Human Molecular Genetics Advance Access originally published online on May 29, 2009
Human Molecular Genetics 2009 18(17):3194-3205; doi:10.1093/hmg/ddp257
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Mitochondrial abnormalities, energy deficit and oxidative stress are features of calpain 3 deficiency in skeletal muscle
1 Department of Neurology and 2 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at University of California, Los Angeles, CA 90095, USA 3 Department of Physical Therapy, University of Florida, Gainesville, FL 32610, USA 4 Institute of Exercise and Environmental Medicine, Dallas, TX 75231, USA 5 Department of Neurology, University of Texas Southwestern Medical School, Dallas, TX 75390, USA 6 Institute of Exercise and Environmental Medicine, Dallas, TX 75231, USA 7 Dallas VA Medical Center, Dallas, TX 75216, USA
* To whom correspondence should be addressed at: 635 Charles Young Drive, NRB, Room 401, Los Angeles, CA 90095, USA. Tel: +1 3107945225; Fax: +1 3102061998; Email: mspencer{at}mednet.ucla.edu
Received April 30, 2009; Accepted May 27, 2009
Mutations in the non-lysosomal cysteine protease calpain-3 cause autosomal recessive limb girdle muscular dystrophy. Pathological mechanisms occurring in this disease have not yet been elucidated. Here, we report both morphological and biochemical evidence of mitochondrial abnormalities in calpain-3 knockout (C3KO) muscles, including irregular ultrastructure and distribution of mitochondria. The morphological abnormalities in C3KO muscles are associated with reduced in vivo mitochondrial ATP production as measured by 31P magnetic resonance spectroscopy. Mitochondrial abnormalities in C3KO muscles also correlate with the presence of oxidative stress; increased protein modification by oxygen free radicals and an elevated concentration of the anti-oxidative enzyme Mn-superoxide dismutase were observed in C3KO muscles. Previously we identified a number of mitochondrial proteins involved in β-oxidation of fatty acids as potential substrates for calpain-3. In order to determine if the mitochondrial abnormalities resulted from the loss of direct regulation of mitochondrial proteins by calpain-3, we validated the potential substrates that were identified in previous proteomic studies. This analysis showed that the β-oxidation enzyme, VLCAD, is cleaved by calpain-3 in vitro, but we were not able to confirm that VLCAD is an in vivo substrate for calpain-3. However, the activity of VLCAD was decreased in C3KO mitochondrial fractions compared with wild type, a finding that likely reflects a general mitochondrial dysfunction. Taken together, these data suggest that mitochondrial abnormalities leading to oxidative stress and energy deficit are important pathological features of calpainopathy and possibly represent secondary effects of the absence of calpain-3.