Metrnl ameliorates myocardial ischemia-reperfusion injury by activating AMPK-mediated M2 macrophage polarization

Abstract

Background: Meteorin-like hormone (Metrnl) is prominently expressed in activated M2 macrophages and has demonstrated potential therapeutic effects in a range of cardiovascular diseases by modulating inflammatory responses. Nevertheless, its precise role and the underlying mechanisms in myocardial ischemia/reperfusion injury (MI/RI) are not fully understood. This study examined whether Metrnl can mitigate MI/RI through the AMPK-mediated polarization of M2 macrophages.

Methods: In vivo, adeno-associated virus 9 containing the F4/80 promoter (AAV9-F4/80) was utilized to overexpress Metrnl in mouse cardiac macrophages before MI/RI surgery. In vitro, mouse bone marrow-derived macrophages (BMDMs) were treated with recombinant protein Metrnl, and the human cardiomyocyte cell line AC16 was subjected to hypoxia/reoxygenation (H/R) after co-culture with the supernatant of these macrophages. Cardiac function was assessed via echocardiography, H&E staining, and Evans blue-TTC staining. Inflammatory infiltration was evaluated by RT-qPCR and ELISA, apoptosis by Western blotting and TUNEL staining, and macrophage polarization by immunofluorescence staining and flow cytometry.

Results: In vivo, Metrnl overexpression in cardiac macrophages significantly attenuated MI/RI, as evidenced by reduced myocardial infarct size, enhancement of cardiac function, diminished inflammatory cell infiltration, and decreased cardiomyocyte apoptosis. Furthermore, Metrnl overexpression promoted M1 to M2 macrophage polarization. In vitro, BMDMs treated with Metrnl shifted towards M2 polarization, characterized by decreased expression of inflammatory cytokines (IL-1β, MCP-1, TNF-α) and increased expression of the anti-inflammatory cytokine IL-10. Additionally, supernatant from Metrnl-treated macrophages protected AC16 cells from apoptosis under H/R conditions, as evidenced by decreased BAX expression and increased BCL-2 expression. However, these effects of Metrnl were inhibited by the AMPK inhibitor Compound C.

Conclusions: Metrnl alleviates MI/RI by activating AMPK-mediated M2 macrophage polarization to attenuate inflammatory response and cardiomyocyte apoptosis. This study highlights the therapeutic potential of Metrnl in MI/RI, and identifies it as a promising target for the treatment of ischemic heart disease.

Keywords: AMPK; Apoptosis; Inflammation; Macrophage polarization; Metrnl; Myocardial ischemia–reperfusion injury.

Fig. 1

Metrnl in macrophages is downregulated after MI/RI, and overexpression of Metrnl attenuates MI/RI. A Immunofluorescence co-localization of F4/80 (red) and AAV9 (GFP) in mouse hearts at 4 weeks post-injection of AAV9-F4/80-Metrnl virus. Scale bar = 50 μm, magnification = 400x (n = 5). B qRT-PCR was employed to detect the expression of Metrnl in the infarct zone of mice in each group following I/R surgery (n = 5). C WB was used to compare the expression of Metrnl protein in the infarct zone of mice (n = 4). D Quantification of panel C.The Metrnl protein expression is the ratio of Metrnl protein to β-actin (n = 4). E Serum cTnT levels of mice in each group were measured by ELISA kits (n = 6). F Evans blue-triphenyltetrazolium chloride (TTC) staining was utilized to determine the infarct area. Area-at-risk area is red and infarct area is white (n = 5). G Infarct area (%) was expressed as the percentage of white area to total area (n = 5). H Echocardiography was performed to assess cardiac function. I Left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS) were compared between groups of mice (n = 5). J H&E staining revealed the morphological changes of myocardial tissue in each group of mice (n = 6). Scale bar = 20 μm, magnification = 400x. Data are presented as means ± SD, ns not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 2

