Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation

Abstract

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.

Mitochondrial Complex I Deficiency Enhances Skeletal Myogenesis but Impairs Insulin Signaling through SIRT1 Inactivation

“…Low glucose has been shown to prevent skeletal myogenesis by increasing the ratio of NAD ϩ to NADH and activating SIRT1, and low glucose-induced myogenesis inhibition is released by SIRT1 knockdown (38). Ironically, PGC-1␣, a SIRT1 deacetylase substrate, is up-regulated and deacetylated despite SIRT1 inactivation during skeletal myogenesis (2,39). These findings create a paradox for the SIRT1-PGC-1␣ pathway in mitochondrial biogenesis during skeletal myogenesis.…”

Mitochondria hyperfusion and elevated autophagic activity are key mechanisms for cellular bioenergetic preservation in centenarians

“…In addition, we also found that cell treatment with Mdivi-1 (black columns) provoked a similar slight decrease of autophagy in DFs derived from Young (-7.7±1.6%), Old (-7.6±1.1%) and LLI (-6.9±1.3%). Conversely, quantitative analysis of mitochondrial mass by using MitoTracker green [28] dye revealed that Mdivi-1 induced a significant (p<0.01) increase of mitochondrial mass in DFs from Young (+48.3±4.7%) and Old individuals (29.3±2.9%) only (Fig. 5B, black columns).…”

Creatine Prevents the Structural and Functional Damage to Mitochondria in Myogenic, Oxidatively Stressed C2C12 Cells and Restores Their Differentiation Capacity

“…Pellets were fixed again in a new fixative solution for 30 min, OsO 4 postfixed, alcohol-dehydrated, and araldite embedded as reported in [33]. Thin sections were collected on nickel 300-mesh grids and stained with uranyl acetate and lead citrate.…”