Multi-omic analysis reveals VEGFR2, PI3K, and JNK mediate the small molecule induction of human iPSC-derived cardiomyocyte proliferation

Figures

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  Figure 1. Five compounds targeting distinct pathways induce proliferation in hiPSC-CMs with minimal multinucleation and nuclear polyploidy (A) Schematic of study. (B) Representative images of 48 h EdU assay measuring DNA synthesis rates of hiPSC-CMs treated with a combination of compound and 1 μM EdU. Cells were fixed and stained with DAPI (blue) and EdU (magenta). (C) Representative images of live-cell assay tracking changes in the number of cells over 6 days. Cells were stained with 0.02 μg/mL Hoechst and imaged on days 0 and 6. (D and E) Quantification of B and C. (F and G) Rate of multinucleation and (G) DNA content analysis, and (H) increase in number of cells after 6 days of treatment. Colors of stacked bar plots indicate ploidy states (2c, 4c, >4c) measured by the integrated intensity of Hoechst 33342. Treatments for (B-H) are as follows: negative control (0.1% DMSO), positive control (1 μM CHIR99021 aka CHIR), and maximum-effect concentrations for top five compounds (1 μM C1, 3.2 μM C2-C4, 10 μM C5). All experiments were performed using lot 3 of CDI iCell Cardiomyocytes. Scale bars are 100 μM. Error bars represent mean ± s.e.m. Stats for (D-G) were assessed by one-way ANOVA with…

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  Figure 2. Functional analogs support putative targets of five lead compounds (A) 5-point concentration responses (0.1–10 μM) of functional analogs to each of the top five compounds measuring the change in the number of cells over 6 days of treatment. (B–F) Change in the number of cells, (C) rate of multinucleation, (D) fraction of diploid nuclei over 6 days of treatment at the maximum-effect concentrations for each compound, (E) area under the concentration response curve, and (F) concentration at 50% maximum-effect proliferation for each compound. (A–F) Metrics are normalized to the negative control (0.1% DMSO) on each plate. Functional analog compounds (gray) are identified by appending consecutive lower-case letters after lead compound (black) names. All plots are grouped by lead compound. Error bars represent mean ± s.e.m. Stats for (B–D) were assessed by one-way ANOVA with Dunnett multiple comparisons test; n = 2–24; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

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  Figure 3. Transcriptomic analysis of proliferation-inducing compounds reveals a common and robust cell cycle response (A) Hierarchical clustering of gene expression profiles and replicates of hiPSC-CMs treated with negative control (0.1% DMSO) and the top five compounds at maximum-effect concentrations (1 μM C1, 3.2 μM C2–C4, 10 μM C5). Only genes that are differentially expressed (FDR ≤0.05) compared to control in at least one compound are shown. (B) Principal component analysis of data shown in (A). (C) Distribution of DEGs that are unique (red) to each compound or common among 2–5 compounds. (D) Enrichr’s Reactome pathway enrichment analysis of the 1764 DEGs common to all five compounds (circles) and 1540 DEGs excluding the Reactome Cell Cycle gene set (squares). Top 15 pathways with FDR ≤0.1 are shown. (E) Heatmap of log2 fold change of genes in Reactome’s Cell Cycle pathway. Genes that are differentially expressed (FDR ≤0.05) in at least one compound are shown. (F) ChEA3 transcription factor enrichment analysis of the 1764 common DEGs. Top 15 TFs rank-ordered by FDR with the corresponding mRNA expression profiles are shown. RNAseq was performed with n = 3 replicates.

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  Figure 4. Functional proteomic profiling identifies a mechanistic role for receptor tyrosine kinases in response to pro-proliferative compounds (A) Venn diagram of 109 significantly differentially expressed RPPA probes (FDR ≤0.05). (B) Heatmap of unidirectional differentially expressed RPPA probes common to all 4 lead compounds. Log2(fold change expression relative to DMSO control) is shown. (C) Heatmap of 49 RTK probes measured using R&D’s Human RTK array. Proteomic data shown in (A–C) were measured in hiPSC-CMs treated with the top 4 lead compounds (C1, C3, C4, and C5) for 12 h. (D) 48 h EdU assay measuring DNA synthesis rates. (E) 6-day live-cell proliferation assay tracking cell counts. (F and G) Rate of multinucleation and G) DNA content after 6 days of treatment. Colors of stacked bar plots indicate DNA content levels (2c, 3c, 4c, >4c) measured by the integrated intensity of DAPI. Stats indicate significance for % 2c nuclei. Treatments for (D–G): negative control (0.1% DMSO), positive control (1 μM CHIR99021), and tyrosine kinase inhibitors targeting ERBB2 (1 μM lapatinib), IGF1R (0.1 μM linsitinib), VEGFR1/2/3 (10 μM axitinib) alone and in combination with 3.2 μM C3 or C4. Error…

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  Figure 5. Compound-induced expression of VEGF receptors limits increases in ploidy (A) Ki67 staining measuring cell cycle activity after 6 days of treatment. (B) Bivariate analysis of DNA content (x axis) and Ki67 staining (y axis) to separate out cell cycle phase and nuclear ploidy levels. DNA content thresholds separating 2c, 3c, and 4c nuclei are shown as blue horizontal lines. Ki67 positive threshold is shown as a red horizontal line. Nuclei with <2c or >4c DNA content are not shown. Replicate with median G0 measurement is shown. (C) Quantification of B. Treatments for (A–C): negative control (0.1% DMSO), positive control (1 μM CHIR99021), and small molecule inhibitors targeting ERBB2 (1 μM lapatinib), IGF1R (0.1 μM linsitinib), and VEGFR1/2/3 (10 μM axitinib) alone and in combination with 3.2 μM C3 or C4. Error bars represent mean ± s.e.m. Stats for (A and C) was assessed by one-way ANOVA with Benjamini-Hochberg FDR correction for select comparisons (∗ = all single treatments were compared to DMSO control; + = all combination treatments were compared to the respective lead compound C3 or C4); n = 3–9; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

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  Figure 6. Network integrating transcriptomic and proteomic data identifies JNK and PI3K pathways mediate compound-induced proliferation of hiPSC-CMs (A) Directed molecular network integrating common factors identified in the proteomic and transcriptomic data. Node colors represent the sum of the qualitative significance (up = 1, down = −1, not significant = 0) across all 4 cpds based on data from the RPPA or RTK array. Gray nodes indicate nodes that were not measured in the RPPA or RTK arrays. (B) 48 h EdU assay measuring DNA synthesis rates. (C) 6-day live-cell proliferation assay tracking cell counts. (D) Ki67 staining measuring cell cycle activity. Treatments for (B–D): neg control (0.1% DMSO), pos control (1 μM CHIR99021), and small molecule inhibitors targeting JNK (10 μM SP600125), MEK (10 μM PD98059), and PI3K (10 μM LY294002) alone and in combination with 3.2 μM C3 or C4. Error bars represent mean ± s.e.m. Stats for (D–G) were assessed by one-way ANOVA with Benjamini-Hochberg FDR correction for select comparisons (∗ = all single treatments were compared to DMSO control; + = all combination treatments were compared to the respective lead compound C3 or C4); n = 3–9; ∗p < 0.05; ∗∗p <…