Skeletal muscle fatigue is defined as a loss in the force/velocity generating capacity of a muscle. A portion of the loss in function is attributable to effects of acidosis (i.e. low pH) on the regulatory proteins, troponin and tropomyosin (Tm), which regulate the binding of myosin and actin in a calcium (Ca++) dependent manner. However, the relative role of troponin and Tm on myosin-actin function during acidosis is not clear, nor are the mechanisms underlying these effects. PURPOSE: To determine the role of Tm in the acidosis-induced depression of muscle function using isolated muscle proteins in an in vitro motility assay. METHODS: Three mutant constructs of Tm were expressed by replacing the two amino acid (histidine) residues most likely affected by low pH with alanine residues (H153A, H276A, H153A/H276A). These mutant constructs were compared to wild-type Tm (wt-Tm) in order to test whether the acidosis-induced charge change of the histidine amino acid governs the pH-dependent alteration of tropomyosin and therefore the decrease in maximal RTF velocity and Ca++-sensitivity. The effect of acidosis on regulated thin filament (RTF) function was determined by assessing the impact of low pH (pH 6.8) versus neutral pH (pH 7.4) on myosin’s ability to move RTFs in the motility assay as a function of increasing levels of Ca++. This was done separately for the wt-Tm and each structural variant. RESULTS: A two-way ANOVA (pH x Tm construct) revealed that acidosis significantly (p<0.05) depressed the maximal sliding velocity of the RTFs across all versions of Tm, but that the magnitude of the depression was similar among the wt and all of the Tm mutants. Acidosis did not significantly depress the sensitivity to Ca++ under the unloaded conditions of this assay (p>0.05). CONCLUSIONS: These data suggest that the histidine residues in tropomyosin do not mediate the acidosis-induced depression in contraction velocity observed during muscle fatigue. However, since these residues may be more important in mediating the depression of force, we are currently testing the impact of the three mutant Tm constructs on the acidosis-induced depression in Ca++-sensitivity using a loaded in vitro motility assay.