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

SHEP1 alleviates cardiac ischemia reperfusion injury via targeting G3BP1 to regulate macrophage infiltration and inflammation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SHEP1 alleviates cardiac ischemia reperfusion injury via targeting G3BP1 to regulate macrophage infiltration and inflammation Junbo Ge, Zhong Xin, Hua Li, Gao Tingwen, Guo Zhenyang, Xinyu Weng, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3964475/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Cell Death & Disease → Version 1 posted 9 You are reading this latest preprint version Abstract Background: The macrophage-associated inflammation response plays an important role in myocardial ischemia-reperfusion injury (MIRI). During MIRI, the role and molecular mechanism of SHEP1 regulating macrophage remains unclear. Methods: By co-cultured with hypoxia reoxygenation cardiomyocytes in vitro , macrophages with SHEP1 knockout or overexpression were detected cell migration ability and related proinflammatory factors; and the molecular network regulated by SHEP1 was identified through transcriptome-wide analysis; then its target molecules were verified by co-immunoprecipitation method. In vivo , an ischemia-reperfusion heart model was established to observe the changes in cardiac function, cardiac tissue injury and inflammation of macrophage-specific deficiency of SHEP1 mice, and to analyze the improvement of cardiac function by administrating inhibitors for targeted molecules of SHEP1. Findings: 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 . RNA-sequencing and co-immunoprecipitation mass spectrometry showed that macrophage-derived SHEP1 competitively bound to G3BP1 to suppress inflammation via the MAPK pathway. And administrating inhibitor of G3BP1 could improve cardiac function in macrophage-specific deficiency of SHEP1 mice under MIRI. Conclusions: SHEP1 targeted G3BP1 to antagonize cardiac ischemia-reperfusion injury by inhibiting infiltration and proinflammatory responses of macrophages, which provided a potential and clinically significant therapeutic target for MIRI. Biological sciences/Cell biology/Cell migration/Integrin signalling Biological sciences/Immunology/Cell death and immune response Myocardial ischemia-reperfusion injury macrophage SHEP1 G3BP1 cGAS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Acute myocardial infarction (AMI) is a major threat to human health worldwide. Revascularization therapy significantly improves the prognosis of AMI, but death rates due to secondary reperfusion injury remain persistently high. Accumulating evidence suggests that inflammatory mediators released from the innate and adaptive immune responses contribute to the progression of myocardial ischemia-reperfusion injury (MIRI) [ 1 , 2 ]. MIRI induces a vigorous immune response, augmented by the generation and release of various inflammatory cytokines and chemokines that accelerate peripheral monocyte mobilization and infiltration to exacerbate myocardial injury [ 3 , 4 ]. This ultimately leads to adverse outcomes including infarct expansion, left ventricular (LV) systolic dysfunction, and LV dilation [ 5 ]. Macrophages involved in MIRI exhibit functional heterogeneity, with pro-inflammatory macrophages infiltrating initially to clear the irreversibly damaged tissues, followed by a reparative phase of anti-inflammatory macrophages that drives the resolution of inflammation, tissue repair, and coronary angiogenesis [ 6 ]. Both migration and differentiation play vital roles in inflammatory processes. However, little is known about the initial mechanisms by which monocytes are recruited to the sites of injury, the diversity of monocyte-derived macrophages, or the instructive cues that specify their fate. To explore the regulators that modulate monocyte recruitment and fate specification during MIRI, we previously sequenced transcriptomes of peripheral blood mononuclear cells derived from 10 patients assigned to percutaneous coronary intervention due to acute ST-segment elevation myocardial infarction at successive timepoints launching around the intervention. The results demonstrated that the mRNA levels of SHEP1 were significantly associated with revascularization [ 7 ], suggesting that it may play a role in MIRI. SHEP1, encoded by the SH2D3C gene, is an Src homology 2 (SH2) -containing NSP scaffold that mediates protein-protein interactions through conserved