Project Summary Cardiac myocyte number is a fundamental determinant of heart function which, due to progressive cell loss, becomes especially important when the heart is stressed, such as when there is pediatric or adult heart disease. Although intensive medical effort is focused on sparing myocytes during the disease process and there is intense (as-yet unrealized) interest in restoring numbers through stem cell therapies, no work has been done to address the large perinatal loss of cardiac myocytes. This is a therapeutic opportunity to increase life-long cardiac myocyte number substantially. What is not known are the exact timing of the cell loss in the peripartum period, and the signals in the near-birth cardiac milieu driving the cell loss. In the same window during which myocyte loss occurs, the fetus faces increasing hypoxia, the fetal heart undergoes metabolic maturation in preparation for the high-fat milk diet to come immediately after birth, and thyroid hormone increases many-fold. The interaction of these three factors likely contribute to cardiac myocyte loss via oxidative stress and apoptosis. Further, male fetuses are notable for having “risky” in utero growth strategies which may lead to increased sensitivity to pathological developmental programming. We suspect that the interactions of these signaling pathways render hearts susceptible before birth, but that oxidative stress and cell death can be suppressed by an antioxidant. In this study we will use a large animal model (sheep) to examine both isolated (in culture) effects of these regulators on metabolism and apoptosis, as well as their role in the more complex in utero environment. We will use the Seahorse Bioanalyzer to measure cardiomyocyte respiration with different metabolic substrates and under different test conditions. We will also measure cell death, as assessed by cell number, enzyme release and activity, and apoptotic and autophagic pathway activation. We will tie these results in to reactive oxygen species generation and activation of cell death pathways. We will also determine if melatonin supplementation to reduce oxidative stress prevents the near-term loss of cardiac myocyte number. Significance: Heart failure can be reduced by increasing the number of healthy cardiac myocytes. Myocyte endowment is substantially reduced around the time of birth in an event that represents a powerful opportunity for intervention. We aim to increase myocyte number at birth by targeting the connection between metabolic maturation, hypoxia and cell loss in a translational model applicable to human physiology.