Phosphate binding induced force-reversal occurs via slow backward cycling of cross-bridges

Abstract

ABSTRACTThe release of inorganic phosphate (Pi) from the cross-bridge is a pivotal step in the cross-bridge ATPase cycle leading to force generation. It is well known that Pirelease and the force-generating step are reversible, thus increase of [Pi] decreases isometric force by product inhibition and increases the rate constantkTRof mechanically-induced force redevelopment due to the reversible redistribution of cross-bridges among non-force-generating and force-generating states. The experiments on cardiac myofibrils from guinea pig presented here show that increasing [Pi] increaseskTRalmost reciprocally to force, i.e.,kTR≈ 1/force. To elucidate which cross-bridge models can explain the reciprocalkTR-force relation, simulations were performed for models varying in sequence and kinetics of 1) the Pirelease-rebinding equilibrium, 2) the force-generating step and its reversal, and 3) the transitions limiting forward and backward cycling of cross-bridges between non-force-generating and force-generating states. Models consisting of fast reversible force generation before/after rapid Pirelease-rebinding fail to describe thekTR–force relation observed in experiments. Models consistent with the experimentalkTR-force relation have in common that Pibinding and/or force-reversal are/is intrinsically slow, i.e., either Pibinding or force-reversal or both limit backward cycling of cross-bridges from force-generating to non-force-generating states.STATEMENT OF SIGNIFICANCEPrevious mechanical studies on muscle fibers, myofibrils and myosin interacting with actin revealed that force production associated to phosphate release from myosin’s active site presents a reversible process in the cross-bridge cycle. The correlation of this reversible process to the process(es) limiting kinetics of backward cycling from force-generating to non-force-generating states remained unclear.Experimental data of cardiac myofibrils and model simulations show that the combined effects of [Pi] on force and the rate constant of force redevelopment (kTR) are inconsistent with fast reversible force generation before/after rapid Pirelease-rebinding. The minimum requirement in sequential models for successfully describing the experimentally observed nearly reciprocal change of force andkTRis that either the Pibinding or the force-reversal step limit backward cycling.