Project Summary Heart failure (HF) is the long term effect of the mechanical stress and chronic inflammation that occurs following an acute ischemic event in cardiac tissue. Current pharmacological agents can only manage the symptoms of HF, and are not reparative. Importantly, cardiovascular disease perpetuates as the leading cause of death worldwide. Therefore, it is crucial to elucidate the mechanisms that govern cardiac injury responses in order to realize effective HF therapies. Although extensive studies have strongly focused on the replenishment of cardiac muscle cells, cardiomyocytes, it has been shown that other cell types in the heart, including resident immune cells and endothelial cells (ECs), are also essential for maintaining tissue homeostasis and orchestrating injury responses. Thus, understanding how these different cardiac cell types govern the injury response leading up to HF is crucial for combatting this deadly disease. Adult mouse myocardial infarction (MI) models of left anterior descending arterial occlusion (LAD-O) exhibit very similar cardiac remodeling to the failing human heart. However, neonatal mice less than 8 days old possess a robust regenerative capacity when subjected to MI and are able to achieve an almost complete functional recovery with scar resolution. Our preliminary single-cell RNA sequencing (scRNA-seq) data uncovered a unique endothelial cell population that emerges post-MI in regenerative postnatal day 1.5 (P1.5) hearts but not in non- regenerative P8 hearts. Moreover, immune cells such as macrophages are expanded in the heart during an injury response to promote a pro-reparative environment. Transcriptome analysis of this regenerative EC population revealed an immune signature, suggesting that ECs may facilitate a pro-regenerative immune response via direct myeloid cell recruitment. The objective of this study is to determine the role of ECs in heart regeneration. Given the above findings, we therefore hypothesize that neonatal cardiac regenerative endothelial cells (rECs) promote regeneration by expanding a pro-reparative immune cellular composition. Utilizing mouse genetics and multi-omics, we aim to uncover the role of rECs and their immune signature in cardiac regeneration. We will study whether rECs expand local reparative immune cells to promote mammalian cardiac regeneration utilizing a combination of in vitro culturing experiments and in vivo transplantation studies. We will then further investigate whether the immune signature of rECs facilitate mammalian heart regeneration by promoting the expansion of reparative macrophages after MI utilizing mouse genetics and in vivo gene editing. These experiments will provide novel insights into how rECs regulate regeneration and help uncover new therapeutic targets in immunomodulation for heart failure.