Title page for ETD etd-04162004-105733

Type of Document Master's Thesis
Author Draper, Kati Elizabeth
Author's Email Address draperke@vt.edu
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
  • muscular dystrophy
  • calcium
  • calpain
Date of Defense 2004-04-02
Availability unrestricted
Increased structure-bound proteolytic activity in maturing dystrophic

skeletal muscles

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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