PROJECT SUMMARY Dysregulation of autonomic control of the heart can provoke arrhythmias, predisposing individuals to potentially life-threatening medical problems such as myocardial infarction and stroke. Many of the medications currently available for treating arrhythmias exhibit limited efficacy and/or the potential for long-term pro-arrhythmic side effects. Accordingly, it is important to gain a more comprehensive understanding of the molecular mechanisms underlying cardiac rhythmicity, so that new and safer approaches for the treatment of arrhythmias can be developed. A long-term goal of our research is to better understand the mechanisms underlying the parasympathetic regulation of the heart and to investigate the therapeutic potential of manipulating relevant signaling pathways for the treatment of rhythm disorders. To this end, we and others have shown that a signaling pathway consisting of the M2 muscarinic receptor (M2R) and IKACh, a G protein-gated K channel + formed by GIRK1 and GIRK4 subunits, mediates much of the parasympathetic influence on heart rate and atrial physiology. While recent evidence suggests that the parasympathetic system also regulates ventricular physiology, the molecular mechanisms underlying this influence are less clear. One goal of this project is to delineate the mechanisms and relevance of the parasympathetic influence on ventricular physiology (AIM 1), testing the working hypothesis that this influence is mediated by an M2R-IKACh signaling pathway in ventricular myocytes. I will test this hypothesis using a unique array of constitutive and tissue-specific knockout mice, together with whole-cell patch-clamp electrophysiology and in vivo electrocardiogram (ECG) approaches. A second goal of this project is to begin exploring the therapeutic potential of IKACh modulators in the context of cardiac rhythm disorders (AIM 2). While numerous studies support the contention that IKACh agonists and antagonists could be used to treat certain types of arrhythmias, including atrial fibrillation, sick sinus syndrome, atrio-ventricular block, and one hereditary type of long QT syndrome (LQTS13), attempts to test this prospect have been impeded by the lack of direct-acting and selective IKACh modulators. Recent high-throughput screening efforts have led to the development of a novel family of GIRK channel agonists and antagonists. Using whole-cell electrophysiological approaches, I will evaluate the selectivity, potency, and efficacy profiles of these compounds. Those exhibiting superior pharmacodynamic characteristics, including selectivity for IKACh relative to neuronal GIRK channels, will be further evaluated in the isolated heart preparation, exploiting established genetic and pharmacologic arrhythmia models. Successful completion of the proposed project will shed new light on the mechanisms and relevance of parasympathetic regulation of the heart, and will yield critical preclinical insights required to assess the potential efficacy associated with manipulation of a key parasympathetic effector (IKACh) in the context of cardiac rhythm disorders.