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Creatine (Cr), one of the most popular nutritional supplements among athletes, has been recently shown to prevent the cytotoxicity caused by different oxidative stressors in various mammalian cell lines, including C2C12 myoblasts, via a direct antioxidant activity. Here, the effect of Cr on the differentiating capacity of C2C12 cells exposed to H(2)O(2) has been investigated. Differentiation into myotubes was monitored using morphological, ultrastructural, and molecular techniques. Treatment with H(2)O(2) (1 h) not only caused a significant (30%) loss of cell viability, but also abrogated the myogenic ability of surviving C2C12. Cr-supplementation (24 h prior to H(2)O(2) treatment) was found to prevent these effects. Interestingly, H(2)O(2)-challenged cells preconditioned with the established antioxidants trolox or N-acetyl-cysteine, although cytoprotected, did not display the same differentiating ability characterizing oxidatively-injured, Cr-supplemented cells. Besides acting as an antioxidant, Cr increased the level of muscle regulatory factors and IGF1 (an effect partly refractory to oxidative stress), the cellular availability of phosphocreatine and seemed to exert some mitochondrially-targeted protective activity. It is concluded that Cr preserves the myogenic ability of oxidatively injured C2C12 via a pleiotropic mechanism involving not only its antioxidant capacity, but also the contribution to cell energy charge and effects at the transcriptional level which common bona fide antioxidants lack.

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This study describes mitochondrial behaviour during the C2C12 myoblast differentiation program and proposes a proteomic approach to mitochondria integrated with classical morphofunctional and biochemical analyses. Mitochondrial ultrastructure variations were determined by transmission electron microscopy; mitochondrial mass and membrane potential were analysed by Mitotracker Green and JC-1 stains and by epifluorescence microscope. Expression of PGC1α, NRF1α, and Tfam genes controlling mitochondrial biogenesis was studied by real-time PCR. The mitochondrial functionality was tested by cytochrome c oxidase activity and COXII expression. Mitochondrial proteomic profile was also performed. These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes. The proteomic profile identifies 32 differentially expressed proteins, mostly involved in oxidative metabolism, typical of myotubes formation. Other notable proteins, such as superoxide dismutase (MnSOD), a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1) involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process. The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells.

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