e f t v e n t r i c u l a r developed pressure (LVDP) s i x weeks a f t e r i n d u c t i o n of diabetes 43 4. E f f e c t of s i x week i n s u l i n treatment on diabetes-induced depression of p o s i t i v e dP/dt 45 5. E f f e c t of s i x week i n s u l i n treatment on diabetes-induced depression of negative dP/dt 47 6. Diabetes-induced depression of calcium uptake i n SR and e f f e c t of i n s u l i n treatment on the depression 49 7. Long chain a c y l c a r n i t i n e l e v e l s i n SR of untreated 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 51 8. Levels of free c a r n i t i n e s i n c a r d i a c SR of i n s u l i n t r e a t e d and untreated d i a b e t i c r a t s 53 9. E f f e c t of i n s u l i n treatment on LVDP i n d i a b e t i c r a t myocardium. I n s u l i n treatment was i n i t i a t e d s i x weeks a f t e r i n d u c t i o n of diabetes 56 10. E f f e c t i v e n e s s of i n s u l i n treatment i n r e v e r s i n g diabetes-induced depression of p o s i t i v e dP/dt 58 11. E f f e c t of i n s u l i n treatment on negative dP/dt i n s i x week d i a b e t i c r a t s 60 12. Diabetes-induced depression of calcium uptake i n the c a r d i a c SR of ten week d i a b e t i c animals and the a b i l i t y of i n s u l i n to reverse the depression 62 i x PAGE 13. Long chain a c y l c a r n i t i n e l e v e l s i n SR of d i a b e t i c r a t hearts and the e f f e c t of i n s u l i n treatment i n d i a b e t i c r a t s 64 14. Levels of free c a r n i t i n e s i n c a r d i a c SR of four week 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 66 15. E f f e c t of i n s u l i n treatment i n i t i a t e d f i v e months a f t e r i n d u c t i o n of diabetes on LVDP 69 16. P o s i t i v e dP/dt i n four week i n s u l i n t r e a t e d f i v e month d i a b e t i c r a t s 71 17. Negative dP/dt i n s i x month d i a b e t i c r a t s and the e f f e c t of four week i n s u l i n treatment i n f i v e month d i a b e t i c r a t s on the parameter 73 18. E f f e c t o f s i x weeks of T3 treatment i n d i a b e t i c r a t s on diabetes-induced depression of LVDP 76 19. Diabetes-induced depression of p o s i t i v e dP/dt and the e f f e c t of T3 treatment on the depression 78 20. E f f e c t of T3 treatment i n d i a b e t i c r a t s on diabetes induced depression of negative dP/dt 80 21. Diabetes-induced depression of calcium uptake i n SR and e f f e c t of i n s u l i n treatment on the depression 82 X ACKNOWLEDGMENT I take t h i s opportunity to express my h e a r t f e l t g r a t i t u d e to my guide Dr. John H. M c N e i l l , Ph.D. whose knowledgeable d i r e c t i o n , sustained i n t e r e s t and patience has enabled me to complete t h i s work. I would al s o l i k e to express s i n c e r e thanks to Mr. Rao Vadlamudi and Mr. Gary Lopaschuk f o r t h e i r genuine i n t e r e s t and encouragement throughout the course of the study. I am g r a t e f u l to Mr. David H a r r i s and Ms. Lynne M a r r i o t t f o r t h e i r i n v a l u a b l e help i n data a c q u i s i t i o n and a n a l y s i s . My thanks are a l s o due to a l l my colleagues and assoc i a t e s who have helped i n no small measure to e x p i d i t e my work. I am e s p e c i a l l y g r a t e f u l to Ms. Judy Wyne f o r t y p i n g t h i s t h e s i s . Arun G. T a h i l i a n i 1. INTRODUCTION A. The disease diabetes m e l l i t u s Diabetes m e l l i t u s has been defined as a d i s o r d e r of metabolism i n which d e f i c i e n c y of i n s u l i n , which may be absolute or r e l a t i v e , leads to a chronic s t a t e of hyperglycemia with or without g l y c o s u r i a . The apparent d e f i c i e n c y of i n s u l i n a c t i o n may be the r e s u l t of inadequate s e c r e t i o n of i n s u l i n from the pancreas or a poor response to endogenous i n s u l i n i n the major t a r g e t t i s s u e s . Various f a c t o r s have been suggested to be involved i n the pathogenesis of the disease. Immuno-l o g i c a l l y , c i r c u l a t i n g i s l e t c e l l a n tibodies which are capable of mediating humoral and c e l l u l a r responses that i n j u r e or destroy pancreatic B - c e l l s are found i n r e a l t i v e l y high t i t r e s i n the i n s u l i n dependent (IDDM) or type I d i a b e t i c s (MacLaren, 1977; Lernmark et a l . , 1978). Epidemiological data from animal models has ass o c i a t e d v i r u s e s with diabetes (Notkins, 1977), the one most c l o s e l y a s s o c i a t e d being Coxsackie B4 v i r u s (Yoon et aJL 1979). There seems to be a p o s i t i v e c o r r e l a t i o n between the disease and h i s t o c o m p a t i b i l i t y antigen genotypes, p a r t i c u l a r l y HLA-B8, BW15 and B18 (Cudworth and Woodrow, 1975). These antigens are determined by l o c i on the short arm of chromosome 6. I t has been suggested that the antigens are determinants of the molecular nature of c e r t a i n 3-cel1 surface p r o t e i n s , some of which may have c e r t a i n unique defects that render the c e l l more s u c e p t i b l e to c e r t a i n i n s u l t s (Cudworth and Woodrow, 1975; Nerup, 1978). On the other hand, i n n o n - i n s u l i n dependent (NIDDM) or Type I I d i a b e t i c s ( p r e v i o u s l y known as maturity onset diabetes m e l l i t u s ) , there seems to be a "down-regulation" of i n s u l i n receptors ( F l i e r 2. et aj_., 1 979) which may be a consequence of higher c i r c u l a t i n g l e v e l s of i n s u l i n (Kahn e_t aj_., 1 977). I n s u l i n r e s i s t a n c e may be due to a decreased a f f i n i t y of the receptors f o r i n s u l i n . For example, i n rare types of diabetes m e l l i t u s which occur i n a s s o c i a t i o n with acanthosis n i g r i c a n s type B ( i n which there i s a c i r c u l a t i n g a n t i r e c e p t o r antibody) and with a t a x i a t e l a n g i e c t a s i a where no antibody i s present but there i s a diminished a f f i n i t y of the receptors f o r i n s u l i n . Bar e_taj_. (1978) suggested that i n s u l i n r e s i s t a n c e could also be due to. a "post receptor d e f e c t " . Obesity seems to r e s u l t i n a s t a t e of peri p h e r a l i n s u l i n r e s i s t a n c e (Ganda and Soeldener, 1977) and i s an important determinant i n NIDDM. In the NIDDM group of d i a b e t i c s , a p o s i t i v e f a m i l y h i s t o r y i s much more frequent than i n the IDDMs and the importance o f he r e d i t y i s f u r t h e r emphasized by studies of t h i s d i s o r d e r i n monozygotic twins ( T a t t e r s a l l and Pyke, 1972). Some other e t i o l o g i c a l concepts involved i n diabetes m e l l i t u s are summarized i n Table I. A t h i r d type of diabetes m e l l i t u s , a l s o known as g e s t a t i o n a l diabetes i s ass o c i a t e d w i t h pregnancy. The term i s r e s t r i c t e d only to those women who become d i a b e t i c during pregnancy and who were not d i a b e t i c p r i o r to i t . As a consequence of diabetes m e l l i t u s a v a r i e t y of p h y s i o l o g i c a l responses are a l t e r e d . On the basis of our knowledge about the e f f e c t s of i n s u l i n on important t a r g e t t i s s u e s (which are summarized i n Table I I ) the abnormalities that occur due to i n s u l i n l ack could be p r e d i c t e d . C l i n i c a l l y , the disease i s c h a r a c t e r i z e d by considerable discomfort and f a t i g u e , weight l o s s , marked t h i r s t , excessive u r i n a t i o n and/or severe s k i n i r r i t a t i o n s such as v a g i n i t i s i n females and b a l a n i t i s 3. TABLE I. ETIOLOGICAL CONCEPTS IN DIABETES MELLITUS FACTOR EFFECT NET RESULT 1. HLA antigens B8, BW15, Bl8 2. V i r a l i n f e c t i o n s 3. Heredity I s l e t c e l l antibody production Beta c e l l d e s t r u c t i o n 4. Obesity 5. A l t e r e d i n s u l i n : glucagon r a t i o Hyperinsulinemia GIucose metabolism Glucose release "Down r e g u l a t i o n " of i n s u l i n receptor Hyperglycemia 6. Presence of serum albumin -synalbumen antagonist (SAA) Glucose uptake by muscle; Glucose uptake by adipose t i s s u e Obesity 7. B i o l o g i c a l d e f e c t i v e i n s u l i n Less uptake of glucose Hyperglycemia 8. Defective adipose l i p a s e Increased l e v e l s of free f a t t y acids I n s u l i n antagonism 9. Accumulation of mucopoly-saccharide i n basement membrane of c a p i l l a r i e s Defective clearance of "Down r e g u l a t i o n " of i n s u l i n from c i r c u l a t i o n i n s u l i n receptor lea d i n g to hyperinsulinemia 4. TABLE I I . EFFECTS OF INSULIN ON MAJOR TARGET TISSUES EFFECT TARGET TISSUE .LIVER MUSCLE ADIPOSE 1. Glycogen synthesis increased increased increased 2. Glucose and amino a c i d uptake 3. P r o t e i n synthesis 4. RNA synthesis 5. Fatty acids and " t r i g l y c e r i d e ' synthesis 6. Glycogenolysis decreased 7. Gluconeogenesis 8. P r o t e i n catabolism decreased and amino a c i d output 9. L i polys i s decreased 5. i n males. The high blood glucose l e v e l s r e s u l t i n elevated l e v e l s of g l y c o s y l a t e d hemoglobins. Since the m o d i f i c a t i o n of . hemoglobin, which i s due to nonenzymatic glycosylation of amino acids i n the hemoglobin, molecule, occurs c o n t i n u a l l y during the l i f e span of the e r y t h r o c y t e , glycohemoglobin l e v e l s can provide a time averaged index of the degree of hyperglycemia during the previous few weeks depending on the species i n c o n s i d e r a t i o n (MacDonald and Davis, 1979). In a d d i t i o n , there are a large number of secondary complications associated with the disease. In extreme forms, k e t o a c i d o s i s r e s u l t s . This i s probably due to a s h i f t i n the u t i l i z a t i o n of free f a t t y acids from synthesis of t r i g l y c e r i d e s and phospholipids to o x i d a t i o n and generation of ketone bodies which may be due to increased a c t i v i t y of the 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 system i n the l i v e r (McGarry and Foster, 1976; 1 977) (Fig;- 1). D i a b e t i c k e t o a c i d o s i s may r e s u l t i n l a c t i c a c i d o s i s (Watkins et aj_., 1 969), a l c o h o l i c k e t o a c i d o s i s ( F u l l o p and Haberman, 1975) and r a r e l y i n cerebral edema (Young and Bradley, 1967). Renal disease i s a s i g n i f i c a n t cause of death i n d i a b e t i c s (Lundbaek, 1965; Balodimos, 1971). The abnormalities so induced include decreased renal f u n c t i o n , glomerular l e s i o n s , l e s i o n s i n the renal v e s s e l s , t u b u l o i n t e r s t i t i a l l e s i o n s and basement membrane changes. Decreased adrenergic nervous a c t i v i t y may occur e v e n t u a l l y r e s u l t i n g i n the syndrome of hyporenemic hypoaldosteronism (Perez et aj_. 1977) which causes hyperkalemia and renal t u b u l a r a c i d o s i s . Retinopathy i s another common mani f e s t a t i o n of diabetes. P r o l i f -e r a t i v e d i a b e t i c retinopathy i s a response of hypoxic r e t i n a r e s u l t i n g i n the formation of a d d i t i o n a l blood vessels and accompanying 6. f r e e f a t t y a c i d s \ f a t t y a c y l c a r n i t i n e m i t r o c h o n d r i a l m e m b r a n e m i t o c h o n d r i a c a r n i t i n e ^ (B o x y g e n a t i o n a c e t y l C o A k e t o n e b o d i e s FIGURE 1: Free f a t t y acids which are normally converted to t r i g l y c e r i d e s and phospholipids, are metabolized to ketones i n the mitochondria during diabetes. I t has been suggested that the s h i f t may p a r t i a l l y be due to an increase i n c a r n i t i n e acyl t r a n s f e r a s e system. 7. fi b r o u s t i s s u e (Davis, 1974). N o n - p r o l i f e r a t i v e d i a b e t i c r e t i n o p a t h y , on the other hand, i s c h a r a c t e r i z e d by increased v a s c u l a r p e r m e a b i l i t y , hemorrhages, microaneurysms, cotton wool spots, hard exudates, venous i r r e g u l a r i t i e s , macular edema and generalized ischemia of the r e t i n a (Davis, 1974). E v e n t u a l l y , ischemia of the macula and o p t i c nerves occurs r e s u l t i n g i n v i s i o n l o s s . Other complications associated with diabetes include hyperosmolar coma without s i g n i f i c a n t k e t o a c i d o s i s ( A r i e f f and C a r o l ! , 1972) u s u a l l y seen i n IDDM i n i n d i v i d u a l s with enough i n s u l i n to c o n t r o l k e t o a c i d o s i s but not enough to c o n t r o l hyperglycemia. Di a b e t i c s seem to be more susceptible to u r i n a r y t r a c t i n f e c t i o n s (Kass, 1956; Ooi et. a l _ . , 1 974); i n f e c t i o n s o f foot u l c e r s (Bessman and Wagner, 1975) and to mucormycosis (Baker, 1971). The incidence of neuropathies i s also much greater i n d i a b e t i c s and these may be e i t h e r metabolic or ischemic and compression type ( S i b l e y , 1982). F i n a l l y , another major complication of diabetes i s the c a r d i o v a s c u l a r disease which w i l l be discussed i n d e t a i l a t a l a t e r stage. B. Regulation of myocardial c o n t r a c t i l i t y Myocardial c o n t r a c t i l i t y i s b a s i c a l l y regulated by two mechanisms. One i s a t o n i c form of c o n t r o l represented by changes i n myosin ATPase a c t i v i t y . In t h i s case, myocardial f u n c t i o n changes evolve over long periods measured i n hours, days or longer. The other, a phasic c o n t r o l mechanism, i s represented by changes i n calcium d e l i v e r y which can vary from one beat to the next. Evidence f o r the involvement of myosin ATPase i n c a r d i a c contrac-8. t i l i t y i s provided by d i f f e r e n t experimental s e t t i n g s . Changes i n endocrine balance, e.g. hypophysectomy or chronic hypothyroidism, in which c a r d i a c f u n c t i o n i s depressed have been c o r r e l a t e d with low l e v e l s of c a r d i a c myosin ATPase a c t i v i t y , whereas i n experimental hyperthyroidism, myosin ATPase a c t i v i t y i s high and myocardial a c t i v i t y i s increased (Thyrum ejt a l _ . , 1970, Goodkind et a l . ,1974'; Korecky et_ a l _ . , 1972). In the aging heart, myosin ATPase a c t i v i t y i s decreased and t h i s decrease i s reported to p a r a l l e l depressed ca r d i a c f u n c t i o n (Katz, 1977). F i n a l l y , hemodynamic overloading r e s u l t s i n an a l t e r a t i o n i n myosin ATPase which i s accompanied by decreased myocardial c o n t r a c t i l i t y (Swynghedauw e_t aj_., 1 976). Thus a l t e r a t i o n of c a r d i a c f u n c t i o n which occurs over longer periods of time seems to r e s u l t , at l e a s t p a r t i a l l y , from an a l t e r a t i o n of myosin ATPase a c t i v i t y . The a l t e r a t i o n could be a r e s u l t of s y n t h e s i s of a new type of myosin with a l t e r e d ATPase a c t i v i t y or from syn t h e s i s of a p r o t e i n which a s s o c i a t e s with myosin le a d i n g to enhanced or diminished ATPase a c t i v i t y depending on the circumstances (Goodkind e t _ a l _ . , 1974; Yazaki and Raben, 1975). The other r e g u l a t o r y f a c t o r , probably operating on a more acute b a s i s , i n v o l v e s calcium. Changes i n the a v a i l a b i l i t y of calcium a v a i l a b l e f o r binding to c o n t r a c t i l e p r oteins have the p o t e n t i a l to modulate myocardial c o n t r a c t i l i t y on a beat-to-beat b a s i s . Normally, as a r e s u l t of e x c i t a t i o n , calcium i s released i n t o _5 the sarcoplasm and the concentration r i s e s to about 10 M. Troponin C binds i o n i c calcium and changes are i n i t i a t e d i n the t r o p o n i n -tropomyosin system r e s u l t i n g i n actin-myosin i n t e r a c t i o n ( P e r r y , 1975). 9. Thus, changes i n the i n t e n s i t y of i n t e r a c t i o n s between c o n t r a c t i l e proteins can be brought about i f the amount of calcium released i s a l t e r e d , i f the binding a b i l i t y of calcium to the troponin complex i s a l t e r e d or i f there i s a change i n the i n t r a c e l l u l a r environment which a l t e r s the i n t e r a c t i o n between c o n t r a c t i l e proteins (Katz, 1977) . The amount of calcium released may vary as a r e s u l t of a l t e r e d a b i l i t y of the s a r c o t u b u l a r network to t r a n s p o r t calcium (Figure 2). A number of i n v e s t i g a t o r s have shown that the calcium t r a n s p o r t i n g a b i l i t y of s u b c e l l u l a r o r g a n e l l e s i s decreased during various types of heart f a i l u r e (Sulakhe and D h a l l a , 1971; Schwartz et aj_., 1973; Sordahl e_t a l _ . , 1 973). A number of f a c t o r s may a f f e c t the a b i l i t y of the sarcotubular network to t r a n s p o r t calcium, one of them being the l e v e l s of long chain acyl c a r n i t i n e s (L.C.A.C.). Elevated l e v e l s of p a l m i t y l c a r n i t i n e , which i s the most abundant of L.C.A.C. has been reported to be a potent time dependent i n h i b i t o r of calcium t r a n s p o r t i n the sarcoplasmic r e t i c u l u m (SR) (Adam et a l _ . , 1978; P i t t s et a l . , 1978) . Thus c a r d i a c c o n t r a c t i l i t y seems to be dependent mainly on two parameters - the a v a i l a b i l i t y of calcium f o r e x c i t a t i o n - c o n t r a c t i o n coupling and a myosin ATPase a c t i v i t y . However, t h i s does not r u l e out the p o s s i b i l i t y of the presence of other biochemical f a c t o r s . C. Diabetes and the heart As mentioned e a r l i e r , one of the major complications of diabetes i s c a r d i o v a s c u l a r disease. There now e x i s t a large number of s t u d i e s 10. EXTRACELLULAR SPACE i •* calcium influx activation Na- Ca exchange ACTIVATOR POOL (subsarcolemmal cisternae ??) INTRACELLULAR POOL (sarcotubular network ??) contraction (positive dP/dt ??) relaxation (negative dP/dt ??) CONTRACTILE ELEMENTS (troponin-tropomyosin system) FIGURE 2: A hyp o t h e t i c a l schemem of calcium f l u x e s i n the car d i a c c e l l , a l t e r a t i o n s i n any of which may lead to a l t e r e d myocardial c o n t r a c t i o n . (Modified from Katz, 1977) 11. which s u b s t a n t i a t e t h i s statement. The Framingham study revealed the development of congestive c a r d i a c disease i n 17% of the d i a b e t i c population compared to 9% i n the t o t a l p opulation. A l s o , deaths due to c a r d i o v a s c u l a r problems i n d i a b e t i c s were t h r i c e as many as those i n the t o t a l population (Garcia ejt aj_., 1974). S i m i l a r l y , the Rochester diabetes p r o j e c t (Palumbo e_t a l _ . , 1981), a study c a r r i e d out by the U n i v e r s i t y Group Diabetes Program (1976) and a r e p o r t by Boyle et al_. (1972) demonstrate the increased incidence of c a r d i o v a s c u l a r problems i n d i a b e t i c s . I t was thought e a r l i e r that diabetes r e s u l t e d i n a t h e r o s c l e r o s i s and that a t h e r o s c l e r o s i s so developed was the cause of c a r d i a c problems i n d i a b e t i c s . Various studies support the hypothesis that diabetes augments a t h e r o s c l e r o s i s to some degree (Robertson and Strong, 1968; M c G i l l , 1968; Sternby, 1968). However, i t had been proposed long before that diabetes a f f e c t s the small and l a r g e blood vessels l e a d i n g to the concept of d i a b e t i c angiopathy (Lundbaek, 1954). I t was then recognized that diabetes a f f e c t e d the small blood vessels and the term " d i a b e t i c microangiopathy" was introduced (Berkman and R i f k i n , 1966). L a t e r , the a s s o c i a t i o n of diabetes with l a r g e vessel disease was recognized, and thus " d i a b e t i c macroangiopathy" emerged (Lundbaek, 1971). The coexistence of diabetes and coronary a r t e r y disease i s very well documented and i t was thought that c a r d i a c problems i n d i a b e t i c p a t i e n t s occured as a r e s u l t of damaged coronary a r t e r i e s . However, V i l h e r t et aJL (1969) reported that the incidence of coronary heart disease was s i m i l a r i n d i a b e t i c and non-diabetic populations. Rubier et a l . (1972) then demonstrated h i s t o l o g i c abnormalities i n 12. d i a b e t i c hearts with normal coronary a r t e r i e s and introduced the concept of " d i a b e t i c cardiomyopathy". Further evidence showing that diabetes-induced c a r d i a c problems were not e x c l u s i v e l y the r e s u l t of coronary abnormalities was provided by Hamby ejt aj_. (1974), where they showed the absence of coronary a r t e r y disease i n a high percentage of d i a b e t i c s with symptoms of heart disease. Since then, a considerable amount of work has been c a r r i e d out towards a b e t t e r understanding of diabetes-induced a l t e r a t i o n s i n the heart i n both c l i n i c a l as well as experimental s e t t i n g s . Another f a c t o r which was thought to be r e s p o n s i b l e f o r c a r d i a c problems i n d i a b e t i c s was autonomic neuropathy. However, u n l i k e coronary a r t e r y disease, the i m p l i c a t i o n s of t h i s d i s o r d e r are as yet c o n t r o v e r s i a l . The neuropathy r e s u l t s i n tachycardia f o r prolonged periods (Keen, 1959); Wheeler and Watkins, 1973). C y c l i c a l heart r a t e v a r i a t i o n which depends on vagal i n n e r v a t i o n i s reduced (Wheeler and Watkins, 1973; Gundersen and Neubauer, 1977). The most s t r i k i n g feature i s postural hypotension which i s probably due to l e s i o n s i n the e f f e r e n t limb of the b a r o r e f l e x arc (Berner, 1952; Odel et a j _ M 1955). Myocardial i n f a r c t i o n i n diabetes has been reported to be p a i n l e s s i n an i n c r e a s i n g number of p a t i e n t s and t h i s has been a t t r i b u t e d to autonomic neuropathy (Bradley and S c h o n f i e l d , 1962; Ewing et aj_. •, 1 976; Clarke et a l _ . , 1979). .'There are a number of consequences r e s u l t i n g from such a b n o r m a l i t i e s but they are only s p e c u l a t i v e and remain to be proved. They include cerebral and p o s s i b l y coronary hypoperfusion as a r e s u l t of postural hypotension. Tachycardia may r e s u l t i n increased heart work, thereby 13. i n c r e a s i n g s u s c e p t i b i l i t y of the heart to c a r d i a c d i s o r d e r s . Damaged sympathetic supply might prevent a compensatory tac h y c a r d i a i n a p a t i e n t with compromised c a r d i a c output due to myocardial n e c r o s i s . F i n a l l y , a f i x e d heart r a t e may reduce e x e r c i s e t o l e r a n c e and might remove one of the mechanisms important to combat heart f a i l u r e (Hampton et aj_., 1981). As mentioned e a r l i e r , ample evidence e x i s t s supporting the concept of d i a b e t i c cardiomyopathy. Regan et aJL (1974) studied v e n t r i c u l a r f u n c t i o n and compliance i n d i a b e t i c dogs. They found that end d i a s t o l i c volume and stroke volume i n the d i a b e t i c hearts were s i g n i f i c a n t l y l e s s than those'nn c o n t r o l s i n response to i n c r e a s i n g a f t e r loads suggesting a decreased.compliance of these hearts. The diminished v e n t r i c u l a r f u n c t i o n was a t t r i b u t e d to increased v e n t r i c u l a r s t i f f n e s s and was apparently due to accumulation of p e r i o d i c a c i d -S c h i f f (PAS) p o s i t i v e g l y c o p r o t e i n i n the myocardium and increased c e l l t r i g l y c e r i d e l e v e l s . Abnormal 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 human d i a b e t i c s has a l s o been demonstrated (Ahmed e_t a l _ . , 1975; Seneviratne, 1977). An experimental model that has been e x t e n s i v e l y s t u d i e d with respect to diabetes-induced myocardial a l t e r a t i o n s i s the d i a b e t i c r a t . Diabetes i s induced using 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 (STZ). In a study on acute (3 day) a l l o x a n d i a b e t i c r a t s , M i l l e r (1979) reported a decreased a b i l i t y of the i s o l a t e d perfused working heart to. respond to increased a t r i a l f i l l i n g pressures at a normal p h y s i o l o g i c glucose concentration of 5 mM. However, when the glucose concentration was increased to 10 mM, c a r d i a c f u n c t i o n was normalized 14. suggesting that the abnormal f u n c t i o n was due to a decreased uptake of glucose by d i a b e t i c hearts r e s u l t i n g i n inadequate q u a n t i t i e s of ATP being generated under working or s t r e s s f u l c o n d i t i o n s . In another study, Fein e t aj_. (1980) examined the e f f e c t s of drug-induced diabetes on mechanics of the i s o l a t e d v e n t r i c u l a r p a p i l l a r y muscle. They observed ab n o r m a l i t i e s i n the r e l a x a t i o n process which included a delayed onset of r e l a x a t i o n , a slower r a t e of r e l a x a t i o n and a delay i n reaching peak r e l a x a t i o n r a t e s . The shortening v e l o c i t y was depressed at various loads, but the peak developed tension remained unchanged. Feuvray e_t aJL (1 979) reported that d i a b e t i c hearts were more s u s c e p t i b l e to severe reductions i n the oxygen supply: demand r a t i o as compared to c o n t r o l s . Using the i s o l a t e d working heart p r e p a r a t i o n , various f u n c t i o n a l a l t e r a t i o n s have been observed (Penparkgul et a l _ . , 1980; Vadlamudi et a l _ . , 1982). Reduction of c a r d i a c output, stroke work, peak l e f t v e n t r i c u l a r developed pressure and p o s i t i v e and negative dP/dt were seen i n d i a b e t i c hearts when they are subjected to higher f i l l i n g pressures. The a b n o r m a l i t i e s i n c a r d i a c f u n c t i o n were reported,to occur at a time point between four weeks and three months of diabetes (Vadlamudi et a l _ . , 1982). S i m i l a r l y , Fein e t al_. (1980) observed that the depression i n p a p i l l a r y muscle mechanics occured about f i v e weeks a f t e r onset of the d i a b e t i c s t a t e . In a recent study, Ku and S e l l e r s (1982) reported t h a t the basal f o r c e of c o n t r a c t i o n of i s o l a t e d e l e c t r i c a l l y d r i v e n a t r i a was lower than the c o n t r o l s and that the magnitude.of the i n o t r o p i c response to ouabain was higher i n d i a b e t i c a t r i a . I t thus seems c l e a r that c a r d i a c f u n c t i o n i s s i g n i f i c a n t l y depressed i n d i a b e t i c rats - the depression occuring four to e i g h t weeks a f t e r the disease 15. i s induced. Using the same model f o r diabe t e s , a number of biochemical studies have been c a r r i e d out f o r a bett e r understanding of the abnormalities at a more basic c e l l u l a r l e v e l . Modrak (1980) studied the c o l l a g e n content i n d i a b e t i c r a t s but d i d not observe any s i g n i f i c a n t change i n twenty-six week d i a b e t i c animals. This i s i n c o n t r a s t to other studies where increases i n c o l l a g e n content have been reported (Regan et aj_., 1 974; Yeh et aj_., 1978) , i n other species. The apparent discrepancy has been suggested to be due to d i f f e r e n c e s i n s p e c i e s , length of diabetes and the type of diabetes produced (Modrak, 1980). As mentioned e a r l i e r , the sarcoplasmic r e t i c u l u m appears to be the most a c t i v e s u b c e l l u l a r o r g a n e l l e involved with s e q u e s t r a t i o n of a c t i v a t o r calcium. Penpargkul et a l . (1979)rreported that the calcium binding and uptake were depressed i n d i a b e t i c r a t hearts and t h i s has since been confirmed by other workers (Lopaschuk et a l _ . , 1983; .