Cross-bridge Turnover Research Articles

Force-velocity relation and rate of ATP hydrolysis in osmotically compressed skinned smooth muscle of the guinea pig.

Chemically skinned guinea pig taenia coli fibre bundles showed a concentration-dependent decrease in width when incubated in media containing Dextran T500 (0-0.2 g ml-1). The maximal reduction in width, observed at 0.2 g ml-1 dextran, was 32%. The effect was reversible upon removal of dextran. Isometric force was slightly increased (about 10%) at the lowest dextran concentration (0.025 g ml-1) but decreased at higher concentrations (40% decrease at 0.2 g/ml-1). The energetic tension cost (ATP turnover/force) was decreased by about 40% after dextran addition. Force development and relaxation were markedly slower in 0.1 g ml-1 and absent in 0.2 g ml-1 dextran. In isotonic quick-release experiments 0.025 g ml-1 dextran did not influence maximal shortening velocity (Vmax) and relative stiffness, whereas 0.1 g ml-1 markedly increased stiffness and decreased Vmax to about 27%. Vanadate induced relaxation in the activated muscle (pCa 4.5) both in the absence and presence (0.1 g ml-1) of dextran and increased the rate of relaxation (pCa 9) at 0.1 g ml-1 dextran. The isometric rate of crossbridge turnover, as reflected by the energetic tension cost and the rate of relaxation, was decreased at all degrees of osmotic compression. Crossbridge turnover rate during shortening (Vmax) was unaffected at an osmotic compression of 12% (width) but was decreased at higher compression (32%).

Myosin light chain phosphorylation and the cross-bridge cycle at low substrate concentration in chemically skinned guinea pig Taenia coli.

Force-velocity relations, rate of ATP turnover (JATP), and phosphorylation of the 20,000 D myosin light chains (LC20) were measured in chemically skinned guinea pig Taenia coli. Relative LC20 phosphorylation at 3.2 mM MgATP was 17% in relaxed tissues at pCa 9, and increased with force at increasing [Ca2+] to a maximum of 67% at pCa 4.5. Force at pCa 4.5 was dependent on the MgATP concentration with a half-maximal response at about 0.1 mM. At 0.1 mM MgATP LC20 phosphorylation at pCa 4.5 was 38%. Both JATP and the maximal shortening velocity (Vmax) were reduced in 0.1 mM MgATP, to 32% and 43%, respectively, of their values at 3.2 mM MgATP. Low-MgATP thus inhibits both LC20 phosphorylation and the extent and rate of cross-bridge interaction. High levels of LC20 phosphorylation, independent of Ca2+ and MgATP concentrations, were obtained by treatment with ATP-gamma-S. Maximal force at 3.2 mM MgATP after LC20 thiophosphorylation was unchanged, whereas halfmaximal force occurred at 0.065 mM MgATP after thiophosphorylation, compared to 0.13 mM after activation by Ca2+. The contraction in thiophosphorylated preparations at low-MgATP (0.1 mM) was associated with submaximal Vmax (60%) and JATP (27%). The results show that LC20 phosphorylation is correlated with the degree of force development in the Ca2+ activated contraction, both when Ca2+ and MgATP concentrations are varied. The reduced force and rate of crossbridge turnover in low MgATP are however primarily mediated by an influence of MgATP on the cross-bridge cycle, which is separate from the effect on LC20 phosphorylation.

Ca 2+ sensitivities and transient tension responses to step-length stretches in feline mechanically-stripped single-fibre jaw-muscle preparations

