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

Debold EP, Longyear TJ, Turner MA. The effects of phosphate and acidosis on regulated thin-filament velocity in an in vitro motility assay. J Appl Physiol 113: 1413-1422, 2012. First published September 27, 2012; doi:10.1152/japplphysiol.00775.2012.-Muscle fatigue from intense contractile activity is thought to result, in large part, from the accumulation of inorganic phosphate (P-i) 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 P-i 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 (V-RTF) by similar to 90% (6.2 +/- 0.3 vs. 0.5 +/- 0.2 mu m/s at pH 7.4 and 6.5, respectively). However, the addition of 30 mM P-i caused V-RTF to increase fivefold, from 0.5 +/- 0.2 to 2.6 +/- 0.3 mu m/s at pH 6.5. Similarly, at all subsaturating Ca+ (+) levels, acidosis slowed V-RTF, but the addition of P-i significantly attenuated this effect. We also manipulated the [ADP] in addition to the [P-i] 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 P-i rebinding, and the associated detachment from actin, this finding may also explain the P-i-induced enhancement of V-RTF 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.