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

Abstract

μ-calpain and calpain-3 are Ca²⁺-dependent proteases found in skeletal 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 μ-calpain becomes proteolytically active in the presence of 2–200 μM 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; ∼1 μM) than the levels at which μ-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 μM). Autolysis of calpain-3 was found to occur across a [Ca²⁺] range similar to that for μ-calpain, and both calpains displayed a seemingly higher Ca²⁺sensitivity in human than in rat muscle homogenates, with ∼15% autolysis observed after 1-min exposure to 2.5 μM Ca²⁺in human muscle and almost none after 1- to 2-min exposure to the same [Ca²⁺]_ilevel in rat muscle. During muscle activity, [Ca²⁺]_imay transiently peak in the range found to autolyze μ-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% V̇o_{2 peak}until fatigue, n = 3) on the amount of autolyzed μ-calpain or calpain-3 in human muscle. No significant autolysis of μ-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca²⁺]_ithat occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo.