Apoptosis inhibitor of macrophage depletion decreased M1 macrophage accumulation and the incidence of cardiac rupture after myocardial infarction in mice

Abstract

Background: Cardiac rupture is an important cause of death in the acute phase after myocardial infarction (MI). Macrophages play a pivotal role in cardiac remodeling after MI. Apoptosis inhibitor of macrophage (AIM) is secreted specifically by macrophages and contributes to macrophage accumulation in inflamed tissue by maintaining survival and recruiting macrophages. In this study, we evaluated the role of AIM in macrophage accumulation in the infarcted myocardium and cardiac rupture after MI.

Methods and results: Wild-type (WT) and AIM‒/‒ mice underwent permanent left coronary artery ligation and were followed-up for 7 days. Macrophage accumulation and phenotypes (M1 pro-inflammatory macrophage or M2 anti-inflammatory macrophage) were evaluated by immunohistological analysis and RT-PCR. Matrix metalloproteinase (MMP) activity levels were measured by gelatin zymography. The survival rate was significantly higher (81.1% vs. 48.2%, P<0.05), and the cardiac rupture rate was significantly lower in AIM‒/‒ mice than in WT mice (10.8% vs. 31.5%, P<0.05). The number of M1 macrophages and the expression levels of M1 markers (iNOS and IL-6) in the infarcted myocardium were significantly lower in AIM‒/‒ mice than in WT mice. In contrast, there was no difference in the number of M2 macrophages and the expression of M2 markers (Arg-1, CD206 and TGF-β1) between the two groups. The ratio of apoptotic macrophages in the total macrophages was significantly higher in AIM‒/‒ mice than in WT mice, although MCP-1 expression did not differ between the two groups. MMP-2 and 9 activity levels in the infarcted myocardium were significantly lower in AIM‒/‒ mice than in WT mice.

Conclusions: These findings suggest that AIM depletion decreases the levels of M1 macrophages, which are a potent source of MMP-2 and 9, in the infarcted myocardium in the acute phase after MI by promoting macrophage apoptosis, and leads to a decrease in the incidence of cardiac rupture and improvements in survival rates.

Fig 1. Survival rates for the WT and AIM^‒/‒ groups after MI.

Kaplan-Meier survival curves for the WT and AIM^‒/‒ groups after MI. *P<0.05 compared with WT mice.

Fig 2. Cardiac rupture in WT and AIM^‒/‒ mice after MI.

The number of animals that died of cardiac rupture in the WT and AIM^‒/‒ groups (A), and the percentages of mice in each group that suffered cardiac rupture after MI (B). *P<0.05 compared with WT mice.

Fig 3. Macrophage accumulation in the infarcted myocardium of WT and AIM^‒/‒ mice.

Representative images of immunohistochemical staining for MAC-3 positive cells in the infarcted myocardium of WT and AIM^‒/‒ mice at 3 days after MI (A). The scale bars indicate 200 μm. The number of MAC-3 positive cells in the infarcted myocardium of WT and AIM^‒/‒ mice at 3 days after MI (B). *P<0.05 compared with WT mice.

Fig 4. M1 and M2 macrophages in the infarcted myocardium of WT and AIM^‒/‒ mice at 3 days after MI.

Representative images of immunofluorescence staining for MAC-3, iNOS (A) and CD206 (B). The number of MAC-3/iNOS double-positive cells (C) and MAC-3/CD206 double-positive cells (D) in the infarcted myocardium. *P<0.05 compared with WT mice.

Fig 5. M1 and M2 macrophage marker expression levels in the infarcted myocardium of WT and AIM^‒/‒ mice.

The mRNA levels of the indicated M1 (iNOS, IL-6 and IL-1β; A) and M2 (Arg-1, CD206 and TGF-β1; B) macrophage markers in the infarcted myocardium of WT and AIM^‒/‒ mice at 3 and 7 days after MI. *P<0.05 compared with sham-operated WT mice, ^#P<0.05 compared with WT-MI mice, n = 7 per group.

Fig 6. Macrophage apoptosis in the infarcted myocardium of WT and AIM^‒/‒ mice at 3 days after MI.

Representative images of TUNEL/MAC-3 double-positive cells in the infarcted myocardium of WT and AIM^‒/‒ mice at 3 days after MI (A). Mirror sections were stained for TUNEL and MAC-3, respectively and yellow arrows indicate TUNEL/MAC-3 double-positive cells. The ratio of TUNEL/MAC-3 double-positive cells in total MAC-3 positive cells in the infarcted myocardium of WT and AIM^‒/‒ mice at 3 days after MI (B). The mRNA levels of MCP-1 in the infarcted myocardium of WT and AIM^‒/‒ mice at 3 and 7 days after MI (C). *P<0.05 compared with sham-operated WT mice, ^#P<0.05 compared with WT-MI mice. n = 6–7 per group.

Fig 7. MMP-2 and 9 activity levels in the infarcted myocardium of WT and AIM^‒/‒ mice at 7 days after MI.

A representative image of gelatin zymography (A), and quantitative analyses of MMP-2 (B) and 9 (C) activity levels in the infarcted myocardium of WT and AIM^‒/‒ mice at 7 days after MI. *P<0.05 compared with sham-operated WT mice, ^#P<0.05 compared with WT-MI mice, n = 6 per group.