Effects of Pharmacological Polyploidization in Human iPSC‐Derived Cardiomyocytes

ell polyploidization, an increase in cellular DNA content without cytokinesis, is closely linked with aspects of cellular function in various tissues 1 and is a defining characteristic of postnatal heart development in many species.2 However, investigation into how polyploidy affects cardiomyocytes (CMs) has been limited due to difficulties in directly comparing diploid to polyploid CMs in vivo as well as inability to culture isolated adult CMs long term without affecting their phenotype.Despite these challenges, CM polyploidy has been directly shown to attenuate proliferation in zebrafish CMs, 3 and diploid CM abundance correlates with recovery following myocardial infarction in mice, 4 highlighting the importance of understanding this biological process in CMs.Unlike in vivo CMs, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are largely diploid and are a valuable system to test how induction of polyploidy alone, independent from other stimuli, affects CM proliferation and function.RNA sequencing data are available on the Gene Expression Omnibus (GEO accession number: GSE293429), and all other data needed to evaluate the conclusions in the article are present in the article.The work performed in this article does not require Institutional Review Board approval and no animals were used in this work.ARRIVE reporting guidelines were followed for article submission (Percie du Sert et al., Br J Pharmacol).To induce CM polyploidy, we targeted Aurora Kinase B (AURKB), an important regulator of mitosis.hiPSC-CMs were treated with the AURKB ATP-competitive inhibitor, AZD1152 (AURKBi), for 4 days followed by a 7day washout period and downstream analysis to study the steady-state consequences of treatment.Flow cytometric analysis showed that AURKBi treatment increased the abundance of polyploid CMs (>2N by DNA intensity, Figure [A]).In culture, AURKBi-treated CMs exhibited larger area (Figure [B]).Expectedly, flow cytometry analysis revealed that polyploid CMs were larger than their diploid counterparts in both control (Ctrl) and AURKBi conditions, with higher fraction of polyploid CMs in the AURKBi group resulting in larger CMs size at a population level (Figure [B]).Importantly, AURKBi-induced polyploidy was not associated with increased apoptosis (Figure [B′]).We then performed bulk RNA-sequencing to compare the transcriptome of Ctrl and AURKBi hiPSC-CMs and found 318 differentially expressed genes (Figure [C]).Gene ontology analysis showed decreased expression of genes related to cell cycling and glycolysis, while some lipid metabolism genes were increased (Figure [1C′-C″]).Based on these results, we first performed flow cytometry measurements of transcription and translation rates and found that while transcription was increased, translation rates were decreased by AURKBi (Figure [1D]), likely indicative of a less proliferative cell population.Interestingly, AURKBi-treated CMs exhibited increased nucleolin abundance, consistent with the higher transcription present in the polyploid CMs (Figure [D]).We also observed increased mitochondrial abundance in AURKBi CMs relative to Ctrl, which along with decreased expression of glycolysis