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Studies on the effect of experimental insulin-dependent diabetes mellitus and hypothyroidism on rat cardiac… Black, Shawn Clive 1990

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STUDIES ON THE EFFECT OF EXPERIMENTAL INSULIN-DEPENDENT DIABETES MELLITUS AND HYPOTHYROIDISM ON RAT CARDIAC AND SARCOPLASMIC RETICULUM FUNCTION by SHAWN CLIVE BLACK B.Sc.(Pharm.), The U n i v e r s i t y o f B r i t i s h Columbia, 1984 M . S c , The U n i v e r s i t y o f B r i t i s h Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES THE DIVISION OF PHARMACOLOGY AND TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES We accept t h i s t h e s i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA August 1990 © S h a w n C l i v e B lack , 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada DE-6 (2/88) i i A b s t r a c t . The o b j e c t i v e o f these s tud ie s was to i n v e s t i g a t e mechanisms whereby c a r d i a c sa rcop lasmic r e t i c u l u m (SR) ca l c i um t r an spo r t a c t i v i t y may be i n f l uenced by changes i n the l i p i d environment o f the SR membrane i n the exper imental endocr ine d i sease s t a te s hypothyro id i sm and in su l i n -dependent d i abetes m e l l i t u s . These endocr ine d i sease s t a t e s were s tud ied to determine, r e s p e c t i v e l y , i f SR f u n c t i o n i s i n f l uenced by endogenous a c y l c a r n i t i n e a s soc i a ted w i th the SR membrane and i f SR pho spho l i p i d acy l compos i t ion p lays a r o l e i n d iabetes - i nduced cardiomyopathy. The e f f e c t s of endogenous a c y l c a r n i t i n e s on SR ca l c i um t r an spo r t i n hypothyro id i sm were o f i n t e r e s t s i nce i t has p r e v i o u s l y been i m p l i c a t e d t ha t acy l c a r n i t i n e s p lay a r e gu l a t o r y r o l e i n SR f u n c t i o n . SR ca l c i um t r an spo r t was not a f f e c t e d at two weeks, but was s i g n i f i c a n t l y reduced at f o u r , s i x and e i gh t weeks f o l l o w i n g thyro idectomy. Endogenous acy l c a r n i t i n e s were de tec t ab l e i n the SR membrane f r a c t i o n i s o l a t e d from both eu thy ro i d c o n t r o l and thy ro idectomized an imals . The l e v e l o f acy l c a r n i t i n e a s soc i a ted w i th the SR d i d not c o r r e l a t e w i th ca l c i um t r an spo r t a c t i v i t y . S ince a c y l c a r n i t i n e d i d not appear to p lay a r o l e i n the reduced SR ca l c i um t r a n s p o r t , SR ca l c i um pump p r o t e i n was q u a n t i f i e d . The reduced SR ca l c i um t r an spo r t o f thy ro idectomized an imals , man i fes t at f ou r weeks, was shown to c o r r e l a t e w i th a reduc t i on i n SR acy lphosphoprote in l e v e l . Therefore the reduced SR ca l c ium t r an spo r t a c t i v i t y o f hypothyro id i sm i s not r e l a t e d to the l e v e l of SR acy l c a r n i t i n e , but r a t h e r a hypo thy ro id -induced r educ t i on i n SR ca l c i um pump s i t e s . S ince omega-3 f a t t y ac ids a f f e c t parameters r e l e van t to d i a b e t e s -induced cardiomyopathy, i t was o f i n t e r e s t to determine the c a r d i a c e f f e c t s of omega-3 f a t t y a c i d treatment o f s t r e p t o z o c i n (STZ)- induced d i a b e t i c an imal s . Omega-3 f a t t y ac id treatment s i g n i f i c a n t l y reduced the development o f d i a b e t i c cardiomyopathy and improved i s o l a t e d c a r d i a c SR ca l c i um t r an spo r t a c t i v i t y o f STZ-induced d i a b e t i c an imal s . To determine i f the c a r d i a c and SR changes were r e l a t e d to membrane changes induced by omega-3 f a t t y a c i d s , the f a t t y acy l compos i t ion o f pho spho l i p i d was determined. Phospho l ip id ana l y s i s of c a r d i a c pho spha t i d y l c ho l i n e and phosphat idy lethano lamine, and t o t a l SR pho spho l i p i d i n d i c a t e d modest changes i n the omega-3 f a t t y ac id component. Omega-3 f a t t y a c i d treatment produced s l i g h t ( s t a t i s t i c a l l y i n s i g n i f i c a n t ) changes i n SR c h o l e s t e r o l l e v e l s . Therefore a change i n membrane phospho l i p id acy l compos i t ion may not account f o r the observed ca rd i a c and SR f u n c t i o n a l changes. John H. M c N e i l l , Ph.D. Sidney Ka tz , Ph.D. Dean and P ro fe s so r P ro fe s so r F acu l t y o f Pharmaceutical Sciences Facu l t y of Pharmaceut ical Sc iences i v Table o f Contents Ab s t r a c t i i Table o f Contents i v L i s t o f Tables v i i i L i s t o f F igures i x Acknowledgments x i i Ded i ca t i on x i i i 1. I n t r oduc t i on 1 1.1 Diabetes M e l l i t u s 3 1.1a E t i o l o g y and C l i n i c a l D e s c r i p t i o n o f Diabetes M e l l i t u s 3 1.1b Ca rd i ova scu l a r Disease i n Diabetes M e l l i t u s 4 1.1c D i a b e t i c Cardiomyopathy 9 S t r u c t u r e and Funct ion of Myosin i n Diabetes M e l l i t u s 11 Funct ion of Sarcolemma i n Diabetes M e l l i t u s 13 Funct ion o f Sarcoplasmic Ret icu lum in Diabetes M e l l i t u s . . . 14 1.2 Hypothyroid ism 19 1.2a E t i o l o g y and C l i n i c a l D e s c r i p t i o n o f Hypothyroid ism 19 1.2b Card iac Disease i n Hypothyroidism 19 Thyro id Hormone and Card iac Myosin 21 Adrenerg ic Receptors i n Hypothyroidism 22 Funct ion of Sarcoplasmic Ret icu lum i n Hypothyroid ism 23 V 1.3 The Role o f Myocardia l Membranes i n Card iac Funct ion 26 1.3i Omega-3 Fa t t y Ac i d Nomenclature 29 1.3a Omega-3 Fa t t y Ac id s and Myocard ia l Membrane Funct ion 30 1.3b A c y l c a r n i t i n e and Myocard ia l Membrane Funct ion 33 1.4 Membrane A l t e r a t i o n s i n Diabetes M e l l i t u s 37 1.5 Membrane A l t e r a t i o n s i n Hypothyroidism 39 1.6 Purpose o f the Study and Approach to the Problem 41 1.6a Ob jec t i ve s o f S tud ies on the E f f e c t s of Thyroidectomy on Card iac Sarcoplasmic Ret iculum Calcium Transport A c t i v i t y and the Role o f Endogenous A c y l c a r n i t i n e 43 1.6b Ob jec t i ve s o f S tud ies on the E f f e c t s of Omega-3 Fa t t y a c i d Treatment o f S t reptozoc in - Induced D i a b e t i c Cardiomyopathy . 44 2. Methods 45 2.1 S tud ies on the Thyroidectomized Wis ta r Rat 45 2.1a Animal Model and Study P rotoco l 45 2.1b I s o l a t i o n o f Card iac Sarcoplasmic Ret icu lum 46 2.1c Determinat ion o f Card iac SR Calc ium Transport A c t i v i t y 47 2.Id Determinat ion o f Card iac SR Acylphosphate Leve l s 48 2.1e Hydroxylamine Treatment o f SR Acy lphosphoprote in 49 2 . I f Sarcoplasmic Ret icu lum C a r n i t i n e E x t r a c t i o n Procedure 49 2.1g Determinat ion of Card iac SR C a r n i t i n e 50 2.1h Determinat ion o f Myocardia l Homogenate and SR P r o t e i n Concent ra t ion 50 2.1 i Determinat ion o f Myocard ia l Homogenate and SR Cytochrome C Oxidase A c t i v i t y 51 2.1j Plasma T3 Ana l y s i s 51 2.1k S t a t i s t i c a l Methods 51 v i 2.2 S tud ies on the E f f e c t s of Omega-3 Fa t ty A c i d Treatment on the S t reptozoc in - Induced D i a b e t i c Wi s ta r Rat 52 2.2a Animal Model and Study Protoco l 52 2.2b Determinat ion o f I s o l a ted Working Rat Heart Funct ion 53 2.2c Card iac L i p i d E x t r a c t i o n Procedure 55 2.2d Thin Layer Chromatographic Separat ion o f Myocard ia l Phospho l ip id s 55 2.2e SR Phospho l i p id E x t r a c t i o n Procedure 56 2.2f D e r i v a t i z a t i o n and Gas Chromatographic A n a l y s i s o f Card iac and SR Phospho l ip id Ex t r ac t s 57 2.3 Determinat ion o f Plasma Glucose, T r i g l y c e r i d e , Cho l e s t e ro l and I n s u l i n 58 2.4 E l e c t r o p h o r e t i c A n a l y s i s of SR P r o t e i n 58 2.5 S t a i n i n g and Des ta in ing Procedures 59 3. Resu l t s 60 3.1 S tud ies on the e f f e c t s o f thyro idectomy on c a r d i a c sa rcop lasmic r e t i c u l u m 60 3.1a General phy s i ca l c h a r a c t e r i s t i c s of eu thy ro id and thy ro idec tomized r a t s 60 3.1b C h a r a c t e r i z a t i o n o f the SR P repa ra t i on 67 3.1c The E f f e c t o f Thyroidectomy on SR Calc ium t r an spo r t 75 3.Id The E f f e c t o f Thyroidectomy on SR A c y l c a r n i t i n e Level 84 3.1e The E f f e c t o f Thyroidectomy on SR Phosphoprotein Level 93 3.2 S tud ies on the e f f e c t s of omega-3 f a t t y a c i d treatment o f STZ-induced d i a b e t i c r a t s 103 3.2a General c h a r a c t e r i s t i c s of the omega-3 f a t t y a c i d t r e a t e d and untreated non -d i abe t i c con t r o l and s t r ep t o zoc i n - i nduced d i a b e t i c r a t s 103 3.2b I s o l a ted working heart f u n c t i o n of omega-3 f a t t y a c i d t r e a t e d and untreated non -d i abe t i c c on t r o l and s t r ep to zoc i n - i nduced d i a b e t i c r a t s 113 v i i 3.2c SR ca l c i um t r an spo r t of omega-3 f a t t y a c i d - t r e a t e d and unt reated non -d i abe t i c c on t r o l and STZ-induced d i a b e t i c animals 125 3.2d Card iac and sarcop lasmic r e t i c u l u m pho spho l i p i d acy l compos i t ion o f non -d i abe t i c and d i a b e t i c animals t r e a t e d w i th omega-3 f a t t y ac ids 127 4. D i s cu s s i on 145 4.1 The E f f e c t o f Hypothyroidism on SR Ca lc ium Transport and the Role of Endogenous Long Chain A c y l c a r n i t i n e 147 4.2 The E f f e c t s of Omega-3 Fa t ty a c i d Treatment on Card iac and SR Funct ion i n the S t reptozoc in - Induced D i a b e t i c Rat . 155 5. Conc lus ions 169 5.1 The e f f e c t o f hypothyro id i sm on SR ca l c i um t r an spo r t and the r o l e of endogenous long cha in a c y l c a r n i t i n e 169 5.2 The e f f e c t s of omega-3 f a t t y a c i d treatment on c a r d i a c and SR f u n c t i o n i n the s t r ep to zoc i n - i nduced d i a b e t i c r a t . 171 6. O r i g i n a l Con t r i b u t i o n s 173 7. B i b l i o g r aphy 174 v i i i L i s t o f Tables Table 1. C h a r a c t e r i z a t i o n of the c a rd i a c sarcop lasmic r e t i c u l u m p r e p a r a t i o n . Determinat ion o f the percentage decrease i n cytochrome c ox idase a c t i v i t y o f the SR p repa ra t i on vs the v e n t r i c u l a r homogenate 72 Table 2. C h a r a c t e r i z a t i o n o f the c a r d i a c sarcop lasmic r e t i c u l u m p r e p a r a t i o n . Determinat ion of the r e l a t i v e i nc rea se i n s p e c i f i c ca l c ium t r an spo r t a c t i v i t y o f the SR p repa ra t i on vs the v e n t r i c u l a r homogenate 74 Table 3. The e f f e c t of d iabetes and omega-3 f a t t y a c i d t reatment on i s o l a t e d c a r d i a c sarcop lasmic r e t i c u l u m ca l c i um t r a n s p o r t a c t i v i t y 126 Table 4. E f f e c t of omega-3 f a t t y a c i d treatment on the r e l a t i v e and abso lute changes i n the amount o f omega-6 and omega-3 f a t t y a c i d i n v e n t r i c u l a r pho spha t i dy l cho l i ne and phosphat idy lethanolamine and t o t a l SR pho spho l i p i d 139 ix L i s t o f F igures F igure 1. Reac t i on/ t ran spo r t c y c l e o f the sarcop lasmic r e t i c u l u m C a 2 + -ATPase ca l c i um pump 15 F igure 2. The e f f e c t o f thyro idectomy on plasma T3 concen t r a t i on over the t ime course o f the study ( 2 - 8 weeks) 61 F igure 3. Body weight o f the eu thy ro id c on t r o l and thy ro idec tomized animals over the t ime course of the study ( 2 - 8 weeks) 63 F igure 4. The e f f e c t o f thyro idectomy on wet v e n t r i c u l a r weight o f eu thy ro i d and thy ro idectomized animals over the t ime course o f the study ( 2 - 8 weeks) 65 F igure 5. The e f f e c t of thyro idectomy on the y i e l d o f v e n t r i c u l a r sa rcop lasmic r e t i c u l u m 68 F igure 6. The e f f e c t o f thyroidectomy on the r a t i o o f SR i s o l a t e d to wet v e n t r i c u l a r weight 70 F igure 7. SR ca l c i um t r an spo r t a c t i v i t y determined two weeks f o l l o w i n g removal o f the t h y r o i d g land. Inset: Ead ie -Hofs tee p l o t o f the SR ca l c i um t r an spo r t data o f two weeks pos t -thyro idectomy 76 F igure 8. SR ca l c i um t r an spo r t a c t i v i t y determined fou r weeks f o l l o w i n g removal of the t h y r o i d g land. Inset: Ead ie -Hofs tee p l o t o f the SR ca l c ium t r an spo r t data fou r weeks post - thyro idectomy 78 F igure 9. SR ca l c i um t r an spo r t a c t i v i t y determined s i x weeks f o l l o w i n g removal of the t h y r o i d g l and . Inset: Ead ie -Hofs tee p l o t o f the SR ca l c ium t r an spo r t data s i x weeks pos t - thyro idectomy 80 F igure 10. SR ca l c i um t r an spo r t a c t i v i t y determined e i gh t weeks f o l l o w i n g removal o f the t h y r o i d g land. Inset: Ead ie -Hofs tee p l o t o f the SR ca l c i um t r an spo r t data e i gh t weeks pos t - thyro idectomy 82 F igure 11. The e f f e c t o f t ime (week of study) on the r a t e o f SR ca l c i um t r an spo r t determined at 2.0 /zM [ C a ^ + ] p r e e (a) and at 5.3 /xM [ C a Z + ] F r e e (b) 85 F igure 12. The e f f e c t o f thyro idectomy on the concen t r a t i on o f f r e e c a r n i t i n e present i n the SR f r a c t i o n 87 F igure 13. The e f f e c t of thyroidectomy on the concen t ra t i on o f shor t cha in a c y l c a r n i t i n e present i n the SR f r a c t i o n 89 X F igure 14. The e f f e c t o f thyro idectomy on the concen t r a t i on o f long cha in a c y l c a r n i t i n e present i n the SR f r a c t i o n 91 F igure 15. Calc ium [[Ca^+]fr^ee) dependence o f sarcop lasmic r e t i c u l u m phosphory la t ion by [ 3 Z P ] -ATP 94 F igure 16. The e f f e c t o f thyro idectomy on the calc ium-dependent phosphory la t ion o f SR determined at 10 mM [ C a z + ] p r e e i n SR from euthy ro id c on t r o l and SR i s o l a t e d two weeks f o l l o w i n g thyro idectomy 97 F igure 17. The e f f e c t o f thyro idectomy on the calc ium-dependent phosphory la t ion o f SR determined at 10 mM [ C a z + ] p r e e i n SR from eu thy ro id c on t r o l and SR i s o l a t e d fou r weeks f o l l o w i n g thyro idectomy 99 F igure 18. K inase mediated phosphory la t ion o f phospholamban i n eu thy ro i d c on t r o l and hypothyro id animals at 2 and 4 weeks po s t -thyro idectomy 101 F igure 19. Plasma i n s u l i n and g lucose concent ra t i on s o f c o n t r o l , c on t r o l t r e a t e d , d i a b e t i c and d i a b e t i c t r e a t e d animals .. 104 F igure 20. Average d a i l y body weight ( i n grams) o f c o n t r o l , c o n t r o l - t r e a t e d , d i a b e t i c and d i a b e t i c - t r e a t e d male Wi s ta r r a t s 106 F igure 21. Plasma t r i g l y c e r i d e concent ra t i on o f c o n t r o l , c o n t r o l - t r e a t e d , d i a b e t i c and d i a b e t i c - t r e a t e d male Wi s ta r r a t s 109 F igure 22. Plasma c h o l e s t e r o l concen t ra t i on of c o n t r o l , c o n t r o l - t r e a t e d , d i a b e t i c and d i a b e t i c - t r e a t e d male Wi s ta r r a t s I l l F i gure 23. Peak l e f t v e n t r i c u l a r developed pressure vs l e f t a t r i a l f i l l i n g pressure of c o n t r o l , c o n t r o l - t r e a t e d , d i a b e t i c and d i a b e t i c - t r e a t e d male Wi s ta r r a t s 114 F igure 24. Maximum r a t e of change o f l e f t v e n t r i c u l a r pressure du r ing s y s t o l e ( + d P / d t m a x ) vs l e f t a t r i a l f i l l i n g pressure o f c o n t r o l , c o n t r o l - t r e a t e d , d i a b e t i c and d i a b e t i c - t r e a t e d male Wi s ta r r a t s 116 F igure 25. Maximum r a t e o f change o f l e f t v e n t r i c u l a r pressure du r ing d i a s t o l e ( ~ d P / d t m a x ) o f c o n t r o l , c o n t r o l - t r e a t e d , d i a b e t i c and d i a b e t i c - t r e a t e d male Wi s ta r r a t s 118 F igure 26. The e f f e c t of s t r ep tozoc i n - i nduced d iabetes and omega-3 f a t t y a c i d treatment on a o r t i c output o f i s o l a t e d working hearts 121 x i F igure 27. The e f f e c t of s t r ep to zoc i n - i nduced d iabetes and omega-3 f a t t y a c i d treatment on pulmonary f l ow o f i s o l a t e d working hearts 123 F igure 28. Plasma g lucose concen t ra t i on of non -d i abe t i c and d i a b e t i c t r e a t e d and untreated animals of f o l l ow -up study 128 F igure 29. Average d a i l y body weight of c o n t r o l , c o n t r o l t r e a t e d , d i a b e t i c and d i a b e t i c t r e a t ed male Wi s ta r r a t s 130 F igure 30. The e f f e c t o f omega-3 f a t t y a c i d treatment on plasma t r i g l y c e r i d e (A) and c h o l e s t e r o l (B) concen t ra t i on s 132 F igure 31. V e n t r i c u l a r pho spha t i dy l cho l i ne acy l compos i t ion o f c o n t r o l , c on t r o l t r e a t e d , d i a b e t i c and d i a b e t i c t r e a t e d male Wi s ta r r a t s 135 F igure 32. V e n t r i c u l a r phosphat idy lethanolamine acy l compos i t ion o f c o n t r o l , c on t r o l t r e a t e d , d i a b e t i c and d i a b e t i c t r e a t e d male Wi s ta r r a t s 137 F igure 33. Sarcoplasmic r e t i c u l u m phospho l i p id acy l compos i t ion o f c o n t r o l , c on t r o l t r e a t e d , d i a b e t i c and d i a b e t i c t r e a t e d male Wi s ta r r a t s 140 F igure 34. Card iac SR membrane c h o l e s t e r o l concen t ra t i on o f c o n t r o l , c o n t r o l t r e a t e d , d i a b e t i c and d i a b e t i c t r e a t e d male Wi s ta r r a t s 143 x i i Acknowledgements. I would l i k e to express my s i nce re g r a t i t u d e to my c o - s upe r v i s o r s , Dean John H. McNe i l l and P ro fe s so r Sidney Ka tz , f o r t h e i r encouragement, support, knowledgeable guidance and t r u s t . The c o - s u p e r v i s i o n o f Drs. McNe i l l and Katz was s y n e r g i s t i c and prov ided an u n p a r a l l e l e d environment f o r s tudy. The d i r e c t i o n and c o n s t r u c t i v e c r i t i c i s m prov ided by the members o f my research committee, Dr. R.W. Brownsey, Dr. P. Hahn, Dr. K.M. MacLeod and Dr. M. Levine (Chairman) i s g r a t e f u l l y acknowledged. I would l i k e to thank a l l F acu l t y members o f the D i v i s i o n o f Pharmacology and Tox ico logy f o r t h e i r e x c e l l e n t i n s t r u c t i o n i n pharmacology at both the undergraduate and graduate l e v e l s . Such i n s t r u c t i o n was i n t e g r a l to my d e c i s i o n to pursue and cont inue graduate educat ion i n pharmacology. I would a l so l i k e to thank these Facu l t y members f o r p r o v i d i n g me w i th s i g n i f i c a n t and i n va l uab l e l e c t u r i n g exper ience wh i l e a graduate student i n the D i v i s i o n o f Pharmacology and Tox i co l ogy . I would l i k e to s i n c e r e l y acknowledge the moral support and a s s i s t ance o f the graduate students w i th whom I had d a i l y i n t e r a c t i o n va ry ing from commiseration to data i n t e r p r e t a t i o n to e l a t i o n as we progressed through graduate school together . P a r t i c u l a r thanks to Bruce A l l e n f o r h i s humor and f r i e n d s h i p . I would l i k e to express my g r a t i t u d e to the B r i t i s h Columbia Heart Foundation and the Canadian Heart and Stroke Foundation f o r p r o v i d i n g f i n a n c i a l support dur ing my graduate educat ion . The support , pat ience and understanding o f L e s l i e B lack encouraged the succes s fu l complet ion of t h i s t h e s i s . x i i i Ded i ca t i on To L e s l i e E l i z a b e t h B lack , w i th l o v e , f o r every th ing that you have brought to my l i f e . 1 1. Introduction That endocr ine d i sease can a f f e c t the heart and c a r d i o v a s c u l a r system has been recogn ized f o r a con s ide rab le pe r iod o f t ime . In 1825 Parry de sc r i bed the behav ior o f the heart i n hyper thyro id i sm (Pa r r y , 1825), and Zondek i n 1918 desc r ibed ca rd i a c a l t e r a t i o n s observed i n hypothyro id i sm (Zondek, 1918). As w e l l , E i c ho r s t i n 1892 observed a p e r s i s t e n t t a c h y c a r d i a i n d i a b e t i c p a t i e n t s , i n d i c a t i v e o f a l t e r e d c a r d i o v a s c u l a r f u n c t i o n i n d iabetes ( E i c h o r s t , 1892). S ince these ob se r va t i on s , which e s s e n t i a l l y remain v a l i d today, i t has become i n c r e a s i n g l y c l e a r t ha t the heart i s a f f e c t e d by hormones under normal p h y s i o l o g i c a l c o n d i t i o n s as we l l as under c e r t a i n endocr ine d i sease s t a t e s . Disease s t a t e s a s soc i a ted w i th a d i s tu rbance i n the normal p h y s i o l o g i c a l c on t r o l o f e i t h e r i n s u l i n or t h y r o i d hormone, the hormones of s p e c i f i c i n t e r e s t to t h i s p r o j e c t , a f f e c t va r i ou s organ systems i n the body (U t i g e r , 1987; S h a f r i r et a l , 1987). Indeed, the pr imary r o l e s o f i n s u l i n and t h y r o i d hormone r e l a t e to whole body metabo l i c r e g u l a t i o n r a t he r than r e g u l a t i o n o f myocardia l metabolism or f u n c t i o n a lone. However, endocr inopath ies which d i s t u r b e i t h e r i n s u l i n or t h y r o i d hormone homeostasis have an impact upon the p h y s i o l o g i c a l f u n c t i o n of the hea r t . Diabetes m e l l i t u s i s a d e s c r i p t i v e term, l i t e r a l l y , of a s i gn i n d i c a t i v e o f severa l forms of s i m i l a r d i seases having i n common a l t e r e d i n s u l i n s yn the s i s , s e c r e t i o n or a c t i o n . The form o f d iabetes m e l l i t u s r e l e van t to t h i s t h e s i s i s i n su l in -dependent d i abetes m e l l i t u s (IDDM, or Type I d i a b e t e s ) . Unless otherwise s t a t e d , t h i s i s the form of d iabetes under d i s c u s s i o n . Hypothyroidism i s s i m i l a r l y a d e s c r i p t i v e term of the d i r e c t i o n of change i n c i r c u l a t i n g t h y r o i d hormone l e v e l s o c cu r r i n g 2 as a r e s u l t o f one of severa l d i seases a f f e c t i n g t h y r o i d hormone s yn thes i s and/or s e c r e t i o n . In d i abetes m e l l i t u s and hypothyro id i sm there are p a t h o l o g i c a l a l t e r a t i o n s o f the c a r d i o v a s c u l a r system in general and, more s p e c i f i c a l l y , d i r e c t pa thophy s i o l o g i c a l changes i n the hea r t . A b r i e f c l i n i c a l d e s c r i p t i o n o f the c a rd i a c man i f e s t a t i on s o f i n s u l i n and t h y r o i d hormone d e f i c i e n c y w i l l t h e r e f o r e be presented. S ince the heart i s p ro found ly a f f e c t e d by a d e f i c i e n c y of e i t h e r o f these hormones, i t i s o f i n t e r e s t to determine the f u n c t i o n a l , s u b c e l l u l a r and b iochemical bases f o r such a l t e r a t i o n s . Fo l l ow ing the c l i n i c a l d e s c r i p t i o n s o f c a r d i a c d i sease i n d iabetes m e l l i t u s and hypothyro id i sm, the d i r e c t e f f e c t s o f these d i sease s t a t e s upon the heart w i l l be reviewed i n the context o f the mechanisms i nvo l ved and w i t h a pe r spec t i ve towards d e f i n i n g the o v e r a l l research o b j e c t i v e o f t h i s p r o j e c t , i . e . to i n v e s t i g a t e c a r d i a c and sarcop lasmic r e t i c u l u m f u n c t i o n i n exper imental d iabetes m e l l i t u s and hypothyro id i sm. The p r o j e c t i s s p e c i f i c a l l y concerned w i th i n the e f f e c t s o f l i p i d s and l i p i d metabo l i c i n te rmed ia tes on i s o l a t e d c a rd i a c f u n c t i o n and sarcop lasmic r e t i c u l u m ca l c i um t r an spo r t a c t i v i t y . There fo re , subsequent to the d i s c u s s i o n o f the mechanisms i nvo l ved i n the c a r d i a c a l t e r a t i o n s o c cu r r i n g i n d i abetes m e l l i t u s and hypothyro id i sm, d i sease s t a te - i nduced a l t e r a t i o n s i n l i p i d metabolism w i l l be presented to prov ide the r a t i o n a l e f o r the approach taken i n t h i s p r o j e c t . 3 1.1 Diabetes Mellitus 1.1a Etiology and Cl in ica l Description of Diabetes Mell itus. IDDM represents approx imately 10% o f a l l cases o f d i abetes m e l l i t u s (Co lwe l l et a l , 1990). The p r e c i s e e t i o l o g y of IDDM i s not known although con s i de rab le i n s i g h t i n t o the mechanisms i nvo l ved i n i t s pathogenesis have been d e f i n e d . IDDM i s c u r r e n t l y cons idered an autoimmune d i sease ( E i s enba r th , 1986). A genet i c p r e d i s p o s i t i o n i s cons idered e s s e n t i a l , but alone i n s u f f i c i e n t f o r the d i sease to mani fest i t s e l f . Genes w i t h i n the major h i s t o c o m p a t i b i l i t y complex c o n t r i b u t e to t h i s gene t i c p r e d i s p o s i t i o n as 95% o f wh i te IDDM p a t i e n t s express the h i s t o c o m p a t i b i l i t y genes DR3 or DR4, or both (Rimoin and Ro t t e r , 1984). S ince concordance o f IDDM i s approx imately 60% amongst i d e n t i c a l tw in s , i t i s cons idered t ha t an a d d i t i o n a l , p o s s i b l y environmental, f a c t o r ( s ) t r i g g e r s i n s u l i t i s w i t h i n the /} c e l l s o f the pancreas. The r e s u l t i n g autoimmune process u l t i m a t e l y leads to a complete l o s s o f f u n c t i o n i n g /J c e l l s w i t h i n the pancreas. The c l i n i c a l man i fe s ta t i on s o f IDDM occur a f t e r s u b s t a n t i a l panc rea t i c ft c e l l l o s s and when i n s u l i n s e c r e t i o n i s inadequate to meet metabo l i c demand (Kro lewsk i et a l , 1985). The c l i n i c a l p r e sen ta t i on at the onset o f ove r t d iabetes can be a s soc ia ted w i th hyperg lycemia, g l y c o s u r i a , k e t oac i do s i s and/or k e t onu r i a . These s igns o f IDDM occur as a r e s u l t o f i n s u l i n d e f i c i e n c y . C h r o n i c a l l y , IDDM i s a d i s o r d e r o f carbohydrate, l i p i d and p r o t e i n metabolism r e s u l t i n g from a d e f i c i e n c y o f endogenous i n s u l i n . 4 1.1b Cardiovascular Disease in Diabetes Mell itus. Desp i te treatment o f d iabetes m e l l i t u s w i th exogenous i n s u l i n , the inc idence and sequelae o f c a r d i o va s cu l a r d i sease remain high i n the d i a b e t i c p o p u l a t i o n . Rubier e t a l (1972), based on observat ions o f pos t -mortem f i n d i n g s o f d i a b e t i c s who d ied o f congest i ve heart f a i l u r e i n the absence o f a t h e r o s c l e r o t i c , v a l v u l a r , c o n g e n i t a l , hyper tens i ve or a l c o h o l i c d i s ea se , were p r e s c i e n t i n t h e i r c on s i de r a t i on t ha t d i abetes may independent ly c o n t r i b u t e to cardiomyopathy. Data from the p ro spec t i ve Framingham study prov ided ep idemio l og i ca l evidence tha t d i abetes was an independent r i s k f a c t o r f o r c a r d i o va s cu l a r d i sease (Kannel e t a l , 1974). Several r i s k f a c t o r s which c o n t r i b u t e to the increased i nc idence o f c a r d i a c d i sease i n IDDM, and c e r t a i n p a t ho l o g i c a l processes which c o n t r i b u t e to the c a r d i a c man i f e s t a t i on s of d iabetes have been i d e n t i f i e d . Macrovascular and mic rova scu la r d i sease o f the heart are two p a t h o l o g i c a l processes which c o n t r i b u t e to c a r d i a c d i sease i n the d i a b e t i c popu l a t i on . Macrovascular d i sease i s p r i m a r i l y mani fes t as coronary a t h e r o s c l e r o t i c d i sease and d i a b e t i c s have a two- to t h r e e - f o l d increased r e l a t i v e r i s k ( r e l a t i v e to age-matched non -d i abe t i c s ) o f deve lop ing coronary a t h e r o s c l e r o t i c d i sease (Kannel and McGee, 1979). R i sk f a c t o r s f o r the development o f coronary a t h e r o s c e r o t i c d i sease i n d i a b e t i c s i n c l ude e l eva ted blood l i p i d s such as hype rcho le s te ro lemia and/or h y p e r t r i g l y c e r i d e m i a (Lau, 1989). H y p e r t r i g l y c e r i d e m i a i s the most f r e q u e n t l y observed l i p i d abnormal i ty i n d i a b e t i c s (Ba r re t t -Conner et a l , 1982) and has r e c e n t l y been shown to be an independent r i s k f a c t o r f o r m o r t a l i t y from coronary a r t e r y d i sease i n d i a b e t i c s (Fontbonne e t a l , 1989). The p a t h o l o g i c a l sequelae o f the increased macrovascular d i sease i s 5 t ha t t h i s remains a major cause o f the increased c a r d i a c morb i d i t y and m o r t a l i t y i n d i a b e t i c s (Garc ia and McNamara, 1974). M i c r ova s cu l a r d i sease o f the heart has been shown to be mani fes t as inc reased basement membrane t h i c k e n i n g ( F i s he r e t a l , 1979) and/or microaneurysms (Factor et a l , 1980). M i c rova s cu l a r p e r m e a b i l i t y i s a l so e l eva ted i n d i a b e t i c s (determined in vivo), as shown by the increased t r a n s c a p i l l a r y escape r a t e of albumin (Jensen et a l , 1989). These m i c rova scu l a r a l t e r a t i o n s may have i m p l i c a t i o n s i n the development o f cardiomyopathy i n d iabetes by p o t e n t i a l l y a f f e c t i n g m i c rova scu l a r blood f l ow, the p r o b a b i l i t y o f thromboembolism and/or t i s s u e oxygenat ion. An a d d i t i o n a l r i s k f a c t o r f o r the development of c a r d i a c d i sease amongst d i a b e t i c s i s the repor ted two - f o l d inc rease i n the i nc i dence of hyper tens ion , which i f untreated i s a pr imary cause o f v e n t r i c u l a r hypertrophy and, i f o f s u f f i c i e n t d u r a t i o n , subsequent conges t i ve heart f a i l u r e . Card iac autonomic neuropathy, o ccu r r i n g to some degree i n 17-40% o f d i a b e t i c s (Ewing, 1984), i s a l so cons idered to c o n t r i b u t e to c a r d i a c d i sease i n d i a b e t i c s (Ewing et a l , 1974; Kahn et a l , 1986). Man i f e s t a t i on s o f the c a r d i a c autonomic neuropathy v a r i e d . Impaired beat to beat v a r i a t i o n i n the heart r a t e or abnormal heart r a t e responses may occur (Bennett e t a l , 1975; Ewing et a l , 1978). D i a b e t i c s w i t h autonomic neuropathy may l a c k the c h a r a c t e r i s t i c t a chyca rd i a and r e l a t i v e b radycard ia observed upon s h i f t i n g from a prone to s tanding p o s i t i o n (Ewing et a l , 1978). The preva lence of abnormal blood pressure responses, i . e . d i a b e t i c s e x h i b i t i n g po s tu ra l hypotension upon s tand ing , i s es t imated at 40% (Canal et a l , 1978). Card iac autonomic neuropathy i s a l s o man i fes t i n 6 exper imenta l d i abe te s . Changes i n the f u n c t i o n a l s t a tu s o f myocardia l a-ad rene rg i c , ^ - ad rene rg i c and c h o l i n e r g i c r ecep to r systems have been demonstrated i n exper imental d i abe te s . The ^ - ad rene rg i c recepto r system changes were i n i t i a l l y shown by Foy and Lucas (1976) u t i l i z i n g p i thed a l l o x a n - or s t r e p t o z o c i n (STZ)- induced d i a b e t i c r a t s and i n d i c a t e d a reduced s e n s i t i v i t y t o the pres so r e f f e c t s o f noradrena l i ne and to the p o s i t i v e i n o t r o p i c and ch rono t rop i c e f f e c t s o f i s o p r o t e r e n o l . Receptor b ind ing s tud ie s o f Savarese and Berkowitz (1979) us ing STZ-induced d i a b e t i c r a t s , subsequently demonstrated a s i g n i f i c a n t 28% r educ t i on i n the number of v e n t r i c u l a r ^ - r e c e p t o r s i t e s w i t h no change i n antagon i s t a f f i