At a muscle length, Lo (just taut), isometric tension at constant levels of various Ca 2+ activations and transient tension responses to rapid length stretches (less than 1 per cent of Lo within 2 ms) at maximal Ca 2+ activation level were measured in temporal, masseter and digastric (anterior belly) muscles (2–3 mm long and 24–48 μm in diameter). Steady isometric tension increased in a sigmoid fashion with increasing Ca 2+ concentration from about pCa 7.28 to 4.49 in temporalis, from about pCa 6.18 to 4.40 in masseter, and from about pCa 5.82 to 4.40 in digastric. The maximum tension was 75.5 ± 10.2 g/mm 2 in temporalis, 44.7 ± 14.1 g/mm 2 in masseter, and 46.1 ± 20.1 g/mm 2 in digastric. In the resting state, the sarcomere length at Lo was 2.34 ± 0.06 μm in temporalis, 2.20 ± 0.08 μm in masseter, and 2.20 ± 0.00 μm in digastric. When the sarcomere length was stretched from 2.20 to 2.34 μm (the sarcomere length of temporalis at Lo) in the masseter and digastric, the Ca 2+ sensitivity increased without significant change of the maximum tension in either muscle. The transient tension responses in all three muscles showed two distinct phases; an immediate tension increase coincident with the length stretch followed by an exponential tension decrease. The mean value of the time constant in the second phase was 58.5 ± 19.7 ms in temporalis, 58.5 ± 12.6 ms in masseter, and 362.6 ± 16.8 ms in digastric. Thus temporalis showed a higher Ca 2+ sensitivity at Lo and a greater maximum tension-producing capability than the other muscles and the cross-bridge turnover rate appears to be slower in digastric than in the others.

Magnesium ion-dependent contraction of skinned frog muscle fibers in calcium-free solution

Skinned frog fibers were reversibly activated in Ca-free solutions containing 0 mM KCl, 23 microM free Mg, and having an ionic strength of approximately 50 mM. Contractile force was nearly maximal at 22 degrees - 25 degrees C and decreased at lower temperatures. Maximal force in Ca-free solution at 50 mM ionic strength was close to twice the calcium-activated force with pCa 5 and 190 mM ionic strength. The force in Ca-free solution could be reduced to zero by raising the concentration of free Mg from 23 microM to 1.0 mM at the same ionic strength (50 mM). On stretching the fiber from 2.0 to 3.2 micron the force decreased; this effect was similar to that seen with Ca-activated fiber and the data support the idea that Ca-free tension is made at the cross-bridge level. Isotonic contraction during Ca-free activation showed a velocity transient as in Ca-activated fiber at 190 mM ionic strength, but the transient in the present case was very much prolonged. This finding suggests that contraction mechanisms for force generation and for shortening are essentially the same in the two conditions, but that certain rate constants of cross-bridge turnover are slower for the Ca-free contraction. Also, the results indicate that, in low ionic strength, Ca binding to thin filaments is not essential for unmasking the cross-bridge attachment sites, which suggests that the steric blocking mechanism is modified under these conditions.

Myosin light chain phosphorylation does not modulate cross-bridge cycling rate in mouse skeletal muscle.

An attempt was made to determine whether phosphorylation of the myosin light chain represents a thick filament-associated mechanism for modulating the rate of cross-bridge cycling in mouse skeletal muscle. When the degree of light chain phosphorylation was varied independently of tetanus duration, there was no correlation of phosphorylation with cross-bridge turnover rate, as measured by the shortening velocity of the muscle. It is concluded that in intact skeletal muscle phosphorylation of the myosin light chain does not in itself modulate cross-bridge cycling rate and that previously reported changes in cycling rate were due to other factors that may vary with tetanus duration.

Effects of fatigue and altered pH on isometric force and velocity of shortening at zero load in frog muscle fibres

