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Altered drug responses in diabetic and hypertensive-diabetic cardiomyopathy Yu, Zhen 1990

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ALTERED DRUG RESPONSES IN DIABETIC AND HYPERTENSIVE-DIABETIC CARDIOMYOPATHY By ZHEN YU Bachelor of Medicine, Tanshan Medical School, 1983 Master of Medicine, China Academy of Traditional Medicine, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Div i s i o n of Pharmacology & Toxicology i n the Faculty of Pharmaceutical Sciences We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January 1990 ©Zhen Yu, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Faculty o f Pharmaceutical Sciences The University of British Columbia Vancouver, Canada January, 1990 DE-6 (2/88) i i ABSTRACT Diabetes m e l l i t u s has been assoc iated with both c l i n i c a l and experimental card iac dys funct ion . D i a b e t i c cardiomyopathy which i s charac ter i zed by depressed cardiac c o n t r a c t i l i t y i s accompanied by a v a r i e t y o f biochemical changes i n C a + + metabolism. T h i s cardiomyopathy may occur i n the presence of normal coronary a r t e r i e s and normal blood pressure . However, some studies have shown that hypertension i s more prevalent among d i a b e t i c s and can aggravate the card iovascu lar abnormal i t ies assoc iated with d iabetes . To understand the mechanisms of d i a b e t i c cardiomyopathy and consequences o f combined hypertension and diabetes , experiments were designed to measure c a r d i a c t i s sue responses to var ious i n o t r o p i c agents i n experimental d iabetes . S ix weeks fo l lowing s t rep tozo toc in (STZ) a d m i n i s t r a t i o n , Wistar , spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) ra t s exh ib i t ed the ' c l a s s i c a l s igns ' o f diabetes which i n c l u d e d : hyperglycemia, hypoinsul inemia , hyper l ip idemia (except i n WKY), and hypothyroidism. Decreased basa l a t r i a l rate and increased basa l developed force (BDF) suggest a depressed SA node funct ion and an a l t e r a t i o n o f C a + + u t i l i z a t i o n by d i a b e t i c v e n t r i c l e s . Decreased post quiescent p o t e n t i a t i o n (PQP) values (except i n WKY) i n v e n t r i c u l a r t i s sues suggest a diminished amount of re l easab le C a + + from sarcoplasmic re t i cu lum (SR). Decreased post s t i m u l a t i o n p o t e n t i a t i o n (PSP) values i n SHR p a p i l l a r y muscles (PM) are probably suggest ive o f a depressed sarcolemmal N a + - C a + + exchange funct ion i n t h i s t i s s u e . D iabet i c ra t s show s u b s e n s i t i v i t y to ^-adrenergic s t i m u l a t i o n i n v e n t r i c u l a r t i s s u e s , s u p e r s e n s i t i v i t y and hyperresponsiveness to C a + + and a-adrenergic s t i m u l a t i o n (except i n WKY) i n i i i v e n t r i c u l a r t i s sues and l e f t a t r i a (LA) and s u p e r s e n s i t i v i t y to BAY K 8644 i n SHR LA and hyperresponsiveness to verapamil i n v e n t r i c u l a r s t r i p s . These a l t e r a t i o n s may be a t t r i b u t e d to a change i n receptor number and/or a post receptor a l t e r a t i o n . Ryanodine decreased the PQP of Wistar and SHR PM and SHR LA i n both c o n t r o l s and d i a b e t i c s . I t e s p e c i a l l y abol i shed PQP i n SHR d i a b e t i c t i s s u e s , but had no e f f e c t on WKY t i s s u e s , which may suggest a d i f f erence i n the SR funct ion i n these t i s s u e s . SR with impaired C a + + uptake may contr ibute to these phenomena i n d i a b e t i c r a t s . Ryanodine a l so diminished (PQP + BDF) o f SHR LA and (PQP/BDF) of Wistar and SHR PM, 'but had no e f f e c t s on c o n t r o l and other d i a b e t i c t i s s u e s . I t appears that ryanodine has some inf luence on the N a + - C a + + exchange generated by sarcolemma (SL) of c e r t a i n d i a b e t i c t i s s u e s . Further experiments are requ ired to c l a r i f y t h i s . SHR d i a b e t i c ra t s had greater changes i n most o f the measurements such as hyper l ip idemia , depressed PQP and PSP va lues , and a l t e r e d drug responses. T h i s model exh ib i t ed very high m o r t a l i t y as compared to Wistar and WKY d i a b e t i c r a t s . As has been shown p r e v i o u s l y , the combination o f hypertension and diabetes exerts a s y n e r g i s t i c e f f e c t on the card iac dys funct ion i n t h i s model, and that a l t e r e d l i p i d metabolism, SL and SR funct ion are a l l invo lved i n the development o f cardiomyopathy. WKY d i a b e t i c r a t s , on the other hand, exh ib i t ed no s i g n i f i c a n t changes i n blood l i p i d s , or i n response to phenylephrine or to C a + + (LA) s t i m u l a t i o n . Lack o f change i n these fac tors may e x p l a i n the r e l a t i v e l y normal card iac funct ion o f t h i s model as measured p r e v i o u s l y . John H. M c N e i l l , PhD Thes i s Superv i sor iv TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES ' v i i LIST OF ABBREVIATION i x ACKNOWLEDGEMENTS x INTRODUCTION 1 I . D iabe t i c Cardiomyopathy and Hypertensive D iabe t i c Cardiomyopathy 1 I I . Animal Models of Genetic Hypertension, Diabetes and Hypertensive-Diabetes 3 A. Spontaneously hypertensive r a t s (SHR) 3 B. S treptozotoc in (STZ) induced d i a b e t i c ra t s 7 C. Hypertensive d i a b e t i c r a t model 7 I I I . Diabetes and A l t e r e d C a + + Metabolism i n Heart 10 IV. The Force-Frequency Re la t ionsh ip and the A c t i v i t i e s of Ryanodine 12 V. S p e c i f i c Aims 13 MATERIAL AND METHODS 15 I . M a t e r i a l 15 A. Animals 15 B. Chemicals and Assay K i t s 15 I I . Methods 16 A. Induction of Experimental Diabetes . . 16 B. V e r i f i c a t i o n o f the D i a b e t i c State 16 C. Pharmacological Experiments 16 1. I so la ted t i s sue preparat ion 16 2. Dose responses o f i s o l a t e d t i s sues to card iac drugs 17 3. The e f f ec t s o f ryanodine on PQP and PSP . . .18 D. S t a t i s t i c a l A n a l y s i s 18 V Page RESULTS I . I n d i c a t i o n o f Diabetes M e l l i t u s 21 I I . Basa l Parameters from I so la ted Card iac Preparat ions . . .21 I I I . Pre-drug Measurements of PQP and PSP 29 A. The time courses o f PQP and PSP 29 B. Pre-drug PQP and PSP i n d i f f e r e n t c a r d i a c preparat ions 36 IV. Inotrop ic and Chronotropic Responses to Card iac Drugs o f I so la ted Heart Preparat ion 36 A. Dose-response curves to c a r d i a c drugs 36 B. The e f f ec t s o f ryanodine on BDF, PQP and PSP. . . .53 DISCUSSION 64 I . General Ind icat ions o f Diabetes M e l l i t u s 64 1. Body weights and m o r t a l i t y 64 2. Plasma glucose and i n s u l i n 65 3 . Blood l i p i d s 65 4. Plasma t r i i o d o t h y r o n i n e (T^) and Thyroxine (T^) . .66 I I . Basal Chronotropic and Ino trop ic Responses from Various Heart Preparat ions 67 I I I . Pre-drug Measurement of PQP and PSP 68 IV. Cardiac Drug Responses i n D i a b e t i c Myocardium 71 1. fS-adrenergic s t i m u l a t i o n 71 2 . a-adrenergic s t i m u l a t i o n ; 74 3 . C a + + . 77 4. BAY K 8644 and verapamil 79 5. The e f f ec t s o f Ryanodine 81 SUMMARY AND CONCLUSIONS 84 REFERENCES 86 vi LIST OF TABLES Table Page 1 Body weight, plasma glucose and blood l i p i d s l e v e l s o f c o n t r o l and 6-week d i a b e t i c r a t s 26 2 I n s u l i n and t h y r o i d hormone l e v e l s o f c o n t r o l and 6-week d i a b e t i c ra t s 27 3 Basa l i n o t r o p i c and chronotropic parameters of c a r d i a c preparat ions from c o n t r o l and d i a b e t i c ra t s . . .31 4 PQP r e l a t e d parameters from c o n t r o l and 6-week d i a b e t i c ra t s 37 5 PSP r e l a t e d parameters from c o n t r o l and 6-week d i a b e t i c ra t s 38 6 EC^O values of various i n o t r o p i c agents i n heart preparat ions of c o n t r o l and 6-week Wistar d i a b e t i c ra t s .39 7 v a l u e s ° f var ious i n o t r o p i c agents i n heart preparat ions of c o n t r o l and 6-week SHR d i a b e t i c r a t s . . .40 8 v a ^ - u e s ° f var ious i n o t r o p i c agents i n heart preparat ions of c o n t r o l and 6-week WKY d i a b e t i c r a t s . . .41 9 Concentrat ions o f verapamil requ ired to decrease 50% c o n t r a c t i l e force from c a r d i a c preparat ions o f c o n t r o l and d i a b e t i c ra t s 54 10 E f f e c t s of ryanodine on PQP and PSP o f c o n t r o l and d i a b e t i c card iac preparat ions 55 11 E f f e c t s o f ryanodine on BDF o f c o n t r o l and d i a b e t i c c a r d i a c preparat ions 56 vi i LIST OF FIGURES Figure Page 1 Measurement o f post quiescent p o t e n t i a t i o n (PQP, A ) , and post s t i m u l a t i o n p o t e n t i a t i o n (PSP, B) i n r a t p a p i l l a r y muscle 19 2 Body weights of c o n t r o l and 6-week d i a b e t i c r a t s 22 3 Plasma concentrat ions o f glucose and i n s u l i n i n c o n t r o l and 6-week d i a b e t i c r a t s 23 4 Plasma concentrat ions of l i p i d s i n c o n t r o l and 6-week d i a b e t i c ra t s 24 5 Plasma concentrat ions of i n c o n t r o l and 6-week d i a b e t i c ra t s 25 6 Basal developed force o f heart preparat ions from c o n t r o l and 6-week d i a b e t i c ra t s 28 7 Basal a t r i a rates of c o n t r o l and 6-week d i a b e t i c ra t s 30 8 E f f e c t of the durat ion o f the tes t i n t e r v e n t i o n on the magnitude o f post quiescent p o t e n t i a t i o n i n Wistar r a t p a p i l l a r y muscles 32 9 E f f e c t o f the durat ion o f the t e s t i n t e r v e n t i o n on the magnitude of post quiescent p o t e n t i a t i o n i n Wistar r a t l e f t a t r i a 33 10 E f f e c t of the durat ion of the tes t i n t e r v e n t i o n on the magnitude of post s t i m u l a t i o n p o t e n t i a t i o n i n Wistar ra t p a p i l l a r y muscle 34 11 E f f e c t of the durat ion o f the t e s t i n t e r v e n t i o n on the magnitude o f post s t i m u l a t i o n p o t e n t i a t i o n i n Wistar r a t l e f t a t r i a 35 12 Isoproterenol dose response curves obtained i n r i g h t v e n t r i c l e t i s sues o f c o n t r o l and d i a b e t i c ra t s 42 13 PD2 values for i s o p r o t e r e n o l i n heart preparat ions from c o n t r o l and 6-week d i a b e t i c r a t s 43 14 Phenelephrine dose response curves obtained i n r i g h t v e n t r i c l e t i s sues o f c o n t r o l and d i a b e t i c r a t s 44 15 PD2 values for phenylephrine i n heart preparat ions from c o n t r o l and 6-week d i a b e t i c r a t s 45 vi i i Figure Page 16 Calcium dose response curves obtained i n r i g h t v e n t r i c l e t i s sues o f c o n t r o l and d i a b e t i c r a t s 46 17 pD£ values for calc ium i n heart preparat ions from c o n t r o l and 6-week d i a b e t i c r a t s 47 18 BAY K 8644 dose response curves obtained i n r i g h t v e n t r i c l e t i s sues o f c o n t r o l and d i a b e t i c r a t s 48 19 pD£ values for BAY K 8644 i n heart preparat ions from c o n t r o l and 6-week d i a b e t i c r a t s 49 20 Verapamil dose response curves obtained i n r i g h t v e n t r i c l e t i s sues o f c o n t r o l and d i a b e t i c r a t s 50 21 P D 2 values for verapamil i n heart preparat ions from c o n t r o l and 6-week d i a b e t i c r a t s 51 22 The e f f ec t s of ryanodine on post quiescent p o t e n t i a t i o n of p a p i l l a r y muscles i n c o n t r o l and 6-week d i a b e t i c ra t s 57 23 The e f f ec t s of ryanodine on post quiescent p o t e n t i a t i o n of l e f t a t r i a i n c o n t r o l and 6-week d i a b e t i c r a t s 58 24 The e f f ec t s o f ryanodine on post s t i m u l a t i o n p o t e n t i a t i o n (PSP+BDF) of p a p i l l a r y muscles i n c o n t r o l and 6-week d i a b e t i c r a t s 60 25 The e f f ec t s of ryanodine on post s t i m u l a t i o n p o t e n t i a t i o n (PSP+BDF) of l e f t a t r i a i n c o n t r o l and 6-week d i a b e t i c ra t s 6 l 26 The e f f ec t s of ryanodine on post s t i m u l a t i o n p o t e n t i a t i o n (PSP/BDF) of p a p i l l a r y muscles i n c o n t r o l and 6-week d i a b e t i c ra t s 62 27 The e f f ec t s of ryanodine on post s t i m u l a t i o n p o t e n t i a t i o n (PSP/BDF) o f l e f t a t r i a i n c o n t r o l and 6-week d i a b e t i c ra t s 63 ix LIST OF ABBREVIATIONS BDF basa l developed force Bmax maximal number of b inding s i t e s CON Wistar c o n t r o l DIA Wistar d i a b e t i c 1,4-DHP 1 ,4 , -d ihydropyridine EC^g the c a l c u l a t e d concentrat ion o f an agonist that produces 50% o f maximum e f f e c t IDDM i n s u l i n dependent diabetes m e l l i t u s ISO i sopro tereno l Kd d i s s o c i a t i o n constant LA l e f t a t r i a LVDP l e f t v e n t r i c u l a r developed pressure n number o f observations p s t a t i s t i c a l p r o b a b i l i t y value PD2 negative l og concentrat ion of an agonist which produce 50% o f maximum e f f e c t PE phenylephrine PM p a p i l l a r y muscle PQP post quiescent p o t e n t i a t i o n PSP post s t i m u l a t i o n p o t e n t i a t i o n RA r i g h t a t r i a RV r i g h t v e n t r i c l e SHC SHR c o n t r o l SHD SHR d i a b e t i c SHR spontaneously hypertensive r a t SL sarcolemma SR sarcoplasmic ret iculum STZ s t rep tozo toc in T^ t r i i o d o t h y r o n i n e Tj| thyroxine WKC WKY c o n t r o l WKD WKY d i a b e t i c WKY Wistar Kyoto ra t X ACKNOWLEDGEMENTS I would l i k e to express my deepest g r a t i t u d e to Dr. John H. M c N e i l l , my superv i sor and academic adv i sor , f or h i s pa t i en t guidance and continuous support . I wish to thank a l l members of the superv i sory committee, Dr. Frank S. Abbott , Dr. Jack Diamond, Dr. Clayton H e y l i g e r , Dr. Kathleen MacLeod and Dr. K e i t h M. McErlane, for t h e i r c o n s t r u c t i v e comments and c r i t i c i s m s . S p e c i a l thanks are given to Dr. Sasanka Ramanadham for h i s va luable help and s t i m u l a t i n g d i scuss ion of t h i s work. I would a l so l i k e to express s incere thanks to Ms. Mary B a t t e l l , Mr. Yong- j iang H e i , and a l l my laboratory col leagues for t h e i r valuable suggestions and encouragement throughout t h i s s tudy. I am e s p e c i a l l y g r a t e f u l to Mrs. S y l v i a Chan for her exce l l en t s e c r e t a r i a l a s s i s tance . xi I wish to dedicate t h i s thes i s to my parents , Professors Yu Zebo and J i a n g Deai . 1 INTRODUCTION I. Diabetic Cardiomyopathy and Hypertensive Diabetic Cardiomyopathy Diabetes m e l l i t u s has been assoc ia ted with both c l i n i c a l and experimental card iac dys funct ion . D i a b e t i c cardiomyopathy which i s charac ter i zed as myocardial f a i l u r e independent of a t h e r o s c l e r o s t i c coronary a r t e r y disease , v a l v u l a r disease or hypertens ion has been s trong ly suggested by epidemological , c l i n i c a l and experimental research (Kannel 1978, Regan et a l . 1977, Fe in et a l . I98O, D h a l l a e t a l . 1985)• Most c l i n i c a l s tudies on d i a b e t i c cardiomyopathy have inc luded pat i ents with diabetes of considerable d u r a t i o n . In these s tud ies , a d i s t i n c t i o n was seldom drawn between Type I and Type II d iabetes . I t i s u n l i k e l y that a t h e r o s c l e r o s i s , microangiopathy or neuropathy cause cardiomyopathy i n diabetes (Gotzsche 1986; F e i n and Sonnenblick 1985)• However, other authors suggested the exis tence o f a card iac disease p e c u l i a r to diabetes as a r e s u l t and combination of microangiopathy, macroangiopathy, neuropathy and metabolic cardiomyopathy, which they pre ferred to c a l l ' d i a b e t i c cardiopathy 1 ra ther than ' d i a b e t i c cardiomyopathy' - which gives the idea only o f non-vascular heart disease (Crepa ld i and Nosadini 1988; Ledet e t a l . 1979)- The main cardiac compl icat ion i n d i a b e t i c pat ients with or without myocardial i n f a r c t i o n cons i s t s o f decreased c o n t r a c t i l i t y o f the l e f t v e n t r i c l e (Jaffe et a l . 1984, Smith et a l . 1984). I t i s a lso suggested that both l e f t v e n t r i c u l a r s y s t o l i c and d i a s t o l i c dysfunct ion c h a r a c t e r i z e d i a b e t i c cardiomyopathy without evidence of ongoing microvascular ischemia (Regan et a l . 1977. Shapiro et a l . 1980). The mechanisms involved i n t h i s cardiomyopathy are not very c l e a r . Considerable data are present ly a v a i l a b l e with regard to the metabolism, 2 mechanics and funct ion of heart preparat ions from experimental d i a b e t i c animals . Rats treated with STZ or a l l oxan expressed depressed v e n t r i c u l a r c o n t r a c t i l i t y , diminished v e n t r i c u l a r compliance and genera l l y decreased i n o t r o p i c and chronotropic responses to c e r t a i n drugs i n i s o l a t e d cardiac preparat ion (Vadlamudi et a l . 1982, Penpargkul et a l . 1980, Fe in et a l . 1980). S ince the ra t u s u a l l y does not develop a t h e r o s c l e r o s i s , the i n v i v o / i n v i t r o abnormal i t ies i n c o n t r a c t i l i t y and metabolism r e f l e c t changes i n the funct ion of the myocardial c e l l (Baandrup et a l . 198I). The i s o l a t e d muscles from d i a b e t i c hearts showed delayed r e l a x a t i o n , slowed r e l a x a t i o n rate and increased peak r e l a x a t i o n time (Fein et a l . I 9 8 O ) . Diabet i c ra t s treated with i n s u l i n , by both 'prevent ion' and ' r e v e r s a l ' regimens ( T a h i l i a n i et a l . 1983) e x h i b i t e d normal myocardial funct ion while the untreated d i a b e t i c showed depressed f u n c t i o n . A l t e r e d p a p i l l a r y muscle mechanics can a lso be res tored by i n s u l i n treatment (Fein et a l . I98O). A m e t a b o l i c a l l y determined abnormality i n contrac t ion thus seems to e x i s t i n experimental d iabetes . C l i n i c a l evidence obtained i n a few prospect ive s tudies suggested that hyperglycemia disposes towards the i n c i p i e n t congest ive heart f a i l u r e i n a large populat ion of IDDM pat ients and that there i s a metabolic f a c t o r i n myocardial mechanical funct ion (Gotzsche 1986; Shapiro et a l . I98I). The increased dependence on l i p i d metabolism with i t s poss ib l e consequences on i n t r a c e l l u l a r C a + + homeostasis could serve as an explanat ion for t h i s myocardial dys funct ion (Katz and Messineo 198l, Rodrigues et a l . 1985). Hypertension i s be l i eved to be more prevalent among d i a b e t i c s and i s known to aggravate the card iovascu lar compl icat ions assoc iated with diabetes ( B e l l 1989)- Factor et a l . (I98O) descr ibed the postmortem feature of d i a b e t i c s with severe congestive heart f a i l u r e and hypertension 3 as w e l l . I n t e r s t i t i a l and replacement f i b r o s i s with myocyto lyt ic necros is which was def ined as a n e c r o t i c process i n myocardial c e l l s demonstrating l o s s o f cytoplasmic const i tuents (Factor et a l . 