n i t y . The reduc t i on i n /J-receptor number c o r r e l a t e d we l l w i t h a 24% decrease i n in vivo heart r a t e (Savarese and Berkowi t z , 1979). Reduced c a r d i a c ^ - ad rene rg i c recepto r number has been conf i rmed i n the STZ-induced d i a b e t i c r a t , (Hey l i ge r et a l , 1982; W i l l i ams e t a l , 1983; A t k i n s et a l , 1985; Ramanadham and Tenner, 1986) and the spontaneously d i a b e t i c B io -B reed ing r a t (Durante et a l , 1989), although the e f f e c t has not always been demonstrable (Gotzsche, 1983). The apparent a f f i n i t y o f the 0-r e cep to r , KQ , has un i fo rmly been shown to be una f fec ted by the d i a b e t i c s t a t e . An except ion has been repor ted where the d i a b e t i c s t a t e was o f very short du r a t i on (72 hours post-STZ treatment) (Wald et a l , 1989). Reduced v e n t r i c u l a r s e n s i t i v i t y t o the i n o t r o p i c e f f e c t o f i s op ro te reno l has been shown in vitro, us ing i s o l a t e d v e n t r i c u l a r t i s s u e (Hey l i g e r et a l , 1982; Ramanadham and Tenner, 1986), and in vivo, as assessed by a reduced response o f the i s op ro te reno l - i nduced inc rease i n the maximum r a t e o f r i s e o f l e f t v e n t r i c u l a r pressure i n anes thet i zed r a t s (A tk in s et a l , 1985). A reduced i n o t r o p i c respons iveness to noradrena l ine (Jackson and C a r r i e r , 7 1983) and a reduced respons iveness to the p o s i t i v e l u s i t r o p i c e f f e c t of i s op ro te reno l has been shown i n STZ-induced d i a b e t i c r a t s (Vadlamudi and M c N e i l l , 1984). S ince membrane recepto r s c o n s t i t u t e on ly the f i r s t step i n the /J-receptor-mediated s i gna l t r an sduc t i on pathway, the i n t e g r i t y o f the system d i s t a l to the recep to r may be a f f e c t e d by d iabetes and i n f l u e n c e i n o t r o p i c and ch rono t rop i c f u n c t i o n . Basal c a r d i a c adenylate c y c l a se a c t i v i t y and a c t i v i t y determined i n the presence o f NaF, f o r s k o l i n , GTP or the non-hydro lyzab le GTP analog, Gpp(NH)p i s not i n f l uenced by d iabetes (A tk in s et a l , 1985; Wald e t a l , 1989; N i s h i o et a l , 1988; Vadlamudi and M c N e i l l , 1983); however, i s o p r o t e r e n o l - s t i m u l a t e d adenylate c y c l a s e a c t i v i t y has been shown to be attenuated i n hearts o f d i a b e t i c animals (Gotzsche, 1983; A t k i n s et a l , 1985). The l a t t e r s t ud i e s i n d i c a t e tha t there may be a f u n c t i o n a l uncoupl ing of the /3-adrenergic r e cep to r from adenylate c y c l a s e . Th is idea i s supported by the r e s u l t s o f Cros e t a l (1986), who found an a l t e r a t i o n o f the coup l i ng c a p a c i t y o f c a r d i a c 0-adrenerg ic recepto r s a f t e r four months o f d i abe te s . The de fec t l ead i ng to the uncoupl ing i s not known; however, G-prote ins may be i m p l i c a t e d s i nce d i abe te s - a s s o c i a t ed G-p ro te in dy s func t i on has been demonstrated by the l o s s i n the hepa t i c express ion o f G i - p r o t e i n (Gawler, e t a l , 1987), and the obse rva t i on t ha t r e s i d u a l G^ p r o t e i n i s i n a c t i v e (Houslay, 1989) i n expe r imenta l l y - i nduced d iabetes i n r a t s . I t has a l s o been shown tha t sarcolemmal GTP-binding p r o t e i n s , G s and G-j, are increased i n STZ-induced d iabetes (N i sh i o et a l , 1988). I t i s p o s s i b l e t ha t the c a r d i a c G s i n d i abetes may be n o n - f u n c t i o n a l , p o s s i b l y through non-enzymatic g l y c o s y l a t i o n of the p r o t e i n , as may occur i n d i abetes (Bunn, 1981). I f G s was f u n c t i o n a l l y i n a c t i v e i n the presence of increased G^, t h i s would 8 support the data i n d i c a t i n g a reduced i s o p r o t e r e n o l - s t i m u l a t e d adenylate c y c l a s e . T i s sue s p e c i f i c d i f f e r e n c e s i n the expres s ion o f G s and G^ may a l s o be i n v o l v e d . Changes i n the f u n c t i o n a l s ta tu s o f the a -ad renerg i c system may a l so c o n t r i b u t e to c a r d i a c dy s func t i on i n d iabetes as i n i t i a l l y demonstrated by a l t e r e d drug responses i n c a r d i a c t i s s u e . The c o n t r a c t i l e response o f i s o l a t e d v e n t r i c u l a r p a p i l l a r y muscles from d i a b e t i c r a t t o the a - r e cep to r agon i s t methoxamine has been repor ted to be reduced concomitant w i th a reduced a - r e cep to r popu la t i on (Hey l i ge r et a l , 1982). However, o ther s t ud i e s i n d i c a t e tha t a - r e cep to r respons iveness i n the d i a b e t i c heart i s enhanced. L e f t a t r i a o f STZ-induced d i a b e t i c r a t s were s u p e r s e n s i t i v e and hyper - respons ive to a j - r e c e p t o r s t i m u l a t i o n by phenylephr ine (Jackson et a l , 1986). S u p e r s e n s i t i v i t y to a -adrenerg i c s t i m u l a t i o n has been shown by others (Downing et a l , 1983; Canga and S t e r i n - Bo rda , 1986; Wald e t a l , 1988) and i n agreement w i th Hey l i g e r e t a l (1982), a - ad rene rg i c r ecep to r number i s reduced i n d i a b e t i c heart ( L a t i f p o u r and M c N e i l l , 1984; Wald e t a l , 1988). The s u p e r s e n s i t i v i t y o f d i a b e t i c c a r d i a c t i s s u e to a -ad rene rg i c agon i s t s has been shown to be accompanied by an apparent i nc rease i n r e cep to r a f f i n i t y (Wald et a l , 1988); however, the m a j o r i t y o f s t ud i e s do not support t h i s view (Hey l i ge r et a l , 1981; W i l l i ams et a l , 1983; L a t i f p o u r and M c N e i l l , 1984; Durante et a l , 1989). The increased respons iveness t o , or e f f i c a c y o f , a -adrenerg i c agon i s t s observed i n the absence o f a - r e cep to r a f f i n i t y changes may i n d i c a t e changes i n po s t -r ecep to r f u n c t i o n i n the d i a b e t i c s t a t e . ^ - ad rene rg i c receptor -mediated s t i m u l a t i o n o f myocardia l adeny late c y c l a s e can be i n f l uenced by the a c t i v i t y o f the parasympathet ic branch o f 9 the autonomic nervous system. The parasympathet ic system i s cons idered to i n f l u e n c e adenylate c y c l a se through c h o l i n e r g i c r e cep to r s t i m u l a t i o n of sarcolemmal G i - p r o t e i n s (Kurose and U i , 1983), which subsequent ly i n t e r a c t w i th and i n h i b i t adenylate c y c l a se a c t i v i t y . I t has been determined t ha t there are a l t e r a t i o n s i n the c a r d i a c c h o l i n e r g i c r e cep to r system i n exper imental d i abe te s . Card iac membrane muscar in i c c h o l i n e r g i c r e cep to r number and a f f i n i t y was una f fec ted i n an STZ model o f IDDM of e i gh t weeks du r a t i on (W i l l i ams et a l , 1983). In longer term STZ-induced d iabetes ( s i x months du ra t i on ) a s i g n i f i c a n t r educ t i on i n v e n t r i c u l a r c h o l i n e r g i c r e cep to r number i n the absence of changes i n c h o l i n e r g i c r ecep to r antagon i s t a f f i n i t y has been repor ted ( L a t i f p o u r and M c N e i l l , 1984). I t i s apparent t he r e f o r e that there are a l t e r a t i o n s i n the f u n c t i o n a l s t a tu s o f the c a r d i a c a - ad rene rg i c , /^adrenerg ic and c h o l i n e r g i c r e cep to r systems i n d i abe te s . These changes may impinge upon c a r d i a c f u n c t i o n i n both c l i n i c a l and expe r imenta l l y - i nduced d iabetes m e l l i t u s . 1.1c D i a b e t i c Cardiomyopathy I t has been recogn ized s i nce 1974 tha t there e x i s t s a s p e c i f i c diabetic cardiomyopathy c o n t r i b u t i n g to heart d i sease i n the d i a b e t i c popu l a t i on separate from ca rd i a c d i sease i n d iabetes a s soc i a ted w i th coronary a t h e r o s c l e r o s i s , c a rd i a c microang iopath ies and/or d i a b e t i c c a r d i a c autonomic neuropathy (Kannel et a l , 1974). D i a b e t i c cardiomyopathy i s not as p reva len t as c e r t a i n other forms of c a rd i a c d i s ea se ; however, c l i n i c a l and exper imental research l ed to i t s i d e n t i f i c a t i o n as d i s t i n c t from other forms o f heart d i s ea se . C l i n i c a l s tud ie s employing non - inva s i ve techniques such as echocardiography and r a d i o n u c l i d e s c i n t i g r a p h y have demonstrated 10 impaired s y s t o l i c (S inger et a l , 1989) and d i a s t o l i c (Ledet et a l , 1984; Uus i tupa e t a l , 1988; P a i l l o e et a l , 1989) f u n c t i o n i n d i a b e t i c p a t i e n t s f r ee o f coronary a r t e r y d i sease and other r i s k f a c t o r s f o r c a r d i a c d i s ea se . The sequelae o f impaired s y s t o l i c and d i a s t o l i c f u n c t i o n i n d i a b e t i c s i n c l ude impaired myocardia l f u n c t i o n dur ing s t r e s s t e s t s (Kahn e t a l , 1986; Abenavol i et a l , 1981) and these changes may be e a r l y markers f o r the development o f cardiomyopathy ( P a i l l o e et a l , 1989). D i a b e t i c cardiomyopathy has a l so been i n v e s t i g a t e d us ing exper imental animal models of d iabetes m e l l i t u s . These models t y p i c a l l y employ e i t h e r a l l o x a n or s t r e p t o z o c i n as the diabetogen to induce the d i a b e t i c s t a t e , and, more r e c e n t l y , animals g e n e t i c a l l y predisposed to IDDM have been s t u d i e d . The d i a b e t i c cardiomyopathy i s mani fest i n a l l o xan - i nduced and s t r ep t o zoc i n - i nduced d i a b e t i c r a t s ( M i l l e r , 1979; Fe in e t a l , 1980; Vadlamudi e t a l , 1982), the g e n e t i c a l l y p red i sposed, spontaneously d i a b e t i c B io -B reed ing r a t (Rodrigues et a l , 1988), a l l o xan - i nduced d i a b e t i c r a b b i t s ( B h i m i j i et a l , 1985) and dogs (Regan et a l , 1974)). Experimental d i a b e t i c cardiomyopathy resembles tha t seen c l i n i c a l l y i n tha t there i s impaired s y s t o l i c and d i a s t o l i c f unc t i on of hearts o f d i a b e t i c animals ( M i l l e r , 1979; Fe in e t a l , 1980; Vadlamudi e t a l , 1982). S y s t o l i c f u n c t i o n i s t y p i c a l l y determined by measurement o f the maximal r a t e o f change of pressure i n the l e f t v e n t r i c l e dur ing c o n t r a c t i o n ( + d P / d t ) , and d i a s t o l i c f u n c t i o n determined by measurement o f the maximal r a t e of change of pressure dur ing d i a s t o l e ( "dP/dt ) . Ex vivo s t ud ie s r e f e r r e d to above a l so demonstrate a reduced peak l e f t v e n t r i c u l a r developed pressure i n heart s of d i a b e t i c an imal s . In vivo s t ud ie s have shown peak s y s t o l i c pressure to be reduced i n a l l o xan - i nduced d i a b e t i c r a b b i t s and STZ-induced d i a b e t i c r a t s . 11 Subsequent to the c l i n i c a l and exper imental r e c o g n i t i o n o f d i a b e t i c cardiomyopathy, severa l b iochemical and s u b c e l l u l a r a l t e r a t i o n s o f the d i a b e t i c heart which may c o n t r i b u t e to the impaired c a r d i a c f u n c t i o n o f d i a b e t i c s have been d e f i n e d . An inc rease i n myocardia l c o l l a gen has been de sc r i bed i n both c l i n i c a l and exper imental d iabetes ( J a r r e t t , 1984) which may c o n t r i b u t e to an increased " s t i f f n e s s " o f the myocardium and i t s at tendant e f f e c t s on s y s t o l e and d i a s t o l e . A d d i t i o n a l l y and more s p e c i f i c a l l y , changes i n ( i ) c a rd i a c c o n t r a c t i l e p r o t e i n s , ( i i ) sarcolemmal f u n c t i o n and ( i i i ) sarcop lasmic r e t i c u l u m f u n c t i o n have been d e s c r i b e d . In the f o l l o w i n g s e c t i o n s , these th ree areas w i l l be d i scus sed to p rov ide the foundat ion f o r the cu r ren t s t u d i e s , and i n a l a t t e r s e c t i o n , d i a b e t e s -induced a l t e r a t i o n s i n myocardial l i p i d s w i l l be presented to p rov ide the r a t i o n a l e f o r the cu r ren t s t u d i e s . Structure and Function of Myosin in Diabetes Mell itus. S ince v e n t r i c u l a r c o n t r a c t i l e performance i s depressed i n both c l i n i c a l and exper imental d i a b e t i c cardiomyopathy, the e f f e c t o f d iabetes upon the c o n t r a c t i l e apparatus o f the heart has been s t u d i e d . Card iac myosin i s a major component of the c o n t r a c t i l e apparatus d i r e c t l y i nvo l ved i n c o n t r a c t i o n . Myosin i s a hexameric p r o t e i n c o n s i s t i n g o f two heavy cha ins (200,000 kDa), two phosphory latab le l i g h t cha ins (18,000-22,000 kDa) and two nonphosphory latable l i g h t cha ins (16,000-27,000 kDa) (Lompre, et a l , 1984). There are two d i f f e r e n t types o f myosin heavy cha in (MHC) i n the hea r t , a and 0, which together w i th the fou r l i g h t cha i n s , combine to form th ree d i f f e r e n t myosin p ro te i n s ( i sozymes) , termed V i , V2 and V3. The d i f f e r e n t MHC compos i t ion o f the th ree d i f f e r e n t myosin isozymes confer s 12 uniqueness: the isozymes have been c h a r a c t e r i z e d on the ba s i s o f t h e i r i n t r i n s i c ca l c i um ATPase a c t i v i t y and the p r o t e i n compos i t ion o f t h e i r myosin heavy cha in s . The Vj isozyme c o n s i s t s o f two a cha ins and expresses the h ighes t i n t r i n s i c myosin ca l c i um ATPase a c t i v i t y . The V3 isozyme c o n s i s t s o f two /J cha ins and expresses the lowest i n t r i n s i c myosin ca l c i um ATPase a c t i v i t y and the V2 isozyme c o n s i s t s o f a s i n g l e a and a s i n g l e /J cha in and expresses an in te rmed ia te i n t r i n s i c ca l c i um ATPase a c t i v i t y . I n t r i n s i c ca l c i um ATPase a c t i v i t y of myosin i s p h y s i o l o g i c a l l y r e l e van t s i nce c a r d i a c myosin ca l c i um ATPase a c t i v i t y i s d i r e c t l y p r opo r t i o na l to the i n t r i n s i c v e l o c i t y of muscle c o n t r a c t i o n (Schwartz et a l , 1981). The d i f f e r e n c e s i n myosin ca l c ium ATPase a c t i v i t y were i n i t i a l l y determined i n myosin e x t r a c t s o f hypothyro id and hyper thy ro id r a t heart (Hoh et a l , 1978). These s tud ie s a l so demonstrated t ha t d i f f e r e n t myosin ATPase a c t i v i t i e s were a consequence o f isoenzymes w i t h d i f f e r e n t p r o t e i n compos i t i on , based on the d i f f e r e n t e l e c t r o p h o r e t i c m o b i l i t i e s i n non-denatur ing ge l s o f myosin ex t r a c ted from hyper- , eu- and hypo - thy ro id r a t hearts (Hoh et a l , 1978). In d i a b e t i c r a t s , there i s a s h i f t i n the expres s ion o f c a r d i a c myosin ATPase from one o f high i n t r i n s i c a c t i v i t y ( V j : a-MHC) to one of lower i n t r i n s i c a c t i v i t y (D i l lmann, 1980; Malhotra e t a l , 1981; P i e r c e and D h a l l a , 1981). S ince the speed o f v e n t r i c u l a r c o n t r a c t i o n i s r e l a t e d to the predominant myosin spec ies present i n the heart (Schwartz et a l , 1981), the s h i f t from Vi to V3 i n the d i a b e t i c v e n t r i c l e may c o n t r i b u t e to the c o n t r a c t i l e a l t e r a t i o n s observed i n d i a b e t i c cardiomyopathy. Although expe r imenta l l y - i nduced d iabetes i s o f ten a s soc i a ted w i th a reduced c i r c u l a t i n g t h y r o i d hormone concen t r a t i o n , treatment o f d i a b e t i c animals 13 wi th T4 inc reases actomyosin ATPase a c t i v i t y above the unt reated d i a b e t i c l e v e l but f a i l s to b r i ng the a c t i v i t y to the non -d i abe t i c l e v e l (Malhotra e t a l , 1981). A d d i t i o n a l l y , c a r d i a c dy s func t i on o f d i abetes cannot be re tu rned to normal by t h y r o i d hormone treatment (Fe in e t a l , 1980; Barbee et a l , 1988). There fo re , although t h y r o i d hormone concen t ra t i on s may be reduced i n exper imental d i abe te s , t h i s "hypothyroid" s t a t e o f d i abetes a lone cannot e x p l a i n the a l t e r e d myosin expres s ion o c c u r r i n g i n d i a b e t e s . Funct ion o f Sarcolemma i n Diabetes M e l l i t u s . The c a r d i a c sarcolemma p lays an important r o l e i n the r e g u l a t i o n o f the c o n t r a c t i o n - r e l a x a t i o n c y c l e o f the hea r t . Hence, t h i s s t r u c t u r e has been i n v e s t i g a t e d f o r p o s s i b l e de fec t s i n d iabetes m e l l i t u s . The sarcolemma possesses a ca l c i um pumping ATPase r e spon s i b l e f o r e x t r u s i o n o f ca l c i um from the myoplasm to the e x t r a c e l l u l a r space dur ing d i a s t o l e (Caroni and C a r a f o l i , 1981). The ca l c ium pumping a c t i v i t y o f the sarcolemmal ca l c i um ATPase has been shown to be depressed i n exper imenta l d i abetes m e l l i t u s (Hey l i ge r et a l , 1987; Makino et a l , 1985). The N a + - C a 2 + exchanger o f the sarcolemmal has a l s o been shown to be depressed i n d iabetes (Makino et a l , 1985) as i s sarcolemmal Na + /K + -ATPase a c t i v i t y ( P i e r ce and D h a l l a , 1983). These a l t e r a t i o n s i n sarcolemmal t r a n s p o r t e r s may c o n t r i b u t e to the impaired s y s t o l i c and d i a s t o l i c f u n c t i o n o f the d i a b e t i c hea r t , although the mechanism re spon s i b l e f o r the reduced a c t i v i t i e s remains to be d e f i n e d . 14 Function of Sarcoplasmic Reticulum in Diabetes Mellitus The c a r d i a c sarcop lasmic r e t i c u l u m (SR) p lays an important r o l e i n myocardia l r e l a x a t i o n through a c t i v e seques t ra t i on o f myoplasmic ca l c i um (Tada e t a l , 1978). The enzyme re spon s i b l e f o r a c t i v e t r an spo r t o f ca l c i um i n t o the SR lumen i s the C a 2 + , M g 2 + - a d e n o s i n e t r i phosphata se (ATPase, ca l c i um pump), a p r o t e i n of approx imately 100 kDa compr i s ing about 40% of the t o t a l SR p r o t e i n (Tada et a l , 1978). The ATP h y d r o l y t i c and ca l c i um t r an spo r t a c t i v i t y o f the enzyme show ca l c ium dependency, such tha t i n t r a c e l l u l a r f r e e C a 2 + r egu la te s the ca l c i um t r an spo r t a c t i v i t y o f the ca l c i um pump p r o t e i n (Hasselbach, 1964). Calc ium t r an spo r t by the ca l c i um pump d i s p l a y s M ichae l i s -Menten k i n e t i c s w i th a Kc a i n the low micromolar range (Kc a * 1 /*M). The r e a c t i o n / t r a n s p o r t c y c l e o f the SR ca l c i um ATPase pump i s represented i n F igure 1. Knowledge of the r e a c t i o n mechanism of the ca l c i um pump has been u t i l i z e d expe r imenta l l y i n order to q u a n t i f y the number o f SR ca l c i um ATPase (ca lc ium pump) s i t e s in vitro. The t h e o r e t i c a l bas i s f o r such experiments can be ev inced from the f i g u r e as i t i s apparent tha t as [ C a 2 + ] p r e e i s i nc rea sed , dephosphory lat ion o f the enzyme w i l l be i n h i b i t e d . Th i s may be cons idered end-product i n h i b i t i o n of the enzyme t r an spo r t c y c l e . Under s u i t a b l e r e a c t i o n c ond i t i o n s the ca l c i um pump may be "frozen" i n the phosphorylated E-P s t a t e and, through the use o f - y - 3 2 P -ATP, the l e v e l o f the enzyme in te rmed ia te q u a n t i f i e d . The i n te rmed ia te spec i e s , E-P, i s s e n s i t i v e to hydroxylamine which c leaves the acylphosphate bond and re lea se s P i . The hydroxylamine s e n s i t i v i t y o f E-P i s used to d i f f e r e n t i a t e the ca lc ium pump in te rmed ia te from other phosphate bonds i n the SR ( i . e phosphorylated phospholamban, see below). 15 F igure 1. Reac t i on/ t ranspor t c y c l e o f the sarcop lasmic r e t i c u l u m c a l c i u m -ATPase pump. The f i g u r e shows the sequent i a l steps o f the enzyme ca t a l y zed t r an spo r t o f ca l c i um from the myoplasm, across the SR membrane to the SR lumen. 16 2+ 2Ca ( v. E Ca ATP 1. Ca. + ADP E -P r „ 2+ ~P 2 Ca2 2Ca IN from Jencks, 1989 17 Calc ium t r an spo r t by the ATPase i s modulated by endogenous p r o t e i n k inase a c t i v i t i e s w i t h i n the myocyte. The r a t e o f SR ca l c i um t r an spo r t i s r egu la ted by ca lmodu l in (Katz and Remtu l la , 1978), c y c l i c 3 ' , 5 ' - a d e n o s i n e monophosphate (cAMP)-dependent p r o t e i n k inase (Tada et a l , 1974; Kran ias e t a l , 1980), and p h o s p h o l i p i d - s e n s i t i v e calcium-dependent p r o t e i n k inase (Limas, 1980; Movsesian et a l , 1984). Calc ium t r an spo r t i s augmented by these endogenous r e gu l a t o r s v i a phosphory la t ion o f phospholamban, a 22,000 kDa p r o t e i n o f the SR (LePeuch et a l , 1979), which i nc reases the i n i t i a l r a t e s o f ca l c i um t r an spo r t and Ca 2 + -ATPase a c t i v i t y and i nc reases the a f f i n i t y o f the enzyme f o r ca l c i um (Gupta e t a l , 1988). S t i m u l a t i o n o f phospholamban phosphory la t ion w i th i s op ro te reno l has been shown to c o r r e l a t e w i t h an increased maximal r a t e of r e l a x a t i o n i n the i s o l a t e d perfused r a t heart (de Weilenmann et a l , 1987), an e f f e c t c o n s i s t e n t w i th enhanced SR ca l c i um t r a n s p o r t . S ince impaired d i a s t o l i c f unc t i on has been de sc r i bed i n c l i n i c a l and exper imental d i abe te s , c a r d i a c SR f u n c t i o n has been i n v e s t i g a t e d to determine whether the ca l c i um t r an spo r t a c t i v i t y o f the o r g a n e l l e i s a f f e c t e d by d i abe te s . SR ca l c i um t r an spo r t and Ca 2 + ,Mg 2 + - ATPa se a c t i v i t i e s , determined at a s i n g l e t o t a l ca l c i um concen t r a t i on o f 0.1 mM (2.94 - 5.4 /iM [ C a 2 + ] p r e e ) , have been shown to be s i g n i f i c a n t l y reduced i n male and female STZ-induced d i a b e t i c r a t s (d iabetes o f 4-5 and 9 weeks d u r a t i o n , r e s p e c t i v e l y ; Penparkgul et a l , 1981). Others have conf i rmed tha t SR ca l c i um t r an spo r t a c t i v i t y i s reduced i n STZ-induced d i a b e t i c r a t s (Lopaschuk et a l , 1982; Ganguly et a l , 1983). The s tud i e s o f Lopaschuk e t a l (1982) showed SR ca l c i um t r an spo r t to be s i g n i f i c a n t l y reduced i n d i a b e t i c animals over a [ C a 2 + ] p r e e range o f 0.5 - 5 /iM. These s t ud i e s a l so 18 showed tha t Ca -ATPase a c t i v i t y was s i g n i f i c a n t l y reduced i n SR o f d i a b e t i c an imal s . The mechanism f o r the reduced SR ca l c i um t r an spo r t a c t i v i t y o f d i a b e t i c animals i s not due to d i f f e r e n t degrees o f non-SR contaminat ion or d i f f e r e n c e s i n the SR y i e l d (Penparkgul e t a l , 1981; Lopaschuk et a l , 1983b). I t has been shown tha t the re i s an increased l e v e l o f endogenous a c y l c a r n i t i n e a s soc i a ted w i th SR i s o l a t e d from d i a b e t i c animals (Lopaschuk et a l , 1982), and tha t d i a b e t i c SR ca l c i um t r an spo r t i s l e s s s e n s i t i v e to the i n h i b i t o r y e f f e c t o f exogenous L - p a l m i t o y l c a r n i t i n e (Lopaschuk et a l , 1984). Th i s l a t t e r r e s u l t suggests t ha t i n t r i n s i c ca l c i um t r an spo r t a c t i v i t y o f d i a b e t i c animals may be t o n i c a l l y i n h i b i t e d by endogenous a c y l c a r n i t i n e , such tha t a d d i t i o n a l (exogenous) a c y l c a r n i t i n e i s l e s s e f f i c a c i o u s . In c a r d i a c SR o f STZ-induced d i a b e t i c r a t s , n e i t h e r ca lmodu l in nor cAMP-dependent p r o t e i n k inase s t i m u l a t i o n o f ca l c i um t r an spo r t i s a f f e c t e d s i nce c on t r o l and d i a b e t i c p repa ra t i on s were s t imu l a ted to a s i m i l a r degree (Lopaschuk et a l , 1984). Other p o t e n t i a l mechanisms c o n t r i b u t i n g to the reduced SR ca l c i um t r an spo r t and Ca^ +-ATPase a c t i v i t i e s o f d i a b e t i c animals remain to be d e f i n e d . S ince the e t i o l o g y o f d i a b e t i c cardiomyopathy i s unknown and because i t i s conce i vab l y m u l t i -f a c t o r i a l i n na tu re , f u r t h e r s tud ie s are warranted. In t h i s rega rd , s i n ce there are changes i n membrane l i p i d compos i t ion i n d i abetes i n general (Holman et a l , 1983; Faas and C a r t e r , 1980) and i n the heart s p e c i f i c a l l y (Gudbjarnason at a l , 1987) the po s s i b l e r e gu l a t o r y r o l e o f such changes on c a r d i a c SR f u n c t i o n i s of p a r t i c u l a r i n t e r e s t . 19 1.2 Hypothyroidism. 1.2a Etiology and Cl in ical Description of Hypothyroidism. Hypothyro id i sm, o f v a r i e d e t i o l o g y , i s a term d e s c r i p t i v e o f the c l i n i c a l c o n d i t i o n where there i s a reduced c i r c u l a t i n g concen t r a t i on o f t h y r o i d hormones. Primary hypothyro id i sm r e f e r s to some form of i n t r i n s i c f a i l u r e o f the t h y r o i d g land to s yn the s i ze , s t o re and/or s ec re te hormones (DeGroot et a l , 1984). C l i n i c a l l y , the man i f e s t a t i on s o f hypothyro id i sm depend on the age o f the p a t i e n t , the du ra t i on and the s e v e r i t y o f the d i s ea se . Untreated neonatal hypothyro id i sm can lead to c r e t i n i s m , an i r r e v e r s i b l e syndrome a s soc ia ted w i th mental and phy s i c a l growth f a i l u r e . In the a d u l t , the c l i n i c a l syndrome o f hypothyro id i sm i s r e f e r r e d to as myxedema. T yp i c a l s igns o f hypothyro id i sm i nc l ude l e t h a r g y , slowed speech, impaired mentat ion, coarse h a i r , th ickened s k i n , reduced basal metabo l i c r a t e and a reduced ch rono t rop i c and i n o t r o p i c s t a t e o f the hea r t . 1.2b Cardiac Disease in Hypothyroidism. As mentioned p r e v i o u s l y , the c a r d i o v a s c u l a r e f f e c t s o f t h y r o i d d e f i c i e n c y have been known f o r a con s ide rab le pe r i od o f t ime (Zondek, 1918). C l i n i c a l d e s c r i p t i o n s o f hypothyro id i sm c o n s i s t e n t l y i n c l ude a l t e r a t i o n s o f the c a r d i o v a s c u l a r system. The c a r d i a c s igns of hypothyro id i sm i nc l ude a negat ive i n o t r o p i c and ch rono t rop i c s t a t e (Werner and Braverman, 1986). Noninvas ive s tud ie s have shown l e f t v e n t r i c u l a r s y s t o l i c f u n c t i o n to be impaired i n hypothyro id i sm (Burchardt e t a l , 1978). A prolonged p r e - e j e c t i o n per iod and reduced LVEF have a l s o been de sc r i bed ( F o r f a r et a l , 1982). Impaired d i a s t o l i c f u n c t i o n , assessed by a 20 s i g n i f i c a n t l y prolonged r a t e o f t h i n n i n g o f the l e f t v e n t r i c u l a r p o s t e r i o r w a l l , has been desc r ibed i n the hypothyro id p a t i e n t (Vora e t a l , 1985). C l e a r l y , the heart i s a f f e c t e d by a d e f i c i e n c y o f t h y r o i d hormone. Experimental t h y r o i d d i sease i s r e a d i l y reproduced i n the l a b o r a t o r y w i t h c a r d i a c changes resembl ing c l i n i c a l ob se rva t i on s . Hypothyro id i sm can be induced by the a n t i t h y r o i d drugs p r o p y l t h i o u r a c i l (McConnaughey et a l , 1979) or methimazole ( Ishac and Pennefather, 1983), by s u r g i c a l removal o f the t h y r o i d g land (Simpson et a l , 1981) or r a d i o i o d i n e treatment (MacLeod and M c N e i l l , 1981). The c a r d i a c man i fe s ta t i on s o f t h y r o i d d i sea se have been i n v e s t i g a t e d expe r imenta l l y in vitro us ing i s o l a t e d v e n t r i c u l a r and a t r i a l t i s s u e s and i s o l a t e d hearts and in vivo w i th whole animal p r epa r a t i on s . The in vitro work o f Buccino e t a l (1967) us ing i s o l a t e d cat v e n t r i c u l a r p a p i l l a r y muscles i s demonstrat ive o f the e f f e c t s o f hypothyro id i sm on i s o l a t e d c a r d i a c t i s s u e . Compared to the eu thy ro i d t i s s u e , p a p i l l a r y muscle from hypothyro id cat has a decreased r a t e and t ime to peak developed t e n s i o n . That these are c h a r a c t e r i s t i c a l t e r a t i o n s o f the hypothyro id s t a t e i s supported by the recent work o f MacKinnon et a l (1988) which e s s e n t i a l l y conf i rmed the experiments o f Buccino et a l (1967) us ing hyper- and hypo- thy ro id f e r r e t v e n t r i c u l a r p a p i l l a r y muscle. I s o l a t ed working r a t heart experiments have shown tha t the spontaneous heart r a t e and the maximal r a t e o f r i s e o f l e f t v e n t r i c u l a r pressure are reduced i n hypothyro id i sm (Brooks et a l , 1985). Thus, as the above in vitro s t ud i e s i n d i c a t e , expe r imenta l l y - i nduced hypothyro id i sm prov ides a s u i t a b l e model system i n which to study the e f f e c t s o f a l a c k o f t h y r o i d hormone on the hea r t . 