Increased structure-bound proteolytic activity in maturing dystrophic skeletal muscle
Draper, Kati Elizabeth
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Duchenne Muscular Dystrophy (DMD) is a severe X-linked progressive muscle wasting disease resulting from the absence of the membrane-associated protein dystrophin and the secondary components of the dystrophin-glycoprotein complex. Although the genetic basis of the disease has been known for over 15 years, the onset mechanism of the disease is not yet known and no treatment is yet available to significantly increase the lifespan of DMD patients. Increased levels of intracellular calcium have been noted in dystrophic muscle (Turner et al., 1991) and increased intracellular levels of calcium in skeletal muscle lead to increased levels of calcium-dependent proteolysis (Zeman et al., 1985). Increased levels of calpain, a calcium-dependent protease have been reported as early as age 4 weeks in mdx (dystrophin-deficient) mice (Spencer et al., 1995). Increased calpain activity has been demonstrated in mdx myotubes (Alderton et al., 2000a). There is also evidence of a role for calpain in DMD, but the contribution of calpain activity to the onset of DMD has not yet been determined. The purpose of this study was to test the hypothesis that increased calpain activity contributes to the onset of DMD in maturing (birth to weaning) dystrophic skeletal muscles and to determine if increased calpain activity was due to the relative distribution of calpain and calpastatin, calpainâ s endogenous inhibitor. Calpain activity was assessed in quadriceps and diaphragm muscle homogenate supernatant and pellet fractions from C57BL/6 control and mdx mice at ages 7, 14, and 21 days. Total calpain and calpastatin content were determined by Western analysis. In both the quadriceps and diaphragm samples, calpain activity in the supernatant increased with age. There was a significant increase (47.7%; p<0.05) in calciumdependent calpain activity in mdx quadriceps pellet compared to control at age 7 days. In the quadriceps at age 7 days, calpain activity in the pellet in the presence of calcium was significantly greater than at age 14 (61.2%) and 21 days (52.6%; p<0.05). In the diaphragm, there were no significant differences in pellet activity in either the presence or absence of calcium at any age between control and mdx samples. In both control and mdx diaphragms, pellet calpain activity in the absence compared with the presence of calcium was significantly greater at both age 7 (control, 46.4%; mdx, 45.4%) and 14 days (control, 42.4%; mdx, 43.6%; p<0.05). At age 21 days, both control and mdx calpain activities in the diaphragm supernatants in the presence of calcium were significantly greater than those at ages 7 (control, 66.7%; mdx, 72.1%) and 14 days (control, 39.9%; mdx 49.5%; p<0.05). In general, there were no differences in total calpain and calpastatin content that would account for the differences in calpain activity. There were similar patterns of calpain activity and total calpain and calpastatin content in both control and mdx samples, suggesting a similar pattern of development in control and mdx muscle from ages 7-21 days. The increase in calcium-dependent calpain activity in mdx quadriceps pellet compared to control at age 7 days may be due to differences in regulation and/or distribution of the calpain system early in mdx maturation compared to control. From the present study, the role of calpain in the onset of DMD appears to be minor if global calcium-dependent activity is evaluated.
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