Transcriptomics reveal stretched human pluripotent stem cell-derived cardiomyocytes as an advantageous hypertrophy model

Figures

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  Fig. 1. Cyclic mechanical stretching induces natriuretic peptide expression in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). A. Schematic representation of the experimental design. RB−, RPMI 1640 medium supplemented with B-27 without insulin; RB+, RPMI 1640 supplemented with B-27; FBS, fetal bovine serum. Figure was created with BioRender.com. For further details, see Methods section. B–C. hiPSC-CMs respond to stretch by increased expression of hypertrophy-associated genes NPPA (natriuretic peptide A; B) and NPPB (natriuretic peptide B; C). mRNA expression of 24-h, 48-h and 72-h stretched hiPSC-CMs measured with qRT-PCR was normalized to the unstretched control. The data are shown as mean ± standard error of the mean, and values from individual experiments are presented as dots (n = 3 for 24 h, n = 4 for 48 h and 72 h; where n represents biological replicates from individual differentiations). *p < 0.05, ***p < 0.001 vs. unstretched control, Student's t-test for independent samples. D. Average perinuclear proBNP intensity analyzed from 9000 stained cardiomyocytes/sample, from three individual differentiations. *p < 0.05 vs. unstretched control, Student's t-test…

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  Fig. 2. Mechanical stretch-induced differential gene expression of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). A–B. Venn diagrams show the number of upregulated (A) and downregulated (B) genes after 24 h, 48 h and 72 h of cyclic stretch measured with RNA sequencing. Differential expression was defined as a >1.5-fold change compared to the unstretched control. C–D. The expression of the top 12 up- (C) and downregulated (D) genes is presented as log2-fold change relative to the unstretched control ± standard error of the mean (n = 3 for 24 h, n = 4 for 48 h and 72 h; where n represents biological replicates of cells from individual differentiations). Only statistically significant (Benjamini-Hochberg adjusted p < 0.05) results are presented.

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  Fig. 3. Time-dependent changes in gene expression of selected genes. Differential expression of 11 selected genes from RNA sequencing was validated by qRT-PCR after 24 h, 48 h and 72 h of stretch. (A) Upregulated genes; (B) downregulated genes. The results are presented as log2-fold change relative to the unstretched control ± standard error of the mean (n = 3 for 24 h, n = 4 for 48 h and 72 h; where n represents biological replicates of cells from individual differentiations). *p < 0.05, **p < 0.01, ***p < 0.001 vs. unstretched control, Student's t-test for independent samples (qRT–PCR), Wald test (RNAseq).

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  Fig. 4. Comparison of differentially expressed genes in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) after 24 h and 48 h of stretch. NRVM gene expression data are from Rysä et al. [12]. A–B. Venn diagrams show the number of upregulated (A) and downregulated (B) genes after 24 h and 48 h of stretch in hiPSC-CMs and NRVMs. C–D. Expression of genes differentially regulated in both cell types after 24 h (C) and 48 h (D) of stretching normalized to the unstretched control (n = 3 for 24 h hiPSC-CMs, n = 4 for 48 h and 72 h hiPSC-CMs, and n = 5 for NRVMs).

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  Fig. 5. Enriched biological processes based on upregulated genes after cyclic stretch in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs; A) and neonatal rat ventricular myocytes (NRVMs; B). Gene Ontology (GO) enrichment analysis was performed with GOrilla. For each significantly enriched GO term, enrichment values are presented as bars, and FDR-adjusted p values are presented as dots. The number of upregulated genes associated with each GO term is shown on the right. The upregulated genes in the NRVMs used for the analysis are from Rysä et al. [12]. The top 30 terms for NRVMs are shown.

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  Fig. 6. Enriched pathways in upregulated genes after cyclic stretch in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs). A–B. KEGG pathway analyses of upregulated genes of stretched hiPSC-CMs (A) and NRVMs (B). C–D. Reactome pathway analyses of upregulated genes of stretched hiPSC-CMs (C) and NRVMs (D). For each significantly enriched pathway term, enrichment values are presented as bars, and FDR-adjusted p values are presented as dots. The number of upregulated genes associated with each term is shown on the right. The upregulated genes in the NRVMs used for the analysis are from Rysä et al. [12].

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  Fig. 7. Enriched transcription factor target sites in the upregulated genes of the stretched human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs; A) and neonatal rat ventricular myocytes (NRVMs; B). Enrichment values for each significantly enriched target site are presented as bars, and FDR-adjusted p values are presented as dots. The number of upregulated genes associated with each target site is shown on the right. The upregulated genes in NRVMs used for the analysis are from Rysä et al. [12].

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  Fig. 8. Effects of p38 mitogen-activated protein kinase (p38 MAPK), mitogen-activated protein kinase kinase 1/2 (MEK1/2) and protein kinase C (PKC) inhibitors on stretch-induced hypertrophic gene expression in human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The following inhibitors were utilized: SB203580 at 10 μM to inhibit p38 MAPK, U0126 at 10 μM to inhibit MEK1/2, Gö6983 at 1 μM to inhibit all PKC isoforms and Gö6976 at 1 μM to inhibit classical PKC isoforms. Natriuretic peptide B (NPPB; A), skeletal muscle alpha actin 1 (ACTA1; B), galanin (GAL; C) and long intergenic non-protein coding RNA 648 (LINC00648; D) RNA expression was measured with qRT–PCR after a 24-h cyclic mechanical stretch. The results are presented as fold change relative to the unstretched control. The data are shown as mean ± standard error of the mean, and values from individual experiments are presented as dots (n = 5, except for SB203580 and Gö6976 n = 4; where n represents biological replicates of cells from individual differentiations). *p < 0.05, **p < 0.01 vs. unstretched DMSO, †p < 0.05 vs. stretched DMSO, Mann–Whitney U test.