Phosphate rebinding induces force reversal via slow backward cycling of cross-bridges.

Objective: Previous studies on muscle fibers, myofibrils, and myosin revealed that the release of inorganic phosphate (P_i) and the force-generating step(s) are reversible, with cross-bridges also cycling backward through these steps by reversing force-generating steps and rebinding P_i. The aim was to explore the significance of force redevelopment kinetics (rate constant k _TR) in cardiac myofibrils for the coupling between the P_i binding induced force reversal and the rate-limiting transition f ^- for backward cycling of cross-bridges from force-generating to non-force-generating states.

Methods: k _TR and force generation of cardiac myofibrils from guinea pigs were investigated at 0.015-20 mM P_i. The observed force-[P_i], force-log [P_i], k _TR-[P_i], and k _TR-force relations were assessed with various single-pathway models of the cross-bridge cycle that differed in sequence and kinetics of reversible P_i release, reversible force-generating step and reversible rate-limiting transition. Based on the interpretation that k _TR reflects the sum of rate-limiting transitions in the cross-bridge cycle, an indicator, the coupling strength, was defined to quantify the contribution of P_i binding induced force reversal to the rate-limiting transition f ^- from the [P_i]-modulated k _TR-force relation.

Results: Increasing [P_i] decreased force by a bi-linear force-log [P_i] relation, increased k _TR in a slightly downward curved dependence with [P_i], and altered k _TR almost reciprocally to force reflected by the k _TR-force relation. Force-[P_i] and force-log [P_i] relations provided less selectivity for the exclusion of models than the k _TR-[P_i] and k _TR-force relations. The k _TR-force relation observed in experiments with cardiac myofibrils yielded the coupling strength +0.84 ± 0.08 close to 1, the maximum coupling strength expected for the reciprocal k _TR-force relationship. Single pathway models consisting of fast reversible force generation before or after rapid reversible P_i release failed to describe the observed k _TR-force relation. Single pathway models consistent with the observed k _TR-force relation had either slow P_i binding or slow force reversal, i.e., in the consistent single pathway models, f ^- was assigned to the rate of either P_i binding or force reversal.

Conclusion: Backward flux of cross-bridges from force-generating to non-force-generating states is limited by the rates of P_i binding or force reversal ruling out other rate-limiting steps uncoupled from P_i binding induced force reversal.