Intracellular calcium movements in skinned muscle fibres

1. Force development and (45)Ca movement between the internal membranes and the myofilament space were measured in segments of skinned muscle fibres.2. At 20 degrees C calcium ions in the myofilament space of calcium loaded fibres caused the internal membranes to release a substantial amount of calcium which produced a large force spike. Calcium produced a similar force spike in fibres containing only the physiological level of calcium when the temperature was lowered to about 6 degrees C.3. The force spike at 6 degrees C was inhibited when the magnesium concentration in the bathing solution was increased from 1 to 6 mM. The pattern of force development in preparations transferred directly from oil to a free calcium solution indicated that the normal concentration of Mg(2+) in the myofilament space of intact fibres is of the order of 10(-3)M.4. A possible role for the regenerative calcium release mechanism in the physiological activation of muscle fibres is suggested.     

The relation between calcium and contraction kinetics in skinned muscle fibres

1. Segments of skinned frog muscle fibres were immersed in solutions in which the calcium ion concentration, and therefore the steady isometric force, was controlled with an EGTA buffer system. The contraction kinetics were measured when the load was quickly reduced to values less than the isometric force.2. The velocity of shortening following release from steady force was a hyperbolic function of relative load. The minimum quick displacement required to reduce the force from the steady isometric value to half this value was about 1% of the fibre segment length.3. The relative force-velocity relation was independent of pCa in the range 5.0-6.75. Thus calcium ions appear to control the number of sites at which cross-bridges can be formed but have no significant effect on the kinetic properties of an individual bridge.4. Cross-bridges along the length of a myofilament appear to act independently of each other.5. The force-velocity relations reported for intact muscle fibres during relaxation, when compared with those of skinned fibres at different calcium levels, indicate that the time course of the fall in force after a stimulus is essentially that of calcium removal from the myofilaments.     

Tension development and calcium sensitivity in skinned muscle fibres of the frog

Calcium activated isometric tension development was measured in single skinned muscle fibres of the ileofibularis muscle of the frog. The experiments were carried out at 5°C, pH=6.9, 1 mM free Mg²⁺ and an ionic strength of 160 mM. A Hill curve was fitted to the isometrically developed tension at different Ca²⁺ concentrations by means of a non-linear least mean square approximation. At a sarcomere length of 2.15 m, the Ca²⁺ concentration for half maximum tension (K) was 1.6 M. This Ca²⁺ concentration decreased with increasing sarcomere length; at 2.7 m, K was 1.1 M and at 3.1 m, K was 0.9 M. Therefore, Ca sensitivity is increased at larger sarcomere lengths. Consequently, the optimal sarcomere length for tension development shifted to larger values when the Ca²⁺ concentration was lowered. Osmotic compression of the fibre at 2.15 m by means of 5% Dextran also caused an increase in Ca sensitivity (K was 1.0 M). At 2.7 m, addition of 5% Dextran hardly affected the Ca sensitivity. The possible role of the interfilament spacing in the explanation of these results discussed.     

Deterioration induced by physiological concentration of calcium ions in skinned muscle fibres

Summary The deteriorating effect of m order of Ca²⁺ on skinned frog skeletal muscle fibres was studied from the view point of the digestion of proteins by calcium-activated neutral protease (CANP). Tension developed in solutions containing no MgATP (rigor solution) decreased irreversibly with the addition of Ca²⁺ in quantities of more than 0.1 m. Low temperature was seen to suppress (Q₁₀>4), and neutral pH to maximize, this decrease in tension. In rigor solution containing Ca²⁺, SDS electrophoresis indicated that a 95 k dalton component (-actinin) was released from the fibre; electron micrography showed the disappearance of Z-lines. These results suggest that one of the causes for decrease in rigor tension is the proteolytic activity of CANP, and its inhibitors were shown to be quite useful in experiments on skinned fibre.     

Force measurements in skinned muscle fibres

1. Isometric force was measured in skinned segments of frog semitendinosus muscle fibres exposed to solutions in which the calcium ion concentration was controlled with EGTA.2. The threshold for force development, calculated from an apparent stability constant for the CaEGTA complex of 10(6.69)M(-1) at pH 7.0, was generally close to pCa 7.5. Maximum force was reached at about pCa 6.0.3. Maximum force is proportional to the cross-sectional area of the fibres.4. The rate of force development was slower than that expected from simple diffusion of a substance from the bathing solution into the fibre. The delay appears to be due to slow equilibration of the EGTA buffer system during calcium uptake by the sarcoplasmic reticulum.5. Addition of deoxycholate (DOC) to the bathing solution produced a reversible increase in the rate of force development. The steady force was also increased for values of pCa that gave less than maximum force, which shifted the force-pCa relation toward lower calcium concentrations by about 0.5 pCa unit.6. The length-force relation in partially activated preparations is similar to that reported for electrically activated intact fibres. This result suggests that in the region of myofilament overlap the affinity of the binding sites for calcium is uniform along the length of the calciumbinding myofilament.     

