Ubiquitin Specific Protease 9X Regulates the Activation of ARK5 and Promotes Progression of Fibrotic Remodeling

Figure 1. USP9X Expression is Elevated in the Ischemic Mice Heart (A) UMAP visualization of single-cell RNA-sequencing data for heart tissues 14 days after myocardial infarction (MI) surgery (n = 1) or sham surgery (n = 4) from the GSE136088 data set. UMAP visualization showing 7 clusters: endothelial cells, smooth muscle cells, macrophages, monocytes, B cells, CD8 NKT cells, and fibroblasts. (B) Volcano plot of DEGs of fibroblasts according to the GSE136088 data set, red indicates up-regulated genes, and blue indicates down-regulated genes. (C) RNA levels of DUBs from tissue-resident cardiac fibroblasts in the healthy and wounded adult mouse heart from GSE111059 data set. (D) Real-time quantification polymerase chain reaction of 6 DUBs was done on the 14-day post-MI mice heart (n = 5). (E) USP9X, α-SMA, and Collagen I expressions in infarcted mouse hearts were detected via Western blotting and quantified according to the immunoblots (n = 5). (F) Immunofluorescence staining images of USP9X (red) and α-SMA (green) in sham and MI mice at 7 and 14 days, n = 5 mice per group. Scale bar: 50 μm. Data represent the mean ± SEM. Mann-Whitney U test (D) and 2-way analysis of variance followed by…

Figure 2. USP9X MF-KO Improved Cardiac Function and Attenuated Organ Fibrosis In Vivo (A) Representative M-mode images from 2 weeks after myocardial infarction (MI) are shown (n = 15 per group). Fibrosis was investigated using histological Masson's trichrome (B) and Sirius red staining (C), quantified at the right (n = 8-13 in each group). Scale bar: 50 μm. (D) Representative hematoxylin-eosin (HE) staining of heart slices (n = 8-13 in each group). Scale bar: 50 μm. (E) Representative images of immunohistochemical staining of a-SMA (n = 8-13 in each group). Scale bar: 50 μm. (F) Collagen I and α-SMA expression levels were quantified by Western blotting (n = 5). (G) USP9X-wild type (WT) or myofibroblast-specific USP9X knockout (MF KO) mice were given a single intratracheal phosphate buffered saline or Bleomycin injection. Lung sections were stained with: Masson's trichrome and Sirius red staining, quantified at the right (n = 8). Scale bar: 50 μm. (H) USP9X-WT or USP9X MF-KO animals underwent sham surgery or unilateral ureteral obstructed. Fibrosis of the kidney was assessed by Masson's trichrome and Sirius red staining, quantified at the right (n = 8). Scale bar: 50 μm. Data represent the…

Figure 3. USP9X Inhibitor Degrasyn Prohibits MI-Induced Fibrosis (A) Representative M-mode echocardiographic images from each group in mice (n = 10-14 in each group). Myocardial fibrosis was assessed by staining with (B) Masson's trichrome, (C) Sirius red staining, and (D) immunohistochemical of a-SMA (n = 8). Scale bar: 50 μm. (E) Fibrosis of the kidney was assessed by Masson's trichrome and Sirius red staining (n = 8), quantified at the right. Scale bar: 50 μm. (F) Lung sections were stained with: Masson's trichrome and Sirius red staining, quantified at the right (n = 8). Scale bar: 50 μm. Values represent the mean ± SEM. One-way analysis of variance followed by Tukey's post hoc test was used for statistical analysis. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Deg = Degrasyn; PBS = phosphate buffered saline; UUO = unilateral ureteral obstructed; other abbreviations as in Figure 2.

Figure 4. USP9X Regulates TGF-β1–Induced Myofibroblast Transformation (A) USP9X, α-SMA, and Collagen I levels in cultured cardiac fibroblasts were detected via Western blotting and quantified by immunoblots. mRNA expression of USP9X (B), Col1a1 (C), and Acta2 (D) were measured by Real-time quantification polymerase chain reaction (n = 5). (E) Cultured cardiac fibroblasts transfected with si-NC or si-USP9X and treated with transforming growth factor (TGF)-β1 for 24 hours, the protein levels of USP9X, a-SMA and Collagen I were measured by Western blot and quantified by immunoblots. (F) Immunofluorescence staining was performed to detect the intensity of α-SMA. Scale bar: 50 μm. (G) Representative images and quantification of wound closure at 24 hours (n = 5). Values represent the mean ± SEM. Two-tailed unpaired t-test (A to D) and 1-way analysis of variance followed by Tukey’s post hoc test (E to G) were used for statistical analysis. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figures 2 and 3.