^Ganguly et $h..t 1.983). The depression could be a r e s u l t of elevated long chain acyl c a r n i t i n e l e v e l s i n the d i a b e t i c c a r d i a c c y t o s o l (Feuvray e_t al_. ,1 979) which 2+ are known to depress Ca uptake by the SR,J' (Cohen et aj_., 1978; Adam et aj_., 1978). The e l e v a t i o n of long-chain acyl c a r n i t i n e s i n car d i a c SR has r e c e n t l y been confirmed (Lopaschuk et aj_., 1983). High l e v e l s of long-chain acyl c a r n i t i n e s are also known to i n h i b i t Na +, K + - ATPase (Wood et a l _ . , 1 977) and Ca 2 +-ATPase i n the SR (Cohen et aj_., 1978). Biochemical studies i n d i a b e t i c r a t s have demonstrated that actomyosin ATPase i s depressed i n the myocardium from these 2+ animals (Maihotra et a l _ . , 1981). M y o f i b r i l l a r basal (Mg dependent) 16. and Ca - s t i m u l a t e d ATPase a c t i v i t i e s i n d i a b e t i c r a t hearts are reported to be s i g n i f i c a n t l y lower than c o n t r o l s ( P i e r c e and D h a l l a , 1981). S i m i l a r l y , Dillman (1980) and Malhotra et al_. (1981) have observed a depression of c a r d i a c myosin ATPase a c t i v i t y i n the d i a b e t i c r a t heart. In a recent study, Ku and S e l l e r s (1982) reported that Na +, K + ATPase a c t i v i t y , estimated by s p e c i f i c ouabain-s e n s i t i v e 86p,b uptake was s i g n i f i c a n t l y decreased i n four to s i x week d i a b e t i c myocardial v e n t r i c u l a r s l i c e s . I t thus appears that a number of biochemical parameters which are apparently important f o r maintenance of normal myocardial performance are a l t e r e d by the d i a b e t i c s t a t e . Furthermore, there a l s o seems to be a c l o s e p a r a l l e l between these parameters and the f u n c t i o n a l and mechanical parameters with respect to the duration of diabetes a f t e r which the a l t e r a t i o n s are seen. Although a s p e c i f i c cardiomyopathy associated with a lack of i n s u l i n and a reasonable amount of biochemical evidence supporting the presence of the d i s o r d e r was e s t a b l i s h e d , l i t t l e was known about the prevention or r e v e r s a l of these a b n o r m a l i t i e s . Wu e_t aj_. (1 977) studied the e f f e c t s of chronic tolbutamide a d m i n i s t r a t i o n on the 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 dogs. They found that the treatment caused a f u r t h e r reduction 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 and enhanced the v e n t r i c u l a r s t i f f n e s s . However, i n a l a t e r study, Pogatsa et al_. (1978) found that hypoglycemic therapy i n more severely d i a b e t i c dogs co u l d , at l e a s t p a r t i a l l y , normalize the v e n t r i c u l a r s t i f f n e s s . Fein e_t aj_. (1981) used i n s u l i n to c o n t r o l hyperglycemia i n d i a b e t i c r a t s and to study i t s a b i l i t y to reverse the abnormalities 17. induced by diabetes on p a p i l l a r y muscle mechanics. They found that i n s u l i n treatment could completely reverse the diabetes-induced a l t e r a t i o n s when i t was i n i t i a t e d s i x to e i g h t weeks a f t e r i n d u c t i o n of the disease. M i l l e r (1 979) and Penparkgul et al_. (1980) s t u d i e d the e f f e c t s of i n s u l i n p erfusion i n v i t r o on d i a b e t i c r a t hearts. M i l l e r ' (1979) used?three day d i a b e t i c r a t s and was able to reverse the depressed myocardial f u n c t i o n by p e r f u s i n g i n s u l i n through the i s o l a t e d working hearts. However, i n c o n t r a s t , Penparkgul et ajL (1980) perfused hearts from c h r o n i c a l l y d i a b e t i c r a t s with i n s u l i n and were unable to reverse the c a r d i a c a b n o r m a l i t i e s . This suggests that the i n i t i a l cause of diabetes-induced myocardial a l t e r a t i o n s i s probably due to i n s u f f i c i e n t glucose t r a n s p o r t , while chronic hyperglycemia r e s u l t s i n changes at more basic l e v e l s , such as the s u b c e l l u l a r l e v e l . These l a t t e r changes could be reversed only by chronic i n s u l i n therapy as suggested by the study of Fein et_al_. (1981). A number of studies have been c a r r i e d out i n attempts to study the e f f e c t s of i n s u l i n treatment on the biochemical and s u b c e l l u l a r parameters. Dillman (1980) s t u d i e d the e f f e c t o f i n s u l i n treatment on myosin ATPase from hearts of four week d i a b e t i c r a t s . I n s u l i n was i n i t i a t e d four weeks a f t e r i n d u c t i o n of diabetes and was continued f o r another four weeks. This r e s u l t e d i n complete r e v e r s a l of depressed myosin ATPase. S i m i l a r l y , Fein et al_. (1981) s t u d i e d the 2+ 2+ e f f e c t of s i m i l a r i n s u l i n therapy on Ca -actomyosin ATPase and Ca -myosin ATPase a c t i v i t i e s . While only trends of r e v e r s a l were seen s i x and ten days a f t e r i n i t i a t i n g i n s u l i n therapy, complete r e v e r s a l 18. was seen by twenty-eight days of treatment. Fein ejt aj_. (1981) a l s o found that i n s u l i n therapy was capable of r e v e r s i n g diabetes-induced depression of actomyosin and myosin ATPase a c t i v i t i e s . In a recent study, Ku and S e l l e r s (1982) reported the a b i l i t y of i n s u l i n to reverse depressed c a r d i a c Na +, K +-ATPase a c t i v i t y as well as myocardial force of c o n t r a c t i o n . H i s t o l o g i c a l s tudies have demonstrated that i n s u l i n treatment can prevent the diabetes-induced increase i n connective t i s s u e (Baandrup e_t a l _ . , 1980). I t thus appears that diabetes induces a s p e c i f i c type of c a r d i o -myopathy which r e s u l t s i n a l t e r e d c a r d i a c muscle mechanics and ev e n t u a l l y decreases c a r d i a c f u n c t i o n . The biochemical basis f o r such a l t e r a t i o n s could be depressed enzyme systems and a l t e r e d s u b c e l l -u l a r o r g a n e l l e f u n c t i o n . The abnormal enzyme a c t i v i t i e s could e i t h e r be a r e s u l t of synthesis of enzymes with lower a c t i v i t i e s , or a r e s u l t of an a l t e r e d environment i n which the enzymes cannot f u n c t i o n properly. Furthermore, i n s u l i n treatment seems to be e f f e c t i v e i n normalizing the mechanical and some biochemical parameters. This could again be a r e s u l t of e i t h e r normal f u n c t i o n due to a normal environment or due to synthesis of enzymes with normal a c t i v i t i e s . The l a t t e r seems to be a more probable hypothesis as i n s u l i n therapy could reverse the a l t e r a t i o n s only a f t e r about four weeks of treatment. D. Thyroid hormones: Their a s s o c i a t i o n with diabetes m e l l i t u s and heart f u n c t i o n . Thyroid hormones are synthesized i n the t h y r o i d gland v i a a s e r i e s of complex steps. 3, 5, 3" t r i i o d o t h r o n i n e (T3) i s g e n e r a l l y 19. estimated to be nearly four times as potent as thyroxine and the l a t t e r u s u a l l y predominates by a f a c t o r of several f o l d . However, i n case of iodi d e d e f i c i e n c y , the r a t i o i s nearly reversed (Greer et aj_., 1968). Thyroxine i s degraded v i a the monodeiodination pathway to give r i s e to e i t h e r T3 or 3, 3', 5 ' - t r i i o d o t h y r o n i n e (.'reverse' or 'R' T3) (Braverman et aj_., 1970; Gavin et a^., 1 977). D i f f e r e n t d e b i l i t a t i n g d isorders are associated with reduced serum T3 and elevated RT3 l e v e l s due to changes i n thyroxine metabolism (Suda et a l _ . , 1 977; Chopra and Smith, 1975; Burger et al_. 1 976). Pittman e_t aj_. (1979) have shown that production of T3 i s s i g n i f i c a n t l y impaired i n diabetes m e l l i t u s . Various suggestions regarding the mechanism of such reduction have been forwarded. D e f i c i e n t t h y r o t r o p i n (TSH) s e c r e t i o n has been demonstrated i n d i a b e t i c animals during a low iodine d i e t regime (Pericas and J o l i n , 1977). More r e c e n t l y , Wilber et ajl_. (1981) demonstrated that diabetes m e l l i t u s i s a s s o c i a t e d with a reduction i n c i r c u l a t i n g t h y r o t r o p i n r e l e a s i n g hormone (TRH). Reduction of T3 l e v e l s during diabetes has a l s o been a t t r i b u t e d to reduced e x t r a t h y r o i d a l conversion of thyronine to T3 (Pittman e_t a l _ . , 1979; Balsam et_ a l _ . , 1 978). This may be due to diminution i n hepatic 5'/monodeiodinase- r e s u l t i n g i n depressed d e i o d i n a t i o n r e a c t i o n s (Chopra et a l . , 1981). While i t i s c l e a r from the above d i s c u s s i o n that diabetes causes t h y r o i d hormone a l t e r a t i o n s , the converse too seems to be t r u e . Hypothyroidism r e s u l t s i n impairment of e a r l y phase i n s u l i n r e l e a s e s i m i l a r to that seen i n NIDDM (Shah and Cerchio, 1973). Besides t h i s carbohydrate homeostasis i s in f l u e n c e d by t h y r o i d hormones 20. (Burman et aj_., 1980). The c o r r e l a t i o n of diabetes with t h y r o i d status i s f u r t h e r supported by studies which demonstrate that diabetes-induced reduction of t h y r o i d hormones can be normalized by i n s u l i n a d m i n i s t r a t i o n ( S e r i f and Sihotang, 1962; Kumaresan and Turner, 1966). Thyroid hormones have a profound e f f e c t on the mammalian ca r d i o v a s c u l a r system. Various parameters are a f f e c t e d by a l t e r e d t h y r o i d hormone l e v e l s r e s u l t i n g i n c a r d i a c complications i n otherwise healthy hearts (Sandler and Wilson, 1959). D i f f e r e n t myocardial enzyme systems seem to be a f f e c t e d and t h i s probably leads to a l t e r e d c a r d i a c c o n t r a c t i l i t y . A d d i t i o n of thyroxine to a p a r t i c u l a t e preparation o f the cat l e f t v e n t r i c l e r e s u l t e d i n s t i m u l a t i o n of adenylate c y c l a s e (Levey and E p s t e i n , 1968). However, a number of other reports suggest that the adenylate c y c l a s e - c y c l i c AMP system may not be very c r u c i a l i n determining the c a r d i o t o n i c e f f e c t s of t h y r o i d hormones (Sobel et a_l_., 1 969; McNeill ejt aj_. 1969; M c N e i l l , 1978). Another enzyme system which i s a c t i v a t e d by the hormones and could be important f o r cardiac c o n t r a c t i l i t y i s the phosphorylase system. The enhanced a c t i v i t y of ca r d i a c phosphorylase a_ i n hyper-t h y r o i d i s m i s not accompanied by increases i n c y c l i c AMP l e v e l s (McNeill ejt aji_., 1 969) or by enhanced phosphorylase b_ kinase a c t i v i t y (Frazer et_ al_. 1969) I t i s not a r e s u l t of increased s e n s i t i v i t y of adenylate c y c l a s e (McNeill et aj_,, 1 969) and i s independent of increased coronary blood flow observed during hyperthyroidism (Longhurst and M c N e i l l , 1979). I t i s not c l e a r as yet as to how t h y r o i d hormone excess r e s u l t s i n an a c t i v a t i o n of phosphorylase. 21. The p o s i t i v e inotropy a s s o c i a t e d with hyperthyroidism could a l s o , at l e a s t i n p a r t , be due to the e f f e c t s of t h y r o i d hormones on myocardial ATPase such as myosin and actomyosin ATPases, Na +, + 2+ 2+ K -ATPase and Mg dependent Ca ATPase. The a s s o c i a t i o n of some of these ATPases with c a r d i a c c o n t r a c t i l i t y i s discussed at a l a t e r stage. Evidence suggests that c a r d i a c c o n t r a c t i l i t y depends on the s t a t e of the ATPases. Calcium s t i m u l a t e d myosin ATPase has been shown to be elevated during hyperthyroidism (Thyrum e_t a l _ . , 1 970). Various studies suggest t h a t the enhanced a c t i v i t y seen i s probably a r e s u l t of synthesis of a new type of myosin, or synthesis of a new p r o t e i n which ass o c i a t e s with myosin leading to greater a c t i n a c t i v a t e d ATPase a c t i v i t y (Goodkind et a l . , 1974; Yazaki and Raben, 1975; Thrum et aV., 1970). However, the a c t i v i t y of t h i s enzyme i s extremely species dependent with r a b b i t s and guinea pigs e x h i b i t i n g low basal a c t i v i t y . Rats, on the other hand have high basal a c t i v i t y i n the euthyroid s t a t e and t h i s may e x p l a i n why studies i n r a t s have been unable to demonstrate augmentation of myosin ATPase i n hyper-t h y r o i d animals (Rovetto et aj_., 1972; Yazaki and Raben, 1975). The e f f e c t of t h y r o i d hormone on calcium ATPase has been su b s t a n t i a t e d i n a number of s t u d i e s . An enhancement of calcium ATPase a c t i v i t y has been reported (Limas, 1978). McConnaughey et a l . ( 1 9 7 8 ) , however, were unable to show any increase i n a c t i v i t y of t h i s enzyme i n thyroxine t r e a t e d r a t s . Most s t u d i e s , i n which thyroxine treatment was c a r r i e d out f o r longer periods of time (about 10 days) demonstrate increased a c t i v i t y , while most others i n which the treatment was f o r s h o r t e r periods (3-5 days) were unable to 22. demonstrate s i g n i f i c a n t increases i n the a c t i v i t y of Ca ATPase. This suggests that the enhanced a c t i v i t y i s probably due to synthesis of e i t h e r a new, more a c t i v e enzyme or of greater amounts of the same enzyme which i s apparent only a f t e r chronic treatment with t h y r o i d hormone. I t thus appears that myocardial c o n t r a c t i l i t y depends, at l e a s t i n p a r t , on the t h y r o i d status of the animal. As myocardial c o n t r a c t i l i t y and t h y r o i d hormone l e v e l s are depressed i n di a b e t e s , we looked a t myocardial c o n t r a c t i l i t y i n d i a b e t i c r a t s a f t e r t r e a t i n g them with t h y r o i d hormone i n an attempt to make them e u t h y r o i d . 23. SPECIFIC GOALS OF THE PRESENT INVESTIGATION This study was designed on the f o l l o w i n g l i n e s : 1. To l o c a t e a time point at which myocardial a l t e r a t i o n s are induced by experimental diabetes. 2. To study the a b i l i t y of i n s u l i n to prevent the diabetes-induced myocardial changes, both f u n c t i o n a l and biochemical, from o c c u r i n g . 3. To study the a b i l i t y of i n s u l i n to. reverse the depression of myocardial f u n c t i o n and the calcium uptake i n SR as well as the a b i l i t y of i n s u l i n treatment to reverse e l e v a t i o n of long chain a c y l c a r n i t i n e s i n SR. The r e v e r s a l study was c a r r i e d out s i x weeks and f i v e months a f t e r i n d u c t i o n of d i a b e t e s , i n an attempt to l o c a t e a point by which the diabetes-induced changes become i r r e v e r s i b l e . 4. To examine whether the diabetes-induced myocardial changes are d i r e c t or whether they are a r e s u l t of diabetes-induced . hypothyroidism. This was done by t r e a t i n g the d i a b e t i c animals with T3 and then studying the f u n c t i o n . Treatment was i n i t i a t e d as soon as diabetes was detected i . e . about 3 days a f t e r the i n j e c t i o n of s t r e p t o z o t o c i n . 