Project Summary Abstract Heart disease remains the number one cause of death worldwide, due to the inability of the injured adult heart to regenerate. We seek to delineate the mechanisms that govern development, disease and regeneration of the heart and to build upon this knowledge to restore cardiac function during injury, disease and aging. In contrast to the adult mammalian heart, which lacks regenerative capacity, the neonatal heart can efficiently regenerate following severe injury. To explore the molecular underpinnings of neonatal cardiac regeneration, we have analyzed global changes in gene expression and the epigenome during regeneration of the neonatal mouse heart in comparison to later stage hearts that cannot regenerate. We have also performed single-cell RNA sequencing of cardiomyocytes and the major non-myocyte cell types from neonatal regenerative and non- regenerative hearts with or without injury. Integration of these comprehensive datasets has begun to reveal a “regenerative” chromatin landscape of the heart and the transcriptional activators and repressors of this process. The overarching goal of this project is to build upon this body of information to elucidate the mechanisms that control the responses of the neonatal heart to injury and to harness these mechanisms to promote adult cardiac regeneration and repair. By focusing on regenerative transcriptional circuitry and paracrine signaling mechanisms, we intend to devise new strategies to enhance cardiomyocyte proliferation and survival, angiogenesis and other regenerative processes mediated by various cellular constituents of the heart. Ultimately, the molecular decoding of cardiac regeneration will provide a molecular blueprint for activating endogenous pathways for cardiac repair and facilitate new strategies for restoring function to the injured and aging heart.