mu-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans

mu-calpain and calpain-3 are Ca2+-dependent proteases found in muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that mu-calpain becomes proteolytically active in the presence of 2-200 mu M Ca2+. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca2+ concentration levels ([Ca2+](i); similar to 1 mu M) than the levels at which mu-calpain activation occurs. We have demonstrated the Ca2+-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca2+] levels (0, 2.5, 10, and 25 mu M). Autolysis of calpain-3 was found to occur across a [Ca2+] range similar to that for mu-calpain, and both calpains displayed a seemingly higher Ca2+ sensitivity in human than in rat muscle homogenates, with similar to 15% autolysis observed after 1-min exposure to 2.5 mu M Ca2+ in human muscle and almost none after 1- to 2-min exposure to the same [Ca2+](i) level in rat muscle. During muscle activity, [Ca2+](i) may transiently peak in the range found to autolyze mu-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s all- out cycling, n = 8; and 70% V-O2peak until fatigue, n = 3) on the amount of autolyzed mu-calpain or calpain-3 in human muscle. No significant autolysis of mu-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca2+](i) that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo.