Ivabradine Prevents Low Shear Stress Induced Endothelial Dysfunction Via MTORC2/eNOS Pathway

Objective:Ivabradine not only reduces heart rate but has other cardiac and vascular protective effects including anti-inflammation and anti-oxidation.Endothelial nitric oxide synthase(eNOS)plays a crucial role in maintaining endothelial activity.Accrodingly,we designed this study aimed to investigate the impact of ivabradine on low shear stress(LSS)induced inflammation and endothelial injury along with the involvement of eNOS.Methods:Human umbilical vein endothelial cells(HUVECs)were subjected to LSS at 2 dyne/cm2,with 1 hour of ivabradine(0.04 ?M)pre-treatment.After been exposed to LSS for 0 min,30 min and 120 min,the mRNA expression of IL-6,VCAM-land eNOS were measured by q-PCR,while their protein expression were measured by immunofluorescence.Reactive oxygen species(ROS)was detected before and after LSS exposure by dihydroethidium(DHE)and DCF,while NO was detected by DAF-FM.Total proteins were collected prior and post LSS exposure,phosphorylation of eNOS,Akt,mTORC1 and mTORC2 pathway were detected by western blot,respectively.Then cells that pre-treated with ivabradine were subjected to LSS for 30 min and 120 min,phosphorylation of p70S6K,S6RP,raptor as well as Akt were detected again by western blot.LY294002(10 ?M)was applied to cells before ivabradine pre-treatment,the expression of mTORC1 and mTORC2 pathway were measured by western blot.Immunofluorescence was conducted to detect IL-6,VCAM-1 and eNOS expression.ROS was revealed by DHE and DCF.NO was indicated by DAF-FM.Results:It demonstrated that the mRNA and protein expression of IL-6,VCAM-1 as well as ROS generation were up-regulated by LSS 30 min and 120 min,while eNOS was down-regulated.NO content was reduced only when LSS applied for 120 min.And ivabradine inhibitied LSS-induced inflammation and oxidative stress in endothelial cells.The phosphorylation of raptor,p70S6K and S6RP were increased by LSS and reach the peak at 30 min,then dropped back to baseline value when LSS applied for 120 min.Akt-Ser473 was gradually reduced within 30 minutes of LSS exposure,and reached its baseline value at LSS 120 min.The eNOS-Thr495 phosphorylation was continuously up-regulated though 120 minutes LSS exposure.These effects were reversed by ivabradine.However,the protective effects of ivabradine were blocked by LY294002,revealed as IL-6,VCAM-1,ROS augument and eNOS,NO depression.By inhibiting mTORC2/Akt phosphorylation,LY294002 provoked mTORC1/eNOS-Thr495 activity.Conclusion:These results together suggest that LSS induced endothelial inflammation and oxidative stress are suppressed by ivabradine via mTORC2/Akt activation mediated mTORC1/eNOS reduction.Objective:Studies have shown that ivabradine revealed its cardiac protective effect by reducing heart rate.However,endothelial protective effect of ivabradine in vivo has not been fully studied.In this study,transverse aortic constriction(TAC)model of mice were used to mimic the low shear stress(LSS)in vivo,in order to verify the anti-inflammation and endothelial protective effect and corrected molecular mechanisms of ivabradine,as well as signal pathways in it.Methods:A 27G needle was tied on the aorta arch between truncus brachiocephalicus and left common carotid artery to create a narrow of 70%to the aorta.Three days after the surgery,survival mice were randomized into 5 group,which were sham,TAC,Ivabradine 10 mg/kg/d(Iva-10),Ivabradine 20 mg/kg/d(Iva-20)and LY294002 30 mg/kg/d(LY)group.After 4 weeks feeding,echocardiography was conducted to evaluate left ventricular function.Mice conscious heart rate and blood pressure were recorded.HE staining was used to observe cardiac hypertrophy and Masson staining was to determine fibrosis of cardiomyocytes and vessels.Endothelial function was evaluated by detecting the sensitivity of mice isolated aorta rings to adenosine.Immunofluorescence was applied to assess the expression of VCAM-1 and eNOS-Thr495 in vessels.And mTOR/Akt as well as eNOS pathway were measured by western blot.Results:It illustrated that TAC caused left ventricular dysfunction,cardiac hypertrophy,cardiomyocyte and vessel fibrosis in mice,which were significantly improved by ivabradine.There was no significant change in blood pressure among each group,whereas heart rate was dramatically decreased by ivabradine.Mice isolated aorta rings obtained from Iva-20 group had higher sensitivity to adenosine compared with TAC and LY groups.Immunofluorescence showed increased expression of VCAM-1 and eNOS-Thr495 in mice aorta of TAC group,which were reduced by ivabradine.Western blot revealed that ivabradine activated mTORC2/Akt with resultant eNOS-Thr495 dephosphorylation in mice aorta.And all the anti-inflammation and endothelial protective effect of ivabradine were inhibited by LY294002.Conclusion:All the results demonstrated that TAC model can be successfully established and used as an in vivo model of low shear stress.Ivabradine achieved its endothelial protective effect by provoking mTORC2/Akt pathway and blocking eNOS-Thr495 phosphorylation.