No abstract

Somatic polyploidization, an adaptation by which cells increase their DNA content to support growth, is observed in many cell types, including cardiomyocytes. Although polyploidization is believed to be beneficial, progression to a polyploid state is often accompanied by loss of proliferative capacity. Recent work suggests that genetics heavily influence cardiomyocyte ploidy. However, the developmental course by which cardiomyocytes reach their final ploidy state has only been investigated in select backgrounds. Here, we assessed cardiomyocyte number, cell cycle activity, and ploidy dynamics across two divergent mouse strains: C57BL/6J and A/J. Both strains are born and reach adulthood with comparable numbers of cardiomyocytes, however the end composition of ploidy classes and developmental progression to reach the final state differ substantially. We expand on previous findings that identified Tnni3k as a mediator of cardiomyocyte ploidy and uncover a novel role for Runx1 in ploidy dynamics and cardiomyocyte cell division, both in developmental and injury contexts. These data provide novel insight into the developmental path to cardiomyocyte polyploidization and challenge the paradigm that hypertrophy is the only mechanism for growth in the postnatal heart.

Outcomes following cardiac injury are influenced by lifestyle and environment, as well as genetic background. Our interest lies in the latter, where very few genes have successfully been linked to heart failure. Contributors to this low success rate include high genetic diversity of patient populations, inability to control for non-genetic factors, and cellular complexities that contribute to outcomes. We propose utilizing the Hybrid Rat Diversity Panel (HRDP) to assess high throughput surrogate phenotypes, which can be predictive of outcomes after injury when measured in the basal state. This approach can offer a solution to the limitations of association studies for complex diseases like heart failure. Our surrogate phenotype of interest is cardiomyocyte ploidy, where high frequency of diploid cardiomyocytes is predictive of ability to regenerate and functional recovery. Conversely, an increased proportion of polyploid (≥8N) cardiomyocytes is associated with adverse ventricular remodeling and dilated cardiomyopathy. Having analyzed 50 of the 96 strains, we see that frequency of the MNDCMs varies from 1.3-20.3% across strains, while frequency of the highly polyploid CMs (≥8N) varies from 0.8-20.9%. Four strains have been selected for myocardial infarction (MI) studies, including M520 (17.4%) and F344 (20.3%) with high MNDCM content and GK (2.8%) and HXB23 (1.8%) with low MNDCM content. Following MI, both M520 and F344 rats exhibit a higher percentage of BrdU+ cardiomyocytes in the border zone region compared to HXB23 rats, supporting the hypothesis that strains with high MNDCM frequency would be better at mounting a proliferative response. This also translated to a greater functional improvement and smaller scars in M520 rats, but not in F344. Additionally, we looked at the size of cardiomyocytes following MI and found that HXB23 rats (20.9% ≥8N CMs) have an increased cross-sectional area in comparison to the other strains, which may be indicative of concentric hypertrophy in response to injury. The HRDP could serve as a powerful tool for mapping traits related to outcomes following MI, allowing us to identify potential gene candidates that may be interrogated further for future use in precision medicine-based therapies. Mapping studies are ongoing.

Polyploidization is a normal cellular adaptation for a variety of cell types in mammals, including cardiomyocytes (CMs); however, the development of polyploidization understudied. Recent work suggests that genetics contribute to diverse displays of ploidy in the adult murine heart. Therefore, we hypothesized that the developmental progression to reach differing end-states may similarly be affected. Here, we assessed CM endowment, cell cycle, and ploidy temporally across two diverse inbred mouse strains, A/J and C57Bl/6J. Consistent with previous work, C57Bl/6J hearts displayed rapid cell cycle activation in the first postnatal week largely coinciding with the first round of endomitosis. Total CM numbers are relatively unchanged after P7, while final ploidy states are generally constant from P14 on. In contrast, A/J mice displayed depressed cell cycle activation in the first week of life. Polyploidy in A/Js reaches its peak at P21, whereby only ~3.5% of CMs remained mononuclear and diploid. Interestingly, from 3-6 weeks of age, we observed a dramatic expansion of total CM numbers and of the 1x2N subpopulation to ~8%, which could not be explained by proliferation of the residual diploid population. Instead, the expanded diploid population appears to come from a polyploid CM. This finding was confirmed by analysis identifying a population of CMs which had definitively completed cytokinesis by 6 weeks which was not present at 3 weeks. We believe this is the first report of ploidy reversal by a mammalian CM, a phenomenon first observed by the hepatocyte field. Ongoing single nuclear RNA seq is examining the transcriptomic differences between A/J and C57Bl/6J CMs at P21 to identify this unique “primed” population competent to undergo ploidy reversal. Preliminary results from this study suggest that AJ CM nuclei, regardless of their ploidy, are more likely to be in the cell cycle in comparison to C57. Understanding the developmental paths to end state CM endowment and ploidy states can help us understand the biology of organ size and reciprocally can be applied to stimulating regeneration.

The longstanding belief in the field is the mammalian heart is incapable of regenerating. However, findings regarding the variability of regenerative capacity of mammalian hearts are opening the door for such a possibility. Mononuclear diploid cardiomyocytes (MNDCMs) are a subpopulation of CMs believed to be capable of mounting a regenerative response and the prevalence of the MNDCM population in mammalian hearts is a variable trait. Thus, if we can understand the genetic mechanisms governing the MNDCM variability, it could indicate strategies to stimulate an endogenous regenerative response. Using a genome-wide association approach across a panel of genetically diverse inbred mice, Dr. Patterson identified genes associated with the frequency of MNDCMs in the uninjured adult heart. From this genetic screen, she validated the gene Tnni3k as one regulator of CM ploidy and regenerative competence after injury. Here, we follow a second novel locus identified from this screen found on chromosome 15, which includes the gene Hdac7. Previously published RNA-seq data on isolated CMs indicate that Hdac7 expression levels increase during the first postnatal week, a time that coincides with the onset of cellular senescence, CM polyploidization, and the loss of innate regenerative capacity in the heart. Using a CM specific Hdac7 conditional knockout mouse, we found that loss of Hdac7 in CMs from birth increases CM DNA synthesis, increases CM ploidy, and increases average CM cell length. Moreover, we identified a protein coding variant of Hdac7 that corresponds to differences in frequency of MNDCM between members of the mouse diversity panel. Finally, we have gathered preliminary findings that suggest genetic epistasis between Hdac7 and Tnni3k. Specifically, Hdac7;Tnni3k double knockout animals display more MNDCMs than Tnni3k knockout did on its own. Our findings suggest Hdac7 negatively regulates DNA synthesis; a key part of the cell cycle that has the potential as a therapeutic target for the stimulation of a proliferation response. In addition, these findings showcase complex genetic interactions regulating CM regeneration, suggesting that combinatorial approaches can enhance regenerative responses.