Type of Document Master's Thesis Author Draper, Kati Elizabeth Author's Email Address email@example.com URN etd-04162004-105733 Title Increased structure-bound proteolytic activity in maturing dystrophic skeletal muscle Degree Master of Science Department Human Nutrition, Foods, and Exercise Advisory Committee
Advisor Name Title Grange, Robert W. Committee Chair Newton, William E. Committee Member Ward, Christopher W. Committee Member Williams, Jay H. Committee Member Keywords
- muscular dystrophy
Date of Defense 2004-04-02 Availability unrestricted AbstractIncreased structure-bound proteolytic activity in maturing dystrophic
Kati E. Draper
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, calpain’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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