OSMR deficiency aggravates pressure overload-induced cardiac hypertrophy by modulating macrophages and OSM/LIFR/STAT3 signalling

Figures

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  Fig. 1. OSMR deficiency aggravates pressure overload-induced cardiac hypertrophy and reduces survival. A Representative gross morphology and haematoxylin and eosin-stained heart sections of wild-type (WT) or OSMR-KO mice subjected to sham or AB surgery at the indicated times (n = 8 mice per experimental group). B Heart weight to body weight (HW/BW) ratios, lung weight to body weight (LW/BW) ratios and heart weight to tibia length (HW/TL) ratios of WT or OSMR-KO mice at the indicated times (n = 8 mice per experimental group). C Quantification of the cross-sectional area in each group (n = 100–200 cells per experimental group). D Survival rates of mice in each group at eight weeks after sham or AB surgery (n = 12 mice per experimental group). Data are presented as the mean ± SEM. *P < 0.05 compared with the matched control

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  Fig. 2. OSMR deficiency exacerbates cardiac dysfunction and fibrotic remodelling in pressure-overloaded hearts. A Echocardiographic parameters and haemodynamic parameters of mice with or without OSMR knockdown at the indicated times (n = 8 mice per experimental group). B Representative images of picrosirius red (PSR) staining of transverse heart sections in each group (n = 8 per experimental group). (C-D) RT‒PCR analysis of ANP, BNP, α-MHC and β-MHC (C) and Col1, Col3, Ctgf and fibronectin (FN). D mRNA expression in mouse hearts at 4 weeks after sham or AB surgery (n = 6 per experimental group). mRNA expression was quantified and normalized to GAPDH (fold change). Data are presented as the mean ± SEM. *P < 0.05 compared with the matched control. #P < 0.05 compared with WT/AB

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  Fig. 3. OSMR deficiency activates STAT3 signalling associated with OSM/LIFR in pressure-overloaded hearts. A RT‒PCR analysis of OSM, LIFR, IL-31, IL-31RA and LIF mRNA expression in mouse hearts at 4 weeks after sham or AB surgery (n = 6 per experimental group). mRNA expression was quantified and normalized to GAPDH (fold change). B Immunofluorescence images of OSM in mouse hearts in each group at 4 weeks after sham or AB surgery (scale bar = 100 μm for 200 × magnification). C Immunofluorescence images of LIFR in mouse hearts in each group at 4 weeks after sham or AB surgery (scale bar = 100 μm for 400 × magnification). D Representative blots and quantitative results for OSM, LIFR, phospho-STAT3 (P-STAT3) and total STAT3 protein expression in the myocardium in each group at 4 weeks after sham or AB surgery (n = 6 per experimental group). Data are presented as the mean ± SEM. *P < 0.05 compared with WT/Sham. #P < 0.05 compared with WT/AB

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  Fig. 4. OSMR deficiency mediates inflammation and macrophage polarization in vivo. A Flow cytometric analysis of F4/80+ and LIFR+ cells in the heart tissue (n = 4 per experimental group). B Flow cytometric analysis of CD45+LIFR+Ly6C+ and CD45+LIFR+F4/80+ cells in mouse hearts at 4 weeks after sham or AB surgery (n = 4 per experimental group). C RT‒PCR analysis of TNF-α, MCP-1, CXCL1, VCAM1, P-selectin, IL-6, IL-1β, iNOS, Arg1 and IL-10 mRNA expression in mouse hearts at 4 weeks after sham or AB surgery (n = 6 per experimental group). mRNA expression was quantified and normalized to GAPDH (fold change). Data are presented as the mean ± SEM. *P < 0.05 compared with WT/Sham. #P < 0.05 compared with WT/AB

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  Fig. 5. Gating and quantification of immune cells in murine hearts and blood at 4 weeks after sham or AB surgery. A Gating strategy for flow cytometric analysis of the leukocyte populations isolated from the blood of WT and OSMR-KO mice at 4 weeks after sham or AB surgery. Flow cytometry-based quantification of the percentage of Ly6Chigh or Ly6Clow cells relative to the total monocyte/macrophage (Mo/MΦ) population and the percentage of F4/80+Ly6Chigh cells relative to the total Mo/MΦ population in WT and OSMR-KO mice at 4 weeks after sham or AB surgery (n = 4 per experimental group). B Gating strategy for flow cytometric analysis of the leukocyte populations isolated from the hearts of WT and OSMR-KO mice at 4 weeks after sham or AB surgery. Flow cytometry-based quantification of the percentage of Ly6Chigh or Ly6Clow cells relative to the total Mo/MΦ population in WT and OSMR-KO mice at 4 weeks after sham or AB surgery (n = 4 per experimental group). Data are presented as the mean ± SEM

