In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes.

CAPN3/calpain-3/p94, a muscle-specific Ca(2+)-dependent cysteine protease, is responsible for limb-girdle muscular dystrophy R1 (LGMDR1), an autosomal recessive muscular dystrophy. However, the activation mechanism and physiological function of CAPN3 in skeletal muscles remain unknown. Here, we capture the in situ activation of CAPN3 in cultured mouse skeletal myotubes. Using our newly developed antibody, which specifically recognizes CAPN3 autolytic processing, we succeeded in differentiating WT CAPN3 from a protease-inactive CAPN3 mutant by immunostaining. We further demonstrated that CAPN3 predominantly localized at the M-bands of cultured skeletal myotubes at rest and translocated to the cytoplasm after activation by stimulation with ouabain, a cardiotonic steroid. This event requires a small but long-lasting cytoplasmic increase in Ca(2+) levels, which is sufficient for the activation of CAPN3 but not of calpain-1/CAPN1. Activated CAPN3 digests the cytoskeletal proteins spectrin and talin. Thus, we successfully visualized the intracellular dynamics of endogenous CAPN3 in cultured skeletal muscles after activation by ouabain and demonstrated the subsequent processing of endogenous substrates in living cells. Our study will help understand the physiological functions of CAPN3 in skeletal muscles and the pathophysiological mechanisms of limb-girdle muscular dystrophy R1.