Role of PMNL, PAF and KCnk3 in Cardiac Arrhythmias

描述(申请人提供)：多形核白细胞(PMNL)在缺血/再灌流后迅速积聚在心脏中，并在随后的组织损伤和心律失常的产生中发挥关键作用。我们重点研究了炎性磷脂、血小板激活因子(PAF)在这一过程中的作用。PAF在分离的心室肌细胞上引起心律失常，并影响体外灌流的梗死心脏的节律。这项应用的目的是确定PAF诱发心律失常的发生机制，从而确定抗心律失常治疗的新靶点。 目的1：PAF诱发心律失常的离子和信号机制是什么？ 我们认为，PAF诱导的心律失常是由两孔结构域K+通道Kcnk3(最初称为cTBAK-1或TASK-1)的蛋白激酶C-epsilon(PKC-epsilon)阻断引起的。在表达Kcnk3的小鼠心室肌细胞或CHO细胞中，采用生化和电生理相结合的方法，我们将确定Kcnk3通道是否是PKC-epsilon或下游激酶磷酸化的目标，如果是，我们将确定磷酸化位点(S)，并制备定点和截断突变体。将表征所表达的通道的功能属性。我们进一步提出，大麻素受体的脂质配体阿南达胺阻断Kcnk3的细胞内信号通路与PAF途径重叠，并模拟PAF的致心律失常作用。我们假设PAF和花生胺之间存在生化关系。 目的2：PAF在完整心脏中是否会导致心律失常？缺血是否会改变心脏对PAF的反应性？PAF受体(PAFR)拮抗剂在脑梗塞动物模型中是有益的。我们认为，梗塞心脏对PAF诱发的心律失常的敏感性高于正常心脏。使用完整的动物，或PAFR基因敲除的完整心脏和在朗宁多夫灌流装置中过度表达PAFR的转基因小鼠，以及分离的肌细胞与PMNL或细胞因子孵育，我们将确定心脏敏感性的调节机制。我们将比较PAF和Anandamide在对照心脏或缺血/再灌流后诱发心律失常的能力。我们假设，激活心肌细胞的条件将上调PAFR的表达，并使心脏对心律失常敏感。我们将测量电生理参数并量化PMNL与分离的心肌细胞结合并诱导心律失常的能力。

DESCRIPTION (provided by applicant): Polymorphonuclear leukocytes (PMNL) accumulate rapidly in the heart after ischemia/reperfusion and play a critical role in the ensuing tissue damage and generation of arrhythmias. We have focused on the role of the inflammatory phospholipid, platelet-activating factor (PAF) in this process. PAF causes arrhythmias in isolated ventricular myocytes, and affects the rhythm of infarcted hearts perfused ex vivo. The goal of this application is to determine the mechanisms by which PAF-induced arrhythmias occur and thus, identify novel targets for antiarrhythmic therapy. Aim 1: What are the ionic and signaling mechanisms by which PAF induces arrhythmias? We propose that PAF-induced arrhythmias are caused by a protein kinase C-epsilon (PKC-epsilon) block of the two-pore domain K+ channel, Kcnk3 (first known as cTBAK-1 or TASK-1). Using a combination of biochemical and electrophysiological methods in murine ventricular myocytes or CHO cells expressing murine Kcnk3, we will determine if the Kcnk3 channel is the target for phosphorylation by PKC-epsilon or a downstream kinase and if so, we will determine the phosphorylation site(s) and prepare site-directed and truncation mutants. The functional properties of the expressed channels will be characterized. We further propose that the intracellular signaling pathway by which anandamide, the putative lipid ligand of the cannabinoid receptors, blocks Kcnk3 overlaps with the PAF pathway and mimics the arrhythmogenic effects of PAF. We hypothesize that there is a biochemical relationship between PAF and anandamide. Aim 2: Is PAF arrhythmogenic in intact heart and does ischemia alter the responsiveness of heart to PAF? PAF receptor (PAFR) antagonists are beneficial in animal models of infarction. We suggest that the sensitivity of infarcted heart to PAF-induced arrhythmias is greater than normal heart. Using whole animals, or intact hearts from PAFR knockout and transgenic mice that overexpress the PAFR in a Langendorff perfusion set-up and isolated myocytes incubated with PMNL or cytokines, we will determine the mechanism by which the sensitivity of the heart is modulated. We will compare the ability of PAF and anandamide to induce arrhythmias in control heart or after ischemia/reperfusion. We hypothesize that conditions that activate myocytes will upregulate PAFR expression and sensitize the heart to arrhythmias. We will measure electrophysiological parameters and quantify the ability of PMNL to bind to isolated myocytes and induce arrhythmias.