Contraction Transients of Skinned Muscle Fibers" Effects of Calcium and Ionic Strength J A G D I S H G U L A T I and R I C H A R D J. P O D O L S K Y From the Laboratory of Physical Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014. Dr. Gulati's present address is Division of Cardiology, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.
A B S TRA C T Calcium and ionic strength are both known to modify the force developed by skinned frog muscle fibers. To determine how these parameters affect the cross-bridge contraction mechanism, the isotonic velocity transients following step changes in load were studied in solutions in which calcium concentration and ionic strength were varied. Analysis of the motion showed that calcium has no effect on either the null time or the amplitude of the transients. In contrast, the transient amplitude was increased in high ionic strength and was suppressed in low ionic strength. These results are consistent with the idea that calcium affects force in skeletal muscle by modulating the number of force generators in a simple switchlike "on-off' manner and that the steady force at a given calcium level is proportional to cross-bridge number. On the other hand, the effect of ionic strength on force is associated with changes in the kinetic properties of the crossbridge mechanism. INTRODUCTION
T h e force d e v e l o p e d by f r o g skinned muscle fibers is m o d u l a t e d by both the calcium ion c o n c e n t r a t i o n (Hellam a n d Podolsky, 1969) a n d the ionic s t r e n g t h ( G o r d o n et al., 1973) o f the b a t h i n g solution. T h e m e c h a n i s m s o f these effects have b e e n e x a m i n e d by studying the steady-state force-velocity relations because these relations contain i n f o r m a t i o n a b o u t the cross-bridge kinetics ( H u x l e y , 1957). I n the case o f calcium, the m o t i o n at p C a 5, which gives full isometric force, was c o m p a r e d with that at lower calcium levels. T h e relative force-velocity relation was f o u n d to be the same at d i f f e r e n t calcium levels, p r o v i d e d the ionic strength was 190 m M or m o r e (Podolsky a n d Teichholz, 1970; T h a m e s et al., 1974). T h i s observation is consistent with the idea that in these fibers calcium regulates force by controlling the n u m b e r of actin sites at which actomyosin cross-bridges can be f o r m e d (Ebashi a n d E n d o , 1968; W e b e r a n d M u r r a y , 1973). As r e g a r d s the effect of ionic s t r e n g t h , the m o t i o n is c o m p l e x at ionic strengths below 190 m M u n d e r conditions used in previous studies ( T h a m e s et al., 1974). At ionic strengths above this value, which r e d u c e the full isometric force, the relative force-velocity relation a p p e a r s to be " e same at d i f f e r e n t force levels, which is similar to the calcium effect. H o w e v e r , in biochemical J. GEN. PHYSIOL. 9 The Rockefeller University Press. 0022-1295/78/1101-070151.00 Volume 72 November 1978 701-716
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studies ionic strength affects the actomyosin A T P a s e kinetics in the absence o f the calcium-sensitive r e g u l a t o r y proteins (Rizzino et al., 1970). T h u s , the lack o f influence o f ionic s t r e n g t h on velocity in skinned fibers could be e x p l a i n e d if (a) ionic strength has an on-off, switchlike effect on the activity o f one o f the c o m p o n e n t s o f the contraction m e c h a n i s m , a n d / o r (b) ionic strength changes certain kinetic p r o p e r t i e s o f the cross-bridge m e c h a n i s m , but the steady-state force-velocity relation in skinned f r o g fibers is insensitive to these changes. T o e x a m i n e these effects in g r e a t e r detail, the isotonic velocity transients o f skinned f r o g fibers (Podolsky et al., 1974) were studied as a function of b o t h calcium ion c o n c e n t r a t i o n a n d ionic s t r e n g t h because the p r e s t e a d y m o t i o n is generally a m o r e sensitive m e a s u r e o f cross-bridge p r o p e r t i e s t h a n the steadystate m o t i o n (Civan a n