TRIM26 exacerbates pathological cardiac hypertrophy by activating TAK1

Figures

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  Fig. 1. TRIM26 aggravate PE-induced cardiomyocyte hypertrophy in vitro. (A) Representative H&E stained images of mouse hearts for 4 weeks after TAC or sham surgery. Scale bar, 25 μm. (B) mRNA levels of TRIM26, Anp, and Bnp in mouse hearts for 4 weeks after TAC or sham surgery (n = 4). (C) The protein levels of TRIM26, Anp, and Bnp in mouse hearts for 4 weeks after TAC or sham surgery (n = 4 mice/group). (D) Representative α-actinin immunofluorescence images of neonatal rat cardiomyocytes (NRCMs) treated with PBS or PE for 24 h. Scale bar, 20 μm. (E) mRNA levels of Anp, Bnp, and TRIM26 in NRCMs treated with PBS or PE for 24 h (n = 3 independent experiments). (F) The protein levels of TRIM26 in NRCMs treated with PBS or PE for 24 h and statistical results (n = 4 independent experiments). (G, H) mRNA (G) and protein (H) levels of TRIM26 in NRCMs infected with adenovirus overexpressing Trim26 (AdTrim26) or GFP vector (AdVector) and statistical results (n = 3 independent experiments). (I) Representative immunofluorescence images of α-actinin staining and its statistical results in NRCMs infected with AdVector or AdTrim26 and treated for 24 h with PBS or PE and statistical analysis (n > 50…

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  Fig. 2. TRIM26 knockdown alleviate PE-induced cardiomyocyte hypertrophy in vitro. (A, B) mRNA (A) and protein (B) levels of TRIM26 in NRCMs infected with adenovirus knock-down Trim26 (AdshTrim26) or control adenovirus (AdshRNA) (n = 3 independent experiments) and statistical analysis. (C) Representative immunofluorescence images of α-actinin staining and its statistical results in NRCMs infected with AdshRNA or AdshTrim26 treated with PBS or PE for 24 h and statistical analysis (n > 50 cells per group in each independent experiment). Scale bar, 20 μm. (D) The mRNA levels of Anp, Bnp, and Myh7 in NRCMs infected with AdshRNA or AdshTrim26 and treated for 24 h with PBS or PE (n = 3 independent experiments). (E) Anp, Bnp, and Myh7 protein levels in NRCMs infected with AdshRNA or AdshTrim26 and treated for 24 h with PBS or PE and statistical results (n = 3 independent experiments). (F) mRNA levels of Tnf,Il6, and Il1b in NRCMs infected with AdshRNA or AdshTrim26 and treated for 24 h with PBS or PE (n = 3 independent experiments). GAPDH was a loading control in B and E. ∗, P < 0.05, ∗∗, P < 0.01 vs. the AdshRNA PE group. #, P < 0.05, ##, P < 0.01 vs. AdshRNA PBS group. Statistical analysis was…

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  Fig. 3. TRIM26 deficiency improves cardiac dysfunction and cardiomyocyte hypertrophy caused by TAC surgery. (A) The protein levels of TRIM26 in WT and Trim26-KO mice (n = 3 mice/group). (B–E) Heart weight (B), HW/BW (C), LW/BW (D), HW/TL (E) of WT and Trim26-KO mice subjected to TAC or sham surgery (n = 10 mice/group). (F–I) Measurements of LVEDd (F), LVESd (G), EF (H), and FS (I) in the indicated groups (n = 10 mice/group). (J) Representative images of gross morphology (Scale bar, 0.3 cm), H&E staining (Scale bar, 50 μm) (n = 6 mice/group), and the statistical result of cell surface area from the indicated groups (n > 300 cells per group). (K) mRNA levels of Anp, Bnp, and Myh7 in the indicated groups (n = 4 mice/group). GAPDH was a loading control in A. ∗, P < 0.05, ∗∗, P < 0.01 vs. WT TAC group. ##, P < 0.01 vs. WT sham group. Statistical analysis was conducted using One -way ANOVA (B-K).

