Notch Pathway Activation Contributes to Inhibition of C2C12 Myoblast Differentiation by Ethanol

Abstract

The loss of muscle mass in alcoholic myopathy may reflect alcohol inhibition of myogenic cell differentiation into myotubes. Here, using a high content imaging system we show that ethanol inhibits C2C12 myoblast differentiation by reducing myogenic fusion, creating smaller and less complex myotubes compared with controls. Ethanol administration during C2C12 differentiation reduced MyoD and myogenin expression, and microarray analysis identified ethanol activation of the Notch signaling pathway target genes Hes1 and Hey1. A reporter plasmid regulated by the Hes1 proximal promoter was activated by alcohol treatment in C2C12 cells. Treatment of differentiating C2C12 cells with a gamma secretase inhibitor (GSI) abrogated induction of Hes1. On a morphological level GSI treatment completely rescued myogenic fusion defects and partially restored other myotube parameters in response to alcohol. We conclude that alcohol inhibits C2C12 myoblast differentiation and the inhibition of myogenic fusion is mediated by Notch pathway activation.

Chronic Alcohol Dysregulates Skeletal Muscle Myogenic Gene Expression after Hind Limb Immobilization in Female Rats

“…These alcohol-mediated changes in myogenic gene expression are consistent with our recent work in myoblasts, showing that in vivo chronic binge alcohol administration decreased MyoD and Mef2C expression in isolated myoblasts after seven days of differentiation in quadriceps muscles from SIV-infected rhesus macaques [23,24,39]. Furthermore, 100 mM alcohol in vitro decreased MyoD mRNA expression during 1-3 days of differentiation in C2C12 cells [40]. In vitro findings also demonstrate that alcohol suppresses myoblast fusion under differentiation conditions [23,24,39,40].…”

“…Furthermore, 100 mM alcohol in vitro decreased MyoD mRNA expression during 1-3 days of differentiation in C2C12 cells [40]. In vitro findings also demonstrate that alcohol suppresses myoblast fusion under differentiation conditions [23,24,39,40]. In mice, MyoD mRNA was significantly increased after seven days of immobilization and went back to control levels by seven days of post-immobilization recovery [41].…”

Effects of Low-level Laser Therapy on Muscle Repair in Rats with Chronic Alcohol Intake

“…The results of the present work showed that alcohol intake induced a decrease in MyoD and myogenin expression at 3 and 7 days after cryolesioning. According to Arya et al 23 , the loss of muscle mass in alcoholic myopathy may reflect inhibition of myogenic cell differentiation into myotubes. The authors demonstrated that ethanol administration during differentiation of the skeletal muscle reduced MyoD and myogenin expression.…”

Roles of Notch1 Signaling in Regulating Satellite Cell Fates Choices and Postnatal Skeletal Myogenesis

“…Hypoxia treatment or deletion of Lkb1 increased levels of Pax7 through activation of Notch1 in myoblasts (Liu et al, ; Shan et al, ). Ethanol administration during C2C12 differentiation decreases MyoD and MyoG expression and inhibits C2C12 myoblast differentiation through activating Notch signaling pathway (Arya et al, ). Sex hormones activate Notch signaling in cycling juvenile satellite cells by inducing Mind bomb 1 expression in myofibers at puberty (Kim et al, ).…”

“…Notch1 activation promotes Pax7‐null satellite cells undergo brown adipogenesis in injured skeletal muscle (Pasut et al, ). Several genes and factors, including hypoxia treatment (Liu et al, ), Lkb1 (Shan et al, ), ethanol administration (Arya et al, ), sex hormones (Kim et al, ), TNFα (Acharyya et al, ), Stra13 deficient (Acharyya et al, ), FOXO3 (Gopinath et al, ), and Fas‐associated death domain (FADD‐D) (Zhang et al, ), have been identified to regulate muscle satellite cells fates and myogenesis through regulating Notch1 signaling pathway.…”