PRDM16 modulates aspects of cell cycle dynamics and maturation in human iPSC-derived cardiomyocytes

Figures

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  Figure 1. Establishment of ventricular cardiomyocyte identity and FUCCI-based analysis reveal cell-cycle-coupled PRDM16 expression in hiPSC-CMs (A) Schematic of ventricular cardiomyocytes differentiation from day 0 to day 14. (B) Flow cytometry plot showing enrichment of cTNT+ cardiomyocytes following sorting, with representative forward scatter (FSC) versus cardiac troponin T (cTNT) gating. (C) Quantitative RT-PCR analysis of ventricular (MYL2) and atrial (NR2F2) marker expression in ventricular versus atrial hiPSC-derived cardiomyocytes. Data are shown as mean ± SEM; ∗∗p < 0.01, ∗∗∗p < 0.001, n = 3 independent cultures per condition. (D) Diagram of the FUCCI cell-cycle reporter system used to identify G1 (Fucci-G1, orange) and S/G2/M (Fucci-S/G2/M, green) phases in live cardiomyocytes. (E) Relative PRDM16 mRNA expression in FUCCI-sorted hiPSC-derived cardiomyocytes in G1 (F-red) and S/G2/M (F-green) phases. Data are shown as mean ± SD; ∗∗∗p < 0.001, n = 3 independent cultures per condition. (F) Heatmap showing log fold change of cell-cycle-related genes in F-red versus F-green cardiomyocytes, indicating enrichment of proliferative markers in F-green cells. (G) Relative mRNA expression of…

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  Figure 2. PRDM16 is required for AKT-dependent cell-cycle progression in human iPSC-derived cardiomyocytes (A) Schematic of experimental timeline used to generate beating monolayer hiPSC-derived cardiomyocytes, followed by siPRDM16 knockdown and mTOR inhibition (Torin1, 200 nM). Time points for cell collection for imaging, RNA extraction and flow cytometry (FACS) are indicated. (B) Immunoblot confirming PRDM16 knockdown efficiency in hiPSC-CMs treated with control or siPRDM16. β-actin serves as a loading control. (C) Quantification of cell number across treatment conditions shown in Figure S2E. Data are shown as mean ± SEM. Statistical significance is indicated (n = 10 independent experiments; repeated measures one-way ANOVA, ∗p < 0.05; ns, not significant). (D) Flow cytometric quantification of FUCCI reporters showing the proportion of cells in S/G2/M phase under each experimental condition, highlighting increased cycling upon PRDM16 depletion and partial rescue by Torin1. (E) Representative fluorescence images of FUCCI-labeled hiPSC-CMs showing G1 (orange), S/G2/M (green), and merged signals under control, Torin1, siPRDM16, and siPRDM16 + Torin1 conditions. Arrowheads indicate cells in…

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  Figure 3. Transcriptomic profiling of PRDM16-deficient hiPSC-CMs reveals activation of cell cycle programs (A) Principal-component analysis (PCA) of bulk RNA-seq datasets from control and siPRDM16-transfected hiPSC-CMs, showing clear segregation between conditions (PC1: 28.6% variance, PC2: 22.7% variance). (B) Hierarchical clustering dendrogram (Manhattan distance) confirming distinct clustering of independent samples by condition. (C) Volcano plot of differentially expressed genes between control and siPRDM16 groups, highlighting upregulated (red, n = 1,498) and downregulated (blue, n = 40) transcripts, including enrichment of proliferation-associated factors. (D) Heatmap of selected cell cycle regulators (AURKB, CCNB1, CDK1, CDK4, ESPL1, MAD2L1, PCNA, and PLK1), demonstrating consistent upregulation in siPRDM16 hiPSC-CMs compared with controls.

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  Figure 4. PRDM16 constrains metabolic maturation while limiting proliferative re-entry of human iPSC-derived cardiomyocytes (A and C) Seahorse XF analysis of mitochondrial respiration in hiPSC-derived cardiomyocytes under control and Torin1 treatment, siPRDM16 cardiomyocytes tested with or without Torin1. Oxygen consumption rate (OCR) is plotted over time following sequential injection of mitochondrial stressors. Data are shown as mean ± SD (n = 5 wells from a single representative differentiation experiment). (B and D) Quantification of basal respiration, maximal respiration, spare respiratory capacity, and ATP-linked respiration from Seahorse measurements shown in (A). Data are presented as mean ± SD (n = 5 wells from a single representative differentiation experiment); statistical significance was determined by Welch’s t test (∗p < 0.05, ∗∗p < 0.01, and “ns” denotes not significant). (E) Immunofluorescence staining of hiPSC-CMs for TNNI1 (red; immature isoform) and TNNI3 (green; mature isoform) under control, Torin1, siPRDM16, and siPRDM16 + Torin1 conditions. Nuclei are counterstained with Hoechst (blue). Scale bars, 50 μm. (F) Immunofluorescence staining for phospho-histone H3 (pH3)…

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  Figure 5. PRDM16 overexpression promotes cardiomyocyte maturation while suppressing cell cycle activity (A) Experimental schematic showing differentiation of hiPSCs into cardiomyocytes followed by ectopic overexpression of PRDM16 using a lentiviral vector (pLenti6.3-PRDM16). Cells were harvested for RNA, protein, imaging, and three-dimensional engineered heart tissue (3D-EHT) analysis at the indicated time points. (B) Quantitative RT-PCR analysis of PRDM16 mRNA expression in control and PRDM16-overexpressing hiPSC-CMs. Statistical significance was determined by Welch’s t test. Data are shown as mean ± SEM; ∗∗∗p < 0.001, n = 3 independent experiments. (C) Immunoblot confirming PRDM16 protein overexpression in hiPSC-CMs. β-actin serves as a loading control. (D) Representative phase-contrast images of hiPSC-CM monolayers under control and PRDM16-overexpressing conditions. Scale bars, 100 μm. (E) Quantification of single-cell surface area in control and PRDM16-overexpressing hiPSC-CMs. Each dot represents one cell; statistical significance was determined by Welch’s t test, ∗∗∗p < 0.001, n = 100 cells per group. (F) Immunoblot analysis of cell-cycle and signaling regulators (CDK1, phospho-AKT,…

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  Figure 6. Model of PRDM16 as a regulator of the proliferation-maturation axis switch in hiPSC-CMs Schematic representation summarizing the dual role of PRDM16 in cardiomyocyte biology. Low PRDM16 expression promotes proliferation through upregulation of CDK1 and phospho-AKT and activation of metabolic programs associated with intrauterine-like states, resulting in increased mitotic index (pH3+, Ki-67+, and AURKB+) and sustained cell cycle activity. In contrast, high PRDM16 expression suppresses proliferation via CDK1 downregulation and phospho-AKT suppression, driving cardiomyocyte maturation characterized by cell-cycle arrest, organized sarcomere structure, and increased cell size.