Roles And Protective Mechanisms Of Apelin In Acute Ischemic Myocardial Injury

BackgroundAcute myocardial infarction is one of most important disease threatening human life and health.In addition to sudden death,Sever and sustained myocardial ischemia triggers cell death and irreversible loss of cardialmyocytes,which may lead to reduced ability to sustain contractile function and progression to heart failure.Programmed cell death,in the forms of apoptosis,necrosis and possibly,autophagic cell death are the final arbiters of cardiomyocyte numbers following myocardial infarction.While the core of the infarct with anoxia and near-total energy depletion displays necrotic changes,the periphery with impaired but somewhat intact oxygen supply display changes suggestive of varying amounts of apoptosis and autophagy.Reperfusion therapy is the most common treatment for myocardial infarction by decreasing the quantum of necrosis,which does however cause myocardial injury including increasing apoptosis in its own right.Studies in both animal models of ischemia and ischemia/reperfusion,as well as human myocardial infarction,demonstrate apoptosis in 5～30%of cells in the infarct zone and the immediate peri-infarct penumbra acutely.A sustained increase in apoptotic rates ultimately leads to clinically relevant loss in cell numbers and ultimately heart failure.Inhibition of apoptosis can significantly decrease infarct size.As a result,improving the tolerance of cardiomyocyte to ischemia and reperfusion injury and blocking irreversible cell death appear to ameliorate the impact of myocardial infarction and thus,arrest the progression to heart failure.Apelin,the endogenous ligand of the human orphan G-protein-coupled APJ receptor,has been isolated from bovine stomach tissue extracts by reverse pharmacology in 1998.Apelin is secreted as a 77 amino acid pre-proprotein which is cleaved to form several active peptides denoted by their length as Apelin-10,11,12,13,15,17,19,28 and -36.Of all the active fragments identified to date,Apelin-13 may represent the most potent biological ligand.The Apelin/APJ system was expressed in the central nervous system and a variety of peripheral tissues and involved in the regulation of immune response,brain signalling,hemodynamic homeostasis, vaso-dilatation,inotropic effect,angiogenesis as well as inhibition of insulin secretion, and so on.In cardiovascular system,high expression of APJ mRNA has been observed in heart.In contrast,Apelin expression is restricted to endothelial cells and negligible in cardiomyocytes in normal myocardium,but detectable in failing hearts.Current studies suggest that Apelin expression is at least maintained and possibly augmented in mild,compensated chronic heart failure but declines in severe disease. However,data of Apelin expression in acute myocardial injury are conflicted.In isolated cardiomyocytes Apelin expression is increased by acute hypoxia through the hypoxia inducible factor pathway;Accordingly,Apelin expression is upregulated in acute myocardial infarction.Endogenous cardiac Apelin and APJ are increased in rats with ischemic heart failure 6 weeks postmyocardial infarction;In contrast,both Apelin and APJ expression fall in a further rodent model of ischemic myocardial injury caused by repeated isoprotenerol administration;In ischemic myocardium of isolated rat hearts Apelin mRNA is upregulated but returns back to baseline after reperfusion,which is considered that reperfusion itself causes myocardial injury.Exogenous Apelin administration during myocardial injury can preserved cardiac function.However,the protective mechanism by which Apelin exerts remains to be elucidated.Some researches suggested that Apelin reduces infarct size and protects myocardial cell against ischemia-reperfusion(I/R) injury by activating the reperfusion injury salvage kinase(RISK) pathway,which incorporates phosphatidylinositol 3-OH kinase(PI3K)-cellμlar Akt/protein kinase B(Akt) and p44/42 mitogen-activated protein kinase(MAPK) extracellular signal-regulated MAPK(Erk1/2).Others believed that the cardioprotective activity of Apelin against myocardial reperfusion injury was independent of PI3K/Akt and P70S6 kinase.Recently,one study showed that Apelin suppresses apoptosis of mouse osteoblastic MC3T3-E1 cells throμgh the JNK and PI3K/Akt signaling pathways.In addition,The recent literature points to an emerging role for the mammalian target of rapamycin(mTOR) pathway in the resistance against cell death during cardiac hypoxia,ischemia/reperfusion and heart failure.So we hypothesised that Apelin can exhibit cardioprotective effect and