d Podolsky, 1966). T h e results show that p C a does not affect the transient, which is direct evidence that calcium regulates force t h r o u g h a switchlike m e c h a n i s m that controls the n u m b e r o f active cross-bridges without affecting the kinetic or mechanical p r o p e r t i e s o f the active crossbridges. In contrast, the transient was significantly c h a n g e d by ionic s t r e n g t h , which implies that the p r o p e r t i e s o f the active cross-bridges are sensitive to this p a r a m e t e r . Additional observations r e g a r d i n g the influence o f these p a r a m e t e r s on the steady-state contraction p r o p e r t i e s o f skinned f r o g muscle fibers will be r e p o r t e d in a s u b s e q u e n t publication. Preliminary accounts of this w o r k have b e e n r e p o r t e d briefly (Gulati a n d Podolsky, 1974, 1976). METHODS
Fiber Preparation Northern frogs, Rana pipiens pipiens, and tropical frogs, Rana pipiens berlindieri, were used. The former were stored in a cold room at 5~ and the latter were kept at room temperature. The dorsal head of the semitendinosus muscle was dissected out and mounted in a chamber containing cold Ringer solution of the following composition (millimolar): KCI, 2.5; NaC1, 115; CaCI2, 1.8; NaH2PO4 + Na2HPO4, 3 (pH = 7.0). A bundle of about 10 fibers was cut from the muscle, blotted lightly, and laid on a glass cover slip. The cover slip was quickly submerged in ice-cold paraffin oil (viscosity 125/ 135). The largest fiber was very carefully isolated from the bundle in oil and skinned with fine stainless steel needles. The length of the skinned region varied between 5-10 mm in length. The displacement and force transducers were those described by Civan and Podolsky (1966). They were adapted for use with skinned fiber preparations by cementing a short attachment wire to the end of each transducer. The fiber was tied to the wires with square knots of 5-0 surgical silk. The fiber length between the ties in different experiments ranged from 0.8 to 3 mm; this was kept short to reduce the compliance of the moving system. The equivalent mass of the displacement transducer was about 3 mg and the natural period for moderate force steps was
A B S TRA C T Calcium and ionic strength are both known to modify the force developed by skinned frog muscle fibers. To determine how these parameters affect the cross-bridge contraction mechanism, the isotonic velocity transients following step changes in load were studied in solutions in which calcium concentration and ionic strength were varied. Analysis of the motion showed that calcium has no effect on either the null time or the amplitude of the transients. In contrast, the transient amplitude was increased in high ionic strength and was suppressed in low ionic strength. These results are consistent with the idea that calcium affects force in skeletal muscle by modulating the number of force generators in a simple switchlike "on-off' manner and that the steady force at a given calcium level is proportional to cross-bridge number. On the other hand, the effect of ionic strength on force is associated with changes in the kinetic properties of the crossbridge mechanism. INTRODUCTION
T h e force d e v e l o p e d by f r o g skinned muscle fibers is m o d u l a t e d by both the calcium ion c o n c e n t r a t i o n (Hellam a n d Podolsky, 1969) a n d the ionic s t r e n g t h ( G o r d o n et al., 1973) o f the b a t h i n g solution. T h e m e c h a n i s m s o f these effects have b e e n e x a m i n e d by studying the steady-state force-velocity relations because these relations contain i n f o r m a t i o n a b o u t the cross-bridge kinetics ( H u x l e y , 1957). I n the case o f calcium, the m o t i o n at p C a 5, which gives full isometric force, was c o m p a r e d with that at lower calcium levels. T h e relative force-velocity relation was f o u n d to be the same at d i f f e r e n t calcium levels, p r o v i d e d the ionic strength was 190 m M or m o r e (Podolsky a n d Teichholz, 1970; T h a m e s et al., 1974). T h i s observation is consistent with the idea