Abstract Body: Background: Most ventricular cardiomyocytes (CMs) are tetraploid (2x2N in rodents and 1X4N in humans). However, hyperpolyploidy (≥8N) is noted in various disease states and arises by activating an alternative cell cycle whereby cells replicate their DNA but fail to divide. We purported that prevalence of hyperpolyploid CMs could be used as a predictive phenotype for cardiomyopathy and thereby leveraged as a surrogate, quantifiable trait for genome-wide association (GWA) mapping.

Methods: We utilize a novel genetic resource, the Hybrid Rat Diversity Panel (HRDP) consisting of 96 inbred rat strains to perform GWA mapping on CM hyperpolyploidy. Across 13 strains, we also perform echocardiography to establish which functional parameters are associated with divergent ploidy phenotypes. Through gene knockout and viral approaches, we investigate the impact of candidate gene, Shroom3, on CM ploidy, cardiac function, and SHROOM3 protein binding interactions.

Results: Across 78 strains of the HRDP, we observe wide phenotypic variation in the frequency of the hyperpolyploid CMs, ranging from 0.8-20.7%. The frequency of hyperpolyploid CMs displayed a strong correlation with ventricular dilation and negative correlation with ejection fraction, indicative of dilated cardiomyopathy (DCM). GWA mapping identified 19 loci associated with hyperpolyploidy. Within the Chr14 locus, lies Shroom3, a gene highly specific to CMs with 7 single amino acid variants across rat strains predicted to be damaging to protein function. CM-specific Shroom3 knockout mice display exacerbated hyperpolyploidy, ventricular dilation, and reduced ejection fractions by 6-weeks of age. Co-immunoprecipitation analysis in HEK293T cells establish the functional role of SHROOM3 amino acid variants on ACTIN binding, while in vivo adenovirus rescue experiments delineate a role for Shroom3 variants in driving postnatal hyperpolyploidy.

Conclusions: Our data identify CM hyperpolyploidy as a genetically inherited trait and an early indicator of DCM. The HRDP represents a novel, powerful, and renewable genetic resource to understand idiopathic cardiomyopathies and other cardiovascular traits of interest. We are working to extend this resource into systems biology capabilities.