Intracellular acidosis is a putative agent of skeletal muscle fatigue, in part, because it depresses the calcium (Ca²⁺) sensitivity of the myofilaments. However, the molecular mechanism behind this depression in Ca²⁺ sensitivity is unknown, providing a significant challenge to a complete understanding of the fatigue process. To elucidate this mechanism, we directly determined the effect of acidosis on the ability of a single myosin molecule to bind to a regulated actin filament in a laser trap assay. Decreasing pH from 7.4 to 6.5 significantly (P < 0.05) reduced the frequency of single actomyosin-binding events at submaximal (pCa 8-pCa 6) but not at maximal Ca²⁺ concentration (pCa 5-pCa 4). To delineate whether this was due to a direct effect on myosin versus an indirect effect on the regulatory proteins troponin (Tn) and tropomyosin (Tm), binding frequency was also quantified in the absence of Tn and Tm. This revealed that acidosis did not significantly alter the frequency of actomyosin binding events in the absence of regulatory proteins (1.4 ± 0.15 vs. 1.4 ± 0.15 events/s for pH 7.4 and 6.5, respectively). Acidosis also did not significantly affect the size of myosin's powerstroke or the duration of binding events in the presence of regulatory proteins, at every [Ca²⁺]. These data suggest acidosis impedes activation of the thin filament by competitively inhibiting Ca²⁺ binding to TnC. This slows the rate at which myosin initially attaches to actin; therefore, less cross bridges will be bound and generating force at any given submaximal [Ca²⁺]. These data provide a molecular explanation for the acidosis-induced decrease in force observed at the submaximal Ca²⁺ concentrations that might contribute to the loss of force during muscle fatigue.

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