Ubiquitin like protein FAT10 repressed cardiac fibrosis after myocardial ischemic via mediating degradation of Smad3 dependent on FAT10-proteasome system

Figures

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  Fig 1. FAT10 deficient mice exhibit increased cardiac fibrotic after myocardial infraction. A Masson staining of whole heart fibrosis (up, bar 1mm) and infarct border fibrosis (down, bar 50μm) (n=4 per group) in 14-days and 28-days. B Western blot analysis of α-SMA, collagen I and collagen III in heart tissue from 14day-MI-FAT10-/- and 14day-MI-WT (n=5 per group). C Immunofluoresence staining of myocardial sections showing α-SMA (in red), collagen I (in green) and collagen III (in yellow) in 14day-MI-FAT10-/- and 14day-MI-wild type mice (n=4 per group). Nuclei were counterstained with DAPI (blue). D IHC staining of α-SMA, collagen I and collagen III of heart tissue from 14day-MI-FAT10-/- and 14day-MI-wild-type (bar 50μm) (n=4 per group). E Echocardiographic analysis to evaluate left ventricular ejection fraction (LVEF), fractional shortening (FS), left ventricular internal diameter at end-diastole (LVIDd) and left ventricular internal diameter at end-systole (LVIDs) in WT and Fat10-/- after sham and 2 weeks post-MI surgery (n=5 per group). F Representative M-mode echocardiographic images of WT and Fat10-/- hearts. *p<0.05, **p<0.01, ***p<0.001 and ns=not significant. All data presented…

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  Fig 2. FAT10 overexpression alleviated MI-induced cardiac fibrosis. A Experimental procedure for injecting mice with ad-Fat10 virus. B Masson stainin of whole heart fibrosis (bar 1mm) and infarct border fibrosis (bar 50μm) (n=6 per group). C Immunofluoresence staining of myocardial sections showing collagen I (in green). Nuclei were counterstained with DAPI (blue). D IHC staining of α-SMA, collagen I and collagen III of heart tissue from per group. E Western blot analysis of α-SMA, collagen I and collagen III in heart tissue from per group. F Representative M-mode echocardiographic images. *p<0.05, **p<0.01, ***p<0.001 and ns=not significant. All data presented as mean±SEM.

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  Fig 3. FAT10 negatively regulates the TGF-β1-induced fibrosis in vitro. A NRCFs were transfected with ad-Fat10 or sh-Fat10 and then subjected to treated with TGF-β1(10ng/ml), the level of α-SMA, collagen I and collagen III were detected by western blot analysis (n=4 per group). B Representative immunofluorescence images of EdU(red) and DAPI (blue)(left) and quantitative data (right) in the different experimental groups of NRCFs (bar 50μm) (n=6 per group). C Migration of NRCFs measured by Transwell assay (bar 100μm) (n=4 per group) and wound-healing assay (n=5 per group) (bar 100μm). Statistical results of relative migration of speed were analyzed (n=4 per group). D Migration of NRCFs measured by RTCA assay. *p<0.05, **p<0.01, ***p<0.001 and ns=not significant. All data presented as mean±SEM.

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  Fig 4. FAT10 regulates MI-induced cardiac fibrosis by targeting Smad3. A The interaction of FAT10 and its potential substrates. B Western blot analysis of Smad3 and P-Smad3 in heart tissue from MI-Fat10-/- and MI-WT. C NRCFs were transfected with ad-Fat10 or sh-Fat10 and then exposed to TGF-β1, the expression of Smad3 and P-Smad3 was measured by western blot analysis. D NRCFs were co-transfected with ad-Fat10 and ad-Smad3 or ad-con and then exposed to TGF-β1; α-SMA, Smad3, P-Smad3, collagen I and collagen III were detected by western blot analysis. E NRCFs were co-transfected with sh-Fat10 and sh-Smad3 or sh-con and then exposed to TGF-β1; α-SMA, Smad3, P-Smad3, collagen I and collagen III were detected by western blot analysis. F Fat10-/- and WT primary CF were transfected with sh-Smad3 then exposed to TGF-β1. α-SMA, Smad3, P-Smad3, collagen I and collagen III were detected by western blot analysis. G-I Representative immunofluorescence images of EdU (red) and DAPI (blue)(left) and quantitative data (right) in the different experimental groups of NRCFs (n=3 per group) (bar 50μm). J-L Migration of NRCFs measured by Transwell assay (bar 100μm), wound-healing assay (bar 100μm) and RTCA…

