ABSTRACT/PROJECT SUMMARY Ischemic heart disease presents a substantial burden of disease, partially due to cardiomyocytes’ low turnover rate. Neonatal mice exhibit a transient cardiac regenerative capacity and serve as a valuable model to study cardiac regeneration. Many in the field theorize that the driver of the regenerative to non-regenerative shift is the reactive oxygen species (ROS) increase that occurs after birth as pups shift from a low to a relatively high- oxygen environment. These ROS are thought to lead to DNA damage, resulting in cell cycle arrest. Although it is well established that high levels of ROS can drive pathology in a variety of tissue types, ROS serve as critical signaling molecules in a wide range of biological processes. One critical developmental process that occurs during the early neonatal period is the metabolic shift to fatty acid oxidation, which is a hallmark of cardiomyocyte maturation. The primary goal of this F31 research proposal is to determine the necessity of neonatal ROS increase in establishing oxidative tolerance in cardiomyocytes prior to ischemic injury (Aim 1) and in driving a metabolic shift to fatty acid oxidation that is characteristic of mature cardiomyocytes (Aim 2). This study includes an AAV9- delivered constitutively active Nrf2 as well as a high-dose antioxidant model to suppress ROS during the early neonatal period. Preliminary RNA sequencing data showed a decrease in genes associated with fatty acid oxidation in ROS-depleted cardiomyocytes compared to controls, suggesting that ROS are required for cardiomyocyte metabolic maturation. To further study the impact of abnormal metabolic shifts on cardiomyocyte regeneration, a Cd36 knockout and AAV9-delivered overexpression model will be utilized in combination with human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (iCMs) studies to manipulate cell metabolism. The proposed study focuses on mitigation of neonatal development ROS to understand their role in cardiomyocyte maturation and establishing oxidative tolerance, in contrast to studies that have aimed to lower injury-related ROS. While cardiac pathology has mainly been attributed to oxidative stress, recent findings suggest that reductive stress may also have pathological implications. Elucidating the temporal regulation of ROS signaling and its influence on cardiomyocyte injury response and metabolism will help inform future therapeutics for the treatment of heart failure.