Endoplasmic Reticulum Stress (ERS)-autophagy Pathway Involved In Cardiomyocytes Hypertrophy Induced By Apelin-13

Aims:Apelin is the endogenous ligand for the G protein coupled receptor APJ. It hasreported that apelin-13promotes vascular smooth muscle cell proliferation andmonocyte adhesion to endothelial cells. In our previous research, we found thatapelin-13promotes H9c2rat cardiomyocytes hypertrophy by PI3K-Akt-ERK1/2-p70S6K and PI3K-autophagy pathway. The objective of this study was to explorethe effect of apelin on the expression of NOX4and endoplasmic reticulum stress (ERstress). For the further study to discuss the mechanism of ER stress-autophagypathway mediating cardiomyocytes hypertrophy induced by apelin-13. To reveal thenew mechanism of apelin-13on the myocardial hypertrophy, and provide a newtheoretical basis for APJ acting as a drug target in cardiovascular disease.Methods:1. The expression of APJ, NOX4, Bip, CHOP, LC3-II/I, beclin-1, p62and β-tubulinwere detected by Western Blot;2. APJ shRNA to interfere the expression of APJ, Bip siRNA and CHOP siRNA tointerfere the expression of Bip and CHOP;3. The formation of autophagosome was observed by using immunofluorescence tolabel the LC3and using chloroquine to block autophagosome degradation; it alsowas detected by GFP-LC3transfection;4. The morphology of endoplasmic reticulum was observed by ER tracker-red;5. The diameter and volume of H9c2rat cardiomyocytes detected by ScepterTMHandheld Automated Cell Counter (Millipore);6. The protein content of H9c2rat cardiomyocytes was detected by quantitativemethod of BCA;7. The H9c2rat cardiomyocytes proliferation was measured by CCK-88. The H9c2rat cardiomyocytes apoptosis was observed by hoechst33258; Results:1. Apelin-13promotes the expression of NOX4in dose and time dependentmanners;2. Apelin-13increases the expression of Bip and CHOP in dose and time dependentmanners;3. APJ shRNA blocks the increase of Bip and CHOP induced by apelin-13;4. Apelin-13induces the formation of autophagosome incardiac cells of newborn SD rats and APJ shRNA inhibits the overexpression ofLC3-II/I, beclin1induced by apelin-13;5. NOX4inhibitor DPI attenuates the increase of Bip and CHOP induced byapelin-13;6. NOX4inhibitor DPI recedes the increase of LC3-II/I, beclin1and the decreaseof p62induced by apelin-13;7. Endoplasmic reticulum stress inhibitor Salubrinal reduces the increase of LC3-II/I, beclin1and the decrease of p62induced by apelin-13; Endoplasmic reticulumstress revulsant Tunicamycin has no effect on the increase of LC3-II/I, beclin1and the decrease of p62induced by apelin-13；Bip siRNA or CHOP siRNAreduces the increase of LC3-II/I, beclin1and the decrease of p62induced byapelin-13;8. Apelin-13induces the expression of LC3in Endoplasmic reticulum;9. APJ shRNA inhibits the increase of cell diameter, volume and intracellular proteincontent induced by apelin-13; Endoplasmic reticulum stress inhibitor Salubrinalweakens the increase of cell diameter, volume and intracellular protein contentinduced by apelin-13; Endoplasmic reticulum stress revulsant Tunicamycinenhanced the increase of cell diameter, volume and intracellular protein contentinduced by apelin-13; Autophagy inhibitor3MA blocks the increase of celldiameter, volume and intracellular protein content induced by apelin-13; BipsiRNA or CHOP siRNA also weaken the increase of cell diameter, volume andintracellular protein content induced by apelin-13; 10. Apelin-13promotes H9c2rat cardiomyocytes proliferation in a dose dependentmanner;11. Apelin-13promotes H9c2rat cardiomyocytes apoptosis in dose and timedependent manners;12. Endoplasmic reticulum stress inhibitor Salubrinal, autophagy inhibitor3MA, BipsiRNA or CHOP siRNA all inhibit the cardiomyocytes apoptosis induced byapelin-13.Conclusion: These findings show that ER stress-autophagy involved in the H9c2rat cardiomyocytes hypertrophy and apoptosis induced by apelin-13.