The importance of connexin 43 and its phosphorylation for mitochondrial function and myocardial ischemia/reperfusion injury

连接蛋白43及其磷酸化对线粒体功能和心肌缺血/再灌注损伤的重要性

• 批准号: 411490269
• 负责人: Privatdozentin Dr. Kerstin Böngler
• 金额: --
• 依托单位: Physiologisches Institut
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/411490269?language=en
• 关键词: importance; connexin; 43; its; phosphorylation

Connexin 43 (Cx43) is localized at gap junctions of the cardiomyocyte sarcolemma, but is also present at the inner membrane of subsarcolemmal mitochondria. Here, the protein is involved in dye uptake and potassium influx, suggesting that Cx43 also forms a channel at the mitochondria. Furthermore, mitochondrial Cx43 influences oxygen consumption and calcium handling. A reduction in mitochondrial Cx43 is associated with a loss of cardioprotection following ischemic preconditioning (IPC). Gap junctional Cx43 is phosphorylated at multiple sites by different kinases, and also mitochondrial Cx43 is phosphorylated. The exact residues, which are phosphorylated in mitochondrial Cx43 under physiological conditions, after ischemia/reperfusion without and with IPC are unknown and will be identified using phospho-specific antibodies. Cx43 phosphorylation will be studied in wildtype mice as well as in mice, in which specific phosphorylation sites within Cx43 (targeted by protein kinase C (Cx43PKCmut mice), casein kinase 1 (Cx43CK1mut mice), and mitogen-activated protein kinase (Cx43MK4mut mice)), are rendered phosphorylation-insensitive. We aim to identify proteins which interact with mitochondrial Cx43. Mitochondrial function will be analyzed in mitochondria from Cx43PKCmut, Cx43CK1mut und Cx43MK4mut mice, and will be compared to that of wildtype mice. The importance of Cx43 phosphorylation will be studied by subjecting wildtype and Cx43 phosphorylation-insensitive mice to myocardial ischemia/reperfusion without or with ischemic preconditioning and myocardial infarct size will be quantified.Prior data on the importance of Cx43 for mitochondrial function and ischemia/reperfusion injury were obtained in models with genetic reduction or pharmacological inhibition of Cx43. To evaluate the effect of enhanced amounts of Cx43, we generated mice, in which the expression of Cx43 can be induced in cardiomyocytes by the administration of tamoxifen. The amount of gap junctional and mitochondrial Cx43 will be analyzed in these mice and compared to that of wildtype mice. The importance of Cx43 for heart function will be characterized by echocardiography. In addition, mitochondrial function will be studied in Cx43-overexpressing mice. In mitochondria from aged myocardium, the amount of mitochondrial Cx43 is reduced. This Cx43 reduction is associated with a loss of infarct size reduction by ischemic preconditioning. In a rescue experiment, Cx43 overexpression will be induced in aged mice and infarct size will be determined after ischemia/reperfusion without and with ischemic preconditioning.

连接蛋白43（Cx43）定位于心肌细胞肌膜的间隙连接处，但也存在于肌膜下线粒体的内膜。在这里，蛋白质参与染料摄取和钾离子内流，表明Cx43也在线粒体中形成通道。此外，线粒体Cx43影响氧消耗和钙处理。线粒体Cx43的减少与缺血预处理（IPC）后心脏保护的丧失相关。间隙连接Cx43在多个位点被不同的激酶磷酸化，并且线粒体Cx43也被磷酸化。确切的残基，这是磷酸化的线粒体Cx43在生理条件下，缺血/再灌注后，没有和有IPC是未知的，将使用磷酸化特异性抗体进行鉴定。将在野生型小鼠以及小鼠中研究Cx43磷酸化，其中Cx43内的特异性磷酸化位点（由蛋白激酶C（Cx43 PKCmut小鼠）、酪蛋白激酶1（Cx43 CK 1 mut小鼠）和促分裂原活化蛋白激酶（Cx43 MK 4 mut小鼠）靶向）对磷酸化不敏感。我们的目标是确定与线粒体Cx43相互作用的蛋白质。将在来自Cx43 PKCmut、Cx43 CK 1 mut和Cx43 MK 4 mut小鼠的线粒体中分析线粒体功能，并将其与野生型小鼠的线粒体功能进行比较。将通过使野生型和Cx43磷酸化不敏感小鼠经受心肌缺血/再灌注而不进行或进行缺血预处理来研究Cx43磷酸化的重要性，并且将定量心肌梗塞大小，在Cx43的遗传减少或药理学抑制的模型中获得关于Cx43对线粒体功能和缺血/再灌注损伤的重要性的先前数据。为了评估增强量的Cx43的作用，我们产生小鼠，其中Cx43的表达可以通过施用他莫昔芬在心肌细胞中诱导。将在这些小鼠中分析间隙连接和线粒体Cx43的量，并与野生型小鼠的量进行比较。将通过超声心动图表征Cx43对心脏功能的重要性。此外，将在Cx43过表达小鼠中研究线粒体功能。在来自老化心肌的线粒体中，线粒体Cx43的量减少。这种Cx43减少与缺血预处理减少梗死面积的损失有关。在挽救实验中，将在老年小鼠中诱导Cx43过表达，并在缺血/再灌注后测定梗死面积，不进行和进行缺血预处理。