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Biochemical, morphological and functional alterations in acute myocardial ischaemia 1984

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BIOCHEMICAL, MORPHOLOGICAL AND FUNCTIONAL ALTERATIONS IN ACUTE MYOCARDIAL ISCHAEMIA by MARGO MARIE MOORE P h . D . , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Depar tment o f Pha rmaco l ogy & T h e r a p e u t i c s , F a c u l t y o f M e d i c i n e We a c c ep t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J a n u a r y 1984 (c) Margo M a r i e Moo re , 1984 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f ^hfiA^V^CsrHo^ Cnyv^l "^J^^I^e^Jl^ The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date ^J^wiA^s^jj l^t^ ( i i ) ABSTRACT Changes i n b i o c h e m i c a l , f u n c t i o n a l and m o r p h o l o g i c a l i n d i c e s o f m y o c a r d i a l c e l l i n j u r y have been ob s e r v ed a f t e r s h o r t p e r i o d s o f i s c h a e m i a p roduced by c o r o n a r y o c c l u s i o n . These a l t e r a t i o n s a r e g e n e r a l l y r e v e r s i b l e i f b l o o d f l o w i s r e - i n s t a t e d w i t h i n a p p r o x i m a t e l y 30 min p o s t - 1 i g a t i o n ; howeve r , r e p e r f u s i o n a f t e r t h i s t i m e c a n n o t r e v e r s e t h e damage and a c t u a l l y e x a c e r b a t e s t h e e x i s t - i n g i n j u r y . These s t u d i e s have i n v e s t i g a t e d i s c h a e m i a / r e p e r f u s i o n i n j u r y , a s s e s s i n g c e l l damage u s i n g s e v e r a l d i f f e r e n t t e c h n i q u e s i n o r d e r t o a v o i d t h e i n h e r e n t b i a s o f any s i n g l e me thod . The e x p e r i m e n t s c on ce rned t h e m o l e c u l a r changes t h a t o c c u r d u r i n g t h e t r a n s i t i o n f r om r e v e r s i b l e t o i r r e v e r s i b l e damage and t h e i r m o d i f i c a t i o n by a v a r i e t y o f d r u g s , e s p e c i a l l y t h e c a l c i u m c h a n n e l - b l o c k e r , D-600 and t h e B - a d r e n e r g i c a n t a g o n i s t , p r o p r a n o l o l . I s c h a e - mic o r i s c h a e m i c / r e p e r f u s e d t i s s u e was o b t a i n e d by l i g a t i o n ( w i t h o r w i t h o u t r e l e a s e ) o f t h e l e f t c i r c u m f l e x c o r o n a r y a r t e r y o f t h e o p e n - c h e s t , a n a e s t h e - t i z e d r a b b i t . T h i s a r e a was i s o l a t e d and samp les removed f o r t he v a r i o u s b i o c h e m i c a l , c h e m i c a l and u l t r a s t r u c t u r a l a n a l y s e s . The f u n c t i o n a l i n t e g r i t y o f m i t o c h o n d r i a l (MITO) and s a r co l emma l (SL ) membranes was a s s e s s e d u s i n g membrane f r a c t i o n s p u r i f i e d by d i f f e r e n t i a l and d e n s i t y g r a d i e n t c e n t r i f u g a - t i o n . M y o c a r d i a l i s c h a e m i a was shown t o p roduce p r o g r e s s i v e d e c r e a s e s i n t h e a c t i v i t i e s o f t h e membrane-bound MITO ATPase and SL N a + , K + - A T P a s e enzyme as w e l l as a d e c l i n e i n t i s s u e ATP. I n t e r e s t i n g l y , r e p e r f u s i o n f u l l y r e s t o r e d o n l y t h e MITO enzyme a c t i v i t y . The enzyme i n h i b i t i o n was not a g e n e r a l i z e d phenomenon as shown by t h e l a c k o f e f f e c t o f i s c h a e m i a on a n o t h e r MITO enzyme, ( i i i ) namely c y t o ch rome c o x i d a s e . The i r r e v e r s i b l e d e c r e a s e i n N a + , K + - A T P a s e a c t i v i t y was no t accompan ied by changes i n K and Mg a f f i n i t y o r a l t e r a - t i o n s t o t h e SL p r o t e i n o r p h o s p h o l i p i d p r o f i l e s a l t h o u g h more s u b t l e changes i n t h e l i p i d o r p r o t e i n env i r onmen t may have o c c u r r e d . The s p e c i f i c i t y o f t h e d e c r e a s e t o t h e SL membrane was shown by t he c o n c o m i t a n t i n c r e a s e i n t he + + 2 + 2 + t i s s u e Na /K r a t i o i n t he absence o f Ca /Mg c h a n g e s . R e s u l t s f r om t h e 20 min l i g a t i o n s t u d y i n wh i ch D-600 p r e t r e a t m e n t p r o v i - ded s i g n i f i c a n t p r o t e c t i o n a g a i n s t i s c h a e m i a - i n d u c e d ATP d e p l e t i o n and MITO ATPase i n h i b i t i o n prompted t h e e x a m i n a t i o n o f t h e e f f i c a c y o f D-600 p r e t r e a t - ment p r i o r t o a 40 min l i g a t i o n and 60 min r e p e r f u s i o n . P r o p r a n o l o l was a l s o i n c l u d e d i n t h i s s t u d y w i t h t h e aim o f d e t e r m i n i n g t h e r e l a t i v e impo r t an c e o f d e c r e a s e d ene r g y c on sump t i on v s . c a l c i u m c h a n n e l - b l o c k a d e i n a t t e n u a t i n g i s c h a e m i a / r e p e r f u s i o n i n j u r y . Bo th agen t s were shown t o s i g n i f i c a n t l y r educe 2+ t h e b i o c h e m i c a l and m o r p h o l o g i c a l damage, i n c l u d i n g Ca a c c u m u l a t i o n , t h a t r e s u l t e d f rom r e p e r f u s i o n o f i r r e v e r s i b l y - d a m a g e d t i s s u e , a l t h o u g h D-600 was c o n s i s t e n t l y s u p e r i o r . D e s p i t e t h e b e n e f i c i a l e f f e c t s on MITO and SL i n t e g r - i t y , n e i t h e r d rug p r e s e r v e d ATP l e v e l s o r c o n t r a c t i l e f u n c t i o n . These d a t a i n d i c a t e t h a t t h e p r edom inan t b e n e f i c i a l e f f e c t s a r e p r o b a b l y t h e r e s u l t o f d e c r e a s ed ene r g y r e q u i r e m e n t s d u r i n g i s c h a e m i a . T h i s h y p o t h e s i s was f u r t h e r s u p p o r t e d by o b s e r v a t i o n s f r om t h e s t u d y i n wh i ch D-600 was a d m i n i s t e r e d j u s t p r i o r t o r e p e r f u s i o n . T h i s method o f d rug a d m i n i s t r a t i o n r educed c a l c i u m i n f l u x t o t h e same deg ree as when t h e d rug was g i v e n p r i o r t o i s c h a e m i a . However , t h i s i n t e r v e n t i o n s i g n i f i c a n t l y p r e s e r v e d o n l y MITO ATPase a c t i v i t y w i t h no p r o t e c t i o n o f SL f u n c t i o n . MITO p r o t e c t i o n i n t h e absence o f p r e s e r - ( i v ) v a t i o n o f SL i n t e g r i t y wou ld no t be e x p e c t e d t o p r o v i d e s u s t a i n e d b e n e f i t t o t he i s c h a e m i c / r e p e r f u s e d myoca rd i um. Even t h e ob s e r v ed p r o t e c t i o n o f SL i n t e g r i t y i n t h e d r u g - p r e t r e a t e d h e a r t s was no t accompan ied by an improvement i n c o n t r a c t i l i t y . Thus , the ma i n t enance o f i n t a c t p e r m e a b i l i t y b a r r i e r s does not n e c e s s a r i l y r e s u l t i n an improvement o f c o n t r a c t i l e f u n c t i o n . L ong - t e rm s t u d i e s wou ld be n e c e s s a r y t o d e t e r m i n e t h e u l t i m a t e b e n e f i t o f d rug t r e a t m e n t i n p r e v e n t i n g o r r e d u c i n g t h e d e l e t e r i o u s s e q u e l a e o f m y o c a r d i a l i s c h a e m i a . ( v ) TABLE OF CONTENTS A b s t r a c t i i L i s t o f T a b l e s v i i L i s t o f F i g u r e s i x L i s t o f A p p e n d i c e s x Acknowledgement x i Q u o t a t i o n x i i HISTORICAL OVERVIEW 1 INTRODUCTION 11 METHODS I . S u r g i c a l T e chn i que 23 Drug i n f u s i o n 24 C o r o n a r y l i g a t i o n , r e p e r f u s i o n and i s o l a t i o n o f t h e i s c h a e m i c a r e a 24 I I . S u b c e l l u l a r F r a c t i o n a t i o n 27 I I I . C h a r a c t e r i z a t i o n o f Sa rco lemma l and M i t o c h o n d r i a l 28 Membrane F r a c t i o n s 30 IV . Enzyme As say s N a + , K + - A T P a s e 30 K + - NPPa s e 33 Cy tochrome c o x i d a s e 34 A z i d e - s e n s i t i v e ATPase 34 T r i t o n X -100 a d d i t i o n 35 V. Chem i ca l A s s a y s Water c o n t e n t 35 ATP 35 Ions 36 SDS-ge l e l e c t r o p h o r e s i s 36 V I . B l ood F l ow S t u d i e s 37 M i c r o s p h e r e p r e p a r a t i o n 38 V I I . E l e c t r o n M i c r o s c o p y 39 Sample p r e p a r a t i o n 41 L i g h t m i c r o s c o p y 42 ( v i ) V I I I . P h o s p h o l i p i d E x t r a c t i o n and S e p a r a t i o n 43 O n e - d i m e n s i o n a l sys tem 43 Phospho rus a n a l y s i s 44 Two - d imen s i o n a l s y s tem 44 S t a t i s t i c a l a n a l y s e s 44 MATERIALS 44 RESULTS I . P r e l i m i n a r y S t u d i e s 45 I I . 20 M i n u t e L i g a t i o n w i t h Drug P r e t r e a t m e n t 53 I I I . 40 M i n u t e L i g a t i o n and 60 M i n u t e R e p e r f u s i o n w i t h D-600 and P r o p r a n o l o l P r e t r e a t m e n t 65 M i t o c h o n d r i a l ATPase a c t i v i t y 65 Sa r co l emma l N a + , K + - A T P a s e and K + -NPPa s e a c t i v i t i e s 66 ATP 70 Water c o n t e n t 70 Ion l e v e l s 73 SDS -ge l e l e c t r o p h o r e s i s 75 C o n t r a c t i l i t y 77 U l t r a s t r u c t u r e 81 IV . 40 M i n u t e L i g a t i o n and 60 M i n u t e R e p e r f u s i o n w i t h D-600 A d m i n i s t r a t i o n 5 M i n u t e s P r i o r t o R e p e r f u s i o n 102 V. 0 -60 M i n u t e L i g a t i o n S t u d y - Enzyme A c t i v i t y v_s. Ion L e v e l s 109 DISCUSSION Aims 117 Advan tages and D i s a d v a n t a g e s o f t h e Model 117 Drug S t u d i e s 130 REFERENCES 145 BIBLIOGRAPHY 158 APPENDICES 159 ( v i i ) L IST OF TABLES T ab l e I . Compa r i s on o f ATP c o n t e n t s i n t i s s u e t a k e n f r om t he b e a t i n g h e a r t v_s. K C l - E D T A - T r i s p e r f u s e d h e a r t 26 T a b l e I I . P e r c e n t c o n t a m i n a t i o n o f sa r co l emma l f r a c t i o n (F^) w i t h m i t o c h o n d r i a (F-^) as a s s e s s e d by c y t o ch rome c o x i d a s e a c t i v i t y 29 T a b l e I I I . P u r i t y o f t he m i t o c h o n d r i a l f r a c t i o n (F-^) a f t e r v a r i o u s t r e a t m e n t s 31 T a b l e IV . P u r i f i c a t i o n o f m i t o c h o n d r i a l and s a r co l emma l p r o t e i n f rom wet h e a r t w e i g h t 32 T a b l e V . B l o o d f l o w i n the i s c h a e m i c and r e p e r f u s e d l e f t v e n t r i c l e 40 T a b l e V I . Degree o f r e c o v e r y o f m i t o c h o n d r i a l and sa r co l emma l ATPase a c t i v i t i e s a f t e r v a r i o u s i n t e r v a l s o f i s c h a e m i a f o l l o w e d by a 60 m i nu t e p e r i o d o f r e p e r f u s i o n 48 T a b l e V I I . ATP c o n t e n t s o f h e a r t s s u b j e c t e d t o v a r i o u s p e r i o d s o f l i g a t i o n and l i g a t i o n p l u s r e p e r f u s i o n 52 T a b l e V I I I . P h o s p h o l i p i d c o n t e n t o f s a r co l emma l membranes i s o l a t e d f r om c o n t r o l , l i g a t e d , and l i g a t e d and r e p e r f u s e d m y o c a r d i a 54 T a b l e I X . Drugs used as p r e t r e a t m e n t p r i o r t o a 20 m i nu t e p e r i o d o f c o r o n a r y l i g a t i o n 56 T a b l e X . Drug e f f e c t s on b i o c h e m i c a l v a r i a b l e s a f t e r 20 min sham l i g a t i o n . "Drug c o n t r o l s " 57 T a b l e X I . T i s s u e w a t e r c o n t e n t i n h e a r t s s u b j e c t e d t o 20 m i nu t e s o f l i g a t i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t 63 T a b l e X I I . I n c i d e n c e o f v e n t r i c u l a r f i b r i l l a t i o n i n r a b b i t s s u b j e c t e d t o 20 m i nu t e s o f i s c h a e m i a w i t h o r w i t h o u t d rug p r e t r e a t m e n t 64 T a b l e X I I I . Membrane a c t i v i t i e s o f h e a r t s s u b j e c t e d t o 10 DC c o u n t e r - shock s o f 0 . 5 W a t t - s e c each d u r i n g s h a m - 1 i g a t i o n and r e p e r f u s i o n 69 T a b l e X I V . T i s s u e w a t e r c o n t e n t i n h e a r t s s u b j e c t e d t o 40 min o f l i g a t i o n and 60 min o f r e p e r f u s i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t 72 T a b l e XV. S y s t o l i c b l o o d p r e s s u r e a f t e r v a r i o u s p e r i o d s o f r e p e r - f u s i o n f o l l o w i n g 40 m i n u t e s o f c o r o n a r y l i g a t i o n 79 ( v i i i ) T a b l e X V I . O b j e c t i v e a s se s smen t o f u l t r a s t r u c t u r a l i n t e g r i t y o f h e a r t s s u b j e c t e d t o i s c h a e m i a / r e p e r f u s i o n i n j u r y w i t h o r w i t h o u t d rug p r e t r e a t m e n t T a b l e X V I I . T i s s u e i o n and w a t e r c o n t e n t i n h e a r t s g i v e n 0 . 10 mg/kg D-600 HCl 5 m i nu t e s p r i o r t o r e p e r f u s i o n T a b l e X V I I I . P e r c e n t i n i t i a l ( d p / d t ) m a x o f h e a r t s d u r i n g r e p e r - f u s i o n p e r i o d a f t e r 40 m i n u t e s o f l i g a t i o n w i t h o r w i t h o u t D-600 p r i o r t o r e p e r f u s i o n T a b l e X I X . Ion and wa te r c o n t e n t o f h e a r t s s u b j e c t e d t o 0 -60 m i n u t e s o f 1 i g a t i on T a b l e XX . P h o s p h o l i p i d p r o f i l e s o f h e a r t s s u b j e c t e d t o v a r i o u s p e r i o d s o f l i g a t i o n ( i x ) L IST OF FIGURES F i g u r e 1 . M i t o c h o n d r i a l membrane-bound enzyme a c t i v i t y a f t e r 0-60 min o f c o r o n a r y l i g a t i o n 46 F i g u r e 2 . Sa r co l emma l enzyme a c t i v i t y a f t e r 0 -60 min o f c o r o n a r y l i g a t i o n 47 F i g u r e 3 . S t i m u l a t i o n by K o f Mg - dependen t NPPase a c t i v i t y i n sa r co l emma l v e s i c l e s i s o l a t e d f r om c o n t r o l , l i g a t e d and l i g a t e d and r e p e r f u s e d m y o c a r d i a 50 2+ + F i g u r e 4 . A c t i v a t i o n by Mg o f K - s t i m u l a t e d NPPase a c t i v i t y i n sa r co l emma l v e s i c l e s p r e p a r e d f r om c o n t r o l , l i g a t e d and l i g a t e d and r e p e r f u s e d m y o c a r d i a 51 F i g u r e 5 . M i t o c h o n d r i a l a z i d e - s e n s i t i v e ATPase a c t i v i t y i n m i t o - c h o n d r i a i s o l a t e d f rom h e a r t s s u b j e c t e d t o 20 min o f l i g a t i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t 59 F i g u r e 6 . N a + , K + - A T P a s e a c t i v i t y i n sa rco lemma i s o l a t e d f r om h e a r t s s u b j e c t e d t o 20 min o f l i g a t i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t 60 F i g u r e 7 . ATP c o n t e n t o f h e a r t s s u b j e c t e d t o 20 min o f l i g a t i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t 61 F i g u r e 8 . M i t o c h o n d r i a l a z i d e - s e n s i t i v e ATPase a c t i v i t y i n m i t o - c h o n d r i a i s o l a t e d f rom c o n t r o l and 1 i g a t e d / r e p e r f u s e d h e a r t s w i t h o r w i t h o u t d rug p r e t r e a t m e n t 67 F i g u r e 9 . N a + , K + - A T P a s e and K + - N P P a s e a c t i v i t y o f sa rco lemma i s o l a t e d f rom h e a r t s s u b j e c t e d t o l i g a t i o n f o l l o w e d by r e p e r f u s i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t 68 F i g u r e 1 0 . ATP c o n t e n t o f h e a r t s s u b j e c t e d t o l i g a t i o n f o l l o w e d by r e p e r f u s i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t 71 F i g u r e 1 1 . Ca , Na , and Mg l e v e l s i n h e a r t s s u b j e c t e d t o l i g a t i o n p l u s r e p e r f u s i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t 74 (x ) F i g u r e 1 2 . F i g u r e 1 3 . F i g u r e 14 . F i g u r e 1 5 . F i g u r e 1 6 . F i g u r e 17 . F i g u r e 1 8 . F i g u r e 1 9 . F i g u r e 2 0 . F i g u r e 2 1 . F i g u r e 2 2 . F i g u r e 2 3 . F i g u r e 2 4 . F i g u r e 2 5 . SDS-ge l e l e c t r o p h o r e s i s o f m i t o c h o n d r i a l f r a c t i o n s i s o l a t e d f rom c o n t r o l , l i g a t e d and 1 i g a t e d / r e p e r f u s e d h e a r t s 76 P e r c e n t i n i t i a l ( d D / d t ) m a x ° f h e a r t s d u r i n g t h e p e r i o d o f d rug t r e a t m e n t , l i g a t i o n and r e p e r f u s i o n 78 L i g h t m i c r o g r a p h s o f s e c t i o n s f rom c o n t r o l and 1 i g a t e d / r e p e r - f u s e d myocard ium 82 L i g h t m i c r o g r a p h s o f 1 i g a t e d / r e p e r f u s e d h e a r t s p r e t r e a t e d w i t h D-600 o r p r o p r a n o l o l 83 E l e c t r o n m i c r o g r a p h s o f c o n t r o l m y o c a r d i a 84 -87 E l e c t r o n m i c r o g r a p h s o f 1 i g a t e d / r e p e r f u s e d m y o c a r d i a 88 -91 E l e c t r o n m i c r o g r a p h s o f 1 i g a t e d / r e p e r f u s e d m y o c a r d i a p r e t r e a t e d w i t h D-600 93-96 E l e c t r o n m i c r o g r a p h s o f 1 i g a t e d / r e p e r f u s e d m y o c a r d i a p r e t r e a t e d w i t h p r o p r a n o l o l 97 -100 M i t o c h o n d r i a l a z i d e - s e n s i t i v e ATPase a c t i v i t y i n CONT, L+R and D-600 REP g roups 103 Sarco lemmal N a + , K + - A T P a s e and K + - N P P a s e a c t i v i t y i n CONT, L+R and D-600 REP g roups 104 ATP c o n t e n t o f h e a r t s i n CONT, L+R, and D-600 REP g roups 106 Sa rco lemma l Na ,K -ATPase and K + - N P P a s e a c t i v i t y o f h e a r t s s u b j e c t e d t o 0 -60 min o f l i g a t i o n 110 + + 2 + 2 + Na /K and Ca /Mg r a t i o s i n t i s s u e o b t a i n e d f r om h e a r t s s u b j e c t e d t o 0 -60 min o f i s c h a e m i a 113 Supe r imposed g r aphs o f t h e a l t e r a t i o n s i n N a + / K + r a t i o , N a + , K + - A T P a s e a c t i v i t y and ATP c o n t e n t w i t h i n c r e a s i n g l i g a t i o n t i m e 114 L IST OF APPENDICES Append i x I . F r e quen c y Response o f G r a s s D i f f e r e n t i a t o r Model 7P20C 159 Append i x I I . S o l u t i o n P r e p a r a t i o n f o r T i s s u e F i x a t i o n f o r E l e c t r o n M i c r o s c o p y 160 Append i x I I I . R a d i o a c t i v e M i c r o s p h e r e Te chn i que f o r B l o o d F low D e t e r m i n a t i o n 161 ( x i ) ACKNOWLEDGEMENTS I w i s h t o thank Dav i d God in f o r h i s p a t i e n c e , k i n d n e s s and s en se o f humour i n g u i d i n g me t h r o u g h t h i s p r o j e c t . H i s e n t h u s i a s m and s u p p o r t made my s t a y i n h i s l a b o r a t o r y an e x c i t i n g and c h a l l e n g i n g o n e . I am a l s o g r a t e f u l t o Maureen G a r n e t t and The r e s e Ng f o r t h e i r supe rb t e c h n i c a l a s s i s t a n c e and i r r e p r e s s i b l e f l o w o f i d e a s wh i ch g e n e r a l l y f e l l o u t s i d e t he domain o f s c i e n c e . My s i n c e r e a p p r e c i a t i o n t o Mr . L. Ve to o f t he B i o s c i e n c e s Depar tment f o r h i s t e c h n i c a l a d v i c e and a s s i s t a n c e w i t h t h e e l e c t r o n m i c r o s c o p y . D r s . W. K. O v a l l e , P. Dow and M. Todd o f t he Depar tment o f Anatomy, a l s o p r o v i d e d v a l u a b l e d i s c u s s i o n s r e g a r d i n g t h e i n t e r p r e t a t i o n o f t h e p h o t o m i c r o g r a p h s . The t y p i n g and o r g a n i z i n g o f t h i s t h e s i s was done by T r a c y S locombe who d e s e r v e s f u l l c r e d i t f o r her s p e e d , a c c u r a c y , and above a l l , p a t i e n c e . I wou ld a l s o l i k e t o thank t he Canad i a n Hea r t F o u n d a t i o n and the U n i v e r - s i t y o f B r i t i s h Co l umb i a f o r t h e i r f i n a n c i a l s u p p o r t . F i n a l l y , I wou ld l i k e t o thank my p a r e n t s who have a lway s encou raged me i n my e n d e a v o u r s . ( x i i ) Death and l i f e a r e l o o k ed on as bu t t r a n s f o r m a t i o n s ; t h e my r i a d c r e a t i o n i s a l l o f a k i n d , t h e r e i s a k i n s h i p t h r o u g h a l l . Hua i Nan Tzu 2 n d C e n t u r y B .C . - 1 - HISTORICAL OVERVIEW D e s c r i p t i o n s o f t h e v a r i o u s c l i n i c a l m a n i f e s t a t i o n s o f c o r o n a r y h e a r t d i s e a s e such as c h e s t p a i n and syncope d a t e f r om t h e e a r l i e s t c i v i l i z a t i o n s . However , u n t i l r i g o r o u s i n v e s t i g a t i o n s o f i n t e r n a l anatomy and p h y s i o l o g y were i n i t i a t e d , t h e a e t i o l o g y o f t h i s mo rb i d c o n d i t i o n c o u l d no t be u n d e r - s t o o d . T r a n s l a t i o n o f s e v e r a l E g y p t i a n p a p y r i t h a t d a t e f r om c a . 3000 B . C . t o c a . 1500 B .C . has r e v e a l e d t h a t t h i s c u l t u r e h e l d some i n a c c u r a t e and e r r o n e o u s i d e a s c o n c e r n i n g h e a r t f u n c t i o n i n h e a l t h and d i s e a s e ; however , t h e E g y p t i a n s d i d r e c o g n i z e t h a t t h e h e a r t was t h e c e n t r a l o rgan f rom wh i ch t h e b l o o d v e s s e l s emanated and t h a t t h e s e were c onne c t e d t o a l l p a r t s o f t h e body . In a d d i t i o n , t h e y e v a l u a t e d a b n o r m a l i t i e s o f t h e p u l s e t o a s c e r t a i n t h e s t a t u s o f t h e h e a r t - a p r a c t i c e known as " p u l s e l o r e " . Du r i ng t h e p e r i o d o f L a o - T s e and C o n f u c i u s ( c a . 300 B . C . ) , t h e C h i n e s e f u r t h e r advanced knowledge o f c a r d i o v a s c u l a r m e d i c i n e . However , by 1000 A . D . m y s t i c i s m and p an t h e i sm f l o u r i s h e d i n C h i n e s e m e d i c a l p r a c t i c e and t h i s t r e n d , c o u p l e d w i t h t h e i r poo r g r a s p o f anatomy, r e s u l t e d i n a r e t r o g r e s s i o n o f s c i e n t i f i c t h o u g h t . In t h e absence o f an u n d e r s t a n d i n g o f i n t e r n a l mechan i sms , t h e C h i n e s e , l i k e t h e E g y p t i a n s , r e l i e d upon a complex p u l s e - l o r e t o a s s e s s t h e s t a t u s o f t h e c a r d i o v a s c u l a r s y s t e m . In c o n t r a s t t o t h e s e e a r l i e r c u l t u r e s , Greek s c h o l a r s s t u d i e d anatomy c a r e f u l l y and i t was p r o b a b l y t h e i r p r a c t i c e o f human and an ima l d i s s e c t i o n t h a t gave them an u n d e r s t a n d i n g o f c a r d i o v a s c u l a r p h y s i o l o g y t h a t was h i t h e r t o u n p a r a l l e l e d . However , t h e po s t -mo r t em d i s s e c t i o n s may a l s o have been t h e s o u r c e o f some o f t h e i r m i s t a k e n i d e a s c o n c e r n i n g t h e mechanisms o f h e a r t f u n c t i o n and b l o o d c i r c u l a t i o n . These e r r o r s were p e r p e t u a t e d f o r c e n t u r i e s by u n c r i t i c a l s c h o l a r s who r e g a r d e d t h e Greek t r e a t i s e s as f a c t . - 2 - N e v e r t h e l e s s , t h e deve lopment o f a r i g o r o u s and r a t i o n a l app roach t o t h e s t u d y o f c a r d i o v a s c u l a r s y s t em i n h e a l t h and d i s e a s e p r o v i d e d a f ramework w i t h i n wh i c h l a t e r i n v e s t i g a t o r s c o u l d e v a l u a t e t h e v a l i d i t y o f t h e Greek f i n d i n g s . P r o b a b l y t h e e a r l i e s t s i g n i f i c a n t c o n t r i b u t i o n d u r i n g t h e Greek c i v i l i z - a t i o n came f r om A lcmaeon o f C r o t o n a ( c a . 500 B . C . ) , who p r a c t i s e d ana tom i c d i s s e c t i o n . He p roposed t h a t t h e b r a i n was t h e s e a t o f c o n s c i o u s n e s s , t h e r e b y c o n t r a d i c t i n g t h e common l y -he l d b e l i e f t h a t t h e h e a r t f u l f i l l e d t h i s r o l e . The l a t e r H i p p o c r a t i c t r e a t i s e s (460 -370 B . C . ) , a c o l l e c t i o n o f manu- s c r i p t s t h a t were w r i t t e n m o s t l y by e x p e r i e n c e d p h y s i c i a n s , a l s o propounded t h i s v i e w . In a d d i t i o n , t h i s i n f l u e n t i a l s e t o f documents expanded t he know ledge o f e x t e r n a l , and t o a l e s s e r e x t e n t , i n t e r n a l anatomy - i n c l u d i n g t h e h e a r t . I n t e r e s t i n g l y , t h e H i p p o c r a t i c d o c t r i n e m a i n t a i n e d t h a t t h e h e a r t was not s u s c e p t i b l e t o d i s e a s e owing t o i t s "mas s i v e and compac t " c o m p o s i t i o n . The work o f A r i s t o t l e (384-332 B . C . ) a l s o had g r e a t i n f l u e n c e and a l t h o u g h he made some v a l u a b l e c o n t r i b u t i o n s t o a n a t o m i c a l k now l ege , he made no a t t emp t t o i n v e s t i g a t e t h e a e t i o l o g y o f m y o c a r d i a l d i s e a s e . H i s b e l i e f s were i n f l u e n c e d t o a l a r g e e x t e n t by t h e H i p p o c r a t i c t r e a t i s e s i n c l u d i n g t h e c on cep t t h a t t h e h e a r t was t h e s t r o n g e s t o f a l l o rgans and was t h e r e f o r e t h e l e a s t s u s c e p t i b l e t o d i s e a s e . In a d d i t i o n , A r i s t o t l e had no c on cep t o f t h e a c t i o n o f t h e h e a r t as a pump and he put f o r w a r d t h e f o l l o w i n g t h e o r y o f how t h e h e a r t f u n c t i o n s : t h e h e a r t was t h e i n n a t e s o u r c e o f hea t ( hea t d i s t i n - g u i s h e d l i f e f r om dea th ) and t h i s hea t l i q u i f i e d t h e b l o o d and expanded i t . T h i s a c c oun t ed f o r t h e movement o f t h e chambers o f t h e h e a r t . R e s p i r a t i o n no t o n l y n o u r i s h e d t h e v i t a l s p i r i t bu t a l s o quenched t h e e x c e s s hea t so t h a t b r e a t h i n g was a l s o a s s o c i a t e d w i t h t h e p a s s i v e movement o f t h e h e a r t - 3 - because t h e c o o l i n g r educed b l o o d vo l ume . O d d l y , he b e l i e v e d t h a t t h e h e a r t , no t t h e b r a i n , was t h e s e a t o f c o n c i o u s n e s s , r e v i v i n g an i d e a t h a t had l o ng s i n c e been r e j e c t e d . G i v en t h e above p r o p o s a l f o r t h e mechan ism of h e a r t a c t i o n , t h i s h e a r t - c e n t r e d v i ew was , i n some s e n s e , a l o g i c a l n e c e s s i t y . Many o f A r i s t o t l e ' s i d e a s were r e f u t e d d u r i n g t h e A l e x a n d r i a n p e r i o d ( c a . 331 B . C . ) . Du r i ng t h i s p e r i o d , human d i s s e c t i o n was p e r m i t t e d by t h e a u t h o r i t i e s and , as a r e s u l t o f t h e s e d i s s e c t i o n s , t h e A l e x a n d r i a n s c h o l a r E r i s t r a t u s made t h e s u g g e s t i o n t h a t t h e h e a r t f u n c t i o n e d as a pump. U n f o r - t u n a t e l y , t h e empty a r t e r i e s o f t h e po s t -mo r t em s u b j e c t s l e d h im t o c o n c l u d e t h a t o n l y t h e v e i n s c o n t a i n e d b l o o d and t h a t t h e a r t e r i e s were f i l l e d w i t h "pneuma" ( v i t a l s p i r i t ) - a l i f e - g i v i n g s u b s t a n c e d e r i v e d f r om t h e a tmos - p h e r e . S u b s e q u e n t l y , P t o l emy Physkon (146-117 B . C . ) banned human d i s s e c t i o n and e x p e l l e d t h e p h y s i c i a n s f r om A l e x a n d r i a . T h i s e r a d i c a t i o n o f a c e n t r a l i z e d , s c h o l a r l y i n s t i t u t i o n f r agmen ted t h e s c i e n t i f i c commun i t y , and i n t h e p e r i o d t h a t f o l l o w e d , t h e pneuma t h e o r y became more complex and e n t r e n c h e d and p u l s e - l o r e , no t ana tom i c o b s e r v a t i o n , become t h e more common m e d i c a l p r a c - t i c e . T h e r e f o r e , i n t h e m i d s t o f t h i s s c i e n t i f i c r e g r e s s i o n , i t i s no t s u r p r i s i n g t h a t t he s c h o l a r l y work o f C l a u d i u s Ga l en (131 -201 A . D . ) had a g r e a t impac t no t o n l y on Greek m e d i c i n e bu t on m e d i c i n e i n t h e c e n t u r i e s t h a t f o l l o w e d . He r e f u t e d t h e e x i s t i n g t h e o r i e s o f p n e u m a - f i l l e d a r t e r i e s u s i n g e x p e r i m e n t a l and r a t i o n a l i n v e s t i g a t i o n and showed t h a t t h e y c o n t a i n e d b l o o d . (However , he s t i l l i n c o r p o r a t e d pneuma i n t o h i s c on c ep t o f b l o o d f l o w ) . G i v en t h a t t h e u n d e r s t a n d i n g o f normal c a r d i o v a s c u l a r p h y s i o l o g y was s k e t c h y , h i s i d e a s r e g a r d i n g h e a r t p a t h o l o g y were no t w e l l - d e v e l o p e d . L i k e A r i s t o t l e , G a l e n f e l t t h a t t h e h e a r t was t h e s o u r c e o f i n n a t e h e a t , t h e r e - f o r e , he c o n c l u d e d t h a t d e a t h must i n some way be due t o h e a r t f a i l u r e , - 4 - a l t h o u g h he d i d no t s p e c i f i c a l l y i n v e s t i g a t e t h e n a t u r e o f t h i s f a i l u r e . He had a s u p e r i o r knowledge o f anatomy, y e t h i s p h y s i o l o g i c a l i n t e r p r e t a t i o n s were o f t e n w r o n g . The i d e a o f t h e c i r c u l a t i o n o f t h e b l o o d e l u d e d him and h i s own t h e o r y o f b l o o d f l o w f o r c e d h im t o p o s t u l a t e t h e e x i s t e n c e o f " i n v i s i b l e p o r e s " t h a t c onne c t e d t h e l e f t and r i g h t s i d e s o f t h e h e a r t . In r e t r o s p e c t , i t seems d i f f i c u l t t o imag i ne t h a t some o f h i s e r r o n o u s i d e a s were uphe l d u n t i l t h e 16 th and 17 th c e n t u r i e s . However , h i s a p p a r e n t l y r i g o r o u s i n v e s t i g a t i o n s spawned a w o r k a b l e model o f c a r d i o v a s c u l a r f u n c t i o n , a l b e i t one t h a t c o n t a i n e d many i n c o n s i s t e n c i e s . H i s t h e o r i e s were r e ga r d ed as f a c t and t h u s l a t e r s c h o l a r s d i d no t f e e l t h a t i t was n e c e s s a r y t o i n v e s - t i g a t e t h e v a l i d i t y o f h i s c l a i m s . A comment a t t r i b u t e d t o a c on t empo r a r y o f W i l l i a m Ha rvey r e v e a l s how much i n f l u e n c e Ga l en had on m e d i c a l t h ough t u n t i l t h e 17 th c e n t u r y . Somewhat f a c e t i o u s l y , J ean R i o l a n w r o t e t h a t " i f d i s s e c t i o n s and o b s e r v a t i o n s d i f f e r e d f r om t h o s e o f G a l e n , t hen n a t u r e must have c h a n g e d " . The i n e v i t a b l e r e j e c t i o n o f G a l e n ' s v i ew o f t h e c a r d i o v a s c u l a r s y s t em was i n i t i a t e d by t h e c a r e f u l a n a t o m i c a l s t u d i e s o f Leona rdo da V i n c i ( 1452 -1519) and V e s a l i u s ( 1 5 1 4 - 1 5 6 4 ) . By u n c o v e r i n g many o f t he e r r o r s o f G a l e n ' s work , t h e i r f i n d i n g s l a i d t h e groundwork f o r t h e subsequen t e l u c i d a - t i o n o f t h e c i r c u l a t i o n o f t h e b l o o d by W i l l i a m Ha rvey i n 1649 . U s i n g e x p e r i m e n t a l and s c i e n t i f i c me thod , Ha r vey no t o n l y d emons t r a t e d t h e c o n t i n - uous c i r c u l a t i o n o f t h e b l o o d - r e f u t i n g t h e t h e o r y o f t h e pe rmeab le i n t r a - v e n t r i c u l a r septum - bu t he a l s o d e s c r i b e d a " t h i r d , e x t r e m e l y s h o r t c i r c u - l a t i o n " , i . e . , t he c o r o n a r y c i r c u l a t i o n . A new u n d e r s t a n d i n g o f t h e normal anatomy and p h y s i o l o g y o f t h e h e a r t p r o v i d e d a f ramework w i t h i n wh i ch a bno r - mal c o n d i t i o n s c o u l d t hen be i d e n t i f i e d and s t u d i e d . W i t h i n 50 y e a r s o f H a r v e y ' s d i s c o v e r i e s , L o r en zo B e l l i n i ( 1643 -1704 ) documented c a l c i f i c a t i o n - 5 - o f t h e c o r o n a r y a r t e r i e s and i n 1698 , P i e r r e C h i r a c p roduced c a r d i a c s t a n d - s t i l l i n t he dog a f t e r p e r f o r m i n g an e x p e r i m e n t a l c o r o n a r y l i g a t i o n . A t e x t b o o k p u b l i s h e d i n 1703 by Jean B a p t i s t de Senac , s e r v e d as a comprehen - s i v e r e f e r e n c e i n t h a t i t c o r r e l a t e d ca se h i s t o r i e s w i t h po s t -mo r t em p a t h o - l o g i c f i n d i n g s i n t h e h e a r t . By t h e 18 th c e n t u r y , t h e e x i s t e n c e o f c o r o n a r y c a l c i f i c a t i o n was common know l edge ; howeve r , t h e c o n n e c t i o n between t h e s e po s t -mo r t em o b s e r v a t i o n s and t h e c l i n i c a l s i g n s o f d i s e a s e as w e l l as an u n d e r s t a n d i n g o f t h e g e n e s i s o f t h e c a l c i f i c a t i o n were s t i l l t o be d i s c o v e r e d . The c l a s s i c d e s c r i p t i o n o f a n g i n a l c h e s t p a i n and i t s r e l a t i o n t o t h e h e a r t was f i r s t e x p r e s s e d by W i l l i a m Heberden i n 1768 , a l t h o u g h he d i d no t a s s o c i a t e " p e c t o r i s d o l o r " , l a t e r named a n g i n a , w i t h any s p e c i f i c mo rb i d p a t h o l o g y o f t h e h e a r t . T h i s a s s o c i a t i o n was made by two o f h i s c on t empo r - a r i e s , C . H. P a r r y and W. J e n n e r , who, u s i n g c a r e f u l b e d s i d e and pos t -mo r t em i n v e s t i g a t i o n , i n i t i a t e d t h e i d e a t h a t a n g i n a was i n some way r e l a t e d t o t h e " o s s i f i c a t i o n " o f t h e c o r o n a r y a r t e r i e s . In a d d i t i o n , r e a l i z i n g t h a t t h e p a i n was r e l a t e d t o e x e r t i o n , P a r r y s ugge s t e d t h a t t h e d i s e a s e d h e a r t was o n l y c a p a b l e o f s u s t a i n i n g modera te w o r k l o a d s . In 1809 , A l l a n Burns o f G l a sgow f u r t h e r r e f i n e d t h i s c on cep t o f an a l t e r e d supp l y /demand r a t i o i n a n g i n a w i t h h i s e l e g a n t e x p e r i m e n t s i n wh i ch he l i g a t e d an e x e r c i s i n g l i m b and no t ed t h a t p a i n was evoked when t h e mus c l e was f a t i g u e d . E x t r a p o l a t i n g t h e s e r e s u l t s t o c a r d i a c m u s c l e , he t h e r e f o r e c o n c l u d e d t h a t p a i n was t h e r e s u l t o f i n s u f f i c i e n t b l o o d s u p p l y t o t h e h e a r t under c o n d i t i o n s o f s t r e s s . Burns w r o t e t h e f o l l o w i n g t o d e s c r i b e h i s f i n d i n g s : " I f , howeve r , we c a l l i n t o v i g o u r o u s a c t i o n , a l imb round wh i ch we have w i t h a modera te deg ree o f t i g h t n e s s a p p l i e d a l i g a t u r e , we f i n d t hen t h e member can o n l y s u p p o r t i t s a c t i o n f o r a v e r y s h o r t t i m e ; f o r - 6 - now i t s s u p p l y o f ene rgy and i t s e x p e n d i t u r e do no t b a l a n c e each o t h e r : c o n s e q u e n t l y , i t soon f r om a d e f i c i e n c y o f ne r vous i n f l u e n c e and a r t e r - i a l b l o o d f a i l s and s i n k s i n t o a s t a t e o f q u i e s c e n c e . A h e a r t , t h e c o r o n a r y v e s s e l s o f wh i ch a r e c a r t i l a g i n o u s o r o s s i f i e d i s i n n e a r l y a s i m i l a r c o n d i t i o n ; i t can l i k e t h e l i m b , be g i r t w i t h a m o d e r a t e l y l i g h t l i g a t u r e , d i s c h a r g e i t s f u n c t i o n s so l o ng as i t s a c t i o n i s modera te and e q u a l . I n c r e a s e , howeve r , t h e a c t i o n o f t h e who le body and a l o ng w i t h t h e r e s t , t h a t o f t h e h e a r t , and you w i l l soon see e x e m p l i f i e d t h e t r u t h o f what has been s a i d ; w i t h t h i s d i f f e r e n c e , t h a t as t h e r e i s no i n t e r - r u p t i o n t o t h e a c t i o n o f t h e c a r d i a l n e r v e s , t h e h e a r t w i l l be a b l e t o h o l d ou t a l i t t l e longer"- '- D e s p i t e t h e p a t h o l o g i c a l e v i d e n c e o f c o r o n a r y o c c l u s i v e d i s e a s e i n some p a t i e n t s w i t h a n g i n a , t h e p r e v a i l i n g v i ew was t h a t t h e symptoms a r o s e f r om a " ne r vou s a f f l i c t i o n " o f t h e h e a r t , p o s s i b l y spasm. However , t h e c on c ep t t h a t an o c c l u s i v e d i s e a s e ( e . g . c a l c i f i c a t i o n ) o f t h e c o r o n a r y a r t e r i e s was r e s p o n s i b l e f o r t h e p a t i e n t s ' symptoms was no t u n i v e r s a l l y a c c ep t e d because c a r e f u l po s t -mo r t em e x a m i n a t i o n o f t h e h e a r t s o f p a t i e n t s who had had symptoms o f " p e c t o r i s d o l o r " d i d no t a lways r e v e a l c o r o n a r y " o s s i f i c a t i o n " . These i n c o n s i s t e n t p a t h o l o g i c a l f i n d i n g s l e d many 19 th c e n t u r y s c i e n t i s t s i n c l u d i n g John Warren ( 1 7 5 3 - 1 8 1 5 ) , h i s s o n , John C o l l i n s Warren ( 1 7 7 8 - 1 8 5 6 ) , and S i r John Fo rbe s (1787 -1861 ) t o v o c i f e r o u s l y c r i t i c i z e t h e c o r o n a r y c a l c i f i c a t i o n t h e o r y o f p a i n . As l a t e as 1854 , W i l l i a m S t o ke s w ro t e t h a t " a n g i n a i s a symptom o f a weakened h e a r t ; t h e o b s t r u c t i o n o f t h e c o r o n a r y a r t e r i e s was a d i s e a s e whose a c t i o n s a r e remote and whose e x i s t e n c e unne ce s - s a r y " . The a l t e r n a t i v e h y p o t h e s i s , wh i ch was s u p p o r t e d by most c r i t i c s , m a i n t a i n e d t h a t va sospasm, not me chan i c a l o b s t r u c t i o n , was t h e cause o f t h e c h e s t p a i n . I n 1867 , S i r Thomas Laude r B run ton p u b l i s h e d an a r t i c l e i n t h e L a n c e t d e s c r i b i n g t h e e f f i c a c y o f t h e v a s o d i l a t o r , amyl n i t r i t e , i n r e l i e v - i n g a n g i n a l p a i n . He a s c r i b e d t h e b e n e f i t s o f d rug a d m i n i s t r a t i o n t o t h e a b i l i t y o f t h e n i t r i t e s t o r e l i e v e v a so spa sm . E a r l y h i s t o r i a n s o f m e d i c i n e o f t e n m i n i m i z e d t h e impo r t a n c e o f t h e o b s e r v a