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  Fig. 6. OSMR deficiency mediates inflammation and macrophage polarization in vitro. A Immunofluorescence images of F4/80 (red) and LIFR (green) in WT and KO bone marrow-derived macrophages (BMDMs) treated with LPS (scale bar = 100 μm for 400 × magnification. B Representative blots and quantitative results for OSM, LIFR, phospho-STAT3 (P-STAT3) and total STAT3 protein expression in BMDMs treated with LPS (n = 6 per experimental group). C RT‒PCR analysis of M1-like (IL-6, IL-1β, iNOS, LFA-1α) and M2-like (Arg1, IL-10, CD206, CD163) macrophage marker mRNA expression in BMDMs treated with LPS (n = 6 per experimental group). mRNA expression was quantified and normalized to GAPDH (fold change). Data are presented as the mean ± SEM. *P < 0.05 compared with WT MΦ/PBS. #P < 0.05 compared with WT MΦ/LPS

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  Fig. 7. Adoptive transfer of OSMR-KO macrophages into WT mice aggravates pressure overload-induced cardiac remodelling and cardiac dysfunction. A Schematic diagram outlining the adoptive transfer experiment. PBS or BMDMs were obtained from WT or OSMR-KO mice and adoptively transferred via the tail vein into WT mice 3 days after AB surgery. B Representative gross morphology and haematoxylin and eosin-stained heart sections of mice subjected to AB surgery and adoptive transfer (n = 8 mice per experimental group). C Heart weight to body weight (HW/BW) ratios, lung weight to body weight (LW/BW) ratios, cross-sectional areas and echocardiographic parameters (LVEF) of mice subjected to AB surgery and adoptive transfer (n = 8 mice/100 + cells per experimental group). D RT‒PCR analysis of ANP, BNP, and β-MHC mRNA expression in murine hearts (n = 6 per experimental group). mRNA expression was quantified and normalized to GAPDH (fold change). E Representative blots and quantitative results for OSM, LIFR, phospho-STAT3 (P-STAT3) and total STAT3 protein expression in murine hearts (n = 6 per experimental group). Data are presented as the mean ± SEM. *P < 0.05 compared with PBS to WT. #P < 0.05…

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  Fig. 8. Gating and quantification of immune cells in the heart and blood of mice that underwent adoptive transfer. A Gating strategy for flow cytometric analysis of the leukocyte population isolated from the blood of WT mice that underwent adoptive transfer of PBS, WT and OSMR-KO BMDMs after AB surgery. Flow cytometry-based quantification of the percentage of Ly6Chigh or Ly6Clow cells relative to the total monocyte/macrophage (Mo/MΦ) population and the percentage of F4/80+Ly6Chigh cells relative to the total Mo/MΦ population. (n = 4 per experimental group). B Gating strategy for flow cytometric analysis of the leukocyte population isolated from the hearts of WT mice that underwent adoptive transfer of PBS, WT and OSMR-KO BMDMs after AB surgery. Flow cytometry-based quantification of the percentage of Ly6Chigh or Ly6Clow cells relative to the total Mo/MΦ population. (n = 4 per experimental group). Data are presented as the mean ± SEM

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  Fig. 9. LIFR knockdown mitigates the hypertrophic effect of OSMR deficiency after AB surgery. A Schematic diagram outlining the intramyocardial injection experiment. Adenovirus-mediated short hairpin RNA (shRNA) transfer was performed through direct intramyocardial injection in the left ventricular free wall. B Representative gross morphology and haematoxylin and eosin-stained heart sections of mice subjected to AB surgery (n = 8 mice per experimental group). C Heart weight to body weight (HW/BW) ratios, lung weight to body weight (LW/BW) ratios, cross-sectional areas and echocardiographic parameters (LVEDd, LVEF, FS) of mice subjected to AB surgery (n = 8 mice/100 + cells per experimental group). D RT‒PCR analysis of ANP, BNP, and β-MHC mRNA expression in murine hearts (n = 6 per experimental group). mRNA expression was quantified and normalized to GAPDH (fold change). E Representative blots and quantitative results for OSM, LIFR, phospho-STAT3 (P-STAT3) and total STAT3 protein expression in murine hearts (n = 6 per experimental group). Data are presented as the mean ± SEM. *P < 0.05 compared with WT + AdGFP. #P < 0.05 compared with KO + AdGFP

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  Fig. 10. Gating and quantification of immune cells in the heart and blood of mice that received intramyocardial transfer of adenovirus. A Gating strategy for flow cytometric analysis of the leukocyte population isolated from the blood of WT and OSMR-KO mice that received intramyocardial transfer of AdGFP and AdLIFRshRNA after AB surgery. Flow cytometry-based quantification of the percentage of Ly6Chigh or Ly6Clow cells relative to the total monocyte/macrophage (Mo/MΦ) population and the percentage of F4/80+Ly6Chigh cells relative to the total Mo/MΦ population. (n = 4 per experimental group). B Gating strategy for flow cytometric analysis of the leukocyte population isolated from the hearts of WT and OSMR-KO mice that received intramyocardial transfer of AdGFP and AdLIFRshRNA after AB surgery. Flow cytometry-based quantification of the percentage of Ly6Chigh or Ly6Clow cells relative to the total Mo/MΦ population. (n = 4 per experimental group). Data are presented as the mean ± SEM

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  Fig. 11. Schematic images of the regulatory mechanisms of Osmr deficiency on pathological cardiac hypertrophy