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Modulation of acto-myosin interaction under pathological conditions. A study using in vitro motility assays
WS5-I-I-01
MODULATION OF ACTO-MYOSIN INTERACTION UNDER PATHOLOGICALCONDITIONS. A STUDY USING IN VITRO MOTILITY ASSAYS. S. Sugiura, M. Sata, H. Fujita, H. Yameshita, S. Momomura, T. Serizawa 2nd Dept. of Internal Medicine, School of Medicine, Univ. of Tokyo, Tokyo, Japan We studied the effect of pathological conditions on acto-myosin interaction using two different types of in vitro motility assay techniques. First, the effect of MgADP was studied in an assay in which fluorescently labeled actin filament was made to slide on rat cardiac myosin layer fixed on a glass cover slip in the presence of ATP. The filament velocity depended on MgATP concentration following classical Mechaelis-Menten kinetics with maximum velocity of 5.6 prn/sec. An addition of as low as 25 p.m ol/L of MgATP significantly inhibited the velocity in the presence of 2 mmol/L MgATP suggesting that increased intracallular MgADP observed under ischemia may reduce the rate of crossbridge detachment, resulting in a decreased ATP consumption. Second, the effect of acidosis was studied in another type of assay in which Ca-regulation was introduced by using cardiac thin filament. The sliding movement of thin filament reconstituted from cardiac actin and tropomyosin-troponin complex was regulated by the calcium concentration of the medium in an on-off manner. Acidosis (pH 6.8) sifted the threshold calcium concentration to a higher level thus desensitized the cardiac contractile apparatus. Furthermore, the maximum sliding velocity was reduced significantly. Calcium sensitizer could reverse the desensitization effect but did not affect the maximum sliding velocity. Since accumulation of ADP and acidosis are known to accompany cardiac ischernia, these findings could account for the contractile dysfunction observed under such a condition.
WS5-I-1-02 MECHANO-ELECTRICAL ABNORMALITIES IN CARDIAC MUSCLE UNDER CALCIUM OVERLOAD. N. Ishide, M. Miura, T. Shinozaki, H. Oda, M. Sato, tl. Numaguchi The F i r s t Department of I n t e r n a l Medicine, Tohoku U n i v e r s i t y School of Medicine, Sendal, Japan. An excess of i n t r a c e l l u l a r calcium ions i s a common path in the process of c e l l injury. We have i n v e s t i g a t e d the e f f e c t s of calcium overload on the mechanical and e l e c t r i c a l a c t i v i t i e s in c a rdi a c v e n t r i c u l a r muscle. The followin g models were employed; 1) s i n g l e cardi a c myocytes i s o l a t e d from r a t s or guinea pi gs loaded with Fura-2, 2) r i g h t v e n t r i c u l a r t r a b e c u l a r muscle from r a t s , 3) i s o v o l u m i c a l l y c o n t r a c t i n g l e f t v e n t r i c l e i s o l a t e d from hamsters. In i s o l a t e d myocytes, we recorded c e l l length, membrane p o t e n t i a l , r e l a t i v e changes in calcium c o n c e n t r a t i o n ([Ca2+]i) and s p a t i a l h e t e r o g e n e i t y of [Ca2+]i. In t r a b e c u l a r muscles, we recorded i s o m e t r i c tension and asynchronous c o n t r a c t i l e a c t i v i t i e s which were d e t e c t e d by l a s e r s c a t t e r i n g methods. In i s o l a t e d heart, we recorded v e n t r i c u l a r p r e s s u r e and asynchronous c o n t r a c t i o n s t h a t were m i c r o s c o p i c a l l y d e t e c t e d by measuring f l u c t u a t i o n s of the d i s t a n c e s between c a p i l l a r y e n d o t h e l i a l c e l l bodies which were s t a i n e d with Fura-2/Indo-1 AM. I s o l a t e d myocytes showed l o c a l i z e d i n c r e a s e s in [Ca2+]i which p e r i o d i c a l l y or s p o r a d i c a l l y occurred and propagated along the myocyte (calcium wave ) before the deve]opment of c o n t r a c t u r e and c e l l death. The mechanism of the wave propagation i s b e l i e v e d to be the calcium induced calcium r e l e a s e (CICR) from the sarcoplasmic r e t i c u l u m (SR). The mechanism for the i n i t i a t i o n of waves can be explained by the calcium s p i l l over from the sarcoplasmic r e t i c u l u m s a t u r a t e d with calcium ions. The f a c t t h a t calcium waves in the oppos i t e d i r e c t i o n disappeared a f t e r they c o l l i d e d i n d i c a t e s the e x i s t e n c e of a r e f r a c t o r y period a f t e r the CICR occurred. Action p o t e n t i a l s , however, could e l i c i t synchronous calcium t r a n s i e n t s throughout