ERK1/2 Signal Pathway Mediate Cardioprotection By Morphine Postconditioning

BackgroundMyocardial infaction and the complications have become a major cause of death in our country. The best treatment of myocardioal infartion is reperfusion the coronary artery as soon as possible, which may be partly abolished by ischemia/reperfusion injury. Ischemia preconditioinig and postconditioning are two important endougous cardioprotective methods to reduce the reperfusion injury. Some targets were found for drug treatments form the mechanism researches. Morphine is an important opioid-receptor agonist, usually used for aneshethsia and analgesia, and for the treatment of acute heart failure. The effects of morphine mimic the cardioprotection of ischemia precondionting have been identify by animal experiments and clinical researches. Whether morphine could minic the ischemia postconditioning effects against reperfusion injury, there was little research reported. It is well know that apoptosis plays an important role in the myocyte cell death after ischemia/reperfusion. The reduction in apoptotic myocyte death induced by myocardial ischemia/reperfusion injury has been demonstrated to contribute to the protection of ischemia precondionting and postconditioning mediated by ERK1/2. Whether ERK1/2 is mediated morphine postconditioning which would be expected to reduce cell death through anti-apoptotic mechanisms has not been identify. This research aimed to invistgate the cardioprotection of morphine postconditioning and the role of ERK1/2.Objectives1. To determine the cardioprotective effects of reducing MI/R injury by morphine postconditioning in vivo rat heart.2. To investigate whether the protection effect of morphine is mediated by enhancing the activation of extracellular signal regulated kinase (ERK1/2) pathway.3. To know the effect of morphine in the aspect of MI/R injury induced apoptosis, and the possible signaling mechanisms.Methods1. Cardioprotective effect induced by morphine postconditioning. Thirty-two healthy male Sprague-Dawley (SD) rats were used for in vivo model of ischemia/reperfusion injury. Anesthetized rats were randomly assigned to the following four groups with 30min of left coronary artery ischemia and 2h reperfusion:①Sham group, rats were opened chest without ligating the left coronary artery;②Ischemia/reperfusion(I/R) Group, as the control group with saline;③Morphine(MOR) group, 0.3mg/kg morphine was given intravenously 5min before reperfusion;④Ischemic preconditioning (IPC) group, 5min ischemia-5min reperfusion for three cycles before the long time ischemia. Hemodynamics, including the heart rate (HR), mean arterial pressure (MAP), and rate pressure product (RPP) were compared for each group. Myocardium was dyed with Evan's blue and TTC to determine the risks area and the area of infarction. Plasma was kept to detect the contents of AST, CK-MB and LDH after 120min reperfusion. The tissue and cell injury of myocardium was examined with optical microscope.2 . ERK1/2 pathway mediated the cardioprotective effect of morphine postconditioning. Forty male SD rats were subjected to 30min myocardial ischemia and 2h reperfusion. Anesthetized rats were randomly assigned to the following groups: (1) Sham group without ischemia and reperfusion; (2) I/R group with saline; (3) MOR group with 0.3mg/kg morphine given intravenously 5min before reperfusion; (4) ERK1/2 inhibitor PD98059 (PD) group, 0.3mg/kg PD98059 was given 10 min before reperfusion; (5) Morphine+PD98059 (MOR+PD) group, 0.3mg/kg PD98059 given 5 min before morphine. Hemodynamics and myocardial infarct size were determined to evaluate the injury severity of MI/R. Phosphorylation of ERK1/2(p-ERK1/2) was analysised by Western Blotting in samples of myocardium obtained after 5min of reperfusion. 3. The effect of morphine on apoptosis induced by MI/R injury and the related mechanisms. Forty male SD rats were randomly divided into 5 groups, 8 ones for each group. The groups were: Sham group, I/R group, MOR group, PD group and MOR+PD group. The myocardial apoptotic index (AI) was detected by terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method; the mRNA of Bcl-2 and Bax was detected by RT-PCR method; and theactivity of Caspase-3 was measured using a fluorospectrophotometer.Results1. The MAP decreased during ischemia and reperfusion period, butthe HR was not significant different among groups at any period. The risk area is roughly identical among the groups. Infarct size (%risk area) of MOR group decreased obviously compared to I/R group (31.1%±2.1% vs. 49.2%±2.1%, P<0.05). This significant difference also appeared between IPC group and I/R group (27.3%±5.1% vs. 49.2%±2.1%, P<0.05). The infarct-sparing effect was a little more in IPC group than in MOR group, although there was no statistical significance between the two groups. The decreases of the CK-MB, AST, and LDH paralleled with infart size. Compared with I/R group, the concentration of CK-MB, AST, and LDH in MOR group significantly decreased(P<0.05), which was the same tendency in the IPC group. Optical microscopy showed the morphological alterations of I/R injury, which could be moderated by morphine or ischemia preconditioning. 2. There was no significant difference in hemodynamics among disposal groups. The area at risk was comparable among groups. Infarct size (%risk area) was reduced from (49.2±2.1) % in I/R group to (31.1±2.1) % in the MOR group (P<0.05). PD98059 alone did not change the infarct size, but it blocked the infarct-sparing effect by morphine, as shown by increase into (43.3±4.2) % respectively. Western Blot showed that the total ERK1/2 protein expression was comparable among groups, but not the p-ERK1/2. Compared with Sham group, the protein expression of p-ERK1/2 was (5.6±0.3) fold in I/R group and (9.5±0.2) fold in MOR group. Morphine strongly enhanced the activation of ERK1/2 induced by I/R (P<0.05). PD98059 inhibited the activity of ERK1/2, also abolished the infarct-sparing effect of morphine.3. Apoptosis index (AI %) revealed by TUNEL was obviously increased in I/R group compared to Sham group (0.8%±0.16% vs. 18.7%±0.12%, P<0.05). Morphine significantly decreased apoptosis induced by M/R (9.8%±0.21% vs. 18.7%±0.12%, P<0.05). Compared with I/R group, MOR group also increased the ratio of Bcl-2/Bax mRNA expression (0.66±0.05 vs. 1.40±0.06, P<0.05), and inhibited Caspase-3 activity (3.1±0.13 vs. 1.8±0.31, P<0.05). Application of PD98059 strongly antagonized the effects of morphine.Conclusions1. In vivo rat heart, reduction of MI/R injury appeared in morphine postcondtioning, which was as powerful as ischemia preconditioning.2. ERK1/2 signaling pathway mediated the cardioprotective effects of morphine postconditioning.3 . Morphine postconditioning prevents MI/R injury induced apoptosis through activating ERK1/2 signaling pathway, up-regulating the ratio of Bcl-2/Bax gene expression, and reducing Caspase-3 activity.