PfliJgersArchiv
PflfigersArch. 382, 165- I70 (1979)
EuropeanJournal of P h ~ y
9 by Springer-VerlagJ979
Rate of Isometric Tension Development in Relation to Calcium Binding of Skinned Muscle Fibres P. J. Griffiths, H. J. Kuhn, K. Gfith, and J. C. Rfiegg II. PhysiologischesInstitut der UniversitfitHeidelberg,Im NeuenheimerFeld 326, D-6900 Heidelberg, Federal Republic of Germany
Abstract. Isolated muscle fibres from the sartorius or
semitendinosus muscles of frogs were mechanically skinned and kept in a relaxed state in a medium containing Mg-ATP and EGTA. When subjected to a rapid increase in internal calcium ion concentration tension rose relatively slowly in comparison to the time course of establishment of the new calcium concentration. Stiffness measurements made during the rise of tension yielded the same stiffness to tension ratio as that observed at steady state force. The linear force extension curve of the activated fibres (Tl-Curves) measured at various moments during the rise of tension extrapolated to zero tension intersected the base line at the same length ( - 0 . 8 ~ L0). This suggests that the extent of myosin interaction increases with the same time course as tension. The rate of tension development accompanying a 'Ca-jump' was strongly increased by an increase in calcium ion concentration and there was a linear relationship between the logarithm of the rate tension development and pCa. The rate of recovery of tension following a large quick release > 2% L0 was not calcium sensitive, and occurred at a rate more than an order of magnitude faster than the corresponding calcium activation in the range of pCa's studied. We suggest that the slowness of tension development accompanying a rapid calcium activation reflects slow reactions occurring after a single Ca-ion has bound to a myofllament binding site and does not reflect the slowness of actin and myosin interaction. Key words: Calcium activation fibres - Contraction velocity.
Skinned muscle
This workwas supported by a grant fromthe DeutscheForschungsgemeinschaft Gu 160/3. One of us (P.J.G.) was supported by the Royal SocietyEuropean ScienceExchangeProgramme
Introduction
The kinetics of the release of calcium into the sarcoplasm of excited muscle have been studied recently (Ashley and Ridgway, 1970; Blinks et al., 1978) and it has been shown that the increase in intracellular free calcium always occurs much faster than the force development. The latter must therefore be rate limited either by the reaction of the calcium ions with the myofibrils and related activation processes or by the subsequent contraction of the myofibrils occurring after the capacity for actin myosin interaction has been fully developed. With the development of the skinned fibre preparation, it is possible to study the kinetics of the contractile and activation processes directly, following an increase in internal calcium concentration. Rapid alterations in the free Ca 2 +-concentration inside the skinned fibre preparation may be achieved by two methods. Ca 2+ ions may be iontophoretically passed into solution in the region of a small segment of skinned fibre (De Clerck et al., 1977). Alternatively Ashley and Moisescu (1973, 1975) and Moisescu and Thieleczek (1978) developed a technique by which it was possible to raise Ca-ion concentration in skinned frog muscle fibres within about 200 ms to the desired value by using a Ca buffer system. The rate of tension development was slow in comparison and it was hardly affected by Ca-diffusion but strongly dependent on pCa. These findings have been interpreted in terms of a theory involving the binding of four Ca-ions to troponin, one of which is assumed to bind slowly and to limit the rate of tension development (Moisescu, 1976; Ashley and Moisescu, 1972). The theory implies, however, that tension development reflects formation of actin myosin linkages. This assumption will be tested by measuring the Hookean instantaneous stiffness during tension development. Stiffness is related to the number of myosin cross bridges attached to actin at any
9 0031-6768/79/0382/0165/$01.20
166 one m o m e n t in b o t h intact muscle ( H u x l e y a n d S i m m o n s , 1973) a n d skinned fibres ( G o l d m a n a n d S i m m o n s , 1977; Y a m a m o t o a n d Herzig, 1978). t t is also o f interest to c o m p a r e the rate o f tension developm e n t following a C a - j u m p to the rate o f tension (and stiffness) recovery following a quick release o f Caa c t i v a t e d muscle. In the latter case Ca-ions s h o u l d have e q u i l i b r a t e d with t r o p o n i n so t h a t the rate o f redevelopm e n t o f force s h o u l d n o t then be limited by the rate o f calcium t r o p o n i n interaction. A p r e l i m i n a r y a c c o u n t o f this w o r k has a l r e a d y been p u b l i s h e d (Griffiths et al., 1978).
Methods Single fibres (diameter 50 - 70 gm) were isolated from the sartorius or semitendinosus muscle of either Rana esculentaor R. temporariaand were rapidly skinned with needles under a drop of ATP solution containing a high concentration of free EGTA [ethyleneglycol-bis (flaminoethylether) N,N'-tetraacetic acid] and then glued between a length step generator and a tension transducer. Fibre length was typically 3 mm (sarcomere length 2.4~m, as measured by He-Ne-laser diffraction), Solutions contained (in mM): imidazole, 60; Na, 15; K, 250; ATP 7.5 ; Mg, 7.5; C1, 192; creatine phosphate, 10; creatine kinase, 25 U. ml- 1; X 2-, 50, where X 2- represents the total EGTA of 0.2 mM total EGTA and 49.8mM HDTA (hexamethylenediamineN,N,N',N'-tetraacetic acid). HDTA can be used as a substitute for EGTA to maintain ionic strength (Moisescn, 1976). Solutions were adjusted to pH 6.7. Free Ca 2+ was calculated assuming an apparent binding constant for EGTA of 1.2 x 106 M-1 (estimated from Anderegg, 1964) at this pH. 10mM caffeine was present in all solutions. A calcium contamination of 3 x 10 -6 M in the experimental solution was calculated from spectrophotometric measurements. Tension was measured using a commercially available tension transducer (Aksjeselskapet 802, 6 kHz resonance frequency, stability at room temperature _+20 pN, compliance adjusted to I lam. mN- t). The transducer was elongated using a piece of carbon fibre to which the preparations were fixed. Changes in length could be administrated to the preparation using a Ling dynamics model 101 vibrator or using the relay type length step generator as described by Gtith and Kuhn (1978). Changes in length were measured either by the change in impedance of germanium field plates displaced in a magnetic field (Steiger and Riiegg, 1968) or by use of a phototransistor device (Gfith and Kuhn, AT 1978). Measurements of immediate stiffness - - were made by AL applying small sinusoidal length oscillations to the preparation (1 kHz, 0.05 % L0). Alternativelystiffness was also evaluated from the linear portion of a force extension diagram obtained (on a digital oscilloscope type Nicolet 1090A Explorer) during quick stretch or release applied within 0.3 ms by means of the relay type length step generator (for details of the method see G~th and Kuhn, 1978). Single fibres attached to the apparatus were exposed to a high EGTA (50 mM) concentration solution free of Ca 2+ after being fixed to the apparatus, in which 10mM caffeine was present to remove Ca 2+ ions present in the sarcoplasmic reticulum (Ashley etal., 1975). After 3 rain, fibres were transferred to the relaxing solution containing the low concentration of EGTA (0.1 mM) in which they remained for further 3 rain, On transfer to the Ca 2+ containing EGTA solution of high concentration (50 mM) tension developed rapidly.
