Triad3A Participates In Pressure Overload-induced Cardiac Hypertrophy

BackgroundClinic pathological cardiac hypertrophy is often the early manifestation of heart failure,has been listed as the impact of cardiovascular disease morbidity and mortality increased independent risk factors.clear its development mechanism and find the intervention target to prevent heart failure is of great significance.Pathological cardiac hypertrophy is a critical compensatory phase of pressure or capacity overload response,which includes a variety of signaling pathways and transcription factor regulation.TLRs/IL-1 signaling pathway plays an important role in the regulation of pathological cardiac hypertrophy,while Triad3A acts as an E3 ubiquitin ligase can catalyse ubiquitination-dependent degradation of TLR4 as a substrate,thus negatively regulating TLRs/IL-1 pathway.It is unclear whether Triad3A is involved in the development of cardiovascular disease and whether it has a regulatory role in the development of pressure overload-induced cardiac hypertrophy.This study was to investigate the role of Triad3A in the regulation of this disease and to further explore its mechanism.Objective1.To clarify the regulation of Triad3A in pressure overload-induced cardiac hypertrophy.2.To clarify the regulation of Triad3A in angiotensin Ⅱ-induced cardiomyocyte hypertrophy.3.To elucidate the molecular mechanism of Triad3A in pressure overload-induced cardiac hypertrophy.Methods1.In vivo animal experimentsSurgical myocardium hypertrophy was established by TAC(transverse aortic constriction).Healthy C56BL/6 male mice aged 7-8 weeks were randomly divided into 4 groups:sham,TAC,TAC+advGFP and TAC+advTriad3A.In order to achieve heart overexpressed Triad3A animal model,we inject 5×108 pfu adenovirus from the apical into the left ventricule under the condition that temporarily blocking the left common carotid artery and the pulmonary artery,the blocking time would last for 10s after injection.With a week after sugery,the left and right carotid artery was measured to observe the degree of narrowing of the operation,and the rates range from 7 to 10.2 weeks after sugery take the specimen2.In vitro cell experimentsIn vitro experiments were conducted using AngⅡ as a stimulus to induce cardiomyocyte hypertrophy for phenotypic and mechanism studies.Neonatal rat ventricular myocytes were isolated from 1-2-day-old Sprague-Dawley rats.The protein and mRNA levels of Triad3A were measured at different concentrations and different stimulating times of AngⅡ,and the correlation between AngⅡ-induced cardiomyocyte hypertrophy was determined.In vitro experiments were divided into four groups:con,AngⅡ,AngII+advTriad3A,AngⅡ+advGFP.The expression of AdvTriad3A or advGFP were transfected into cardiomyocyte.48h later,AngⅡ stimulation was administrated to cardiomyocyte for next 24h.To observe:①the effect of Triad3A on AngⅡ-induced cardiomyocyte area enlargement;②Triad3A regulates AngⅡ-induced hypertrophy gene expression;③The molecular mechanism of Triad3A regulating AngⅡ-induced cardiomyocyte hypertrophy.3.Detection indicatorThe thickness of ventricular wall,ventricular septum and the change of cardiac function were measured by echocardiography.The specimens were calculated by HW/BW and LVW/TL.The expression of Triad3A,TLR4,AKT,IκB,P65 and other proteins were detected by immunoblotting.The combination of Triad3A and TLR4 or the combination of TLR4 and the downstream MyD88 protein were detected by immunoprecipitation.NF-κB activity was measured by electrophoretic mobility shift assay(EMSA).Results1.Triad3A is associated participate the development of pressure overload-induced cardiac hypertrophy1.1 Compared with the sham group,the indicators of heart structure,such as ventricular wall thickness and ventricular septal thickness were significantly higher.The ratio of HW/BW and LVW/TL were also high in the TAC group than the sham group.The above indexes