USP18 Serves as a Key Mediator of cGAS‐STING for Cardiac Aging in Diabetes

Figures

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  Figure 1. The diabetic mice show manifest aging changes. (A) Representative M‐mode echocardiographic imaging of the heart in two groups of mice. n = 6 mice per group (B, C) Analysis of fractional shortening and left ventricular ejection fraction of the heart. (D–F) Analysis of Tei index, IVS;d and LVID;d. Significance was assessed by unpaired t‐test. Data are shown as the mean ± SEM. IVS;d indicates diastolic interventricular septal thickness; LVID;d, left ventricular internal diameter. (G–I) Representative immunoblot and quantification of p21 and p53 in myocardial tissue in the indicated group. GAPDH is shared among Figures 1G, 4G and 4J as these targets were analyzed on the same blot (J) Representative images of H&E‐stained sections. (K) Representative images of general hearts. (L) Representative Masson staining of left ventricular collagen volume. Scale bar, 20 μm. H&E indicates hematoxylin and eosin; LV, left ventricle; FS, fractional shortening; LVEF, Left Ventricular Ejection Fraction; LVID;d, Left Ventricular Internal Diameter at End‐Diastole; IVS;d interventricular septum at End‐Diastole; Control, normal mice group; DM, diabetic mice group. These data represent two independent…

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  Figure 2. Single‐cell RNA‐sequencing (scRNA‐seq) predicts gene expression differences between the diabetic mice and normal mice. (A) The volcano plot shows the top 10 genes with the most significant up‐regulation and down‐regulation. Each point represents a gene. Up‐regulated genes are in red and the down‐regulated genes are in blue. Threshold for significance was set at q < 0.05, |log2FC | > 1. (B) Heatmapdisplays significantly differentially expressed genes between diabetic mice and normal mice (D–N, diabetic group; N–N, normal group; blue, lower expression; red, higher expression) (C,D) GO term analysis and KEGG enrichment analysis. (C) The top 30up‐regulated genes. (D) The top 20 up‐regulated genes or down‐regulated genes. BP—biological process, CC—cellular component, MF—molecular function.

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  Figure 3. Expression of USP18 is upregulated in hearts of diabetic mice. (A, B) Representative immunoblot and quantification of USP18 in hearts of diabetic mice (n = 3) and normal mice (n = 3). (C–E) Representative images of immunofluorescence staining of USP18 (red) in the hearts of diabetic mice and normal mice. (F). qPCR analysis of USP18 between diabetic mice(n = 7) and norma mice(n = 9). These data represent two independent experiments; mean ± SEM from at least triplicates of biological duplication, unpaired t‐test; * p < 0.05; **Ρ < 0.01; Control, normal mice group; DM, diabetic mice group. USP18, ubiquitin‐specificprotease 18.

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  Figure 4. USP18 promotes the deubiquitination modification and stability of cGAS, inducing cardiac aging in diabetic mice. (A–D) The expression of cGAS‐STING proteins was up‐regulated in diabetic mice by immunoblotting analysis. n = 6 mice per group (E) Immunofluorescence (IF) staining for STING (red) and nuclei (DAPI, blue) was performed in cardiac tissue. (F) Quantification of STING in hearts of diabetic mice and normal mice. There were three mice in each experimental group. STING and USP18 were sequentially detected on the same membrane after stripping and reprobing. GAPDH served as the common loading controlThese data represent two independent experiments; mean ± SEM from at least triplicates of biological duplication, unpaired t‐test; * p < 0.05; **Ρ < 0.01; ***Ρ < 0.001. Control, normal mice group; DM, diabetic mice group.

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  Figure 5. Rat cardiomyocytes were cultivated in high glucose, which performed inflammatory senescence. (A, B) Representative immunoblot and quantification of p53 protein levels for the control group and high glucose group. (C, D) Immunoblot analysis of USP18 in the indicated group. (E, F) The expression of cGAS‐STING proteins was up‐regulated in high glucose treatment. (G, H) The downstream molecules of cGAS‐STING were increased in high glucose treatment. These data represent two independent experiments; mean ± SEM from at least triplicates of biological duplication, unpaired t‐test; * p < 0.05; **Ρ < 0.01;***Ρ < 0.001. Control, normal mice group; group. Control, control group which was cultured with normal glucose; HG, high glucose group. USP18, ubiquitin‐specific protease 18.