24. MATERIALS AND METHODS A) MATERIALS ( i ) Radioisotopes: 4 5 C a C l 2 (10 Ci/ymole), a 3 2P-ATP (16.4 Ci/ymole) and (1 - 1 4 C ) ac e t y l CoA were purchased from Amersham Radiochemicals (Toronto, O n t a r i o ) . ( i i ) Enzyme: 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 was purchased from Sigma Chemical Co. ( i i i ) Chemicals: The f o l l o w i n g chemicals were purchased from Sigma Chemical Co.: Trizma adenosine triphosphate ( T r i s - A T P ) , 1 x 8-400 Dowex - 1 Anion Exchange Resin, Bovine Serum Albumin, L - h i s t i d i n e free 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 , sucrose, Trizma base, Trizma HC1, T r i t o n X-100, Trizma m a l e a t e , d i t h i o t h e r e t o l , D , 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 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-hydroxymethylfurfuraldehyde, t r i c h l o r o a c e t i c a c i d , sodium f l u o r i d e , potassium hydronide, copper s u l f a t e , deoxycholate, potassium phosphate, 3, 5, 3' t r i i o d o t h y r o n i n e . Calcium c h l o r i d e d i h y d r a t e , L-ascorbate, Trizma o x a l a t e , sodium carbonate, D-glucose, sodium bicarbonate and c i t r i c a c i d were purchased from Avalar Chemicals. Sodium t e t r a t h i o n a t e was purchased from Pierce Chemical Co. Aquasol s c i n t i l l a t i o n f l u i d was purchased from New England Nuclear. PZI i n s u l i n (100 y/ml) was purchased from Connaught L a b o r a t o r i e s . 25. ( i v ) Apparatus: TM R A Glucometer and D e x t r o s t i x were purchased from Ames Co., ( E l k h a r t , Indiana). (v) Assay k i t s : Glucose assay k i t s were purchased from Sigma Chemicals. I n s u l i n assay k i t s were obtained from Becton Dickinson. Thyroid assay k i t s were obtained from Abbott L a b o r a t o r i e s . ( v i ) Animals: Female Wistar r a t s (Canadian Breeding Farms, Montreal) weighing 150-175 g were used throughout the study. They received food and water ad_ 1 ibitum. B) METHODS I Preparation of animals; a) Induction of diabetes: The ra t s were made d i a b e t i c by a s i n g l e intravenous i n j e c t i o n of s t r e p t o z o t o c i n (STZ) (55 mg/kg) di s s o l v e d i n 0.1 M c i t r a t e b u f f e r (pH 4.5). Control r a t s were i n j e c t e d with the buffe r alone. The onset of diabetes was assessed by t e s t i n g f o r urine glucose using TesTape , and s e v e r i t y of the disease was estimated by measuring non-fasting blood glucose concentrations (blood being obtained from the t a i l vein during ether anesthesia) using D e x t r o s t i x and a Glucometer. b) Treatment p r o t o c o l s : ( i ) s i x week study: Having a s c e r t a i n e d that the animals were d i a b e t i c , t h e i r blood glucose l e v e l s were measured on the t h i r d 26. day a f t e r i n j e c t i n g them with STZ. Those with blood glucose values >300 mg % were subdivided i n t o two weight-matched groups. In one study, one group of animals received protamine z i n c i n s u l i n (PZI) subcutaneously once d a i l y , while the other was l e f t untreated. Blood glucose was monitored using a D e x t r o s t i x and a Glucometer once every two days i n the f i r s t week, a f t e r which i t was monitored every week. The dose of i n s u l i n was adjusted i n order to achieve blood glucose l e v e l s i n the range of 100-200 mg %. The approximate dose of i n s u l i n used was 0.9 U/100 g body weight/day. In another study, one group of d i a b e t i c animals was t r e a t e d with t r i i o d o t h y r o n i n e (T3) i n . a l k a l i n e s o l u t i o n (30 yg/kg/day s . c ) . In t h i s l a t t e r study, one group of c o n t r o l s was a l s o t r e a t e d with T3. Treatment i n e i t h e r case was c a r r i e d out f o r s i x weeks a f t e r which period the animals were s a c r i f i c e d . Hearts were excised and e i t h e r perfused f o r f u n c t i o n studies or used f o r the preparation of sarcoplasmic r e t i c u l u m (SR). Blood was c o l l e c t e d and the plasma was analyzed f o r glucose, i n s u l i n , g l y c o s y l a t e d hemoglobin and T3 l e v e l s . ( i i ) Ten week study: In t h i s case, the d i a b e t i c animals were subdivided i n t o two groups s i x weeks a f t e r i n j e c t i n g STZ. One group received PZI as i n the above protocol f o r four weeks. Blood glucose determinations were made three times i n the f i r s t week of treatment and once each week t h e r e a f t e r . In e f f e c t , the animals were s a c r i f i c e d ten weeks a f t e r i n d u c t i o n of diabetes. The hearts were used f o r e i t h e r f u n c t i o n or biochemical s t u d i e s as p r e v i o u s l y described and blood was c o l l e c t e d and used f o r the above mentioned analyses. ( i i i ) S i x month study: D i a b e t i c animals were subdivided f i v e months a f t e r they were made d i a b e t i c . One subgroup was t r e a t e d with 27. PZI i n a manner i d e n t i c a l to the protocol described i n the "ten week study". In t h i s study the r a t s were s a c r i f i c e d s i x months a f t e r they were made d i a b e t i c and the hearts perfused f o r f u n c t i o n s t u d i e s . II I s o l a t e d working perfused heart preparation. The method used f o r perfusing hearts i s a m o d i f i c a t i o n of Neely's working heart p r e p a r a t i o n , as described by Rodgers et aj_. (1981). The perfusion f l u i d used was Chenoweth-Koelle (1946) b u f f e r which has the f o l l o w i n g composition (mM concentrations of s o l u t e s ) : NaCl, 120: KC1, 5.6: CaCT 2, .2.18; MgCl 2, 2.1; NaHC0 3 > 19; glucose, 10 and EDTA 0.03. The b u f f e r was oxygenated continuously with a 95% 0£ and 5% CO2 mixture and was maintained at 37°±1°C (pH 7.4). A 3 cm piece of PE 90 tubing was attached on one side to a Statham P23AA transducer (Gould Statham Instruments) and to a 20 gauge needle on the other. L e f t v e n t r i c u l a r developed pressue (LVDP) was measured by i n s e r t i n g the needle through the apex of the heart i n t o the l e f t v e n t r i c l e . I n t r a -a o r t i c pressure was measured by means of another Statham P23AA t r a n s -ducer connected to a side arm of the a o r t i c outflow system. LVDP, i n t r a a o r t i c pressure and the f i r s t d e r i v a t i v e of LVDP were recorded on a Grass Model 79D polygraph. The hearts were st i m u l a t e d by means of a platinum e l e c t r o d e placed on the l e f t atrium at twice the threshold voltage with square pulses of 5 msec duration from a Grass Model SD9D s t i m u l a t o r to give a rate of 300 beats/min. The data were c o l l e c t e d and analyzed with a microcomputer according to a method by H a r r i s et a l . (1983) (Apple 11+ with a Mountain hardware board). The pressure transducer s i g n a l from the polygraph was sampled at 667 Hz over 1.5 28. seconds at each point where data were recorded. S i x complete c a r d i a c pulses were thus obtained and analyzed using various curve f i t t i n g techniques to determine pulse height, slopes of the curves, area, s t a r t , f i n i s h , e t c . Values from three pulses were averaged to produce data values at each p o i n t . Function curves were performed on each heart a f t e r i t had e q u i l i b r a t e d f o r about 5-7 minutes at an a t r i a l f i l l i n g pressure of 15 cm H^O. Studies were performed by varying 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 (and thus varying the l e f t a t r i a l f i l l i n g pressure) from 5 cm iSCO to 22.5 cm H^O i n 2.5 cm steps. Reservoir height was a l t e r e d stepwise from 15 cm H^O to 5 cm H^O, increased to 22.5 cm F ^ O and f i n a l l y reduced to 15 cm F^O, data being recorded at each point a f t e r pressure development s t a b i l i z e d . A complete f u n c t i o n curve took about 30 mins to perform, the t o t a l perfusion time being 35 to 45 minutes. At the end of the f u n c t i o n curve, wet v e n t r i c l e weight was recorded. I l l Preparation of c a r d i a c microsomes enriched with sarcoplasmic Reticulum. This preparation was made by a m o d i f i c a t i o n of the method of Sumida et al_. (1978). Hearts were excised and placed i n c o l d 10 mM T r i s maleate buf f e r (pH 6.8). The v e n t r i c l e s were separated, weighed and homogenized i n about 15 ml of the above b u f f e r with a t e f l o n p e s t l e f o r 15 seconds at 1500 rpm. The r e s u l t i n g homogenate was D placed i n a Korex tube and c e n t r i f u g e d at 4000 times g f o r 10 minutes. 29. The supernatent was passed through four l a y e r s of cheesecloth and c e n t r i f u g e d at 9000 times g f o r twenty minutes. Supernatent was again passed through four l a y e r s of cheese c l o t h and c e n t r i f u g e d at 40,000 times g f o r 100 minutes. The r e s u l t i n g p e l l e t was resuspended in a 10 mM T r i s maleate buff e r c o n t a i n i n g 0.6 M KC1 and c e n t r i f u g e d . at 40,000 times g f o r 100 minutes. The supernatent was discarded and the p e l l e t gently washed with 10 mM T r i s maleate b u f f e r . I t was then resuspended i n 10 mM T r i s Maleate b u f f e r c o n t a i n i n g 40% sucrose, quick frozen i n acetone on dry i c e and stored at -70°C u n t i l f u r t h e r use. IV Measurement of calcium uptake by ca r d i a c microsomes enriched i n sarcoplasmic r e t i c u l u m v e s i c l e s . a) Measurement of calcium t r a n s p o r t a c t i v i t y i n the SR: ATP-dependent calcium uptake a c t i v i t y was measured by the method of Tada et aj_. (1974) with a few m o d i f i c a t i o n s . The incubation medium contained 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 MgCl2» 110 mM KC1, 5 mM Tris-ATP, 2.5 mM T r i s - o x a l a t e and d i f f e r e n t concentrations of 45 CaCl2 c o n t a i n i n g CaC^. About 50 yg of microsomal sarcoplasmic r e t i c u l u m was used at each calcium c o n c e n t r a t i o n . The desired free calcium concentrations were obtained by adding ethylene g l y c o l T r i s (-aminoethyl ether) - N,N'-tetraacetate (EGTA) to a C a C l 2 s o l u t i o n , the free calcium being c a l c u l a t e d by the equation of Katz et a l . (1970). 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 Co.). The f i l t e r was then washed with about 20 ml of 40 mM T r i s c h l o r i d e , pH 7.2, d r i e d and counted f o r r a d i o a c t i v i t y i n Aquasol using standard 30. l i q u i d s c i n t i l l a t i o n counting techniques. The r a t e of C a C T uptake 2+ by the microsomal preparation i s expressed as nmoles Ca taken up per mg p r o t e i n per minute. V Determination of long chain a c y l c a r n i t i n e l e v e l s i n SR. An a l i q u o t of the microsomal suspension c o n t a i n i n g about 0.5 mg p r o t e i n was d i l u t e d to 4 ml with 10 mM T r i s maleate and c e n t r i f u g e d at 40,000 x g f o r 45 minutes. The p e l l e t was resuspended i n 0.5 ml of c o l d 6% (0.6 y l ) p e r c b l ; o r i C i a c i d . The suspension was c e n t r i f u g e d at 12,000 x g f o r 10 minutes. To a 200 y l a l i q u o t of the supernatent, 150 y l of a 2 M T r i s base s o l u t i o n was added. 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 s o l u t i o n , suspended i n 100 y l d i s t i l l e d water and subjected to a l k a l i n e h y d r o l y s i s by incubating i t with 100 y l of 1 1 T r i s base and 50 y l of 0.4N- KOH at 70° C f o r 1 hour. Following i n c u b a t i o n , 0.575N HC1 (200 y l ) was added to n e u t r a l i z e the base. The actual determination of c a r n i t i n e was done according to a r a d i o i s o t o p i c procedure developed by McGarry and Foster (1976). A medium c o n t a i n i n g 120 yM T r i s c h l o r i d e (pH 7.3), 2 yM sodium t e t r a -thionate and 25 nmoles (0.025 yCi) of 1-^4C a c e t y l coenzyme A was prepared and 1.05 ml added to each of the supernatent or hydrolyzed samples c o n t a i n i n g c a r n i t i n e . Free c a r n i t i n e i n the sample was converted to l a b e l l e d a c e t y l c a r n i t i n e by the a d d i t i o n of 0.5 U of the enzyme 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 . The r e a c t i o n mixture was incubated at room temperature f o r 30 minutes. To t h i s was added 0.3 ml of Dower 1x8-400 anion exchange r e s i n (200-400 mesh i n c h l o r i d e form) suspension 31. (c o n t a i n i n g 0.08 ml of the r e s i n and 0.22 ml of water) and the sample was 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 x g f o r 5 minutes. To an 0.7 ml a l i q u o t of the supernatent, 10 ml of aquasol was added and the sample counted using standard s c i n t i l l a t i o n techniques. Levels of free 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 expressed as nmoles/mg p r o t e i n sarcoplasmic r e t i c u l u m . VI P r o t e i n assay. To a 20 ul. a l i q u o t of the microsomal sample, d i s t i l l e d water was added. Ten til of 2% deoxycholate s o l u t i o n were added to the suspension and a f t e r 10 minutes of i n c u b a t i o n , 0.5 ml 24% t r i c h l o r o a c e t i c a c i d were added to p r e c i p i t a t e the p r o t e i n s . The tubes were c e n t r i f u g e d at 3000 x g f o r 30 minutes and the p r e c i p i t a t e d p r o t e i n assayed using the standard Lowry (1951) p r o t e i n assay. Bovine serum albumin was used as a standard. VII Blood a n a l y s i s . ( i ) G lycosylated hemoglobin i n blood was assayed by the method of Subramanian et al_. (1980). C e l l s obtained a f t e r separation of plasma were washed t h r i c e with s a l i n e at 4°C and were then hemolysed with d i s t i l l e d water. To the r e s u l t a n t supernatent, a c i d i f i e d acetone was added to separate out the g l o b i n . The g l o b i n p e l l e t obtained by c e n t r i f u g i n g the suspension was washed once with a c i d i f i e d acetone and then with acetone to remove the a c i d . F i n a l l y , the 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 . Globin thus 32. obtained was stored at -20°C u n t i l the assay was c a r r i e d out. Globin was assayed by d i g e s t i n g 75 mg with 4 ml 10 M a c e t i c a c i d f o r 16 hours at 80°C. To a 1 ml a l i q u o t of the dig e s t , 1 ml of .2% t r i c h l o r o a c e t i c a c i d was added and the mixture allowed to s i t f o r 5 minutes. The suspension was c e n t r i f u g e d and t h i o b a r b i t u r a t e reagent was added to the p e l l e t . The r e a c t i o n mixture was incubated f o r 50 minutes a t 40°C and the absorbance measured at 443 nM. Hydroxymethylfurfuraldehyde (HMF) was used as a standard. The degree of g l y c o s y l a t i o n i s expressed as ymol HMF/g g l o b i n p r o t e i n . ( i i ) Plasma immunoreactive i n s u l i n was determined by the r a d i o -immunoassay method of Herbert et al_. (1965). Reagents f o r the assay were obtained from Becton Dickinson and Co. The i n s u l i n assay reagents 1 2 5 consisted of human i n s u l i n standards. [ I] porcine i n s u l i n and an i n s u l i n antibody r a i s e d a gainst porcine i n s u l i n , f o r which human i n s u l i n e x h i b i t s 100% and r a t i n s u l i n 90% cross r e a c t i v i t y . Human i n s u l i n standards were used since the e r r o r made i n the est i m a t i o n of r a t i n s u l i n l e v e l s was minimal owing to the very high cross r e a c t i v i t y with r a t i n s u l i n . ( i i i ) Plasma glucose l e v e l s were determined by the glucose oxidase method using a Sigma glucose k i t . ( i v ) Thyroid status of the r a t s was determined by measuring t h e i r T3B index which shows how much excess binding c a p a c i t y the serum thyroxine binding g l o b u l i n has a v a i l a b l e . The T3B index was determined using a Triobead - 125 T3 uptake k i t which was obtained from Abbott Laboratories. 