Phosphate has dual roles in cross-bridge kinetics in rabbit psoas single myofibrils

In this study, we aimed to study the role of inorganic phosphate (P_i) in the production of oscillatory work and cross-bridge (CB) kinetics of striated muscle. We applied small-amplitude sinusoidal length oscillations to rabbit psoas single myofibrils and muscle fibers, and the resulting force responses were analyzed during maximal Ca²⁺ activation (pCa 4.65) at 15°C. Three exponential processes, A, B, and C, were identified from the tension transients, which were studied as functions of P_i concentration ([P_i]). In myofibrils, we found that process C, corresponding to phase 2 of step analysis during isometric contraction, is almost a perfect single exponential function compared with skinned fibers, which exhibit distributed rate constants, as described previously. The [P_i] dependence of the apparent rate constants 2πb and 2πc, and that of isometric tension, was studied to characterize the force generation and P_i release steps in the CB cycle, as well as the inhibitory effect of P_i. In contrast to skinned fibers, P_i does not accumulate in the core of myofibrils, allowing sinusoidal analysis to be performed nearly at [P_i] = 0. Process B disappeared as [P_i] approached 0 mM in myofibrils, indicating the significance of the role of P_i rebinding to CBs in the production of oscillatory work (process B). Our results also suggest that P_i competitively inhibits ATP binding to CBs, with an inhibitory dissociation constant of ∼2.6 mM. Finally, we found that the sinusoidal waveform of tension is mostly distorted by second harmonics and that this distortion is closely correlated with production of oscillatory work, indicating that the mechanism of generating force is intrinsically nonlinear. A nonlinear force generation mechanism suggests that the length-dependent intrinsic rate constant is asymmetric upon stretch and release and that there may be a ratchet mechanism involved in the CB cycle.