The Cardioprotective Effects Of Diazoxide And The Effects On ERK1/2,STAT3 Pathways

[Background] Ischemia reperfusion injury is a major pathophysiologic mechanism leading to myocardial dysfunction after myocardial infarction or cardiac surgery. It is necessary to find effective methods to limit ischemia reperfusion injury. Many effective methods, such as cardioplegia, ischemia or pharmacological preconditioning and postconditioning, heat shock, garners more and more attention. Cardioplegia can’t be used without CPB, and the ischemia postconditioning also has the risk when clip the vessels, so compared with those two strategy, pharmacological postconditioning has more benefit in the clinical practice. By regulating the transcription, ERK1/2 and STAT3 pathways play essential roles in ischemia reperfusion injury and myocardial protection. Diazoxide, a selective MitoK+ channel opener, has been reported to reduce the infarction area and inflammation after ischemia reperfusion, demonstrating the cardioprotective effect. However, the mechanism of the cardioproteciton remains unclear.[Objective] To investigate the influence of diazoxide on myocardial function in patients undergoing CABG and the cardioprotective effect against ischemia reperfusion in rat. Moreover, in this study, we also want to evaluate the role of ERK1/2 and STAT3 pathways in diaxoxide postconditioning. The study included three parts:(1) To compare the effects on myocardial function in the patients undergoing CABG between cardioplegia and DZX cardioplegia. (2) To examine the effects of DZX cardioplegia on infarction area and myocardial apoptosis following ischemia reperfusion injury. (3) To investigate the cardioprotective effects of dizoxide postconditioning against ischemia reperfusion injury and the role of ERK1/2 and STAT3 pathways in dizoxide postconditioning in vivo rat model, as well as the interaction of these two pathways.[Methods] First part:This clinical trial is a random, controlled, double-blinded research. Forty patients with TnT>0.05 (IU/L) who were scheduled for isolated elective CABG operations were randomized into placebo group (C group) and diazoxide group (D group). In the D group,50μmol/L diazoxide was infused together with cardioplegia. In the C group, the placebo infusion (0.9%NS) was given. Hemodynamic data, such as HR, MAP、CI、PAP、PCWP、CVP、SVR. PVR、LVSWI、RVSWI were measured before the operation, lh after CBP,6h,12h,24h after the operation respectively and biochemical markers of myocardial injury, CK-MB and TnT, were measured before the operation,6h,24h after the operation. The second part:48 isolated and perfused rat hearts were prepared first, and then randomly assigned to the following groups:sham group, ischemia reperfusion group (IR group), cardioplegia group (P group), cardioplegaia containing diazoxide group (DZX group). (1) Sham group:no ischemia; (2)IR group:30 min of ischemia and 60 min of reperfusion. (3) P group:after prepared, cardioplegia containing 0.4%DMSO was administrated for 5 min prior to ischemia, and then 30 min ischemia followed by 60min reperfusion. (4) DZX group:cardioplegia containing 50μmol/L diazoxide was administrated for 5 min prior to ischemia, and then 30 min ischemia followed by 60min reperfusion. Infarct size after reperfusion was determined by TTC staining. TUNEL analysis was used to detect the apoptosis after reperfusion. The third part: 72 adult male SD rats were randomly divided into following groups:(1)sham group, place silk under the coronary artery, but don’t occlude the artery. (2) ischemia-reperfusion injury group (IR group):LAD was occluded for 30min and then reperfusion 120min. (3)Vehicle group (Ve group):After LAD was occluded for 30min,1ml 0.4%DMSO was administrated through femoral vein at the onset of the reperfusion. (4)Diazoxide postconditioning group (DZX group):After LAD was occluded for 30min, lml diazoxide was administrated through femoral vein at the onset of the reperfusion. (5) Stattic group:During the ischemia,10 minutes before reperfusion, rats were treated with stattic through femoral vein, followed by diazoxide postconditioning at the onset of the reperfusion. (6)U0126 group:During the ischemia,10 minutes before reperfusion, rats were treated with U0126 through femoral vein rest procedure was same as that in static group. TTC staining and TUNEL analysis was detected after reperfusion. Western-blot and RT-PCR were used to detect the phosphorylated ERK1/2, STAT3 levels and the expression of ERK1/2, STAT3 mRNA.[Results] (1) CI increased postoperatively as compared with baseline. In the DZX group, the improvement of CI was better than that in the placebo group (p<0.05). LVSWI and RVSWI decreased 1h after CPB, and recovered much faster in the DZX group than those in placebo group (p<0.05). There were no statistically significant differences in the other hemodynamic parameters. CK-MB increased postoperatively, and the increase was remarkably faster in DZX group than that in placebo group (p<0.05). TnT decreased after the operation, and the decrease was greater in DZX group (p<0.05). (2) Cardioplegia could significantly reduce the infarction area and apoptosis index (infarction area:IR group 46.31%±5.21% vs P group 19.50%±6.19%; apoptosis index:IR group:25.91%±6.38% vs P group:11.65%±3.49%, p<0.01). However, the effect of DZX cardioplegia on myocardial infarction and apoptosis were greater than those in cardioplegia alone (infarction area:DZX group 8.50%±2.45% vs P group 19.50%±6.19%, p<0.05; apoptosis index:DZX group 6.20%±1.78% vs P group 11.65%±3.49%, p<0.05). (3) DZX postconditioning significantly reduced the infarction area and myocardial apoptosis (infarction area from 39.07%to 9.18%; apoptosis index from 46.63%to 12.43%, respectively, p<0.01), and increased the phosphorylation of both ERK1/2 and STAT3 as well as the expression of ERK1/2 and STAT3 mRNA. Stattic, the inhibitor of STAT3, and U0126, the inhibitor of ERK1/2, could abolish both the limitation of the infarction area and the anti-apoptosis effect of DZX postconditioning. (Compared with DZX postconditioning, p<0.01, respectively). Startic could downregulate the phospholation of ERK1/2 and the expression of ERK1/2 mRNA; however, U0126 did nothing on phospholated STAT3 level and STAT3 mRNA expression.[Conclusion] (1) Cardioplegia containing diazoxide confers additional myocardial protection beyond that provided by the cardioplegia alone by improving the contractibility of the heart and attenuating the release of CK-MB and TnT after CABG operations. (2) Attenuating the myocardial apoptosis maybe the possible mechanism of cardioplegia containing diazoxide on cardioprotection. (3) Diazoxide postconditioning showed remarkable cardioprotection by limiting myocardial infarction area and attenuating myocardial apoptosis, in which the activation of ERK1/2 and STAT3 pathways played important roles. (4) In the cardioprotective effects of diazoxide postconditioning, STAT3 pathway maybe the upstream of ERK1/2 pathway.