Long-Term Administration of Eicosapentaenoic Acid Improves Post-Myocardial Infarction Cardiac Remodeling in Mice by Regulating Macrophage Polarization

Abstract

Background: Consumption of n-3 fatty acids reduces the incidence of cardiovascular mortality in populations that consume diets rich in fish oil. Eicosapentaenoic acid (EPA) is an n-3 fatty acid known to reduce the frequency of nonfatal coronary events; however, the frequency of mortality after myocardial infarction (MI) is not reduced. The aims of this study were to determine whether long-term administration of EPA regulated cardiac remodeling after MI and to elucidate the underlying therapeutic mechanisms of EPA.

Methods and results: C57BL/6J mice were divided into control (phosphate-buffered saline-treated) and EPA-treated groups. After 28 days of treatment, the mice were subjected to either sham surgery or MI by left anterior descending coronary artery ligation. Mortality due to MI or heart failure was significantly lower in the EPA-treated mice than in the phosphate-buffered saline-treated mice. However, the incidence of cardiac rupture was comparable between the EPA-treated mice and the phosphate-buffered saline-treated mice after MI. Echocardiographic tests indicated that EPA treatment attenuated post-MI cardiac remodeling by preventing issues such as left ventricular systolic dysfunction and left ventricle dilatation 28 days after MI induction. Moreover, during the chronic remodeling phase, ie, 28 days after MI, flow cytometry demonstrated that EPA treatment significantly inhibited polarization toward proinflammatory M1 macrophages, but not anti-inflammatory M2 macrophages, in the infarcted heart. Furthermore, EPA treatment attenuated fibrosis in the noninfarcted remote areas during the chronic phase.

Conclusions: Long-term administration of EPA improved the prognosis of and attenuated chronic cardiac remodeling after MI by modulating the activation of proinflammatory M1 macrophages.

Keywords: acute myocardial infarction; eicosapentaenoic acid; inflammation; macrophage; macrophage polarization; mouse; remodeling.

© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Figures

Figure 1

EPA improved mortality from heart failure after MI, but it did not improve mortality from cardiac rupture. A, Kaplan‐Meier survival plot representing the percentage of surviving mice subjected to sham surgery with PBS treatment (sham+PBS, n=5), sham surgery with EPA treatment (sham+EPA, n=5), coronary ligation with PBS treatment (MI+PBS, n=68), or coronary ligation with EPA treatment (MI+EPA, n=59). Mortality from congestive heart failure (B) and cardiac rupture (C) after MI induction in mice by coronary artery ligation. In the survival analysis we did not include the mice that were sacrificed at intermediate time points. Differences between groups were tested by the log‐rank test. *P<0.05, comparing the MI+PBS and MI+EPA groups. EPA indicates eicosapentaenoic acid; MI, myocardial infarction; PBS, phosphate‐buffered saline.

Figure 2

EPA attenuated cardiac remodeling and congestive heart failure after MI. Ratio of (A) heart weight to tibial length (HW/TL) and (B) lung weight to tibial length (LW/TL), calculated 28 days after the MI and sham operations (n=5‐10 biological replicates per group). Cardiac mRNA expression levels of (C) ANP and (D) BNP 28 days after coronary artery ligation and the sham operations were determined by RTD‐PCR (n=4‐5 biological replicates per group). E, Echocardiographic analyses of the EPA‐ and PBS‐treated groups 28 days after coronary artery ligation and the sham operations. Data are presented as mean±standard error of the mean (n=3‐7 biological replicates per group). Values were compared between the MI+PBS and MI+EPA groups using the nonparametric Mann‐Whitney U test. *P<0.05, **P<0.01, comparing the MI+PBS and MI+EPA groups. %FS indicates percentage fractional shortening; ANP, atrial natriuretic peptide; BNP, B‐type natriuretic peptide; EPA, eicosapentaenoic acid; LVEDD, left ventricular end‐diastolic diameter; LVEF, left ventricular ejection fraction; LVESD, left ventricular end‐systolic diameter; MI, myocardial infarction; PBS, phosphate‐buffered saline; RTD‐PCR, real‐time detection polymerase chain reaction.

Figure 3

EPA treatment inhibited polarization toward proinflammatory M1‐like macrophages in myocardial tissue after MI. A, Gating strategy after purification of CD45+ cells by MACS separation of leukocytes (identified as SSC low and FSC high), single cells (doublet discrimination), and heart macrophages (identified as CD11b+F4/80+). B and E, Representative images of flow cytometric analysis of the sham, MI+PBS, and MI+EPA treatment groups 28 days after surgery. B, Scatter plots identifying cardiac‐residing CD11b+F4/80+ macrophages. E, Scatter plots identifying cardiac‐residing M1 macrophages (proinflammatory; CD11c+ CD206−) and M2 macrophages (anti‐inflammatory; CD11c− CD206+). Flow cytometric quantification of (C) total CD45+ cell counts, (D) total macrophage (CD11b+F4/80+) cell counts, (F) the percentage of M1 macrophage (proinflammatory, CD11c+ CD206−), (G) the ratio of M1 (proinflammatory, CD11c+ CD206−) to M2 (anti‐inflammatory, CD11c− CD206+) macrophages, (H) the percentage of total macrophage (CD11b+F4/80+), and (I) the percentage of M2 macrophage (anti‐inflammatory, CD11c− CD206+) in the hearts of the sham, MI+PBS, and MI+EPA groups 28 days after the MI and sham operations. Data are presented as mean±standard error of the mean (n=2‐5 biological replicates per group). Values were compared between the MI+PBS and MI+EPA groups using the nonparametric Mann‐Whitney U test. *P<0.05, comparing the MI+PBS and MI+EPA groups. EPA indicates eicosapentaenoic acid; FSC, forward scatter; MI, myocardial infarction; PBS, phosphate‐buffered saline; SSC, side scatter.