1980) was prominent i n these p a t i e n t s ' d i l a t e d and hypertrophied h e a r t s . These changes were s u b s t a n t i a l l y more prominent than i n pat i en t s with e i t h e r diabetes or hypertension alone. The concept that two added s tresses leads to increased p a t h o l o g i c a l changes i s cons is tent with s tud ies on the e f f ec t s of hypertension on d i a b e t i c ret inopathy and nephropathy ( B e l l 1989). Furthermore, p a t h o l o g i c a l and c l i n i c a l f ind ings i n hyper tens ive -d iabe t i c pa t i en t s c o r r e l a t e s with the extensive myocardial damage i n hypertens ive-d i a b e t i c ra t s (Shapiro et a l . 1981). Perhaps diabetes s e n s i t i z e s the myocardium so that superimposed hypertension, with the attendant vascular changes, r e s u l t s i n progress ive myocyte damage l ead ing u l t i m a t e l y to congestive heart f a i l u r e . One example of the s e n s i t i z i n g e f f e c t of diabetes i s the greater tendency for i s o l a t e d muscle from d i a b e t i c rats to develop contracture when exposed to high concentrat ions o f e x t r a c e l l u l a r C a + + (Fein et a l . I98O). However, the features of the hypertensive-d i a b e t i c r a t model need to be further c h a r a c t e r i z e d by measurement of more parameters. I I . Animal Models of Genetic Hypertension, Diabetes and Hypertensive-Diabetes A. Spontaneously hypertensive rat s (SHR) Since the Japanese s t r a i n of spontaneously hypertens ive rat s (SHR) was e s tab l i shed i n the e a r l y 1960's (Okamoto and Aoki 1963), SHR have been d i s t r i b u t e d to researchers a l l over the world and numerous inves t iga t ions on t h i s genet ic model o f hypertension have been undertaken (Yamori 1984). 4 The s t a r t o f s e l e c t i o n o f hypertensive r a t s was made by mating a male Wistar-Kyoto (WKY, Wistar colony at Kyoto U n i v e r s i t y ) r a t with mild hypertension ( s y s t o l i c pressure 145-175 mmHg) and a female with mild hypertension ( s y s t o l i c pressure 130-140 mmHg) i n 1959 (Okamoto and Aoki 1963). By repeatedly checking the blood pressure to s e l e c t hypertensive o f f s p r i n g , a colony o f rats which developed hypertension without exception was f i n a l l y e s tab l i shed , and reported as spontaneously hypertensive rats (SHR) i n I963. Moreover, sub- s t ra ins have been e s t a b l i s h e d i n the l a s t decade. Stroke-prone SHR serve as a model o f hypertensive vascular d isease , a r t e r i o l i p i d o s i s - p r o n e rats are a model o f atherogenesis , spontaneous thrombogenic ra t s provide a model o f c e r e b r a l thrombosis and myocardial ischemic rats as a model o f myocardial i n f a r c t i o n were developed (Yamori 1984) . SHR genera l ly develop moderate to severe hypertension spontaneously without exception at the age o f 7~15 weeks with a s y s t o l i c blood pressure p lateau o f about 200 mmHg. The fact that a 100% hypertensive s t r a i n of ra t s was developed by s e l e c t i v e i n - b r e e d i n g demonstrates that heredi ty i t s e l f i s an extremely important f a c t o r . Based on the cross -breeding experiments, i t has become c l e a r that about three major genes are involved i n the development of hypertension i n SHR (Yamori 1984). In a d d i t i o n , experimental fac tors such as exposure to s t re s s or s a l t l o a d i n g a l so play a r o l e i n the development of hypertension (Yamori I98I). As observed by microscopy, the i n i t i a l v a s o c o n s t r i c t i o n i n the p e r i p h e r a l r e s i s t a n t vesse ls i s probably of neurogenic o r i g i n as there i s a r i s e i n plasma norepinephrine l e v e l s i n SHR, which i s s i m i l a r to the increase seen i n l i g h t l y s tressed normotensive rat s (Nakamura and Nakamura 1978). In young SHR, when plasma norepinephrine i s e l evated , norepinephrine turnover i n the 5 heart i s acce lerated concomitantly with increased catecholamine s y n t h e s i z i n g enzyme a c t i v i t i e s i n blood v e s s e l s . Des truct ion of the c e n t r a l nervous system decreased blood pressure markedly. Furthermore, i n SHR the frequency of sympathetic discharge i s increased at splanchnic nerve endings (Yamori 1984). On the other hand, v a s c u l a r hypertrophy i s noted morphologica l ly three months a f t e r b i r t h . The synthes i s o f both co l lagen and non-col lagen p r o t e i n i s increased i n a r t e r i e s as e a r l y as 4 weeks a f t er b i r t h and i s i n d i c a t e d by increased amino a c i d i n c o r p o r a t i o n , which supposedly i s the biochemical express ion o f an e a r l y s t r u c t u r a l vascu lar change. I t i s probable that an abnormality o f a c e n t r a l mechanism of blood pressure r e g u l a t i o n r e s u l t s i n increased sympathetic outflow and elevated blood pressure (Lovenberg, 1987). Both the e l e v a t i o n o f blood pressure i t s e l f and the increased sympathetic tone acce l era te vascu lar p r o t e i n synthes i s . These processes, from a c t i v a t i o n o f v a s c u l a r p r o t e i n synthesis to s t r u c t u r a l vascu lar changes, which are a l s o observed i n other forms of experimental hypertension (Lovenberg, 1987), are considered to be the f i n a l common pathway, whatever the i n i t i a t i n g mechanism o f hypertension may be (Yamori 1984). The mechanism of the development of sus ta ined hypertension poss ib ly invo lves n e u r a l l y induced s t r u c t u r a l changes o f blood vesse l s as we l l as a g e n e t i c a l l y determined a l t e r a t i o n o f blood v e s s e l s . The l a t t e r i s suggested by the increased growth and migrat ion ra te o f SHR smooth muscle c e l l s . The occurrence of card iac hypertrophy i n hypertension was genera l ly considered to be secondary to hypertension both i n man and i n animal models. Since vascu lar wal l hypertrophy and c a r d i a c hypertrophy occur at an e a r l y stage i n SHR, the poss ib l e involvement o f genet ic fac tors has to be cons idered. During the observat ion of SHR and WKY i n the age range of 6 4-16 weeks, i t became evident that the heart /body weight r a t i o i n SHR tended to be higher even at the age of 4 weeks, i . e . before the development o f hypertension (Yamori 1984). There are severa l areas i n which d i f f erences between the SHR and i t s normotensive c o n t r o l are s i g n i f i c a n t with regard to the pathogenesis of hypertens ion . These areas are at the l e v e l o f sympathetic nerve a c t i v i t y , the vascu lar s t ruc ture and rate of p r o t e i n synthes is i n the vasculature , and some defect i n the system for mainta in ing i o n i c homeostasis with var ious c e l l types (Lovenberg 1987, Yamori 1984, E l - M a l l a k h 1986). The r i s e i n blood pressure i n SHR i s due p r i m a r i l y to an increase i n general v a s o c o n s t r i c t i o n of a r t e r i o l e s without any l a r g e drop i n card iac output (Tobian I 9 8 I ) . Genetic inf luences make some humans and some rats e s p e c i a l l y suscept ib le to NaCl- induced hypertens ion . These genetic features would cause the kidney to have a slow rate o f n a t r i u r e s i s and the blood pressure to r i s e r a p i d l y when the load of body Na i s increased (Tobian 1981). Rodrigues and McNe i l l (1986) observed no d i f f e r e n c e i n cardiac funct ion between normotensive Wistar , WKY and spontaneously hypertensive r a t s . The 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 at the same values i n i s o l a t e d perfused working heart preparat ions from these r a t s . Previous r e s u l t s (Noresson et a l . 1979. F r i b e r g et a l . 1985. F r i b e r g and Noridborg 1986) showed i s o l a t e d hypertrophied SHR l e f t v e n t r i c l e s with an improved v e n t r i c u l a r performance at high a f ter loads compared with non-hypertrophied normotensive c o n t r o l s , and l a r g e l y i n proport ion to the degree o f hypertrophy. These authors suggested that SHR associated card iovascu lar s t r u c t u r a l adaptations develop 7 more gradua l ly than adaptations i n experimental secondary renal hypertension i n r a t s . B. S treptozotoc in (STZ) induced d i a b e t i c r a t s The most commonly used models i n d i a b e t i c s tudies inc lude STZ and a l loxan induc t ion of diabetes i n r a t s . STZ i s the most widely used diabetogenic agent. I t s e l e c t i v e l y destroys pancreat i c fJ -ce l l and produces a d i a b e t i c s ta t e , the s e v e r i t y of which can be v a r i e d by a l t e r i n g the dose of STZ. With time, e .g . a f t e r 4-6 weeks o f i n j e c t i o n , ra t s so treated develop biochemical and func t iona l myocardial abnormal i t ies which appear to be a r e s u l t o f the drug-induced hypoinsul inemia ra ther than a d i r e c t e f f ec t o f the drug i t s e l f (McNei l l and T a h i l i a n i 1986) . STZ contains a N-methylni trosourea moiety and i s an a l k y l a t i n g agent. I t i s suggested that STZ i n i t i a t e s i t s cy to tox ic ac t i on by a l k y l a t i n g e f f ec t s (F i scher 1985). The glucose moiety i n the STZ molecule may have a s p e c i f i c a f f i n i t y for the pancreat i c f i - c e l l , because STZ concentrates i n pancreat i c i s l e t s (F i scher 1985)• Some evidence i n d i c a t e s that oxygen-free r a d i c a l s may also be invo lved i n STZ induced diabetes (Wilson et a l . 1984, F i s c h e r 1985, Sandler et a l . I983). The abnormal i t ies o f myocardial func t ion i n STZ d i a b e t i c ra t s have been addressed i n the l a s t s e c t i o n . C . Hypertensive d i a b e t i c r a t model The inc idence o f hypertension i s h igher i n pat i en t s with diabetes , and diabetes i s frequent ly assoc iated with hypertension which increases the frequency and s e v e r i t y of the vascu lar l e s i o n s (Kannel 1978, Kannel and McGee 1979)• Since comparable pathophys io log ic s tud ies on a large number o f hypertensive d i a b e t i c humans are d i f f i c u l t , adequate animal models with 8 both diabetes and hypertension are needed f o r experimental s tudies on i n t e r a c t i o n between hypertension and d iabetes . Factor and co-workers superimposed STZ induced diabetes (8 weeks) on renovascular hypertension and found that the combination had more pronounced h i s t o l o g i c , u l t r a - s t r u c t u r a l , and mechanical e f f ec t s on the myocardium than d i d hypertension alone (Factor et a l . 198l, 1983; Fe in et a l . 1984). These f indings suggested that hypertension may be c a u s a l l y r e l a t e d to d i a b e t i c cardiomyopathy. They provided evidence that the combination o f diabetes and hypertension produces greater myocardial f i b r o s i s and degeneration than was seen with e i t h e r disease alone, f indings which were s i m i l a r to t h e i r e a r l i e r s tudies on h y p e r t e n s i v e - d i a b e t i c pat i ents (Factor et a l . 1980). The v e n t r i c u l a r p a p i l l a r y muscles from hyper tens ive -d iabe t i c ra t s showed a marked slowing of i sometr ic and i s o t o n i c contract ions and profound e l e c t r o p h y s i o l o g i c a l changes (Fein et a l . 1984). These authors considered that the combination o f renovascular hypertension and diabetes m e l l i t u s i n the r a t represented a preparat ion of congestive cardiomyopathy assoc iated with s i g n i f i c a n t m o r t a l i t y and the development of congestive heart f a i l u r e . In ra t s s i m i l a r l y treated with STZ but a l so subjected to two-kidney, one c l i p renohypertension there was an at tenuat ion i n a r t e r i a l blood presure l e v e l s , accompanied by lower plasma aldosterone l e v e l s (Ramos 1988). The hypertensive d i a b e t i c model developed by Sato et a l . (1987) by i n j e c t i n g STZ subcutaneously i n t o neonatal SHR and WKY, showed mi ld i n s u l i n d e f i c i e n c y and mi ld hyperglycemia at the age o f three to four months. SHR showed higher glucose l e v e l s than WKY, and a greater s e n s i t i v i t y to the tox i c e f f e c t s o f STZ as suggested by morphologic s tudies of pancreas. The development of hypertension was not acce l era ted i n the SHR compared with 9 the respec t ive nontreated c o n t r o l s . D i a b e t i c nephropathy was produced p a r t i c u l a r l y i n STZ treated SHR at the age o f s i x months, as shown by increased kidney weight/body weight r a t i o , increased plasma urea n i trogen and hypoalbuminemia due to p r o t e i n u r i a p lus an e a r l y stage of d i a b e t i c g lomeru losc l eros i s i n almost a l l g lomerul i o f these r a t k idneys . By using a s i m i l a r model, Iwase et a l . (1989) showed that i n s u l i n p o s i t i v e ( i -ce l l s and pancreat ic i n s u l i n content were markedly reduced i n neonata l ly STZ-treated SHR, whereas i n WKY these changes were l e s s severe. These f indings were compatible with t h e i r plasma glucose (SHR > WKY) and i n s u l i n (SHR < WKY) l e v e l s , r e s p e c t i v e l y . The mechanisms of these s t r a i n d i f f erences have not been e l u c i d a t e d . These authors (Sato et a l . 1987, Iwase et a l . 1987. 1989) considered t h i s neonatal STZ treated SHR model as a model for non-insul in-dependent diabetes m e l l i t u s , s ince these STZ treated SHR developed mi ld d i a b e t i c symptoms with hypertension and mi ld d i a b e t i c g l o m e r u l o s c l e r o s i s . They thus provide a model for s tudying the synergism between hypertension and diabetes m e l l i t u s . These s tudies i n d i c a t e d that SHR are more suscept ib l e to STZ-induced diabetes than WKY. The reduced capac i ty f o r p - c e l l regenerat ion i n SHR might account f o r t h i s d i f f erence (Iwase et a l . 1 9 8 7 ) . Cardiac funct ion i n t h i s model has not been s tud ied . Another hyper tens ive -d iabe t i c model was developed by i n j e c t i n g STZ to adul t (9 -15 week old) SHR ra t s (Rodgers 1985, Rodrigues and M c N e i l l I986). Twelve weeks a f t e r STZ i n j e c t i o n SHR and Wistar d i a b e t i c r a t s exh ib i ted a depressed 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 when compared with c o n t r o l s . Various parameters o f heart funct ion showed h i g h l y s i g n i f i c a n t d i f ferences between SHR d i a b e t i c ra t s and a l l other groups and there was an increased m o r t a l i t y (46% i n 12 weeks) i n SHR i n t h i s group. However, WKY d i a b e t i c ra t s d i d not show t h i s depression i n 10 l e f t v e n t r i c u l a r developed pressure (LVDP). I t was suggested that the normal l i p i d metabolism and thyro id s tatus could e x p l a i n the lack of card iac dysfunct ion i n WKY d i a b e t i c r a t s (Rodrigues and M c N e i l l 1986). Other r e s u l t s a l so showed that diabetes s e l e c t i v e l y reduced contrac t ion e f f i c i e n c y , c o n t r a c t i l i t y and LVDP o f the hearts o f SHR but not o f WKY. The p o s s i b l e c o n t r i b u t i o n of hypothyroidism to t h i s observat ion was discussed (Rodgers 1 9 8 5 ) . I I I . Diabetes and A l t e r e d C a + + Metabolism i n Heart Evidence for an abnormal myocardial c e l l func t ion i n diabetes m e l l i t u s , in f luenced by metabolic changes, has appeared wi th in recent years . Experimental s tudies at the c e l l u l a r l e v e l have provided data for severa l p o s s i b l e explanat ions . These are concerned with the i n t r a c e l l u l a r C a + + homeostasis and transsarcolemmal receptor s i g n a l l i n g (Dhal la et a l . 1 9 8 5 , T a h i l i a n i and M c N e i l l 1 9 8 6 ) . Calcium movements are c l o s e l y r e l a t e d to c a r d i a c e l e c t r o -p h y s i o l o g i c a l events , c o n t r a c t i l e f u n c t i o n , membrane i n t e g r i t y and energy metabolism. During card iac e x c i t a t i o n the e x t r a c e l l u l a r C a + + d i f fuses in to the c e l l s through a c t i v a t e d C a + + channels , and p o s s i b l y by means of reversed N a + - C a + + exchange (Noble 1 9 8 3 , Sheu et a l . 1 9 8 6 ) . Th i s s h i f t br ings about a t rans i en t increase i n sarcoplasmic C a + + concentrat ion which may a c t i v a t e d i r e c t l y the c o n t r a c t i l e p r o t e i n s , but which a l so induce re lease o f C a + + s tored i n SR (Fabiato 1 9 8 3 ) . Hence the t r a n s i e n t C a + + increase o r i g i n a t e s from two sources: e x t r a c e l l u l a r and i n t r a c e l l u l a r . C o n t r a c t i l e force i s p r o p o r t i o n a l to the amount o f C a + + re leased in to the sarcoplasm from both sources (Yue 1 9 8 7 ) • The re leased C a + + i s qu ick ly pumped back i n t o the SR and/or out o f the c e l l to the e x t r a c e l l u l a r space. 11 The l a t t e r i s accomplished by the N a + - C a + + exchange system working i n an 'out' d i r e c t i o n and by the SL C a + + - A T P a s e . These processes act i n concert both to regulate i n t r a c e l l u l a r C a + + concentrat ion and to modulate C a + + dependent events (Janis e t a l . 1987). Several abnormal i t ies i n the i n t r a c e l l u l a r handl ing o f C a + + have been suggested i n myocardium from d i a b e t i c r a t s . Myocardia l enzyme systems and s u b c e l l u l a r organel les are a f f ec ted . SR, which acts as a C a + + s tore and which takes up and releases C a + + on a beat - to-beat b a s i s , i s impaired (Lopaschuk et a l . 1983) i n diabetes . Th i s may be explained by the increased myocardial cardiac l e v e l o f long chain a c y l c a r n i t i n e , a metabolic intermediate which transports f a t t y ac ids i n t o the mitochondria . Long chain a c y l c a r n i t i n e s have a l so been shown to s t rong ly i n h i b i t C a + + uptake i n i s o l a t e d SR and also to i n h i b i t N a + / K + ATPase. The sarcolemmal C a + + pump a c t i v i t y i s a l so depressed (Heyl iger et a l . 1987; T a h i l i a n i and M c N e i l l 1986). Thus the e f f l u x of C a + + through SL i s decreased. Mi tochondr ia l abnormal i t ies have a l so been reported (Dhal la et a l . 