Thyroid hormones regulate cardiac repolarization and QT-interval related gene expression in hiPSC cardiomyocytes

Figures

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  Figure 1. Schematic representation of MEA experimental timeline. (a) scheme of MEA probe plating and maturation protocol of hiPSC-CMs. (b) schematic illustration of treatments and field potential (FP) MEA recordings time (red box). For a detailed description see “MEA experimental protocol”.

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  Figure 2. Effect of T3 treatment on FPDc in hiPSC-CMs. Upper panel: analysis of FPDc in hiPSC-CMs treated for 3, 6, 12 and 24 h with 10 nM and 100 nM T3 compared with vehicle treated cells (ctr); data are expressed as the percentage change of FPDc compared to baseline values; lower panel: representative extracted FPD waveforms of hiPSC-CMs treated with T3 at the indicated doses. *p < 0.05 and **p < 0.005.

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  Figure 3. Effect of physiological doses of THs on FPDc in hiPSC-CMs. (a) left panel: analysis of FPDc in hiPSC-CMs treated for 3, 6, 12 and 24 h with T3 at different doses (0.01 nM, 0.1 nM, 1 nM) compared with vehicle treated cells (ctr); data are expressed as the percentage change of FPDc compared to baseline values; right panel: representative extracted FPD waveforms of hiPSC-CMs treated with 1 nM T3 at the indicated times. (b) left panel: analysis of FPD in hiPSC-CMs treated for 3, 6, 12 and 24 h with T4 at different doses (0.0002 nM, 0.02 nM, 2 nM), compared with vehicle treated cells (ctr); data are expressed as the percentage change of FPDc compared to baseline values; right panel: extracted FPD waveforms of hiPSC-CMs treated with 2 nM T4 at the indicated times. *p < 0.05 and **p < 0.005.

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  Figure 4. LEAP analysis of hiPSC-CM treated with T3. (a) Local extracellular APD analysis of iPSC-CM treated for 24 h with T3 1 nM. The amplitude normalized LEAP waveforms (averaged across 5 beats) from representative wells are overlaid for the vehicle control (grey) and the T3 1 nM (black) treated cardiomyocytes. (b) The histogram represents the mean ± standard deviation of APD30, 50, and 90 measured in four wells. *p < 0.05.

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  Figure 5. Reverse T3 blocks the T3-dependent FPDc prolongation. Analysis of FPDc T3-treated cells after the preincubation with 1 nM rT3 or rT3 vehicle (ammonia solution) at the indicated times. Data are expressed as the percentage change of FPDc compared to baseline values; lower panel: representative extracted FPD waveforms of hiPSC-CMs treated as previously described. *p < 0.05.

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  Figure 6. RNAseq analysis identifies T3-induced gene expression modifications in hiPSC-CMs. (a) Multidimensional Scaling (MDS) plot showed a clear separation between three replicates of control (A1-A3) and T3-treated (B1-B3) hiPSC-CMs samples. (b) Smear Plot of RNA-seq data of different gene expression profile between two groups. Average signal intensity (log CPM versus logFC) of DEGs between ctr and T3 treated iPSC-CMs are shown. The FDR cut-off 0.05 was applied to display the significant DEGs, which are highlighted in red, while non-significant changes are shown in black. Blue horizontal lines represent log2FC. (c) GO.BP functional enrichment results bar plot. Length of bar indicates the percentage of genes in each biological process.