Meteorin-like regulates cardiac repair via macrophage modulation following myocardial infarction

Figures

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  Fig. 1. Metrnl is expressed and secreted during the acute phase post-MI. 17-week-old wt mice were subjected to sham operation (Sham, white bars) or permanent coronary artery ligation (MI, grey bars) and samples were analyzed 4 days after surgery. A Circulating METRNL protein levels; n = 10–13 mice/group. B Metrnl expression levels in the myocardium of sham or MI mice in the remote zone (RZ), border zone (BZ), and ischemic zone (IZ); n = 6–7 mice/group. C Correlation between cardiac Metrnl expression levels in the IZ and METRNL circulating levels in wt, Metrnl−/−, and AAV9Metrnl mice 4 days post-MI. D Circulating levels of METRNL in control individuals (mean age 60 ± 2.63) and MI patients (mean age 60 ± 11.70). E Violin-plot representation of normalized Metrnl counts from single-cell RNA-seq (sc-RNAseq) macrophage sub-cluster at days 0, 1, 3, 5, and 7 post-MI. F–J Single-nucleus RNA-seq (snRNA-seq) of wt, Metrnl⁻/⁻, and Metrnl⁻/⁻ + AAV9-Metrnl mouse hearts at day 4 post-MI (n = 2 mice/group). F Two-dimensional UMAP visualization of cardiac nuclei identifying nine major cell types. G Two-dimensional UMAP showing expression levels of Metrnl and (H) violin-plot and heatmap displaying the…

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  Fig. 2. Metrnl prevents cardiac dysfunction and enhances remodeling after MI. 17-week-old wt (white bars), Metrnl−/− mice injected i.p with AAV9-null (Metrnl−/−, grey bars) or with AAV9 carrying Metrnl (AAV9Metrnl, orange bars) were subjected to permanent coronary artery ligation for 4 and 21 days. The myocardium of MI mice was divided in three zones: remote zone (RZ), border zone (BZ), and ischemic zone (IZ) (A) Metrnl expression levels in mouse heart post-MI; n = 4–8 mice/group (B) Circulating METRNL protein levels post-MI; n = 6–10 mice/group (C) Echocardiographic data from wt, Metrnl−/− and AAV9Metrnl mice after 2, 4, and 21 days post-MI induction. EF: ejection fraction, FS: fractional shortening, FAC: Fractional area change. n = 12–15 mice/group. D Quantification of fibrotic area in histological heart sections stained with Picrosirius red 4 and 21 days post-MI. Scale bar 1 mm. Lines show the fibrotic tissue. Results are expressed as a percentage of total myocardium; n = 4–7 mice/group. E Gene expression of fibrotic markers Col3a1, Col1a1, Mmp9, and Timp1 in RZ, BZ, and IZ at day 4 post-MI; n = 5–8 mice/ group. F Single-nucleus RNA-seq (snRNA-seq) of wt, Metrnl⁻/⁻, and Metrnl⁻/⁻ +…

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  Fig. 3. Metrnl deficiency promotes recruitment of inflammatory cells after MI. 17-week-old wt (white bars), Metrnl−/− mice injected i.p with AAV9-null (Metrnl−/−, grey bars) or with AAV9 carrying Metrnl (AAV9Metrnl, orange bars) were subjected to permanent coronary artery ligation for 4 days. A Determination of vascular density by immunofluorescence staining of CD31 protein (red) in IZ and BZ. Scale bar 20 µm. DAPI was used to stain nuclei (blue). Results are expressed as the percentage of the area occupied by the CD31 signal; n = 4–8 mice/group. B Histograms representing the percentage of macrophages, Ly6C High, Ly6C Low and expression of CD86 (MFI) in spleen 4 days post-MI; n = 4–8 mice. C Representative graph of CCR2+ or CCR5+ Ly6C populations in blood 4 days post-MI. D Histograms representing the percentage of macrophages CCR2+ or CCR5+ Ly6C in blood 4 days post-MI; n = 4–8 mice/group. Data are expressed as mean ± SD. Data were analyzed by t test and one-way ANOVA as appropriated. *p < 0.05 compared with wt mice; #p < 0.05, ##p < 0.01 compared with Metrnl−/− mice