1 9 8 5 ) . Mitochondria serve as a r e s e r v o i r f or C a + + accumulation, but t h e i r r o l e i n diabetes i s not c l e a r . SR and SL organel les are i n t i m a t e l y involved with the c o n t r o l o f C a + + m o b i l i t y , and i f they are impaired , an abnormal b u i l d -up o f C a + + i n the myocardium may occur a long with the a l t e r a t i o n s i n e x c i t a t i o n , c o n t r a c t i o n , and r e l a x a t i o n . The i n o t r o p i c response to C a + + was reported to be increased i n v e n t r i c u l a r s t r i p s from d i a b e t i c Sprague Dawley r a t s (Ramanadham and Tenner 1986). Whether t h i s phenomenon ex i s t s i n other card iac t i s sues or other animal models i s unknown. The mechanism of these events requires fur ther i n v e s t i g a t i o n . Cardiac ^-adrenoceptor funct ion i n the d i a b e t i c animals i s af fected i n terms o f depressed p-receptor number and response to agonist s t imula t ion 12 (Ramanadham and Tenner 1987, Hey l iger et a l . 1982, Gotzsche 1983). An i n t e r e s t i n g phenomenon i s the increase i n myocardial s e n s i t i v i t y to C a + + from d i a b e t i c r a t s o c c u r r i n g independently o f s e n s i t i v i t y changes to $-adrenoceptor agonist i soprotereno l s t i m u l a t i o n (Ramanadham et a l . , unpublished observat ion) . On the other hand, the response to ct-agonists has been shown to increase (Jackson et a l . 1985. Canga and Borda 1986, Ramanadham and Tenner 1987) or to decrease (Heyl iger et a l . 1982) while the receptor number decreased (Williams et a l . 1983. H e y l i g e r et a l . 1982, L a t i f p o u r and M c N e i l l 1984). Recent evidence shows that card iac a-adrenergic a c t i v a t i o n by catecholamines r e s u l t s i n s t i m u l a t i o n of phosphoinos i t ide h y d r o l y s i s , which i n t u r n , may t r i g g e r C a + + m o b i l i z a t i o n i n myocytes (van Zwieten and Timmermans 1987, Nicho l s e t a l . 1988). The exact metabolic steps involved i n t h i s process are not c l e a r although there has been tremendous i n t e r e s t i n t h i s f i e l d . I t i s o f i n t e r e s t to i n v e s t i g a t e the r e l a t i o n s h i p between the a-adrenoceptor and the C a + + response s ince i t w i l l enhance our understanding of the mechanism(s) of a-adrenergic a c t i v a t i o n i n the heart as w e l l as p r o v i d i n g f u r t h e r information regarding diabetes- induced cardiomyopathy. IV. The Force-Frequency Re la t ionsh ip and A c t i v i t i e s o f Ryanodine The force-frequency r e l a t i o n s h i p seems to be an i n t r i n s i c mechanism r e g u l a t i n g the mechanics of the hear t . I t i s accomplished under p h y s i o l o g i c a l condi t ions and without any important c o n t r i b u t i o n on the part of the e x t r a c a r d i a c regulatory system. Post quiescent p o t e n t i a t i o n (PQP) and post s t i m u l a t i o n p o t e n t i a t i o n (PSP), two parameters to be used i n our experiment, i n fac t are assoc iated with two very o l d concepts, the Woodworth s t a i r c a s e and Bowditch s t a i r c a s e (Woodworth 1902, Bowditch I87I). 1 3 PQP, or Woodworth s t a i r c a s e which i s a l so named r e s t p o t e n t i a t i o n , i s an enhanced c o n t r a c t i o n fo l lowing a long pause. PSP, or Bowditch s t a i r c a s e which i s a l so named p o s t - e x t r a s y s t o l i c p o t e n t i a t i o n i s expressed by an enhanced contrac t ion r e s u l t i n g from a r a p i d success ion of contract ions and i s due to an increase i n the i n f l u x of e x t r a c e l l a r C a + + . Woodworth (1902) descr ibed the two phenomena which inf luence the c o n t r a c t i o n of mammalian heart : The strength of the heart beat i s the r e s u l t o f the s t i m u l a t i n g e f f e c t o f r a p i d successions o f contract ions and the recuperat ive e f f e c t of a long pause. These two phenomena i n d i c a t e C a + + re lease from i n t r a c e l l u l a r s tores and i n f l u x of e x t r a c e l l u l a r C a + + . T h i s mechanism was supported by l a t e r s tudies (Hajdu 1969, Langer 1983). Therefore they provide i n s i g h t i n t o the most important funct ion of the card iac myocytes with poss ib le p r a c t i c a l i m p l i c a t i o n s . The t r a n s i t i o n between the two steady s tates ( in p a r t i c u l a r a c t i v i t y and rest ) proved to be a very u s e f u l experimental too l i n the d i a b e t i c study. Ryanodine, a s p e c i f i c SR antagonist (see D i s c u s s i o n ) , i s a l so known to be a very use fu l too l i n e l u c i d a t i n g SR funct ion and C a + + m o b i l i z a t i o n i n d i f f e r e n t c e l l s . I t e i t h e r ac t iva tes or i n h i b i t s the C a + + re lease channel i n c a r d i a c SR at d i f f e r e n t concentrat ions (Meissner 1986). In the presence o f ryanodine, i t i s suggested that the C a + + c o n t r a c t i o n i n ra t myocardium i s 90% from SR (Lucas and Bose 1985) . In our experiments, ryanodine was used to determine the a b i l i t y o f SR to re lease C a + + for c o n t r a c t i o n i n c o n t r o l and d i a b e t i c r a t heart p r e p a r a t i o n s . V. Spe c i f i c Aims The o b j e c t i v e of the present study i s to descr ibe the features of the a l t e r e d C a + + m o b i l i z a t i o n i n d i a b e t i c myocardium, i n order to further 14 understand the mechanism of d i a b e t i c cardiomyopathy. The e l u c i d a t i o n of the mechanism(s) of many important b i o l o g i c a l process has been acce lerated by the use o f pharmacological agents that s e l e c t i v e l y a f f e c t s p e c i f i c steps of the process . Therefore , card iac drugs, with d i f f e r e n t mechanism(s) of a c t i o n that in f luence C a + + m o b i l i t y were chosen to i n v e s t i g a t e i s o l a t e d myocardial t i s sue responses from d i a b e t i c and hypertens ive d i a b e t i c r a t s . The aims o f th i s study were to address the f o l l o w i n g quest ions: 1 . Is the a b i l i t y of i s o l a t e d card iac preparat ion obtained from d i a b e t i c animals to handle and/or to u t i l i z e C a + + changed? 2. Does t h i s change have any relevance on the adrenoceptor response? 3. Is the changed response to C a + + and to c a r d i a c drugs also evident i n other c l o s e l y r e l a t e d card iac dysfunct ion s tates? Attempts were made to: 1. Estimate the amount o f i n t r a c e l l u l a r s tores o f re l easab le C a + + and C a + + i n f l u x by SL N a + - C a + + exchange. 2. Obtain dose-response curves to var ious agonists which promote C a + + movement and c o n t r a c t i o n . 3. Determine the a b i l i t y of a C a + + channel antagonist to i n h i b i t response to C a + + . k. Assess the e f f ec t s o f an SR antagonist on SR C a + + re lease and contrac t ion generat ion. 5- Invest igate the occurrence o f a s i m i l a r s e n s i t i v i t y and response i n other models of cardiac dys funct ion . 15 MATERIALS AND METHODS I. MATERIALS A. Animals Male Wis tar , SHR and WKY r a t s , obtained from Charles R i v e r , Canada, 200-225 gm were used i n the study. B. Chemicals and assay k i t s Chemicals and assay k i t s were purchased from the f o l l o w i n g sources: 1. BDH Chemicals, Vancouver. Buf fer chemicals: calc ium c h l o r i d e , magnesium c h l o r i d e , potassium c h l o r i d e , sodium bicarbonate , sodium c h l o r i d e , and d-glucose . 2. Sigma Chemical Company. s t r e p t o z o t o c i n , d ,1 - i soprotereno l h y d r o c h l o r i d e , 1-phenylephrine h y d r o c h l o r i d e , d , l - p r o p r a n o l o l h y d r o c h l o r i d e , and ( ± ) - v e r a p a m i l hydroch lor ide . 3. Amersham Company. I n s u l i n , T^ and T/j RIA k i t . 4. Boehringer-Mannheim. Peridochrom^ Glucose GOD-PAP assay k i t , Peridochrom R t r i g l y c e r i d e s GPO-PAP assay k i t , C-system c h o l e s t e r o l CHOP-PAP assay k i t , and Test-combinat ion phosphol ip id assay k i t . 5. Merck Sharp and Dohme Research Lab, D i v i s i o n of Merck and Company. Ryanodine. 6. Bayer Leverkusen. BAY K 8644 (generous g i f t from Dr. G Franckowiak). 7. E l i L i l l y Canada Inc. T e s - T a p e R . 16 I I . METHODS A. Induction of Experimental Diabetes Diabetes was induced by a s i n g l e t a i l ve in i n j e c t i o n of STZ (55 mg/kg) d i s s o l v e d i n 0.3% NaCl s o l u t i o n . C o n t r o l r a t s were i n j e c t e d with v e h i c l e a lone . The i n j e c t i o n was performed a f t e r anaes the t i z ing the ra t by halothane inhalation. Animals were maintained with free access to food and water f o r up to s i x weeks. B. V e r i f i c a t i o n of the Diabetic State Animals were tested for diabetes three days a f t e r i n j e c t i o n by checking for g l u c o s u r i a us ing the ezymatic t e s t s t r i p , L i l l y Tes-Tape . Other i n d i c a t i o n s of diabetes which were monitored were as fo l lows: body weight, plasma glucose and i n s u l i n . Plasma t r i g l y c e r i d e , c h o l e s t e r o l , phospho l ip id , T^ and were a l so measured. Blood samples were c o l l e c t e d at the time o f s a c r i f i c e by d e c a p i t a t i o n . The radioimmunoassay or biochemical measurement by us ing commercial assay k i t was performed according to the corresponding manual. C. Pharmacological Experiments 1. I s o l a t e d t i s sue preparat ions At the time of s a c r i f i c e , the hearts were exc i sed and immediately placed i n cold-oxygenated Chenoweth-Koelle (CK) buf fer of fo l lowing composit ion (mm): NaCl 120, KC1 5.6, MgCl-6H 2 0 2.1, C a C l 2 - 2 H 2 0 2.2, NaHCO^ 1 .9. and glucose 10. A f t e r express ing blood from the hear t , the r i g h t and l e f t a t r i a were c a r e f u l l y excised from v e n t r i c l e s . The r i g h t v e n t r i c u l a r s t r i p s and l e f t v e n t r i c u l a r p a p i l l a r y muscles were obtained as w e l l . Each t i s sue was then mounted on a s t i m u l a t i n g e l ec t rode ( r i g h t a t r i a were mounted on a t i s sue h o l d e r ) . The other end o f the t i s sue was attached to a Grass FT3 tens ion transducer connected to a Grass Model 7D polygraph e ight -17 channel recorder . Perfus ion was c a r r i e d out i n a 20 ml t i s sue bath with CK b u f f e r , pH 7.4 (changed every 15 min), at 37°C, oxygenated with 95% 0 2/5# CO2, under the fo l lowing r e s t i n g tens ions: a t r i a 1.0 gm, p a p i l l a r y muscle 1.5 gm, and r i g h t v e n t r i c u l a r s t r i p 2 . 0 gm. At the end of a one-hour e q u i l i b r a t i o n p e r i o d , the t i s sues were s t imulated by a Grass SD9 s t imulator at a frequency o f 1.0 Hz, 1-5 v o l t s , and a durat ion of 5 msec, except that the r i g h t a t r i a was allowed to beat spontaneously. A f t e r 15 min of s t i m u l a t i o n var ious experimental procedures were i n i t i a t e d . Basal developed force and basa l a t r i a l rate were recorded before the experimental procedures. 2. Dose responses of i s o l a t e d t i s sues to card iac drugs The card iac t i s sue preparat ions were exposed to var ious i n o t r o p i c and chronotropic agents, which i n c l u d e : (1) i s o p r o t e r e n o l , a ^-adrenergic agonist producing i n o t r o p i c e f f ec t s through a cAMP dependent mechanism. (2) phenylephrine i n the presence o f p r o p r a n o l o l (10~^M with pretreatment of 10 min.) an a^-adrenergic agonist producing c o n t r a c t i o n by enhancing the i n f l u x o f ca lc ium. (3) ca lc ium, which produces c o n t r a c t i o n by en ter ing the c e l l v i a a voltage-dependent mechanism. (4) BAY K 8644, a d ihydropyr id ine d e r i v a t i v e with n i f e d i p i n e -antagonist p r o p e r t i e s , which promotes calc ium i n f l u x l ead ing to c o n t r a c t i o n . (5) verapami l , an L-type calcium channel b locker (See D i scuss ion ) . Drugs (1) to (4) were tested for p o s i t i v e i n o t r o p i c and chronotropic e f f e c t s . Verapamil was tested for i t s negative i n o t r o p i c e f f e c t s . Force and rate developed with each dose o f the agent were recorded at 3 min 1 8 i n t e r v a l s except for BAY K 8644 and verapamil which were monitored every 20 min. The dose range and i n t e r v a l s were determined by pre l iminary experiments. 3- The e f f ec t s of ryanodine on the post -quiescent p o t e n t i a t i o n (PQP) and  p o s t - s t i m u l a t i o n p o t e n t i a t i o n (PSP) (1) Recording o f post -quiescent p o t e n t i a t i o n (PQP) PQP has been used to r e f l e c t the degree o f s tored calcium a v a i l a b l e for contrac t ion (Lukas and Bose, 19-86) . To obta in PQP's, s t i m u l a t i o n of t i s sues was terminated a f t e r 15 min o f e l e c t r i c a l s t i m u l a t i o n . The increase from the BDF at f i r s t c o n t r a c t i o n a f t e r 1 min of r e s t fo l l owing the resumption o f s t i m u l a t i o n i s recorded as PQP ( F i g . 1A). (2) Recording of p o s t - s t i m u l a t i o n p o t e n t i a t i o n (PSP) PSP was used as an i n d i c a t o r o f calc ium i n f l u x a f t e r high frequency s t i m u l a t i o n and as an index for N a + - C a + + exchange a c t i v i t y i n sarcolemma (Lukas and Bose, 1986). To record the PSP the frequency of s t i m u l a t i o n was changed from 1 Hz to 10 Hz f o r 15 sec . The p o t e n t i a t i o n from the BDF at the f i r s t contrac t ion a f t e r changing the frequency back to 1 Hz was recorded as the PSP ( F i g . I B ) . (3) The e f f ec t s o f ryanodine Ryanodine i s a p lant a l k a l o i d which s p e c i f i c a l l y increases or block the re lease of calcium from SR at d i f f e r e n t concentrat ions (Meissner 1986). Cardiac t i s sues were exposed to 3xl0~^M f o r 20 min (concentrat ion determined by a pre l iminary experiment) before r e c or d in g PQP and PSP. D. S t a t i s t i c a l a n alysis The data are expressed as mean ± standard e r r o r of the mean (SE). S t a t i s t i c a l s i g n i f i c a n c e was determined by a two-way a n a l y s i s of variance 19 Figure 1. Measurement o f post quiescent p o t e n t i a t i o n (PQP, A ) , and post s t i m u l a t i o n p o t e n t i a t i o n (PSP, B) i n r a t p a p i l l a r y muscle. A. The p o t e n t i a t i o n from basa l developed force at the f i r s t contrac t ion a f t e r r e s t f o l l o w i n g the resumption of s t imula t ion was recorded as PQP. B. To record PSP, the frequency o f s t i m u l a t i o n was changed from 1 Hz to 10 Hz f o r 15 sec . The p o t e n t i a t i o n from basal developed force o f the f i r s t c o n t r a c t i o n a f t er changing the frequency back to 1 Hz was recorded as PSP. 20 fol lowed by a one-way ANOVA and the Newman-Keuls m u l t i p l e range t e s t . The l e v e l o f s t a t i s t i c a l s i g n i f i c a n c e was set at a p r o b a b i l i t y of l e s s than 0.05 (P < 0.05). 21 RESULTS I . INDICATION OF DIABETES MELLITUS STZ-trea ted animals exh ib i t ed p o l y u r i a , p o l y d i p s i a , and hyperphagia. G l y c o s u r i a was greater than 2% (w/v)in a l l STZ i n j e c t e d r a t s . Body weight and plasma i n s u l i n decreased and blood glucose increased (Figures 2,3) i n S T Z - d i a b e t i c animals compared with age-matched c o n t r o l s . These changes were more prominent i n SHR d i a b e t i c r a t s . S i g n i f i c a n t d i f f erences could be seen i n body weight, blood glucose and i n s u l i n between SHR and Wistar d i a b e t i c r a t s . Hyper l ip idemia was found i n Wistar and SHR d i a b e t i c ra t s (Figure 4) . WKY d i a b e t i c ra t s d i d not show any s i g n i f i c a n t change i n blood l i p i d s measured i n comparison with WKY c o n t r o l s . The l e v e l s of plasma t r i g l y c e r i d e and c h o l e s t e r o l i n SHR were much higher than those i n e i t h e r i t s own c o n t r o l s or other d i a b e t i c r a t s . However phosphol ip id l e v e l s i n the SHR d i a b e t i c group were not changed when compared to the SHR contro l s and were lower than those found i n Wistar d i a b e t i c r a t s . These data are i n good agreement with previous work done i n our l a b o r a t o r y (Rodrigues et a l . 1985) • Plasma concentrat ions of T^ and T/j were decreased i n a l l three s t r a i n s o f d i a b e t i c ra t s (Figure 5)- C e r t a i n features of c o n t r o l and 6-week d i a b e t i c r a t s are shown i n Tables 1 and 2. I I . BASAL PARAMETERS FROM ISOLATED CARDIAC PREPARATIONS Basal developed force (BDF) i n three kinds o f preparat ions are shown i n F igure 6. BDF was higher i n d i a b e t i c Wistar and WKY r i g h t v e n t r i c l e (RV) than i n i t s corresponding c o n t r o l i n accordance with the f indings by Ramanadham and Tenner (1983) i n Sprague Dawley r a t s . However, there were 22 Figure 2. Body weights of c o n t r o l and 6-week d i a b e t i c r a t s . con c o n t r o l , d i a d i a b e t i c , p < 0.05: * vs c o n t r o l of same s t r a i n ; * # vs Wistar d i a b e t i c ; * * * vs WKY d i a b e t i c ; # vs Wistar c o n t r o l , n = 20-27-23 O 60 E WISTAR S H R W K Y WISTAR S H R W K Y G L U C O S E INSULIN Figure 3. Plasma concentrat ions o f glucose and i n s u l i n i n c o n t r o l and 6-week d i a b e t i c r a t s . con c o n t r o l , d i a d i a b e t i c , p < 0.05: * vs c o n t r o l o f same s t r a i n ; * * vs Wistar d i a b e t i c ; * * * vs WKY d i a b e t i c ; # vs Wistar c o n t r o l ; ## vs WKY c o n t r o l , n =20-27. 24 • T R I G L Y C E R I D E S • • C H O L E S T E R O L ( S 3 P H O S P H O L I P I D S "I WIC 1 WID J , I S H C ** * * * l * * S H D I W K C A W K D Figure 4. Plasma consentrat ions of l i p i d s i n c o n t r o l and 6-week d i a b e t i c r a t s . WIC Wistar c o n t r o l , WID Wistar d i a b e t i c , SHC SHR c o n t r o l , SHD SHR d i a b e t i c , WKC WKY c o n t r o l , WKD WKY d i a b e t i c , p < 0.05: * vs c o n t r o l o f same s t r a i n ; * * vs Wistar d i a b e t i c ; * * * vs WKY d i a b e t i c , n = 20-27-25 60 2.0 45 X o 30 15 0 WISTAR S H R W K Y WISTAR S H R W K Y T z Figure 5. Plasma concentrat ions of T^/T/j i n c o n t r o l and 6-week d i a b e t i c r a t s . con c o n t r o l , d i a d i a b e t i c , p < 0.05: * vs same s t r a i n c o n t r o l ; ** vs Wistar d i a b e t i c ; * * * vs WKY d i a b e t i c . ' n = 20-27. Table 1. Body Weight, Plasma Glucose and Blood L i p i d s Levels of Contro l and 6-week Diabe t i c Rats . WISTAR SHR WKY Contro l (n=23) Diabet ic (n=24) Contro l (n=20) Diabet i c C o n t r o l (n=27) (n=20) Diabet i c (n=20) Body Weight (gm) 439*7 311*7* 334±7 170±6* 3l8±6 230±6* Plasma Glucose (mM) 6.43±0.4o 19.21±0.39* 6.67±0.43 2i .43±0.37* 5-85*0.33 16.53*0.33 T r i g l y c e r i d e s (mM) l . 6 l ± 0 . 4 5 3-69*0.43* l .27±0 .48 9-73*0.41* 1 .52*0.33 2.36±0.33 C h o l e s t e r o l (mM) 1.48±0.12 2.01±0.11* 1.37*0.12 2.52±0.11* 1 .6 l±0.05 l . 9 l ± 0 . 0 5 Phosphol ipids (mM) 0.80±0.13 1 .24*0.13* 0.54±0.l4 0 .61±0.12 0.59*0 .14 0-30±0.l4 Values are expressed by M±SE; n , number of animals; * 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 o f the same s t r a i n , p < 0 . 