ECSIT Participates In Pressure Overload-induced Cardiac Hypertrophy Via Modulating Activation Of NF-?B And MAPK

BackgroundHeart failure,a leading cause of morbidity and mortality in industrialized countries worldwide,frequently is a unfavorable outcome of pathological heart hypertrophy.In order to have a better understanding of heart failure and find out the possible therapies,it is very important to study the mechaisms of pathological heart hypertrophy.Our previous research has found out that TLR4/IL-1R-mediated MyD88-dependent signaling pathway predominately could activate NF-?B and contribute to pressure overload-induced cardiac hypertrophy.Nevertheless,the underlying molecular mechanism remains unclear.Recent studies claims that ECSIT could particapte in TLR4/IL-1R-mediated activation of NF-?B via binding with TRAF6.ECSIT mainly expresses in heart and skeletal muscle.However,it is not clear whether and if so how ECSIT might contribute to cardiac diseases.Therefore,in the present study,we investigate the role of ECSIT in pressure overload-induced cardiac hypertrophy.Objective1.To identify the role of ECSIT in pressure overload-induced cardiac hypertrophy.2.To identify the role of ECSIT in Ang ?-induced cardiac myocyte.3.To clarify the molecular mechanism of ECSIT in pressure overload-induced cardiac hypertrophy.Methods1.Animal modelsTAC is used to induce cardiac hypertrophy.To illuminate whether ECSIT participate in pressure overload-induced cardiac hypertrophy,the animals were divided into 2 groups:TAC group and the sham control group.To illuminate whether knock down of ECSIT expression could affect pressure overload-induced cardiac hypertrophy,we developed AAV9 of shECSIT to knock down the expression of ECSIT.Adv-shECSIT or Adv-shScr was transfered into mice via caudal vein injection.4 weeks later,TAC or sham operations were performed.The animals were divided into 4 groups:sham,TAC,TAC+shScr and TAC+shEC.2.Cell culturingNeonatal rat ventricular myocytes were isolated from 1 to 3-day-old Sprague-Dawley rats.Ang ? stimulation were given to cardiomyocytes.To illuminate whether ECSIT participate in Angll-induced cardiac myocyte,the cells were divided into 2 groups:the control group and Ang ? stimulation group.To illuminate whether knock down of ECSIT expression could affect cardiomyocyte hypertrophy induced by Ang ?,we constructed siRNA of ECSIT to knock down the expression of ECSIT.siEC or siNC was transfected into cardiomyocytes.72 h later,Ang ? stimulation were given to cardiomyocytes.The cells were divided into 4 groups:control,Ang ?,Ang ?+siNC and Ang ?+siEC.3.methodsUltrasonic Doppler was used to assess the mouse models.Echocardiogram,HW/BW,LVW/TL and WGA staining was used to assess the cardiac construction and function of mice.Western Blot and qRT-PCR were used to assess the protein and mRNA levels of ECSIT.qRT-PCR was used to assess the expression of hypertrophic marker genes ANP,BNP and ?-MHC.?-actinin staining was used to assess the cross section area of cardiomyocyte.Co-immunoprecipitation was used to assess the binding of ECSIT with TRAF6 and the level of polyubiquitinated ECSIT.EMSA was used to assess the activation of NF-?B.Western Blot was used to assess the phosphorylation of p38,ERK1/2 and JNK.Results1.ECSIT participates in pressure overload-induced cardiac hypertrophy2 weeks after TAC,echocardiogram showed that IVSd,LVIDd and LVPWd were increased significantly compared with the age-matched sham control group;while EF%and FS%were decreased apparently.HW/BW and LVW/TL also multiplied in TAC-treated mice.The mRNA levels of ANP,BNP and ?-MHC were remarkably increased after TAC.WGA staining revealed TAC significantly increased cross section area of cardiomyocyte,indicating TAC induced cardiac hypertrophy.The protein and mRNA levels of ECSIT were increased after TAC,showing that ECSIT participated in pressure overload-induced cardiac hypertrophy.2.ECSIT participates in cardiomyocyte hypertrophy induced by Angll24 h after Ang? stimulation,the mRNA levels of ANP,BNP and ?-MHC were remarkably increased compared with the control group,showing Ang? induced cardiac myocyte hypertrophy.Also,the protein and mRNA levels of ECSIT were increased notably after Ang? stimulation,indicating that ECSIT participated in cardiomyocyte hypertrophy induced by Ang?.3.Inhibition of ECSIT attenuates pressure overload-induced cardiac hypertrophyAAV9 of shECSIT was transfered into mice via caudal vein injection to knock down the expression of ECSIT in heart.4 weeks later,mice were given TAC operations.2 weeks after TAC,echocardiogram showed that IVSd,LVIDd and LVPWd were decreased significantly compared with TAC group;while EF%and FS%were increased apparently.The increase of HW/BW and LVW/TL induced by TAC were also ameliorated in TAC+shEC group.The data suggest that silence of ECSIT attenuated pressure overload-induced cardiac hypertrophy.4.Inhibition of ECSIT attenuates cardiomyocyte hypertrophy induced by AngllNRVMs were transfected with siRNA of ECSIT to knock down the expression of ECSIT.48 or 72 h later,Ang? stimulation was applied to cardiomyocytes.Western Blot and qRT-PCR showed that the increase of protein and mRNA levels of ECSIT induced by Ang? were significantly reversed after transfection of siECSIT.silence of ECSIT suppressed the increase of the markers of myocyte hpertrophy(ANP,BNP and ?-MHC)induced by Ang?.?-actinin staining also showed the cross section area of cardiomyocyte were decreased in Ang?+siEC group compared with the Ang? group,indicating inhibition of ECSIT attenuated cardiomyocyte hypertrophy induced by Ang?.To further study the mechanism of how ECSIT modulate cardiomyocyte hypertrophy,we investigated the binding of ECSIT with TRAF6 and the level of polyubiquitinated ECSIT.Angll increased the conjugation of TRAF6 and ECSIT,as well as the level of polyubiquitinated ECSIT,while siEC reversed the change.Ang?also increased the activation of NF-?B compared with the control group,while silence the expression of ECSIT reversed it.Moreover,increase of p38,ERK1/2 and JNK phosphorylation induced by Ang? were abolished after inhibiting of ECSIT.ConclusionTaken together,we demonstrated that after Ang? stimulation,ECSIT induced activation of NF-?B and MAPK via increase of the levels of binding with TRAF6 and polyubiquitinated ECSIT,resulting in cardiac hypertrophy.