The Acidosis-Induced Slowing of Regulated Thin Filament Velocity in a Motility Assay Disappears at Low ATP

Abstract

Acidosis (low pH) is a putative agent of muscle fatigue that slows contraction velocity but the mechanisms underlying this effect are unclear. It may directly affect myosin by slowing a step associated with detachment or it may act indirectly by affecting the ability of Ca++ to activate the thin filament, through effects on the regulatory proteins troponin/tropmyosin. We measured the effect of manipulating Ca++ and the lifetime rigor binding (by lowering the [ATP]) to examine the relative role of each of these mechanisms. At saturating Ca++ (pCa 4) and ATP concentrations (1mM) decreasing the pH from 7.4 to 6.8 decreased regulated actin filament velocity (Vrtf) by 50%, however at low [ATP] (<200 μM) Vrtf was unaffected by acidosis. Repeating these experiments in the absence of Ca++ (pCa 9) confirmed the phenomenon of rigor activation at normal pH (7.4), with filaments moving fastest at 50 μM ATP (0.5 μm/s). At low pH the filaments moved slower but still demonstrated rigor activation at [ATP] <75 μM. Single molecule laser trap assay experiments are under way to determine whether the slowed velocity was due slowed attachment or detachment kinetics. The acidosis-induced decrease in Vrtf at saturating Ca++ and ATP are consistent with a slowing of the steps in the cross-bridge cycle associated with ADP-release. However this effect disappears at low ATP where the attached lifetime is more dependent on the duration of rigor. Therefore the data suggest that acidosis does not affect the formation of a rigor bond or its ability to activate the thin filament. Inhibition of Ca++ binding to troponin might still contribute to slowing at saturating ATP and submaximal Ca++ levels.