The Role And Mechanism Of GCN5 In Pressure Overload-induced Cardiac Hypertrophy

1 Correlation between GCN5 and pressure overload-induced cardiac hypertrophy Objective: Pressure overload-induced cardiac hypertrophy is a very common disease,which may progress to heart failure(HF).However,the pathogenesis of pressure overload-induced cardiac hypertrophy is still unclear.There are no effective drugs available for treatment in pressure overload-induced cardiac hypertrophy.As a histone lysine acetyltransferase,GCN5 is involved in a variety of physiological and pathological processes,while the role of GCN5 in pressure overload-induced cardiac hypertrophy remains unclear.This part aims to explore the expression of GCN5 in the model of pressure overload-induced cardiac hypertrophy.Methods:(1)Transverse aortic constriction(TAC)surgery was performed in C57BL/6J mice to establish pressure overload-induced cardiac hypertrophy model,cardiac function was detected by cardiac ultrasound,cardiomyocyte size and myocardial fibrosis were detected by HE,MASSON and WGA staining.Western Blot and RT-PCR were used to examine HF related indicators(ANP 、 β-MHC).(2)Neonatal rats cardiomyocytes(NRCMs)was stimulated with PE,immunofluorescence staining was performed to indicate the cell surface,Western Blot and RT-PCR were used to examine HF related indicators(ANP、β-MHC).(3)The expressions of GCN5 m RNA and protein levels were detected in vivo and in vitro via Western Blot and RT-PCR.Results:(1)Compared with SHAM group,the cardiac function at 2 and 4 weeks after TAC surgery was gradually decreased,the cardiomyocytes size and cardiac fibrosis was gradually increased in a time-dependent manner.The m RNA and protein levels of GCN5 were increased in the myocardium of pressure overload-induced cardiac hypertrophy.(2)In vitro,compared with PBS group,surface of NRCMs stimulated with PE was increased.The m RNA and protein levels of GCN5 in NRCMs were increased under PE stimulation.Conclusions: The m RNA and protein expression of GCN5 was increased in pressure overload-induced cardiac hypertrophy model in mice after TAC surgical intervention and NRCMs after PE stimulation in vivo and in vitro.2 Role of GCN5 in pressure overload-induced cardiac hypertrophyObjective: The experiments of Part Ⅰ have demonstrated that GCN5 was increased in the model of pressure overload-induced cardiac hypertrophy.Next,we will explore the role of GCN5 in the development of pressure overload-induced cardiac hypertrophy.Methods:(1)In vivo,Adeno-associated virus 9(AAV9)was injected into C57BL/6J mice by the tail vein to overexpress GCN5.GCN5 specific inhibitor MB3 was given by intraperitoneal injection to inhibit the activity of GCN5.After 4 weeks of TAC surgery,Body weight(BW),Heart weight(HW)and Lung weight(LW)were monitored,cardiac function was detected by ultrasound,cardiomyocyte size and the peritubular and interstitial fibrosis were detected by HE,WGA and MASSON staining,and the indexes of heart failure(ANP,BNP,β-MHC,α-MHC)and myocardial fibrosis indexes(Collagen-1、Collagen-3、TGF-β)were examined by Western Blot and RT-PCR.In addition,indexes related to apoptosis(Bcl-2,Bax,Cleaved-caspase3,Caspase3)was detected by Western blot.(2)In vitro,adenovirus was selected to overexpress and knockdown GCN5 in NRCMs.Immunofluorescence staining of α-actinin was used to reflect the surface area of NRCMs,RT-PCR and Western Blot was used to detect the indexes of HF(ANP、β-MHC)and apoptosis(Bax、Bcl2、Cleavedcaspase3、Caspase3).TUNEL staining was used to examine the effect of GCN5 on cardiomyocyte apoptosis.Results:(1)Compared with the AAV-Vector+TAC group,the cardiac function of AAVGCN5+TAC group was decreased,and HW/BW,LW/BW was significantly increased.The cross-sectional area of cardiomyocytes was larger and the degree of myocardial fibrosis was more serious in AAV-GCN5+TAC group.Compared with DMSO+TAC group,the cardiac function of MB3+TAC was improved,the degree of cardiac hypertrophy and fibrosis was significantly reduced.(2)In vitro,under PE stimulation,overexpression of GCN5 aggravated cardiomyocyte hypertrophy and cardiomyocyte apoptosis.In contrast,knockdown of GCN5 reduced cardiomyocyte hypertrophy and cardiomyocyte apoptosis.Conclusions:(1)GCN5 overexpreesion promotes pressure overload-induced cardiac hypertrophy.(2)Knockdown or inhibition of GCN5 alleviates pressure overload-induced cardiac hypertrophy.3 Mechanism of GCN5 in regulating pressure overload-induced cardiac hypertrophyObjective: After clarifying the role of GCN5 in the development of pressure overloadinduced cardiac hypertrophy,we aim to further explore the mechanism of GCN5 in promoting pressure overload-induced cardiac hypertrophy.Methods:(1)Western Blot was used to detect the phosphorylation levels of MAPK signal pathway(p-JNK、JNK、p-ERK、ERK、p-p38、p38)and transforming growth factor β active kinase 1(TAK1)in the myocardium of GCN5-overexpressed mice and MB3-treated mice at 4 weeks after TAC operation.Besides,Western Blot was also used to examine the phosphorylation levels MAPK signal pathway and TAK1 in NRCMs with GCN5 overexpressed and knockdown followed by PE stimulation.(2)To determine the relationship between GCN5 and TAK1 and the key role of TAK1 in GCN5 on myocardial hypertrophy,co-immunoprecipitation(Co-IP)was used to detect the binding of TAK1 between GCN5,TAK1 inhibitor 5Z-7-oxozeaenol and NG25 were used to verify the role of TAK1 in GCN5 promoting pressure overload-induced cardiac hypertrophy.(3)In the disease model,GCN5 inhibition was used to detect GCN5 expression level the interaction between TAK1 and GCN5,and the acetylation level of TAK1.(4)According to lots of literatures,TAK1-binding protein 1(TAB1)and TAK1-binding protein 2(TAB2)was selected for determining TAK1 phosphorylation activation.Further,the effect of MB3 on the binding between TAK1 and TAB1 or TAB2 was detected by Co-IP experiment.Results:(1)Compared with AAV-Vector+SHAM group,the phosphorylation levels of MAPK signaling pathway and TAK1 were not changed in AAV-GCN5+SHAM group.However,compared with AAV-Vector+TAC group,AAV-GCN5+TAC group had higher phosphorylation levels of TAK1,JNK and p38.Compared with DMSO+TAC group,the phosphorylation levels of TAK1,JNK and p38 were decreased in MB3+TAC group,while the level of p-ERK was not changed.These results in vitro were consistent both those in vivo.(2)TAK1 inhibitor blocked the effect of GCN5 on pressure overload-induced cardiac hypertrophy,which was confirmed by HE,MASSON and WGA staining.The results in vitro were consistent both those in vivo.(3)Co-IP experiment confirmed that GCN5 could bind with TAK1 in cardiomyocytes.In the presence of pathological stimulation,GCN5 inhibition did not influence the interaction between GCN5 and TAK1,and the acetylation level of TAK1.(4)Under PE stiulation,MB3 reduced the binding between TAB1 and TAK1,however,the binding of TAB2 and TAK1 was not unaffected by MB3.Conclusions: GCN5 aggravated the development of pressure overload-induced cardiac hypertrophy by activiting TAK1-JNK/p38 pathway.