Beardslee M A, Lerner D L, Tadros P N, Laing J G, Beyer E C, Yamada K A, Kléber A G, Schuessler R B, Saffitz J E
Department of Medicine, Center for Cardiovascular Research, Washington University School of Medicine, St. Louis, MO 63110, USA.
Circ Res. 2000 Oct 13;87(8):656-62. doi: 10.1161/01.res.87.8.656.
Electrical uncoupling at gap junctions during acute myocardial ischemia contributes to conduction abnormalities and reentrant arrhythmias. Increased levels of intracellular Ca(2+) and H(+) and accumulation of amphipathic lipid metabolites during ischemia promote uncoupling, but other mechanisms may play a role. We tested the hypothesis that uncoupling induced by acute ischemia is associated with changes in phosphorylation of the major cardiac gap junction protein, connexin43 (Cx43). Adult rat hearts perfused on a Langendorff apparatus were subjected to ischemia or ischemia/reperfusion. Changes in coupling were monitored by measuring whole-tissue resistance. Changes in the amount and distribution of phosphorylated and nonphosphorylated isoforms of Cx43 were measured by immunoblotting and confocal immunofluorescence microscopy using isoform-specific antibodies. In control hearts, virtually all Cx43 identified immunohistochemically at apparent intercellular junctions was phosphorylated. During ischemia, however, Cx43 underwent progressive dephosphorylation with a time course similar to that of electrical uncoupling. The total amount of Cx43 did not change, but progressive reduction in total Cx43 immunofluorescent signal and concomitant accumulation of nonphosphorylated Cx43 signal occurred at sites of intercellular junctions. Functional recovery during reperfusion was associated with increased levels of phosphorylated Cx43. These observations suggest that uncoupling induced by ischemia is associated with dephosphorylation of Cx43, accumulation of nonphosphorylated Cx43 within gap junctions, and translocation of Cx43 from gap junctions into intracellular pools.
急性心肌缺血期间，间隙连接处的电脱耦联会导致传导异常和折返性心律失常。缺血期间细胞内Ca(2+)和H(+)水平升高以及两亲性脂质代谢产物的积累会促进脱耦联，但其他机制可能也起作用。我们检验了这样一个假设，即急性缺血诱导的脱耦联与主要心脏间隙连接蛋白连接蛋白43（Cx43）的磷酸化变化有关。在Langendorff装置上灌注的成年大鼠心脏经历缺血或缺血/再灌注。通过测量全组织电阻来监测耦联变化。使用亚型特异性抗体，通过免疫印迹和共聚焦免疫荧光显微镜测量Cx43磷酸化和非磷酸化亚型的数量和分布变化。在对照心脏中，几乎所有在明显细胞间连接处通过免疫组织化学鉴定的Cx43都是磷酸化的。然而，在缺血期间，Cx43经历了逐渐的去磷酸化，其时间进程与电脱耦联相似。Cx43的总量没有变化，但细胞间连接处的总Cx43免疫荧光信号逐渐减少，同时非磷酸化Cx43信号积累。再灌注期间的功能恢复与磷酸化Cx43水平升高有关。这些观察结果表明，缺血诱导的脱耦联与Cx43的去磷酸化、间隙连接内非磷酸化Cx43的积累以及Cx43从间隙连接向细胞内池的转运有关。