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  Fig. 4. TRIM26 deficiency ameliorates fibrosis and inflammation in myocardial hypertrophy. (A) Representative images of PSR staining (Scale bar, 50 μm) (n = 6 mice/group), and the statistical result of relative LV collagen volume from the indicated groups (n > 50 images per experimental group). (B, C) mRNA levels of Collagen Iα1, Collagen IIIα1, Ctgf, Tnf,Il6, and Il1b in the indicated groups (n = 4 mice/group). (D) The protein levels of p-IKKβ, IKKβ, p-P65, and P65 from the indicated groups and statistical analysis (n = 3 mice/group). GAPDH was a loading control in D. ∗, P < 0.05, ∗∗, P < 0.01, vs. WT TAC group. #, P < 0.05, ##, P < 0.01, vs. WT sham group. Statistical analysis was conducted using One-way ANOVA (A-C) or a two-tailed Student's t-test (D).

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  Fig. 5. The RNA-Seq results of WT and Trim26-KO mice heart samples after TAC surgery. (A) Principal component analysis (PCA) showing global sample distribution profiles from WT and Trim26-KO mice heart samples subjected to TAC for 4 weeks (n = 4 mice/group). (B) Gene set enrichment analysis (GSEA) analysis showed the molecular events involved in heart function, protein processing, fibrosis, and inflammation in RNA-Seq data (n = 4 mice/group). (C) Heatmap of significantly altered genes related to heart function, protein processes, fibrosis, and inflammation in the WT TAC and Trim26-KO TAC groups (n = 4 mice/group). (D) Top enriched pathways contributing to the Trim26 function were identified using reactome enrichment analysis(n = 4 mice/group).

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  Fig. 6. TRIM26 promotes the activation of TAK1-JNK/P38 signaling pathway. (A) Protein levels of total and phosphorylated TAK1, ERK, JNK, and P38 in heart tissues of WT and Trim26-KO mice treated with TAC or sham surgery and statistical analysis (n = 4 mice/group). (B) Protein levels of total and phosphorylated TAK1, ERK, JNK, and P38 in PE-stimulated AdTrim26 and AdVector NRCMs treated with PBS or PE for 24 h and statistical analysis (n = 3 independent experiments). (C) Representative endogenous co-immunoprecipitation (IP) analyses showing the binding of TRIM26 to TAK1 in NRCMs; IgG was used as a control (n = 3 independent experiments). (D) IP analysis of TAK1 ubiquitination in HEK 293T cells transfected with the indicated plasmids (n = 3 independent experiments). (E) IP analysis of TAK1 K63-linked ubiquitination in HEK 293T cells transfected with the indicated plasmids (n = 3 independent experiments). GAPDH was a loading control in E and F. ∗, P < 0.05, ∗∗, P < 0.01, n.s., no significance. Statistical analysis was conducted using One-way ANOVA.

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  Fig. 7. TRIM26 regulate cardiomyocyte hypertrophy through TAK1 activation. (A) Protein levels of TRIM26, p-TAK1, and TAK1 in NRCMs in the indicated groups (n = 3 independent experiments). (B) Representative immunofluorescence images of α-actinin staining and its statistical results in PE-stimulated AdTrim26 and AdVector NRCMs with iTAK1 or DMSO as control (n > 50 cells per group in each independent experiment). Scale bar, 20 μm. (C, D) mRNA levels of Anp, Bnp, Myh7, Tnf, Il6, and Il1b in PE-stimulated AdTrim26 NRCMs and AdVector with iTAK1 or DMSO as control (n = 3 independent experiments). GAPDH served as a loading control in A. ∗∗, P < 0.01 vs. AdTrim26 CT PE group. #, P < 0.05, ##, P < 0.01, n.s., no significance vs. AdVector CT PE group. Statistical analysis was conducted using One-way ANOVA (A-D).

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  Fig. 8. TRIM26 regulate cardiomyocyte hypertrophy through TAK1 activation. (A) Protein levels of TRIM26, p-TAK1, and TAK1 in NRCMs in the indicated groups (n = 3 independent experiments). (B) Representative immunofluorescence images of α-actinin staining and its statistical results in PE-stimulated AdshTrim26 and AdshRNA NRCMs infected with AdTAK1 or AdVector (n > 50 cells per group in each independent experiment). Scale bar, 20 μm. (C, D) mRNA levels of Anp, Bnp, Myh7, Tnf, Il6 and Il1b in PE-stimulated AdshTrim26 and AdshRNA NRCMs infected with AdTAK1 or AdVector (n = 3 independent experiments). GAPDH served as a loading control in A. ∗∗, P < 0.01 vs. AdshTrim26 AdVector PE group. ##, P < 0.01, n.s., no significance vs. AdshRNA AdVector PE group. Statistical analysis was conducted using One-way ANOVA.

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