Deletion of activating transcription factor 3 (Atf3) promotes cardiomyocyte differentiation from embryonic stem cells

Figures

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  Fig. 1. Confirmation of activating transcription factor 3 (Atf3) gene knockout in mouse embryonic stem cells (mESCs). (A) Relative gene expression levels for Atf3 between wild-type (WT) and Atf3-KO mESCs. (B) Representative immunofluorescence image of Atf3 protein expression between the 2 groups. (C) Representative Western blot image of Atf3 protein expression between the 2 groups. (D) Atf3-KO did not affect the morphology of monoclonal cells in the 2 groups. (E) Relative expression levels for pluripotency genes Oct4, Sox2, Nanog, and estrogen-related receptor-β (Esrrb), between the 2 groups. (F) Representative images of alive and dead embryoid bodies (EBs), as well as (G) EB formation percentages, between the 2 groups. Data represented as mean ± standard error of the mean (SEM). n = 3/group for all experiments. ***p < 0.001, ns is no statistical significance. Statistical analysis was conducted using the unpaired 2-tailed Students t-test.

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  Fig. 2. Atf3-KO mESCs yield more beating EBs with higher beating frequencies. (A) Schematic diagram of the timeline for EB formation and mESC differentiation. (B) Images of EBs for both WT and Atf3-KO groups in Days 3, 6, 9 and 12 of differentiation. Red dotted line indicated areas of beating cardiomyocytes. (C) Percentages and (D) frequencies of beating EBs for WT and Atf3-KO mESCs in Days 7–12 of differentiation. Data represented as mean ± SEM. n = 3/group for all experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns is no statistical significance. Statistical analysis was conducted using 2-way analysis of variance (ANOVA).

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  Fig. 3. Atf3-KO promotes mESC differentiation into cardiomyocytes. (A) Relative gene expression levels for cardiomyocyte markers actin-α cardiac muscle 1 (Actc1), α-actinin 2 (Actn2), Tnnt2 (cardiac troponin T [cTnT]), myosin heavy chain, α isoform (Myh6) and GATA Binding Protein 4 (Gata4), between WT and Atf3-KO mESCs, in Days 0, 9, and 12 of differentiation. (B) Relative gene expression levels for cardiac progenitor marker platelet-derived growth factor receptor-α (Pdgrfa) between the 2 groups, in Days 0, 3, 6, and 9 of differentiation. (C) Representative immunocytochemical images for cTnT, between the 2 groups, in Day 9 of differentiation. Insets show high-magnification views, in which Atf3-KO had obvious sarcomere structures. (D) Flow cytometry analyses and (E) quantification of cTnT+ cells, between the 2 groups, in Day 9 and 12 of differentiation. Data represented as mean ± SEM. n = 3/group for all experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns is no statistical significance. Statistical analysis was conducted using 2-way ANOVA.

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  Fig. 4. Atf3-KO promotes mesodermal germ layer development. (A) Relative gene expression levels for mesoderm markers T-box transcription factor T (T), fibroblast growth factor 8 (Fgf8), Flk-1, (B) endoderm markers SRY-box transcription factor 17 (Sox17), Amn1, tetraspanin 7 (Tm4sf2), and (C) ectoderm markers Nestin, SOX1, and paired box 6 (Pax6), between WT and Atf3-KO mESCs in Days 0, 3, and 6 of differentiation. Data represented as mean ± SEM. n = 3/group for all experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001, ns is no statistical significance. Statistical analysis was conducted using 2-way ANOVA.

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  Fig. 5. RNA-sequencing (RNA-seq) analysis of WT and Atf3-KO mESCs at Days 0–9 of differentiation. (A) Volcano plots of differentially-expressed genes (DEGs) between WT and Atf3-KO groups, at Days 0, 3, 6, and 9 of differentiation. Downregulated DEGs were blue, upregulated were red, and those with no statistically significant expression changes were grey. (B) Heat maps of the 29 DEGs (15 upregulated, 14 downregulated) with the most significant expression level changes between WT and Atf3-KO groups, at Day 0, 3, 6, and 9 of differentiation. Data represented as mean ± SEM. Statistical analysis was conducted using the unpaired 2-tailed Students t-test.

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  Fig. 6. RNA-seq analysis revealed that the signaling pathways related to myocardial differentiation were upregulated after Atf3-KO. Kyoto Genes and Genomes (KEGG) enrichment analysis of DEGs (p < 0.05) at (A) Days 6 and (B) 9 of differentiation. (C) Bubble, (D) pathway, and (E) ridge plots of GSEA enrichment analysis of DEGs on Day 9 of differentiation. Data represented as mean ± SEM. Statistical analysis was conducted using the unpaired 2-tailed Students t-test.

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  Fig. 7. Restoration of Atf3 expression in Atf3-KO mESCs reversed the effects of Atf3-KO towards that of WT cells. (A) Relative mRNA expression levels for Atf3 among WT, Atf3-KO, and Atf3-KO mESCs who had Atf3 expression restored (Atf3-KO+Rescue). Relative gene expression levels for (B) Actc1, (C) Actn2, (D) Tnnt2, (E) Myh6, and (F) Gata4, among the 3 groups, on Day 9 of differentiation. (G) Representative immunocytochemical images for cTnT, among the 3 groups, on Day 9 of differentiation. (H) Flow cytometry analyses and (I) quantification of cTnT+ cells, among the 3 groups, on Day 9 of differentiation. Data represented as mean ± SEM. n = 3/group for all experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns is no statistical significance. Statistical analysis was conducted using 1-way ANOVA, followed by Tukey’s post hoc test.