Ubiquitin-specific protease 38 exacerbates diabetic cardiomyopathy via post-translational modification of ACAD11

Figures

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  Fig. 1. The expression of USP38 is upregulated in diabetic myocardium. (A) Heatmap of differentially expressed deubiquitinating enzyme genes in GSE197850. (B) Heatmap of differentially expressed deubiquitinating enzyme genes in GSE202418. (C) Venn diagram of differentially expressed deubiquitinating enzyme genes in GSE197850 and GSE202418. (D–E) Representative immunoblots and corresponding quantification showing cardiac USP38 in ventricular tissues (n = 6). (F–G) Representative images and corresponding quantification showing USP38 in ventricular tissues (magnification × 40) (n = 3). (H–I) Representative immunoblots and quantification showing USP38 in NRCMs (n = 6). (J–K) Representative immunoblots and corresponding quantification showing USP38 in NRCFs (n = 6). Data was calculated by student's t-test (unpaired, two-tailed, two groups). ∗P < 0.05, ∗∗P < 0.01.

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  Fig. 2. Cardiomyocyte-specific USP38 overexpression aggravates cardiac dysfunction and cardiac structural remodeling in diabetic mice. (A) Change in body weight in each group after 0w, 1w, 4w, 8w and 12w (n = 6–7). (B) Statistical analysis of body weight in each group after 12 w (n = 6–7). (C) Change in blood glucose in each group after 0w, 1w, 4w, 8w and 12w (n = 6–7). (D) Statistical analysis of blood glucose in each group after 12 w (n = 6–7). (E–J) Representative images and corresponding quantification for echocardiography(n = 6–7). (K–L) Representative images of H&E stained and Masson stained, and corresponding quantification based on myocardial fibrosis (magnification × 40) (n = 6). (M–O) Representative immunohistochemical images and corresponding quantification of Collagen I and Collagen III (magnification × 40) (n = 6). (P–Q) Representative immunoblots and corresponding quantification for Collagen I, Collagen III and TGF-β (n = 4). Data was calculated by one-way analysis of variance (Tukey's multiple comparisons test). ∗P < 0.05, ∗∗P < 0.01.

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  Fig. 3. Cardiomyocyte-specific USP38 overexpression aggravates cardiac inflammation and cardiomyocytes apoptosis in diabetic mice. (A–B) Representative immunoblots and corresponding quantification for TNF-α, IL-1-β and IL-6 (n = 4). (C–D) Representative images and corresponding quantification for TUNEL staining (magnification × 40) (n = 6). (E–F) Representative immunoblots and corresponding quantification for Bax and Bcl-2 (n = 4). Data was calculated by one-way analysis of variance (Tukey's multiple comparisons test). ∗P < 0.05, ∗∗P < 0.01.

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  Fig. 4. Cardiomyocyte-specific USP38 overexpression disturbs fatty acid oxidation and aggravates mitochondrial dysfunction. (A) Gene ontology (GO) analysis of DEGs between Usp38-TG STZ group and NTG STZ group. (B) Representative images of Oil Red O staining (magnification × 40) (n = 6). (C–D) Representative images and corresponding quantification for transmission electron microscope based on lipid droplet numbers/100 μm2 (n = 6). (top, magnification × 15000, scale bar = 10 μm). (E–F) mRNA level of Mfn2, Opa1, Drp1 and Fis1 (n = 5–6). (G–H) Representative images and corresponding quantification for Mito Tracker staining in NRCMs (n = 15), (top, magnification × 1200, scale bar = 10 μm). (I–J) Representative images and corresponding quantification for MitoSOX staining in NRCMs (n = 8), (magnification × 600, scale bar = 20 μm). (K–L) Representative images and corresponding quantification for TMRE staining in NRCMs (n = 8), (magnification × 1200, scale bar = 10 μm). (M) ATP content in NRCMs (n = 5–6). Data was calculated by one-way analysis of variance (Tukey's multiple comparisons test). ∗P < 0.05, ∗∗P < 0.01.

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  Fig. 5. Cardiomyocyte-specific USP38 deficiency improves cardiac function and cardiac structural remodeling in diabetic mice. (A) Change in body weight in each group after 0w, 1w, 4w, 8w and 12w (n = 6–7). (B) Statistical analysis of body weight in each group after 12 w (n = 6–7). (C) Change in blood glucose in each group after 0w, 1w, 4w, 8w and 12w (n = 6–7). (D) Statistical analysis of blood glucose in each group after 12 w (n = 6–7). (E–J) Representative images and corresponding quantification for echocardiography (n = 6–7). (K–L) Representative images of H&E stained and Masson stained, and corresponding quantification based on myocardial fibrosis (magnification × 40) (n = 6). (M–N) Representative immunoblots and corresponding quantification for Collagen I, Collagen III and TGF-β (n = 4). (O–P) Representative immunoblots and corresponding quantification for TNF-α, IL-1-β and IL-6 (n = 4). (Q–R) Representative immunoblots and corresponding quantification for Bax and Bcl-2 (n = 4). Data was calculated by one-way analysis of variance (Tukey's multiple comparisons test). ∗P < 0.05, ∗∗P < 0.01.