Effects of amrinone on shortening velocity and force development in skinned skeletal muscle fibres

Summary The effects of amrinone were studied on single skinned fibres isolated from rat hindlimb muscles. In each fibre a force-velocity relation was determined during maximal calcium activation (pCa=4.45) in control conditions and in the presence of amrinone. The MgATP concentration was 3.93 mm, close to the physiological value. After the experiment the fibre was classified as fast or slow on the basis of its reactivity with anti-myosin monoclonal antibodies. In fast fibres amrinone (3 mm) potentiated isometric tension (P ₀) by 13.8±2.9% (n=13), reduced maximum shortening velocity (V _max ) by 32.6±3.2% and the curvature of the force-velocity relation (a/P ₀) was increased by 98.9±46.0%. All these effects were less pronounced in slow fibres, where V _max was reduced only by 11.4±3.6 (n=16). The effects of amrinone (0.3–6 mm) on the ATPase activity of myofibrils and myosin prepared from fast (tibialis anterior) and slow (soleus) rat skeletal muscles were studied. Amrinone was found to depress Ca–Mg dependent ATPase activity of myofibrillar preparations of the tibialis anterior (up to 16.6±2%) and, to a lesser extent, of the soleus (up to 7.2±1.2%). On the contrary, Ca-stimulated myosin ATPase activity was significantly increased by amrinone in myosin preparations from the tibialis anterior. Experiments were carried out to test whether amrinone (3 mm) might affect the sensitivity of the contractile system to MgATP concentration ([MgATP]). The results obtained showed that (1) the [MgATP] value at which isometric tension reached its maximum was shifted by amrinone from 0.1 mm to 0.3 mm, (2) the slope of the negative relation between [MgATP] and a/P ₀ was made more steep by amrinone, and (3) the Km of the hyperbolic relation between [MgATP] and V _max was increased from 0.39 to 1.71 mm by amrinone, thus indicating a reduced affinity of myosin for MgATP. These results are in accordance with the hypothesis that amrinone exerts a direct effect on the contractile mechanism.     

Relationship between sarcoplasmic reticulum volume and calcium capacity in skinned frog skeletal muscle fibres

Semitendinosus fibres from Rana pipiens were examined in the electron microscope. When the aqueous solutions in which the fibres were prepared contained no sucrose, the sarcoplasmic reticulum was markedly swollen and possibly fragmented. When as little as 50 mM sucrose was included in the aqueous solution, the sarcoplasmic reticulum retained the flattened appearance characteristic of intact fibres. The degree of flattening was not substantially different with concentrations of sucrose ranging from 50 to 300 mM, suggesting that the flattened volume may be determined by structural elements. In all the fibre segments, the regularity of the hexagonal filament lattice was disrupted and the disruption was not affected by sucrose. Radioactive calcium uptake was measured in the presence and absence of 200 mM sucrose. The capacity of the sarcoplasmic reticulum (SR) was depressed by about 30% by the sucrose but the initial uptake was not significantly affected. This finding suggests that most of the calcium within the SR is bound and not free in solution.     

The effect of procaine on snake twitch muscle fibres

1. The effect of procaine on the contractile responses of voltage clamped snake twitch muscle fibres has been investigated. Procaine hydrochloride shortened the duration of contractures produced by long depolarizing pulses. It also reduced the amount of inactivating outward current.2. The rate of repriming was greatly slowed by procaine. At -80 mV, for example, the time to half restoration of contractile response was 6 sec in procaine (5 x 10(-3), w/v) as compared to 1.15 sec in tetrodotoxin (TTX), (1 x 10(-7) w/v). At -100 mV, repriming was still much slower in procaine. However, at -150 mV, repriming in procaine was quite rapid, the time to half recovery being about 0.4 sec.3. After prolonged exposure to procaine, subthreshold depolarizing pulses produced a marked diminution in the contractile response to a subsequent supermaximal depolarization. In some fibres complete inactivation could be produced in the presence of procaine without any contractile response having occurred.     