pfltigers Arch. 382 (1979)
Results The rate o f tension d e v e l o p m e n t resulting f r o m a ' C a j u m p ' was influenced by free C a 2 + c o n c e n t r a t i o n a n d t e m p e r a t u r e . F i g u r e 1 shows a time course o f tension rise following a s u d d e n increase o f C a 2 § to 1 0 - 6 M (a ' C a - j u m p ' ) . T y p i c a l h a l f times for tension rise were 15 s a n d 1.5 s at p C a ' s 5 a n d 6 respectively (9~ p H 7.0), or 3.8 s a n d 0.45 s at these p C a ' s (22~ p H 7.0). R e v e r s e d ' C a - j u m p s ' f r o m low p C a values to high ones were f o u n d to occur with a very r a p i d halftime (120 ms at 12~ . Q10 for the halftime o f tension rise following a C a - j u m p was 2.6. T h e e x t e n t o f calcium i n d u c e d tension was dep e n d e n t on the c a l c i u m ions c o n c e n t r a t i o n , the calcium c o n c e n t r a t i o n r e q u i r e d for h a l f m a x i m u m a c t i v a t i o n being o f a b o u t p C a 6.0. M a x i m u m tension at full a c t i v a t i o n ( p C a a b o u t 5.5) was c o m p a r a b l e to tension o b t a i n e d on tetanic s t i m u l a t i o n o f intact single fibres being o f the o r d e r o f 25 N . c m - 2 at 12~
Time Course o f Stiffness-Changes In o r d e r to m e a s u r e fibre stiffness, the sinusoidal oscillations described in the m e t h o d s section were i m p o s e d on the fibre length d u r i n g the Ca 2 § i n d u c e d tension rise a n d the c o r r e s p o n d i n g tension changes recorded. A s can be seen in Fig. 1, the presence o f the length oscillations h a d little effect u p o n the time course or extent o f the c a l c i u m i n d u c e d tension increase. R e c o r d s were o b t a i n e d at p C a values o f activating solutions in the r a n g e 6.5 to 5, a n d in all cases stiffness a n d tension increased in parallel to one another, as s h o w n in Fig. 2. W h e n stiffness was p l o t t e d versus force d u r i n g the rising p h a s e o f tension a straight line was o b t a i n e d which p a s s e d t h r o u g h the origin (Fig. 2). This g r a d i e n t o f stiffness a n d tension equals the stiffness to tension r a t i o o b t a i n e d at steady state o f force m a i n t e n a n c e . In o r d e r to find o u t whether the elasticity o f c o n t r a c t e d fibres obeys H o o k e ' s law fibres have been stretched a n d released (by 0 . 3 % ) d u r i n g a calcium i n d u c e d tension d e v e l o p m e n t , a n d tension was p l o t t e d versus the length change d u r i n g stretch or release. As s h o w n in Fig. 3 the force extension d i g r a m s o b t a i n e d d u r i n g stretch were fairly linear indicating t h a t H o o k e ' s law is obeyed. D u r i n g the g r a d u a l b u i l d - u p o f contractile tension after i m m e r s i n g into the c a l c i u m sol u t i o n the slope o f a force extension d i a g r a m b e c a m e steeper i n d i c a t i n g t h a t stiffness grows in p r o p o r t i o n to tension. A similar force length r e l a t i o n s h i p was obt a i n e d in e x p e r i m e n t s in which the fibres were released r a t h e r t h a n stretched d u r i n g different times o f tension d e v e l o p m e n t . In all cases the linear p a r t o f the force extension curve intercepted the abscissa at a b o u t 0.7 to
P. J. Griffiths et al. : Calcium Activation Kinetics in Muscle
167 -
200
A
[
O
~
,s0 "
100
100pN
J 9~
-
-
m
uJ
1,
Z
ss
"
LI-
o
s'o
lbo
lgo
2bo
2go
5 ,,ob,%,o-I
.
10 s _
B / 1
B
IIIII
1/
,r
100
D
501
mN
.I~
100pN
5oom 10s
2
pH 6.7 , 12~ , pea 6
o
s'o
16o
{..]
2bo
TENSION
Fig. 1. Calcium activated tension development following a 'Ca-jump' in a skinned single muscle fibre from frog sartorius in the presence (B) and absence (A) of sinusoidal length oscillations (0.05 %Lo, I kHz). Stiffness is indicated from the band width of the signal in record B. Temperature 9~ At arrow, Ca-jump: fibre transferred from relaxing solution containing 0.2 mM EGTA (pCa > 8) to activating solution [pCa 6, calcium buffer (EGTA total) 50mM] at 9~ Fig. 2. The relationship between stiffness and tension (C) during the tension development resulting from a 'Ca-jump' (A) and the relationship between stiffness and tension (D) during recovery from a quick release (B), both performed at pCa 6, 9~ Stiffness was measured using small amplitude oscillations in length for both forms of tension development. All records were obtained from one experiment on a skinned fibre from sartorius muscle. Note the delayed tension rise following the restoration of the fibre length to its original value in B
0.9% Lo (as found with a different technique by Yamamoto and Herzig in skinned muscle fibres). The intercept is slightly larger than that obtained in releasing contracted living muscle fibres (C. F. Ford et al., 1977).
Recovery After Quick Release The question arises whether a slow increase in tension and stiffness induced by Ca is rate-limited by activation processes (e.g. binding of Ca z + to troponin) or by the actin myosin interaction per se. The latter may be judged from the rate of tension (and stiffness) recovery following a quick release of a fibre which is already in a state of activation and tension development. A quick release of isometrically contracted single fibres of about 4 %L o was performed within 5 ms. Tension fell to almost zero after which tension recovered. Stiffness and tension were rising at a much faster rate during the recovery following the quick release than during the tension rise following the 'Ca-jump'. In both cases, however, stiffness and tension rise in proportion and in the same relation to one another (Fig. 2). It was of interest to know if the rate of tension and stiffness rise following a quick release showed a similar Ca 2 + sensitivity as the tension rise of a Ca-jump. A comparison of the effects of Ca 2 + on the two types of
tension rise is shown in Fig. 4. Recovery from a quick release shows little Ca 2 § sensitivity in the pCa range examined, and in all cases the stiffness to tension ratios were constant during the tension rise. In Fig. 5, the Ca 2 + sensitivity of isometric tension and ofhalftimes of tension rise following quick release and 'Ca-jump' are shown. It can be seen that the range of Ca 2 + sensitivity of the rate of tension development following a 'Cajump' extends outside the range of sensitivity of isometric tension. At pCa 5.8 tension production is already fully activated whereas the rate of force development is only 10 ~ of the rate at pCa 5. There was a linear relationship between the logarithm of the rate of tension development and pCa in the range from 6 to 5. The recovery of tension following a quick release shows little Ca 2+ sensitivity in the range of Ca 2§ concentrations examined.
