MOLECULAR APPROACHES TO STUDY THE MITOCHONDRIAL BEHAVIOUR DURING A MYOGENIC PROGRAM

Numerous data suggest that mitochondria are involved in the regulation of the skeletal muscle physiology and play a critical role in the cell growth, proliferation, death and differentiation. In particular, mitochondrial activity is involved in the regulation of myoblast differentiation through myogenin expression, activity of myogenic factors and by control of c-Myc expression (1,2). Furthermore, differentiation appears to be a program dependent on both mitochondrial function and mitochondrial biogenesis as indicated by a rapid increase in mitochondrial mass/volume, mtDNA copy number, enzyme activities and mitochondrial mRNA levels within the first 48hrs of myoblasts differentiation (3). We have studied the changes in mitochondrial biogenesis and activity in murine myoblast cultures (C2C12 line) undergoing differentiation: myoblasts were grown and induced to differentiation. Staining of mitochondria by Mitotracker green was used to evaluate their mass/volume by epifluorescence microscope. Real time RT-was used to study the expression level of PGC1-a, TFamA, and COXII, target genes for mitochondrial biogenesis and activity. The mitochondrial functionality was also tested by their ATP production and cytochrome c oxidase activity using HPLC, HPCE and spectrophotometer approaches respectively. These assays show that the mitochondrial biogenesis and activity significantly increase in differentiating myotubes, also compared to undifferentiated and quiescent cells. Intriguingly, using a proteomic approach, changes appear also in mitochondria proteomic pattern during differentiation. Indeed a significant number of new and up-regulated proteins were reveled in mitochondria isolated from myotubes respect to those isolated from myoblasts. Such data are also supported by a preliminary study in which an increase of energy production during the differentiation, is observed. Thus, the mitochondrial biogenesis seems to be correlated to a change in cellular metabolism during myogenesis.