Role of ATOH8 in Mitigating Cardiac Fibrosis in Heart Failure via Inhibition of the Transcriptional Repression of Smad7 by RUNX2

Figures

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  Fig. 1. Cardiac fibrosis and downregulation of ATOH8 expression in the left ventricular cardiac tissue of patients with DCM. (A) Representative hematoxylin-eosin (HE) staining, Picrosirius red staining, and immunohistochemical staining of ATOH8 (scale bar=20 µm). (B, C) Quantification of fibrotic and ATOH8-positive areas (n=6 for each group, ≥40 fields per group). (D) Quantitative polymerase chain reaction analyses of mRNA expression of ATOH8 and α-SMA in the left ventricular tissue samples obtained from patients with DCM and the control group (n=6 for each group). (E) Representative western blotting and quantification data showing ATOH8 and α-SMA expression levels in the left ventricular tissues of patients with DCM and control individuals (n=6 for each group). Data are presented as mean ± SEM. **p<0.01 vs. the normal group, ***p<0.001 vs. the normal group.

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  Fig. 2. Downregulation of ATOH8 in the left ventricular tissues obtained from cardiac fibrosis model mice subjected to the TAC surgery. (A) Representative M-mode images of TAC and sham group mice. Ejection fraction (EF%) was quantified via echocardiography (n=6 for each group, scale bar=100 µm). (B) Representative Masson staining results were presented and quantified to demonstrate the extent of cardiac fibrosis (n=6 for each group). (C) Representative immunofluorescence images of ATOH8 expression in the left ventricles (n=6 for each group; red, ATOH8; green, vimentin; blue, DAPI; scale bar=50 µm). (D) The levels of ATOH8 and α-SMA protein in the sham group and TAC group (n=6 for each group). Data are presented as the mean ± SEM. **p<0.01, ***p<0.001 vs. the sham group.

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  Fig. 3. TGF-β1-induced cardiac fibrosis and myofibroblast activation in cultured CFs transfected with siRNA against ATOH8. (A, B) Knockdown efficiency of three different interference sequences of ATOH8 were verified by q-PCR and western blotting. (C, D) Relative expression levels and quantification of fibrosis and extracellular matrix synthesis proteins in cultured CFs of the indicated groups. (E) Wound repair assays were performed to assess the migratory capacities of cells that had been transfected with either scramble-siRNA or ATOH8-siRNA and stimulated by TGF-β1. The percentages of recovered areas were analyzed (n=4 for each group, scale bar=500 µm). (F) Representative images of results from the Transwell migration assay and quantification of migrated CFs of the indicated groups (n=4 for each group; scale bar=100 µm). Data are presented as the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. NS indicates that there was no significant difference between the indicated groups. ‘Ad-A8’ is used as the abbreviation for ‘Ad-ATOH8’.

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  Fig. 4. Amelioration of cardiac fibrosis in cultured CFs injected with ATOH8-overexpression adenovirus. (A) The overexpression efficiency of Ad-ATOH8 was assessed by western blotting. (B) Representative western blotting and quantitative analysis results for α-SMA are shown for the indicated groups. (C, D) Representative western blotting and quantification of ECM-related proteins in CFs for the indicated groups. (E) Wound repair assays were performed to evaluate the migratory capacities of cells of the indicated groups (n=4 for each group, scale bar=500 µm). (F) Representative images of results from the Transwell migration assay and quantification of migrated CFs of the indicated groups (n=4 for each group; scale bar=100 µm). Data are presented as the mean ± SEM. **p<0.01, ***p<0.001. NS indicates that there was no significant difference between the indicated groups. ‘Ad-A8’ is used as the abbreviation for ‘Ad-ATOH8’.

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  Fig. 5. The in vivo fibroblast-specific knockdown of ATOH8 exacerbates cardiac fibrosis and impairs cardiac function. (A) Schematic representation of the procedure for the injection of AAV9-Postn containing either GFP or ATOH8 shRNA. (B) The knockdown efficiency of ATOH8 was verified by western blotting. (C) Representative M-mode images of the indicated groups and EF% quantified by echocardiography. (D) Representative Masson staining images are presented and quantified to show the extent of cardiac fibrosis (n=8 for each group, scale bar=100 µm). (E) Representative western blotting and quantitative results of the cardiac fibrosis-related protein levels of heart tissue samples obtained from the indicated groups. Data are presented as the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. NS indicates that there was no significant difference between the two indicated groups. ‘Ad-A8’ is used as the abbreviation for ‘Ad-ATOH8’.

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  Fig. 6. Protective role of ATOH8 against cardiac fibrosis via the RUNX2/Smad7 signaling pathway. (A) Differentially expressed genes identified through RNA sequencing are represented by clustering heat maps (blue, downregulated genes; red, upregulated genes) for CFs transfected with SCR or si-ATOH8 and stimulated with TGF-β1 (n=4 for each group). (B, C) KEGG and Gene Ontology Cellular Component (GO-CC) pathway analysis. (D) q-PCR analysis of the expression of Smad7 in the indicated groups. (E) Protein coverage of IP-mass spectrometry sequencing by targeting ATOH8. (F) KEGG pathways analysis of proteins binding to ATOH8 sequenced by IP-mass spectrometry. (G, H) Immunoprecipitation experiments demonstrating the interaction of ATOH8 with RUNX2. (I) Promoter sequence of Smad7 predicted by the JASPAR database for RUNX2 binding. (J) Enrichment of RUNX2 on the Smad7 promoter region determined by ChIP-qPCR (n=5 for each group). Data are presented as the mean ± SEM. **p<0.01 versus the indicated group.

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  Fig. 7. The protective role of fibroblast-specific ATOH8 overexpression in mice with TAC-induced cardiac fibrosis. (A) Schema of the procedure for injection of AAV9-Postn carrying ATOH8-overexpression plasmids. (B) The overexpression efficiency of ATOH8 was verified by western blotting. (C) Representative M-mode images of the indicated groups and EF% quantified by echocardiography. (D) Representative Masson staining images are presented and quantified to show the extent of cardiac fibrosis (n=8 for each group, scale bar=100 µm). (E) Representative western blotting and quantitative analysis of the levels of cardiac fibrosis-related proteins in the tissues obtained from the indicated groups. Data are presented as the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. NS indicates that there was no significant difference between the indicated groups. ‘Ad-A8’ is used as the abbreviation for ‘Ad-ATOH8’.