Role Of JNK/MAPK Signaling-dependent Regulation Of Histone Acetylation On The Attenuation Of Anacardic Acid For Cardiomyocyte Hypertrophy Induced By Phenylephrine

Part Ⅰ Anacardic Acid Attenuates Cardiomyocyte Hypertrophy Induced by Phenylephrine through Inhibiting Histone H3K9 ac acetylationObjective:To investigate the regulation of histone acetylation modification on anacardic acid(AA)attenuates cardiomyocyte hypertrophy induced by phenylephrine(PE)in mouse.Methods:Primary cultures of myocardial cells were from new born Kunming mouse(< 3d).According to random number table method,experiments were designed in four groups as following: Blank group,Vehicle group(PE,100 μmol/L + DMSO),PE group(PE,100 μmol/L),AA group(PE,100 μmol/L + AA 50 μmol/L).Mouse myocardial cells were collected after intervention 48 hours for analysis.(1)Colorimetry was used to detect HATs activity.(2)The protein levels expression of brain natriuretic peptide(BNP),atrial natriuretic peptide(ANP),β-myosin heavy chain(β-MHC),histone H3K9 ac,P300/CBP-associated factor(PCAF),E1A-binding protein 300kD(P300),T-JNK,p-JNK were determined by western blot.(3)The mRNA expression levels of myocyte enhancer factor-2A(MEF-2A)were tested by Real-Time PCR.(4)Mouse myocardial cells surface area was observed by immunofluorescence staining.Results:(1)Colorimetric assays revealed significantly increased HATs activity in PEgroup compared to that in Blank group(P < 0.05),while HATs activity in AA group was significantly lower than that in PE group(P < 0.05).There was no significant difference between PE group and Vehicle group(P > 0.05).(2)Western blot results showed that the expression levels of p-JNK and histone H3K9 ac in PE group were significantly increased compared with Blank group(all P <0.05),and the expression levels of cardiomyocyte hypertrophy markers ANP,BNP,β-MHC,and PCAF-HAT,P300-HAT in PE group were apparently increased compared with Blank group(all P < 0.05).Compared with PE group,the expression levels of p-JNK and histone H3K9 ac were significantly decreased in AA group(all P< 0.05),while there were not significantly changed between the Vehicle group and the PE group(all P > 0.05).At the same time,the expression levels of cardiomyocyte hypertrophy markers ANP,BNP,β-MHC,and PCAF-HAT,P300-HAT were significantly suppression compared with those of the PE group in the AA group(all P< 0.05),while there were no significant changed between the Vehicle group and the PE group(all P > 0.05).(3)Real-time PCR results showed that compared with the Blank group,the mRNA expression of cardiac nucleus transcription factor MEF-2A was significantly increased in the PE group,while AA could significantly down-regulate the mRNA expression level of the heart nucleus transcription factor MEF-2A in PE-treated myocardial cells(P < 0.05).There was no significantly changed between the Vehicle group and the PE group(P > 0.05).(4)The results of immunofluorescence staining showed that phenylephrine apparently increased mouse myocardial cells surface area(P < 0.05),while AA attenuated cardiomyocyte hypertrophy induced by phenylephrine(P < 0.05).There was no significant difference between the Vehicle group and the PE group(P > 0.05).Conclusions:(1)The acetylation of histone H3K9 ac mediated by PCAF-HAT and P300-HAT is involved cardiomyocyte hypertrophy of mouse caused by PE.(2)HATs inhibitor AA can attenuate the cardiomyocyte hypertrophy induced byPE through inhibiting acetylation of histone H3K9 ac mediated by PCAF-HAT and P300-HAT to regulate the expression level of cardiomyocyte hypertrophy related genes.Part Ⅱ Role of JNK/MAPK Signaling-Dependent Regulation of Histone Acetylation on the Attenuation of Anacardic Acid for Cardiomyocyte Hypertrophy Induced by PhenylephrineObjective:To investigate the effects of C-Jun N-terminal kinase mitogen-activated protein kinase(JNK/MAPK)on histone acetylases(HATs)inhibitor anacardic acid(AA)attenuates cardiomyocyte hypertrophy induced by phenylephrine(PE).Methods:Primary cultures of myocardial cells were from new born kunming mouse(< 3 d).According to random number table method,experiments were designed in four groups as following: Blank group,Vehicle group(PE,100 μmol/L + DMSO),PE group(PE,100 μmol/L),AA group(PE,100 μmol/L + AA 50 μmol/L),AA + SP group(PE 100μmol/L + AA 50 μmol/L + SP600125 20 μmol/L),SP group(PE 100 μmol/L +SP600125 20 μmol/L).Mouse myocardial cells were collected after intervention 48 hours for analysis.