that in these fibers calcium regulates force by controlling the n u m b e r of actin sites at which actomyosin cross-bridges can be f o r m e d (Ebashi a n d E n d o , 1968; W e b e r a n d M u r r a y , 1973). As r e g a r d s the effect of ionic s t r e n g t h , the m o t i o n is c o m p l e x at ionic strengths below 190 m M u n d e r conditions used in previous studies ( T h a m e s et al., 1974). At ionic strengths above this value, which r e d u c e the full isometric force, the relative force-velocity relation a p p e a r s to be " e same at d i f f e r e n t force levels, which is similar to the calcium effect. H o w e v e r , in biochemical J. GEN. PHYSIOL. 9 The Rockefeller University Press. 0022-1295/78/1101-070151.00 Volume 72 November 1978 701-716
701
702
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E 7 2 " 1 9 7 8
studies ionic strength affects the actomyosin A T P a s e kinetics in the absence o f the calcium-sensitive r e g u l a t o r y proteins (Rizzino et al., 1970). T h u s , the lack o f influence o f ionic s t r e n g t h on velocity in skinned fibers could be e x p l a i n e d if (a) ionic strength has an on-off, switchlike effect on the activity o f one o f the c o m p o n e n t s o f the contraction m e c h a n i s m , a n d / o r (b) ionic strength changes certain kinetic p r o p e r t i e s o f the cross-bridge m e c h a n i s m , but the steady-state force-velocity relation in skinned f r o g fibers is insensitive to these changes. T o e x a m i n e these effects in g r e a t e r detail, the isotonic velocity transients o f skinned f r o g fibers (Podolsky et al., 1974) were studied as a function of b o t h calcium ion c o n c e n t r a t i o n a n d ionic s t r e n g t h because the p r e s t e a d y m o t i o n is generally a m o r e sensitive m e a s u r e o f cross-bridge p r o p e r t i e s t h a n the steadystate m o t i o n (Civan a n d Podolsky, 1966). T h e results show that p C a does not affect the transient, which is direct evidence that calcium regulates force t h r o u g h a switchlike m e c h a n i s m that controls the n u m b e r o f active cross-bridges without affecting the kinetic or mechanical p r o p e r t i e s o f the active crossbridges. In contrast, the transient was significantly c h a n g e d by ionic s t r e n g t h , which implies that the p r o p e r t i e s o f the active cross-bridges are sensitive to this p a r a m e t e r . Additional observations r e g a r d i n g the influence o f these p a r a m e t e r s on the steady-state contraction p r o p e r t i e s o f skinned f r o g muscle fibers will be r e p o r t e d in a s u b s e q u e n t publication. Preliminary accounts of this w o r k have b e e n r e p o r t e d briefly (Gulati a n d Podolsky, 1974, 1976). METHODS
Fiber Preparation Northern frogs, Rana pipiens pipiens, and tropical frogs, Rana pipiens berlindieri, were used. The former were stored in a cold room at 5~ and the latter were kept at room temperature. The dorsal head of the semitendinosus muscle was dissected out and mounted in a chamber containing cold Ringer solution of the following composition (millimolar): KCI, 2.5; NaC1, 115; CaCI2, 1.8; NaH2PO4 + Na2HPO4, 3 (pH = 7.0). A bundle of about 10 fibers was cut from the muscle, blotted lightly, and laid on a glass cover slip. The cover slip was quickly submerged in ice-cold paraffin oil (viscosity 125/ 135). The largest fiber was very carefully isolated from the bundle in oil and skinned with fine stainless steel needles. The length of the skinned region varied between 5-10 mm in length. The displacement and force transducers were those described by Civan and Podolsky (1966). They were adapted for use with skinned fiber preparations by cementing a short attachment wire to the end of each transducer. The fiber was tied to the wires with square knots of 5-0 surgical silk. The fiber length between the ties in different experiments ranged from 0.8 to 3 mm; this was kept short to reduce the compliance of the moving system. The equivalent mass of the displacement transducer was about 3 mg and the natural period for moderate force steps was