21 S ince c l i n i c a l and exper imental s tud ie s have demonstrated t ha t the c a r d i a c e f f e c t s o f t h y r o i d hormone d e f i c i e n c y are prominent, research has focused on the mechanism by which t h y r o i d hormone induces these changes. Three areas have been e x t e n s i v e l y i n v e s t i g a t e d : ( i ) c a r d i a c myosin ATPase, ( i i ) c a r d i a c adrenerg ic recepto r s and ( i i i ) , c a r d i a c sa rcop lasmic r e t i c u l u m . These th ree areas w i l l be d i scus sed i n some d e t a i l t o prov ide a background f o r the r a t i o n a l e o f the cu r ren t s t u d i e s . In a l a t e r s e c t i o n , a l t e r a t i o n s i n myocardia l l i p i d s and l i p i d metabolism i n hypothyro id i sm w i l l be presented. Thyro id Hormone and Card iac Myosin. The obse rva t i on tha t exper imental hyper thyro id i sm i s a s s oc i a ted w i th an i nc rease and hypothyro id i sm w i th a decrease i n the i n t r i n s i c v e l o c i t y o f c a r d i a c c o n t r a c t i o n (Buccino et a l , 1967; MacKinnon et a l , 1988) l e d to the i n v e s t i g a t i o n o f changes i n the c o n t r a c t i l e p r o t e i n s o f the heart i n c l u d i n g the major component o f the c o n t r a c t i l e apparatus, myosin. Hoh et a l (1978) demonstrated tha t d i f f e r e n t myosin ATPase a c t i v i t i e s were the consequence o f isoenzymes w i th d i f f e r e n t p r o t e i n compos i t ion , based on the d i f f e r e n t e l e c t r o p h o r e t i c m o b i l i t i e s i n non-denatur ing ge l s o f myosin e x t r a c t ed from hyper - , eu- and hypo- thy ro id r a t hea r t s . Fu r ther work has shown tha t t h y r o i d hormone c o n t r o l s the express ion o f the a - and 0-MHC genes a n t i t h e t i c a l l y , and tha t i n the hypothyro id r a t , /J-MHC mRNA i s the s o l e d e t e c t a b l e MHC t r a n s c r i p t (Lompre et a l , 1984). Thyro id hormone i s known to i n f l u e n c e the express ion of growth hormone (Ya f fe and Samuels, 1984); however the e f f e c t s o f t h y r o i d hormone on c a r d i a c myosin expres s ion are 22 independent o f these ac t i on s (Gustafson e t a l , 1987; But ler-Browne et a l , 1987). The e f f e c t o f t h y r o i d hormone on the myocardia l expres s ion o f MHC i s a d i r e c t c a r d i a c e f f e c t and independent o f secondary i n f l u e n c e s o f the hormone, such as increased c a r d i a c work or i n t a c t i n n e r v a t i o n ( K l e i n and Hong, 1986; Korecky et a l , 1987). There fo re , based on s tud ie s o f myosin isoenzyme exp re s s i on , i t i s apparent t ha t t h y r o i d hormone exe r t s a s p e c i f i c e f f e c t upon the hea r t , the b iochemical man i f e s t a t i on s of which are c o n s i s t e n t w i t h the pa thophy s i o l o g i c a l change observed in vivo. Adrenerg i c Receptors i n Hypothyro id i sm. The e f f e c t s o f hypothyro id i sm on the heart c l o s e l y resemble those o f catecholamine d e f i c i e n c y . Th is s i m i l a r i t y suggested t ha t c a r d i a c adrenerg ic r ecep to r a l t e r a t i o n s may be r e spon s i b l e f o r the c a r d i o v a s c u l a r man i f e s t a t i on s of hypothyro id i sm. P a r a d o x i c a l l y , the plasma l e v e l s o f no rad rena l i ne are t y p i c a l l y e leva ted i n hypothyro id i sm, and plasma ad rena l i ne concent ra t i on s are g e n e r a l l y not a f f e c t e d (Coulumbe et a l , 1976). S ince plasma catecholamine l e v e l s are at va r i ance w i t h observed c a r d i a c f u n c t i o n a l a l t e r a t i o n s , research has focused on a l t e r a t i o n s i n c a r d i a c adrenerg ic recepto r s e n s i t i v i t y or respons iveness to endogenous catecholamines as a means of e x p l a i n i n g the c a r d i a c a l t e r a t i o n s o f t h y r o i d d i s ea se . There have been a number o f s tud ie s o f man and exper imental animals (Benfey and Varma, 1963; Margo l ius and Gaffney, 1965; van der Schoot and Moran, 1965) i n d i c a t i n g tha t adrenerg ic respons iveness i s una l te red by the t h y r o i d s t a t e , although more recent in vitro experiments 23 i n d i c a t e o therw i se . Hearts from hypothyro id r a t s and guinea p igs show decreased s e n s i t i v i t y to the i n o t r o p i c and ch rono t rop i c e f f e c t s o f i s op ro te reno l (MacLeod and M c N e i l l , 1981; Handberg, 1984). A r educ t i on i n the number o f myocardia l /J-receptors and an inc rease i n a - r e cep to r s i s a c o n s i s t e n t f i n d i n g i n hypothyro id i sm ( B i l e z i k i a n and Loeb, 1983). I t i s apparent tha t changes i n c a r d i a c f u n c t i o n seen i n hypothyro id i sm are a s soc i a ted w i th changes i n t i s s u e s e n s i t i v i t y to adrenerg ic s t i m u l a t i o n and the number o f myocardia l 0 - recepto r s . These r e c e p t o r - r e l a t e d a l t e r a t i o n s may c o n t r i b u t e to the c a r d i a c a l t e r a t i o n s of hypothyro id i sm in vivo. Funct ion o f Sarcoplasmic Ret icu lum i n Hypothyro id i sm. V e n t r i c u l a r r e l a x a t i o n i s c o n s i s t e n t l y a l t e r e d i n t h y r o i d d i s ea se . Changes i n the r a t e o f v e n t r i c u l a r r e l a x a t i o n i n hypo- and hype r - thy ro i d i sm i s a s soc i a ted w i th complementary a l t e r a t i o n s o f ca l c i um f l u x i n the sarcop lasmic r e t i c u l u m . Using the c a l c i u m - s e n s i t i v e dye aequor in i t has been shown tha t ca l c i um removal from ca rd i a c myoplasm dur ing r e l a x a t i o n i s enhanced i n hyper thy ro id and impaired i n hypothyro id f e r r e t p a p i l l a r y muscle (MacKinnon et a l , 1988). S ince c a r d i a c sa rcop lasmic r e t i c u l u m i s cons idered to p lay a p i v o t a l r o l e i n v e n t r i c u l a r r e l a x a t i o n (Tada et a l , 1978), ca l c i um t r an spo r t a c t i v i t y o f the SR under d i f f e r e n t t h y r o i d s t a t e s has been e x t e n s i v e l y s tud ied in vitro. SR ca l c ium t r an spo r t and ca l c i um ATPase a c t i v i t y are increased i n hyper thyro id i sm (Suko, 1973; Limas, 1978; Takacs et a l , 1985; B lack et a l , 1988) and decreased i n hypothyro id i sm (Suko, 1973; Takacs et a l 1985). The mechanism f o r the e f f e c t o f hypothyro id i sm on c a r d i a c SR has not been determined. 24 A number o f mechanisms by which t h y r o i d hormone may a f f e c t c a r d i a c SR calcium-ATPase and ca l c i um t r an spo r t a c t i v i t i e s have been i n v e s t i g a t e d . Thyro id hormone inc reases the abso lute amount o f SR i n r a t heart a f t e r 24 days o f t h y rox i ne treatment ( M c C a l l i s t e r and Page, 1973). There i s a l s o an inc reased y i e l d o f SR membranes i s o l a t e d from hyper thy ro id r a t heart (B lack et a l , 1988). I n d i r e c t evidence t ha t t h y r o i d hormone may s p e c i f i c a l l y a f f e c t SR p r o t e i n s ynthes i s i s t ha t a l t e r e d ca l c i um transport/ATPase a c t i v i t i e s are t y p i c a l l y expressed on a per mg SR p r o t e i n b a s i s , thereby no rma l i z i n g the amount o f ca l c ium transported/ATP hydro lyzed to the t o t a l amount o f p r o t e i n (Suko, 1973; Limas, 1978; Takacs e t a l , 1985; B lack et a l , 1988). Increased l e v e l s of the phosphoprotein i n te rmed ia te o f the SR ca l c i um ATPase i n SR i s o l a t e d from hearts o f hyper thy ro id r a t lend support to an i n d u c t i v e e f f e c t of t h y r o i d hormone on the ca l c i um pump (Limas, 1978; B lack et a l , 1988). Fur ther evidence o f a t h y r o i d hormone-induced p r o t e i n s y n t h e t i c mechanism i s t ha t increased c a r d i a c SR ca l c i um t r an spo r t and a concomitant i nc rease i n ca l c ium ATPase phosphoprotein i n te rmed ia te i n hype r thy ro id r a t s i s b locked by the a d m i n i s t r a t i o n of act inomyc in D, cyclohexamide or puromycin ( i n h i b i t o r s o f RNA and p r o t e i n s yn thes i s ) concurrent w i t h T4 treatment (Limas, 1978). Thyro id hormone-st imulated inc reases i n SR ca l c ium ATPase mRNA in hypothyro id r a t heart i s d i r e c t evidence t ha t t h y r o i d hormone may d i r e c t l y c on t r o l SR ca l c i um t r an spo r t a c t i v i t y v i a i nduc t i on o f ca l c i um pump p r o t e i n s yn thes i s (Rohrer and D i l lmann, 1988). In s k e l e t a l muscle SR, i n which ca l c i um t r an spo r t a c t i v i t y responds to t h y r o i d hormone analogous ly to the hea r t , i t has been shown tha t developmental increases i n SR ca l c i um t r an spo r t are d i r e c t l y r e l a t e d to the l e v e l of t h y r o i d hormone (Simonides and van Hardeve ld, 25 1989). These s t ud i e s a l so demonstrated t ha t t h y r o i d hormone e f f e c t s on s k e l e t a l muscle SR were independent o f growth hormone, an obse rva t i on s i m i l a r t o the t h y r o i d hormone-mediated (growth hormone independent) expres s ion and i n h i b i t i o n o f c a r d i a c a- and /3-MHC genes. In a d d i t i o n to a d i r e c t e f f e c t on the SR v i a an inc rease i n the number o f ca l c i um pump s i t e s , o ther mechanisms f o r the e f f e c t s o f t h y r o i d hormone on c a r d i a c SR which i n vo l ve the r e g u l a t i o n o f the ca l c i um t r a n s p o r t e r by k inase-mediated phosphory la t ion have been i n v e s t i g a t e d . Card iac SR from hyper thy ro id r a t i s phosphorylated ( t h i s may be l a r g e l y accounted f o r by phospholamban phosphory la t ion) to a g r ea te r extent i n the presence o f exogenous cAMP than SR o f eu thy ro id r a t (Limas, 1978b). However cAMP-dependent p r o t e i n k inase-mediated s t i m u l a t i o n o f hype r thy ro id c a r d i a c SR ca l c i um t r an spo r t i s not enhanced above tha t o f SR o f eu thy ro i d c o n t r o l s (Gua rn ie r i et a l , 1980). The r e g u l a t i o n o f SR ca l c i um t r an spo r t i n hypothyro id i sm by k inase-mediated phosphory la t ion o f phospholamban remains to be d e f i n e d . P h y s i o l o g i c a l concent ra t i on s o f t hy rox i ne and T3 ( 1 0 " ^ - 1 0 " M ) have a l s o been shown to d i r e c t l y s t i m u l a t e , in vitro, e r y t h r o c y te membrane ca l c i um ATPase (Galo e t a l , 1981), c a r d i a c sarcolemmal ca l c i um ATPase (Rudinger et a l , 1984), thymocyte plasma membrane ca l c i um uptake (Sega l , 1988) and c a l c i u m - s t i m u l a t e d ATPase (Segal et a l , 1989) a c t i v i t i e s . D i r e c t e f f e c t s o f t h y r o i d hormone on enzymes mediat ing ca l c i um f l u x across the c a r d i a c c e l l membrane, and p o s s i b l y the SR, represents an a d d i t i o n a l mechanism whereby t h y r o i d hormone may con t r o l c a r d i a c f u n c t i o n . I t i s ev ident tha t t h y r o i d hormone may a f f e c t SR f u n c t i o n through a number o f p o s s i b l e mechanisms. Thyro id hormone i s cons idered to exe r t i t s 26 e f f e c t s on the c e l l v i a s p e c i f i c nuc lea r recepto r s (Oppenheimer, 1985). An inc reased number o f SR ca l c i um pumps i n hyper thyro id i sm would conform to a p r o t e i n i n d u c t i v e s i t e o f a c t i o n mediated by nuc lea r r e c e p t o r s . The inc reased SR mRNA f o l l o w i n g T3 treatment (Rohrer and D i l lmann, 1988) a l s o supports a nuc lea r receptor -mediated s i t e o f a c t i o n f o r the e f f e c t of t h y r o i d hormone on c a rd i a c SR. Ex t ranuc lea r s i t e s o f a c t i o n o f t h y r o i d hormone have a l s o been proposed (Mu l l e r and S e i t z , 1984) and the d i r e c t e f f e c t s o f t h y r o i d hormone desc r ibed above i n d i c a t e t ha t the hormone may i n t e r a c t w i th the SR d i r e c t l y , independent of a nuc l ea r i n t e r a c t i o n . A d d i t i o n a l l y t h y r o i d hormone-induced a l t e r a t i o n s i n l i p i d metabol ism, i n c l u d i n g changes i n the ^ - o x i d a t i o n o f l i p i d s and the de s a t u r a t i o n o f pho spho l i p i d f a t t y acy l s pec ie s , may i n f l u e n c e SR ca l c i um t r an spo r t through changes i n the membrane l i p i d environment. These aspects o f t h y r o i d hormone a c t i o n on the heart are presented w i th a view t o p r o v i d i n g the r a t i o n a l e f o r the approach taken i n t h i s t h e s i s p r o j e c t . 1.3 The Role o f Myocardia l Membranes i n Ca rd iac Func t i on . The phys icochemical nature of the c a r d i a c sarcolemma (SL) and SR p lay s a s i g n i f i c a n t r o l e i n the i n t r i n s i c f u n c t i o n o f these membranes. Changes i n the l i p i d compos i t ion o f c a r d i a c SL and/or SR can a f f e c t the a c t i v i t i e s o f va r i ous transmembrane p r o te i n s mediat ing a c t i v e ion t r a n s p o r t , membrane-bound enzymes and membrane-associated recep to r mechanisms. Card iac f u n c t i o n and adaptat ion r equ i r e s t ha t these membrane-a s soc i a ted p r o t e i n a c t i v i t i e s remain w i t h i n p h y s i o l o g i c a l l i m i t s . SL and SR membranes are comprised of pho spho l i p i d s , f r ee c h o l e s t e r o l , c h o l e s t e r o l e s t e r s and f r e e f a t t y a c i d s . In the SR, phospho l ip id s account f o r 27 approx imate ly 73% and neut ra l l i p i d s 19% o f the membrane l i p i d (Limas, 1978). Of an in vitro i s o l a t e d SR membrane f r a c t i o n , t r i g l y c e r i d e , c h o l e s t e r o l and f r e e f a t t y ac ids account f o r 60%, 25% and 15%, r e s p e c t i v e l y , o f the neut ra l l i p i d f r a c t i o n (Limas, 1978). These d i f f e r e n t l i p i d c l a s s e s and s p e c i f i c molecu lar s t r u c t u r e s w i t h i n c l a s s e s con fe r upon the membrane the r e q u i s i t e phys icochemical nature which supports the membrane's b iochemica l and, hence, p h y s i o l o g i c a l f u n c t i o n . The concepts o f l i p i d f l u i d i t y and membrane i n t e g r i t y are we l l e s t a b l i s h e d and pa t ho l o g i c a l pe r tu rba t i on s o f the l i p i d cha rac te r o f c e l l membranes have been i m p l i c a t e d i n c a r d i a c d i sease and c a r d i a c f u n c t i o n a l abno rma l i t i e s (Katz and Messineo, 1981; Corr e t a l , 1984). Var ious l i p i d spec ies have been cons idered to have pa thophy s i o l o g i ca l (arrhythmogenic) e f f e c t s upon c a r d i a c f u n c t i o n f o l l o w i n g an i schemic ep i sode. E levated c i r c u l a t i n g f r ee f a t t y a c i d s , increased myocardia l pho spho l i p i d ca tabo l i sm l e ad i n g to pho spho l i p i d d e p l e t i o n and lysophosphat ide accumulat ion and the accumulat ion of f a t t y ac id s and t h e i r metabo l i c d e r i v a t i v e s w i t h i n the heart have been imp l i c a t ed as arrhythmogenic f o l l o w i n g an i schemic episode (Katz and Messineo, 1981). M e c h a n i s t i c a l l y , e l eva ted plasma f r e e f a t t y ac id s may a f f e c t the heart through negat ive i n o t r o p i c e f f e c t s ( L i ed tke et a l , 1978) or through arrhythmogenic e l e c t r o p h y s i o l o g i c a l e f f e c t s (Corr et a l , 1978). I t i s e s t a b l i s h e d t ha t the c a r d i a c SR p lays a s i g n i f i c a n t r o l e i n c a r d i a c f u n c t i o n . As d i scussed e a r l i e r , SR ca l c i um transport/ATPase a c t i v i t y i s r egu la ted by endogenous p r o t e i n k inase a c t i v i t i e s (cAMP-dependent p r o t e i n k inase , Ca z + - ca lmodu l i n -dependent p r o t e i n k inase and C a ^ + - a c t i v a t e d , phosphol ip id-dependent p r o t e i n k inase) which phosphory late 28 phosphol amban and s t imu l a t e the a c t i v i t y o f the Ca* -ATPase/ pump o f the SR. In a d d i t i o n to p r o t e i n k inase-mediated r e g u l a t i o n , SR ca l c i um pump f u n c t i o n i s a l s o regu la ted by the p r o p e r t i e s o f the SR membrane. Such r e g u l a t i o n may occur through membrane l i p i d e f f e c t s on the ca l c i um pump p r o t e i n (1 i p i d - p r o t e i n i n t e r a c t i o n s ) or through changes i n membrane f l u i d i t y (H ida lgo , 1987). I ncorpora t ion o f c h o l e s t e r o l i n t o SR v e s i c l e s reduces the f l u i d i t y of the membrane and i s a s soc i a ted w i t h a r educ t i on i n both Ca 2 + -ATPase a c t i v i t y and the coup l i ng e f f i c i e n c y o f the ca l c i um pump (Almedia e t a l , 1984). The changes i n s u s c e p t i b i l i t y o f the SR Ca t T ATPase to t r y p t i c c leavage over a range o f temperatures known to i n f l u e n c e the f l u i d i t y o f the l i p i d microenvironment has been suggested as evidence f o r s t r u c t u r a l e f f e c t s of the l i p i d environment on the Ca 2 + ATPase p r o t e i n (B lazyk e t a l , 1985). Membrane f l u i d i t y a l so d i r e c t l y a f f e c t s r o t a t i o n a l m o b i l i t y of the SR Ca 2 + -ATPase which appears to be the r a t e - l i m i t i n g step of ca l c i um t r an spo r t (Squ ier e t a l , 1988). The N a + - C a 2 + exchanger o f c a r d i a c sarcolemma i s a l s o i n f l uenced by the l i p i d microenvironment i n which i t i s embedded. R e c o n s t i t u t i o n o f p u r i f i e d exchange p r o t e i n w i th an i on i c phospho l i p id s such as phosphat idy l s e r i n e , c a r d i o l i p i n or phosphat id i c a c i d ( to 50% by weight) promote opt imal a c t i v i t y of the exchanger, as does c h o l e s t e r o l (at 20% by we i gh t ) , whereas phosphat idy l i n o s i t o l and pho spha t i d y l g l y ce r o l p rov ide a poor environment f o r the a c t i v i t y o f the exchanger (Vemuri and P h i l i p s o n , 1987). The s p e c i f i c observat ions desc r ibed above i n d i c a t e t ha t the in vitro a c t i v i t y o f ca l c i um t r an spo r t systems i s i n f l uenced by the phys icochemica l nature o f the membrane, a concept which i s w ide l y accepted f o r many 29 membrane-associated p r o t e i n s . I t i s t he r e f o r e l o g i c a l to p o s i t t ha t i f SR membrane ca l c i um pump f u n c t i o n i s a l t e r e d i n d i sease s t a t e s where c a r d i a c f u n c t i o n i s a f f e c t e d , SR membrane l i p i d compos i t ion should be s tud ied to determine i f i t p lays a r o l e i n the f u n c t i o n a l a l t e r a t i o n s . 1.31 Omega-3 F a t t y A c i d Nomenclature The phrase omega-3 f a t t y a c i d r e f e r s to a s p e c i f i c c l a s s o f po lyunsaturated f a t t y a c i d (PUFA). Omega-3 r e f e r s to the double bond s t r u c t u r e o f the f a t t y a c i d molecu le , such t h a t , when count ing from the methyl end o f the acy l cha in the f i r s t double bond i s p o s i t i o n e d between the t h i r d and f ou r t h carbon atoms. S i m i l a r l y , an omega-6 f a t t y a c i d i s desaturated from the s i x t h carbon atom d i s t a l to the methyl end o f the molecu le . Long cha in omega-3 f a t t y ac id s are p r i n c i p a l l y o f marine o r i g i n , en te r i n g the food cha in f o l l o w i n g s yn thes i s by zooplankton and phytoplankton (Moreno et a l , 1979). O i l i s o l a t e d from f i s h i s p a r t i c u l a r l y abundant i n the omega-3 f a t t y ac id s 5 ,8,11,14,17-e icosapentaenoic a c i d (EPA), and 4,7,10,13,16,19-docosahexaenoic a c i d (DHA). EPA and DHA (alone or i n combinat ion) are the p r i n c i p a l omega-3 f a t t y ac id s o f research i n t e r e s t . F a t t y a c i d s t r u c t u r e i s abbrev iated accord ing to the number o f carbon atoms i n the molecule (C20 imp l i e s twenty carbon atoms), the number o f double bonds (C20:5 imp l i e s f i v e double bonds i n a twenty carbon cha in) and the p o s i t i o n o f the f i r s t double bond d i s t a l to the methyl end o f the molecule (C20:5, n-3 imp l i e s an omega-3 f a t t y a c i d w i th f i v e double bonds i n a twenty carbon c h a i n ) . EPA and DHA are t h e r e f o r e abbrev ia ted as C20:5, n-3 and C22:6, n-3, r e s p e c t i v e l y . 30 1.3a Omega-3 F a t t y A d d s and Myocardia l Membrane Func t i on . The p o t e n t i a l importance o f omega-3 f a t t y ac id s i n c a r d i a c f u n c t i o n i s i n d i r e c t l y suggested by the amount of these l i p i d s i n the heart and SR. C e r t a i n omega-3 f a t t y a c i d s , p a r t i c u l a r l y DHA, comprise a s i g n i f i c a n t p r opo r t i on o f the phospho l i p id spec ies i n the hea r t . In the r a t , DHA comprises between 7.8% and 12.6% o f the t o t a l pho spho l i p i d FA compos i t ion (Swanson and K i n s e l l a , 1986; Moonfoort e t a l , 1986; Charnock e t a l , 1986). Of the v e n t r i c u l a r pho spha t i d y l cho l i ne , DHA accounts f o r 6.7%, and o f the phosphat idy lethano lamine, DHA accounts f o r 25.8% o f the FA mo ie t i e s o f these phospho l i p id s (Charnock et a l , 1986). Other omega-3 f a t t y ac id s are a l s o present i n the hea r t : EPA accounts f o r 0.1% and C22:5, n-3 accounts f o r 1.5% to 2.6% o f the t o t a l phospho l i p id FA compos i t ion (Swanson and K i n s e l l a , 1986; Moonfoort et a l , 1986; Charnock et a l , 1986). The l i p i d compos i t ion o f the SL and SR are d i f f e r e n t (as determined i n r a t s k e l e t a l muscle) as DHA accounts f o r 1.2% and 23.2% o f the pho spho l i p i d FA compos i t ion o f the SL and SR, r e s p e c t i v e l y (F iehn et a l , 1971). EPA accounts f o r approx imately the same percentage o f the t o t a l pho spho l i p i d FA compos i t ion (1.3% and 1% i n SL and SR, r e s p e c t i v e l y ) . In SL and SR, C22:5,n3 accounts f o r 3.2% and 3.5%, r e s p e c t i v e l y , o f the t o t a l pho spho l i p i d f a t t y a c i d s . A f u n c t i o n a l r o l e f o r omega-3 f a t t y ac id s i n the heart i s f u r t h e r suggested by the c o r r e l a t i o n o f i n c r ea s i n g heart r a t e i n a number o f d i f f e r e n t spec ies w i th i n c r ea s i n g c a rd i a c pho spho l i p i d DHA content (Gudbjarnason et a l , 1978). A s p e c i f i c myocardia l e f f e c t r e s u l t i n g from a d e f i c i e n c y o f omega-3 f a t t y ac id s (EPA and/or DHA) has not been i d e n t i f i e d . However, evidence f o r a p u t a t i v e r o l e o f omega-3 f a t t y ac id s i n c a r d i a c 31 f u n c t i o n comes from s tud ie s which have i n v e s t i g a t e d the e f f e c t s on va r ious c a r d i a c parameters o f omega-3 f a t t y a c i d supplementat ion (enr iched d i e t a r y i n t ake o f omega-3 f a t t y ac ids vs omega-6 f a t t y a c i d s ) . Omega-3 f a t t y ac id s have been shown to suppress/slow the development o f a t h e r o s c l e r o t i c l e s i o n s i n man and exper imental an imals . In man, d i e t a r y supplementat ion w i th omega-3 f a t t y ac id s has been shown to reduce the r a t e o f r e - s t e n o s i s f o l l o w i n g coronary a r t e r y bypass surgery (Dehmer et a l , 1988; M i l n e r et a l , 1989). In d i e t - i n d u c e d hypercho le s te ro lemia i n p i g s , omega-3 f a t t y a c i d supplementat ion i n h i b i t e d the development o f coronary a t h e r o s c l e r o s i s (Weiner et a l , 1986). The reduc t i on i n a t h e r o s c l e r o s i s occurred w i th no apparent changes i n plasma t r i g l y c e r i d e , t o t a l c h o l e s t e r o l o r l i p o p r o t e i n c oncen t r a t i on s , suggest ing tha t the a n t i - a t h e r o s c l e r o t i c e f f e c t s were independent o f the h ype r l i p i dem i c s t a t e o f the p i g . Such e f f e c t s are not observed i n a l l models however, s i n ce i n the development o f a t h e r o s c l e r o s i s due to a gene t i c de fec t i n low den s i t y l i p o p r o t e i n recepto r s ( h ype r l i p i dem i c Watanabe r a b b i t ) , s tud ie s have shown no b e n e f i c i a l e f f e c t o f omega-3 f a t t y ac id s on the development o f the a t h e r o s c l e r o t i c l e s i o n s (Clubb et a l , 1988; R ich e t a l , 1989). D i r e c t myocardia l e f f e c t s a t t r i b u t a b l e to omega-3 f a t t y a c i d treatment i n c l ude a r educ t i on i n po s t -i schemia- induced arrhythmias (McLennan et a l , 1987) and a r educ t i on i n l o s s o f myocardia l c r e a t i n e k inase f o l l o w i n g coronary a r t e r y l i g a t i o n , i n d i c a t i v e o f a p r e se r va t i on o f c e l l u l a r i n t e g r i t y (Hock et a l , 1987). Changes i n the f a t t y a c i d compos i t ion o f c e l l membranes may be a s soc i a ted w i th changes i n membrane v i s c o s i t y which i n f l u e n c e the a c t i v i t y o f membrane-bound enzymes such as the ca l c ium ATPase and membrane s i gna l t r a n s d u c t i o n systems such as the /J- receptor -adeny late c y c l a s e system 32 (Stubbs and Smith, 1984). In r a t , f o l l o w i n g fou r months o f omega-3 f a t t y a c i d supplementat ion the spontaneous r a t e , c o n t r a c t i l e f o r c e and cAMP l e v e l o f i s o l a t e d a t r i a are reduced ( L a u s t i o l a et a l , 1986). Concomitant w i th these changes was a s i g n i f i c a n t i nc rease i n a t r i a l DHA and EPA i n both PC and PE. The inc rease i n cAMP a f t e r no radrena l i ne s t i m u l a t i o n was g rea te r i n the a t r i a from the omega-3 supplemented group. Th i s may i n d i c a t e an increased respons iveness o f adenylate c y c l a se i n a t r i a f o l l o w i n g omega-3 supplementat ion. The i n t r i n s i c FA compos i t ion of r a t heart phospho l i p id s has been shown to be i n f l uenced by omega-3 f a t t y a c i d supplementat ion. On an abso lute b a s i s , the g rea te s t change i s i n the content o f DHA, as an approximate doub l ing o f t h i s f a t t y a c i d has been shown to occur (Swanson and K i n s e l l a , 1986; Montfoort et a l , 1986; Charnock et a l , 1986). EPA content inc reased from 0.1% or l e s s to between 1.3% and 4.8%. Increased omega-3 FA content o f r a t heart phospho l ip id s g e n e r a l l y occurs at the expense of AA (Montfoort et a l , 1986; Charnock et a l , 1986). The degree of un sa tu ra t i on o f the f a t t y acy l cha in o f a phospho l i p id molecule i n f l uence s the phys icochemical nature o f the membrane. As the degree of pho spho l i p i d un sa tu ra t i on i n c rea se s , the v i s c o s i t y of the membrane decreases (Stubbs and Smith, 1984). Fo l l ow ing omega-3 f a t t y a c i d t reatment, the omega-6:omega-3 r a t i o o f r a t heart phospho l i p id f a t t y ac ids decreases (Charnock e t a l , 1986), and the unsa tu ra t i on index increases (Swanson and K i n s e l l a , 1986). S ince phospho l i p id s are p r i m a r i l y components o f membranes, these r e s u l t s suggest a change i n membrane v i s c o s i t y o f SL, SR and/or o ther membrane bound o r g a n e l l e s . 33 The above s tud i e s have demonstrated tha t omega-3 f a t t y ac id s present i n the heart may i n f l u e n c e c a r d i a c f u n c t i o n under c e r t a i n c l i n i c a l and exper imental c o n d i t i o n s . Card iac f u n c t i o n may be a f f e c t e d through changes i n a t h e r o s c l e r o t i c d i s ea se , through changes i n the r e l a t i v e amount o f omega-3 f a t t y ac id s i n t i s s u e pho spho l i p i d s , and/or through changes i n b iochemica l processes dependent ( to a degree) on membrane l i p i d compos i t i on . Omega-3 f a t t y ac id s may t he r e f o r e p lay a r o l e i n myocardia l r e g u l a t i o n i n c e r t a i n d i sease s t a t e s . 1.3b A c y l c a r n i t i n e and Myocardia l Membrane Func t i on . The pr imary metabo l i c r o l e of a c y l c a r n i t i n e , an i n te rmed ia ry i n f a t t y a c i d metabol ism, i s to s p e c i f i c a l l y serve as a t r a n s p o r t a b l e form of long cha in f a t t y ac id s i n t o the m i tochondr i a l mat r i x f o r subsequent ^ - o x i d a t i o n (Norum and Bremer, 1963). Long cha in f a t t y ac ids en t e r i n g the c a r d i a c myocyte are i n i t i a l l y " a c t i v a t e d " by an ATP dependent process c a t a l y z e d by acyl-coenzyme (CoA) synthetase, y i e l d i n g acyl -CoA t h i o e s t e r s . Acy l CoA i s the sub s t r a te f o r fl o x i d a t i o n but i s t r an spo r ted extremely s l ow l y ( i f at a l l ) across the inner m i tochondr i a l membrane. T r a n s l o c a t i o n i n t o the mi tochondr ia i s t he r e f o r e accomplished by a t r a n s a c y l a t i o n r e a c t i o n c a t a l y zed by c a r n i t i n e p a l m i t o l y t r a n s f e r a s e I ( oute r ) (CPT ! ) • CPT I c a t a l y z e s the format ion of a c y l c a r n i t i n e ( long cha in acy l c a r n i t i n e : LCAC) and f r e e CoA. LCAC thus formed crosses the inner m i tochondr i a l membrane through coun te r - t r an spo r t w i th f r ee c a r n i t i n e l o c a t e d on the lumenal su r face o f the inner m i tochondr i a l membrane. Coun te r - t r an spo r t i s f a c i l i t a t e d by a c a r n i t i n e t r an s l o ca se p r o t e i n spanning the inner m i tochondr i a l membrane. C a r n i t i n e p a l m i t o l y t r a n s f e r a s e 11 ( i nnev~) ( c p T I I ) , 34 o r i e n t e d to the lumenal s i de of the inner m i tochondr i a l membrane, c a t a l y z e s the rever se a c y l a t i o n r e a c t i o n y i e l d i n g f r e e c a r n i t i n e and acy l -CoA. C a r n i t i n e w i t h i n the mi tochondr i a l mat r i x i s removed through the c a r n i t i n e t r a n s l o c a s e , such tha t as acy l c a r n i t i n e enter s the m a t r i x , f r e e c a r n i t i n e l eave s . Myocard ia l a c y l c a r n i t i n e has a l so been shown to exe r t o ther r e g u l a t o r y and/or p a t ho l o g i c a l e f f e c t s upon the hea r t . The p o t e n t i a l r e g u l a t o r y r o l e o f a c y l c a r n i t i n e i n c a r d i a c or c a r d i a c s u b c e l l u l a r f u n c t i o n was i n i t i a l l y suggested by Wood et a l (1977) who demonstrated tha t exogenous p a l m i t o y l c a r n i t i n e was a potent (IC50 « 45 /xM) i n h i b i t o r o f bovine c a r d i a c Na + /K + -ATPase. Exogenous p a l m i t o y l c a r n i t i n e a l s o i n h i b i t s canine c a r d i a c sarcop lasmic r e t i c u l u m ca l c ium ATPase a c t i v i t y (IC50 * 20 /xM) and ca l c i um t r an spo r t a c t i v i t y (complete i n h i b i t i o n at 10 /iM; P i t t s et a l , 1978). P a l m i t o l y c a r n i t i n e i n h i b i t i o n o f c a r d i a c Na + ,K + -ATPase a c t i v i t y has been conf i rmed (Adams e t a l , 1979; P i t t s and Okhuysen, 1984) as has p a l m i t o l y c a r n i t i n e i n h i b i t i o n of canine (Adams e t a l , 1979) and r a t (Lopaschuk et a l , 1983a) c a r d i a c sarcop lasmic r e t i c u l u m ca l c i um ATPase and ca l c ium t r a n s p o r t . I n h i b i t i o n of c a r d i a c sarcolemmal and sarcop lasmic r e t i c u l u m ion t r a n s p o r t i n g enzymes by p a l m i t o y l c a r n i t i n e appears to be b i p h a s i c , w i t h i n i t i a l s t i m u l a t i o n at 5-50 itM and i n h i b i t i o n at 50-200 /iM (Adams et a l , 1979, P i t t s and Okhuysen, 1984). These e f f e c t s are c o n s i s t e n t w i th i n s e r t i o n i n t o the membrane o f the l i p i d s o l ub l e acy l cha in o f p a l m i t o y l c a r n i t i n e w i th subsequent d i s r u p t i o n o f the membrane. Mechanisms f o r the p u t a t i v e i n h i b i t o r y e f f e c t s of the a c y l c a r n i t i n e have been con s ide red . The cu r ren t consensus i s t ha t these metabo l i c i n te rmed ia te s i n h i b i t c e l l u l a r membrane f u n c t i o n through a membrane 35 pe r tu rb i ng e f f e c t , a c t i n g as detergents to d i s r u p t the membrane. P a l m i t o y l c a r n i t i n e causes a dose-dependent i nc rease i n the f l uo re scence i n t e n s i t y o f a l i p i d - b o u n d f l u o r e s c e n t probe i n sa rcop lasmic r e t i c u l u m or sarcolemmal membranes, i n d i c a t i n g a membrane pe r tu rb i ng e f f e c t (Adams et a l , 1979). S ince both sarcolemmal Na + /K + -ATPase a n c | sa rcop lasmic r e t i c u l u m ca l c ium ATPase/transport demonstrate a l i p i d dependency, a d i s r u p t i v e detergent mechanism i s p l a u s i b l e . Myocard ia l a c y l c a r n i t i n e s accumulate i n the heart dur ing i schemia ( I de l l -Wenger ,e t a l ,1978; L i e d t k e , et a l , 1978) and, through membrane p e r t u r b a t i o n e f f e c t s , d i s t u r b ion f l u x e s i n the heart which may c o n t r i b u t e to the po s t - i s chemic d y s f u n c t i o n . A c y l c a r n i t i n e e f f e c t s on the myocyte are not r e s t r i c t e d to i n t e r a c t i o n s w i th i o n - t r a n s p o r t i n g pumps. A c y l c a r n i t i n e s a l s o reduce the maximal r a t e o f r i s e of a c t i o n p o t e n t i a l s , i nc rease a c t i o n p o t e n t i a l d u r a t i on o f v e n t r i c u l a r muscle (Inoue and Pappano, 1983) and a c t i v a t e vo l tage -opera ted ca l c ium channels i n smooth muscle p repa ra t i on s (Spedding and M i r , 1987). The l a t t e r , channel -modulat ing e f f e c t s o f a c y l c a r n i t i n e were cons idered to be independent o f membrane p e r t u r b a t i o n and a d i r e c t e f f e c t upon the channel (Spedding and M i r , 1987). The above e l e c t r o p h y s i o l o g i c a l e f f e c t s o f exogenous a c y l c a r n i t i n e are r e l e van t to the p u t a t i v e e f f e c t s o f endogenous a c y l c a r n i t i n e as shown by Knabb et a l (1986). Sub jec t i ng c u l t u r e d r a t myocytes to a hypoxic pe r i od inc reases endogenous sarcolemmal a c y l c a r n i t i n e 7 0 - f o l d and produces d i s t i n c t e l e c t o p h y s i o l o g i c a l d i s tu rbances (e .g . decreased maximum d i a s t o l i c p o t e n t i a l and maximum upstroke v e l o c i t y ) . Accumulat ion o f a c y l c a r n i t i n e i n the sarcolemma and the e l e c t r o p h y s i o l o g i c a l d i s tu rbances induced by the hypoxic pe r i od are prevented by i n h i b i t i o n o f CPT by POCA, a s p e c i f i c 36 i n h i b i t o r o f CPT (Knabb et a l , 1986). Fo l l ow ing an i schemic p e r i o d , there i s an i nc rease i n myocardia l a i - r e c e p t o r s which may c o n t r i b u t e to i s chemia -induced arrhythmias (Sheridan et a l , 1980). Hypoxia- induced accumulat ion o f a c y l c a r n i t i n e i n the sarcolemma i s c o i n c i d e n t w i th an i nc rease i n a i - a d r e n e r g i c r ecep to r number (Heathers et a l , 1987). That a c y l c a r n i t i n e causes the increased a j - a d r e n e r g i c r ecep to r number i s ev inced by data showing t ha t i n h i b i t i o n of i schemia- induced a c y l c a r n i t i n e v i a CPT i n h i b i t i o n prevents the increase i n sarcolemmal a i - a d r e n e r g i c recepto r s (Heathers et a l , 1987). A c y l c a r n i t i n e a l s o accumulates i n the heart i n d i abetes (Feuvray et a l , 1979) and increased endogenous a c y l c a r n i t i n e of r a t c a r d i a c sarcop lasmic r e t i c u l u m i s c o r r e l a t e d w i th impaired ca l c i um t r an spo r t a c t i v i t y (Lopaschuk et a l , 1983a). I t has a l s o been shown tha t i n exper imenta l hyper thyro id i sm increased ca l c i um t r an spo r t a c t i v i t y i s we l l c o r r e l a t e d w i th a reduc t i on i n the l e v e l o f endogenous a c y l c a r n i t i n e a s soc i a ted w i t h the sarcop lasmic r e t i c u l u m membrane (B lack et a l , 1988). The s t ud i e s c i t e d i n d i c a t e tha t a c y l c a r n i t i n e s may a f f e c t myocardia l f u n c t i o n through pe r t u rba t i on o f c e l l membranes and thereby cause i n h i b i t i o n of s p e c i f i c ion t r a n s p o r t i n g ATPases, e l e c t r o p h y s i o l o g i c a l d i s tu rbances and i n c r ea s i n g a i - a d r e n e r g i c r ecep to r number. A d d i t i o n a l p o t e n t i a l r e g u l a t o r y / p a t h o l o g i c a l e f f e c t s o f a c y l c a r n i t i n e s i n c l ude an i n h i b i t i o n o f c a r d i a c p h o s p h o l i p i d - s e n s i t i v e calc ium-dependent k inase ( p o s s i b l y p r o t e i n k inase C)-mediated phosphory la t ion o f endogenous myocardia l s ub s t ra te s (Katoh et a l , 1981) through compet i t i v e i n h i b i t i o n w i t h pho spha t i d y l s e r i ne , a phospho l i p id c o f a c t o r o f the enzyme (Wise and Kuo, 1983). A c y l c a r n i t i n e a l s o i n h i b i t s p r o t e i n k inase C a c t i v i t y o f i n t a c t r a t p anc rea t i c a c i n i (Brockenbrough and Korc, 1987) and i n h i b i t s 12-37 O-te t radecanoy lphorbo l -13 -aceta te (TPA)-induced blockade o f gap j u n c t i o n a l i n t e r c e l l u l a r communication i n WB-F344 c e l l s ( r a t e p i t h e l i a l c e l l s , Oh et a l , 1988). Although a c y l c a r n i t i n e does not a f f e c t the a c t i v i t y o f c a r d i a c c y c l i c AMP-dependent or c y c l i c GMP-dependent p r o t e i n k inases (Katoh e t a l , 1981) the l i p i d metabo l i c i n te rmed ia te has been shown to i n h i b i t C a 2 + -independent phosphodiesterase and s t imu l a t e Ca 2 + -dependent phosphodiesterase from bovine heart (Katoh et a l , 1982). The above l i t e r a t u r e rev iew c l e a r l y i n d i c a t e s t ha t i n a d d i t i o n to i t s pr imary r o l e as a t r an spo r t ab l e form of long cha in f a t t y a c i d s , a c y l c a r n i t i n e may a l s o p lay a r o l e i n myocardia l r e g u l a t i o n i n c e r t a i n d i sease s t a t e s . 