Roles of connexin-43 in cardiomyocyte tolerance against ischemic injury

心肌缺血诱导大鼠心脏缝隙连接蛋白-43 （Cx43）与Src、PKC-ε和p38MAPK-α复合物的形成。缺血预处理（PC）不改变缺血期间Cx34-Src的相互作用，但显著增加Cx43与PKC-ε的结合。PC抑制Cx43免疫沉淀中Cx43-p38MAPK结合和p38MAPK活性。路西法黄评价的间隙结渗透率被PC显著抑制，而PC的这种影响被PKC-ε的抑制所消除。相反，抑制p38MAPK可适度增加心肌缺血时间隙连接的通透性。通过δ-阿片受体的激活来评估间隙连接通透性抑制对心肌修复的贡献，δ-阿片受体是触发PC机制的受体之一。δ-阿片受体激动剂DADLE模拟了PC对间隙连接通透性和梗死面积的影响，PKC-ε抑制剂消除了它对间隙连接的影响，使DADLE的梗死面积限制作用减弱了35%。在心肌细胞系（H9c2细胞）中，δ-阿片受体或IGF受体的激活可诱导Akt和GSK-3β的磷酸化，并对氧化应激诱导的细胞坏死提供细胞保护。使用Cx43的siRNA抑制其表达，可以消除PI3K-Akt-GSK-3β信号传导和δ-阿片受体激动剂诱导的细胞保护作用。然而，即使在敲除Cx43后，IGF-1对Akt和GSK-3β的磷酸化和细胞保护作用也得以保留。本研究结果表明，Cx43至少在心肌耐受缺血/再灌注损伤方面起着两个重要作用。首先，间隙连接Cx43是PC机制的靶点，该机制通过间隙连接抑制细胞损伤的传播。PKC-ε和p38MAPKα分别是PC缝隙连接调节的主要抑制因子和拮抗调节因子。其次，Cx43在活化的g蛋白偶联受体传递细胞保护PI3K-Akt-GSK-3β信号中起关键作用。少

Myocardial ischemia induced complex formation of gap junction connexin-43 (Cx43) with Src, PKC-ε and p38MAPK-α in rat hearts. Ischemic preconditioning (PC) did not modify Cx34-Src interaction during ischemia but significantly increased binding of Cx43 with PKC-ε. PC suppressed Cx43-p38MAPK binding and p38MAPK activity in the Cx43 immunoprecipitates. Gap junction permeability assessed by Lucifer yellow was significantly suppressed by PC, and this effect of PC was abolished by inhibition of PKC-ε. In contrast, inhibition of p38MAPK modestly increased gap junction permeability during myocardial ischemia. Contribution of suppressed gap junction permeability to myocardial salvage was assessed by activation of the δ-opioid receptor, which is one of receptors responsible for triggering PC mechanisms. A δ-opioid receptor agonist, DADLE, mimicked the effect of PC on gap junction permeability and infarct size, and elimination of its effect on gap junction by a PKC-ε inhibitor attenuated infarct … More size-limiting effect of DADLE by 35%. In cardiomyocyte cell line (H9c2 cell), activation of the δ-opioid receptor, or the IGF receptor induces phosphorylation of Akt and GSK-3β and afford cytoprotection against oxidant stress-induced cell necrosis. Suppression of Cx43 expression by use of its siRNA abolished both PI3K-Akt-GSK-3β signaling and cytoprotection induced by a δ-opioid receptor agonist. However, Akt and GSK-3β phosphorylation and cytoprotection against necrosis by IGF-1 were preserved even after knock-down of Cx43. The results of the present study indicate that Cx43 plays two, at least, important roles as a determinant of myocardial tolerance against ischemia/reperfusion injury. First, gap junction Cx43 is a target of PC mechanisms which suppress propagation of cell injury via the gap junction. PKC-ε and p38MAPKα are a primary inhibitory factor and a counter-acting modulation factor, respectively, of the gap junction regulation by PC. Second, Cx43 plays a crucial role in transmission of cytoprotective PI3K-Akt-GSK-3β signaling from activated G-protein coupled receptors. Less