Rate of isometric tension development in relation to calcium binding of skinned muscle fibres

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 (T₁-curves) measured at various moments during the rise of tension extrapolated to zero tension intersected the base line at the same length (–0.8% L_o). 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% L_o 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 myofilament binding site and does not reflect the slowness of actin and myosin interaction.This work was supported by a grant from the Deutsche Forschungs-gemeinschaft Gu 160/3. One of us (P.J.G.) was supported by the Royal Society European Science Exchange Programme     

Alteration of calcium sensitivity of skinned frog skeletal muscle fibres by inositol triphosphate and calmodulin antagonists

We investigated the influence of inositol triphosphate (IP₃), trifluoperazine (TFP), and perhexiline on the calcium sensitivity of freeze-dried frog semitendinosus muscle fibres. Further, the effect of IP₃ on calcium release from the sarcoplasmic reticulum (SR) of frog semitendinosus fibres skinned by saponin was studied. IP₃ decreased the calcium sensitivity of freeze-dried frog skeletal muscle fibres and failed to induce a calcium release from SR of saponin-skinned fibres. Freeze-dried frog skeletal muscle fibres were strongly sensitized for calcium by TFP and perhexiline.     

Effects of myofibrillar bundle diameter on the unloaded shortening velocity of skinned skeletal muscle fibres

Using both slack tests and force clamp experiments, the velocity of unloaded shortening (Vu; Vu(st), slack test; Vu(fc), force clamp) was determined for maximally Ca2+-activated myofibrillar bundles. These were obtained by mechanically splitting single muscle fibres of rat, rabbit, crab and lobster skeletal muscles. A comparison was made between the Vu of thick (mammalian: 45-70 mum mean diameter; crustacean: 90-175 mum) and thin (mammalian: 25-40 mum; crustacean: 35-85mum) preparations of the same muscle fibre. The bundle diameter had opposite effects on Vu in mammalian and crustacean muscle fibres. The Vu of thin mammalian bundles was about 0.6times that of the thick ones, whereas in crustacean preparations this ratio was about 1.5. The kinetics of stretch-induced delayed force increase of maximally Ca2+-activated fibres (stretch activation) appeared not to differ between the thick and thin bundles from any animal preparation. Control experiments showed that the observed diameter effects on Vu are not due to differences in the chemical environment of the myofilaments. One possible explanation is that the intrinsic physical factors of the myofibrils modify Vu differently during progressive shortening in mammalian and crustacean preparations. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effect of calcium on the maximum velocity of shortening in skinned skeletal muscle fibres of the rabbit

Summary The relationship between maximum shortening velocity (V _max) and free calcium concentration has been studied in skinned single fibres from rabbit psoas and soleus muscles. At both 10 and 15° C,V _max measured in the psoas fibres was found to decrease by 40% when the pCa (-log[Ca²⁺]) was increased from 5.49 (maximally activating) to 6.21. Further decreases inV _max were observed when the pCa was increased to 6.32.V _max measured in soleus fibres at 15° C also decreased when the Ca²⁺ concentration was lowered, though the magnitude of this effect was slightly less than in the psoas fibres. Thus, a distinct effect of Ca²⁺ uponV _max has been shown to occur in mammalian skeletal muscle. The occurrence of this effect in both fast and slow muscle types may indicate that the underlying mechanism in the two cases is similar.     

Filament overlap affects TnC extraction from skinned muscle fibres

Summary Recent studies on calcium regulation of muscle contraction selectively extract troponin C (TnC) from skinned skeletal muscle fibres with a low ionic strength rigor solution containing a Ca²⁺/Mg²⁺ chelator. As previous results from this laboratory and others demonstrate a crossbridge effect, especially rigor, on many of the properties of TnC, the effects of filament overlap on TnC extraction from skinned rabbit psoas muscle fibres were investigated. Tension-pCa relationships at a sarcomere length of 2.7 m were determined before and after a 5 min TnC extraction at sarcomere lengths of 2.3, 2.5, 2.7, 3.1, 3.3 or 3.5 m with 20 mm Tris, pH 7.8, 5 mm EDTA. The decrease in the post-extraction maximum Ca²⁺ activated tension, an indicator of the amount of TnC extracted, was linearly related to the overlap of the thick and thin filaments with decreases in tension being associated with a decrease in filament overlap. The smaller fibre diameter at the longer sarcomere length could facilitate diffusion of TnC from fibre segments. However, the wide range of measured diameters, 40–120 m, accounted for only 14% of the observed tension decrement and shrinking the fibre with polyvinylpyrrolidone did not increase the tension decrement. Increasing the sarcomere length before extraction was also found to decrease the TnC content of fibre segments along with the post-extraction maximum tension. Thus, TnC appears to be preferentially extracted from non-overlap than overlap regions of the sarcomere. These results further indicate that rigor crossbridges affect TnC other than through increased Ca²⁺ binding and that under the conditions used here, they retard its extraction.     

