THE POTENTIAL OF CONTRACTILE ACTIVITY TO REVERSE THE SEVERITY OF MTDNA DEFECTS

Abstract

Defects in mitochondrial DNA (mtDNA) evoke adaptive responses in the nuclear genome, leading to altered mitochondrial biogenesis. PURPOSE To examine adaptations in the protein import machinery, and transcription factors involved in mitochondrial biogenesis. METHODS We used immunoblotting and biochemical techniques to evaluate protein levels and protein import in C2C12 muscle cells depleted of mtDNA (rho- cells), as well as in fibroblasts from a patient with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS). RESULTS In rho- cells, Tom20, Tom34 and Tim23 protein levels were reduced by 25%, 12% and 59% of control, respectively. No change was observed in HSP60, but mtHSP70 was induced by 2-fold in rho-, relative to control cells. These changes were accompanied by a 21% increase in import into the mitochondrial matrix in rho-cells (p < 0.05). In contrast, in MELAS cells mtHSP70 was elevated by 70% while Tom20 and Tom34 protein levels were increased by 45% and 112%, respectively, while HSP60 was reduced by 25%. Matrix protein import was not impaired in MELAS cells. Overexpression of Tom34 in C2C12 cells resulted in a 13% (p < 0.05) increase in import into the matrix. To evaluate the effect of contractile activity, we stimulated C2C12 myotubes for 4 days. A 67% (p < 0.05) increase in Tom34 was observed. In rho- cells, NRF-1 and Tfam protein levels declined by 33% and 54%, respectively, while no change was observed in PGC-1. Rho- cells had a lower rate of oxygen consumption (VO2) and reduced ATP, along with a 2-fold increase in lactate (p < 0.05). In contrast, in stimulated cells, 42% and 78% increases were observed in mtDNA and VO2, respectively (p < 0.05). CONCLUSION Our findings suggest that in response to mtDNA defects, compensatory adaptations occur in the mitochondrial import pathway to maintain a normal rate of import. These data support a role for exercise in reducing the pathological phenotype associated with mtDNA depletion. Supported by CIHR.