Overexpression of Metrnl in macrophages attenuates I/R-induced inflammatory response and cardiomyocyte apoptosis. A–C qRT-PCR was performed to detect the expression levels of inflammatory factors IL-1β, TNF-α, IL-6, MCP-1 and anti-inflammatory factors IL-10 and TGF-β in each group, respectively (n = 5). D Immunohistochemistry was used to compare the expression levels of p-NF-κB in each group (n = 6). Scale bar = 20 μm, magnification = 400x. E WB was employed to detect the expression of anti-apoptotic protein BCL-2 and its quantitative comparison (n = 6). F WB was used to detect the expression of apoptotic protein Bax and its quantitative comparison (n = 6). G TUNEL staining was conducted to compare the apoptosis rate of each group (n = 6). Scale bar = 50 μm, magnification = 400x. H Quantification of panel G. The apoptosis rate (%) was calculated as the number of nuclei stained by green fluorescence divided by the number of all nuclei (n = 6). Data are presented as means ± SD, ns not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

Metrnl overexpression promotes macrophage polarization from the M1 to the M2 subtype. A, B qRT-PCR was performed to detect CD86,CD206,ARG1,NOS2 mRNA levels in the infarct zone of each group (n = 5). C Immunofluorescence staining was performed to determine the expression of CD206 (green). Scale bar = 20 μm, magnification = 400x (n = 6). D Quantification of panel C. CD206/CD68 indicates the percentage of CD206 fluorescence area to CD68 fluorescence area (n = 6). E Immunofluorescence staining was performed to determine the expression of CD86 (green). Scale bar = 20 μm, magnification = 400x (n = 6). F Quantification of panel E. CD86 mean fluorescence intensity indicates the percentage of CD86 gray value and fluorescence area (n = 6). Data are presented as means ± SD, ns not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

Effect of Metrnl on macrophage polarization in vitro. A Diagram of the experimental protocol for the effect of Metrnl on macrophage polarization. B–D qRT-PCR compared mRNA levels of inflammatory and anti-inflammatory factors in bone marrow-derived macrophages (BMDMs) and after Metrnl intervention. The recombinant protein Metrnl intervention was 200 ng/mL for 24 h, as below. E–G qRT-PCR detected mRNA levels of inflammatory and anti-inflammatory factors in M1-type macrophages and after Metrnl intervention. M1-type macrophages were obtained by treating BMDMs with LPS (100 ng/ml) and IFN-γ (20 ng/ml) for 24 h. H–J qRT-PCR for mRNA levels of inflammatory and anti-inflammatory factors in M2-type macrophages and after Metrnl intervention. M2-type macrophages were obtained by treating BMDMs with IL-4 (20 ng/ml) for 24 h. (K-M) qRT-PCR detected mRNA levels of inflammatory and anti-inflammatory factors in each group. (N-Q) ELISA kits detected levels of inflammatory factors IL-1β, IL-6, TNF-α and anti-inflammatory factor IL-10 in macrophage supernatants. “rMetrnl”: recombinant protein Metrnl. NCMs: necrotic cardiomyocytes. CC Compound C. Control group: BMDMs. NCM group: stimulated with NCMs for 24 h. NCM + Metrnl group: intervened with NCMs and recombinant protein Metrnl. NCM + Metrnl + Compound C group: stimulated for 2 h by adding Compound C (100 μM/L) to NCM + Metrnl group. All tests were repeated independently at least three times. Data are presented as means ± SD, ns: not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

Metrnl attenuates H/R-induced cardiomyocyte apoptosis via AMPK-mediated M1-to-M2 macrophage polarization. A Immunofluorescence staining detected CD86 (green) expression in each group. Scale bar = 50 μm, magnification = 200 × . B Quantification of panel A. Mean fluorescence intensity of CD86 = CD86 gray value/fluorescence area. C Immunofluorescence staining detected CD206 (red) expression in four groups. Scale bar = 50 μm, magnification = 200 × . D Quantification of panel C. CD206 mean fluorescence intensity = CD206 gray value/fluorescence area. E Flow cytometry detected macrophage polarization in four groups. F Quantification of panel E. G WB experiments verified p-AMPK and AMPK protein expression in four groups. H Quantification of panel G. Relative expression of p-AMPK = p-AMPK/AMPK. I WB detected BCL-2 and BAX protein expression in AC16 cells in each group. J Quantification of panel L. Relative expression of BCL-2 = BCL-2/GAPDH; relative expression of BAX = BAX/GAPDH. All tests were repeated independently at least three times. Data are presented as means ± SD, ns not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

Molecular mechanism diagram. It revealed that Metrnl alleviated MI/RI by activating AMPK-mediated M2 macrophage polarization to attenuate inflammatory response and cardiomyocyte apoptosis