Cardiomyocyte ploidy has been described but remains obscure in cardiac interstitial cells. Ploidy of c-kit+ cardiac interstitial cells was assessed using confocal, karyotypic, and flow cytometric technique. Notable differences were found between rodent (rat, mouse) c-kit+ cardiac interstitial cells possessing mononuclear tetraploid (4n) content, compared to large mammals (human, swine) with mononuclear diploid (2n) content. In-situ analysis, confirmed with fresh isolates, revealed diploid content in human c-kit+ cardiac interstitial cells and a mixture of diploid and tetraploid content in mouse. Downregulation of the p53 signaling pathway provides evidence why rodent, but not human, c-kit+ cardiac interstitial cells escape replicative senescence. Single cell transcriptional profiling reveals distinctions between diploid versus tetraploid populations in mouse c-kit+ cardiac interstitial cells, alluding to functional divergences. Collectively, these data reveal notable species-specific biological differences in c-kit+ cardiac interstitial cells, which could account for challenges in extrapolation of myocardial from preclinical studies to clinical trials.

Ploidy for cardiomyocytes is well described but remains obscure in cardiac interstitial cells (CICs). Ploidy of c-kit+CICs (cCICs) were assessed using a combination of confocal, karyotypic, and flow cytometric assessments coupled with molecular and bioinformatic analyses. Fundamental differences were found between cultured rodent (rat, mouse) cCICs possessing mononuclear tetraploid (4n) content versus large mammal (human, swine) with mononuclear diploid (2n) content. In-situ analysis, confirmed with fresh isolates, revealed diploid content in cCICs from human and a mixture of diploid and tetraploid nuclei in mouse. Molecular assessment of the p53 signaling pathway provides a plausible explanation for escape from replicative senescence in rodent but not human cCICs. Single cell transcriptional profiling reveals distinctions between diploid versus tetraploid populations in mouse cCICs, alluding to functional divergences. Collectively, these data reveal fundamental species-specific biological differences in cCICs that could account for challenges in extrapolation of myocardial preclinical studies from rodent to large animal models.

No abstract

Discovery of endogenous stem cells found within the heart, cardiac progenitor cells (CPC), has prompted intense basic discovery in multiple experimental animal models and clinical trials in heart failure patients. A survey of the literature reveals animal or cellular models exhibiting regenerative properties are also characterized by genome duplication or polyploidization. Our lab recently discovered a fundamental difference between large and small mammalian CPCs through karyotype analysis: rodent (rat, mouse) CPCs possess mononuclear tetraploid (4n) chromosome content, whereas large animal (human, swine) CPCs are mononuclear diploid (2n). This primary distinction between large and small animals prompts provocative questions regarding regenerative potential as well as the translational applicability of regenerative studies performed in rodent models. To expand on this finding, CPCs were isolated from eight human tissue samples (normal and heart failure), two swine strains (Gottingen and Yorkshire) six mice strains (FVB, C57, CAST, SPRET, SAMP6, and SAMR1), and seven rat clonal lines (Sprague Dawley), which confirmed karyotyped ploidy content per species as characterized through flow cytometry and confocal microscopy techniques. The large (human, swine) and small (rat, mouse) mammal CPCs maintained stable chromosome content through increased passaging and positive correlations appears between chromosome content and growth rate through increased passages as well as chromosome content and unrestrained growth through increased passages. The ploidy content of endogenous CPCs was investigated in situ with heart tissue sections with comparable ploidy determinations to that of cultured CPCs. Furthermore, the unique tetraploid content of murine CPCs is reinforced by comparison with ckit+ progenitor cells of secondary tissues (intestine and bone marrow) and cardiomyocytes in situ and in vitro that exhibit mostly diploid (2n) chromosome content per nuclei. These studies suggest ploidy may play a significant role in the biological capacity of the CPC and the endogenous stem cell pool may influence the regenerative capacity of the heart. Future directions will focus on defining the advantage of polyploid content in mediating regeneration.