modular domains [ 8 ]. Its architectures include an amino-terminal SH2 domain that binds to phosphorylated tyrosine residues, followed by a proline/serine-rich region, and a conserved carboxyl-terminal domain with homology to CDC25-related guanine nucleotide exchange factors (GEFs), which promote guanine nucleotide exchange on Ras family GTPases. Although the GEF domain of SHEP1 binds to several Ras proteins, it appears to lack enzymatic activity [ 9 ]. It has been shown that NSP family members affect the migration and adhesion of T cells and B cells by regulation of cell signaling [ 10 , 11 ]. In addition, they act as essential regulators of monocyte migration during atherosclerosis development [ 12 ]. However, whether and how SHEP1 participates in macrophage function during MIRI remains unclear. To ascertain the role of SHEP1 in macrophages during MIRI, we examined cardiac repair in mice genetically engineered to lack SHEP1 in macrophages. Our findings reveal that a deficiency of SHEP1 in macrophages antagonizes optimal cardiac rehabilitation by promoting macrophage infiltration while fostering pro-inflammatory responses, thus providing an attractive new approach to preventing adverse events in MIRI. Materials and Methods Isolation and culture of adult mouse primary cardiomyocytes Mouse cardiomyocytes were isolated from WT mice using Liberase enzymatic digestion as previously described [ 14 ]. Cell co-culturing The cell co-culturing system was performed using Corning Transwell chambers (0.4 µm pores, 6.5 mm diameter) where primary cardiomyocytes from adult wild-type C57 mouse were hypoxia-injured (in low-glucose DMEM without FBS, incubated in a 37°C air-tight incubator with 0.1%O2, 5% CO2 balanced by N2 for 6 hours) in the upper well, subsequently reoxygenated (back to high-glucose DMEM with 10% FBS), and concurrently inserted into lower well laid with RAW264.7 followed by incremental periods of co-culturing. RNA isolation and quantitative PCR (qPCR) Total RNA was extracted with TRIZOL reagent (93289, Sigma) according to the manufacturer’s protocol and quantified using a Nanodrop 2000. Total RNAs (1000ng) were used to perform the reverse transcription with the PrimeScript RT reagent kit (Takara, DRR037A). To determine relative mRNA abundance, qPCR was performed with SYBR Green Master Mix (Yeason) on an ABI StepOnePlus system according to the manufacturer’s protocol. Data were analyzed with StepOnePlus software. The primers were synthesized from Tsingke Biotechnology and sequence information is provided in Supplementary Table 1. Mouse 18S was used for normalization. The experiment was repeated three times. For RNA-seq, after RNA extraction and quantification, Oliago(dT)-attached magnetic beads were used to purify mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at an appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by second-strand cDNA synthesis. Afterward, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from the previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double-stranded PCR products from the previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single-strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make a DNA nanoball (DNB) which had more than 300 copies of one molecular. DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on the BGIseq500 platform (BGI-Shenzhen, China). The sequencing data was filtered with SOAPnuke (v1.5.2) by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterward clean reads were obtained and stored in FASTQ format. The clean reads were mapped to the reference genome using HISAT2 (v2.0.4). Bowtie2 (v2.2.5) was applied to align the clean reads to the reference coding gene set, then the expression level of the gene was calculated by RSEM (v1.2.12). The heatmap was drawn by heatmap (v1.0.8) according to the gene expression in different samples. Essentially, differential expression analysis was performed using the DESeq2 (v1.4.5) [with Qvalue ≤ 0. 05. To take an insight into the change of phenotype, GO ( http://www.geneontology.org/ ) and KEGG( https://www.kegg.jp/ ) enrichment analysis of annotated different expressed genes was performed by Phyper ( https://en.wikipedia.org/wiki/Hypergeometric_distribution ) based on Hypergeometric test. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold (Qvalue ≤ 0. 