SHEP1 alleviates cardiac ischemia reperfusion injury via targeting G3BP1 to regulate macrophage infiltration and inflammation

Abstract

The macrophage-associated inflammation response plays an important role in myocardial ischemia-reperfusion injury (MIRI). SHEP1(SH2 domain-containing Eph receptor-binding protein 1) has been implicated in adhesion and migration of inflammatory cells. However, the role and molecular mechanism of SHEP1 regulating macrophage remains unclear during MIRI. Here, the expression of SHEP1 was increased in macrophages co-cultured with hypoxia-reoxygenated cardiomyocytes and within ischemia-reperfusion injured myocardium at the early stage of injury. Cell migration and inflammation were also enhanced in SHEP1 knock-out macrophages and macrophage-specific deficiency of SHEP1 mice under MIRI, which further led to deteriorated cardiac injury and cardiac function in vivo. Mechanistically, macrophage-derived SHEP1 competitively bound to G3BP1 to suppress inflammation via the MAPK pathway. In addition, administrating inhibitor of G3BP1 could improve cardiac function in macrophage-specific deficiency of SHEP1 mice under MIRI. Our results demonstrate that SHEP1 deficiency in macrophages exacerbates MIRI through G3BP1-dependent signaling pathway. SHEP1-G3BP1 interaction are therefore indispensable for SHEP1 regulated- infiltration and proinflammatory responses of macrophages, which provided a potential and clinically significant therapeutic target for MIRI.

Fig. 1. SHEP1 selectively expressed in macrophages co-cultured with H/R cardiomyocytes fluctuates over time.

A Representative dual-immunofluorescence staining of SHEP1(green), F4/80(red), and nuclei (4,6-diamidino-2-phenylindole, blue) in mice hearts 1 day after IR. Scale bar, 10 μm. B Flow chart of the co-culture system. SHEP1 mRNA levels (C) and protein levels (D) in RAW264.7 co-cultured with hypoxia-reoxygenated cardiomyocytes for different time periods (n = 3 sets of cells). Data are presented as mean ± SEM.

Fig. 2. SHEP1 deletion in RAW264.7 co-cultured with H/R cardiomyocytes enhances macrophages migration and inflammation in vitro.

A SHEP1 mRNA levels in SHEP1 knocked-out and over-expressed RAW264.7 as well as their respective negative controls (NC-KO and NC-OE) analyzed by RT-qPCR. B SHEP1 protein levels in SHEP1 knocked-out and over-expressed RAW264.7 as well as their respective negative controls (NC-KO and NC-OE) analyzed by western-blot. C Representative images of transwell migration assay in which upper-well RAW264.7 migrated towards lower-well hypoxia-reoxygenated (H/R) cardiomyocytes. Cell numbers were calculated and compared (n = 6). D Representative images of cell scratch assay in which RAW264.7 monolayers were scratched and afterwards exposed to the supernatant derived from H/R cardiomyocytes. Cell numbers were calculated and compared (n = 6). E mRNA levels of IL-1β, MCP-1, and CCR2 in SHEP1 knocked-out/over-expressed RAW264.7 and their respective negative controls (NC-KO and NC-OE) co-cultured with H/R primary cardiomyocytes from adult mouse for 9 or 24 h in our co-culture system described above (n = 3 sets of cells). All data are presented as mean ± SEM.

Fig. 3. SHEP1 regulates macrophages activities primarily through the MAPK signaling pathway.

A RNA-sequencing (RNA-seq) for total RNA extracted from RAW264.7 cells (SHEP1 KO and WT) co-cultured for 0 h (SH) or 6 h (H6) with H/R primary cardiomyocytes isolated from adult mice. VENN, volcano blots, KEGG enrichment analysis and Heatmap are displayed. B WAVE2 and p-ERK1/2 protein levels in SHEP1 knocked-out (KO)/over-expressed (OE) RAW264.7 and their respective negative controls (NC-KO and NC-OE) co-cultured with H/R primary cardiomyocytes from adult mice for 9 or 24 h (n = 3 sets of cells). All data are presented as mean ± SEM.

Fig. 4. SHEP1 targets G3BP1 to attenuate macrophages migration and inflammation via MAPK signaling pathway.