t i o n s o f t h e c r i t i c s and even s ugge s t e d t h a t t h e y impeded t h e u n i v e r s a l a c c e p t a n c e o f t h e c o r o n a r y o c c l u - - 7 - s i o n t h e o r y . I t i s i n t e r e s t i n g t o v i ew t h i s a t t i t u d e i n t h e c o n t e x t o f t h e c u r r e n t c o n t r o v e r s i e s r e g a r d i n g t h e r o l e o f vasospasm i n t h e g e n e s i s o f bo th a n g i n a and m y o c a r d i a l i n f a r c t i o n . In r e t r o s p e c t , t h e r e s t r a i n t o f some s c h o l a r s i n u n c r i t i c a l l y a c c e p t i n g t h e c o r o n a r y o c c l u s i o n t h e o r y was , i t seems, t h e r e s u l t o f c a r e f u l o b s e r v a t i o n and no t a s t u b b o r n r e f u s a l t o " s ee t h e t r u t h " . A no t h e r c o n t r o v e r s y t h a t a r o s e d u r i n g t h e 19 th c e n t u r y c on ce r ned t h e e l u c i d a t i o n o f t h e i s c h a e m i a - t o - i n f a r c t p r o g r e s s i o n i n t h e myoca rd i um. In 1852 , t h e renowned German p a t h o l o g i s t , R u d o l f V i r c h o w , a t t r i b u t e d t h e f i b r o - s i s i n i n f a r c t e d h e a r t s t o c h r o n i c m y o c a r d i t i s . T h i s v i ew was a c c e p t e d by many p r om inen t i n v e s t i g a t o r s u n t i l 1880 when V i r c h o w ' s s t u d e n t , C a r l W e i g a r t , a f t e r c a r e f u l m i c r o s c o p i c e x a m i n a t i o n o f h e a r t s t h a t had v a r y i n g deg r ee s o f m y o c a r d i a l damage, d e s c r i b e d t h e e n t i r e i n f a r c t i o n p r o c e s s , f r om c o r o n a r y o b s t r u c t i o n and musc l e a t r o p h y t o t h e consequen t n e c r o s i s and onse t o f f i b r o s i s . Thus , by t h e end o f t h e c e n t u r y , t h e g e n e s i s o f m y o c a r d i a l i n f a r c t i o n f r om c o r o n a r y o c c l u s i o n had been d e s c r i b e d i n d e t a i l , and t h e r e l a t i o n s h i p between c o r o n a r y n a r r o w i n g ( e i t h e r by me chan i c a l o b s t r u c t i o n o r by spasm) and ang i n a was known amongst t h e p r om inen t i n v e s t i g a t o r s o f t h e p e r i o d . D e s p i t e t h e r e l a t i v e l y c omp l e t e d e s c r i p t i o n o f t h e d i s e a s e by t h e end o f t h e 19 th c e n t u r y , i t s p r e v a l e n c e was s t i l l u n r e c o g n i z e d u n t i l w e l l i n t o t h e 20 th c e n t u r y . The d e t a i l s o f t h e c l i n i c a l s i g n s o f c o r o n a r y h e a r t d i s e a s e and t h e po s t -mo r t em p a t h o l o g y , as w e l l as t h e d i s t i n c t i o n between a n g i n a and m y o c a r d i a l i n f a r c t i o n , were no t commonly known amongst t h e m a j o r i t y o f p r a c t i s i n g p h y s i c i a n s . Thu s , i n t h e f i r s t y e a r s o f t h e 20 th c e n t u r y , t he d i s e a s e was no t f e l t t o be t oo i m p o r t a n t . Two p a p e r s , t h e f i r s t by W.P. Ob ras t zow and N.D. S t r a s c h e s k o (1910) and t h e se cond by James H e r r i c k - 8 - ( 1 9 1 2 ) , were e s p e c i a l l y i m p o r t a n t i n i n i t i a t i n g t h e u n d e r s t a n d i n g o f myoca r - 2 3 d i a l i n f a r c t i o n as a c l i n i c a l l y r e c o g n i z a b l e e n t i t y . ' These gave c o h e r - en t a c c o u n t s o f t h e c u r r e n t knowledge o f h e a r t d i s e a s e and , mo r eo ve r , t h e paper by Ob ras to zow and S t r a s c h e s k o c l e a r l y d i f f e r e n t i a t e d between ang i n a p e c t o r i s and m y o c a r d i a l i n f a r c t i o n . C l i n i c a l d i a g n o s i s was a l s o a i d e d by t h e deve lopment o f t h e E i n t h o v e n s t r i n g e l e c t r o c a r d i o g r a p h (1908) and t h e a s s o c i a t i o n o f m y o c a r d i a l i s c h a e m i a w i t h e a s i l y measured ECG a b n o r m a l i t i e s p r o b a b l y p l a y e d t h e g r e a t e s t r o l e i n f a c i l i t a t i n g t h e c l i n i c a l r e c o g n i t i o n 4 o f c o r o n a r y h e a r t d i s e a s e . D u r i n g t h e 1930s t he i s c h a e m i a / i n f a r c t i o n p r o c e s s was s t u d i e d more r i g o r o u s l y w i t h t h e aim o f e l u c i d a t i n g t h e e a r l y f u n c t i o n a l s e q u e l a e o f c o r o n a r y o c c l u s i o n . The c o r o n a r y l i g a t i o n model o f m y o c a r d i a l i s c h a e m i a was chosen by R. Tennant and C . W igge rs (1935) f o r t h e i r c l a s s i c s t u d y o f t h e 5 c o n t r a c t i l e p r o p e r t i e s o f t h e i s c h a e m i c c a n i n e l e f t v e n t r i c l e . In a d d i - t i o n t o n o t i n g t h e immed ia te d e c l i n e i n c o n t r a c t i l e f o r c e caused by c o r o n a r y o c c l u s i o n , t h e y a l s o r e c o g n i z e d t h a t r e p e r f u s i o n c o u l d r e v e r s e t h e myog ra - p h i c a b n o r m a l i t i e s i f t h e p e r i o d o f i s c h a e m i a was no t t o o p r o l o n g e d . In 1941 , t h i s p e r i o d o f r e v e r s i b l e c o r o n a r y damage i n dogs was more t h o r o u g h l y a s s e s s e d by B l u m g a r t , G i l l i g a n and S c h l e s i n g e r . ^ L i k e Tennant and W i g g e r s , t h e y r e c o g n i z e d t h a t t empo r a r y i s c h a e m i a may l e a d t o m y o c a r d i a l n e c r o s i s and t h e i r s t u d y a t t emp ted t o d e f i n e t h e p e r i o d o f o c c l u s i o n t h a t p roduced i r r e v e r s i b l e m y o c a r d i a l damage, as a s s e s s e d by t h e deve lopment o f s c a r t i s s u e . They showed t h a t 5-20 m inu t e s o f o c c l u s i o n p roduced i n j u r y t h a t was r e v e r s e d by r e p e r f u s i o n , whereas l o n g e r l i g a t i o n p e r i o d s (>30 m i n u t e s ) r e s u l t e d i n sub sequen t f i b r o s i s t h e r e b y c o n f i r m i n g t h e s u g g e s t i o n t h a t s h o r t p e r i o d s o f i s c h a e m i c damage were r e v e r s i b l e . - 9 - W h i l e t h e b a s i c s c i e n t i f i c r e s e a r c h i n t o t h e i s c h a e m i a / i n f a r c t i o n p r o c e s s was p r o c e e d i n g , s t u d i e s a imed a t r e d u c i n g i s c h a e m i a - i n d u c e d myoca r - d i a l i n j u r y were a l r e a d y i n p r o g r e s s . In 1932 , C . S . Beck began p e r f o r m i n g r e v a s c u l a r i z a t i o n s u r g e r y on dogs and t h e work c u l m i n a t e d i n h i s f i r s t such o p e r a t i o n on a p a t i e n t i n 1949 .^ F u r t h e r m o r e , he c o r r e c t l y h y p o t h e s i z e d t h a t t h e " l o c a l i z e d a n o x i a " p roduced by c o r o n a r y o c c l u s i o n c o u l d t r i g g e r v e n t r i c u l a r f i b r i l l a t i o n a l t h o u g h he d i d no t u n d e r s t a n d t h e u n d e r l y i n g mechan i sm. Work i s c o n t i n u i n g i n t h i s f i e l d t o f u r t h e r improve t h e c o n d i - t i o n s o f bypas s s u r g e r y i n o r d e r t o r e du ce t h e i n c i d e n c e o f i n t r a - o p e r a t i v e , as w e l l as p o s t - o p e r a t i v e m y o c a r d i a l damage. In t h e 1930s , t r e a t m e n t s o t h e r than r e v a s c u l a r i z a t i o n s u r g e r y were a t t emp t ed t o l i m i t t h e c o u r s e o f c o r o n a r y a r t e r y d i s e a s e . These i n c l u d e d t h y r o i d e c t o m y and sympathectomy and , no t s u r p r i s i n g l y , t h e s e r a t h e r h a r s h t r e a t m e n t s were q u i c k l y abandoned . Sympathectomy was l a t e r r e p l a c e d by a l c o h o l i n f u s i o n p a r a v e r t e b r a l ^ a l o n g t h e s y m p a t h e t i c c h a i n and even t o d a y , d r ug a b l a t i o n o f t h e l e f t s t e l l a t e g a n g l i o n i s s t i l l r a r e l y a used t r e a t m e n t f o r i n t r a c t a b l e a n g i n a . Thus , e a r l y i n v e s t i g a t o r s s u s p e c t e d t h a t t h e sympa- t h e t i c n e r vou s s y s t em was i n v o l v e d i n t h e deve lopment o f i s c h a e m i c damage. In f a c t , t h e a s s o c i a t i o n between e m o t i o n a l s t r e s s and a n g i n a l p a i n was r e c o g n i z e d even a t t h e t ime o f Heberden whose c o n t e m p o r a r y , John H u n t e r , s u f f e r e d f r om s t r e s s - i n d u c e d a n g i n a . ( I t was s a i d t h a t Hun te r j o k i n g l y c l a i m e d t h a t h i s l i f e was i n t h e hands o f any r a s c a l who chose t o annoy o r p r ovoke h i m . I r o n i c a l l y , he d i e d ( p r e sumab l y o f a h e a r t a t t a c k ) d u r i n g a p a r t i c u l a r l y v i o l e n t d i s c u s s i o n a t a boa rd m e e t i n g ) . The adven t o f t h e B - a d r e n e r g i c a n t a g o n i s t s i n t he 1960s and 1970 s , meant t h a t s y m p a t h e t i c b l o c k a d e became l e s s t r a u m a t i c and t h e a d m i n i s t r a t i o n o f e - a d r e n e r g i c a n t a g - o n i s t s i n p o s t - m y o c a r d i a l i n f a r c t i o n has r e c e n t l y been shown t o s i g n i f i c a n t - - 10 - l y r educe one - y ea r m o r t a l i t y r a t e s . A v a r i e t y o f d r ug i n t e r v e n t i o n s w i t h v e r y d i f f e r e n t p h a r m a c o l o g i c a l p r o p e r t i e s a re c u r r e n t l y b e i n g a s s e s s ed f o r t h e i r a b i l i t y t o l i m i t m y o c a r d i a l i s c h a e m i c damage. In summary, t h e know ledge o f t h e norma l p h y s i o l o g y o f t h e human c a r d i o - v a s c u l a r s y s t em a t t a i n e d by t h e 17 th c e n t u r y s e t t h e s t a ge f o r t h e s u b s e - quen t i n t e g r a t i o n o f i ndependen t p a t h o l o g i c a l and c l i n i c a l o b s e r v a t i o n s o f c o r o n a r y h e a r t d i s e a s e . The c o n n e c t i o n between a n g i n a p e c t o r i s and c o r o n a r y p a t h o l o g y was l a r g e l y a c c ep t e d by t h e 20 th c e n t u r y a l t h o u g h no t w i t h o u t a c o n t i n g e n t o f i n v e s t i g a t o r s who f e l t t h a t vasospasm was t h e o n l y s o u r c e o f t h e p r e - c o r d i a l p a i n . As t h e d i s t i n c t i o n between a n g i n a and m y o c a r d i a l i n f a r c t i o n became c l e a r , t h e r e was a r e a l i z a t i o n t h a t t h e " o s s i f i c a t i o n " was no t n e c e s s a r i l y a t e r m i n a l e v e n t , i . e . , t h e p a t i e n t m igh t s u r v i v e such an i n s u l t . U s i n g improved c l i n i c a l and l a b o r a t o r y d i a g n o s t i c t e c h n i q u e s , p a t i e n t s w i t h t h e d i s e a s e c o u l d be i d e n t i f i e d and w i t h t h i s advance , came the r e c o g n i t i o n o f t he p r e v a l e n c e o f t h e d i s e a s e . Subsequen t r e s e a r c h on t h e compromised myocard ium has r e s u l t e d no t o n l y i n improved methods o f t r e a t m e n t bu t i t has a l s o expanded t h e body o f knowledge c o n c e r n i n g t h e c e l l u l a r p h y s i o l o g y o f bo th normal and i s c h a e m i c c a r d i a c m u s c l e . INTRODUCTION T w e n t i e t h c e n t u r y s t u d i e s o f i s c h a e m i c h e a r t d i s e a s e have examined numerous i n d i c e s o f c e l l u l a r damage and a l t e r a t i o n s i n a lmos t e v e r y a s p e c t o f c e l l u l a r f u n c t i o n have been measu red . Because i r r e v e r s i b l e i s c h a e m i c i n j u r y r e s u l t s i n n e c r o s i s and f i b r o s i s o f t h e myoca rd i um , c e s s a t i o n o f b l o o d f l o w w i l l u l t i m a t e l y a f f e c t a l l c e l l u l a r p r o c e s s e s . The i m p o r t a n t q u e s t i o n then becomes: wh i ch e f f e c t s o f c o r o n a r y l i g a t i o n a r e r e s p o n s i b l e f o r t he deve l opmen t o f i r r e v e r s i b l e m y o c a r d i a l damage? I f i t were p o s s i b l e t o s u f f i c i e n t l y d e l a y o r p r e v e n t t h e s e c r i t i c a l c h ange s , i t m igh t a l s o be p o s s i b l e t o r educe t h e e x t e n t o f n e c r o s i s . T h i s c o u l d be a c c o m p l i s h e d e i t h e r by an i n t e r v e n t i o n wh i c h a f f e c t s a s p e c i f i c c e l l u l a r p r o c e s s o r by a g e n e r a l t r e a t m e n t wh i ch a t t e n u a t e s t h e e f f e c t s o f i s c h a e m i a by a more g e n e r a l a c t i o n on c e l l u l a r m e t a b o l i s m . A t t e m p t s have been made t o u n d e r s t a n d more f u l l y t h e consequence o f c o r o n a r y l i g a t i o n , i n c l u d i n g i t s e f f e c t s on t h e c o n t r a c t i l e p r o p e r t i e s o f t h e h e a r t . The 17 th and 18 th c e n t u r y i n v e s t i g a t o r s o f t e n o b s e r v ed t h a t c o r o n a r y l i g a t i o n p roduced c a r d i a c s t a n d s t i l l ; howeve r , i t w a s n ' t u n t i l 1935 t h a t a d e t a i l e d a c coun t o f t h e i s c h a e m i a - i n d u c e d c o n t r a c t i l e a b n o r m a l i t i e s was p u b l i s h e d by Tennan t and W i g g e r s . By mea su r i n g t h e c o n t r a c t i l e f o r c e o f t h e i s c h a e m i c c a n i n e l e f t v e n t r i c l e , t h e y o b s e r v e d t h a t c o r o n a r y o c c l u s i o n p roduced a r a p i d d e c r e a s e i n c o n t r a c t i o n t h a t o n l y s l o w l y r e v e r s e d upon 5 r e p e r f u s i o n . Subsequen t s t u d i e s have c o n f i r m e d t h e v a l i d i t y o f t h e s e o b s e r v a t i o n s and , i n a d d i t i o n , have e v a l u a t e d bo th t h e i s c h a e m i c and t h e normal l e f t v e n t r i c l e and i n t h e f o rme r no ted t h e p r e s en c e o f a t r a n s i e n t m a r g i n a l zone o f p a r t i a l l y i m p a i r e d f u n c t i o n i n a d d i t i o n t o t h e c e n t r a l zone 9 10 o f n o n - c o n t r a c t i n g f i b r e s . ' Thu s , c o r o n a r y l i g a t i o n has been found t o - 12 - p roduce an immed ia te d e c l i n e i n c o n t r a c t i o n t h a t o n l y s l o w l y r e v e r s e s w i t h r e p e r f u s i o n As men t i oned p r e v i o u s l y , t h e e l e c t r o c a r d i o g r a m p r o v i d e d t h e f i r s t r e l i a - b l e and s e n s i t i v e c l i n i c a l method o f d e t e c t i n g i s c h a e m i c damage. ' ' However , a l t h o u g h q u a n t i t a t i o n o f t h e ECG wave changes ( s p e c i f i c a l l y S-T segment e l e v a t i o n ) i s a u s e f u l t o o l i n a s s e s s i n g t h e e f f i c a c y o f c e r t a i n i n t e r v e n t i o n s on m y o c a r d i a l i s c h a e m i c i n j u r y , d i f f i c u l t y a r i s e s i n r e l a t i n g t h e s e e l e c t r i c a l a b n o r m a l i t i e s t o a l t e r a t i o n s i n i n t r a c e l l u l a r p o t e n t i a l c h a n g e s . In c ompa r i s on t o t he e l e c t r o c a r d i o g r a m , t h e m i c r o s c o p i c e x a m i n a t i o n o f t h e i s c h a e m i c myocard ium r e v e a l s more d i r e c t l y t h e i n t e g r i t y o f t h e c e l l s , a nd , i n s p i t e o f i t s d i s a d v a n t a g e s , m i c r o s c o p i c a n a l y s i s o f m y o c a r d i a l t i s s u e has been a v a l u a b l e t e c h n i q u e i n t h e e l u c i d a t i o n o f t h e e v e n t s f o l l o w i n g c o r o n a r y o c c l u s i o n . The use o f t h e e l e c t r o n m i c r o s c o p e r e v e a l e d more s u b t l e s t r u c t u r a l changes t han had been d e t e c t e d by t h e 19 th c e n t u r y p a t h o l o g i s t s . J e n n i n g s e t a l . o b s e r v e d e a r l y a l t e r a t i o n s i n m i t o c h o n d r i a l 14 i n t e g r i t y , i n c l u d i n g c l e a r i n g o f t h e m a t r i x and c r i s t a e f r a g m e n t a t i o n . G l y c ogen d e p l e t i o n , t h e appea rance o f m a t r i x d e n s i t i e s , s a r co l emma l d i s r u p - t i o n and n u c l e a r c h r o m a t i n c l u m p i n g became appa r en t i n t i s s u e t h a t had been s u b j e c t e d t o l o n g e r p e r i o d s o f i s chaem i a . ^ - " ^ T h i s t e c h n i q u e a l l o w e d i n v e s t i g a t o r s t o s t u d y t h e t i m e - c o u r s e o f m o r p h o l o g i c changes i n t h e o r g a n - e l l e s ; howeve r , S chape r e t a l . have shown t h a t t h e appea rance o f u l t r a s t r u c - t u r a l d e f e c t s l a gged b eh i n d t h e immed ia te d e c r e a s e i n c o n t r a c t i l i t y and , c o n v e r s e l y , c e l l mo rpho l ogy n o r m a l i z e d w i t h r e p e r f u s i o n d e s p i t e t h e p e r s i s - t e n c e o f d e f e c t s i n b i o c h e m i c a l and c o n t r a c t i l e f u n c t i o n . ^ T h u s , a l t h o u g h m i c r o s c o p i c a n a l y s i s y i e l d s u s e f u l i n f o r m a t i o n r e g a r d i n g t h e e f f e c t s o f i s c h a e m i a on s u b c e l l u l a r s t r u c t u r e , i t does no t e v a l u a t e t h e f u n c t i o n a l - 13 - c a p a b i l i t y o f t h e c e l l , e . g . t h e normal appea rance o f t h e m i t o c h o n d r i o n on an e l e c t r o n m i c r o g r a p h does no t p r e c l u d e t h e e x i s t e n c e o f a d e f e c t i n o x i d a - t i v e p h o s p h o r y l a t i o n . Such i s c h a e m i a - i n d u c e d d e c r e a s e s i n m i t o c h o n d r i a l o x i d a t i v e p h o s p h o r y l a t i o n have been measured i n v i t r o by many i n v e s t i g a - t o r s . 1 8 > 1 9 i n a d d i t i o n , R o u s l i n and M i l l a r d have ob s e r v ed t h a t i s c h a e m i a i n h i b i t s t h e a c t i v i t i e s o f s e v e r a l o f t h e e l e c t r o n t r a n s f e r c h a i n 20 enzymes . The d y s f u n c t i o n o f o x i d a t i v e p h o s p h o r y l a t i o n wou ld be e x p e c t e d t o l i m i t t h e s u p p l y o f a d e n o s i n e - 5 ' - t r i p h o s p h a t e (ATP) a nd , i n d e e d , t h e r a p i d d e p l e t i o n o f ATP a f t e r c o r o n a r y o c c l u s i o n i s a w e l l - d o c u m e n t e d pheno- 21 22 menon. ' I t has been s p e c u l a t e d t h a t measurement o f t i s s u e l e v e l s o f ATP may no t r e f l e c t t h e a c t u a l a v a i l a b i l i t y o f ATP t o t h e ATP-dependen t p r o c e s s e s i n t h e c e l l because ATP i s l i m i t e d t o c e r t a i n s u b c e l l u l a r a r ea s o r c ompa r tmen t s . Such a c o m p a r t m e n t a l i z a t i o n o f ATP has been i n voked t o e x p l a i n t h e f a c t t h a t c o n t r a c t i l i t y d e c l i n e s b e f o r e a f a l l i n ATP l e v e l s can 23 be d e t e c t e d . S e v e r a l i n v e s t i g a t o r s have s ugge s t ed t h a t ATP a v a i l a b i l i t y c o u l d be r e du ced as a r e s u l t o f t h e i s c h a e m i a - i n d u c e d i n h i b i t i o n o f t h e m i t o c h o n d r i a l a den i ne n u c l e o t i d e t r a n s l o c a s e enzyme wh i ch exchanges ATP f o r e x t r a m i t o c h o n d r i a l ADP. T h i s i n h i b i t i o n may be med i a t ed by a c cumu l a t ed 24 m e t a b o l i t e s o f f a t t y a c i d o x i d a t i o n , e s p e c i a l l y a c y l - C o A d e r i v a t i v e s . O t he r f a c t o r s t h a t a r e t h ough t t o l i m i t ATP s u p p l y i n t h e i s c h a e m i c h e a r t i n c l u d e d e c r e a s e d g l y c o g e n s t o r e s , d e c r e a s e d g l u c o s e s u p p l y and compromised a n a e r o b i c m e t a b o l i s m ( g l y c o l y s i s ) . The l a t t e r may be due t o H + - m e d i a t e d 22 25 i n h i b i t i o n o f g l y c o l y t i c enzymes . ' I t i s e v i d e n t f rom t he f o r e g o i n g t h a t many o f t h e d e l e t e r i o u s e f f e c t s o f c o r o n a r y o c c l u s i o n a r i s e no t o n l y f r om t he d e p l e t i o n o f O2 and s u b s t r a t e s , bu t a l s o f r om t h e a c c u m u l a t i o n o f m e t a b o l i c p r o d u c t s such as H and f a t t y a c i d i n t e r m e d i a t e s . T h e r e f o r e , i t i s i m p o r t a n t t o d i s t i n g u i s h between t h e i n j u r y p roduced by i s c h a e m i a and - 14 - t h a t a s s o c i a t e d w i t h a n o x i a . A n o x i a i s t h e c o n d i t i o n t h a t a r i s e s when t h e PC>2 i s n e g l i g i b l e bu t b l o o d f l o w t o t h e myocard ium i s m a i n t a i n e d , whereas i s c h a e m i a r e s u l t s f r om bo th low and a c r i t i c a l d e c r e a s e i n b l o o d f l o w . I s c h a e m i c and a n o x i c h e a r t s bo th depend on a n a e r o b i c m e t a b o l i s m f o r ene r g y bu t a n o x i c i n j u r y i s no t as s e v e r e because s u b s t r a t e s u p p l y and m e t a b o l i t e remova l a r e p r e s e r v e d . T h u s , t h e r e s u l t s o f s t u d i e s e x a m i n i n g a n o x i c i n j u r y c anno t a lways be d i r e c t l y a p p l i e d t o t h e i n v e s t i g a t i o n o f t h e m o l e c u l a r mechanisms o f i s c h a e m i c i n j u r y . C o r o n a r y o c c l u s i o n no t o n l y d i s r u p t s m i t o c h o n d r i a l f u n c t i o n bu t a l s o appea r s t o a f f e c t t h e sa r co l emma l membrane. U s i n g e x t r a c e l l u l a r K + e l e c - t r o d e s , We i ss and Sh i ne have demons t r a t ed t h e e x i s t e n c e o f a s m a l l and r a p i d l y r e v e r s i b l e e f f l u x o f K + f r om t h e i s c h a e m i c p o r t i o n o f t h e myoca r - dium i n t h e f i r s t few m i nu t e s f o l l o w i n g c o r o n a r y l i g a t i o n . I f t h e i s c h a e m i c a r e a i s r e p e r f u s e d a f t e r a p p r o x i m a t e l y 15 -30 min o f c o r o n a r y + ? + 2+ o c c l u s i o n , t h e c e l l s g r a d u a l l y a c cumu l a t e Na and Ca and l o s e Mg + 0 7 28 and K . The r e i s a l s o a c o n c o m i t a n t g a i n i n i n t r a c e l l u l a r w a t e r . ' In c o n t r a s t t o t he i n i t i a l K + e f f l u x , t h e s e i o n i c changes a re no t e a s i l y r e v e r s e d s u g g e s t i n g t h a t t h e d e f e c t s i n s a r co l emma l p e r m e a b i l i t y a r e i r r e - v e r s i b l e . The r e - i n s t a t e m e n t o f b l o o d f l o w t o i r r e v e r s i b l y - d a m a g e d t i s s u e p r o b a b l y i n du ce s t h e s e i o n i c a b n o r m a l i t i e s because t h e sarco lemma i s no l o n g e r a b a r r i e r t o t h e p a s s i v e f l o w o f i o n s down t h e i r c o n c e n t r a t i o n g r a d i - e n t s . C a l c i u m i n f l u x i s t hough t t o be e s p e c i a l l y d e l e t e r i o u s i n t h a t h i g h c y t o s o l i c l e v e l s o f c a l c i u m i n h i b i t a v a r i e t y o f c e l l p r o c e s s e s . I t has been s ugge s t e d t h a t t h i s c a l c i u m a c c u m u l a t i o n , i n a d d i t i o n t o t h e c e l l s w e l l i n g , c o n t r i b u t e s t o t h e r a p i d d e t e r i o r a t i o n o f c e l l u l a r i n t e g r i t y t h a t o c c u r s w i t h r e p e r f u s i o n . 3 0 , 3 1 - 15 - Wi th i n c r e a s i n g p e r i o d s o f c o r o n a r y o c c l u s i o n , t h e sa r co l emma l d i s r u p - t i o n p r o g r e s s e s u n t i l i t i s o f a magn i tude t h a t a l l o w s l e a k age o f l a r g e r components o f t h e c y t o s o l , i n c l u d i n g s o l u b l e enzymes . The phenomenon has been e x p l o i t e d c l i n i c a l l y i n t h a t measurement o f enzyme l e a kage i s now used as an i n d i c a t o r o f i n f a r c t s i z e . La Due and Wrob l ewsk i showed t h a t serum g l u t a m i c o x a l o a c e t i c t r a n s a m i n a s e (SGOT) l e v e l s were i n c r e a s e d i n t h e p lasma o f p a t i e n t s who had r e c e n t l y s u f f e r e d a m y o c a r d i a l i n f a r c t i o n . More r e c e n t l y , t h e p l a sma l e v e l o f t h e m y o c a r d i a l i soenzyme o f SGOT as w e l l as s e v e r a l o t h e r c y t o s o l i c enzymes , e . g . , t h e c r e a t i n e pho sphok i n a s e and l a c t a t e dehyd rogenase MB - i s o en z ymes , have been used t o c o n f i r m t h e p r e s en ce -3-3 o f a m y o c a r d i a l i n f a r c t . A l t h o u g h t h i s t e c h n i q u e i s o f c l i n i c a l impo r - t a n c e , t h e f a c t t h a t enzyme l e a k a g e does no t r e a c h a maximum u n t i l s e v e r a l hou r s a f t e r c o r o n a r y v e s s e l o c c l u s i o n i n d i c a t e s t h a t i t i s a r e l a t i v e l y l a t e e ven t i n t h e deve lopment o f i s c h a e m i c damage and h e r a l d s t h e deve lopment o f an i n f a r c t i n t h e i s c h a e m i c z o n e . Thu s , a l t h o u g h t h e measurement o f p l asma enzymes can be o f c l i n i c a l use t o d e t e r m i n e t h e p r e s en ce o f an i n f a r c t , i t p r o v i d e s m i n ima l i n f o r m a t i o n r e g a r d i n g t h e b i o c h e m i c a l mechan ism o f s a r c o - lemmal damage. I t i s a p p a r e n t t h a t t h e c r i t i c a l e v e n t s wh i c h d e t e r m i n e t h e u l t i m a t e v i a b i l i t y o f t h e i s c h a e m i c m y o c a r d i a l c e l l o c c u r w i t h i n t h e f i r s t hour f o l l o w i n g c o r o n a r y l i g a t i o n and t h a t t h e r e s u l t i n g p l a sma membrane damage wou ld be e x p e c t e d t o s e r i o u s l y compromise c e l l u l a r h o m e o s t a s i s . The p r e c i s e m o l e c u l a r e v e n t s t h a t a r e r e s p o n s i b l e f o r s a r co l emma l damage a r e c u r r e n t l y unknown, a l t h o u g h s e v e r a l h y po t h e s e s have been f o r m u l a t e d . F o r e xamp l e , i t has been s p e c u l a t e d t h a t t h e a c c u m u l a t i o n o f t h e m e t a b o l i t e s o f f a t t y a c i d 34 35 o x i d a t i o n may c o n t r i b u t e t o c e l l i n j u r y . ' A t low c o n c e n t r a t i o n s , t h e s e d e t e r g e n t - l i k e , a m p h i p a t h i c compounds have been shown t o d i s r u p t a i - 16 - v a r i e t y o f c e l l u l a r f u n c t i o n s i n v i t r o , i n c l u d i n g sa r co l emma l N a + , K + - 36 37 33 ATPase a c t i v i t y , ' s a r c o p l a s m i c r e t i c u l u m f u n c t i o n a nd , as no t ed 38 39 e a r l i e r , m i t o c h o n d r i a l f u n c t i o n . ' I t i s c o n c e i v a b l e t h a t even low c o n c e n t r a t i o n s o f t h e s e agen t s may have w i d e s p r e a d i n h i b i t o r y e f f e c t s on c e l l u l a r m e t a b o l i s m . O the r compounds have been i m p l i c a t e d i n t h e p r o d u c t i o n o f i s c h a e m i a - i n d u c e d membrane d i s r u p t i o n . C h i e n e t a l . have demons t r a t ed an i s c h a e m i a - i n d u c e d i n c r e a s e i n l y s o s o m a l enzyme a c t i v i t y as w e l l as an a c c e l - 40 -42 e r a t e d p h o s p h o l i p i d d e g r a d a t i o n i n bo th h e a r t and l i v e r t i s s u e . These d a t a have l e d them t o s ugge s t t h a t a c t i v a t i o n o f l y s o s o m a l p h o s p h o l i p - a ses may be r e s p o n s i b l e f o r t h e o b s e r v e d membrane damage. M o r e o v e r , f u r t h e r i n d i r e c t e v i d e n c e f o r p h o s p h o l i p i d d e g r a d a t i o n has come f r om e xpe r imen t s d e m o n s t r a t i n g e l e v a t i o n s i n t h e t i s s u e c o n t e n t o f p h o s p h o l i p i d b r eakdown- p r o d u c t s , e s p e c i a l l y l y s o p h o s p h a t i d y l c h o l i n e and l y s o p h o s p h a t i d y l e t h a n o l a - m i n e . These agen t s a r e a l s o p o t e n t d e t e r g e n t s a nd , i f p r e s e n t i n c r i t i c a l c o n c e n t r a t i o n s i n t h e membrane, c o u l d p o t e n t i a l l y d i s r u p t m e t a b o l i c p r o c e s - s e s . However , t h e magn i t ude and s i g n i f i c a n c e o f t h e i n c r e a s e i n 43 -45 p h o s p h o l i p i d breakdown p r p d u c t s i s c u r r e n t l y i n d i s p u t e . T h u s , t h e r o l e o f pho s - p h o l i p i d d e g r a d a t i o n a n d / o r t h e a c c u m u l a t i o n o f m e t a b o l i c b y - p r o d u c t s i n t h e g e n e s i s o f i s c h a e m i c membrane damage i s c u r r e n t l y u n c l e a r . From t h e f o r e g o i n g , i t i s a ppa r en t t h a t t h e a l t e r a t i o n s i nduced by i s c h a e m i a must u l t i m a t e l y i n v o l v e v i r t u a l l y e v e r y a s p e c t o f c e l l u l a r me t abo - l i s m . C o n s e q u e n t l y , a d i v e r s i t y o f t h e r a p e u t i c i n t e r v e n t i o n s have been t r i e d e m p i r i c a l l y i n an a t t emp t t o l i m i t t h e damage. M o r e o v e r , t h e e x i s - t e n c e o f t i s s u e w i t h i n t e r m e d i a t e damage b o r d e r i n g t h e s e v e r e l y i s c h a e m i c z o n e ^ ' ^ has m o t i v a t e d r e s e a r c h e r s t o i d e n t i f y t r e a t m e n t s wh i c h m igh t " s a l v a g e " t h i s t i s s u e , t h e r e b y r e d u c i n g t h e e x t e n t o f t h e i n f a r c t i o n . In e x p e r i m e n t a l m y o c a r d i a l i s c h a e m i a i n t h e r a t , F i s h b e i n e t a l . showed t h a t - 17 - t h i s " b o r d e r z one " i s p r e s e n t f o r a p p r o x i m a t e l y 9 hou r s p o s t - 1 i g a t i o n a f t e r 47 wh i ch t h e c e l l s become i r r e v e r s i b l y i n j u r e d . An appa r en t zone of i n t e r - med i a t e damage may be fo rmed when zones o f normal and i s c h a e m i c myocard ium i n t e r d i g i t a t e a t t h e b o r d e r o f t h e o c c l u d e d a r e a . T h i s p a t t e r n i s seen i n 48 t h e i s c h a e m i c c a n i n e myoca rd i um. Thus , t h e t i m i n g o f i n t e r v e n t i o n s a imed a t r e d u c i n g i n f a r c t s i z e i s c r u c i a l t o t h e i r u l t i m a t e e f f i c a c y . R e p e r f u s i o n a l o n e w i l l r e v e r s e some o f t h e d e l e t e r i o u s e f f e c t s o f s h o r t 6 49 50 (up t o 15-20 min) p e r i o d s o f c o r o n a r y o c c l u s i o n . ' ' However , r e p e r f u - s i o n a f t e r more p r o l o n g e d p e r i o d s o f i s c h a e m i a (>20-30 m in) no t o n l y f a i l s t o r e v e r s e many o f t h e b i o c h e m i c a l and f u n c t i o n a l a b n o r m a l i t i e s bu t a c t u a l l y 51 e x a c e r b a t e s t h e t i s s u e damage. T h i s s o - c a l l e d " r e p e r f u s i o n i n j u r y " does no t o c c u r u n l e s s t h e t i s s u e has a l r e a d y been made i s c h a e m i c , t h u s t h e l a b e l " r e p e r f u s i o n i n j u r y " i s a m i snomer . A more s e m a n t i c a l l y - c o r r e c t t e rm wou ld be i s c h a e m i c / r e p e r f u s i o n i n j u r y . As men t i oned w i t h r e g a r d t o i o n i c changes a f t e r c o r o n a r y o c c l u s i o n , t h e a c c e l e r a t e d damage i s p r e sumab l y caused by t h e sudden r e i n s t a t e m e n t o f b l o o d f l o w t o c e l l s wh i ch have n o n - f u n c t i o n a l pe rme- a b i l i t y b a r r i e r s . A v a r i e t y o f t r e a t m e n t s have been examined f o r t h e i r a b i l i t y t o l i m i t t h e i n j u r y p roduced by bo th i s c h a e m i a and r e p e r f u s i o n . G e n e r a l l y , t h e y p o s s e s s a t l e a s t one o f t h e f o l l o w i n g a c t i o n s ; i n c r e a s i n g b l o o d f l o w t o t h e i s c h a e m i c z o n e , a l t e r i n g i s c h a e m i c m e t a b o l i s m t o r e du c e t h e p r o d u c t i o n of d e l e t e r i o u s m e t a b o l i t e s , d e c r e a s i n g t h e w o r k - l o a d o f t h e h e a r t o r d i r e c t l y m o d i f y i n g t h e c o n d i t i o n s o f r e p e r f u s i o n . B - A d r e n e r g i c a n t a g o n i s t s po s s e s s s e v e r a l a c t i o n s some o r a l l o f wh i ch m igh t a t t e n u a t e i s c h a e m i c damage and t h e s e agen t s have been r e p o r t e d t o r e du ce i s c h a e m i c i n j u r y w i t h o r w i t h o u t s ub sequen t r e p e r f u s i o n i n a v a r i e t y 52 -54 o f e x p e r i m e n t a l m o d e l s . The r e d u c t i o n o f m y o c a r d i a l oxygen consump- - 18 - t i o n (MVC^) wh i ch r e s u l t s f r om t h e b e t a - a d r e n e r g i c b l o c k i n g p r o p e r t i e s i s t h ough t t o be t he most i m p o r t a n t d e t e r m i n a n t o f t h e b e n e f i c i a l e f f e c t s o f 55 56 t h i s c l a s s o f d r u g s ; ' howeve r , some i n v e s t i g a t o r s have shown t h a t t h e n o n - B - b l o c k i n g p r o p e r t i e s o f t h e s e agen t s a l s o c o n t r i b u t e t o t h e r e d u c t i o n • 57 58 i n MVC^. ' In a d d i t i o n , H i l l i s e t al_. showed t h a t e - a d r e n e r g i c a n t a g o n i s t t r e a t m e n t c o u l d a l s o i n c r e a s e b l o o d f l o w t o t h e i s c h a e m i c a r e a - 59 an a c t i o n wh i ch c o u l d p o t e n t i a l l y r e du ce i n f a r c t s i z e . However , t h i s c o n c l u s i o n i s no t s u p p o r t e d by t h e f i n d i n g s o f K l o n e r , Re imer and J e n n i n g s who showed t h a t t h e r e d u c t i o n i n i n f a r c t s i z e by e - b l o c k e r s was p r o b a b l y no t a r e s u l t o f i n c r e a s e d c o l l a t e r a l f l o w . ^ T h u s , a l t h o u g h t h e s e agen t s appear t o s i g n i f i c a n t l y r educe t i s s u e damage i n t h e a c u t e model o f myoca r - d i a l i s c h a e m i a , t h e r e l a t i v e c o n t r i b u t i o n o f t h e v a r i o u s d rug a c t i o n s t o t h e b e n e f i c i a l e f f e c t s i s p r e s e n t l y i n d i s p u t e . The as sessmen t o f d r ug a c t i o n i s f u r t h e r c o m p l i c a t e d by p rob l ems i n t h e d e f i n i t i o n and measurement o f " i n f a r c t s i z e " y_s. " i s c h a e m i c z one " v_s. " a r e a o f r i s k " . The o r g a n i c c a l c i u m c h a n n e l - b l o c k e r s have a l s o been shown t o improve t h e 61—65 f u n c t i o n a l , b i o c h e m i c a l and m o r p h o l o g i c a l i n d i c e s o f i s c h a e m i c i n j u r y . The c a l c i u m c h a n n e l - b l o c k i n g p r o p e r t i e s o f t h e s e agen t s were f i r s t d e s c r i b e d i n 1964 by F l e c k e n s t e i n who s t a t e d t h a t t h e i r e f f e c t s on t h e h e a r t were 66 m im i cked by e x p o s i n g t h e h e a r t t o a h y p o c a l c a e m i c p e r f u s a t e . The m o s t - s t u d i e d d rugs i n c l u d e v e r a p a m i l , d i l t i a z e m and n i f e d i p i n e . T h i s c h e m i c a l l y h e t e r ogeneou s g roup o f compounds a r e a l l c l a s s i f i e d as c a l c i u m c h a n n e l - b l o c k e r s by v i r t u e o f t h e i r r e d u c t i o n i n t h e c a l c i u m conduc t an ce wh i c h i s p a r t l y r e s p o n s i b l e f o r t h e p l a t e a u phase o f t h e a c t i o n p o t e n t i a l i n t h e h e a r t ; ^ - ^ howeve r , t h e y have d i f f e r e n t e f f e c t s on m y o c a r d i a l c o n t r a c t i l i t y and smooth mus c l e r e a c t i v i t y i n t h e i n t a c t a n i m a l . Fo r e xamp l e , a l t h o u g h a l l t h r e e d rugs s upp r e s s a t r i o v e n t r i c u l a r noda l f u n c t i o n - 19 - i n t h e i s o l a t e d r a b b i t h e a r t , o n l y v e r a p a m i l and d i l t i a z e m a r e c l i n i c a l l y u s e f u l i n t h e t r e a t m e n t o f a t r i a l a r r h y t h m i a s . In e x p l a n a t i o n , Kawai e t a l . have p o s t u l a t e d t h a t t h e g r e a t e r v a s o d i l a t o r y p r o p e r t i e s o f n i f e d i p i n e p roduce a r e f l e x a c t i v a t i o n o f t h e s y m p a t h e t i c ne r vous s y s t em wh i ch o v e r - 71 r i d e s any d i r e c t e f f e c t on t h e A-V node . A consequence o f t h e i r c a l c i u m c h a n n e l - b l o c k i n g a b i l i t y i s n e g a t i v e i n o t r o p i s m and t h i s , i n t u r n , r e s u l t s i n a c o n c o m i t a n t r e d u c t i o n i n myoca r - d i a l oxygen consump t i on i n norma l myoca rd i um. Thu s , one e x p l a n a t i o n f o r t h e i r b e n e f i c i a l e f f e c t s i n e x p e r i m e n t a l i s c h a e m i c i n j u r y i s t h a t , l i k e t h e 6 - a d r e n e r g i c a n t a g o n i s t s , c a l c i u m c h a n n e l - b l o c k e r s a r e c a p a b l e o f r e d u c i n g t h e ene r g y r e q u i r e m e n t o f t h e i s c h a e m i c c e l l t h e r e b y p r e s e r v i n g h i gh ene rgy phospha te l e v e l s f o r m a i n t a i n a n c e o f c e l l u l a r i n t e g r i t y . In a d d i t i o n , t h e v a s o d i l a t o r y p r o p e r t i e s o f f - l o a d t h e h e a r t , f u r t h e r r e d u c i n g MVO^. The smooth m u s c l e r e l a x a t i o n may a l s o c o n t r i b u t e t o t h e b e n e f i c i a l e f f e c t s o f d rug t r e a t m e n t v i a c o r o n a r y v e s s e l d i l a t i o n w i t h c on sequen t i n c r e a s e s i n 72 c o r o n a r y b l o o d f l o w . B e s i d e s t h e s e ma jo r e f f e c t s on c a r d i a c f u n c t i o n and haemodynamics , wh i c h a r e f o r t h e most p a r t a s s o c i a t e d w i t h t h e d e c r e a s e d c a l c i u m c o n d u c t a n c e , t h e s e d rugs have been shown t o a f f e c t o t h e r a s p e c t s o f c e l l f u n c t i o n . Pang - 6 - 5 and S p e r e l a k i s have shown t h a t 10 - 1 0 M v e r a p a m i l ( bu t no t n i f e d i p i n e o r d i l t i a z e m ) i n h i b i t e d c a l c i u m b i n d i n g t o t h e g u i n e a p i g sa r co l emma l 73 membrane. E a r l i e r e x p e r i m e n t s by M a s - O l i v a and N a y l e r had a l s o demon- 45 2+ s t r a t e d t h a t v e r a p a m i l i n h i b i t e d p a s s i v e Ca - b i n d i n g t o t h e s a r c o l e m - ma and , i n a d d i t i o n , showed t h a t v e r a p a m i l a l s o i n t e r f e r e d w i t h t h e A T P - d e - 45 2+ 2+ pendent t r a n s p o r t o f Ca . M o r e o v e r , t h e d e c r e a s e i n Ca t r a n s p o r t 2+ 74 was a s s o c i a t e d w i t h a d e c r e a s e i n s a r co l emma l Ca - ATPa se a c t i v i t y . - 20 - N i f e d i p i n e has a l s o been shown t o a f f e c t m e t a b o l i s m i ndependen t f rom i t s e f f e c t s on c a l c i u m c o n d u c t a n c e . In e x p e r i m e n t s e x am i n i n g t h e e f f e c t o f n i f e d i p i n e on r e g i o n a l m y o c a r d i a l f u n c t i o n i n a n a e s t h e t i z e d p i g s , Verdouw e t a l . f o und t h a t i n t r a c o r o n a r y a d m i n i s t r a t i o n o f n i f e d i p i n e p roduced d e c r e a s e s i n r e g i o n a l 0^ c on sump t i on wh i ch were unaccompan ied by a d e c r e a s e i n 75 r e g i o n a l m y o c a r d i a l c o n t r a c t i l e f u n c t i o n . The a u t h o r s p o s t u l a t e d t h a t t h e d rug r e du ced O2 c on sump t i on by a mechanism u n r e l a t e d t o i t s n e g a t i v e i n o t r o p i c e f f e c t s . O the r e v i d e n c e f o r d i r e c t e f f e c t s o f v e r a p a m i l and d i l t i a z e m on m i t o c h o n d r i a l f u n c t i o n has been r e p o r t e d by Vaghy e t a l . . C a l c i u m c h a n n e l - b l o c k e r s have been shown t o be e f f e c t i v e no t o n l y i n l i m i t i n g t h e damage i n c u r r e d d u r i n g t h e i s c h a e m i c p e r i o d , bu t a l s o i n p r e v e n t i n g t h e sudden l a r g e i n f l u x o f c a l c i u m t h a t i s a s s o c i a t e d w i t h r e p e r - f u s i o n . Whether t h e s e d rugs p r e v e n t c a l c i u m i n f l u x v i a p r o t e c t i v e e f f e c t s d u r i n g t h e i s c h a e m i c p e r i o d , d i r e c t c a l c i u m c h a n n e l - b l o c k a d e upon r e p e r f u - s i o n o r a c o m b i n a t i o n o f b o t h o f t h e s e mechanisms i s a s u b j e c t o f c u r r e n t c o n t r o v e r s y . 