Cardioprotection Of L-NAME And L-Arg Preconditioning In Isolated Rat Hearts After Ischemia-reperfusion Injury

BACKGROUNDPrevious investigations have shown that the second window of myocardial protection(SWOP)were induced by preconditioning with L-arginine(L-Arg)and NG-nitro-L-arginine methyl ester(L-NAME,nitric oxide synthase inhibitors)after 24h respectively,ameliorating the function of isolated heart after ischemia-reperfusion injury(IRI).Before L-NAME preconditioning or reperfusion,the protection was blocked by adenosine(Ado)receptor antagonist DPCPX,indicating that Ado may be related to this protection.As a reactive production under hypoxia and regulated by hypoxia-inducible factor-1?(HIF-1?),Ado can inhibit the activation of the intrnnsic apoptotic pathways,which mediated by Bax and Caspase-3 im IRI.However,the mechanism of L-NAME and L-Arg preconditioning is not clear.OBJECTIVE1.To investigate whether endogenous Ado levels and expression of HIF-1? are related to SWOP induced by L-NAME and L-Arg.2.To assess the influence of Bax and caspase-3 in L-NAME and L-Arg preconditioning after IRI,exploring the relationship between preconditioning and apoptosis.3.To clarify the mechanism of L-NAME preconditioning and the difference with L-Arg pretreatment in myocardial protection.METHODSTwenty-four male Sprague-Dawley rats aged 8-10 weeks were randomly divided into three groups(n=8)5 which were pretreated by intraperitoneal injection of the corresponding drugs:L-NAME group(10 mg/kg)and L-Arg group(500mg/kg),Control group(normal saline 3.3ml/kg),blood samples were taken after 24h preconditioning to detect plasma Ado concentration by high performance liquid chromatography while IRI model of isolated heart were established.When stable beating for 15 min,hearts were arrested by perfusion of modified cardioplegic solution at 4? into the aortic root.All hearts were subjected to global ischemia at 20°C for 60 minutes.At the end stage of the global ischemia,they were reperfused for 120 minutes by the K-H buffer perfusion solution at 37? with the same pressure(80 mmHg).Heart rate(HR),left ventricular development pressure(LVDP)and coronary flow(CF)were measured in all groups before cardiac arrest(S15)and 1,60 and 120 minutes(Rl,R60,R120)after the start of reperfusion.The infarct size was assessed by TTC and HE staining at the end of perfusion.Immunohistochemical analysis of Bax expression were performed to evaluate the apoptosis.RT-PCR was used to assess the mRNA levels of HIF-1? and Caspase3 in myocardium.RESULTSThere were no significant differences in hemodynamic parameters between the groups before arrest.The L-NAME group was able to restore LVDP and CF in the whole process of reperfusion(P<0.05).The L-Arg group can recovery LVDP and CF better in the early stage of reperfusion(Rl,R60),but the suddenly decline of CF in the late stage(R120),which is not significantly different vs Control group(P>0.05).There is no significant difference in HR between the groups before and after reperfusion(P>0.05).The plasma Ado concentrations in each group were similarity(P>0.05):L-NAME group(84.121±29.533?g/L),L-Arg group(80.670±17.170?g/L),Control group(83.756±24.979?g/L).The infarct size in L-NAME group were comparable with L-Arg group(23.50±4.51%VS 29.05±5.83%,P>0.05),but that were severe in the Control group(40.55±6.16%)with morphological damage.In immunohistochemistry,the decrease of Bax in L-NAME group was obviously than that in L-Arg group(P<0.05),and the apoptosis-related proteins in the two groups were significantly lower than that in Control group(P<0.05).Vs Control group,L-NAME and L-Arg preconditioning both reduce the expression of Caspase-3 after IRI,but only increase the expression of HIF-1? in L-NAME group(P<0.05).CONCLUSION1.Preconditioning with L-NAME can mimetic the second window of myocardial protection,reducing myocardial infarct size,improving cardiac function and protecting cardiomyocytes morphology after ischemia-reperfusion.The mechanism may involve up-regulation of HIF-la inducing pseudohypoxia and suppression of apoptotic signaling pathways associating with Bax and Caspase-3.2.L-Arg preconditioning exhibited a certain protection but weaker than L-NAME,indicating that the mechanism of them were difference where HIF-la may play an important role in.3.After 24 hours preconditioning,the change of plasma Ado concentration was not obvious.It is necessary to further improve the detection technology and monitor the levels of Ado at multiple time points within 24 hours dynamically.