Tirasemtiv and dATP Synergistically Reverse the Acidosis-induced Depression of Myosin’s Force and Motion Generating Capacity

Abstract

In response to repeated, intense contractions muscle reduces its ability to generate force and velocity, characterizing fatigue. This reduction is due, in part, to the depressive direct effects of acidosis on myosin and thin-filament function, particularly at sub-saturating calcium concentrations. We sought to reverse these effects in vitro by 1) replacing ATP with 2-deoxy ATP (dATP), and 2) the addition of the troponin activator tirasemtiv (TR). We used a mini-ensemble laser trap assay to measure the force generating capacity with a regulated thin filament at pCa 7. Acidosis (pH 6.8 vs 7.4) significantly decreased myosin's force-generating capacity (0.80 ± 0.23 vs. 0.93 ± 0.29 pN; p < 0.05, at 6.8 and 7.4 respectively) as measured in a mini-ensemble laser trap assay. However, replacing the ATP with dATP and adding TR at pH 6.8 caused force to not only recover but increase above the control value at pH 7.4 (0.93 ± 0.007 vs. 1.32 ± 0.001; p < 0.05, for 7.4 and 6.8 respectively). The increased force at low pH (6.8) appeared to be due to an increased rate of myosin's attachment to the thin filament because the frequency of binding events was significantly increased under acidic conditions with dATP and TR (2.34 ± 2.94) added compared to ATP (0.47 ± 1.16; p < 0.05). Overall, these findings suggest that acidosis directly inhibits myosin's ability to bind and translocate a regulated actin filament, and that these agents that increase myosin's rate of attachment to actin can reverse these effects in vitro. Thus, these results suggest that these agents might offer hope in mitigating the deleterious effects of muscle fatigue in clinical populations.