temporaria (0.9-2.5degrees C) were stimulated to produce a 1 s isometric tetanus at regular intervals until constant mechanical responses were attained. Various degrees of force depression ("fatigue") were produced by decreasing the contraction interval from 30 or 15 min (control to 120, 60, 30 and 15s, respectively. In this was the steady-state tetanic force could be reversibly reduced to approximately 70% of the control value. The velocity of shortening at zero load, V0, was determined at each level of fatigue using an approach for direct measurement of V0. V0 was not significantly affected as long as the decrease in force was less than 10%. With further reduction of the isometric tension there was a progressive decline of V0 according to the following empirical relationship between percentage depression of force (delta P0) and maximum speed (delta V0) of shortening: delta V0 = 0.006 delta P02.48- 1.0 (correlation coefficient, 0.86). Cine photographic recording of nylon markers on the fibre surface provided evidence that fatigue developed uniformly along the fibre with no sign of failure of excitation in any segment. The change in mechanical performance during fatigue could be reproduced inthe non-fatigued fibre by reducing the pH of the external medium within the range 8.0-6.6 using a bicarbonate-CO2 buffer. A decrease in pH thus reduced both the rate of rise and the total amplitude of isometric force and prolonged the relaxation phase. Furthermore, there was a drop in V0 that was related to the force decline in approximately the same way as observed during fatigue. The results support the idea the fatigue involves both a reduced state of activation of the contractile system and a specific (activation independent) inhibition of crossbridge turnover. Increased intracellular H+ concentration is likely to contribute to the development of both these effects during fatigue.

Vanadare and phosphate ions reduce tension and increase cross-bridge kinetics in chemically skinned heart muscle

Tension development, immediate stiffness and ATPase of chemically skinned myocardial strips were measured in solutions with varying concentrations of phosphate (P i) or vanadate (predominantly H 2VO 4 − at pH 7) ion. Vanadate and P i decreased stiffness in proportion to tension. The results show that, like P i, vanadate accelerates the turnover rate of cross-bridges, but is effective at about 1/500 the concentration required for the P i effect. Both P i and vanadate increased the energy cost of isometric tension maintenance (that is, the ratio of ATPase to tension) and increased the velocity of delayed tension development following quick stretch of the chemically skinned myocardial strips. The results also show that changes in the rate of rise of delayed tension during stretch activation probably reflect changes in the kinetics of the biochemical cycle of the cross-bridges.

The passive elastic force-velocity relationship of cat skeletal muscle: Influence upon the maximal contractile element velocity

The medial gastrocnemius muscle of four cats was studied and two force-velocity relationships were determined: (1) the classical Hill force-velocity curve was derived under isotonic, afterloaded, activated conditions, and (2) a passive elastic force-velocity curve was derived under isotonic, quick-release, passive conditions. The velocity of contraction due to active cross-bridge turnover (pulling the actomyosin filaments over each other), i.e. ( V CE ) a , is defined mathematically as the difference between the overall muscle velocity ( V M ) and the velocity of contractile element shortening due to passive recoil of the parallel elastic spring (pushing the actomyosin filaments over each other), i.e. ( V CE ) p . ( V CE ) p was seen to be nearly equal to V M at zero afterload ( P P 0 = 0 ). ( V CE ) p fell rapidly to zero at P P 0 between 0.15 and 0.20, while V M only fell to zero at P P 0 = 1 . The result was that ( V CE ) a was very low at P P 0 = 0 , rose to a maximum at P P 0 between 0.10 and 0.15, then fell back to zero at P P 0 = 1 . A physiological explanation for this analytical result is proposed.

The force-velocity relation of isolated twitch and slow muscle fibres of Xenopus laevis.

