| Background Heart failure is the end-stage of a variety of cardiovascular diseases including hypertension, valvular heart disease and dilated cardiomyopathy. However, the failing hearts may be more vulnerable to ischemia-reperfusion injury(IRI) due to CPB or noncardiac surgery. Numerous treatment strategies have been investigated to protect myocardium against IR injury, among which ischemic preconditioning(IPC) is well known as a powerful innate protective mechanism. Unfortunately, the cardioprotective effect of IPC is weakened or abolished in some pathological conditions such as aging, diabetes and hyperglycaemia. It has been speculated that pathological processes result in fundamental signaling molecular alterations that may affect the responses to IPC. The activation of reperfusion injury salvage kinase(RISK) pathways at reperfusion, including phosphatidylinositol-3 kinase/protein kinase B(PI3K/Akt) and extracellular signal-regulated kinase(ERK), plays a critical role in IPC induced cardioprotection by phosphorylating the downstream glycogen synthase kinase(GSK)-3β and thus inhibiting its activity. In previous in vivo studies, we have demonstrated that IPC failed to confer cardioprotection in rats with heart failure, while morphine preconditioning(MPC) retained its protection. However, the exact mechanisms are not known. This study was designed to examine the role of RISK pathways in MPC induced protection against IRI in isolated failing rat hearts by using the DOX-induced rat chronic heart failure(CHF) model, and the normal rats were served as the control.Methods1. To induce the rat model of chronic heart failureThe adult male rats(200±30 g) were randomly divided into doxorubicin(DOX)and normal saline(NS) groups. Chronic heart failure model was induced by intravenous administration of doxorubicin(DOX) weekly via tail vein in 6 equal injections(each containing 2.0 mg/kg in normal saline) over a period of 6 weeks for a total cumulative dose of 12 mg/kg, and the control rats were injected normal saline(NS). At the end of 8th week of injection, Echocardiography was performed to evaluate cardiac function, and histological examination was conducted to examine morphological changes in myocardium.2. To study the role of RISK pathway in MPC induced cardioprotection in normal ratsAll NS injected normal rat hearts were randomly divided into 8 groups: Sham group(sham, n=9), ischemia reperfusion group(IR, n=10), ischemic preconditioning group(IPC, n=10), morphine preconditioning group(MPC, n=9), MPC+ wortmannin group(MWT, n=9), MPC+PD98059 group(MPD, n=9), wortmannin group(WT, n=6) and PD98059 group(PD, n=6). All hearts were stabilized for 15 min and subjected to 30 min ischemia followed by 120 min reperfusion except that the hearts in sham group were perfused KH solution continuously until the end of experiment. IPC was elicited with 3 cycles of 5 min of ischemia and 5 min of reperfusion before lethal ischemia. MPC was performed with three cycles of 5 min infusion of 1μmol/L morphine hydrochloride and 5 min drug-free infusion. PI3 K inhibitor wortmannin(100 nmol/L) and ERK inhibitor PD98059(10 μmol/L) were perfused for a period of 10 min before MPC until 5 min after the end of MPC, respectively. The coronary flow was collected to detect the activity of lactate dehydrogenase(LDH) and Infarct size(IS) were determined by 2,3,5-triphenyl-tetrazolium(TTC) staining at the end of reperfusion. Tissue samples(n=3/group) were taken from area at risk of left ventricular myocardium 10 min after reperfusion. The protein levels of p-ERK/ERK, p-Akt/Akt, p-GSK-3β/GSK-3β and β-actin were detected by western blot.3. To study the role of RISK pathways in MPC induced cardioprotection in isolated failing rat heartsIn the first series of experiments, the cardioprotective effects of morphinepreconditioning at different concentrations in isolated failing rat hearts were observed. All rats were confirmed chronic heart failure through echocardiography and randomly divided into 8 groups: Sham group(n=9), IR group(n=10), IPC group(n=10), MPC groups(0.3, 1, 3, 10, 30 mmo/L, n=6-9). In the second series of experiments, the role of RISK pathways in MPC induced cardioprotection against IRI in isolated failing rat hearts was investigated. The chronic heart failure rats were randomly divided into 4 groups: MPC+ wortmannin group(MWT, n=9), MPC+PD98059 group(MPD, n=9), wortmannin group(WT, n=6) and PD98059 group(PD, n=6). All experimental procedures were conducted as described above. The protein levels of p-ERK/ERK, p-Akt/Akt, p-GSK-3β/GSK-3β, β-actin and the m RNA