0 5 -Table 2. I n s u l i n and Thyro id Hormone Levels of Contro l and 6-week D i a b e t i c Rats . WISTAR SHR WKY Control Diabet ic Contro l D i a b e t i c Contro l D iabet i c (n=23) (n=24) (n=20) (n=27) (n=20) (n=20) I n s u l i n (uunit/ml) 48.5*2.5 24.1±2* 3^.1*2.4 18.5*2.1* 45-8±2 .5 21.9*2.3* T 3 (nM) 0.94*0.04 0 .67*0 .05* 0 .91*0 .05 0.66*0.04* 0 .90±0 .05 0.73*0.05* T 4 (nM) 46.9*1-5 26.1*1.5* 35-9*1.6 17.3*1-4* 33-1*1-6 26.6*1.6* Values are expressed by M±SE; n , number of animals; * 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 of same s t r a i n , p < 0 . 0 5 -28 2.500 o 1.500 a: o d 1.000--0.500 0.000 WIS S H R W K Y RV WIS S H R W K Y P M WIS S H R W K Y LA Figure 6. Basa l developed force of heart preparat ions from c o n t r o l and 6-weeks d i a b e t i c r a t s . con c o n t r o l , d i a d i a b e t i c , p < 0.05: * vs same s t r a i n c o n t r o l ; * * vs Wistar d i a b e t i c ; ## vs WKY c o n t r o l , n = 8-21. 2 9 no d i f f erences i n BDF o f PM and LA obtained from between d i a b e t i c and c o n t r o l r a t s . Bradycardia was found i n a l l three s t r a i n s o f d i a b e t i c ra t s (Figure 7)- BDF and basal a t r i a l rate (BAR) values are shown i n Table 3-I I I . PRE-DRUG MEASUREMENTS OF PQP AND PSP A. The Time Courses of PQP and PSP The amplitude of PQP was measured as a func t ion o f d i f f e r e n t durat ion of r e s t p e r i o d s . PQP was expressed by the r a t i o (PQP/BDF) to normalize d i f f e r e n t pre te s t contrac t ion amplitudes. F igures 8 and 9 show the PQP time course of PM and LA i n c o n t r o l and d i a b e t i c Wistar r a t s . BDF was not changed s i g n i f i c a n t l y during the tes t p e r i o d . In both c o n t r o l and d i a b e t i c t i s s u e s , the maximum PQP/BDF values were reached at time po ints between one and two minutes. One minute was chosen as the opt imal po in t o f measurement f o r PQP. Both PM and LA preparat ions showed a s i m i l a r tendency to increase PQP with increases i n the durat ion o f r e s t p e r i o d and to dec l ine a f t e r the opt imal p o i n t , except that PM appears to have a higher maximum tension and to d e c l i n e more q u i c k l y than LA. D iabe t i c PM showed s i g n i f i c a n t l y lower PQP/BDF r a t i o s than contro l s dur ing the whole time course t e s t i n g . The time courses o f absolute peak tens ion (PSP + BDF) and change i n PSP (PSP/BDF) are shown i n Figures 10 and 11. With an increase o f s t i m u l a t i o n (10 Hz) d u r a t i o n , the amplitude o f peak tens ion dec l ined g r a d u a l l y i n both PM and LA t i s sues . The changes i n PSP stayed at a r e l a t i v e l y constant l e v e l a f t e r a 5 second s t i m u l a t i o n d u r a t i o n . The d e c l i n e o f BDF was a l so observed i n the experiment with increase i n the durat ion o f s t i m u l a t i o n . Cons ider ing t h i s gradual b a s a l tens ion change, i t appears that an increase o f the s t i m u l a t i o n time has no s i g n i f i c a n t in f luence on the amplitude of PSP. F i f t e e n seconds was chosen as the tes t 30 Figure 7. Basa l a t r i a rates o f c o n t r o l and 6-week d i a b e t i c r a t s . con c o n t r o l , d i a d i a b e t i c , p < 0.05: * vs c o n t r o l o f same s t r a i n , n = 6-13-Table 3« Basal Inotropic and Chronotropic Parameters of Cardiac Preparat ions from Contro l and Diabet i c Rats . WISTAR SHR WKY Contro l Diabet ic Control D i a b e t i c Contro l D iabe t i c (n=23) (n=24) (n=20) (n=27) (n=20) (n=20) RV (BDF, gm) 0.67±0.08 1.18±0.09* 0.75*0.10 0.96±0.07 0.68±0.09 0 . 9 9 * 0 . 0 9 * n 23 19 16 27 20 20 PM (BDF, gm) 1.04±0.12 0.95*0.11 0 .83*0.18 0.62±0.21 1 .21±0.18 1 .25*0.22 n 17 21 8 6 8 8 LA (BDF, gm) 0 . 8 6 ± 0 . l l 1.00±0.11 1.05*0.12 0 . 6 3 * 0 . 0 9 * 0.76±0.09 0 . 7 7 * 0 . 0 9 n 12 12 11 8 6 6 RA (BAR,beats/min) 255±11 198±11* 226±12 I88±l4* 240±16 170±l6* n 13 12 11 8 6 6 A l l values are expressed by mean ± standard e r r o r . * p < 0 .05 versus contro l of the same s t r a i n . RV, r i g h t v e n t r i c u l a r s t r i p s PM, p a p i l l a r y muscle LA, l e f t a t r i a RA, r i g h t a t r i a n, number of animals BDF, basal developed force i n grams BAR, basal a t r i a l rate i n beats/min 4 3 2 O •O CON - O DIA 0< - 1 4 1 1 1 1 0 . 0 1 0 0 . 1 0 0 1 . 0 0 0 1 0 . 0 0 0 1 0 0 . 0 0 0 Duration of Rest (min.) Figure 8. E f f e c t of the durat ion o f the t e s t i n t e r v e n t i o n on the magnitude of post quiescent p o t e n t i a t i o n i n Wistar rat p a p i l l a r y muscles. PQP post quiescent p o t e n t i a t i o n , BDF basa l developed force . Resul t s obtained i n Wistar c o n t r o l and d i a b e t i c r a t s , n = 6-12. 33 4 - 1 A 1 1 1 1 0 . 0 1 0 0 . 1 0 0 1 . 0 0 0 1 0 . 0 0 0 1 0 0 . 0 0 0 Duration of Rest (min.) Figure 9. E f f e c t o f the durat iaon o f the t e s t i n t e r v e n t i o n on the magnitude o f post quiescent p o t e n t i a t i o n i n Wistar r a t l e f t a t r i a . PQP post quiescent p o t e n t i a t i o n , BDF basa l developed force . Resul ts obtained i n Wistar c o n t r o l and d i a b e t i c r a t s , n = 6-12. 34 m Q CL CO 00 \ Q _ CL v ~ ' oo -^ £ C L ^ 00 . E 1* ° CL 4 3 2 1 D 0< o A -- O C 0 N , PSP/BDF - • DIA, PSP/BDF A C O N , Peak Tension } O 0 . 0 1 0 0 . 1 0 0 1 . 0 0 0 Duration of Stimulation (min.) 1 0 . 0 0 0 Figure 1 0 . E f f e c t of the durat ion o f the t e s t i n t e r v e n t i o n on the magnitude o f post s t i m u l a t i o n p o t e n t i a t i o n i n Wistar r a t p a p i l l a r y muscle. PSP post s t i m u l a t i o n p o t e n t i a t i o n , BDF basa l developed force . Resul ts obtained i n Wistar c o n t r o l and d i a b e t i c r a t s , n = 6-12. 35 4 3-O OCON, PSP/BDF • 0DIA, PSP/BDF A A C O N , Peak Tension 0 . 1 0 0 1 . 0 0 0 Duration of Stimulation (min.) 1 0 . 0 0 0 Figure 11. Effect of the duration of the test intervention on the magnitude of post stimulation potentiation in Wistar rat left atria. PSP post stimulation potentiation, BDF basal developed force. Results obtained in Wistar control and diabetic rats, n = 6-12. 36 i n t e r v a l i n the l a t e r experiments. There was no s i g n i f i c a n t d i f f e r e n c e of P S P / B D F values between d i a b e t i c and c o n t r o l t i s sues from Wistar r a t s . B. Pre-Drug PQP and PSP i n Different Cardiac Preparations Basal amplitudes of PQP and P S P measured before the incubat ion with ryanodine can be seen i n Tables 4-5, F igures 22-27. Pre -drug P Q P , expressed by i t s r a t i o to BDF ( P Q P / B D F ) , was decreased s i g n i f i c a n t l y i n d i a b e t i c t i s sues o f W i s t a r - P M and S H R - L A but not i n other t i s sues tes ted (Figures 22 and 23, Table 4). Pre-drug P S P , expressed by P S P / B D F , was s i g n i f i c a n t l y decreased only i n the SHR d i a b e t i c p a p i l l a r y muscles (Figures 26 and 27, Table 5). The amplitude of peak tension ( P S P + BDF) was decreased i n SHR d i a b e t i c p a p i l l a r y muscle and WKY d i a b e t i c l e f t a t r i a (Figures 24 and 25, Table 5) showing that these t i s sues have l e s s a b i l i t y to generate P S P , s ince 10 Hz s t imula t ion for 15 seconds had no s i g n i f i c a n t e f f e c t on basal developed force i n a l l the t i s sues tes ted . IV. INOTROPIC AND CHRONOTROPIC RESPONSES TO CARDIAC DRUGS OF ISOLATED HEART PREPARATION A . Dose-Response Curves to Cardiac Drugs E C ^ Q values o f various agonists c a l c u l a t e d from i n o t r o p i c responses i n RV, PM and LA and chronotropic responses i n RA are shown i n Tables 6-8 and the comparison of the drug e f f ec t s on the d i f f e r e n t myocardial t i s sues expressed by pD2 values are shown i n F igures 13, 15, 17, 19 and 21 r e s p e c t i v e l y . Dose-response expressed as change i n developed tension o f RV, to ISO, P E , C a + + , and BAY K 8644 are shown i n F igures 12, 14, 16 and 18, r e s p e c t i v e l y . Responses to the negative i n o t r o p i c e f f ec t s of Table 4 . PQP Related Parameters from Contro l and 6-week Diabet ic Rats . WISTAR SHR WKY Control Diabet ic Control D iabe t i c Contro l D iabe t i c (n=23) (n=24) (n=20) (n=27) (n=20) (n=20) 15 min PQP RV (PQP/BDF) l . l 8 ± 0 . 1 3 0.75*0.15* 1 .43±0.l4 0 .57*0.12* n 23 18 19 26 PM (PQP/BDF) 1 .55±0. l4 0.97±0.15* 0.97±0.25 0.27±0.22* n 18 17 6 7 LA (PQP/BDF) 1 . 9U0 .44 1.46±0.38 0.83±0.40 0 .79*0.44 n 9 12 11 9 1 min PQP PM (PQP) 1.92±0.26 1.10±0.25* 0.84±0.26 0.29±0.28 0.57*0.25 0.93±0.26 (PQP/BDF) 1.23±0.16 0.68±0.15* 0.79*0 .14 0.43±0.15 0.49±0.09 0.68±0.09 n 8 9 8 8 8 8 LA (PQP) 1.17±0 .16 0.93*0.15 0.66±0.l6 0.19*0.16 I . l 4 ± 0 . l 6 0.65*0 .16 (PQP/BDF) 0 .99*0.23 1.00±0.22 0.71*0 .10 0.30±0.11* 2. l4±0.30 1.32±0.30 n 8 9 8 7 8 8 A l l values are obtained by pre-drug measurements and expressed by mean ± standard e r r o r . * p < 0.05 versus contro l of the same s t r a i n . PQP, post quiescent p o t e n t i a t i o n , measured af ter 15 min & 1 min res t r e s p e c t i v e l y as noted. RV, r i g h t v e n t r i c u l a r s t r i p s PM, p a p i l l a r y muscle LA, l e f t a t r i a n, number of animals BDF, basal developed force Table 5« PSP Related Parameters from Contro l and 6-week Diabet i c Rats . WISTAR SHR WKY Contro l Diabet ic Contro l D iabet i c C o n t r o l D iabe t i c PM (PSP) 0.85*0 .14 0.56±0.l4 0.77*0.14 0. ,10+0.15* 0.23±0.l4 0 . 4 3 * 0 . ,14 (PSP+BDF) 2.26±0.38 2.06±0.34 1.57*0.25 0. ,69*0.26* 1 .59*0.26 1.86*0. ,26 (PSP/BDF) 0.55*0.17 0.36*0.16 0.83±0.11 0. ,06±0.04* 0.18*0.11 0 . 3 3 * 0 . 11 n 8 9 • 8 7 7 7 LA (PSP) 0.57*0.10 0.68±0.09 0.43±0.10 0. ,25±0 .09 0.74*0.10 0 . 3 9 * 0 . ,10* (PSP+BDF) 1.75*0.20 1.77*0.19 1.32±0.15 0. ,94*0.16 1.33*0.10 0 . 9 5 * 0 . ,11* (PSP/BDF) 0.73*0.23 0.85*0.23 0.54*0.37 0. ,42±0.39 1.35*0.45 0.68±0. • 52 n 8 9 8 7 8 6 A l l values are obtained by pre-drug measurements and expressed by mean ± standard e r r o r . * p < 0 .05 versus contro l of the same s t r a i n . PSP, post s t imulat ion p o t e n t i a t i o n , measured a f ter 15 sec 10 Hz s t imula t ion PM, p a p i l l a r y muscle LA, l e f t a t r i a n, number of animals BDF, basal developed force . Table 6. E C ^ Q Values of Various Inotropic Agents i n Heart Preparations of Control and 6-week Wistar Diabetic Rats. C O N T R O L D I A B E T I C n=8-15 jm *3M) RV PM LA RA RV PM LA RA Isoproterenol 6.46 3-98 0.87 0.71 11.48* 9-77* 0.74 O.98 (10"BM) (4.90-8.51) (3 .24-4 .90) (0.65-1.17) (0.51-1-02) (8.32-16.20) (7 .76-12.59) (0.55-1-58) (0.69-1-38) n=6-12 Phenylephrine 3-89 2.18 1.29 1-91* * O.58* 0.24* (10~5M) (2 .63-10.00) (1 .23-3 .80) (0 .83-1 .95) (1 .38-2 .63) (O .30-I .18) (0 .16-0.37) Calciu 2.2 3-2 1.7* 2 .5* (10"3 ) (1-9-2.5) ( 2 . 8 - 3 - 6 ) (1-5-1-9) ( 2 . 1 - 2 . 9 ) n=8-l4 BAY K 8644 2.69 1.00 0.51 2.09 (10" 'M) (0.85-8.31) (0.62-1.66) (0.15-1 .70) (1 .29-3-47) n=8-10 A l l values are expressed by geometric mean and 95X confidence interval. * p < 0.05 versus control of the same tissue; blank space, not measured. RV, right ventricular strips PM, papillary muscle LA, l e f t a t r i a RA, right a t r i a n, number of experiments CO Table 7- E C c n Values of Various Inotropic Agents i n Heart Preparations o f C o n t r o l and 6-week SHR Diabet i c Rats . C O N T R O L D I A B E T I C RV LA RA RV LA RA Isoproterenol 2 . 8 8 0.26 0 .46 8 . 1 3 * 0 . 3 2 0 . 7 1 (10"aM) (2 .04-4 .07) (0 .20-0 .34) ( 0 . 3 2 - 0 . 6 8 ) ( 5 . 7 5 - H - 4 8 ) ( 0 . 2 4 - 0 . 4 2 ) ( 0 . 5 0 - 1 . 0 2 ) n=7-9 Phenylephrine 3 - 7 2 0 .68 1 . 7 9 * 0 . 1 2 * ( 1 0 ~ 5 M ) ( 2 . 3 4 - 1 0 . 0 0 ) ( 0 . 4 4 - 1 . 0 2 ) ( 1 . 2 6 - 2 . 5 1 ) ( 0 . 0 8 - 0 . 1 8 ) n = 8 - 1 3 Calcium 2 . 8 3 - 4 2 . 3 * 2 . 6 * (10' 3M) ( 2 . 5 - 3 . 2 ) ( 2 . 9 - 4 . 1 ) ( 1 - 3 - 2 . 6 ) ( 2 . 3 - 3 - 1 ) n=6-12 BAY K 8644 2 . 0 9 2 . 0 9 1 - 2 0 1.20 (10"7M) (0 .63-7-08) (1 .38-3-16) ( 0 . 4 1 - 3 - 6 3 ) ( O . 8 3 - I . 7 8 ) n=8-10 A l l values are expressed by geometric mean and 9 5 $ confidence i n t e r v a l . * p < 0 . 0 5 versus contro l of the same t i ssue; blank space, not measured. RV, r i g h t v e n t r i c u l a r s t r i p s LA, l e f t a t r i a RA, r i g h t a t r i a n, number of experiments o Table 8. ECco Values of Various Inotropic Agents i n Heart Preparat ions of C o n t r o l and 6-week WKY Diabet i c Rats C O N T R O L D I A B E T I C RV LA RA RV LA RA Isoproterenol 4.47 0.28 0.33 11.7* 0.24 0.35 (10"°M) (2.40-8.18) (0180-0.42) (0.21-0.50) (6.48-21.10) (0.16-0.37) (0.22-0.53) n=8-9 Phenylephrine 2.69 0 .91 0.83 0.45 (10"5M) (1.15-10.00) (0.48-1.74) (0.34-2.04) (0.24-0.87) n=7-ll Calcium 3-5 2.7 2.8* 2.3 ( 1 0 ~ 3 M ) (3.2-4.1) (2.2-3.3) (2.5-3-2) (1.9-2.8) n=8-9 BAY K 8644 0.49 2 . 19 0 . 3 0 2 . 09 (10"7M) (0.24-1.02) (1.51-4.27) ( 0 . 1 5 - 0 . 6 2 ) (1.26-3.47) n=8-10 A l l values are expressed by geometric mean and 9 5 $ confidence i n t e r v a l . * p< 0 . 0 5 versus contro l of the same t i s sue; blank space not measured. RV, r i g h t v e n t r i c u l a r s t r i p s LA, l e f t a t r i a RA, r i g h t a t r i a n, number of experiments 42 1.500 o> 1 . 0 0 0 -£ 0.500 c (D o 0 .000 o - 0 . 5 0 0 - 1 0 T O ' X i T . O ' i T X : « : T •o 1 T o 1 T T 0 — # A — A 1 X A * — A * - 8 - 6 L O G M ( ISO) - 4 Figure 12. I soproterenol dose response curves obtained i n r i g h t v e n t r i c l e t i s sues of c o n t r o l and d i a b e t i c r a t s . ISO i s o p r o t e r e n o l , 0 0 Wistar c o n t r o l , • § Wistar d i a b e t i c , A A SHR c o n t r o l , A • SHR d i a b e t i c , * p < 0.05 vs same s t r a i n c o n t r o l , n = 8-10. 43 1 0 8 C O N D I A RV P M LA RA WISTAR RV LA RA SHR RV LA RA WKY Figure 1 3 . p D 2 values for i s o p r o t e r e n o l i n heart preparat ions from c o n t r o l and 6-week d i a b e t i c r a t s . ISO i s o p r o t e r e n o l , p D 2 negat ive l o g concentrat ion o f the agonist to produce 50% of maximum response, con c o n t r o l , d i a d i a b e t i c , RV r i g h t v e n t r i c l e , PM p a p i l l a r y muscle, LA l e f t a t r i a , RA r i g h t a t r i a * p < 0.05 vs c o n t r o l o f same s t r a i n t i s s u e , n = 1-12. 44 0.800 £ 0.600 c ~ 0.400 c CD •- 0.200 CP CD C D 5 o.ooo t •o- •o-- O - -o-• o — o - 0 . 2 0 0 - 7 - 5 - 4 L O G M ( P E ) - 3 - 2 Figure lM. Phenylephrine dose response curves obtained i n right v e n t r i c l e tissues of control and diabetic r a t s . PE phenylephrine, 0 0 Wistar control, • • Wistar diabetic, A A SHR control, A A SHR diabetic, * p < 0.05 vs same s t r a i n c o n t r o l , n = 10. 45 8 . 0 0 0 5.500 3.000 RV P M LA WISTAR RV LA S H R RV LA W K Y Figure 15- Pn2 v a l u e s f° r phenylephrine i n heart preparations from control and 6-week diabetic rats. pD£ negative log concentration of the agonist to produce 50% of maximum response. con control, d i a diabetic, RV right v e n t r i c l e , PM p a p i l l a r y muscle, LA l e f t a t r i a . * p < 0.05 vs control of same s t r a i n tissue, n = 7-15-46 1 . 5 0 0 2 . 5 0 0 — 2 . 0 0 0 E cn c o £ 1 . 0 0 0 c S) 0 . 5 0 0 + c D ° 0 . 0 0 0 - 0 . 5 0 0 + - 3 L O G M ( C a ) - 2 Figure 16. Calcium dose response curves obtained i n right v e n t r i c l e tissues of control and diabetic r a t s . Ca calcium, 0 0 Wistar control, • 0 Wistar diabetic, A A SHR control, A A SHR diabetic, * p <0.05 vs same s t r a i n control, n = 8-10. 47 3.500 E D JJ D O O in CD O > C N Q C L 2 . 7 5 0 -2.000 RV P M LA WISTAR RV LA S H R RV LA W K Y Figure 17- pD 2 values for calcium i n heart preparations from control and 6-week diabetic rats. p n2 negative log concentration of the agonist to produce 50% of maximum response, con control, d i a diabetic, RV right v e n t r i c l e , PM p a p i l l a r y muscle, LA l e f t a t r i a . * p < 0.05 vs control of same s t r a i n tissue, n = J-lk. 48 1 . 0 0 0 ^ 0 . 8 0 0 E cn ^ 0 . 6 0 0 o 00 u 0 . 4 0 0 h— c g, 0 . 2 0 0 + c D o 0 . 0 0 0 - 0 . 2 0 0 - 1 0 T , O 4 I -o-o 1. I 1=1 - 8 - 6 L o g M ( B A Y K 8 6 4 4 ) - 4 Figure 18. BAY K 8644 dose response curves obtained i n right v e n t r i c l e tissues of control and diabetic r a t s . 0 0 Wistar control, • • Wistar diabetic, A A SHR control, A A SHR diabetic. (n = 9-10). 49 1 0 00 >-< m c/) CD Z5 O > C M Q C L 8 -C O N !DIA T 1 RV LA WISTAR R V LA S H R R V LA W K Y Figure 19- pn2 values for BAY K 8644 i n heart preparat ions from c o n t r o l and 6-week d i a b e t i c r a t s . p D 2 negative l og concentrat ion o f the agonist to produce 50% of maximum response, con c o n t r o l , d i a d i a b e t i c , RV r i g h t v e n t r i c l e , LA l e f t a t r i a . * p < 0.05 vs c o n t r o l o f same s t r a i n t i s s u e , n = 8-10 (RV), 6-10 (LA) . 50 C D C o "(/) c <D t— " O Q) CL _o > Q 0 ) - + -_ 3 ~6 V) < c o "(/) c Q) O 2 . 0 0 0 1 . 5 0 0 + 1 . 0 0 0 0.500 0 . 0 0 0 2.000 E 1 . 5 0 0 1 . 0 0 0 0.500 + » 0.000 Q - 0 . 5 0 0 - 1 0 I T I • -• 1 1 1 T • X T - A T - A X 6 -6 -6 A - A - A T 1^1 -o-6. A -i \ T A ^ --ex - A -* T T / l * • A - + -8 -6 -4 L O G M ( V E R A P A M I L ) Figure 20. Verapamil dose response curves obtained i n r igh t ven t r i c l e t issues of control and d iabe t ic r a t s . 0 0 Wistar con t ro l , 0 0 Wistar d i abe t i c , A A SHR con t ro l , A A SHR d i abe t i c , n = 8-10. 51 o m CD (/) D CD 8 . 0 0 0 (D C_ X ! O (D £ 5 . o D O CD O O CO o 500 I 3.000 RV LA WISTAR RV LA S H R RV LA W K Y Figure 21. pD 2 values for verapamil i n heart preparations from control and 6-week diabetic rats. con control, dia diabetic, RV r i g h t v e n t r i c l e , LA l e f t a t r i a , * p < 0.05 vs control of same s t r a i n tissue, n = 8-14 (RV), n = 6-7 (LA). 52 verapami l , expressed as absolute developed force and decrease i n tens ion, are shown i n F igure 20. 1. I soproterenol (ISO) Increased E C ^ Q values o f ISO were found i n v e n t r i c u l a r t i s sues i n three kinds of d i a b e t i c ra t s but not i n a t r i a l t i s s u e s . Comparing the s e n s i t i v i t i e s o f d i f f e r e n t t i s s u e s , a t r i a were more s e n s i t i v e to (5-agonis t s , but no a l t e r a t i o n i n terms o f d i a b e t i c s ta te was shown i n th i s t i s sue (Figure 13)- The maximum response to ISO was a l so decreased i n d i a b e t i c r i g h t v e n t r i c u l a r s t r i p s (Figure 12). 2. Phenylephrine (PE) i n the Presence of Proprano lo l The i n o t r o p i c responses to PE i n v e n t r i c u l a r and a t r i a l t i s sues a l l showed an enhanced s e n s i t i v i t y to PE i n d i a b e t i c ra t s as compared to c o n t r o l s , which i n d i c a t e d a s u p e r s e n s i t i v i t y o f Wistar and SHR d i a b e t i c rat hearts to a-agonist s t i m u l a t i o n . The maximum response to PE i n d i a b e t i c RV was s i g n i f i c a n t l y increased , e s p e c i a l l y i n SHR d i a b e t i c RV (Figures 14 and 15). The s e n s i t i v i t y to PE i n RV and LA t i s sues of WKY d i a b e t i c ra t s was not changed i n comparison with same s t r a i n c o n t r o l s . 