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Key Points: Actomyosin ATP hydrolysis occurring during muscle contraction releases inorganic phosphate [P i ] in the myoplasm. High [P i ] reduces force and affects force kinetics in skinned muscle fibres at low temperature. These effects decrease at high temperature, raising the question of their importance under physiological conditions. This study provides the first analysis of the effects of P i on muscle performance in intact mammalian fibres at physiological temperature. Myoplasmic [P i ] was raised by fatiguing the fibres with a series of tetanic contractions. [P i ] increase reduces muscular force mainly by decreasing the force of the single molecular motor, the crossbridge, and alters the crossbridge response to fast length perturbation indicating faster kinetics. These results are in agreement with schemes of actomyosin ATPase and the crossbridge cycle including a low- or no-force state and show that fibre length changes perturb the P i -sensitive force generation of the crossbridge cycle., Abstract: Actomyosin ATP hydrolysis during muscle contraction releases inorganic phosphate, increasing [P i ] in the myoplasm. Experiments in skinned fibres at low temperature (10-12°C) have shown that [P i ] increase depresses isometric force and alters the kinetics of actomyosin interaction. However, the effects of P i decrease with temperature and this raises the question of the role of P i under physiological conditions. The present experiments were performed to investigate this point. Intact fibre bundles isolated from the flexor digitorum brevis of C57BL/6 mice were stimulated with a series of tetanic contractions at 1.5 s intervals at 33°C. As show previously the most significant change induced by a bout of contractile activity similar to the initial 10 tetani of the series was an increase of [P i ] without significant Ca 2+ or pH changes. Measurements of force, stiffness and responses to fast stretches and releases were therefore made on the 10th tetanus of the series and compared with control. We found that (i) tetanic force at the 10th tetanus was ∼20% smaller than control without a significant decrease of crossbridge stiffness; and (ii) the force recovery following quick stretches and releases was faster than in control. These results indicate that at physiological temperature the increase of [P i ] occurring during early fatigue reduces tetanic force mainly by depressing the individual crossbridge force and accelerating crossbridge kinetics., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Fatigue occurring during exercise can be defined as the inability to maintain the initial force or power output. As fatigue becomes pronounced, force and maximum velocity of shortening are greatly reduced and force relaxation is prolonged. In principle, force loss during fatigue can result from a decrease in the number of cross-bridges generating force or a decrease of the individual cross-bridge force or to both mechanisms. The present experiments were made to investigate this point in single fibres or small fibre bundles isolated from flexor digitorum brevis (FDB) of C57BL/6 mice at 22-24◦C. During a series of 105 tetanic contractions, we measured force and fibre stiffness by applying small sinusoidal length oscillations at 2.5 or 4 kHz frequency to the activated preparation and measuring the resulting force changes. Stiffness data were corrected for the influence of compliance in series with the cross-bridge ensemble. The results show that the force decline during the first 20 tetani is due to the reduction of force developed by the individual cross-bridges and thereafter as fatigue becomes more severe, the number of cross-bridges decreases. In spite of the force reduction in the early phase of fatigue, there was an increased rate of tetanic force development and relaxation. In the latter stages of fatigue, the rate of force development and relaxation became slower. Thus, the start of fatigue is characterised by decreased cross-bridge force development and as fatigue becomes more marked, the number of cross-bridges decreases. These findings are discussed in the context of the current hypotheses about fatigue mechanisms.

The mechanism of force enhancement during lengthening was investigated on single frog muscle fibres by using fast stretches to measure the rupture tension of the crossbridge ensemble. Fast stretches were applied to one end of the activated fibre and force responses were measured at the other. Sarcomere length was measured by a striation follower device. Fast stretching induced a linear increase of tension that reached a peak and fell before the end of the stretch indicating that a sudden increase of fibre compliance occurred due to forced crossbridge detachment induced by the fast loading. The peak tension (critical tension, Pc) and the sarcomere length needed to reach Pc (critical length, Lc) were measured at various tensions during the isometric tetanus rise and during force enhancement by slow lengthening. The data showed that Pc was proportional to the tension generated by the fibre under both isometric and slow lengthening conditions. However, for a given tension increase, Pc was 6.5 times greater during isometric than during lengthening conditions. Isometric critical length was 13.04 +/- 0.17 nm per half-sarcomere (nm hs(-1)) independently of tension. During slow lengthening critical length fell as the force enhancement increased. For 90% enhancement, Lc reduced to 8.19 +/- 0.039 nm hs(-1). Assuming that the rupture force of the individual crossbridge is constant, these data indicate that the increase of crossbridge number during lengthening accounts for only 15.4% of the total force enhancement. The remaining 84.6% is accounted for by the increased mean strain of the crossbridges.

