Project Summary In contrast to the cardiomyocytes (CMs) from lower vertebrates, adult mammalian CMs possess very limited regenerative potential as a result of cell cycle exit. Interestingly, neonatal mice retain cardiac regenerative capacity, which is lost by postnatal day 7. We have recently shown that 1-day-old pigs can also regenerate lost myocardium in response to myocardial infarction (MI). This regeneration is mediated by the proliferation of preexisting CMs, which does not occur when CMs permanently exit the cell cycle. Mechanisms underlying the injury-mounted regenerative response especially in large mammals are not fully understood. However, investigating underlying mechanisms is likely to identify novel targets for future therapeutic interventions. Recent studies in fish and rodents emphasized the critical importance of vascularization and autonomic innervation of the regenerating myocardium in zebrafish and neonatal mouse hearts. Besides their function to provide nutrients, transport metabolites and enable adaptation to stress, it is unknown whether vascular and neuronal cells, via paracrine interactions, also promote CM proliferation. Intriguingly, our preliminary data support the idea that soluble factors, e.g., cytokines, secreted from vascular endothelial cells and peripheral sympathetic neurons significantly stimulate cell cycle activity of co-cultured human induced pluripotent stem cells-derived CMs, suggesting a critical role of nonmyocyte-CM interactions in modulating CM proliferation in hearts of larger mammals post injury. In this project, we will exploit the established high regenerative capacity of the neonatal pig heart model to experimentally address the role of nonmyocytes in injury-induced cardiac regeneration in large mammals. Two specific aims are proposed. Aim 1 is to define the role of early revascularization in injury-mounted cardiac regeneration. We will test the hypothesis that early revascularization is essential for cardiac regeneration in neonatal pigs, and determine whether angiogenesis promotes cardiac regeneration through the release of pro-myogenic factors from endothelial and/or smooth muscle cells and/or via de novo formation of functional vessels for maintenance of CM viability. Aim 2 is to delineate the role of autonomic innervation in injury-mounted cardiac regeneration. We will test the hypothesis that innervation is essential for post injury cardiac regeneration in neonatal pigs, and determine if biomaterial-mediated epicardial delivery of angiogenic and neurotrophic factors enhances cardiac regeneration in neonatal pigs post MI.