Tropomyosin-based Effects Of Acidosis On Thin-filament Regulation During Muscle Fatigue

Abstract

Skeletal muscle fatigue is defined by a loss in the force and 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 the regulatory proteins is not clear, nor are the mechanisms underlying the effect acidosis has on them. 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: To test this idea we expressed 3 mutant constructs of Tm with the 2 amino acid residues affected by low pH (histidine residues) replaced with alanine residues (H153A, H276A, H153A/H276A). These constructs were compared to a wild-type Tm, to test the hypothesis that acidosis-induced charge changes of the histidine amino acid governs tropomyosin’s pH-dependent decrease in maximal velocity and Ca++-sensitivity. The effect on RTF function was determined by assessing the impact of acidosis on myosin’s ability to move regulated actin filaments (RTF) in the motility assay as a function of increasing level 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, it is possible that these residues are more important in mediating the depression in force, therefore we are currently testing the impact of these mutations in Tm on the acidosis-induced depression in the Ca++-sensitivity using a loaded in vitro motility assay. Supported by: 2018 UMass UMOVE Initiative