Effect of low pH on single skeletal muscle myosin mechanics and kinetics

Abstract

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity (V(actin)) in the IVM by 36%. Single molecule experiments, at 1 microM ATP, decreased the average unitary step size of myosin (d) from 10 +/- 2 nm (pH 7.4) to 2 +/- 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 microM to 1 mM at pH 6.4 restored d (9 +/- 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. V(actin), however, was not restored by raising the [ATP] (1-10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding (t(on)). Measurement of t(on) as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs t(on) by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., V(actin) approximately d/t(on)), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in V(actin), suggesting a molecular explanation for this component of muscular fatigue.

Phosphate enhances myosin-powered actin filament velocity under acidic conditions in a motility assay

“…Our own previous work, using a single-molecule laser trap assay, indicates that acidosis prolongs t on by prolonging the lifetime of the AM.ADP state, with little effect on the k ATP rate (8). It is also well established that the rigor lifetime of myosin is ATP dependent (2).…”

“…This has been a particularly challenging issue to address, because skeletal muscle myosin has an inherently short ADP lifetime when bound to actin (23). Fortuitously, our laboratory's own recent work demonstrated that acidosis prolongs actomyosin's ADP-bound state (AM.ADP) under the unloaded conditions of the motility assay (8). This finding provides a unique opportunity to determine whether P i can rebind to the ADP state under these conditions and what effects it might have on myosin function.…”

Muscle Fatigue from the Perspective of a Single Crossbridge

“…Therefore, investigators turned to the skinned muscle fiber preparation where they observed that decreasing the pH from 7.0 to 6.2 reduces unloaded shortening velocity by ~20%-30% even near physiological temperatures (30°C). 42,43,46 Consistent with the reduction in unloaded shortening velocity, in an in vitro motility assay (a measure analogous to unloading shortening velocity), Debold et al 23 observed that reducing the pH from 7.4 to a value reached during fatigue (6.4) slowed the actin filament velocity by roughly 67% at 20°C. A similar reduction in velocity is observed at 30°C, 27 suggesting that the effect on velocity is not as temperature dependent as the effect on force.…”

“…The decrements in fiber shortening velocity are thought to be due to slowed myofibrillar ATPase activity, 15 secondary to the H + -mediated reduction in the rate that ADP dissociates from the myosin head. 23 The effects of acidosis on both fiber force and velocity resulted in an 18%-34% reduction in peak power of rat fibers at 30°C 46 ( Fig. 2B).…”

“…Fortunately, recent technological advances that have enabled the direct observation of the mechanics and kinetics of a single actomyosin crossbridge under fatigue-like conditions are providing the first opportunities to observe how a single crossbridge responds to these fatiguing ions. 23,28 In addition, parallel advancements in single muscle fiber technology and transgenic manipulation of the contractile proteins are also providing important new insights into the molecular basis of the fatigue process. 20,24,26,46,[58][59][60] Characterizing the mechanisms of fatigue at the cellular and molecular levels is important because many of the most promising therapeutic interventions act at this level to attenuate the debilitating effects of fatigue in clinical populations.…”