33. VII S t a t i s t i c a l analyses. A l l r e s u l t s f o r any p a r t i c u l a r group were averaged and the standard e r r o r of the mean (S.E.M.) determined at each experimental p o i n t . One way a n a l y s i s of variance (ANOVA) followed by Newman-Keuls t e s t was used f o r comparing r e s u l t s from a given set of groups. A p r o b a b i l i t y of l e s s than 0.05 (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 . 34. RESULTS E f f e c t of diabetes on myocardial f u n c t i o n , calcium uptake a c t i v i t y of c a r d i a c SR and c a r n i t i n e l e v e l s of c a r d i a c SR i n r a t s . The general features of d i a b e t i c r a t s were qu i t e c h a r a c t e r i s t i c of the disease and are summarized i n Tables 111-1X. Di a b e t i c animals weighed s i g n i f i c a n t l y l e s s than the buffe r i n j e c t e d c o n t r o l s and the plasma glucose l e v e l s of d i a b e t i c r a t s were exceedingly high (between 300% and 500% over c o n t r o l ) . Other features e x h i b i t e d by d i a b e t i c r a t s were low i n s u l i n l e v e l s ( i n most cases l e s s than 50% of c o n t r o l ) and elevated g l y c o s y l a t e d hemoglobin l e v e l s . Upon s a c r i f i c e , i t was found that d i a b e t i c r a t hearts were smaller and weighed l e s s than c o n t r o l s , but i n mose cases the heart weight/body weight r a t i o s were s i g n i f i c a n t l y greater than c o n t r o l s . In a d d i t i o n , i t was observed that d i a b e t i c animals e x h i b i t e d p o l y d i p s i a and p o l y u r i a . I t was thus ascertained that the animals were indeed moderately to severely d i a b e t i c . M o r t a l i t y r a t e w i t h i n the d i a b e t i c groups was very low (about 2%). When the hearts were i s o l a t e d and perfused i n the working heart mode, i t was observed that by s i x weeks of diabe t e s , the f u n c t i o n a l parameters measured were s i g n i f i c a n t l y depressed i n r e l a t i o n to c o n t r o l s . L e f t v e n t r i c u l a r developed pressure (LVDP), rat e of pressure development (+dP/dt) and rate of pressure d e c l i n e (-dP/dt) were found to be s i g n i f i -c a n t l y lower than c o n t r o l s at f i l l i n g pressures greater than 17.5 cm H^ O (Figures 3, 4, 5). Previous studies have suggested that myocardial changes induced by s t r e p t o z o t o c i n diabetes i n r a t s occurred between four weeks and twelve weeks a f t e r i n j e c t i o n of the drug (Vadlamudi et a l . 35. 1982). From our study i t seems that changes i n myocardial f u n c t i o n set i n by s i x weeks of diabetes. Results s i m i l a r to these were obtained when longer durations of diabetes were s t u d i e d . Hearts obtained from r a t s d i a b e t i c f o r ten weeks als o e x h i b i t e d depressed c a r d i a c f u n c t i o n . The LVDP was s i g n i f i c a n t l y depressed at higher f i l l i n g pressures, namely, those above 17.5 cm HgO (Figure 9). S i m i l a r l y , p o s i t i v e dP/dt was depressed i n the d i a b e t i c hearts at f i l l i n g pressures greater than 12.5 cm H^O (Figure 10). Negative dP/dt was also depressed at higher f i l l i n g pressures (Figure 11). The depression, of these parameters i n ten week d i a b e t i c animals d i d not appear to be any worse than the depression seen i n s i x week d i a b e t i c animals. However, i n the ten week d i a b e t i c r a t h e a r t s , depression of p o s i t i v e and negative dP/dt seemed to occur at r e l a t i v e l y lower f i l l i n g pressures. In another set of experiments we studi e d myocardial f u n c t i o n o f rats s i x months a f t e r they were i n j e c t e d with s t r e p t o z o t o c i n . As i n the above s t u d i e s , depression of the parameters studied was observed. LVDP was depressed a t f i l l i n g pressures greater than 17.5 cm H2O (Figure 15). P o s i t i v e dP/dt was al s o depressed at f i l l i n g pressures greater than 17.5 cm H^ O (Figure 16) while negative dP/dt was depressed at f i l l i n g pressures greater than 15 cm H^ O (Figure 17). The values of LVDP, p o s i t i v e dP/dt and negative dP/dt obtained i n s i x month co n t r o l hearts were lower than those obtained i n e i t h e r s i x week or ten week c o n t r o l s . However, f u n c t i o n of s i x month d i a b e t i c hearts did not appear to be more depressed than the s i x week or ten week d i a b e t i c hearts. 36. Calcium uptake a c t i v i t y i n ca r d i a c SR was a f f e c t e d s i g n i f i c a n t l y by diabetes. S i x weeks and ten weeks of diabetes a f f e c t e d the calcium t r a n s p o r t i n g a b i l i t y of SR at a l l calcium concentrations used (Figures 6 and 12). A s i g n i f i c a n t e l e v a t i o n of long chain a c y l c a r n i t i n e l e v e l s i n the SR which p a r a l l e l e d the depression of calcium uptake was seen both s i x weeks (Figure 7) and ten weeks (Figure 13) a f t e r the ind u c t i o n of diabetes. However, the free c a r n i t i n e l e v e l s were not a f f e c t e d by diabetes a t e i t h e r of the time points studies (Figures 8 and 14). E f f e c t s of i n s u l i n treatment on diabetes-induced a l t e r a t i o n o f myocardial f u n c t i o n and biochemistry. a) S i x week prevention study. I n s u l i n treatment i n a subgroup of d i a b e t i c r a t s was i n i t i a t e d three days a f t e r STZ i n j e c t i o n . These rats d i d not los e weight, had plasma glucose values not s i g n i f i c a n t l y higher than c o n t r o l s , and were normoinsulinemic (Tables I I I and IV). G l y c o s y l a t i o n of hemoglobin was normal and they d i d not e x h i b i t p o l y d i p s i a or p o l y u r i a . There were no m o r t a l i t i e s during the course of treatment. I t thus seemed that t h i s group of d i a b e t i c r a t s was r e c e i v i n g adequate i n s u l i n treatment. As mentioned above, s i x weeks of diabetes were found to be s u f f i c i e n t to induce changes i n the r a t myocardium. By t r e a t i n g a subgroup of d i a b e t i c r a t s with i n s u l i n immediately a f t e r the onset of d i a b e t e s , i t appeared that the abnormalities could be prevented from oc c u r i n g . The LVDP, p o s i t i v e dP/dt and negative dP/dt were 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 37. values (Figures 3, 4 and 5) and were s i g n i f i c a n t l y higher than values obtained from the d i a b e t i c group. I t was a l s o seen that at a l l f i l l i n g pressures these parameters were s l i g h t l y , though not s i g n i f i c a n t l y higher i n the tr e a t e d d i a b e t i c s than i n normal c o n t r o l s . S i m i l a r l y , i n s u l i n treatment was e f f e c t i v e i n preventing biochemi-cal changes from occuring i n d i a b e t i c r a t s . Calcium t r a n s p o r t i n g a c t i v i t y of c a r d i a c SR, which was depressed i n d i a b e t i c animals, was found to be normal i n tr e a t e d d i a b e t i c s (Figure 6). Levels of long chain acyl c a r n i t i n e s i n c a r d i a c SR were al s o found to be normal i n i n s u l i n t r e a t e d r a t s (Figure 7). Free c a r n i t i n e l e v e l s i n SR remained unaffected (Figure 8 ) . b) Ten week r e v e r s a l study: In t h i s study, i n s u l i n treatment was i n i t i a t e d s i x weeks a f t e r the r a t s were made d i a b e t i c with STZ - a period by which the myocardial abnormalities supposedly occur. The d i a b e t i c animals weighed l e s s than c o n t r o l s when treatment was i n i t i a t e d but gained weight r a p i d l y during the course of i n s u l i n treatment. At the time of s a c r i f i c e , the tr e a t e d d i a b e t i c s weighed s i g n i f i c a n t l y more than the untreated d i a b e t i c s , had normal plasma glucose and plasma i n s u l i n l e v e l s and d i d not e x h i b i t p o l y d i p s i a and p o l y u r i a . However, though g l y c o s y l a t e d hemoglobin l e v e l s were s i g n i f i -c a n t l y lower than untreated d i a b e t i c s , the l e v e l s were s i g n i f i c a n t l y elevated as compared to c o n t r o l s (Tables V and V I ) . As i s evident from Figure 9, LVDP could be reversed to normal by t r e a t i n g the d i a b e t i c animals with i n s u l i n even a f t e r the myocardial abnormalities had occured., S i m i l a r l y p o s i t i v e dP/dt (Figure 10) and negative dP/dt (Figure 11) could be brought back to normal. I t thus 38. seems that myocardial f u n c t i o n changes induced by diabetes can be normalized i f i n s u l i n treatment i s i n i t i a t e d soon a f t e r the changes occur. This view i s f u r t h e r supported by the biochemical observations. Calcium uptake a c t i v i t y i n c a r d i a c SR could be reversed to normal by four weeks of i n s u l i n treatment (Figure 12). Long chain acyl c a r n i t i n e l e v e l s i n the SR seemed to p a r a l l e l the n o r m a l i z a t i o n of f u n c t i o n and calcium uptake a c t i v i t y as they were brought back to normal l e v e l s i n the c a r d i a c SR of t r e a t e d d i a b e t i c s (Figure 13). However, as i n the previous study, free c a r n i t i n e l e v e l s remained unaffected (Figure 14). c) S i x month r e v e r s a l study: In t h i s study, d i a b e t i c r a t s were treated with i n s u l i n f o r four weeks, f i v e months a f t e r they were made d i a b e t i c . While d i a b e t i c animals weighed s i g n i f i c a n t l y l e s s than c o n t r o l s , the t r e a t e d d i a b e t i c s gained weight and weighed nearly the same as c o n t r o l s . Plasma glucose and plasma i n s u l i n i n the i n s u l i n t r e a t e d group were normal but the g l y c o s y l a t e d hemoglobin showed only a p a r t i a l recovery towards normal (Table V I I ) . Figure 15 demonstrates the e f f e c t of i n s u l i n treatment on LVDP i n d i a b e t i c hearts. Though a trend towards normalization was seen, LVDP i n t r e a t e d d i a b e t i c 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 e i t h e r from normal c o n t r o l s or from d i a b e t i c s at a l l f i l l i n g pressures. In the case of p o s i t i v e dP/dt, a s i m i l a r trend was seen. However, here the values f o r t r e a t e d d i a b e t i c s were s i g n i f i c a n t l y lower than c o n t r o l s but s i g n i f i c a n t l y greater than the untreated d i a b e t i c s (Figure 16). The only parameter that seemed to be a p p r e c i a b l y reversed to normal was negative dP/dt (Figure 1.7). At a l l f i l l i n g pressures above 17.5 cm H2O, there was no d i f f e r e n c e between c o n t r o l s and t r e a t e d d i a b e t i c s while values f o r untreated d i a b e t i c s were 39. s i g n i f i c a n t l y depressed. E f f e c t of T3 treatment on diabetes-induced myocardial a l t e r a t i o n s . treatment was i n i t i a 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 3 days a f t e r i n j e c t i n g them with b u f f e r or with STZ. While the d i a b e t i c s , t r e a t e d and untreated l o s t weight r a p i d l y , the tre a t e d c o n t r o l s d i d not lose weight s i g n i f i c a n t l y . Both the d i a b e t i c groups e x h i b i t e d hyperglycemia and hypoinsulinemia. Glycosylated hemoglobin values were elevated i n both the d i a b e t i c groups (Tables VIII and I X ) . I t thus seemed that T3 treatment did not a l t e r the d i a b e t i c status of r a t s . However, when t h y r o i d status was s t u d i e d , untreated d i a b e t i c s e x h i b i t e d hypothyroidism and T3 t r e a t e d d i a b e t i c r a t s had normal t h y r o i d s t a t u s . Thus i s appeared that the T^ treatment regimen used was s u f f i c i e n t to normalize the diabetes-induced hypothyroidism when the heart f u n c t i o n of these animals was s t u d i e d . T^ treatment d i d not appear to have any s i g n i f i c a n t e f f e c t on f u n c t i o n . Figure 18 shows the LVDP and untreated d i a b e t i c s showed depressed LVDP. In the T3 t r e a t e d c o n t r o l s or d i a b e t i c s no s i g n i f i c a n t e l e v a t i o n or depression was seen as compared to untreated c o n t r o l s or d i a b e t i c s r e s p e c t i v e l y . S i m i l a r r e s u l t s were obtained f o r p o s i t i v e dP/dt (Figure 19) and negative dP/dt (Figure 20). F i n a l l y , we studied the e f f e c t of T 3 treatment on calcium uptake a c t i v i t y i n d i a b e t i c r a t s . As i s seen i n Figure 21, the d i a b e t i c c a r d i a c SR was s i g n i f i c a n t l y depressed as compared to c o n t r o l . As i n the fu n c t i o n s t u d i e s , T 3 treatment d i d not s i g n i f i c a n t l y a f f e c t the SR a c t i v i t y i n e i t h e r c o n t r o l or d i a b e t i c hearts. 40. Thus i t appears that c o r r e c t i n g the t h y r o i d status i n d i a b e t i c r a t s i s not b e n e f i c i a l with respect to myocardial changes while i n s u l i n seems to be very e f f e c t i v e at l e a s t i n the i n i t i a l phases of the disease. 