Rev-erb agonist improves adverse cardiac remodeling and survival in myocardial infarction through an anti-inflammatory mechanism

Figures

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  Fig 1. Gene and protein expression levels of Rev-erb in the left ventricular (LV) border area after myocardial infarction (MI). (A) The expression level of Rev-erb α mRNA in the LV was not significantly different between sham, 3 days, and 7 days after MI (n = 4 animals for each time point). (B) The expression level of Rev-erb β mRNA in the LV was significantly decreased 3 and 7 days after MI (n = 4 animals for each time point). (C) Western blotting analysis showed a significant decrease in the expression level of Rev-erb α 7 days after MI (n = 3 animals for each time point). The bar graphs show the group mean±SEM. *p<0.05 vs Sham.

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  Fig 2. Kaplan-Meier survival curves after myocardial infarction. Survival rate was significantly higher in MI+SR than in MI+V (60.0% vs 31.6% at day 14 after myocardial infarction; log-rank test, p = 0.032 compared between MI+SR and MI+V). Solid line denotes the survival curve of MI+V and dashed line denotes of MI+SR.

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  Fig 3. M-mode echocardiographic analysis. The representative M-mode findings of the left ventricular short-axis view on day7 after MI: (A) Sham+V, (B) Sham+SR, (C) MI+V, and (D) MI+SR.

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  Fig 4. Representative photographs of the left ventricles at papillary muscle level. (A) Sham+V, (B) Sham+SR, (C) MI+V, and (D) MI+SR. (E) shows the ratio of collagen depositing area to total area. There was no significant difference in collagen deposition between MI+V and MI+SR (n = 4 animals in each group). (F) The mRNA expression level of collagen type Ia1 (Col1a) was significantly increased in MI+V, and was tend to be decreased in MI+SR, but not significantly different between MI+V and MI+SR. (G) The mRNA expression level of collagen type IIIa1 (Col3a) was also significantly increased in MI+V, and was not significantly different between MI+V and MI+SR (n = 4 animals in each group).

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  Fig 5. Gene expression levels of the left ventricles analyzed by real-time PCR and plasma BNP concentration. (A) The mRNA expression level of natriuretic peptide precursor B (Nppb) was significantly higher in MI+V than in sham, and was significantly lower in MI+SR than in MI+V (n = 4 animals in each group). The expression levels of (B) Il6, (C) Mcp1, (D) Ly6g, and (E) Cd11b were significantly increased in MI+V and were markedly decreased in MI+SR. (F) Matrix metallopeptidase (Mmp)9 was also significantly lower in MI-SR than in MI-V. (G) Plasma BNP concentration was also significantly higher in MI than in sham, and was significantly lower in MI+SR than in MI+V. The bar graphs show the group mean±SEM. *p<0.05 vs Sham+V; †p<0.05 vs MI+V.

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  Fig 6. Representative immunofluorescence images of the left ventricles stained with MMP-9. (A) Sham+V, (B) Sham+SR, (C) MI+V, and (D) MI+SR. MMP-9 was stained with Alexa Fluor 594 (red) and nuclei were stained with DAPI (blue). All images show 40× magnification. Sham groups showed low immunofluorescence intensity, and MMP-9 was strongly detected in the infarct and border areas in the MI groups. MI+SR showed less intensity compared with MI+V. (E) The ratio of immunofluorescence-positive area to total area was significantly increased in MI+V, and was markedly decreased in MI+SR (n = 4 animals in each group). The bar graphs show the group mean±SEM. *p<0.05 vs Sham+V; †p<0.05 vs MI+V.

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  Fig 7. Flow cytometric analysis. Flow cytometric analysis of the infiltration of inflammatory cells into the infarct and border areas at 1 day after myocardial infarction (MI) (n = 5 in each group). The representative plots (gated on CD45+ and expanded into Ly6G and CD11b) of (A) Sham+V, (B) MI+V, and (C) MI+SR9009 are shown. (D) The percentage of CD45+CD11b+Ly6G+ cells (neutrophils) to CD45+ cells was significantly lower in MI+SR than in MI+V, suggesting that neutrophil infiltration was reduced by SR9009 treatment. Flow cytometric analysis of M1/M2 macrophage infiltration into the infarct and border areas at 5 days after MI (n = 5 in each group). The representative plots (gated on CD11b and expanded into F4/80 and CD206) of (E) MI+V, and (F) MI+SR9009 are shown. (G) The percentage of F4/80+CD206- cells to live cells was significantly lower in MI+SR than in MI+V although the percentage of F4/80+CD206+ cells to live cells was not significantly different between MI+V and MI+SR, suggesting that proinflammatory M1 macrophage infiltration was reduced by SR9009 treatment. The bar graphs show the group mean±SEM. *p<0.05 vs Sham+V; †p<0.05 vs MI+V.

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  Fig 8. Western blotting analysis. (A) Representative images of western blotting for phospho-NF-κB p65 (S468 and S536), NF-κB, phospho-p38, p38, phospho-ERK, ERK, and β-actin. (B) The ratio of phospho-NF-κB (S468) to NF-κB was significantly lower in MI+SR than in MI+V. (C) The ratio of phospho-NF-κB (S536) to NF-κB had a similar tendency, but no significant difference. (D) The ratio of phospho-p38 to p38 was significantly lower in MI+SR than in MI+V. (E) The ratio of phospho-ERK to ERK was also significantly lower in MI+SR than in MI+V. (n = 3 animals in each group). The bar graphs show the group mean±SEM. *p<0.05 vs Sham+V; †p<0.05 vs MI+V.