1.4 Membrane A l t e r a t i o n s i n Diabetes M e l l i t u s Although d iabetes m e l l i t u s i s p r i m a r i l y cons idered a d i sease o f carbohydrate, l i p i d and p r o t e i n metabolism ( c a t a b o l i c and anabo l i c changes) there i s c on s i de rab l e evidence f o r impaired f a t t y a c i d de s a tu r a t i on i n both human and animal models. E r y th rocy te membrane phospho l ip id s o f IDDM human sub jec t s have a reduced po lyunsaturated f a t t y a c i d compos i t ion and a decreased po lyunsaturated to sa tu ra ted (P/S) f a t t y a c i d r a t i o (Kamada and O t s u j i , 1983; Kamada et a l , 1986; T i l v i s and M i e t t i n e n , 1985). Reduced e r y t h r o c y te membrane po lyunsaturated f a t t y ac ids and P/S r a t i o have been i m p l i c a t e d i n the increased membrane v i s c o s i t y o f e r y t h r ocy te s from d i a b e t i c s (Kamada and O t s u j i , 1983). The s tud i e s o f Kamada and O t s u j i (1983) i n d i c a t e d a s i g n i f i c a n t l y h igher C16:0 and lower C22:6,n-3 (docosahexaenoic acid,DHA) content o f e r y th rocy te membranes. Reduced C22:6, n-3 has a l so been repor ted i n p l a t e l e t s o f d i a b e t i c s ( P r i s c o e t a l , 38 1989). P l a t e l e t s from poor l y c o n t r o l l e d d i a b e t i c sub jec t s demonstrated an inc reased l i p i d s t r u c t u r a l order and a decreased membrane f l u i d i t y (Winocour e t a l , 1990). The e f f e c t of d iabetes on membrane pho spho l i p i d f a t t y a c i d compos i t ion i s somewhat e q u i v o c a l , however, s i n ce i t has been repor ted t h a t , i n poor l y c o n t r o l l e d IDDM p a t i e n t s , e r y t h r o c y t e membrane pho spho l i p i d compos i t ion i s not a l t e r e d (Freyburger e t a l , 1989). In a l l o x a n - and s t r ep to zoc i n - i nduced d iabetes i n the r a t , changes i n pho spho l i p i d f a t t y a c i d compos i t ion have been desc r ibed f o r hea r t , l i v e r , k idney, a o r t a , p l a t e l e t s and serum (Holman et a l , 1983; Faas and C a r t e r , 1980; Gudbjarnason et a l , 1987; Kanazaki e t a l , 1987; Dang et a l , 1988). The e f f e c t s o f chemica l l y - i nduced d iabetes on membrane pho spho l i p i d f a t t y a c i d compos i t ion resemble the changes seen i n e r y t h r ocy te s o f human d i a b e t i c s . Increased l i n o l e i c a c i d (C18:2, n-6) and DHA, and decreased a rach idon i c a c i d (AA, C20:4, n-6), p a l m i t o l e i c and o l e i c ac id s have been repor ted f o r the organs and t i s s u e s desc r ibed above. Increased DHA and reduced AA content o f t i s s u e phospho l ip id s are r e l a t i v e changes. When pho spho l i p i d acy l compos i t ion i s cons idered on an abso lute bas i s a l l po lyunsatura tes o f d i a b e t i c s are reduced (both omega-6 and omega-3 c l a s s e s ) . To ta l po lyunsaturates f a l l from 13.12 ± 1.16 mg PUFA/g t i s s u e i n non -d i abe t i c c o n t r o l s to 6.33 ± 0.75 mg PUFA/g t i s s u e i n d i a b e t i c s (Holman et a l , 1983). The changes i n phospho l i p id f a t t y a c i d compos i t ion i n d i abe te s have been a s c r i bed to reduced a c t i v i t y o f the enzymes r e spon s i b l e f o r de sa tu r a t i on o f the f a t t y a c i d s . De l ta -9 desaturase a c t i v i t y o f d i a b e t i c r a t s , r e spon s i b l e f o r conve r t i ng sa tu ra ted f a t t y ac id s to monounsaturated f a t t y a c i d s , has been repor ted to be 22% of non -d i abe t i c c o n t r o l a c t i v i t y 39 (Eck et a l , 1979). De l ta -6 desaturase a c t i v i t y o f d i a b e t i c r a t s , r e spon s i b l e f o r conve r t i ng l i n o l e i c a c i d (C18:2,n-6) to 7 - l i n o l e n i c a c i d (C18:3,n-6) , the r a t e l i m i t i n g step i n the synthes i s o f AA, i s 64% o f non-d i a b e t i c c o n t r o l s i n s t r ep tozoc i n - i nduced d iabetes (Eck e t a l , 1979). A d d i t i o n a l l y , d e l t a - 5 desaturase a c t i v i t y , r e spon s i b l e f o r conve r t i ng d ihomo-Y -1 ino len i c a c i d (C20:3,n-6) to AA, i s suppressed i n d iabetes (Holman et a l , 1983). The d e l t a - 9 , - 6 , - 5 desaturase enzymes a l s o convert omega-3 f a t t y a c i d s , such tha t a - l i n o l e n i c a c i d (C18:3,n-3) i s metabo l i zed to EPA (C20:5, n-3) . The f i n a l convers ion o f EPA to DHA i s mediated v i a e l onga t i on and a d e l t a - 4 desaturase. The a l t e r e d desaturase a c t i v i t i e s are c o n s i s t e n t w i t h a l t e r e d membrane phospho l i p id f a t t y a c i d compos i t ion seen i n d i abe te s , and i t i s t he r e f o r e apparent t ha t f a t t y a c i d de s a tu r a t i on i s a f f e c t e d by d i abe te s , l ead ing to a general d e f i c i e n c y o f omega-6 and omega-3 f a t t y a c i d s . A l t e r e d pho spho l i p i d acy l compos i t ion may account f o r the reduced e r y t h r o c y t e membrane (Kamada and O t i s u j i , 1983) and p l a t e l e t membrane (Winocour e t a l , 1980) f l u i d i t y desc r ibed i n d i a b e t i c s u b j e c t s . That d i a b e t e s - r e l a t e d membrane pe r tu rba t i on s are amenable to omega-3 f a t t y a c i d treatment has been shown i n s tud ie s where the reduced e r y t h r o c y t e membrane f l u i d i t y o f d i a b e t i c sub jec t s (Kamada and O t i s u j i , 1983) was r e v e r s i b l e f o l l o w i n g d i e t a r y sa rd ine o i l supplementation (Kamada et a l , 1986). 1.5 Membrane A l t e r a t i o n s i n Hypothyro id i sm. The a c t i o n of t h y r o i d hormone upon l i p i d metabolism may be cons idered p l e i o t r o p i c s i n ce severa l d i f f e r e n t enzymes and d i f f e r e n t steps o f va r i ous pathways of l i p i d metabolism are a f f e c t e d by e i t h e r t h y r o i d hormone 40 d e f i c i e n c y or excess . I t has been suggested t ha t the p l e i o t r o p i s m o f t h y r o i d hormone a c t i o n may be p a r t l y a t t r i b u t a b l e to the i n i t i a l i n d u c t i o n , v i a nuc lea r receptor -med iated events, o f l i p o g e n i c enzymes and p o s s i b l y mod i f i e r s o f these enzymes, r e s u l t i n g i n membrane l i p i d compos i t iona l changes which mediate processes tha t u n d e r l i e the d i v e r s e spectrum of t h y r o i d hormone a c t i o n (Hoch, 1988). Enzymes i nvo l ved i n the de sa tu r a t i on o f f a t t y a c i d s , and hence the mode l l i ng o f membrane pho spho l i p i d compos i t i on , are known to be a f f e c t e d by the t h y r o i d s t a t e (Hoch, 1988). Card iac SR membrane phospho l i p id acy l compos i t ion has not been e x t e n s i v e l y i n v e s t i g a t e d i n hypothyro id i sm; however, i n the hype r thy ro id s t a t e c a r d i a c SR changes i n c l ude increases i n t o t a l phospho l i p id s and i n pho spha t i d y l cho l i ne C16:0, C18:0 and C22:5,n-6 and decreases i n C18:2,n-6 and C20:4,n-6 (Limas, 1978). S ince SR ca l c i um pump a c t i v i t y i s dependent upon the phys icochemical nature o f the l i p i d environment i n which i t i s l o ca ted (Stubbs and Smith, 1984), these l i p i d a l t e r a t i o n s may c o n t r i b u t e to the increased SR ca l c ium t r an spo r t a c t i v i t y observed i n the hyper thy ro id s t a t e . In hypothyro id i sm, the a c t i v i t y o f enzymes mediat ing pho spho l i p i d f a t t y a c i d de s a tu r a t i on are reduced (Faas and C a r t e r , 1982). A l t e r a t i o n s i n the f a t t y acy l compos i t ion o f s k e l e t a l muscle SR PC and PE have been shown and suggested to p lay a r o l e i n the a l t e r e d ca l c i um t r an spo r t a c t i v i t y o f s k e l e t a l muscle SR (Simonides and van Hardeveld, 1987). The l i p i d compos i t ion of the SR membrane i n hypothyro id i sm may a l s o i n f l u e n c e C a 2 + - t r a n s p o r t by the presence o f endogenous a c y l c a r n i t i n e a s soc i a ted w i th the SR. As desc r ibed above, long cha in a c y l c a r n i t i n e s are potent i n h i b i t o r s o f SR ca l c ium t r an spo r t and ATPase a c t i v i t i e s . In the hyper thy ro id s t a t e i t has been shown tha t the l e v e l o f endogenous 41 acylcarnitine associated with cardiac SR is reduced concomitant with increased calcium transport act iv i ty (Black et a l , 1988). Those studies suggested the poss ib i l i ty that there may be a reduction in endogenous (tonic) inhibit ion of the SR calcium pump in the hyperthyroid state by removal of acylcarnitine from the SR membrane. The potential role of endogenous myocardial acylcarnitine in influencing previously described reductions in SR calcium transport has not been investigated. Lipid metabolism is influenced by the thyroid state such that in hypothyroidism the rate of palmitate and oleate oxidation is reduced (Stakkestad and Bremer, 1983). Fatty acid ester i f icat ion to acylcarnitines and phospholipid is also reduced in hypothyroidism; however, the ratio of ester i f ied to oxidized l i p i d is considerably increased in the hypothyroid state (Stakkestad and Bremer, 1983). Therefore, since the ratio of ester i f ied to oxidized l i p i d is increased approximately three-fold in hypothyroidism, the amount or distribution of myocardial acylcarnitine into the SR membrane may be affected by such l i p i d metabolic changes. 1.6 Purpose of the Study and Approach to the Problem. The overall objective of the study was to investigate mechanisms whereby cardiac SR calcium transport act iv i ty may be influenced by changes in the l i p i d environment of the SR membrane. Two different endocrine disease states, hypothyroidism and diabetes mellitus, were employed. The hypothyroid state was employed to determine i f SR calcium transport act iv i ty is influenced by endogenous acylcarnitine associated with the SR membrane and the diabetic model to determine i f SR phospholipid acyl 42 compos i t ion p lays a r o l e i n d i abetes - i nduced cardiomyopathy. The e f f e c t s o f endogenous a c y l c a r n i t i n e s on c a r d i a c SR ca l c i um t r an spo r t were of i n t e r e s t s i n ce these l i p i d metabo l i c i n te rmed ia tes may f u n c t i o n as endogenous r e g u l a t o r s o f c a rd i a c sarcop lasmic r e t i c u l u m f u n c t i o n . Omega-3 f a t t y ac id s have been shown to a f f e c t va r i ous parameters r e l e v a n t to d i abe te s - i nduced cardiomyopathy; t h e r e f o r e , i t was o f i n t e r e s t to determine the c a r d i a c e f f e c t s of omega-3 f a t t y a c i d treatment o f STZ-induced d i a b e t i c an imal s . The approach taken to a t t a i n the o v e r a l l o b j e c t i v e s i s o u t l i n e d on the f o l l o w i n g pages. 43 1.6a Ob jec t i ve s of S tud ies on the E f f e c t s of Thyroidectomy on Ca rd iac SR Calc ium Transport A c t i v i t y and the Role of Endogenous A c y l c a r n i t i n e . 1) Determine i f SR ca l c i um t r an spo r t a c t i v i t y i s i n f l uenced by removal o f the t h y r o i d g land and, i f ca l c i um t r an spo r t a c t i v i t y i s a f f e c t e d , to determine the time po in t f o l l o w i n g thyro idectomy when SR ca l c i um t r an spo r t i s a f f e c t e d . 2) To determine the mechanism re spon s i b l e f o r a l t e r e d SR ca l c i um t r an spo r t a c t i v i t y by i n v e s t i g a t i n g : i ) the l e v e l of a c y l c a r n i t i n e a s soc ia ted w i t h the i s o l a t e d SR membrane p r e p a r a t i o n . Th i s approach w i l l be taken to determine i f the endogenous l e v e l of these l i p i d metabo l i c i n te rmed ia te s i s r e l a t e d to SR ca l c i um t r an spo r t a c t i v i t y . The r e l a t i o n s h i p between SR ca l c i um t r an spo r t a c t i v i t y and the l e v e l o f SR a c y l c a r n i t i n e w i l l be determined i n eu thy ro id and thy ro idec tomized animals . i i ) the e f f e c t of thyro idectomy on SR acy lphosphoprote in l e v e l s i n eu thy ro i d and thy ro idectomized animals to determine i f the acy lphosphoprote in ( p u t a t i v e l y SR ca l c i um pump p r o t e i n ) l e v e l i s a f f e c t e d at t ime po in t s when ca l c i um t r an spo r t may be i n f l u e n c e d . 44 1.6b Objectives of Studies on the Effects of Omega-3 Fatty acid Treatment of Streptozocin-Induced Diabetic Cardiomyopathy. 1) To determine the e f f e c t s o f omega-3 f a t t y a c i d on plasma l i p i d parameters cons idered to be important i n the development o f long term comp l i ca t i on s o f d iabetes m e l l i t u s . 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 l e v e l s w i l l be determined. 2) To determine i f treatment of STZ-induced d i a b e t i c animals w i t h an omega-3 f a t t y a c i d p repa ra t i on a f f e c t s the development o f the d i a b e t e s -a s soc i a ted cardiomyopathy. Card iac f u n c t i o n w i l l be s t ud i ed us ing the i s o l a t e d working heart apparatus. LVDP, + d P / d t , "dP/dt , a o r t i c f l ow and coronary f l ow w i l l be monitored. 3) To determine the e f f e c t o f omega-3 f a t t y a c i d treatment on i s o l a t e d c a r d i a c SR ca l c i um t r an spo r t a c t i v i t y . 4) To determine the e f f e c t of the omega-3 f a t t y a c i d treatment on c a r d i a c and SR pho spho l i p i d acy l compos i t ion and SR membrane c h o l e s t e r o l l e v e l s . 45 2 METHODS 2.1 S tud ies on the Thyro idectomized Wis ta r Rat 2.1a Animal Model and Study P r o t o c o l . Male Wi s ta r r a t s used i n a l l experiments were obta ined from Char les R i ve r l a b o r a t o r i e s , Montreal P.Q.. Two groups o f animals were ob ta i ned : a c o n t r o l , eu thy ro id group and a thy ro idectomized group. The animals i n the l a t t e r group underwent s u r g i c a l thyro idectomy at the Char les R i ve r Laboratory f a c i l i t i e s . The eu thy ro id c on t r o l group were age and sex matched. Fo l l ow ing surgery the animals were a l lowed to recover f o r a pe r i od o f one week p r i o r to being shipped to Vancouver. Upon a r r i v a l the animals were housed i n the Facu l t y o f Pharmaceutical Sc iences animal care f a c i l i t i e s . A l l r a t s were a l lowed access to food and water ad libitum, were kept at 21°c and were on a twelve hour l i g h t / d a r k c y c l e . The e f f e c t o f thyroidectomy was s tud ied as a t ime course over a pe r i od o f e i gh t weeks. Animals from both the eu thy ro i d and thy ro idec tomized groups were s tud ied two, f ou r , s i x and e i gh t weeks f o l l o w i n g thyro idectomy. At each t ime p o i n t , the f o l l o w i n g p ro toco l was employed. Animals were k i l l e d by a blow to the head and c e r v i c a l d i s l o c a t i o n . The heart was immediately and r a p i d l y e x c i s e d , and a blood sample ob ta i ned . Card iac sarcop lasmic r e t i c u l u m (SR) was immediately prepared as desc r ibed below. On the day subsequent to SR i s o l a t i o n , SR ca l c i um t r an spo r t a c t i v i t y was determined. Two to th ree days subsequent to the SR ca l c i um t r an spo r t assay, SR c a r n i t i n e l e v e l s were determined as de sc r i bed below. In a separate s e r i e s of exper iments, us ing the i d e n t i c a l 46 study p r o t o c o l , SR was prepared and calcium-dependent acylphosphate l e v e l s determined as de sc r i bed below. 2.1b I s o l a t i o n o f Ca rd iac Sarcoplasmic Ret i cu lum. The heart was r a p i d l y exc i sed and p laced i n i c e c o l d i s o l a t i o n Medium I (10 mM NaHC03, 4 mM d i t h i o t h r e i t o l , pH 7.4) and trimmed o f p e r i c a r d i a l f a t , a o r t i c and pulmonary remnants and a t r i a . The v e n t r i c u l a r t i s s u e was r a p i d l y b l o t t e d dry and weighed. Microsomes enr i ched i n sarcop lasmic r e t i c u l u m (SR) were immediately prepared from f r e sh t i s s u e . V e n t r i c u l a r t i s s u e (600-900 mg, wet weight) was r a p i d l y minced w i t h s c i s s o r s under 15 mL i c e c o l d medium I f o l l owed by two 10 second homogenizations separated by a 5 second pause us ing a K inemat ica^ v a r i a b l e speed homogenizer w i t h a Brinkman PT-10 homogenizer b lade. The homogenate was d i l u t e d to 25 mL w i th i c e c o l d homogenization b u f f e r (Medium I ) , and c e n t r i f u g e d at 500 x g f o r 15 minutes ( a l l c e n t r i f u g a t i o n at 4°c) us ing a Beckmann J2-21 c e n t r i f u g e and a JA-20 r o t o r (the Beckman J2-21 c e n t r i f u g e and JA-20 r o t o r were used throughout the p r e p a r a t i o n ) . The r e s u l t i n g p e l l e t was d i s ca rded and the supernatant c e n t r i f u g e d at 7,000 x g f o r 15 minutes. The p e l l e t was again d i s ca rded and the supernatant c en t r i f u ged at 31,000 x g f o r 30 minutes. The r e s u l t i n g p e l l e t was resuspended i n 12 mL o f i c e c o l d Medium II (0.6 M KCL, 4 mM d i t h i o t h r e i t o l , 30 mM h i s t i d i n e c h l o r i d e , pH 7.0) us ing a precoo led Po t te r -E l ve jehm homogenizer, and c e n t r i f u g e d at 31,000 x g f o r 30 minutes. The f i n a l p e l l e t was gen t l y resuspended i n i c e c o l d Medium I I I (0.75 mL o f 0.25 M sucrose, 0.3 M KC1 and 0.1 M T r i s - c h l o r i d e , pH 7.2) , us ing a 1 mL c apac i t y precooled Po t te r -E l ve jehm homogenizer. The SR 47 enr iched suspension was immediately f rozen i n l i q u i d n i t r ogen and s to red at -80 °C p r i o r to use. 2.1c Determination of Cardiac Sarcoplasmic Reticulum Calcium Transport Act iv i ty. SR ca l c i um t r an spo r t a c t i v i t y was determined by a f i l t r a t i o n techn ique : The SR (10 ug) was pre incubated f o r 3 minutes at the r e a c t i o n temperature of 30 °C i n a medium c o n s i s t i n g of ( i n mM): h i s t i d i n e c h l o r i d e , 40, pH 6.8; KC1, 110; MgCl2, 5; NaN3 5; T r i s - o x a l a t e , 2.5; T r i s -ATP 5. The r e a c t i o n was i n i t i a t e d by the a d d i t i o n o f EGTA-buffered ca l c i um ( y i e l d i n g f r e e ca l c i um concent ra t i on s ranging from 0.1 /iM to 5.3 /iM, from 63 nmoles t o t a l added c a l c i um, con ta i n i n g 4 5 C a C l 2 (200,000 dpm/tube) to monitor ca l c i um t r a n s p o r t ) . Free ca l c ium concent ra t i on s were c a l c u l a t e d us ing a FORTRAN program, TCATI0NS.BC, mod i f ied s l i g h t l y from Go l d s t e i n (1979). The t r an spo r t r e a c t i o n proceeded f o r 3 minutes and was terminated by f i l t r a t i o n of an a l i q u o t o f the r e a c t i o n mixture through a M i l l i p o r e R HA 0.45 /i f i l t e r . The f i l t e r was washed once w i th 15 mL o f 40 mM T r i s - c h l o r i d e , pH 7.2, to remove low a f f i n i t y , n o n - s p e c i f i c bound c a l c i um. A f t e r d r y i n g , the f i l t e r s were counted f o r r a d i o a c t i v i t y i n 5 mL l i q u i d s c i n t i l l a t i o n f l u i d . Calc ium t r an spo r t a c t i v i t y was determined accord ing to the f o l l o w i n g fo rmu la : Sample counts - Background x D.F. x Tota l ca l c i um = nmoles/mg/min Tota l counts - Background R.T. mg SR p r o t e i n where, Sample counts = cpm of i n d i v i d u a l sample f i l t e r Tota l counts = t o t a l added cpm f o r i n d i v i d u a l [Ca^ ] f 48 Background = cpm of l i q u i d s c i n t i l l a t i o n f l u i d a lone D.F. = d i l u t i o n f a c t o r to account f o r volume o f assay f i l t e r e d / c o u n t e d R.T. = r e a c t i o n t ime ( three minutes) To ta l ca l c i um = t o t a l added ca l c i um (63 nmoles) mg SR p r o t e i n = amount SR p r o t e i n assayed ( u s ua l l y 10 /ig) 2.ld Determination of Cardiac Sarcoplasmic Reticulum Acylphosphate Levels. The SR was i n i t i a l l y pre incubated f o r 10 minutes at 10 °C. The phosphory la t ion r e a c t i o n was i n i t i a t e d by the a d d i t i o n o f SR (10 - 20 fig) to a r e a c t i o n medium (precooled to 10 °C) c on ta i n i n g 40 mM h i s t i d i n e c h l o r i d e , pH 6.8, 10 /iM MgCl2, 100 /iM EGTA and 2.0 /iM T r i s -ATP ( con ta i n i n g •so J t P - A T P , 2000 dpm/pmole). The r e a c t i o n proceeded f o r 20 seconds and was terminated by the a d d i t i o n of 0.4 mL o f a s o l u t i o n c on t a i n i n g 5% w/v t r i c h l o r o a c e t i c a c i d , 5 mM Na2ATP and 2 mM KH2PO4, and immediately p laced i n an i c e bath . An a l i q u o t o f the r e a c t i o n mixture was f i l t e r e d through a Whatman GF/C f i l t e r , washed tw ice w i th 5 mL 5% w/v t r i c h l o r o a c e t i c a c i d . The f i l t e r s were dryed and counted. Calcium-dependent acylphosphate l e v e l s were c a l c u l a t e d accord ing to the fo rmula : S. C. - B. C. x R.V. x [ P r o te i n ] = fmol/mg S.A. Where, S.C. = sample counts, cpm from i n d i v i d u a l ^ Z P con t a i n i n g f i l t e r s B.C. = background counts , cpm from l i q u i d s c i n t i l l a t i o n f l u i d a lone S.A. = s p e c i f i c a c t i v i t y = (medium counts - B.G.)/Total ATP 49 R.V. = r e a c t i o n volume [ P r o t e i n ] = p r o t e i n c oncen t r a t i o n , mg SR/mL 2.1e Hydroxylamine Treatment o f SR Acy lphosphoprote in . The SR was phosphory lated as desc r ibed above and the r e a c t i o n stopped by the a d d i t i o n o f 0.4 mL o f i c e c o l d 15% w/v t r i c h l o r o a c e t i c a c i d and p l a c i n g the r e a c t i o n vesse l i n an i c e bath . The SR was c e n t r i f u g e d at 2,500 x g f o r 10 minutes and the supernatant d i s c a r ded . The p e l l e t was resuspended i n 0.5 mL o f e i t h e r (a) 0.6 M hydroxylamine and 0.68 M sodium acetate or (b) 0.6 M NaCl and 0.68 M sodium ace ta te , incubated at room temperature f o r 10 minutes. The SR was c e n t r i f u g e d at 2,500 x g f o r 10 minutes, the supernatant d i s ca rded and the p e l l e t resuspended i n 0.5 mL of 5% w/v t r i c h l o r o a c e t i c a c i d . An a l i q u o t o f the r e a c t i o n mixture was f i l t e r e d through a Whatman GF/C f i l t e r , washed tw ice w i t h 5 mL 5% w/v t r i c h l o r o a c e t i c a c i d and the f i l t e r s dryed and counted. 2 . I f Sarcoplasmic Ret icu lum C a r n i t i n e E x t r a c t i o n Procedure. An a l i q u o t o f SR ( con ta in i ng 200 - 400 fig SR p r o t e i n ) was c e n t r i f u g e d at 26000 x g i n a B iofuge 17R c e n t r i f u g e . The p e l l e t was resuspended i n 550 ul o f i c e c o l d 6% p e r c h l o r i c a c i d . The homogenate was c e n t r i f u g e d at 26,000 x g f o r 10 minutes. A 200 juL a l i q u o t of the supernatant was i s o l a t e d f o r both the f r e e and t o t a l a c i d s o l ub l e c a r n i t i n e de te rm ina t i on s . The a c i d i n s o l u b l e p e l l e t c on ta i n i n g long cha in a c y l c a r n i t i n e was resuspended i n 400 fil o f de i on i zed water. KOH was added to the t o t a l a c i d s o l u b l e and a c i d i n s o l u b l e f r a c t i o n s (pH 11.5 - 12) and the samples hydro lyzed at 70 °C f o r 1 hour. To each o f the th ree f r a c t i o n s T r i s - C l , pH 50 7.8 was added ( 7 - 8 mM) and n e u t r a l i z e d ( f r ee f r a c t i o n n e u t r a l i z e d w i t h 2 M KOH, t o t a l a c i d s o l ub l e and i n s o l u b l e f r a c t i o n s w i t h 12% p e r c h l o r i c a c i d ) . The f i n a l volume o f each n e u t r a l i z e d f r a c t i o n was determined, and a 250 ul a l i q u o t assayed f o r the presence o f f r e e c a r n i t i n e . 2.lg Determinat ion o f Ca rd iac Sarcoplasmic Ret icu lum C a r n i t i n e . SR c a r n i t i n e was determined us ing a p r e v i o u s l y de sc r i bed (McGarry and Fo s te r , 1977) r ad i ome t r i c assay procedure. To the 250 /iL a l i q u o t c on t a i n i n g f r e e c a r n i t i n e was added 950 /zL o f a r e a c t i o n medium con ta i n i n g ( f i n a l concen t ra t i on ) T r i s - C l , pH 7.8 3 mM, sodium t e t r a t h i o n a t e 1.65 mM and acety l -CoA 20 JJM ( con ta in i ng l - 1 4 C - a c e t y l CoA, 0.025 /tCi/tube). The r e a c t i o n was s t a r t e d by the a d d i t i o n o f 1 u n i t o f c a r n i t i n e a ce t y l t r a n s f e r a s e , proceeded f o r 30 minutes at 37 °c, and was stopped by the a d d i t i o n o f an a l i q u o t o f the r e a c t i o n mixture to a column con t a i n i n g approx imately 1 mL o f Dowex 1X8-400 anion exchange r e s i n . The column was washed tw ice w i th 0.5 mL de i on i zed water, the e l ua te s combined and r a d i o a c t i v i t y counted i n a t o t a l volume o f 5 mL l i q u i d s c i n t i l l a t i o n f l u i d . 2.1h Determinat ion o f Myocardia l Homogenate and Sarcop lasmic Ret icu lum P r o t e i n Concen t r a t i on . The p r o t e i n content of the myocardia l homogenates and c a r d i a c SR was q u a n t i f i e d us ing the method of Lowry (Lowry e t . a l . , 1951) w i th bovine serum albumin as the re fe rence s tandard. 51 2.11 Determinat ion o f Myocardia l Homogenate and Sarcoplasmic Ret icu lum Cytochrome C Oxidase A c t i v i t y . M i t ochond r i a l membrane cytochrome C ox idase a c t i v i t y o f c a r d i a c homogenate and SR was assayed accord ing to the method o f Smith (1955) as de sc r i bed by Wharton and T zago l o f f (1966). The r e a c t i o n was i n i t i a t e d by the a d d i t i o n o f homogenate or SR sample to a cuve t te incubated at 37 °C c on t a i n i n g potass ium phosphate b u f f e r (0.1 M, pH 7.0) and 20 - 50 ul o f 1% ferrocytochrome C. Cytochrome C ox idase a c t i v i t y was determined by mon i to r ing the decrease i n absorbancy at 550 nm over a pe r i od o f one minute. The r a t e o f cytochrome C ox idase a c t i v i t y was determined accord ing to the f o l l o w i n g fo rmula : S.A. = k ( concent ra t i on of Cytochrome C) ( concen t ra t i on o f homogenate/SR p r o t e i n ) where, S.A. = s p e c i f i c a c t i v i t y (itMoles/mg/min) k = In Absorbance 5- 5- 0- t = 0 ' Absorbance55g,t = 1 ' 2 .1 j Plasma T3 A n a l y s i s . Plasma T3 concent ra t i on s were determined us ing an Amer lex-M R T3 RIA k i t (Amersham). 2.1k S t a t i s t i c a l Methods. A l l data are presented as mean ± standard e r r o r o f the mean (S.E.M.). Data were analyzed by two-way ana l y s i s of v a r i ance . Where group, treatment 52 or i n t e r a c t i v e d i f f e r e n c e s were found, data were analyzed by one-way a n a l y s i s o f v a r i a n ce , f o l l owed by Newman-Keul's t e s t . Where comparisons between two means were made, S tudent ' s t - t e s t was used. Data were cons idered d i f f e r e n t at p < 0.05. 