41. TABLE I I I GENERAL FEATURES OF ANIMALS USED FOR HEART FUNCTION ANALYSIS (SIX WEEK INSULIN PREVENTION STUDY) BODY WEIGHT PLASMA GLUCOSE GLYCOSYLATED PLASMA HEART HEART WT/ (g) (mg %) HEMOGLOBIN INSULIN WEIGHT BODY WT (ymol HMF/g globin) (pu/ml) (g) (mg/g) 1. CONTROL 220±5 129±8 1.38+0.21 17.2±3.2 0.69+0.01 3.69±0.06 2. DIABETIC 162±7* 549±14* 2.98±0.14* 1.4±0.2* 0.50±0.02* 4.08±0.09* 3. DIABETIC 237+7 175x20 1.51+0.19 13.8+4.6 0.66±0.02 3.72±0.12 TREATED V 0 . 0 5 TABLE IV GENERAL FEATURES OF ANIMALS USED FOR CARDIAC BIOCHEMICAL ANALYSIS (SIX WEEK INSULIN PREVENTION STUDY) BODY WEIGHT PLASMA GLUCOSE GLYCOSYLATED PLASMA HEART HEART WT/ (g) (ng %) HEMOGLOBIN INSULIN WEIGHT BODY WT (pmol HMF/g globin) (uu/ml) (g) (mg/g) 1. CONTROL 218+3 130+6 1.36±0.11 29.3±0.6 0.59±0.01 2.69±0.05 2. DIABETIC 176±10* 388±19* 2.89±0.13* 14.0+0.5* 0.50+0.02 2.88±0.08 3. DIABETIC 218±2 190±7 1.56+0.09 23.5+0.7 0.64±0.02 2.95±0.06 TREATED p<0.05 42. FIGURE 3: E f f e c t of i n s u l i n treatment on myocardial f u n c t i o n i n s i x week animals. L e f t v e n t r i c u l a r developed pressure (LVDP) i s p l o t t e d against l e f t a t r i a l f i l l i n g pressure. I n s u l i n treatment was i n i t i a t e d three days a f t e r i n j e c t i o n of s t r e p t o z o t o c i n (STZ) and continued f o r s i x weeks. The depression of LVDP seen i n d i a b e t i c r a t s could be prevented by i n s u l i n treatment. Results are expressed as mean ± S.E.M. (*p<0.05). 43. SIX WEEK STUDY {*) CONTROL (N*6) (o) DIABETIC CN«=7) (") INSULIN TREATED (N«5) \5Qn 44. FIGURE 4: Rate of pressure development ( p o s i t i v e dP/dt) i s p l o t t e d against l e f t a t r i a l f i l l i n g pressure. Conditions were s i m i l a r to those i n Figure 3. A depression was seen at f i l l i n g pressure greater than 15.0 cm iS^O i n d i a b e t i c animals and the depression could be prevented by i n s u l i n treatment (*p<0.05). 45. SIX WEEK STUDY (x) CONTROL (N=6) (o) DIRBETIC (N=7) (H) INSULIN TREATED (N*5) 4500-1 500 +-2.5 T 1 1 1 1 1 r 5 7.5 10 12.5 15 17.5 20 FILLING PRESSURE (CM H20) 2?. 5 46. FIGURE 5: Changes i n the rate of r e l a x a t i o n are shown. I n s u l i n treatment proved to be e f f e c t i v e i n preventing the depression of negative dP/dt seen i n d i a b e t i c hearts a t higher f i l l i n g pressures (*p<0.05). 47. SIX WEEK STUDY (x) CONTROL (N=6) (o) DIABETIC (N=7) (H) INSULIN TREATED (N*=5) 4500-1 48. FIGURE 6: The f i g u r e demonstrates the e f f e c t o f s i x weeks of diabetes on calcium uptake a c t i v i t y i n the car d i a c sarcoplasmic r e t i c u l u m (SR). The a c t i v i t y was depressed over the e n t i r e range of calcium concentrations used i n d i a b e t i c r a t s . I n s u l i n treatment prevented such a depression from occuring i n d i a b e t i c r a t s (*p<0.05). 49. SIX WEEK. CA UPTAKE STUDY (x) CONTROL (N=8) (a) D I A B E T I C (N=8) (o) DIABETIC (INSULIN TREATED) (N«=8) 60 n i i i " T ~ - 1 i i i i I FREE CA LOG CONC (UM) 50. FIGURE 7: The 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 SR of c o n t r o l (CON), d i a b e t i c (DIAB) and d i a b e t i c animals tr e a t e d with i n s u l i n f o r s i x weeks (DIAB + INS) are shown. Though the l e v e l s were elevated i n s i x week d i a b e t i c r a t s , the e l e v a t i o n could be prevented by i n s u l i n treatment (*p<0.05). 51. 52. FIGURE 8: Free c a r n i t i n e l e v e l s i n the SR i n s i x week d i a b e t i c animals (DIAB) d i d not appear to be elevated as compared to c o n t r o l (CON). I n s u l i n treatment d i d not a f f e c t the free c a r n i t i n e l e v e l s . 53. SIX WEEK STUDY CN*=8) 1 0 n 2 CON DIflB DIRB+INS 54. TABLE V GENERAL FEATURES OF ANIMALS USED FOR HEART FUNCTION ANALYSIS (TEN WEEK INSULIN REVERSAL STUDY) BODY WEIGHT PLASMA GLUCOSE 1 . CONTROL 2. DIABETIC 3. DIABETIC TREATED (9) 263r5 206^5* 271-6.0 (mg %) 117±19 584^26* 168-17 GLYCOSYLATED PLASMA HEART HEART WT/ HEMOGLOBIN INSULIN WEIGHT BODY WT (pmol HMF/g globin) (pu/ml) (g) (mg/g) 1.15±0.09 14.8+3.2 0.72+0.02 3.57+0.10 2.60+0.22* 2.8±0.7* 0.61±0.01 * 4.12±0.08* 1.72+0.10* 18.4±3.9 0.76+0.02 3.7U0.15 *p<0.05 TABLE VI GENERAL FEATURES OF ANIMALS USED FOR CARDIAC BIOCHEMICAL ANALYSIS (TEN WEEK INSULIN REVERSAL STUDY) BODY WEIGHT PLASMA GLUCOSE 1 . CONTROL 2. DIABETIC 3. DIABETIC TREATED (g) 225±2 154-10* 232 + 4 (mg X) 133±9 366±28* 193+16 GLYCOSYLATED PLASMA HEART HEART WT/ HEMOGLOBIN INSULIN WEIGHT BODY WT (pmol HMF/g globin) (jju/ml) (g) (mg/g) 1.38+0.09 34.1±0.3 0.61+0.01 2.73+0.06 2.99±0.08* 19.4±0.46* 0.49±0.02* 3.2U0.09 2.15±0.07 32.5±0.99 0.64±0.02 2.7U0.04 *p<0.05 55. FIGURE 9: The f i g u r e demonstrates the e f f e c t i v e n e s s of i n s u l i n treatment i n r e v e r s i n g diabetes-induced depression of LVDP. Treatment with i n s u l i n was i n i t i a t e d s i x weeks a f t e r i n j e c t i o n of STZ, and was continued f o r four weeks. The depression was reversed by i n s u l i n treatment. Values are expressed as mean ± S.E.M. (*p<0.05). 56. TEN WEEK STUDY (x) TEN WEEK CONTROL (N=7) (o) TEN WEEK DIABETIC (N=6) in) FOUR WEEK TREATED (N«6) 150n 57. FIGURE 10: Rate of pressure development at various l e f t a t r i a l f i l l i n g pressure i s shown. The c o n d i t i o n of treatment were s i m i l a r to those i n Figure 9. I n s u l i n treatment was capable of r e v e r s i n g the depression of p o s i t i v e dP/dt induced by s i x weeks of diabetes (*p<0.05). 58. TEN WEEK STUDY (x) CONTROL (N=7) (o) D I A B E T I C (N=6) (H) INSULIN TREATED (N=6) 1000-f-2.5 T 1 1 1 — I 1 — i — r 5 7.5 10 12.5 15 17.5 20 22.5 FILLING PRESSURE (CM H20). 59. FIGURE 11: Rate of pressure d e c l i n e (negative dP/dt) was depressed i n d i a b e t i c hearts at f i l l i n g pressures greater than 12.5 cm H^O. I n s u l i n treatment seemed to reverse the depression and the values in t reated animals were s i m i l a r to c o n t r o l (p<0.05). 60. TEN WEEK. STUDY (x) TEN WEEK CONTROL (N*7) (o) TEN WEEK DIABETIC (N=6) (") FOUR WEEK TREATED (N«6) 3530 -i 500 H 1 1 1 1 1 1 1 r 2.5 5 7.5 10 12.5 15 17.5 20 22.5 FILLING PRESSURE (CM H20) 61. FIGURE 12: Calcium uptake a c t i v i t y i n the SR i s demonstrated i n t h i s f i g u r e . As i n s i x week d i a b e t i c r a t s , the a c t i v i t y was depressed i n ten week d i a b e t i c s over almost the e n t i r e concentration range used. By t r e a t i n g the animals with i n s u l i n , i t was poss i b l e to reverse the depression (p<0.05). 62. TEN WEEK. CALCIUM UPTAKE (INSULIN & DIABETES) (x) CONTROL (N=10) (•) DIABETIC (N=9) (o) DIABETIC TREATED (N=9) 35n 63. FIGURE 13: The histogram demonstrates the a b i l i t y of i n s u l i n to reverse the diabetes-induced e l e v a t i o n of long chain a c y l c a r n i t i n e l e v e l s i n the SR. Each bar represents mean ± S.E.M. of a group of animals (*p<0.05). 64. TEN WEEK REVERSAL STUDY (N*5-8) 3.2-1 in LD 2.4H 5 u cc LO .8-CON DIAB DIAB*INS 65. FIGURE 14: Free c a r n i t i n e l e v e l s i n the SR of c o n t r o l , d i a b e t i c and i n s u l i n treated animals are shown. Neither diabetes nor i n s u l i n treatment i n d i a b e t i c r a t s a f f e c t e d these l e v e l s . TEN WEEK REVERSAL STUDY (N=5-8) 8-i tn LO 2H 0 - L 1 1 1 I I L _ CON DIA6 DIAB+INS 67 . TABLE VII GENERAL FEATURES OF ANIMALS USED FOR HEART FUNCTION ANALYSIS (SIX MONTH INSULIN REVERSAL STUDY) BODY WEIGHT PLASMA GLUCOSE GLYCOSYLATED PLASMA HEART HEART WT/ (g) (mg %) HEMOGLOBIN INSULIN WEIGHT BODY WT (pmol HMF/g globin) (pu/ml) (g) (mg/g) 1. CONTROL 297x8 130x6 1 . 6 3 ± 0 . 0 9 23.8+0.7 1 . 0 6 ± 0 . 0 4 3.49=0.12* 2. DIABETIC 234-10* 281 + 19* 3.32-0.11 * 13.25+0.18 0 . 9 8 ± 0 . 0 3 4 . 2 U 0 . 1 3 3. DIABETIC 301 ±6 182=1 2 2.72 + 0.14* 24.5+0.3 1 . 2 4 ± 0 . 0 5 * 4.09+0.10 TREATED 68. FIGURE 15: E f f e c t of chronic ( s i x month) diabetes on LVDP i s shown. At a t r i a l f i l l i n g pressures greater than 17.5 cm ^ 0 , the LVDP was depressed i n d i a b e t i c r a t s . Four weeks of i n s u l i n treatment i n i t i a t e d f i v e months a f t e r i n d u c t i o n of diabetes did not s i g n i f i c a n t l y reverse the depression. However, the depression was a f f e c t e d to an extent that i t was no longer s i g n i f i c a n t l y depressed as compared to c o n t r o l s . This suggests that i n s u l i n treatment at t h i s stage showed only a trend towards rev e r s a l (*p<0.05). 69. FUNCTION STUDIES (x) SIX MONTH CONTROL (N*7) (o) SIX MONTH DIABETIC (N«=6) «) FOUR WEEK. INSULIN TREATED (N»7) * T 1 1 1 1 1 ~L 0 5 10 15 20 25 FILLING PRESSURE (CM H20) 70. FIGURE 16: P o s i t i v e dP/dt i s p l o t t e d against l e f t a t r i a l f i l l i n g pressure. In c o n t r a s t to the r e s u l t s obtained i n LVDP, p o s i t i v e dP/dt depression which was s i g n i f i c a n t i n s i x month d i a b e t i c s was s i g n i f i c a n t l y reversed by i n s u l i n treatment. However, i t remained s i g n i f i c a n t l y depressed as compared to c o n t r o l s suggesting a p a r t i a l r e v e r s a l towards normal (*p<0.05). 71. FUNCTION STUDIES (x) SIX MONTH CONTROL (N=7) Co) SIX MONTH DIABETIC (N«6) t<) FOUR WEEK INSULIN TREATED (N«7) 3500-1 T T 1 1 1 1 0 5 10 15 20 25 FILLING PRESSURE (CM H20). 72. FIGURE 17: Si x months of diabetes induced s i g n i f i c a n t reduction i n the negative dP/dt. Unlike the e f f e c t o f i n s u l i n treatment on LVDP and p o s i t i v e dP/dt, the depression of negative dP/dt was completely reversed i n s i x month d i a b e t i c animals by four weeks of i n s u l i n treatment (*p<0.05). 73. FUNCTION STUDIES (x) SIX MONTH CONTROL (N=7) (o) SIX MONTH DIABETIC (N*6) t<) FOUR WEEK INSULIN TREATED (N*7) 3O0O-. FILLING PRESSURE (CM H20) 7 4 . TABLE VIII GENERAL FEATURES OF ANIMALS USED FOR HEART FUNCTION ANALYSIS (SIX WEEK THYROID PREVENTION STUDY) BODY WEIGHT PLASMA GLUCOSE GLYCOSYLATED PLASMA HEART HEART WT/ T, B (9) (mg 1) HEMOGLOBIN INSULIN WEIGHT BODY WT INDEX (pmol HMF/g globin) (iiu/m)) (g) (mg/g) [% 1. CONTROL 218±6 129±8 1 . 5 9 ± 0 . 1 2 1 8 . 7 ± 0 . 6 0.6+0.005 2 . 7 3 ± 0 . 0 3 5 3 . 3 ± 2 . 7 2. CONTROL TREATED 211±4 119±8 1 . 3 3 ± 0 . 0 9 1 9 . 1 ± 0 . 7 0 . 6 6 ± 0 . 0 1 2 . 9 7 ± 0 . 0 3 68.8+6.0 3 . DIABETIC 70+7* 403+25* 3.04+0.17* 5 . 6 ± 0 . 4 * 0.55+0.02* '3.25+0.07 45. 9+0. 4. DIABETIC TREATED 1 7 6 ± 7 * 339x29 3.32+0.11* 7 . 1 2 ± 0 . 6 * 0 . 5 9 ± 0 . 0 2 3.35+0.06* 5 4 . 7 ± 4 . 1 TABLE IX GENERAL FEATURES OF ANIMALS USED FOR CARDIAC BIOCHEMICAL ANALYSIS (SIX WEEK THYROID PREVENTION STUDY) BODY WEIGHT PLASMA GLUCOSE GLYCOSYLATED PLASMA HEART HEART WT/ T 3 B ( 9 ) (mg 1) HEMOGLOBIN INSULIN WEIGHT BODY UT INDEX (pmol HMF/g globin) (pu/ml) (g) (mg/g) (a) 1. CONTROL 2 1 4 ± 5 120+5 1.55+0.1 3 31 .1+4.1 0.8 ± 0 . 0 2 3.79+0.05 53.3x2.6 2. CONTROL TREATED 217+4 121+6 1.47x0.11 31.1+5.7 0.83+0.01 3 . 8 2 ± 0 . 3 1 6 8 . 7 ± 6 . 0 * 3. DIABETIC 1 5 8 ± 1 1 * 3 8 5 ± 2 2 * 3.08+0.11* 11.5+2.9* 0.66+0.05 4 . 1 4 ± 0 . 5 * 4 5 . 8 ± 0 . 8 * 4. DIABETIC TREATED 1 5 8 ± 6 * 376+35* 3.26+0.05* 10.8+1.9* 0.76+0.04 4.81+0.5* 54.69+4.15 75. FIGURE 18: E f f e c t of t r i i o d o t h y r o n i n e (T3) on myocardial f u n c t i o n of s i x week animals. T3 treatment (30 ug/kg/day s.c.) was i n i t i a t e d three days a f t e r the animals were made d i a b e t i c and was continued f o r s i x weeks. LVDP, which was depressed i n s i x week d i a b e t i c r a t s was not s i g n i f i c a n t l y a f f e c t e d by the treatment (*p<0.05). 76. FUNCTION DATA (T3 & DIABETES) (x) CONTROL (N=8) la) CONTROL (T3 TREATED) (N«=7) (o) DIABETIC (N=7) 120-1 H) DIABETIC (T3 TREATED) (N=6) 30-|- \ 1 1 1 1 0 S 10 15 20 25 FILLING PRESSURE tCM H20) 7 7 . FIGURE 19: P o s i t i v e dP/dt i s p l o t t e d a g i n s t various l e f t a t r i a l f i l l i n g pressures. The parameter was depressed i n d i a b e t i c animals and the depression could not be prevented by T^ treatment (*p<0.05). 78. FUNCTION DATA (T3 & DIABETES) (x) CONTROL (N=8) (o) CONTROL (T3 TREATED) (N=7) (o) DIABETIC (N«=7) 4500-1 t<) DIABETIC (T3 TREATED) (N=6) 79. FIGURE 20: The changes i n negative dP/dt are shown. T 3 treatment d i d not prove to be e f f e c t i v e i n preventing the depression of the rate of r e l a x a t i o n i n d i a b e t i c r a t s (*p<0.05). 80. (x) CONTROL (N«8) (a) CONTROL (T3 TREATED) (N=7) to ) DIABETIC (N=7) 3090n W DIABETIC (T3 TREATED) (N=6) 81. FIGURE 21 : The Figure demonstrates the e f f e c t of s i x weeks of diabetes on calcium uptake a c t i v i t y i n the ca r d i a c SR. The depression of a c t i v i t y was seen over the e n t i r e concentration range i n d i a b e t i c animals and was not s i g n i f i c a n t l y a f f e c t e d by T, treatment (*p<0.05). 