suggested the operation is successed.1.2 The expression of Triad3A protein was detected by immunoblotting assay.The results showed that Triad3A protein was significantly lower than that of sham group after TAC operation.The mRNA of Triad3A was detected by real-time quantitative PCR.The results were consistent with the protein trend.Triad3A was associated with stress overload induced cardiac hypertrophy by the above experiments.2.Triad3A expression is associated with angiotensin Ⅱ-induced cardiomyocyte hypertrophy2.1 Cardiomyocytes were cultured in vitro followed by AngⅡ stimulation for 24h to induce cardiomyocyte hypertrophy.The levels of ANP,BNP and β-MHC mRNA were significantly higher than those of the control group(P<0.05)2.2 AngⅡ stimulation can induce the decrease of Triad3A protein and mRNA expression in cardiomyocytes,indicating that Triad3A is associated with angiotensinⅡ-induced cardiomyocyte hypertrophy.3.Overexpression of Triad3A inhibits TAC-induced cardiac hypertrophy3.1 Cardiac local injection of adenovirus and aortic arch constriction 2 weeks after surgery,interventricular septum thickness,ventricular wall thickness,Left ventricular diameter induced by TAC were significantly inhibited in the overexpression of Triad3A group.At the same time,Triad3A can improve the TAC 2 week-induced reduction in cardiac function markers EF%and FS%.3.2 Cardiac local injection of adenovirus and aortic arch constriction 2 weeks after surgery,HW/BW and LVW/TL values induced by TAC were significantly inhibited in the overexpression of Triad3A group,and the cross-sectional area of myocardial cells were also showed the same trend.3.3 Cardiac local injection of adenovirus and aortic arch constriction 2 weeks after surgery,TAC induced the up-regulation of ANP,BNP,β-MHC,which was inhibited when overexpression of Triad3 A.4.Overexpression of Triad3A inhibits angiotensin Ⅱ-induced cardiomyocyte hypertrophy4.1 Transfected cardiomyocytes with advTriad3A or advGFP for 0h,24h and 48h,the transfection efficiency was observed by fluorescence and the protein level of Triad3A was detected increased by immunoblotting.4.2 Adenovirus transfected cardiomyocytes were administered for 48h followed by AngⅡ sitimulation at a concentration of 10-6 μmol/l for another 24h.Compared with advGFP group,advTriad3A group showed lower mRNA level of ANP,BNP,β-MHC(P<0.05).Alpha-actinin staining also showed the same trend.These results suggest that the expression of Triad3A can inhibit angiotensin II-induced cardiomyocyte hypertrophy.5.Molecular mechanism of Triad3A in regulation of pressure overload-induced cardiac hypertrophy5.1 Triad3A has E3 ubiquitin ligase activity,the ubiquitination level of TLR4 was examined.Compared with the control virus group,overexpression of Triad3A significantly increased the level of TLR4 ubiquitination in cardiomyocytes.Overexpression of Triad3A,AngⅡ-induced the interaction of TLR4 and downstream adapter protein MyD88(myeloiddifferentiationfactor88,myeloid differentiation factor)was inhibited5.2 The level of phosphorylation of IκB,downstream of TLR4 signaling pathway,increased significantly after TAC/AngⅡ induction,while NF-κB double subunits P65 and 50 increased in the nuclei and NF-κB transcription activity increased.These above trends waere inhibited after overexpression of Triad3A;Suggesting that Triad3A can promote the development of cardiac hypertrophy by promoting TLR4 ubiquitination degradation and inhibiting NF-κB transcriptional activity.5.3 TAC/AngⅡ induced elevated phosphorylation of AKT(protein kinase B),which was inhibited after overexpression of Triad3A,suggesting that Triad3A can inhibit cardiac hypertrophy by reducing AKT phosphorylation.ConclusionIn summary,Triad3A is involved in pressure overload-induced cardiac hypertrophy and plays a negative role in this pathogenesis.Its regulatory role is mainly through the negative regulation of TLR signal and inhibition AKT phosphorylation.