Alcoholexposure Increases Histone H3Acetylation Levels To Overexpress Cardiac Transcription Factors In H9c2Cells Viabmp Signaling Pathway

Objective:Dorsomorphin(DM),the specific inhibitor of BMPs signaling pathwaywas used to research the role of it in alcohol-induced histonehyperacetylation,to verify the hypothesis that BMPs are involved in thealcohol-induced histone hyperacetylation in H9c2cells.Methods:（1）H9c2cells were treated by different concentrations (0mM,10mM,50mM,100mM,200mM,500mM and1000mM) of alcohol, MTTassay was used to determine the cells viabilities after36h.（2） Real-Time qRT-PCR assay was used to detect mRNA expressionlevels of subtypes of BMPs.Cells were treated by differentconcentrations of DM (0μM,1μM,2.5μM,5μM,10μM and20μM)to block the influence of alcohol, Real-Time qRT-PCR assay wasused to detect expression levels of GATA4.（3）H9c2cells were treated by alcohol and DM.Real-time PCR was used to measure the expression of Mef2c, GATA4, Nkx2.5, TBX5andSmad4. Westernbolt analysis was used to detected the heartdevelopment-related gene Cx43and histone H3acetylation level inthe whole chromatin. ChIP qPCR assay was used to detect thehistone H3acetylation level in the promoter regions ofMEF2C,GATA4,Nkx2.5and Tbx5.Results:（1）0mM,10mM,50mM and100mM concentrations of alcohol haveno effect for H9c2cell growth(P>0.05),200mM,500mM, and1000mM concentrations of alcohol inhibited growth (P <0.05)（2）Alcohol induced the expressions of BMP2, BMP4, BMP6and BMP7(P<0.05); BMP5and BMP10compared with the control group hadelevated, but the difference was not statistically significant (P>0.05)（3）5μM DM blocked the effects of alcohol increasing the expression ofGATA4（4）At100mM alcohol the expressions of Mef2c, GATA4, NKX2.5andSmad4were increased (P<0.05), meanwhile, the expression of Cx43was increased too(P<0.05). DM at5μM decreased expressions ofMef2c, GATA4, NKX2.5, Smad4and Cx43to the level of controlgroup (P>0.05). Alcohol at100mM and DM at5μM had no effecton the expression of TBX5. （5）100mM Alcohol increased total histone H3acetylation (P<0.05), thatdecreased after adding5μM DM (P>0.05).DM increased totalhistone H3acetylation independently.（6）Alcohol at100mM induced histone H3acetylation levels in promoterregion of Mef2c, GATA4and Nkx2.5(P<0.05).Adding5μM DM,Mef2c and GATA4promoter region acetylation levels falled tocontrol group (P>0.05), Nkx2.5acetylation levels in promoter regionhad down, but did not falled back to control group (P<0.05). Alcoholat100mM and DM at5μM had no effect on TBX5promoter regionacetylation levels(P>0.05).Conclusions（1）Alcohol at100mM increases the expression of BMPs.（2）Alcohol via BMPs signaling pathway increased histone H3acetylation in the promoter region role in promoting of MEF2C,GATA4and Nkx2.5that may one of the mechanisms of alcoholincreases the MEF2C, GATA4and Nkx2.5expression.