82. T3 -TREATMENT & CALCIUM UPTAKE IN DIABETIC RATS (x) CONTROL (n=7) <o) CONTROL TREATED (n«=7) (") DIABETIC (n«=7) 60 n i<) DIABETIC TREATED (n«=7) • i i i i i i - i • 1 i i—I—r—] - 1 FREE CA CONC (LOG UM) 83. DISCUSSION The present s e r i e s of experiments was designed to l o c a t e a point i n the duration of diabetes at which myocardial a l t e r a t i o n s are seen and to i n v e s t i g a t e the e f f e c t of various treatments on such a l t e r a t i o n s . Previous s t u d i e s c a r r i e d out on myocardial f u n c t i o n as well as biochemistry were c a r r i e d out at various times f o l l o w i n g the onset of diabetes. M i l l e r (1979) reported that myocardial f u n c t i o n changes could be seen three days a f t e r r a t s were i n j e c t e d with a l l o x a n . However, Vadlamudi et al_. (1982) f a i l e d to confirm t h i s observation and suggested that changes i n myocardial f u n c t i o n of d i a b e t i c r a t s occur between t h i r t y days .and three months a f t e r r a t s are i n j e c t e d with STZ. S i m i l a r l y , Fein e_t aj_. (1980) studied the e f f e c t o f STZ - diabetes on r a t p a p i l l a r y mechanics and reported that the changes were seen f i v e weeks a f t e r the onset of diabetes. Results from our study are i n general agreement with the studies of Vadlamudi ejt al_. and Fein et a l . as depression of heart f u n c t i o n was seen a f t e r s i x weeks of diabetes (Figures 3 and 4 ) . The diabetes-induced depression d i d not appear to worsen p e r c e p t i b l y with progression of the disease. The values f o r LVDP, p o s i t i v e dP/dt and negative dP/dt di d not appear to be depressed any f u r t h e r i n the ten week (Figures 9, 10, 11) or s i x month (Figures 14, 15, 16) d i a b e t i c r a t s as compared to the s i x week d i a b e t i c r a t s . This observation i s supported by Fein et aj_. (1980) where they studied the p a p i l l a r y muscle mechanics abnormalities at f i v e , ten and t h i r t y weeks a f t e r inducing diabetes. They found that the changes d i d not d e t e r i o r a t e f u r t h e r as the disease progressed. I t thus seems that depression of heart f u n c t i o n occurs by about s i x weeks and the depression 84. apparently plateaus soon a f t e r i t sets i n . This i s i n agreement with the study by Vadlamudi et_ aj_. (1982) where a plateauing phenomenon! with respect to the depression of heart f u n c t i o n was seen. S e v e r i t y of diabetes may play a r o l e i n the depression of heart f u n c t i o n as w e l l . The model we used was a moderately d i a b e t i c one ^ while that used by Fein e_t aj_. (1980) appeared to be s e v e r e l y hyper-glycemic which may be why they observed depression at an e a r l i e r time point i n r a t s of same s t r a i n , sex and age as were used i n our study. However, i t should be pointed out that we d i d not study the f u n c t i o n at f i v e weeks and the p o s s i b i l i t y t h a t we would have seen depressed f u n c t i o n at f i v e weeks cannot be r u l e d out. Secondly, we used the whole working heart and changes i n o v e r a l l c a r d i a c function.may not n e c e s s a r i l y c o i n c i d e with changes i n i n d i v i d u a l components of the heart such as the p a p i l l a r y muscle.;, From our s t u d i e s , i t i s a l s o apparent that calcium uptake i n cardiac SR i s depressed by s i x weeks of diabetes (Figure 6). Lopaschuk jet a]_. (1983) reported that calcium uptake a c t i v i t y i n d i a b e t i c c a r d i a c SR i s depressed between four weeks and three months of diabetes. This observation i s i n agreement with our r e s u l t s . There a l s o seems to be a c l o s e p a r a l l e l between the calcium uptake a c t i v i t y and heart f u n c t i o n . Diminished accumulation of calcium from the cytoplasmic space by SR has been suggested to be a detrimental f a c t o r i n the decreased a b i l i t y of the heart to r e l a x ,(Dhalla et al_. 1982). This suggestion could e x p l a i n the depression of negative dP/dt which we observe i n d i a b e t i c hearts. Regarding the time course of a l t e r e d f u n c t i o n and biochemistry, the a l t e r e d calcium uptake a c t i v i t y seems to precede the a l t e r e d 85. c o n t r a c t i l i t y . Such a conclusion could be drawn on the basis of f i n d i n g s by Vadlamudi et al_. (1982) and Ganguly et aj_. (1983), While Vadlamudi et_ al_. (1982) did not f i n d a depression of negative dP/dt four weeks a f t e r the onset of d i a b e t e s , Ganguly et a l . (1983) reported a l t e r e d calcium uptake i n SR by four weeks of diabetes. On the other hand we found that c a r d i a c f u n c t i o n was depressed by s i x weeks of diabetes and Fein et aJL (1980) reported that p a p i l l a r y muscle mechanics were a l t e r e d by f i v e weeks. Dillman (1980) and Malhotra 2+ et_ al_. (1981) have demonstrated a depression i n Ca s t i m u l a t e d c a r d i a c ATPase a c t i v i t y due to diabetes. This f i n d i n g has been confirmed i n a number of other studies (Fein et al_. 1981; Dillman, 1982; Ganguly et a l _ . , 1983). I t may thus be hypothesized that diabetes induces a 2+ depression of Ca ATPase a c t i v i t y which i n turn causes a depression of calcium uptake a c t i v i t y i n the SR. Such depression of calcium t r a n s p o r t may then c o n t r i b u t e towards an a l t e r e d myocardial f u n c t i o n . One of the f a c t o r s that have been i m p l i c a t e d as a p o s s i b l e cause of depressed calcium uptake by the SR i s elevated l e v e l s o f long chain acyl c a r n i t i n e s . Various workers have shown that long chain acyl c a r n i t i n e s are potent i n h i b i t o r s of membrane t r a n s p o r t proteins (Woods ejb a l _ . , 1977; Adams et aj_., 1979; P i t t s et aj_., 1978). Our r e s u l t s (Figure 7) as well as studies from other l a b o r a t o r i e s demonstrate that long chain acyl c a r n i t i n e s are s i g n i f i c a n t l y elevated during diabetes (Feuvray ejt aj_-» 1979). Moreover, the e l e v a t i o n of long chain acyl c a r n i t i n e s i n c a r d i a c SR seems to p a r a l l e l the depression of calcium t r a n s p o r t as well as a l t e r e d myocardial f u n c t i o n . Studies c a r r i e d out under other experimental c o n d i t i o n s such as ischemia have a l s o demonstrated s i g n i f i c a n t e l e v a t i o n s of long chain a c y l c a r n i -86. t i n e s i n c y t o s o l of heart c e l l s (Idell-Wenger e_t a]_., 1 979). Such observations support the suggestion that a l t e r e d long chain acyl c a r n i t i n e l e v e l s may indeed be involved with changes i n myocardial c o n t r a c t i l i t y . -This'suggestion i s supported by the work of Lopaschuk et a l . (1983) which was c a r r i e d out i n p a r a l l e l with the experiment of Vadlamudi et aj_. (1982). Lopaschuk et a l (.1983) found that the depression of calcium t r a n s p o r t i n g a c t i v i t y of SR and the e l e v a t i o n of long chain c a r n i t i n e s i n SR was apparent three months a f t e r diabetes was induced and was not seen at four weeks of diabetes. Thus there seems to be a c l o s e c o r r e l a t i o n between ca r d i a c f u n c t i o n and these biochemical parameters. However, i n a very recent study, Lopaschuk e t al_. (1983)* have presented evidence d i s s o c i a t i n g the c a r d i a c f u n c t i o n from calcium uptake a c t i v i t y and l e v e l s of long chain acyl c a r n i t i n e s and the r e s u l t s of that study are discussed at a l a t e r stage. Besides the biochemical f a c t o r s which we have s t u d i e d , other biochemical f a c t o r s have a l s o been suggested to play a r o l e i n diabetes-induced a l t e r a t i o n s of myocardial c o n t r a c t i l i t y . Regan e_t al_. (1979) have suggested that a l t e r e d myocardial c o n t r a c t i l i t y during diabetes could be a r e s u l t of increased v e n t r i c u l a r s t i f f n e s s , which was a t t r i b u t e d to an increase i n i n t e r s t i t i a l g l y c o p r o t e i n i n the myocardium. However, t h i s study was c a r r i e d out i n dogs and no such f i n d i n g s were reported when d i a b e t i c r a t myocardium was h i s t o l o g i c a l l y examined (Bhan et a l _ . , 1 978). Myocardial c o l l a g e n has been shown to be increased i n d i a b e t i c rhesus monkeys eighteen months a f t e r i n d u c t i o n of a l l o x a n diabetes (Yeh et a l . , 1978). However, Modrak (1980) was unable to 87. demonstrate s i g n i f i c a n t increases i n myocardial c o l l a g e n content i n twenty-six week STZ d i a b e t i c r a t s . The c o n t r a s t i n g r e s u l t s have been suggested to be due e i t h e r to the d i f f e r e n c e i n species or the d i f f e r e n c e i n duration of diabetes i n the two studies (Modrak, 1980). C o n t r a c t i l e proteins i n the heart may als o be resp o n s i b l e f o r a l t e r e d myocardial c o n t r a c t i l i t y . In r a t , myosin ATPase has three isoenzymes which d i f f e r i n i n t r i n s i c a c t i v i t y (Hoff et aj_., 1 977). Diabetes seems to cause a r e d i s t r i b u t i o n of the isoenzymes and t h i s r e s u l t s i n an o v e r a l l decrease of myosin ATPase a c t i v i t y ( D i l l m a n , 1980; Malhotra et_ a l . 1981). Sodium pump a c t i v i t y was als o found to be depressed four to s i x weeks a f t e r i n d u c t i o n of diabetes (Ku and S e l l e r s , 1982). Depression of the sodium pump was i n d i c a t e d by a decrease i n 86 the o u a b a i n - s e n s i t i v e Rb uptake by the sarcolemma as wel l as a decrease i n production of inorganic phosphate." I t seems u n l i k e l y that depression of myocardial f u n c t i o n by diabetes i s a r e s u l t of the depression of a s i n g l e biochemical parameter but i s probably the net r e s u l t o f a number of f a c t o r s a c t i n g together. In the d i a b e t i c s t a t e which i s an abnormal environment, the preformed normal systems may not f u n c t i o n as w e l l . I t could a l s o be that the systems which are a l t e r e d i n the d i a b e t i c s t a t e - such as c e r t a i n enzymes - have lower e f f i c i e n c y than normal and thus the heart as a whole cannot f u n c t i o n as w e l l . Lack of i n s u l i n i s the c h a r a c t e r i s t i c feature of diab e t e s . There i s now a growing i n t e r e s t regarding the a b i l i t y of i n s u l i n to normalize diabetes-induced myocardial changes. We attempted to study the a b i l i t y of i n s u l i n to prevent and reverse the a l t e r e d myocardial f u n c t i o n as 88. we l l as calcium uptake a c t i v i t y and 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 c a r d i a c SR. By t r e a t i n g d i a b e t i c animals with a long a c t i n g porcine i n s u l i n (PZI) (duration of a c t i o n about 36 h) d a i l y , we were able to c o n t r o l the diabetes q u i t e e f f e c t i v e l y . The t r e a t e d animals d i d not lose any weight and at the time of s a c r i f i c e , e x h i b i t e d normal plasma glucose and i n s u l i n l e v e l s . The heart weight to body weight r a t i o s which were higher i n d i a b e t i c r a t s were normal i n the t r e a t e d group (Tables I I I and IV). However, the most convincing evidence suggesting e f f e c t i v e c o n t r o l of diabetes was the degree of g l y c o s y l a t i o n of hemoglobin. In the prevention study, g l y c o s y l a t e d hemoglobin l e v e l s d i d not increase as compared to normal while the d i a b e t i c l e v e l s increased s i g n i f i c a n t l y a f t e r a period of s i x weeks. I t was thus apparent that blood glucose l e v e l s i n the t r e a t e d group were not elevated to l e v e l s high enough to increase g l y c o s y l a t i o n f o r any length of time at any point during treatment. We observed a large v a r i a t i o n of i n s u l i n values between groups (Tables I I I and IV) which may be due to the non-fasted s t a t e of the r a t s . When heart f u n c t i o n was s t u d i e d , we found that the depression i n c a r d i a c f u n c t i o n d i d not occur i n the t r e a t e d group of d i a b e t i c s (Figures 3, 4 and 5). This suggests that by c o r r e c t i n g the l a c k of i n s u l i n , the myocardial changes may be prevented. The observation a l s o suggests that myocardial a l t e r a t i o n s r e s u l t i n g from STZ-induced diabetes are not due to a d i r e c t t o x i c e f f e c t of STZ but r e s u l t from hyperglycemia or other metabolic changes induced by the drug. Such a view i s supported by the observation that myocardial changes do not occur i n r a t s t r e a t e d with 3-0-methyl glucose p r i o r to a d m i n i s t r a t i o n of STZ (Fein et aj_., 1981). 3-0-methyl glucose i s a non-metabol i z a b l e 89. analog of glucose and prevents development of diabetes i n STZ t r e a t e d r a t s . Penparkgul et aj_. (1981) al s o found that treatment with the analog could prevent depression of c a r d i a c SR f u n c t i o n by preventing the development of diabetes. In .close p a r a l l e l to the f u n c t i o n s t u d i e s we found that the calcium uptake a c t i v i t y i n c a r d i a c SR was normal i n the t r e a t e d group of d i a b e t i c r a t s (Figure 6). S i m i l a r l y , long chain acyl c a r n i t i n e l e v e l s were not s i g n i f i c a n t l y elevated i n the t r e a t e d d i a b e t i c s (Figure 7). Baandrup et aj_. (1981) stud i e d the a b i l i t y of i n s u l i n to prevent h i s t o l o g i c a l changes i n the myocardium of d i a b e t i c r a t s . They found that proper c o n t r o l of hyperglycemia with i n s u l i n could prevent the changes such as increases i n the amount of connective t i s s u e . I t thus seems that i n s u l i n treatment i s an e f f e c t i v e way of preventing diabetes induced changes i n f u n c t i o n , biochemistry and s t r u c t u r e of the r a t myocardium. In the r e v e r s a l study, we t r e a t e d d i a b e t i c r a t s s i x weeks a f t e r i n d u c t i o n of diabetes i . e . a period by which the a l t e r a t i o n s had supposedly occured. Treatment of the d i a b e t i c animals f o r four weeks with i n s u l i n r e s u l t e d i n a dramatic change i n t h e i r p h y s i c a l condition.. They regained weight s i g n i f i c a n t l y , t h e i r plasma glucose l e v e l s were lowered to normal and plasma i n s u l i n l e v e l s elevated to normal (Tables V and V I ) . However, the g l y c o s y l a t i o n of hemoglobin was not completely reversed by such treatment. The values f o r g l y c o s y l a t e d hemoglobin were between normal and d i a b e t i c values and s i g n f i c a n t l y d i f f e r e n t from both. G l y c o s y l a t i o n of hemoglobin occurs throughout the l i f e span of the e r y t h r o c y t e (McDonald and Davis, 1979), and by s i x weeks of diabetes there probably i s a high l e v e l of g l y c o s y l a t i o n of 90. hemoglobin. Four weeks of i n s u l i n treatment d i d not reverse the g l y c o s y l a t i o n completely and t h i s can be explained on the basis of the, l i f e span of,, the mammal iaru e r y t h rocyte which i s about 120 days. I n s u l i n treatment would e f f e c t i v e l y prevent f u r t h e r g l y c o s y l a t i o n of c e l l s but the treatment would have to be continued longer i n order to allow f o r t o t a l replacement by unglycosylated e r y t h r o c y t e s . Studying the heart f u n c t i o n , we found that four weeks of i n s u l i n treatment was capable of r e v e r s i n g the diabetes-induced a b n o r m a l i t i e s completely (Figures 9, 10, 11). Our study i s i n agreement with a study c a r r i e d out by Fein ejt al_. (1981) where they examined the e f f e c t of i n s u l i n t r e a t e d diabetes-induced a l t e r a t i o n s i n myocardial muscle mechanics. They i n i t i a t e d i n s u l i n treatment about e i g h t weeks a f t e r i n d u c t i o n of diabetes and studied the a b i l i t y of i n s u l i n to reverse the abnormalities at various time points during the course of treatment. Complete r e v e r s a l of a l t e r e d p a p i l l a r y muscle mechanics was seen