The aims of this study were to investigate the effects of solution tonicity on muscle properties, and to verify their consistence with the lever arm theory of force generation. Experiments were made in single muscle fibres and in fibre bundles from the frog, using both fast stretches and time-resolved X-ray diffraction, in isotonic Ringer solution (1T), hypertonic (1.4T) and hypotonic (0.8T) solutions. Fast stretches (0.4-0.6 ms duration and 16-25 nm per half-sarcomere (nm hs(-1)) amplitude) were applied at various tensions during the force development in isometric tetani. Force increased during the stretch up to a peak (critical tension, Pc) at which it started to fall, in spite of continued stretching. In all solutions, Pc was proportional to the initial isometric tension developed. For a given isometric tension, Pc increased with solution tonicity and occurred at a precise sarcomere elongation (critical length, Lc) which also increased with tonicity. M3 meridional layer line intensity (I M3) was measured during the application of sinusoidal length oscillations (1 kHz frequency, and about 2% fibre length amplitude) at tetanus plateau. I M3 changed during the length oscillations in a sinusoidal manner in phase opposition to length changes, but a double peak distortion occurred at the peak of the release phase. The presence of the distortion, which decreased with tonicity, allowed calculation of the mean position of the myosin head (S1) during the oscillation cycle. In agreement with the lever arm theory, both X-ray diffraction and mechanical data show that solution tonicity affects S1 mean position and consequently crossbridge individual extension and force, with no effect on crossbridge number. The force needed to break the single crossbridge was insensitive to solution tonicity suggesting a non-ionic nature of the actomyosin bond.

Very fast ramp stretches at 9.5-33 sarcomere lengths s(-1) (l0 s(-1)) stretching speed, 16-25 nm per half-sarcomere (nm hs(-1)) amplitude were applied to activated intact frog muscle fibres at tetanus plateau, during the tetanus rise, during the isometric phase of relaxation and during isotonic shortening. Stretches produced an almost linear tension increase above the isometric level up to a peak, and fell to a lower value in spite of continued stretching, indicating that the fibre became suddenly very compliant. This suggests that peak tension (critical tension, P(c)) represents the tension at which crossbridges are forcibly detached by the stretch. The ratio of P(c) to the isometric tension at tetanus plateau (P0) was 2.37 +/- 0.12 (S.E.M.). This ratio did not change significantly at lower tension (P) during the tetanus rise but decreased with time during the relaxation and increased with speed during isotonic shortening. At tetanus plateau P(c) occurred when sarcomere elongation attained a critical length (L(c)) of 10.98 +/- 0.13 nm hs(-1), independently of the stretching speed. L(c) remained constant during the tetanus rise but decreased on the relaxation and increased during isotonic shortening. Length-clamp experiments on the relaxation showed that the lower values of P(c)/P ratio and L(c), were both due to the slow sarcomere stretching occurring during this phase. Our data show that P(c) can be used as a measure of crossbridge number, while L(c) is a measure of crossbridge mean extension. Accordingly, for a given tension, crossbridges on the isometric relaxation are fewer than during the rise, develop a greater individual force and have a greater mean extension, while during isotonic shortening crossbridges are in a greater number but develop a smaller individual force and have a smaller extension.

1. Passive force responses to ramp stretches at various velocities were measured in intact and skinned single muscle fibres isolated from the lumbricalis muscle of the frog. Force was measured using a fast capacitance transducer and sarcomere length was measured using a laser light diffraction technique at a point very close to the fixed end so as to avoid effects of fibre inertia. Experiments were performed at 15 degrees C with sarcomere length between 2.13 and 3.27 microns under high (170 mM) and low (20 mM) ionic strength. 2. The analysis shows that the force response is the sum of at least three components: (i) elastic (force proportional to the amount of stretch), (ii) viscous (force proportional to rate of stretch), and (iii) viscoelastic (resembling the response of a pure viscous element in series with an elastic element). 3. The amplitude of all these components increased progressively with sarcomere length in the whole range measured. 4. A further component, attributable to the short-range elasticity (SREC), was present in the force response of the intact fibres. 5. The amplitude of the force response decreased substantially upon skinning at high ionic strength but increased again at low ionic strength. The SREC was completely abolished by skinning. 6. None of the components of the force response was found to have the properties expected from the previously postulated 'weakly binding bridges'.