6 1 , 7 7 - 7 9 In c o n t r a s t t o t he l a r g e l y p o s i t i v e f i n d i n g s i n t h e l i t e r a t u r e r e g a r d i n g t h e a b i l i t y o f c a l c i u m c h a n n e l - b l o c k e r s o r e - a d r e n e r g i c a n t a g o n i s t s t o r educe i s c h a e m i c / r e p e r f u s i o n i n j u r y , t h e e f f e c t i v e n e s s o f a v a r i e t y o f o t h e r agen t s i n l i m i t i n g i s c h a e m i c damage i s l e s s c l e a r l y e s t a b l i s h e d . Fo r exam- p l e , t h e g l u c o c o r t i c o i d s have been shown t o p r e s e r v e membrane s t r u c t u r e under c e r t a i n e x p e r i m e n t a l c o n d i t i o n s ; howeve r , t h e i r a b i l i t y t o p r e s e r v e m y o c a r d i a l s t r u c t u r e and f u n c t i o n d u r i n g i s c h a e m i a i s , a t mos t , m a r g i n - on_o? a l . S i m i l a r l y , t h e r e s u l t s o b t a i n e d u s i n g p r o s t a g l a n d i n (PG) s y n t h e - t a s e i n h i b i t o r s ( such as i n d ome t ha c i n ) have been i n c o n c l u s i v e . Sm i th e t a l . p o s t u l a t e d t h a t t h e p r o t e c t i v e e f f e c t s o f PG s y n t h e t a s e i n h i b i t o r s r e s u l t e d 83 f r om i n h i b i t i o n o f th romboxane ^ s y n t h e s i s whereas M i y a z a k i e t a l . - 21 - a t t r i b u t e d t h e b e n e f i t t o a r e d u c t i o n i n t h e f o r m a t i o n o f p r o s t a g l a n d i n 84 E^. C o n v e r s e l y , J u d g u t t e t a l . h a v e d e m o n s t r a t e d t h a t t h e i n h i b i t i o n o f p r o s t a g l a n d i n s y n t h e s i s may a c t u a l l y i n c r e a s e i n f a r c t s i z e , p o s s i b l y b y a OC d i r e c t e f f e c t on C^ c o n s u m p t i o n . T h u s , i t i s n o t c l e a r w h e t h e r o r n o t t h e n o n - s p e c i f i c i n h i b i t i o n o f p r o s t a g l a n d i n s y n t h e s i s d u r i n g a c u t e m y o c a r - d i a l i s c h a e m i a i s b e n e f i c i a l . O t h e r m i s c e l l a n e o u s a g e n t s w h i c h h a v e b e e n s h o w n t o a f f o r d l i m i t e d b e n e f i t t o t h e i s c h a e m i c h e a r t i n c l u d e : g l u c o s e - i n - s u l i n - p o t a s s i u m s o l u t i o n s , ' h y a l u r o n i d a s e , a n d h y p e r o s m o l a r m a n n i - t o l . 8 9 T h e u s e o f t h i s a r r a y o f a g e n t s h a s n o t o n l y p r o v i d e d i n f o r m a t i o n r e g a r d i n g p o t e n t i a l t h e r a p e u t i c b e n e f i t s o f c e r t a i n d r u g i n t e r v e n t i o n s b u t h a s i n d i c a t e d w h i c h o f t h e s e q u e l a e o f c o r o n a r y o c c l u s i o n may b e i n v o l v e d i n t h e p r o d u c t i o n o f c e l l d a m a g e. H o w e v e r , t h e " t r i g g e r " o f i r r e v e r s i b l e c e l l d amage i s s t i l l u n k n o w n . A d i f f i c u l t y a r i s e s i n s e p a r a t i n g t h e p r i m a r y e v e n t ( s ) o f i s c h a e m i c i n j u r y f r o m t h e s e c o n d a r y a n d t e r t i a r y e v e n t s . T h e e x p e r i m e n t s d i s c u s s e d i n t h i s t h e s i s h a v e f o c u s s e d on t h e e l u c i d a - t i o n o f t h e e a r l y m o l e c u l a r a l t e r a t i o n s t h a t r e s u l t f r o m e x p e r i m e n t a l a c u t e m y o c a r d i a l i s c h a e m i a a n d r e p e r f u s i o n i n t h e a n a e s t h e t i z e d r a b b i t . T h e p r e l i m i n a r y s t u d i e s e x a m i n e d t h e t i m e c o u r s e o f i s c h a e m i c c h a n g e s on s e v e r a l i n d i c e s o f c e l l u l a r damage i n o r d e r t o d e t e r m i n e t h e e f f e c t s o f c o r o n a r y l i g a t i o n o n i n d i r e c t m e a s u r e s o f b o t h s a r c o l e m m a l a n d m i t o c h o n d r i a l f u n c - t i o n a l i n t e g r i t y a n d t h e i r r e l a t i o n s h i p t o c e l l u l a r e n e r g y l e v e l s . S u b s e - q u e n t e x p e r i m e n t s w e r e u n d e r t a k e n t o d e f i n e t h e i n v i v o s i g n i f i c a n c e o f t h e i s c h a e m i a - i n d u c e d s a r c o l e m m a l e n z y m e c h a n g e s t h a t w e r e m e a s u r e d i n v i t r o . H a v i n g e s t a b l i s h e d t h e t i m e - c o u r s e a n d r e l a t i v e r e v e r s i b i l i t y o f t h e s e b i o c h e m i c a l a l t e r a t i o n s , t h e e f f e c t o f v a r i o u s p h a r m a c o l o g i c a l a g e n t s on t h e - 22 - changes p roduced by a 20 m i nu t e l i g a t i o n p e r i o d has been e x p l o r e d . The s i g n i f i c a n t r e d u c t i o n i n i s c h a e m i c damage by t h e c a l c i u m c h a n n e l - b l o c k e r , D-600 (methoxyve rapami 1 ) , as w e l l as a d e s i r e t o compare and c o n t r a s t t h e e f f e c t s o f D-600 w i t h t h e b e t a - b l o c k i n g agent p r o p r a n o l o l prompted us t o s t u d y t h e a b i l i t y o f t h e s e d rugs t o a t t e n u a t e i s c h a e m i c / r e p e r f u s i o n damage when a d m i n i s t e r e d p r i o r t o t h e l i g a t i o n p e r i o d . I n t h i s s t u d y , s e v e r a l l e v e l s o f c e l l u l a r o r g a n i z a t i o n r a n g i n g f r om who le o rgan f u n c t i o n t o s a r c o - lemmal membrane i n t e g r i t y were m o n i t o r e d i n t h e same an ima l t h u s e n a b l i n g us t o a s s e s s t h e e f f e c t s o f d rug t r e a t m e n t w i t h o u t b e i n g r e s t r i c t e d t o t h e r e s u l t s o f one t e c h n i q u e . The a im o f t h e s e s t u d i e s c o n c e r n i n g t h e e f f e c t s o f d rug t r e a t m e n t was no t o n l y t o i n v e s t i g a t e t h e mechanisms o f b e n e f i c i a l d rug a c t i o n bu t a l s o t o a t t emp t t o i d e n t i f y t h e c r i t i c a l m o l e c u l a r e v e n t s t h a t a r e r e s p o n s i b l e f o r t h e p r o d u c t i o n o f i r r e v e r s i b l e c e l l i n j u r y . - 23 - METHODS I . S u r g i c a l T e chn i que M a l e , New Zea l a nd w h i t e r a b b i t s 2-3 k g , were a n a e s t h e t i z e d by i n j e c t i n g 60 mg/kg o f sod ium p e n t o b a r b i t a l i n t o t h e m a r g i n a l e a r v e i n . A t r a c heo t omy was s u b s e q u e n t l y pe r f o rmed t o p e r m i t p o s i t i v e - p r e s s u r e v e n t i l a t i o n u s i n g a Pa lme r pump. In t h e e a r l i e r s t u d i e s , t h e a n i m a l s were v e n t i l a t e d w i t h room a i r bu t t h i s was changed t o room a i r e n r i c h e d w i t h 95% 0 2 /5% C 0 2 i n t h e l a s t e x p e r i m e n t s c o n c e r n i n g t h e e f f e c t s o f 0 -60 min o f o c c l u s i o n on t h e sa r co l emma l p e r m e a b i l i t y . B l o od gas measurements were done p e r i o d i c a l l y and were i n t h e normal r a n g e . A p o l y e t h y l e n e c a n n u l a was i n s e r t e d i n t o t h e c a r o t i d a r t e r y and c onne c t ed t o a B e l l and Howe l l p r e s s u r e t r a n s d u c e r (model 4 - 327 - 0010 ) i n o r d e r t o m o n i t o r b l o o d p r e s s u r e . The c h e s t was opened by a s t e r n o t o m y , t h e r i b cage r e t r a c t e d , and t h e h e a r t suspended i n a p e r i c a r d i a l c r a d l e . I n most o f t h e l a t e r e x p e r i m e n t s , a 20 -gauge J e l c o c a t h e t e r was i n s e r t e d i n t o t h e l e f t v e n t r i c l e t h r o u g h t h e apex and c o n n e c t e d v i a p o l y e t h - y l e n e t u b i n g t o a S t a tham P23 ID p r e s s u r e t r a n s d u c e r . The o u t p u t o f t h i s l e f t v e n t r i c u l a r p r e s s u r e r e c o r d i n g was f e d i n t o a c a l i b r a t e d d i f f e r e n t i a t o r ( G r a s s I n s t r u m e n t s 7P20C) t o o b t a i n t h e d i f f e r e n t i a t e d waveform - an i n d i r - 90 e c t measure o f c a r d i a c c o n t r a c t i l i t y (^P/d t ) x . Max ima l r a t e s o f r i s e o f l e f t v e n t r i c u l a r p r e s s u r e were w e l l w i t h i n t h e r e s pon se r ange o f t h e pen r e c o r d e r ( see Append i x I ) . A l l p r e s s u r e r e c o r d i n g s and t h e e l e c t r o c a r - d i og ram were r e c o r d e d on a G r a s s I n s t r u m e n t s P o l y g r a p h (model 7PCPB ) . Subcu taneous e l e c t r o d e s measured l e a d I I o f t h e ECG and changes c h a r a c - t e r i s t i c o f i s c h a e m i a ( e . g . S-T segment e l e v a t i o n , R wave en l a r g emen t ) were m o n i t o r e d i n o r d e r t o a s s e s s whe the r o r no t t h e l e f t c i r c u m f l e x c o r o n a r y a r t e r y had been s u c c e s s f u l l y l i g a t e d . The ECG a l s o p r o v i d e d i n d i r e c t e v i - dence o f t h e o c c u r r e n c e o f r e p e r f u s i o n i n our model because l o o s e n i n g t h e - 24 - l i g a t u r e caused a g r a dua l r e v e r s a l o f t h e ECG a b n o r m a l i t i e s i nduced by c o r o n a r y o c c l u s i o n . Drug I n f u s i o n A l l d rugs were d i s s o l v e d i n s a l i n e and i n j e c t e d i n t o t h e an ima l i n 2 -3 ml vo lumes v i a t h e m a r g i n a l e a r v e i n . Sham-ope ra ted a n i m a l s r e c e i v e d s a l i n e o n l y . No d rug was a d m i n i s t e r e d u n t i l t h e b l o o d p r e s s u r e had s t a b i l i z e d ( a t l e a s t 15 min a f t e r a l l s u r g e r y was c o m p l e t e d ) . Drugs were i n f u s e d o v e r a 20 m i nu t e p e r i o d p r i o r t o l i g a t i o n . C o r o n a r y L i g a t i o n , R e p e r f u s i o n and I s o l a t i o n o f t h e I s c h a e m i c A r e a J u s t p r i o r t o i n s e r t i o n o f t h e l e f t v e n t r i c u l a r c a t h e t e r , a 4 : 0 s i l k c a r d i o v a s c u l a r s u t u r e was p l a c e d a round t h e l e f t c i r c u m f l e x c o r o n a r y a r t e r y and s u r r o u n d i n g mus c l e at i t s o r i g i n . The two f r e e ends o f t h e l i g a t u r e were pas sed t h r o u g h a s h o r t l e n g t h o f p o l y e t h y l e n e t u b i n g and i s c h a e m i a was p roduced by p u l l i n g up and s e c u r i n g t h e ends w i t h a h a e m o s t a t . C l amp i ng t h e s u t u r e i n t h i s way made t h e o c c l u s i o n r e a d i l y r e v e r s i b l e when r e p e r f u s i o n was p a r t o f t h e s t u d y p r o t o c o l . T i g h t e n i n g o f t h e l i g a t u r e r e s u l t e d i n an o c c l u d e d zone o f 55 ± 3 % (mean * SE , n = 15) o f t h e l e f t v e n t r i c l e . The o c c l u d e d zone was measured as wet we i g h t o f o c c l u d e d zone v 1 n n ( y t o t a l l e f t v e n t r i c u l a r w e i g h t x i W % R e p e r f u s i o n was accompan ied by a d e c l i n e i n ( d P / d t ) . marked hype raem ia n i c i x and , as s t a t e d p r e v i o u s l y , a g r a dua l r e v e r s a l o f ECG a b n o r m a l i t i e s . E x p e r i - ments w i t h r a d i o a c t i v e l y - 1 a b e l l e d m i c r o s p h e r e s p r o v i d e d a q u a n t i t a t i v e a n a l y s i s o f t h e e x t e n t o f o c c l u s i o n and r e p e r f u s i o n . These s t u d i e s a r e d e s c r i b e d i n a l a t e r s e c t i o n . I f f i b r i l l a t i o n o c c u r r e d d u r i n g t h e l i g a t i o n or r e p e r f u s i o n p e r i o d s , b r a s s e l e c t r o d e s were p l a c e d d i r e c t l y on t h e h e a r t and a 0 .5 Wa t t - s e c ond - 25 - D.C . c o u n t e r s h o c k was d i s c h a r g e d f r om a d e f i b r i l l a t o r ( Ame r i c an O p t i c a l C o . ) . T h i s low l e v e l o f s t o r e d ene rgy was u s u a l l y e f f e c t i v e i n r e s t o r i n g s i n u s r h y t hm and d i d so a t a l e v e l w e l l be l ow t h e t h r e s h o l d shown by Kon i ng e t a l . t o p r oduce t i s s u e damage i n i s o l a t e d r a b b i t h e a r t s . ^ When t he e xpe r imen t was c o m p l e t e d , t h e h e a r t was q u i c k l y removed by c u t t i n g t h e a o r t a and o t h e r ma jo r v e s s e l s and t h e h e a r t was i m m e d i a t e l y d ropped i n t o i c e - c o l d b u f f e r ( . 25 M KC1 , 1 mM EDTA, 50 mM T r i s , pH 7 . 4 ) . B e a t i n g ceased w i t h i n s e c o n d s . A f t e r remova l o f t h e h e a r t , a l l p r o c edu r e s were c a r r i e d ou t a t 4 °C . To v i s u a l i z e t he o c c l u d e d z o n e , t h i s same b u f f e r was p e r f u s e d r e t r o g r a d e l y v i a t h e a o r t i c r o o t ( t h e l i g a t u r e was r e - c l a m p e d i f t h e h e a r t was removed d u r i n g r e p e r f u s i o n ) . Thus , t h e l i g a t e d zone r ema i ned u n p e r f u s e d and c o u l d be s e p a r a t e d f r om t h e s u r r o u n d i n g , b l a n c h e d , p e r f u s e d t i s s u e . An e x c e s s o f t i s s u e was t r immed f r om t h e b o r d e r o f t h e i s c h a e m i c zone t o e n su r e t h a t t h e i s o l a t e d a r e a c o n t a i n e d o n l y o c c l u d e d t i s s u e . Two t r a n s m u r a l samp les o f i s c h a e m i c t i s s u e ( a p p r o x i m a t e l y 50 mg) were q u i c k l y f r o z e n w i t h l i q u i d n i t r o g e n f o r s ub sequen t a n a l y s i s o f ATP, wa t e r and i o n c o n t e n t s . Because o f t h e s m a l l s i z e o f t h e r a b b i t h e a r t , t i s s u e f o r ATP a n a l y s i s c o u l d no t be r o u t i n e l y t a k en f r om t h e b e a t i n g h e a r t w i t h o u t some compromise i n f u n c t i o n . T h e r e f o r e , samp les f o r ATP and i o n a n a l y s i s f r om a l l g roups were f r o z e n i n l i q u i d n i t r o g e n a t t h e same t ime a f t e r remova l o f t h e h e a r t . I t has r e c e n t l y been demons t r a t ed by J e n n i n g s e t a l . t h a t t h e ATP c o n t e n t o f t h e myocard ium does no t d e c r e a s e when t he t i s s u e i s s t o r e d i n c o l d b u f f e r f o r s e v e r a l m i nu t e s b e f o r e f r e e z i n g ( a l t h o u g h 92 c r e a t i n e phospha t e l e v e l s a r e a f f e c t e d ) . A s t u d y compa r i ng t h e ATP l e v e l s i n samp les removed u s i n g t h e above p r o t o c o l w i t h samp les removed d i r e c t l y f r om t h e b e a t i n g h e a r t c o n f i r m e d J e n n i n g s ' f i n d i n g s . The r e s u l t s o f t h i s s t u d y a r e shown i n T a b l e 1. - 26 - T a b l e I . . Compa r i son o f ATP c o n t e n t s i n t i s s u e t a k e n f r om t h e b e a t i n g h e a r t v s . KC1 E D T A - T r i s p e r f u s e d h e a r t L i g a t i o n Time ATP c o n t e n t (min) nmoles ATP mg d r y w e i g h t P e r f u s e d H e a r t - B e a t i n g H e a r t - C o n t r o l 13 .7 ± 2 .7 (2) 9 . 2 ± 1.2 (3) 10 min p o s t - l i g a t i o n 3 . 9 ± 0 . 4 (2) 4 . 3 ± 0 . 6 (2) 60 min p o s t - l i g a t i o n 0 . 8 (1) 0 . 2 ± 0 . 1 (2) A l l v a l u e s a r e e x p r e s s e d as mean ± S . E . Number o f a n i m a l s pe r g roup shown i n b r a c k e t s — P e r f u s e d h e a r t - who le h e a r t r emoved , p l a c e d i n c o l d b u f f e r , and p e r f u s e d as d e s c r i b e d i n t h e Methods s e c t i o n . Sample t a k en a p p r o x i m a t e l y 7 min a f t e r r e m o v a l . — B e a t i n g h e a r t - t i s s u e c u t f r om i s c h a e m i c p o r t i o n o f l e f t v e n t r i c l e o r compa rab l e a r e a i n c o n t r o l h e a r t , b l o t t e d t o remove e x c e s s b l o o d and i m m e d i a t e l y f r o z e n i n l i q u i d n i t r o g e n . F r e e z i n g o c c u r r e d w i t h i n 20 se c o f r e m o v a l . - 27 - In h e a r t s t h a t were t o be a n a l y z e d by e l e c t r o n m i c r o s c o p y , a sample o f 3 a p p r o x i m a t e l y 3 mm f rom the subendoca rd i um was f i x e d i n c o l d , b u f f e r e d 3% g l u t a r a l d e h y d e s o l u t i o n . F u r t h e r d e t a i l s r e g a r d i n g t h e p r o c e d u r e s used i n t he u l t r a s t r u c t u r a l a n a l y s i s a r e c o n t a i n e d i n a l a t e r s e c t i o n and i n Appen - d i x I I . The r ema i nde r o f t h e t i s s u e f r om t h e o c c l u d e d zone was s u b j e c t e d t o a s u b c e l l u l a r f r a c t i o n a t i o n as o u t l i n e d b e l o w . C o n t r o l t i s s u e a lways was o b t a i n e d f r om sham-ope ra t ed an ima l s wh i ch were s u b j e c t e d t o a l l s u r g i c a l p r o c e d u r e s e x c e p t t i g h t e n i n g o f t h e l i g a t u r e . The compa rab l e a r e a o f t h e l e f t v e n t r i c l e was used f o r t i s s u e s a m p l i n g a f t e r t h e a p p r o p r i a t e t ime l a g between remova l and f r e e z i n g o f t h e t i s s u e . N e i t h e r t h e c o n t r o l t i s s u e no r t h e o c c l u d e d zone were d i r e c t l y p e r f u s e d v i a t h e c o r o n a r i e s w i t h t h e K C l / E D T A / T r i s b u f f e r s o l u t i o n . 1 1 . S u b c e l l u l a r F r a c t i o n a t i o n The p r e p a r a t i o n o f m i t o c h o n d r i a l and s a r c o l e m m a l - e n r i c h e d f r a c t i o n s was 93 94 pe r f o rmed u s i n g m o d i f i c a t i o n s o f two p r e v i o u s l y p u b l i s h e d methods ' wh i c h a r e summar i zed as f o l l o w s . A l l p r o c edu r e s were pe r f o rmed a t 4 °C . A p p r o x i m a t e l y 1 -1 .5 g o f l e f t v e n t r i c u l a r normal o r i s c h a e m i c myocard ium was m inced and homogen i zed as a l(% s u s p e n s i o n (w/v) i n b u f f e r ( 1 . 2 5 M KC1 , 0 . 5 mM C a C l 2 , 2 mM d i t h i o t h r e i t o l and 10 mM T r i s , pH 7 . 4 ) u s i n g a P o l y t r o n PT-10 homogen i ze r (2 b u r s t o f 5 sec each a t 1/4 maximum s p e e d ) . The s u s p e n - s i o n was f i l t e r e d t h r o u g h n y l o n mesh ( 0 . 5 mm ) and r i n s e d t h r o u g h w i t h an a d d i t i o n a l 10 ml o f b u f f e r . The f i l t r a t e was c e n t r i f u g e d f o r 10 min a t 1200 g and t h e r e s u l t i n g p e l l e t r e su spended t o t h e o r i g i n a l v o l ume . A second h o m o g e n i z a t i o n was pe r f o rmed u s i n g a T e f l o n - i n - g l a s s homogen i ze r (5 s t r o k e s ) , f o l l o w e d by c e n t r i f u g a t i o n a t 500g f o r 10 m i n . T h i s second p e l l e t - 28 - was r e s u spended i n 10% (w/v) s u c r o s e d i s s o l v e d i n 10 mM T r i s , 2 mM d i t h i o - t h r e i t o l , pH 8 . 2 , and hand-homogen i zed w i t h a g r o u n d - g l a s s homogen i ze r ( P y r e x 7727) u n t i l a l l v i s i b l e p a r t i c u l a t e m a t t e r was d i s r u p t e d . One ml o f t h i s m i x t u r e was l a y e r e d on each o f s i x s u c r o s e g r a d i e n t s c o n s i s t i n g o f 2 .5 ml o f 50 , 5 2 . 5 , and 60% (w/v) b u f f e r e d s u c r o s e , pH 8 . 2 . F o l l o w i n g c e n t r i f u - g a t i o n a t 4 0 , 000 g f o r 60 m i n , t h e s a r c o l e m m a l - e n r i c h e d f r a c t i o n appea red a t t h e 55-60% i n t e r f a c e and t h e m i t o c h o n d r i a l f r a c t i o n appea red a t t h e 10-50% i n t e r f a c e . These two f r a c t i o n s were c o l l e c t e d w i t h a P a s t e u r p i p e t t e , d i l u t e d 5 - f o l d w i t h 10 mM T r i s , pH 7 .4 and c e n t r i f u g e d a t 23 , 000g f o r 15 m i n . The f i n a l membrane p e l l e t s were r e su spended i n d o u b l e - d i s t i l l e d w a t e r . P r o t e i n c o n c e n t r a t i o n s ( a p p r o x i m a t e l y 3 mg/ml) were e s t i m a t e d by t h e 95 method o f Lowry e t a l . . Membrane f r a c t i o n s were a l s o a s s ayed t h e same day f o r c y t o ch rome c o x i d a s e a c t i v i t y ( see P a r t I I I ) and t hen f r o z e n immed i - a t e l y i n v i a l s i n d r y i c e / a c e t o n e and s t o r e d o v e r n i g h t a t 20 °C . A l l o t h e r enzyme a s s a y s were done on t h e f o l l o w i n g d a y . I l l . C h a r a c t e r i z a t i o n o f Sa r co l emma l and M i t o c h o n d r i a l Membrane F r a c t i o n s The a c t i v i t y o f t he m i t o c h o n d r i a l marker enzyme cy toch rome c o x i d a s e , was measured i n bo th t h e sa r co l emma l and m i t o c h o n d r i a l f r a c t i o n s i n o r d e r t o a s s e s s t h e c o n t a m i n a t i o n o f s a r co l emma l f r a c t i o n w i t h m i t o c h o n d r i a . S p e c i - f i c d e t a i l s o f t h e a s s a y method a r e o u t l i n e i n S e c t i o n I V . T a b l e I I d emons t r a t e s t h a t even p r o l o n g e d i s c h a e m i a w i t h o r w i t h o u t r e p e r f u s i o n does no t a f f e c t t h e f r a c t i o n a t i o n c h a r a c t e r i s t i c s o f t h e s a r c o - lemma as d emons t r a t e d by t he c o n s i s t e n t p e r c e n t a g e c o n t a m i n a t i o n o f t h e sa r co l emma l f r a c t i o n w i t h m i t o c h o n d r i a . S i m i l a r l y , t i s s u e i n j u r y does no t a f f e c t t h e p u r i t y o f t h e m i t o c h o n d r i a . - 29 - T a b l e I I . P e r c e n t c o n t a m i n a t i o n o f s a r co l emma l f r a c t i o n (F^) w i t h m i t o c h o n d r i a (F^) as a s s e s s ed by c y t o ch rome c o x i d a s e a c t i v i t y T r ea tmen t n P e r c e n t c o n t a m i n a t i o n a c t i v i t y F . _a a c t i v i t y F J X 1 0 ° C o n t r o l 9 12 . 7 ± 1.0 1 min l i g a t i o n 5 10 . 8 ± 1.8 5 min l i g a t i o n 10 12 .1 0 .7 10 min l i g a t i o n 5 13 .4 ± 0 . 9 20 min l i g a t i o n 3 14 .2 ± 2 .9 60 min 1 i g a t i o n 2 15 .6 ± 2.5 10 min l i g a t i o n + 60 min r e p e r f u s i o n 2 11 . 5 ± 0 .9 20 min l i g a t i o n + 60 min r e p e r f u s i o n 2 9 .4 ± 2 .6 60 min l i g a t i o n + 60 min r e p e r f u s i o n 2 10 . 8 ± 3 . 0 Mean ± S . E . 1 a c t i v i t y = s l o p e mg p r o t e i n where ' s l o p e ' e q u a l s t h e s l o p e o f t h e r e g r e s s i o n l i n e , l o g ab so r bance v s . t i m e ; T h i s e q u a l s t h e r a t e o f o x i d a t i o n o f r educed cy to ch rome C . See enzyme a s say s e c t i o n i n Methods f o r d e t a i l s . - 30 - The f o l l o w i n g c a l c u l a t i o n was used t o e s t i m a t e t h e p u r i t y o f t h e m i t o c h o n - d r i a l f r a c t i o n : a z i d e - i n s e n s i t i v e ATPase (N0N-MIT0) x 100$ a z i d e - s e n s i t i v e ATPase a c t i v i t y (MITO) The d a t a shown i n T a b l e I I I d emons t r a t e t h a t n e i t h e r t h e deg ree o f t i s s u e i n j u r y nor t h e p r e s en c e o f d r ug s a f f e c t e d t h e p u r i t y o f t h e m i t o c h o n d r i a l f r a c t i o n . The r e s u l t s shown i n T ab l e IV d emons t r a t e t h a t t h e f r a c t i o n a t i o n o f t h e membranes i s u n a f f e c t e d by i s c h a e m i a a l o n e . T h u s , changes i n s p e c i f i c enzyme a c t i v i t y o f a p a r t i c u l a r ma rke r enzyme d u r i n g i s c h a e m i a a re no t caused by changes i n t h e f r a c t i o n a t i o n p a t t e r n o f t h e o r g a n e l l e . T h i s i s i n agreement w i t h r e c e n t r e s u l t s o f Be r sohn e t a l . . U s i n g r a b b i t myocard ium made i s c h a e m i c i n v i t r o , t h e y showed t h a t t h e r e was no d i f f e r e n c e i n t h e p e r c e n t i n c r e a s e i n enzyme a c t i v i t y o f t h e p a r t i a l l y - p u r i f i e d f r a c t i o n s f rom 96 t h e c r u d e homogenate between c o n t r o l and i s c h a e m i c h e a r t s . I n t e r e s t i n g - l y , t h e r e i s a r e d u c t i o n i n t h e y i e l d o f m i t o c h o n d r i a a f t e r r e p e r f u s i o n o f myocard ium t h a t has been made i s c h a e m i c f o r 40 m i n u t e s . I V . Enzyme A s s a y s N a + , K + - A T P a s e N a + , K + - A T P a s e i s a r e l i a b l e marke r enzyme f o r t h e sa rco lemma and t h e a c t i v i t y o f t h i s enzyme was measured i n t h e s a r c o l e m m a - e n r i c h e d f r a c t i o n 97 a c c o r d i n g t o t h e method o f God i n and S c h r i e r e x c e p t t h a t t h e r e a c t i o n t i m e was 15 min and each t ube c o n t a i n e d 150 ug o f membrane p r o t e i n . The r e a c t i o n was s t a r t e d w i t h t h e a d d i t i o n o f t h e membranes t o 3 ml ( f i n a l vo lume) o f a s s a y medium a t 37°C (55 mM T r i s , 3 mM ATP, 3 rrM M g C ^ , .03 mM EGTA, 80rrfl N a C l , 20 nM KC1 , 5 rrM NaN,). N o n - s t i m u l a t e d a c t i v i t y was - 31 - T ab l e I I I . P u r i t y o f t h e m i t o c h o n d r i a l f r a c t i o n (F^) a f t e r v a r i o u s t r e a t m e n t s T r ea tmen t P e r c e n t r e s i d u a l ATPase a c t i v i t y i n t h e F j f r a c t i o n ! C o n t r o l 40 min l i g a t i o n + 60 min r e p e r f u s i o n (L + R) D-600 p r e t r e a t m e n t + L + R P r o p r a n o l o l p r e t r e a t m e n t + L + R 3 .0 ± 2 . 0 4 . 1 ± 0 .4 3 .0 ± 0 . 3 3 .1 ± 0 . 3 r e s i d u a l ATPase a z i d e - s e n s i t i v e ATPase s p e c i f i c a c t i v i t y ( m i t o ) a c t i v i t y a z i d e - i n s e n s i t i v e ATPase s p e c i f i c a c t i v i t y ( n o n - m i t o ) X 100% T a b l e I V . P u r i f i c a t i o n o f m i t o c h o n d r i a l and sa r co l emma l p r o t e i n f r om wet h e a r t w e i g h t T r ea tmen t n p r o t e i n i n p u r i f i e d f r a c t i o n ^ 3 wet w e i g h t o f o c c l u d e d zone M i t o c h o n d r i a Sarcolemmma C o n t r o l 10 3 .2 ± .2 7 .2 ± .5 2 0 ' L 7 2 .8 ± .2 8 . 4 .6 4 0 1 L 7 2 .6 ± .3 7 . 6 ± .6 6 0 ' L 8 3 .4 ± .3 9 . 0 zfc 1.1 4 0 ' L + 6 0 ' R 9 1.6 ± .2 6 . 8 ± .9 P r o p r a n o l o l + 7 L+R 2 . 0 ± .2 6 .6 ± .6 D-600 + L+R 6 2 .8 ± .4 8 . 2 ± .7 A l l v a l u e s a r e e x p r e s s e d as mean ± S . E . - 33 - measured i n t ube s w i t h o u t NaCl and KC1 . A t ube c o n t a i n i n g 0 . 65 mM i n o r g a n i c phospha te was used as a s t a n d a r d . A f t e r t h e 15 min i n c u b a t i o n , t h e r e a c t i o n was s t opped w i t h c o l d 10 t r i c h l o r o a c e t i c a c i d (TCA) a n d , a f t e r c e n t r i f u g a - t i o n a t 1000 g t o remove t h e membranes, an a l i q u o t o f t h e s u p e r n a t a n t was 98 a s s a y ed f o r i n o r g a n i c phospho rus by t h e method of F i s k e and Subbarow. The a c t i v i t y i s e x p r e s s e d i n umoles P^/mg p r o t e i n / h o u r . A l l t u be s were k e p t on i c e and t h e s u p e r n a t a n t a s s a yed as soon as p o s s i b l e t o p r e v e n t n o n - s p e c i f i c h y d r o l y s i s o f ATP i n t h e a c i d i c s o l u t i o n . As a f u r t h e r p r e c a u t i o n , b l a n k s w i t h no membrane a d d i t i o n were s u b t r a c t e d f r om a l l r e a d i n g s t o a c c oun t f o r any n o n - s p e c i f i c h y d r o l y s i s . K + - NPPa s e The K - s t i m u l a t e d p - n i t r o p h e n y l p h o s p h a t a s e (K -NPPa se ) enzyme i s f u n c t i o n a l l y - r e l a t e d t o t h e N a + , K + - A T P a s e i n t h a t i t c a t a l y z e s t h e t e r m i n a l K + - d e p e n d e n t d e p h o s p h o r y l a t i o n s t e p o f t h e sod ium pump a c t i v i - 99 t y . The enzyme a c t i v i t y was measured a c c o r d i n g t o t h e method o f God in 97 and S c h r i e r . The r e a c t i o n t i m e and amount o f p r o t e i n a s sayed was t h e same as f o r t h e N a + , K + - A T P a s e bu t t h e c o m p o s i t i o n o f t h e i n c u b a t i o n medium d i f f e r e d (50 mM i m i d a z o l e , pH 7 . 4 , 3 rrM M g C l 2 , 3 rrM p - n i t r o p h e n o l - p h o s p h a t e , 30 rrM K C 1 ) . The r e a c t i o n was s t opped w i t h c o l d 20 TCA and t h e c o l o u r i m e t r i c r e a c t i o n was i n i t i a t e d by a l k a l i n i z a t i o n w i t h 1 ml o f 1.5 M T r i s t o each t u b e . I n b o t h t h e N a + , K + - A T P a s e and K + - N P P a s e a s s a y s , 30 rrM o u a b a i n was added t o one t u be t o p r o v i d e a measure o f t h e o u a b a i n - s e n s i t i v i t y o f t h e enzyme. O u a b a i n - i n s e n s i t i v e ATPase o r NPPase a c t i v i t y was no t s i g n i f i c a n t l y d i f f e r e n t f r om t h e a c t i v i t y measured i n t h e absence o f added N a + and K + ( b a s a l a c t i v i t y ) . Thus , t he s p e c i f i c a c t i v i t y o f t h e enzyme (umoles NPP/mg p r o t e i n / h o u r ) c o u l d be e x p r e s s e d as t h e d i f f e r e n c e between t h e i o n - s t i m u l a - t e d and b a s a l enzyme a c t i v i t y . - 34 - Cy tochrome c o x i d a s e The a c t i v i t y o f a m i t o c h o n d r i a l ma rke r enzyme, c y t o ch r ome c o x i d a s e was measured i n bo th t h e m i t o c h o n d r i a l and sa r co l emma l f r a c t i o n s f o r two r e a s o n s : f i r s t l y , t o a s s e s s t h e f u n c t i o n o f t h i s m i t o c h o n d r i a l membrane enzyme; and s e c o n d l y , t o p r o v i d e an e s t i m a t e o f t h e c o n t a m i n a t i o n o f t h e s a r co l emma l f r a c t i o n w i t h m i t o c h o n d r i a ( see T a b l e I I ) . Measurement o f s p e c i f i c a c t i v i t y was pe r f o rmed u s i n g t h e s p e c t r o p h o t o m e t r i c method of C o o p e r s t e i n and L a z a r o w 1 ^ i n wh i c h a l i q u o t s o f t h e d i l u t e d membrane f r a c t i o n s were added t o b u f f e r e d , r educed c y t o ch r ome c , pH 7 . 4 , and t h e o x i d a t i o n r a t e was f o l l o w e d o v e r a 3 m i nu t e p e r i o d by r e c o r d i n g t h e d e c r e a s e i n ab so r bance a t 550 nm. The s l o p e o f t h e r e g r e s s i o n l i n e , l o g ab so rbance v s . t i m e , was e x p r e s s e d pe r mg p r o t e i n . The r a t i o o f a c t i v i t y ( l o g o x i d a - t i o n r a t e / m g p r o t e i n ) i n t h e sa r co l emma l f r a c t i o n t o t h e a c t i v i t y i n t h e m i t o c h o n d r i a l f r a c t i o n was used as an i n d e x o f c o n t a m i n a t i o n o f t h e s a r c o - lemmal f r a c t i o n . S p e c i f i c a c t i v i t y was e x p r e s s e d as umoles c y t o ch r ome c o x i d i z e d / m i n / m g p r o t e i n . A z i d e - s e n s i t i v e ATPase M i t o c h o n d r i a l ATPase a c t i v i t y was a s sayed i n t h e p r e s en ce and absence o f sod ium a z i d e (NaNg, 5 mM) as d e s c r i b e d by God i n and S c h r i e r 9 7 e x c e p t t h a t t h e r e a c t i o n t i m e was 15 min and each t ube c o n t a i n e d 15-30 ug o f p r o t e i n . The a s s a y medium had t h e same c o m p o s i t i o n as t h a t f o r t h e N a + , K + - A T P a s e a s s a y e x c e p t t h a t NaCl and KC1 were o m i t t e d . O l i g o m y c i n i s known t o s p e c i f i c a l l y i n h i b i t t h e m i t o c h o n d r i a l ATPase a c t i v i t y 1 ^ 1 a n d , i n one e x p e r i m e n t , e i t h e r 0 , . 3 , 1 . 0 , o r 3 . 0 ug/ml o l i g o m y c i n was added t o t h e a s s a y m i x t u r e . The r e s i d u a l a c t i v i t y a f t e r a d d i t i o n o f 3 .0 yg/ml o l i g o - myc in was no t s i g n i f i c a n t l y d i f f e r e n t f r om t h e l e v e l measured a f t e r NaNg t r e a t m e n t . Thu s , t h e a z i d e - s e n s i t i v e ATPase a c t i v i t y i s e q u i v a l e n t t o t h e - 35 - o l i g o m y c i n - s e n s i t i v e ATPase and s h a l l be h e n c e f o r t h r e f e r r e d t o as a z i d e - s e n s i t i v e ATPase a c t i v i t y . T r i t o n X -100 A d d i t i o n F o r a l l bu t t h e e a r l i e s t e x p e r i m e n t s ( p r e l i m i n a r y and 20 min l i g a t i o n s t u d i e s ) , 0.005% and 0.01% T r i t o n X -100 was added t o t h e a s s a y medium f o r t he m i t o c h o n d r i a l and sa r co l emma l f r a c t i o n s , r e s p e c t i v e l y , i n o r d e r t o 102 103 e l i m i n a t e t h e e f f e c t s o f v e s i c u l a t i o n on measured enzyme a c t i v i t y . ' The e f f e c t s o f d e t e r g e n t on enzyme a c t i v i t y f o l l o w e d a b e l l - s h a p e d c u r v e , i . e . a c t i v a t i o n a t low c o n c e n t r a t i o n s f o l l o w e d by i n h i b i t i o n a t h i g h e r c o n c e n t r a t i o n s t han t h o s e l i s t e d above . V . C hem i c a l A s s a y s Water c o n t e n t T r an smu r a l samp les were b l o t t e d l i g h t l y w i t h paper and f r o z e n a t - 8 0 ° C . The f r o z e n l e f t v e n t r i c u l a r t i s s u e ( a p p r o x i m a t e l y 50 mg) was we ighed i n p o r c e l a i n c r u c i b l e s , d r i e d i n t h e oven a t 200°F and t h e d r y w e i g h t d e t e r - mined by s u b t r a c t i o n . Water c o n t e n t i s e x p r e s s e d as a p e r c e n t o f t o t a l t i s s u e w e i g h t . ATP A 50 mg t r a n s m u r a l m y o c a r d i a l samp le was we ighed w h i l e s t i l l f r o z e n , p u l v e r i z e d i n a p r e - c o o l e d mo r t a r w i t h 4 vo lumes (w/v) o f 6% p e r c h l o r i c a c i d and thawed on i c e . F o l l o w i n g t h e p r o c e d u r e o f Jaworek e t a l . , t h e e x t r a c t was n e u t r a l i z e d w i t h 1.4 M KHCOg, t h e p r e c i p i t a t e c e n t r i f u g e d and t h e 104 s u p e r n a t a n t a s sayed f o r ATP c o n t e n t . T h i s was done s p e c t r o p h o t o m e t r i - c a l l y a t 340 nm u s i n g a c o u p l e d enzyme r e a c t i o n i n wh i ch t h e amount o f ATP p r e s e n t i s p r o p o r t i o n a l t o t h e amount o f NADH o x i d i z e d . The l e v e l s a r e e x p r e s s e d as nmole ATP/mg d r y w e i g h t . - 36 - Ions T i s s u e l e v e l s o f s od i um , p o t a s s i u m , magnesium and c a l c i u m were measured by a t om i c a b s o r p t i o n s p e c t r o s c o p y u s i n g a m o d i f i c a t i o n o f t h e t e c h n i q u e o f James and R o u f o g a l i s . The d r i e d samp les f r om t h e o c c l u d e d zone o r t h e compa rab l e a r e a o f c o n t r o l h e a r t s ( a p p r o x . 10 mg) were d i s s o l v e d i n b o i l i n g 100% t r i c h l o r o a c e t i c a c i d r g l a c i a l a c e t i c a c i d ( v / v , 1:1) and 1 mM L a C l g was added t o p r e c i p i t a t e p h o s p h a t e . A f t e r c e n t r i f u g a t i o n , t h e s u p e r n a t a n t was a n a l y z e d u s i n g a V a r i a n T e c h t r o n a t om i c a b s o r p t i o n s p e c t r o p h o t o m e t e r (Model A A 5 ) , c a l i b r a t e d w i t h f r e s h l y p r e p a r e d s t a n d a r d s o l u t i o n s . S t a n d a r d s o l u t i o n s c o n t a i n e d t h e same p r o p o r t i o n o f a c e t i c a c i d , L a C l g , KC1 and C sC l as d i d t h e sample s o l u t i o n s as w e l l as t h e a p p r o p r i a t e vo lume o f a t om i c a b s o r p t i o n s t o c k s o l u t i o n f o r each i o n ( F i s h e r Chem i ca l C o . , 1000 y g / m l ) . A l l s o l u t i o n s were p r e p a r e d u s i n g f r e s h l y d e - i o n i z e d w a t e r . Sample and s t a n d a r d b l ank s o l u t i o n s were a l s o p r e p a r e d i n o r d e r t o s u b t r a c t t h e backg round r e a d i n g s . A i r : a c e t y l e n e f u e l was used f o r t h e measurement o f Na, K and Mg . C a l c i u m abso rbance was measured i n a n i t r o u s o x i d e : a c e t y l e n e f l a m e . S u p p r e s s i o n o f i o n i z a t i o n was e f f e c t e d by t h e a d d i t i o n o f KC1 ( 2 . 5 mg/ml) t o t h e s o l u t i o n s f o r Mg and Ca a n a l y s i s and C sC l ( 1 . 0 mg/ml) t o t h e s o l u t i o n s f o r K a n a l y s i s . The amount o f t h e i o n i n each sample was d e t e rm i n ed by i n t e r p o l a t i o n f r om t h e s t a n d a r d c u r v e and a l l r e s u l t s a r e e x p r e s s e d as nmoles e l ement /mg d r y w e i g h t . SDS -ge l e l e c t r o p h o r e s i s An a l i q u o t o f t h e m i t o c h o n d r i a l o r s a r co l emma l f r a c t i o n (100 y l ) was s o l u b i l i z e d i n t h e f o l l o w i n g s o l u t i o n : 30 mM T r i s , pH 8 . 