2.2 S tud ies on the E f f e c t s o f Omega-3 F a t t y A c i d Treatment on the S t r ep tozoc i n - i nduced D i a b e t i c Wi s ta r Rat 2.2a Animal Model and Study P r o t o c o l . Male Wi s ta r r a t s used i n a l l experiments were obta ined from the U n i v e r s i t y o f B r i t i s h Columbia animal care f a c i l i t i e s at a weight o f 250-275g. Diabetes was induced by a s i n g l e i . v . i n j e c t i o n o f STZ (Upjohn) at 55 mg/kg. Three days f o l l o w i n g STZ t reatment, d i abetes was conf i rmed by the presence of g l y c o s u r i a . A l l animals t r e a t ed w i th STZ became d i a b e t i c . Cont ro l and d i a b e t i c animals were randomly ass igned to e i t h e r unt reated or t r e a t e d groups two weeks f o l l o w i n g STZ (or v e h i c l e a lone) t reatment . Animals i n each exper imental group were fed Rodent Laboratory Chow R #5001 (Pur ina M i l l s , S t . Lou i s , MI). Food and water were s upp l i ed to animals i n each o f the fou r exper imental groups ad libitum, and the animals were kept on a 12 hour l i g h t / d a r k c y c l e . The treatment p ro toco l chosen to exp lo re e f f e c t s o f omega-3 f a t t y a c i d supplementat ion i n the d i a b e t i c and con t r o l animals employed the use o f Promega R (Warner Lambert, Mor r i s P l a i n s , NJ ) , a r e f i n e d f i s h o i l product concent rated i n long cha in po lyunsaturated omega-3 f a t t y a c i d s . The p r i n c i p a l omega-3 f a t t y ac ids i n the p repa ra t i on are 5,8,11,14,17-e icosapentaeno ic a c i d , C20:5,n-3 (EPA), and 4,7,10,13,16,19-docosahexaenoic 53 a c i d , C22:6,n-3 (DHA). An i n i t i a l dose-response study i n c o n t r o l and d i a b e t i c r a t s o f the e f f e c t o f Promega^ treatment demonstrated an a m e l i o r a t i v e e f f e c t o f Promega^ on i s o l a t e d heart f u n c t i o n at 0.5 mL/kg/day and 1.0 mL/kg/day (B lack et a l , 1988). Based on those da t a , Promega^ was admin i s tered to c on t r o l and d i a b e t i c - t r e a t e d r a t s at a dose o f 0.5 mL/kg/day. Promega^ was obta ined i n bulk form and to prevent o x i d a t i o n of the h i g h l y unsaturated l i p i d s , a l i q u o t s o f the s tock were drawn and s to red i n g l a s s v i a l s under n i t rogen at -80 °C p r i o r to use. Cont ro l and STZ -d i abe t i c t r e a t ed animals were admin i s tered Promega R between 0830 and 0930 d a i l y f o r the du ra t i on o f the treatment p e r i o d . Promega^ was admin i s tered to the r a t s v i a o ra l gavage us ing a 75 mm s t a i n l e s s s t e e l tube. Animals o f e i t h e r group t r e a t ed by gavage d i d not demonstrate any apparent s igns o f d i s comfo r t/pa in dur ing the course o f the study. Promega* treatment was i n i t i a t e d two weeks f o l l o w i n g STZ/veh ic le i n j e c t i o n and cont inued f o r a pe r i od of f ou r weeks. Body weight and water consumption were recorded d a i l y . 2.2b Determinat ion o f I s o l a t ed Working Rat Heart Func t i on . Card iac f u n c t i o n was determined us ing an i s o l a t e d working heart apparatus. Fo l l ow ing d e c a p i t a t i o n of the r a t , the heart was r a p i d l y e x c i s e d , p laced i n c o l d pe r fu s i on bu f f e r and trimmed of p e r i c a r d i a l f a t and extraneous t i s s u e . The Chenoweth-Koelle pe r fu s i on b u f f e r ( c o n s i s t i n g o f g lucose 10 mM, MgC l 2 2.1 mM, KC1 5.6 mM,NaCl 120 mM, C a C l 2 2.2 mM and NaHC03 19 mM) was aerated w i th 95% C02/5% 0 2 at 37 °C. The heart was i n i t i a l l y perfused i n the Langendorf mode (non-working) f o r ten minutes. A s t a i n l e s s s t e e l cannula (16 gauge) was f i x e d to the l e f t a t r i a l appendage. 54 Fo l l ow ing cannu la t i on o f the l e f t a t r i um, the heart was s h i f t e d to the working heart mode. A second s t a i n l e s s s t e e l cannula (20 gauge) was then i n s e r t e d i n t o the apex o f the l e f t v e n t r i c l e and f i x e d t o a Statham P23AA pressure t ransducer by means o f an i n t e r connec t i n g 3 cm length o f PE 90 t u b i n g . I n t r i n s i c working heart f u n c t i o n was determined by mon i to r ing peak l e f t v e n t r i c u l a r developed pressure and the maximal r a te s o f change o f pressure du r ing l e f t v e n t r i c u l a r s y s t o l e and d i a s t o l e wh i l e imposing upon the heart va r i ous l e f t a t r i a l f i l l i n g pressures (LAFP). LAFP was regu la ted by means o f an ad ju s t ab l e r e s e r v o i r connected to the l e f t a t r i a l cannu la . The he ight o f the r e s e r v o i r was c a l i b r a t e d to e f f e c t changes i n LAFP from 7.5 cm to 20.0 cm H2O. Contro l and d i a b e t i c hearts were paced at 300 beats per minute at tw ice th re sho ld vo l tage by square wave pu l ses o f 5 ms d u r a t i o n . The v e n t r i c u l a r pressure pu l se and i t s f i r s t d e r i v a t i v e were recorded by a Grass model 79D polygraph i n t e r f a c e d w i th an Apple II microcomputer curve f i t t i n g program (Ha r r i s et a l , 1983). To determine the a o r t i c f low r a t e ( f o l l o w i n g s t a b i l i z a t i o n o f v e n t r i c u l a r f u n c t i o n at each l e f t a t r i a l pressure) the pe r f u s i on out f l ow from the a o r t i c cannula was fed i n t o a 10 mL p i p e t t e and the r a t e determined by c a l c u l a t i n g the pe r iod o f t ime r equ i r ed to charge the p i p e t t e . In a s i m i l a r f a s h i on , the pulmonary a r t e r y was cannulated w i th a 16 gauge s t a i n l e s s s t ee l cannula and the coronary f l ow determined by the pe r i od of t ime requ i red to charge a separate 10 mL p i p e t t e . Fo l l ow ing t h i s procedure, hearts were f rozen i n l i q u i d n i t r ogen and s to red at -80 °c u n t i l p repa ra t i on of microsomes enr i ched i n SR. SR was prepared as desc r ibed f o r the thyroidectomized Mi star rat studies. The f i n a l p e l l e t was resuspended i n Medium I I I , f r ozen i n l i q u i d n i t r ogen and 55 s to red at -80°c u n t i l use. The y i e l d of SR p r o t e i n was q u a n t i f i e d on the ba s i s o f p r o t e i n content est imated us ing the method o f Lowry e t a l (1957). SR ca l c i um t r an spo r t a c t i v i t y from each o f the fou r exper imental groups was determined as desc r ibed f o r the thyroidectomized Mi star rat studies, us ing the f i l t r a t i o n techn ique. Calc ium t r an spo r t a c t i v i t y was determined at 0.1 /iM and 5.3 /iM [ C a 2 + ] p r e e . 2.2c Ca rd iac L i p i d E x t r a c t i o n Procedure. Hearts were i n i t i a l l y f rozen i n l i q u i d n i t r ogen f o l l o w i n g r a p i d removal and r i n s i n g . Hearts were p u l v e r i z e d under N 2 ( l ) , decanted i n t o 30mL g l a s s corex tubes and homogenized i n ch lo ro fo rm (CHCI3)rmethanol (MeOH):water (1:2:0.8) ( con ta in i ng BHT 0.05% w/v) tw ice f o r 10 seconds each. The homogenate was c e n t r i f u g e d at 1500 xg f o r 10 minutes and the supernatant f i l t e r e d through g l a s s wool i n t o 50mL c o n i c a l tubes s to red on i c e . The p e l l e t was r e - e x t r a c t e d tw ice as above, and the supernatants combined. To the combined supernatants equal volumes o f CHCI3 and H2O were added, and shaken severa l t imes over 10-15 minutes. The mixture was c e n t r i f u g e d at 1500xg f o r 10 minutes and the lower phase i s o l a t e d and evaporated w i th N 2 (g) to approximately 1 mL, t r a n s f e r r e d to an amber g l a s s v i a l and s to red at -80°C under N 2 ( g ) . 2.2d Th in Layer Chromatographic Separat ion o f Myocardia l Pho spho l i p i d s . Merck s i l i c a gel 60 p l a t e s (20 cm x 20 cm) were used. The p l a t e s were i n i t i a l l y heated to ~ 150 °C f o r 30 minutes, a f t e r which the p l a t e s were covered w i th an a p p r o p r i a t e l y s i z ed g l a s s p l a t e and a l lowed to cool to room temperature before the c a rd i a c l i p i d e x t r a c t was app l i ed to the p l a t e . 56 Phospho l ip id s were separated i n one dimension us ing a so l ven t system con t a i n i n g ch lo ro fo rm, methanol, a c e t i c a c i d and water , i n r a t i o s o f 60:50:1:4, r e s p e c t i v e l y . Fo l l ow ing separa t i on ( so l ven t f r o n t « 1 - 2 cm from the top o f the p l a t e ) of the pho spho l i p i d s , the p l a t e was d r i e d under a stream of N2(g). The p l a t e s were then sprayed w i th 2 ' , 7 ' - d i c h l o r o f l u o r e s c e i n and v i s u a l i z e d and marked under long wave uv l i g h t . To remove the f l u o r e s c e n t dye and i s o l a t e the pho spho l i p i d spec ies o f i n t e r e s t , the s i l i c a gel was scraped from the p l a t e i n t o capped v i a l s (N2(g) sa tu ra ted) and the l i p i d s e x t r a c t ed w i th ch lo ro fo rm:methano l :wate r :ace t i c a c i d (50:39:10:1) over 10 minutes on i c e . Fo l l ow ing c e n t r i f u g a t i o n at 1500xg f o r 10 minutes, the supernatants were i s o l a t e d and the p e l l e t s r e - e x t r a c t e d as above. The supernatants were combined, 4 M NH4OH added, the mixture vortexed v i g o r ou s l y and c e n t r i f u g e d to achieve phase s epa ra t i on . The lower phase was p laced i n an amber v i a l and 1 mL o f CHCI3 ( con ta i n i ng 0.05% w/v BHT) was added and s to red at -80°C under n i t r o g e n . 2.2e SR Pho spho l i p i d E x t r a c t i o n Procedure. To 1 mL o f SR suspens ion, 3.75 mL chloroform:methanol (1:2, 0.05% w/v BHT) was added and the mixture vortexed severa l t imes over 45 minutes wh i l e on i c e . The suspension was c e n t r i f u g e d f o r 5 minutes at 1500xg and the supernatant i s o l a t e d to a capped tube (N2(g) sa turated) on i c e . The p e l l e t was resuspended i n ch loroform:methanol :water ( 1 :2 :0 .8 ) , vortexed over 15 minutes and c e n t r i f u g e d f o r 5 minutes at 1500xg. The supernatants were combined, equal volumes of ch lo ro form and water added, and c e n t r i f u g e d to separate the two phases . The lower phase was i s o l a t e d f o r TLC o f 57 pho spho l i p i d s . Phospho l ip id s were i s o l a t e d us ing a one d imens ional TLC techn ique. Merck s i l i c a gel 60 p l a t e s (20 cm x 20 cm) were used. The p l a t e s were prepared as above. The so l vent system used was n -hexane :e the r : ace t i c a c i d (80:40:1). Under these c o n d i t i o n s phospho l ip id s remained at the o r i g i n and were v i s u a l i z e d and i s o l a t e d from the s i l i c a gel as de sc r i bed above. 2.2f D e r i v a t i z a t i o n and Gas Chromatographic A n a l y s i s o f Ca rd i ac and SR Pho spho l i p i d E x t r a c t s . P r i o r to gas chromatographic separa t i on and i d e n t i f i c a t i o n , f a t t y a c i d methyl e s t e r s o f the phospho l i p id spec ies were prepared. Phospho l i p id e x t r a c t was added to an amber R e a c t i - v i a l R and evaporated to dryness w i th N 2 ( g ) . The r e a c t i o n was s t a r t e d w i th BF3-methanol, vortexed and heated at 100°C f o r 3 minutes us ing a Reacti-Therm heat ing u n i t and aluminum heat ing b l ock . The r e a c t i o n was terminated on i c e by the a d d i t i o n o f n-hexane and water (2 :1 ) . The v i a l s were immediately c e n t r i f u g e d at 1500xg (0°C) f o r 5 minutes, the upper phase i s o l a t e d and s to red i n amber v i a l s under N 2 ( g ) . D e r i v a t i z e d phospho l ip id s were separated on a Supelco SP-2330 column (30 m x 0.32 mm i . d . ) us ing a Hewlett -Packard model 5880A gas chromatograph. The i n j e c t i o n temperature was 200°C, the i n i t i a l column temperature was 150°C f o r a pe r i od o f e i gh t minutes, a f t e r which the column temperature was programmed to i nc rease at a r a t e of 2°C/minute up to the f i n a l temperature o f 190 °C. The column running time at 190 °C was seven minutes. The d e t e c t i o n temperature was 240 °C. 58 2.3 Determinat ion o f Plasma Glucose, T r i g l y c e r i d e , Cho l e s t e r o l and I n s u l i n . Plasma g lucose , t r i g l y c e r i d e and c h o l e s t e r o l were determined us ing commerc ia l l y a v a i l a b l e (Boehr inger Mannheim GmbH, D iagnos t i ca ) enzymatic c o l o r i m e t r i c assay k i t s . Plasma i n s u l i n concen t ra t i on s were determined us ing a commerc ia l l y a v a i l a b l e radioimmunnoassay k i t (Amersham). 2.4 E l e c t r o p h o r e t i c A n a l y s i s o f SR P r o t e i n . P r o te i n s o f sarcop lasmic r e t i c u l u m were r e so l ved by sodium d o d e c y l s u l f a t e po lyac ry lamide gel e l e c t r o p h o r e s i s (SDS-PAGE) accord ing to the method o f Laemmli and Favre (1973). Latex g loves were worn throughout SDS-PAGE p r o t o c o l s . A l l c a s t i n g apparatus were i n i t i a l l y c leaned w i t h 95% ethanol us ing a l i n t f r ee paper t o w e l . The 1.5 mm r e s o l v i n g ge l (5% -20%/0.13% - 0.53% acry lamide/BIS, 375 mM t r i s - c h l o r i d e , pH 8.8, 0.1% SDS, 3.56 % v/v g l y c e r o l ) was c a s t , 400 jiL H2O over layed and the acry lamide a l lowed to po lymer ize 2.5 hours (minimum). The H2O was decanted and the s t a ck i n g gel (3.5%/0.09% acry lamide/BIS, 318 mM T r i s - c h l o r i d e , pH 6.8 and 0.1% SDS) was cas t and a l lowed to po lymer ize f o r at l e a s t 1.5 hours. SR samples (10 - 25 /xg p r o te i n ) were added to sample b u f f e r (0-mercaptoethanol , SDS, g l y c i n e , t r i s - c h l o r i d e , pH 6.8 and Bromophenol b lue) and dispensed i n t o the s t ack i ng gel w e l l s by under lay ing i n the running b u f f e r (25 mM T r i s - c h l o r i d e , pH 8.3, 192 mM g l y c i n e and 0.1% SDS). The gel apparatus was set i n a lower r e s e r v o i r b u f f e r (25 mM T r i s - c h l o r i d e , pH 8.0, 20°c) . The gel was run through the s tack at 25 mA/slab and the r e s o l v i n g ge l at e i t h e r 6 mA/slab (overn ight ) or 45 mA/slab (day r un ) . 59 2.5 S t a i n i n g and Des ta in ing Procedures. SDS-PAGE ge l s were s t a i ned w i th Coumassie Blue s t a i n (0.25% Coumassie B r i l l i a n t b lue R-250, 45% methanol) . The t r a y c on t a i n i n g the ge l and s t a i n was p laced on a t i l t / s w i v e l r o t a t i n g p l a t f o r m , and s t a i ned f o r a minimum of 30 minutes. The s t a i n was decanted and de s t a i n I (methanol 50%, a c e t i c a c i d 10% i n de i on i zed water) added to the t r a y . Desta in I was changed fou r to s i x t imes over an e i gh t hour p e r i o d . Desta in II (20% methanol, 5% a c e t i c a c i d i n de i on i zed water) rep laced de s t a i n I and the gel des ta ined u n t i l the background was c l e a r . Gels were dryed and preserved us ing B ioGelWrap R . 60 3. Results: 3.1 Studies on the effects of thyroidectomy on cardiac sarcoplasmic reticulum. 3.1a General physical characteristics of euthyroid and thyroidectomized rats. The e f f e c t o f s u r g i c a l thyro idectomy on the phy s i c a l c h a r a c t e r i s t i c s of the exper imental animals was t y p i c a l o f t h y r o i d g land removal. The thy ro idec tomized animals d i s p l a yed s igns i n d i c a t i v e o f hypothyro id i sm. The plasma T3 concent ra t i on s o f each of the eu thy ro id and thy ro idec tomized groups s tud ied are shown i n F igure 2. Although two way a n a l y s i s o f va r i ance d i d not d i s c e r n a s t a t i s t i c a l l y s i g n i f i c a n t group/time i n t e r a c t i o n , the plasma T3 concent ra t i on s o f the thy ro idec tomized r a t s were between 28% and 65% l e s s than r e s p e c t i v e eu thy ro id c o n t r o l va lue s . Compared to age matched c o n t r o l s , whole body weights o f thy ro idec tomized animals were s i g n i f i c a n t l y (p < 0.05) lower at 4, 6, and 8 weeks (F igure 3 ) . A d d i t i o n a l l y , whereas the body weights of the eu thy ro i d c o n t r o l animals inc reased (p < 0.05) w i th t ime (euthy ro id 2 week < 4 week < 6 week = 8 week), body weight o f the thy ro idectomized animals d i d not , except by e i gh t weeks ( thy ro idectomized 2 week < 8 week, p < 0 .05) . The wet v e n t r i c u l a r weights of thy ro idectomized animals were not d i f f e r e n t from age-matched c o n t r o l s at 2 weeks; however, wet v e n t r i c u l a r weights of thy ro idec tomized r a t s were s i g n i f i c a n t l y (p < 0.05) lower than r e s p e c t i v e age-matched c o n t r o l s 4, 6 and 8 weeks f o l l o w i n g thyro idectomy (F igure 4). The y i e l d o f c a r d i a c SR p r o t e i n from eu thy ro id c on t r o l and thy ro idec tomized 61 F igure 2. The e f f e c t o f thyro idectomy on plasma T3 concen t r a t i on over the t ime course o f the study ( 2 - 8 weeks). Re su l t s shown are the mean ± S.E.M., w i th an n = 5-6 animals/group/week assayed. Blood was c o l l e c t e d at s a c r i f i c e and plasma i s o l a t e d immediately, plasma [T3] was determined as de sc r i bed i n Methods. The plasma [T3] of thy ro idec tomized animals ( f l ) at 2, 4, 6 and 8 weeks were r e s p e c t i v e l y , 65%, 28%, 47% and 51% o f the eu thy ro id c o n t r o l s ( O ) . 62 1.25-r of 1.00 + O E 0.75 + i—i t-| 0.50 4-tn _a CL •—1 0.25 0.00 2 4 6 8 Time (Week Post—Thyroidectomy) 63 F igure 3. The e f f e c t o f s u r g i c a l thyro idectomy on body weight over the t ime-course o f the study ( 2 - 8 weeks). Euthyro id c o n t r o l r a t s ( 0 ) , thy ro idectomized ( • ) r a t s , n = 6/group/week assayed, data shown are mean ± S.E.M.. * , s i g n i f i c a n t l y d i f f e r e n t from eu thy ro id c o n t r o l , p < 0.05. 64 550 -r 1 , , r— 2 4 6 8 Time (Week Post-Thyroidectomy) V 65 F igure 4. The e f f e c t of s u r g i c a l thyro idectomy on wet v e n t r i c u l a r weight o f eu thy ro id and thy ro idectomized animals over the t ime-course o f the study. Euthyro id c on t r o l animals ( • ) , thy ro idec tomized animals ( M ) . n = 6/group/week assayed, data shown are mean ± S.E.M.. * , s i g n i f i c a n t l y d i f f e r e n t from eu thy ro id c o n t r o l , p < 0.05. 66 Time (Week Post-Thyroidectomy) 67 r a t s at each t ime po i n t s tud ied i s represented by F igure 5. There was a s i g n i f i c a n t l y (p < 0.05) lower y i e l d o f SR from the 2 week hypothyro id group. There was no d i f f e r e n c e i n the y i e l d o f SR p r o t e i n at the other t ime po i n t s s t u d i e d . An i n d i c a t o r o f a hypothyro id-mediated e f f e c t on c a r d i a c SR, independent o f gross c a r d i a c change, i s the r a t i o o f SR y i e l d r w e t heart weight (/xg:mg). These data were c a l c u l a t e d f o r each group at each week o f the study. As shown i n F igure 6, the SR y i e l d r w e t v e n t r i c u l a r weight r a t i o (/xg:mg) was not a f f e c t e d by thyro idectomy and remained e s s e n t i a l l y constant over the t ime course o f the study. The data of F igures 2 - 5 t he r e f o r e i n d i c a t e tha t s u r g i c a l thyro idectomy rendered the animals hypothyro id as determined by a number of ph y s i c a l i n d i c e s , p r i m a r i l y growth f a i l u r e (manifest as reduced body weights o f the thy ro idec tomized an ima l s ) , reduced heart weights and reduced c i r c u l a t i n g plasma T3 c oncen t r a t i on s . 3.1b C h a r a c t e r i z a t i o n o f the SR P r e p a r a t i o n . The r a t c a r d i a c SR p repa ra t i on was i n i t i a l l y c h a r a c t e r i z e d to determine the degree of non-SR membrane contaminat ion and the degree o f enrichment o f ca l c i um t r an spo r t a c t i v i t y . M i t ochond r i a l contaminat ion o f the SR p repa ra t i on was assessed by the presence of cytochrome C ox idase a c t i v i t y . As shown i n Table 1, when the SR p repa ra t i on was compared to a v e n t r i c u l a r homogenate there was a c on s i s t en t r educ t i on i n cytochrome C ox idase a c t i v i t y . The percentage reduc t i on i n cytochrome C ox idase a c t i v i t y i n the eu thy ro id groups was 97.8% to 99.2%. 68 F igure 5. The e f f e c t o f s u r g i c a l thyro idectomy on the y i e l d o f v e n t r i c u l a r sarcop lasmic r e t i c u l u m . Euthyro id c o n t r o l animals (Q), thy ro idec tomized animals ( • ) . n = 6/group/week assayed, data shown are mean ± S.E.M.. * , s i g n i f i c a n t l y d i f f e r e n t from eu thy ro id c o n t r o l , p < 0.05. 69 1 0 0 0 -^ 9 0 0 -•Q3 8 0 0 O 7 0 0 ° - 6 0 0 A 5 0 0 33 >-C O 4 0 0 3 0 0 2 0 0 1 0 0 0 2 4 6 8 Time (Week Post—Thyroidectomy) 70 F igure 6. The e f f e c t of s u r g i c a l thyro idectomy on the r a t i o o f SR i s o l a t e d to wet v e n t r i c u l a r weight (/xg:mg). Euthyro id c o n t r o l animals ( P ) , thy ro idectomized animals ( • ) . n = 6/group/week assayed. 71 Time (Week Post Thyroidectomy) 72 Table 1. C h a r a c t e r i z a t i o n o f the c a r d i a c sa rcop lasmic r e t i c u l u m p r e p a r a t i o n . Determinat ion o f the r e l a t i v e decrease i n cytochrome C oxidase a c t i v i t y o f the SR p repa ra t i on vs the v e n t r i c u l a r homogenate. Resu l t s shown are mean o f n=6/group. Cytochrome C Oxidase A c t i v i t y (itmoles/minute) Homogenate SR Euthy ro id Two Week Four Week S i x Week E ight Week 16.61 43.65 44.29 42.33 Thyro idectomized Two Week 13.33 Four Week 18.57 S i x Week 21.96 E ight Week 22.35 0.363 0.527 0.467 0.343 0.185 0.309 0.349 0.272 Percent Decrease SR vs Homogenate 97.8 98.8 98.9 99.2 98.6 98.3 98.4 98.8 73 The magnitude o f the percentage reduc t i on i n cytochrome C ox idase a c t i v i t y of the SR compared to the homogenate of the thy ro idec tomized groups was e s s e n t i a l l y the same (as t ha t o f the eu thy ro id groups) at 98.3% to 98.8%. The data o f Table 1 t he r e f o r e i n d i c a t e t ha t there was a c o n s i s t e n t and s i g n i f i c a n t r educ t i on i n the amount o f the m i tochondr i a l marker enzyme a c t i v i t y i n the SR compared to the homogenate. The r e l a t i v e enrichment o f ca l c i um t r an spo r t a c t i v i t y was determined by comparing the ca l c i um t r an spo r t a c t i v i t y of the myocardia l homogenate to the ca l c i um t r an spo r t a c t i v i t y o f the SR p r e p a r a t i o n . Calc ium t r an spo r t a c t i v i t y o f c a r d i a c homogenate and SR was determined at 5.3 /xM [Ca ] f r e e . The data of t a b l e 2 show tha t there was enrichment o f s p e c i f i c ca l c i um t r an spo r t a c t i v i t y i n the SR p repara t i on of both the eu thy ro i d (97- to 142-f o l d i nc rease ) and thy ro idectomized (84- to 116 - fo ld i nc rease ) groups. Sarcolemmal contaminat ion was determined based on the presence of Na + /K + -ATPase a c t i v i t y . S tud ies o f the Na + /K + - ATPase enzyme showed very low a c t i v i t y , which was not s t imu la ted by i ncubat i on o f the SR w i th detergent (sodium d o d e c y l s u l f a t e , data not shown), i n d i c a t i n g the absence of l a t e n t a c t i v i t y (masked s i t e s ) . 74 Table 2. C h a r a c t e r i z a t i o n o f the c a r d i a c sa rcop lasmic r e t i c u l u m p r e p a r a t i o n . Determinat ion of the r e l a t i v e i nc rease i n s p e c i f i c ca l c i um t r an spo r t a c t i v i t y o f the SR p repa ra t i on vs the v e n t r i c u l a r homogenate. Resu l t s shown are the mean ± S.E.M., n=6/group. Ca^ -Transport A c t i v i t y (nmoles/mg/minute) Homogenate SR Euthyro id Two Week Four Week S i x Week E ight Week 1.3 ± 0.1 1.1 ± 0.2 1.6 ± 0.1 1.9 ± 0.2 Thyro idectomized Two Week 1.1 ± 0.1 Four Week 1.5 ± 0.2 S i x Week 1.4 ± 0.2 E ight Week 1.9 ± 0.3 129 ± 7 162 ± 10 225 ± 4 243 ± 7 116 ± 9 122 ± 11 162 ± 17 206 ± 12 Fo ld Increase SR vs Homogenate 97 ± 5 143 ± 9 142 ± 3 126 ± 4 107 ± 8 84 ± 8 116 ± 12 107 ± 6 75 3.1c The E f f e c t o f Thyroidectomy on SR Calc ium Transport A c t i v i t y . The e f f e c t o f hypothyro id i sm on SR ca l c i um t r an spo r t a c t i v i t y was determined 2, 4, 6 and 8 weeks f o l l o w i n g thyro idectomy. SR C a z + - t r a n s p o r t a c t i v i t y was assessed at [ C a z + ] p r e e ranging from 0.1 /iM to 5.3 /iM (as o u t l i n e d i n the Methods). The r e s u l t s o f these ca l c i um t r an spo r t a c t i v i t y exper iments, determined i n eu thy ro id c on t r o l and thy ro idec tomized r a t s at the 2, 4, 6, and 8 week time p o i n t s , are represented i n F igures 7 - 10, r e s p e c t i v e l y . SR C a 2 + - t r a n s p o r t a c t i v i t y i n eu thy ro i d c o n t r o l and thy ro idec tomized r a t s were not d i f f e r e n t at 2 weeks (F igure 7 ) . There was however, a s i g n i f i c a n t (p <0.05) depress ion i n SR C a z + - t r a n s p o r t at a l l [ C a z + ] p r e e t e s t ed except 2.0 /iM at 4 weeks (F igure 8 ) . The SR C a z + -t r an spo r t r a t e remained s i g n i f i c a n t l y (p < 0.05) depressed i n the thy ro idec tomized r a t s at the 6 and 8 week t ime po i n t s ( F i gu re 9 and 10, r e s p e c t i v e l y ) . The C a z + - t r a n s p o r t data o f both the eu thy ro i d and thy ro idec tomized groups at each time po in t were transformed f o r an Ead ie -Hofstee p l o t a n a l y s i s . These data are represented i n the i n s e t s to F igures 7 - 1 0 f o r the 2 - 8 week time p o i n t s , r e s p e c t i v e l y . The Ead ie -Hofs tee p l o t s i n d i c a t e tha t the changes i n C a 2 + - t r a n s p o r t a c t i v i t y o f SR i s o l a t e d from the eu thy ro id and thy ro idectomized r a t s were not a s soc i a ted w i th a l t e r a t i o n s i n the apparent Kr, a o f the ca l c i um t r an spo r t r e a c t i o n , s i nce the s lopes from these p l o t s f o r both groups remained comparable w i t h i n the sub-micromolar range (0.42 /iM - 0.80 /iM). Although SR C a z + - t r a n s p o r t was s i g n i f i c a n t l y reduced i n the thy ro idec tomized animals at 4, 6 and 8 weeks, i t became apparent t ha t there was a l s o a time-dependent inc rease i n the SR C a 2 + - t r a n s p o r t a c t i v i t y o f both the eu thy ro i d and thy ro idectomized groups. The data represented i n 76 F igure 7 . SR ca l c i um t r an spo r t a c t i v i t y determined two weeks f o l l o w i n g s u r g i c a l removal of the t h y r o i d g l and . Euthy ro id c on t r o l animals ( o ) , thy ro idectomized animals ( • ) . n = 6/group, data shown are mean ± S.E.M.. Inset: Ead ie -Hofs tee p l o t o f the SR ca l c i um t r an spo r t data o f two weeks pos t - thy ro idec tomy. 77 78 F igure 8. SR ca l c i um t r an spo r t a c t i v i t y determined f ou r weeks f o l l o w i n g s u r g i c a l removal o f the t h y r o i d g l and . Euthy ro id c on t r o l animals ( o ) , thy ro idectomized animals ( • ) . n = 6/group, data shown are mean ± S.E.M.. * , s i g n i f i c a n t l y d i f f e r e n t from r e s p e c t i v e eu thy ro id c on t r o l va l ue , p < 0.05. Inset: Ead ie -Hofstee p l o t of the SR ca l c i um t r an spo r t data o f f ou r weeks pos t - thy ro idectomy. 7 9 200 T 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 [Caz+]free (fiM) 80 F igure 9. SR ca l c i um t r an spo r t a c t i v i t y determined s i x weeks f o l l o w i n g s u r g i c a l removal o f the t h y r o i d g l and . Euthy ro id c o n t r o l animals ( O ) , thy ro idectomized animals ( • ) . n = 6/group, data shown are mean ± S.E.M.. * , s i g n i f i c a n t l y d i f f e r e n t from r e s p e c t i v e eu thy ro id c on t r o l va l ue , p < 0.05. Inset: Ead ie -Hofstee p l o t o f the SR ca l c i um t r an spo r t data o f s i x weeks pos t - thy ro idectomy. 81 "E C P E \ tn _cu o E c [ C a 2 + ] F r e e ^ M ) 82 F igure 10. SR ca l c i um t r an spo r t a c t i v i t y determined e i g h t weeks f o l l o w i n g s u r g i c a l removal o f the t h y r o i d g l and . Euthyro id c o n t r o l animals ( O ) , thy ro idectomized animals ( • ) . n = 6/group/week assayed, data shown are mean ± S.E .M.. * , s i g n i f i c a n t l y d i f f e r e n t from r e s p e c t i v e eu thy ro id c o n t r o l v a l ue , p < 0.05. Inset: Ead ie-Hofstee p l o t of the SR ca l c i um t r an spo r t data o f e i g h t weeks pos t - thyro idectomy. 83 84 F igure 11 (a and b) i n d i c a t e the time-dependent i nc rease i n SR Ca -t r an spo r t a c t i v i t y determined at 2.0 juM and 5.3 /JM [Ca^ ] p r e e , r e s p e c t i v e l y , f o r both the eu thy ro id and thy ro idec tomized groups. 3.1d The E f f e c t o f Thyroidectomy on SR A c y l c a r n i t i n e L e ve l s . Endogenous c a r n i t i n e i n the SR was found to be present i n d i f f e r e n t forms: f r e e , e s t e r i f i e d w i th short cha in acy l mo ie t i e s and e s t e r i f i e d w i th long cha in acy l m o i e t i e s . Each form of the c a r n i t i n e present was q u a n t i f i e d f o l l o w i n g i s o l a t i o n as desc r ibed i n the Methods s e c t i o n . The e f f e c t of thyro idectomy on the concen t ra t i on of f r e e c a r n i t i n e present i n the SR f r a c t i o n i s shown i n F igure 12. Thyroidectomy d i d not a f f e c t the l e v e l o f f r e e c a r n i t i n e present i n the SR at the time po i n t s s t u d i e d . The concen t r a t i on o f short cha in c a r n i t i n e i n the SR i s shown i n F igure 13. As was the case w i th f r ee c a r n i t i n e , there was no e f f e c t o f thyro idectomy at the t ime po i n t s s t u d i e d . I t appears from F igure 13 t ha t the concen t r a t i on o f short cha in c a r n i t i n e present i n the SR of thy ro idec tomized animals at the fou r week t ime po in t i s g r ea te r than tha t of the r e s p e c t i v e eu thy ro id SR; however, two way ana l y s i s o f va r iance revea led no s i g n i f i c a n t d i f f e r e n c e s . The l e v e l of endogenous long cha in a c y l c a r n i t i n e a s soc i a ted w i th the SR f r a c t i o n i s shown i n F igure 14. There was no e f f e c t o f thyro idectomy on the concen t ra t i on o f long cha in a c y l c a r n i t i n e a s soc i a ted w i t h the SR f r a c t i o n at any of the fou r t ime po i n t s s t u d i e d . The data represented i n F igures 12 - 14, t h e r e f o r e , i n d i c a t e tha t thyro idectomy i n f l u ence s n e i t h e r the l e v e l of f r ee c a r n i t i n e nor the l e v e l o f short cha in or long cha in a c y l c a r n i t i n e a s soc ia ted w i th the SR membrane. 85 F igure 11. The e f f e c t o f t ime (week of study) on the r a t e o f SR ca l c i um t r an spo r t determined at 2.0 /zM [ C a 2 + ] p r e e (a) and at 5.3 /iM [ C a 2 + ] p r e e (b). Euthyro id c o n t r o l animals (O), thy ro idec tomized animals ( • ) . n = 6/group/week assayed, data shown are mean ± S.E.M.. * , s i g n i f i c a n t l y d i f f e r e n t from r e s p e c t i v e eu thy ro id c on t r o l va l ue , p < 0.05. Calcium Transport Rate -(nmoles/mg/min) (nmoles/mg/min) CD > 87 F igure 12. The e f f e c t o f thyro idectomy on the concen t r a t i on o f f r e e c a r n i t i n e present i n the SR f r a c t i o n . Euthyro id c o n t r o l animals ( O ) , thy ro idec tomized animals ( • ) . n = 3-6/group/week assayed, data shown are mean ± S.E.M.. 8 8 300-r 2 4 6 8 Time (Week Post—Thyroidectomy) 89 F igure 13. The e f f e c t o f thyro idectomy on the concen t r a t i on o f shor t cha in a c y l c a r n i t i n e present i n the SR f r a c t i o n . Euthy ro id c o n t r o l animals ( • ) , thy ro idectomized animals ( f l ) . n = 3-6/group/week assayed, data shown are mean ± S.E.M.. 90 0) c D 2 O o-c o _c O o J Z 00 or: oo co cn E \ tn o E C L 700 600 500-400-300 200-100 Time (Week Post—Thyro idectomy) 91 F igure 14. The e f f e c t of thyroidectomy on the concen t r a t i on o f long cha in a c y l c a r n i t i n e present i n the SR f r a c t i o n . Euthy ro id c on t r o l animals (CD), thy ro idectomized animals ( • ) . n = 3-6/group/week assayed, data shown are mean ± S.E.M.. 92 350 (D C o o < cn -4-> o i_ tr: co cn E O E C L 300 250 200 150 100--50 0 2 4 6 8 Time (Week Post—Thyro idectomy) 93 3.1e The E f f e c t o f Thyroidectomy on SR Phosphoprotein L e v e l . S ince the r e s u l t s de sc r ibed above i n d i c a t e d t ha t the l e v e l o f endogenous long cha in a c y l c a r n i t i n e a s soc i a ted w i th the SR membrane was not i n f l uenced by the hypothyro id s t a t e , and t he r e f o r e not r e l a t e d to the observed decrease i n SR C a z + - t r a n s p o r t , ca l c i um pump s i t e s i n the SR were q u a n t i f i e d . The t h e o r e t i c a l bas i s f o r these experiments has been d i scus sed i n the I n t r o d u c t i o n . I t was i n i t i a l l y o f i n t e r e s t to determine i f a calcium-dependent inc rease i n SR phosphory la t ion cou ld be demonstrated i n the SR p repa ra t i on employed i n the cu r ren t s t u d i e s . The data represented i n F igure 17 show tha t there was a calcium-dependent i nc rease i n the phosphory la t ion s t a t e o f the SR ca l c ium pump i n SR from eu thy ro id c o n t r o l an imals . The F igure i n d i c a t e s that the SR was maximal ly phosphory lated at 5 mM [ C a 2 + ] p r e e and tha t the phosphory la t ion s t a t e o f the SR was not f u r t h e r increased at 10 or 12 mM [ C a 2 + ] p r e e . The SR phosphory la t ion experiments were performed w i th 2 /iM t o t a l ATP ( non - r ad i oac t i v e (co7tf) and op J t P - A T P ) and a r e a c t i o n time o f 20 seconds to maximize acylphosphate format ion (ca lc ium pump p r o t e i n ) and minimize k inase-mediated phophory la t ion o f the SR membrane p r o t e i n s . That the pr imary s i t e phosphory lated under the r e a c t i o n cond i t i on s employed was the ca l c i um pump was f u r t h e r evidenced by experiments which determined t ha t 88.2% - 95.5% o f the calc ium-dependent phosphory la t ion was hyd roxy l am ine - sen s i t i ve (at 0.1 -10 mM [ C a 2 + ] p r e e , r e s p e c t i v e l y ) , i n d i c a t i v e o f acylphosphate f o rmat i on . There fo re , s i n ce the SR was maximally phosphory lated at 5 - 12 mM [ C a 2 + ] p r e e , i n subsequent s tud ie s on the e f f e c t of thyro idectomy on SR calc ium-dependent phosphory la t ion 10 mM [ C a 2 + ] p r e e was used. 94 F igure 15. Calc ium {[Ca*JFree) dependence o f sa rcop lasmic r e t i c u l u m phospho ry l a t i on . SR was phosphorylated i n the presence o f 7~3 2P-ATP a s d e s c n b e d i n the Methods. Data shown (mean ± S.E.M., n = 2-4 o f three separate experiments i n t r i p l i c a t e ) are calcium-dependent phosphory la t ion c a l c u l a t e d by s u b t r a c t i o n of ca lc ium-independent phosphory l a t i on . 