05) by Bonferroni. Protein sample preparation, IP-MS, and Western blotting assay Total proteins were extracted in RIPA lysis buffer (Beyotime, China) supplemented with a Complete Mini protease inhibitor cocktail (Roche, 11836153001). The protein concentration was determined by the BCA protein assay kit (Bio-Rad, 5000006JA). For immunoprecipitation (IP), the Pierce Classic IP Kit (Thermo Scientific™, 26146) was utilized according to the manufacturer’s protocol. For identification of shep1-interacting proteins, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed. Immunoprecipitates were separated with SDS–PAGE and the gel was stained with Coomassie brilliant blue for protein band visualizing. The entire lane was cut into 1cm gel slices and subjected to in-gel digestion. The mass spectrometry analysis was conducted by PTM-BIO (China). The ranking of the identified proteins was based on the peptide coverage of each identification. The related proteins were selected and subjected to further validation by immunoprecipitation and immunoblot. This study focused on G3BP1. After quantified, protein samples (10–20 µg) were loaded on SDS-polyacrylamide gels for electrophoresis and then transferred to PVDF membranes. After blocking with 5% BSA for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. After the subsequent washes, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Weiao Biological Company, China) at room temperature for 1 h. The blots were visualized and detected by Chemiluminescence Reaction (LuminataTM Forte, Millipore, USA) and ChemiDocTM Imaging System (Bio-Rad, CA, US). The density of the protein blots was analyzed by ImageJ (Version 1.53c, NIH, USA). Detailed information on the primary antibodies used in immunoblotting, including source, catalog number, and dilution, is listed in Supplementary Table 2. We also provide full scans of all the blots as Supplementary Dataset. Migration wound healing Migration assays were performed using Corning Transwell chambers (8 µm pores, 6.5 mm diameter, USA). Primary cardiomyocytes were isolated from adult wild-type C57 mice, laid in the lower wells, and exposed to H/R injury. After reoxygenation, RAW264.7 cells were seeded in the upper chambers containing FBS-free DMEM. After 24 h of co-culture, cells that traversed through the membrane were fixed with 4% PFA for 30 min and stained with 0.05% crystal violet solution (Solarbio, China) for 20 min. A wound-healing assay was conducted in 6-well plates by scratching the cell monolayer with a 200 µl pipette tip. Cells were washed with PBS twice and cultured in conditional media of H/R cardiomyocytes. Images were collected at the beginning and 24 h after scratching. The wound-healing speed was calculated as the ratio of the healed areas at 24 h to the original injured areas. RNA interference RAW264.7 cells were transfected with siRNAs by Lipofectamine™ 3000 transfection reagent (Invitrogen™, L3000150) for 48h before stimulation according to the manufacturer’s instruction. The siRNAs used were as follows: si-cGAS (5'-CGGCAGCUAUUAUGAACAU-3'), si-G3BP1(5′-CCCTATGGAAATCATTCCT-3′). Mice Mice were fed with a standard rodent chow diet and housed in micro isolator cages in a special pathogen-free facility. The number of animals in each experimental group is indicated in the tables or figure legends. This study and all animal procedures conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH publication NO. 85 − 23, revised 1996), and were approved by the animal care and use committee of Fudan University. Wild-type (WT) C57BL6/J mice (male, 14–16 weeks old; SLRC Laboratory Animal, Shanghai, China) were used in this study. Myeloid-specific SHEP1-deficient mice were generated by crossing SHEP1 fl/fl mice with LysM-Cre mice and are designated as LysM Cre + SHEP1 fl/fl (CKO) in this paper (constructed by Cyagen Biosciences Inc. China). For experiments described in this paper, adult male mice (14 to 16 weeks of age) were used. EGCG administration: Mice were intraperitoneally injected with EGCG (40mg/kg) right after reperfusion, and afterward once a day for 3 days. Cardiac I/R injury model Mice underwent surgical cardiac I/R injury as described previously [ 13 ]. Briefly, the mice were anesthetized with 2% isoflurane inhalation using an isoflurane delivery system. Subsequently, a small skin cut (1.2 cm) was made over the left chest, and the left anterior descending coronary artery (LAD) was ligated via a slipknot using 6–0 silk for 45 min before the slipknot was gently loosened to induce reperfusion injury. The suture was passed 2–3 mm below the