Fig 2: OTUD1 is induced in Ang II-mediated cardiac hypertrophy and fibrosis model. (A) RNA sequencing was performed in heart tissues of C57BL/6 mice infused with saline (Ctrl) or Ang II for 4 weeks. Expression profile of deubiquitylating enzymes (DUBs) showing up- (red) and down-regulated (green) genes. Grey points represent DUBs showing no statistical difference compared to Ctrl. Fold change ? 2 times and p ? 0.05 indicate statistically significant differences. (B) Representative western blot analysis for OTUD1 levels in Ctrl and Ang II-infused heart tissues. GAPDH was used as loading control. (C) Immunofluorescence staining of mouse heart tissues for OTUD1 (red), vimentin (green), and sarcomeric alpha actinin (green). Sections were counterstained with DAPI (blue). Arrows showing OTUD1-positive cells [scale bar = 50 µm]. (D) Representative western blot analysis for OTUD1 protein levels in macrophages, primary cardiomyocytes (neonatal ventricular myocytes), cardiac fibroblast and H9c2 cells. GAPDH was used as loading control.

Fig 3: OTUD1 expression protects against Ang II-induced cardiac hypertrophy and fibrosis by regulating STAT3. C57BL/6 mice received two injections of AAV9 encoding OTUD1, one month apart. Saline or Ang II was then infused for 2 weeks. (A) Ang II levels in serum samples obtained from mice. (B) Representative echocardiographic images from mice in each experimental group. (C, D) Cardiac functional tests showing ejection fraction (EF; panel C) and isovolumic relaxation time (IVRT; panel D). (E) CK-MB levels in serum obtained from mice. (F, G) Hematoxylin and eosin (H&E) staining of heart tissues [scale bar = 5 mm (F) and 50 µm (G)]. (H) Staining of heart tissues with fluorophore-conjugated wheat germ agglutinin (WGA) to measure cardiomyocyte hypertrophy [scale bar = 50 µm]. (I, J) Fibrosis in cardiac tissues was determined by Masson's Trichrome (I) and Picro Sirius Red (J) staining [scale bar = 50 µm]. (K) Western blot analysis of fibrosis-associated proteins ß-MyHC, COL-1, and TGF-ß1 in heart tissues. GAPDH was used as the loading control. (L, M) mRNA levels of fibrosis-associated genes (L) and inflammatory genes (M) in heart tissues of mice. Data was normalized to Actb. (N, O) Levels of p-STAT3 (Tyr705) in whole heart lysates, and nuclear and cytosolic STAT3 in fractionated heart lysates from mice. All quantitative data is presented as Mean ± SEM; n = 6; ns = not significant; * = p < 0.05 and ** = p < 0.01.

Fig 4: OTUD1 suppression reduces Ang II-induced cardiomyocyte hypertrophy and fibrogenic responses in vitro. Neonatal rat ventricular myocytes (NVRMs) were used to model Ang II effects in culture. Cells were transfected with siRNA against OTUD1 (panels A-D) or OTUD1 expressing vectors (panels E-H). Scrambled siRNA and empty vectors were used as control. Following transfections, cells were exposed to 1 µM Ang II for 48 h. (A) Following Ang II exposure, cells were stained with TRITC Phalloidin to assess hypertrophic responses [scale bar = 50 µm]. (B) Representative western blotting for ß-MyHC, COL-1, and TGF-ß1 in NVRMs. GAPDH was used as loading control. (C, D) mRNA levels fibrosis-associated genes (C) and inflammatory genes (D) were measured in NVRMs. Data was normalized to Actb. (E) Hypertrophic response to Ang II in cells expressing OTUD1. Figure showing TRITC Phalloidin staining [scale bar = 50 µm]. (F) Representative western blotting for ß-MyHC, COL-1, and TGF-ß1 in cells transfected with OTUD1 vector. (G, H) mRNA levels fibrosis-associated genes (G) and inflammatory genes (H) were measured in NVRMs. Data was normalized to Actb. All quantitative data is presented as Mean ± SEM; n = 3; ns = not significant; * = p < 0.05 and ** = p < 0.01.

Fig 5: OTUD1 deficiency protects against Ang II-induced myocardial hypertrophy and fibrosis in mice. Wildtype and OTUD1 knockout mice were infused with saline (Sham) or Ang II for 4 weeks. (A) Systolic blood pressure was determined by non-invasive tail-cuff on a weekly basis. (B) Ang II levels were measured in serum samples from mice. (C) Representative echocardiographic images from mice in each group. (D, E) Cardiac functional tests showing ejection fraction (EF; panel D) and isovolumic relaxation time (IVRT; panel E). (F) Creatine kinase-MB (CK-MB) levels in serum of mice. (G, H) Hematoxylin and eosin (H&E) staining of heart tissues [scale bar = 5 mm (G) and 50 µm (H)]. (I) Staining of heart tissues with fluorophore-conjugated wheat germ agglutinin (WGA) was performed to measure cardiomyocyte hypertrophy [scale bar = 50 µm]. (J, K) Fibrosis in cardiac tissues was determined by Masson's Trichrome (J) and Picro Sirius Red (K) staining [scale bar = 50 µm]. (L, M) Western blot analysis of fibrosis-associated proteins ß myosin heavy chain (ß-MyHC), collagen 1(COL-1), and transforming growth factor- ß1 (TGF-ß1) in heart tissues. GAPDH was used as the loading control. Densitometric quantification is shown in panel M. (N, O) mRNA levels of fibrosis-associated genes (N) and inflammatory genes (O) in heart tissues of mice. Data was normalized to Actb. All quantitative data is presented as Mean ± SEM; n = 6; ns = not significant; * = p < 0.05 and ** = p < 0.01.