the myocyte and r e s e t the pe ri od of the wave i n i t i a t i o n . Trabecular muscle exposed to low sodium pe rfus i on which induces an i n c r e a s e in [Ca2+]i by i n h i b i t i n g the Na/Ca exchange mechanism showed increased d i a s t o l i c t e n s i o n , l e s s developed tension, t r a n s i e n t h y p e r r e l a x a t i o n and a f t e r c o n t r a c t i o n s . During r e s t i n g periods, asynchronous spontaneous c o n t r a c t i l e a c t i v i t i e s as observed in i s o l a t e d myocytes appeared. These a c t i v i t i e s could be synchronized with a t w i t c h c o n t r a c t i o n . Thus, calcium excess and the r e s u l t a n t calcium o s c i l l a t i o n can e l i c i t s y s t o l i c f a i l u r e and d i a s t o l i c f a i l u r e . In c o n t r a s t , the v e l o c i t y of t e n s i o n d e c l i n e was not decreased; no r e l a x a t i o n f a i l u r e occurred. Calcium t r a n s i e n t s in i s o l a t e d myocytes t h a t showed a f t e r c o n t r a c t i o n s possessed higher r e s t i n g [Ca2+]i and f a s t e r de c l i ne , i n d i c a t i n g t h a t the SR Ca-ATPase a c t i v i t i e s are enhanced under these conditions . Thus, calcium overload i s not the s o l e f a c t o r r e s p o n s i b l e for n e g a t i v e l u s i t r o p y which i s f r e q u e n t l y observed in damaged myocardium. In i s o l a t e d v e n t r i c l e , spontaneous asynchronous c o n t r a c t i o n could be observed on the epicardium under the c o n d i t i o n of high calcium perfusion. Developed pre s s ure d e c l i n e d and d i a s t o l i c pre s s ure i nc re a s e d with high calcium p e r f u s i o n (>4raM). Dantrolene, which i n h i b i t s calcium r e l e a s e from SR and shows ne ga t i ve inotropism at normal calcium perfusion, showed r a t h e r p o s i t i v e inotropism, e l i m i n a t i n g calcium o s c i l l a t i o n s . In i s o l a t e d myocytes, a f t e r c o n t r a c t i o n s t h a t occurred a f t e r the f i n i s h of t w i t c h c o n t r a c t i o n were generated by calcium waves. They were accompanied by delayed a f t e r d e p o l a r i z a t i o n s , which sometimes r e s u l t e d in t r i g g e r e d a c t i v i t y type arrythmias. Most of the e a r l y a f t e r d e p o l a r i z a t i o n s , except those t h a t arose from a near r e s t i n g p o t e n t i a l , were not accompanied by calcium waves. Spontaneous calcium r e l e a s e from SR causes s y s t o l i c f a i l u r e , d i a s t o l i c f a i l u r e and g e n e s i s of arrythmias. Modification of t h i s r e l e a s e could be an approach t o t r e a t these conditions.
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MODULATION OF ACTO-MYOSIN INTERACTION UNDER PATHOLOGICALCONDITIONS. A STUDY USING IN VITRO MOTILITY ASSAYS. S. Sugiura, M. Sata, H. Fujita, H. Yameshita, S. Momomura, T. Serizawa 2nd Dept. of Internal Medicine, School of Medicine, Univ. of Tokyo, Tokyo, Japan We studied the effect of pathological conditions on acto-myosin interaction using two different types of in vitro motility assay techniques. First, the effect of MgADP was studied in an assay in which fluorescently labeled actin filament was made to slide on rat cardiac myosin layer fixed on a glass cover slip in the presence of ATP. The filament velocity depended on MgATP concentration following classical Mechaelis-Menten kinetics with maximum velocity of 5.6 prn/sec. An addition of as low as 25 p.m ol/L of MgATP significantly inhibited the velocity in the presence of 2 mmol/L MgATP suggesting that increased intracallular MgADP observed under ischemia may reduce the rate of crossbridge detachment, resulting in a decreased ATP consumption. Second, the effect of acidosis was studied in another type of assay in which Ca-regulation was introduced by using cardiac thin filament. The sliding movement of thin filament reconstituted from cardiac actin and tropomyosin-troponin complex was regulated by the calcium concentration of the medium in an on-off manner. Acidosis (pH 6.8) sifted the threshold calcium concentration to a higher level thus desensitized the cardiac contractile apparatus. Furthermore, the maximum sliding velocity was reduced significantly. Calcium sensitizer could reverse the desensitization effect but did not affect the maximum sliding velocity. Since accumulation of ADP and acidosis are known to accompany cardiac ischernia, these findings could account for the contractile dysfunction observed under such a condition.