9 5 2 2 5 T c n £ 2 0 0 - -0 4 1 1 1 1 1 0.1 1.0 1 0 . 0 1 0 0 . 0 1 0 0 0 . 0 1 . 0 E 4 [ C a ^ + ] F R E E (JJLM) 96 The l e v e l of SR acy lphosphoprote in was determined at two and fou r weeks f o l l o w i n g thyro idectomy. F igure 16 shows there was no d i f f e r e n c e i n the l e v e l o f SR calcium-dependent acylphosphate format ion two weeks f o l l o w i n g thyro idectomy. These data are i n accord w i t h F i gu re 7. Four weeks f o l l o w i n g thyro idectomy there was a s i g n i f i c a n t (p < 0.05) r educ t i on i n SR acy lphosphoprote in l e v e l s i n thy ro idec tomized animals ( F i gu re 17). S ince SR ca l c i um t r an spo r t was reduced at four weeks (F igure 8) the data o f F igure 17 may e x p l a i n the d e c l i n e i n ca l c ium t r an spo r t a c t i v i t y . A d i f f e r e n c e i n endogenous k inase-mediated phosphory la t ion o f the SR membrane between eu thy ro id and thy ro idectomized animals cou ld c o n t r i b u t e to the de sc r i bed changes i n SR ca l c ium t r an spo r t and SR acy lphosphoprote in l e v e l . As shown i n F igure 18, there was no d i f f e r e n c e i n non-exogenous k inase-mediated phosphory la t ion of the pr imary phosphate acceptor i n c a r d i a c SR, phospholamban, between eu thy ro id c o n t r o l and hypothyro id animals at e i t h e r 2 or 4 weeks pos t - thy ro idectomy. Indeed, the non-k inase-mediated phosphory la t ion o f the SR o f eu thy ro id and thy ro idec tomized groups was e s s e n t i a l l y blank ( lanes A, B, and C ) . Lanes B and C represent the e f f e c t o f ca l c i um i n the r e a c t i o n medium. In the absence o f exogenous ca lmodu l i n , phospholamban was not phosphory lated, i n d i c a t i n g the absence o f endogenous ca lmodu l in i n the SR p r e p a r a t i o n . Phospholamban was present i n the SR p repa ra t i on and was phosphorylated by e i t h e r the c a t a l y t i c subuni t o f cAMP-dependent p r o t e i n k inase or calmodul in-dependent p r o t e i n k i na se . That the p r o t e i n phosphorylated under the present c o n d i t i o n s was phospholamban was ev inced by the c h a r a c t e r i s t i c s h i f t i n mo lecu la r weight f o l l o w i n g heat ing o f the sample. 9 7 F igure 16. The e f f e c t o f thyro idectomy on the calc ium-dependent phosphory la t ion o f SR determined at 10 mM [ C a 2 + ] p r e e i n S R i s o l a t e d two weeks f o l l o w i n g thyro idectomy. Euthy ro id c on t r o l animals ( D ) , thy ro idectomized animals ( • ) . Re su l t s shown are expressed as the mean ± S.E.M. as percentage o f eu thy ro id c o n t r o l (100 % ) , n = 7-8. The acylphosphate va lues obta ined were 75 ± 9 and 65 ± 6 pmoles P/mg SR p r o t e i n , i n the eu thy ro i d and thy ro idectomized groups, r e s p e c t i v e l y . SR Phosphorylation Level % of Euthyroid Control o o o o o o o o o o o o b I— I—I—I—I—I— I— I—I—I—I—I—I 99 F igure 17. The e f f e c t o f thyro idectomy on the calc ium-dependent phosphory la t ion o f SR determined at 10 mM [ C a 2 + ] p r e e i n SR i s o l a t e d four weeks f o l l o w i n g thyro idectomy. Euthy ro id c o n t r o l animals ( • ) , thy ro idectomized animals ( • ) . Resu l t s shown are expressed as the mean ± S.E.M. as percentage o f eu thy ro i d c on t r o l (100 % ) , n = 8/group. The acylphosphate va lues obta ined were 148 ± 24 and 77 ± 13 pmoles P/mg SR p r o t e i n , i n the eu thy ro id and thy ro idectomized groups, r e s p e c t i v e l y . * , s i g n i f i c a n t l y d i f f e r e n t from eu thy ro i d c o n t r o l v a l ue , p < 0.05. SR Phosphorylation Level % of Euthyroid Control - • M U - K U i m v l O O I O O - ' f O O O O O O O O O O O O O O 101 F igure 18. Phosphory la t ion of phospholamban i n eu thy ro i d c o n t r o l (a and b) and hypothyro id (c and d) animals at 2 weeks (a and c) and 4 weeks (b and d ) . Lane A, c o n t r o l , no added ca l c i um or k i na se ; lane B, added C a 2 + no k ina se ; lane C, added C a 2 + no k ina se , b o i l e d * ; lane 0, i n the presence o f the c a t a l y t i c subun i t of cAMP-dependent p r o t e i n k i n a s e 2 ; lane £", p lus c subun i t , b o i l e d ; lane F i n the presence o f exogenous ca lmodul in " * ; and 7ane G, plus ca lmodu l i n , b o i l e d . Mo lecu la r weight standards • r ep re sen t i ng 8.45 kDa, 21.5 kDa and 31.0 kDa were u b i q u i t i n , soybean t r y p s i n i n h i b i t o r and ca rbon ic anhydrase, r e s p e c t i v e l y . * Samples were b o i l e d f o r a pe r iod o f 90 seconds f o l l o w i n g the phosphory la t ion r e a c t i o n . 2 The c a t a l y t i c subunit of cAMP-dependent p r o t e i n k inase was present at 625 pmol/min/mL. Exogenous ca lmodul in was present at 50 units/mL. 102 103 3.2 Studies on the effects of omega-3 fatty acid treatment of STZ-induced diabetic rats. 3.2a General characteristics of the omega-3 fatty acid-treated and untreated non-diabetic control and streptozocin-induced diabetic rats. The animal model o f i n su l in -dependent d i abetes m e l l i t u s chosen f o r these s tud ie s was the s t r e p t o z o c i n - (STZ) induced d i a b e t i c male Wi s ta r r a t model. The animals were t r e a t ed w i th STZ and made d i a b e t i c as de sc r i bed i n the Methods. C h a r a c t e r i s t i c a l l y , the untreated d i a b e t i c r a t s e x h i b i t e d s igns t y p i c a l of t h i s model o f IDDM d i abe te s ; po l yphag ia , po l ydyp s i a and p o l y u r i a were apparent throughout the study p e r i o d . Treated d i a b e t i c animals e x h i b i t e d p a r a l l e l s igns of un con t r o l l ed IDDM. The data represented i n F igure 19 i n d i c a t e that the dose o f STZ chosen was app rop r i a te and e f f i c a c i o u s f o r the i nduc t i on o f a d i a b e t i c s t a t e . I n s u l i n and g lucose concent ra t i on s of plasma c o l l e c t e d at s a c r i f i c e are shown i n F igure 19. The data c l e a r l y i n d i c a t e tha t a l l animals t r e a t e d w i th STZ were rendered d i a b e t i c . I n s u l i n concent ra t i on s o f both groups o f STZ- t reated r a t s were s i g n i f i c a n t l y lower than c o n t r o l . Two way a n a l y s i s o f va r i ance i n d i c a t e d a s i g n i f i c a n t (p < 0.05) group e f f e c t between c o n t r o l and d i a b e t i c groups when e i t h e r i n s u l i n or g lucose concent ra t i on s were con s ide red . The data a l s o show tha t omega-3 f a t t y a c i d treatment d i d not a f f e c t g lucose homeostasis o f c o n t r o l s and d i d not exacerbate the hyperglycemia o f STZ-induced d i abe te s . The data represented i n F igure 20 show tha t STZ-induced d i a b e t i c r a t s f a i l e d to grow to the same extent as age matched n o n - d i a b e t i c c o n t r o l 104 F igure 19. Plasma i n s u l i n and g lucose concent ra t i on s o f c o n t r o l ( O ) , c o n t r o l t r e a t e d (13), d i a b e t i c ((•) and d i a b e t i c t r e a t e d (B9) an imal s . Resu l t s shown are the mean ± S.E.M., n = 11-12/group, * , s i g n i f i c a n t l y d i f f e r e n t from non -d i abe t i c c o n t r o l va l ue , p < 0.05. P l a sma Glucose (mM) P la sma Insulin (/xU/mL) 106 F igure 20. Average d a i l y body weight ( i n grams) o f c o n t r o l ( o ) , c on t r o l t r e a t e d (•), d i a b e t i c (&) and d i a b e t i c t r e a t e d (A) male Wi s ta r r a t s . Day 1 represents the day o f e i t h e r v e h i c l e ( c o n t r o l ) or s t r e p t o z o c i n ( d i a b e t i c ) i n j e c t i o n , n = 11-12 animals/group. Data shown are the means. 107 550 T 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 Time (Day of Study) 108 animals dur ing the exper imental p e r i o d . The f i g u r e a l s o shows t ha t omega-3 f a t t y a c i d treatment d i d not a f f e c t body weights o f e i t h e r c o n t r o l - or d i a b e t i c - t r e a t e d r a t s . The body weight o f the c o n t r o l and c o n t r o l - t r e a t e d animals at s a c r i f i c e were 515 ± 15g and 532 ± 9g, r e s p e c t i v e l y . The body weights o f the d i a b e t i c and d i a b e t i c - t r e a t e d r a t s at s a c r i f i c e were s i g n i f i c a n t l y (p < 0.05) lower than con t r o l at 385 ± 25g and 403 ± 14g, r e s p e c t i v e l y . The s i m i l a r body weights o f the t r e a t e d and unt reated animals i s i n accord w i th the observat ion made dur ing the study t ha t omega-3 f a t t y a c i d treatment a f f e c t e d n e i t h e r food nor water consumption of e i t h e r c o n t r o l or d i a b e t i c groups. H y p e r t r i g l y c e r i d e m i a i s t y p i c a l l y man i fes t i n the STZ-induced model of IDDM; i n t h i s s tudy, plasma t r i g l y c e r i d e concent ra t i on s of d i a b e t i c s were s i g n i f i c a n t l y (p < 0.05) e l eva ted above non -d i abe t i c c on t r o l va lues (F igure 21). Omega-3 f a t t y a c i d treatment was a s soc i a ted w i th a h y p o t r i g l y c e r i d e m i c response i n the d i a b e t i c r a t s , although t h i s e f f e c t was not s i g n i f i c a n t . F igure 22 shows t ha t the plasma c h o l e s t e r o l concen t ra t i on o f untreated d i a b e t i c animals was h igher than non -d i abe t i c c o n t r o l s . In omega-3 f a t t y a c i d - t r e a t e d d i a b e t i c r a t s , plasma c h o l e s t e r o l concent ra t i on s were e leva ted above non -d i abe t i c c on t r o l and not a f f e c t e d by the omega-3 f a t t y a c i d t reatment . There was no e f f e c t o f omega-3 f a t t y ac id on plasma c h o l e s t e r o l concen t ra t i on s o f the n o n - d i a b e t i c c o n t r o l t r e a t ed an imals . 109 F igure 21. Plasma t r i g l y c e r i d e concen t ra t i on (mM) o f c o n t r o l ( • ) , c on t r o l t r e a t e d ( S I ) , d i a b e t i c ( • ) and d i a b e t i c t r e a t e d ( S i ) male Wi s ta r r a t s . Resu l t s shown are the mean ± S.E.M., n = 11-12 animals/group. * , s i g n i f i c a n t l y d i f f e r e n t from non -d i abe t i c c on t r o l va l ue , p < 0.05. 1 1 0 10.0 9.0 8.0 7.0 6.0 5.0 4.04-3.0 2.0 1.0 0.0 i l l l l l 111111 I l l F igure 22. Plasma c h o l e s t e r o l concen t ra t i on (mM) o f c o n t r o l ( Q ) , c on t r o l t r e a t e d (G3), d i a b e t i c ( • ) and d i a b e t i c t r e a t e d (B8) male Wi s ta r r a t s . Resu l t s shown are the mean ± S.E.M., n = 11-12 animals/group. * , s i g n i f i c a n t l y d i f f e r e n t from n o n - d i a b e t i c c on t r o l va l ue , p < 0.05 1 1 2 113 3.2b Isolated working heart function of omega-3 fatty acid-treated and untreated non-diabetic control and streptozocin-induced diabetic rats. The e f f e c t o f d iabetes and o f omega-3 f a t t y a c i d treatment on i s o l a t e d working heart f u n c t i o n i s shown i n F igures 23 - 25. Peak l e f t v e n t r i c u l a r developed pressure (LVDP, F igure 23) o f unt reated d i a b e t i c r a t s was s i g n i f i c a n t l y lower than tha t o f c o n t r o l s at each LAFP assayed (7.5 cm H2O - 20 cm H2O). S t a t i s t i c a l a na l y s i s o f the data by two-way a n a l y s i s o f va r i ance conf i rmed a s i g n i f i c a n t group e f f e c t at each o f these LAFP, and a l so e s t a b l i s h e d t ha t there was a s i g n i f i c a n t group-treatment i n t e r a c t i o n at each LAFP above 12.5cm H2O. Omega-3 f a t t y a c i d treatment o f STZ-induced d i a b e t i c r a t s f o r f ou r weeks reduced the degree to which the c a r d i a c dy s func t i on developed. Th i s i s apparent i n F igure 23 i n view o f the f a c t t ha t the c a r d i a c f u n c t i o n curve of the d i a b e t i c - t r e a t e d animals i s s upe r i o r to tha t o f the untreated d i a b e t i c s . F igure 23 a l s o shows tha t omega-3 f a t t y a c i d treatment d i d not a f f e c t peak LVDP o f non -d i abe t i c c o n t r o l W i s ta r r a t s . The data represented i n F igures 24 and 25 i l l u s t r a t e the e f f e c t o f d iabetes and the omega-3 treatment p ro toco l on the maximal r a t e o f change o f l e f t v e n t r i c u l a r pressure dur ing s y s t o l e ( + d P / d t m a x > ) , and the maximal r a t e o f change o f l e f t v e n t r i c u l a r pressure dur ing d i a s t o l e ( - d P / d t m a X i ) , r e s p e c t i v e l y . Two way ana l y s i s of va r i ance o f these data i n d i c a t e s tha t the re was a s i g n i f i c a n t group-treatment e f f e c t at each LAFP above 12.5 cm H2O. The data o f F igures 24 and 25 suggest t ha t the improved c a r d i a c f u n c t i o n o f the t r e a t ed d i a b e t i c animals i s a s soc i a ted w i t h i nc reases i n both s y s t o l i c and d i a s t o l i c f u n c t i o n . 114 F igure 23. Peak l e f t v e n t r i c u l a r developed pressure (mm Hg) vs l e f t a t r i a l f i l l i n g pressure (cm H2O) of c on t r o l ( 0 ) , c o n t r o l t r e a t e d (•), d i a b e t i c (A) and d i a b e t i c t r e a t e d (A) male Wi s ta r r a t s , n = 11-12 animals/group. Data shown are mean ± S.E.M.. * , s i g n i f i c a n t l y d i f f e r e n t from con t r o l un t rea ted , p< 0.05. * * , s i g n i f i c a n t l y d i f f e r e n t from d i a b e t i c un t rea ted , p < 0.05. 1 1 5 175-, 7 5 -I 1 1 1 1 1 1 : 1 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 Left Atrial Fill ing P re s su re ( c m H 2 0 ) 116 F igure 24. Maximum r a t e of change o f l e f t v e n t r i c u l a r pressure dur ing s y s t o l e ( + dP/dt ) vs l e f t a t r i a l f i l l i n g pressure of c o n t r o l ( o ) , c o n t r o l t r e a t ed (•), d i a b e t i c (A) and d i a b e t i c t r e a t e d (A) male Wi s ta r r a t s , n = 11-12 animals/group. Data shown are mean ± S.E.M.. * , 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 un t rea ted , p < 0.05. , s i g n i f i c a n t l y d i f f e r e n t from d i a b e t i c un t rea ted , p < 0.05. 1 1 7 118 F igure 25. Maximum r a t e o f change of l e f t v e n t r i c u l a r pressure dur ing d i a s t o l e o f c on t r o l ( o ) , c on t r o l t r e a t e d (•), d i a b e t i c (A) and d i a b e t i c t r e a t ed (•) male Wi s ta r r a t s , n = 11-12 animals/group. Data shown are mean ± S.E.M.. * , s i g n i f i c a n t l y d i f f e r e n t from con t r o l un t rea ted , p < 0.05. , s i g n i f i c a n t l y d i f f e r e n t from d i a b e t i c un t rea ted , p < 0.05. 119 "2000-1 1 1 1 , , . 1 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 Left Atrial Riling Pressure (cm h^O) 120 To f u r t h e r assess the e f f e c t of the omega-3 f a t t y a c i d treatment and d iabetes on myocardia l f u n c t i o n , a o r t i c output and pulmonary a r t e r y output were determined. These determinat ions were made i n p a r a l l e l w i t h the l e f t v e n t r i c u l a r f u n c t i o n a l assessments desc r ibed above. The a o r t i c output of t r e a t e d and untreated non -d i abe t i c c on t r o l and STZ-induced d i a b e t i c groups i s shown in F igure 26. The data of F igure 26 i n d i c a t e t ha t the re was a s i g n i f i c a n t (p < 0.05) depress ion i n the a o r t i c output o f hear t s i s o l a t e d from untreated STZ -d i abe t i c an imals , c on s i s t en t w i th the depressed myocardia l f u n c t i o n of these an imals . A o r t i c output o f omega- 3 f a t t y a c i d - t r e a t e d animals was s i g n i f i c a n t l y (p < 0.05) g r ea te r than t ha t o f the untreated d i a b e t i c an imals . When the i s o l a t e d heart i s perfused i n the working mode (see Methods f o r d e s c r i p t i o n ) the coronary venous f l ow d r a i n s i n t o the r i g h t v e n t r i c l e v i a the coronary s inus (except f o r a small f r a c t i o n en te r i n g the v e n t r i c l e s d i r e c t l y v i a Thebesian v e i n s ) . The coronary f l ow i s subsequently emptied from the r i g h t v e n t r i c l e v i a the pulmonary a r t e r y , cannulated (as desc r ibed i n the Methods) to monitor coronary f l ow dur ing the working heart exper iments. Pulmonary {coronary) f l ow o f the fou r groups s tud ied i s represented by the data o f F igure 27. The coronary f low r a t e was s i g n i f i c a n t l y (p < 0.05) depressed i n the unt reated d i a b e t i c hea r t s , and s i g n i f i c a n t l y (p < 0.05) improved i n the t r e a t e d d i a b e t i c an imals . Indeed, omega-3 f a t t y a c i d treatment o f the d i a b e t i c animals c o r r e c ted the coronary f low r a t e to the non -d i abe t i c c o n t r o l l e v e l . There was no e f f e c t o f the omega-3 f a t t y a c i d treatment on e i t h e r a o r t i c or pulmonary (coronary) f l ow o f non -d i abe t i c an imal s . 121 F igure 26. The e f f e c t of STZ-induced d iabetes and omega-3 f a t t y a c i d treatment on a o r t i c output (mL bu f fe r/m inu te ) . c o n t r o l ( o ) , c o n t r o l t r e a t e d (•), d i a b e t i c (A) and d i a b e t i c t r e a t e d (A) male Wi s ta r r a t s . Data shown are mean ± S.E.M., n = 8-11 animals/group. * , s i g n i f i c a n t l y d i f f e r e n t from con t r o l ** unt rea ted , p < 0.05. , s i g n i f i c a n t l y d i f f e r e n t from d i a b e t i c un t rea ted , p < 0.05. 1 2 2 2 0 . 0 17.5 15.0 o LZ 12.5 o *-£ o < 10.0 7.5 5.0 7 .5 10.0 1 2 . 5 15 .0 1 7 . 5 20.0 2 2 . 5 Left Atrial Filling Pressure (cm H2O) 123 F igure 27. The e f f e c t o f STZ-induced d iabetes and omega-3 f a t t y a c i d treatment on pulmonary (coronary) f l ow (mL bu f fe r/m inu te ) , c o n t r o l ( o ) , c on t r o l t r e a t ed (•), d i a b e t i c (£) and d i a b e t i c t r e a t e d (4) male Wi s ta r r a t s , n = 8-11 animals/group. Data shown are mean ± S.E.M.. * , 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 un t rea ted , p < 0.05. * * , s i g n i f i c a n t l y d i f f e r e n t from d i a b e t i c un t rea ted , p < 0.05. 124 2 0 . 0 - r 7 . 5 A 1 1 1 1 1 r-7 . 5 1 0 1 2 . 5 1 5 1 7 . 5 2 0 Left Atrial Filling Pressure (cm H2O) 125 3.2c Sarcop lasmic r e t i c u l u m ca l c i um t r a n s p o r t o f omega-3 f a t t y a c i d t r e a t e d and untreated n o n - d i a b e t i c c o n t r o l and STZ-induced d i a b e t i c an imal s . The SR i s cons idered to p lay an important r o l e i n c a r d i a c r e l a x a t i o n through a c t i v e s eque s t r a t i on o f myoplasmic ca l c i um dur ing d i a s t o l e . S ince d i a s t o l i c f u n c t i o n o f omega-3 f a t t y a c i d - t r e a t e d d i a b e t i c r a t s was improved above t ha t o f untreated d i a b e t i c s (F igure 25), SR ca l c i um t r an spo r t a c t i v i t y was determined. The e f f e c t o f the d i a b e t i c s t a t e and omega-3 f a t t y a c i d treatment on SR ca lc ium t r an spo r t i s shown i n Table 3. The r a t e of ca l c i um t r an spo r t was determined at two f r e e ca l c i um concen t r a t i o n s , 0.1 /iM and 5.3 /iM. These ca l c ium concent ra t i on s were used s i n ce 0.1 /iM i s i n the range o f the Kca2+ and 5.3 /iM i s a concen t ra t i on at which V m a x can be determined (data not shown). Card iac SR C a 2 + - t r a n s p o r t at 0.1 /iM C a 2 + p r e e was s i g n i f i c a n t l y reduced i n untreated d i a b e t i c r a t s . Th i s depress ion i n SR Ca - t r a n s p o r t was prevented by the omega-3 f a t t y a c i d t reatment . Table 3 i n d i c a t e s t ha t s i m i l a r changes i n ca l c ium t r an spo r t were observed when C a 2 + - t r a n s p o r t was determined at 5.3 /iM C a 2 + p r e e . The d i f f e r e n c e s i n ca l c i um t r an spo r t r a te s amongst the fou r groups at 5.3 /iM were not due to d i f f e r e n c e s i n n o n - s p e c i f i c oxa late- independent ca l c i um b ind ing (data not shown). These va lues averaged < 3.5% of t o t a l oxalate-dependent ca l c i um t r an spo r t i n a l l cases. I t was a l so determined tha t non-ATP-dependent ca l c i um b ind ing was n e g l i g i b l e and d i d not c o n t r i b u t e to the observed ca l c i um t r an spo r t r a te s (data not shown). 126 Table 3. The e f f e c t o f d iabetes and omega-3 f a t t y a c i d treatment on i s o l a t e d c a r d i a c sarcop lasmic r e t i c u l u m ca l c i um t r an spo r t a c t i v i t y . C a 2 + - T r a n s p o r t A c t i v i t y (nmoles/mg/minute) Group (n) Contro l (6) Cont ro l Treated (6) D i a b e t i c (6) D i a b e t i c Treated (6) 0.1 /zM [ C a 2 + ] F r e e 16.13 ± 0.76 15.33 ± 0.76 11.65 ± 0.76* 5.3 /iM [ C a 2 + ] F r e e 59.22 ± 2.80 60.82 ± 2.80 50.26 ± 1.55* 14.63 ± 0.76 ** 66.55 ± 2.80 ** Resu l t s shown are the mean ± S.E.M.. , s i g n i f i c a n t l y d i f f e r e n t from * * c o n t r o l . , s i g n i f i c a n t l y d i f f e r e n t from untreated d i a b e t i c , (p < 0.05) 127 3.2d Card iac and sa rcop lasmic r e t i c u l u m phospho l i p id acy l compos i t ion o f n o n - d i a b e t i c and d i a b e t i c animals t r e a t e d w i t h omega-3 f a t t y a c i d . S ince myocardia l membrane l i p i d compos i t ion can i n f l u e n c e c a r d i a c f u n c t i o n , i t was o f i n t e r e s t to a s c e r t a i n i f the observed a l t e r a t i o n s i n i s o l a t e d working heart f u n c t i o n , a o r t i c and pulmonary f l ow and SR ca l c i um t r an spo r t a c t i v i t y cou ld be a t t r i b u t e d to phospho l i p id acy l compos i t iona l changes. There fo re , a subsequent s e r i e s of experiments were designed to i n v e s t i g a t e t h i s p o s s i b i l i t y . The animals used were subjected to i d e n t i c a l STZ/vehic le dosage and a d m i n i s t r a t i o n , and the i d e n t i c a l Promega R (omega-3 f a t t y a c i d ) treatment p ro toco l (see Methods). The plasma g lucose data o f F i gu re 28 c l e a r l y demonstrate tha t the STZ- t reated animals were hyperglycemic ( s i g n i f i c a n t l y increased plasma g lucose c oncen t r a t i o n s , p < 0.05) and tha t g lucose homeostasis of e i t h e r the n o n - d i a b e t i c or the d i a b e t i c animals was not a f f e c t e d by the omega-3 f a t t y a c i d t reatment . The average d a i l y body weight data of F igure 29 a l s o i n d i c a t e t ha t the STZ-induced d i a b e t i c animals f a i l e d to t h r i v e to the same extent as age-matched non -d i abe t i c c o n t r o l s . As w e l l , omega-3 f a t t y a c i d t reatment d i d not a f f e c t the growth o f the non -d i abe t i c c o n t r o l s nor the d i a b e t i c an imal s . D iabetes - induced h y p e r t r i g l y c e r i d e m i a (F igure 30 A) was mani fes t i n both of the d i a b e t i c groups (p <0.05), and although omega-3 f a t t y a c i d treatment was a s soc i a ted w i th a 61% reduc t i on i n the mean plasma t r i g l y c e r i d e c o n c e n t r a t i o n , t h i s r educ t i on was not s i g n i f i c a n t . Omega-3 f a t t y a c i d treatment d i d not a f f e c t the plasma t r i g l y c e r i d e concen t r a t i on of the non-d i a b e t i c c o n t r o l an imals . In agreement w i th the r e s u l t s o f the study i n v o l v i n g c a r d i a c and SR f u n c t i o n , plasma c h o l e s t e r o l concen t ra t i on s o f both d i a b e t i c groups were s i g n i f i c a n t l y (p < 0.05) inc reased above the non-128 F igure 28. Plasma g lucose concen t ra t i on (mM) o f n on -d i abe t i c and d i a b e t i c t r e a t e d and untreated an imals . Cont ro l ( • ) , c o n t r o l t r e a t e d ( K l ) , d i a b e t i c ((•) and d i a b e t i c t r e a t e d ({§§) male Wi s ta r r a t s . Data shown are mean ± S.E.M., n = 11-12 animals/group, * , s i g n i f i c a n t l y d i f f e r e n t from non -d i abe t i c c o n t r o l . 1 2 9 30 25 20 154-10 0 V •£ „ TJ ^ V ^ 130 F igure 29. Average d a i l y body weight ( i n grams) o f c o n t r o l ( o ) , c o n t r o l t r e a t e d (•), d i a b e t i c (&) and d i a b e t i c t r e a t e d (A) male Wi s ta r r a t s . Day 1 represents the day o f e i t h e r v e h i c l e ( c o n t r o l ) or s t r e p t o z o c i n ( d i a b e t i c ) i n j e c t i o n , n = 11-12 animals/group. Data shown are mean ± S.E.M.. 1 3 1 500 -r 450 400 "<D 350 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 Time ( D a y s ) 132 F igure 30. The e f f e c t of omega-3 f a t t y a c i d treatment on plasma t r i g l y c e r i d e (A) and c h o l e s t e r o l (B) c oncen t r a t i on s . Cont ro l ( Q ) , c on t r o l t r e a t e d (63), d i a b e t i c ( • ) and d i a b e t i c t r e a t e d (j&) male Wi s ta r r a t s . Data shown are mean ± S.E.M., n = 11-12 animals/group. * , s i g n i f i c a n t l y d i f f e r e n t from non-d i a b e t i c c o n t r o l . 1 3 3 10.0 9.0 8.0 7.0 ^ 6 . 0 "I 5.0 ^ 4 . 0 3.0 2.0 1.0-0.0-3.0 2.5 2.0 1.5 1.0 0.5 4 0.0 134 d i a b e t i c concen t r a t i on (F igure 30 B) and there was no e f f e c t o f omega-3 f a t t y a c i d on the plasma c h o l e s t e r o l l e v e l s o f e i t h e r n o n - d i a b e t i c or d i a b e t i c an imal s . Pho spha t i d y l cho l i ne (PC) and phosphat idy lethanolamine (PE) were i s o l a t e d from l e f t v e n t r i c u l a r t i s s u e and the pho spho l i p i d acy l compos i t ion determined. The r e s u l t s o f these determinat ions are presented i n F igures 31 and 32, r e s p e c t i v e l y . These are histograms o f the r e l a t i v e amount o f the i n d i v i d u a l phospho l i p id acy l s pec i e s . The f i g u r e s demonstrate t ha t there was no s i g n i f i c a n t e f f e c t o f the omega-3 f a t t y a c i d treatment on the amount of omega-3 f a t t y a c i d i nco rpora ted i n t o c a r d i a c PC and PE. The changes i n the r e l a t i v e and abso lute amounts o f omega-6 and omega-3 f a t t y ac id s i n PC and PE are summarized i n Table 4. The phospho l i p id acy l compos i t ion o f c a r d i a c SR was a l s o determined i n each o f the fou r groups s t ud i ed . In these exper iments, the re was i n s u f f i c i e n t s t a r t i n g mate r i a l (SR) to a f f o r d separa t i on o f the i n d i v i d u a l phospho l i p id s pec i e s ; t h e r e f o r e , the phospho l ip id s were i s o l a t e d as a group and the acy l compos i t ion determined. As shown i n F igure 33, the omega-3 f a t t y a c i d treatment of the non -d i abe t i c c on t r o l and STZ-induced d i a b e t i c animals a f f e c t e d the omega-3 f a t t y a c i d content of the phospho l i p id s o f the SR membrane. I t i s apparent from F igure 33 tha t EPA (C20:5, n-3) was not de t e c t ab l e i n the c a r d i a c SR of untreated con t r o l and d i a b e t i c an imals . Although the changes were r e l a t i v e l y s m a l l , EPA was de t e c t ab l e i n the SR o f the omega-3 f a t t y a c i d - t r e a t e d con t r o l and d i a b e t i c an imal s , account ing f o r 1.58% and 0.65% o f the t o t a l phospho l i p id spec ies p resent , r e s p e c t i v e l y . DHA was de t e c t ab l e i n the SR o f untreated c o n t r o l and d i a b e t i c an imal s , 135 F igure 31. V e n t r i c u l a r pho spha t i dy l cho l i ne acy l compos i t ion o f c o n t r o l ( O ) , c on t r o l t r e a t ed (C3), d i a b e t i c ( • ) and d i a b e t i c t r e a t e d ( 0 ) male Wis tar r a t s . Data shown are mean ± S.E.M. ( e r r o r bars not v i s i b l e on the graph are present but not r e so l ved by the f i g u r e ) . 1 3 6 137 F igure 32. V e n t r i c u l a r phosphat idy lethanolamine acy l compos i t ion o f c o n t r o l ( O ) , c on t r o l t r e a t ed ( I S ) , d i a b e t i c ( • ) and d i a b e t i c t r e a t e d ( 0 ) male Wi s ta r r a t s . Data shown are mean ± S.E.M. ( e r r o r bars not v i s i b l e on the graph are present but not r e so l ved by the f i g u r e ) . 1 3 8 CJ—6 CJ—3 GJ-6 CJ—3 co 3 139 Table 4. E f f e c t of omega-3 f a t t y a c i d treatment on the r e l a t i v e 1 and a b s o l u t e 2 changes i n the amount o f omega-6 and omega-3 f a t t y a c i d i n v e n t r i c u l a r pho spha t i d y l cho l i ne and phosphat idy lethanolamine and SR t o t a l p h o s p h o l i p i d . Cont ro l vs Contro l Treated D i a b e t i c vs D i a b e t i c Treated D i a b e t i c vs Contro l PC n-6 n-3 -17.9/7.0 +28.6/2.5 +26.6/8.0 +27.2/1.8 •22.7/8.9 -24.7/2.2 PE n-6 n-3 -22.2/7.0 +10.2/3.6 +6.7/2.2 +32.9/9.9 N.C. •15.9/5.7 SR n-6 n-3 -8.9/3.3 +27.1/3.4 -5.5/2.0 +20.7/2.9 N.C. +13.8/1.7 R e l a t i v e Amount represents the % change i n the t o t a l amount o f e i t h e r omega-6 or omega-3 f a t t y a c i d between groups. (-) = r e l a t i v e r e d u c t i o n . (+) = r e l a t i v e inc rease Abso lute amount represents the ac tua l % d i f f e r e n c e i n the t o t a l amount o f e i t h e r omega-6 or omega-3 f a t t y a c i d . No change. 140 F igure 33. Sarcoplasmic r e t i c u l u m phospho l i p id acy l compos i t ion o f c on t r o l ( • ) , c on t r o l t r e a t ed (£3), d i a b e t i c ( • ) and d i a b e t i c t r e a t e d (£3) male Wi s ta r r a t s . Data shown are mean ± S.E.M. ( e r r o r bars not v i s i b l e on the graph are present but not r e so l ved by the f i g u r e ) . 