The Function And Mechanism Research Of CD38 Promotes Angiotensin ?-induced Cardiac Hypertrophy

Background and aims: Myocardial hypertrophy is a kind of heart disease caused by a variety of heart diseases such as high blood pressure,heart valve disease and myocarditis,with the main features characterized by the increase of myocardial cell volume and the increase of extracellular matrix which may further induce heart fibrosis.The long-term persistent cardiac hypertrophy is an important cause of heart failure or even sudden death.However,there is still no effective method for prevention and treatment.According the literatures,several signal pathways are involved in the regulation of cardiac hypertrophy,while the specific mechanisn of regulation of these pathways is not completely clear.Therefore,the discovery of specific moleculars mediated cardiac hypertrophy and its related signaling pathway preserve importante clinical significance for further clarify the pathogenesis of cardiac hypertrophy and exploring new targets for the prevention and treatment of heart hypertrophy.Rescently,we observed that mouse embryonic fibroblasts from CD38 knockout mice were significantly resistant to oxidative stress such as H2O2-induced injury and hypoxia/reoxygenation-induced injury.In addition,we also found that CD38 knockout mice protected heart from ischemia reperfusion injury through activating SIRT1/FOXOs-mediated anti-oxidative stress pathway.However,the role of CD38 in cardiac hypertrophy was not explored.Here,this study aimed to investigat the roles and mechanisms of CD38 in angiotensin II(AngII)induced cardiac hypertrophy,and provide new targets and research ideas for the treatment and prevention of cardiac hypertrophy.Methods:1.Preparation and analysis of AngII induced cardiac hypertrophy model in mice.A.Preparing AngII induced heart hypertrophy mice model: Ten to twelve weeks old male wild-type and CD38 knockout mice were randomly assigned to the control or Ang-II treated group,which were infused with saline or Ang-II(1500 ng/kg/day)for 14 days by implanting osmotic mini-pumps(ALZET model 2002)in theperitoneal cavity,respectively.B.Elevation of systolic blood pressure was measured by tail-cuff system(softron BP-98A).C.Analysis of mouse body weight and LV weight radio.D.Heart tissues were fixed in 4% paraformaldehyde,embedded in paraffin and sectioned at 3?m intervals.A 3?m section was de-paraffinized and incubated with 100?g/mL FITC-labeled wheat germ agglutinin for 30 minutes at 37°C.E.Masson trichrome staining and picrosiriusred staining were used for examining the cardiac fibrosis area,and the collagen types I and III were differentiated by a polarizer.F.The vevo770 High-Resolution Imaging System was used for the echocardiography analysis.2.Preparation and analysis of AngII induced cardiac hypertrophy cell model with H9c2 cell line.A.cell line preparation: The stable CD38 knockdown H9c2 cell lines were prepared with CD38 specific shRNA construct and the reduced expression of CD38 in the cell lines was confirmed by western blot.B.preparation of hypertrophy in vitro model.The stable cell lines were subjected to 48 hours Ang-II stimulation with different concerntraion,and mRNA levels of BNP was detected by Q-PCR.C.Biomarker detection of cardiac hypertrophy.The hypertrophy marker moleculars ANP and BNP was detected by Q-PCR after 48 hours 2 ?moL AngII stimulation.D.cell size detection with crystal violet staining: H9c2 cells were stained with crystal violet after treated with 2 ?mo L AngII for 48 hours and further fixed with 90% ethanol for 20 min.E.ROS detection: H9c2 cells were treated by Ang-II for 20 minutes,and then stained with CM-H2 DCFDA for ROS detection.3.The mechanism study of CD38 mediated hypertrophy.A.Western blot analysis was performed to detect the protein expression of SIRT3 after AngII stimulation.B.The mRNA levels of FOXO3 and its downstream targets ARC,Bcl2 and SOD2 were detected by Q-PCR.C.The protein phosphorylation of AKT1,ERK and GSK3? were detected by westerin blot after 20 minutes AngII stimulation in control and CD38 knockdown H9c2 cells.D.The intracellular Ca2+ was detected with fluro-3am after Ang-II stimulation.E.To obtain additional evidence for the effect of CD38 knockdown on the localization of NFATc4,we analyzed cytoplasmic and nuclear fractions of cardiomyocytes by western blotting.Results:1.CD38 gene deficiency protect hearts from AngII induced heart hypertrophy and fibrosis in vivo.1)After 14 days AngII infusion,the systolic blood pressures were markedly increased in both mouse models.Meanwhile,there was no fifference in the blood pressures between the WT and CD38 KO mice.2)Ang-II infusion significantly increased the ratio of left ventricle/body weight(LV/BW)by almost 24% in wild-type mice,but not in CD38 deficient mice.3)The wheat germ agglutinin staining showed that the cardiomyocyte size for WT and CD38 KO mice was comparable after saline infusion,whereas AngII infusion significantly increased the cell size of WT mice,in which the cardiomyocyte size of CD38 KO mice was attenuated by 12% compared with that of wild type mice.4)Picrosirius red staining and masson trichrome staining was used for analysis of collagen accumulation,and the results all showed that the heart fibrosis was greater in the interstitial and perivascular sites in WT hearts than CD38-/-hearts after AngII infusion.2.CD38 gene knockdown attenuated AngII induced hypertrophy in vitro.A.The protein expression of CD38 was knockdown to 40% of that in control cells.B.The mRNA expression of BNP was upregulated by Ang-II in a dose-dependent manner.C.Knockdown of CD38 significantly decreased Ang-II induced expressions of ANP and BNP which serve as the markers of cardiac hypertrophy.D.The cardiomyocyte sizes induced by Ang II were much larger than that of the untreated cells in normal cell lines,whereas CD38 knockdown cells were not enlarged after AngII stimulation.E.ROS generation induced by Ang-II was attenuated by CD38 knockdown.3.CD38 gene knockdown attenuated AngII induced heart hypertrophy through actiation the SIRT3-FOXO3 signaling pathway.A.SIRT3 expression was increased in CD38 knockdown H9c2 cells and Ang-II further increased SIRT3 expression levels in CD38 knockdown cells compared with normal H9c2 cells.B.The mRNA expression of FOXO3 was proved to be elevated by CD38 knockdown,and the increased expression of its downstream molecules ARC,Bcl2 and SOD2.C.The phosphorylation of AKT1,ERK and GSK3? were increased in normal H9c2 cells,but not in CD38 knockdown H9c2 cells.4.Knockdown of CD38 alleviated Ang-II induced intracellular Ca2+ overload and NFATc4 nucleus expression.A.The result showed that there was an markedly elevation in normal H9c2 cells,however,the intracellular Ca2+ in CD38 knockdown cells was much lower than that of control.B.CD38 knockdown caused the decrease of NFATc4 nucleus expression.Conclusion:1.CD38 deficiency prevented mice from Ang-II induced cardiac hypertrophy in vivo.2.RNAi-mediated knockdown of CD38 prevented Ang-II induced cardiac hypertrophy and oxidative stress in vitro.3.Knockdown of CD38 inhibited Ang-II induced cardiac hypertrophy through activation of SIRT3-FOXO3 signaling.4.Knockdown of CD38 inhibited Ang-II induced cardiac hypertrophy through inhibiting Ca2+-calcineurin-NFAT pathway.