only a f t e r four weeks of i n s u l i n as was observed i n our case. I t could be argued t h a t heart f u n c t i o n improvement could simply be the r e s u l t o f elevated i n s u l i n l e v e l s i n blood and a d d i t i o n of i n s u l i n to the perfusion medium could normalize heart f u n c t i o n as w e l l . This view would be supported by s t u d i e s i n which a d d i t i o n of i n s u l i n to the perfusate has been shown to r e s u l t i n improved f u n c t i o n ( M i l l e r , 1979). However, i t should be pointed out that M i l l e r ' s study was c a r r i e d out three days a f t e r i n j e c t i o n of a l l o x a n , at which stage a depression of c a r d i a c f u n c t i o n was observed when 5.5 mM glucose was used and the depression was overcome e i t h e r by i n c r e a s i n g the glucose concentration to 10 mM or by i n c l u d i n g i n s u l i n i n the medium. The defects observed by M i l l e r 91. could be the r e s u l t of acute metabolic derangements causing defects i n energy u t i l i z a t i o n due to lack of i n s u l i n and at t h i s stage, the c a r d i a c f u n c t i o n abnormalities could be overcome simply by i n c l u d i n g i n s u l i n or i n c r e a s i n g the substrate c o n c e n t r a t i o n . Chronic d i a b e t i c states such as the model we used may i n v o l v e a l t e r e d biochemical changes. Moreover, Penparkgul e_t al_. (1980) have demonstrated that perfusion of working hearts with i n s u l i n - c o n t a i n i n g b u f f e r d i d not improve f u n c t i o n of e i g h t week d i a b e t i c hearts. S i m i l a r l y , Fein et a l . (1981) were unable to reverse depression of p a p i l l a r y muscles by i n c l u d i n g i n s u l i n i n the bathing s o l u t i o n . We a l s o found that i n s u l i n treatment could reverse the depression of calcium uptake a c t i v i t y i n SR (Figure 12) and normalized the elevated l e v e l s of long chain acyl c a r n i t i n e s i n SR (Figure 13). Once again we were able to demonstrate a p a r a l l e l between the c a r d i a c c o n t r a c t i l i t y and biochemistry of the heart. Studies from other l a b o r a t o r i e s have also i n d i c a t e d that i n s u l i n treatment i s capable of r e v e r t i n g c a r d i a c SR f u n c t i o n to normal (Penparkgul et aj_., 1981). Myosin ATPase has a l s o been reported to be normalized by four weeks of i n s u l i n i n d i a b e t i c r a t s (Dillman, 1980; Fein e_t a l _ . , 1981). I t thus appears that i n s u l i n treatment can reverse a l l the diabetes-induced myocardial changes that have been s t u d i e d . The r e v e r s a l i s gradual but d e f i n i t e and i s complete by four weeks of treatment as pointed out by Fein e_t aj_. f (1981) and by our study. These f i n d i n g s are of s i g n i f i c a n t importance but before these r e s u l t s are a p p l i e d more wi d e l y , the features of t h i s model should be considered. The model we have used, as have others who reported r e s u l t s s i m i l a r to ours, i s a moderate to severe 92. d i a b e t i c r a t which has diabetes f o r only s i x weeks (and up to ten weeks i n other s t u d i e s ) before i n s u l i n treatment i s i n i t i a t e d . The duration of diabetes would obviously have an i n f l u e n c e on the r e s u l t s obtained. I t i s p o s s i b l e that at longer d u r a t i o n s , the abnormalities become more pronounced and. a f t e r a p a r t i c u l a r stage, they probably become i r r e v e r s i b l e . Pogatsa et aj_. (1979) reported that i n s u l i n therapy could not completely reverse myocardial a l t e r a t i o n s i n c h r o n i c a l l y d i a b e t i c dogs. In order to examine the above problem we studied the e f f e c t of i n s u l i n treatment i n r a t s d i a b e t i c f o r a longer duration of time. Diabetic r a t s were t r e a t e d with i n s u l i n f o r four weeks f i v e months a f t e r diabetes was induced with STZ. The animals t r e a t e d with i n s u l i n gained weight, had plasma glucose and i n s u l i n l e v e l s s i m i l a r to those of c o n t r o l s but the g l y c o s y l a t e d hemoglobin l e v e l s d i d not come back to c o n t r o l values (Table V I I ) . When the heart f u n c t i o n was s t u d i e d , the r e s u l t s obtained were not as promising as i n the e a r l i e r s t u d i e s . The LVDP i n d i a b e t i c t r e a t e d r a t s was not completely reversed to normal. Although i t was higher than i n the d i a b e t i c animals and lower than i n the c o n t r o l animals i t d i d not d i f f e r s i g n i f i c a n t l y from e i t h e r (Figure 15). On the other hand, p o s i t i v e dP/dt i n the t r e a t e d group of animals was higher than the d i a b e t i c s and lower than the c o n t r o l s , and the d i f f e r e n c e was s i g n i f i c a n t i n both cases (Figure 16). This suggests that even at t h i s stage the changes are not completely i r r e v e r s i b l e and i t may be p o s s i b l e to normalize the changes by using a longer duration of i n s u l i n treatment. The view that changes are not i r r e v e r s i b l e even at s i x months of diabetes i s supported by our r e s u l t s 93. obtained on the r a t e of r e l a x a t i o n . Negative dP/dt was completely reversed to normal by i n s u l i n treatment (Figure 17). Studies have not p r e v i o u s l y been c a r r i e d out on 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 at t h i s stage of diabetes and i t would be i n t e r e s t i n g to see whether or not i n s u l i n i s capable of r e v e r s i n g the abnormalities to normal. From our study i t would seem that i n s u l i n treatment does a f f e c t myocardial f u n c t i o n even at t h i s stage and perhaps a longer treatment regimen would e f f e c t i v e l y normalize the f u n c t i o n . As mentioned i n the I n t r o d u c t i o n , diabetes causes a s i g n i f i c a n t depression of t h y r o i d hormone l e v e l s . Hypothyroidism r e s u l t i n g from diabetes has been suggested to be the cause f o r diabetes-induced myocardial changes because hypothyroidism i s known to slow r e l a x a t i o n and to depress the f o r c e - v e l o c i t y r e l a t i o n i n p a p i l l a r y muscles (Buccino et a l _ . , 1967). In a recent study, Dillman (1982) stu d i e d the e f f e c t of t h y r o i d replacement therapy or diabetes-induced depression of myosin ATPase. The treatment e f f e c t i v e l y restored depressed c a r d i a c myosin ATPase a c t i v i t y to normal i n d i a b e t i c r a t s and when the treatment was i n i t i a t e d e a r l y enough, the depression could be prevented. However, r e s u l t s from our study i n d i c a t e that diabetes-induced myocardial f u n c t i o n a l a l t e r a t i o n s are u n l i k e l y to be a r e s u l t of depressed t h y r o i d status as replacement with T3 was unable to cause any improvement i n the myocardial f u n c t i o n (Figures 18, 19, 20). Again, i n p a r a l l e l to the f u n c t i o n study, we found that depression of calcium uptake a c t i v i t y i n the SR could not be prevented by T3 treatment (Figure 21). Fein e_t al_. (1981) a l s o found that t h y r o i d replacement therapy was unable to reverse diabetes-induced a l t e r a t i o n s 94. of p a p i l l a r y muscle mechanics. In a very recent r e p o r t , Ganguly et al_. (1983) were unable to normalize the calcium uptake i n the SR of d i a b e t i c hearts by t r e a t i n g the d i a b e t i c r a t s with t h y r o i d hormone. I t thus seems that hypothyroidism does not mediate the diabetes-induced myocardial a l t e r a t i o n . I t a l s o appears t h a t myosin ATPase i s not s o l e l y r e s p o n s i b l e f o r the depression of heart f u n c t i o n because while 2+ the Ca s t i m u l a t e d myosin ATPase can be normalized by t h y r o i d replacement (Dillman, 1982) muscle f u n c t i o n cannot be normalized. From our s t u d i e s , i t seems l i k e there i s a c l o s e p a r a l l e l between the l e v e l s of long chain acyl. c a r n i t i n e s , the calcium uptake a c t i v i t y of SR as well as the myocardial f u n c t i o n . However, i n a recent study from our l a b o r a t o r y , Lopaschuk e_t aj_. (1983) have demonstrated a d i s s o c i a t i o n between calcium uptake a c t i v i t y i n SR and c a r d i a c f u n c t i o n . By t r e a t i n g d i a b e t i c animals with c a r n i t i n e i n an attempt to decrease long chain acyl c a r n i t i n e l e v e l s i n the SR, they were able to normalize the calcium uptake a c t i v i t y i n SR to normal. However, when c a r d i a c f u n c t i o n i n these animals was s t u d i e d , the myocardial f u n c t i o n remained depressed i n the c a r n i t i n e t r e a t e d r a t s i n d i c a t i n g a p o s s i b l e d i s s o c i a t i o n between the calcium uptake i n SR and the myocardial f u n c t i o n . To gain a c l e a r e r perspective as to the s i g n i f i c a n c e of biochemical parameters i n myocardial f u n c t i o n , i t would be e s s e n t i a l to study a l l these parameters under a s e r i e s of d i f f e r e n t c o n d i t i o n s . At present, i t seems more l i k e l y t hat myocardial c o n t r a c t i l i t y i s the end r e s u l t of a number of biochemical f a c t o r s put together r a t h e r than any one f a c t o r and that most or a l l of these biochemical f a c t o r s have to be f u n c t i o n i n g normally f o r the heart to f u n c t i o n normally. 95. F i n a l l y , the sarcolemmal calcium pump may al s o be an important c o n t r i b u t o r towards c a r d i a c c o n t r a c t i l i t y (Caroni and C a r i f o l i , 1980; Trumble e_t a l _ . , 1980). This parameter remains to be stud i e d i n the d i a b e t i c s t a t e . However, studies i n our l a b o r a t o r y c a r r i e d out i n conjunction with D •. Godin (unpublished r e s u l t s ) i n d i c a t e the sarco-lemmal N a % K + ATPase i s not a f f e c t e d by diabetes. S i m i l a r l y p - n i t r o phenyl phosphate (PNPP) ATPase which i s an i n d i c a t o r of Na +, K + ATPase a c t i v i t y was not a f f e c t e d by diabetes. I t i s obvious that myocardial c o n t r a c t i l i t y , even though depressed i n di a b e t e s , i s compatible with l i f e to a c e r t a i n degree. Probably, the sarcolemmal pumps are more r e s i s t a n t to the e f f e c t s of diabetes and perhaps when these pumps are a f f e c t e d , the heart becomes sev e r e l y damaged. A s i m i l a r hypothesis could be forwarded f o r mitochondrial enzymes 2+ 2+ as the mitochondrial Ca -Mg ATPase was unaffected by four weeks of diabetes (unpublished r e s u l t s from our l a b o r a t o r y ) . 96. SUMMARY AND CONCLUSIONS 1. Streptozotocin-induced diabetes caused a depression of the l e f t v e n t r i c u l a r developed pressure (LVDP), ra t e of pressure r i s e and the rate of pressure d e c l i n e i n the r a t i s o l a t e d working heart. The depression was apparent by s i x weeks and did not worsen p e r c e p t i b l y with progression of the disease. 2. In c l o s e p a r a l l e l to the depression of heart f u n c t i o n , a depression of 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 (SR) to t r a n s p o r t calcium.was seen. An accompanying e l e v a t i o n of long chain acyl c a r n i t i n e s i n the SR was seen. However, the l e v e l s of free c a r n i t i n e s were not s i g n i f i c a n t l y a f f e c t e d by diabetes. 3. By t r e a t i n g d i a b e t i c r a t s with i n s u l i n immediately a f t e r diabetes was detected, the depression of heart f u n c t i o n could be prevented. The treatment was a l s o e f f e c t i v e i n preventing depression o f calcium uptake a c t i v i t y i n the SR as well as e l e v a t i o n of long chain acyl c a r n i t i n e s i n the SR. 4. In the re v e r s a l study, i . e . when i n s u l i n treatment was i n i t i a t e d s i x weeks a f t e r i n d u c t i o n of di a b e t e s , the myocardial f u n c t i o n could be reversed to normal. S i m i l a r l y , calcium uptake depression and e l e v a t i o n of long chain acyl c a r n i t i n e s i n SR could be normalized by i n s u l i n treatment. 5. When diabetes was allowed to progress f o r f i v e months before i n i t i a t i n g i n s u l i n treatment, the r e v e r s i b i l i t y was not seen as r e a d i l y . Though the rate of r e l a x a t i o n was completely normalized, the r e v e r s i b i l i t y was not seen as r e a d i l y . Though the rate of 97. r e l a x a t i o n was completely normalized, r a t e of c o n t r a c t i o n and LVDP showed only trends towards normal. 6. Replacement of t h y r o i d hormone with e f f e c t i v e l y elevated the t h y r o i d index to normal. However, n e i t h e r f u n c t i o n nor calcium uptake were normal i n the t r e a t e d group of d i a b e t i c s suggesting that the changes observed during diabetes do not occur as a r e s u l t o f hypothyroidism. In c o n c l u s i o n , i t i s apparent t h a t i n s u l i n treatment i s an e f f e c t i v e method to prevent as well as reverse c a r d i a c abnormalities induced by diabetes. However, i t i s c r i t i c a l t hat the therapy be i n i t i a t e d without a l l o w i n g the disease to progress f o r very long periods of time as the e f f e c t i v e n e s s of i n s u l i n treatment to reverse the myocardial abnormalities seemsto decrease as the disease progresses. While diabetes does cause hypothyroidism, the lowered t h y r o i d s t a tus does not seem to be r e s p o n s i b l e f o r the myocardial changes. This view i s based on our f i n d i n g s that replacement therapy with T^ does not prevent the myocardial damage from occuring i n d i a b e t i c r a t s . 98. REFERENCES Adam, R.J., Cohen, D.W., Gupte, J . , Johnson, D., W a l l i c k , E.T., Wang, T. and Schwartz, A. (1978): 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 C a + 2 -ATPase and C a + 2 - t r a n s p o r t . J . B i o l . Chem. 254: 12404-12410. Ahmed, S.S., A.J. Ghazanfar, R.M. 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( 1 9 8 3 ) 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 i n s u l i n a n d c a r n i t i n e t r e a t e d d i a b a t i c r a t s . A m e r . J . P h y s i o l . ( S u b m i t t e d ) . 

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