TRIM21 aggravates cardiac injury after myocardial infarction by promoting M1 macrophage polarization

Figure 1. Increased TRIM21 expression after MI and in macrophages by LPS. (A) Comparison of upregulated DEGs in GSE147310 and GSE158889 microarray indicates the intersecting gene of TRIM21. (B) TRIM21 expression treated with or without LPS in GSE147310. Relative TRIM21 mRNA (C) and protein (D) levels in the infarcted areas of heart tissues following MI at day 1, 3, 5, and 7, as compared with Sham group. n = 2 mice/group pooled from 3 independent experiments. (E) Representative Masson Trichrome staining (left) and immunostaining (right) of TRIM21 (red), F4/80 (green) and DAPI (blue) in the hearts of sham and MI groups (3 days after MI). Scale bar: 20 μm. n = 2 mice/group pooled from 3 independent experiments. (F) Representative images of BMDMs stimulated by PBS and LPS. Relative iNOS and TRIM21 mRNA (G) and protein (H) levels in BMDMs with LPS treatment for 1h, 3h, 6h, 12h, and 24h as compared with control group. Data are representative of 3 independent experiments. n = 3 sets of cells/group. *P<0.05, **P<0.01, and ***P<0.001.

Figure 2. TRIM21 deficiency alleviates MI-induced changes in the cardiac structure and preserves cardiac function. (A) Survival rates after MI in WT and TRIM21 knockout (TRIM21-/-) mice. (B) Representative echocardiographic images of WT mice and TRIM21-/- mice 3 days after sham operation or LAD ligation. (C) Quantification of the left ventricular ejection fraction, left ventricular fractional shortening, left ventricular end-systolic internal diameter, and end-diastolic internal diameter. For (B, C), n = 7–11 mice/group pooled from 2 independent experiments. (D) Representative H&E staining and Masson Trichrome staining of the cardiac tissue sections of WT mice and TRIM21-/- mice 3 days after sham operation or LAD ligation. Scale bar: 1 mm. Data are representative of 3 independent experiments. n = 2 mice/group. *P<0.05, **P<0.01.

Figure 3. TRIM21 deficiency decreased MI-induced apoptosis and DNA damage. (A) Immunofluorescence staining and positive cell-count results of TUNEL in the heart tissues. (B) Representative double-fluorescent immunostaining images of γ-H2AX and α-actinin. The positive γ-H2AX (γ-H2AX+) cell areas were quantified (right). (C) Bcl-2, Bax, and γ-H2AX protein expressions at day 3 post-MI in the infarct zone isolated from WT and TRIM21-/- mice. For A-C, data are representative of 3 independent experiments. n = 2 mice/group. *P<0.05, **P<0.01, and ***P<0.001.

Figure 4. TRIM21 deficiency mediates macrophage polarization in the infarct infarcted zone at day 3 post‐MI. Representative double-fluorescent immunostaining images of F4/80 and iNOS (A) and F4/80 and Arg1 (C). The double‐positive iNOS and F4/80 (iNOS+/F4/80+) areas of M1 macrophages and Arg1 and F4/80 (Arg1+/F4/80+) M2 macrophages were quantified (right) Scale bar:50 mm. (B) Gene expression of iNOS, TNF-α, and IL-6 in heart tissues isolated from infarct border zone at day 3 after MI. n = 2 mice/group pooled from 3 independent experiments. (D) Gene expression of Arg1, Ym-1, and IL-10 in heart tissues isolated from infarct zone at day 3 after MI. n = 2 mice/group pooled from 3 independent experiments. (E) iNOS and Arg1 protein expression at day 3 post-MI in the infarct zone isolated from WT and TRIM21-/- mice. n = 2 mice/group pooled from 3 independent experiments. *P<0.05, **P<0.01, and ***P<0.001.

Figure 5. TRIM21 overexpression induces macrophage polarized into M1 phenotype in RAW264.7 cells. (A) Representative images of RAW264.7 cells transfected with lentivirus targeting the TRIM21 gene (LV-TRIM21) or negative lentivirus (LV-GFP). The protein levels of TRIM21 and Flag (A) and the mRNA of TRIM21 (B) in RAW264.7 cells. (C) Representative images of iNOS staining in LPS-stimulated RAW264.7 cells transfected with LV-TRIM21 or LV-GFP. Scale bar: 200 mm. (D) The protein level of iNOS and mRNA levels of iNOS, IL-6 and TNF-α (E) in LPS-stimulated RAW264.7 cells transfected with LV-TRIM21 or LV-GFP. For (A-E), data are representative of 3 independent experiments. n = 3 sets of cells/group. *P<0.05, **P<0.01, and ***P<0.001.

Figure 6. RNA-seq results of LPS-treated BMDMs with or without TRIM21 depletion (A) Volcano plot of DEGs between WT and TRIM21-/- in BMDMs treated with LPS for 12 h. Red color indicates genes that are upregulated and blue color indicates downregulated genes. (B) Heatmap of color-coded expression levels of DEGs. (C) Top 10 KEGG down regulated signaling pathways.

Figure 7. TRIM21 regulates macrophage polarization by the PI3K/Akt pathway. (A) The protein levels of TRIM21, p-PI3K, PI3K, p-AKT and AKT on day 3 post-MI in the infarct zone isolated from WT and TRIM21-/- mice. (B) The protein levels of TRIM21, p-PI3K, PI3K, p-AKT and AKT in LPS-stimulated RAW264.7 cells transfected with LV-GFP or LV-TRIM21, (C) and in LV-GFP and LV-TRIM21 group treated with LY294002 or PBS after LPS exposure. (D) The protein levels of iNOS (left) and mRNA levels of iNOS, TNF-α, and IL-6 (right) in LV-GFP and LV-TRIM21 group treated with LY294002 or PBS. (E) Representative images and quantified results of gap closure of cardiac fibroblast cultured in conditioned medium from LV-GFP and LV-TRIM21 macrophages treated with LY294002 or PBS macrophages after LPS exposure. For (A-E), data are representative of 3 independent experiments. n = 3 sets of cells/group. *P<0.05, **P<0.01, and ***P<0.001.

Figure 8. Schematic diagram illustrates that TRIM21 mediates macrophage polarization via PI3K/Akt pathway after myocardial infarction.