Effect Of BMP2on Histone Acetylation And The Underlying Mechanisms In H9c2Cells

ObjectiveBuilding the myocardial cell model that overexpressing BMP2to studythe effects of BMP2on the expression of cardiac core transcription factorsGATA4, MEF2C and Tbx5, the histone acetylation level both in the wholechromatin and in the promoter regions of GATA4, MEF2C and Tbx5, andthe expression of histone deacetylase (HAT) p300and GCN5. In the presentstudy, we will test the hypothesis that BMP2acts as one of the upstreamregulators of histone acetylation in cardiomyocytes.Methods（1）Rat H9c2myocardial cells(H9c2cells) were transfected withAdBMP2or AdGFP adenovirus that amplified in HEK293cells, theexpression of green fluorescent protein(GFP) were detected under aninverted fluorescence microscope, and the transfection efficiency weredetected by flow cytometry24h after transfection. （2）24h,48h and72h after transfected with AdBMP2, H9c2cells werecollected to extract mRNA, Real-Time qRT-PCR were used to detectthe mRNA expression levels of BMP2, MEF2C, GATA4, Tbx5, p300and GCN5.（3）48h after transfected with AdBMP2, H9c2cells were collected,colorimetric assay were used to detect HAT activity, Western-blottingwere used to detect the histone H3acetylation levels in the wholechromatin. ChIP-Real-Time qPCR were used to detect the histone H3acetylation level in the promoter regions of GATA4, MEF2C andTbx5.Results（1）24h after AdBMP2transfection, high expression level of GFP werevisible in the cells under the inverted fluorescence microscope. Theresults of flow cytometry showed that the transfection efficiency wasup to90%.（2）24h,48h,72h after AdBMP2transfection, the mRNA expression levelof BMP2and the cardiac core transcription factor MEF2C andGATA4were significantly increased compared with the controlcells(P<0.05) and reached the peak at48h after transfection inH9c2cells. However the expression of Tbx5was not affected in thesame cells after AdBMP2transfection.（3）48h after AdBMP2transfection, the mRNA expression level of HAT p300was upregulated compared with the control cells(P<0.05), whilethe expression of another HAT GCN5was not changed.（4）48h after AdBMP2transfection, HAT activity and the histone H3acetylation level in the whole chromatin were significantly increasedcompared with the control H9c2cells(P<0.05). In addition, thehistone H3acetylation level in the promoter regions of GATA4andMEF2C were also increased compared with the control cells(P<0.05).However the histone H3acetylation level in the promoter region ofTbx5was not changed.Conclusions（1）BMP2upregulates the expression of cardiac core transcription factorsMEF2C and GATA4, but not Tbx5.（2）BMP2-induced histone H3hyperacetylation in H9c2cells revealedthat BMP2acts as one of the upstream regulators of histoneacetylation in cardiomyocytes.（3）BMP2-induced histone H3hyperacetylation in H9c2cells maybeassociated with the HAT p300.（4）Histone H3acetylation may be one of the molecular mechanisms bywhich BMP2upregulates the expression of GATA4and MEF2C.However, the unaffected expression of Tbx5maybe associated withthe unchanged histone H3acetylation in the promoter region of Tbx5in H9c2cells overexpressing BMP2. ObjectiveHAT p300inhibitor curcumin was used to research the effect of p300on the BMP2-induced histone hyperacetylation and the BMP2-inducedupregulation of cardiac core transcription factors GATA4and MEF2C, totest the hypothesis that p300is involved in the regulation of cardiac coretranscription factors and the histone H3acetylation by BMP2in H9c2cells.Methods（1）The AdBMP2transfected H9c2cells were treated with differentconcentrations(10μM,20μM,30μM,40μM) of the HAT p300inhibitorcurcumin for various treatment times (6h,12h,24h,48h), colorimetricassay were used to detect HAT activity and to find an optimumconcentration and time duration.