A critical role for IL-21/IL-21 receptor signaling in isoproterenol-induced cardiac remodeling

Figures

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  Fig. 1. Increased expression of IL-21/IL-21R in hypertrophic myocardium in mice. (A) Heart weight/tibia length (H/T) and heart weight/body weight (H/B) were calculated. n = 6. (B) Relative quantification of IL-21R, IL-21, and ANF mRNA levels in cardiac tissue after subcutaneous injection of isoprenaline in mice for 3 and 7 days, n = 5. (C,D) Western blot analysis of IL-21R, IL-21 and ANF in myocardial tissue and their quantification, n = 6. (E,F) Representative immunofluorescence images showing IL-21R and IL-21 expression. IL-21-positive areas are indicated by white arrows; scale bar: 10 μm. n = 6. (G) Flow cytometry analysis of lymphocytes in mediastinal lymph nodes (mLNs) and their quantification, n = 5. Figure 1 was created using Adobe Illustrator 2025. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Fig. 2. Knockout of the IL-21R reduces cardiac hypertrophy and apoptosis caused by ISO. (A) Representative echocardiograms for each group after ISO subcutaneous injection. (B) Representative WGA staining images, scale bar: 20 μm. (C) Representative TUNEL staining images, scale bar: 100 μm. (D) Quantification of ejection fraction (EF) and fractional shortening (FS) for each group, n = 5. (E) Quantification of WGA staining, n = 6. (F) Quantification of TUNEL staining, n = 4. (G,H) Western blot analysis of ANF and BAX in myocardial tissue and their quantification, n = 6. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Fig. 3. Knocking out the IL-21R improved ISO-induced cardiac fibrosis in mice. (A) Representative hematoxylin and eosin (HE) staining images, scale bar: 100 μm. (B) Representative Masson staining images, scale bar: 100 μm. (C) Representative Picrosirius Red staining images, scale bar: 100 μm. (D) Quantification of Masson trichrome staining, n = 4. (E) Quantification of Picrosirius Red staining, n = 5. (F,G) Relative quantification of mRNA levels of TGF-β (n = 5), α-SMA (n = 6), Fn1 (n = 8), Collagen I (n = 8), Collagen III (n = 6), and Collagen IV (n = 7) in myocardial tissue. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Fig. 4. Mice lacking the IL-21R showed diminished Th1 responses following ISO injection. (A) Relative quantification of STAT4 (n = 6), T-bet (n = 6), and IFN-γ (n = 5) mRNA levels in mLNs. (B,C) Flow cytometry analysis of mLNs and its quantification, n = 5. (D) CD4+ T cells were isolated from the mediastinal lymph nodes (mLNs) of control, ISO-treated WT, and ISO-treated IL-21R−/− mice. Another group consisted of CD4+ T cells from ISO-treated WT mice co-cultured with CFBs in the presence of exogenous IFN-γ antibody (0.5 μg/ml). After 24 h of co-culture, adherent CD4+ T cells on CFBs were imaged. Cells were co-cultured at a 5:1 ratio (CFB/T cells) and photographed after washing, scale bar: 100 μm. (E) Immunofluorescence analysis of α-SMA in CFBs after 24-h co-culture with CD4+ T cells from control or ISO-treated WT and IL-21R−/− mice, scale bar: 50 μm. (F) Quantification of CD4+ T cells adhered to CFBs, n = 6. (G) Quantification of α-SMA immunofluorescence staining, n = 4. (H–I) Western blot analysis of α-SMA and Collagen I expression in CFBs after 24 h co-culture and its quantification, n = 3. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Fig. 5. IL-21 induces hypertrophy and apoptosis in cardiomyocytes. Primary mouse cardiomyocytes were treated with IL-21 (100 ng/ml) for subsequent analyses. (A) Relative quantification of IL-21R, ANF, and BNP mRNA levels in primary mouse cardiomyocytes, n = 5. (B) Representative phalloidin staining of primary cardiomyocytes, scale bar: 50 μm. (C) Representative TUNEL staining of primary cardiomyocytes, scale bar: 100 μm. (D) Quantification of cell area after phalloidin staining, n = 6. (E) Quantification of TUNEL staining, n = 6. (F,G) Western blot analysis of, ANF, and BAX expression in primary mouse cardiomyocytes after IL-21 treatment for different durations, and their quantification, n = 5. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Fig. 6. IL-21 controls cardiomyocyte hypertrophy and apoptosis by activating the STAT3 pathway. Primary mouse cardiomyocytes were treated with IL-21 (100 ng/ml) and WP1006 (3 μmol/L) for 24 h for subsequent analyses. (A) Western blot analysis of phosphorylated STAT3 (p-STAT3) and total-STAT3 in myocardial tissue and their quantification, n = 6. (B) Western blot analysis of p-STAT3, total-STAT3, p-ERK and ERK expression in primary mouse cardiomyocytes after IL-21 treatment for different durations and their quantification, n = 5. (C) Representative phalloidin staining images of primary cardiomyocytes and quantification of cell area, n = 6, scale bar: 50 μm. (D) Representative TUNEL staining of primary cardiomyocytes and quantification of TUNEL staining, n = 6. scale bar: 100 μm. (E) Western blot analysis of p-STAT3, total-STAT3, ANF, and BAX expression in primary mouse cardiomyocytes after 24-h treatment with IL-21 and WP1006 and their quantification, n = 5. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Fig. 7. Schematic representation of IL-21/IL21R-mediated cardiac remodeling in HF mice. Pressure overload induces upregulation of IL-21 and the IL-21 receptor in the myocardium. On the one hand, IL-21 promotes an increase in IFN-γ secreting T cells, which subsequently increases the adhesion of T cells to fibroblasts, thereby aggravating myocardial fibrosis. On the other hand, IL-21 directly binds to IL21R on the surface of cardiomyocytes, which promotes gene expression and signal transduction related to cardiac hypertrophy and apoptosis, leading to these pathological changes. These two different processes combine to exacerbate myocardial injury in mice. This figure was created using Figdraw (URL: https://www.figdraw.com/static/index.html#/).