The Calpain System in Muscular Dystrophy

The muscular dystrophies cause progressive muscle wasting leading to decreased ability to function physically and even death. A large amount of evidence suggests that the calpain system is involved in the muscle wasting associated with these diseases. Calpains are Ca 2+ ‐activated proteases and intracellular [Ca 2+ ] is elevated 2‐4‐fold in dystrophic muscle. The increased [Ca 2+ ] may result in unregulated calpain activity and increased protein degradation. The calpain system in skeletal muscle consists of 3 proteins: 1) μ‐calpain, a protease requiring 3–50 μM Ca 2+ for half‐maximal activity; 2) m‐calpain, a protease requiring 400–800 μM Ca 2+ for half‐maximal activity; and 3) calpastatin, a specific protein inhibitor of μ‐ and m‐calpain. The goal of this project is to characterize these proteins in human dystrophic skeletal muscle. We have developed a protocol for analysis of μ‐calpain, m‐calpain, and calpastatin from a 200–800 mg biopsy sample. Samples were obtained at the University Medical Center. Biochemical analysis of the soluble proteins included casein zymography, western blotting, enzymatic activity assays, inhibitor assays, and ELISA. In nearly every sample analyzed so far, the calpastatin activity in the crude supernatant is 5–20‐fold higher than the calpain activity. However, once separated, the calpain activity appears to be higher than the calpastatin activity. This may indicate that the calpastatin in the dystrophic muscle samples is unable to inhibit the endogenous calpain activity. Supported by NRI 2002‐35206‐11630, 2004‐04338, and the MDA.