Genome-wide association mapping and targeted loss of function studies identify <i>Shroom3</i> as a driver of hyperpolyploidy and ventricular dilation

Various states of cardiomyocyte (CM) polyploidy have been associated with cardiac injury responses, including regeneration and heart failure. However, our understanding of the comprehensive mechanisms governing CM ploidy and its relationship with heart physiology is limited. To address this issue and uncover genetic regulators, we surveyed CM ploidy across a new genetic resource known as the Hybrid Rat Diversity Panel (HRDP) and found significant variation in ploidy phenotypes across the panel. Using select rat strains with divergent displays of CM ploidy, we found that CM hyperpolyploidization (≥8 N) positively correlates with various physiological parameters, namely left ventricular dilation and reduced ejection fraction. Genome-wide association mapping identified several loci significantly associated with frequency of hyperpolyploid CMs. Investigation of genes harboring damaging protein coding variants within these loci identified enrichment of cytoarchitectural genes, of which the ACTIN-binding protein, Shroom3 , was found to be strongly and specifically expressed in CMs and harbors 7 damaging protein coding variants. CM-specific deletion of Shroom3 resulted in increased hyperpolyploidization and left ventricular dilation with reduced ejection fraction. Furthermore, functional characterization of single-nucleotide variants resulting in amino acid changes within SHROOM3 confirmed two protein coding variants that disrupted SHROOM3–ACTIN interaction and led to altered expression of genes involved in DNA replication. This study elucidates the genetic determinants of CM ploidy phenotypes and solidifies a correlative relationship between CM ploidy and left ventricular function. Importantly, CM intrinsic expression of at least one gene mapped in this study, Shroom3 , is confirmed to regulate CM hyperpolyploidization and cardiac function.