0 , 0.3% m e r c a p t o e t h - a n o l , 3% sod ium d o d e c y l s u l p h a t e ( SDS ) , 10% s u c r o s e , 3 mM EDTA, 20 yg/ml brompheno l b l u e ) and i n c u b a t e d a t 37°C f o r 20 min t o r e du ce s u l f h y d r y l g r o u p s . The samp les were l o aded i n t o one o f s i x s l o t s o f a 5% p o l y a c r y l a - - 37 - mide s l a b ge l (75 ug p r o t e i n / s l o t ) (E-C Appa r a t u s C o r p . ) . The samp les were e l e c t r o p h o r e s e d a t 150 V f o r a p p r o x i m a t e l y 3 hou r s by wh i ch t i m e t h e marke r dye had moved 10 cm. The SDS was removed by r i n s i n g t h e ge l o v e r n i g h t w i t h d i s t i l l e d w a t e r : m e t h a n o l : g l a c i a l a c e t i c a c i d , 4 5 : 4 5 : 1 0 ( v / v / v ) and t h e ge l was t hen s t a i n e d w i t h Coomass i e B l u e . Q u a l i t a t i v e a n a l y s i s was done by i n s p e c t i o n o f b l a c k and w h i t e pho t og r aph s o f t h e g e l s . Fo r more q u a n t i t a - t i v e a n a l y s i s , t h e g e l s were scanned u s i n g a Gelman DCD 16 d e n s i t o m e t e r wh i ch r e c o r d e d i n c r e a s e d band d e n s i t y by g r e a t e r pen d e f l e c t i o n . Sub se - q u e n t l y , t h e s e peaks were i n t e g r a t e d u s i n g a T e c h n i c o n I n t e g r a t o r Model AAG. Scans o f t h e p r o t e i n p r o f i l e s f rom bo th o f t h e m i t o c h o n d r i a l and sa r co l emma l membrane f r a c t i o n s showed c o n s i d e r a b l e v a r i a b i l i t y i n t h e a b s o l u t e a r ea s o f t h e peaks a l t h o u g h t h e p a t t e r n o f p o l y p e p t i d e d i s t r i b u t i o n r ema ined c o n s t a n t . As a c on s equen ce , a l l peak a r e a s were n o r m a l i z e d by e x p r e s s i n g a p a r t i c u l a r peak a r e a as a f r a c t i o n o f t h e a r e a o f a peak t h a t was u n a f f e c t e d by t he t r e a t m e n t , i . e . Peak a r e a X x 100% Peak a r e a B Peak B was chosen as a r e f e r e n c e f o r bo th f r a c t i o n s because i t r ema ined c o n s t a n t r e g a r d l e s s o f t h e t r e a t m e n t . V I . B l ood F low S t u d i e s In o r d e r t o measure t h e e x t e n t o f b l o o d f l o w r e d u c t i o n a f t e r l i g a t i o n a nd , more i m p o r t a n t l y , t h e l e v e l o f r e p e r f u s i o n when t h e l i g a t u r e i s r e l e a s e d , r a d i o a c t i v e l y - 1 a b e l l e d m i c r o s p h e r e s were i n j e c t e d d u r i n g t h e d i f f e r e n t t r e a t m e n t p e r i o d s . Two i s o t o p e s were u s e d , ^ S n and ^ C o . In o r d e r f o r each an ima l t o s e r v e as i t s own c o n t r o l , we d i v i d e d t h e s t u d y i n t o two s e c t i o n s . The f i r s t s t u d i e d t h e r e d u c t i o n o f b l o o d f l o w as a - 38 - r e s u l t o f l i g a t i o n and t h e second measured t h e e x t e n t o f r e p e r f u s i o n a f t e r 40 min o f l i g a t i o n . In t h i s way , t h r e e t r e a t m e n t p e r i o d s c o u l d be s t u d i e d ( c o n t r o l , l i g a t i o n , r e p e r f u s i o n ) u s i n g o n l y two i s o t o p e s . Two g roups o f 3 a n i m a l s were s u b j e c t e d t o t he same s u r g i c a l p r o c e d u r e s as o u t l i n e d i n t h e s e c t i o n e n t i t l e d " O p e r a t i v e T e c h n i q u e " . M i c r o s p h e r e p r e p a r a t i o n ^ 7 Co and ^ S n - 1 a b e l l e d m i c r o s p h e r e s (15 ± 3 y ) were o b t a i n e d f r om New Eng l and N u c l e a r suspended i n p h y s i o l o g i c a l s a l i n e . These were v o r t e x e d f o r 20 min b e f o r e add i ng them t o a 10% F i c o l l s o l u t i o n ( w i t h t h e a d d i t i o n o f 0.05% c h l o r b u t a n o l as a p r e s e r v a t i v e ) c o n t a i n i n g a d r op o f Tween 80 t o 113 p r e v e n t a g g r e g a t i o n . I m m e d i a t e l y b e f o r e u s e , t h e Sn m i c r o s p h e r e s were d i l u t e d a g a i n w i t h t h e same v e h i c l e so t h a t each i n j e c t i o n c o n t a i n e d a p p r o x - i m a t e l y 126 ,000 m i c r o s p h e r e s . Because o f t h e i r h i g h e r a c t i v i t y , t h e 57 C o - l a b e l l e d m i c r o s p h e r e s were d i l u t e d t o a l e s s e r e x t e n t r e s u l t i n g i n a t o t a l o f 2 5 , 000 m i c r o s p h e r e s per i n j e c t i o n . When t h e haemodynamic v a r i a b l e s had s t a b i l i z e d a f t e r s u r g e r y , 113 S n - l a b e l l e d m i c r o s p h e r e s , p r e p a r e d as d e s c r i b e d above , were i n j e c t e d d i r e c t l y i n t o t h e l e f t v e n t r i c u l a r c a n n u l a and r i n s e d t h r ough w i t h s a l i n e . J u s t p r i o r t o i n j e c t i o n , r e f e r e n c e b l o o d f r om t he c a r o t i d a r t e r y was w i t h - drawn a t a c o n s t a n t r a t e ( 0 . 7 m l /m in ) w i t h a H a r v a r d Appa r a t u s pump and t h i s was c o n t i n u e d f o r a t l e a s t one m inu t e a f t e r c o m p l e t i o n o f m i c r o s p h e r e i n j e c - t i o n . T h i s p r o c e d u r e p r o v i d e d t h e e s t i m a t e o f c o n t r o l b l o o d f l o w . A f t e r 3-5 min had e l a p s e d , t he l e f t c i r c u m f l e x c o r o n a r y a r t e r y was o c c l u d e d . Group I ( l i g a t i o n s t u d y ) - a f t e r 10 min o f c o r o n a r y l i g a t i o n r e f e r e n c e 57 b l o o d f l o w was w i t hd r awn and t h e second i s o t o p e was i n j e c t e d ( Co) i n t h e same manner as t h e f i r s t . Th r ee m i nu t e s l a t e r t h e h e a r t was removed and p l unged i n t o c o l d , b u f f e r e d h i g h K + s o l u t i o n . - 39 - Group 2 ( r e p e r f u s i o n s t u d y ) - a f t e r 40 min o f l i g a t i o n and 10 min o f 57 r e p e r f u s i o n , t h e Co was i n j e c t e d v i a t he l e f t v e n t r i c u l a r c a n n u l a j u s t a f t e r t h e w i t h d r a w a l pump was s w i t c h e d o n . A few m i nu t e s l a t e r , t h e h e a r t was removed and immersed i n c o l d , b u f f e r e d , h i g h K + s o l u - t i o n . A f t e r r e m o v a l , bo th g roups o f h e a r t s were r e - l i g a t e d and p e r f u s e d r e t r o - g r a d e l y w i t h c o l d b u f f e r t o v i s u a l i z e t h e o c c l u d e d z o n e . The r a d i o a c t i v i t y o f t h e w e l l - t r i m m e d o c c l u d e d z o n e , t h e n o n - o c c l u d e d l e f t v e n t r i c l e , t h e r i g h t v e n t r i c l e and t h e k i d n e y s was coun t ed on a Beckmann Gamma 8000 gamma r a d i a t i o n c o u n t e r . A summary o f t he p r o t o c o l and a sample c a l c u l a t i o n a r e shown i n Append i x I I I . As shown i n T a b l e V, t h i s method o f c o r o n a r y l i g a t i o n r educed t h e f l o w i n t h e t i s s u e s u p p l i e d by t h e l e f t c i r c u m f l e x a r t e r y ( i . e . , t h e o c c l u d e d zone) t o 6 .2 * 2.2% (mean ± S . E . n = 3) o f t he f l o w measured i n n o n - i s c h a e - mic l e f t v e n t r i c l e . R e l e a s e o f t h e l i g a t u r e p e r m i t t e d a r e t u r n o f b l o o d f l o w ; r e p e r f u s i o n r e s u l t e d i n f l o w r a t e s o f 110 ± 15% (mean ± S . E . , n = 3) i n t h i s e x p e r i m e n t a l m o d e l . V I I . E l e c t r o n M i c r o s c o p y Sample p r e p a r a t i o n f o r e l e c t r o n m i c r o s c o p y r e q u i r e s c o n s i s t e n t t e c h n i q u e and c a r e f u l l y p r e p a r e d s o l u t i o n s i n o r d e r t o a v o i d g e n e r a t i o n o f t i s s u e a r t i f a c t s . The t e c h n i q u e s used i n t h e u l t r a s t r u c t u r a l s t u d y ( t h e e f f e c t o f D-600 and p r o p r a n o l o l p r e t r e a t m e n t on r e p e r f u s i o n i n j u r y ) were a s y n t h e s i s o f p r o c e d u r e s f r om s e v e r a l c u r r e n t r e f e r e n c e s d e s c r i b i n g methods o f t i s s u e f - a + . . n 106-108 f l x a t i o n . A d d i t i o n a l i n f o r m a t i o n was r e c e i v e d f r om M r . L a s z l o V e t o , S e n i o r E l e c t r o n M i c r o s c o p y T e c h n i c i a n a t t h e Depar tment o f B i o l o g y , U n i v e r s i t y o f - 40 - T a b l e V . B l o od f l o w i n t he i s c h a e m i c and r e p e r f u s e d l e f t v e n t r i c l e T r ea tmen t B l o od F low^ A . C o n t r o l ( 1 1 3 S n ) — L i g a t i o n ( 5 7 C o ) 85 ± 6 . 4 6 .2 ± 2 .2 B. C o n t r o l ( 1 1 3 S n ) — R e p e r f u s i o n ( 5 7 C o ) 91 ± 17 110 ± 15 V a l u e s a r e e x p r e s s e d as mean ± S . E . n = 3 f o r each t r e a t m e n t BF i s c h a e m i c LV a B l o od f l o w = BF normal L V — x 1 0 ° * - 41 - B r i t i s h C o l u m b i a . M r . Ve t o k i n d l y d i d t h e subsequen t embedd ing , s e c t i o n i n g , s t a i n i n g and pho t og r aphy o f t h e s a m p l e s . He was g i v e n no i n f o r m a t i o n r e g a r d i n g t h e t r e a t m e n t s and a l l samp les were coded so t h a t t h e pho t og r aphy was done i n a b l i n d manner . Sample P r e p a r a t i o n ( Fo r f u r t h e r d e t a i l s r e g a r d i n g s o l u t i o n c o m p o s i t i o n and p r e p a r a t i o n , see Append i x I I ) A f t e r t h e h e a r t s were r emoved , p e r f u s e d , and t h e o c c l u d e d zone i s o l a t e d , 3 a s m a l l s e c t i o n ( a p p r o x . 3 mm ) o f i s c h a e m i c o r c o n t r o l s ubendoca rd i um was p l a c e d i n a P e t r i d i s h f i l l e d w i t h 4°C b u f f e r e d 3% g l u t a r a l d e h y d e and m inced w i t h a s c a l p e l . A f t e r one hou r a t 4°C t h e t i s s u e was t r a n s f e r r e d t o s m a l l v i a l s c o n t a i n i n g f r e s h 3% b u f f e r e d g l u t a r a l d h y d e u s i n g a w ide -mou th P a s t e u r p i p e t t e and t h e v i a l was a l l o w e d t o warm t o room t e m p e r a t u r e f o r t h e second hour o f f i x a t i o n . A f t e r t r a n s f e r , t h e t i s s u e was m inced aga i n u n t i l t h e b l o c k s were a p p r o x i m a t e l y 0 . 5 mm. The s m a l l s i z e o f t h e t i s s u e b l o c k s p e r m i t s maximum a b s o r p t i o n o f t h e p o o r l y - p e n e t r a t i n g s e c o n d a r y f i x a t i v e , osmium t e t r o x i d e ( 0 s 0 4 ) . A t l e a s t 20 samp les per h e a r t were p r o c e s s e d . The t i s s u e was washed w i t h 3-4 changes o f 4°C b u f f e r (no g l u t a r a l d e h y d e ) and s t o r e d i n t h e r e f r i g e r a t o r u n t i l a l l samp les were r e a d y f o r 0 s 0 4 f i x a t i o n ( a p p r o x i m a t e l y 3 w e e k s ) . The b u f f e r was changed each week t o p r e v e n t any m i c r o o r g a n i s m g r o w t h , a l t h o u g h t h i s p r e c a u t i o n may have been u n n e c e s s a r y because c a c o d y l a t e i s an a r s e n i c compound and does no t s u p p o r t b a c t e r i a l g r o w t h . A f t e r a l l 8 samp les had been c o l l e c t e d (2 per t r e a t m e n t g r o u p ) , a l l t h e t i s s u e b l o c k s were p o s t - f i x e d i n OsO^, d e h y d r a t e d and embedded i n one day . A l l were s u b j e c t e d t o t h e f o l l o w i n g t r e a t m e n t s w i t h t h e e x c e p t i o n t h a t one h a l f o f t h e t i s s u e b l o c k s ( a p p r o x . 10) o f each samp le were s t a i n e d f o r 45 min a t room t e m p e r a t u r e w i t h 0.8% r u t h e n i u m r e d - a dye t h a t has an - 42 - a f f i n i t y f o r m u c o p o l y s a c c h a r i d e . The r u t h e n i u m r e d was removed w i t h t h r e e washes o f b u f f e r , 30 min pe r wash . A l l samp les were t hen p o s t - f i x e d w i t h t h e a d d i t i o n o f c a c o d y l a t e - b u f f e r e d 1% OsO^ f o r 30 min on i c e f o l l o w e d by 30 min a t room t e m p e r a t u r e . Ex ces s OsO^ was removed w i t h two , 5 m in changes o f w a t e r . D eh yd r a t i o n was a c c o m p l i s h e d by r e p l a c i n g t h e wa t e r w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f e t h a n o l ; 10 , 2 0 , 3 0 , 4 0 , 5 0 , 6 0 , 7 0 , 8 5 , 95% each f o r 2-3 m i n . Fo r c omp l e t e d e h y d r a t i o n , t h e 95% ETOH was r e p l a c e d w i t h 2 changes o f 100% ETOH f o r 30 min e a c h . The embedding p l a s t i c i s m i s c i b l e w i t h p r o p y l e n e o x i d e t h e r e f o r e 3 : 1 , 1 : 1 , 1:3 ETOH(100%):PrOX(100%) ( v / v ) s o l u t i o n s were added f o r 2 -3 min each a f t e r t h e 100% ETOH. F i n a l l y 2 changes o f 100% p r o p y l e n e o x i d e f o r 20 min each c omp l e t ed t h e t i s s u e f i x a t i o n . Samples were g i v e n t o L. Ve t o w i t h i n one hour a f t e r wh i ch t h e y were embedded i n Epon 812 . From t h e 10 b l o c k s , two were r andom l y chosen and s e c t i o n s were c u t w i t h a P o r t e r - B l u m MT-2 u l t r a m i c r o t o m e , s t a i n e d w i t h u r a n y l a c e t a t e and examined on a K a r l Z e i s s EM-10 e l e c t r o n m i c r o s c o p e a t 8 KV. F i v e g r i d s were r andom l y s e l e c t e d and pho tog r aphed a t v a r i o u s m a g n i f i c a t i o n s . A s ses smen t o f t h e pho tog raphs was pe r f o rmed by Dr . M. Todd , Depar tment o f Anatomy, U n i v e r - s i t y o f B r i t i s h C o l u m b i a , u s i n g a r a n d o m i z e d , b l i n d p r o t o c o l . V a r i o u s f e a t u r e s on t h e m i c r o g r a p h s were s c o r e d a c c o r d i n g t o a p r e - a r r a n g e d s c o r i n g s y s t em i n wh i ch 0 = n o r m a l , 1 = m i l d a b n o r m a l i t y , 2 = mode ra te a b n o r m a l i t y , and 3 = s e v e r e a b n o r m a l i t y . L i g h t M i c r o s o p y In o r d e r t o d e t e r m i n e t he u n i f o r m i t y o f t i s s u e sample p r e p a r a t i o n , i . e . , t h a t t h e m y o c a r d i a l s e c t i o n s d i d n o t c o n t a i n m ixed a r e a s o f damaged and norma l t i s s u e , t h i c k s e c t i o n s were c u t and s t a i n e d w i t h t o l u i d i n e b l u e t o be examined w i t h a l i g h t m i c r o s c o p e . T i s s u e b l o c k s o t h e r t h an t h o s e used f o r e l e c t r o n m i c r o s c o p i c a n a l y s i s were s e c t i o n e d . The s e c t i o n i n g and s t a i n i n g - 43 - were done by M r . L. Ve to o f t he Depar tment o f Z o o l o g y and t h e l i g h t m i c r o - s c o p i c pho t og r aphy by D r . W. K. O v a l l e , Depar tment o f Anatomy, U n i v e r s i t y o f B r i t i s h C o l u m b i a . The s e c t i o n s were pho tog raphed ( n e g a t i v e m a g n i f i c a t i o n s 80X) on Kodak b l a c k and w h i t e f i l m and p r i n t e d on 3" x 5" paper f o r a f i n a l m a g n i f i c a t i o n o f X260 . V I I I . P h o s p h o l i p i d E x t r a c t i o n and S e p a r a t i o n Two d i f f e r e n t p r o c edu r e s were used t o s e p a r a t e t h e v a r i o u s c l a s s e s o f p h o s p h o l i p i d s . I n t h e p r e l i m i n a r y e x p e r i m e n t s , e x t r a c t i o n was f o l l o w e d by a o n e - d i m e n s i o n a l t h i n l a y e r c h r o m a t o g r a p h i c (TLC) p r o c e d u r e . Fo r t h e D-600 and p r o p r a n o l o l and t h e 0 -60 min l i g a t i o n s t u d y , a t w o - d i m e n s i o n a l TLC sys tem was adop ted i n o r d e r t o s e p a r a t e p h o s p h a t i d y l s e r i n e and p h o s p h a t i d y l - i n o s i t o l . Both p r o c e d u r e s a r e o u t l i n e d b e l o w . O n e - d i m e n s i o n a l s y s tem E x t r a c t i o n - One ml o f membrane s u s p e n s i o n was e x t r a c t e d w i t h 2 ml c h l o r o f o r m : m e t h a n o l (CHC lg iMeOH, 2 :1 ) ( v / v ) f o r 30 min on i c e . The s u s p e n s i o n was c e n t r i f u g e d a t 12 ,000 g x 5 min and t h e s u p e r n a t a n t d e c an t ed i n t o a g l a s s c e n t r i f u g e t u b e . The p e l l e t was e x t r a c t e d w i t h an a d d i t i o n a l 2 ml o f CHCl-^MeOH f o r 30 min on i c e , and , a f t e r c e n t r i f u g a t i o n , t h e o r g a n i c phases were p oo l e d and washed t h r e e t i m e s u s i n g 0 . 5 ml o f 0.75% N a C l . Each wash was f o l l o w e d by c e n t r i f u g a t i o n and t h e aqueous phase d i s c a r d e d . The CHCl-^MeOH e x t r a c t was t hen e v a p o r a t e d t o d r y n e s s under a s t r e am o f n i t r o g e n and t h e r e s i d u e r e su spended w i t h 200y l CHClg:Me0H and s p o t t e d on an a l um inum-backed s i l i c a ge l t h i n l a y e r c h r o m a t o g r a p h i c p l a t e (Me r c k , S i l i c a g e l 60 F^^, 0 .2 mm t h i c k n e s s ) . The s o l v e n t s y s t em c o n s i s t e d o f C H C l 3 : M e 0 H : g l a c i a l CH 3 C00H :H 2 0 ( v / v / v / v , 2 5 : 1 5 : 4 : 1 ) . N i n h y d r i n and i o d i n e vapou r were used s e q u e n t i a l l y t o v i s u a l i z e t he s p o t s . - 44 - Phospho rus A n a l y s i s The p l a t e s were s c r a p ed and t h e ge l a n a l y z e d f o r i n o r g a n i c phospho rus 109 u s i n g a m o d i f i c a t i o n o f t h e method o f B a r t l e t t . T w o - d i m e n s i o n a l Sys tem The e x t r a c t i o n p r o c edu r e was i d e n t i c a l t o t h a t d e s c r i b e d above bu t t h e e x t r a c t was ch roma tog raphed i n 2 d i m e n s i o n s u s i n g a m o d i f i c a t i o n o f t h e t e c h n i q u e o f Y a v i n and Z u t r a . 1 1 0 A f t e r t h e f i r s t s o l v e n t - CHC l 3 :MeOH: NHg ( v / v / v , 3 5 : 1 5 : 2 . 5 ) - had e v a p o r a t e d , t h e p l a t e was pu t i n t h e second s o l v e n t sy s tem 90° f r om t h e f i r s t d i r e c t i o n . The second s o l v e n t sy s tem c o n s i s t e d o f C H C l 3 : C O ( C H 3 ) 2 : C H 3 O H : g l a c i a l CH 3 C00H :H 2 0 ( v / v / v , 2 5 : 1 0 : 5 : 7 . 5 : 2 . 5 ) . The s p o t s were v i s u a l i z e d and a n a l y z e d f o r i n o r g a n i c phospho rus as o u t l i n e d i n t h e p r e v i o u s s e c t i o n . S t a t i s t i c a l A n a l y s e s R e s u l t s were a n a l y z e d u s i n g a one-way a n a l y s i s o f v a r i a n c e f o l l o w e d by T u k e y ' s t e s t t o a s s e s s s p e c i f i c g roup d i f f e r e n c e s a t P < 0 . 0 5 . P e r c e n t a g e d a t a were t r a n s f o r m e d w i t h t he a r c s i n s qua r e r o o t t r a n s f o r m a t i o n b e f o r e any 111 a n a l y s i s was done . The c h i - s q u a r e t e s t f o r k i n dependen t v a r i a b l e s was used t o a n a l y z e t h e f i b r i l l a t i o n d a t a f r om t h e 20 m i nu t e l i g a t i o n s t u d y . MATERIALS P e n t o b a r b i t o n e s od i um , OsO^ ampou le s , and p r o p y l e n e o x i d e were o b t a i n e d f r om B . D . H . . A c i d s , s o l v e n t s and s t a n d a r d i o n s o l u t i o n s f o r a t om i c a b s o r p t i o n s p e c t r o m e t r y were a l l r e a g e n t g rade ( e x c e p t t h e CHC1 3 and CH 3 0H wh i ch were s p e c t r o p h o t o m e t r y g r a d e ) . Enzyme-grade s u c r o s e ( U l t r a - pu re ) was f rom Schwar z -Mann . Ge l e l e c t r o p h o r e s i s m a t e r i a l s came f r om E-C a ppa r a t u s and t h e S i l i c a g e l p l a t e s (60 F254) f o r t h i n - l a y e r c h r oma tog r aphy and e l e c t r o n m i c r o s c o p y - g r a d e g l u t a r a l d e h y d e were o b t a i n e d f r om Merck (Darm- s t a d t ) . J . B . EM S e r v i c e s was t h e s o u r c e o f t h e c a c o d y l a t e . A l l o t h e r c h e m i c a l s were r e a g e n t g r ade f r om S igma Chem i c a l C o . - 45 - RESULTS I . P r e l i m i n a r y S t u d i e s A p r e l i m i n a r y s t u d y was unde r t a k en i n o r d e r t o e s t a b l i s h t h e e f f e c t s o f i s c h a e m i a on t h e a c t i v i t y o f membrane-bound marke r enzymes . The e f f e c t s o f i s c h a e m i a on two m i t o c h o n d r i a l marke r enzymes , name ly c y t o ch rome c o x i d a s e and m i t o c h o n d r i a l a z i d e - s e n s i t i v e ATPase a r e shown i n F i g u r e 1 . The i n s e n s - i t i v i t y o f t h e c y t o ch rome c o x i d a s e a c t i v i t y t o i s c h a e m i a c o n t r a s t s m a r k e d l y w i t h t h e r a p i d d e c r e a s e i n t h e ATPase a c t i v i t y f o l l o w i n g c o r o n a r y l i g a t i o n . The d e c r e a s e i n m i t o c h o n d r i a l ATPase a c t i v i t y r e a ched a p l a t e a u o f 40% o f c o n t r o l a t a p p r o x i m a t e l y 20 m i nu t e s p o s t - 1 i g a t i o n . A s i m i l a r p a t t e r n o f p r o g r e s s i v e enzyme i n h i b i t i o n w i t h i n c r e a s i n g p e r i o d s o f l i g a t i o n was seen w i t h bo th t h e s a r co l emma l N a + , K + - A T P a s e and K + - N P P a s e a c t i v i t i e s ( F i g u r e 2 ) . These measurements r e f l e c t t h e f u n c t i o n - a l c a p a b i l i t y o f t h e o u a b a i n - s e n s i t i ve sod ium pump - t h e N a + and K + - s t i m u l a t e d ATP h y d r o l y s i s and t h e t e r m i n a l K + - d e p e n d e n t s t e p o f ATP 99 h y d r o l y s i s , r e s p e c t i v e l y . Bo th were a f f e c t e d t o a p p r o x i m a t e l y t h e same e x t e n t by i s c h a e m i a . In o r d e r t o i n v e s t i g a t e t he r e v e r s i b i l i t y o f t h e s e enzyme a l t e r a t i o n s , a no t h e r g roup o f a n i m a l s were s u b j e c t e d t o 60 m i nu t e s o f r e p e r f u s i o n o f t h e i s c h a e m i c a r e a a f t e r v a r i o u s p e r i o d s o f c o r o n a r y o c c l u s i o n . The r e s u l t s , shown i n T a b l e V I , i n d i c a t e t h a t t h e e f f e c t s o f i s c h a e m i a on t h e m i t o c h o n - d r i a l ATPase a c t i v i t y a r e l a r g e l y r e v e r s i b l e . Even a f t e r 60 m i nu t e s o f c o r o n a r y l i g a t i o n , r e p e r f u s i o n f o r one hour r e s t o r e d 80% o f c o n t r o l m i t o - c h o n d r i a l ATPase a c t i v i t y . In c o n t r a s t , r e c o v e r y o f t h e sa r co l emma l N a + , K + - A T P a s e and K + - N P P a s e a c t i v i t i e s w i t h r e p e r f u s i o n was no t as g r e a t as f o r t h e m i t o c h o n d r i a l ATPa se . I n a d d i t i o n , as t h e p e r i o d o f i s c h a e m i a i n c r e a s e d , t h e p e r c e n t r e c o v e r y o f t h e sa rco lemma enzyme a c t i v i t y - 46 - C Y T O C H R O M E C OX IDASE ( « M O L E s / m i n / m g PROTEIN ) >- h h u Ul 2 5 0 2 O O N ' 5 ° Z u A Z I D E - S E N S I T I V E A T P a s e ( JJ M O L E S P i / h r / m g PROTEIN ) 50 SO 4 0 SO L I G A T I O N T I M E ( M I N ) F i g u r e 1 . M i t o c h o n d r i a l membrane-bound enzyme a c t i v i t y a f t e r 0 -60 min o f l i g a t i o n . A s s a y s were pe r f o rmed on p u r i f i e d m i t o c h o n d r i a and i s o l a t i o n p r o c edu r e s and d e t a i l s o f t he a s s a y s a re d e s c r i b e d i n t h e Methods s e c t i o n . No T r i t o n X -100 a d d i t i o n was made t o t he a s s a y medium. The t op s e c t i o n shows t he l a c k o f e f f e c t o f i s c h a e m i a on cy to ch rome c o x i d a s e a c t i v i t y . In c o n t r a s t , t h e i s c h a e m i a - i n d u c e d i n h i b i t i o n o f a z i d e - s e n s i t i v e ATPase a c t i v i - t y i s shown on t h e bo t tom h a l f . A l l v a l u e s a re e x p r e s s e d as mean ± S . E . , n = 4 - 6 f o r each p o i n t . - 47 - LIGATION TIME (MUM) F i g u r e 2 . Sa rco lemmal enzyme a c t i v i t y i n r e s pon s e t o i s c h a e m i a . Bo th Na , K + - A T P a s e a c t i v i t y ( • — • ) and K + - NPPa s e (*™--*) d e c r e a s e w i t h i n c r e a s i n g p e r i o d s o f l i g a t i o n . R e s u l t s a r e e x p r e s s e d as mean ± S . E . , and n = 4 -6 f o r each g r o u p . No T r i t o n X-100 was added t o t h e a s s a y medium. - 48 - T a b l e V I . Degree o f r e c o v e r y o f m i t o c h o n d r i a l and sa r co l emma l ATPase a c t i v i t i e s a f t e r v a r i o u s i n t e r v a l s o f i s c h a e m i a f o l l o w e d by a 60 m inu t e p e r i o d o f r e p e r f u s i o n % Re cove r y on r e p e r f u s i o n L i g a t i o n P e r i o d M i t o c h o n d r i a Sarco lemma (min) a z i d e - s e n s i t i v e N a + K + - A T P a s e K + -NNPase ATPase 0 100 100 100 10 91 79 58 20 113 60 42 30 104 37 37 40 82 35 33 60 80 25- 16 Each v a l u e r e p r e s e n t s t h e mean o f 2 a n ima l s pe r g roup Fo r a b s o l u t e v a l u e s o f enzyme a c t i v i t i e s , see F i g . 1 and 2 . - 49 - d e c r e a s e d . Ba sa l (Mg - d ependen t ) enzyme a c t i v i t i e s a l s o showed p r o g r e s - s i v e , i r r e v e r s i b l e d e c r e a s e s i n a c t i v i t y ; however , t h e s e were no t as s e v e r e as t h o s e seen w i t h t h e N a + and K + - s t i m u l a t e d a c t i v i t y ( d a t a no t shown) . Fo r e x amp l e , t h e i n h i b i t i o n o f m i t o c h o n d r i a l ATPase a c t i v i t y p roduced by 30 m inu t e s o f c o r o n a r y l i g a t i o n was c o m p l e t e l y r e v e r s e d by a 60 m inu t e r e p e r f u - s i o n p e r i o d , whereas a f t e r s i m i l a r r e p e r f u s i o n M g + + - d e p e n d e n t , sa r co l emma ATPase and NPPase a c t i v i t i e s were r e s t o r e d t o 70% and i o n - s t i m u l a t e d ATPase a c t i v i t i e s r e c o v e r e d o n l y 37% o f c o n t r o l a c t i v i t y . ++ + + + Mg and K i n t e r a c t w i t h t h e Na ,K -ATPase enzyme complex a t 99 t h e i n n e r and o u t e r s i d e s o f t h e membrane r e s p e c t i v e l y , so t h a t changes i n t h e k i n e t i c p r o p e r t i e s o f i o n s t i m u l a t i o n c o u l d r e v e a l t h e l o c a t i o n o f s a r co l emma l enzyme a l t e r a t i o n s . F i g u r e 3 ( K + ) and F i g u r e 4 ( M g + + ) show t h a t a l t h o u g h 60 m i nu t e s o f i s c h a e m i a r educed NPPase a c t i v i t y ( V m a x ) a t a l l c o n c e n t r a t i o n s o f t h e s u b s t r a t e when compared w i t h c o n t r o l , i t d i d no t + ++ cause any change i n t h e appa r en t a f f i n i t y (K^) f o r e i t h e r K o r Mg ( see i n s e t s o f F i g u r e s 3 and 4 ) . F u r t h e r m o r e , t h e r e was no s i g n i f i c a n t d i f f e r e n c e between t he l i g a t e d and t h e 1 i g a t e d / r e p e r f u s e d g roups f u r t h e r s u p p o r t i n g t h e h y p o t h e s i s t h a t t h e d e c r e a s e i n s a r co l emma l enzyme a c t i v i t y a f t e r 60 m i nu t e s o f o c c l u s i o n i s i r r e v e r s i b l e . The t i s s u e c o n t e n t o f ATP was a s s e s s e d t o d e t e r m i n e t h e t i m e c o u r s e o f ATP d e p l e t i o n r e l a t i v e t o a l t e r a t i o n s i n t h e i n t e g r i t y o f t h e s e sa r co l emma l and m i t o c h o n d r i a l enzymes . ATP c o n t e n t ( e x p r e s s e d as nmoles ATP/mg d r y w e i g h t ) was measured i n h e a r t s s u b j e c t e d t o 0-60 m inu t e s o f l i g a t i o n w i t h o r w i t h o u t r e p e r f u s i o n . The r e s u l t s i n d i c a t e t h a t i s c h a e m i a c au se s a f a l l i n ATP l e v e l s t h a t r e a c h e s a p l a t e a u a t a p p r o x i m a t e l y 30 m i nu t e s p o s t - l i g a t i o n ( T a b l e V I I ) . R e p e r f u s i o n does no t r e s t o r e ATP and a f t e r 20 t o 40 m i nu t e s o f i s c h a e m i a , ATP l e v e l s were even l owe r i n t h e r e p e r f u s e d a n ima l s ( a l t h o u g h t he d i f f e r e n c e was no t s t a t i s t i c a l l y s i g n i f i c a n t ) . - 50 - > > • C O N T R O L h • L I G A T E D U a . - L I G A T E D AIMO R E P E R F U S E D ^ [ K ] ( m M ) F i g u r e 3 . S t i m u l a t i o n by K + o f M g 2 + - d e p e n d e n t NPPase a c t i v i t y i n s a r c o - lemmal v e s i c l e s i s o l a t e d f r om c o n t r o l , 60 min l i g a t e d and 60 min l i g a t e d and r e p e r f u s e d m y o c a r d i a . Each p o i n t r e p r e s e n t s t h e mean o f two s e p a r a t e membrane p r e p a r a t i o n s . I n s e t : d oub l e r e c i p r o c a l p l o t s o f t h e same d a t a . - 51 - F i g u r e 4 . A c t i v a t i o n by M g 2 + o f K - s t i m u l a t e d NPPase a c t i v i t y i n s a r c o - lemmal v e s i c l e s p r e p a r e d f r om c o n t r o l ; 60 min l i g a t e d and 60 min l i g a t e d and r e p e r f u s e d m y o c a r d i a . Each p o i n t r e p r e s e n t s t h e mean o f two s e p a r a t e membrane p r e p a r a t i o n s . I n s e t : d oub l e r e c i p r o c a l p l o t s o f t h e same d a t a . - 52 - T a b l e V I I . ATP c o n t e n t s o f h e a r t s s u b j e c t e d t o v a r i o u s p e r i o d s o f l i g a t i o n and l i g a t i o n p l u s r e p e r f u s i o n mg d r y w e i g h t L i g a t i o n p e r i o d (min) L i g a t i o n L i g a t i o n + R e p e r f u s i o n ^ C o n t r o l 11.6 ± 2.0 5 9.8 ± 0.3 (2) 12.1 ± 1.4 (2) 10 6.9 ± 1.2 (3) 7.0 ± 1.8 (2) 20 4.9 ± 0.5 (4) 3.1 ± 0.3 (2) 40 3.1 ± 0.3 (3) 1.4 ± 0.2 (2) 60 0.6 ± 0.3 (3) 1.7 ± 0.3 (3) A l l number a r e e x p r e s s e d as mean ± s . E . ( )= number o f a n ima l s pe r g roup £ R e p e r f u s i o n p e r i o d was 60 min f o r a l l g roups - 53 - The l a c k o f r e s t o r a t i o n o f t h e sa r co l emma l enzyme a c t i v i t i e s s ugge s t e d t h a t t h e y may have undergone an i r r e v e r s i b l e c h e m i c a l m o d i f i c a t i o n d u r i n g i s c h a e m i a ; howeve r , p o l y a c r y l a m i d e ge l e l e c t r o p h o r e s i s o f bo th m i t o c h o n d r i a l and s a r co l emma l membranes p r e p a r e d f r om l i g a t e d o r 1 i g a t e d / r e p e r f u s e d m y o c a r d i a r e v e a l e d no o b v i o u s q u a l i t a t i v e d i f f e r e n c e s i n p o l y p e p t i d e p r o f i l e s r e l a t i v e t o c o n t r o l ( d a t a no t shown) . The 1 i p i d - d e p e n d e n c e o f bo th t h e sa r co l emma l and m i t o c h o n d r i a l ATPase 112 113 a c t i v i t y » prompted t he e x a m i n a t i o n o f the e f f e c t s o f i s c h a e m i a on two o f t h e ma jo r p h o s p h o l i p i d c l a s s e s o f t h e m y o c a r d i a l membrane, namely p h o s p h a t i d y l c h o l i n e and p h o s p h a t i d y l e t h a n o l a m i n e . S i x t y m i nu t e s o f l i g a t i o n w i t h o r w i t h o u t r e p e r f u s i o n p roduced no q u a n t i t a t i v e change i n e i t h e r o f t h e s e p h o s p h o l i p i d s i n e i t h e r t h e s a r co l emma l o r t h e m i t o c h o n d r i a l membrane f r a c t i o n s ( T ab l e V I I I - o n l y s a r co l emma l d a t a shown) . I I . 20 M i nu t e L i g a t i o n w i t h Drug P r e t r e a t m e n t The e f f e c t s o f s e v e r a l d i f f e r e n t agen t s on t he b i o c h e m i c a l a l t e r a t i o n s p roduced by 20 m i nu t e s o f c o r o n a r y o c c l u s i o n w i t h o u t subsequen t r e p e r f u s i o n was i n v e s t i g a t e d i n t h i s g roup o f e x p e r i m e n t s . A 20 m i nu t e l i g a t i o n p e r i o d was chosen be cause t h e r e s u l t s o f t h e p r e v i o u s s t u d y and work by o t h e r 6 31 i n v e s t i g a t o r s ' had s ugge s t e d t h a t t h i s p e r i o d o f i s c h a e m i a i s a t t h e t r a n s i t i o n between r e v e r s i b l e and i r r e v e r s i b l e c e l l i n j u r y and , i n d e e d , marked membrane a b n o r m a l i t i e s were e v i d e n t i n bo th m i t o c h o n d r i a l and s a r c o - lemmal membranes a f t e r 20 m i n u t e s o f c o r o n a r y l i g a t i o n . The a b i l i t y o f - 54 - T a b l e V I I I . P h o s p h o l i p i d c o n t e n t o f s a r co l emma l membranes i s o l a t e d f rom c o n t r o l , l i g a t e d , and l i g a t e d and r e p e r f u s e d m y o c a r d i a T r ea tmen t % T o t a l L i p i d Phospho rus P h o s p h a t i d y l e t h a n o l a m i n e P h o s p h a t i d y l c h o l i n e C o n t r o l 4 5 . 4 ± 3 .4 3 7 . 2 ± 6 . 9 L i g a t i o n 4 7 . 8 ± 6 . 8 4 0 . 8 ± 6 .1 L i g a t i o n and R e p e r f u s i o n 5 1 . 0 ± 3 .2 32 .2 ± 3.7 A l l v a l u e s a r e e x p r e s s e d as mean ± S .D . n = 2 f o r each g r o u p . - 55 - p r e t r e a t m e n t w i t h each o f t h e f o l l o w i n g agen t s t o r educe t h e s e b i o c h e m i c a l a l t e r a t i o n s was s t u d i e d : 3 . 0 mg/kg d l - p r o p r a n o l o l HCl (PROP) 3 . 0 mg/kg p r a n o l i u m C l (PRAN) 0 .15 mg/kg (±) -D -600 HCl (D-600) 30 mg/kg i m i d a z o l e ( IMID) 2 . 0 mg/kg c h l o r p r o m a z i n e (CPZ) The v a r i o u s r a t i o n a l e f o r t h e use o f t h e s e agen t s i n t h e t r e a t m e n t o f m y o c a r d i a l i s c h a e m i c i n j u r y a r e l i s t e d i n T a b l e I X . I n o r d e r t o e l i m i n a t e t h e c o n f o u n d i n g e f f e c t s o f d rug a c t i o n on t h e v a r i a b l e s i ndependen t o f t h e i r p o s s i b l e b e n e f i t i n r e d u c i n g c e l l i n j u r y , t h e above dose o f each d rug was a d m i n i s t e r e d t o one o f 5 g roups o f a n ima l s (n = 4 - 6 ) . These d rug t r e a t e d a n i m a l s underwent a l l s u r g i c a l p r o c edu r e s e x c e p t l i g a t i o n and a r e h e r e t o f o r e r e f e r r e d t o as " d r ug c o n t r o l s " . The r e s u l t s o f t h e d rug c o n t r o l s t u d y a r e shown i n T a b l e X . I n t e r e s t i n g l y , t h e a d m i n i s t r a t i o n o f p r o p r a n o l o l i n t h e absence o f m y o c a r d i a l i s c h a e m i a caused an i n c r e a s e i n bo th t he m i t o c h o n d r i a l ATPase a c t i v i t y and t h e ATP c o n t e n t o f t h e t i s s u e , a l t h o u g h o n l y t h e r i s e i n ATP c o n t e n t was s t a t i s t i c a l l y s i g n i f i - c a n t . The r e d u c t i o n o f ( d P / d t ) m a x f o r c e and h e a r t r a t e by p r o p r a n o l o l p r e t r e a t m e n t wou l d be e x p e c t e d t o r e du ce m y o c a r d i a l oxygen consump t i on 55 (MVO2) a n d t h l s m a y a c c o u n t f o r t h e appa r en t " A T P - s p a r i n g " e f f e c t . The r e d u c t i o n i n h e a r t r a t e may be t h e ma jo r d e t e r m i n a n t because D-600 p r e t r e a t m e n t a l s o r educed c o n t r a c t i l i t y bu t d i d no t i n c r e a s e ATP . No i n c r e a s e was seen w i t h p r a n o l i u m t h e r e f o r e n o n - e - b l o c k i n g e f f e c t s o f p r o p r a n o l o l were a l s o no t t h e cause o f s a l u t a r y e f f e c t s on ATP c o n t e n t . Inasmuch as t h e s e d rugs ( w i t h t h e e x c e p t i o n o f p r o p r a n o l o l ) c aused no a l t e r a t i o n i n t h e b a s e l i n e b i o c h e m i c a l measu remen t s , we examined t h e i r - 56 - T a b l e I X . Drugs used as p r e t r e a t m e n t p r i o r t o a 20 m inu t e p e r i o d o f c o r o n a r y l i g a t i o n Agent R a t i o n a l e P r o p r a n o l o l B - a d r e n e r g i c a n t a g o n i s t , "membrane s t a b i l i z e r " , a n t i a r r h y t h m i c , may b l o c k e f f e c t s o f c a t e c h o l a m i n e r e l e a s e i n M . I . P r a n o l i u m Q u a t e r n a r y d e r i v a t i v e o f p r o p r a n o l o l , s h a r e s p r o p e r t i e s o f p a r e n t compound e x c e p t B - b l o c k a d e . I m i d a z o l e th romboxane s y n t h e s i s i n h i b i t o r ( th romboxanes may i n i t i a t e c o r o n a r y v a s o c o n s t r i c t i o n and p l a t e l e t a g g r e g a t i o n ) . C h l o r p r o m a z i n e D-600 (methoxyve rapami 1) " l y s o s o m a l membrane s t a b i l i z a t i o n " , i n h i b i t o r o f i n t r a c e l l u l a r p h o s p h o l i p a s e s . 2+ Ca c h a n n e l - b l o c k e r , p e r i p h e r a l v a s o d i l a t o r , c o r o n a r y d i l a t o r . - 57 - T a b l e X . Drug e f f e c t s on b i o c h e m i c a l v a r i a b l e s a f t e r 20 min sham l i g a t i o n . "Drug c o n t r o l s " T r ea tmen t M i t c £ K + - N P P a s e £ ATP Water ATPase nmoles ATP Con t en t mg d r y wt ^ ̂ C o n t r o l - S a l i n e 193 ± 6 Sham 20 min l i g a t i o n , p r e t r e a t m e n t w i t h : P r o p r a n o l o l (3 mg/kg) 222 ± 5 D-600 HCI ( . 15 mg/kg) 209 ± 3 I m i d a z o l e (30 mg/kg) 208 ± 8 C h l o r p r o m a z i n e HCI (2 mg/kg) 208 ± 14 P r a n o l i u m CI (3 mg/kg) 196 ± 7 .99 .08 13 .6 ± 1.9 79 . 3 ± .4 .95 ± .05 25 . 1 2 . 3 - 79 .1 ± .4 1.00 .07 17 . 0 ± 2.5 8 0 . 0 .2 .93 .07 19 .7 ± . 88 8 0 . 5 ± .6 .86 .07 16 .4 ± 1.4 8 0 . 3 .2 .91 ± .05 15 . 4 ± 1.6 8 0 . 