1. Force responses to ramp stretches were recorded in single muscle fibres isolated from the lumbricalis muscle of the frog. Stretches were applied at rest and at progressively increasing times after a single stimulus. 2. The increase of fibre stiffness that precedes tension development has a 'static' component that accounts for the whole fibre stiffness increase during the latent period and at very low tension at the beginning of the twitch. 3. Static stiffness increase was not affected by 2,3-butanedione-2-monoxime, a drug that almost completely inhibited twitch tension. 4. Static stiffness increased approximately 5-fold as the sarcomere length was increased from 2.1 to 2.84 microns. 5. These results suggest that static fibre stiffness increase is not attributable to the formation of non-force-generating cross-bridges.

1. Apparatus for recording the small tensions developed by electrically stimulated single intact myocytes of frog heart is described. A laser-light optoelectronic transducer was used. The compliance of the force probes was 10-20 nm/nN, with a frequency response of 600-900 Hz in Ringer solution. The myocyte shortening during an ordinary twitch contraction was no greater than 1% of the slack length. The overall sensitivity of the transducer system was 5-10 mV/nN, with a total noise of 0.5-1 nN peak to peak. The experiments were performed at 20-23 degrees C on either atrial or ventricular myocytes at 2.15-2.2 microns sarcomere length, in 1 mM-Ca2+ Ringer solution. 2. Isoprenaline (5 microM), increases in external Ca2+ concentration ([Ca2+]o), and shortening of stimulus interval potentiated the myocyte twitch tension. The dependence of twitch characteristics on these inotropic interventions for all the atrial and ventricular myocytes tested was comparable to that of multicellular preparations under similar experimental conditions. This implies that the enzymatic isolation procedure had not altered the physiological properties of the myocytes. 3. The stimulus interval-tension relation for premature twitches of atrial and ventricular myocytes showed (i) a very steep rising phase in the region of intervals just longer than 0.52 and 0.66 s (the duration of the mechanical refractoriness in atrial or ventricular cells), (ii) a peak, at intervals of 0.7-0.8 s, where the twitch tension was strongly potentiated compared to that of the controls, and (iii) as the stimulus interval was further increased, a progressive return to the control level. The stimulus interval-tension relation for steady-state conditions exhibited similar characteristics. 4. The degree of tension potentiation by isoprenaline was greater in the controls than in the earliest test twitches. The result was that the stimulus interval-tension relations for isoprenaline-treated myocytes showed a gentler rise and a lower peak than for untreated cells. 5. The stimulus interval-tension relation of the heart is a basic property of its cells. It is related to changes in the activation level. The higher the activation level reached in control twitches, the lower the stimulus interval-dependent potentiation capability.

1. The relation between sarcomere length and steady tetanic tension was determined at 10-12 degrees C for 70-80 microns long length-clamped segments of single fibres isolated from the tibialis anterior muscle of the frog, in normal and hypertonic or hypotonic Ringer solutions. 2. The tension depression and potentiation observed in hypertonic and hypotonic Ringers solutions varied with sarcomere length, so that, as opposed to myofilament overlap predictions, the optimum length for tension development was shorter in hypertonic Ringer solution and longer in hypotonic Ringer solution than in normal Ringer solution. As the fibres were stretched from 1.96 to 2.24 microns sarcomere length, both tension depression in hypertonic Ringer solution and tension potentiation in hypotonic Ringer solution increased by 9 and 5%, respectively. 3. Within this range of sarcomere lengths the length-stiffness relation in hypotonic and in hypertonic Ringer solutions exhibit little or no change relative to that in normal Ringer solution. 4. The results indicate that separation between the thick and the thin myofilaments influences the mechanism of force generation. There is an optimum interfilament distance (10-12 nm surface to surface between the thick and the thin filaments) for tension production. In isotonic Ringer solution, this corresponds to the interfilament distance at sarcomere lengths around 2.10 microns. The force per attached cross-bridge, rather than their number, appears to decrease as the interfilament distance is brought above or below the optimum length. Even if this effect is moderate in isotonic Ringer solution, it should be taken into account in models of the force-generation mechanism.

