DNA demethylation enhances myoblasts hypertrophy during the late phase of myogenesis activating the IGF-I pathway

Abstract

Skeletal muscle regeneration and hypertrophy are important adaptive responses to both physical activity and pathological stimuli. This research was performed to investigate DNA demethylation action on the late phase of muscle differentiation and early stage of hypertrophy. The epigenetic process involved in myogenesis was studied with the DNA-demethylating agent 5-azacytidine (AZA). We induced muscle differentiation in C2C12 mouse myoblasts in the presence of 5 μM AZA and growth or differentiation medium for 48, 72, and 96 h. To study a potential AZA hypertrophic effect, we stimulated 72 h differentiated myotubes with AZA for 24 h. Unstimulated cells were used as control. By western blot and immunofluorescence analysis, we examined AZA action on myogenic regulatory factors expression, hypertrophic signaling pathway and myotube morphology. During differentiation, protein levels of myogenic markers, Myf6 and Myosin Heavy Chain (MyHC), were higher in AZA stimulated cells compared to control. Myostatin and p21 analysis revealed morphological changes which reflect a tendency to hypertrophy in myotubes. In AZA stimulated neo formed myotubes, we observed that IGF-I pathway, kinases p70 S6, 4E-BP1, and ERK1/2 were activated. Furthermore, AZA treatment increased MyHC protein content in stimulated neo myotubes. Our work demonstrates that DNA demethylation could plays an important role in promoting the late phase of myogenesis, activating endocellular pathways involved in protein increment and stimulating the hypertrophic process.

Metformin Treatment Prevents Sedentariness Related Damages in Mice

“…Cell differentiation was initiated by placing 70% confluent cell cultures in differentiation medium (DM), containing DMEM supplemented with 1% horse serum (HS), antibiotics, and 1% l-glutamine. In our in vitro differentiation model, early myotubes appeared 24–48 hours (h) after serum starvation and neomyotubes formation was completed after 72 h [ 19 ]. Proliferating cells, myoblasts during differentiation process, and neomyotubes were treated with 400 μ m METF.…”

Potential Therapeutic Role of L-Carnitine in Skeletal Muscle Oxidative Stress and Atrophy Conditions

“…Quantitative measurement of immunoreactive bands intensities, visualized by an enhanced chemiluminescence method (Amersham Pharmacia Biotech, Piscataway, NJ, USA), was performed by densitometric analysis using the Scion Image software (Scion Corporation, Frederick, MD, USA). Data were then converted into fold-changes (FC) of the controls [ 18 ].…”

MSTN Mutant Promotes Myogenic Differentiation by Increasing Demethylase TET1 Expression via the SMAD2/SMAD3 Pathway

“…Various studies have demonstrated that DNA methylation is a major repressive mechanism of muscle satellite cell differentiation [11,13], whereas demethylation, as well as MyoD and MyoG, are required for the initiation of the differentiation program [11]. In C2C12 cells, treatment with 5-azacytidine resulted in an enhanced expression of muscle-specific genes (including myogenin) and increased myotube maturation [18,19], which suggests that DNA methylation plays an important role in the regulation of the differentiation of muscle satellite cells. In the present study, we found that the MSTN mutant promoted the expression of the demethylase TET1, but reduced the expression of TET2 and TET3.…”

“…The modulation of methylation via prolonged treatment with 5-azacytidine, an inhibitor of DNA methylation, has been associated with an increased myogenic commitment of fibroblasts, mature adipocyte-derived dedifferentiated fat cells, and cardiac cells [14][15][16][17]. Furthermore, in C2C12 cells, treatment with 5-azacytidine resulted in an enhanced expression of muscle-specific genes (including myogenin) and increased myotube maturation, which suggested that the inhibition of methylation was a suitable tool to further boost myogenic differentiation in already committed precursors [18,19].…”