Big MAP Kinase 1-Cx 43 Signaling in Ischemia Injury

描述（申请人提供）：钙超载是缺血性心脏病中心肌细胞损伤和心功能不全的主要原因。间隙连接偶联抑制剂和连接蛋白（Cx）43基因敲除小鼠的研究表明，Cx 43在调节缺血/再灌注（IR）诱导的细胞Ca 2+超载中是重要的。Cx43间隙连接偶联受包括丝裂原活化蛋白（MAP）激酶在内的几种激酶调节。由于缺血预处理改善了IR损伤后心功能的恢复，并且还激活了MARK的成员Big-MAPK 1（BMK 1），我们假设预处理诱导的BMK 1的激活磷酸化Cx43，减少间隙连接偶联，并减少缺血/再灌注诱导的细胞Ca 2+超载。支持我们假设的关键数据包括：1）BMK 1磷酸化并与Cx43结合，并导致间隙连接解偶联，2）在表达特异性激活BMK 1的上游激酶MEK 5（CA-MEK 5）的组成型活性形式的心脏特异性转基因小鼠中，Cx43磷酸化增加，3）在Langendorff模型中，CA-MEK 5转基因心脏在IR损伤后表现出泵功能的显著加速恢复。我们将研究BMK 1和Cx43磷酸化在缺血后心功能障碍中的作用，具体目标如下：目的1：确定BMK 1在Cx43磷酸化和随后的Cx43间隙连接解偶联中的作用。目标二：确定BMK 1在Cx43磷酸化中的作用以及Cx43磷酸化对IR诱导的Ca 2+过载、随后的钙蛋白酶活性和线粒体功能障碍的影响。目标3：确定BMK 1和随后的Cx43磷酸化在体内IR损伤中的作用，目的4：研究BMK 1-Cx43信号传导在化学预处理中的作用。基础广泛的实验方法应使我们能够得出结论的重要性，BMK 1-Cx43信号在IR损伤。这一系列研究的长期目标是发现一种减少IR损伤的新治疗策略。

DESCRIPTION (provided by applicant): Ca2+ overload is a major cause of myocardial cell damage and cardiac dysfunction in ischemic heart diseases. Studies with gap junction coupling inhibitors and connexin (Cx) 43 knock out mice suggest that Cx43 is important in regulating ischemia/ reperfusion (IR)- induced cellular Ca2+ overload. Cx43 gap junction coupling is regulated by several kinases including mitogen-activated-protein (MAP) kinases. Since ischemic preconditioning improves the recovery of cardiac function after IR-injury and also activates Big- MAPK 1 (BMK1), a member of MARK, we hypothesized that activation of BMK1 induced by preconditioning phosphorylates Cx43, decreases gap junction coupling, and reduces ischemia/ reperfusion-induced cellular Ca2+ overload. Key data in support of our hypothesis include: 1} BMK1 phosphorylates and associates with Cx43, and leads to gap junction uncoupling, 2) Cx43 phosphorylation is increased in cardiac-specific transgenic mice expressing the constitutively active form of the upstream kinase MEK5 (CA-MEK5) which specifically activates BMK1, 3) CA-MEK5 transgenic hearts exhibit a profoundly accelerated recovery of pump function after IR-injury in a Langendorff model. We will study the role of BMK1 and Cx43 phosphorylation in post-ischemic cardiac dysfunction with the following specific aims: Aim 1: Define the role of BMK1 in Cx43 phosphorylation and subsequent Cx43 gap junction uncoupling. Aim 2: Define the role of BMK1 in Cx43 phosphorylation and the effect of Cx43 phosphorylation on IR- induced Ca2+ overload, subsequent calpain activity, and mitochondrial dysfunction. Aim 3: Determine the role of BMK1 and subsequent Cx43 phosphorylation in IR-injury, in vivo, and Aim 4: Investigate the role of BMK1-Cx43 signaling in chemical preconditioning. The broad-based experimental approach should allow us to conclude the importance of BMK1-Cx43 signaling in IR-injury. The long-range goal of this line of investigation is to discover a novel therapeutic strategy for reducing IR-injury.