3- Calcium S u p e r s e n s i t i v i t y to calc ium was found i n v e n t r i c u l a r and a t r i a l t i s sues from a l l three s t r a i n s of d i a b e t i c r a t tes ted (except i n WKY l e f t a t r i a ) with increased p D 2 values and increased maximum responses compared to the corresponding contro l s (Figures 16 and 17)-4. BAY K 8644 The s e n s i t i v i t y to BAY K 8644 was increased i n SHR LA expressed by a increased p D 2 va lue . No other d i f f e r e n c e i n p D 2 values was found between d i a b e t i c and c o n t r o l t i s sues (Figure 19)• Maximum response was the same i n d i a b e t i c and c o n t r o l t i s sues (Figure 18). 53 5. Verapamil The concentrat ion o f verapamil requ ired to produce a 50% reduct ion i n BDF was not d i f f e r e n t between c o n t r o l and d i a b e t i c t i s sues (Figure 21, Table 9)• However the maximum response to verapamil i n terms o f decreased BDF was higher i n the d i a b e t i c RV than the corresponding contro l s (Figure 20) . B. The E f f e c t s o f Ryanodine on BDF, PQP and PSP T h i s study was done i n PM and LA from Wis tar , SHR and WKY r a t s . Ryanodine 3 x l 0 _ ^M, i n a 20 min incubat ion , s i g n i f i c a n t l y diminished BDF i n both c o n t r o l and d i a b e t i c t i s sues . In LA, ryanodine decreased basal tens ion (expressed as a percentage decrease o f BDF) from 32-5% to 52.9% and i n PM from 20% to 40% (Table 11). No s i g n i f i c a n t d i f f e r e n c e i n pre-drug BDF was found between c o n t r o l and d i a b e t i c r a t s i n PM and LA from Wis tar , SHR and WKY rat s (Table 3. F igure 6 ) . T h i s same observat ion was found i n pos t -drug BDF measurements, i n d i c a t i n g that the negat ive i n o t r o p i c e f f ec t s o f ryanodine were s i m i l a r i n both d i a b e t i c and c o n t r o l ra t s i n the d i f f e r e n t s t r a i n s . However, the responses i n LA were more s e n s i t i v e than the responses i n PM (percentage decrease o f BDF i n c o n t r o l r a t s , LA vs PM p < 0.05) showing a d i f f e r e n t e f f i c a c y o f ryanodine i n these two t i s sues (Table 11). An o v e r a l l e f f e c t of ryanodine i s shown i n Table 10. The PQP response o f PM to ryanodine (Figure 22) was obvious i n Wistar and SHR r a t s . Both c o n t r o l and d i a b e t i c PM showed a decrease o f PQP. The decrease o f PQP i n SHR was s i g n i f i c a n t ; ryanodine almost t o t a l l y abo l i shed PQP i n SHR d i a b e t i c r a t s . However there was no s i g n i f i c a n t e f f e c t s o f ryanodine on PQP found i n WKY c o n t r o l and d i a b e t i c PM. In LA (Figure 23) , ryanodine Table 9- Concentrations o f Verapamil Required to Decrease 50% C o n t r a c t i l e Force from Cardiac Preparations of Contro l and Diabet ic Rats. C O N T R O L RV LA D I A B E T I RV C LA WISTAR 2 . 3 0 2.04 2 . 8 6 1.41 n = 6 - 9 ( 1 . 2 5 - 4 . 2 1 ) ( 0 . 5 9 - 1 0 . 0 ) ( 2 . 9 5 - 1 0 . 0 ) ( 0 . 4 1 - 4 . 9 0 ) SHR 1 . 5 9 2 . 1 9 2 . 8 3 0 . 4 5 n = 6 - 1 2 (0.96-2 .64) ( 0 . 6 3 - 1 0 . 0 ) ( 1 . 4 9 - 1 0 . 0 ) ( 0 . 1 4 - 1 . 4 1 ) WKY 3 . 0 2 1 . 0 2 2.24 1 . 0 7 n = 8 - 1 0 ( 1 . 8 6 - 4 . 7 9 ) ( 0 . 5 3 - 2 . 0 0 ) (1.41-3-55) ( 0 . 5 5 - 2 . 0 9 ) A l l values are expressed by geometric mean and 95% confidence i n t e r v a l . * p < 0 . 0 5 versus contro l of the same t i s sues . RV, r i g h t v e n t r i c u l a r s t r i p s LA, l e f t a t r i a n, number of experiments. Table 10. Effects of Ryanodine on PQP and PSP of Control and Diabetic Cardiac Preparations. Control WISTAR SHR WKY Diabetic Control Diabetic Control Diabetic PM PQP/BDF n=7~9 a,b - b - -LA PQP/BDF n=7"9 a,b -PM PSP+BDF n=7-9 - a,b -LA PSP+BDF n=6-9 - b - a,b PM PSP/BDF n=7-9 b a.b LA PSP/BDF n=7-9 a, pre-drug value s i g n i f i c a n t l y different from corresponding control, p < 0 .05 b, pro-drug value s i g n i f i c a n t l y different from corresponding control, p < 0 .05 PM, p a p i l l a r y muscle LA, l e f t a t r i a n, number of animals Ryanodine 3xl0~^M, 20 min incubation Drug effects on PQP or PSP: , increase, p < 0.05 vs pre-drug value. , decrease, p < 0 .05 vs pre-drug value. -, no effects, p > 0.05 versus pre-drug value. Table 11 . Effects of Ryanodine on BDF of Control and Diabetic Cardiac Preparations. CONTROL DIABETIC PM LA PM LA WISTAR 34±5# 49±7% 2B±5% 49±7% n 8 8 9 9 SHR ko±8% 33±9% 34±9% 53±10% n 8 8 7 7 WKY 20±7% 44±7% 3^±7% 43±7% n 8 8 8 8 A l l values are expressed as the percentage decrease of BDF when exposed to ryanodine, (calculation: BDF - Post-drug Tension / BDF x 100%) by mean ± standard error. No difference was found between diabetic and control i n the same tissues of same s t r a i n . BDF basal developed force PM, p a p i l l a r y muscle LA, l e f t a t r i a n, number of experiments. 57 2 . 0 0 0 Q 1.500 m \ Q_ o ^ 1 . 0 0 0 Q_ O Q_ c 0 . 5 0 0 [• CD C D c 0 . 0 0 0 o T O 1 T 1 T O 1 * T 1 •0 .500 C T WISTAR T o . 1 T T •o 1 o O con - • d i d C T S H R C T W K Y Figure 22. The effects of ryanodine on post quiescent potentiation of pa p i l l a r y muscles i n control and 6-week diabetic rats. PQP and BDF were measured before and af t e r the incubation of tissues with ryanodine (3x10" 20 min). BDF basal developed force, PQP post quiescent potentiation, con control, dia diabetic, C pre-drug, T post-drug, * p < 0.05 vs same s t r a i n control, n = 7 - 9-58 CO Q_ o Q_ D_ a O ) C D SZ o 4 0 O O con - • dia T o X o X -1 C T WISTAR C T S H R T W K Y Figure 23. The e f fec t s o f ryanodine on post quiescent p o t e n t i a t i o n of l e f t a t r i a i n c o n t r o l and 6-week d i a b e t i c r a t s . PQP and BDF were measured before and a f t e r the incubat ion of t i s sues with ryanodine (3xlO~^M, 20 min) . BDF basa l developed force , PQP post quiescent p o t e n t i a t i o n , con c o n t r o l , d i a d i a b e t i c , C pre -drug , T p o s t - d r u g , * p < 0.05 vs same s t r a i n c o n t r o l , n = 7~9> 59 decreased PQP i n SHR control and diabetic rats with the same potency, but the same concentrations of ryanodine had no effects on PQP of LA i n Wistar and WKY rats. Pre-drug peak tension (PSP + BDF) after 10 Hz 15 second stimulation were lower only i n SHR PM and WKY LA but not other tissues (Figures 2k and 25). After the incubation with ryanodine, the decline of peak tension i n certain tissues can be accounted for by i t s negative inotropic effects on BDF. Generally however, the PSP was not influenced by the effects of ryanodine. The peak tension of SHR diabetic LA was decreased s i g n i f i c a n t l y . Because BDF was decreased by ryanodine and post-stimulation peak tension (PSP + BDF) was r e l a t i v e l y persistent, the r a t i o of PSP/BDF was increased i n a l l tissues tested (Figures 26 and 27). The special s i t u a t i o n i s i n PM of Wistar and SHR, these r a t i o s increased less than corresponding controls, and th i s causes the r a t i o of PSP/BDF af t e r the high frequency stimulation to be s i g n i f i c a n t l y lower than controls. 60 Q + 4 Q_ 00 3 E C J ) c o (0 c Q) D CD Q . 0 L C T WISTAR T o -* T T - o 1 T 1 C T S H R O O c o n • • d i a i • © i C T W K Y Figure 2k. The effects of ryanodine on post stimulation potentiation (PSP+BDF) of p a p i l l a r y muscles i n control and 6-week diabetic r a t s . PSP and BDF were measured before and af t e r the incubation of tissues with ryanodine (3xl0~^M, 20 min). BDF basal developed force, PSP post stimulation potentiation, con control, dia diabetic, C pre-drug, T post-drug, * p < 0.05 vs same s t r a i n control, n = 7 -9-61 3.000 g 2.500 + Q_ Ul Q- 2.000 °> 1.500 T T * 1 c o £ 1.000 (1) o 0.500 Q) 0_ 0.000 C ' T WISTAR o O con • did O-T T • o o- •o C T S H R C T W K Y Figure 25- The e f f ec t s of ryanodine on post s t i m u l a t i o n p o t e n t i a t i o n (PSP+BDF) o f l e f t a t r i a i n c o n t r o l and 6-week d i a b e t i c r a t s . PSP and BDF were measured before and a f t e r the incubat ion of t i s sues with ryanodine (3xl0~-'M, 20 min) . BDF basa l developed f o r c e , PSP post quiescent p o t e n t i a t i o n , con c o n t r o l , d i a d i a b e t i c , C pre -drug , T p o s t - d r u g , * p < 0.05 vs same s t r a i n c o n t r o l , n = 6-9. 62 Figure 26. The effects of ryanodine on post stimulation potentiation (PSP/BDF) of p a p i l l a r y muscles i n control and 6-week diabetic rats. PSP and BDF were measured before and af t e r the incubation of tissues with ryanodine (3X10 20 min). BDF basal developed force, PSP post stimulation potentiation, con control, dia diabetic, C pre-drug, T post-drug, * p < 0.05 vs same s t r a i n control, n = 7_9-63 Figure 27. The effects of ryanodine on post stimulation potentiation (PSP/BDF) of l e f t a t r i a i n control and 6-week diabetic rats. PSP and BDF were measured before and a f t e r the incubation of tissues with ryanodine (3xlO~^M, 20 min). BDF basal developed force, PSP post quiescent potentiation, con control, dia diabetic, C pre-drug, T post-drug, n = 7-9. 64 DISCUSSION I. GENERAL INDICATIONS OF DIABETES MELLITUS The c l a s s i c a l symptoms of Type I, i n s u l i n dependent diabetes mellitus (IDDM), include glucosuria, polyuria, polydipsia, hyperphagia and weight los s . Examination of blood from diabetics reveals hyperglycemia, hypoinsulinemia, hyperlipidemia, and a decrease i n thyroid hormone (Foster 1983, Pittman et a l , 1979)' These alterations were successfully induced i n STZ-treated rats. The mechanism by which STZ e l i c i t s i t s s p e c i f i c necrotic action on the p - c e l l i s largely unknown. I t probably involves the a b i l i t y of STZ to generate free radicals (Fischer, 1985)• However the close approximation of STZ-induced diabetes to the human IDDM state warrants i t s use i n diabetic studies. 1. Body Weights and Mortality Comparing the three strains of control r a t s , Wistar rats had higher body weights than SHR and WKY (Figure 2) , which indicated a s t r a i n s p e c i f i c i t y . Six weeks after STZ i n j e c t i o n , the three st r a i n s of diabetic rats exhibited substantially decreased body weights i n comparison with same s t r a i n control. SHR diabetic rats l o s t the greatest weight during the weeks following STZ in j e c t i o n . This dramatically decreased body weight was intimately associated with the most severe hyperglycemia, hypoinsulinemia, hyperlipidemia, and unique mortality (38.8±15>6#, calculated i n s i x groups of s i x t y three SHR diabetic r a t s ) . The higher mortality associated with SHR-diabetic rats indicates a synergistic detrimental effect of these two disease states. Considering the high incidence of mortality i n diabetic hypertensive patients (Factor et a l , 198O, B e l l 1989). the SHR diabetic rat 65 i s a promising model to study the pathophysiological process of these combined diseases. 2. Plasma Glucose and Insulin There were no si g n i f i c a n t differences between plasma glucose concentrations i n the three strains of control rats. However the order of elevated glucose concentrations i n diabetic rats was SHR > WISTAR > WKY (Figure 3)• This may suggest d i f f e r e n t degrees of metabolic alterations i n these rats. The control levels of plasma i n s u l i n were not s i g n i f i c a n t l y d i f f e r e n t i n the three st r a i n s , and i n s u l i n concentrations were a l l decreased by about the same percentage of the same s t r a i n controls when diabetes was induced. In the l i t e r a t u r e the plasma i n s u l i n concentrations range from 18-96 (control) to 6-26 (diabetic) uunit/ml (Rodrigues and McNeill 1986, Pierce et a l , 1983, T a h i l i a n i and McNeill 1984). The diff e r e n t concentrations of plasma i n s u l i n i n the measurements above may be attributed to differences i n sex and method used. 3. Blood Lipids Increased concentrations of t r i g l y c e r i d e s and cholesterol appear to be very important events i n diabetic r a t s , because these increased blood l i p i d s have been related to the depressed cardiac mechanical and SR functions reported by some investigators (Rodrigues and McNeill 1986, Katz and Messineo I98I). The present data are i n a good agreement with the observations by Rodrigues and McNeill. At s i x weeks, WKY diabetic rats showed no si g n i f i c a n t change i n blood l i p i d s , while SHR diabetic rats demonstrate markedly enhanced t r i g l y c e r i d e s and cholesterol l e v e l s . In Wistar diabetic rat t r i g l y c e r i d e and cholesterol l e v e l s were between those 66 found i n SHR and WKY diabetics. The phospholipid content i n our measurement can not be discriminated as major phospholipids, such as phosphatidyl-ethonalamine or phosphatidylcholine, and minor phospholipids, sphingomyeline and lysophosphatidylcholine. I t has been reported that the major phospholipids decreased whereas t o t a l phospholipids, l e v e l elevated i n diabetic heart (Okumura et a l . 1988). Further the correlation of changes between plasma phospholipid content and the cardiac function requires analysis of phospholipids i n myocardium or cardiac membrane. The alt e r a t i o n i n myocardial phospholipid content may be associated with the development of myocardial dysfunction. 4. Plasma Triiodothyronine (T^) and Thyroxine (T/j) 'Hypothyroidism' i n diabetic animals has been documented i n many reports (Rodgers and McNeill 1986, Grassby and McNeill 1988). There are differences i n the various reports with regard to the active T^/Tjj concentrations, which may be explained by the duration of diabetes, species v a r i a t i o n and assay accuracy. However, the basic finding i n diabetic animals i s a decrease i n blood thyroid hormone l e v e l s . I t has been reported that i n s u l i n deficiency can cause changes of many hormone levels i n diabetic animals. Thyroid hormone, thyroid stimulating hormone (TSH) and thyrotropin releasing hormone (TRH) have a l l been reported to be decreased i n plasma from STZ diabetic rats (Gonzalez et a l . 1980). This suggests a hypothalamic-pituitary-thyroid axis impairment and possibly the primary cause of p i t u i t a r y - t h y r o i d a l t e r a t i o n i n STZ diabetic rats l i e s i n the hypothalamus. Thyroid hormone i s known to regulate various biochemical parameters such as myosin ATPase (Thyrum et a l . 1979) and" SR Ca + + transport (Suko 67 1971)' I t i s also known that diabetes results i n a state of mild hypothyroidism i n some humans (Pittman et a l . 1979)- The relationship of hypothyroidism and cardiac function, i n terms of changes i n a- and f i -adrenoceptor system and myosin isoenzymes w i l l be discussed under the section on cardiac drug responses. I I . BASAL CHRONOTROPIC AND INOTROPIC RESPONSES FROM VARIOUS HEART PREPARATIONS Bradycardia was found i n a l l strains of diabetic rats i n the study. Basal a t r i a l rates are si m i l a r i n the three kinds of control rats, and STZ produces s i m i l a r effects on the right a t r i a of these rats. Therefore the s t r a i n factor did not influence bradycardia i n STZ induced diabetes. I t has been proposed that bradycardia i s due to alterations i n SA node e l e c t r i c a l a c t i v i t y as a result of hyperglycemia (Senges et a l , 1980), hyperlipidemia (Rodrigues et a l , 1985) or hypothyroidism (Garber et a l , 1983). On the whole animal l e v e l , diabetic rats also have bradycardia which may be a consequence of reduced number of (J-adrenergic receptors and defective adrenergic receptor adenylate cylase coupling i n diabetic heart due to down regulation of the system. Another speculated interpretation of the bradycardia i s sympathetic nerve denervation. The increased BDF observed i n RV from Wistar and WKY diabetic rats i s i n agreement with results previously reported from Sprague-Dawley rats (Ramanadham and Tenner 1987). The authors suggested a relationship between thi s increased BDF and C a + + overload i n diabetic myocardial tissues. This needs to be established by p a r a l l e l measurements of BDF and i n t r a c e l l u l a r calcium concentration. I t i s also possible that the a b i l i t y of the diabetic v e n t r i c l e to u t i l i z e C a + + increases, which may be associated with 68 a supersensitivity to C a + + (see below). However, why the increased BDF only occurred i n ventricular s t r i p s but not i n p a p i l l a r y muscles i s not clear. Fein (I98O) reported no difference i n BDF from Wistar diabetic and control PM i n agreement with our observations. Other investigators have also described an increased BDF i n l e f t / r i g h t a t r i a (Foy and Lucas,1978). Because of the size difference i n the tissues, some units of measurement need to be normalized. For example, i n our experiments SHR diabetic LA had a lower BDF compared with same s t r a i n controls. Because the small size of this preparation may influence the re s u l t s , further calculations were done to normalize the units used. I f the BDF i s expressed as mg force/mg tissue weight, instead of only as force, the data showed no difference between diabetic and control LA. I t should be mentioned here that the exact expression of force should be N (Newton) or mN, gram i s the mass-unit recorded d i r e c t l y from the polygraph recorder. I I I . PRE-DRUG MEASUREMENT OF PQP AND PSP PQP and PSP occur by diff e r e n t mechanisms and may involve d i f f e r e n t sources of C a + + i n isolated rat PM and LA. Lukas and Bose (1986) suggested mechanisms for PQP and PSP based on the evidence available. PQP appears to depend primarily on the release of C a + + from i n t r a c e l l u l a r stores. I t was proposed that the rest period provides a longer i n t e r v a l for the transfer of C a + + from uptake to release s i t e s i n SR, thereby increasing the quantity of C a + + available for release by the post-rest beats. Moreover, the rapid decay of PQP probably r e f l e c t s depletion of the 'extra' C a + + i n the release s i t e s . PSP occurs through the increased trans-sarcolemmal i n f l u x of Ca+ + during potentiated contraction. The mechanism mediating the increased i n f l u x of C a + + i s l i k e l y to be Na +-Ca + + exchange. High frequency 69 stimulation i s known to increase the i n t r a c e l l u l a r concentration of sodium ([Na+]£), the so call e d sodium pump l a g phenomenon (Cohen et a l . 