Epigenetics: DNA demethylation promotes skeletal myotube maturation

Abstract

Mesenchymal progenitor cells can be differentiated in vitro into myotubes that exhibit many characteristic features of primary mammalian skeletal muscle fibers. However, in general, they do not show the functional excitation-contraction coupling or the striated sarcomere arrangement typical of mature myofibers. Epigenetic modifications have been shown to play a key role in regulating the progressional changes in transcription necessary for muscle differentiation. In this study, we demonstrate that treatment of murine C2C12 mesenchymal progenitor cells with 10 μM of the DNA methylation inhibitor 5-azacytidine (5AC) promotes myogenesis, resulting in myotubes with enhanced maturity as compared to untreated myotubes. Specifically, 5AC treatment resulted in the up-regulation of muscle genes at the myoblast stage, while at later stages nearly 50% of the 5AC-treated myotubes displayed a mature, well-defined sarcomere organization, as well as spontaneous contractions that coincided with action potentials and intracellular calcium transients. Both the percentage of striated myotubes and their contractile activity could be inhibited by 20 nM TTX, 10 μM ryanodine, and 100 μM nifedipine, suggesting that action potential-induced calcium transients are responsible for these characteristics. Our data suggest that genomic demethylation induced by 5AC overcomes an epigenetic barrier that prevents untreated C2C12 myotubes from reaching full maturity.

Exercise training alters DNA methylation patterns in genes related to muscle growth and differentiation in mice

“…Noteworthy, treatment of myoblasts with the DNA methylation inhibitor 5-azacytidine was shown to promote myogenesis (19), which is consistent with our observation that the majority of growth-and differentiation-related genes in our list show decreased methylation.…”

LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration

“…To confirm that Dppa2 transcription is regulated by DNA methylation in myoblasts, we treated C2C12 cells with 5-Aza. Expectedly, the treatment induced Dppa2 expression ( Figure 5A); as a positive control, myogenin gene was also induced as previously reported [23]. Several CpG sites were identified in two regions of Dppa2 promoter by computational prediction (EMBOSS Cpgplot; http://www.ebi.ac.uk) ( Figure 5B).…”

Striated muscle activator of Rho signaling is required for myotube survival but does not influence basal protein synthesis or degradation

“…The C2C12 mouse myotubes used in the present study display similarities to skeletal muscle, such as the expression of metabolic, myogenic, and sarcomeric proteins, and similarities in their response to anabolic and catabolic compounds (5,32,49). C2C12 myotubes can spontaneously contract; however, they lack neural innervation and the contractile synchronicity of multiple muscle fibers (18). Therefore, the effect of STARS as a regulator of protein synthesis was investigated under basal conditions and following intracellular anabolic and catabolic stress.…”