tip of the left auricle. Sham-operated animals underwent the same procedure without tying the slipknot. Triphenyltetrazolium chloride (TTC) staining TTC staining was conducted for evaluation of cardiac infarction size. After the mice were anesthetized with 2% isoflurane, the slipknot was tied again, and 1% Evans blue (w/v, Sigma, USA) was injected into the aortic root to perfuse the left ventricle. Then, the heart was rapidly excised, cut into 1 mm slices, and incubated with 1% TTC solution (Sigma, USA) at 37°C for 10 min. Consecutive slices were scanned by a white light scanner (Canon, Japan). LV area, AAR, and infarct area were determined by computerized planimetry and comprehensively analyzed in serial sections of each mouse) using ImageJ software. The infarction degree was calculated as the ratio of the infarction area to the AAR. Transthoracic echocardiography Cardiac function was assessed with a Vevo2100 Ultrasound system (VisualSonics, Toronto, Canada). Mice were mildly anesthetized with 0.5% isoflurane and placed on a heated ECG platform. The left ventricle and the aortic outflow tract were observed under the two-dimensional B-Mode, and the sample line was placed at the maximum cross-section of the left ventricle to guide the recording of serial M-Mode echocardiographic images. The left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), fractional shortening (FS), left ventricular end-systolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD) were measured from at least three distinct frames for each mouse, and the average was used for data analysis. IHC and immunofluorescence staining Analysis was performed using paraffin-embedded sections of the heart tissue samples fixed with 4% paraformaldehyde. All tissue samples were characterized using hematoxylin and eosin (H&E) staining before further immunostaining. After the inhibition of endogenous peroxidase activity, the sections were incubated with primary anti-SHEP1 (sc-100792, Santa Cruz) at 4°C overnight. Following the incubation, a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) was used according to the manufacturer’s instructions. After the visualization with 3,3’-diaminobenzidine, the sections were counterstained with hematoxylin. As for immunofluorescence analysis, after deparaffinization, heat-mediated antigen retrieval in citrate buffer pH 6.0, and blocking with 5% horse serum at room temperature for 30 min, samples were incubated with primary antibodies overnight at 4°C. The primary antibodies anti-SHEP1 (Santa Cruz, 1:100, sc-100792) and anti-F4/80 (AbD Serotec, 1:200, MCA497GA) were utilized for these experiments. Signals were visualized with Alexa fluorescence-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). TUNEL staining was performed with an In Situ Apoptosis Detection kit (Yeasen, Shanghai, China), according to the manufacturer’s instructions. Cell nuclei were stained with DAPI. Five fields or more in > 3 different sections/animals were examined by a technician who was blinded to the treatment groups. Images were obtained using an Olympus microscope and quantified using Image J. For each heart sample, at least five random fields were measured. Cell extract, flow cytometry, and magnetic beads sorting The hearts were dissected, carefully cut into small pieces with fine scissors, and enzymatically digested with type II collagenase (1.5 mg mL-1, Worthington Biochemical, Lakewood, NJ, USA), elastase (0.25 mg mL-1, Worthington Biochemical), and DNase I (0.5 mg mL-1, Worthington Biochemical) for 1 h at 37°C. Following digestion, the tissues were passed through 70-µm cell strainers, washed, and stained with antibodies for fluorescence-activated cell sorter (FACS) analysis. To block the non-specific binding of antibodies to Fcγ receptors, isolated cells were incubated first with anti-CD16/32 antibody (BD Biosciences, #553142) at 4°C for 5 min. The primary antibodies against the following were obtained from BD Biosciences (Franklin Lakes, NJ, USA): CD45.2 (#564616), CD11b (#552850), Ly-6G (#560599), Ly6C (#563011), CD64 (Biolegend, #139309), CD206 (Thermo, 17-2061-82). The obtained results are expressed as cell number/µg tissue. Flow cytometric analysis was performed using a FACSAria™ flow cytometer (BD Biosciences), and the obtained data were analyzed with FlowJo 7.6.1 software (Tree Star, Ashland, OR, USA). Magnetic cell sorting was performed using QuadroMACS (Miltenyi Biotec, 130-091-051) according to the manufacturer’s