1982) i n cardiac tissue. As [Na +]^ r i s e s , the i n f l u x of C a + + would be increased through Na +-Ca + + exchange, because of competition between Na + and C a + + for active s i t e s on the exchange c a r r i e r (Reeves and Sutko 1983). This would promote increased Na +-Ca + + exchange a c t i v i t y upon return to basal rate stimulation. These assumptions were supported by measurement of ^Ca++ content, which was increased during PSP ind i c a t i n g a net gain i n i n t r a c e l l u l a r C a + +, and was not changed during PQP (Lukas and Bose 1986). I t i s suggested that the f i r s t post rest beat (including PQP), i n rat v e n t r i c l e i s highly dependent on the amount of C a + + released by SR (Bers I985). As we can see from the time course of PQP (Figures 8 and 9). when the rest i n t e r v a l increases up to 1-2 minutes, the tension of the f i r s t post rest beat increases to a maximum, as a result of the rate-dependent f i l l i n g of i n t r a c e l l u l a r C a + + stores. As the rest duration increases, the tension of f i r s t post rest beat decreases. This decline of tension r e f l e c t s a progressive f a l l i n the C a + + content of SR. The time course of PSP shows that the amplitude of PSP i s not a function of increased stimulation time. This may suggest that C a + + i n f l u x upon the resumption of normal rate i s not influenced by the time i n t e r v a l of the high frequency stimulation which has already saturated the Na + pump, maximizing the concentration of internal sodium and increased the rate of Na +-Ca + + exchange. The amount of Ca + + released from SR i s a function of the amount available for release (Fabiato 1983). Diabetic cardiac tissues, such as Wistar and SHR pa p i l l a r y muscles and SHR a t r i a showed a depressed PQP i n our experiments (Figures 22, 23), which may indicate less C a + + released 70 from SR to activate contraction and less C a + + stored i n the SR of diabetic myocardium. The impaired function of C a + + uptake by diabetic SR (Lopaschuk et a l . 1982) may explain this phenomenon. Another prominent event i s that there i s no PQP change i n WKY diabetic r a t s . The unchanged cardiac function and blood l i p i d s i n WKY diabetic model have been reported before (Rodrigues and McNeill, 1986). Hyperlipidemia i n diabetes i s thought to cause cardiac deficiency because altered membrane phospholipids may play an important role i n impaired C a + + transport and thus they could be involved i n the regulation of myocardial c o n t r a c t i l i t y (Katz and Messineo 198l, Langer and Rich 1986). There may be a relationship between the normal PQP, l i p i d metabolism and cardiac function i n WKY diabetic r a t s , which suggests that the WKY diabetic rat i s not a good model to study cardiomyopathy. The SHR diabetic PM show a decreased PSP i n comparison with control. I t seems that t h i s tissue i s not able to generate s u f f i c i e n t Na +-Ca + + exchange i n SL. The function of Na +-Ca + + exchange was reported as decreased i n diabetic heart (Makino et a l 1987). We examined Na +-Ca + + exchange a c t i v i t i e s i n pu r i f i e d cardiac membrane from Wistar and SHR diabetic ventricular tissues (unpublished data). The V m o v and Km for C a + + were s i g n i f i c a n t l y decreased i n the SHR diabetic group (p < 0.05, n=8) suggesting an increased a f f i n i t y to C a + + of the exchangers and a decreased exchanger number i n this tissue. The values were not changed i n Wistar diabetic rats. These results showed a good cor r e l a t i o n with the PSP values of corresponding tissues. Some investigators (Bose et a l . 1988) have argued that i t i s u n l i k e l y that an increase i n C a + + i n f l u x can account for what they c a l l e d the post-ex t r a s y s t o l i c potentiation (PESP) which was measured by s i m i l a r method with PSP. According to t h e i r calculation the additional C a + + entering during 71 the extrasystole can account for only 25% of the potentiation, which i s less than that observed during PESP. There may be a fr a c t i o n of Ca + + entering the c e l l during an extrasystole which does not contribute to the beat occurring immediately but i s stored i n the SR and released during PESP (Bose et a l . 1988). They also suggested that PESP i s associated with a faster 'repriming' of the release pool i n the SR. Consequently i f this process i s impaired there w i l l be a decrease i n the PESP. However this measurement i s an estimation of the maximum increase i n contraction possible through enhanced transmembrane C a + + entry. IV. CARDIAC DRUG RESPONSES IN DIABETIC MYOCARDIUM As was mentioned before, tissue responses to cardiac drugs with d i f f e r e n t mechanisms may provide certain information about C a + + mobilities i n these tissue. Using c l a s s i c a l pharmacological measurements, the s e n s i t i v i t y of a tissue (expressed by E C ^ Q value) for an agonist as derived from the concentration-effect relationship i s an estimate of the apparent a f f i n i t y , usually defined as s e n s i t i v i t y , of the receptor for the agonist, and the maximum response i s the i n t r i n s i c a c t i v i t y of a drug, i . e . the effe c t produced after receptor occupancy (Ariens and Simonis 1964). 1. B-Adrenergic Stimulation Isoproterenol, a p-adrenocepter stimulator, causes p o s i t i v e inotropic and chronotropic effects i n mammalian heart by a cAMP dependent pathway. The mechanism of ^ -stimulation has been intensively investigated and i s believed to include the following steps: Isoproterenol acts on a regulatory component (guanine nucleotide-binding protein) of the adenylate cyclase complex at the SL to stimulate cAMP production ( L e v i t z k i 1987)• 72 The cAMP may subsequently bind to and activate a cAMP-dependent protein kinase, which then can phosphorylate a number of functional proteins, including SL (C a + + channel or a closely-associated regulatory protein), SR (phospholamban), and thin filaments (troponin I ) . This results i n stimulation of the C a + + channels, greater C a + + i n f l u x , C a + + uptake and release from SR, and increased c o n t r a c t i l i t y associated with acceleration of relaxation (Sperelakis and Wahler 1988). In th i s process, the proper performance of normal cardiac function depends on a series of factors: the number and a f f i n i t y of ^ -receptors, the a c t i v i t i e s of adenylate cyclase, cAMP-dependent protein kinase and phosphorylase, the concentration of cAMP, the basal and stimulated a c t i v i t i e s of C a + + channels, the function of SR i n terms of C a + + mobilization, C a + + binding proteins, such as calmodulin, and the function of m y o f i b r i l l a r ATPase. The diminished ^-response from diabetic heart, represented by increased E C ^ Q values and decreased maximum c o n t r a c t i l e force, as observed i n our experiment, has been reported by many investigators. The data include depressed inotropic response i n rat l e f t a t r i a (Foy and Lucas, 1978) i n l e f t ventricular p a p i l l a r y muscle (Heyliger et a l . 1982) and i n rig h t ventricular s t r i p s (Ramanadham and Tenner 1987). In accordance with the functional response, a diminished p-receptor density without altered a f f i n i t y has been reported (Heyliger et a l . 1982, Williams et a l . 1983. Ingebretsen et a l . 1983. Ramanadham and Tenner 1987 and Nishio et a l . 1988). Two groups however, found no change i n (J-receptor density i n diabetic rats despite a diminished myocardial cAMP (Gotzsche 1983) and adenylate cyclase (Mooradian et a l . 1988). In view of the variable reports of p-receptor density, the mechanism of decreased s e n s i t i v i t y of cardiac P>-adrenoceptors cannot be sole l y attributed to decreased receptor number. 73 Vadlamudi and McNeill ( 1 9 8 3 ) demonstrated no correlat i o n between cardiac responses to p*-agonist and the binding of ligand to the receptor i n diabetic rats. A functional uncoupling of the myocardial ^-adrenoceptor from productive adenylate cyclase a c t i v a t i o n or other following steps may exi s t i n experimental diabetes (Cros et a l . 1 9 8 6 ) . I t i s interesting that when the ^-receptor density and isoproterenol stimulated response were measured i n human f a i l i n g heart the same observation was obtained, that i s a decrease i n both {5-receptor numbers and isoproterenol effects. This suggests that a decrease i n (5-receptor density leads to subsensitivity of the fJ-adrenergic pathway and decreased p-agonist stimulated muscle contraction (Bristow et a l . 1 9 8 2 ) . In addition the alt e r a t i o n of the fi-adrenergic system i n diabetic heart may be a general end point of heart dysfunction. I t has been argued that t h i s cardiac deficiency was s p e c i f i c to diabetes since the depressed fi-receptor mediated C a + + uptake i n perfused diabetic rat heart was reversed by i n s u l i n treatment (Gotzsche 1 9 8 6 ). However i t s exact mechanism requires further inves t i g a t i o n . The d i f f e r e n t response to isoproterenol between diabetic v e n t r i c l e and a t r i a obtained i n our experiment i s i n agreement with the observation of Ramanadham and Tenner ( 1 9 8 7 ) but not with the finding of Foy and Lucas ( 1 9 7 8 ) . From a h i s t o l o g i c a l point of view, ven t r i c u l a r tissue has a more developed calcium transport system than a t r i a (Dhalla et a l . I98O), and higher C a + + binding, C a + + uptake, and C a + + ATPase a c t i v i t y of SR. I t i s also reported that the C a + + content was higher > i n a t r i a than i n ve n t r i c l e i n rat (Fukuda 1 9 7 5 ) and that a t r i a l tissue was more resistant to hypoxia (McDonald and MacLeod 1 9 7 3 ) - These tissue differences and s p e c i f i c i t i e s may contribute to the different s e n s i t i v i t i e s to adrenergic stimulation i n 74 rat a t r i a and ve n t r i c l e i n both control and the diabetic tissue. I t i s also possible that the a l t e r a t i o n of the ^-adrenergic system i s more prominent i n ve n t r i c l e than i n a t r i a . An influence of s t r a i n on the ^-adrenergic responses was not detected. The ventricular tissues from a l l diabetic ra t s , Wistar, SHR and WKY, showed s i m i l a r diminished responses. 2. a-Adrenergic Stimulation Phenylephrine, i n the presence of propranolol, represents a a-^ -agonist. The exact pathway following cardiac a-adrenoceptor stimulation i s not very clear. According to Endoh et a l . ( 1 9 8 6 ) , a-adrenergic stimulation may play a functional role i n the regulation of myocardial c o n t r a c t i l i t y . Alpha-adrenergic agonists catalyze polyphosphoinositide turnover i n rat myocardial c e l l s (Brown et a l . 1 9 8 5 ) and rat a t r i a (Sekar and Roufogalis 1 9 8 4 ) . In other b i o l o g i c a l systems, i n o s i t o l 1 , 4 , 5 - t r i p h o s p h a t e (IP3), one of the breakdown products of polyphosphinositides, mobilizes C a + + from i n t r a c e l l u l a r C a + + stores, and diacylglyceride, another product, activates protein kinase C and thereby modifies membrane transport of C a + + (Berridge and Irvine 1 9 8 4 ) . Schmitz et a l . ( 1 9 8 7 ) reported that phenylephrine increased i n o s i t o l triphosphate (IP3) p r i o r to the increase i n force of contraction i n rat isolated l e f t v e n t r i c l e s , and suggested that the increase i n IP^ and force of contraction may be causally related. The character of the cardiac responses to a- and ^-adrenergic stimulation are quite d i f f e r e n t . The f a c i l i t a t i o n of transmembrane Ca + + i n f l u x r e s u l t i n g from stimulation of a-adrenoceptors i s much less than that induced by ^-adrenoceptor stimulation (Endon 1 9 8 6 ) . Stimulation of p-adrenoceptors leads to a prominent abbreviation of both the contractions 75 and Ca + +-aequorin, as an i n t r a c e l l u l a r C a + + indicator, signals. However, stimulation of a-adrenoceptors prolongs the contraction, but abbreviates aequorin signals (Blinks and Endoh 1986). This finding can be explained by the fact that the C a + + a f f i n i t y of troponin C i s increased. The authors concluded that the stimulation of myocardial a-adrenoceptors may increase the C a + + s e n s i t i v i t y of the c o n t r a c t i l e apparatus, while ^-adrenoceptor stimulation decreases the s e n s i t i v i t y . Enhanced a-adrenergic responses have been shown i n l e f t a t r i a from Sprague-Dawley (Jackson et a l . 1985) or Wistar diabetic rats (Canga and Sterin-Berda 1986), right v e n t r i c l e from Sprague-Dawley rats (Ramanadham and Tenner 1987) and perfused heart from neonatal lamb (Downing and Lee 1985). However, an opposite result was found i n diabetic p a p i l l a r y muscles exposed to methoxamine (Heyliger et a l . 1982). A reduced number of a-receptor binding s i t e s with or without change i n the receptor a f f i n i t y has been shown by several investigators (Heyliger et a l . 1982, Latifpour and McNeill 1984, Williams et a l . 1983, Ramanadham and Tenner 1987). In theory the increased s e n s i t i v i t y could be due to an increased a-adrenoceptor density (not l i k e l y i n this case) or to an al t e r a t i o n i n the calcium u t i l i z a t i o n by the myocyte after receptor occupancy for activation of contraction. The observation from our study indicates increased responses to PE i n both ventricular and a t r i a l tissues from Wistar and SHR diabetic rats. Isolated cardiac tissues from these diabetic rats were supersensitive (decrease i n EC50 values) and hyperresponsive (increase i n maximum c o n t r a c t i l i t y ) to a^-adrenoceptor stimulation by PE. No experimental data showing a-adrenoceptor response from ven t r i c u l a r tissues of Wistar diabetic rats have previously been reported . The results from LA are i n accordance 76 with the report of Jackson et al. (1985) who also indicated that in response to PE, diabetic left atria exchanged more ^ C a + + than control left atria and PE had no effect on the efflux of ^Ca++ from either diabetic or control left atria. These data suggest that an alteration in Ca+ + mobilization may contribute to the enhanced receptor activation. The increase in exchangeable Ca + + could be provided by either an increase in the influx of Ca + + from extracellular sites and/or the release of Ca + + from intracellular storage sites (i.e. SR, mitochondria, Ca + + binding proteins). Some investigators attribute the enhanced a-adrenergic response to the influence of the 'hypothyroidism' which accompanies the diabetic state. Hypothyroidism has been associated with an enhanced response of the rat myocardium to a-adrenergic stimulation, a decreased response to adrenergic stimulation (Simpson et al. 1981), and a shift from a (J- to a-receptor type response (Simpson et al. 1981, Kunos et al. I98O). There is also a report that hypothyroid rat papillary muscle is more sensitive to Ca + + and that the response to BAY K 8644 is increased (Hawthorn et al. 1988). T^ treatment was shown to prevent the enhanced a-adrenergic response in diabetic rat atria (Goyal et al. 1987). However, the depression of the ^-adrenoceptor response, cardiac contraction, and Ca + + uptake activity by SR could not be prevented by T^ treatment (Goyal et al. 1987, Tahiliani and McNeill 1984). Our results show that a significant decrease in the T^/T^ plasma concentrations in diabetic rats accompanied an enhanced a-adrenoceptor sensitivity/response in cardiac preparations, especially in ventricular tissues. Coupled with the information stated above, we suggest that the enhanced a-response and hypothyroidism in diabetic rats are related, but some other diabetic-induced alterations in adrenoceptor activity and myocardial dysfunction itself cannot be 77 associated with hypothyroidism. Furthermore the role of increased a-response i n diabetic cardiomyopathy i s not clear. 3. Ca + + I t i s well known that cardiac function i s dramatically influenced by the concentration of ex t r a c e l l u l a r C a + + (Ringer 1 8 8 3 ) . The mechanism of the positive inotropic effect induced by e x t r a c e l l u l a r calcium i s based on the cross membrane potential maintained by the ion gradients of Na +, K + and Ca + +. Upon depolarization, e l e c t r i c a l l y or chemically, the e x t r a c e l l u l a r C a + + enters the c e l l through Ca + +-channels and an increase i n the a v a i l a b i l i t y of C a + + to the c o n t r a c t i l e protein could enhance the a b i l i t y of the tissue to develop force. In a t y p i c a l mammalian c e l l under physiological conditions, the e x t r a c e l l u l a r and i n t r a c e l l u l a r C a + + concentration would be i n the range of 10~^M and 10~^M (Carefoli and Penniston 1985)• I t has been suggested that diabetes results i n a C a + + overload i n the heart (Dhalla et a l . 1985, McNeill and T a h i l i a n i 1986) . Several l i n e s of evidence support such hypothesis. An increased content of C a + + i n diabetic heart (Regan et a l . 1981, Dhalla et a l . 1985) has been reported and related to the increased permeability of SL (Pierce et a l . 1983) and depressed C a + + ATPase, Na +-Ca + + exchange (Dhalla et a l . 1985. Makino et a l . 1987) and C a + + uptake a c t i v i t y of SR (Lopaschuk et a l . 1983)-Increased electron density of mitochondria and mitochondria swelling have been found i n diabetic animal heart by l i g h t and electron microscopic analysis (Seager et a l . 1984 ) . Cytoplasmic enzymes such as phosphorylase exhibit higher basal a c t i v i t i e s and an abnormal s e n s i t i v i t y to phosphorylase kinase i n diabetic hearts ( M i l l e r 1984) which has been suggested to be the result of an increased i n t r a c e l l u l a r free C a + + 78 concentration i n diabetic myocardium. I t i s suspected that i f the i n t r a c e l l u l a r C a + + concentration i s high, the membrane resting potential may be closer to the threshold of triggeri n g normal contraction (more depolarized), and more C a + + would cause a stronger contraction as well. This may explain the supersensitivity and hyperresponsiveness of the cardiac tissues observed i n our experiments. From different cardiac tissues, supersensitivity to C a + + was reported by Ramanadham (1°>83, 1987) and Borda (1988), but there are some negative results as well (Downing et a l . 1983). The subcellular organelles, S L , SR and mitochondria, which control C a + + mobilization i n diabetic rats, with depressed function (Dhalla et a l . 1985) may at least p a r t l y play a role i n the increased C a + + s e n s i t i v i t y and response. The p a r a l l e l enhancement of the a-adrenergic and C a + + responses shown by our data may be related. Because a-stimulation i s mediated by Ca + + i n f l u x , we f e e l that the al t e r a t i o n i n the a-adrenergic-Ca + + response pathway may occur after the a-receptor i s occupied. Okumura et a l (1988) documented a high 1 , 2-diacylglycerol l e v e l i n diabetic myocardium which may res u l t i n the activation of protein kinase C. I n s u l i n , one of the agonists which generate 1 , 2-diacylglycerol, however did not influence 1,2-d i a c y l g l y c e r o l content i n diabetic myocardium. Recent work i n our laboratory has shown that i n o s i t o l 1 , 4 , 5 -triphosphate, which l i k e d i a c y l g l y c e r o l i s another important second messenger, concentrations i n rig h t v e n tricles of Wistar diabetic rats were s i g n i f i c a n t l y increased (Xiang 1990)• The exact mechanism of these a l t e r a t i o n requires further investigation. As a whole, increased a-adrenergic and C a + + s e n s i t i v i t i e s suggest an enhanced a b i l i t y of diabetic heart to u t i l i z e C a + + for contraction. The 79 a l t e r a t i o n of the movement of e x t r a c e l l u l a r C a + + across the myocardial membrane i s probably an important event i n the abnormal co n t r a c t i l e r e a c t i v i t y of diabetic heart to a/f> adrenoceptor agonists, and to ex t r a c e l l u l a r Ca + +. 