Discussion Under Ca-jump conditions using the Ca-jump we find that a sudden increase in external Ca2+-ion concentration causes a rapid force development in skinned fibre, the rate of which is similar to that reported previously by Moisescu and Thieleczek (1978). This rate is much smaller than the rate of tension rise on
168
Pfltigers Arch. 382 (1979)
10-5M
A //"
,mN I 1
j
//" I
i
I
2s
10-SM
C a ++
C a ++
C !
, ,
T/2=17 ms
2s
B
20 prn ]
I 0 -5"8 M Ca++
10-5"8M C a ++
B
D
1mN[ T/2:23ms I
I0
ms
I
I
I0 pN
,.-%iL.~%
-,To
5"-'::
-o3s
~
-o's
C
..
~
-o72s
ms
~
o
2s ' ~ 200ms Fig, 4. Comparison of the velocity of tension development in a 'Cajump' (A and B) and the recovery from a quick release of 4 % Lo (C and D) at different Ca + concentrations, 10- 5 M in A, C; 10- s.8 M in B, D. Half times of tension rise are shown accompanying the relevant transients. All transients were obtained from a single skinned frog sartorius muscle fibre during the same experiment at 12~
.-~
o'25
% Lo Fig.3A--C. Increase of immediate stiffness during isometric contraction following 'Ca-jump' from pCa 8 to pCa 5.6; single frog sartorius muscle fibre at 4 ~C. (A) Time course of force development during which stiffness was measured using square wave stretches (left) or releases (right). (B) Typical tension response to quick stretch (left) or quick release (right); force before length step: 0,2 mN. (C) Force extension curves (Tl-curves) obtained during length steps applied at various moments during the tension rise following a 'Cajump'. Note that stiffness (slope of Tl-curve) increases in proportion to contractile force during contraction, the linear portions of the force extension curves extrapolating to zero tension to intercept the abscissa at - 0 . 9 % L0
ul
g .,d_
0-
-
-1.5'
~: -t0-
-';----'-
"--'-
- -" .....
: .....
~-
B
-- -OS"-" &
tetanizing intact fibres and the rate of tension recovery following a quick release of isometrically contracted skinned fibres. The possibility must therefore be considered that following a Ca-jump the rise in internal Ca 2 +-ion concentration may be delayed partly because of diffusional hindrances and partly because of the buffering action of intracellular calcium binding sites (e.g.: sarcoplasmic reticulum, mitochondria) which might compete with the myofilaments for Ca 2 § ions. The principle of the Ca-jump technique is to reduce the concentration of these binding sites prior to entering the activating solution, with its high concentration of calcium buffer. The EGTA concentration of the binding sites prior to activation is reduced by preequilibration in 0.1 mm EGTA solution. The buffering action of the sarcoplasmic reticulum may be reduced by the presence of caffeine in the solution, which is known to reduce the capacity for retaining calcium in the
50-
9
C
o051.0
9
9 ~
C5
~ }pco}
Fig. 5. Comparison of calcium ion dependence of (A) isometric tension (mean values of 7 experiments) (B) halftime of recovery following a quick release of 4 % Lo and (C) halftime of a 'Ca-jump' induced tension development. Halftimes of tension development (T) were all normalised with respect to the halftime of tension development as a result of a 'Ca-jump' from pCa 8 to pCa 5, p H 6.7: (Ordinate = log Ta Tb, where Ta is halftime of observed tension rise and Tb is halftime of tension rise following a ~ to pCa 5)
compartment (Caputo, 1976). In similar experiments in which the SR disrupting non ionic detergent Brij 58 (Orentlicher et al., 1974) has been used instead of caffeine in our solutions, the results were quantitatively similar to those obtained in the presence of caffeine. The velocity of binding of Ca ions by mitochondria
P. J. Griffithset al.: CalciumActivationKineticsin Muscle appears to be too small to significantly compete with EGTA as a Ca 2 § buffering compartment (Batra, 1973). Moisescu and Thieleczek (1978) have calculated the time course of establishment of a new free calcium concentration inside the fibre following a Ca-jump, allowing for diffusional delays and the relatively low dissociation rate of Ca EGTA. Their predicted rate of change of free Ca inside the fibre agreed well with measurements they obtained using the photoprotein aequorin. These showed that the Ca-ion concentration inside the fibre approached that of the buffer within 200 ms when the single fibre was immersed into CaEGTA-buffer-solution. Since our experiments were conducted on the same type of fibre under similar experimental conditions, we feel justified in assuming that the new Ca 2 + ion concentration was established within 0.5 s in our experiments, while the development of tension took a much longer time. The rate of tension development was not altered by changing the total Ca buffer content of the activating solution at a given pCa, but was increased on reducing the ionic strength. Diffusion processes seem, then, unlikely to be the cause for slowness of tension development, in particular because of the high Q10 value for the speed of tension development and the tenfold increase in rate of tension development accompanying a tenfold increase in free Ca 2§ ion concentration (compared to a 1.7 fold increase in the total calcium content of the activating solution). Moisescu (1976) has provided evidence suggesting that the force development resulting from a 'Ca-jump' is rate-limited by the binding of Ca 2 § ions to troponin. Our experiments indicate that there is a linear relationship between the logarithm of the rate of contraction and the pCa. From the size of the slope of this relation it appears that the binding of only one of the postulated four Ca 2 + ions binding to troponin is rate limiting. As pointed out already, the theory of Ashley and Moisescu assumes that the Ca-dependent time course of tension increase does reflect the time course by which the number of actin myosin linkages increases. The observed proportionality between stiffness and tension (measured using small amplitude sinusoidal length oscillations) supports this notion, since stiffness is related to the extent of actin myosin interaction according to Huxley and Simmons. However, one might object to this interpretation on the grounds that the observed proportionality of stiffness and tension increase might be also accounted for by the presence of additional series elasticity. For example, supposing the central part of the fibre were to shorten during activation at the expense of the tendon regions, the system would behave as a two component model consisting of a contractile element connected to a series elastic element (cf. Carlson and Wilkie, 1974). If the
