Immunosuppressants Tacrolimus and Sirolimus revert the cardiac antifibrotic properties of p38-MAPK inhibition in 3D-multicellular human iPSC-heart organoids

Figures

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  FIGURE 1. Retrospective analysis of transcriptomic data from mouse and human cardiac fibroblasts activated with TGF-β1. (A) schematic representation of the activated nodes of the Ras signaling pathway. (B) Expression analysis of the mouse cardiac fibroblasts dataset GSE132146 showing transcriptional levels of pro-fibrotic and RAS pathway-related genes (*, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to control). (C) Violin plots showing pro-fibrotic and RAS pathway related genes derived from a retroactive analysis of GSE97458 generated from single-cell RNA-seq of serum-free cultured human cardiac fibroblasts activated with TGF-β1 (*, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to control).

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  FIGURE 2. Chemical evaluation of antifibrotic compounds in cardiac fibroblast primary human line activated with TGF-β1. (A) Schematic representation of the experimental workflow. (B) IC50 assay (96 h) for the evaluation of the cytotoxic profile of SB202190, Tacrolimus and Sirolimus in NHCF-V cell line. (C) Western blot analysis in NHCF-V cell line for pathway targets upon different treatments and TGF-β1 stimulation [pS6K, p4E-BP1, 4E-BP1, p-p38, p38, pAkt; β-actin is used as a loading control derived from the same experiments in Figure 3D]. (D) Western blot analysis in NHCF-V cell line for evaluation of cardiac fibrosis and TGF-β1 responsive genes [COL1A1, IL-11]. (E) Immunocytochemistry analysis of COL1A1 expression in NHCF-V upon 5 ng/ml TGF-β1 stimulation for 24 h. (F) Immunocytochemistry analysis of COL1A1 expression in NHCF-V treated with SB202190 (10 μM), Tacrolimus (10 μM) and Sirolimus (200 nM) and 5 ng/ml TGF-β1 stimulation for 24 h [scale bar: 50 μm].

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  FIGURE 3. Cardiotoxicity of antifibrotic compounds in human heart organoids (hHOs) for disease modeling. (A) MTT assay showing hHO cellular viability and IC50 after 96 h of treatment with SB202190 in decreasing concentrations at fixed 1:2 dilutions from 10 mM. (B) compound dose-profiling for electrophysiological beating perturbation in hHO under decreasing concentrations of SB202190 for 96 h. (C) MTT assay showing hHO cellular viability and IC50 after 96 h of treatment with Tacrolimus in decreasing concentrations at fixed 1:2 dilutions from 10 mM. (D) compound dose-profiling for electrophysiological beating perturbation in hHO under decreasing concentrations of Tacrolimus for 96 h. (E) MTT assay showing hHO cellular viability and IC50 after 96 h of treatment with Sirolimus in decreasing concentrations at fixed 1:2 dilutions from 2 mM. (F) compound dose-profiling for electrophysiological beating perturbation in hHO under decreasing concentrations of Sirolimus for 96 h.

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  FIGURE 4. Monotherapy and combo-based chemical evaluation of antifibrotic compounds in human heart organoid modeling reactive fibrosis. (A) Schematic representation of the experimental workflow in hHOs. (B) Phase contrast microscopy for hHOs d15 + 3+1 (scale bar: 500 μm). (C) hHO area size after 96 h of compound treatment [relative to control [FC], n ≥ 11, two-tailed, unpaired t-test]. (D) Western blot analysis in hHOs for pathway targets upon different treatments and TGF-β1 stimulation [pS6K, pAkt, p-p38, p38] and TGF-β1 fibrosis-related responsive genes [COL1A1, IL-11]. (E) Immunocytochemistry analysis of COL1A1 expression in hHOs treated with SB202190 (10 μM) alone or in combination with Tacrolimus (10 μM) and Sirolimus (200 nM) and 5 ng/ml TGF-β1 stimulation for 24 h [scale bar: 500 μm; inset: 100 μm].

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  FIGURE 5. The biological model for the effects of Sirolimus and Tacrolimus effects in fibroblast and non-fibroblast populations. Illustration representing the 2D and 3D models explaining the non-cell-autonomous molecular phenotypic divergences with Sirolimus and Tacrolimus reverting the antifibrotic-response of p38-MAPK inhibition derived from SB202190.