Roles of connexin-43 in cardiomyocyte tolerance against ischemic injury

心肌缺血可诱导大鼠心肌缝隙连接蛋白43（Cx43）与Src、PKC-ε和p38 MAPK-α形成复合物。缺血预处理（PC）不改变缺血时Cx 34-Src的相互作用，但显著增加Cx43与PKC-ε的结合。PC抑制Cx43免疫沉淀物中Cx43-p38 MAPK结合和p38 MAPK活性。PC可显著抑制荧光黄法测定的缝隙连接通透性，抑制PKC-ε可消除PC的这种作用。相反，抑制p38 MAPK适度增加心肌缺血期间间隙连接通透性。通过激活δ-阿片受体（负责触发PC机制的受体之一）评估间隙连接通透性抑制对心肌挽救的贡献。δ-阿片受体激动剂DADLE模拟PC对缝隙连接通透性和梗死面积的影响，PKC-ε抑制剂消除其对缝隙连接的影响可减轻梗死。 ...更多信息 DADLE的尺寸限制效果为35%。在心肌细胞系（H9 c2细胞）中，δ-阿片受体或IGF受体的激活诱导Akt和GSK-3β的磷酸化，并提供针对氧化应激诱导的细胞坏死的细胞保护。通过使用Cx43的siRNA抑制Cx43的表达，可消除PI 3 K-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，Akt和GSK-3β磷酸化以及针对IGF-1引起的坏死的细胞保护作用即使在Cx43敲低后也得以保留。本研究的结果表明，Cx43起着两个，至少，重要的作用，作为一个决定性的心肌缺血/再灌注损伤的耐受性。首先，间隙连接Cx43是PC机制的靶点，其通过差距连接抑制细胞损伤的传播。PKC-ε和p38 MAPK α分别是PC调节差距连接的主要抑制因子和对抗调节因子。第二，Cx43在来自活化的G蛋白偶联受体的细胞保护性PI 3 K-Akt-GSK-3β信号传导中起关键作用。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