Apelin Attenuates Phenylephrine-induced Cardiac Hypertrophy Via Activation Of PI3K-AKT-mTOR Signaling Pathway

ObjectiveTo investigate whether Apelin affects phenylephrine（PE）-induced cardiomy--ocyte hypertrophy and cardiomyocyte autophagy through PI3K-AKT-mTOR sig--naling pathway.Method1.H9C2 cells was interfered by PE to establish an model of cardiomyocyte hypertrophy in vitro.Myocardial cytoskeleton was visualized by Rhodamine-labeled phalloidin staining,and analysis of myocardial cell area was measured by high-content imaging system.Determination of total cell protein expression level was measured by BCA method,and RT-PCR was used to detect the m RNA expression of myocardial hypertrophy marker myosin heavy chain 7（MYH7）,and the cell viability was detected by cell counting kit-8（CCK-8）.2.Electroporation transfection technology was used to screen the low-expression cells（Si-APJ）of the angiotensin typeⅠreceptor-related protein（APJ）constructed by the plasmid,and the stable transfer of their corresponding empty plasmid Cells（Vector-Si-APJ）3.Experimental grouping:H9C2 cells,and Si-APJ cells were divided into the control group,the PE group,the PE+Apelin group,the PE+Rapa group and the PE+Apelin+Rapa group according to different intervention methods.4.The expression levels of MYH7,phosphoinositide 3 kinase（PI3K）,AKT,mTOR,Beclin-1,microtubule associated protein 1 light chain 3（LC3）B/A,P62protein were measured by the western blot.Dansylcadaverine（MDC）was used to analyze fluorescence intensity to indicate changes in autophagosomes by the high-content imaging analysis system.Rhodamine-labeled phalloidin staining was used to stain myocardial cytoskeleton and the area of cardiomyocytes was measured by high-content imaging analysis system.The total protein expression level was determined by BCA method.Result1.The concentration gradient method explores that the best working concentration of PE is 50umol/L,and the best working concentration of Apelin is10^-7mol/L.2.Effect of Apelin on PI3K-AKT-mTOR signaling pathway:In the H9C2 cells,compared with PE group,the expression levels of PI3K,AKT,and mTOR phosphorylated protein in PE+Apelin group were significantly increased（P<0.05）.In the Si-APJ H9C2 cells,compared with PE group,the expression of PI3K,AKT,and mTOR phosphorylated protein in PE+Apelin group decreased（P<0.05）.3.Effect of Apelin on myocardial hypertrophy:In the H9C2 cells,compared with the blank control group,the expression of MYH7 protein,the cell surface area,and total cell protein in the PE group were increased（P<0.05）.Compared with the PE group,the MYH7 protein expression,the cell surface area,and total cell protein in the PE+Apelin group were decreased significantly（P<0.05）.In the Si-APJ H9C2 cells,compared with the blank control group,the relative expression level of MYH7protein,the cell surface area,and total cell protein in the PE group were significantly increased（P<0.05）.Compared with the PE group,the relative expression of MYH7protein and total cell protein in the PE+Apelin group increased significantly（P<0.05）.In the H9C2 cells,compared with the PE+Apelin group,MYH7 protein expression,cell surface area,and total cell protein expression in the PE+Apelin+Rapa group were significantly increased（P<0.05）.4.Effect of Apelin on cardiomyocyte autophagy:In the H9C2 cells,compared with the blank control group,the relative expression of the Beclin-1 protein and the relative fluorescence intensity of MDC staining in PE group were significantly increased（P<0.05）,and the relative expression of the P62 protein was decreased（P<0.05）.In the H9C2 cells,compared with the PE group,the relative expression level of the Beclin-1 protein and LC3B/A in the PE+Apelin group decreased（P<0.05）,and the relative expression level of the P62 protein increased（P<0.05）,The relative fluorescence intensity of MDC staining was reduced（P<0.05）.In Si-APJ H9C2 cells,the relative fluorescence intensity of MDC staining was enhanced compared with the blank control group（P<0.05）.Compared with the PE group,in the PE+Apelin group,LC3B/A increased（P<0.05）,the relative expression of P62 protein decreased（P<0.01）,the relative expression of Beclin-1 protein,the relative fluorescence intensity of MDC staining was not showed difference Statistical significance（P>0.05）.In the H9C2 cell,Compared with the PE+Apelin group,LC3B/A in the PE+Apelin+Rapa group was significantly increased（P<0.05）,the relative expression of the P62 protein was significantly reduced（P<0.05）,and the relative fluorescence intensity of MDC staining was enhanced（P<0.05）.ConclusionApelin improves PE-induced cardiomyocyte hypertrophy and reduces PE-induced hypertrophy of cardiomyocytes via activating the PI3K-AKT-mTOR signaling pathway.