Progranulin deficiency exacerbates cardiac remodeling after myocardial infarction

Figures

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  FIGURE 1. Dynamic alterations of PGRN expression associated with macrophages throughout infarcted myocardium. (A) Immunoblots exhibit PGRN (58–68 kDa) and GAPDH (37 kDa) in whole hearts at 1, 3, 7, and 14 days after myocardial infarction (MI). (B) Quantitative analysis of PGRN expression level normalized to GAPDH in the entire infarcted myocardium. Data are the means ± SEM (Sham group: n = 5, MI groups; 1 d: n = 6, 3 d: n = 6, 7 d: n = 5, 14 d: n = 6). *p < 0.05, **p < 0.01 versus Sham group (one‐way ANOVA followed by Dunnett T3 test). (C) Scheme of a myocardial transverse section after MI. The myocardium was classified into three different regions as follows: infarct, border, and remote areas. (D) Confocal microscopic images (×10 magnification) of PGRN at 3 days after MI. Scale bar: 500 μm. (E) Quantitative analysis of fluorescence intensity for PGRN at 3 days after MI. Data are the means ± SEM (Normal: n = 5, Sham: n = 5, Infarct: n = 5, Border: n = 5, Remote: n = 5). **p < 0.01 versus Sham group (one‐way ANOVA followed by Dunnett T3 test). (F) Enlarged images of a cell expressing PGRN. The white arrows indicate one of the CD68+ PGRN+ cells. Scale bar: 10 μm. (G) Typical confocal…

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  FIGURE 2. Adverse histological and electrophysiological cardiac remodeling after MI in PGRN‐KO mice. (A) Typical images of the whole hearts in WT and PGRN‐KO mice at 14 days post‐MI. Scale Bars: 1 mm. (B) Quantitative analysis of heart weight per body weight (HW/BW). (C) Typical images of the myocardial transverse sections from apex to ligature visualized by Masson's trichrome staining in WT and PGRN‐KO mice at 14 days after MI. Scale Bars: 1 mm. (D, E) Quantitative analysis of fibrosis size (%) in left and right ventricular (LV and RV). Data are the means ± SEM (WT: n = 8, KO: n = 4). *p < 0.05 versus WT (Levene's test followed by Student's two‐tailed t‐test). (F) Kaplan–Meier survival curves in WT and PGRN‐KO mice following MI. Data are the means ± SEM (WT: n = 33, PGRN‐KO: n = 28). **p < 0.01 versus WT (Log‐rank test). (G) Observed days of cardiac rupture post‐MI. Data are the means ± SEM (WT: n = 11, PGRN‐KO: n = 13). *p < 0.05 versus WT (Mann–Whitney U test). (H) Electrocardiograms (ECGs) in WT and PGRN‐KO mice at 14 days after MI. (I–K) Quantitative analysis of corrected QT interval (QTc), QT interval, and RR interval, respectively. Data are the means ± SEM (WT: n = 9, PGRN‐KO: n =…

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  FIGURE 3. Alteration of protein expression and macrophage accumulation affected by PGRN deficiency following MI. (A) Immunoblots exhibit PGRN (58–68 kDa), CD206 (190 kDa), and MerTK (175 kDa) in whole hearts of WT and PGRN‐KO mice at 3 days after MI. (B) Quantitative analysis of CD206 and MerTK expression levels normalized to GAPDH, in the entire myocardium at 3 days post‐MI. Data are the means ± SEM (WT: n = 10, PGRN‐KO: n = 7). *p < 0.05, ***p < 0.001 versus WT (Levene's test followed by Student's or Welch's two‐tailed t‐test). (C) Typical confocal microscopic images (×20 magnification) of macrophages at border areas in WT and PGRN‐KO mice at 3 days after MI. Blue: nuclei stained with Hoechst 33342, Red: CD68, Gray: Troponin‐I. Scale bar: 100 μm. Quantitative analysis of fluorescence intensity of CD68 and the number of CD86+ cells at border areas in WT and PGRN‐KO mice at 3 days post‐MI. Data are the means ± SEM (WT: n = 6, PGRN‐KO: n = 4). *p < 0.05, **p < 0.01 versus WT (Levene's test followed by Student's or Welch's two‐tailed t‐test). (D) Representative confocal microscopic images (×10 magnification) of neutrophils at border areas in WT and PGRN‐KO mice at 3 days after MI. Cyan:…

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  FIGURE 4. Abnormal subpopulation of cardiac macrophages in PGRN‐KO mice post‐MI. (A, B) Representative FACS plots of CD11b+ cells and quantified ratio of CD11b+ cells in total singlet cells in WT and PGRN‐KO mice 3 days following MI. (C) Representative FACS plots of cells distinguished by expression levels of Ly6C and CCR2. (D) Quantified proportion of Ly6C− CCR2+ CD11b+ cells in singlet cells. (E, F) Representative histograms of CD206+ and MerTK+ cells in Ly6C− CCR2+ macrophages. (G, H) Quantified proportion of CD206+ Ly6C− CCR2+ CD11b+ and MerTK+ Ly6C− CCR2+ CD11b+ cells in singlet cells. Data are the means ± SEM (WT: n = 4, PGRN‐KO: n = 3). *p < 0.05, **p < 0.01 versus WT (Levene's test followed by Student's two‐tailed t‐test).

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  FIGURE 5. Abnormal polarization of PGRN‐deficient bone marrow‐derived macrophages in inflammatory responses. (A) Granulin (GRN) mRNA levels in bone marrow‐derived macrophages (BMDMs), treated with LPS + IFN‐γ and IL‐4 for 48 h. Data are the means ± SEM (Normal: n = 4, LPS + IFN‐γ: n = 4, IL‐4: n = 4). ****p < 0.0001 versus LPS+ IFN‐γ (one‐way ANOVA followed by Dunnett T3 test). (B–I) mRNA levels of IL‐1β, IL‐6, TNF‐α, CCL2, IL‐10, IL‐4R, TGF‐β, and CD206 in WT and PGRN‐deficient BMDMs treated with LPS + IFN‐γ and IL‐4 for 48 h, respectively. Data are the means ± SEM (WT; Normal: n = 4, LPS + IFN‐γ: n = 4, IL‐4: n = 4, PGRN‐KO; Normal: n = 4, LPS + IFN‐γ: n = 4, IL‐4: n = 4). **p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one‐way ANOVA followed by Tuckey's test). (J–L) Phagocytosis activities quantified by fluorescence intensity of latex‐microbeads corrected by the number of cells in WT and PGRN‐deficient BMDMs treated with LPS for 1, 6, and 24 h. Data are the means ± SEM (WT; Normal: n = 5, LPS: n = 5, PGRN‐KO; Normal: n = 5, LPS: n = 5). **p < 0.01, ***p < 0.001, ****p < 0.0001 (one‐way ANOVA followed by Tuckey's test).