Acidosis and Phosphate Directly Reduce Myosin's Force-Generating Capacity Through Distinct Molecular Mechanisms

Elevated levels of the metabolic by-products, including acidosis (i.e., high [H⁺]) and phosphate (P_i) are putative agents of muscle fatigue; however, the mechanism through which they affect myosin's function remain unclear. To elucidate these mechanisms, we directly examined the effect of acidosis (pH 6.5 vs. 7.4), alone and in combination with elevated levels of P_i on the force-generating capacity of a mini-ensemble of myosin using a laser trap assay. Acidosis decreased myosin's average force-generating capacity by 20% (p < 0.05). The reduction was due to both a decrease in the force generated during each actomyosin interaction, as well as an increase in the number of binding events generating negative forces. Adding P_i to the acidic condition resulted in a quantitatively similar decrease in force but was solely due to an elimination of all high force-generating events (>2 pN), resulting from an acceleration of the myosin's rate of detachment from actin. Acidosis and P_i also had distinct effects on myosin's steady state ATPase rate with acidosis slowing it by ∼90% (p > 0.05), while the addition of P_i under acidic conditions caused a significant recovery in the ATPase rate. These data suggest that these two fatigue agents have distinct effects on myosin's cross-bridge cycle that may underlie the synergistic effect that they have muscle force. Thus these data provide novel molecular insight into the mechanisms underlying the depressive effects of P_i and H⁺ on muscle contraction during fatigue.