Calpain-1 is required for hydrogen peroxide-induced myotube atrophy.
Recent reports suggest numerous roles for cysteine proteases in the progression of skeletal muscle atrophy due to disuse or disease. Nonetheless, a specific requirement for these proteases in the progression of skeletal muscle atrophy has not been demonstrated. Therefore, this investigation determined whether calpains or caspase-3 is required for oxidant-induced C2C12 myotube atrophy. We demonstrate that exposure to hydrogen peroxide (25 microM H2O2) induces myotube oxidative damage and atrophy, with no evidence of cell death. Twenty-four hours of exposure to H2O2 significantly reduced both myotube diameter and the abundance of numerous proteins, including myosin (-81%), alpha-actinin (-40%), desmin (-79%), talin (-37%), and troponin I (-80%). Myotube atrophy was also characterized by increased cleavage of the cysteine protease substrate alphaII-spectrin following 4 h and 24 h of H2O2 treatment. This degradation was blocked by administration of the protease inhibitor leupeptin (10 microM). Using small interfering RNA transfection of mature myotubes against the specific proteases calpain-1, calpain-2, and caspase-3, we demonstrated that calpain-1 is required for H2O2-induced myotube atrophy. Collectively, our data provide the first evidence for an absolute requirement for calpain-1 in the development of skeletal muscle myotube atrophy in response to oxidant-induced cellular stress.
McClung
J M
JM
Dept. of Applied Physiology and Kinesiology, Univ. of Florida, Rm. 25 Florida Gym, Gainesville, FL 32611, USA. joseph.mcclung@duke.edu
Judge
A R
AR
Talbert
E E
EE
Powers
S K
SK
D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D009124:Muscle Proteins / Q000378:metabolism; D009133:Muscular Atrophy / Q000201:enzymology* / Q000473:pathology; D032448:Myoblasts, Skeletal / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects; D034622:RNA Interference; D012518:Sarcomeres / Q000201:enzymology; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009124:Muscle Proteins; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D002154:Calpain; C505409:Casp3 protein, mouse; D053148:Caspase 3; C112682:Capn1 protein, mouse; C032854:leupeptin