PROJECT SUMMARYAbnormal activity of the cardiac ryanodine receptor (RyR2) leads to increased and untimely release of Ca2+ fromthe sarcoplasmic reticulum (SR) driving Ca2+-dependent arrhythmogenesis that can lead to sudden death inmany cardiac disorders. Oxidative modification of RyR2 by reactive oxygen species (ROS) has long beenestablished to enhance the sensitivity of the channels to Ca2+ within the SR (intraluminal Ca2+) in the failing heart.However both the intracellular source of ROS as well as the specific redox-sensitive residues of RyR2 whichcontrol intraluminal Ca2+ sensitivity remain elusive. Our initial studies implicate the role of the SR oxidoreductasesystem in this control whereby molecular chaperones and enzymes that facilitate protein folding also modulateactivity of RyR2. We have identified intraluminal cysteines of RyR2 that elicit functional effects on the channelas well as an oxidoreductase chaperone that associates with the channel in a redox-dependent manner.Moreover we found upregulation of oxidoreductase enzyme in rodent models of cardiac disease and observedRyR2 activity stabilization with pharmacological inhibition of this enzyme. We therefore hypothesize thatdysregulation of the SR oxidoreductase system impairs luminal Ca2+ regulation of RyR2 via an intraluminal SRredox sensor and promotes arrhythmogenesis. We will test our hypothesis by 1) defining the molecularcomponents of the SR redox sensor that control luminal Ca2+ sensitivity of RyR2 and 2) determining the role ofdysregulated SR redox homeostasis in Ca2+-dependent arrhythmogenesis. To address these aims we willemploy a multilevel experimental approach investigating at the molecular cellular and whole heart level. Wepropose to use heterologous systems biochemical approaches and human induced pluripotent stem cellcardiomyocyte (hiPSC-CM) technology to identify the RyR2 redox sensor. We also propose to study disease-associated perturbations of the SR oxidoreductase system in rodent models of inherited and acquired Ca2+-dependent arrhythmia utilizing novel genetic biosensors as well as adenoviral (AV) and adeno-associated viral(AAV) gain- and loss- of function approaches. With renowned experts in cardiac EC coupling proteinbiochemistry and hiPSC-CM technology The Ohio State University offers an exceptional training environmentfor the mentored phase of the award to reach these goals. Furthermore building on my strong background inmolecular biology I will collaborate with an expert in CRISPR-mediated gene editing of hiPSC-CMs to studythese mechanisms in a relevant human model. The achievement of the proposed aims will uncover novelregulatory mechanisms of RyR2 regulation with potential to be therapeutically exploited. This proposal thereforeaddresses a fruitful and unexplored research area relevant to a spectrum of cardiovascular diseases which willlay strong foundations for an independent research career in cardiovascular physiology.