Figure 5. USP9X Interacts Directly With ARK5 (A) Western blot analysis of SMAD4 in TGF-β1–treated cardiac fibroblasts (CFs) for 24 hours, representative blot and quantification, n = 3. (B) Diagrammatic representation of a quantitative proteomic screen used to find proteins that bind to USP9X. (C) Table showing the candidates of USP9X interactions. (D, E) Immunoprecipitation of rabbit/mouse IgG or USP9X/ARK5 antibody followed by immunoblotting with USP9X or ARK5 antibody in whole cell lysates of National Institutes of Health-3T3 cells was performed using Western blot analysis of the specified proteins. (F) HEK293 cells cotransfected with FLAG-labeled ARK5 and HA-Ub in the presence of siNC or siUSP9X plus the proteasome inhibitor MG132 (10 μmol/L) before IP of entire cell lysates with FLAG magnetic beads were subjected to Western blot analysis of the indicated proteins. (G) HEK293T cells were transfected with a plasmid (USP9X WT or USP9X C1566S mutant), and treated with MG132 before IP of whole cell lysates with FLAG magnetic beads. (H) HA-ubiquitin mutants with only the indicated lysine residue were used, and an in vivo ubiquitin assay was performed. (I) The in vivo ubiquitin experiment was…

Figure 6. USP9X Mediates Profibrotic Effects by Regulating ARK5 (A) The effect of USP9X siRNA on the expression of ARK5 in TGF-β1–treated CFs, representative blot and quantification (n = 3). (B) Lysates from CFs transfected with USP9X siRNA and probed phospho- and total MYPT1. (C) Whole-cell extracts from CFs treated with USP9X siRNA and probed with the indicated antibodies. (D) Immunofluorescence staining was performed to detect the intensity of α-SMA (n = 5). Scale bar: 50 μm. (E) CFs were infected with siRNA-control, siRNA-USP9X, Ad-GFP, or Ad-ARK5. Representative Western blot analysis of ARK5, p-MYPT1, and α-SMA protein levels for each group and quantification level (n = 5), Values represent the mean ± SEM. Two-tailed unpaired Student's t-test (A, B, and D), One-way analysis of variance followed by Tukey’s post hoc test (C), and 2-way analysis of variance followed by the Tukey’s post hoc test (E) were used for statistical analysis were used for statistical analysis. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figures 2, 3, and 5.

Figure 7. ARK5 Facilitates TGF-β1–Induced Myofibroblast Transformation via Activating RhoA/MRTF-A Pathway (A) Fibroblasts were infected with USP9X siRNA, then subjected to TGF-β1 stimulation. Total RNA samples were collected and sequenced. The most significantly altered KEGG pathways were displayed. (B) RhoA activity was assessed using western blot analysis after transfecting CFs with ARK5 siRNA or siRNA-control. (C) CFs were infected with siRNA-control, siRNA-USP9X, Ad-GFP, or Ad-ARK5, and then tested for RhoA activity. (D) MRTF-A protein levels in the nuclear and cytosolic fractions were detected by western blot analysis. (E) Immunofluorescence staining was performed to detect the intensity of MRTF-A. Scale bar: 50 μm. (F) CFs were infected with siRNA-control, siRNA-USP9X, Ad-GFP, or Ad-ARK5, and then tested for MRTF-A protein levels. (G) Representative immunofluorescence staining of MRTF-A for each group. Scale bar: 50 μm. n = 3-5, Values represent the mean ± SEM. One-way analysis of variance followed by Tukey’s post hoc test was used for statistical analysis. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figures 2, 3, and 5.

Figure 8. The Activation of ARK5 Is Critical for USP9X-Mediated Cardiac Fibrosis In Vivo USP9X MF-KO or WT mice were injected with AAV9-GFP or AAV9-ARK5 for 2 weeks, followed by MI for another 2 weeks. (A) Representative immunoblotting analysis and quantification of ARK5, Collagen I, and α-SMA protein levels in the hearts (n = 5). (B) Representative M-mode echocardiographic images of the indicated groups (n = 8 per group). (C and D) Histological examination of myocardial fibrosis detected by Sirius red staining (Scale bar: 500 μm) and (D) Masson’s trichrome staining (Scale bar: 50 μm), quantified at the right (n = 8). (E) Representative images of immunohistochemical staining of a-SMA (n = 8). Scale bar: 50 μm. (F) Representative HE staining of heart slices (n = 8). Scale bar: 50 μm. One-way analysis of variance followed by Tukey's post hoc test was used for statistical analysis. Values represent the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figures 2, 3, and 5.