Elevated Ionic Strength Diminishes Force/Cross-Bridge and the Number of Force-Generating Cross-Bridges

Abstract

Ionic strength (IS) is an important parameter to govern the inter- and intra-molecular interactions. In muscle, it has been known that an increase in IS lowers Ca2+ activated tension, however, its molecular mechanism is not well understood. Our aim was to determine whether force/cross-bridge or the number of force-generating cross-bridges changes with IS. Stiffness during rigor was studied on single fibers from rabbit psoas, which showed that there was no effect of IS, demonstrating that in-series compliance is not affected by IS. This observation indicates that stiffness of thick filament, thin filament, myosin head, and actomyosin interface are not affected by IS. Sinusoidal analyses were performed during Ca2+ activation, and the effects of ATP, phosphate (Pi), and ADP on three rate constants were studied at IS ranging 150mM-300mM to characterize elementary steps of the cross-bridge cycle. Both ATP binding (K1) and ADP binding (K0) increased to 2x, and the Pi binding (K5) decreased to 1/2 when IS was increased from 150mM to 300mM. The effect of Pi can be explained by the electrostatic interaction with the Pi binding site on myosin. The effect on ATP/ADP can be attributed to improved stereoscopic and hydrophobic interaction with the nucleotide binding site. The increase in IS increased cross-bridge detachment steps (k2 and k(-4)), indicating that electrostatic force counteracts these steps. However, IS did not affect attachment steps (k(-2) and k4). Consequently, the equilibrium constant of the detachment step (K2) increased to 2x, and the force generation step (K4) decreased to 0.7x. These effects together diminished the number of force-generating cross-bridges by ∼10%. Because associated decrease of tension was ∼40%, the major effect of IS is a decrease in force/cross-bridge, but also a decrease in the number of force generating cross-bridge occurs.