Epigenomic mechanisms of alcohol-induced impaired differentiation of skeletal muscle stem cells; role of Class IIA histone deacetylases

Abstract

Loss of functional metabolic muscle mass remains a strong and consistent predictor of mortality among people living with human immunodeficiency virus (PLWH). PLWH have a higher incidence of alcohol use disorder (AUD), and myopathy is a significant clinical comorbidity due to AUD. One mechanism of skeletal muscle (SKM) mass maintenance and repair is by differentiation and fusion of satellite cells (SCs) to existing myofibers. Previous studies demonstrated that chronic binge alcohol (CBA) administration decreases SC differentiation potential, myogenic gene expression, and miR-206 expression in simian immunodeficiency virus (SIV)-infected male rhesus macaques and that miR-206 targets the Class IIA histone deacetylase, HDAC4. The aim of this study was to determine whether alcohol-induced increases in Class IIA HDACs mediate the observed decrease in differentiation potential of SCs. Data show that CBA dysregulated HDAC gene expression in SKM and myoblasts of SIV-infected macaques. CBA and antiretroviral therapy increased HDAC activity in SKM and this was positively correlated with HDAC4 gene expression. In vitro ethanol (ETOH) treatment increased HDAC expression during differentiation and decreased differentiation potential of myoblasts. HDAC expression was negatively correlated with fusion index and myotube formation, indicators of differentiation potential. Treatment with a Class II HDAC inhibitor, TMP195, restored differentiation in ETOH-treated myoblasts. MEF2C expression at day 3 of differentiation was positively correlated with fusion index and myotube formation. These findings suggest that an alcohol-mediated increase in Class IIA HDAC expression contributes to decreased myoblast differentiation by downregulating MEF2C, a transcription factor critical for myogenesis.

Keywords: chronic alcohol; histone deacetylases; stem cell differentiation.

Fig. 1.

Relative gene expression of Class IIA histone deacetylases, HDAC4, HDAC5, and HDAC7 in skeletal muscle. A: there was a main effect of chronic binge alcohol (CBA) to significantly increase HDAC4 expression. Control (white bars); sucrose (VEH)/simian immunodeficiency virus (SIV)/antiretroviral therapy (ART)− (light gray bars), VEH/SIV/ART+ (light gray diagonal hatched bars), chronic binge alcohol (CBA)/ SIV/ ART− (dark gray bars), and CBA/SIV/ART+ (dark gray diagonal hatched bars). B: there was no significant effect of CBA, SIV, and ART on HDAC5 expression in skeletal muscle. C: there was no significant effect of CBA, SIV, and ART on HDAC7 expression in skeletal muscle. 2-way ANOVA. Values are means ± SE.

Fig. 2.

Histone deacetylase (HDAC) activity in skeletal muscle of SIV-infected macaques. A: there was a main effect of chronic binge alcohol (CBA) and antiretroviral therapy (ART) to significantly increase total nuclear HDAC activity in skeletal muscle. Control (white bars); sucrose (VEH)/simian immunodeficiency virus (SIV)/ART− (light gray bars), VEH/SIV/ART+ (light gray diagonally hatched bars), CBA/ SIV/ ART− (dark gray bars) and CBA/SIV/ART+ (dark gray diagonally hatched bars). B: there was a significant positive correlation of HDAC4 expression with total nuclear HDAC activity. RFU, relative fluorescence units. 2-way ANOVA and Pearson correlation. Values are means ± SE.

Fig. 3.

Relative gene expression of Class IIA histone deacetylases, HDAC4, HDAC 5, and HDAC7 in myoblasts. A: there was significant increase in HDAC4 expression in myoblasts of chronic binge alcohol (CBA)/simian immunodeficiency virus (SIV)/antiretroviral (ART)+ (dark gray hatched bars) macaques compared with VEH/SIV/ART+ (light gray hatched bars) and CONTROL (white bars) myoblasts during proliferation. B: there was a significant increase in HDAC5 expression in myoblasts of VEH/SIV/ART+ compared with CONTROL macaques. C: there were no significant differences in HDAC7 expression in myoblasts of CONTROL, VEH/SIV/ART+, and CBA/SIV/ART+ macaques. *P < 0.05 (1-way ANOVA). Values are means ± SE.

Fig. 4.

Class IIA HDAC expression in ethanol (ETOH)-treated myoblasts. A: there was no significant change in HDAC4 expression in ETOH-treated (dark gray bar) compared with CTRL (white bars) myoblasts at day 5 of differentiation. B: there was a significant increase in HDAC5 expression in ETOH-treated compared with CTRL myoblasts. C: there was a significant increase in HDAC7 expression in ETOH-treated compared with CTRL myoblasts. *P < 0.05 (t test). Values are means ± SE.

Fig. 5.

In vitro differentiation potential of ethanol (ETOH) and Class IIA histone deacetylase inhibitor, TMP195-treated myoblasts. A: representative images of Jenner-Giemsa stained myoblast cultures from CTRL/VEH, CTRL/TMP195, ETOH/VEH, and ETOH/TMP195 at day 5 of differentiation. B: there was a main effect of ETOH to decrease and TMP195 to increase fusion index. CTRL/VEH (white bars), CTRL/TMP195 (white horizontal hatched bars), ETOH/VEH (dark gray bars), and ETOH/TMP195 (dark gray horizontal hatched bars). C: there was a main effect of ETOH to decrease and a main effect of TMP195 to increase number of myotubes per field at day 5 of differentiation. For statistical purposes, treatment group myoblasts are normalized to their respective controls due to variability between primary macaque cell lines. 2-way ANOVA. Values are means ± SE.

Fig. 6.

Relative gene expression of MEF2C in ethanol (ETOH) and Class IIA histone deacetylase inhibitor, TMP195-treated myoblasts. A: there was a significant decrease in MEF2C expression during proliferation in ETOH-treated (dark gray bars) compared with CTRL (white bars) myoblasts. B: MEF2C expression at day 3 of differentiation in CTRL/VEH (white bars), CTRL/TMP195 (white horizontally hatched bars), ETOH/VEH (dark gray bars), ETOH/TMP195 (dark gray horizontally hatched bars). Main effect of TMP195, P = 0.09, Interaction of ETOH and TMP195, P = 0.08. C: there was a significant decrease in MEF2C expression at day 5 of differentiation in ETOH/VEH and ETOH/TMP195 groups compared with CTRL/VEH and CTRL/TMP195 groups. D, E: there was a significant positive correlation of MEF2C expression at day 3 of differentiation with number of myotubes per field and fusion index in all treatment groups. Values are means ± SE 2-way ANOVA and Pearson correlation.

Fig. 7.

Working model of how alcohol-mediated dysregulation of Class IIA histone deacetylases decreases differentiation potential of satellite cells.