Myocardial connexin 43 (Cx43) forms gap junctions and hemichannels, and is also present within subsarcolemmal mitochondria. The protein is phosphorylated by several kinases including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and casein kinase 1 (CK1). A reduction in Cx43 content abrogates myocardial infarct size reduction by ischemic preconditioning (IPC). The present study characterizes the contribution of Cx43 phosphorylation towards mitochondrial function, hemichannel activity, and the cardioprotection by IPC in wild-type (WT) mice and in mice in which Cx43-phosphorylation sites targeted by above kinases are mutated to non-phosphorylatable residues (Cx43^MAPKmut, Cx43^PKCmut, and Cx43^CK1mut mice). The amount of Cx43 in the left ventricle and in mitochondria was reduced in all mutant strains compared to WT mice and Cx43 phosphorylation was altered at residues not directly targeted by the mutations. Whereas complex 1 respiration was reduced in all strains, complex 2 respiration was decreased in Cx43^CK1mut mice only. In Cx43 epitope-mutated mice, formation of reactive oxygen species and opening of the mitochondrial permeability transition pore were not affected. The hemichannel open probability was reduced in Cx43^PKCmut and Cx43^CK1mut but not in Cx43^MAPKmut cardiomyocytes. Infarct size in isolated saline-perfused hearts after ischemia/reperfusion (45 min/120 min) was comparable between genotypes and was significantly reduced by IPC (3 × 3 min ischemia/5 min reperfusion) in WT, Cx43^MAPKmut, and Cx43^PKCmut, but not in Cx43^CK1mut mice, an effect independent from the amount of Cx43 and the probability of hemichannel opening. Taken together, our study shows that alterations of Cx43 phosphorylation affect specific cellular functions and highlights the importance of Cx43 phosphorylation by CK1 for IPC’s cardioprotection.

心肌连接蛋白 43 (Cx43) 形成间隙连接和半通道，并且也存在于肌膜下线粒体中。该蛋白被几种激酶磷酸化，包括丝裂原活化蛋白激酶 (MAPK)、蛋白激酶 C (PKC) 和酪蛋白激酶 1 (CK1)。Cx43 含量的减少通过缺血预处理 (IPC) 消除了心肌梗死面积的减少。本研究描述了 Cx43 磷酸化对线粒体功能、半通道活性和 IPC 在野生型 (WT) 小鼠和上述激酶靶向的 Cx43 磷酸化位点突变为非磷酸化残基的小鼠中的心脏保护的贡献。 Cx43 ^MAPKmut、 Cx43 ^PKCmut和 Cx43 ^CK1mut老鼠）。与 WT 小鼠相比，所有突变株的左心室和线粒体中 Cx43 的数量减少，并且 Cx43 磷酸化在突变未直接靶向的残基处发生了改变。尽管所有菌株的复合物 1 呼吸减少，但复合物 2 呼吸仅在 Cx43 ^CK1mut小鼠中减少。在 Cx43 表位突变的小鼠中，活性氧的形成和线粒体通透性转换孔的开放不受影响。^{Cx43 PKCmut}和 Cx43 ^CK1mut的半通道开放概率降低，但 Cx43 ^{MAPKmut没有}心肌细胞。缺血/再灌注（45 分钟/120 分钟）后孤立的盐水灌注心脏的梗死面积在基因型之间相当，并且在 WT、Cx43 ^MAPKmut和 Cx43 ^PKCmut中通过 IPC（3 × 3 分钟缺血/5 分钟再灌注）显着减少，但在 Cx43 ^CK1mut小鼠中没有，这种影响与 Cx43 的量和半通道开放的概率无关。总之，我们的研究表明 Cx43 磷酸化的改变会影响特定的细胞功能，并强调 CK1 磷酸化 Cx43 对 IPC 心脏保护的重要性。