169 elastic elements were non linear (i.e. : exponential) the shortening of the contractile elements would of course also lead to a proportional increase in tension and stiffness (when measured by small length changes 0.05 ~ Lo), though the latter would not then be directly related to crossbridge attachment. To exclude this possibility immediate stiffness (Huxley and Simmons, 1973) has also been measured using force extension curves (T1-curves) obtained by recording tension and muscle length during length steps (0.3 ~ Lo, i.e. : a large length change) applied at various moments during the tension rise. The curves show (1) a linear relation between length change and tension indicating a Hookean elasticity for the elastic component for small stretches and releases; (2) they extrapolate to the same abscissa intercept of about - 0 . 8 ~ L 0 regardless of tension; (3) the slope (stiffness) is proportional to tension before the length change. If stiffness is a relative measure of the number of crossbridges attached to the actin filament as suggested by Huxley and Simmons (1973) it follows that the tension rise following a 'Cajump' does reflect the time course of the increase in actin myosin interaction possibly by the recruitment of crossbridges as assumed in the theory of Ashley and Moisescu. Isometrically contracted fibres in which Ca 2 + ions were already equilibrated with troponin were released in 5 ms by a length change sufficient to bring the tension at the end of the release close to zero (between 2 ~ L0 and 4 ~ L0). At the end of the release, tension recovered at a rate much faster than that of the tension rise accompanying a Ca jump. Furthermore, this rate of tension recovery following a quick release was insensitive to the free Ca 2+ ion concentration. The stiffness to tension ratio was maintained constant during the tension rise, just as in the measurements obtained during tension development accompanying a Ca jump. In this type of experiment the regulatory system is already at equilibrium with the free calcium concentration, and so the recovery of stiffness and tension is presumably determined by the rate of actin myosin interaction, i.e.: crossbridge attachment and detachment, rather than by Ca binding. These experiments show that actin myosin interaction is so fast that it cannot be rate limiting during the relatively slow tension and stiffness increase following a 'Ca-jump'. From such measurements on the rate of stiffness and tension increase following a 'Ca-jump' the rate of Ca binding can be determined and may be compared with biochemical studies on cation binding by troponin (e.g. : Potter 1975). On lowering the ionic strength or reducing the free magnesium ion concentration in the solution, it is possible to increase the velocity of a 'Cajump' activation to approach that of a recovery from a quick release (unpublished experiments). Since the rate
170 o f t e n s i o n d e v e l o p m e n t d u r i n g t e t a n u s in the i n t a c t fibre is v e r y similar to the r a t e o f r e c o v e r y f o l l o w i n g a q u i c k release, it w o u l d s e e m likely t h a t the r a t e o f t e n s i o n d e v e l o p m e n t in the t e t a n i z e d i n t a c t fibre is n o t l i m i t e d b y C a b i n d i n g k i n e t i c s b u t p r o b a b l y b y the r a t e of actin myosin interaction.
References Anderegg, G. : Reaktionsenthalpie und -entropie bei der Bildung der Metallkomplexe der h6heren EDTA-Homologen. Helv. Chim. Acta 47, 1801-1814 (1964) Ashley, C. C., Griffiths, P. J., Moisescu, D. G., Rose, R. M. : The use of aequorin and the isolated myofibrillar bundle preparation to investigate the effect of SR calcium releasing agents. J. Physiol. (Loud.) 245, 12-14P (1975) Ashley, C. C., Moisescu, D. G. : Model for the action of calcium in muscle. Nature New Biol. 237, 208-211 (1972) Ashley, C. C., Moisescu, D. G. : Tension changes in isolated bunclles of frog and barnacle myofibrils in response to sudden changes in external free calcium concentration. J. Physiol. (Lond.) 233, 8 9P (1973) Ashley, C. C., Moisescu, D. G.: The part played by Ca 2+ in the contraction of isolated bundles of myofibrils. In: Calcium transport in contraction and secretion (E. Carafoli, ed.), pp. 517-525. Amsterdam: North-Holland Publishing Company 1975 Ashley, C. C., Ridgway, E. B. : On the relationship between membrane potential, calcium transient and tension in single barnacle muscle fibres. J. Physiol. (Lond.) 209, 105 - 130 (1970) Batra, S. : The effects of zinc and lanthanum on calcium uptake by mitochondria and fragmented sarcoplasmic reticulum of frog skeletal muscle. J. Cell. Physiol. 82, P245-256 (1973) Blinks, J. R., Rtidel, R., Taylor, S. R. : Calcium transients in isolated amphibian skeletal muscle fibres. Detection with aequorin. J. Physiol. (Loud.) 277, 291 - 334 (1978)
Pflfigers Arch. 382 (1979) Caputo, C. : The effect of caffeine and tetracaine on the time course of potassium contractures of single muscle fibres. J. Physiol. (Lond.) 255, P191-207 (1976) Carlson, F. D., Wilkie, D. R. : Muscle physiology. Englewood Cliffs, N.J, : Prentice-Hall, Inc., 1974 De Clerck, N. M., Claes, V. A., Brutsaert, D. L.: Force velocity relations of single cardiac muscle cells. J. Gen. Physiol. 69, P221-241 (1977) Ford, L. E., Huxley, A. F., Simmons, R. M.: Tension responses to sudden length changes in stimulated frog muscle fibres near slack length. J. Physiol. (Lond.) 269, 441-517 (1977) Goldman, Y. E., Simmons, R. M. : Active and rigor muscle stiffness. J. Physiol. (Lond.) 269, 5 5 - 5 7 P (1977) Griffiths, P. J., Kuhn, H. J., Riiegg, J. C. : Rapid calcium activation of skinned fibres. J. Physiol. (Loud.) 284, 141P (1978) Giith, K., Kuhn, H. J. : Stiffness and tension during and after sudden length changes of glycerinated rabbit psoas muscle fibres. Biophys. Struct. Mech. 4, 223-236 (1978) Huxley, A. F., Simmons, R. M. : Mechanical transients and the origin of muscular force. Cold Spring Harbor Symp. Quant. Biol. 37, 669- 680 (1973) Moisescu, D. G. : Kinetics of reaction in Ca-activated skinned muscle fibres. Nature 262, 610-613 (1976) Moisescu, D. G., Thieleczek, R. : Calcium and strontium concentration changes within skinned muscle preparations following a change in the external bathing solution. J. Physiol. (Loud.) 275, 241-262 (1978) Orentlicher, M., Reuben, J. P., Grumpert, G., Brandt, P. W.: Calcium binding and tension development in detergent-treated muscle fibres. J. Gen. Physiol. 63, 168-187 (1974) Potter, J. D., Gergely, J. : The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphospfiate. J. Biol. Chem. 250, 4628-4633 (1975) Yamamoto, T., Herzig, J. W.: Series elastic properties of skinned muscle fibres in contraction and rigor, Pfltigers Arch. 373, 21 24 (1978)
Received July 7/Accepted July 13, 1979
PflfigersArch. 382, 165- I70 (1979)
EuropeanJournal of P h ~ y
9 by Springer-VerlagJ979
Rate of Isometric Tension Development in Relation to Calcium Binding of Skinned Muscle Fibres P. J. Griffiths, H. J. Kuhn, K. Gfith, and J. C. Rfiegg II. PhysiologischesInstitut der UniversitfitHeidelberg,Im NeuenheimerFeld 326, D-6900 Heidelberg, Federal Republic of Germany
Abstract. Isolated muscle fibres from the sartorius or
semitendinosus muscles of frogs were mechanically skinned and kept in a relaxed state in a medium containing Mg-ATP and EGTA. When subjected to a rapid increase in internal calcium ion concentration tension rose relatively slowly in comparison to the time course of establishment of the new calcium concentration. Stiffness measurements made during the rise of tension yielded the same stiffness to tension ratio as that observed at steady state force. The linear force extension curve of the activated fibres (Tl-Curves) measured at various moments during the rise of tension extrapolated to zero tension intersected the base line at the same length ( - 0 . 8 ~ L0). This suggests that the extent of myosin interaction increases with the same time course as tension. The rate of tension development accompanying a 'Ca-jump' was strongly increased by an increase in calcium ion concentration and there was a linear relationship between the logarithm of the rate tension development and pCa. The rate of recovery of tension following a large quick release > 2% L0 was not calcium sensitive, and occurred at a rate more than an order of magnitude faster than the corresponding calcium activation in the range of pCa's studied. We suggest that the slowness of tension development accompanying a rapid calcium activation reflects slow reactions occurring after a single Ca-ion has bound to a myofllament binding site and does not reflect the slowness of actin and myosin interaction. Key words: Calcium activation fibres - Contraction velocity.