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  Fig. 4. Metrnl−/− mice develop worse inflammatory response in the myocardium after MI. 17-weeks old wt (white bars), Metrnl−/− mice injected i.p. with AAV9-null (Metrnl−/−, grey bars) or with AAV9 carrying Metrnl (AAV9Metrnl, orange bars) were subjected to permanent coronary artery ligation for 4 days. The myocardium of MI mice was divided in three zones: remote zone (RZ), border zone (BZ), and ischemic zone (IZ). A Quantification of immune cell infiltration in the IZ 4 days post-MI by immunofluorescence staining of macrophages (F4/80, red) and neutrophils (Ly6B, white) in heart. DAPI was used to stain nuclei (blue). Scale bar: 20 µm. Results are expressed as number of cells/field; n = 4–8 mice/group. B mRNA expression levels of inflammatory marker TNFα and ligands of chemokine receptors Ccl2 and Ccl5; n = 5–8 mice/group. C mRNA expression levels of anti-inflammatory markers Arg1, Cd163, Mrc1; n = 5–8 mice/group. D Pro-inflammatory/anti-inflammatory macrophage ratio, expressed as iNos/Arg1 expression level ratio; n = 5–8 mice/group. E–F Single-nucleus RNA-seq (snRNA-seq) of wt, Metrnl⁻/⁻, and Metrnl⁻/⁻ + AAV9-Metrnl mouse hearts at day 4 post-MI (n = 2 mice/group). Two-dimensional UMAP…

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  Fig. 5. Oncostatin M (Osm) levels are regulated by Metrnl in mice post-MI. 17-week-old wt (white bars), Metrnl−/− mice injected i.p with AAV9-null (Metrnl−/−, grey bars) or with AAV9 carrying Metrnl (AAV9Metrnl, orange bars) were subjected to permanent coronary artery ligation for 4 days. The myocardium of MI mice was divided in three zones: remote zone (RZ), border zone (BZ), and ischemic zone (IZ). A mRNA levels of Osm in RZ, BZ, and IZ; n = 5–8 mice/group. B Representative images and quantification of OSM protein levels in IZ 4 days post-MI induction by western blot analysis. Ponceau S were used as loading control; n = 4–5 mice/group. C mRNA levels of Osmr in RZ, BZ, and IZ; n = 5–8 mice/group. D OSM circulating protein levels; n = 5–13 mice/group. E Plasmatic OSM protein levels in control individuals (Control) (59 ± 6.63) and MI patients (MI) (70 ± 10.2). F, G Single-nucleus RNA-seq (snRNA-seq) of wt, Metrnl⁻/⁻, and Metrnl⁻/⁻ + AAV9-Metrnl mouse hearts at day 4 post-MI (n = 2 mice/group). F Heatmap displaying the expression pattern of Metrnl and Osm across the three macrophage subtypes. G Violin-plot of Kit (c-kit) expression across the different cell types. Results are expressed as…

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  Fig. 6. Metrnl regulates Osm in macrophages in vitro. A Osm mRNA and OSM protein levels in cell culture media of pro- and anti-inflammatory-activated RAW264.7 macrophages treated with PBS (control, white bars) or Metrnl (orange bars) for 24 h. B Expression levels of anti-inflammatory (Clec10a, Mrc1, Arg1) and pro-inflammatory (Tnf, Il6, Ccl2) markers in activated RAW264.7 macrophages treated with PBS (control, white bars) or Metrnl (orange bars) for 12 h. Results are expressed as fold induction versus their corresponding non-activated cells. C Expression levels of Osm in activated bone marrow-derived macrophages (BMDM) obtained from wt (white bars) and Metrnl−/− mice (grey bars). D Expression levels of anti-inflammatory (Clec10a, Mrc1, Arg1) and pro-inflammatory (Tnf, Nos2, Ccl2) markers in activated BMDM obtained from wt (white bars) and Metrnl−/− mice (grey bars). Results are expressed as fold induction versus their corresponding non-activated cells and set at 1 for graphical representation. E Expression levels of Metrnl in primary neonatal cardiomyocytes in culture (NCMs) subjected to the hypoxia mimetic CoCl2 or hypoxic conditions (1% Oxygen, 5% CO2 and 94% N2 v/v). F Migration assay…

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  Fig. 7. Reg3β secreted by cardiomyocytes and regulated by the Metrnl–Osm axis inhibits Metrnl. A mRNA transcript levels of Reg3β and REG3β protein levels in cell culture media of neonatal cardiomyocytes (NCMs) previously infected with null adenovirus (CT) or Metrnl adenovirus (AdMetrnl) and treated with/without Osm for 24 h. B Metrnl mRNA levels in RAW264.7 macrophages (pro-inflammatory or anti-inflammatory-activated) in the presence or absence of recombinant Reg3β protein for 12 h during the activation process. C Expression levels of anti-inflammatory (Clec10a, Mrc1, Arg1) and pro-inflammatory (Tnf, Il6, Ccl2) markers in activated RAW264.7 macrophages treated with PBS (control, white bars) or Reg3β (pink bars) for 12 h. Results are expressed as fold induction versus their corresponding non-activated cells and set at 1 for graphical representation. D Reg3β expression levels in the myocardium of wt (white bars), Metrnl−/− mice injected i.p. with AAV9-null (Metrnl−/−, grey bars) or with AAV9 carrying Metrnl (AAV9Metrnl, orange bars) subjected to permanent coronary artery ligation for 4 days. Cell culture experiments were conducted on at least three independent cardiomyocyte isolations.…