1. A study has been made of the relation between force and speed of shortening, or lengthening, in isolated twitch and slow muscle fibres, dissected from the iliofibularis muscle of Xenopus laevis. Both after-loaded and quick-release contractions were studied. Twitch fibres were stimulated electrically to give tetanic contractions (5-20 degrees C); slow fibres were activated by a rapid change to solutions with high K concentration (30-75 mM; experiments at 21-24 degrees C).2. The velocity of slow fibres was constant during shortening over 10% length change in after-loaded contractions, except at forces exceeding about 0.8 of isometric tension, P(0). In quick-release experiments, shortening velocity was found to depend not only on the relative load, P/P(0), but also on the instant when the release was made. With increasing time after onset of contraction the initial rate of shortening decreased; also, a progressive fall in speed during shortening became more marked.3. The fall in initial shortening speed with time of release from the onset of a contracture was more pronounced at high [K](o) than at low.4. The relation between the relative force, P/P(0), and shortening velocity, V, in after-loaded contractions (75 mM-K) and quick-release contractions (45 mM-K, early releases) in slow fibres could be represented by a hyperbola with the constants a = 0.10P(0), b = 0.11 lengths/sec; extrapolated V(max.) was 1.10 lengths/sec.5. Isometric tension and maximum shortening velocity in slow fibres were very nearly constant between 32 and 75 mM-K. a/P(0), however, was clearly reduced at 32 mM-K, representing a more curved P-V relation.6. Force-velocity data for twitch fibres (quick-release contractions, 20 degrees C) were reasonably well fitted by a hyperbola (a = 0.38P(0), b = 1.97 lengths/sec, V(max.) = 5.20 lengths/sec), but a systematic deviation was observed for forces exceeding 0.6P(0).7. a/P(0) for twitch fibres was found to be independent of temperature in the range 5-20 degrees C. Q(10) for b was 2.24 (10-20 degrees C), and 2.86 (5-10 degrees C).8. V(max.) for twitch fibres was calculated to be 6.34 lengths/sec at 22.5 degrees C, the average temperature in the slow fibre experiments. The maximum shortening velocity in twitch fibres is thus 6 times higher than in slow fibres.9. When loads in the range 1.1-1.4P(0) were quickly applied to an actively contracting slow fibre, lengthening of the fibre occurred in two phases, an initial rapid phase, followed by a phase of extremely slow lengthening. In corresponding experiments on twitch fibres lengthening was rapid at first and then gradually became slower.10. Factors affecting the shape of the force-velocity curve are discussed. Calculations based on A. F. Huxley's (1957) model for muscle contraction indicated that cross-bridge turnover rate is about 15 times lower in slow than in twitch fibres.

The stiffness of amphibian slow and twitch muscle during high speed stretches.

Experiments were carried out to compare the stiffness of cross-bridges in amphibian slow and twitch muscle. An isolated iliofibularis muscle was subjected to rapid, small stretches during contraction of either slow or twitch fibres at a number of different isometric tensions. The method of analysis allowed the compliance of the cross-bridges to be distinguished from other sources of compliance. Provided that the muscle was stretched sufficinetly rapidly to obtain limiting values of stiffness, little difference was found between the mechanical properties of the cross-bridges in slow and twitch muscle. It is concluded that the difference in observed stiffness of the two muscle types is due to a lower turnover rate of cross-bridges and a smaller number of sarcomeres in slow fibres.

Oxygen consumption and lactate production of the rat portal vein in relation to its contractile activity.

Energy turnover in the isolated rat portal vein was investigated by measurement of oxygen consumption (JO2) and lactate production (JLA) under simultaneous recording of mechanical activity. In spontaneous activity under aerobic conditions and at optimal muscle length JO2 and JLA were 0.55 and 0.62 micromol/min X g, respectively, corresponding to an ATP-production of 4.3 micromol/min X G. When muscle length was changed, an approximately linear relation was found between energy turnover and mean isometric tension. The tension-indpendent part of ATP-production was 3.0 micromol/min X g. In Ca2+-free solution the metabolic rate was 20% lower still. JO2 was nearly equal in isometric contractions and in afterloaded isotonic contractions from the same initial muscle length. During a maximal tonic contracture in 5+-depolarized portal vein JO2 increased to about twice that in spontaneous activity. Changes in contracture force by variations in muscle length or in [Ca2+]0 were associated with identical linear relations between JO2 and active tension. This relation was less steep than the corresponding relation for spontaneous activity. The anaerobic lactate production of the portal vein was 2.7 times theaerobic leve. The accelerated glycolysis did not compensate for eliminated oxidative metabolism. Under substrate-free aerobic conditions no lactate was produced by the muscle and compared to the control situation JO2 declined more than could be accounted for by reduced mechanical activity alone. The metabolic turnover rate in relation to isometric tension is high in the rat portal vein compared to that of tonic vascular smooth muscle from larger vessels. This correlates with differences in dyanmic mechanical properties. At comaparable tension levels in the portal vein, the rate of cross-bridge turnover may be higher in spontaneous phasic activity than in sustained contracture.