3 ± .2 A l l v a l u e s a r e e x p r e s s e d as mean ± S . E . n = 4 -6 per group e x c ep t C o n t r o l a n i m a l s , w h e r e n = 7 - 8 . — enzyme a c t i v i t y = umoles P-j/hr/mg p r o t e i n . — enzyme a c t i v i t y = umoles n i t r o p h e n o l / h r / m g p r o t e i n . — S i g n i f i c a n t l y g r e a t e r t h an c o n t r o l a t P < 0 .05 - 58 - a c t i o n s on t h e e s t a b l i s h e d i n d i c e s o f i s c h a e m i c i n j u r y . D-600 a d m i n i s t r a - t i o n p r i o r t o l i g a t i o n was t h e o n l y t r e a t m e n t t h a t s i g n i f i c a n t l y r e du ced t h e i s c h a e m i a - i n d u c e d d e c r e a s e i n t h e m i t o c h o n d r i a l ATPase a c t i v i t y as compared w i t h t h a t o f s a l i n e - t r e a t e d l i g a t e d a n i m a l s . Enzyme a c t i v i t y was m a i n t a i n e d a t 75% o f c o n t r o l l e v e l s compared w i t h t he s a l i n e - t r e a t e d g roup wh i c h r e t a i n e d o n l y 35% of c o n t r o l a c t i v i t y ( F i g u r e 5 ) , c o n f i r m i n g t h e r e s u l t s o f ou r e a r l i e r e x p e r i m e n t s . P r o p r a n o l o l and c h l o r p r o m a z i n e p r e t r e a t m e n t r e s u l - t e d i n s l i g h t bu t n o n - s i g n i f i c a n t p r o t e c t i o n o f m i t o c h o n d r i a l ATPase a c t i v i - t y f r om t he e f f e c t s o f i s c h a e m i a . Drug e f f e c t s on t he sa r co l emma l enzyme a c t i v i t i e s were more c omp l e x . D-600 s i g n i f i c a n t l y p r e v e n t e d t h e d e c r e a s e i n t h e M g + + - A T P a s e a c t i v i t y ( n o n - N a + and K + - s t i m u l a t e d ) o f t h e sarco lemma ( d a t a no t shown) ; howeve r , i t o n l y s l i g h t l y p r o t e c t e d N a + and K + - s t i m u l a t e d ATPase a c t i v i - t y ( F i g u r e 6 ) . I n t e r e s t i n g l y , d e s p i t e t h e f u n c t i o n a l r e l a t i o n s h i p between t he N a + , K + - A T P a s e and K + - N P P a s e enzymes , D-600 p r e t r e a t m e n t d i d no t r e d u c e t h e i s c h a e m i a - i n d u c e d d e c r e a s e i n K + - NPPa s e a c t i v i t y . As seen i n t he p r e v i o u s s t u d y , t h e ATP l e v e l o f t he myocard ium i s s e v e r e l y r educed a f t e r 20 min o f o c c l u s i o n . As shown i n F i g u r e 7 , D-600 was t h e o n l y d rug wh i ch s i g n i f i c a n t l y p r e s e r v e d ATP l e v e l s i n i s c h a e m i c myoca r - d ium; u n t r e a t e d h e a r t s c o n t a i n e d 30% o f c o n t r o l l e v e l s whereas D - 6 0 0 - p r e - t r e a t e d h e a r t s m a i n t a i n e d t h e l e v e l a t 60% o f c o n t r o l . In c o n t r a s t , d e s p i t e t h e h i g h e r p r e - o c c l u s i o n l e v e l s o f ATP i n p r o p r a n o l o l - t r e a t e d h e a r t s , i s c h a e m i a r e du ced t h e ATP c o n t e n t t o t h e same e x t e n t as i n t h e s a l i n e - t r e a - t e d a n i m a l s . T h u s , t h e i n c r e a s e i n h i g h ene rgy phospha t e i n t h e p r o p r a n o l - o l - t r e a t e d d rug c o n t r o l s appea red t o c o n f e r no o b v i o u s b e n e f i t d u r i n g t h e subsequen t l i g a t i o n p e r i o d . - 59 - Ul |» saa • II a OJ i s o oo c _L (fl ui j 0 s o II It c _L C O N T P R O P I M I O P B A N L I B D - S D D C P Z F i g u r e 5 . M i t o c h o n d r i a l a z i d e - s e n s i t i v e ATPase a c t i v i t y i n m i t o c h o n d r i a i s o l a t e d f r om h e a r t s s u b j e c t e d t o 20 min o f l i g a t i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t . CONT = sham-ope ra t ed c o n t r o l ; L IG = l i g a t e d w i t h s a l i n e p r e t r e a t m e n t , 3 mg; D-600 = D-600 p r e t r e a t m e n t , 0 . 15 mg/kg; IMID = i m i d a z o l e p r e t r e a t m e n t , 30 mg/kg ; CPZ = c h l o r p r o m a z i n e p r e t r e a t m e n t , 2 mg/kg; PRAN = p r a n o l i u m p r e t r e a t m e n t , 3 mg /kg . Drugs were a d m i n i s t e r e d as d e s c r i b e d i n t h e Methods s e c t i o n . Ba r s r e p r e s e n t mean ± S . E . C o n t r o l and D-600 v a l u e s a re s i g n i f i c a n t l y d i f f e r e n t f r om a l l o t h e r g roups bu t no t s i g n i f i c a n t l y d i f f e r e n t f rom each o t h e r (P < 0 . 0 5 ) . S t a t i s t i c a l methods a r e o u t l i n e d i n t h e Methods s e c t i o n . - 60 - CO C O I M T P R O P I M I D P R A N 1-1(3 D - 6 0 0 C P Z F i g u r e 6 . N a +,K + - A T P a s e a c t i v i t y i n sarco lemma i s o l a t e d f r om h e a r t s s u b j e c t e d t o 20 min o f l i g a t i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t . A b b r e v - i a t i o n s a r e t h e same as i n F i g u r e 5 . O n l y LIG and IMID a re s i g n i f i c a n t l y d i f f e r e n t f r om c o n t r o l (P < 0 . 0 5 ) . Ba r s r e p r e s e n t mean * S . E . - 61 - C O I M T P R O P I M i n P R A N LI G D - 6 0 0 C P Z F i g u r e 7 . ATP c o n t e n t o f h e a r t s s u b j e c t e d t o 20 min o f l i g a t i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t . A b b r e v i a t i o n s a re t h e same as i n F i g u r e 5 . A l l g roups e x c e p t D-600 have s i g n i f i c a n t l y d e c r e a s e d ATP l e v e l s compared t o c o n t r o l a n i m a l s . Ba r s r e p r e s e n t mean ± S . E . - 62 - I n c r e a s e s i n t i s s u e w a t e r were no t appa r en t a f t e r t h i s r e l a t i v e l y s h o r t p e r i o d o f i s c h a e m i a , as shown by t h e e q u i v a l e n t wa t e r c o n t e n t o f a l l g roups ( T a b l e X I ) . However , t h i s does no t e x c l u d e t he p o s s i b i l i t y t h a t t h e r e was a r e - d i s t r i b u t i o n o f wa t e r f r om t h e e x t r a c e l l u l a r t o i n t r a c e l l u l a r space o r v i c e v e r s a . U n e x p e c t e d l y , t h e s e agen t s had v a r i a b l e e f f e c t s on t he i n c i d e n c e o f v e n t r i c u l a r f i b r i l l a t i o n d u r i n g t h e l i g a t i o n p e r i o d . V e n t r i c u l a r f i b r i l l a - t i o n was i d e n t i f i e d by t he c h a r a c t e r i s t i c random e l e c t r i c a l s i g n a l on l e a d I I o f t h e e l e c t r o c a r d i o g r a m c o u p l e d w i t h a p r e c i p i t o u s f a l l i n s y s t e m i c b l o o d p r e s s u r e t o 0 mm Hg . An a r b i t r a r y i n c i d e n c e s c a l e was d e v i s e d and a s c o r e was g i v e n t o each an ima l a t t h e end o f t h e e x p e r i m e n t a c c o r d i n g t o t h e f o l l o w i n g p r o t o c o l : 0 = no f i b r i l l a t i o n 1 = 1 e p i s o d e 2 = 2-5 e p i s o d e s 3 = >5 e p i s o d e s The mean s c o r e ± S . E . M . f o r each g roup i s shown i n T a b l e X I I . Of p a r t i c u l a r s i g n i f i c a n c e a r e t h e a c t i o n s o f D-600 and p r o p r a n o l o l . Whereas p r e t r e a t m e n t w i t h D-600 c o m p l e t e l y a b o l i s h e d v e n t r i c u l a r f i b r i l l a t i o n , p r o p r a n o l o l - t r e a - t e d a n i m a l s c o n s i s t e n t l y had g r e a t e r t h an 5 e p i s o d e s o f t h i s a r r h y t h m i a d u r i n g t he i s c h a e m i c p e r i o d . T h i s i s s i g n i f i c a n t l y g r e a t e r t han t h e i n c i - dence o f f i b r i l l a t i o n i n t h e a n i m a l s wh i ch r e c e i v e d o n l y s a l i n e . C h l o r p r o - maz i ne a l s o s i g n i f i c a n t l y i n c r e a s e d t h e number o f e p i s o d e s ; howeve r , t h e f r e q u e n c y per an ima l was l e s s and t h e r e s u l t s more v a r i a b l e when compared t o p r o p r a n o l o l . I m i d a z o l e and p r a n o l i u m had no appa r en t e f f e c t on t h e mechan- ism o f t h i s . The f r e q u e n c y o f f i b r i l l a t i o n was no t s i m p l y r e l a t e d t o a l owe r s y s t o l i c p r e s s u r e i n t he p r o p r a n o l o l g roup because t he D-600 g roup had e q u i v a l e n t r e d u c t i o n s i n b l o o d p r e s s u r e and t h e i n c i d e n c e o f f i b r i l l a t i o n i n t h i s g roup was z e r o . T a b l e X I . T i s s u e wa t e r c o n t e n t i n h e a r t s s u b j e c t e d t o 20 m i nu t e s o f l i g a t i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t T r ea tmen t n Water c o n t e n t {%) C o n t r o l 7 79 . 3 0 . 4 20 min 1 i g a t i o n 6 8 1 . 0 0 . 5 20 min l i g a t i o n , p r e t r e a t m e n t w i t h : d l - p r o p r a n o l o l HCI 7 8 0 . 0 ± 1.2 D-600 HCI 8 81 . 7 1.2 I m i d a z o l e 6 8 1 . 0 ± 0 .4 C h l o r p r o m a z i n e HCI 8 81 . 6 ± 0 . 4 P r a n o l i u m CI 6 8 0 . 8 ± 0 . 8 A l l v a l u e s a r e e x p r e s s e d as mean ± S . E . - 6 4 - Table XII. Incidence of ventricular f i b r i l l a t i on in rabbits subjected to 20 minutes of ischaemia with or without drug pretreatment Treatment n Incidence of f i b r i l l a t i on (arbitrary scale)— Control 8 0.0 ± 0.0 20 min 1igation 7 0.4 ± 0.2 20 min l igat ion, pretreatment with: dl-Propranolol HCl 7 3.0 ± 0. (£ D-600 HCl 8 0.0 * 0.<£ Imidazole 7 0.7 ± 0.4 Chlorpromazine HCl 8 2.1 * 0.4^ Pranolium Cl 7 0.4 ± 0.2 Al l values are mean ± S.E. - scale: 0 = 0 episodes of f i b r i l l a t i on during 1igation 1 = 1 episodes of " II II 2 = 2-5 episodes of II H 3 = >5 episodes of " II n Signif icantly different from the expected value after 20 min ligation (0.4) at P < 0.05 using chi-squared test for k independent samples. - 65 - I I I . 40 M i n u t e L i g a t i o n and 60 M i n u t e R e p e r f u s i o n w i t h D-600 and P r o p r a n o l o l P r e t r e a t m e n t T h i s s t u d y c on ce rned t h e a b i l i t y o f D-600 t o a t t e n u a t e t he a d d i t i o n a l damage caused by 60 m i nu t e s o f r e p e r f u s i o n a f t e r 40 min o f i s c h a e m i a i n v i ew o f t h i s d r u g ' s s a l u t a r y e f f e c t s on 20 m i nu t e s o f i s c h a e m i c i n j u r y . F u r t h e r - more , be cause r a p i d a c c u m u l a t i o n o f c a l c i u m has been shown t o a c c e l e r a t e i s c h a e m i c damage, t h e c a l c i u m c h a n n e l - b l o c k i n g p r o p e r t i e s o f D-600 m igh t be e x p e c t e d t o p r o v i d e a d d i t i o n a l b e n e f i t d u r i n g t h e r e p e r f u s i o n p e r i o d . I n a d d i t i o n , many o f t h e mechanisms whereby t h e c a l c i u m channe l a n t a g o n i s t s a re t h o u g h t t o b e n e f i t t h e i s c h a e m i c h e a r t a r e s ha r ed by t h e b e t a - a d r e n e r g i c b l o c k e r , p r o p r a n o l o l . C o n s e q u e n t l y , we a l s o examined t h e a c t i o n s o f t h i s agen t on i s c h a e m i c / r e p e r f u s i o n damage u s i n g t h e same model o f m y o c a r d i a l i s c h a e m i a as i n ou r p r e v i o u s s t u d i e s ; howeve r , a l owe r dose o f t h i s d rug was u s e d . The a n i m a l s were r andom ly a l l o c a t e d i n t o t h e f o l l o w i n g g r o u p s : Sham-ope ra ted c o n t r o l (CONT) 40 min l i g a t i o n f o l l o w e d by 60 min o f r e p e r f u s i o n - s a l i n e p r e t r e a t m e n t (L+R) 40 min l i g a t i o n and 60 min r e p e r f u s i o n - 0 . 1 mg/kg D-600 HCI a d m i n i s t e r e d 20 min p r i o r t o l i g a t i o n (D-600) 40 min l i g a t i o n and 60 min r e p e r f u s i o n - 1.5 mg/kg p r o p r a n o l o l HCI a d m i n i s t e r e d 20 min p r i o r t o l i g a t i o n (PROP) M i t o c h o n d r i a l ATPase a c t i v i t y P r e v i o u s e x p e r i m e n t s have demons t r a t ed t h a t 60 min o f r e p e r f u s i o n r e s t o r e s 75% o f m i t o c h o n d r i a l ATPase a c t i v i t y a f t e r a 40 min l i g a t i o n and t h i s was c o n f i r m e d by t he p r e s e n t r e s u l t s (L+R; F i g u r e 8 ) . In t h e s e e x p e r - i m e n t s , t h e d e c r e a s e i n m i t o c h o n d r i a l a z i d e - s e n s i t i v e a c t i v i t y was p r e v e n t e d by p r e - t r e a t m e n t w i t h e i t h e r D-600 o r p r o p r a n o l o l . Drug p r e t r e a t m e n t - 66 - m a i n t a i n e d t h e m i t o c h o n d r i a l enzyme a c t i v i t y a t 100% o f c o n t r o l v a l u e s , i n d i c a t i n g s i g n i f i c a n t p r e s e r v a t i o n o f m i t o c h o n d r i a l membrane enzyme i n t e g - r i t y ( F i g u r e 8 ) . R e c a l l t h a t n e i t h e r D-600 nor p r o p r a n o l o l had any e f f e c t on t he enzyme a c t i v i t y i n dependen t o f t h e i r a c t i o n s on t h e r e p e r f u s e d myoca rd ium, i . e . , a d m i n i s t r a t i o n o f t h e d rugs t o t h e CONT group d i d no t change t h e b a s e l i n e enzyme a c t i v i t i e s . Sa r co l emma l N a + , K + - A T P a s e and K + - N P P a s e a c t i v i t i e s I s c h a e m i a - i n d u c e d d e c r e a s e s i n s a r co l emma l membrane-bound enzyme a c t i v i - t i e s were shown t o be l e s s e a s i l y r e v e r s e d by r e p e r f u s i o n compared t o t h e m i t o c h o n d r i a l ATPase . R e s u l t s f rom t h e p r e s e n t e x p e r i m e n t s f u r t h e r s u p p o r t t h e h y p o t h e s i s t h a t t h e sa rco lemma i s more s u s c e p t i b l e t o i r r e v e r s i b l e i s c h a e m i c damage i n t h a t enzyme a c t i v i t y i s m i n i m a l l y r e s t o r e d even a f t e r 60 min o f r e p e r f u s i o n . I n c o n t r a s t t o t h e b e n e f i c i a l e f f e c t s o f bo th d rugs on t h e m i t o c h o n d r i a l ATPase , t he s p e c i f i c a c t i v i t y o f t h e s a r co l emma l enzymes i s p r e s e r v e d o n l y by D-600 ( F i g u r e 9 ) . W i t h no d rug p r e - t r e a t m e n t ( L+R) , t h e N a + , K + - A T P a s e a c t i v i t y was o n l y 40% o f t h e v a l u e o f t h e CONT g r o u p , whereas D-600 p r e - t r e a t m e n t r e s u l t e d i n a g r e a t e r sa r co l emma l enzyme a c t i v i - t y - 73% o f t h e CONT v a l u e . A l t h o u g h t h e e f f e c t s o f p r o p r a n o l o l q u a l i t a - t i v e l y p a r a l l e l e d t h o s e o f D-600 ( a c t i v i t y = 60% o f CONT), t h e y d i d no t r e a c h s t a t i s t i c a l s i g n i f i c a n c e . However , p r o p r a n o l o l - t r e a t e d h e a r t s c o n s i s - t e n t l y r e q u i r e d a g r e a t e r number o f d e f i b r i l l a t i n g c o u n t e r s h o c k s i n c o m p a r i - son w i t h any o f t he o t h e r g roups wh i c h m igh t have o b s c u r ed a b e n e f i c i a l e f f e c t o f t h e d r u g . C o n s e q u e n t l y , a n o t h e r g roup o f s ham-ope ra t ed h e a r t s (n = 5) were g i v e n 10 c o u n t e r s h o c k s o v e r a p e r i o d o f 10 m i nu t e s (one pe r m i n u t e ) i n o r d e r t o a s s e s s t h e e x t e n t o f s h o c k - i n d u c e d t i s s u e damage. The low l e v e l s o f s t o r e d e n e r g y i n v o l v e d i n t h e D.C. c o u n t e r s h o c k s ( 0 . 5 W a t t - s e c ) a r e w e l l be low t h e t h r e s h o l d r e q u i r e d t o p r oduce t i s s u e damage - 67 - F i g u r e 8 . M i t o c h o n d r i a l a z i d e - s e n s i t i ve ATPase a c t i v i t y i n m i t o c h o n d r i a i s o a t e d f r om h e a r t s s u b j e c t e d t o a sham 40 min l i g a t i o n p l u s 60 min r e p e r f u - s i o n (CONT), 40 min l i g a t i o n p l u s r e p e r f u s i o n w i t h s a l i n e p r e t r e a t m e n t ( L+R) , 40 min l i g a t i o n p l u s r e p e r f u s i o n w i t h D-600 (D-600) o r p r o p r a n o l o l (PROP) p r e t r e a t m e n t . Ba r s r e p r e s e n t t h e mean ± S . E . f o r each g r oup , n = 8 f o r a l l g r o u p s . On l y L+R i s s i g n i f i c a n t l y d e c r e a s ed f rom CONT, # = no s i g n i f i c a n t d i f f e r e n c e f r om CONT a t P < 0 . 0 5 . - 68 - • ATPase C O N T L + R O - B O O P R O P F i g u r e 9 . N a + , K + - A T P a s e and K + - N P P a s e a c t i v i t y o f sa rco lemma i s o l a t e d f rom h e a r t s s u b j e c t e d t o 40 min l i g a t i o n f o l l o w e d by 60 min o f r e p e r f u s i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t . Ba r s r e p r e s e n t mean ± S . E . f o r each g r o u p , and n = 8-9 f o r each g r o u p . L+R and PROP s i g n i f i c a n t l y r educed f r om CONT. * = D-600 s i g n i f i c a n t l y d i f f e r e n t f r om L+R (P < 0 . 0 5 ) . - 6 9 - T a b l e X I I I . Membrane a c t i v i t i e s o f h e a r t s s u b j e c t e d t o 10 D.C. c o u n t e r s h o c k s o f 0 .5 W a t t - s e c each d u r i n g s h a m - 1 i g a t i o n and r e p e r f u s i o n . T r ea tmen t M i t o c h o n d r i a Sarco lemma a z i d e - s e n s i t i v e ATPase- 9- N a + K + - A T P a s e a K + - N P P a s e b C o n t r o l 206 ± 4 4 . 75 ± .79 1.09 ± .07 C o u n t e r s h o c k e d 199 ± 8 5 .58 ± .22 1.19 ± .18 A l l v a l u e s a r e e x p r e s s e d as mean ± S . E . , n=5 — enzyme a c t i v i t y = ymoles P i / h r / m g p r o t e i n . — enzyme a c t i v i t y = umoles n i t r o p h e n o l / h r / m g p r o t e i n . - 70 - 91 d i r e c t l y . The s a r co l emma l enzyme a c t i v i t i e s were u n a f f e c t e d by t h e c o u n t e r s h o c k s ( T ab l e X I I I ) , so t h a t t h e l a c k o f p r o t e c t i o n o f p r o p r a n o l o l was no t t h e r e s u l t o f an a r t i f a c t u a l d e c r e a s e caused by e xposu r e t o t h e d e f i b r i 1 l a t i n g c o u n t e r s h o c k s . ATP The n e g a t i v e i n o t r o p i s m and o f f - l o a d i n g t h a t r e s u l t s f r om a d m i n i s t r a t i o n o f bo th o f t h e s e d r ug s wou ld be e x p e c t e d t o d e c r e a s e t h e 0 2 c on sump t i on o f t h e normal and i s c h a e m i c h e a r t , t h e r e b y p r e s e r v i n g s t o r e s o f h i g h e ne r g y p h o s p h a t e s . T h i s , i n t u r n , s h o u l d be e x p e c t e d t o a l l o w t h e c e l l t o m a i n t a i n h o m e o s t a s i s w i t h a c o n c o m i t a n t p r o t e c t i o n o f o r g a n e l l e i n t e g r i t y . I n t e r e s t - i n g l y , d e s p i t e t h e b e n e f i c i a l e f f e c t o f t h e s e d rugs on membrane enzyme a c t i v i t i e s , n e i t h e r D-600 no r p r o p r a n o l o l s i g n i f i c a n t l y p r e s e r v e d m y o c a r d i a l l e v e l s o f ATP ( F i g u r e 1 0 ) . Marked h i g h ene r g y phospha te d e p l e t i o n o c c u r s 17 114 v e r y e a r l y i n i s c h a e m i c i n j u r y ' a nd , i n ou r e x p e r i m e n t s , u n t r e a t e d h e a r t s (L+R) r e t a i n o n l y 6% o f c o n t r o l ATP l e v e l s , i . e . , o v e r 90% o f t h e ATP has been l o s t . The ATP l e v e l s o f t h e D-600 and PROP g roups were no t s i g n i f - i c a n t l y i n c r e a s e d o v e r t h o s e o f t h e u n t r e a t e d (L+R) a n i m a l s a l t h o u g h D-600 p r e t r e a t m e n t p roduced a s l i g h t l y g r e a t e r e f f e c t when compared w i t h p r o p r a n o l o l a t t a i n i n g 15% and 9% of CONT v a l u e s , r e s p e c t i v e l y . Water c o n t e n t I n t r a c e l l u l a r oedema can r e s u l t f r om t h e r e p e r f u s i o n o f damaged myoca r - d i a l t i s s u e and h e a r t s f r om t h e L+R, D-600 and PROP g roups a l l had some wa t e r g a i n upon r e p e r f u s i o n when compared t o t h e CONT an ima l s ( a s s e s s e d by g r a v i m e t r i c a n a l y s i s ) . However , o n l y t h e p r o p r a n o l o l - t r e a t e d h e a r t s showed s t a t i s t i c a l l y s i g n i f i c a n t i n c r e a s e s i n t o t a l t i s s u e wa t e r ( T ab l e X I V ) . The wa t e r c o n t e n t o f t h e CONT h e a r t s was 8 0 . 3 ± 0.57%, i n c o n t r a s t t o t h e PROP group - 84 .7 ± 1.2%. The d e f i b r i 1 l a t o r y c o u n t e r s h o c k s do no t a c coun t f o r - 71 - F i g u r e 1 0 . ATP c o n t e n t o f h e a r t s s u b j e c t e d t o 40 min o f l i g a t i o n f o l l o w e d by 60 min o f r e p e r f u s i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t . Ba r s r e p r e s e n t mean ± S . E . f o r each g r o up , and n = 8-9 f o r each g r o u p . A l l g roups a re s i g n i f i c a n t l y d e c r e a s e d f r om CONT a t P < 0 . 0 5 . - 7 2 - T a b l e X I V . T i s s u e wa t e r c o n t e n t i n h e a r t s s u b j e c t e d t o 40 min o f l i g a t i o n and 60 min o f r e p e r f u s i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t T r ea tmen t n Water c o n t e n t {% ) CONT 8 8 0 . 3 0 . 6 L+R 7 8 2 . 3 ± 1.3 D-600 + L+R 8 82 . 6 0 . 8 PROP + L+R 8 8 4 . 7 ± 1 . 2* SHOCKS + L+R 5 81 . 6 ± 0 .9 A l l v a l u e s a r e e x p r e s s e d as mean ± S . E . S i g n i f i c a n t l y i n c r e a s e d above c o n t r o l v a l u e s a t P < 0 . 0 5 . - 73 - t he e l e v a t e d wa t e r c o n t e n t o f t h e s e h e a r t s because t h e wa te r c o n t e n t i n t he SHOCKS g roup was no t s i g n i f i c a n t l y i n c r e a s e d above CONT v a l u e s ( 8 1 . 6 ± 0 .79%) . The p r e s en ce o f l a r g e membrane-bound v a c u o l e s w i t h i n t he musc l e f i b r e s seen i n t h e e l e c t r o n m i c r o g r a p h s o f t h e s e t i s s u e samp les a l s o s ugge s t t h a t t h e p r o p r a n o l o l - t r e a t e d h e a r t s had wa t e r g a i n upon r e p e r f u s i o n ( see F i g u r e 1 9 ) . Ion l e v e l s I n t a c t membrane s t r u c t u r e i s i n t i m a t e l y c o n n e c t e d w i t h t h e ma i n t enance o f i o n l e v e l s w i t h i n t h e m y o c a r d i a l c e l l . T h e r e f o r e , d rug i n t e r v e n t i o n s wh i c h p r e s e r v e membrane i n t e g r i t y s h o u l d r educe t he p a s s i v e f l u x o f i o n s down t h e i r c o n c e n t r a t i o n g r a d i e n t s o c c u r r i n g as a r e s u l t o f i s c h a e m i c / r e p e r - f u s i o n i n j u r y . In a d d i t i o n , bo th D-600 and p r o p r a n o l o l wou ld be e x p e c t e d t o r e du ce t h e i n f l u x o f c a l c i u m t h r o u g h t h e I^j c h a n n e l s : D-600 by a d i r e c t b l o c k a d e o f t h e s l ow c h a n n e l s and p r o p r a n o l o l by an an t agon i sm o f e - a d r e n e r - g i c r e c e p t o r a c t i v a t i o n . C a l c i u m - P r e - t r e a t m e n t o f r a b b i t s w i t h e i t h e r D-600 or d l - p r o p r a n o l o l s i g n i f i c a n t l y r educed t h e c a l c i u m e n t r y a s s o c i a t e d w i t h r e p e r f u s i o n i n j u r y ( F i g u r e 1 1 ) . CONT c a l c i u m l e v e l s were 12 .2 ± 2 . 0 nmoles/mg d r y we i g h t (n = 8 ) . L i g a t i o n f o l l o w e d by r e p e r f u s i o n r e s u l t e d i n a 4 - f o l d i n c r e a s e i n c a l c i u m l e v e l s t o 4 8 . 5 ± 5 . 9 , i n c o n t r a s t t o t he modera te i n c r e a s e s seen i n t h e D-600 and PROP h e a r t s t o l e v e l s o f 23 . 2 ± 2 .5 and 2 9 . 8 ± 6 .2 n m o l e s / d r y w e i g h t , r e s p e c t i v e l y . Thu s , D-600 and p r o p r a n o l o l a d m i n i s t r a t i o n r e s u l t e d i n a 50% r e d u c t i o n i n t h e amount o f c a l c i u m t a k en up by t h e i s c h a e m i c myoca r - d i um . Sodium - In c o n t r a s t t o t he s i m i l a r e f f e c t s o f t he d rugs on r e d u c i n g t he c e l l c a l c i u m c o n c e n t r a t i o n , o n l y D-600 a d m i n i s t r a t i o n was e f f e c t i v e i n s i g n i f i c a n t l y p r e v e n t i n g sod ium a c c u m u l a t i o n ( F i g u r e 1 1 ) . T h i s may be - 74 - F i g u r e 1 1 . Ca , Na , and Mg l e v e l s o f h e a r t s s u b j e c t e d t o 40 min o f l i g a t i o n p l u s 60 min o f r e p e r f u s i o n w i t h o r w i t h o u t d rug p r e t r e a t m e n t . Ba r s r e p r e s e n t mean ± S . E . , and n = 8 -9 f o r each g r o u p . Ca - * = s i g n i f i - c a n t l y r educed f r om L+R v a l u e . Na -D -600 g roup i s no t s i g n i f i c a n t l y d i f f e r e n t f r om c o n t r o l and L+R and PROP a re s i g n i f i c a n t l y i n c r e a s e d f rom c o n t r o l l e v e l s (P < 0 . 0 5 ) . Mg -same as f o r N a + . - 75 - + + a t t r i b u t a b l e t o e i t h e r i t s s u p e r i o r p r e s e r v a t i o n o f Na ,K -ATPase a c t i v - i t y o r s a r co l emma l i n t e g r i t y o r b o t h . Magnes ium - The e f f e c t s o f t h e d rugs on t h e magnes ium c o n t e n t s o f t h e s e h e a r t s were q u a l i t a t i v e l y s i m i l a r t o t h o s e o b t a i n e d f o r t h e sod ium i o n ( F i g u r e 1 1 ) . R e p e r f u s i o n i n j u r y r e s u l t s i n a l o s s o f magnesium ( see L+R) wh i ch was p r e v e n t e d by D-600 p r e t r e a t m e n t bu t no t by p r e t r e a t m e n t w i t h p r o p r a n o l o l . P o t a s s i u m - T i s s u e l e v e l s o f p o t a s s i u m d i d no t d i f f e r f rom c o n t r o l , a l t h o u g h t h e l a r g e r e r r o r seen i n t h e t r e a t m e n t g r o u p s , i n c ompa r i s on w i t h t h e c o n t r o l , may have masked s u b t l e changes i n p o t a s s i u m c o n t e n t . G i v en t h e + + s i g n i f i c a n t d e c r e a s e i n sa r co l emma l Na ,K -ATPase a c t i v i t y due t o r e p e r f u s i o n i n j u r y , one m igh t have e x p e c t e d t o see a s i g n i f i c a n t d e c r e a s e i n t h e p o t a s s i u m c o n c e n t r a t i o n s . However , i s c h a e m i a - i n d u c e d changes i n t i s s u e l e v e l s o f K + (as opposed t o changes i n t h e e x t r a c e l l u l a r c o n c e n t r a t i o n o f t h i s i o n ) have been shown by o t h e r i n v e s t i g a t o r s t o be o f a much s m a l l e r 29 115 magn i t ude t han t h o s e o f sod ium o r c a l c i u m . ' SDS -ge l e l e c t r o p h o r e s i s The i r r e v e r s i b l e d e c r e a s e s i n enzyme a c t i v i t y and a l t e r a t i o n s i n myo ca r - d i a l i o n measurements s ugge s t e d t h e p o s s i b l y t h a t t h e membrane p r o t e i n and p o l y p e p t i d e p r o f i l e s o f t h e s e i s c h a e m i c h e a r t s m i gh t be a l t e r e d . U s i n g more q u a n t i t a t i v e methods t h e r e was s t i l l no d e t e c t a b l e change i n t h e p o l y p e p t i d e p r o f i l e s o f t h e sa rco lemma f r om any o f t h e g r o u p s . However , an a l t e r a t i o n was no t ed i n t h e m i t o c h o n d r i a l p r o t e i n p r o f i l e o f t h e L+R g r o u p . The r e s u l t s o f t h e d e n s i t o m e t r i c s c an s o f t h e g e l s c o n t a i n i n g t h e m i t o c h o n d r i a l membrane f r a c t i o n a r e shown i n F i g u r e 1 2 . S o l u b i l i z e d p r o t e i n f r om m i t o - c h o n d r i a l membranes f rom t h e i s c h a e m i c a r e a show a s i g n i f i c a n t d e c r e a s e i n - 76 - 4 0 M I N L I G L + R C O N T F r a c t i o n a l P e a k A r e a ( P e a k a r e a X / P e a k a r e a 6 x 1 0 0 ) G R O U P n f C 0 E F G 1 F O N T 5 1 6 5 * 1 6 2 4 * ? . ? 9 . 4 . 3 . 7 5 8 « 2 . 8 2 0 * 1 . 1 1 6 • 2 . 1 L * p 5 1 6 4 . 3 8 5 . 0 * 2 . 2 * 8 . 2 * 1 . 3 5 5 * 3 . 8 1 6 * 3 . 7 1 5 * 2 . 9 D - 6 0 0 4 1 8 3 * 1 7 9 . 5 * 2 . 9 * 6 . 0 * 1 . 9 5 3 * 4 . 2 1 7 * 3 . 3 9 . 8 * 1 . 1 ' 1 8 9 * 1 . 3 3 . 2 * 0 . 7 * 1 1 * 3 . 1 5 0 * 5 . 5 2 1 * 3 . 5 1 5 * 1 . 3 F i g u r e 1 2 . S D S - g e l e l e c t r o p h o r e s i s o f m i t o c h o n d r i a l f r a c t i o n s i s o l a t e d f r o m h e a r t s s u b j e c t e d t o 4 0 m i n l i g a t i o n o n l y ( 4 0 MIN L I G ) , 4 0 m i n l i g a t i o n p l u s 6 0 m i n r e p e r f u s i o n ( L + R ) , sham 4 0 m i n l i g a t i o n a n d r e p e r f u s i o n ( C O N T ) . P r o t e i n p r o f i l e s a r e shown a t t h e t o p l e f t o f t h e f i g u r e . T h e a r r o w s show t h e b a n d w h i c h i s r e d u c e d a f t e r r e p e r f u s i o n ( C ) on b o t h t h e g e l a n d t h e d e n s i t o m e t r i c s c a n ( t o p r i g h t ) . T h e t a b l e a t t h e b o t t o m g i v e s t h e n o r m a l i z e d a r e a s u n d e r t h e p e a k s o f t h e s c a n f o r h e a r t s s u b j e c t e d t o l i g a t i o n a n d r e p e r f u s i o n w i t h o r w i t h o u t d r u g p r e t r e a t m e n t . N e i t h e r D-600 o r PROP p r e v e n t e d t h e l o s s o f b a n d C. - 77 - t he a rea o f one o f t he p r o t e i n peaks d e s p i t e t h e equa l amounts o f p r o t e i n a p p l i e d to t h e g e l . N e i t h e r D-600 no r p r o p r a n o l o l p r e t r e a t m e n t p r e v e n t e d t he l o s s o f t h i s p r o t e i n , whose m o l e c u l a r we i g h t was e s t i m a t e d t o be 55 ,000 by c o n c u r r e n t e l e c t r o p h o r e s i s o f p r o t e i n s t a n d a r d s o f known m o l e c u l a r w e i g h t . As s t a t e d i n the Methods s e c t i o n , t h e c o n t a m i n a t i o n o f the m i t o c h o n d r i a l membranes w i t h sarco lemma was c o n s i s t e n t l y 3% f o r a l l g r o u p s , t h e r e b y e l i m i - n a t i n g t he p o s s i b i l i t y t h a t d i f f e r e n t i a l s a r co l emma l c o n t a m i n a t i o n c o u l d a c c oun t f o r t h e ob s e r v ed d e c r e a s e i n a p a r t i c u l a r p o l y p e p t i d e . C o n t r a c t i 1 i t y An i n d i r e c t measure o f t he c a r d i a c c o n t r a c t i l e f o r c e was o b t a i n e d f rom t he d i f f e r e n t i a t e d p r e s s u r e wave s i g n a l o f t h e l e f t v e n t r i c l e — d P / d t m a x = t he 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 p r e s s u r e d e ve l o pmen t . F i g u r e 13 shows t h e e f f e c t o f d rug t r e a t m e n t , l i g a t i o n and r e p e r f u s i o n on d P / d t m a x as a p e r c e n t a g e o f t h e o r i g i n a l c o n t r a c t i l i t y where i n i t i a l d P / d t m a x = 100%. Bo th d r ug s r e du ced c o n t r a c t i l e f o r c e p r i o r t o l i g a t i o n : D-600 t o 55% o f p r e - d r u g v a l u e and p r o p r a n o l o l t o 80% o f p r e - d r u g d P / d t m a x . I n t e r e s t i n g l y , d e s p i t e t h e a b i l i t y o f t h e d rugs t o m a i n t a i n s u b c e l l u l a r s t r u c t u r a l i n t e g r i t y , t h e r e was no improvement i n c o n t r a c t i l i t y upon r e p e r f u s i o n o f i s c h a e m i c h e a r t s . I n a l l g roups t h e d P / d t v r e a ched III O A o n l y 50-60% o f t h e CONT d P / d t m f l x , i . e . , t h e r e was no s i g n i f i c a n t d i f f e r - ence i n t he c o n t r a c t i l i t y between h e a r t s f r om t he L+R, D-600 and PROP g roups a f t e r 60 min o f r e p e r f u s i o n . D-600 d i d seem t o have a s l i g h t l y s a l u t a r y e f f e c t on c o n t r a c t i l i t y when compared w i t h t h e o t h e r t r e a t m e n t g r o u p s , bu t t h i s was no t s t a t i s t i c a l l y s i g n i f i c a n t . T a b l e XV shows t h a t t h e s y s t o l i c a r t e r i a l p r e s s u r e s o f t h e L+R, D-600 and PROP g roups were no t s i g n i f i c a n t l y d i f f e r e n t so t h a t changes i n a f t e r l o a d p r o b a b l y d i d no t a f f e c t t h e ou t come . A c o n f o u n d i n g f a c t o r i n t h i s e x p e r i m e n t a r i s e s when one c o n s i d e r s t h a t t h e - 78 - S O 4 0 B O IO S O 3 0 4 0 - S O O r t r D R U G L I G A T I O N R E P E R F LIS I O N T I M E ( I V I I I M ) F i g u r e 1 3 . P e r c e n t i n i t i a l ( d P / d t ) r n a x o f h e a r t s d u r i n g t h e p e r i o d o f d rug t r e a t m e n t , l i g a t i o n and r e p e r f u s i o n . Four g roups a r e shown: • — • (CONT), A- A ( D - 6 0 0 ) , o-• -o (PROP) and A — - A ( L+R) . No s i g n i f i c a n t d i f f e r e n c e i n c o n t r a c t i l i t y was f ound between t h e L+R, D-600 and PROP g roups a t t he end o f t h e r e p e r f u s i o n p e r i o d . - 7 9 - T a b l e XV. S y s t o l i c b l o o d p r e s s u r e a f t e r v a r i o u s p e r i o d s o f r e p e r f u s i o n f o l l o w i n g 40 m i nu t e s o f c o r o n a r y l i g a t i o n B l ood p r e s s u r e (mm Hg) M i n u t e s o f r e p e r f u s i o n T r e a tmen t n 3 10 20 60 C o n t r o l 8 109 ± 4 109 ± 4 107 ± 4 104 ± 5 L+R 8 64 5 69 ± 4 69 ± 3 70 ± 5 D-600 8 63 6 66 ± 6 74 ± 6 74 ± 4 PROP 8 64 4 69 ± 5 70 ± 6 73 ± 6 A l l v a l u e s a r e e x p r e s s e d as mean ± S . E . - 80 - n e g a t i v e i n o t r o p i c a c t i o n s o f t h e s e agen t s may d i r e c t l y a f f e c t t h e c o n t r a c - t i l i t y o f t h e h e a r t s a t t h e end o f t h e r e p e r f u s i o n p e r i o d . I n o r d e r t o d e t e r m i n e t h e t i m e c o u r s e o f t h e n e g a t i v e i n o t r o p i c e f f e c t o f D-600 i n ou r m o d e l , we a d m i n i s t e r e d D-600 (n = 5) t o sham-ope ra t ed r a b b i t s u s i n g t h e same p r o t o c o l as d e s c r i b e d i n t he Methods s e c t i o n . The s t u d y showed t h a t d P / d t m , „ had r e t u r n e d t o c o n t r o l v a l u e s 40 m i nu t e s a f t e r a d m i n i s t r a t i o n , max Thu s , f o r D-600 a t l e a s t , t h e r e d u c t i o n i n c o n t r a c t i l i t y a t t h e end o f t he r e p e r f u s i o n p e r i o d was p r o b a b l y no t a r e s u l t o f d i r e c t e f f e c t s o f t h e d rug on t h e myocard ium ( d a t a no t shown) . However , i t has been p o s t u l a t e d t h a t c a l c i u m c h a n n e l - b l o c k a d e i s more e f f e c t i v e on d e p o l a r i z e d , i s c h a e m i c t i s s u e . * ^ T h e r e f o r e , t h e r e v e r s a l o f t he n e g a t i v e i n o t r o p i c e f f e c t s o f D-600 i n t h e s e sham-ope ra t ed a n i m a l s may have been more r a p i d t h an i n t h e i s c h a e m i c r a b b i t s . I f D-600 was s e l e c t i v e l y d e p r e s s i n g t he c o n t r a c t i l i t y o f t h e i s c h a e m i c z o n e , t h i s e f f e c t may have masked any improvement in. dP/dW D e s p i t e t h e r educed dose o f p r o p r a n o l o l i n t h e s e an ima l s when compared t o t h e 20 min l i g a t i o n s t u d y ( 1 . 5 mg/kg v s . 3 . 0 mg / kg ) , t h i s g roup s t i l l had a g r e a t l y i n c r e a s e d i n c i d e n c e o f f i b r i l l a t i o n t h a t c h a r a c t e r i s t i c a l l y began a t a p p r o x i m a t e l y 12 min p o s t - l i g a t i o n ( s i m i l a r t o t h e L+R a n i m a l s ) and c o n t i n u e d as an u n s t a b l e f l u c t u a t i o n o f s i n u s rhy thm and f i b r i l l a t i o n f o r a p p r o x i m a t e l y 10 m i n . T h i s a r r h y t h m i a was no t e a s i l y r e v e r s e d by c o u n t e r - s h o c k ( i n c o n t r a s t t o t he L+R a n i m a l s ) . S i m i l a r t o t h e p r e v i o u s s t u d y , t h e f i b r i l l a t i o n was p r o b a b l y no t t h e r e s u l t o f low p e r f u s i o n p r e s - s u r e because t h e D-600 group had e q u a l l y low s y s t o l i c p r e s s u r e s and none o f t h e h e a r t s i n t h e D-600 group f i b r i l l a t e d . - 81 - U l t r a s t r u c t u r e A p o t e n t i a l p r ob l em i n i n t e r p r e t i n g u l t r a s t r u c t u r a l d a t a a r i s e s when t he e x p e r i m e n t a l p r o c e d u r e c r e a t e s inhomogeneous t i s s u e damage. An advan tage o f u s i n g t h e r a b b i t model i s t h a t , u n l i k e t he dog , c o r o n a r y l i g a t i o n r e s u l t s i n an i s c h a e m i c zone w i t h a c l e a r l y d e f i n e d b o r d e r w i t h o u t i n t e r d i g i t a t i o n o f 118 normal t i s s u e and i s c h a e m i c t i s s u e . T h i s u n i f o r m i t y o f damage was c o n f i r m e d i n ou r samp les o f t h i c k s e c t i o n s examined by l i g h t m i c r o s c o p y ( F i g u r e 14 and 1 5 ) . The deg ree o f i s c h a e m i c membrane damage d e t e rm i n ed by t he b i o c h e m i c a l measurements l i s t e d above was compa rab l e w i t h t h e appea rance o f t h e h e a r t s i n t h e e l e c t r o n m i c r o g r a p h s . Sham-ope ra ted c o n t r o l h e a r t s showed t i g h t l y - packed r e g u l a r m i t o c h o n d r i a w i t h dense i n t r a m i t o c h o n d r i a l m a t r i c e s c o n t a i n - i n g s m a l l d i s t i n c t g r a n u l e s a l o n g t h e c r i s t a e and no e v i d e n c e o f oedema f o r m a t i o n . The s a r c o t u b u l a r s y s tem appeared norma l ( F i g u r e s 1 6a , 1 6 b ) . In a d d i t i o n , t h e sa r co l emma l membrane and g l y c o c a l y x were no t s e p a r a t e d f rom t h e u n d e r l y i n g s t r u c t u r e s and i n t e r c a l a t e d d i s k s were c l o s e l y apposed ( F i g u r e 1 6 c ) . L+R h e a r t s showed g r e a t d i s r u p t i o n w i t h c l e a r i n g o f t he m i t o c h o n d r i a l s pace and f r a g m e n t a t i o n o f t h e c r i s t a e and t h e r e were f o c i where c omp l e t e d e s t r u c t i o n o f t he m i t o c h o n d r i o n had t a k en p l a c e , as was e v i d e n t by b roken o u t e r membranes ( F i g u r e 1 7 a ) . I n a d d i t i o n , l i g a t i o n f o l l o w e d by r e p e r f u s i o n r e s u l t e d i n t he appea rance o f amorphous i n t r a m i t o c h o n d r i a l p r e c i p i t a t e s , wh i ch may be c a l c i u m s a l t s o r p o s s i b l y d ena t u r ed p r o t e i n . M y o f i b r i l l a r d e g e n e r a t i o n i s e v i d e n t and t h e s a r come re s a re a b n o r m a l l y r e l a x e d g i v i n g r i s e t o p r om inen t I - b and s ( F i g u r e 1 7 b ) . F i g u r e 17c shows t h e a r ea s where t h e g l y c o c a l y x had l i f t e d e n t i r e l y away f r om t he c e l l s u r f a c e . The re i s a l s o s e v e r e c l ump i ng o f n u c l e a r c h r o m a t i n . - 82 - F i g u r e 14a) L i g h t m i c r o g r a p h o f t h i c k s e c t i o n f r om CONT t i s s u e . X 2 6 0 . b) Pho t og r aph (as above) o f s e c t i o n o f l i g a t e d and r e p e r f u s e d t i s s u e (L+R) T i s s u e has u n i f o r m appea rance i n bo th c a s e s . Note t he appa ren t l o s s o f m y o f i b r i l s i n ( b ) . - 83 - F i g u r e 1 5 a ) L i g h t m i c r o g r a p h o f l i g a t e d a n d r e p e r f u s e d t i s s u e p r e t r e a t e d w i t h D-600. S t a i n i n g was m o r e d i f f i c u l t i n t h i s s e c t i o n . X 2 6 0 . b ) L i g h t m i c r o g r a p h ( a s a b o v e ) e x c e p t t h a t t h i s h e a r t was p r e t r e a t e d w i t h p r o p r a n o l o l . F a i r l y h o m o g e n o u s damage t o t h e t i s s u e . C o n t r a c t i o n b a n d s a r e a p p a r e n t i n ( b ) . - 84 - F i g u r e 16 . (CONT) a) E l e c t r o n m i c r o g r a p h o f c o n t r o l t i s s u e showing n o r m a l , t i g h t l y - p a c k e d m i t o c h o n d r i a c o n t a i n i n g s m a l l , dense g r a n u l e s . S a r c o t u b u l a r sys tem i s n o r m a l . M y o f i b r i l s do no t appear t o be a b n o r m a l l y c o n t r a c t e d o r r e l a x e d and no m y o f i b r i l l a r d e g e n e r a t i o n i s a p p a r e n t . X 3 0 , 0 0 0 . b) C o n t r o l h e a r t showing a l a r g e normal n u c l e u s ( c e n t r e ) w i t h d i f f u s e c h r o m a t i n . Sarco lemma and g l y c o c a l y x a r e a t t a c h e d t o t h e u n d e r l y i n g s t r u c t u r e . M i t o c h o n d r i a have dense m a t r i c e s and unbroken c r i s t a e . The da rk m y e l i n f i g u r e s a r e a r t i f a c t s p roduced by t h e r u t h e n i u m r e d s t a i n . X 6 , 0 0 0 . c ) ( t o p ) H i g h e r m a g n i f i c a t i o n o f t h e sa rco lemma and g l y c o c a l y x . X 2 0 , 0 0 0 . (bo t tom) Normal i n t e r c a l a t e d d i s k o f a c o n t r o l h e a r t . No s e p a r a t i o n o f t he c e l l s i s a p p a r e n t . X 2 0 , 0 0 0 . - 85 - -f fi.   - 88 - F i g u r e 1 7 . (L+R) a) E l e c t r o n m i c r o g r a p h o f h e a r t t h a t r e c e i v e d o n l y s a l i n e p r e t r e a t m e n t b e f o r e 40 min l i g a t i o n and 60 min r e p e r f u s i o n . M y o f i b r i l l a r damage i s e v i d e n t . M i t o c h o n d r i a a r e g r o s s l y d i s r u p t e d and some have been e n t i r e l y l o s t . I n t r a c e l l u l a r oedema may be r e s p o n s i b l e f o r t h e i n c r e a s e i n i n t r a m y o f i b r i 1 1 a r s p a c e . The i n t e r c a l a t e d d i s k ( t op r i g h t c o r n e r ) appea r s n o r m a l . X6000 . b) H i g h e r m a g n i f i c a t i o n show ig t he p r e s en ce o f amorphous i n t r a m i t o c h o n d r i a l d e n s i t i e s and r e l a x e d m y o f i b r i l s . X 3 0 , 0 0 0 . c ) In t h i s p h o t o m i c r o g r a p h , t h e s a r c o l e m m a / g l y c o c a l y x has p u l l e d away f r om t h e u n d e r l y i n g s t r u c t u r e s . The r e i s a s e v e r e l y damaged n u c l e u s w i t h c lumped c h r o m a t i n shown i n t h e c e n t r e o f t he page . M y o f i b r i l l a r l o s s i s a p p a r e n t . X 6 , 0 0 0 .    - 92 - Samples f r om t h e D-600 t r e a t e d h e a r t s showed a marked improvement , when compared w i t h t h e u n t r e a t e d h e a r t s w i t h d r a m a t i c a l l y l e s s m i t o c h o n d r i a l d e s t r u c t i o n and no s e p a r a t i o n o f t h e sa r co l emma l membrane f r om t h e u n d e r l y - i n g s t r u c t u r e s ( F i g u r e 18a and 1 8 b ) . The r e was a n o t i c e a b l e c l e a r i n g o f t h e m i t o c h o n d r i a l m a t r i x i n some c a s e s , bu t t h e o v e r a l l i n t e g r i t y was w e l l p r e s e r v e d . I n agreement w i t h t h e d e c r e a s e d c a l c i u m l e v e l s i n t h e s e h e a r t s , t h e r e appea red t o be f ewe r p r e c i p i t a t e s i n t he m i t o c h o n d r i a o f a n ima l s p r e - t r e a t e d w i t h D-600 a l t h o u g h m y o f i b r i l c o n t r a c t i o n and r e l a x a t i o n was no t e n t i r e l y normal ( F i g u r e 1 8 c ) . The p r o p r a n o l o l - t r e a t e d r a b b i t s a l s o e x h i b i t e d l e s s m y o c a r d i a l damage f rom l i g a t i o n and r e p e r f u s i o n t h an u n t r e a t e d a n i m a l s ( F i g u r e 1 9 a , 1 9 b ) . The ma i n t enance o f s t r u c t u r a l i n t e g r i t y appea red v e r y s i m i l a r t o t h a t o f the D-600 h e a r t s , i . e . , n e a r - n o r m a l m i t o c h o n d r i a l s t r u c t u r e , u n d i s r u p t e d s a r c o - lemmal membrane, no i n t r a - m i t o c h o n d r i a l d e n s i t i e s . However , more c o n t r a c - t i o n bands appea red i n t h i s t i s s u e . The f e a t u r e o f t h e s e h e a r t s no t seen i n any o f t he o t h e r g roups was t h e p r e s en ce o f membrane-bound v a c u o l e s a l o n g t h e Z - l i n e s ( F i g u r e 19c) i m p l y i n g an a b n o r m a l i t y o f t h e s a r c o t u b u l a r s y s t em and t h i s was c o n s i s t e n t w i t h t he i n c r e a s e d t i s s u e wa t e r i n t he p r o p r a n o l o l - t r e a t e d h e a r t s . The q u a l i t a t i v e a n a l y s i s was c o n f i r m e d by t he more o b j e c t i v e a s sessmen t o f s t r u c t u r a l i n t e g r i t y . These r e s u l t s a r e summar ized i n T a b l e XVI and t h e s e demons t r a t e a s i m i l a r l e v e l o f t i s s u e p r e s e r v a t i o n as t h a t e s t i m a t e d by b i o c h e m i c a l me thods . - 93 - F i g u r e 1 8 . (D-600) a) P h o t o m i c r o g r a p h o f D-600 p r e t r e a t e d 1 i g a t - ed / r epe r f used h e a r t . E s s e n t i a l l y normal m i t o c h o n d r i a l s t r u c t u r e w i t h some c l e a r i n g o f m a t r i c e s and o c c a s i o n a l l o s s o f t h i s o r g a n e l l e ( see m y e l i n f i g u r e , bo t t om r i g h t ) . Nu c l e u s e s s e n t i a l l y undamaged and m y o f i b r i l l a r d e g e n e r a t i o n i s no t a p p a r e n t . Amorphorus i n t r a m i t o c h o n d r i a l d e n s i t i e s a re no t i n e v i d e n c e . X 3 0 , 0 0 0 . b) Lower m a g n i f i c a t i o n o f D-600 h e a r t . Normal i n t e r c a l a t e d d i s k appea r s i n t h e c e n t r e o f t h e p h o t o g r a p h . M i t o c h o n d r i a l s t r u c t u r e and numbers a r e s i m i l a r t o c o n t r o l h e a r t s . M y o f i b r i l s a r e no t d eg r aded ; howeve r , a r ea s o f dense c o n t r a c t i o n and r e l a x a t i o n e x i s t w i t h i n t h e same m y o f i b r i l l a r b u n d l e . X 6 , 0 0 0 . c ) H igh m a g n i f i c a t i o n show ing m i t o c h o n d r i a w i t h s l i g h t l y c l e a r e d m a t r i c e s . The i n t e r c a l a t e d d i s k i s a b n o r m a l l y w i dened on t h e r i g h t s i d e o f t he p h o t o m i c r o g r a p h . X 3 0 , 0 0 0 .  - 95 -  - 97 - F i g u r e 1 9 . (PROP) a) E l e c t r o n m i c r o g r a p h o f p r o p r a n o l o l p r e t r e a t e d h e a r t s u b j e c t e d t o 40 min l i g a t i o n f o l l o w e d by r e p e r f u s i o n . M y o f i b r i l s and m i t o c h o n d r i a appea r f a i r l y norma l e x c e p t t h e r e a re a few amorphous d e n s i t i e s i n some m i t o c h o n d r i a ( t o p r i g h t c o r n e r ) . The i n t e r c a l a t e d d i s k ( bo t t om l e f t ) i s n o r m a l . The s a r c o l e m m a / g l y c o c a l y x i s no t d e t a ched f r om u n d e r l y i n g s t r u c t u r e s . The t e r m i n a l c i s t e r n a e o f t h e s a r c o t u b u l a r s y s t em appear d i l a t e d . X 3 0 . 0 0 0 . b) Lower m a g n i f i c a t i o n wh i c h shows some c l e a r i n g o f m i t o c h o n d r i a l m a t r i c e s and p r om inen t c o n t r a c t i o n b and s . X 6 , 0 0 0 . c ) H i g h e r m a g n i f i c a t i o n o f p r o p r a n o l o l - t r e a t e d h e a r t s show ing r e l a t i v e l y norma l m i t o c h o n d r i a l s t r u c t u r e . P r om inen t d i l a t i o n o f c i s t e r n a e i s seen a t t he Z - l i n e . X 6 0 . 0 0 0 .  - 99 -  T a b l e X V I . O b j e c t i v e a s s e s s m e n t ! o f u l t r a s t r u c t u r a l i n t e g r i t y o f h e a r t s s u b j e c t e d t o i s c h a e m i a / r e p e r f u s i o n i n j u r y w i t h o r w i t h o u t d rug p r e t r e a t m e n t T r ea tmen t M i t o c h o n d r i a l M i t o c h o n d r i a l Sarco lemma& M y o f i b r i l I n t e r c a l a t e d Abnormal Membranes D e n s i t i e s G l y c o c a l y x I n t e g r i t y D i s k C o n t r a c t i o n o r R e l a x a t i o n C o n t r o l 0 . 4 ± 0 . 1 ^ 0 . 3 ± 0 .3 0 .2 ± 0.3 0 . 3 ± 0 . 3 0 0 . 6 0 . 4 L+R 2 . 3 ± 0 .3 2 .9 ± 0 .2 3 .0 0 .0 2 . 8 ± 0 . 3 1.5 0 .2 2 . 2 ± 0 . 5^ D-600 0 . 8 ± 0 .7 0 . 8 ± 0 .7 0 .3 0 .3 0 . 5 ± 0 . 6 0 . 4 0 . 8 0 . 9 ± 1.0 PROP 1.2 ± 0 . 8 1.4 ± 0 .7 1.5 ± 1.1 1.1 ± 1.4 0 . 6 1.3 1.4 ± 1.1 A l l v a l u e s e x p r e s s ed as mean ± S . D . n = 2 f o r each g roup ; number o f pho tog raphs a s s e s s ed pe r t r e a t m e n t g roup = 1 5 - 2 0 . — S c o r i n g s y s t em : 0 = normal appearance 1 = m i l d a b n o r m a l i t y 2 = modera te a b n o r m a l i t y 3 = s e ve r e a b n o r m a l i t y — The S . D . was i n c l u d e d t o i n d i c a t e t h e range o f damage w i t h i n each g r o u p . — Abnormal r e l a x a t i o n w i t h l i t t l e e v i d e n c e o f c o n t r a c t i o n . - 102 - I V . 40 M i n u t e L i g a t i o n and 60 M i n u t e R e p e r f u s i o n w i t h D-600 A d m i n i s t r a t i o n 5 M i n u t e s P r i o r t o R e p e r f u s i o n The r e s u l t s f r om t h e p r e v i o u s s t u d y s ugge s t e d t h a t D-600 p r e t r e a t m e n t r educed t h e s e v e r i t y o f t h e measured b i o c h e m i c a l and u l t r a s t r u c t u r a l abnorm- a l i t i e s o f t h e i s c h a e m i c / r e p e r f u s e d h e a r t . I f t h e s e a c t i o n s were p r e dom i n - a n t l y due t o t h e a b i l i t y o f D-600 t o b l o c k c a l c i u m e n t r y d u r i n g r e p e r f u s i o n one m igh t e x p e c t t h a t t h e p r e s en ce o f t he d rug i n t h e r e p e r f u s a t e wou ld r e s u l t i n a s i m i l a r p a t t e r n o f p r o t e c t i o n as t h a t seen when t he d rug i s g i v e n p r i o r t o l i g a t i o n . We t h e r e f o r e compared t h e b i o c h e m i c a l and f u n c - t i o n a l consequences o f 40 m i nu t e l i g a t i o n p e r i o d f o l l o w e d by 60 m i nu t e s o f r e p e r f u s i o n on t h e f o l l o w i n g t h r e e g roups o f a n i m a l s : 1 Sham-ope ra ted c o n t r o l s , as d e s c r i b e d p r e v i o u s l y (CONT) L i g a t e d and r e p e r f u s e d w i t h s a l i n e a d m i n i s t r a t i o n a t 35 m i nu t e s p o s t - l i - g a t i o n (L+R) L i g a t i o n and r e p e r f u s e d w i t h 0 .10 mg/kg D-600 HCl a d m i n i s t e r e d a t 35 m inu t e s p o s t - l i g a t i o n ( i . e . 5 m i nu t e s p r i o r t o r e p e r f u s i o n ) (D-600 REP) The d e c r e a s e i n a z i d e - s e n s i t i v e m i t o c h o n d r i a l ATPase a c t i v i t y was s i g n i - f i c a n t l y a t t e n u a t e d by D-600 a d m i n i s t r a t i o n j u s t p r i o r t o r e p e r f u s i o n ( F i g u r e 2 0 ) . However , u n l i k e t h e r e s u l t s o b t a i n e d when D-600 was g i v e n b e f o r e c o r o n a r y o c c l u s i o n , t h i s method o f d rug a d m i n i s t r a t i o n d i d no t c o m p l e t e l y r e s t o r e t h e enzyme a c t i v i t y t o c o n t r o l l e v e l s ( s ee F i g u r e 8 ) . I n c o n t r a s t t o t h e b e n e f i c i a l e f f e c t o f D-600 on m i t o c h o n d r i a l ATPase and on s a r co l emma l enzymes when D-600 was g i v e n p r i o r t o l i g a t i o n , n e i t h e r o f t h e s a r co l emma l marke r enzyme a c t i v i t i e s was i n c r e a s e d by t h i s i n t e r v e n - t i o n - b o t h s a l i n e - t r e a t e d (L+R) and D - 6 0 0 - t r e a t e d h e a r t s had a c t i v i t i e s wh i ch r ema ined a t a p p r o x i m a t e l y 60% o f s ham-ope ra t ed c o n t r o l ( F i g u r e 2 1 ) . S i m i l a r l y , t h e i s c h a e m i a - i n d u c e d a l t e r a t i o n s i n o t h e r a s se s smen t s o f s a r c o - - 103 - F i g u r e 2 0 . M i t o c h o n d r i a l a z i d e - s e n s i t i v e ATPase a c t i v i t y i n sham-ope ra t ed h e a r t s (CONT), 40 min l i g a t e d and 60 min r e p e r f u s e d h e a r t s ( L+R) , and l i g a t e d and r e p e r f u s e d h e a r t s w i t h D-600 a d m i n i s t r a t i o n 5 min p r i o r t o r e p e r f u s i o n (D-600 R E P ) . Enzyme a c t i v i t y o f D-600 REP i s s i g n i f i c a n t l y i n c r e a s e d o v e r t he L+R group (P < 0 . 0 5 ) . Ba r s r e p r e s e n t mean ± S . E . and n = 4 -6 f o r each, g r o u p . - 104 - C O N T L. • R D-600 REP F i g u r e 2 1 . Sa r co l emma l N a + , K + - A T P a s e and K + - NPPa s e a c t i v i t y i n CONT, L+R and D-600 REP g r o u p s . Ba r s r e p r e s e n t mean ± S . E . and n = 4 -6 f o r each g r o u p . Drug a d m i n i s t r a t i o n d i d no t s i g n i f i c a n t l y i n c r e a s e t he l e v e l o f enzyme a c t i v i t y o v e r t h a t i n t h e 1 i g a t e d / r e p e r f u s e d g roup ( L+R ) . - 105 - lemmal i n t e g r i t y (L+R) were a l s o unchanged by d rug t r e a t m e n t j u s t b e f o r e 2+ + r e p e r f u s i o n (D-600 R E P ) . L o s s e s o f Mg and K as w e l l as a c c u m u l a t i o n + o f Na o c c u r r e d r e g a r d l e s s o f t h e p r e s en ce o f D-600 upon r e p e r f u s i o n . In a d d i t i o n , t i s s u e wa t e r c o n t e n t i n c r e a s e d s i g n i f i c a n t l y i n t h e t r e a t e d as w e l l as t h e u n t r e a t e d h e a r t s ( T a b l e X V I I ) . I n t e r e s t i n g l y , d e s p i t e t h e l a c k + + 2+ o f e f f e c t o f t h e c a l c i u m c h a n n e l - b l o c k e r on Na , K and Mg l e v e l s , t he p r e s en c e o f D-600 d u r i n g r e p e r f u s i o n s i g n i f i c a n t l y r educed t h e c a l c i u m a c c u m u l a t i o n t h a t i s t h e h a l l m a r k o f i r r e v e r s i b l y damaged, r e p e r f u s e d m y o c a r d i a l t i s s u e . However , a l t h o u g h a s i g n i f i c a n t l y l owe r a c c u m u l a t i o n o f 2+ Ca o c c u r r e d w i t h d rug t r e a t m e n t - 4 2 . 8 ± 4 . 5 ( s a l i n e t r e a t e d ) compared w i t h 2 5 . 3 ± 4 . 5 nmoles/mg d r y w e i g h t i n t h e D-600 g r oup , t h e c o n c e n t r a t i o n o f c a l c i u m i n t he D - 6 0 0 - t r e a t e d h e a r t s was s t i l l 5X t h a t i n t h e n o n - i s c h a e - mic myocard ium ( 5 . 4 ± 0 .5 nmoles/mg d r y w e i g h t ) . I t was , howeve r , compa rab l e t o t h e l e v e l f ound i n h e a r t s r e c e i v i n g D-600 p r i o r t o i s c h a e m i a . In v i ew o f t h e r e s u l t s d e m o n s t r a t i n g t h a t D-600 p r e s e r v e s t h e m i t o c h o n - d r i a l ATPase a c t i v i t y and r e du ce s i n t r a c e l l u l a r c a l c i u m i n f l u x , i t seemed p o s s i b l e t h a t t h e d rug c o u l d have a s a l u t a r y e f f e c t on t h e r e d u c t i o n o f h i g h ene r g y phospha te l e v e l s o f t h e i s h a e m i c / r e p e r f u s e d h e a r t s . However , F i g u r e 22 shows t h a t t h e l a r g e r e d u c t i o n i n ATP i s no t s i g n i f i c a n t l y p r e v e n t e d by t he p r e s en ce o f D-600 d u r i n g r e p e r f u s i o n . The re was a s m a l l , n o n - s i g n i f i - c a n t enhancement i n ATP l e v e l s t o 3 . 0 ± 1.2 nmoles/mg d r y we i g h t i n t h e d rug t r e a t e d a n ima l s (D-600 REP) when compared t o t he s a l i n e - t r e a t e d (L+R) a n i m a l s , 3 .04 ± 1.2 compared t o - . 8 6 ± 0 .33 nmoles/mg d r y w e i g h t , a l t h o u g h t h e s e v a l u e s a r e bo th be low 20% o f c o n t r o l l e v e l s . The s m a l l enhancement o f ATP by d rug t r e a t m e n t upon r e p e r f u s i o n was no t accompan ied by any i n c r e a s e i n c o n t r a c t i l e f o r c e ( d p / d t ) m a x o f t h e D-600 REP group compared t o L+R a l o n e ( T a b l e X V I I I ) . - 106 - F i g u r e 2 2 . ATP c o n t e n t o f h e a r t s i n CONT, L+R, and D-600 REP g r o u p s . D-600 REP i s no t s i g n i f i c a n t l y g r e a t e r t han L+R a t P < 0 . 0 5 . Ba r s r e p r e s e n t mean ± S . E . and n = 4 -6 f o r each g r o u p . - 1 0 7 - T a b l e X V I I . T i s s u e i o n and wa t e r c o n t e n t i n h e a r t s g i v e n 0 . 10 mg/kg D-600 HCl 5 m i nu t e s p r i o r t o r e p e r f u s i o n . Ion Con t en t (nmole/mg d r y w e i g h t ) + + +2 T r ea tmen t n Water c o n t e n t (%) Na K Mg Ca C o n t r o l 5 8 0 . 8 ± 0 .5 196 ± 17 492 ± 10 3 4 . 0 ± 0 .9 5.4 ± 0 . 5 L+R- 4 8 4 . 8 ± 0 . 3 455 ± 19 360 ± 20 2 1 . 3 ± 1.0 42 . 8 ± 4 . 6 D-600 REP 6 84 . 2 ± 0 . 3 415 ± 28 422 ± 161 22 . 2 ± 0 . 6 2 5 . 3 ± 4 . 5 1 A l l v a l u e s a r e e x p r e s s e d as mean ± S . E . - V a l u e s o f D-600 REP s i g n i f i c a n t l y d i f f e r e n t f rom L+R a t P < 0 . 0 5 . — A l l L+R measurements a r e s i g n i f i c a n t l y d i f f e r e n t f r om c o n t r o l a t P < 0 . 0 5 . - 108 - T a b l e X V I I I . P e r c e n t i n i t i a l ( d P / d t ) - o f h e a r t s d u r i n g 'max 3 r e p e r f u s i o n p e r i o d a f t e r 40 m i nu t e s o f l i g a t i o n w i t h o r w i t h o u t D-600 p r i o r t o r e p e r f u s i o n M i n u t e s o f r e p e r f u s i o n T r e a tmen t (n) 3 5 10 20 40 60 C o n t r o l (5) 103 ± 4 102 ± 4 101 ± 3 102 ± 5 98 ± 9 97 ± 8 L+R (4 ) 36 ± 3 43 ± 4 45 ± 5 56 ± 5 61 ± 5 63 ± 4 D-600 Rep (5) 29 ± 4 33 ± 4 31 ± 5 49 ± 8 61 ± 5 60 ± 5 A l l v a l u e s a r e e x p r e s s e d as mean ± S . E . — I n i t i a l ( d P / d t ) f o r each g roup (mm H g / s e c ) ; mean ± S . E . msx C o n t r o l 3550 ± 320 = 100% L+R 3688 ± 426 = 100% D-600 REP 3650 ± 400 = 100% - 109 - V. 0 -60 M i n u t e L i g a t i o n S t udy - Enzyme A c t i v i t y v s . Ion L e v e l s The a c t i v i t y o f a z i d e - s e n s i t i v e m i t o c h o n d r i a l ATPase d e c r e a s e s r a p i d l y w i t h i n c r e a s i n g p e r i o d s o f l i g a t i o n ( d a t a no t shown) as was seen i n t h e p r e l i m i n a r y s t u d y . The r e d u c t i o n i s s t a t i s t i c a l l y s i g n i f i c a n t as e a r l y as 5 m i nu t e s p o s t - l i g a t i o n and d e c r e a s e s f u r t h e r t o a p l a t e a u a t 40% o f c o n t r o l a c t i v i t y a f t e r 20 m i nu t e s o f i s c h a e m i a . In c o n t r a s t , N a + , K + - A T P a s e a c t i v i t y d i d no t d e c r e a s e i n a c o n t i n u o u s f a s h i o n bu t r a t h e r showed a s m a l l i n i t i a l r e d u c t i o n a f t e r wh i ch t h e enzyme a c t i v i t y r ema ined c o n s t a n t u n t i l 20 m i nu t e s p o s t - l i g a t i o n ( F i g u r e 2 3 ) . On l y a f t e r t h i s t i m e , when t h e m i t o c h o n d r i a l ATPase a l r e a d y was m a x i m a l l y i n h i b i - t e d , d i d s a r co l emma l enzyme a c t i v i t y d e c r e a s e f u r t h e r . S i m i l a r b e h a v i o u r was o b s e r v ed f o r t h e f u n c t i o n a l l y - r e l a t e d K + - s t i m u l a t e d p - n i t r o p h e n y l p h o s - pha t a s e ( K + - N P P a s e ) s y s t e m . Recen t e x p e r i m e n t s by Be r sohn e t a l . u s i n g r a b b i t v e n t r i c u l a r s l i c e s made i s c h a e m i c i n v i t r o , a l s o r e v e a l e d a . s l i g h t p l a t e a u i n t he f a l l i n N a + , K + - A T P a s e a c t i v i t y w i t h i n t h e f i r s t 20 96 minu t e s o f i s c h a e m i a . A c ompa r i s on o f F i g u r e s 2 and 23 show how t h e a d d i t i o n o f T r i t o n X -100 t o t h e a s s a y medium a l t e r e d t h e p a t t e r n o f enzyme a c t i v i t y w i t h l i g a t i o n t i m e . The g e n e r a l d e c r e a s e was s t i l l o b v i o u s bu t a p l a t e a u i n a c t i v i t y u n t i l 20 m i nu t e s p o s t - l i g a t i o n was r e v e a l e d by d e t e r g e n t t r e a t m e n t . Most l i k e l y t h e m o d i f i c a t i o n t o t he a s s a y p r o c edu r e caused an open i n g o f v e s i c l e s i n o r d e r t o a l l o w s u b s t r a t e a v a i l a b i l i t y t o e n z y m a t i c s i t e s s e a l e d w i t h i n an i n s i d e - o u t v e s i c l e ; howeve r , t h e p o s s i b i l i t y t h a t t h e T r i t o n X -100 was i t s e l f h a v i n g a d i r e c t e f f e c t on enzyme a c t i v i t y s h o u l d no t be d i s c o u n t e d . T a b l e XIX shows t h e r e s u l t s o f t h e t i s s u e i on and wa t e r measu remen t s . No appa r en t a c c u m u l a t i o n o f wa t e r i s e v i d e n t e v e r a f t e r 60 m i nu t e s 2+ 2+ p o s t - l i g a t i o n bu t t h e r e was no change i n Mg o r Ca l e v e l s a t t h e - 110 - o 10 so 3 0 4 0 a o B O 3, L I G A T I O N T I M E ( M I N ) F i g u r e 2 3 . Sa rco lemmal N a + , K + - A T P a s e and K -NPPase a c t i v i t y o f h e a r t s s u b j e c t e d t o 0 -60 min o f l i g a t i o n . E r r o r b a r s were o m i t t e d f o r c l a r i t y - p o i n t s r e p r e s e n t mean o f n = 5-7 a n i m a l s . Enzyme a c t i v i t y i s s i g n i f i c a n t l y r educed by 40 and 60 min p o s t - l i g a t i o n f o r bo th enzymes (P < 0 . 0 5 ) . T r i t o n X-100 was added t o t he a s s a y medium ( see Methods f o r f u r t h e r d e t a i l s ) . T a b l e X I X . Ion and wa te r c o n t e n t o f h e a r t s s u b j e c t e d t o 0 -60 m i n u t e s o f l i g a t i o n T rea tmen t n Water c o n t e n t Na K Mg Ca (min o f l i g a t i o n ) (%) (nmoles/mg d r y w e i g h t ) 0 6 79 .4 ± 0 .5 217 ± 13 462 14 3 1 . 4 ± 0 . 9 7 .84 ± 0 . 58 1 2 8 0 . 3 1.8 199 10 430 ± 57 3 3 . 0 ± 2 .9 N l £ 5 6 79 . 8 0 .7 218 17 484 38 3 2 . 0 1.5 8 . 26 ± 1.10 10 6 80 . 4 0 .3 231 ± 16 454 ± 15 3 2 . 3 0 . 8 7 .41 ± 0 .86 20 7-9 8 0 . 4 ± 0 .6 209 ± 16 407 24 2 9 . 3 2 .5 8 . 12 ± 1.28 40 6 8 0 . 2 0 .7 262 ± 26 399 ± 26 2 8 . 4 1.6 8 . 18 ± 0 .94 60 6 79 .6 0 .7 309 ± 1 4 ! 389 29 2 9 . 4 ± 1.4 8 . 95 ± 1.0 A l l v a l u e s are e xp r e s s ed as mean ± S . E . - S i g n i f i c a n t l y d i f f e r e n t f rom c o n t r o l a t P < 0 . 0 5 . - ND = no t done . - 112 - t i m e . K + l o s s was mea su r ab l e bu t t h i s d i d no t a c h i e v e s t a t i s t i c a l s i g n i - f i c a n c e . I n o r d e r t o see t h e e f f e c t s o f i s c h a e m i a on o v e r a l l N a + and K 2 + 2 + + + 2+ 2+ v s . Ca and Mg h o m e o s t a s i s , t h e Na /K and Ca /Mg were c a l c u l a t e d f o r up t o 60 m i nu t e s p o s t - o c c l u s i o n . The r e s u l t s a r e shown i n 2+ 2+ F i g u r e 24 . I n c o n t r a s t t o t h e m i n i m a l changes i n t h e Ca /Mg r a t i o , a p r o g r e s s i v e i n c r e a s e i n t h e N a + / K + r a t i o was o b s e r v e d a f t e r 20 m i nu t e s o f i s c h a e m i a . I n t e r e s t i n g l y , t h i s i n c r e a s e p a r a l l e l e d t h e f a l l i n N a + , K + - A T P a s e a c t i v i t y ( F i g u r e 2 1 ) , s u g g e s t i n g t h a t t h e d e c r e a s e d sod ium pump a c t i v i t y measured i n v i t r o , r e f l e c t s an i n v i v o phenomenon wh i ch i s a s s o c i a t e d w i t h an i n t r a c e l l u l a r a c c u m u l a t i o n o f sod ium and a l o s s o f p o t a s - s i u m . H i gh e ne r g y phospha te l e v e l s a r e known t o d e c r e a s e r a p i d l y f o l l o w i n g c o r o n a r y l i g a t i o n and ATP l e v e l s d e c r e a s e t o 50% o f c o n t r o l a f t e r o n l y 1 m i nu t e o f c o r o n a r y o c c l u s i o n . T h i s v a l u e i s m a i n t a i n e d f o r a f u r t h e r 10 m i nu t e s a f t e r wh i c h t h e r e i s a n o t h e r d r op i n ATP t o 17% o f c o n t r o l , f o l l o w e d by a more g r a d u a l d e c l i n e f r om 20 m i nu t e s u n t i l 60 m i nu t e s o f l i g a t i o n . T h u s , most o f t h e marked r e d u c t i o n i n c e l l u l a r ATP l e v e l s p r e c ede s t h e i s c h a e m i a - i n d u c e d a l t e r a t i o n s i n t h e sa r co l emma l enzyme a c t i v i t y and t h e N a + / K + r a t i o . In o r d e r t o i l l u s t r a t e t h e s e t empo ra l r e l a t i o n s h i p s more c l e a r l y , t h e i n d i v i d u a l g r aphs have been supe r imposed ( F i g u r e 2 5 ) . The + + + + c l o s e a s s o c i a t i o n between Na ,K -ATPase a c t i v i t y and t h e Na /K r a t i o and t h e i r d i s s o c i a t i o n f rom t he e a r l i e r f a l l i n ATP a re c l e a r l y a p p a r - e n t . In o r d e r t o e x p l o r e t h e p o s s i b l e m o l e c u l a r b a s i s o f t h e o b s e r v ed membrane f u n c t i o n a l a l t e r a t i o n s , we have i n v e s t i g a t e d t h e e f f e c t of i s c h a e m i c i n j u r y on m y o c a r d i a l p h o s p h o l i p i d p r o f i l e s . T w o - d i m e n s i o n a l t h i n - l a y e r c h r o m a t o - g r aphy p e r m i t t e d t h e a s se s smen t o f t h e ma jo r c l a s s e s o f p h o s p h o l i p i d s . - 113 - ' • • 1 • ' • a 10 so 3 0 40 B O B O L I G A T I O N T I M E ( M U M ) F i g u r e 2 4 . N a / K and C a 2 / M g 2 r a t i o s i n t i s s u e o b t a i n e d f r om h e a r t s s u b j e c t e d t o 0 -60 min o f i s c h a e m i a . The r i s e i n t h e N a + / K + r a t i o i s s t a t i s t i c a l l y s i g n i f i c a n t by 40 min p o s t - l i g a t i o n . C a 2 + / M g 2 + i s no t s i g n i f i c a n t l y d i f f e r e n t f r om c o n t r o l even a f t e r 60 min o f l i g a t i o n (P < 0 . 0 5 ) . P o i n t s r e p r e s e n t mean ± S . E . - 114 - F i g u r e 2 5 . Supe r imposed g raphs o f t h e a l t e r a t i o n i n N a + / K + r a t i o , Na , K + - A T P a s e a c t i v i t y and ATP c o n t e n t w i t h i n c r e a s i n g l i g a t i o n t i m e . Note t he c o n c u r r e n t r i s e o f t h e N a + / K + i o n r a t i o w i t h t he d e c r e a s e i n sod ium pump a c t i v i t y . ( E r r o r ba r s have been o m i t t e d f o r c l a r i t y ) . - 115 - P r e v i o u s l y , no r e d u c t i o n i n PC and PE were seen even a f t e r 60 m i nu t e s o f l i g a t i o n . T h i s was c o n f i r m e d u s i n g more s e n s i t i v e a n a l y s i s and f u r t h e r - more , no s i g n i f i c a n t a l t e r a t i o n was seen i n any o f t h e o t h e r p h o s p h o l i p i d components ( T a b l e X X ) . The s t a b i l i t y o f t he two ma jo r p h o s p h o l i p i d s , pho s - p h a t i d y l c h o l i n e (PC) and p h o s p h a t i d y l e t h a n o l a m i n e ( P E ) , i s s i g n i f i c a n t i n t h a t t h e s e a r e t h ough t t o be t he most s u s c e p t i b l e t o d e g r a d a t i o n under c o n d i t i o n s o f i s chaemia^ ' ' ' . The p o s s i b l e i n v o l v e m e n t o f membrane p r o t e i n a l t e r a t i o n s i n t h e ob s e r v ed e f f e c t s o f i s c h a e m i a was e x p l o r e d by means o f p o l y a c r y l a m i d e ge l e l e c t r o p h o - r e s i s wh i c h was q u a n t i t a t i v e l y a s s e s s e d by d e n s i t o m e t r y . The l a c k o f d e t e c - t a b l e changes i n t h e p r o t e i n p r o f i l e s o f t h e m i t o c h o n d r i a l and sa r co l emma l f r a c t i o n s i n t h e p r e s e n t s t u d y ( d a t a no t shown) c o n t r a s t s w i t h t h e r e s u l t s f r om t h e r e p e r f u s i o n s t u d y and s ugge s t t h a t t h e a l t e r a t i o n s t h a t r e s u l t f r om r e p e r f u s i o n a f t e r i s c h a e m i a a re r e s p o n s i b l e f o r t h e m i t o c h o n d r i a l p o l y p e p t i d e a l t e r a t i o n r a t h e r than t h e i s c h a e m i c i n s u l t p e r s e . T a b l e XX . P h o s p h o l i p i d p r o f i l e s o f h e a r t s s u b j e c t e d t o v a r i o u s p e r i o d s o f l i g a t i o n P h o s p h o l i p i d c on t e n t (% t o t a l l i p i d phospho ru s ) T r ea tmen t n Sph PI PS PC PE CL C o n t r o l 2 4 .52 .18 2 .95 .08 2 .10 .40 4 1 . 0 ± .6 3 4 . 8 1.7 14 .6 ± .4 10 ' L i g a t i o n 4 3 .69 ± .94 3 .05 ± .78 2 .29 ± .33 4 3 . 2 1.9 34 . 4 2 . 3 13 . 0 ± 1.3 4 0 ' L i g a t i o n 3 3 .55 .29 2.87 .88 1.93 .29 4 3 . 0 ± 1.8 3 4 . 5 1.7 13 .1 ± 1.3 6 0 ' L i g a t i o n 6 3 .94 ± .85 2 .79 ± .85 1.94 ± .57 4 4 . 0 ± 1.9 3 4 . 5 2 . 1 13 .1 ± .7 A l l v a l u e s a r e e xp r e s s ed as mean ± S . E . Sph = sph ingomye. l i n; • PI = p h o s p h a t i d y l i n o s i t o l PS = p h o s p h a t i d y l s e r i n e PC = p h o s p h a t i d y l c h o l i n e PE = p h o s p h a t i d y l e t h a n o l a m i n e CL = c a r d i o l i p i n - 117 - DISCUSSION Aims The pu rpose o f t h e work i n t h i s t h e s i s was t o i n v e s t i g a t e t h e b i o c h e m i - c a l a l t e r a t i o n s t h a t o c c u r d u r i n g a c u t e m y o c a r d i a l i s c h a e m i a and r e p e r f u - s i o n . S p e c i f i c a l l y , t h e s e e x p e r i m e n t s have a t t emp t ed t o i d e n t i f y t h e on s e t o f i r r e v e r s i b l e damage t o s a r co l emma l and m i t o c h o n d r i a l membranes by e x a m i n - i n g t h e t i m e c o u r s e o f d e c r e a s e s i n membrane-bound enzyme a c t i v i t y and r e l a t i n g t h e s e t o changes i n h i g h ene rgy phospha t e d e p l e t i o n , p r o t e i n and p h o s p h o l i p i d d e g r a d a t i o n and t i s s u e i o n l e v e l s . As d i s c u s s e d i n t h e I n t r o - d u c t i o n , i t i s e v i d e n t t h a t a l l c e l l u l a r p r o c e s s e s w i l l e v e n t u a l l y be a f f e c t e d as i s c h a e m i a p r o g r e s s e s t o i n f a r c t i o n . Thu s , t h e s e e x p e r i m e n t s c on ce r ned o n l y t h e b i o c h e m i c a l changes o c c u r r i n g w i t h i n t h e f i r s t 60 m i nu t e s f o l l o w i n g c o r o n a r y a r t e r y l i g a t i o n . A n o t h e r a s p e c t o f t h e e x p e r i m e n t s d e s c r i b e d h e r e i n was t o examine t h e e f f e c t s o f s e v e r a l d r ug s on our model o f i s c h a e m i a / r e p e r f u s i o n i n j u r y i n o r d e r t o i n v e s t i g a t e t h e e f f e c t s o f s p e c i f i c p h a r m a c o l o g i c a l i n t e r v e n t i o n on t h e b i o c h e m i c a l i n d i c e s o f c e l l damage. Advan tages and D i s a d v a n t a g e s o f t he Model The a c u t e o p e n - c h e s t r a b b i t p r o v i d e s a s t a b l e , p h y s i o l o g i c a l p r e p a r a t i o n wh i c h a l l o w s bo t h f u n c t i o n a l and b i o c h e m i c a l measurements t o be done i n t h e same a n i m a l . The y i e l d o f i s c h a e m i c t i s s u e i s s u f f i c i e n t t o a l l o w s a m p l i n g f o r ATP , i o n s , e l e c t r o n m i c r o s c o p y as w e l l as s e p a r a t i o n i n t o m i t o c h o n d r i a l and s a r co l emma l f r a c t i o n s w i t h o u t h a v i n g t o poo l t i s s u e f rom s e v e r a l a n i m a l s . F r a c t i o n a t i o n p e r m i t t e d t h e e v a l u a t i o n o f t h e dynamic f u n c t i o n o f membrane-bound sa r co l emma l and m i t o c h o n d r i a l enzymes . However , one must be c a u t i o u s i n e x t r a p o l a t i n g r e s u l t s f r om i n v i t r o a s s a y s t o t h e f u n c t i o n o f - 118 - t h e enzyme i n v i v o because o r g a n e l l e p u r i f i c a t i o n may ob s cu r e o r a l t e r t h e c h a r a c t e r o f i s c h a e m i a - i n d u c e d membrane a l t e r a t i o n s . T o t a l o c c l u s i o n w i t h sub sequen t r e l e a s e o f t h e l i g a t u r e a round the l e f t c i r c u m f l e x c o r o n a r y a r t e r y was used as a model of m y o c a r d i a l i s c h a e m i a and r e p e r f u s i o n i n man. Human pos t -mo r t em s t u d i e s have no t a lways demons t r a t ed t h e e x i s t e n c e o f t h r omb i i n c o r o n a r y v e s s e l s s u p p l y i n g t h e i n f a r c t e d 119 t i s s u e . A n g i o g r a p h i c e v i d e n c e , howeve r , has shown t h a t a l a r g e p e r c e n t a g e o f p a t i e n t s w i t h t r a n s m u r a l i n f a r c t i o n have t h r omb i i n t h e 120 i m p l i c a t e d c o r o n a r y v e s s e l a l t h o u g h whe the r o r no t t h e s e p l a t e l e t a g g r e g a t e s p r e c i p i t a t e i s c h a e m i a / i n f a r c t i o n o r a r e fo rmed as a r e s u l t of 121 i s c h a e m i a - i n d u c e d l o c a l e n d o t h e l i a l changes i s p r e s e n t l y u n c l e a r . An e x p l a n a t i o n f o r t h e absence o f t h r omb i upon pos t -mo r t em e x a m i n a t i o n has a r i s e n f r om r e c e n t s t u d i e s t h a t s t r o n g l y s ugge s t t h a t spon taneous r e p e r f u s i o n o f o c c l u d e d c o r o n a r y v e s s e l s o c c u r s i n p a t i e n t s as e a r l y as 6 122 123 hou r s a f t e r t h e i n i t i a l symptoms. ' Thus , l i g a t i o n o f a c o r o n a r y a r t e r y f o l l o w e d by r e l e a s e i s p r o b a b l y an adequa te model o f t h e human d i s e a s e p r o c e s s . A f u r t h e r advan tage o f u s i n g t h e r a b b i t as t h e e x p e r i m e n t a l an ima l i s t h a t l e f t c i r c u m f l e x c o r o n a r y a r t e r y o c c l u s i o n p roduces a w e l l - d e f i n e d zone 48 o f i s c h a e m i a w i t h o u t i n t e r - d i g i t a t i n g normal t i s s u e t h u s mak ing f o r more r e p r o d u c i b l e and m e a n i n g f u l t i s s u e s a m p l i n g . A l t h o u g h t h e i n s i t u model i s more p h y s i o l o g i c a l t h an t h e i s o l a t e d h e a r t , t he p e n t o b a r b i t o n e a n a e s t h e t i c b l u n t s r e f l e x r e s p o n s e s a nd , as a c on sequence , t h e c o n c l u s i o n s r e g a r d i n g t h e e f f e c t s o f any t r e a t m e n t c anno t be u n r e s e r v e d l y a p p l i e d t o t h e c o n s c i o u s a n i m a l . F u r t h e r m o r e , t h e haemody- namics were no t c o n t r o l l e d i n t h e s e e x p e r i m e n t s . A l t h o u g h t h i s a v o i d e d c o m p l i c a t i o n s f r om a d d i t i o n a l p h y s i o l o g i c a l o r p h a r m a c o l o g i c a l m a n i p u l a - t i o n s , i t was more d i f f i c u l t t o d i s s o c i a t e d i r e c t and i n d i r e c t e f f e c t s of - 119 - d r ug t r e a t m e n t . The use o f t h e i n v i v o p r e p a r a t i o n a l s o d i d no t r e a d i l y p e r m i t t h e e s t i m a t i o n o f d rug c o n c e n t r a t i o n i n t h e myoca rd i um . E f f e c t i v e c o n c e n t r a t i o n s were a s c e r t a i n e d i n d i r e c t l y by m e a s u r i n g t h e d r u g - i n d u c e d a l t e r a t i o n s i n h e a r t r a t e and ( d P / d t ) m a x . C o r o n a r y l i g a t i o n p roduced a v a r i e t y o f e f f e c t s on t h e b i o c h e m i c a l , f u n c t i o n a l and m o r p h o l o g i c a l i n d i c e s used t o a s s e s s c e l l u l a r damage. Bo th m i t o c h o n d r i a l and s a r co l emma l ATPase a c t i v i t i e s were r educed by s h o r t p e r i o d s o f c o r o n a r y l i g a t i o n ; howeve r , t h e r e were s e v e r a l i m p o r t a n t d i f f e r - ences i n t h e t i m e c o u r s e and r e v e r s i b i l i t y o f t h e a l t e r a t i o n s . The r a p i d f a l l i n m i t o c h o n d r i a l ATPase was max ima l a t 20 m i nu t e s p o s t - l i g a t i o n , a t ime when no s i g n i f i c a n t d e c r e a s e i n Na ,K -ATPase a c t i v i t y was a p p a r e n t . The b e h a v i o u r o f t h e s e two enzymes upon r e p e r f u s i o n was o f p a r t i c u l a r s i g n i - f i c a n c e — o n l y t h e m i t o c h o n d r i a l enzyme a c t i v i t y c o u l d be r e a d i l y r e v e r s e d by r e p e r f u s i o n . Even a f t e r 30 m i nu t e s o f i s c h a e m i a , r e p e r f u s i o n r e v e r s e d t h e d e c r e a s e i n m i t o c h o n d r i a l ATPase a c t i v i t y i n c o n t r a s t t o t h e s m a l l r e c o v e r y o f s a r co l emma l Na ,K -ATPase a c t i v i t y . Thu s , i t appea r s t h a t t h e sa r co l emma l ATPase i s i r r e v e r s i b l y damaged b e f o r e any permanent d e p r e s - s i o n i n m i t o c h o n d r i a l ATPase has o c c u r r e d , i n d i c a t i n g t h a t i s c h a e m i a - i n d u c e d d e c r e a s e s i n t h e two enzyme a c t i v i t i e s may i n v o l v e d i f f e r e n t mechan i sms . O t h e r i n v e s t i g a t o r s have a l s o d emons t r a t ed t h e r e l a t i v e r e v e r s i b i l i t y o f i s c h a e m i a - i n d u c e d damage t o m i t o c h o n d r i a l s t r u c t u r e and f u n c t i o n . ^ ' ^ ' 1 ^ In c o n t r a s t , N a y l e r and S c o t t were no t a b l e t o r e v e r s e t h e d e f e c t i n m i t o - c h o n d r i a l o x i d a t i v e p h o s p h o r y l a t i o n w i t h r e p e r f u s i o n a f t e r 90 m i n u t e s o f g l o b a l i s c h a e m i a i n i s o l a t e d r a b b i t h e a r t s . I t i s p o s s i b l e t h a t t h i s v e r y l ong i s c h a e m i c p e r i o d caused i r r e v e r s i b l e damage t o t h e m i t o c h o n d r i o n , p r e v e n t i n g t h e r e s t o r a t i o n o f f u n c t i o n . On t h e o t h e r hand , o x i d a t i v e p h o s - - 120 - p h o r y l a t i o n w i l l no t n e c e s s a r i l y r e t u r n t o c o n t r o l v a l u e s even i n t h e p r e s en c e o f normal ATPase a c t i v i t y . The c o - o r d i n a t e d a c t i v i t y o f many enzymes i s r e q u i r e d f o r o x i d a t i v e p h o s p h o r y l a t i o n and t h e d i s t u r b a n c e o f a s i n g l e p r o c e s s wou l d a f f e c t t h e o v e r a l l r a t e o f ATP f o r m a t i o n . A z i d e - s e n s i - t i v e ATPase a c t i v i t y and c e l l u l a r r e s p i r a t i o n a r e f u n c t i o n a l l y r e l a - 1nc I 07 t e d ' so t h a t t h e d e p r e s s i o n o f ATPase a c t i v i t y as w e l l as t h e low oxygen t e n s i o n d u r i n g t h e i s c h a e m i c p e r i o d wou ld p r o b a b l y s upp r e s s o x i d a t i v e p h o s p h o r y l a t i o n i n v i v o . In v i t r o , t h e ATPase a c t i v i t y i s s t i l l d e p r e s s e d , even i f a s s ayed i n t h e p r e s en c e o f o p t i m a l c o n c e n t r a t i o n s o f s u b s t r a t e s . T h i s i n d i c a t e s t h a t t he i s c h a e m i a - i n d u c e d a l t e r a t i o n i s no t s i m p l y due t o a l a c k o f s u b s t r a t e . The r e s t o r a t i o n o f enzyme a c t i v i t y w i t h r e p e r f u s i o n s u g g e s t s t h e i n v o l v e m e n t o f a r e v e r s i b l e i n h i b i t o r a c c u m u l a t i n g d u r i n g t h e c o u r s e o f i s c h a e m i a . ( I t s h o u l d a l s o be no t ed t h a t t h e o b s e r v e d c o n t r o l v a l u e s f o r enzyme a c t i v i t y measured i n v i t r o does no t p r o ve t h a t ATPase a c t i v i t y has been r e s t o r e d i n v i v o as many c o f a c t o r s and s u b s t r a t e s may have been d e p l e