instructions, anti-F4/80 MicroBeads (Miltenyi Biotec, 130-110-443) were utilized for separating macrophages. Statistical analysis Data were collected and expressed as average ± standard error of the mean (SEM). T-test were conducted when comparing 2 independent groups with the normalized distribution of values. For n = 3–4 per group, Mann-Whitney tests were used. ANOVA-test or Kruskal-Wallis (with low numbers) were carried out when comparing more than 2 groups. For two groups and multiple time points, a two-way ANOVA was used. Statistical analysis was conducted using GraphPad Prism 8.0 (GraphPad Prism Software Inc., San Diego, CA, USA). Results SHEP1 selectively expressed in macrophages co-cultured with H/R cardiomyocytes fluctuates over time We first co-stained heart tissue slices from wild-type C57BL6 mice that had undergone cardiac IR operation with fluorescent-labeled antibodies for macrophages (F4/80, red) and SHEP1 (green) to investigate whether SHEP1 selectively expressed in macrophages. And DAPI (blue) was used to stain the nuclei. The results demonstrated that SHEP1 was specifically localized in the cytoplasm of macrophages in merged confocal images (Fig. 1 a). To mimics microenvironment in vivo under the condition of MIRI, we established co-culture systems with transwells between macrophages and hypoxia-reoxygenated (H/R) cardiomyocytes (flow chart shown in Fig. 1 b) to investigate the evolution of SHEP1 expression in RAW264.7 cells over time, with the results that SHEP1 mRNA levels peaked at 3 hours and protein levels peaked at 6 hours after co-culture, then both gradually declined to baseline level within 24 hours (Fig. 1 c & d ). SHEP1 deletion in RAW264.7 co-cultured with H/R cardiomyocytes enhances macrophages migration and inflammation in vitro To observe the effect of SHEP1 on the biological function of monocyte in vitro , we thereupon constructed SHEP1 knock-out (KO) and over-expressed (OE) RAW264.7 monoclonal cell lines for verification by qPCR and western-blot (Fig. 2 a & b) . By the above transwell co-culture systems, the migration of SHEP1-KO RAW264.7 group was significantly promoted than that of NC-KO group, whereas it showed the opposite trend between the SHEP1-OE group and NC-OE group (Fig. 2 c). And another result with the same biological characteristics was confirmed by wound healing in vitro among these 4 groups (Fig. 2 d). Simultaneously, the effect of SHEP1 regulating inflammation in macrophage was also investigated through using the co-culture system described above. qRT-PCR showed that the pro-inflammatory cytokine IL-1β, chemokine MCP-1 and CCR2 were obviously upregulated in the SHEP1-KO RAW264.7 group (lower-chamber), while down-regulated in the SHEP1-OE group, compared with their corresponding control groups. Unlike that CCR2 showed significant differences at the baseline level, the differences of IL-1β and MCP-1 were gradually occurred after co-culture stimulation, which might mainly be due to that IL-1β and MCP-1 are inflammatory factors produced by macrophages in response to the stimulation of an inflammatory environment, whereas CCR2 represents the susceptibility of macrophages to chemokines (Fig. 2 e). SHEP1 regulates macrophages activities primarily through the MAPK signaling pathway To elucidate the molecular mechanisms of SHEP1 in various cell activities, we carried out RNA-sequencing (RNA-seq) for total RNA extracted from RAW264.7 cells (SHEP1-KO group and control group, respectively) co-cultured for 0 h or 6 h with H/R primary cardiomyocytes isolated from adult wild-type C57 mice. The transcriptome-wide role of SHEP1 in RAW264.7 cells was evaluated in the co-culture system to identify the key pathways that may be affected by the loss of SHEP1. In total, 720 differential genes were obtained from a Venn diagram analysis of different cell groups for the same co-culture duration and different co-culture durations from the same cell group (Fig. 3 a). Subsequently, KEGG pathways analysis among these differential genes indicated that the primary RAS-MAPK-ERK pathway was identified from top 20 pathways and partially involved in cell migration (Fig. 3 a). To illuminate the regulatory role of SHEP1 in RAS-MAPK-ERK signaling pathways, western blotting was used to detect the expression of ERK phosphorylation and its downstream protein WAVE2. The results showed that it was a negative regulation of this pathway by SHEP1, with increased expression of ERK phosphorylation