1 4 1 C16:0 C18:0 C18:1 C18:1 C18:2 C20:4 C20:5 C22:6 G)—6 CJ — 6 G)—3 CJ —3 142 account ing f o r 12.51% and 14.31% of the t o t a l pho spho l i p i d s pec i e s , r e s p e c t i v e l y . The omega-3 f a t t y a c i d treatment inc reased the abso lute amount o f DHA i n both the c on t r o l and d i a b e t i c groups by 1.81% and 2.29%, r e s p e c t i v e l y , o f the t o t a l f a t t y a c i d p o o l . The e f f e c t o f d i abetes and the omega-3 f a t t y a c i d treatment on c a rd i a c SR pho spho l i p i d acy l spec ie s i s summarized i n Table 4, which shows tha t there was a 27.1% and 20.7% inc rease i n the r e l a t i v e amount o f t o t a l omega-3 f a t t y acy l content of c o n t r o l - t r e a t e d and d i a b e t i c - t r e a t e d c a r d i a c SR pho spho l i p i d , r e s p e c t i v e l y , when compared to t h e i r r e s pec t i v e untreated c o n t r o l s . The e f f e c t o f d iabetes and the omega-3 f a t t y a c i d treatment on the c a r d i a c SR c h o l e s t e r o l concen t ra t i on are shown i n F igure 34. The data i n d i c a t e tha t there was no e f f e c t of d iabetes or omega-3 f a t t y a c i d treatment on SR c h o l e s t e r o l 143 F igure 34. Sarcoplasmic r e t i c u l u m membrane c h o l e s t e r o l concen t ra t i on s o f the c on t r o l (O), c on t r o l t r e a t ed (E3), d i a b e t i c ( • ) and d i a b e t i c t r e a t ed (fig) male Wi s ta r r a t s . Resu l t s shown are the mean o f 6 determinat ions/group. Standard e r r o r bars have been removed f o r c l a r i t y o ro o SR [Cholesterol] (nmoles/mg protein) o O CO O O O ro o H 145 4. Discussion The s t ud i e s presented are concerned w i th the e f f e c t s o f two d i f f e r e n t endocr ine d i sease s t a t e s , d iabetes m e l l i t u s and hypothyro id i sm, on c a r d i a c sa rcop lasmic r e t i c u l u m (SR) ca l c i um t r an spo r t a c t i v i t y from the pe r spec t i ve o f the r e g u l a t i o n o f t h i s a c t i v i t y by changes i n the l i p i d compos i t ion o f the SR membrane. The f u n c t i o n a l importance of the l i p i d b i l a y e r i n the r e g u l a t i o n o f i n t e g r a l membrane p r o t e i n a c t i v i t y has become i n c r e a s i n g l y apparent s i n ce S inger and N ico l son (1971) in t roduced the f l u i d mosaic l i p i d b i l a y e r concept of c e l l membrane s t r u c t u r e . The a c t i v i t y of i n t e g r a l membrane p r o te i n s can be i n f l uenced by the compos i t ion of the membrane such tha t membrane fluidity (or i t s i n ve r se , viscosity) impinges upon enzyme/transporter a c t i v i t y . In t h i s rega rd , membrane f l u i d i t y has been s tud ied from the pe r spec t i ve of the e f f e c t s o f membrane c h o l e s t e r o l (Almedia et a l , 1984) and temperature (B lazyk et a l , 1985). For example, i n c r e a s i n g membrane c h o l e s t e r o l or l ower ing of r e a c t i o n temperature reduces membrane f l u i d i t y and reduces ATPase a c t i v i t y . Membrane f l u i d i t y a l so a f f e c t s Ca 2 + -pump p r o t e i n r o t a t i o n w i t h i n the membrane (Squ i re e t a l , 1988 ) . A more d i s c r e t e and s p e c i f i c r o l e f o r c e r t a i n membrane pho spho l i p i d spec ies as mediators o f s i gna l t r an sduc t i on mechanisms has been ev inced by the r o l e o f phosphat idy l i n o s i t o l i n a i - a d r e n e r g i c r ecep to r s i g n a l l i n g (Gi lman, 1986). There fo re , i t i s apparent tha t membrane phospho l i p id s not on ly p rov ide a b a r r i e r between 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 environments, or between lumenal spaces o f i n t r a c e l l u l a r o r gane l l e s and the i n t r a c e l l u l a r m a t r i x , but p lay a more dynamic and f u n c t i o n a l r o l e by p r o v i d i n g the proper phys icochemical environment f o r i n t e g r a l membrane p r o t e i n s to f u n c t i o n . 146 As p r e v i o u s l y i n d i c a t e d , the endocr ine d i sease s t a t e s o f i n t e r e s t to t h i s p r o j e c t are a s soc i a ted w i th we l l de sc r ibed c a r d i a c f u n c t i o n a l changes. S u b c e l l u l a r and biochemical a l t e r a t i o n s w i t h i n the cardiomyocyte proposed to c o n t r i b u t e , or be r e spon s i b l e f o r , the f u n c t i o n a l changes observed i n each d i sease s t a t e are d i v e r s e . Although there has been ex ten s i ve study o f b iochemical parameters known to i n f l u e n c e c a r d i a c f u n c t i o n , the r e l a t i v e importance o f membrane a l t e r a t i o n s has not been complete ly de f i ned f o r e i t h e r hypothyro id i sm or d iabetes m e l l i t u s . There fo re , t h i s p r o j e c t was concerned w i t h the e f f e c t s of membrane l i p i d changes on c a r d i a c SR ca l c i um t r an spo r t f u n c t i o n from two d i f f e r e n t pe r s pec t i v e s : i ) the i n f l u e n c e o f hypothyro id i sm on SR ca l c i um t r an spo r t and endogenous long cha in acy l c a r n i t i n e a s soc i a ted w i th the SR, i n order to d i s c e r n the p o t e n t i a l r e gu l a t o r y r o l e o f these l i p i d metabo l i c i n te rmed ia te s on SR ca l c i um t r a n s p o r t , and i i ) the i n f l u e n c e o f an omega-3 f a t t y a c i d treatment on c a r d i a c and SR ca l c i um t r an spo r t f u n c t i o n i n an animal model o f IDDM, i n order to d i s c e r n whether the d iabetes - i nduced cardiomyopathy i s i n f l uenced by the t reatment . The omega-3 f a t t y a c i d s t ud i e s f u r t h e r proposed to d e l i n e a t e the e f f e c t s o f the t reatment, and the p o s s i b i l i t y t ha t pho spho l i p i d a l t e r a t i o n s may be invo l ved i n p r e v i ou s l y de sc r i bed a l t e r e d SR ca l c ium t r an spo r t i n d i abe te s , by s tudy ing SR ca l c i um t r an spo r t and phospho l i p i d acy l compos i t ion . The r e s u l t s from these s t ud i e s w i l l be d i scus sed w i th a view to an understanding o f the r o l e o f l i p i d s i n i n t e g r a l membrane p r o t e i n f u n c t i o n and SR ca l c i um t r an spo r t f u n c t i o n . 147 4.1 The E f f e c t o f Hypothyroid ism on SR Calc ium Transport and the Role o f Endogenous Long Chain A c y l c a r n i t i n e . Myocard ia l changes are amongst the more profound p h y s i o l o g i c a l a l t e r a t i o n s observed i n hypothyro id i sm and have been recogn ized to occur i n the d i sease s i nce the observat ions of Zondek (1918). In g e n e r a l , t h y r o i d hormone a c t i o n i n human phys io logy i s p l e i o t r o p i c and the hormone's myocardia l e f f e c t s are s i m i l a r l y d i v e r s e . Changes i n c a r d i a c autonomic r e cep to r s , v e n t r i c u l a r myosin ATPase and isoenzyme d i s t r i b u t i o n are we l l c h a r a c t e r i z e d parameters a f f e c t e d by a l a c k o f c i r c u l a t i n g t h y r o i d hormone, and cons idered r e l e van t to observed changes i n c a r d i a c f u n c t i o n . The e f f e c t o f hypothyro id i sm on c a r d i a c SR ca l c i um t r an spo r t and ATPase a c t i v i t i e s has been the sub ject o f prev ious i n v e s t i g a t i o n s . Hypothyroid ism leads to a s i g n i f i c a n t r educ t i on i n the r a t e o f c a r d i a c SR ca l c i um t r an spo r t in situ i n hypothyro id p a p i l l a r y muscle (MacKinnon et a l , 1988) and in vitro us ing i s o l a t e d SR membrane p repara t i ons (Suko, 1971; Suko, 1973; Takacs et a l 1985; Rodgers et a l , 1986). The mechanism re spon s i b l e f o r at tenuated SR ca l c i um t r an spo r t a c t i v i t y has not been d e f i n e d . The aim o f the present s t ud i e s was to determine i f endogenous myocardia l a c y l c a r n i t i n e a s soc ia ted w i th the SR i s i nvo l ved i n the p r e v i o u s l y desc r ibed SR ca l c i um t r an spo r t a c t i v i t y . The animal model used to exp lo re the e f f e c t s o f hypothyro id i sm on c a r d i a c SR ca l c i um t r an spo r t and the po s s i b l e r e g u l a t o r y r o l e o f endogenous acy l c a r n i t i n e i n SR ca l c i um t r an spo r t was the s u r g i c a l l y thy ro idec tomized 148 male Wi s ta r r a t . I t was f i r s t important to e s t a b l i s h t ha t the commercial thyro idectomy procedure rendered the animals hypothy ro id . As presented i n the Results, thyro idectomy produced s igns i n d i c a t i n g t ha t the animals were hypothy ro id : animals had reduced body weights and v e n t r i c u l a r we ight s . Based on these gross phy s i ca l a l t e r a t i o n s , which are c o n s i s t e n t w i th s igns and symptoms o f hypothyro id i sm, the s u r g i c a l l y thy ro idec tomized -hypothy ro id model chosen was cons idered s u i t a b l e f o r the present i n v e s t i g a t i o n s . S ince the study i nvo l ved a comparison o f ca l c i um t r an spo r t a c t i v i t y o f an i s o l a t e d membrane p repara t i on enr i ched i n SR from eu thy ro id and thy ro idec tomized groups, i t was necessary to a s c e r t a i n whether the re were s i g n i f i c a n t d i f f e r e n c e s i n the cha rac te r ( i . e . contaminat ion by other c e l l u l a r membranes) o f the membrane p repara t i ons i s o l a t e d . M i t ochond r i a l cytochrome c ox idase a c t i v i t y was determined i n v e n t r i c u l a r homogenates and the SR o f the eu thy ro id and hypothyro id groups. The r e s u l t s o f these determinat ions (Table 1) i n d i c a t e d tha t the SR p repa ra t i on was not devoid o f m i tochondr i a l membrane contaminat ion . However, the percentage reduc t i on (97%-99%) i n m i tochondr i a l membrane contaminat ion determined by comparison o f homogenate to SR cytochrome c ox idase a c t i v i t y , was o f the same order f o r both the eu thy ro id and thy ro idectomized groups. The SR p repa ra t i on was a l s o c h a r a c t e r i z e d i n terms o f the r e l a t i v e inc rease i n a s p e c i f i c ca l c i um t r an spo r t a c t i v i t y . Experiments (Table 2) showed tha t the i nc rease i n ca l c i um t r an spo r t a c t i v i t y o f the SR r e l a t i v e t o the homogenate was o f the same order f o r the eu thy ro id and thy ro idectomized groups. The r e l a t i v e inc rease tended to be lower i n the thy ro idectomized an imal s . Th i s was a t t r i b u t a b l e to the lower s p e c i f i c a c t i v i t y o f the SR f r a c t i o n o f these an imals . The r e l a t i v e decrease i n cytochrome c ox idase a c t i v i t y and 149 i nc rease i n ca l c i um t r an spo r t were o f the same order i n the eu thy ro i d or hypothyro id groups. There fo re , i t i s u n l i k e l y t ha t the SR ca l c i um t r an spo r t a c t i v i t y would be a r t i f a c t u a l l y increased or decreased due to the presence o f "contaminating" ma te r i a l i n the SR p r e p a r a t i o n . The SR p repa ra t i on s were t h e r e f o r e cons idered s u i t a b l e f o r the proposed s t u d i e s . Although i t i s e s t a b l i s h e d tha t hypothyro id i sm e f f e c t s a r educ t i on i n c a r d i a c SR ca l c i um t r an spo r t a c t i v i t y , prev ious s tud ie s had not determined the mechanism f o r t h i s r e d u c t i o n . Th is study cons idered t ha t endogenous myocardia l a c y l c a r n i t i n e s , potent i n h i b i t o r s o f SR ca l c i um t r a n s p o r t , may p lay a r o l e i n the reduced c a r d i a c SR ca l c i um t r a n s p o r t a c t i v i t y of hypothyro id an imal s . As d i scussed above, the s u r g i c a l l y thy ro idec tomized male Wi s ta r r a t demonstrated s igns i n d i c a t i n g t h i s to be a s u i t a b l e exper imental paradigm f o r the proposed s t u d i e s . I t was, however, a l s o necessary to determine i f SR ca l c ium t r an spo r t was reduced i n the hypothyro id Wi s ta r r a t used i n the cu r ren t s t u d i e s . S ince the study aimed to determine the r o l e o f endogenous myocardial a c y l c a r n i t i n e , i t was a l s o necessary to determine a time po in t f o l l o w i n g thyro idectomy when SR ca l c i um t r an spo r t was not a f f e c t e d , as we l l as t ime po in t s when ca l c i um t r an spo r t was a l t e r e d . There fo re , SR ca l c ium t r an spo r t a c t i v i t y was determined i n SR i s o l a t e d from the hearts of animals two, f ou r , s i x and e i gh t weeks f o l l o w i n g removal o f the t h y r o i d g l and . The r e s u l t s o f these dete rminat ions (F igures 7 - 10) show tha t SR ca l c i um t r an spo r t was not a f f e c t e d u n t i l f ou r weeks a f t e r thyro idectomy and then remained depressed u n t i l the f i n a l t ime po in t s t ud i ed . The magnitude o f the r educ t i on i n SR ca l c i um t r an spo r t a c t i v i t y i n the cu r ren t sudies i s o f a s i m i l a r order to prev ious r epo r t s (Suko, 1973, Rodgers et a l , 1985, Takacs e t a l , 1985). I t 150 i s o f p a r t i c u l a r i n t e r e s t t ha t the magnitude o f the r educ t i on i n SR ca l c i um t r an spo r t a c t i v i t y i s such tha t i t i s reasonable to suggest t ha t the a l t e r e d f u n c t i o n o f t h i s o r gane l l e may be r e spon s i b l e f o r the impaired r e l a x a t i o n o f the v e n t r i c l e i n hypothyro id i sm. To address the p o s s i b i l i t y t ha t endogenous myocardia l a c y l c a r n i t i n e a s soc i a ted w i t h the SR was r e spon s i b l e f o r the reduced ca l c i um t r an spo r t r a t e , the l e v e l o f these l i p i d metabo l i c i n te rmed ia tes was determined. Long cha in acy l c a r n i t i n e was present i n SR o f eu thy ro i d and thy ro idec tomized animals at each time po in t s t ud i ed . The concen t r a t i on of acy l c a r n i t i n e present i n the SR of the eu thy ro id groups was somewhat v a r i a b l e (but not d i f f e r e n t , p < 0.05), averaging between 150 and 224 pmoles/mg SR p r o t e i n . These values f o r endogenous a c y l c a r n i t i n e a s soc i a ted w i th c a r d i a c SR are o f the same magnitude as those o f Lopaschuk et a l (960 pmoles/mg SR p r o t e i n , 1982), but somewhat lower than p r e v i o u s l y desc r ibed va lues (6 - 8 nmoles/mg SR p r o t e i n , B lack et a l , 1988). The concen t ra t i on o f endogenous a c y l c a r n i t i n e i n sarcolemma of c u l t u r e d c a r d i a c c e l l s has been repor ted to be 1.3 nmoles/mg p r o t e i n , and o f i n t e r n a l cy top la smic membranes 4.2 nmoles/mg p r o t e i n (Knabb et a l , 1986). C a l c u l a t e d est imates of endogenous SR a c y l c a r n i t i n e concen t ra t i on have suggested 30 nmoles/mg SR p r o t e i n ( P i t t s et a l , 1978). The reason f o r the d i f f e r e n c e i n the va lues repor ted f o r the l e v e l o f endogenous long cha in acy l c a r n i t i n e a s soc i a ted w i th SR i s not known; however, methodolog ica l d i f f e r e n c e s may be cons ide red . With respect to the d i f f e r e n c e s found between t h i s study and prev ious s t ud i e s (B lack et a l , 1988), t e rm ina t i on o f the r e a c t i o n and sepa ra t i ng product from reac tant r equ i red f i l t e r i n g the r e a c t i o n media through a column of Dowex anion 1X8 exchange r e s i n . In prev ious s t u d i e s , a 151 batch b ind ing technique was employed (B lack e t a l , 1988). The column f i l t e r i n g techn ique was found to produce a h igher and more c o n s i s t e n t s i gna l to no i se r a t i o i n comparison to the prev ious batch b ind ing procedure. The d i f f e r e n t separat ion techniques may have c o n t r i b u t e d to the d i f f e r e n t l e v e l s o f SR acy l c a r n i t i n e r epo r ted . The acy l c a r n i t i n e l e v e l s o f c u l t u r e d c a r d i a c c e l l s were determined us ing an au to rad iog raph i c technique on the i n t a c t myocyte, w i th c a l c u l a t i o n o f an est imated amount o f membrane-associated acy l c a r n i t i n e (Knabb et a l , 1986). Th i s technique a f f o rded the advantage of avo id ing p o s s i b l e changes i n the d i s t r i b u t i o n , or l o s s , o f acy l c a r n i t i n e dur ing e x t r a c t i o n and i s o l a t i o n , but d i d not determine the amount o f acy l c a r n i t i n e i n the SR compartment alone ( r i bo soma l , nuc lea r and lysosomal membranes were not exc luded ) . The value repor ted f o r endogenous sarcolemmal acy l c a r n i t i n e by Knabb et a l (1986), 1.3 nmoles/mg p r o t e i n , was o f s i m i l a r order to the l e v e l i n the SR as repor ted i n the cu r ren t study and by Lopaschuk et a l (1982). There fo re , although there i s a range of va lues repor ted (high p icomolar to low nanomolar) f o r the l e v e l o f endogenous acy l c a r n i t i n e a s soc i a ted w i t h the SR membrane, s t ud i e s employing d i f f e r e n t d e t e c t i o n techn iques suggest ( d i r e c t l y and i n d i r e c t l y ) t ha t the l i p i d metabo l i c i n te rmed ia te does a s s o c i a t e w i th the c a rd i a c SR membrane. The technique used f o r the cu r ren t s t u d i e s , however, cannot determine whether the acy l c a r n i t i n e measured i n the i s o l a t e d SR f r a c t i o n i s r e s t r i c t e d to tha t of n a t i v e SR o r i g i n . There was m i tochond r i a l cytochrome c ox idase a c t i v i t y i n the SR p r e p a r a t i o n , i n d i c a t i n g t ha t a c e r t a i n degree o f m i tochondr i a l membrane d i d occur dur ing i s o l a t i o n o f the SR. However, s i nce the acy l c a r n i t i n e va lues repor ted i n the cu r ren t s tud ie s are approximately o f the same order o f magnitude as 152 others have r epo r ted , i t does not seem unreasonable tha t the acy l c a r n i t i n e measured i s p r i m a r i l y o f SR o r i g i n . As shown i n the Results s e c t i o n , the l e v e l o f long cha in acy l c a r n i t i n e a s soc i a ted w i th the SR membrane was not i n f l uenced by t ime or thyro idectomy (F igure 14). There was no d i f f e r e n c e i n the amount o f acy l c a r n i t i n e a s soc i a ted w i th the SR o f hypothyro id animals compared to eu thy ro i d c o n t r o l s . These r e s u l t s show tha t endogenous a c y l c a r n i t i n e a s soc i a ted w i t h the SR p repa ra t i on from hypothyro id animals was not r e spon s i b l e f o r the reduced in vitro SR ca l c i um t r an spo r t observed at 4, 6 and 8 weeks f o l l o w i n g thyro idectomy. Of p a r t i c u l a r re levance to the hypothes i s was the obse rva t ion tha t the SR a c y l c a r n i t i n e concen t r a t i on was not d i f f e r e n t between euthy ro id c on t r o l and thy ro idec tomized animals at f ou r weeks pos t - thy ro idectomy, a t ime at which SR ca l c i um t r an spo r t a c t i v i t y was s i g n i f i c a n t l y (p < 0.05) depressed i n the thy ro idec tomized group (F igure 8 ) . The l a c k o f a s i g n i f i c a n t c o r r e l a t i o n between endogenous SR acy l c a r n i t i n e and ca l c ium t r an spo r t r e f u t e s the proposal t ha t acy l c a r n i t i n e may be i nvo l ved i n the reduced in vitro SR ca l c i um t r an spo r t a c t i v i t y o f hypothyro id an imals . However, endogenous SR a c y l c a r n i t i n e l e v e l s may be a f f e c t e d ( r e d i s t r i b u t i o n upon homogenization) du r i ng the i s o l a t i o n o f the membrane f r a c t i o n , and t h e r e f o r e , in vivo these l i p i d metabo l i c i n te rmed ia te s may exer t an i n h i b i t o r y e f f e c t upon SR ca l c i um t r an spo r t f u n c t i o n i n the hypothyro id s t a t e . As ev inced from the Ead ie-Hofstee p l o t ana ly ses , the maximal r a t e o f SR ca l c i um t r an spo r t ( V m a x ) was i n f l uenced by the hypothyro id s t a t e whereas no change i n the a f f i n i t y of the ca l c ium t r a n s p o r t e r f o r ca l c i um (Kr, a) was apparent. Th i s suggests tha t the lower SR ca l c i um t r an spo r t r a t e o f 153 thy ro idec tomized animals cou ld be the r e s u l t o f a r educ t i on i n the number of f u n c t i o n a l ca l c i um pump s i t e s , or a lower i n t r i n s i c ca l c i um pumping r a t e o f an equal number o f f u n c t i o n a l ca l c i um t r a n s p o r t e r s . To determine, i f i n f a c t , the re was a r educ t i on i n the f u n c t i o n a l ca l c i um pump s i t e s c a l c i u m -dependent acy lphosphoprote in l e v e l s were q u a n t i f i e d as an index o f the amount o f SR ca l c i um pump p r o t e i n . The phosphorylated enzyme i n te rmed ia te o f the ca l c i um t r an spo r t r e a c t i o n (F igure 1, E-P) r e s u l t s from the t r a n s f e r o f the TT-phosphate group o f ATP to a /J-aspartyl group o f the enzyme p r o t e i n ( P i f l et a l , 1984). The acy l phosphate bond so formed i s s e n s i t i v e to hydroxylamine and ba s i c c ond i t i o n s (Suko and Hasselbach, 1976), and i s thus d i f f e r e n t i a t e d from hyd roxy l am ine - i n sen s i t i v e s e r i ne phosphoprotein bonds ( i . e . phosphory lated phospholamban; LePeuch et a l , 1979; K i r chbe rge r and Antonez, 1982). The SR acy lphosphoprote in measured at 10 mM [ C a 2 + ] p r e e i n theses s t ud i e s was 95.5% hydroxylamine s e n s i t i v e , t h e r e f o r e i n d i c a t i v e o f ca l c i um pump p r o t e i n . The ca l c ium t r an spo r t r e s u l t s showed t ha t the re was a s i g n i f i c a n t depress ion i n the r a t e of SR ca l c ium t r an spo r t at f o u r , but not at two, weeks f o l l o w i n g thyro idectomy (F igures 7 and 8 ) ; t h e r e f o r e , the l e v e l o f SR acy lphosphoprote in was determined at these t imes . There was no d i f f e r e n c e between the acy lphosphoprote in l e v e l o f eu thy ro i d and hypothyro id groups at two weeks, when ca lc ium t r an spo r t was not d i f f e r e n t . There was, however, a s i g n i f i c a n t reduc t i on i n the calc ium-dependent SR acy lphosphoprote in format ion four weeks a f t e r thyro idectomy. These data prov ide the f i r s t d i r e c t evidence that hypothyro id i sm leads to a r educ t i on i n the number o f SR ca l c i um t r an spo r t s i t e s , and suggest t ha t the r educ t i on i n SR ca l c i um t r an spo r t a c t i v i t y at four weeks i s the r e s u l t o f a hypothyro id - induced reduc t i on i n the amount o f f u n c t i o n a l ca l c i um ATPase 154 p r o t e i n i n the SR membrane. I t i s i n t e r e s t i n g to note t ha t Suko (1973) was p r e s c i e n t i n t h i s regard when he suggested tha t " . . . thyroid activity modifies the SR membranes by increasing the amount of transport sites per unit membrane". The experiments of Rohrer and D i l lmann (1988) f u r t h e r support an e f f e c t o f t h y r o i d hormone on c a r d i a c SR ca l c i um pump p r o t e i n . The steady s t a t e l e v e l o f Ca 2 + -ATPase mRNA of hypothyro id r a t heart was 36% o f the eu thy ro id c on t r o l l e v e l (Rohrer and D i l lmann, 1988). Thyro id hormone (T3) treatment (2 mg/kg) o f r a t s made hypothyro id f o r a pe r i od o f f ou r to s i x weeks r e s u l t e d i n r ap i d s t i m u l a t i o n o f the expres s ion o f the mRNA encoding f o r the SR Ca 2 + -ATPase (Rohrer and D i l lmann, 1988). The development o f s k e l e t a l muscle SR ca l c ium t r an spo r t a c t i v i t y p o s t - n a t a l l y has been shown to be dependent on the presence o f t h y r o i d hormone (Simonides and van Hardeveld, 1989). S k e l e t a l muscle SR ca l c i um t r an spo r t a c t i v i t y increased i n eu thy ro id animals over the e i gh t week study pe r iod whereas the t r an spo r t a c t i v i t y o f the hypothyro id animals remained at the one week eu thy ro id l e v e l and d i d not i nc rease beyond t ha t l e v e l . The e f f e c t o f hypothyro id i sm was r e v e r s i b l e upon treatment w i t h exogenous t h y r o i d hormone and was a l so shown to be independent o f growth hormone. There fo re , as suggested by severa l l i n e s o f ev idence, the mechanism r e spon s i b l e f o r the reduced SR ca l c i um t r an spo r t a c t i v i t y o f hypothyro id animals may r e l a t e to a hypothyro id- induced reduc t i on i n the amount o f SR ca l c i um ATPase p r o t e i n . I f one cons ider s the e f f e c t o f t h y r o i d hormone on the expres s ion o f v e n t r i c u l a r myosin heavy cha in isoenzymes (Hoh et a l , 1978; Lompre et a l , 1984), sarcolemmal Na + /K + -ATPase a c t i v i t y ( I s m a i l - B e i g i and Edelman, 1970) and Na + /K + -ATPase a - subun i t mRNA ( I s m a i l - B e i g i , 1986), and the general tenet tha t t h y r o i d hormone a c t i o n i s mediated v i a 155 i n t e r a c t i o n w i t h s p e c i f i c nuc lea r recepto r s w i t h subsequent i nduc t i on o f mRNA expres s ion and p r o t e i n s yn thes i s (Oppenheimer, 1985), i t i s reasonable to suggest t ha t such a t h y r o i d hormone-mediated e f f e c t on the SR ca l c i um pump p r o t e i n may be important i n the observed changes i n c a l c i um t r an spo r t a c t i v i t y . 4.2 The E f f e c t s o f Omega-3 F a t t y A c i d Treatment on Card iac and SR Funct ion i n the S t reptozoc in - Induced D i a b e t i c Rat. The e t i o l o g y o f d i a b e t i c cardiomyopathy remains to be d e f i n e d . The p o t e n t i a l r o l e o f an omega-3 f a t t y a c i d treatment was s tud ied to a s c e r t a i n whether a membrane-associated event(s ) i s i nvo l ved i n the cardiomyopathy. Pharmacologic i n t e r v e n t i o n of d i a b e t i c cardiomyopathy w i th omega-3 f a t t y a c i d treatment prov ides the oppor tun i t y to address the i s sue o f the pathogenesis o f the c a rd i a c d y s f u n c t i o n . Omega-3 f a t t y ac id s are known to p a r t i t i o n i n t o c a r d i a c c e l l u l a r membranes and i n f l u e n c e c a r d i a c and membrane f u n c t i o n . There fo re , i t was po s i t ed tha t i f the development of d i a b e t i c cardiomyopathy was r e l a t e d to known d i abe te s - i nduced reduc t i on s i n pho spho l i p i d de sa tu r a t i on a c t i v i t y , and/or the reduced po lyunsaturated f a t t y acy l content of c a rd i a c phospho l i p i d s , then treatment o f d i a b e t i c animals w i t h long cha in po lyunsaturated omega-3 f a t t y ac id s may c o r r e c t d i abetes induced membrane changes which may be a f f e c t i n g the development o f the cardiomyopathy. To t e s t t h i s hypothes i s i t was f i r s t necessary to determine i f omega-3 f a t t y a c i d treatment a f f e c t e d the c a r d i a c d y s f unc t i on 1 5 6 of d i a b e t i c an imal s . The model of IDDM chosen to exp lo re the e f f e c t s o f the omega-3 f a t t y a c i d treatment was the STZ-induced model o f IDDM. Th i s model o f IDDM i s w e l l e s t a b l i s h e d i n the l a b o r a t o r y o f Dean McNe i l l i n the Facu l t y o f Pharmaceut ical Sc iences , and the d i a b e t i c cardiomyopathy i s known to be mani fes t s i x weeks a f t e r the onset o f d i abetes induced by intravenous a d m i n i s t r a t i o n o f STZ. The STZ treatment p ro toco l employed i n t h i s study rendered the male Wi s ta r r a t s d i a b e t i c , as ev inced by the hypo in su l i nemia , hyperg lycemia, po lyphag ia , p o l y u r i a and growth r e t a r d a t i o n observed i n these an imals . STZ-induced d i a b e t i c animals were hypo in su l i nemic , hyperglycemic (F igures 19 and 28) and f a i l e d to t h r i v e to the same extent as non -d i abe t i c c o n t r o l s (F igures 20 and 29). A d d i t i o n a l l y , i t was observed throughout the s tud i e s t ha t STZ-induced d i a b e t i c r a t s e x h i b i t e d hyperphagia, po l ydyps i a and p o l y u r i a . S ince these s igns are a l s o observed i n human IDDM (poor l y c o n t r o l l e d ) , these data support the use o f the STZ-induced d iabetes as a model o f IDDM. I t should a l s o be noted, however, t ha t the STZ-induced model o f d i abetes does not s t r i c t l y p a r a l l e l IDDM of humans, s ince the animals are hypo in su l i nemic , r a t h e r than an in su l i nemic and do not r equ i r e exogenous i n s u l i n to s u r v i v e . In s p i t e o f these p i t f a l l s of the STZ -d i abe t i c model, there i s s u f f i c i e n t s i m i l a r i t y t ha t the model a l lows f o r d e t a i l e d a n a l y s i s o f va r i ous man i f e s t a t i on s ( ch ron ic comp l i ca t i on s ) of prolonged hyperg lycemia. The e f f e c t s o f omega-3 f a t t y a c i d treatment i n the STZ-induced model of d iabetes were s tud ied from fou r major p e r s p e c t i v e s : the e f f e c t s o f treatment on i ) plasma l i p i d s , i i ) i s o l a t e d working heart f u n c t i o n , i i i ) i s o l a t e d c a r d i a c SR ca lc ium t r an spo r t and, i v ) , c a r d i a c and SR phospho l i p id acy l compos i t i on . The s tud ie s i n d i c a t e d tha t the h y p e r t r i g l y c e r i d e m i a and 157 hypercho le s te ro lemia of the STZ-induced d i a b e t i c r a t were not a f f e c t e d by the omega-3 f a t t y a c i d t reatment. The s tud i e s a l s o showed t ha t omega-3 f a t t y a c i d treatment decreases the degree to which c a r d i a c d y s f unc t i on developed i n the d i a b e t i c r a t s . There was a s i g n i f i c a n t improvement i n the c a r d i a c f u n c t i o n o f the d i a b e t i c t r e a t ed r a t s when peak LVDP, + d P / d t m a x and " d P / d t m a x were assessed. A d d i t i o n a l l y , a o r t i c f l ow ( ca rd i a c output) and pulmonary f l ow (coronary f low) o f omega-3 f a t t y a c i d - t r e a t e d STZ-induced d i a b e t i c r a t s were s i g n i f i c a n t l y improved. The omega-3 f a t t y a c i d treatment of the d i a b e t i c r a t s d i d not improve c a r d i a c f u n c t i o n to the non-d i a b e t i c c o n t r o l l e v e l . The omega-3 f a t t y a c i d treatment had no e f f e c t on plasma l i p i d s , i s o l a t e d working heart f u n c t i o n , a o r t i c f l ow or pulmonary f l ow o f n on -d i abe t i c con t r o l an imals . E levated plasma l i p i d s a s soc ia ted w i th d iabetes are cons idered t o be i nvo l ved i n the development of expe r imenta l l y - i nduced d i a b e t i c cardiomyopathy (Rodrigues et a l , 1986), and i n human d iabetes e l eva ted plasma l i p i d s are an independent r i s k f a c t o r f o r the development o f c a r d i o v a s c u l a r comp l i ca t i on s a s soc ia ted w i th IDDM (Fontbonne et a l , 1989). There fo re , the e f f e c t s o f omega-3 f a t t y ac id s on l i p i d metabol ism i n d iabetes are o f cons ide rab le i n t e r e s t i n view o f the e s t a b l i s h e d h ype r l i p i dem ia o f the d i sease and the c a r d i o v a s c u l a r r i s k con fe r red upon the d i a b e t i c popu la t i on by e leva ted serum l i p i d s . The hypo l i p i demic e f f e c t s o f omega-3 f a t t y ac ids i n d iabetes have been s tud ied i n both expe r imenta l l y - i nduced and human forms of the d i s ea se . Pretreatment o f r a t s w i th an omega-3 f a t t y a c i d supplement i n STZ-induced d iabetes was a s soc i a ted w i th the development o f h y p e r t r i g l y c e r i d e m i a on the second and e leventh days f o l l o w i n g STZ i n j e c t i o n ( I l lman et a l , 1986). In the study 158 of I l lman e t a l (1986), t r i g l y c e r i d e l e v e l s were not f o l l owed f o r longer than e leven days to determine i f the inc rease was o f a ch ron i c or t r a n s i e n t na tu re . The degree o f h y p e r t r i g l y c e r i d e m i a and du ra t i on o f treatment may be important i n the STZ-model o f d iabetes s i nce 20 weeks o f omega-3 f a t t y a c i d treatment reduced d i a b e t e s - r e l a t e d h y p e r t r i g l y c e r i d e m i a from 1162 mg% to 150 mg% (S inha, et a l , 1988). Plasma l i p i d a l t e r a t i o n s r e s u l t i n g from