11/8/11

Thomas Longyear competed in the Masters Student oral presentations at the New England American College of Sports Medicine Conference in Boston, MA.

He discussed "The effect of H+ and Pi on myosin's function: simulating fatigue in vitro"

Abstract: During intense muscular fatigue elevated levels of inorganic phosphate (Pi) and hydrogen ions (H+) are believed to inhibit contraction. Evidence suggests that a portion of the inhibition is due to a direct effect on myosin however the most pronounced losses in muscle force are believed to be mediated through the regulatory proteins Troponin (Tn) and Tropomyosin (Tm) indirectly affecting the actomyosin interaction. To begin to understand the molecular basis of this indirect inhibition we determined the effects of H+ and Pi on velocity-pCa relationship of regulated thin filaments in an in vitro motility assay using chicken skeletal myosin. Decreasing pH from 7.4 to 6.5 significantly (p<0.05) decreased filament velocity (Vactin) saturating Ca2+ (pCa 4) from 6.2±0.3um/s to 0.4±0.2 at pH 6.5. In addition, low pH increased the Ca2+ level required to reach half- maximal velocity (pCa50) from pCa 6.26 to 5.45, suggesting a pronounced decrease in Ca2+-sensitivity. The addition of 30mM Pi at pH 7.4 caused a minor increase in Vactin in the absence of Ca2+ (pCa 10), that was dependent on the presence of excess Tn/Tm in the buffer, but these effects were not present at higher Ca2+ levels (pCa <7.0). In contrast to pH 7.4, the addition of Pi at pH 6.5 increased Vactin at every Ca2+ from pCa 9 to pCa 4, and increased the pCa50 5.45 to 7.23 indicating a heighted sensitivity to Ca2+. In addition, at saturating Ca2+ adding 30mM Pi increased Vactin from 0.4±0.2 to 2.6±0.3 at pH 6.5. These data indicate that H+ alone can profoundly reduce the Ca2+-sensitivity of thin filaments but that a simultaneous increase in Pi, as occurs during fatigue can counteract these depressive effects on regulated filament velocity in vitro.