4. BAY K 8644 and Verapamil BAY K 8644 i s the most potent and most extensively studied 1 ,4 , -dihydropyridine compound with C a + + channel agonist properties. I t was discovered by Schramm et a l . (1983) and was shown to have cardiotonic and vasoconstrictor properties. I t s a c t i v i t y was attributed to i t s ( - ) - (4S) -isomer, while the (+)-(4R)-isomer had C a + + channel antagonistic properties (Franckowiak 1985). The potency of the C a + + antagonistic (+)-(4R)-isomer i s much lower than that of agonistic (-)-(4S)-isomer as an agonist. Therefore, results obtained from investigations with racemic BAY K 8644, as i n our experiments, can be considered as effects of a C a + + agonist. BAY K 8644 acts primarily on the same s i t e , presumably the dihydropyridine receptors of the voltage-dependent C a + + channels, at which nifedipine acts (Aoki and Asano 1987). The BAY K 8644-induced vasopresser and p o s i t i v e inotropic effects appear to be primarily due to the increase i n C a + + entry into vascular smooth muscle or myocardial c e l l s , respectively (Gacia et a l , 1984). A s e n s i t i v i t y to BAY K 8644 as shown by increased pD 2 values was found only i n SHR diabetic l e f t a t r i a i n our studies. No s i g n i f i c a n t difference i n terms of maximum response to BAY K was observed between diabetic and control tissues of a l l st r a i n s of rats tested and both v e n t r i c l e and l e f t a t r i a showed much lower maximum responses than with C a + + i t s e l f . These results could depend upon the density and/or a f f i n i t y of 80 calcium channels and BAY K binding s i t e s , but they need to be examined i n a t r i a and v e n t r i c l e s i n these r a t s . We have measured PN-200-110 ( i s r a d i p i n e ) , a 1,4-DHP calcium channel antagonist (Hof et a l . 1984), binding i n crude cardiac homogenate of Wistar c o n t r o l and d i a b e t i c rats (n=8 r e s p e c t i v e l y ) . No dif f e r e n c e i n B_„-_ and Kd was found between the two groups i n these preparations (unpublished observation) which indicates that no p r o l i f e r a t i o n i n 1,4-DHP binding s i t e s could be found i n d i a b e t i c cardiac SL. Increased response to BAY K, ind i c a t e d by increased pD 2 values and maximum contraction i n SHR femoral a r t e r i e s has also been reported (Aoki and Asano 1987). The authors suggested that the SHR a r t e r i e s were more depolarized than WKY rat femoral a r t e r i e s . On the whole, the reason f o r the increased s e n s i t i v i t y to BAY K 8644 i n SHR d i a b e t i c l e f t a t r i a cannot be explained by present experimental data, but i t presumably i s r e l a t e d to the increased responsiveness to C a + + i n d i a b e t i c cardiac tissues, which may be a more prominent a l t e r a t i o n i n SHR d i a b e t i c model. In addition, the f a c t that the s u p e r s e n s i t i v i t y to BAY K 8644 only e x i s t s i n SHR di a b e t i c a t r i a but not v e n t r i c l e may be due to the d i f f e r e n t C a + + transport system i n these two tis s u e s and a higher concentration of C a + + i n a t r i a than v e n t r i c l e as mentioned above (Dhalla et a l . 1980) Verapamil i s one of the f i r s t members of the C a + + channel antagonists. I t was shown i n 1964 to mimic the cardiac e f f e c t s of C a + + withdrawal i n that i t diminished c o n t r a c t i l e force without producing a major change i n the action p o t e n t i a l and these e f f e c t s could be reversed by the addition of C a + + (Fleckenstein 1983). According to the WHO c l a s s i f i c a t i o n of calcium antagonists, verapamil-like Type I calcium antagonists s e l e c t i v e l y act on the L(long lasting)-channel (Vanhoutt 1987). 81 Verapamil i s also known to interact with adrenergic and muscarinic receptors (Insel 19-88). In our study, verapamil, at 10~^ M - 10~7M concentrations, did not produce a negative inotropic action (Figure 20). This confirms a previous report i n which, at concentrations less than 10~7M verapamil showed l i t t l e i n h i b i t i o n on the increase i n membrane C a + + conductance i n rat ventricular trabeculae (Payet et a l . 1980) . The s e n s i t i v i t y to verapamil i n LA from SHR diabetic rats appears to be increased (Figure 21) but not s i g n i f i c a n t l y . The s e n s i t i v i t y of other diabetic tissues i n a l l strains showed no change. The maximum negative inotropic response i n diabetic ventricular s t r i p s was higher (Figure 20). These phenomena might be related to the supersensitive and hyperesponsiveness responses to C a + + i n diabetic heart. Verapamil binding s i t e s and C a + + binding s i t e s i n SL are closely situated and functionally associated (Janis et a l . 1987). I t i s possible that these binding s i t e s and/or processes after binding are altered especially i n SHR diabetic rats. 5. The E f f e c t s of Ryanodine The primary effect of ryanodine on cardiac muscle i s a negative inotropic e f f e c t . Sutko et a l . (1985) interpreted t h i s e f f e c t as either a slowing of the time course of C a + + release from SR or a decrease i n the extent or magnitude of the C a + + release. Meissner (1986) also reported that ryanodine can either block or enhance C a + + loss from isolated SR vesi c l e s , depending on the experimental conditions. Cardiac preparations yielded non-linear Scatchard plots i n d i c a t i n g the presence of multiple receptor s i t e s . Comparison of the binding data with dose response curves suggests ryanodine stimulated the release of C a + + from SR by binding to 82 high a f f i n i t y s i t e s on the C a + + channel. These s i t e s may be accessible only when the channel i s open. Inactivation of C a + + release was observed only when ryanodine concentrations exceeded 10 uM (Meissner 1986). According to these reports, ryanodine 3xl0~^M as used i n our experiment, would release and deplete C a + + from SR. BDF i n a l l cardiac tissues decreased and there were no differences between strains or among the diabetic groups, except that LA were more sensitive to ryanodine than PM as represented by a more prominent decrease i n BDF (Table 11). These data add to the report by Fabiato (1985) i n which no s i g n i f i c a n t differences were observed i n the effects of ryanodine i n skinned cardiac c e l l s from di f f e r e n t adult mammalian species. The effects of ryanodine on PQP are varied. I t depressed PQP i n diabetic tissues which had diminished PQP before treatment. SHR diabetic PM and LA showed almost complete a b o l i t i o n of PQP with the treatment of ryanodine. I t seems that effects of ryanodine are greater i n diabetic Wistar PM and SHR PM/LA, and that SHR diabetic SR are the most susceptible to t h i s interference. The impaired function of SR i n diabetic rats (see above) may also contribute to t h i s phenomenon. WKY diabetic tissues showed no response to t h i s concentration of ryanodine, which may be related to the unchanged PQP (see above) observed i n t h i s tissue. In comparison with the pretreatment l e v e l , ryanodine had no influence on post stimulation potentiated peak tension, PSP+BDF, no matter whether diabetic or control tissues were used. This may be explained by the fact that ryanodine has no effects on Na +-Ca + + exchange i n SL. The r a t i o of PSP/BDF was increased by ryanodine, i n PM and LA. The increased r a t i o can be attributed to the decrease i n BDF produced by ryanodine which, as we mentioned before, i s more prominent i n LA than PM. In diabetic tissues the 83 r a t i o of PSP/BDF i n Wistar and SHR PM and (PSP+BDF) i n SHD LA were decreased after ryanodine i f we make a comparison with the same treatment control. This cannot be explained d i r e c t l y by the effects of ryanodine. Diabetic tissue may have an i n t r i n s i c deficiency i n generating PSP and the influence of ryanodine may add to t h i s e f f e c t . However these in d i r e c t measurements do not allow us to determine the exact mechanism. 8 4 SUMMARY AND CONCLUSIONS 1. STZ treated Wistar, SHR and WKY rats, as expected, exhibited the 'c l a s s i c a l signs' of diabetes, hyperglycemia, hyperinsulinemia, hyperlipedemia (not i n WKY) and 'hypothyroidism'. 2. SHR diabetic rats showed the greatest changes i n body weight loss hyperglycemia and hyperlipidemia and had a high mortality ( 3 8 . 8 % ) , which suggests that the combined effecs of diabetes mellitus and hypertension produce a greater degree of pathological alterations. 3 - Major myocardial alterations observed i n diabetic include: a. Decreased basal a t r i a l rate and enhanced BDF i n RV which suggested a depressed SA node function and al t e r a t i o n of Ca + + u t i l i z a t i o n . b. Decreased PQP values (not i n WKY) i n ventricular tissues suggesting a diminished degree of releasable C a + + from SR. Decreased PSP value i n SHR LA possibly related to the depressed SL Na +-Ca + + exchange i n t h i s tissue. c. Alterations of the cardiac drug responses: subsensitivity to fj-adrenergic stimulation i n ventr i c u l a r tissues; supersensitivity and hyperresponsiveness to C a + + and a-adrenergic stimulation i n ventricular tissue and l e f t a t r i a ; and supersensitivity to BAY K 8 6 4 4 and hyperresponsiveness to verapamil i n SHR l e f t a t r i a and ventr i c u l a r s t r i p s . These vari a t i o n may be attributed to the changed receptor numbers and/or post receptor alterations. d. Ryanodine (3X10"9M) depressed PQP of Wistar (PM) and SHR (PM, LA) including ccontrols. I t especially abolished PQP i n SHR 85 diabetic tissues, but had no effects on WKY tissues. These results suggest the differences i n the SR function i n the tissues. Diabetic SR with impaired C a + + uptake may contribute to these phenomena. Ryanodine depressed (PSP+BDF) of SHE LA and (PSP/BDF) of Wistar and SHR PM, but had no e f f e c t on other control or diabetic tissues. In view of t h i s , the influence of ryanodine on diabetic SL NA +-Ca + + exchange needs to be further investigated. 4. The experimental data suggest an impairment of C a + + mobility i n diabetic and hypertensive diabetic rats. The inseparable relationship between the enhanced i n t r i n s i c a c t i v i t i e s of the a-adrenergic agonist and C a + + i n diabetic heart tissue, the depressed SR Ca + +-release and SR Na +-Ca + + exchange function, and the diminished (S-adrenergic response as well probably play an important role i n the development of diabetic cardiomyopathy. Additionally, hypertensive diabetic rats expressed greater changes such as hyperlipidemia, depressed PQP and PSP values, and altered drug responses ind i c a t i n g abnormal SR or SL function. These may add to information about t h i s combined disease and i t s high mortality. 86 REFERENCES Aoki, K. and Asano, M. 1987- Increased responsiveness to calcium agonist BAY K 8644 and calcium antagonist nifedipine i n femoral arteries of spontaneously hypertensive rats J . Cardiovascul. Pharmacol. 19(suppl.l0):S62-S64. Ariens, E.J. and Simonis, A.M. 1964. Drug receptor i n t e r a c t i o n : interaction of one or more drugs with a receptor system. In: Molecular Pharmacology, Vol. 1. pp.136-148 (Ed.) E.J. Ariens. Academic Press, Inc., New York. Baandrup, U., Ledet, T. and Rasch, R. 1981. Experimental diabetic cardiomyopathy prevented by i n s u l i n treatment. Lab. Invest. 45:l63 -173. B e l l , D.S.H. 1989- Hypertension i n the person with diabetes. Am. J. Med. Sc i . 297(4):228-232. Berridge, M.J. and Irvine, R . F . 1984. I n o s i t o l triphosphate, a novel second messenger. Biochem. J. 220:345~360. Bers, D.M. 1985- Ca i n f l u x and sarcoplasmic reticulum C a + + release i n cardiac muscle activation during post-rest recovery. Am. J. Physiol 248:H366-H38l. Blinks, J.R. and Endoh, M. 1986. Modification of m y o f i l b r i l l a r responsiveness to Ca + + as an inotropic mechanism. Circulation 73[suppl] l l l . 8 5 - l l l . 9 8 . Borda, E., Pascual, J . , Wald, M. and Borda, L.S. 1988. Hypersensitivity to calcium associated with an increased sarcolemmal C a + + ATPase a c t i v i t y i n diabetic r a t heart. C a n . J . Cardiol. 4(2):97-101• 87 Bose, D., Hryshko, L.V. , King, B.W. and Chau, T. 19-88. Control of interval-force r e l a t i o n i n canine ve n t r i c u l a r myocardium studied with ryanodine. Br. J. Pharmacol. 95:811-820. Bowditch, H.P. 1871- Uber die Eigenthumlichkeiten der Reizbarkeit, welch die Muskelfasern des Herzens zeigen. Arch. Physiol. Leipzig 6:139~ 176. Bristow, M.R., Ginsberg, R. , Minobe, W., C u b i c c i o t t i , R.S., Sageman, W.S., Lurie, K. , Billingham, M. , Harrison, D.C. and Stinson, E.B. I982. Decreased catecholamine s e n s i t i v i t y and fS-adrenergic-receptor density i n f a i l i n g human heart. N. Engl. J. Med. 307:205-211. Bristow, M.R., Kantrowitz, N.E., Ginsberg, R. and Fowler, M.B. 1984. Beta-adrenergic function i n heart muscle disease and heart f a i l u r e . J. Mol. C e l l . Cardiol. 17[Suppl. 2] 41-52. Brown, J.H., Buxton, I.L. and Brunton, L.L. 1985- a^-adrenergic and muscarinic cholinergic stimulation of phosphoinositide hydrolysis i n adult rat cardiomyocytes. Ci r c . Res. 57:532-537-Canga, L. and Sterin-Berda, S. 1986. Hypersensitivity to methoxamine i n a t r i a isolated from streptozotocin-induced diabetic rats. Br. J. Pharmac. 87:157-165-C a r a f o l i , E. and Penniston, J.T. 1985- The calcium s i g n a l . S c i e n t i f i c American 7155:70-78. Chemnitius, J.M., Sasaki, Y. , Burger, W. and Bing, R.J. 1985- The effect of ischemia and reperfusion on sarcolemmal function i n perfused canine hearts. J.Mol. C e l l . Cardiol. 17:1139-1150. Cohen, C.J., Fozzard, H.A. and Sheu, S-S. 1982. Increase i n i n t r a c e l l u l a r sodium ion a c t i v i t y during stimulation i n mammalian cardiac muscle. C i r c . Res. 50:651-662. 88 Cros, G.H., Chanez, P.O., Michel, A., McNeill, J.H. and Serrano, J.J. 1986. Cardiac ^-adrenergic receptors i n diabetic rats: a l t e r a t i o n of guanyl nucleotide regulation. J. Pharmacol. (Paris) 17(4):595_600. Dhalla, N.S., Pierce, G.N., Innes, I.R. and Beamish, R.E. 1985-Pathogenesis of cardiac dysfunction i n diabetes mellitus. Can. J. Cardiol. 1(4):263"28l. Dhalla, N.S., Sulakhe, P.V., Lee, S.L., Singal, P.K., Varley, K.G. and Yates, J.C. 1980. Subcellular Ca + + transport i n different areas of dog heart. Can. J. Physiol. Pharmacol. 58:360-367. Dillman, W.H. 1980. Diabetes mellitus induces changes i n cardiac myosin of rat. Diabetes 29:579~582. Downing, S.E., Lee, J.C. and Fripp, R.R. 1983« Enhanced s e n s i t i v i t y of diabetic hearts to a-adrenoceptor stimulation. Am. J. Physiol. 245:H808-H813. Downing, S.E. and Lee, J.C. 1985. Enhanced adrenergic s e n s i t i v i t y of the diabetic neonatal heart. Am. J. Physiol. 248:H125-H131. El-Mallakh, R.S. 1986. Hypertension and diabetes i n obesity: a review and new ideas on the contributing role of ions. Medical Hypotheses 19:47-55-Endoh, M. and Blinks, J.R. 1988. Action of sympathomimetic amines on the Ca^ + transients and contractions of rabbit myocardium: reciprocal changes i n m y o f i b r i l l a r responsiveness to Ca^ + mediated through alpha- and beta-adrenoceptors. C i r . Res. 62(2):247~265. Endoh, M. , Yanagisawa, T. , Taira, N. and Blinks , J.R. 1986. Effects of new inotropic agents on c y c l i c nucleotide metabolism and calcium transients i n canine ventricular muscle. C i r c u l a t i o n 73 (supple) III . l i 7-HI . i 3 3 . 89 Fabiato, A. 1983- Calcium-induced-release of calcium from cardiac sarcoplasmic reticulum. Am. J. Physiol. 245:C1-Cl4. Fabiato, A. 1985- Effects of ryanodine i n skinned cardiac c e l l s . Federation Proc. 44:2970-2976. Factor, S.M., Bhan, R., Minase, T., Wolinsky, H. and Sonnenblick, E.H. 1 9 8 l . Hypertensive-diabetic cardiomyopathy i n the rat, an experimental model of human disease. Am. J. Pathol. 102:219-228. Factor, S.M., Minase, T. and Sonnenblick, F.H. I98O. C l i n i c a l and Morphological features of human hypertensive-diabetic cardiomyopathy. Am. Heart J. 79:446-458. Factor, S.M., Takashi, M., Bhan, R., Wolinsky, H. and Sonnenblick, E.H. I983. Hypertensive diabetic cardiomyopathy i n the rat: ul t r a s t r u c t u r a l features. Virchows Arch. [Pathol. Anat.] 398:305" 317. Fein, F.S., Capasso, J.M., Aronson, R.S., Cho, S., Nordin, C. , M i l l e r -Green, B., Sonnenblick, E.H. and Factor, S.M. 1984. Combined renovascular hypertension and diabetes i n rats: A new preparation of congestive cardiomyopathy. C i r c u l a t i o n 7 0 ( 2 ) : 3 l 8 ~ 3 3 0 . Fein, F.S., Kornstein, L.B., Strobeck, J.E., Capasso, J.M. and Sonnenblick, E.H. I98O. Altered myocardial mechanics i n diabetic rats. Circ. Res. 49:922-933-Fischer, L.J. 1985- Drugs and chemicals that produce diabetes. Trends Pharmac. S c i . 6 :72-75. Fleckenstein, A. 1983- Calcium antagonism i n heart and smooth muscle. Experimental facts and therapetic prospects. John Wiley and Sons, New York. 90 Foster, D.W. 1983- Diabetes mellitus. In: Harrison's P r i n c i p l e s of Internal Medicine. Tenth editio n . Edited by Adams, R.D., Martin, J.B., Bracenwald, E ., Wilson, J.D., Isselbacher, K.J. and Petersdrof, R.G. pp.661-679. McGram-Hill Book Company, New York. Foy, J.M. and Lucas, P.D. 1978- Comparison between spontaneously beating a t r i a from control and streptozotocin-diabetic rats. J. Pharm. Pharmac. 30:558-562. Franckowiak, G., Bechem, M., Schramm, M. and Thomas, G. 1985- The op t i c a l isomers of the 1 ,4-dihydropyridine BAY K 8644 show opposite effects on Ca channels. Eur. J. Pharmacol. 114:223-226. Friberg, P. and Nordborg, C. 1986. Functional, morphological and metabolic charactgeristics of isolate d hearts from normotensive and spontaneously hypertensive rats before, during and after renal hypertension. Acta. Physiol. Scand. 126:161-171-Friberg, P., Nordlander, M., Lundin, S. and Folkow, B. 1985- Effects of aging on cardiac performance and coronary flow capacity i n spontaneously hypertensive and normotensive control rats. Acta. Physiol. Scand. 125:1-11. Fukuda, Y. 1975- Difference i n calcium content of a t r i a l and ventricular muscle. Jpn. J. Physiol. 