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 PGC1 , NRF1 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. 1. Introduction Skeletal muscle represents an important model for studying mitochondrial behaviour during cell growth and differentiation. Myoblasts cultured in vitro, if induced by cell confluence and serum deprivation, follow a myogenic program, which includes an active proliferation, withdrawal from the cell cycle, synthesis of muscle-specific proteins, and fusion into multinucleated myotubes [1, 2]. This event is accomplished by the activation of specific myogenic regulatory factors (MRFs) [3–5]. Recent studies suggest that mitochondria are involved in the regulation of the skeletal muscle physiology and play a critical role in cell growth, cell proliferation, cell death, and cell differentiation [6–13]. In particular, mitochondrial activity is involved in the regulation of myoblast differentiation through myogenin expression, the activity of myogenic factors, and by control of c-Myc expression [8, 14, 15]. Furthermore, differentiation appears to be a program which is dependent on both mitochondrial function and mitochondrial biogenesis, as indicated by the rapid increase in mitochondrial mass/volume, mtDNA copy number, mitochondrial enzyme activities, and mRNA
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