and WAVE2 in SHEP1-KO RAW264.7 cells, while decreased expression in the SHEP1-OE group, when they co-cultured with H/R cardiomyocytes (Fig. 3 b). SHEP1 targets G3BP1 to attenuate macrophages migration and inflammation via MAPK signaling pathway To further explore interactive proteins targeted by SHEP1, Co-IP mass spectrometry and competition assay were used to elucidate these potential molecular cluster in RAW264.7 cells and their possible regulation of the RAS-MAPK-ERK pathway. Firstly, differentially expressed proteins binding to SHEP1 in RAW264.7 cells between co-cultured with H/R cardiomyocytes for 9 hours and the normoxia group was analyzed (Fig. 4 a). Among these differential proteins, Ras-GTPase-activating protein SH3 domain binding protein1(G3BP1) was an optimal candidate since it could bind to the SH3 domain of Ras-GTPase-activating protein (RasGAP), which might regulate RAS-MAPK-ERK signaling pathway[ 15 ]. Subsequently, the direct interaction between SHEP1 and G3BP1 was confirmed through Co-IP experiment when RAW264.7 cells were co-cultured with H/R cardiomyocytes (Fig. 4 b). Secondly, via gene regulation of targeting protein G3BP1, the effect of SHEP1 on expression of its downstream proteins in MAPK signaling pathway was also observed. The results indicated that siRNA-assisted knockdown of G3BP1 abrogated increased expression of WAVE2 in SHEP1-KO RAW264.7 group and decreased expression of WAVE2 in the SHEP1-OE group, which showed that through G3BP1, SHEP1 had a regulatory effect on the RAS-MAPK-ERK pathway, thereby affecting cell migration (Fig. 4 c). Thirdly, G3BP1 has been revealed to promote the activation of cGAS via DNA binding, and subsequently to activate NF-κB in previous studies [ 16 , 17 ]. To clarify that SHEP1 suppresses the expression of inflammatory factors by competitive binding with G3BP1 to interrupt the G3BP1-cGAS interaction, western blotting was applied to detect the expression of TBK1 phosphorylation and proteins of NF-κB pathway. The following experiments demonstrated that the binding of SHEP1 and G3BP1 was enhanced in macrophages co-culturing with H/R cardiomyocytes, whereas the binding of G3BP1 and cGAS was synchronously abated (Fig. 4 b). Simultaneously, SHEP1 was also found to suppress the activation of cGAS, with evidence of the repression of TBK1 phosphorylation (Fig. 4 e) and the activation of proteins of NF-κB pathway (Fig. 4 f) in SHEP1-KO RAW264.7 group, while an opposite trend was observed in the SHEP1-OE group. Further, blockage of either G3BP1 or cGAS could abolish these effects of SHEP1 on proteins related to NF-κB (Fig. 4 f). The expression of SHEP1 shows sequential characteristic under the condition of MIRI in vivo Immunohistochemistry showed that SHEP1 was expressed mainly in the infarct and border areas after MIRI, with low expression in the non-infarct areas (Fig. 5 a). Through continuous observation of expression of SHEP1 in histological sections for 7 days, SHEP1 area was found to reach its peak at 24 hours after MIRI and afterward experienced a gradual decline (Fig. 5 b). To elucidate the expression of SHEP1 in cardiac tissue under the condition of MIRI at different reperfusion time points, we checked the gene expression levels and protein abundance of SHEP1 in the myocardium. The results uncovered that mRNA level of SHEP1 increased as early as 3 hours after MIRI and peaked at 6 hours, while protein accumulation progressively increased from 9 hours to 24 hours and subsequently declined (Fig. 5 c and 5 d). These data in vivo showed the similar tendency with the western blot results of cells in vitro. Macrophage-specific deficiency of SHEP1 deteriorated cardiac function and cardiac injury under MIRI in vivo To investigate the crucial biological role of SHEP1 on regulation of cardiac function and cardiac injury under the condition of MIRI in vivo, LysM Cre + SHEP1 fl/fl (CKO) and SHEP1 fl/fl (flox) littermates were generated by using gene-editing technology and then underwent the operation of IR. Echocardiography was performed to observe the difference among various groups. The data showed that despite similar baseline echocardiographic parameters, macrophage-specific deficiency of SHEP1 group resulted in worse cardiac function both 4 days and 21 days after MIRI, with a significant reduction in the ejection fraction (EF%) and fractional shortening (FS%), compared with the control group (Figs. 6 a). Subsequently, the pathologic character of