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  Fig 5. Smad3-specific inhibitor (SIS3) inhibited Fat10-/--induced cardiac fibrosis after MI. A Masson staining of whole heart fibrosis (bar 1mm) and infarct border fibrosis (bar 50μm) (n=4 per group). B Western blot analysis of α-SMA, collagen I and collagen III in heart tissue from per group (n=4 per group). C Echocardiographic analysis to evaluate LVEF, FS, LVIDd and LVIDs and representative M-mode echocardiographic images (n=5 per group). *p<0.05, **p<0.01, ***p<0.001 and ns=not significant. All data presented as mean±SEM.

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  Fig 6. Smad3 plays key role of FAT10 regulating cardiac fibrosis after MI. A NRCFs were treated with CHX (20 μM) for the indicated times in the presence or absence of ad-Fat10, and the levels of Smad3 were then detected. B NRCFs were transfected with ad-Fat10, the cells were then exposed to CHX (20 μM), and the Smad3 levels in the supernatants were measured by western blot. C NRCFs were treated with CHX (20 μM) for the indicated times in the presence or absence of MG132 (15 μM), and the levels of Smad3 were then detected (n=3 per group). D Cells in each group were treated with MG132 (15 μM) for 12 h, and the expression of Smad3 was measured by western blot analysis (n = 4 per group). E-F. NRCFs were co-transfected with ad-Fat10 and either a FAT10-specific E1-activated enzyme Uba6 (E) (n = 4 per group) interference sequence or an E2-transferase enzyme Ube2z (F) interference sequence; Smad3 levels in the cell extracts were measured by western blot analysis (n = 5 per group). G NRCFs were co-transfected with ad-Fat10 and sh-Ub, Smad3 levels in the cell extracts were measured by western blot analysis (n = 3 per group). *p<0.05, **p<0.01, ***p<0.001 and ns=not significant. All data presented…

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  Fig 7. Identification of the FAT10-binding site in Smad3. A Co-IP for endogenous Smad3 and FAT10 in NRCFs. B The association between FAT10 and Smad3 in HEK293 cells expressing Flag-Fat10 and HA-Smad3 was detected by Co-IP. C Direct interaction of Smad3 with FAT10 by GST pull down assay. D Co-localization of Smad3 and FAT10 in NRCFs; FAT10(1:100) in red, Smad3 (1:100) in green, and DAPI nuclear counterstaining in blue. E Bimolecular fluorescence complementation assay exhibited interactions of FAT10 (fused with N-terminus of Venus [VN-FAT10]) with Smad3 (fused with C-terminus of Venus [VC-Smad3]. F Co-IP assays were performed to analyze the binding of FAT10 and Smad3 when the Flag-△Fat10 and HA-Smad3 were co-transfected into HEK293T cells. G Western Blot analysis of α-SMA, collagen I and collagen III in the NRCFs after transfection with ad-Fat10 or ad-△Fat10. H Schematic representation of Smad3, which has three functional domains(left). Direct interaction of His-Smad3 domain and GST-FAT10 FAT by GST pull down assay(right). I Schematic distribution of the 4 lysine residues in domains 3 of Smad3(left). HEK293T cells were transfected with indicated constructs. Cells were lysed for Co-IP…

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  Fig 8. FAT10 overexpression could ameliorate TGF-β1-induced fibrotic response in hiPSC-CFs. A Schematic diagram of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CFs) generated. B Representative immunofluorescence assays of α-SMA (left, red), collagen I (middle, green) and collagen III (right, red) (bar 50μm). C hiPSC-CFs were transfected with ad-Fat10 and then subjected to treated with TGF-β1(10ng/ml), the level of α-SMA, collagen I and collagen III were detected by western blot analysis (n=3 per group). D Representative immunofluorescence images of EdU (bar=50μm) and migration of hiPSC-CFs measured by Transwell assay (bar 100μm) (n=3 per group). *p<0.05, **p<0.01, ***p<0.001 and ns=not significant. All data presented as mean ± SEM.