The prevalence of heart failure continues to rise with unacceptable rates of morbidity and mortality. Currently, over 6 million Americans have been diagnosed with heart failure with an associated annual health care expense estimated at 35 billion US dollars and rising. The single most important cause for heart failure is ischemic cardiomyopathy, where loss of cardiomyocytes due to ischemic events is the main driver of disease. Due to limited rates of cardiac regeneration (estimated at 1% new cardiomyocyte formation per year during normal aging), the workload on remaining cardiomyocytes is increased, giving rise to cardiac hypertrophy and ultimately heart failure. Since the most important driver of heart failure is loss of cardiomyocytes, the discovery of cardiac progenitor cells (CPCs) in the adult heart that can give rise to all cardiac lineages when cultured uncovered the possibility of enhancing cardiac regeneration. Up until this point cell therapy using cultured CPCs has been the main method used to enhance cardiac regeneration. However, most injected cells die within weeks and the ultimate improvement in cardiac function is likely mediated by endogenous CPCs. The current proposal will identify ways of stimulating CPCs to enhance endogenous regeneration. Our long-term goal is to understand how endogenous CPCs are activated in response to cardiac injury and what determines CPC fate decisions in vivo with the ultimate objective of finding strategies that enhance endogenous cardiac regeneration. We recently published genetic mouse models that allow lineage tracing of endogenous cardiac progenitor cells and will use these mouse models throughout this proposal. Upon genetic lineage tracing of endogenous CPCs we noted many endothelial cells being generated, but only few cardiomyocytes. However, we hypothesize that endogenous CPCs can be activated to enhance their cardiogenic potential. The proposed genetic mouse models and manipulations of specific signaling pathways will be used to identify strategies to enhance cardiac regeneration. Our specific aims are to 1) Determine whether and how different stimuli activate endogenous cardiac c-kit+ CPCs toward cardiac lineage, 2) Determine whether blocking endothelial fates will activate endogenous c-kit+ CPCs to become cardiomyocytes, and 3) Establish to what extent Notch1 signaling is important for c-kit+ progenitor proliferation and differentiation toward both endothelial cells and cardiomyocytes. The goal of this study is to define pathways that can stimulate endogenous cardiac progenitor cells to produce more cardiomyocytes. Future studies will employ strategies to enhance the proliferation and differentiation of eCPCs to improve cardiac regeneration.