Skinned muscle
This workwas supported by a grant fromthe DeutscheForschungsgemeinschaft Gu 160/3. One of us (P.J.G.) was supported by the Royal SocietyEuropean ScienceExchangeProgramme
Introduction
The kinetics of the release of calcium into the sarcoplasm of excited muscle have been studied recently (Ashley and Ridgway, 1970; Blinks et al., 1978) and it has been shown that the increase in intracellular free calcium always occurs much faster than the force development. The latter must therefore be rate limited either by the reaction of the calcium ions with the myofibrils and related activation processes or by the subsequent contraction of the myofibrils occurring after the capacity for actin myosin interaction has been fully developed. With the development of the skinned fibre preparation, it is possible to study the kinetics of the contractile and activation processes directly, following an increase in internal calcium concentration. Rapid alterations in the free Ca 2 +-concentration inside the skinned fibre preparation may be achieved by two methods. Ca 2+ ions may be iontophoretically passed into solution in the region of a small segment of skinned fibre (De Clerck et al., 1977). Alternatively Ashley and Moisescu (1973, 1975) and Moisescu and Thieleczek (1978) developed a technique by which it was possible to raise Ca-ion concentration in skinned frog muscle fibres within about 200 ms to the desired value by using a Ca buffer system. The rate of tension development was slow in comparison and it was hardly affected by Ca-diffusion but strongly dependent on pCa. These findings have been interpreted in terms of a theory involving the binding of four Ca-ions to troponin, one of which is assumed to bind slowly and to limit the rate of tension development (Moisescu, 1976; Ashley and Moisescu, 1972). The theory implies, however, that tension development reflects formation of actin myosin linkages. This assumption will be tested by measuring the Hookean instantaneous stiffness during tension development. Stiffness is related to the number of myosin cross bridges attached to actin at any
9 0031-6768/79/0382/0165/$01.20
166 one m o m e n t in b o t h intact muscle ( H u x l e y a n d S i m m o n s , 1973) a n d skinned fibres ( G o l d m a n a n d S i m m o n s , 1977; Y a m a m o t o a n d Herzig, 1978). t t is also o f interest to c o m p a r e the rate o f tension developm e n t following a C a - j u m p to the rate o f tension (and stiffness) recovery following a quick release o f Caa c t i v a t e d muscle. In the latter case Ca-ions s h o u l d have e q u i l i b r a t e d with t r o p o n i n so t h a t the rate o f redevelopm e n t o f force s h o u l d n o t then be limited by the rate o f calcium t r o p o n i n interaction. A p r e l i m i n a r y a c c o u n t o f this w o r k has a l r e a d y been p u b l i s h e d (Griffiths et al., 1978).
Methods Single fibres (diameter 50 - 70 gm) were isolated from the sartorius or semitendinosus muscle of either Rana esculentaor R. temporariaand were rapidly skinned with needles under a drop of ATP solution containing a high concentration of free EGTA [ethyleneglycol-bis (flaminoethylether) N,N'-tetraacetic acid] and then glued between a length step generator and a tension transducer. Fibre length was typically 3 mm (sarcomere length 2.4~m, as measured by He-Ne-laser diffraction), Solutions contained (in mM): imidazole, 60; Na, 15; K, 250; ATP 7.5 ; Mg, 7.5; C1, 192; creatine phosphate, 10; creatine kinase, 25 U. ml- 1; X 2-, 50, where X 2- represents the total EGTA of 0.2 mM total EGTA and 49.8mM HDTA (hexamethylenediamineN,N,N',N'-tetraacetic acid). HDTA can be used as a substitute for EGTA to maintain ionic strength (Moisescn, 1976). Solutions were adjusted to pH 6.7. Free Ca 2+ was calculated assuming an apparent binding constant for EGTA of 1.2 x 106 M-1 (estimated from Anderegg, 1964) at this pH. 10mM caffeine was present in all solutions. A calcium contamination of 3 x 10 -6 M in the experimental solution was calculated from spectrophotometric measurements. Tension was measured using a commercially available tension transducer (Aksjeselskapet 802, 6 kHz resonance frequency, stability at room temperature _+20 pN, compliance adjusted to I lam. mN- t). The transducer was elongated using a piece of carbon fibre to which the preparations were fixed. Changes in length could be administrated to the preparation using a Ling dynamics model 101 vibrator or using the relay type length step generator as described by Gtith and Kuhn (1978). Changes in length were measured either by the change in impedance of germanium field plates displaced in a magnetic field (Steiger and Riiegg, 1968) or by use of a phototransistor device (Gfith and Kuhn, AT 1978). Measurements of immediate stiffness - - were made by AL applying small sinusoidal length oscillations to the preparation (1 kHz, 0.05 % L0). Alternativelystiffness was also evaluated from the linear portion of a force extension diagram obtained (on a digital oscilloscope type Nicolet 1090A Explorer) during quick stretch or release applied within 0.3 ms by means of the relay type length step generator (for details of the method see G~th and Kuhn, 1978). Single fibres attached to the apparatus were exposed to a high EGTA (50 mM) concentration solution free of Ca 2+ after being fixed to the apparatus, in which 10mM caffeine was present to remove Ca 2+ ions present in the sarcoplasmic reticulum (Ashley etal., 1975). After 3 rain, fibres were transferred to the relaxing solution containing the low concentration of EGTA (0.1 mM) in which they remained for further 3 rain, On transfer to the Ca 2+ containing EGTA solution of high concentration (50 mM) tension developed rapidly.