1. The force-velocity (P-V) relation for normal or NO-3 treated single fibres isolated from the semitendinosus muscle of the frog was determined at given times during the rise of tension and the plateau of isometric tetani. Experiments were made at about 2.25 micron sarcomere length and at constant temperatures, from 3 to 4.5 degrees C and from 19 to 21 degrees C. The controlled-velocity release method was used. 2. During the rise of tension, at any initial tension higher than about 0.2 P0, the lowest release velocity required to drop the tension to zero was the same as at the tetanic plateau, independent of the temperature and the presence of NO-3 ions in the bathing solution. 3. The degree of activation (measured by the steady force exerted at a given velocity of shortening lower than V0) increased with time, but attained its steady-state level before isometric tension. 4. At about 20 degrees C, frog muscle fibres at about 2.2 micron sarcomere length were only partially activated after a single stimulus. 5. NO-3 ions did not affect the steady-state P-V relation. At about 20 degrees C, NO-3 ions increased the rate of development of activation. Potentiation of the twitch contraction was due at least in part to this mechanism. 6. The 'relative' P-V relation appears to be independent of both the time after start of stimulation and the presence of NO-3 ions in the bathing solution. 7. The results are discussed in terms of the sliding filament model of Huxley (1957), assuming that either the number of actin sites available for cross-bridge formation, or the value of the rate constant for making of cross-bridges, is time dependent.

1. The force-velocity (P-V) relation from a single fibres isolated from the semitendinosus muscle of the frog was determined at pre-set times during the rise of tension and the plateau of isometric tetani. The controlled-velocity release method was used. Experiments were performed at about 2.25 micrometers sarcomere length and at 3-4 degrees C or at 19-21 degrees C. 2. Replacing H2O with D2O resulted in a rapid large reduction of the peak twitch tension and of the speed of development of twitch and tetanic tensions. The tetanic tension (P0) was usually reduced, in certain fibres to as low as 5% of the value in H2O-Ringer solution. 3. The depression of twitch and tetanus characteristics was followed by a recovery, the duration of which varied greatly in different fibres. During the recovery period previous conditioning activity potentiated the tetanus characteristics. 4. After the end of the recovery period in D2O-Ringer solution both the peak twitch tension and the speed of development of tetanic tension was still greatly depressed, whereas the value of P0 was slightly greater than in H2O-Ringer. The speed of rise of isometric tension after a quick release imposed at the tetanus plateau was reduced in D2O-Ringer, usually to about 50% of the value in H2O-Ringer. 5. D2O increased the development time of the P-V relation and produced a conspicuous increase in the degree of its curvature. The value of V0 (the velocity of shortening at zero load) was not significantly depressed by D2O and it was the same independent both of the time after the beginning of stimulation and of the isometric tension at which the measurement was made. The P-V relation attained its final characteristics before the isometric tension reached the plateau. During the recovery period in D2O-Ringer, at the plateau of isometric tetani of different size, the relative force exerted at a given velocity of shortening was constant. 6. In D2O-treated fibres, NO3- and caffeine (i) potentiated the peak twitch tension and the speed of development of tetanic tension without affecting significantly the speed of the redevelopment of tension after a quick release imposed at the tetanus plateau and (ii) reduced the development time of the P-V relation, but did not affect either the degree of its curvature or the value of V0 and P0. 7. The results are discussed by assuming that the release of Ca2+ from the sarcoplasmic reticulum is a rate-limiting process for the development of activation and in turn for the development of isometric tension. In terms of the cross-bridge model of Huxley (1957), the time or Ca2+-dependent factor of activation appears to be the recruitment of actin sites for cross-bridge formation, whereas the value of the rate constants regulating the cross-bridge kinetics appears to be time and Ca2+-independent.

1. The relation between sarcomere length and tetanic tension was determined at 10-12 degrees C for 70-80 microns long segments of single fibres isolated from the tibialis anterior and semitendinosus muscles of the frog. Measurements of segment striation spacings were performed during fixed-end or length-clamp contractions by means of a laser light diffractometer. 2. At sarcomere lengths of around 2.10 microns tetanic tension rose promptly to a steady plateau, independent of the recording conditions. At greater sarcomere lengths under fixed-end conditions the tension rise occurred in two distinct stages: an initial rapid rise followed by a much slower creep. The tension creep was entirely abolished in length-clamp contractions. 3. The sarcomere length-tension diagram of length-clamped segments of tibialis anterior fibres exhibited a definite flat region between about 1.96 and 2.16 microns where tension varied by less than 1.5%. The highly linear descending limb reached zero tension at about 3.53 microns. The shift to the left by about 0.10 microns, with respect to the length-tension diagram of length-clamped segments of semitendinosus fibres, may be tentatively explained by assuming that thin filament lengths vary in different muscles. 4. The results are in agreement with those of a previous work by Gordon, Huxley & Julian (1966) and support the hypothesis (Huxley, 1957, 1980) that muscle tension is produced by simultaneous action of independent force generators, in proportion to the number of myosin bridges overlapped by actin filaments.