Concentration-dependent effects of tetracaine on excitation-contraction coupling in frog skeletal muscle fibres

Summary The effects of low (10–100 m) concentrations of tetracaine on intramembrane charge movement and on the rate of calcium release (R_rel) from the sarcoplasmic reticulum (SR) were studied in cut skeletal muscle fibres of the frog using the voltage clamp technique. The fibres were mounted in a single or double vaseline gap chamber to study the events near the contraction threshold or in a wide membrane potential range. Although the hump component of charge movement (Q) was suppressed to some extent, the voltage dependence and the parameters of the Boltzmann distribution were not modified significantly at tetracaine concentrations below 50 m. At 50 and 100 m of tetracaine the midpoint voltage of the Boltzmann distribution was shifted to higher membrane potentials and the steepness was decreased. The total available charge remained the same at all concentrations tested. Using fura-2 to measure calcium transients at 100 m tetracaine the threshold for calcium release was found to be significantly shifted to more positive membrane potentials. Tetracaine reversibly suppressed both the early inactivating peak and the steady-level of R_rel but the concentration dependence of these effects was markedly different. The inactivating component of calcium release was decreased with a Hill coefficient of approximately 1 and half effective concentration of 11.8 m while the steady-level was decreased with a Hill coefficient of greater than 2 and a half effective concentration of 47.0 m. These results favour two sites of action where tetracaine would suppress the calcium release from the SR.     

Effects of caffeine on Ca-activated force production in skinned cardiac and skeletal muscle fibres of the rat

Skinned fibres prepared by mechanical and/or chemical means from cardiac and skeletal muscles of the rat were activated in solution strongly buffered for Ca²⁺ (with 50 mM EGTA) in the absence or presence of caffeine 5–40 mM. In all preparations caffeine was found to reversibly shift the relation between steady-state force and free [Ca²⁺] toward lower free [Ca²⁺] in a dose dependent manner. This increase in apparent Ca²⁺ sensitivity was not antagonized by procaine and was the same, within each muscle type, irrespective of the manner in which the skinned fibre was prepared, and consequently the degree to which it retained cellular membranes. The effect was more pronounced in cardiac and slow-twitch than in fast-twitch, myofibrillar preparations. At equivalent concentrations theophylline mimicked this effect of caffeine in all preparations, however, addition of exogenous cyclic AMP neither mimicked nor modified, in any way, the effect. Maximum Ca²⁺-activated force production was not affected by caffeine below 20 mM but was depressed by concentrations of 20 mM and above. The increase in apparent Ca²⁺ sensitivity produced by caffeine can not be the result of a mobilization of some cellular store of Ca²⁺ but must arise from a direct effect of caffeine on the myofilaments which leads to a change in the apparent affinity constant of the force controlling sites for Ca²⁺.     

Cyclic-AMP mediated relaxation of chemically skinned fibres of smooth muscle

Smooth muscle from guinea pig taenia coli was chemically skinned with Triton X-100 and stored in ATP-salt solution containing 50% glycerol at –20°C. Fibre bundles were relaxed at Ca²⁺-concentrations below 10^–7 M, but contracted at 10^–6 M Ca²⁺. The isometric tension developed could be partly relaxed by the addition of c-AMP (in the presence of NaF), and it could also be inhibited following preincubation with the catalytic subunit of c-AMP dependent protein kinase. The inhibitory effect was much more pronounced at intermediate Ca²⁺-concentrations (e.g. 10^–6) than at concentrations producing a maximum contraction, suggesting that Ca-sensitivity had been lowered. Sodium fluoride which was required to potentiate the c-AMP effects was found to have a slight relaxing effect per se. The c-AMP effect may be mediated through activation of cyclic AMP-dependent kinase, producing phosphorylation of the myosin light chain kinase which, according to adelstein et al. (1978), may result in a net dephosphorylation of the myosin light chains and a concomittant inhibition of the contractile response.