PROJECT SUMMARY Heart Failure has resisted the downward trend in mortality seen in other diseases and new therapies are needed. Heart failure is driven by the hypertrophy and loss of cardiomyocytes, which comprise the bulk of the heart by volume and provide the main contractile force necessary to move blood throughout the body. Over 90% of cardiomyocytes in mammalian species are polyploidal and/or multinucleated. These extra copies of DNA have been studied in the context of the low regenerative capacity of the heart, yet comparatively little effort has focused on other potential adaptive or maladaptive effects of this increased nuclearity. Historically, the large size of the mature cardiomyocyte has precluded its analysis using unbiased single cell RNA sequencing. Research has focused on RNAseq in isolated nuclei, which by its very nature lacks information about nuclearity within the studied hearts. Recently, new technology has allowed for unbiased, high-throughput single cell RNAseq of whole, mature cardiomyocytes. When the results of these single cell studies are compared to the isolated nuclei studies, researchers find that, using the same clustering algorithms, there are approximately twice as many distinct cellular transcriptome clusters as there are distinct nuclear transcriptome clusters. This result suggests the existence of a form of ‘nuclear code’ in which a multinucleated cell’s transcriptome is ‘encoded’ by nuclei drawn from two or more nuclear clusters, a result supported by observations from other multinucleated cell populations. These encoded cells could display differences in contractility, resistance to apoptosis, or even proliferative potential, with significant implications for cardiac function and the development of heart failure. The goal of this Steven I. Katz proposal is to 1) Utilize cutting edge single cell and single nucleus sequencing technology to identify and confirm this nuclear encoding, 2) Explore the spatial distribution of mono and multi- nucleated cells across the heart, and 3) Identify how this encoding responds to genetic and environmental perturbations in healthy and failing hearts. As requested by the mechanism, in this proposal we set forth a plan for an ambitious new direction for our laboratory’s research, built upon rigorous work documented in the literature and supported by collaborators and subject matter experts. The proposal combines bioinformatic, imaging, and next generation sequencing approaches to identify how changes in the nuclear code of multinucleated cardiomyocytes leads to differences in response to cardiac injury and stress. Upon completion, this grant will result in a more complete understanding of the role of multinuclearity and polyploidy in the mammalian heart, with subsequent revelation of numerous new avenues of research for diagnostic and therapeutic approaches to combat heart failure.