WS5-I-1-02 MECHANO-ELECTRICAL ABNORMALITIES IN CARDIAC MUSCLE UNDER CALCIUM OVERLOAD. N. Ishide, M. Miura, T. Shinozaki, H. Oda, M. Sato, tl. Numaguchi The F i r s t Department of I n t e r n a l Medicine, Tohoku U n i v e r s i t y School of Medicine, Sendal, Japan. An excess of i n t r a c e l l u l a r calcium ions i s a common path in the process of c e l l injury. We have i n v e s t i g a t e d the e f f e c t s of calcium overload on the mechanical and e l e c t r i c a l a c t i v i t i e s in c a rdi a c v e n t r i c u l a r muscle. The followin g models were employed; 1) s i n g l e cardi a c myocytes i s o l a t e d from r a t s or guinea pi gs loaded with Fura-2, 2) r i g h t v e n t r i c u l a r t r a b e c u l a r muscle from r a t s , 3) i s o v o l u m i c a l l y c o n t r a c t i n g l e f t v e n t r i c l e i s o l a t e d from hamsters. In i s o l a t e d myocytes, we recorded c e l l length, membrane p o t e n t i a l , r e l a t i v e changes in calcium c o n c e n t r a t i o n ([Ca2+]i) and s p a t i a l h e t e r o g e n e i t y of [Ca2+]i. In t r a b e c u l a r muscles, we recorded i s o m e t r i c tension and asynchronous c o n t r a c t i l e a c t i v i t i e s which were d e t e c t e d by l a s e r s c a t t e r i n g methods. In i s o l a t e d heart, we recorded v e n t r i c u l a r p r e s s u r e and asynchronous c o n t r a c t i o n s t h a t were m i c r o s c o p i c a l l y d e t e c t e d by measuring f l u c t u a t i o n s of the d i s t a n c e s between c a p i l l a r y e n d o t h e l i a l c e l l bodies which were s t a i n e d with Fura-2/Indo-1 AM. I s o l a t e d myocytes showed l o c a l i z e d i n c r e a s e s in [Ca2+]i which p e r i o d i c a l l y or s p o r a d i c a l l y occurred and propagated along the myocyte (calcium wave ) before the deve]opment of c o n t r a c t u r e and c e l l death. The mechanism of the wave propagation i s b e l i e v e d to be the calcium induced calcium r e l e a s e (CICR) from the sarcoplasmic r e t i c u l u m (SR). The mechanism for the i n i t i a t i o n of waves can be explained by the calcium s p i l l over from the sarcoplasmic r e t i c u l u m s a t u r a t e d with calcium ions. The f a c t t h a t calcium waves in the oppos i t e d i r e c t i o n disappeared a f t e r they c o l l i d e d i n d i c a t e s the e x i s t e n c e of a r e f r a c t o r y period a f t e r the CICR occurred. Action p o t e n t i a l s , however, could e l i c i t synchronous calcium t r a n s i e n t s throughout the myocyte and r e s e t the pe ri od of the wave i n i t i a t i o n . Trabecular muscle exposed to low sodium pe rfus i on which induces an i n c r e a s e in [Ca2+]i by i n h i b i t i n g the Na/Ca exchange mechanism showed increased d i a s t o l i c t e n s i o n , l e s s developed tension, t r a n s i e n t h y p e r r e l a x a t i o n and a f t e r c o n t r a c t i o n s . During r e s t i n g periods, asynchronous spontaneous c o n t r a c t i l e a c t i v i t i e s as observed in i s o l a t e d myocytes appeared. These a c t i v i t i e s could be synchronized with a t w i t c h c o n t r a c t i o n . Thus, calcium excess and the r e s u l t a n t calcium o s c i l l a t i o n can e l i c i t s y s t o l i c f a i l u r e and d i a s t o l i c f a i l u r e . In c o n t r a s t , the v e l o c i t y of t e n s i o n d e c l i n e was not decreased; no r e l a x a t i o n f a i l u r e occurred. Calcium t r a n s i e n t s in i s o l a t e d myocytes t h a t showed a f t e r c o n t r a c t i o n s possessed higher r e s t i n g [Ca2+]i and f a s t e r de c l i ne , i n d i c a t i n g t h a t the SR Ca-ATPase a c t i v i t i e s are enhanced under these conditions . Thus, calcium overload i s not the s o l e f a c t o r r e s p o n s i b l e for n e g a t i v e l u s i t r o p y which i s f r e q u e n t l y observed in damaged myocardium. In i s o l a t e d v e n t r i c l e , spontaneous asynchronous c o n t r a c t i o n could be observed on the epicardium under the c o n d i t i o n of high calcium perfusion. Developed pre s s ure d e c l i n e d and d i a s t o l i c pre s s ure i nc re a s e d with high calcium p e r f u s i o n (>4raM). Dantrolene, which i n h i b i t s calcium r e l e a s e from SR and shows ne ga t i ve inotropism at normal calcium perfusion, showed r a t h e r p o s i t i v e inotropism, e l i m i n a t i n g calcium o s c i l l a t i o n s . In i s o l a t e d myocytes, a f t e r c o n t r a c t i o n s t h a t occurred a f t e r the f i n i s h of t w i t c h c o n t r a c t i o n were generated by calcium waves. They were accompanied by delayed a f t e r d e p o l a r i z a t i o n s , which sometimes r e s u l t e d in t r i g g e r e d a c t i v i t y type arrythmias. Most of the e a r l y a f t e r d e p o l a r i z a t i o n s , except those t h a t arose from a near r e s t i n g p o t e n t i a l , were not accompanied by calcium waves. Spontaneous calcium r e l e a s e from SR causes s y s t o l i c f a i l u r e , d i a s t o l i c f a i l u r e and g e n e s i s of arrythmias. Modification of t h i s r e l e a s e could be an approach t o t r e a t these conditions.
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