A Mass spectrometry analysis of SHEP1 immunoprecipitates. Selected peptide hits and coverage are shown. B RAW264.7 co-cultured with H/R primary cardiomyocytes or sham were subjected to co-immunoprecipitation (co-IP) respectively with anti-SHEP1 and anti-G3BP1 antibodies. C SHEP1 knocked-out (KO)/over-expressed (OE) RAW264.7 cells and their respective negative controls (NC-KO and NC-OE) cells co-cultured with H/R primary cardiomyocytes or sham were subjected to co-immunoprecipitation (co-IP) respectively with anti-G3BP1 antibodies. D SHEP1 KO, OE and NC-KO, NC-OE RAW264.7 cells with or without G3BP1 deficiency were co-cultured with H/R primary cardiomyocytes for 9 or 24 h. Protein levels of WAVE2 were analyzed and compared (n = 3 sets of cells). E TBK1 and p-TBK1 protein levels in SHEP1 knocked-out (KO)/over-expressed (OE) RAW264.7 and their respective negative controls (NC-KO and NC-OE) co-cultured with H/R primary cardiomyocytes from adult mice for 9 or 24 h (n = 3 sets of cells). F P100/52 (NFκB2) and P105/50 (NFκB1) protein levels in SHEP1 knocked-out (KO)/over-expressed (OE) RAW264.7 and their respective negative controls (NC-KO and NC-OE) co-cultured with H/R primary cardiomyocytes from adult mice for 9 or 24 h (n = 3 sets of cells). G SHEP1 knocked-out (KO)/over-expressed (OE) RAW264.7 and their respective negative controls (NC-KO and NC-OE) with or without G3BP1 or cGAS deficiency were co-cultured with H/R primary cardiomyocytes for 9 or 24 h. Protein levels of P100/52 (NFκB2) were analyzed and compared (n = 3 sets of cells). All data are presented as mean ± SEM.

Fig. 5. The expression of SHEP1 shows sequential characteristic under the condition of MIRI in vivo.

A Representative immunohistochemical analyses of SHEP1 at different time points after IR. Scale bar, 500 μm. SHEP1 expression were quantified and compared (n = 4). B Representative immunohistochemical analyses of SHEP1 in remote area, border area, and infarct area on day 1 after IR. Scale bar, 500 μm. SHEP1 expression were quantified and compared (n = 4). C S SHEP1 mRNA levels were analyzed by RT-qPCR at different time points after MIRI (n = 4). D SHEP1 protein levels in myocardium were examined at different time points after MIRI by western blot with SHEP1 antibody, β-actin was used as loading control (n = 4). All data are presented as mean ± SEM.

Fig. 6. Macrophage-specific deficiency of SHEP1 deteriorated cardiac function and cardiac injury under MIRI in vivo.

A Representative cardiac echocardiography images at 3 and 21 days after MIRI with quantification of percentage ejection fraction (% EF) and percentage fractional shortening (% FS) (n = 12) (B) Representative heart sections from LysMCre⁺Shep1^fl/fl or Shep1^fl/fl mice stained with Evans-blue and 2,3,5-triphenyltetrazolium chloride (TTC) at 1 day after MIRI to delineate the area at risk (AAR) and the infarcted region. The ratios of AAR/LV and infarct area/AAR were compared (n = 6). C Representative photomicrographs of terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphate nick-end labeling (TUNEL) and nuclear (DAPI) staining of myocardium obtained 1day after MIRI. TUNEL-positive cell number per ROI were compared (n = 6). D Representative Western blot analysis showing the protein levels of Bax and Bcl-2 in LysMCre⁺Shep1^fl/fl and Shep1^fl/fl mice hearts at 1 day after MIRI or sham operation (n = 6). E Representative Masson trichrome staining of cardiac tissue obtained from LysMCre⁺Shep1^fl/fl and Shep1^fl/fl mice at day 21 after MIRI. Quantitative analysis of fibrotic areas (n = 6). All data are presented as mean ± SEM.

Fig. 7. Macrophage-specific deficiency of SHEP1 exacerbates cardiac injury through amplifying inflammatory process under MIRI in vivo.

A Representative dual-immunofluorescence staining of SHEP1 (green), F4/80 (red), and nuclei (4,6-diamidino-2-phenylindole, blue) in mice hearts (flox and CKO) 1 and 3 days after IR, F4/80⁺ (red) cell numbers were counted and compared (n = 4; scale bar, 500 μm). B, C Flow cytometry analysis for cardiac macrophages within LV myocardium in LysM Cre⁺ Shep1^fl/fl and Shep1^fl/fl mice 1 or 3 days after MIRI. n = 5 mice/group pooled from 5 independent experiments. D RT-qPCR analysis of the mRNA levels of inflammatory mediators in cells sorted by F4/80 magnetic beads from total cardiac cells 1 or 3 days after MIRI from LysM Cre⁺ Shep1^fl/fl mice and their Shep1^fl/fl littermates. All data are presented as mean ± SEM.

Fig. 8. G3BP1 inhibition mproves cardiac function of myeloid-specific SHEP1 deficiency mice under MIRI.

A LysM Cre⁺ Shep1^fl/fl mice and their Shep1^fl/fl littermates were intraperitoneally infused with EGCG or PBS as control right after IR. Echocardiography was conducted 3 days later, and representative images are displayed with quantification of percentage ejection fraction (% EF) and percentage fractional shortening (% FS) (n = 6). All data are presented as mean ± SEM.