myocardial injury was evaluated, according to infarct area/ areas at risk (AAR) ratio and myocardial apoptosis markers. The findings displayed that AAR ratio in CKO mice with SHEP1 deficiency in macrophages was obviously higher than that in control group (Fig. 6 b). Similar results were confirmed by further evidence that the increased TUNEL + cardiomyocytes (Fig. 6 c) in CKO mice group were more obvious, concurrently, higher expression of pro-apoptotic molecule BAX and lower expression of anti-apoptosis molecule Bcl-2 (Fig. 6 d) in LV tissue samples could be observed in CKO mice group 24 hours after MIRI, compared with the control group. Additionally, myocardial fibrosis post myocardial infarction is secondary pathological changes, which could be evaluated by Masson’s trichrome staining. The results showed that myocardial fibrosis 21 days after MIRI increased significantly in the CKO group, compared with the control group (Fig. 6 e). Although the increase in SHEP1 expression in macrophages after MIRI returned to baseline levels within 3 days, the reason why the difference between various groups was still significant might be due to the reflection of the integrated effect of the lack of SHEP1, the early inflammatory and myocardial injury. Macrophage-specific deficiency of SHEP1 exacerbates cardiac injury through amplifying inflammatory process under MIRI in vivo AS an important source for inflammatory cytokines, macrophages play a critical role in the regulation of inflammation caused by myocardial injury. To investigate the role of SHEP1 in macrophage mediated inflammatory response, the operation of IR was conducted in both CKO mice and flox littermates. The results indicated that by the comparison of their differences at timepoints of 1 and 3 days after the initiation of reperfusion, immunofluorescence staining of the myocardium in CKO mice appeared obviously enhanced infiltration of macrophages into the myocardium, compared with that in flox littermates (Fig. 7 a). And the number of CD11b/CD46 double labeled positive macrophages in CKO mice hearts were significantly higher than that of control group whether 1 day or 3 days after MIRI (Fig. 7 b and supplementary Fig. 1 ). Simultaneously, the ratio of M1/M2 of macrophages in CKO mice hearts by flow cytometry was found to be higher than that of hearts in flox littermates 1 day after MIRI, without difference 3 days after MIRI (Fig. 7 c). Besides, the proportion of Ly6c low /macrophages among various groups showed no remarkable difference (Fig. 7 d). These above flow cytometry results implied the positive role of SHEP1-deficiency improving macrophage differentiation under MIRI. Inflammatory cytokines are important regulators of inflammatory reaction under the condition of various injuries. We further conducted magnetic cell sorting for F4/80 + cells from total cardiac cells of different groups of mice at 1 and 3 days after MIRI and then applied RNA analysis of the isolated cells to quantify their pro-inflammatory phenotype. The results showed that compared to the control group, the pro-inflammatory factors IL-1β and MCP-1 were significantly enriched in the CKO mice group at 1 and 3 days after MIRI. And the similar expression trend was consistent with C-C motif chemokine receptor 2 (CCR2), whereas inconsistent with TGF-β and IFN-β (Fig. 7 e). Epigallocatechin gallate, an inhibitor of G3BP1, improves cardiac function of myeloid-specific SHEP1 deficiency mice under MIRI To verify whether SHEP1 also competitively bound to G3BP1 to improve heart function in vivo , we first selected epigallocatechin gallate (EGCG), an inhibitor of G3BP1 extracted from green tea, as targeting reagent, and then administered it to the MIRI model mice once daily, intraperitoneally for 3 days. The results indicated that a significant improvement in cardiac function in CKO mice group receiving EGCG occurred, with increased ejection fraction (EF%) and fractional shortening (FS%), compared with that in CKO mice group receiving PBS. What’s more, the increasing extent in the above two parameters in CKO mice group was significantly higher than those in Flox mice group receiving EGCG and PBS (Fig. 8 a). Discussion In the course of myocardial ischemia-reperfusion injury, large quantities of monocytes invade the heart within 24 h as the dominant immune cells obliterate debris [ 18 ]; however, excessive inflammation is thought to exacerbate adverse remodeling and heart failure pathogenesis. This