The Mechanisms Of Epac/Rap1 Signaling Pathway On Hypoxia/Reoxygenation Injury In H9c2 Cells

ObjectiveThe aim of this study was to detect the mechanism of Epac/Rap1 signaling pathway on hypoxia/reoxygenation(H/R)injury in H9c2 cells,and to provide theoretical and experimental basis for the clinical treatment of myocardial ischemia reperfusion injury.Methods1.Hypoxia/reoxygenation(H/R)in H9c2 cells was used to simulate ischemia reperfusion in vivo.The experiment was falled into control group,OGD(Oxygen Glucose Deprivation)group,OGD+Epacl agonist group(OGD + 8-CPT),OGD+Epacl inhibitor group(OGD + ESI-09),OGD+Epacl agonist+PKA inhibitor group(OGD +8-CPT + H-89),OGD+Epacl inhibitor+PKA inhibitor group(OGD + ESI-09 + H-89)six groups in total.MTT and LDH detect cell vitality and damage,fluo-3 AM respectively detect the contents of intracellular[Ca2+].Western Blot,qRT-PCR and IF observe Epacl expression and its downstream Rapl active form Rap 1-GTP and other related proteins expression.2.Hypoxia/reoxygenation(H/R)was used to simulate ischemia reperfusion.The experiment was divided into control group,model group(OGD),OGD + Epacl agonist group,OGD + Epac1 inhibitors group,Epac1-shRNA interference group,Epac2-shRNA interference group,OGD + Epacl-shRNA interference group,OGD + Epac2-shRNA interference group eight groups in total.MTT assay detect cell vitality,calcium ion fluorescence probe(fluo-3 AM)respectively detected the contents of intracellular[Ca2+].Western Blot detect the changes of Epacl,Rap1-GTP and CaMK?and p-ERK protein expression in H9c2 cells;The expression changes of Epacl and Rap1 mRNA were detected by real-time fluorescence quantitative PCR.The interaction between Epac and Rap1 was detected by Co-IP.Results1.Compared with the control group,cell viability decreased with the release of LDH increased in myocardial cell after hypoxia for 5h and reoxygenation for 1h.Myocardial intracellular signaling molecules Epacl was activated and its expression up-regulated,its downstream of Rapl-GTP activated,promoting myocardial cell injury;8-CPT could further promote Epacl overexpression of myocardial cells and enhance the activation of downstream Rapl-GTP,resulting in overloading of intracellular calcium and aggravating myocardial cell injury;ESI-09 inhibited the activation and the expression of Epacl,inhibited and the downstream Rapl-GTP activation,and curb its downstream CaMK II protein expression,up-regulation the phosphorylation of ERK,inhibiting calcium inside flow and reducing myocardial cell injury.H-89 had a mild inhibitory effect on the expression of Epac1 and the activation of downstream Rap1 in cardiomyocytes.The Epac1 and Epac2 gene were silenced by slow virus stabil in der interference.after OGD,cell viability was higher than that of the OGD group.Epacl and downstream related protein,intracellular calcium ion concentration expression decreased in cardiac myocytes,and inhibited the phosphorylation of ERK,Epac2 protein expression was unchanged.When Epac2 gene was silenced and OGD,there was no significant change in cell activity compared with OGD group.Epac2 expression decreased in cardiomyocytes,Epacl and its downstream proteins,intracellular calcium concentration expression were not significantly changed,and the phosphorylation of ERK decreased.The immunoprecipitation experiment showed that the interaction between Epacl and Rap1 was both detected by Epacl or Rapl antibody respectively.ConclusionThe results of this study suggest that Epacl may be a potential target for the treatment of myocardial ischemia reperfusion injury,and Epac/Rapl signaling pathway is the pathway of hypoxic oxygenation injury of myocardial cells.