Figure 4

EPA modulated gene expression profiles in the heart 28 days after MI. The cardiac mRNA expression level of (A) collagen I, (B) collagen III, (C) TGF‐β1, (D) CCL2, (E) EMR1, (F) IL‐6, (G) MRC1, and (H) VEGF 28 days after coronary artery ligation and the sham operations was determined by RTD‐PCR. Data are presented as mean±standard error of the mean, n=5 to 8 biological replicates per group. Values were compared between the MI+PBS and MI+EPA groups using the nonparametric Mann‐Whitney U test. *P<0.05, **P<0.01, comparing the MI+PBS and MI+EPA groups. CCL2 indicates chemokine (C‐C motif) ligand 2; EMR1, EGF‐like module‐containing mucin‐like hormone receptor‐like 1; EPA, eicosapentaenoic acid; IL‐6, interleukin‐6; MI, myocardial infarction; MRC1, mannose receptor C type 1; PBS, phosphate‐buffered saline; TGF‐β1, transforming growth factor‐β1; VEGF, vascular endothelial growth factor.

Figure 5

EPA modulated cardiac remodeling in the noninfarcted area of the mouse heart 28 days after MI. A, Overview and representative high‐power photographs of picro‐sirius red‐stained remote LV myocardium 28 days after the MI and sham operations at mid‐LV levels. Red stains indicate collagen accumulation. B, The collagen volume fractional percentage of picro‐sirius red‐stained remote LV myocardium. n=3 to 7 biological replicates per group. C, Overview and representative high‐power photographs of AZAN‐stained remote LV myocardium 28 days after the MI and sham operations at mid‐LV levels. Blue stains indicate fibrosis. D, The fibrosis area percentage of AZAN‐stained remote LV myocardium. n=3 to 7 biological replicates per group. E, Overview and representative high‐power photographs of hematoxylin‐eosin–stained remote LV myocardium 28 days after the MI and sham operations at mid‐LV levels. F, Cardiac myocyte cross‐sectional area in the noninfarcted myocardium was obtained at 28 days. n=5 to 9 biological replicates per group. G, Overview and representative immunohistochemical staining of F4/80 in the noninfarcted area of the mouse heart 28 days after the MI and sham operations at mid‐LV levels. Immunohistochemical staining with F4/80 identified macrophages. Brown color indicated F4/80; blue color indicated nuclei. H, Percentage of the total number of cells that represent F4/80‐positive cells in the noninfarcted myocardium. n=3 to 11 biological replicates per group. Scale bar=1000 μm (overview) and 50 μm (high power). Data are presented as mean±standard error of the mean. Values were compared between the MI+PBS and MI+EPA groups using the nonparametric Mann‐Whitney U test. *P<0.05, **P<0.01, ***P<0.001, comparing the MI+PBS and MI+EPA groups. EPA indicates eicosapentaenoic acid; LV, left ventricle; MI, myocardial infarction; PBS, phosphate‐buffered saline.

Figure 6

Upregulation of IRF5 expression 28 days after MI was significantly attenuated by EPA treatment. A, Cardiac mRNA expression level of IRF5 28 days after coronary artery ligation and the sham operations was determined by RTD‐PCR. n=5 to 8 biological replicates per group. B, Protein levels of IRF5 in the heart 28 days after coronary artery ligation and the sham operations were determined by Western blotting. Protein levels were quantified by densitometry and normalized against GAPDH. n=4 to 8 biological replicates per group. Data are presented as mean±standard error of the mean. Values were compared between the MI+PBS and MI+EPA groups using the nonparametric Mann‐Whitney U test. *P<0.05, **P<0.01, comparing the MI+PBS and MI+EPA groups. EPA indicates eicosapentaenoic acid; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; IRF5, interferon regulatory factor 5; MI, myocardial infarction; PBS, phosphate‐buffered saline; RTD‐PCR, real‐time detection polymerase chain reaction.

Figure 7

EPA treatment attenuated TGF‐β/Smad signaling 28 days after MI. A, Representative Western blots and (B) quantitative analysis of total Smad2, phosphorylated Smad2, total Smad3, phosphorylated Smad3, and GAPDH. Quantitative data were expressed as the ratio between phosphorylated and total protein. n=5 to 6 biological replicates per group. Data are presented as mean±standard error of the mean. Values were compared between the MI+PBS and MI+EPA groups using the nonparametric Mann‐Whitney U test. *P<0.05, comparing the MI+PBS and MI+EPA groups. EPA indicates eicosapentaenoic acid; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; MI, myocardial infarction; PBS, phosphate‐buffered saline; TGF‐β, transforming growth factor‐β.