Sodium arsenite delays the differentiation of C2C12 mouse myoblast cells and alters methylation patterns on the transcription factor myogenin

Abstract

Epidemiological studies have correlated arsenic exposure with cancer, skin diseases, and adverse developmental outcomes such as spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. The current study used C2C12 mouse myoblast cells to examine whether low concentrations of arsenic could alter their differentiation into myotubes, indicating that arsenic can act as a developmental toxicant. Myoblast cells were exposed to 20 nM sodium arsenite, allowed to differentiate into myotubes, and expression of the muscle-specific transcription factor myogenin, along with the expression of tropomyosin, suppressor of cytokine signaling 3 (Socs3), prostaglandin I2 synthesis (Ptgis), and myocyte enhancer 2 (Mef2) were investigated using QPCR and immunofluorescence. Exposing C2C12 cells to 20nM sodium arsenite delayed the differentiation process, as evidenced by a significant reduction in the number of multinucleated myotubes, a decrease in myogenin mRNA expression, and a decrease in the total number of nuclei expressing myogenin protein. The expression of mRNA involved in myotube formation, such as Ptgis and Mef2 mRNA, was also significantly reduced by 1.6-fold and 4-fold during differentiation. This was confirmed by immunofluorescence for Mef2, which showed a 2.6-fold reduction in nuclear translocation. Changes in methylation patterns in the promoter region of myogenin (−473 to +90) were examined by methylation-specific PCR and bisulfite genomic sequencing. Hypermethylated CpGs were found at −236bp and −126bp, whereas hypomethylated CpGs were found at −207bp in arsenic exposed cells. This study indicates that 20 nM sodium arsenite can alter myoblast differentiation by reducing the expression of the transcription factors myogenin and Mef2c, which is likely due to changes in promoter methylation patterns. The delay is muscle differentiation may lead to developmental abnormalities.

Role of PRDM16 and its PR domain in the epigenetic regulation of myogenic and adipogenic genes during transdifferentiation of C2C12 cells

“…CpGs − 76 and + 2 on myogenin gene, which were also hypermethylated, are in close proximity to the binding sites of SIX1 (sine oculis homeobox 1) and MEF2 (myocyte-specific enhancer factor) which are proven to be essential for the transactivation of myogenin gene (Palacios et al, 2010). It is surprising that CpG − 205 on myogenin gene promoter was significantly hypomethylated in PRDM16-overexpressing cells, but similar result was reported in sodium arsenite-induced delay in myogenesis (Steffens et al, 2011), thus we speculate that CpG − 205 might be or conformationally close to a binding site of a transcription repressor. PPARγ transcription was validated to be negatively related with the methylation status of its promoter in visceral adipose tissue of a diabetic mouse model (Fujiki et al, 2009) and a fat chicken line (Sun et al, 2014) where PPARγ was preternaturally activated.…”

The methyl-CpG-binding protein CIBZ suppresses myogenic differentiation by directly inhibiting myogenin expression

“…During revision of this manuscript, Steffens et al reported that exposure of C2C12 cells to sodium arsenite delayed DM-induced differentiation [31]. Increased methylation of the same 12 CpG sites at the Myog promoter that we describe here correlated with a reduction in Myog transcription, which may consequently lead to a delay in myogenic differentiation [31].…”

“…During revision of this manuscript, Steffens et al reported that exposure of C2C12 cells to sodium arsenite delayed DM-induced differentiation [31]. Increased methylation of the same 12 CpG sites at the Myog promoter that we describe here correlated with a reduction in Myog transcription, which may consequently lead to a delay in myogenic differentiation [31]. Generally, the number of methylated CpGs and the methylation levels of CpG sites appear to coordinately control gene transcription in cells and tissues, although the molecular mechanism underlying this phenomenon remains poorly understood [13,30].…”

Arsenic induces apoptosis in myoblasts through a reactive oxygen species-induced endoplasmic reticulum stress and mitochondrial dysfunction pathway

“…It has been shown that lower concentrations of inorganic arsenic (0.02-0.5 lM) inhibit the myoblast differentiation (Steffens et al 2011;Yen et al 2010). Li et al (2010) reported that higher concentrations of As 2 O 3 (4-32 lM) obviously reduced smooth muscle cell viability in a concentration-dependent manner ).…”

“…Chronic exposure to arsenic via drinking water has known to be associated with numerous cancers (e.g., skin, lung, and bladder) and non-cancer harmful health (e.g., skin lesions, peripheral vascular disease, cardiovascular disease, and diabetes mellitus) (Navas-Acien et al 2005;Yoshida et al 2004). Previous studies have demonstrated that low dose of inorganic arsenic is capable of inhibiting myoblast differentiation (Steffens et al 2011). Arsenic trioxide is known to be used to treat acute promyelocytic leukemia and can induce apoptosis in various cancer and normal cell lines Florea et al 2007;Li et al 2010;Lu et al 2011;Tang et al 2009).…”