ZEB2 SUMOylation in Cardiac Fibroblast Drives Post-Infarction Cardiac Remodeling through CtBP1-Associated repression of Nr4a1

Figures

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  Figure 1. ZEB2 is increased in ischemic myocardium and cardiac fibroblasts post-MI (A) Immunofluorescence staining for ZEB2 (red), Vimentin (green), and DAPI (blue) in the human infarcted myocardium (MI) ventricular aneurysm tissue undergoing surgical aneurysmectomy (chronic phase after MI) and healthy control myocardium. Scale bars, 20 μm, n = 3. (B) qPCR analysis and quantification of ZEB2 in healthy control and infarcted myocardium with ventricular aneurysms from patients with prior myocardial infarction. Data were normalized to the internal control and expressed relative to healthy control myocardium. (C–H) C57BL/6J mice were subjected to sham or MI surgery for 2 weeks. Western blotting and qRT-PCR were performed on the heart lysates from the infarct zones and non-infarct zones at 2 weeks post-MI to analyze the expression of ZEB2, n = 6 mice per group. Data were normalized and expressed relative to the corresponding sham group. (I) Immunofluorescence co-staining for vimentin (green) with ZEB2 (red) and DAPI (blue) in the heart post-MI. Scale bars, 20 μm, n = 6 mice per group. (J–L) ZEB2 protein expression was measured in cardiomyocytes and fibroblasts isolated from the sham or MI heart…

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  Figure 2. Myofibroblast-specific ZEB2 knockout attenuates adverse remodeling and cardiac dysfunction after MI (A–C) ZEB2f/f and ZEB2MF−KO mice were injected with tamoxifen for 5 continuous days after being subjected to MI surgery for 7 days. At 14 days post-MI, cardiac function indices were measured by echocardiography. EF (ejection fraction) and FS (fractional shortening) were quantified. n = 8 mice per group. (D) Quantification of HW/BW (heart weight/body weight) between ZEB2f/f and ZEB2MF−KO mice. n = 8 mice per group. (E–H) Heart sections were stained with Masson trichrome at 2 weeks after MI. The scar size was measured and quantified as a percentage of the total circumference of the left ventricle. n = 8 mice per group. Scale bars, 500 μm and 50 μm. (I and J) HE staining of cardiomyocyte cell size in left ventricular non-infarct zone. Scale bars, 500 μm and 50 μm. (K and L) Western blot and analysis of α-SMA and Collagen I in the infarct zone at 2 weeks post-MI. n = 6 mice per group. (M) mRNA expression levels of Acta2, Col1α1, Col3α1, Ctgf were detected by real-time quantification PCR. n = 6 mice per group. Data are expressed as mean ± SD; two-way ANOVA followed by Tukey’s multiple…

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  Figure 3. SUMO1 modification enhances ZEB2 stability via conserved lysine residues (A) Schematic illustration of quantitative proteomic screen to identify proteins binding to ZEB2. (B) Lysates from CFs were immunoprecipitated with anti-ZEB2 antibody followed by mass spectrometry analysis to identify the proteins that interact with ZEB2. The graph shows the potentially related proteins. (C) CFs were treated with TGF-β1 (10 ng/mL) for 24 h. Cell lysates were immunoprecipitated with ZEB2 and IgG antibodies, and immunoblot assays were performed using SUMO1 antibodies. (D) Lysates of MI heart tissues were immunoprecipitated with IgG or ZEB2 antibodies, and immunoblot assays were performed using SUMO1 antibody. (E) Pattern diagram indicating the distribution of the consensus SUMOylation of ZEB2. (F) HEK293T cells were transfected with Flag-tagged ZEB2-WT, ZEB2-K462R, ZEB2-K479R, ZEB2-K611R, ZEB2-K774R, ZEB2-4KR and HA-SUMO1 plasmids. Lysates of cells were immunoprecipitated with Flag antibody, and immunoblot assays were performed using SUMO1 antibody. (G and H) Flag-tagged ZEB2-WT, ZEB2-K462R, ZEB2-K479R, ZEB2-K611R, ZEB2-K774R, and ZEB2-4KR plasmids were packaged with a lentivirus vector and…