t e d d u r i n g t h e i s c h a e m i c p e r i o d ) . The d e c r e a s e i n m i t o c h o n d r i a l ATPase a c t i v i t y does no t i n d i c a t e a g e n e r a l i z e d d e p r e s s i o n o f a l l r e s p i r a - t o r y c h a i n enzyme a c t i v i t i e s as shown by t h e l a c k o f e f f e c t o f i s c h a e m i a on a n o t h e r i n n e r m i t o c h o n d r i a l membrane enzyme, name ly c y t o ch r ome c o x i d a s e . S o r d a h l and S t ewa r t have a l s o demons t r a t ed t h a t t h e r e s p i r a t o r y c h a i n damage 1 op caused by m y o c a r d i a l i s c h a e m i a does no t i n v o l v e c y t o ch r ome c o x i d a s e . Measurement o f m i t o c h o n d r i a l ATPase a c t i v i t y may, howeve r , be a r e a s o n a b l e i n d i c a t o r o f o v e r a l l m i t o c h o n d r i a l f u n c t i o n . R o u s l i n and M i l l a r d have measured r e s p i r a t o r y c h a i n enzyme a c t i v i t i e s a f t e r c o r o n a r y l i g a t i o n , and found t h a t t h e o v e r a l l r e d u c t i o n i n o x i d a t i v e p h o s p h o r y l a t i o n can be a lmo s t e n t i r e l y a c coun t ed f o r by r e d u c t i o n s i n NADH-coenzyme Q and o l i g o m y c i n - ( a z - 129 i d e - ) s e n s i t i v e ATPase a c t i v i t i e s . - 121 - A l t h o u g h r e p e r f u s i o n o f i s c h a e m i c m i t o c h o n d r i a a p p a r e n t l y r e v e r s e s t h e d e c r e a s e i n t h e ATPase a c t i v i t y , t h e r educed m i t o c h o n d r i a l r e c o v e r y o f t h e m i t o c h o n d r i a f r a c t i o n f r om i s c h a e m i c / r e p e r f u s e d h e a r t s a l s o s u gge s t s t h a t t h e r e may have been s e l e c t i v e r e c o v e r y o f undamaged m i t o c h o n d r i a d u r i n g t h e p u r i f i c a t i o n p r o c e d u r e . The r e s t o r a t i o n o f ATPase a c t i v i t y t o 75% of c o n t r o l v a l u e s a f t e r a 40 min l i g a t i o n wou ld o n l y be a c h i e v e d i f t h e p r o p o r - t i o n o f r e c o v e r e d m i t o c h o n d r i a was 50% damaged ( a c t i v i t y = 40% o f c o n t r o l ) and 50% norma l m i t o c h o n d r i a ( a c t i v i t y = 100% o f c o n t r o l ) . I f t h i s were s o , one wou ld have e x p e c t e d t o see e qua l p r o p o r t i o n s o f norma l and s e v e r e l y damaged m i t o c h o n d r i a i n t h e same t i s s u e s e c t i o n ; howeve r , t h i s was no t a ppa r en t i n t h e l i g h t and e l e c t r o n m i c r o g r a p h s o f t h e l i g a t e d and r e p e r f u s e d h e a r t s ( L+R) . The deg r ee o f i n j u r y t o m i t o c h o n d r i a was r e l a t i v e l y u n i f o r m w i t h i n t h e L+R g roup and t h e number o f norma l m i t o c h o n d r i a was e x t r e m e l y s m a l l compared t o t h e damaged o r g a n e l l e s . The L+R h e a r t s d i d have an a p p a r - en t l o s s o f m i t o c h o n d r i a when compared t o t h e c o n t r o l and d r u g - t r e a t e d h e a r t s and t h i s may have c o n t r i b u t e d t o t h e d e c r e a s e d y i e l d o f t h i s o r g a n - e l l e f r om s e v e r e l y damaged t i s s u e r a t h e r t h an a s e l e c t i v e r e c o v e r y o f undam- aged m i t o c h o n d r i a . F u r t h e r m o r e , t h e m i t o c h o n d r i a l membrane f r a c t i o n i s o l a - t e d f r om i s c h a e m i c h e a r t s has a m i s s i n g p r o t e i n b and , i n d i c a t i n g t h a t t h e s e p a r a t i o n p r o c edu r e p r o b a b l y does no t p r e f e r e n t i a l l y e l i m i n a t e damaged m i t o c h o n d r i a . V a r i o u s i n v e s t i g a t o r s have a l s o shown t h a t i s c h a e m i a p roduces i r r e v e r s i - b l e damage t o t h e sa r co l emma l membrane, a l t h o u g h most s t u d i e s have f o c u s s e d on changes i n s a r co l emma l p e r m e a b i l i t y r a t h e r t h an membrane-bound enzyme f u n c t i o n . Fo r e xamp l e , We i s s and Sh i n e have d emon s t r a t e d an immed ia te and r e v e r s i b l e i n c r e a s e i n K + e f f l u x f r om t h e i s c h a e m i c , a r t e r i a l l y - p e r f u s e d i n t r a v e n t r i c u l a r r a b b i t s ep tum. T h i s r e a ched a p l a t e a u a t a p p r o x i m a t e l y 12 - 122 - m i nu t e s p o s t - 1 i g a t i o n a f t e r wh i ch t h e y f ound t h a t t h e l o s s o f K + f r om t he c e l l was i r r e v e r s i b l e (25 m i nu t e s p o s t - 1 i g a t i o n ) . H i l l and G e t t e s have 130 o b t a i n e d s i m i l a r r e s u l t s i n i s c h a e m i c sw ine h e a r t s . They measured t h e e x t r a c e l l u l a r K + a c t i v i t y and f ound t h a t t h e r i s e f o l l o w e d a d i s t i n c t t r i p h a s i c p a t t e r n t h a t c l o s e l y p a r a l l e l e d t h e r e s u l t s o f We i ss and S h i n e . The s u b t l e t i e s o f K + e f f l u x a r e somewhat o b s c u r ed when t o t a l t i s s u e K + l e v e l s a r e measured (as was done i n t h e s t u d i e s d e s c r i b e d h e r e i n ) because o f t h e l a r g e K - j n / K o u t r a t i o . D e s p i t e t h i s t e c h n i c a l l i m i t a t i o n , i t i s i n t e r e s t i n g t o no t e t he s i m i l a r i t y o f t h e t ime o f o n s e t o f i r r e v e r s i b l e K + l o s s i n t h e s t u d i e s o f We i ss and Sh i n e and H i l l and G e t t e s (25 -30 m i n u t e s ) w i t h t h e on se t o f a s i g n i f i c a n t d e c r e a s e i n N a + , K + - A T P a s e a c t i v i t y and c o n c u r r e n t i n c r e a s e i n t h e Na /K r a t i o ( 20 -30 m i n u t e s ) shown i n t he p r e s e n t e x p e r i m e n t s . In a d d i t i o n t o t h e l o s s o f p o t a s s i u m , ne t sod ium i n f l u x has a l s o been demons t r a t ed by o t h e r i n v e s t i g a t o r s d u r i n g t h e 115 e a r l y i s c h a e m i c p e r i o d u s i n g t o t a l t i s s u e sod ium measu remen t s . These i o n changes t a k e p l a c e i n t h e absence o f changes i n t h e e x t r a c e l l u l a r space 51 28 ( a s s e s s e d by Cr-EDTA d i s t r i b u t i o n ) . Of p a r t i c u l a r i n t e r e s t i s t he o b s e r v a t i o n o f an i n c r e a s e i n t h e N a + / K + r a t i o i n t h e absence o f changes 2+ 2+ t o t h e Ca /Mg r a t i o i n d i c a t i n g t h a t i s c h a e m i a - i n d u c e d p lasma membrane damage c o n s i s t e d o f a s p e c i f i c a l t e r a t i o n t o t h e p l a sma membrane i n v o l v i n g Na and K p e r m e a b i l i t y ( p r o b a b l y r e l a t e d t o a d e c r e a s e i n t h e N a + , K + - A T P a s e a c t i v i t y ) r a t h e r t han a g e n e r a l i z e d i n c r e a s e i n p e r m e a b i l - i t y t o a l l i o n s . Sa r co l emma l c a l c i u m p e r m e a b i l i t y d u r i n g i s c h a e m i a and r e p e r f u s i o n i s d i s c u s s e d i n more d e t a i l under "Drug S t u d i e s " ( s ee page 1 3 0 ) . O t h e r i n v e s t i g a t o r s have a l s o a t t emp t ed t o r e l a t e d e c r e a s e s i n N a + , K + - ATPase a c t i v i t y t o t h e i o n a b n o r m a l i t i e s ob s e r v ed a f t e r s h o r t p e r i o d s o f c o r o n a r y o c c l u s i o n . M e a s u r e a b l e d e c r e a s e s i n enzyme f u n c t i o n have been - 123 - Q/~ 1 O 1 1 o b s e r v e d ' ' a l t h o u g h S chwa r t z e t a l . were no t a b l e t o show any 133 d e c r e a s e i n enzyme a c t i v i t y even a f t e r 60 m i n u t e s o f i s c h a e m i a . The l a c k o f e f f e c t o f i s c h a e m i a on sod ium pump a c t i v i t y i n t h e s t u d y o f S chwa r t z e t a l . may be a t t r i b u t a b l e t o t h e f a c t t h a t t h e y examined h i g h l y p u r i f i e d s a r co l emma l enzyme. I s c h a e m i a - i n d u c e d d e c r e a s e s i n sod ium pump a c t i v i t y may, t h e r e f o r e , be med i a t e d v i a e f f e c t s on t h e l i p i d env i r onmen t o f t h e enzyme r a t h e r t h an on t h e a c t u a l enzyme p r o t e i n . The r e a r e s e v e r a l l i n e s o f e v i d e n c e i n s u p p o r t o f t h i s h y p o t h e s i s . F i r s t l y , t h e ge l e l e c t r o p h o r e t i c s t u d i e s do no t r e v e a l any g r o s s change i n t h e sa r co l emma l p r o t e i n bands f r om h e a r t s s u b j e c t e d t o 60 m i n u t e s o f i s c h a e m i a . T h i s , o f c o u r s e , does no t p r e c l u d e t h e o c c u r r e n c e o f changes i n m ino r and f u n c t i o n a l l y - i m p o r t a n t membrane p r o t e i n s a nd / o r s u b t l e e f f e c t s on enzyme c o n f o r m a t i o n i n v i v o . S e c o n d l y , t h e N a + , K + - A T P a s e and K + - N P P a s e enzymes were a f f e c t e d t o t h e same deg ree r e g a r d l e s s o f t h e t r e a t m e n t . F i n a l l y , t h e r e d u c t i o n i n V m g x o f t h e N a + , K + - A T P a s e comp lex i s n o t accompan ied by d e c r e a s e s i n t he + 2+ appa r en t a f f i n i t i e s o f K and Mg . The above wou ld i n d i c a t e an e f f e c t on t h e who le enzyme complex r a t h e r t h an an i n t e r f e r e n c e w i t h one o f i t s p a r t i a l r e a c t i o n s . F u r t h e r m o r e , i t i s known t h a t t h e a c t i v i t y o f t h i s 134 enzyme depends on two d i s t i n c t 1 i g a n d - i n d u c e d c o n f o r m a t i o n a l t r a n s i t i o n s and t h i s change i n c o n f o r m a t i o n i s dependent upon t h e l i p i d e n v i r o nmen t o f 112 1 "35 t h e enzyme. ' T hu s , i s c h a e m i a - i n d u c e d a l t e r a t i o n s i n t h e l i p i d b i l a y e r c o u l d be r e s p o n s i b l e f o r t h e ob s e r v ed d e c r e a s e i n sod ium pump a c t i v - i t y . M i t o c h o n d r i a l a z i d e - s e n s i t i v e ATPase has a l s o been shown t o have a 113 s p e c i f i c l i p i d r e q u i r e m e n t and a l t e r a t i o n s i n c e l l u l a r l i p i d c o u l d a l s o a f f e c t i t s a c t i v i t y . Many a t t emp t s have been made t o i d e n t i f y t h e p r e s en ce o f i s c h a e m i a - i n d u c e d l i p i d d i s r u p t i o n . S e v e r a l h ypo t he s e s have been advanced and t h e more t e n a b l e o f t h e s e w i l l be d i s c u s s e d i n c o n n e c t i o n w i t h t he r e s u l t s f r om t he p r e s e n t s t u d i e s . - 124 - I t has been p o s t u l a t e d t h a t l y s o s oma l d i s r u p t i o n o c c u r s as a consequence o f i s c h a e m i a r e s u l t i n g i n r e l e a s e o f p r o t e a s e s and p h o s p h o l i p a s e s i n t o t h e c e l l w h i c h c o u l d p r o d u c e , amongst o t h e r t h i n g s , damage t o t h e l i p i d b i l a y - 136 e r . E a r l y s t u d i e s by Decker e t a l . i n d i c a t e d t h a t l y s o s oma l enzyme 137 r e l e a s e o c c u r r e d d u r i n g i s c h a e m i a and Ch i e n e t a l . have i m p l i c a t e d such r e l e a s e as t h e p o s s i b l e c ause o f t h e p h o s p h o l i p i d d e g r a d a t i o n seen a f t e r 3 42 hou r s o f i s c h a e m i a . M o r e o v e r , work f r om our l a b o r a t o r y has demons t r a t ed t h a t i s c h a e m i a r e s u l t s i n a r e d i s t r i b u t i o n o f two l y s o s oma l enzymes ( c a t h e p s i n D and N - a c e t y l - 3 - g l u c o s a m i n i d a s e ) f r om t h e s e d i m e n t a b l e t o t h e s u p e r n a t a n t p o r t i o n o f i s o l a t e d l y s o s oma l membranes. However , t h i s r e d i s t r i b u t i o n does no t r e a c h s t a t i s t i c a l s i g n i f i c a n c e u n t i l marked d e c r e a - s e s i n bo th t h e sa r co l emma l and m i t o c h o n d r i a l enzyme a c t i v i t i e s have o c c u r - r e d . M o r e o v e r , i t has no t been p o s s i b l e t o d emons t r a t e t he e x i s t e n c e , i n i s c h a e m i c t i s s u e , o f l y sosomes show ing an i n c r e a s e d s u s c e p t i b i l i t y t o h y p o t o n i c l y s i s . No measu r eab l e p h o s p h o l i p i d o r p r o t e i n d e g r a d a t i o n was d e t e c t a b l e , even a f t e r 60 m i n u t e s o f i s c h a e m i a mak ing i t u n l i k e l y t h a t l y s o s o m a l enzyme r e l e a s e , i f i t does o c c u r i n v i v o , i s r e s p o n s i b l e f o r t h e o b s e r v ed d e c r e a s e s i n membrane-bound enzyme a c t i v i t y . I f a c t i v a t e d d u r i n g i s c h a e m i a , e x t r a - l y s o s o m a l p h o s p h o l i p a s e s c o u l d a l s o deg rade membranes and p roduce membrane damage bo th by a r e d u c t i o n i n p h o s - p h o l i p i d c o n t e n t and by t h e c o n c o m i t a n t p r o d u c t i o n o f l y s o p h o s p h o l i p i d s . Such d e g r a d a t i o n m i gh t n o t be appa r en t by measurement o f t o t a l p h o s p h o l i p i d c o n t e n t because t h e p e r c e n t a g e o f l y s o p h o s p h o l i p i d i n c e l l membranes i s v e r y s m a l l i n c ompa r i s on t o t h e o t h e r p h o s p h o l i p i d s ( l e s s t h an 1% o f t o t a l l i p i d pho spho ru s ) and t h e c o n c e n t r a t i o n t h ough t t o p roduce membrane d i s r u p t i o n i s o n l y m a r g i n a l l y g r e a t e r . The i n v o l v e m e n t o f t h e s e a m p h i p a t h i c , d e t e r g e n t - l i k e compounds i n t h e g e n e s i s o f i s c h a e m i c damage was f i r s t s ugge s t ed by - 125 - H a j d u , We i s s and T i t u s as e a r l y as 1957 . The l e v e l o f l y s o l i p i d s i n i s c h a e m i c t i s s u e was no t a c t i v e l y i n v e s t i g a t e d aga i n u n t i l t h e 1970s when e x p e r i m e n t s by Sobe l and h i s c o l l e a g u e s d emons t r a t ed l y s o p h o s p h o l i p i d accum- u l a t i o n i n i s c h a e m i c myoca rd i um. They p o s t u l a t e d t h a t t h e s e agen t s were t h e 44 129 cause o f i s c h a e m i a - i n d u c e d d y s r h y t h m i a s . ' R e c e n t l y , Man e t a l . have a l s o shown t h a t s e v e r a l d e t e r g e n t s (SDS, T r i t o n X-100) as w e l l as l y s o p h o s - p h o g l y c e r i d e s p roduce a r r h y t h m i a s i n t h e i s o l a t e d , p e r f u s e d hams te r h e a r t a t 140 c o n c e n t r a t i o n s be low t h e i r c r i t i c a l m i c e l l e c o n c e n t r a t i o n . The memb rane - a c t i v e p r o p e r t i e s o f t h e s e compounds a re a l s o e v i d e n t i n t h e i r a b i l i t y t o i n h i b i t N a + , K + - A T P a s e i n v i t r o when added t o a d u l t , c a n i n e 141 c a r d i a c m y o c y t e s . I t wou l d appea r t h a t t h e s e compounds a re p o t e n t i a l c a n d i d a t e s i n c a u s i n g i s c h a e m i c membrane damage; howeve r , d e s p i t e t h e c o m p e l l i n g e v i d e n c e f r om i s o l a t e d p r e p a r a t i o n s , o t h e r e v i d e n c e e x i s t s t h a t c a s t s doub t on t h e i m p o r t a n c e o f t h e s e compounds i n c a u s i n g i r r e v e r s i b l e membrane damage. S h a i k h and Downar have d i s c r e d i t e d e a r l i e r pape r s i n wh i c h a c i d i f i c a t i o n o f t h e e x t r a c t i o n med ia was pe r f o rmed because t h i s p r o c e d u r e p roduces a l a r g e a r t i f a c t u a l i n c r e a s e i n l y s o - c ompound s . In t h e i r r i g o u r o u s s t u d y , t h e y f ound no d e g r a d a t i o n o f p h o s p h o l i p i d o f sw ine h e a r t s s u b j e c t e d t o l e f t a n t e r i o r d e s c e n d i n g (LAD) c o r o n a r y a r t e r y o c c l u s i o n u n t i l 8 h ou r s p o s t - l i g a t i o n and , more i m p o r t a n t l y , t h e i n c r e a s e i n l y s o p h o s p h a t i d e l e v e l s ( s t i l l l e s s t han 0 .6% t o t a l l i p i d phospho rus ) was maximal a t 8 m i nu t e s 45 p o s t - o c c l u s i o n w i t h l i t t l e change i n l e v e l s a f t e r t h i s t i m e . I n v i ew o f t he t ime c o u r s e o f membrane enzyme i n h i b i t i o n , t h e n , l y s o p h o s p h a t i d e s a r e an u n l i k e l y cause o f membrane d i s r u p t i o n seen a f t e r 20 -40 m i nu t e s o f c o r o n a r y o c c l u s i o n . In a d d i t i o n , a l t h o u g h l y s o p h o s p h a t i d e - i n d u c e d i n h i b i t i o n o f many c e l l u l a r f u n c t i o n s i s d e m o n s t r a b l e i n v i t r o , what impac t such l e v e l s have i n v i v o i s unknown. F o r e xamp l e , a 350% i n c r e a s e i n c a r d i a c l y s o p h o s p h a t i d e - 126 - l e v e l s i s seen i n h i b e r n a t i n g g round s q u i r r e l s . The s q u i r r e l s awaken each 8-12 days and t h e i r body t e m p e r a t u r e r i s e s f rom 1 t o 37°C , and , a l t h o u g h t h e e r y t h r o c y t e l e v e l s o f t h e s e compounds f a l l r a p i d l y upon a r o u s a l , a v e r y s i g n i f i c a n t c o n t e n t o f l y s o p h o s p h a t i d e s p r o b a b l y r ema i n s i n c a r d i a c t i s s u e 142 and t h i s l e v e l i s c o m p a t i b l e w i t h normal m y o c a r d i a l t i s s u e f u n c t i o n . These l a t t e r c o n s i d e r a t i o n s c h a l l e n g e t h e p r o p o s a l t h a t e a r l y i s c h a e m i c - i n - duced membrane damage i s t h e r e s u l t o f l y s o p h o s p h a t i d e a c c u m u l a t i o n . Hav i ng l a r g e l y d i s c o u n t e d p h o s p h o l i p i d o r p r o t e i n d e g r a d a t i o n as t he ma jo r cause o f e a r l y i s c h a e m i a - i n d u c e d m i t o c h o n d r i a l o r s a r co l emma l damage, a n o t h e r p o s s i b l e s o u r c e o f d i s r u p t i o n t o t he l i p i d b i l a y e r c o u l d a r i s e f r om 24 a c c u m u l a t i o n o f i n t e r m e d i a t e s o f f a t t y a c i d m e t a b o l i s m . Whi tmer e t a l . have f ound t h a t i n c r e a s e s i n a c y l CoA l e v e l s r e a c h a maximum a t 5 m i nu t e s p o s t - 1 i g a t i o n whereas a c y l c a r n i t i n e l e v e l s a r e s t i l l i n c r e a s i n g a f t e r 20 35 m i nu t e s o f i s c h a e m i a . F a t t y a c y l co-enzyme A and f a t t y a c y l c a r n i t i n e a re a m p h i p a t h i c m o l e c u l e s w i t h d e t e r g e n t p r o p e r t i e s a nd , as s u c h , i n h i b i t a v a r i e t y o f c e l l u l a r p r o c e s s e s a t c o n c e n t r a t i o n s compa rab l e t o t he l y s o p h o s - p h a t i d e s and o t h e r d e t e r g e n t s , i . e . , a p p r o x i m a t e l y 50 uM. However , Owens e t a l . u s i n g a m o d i f i e d method o f membrane p u r i f i c a t i o n i n wh i c h no d e t e r g e n t s were added , have been u n a b l e t o d emons t r a t e i n h i b i t i o n o f N a + , K + - A T P a s e a c t i v i t y by e i t h e r p a l m i t y l c a r n i t i n e ( 0 . 4 mM) o r p a l m i t y l CoA ( 0 . 1 mM) i n v i t r o even though t h e s e compounds were shown t o b i n d e x t e n s i v e l y t o s a r c o - lemmal v e s i c l e s I n a d d i t i o n , r e s u l t s f r om t h i s l a b o r a t o r y have shown t h a t i n c u b a t i o n o f membrane f r a c t i o n s w i t h 0 . 5 mM p a l m i t y l c a r n i t i n e ( i n t h e absence o f T r i t o n X -100) had no e f f e c t on m i t o c h o n d r i a l ATPase and o n l y r e du ced N a + , K + - A T P a s e a c t i v i t y by 25% ( d a t a no t shown) . O t he r e v i d e n c e , a l b e i t i n d i r e c t , t h a t f u r t h e r d i s p u t e s t h e impo r t an c e o f p a l m i t y l c a r n i t i n e a c c u m u l a t i o n i n i s c h a e m i a - i n d u c e d N a + , K + - A T P a s e i n h i b i t i o n comes f rom a - 127 - s t u d y on d i a b e t i c r a t s done by Dr . D. V . God in i n c o l l a b o r a t i o n w i t h D r s . J . H. M c N e i l l and G. Lopaschuk ( F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s , U . B . C . ) B r i e f l y , t h e y f ound t h a t s i g n i f i c a n t i n c r e a s e s i n p a l m i t y l c a r n i t i n e l e v e l s i n d i a b e t i c h e a r t s were no t accompan ied by d e c r e a s e s i n e i t h e r m i t o c h o n d r i a l 143 o r s a r co l emma l ATPase a c t i v i t y . Thus , even i f t h e r e i s an i n c r e a s e i n f a t t y a c y l c a r n i t i n e l e v e l s d u r i n g i s c h a e m i a , t h e r e i s no t n e c e s s a r i l y an i n v i v o i n h i b i t i o n o f N a + , K + - A T P a s e a c t i v i t y . These agen t s have no e f f e c t on m i t o c h o n d r i a l ATPase a c t i v i t y i n v i t r o ; however K o t a k a e t a l . have shown t h a t m i t o c h o n d r i a l a c y l CoA l e v e l s c o r r e l a t e w e l l w i t h d e c r e a s e s i n a no t h e r m i t o c h o n d r i a l f u n c t i o n , namely S t a t e 3 oxygen c on sump t i o n ( r = - 0 . 9 7 ) . ^ I n t e r e s t i n g l y , r e p e r f u s i o n a f t e r 30 m i nu t e s r e v e r s e d t h e i n h i b i t i o n o f m i t o - c h o n d r i a l f u n c t i o n as w e l l as l o w e r i n g t h e l e v e l o f a c y l CoA. The a s s o c i a - t i o n between t h e two e v e n t s i s i n t e r e s t i n g , bu t i t does no t p r ove t h a t t h e e v e n t s a r e c a u s a l l y r e l a t e d . A l t h o u g h f a t t y a c y l c a r n i t i n e (and p r o b a b l y f a t t y a c y l CoA) have no e f f e c t on m i t o c h o n d r i a l ATPase a c t i v i t y i n v i t r o , t h e a c c u m u l a t i o n and washout o f an i n h i b i t o r y m e t a b o l i t e does f i t t h e p a t t e r n o f m i t o c h o n d r i a l ATPase i n h i b i t i o n . T h i s i s u n l i k e l y t o be t h e c a s e , however , i n t h e mechanism o f i r r e v e r s i b l e d e c r e a s e s i n N a + , K + - A T P a s e and K + - N P P a s e a c t i v i t y . The re has n o t y e t been any u n e q u i v o c a l e v i d e n c e t h a t some p a r t i c - u l a r memb r ane - a c t i v e agent i s r e s p o n s i b l e and i t i s d o u b t f u l t h a t l y s o p h o s - p h o l i p i d s , f a t t y a c i d m e t a b o l i t e s , o r l y s o s o m a l enzymes a r e i n v o l v e d i n e a r l y s a r co l emma l membrane a l t e r a t i o n s . The a f o r e m e n t i o n e d s t u d i e s have o n l y examined i s c h a e m i c damage i n i s o l a t e d membranes and i t i s p o s s i b l e t h a t t h e s e compounds may have somewhat d i f f e r e n t p r o p e r t i e s i n t h e i n t a c t myoca r - d i um . - 128 - ATPase a c t i v i t y wou ld o b v i o u s l y be r educed i n v i v o because o f t h e u n a v a i l a b i l i t y o f ATP; howeve r , enzyme a c t i v i t i e s were a s sayed i n v i t r o w i t h o p t i m a l c o n c e n t r a t i o n s o f s u b s t r a t e s . T h u s , low ATP l e v e l s r e s u l t i n g f rom c o r o n a r y o c c l u s i o n do no t c au se t h e i r r e v e r s i b l e d e c r e a s e i n sa r co l emma l N a + , K + - A T P a s e bu t may i n i t i a t e a s e c o n d a r y o r t e r t i a r y e v e n t t h a t r e s u l t s i n i n h i b i t i o n o f enzyme f u n c t i o n . The r e d u c t i o n o f ATP c o n t e n t i n our s t u d i e s p r e ceded t h e d e c r e a s e i n s a r co l emma l N a + , K + - A T P a s e a c t i v i t y and t he i n c r e a s e i n t he N a + / K + r a t i o . S i m i l a r l y , Re imer e t a l . , u s i n g p o s t e r i o r p a p i l l a r y mus c l e s f r om d o g s , has shown t h a t d e p r e s s e d h i g h ene rgy phospha te s y n t h e s i s p r e ceded t h e deve lopment o f o v e r t s a r co l emma l membrane 144 damage. A f u r t h e r t empo r a l d i s s o c i a t i o n between low ATP l e v e l s and i n c r e a s e s i n s a r co l emma l p e r m e a b i l i t y has been made by Hawor th e t a l . . U s i n g i n t a c t , q u i e s c e n t m y o c y t e s , t h e y showed t h a t t h e on s e t o f i s c h a e m i a - i n d u c e d c o n t r a c t u r e a lway s f o l l o w e d a c r i t i c a l d e c r e a s e i n ATP 145 c o n t e n t . J e n n i n g s e t a l . have s ugge s t e d t h a t d e p l e t i o n o f h i g h ene rgy phospha t e s t o a c r i t i c a l l e v e l o f <3-8% o f c o n t r o l was a s s o c i a t e d w i t h 21 i r r e v e r s i b l e c e l l damage. Whether o r no t low ATP l e v e l s and subsequen t s a r co l emma l damage a r e c a u s a l l y r e l a t e d i s no t c e r t a i n a l t h o u g h , as s t a t e d e a r l i e r , ATP d e p l e t i o n wou l d p r o b a b l y r e s u l t i n i r r e v e r s i b l e p l a sma membrane damage v i a an i n t e r m e d i a t e s t e p . A l t e r n a t i v e l y , membrane p h o s p h o r y l a t i o n by ATP may d i r e c t l y p r e s e r v e s a r co l emma l membrane i n t e g r i t y . In t h i s c a s e , ATP d e p l e t i o n m igh t have a d i r e c t e f f e c t on sa r co l emma l s t r u c t u r e and f u n c t i o n . The r e d u c t i o n i n m i t o c h o n d r i a l ATPase a c t i v i t y c o u l d a c c o u n t , i n p a r t , f o r t h e f a l l i n ATP l e v e l s be cause t h e enzyme i s a l s o i n v o l v e d i n ATP s y n t h e s i s . However , u n l i k e m i t o c h o n d r i a l ATPase a c t i v i t y , t h e d e c r e a s e i n ATP l e v e l s i s no t r e v e r s e d by r e p e r f u s i o n , even a f t e r s h o r t p e r i o d s o f l i g a - t i o n . S i n c e d e c r e a s e s i n m i t o c h o n d r i a l f u n c t i o n a r e l a r g e l y r e v e r s i b l e w i t h - 129 - r e p e r f u s i o n , e . g . , m i t o c h o n d r i a l ATPase a c t i v i t y , S t a t e 3 oxygen consump- t i o n , and c r e a t i n e phospha t e l e v e l s a t t a i n p r e - o c c l u s i o n v a l u e s , i t has been p o s t u l a t e d t h a t t h e l a c k o f r e g e n e r a t i o n o f ATP i s due t o an e f f l u x o f p r e c u r s o r s f o r t h e s y n t h e s i s o f a den i n e n u c l e o t i d e s f rom t h e i s c h a e m i c 92 147 z o n e . ' The compound a l l o p u r i n o l p r e s e r v e s t h e p u r i n e poo l by b l o c k - i n g t h e i r r e v e r s i b l e c o n v e r s i o n o f x a n t h i n e t o u r i c a c i d by x a n t h i n e o x i d - a s e , and l i m i t e d s u c c e s s i n r e d u c i n g i s c h a e m i c damage has been a c h i e v e d w i t h 148 149 a d m i n i s t r a t i o n o f t h i s d rug p r i o r t o c o r o n a r y l i g a t i o n . ' However , t h e impo r t an c e o f t h i s e f f e c t v_s. t h e v a s o d i l a t o r y p r o p e r t i e s o f t he d rug i n p r o t e c t i n g t h e myocard ium f r om i s c h a e m i c damage i s u n c e r t a i n . De c r ea se s i n c e l l u l a r ATP have a l s o been i m p l i c a t e d as t h e cause o f t he p r o l o n g e d d e p r e s s i o n o f c o n t r a c t i l i t y . 1 7 > 1 5 0 , 1 5 1 j n a g r e e m e n t w i t h t h e r e p o r t s i n t h e l i t e r a t u r e , t he p r e s e n t r e s u l t s showed t h a t t h e d e p r e s s i o n o f b o t h m e c h a n i c a l f u n c t i o n and h i g h ene r g y phospha te l e v e l s was no t r e v e r s e d upon r e p e r f u s i o n d e s p i t e n o r m a l i z a t i o n o f o t h e r s t r u c t u r a l and f u n c t i o n a l i n d i c e s . The appa r en t co -dependence o f ATP c o n t e n t and c o n t r a c t i l e f u n c t i o n has l e d t o t h e s u g g e s t i o n t h a t t h e r a p i d d e c r e a s e i n ATP may be r e s p o n s i b l e f o r t h e immed ia te d e c l i n e i n t e n s i o n seen a f t e r c o r o n a r y l i g a t i o n . However , Hea r se has shown t h a t w i t h i n 5 seconds o f a n o x i a , ATP has been r edu ced by 152 25% w h i l e c o n t r a c t i l i t y r ema i n s c o n s t a n t . As w e l l as d e m o n s t r a t i n g a d i s s o c i a t i o n o f t he two v a r i a b l e s , i t i s i n t e r e s t i n g t o compare t h e s e r e s u l t s f r om a n o x i c i n j u r y w i t h t h o s e o f K l o n e r e t a l . who have shown t h a t t h e o p p o s i t e s i t u a t i o n o c c u r s i n i s c h a e m i a f o l l o w i n g l i g a t i o n o f a c o r o n a r y 23 a r t e r y , i . e . , c o n t r a c t i l i t y has d e c r e a s e d d e s p i t e normal l e v e l s o f ATP . I t i s c l e a r , t h e n , t h a t t h e n a t u r e o f t h e r e l a t i o n s h i p between ATP l e v e l s and c o n t r a c t i l e f o r c e i s no t a s i m p l e one . - 130 - A n o t h e r p o s s i b l e cause o f t h e immed ia te d e c l i n e i n t e n s i o n i s t h e f a l l i n pH w i t h i n t h e i s c h a e m i c c e l l . R ap i d d e c r e a s e s o f pH ( w i t h i n 5 s e conds ) 153 31 have been r e c o r d e d by bo th p H - s e n s i t i v e e l e c t r o d e and phospho rus 22 + n u c l e a r m a g n e t i c r e s onan c e t e c h n i q u e s . The ma jo r s o u r c e o f t h e H 22 p r o d u c t i o n has been a t t r i b u t e d t o g l y c o l y t i c l a c t a t e a c c u m u l a t i o n . A g a i n , a l t h o u g h t h e p a r a l l e l changes i n t e n s i o n and pH bo th w i t h i s c h a e m i a and r e p e r f u s i o n s ugge s t t h a t t h e s e two o c c u r r e n c e s may be r e l a t e d , t h i s does no t p r e c l u d e a co -dependence o f pH and t e n s i o n on some o t h e r f a c t o r . I n t e r e s t i n g l y , t h e f a l l i n pH a l s o c l o s e l y p a r a l l e l s t h e d e c r e a s e s i n m i t o c h o n d r i a l ATPase a c t i v i t y , and bo t h r e c o v e r t o c o n t r o l l e v e l s w i t h r e p e r f u s i o n , i . e . , r e c o v e r y i s a f l o w - d e p e n d e n t r a t h e r t h an ATP-dependent phenomenon. Whether o r no t t h e f a l l i n pH i s r e s p o n s i b l e f o r t he ob s e r v ed d e c r e a s e i n m i t o c h o n d r i a l enzyme a c t i v i t y i s a m a t t e r o f s p e c u l a t i o n and i n v i t r o s t u d i e s on t h e pH dependence o f t h i s enzyme wou l d be o f i n t e r e s t . Drug S t u d i e s Drug p r e t r e a t m e n t p r i o r t o a 20 m i nu t e p e r i o d o f l i g a t i o n had some e f f e c t on t h e measured i n d i c e s o f c e l l u l a r i n j u r y . Of p a r t i c u l a r b e n e f i t was t h e c a l c i u m channe l b l o c k i n g agent D-600 HCI (me thoxyve rapam i1 ) wh i c h s i g n i f i c a n t l y p r e s e r v e d m i t o c h o n d r i a l ATPase a c t i v i t y and ATP l e v e l s . P r o p r a n o l o l , had some e f f e c t on p r e s e r v i n g t h e m i t o c h o n d r i a l enzyme a c t i v i t y a l t h o u g h t h i s d i d no t r e a ch s t a t i s t i c a l s i g n i f i c a n c e . D e s p i t e t h e f a c t t h a t p r o p r a n o l o l d e c r e a s e d r a t e and i n c r e a s e d ATP l e v e l s i n c o n t r o l h e a r t s , t h i s agen t d i d no t p r e s e r v e ATP l e v e l s d u r i n g 20 m i nu t e s o f l i g a t i o n . N e i t h e r p r o p r a n o l o l no r p r a n o l i u m , t h e q u a t e r n a r y d e r i v a t i v e o f p r o p r a n o l o l , had s i g n i f i c a n t e f f e c t s on any o f t h e measured i n d i c e s o f i n j u r y o t h e r t han f i b r i l l a t i o n . The r e l a t i v e l y s h o r t p e r i o d o f i s c h a e m i a i s j u s t a t t h e t r a n - s i t i o n o f r e v e r s i b l e t o i r r e v e r s i b l e s a r co l emma l i n j u r y and c o n s e q u e n t l y t h e - 131 - e f f e c t s o f d rug p r e t r e a t m e n t a r e no t c l e a r c u t . In c o n t r a s t t o t h e r e s u l t s o f t h e 20 m i n u t e l i g a t i o n s t u d y shown h e r e , some i n v e s t i g a t o r s have shown t h a t p r o p r a n o l o l r e du ce s i s c h a e m i c damage ( see I n t r o d u c t i o n ) . However , o t h e r s have a l s o f a i l e d t o d emon s t r a t e any p r o t e c t i v e a c t i o n o f e i t h e r p r a n - 154 155 156 o l i u m ' o r d - p r o p r a n o l o l ( t h e n o n - B - b l o c k i n g o p t i c a l i s omer o f 1 - p r o p r a n o l o l ) i n p r e v e n t i n g i s c h a e m i c i n j u r y . T h u s , any b e n e f i c i a l a c t i o n s o f ^ - b l o c k e r s p r o b a b l y a r i s e f r om e - b l o c k a d e and no t o t h e r memb rane - r e l a t e d p r o p e r t i e s . C o n t r a r y t o e x p e c t a t i o n s , one o f t h e most i n t e r e s t i n g a s p e c t s o f t he 20 m i nu t e l i g a t i o n s t u d y was t h e e f f e c t o f d rugs on t h e i n c i d e n c e o f v e n t r i c u l a r f i b r i l l a t i o n . P r o p r a n o l o l and c h l o r p r o m a z i n e s i g n i f i c a n t l y i n c r e a s e d t h e i n c i d e n c e o f f i b r i l l a t i o n whereas D-600 a b o l i s h e d i t . P r a n o l - ium d i d no t a l t e r t h e f r e q u e n c y o f f i b r i l l a t i o n f r om t h a t o b s e r v ed w i t h s a l i n e p r e t r e a t m e n t . The a b i l i t y o f D-600 t o d e c r e a s e v e n t r i c u l a r a r r h y t h - m ias may be e x p l a i n e d by t h e b l o c k a d e o f s low c a l c i u m c h a n n e l s wh i ch may be opened i n d e p o l a r i z e d , i s c h a e m i c v e n t r i c u l a r t i s s u e t h e r e b y i n i t i a t i n g abnormal c o n d u c t i o n . The e f f e c t s o f p r o p r a n o l o l , c h l o r p r o m a z i n e and p r a n o l i u m a re l e s s e a s i l y e x p l a i n e d . P r o p r a n o l o l and c h l o r p r o m a z i n e bo th have q u i n i d i n e - l i k e e f f e c t s , i . e . , d e c r e a s e d a u t o m a t i c i t y o f P u r k i n j e 157 158 159 t i s s u e and d e c r e a s e d membrane r e s p o n s i v e n e s s . ' ' However , t h e s e e f f e c t s wou ld t end t o oppose t h e deve lopment o f a b e r r a n t c o n d u c t i o n pathways r a t h e r t h an p r e c i p i t a t e t hem. I t i s p o s s i b l e t h a t t h e h i g h doses used i n t h i s s t u d y i n t h e p r e s en ce o f d e p o l a r i z e d , i s c h a e m i c t i s s u e may c r e a t e an env i r onmen t t h a t p romotes v e n t r i c u l a r a r r h y t h m i a s a l t h o u g h a t h i g h e r d o s e s , p r o p r a n o l o l r e du ce s t h e i nwa rd f a s t sod ium c u r r e n t and one wou l d a l s o e x p e c t t h i s a c t i o n t o s upp r e s s abnormal v e n t r i c u l a r r h y t h m . These agen t s a l s o i n c r e a s e t h e P-R i n t e r v a l and r e du c e c o n d u c t i o n t h r o u g h the a t r i o v e n t r i c u l a r (A -V ) node . However , t h i s p r o p e r t y i s a l s o s h a r e d by D-600 wh i ch does no t have the - 132 - d e l e t e r i o u s e f f e c t on e l e c t r i c a l a c t i v i t y . Thus , most o f t he above a c t i o n s o f t h e s e agen t s c anno t r e a d i l y a c coun t f o r t h e i n c r e a s e i n f i b r i l l a t i o n . A p o s s i b l e mechan ism o f p r o p r a n o l o l may be r e l a t e d t o t he f a c t t h a t i t can i n c r e a s e t h e e f f e c t i v e r e f r a c t o r y p e r i o d (ERP) o f t h e A-V node w i t h l i t t l e e f f e c t on normal a t r i a l and v e n t r i c u l a r m u s c l e , w h i l e a t t he same t ime s u b - 1 C I s t a n t i a l l y s h o r t e n i n g t h e t h e ERP o f P u r k i n j e f i b r e s . One m igh t t h e r e - f o r e s p e c u l a t e t h a t t h e r e d u c t i o n i n t h e r e f r a c t o r y p e r i o d o f P u r k i n j e t i s s u e may a l l o w an e s cape phenomenon t o d e v e l o p i n t h e v e n t r i c l e s , e s p e c i - a l l y when c o u p l e d w i t h t h e s l owed c o n d u c t i o n t h r o u g h t h e A-V node . P r a n o l - ium does no t s h a r e t h e b e t a - b l o c k i n g p r o p e r t i e s o f p r o p r a n o l o l a l t h o u g h i t has many o f t h e same a c t i o n s on i s o l a t e d m e m b r a n e s ^ . From t h e d a t a p r e s e n t e d h e r e , one m igh t c o n c l u d e t h a t i t i s t h e b e t a - b l o c k i n g a c t i o n s o f p r o p r a n o l o l t h a t a r e r e s p o n s i b l e f o r t h e a r r h y t h m o g e n e s i s ; howeve r , n o n - b e t a - b l o c k i n g p r o p e r t i e s o f p r o p r a n o l o l may c o n t r i b u t e t o t h e i n c r e a s e d f i b r i l l a - t i o n f r e q u e n c y and whe the r o r no t t h e s e a r e a l s o s ha r ed by p r a n o l i u m i s d i f f i c u l t t o s a y . The r e a l p rob l em l i e s i n t he f a c t t h a t t h e knowledge o f t h e p r e c i s e mechanisms u n d e r l y i n g a r r h y t h m o g e n e s i s a r e l a r g e l y s p e c u l a t i v e i n n a t u r e . An a l t e r n a t i v e e x p l a n a t i o n may be f ound i n t h e e f f e c t s o f t h e s e agen t s on t h e work o f t h e h e a r t . I f p r o p r a n o l o l o r c h l o r p r o m a z i n e r educed t h e supp l y /demand r a t i o t o a c r i t i c a l l e v e l , one c o u l d imag ine f i b r i l l a t i o n r e s u l t i n g f r om t h e i n c r e a s e d damage caused by an e x a c e r b a t i o n o f i s c h a e m i c i n j u r y . P r o p r a n o l o l , a t l e a s t , p r o b a b l y d i m i n i s h e d t h e work o f t h e i s c h a e - mic h e a r t by a r e d u c t i o n o f r a t e and c o n t r a c t i l e f o r c e ( see be l ow) and t h i s , i n c o n j u n c t i o n w i t h t h e improved i n t e g r i t y o f p r o p r a n o l o l - t r e a t e d h e a r t s , make i t u n l i k e l y t h a t t h i s i s t h e mechan ism o f i t s a c t i o n . C h l o r p r o m a z i n e , on t h e o t h e r h and , may have had i t s e f f e c t s by e x a c e r b a t i n g i s c h a e m i c t i s s u e - 133 - damage. I t i s e v i d e n t t h a t t he c au se o f t he i n c r e a s e d f i b r i l l a t i o n f r e q u e n - c y w i t h t h e s e d rugs i s no t e a s i l y e x p l a i n e d and f u r t h e r s t u d i e s a r e r e q u i r e d i n o r d e r t o