calpastatin and Muscular-Dystrophy--Animal

Reduced sarcolemmal integrity in dystrophin-deficient muscles of mdx mice and Duchenne muscular dystrophy (DMD) patients has been reported to result in altered calcium homeostasis. Previous studies have shown a correlative relationship between calcium-dependent protease (calpain) activity in dystrophic muscle and muscle necrosis, but have not tested whether calpain activation precedes cell death or is a consequence of it. To test a causal relationship between calpain activation and muscle cell death in dystrophin deficiency, mdx mice were generated that overexpress a calpastatin transgene in muscle. Calpastatin (CS) is a specific, endogenous inhibitor of m- and micro -calpains that does not inhibit calpain 3 (p94). CS overexpression on a C57/BL 10 background produced no phenotype. Transgenic (Tg) mice crossed with mdx mice were tested for pathological indicators of necrosis, regeneration and membrane damage. Two lines of mice were examined, with different levels of CS overexpression. Both lines of Tg/mdx mice showed reductions in muscle necrosis at 4 weeks of age. These mice had fewer as well as smaller lesions. In addition, one line of mice had significantly less regeneration, indicating a reduction in previous necrosis. The extent of improvement correlated with the level of CS protein expression. Membrane damage, as assessed by procion orange and creatine kinase assays, was unchanged, supporting the idea that calpains act downstream of the primary muscle defect. These data suggest that calpains play an active role in necrotic processes in dystrophic muscle and that inhibition of calpains might provide a good therapeutic option for treatment of DMD.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Membrane; Down-Regulation; Mice; Mice, Inbred mdx; Mice, Transgenic; Muscle Cells; Muscle, Skeletal; Muscular Dystrophy, Animal

Death of dystrophin-deficient muscle purportedly results from increases in [Ca]in that cause the activation of calpains. We have tested whether calpains play a role in this process by assaying for changes in calpain concentration and activation in peak necrotic mdx mice (4 weeks of age) and in completely regenerated mdx mice (14 weeks of age). Biochemical fractionation and immunoblotting with epitope-specific antisera allowed measurement of the concentrations of m- and mu-calpains and the extent of autoproteolytic modification. Our findings show that total calpain concentration is elevated in both 4-week and 14-week mdx mice. This increase in concentration was shown to result primarily from a significant increase in m-calpain concentration at 4 weeks. Northern analysis demonstrated that neither m- nor mu-calpain mRNA concentrations differed between mdx and controls suggesting that the increased calpain concentration results from post-translational regulation. Immunoblotting with antibodies directed against amino-terminal peptides revealed an increase in autoproteolysis of mu-calpain, indicative of increased activation. The extent of autoproteolysis of mu-calpain returns to control levels during regeneration. This is not a consequence of increased calpastatin mRNA or protein. The findings reported here support a role for calpains in both the degenerative and regenerative aspects of mdx dystrophy.

Topics: Age Factors; Animals; Calcium-Binding Proteins; Calpain; Dystrophin; Enzyme Activation; Gene Expression; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Muscular Dystrophy, Animal; Necrosis; RNA, Messenger

1. Two fast-twitch skeletal muscles from normal and dystrophic hamsters were analysed for their calpain and calpastatin contents. 2. Assays of wide-specificity calpain II showed that the activity levels in the two muscles were increased 1.5 and 1.6 times in dystrophic animals. 3. Analysis of calpastatin levels showed that the respective dystrophic muscles had activity levels of 2.2 and 2.8 times those of control muscles. 4. These results contrast with previous studies on denervated hamster muscles which showed that denervation causes an increase in calpain levels but a decrease in calpastatin levels.

Topics: Animals; Calcium-Binding Proteins; Calpain; Muscles; Muscular Dystrophy, Animal; Proteins