improve cardiac function by inhibiting cardiomyocyte apoptosis and activating the PI3K/Akt and mTOR signaling pathways during acute myocardial injury.Objective1.Observe the expression of endogenous Apelin/APJ system during different periods of myocardial ischemia;2.Examine the cardioprotection of exogenous Apelin-13 on acute myocardial injury in rats induced by ligating the left anterior descending coronary artery(LAD);3.Determine the protective effect of Apelin-13 on cardiomyocyte apoptosis induced by acute myocardial ischemia;4.Determine the signaling pathways involved in the protective effect of Apelin-13 on cardiomyocytes survival during acute myocardial ischemia in vitro mimicked by glucose deprivation(GD).Methods1.To detect the expression of endogenous Apelin/APJ system during different period of myocardial ischemia.(1) Cardiomyocytes were cultured with day 1-2 Sprague Dawley neonatal rats, Apelin and APJ mRNA expression were determined by RT-PCR in rat cardiomyocytes during different periods of GD.Cardiomyocyte viability was detected by methyl thiazolyl tetrazolium(MTT) assay.(2) The myocardial contents and plasma concentrations of endogenous Apelin were detected by ELISA in SD rats suffered from acute myocardial ischemia by ligating the left anterior descending coronary artery(LAD) for 30,60,120 min respectively.2.The rats were then randomly divided into sham operated group,model control group and two Apelin-13 groups,to which Apelin-13 of 10 and 100μg/kg was infused via caudal vein 5 min before a 120 min-myocardial ischemia.The cardial function including LVESP,LVEDP and LV±dp/dt_max was measured on electrophysiolograph. Cardiomyocyte apoptosis was detected with TUNEL.Bcl-2,Bax,Caspase-3 protein expression of myocardium were detected with Western blotting.3.To detect the effect of Apelin-13 on cardiomyocyte apoptosis induced by GD as well as the machnism involved.(1) Cardiomyocyte apoptosis was detected by annexin V-FITC/propidium iodide (PI) staining and analyzed using flow cytometer after GD for 12 h with or without Apelin-13(10,100 nM) pretreatment.(2) To determine whether the cardioprotective effects of Apelin-13 involving activation of the phosphatidylinositol 3-OH kinase(PI3K)/Akt and mTOR pathways, Akt and mTOR protein expression as well as cell apoptosis were detected in the presence or absence of LY294002(a PI3K inhibitor) or rapamycin(a mTOR inhibitor).Results1.Apelin mRNA expression increased 0.93,1.32,1.44,1.29-fold(P＜0.05,0.01, 0.01,0.01) when cells suffered from GD for 6,12,18 and 24 h compared with the base level.However,when cells suffered from GD up to 36 h,Apelin mRNA expression was 85%lower than that in base level(P＜0.01).The change curve of APJ mRNA expression was coincident with that of Apelin.2.The content of Apelin in myocardium was markedly increased during myocardial ischemia 30 to 60 min and decreased to below basal level on ischemia 120 min(P＜0.01).The content of Apelin in plasma increased gradually during myocardial ischemia 30 to 120 min(all P＜0.01).3.In myocardial ischemia models induced by ligating LAD for 120 min, Apelin-13(100μg/kg) infused pre-ischemia can significantly improve cardial function,the LV±dp/dt_max and LVESP were higher and LVEDP lower than those in model control group;4.Compared with model control group,cardiomyocyte apoptosis index in high dose Apelin-13 group was markedly decreased(33.3±2.9%vs 45.1±0.7%,P＜0.01). The expressions of Bax and Caspase-3 proteins in high dose Apelin-13 were decreased and Bcl-2 protein increased compared with those in model control group(P＜0.05,0.05,0.01).5.Apelin-13 pretreatment at 100 nM significantly inhibited GD-induced cardiomyocyte apoptosis(apoptosis index:39.16±0.75%vs 50.25±5.9%;P＜0.05) as well as increase Akt and mTOR phosphorylation(P＜0.01,0.01).At the same time, Apelin-13(100 nM) up-regulated Bcl-2 protein expression and down-regulated Bax and Caspase-3 expression(P＜0.01,0.05,0.05).The anti-apoptotic effect of Apelin-13 was prevented by LY294002(apoptosis index:51.48±3.02%vs 39.16±0.75%,P＜0.01) but not by rapamycin(apoptosis index:45.72±2.72%vs 39.16±0.75%,P＞0.05).Conclusions1.The expression of Apelin/APJ system in myocardium is increased in a compensatory role in early myocardial ischemia and decreased in sustained myocardial ischemia.2.Apelin protects against acute ischemic myocardial injury in a dose-dependent mode.3.The protective mechanism of exogenous Apelin-13 on acute ischemic myocardial injury may be related to inhibition of cardiomyocyte apoptosis.4.The protection mechanism of Apelin against cardiomyocyte apoptosis is involved in activation of PI3K/Akt pathway.