Role of PMNL, PAF and KCnk3 in Cardiac Arrhythmias

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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.