25:407-479-Gacia, A.G., Sala, F., Reig, J.A., Viniegra, S., Fr i a s , J . , Fonterz, R. and Gardia, L. 1984. Dihydropyridine BAY K 8644 activates chromaffin c e l l calcium channels. Nature 309:69~71-Ganguly,, P.K., Pierce, G.N., Dhalla, K.S. and Dhalla, N.S. 1983-Defective sarcoplasmic r e t i c u l a r calcium transport i n diabetic cardiomyopathy. Am. J. Physiol. 244:E528-E535. 91 Garber, D.W., Everett, A.W. and Neely, J.R. 1983- Cardiac function and myosin ATPase i n diabetic rats treated with i n s u l i n , T^ and T^. Am. J. Physiol. 244:H592-H598. Gonzalez, C., Montoya, E., J o l i n , T. and Gonzalez, M. I98O. Effect of streptozotocin diabetes on the hypothalamic-pituitary-thyroid axis i n the r a t . Endocrinology 107(6):2099"2103. Gotzsche, 0 . 1983- Decreased myocardial calcium uptake after isoproterenol i n streptozotocin-induced diabetic rats. Studies i n the i n v i t r o perfused heart. Lab. Invest. 48:156-161. Gotzsche, 0 . 1983- The adrenergic f-receptor adenylate cyclase system i n heart and lymphocytes from streptozotocin-diabetic rats. Diabetes 32:1110-1116. Gotzsche, 0 . I986. Myocardial c e l l dysfunction i n diabetes mellitus. Diabetes 35:1158-1162. Goyal, R.K., Rodrigues, B. and McNeill, J.H. 1987. Effects of t r i -iodothyronine on cardiac responses to adrenergic-agonists i n STZ-induced diabetic rats. Gen. Pharmac. 1 8 ( 4 ) : 3 5 7 _ 3 6 2 . Grassby, P.F. and McNeill, J.H. 1988. S e n s i t i v i t y changes to inotropic agents i n rabbit a t r i a a f t e r chronic experimental diabetes. Can. J. Physiol. Pharmacol. 6 6 : l 4 7 5 ~ l 4 8 0 . Gudbjarnason, S., El-Hage, A.N., Whitehurst, V.E., Simental, F. and Balazs, T. 1987. Reduced arachidonic acid i n major phospholipids of heart muscle i n the diabetic r a t . J. Mol. C e l l . Cardiol. 19:1141-1146. Hajdu, S. 1969' Mechanism of woodworth staircase phenomenon i n heart and sk e l e t a l muscle. Am. J. Physiol. 216(1):206-214. 92 Hattori, Y. and Kanno, M. 1 9 8 4 . Influences of e x t r a c e l l u l a r calcium ions, verapamil, and calcium antagonistic cations on the po s i t i v e inotropic effects mediated by a- and ^-adrenoceptors i n the l e f t a t r i a of rats. Gen. Pharmac. 1 5 ( 2 ) : 9 1 - 9 7 . Hawthorne, M.H., Gengo, P., Wei, X.Y., Rutledge, A., Moran, J.F., Gallant, S. and Triggle, D.J. 1 9 8 8 . Effect of thyroid status on B-adrenoceptors and calcium channels i n rat cardiac and vascular tissue. Arch. Pharmacol. 3 3 7 : 5 3 9 _ 5 4 4 . Heyliger, C.E., Pierce, G.N., Singal, P.K., Beamish, R.E. and Dhalla, N.S. 1 9 8 2 . Cardiac alpha- and beta-adrenergic receptor alterations i n diabetic cardiomyopathy. Basic Res. Cardiol. 7 7 : 6 1 0 - 6 1 8 . Hof, R.P., Scholtysik, G. , Lontzenhiser, R. , Vuorela, H. and Neuman, P. 1 9 8 4 . PN 2 0 0 - 1 1 0 , a new calcium antagonist: electrophysiological, inotropic and chronotropic effects on guinea pig myocardial tissue and effects on concentration and calcium uptake. J. Cardiovasc. Pharmacol. 6 : 3 9 9 - 4 0 6 . Ingebretsen, C.G., Haweln-Johnson, C. and Ingebretsen, W.R. J r . 1 9 8 3 -Alloxan-induced diabetes reduces B-adrenergic receptor number without af f e c t i n g adenylate cyclase i n rat v e n t r i c u l a r membranes. J. Cardiovascul. Pharmacol. 5 : 4 5 4 - 4 6 1 . I n s e l , P.A. 1 9 8 8 . Phenylalkylamines are promiscuous receptor blockers. Trends Pharmacol. S c i . 9 : 1 0 . Iwase, M., Kikuchi, M., Nunoi, K., Wakisaka, M., Maki, Y., Sadoshima, S. and Fujishima, M. 1 9 8 7 . Diabetes induced by neonatal streptozotocin tgreatment i n spontaneously hypertensive and normotensive rats. Metabolism 3 6 ( 7 ) : 6 5 4 - 6 5 7 . 93 Iwase, M., Nunoi, K., Kikuchi, M., Maki, Y., Kodama, T., Sadoshima, S. and Fujishima, M. 1989- Mophometrical and biochemical differences of endocrine pancreats between spontaneously hypertensive and normotensive rats with or without neonatal streptozotocin-induced diabetes. Laboratory Investigation 60(1):102-105-Jackson, C.V., McGrath, G.M. and McNeill,J.H. 1985- A l t e r a t i o n i n alpha-^-adrenoceptor stimulation of isolat e d a t r i a from experimental diabetic rats. Can. J. Physiol. Pharmacol. 64:l45~151-Jaffe, A.S., Spadaro, J.J., Schechtma, K., Roberts, R. , Geltma, E.M. and Sobel, B.E. 1984. Increased congestive heart f a i l u r e a f t e r myocardial i n f a r c t i o n of modest extent i n patients with diabetes mellitus. Am. Heart J. 108:31-37-Janis, R.A., S i l v e r , P.J. and Triggle, D.J. 1987- Drug action and c e l l u l a r calcium regulation. In: Adv. Drug Res., (Ed.) Burnard, T. 16:479-508. Academic Press, London. Kannel, W.B. 1978. Role of diabetes i n cardiac disease: conclusion from population studies. In: Diabetes and the Heart. (Ed.) S. Zoneraich, pp.97-112. Charles C. Thomas, Sp r i n g f i e l d . Kannel, W.B. and McGee, D.L. 1979- Diabetes and cardiovascular disease. The Framingham Study. JAMA 24l:2035-2038. Katz, A.M. and Messineo, F.C. I 9 8 I. Lipid-membrane interaction and the pathogenesis of ischemic damage i n the myocardium. C i r c . Res. 48:1-16. Kunos, G., Mucci, L. and 0'Regan. S. 1980. The influence of hormonal and neuronal factors on rat heart adrenoceptors. Br. J. Pharmac. 71:371-386. 94 Kunos, G. , Vermes-Kunos, I. and Nickerson, M. 1974. Effects of thyroid state on adrenoceptor properties. Nature 250:779"78l. Langer, G.A. and Rich, T.L. 1986. Augmentation of sarcolemmal Ca by anionic amphiphile: co n t r a c t i l e response of three ventricular tissues. Am. J. Physiol. 250:H247~H254. Langer, G.A. 1983. The 'sodium pump lag' r e v i s i t e d . J . Mol. C e l l . Cardiol. 15:647-651. Latifpour, J . and McNeill, J.H. 1984. Cardiac autonomic receptors: effect of long-term experimental diabetes. J. Pharmacol. Exp. Ther. 230:242-249. L e v i t z k i , A. 1987- Regulation of hormone-sensitive adenylate cyclase. TIPS 8:299-303. Lopaschuk, G.D., T a h i l i a n i , A.G., Vadlamudi, R.V.S.V., Katz, S. and McNeill, J.H. 1983- Cardiac sarcoplasmic reticulum function i n i n s u l i n or carnitine-treated diabetic rats. Am. J. Physiol. 845:H969-H976. Lovenberg, W. 1987. Animal models for hypertension research. In: Animal models: Assessing the Scope of Their Use i n Biomedical Research. Progress i n C l i n i c a l and B i o l o g i c a l Research, Vol. 29, pp.225-240 (Ed.) Kawamata, J. and Melby, EC J r . , Alan R. L i s s , Inc., New York. Lukas, A. and Bose R. I986. Mechanisms of frequency-induced potentiation of contractions i n isolated a t r i a . Naunyn-Schmiedeberg's Arch. Pharmacol. 334:480-487• Makino, N., Dhalla, K.S., Elimban, V. and Dhalla, H.S. 1987. Sarcolemmal C a + + transport i n streptozotocin-induced diabetic cardiomyopathy i n rats . Am. J. Physiol. 253:F202-F207. 95 Makino, N. , Jasmin, G., Beamish, R.E. and Dhalla, N.S. 1985- Sarcolemmal Na +-Ca + + exchange during the development of genetically determined cardiomyopathy. Biochem. Biophys. Res. Commun. 133:491-497• Makino, N., Phruvarajan, R., Elimban, V., Beamish, R.E. and Dhalla, N.S. I985. Alterations of sarcolemmal Na +-K + exchange i n catecholamine-induced cardiomyopathy. Can. J. Cardiol. 1:225-232. McDonald, T.F. and MacLeod, D.P. 1973- Anoxic a t r i a l and ventricular muscle e l e c t r i c a l a c t i v i t y , c e l l potassium, and metabolism. A comparative study. J. Mol. C e l l . Cardiol. 5:149-159. McNeill, J.H. and T a h i l i a n i , A.G. 1986. Diabetes-induced cardiac changes. Trends Pharmacol. S c i . 7(9):364~367-Meissner, G. 1986. Ryanodine activation and i n h i b i t i o n of the Ca + + release channel of sarcoplasmic reticulum. J . B i o l . Chem. 26(14):6300-6306. M i l l e r , T.B. 1984., Phosphorylase activation hypersensitivity i n hearts of diabetic rats. Am. J. Physiol. 246:E134-l40. Mooradian, A.D., Morley, J.E. and Scarpace, P.J. 1988. The role of zinc status i n altered cardiac adenylate cyclase a c t i v i t y i n diabetic r a t s . Acta Endocrinologica (Copenh) 119:174-180. Morley, J.E., Levine, A.S., Brown, D.M. and Handmerger, B.S. 1982. Calmodulin levels i n diabetic mice. Biochem. Biophys. Res. Commun. 108:1418-1423. Nakamura, K. and Nakamura, K. 1978. Role of brainstem and spinal noradrenergic and adrenergic neurons i n the development and maintenance of hypertension i n spontaneously hypertensive rats. Naunyn-Schemiedeberg's Arch. Pharmacol. 305:127-96 Nishio, Y. , Kashivagi, A., Kida, Y., Kodoma, M., Abe, N. , Saeki, Y. and Shigeta, Y. 1988. Deficiency of cardiac B-adrenergic receptors i n streptozocin-induced diabetic rats. Diabetes 37:1181-1187-Nobel, MIM. 1983' Excitation-contraction coupling. In Cardiac Metabolism pp.49-71 (Eds.) A. Drake-Holland and MIM Noble. John Wiley, Chichester. Noresson, E., Rickston, S.E., Hallback-Nordlander, M. and Thoren, P. 1979-Performance of the hypertrophied l e f t v e n t r i c l e i n spontaneously hypertensive rats. Effects of changes i n preload and afterload. Acta. Physiol. Scand. 107:1-8. Okamota, K. and Aoki, K. 1963- Development of a s t r a i n of spontaneously hypertensive rats. Jpn. C i r c . J . 27:282. Okumura, K., Akiyama, N., Hashimoto, H., Ogawa, K. and Satake, T. 1988. Alteration of 1,2-diacylglycerol content i n myocardium from diabetic ra t s . Diabetes 37:1168-1172. Payet, M.D., Schanne, D.F., Ruiz-Ceretti, E. and Demers, J.M. I98O. Inhibitory a c t i v i t y of blocker of slow inward current i n rat myocardium, a study i n steady state and of rate of action. J . Mol. C e l l and Cardiol. 12:187-200. Penpargkul, S., Schaible, T., Y i p i n s t o i , T. and Scheuer, J . I98O. The effects of diabetes on performance and metabolism of rat hearts. C i r c . Res. 47:911-921. Pierce, G.N. and Dhalla, N.S. 1983. Sarcolemmal Na +-K + ATPase a c t i v i t i e s i n diabetic rat heart. Am. J. Physiol. 245:C24l-C247-Pierce, G.N. and Dhalla, N.S. 1985' Mechanisms of the defect i n cardiac m y o f i b r i l l a r function during diabetes. Am. J. Physiol. 248 :E170-E175-97 Pierce, G.N., Kutryk, M.J.B. and Dhalla, N.S. 1983. Alterations i n C a + + binding by and composition of the cardiac sarcolemmal membrane i n chronic diabetes. Proc. Natl. Acad. S c i . 8 0 : 5 4 l 2 - 5 4 l 6 . Pittman, C.S., Suda, A.K., Chambers, J.B. and Ray, G.Y. 1979- Impaired 3 . 5 . 3 '-triiodothyronine (T^) production i n diabetic patients. Metabolism 28:333-338. Ramanadham, S. and Tenner, T.E. J r . 1983- Alterations i n cardiac performance i n experimentally-induced diabetes. J . Pharmacol. 27:130. Ramanadham, S. and Tenner, T.E. J r . 1986. Chronic effects of streptozotocin diabetes on myocardial s e n s i t i v i t y i n the rat. Diabetologia 27:741-748. Ramanadham, S. and Tenner, T.E. J r . 1987. Alterations i n the myocardial ^-adrenoceptor system of streptozotocin-diabetic r a t s . Eur. J. Pharmacol. 136:337-389-Ramos, O.L. 1988. Diabetes mellitus and hypertension, state of the art lecture. Hypertension l l [ S u p p l I]:I-14-I - 1 8 . Reeves, J.P. and Sutko, J.L. 1979- Sodium-calcium ion exchange i n cardiac membrane vesicles. Proc. Natl. Acad. S c i . , 76:590-594. Regan, T.J., Lyons, M.M. and Ahmed, S.S. 1977- Evidence for cardiomyopathy i n f a m i l i a l diabetes mel l i t u s . J . C l i n . Invest. 60:885-889. Regan, T.J., Wu, CF., Yeh, C.K., Oldewurtel, H.A. and Haider, B. I 9 8 I . Myocardial composition and function i n diabetes: the effects of chronic i n s u l i n use. C i r c . Res. 49:1268-1277-98 Ringer, S. 1 8 8 3 - A further contribution regarding the influence of the different constituents of the blood on the contraction of the heart. J. Physiol. (London) 4:29-42. Rodgers, R.L. 1 9 8 5 - Depresser effect of diabetes in the spontaneously hypertensive rat: associated changes i n heart performance. Can. J. Physiol. Pharmacol. 64 :1177-1184 . Rodrigues, B. and McNeill, J.H. 1 9 8 6 . Cardiac function i n spontaneously hypertensive diabetic rats. Am. J. Physiol. 251:H571-H580, 1 9 8 6 . Rodrigues, B.t Agrawal, D.K. and McNeill, J.H. 1 9 8 5 - Are elevated lipids and diabetic cardiomyopathy related. Fed. Proc.4 4 ( 5 ) : 1 6 5 7 (Abstract). Sandler, S., Welsh, M. and Andersson, A. 1 9 8 3 - Streptozotocin-induced impairment of i s l e t B-cell metabolism and i t s prevention by a hydroxyl radical scavenger and inhibitors of poly(ADP-ribose) synthetase. Acta. Pharmacol. Toxicol. 53^392-400. Sato, T., Nara, Y., Note, S. and Yamori, Y. 1 9 8 7 . New establishment of hypertensive diabetic animal models: neonatally streptozotocin-treated spontaneously hypertensive rats. Metabolism 3 6 ( 8 ) : 7 3 1 ~ 7 3 7 -Seager, M.J., Singal, P.K., Orchard, R., Pierce, G.N. and Dhalla, N.S. 1 9 8 4 . Cardiac c e l l damage: a primary myocardial disease in streptozotocin-induced chronic diabetes. Br. J. Exp. Pathol. 2 7 : 3 9 7 -4 0 1 . Schmitz, W., Scholz, H., Scholz, J. and Steinfath, M. 1 9 8 7 - Increase in IP^ precedes a-adrenoceptor-induced increase i n force of contraction in cardiac muscle. Euro. J. Pharmacol. l 4 0 : 1 0 9 - l l l . 9 9 Schramm, M. , Thomas, G. , Towart, R. and Franckowiak, G. 1983- Novel dihydropyridine with positive inotropic action through activation of Ca + + channels. Nature 303:535-537-Sedlis, S.P., Corr, P.B., Sobel, B.E. and Ahumada, G.G. 1983. Lysophosphatidylcholine potentiates C a + + accumulation i n rat cardiac myocytes. Am. J. Physiol. 244:H32-H38. Sekar, M.C. and Roufogalis, B.D. 1984. Comparison of muscarinic and alpha-adrenergic receptors i n rat a t r i a based on phospho-inositide turnover. L i f e S c i . 35:1527-1533. Senges, J . , Brachmann, J. , Pelzer, D., Hasslacher, C , Weike, E. and Kubler, W. 1980. Altered cardiac automaticity and conduction i n experimental diabetes mellitus. J. Mol. C e l l . Cardiol. 12:1341-1351-Shapiro, L.M., Howat, A.P. and Calter, M.M. I98I. Left ventricular function i n diabetes mellitus: I. Methodology and prevalence and spectrum of abnormalities. Br. Heart J. 45:122-128. Shapiro, L.M., Leatherdale, B.A., Coyne, M.E., Fletcher, R.F. and Makinnon, J. 1980. Prospective study of heart disease i n untreated maturity onset diabetes. Br. Heart J. 44:342-348. Shen, S.S., Virendra, K., Sharma, K. and Uglesity, A. 1986. Na +-Ca 2 + exchange contributes to increase i n cyt o s o l i c C a 2 + concentration during depolarisation i n heart muscle. Am. J. Physiol. 250:C651-C656. Simpson, W.W. and McNeill, J.H. 1980. Effect of adrenergic agonists on tension development and rate i n a t r i a from euthyroid and hypothyroid rats. Adv. Myocardiol. 1:417-435. 100 Simpson, W.W., Rodgers, R.L. and McNeill, J.H. I98I. Cardiac responsiveness to alpha- and beta-adrenergic amines: eff e c t of carbachol and hypothyroidism. J. Pharmacol. Exp. Ther. 219:231-234. Smith, J.M., Marcus, F.I. and Serokman, R. 1984. Prognosis of patients with diabetes mellitus after acute myocardial i n f a r c t i o n . Am. J. Cardiol. 54:718-721. Sperelakis, N. and Wahler, G. 1988. Relation of C a + + i n f l u x i n myocardial c e l l s by beta adrenergic receptors, c y c l i c nucleotides, and phosphorylation. Mol. and C e l l u l a r Biochem. 82:19-28. Suko, J. 1971. Alteration of C a + + uptake and C a + + activated ATPase of cardiac sarcoplasmic reticulum i n hyper- and hypothyroidism. Biochim. Biophys. Acta. 252:324-327. Sutko, J.L., I t o , K. and Kenyon, J.L. 1985. Ryanodine: a modifier of sarcoplasmic reticulum calcium release i n s t r i a t e d muscle. Fed. Proc. 44:2984-2988. T a h i l i a n i , A. and McNeill, J.H. 1984. Lack of e f f e c t of thyroid hormone on diabetic rat heart function and biochemistry. Can. J. Physiol. Pharmacol. 62:617-621. T a h i l i a n i , A.G. and McNeill, J.H. 1986. Diabetes-induced abnormalities i n the myocardium. L i f e S c i . 38:959-974. T a h i l i a n i , A.G., Vadlamudi, R.V.S.V. and McNeill, J.H. 1983. Prevention and reversal of altered myocardial function i n diabetic rats by in s u l i n treatment. Can. J. Physiol. Pharmacol. 6l:516-523. Thyrum, P.T., Kritcher, E.M. and Luchi, R.J. 1970. Effects of L-thyroxin on the primary structure of cardiac myosin. Biochim. Biophys. Acta. 197:335-336. 101 Tobian, L. I 9 8 I . Hypertension mechanisms i n experimental animals and the i r relevance to human. In: Frontiers i n Hypertension Research, (Eds.) Laragh, JH, Buhler, FR, Seldin, DW. Sprnger-Verlag, New York. Vadlamudi, R.V.S.V. and McNeill, J.H. 1 9 8 3 . Effects of experimental diabetse on rat cardiac c y c l i c AMP, phosphodiesterase, and inotropy. Am. J . Physiol. 244:H844-H851. Vadlamudi, R.V.S.V., Rogers, R.L. and McNeill, J.H. 1 9 8 2 . The effect of chronic administration of alloxan- and streptozotocin-induced diabetes on isolated rat heart performance. Can. J. Physiol. 60:902-9 1 1 . Vaghy, P.L., Groupp, I.L., Groupp, G. and Schwarts, A. 1 9 8 4 . Effects of BAY K 8644. A dihydropyridine analog on [3H] nitrendipine binding to canine cardiac sarcolemma and the relationship to a positive inotropic effect. C i r . Res. 5 5 : 5 4 9 - 5 5 3 . van Zwieten, P.A. and Timmermans, P.B.M.W.M. 1 9 8 7 . Alpha-adrenoceptor stimulation and calcium movements. Blood Vessels 24:270-280. Vanhoutte, P.M. and P a o l e t t i , R. 1 9 8 7 . The WHO c l a s s i f i c a t i o n of calcium antagonists. Trends Pharmacol. S c i . 8 : 4 - 5 . Wier, W.G., Yue, D.T. and Marban, E. 1 9 8 5 . Effects of ryanodine on in t r a c e l l u l a r C a + + transients i n mammalian cardiac muscle. Federation Proc. 4 4 : 2 9 8 9 - 2 9 9 3 . Williams, R.S., Schaible, T.F., Scheuer, J. and Kennedy, R. 1 9 8 3 . Effects of experimental diabetes on adrenergic and cholinergic preceptors of rat myocardium. Diabetes 3 2 : 8 8 1 - 8 8 6 . Wilson, G.L., Patton, N.J., McCord, J.M., Mullins, D.W. and Mossman, B.T. 1 9 8 4 . Mechanisms of streptozotocin- and alloxan-induced damage i n rat p" c e l l s . Diabetologia 2 7 : 5 8 7 ~ 5 9 1 -102 Woodworth, R.S. 1 9 0 2 . Maximal contraction, 'staircase' contraction, refractory period, and compensatory pause of the heart. Am. J. Physiol. 8 : 2 1 3 - 2 4 9 . Xiang, H. 1 9 9 0 . Alpha^-adrenoceptor-mediated phosphoinositide breakdown and inotropic responses i n right v e n t r i c l e s of streptozotocin-diabetic rats. Ph.D. Thesis, Faculty of Pharmaceutical Sciences, The University of B r i t i s h Columbia. Yamori, Y. 1 9 8 1 . Environmental influences on the development of hypertensive vascular diseases i n SHR and related models, and their r e l a t i o n to human disease. In: New Trends i n A r t e r i a l Hypertension, PP -305 (Eds.) Worcel, M, Bonvalet, JP, Canger, SZ. Elsevier/North-Holland Biomedical Press, Amsterdam. Yamori, Y. 1 9 8 4 . Development of the spontaneously hypertensive rat (SHR) and of various spontaneous rat models, and t h e i r implications. In: Handbook of Hypertension, Vol. 4(10):224-239. (Ed.) W. de Jong. Elsevier Science Publishers, B.V., Amsterdam. Yue, D. 1 9 8 7 . I n t r a c e l l u l a r [Ca + +] related to rate of force development i n twitch contraction of heart. Am. J. Physiol. 252:H760-H770. 

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