a s c e r t a i n t h e u n d e r l y i n g m o l e c u l a r a l t e r a t i o n s . Bo th D-600 and p r o p r a n o l o l m i gh t be e x p e c t e d t o have s a l u t a r y a c t i o n s on i s c h a e m i a / r e p e r f u s i o n i n j u r y because o f p o t e n t i a l e f f e c t s on t h e s u p p l y / d e - mand r a t i o as w e l l as c a l c i u m channe l b l o c k a d e . D-600 and p r o p r a n o l o l a r e c a p a b l e o f r e d u c i n g c a l c i u m e n t r y a l t h o u g h p r o p r a n o l o l can do t h i s d i r e c t l y CC I CO o n l y a t p h a r m a c o l o g i c a l d o s e s . ' R e d u c t i o n o f c a l c i u m e n t r y by f u n c - t i o n a l a n t agon i sm by p r o p r a n o l o l wou ld be e x p e c t e d a t doses t h a t p roduce B - b l o c k a d e . C a l c i u m i n f l u x i s t h ough t t o be d e l e t e r i o u s f o r t he f o l l o w i n g 2+ r e a s o n s ; m y o f i b r i l l a r a c c u m u l a t i o n can r e s u l t i n c o n t r a c t u r e , Ca - d e p e n - 2+ den t d e g r a d a t i v e enzymes may be a c t i v a t e d , Ca a c c u m u l a t i o n by m i t o c h o n - 1 9 7 d r i a o c c u r s a t t h e expense o f ATP s y n t h e s i s , and v e r y h i g h c e l l u l a r c a l c i u m c o n c e n t r a t i o n w i l l i n h i b i t a v a r i e t y o f e n z y m a t i c r e a c t i o n s . These d r ug s a l s o d e c r e a s e t h e oxygen con sump t i on o f t h e h e a r t by r e d u c i n g some o f t h e d e t e r m i n a n t s o f m y o c a r d i a l e ne r g y c o n s u m p t i o n , s p e c i f i c a l l y h e a r t r a t e , mean a r t e r i a l p r e s s u r e , s t r o k e vo l ume , and w a l l s t r e s s . G i bb s has d e t e r - mined t h a t 6 - a d r e n e r g i c b l o c k i n g agen t s r e du ce ene rgy consumpt i on v i a a r e d u c t i o n i n h e a r t r a t e bu t t h a t t h i s s a v i n g w i l l be o f f - s e t by an i n c r e a s e i n e n d - d i a s t o l i c vo lume ( i n c r e a s i n g w a l l s t r e s s ) . On b a l a n c e , t h o u g h , t h e d e c r e a s e i n h e a r t r a t e , a f t e r l o a d and c o n t r a c t i l i t y p roduced by p r o p r a n o l o l a d m i n i s t r a t i o n w i l l more t han compensate f o r any i n c r e a s e i n e n d - d i a s t o l i c v o l ume . C a l c i u m c h a n n e l - b l o c k e r s a l s o r educe c o n t r a c t i l i t y , d e c r e a s e t h e I C O a f t e r l o a d and d i r e c t l y d i l a t e t h e c o r o n a r y v a s c u l a t u r e ( t h e r e b y i n c r e a - s i n g t he supp l y /demand r a t i o ) . Thu s , a l t h o u g h bo th o f t h e s e agen t s wou ld be e x p e c t e d t o r e t a r d i s c h a e m i c damage, whe the r o r no t t h e y a c t u a l l y r educe t h e f i n a l e x t e n t o f i s c h a e m i c i n j u r y i s p r e s e n t l y no t known. In t h e p r e s e n t - 134 - s t u d i e s , when i r r e v e r s i b l e i s c h a e m i c damage (40 m i n u t e s ) was f o l l o w e d by r e p e r f u s i o n , bo th D-600 and p r o p r a n o l o l p r oduced a p a r a l l e l deg ree o f p r o t e c t i o n t o t he i s c h a e m i c myocard ium a l t h o u g h D-600 was c o n s i s t e n t l y s u p e r i o r . Bo th d rugs p r e v e n t e d t h e d e c r e a s e i n m i t o c h o n d r i a l ATPase a c t i v - i t y as w e l l as r e d u c i n g t h e e x t e n t o f s a r co l emma l damage, i n c l u d i n g Na ,K -ATPase a c t i v i t y and i on a b n o r m a l i t i e s . D e s p i t e t h e s e b e n e f i c i a l e f f e c t s on membrane i n t e g r i t y , ATP l e v e l s were no t s i g n i f i c a n t l y i n c r e a s e d by e i t h e r d rug p r e t r e a t m e n t , a l t h o u g h t h e r e was a n o n - s i g n i f i c a n t i n c r e a s e i n ATP l e v e l s w i t h D-600 p r e t r e a t m e n t . T h u s , t h e p r o t e c t i v e e f f e c t s do no t appea r t o be e x c l u s i v e l y med i a t ed by a p r e s e r v a t i o n o f ATP . T h i s does no t r u l e ou t t h e p o s s i b i l i t y t h a t a s p e c i f i c " compar tment " o f ATP was i n c r e a s e d w i t h d rug t r e a t m e n t o r t h a t t he p r o l o n g e d d e p r e s s i o n o f h i g h ene rgy pho s - pha te l e v e l s i s r e f l e c t i n g a h i g h e r t u r n o v e r o f ATP f o r c e l l h o m e o s t a s i s . The p r e s e r v a t i o n o f c e l l u l a r i n t e g r i t y by d rug p r e - t r e a t m e n t was no t a c compan ied by p r e s e r v a t i o n o f c o n t r a c t i l e f o r c e . T h i s i s i n c o n t r a s t t o t he work o f o t h e r i n v e s t i g a t o r s who have shown t h a t c a l c i u m channe l b l o c k e r s improve c o n t r a c t i l e f u n c t i o n as w e l l as o t h e r measures o f c e l l v i a b i l i - t y 63 ,79 ,164 j n ^ e p r e s e n t e x p e r i m e n t s , i t i s p o s s i b l e t h a t t h e s m a l l n o n - s i g n i f i c a n t i n c r e a s e i n c o n t r a c t i l i t y i n t h e D-600 group may have been enhanced i f a l a r g e r dose o f t he d rug had been u s e d . P r e l i m i n a r y r e s u l t s u s i n g a dose o f D-600 HCl t w i c e t h a t used i n t h e s t u d i e s p r e s e n t e d he re have demons t r a t ed even g r e a t e r p r o t e c t i v e e f f e c t s on some o f t h e i n d i c e s o f i s c h a e m i a / r e p e r f u s i o n i n j u r y ( d a t a no t shown) . The l a c k o f any improvement i n f u n c t i o n by p r o p r a n o l o l a l s o c o n t r a s t s w i t h t h e b i o c h e m i c a l and m o r p h o l - o g i c a l p r o t e c t i o n o b s e r v e d . I n f a c t , i n c o n t r a s t t o t h e c a l c i u m c h a n n e l - b l o c k e r s , most s t u d i e s have d e s c r i b e d b e n e f i t s o f p r o p r a n o l o l a d m i n i s t r a t i o n 62 154 165 a t t h e m o l e c u l a r r a t h e r t h an f u n c t i o n a l l e v e l . ' ' Mo r e o v e r , ou r - 135 - r e s u l t s w i t h D-600 and p r o p r a n o l o l s u p p o r t t h e h y p o t h e s i s t h a t t h e l e v e l o f ATP i n t h e c e l l i s somehow r e l a t e d t o i t s c o n t r a c t i l e a b i l i t y i n l i g h t o f t h e c o n c o m i t a n t ( a l b e i t s m a l l ) i n c r e a s e i n ATP and c o n t r a c t i l i t y w i t h D-600 and t h e l a c k o f ATP p r e s e r v a t i o n and c o n t r a c t i l e f u n c t i o n w i t h p r o p r a n o l o l . P r e s e r v a t i o n o f c e l l i n t e g r i t y by d rug p r e t r e a t m e n t was a l s o v e r i f i e d by e l e c t r o n m i c r o s c o p i c a n a l y s i s . The appea rance o f t h e m y o c a r d i a l s e c t i o n s f r om t h e i s c h a e m i c / r e p e r f u s e d zone g e n e r a l l y ag reed w i t h t h e as sessmen t o f i n j u r y by b i o c h e m i c a l t e c h n i q u e s . F o r e xamp l e , t h e c a l c i u m l e v e l s i n t h e d r u g - t r e a t e d h e a r t s were o n l y h a l f t h e v a l u e o f u n t r e a t e d 1 i g a t e d / r e p e r f u s e d h e a r t s , a l t h o u g h , t h e i r c a l c i u m c o n t e n t was s t i l l 5 t i m e s g r e a t e r t han c o n t r o l l e v e l s . M i t o c h o n d r i a l i n j u r y was appa r en t i n t h e as sessmen t o f membrane s t r u c t u r e and t h e p r e s en ce o f i n t r a - m i t o c h o n d r i a l d e n s i t i e s and t h e e v i d e n c e o f s a r co l emma l damage by u l t r a s t r u c t u r a l a n a l y s i s p a r a l l e l e d t h e r e s u l t s o f t he enzyme a s s a y s . In a d d i t i o n , bo th o f t h e d r u g - t r e a t e d g roups had h i g h e r s c o r e s i n t h e c o n t r a c t i l e a b n o r m a l i t i e s ( p r e s e n c e o f c o n t r a c t i o n band and / o r r e l a x a t i o n ) c o n f i r m i n g t h a t t h e sa r co l emma l p e r m e a b i l i t y t o c a l c i u m was comprom i sed . However , when i n t e r p r e t i n g t h e r e s u l t s o f an e l e c - t r o n m i c r o s c o p i c s t u d y , one must be c a u t i o u s i n mak ing c o n c l u s i o n s abou t t h e s t a t e o f t h e t i s s u e i n v i v o , i . e . , i t i s p r o b a b l e t h a t some o f t h e damage t o t h e c e l l s i s t h e r e s u l t o f f i x a t i o n a r t i f a c t . Fo r e xamp l e , i t i s d i f f i c u l t t o imag i ne t h a t t h e s e v e r e l y damaged m i t o c h o n d r i a f r om t h e L+R group ( s a l i n e p r e t r e a t m e n t ) have an ATPase a c t i v i t y wh i c h i s 75% o f c o n t r o l . The r e i s a f u r t h e r p r ob l em w i t h t h e use o f u l t r a s t r u c t u r a l d a t a a l o n e because t h e r e i s o f t e n a g r e a t d i f f e r e n c e between t he a p p a r e n t l y normal "mummi f i ed " c e l l and i t s v i a b i l i t y as a s s e s s e d by b i o c h e m i c a l t e c h n i q u e s . A s t r i k i n g example o f t he d i s p a r i t y between u l t r a s t r u c t u r a l appea rance and b i o c h e m i c a l a s sessmen t o f c e l l i n j u r y was shown by R u i g r o k e t a l . i n wh i ch t h e y found t h a t i s c h a e - - 136 - m ic m y o c a r d i a l t i s s u e p r e t r e a t e d w i t h DMSO appea red norma l on e l e c t r o n m i c r o g r a p h s even though t he sa rco lemma was pe rmeab le t o t he d ye , t r y p a n 166 b l u e . T h e r e f o r e , a more a c c u r a t e p i c t u r e o f t h e s t r u c t u r a l i n t e g r i t y o f t h e t i s s u e i s o b t a i n e d by a c o r r o b o r a t i o n o f s e v e r a l d i f f e r e n t methods o f e v a l u a t i o n as was done i n t h e p r e s e n t s t u d y . The r e s u l t s f r om t h e r e p e r f u s i o n s t u d i e s p r e s e n t e d he re have i n d i c a t e d t h a t e l e v a t e d c a l c i u m l e v e l s w i t h i n t h e t i s s u e a re a s s o c i a t e d w i t h a d e p r e s - s i o n i n m i t o c h o n d r i a l ATPase a c t i v i t y . The a c c u m u l a t i o n o f c a l c i u m by t h e m i t o c h o n d r i o n i s one o f t he p r o c e s s e s o f c a l c i u m h o m e o s t a s i s t h a t m a i n t a i n c y t o s o l i c c a l c i u m i n t h e r ange o f a p p r o x i m a t e l y 1 0 - 7 M a l t h o u g h m i t o c h o n - d r i a l c a l c i u m up t ake does no t g e n e r a l l y o p e r a t e e x c e p t a t c a l c i u m c o n c e n t r a - t i o n s f a r i n e x c e s s o f norma l l e v e l s ( a p p r o x i m a t e l y 1 0 " ̂  M ) . The m i t o - c h o n d r i o n has a l a r g e c a p a c i t y f o r c a l c i u m , w h i c h , i n t h e p r e s en c e o f pho s - p h a t e , w i l l f o rm c a l c i u m phospha t e c r y s t a l s . As s t a t e d p r e v i o u s l y , c a l c i u m up take a l s o o c c u r s a t t h e expense o f ATP s y n t h e s i s . T h i s wou l d have m y r i a d e f f e c t s on c e l l f u n c t i o n i n a d d i t i o n t o c omp rom i s i ng t h e a c t i v e t r a n s p o r t o f more c a l c i u m ou t o f t h e c e l l . Once t h e c a p a c i t y o f t he m i t o c h o n d r i o n i s e x c e e d e d , howeve r , c a l c i u m e f f l u x o c c u r s and t h e r e i s a c o l l a p s e o f t h e m i t o c h o n d r i a l membrane p o t e n t i a l , p r e sumab l y r e s u l t i n g i n i r r e v e r s i b l e m i t o - c h o n d r i a l d a m a g e . 1 6 7 T h i s t y p e o f damage o c c u r s l o ng a f t e r t h e d e c r e a s e i n o t h e r m i t o c h o n d r i a l f u n c t i o n s , and g i v e n t h e r e l a t i v e r e v e r s i b i l i t y o f m i t o c h o n d r i a l s t r u c t u r a l and f u n c t i o n a l i n j u r y , i t wou l d appea r t h a t t h e u l t i m a t e i r r e v e r s i b l e d e p r e s s i o n o f m i t o c h o n d r i a l f u n c t i o n may r e s u l t f r om an i n c r e a s e i n s a r co l emma l p e r m e a b i l i t y t o c a l c i u m . A d d i t i o n a l l y , sod ium a c c u m u l a t i o n (wh i ch i n t h e p r e s e n t e x p e r i m e n t s o c c u r s much e a r l i e r t han c a l c i u m i n f l u x ) w i l l t e n d t o c r e a t e an i n f l u x o f wa te r i n t o t he c e l l , w h i c h , - 137 - i f s u f f i c i e n t l y s e v e r e , has a l s o been p o s t u l a t e d t o p e r t u r b m i t o c h o n d r i a l f u n c t i o n by c a u s i n g m i s a l i g n m e n t o f s p a t i a l l y - o r i e n t e d enzyme sys tems w i t h i n 1 C O + t h e m i t o c h o n d r i a l m a t r i x . R e p e r f u s i o n per se c au se s m a s s i v e Na and 2+ Ca i n f l u x , t h e r e b y r e d u c i n g m i t o c h o n d r i a l f u n c t i o n by a t l e a s t two p o s s - i b l e mechan i sms . I t wou ld a ppea r , t h e n , t h a t i r r e v e r s i b l e m i t o c h o n d r i a l damage does no t o c c u r u n l e s s t h e sa r co l emma l p e r m e a b i l i t y i s g r e a t enough t o p e r m i t Ca t o a c cumu l a t e i n t r a c e l l u l a r ^ t o l e v e l s t h a t a re beyond t he a c c u m u l a t i n g c a p a c i t y o f t h e m i t o c h o n d r i o n and /o r l a r g e i n f l u x e s o f N a + and w a t e r . The i n f l u e n c e o f N a + and wa t e r i n f l u x p e r se on m i t o c h o n d r i a l f u n c t i o n i s no t p r e c i s e l y known. However , t h e p r e s e n t e x p e r i m e n t s d i d d emons t r a t e a r e l a t i o n s h i p between the l e v e l o f i n t r a c e l l u l a r c a l c i u m and m i t o c h o n d r i a l f u n c t i o n . R e d u c t i o n o f c a l c i u m l e v e l s by p r e t r e a t m e n t w i t h e i t h e r D-600 o r p r o p r a n o l o l r e s u l t e d i n p r e s e r v a t i o n o f m i t o c h o n d r i a l ATPase a c t i v i t y . F u r t h e r m o r e , a d d i t i o n o f D-600 p r i o r t o r e p e r f u s i o n a l s o s i g n i f i - c a n t l y p r e v e n t e d t h e d e c r e a s e i n m i t o c h o n d r i a l ATPase a c t i v i t y and s l i g h t l y p r e s e r v e d ATP l e v e l s d e s p i t e t h e absence o f any b e n e f i c i a l e f f e c t on o t h e r a s p e c t s o f c e l l f u n c t i o n i n c l u d i n g sa r co l emma l p e r m e a b i l i t y t o o t h e r i o n s . I s t h e mechanism o f p r o t e c t i o n o f c e l l u l a r i n t e g r i t y by t h e s e agen t s r e l a t e d t o t h e i r a b i l i t y t o d i r e c t l y p r e v e n t c a l c i u m i n f l u x r a t h e r t han v i a t h e i r a b i l i t y t o r educe t h e m e t a b o l i c r e q u i r e m e n t s o f t h e i s c h a e m i c c e l l ? In t he e x p e r i m e n t s i n v o l v i n g 40 m i nu t e s o f l i g a t i o n f o l l o w e d by 60 m i nu t e s o f r e p e r f u s i o n , one wou ld have e x p e c t e d o n l y D-600 t o r educe t h e c a l c i u m i n f l u x d u r i n g r e p e r f u s i o n v i a a d i r e c t b l o c k a d e o f c a l c i u m c h a n n e l s . P r o p r a n o l o l wou ld o n l y r educe c a l c i u m a c c u m u l a t i o n i f i t p r e s e r v e d s a r c o l e m - mal i n t e g r i t y d u r i n g t he i s c h a e m i c p e r i o d . Both agen t s were f ound t o r educe c a l c i u m i n f l u x t o t h e same e x t e n t and f u r t h e r m o r e , bo th p r o v i d e d some p r o t e c t i o n a g a i n s t N a + i n f l u x . T h i s r e s u l t s u gge s t s t h a t t he mechan ism o f - 138 - p r o t e c t i o n o c c u r s a s a r e s u l t o f d r u g - i n d u c e d d e c r e a s e s i n m y o c a r d i a l e n e r g y r e q u i r e m e n t s d u r i n g t h e i s c h a e m i c p e r i o d w i t h c o n s e q u e n t p r e s e r v a t i o n o f s a r c o l e m m a l i n t e g r i t y . T h e i m p o r t a n c e o f c a l c i u m c h a n n e l b l o c k a d e p e r s e d u r i n g r e p e r f u s i o n was e x a m i n e d i n t h e s t u d y i n w h i c h D-600 was a d m i n i s t e r e d i m m e d i a t e l y p r i o r t o r e p e r f u s i o n a f t e r a 4 0 m i n u t e l i g a t i o n p e r i o d . A s m e n t i o n e d a b o v e , t h i s i n t e r v e n t i o n r e d u c e d c a l c i u m i n f l u x a n d p r o d u c e d a c o n c o m i t a n t p r e s e r v a t i o n o f m i t o c h o n d r i a l A T P a s e a c t i v i t y ( a n d ATP l e v e l s t o a s m a l l e x t e n t ) . How- + + 2+ e v e r , i t h a d no e f f e c t on Na o r w a t e r i n f l u x , K a n d Mg l o s s , N a + , K + - A T P a s e a c t i v i t y , o r ( d p / d t ) m a x > t n e r e D V c o n f i r m i n g t h e h y p o t h e - s i s t h a t t h e d r u g s ' b e n e f i c i a l a c t i o n s o c c u r p r i m a r i l y d u r i n g t h e i s c h a e m i c p e r i o d . T h e r e h a s . b e e n a g r e a t d e a l o f c o n t r o v e r s y i n t h e l i t e r a t u r e r e g a r d i n g t h e m e c h a n i s m o f B - a d r e n e r g i c a n t a g o n i s t a n d c a l c i u m c h a n n e l b l o c k e r p r o t e c - t i o n a g a i n s t i s c h a e m i a / r e p e r f u s i o n i n j u r y . 6 1 , 7 9 ' ' ' ' 6 9 - " ' ' 7 ' ' ' T h e r e s u l t s o f t h e p r e s e n t e x p e r i m e n t s may p r o v i d e some i n s i g h t s i n t o t h e r e a s o n s f o r 2+ d i f f e r i n g o p i n i o n s on t h e r e l a t i v e i m p o r t a n c e o f C a b l o c k a d e y_s. d e c r e a s e d e n e r g y r e q u i r e m e n t s i n p r e v e n t i n g r e p e r f u s i o n d a m a g e . A d m i n i s t r a - t i o n o f c a l c i u m a n t a g o n i s t s a f t e r t h e i s c h a e m i c i n s u l t a n d p r i o r t o r e p e r f u - s i o n d i r e c t l y r e d u c e s c a l c i u m o v e r l o a d t h e r e b y p r e s e r v i n g m i t o c h o n d r i a l f u n c t i o n a n d ATP s t o r e s . I f t h e s e i n d i c e s a r e u s e d a s t h e e x c l u s i v e d e t e r - m i n a n t s o f c e l l u l a r i n t e g r i t y , o n e w o u l d e x p e c t t o o b s e r v e a p r o t e c t i v e e f f e c t o f c a l c i u m c h a n n e l b l o c k e r a d m i n i s t r a t i o n i n r e p e r f u s i o n i n j u r y , e s p e c i a l l y i n t h e s h o r t - t e r m . H o w e v e r , i t i s d o u b t f u l t h a t t h e p r e s e r v a t i o n o f m i t o c h o n d r i a l f u n c t i o n w o u l d b e o f s u s t a i n e d b e n e f i t w h e n , a t t h e same t i m e , t h e r e i s i r r e v e r s i b l e s a r c o l e m m a l m e m b r a n e damage. T h e p r e s e n t r e s u l t s i n d i c a t e t h a t t h e p r e d o m i n a n t b e n e f i c i a l e f f e c t i s d e r i v e d f r o m a - 139 - r e d u c t i o n o f MVO^,. T h i s h y p o t h e s i s r e c e i v e s f u r t h e r s u p p o r t f r om t he work o f R o b b - N i c h o l s o n e t a l . i n wh i c h e i t h e r KC1 (16 mEq/1) o r v e r a p a m i l (2 ug /m l ) was added t o r educe t h e ene r g y r e q u i r e m e n t s o f p e r f u s e d , i s o l a t e d r a t h e a r t s . The r e s u l t s showed t h a t KC1 and v e r a p a m i l p r o v i d e d e q u i v a l e n t 172 p r o t e c t i o n a g a i n s t i s c h a e m i a / r e p e r f u s i o n i n j u r y . F u r t h e r m o r e , c o n s i s - t e n t w i t h t h e i d e a t h a t c a l c i u m channe l b l o c k a d e may s e l e c t i v e l y improve ATP l e v e l s , t h e s e a u t h o r s f ound t h a t D-600 a f f o r d e d s l i g h t l y b e t t e r p r o t e c t i o n t h an KC1 a g a i n s t ATP l o s s e s . S i m i l a r r e s u l t s were o b t a i n e d i n t he i s o l a t e d , i s o v o l u m i c f e l i n e h e a r t by Magee e t a l . who a l s o f ound s i m i l a r p r o t e c t i v e e f f e c t s o f n i f e d i p i n e ( a n o t h e r c a l c i u m channe l b l o c k e r ) and KC1 bu t o n l y 170 when t h e dose o f n i f e d i p i n e was s u f f i c i e n t t o p roduce immed ia te a s y s t o l e . The e x pe r imen t w h e r e i n D-600 was added p r i o r t o r e p e r f u s i o n has - shown t h a t c a l c i u m i n f l u x i s a s s o c i a t e d w i t h i s c h a e m i c / r e p e r f u s i o n i n j u r y d u r i n g t he i s c h a e m i c p e r i o d . The r e s u l t s o f e x p e r i m e n t s p r e s e n t e d h e r e i n have shown t h a t even a f t e r 60 m i nu t e s o f i s c h a e m i a i n wh i ch c o r o n a r y f l o w was r e du ced t o a p p r o x i m a t e l y 5% o f t h e b l o o d f l o w i n t h e n o n - i s c h a e m i c l e f t v e n t r i c l e , t h e o c c l u d e d zone d i d no t a c cumu l a t e c a l c i u m t o any s i g n i f i c a n t deg r ee ( c f . N a + / K + r a t i o ) . More s e n s i t i v e t e c h n i q u e s u s i n g r a d i o a c t i v e - l y - l a b e l l e d 4 5 C a 2 + o r 4 7 C a 2 + have a l s o shown t h a t t he c a l c i u m does 51 174 no t a c cumu l a t e d u r i n g a 60 m i nu t e i s c h a e m i c p e r i o d . ' These s t u d i e s wou ld no t be a b l e t o d e t e c t r a p i d l y e x c h a n g i n g c a l c i u m so i t i s p o s s i b l e t h a t a s t i m u l a t i o n o f e f f l u x c o u l d have compensated f o r s m a l l i n c r e a s e s i n 2+ i n t r a c e l l u l a r Ca l e v e l s . The re a r e a t l e a s t two o t h e r mechanisms f o r ma i n t enance o f t h e c a l c i u m g r a d i e n t and bo th o p e r a t e a t l owe r c a l c i u m 2+ c o n c e n t r a t i o n s t h an t h e m i t o c h o n d r i a l u p t a k e . Sa rco lemma l Ca -ATPase has a h i g h a f f i n i t y f o r c a l c i u m ( K M = .2 - . 6 uM) a l t h o u g h a l owe r t r a n s p o r t r a t e as compared w i t h t h e ATP - i n d ependen t N a + - C a 2 + e x c hange . The - 140 - 2+ l i m i t i n g f a c t o r f o r c a l c i u m e x t r u s i o n by Ca - ATPa se wou ld be t h e a v a i l a - + 2+ b i l i t y o f ATP and f o r Na - C a exchange i t wou ld be a r e d u c t i o n i n t h e + Na g r a d i e n t . Thu s , even w i t h low c o n c e n t r a t i o n s o f ATP, t h e c e l l c o u l d s t i l l u t i l i z e t h e N a + g r a d i e n t t o remove e x c e s s c a l c i u m . T h e r e f o r e , + + 2 + 2 + i n c r e a s e s i n t h e Na /K r a t i o i n t h e absence o f changes i n Ca /Mg c o n t e n t may be e x p l a i n e d e i t h e r by p o s t u l a t i n g t h a t a s p e c i f i c a l t e r a t i o n i n N a + , K + - A T P a s e ( w i t h a c o n c o m i t a n t i n c r e a s e i n t h e N a + / K + r a t i o ) o c c u r s w i t h o u t any i n c r e a s e i n s a r co l emma l c a l c i u m p e r m e a b i l i t y o r t h a t t h e i n f l u x o f c a l c i u m a r i s i n g f r om a g e n e r a l i n c r e a s e i n sa r co l emma l p e r m e a b i l - i t y i s compensated f o r by c a l c i u m e f f l u x . In e i t h e r c a s e , t he i s c h a e m i c m y o c a r d i a l c e l l can m a i n t a i n low i n t r a c e l l u l a r c a l c i u m l e v e l s . However , t h i s does no t t a k e i n t o a c c oun t any r e - d i s t r i b u t i o n o f c a l c i u m w i t h i n t h e c e l l , e . g . , r e l e a s e i n t o t h e c y t o s o l f r om t h e s a r c o p l a s m i c r e t i c u l u m o r m i t o c h o n d r i o n . I t i s u n l i k e l y t h a t c a l c i u m r e l e a s e wou l d o c c u r f rom t h e m i t o c h o n d r i o n because t h e a s s o c i a t e d c o l l a p s e o f t h e m i t o c h o n d r i a l membrane p o t e n t i a l i s more ap t t o be a consequence r a t h e r t han a c au se o f c y t o p l a s m i c c a l c i u m a c c u m u l a t i o n . T h i s a ppa r en t p r e s e r v a t i o n o f t h e c a l c i u m g r a d i e n t i n i r r e v e r s i b l y - i n - j u r e d i s c h a e m i c c e l l s ( e xposed t o p e r i o d s o f i s c h a e m i a g r e a t e r t h an 30 m in) i s r e a d i l y d e s t r o y e d by t h e r e - i n s t a t e m e n t o f b l o o d f l o w t o t h e o c c l u d e d z o n e . Some a u t h o r s have s i m p l y e x p l a i n e d t h i s phenomenon as r e s u l t i n g f r om t he l a r g e i n f l u x o f p l a sma c a l c i u m e n t e r i n g t h e i s c h a e m i c t i s s u e w i t h t h e f r e s h b l o o d o f r e p e r f u s i o n . However , even w i t h z e r o f l o w , t h e r e i s s t i l l a v e r y l a r g e c a l c i u m g r a d i e n t a c r o s s t h e membrane a nd , f r om the above e v i - d en c e , i t appea r s t h a t t h e sa r co l emma l b a r r i e r t o t h e h i gh e x t r a c e l l u l a r c o n c e n t r a t i o n o f t h i s i o n r ema i n s r e l a t i v e l y i n t a c t . Thus , i t i s no t j u s t t h e p r e s en ce o f c a l c i u m i n t h e r e p e r f u s i n g b l o o d t h a t c au se s c a l c i u m o v e r - - 141 - l o a d . R e p e r f u s i o n o f i r r e v e r s i b l y damaged c e l l s p r e c i p i t a t e s some e v e n t ( s ) t h a t d e s t r o y t h e sa r co l emma l p e r m e a b i l i t y b a r r i e r . As y e t , t h e r e has been no s a t i s f a c t o r y answer as t o what c au se s t h e sudden i n c r e a s e i n p e r m e a b i l i - t y . I t i s i n t e r e s t i n g t o no t e t h a t a t l e a s t some o f t he c a l c i u m i n f l u x d u r i n g r e p e r f u s i o n i s t h r o u g h v o l t a g e - s e n s i t i v e c a l c i u m c h a n n e l s t h a t can be b l o c k e d by D-600 . A f t e r c o n s i d e r a t i o n o f t h e s e d a t a , a p o s s i b l e ( a l b e i t s i m p l i f i e d ) mechan ism o f r e p e r f u s i o n - i n d u c e d c a l c i u m a c c u m u l a t i o n m igh t be as f o l l o w s : A f t e r a p p r o x i m a t e l y 20 -30 m i nu t e s o f i s c h a e m i a , i r r e v e r s i b l e damage t o t h e l i p i d e n v i r o nmen t o f t h e p l a sma membrane-bound N a + , K + - A T P a s e o c c u r s v i a some mechan ism t h a t i s p r o b a b l y a s s o c i a t e d w i t h low ATP l e v e l s . As i s c h a e m i a p r o g r e s s e s , t h e g r a d i e n t s f o r Na + and K + r u n down. O the r compensa to r y mechan isms f o r N a + and K + h o m e o s t a s i s a p p a r e n t l y do no t e x i s t , and t h e c e l l g r a d u a l l y d e p o l a r i z e s . 2+ D u r i n g i s c h a e m i a , Ca does no t e n t e r t h e c e l l because s a r c o l e m - mal p e r m e a b i l i t y t o c a l c i u m i s e i t h e r unchanged o r s m a l l i n f l u x e s a r e compensa ted f o r by ( e . g . , Ca -ATPase and Na - C a e x c h a n g e ) . Upon r e p e r f u s i o n a f t e r i r r e v e r s i b l e i n j u r y , t h e r e i s a l a r g e i n f l u x o f w a t e r and N a + as w e l l as e f f l u x o f K + . Water w i l l f l o w i n t o t h e c e l l be cause o f t h e i n c r e a s e d i n t e r n a l o s m o t i c p r e s s u r e wh i ch r e s u l t s f r om t h e a c c u m u l a t i o n o f N a + and H + . Sodium i n f l u x exceeds p o t a s - s ium e f f l u x and t he c e l l wou ld d e p o l a r i z e f u r t h e r . When t he membrane p o t e n t i a l becomes l e s s n e g a t i v e ( a p p r o x . - 5 0 mV) , v o l t a g e - s e n s i t i v e 2+ 2+ Ca c h a n n e l s wou ld open and a l l o w Ca i n f l u x . Low ATP l e v e l s 2+ wou ld r educe up take by t h e s a r c o p l a s m i c r e t i c u l u m Ca -ATPase and 2+ + e f f l u x by t h e sa r co l emma l Ca - A T P a s e . F u r t h e r m o r e , t h e Na g r a d i - + 2+ e n t wou ld be a b o l i s h e d and t h e r e f o r e Na - C a exchange wou ld a l s o be - 142 - i n o p e r a t i v e . M i t o c h o n d r i a w i l l t a k e up c a l c i u m u n t i l t h e i r c a p a c i t y i s 2+ e x c e e d e d a f t e r w h i c h p o i n t t h e d a m a g e d m i t o c h o n d r i a r e l e a s e C a i n t o t h e c y t o s o l . T h i s e v e n t , a c c o m p a n i e d b y i n t r a c e l l u l a r o e d e m a , w o u l d c a u s e i r r e v e r s i b l e damage t o m o s t e n z y m e s y s t e m s . A l o g i c a l i m p l i c a t i o n o f t h e s e d a t a w o u l d b e t h a t i f i s c h a e m i a i s s u f f i - c i e n t l y p r o l o n g e d ( s e v e r a l h o u r s ) o n e m i g h t e x p e c t t o s e e a g r a d u a l i n f l u x o f c a l c i u m a s t h e g r a d i e n t s f o r N a + a n d K + a r e d e s t r o y e d a n d t h e c e l l s d e p o l a r i z e . To my k n o w l e d g e , s e r i a l m e a s u r e m e n t s o f t i s s u e i o n s d u r i n g p e r i o d s o f i s c h a e m i a g r e a t e r t h a n 2 h o u r s h a v e n o t b e e n d o n e . T h e d i s c u s s i o n o f " c a l c i u m - i n d u c e d r e p e r f u s i o n i n j u r y " w o u l d n o t b e c o m p l e t e w i t h o u t t h e m e n t i o n o f t h e s o - c a l l e d " c a l c i u m p a r a d o x " p h e n o m e n o n . I t h a s b e e n s h o w n u s i n g i s o l a t e d h e a r t s t h a t a p e r i o d o f c a l c i u m - f r e e p e r f u - 2+ s i o n f o l l o w e d b y p e r f u s i o n w i t h p h y s i o l o g i c a l C a s o l u t i o n c a u s e s e x t e n - s i v e damage t o m y o c a r d i a l c e l l s , i n c l u d i n g i o n d e r a n g e m e n t s s i m i l a r t o t h o s e s e e n w i t h i s c h a e m i a / r e p e r f u s i o n i n j u r y . 1 7 7 V a r i o u s i n v e s t i g a t o r s h a v e e x a m i n e d t h e e f f e c t s o f c a l c i u m c h a n n e l b l o c k e r s o n c a l c i u m e n t r y d u r i n g t h e " r e p e r f u s i o n " p e r i o d w i t h m o s t l y n e g a t i v e r e s u l t s . 1 7 ^ ' 1 7 9 T h e s e r e s u l t s s h o u l d n o t b e e x t r a p o l a t e d t o t h e s i t u a t i o n w i t h i s c h a e m i a / r e p e r f u s i o n i n j u r y b e c a u s e t h e m e c h a n i s m o f s a r c o l e m m a l damage i s p r o b a b l y q u i t e d i f f e r - e n t . D u r i n g c a l c i u m - f r e e p e r f u s i o n t h e s u p e r f i c i a l l y - b o u n d c a l c i u m o f t h e g l y c o c a l y x i s r e m o v e d . T h e p r e s e n c e o f t h i s c a l c i u m i s n e c e s s a r y f o r t h e m a i n t e n a n c e o f an i n t a c t s a r c o l e m m a / g l y c o c a l y x a n d s u b s e q u e n t r e p e r f u s i o n w i t h c a l c i u m - c o n t a i n i n g s o l u t i o n c a u s e s p r o f o u n d c h a n g e s i n m y o c a r d i a l i o n l e v e l s , m o r p h o l o g y a n d b i o c h e m i s t r y . R e p l e t i o n o f c a l c i u m i n t h e p e r f u s a t e a f t e r a 1 0 m i n u t e p e r i o d o f c a l c i u m - f r e e p e r f u s i o n , r e s u l t e d i n l o s s o f 78 c y t o s o l i c e n z y m e s w i t h i n 1 m i n u t e o f " r e p e r f u s i o n " . T h u s , a l t h o u g h t h e - 1 4 3 - d e g r e e o f c a l c i u m a c c u m u l a t i o n may b e c o m p a r a b l e , t h e t i m e c o u r s e a n d f e a t u r e s o f " c a l c i u m p a r a d o x " d amage d i f f e r m a r k e d l y f r o m i s c h a e m i a / r e p e r f u - s i o n i n j u r y . Some r e s e a r c h e r s h a v e i n v e s t i g a t e d t h e e f f i c a c y o f i n t e r v e n - t i o n s f o r c a l c i u m - i n d u c e d r e p e r f u s i o n i n j u r y b y u s i n g t h e " c a l c i u m p a r a d o x " t e c h n i q u e ; h o w e v e r , i t i s s u g g e s t e d t h a t t h i s i s n o t an a p p r o p r i a t e m o d e l o f i s c h a e m i a / r e p e r f u s i o n i n j u r y . T h e r e s u l t s o f t h e s e e x p e r i m e n t s a n d t h o s e o f many o t h e r i n v e s t i g a t o r s , h a v e s h o w n t h a t a f e w a g e n t s , e . g . p r o p r a n o l o l , c a l c i u m c h a n n e l b l o c k e r s , c a n l i m i t t h e s h o r t - t e r m damage p r o d u c e d b y c o r o n a r y o c c l u s i o n a n d r e l e a s e . H o w e v e r , i t i s n o t c l e a r t h a t t h e s e i n t e r v e n t i o n s u l t i m a t e l y i m p r o v e f u n c - t i o n a l s t a t u s . Few l o n g - t e r m s t u d i e s h a v e b e e n p e r f o r m e d . B u s h e t a l . h a v e s h o w n t h a t o v e r a 4 week p e r i o d o f r e p e r f u s i o n , t h e i n i t i a l l y - b e t t e r f u n c - 180 t i o n a l s t a t u s o f d i l t i a z e m - p r e t r e a t e d h e a r t s was n o t m a i n t a i n e d a n d a l l t r e a t m e n t g r o u p s h a d s i m i l a r f u n c t i o n b y t h e e n d o f a 4 week p e r i o d . I n t h e p r e s e n t s t u d i e s , t h e s l i g h t b e n e f i t o f D-600 on ( d P / d t ) was i n c o n t r a s t III d X t o t h e s i g n i f i c a n t b i o c h e m i c a l a n d m o r p h o l o g i c a l i m p r o v e m e n t . T h e p r e s e n c e o f i n t a c t p e r m e a b i l i t y b a r r i e r s w o u l d b e n e c e s s a r y f o r c o n t r a c t i o n ; how- e v e r , t h e i r e x i s t e n c e d o e s n o t mean t h a t c o n t r a c t i l e f o r c e w o u l d b e a b l e t o r e t u r n t o c o n t r o l l e v e l s . T h u s , i t i s i m p o r t a n t t o e m p h a s i z e t h a t p r e s e r v a - t i o n o f m y o c a r d i a l c e l l i n t e g r i t y d o e s n o t n e c e s s a r i l y mean t h a t t h e f u n c - t i o n w i l l a l s o b e p r e s e r v e d . L o n g - t e r m s t u d i e s o f t h e d o s e a n d t i m e - d e p e n - d e n c e o f p r o t e c t i v e e f f e c t s o f b o t h c a l c i u m c h a n n e l b l o c k e r s a n d b e t a - a d r e n - e r g i c a n t a g o n i s t s i n i s c h a e m i c i n j u r y w o u l d be v a l u a b l e . T h e r e s u l t s o f s u c h s t u d i e s may f i n a l l y r e s o l v e t h e q u e s t i o n a s t o w h e t h e r o r n o t t h e c a r d i o p r o t e c t i v e a c t i o n s s e e n i n a c u t e m o d e l s w i l l u l t i m a t e l y r e s u l t i n s u s t a i n e d i m p r o v e m e n t o f m y o c a r d i a l f u n c t i o n a n d t h e r e f o r e b e o f v a l u e - 144 - c l i n i c a l l y i n p r e v e n t i n g o r r e d u c i n g t h e deg ree o f m y o c a r d i a l f u n c t i o n a l impa i rmen t r e s u l t i n g f r om i s c h a e m i c i n s u l t s s u s t a i n e d e i t h e r i n t r a o p e r a t i v e - l y o r f o l l o w i n g a c u t e m y o c a r d i a l i s c h a e m i a . - 145 - REFERENCES 1 . 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BIBLIOGRAPHY For Historical Overview only Bishop, LF, Neilson J Jr (1927) J_n: History of Cardiology. New York, Medical Life Press. Harris, CRS (1973) The Heart and the Vascular System in Ancient Greek Medicine, London, Oxford University Press. East T (1957) J_n: The Story of Heart Disease. London, Wm. Dawson and Sons. Leibowitz, JO (1970) J_n: The History of Coronary Heart Disease, London, Wm. Clowes and Sons Will i us FA, Dry TJ (1948) J_n: The Heart and the Circulat ion, Philadelphia, W. B. Saunders Co. White PD (1951) Heart Disease New York, The MacMillan Co. - 159 - APPENDIX I FREQUENCY RESPONSES: Grass Differentiator Model 7P20C Input triangular wave: 1-100 volts/sec/cm Output from differentiator on oscilloscope - "D" Output from pen recorder - "P" Input Output "D" Output "P" (v/sec/cm) (v/sec/cm) 1 0.9 0.9 5 4.5 4.8 10 9.0 9.3 50 50 48 100 100 40 a a 54 Hz input frequency exceeds pen limit Maximum setting used experimentally was 25 v/sec/cm - 160 - APPENDIX II Electron Microscopy Procedures Buffered glutaraldehyde: made fresh for each experiment - 25% glutaral - dehyde was diluted with cold (4°C) cacodylate buffer (0.1 M cacodylate, 1 mM CaC12, pH 7.2) to a f inal concentration of 3% glutaraldehyde. Ruthenium red: Dye crystals were diluted with the same buffer as des- cribed above to a f inal concentration of 0.8%. The solution was made one day before use and stored in a dark bottle in the refr igerator. OsO^ (osmium tetroxide): OsO^ is l ight-sensitive and vo lat i le . Consequently, a cleaned ampoule of OsO^ crystals was dropped into an Erlenmeyer flask covered with aluminum and the ampoule broken open with a glass rod. Sufficient cacodylate buffer was added to produce a f inal concentration of 1% OsO^. This was prepared one day in advance to allow al l crystals to dissolve, and stored in the refr igerator. Ethanol and Propylene Oxide: 95% ethanol (v/v) was diluted with double- d is t i l l ed water to make up the desired f inal concentrations. To dehydrate the ethanol, ethanol/propylene oxide mixtures and the propylene oxide, molecular sieves (Davison Chemical Co.; 8-12 mesh) were added to the stock solutions to adsorb water. This precaution allowed these solvents in the tissue to be miscible with the embedding plastic thereby prevent- ing the formation of water pockets within the sample. - 161 - APPENDIX III Radioactive microsphere technique for blood flow determination Injection protocol: Control Ligation Reperfusion Group I H3$n Group II 1 1 3 Sn 57Co 57 C o After adjusting total and reference blood samples cpm values for losses in the injection and withdrawal syringes, as well as correcting the cobalt cpm for spill-over from the t in channel (approximately 17 ), the following c a l - culation for the flow to the individual tissue could be carried out: SAMPLE CALCULATIONS: Experiment - February 2, 1983 Sample calculation: 1st isotope (Sn) 2nd isotope (Co) (Control) (After 10 min of l igation) Amount given (cpm) 33,997 247,528 Withdrawal rate (ml/min) 0.7 0.7 Amount in blood (cpm) 76 700 Total Cardiac Output: Control period = 33997 x 0.7 ml/min 76 = 313 ml/min Ligation period = 247,528 x 0.7 ml/min 700 = 248 ml/min Flow calculations on following page. TISSUE FLOW: Tissue Wt (g) Ch 1 (Co) Ch 2 (Sn) -Blood Flow (ml/min/g) Percent C O . [corrected cpm] Control Ligation Control Ligation Ischaemic L.V. 0.50 95 207 3.82 0.19 0.61 0.04 Normal L.V. 0.87 2143 416 4.40 2.46 1.22 0.86 Right Ventricle 1.15 3203 446 3.57 2.78 1.31 1.29 Left Kidney 8.04 10,737 2012 2.30 1.30 5.90 4.30 Right Kidney 7.71 10,985 2101 2.50 1.40 6.20 4.40 ^Blood flow in ml/min/g = ^'LI^MLH X W I T H D R A W A L R A T E 3 cpm ref. blood tissue weight in g

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