TRIM47, stabilized by YTHDF3-mediated m6A modification, promotes cardiac lipid accumulation and aggravates diabetic cardiomyopathy

Figures

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  Figure 1. TRIM47 is elevated in the cardiac tissues of DCM mice in the GEO database (A) Volcano plot of differentially expressed genes (DEGs) in DCM mice from GSE123975. Blue indicates upregulation and gray indicates downregulation. (B) Heatmap of DEGs. (C–D) GO and KEGG enrichment analyses by GSEA, sorted by NES in descending order; the top 10 terms are shown for each. (E) TRIM47 mRNA expression in cardiac tissues of control and STZ mice from GSE123975. Data are presented as the mean ± SD.

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  Figure 2. TRIM47 is elevated in the cardiac tissues of DCM mice (A) A type 2 DCM mouse model was established by feeding 8-week-old male C57BL/6 mice an HFD for 24 weeks, combined with intraperitoneal STZ injections on three consecutive days beginning at week 8 of HFD. Control mice received a normal diet. (B) Body weight and fasting blood glucose levels were measured every 4 weeks after HFD initiation until the end of the modeling period in both ND and STZ/HFD mice. (C) Representative M-mode echocardiography images of ND and STZ/HFD mice. (D) Quantitative analysis of left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF), fractional shortening (LVFS), and left ventricular (LV) mass in ND and STZ/HFD mice. (E) Triglyceride (TG) levels were measured in both cardiac tissue and serum of ND and STZ/HFD mice using commercial kits. (F–G) Expression of TRIM47 in left ventricular myocardial tissue of ND and STZ/HFD mice was detected by qPCR and Western blotting. (H) Double IF staining of TRIM47 and cTnT was performed to assess TRIM47 expression in cardiomyocytes of ND and STZ/HFD mice. Data are presented as the mean…

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  Figure 3. Cardiomyocyte-specific knockdown of TRIM47 improves cardiac function in DCM mice (A) An AAV9 vector expressing TRIM47 shRNA under the cardiomyocyte-specific cTnT promoter was constructed. C57BL/6 mice were injected via the tail vein with 5 × 1011 vg of AAV9. Two weeks later, DCM was induced by STZ/HFD. After 24 weeks of HFD, serum and cardiac tissues were collected. (B) Representative M-mode echocardiography images of the indicated groups. (C) Quantitative analysis of left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF), fractional shortening (LVFS), and left ventricular (LV) mass. (D–E) Expression of TRIM47 in left ventricular myocardial tissues was detected by qPCR and Western blotting. (F–G) Representative images of whole hearts; heart weight (HW) and tibia length (TL) were measured, and the HW/TL ratio was calculated. (H) Histological assessment of cardiac tissue via hematoxylin and eosin (H&E) staining. (I–J) Wheat germ agglutinin (WGA) staining and Masson staining were performed to evaluate cardiomyocyte hypertrophy and myocardial fibrosis, and the cross-sectional area of cardiomyocytes…

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  Figure 4. Cardiomyocyte-specific knockdown of TRIM47 alleviates lipid accumulation in DCM mice (A) The effect of TRIM47 knockdown on lipid infiltration in left ventricular myocardial tissues was detected by Oil Red O staining. (B) The effect of TRIM47 knockdown on lipid accumulation in left ventricular myocardial tissues was examined by TEM. (C) The effect of TRIM47 knockdown on TG levels in mouse cardiac tissues and serum was measured using commercial kits. (D) The effect of TRIM47 knockdown on CD36 protein expression in left ventricular myocardial tissues was detected by Western blotting. Data were expressed as the mean ± SD.

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  Figure 5. TRIM47 knockdown reduces PA-induced lipid deposition in cardiomyocytes (A) Primary neonatal mouse ventricular cardiomyocytes (NMCMs) were isolated from 1- to 2-day-old C57BL/6 mice, and the purity of primary NMCMs was assessed by IF staining of cTnT. (B) NMCMs were treated with 100 μmol/L palmitic acid (PA) for 72 h before detection. (C–D) TRIM47 expression was detected by qPCR and Western blotting. (E–F) NMCMs were infected with adenovirus expressing TRIM47 shRNA (sh-TRIM47Ad) or a negative control (sh-NCAd) for 24 h, and TRIM47 expression was detected by qPCR and Western blotting. (G–I) After 24 h of infection, NMCMs were treated with 100 μmol/L PA for 72 h. The effect of TRIM47 knockdown on TRIM47 expression in PA-treated NMCMs was detected by qPCR and Western blotting. (J) The effect of TRIM47 knockdown on the mRNA expression of ANP, BNP, and β-MHC in PA-treated NMCMs was detected by qPCR. (K) The effect of TRIM47 knockdown on the mRNA expression of TNFα, IL-6, and IL-1β in PA-treated NMCMs was detected by qPCR. (L) The effect of TRIM47 knockdown on apoptosis in PA-treated NMCMs was detected by TUNEL staining. (M) The effect of TRIM47 knockdown on lipid formation in…

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  Figure 6. YTHDF3 mediates TRIM47 m6A modification and enhances its mRNA stability (A) The online analysis website indicates that TRIM47 has m6A modification sites. (B) NMCMs were treated with 100 μmol/L PA for 72 h, and YTHDF3 expression was detected by Western blotting. (C–D) NMCMs were infected with adenovirus overexpressing YTHDF3 (Ad-YTHDF3) or a negative control (Ad-null) for 24 h. The expression of YTHDF3 and TRIM47 was detected by qPCR and Western blotting. (E) After 24 h of infection, NMCMs were treated with PA for 72 h and then exposed to actinomycin D for the indicated time points, after which TRIM47 mRNA levels were quantified by qPCR. (F) The binding between YTHDF3 and TRIM47 mRNA was detected by RIP-qPCR. (G) To investigate whether YTHDF3 mediates the function of TRIM47 in NMCMs, TRIM47 knockdown adenovirus and YTHDF3 overexpression adenovirus were co-infected into NMCMs. After 24 h, PA treatment was performed for 72 h. (H) The effect of YTHDF3 overexpression on TNF-α mRNA expression in NMCMs. (I) The effect of YTHDF3 overexpression on cardiomyocyte apoptosis was detected by TUNEL staining. (J) The effect of YTHDF3 overexpression on lipid formation in NMCMs was detected by Oil…