PROJECT SUMMARY / ABSTRACT Cardiomyocytes are the working muscle cells of the heart, and their number is intrinsically tied to heart function and resilience. Too few increases the stress on each cardiomyocyte, leading to pathological changes and a vicious cycle of cell death and fibrosis. In hypoplastic left heart syndrome (HLHS), the right ventricle (RV) is the single working ventricle for life. This puts a lot of stress on the cardiomyocytes, often leading to heart failure early in life. Given that the RV is expected to do twice the usual work in these hearts, the usual number of cardiomyocytes is too few in HLHS. The goal of this proposal is to investigate how the cardiomyocytes of the fetal heart with reduced left ventricular inflow grow differently than normal, whether by proliferation, hypertrophy, or apoptosis. This information will help us to develop therapies to increase cardiomyocyte number in hearts of these most vulnerable infants. Although prior studies by experienced investigators have not perfectly modeled clinical HLHS, each has added new and important information. We have newly established a fetal sheep model of impaired left ventricular inflow, similar to the hemodynamic pathology of HLHS. Comparing fetal sheep chronically affected by impaired left heart inflow versus those with normal hemodynamics, we will assess changes in the physical and hormonal factors that we know regulate cardiomyocyte growth, maturation and survival before birth. We will then look at the hearts themselves to determine how they have grown, and how they function. We will study the cardiomyocytes to understand their contribution to heart growth, and to test how they respond to the regulatory factors changed in the fetus. Finally, we will test ways to intervene and increase cell number in the heart of the fetus with reduced LV inflow. Ultimately, this work will help optimize cardiomyocyte number in babies with HLHS in order to strengthen their hearts for life-saving surgery and make them more resilient against heart failure for life.