pfltigers Arch. 382 (1979)
Results The rate o f tension d e v e l o p m e n t resulting f r o m a ' C a j u m p ' was influenced by free C a 2 + c o n c e n t r a t i o n a n d t e m p e r a t u r e . F i g u r e 1 shows a time course o f tension rise following a s u d d e n increase o f C a 2 § to 1 0 - 6 M (a ' C a - j u m p ' ) . T y p i c a l h a l f times for tension rise were 15 s a n d 1.5 s at p C a ' s 5 a n d 6 respectively (9~ p H 7.0), or 3.8 s a n d 0.45 s at these p C a ' s (22~ p H 7.0). R e v e r s e d ' C a - j u m p s ' f r o m low p C a values to high ones were f o u n d to occur with a very r a p i d halftime (120 ms at 12~ . Q10 for the halftime o f tension rise following a C a - j u m p was 2.6. T h e e x t e n t o f calcium i n d u c e d tension was dep e n d e n t on the c a l c i u m ions c o n c e n t r a t i o n , the calcium c o n c e n t r a t i o n r e q u i r e d for h a l f m a x i m u m a c t i v a t i o n being o f a b o u t p C a 6.0. M a x i m u m tension at full a c t i v a t i o n ( p C a a b o u t 5.5) was c o m p a r a b l e to tension o b t a i n e d on tetanic s t i m u l a t i o n o f intact single fibres being o f the o r d e r o f 25 N . c m - 2 at 12~
Time Course o f Stiffness-Changes In o r d e r to m e a s u r e fibre stiffness, the sinusoidal oscillations described in the m e t h o d s section were i m p o s e d on the fibre length d u r i n g the Ca 2 § i n d u c e d tension rise a n d the c o r r e s p o n d i n g tension changes recorded. A s can be seen in Fig. 1, the presence o f the length oscillations h a d little effect u p o n the time course or extent o f the c a l c i u m i n d u c e d tension increase. R e c o r d s were o b t a i n e d at p C a values o f activating solutions in the r a n g e 6.5 to 5, a n d in all cases stiffness a n d tension increased in parallel to one another, as s h o w n in Fig. 2. W h e n stiffness was p l o t t e d versus force d u r i n g the rising p h a s e o f tension a straight line was o b t a i n e d which p a s s e d t h r o u g h the origin (Fig. 2). This g r a d i e n t o f stiffness a n d tension equals the stiffness to tension r a t i o o b t a i n e d at steady state o f force m a i n t e n a n c e . In o r d e r to find o u t whether the elasticity o f c o n t r a c t e d fibres obeys H o o k e ' s law fibres have been stretched a n d released (by 0 . 3 % ) d u r i n g a calcium i n d u c e d tension d e v e l o p m e n t , a n d tension was p l o t t e d versus the length change d u r i n g stretch or release. As s h o w n in Fig. 3 the force extension d i g r a m s o b t a i n e d d u r i n g stretch were fairly linear indicating t h a t H o o k e ' s law is obeyed. D u r i n g the g r a d u a l b u i l d - u p o f contractile tension after i m m e r s i n g into the c a l c i u m sol u t i o n the slope o f a force extension d i a g r a m b e c a m e steeper i n d i c a t i n g t h a t stiffness grows in p r o p o r t i o n to tension. A similar force length r e l a t i o n s h i p was obt a i n e d in e x p e r i m e n t s in which the fibres were released r a t h e r t h a n stretched d u r i n g different times o f tension d e v e l o p m e n t . In all cases the linear p a r t o f the force extension curve intercepted the abscissa at a b o u t 0.7 to
P. J. Griffiths et al. : Calcium Activation Kinetics in Muscle
167 -
200
A
[
O
~
,s0 "
100
100pN
J 9~
-
-
m
uJ
1,
Z
ss
"
LI-
o
s'o
lbo
lgo
2bo
2go
5 ,,ob,%,o-I
.
10 s _
B / 1
B
IIIII
1/
,r
100
D
501
mN
.I~
100pN
5oom 10s
2
pH 6.7 , 12~ , pea 6
o
s'o
16o
{..]
2bo
TENSION
Fig. 1. Calcium activated tension development following a 'Ca-jump' in a skinned single muscle fibre from frog sartorius in the presence (B) and absence (A) of sinusoidal length oscillations (0.05 %Lo, I kHz). Stiffness is indicated from the band width of the signal in record B. Temperature 9~ At arrow, Ca-jump: fibre transferred from relaxing solution containing 0.2 mM EGTA (pCa > 8) to activating solution [pCa 6, calcium buffer (EGTA total) 50mM] at 9~ Fig. 2. The relationship between stiffness and tension (C) during the tension development resulting from a 'Ca-jump' (A) and the relationship between stiffness and tension (D) during recovery from a quick release (B), both performed at pCa 6, 9~ Stiffness was measured using small amplitude oscillations in length for both forms of tension development. All records were obtained from one experiment on a skinned fibre from sartorius muscle. Note the delayed tension rise following the restoration of the fibre length to its original value in B
0.9% Lo (as found with a different technique by Yamamoto and Herzig in skinned muscle fibres). The intercept is slightly larger than that obtained in releasing contracted living muscle fibres (C. F. Ford et al., 1977).
Recovery After Quick Release The question arises whether a slow increase in tension and stiffness induced by Ca is rate-limited by activation processes (e.g. binding of Ca z + to troponin) or by the actin myosin interaction per se. The latter may be judged from the rate of tension (and stiffness) recovery following a quick release of a fibre which is already in a state of activation and tension development. A quick release of isometrically contracted single fibres of about 4 %L o was performed within 5 ms. Tension fell to almost zero after which tension recovered. Stiffness and tension were rising at a much faster rate during the recovery following the quick release than during the tension rise following the 'Ca-jump'. In both cases, however, stiffness and tension rise in proportion and in the same relation to one another (Fig. 2). It was of interest to know if the rate of tension and stiffness rise following a quick release showed a similar Ca 2 + sensitivity as the tension rise of a Ca-jump. A comparison of the effects of Ca 2 + on the two types of
tension rise is shown in Fig. 4. Recovery from a quick release shows little Ca 2 § sensitivity in the pCa range examined, and in all cases the stiffness to tension ratios were constant during the tension rise. In Fig. 5, the Ca 2 + sensitivity of isometric tension and ofhalftimes of tension rise following quick release and 'Ca-jump' are shown. It can be seen that the range of Ca 2 + sensitivity of the rate of tension development following a 'Cajump' extends outside the range of sensitivity of isometric tension. At pCa 5.8 tension production is already fully activated whereas the rate of force development is only 10 ~ of the rate at pCa 5. There was a linear relationship between the logarithm of the rate of tension development and pCa in the range from 6 to 5. The recovery of tension following a quick release shows little Ca 2+ sensitivity in the range of Ca 2§ concentrations examined.