（2）After AdBMP2and/or curcumin treatment, H9c2cells were collected.Real-Time qRT-PCR were used to detect the expression levels of cardiac core transcription factor GATA4, MEF2C and Tbx5,Western-blotting were used to detect histone H3acetylation level inthe whole chromatin, and ChIP-Real-Time qPCR were used to detectthe histone H3acetylation levels in the promoter regions of GATA4,MEF2C and Tbx5.Results（1）24h after treated with different concentrations (10μM,20μM,30μM,40μM) of curcumin, HAT activity was decreased(P<0.05) and reachedits lowest point at40μM in AdBMP2transfected H9c2cells. Aftertreated with40μM curcumin for various treatment times (6h,12h,24h,48h), HAT activity was decreased(P<0.05) and reached its lowestpoint at12h after treatment in AdBMP2transfected H9c2cells.（2）The mRNA expression levels of cardiac core transcription factorsGATA4and MEF2C and HAT p300were decreased(P<0.05) inAdBMP2transfected and curcumin treated cells compared with theAdBMP2transfection group, while the mRNA expression levels ofTbx5and GCN5were not changed.（3）The histone H3acetylation level in the whole chromatin and in thepromoter regions of GATA4and MEF2C were also decreased(P<0.05)in AdBMP2transfected and curcumin treated cells compared with theAdBMP2transfection group, while the histone H3acetylation level inthe promoter region of Tbx5was not changed. Conclusions（1）HAT p300inhibitor curcumin is able to inhibit the expression of p300but not GCN5.（2）BMP2-induced histone hyperacetylation and the upregulation ofcardiac core transcription factors GATA4and MEF2C are antagonizedby curcumin, suggesting that p300is involved in the regulation ofcardiac core transcription factors and the histone H3acetylation byBMP2.（3）The expression and the histone H3acetylation in the promoter regionof Tbx5maybe not regulated by p300in H9c2cells. ObjectiveBMPs signal inhibitor dorsomorphin (DM) was used to research theeffect of BMPs signal on the oxidative stress-induced histonehyperacetylation, to test the hypothesis that BMPs are involved in theoxidative stress-induced histone hyperacetylation in H9c2cells Methods（1）H9c2cells were treated with different concentrations of H2O2(50μM,100μM,150μM,200μM,250μM,300μM,350μM,400μM) for24h,MTT assay were used to detect cell survival rate, and to select theappropriate concentration of H2O2to build the oxidative injury modelof H9c2cells.（2）H9c2cell were treated with5μM BMPs signal inhibitor dorsomorphin(DM) and/or the appropriate concentration of H2O2. Real-TimeqRT-PCR were used to detect the mRNA expression levels of BMP2and cardiac core transcription factors GATA4, MEF2C and Tbx5.Western-blotting assay were used to detect histone H3acetylationlevel in the whole chromatin.Results（1）The H9c2cell survival rates were not changed after50,100and150μM H2O2treatment, and after treatment with200,250,300,350,400and450μM H2O2, the cell survival rate were reduced by10.5%,16.9%,21.9%,32.4%,47.0%and58.6%respectively.（2）In400μM H2O2treated H9c2cells, The histone H3acetylation levelin the whole chromatin and the mRNA expression levels of BMP2,GATA4, MEF2C and Tbx5were significantly increased(P<0.05)compared with blank control cells.（3） The histone H3acetylation level in the whole chromatin and themRNA expression levels of GATA4and Tbx5were decreased(P<0.05) in400μM H2O2and DM treated H9c2cells compared with400μMH2O2treated group. However, the mRNA expression level of MEF2Cwere increased(P<0.05) in DM and400μM H2O2treated H9c2cellscompared with400μM H2O2treated group.Conclusions（1）The oxidative injury model of H9c2cells were successfullyconstructed with H2O2.（2）The expression of BMP2, GATA4, MEF2C and Tbx5are upregulatedby400μM H2O2-induced oxidative stress.（3）400μM H2O2-induced histone H3acetylation and the expression ofGATA4and Tbx5is partially antagonized by BMPs signal inhibitorDM in H9c2cells, suggesting that BMPs(BMP2and other BMPssubtypes) is involved in oxidative stress-induced histone acetylationand the expression of GATA4and Tbx5, indicating that in thepathological state(oxidative stress), BMPs may also act as theupstream signal pathways of histone acetylation in cardiomyocytes,and also indicating that certain BMPs subtypes that upregulate theexpression of Tbx5maybe induced under oxidative stress.（4）The expression of MEF2C is enhanced by BMPs signal inhibitor DMin H9c2cells, suggesting that the expression of MEF2C maybeinhibited by the combined effects of BMPs subtypes, and alsoindicating that other signals maybe in involved in oxidative stress-induced expression of MEF2C.