Abstract Body: Background:
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 are limited. To address this and uncover genetic regulators, we surveyed CM ploidy across a novel genetic resource known as the Hybrid Rat Diversity Panel (HRDP).

Methods:
Langendorff digestions were used to assess CM ploidy (nuclear number and DAPI intensity) and dimension across 75 inbred rat strains of the HRDP (N=3-8/strain). Echocardiography was performed at 8 weeks of age on 13 strains (N=6-12/strain). All strains of the HRDP have been fully sequenced allowing for genetic mapping by GEMMA. Validation studies were carried out using a conditional knockout mouse model. Further, co-immunoprecipitation studies were used to assess the ability to bind ACTIN.

Results:
Broad phenotypic variability in the frequency of various ploidy classes was observed across the HRDP. Using 13 select rat strains with divergent displays of CM ploidy, we found that CM hyperpolyploidization (≥8N) correlates with various physiological parameters, i.e., left ventricular dilation and reduced ejection fraction, and cell dimension indicative of eccentric growth. Genome-wide association mapping identified several loci significantly associated with each ploidy class including the 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.

Conclusions:
This study elucidates the genetic determinants of CM ploidy phenotypes and solidifies a relationship between CM ploidy and left ventricular function. Initiated from an unbiased forward genetics approach using a novel genetic resource, we were able to identify not only one causative gene but also its functional variants.