levels of Bax and Bcl-2 were detected by western blot or quantitative RT-PCR, respectively.Results1. Establishment of the rat model of chronic heart failureThe mortality of rats in DOX group was 35.6% at the end of the 8th weeks, while the NS injected rats all survived during the experiment. DOX injected rats experienced signs of heart failure over time including listlessness, less appetite, growth slowly or weight loss, while the NS rats remained healthy throughout the study. Echocardiography results showed that left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS) were significantly decreased in DOX group as compared to the NS group at the end of 8th week. Histological examination of cardiac sections stained with hematoxylin-eosin revealed that DOX induced cardiomyocytes cytoplasmic vacuolization.2. Role of RISK pathways in MPC induced cardioprotection against myocardial IRI in isolated normal rat hearts2.1 Effects of PD98059 and wortmannin on MPC mediated cardioprotection in normal rat heartsBoth MPC and IPC significantly reduced the infarct size and the elevated lactate dehydrogenase(LDH) activity caused by myocardial IRI in normal rat hearts. However, pretreatment with ERK inhibitor PD98059 or PI3 K inhibitor wortmanninbefore MPC abolished the cardioprotection induced by MPC. These findings suggested that MPC-induced cardioprotective effects in normal rat hearts were dependent upon the activation of ERK and PI3K/Akt pathways.2.2 Effects of PD98059 and wortmannin on the phosphorylation of ERK, Akt, GSK-3b induced by MPC in normal rat heartsMPC and IPC significantly increased the phosphorylation of ERK and Akt, and subsequently elevated the phosphorylation of GSK-3b. However, PD98059 and wortmannin inhibited the phosphorylation of ERK or Akt induced by MPC, respectively. In addition, the phosphorylation of GSK-3b induced by MPC was suppressed by both PD98059 and wortmannin.3. Role of RISK pathways in MPC induced cardioprotection against myocardial IRI in isolated failing rat hearts3.1 The cardioprotective effects of MPC at different concentrations in isolated failing rat heartsMPC significantly reduced IS/AAR and decreased the activity of LDH caused by IR injury at the concentrations of 1 μmol/L and 3 μmol/L. Nevertheless, the cardioprotective effects of MPC at the lower concentration of 0.3μmol/L or the higher concentrations of 10 μmol/L and 30 μmol/L were unconspicuous. Meanwhile, IPC attenuate neither the infarct size nor the activity of LDH in coronary effluent.3.2 Effects of PD98059 and wortmannin on the cardioprotection induced by MPC in failing rat heartsPD98059 significantly abrogated the cardioprotective effects of MPC by increasing the infarct size and the LDH activity at reperfusion. Interestingly, wortmannin did not reverse MPC-induced cardioprotection including infarct size and LDH activity. These findings suggested that MPC-induced cardioprotective effects were dependent upon the activation of ERK but not PI3K/Akt pathway in failing rat hearts.3.3 Effects of PD98059 and wortmannin on the phosphorylation of ERK, Akt and GSK-3b induced by MPC in failing rat heartsMPC markedly elevated the phosphorylation of ERK and the downstream GSK-3β, while failed to increase Akt phosphorylation. PD98059 suppressed the phosphorylation of ERK and GSK-3β induced by MPC. In contrast, although wortmannin inhibited Akt phosphorylation, it did not affect the phosphorylation of GSK-3β induced by MPC. At the same time, IPC was unable to increase the phosphorylation of ERK, Akt or GSK-3β.3.4 Effects of PD98059 and wortmannin on the m RNA levels of Bcl-2 and Bax induced by MPC in failing rat heartsMPC not only increased Bcl-2 m RNA, but also decreased Bax m RNA, leading to a much higher Bcl-2/Bax ratio than IR group, while these effects were abolished by the addition of PD98059, but not by wortmannin. At the same time, IPC did not affect the m RNA levels of Bcl-2 and Bax or the ratio of Bcl-2/Bax induced by IR injury.ConclusionsBoth MPC and IPC induce the activation of ERK and PI3K/Akt pathways and subsequent GSK-3β phosphorylation in isolated normal rat hearts, leading to reduced myocardial IRI. MPC confers cardioprotective effects in failing rat hearts, and its underlying mechanisms involve the activation of ERK/GSK-b pathway independent of PI3K/Akt, resulting in regulation of apoptotic genes. In contrast, the classic endogenous protective strategy of IPC failed to confer cardioprotection in rats with heart failure due to the unresponsive RISK pathways and downstream signals. |
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