mu-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans.
mu-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 mu-calpain becomes proteolytically active in the presence of 2-200 microM 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; approximately 1 microM) 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 microM). 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 approximately 15% autolysis observed after 1-min exposure to 2.5 microM 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% VO2 peak 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.
Murphy
Robyn M
RM
Dept. of Zoology, La Trobe Univ., Victoria 3086, Australia. r.murphy@latrobe.edu.au
Snow
Rodney J
RJ
Lamb
Graham D
GD
D000818:Animals; D001329:Autolysis; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology*; D010807:Physical Endurance / Q000502:physiology*; D005082:Physical Exertion / Q000502:physiology*; D051381:Rats; D020318:Rats, Long-Evans; D013997:Time Factors
D007527:Isoenzymes; D009124:Muscle Proteins; C105884:CAPN3 protein, human; D002154:Calpain; C491372:Capn3 protein, rat; C449002:mu-calpain; D002118:Calcium