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Effects of Pharmacological Polyploidization in Human iPSC-derived Cardiomyocytes

ABSTRACT: Cell polyploidization, an increase in cellular DNA content without cytokinesis, is closely linked with aspects of cellular function in various tissues and is a defining characteristic of postnatal heart development in many species. However, investigation into how polyploidy affects cardiomyocytes (CMs) has been limited. Despite these challenges, CM polyploidy has been directly shown to attenuate proliferation in zebrafish CMs and diploid CM abundance correlates with recovery following coronary artery ligation in mice, highlighting the importance of understanding this biological process in CMs. In this study, we generated polyploid hiPSC-CMs via inhibition of Aurora Kinase B and found this results in increased cell size, reduced baseline cycling rates and responsiveness to mitogenic stimuli, affected the overall transcriptome and rates of transcription and translation, and altered CM Ca2+ handling. Our work suggests that CM polyploidy may not be just an outcome of CM maturation, but rather it serves a functional purpose. Further examination of the differences between diploid and polyploid hiPSC-CMs can expand our understanding of postnatal CM development and how polyploidy impacts CM growth and maturation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE293429 | GEO | 2025/08/23

REPOSITORIES: GEO

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Project description:A defining feature of the mammalian liver is polyploidy, a numerical change in the entire complement of chromosomes. The first step of polyploidization involves cell division with failed cytokinesis. Although polyploidy is common, affecting ~90% of hepatocytes in mice and 50% in humans, the specialized role played by polyploid cells in liver homeostasis and disease remains poorly understood. The goal of this study was to identify novel signals that regulate polyploidization, and we focused on microRNAs (miRNAs). First, to test whether miRNAs could regulate hepatic polyploidy we examined livers from Dicer1 liver-specific knockout mice, which are devoid of mature miRNAs. Loss of miRNAs resulted in a 3-fold reduction in binucleate hepatocytes, indicating that miRNAs regulate polyploidization. Secondly, we surveyed age-dependent expression of miRNAs in wild-type mice and identified a subset of miRNAs, including miR-122, that is differentially expressed at 2-3 weeks, a period when extensive polyploidization occurs. Next, we examined Mir122 knockout mice and observed profound, life-long depletion of polyploid hepatocytes, proving that miR-122 is required for complete hepatic polyploidization. Moreover, the polyploidy defect in Mir122 knockout mice was ameliorated by adenovirus-mediated over-expression of miR-122, underscoring the critical role miR-122 plays in polyploidization. Finally, we identified direct targets of miR-122 (Cux1, Rhoa, Iqgap1, Mapre1, Nedd4l and Slc25a34) that regulate cytokinesis. Inhibition of each target induced cytokinesis failure and promoted hepatic binucleation. Conclusion: Our data demonstrate that miR-122 is both necessary and sufficient in liver polyploidization. Among the different signals that have been associated with hepatic polyploidy, miR-122 is the first liver-specific signal identified. These studies will serve as the foundation for future work investigating miR-122 in liver maturation, homeostasis and disease. Livers from C57Bl/6 mice were isolated at defined ages: embryonic day 15.5 (n=3; mixed gender), 2 weeks (n=3; male), 3 weeks (n=3, male) and 7 weeks (n=3; male). Differential miRNA expression was assessed using the nCounter Mouse miRNA Expression Assay Kit (nanoString).

Project description:Human induced Pluripotent Stem Cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used to identify potential factors capable of inducing endogenous cardiomyocyte proliferation to regenerate the injured heart. L-type calcium channel blockers have previously been identified as a class of factors capable of inducing matured hiPSC-CMs to proliferate. However, the mechanism by which L-type calcium channel blockers promote hiPSC-CM proliferation remains unclear. Here we provide evidence that matured hiPSC-CMs possess plasticity to undergo dematuration in response to certain pharmacological compounds. Consistent with primary cardiomyocyte maturation during perinatal development, we found that centrosome disassembly occurs in hiPSC-CMs during plate-based, temporal, maturation. A small molecule screen identified Nitrendipine, an L-type calcium channel blocker, and 1-NA-PP1, a Src kinase inhibitor, as factors capable of inducing centrosome reassembly in a subpopulation of hiPSC-CMs. Furthermore, centrosome-positive hiPSC-CMs were more likely to exhibit cell cycle activity than centrosome-negative hiPSC-CMs. In contrast, neither Nitrendipine or 1-NA-PP1 induced centrosome reassembly, or cell cycle activity, in neonatal rat ventricular myocytes (NRVMs). Differential bulk transcriptome analysis indicated that matured hiPSC-CMs, but not NRVMs, treated with Nitrendipine or 1-NA-PP1 undergo dematuration. ScRNA transcriptome analysis supported that matured hiPSC-CMs treated with either Nitrendipine or 1-NA-PP1 undergo dematuration. Collectively, our results indicate that matured hiPSC-CMs, but not primary NRVMs, possess plasticity to undergo dematuration in response to certain pharmacological compounds such as L-type calcium channel blockers and Src-kinase inhibitors. This study shows that once mature, hiPSC-CMs may not maintain their maturity under experimental conditions and thus may have implications for experimental systems where the state of hiPSC-CM maturation is relevant.

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Effects of Pharmacological Polyploidization in Human iPSC-derived Cardiomyocytes

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