Discussion Under Ca-jump conditions using the Ca-jump we find that a sudden increase in external Ca2+-ion concentration causes a rapid force development in skinned fibre, the rate of which is similar to that reported previously by Moisescu and Thieleczek (1978). This rate is much smaller than the rate of tension rise on
168
Pfltigers Arch. 382 (1979)
10-5M
A //"
,mN I 1
j
//" I
i
I
2s
10-SM
C a ++
C a ++
C !
, ,
T/2=17 ms
2s
B
20 prn ]
I 0 -5"8 M Ca++
10-5"8M C a ++
B
D
1mN[ T/2:23ms I
I0
ms
I
I
I0 pN
,.-%iL.~%
-,To
5"-'::
-o3s
~
-o's
C
..
~
-o72s
ms
~
o
2s ' ~ 200ms Fig, 4. Comparison of the velocity of tension development in a 'Cajump' (A and B) and the recovery from a quick release of 4 % Lo (C and D) at different Ca + concentrations, 10- 5 M in A, C; 10- s.8 M in B, D. Half times of tension rise are shown accompanying the relevant transients. All transients were obtained from a single skinned frog sartorius muscle fibre during the same experiment at 12~
.-~
o'25
% Lo Fig.3A--C. Increase of immediate stiffness during isometric contraction following 'Ca-jump' from pCa 8 to pCa 5.6; single frog sartorius muscle fibre at 4 ~C. (A) Time course of force development during which stiffness was measured using square wave stretches (left) or releases (right). (B) Typical tension response to quick stretch (left) or quick release (right); force before length step: 0,2 mN. (C) Force extension curves (Tl-curves) obtained during length steps applied at various moments during the tension rise following a 'Cajump'. Note that stiffness (slope of Tl-curve) increases in proportion to contractile force during contraction, the linear portions of the force extension curves extrapolating to zero tension to intercept the abscissa at - 0 . 9 % L0
ul
g .,d_
0-
-
-1.5'
~: -t0-
-';----'-
"--'-
- -" .....
: .....
~-
B
-- -OS"-" &
tetanizing intact fibres and the rate of tension recovery following a quick release of isometrically contracted skinned fibres. The possibility must therefore be considered that following a Ca-jump the rise in internal Ca 2 +-ion concentration may be delayed partly because of diffusional hindrances and partly because of the buffering action of intracellular calcium binding sites (e.g.: sarcoplasmic reticulum, mitochondria) which might compete with the myofilaments for Ca 2 § ions. The principle of the Ca-jump technique is to reduce the concentration of these binding sites prior to entering the activating solution, with its high concentration of calcium buffer. The EGTA concentration of the binding sites prior to activation is reduced by preequilibration in 0.1 mm EGTA solution. The buffering action of the sarcoplasmic reticulum may be reduced by the presence of caffeine in the solution, which is known to reduce the capacity for retaining calcium in the
50-
9
C
o051.0
9
9 ~
C5
~ }pco}
Fig. 5. Comparison of calcium ion dependence of (A) isometric tension (mean values of 7 experiments) (B) halftime of recovery following a quick release of 4 % Lo and (C) halftime of a 'Ca-jump' induced tension development. Halftimes of tension development (T) were all normalised with respect to the halftime of tension development as a result of a 'Ca-jump' from pCa 8 to pCa 5, p H 6.7: (Ordinate = log Ta Tb, where Ta is halftime of observed tension rise and Tb is halftime of tension rise following a ~ to pCa 5)
compartment (Caputo, 1976). In similar experiments in which the SR disrupting non ionic detergent Brij 58 (Orentlicher et al., 1974) has been used instead of caffeine in our solutions, the results were quantitatively similar to those obtained in the presence of caffeine. The velocity of binding of Ca ions by mitochondria
P. J. Griffithset al.: CalciumActivationKineticsin Muscle appears to be too small to significantly compete with EGTA as a Ca 2 § buffering compartment (Batra, 1973). Moisescu and Thieleczek (1978) have calculated the time course of establishment of a new free calcium concentration inside the fibre following a Ca-jump, allowing for diffusional delays and the relatively low dissociation rate of Ca EGTA. Their predicted rate of change of free Ca inside the fibre agreed well with measurements they obtained using the photoprotein aequorin. These showed that the Ca-ion concentration inside the fibre approached that of the buffer within 200 ms when the single fibre was immersed into CaEGTA-buffer-solution. Since our experiments were conducted on the same type of fibre under similar experimental conditions, we feel justified in assuming that the new Ca 2 + ion concentration was established within 0.5 s in our experiments, while the development of tension took a much longer time. The rate of tension development was not altered by changing the total Ca buffer content of the activating solution at a given pCa, but was increased on reducing the ionic strength. Diffusion processes seem, then, unlikely to be the cause for slowness of tension development, in particular because of the high Q10 value for the speed of tension development and the tenfold increase in rate of tension development accompanying a tenfold increase in free Ca 2§ ion concentration (compared to a 1.7 fold increase in the total calcium content of the activating solution). Moisescu (1976) has provided evidence suggesting that the force development resulting from a 'Ca-jump' is rate-limited by the binding of Ca 2 § ions to troponin. Our experiments indicate that there is a linear relationship between the logarithm of the rate of contraction and the pCa. From the size of the slope of this relation it appears that the binding of only one of the postulated four Ca 2 + ions binding to troponin is rate limiting. As pointed out already, the theory of Ashley and Moisescu assumes that the Ca-dependent time course of tension increase does reflect the time course by which the number of actin myosin linkages increases. The observed proportionality between stiffness and tension (measured using small amplitude sinusoidal length oscillations) supports this notion, since stiffness is related to the extent of actin myosin interaction according to Huxley and Simmons. However, one might object to this interpretation on the grounds that the observed proportionality of stiffness and tension increase might be also accounted for by the presence of additional series elasticity. For example, supposing the central part of the fibre were to shorten during activation at the expense of the tendon regions, the system would behave as a two component model consisting of a contractile element connected to a series elastic element (cf. Carlson and Wilkie, 1974). If the
