Role of PMNL, PAF and KCnk3 in Cardiac Arrhythmias

描述（由申请人提供）：多形性白细胞（PMNL）在缺血/再灌注后在心脏中迅速积聚，并在随后的组织损伤和心律失常的产生中起关键作用。我们集中在炎症磷脂，血小板活化因子（PAF）在这一过程中的作用。PAF在离体心室肌细胞中引起心律失常，并影响离体灌注的梗死心脏的节律。本申请的目的是确定PAF诱导的心律失常发生的机制，从而确定抗心律失常治疗的新靶点。 目的1：PAF诱导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蛋白激酶C-κ B（PKC-κ B）阻断双孔结构域K+通道Kcnk 3（最初称为cTBAK-1或ASK-1）引起的。 在表达鼠Kcnk 3的鼠心室肌细胞或CHO细胞中使用生物化学和电生理学方法的组合，我们将确定Kcnk 3通道是否是PKC β或下游激酶磷酸化的靶点，如果是，我们将确定磷酸化位点并制备定点和截短突变体。表达的通道的功能特性将被表征。 我们进一步提出，大麻素受体的假定脂质配体anandamide通过其阻断Kcnk 3的细胞内信号传导途径与PAF途径重叠，并模仿PAF的促肾上腺皮质激素作用。 我们推测PAF和花生四烯酸之间存在生化关系。 目的2：PAF是否在完整心脏中致心律失常？缺血是否改变心脏对PAF的反应性？ PAF受体（PAFR）拮抗剂在梗塞的动物模型中是有益的。 提示梗塞心脏对PAF诱发的心律失常的敏感性高于正常心脏。使用整个动物，或完整的心脏从PAFR基因敲除和转基因小鼠，过表达PAFR在Langendorff灌注设置和分离的肌细胞与PMNL或细胞因子孵育，我们将确定的机制，心脏的敏感性是调制。我们将比较PAF和花生四烯酸酰胺在对照心脏或缺血/再灌注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表达，使心脏对心律失常敏感。 我们将测量电生理参数，并量化PMNL结合分离的心肌细胞和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.