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  Figure 4. SENP1 is the key deSUMOylation that mediates ZEB2 deSUMOylation (A) HEK293T cells were transfected with Flag-tagged ZEB2-WT, Flag-ZEB2-4KR, Myc-SENP1, His-SUMO1 plasmids. Lysates from cells were immunoprecipitated with anti-Flag antibody, and immunoblot assays were performed using SUMO1 antibody. (B) HEK293T cells were transfected with Flag-tagged ZEB2-WT, Flag-ZEB2-4KR, Myc-SENP2, His-SUMO1 plasmids. Lysates from cells were immunoprecipitated with anti-Flag antibody, and immunoblot assays were performed using SUMO1 antibody. (C and D) HEK293T cells were transfected with Flag-tagged ZEB2-WT, Flag-ZEB2-4KR and His-SUMO1 plasmids and si-NC, si-SENP1 or si-SENP2. Lysates from cells were immunoprecipitated with anti-Flag antibody, and immunoblot assays were performed using SUMO1 antibody. n = 3 individual experiments.

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  Figure 5. ZEB2 SUMOylation is essential for TGF-β1-induced myofibroblast transformation in vitro Primary cardiac fibroblasts were isolated from the 6-week-old male ZEB2KO mice and then infected with Adv-GFP, Adv-ZEB2-WT and Adv-ZEB2-4KR, followed by exposure to vehicle or TGF-β1 (10 ng/mL) for 24 h. (A and B) Collagen contractility with representative collagen gels shows contraction 48 h after the gel release, with percent collagen gel contraction quantified over a 48 h period. (C) Immunofluorescence staining was performed to detect the expression of α-SMA (green) and EdU (red). Nuclei were stained with DAPI (blue). Scale bars, 20 μm. (D) The quantification of EdU+/α-SMA+ cells. (E and F) Immunoblots and quantification of α-SMA, Flag. Tubulin was used as a reference. (G) mRNA expression levels Acta2, Col1α1, Col3α1, Ctgf were detected by real-time quantification PCR. n = 3 independent biological. Data are expressed as mean ± SD. Two-way ANOVA followed by Tukey’s multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

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  Figure 6. SUMOylation-deficient ZEB2 attenuates adverse remodeling and cardiac dysfunction post-MI in a ZEB2-null background ZEB2KO mice were injected with rAAV9-EGFP, rAAV9-ZEB2-WT-3×Flag, and rAAV9-ZEB2-4KR-3×Flag and then subjected to sham or MI surgery for 14 days. Cardiac function indices were measured by echocardiography. (A) Representative M-mode echocardiographic images of each group. (B and C) Quantification of cardiac EF: ejection fraction and FS: fractional shortening. n = 8 mice per group. (D) Quantification of HW/BW (heart weight/body weight) between GFP Sham, GFP MI, ZEB2-WT MI, and ZEB2-4KR MI mice. n = 8 mice per group. (E–H) Representative images and quantification of heart sections stained with Masson trichrome staining at 2 weeks post-MI in each group. n = 8 mice per group. Scale bars, 500 μm and 50 μm. (I and J) Representative images and quantification of cell size in the left ventricle. n = 8 mice per group. Scale bars, 50 μm. (K and L) Western blot and analysis of α-SMA and Collagen I in the infarct zone at 2 weeks post-MI. n = 6 mice per group. (M) mRNA expression levels of Acta2, Col1α1, Col3α1, Ctgf were detected by real-time quantification PCR. n = 6 mice per…