(1)The protein expression levels of brain natriuretic peptide(BNP),atrial natriuretic peptide(ANP),β-myosin heavy chain(β-MHC),histone H3K9 ac,P300/CBP-associated factor(PCAF),E1A-binding protein 300 k D(P300),T-JNK,p-JNK was determined by western blot.(2)The interaction between p-JNK and P300,PCAF,histone H3K9 ac protein was verified by Co-immunoprecipitation(Co-IP).(3)The m RNA expression levels of myocyte enhancer factor-2A(MEF-2A)were tested by Real-Time PCR.(4)The interaction of MEF-2A with P300-HAT,PCAF-HAT,and cardiomyocyte hypertrophy markers ANP,BNP,β-MHC was verified by Chromatin immunoprecipitation assay(Ch IP).(5)The expression levels of histone H3K9 ac,P300-HAT,PCAF-HAT,and mouse myocardial cells surface area were observed by immunofluorescence staining.(6)Flou-3/AM fluorescent staining was used to detect intracellular [Ca2+]i in myocardial cells.Results:(1)The results of western blot showed that the expression levels of p-JNK and histone H3K9 ac in PE group were significantly increased compared with Blank group(all P < 0.05),and the expression levels of cardiomyocyte hypertrophy markers ANP,BNP,β-MHC,and PCAF-HAT,P300-HAT in PE group were apparently increased compared with Blank group(all P < 0.05).Compared with PE group,the expression levels of p-JNK and histone H3K9 ac were significantly decreased in AA group and SP group(all P < 0.05),while there were not significantly changed between the Vehicle group and the PE group(all P > 0.05).At the same time,the expression levels of cardiomyocyte hypertrophy markers ANP,BNP,β-MHC,and PCAF-HAT,P300-HAT were significantly suppression compared with those of the PE group in the AA group SP group(all P < 0.05),while there were no significant changed between the Vehicle group and the PE group(all P > 0.05).(2)The results of Co-IP showed that p-JNK directly interacts with PCAF-HAT,P300-HAT and alters histone H3K9 acetylation in primary mouse cardiomyocytes.(3)Real-time PCR results showed that compared with the Blank group,the m RNA expression of cardiac nucleus transcription factor MEF-2A was significantly increased in the PE group,while AA and JNK inhibitor SP600125 could significantly down-regulate the m RNA expression level of the heart nucleus transcription factor MEF-2A in PE-treated myocardial cells(P < 0.05).There was no significantly changed between the Vehicle group and the PE group(P > 0.05).(4)The results of Ch IP showed that the P300-HAT and PCAF-HAT could bind to the in the promoter region of cardiac nucleus transcription factor MEF-2A,while cardiac nucleus transcription factor MEF-2A could bind to the promoter regions of cardiomyocyte hypertrophy markers ANP,BNP,and β-MHC genes.(5)The results of immunofluorescence staining showed that the expression levels of histone H3K9 acetylation and P300-HAT,PCAF-HAT in PE group were significantly increased compared with Blank group(all P < 0.05).Compared with PE group,the expression levels of histone H3K9 acetylation and P300-HAT,PCAF-HAT were significantly decreased in AA group and SP group(all P < 0.05).At the same time,the results of immunofluorescence staining showed that phenylephrine apparently increased mouse myocardial cells surface area(P < 0.05),while AA and JNK inhibitor SP600125 attenuated cardiomyocyte hypertrophy induced by phenylephrine(all P < 0.05).There was no significant difference between the Vehicle group and the PE group(P > 0.05).(6)The results of Flou-3/AM staining showed that the intracellular Ca2+ was clearly increased in PE group compared to that in Blank group(P < 0.05),whereas both HAT inhibition by AA and JNK inhibition by SP600125 reduced this effect(all P< 0.05).There was no significantly changed between the Vehicle group and the PE group(P > 0.05).Conclusions:(1)AA can attenuate cardiomyocyte hypertrophy induced by PE in mouse.(2)JNK signaling-dependent regulation of histone acetylation was involved in AA can attenuate cardiomyocyte hypertrophy induced by PE in mouse.