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

连接蛋白43 （Cx43）定位于心肌细胞肌膜的间隙连接，但也存在于肌膜下线粒体的内膜。在这里，该蛋白参与染料摄取和钾的内流，表明Cx43也在线粒体中形成通道。此外，线粒体Cx43影响氧气消耗和钙处理。线粒体Cx43的减少与缺血预处理（IPC）后心脏保护功能的丧失有关。间隙连接的Cx43在多个位点被不同的激酶磷酸化，线粒体的Cx43也被磷酸化。在缺血/再灌注后，线粒体Cx43在生理条件下磷酸化的确切残基是未知的，将使用磷酸化特异性抗体进行鉴定。Cx43的磷酸化将在野生型小鼠和小鼠中进行研究，其中Cx43中的特定磷酸化位点（由蛋白激酶C （Cx43PKCmut小鼠）、酪蛋白激酶1 （Cx43CK1mut小鼠）和丝裂原活化蛋白激酶（Cx43MK4mut小鼠）靶向）对磷酸化不敏感。我们的目标是鉴定与线粒体Cx43相互作用的蛋白质。我们将分析Cx43PKCmut、Cx43CK1mut和Cx43MK4mut小鼠线粒体的线粒体功能，并与野生型小鼠进行比较。我们将野生型小鼠和Cx43磷酸化不敏感小鼠分别置于心肌缺血/再灌注条件下（无缺血预处理或有缺血预处理），研究Cx43磷酸化的重要性，并量化心肌梗死面积。在基因降低或药理抑制Cx43的模型中，获得了Cx43对线粒体功能和缺血/再灌注损伤重要性的先前数据。为了评估增加Cx43的量的效果，我们制造了小鼠，在心肌细胞中，他莫昔芬可以诱导Cx43的表达。将在这些小鼠中分析间隙连接和线粒体Cx43的数量，并与野生型小鼠进行比较。超声心动图将描述Cx43对心功能的重要性。此外，我们将在过表达cx43的小鼠中研究线粒体功能。在衰老心肌的线粒体中，线粒体Cx43的数量减少。这种Cx43减少与缺血预处理导致的梗死面积减少有关。在一项救援实验中，我们将诱导老年小鼠在缺血/再灌注时Cx43过表达，并测定缺血预处理和缺血预处理后的梗死面积。

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.