Ca ++ -sensitizing mutations in troponin, P i , and 2-deoxyATP alter the depressive effect of acidosis on regulated thin-filament velocity

Repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, resulting in fatigue. The decrease in velocity is thought to be due, in part, to the intracellular build-up of acidosis inhibiting the function of the contractile proteins myosin and troponin; however, the underlying molecular basis of this process remains poorly understood. We sought to gain novel insight into the decrease in velocity by determining whether the depressive effect of acidosis could be altered by 1) introducing Ca ++ -sensitizing mutations into troponin (Tn) or 2) by agents that directly affect myosin function, including inorganic phosphate (P i ) and 2-deoxy-ATP (dATP) in an in vitro motility assay. Acidosis reduced regulated thin-filament velocity ( V RTF ) at both maximal and submaximal Ca ++ levels in a pH-dependent manner. A truncated construct of the inhibitory subunit of Tn (TnI) and a Ca ++ -sensitizing mutation in the Ca ++ -binding subunit of Tn (TnC) increased V RTF at submaximal Ca ++ under acidic conditions but had no effect on V RTF at maximal Ca ++ levels. In contrast, both P i and replacement of ATP with dATP reversed much of the acidosis-induced depression of V RTF at saturating Ca ++ . Interestingly, despite producing similar magnitude increases in V RTF , the combined effects of P i and dATP were additive, suggesting different underlying mechanisms of action. These findings suggest that acidosis depresses velocity by slowing the detachment rate from actin but also by possibly slowing the attachment rate.