Carbamylated Erythropoietin-induced Cardioprotection Against Myocardial Ischemia/reperfusion Injury Is Mediated Through A Phosphatidylinositol 3-kinase/Akt-dependent Mechanism

Carbamylated EPO(CEPO)is a derivative of erythropoietin(EPO) by subjecting it to carbamylation.Unlike EPO,it does not stimulate erythropoiesis,but effectively protects tissue from injury.In the models of long-time myocardial ischemic injury and prolonged myocardial ischemia/reperfusion(I/R)injury,the main pathological characters are great loss of cardiomyocytes,accrementition of fibroblast and rebuild of ECM.The increase of collagen,change ofⅠandⅢcollagen percentage will damage the integrity and concordant,which will be resulted in heart failure.It has been reported that CEPO administration daily can attenuate left ventricle remodeling in the models of long-time myocardial ischemic injury and chronic myocardial I/R injury.While in the model of acute myocardial I/R injury,the main pathological characters are degeneration, necrosis,apoptosis of cardiomyocytes and inflammatory cell infiltration, which will be resulted in the contractile dysfunction.However,the question as to whether a single dose of CEPO at the onset of reperfusion will protect heart from acute I/R injury has not been explored.Nor has the mechanism of CEPO-induced cardioprotection been fully understood.A number of signaling pathways,such as Jak2/STAT5 signaling, PI3K signaling,protein kinase C and MAPK,have been implicated in EPO-induced cardioprotection.Since CEPO signals only through EPOR-βcR,the mechanism of CEPO-induced cardioprotection may be different from that of EPO.It has been reported that CEPO did not effectively activate transcription factors STAT-5 or Jak2,a downstream kinase directly activated upon ligand binding to(EPOR)2.Recently, some studies showed that CEPO protected the kidneys from apoptosis with a concomitant up-regulation of PI3K and phosphorylation of Akt in CEPO-treated kidneys 24 hours until 1 week after I/R injury.Others reported PI3-kinase inhibitor,wortmannin,blocked the beneficial effect of CEPO on mitochondrial permeability transition(MPT)- reactive oxygen species(ROS)threshold in a permanent coronary artery ligation model in rats.Based on these data,we hypothesized that PI3K/Akt pathway might mediate CEPO-induced cardioprotection against acute myocardial I/R injury.In the present study,we examined whether a single dose of CEPO at the onset of reperfusion could protect heart from acute I/R injury in mice and whether PI3K/Akt pathway might mediate CEPO-induced cardioprotection against myocardial I/R injury.BALB/c mice were exposed to a 45-minute period of ischemia by ligating the left anterior descending(LAD)coronary artery followed by 4 hours of reperfusion.A single dose of CEPO or EPO was administered at the onset of reperfusion to observe their effect on acute myocardial I/R injury.Afterwards,a group of mice were pretreated with wortmannin(a PI3K inhibitor)to determine whether PI3K/Akt pathway will mediate CEPO-induced cardioprotection against myocardial I/R injury.The results showed that CEPO treatment decreased infarct area/risk area by 40.08%and IA/LV by 43.20%,whereas it increased ejection fraction and fractional shortening by 26.71%and 21.73%,respectively. There was no significant difference between CEPO and EPO treated group.Wortmannin significantly blocked the beneficial effect of CEPO on infarct size and cardiac function.CEPO promoted the association of erythropoietin receptor andβcommon receptor as evidenced by immunoprecipitation.Western blot analysis showed that CEPO administration enhanced Akt phosphorylation by 58.54%in cardiac tissues and wortmannin significantly attenuated CEPO-induced Akt phosphorylation by 67.69%.Moreover,CEPO increased the protein levels of phospho-GATA-4,GATA-4,myosin heavy chain and troponin I, an effect partly blocked by wortmannin.In conclusion,a single dose of CEPO at the onset of reperfusion significantly attenuated acute myocardial I/R injury,with potency and efficacy comparable to EPO.More importantly,CEPO-induced cardioprotection against myocardial I/R injury is mediated through a PI3K/Akt-dependent mechanism.