Phosphate increase during fatigue affects crossbridge kinetics in intact mouse muscle at physiological temperature

Abstract

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 Ca²⁺ 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.

Keywords: crossbridge kinetics; fatigue; phosphate.

Figure 1. Sample records illustrating the experimental procedure

Superimposed sample records of force (lower traces) and length changes (upper traces) following the application of a short burst of sinusoidal length oscillation (thickened regions on the traces) followed by a stretch or release at tetanus plateau. The expanded burst of 4 kHz oscillations (inset) shows the absence of phase shift between force (lower trace) and length sinusoids (upper trace). To obtain the best resolution of fast and slow parts of the records, a double time base was used. The 10 ms time calibration refers to the part of the record inside the dashed vertical lines, and 1 s to the parts outside.

Figure 2. Time course of fibre force and stiffness at tetanus plateau during the initial part of fatigue

Force and stiffness data are expressed relative to plateau control values. Open circles, stiffness; filled circles, force. Mean values ± SEM (n = 13). Note that plotted fibre stiffness includes stiffness of crossbridge, myofilaments and tendons.

Figure 3. Stiffness and force measurements during the tetanus rise

Sinusoidal length oscillations were applied throughout the tetanus rise to measure stiffness. Trace a, stiffness; b, force; c, force with oscillations superimposed; d, difference between c and b. Force and stiffness traces are shifted upward for clarity. The initial step rise of force visible on the tension trace is due to the short period of slow sampling at 1 ms/point preceding the fast sampling at 10 μs point⁻¹.

Figure 4. Force–stiffness relationships

Force–stiffness relationship on the tetanus rise (noisy continuous line) and during fatigue (open circles). Data from the same 6 fibres. The open circles are individual stiffness data and the grey continuous line is the best fit with a polynomial equation used to find the mean values. The noisy continuous line is the average stiffness measured continuously during tetanus rise. The error bars are the SEM at 4 force levels showing the variability. The dashed line represents the direct proportionality between stiffness and force. The deviation of the stiffness on the tetanus rise from the direct proportionality expected from crossbridge stiffness is due to the presence of myofilament and tendon stiffness.

Figure 5. Calculated crossbridge, myofilament and tendon stiffness as function of force during the tetanus rise

Stiffness is expressed with respect to total fibre stiffness at tetanus plateau taken as 1. Note that tendon stiffness (open triangles) rises with force in a similar, but not equal, way to crossbridge stiffness (filled circles). For the calculation we assumed that filament stiffness (filled squares) was Hookean.

Figure 6. Crossbridge stiffness and force during fatigue and tetanus rise

Crossbridge stiffness (A) and individual crossbridge force (B) during tetanus rise (open triangles) and fatigue (filled circles). Stiffness in A is expressed with respect to total fibre stiffness at tetanus plateau taken as 1. At any tension, crossbridge stiffness, proportional to crossbridge number, is higher during fatigue than during tetanus rise.

Figure 7. Effects of early fatigue on the tetanus rise and fitting with a single exponential equation

A, effects of early fatigue on the tetanus rise. Control, thin line; 10th tetanus, thick line. Both records are normalized for their respective P ₀. B, fit (continuous line) of the control tetanus rise (empty circles). The fitting equation is $P=A_{\mathrm{s}}(1-e^{-k_{\mathrm{s}}t})$, where A _s is amplitude of exponential force component of the tetanus rise and k _s is rate constant of the exponential force component of the tetanus rise. Experimental force data were sampled at 1 ms/point but the number of points plotted on the figure were reduced for clarity.

Figure 8. Responses to stretch and release in control tetani and during fatigue

Sample records of force responses to stretch (A) and release (B) applied in control tetanus (thin line) and at the 10th tetanus of the fatigue protocol (thick line). Traces are vertically shifted to superimpose the tetanic force just before the stretch. Faster time base (10 ms calibration) refers to the record between the two dashed vertical lines, lower time base (1 s calibration) to the record outside the lines. Force recovery is faster in the 10th tetanus than in control.

Figure 9. Fitting with a triexponential equation of force recovery from stretch (A) and release (B) applied at tetanus plateau in control tetani

Top traces, forces (open circles) and fittings (continuous line); horizontal dashed line is the plateau force. Bottom traces, residuals. The noise is mostly due to oscillations of the stretcher lever arm. Stretch amplitude, 0.67% l _f; release amplitude, 0.63% l _f. The fitting equation is show in Table 1. Experimental force data were sampled at 10 μs point⁻¹ but the number of points plotted on figures were reduced for clarity.