PROJECT SUMMARY / ABSTRACT Atrial fibrillation (AF), the most common arrhythmia, is associated with high morbidity and mortality, but remains difficult to treat due to an incomplete understanding of underlying mechanisms. Emerging evidence suggests that nucleoside diphosphate kinases (NDPKs) play an important role in the heart by being able to elevate cAMP levels in a G-protein receptor-independent manner. Our pilot data suggest that increased levels of NDPK-B and NDPK-C isoforms in patients with persistent (chronic) AF are associated with elevated cAMP levels, abnormal sarcoplasmic reticulum Ca2+ releases, ectopic (triggered) activity and inducible AF. The long- term goal of this project is to dissect the molecular and cellular basis of NDPK-dependent arrhythmia mechanisms in AF. The central hypothesis is that enhanced NDPK-B and -C levels promote AF by enhancing cAMP levels in the RyR2 microdomain, resulting in aberrant intracellular Ca2+ signaling that creates a substrate for AF initiation, maintenance, and progression. Three specific aims will be pursued: 1) Determine whether increased NDPK expression is both necessary and sufficient to promote spontaneous AF, 2) Assess whether elevated NDPK levels cause cAMP-dependent SR Ca2+ leak via RyR2, and 3) Determine the role of Ankrd1 within the RyR2-NDPK signaling complex. These studies will be performed in atrial myocytes from patients with AF, a dog model of AF, and various atrial-selective genetic mouse models of AF. Novel targeted cAMP FRET sensors and atrial-selective adeno-associated virus (AAV9)-mediated gene therapy will be utilized to resolve the pro-arrhythmic roles of NDPK-B/C and cAMP within specific cellular microdomains. These studies are expected to establish whether and how NDPK isoforms contributes to AF development and serve as a platform for the validation of NDPKs as novel druggable target for the prevention or treatment of AF.