Birth is the most abrupt transition during life, and the neonatal heart must accommodate to this dramatic change in environment by increasing its output to the body. Exposure to higher levels of oxygen at birth likely activates intracellular pathways that allow cardiac myocytes to rapidly proliferate and then differentiate to cause the final maturation of cardiac structure and function that is required for this increased output and survival. However, major gaps in our understanding of this process remain. It is apparent that mitochondria play an important role in this process. We have found that mitochondria regulate cardiac development in the embryo and neonate and that the mitochondrial chaperone protein, cyclophilin D (CyPD), regulates changes in mitochondrial function and reactive oxygen species (ROS) production that control cardiomyocyte proliferation and differentiation. Our preliminary data have begun to define changes in this CyPD-mitochondrial-ROS-differentiation pathway that occur in the neonatal heart. In addition, these data provide novel models to dissect the mechanisms of this pathway. These findings suggest the hypothesis that increased O2 at birth initiates a rise and then fall in CyPD activity, which regulates mitochondrial function, particularly ROS production, to control neonatal myocyte proliferation and differentiation and cardiac function. The scientific premise of this proposal is supported by data discussed above, but the mechanisms involved have not been fully elucidated. Our overall goal is to use our expertise in cardiac development and mitochondrial biology to dissect the mechanisms that control this important physiologic pathway in the neonatal heart and determine if CyPD inhibition can be used to ameliorate pathology in clinically relevant models. To achieve these goals, we propose 3 Specific Aims: 1. Determine how CyPD controls the neonatal cardiac mitochondrial-ROS-differentiation pathway. 2. Determine effects of disrupting CyPD activity in the neonatal heart. 3. Determine effects of hypoxia on the neonatal CyPD- mitochondria-ROS-differentiation pathway. The proposed experiments use a novel set of pharmacologic and genetic approaches that manipulate oxygen, CyPD, inner mitochondrial membrane coupling, and ROS in the neonatal heart. Specimens will be processed using a battery of assays to measure CyPD expression, acetylation, and activity; mitochondrial structure and function, ETC activity and assembly, ROS production; myocyte proliferation and differentiation; and cardiac function. Our team has unique expertise in cardiac, developmental, and mitochondrial biology and in biostatistics and we employ novel concepts and cutting-edge techniques to study mitochondria during late cardiac development. The anticipated results will significantly change our understanding of bioenergetics in the neonatal heart and will lead to future studies that use mitochondrial targeted therapies to enhance cardiac function and cardiac m...