Study On The Mechanism Of GPR39 Promoting Cardiac Hypertrophy By Regulating AMPK-mTOR Signaling Pathway And Protein Synthesis

Objective: The purpose of this study was to investigate the effect of GPR39 on protein synthesis and expression in the AMPK-mTOR signaling pathway in the induction of cardiac hypertrophy cells,and to clarify the role of GPR39 in the development of cardiac hypertrophy and its possible regulatory pathway transduction mechanism.Pre-intervention and targeted therapy for myocardial hypertrophy and heart failure seek new targets and provide theoretical basis and direction.Methods:1.The mouse myocardial hypertrophy model was constructed by aortic coarctation method,and the cardiac function of the mouse was measured by echocardiography 4 weeks later to confirm the success of the model;2.The myocardial tissue samples of 5 patients with clinical myocardial hypertrophy and 5 healthy people in the control group were collected,and real-time quantitative real-time PCR(q RT-PCR)and Western blot(Western blot,WB)were used to detect the expression of GPR39,respectively Differences in expression between human and mouse hypertrophic myocardium model tissue and control normal myocardial tissue;3.In vitro experiment: Isolate and culture rat neonatal rat cardiomyocytes,construct an adenovirus AAV9-mediated GPR39 overexp-ression vector and transfect it into neonatal rat cardiomyocytes,and knock down the expression of GPR39 by sh RNA,and verify by q RT-PCR and WB experiments.After overexpression and silencing of GPR39,its effects on angiotensin Ang II-induced myocardial cell hypertrophy in mice were respectively verified m RNA and protein expression of ANP,BNP and MYH7;4.In vivo experiment: 1-week-old wild-type C57BL/6 mice(2 groups,n=8)were injected with adenovirus AAV9 carrying sh RNA(control group)and sh GPR39(experimental group)in a single tail vein.After 8 weeks,the two groups of mice received TAC or sham surgery to induce cardiac hypertrophy.After 4 weeks,m RNA analysis was performed to compare the expression differences of cardiac hypertrophy markers ANP and BNP,and the ratio of cardiac wet weight and body weight as well as the length of heart and tibia were calculated.The myocardial surface area was marked by immunofluorescence WGA staining,and the changes of myocardial hypertrophy induced by pressure load were detected in GPR39-deficient mice.5.The rat hypertrophic myocardial model was established by TAC method in vivo experiment,and the in vitro model of myocardial hypertrophy was induced by Ang II in vitro experiment(all were set as normal control group),respectively.Changes in expression levels of AMPK,mTOR,S6K1 and hypertrophic cardiomyocyte-related proteins de novo synthesis(monitoring of leucine incorporation)after GPR39 knockdown were detected respectively;6.Rescue experiments with the mTOR inhibitor rapamycin to verify the effect of GPR39 overexpression on cardiomyocyte size and ANP expression after blocking the mTOR-S6K1 pathway: cardiomyocytes were infected with adenovirus for 24 hours,and then in the absence of(DMSO control)/ Ang II(1μM)treatment in the presence of rapamycin(100 n M)treatment for 48 hours to monitor protein synthesis promoted by GPR39 overexpression in cardiomyocytes.Results:1.In human and mouse hypertrophic myocardium,the m RNA and protein levels of GPR39 were significantly increased compared with the control group;2.GPR39 can promote the hypertrophy of primary rat cardiomyocytes in in vitro cell experiments,and in vivo experiments can aggravate mouse cardiac insufficiency,increase ventricular wall thickness,decrease myocardial short-axis shortening rate and ejection fraction;3.In vitro experiments,overexpression of GPR39 significantly promoted Ang II-induced increase in primary rat cardiomyocytes and the overexpression of hypertrophic genes ANP,BNP,and MYH7;on the contrary,knockdown of CPR39 inhibited Ang II-induced cardiomyocyte enlargement.At the same time inhibit the expression of ANP,BNP,MYH7;4.In vivo experiments in mice suggest that knockdown of CPR39 inhibits Ang II-enhanced de novo protein synthesis,while GPR39 overexpression promotes the effect of Ang II on protein de novo synthesis;5.Knockdown of GRP39 can promote AMPK activation and up-regulate mTOR-S6K1 protein expression in Ang II-induced cardiomyocytes;6.Rapamycin reduced GPR39 overexpression-promoted protein synthesis,cellular hypertrophy,and ANP expression in Ang II-treated cardiomyocytes.Conclusions:1.GPR39 is a pro-cardiac hypertrophy factor,and overexpression of GPR39 can aggravate myocardial hypertrophy and heart failure;2.GPR39 is a negative regulator of AMPK,which can promote cardiac hypertrophy by inhibiting AMPK activation;3.GPR39 may promote S6K1 protein synthesis and cardiac hypertrophy through targeted regulation of AMPK-mTOR signaling pathway activation;4.GPR39 as a target for the treatment of cardiac hypertrophy and heart failure;...