Ca++-sensitizing mutations in troponin, Pi, and 2-deoxyATP alter the depressive effect of acidosis on regulated thin-filament velocity

Repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, resulting in 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 poorly understood. We sought to gain novel insight into the decrease in velocity by determining whether 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 submaximal Ca++ levels in a pH-dependent manner. A truncated construct of the inhibitory subunit of Tn (TnI) and a Ca++-sensitizing mutation in the Ca++-binding subunit of Tn (TnC) increased VRTF at submaximal Ca++ under acidic conditions but had no effect on VRTF at maximal Ca++ levels. In contrast, both Pi and replacement of ATP with dATP reversed much of the acidosis-induced depression of VRTF at saturating Ca++. Interestingly, despite producing similar magnitude increases in VRTF, the combined effects of Pi and dATP were additive, suggesting different underlying mechanisms of action. These findings suggest that acidosis depresses velocity by slowing the detachment rate from actin but also by possibly slowing the attachment rate.

The effects of phosphate and acidosis on regulated thin-filament velocity in an in vitro motility assay

Muscle fatigue from intense contractile activity is thought to result, in large part, from the accumulation of inorganic phosphate (Pi) and hydrogen ions (H+) acting to directly inhibit the function of the contractile proteins; however, the molecular basis of this process remain unclear. We used an in vitro motility assay and determined the effects of elevated H+ and Pi on the ability of myosin to bind to and translocate regulated actin filaments (RTF) to gain novel insights into the molecular basis of fatigue. At saturating Ca++, acidosis depressed regulated filament velocity ( VRTF) by ∼90% (6.2 ± 0.3 vs. 0.5 ± 0.2 μm/s at pH 7.4 and 6.5, respectively). However, the addition of 30 mM Pi caused VRTF to increase fivefold, from 0.5 ± 0.2 to 2.6 ± 0.3 μm/s at pH 6.5. Similarly, at all subsaturating Ca++ levels, acidosis slowed VRTF, but the addition of Pi significantly attenuated this effect. We also manipulated the [ADP] in addition to the [Pi] to probe which specific step(s) of cross-bridge cycle of myosin is affected by elevated H+. The findings are consistent with acidosis slowing the isomerization step between two actomyosin ADP-bound states. Because the state before this isomerization is most vulnerable to Pi rebinding, and the associated detachment from actin, this finding may also explain the Pi-induced enhancement of VRTF at low pH. These results therefore may provide a molecular basis for a significant portion of the loss of shortening velocity and possibly muscular power during fatigue.

Micromechanical Thermal Assays of Ca2+-Regulated Thin-Filament Function and Modulation by Hypertrophic Cardiomyopathy Mutants of Human Cardiac Troponin

Microfabricated thermoelectric controllers can be employed to investigate mechanisms underlying myosin-driven sliding of Ca2+-regulated actin and disease-associated mutations in myofilament proteins. Specifically, we examined actin filament sliding—with or without human cardiac troponin (Tn) and α-tropomyosin (Tm)—propelled by rabbit skeletal heavy meromyosin, when temperature was varied continuously over a wide range (∼20–63C°). At the upper end of this temperature range, reversible dysregulation of thin filaments occurred at pCa 9 and 5; actomyosin function was unaffected. Tn-Tm enhanced sliding speed at pCa 5 and increased a transition temperature (Tt) between a high activation energy (Ea) but low temperature regime and a lowEabut high temperature regime. This was modulated by factors that alter cross-bridge number and kinetics. Three familial hypertrophic cardiomyopathy (FHC) mutations, cTnI R145G, cTnI K206Q, and cTnT R278C, cause dysregulation at temperatures ∼5–8C°lower; the latter two increased speed at pCa 5 at all temperatures.