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  Figure 7. SUMOylation-Deficient ZEB2 attenuates Fibrosis via Enhanced Nr4a1 repression and PI3K/AKT-mTOR pathway inhibition ZEB2KO CFs were infected with Adv-ZEB2-WT and Adv-ZEB2-4KR, followed by exposure to vehicle or TGF-β1 (10 ng/mL) for 24 h. RNA-Seq analysis was performed to reveal differentially expressed genes (DEGs). (A) Volcano plot. (B) Heatmap of differentially expressed genes. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway Analysis. (D) CUT&Tag seq was performed as described in STAR Methods. Heatmap of peaks. (E) Location of ZEB2-WT and ZEB2-4KR binding events respective to various genomic features. (F) Integration of the RNA-seq and CUT&Tag-seq datasets identified five genes whose expression was upregulated by ZEB2-4KR and whose promoters were bound by ZEB2-4KR. (G) CUT&Tag gene plot tracks for the read coverage of Nr4a1. (H) The independent CH-IP assay was performed on Adv-GFP, Adv-ZEB2-4KR and Adv-ZEB2-WT infected CFs to verify ZEB2 binding to the promoter regions of Nr4a1. n = 3 independent biological cultures. (I) Cell lysates were immunoprecipitated with Flag antibody and immunoblot assays were performed using CtBP1 and Smad2/3 antibodies. (J and K) ZEB2KO…

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  Figure 8. ZEB2 SUMOylation critically regulates activation of PI3K/AKT-mTORC1 signaling axis ZEB2KO mice were injected with rAAV9-EGFP, rAAV9-ZEB2-WT-3×Flag and rAAV9-ZEB2-4KR-3×Flag and then subjected to MI surgery for 14 days. Heart tissues were collected and lysed for western blotting analysis. (A) Western blotting analysis for P-AKT (Ser473), P-S6K (Thr389), P-4EBP1(Ser65). (B) Quantification of the protein. n = 6 mice per group. (C and D) ZEB2KO CFs were infected with Adv-ZEB2-WT and Adv-ZEB2-4KR and then treated for TGF-β1 (10 ng/mL) for 24 h. Western blotting analysis for P-AKT (Ser473), P-S6K (Thr389), P-4EBP1 (Ser65). (E and F) ZEB2KO CFs were infected with Adv-ZEB2-4KR-3×Flag in the presence or absence of Adv-Sh-Nr4a1 and then stimulated with TGF-β1 for 24 h. Western blot and analysis and quantification of P-AKT (Ser473), P-S6K (Thr389), P-4EBP1 (Ser65) expression. n = 3 individual experiments. Data are expressed as mean ± SD. two-way ANOVA followed by Tukey’s multiple comparisons test was used for analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

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  Figure 9. Nr4a1 knockdown Abolishes the Cardioprotective Effects of SUMOylation-Deficient ZEB2-4KR in post-MI Hearts ZEB2KO mice were injected with rAAV9-Col1a2-sh-Nr4a1 with or without rAAV9-ZEB2-4KR-3×Flag for 2 weeks and then subjected for MI surgery. Cardiac function indices were measured by echocardiography. (A) Representative M-mode echocardiographic images of each group. (B and C) Quantification of cardiac EF: ejection fraction and FS: fractional shortening. n = 8 mice per group. (D) Quantification of HW/BW (heart weight/body weight) of each group. n = 8 mice per group. (E–H) Representative images and quantification of heart sections stained with Masson trichrome staining of each group. n = 6 mice per group. Scale bars, 500 μm and 50 μm. (I and J) Representative images and quantification of cell size in left ventricular. n = 6 mice per group. Scale bars, 50 μm. (K and L) Western blot and analysis of α-SMA and Collagen I in the fibrotic myocardium. n = 6 mice per group. (M) mRNA expression levels of Acta2, Col1α1, Col3α1, Ctgf were detected by real-time quantification PCR. n = 6 mice per group. Data are expressed as mean ± SD. two-way ANOVA followed by Tukey’s multiple comparisons…