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

Pamela Senesi,Livio Luzi,Ileana Terruzzi,Anna Montesano

doi:10.1007/s12020-013-0142-5

Pamela Senesi, Livio Luzi + Show 2 more

Open Access

https://doi.org/10.1007/s12020-013-0142-5

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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.

Highlights

Skeletal muscle regeneration and hypertrophy are important adaptive responses to both disease and physical activity [1]
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
In the first case (GMAZA), we would delineate the possible ability of DNA demethylation to accelerate myoblast spontaneous differentiation due to cell–cell contact; and in the second (DMAZA), we would determinate the probable role of DNA hypomethylation to improve myogenesis process (Fig. 1)

Summary

Introduction

Skeletal muscle regeneration and hypertrophy are important adaptive responses to both disease and physical activity [1]. Myoblasts withdraw from the cell cycle and form myotubes [3]. This process is principally governed by muscle regulatory factors (MRFs), including MyoD and Myf6 [4, 5]. A member of the transforming growth factor-b superfamily, is an important negative regulator of skeletal muscle development. Mnst occurring mutations, as well as experimental knockout of the Mstn gene, lead to hypermuscular phenotype [8]. In vitro model of skeletal muscle cells, Mstn is predominantly localized in the nuclei of Endocrine (2014) 47:244–254 differentiated, polynucleated myotubes and down regulates the muscle genes expression [9]

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