longer term omega-3 f a t t y a c i d treatment i n the d i a b e t i c may be more r e l e van t c on s i de r i n g the r e l a t i o n s h i p of such ch ron i c changes to the development o f d i a b e t i c cardiomyopathy. In we l l c o n t r o l l e d s t ud i e s o f IDDM p a t i e n t s , va r i ou s omega-3 f a t t y a c i d supplementation regimens have been shown to reduce plasma t r i g l y c e r i d e concent ra t i on s ( M i l l e r at a l , 1987; R i l l a e r t s et a l , 1989; Landgraf-Leurs et a l , 1990; Bagdade e t a l , 1990). The r e s u l t s o f the present s tud ie s i n d i c a t e tha t the h y p e r t r i g l y c e r i d e m i a of STZ-induced d i a b e t i c was not a f f e c t e d by the omega-3 f a t t y a c i d t reatment. Hypo t r i g l y ce r i dem i c e f f e c t s o f the omega-3 f a t t y a c i d may be do se - r e l a t ed and i t remains a p o s s i b i l i t y t ha t a g r ea te r e f f e c t cou ld be shown w i th a h igher dose o f the omega-3 f a t t y a c i d p r e p a r a t i o n . In NIDDM p a t i e n t s , the e f f e c t s of omega-3 f a t t y a c i d treatment have not been as p o s i t i v e as observed i n IDDM: i n p a t i e n t s w i thout over t h y p e r l i p i d e m i a , e i gh t weeks o f omega-3 f a t t y a c i d (180 mg EPA and 120 mg DHA) supplementat ion d i d not reduce plasma t r i g l y c e r i d e (Kasim et a l , 1988). Others have a l s o f a i l e d to show b e n e f i c i a l e f f e c t s o f omega-3 f a t t y ac id s i n NIDDM (Schectman et a l , 1988). The r e s u l t s o f the present s tud ie s i n d i c a t e t ha t there was no s i g n i f i c a n t e f f e c t o f the omega-3 f a t t y a c i d treatment on plasma c h o l e s t e r o l or t r i g l y c e r i d e concent ra t i on s i n the non -d i abe t i c c o n t r o l and 159 STZ-induced d i a b e t i c r a t s . The l a c k of e f f e c t o f the omega-3 f a t t y a c i d treatment on c o n t r o l animal plasma l i p i d s are at va r i ance w i t h s t ud i e s which have shown tha t under non -d i abe t i c c o n d i t i o n s , f i s h o i l - e n r i c h e d d i e t s can s i g n i f i c a n t l y reduce 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 l e v e l s ( H a r r i s e t a l , 1983; Hartog et a l , 1986; Herzberg and Rogerson, 1988). Omega-3 f a t t y a c i d supplementation s i g n i f i c a n t l y reduces plasma t r i g l y c e r i d e concent ra t i on s i n va r ious h ype r l i pop ro te i nem ic s t a t e s a f t e r two weeks and th ree months of treatment (Simons et a l , 1985; S inger et a l , 1985). The h y p o t r i g l y c e r i d e m i c e f f e c t o f omega-3 f a t t y ac id s has a l s o been observed f o r per iods o f up to two years (Saynor et a l , 1984). The l a c k o f e f f e c t o f omega-3 f a t t y ac id s on plasma t r i g l y c e r i d e s and c h o l e s t e r o l i n the present s t ud i e s may be due to the dose of omega-3 f a t t y a c i d employed. The reason f o r the l a c k o f e f f e c t of the omega-3 f a t t y a c i d treatment on the h y p e r t r i g l y c e r i d e m i a o f STZ-induced d iabetes i n the present s t ud i e s i s not known. Others have i n v e s t i g a t e d the mechanism by which omega-3 f a t t y ac id s reduce c i r c u l a t i n g t r i g l y c e r i d e l e v e l s . These s tud i e s have u t i l i z e d i s o l a t e d perfused r a t l i v e r , r a t hepatocytes and c u l t u r e d human hepatoma c e l l s . A two week per iod of omega-3 f a t t y a c i d treatment reduced plasma t r i g l y c e r i d e l e v e l s , the ex vivo r a t e of hepat i c l i p o g e n e s i s and the hepat i c s e c r e t i o n o f t r i g l y c e r i d e (Wong et a l , 1984). Although hepat i c t r i g l y c e r i d e concen t ra t i on of the omega-3 f a t t y a c i d - t r e a t e d group ( r e c e i v i n g a 15% f a t d i e t ) was h igher than a c o n t r o l group r e c e i v i n g a 5% f a t d i e t , the t r i g l y c e r i d e concen t ra t i on was lower compared to a t h i r d group r e c e i v i n g PUFA p r i m a r i l y of the omega-6 type ( i n a 15% f a t d i e t ) . These r e s u l t s exempl i f y the importance o f the f a t t y acy l cha in unique to the omega-3 type o f PUFA, because the r a t e o f t r i g l y c e r i d e s yn thes i s was 160 lower i n the presence o f the omega-3 type r e l a t i v e to f a t t y ac id s o f the omega-6 t ype . P re i ncuba t i on o f i s o l a t e d r a t hepatocytes w i t h a f a t t y a c i d sub s t ra te s t imu l a t e s t r i g l y c e r i d e s yn the s i s . The type o f f a t t y a c i d s u p p l i e d , however, markedly a f f e c t s the degree o f s t i m u l a t i o n . Incubat ion o f i s o l a t e d hepatocytes w i th o l e i c a c i d (omega-9 f a t t y a c i d FA) s t imu l a ted t r i g l y c e r i d e s yn thes i s 7 - f o l d and t r i g l y c e r i d e s e c r e t i o n 4 - f o l d (Nossen et a l , 1986). Under the same cond i t i o n s EPA d i d not have a s t r i k i n g e f f e c t on t r i g l y c e r i d e s yn thes i s (1.3 f o l d inc rease o f unst imulated) and i n h i b i t e d t r i g l y c e r i d e s e c r e t i o n to 40% of unst imulated l e v e l s (Nossen et a l , 1986). In c on t r a s t to the e f f e c t of o l e i c a c i d which augments 1 4 C - t r i g l y c e r i d e s yn thes i s and s e c r e t i o n when co- incubated w i th ^ C - p a l m i t i c a c i d , EPA i n h i b i t s both the s ynthes i s and s e c r e t i o n of 1 4 C - t r i g l y c e r i d e when co -incubated w i th the r a d i o l a b e l e d s ub s t r a te . In a d d i t i o n , EPA i s p r e f e r e n t i a l l y taken up by hepatocytes i n the presence o f f a t t y ac id s o f the omega-6 t ype . S i m i l a r observat ions concerning the e f f e c t o f omega-3 f a t t y ac id s on t r i g l y c e r i d e metabolism have been obta ined w i t h the human hepatoma c e l l l i n e , HEP G2 (Wong and N e s t e l , 1987). R e l a t i v e to o l e i c a c i d , the degree o f accumulat ion of t r i g l y c e r i d e i s s i g n i f i c a n t l y lower and the s e c r e t i o n o f both VLDL-apoprotein B and V L D L - t r i g l y c e r i d e reduced when the hepatoma c e l l s are incubated w i th EPA. D i r e c t i n h i b i t i o n o f hepa t i c a c y l - C o A : l , 2 - d i a c y l g l y c e r o l - 0 - a c y l t r a n s f e r a s e by EPA has been shown and may be the mechanism f o r the observed i n h i b i t o r y e f f e c t o f omega-3 f a t t y a c i d on t r i g l y c e r i d e s ynthes i s (Rustan et a l , 1988). S t r u c t u r a l s p e c i f i c i t y i n f a t t y ac ids on the s ynthes i s o f t r i g l y c e r i d e has been shown i n i s o l a t e d hepatocytes (Davis and Boogarts, 1982) and c l i n i c a l l y i n humans (Nes t le and 161 B a r t e r , 1971) where sa tu ra ted f a t t y ac ids are i nco rpo ra ted i n t o t r i g l y c e r i d e i n preference to unsaturated f a t t y a c i d s . D i e t a r y f i s h o i l has p r e v i ou s l y been shown not to a f f e c t i s o l a t e d working heart f u n c t i o n i n non -d i abe t i c c o n t r o l r a t s f o l l o w i n g f ou r weeks o f supplementat ion (Re ibe l et a l , 1988). The r e s u l t s o f the present s tud ie s con f i rm the l a c k of e f f e c t of omega-3 f a t t y a c i d treatment on i s o l a t e d heart f u n c t i o n o f c on t r o l r a t s . In the STZ-induced d i a b e t i c r a t , however, the present r e s u l t s are the f i r s t to demonstrate tha t an omega-3 f a t t y a c i d treatment e f f e c t e d a s i g n i f i c a n t improvement on i s o l a t e d working heart f u n c t i o n compared to untreated d i a b e t i c r a t s . P r e v i o u s l y , i t has been repor ted t ha t pharmacological treatments o f STZ-induced d i a b e t i c r a t s can improve c a r d i a c performance ( T a h i l i a n i and M c N e i l l , 1985; Rodrigues et a l , 1986). These prev ious s tud ie s suggest that improvements i n d i a b e t i c c a r d i a c performance are a s soc ia ted w i th r e s t o r a t i o n o f d i s t i n c t metabo l i c abno rma l i t i e s induced by the d i a b e t i c s t a t e . The r e s u l t s o f t h i s study i n d i c a t e tha t c a r d i a c performance o f d i a b e t i c r a t s can be improved i n s p i t e o f the d i a b e t i c s t a t e . S ince c a r d i a c performance o f the omega-3 f a t t y a c i d - t r e a t e d d i a b e t i c r a t s was not p a r a l l e l to t ha t o f the non-d i a b e t i c c o n t r o l an imals , t h i s may i n d i c a t e tha t the omega-3 f a t t y a c i d treatment i s able to a f f e c t on ly a d i s c r e t e aspect of the cardiomyopathy. The l i m i t e d a t t enua t i on of the development o f the d i a b e t i c cardiomyopathy by the omega-3 f a t t y a c i d treatment i n d i c a t e s t ha t there are more profound myocardia l a l t e r a t i o n s which the treatment i s i ncapab le o f p r even t i n g . D i r e c t e f f e c t s of omega-3 f a t t y ac ids on the myocardium have been i n v e s t i g a t e d i n d i sease s t a te s other than d i abe te s . S a l u t a r y e f f e c t s o f d i e t a r y f i s h o i l have been a s soc ia ted w i th reduc t i on s i n both i schemic 162 damage (Hock e t a l , 1987) and the inc idence o f spontaneous arrhythmias (McLennan e t a l , 1987) i n rodent models o f myocardia l i s chemia . In a d d i t i o n , i n normal c on t r o l r a t s , c a r d i a c c o n t r a c t i l e respons iveness t o the a -ad renerg i c r ecep to r agon i s t phenylephr ine was reduced by approx imately 50% f o l l o w i n g an omega-3 f a t t y a c i d - en r i c hed d i e t (Re ibe l et a l , 1988). The mechanism f o r these e f f e c t s i s not known. Determinat ion o f the mechanisms by which omega-3 f a t t y ac ids a f f e c t the heart i s confounded by r e s u l t s o f s t ud i e s us ing non-rodent models of myocardia l i schemia which have f a i l e d to demonstrate b e n e f i c i a l e f f e c t s o f omega-3 f a t t y a c i d treatment (Hartog et a l , 1986). Th is study has demonstrated t ha t i n the STZ-induced d i a b e t i c r a t , an omega-3 f a t t y a c i d treatment can ame l i o ra te the development o f the ca rd i a c comp l i ca t i on s a s soc i a ted w i th t h i s model of IDDM. The e t i o l o g y o f the d i a b e t i c cardiomyopathy i s not known and s i m i l a r l y , the mechanism whereby the omega-3 f a t t y a c i d treatment confe r s a degree o f p r o t e c t i o n aga ins t the development o f the cardiomyopathy i s unknown. However, the data from the pulmonary a r t e r y cannu la t i on experiment (F igures 27) i n d i c a t e tha t coronary f l ow o f d i a b e t i c r a t s i s s i g n i f i c a n t l y improved by the omega-3 f a t t y a c i d t reatment . S ince omega-3 f a t t y ac id s have been shown to a f f e c t v a s cu l a r f u n c t i o n , i t i s p o s s i b l e t ha t the improved ex vivo c a r d i a c f u n c t i o n of the t r e a t e d d i a b e t i c animals may r e l a t e to the improved coronary f l ow. An increased coronary f l ow i n t u r n , would conce i vab l y enhance sub s t ra te and oxygen d e l i v e r y , and inc rease the wash out o f t i s s u e me tabo l i t e s . As p r e v i o u s l y noted, pharmacologic i n t e r v e n t i o n s t ud i e s prov ide the oppor tun i t y t o address the i s sue o f pathogenes i s , and the data regard ing the e f f e c t o f the omega-3 f a t t y a c i d treatment on coronary f low i s an example of t h i s s i t u a t i o n . S ince coronary 163 f l ow o f the untreated d i a b e t i c hearts was s i g n i f i c a n t l y reduced, i t i s p o s s i b l e t ha t t h i s c o n t r i b u t e s to the c a rd i a c dy s func t i on observed i n these an imals . The obse rva t ion tha t i s o l a t e d c a r d i a c SR ca l c i um t r an spo r t a c t i v i t y was improved concomitant w i th the improved d i a s t o l i c f u n c t i o n o f the omega-3 f a t t y a c i d - t r e a t e d d i a b e t i c r a t s suggests t ha t the ca l c i um t r an spo r t a c t i v i t y o f t h i s o r gane l l e i s i nvo l ved i n the cardiomyopathy, as has been p r e v i o u s l y shown (Penpargkul et a l , 1981; Lopaschuk et a l , 1983), and a l s o tha t the omega-3 f a t t y a c i d treatment was ab le to e f f e c t an i nc rease i n t h i s a c t i v i t y . The d i a b e t i c cardiomyopathy i s o f complex e t i o l o g y and the augmented SR ca l c ium t r an spo r t a c t i v i t y represent s an a d d i t i o n a l mechanism invo l ved i n the improved c a r d i a c f u n c t i o n o f the omega-3 f a t t y a c i d - t r e a t e d r a t s . Our l abo r a to r y has p r e v i o u s l y shown t h a t , under c e r t a i n c i rcumstances c a rd i a c f unc t i on i s not improved de sp i t e improved SR ca l c i um t r an spo r t f u n c t i o n (Lopaschuk et a l , 1983a). Th i s may i n d i c a t e t ha t the omega-3 f a t t y a c i d treatment has other e f f e c t s , p o s s i b l y on sarcolemmal membrane f u n c t i o n ( i . e . C a 2 + - t r a n s p o r t and/or Na + /K + -ATPase a c t i v i t i e s ) which i n a d d i t i o n to the augmented SR ca l c i um t r an spo r t a c t i v i t y and coronary f l ow, c o n t r i b u t e to the improved ex vivo c a r d i a c f u n c t i o n . Based on the repor ted e f f e c t s o f omega-3 f a t t y a c i d supplementat ion on a l t e r a t i o n s o f c a r d i a c phospho l i p id f a t t y acy l compos i t i on , i t i s conce i vab le t ha t the omega-3 f a t t y a c i d treatment i s a f f e c t i n g d i a b e t i c heart f u n c t i o n v i a a membrane-associated event. The a c t i v i t i e s o f the d e l t a 5 (Holman et a l , 1983), d e l t a 6 and d e l t a 9 (Eck et a l , 1979) desaturase enzymes are impaired i n the STZ d i a b e t i c r a t . Card iac desaturase enzyme 164 a c t i v i t y i s a l s o a f f e c t e d as the a rach idon i c a c i d and p roduc t -p recu r so r r a t i o o f C22:6,n-3/C22:5,n-3 are decreased i n d i a b e t e s , the l a t t e r a l t e r a t i o n p o s s i b l y i n d i c a t i v e o f impaired d e l t a * desaturase a c t i v i t y (Gudbjarnson e t a l , 1987). The e f f e c t o f these enzymatic changes induced by the d i a b e t i c s t a t e may r e s u l t i n a r educ t i on i n the f l u i d i t y o f c a r d i a c membranes. As d i scus sed i n the i n t r o d u c t i o n , the phys icochemica l nature of the l i p i d b i l a y e r i s cons idered to be an important f a c t o r i n determin ing c a r d i a c f u n c t i o n (Katz and Messineo, 1981), and the a c t i v i t y o f membrane a s soc i a ted enzymes (Stubbs and Smith, 1984), i n c l u d i n g the SR C a 2 + -pump/ATPase (Hidalgo et a l , 1982; Almeida et a l , 1984; Navarro et a l , 1984). There fo re , i n view o f a reduced p o t e n t i a l to unsaturate f a t t y ac id s i n the d i a b e t i c myocardium, and s i nce the a c t i v i t y o f the SR i s dependent on the app rop r i a te f l u i d s t a t e , i t i s conce i vab le tha t the omega-3 f a t t y a c i d treatment i s a f f e c t i n g SR f unc t i on by a l t e r i n g the pho spho l i p i d f a t t y acy l s t r u c t u r e . In the cu r ren t s t u d i e s , the omega-3 f a t t y acy l content o f c a r d i a c pho spha t i d y l cho l i ne and phosphat idy lethanolamine was inc reased by approx imately 27.2% and 32.9%, r e s p e c t i v e l y , i n the d i a b e t i c - t r e a t e d animals compared to untreated d i a b e t i c s . A d d i t i o n a l l y , c a r d i a c SR pho spho l i p i d omega-3 f a t t y acy l content increased by 20.7%. These changes, a lthough s t a t i s t i c a l l y i n s i g n i f i c a n t , represent a con s i de rab le i nc rease i n the r e l a t i v e p ropo r t i on o f omega-3 f a t t y a c i d as a component o f membrane pho spho l i p i d . I t i s a l s o apparent tha t there was an i nc rea se i n phosphat idy lethanolamine C16:0, C 1 6 : l , C18:2 and C22:5 acy l spec ies o f unt reated d i a b e t i c an imals . The increased content o f these more sa tu ra ted acy l spec ies were returned to non -d i abe t i c c o n t r o l l e v e l s i n the omega-3 f a t t y a c i d t r e a t e d d i a b e t i c an imals . The increased omega-3 f a t t y a c i d 165 content and reduced C16:0, C 1 6 : l , C18:2 and C22:5 content o f t r e a t e d d i a b e t i c animals may act i n concert to e f f e c t a change i n the SR l i p i d compos i t i on , and hence SR ca l c i um t r an spo r t a c t i v i t y . The acy l spec ies a f f e c t e d by the treatment do not account f o r a major p r opo r t i on o f the t o t a l acy l con tent ; however, i n view o f a p o s s i b l e d i r e c t r o l e o f these spec ie s i n SR ca l c i um t r an spo r t (a d i r e c t 1 i p i d - p r o t e i n i n t e r a c t i o n ) , i t i s p o s s i b l e tha t these a l t e r a t i o n s may c o n t r i b u t e to the observed c a r d i a c and SR f u n c t i o n a l changes. S ince omega-3 f a t t y a c i d treatment d i d not l ead to p e r c e p t i b l e ( s i g n i f i c a n t ) changes i n t o t a l phospho l i p id acy l compos i t ion o f SR membranes the present s tud ie s do not support such a r o l e . However, i t remains to be determined whether i n d i v i d u a l pho spho l i p i d acy l compos i t ion was s i g n i f i c a n t l y a f f e c t e d by the t reatment. A small or minor l i p i d pool may be i n f l uenced which cou ld a f f e c t SR ca l c i um t r a n s p o r t . Although not measured i n these s t u d i e s , the SL may a l so be a f f e c t e d i n a s i m i l a r manner, and hence c o n t r i b u t e to the improved ca rd i a c performance o f the t r e a t e d d i a b e t i c r a t s . The e f f e c t o f omega-3 f a t t y a c i d treatment on c a r d i a c c e l l membranes and the b iochemica l a c t i v i t i e s a s soc ia ted w i th the s p e c i f i c membrane s t r u c t u r e s may i n vo l ve mechanisms other than d i r e c t i n s e r t i o n o f the omega-3 f a t t y acy l moiety, as a component of pho spho l i p i d , i n t o the membrane. The e f f e c t o f omega-3 f a t t y a c i d treatment on plasma c h o l e s t e r o l l e v e l s may a f f e c t c a r d i a c f u n c t i o n through changes i n the r e l a t i v e p r opo r t i on or p a r t i t i o n i n g of c h o l e s t e r o l i n t o c a r d i a c c e l l membranes. Cho l e s t e r o l i n membranes inc reases the v i s c o s i t y of the l i p i d b i l a y e r s t r u c t u r e and can a f f e c t the i n t r i n s i c a c t i v i t y of c e l l u l a r and s u b c e l l u l a r membranes. In the present s t u d i e s , c h o l e s t e r o l concent ra t i on s o f SR membranes were not 166 d i f f e r e n t between non -d i abe t i c and d i a b e t i c groups, and the omega-3 f a t t y a c i d treatment was wi thout e f f e c t i n e i t h e r group. Although c h o l e s t e r o l i s known to a f f e c t SR ca l c i um t r an spo r t (Almedia e t a l , 1984), the cu r ren t r e s u l t s suggest t ha t a r educ t i on i n the l e v e l o f c h o l e s t e r o l i n the membrane does not c o n t r i b u t e to the augmented SR ca l c i um t r an spo r t a c t i v i t y o f the omega-3 f a t t y a c i d - t r e a t e d d i a b e t i c an imals . In c o n s i d e r a t i o n o f o ther po s s i b l e mechanisms c o n t r i b u t i n g to the improved c a r d i a c f u n c t i o n o f the omega-3 f a t t y a c i d - t r e a t e d d i a b e t i c r a t s , b r i e f mention o f p l a t e l e t abno rma l i t i e s i n d iabetes and the e f f e c t s o f omega-3 f a t t y ac id s on p l a t e l e t f u n c t i o n i s warranted. The r o l e o f abnormal p l a t e l e t f unc t i on i n the development o f a t h e r o s c l e r o s i s and microangiopathy i n d i abe te s , and the p o t e n t i a l r o l e o f omega-3 f a t t y ac id s i n these pa tho log ie s was not i n v e s t i g a t e d i n the cu r ren t s t u d i e s . However, research on omega-3 f a t t y a c i d e f f e c t s on p l a t e l e t f u n c t i o n has y i e l d e d i n fo rmat i on which may be r e l e van t to p o s s i b l e mechanisms f o r the observed c a r d i a c e f f e c t s o f the omega-3 f a t t y a c i d s . Fo l l ow ing o r a l a d m i n i s t r a t i o n , in vivo EPA and DHA are i nco rpo ra ted i n t o p l a t e l e t phospho l i p id spec i e s . There i s i n c o r p o r a t i o n o f the omega-3 f a t t y ac id s i n t o PC and PE concomitant w i th reduced omega-6 f a t t y a c i d compos i t ion o f these phospho l i p id spec ies (Von Schacky et a l , 1985). Numerous animal and c l i n i c a l s tud ie s have demonstrated t ha t p l a t e l e t aggregat ion i n response to a v a r i e t y of agon i s t s in vitro, i s a t tenuated f o l l o w i n g an omega-3 f a t t y ac id treatment regimen (Heemskerk e t a l , 1989; Nordoy et a l , 1985; K i r s ten sen et a l , 1987). Increased b leed ing t ime f o l l o w i n g omega-3 f a t t y a c i d supplementation has a l s o been a t t r i b u t e d to reduced p l a t e l e t a g g r e g a b i l i t y (Saynor et a l , 1984). These e f f e c t s , 167 however, are not always demonstrable (Galloway et a l , 1985). Mechanisms f o r the a l t e r e d p l a t e l e t r e a c t i v i t y f o l l o w i n g omega-3 f a t t y a c i d treatment have been exp l o red . P l a t e l e t s incubated in vitro i n the presence o f exogenous a rach idon i c a c i d aggregate but p l a t e l e t s incubated w i t h exogenous EPA do not aggregate (Dyerberg et a l , 1978). P l a t e l e t s metabo l i ze EPA i n t o i ) p r o s t a c y c l i n H3 which possess ant i -aggegatory and v a s o d i l a t o r y p r o p e r t i e s , and i i ) , thromboxane A3 which i s n e i t h e r pro-aggregatory nor v a s o c o n s t r i c t o r y (Needleman et a l , 1979). The pharmacolog ica l p r o p e r t i e s o f EPA m e t a b o l i t e s , p r o s t a c y c l i n H3 and thromboxane A3 may account f o r the a l t e r e d aggregatory response to exogenous EPA, and a l s o the a l t e r e d aggregatory response o f p l a t e l e t s in vitro f o l l o w i n g omega-3 f a t t y a c i d supplementat ion. P l a t e l e t f u n c t i o n i n d iabetes i s a l t e r e d such t ha t p l a t e l e t s o f d i a b e t i c s are h ype r s en s i t i v e to the e f f e c t s o f aggregatory agon i s t s (Butkus et a l , 1980; T i n d a l l et a l , 1981; Winocour e t a l , 1990). Increased p roduct ion o f p l a t e l e t thromboxane A2 by d i a b e t i c sub jec t s has been documented and may c o n t r i b u t e to these changes ( P r i s c o e t a l , 1989). Th i s increased p ropens i t y of p l a t e l e t s to aggregate i n d i abetes has been cons idered to p lay a r o l e i n m ic rova scu la r d i sease i n IDDM. Omega-3 f a t t y a c i d treatment o f IDDM p a t i e n t s reduces p l a t e l e t and whole blood thromboxane product ion and inc reases the l a g t ime f o r c o l l a gen s t imu la ted p l a t e l e t aggregat ion to occur in vitro ( B e i t z e t a l , 1986; Haines e t a l , 1986). In the present s tud ie s i t was determined tha t coronary pe r fu s i on was reduced i n d i a b e t i c hea r t s . The omega-3 f a t t y a c i d t reatment improved the coronary f l ow to the non -d i abe t i c r a t e . I t i s p o s s i b l e , c on s i de r i n g the known a l t e r a t i o n s i n p l a t e l e t f u n c t i o n i n d i abe te s , and the e f f e c t s o f 168 omega-3 f a t t y ac id s on p r o s t a c y c l i n and thromboxane p roduct ion and p l a t e l e t aggregat ion, t ha t the omega-3 f a t t y a c i d treatment used i n the cu r ren t s t ud i e s reduced or prevented m ic rovascu la r l e s i o n s caused by the d i a b e t i c s t a t e . Over the pe r i od o f the s i x week study an improvement i n myocardia l pe r f u s i on mediated by omega-3 f a t t y a c i d treatment may have c o n t r i b u t e d to the observed r educ t i on i n the development o f d i a b e t i c cardiomyopathy. S ince n e i t h e r p l a t e l e t f u n c t i o n , e i co sano id metabolism nor m i c rova scu l a r anatomy were s t u d i e d , t h i s remains a s p e c u l a t i v e p o s s i b i l i t y . 169 5. Conclusions: 5.1 The effect of hypothyroidism on SR calcium transport and the role of endogenous long chain acylcarnitine: 1) Surgical thyroidectomy of male Wistar rats produced gross physical signs indicative of hypothyroidism: thyroidectomized animals had lower plasma T3 concentrations and reduced body and ventricular weights four weeks after thyroidectomy. 2) The cardiac SR calcium transport act iv ity was influenced by removal of the thyroid gland. The SR calcium transport rate was not affected two weeks after thyroidectomy, but was s ignif icantly reduced four, six and eight weeks after thyroidectomy. The changes in SR calcium transport rate were the result of a reduced Vca, with no effect on the Kca of the reaction. 3) The level of long chain acylcarnitine associated with the isolated SR membrane preparation was not affected by thyroidectomy. The level of endogenous SR acyl carnitine was not different between the euthyroid control and hypothyroid groups four weeks after thyroidectomy when SR calcium transport was shown to be s ignif icantly reduced. These results suggest that the endogenous level of these l i p i d metabolic intermediates associated with the SR membrane is not related to SR calcium transport act iv i ty in hypothyroidism. 170 4) Four weeks post - thyro idectomy the l e v e l o f SR acy lphosphoprote in ( p u t a t i v e l y SR ca l c i um pump p ro te i n ) was s i g n i f i c a n t l y reduced. These r e s u l t s are i n agreement w i th the ca l c i um t r an spo r t d a t a , and suggest tha t a hypothyro id - induced reduc t i on i n SR ca l c i um pump p r o t e i n may be r e spon s i b l e f o r the reduced SR ca l c ium t r an spo r t a c t i v i t y . 171 5.2 The e f f e c t s o f omega-3 f a t t y a c i d treatment on c a r d i a c and SR f u n c t i o n i n the s t r ep t o zoc i n - i nduced d i a b e t i c r a t : 1) Treatment o f male Wi s ta r r a t s w i th s t r e p t o z o c i n (55 mg/kg i . v . ) produced d iabetes i n these an imals . STZ-t reated animals e x h i b i t e d s igns t y p i c a l o f t h i s model o f IDDM d i abe te s : po lyphag ia , po l ydyp s i a and p o l y u r i a were apparent throughout the study p e r i o d . Plasma i n s u l i n concen t ra t i on s o f STZ-t reated animals were s i g n i f i c a n t l y lower than c o n t r o l , and plasma g lucose concen t ra t i on s s i g n i f i c a n t l y h igher than c o n t r o l . These r e s u l t s i n d i c a t e d t ha t the STZ-model of IDDM was s u i t a b l e f o r these s t u d i e s . 2) Omega-3 f a t t y a c i d treatment s h i f t e d (not s i g n i f i c a n t l y ) e l eva ted plasma t r i g l y c e r i d e s of d i a b e t i c animals towards n o n - d i a b e t i c l e v e l s . Omega-3 f a t t y a c i d treatment d i d not reduce plasma c h o l e s t e r o l l e v e l s of d i a b e t i c an imals . Ne i the r plasma t r i g l y c e r i d e nor c h o l e s t e r o l were a f f e c t e d by the omega-3 f a t t y a c i d treatment i n c o n t r o l an ima l s . 3) Omega-3 f a t t y a c i d treatment o f STZ-induced d i a b e t i c animals reduced the development o f ( p a r t i a l l y prevented) the d i abe te s - i nduced cardiomyopathy. Improved ca rd i a c f unc t i on was i n d i c a t e d by the increased LVDP, + d P / d t , "dP/dt, a o r t i c f low and coronary f l ow o f omega-3 f a t t y a c i d t r e a t e d an imal s . There was no e f f e c t o f omega-3 f a t t y a c i d treatment on c a r d i a c f u n c t i o n o f non -d i abe t i c c on t r o l an imals . 172 4) Omega-3 f a t t y a c i d treatment of STZ-induced d i a b e t i c animals inc reased i s o l a t e d c a r d i a c SR ca l c ium t r an spo r t a c t i v i t y of d i a b e t i c animals t o the n o n - d i a b e t i c c o n t r o l l e v e l . 5) The omega-3 f a t t y a c i d treatment tended to inc rease the omega-3 f a t t y acy l compos i t ion o f the c on t r o l and d i a b e t i c t r e a t e d groups, a lthough the inc reases were not s i g n i f i c a n t . SR phospho l i p id omega-3 f a t t y acy l compos i t ion was s i m i l a r l y i nc rea sed , but not s i g n i f i c a n t l y . 6) There was a tendency f o r SR c h o l e s t e r o l to be inc reased i n the untreated d i a b e t i c an imals , and t h i s e leva ted SR c h o l e s t e r o l o f d i a b e t i c animals was reduced by the omega-3 f a t t y a c i d t reatment, a lthough these changes were not s i g n i f i c a n t l y d i f f e r e n t . 173 6. O r i g i n a l Con t r i b u t i o n s 1. In c a r d i a c SR i s o l a t e d from hypothyro id r a t s the number o f SR ca l c i um t r an spo r t s i t e s was shown to be s i g n i f i c a n t l y reduced concommitant w i th a r educ t i on i n the ca l c ium t r an spo r t a c t i v i t y . 2 . The c a r d i a c f u n c t i o n of STZ-induced d i a b e t i c r a t s was shown to be s i g n i f i c a n t l y improved by the omega-3 f a t t y a c i d t reatment . 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Response to c a r d i a c r e l a x a t i o n : load s e n s i t i v i t y and sarcop lasmic r e t i c u l u m . Am. J . P h y s i o l . 251: H1285-H1286, 1987 B lack , S.C., M c N e i l l , J . H . and Katz , S. Sarcoplasmic r e t i c u l u m C a 2 + t r an spo r t and long cha in a c y l c a r n i t i n e s i n hype r thy ro id i sm. Can. J . P h y s i o l . Pharmacol. 66: 159-165, 1988 B lack , S . C , Ka t z , S. and M c N e i l l , J . H . Card iac performance and plasma l i p i d s o f omega-3 f a t t y a c i d t r e a t e d s t r e p t o z o t o c i n - i n d u c e d d i a b e t i c r a t s . D iabetes . 38: 969-974, 1989 X iang, H., B lack , S . C , Ka tz , S. and M c N e i l l , J . H . Omega-3 f a t t y a c i d treatment f a i l s to a l t e r a l pha j - ad rene r g i c r e cep to r mediated phospho ino s i t i de turnover i n s t r ep t o zo t o c i n - i nduced d i a b e t i c r a t s . Gen. Pharmacol. (In Press) B lack , S.C. and M c N e i l l , J . H . Mo lecu la r and s u b c e l l u l a r mechanisms d f t h y r o i d hormone induced ca rd i a c a l t e r a t i o n s . In. S u b c e l l u l a r Bas i s  of C o n t r a c t i l e F a i l u r e . D h a l l a , N.S. and Beamish, R.E. (Eds.) (In Press) B lack , S.C. and M c N e i l l , J . H . Funct iona l s ta tu s o f the c a r d i a c sympathetic system i n d iabetes m e l l i t u s . In. Catecholamines and  Heart D i sease. Ganguly, P.K. (Ed.) CRC Pres s , Boca Raton. (In Press) B lack , S . C , M c N e i l l , J . H . and Katz S. Calc ium t r an spo r t a c t i v i t y o f r a t c a r d i a c sarcop lasmic r e t i c u l u m i s o l a t e d i n the presence o f d i t h i o t h r e i t o l . J . Pharmacol. Meth. (In Press) page i i GRADUATE AWARDS 1984-86 B r i t i s h Columbia Heart Foundation Research T ra i nee sh ip 1986-89 Canadian Heart Foundation Research T ra ineesh ip 1987 B r i t i s h Columbia P r o v i n c i a l Government S cho l a r sh i p 1987 W i l l i a m W. Simpson Memorial Award 1988 UBC Graduate Student Trave l Award 1988 UBC Hea l th Sc iences Research Day Award 1989 Facu l t y o f Pharmaceutical Sc iences Research Day Award 1990 Canadian Heart and Stroke Foundation Pos t -Docto ra l Fe l l owsh ip 

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