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Glycogen Synthase Kinase-3 Inhibition and Insulin Synergistically Enhance Proliferation and Inhibit Maturation of Human iPSC-derived Cardiomyocytes via TCF and FOXO Signaling

ABSTRACT: Embryonic signaling pathways exert stage-specific effects during cardiac development, yet the precise cues for proliferation or maturation remain elusive. To address this, we utilized spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) at day 12 of differentiation and performed a combinatory screen for various dosages of the glycogen synthase kinase-3 (GSK3) inhibitor CHIR99021 and Insulin for the analysis of cell cycle in hiPSC-CMs. Our combinatory screen for proliferation, subsequential downstream sarcomere development and RNA-seq analyses for Insulin/Akt and CHIR99021/Wnt demonstrate synergistic effects on proliferation of immature hiPSC-CMs. Conversely, removal of the Wnt and Insulin stimuli leads to rapid cell cycle exit and facilitates the terminal differentiation of immature hiPSC-CMs. Detailed characterization reveals that Wnt/CHIR99021, but not Insulin, regulates sarcomere homeostasis and architecture of immature hiPSC-CMs. Moreover, we further identify a temporal interplay between CHIR99021/Wnt via TCF and Insulin via FoxO signaling as regulators between proliferation and maturation in immature hiPSC-CMs. This work describes the cues that control proliferation versus terminal differentiation in functional immature hiPSC-CMs, and provides molecular mechanistic understanding between proliferation and maturation development of hiPSC-CMs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE278598 | GEO | 2024/12/10

REPOSITORIES: GEO

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Project description:Background ELMSAN1 is a newly identified scaffolding protein of the mitotic deacetylase complex (MiDAC), playing a pivotal role in early embryonic development. Studies on Elmsan1 knockout mice showed that its absence results in embryo lethality and heart malformation. However, the precise function of ELMSAN1 in heart development and formation has remained elusive. To gain insights into its potential role in cardiac lineage, we employed human-induced pluripotent stem cells (hiPSCs) to model early cardiogenesis and investigated the function of ELMSAN1. Methods and Results We generated ELMSAN1-deficient hiPSCs through both knockdown and knockout, revealing that ELMSAN1 appears to be unnecessary for hiPSC maintenance. During the process of cardiac differentiation, ELMSAN1 depletion led to a delay in pluripotency deactivation, decreased expression of cardiac-specific markers, and reduced differentiation efficiency. The impaired expression of genes associated with contractile sarcomere structure, calcium handling, and ion channels were also noted in ELMSAN1-deficient cardiomyocytes derived from hiPSCs (hiPSC-CMs). Additionally, through a series of structural and functional assessments, we found that these cells are immature, exhibiting incomplete sarcomere Z-line structure and length, decreased calcium amplitude, increased time to peak value, faster beat rate with a shorter beat period, decreased field potential duration, and prolonged propagation delay. Of note, we found that the cardiac specific role of ELMSAN1 is likely associated with histone H3K27 acetylation level. The transcriptome analysis provided additional insights, indicating maturation reduction with energy metabolism switch and restored cell proliferation in ELMSAN1 knockout cardiomyocytes. Conclusions In this study, we address the significance of the direct involvement of ELMSAN1 in the differentiation and maturation of hiPSC-CMs. We first report the impact of ELMSAN1 on multiple aspects of hiPSC-CM generation, including cardiac differentiation, sarcomere formation, calcium handling, electrophysiological maturation and proliferation.

Project description:ABSTRACT Background: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing mutant of the coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs. Viral proliferation on hiPSC-CMs was subsequently quantified using bioluminescence imaging. For drug screening, select antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate (PDTC), and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of some of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with the reported drug effects in previous studies. Finally, mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways within these hiPSC-CMs after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to confirm antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that could be used to screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion. For this experiment, human induced pluripotent stem cell derived cardiomyocytes were infected with coxsackievirus at multiplicity of infection (MOI) of 5 for 8 hours. Cells were treated with and without interferon beta 1 in order to determine if treatment activates antiviral response genes and/or viral clearance pathways. 4 total samples (2 for each condition) were analyzed

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Glycogen Synthase Kinase-3 Inhibition and Insulin Synergistically Enhance Proliferation and Inhibit Maturation of Human iPSC-derived Cardiomyocytes via TCF and FOXO Signaling

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