Attentuating the Depressive Effect of Acidosis with Mutations in Troponin and with 2-Deoxy-Atp

Abstract

Repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, which defines fatigue. The decrease in velocity is thought to be due, in part, to the intracellular build-up of acidosis inhibiting the function of the contractile proteins myosin and troponin; however, the underlying molecular basis of this process remains unclear. We sought to gain novel insight into the decrease in velocity by determining if the depressive effect of acidosis could be altered by 1) introducing Ca++-sensitizing mutations into troponin (Tn) or 2) by agents that directly affect myosin function, including inorganic phosphate (Pi) and 2-deoxy-ATP (dATP) in an in vitro motility assay. Acidosis reduced regulated thin filament velocity (VRTF) at both maximal and sub-maximal Ca++ levels in a pH-dependent manner. A truncated construct of the inhibitory subunit of Tn, R156, and a Ca++-sensitizing mutation in the Ca++-binding subunit of Tn, V43Q, increased VRTF at sub-maximal Ca++ under acidic conditions, but had no effect on VRTF at maximal Ca++ levels. In contrast, both 15mM Pi and replacement of ATP with dATP reversed much of the acidosis-induced depression of VRTF at saturating Ca++ (0.7±0.1 control, 2.0±0.3 with Pi, 1.8±0.3 with dATP, 3.8±0.1 with both Pi and dATP), with the combined effect fully restoring the VRTF to the value under control conditions. Interestingly, despite producing similar magnitude increases in VRTF, the combined effects of Pi and dATP were additive, suggesting different underlying mechanisms of action. These results suggest that the major mechanism by which acidosis slows VRTF is through directly slowing myosin's rate of detachment from actin.