169 elastic elements were non linear (i.e. : exponential) the shortening of the contractile elements would of course also lead to a proportional increase in tension and stiffness (when measured by small length changes 0.05 ~ Lo), though the latter would not then be directly related to crossbridge attachment. To exclude this possibility immediate stiffness (Huxley and Simmons, 1973) has also been measured using force extension curves (T1-curves) obtained by recording tension and muscle length during length steps (0.3 ~ Lo, i.e. : a large length change) applied at various moments during the tension rise. The curves show (1) a linear relation between length change and tension indicating a Hookean elasticity for the elastic component for small stretches and releases; (2) they extrapolate to the same abscissa intercept of about - 0 . 8 ~ L 0 regardless of tension; (3) the slope (stiffness) is proportional to tension before the length change. If stiffness is a relative measure of the number of crossbridges attached to the actin filament as suggested by Huxley and Simmons (1973) it follows that the tension rise following a 'Cajump' does reflect the time course of the increase in actin myosin interaction possibly by the recruitment of crossbridges as assumed in the theory of Ashley and Moisescu. Isometrically contracted fibres in which Ca 2 + ions were already equilibrated with troponin were released in 5 ms by a length change sufficient to bring the tension at the end of the release close to zero (between 2 ~ L0 and 4 ~ L0). At the end of the release, tension recovered at a rate much faster than that of the tension rise accompanying a Ca jump. Furthermore, this rate of tension recovery following a quick release was insensitive to the free Ca 2+ ion concentration. The stiffness to tension ratio was maintained constant during the tension rise, just as in the measurements obtained during tension development accompanying a Ca jump. In this type of experiment the regulatory system is already at equilibrium with the free calcium concentration, and so the recovery of stiffness and tension is presumably determined by the rate of actin myosin interaction, i.e.: crossbridge attachment and detachment, rather than by Ca binding. These experiments show that actin myosin interaction is so fast that it cannot be rate limiting during the relatively slow tension and stiffness increase following a 'Ca-jump'. From such measurements on the rate of stiffness and tension increase following a 'Ca-jump' the rate of Ca binding can be determined and may be compared with biochemical studies on cation binding by troponin (e.g. : Potter 1975). On lowering the ionic strength or reducing the free magnesium ion concentration in the solution, it is possible to increase the velocity of a 'Cajump' activation to approach that of a recovery from a quick release (unpublished experiments). Since the rate
170 o f t e n s i o n d e v e l o p m e n t d u r i n g t e t a n u s in the i n t a c t fibre is v e r y similar to the r a t e o f r e c o v e r y f o l l o w i n g a q u i c k release, it w o u l d s e e m likely t h a t the r a t e o f t e n s i o n d e v e l o p m e n t in the t e t a n i z e d i n t a c t fibre is n o t l i m i t e d b y C a b i n d i n g k i n e t i c s b u t p r o b a b l y b y the r a t e of actin myosin interaction.
References Anderegg, G. : Reaktionsenthalpie und -entropie bei der Bildung der Metallkomplexe der h6heren EDTA-Homologen. Helv. Chim. Acta 47, 1801-1814 (1964) Ashley, C. C., Griffiths, P. J., Moisescu, D. G., Rose, R. M. : The use of aequorin and the isolated myofibrillar bundle preparation to investigate the effect of SR calcium releasing agents. J. Physiol. (Loud.) 245, 12-14P (1975) Ashley, C. C., Moisescu, D. G. : Model for the action of calcium in muscle. Nature New Biol. 237, 208-211 (1972) Ashley, C. C., Moisescu, D. G. : Tension changes in isolated bunclles of frog and barnacle myofibrils in response to sudden changes in external free calcium concentration. J. Physiol. (Lond.) 233, 8 9P (1973) Ashley, C. C., Moisescu, D. G.: The part played by Ca 2+ in the contraction of isolated bundles of myofibrils. In: Calcium transport in contraction and secretion (E. Carafoli, ed.), pp. 517-525. Amsterdam: North-Holland Publishing Company 1975 Ashley, C. C., Ridgway, E. B. : On the relationship between membrane potential, calcium transient and tension in single barnacle muscle fibres. J. Physiol. (Lond.) 209, 105 - 130 (1970) Batra, S. : The effects of zinc and lanthanum on calcium uptake by mitochondria and fragmented sarcoplasmic reticulum of frog skeletal muscle. J. Cell. Physiol. 82, P245-256 (1973) Blinks, J. R., Rtidel, R., Taylor, S. R. : Calcium transients in isolated amphibian skeletal muscle fibres. Detection with aequorin. J. Physiol. (Loud.) 277, 291 - 334 (1978)
Pflfigers Arch. 382 (1979) Caputo, C. : The effect of caffeine and tetracaine on the time course of potassium contractures of single muscle fibres. J. Physiol. (Lond.) 255, P191-207 (1976) Carlson, F. D., Wilkie, D. R. : Muscle physiology. Englewood Cliffs, N.J, : Prentice-Hall, Inc., 1974 De Clerck, N. M., Claes, V. A., Brutsaert, D. L.: Force velocity relations of single cardiac muscle cells. J. Gen. Physiol. 69, P221-241 (1977) Ford, L. E., Huxley, A. F., Simmons, R. M.: Tension responses to sudden length changes in stimulated frog muscle fibres near slack length. J. Physiol. (Lond.) 269, 441-517 (1977) Goldman, Y. E., Simmons, R. M. : Active and rigor muscle stiffness. J. Physiol. (Lond.) 269, 5 5 - 5 7 P (1977) Griffiths, P. J., Kuhn, H. J., Riiegg, J. C. : Rapid calcium activation of skinned fibres. J. Physiol. (Loud.) 284, 141P (1978) Giith, K., Kuhn, H. J. : Stiffness and tension during and after sudden length changes of glycerinated rabbit psoas muscle fibres. Biophys. Struct. Mech. 4, 223-236 (1978) Huxley, A. F., Simmons, R. M. : Mechanical transients and the origin of muscular force. Cold Spring Harbor Symp. Quant. Biol. 37, 669- 680 (1973) Moisescu, D. G. : Kinetics of reaction in Ca-activated skinned muscle fibres. Nature 262, 610-613 (1976) Moisescu, D. G., Thieleczek, R. : Calcium and strontium concentration changes within skinned muscle preparations following a change in the external bathing solution. J. Physiol. (Loud.) 275, 241-262 (1978) Orentlicher, M., Reuben, J. P., Grumpert, G., Brandt, P. W.: Calcium binding and tension development in detergent-treated muscle fibres. J. Gen. Physiol. 63, 168-187 (1974) Potter, J. D., Gergely, J. : The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphospfiate. J. Biol. Chem. 250, 4628-4633 (1975) Yamamoto, T., Herzig, J. W.: Series elastic properties of skinned muscle fibres in contraction and rigor, Pfltigers Arch. 373, 21 24 (1978)
Received July 7/Accepted July 13, 1979