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  Fig. 6. USP38 downregulation improves mitochondrial function in HG-treated NRCMs. (A–B) Representative images and corresponding quantification for Mito Tracker staining (n = 10–15). (top, magnification × 1200, scale bar = 10 μm). (C–D) Representative images and corresponding quantification for MitoSOX staining (n = 8), (magnification × 600, scale bar = 20 μm). (E–F) Representative images and corresponding quantification for TMRE staining (n = 8). (magnification × 1200, scale bar = 10 μm). (G) ATP content in NRCM (n = 5–6). Data was calculated by one-way analysis of variance (Tukey's multiple comparisons test). ∗P < 0.05, ∗∗P < 0.01.

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  Fig. 7. USP38 interacts with ACAD11. (A–C) The diagram of immunoprecipitation-mass spectrometry (IP-MS), and USP38-interacting Top 10 proteins. (D–E) Immunoblot showing the endogenous interaction between USP38 and ACAD11 in NRCMs. (F–G) Immunoblot showing the exogenous interaction between USP38 and ACAD11 in HEK293T cells. Cell lysates were extracted and immunoprecipitated with anti-Myc or anti-Flag antibody, followed by the detection of Flag-tag or Myc-tag. (H) Representative images of immunofluorescence staining for USP38 (yellow) and ACAD11 (green) in NRCMs. (magnification × 1200, scale bar = 20 μm). (I) Representative images of immunofluorescence staining for ACAD11 (green) and COV IV (red) in NRCMs. (magnification × 1200, scale bar = 20 μm). (J) Representative images of immunofluorescence staining for ACAD11 (green) and PEX1 (red) in NRCMs. (magnification × 1200, scale bar = 20 μm). (K) Schematic diagram showing the constructs expressing full-length USP38 or its truncated fragments. (L) Myc-tagged full-length or truncated USP38 were co-transfected with Flag-tagged full-length ACAD11 into HEK293T cells, respectively. Cell lysates were immunoprecipitated with anti-Myc antibody, and…

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  Fig. 8. USP38 removes the K63-linked polyubiquitination of ACAD11. (A) Ubiquitination assay of ACAD11 in HEK293T cells transfected with Myc-tagged USP38 or catalytically inactive mutant USP38H857A. (B) Ubiquitination assay of ACAD11 in HEK293T cells transfected with HA-tagged ubiquitin-K63, HA-tagged ubiquitin-K48, HA-tagged ubiquitin, Myc-tagged USP38 and Flag-tagged ACAD11. (C–D) Ubiquitination assay of ACAD11 in NRCMs.

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  Fig. 9. USP38 regulates cardiac remodeling by promoting RAGE signaling pathway in diabetic mice. (A) Bilateral bar diagram of differently lipid metabolites between Usp38-TG STZ group and NTG STZ group. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differently lipid metabolites between Usp38-TG STZ group and NTG STZ group. (C) KEGG analysis of DUBs between Usp38-TG STZ group and NTG STZ group. (D–E) Representative immunofluorescence staining images and corresponding quantification of RAGE (magnification × 40) (n = 4). (F–I) Representative immunoblots and corresponding quantification of RAGE in ventricular tissues(n = 4). (J–K) Representative immunoblots and corresponding quantification of RAGE in NRCMs(n = 4). Data was calculated by one-way analysis of variance (Tukey's multiple comparisons test). ∗P < 0.05, ∗∗P < 0.01.

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  Fig. 10. FPS-ZM1 improves cardiac function and cardiac structural remodeling in Usp38-TG diabetic mice. (A) Change in body weight in each group after 0w, 1w, 4w, 8w and 12w (n = 6–7). (B) Statistical analysis of body weight in each group after 12 w (n = 6–7). (C) Change in blood glucose in each group after 0w, 1w, 4w, 8w and 12w (n = 6–7). (D) Statistical analysis of blood glucose in each group after 12 w (n = 6–7). (E–H) Representative images and corresponding quantification of echocardiography (n = 6–7). (I–J) Representative images of H&E stained and Masson stained, and corresponding quantification based on myocardial fibrosis (magnification × 40) (n = 6). (K–L) Representative immunohistochemical images and corresponding quantification of Collagen I (magnification × 40) (n = 6). (M–N) Representative immunoblots and corresponding quantification for Collagen I, Collagen III and TGF-β(n = 4). (O–P) Representative immunoblots and corresponding quantification for TNF-α, IL-1-β and IL-6 (n = 4). Data was calculated by student's t-test (unpaired, two-tailed, two groups). ∗P < 0.05, ∗∗P < 0.01.