highlights the need for an appropriate extent of anti-inflammatory approaches within the desired time window to effectively suppress injurious processes without negatively interfering with the reparative response. However, little is known about the variety of monocytes and monocyte-derived macrophages recruited to the heart during MIRI, or the mechanisms that modulate monocyte recruitment and fate determination. In our model of clinically relevant MIRI, we observed an increased level of SHEP1 in the myocardium at early time points after injury, with subtotal expression in F4/80 + macrophages. Using myeloid cell-specific SHEP1 knockout mice, we demonstrated for the first time that genetic depletion of SHEP1 in macrophages exacerbated MIRI through enhanced infiltration and pro-inflammatory differentiation of monocytes, revealing a role for SHEP1 in macrophages during MIRI, implying that targeting SHEP1 on macrophages may constitute a therapeutic approach for the prevention of IR injury. As mentioned above, SHEP1 was only highly expressed in the early stage of MIRI and decreased to baseline levels within 3 days. The early expression of SHEP1 could counteract excessive macrophage infiltration and inflammatory factor secretion only at the early stages of MIRI to further reduce myocardial tissue damage and cardiac function deterioration caused by an exaggerated inflammatory response, without directly impeding the reparative function of macrophages in the middle and late stages of injury. This makes it an ideal target and research direction for MIRI therapy. Specialized receptors on the cell surface transduce extracellular stimuli and initiate signaling cascades that culminate in diverse functional outcomes. This organization is achieved through specific high-affinity protein-protein interactions and multiprotein complex formation facilitated by conserved modular domains. Src homology region 2 (SH2) is one of the most prevalent conserved modular domains [ 8 ]. Accordingly, we assumed that SHEP1 manipulates macrophage functionality through protein-protein interactions, and subsequently conducted protein mass spectrometry in the immunoprecipitates of SHEP1. G3BP1 was screened from those identified proteins because it can link SHEP1 to both the migration and inflammation pathways. G3BP1 was initially discovered as a binding protein of the Ras-GTPase-activating protein (RAS-GAP), which is an inactivator of RAS GTPase via hydrolysis of the Ras-bound GTP molecule to GDP [ 19 , 20 ]. With no enzymatic activity, G3BP1 acts on the RAS signaling pathway by interacting with GAP [ 21 ]. Here, we demonstrated the combination of SHEP1 and G3BP1 to a greater extent when co-cultured with hypoxia-reoxygenated cardiomyocytes, indicating a potential role of SHEP1 in RAS signaling pathway manipulation. This was confirmed by RNA-sequencing of RAW264.7, co-cultured with hypoxia-reoxygenated cardiomyocytes, and further confirmed by protein analysis, in which SHEP1 suppressed phosphorylation of ERK1/2 as well as expression of WAVE2, an activator of the Arp2/3 actin nucleator essential for cell migration through actin assembly [ 22 ]. Further blocking of G3BP1 by siRNA abolished SHEP1 influence on WAVE2, confirming that SHEP1 acts on the RAS-MAPK-ERK pathway through G3BP1. Additionally, recent studies have revealed that G3BP1 abrogates cGAS stimulation, subsequently activating NF-κB by enhancing cGAS-DNA association independent of SGs [ 16 , 17 ], despite its essential role in promoting the formation of SGs [ 23 – 26 ]. As a cytosolic DNA sensor, cGAS is activated robustly in myocardial infarction, triggering downstream events mediated by the STING cascade. This, in turn, promotes macrophage transformation toward the M1-like subtype [ 27 ]. Our study confirmed the G3BP1-cGAS combination, ulteriorly disclosing that it was compromised when co-cultured with H/R cardiomyocytes, consistent with the dissociation of G3BP1 from cGAS upon stimulation by DNA as revealed in previous studies. In contrast, the SHEP1-G3BP1 combination escalated resulting from an increase in SHEP1 expression. Together with a lack of direct combination detected between SHEP1 and cGAS, we concluded that SHEP1 might compete with cGAS in binding with G3BP1 to suppress cGAS activation. This competitive binding relationship needs to be confirmed by further molecular protein experiments. Therefore, SHEP1 reduces the binding of cGAS to G3BP1 by binding to G3BP1, thereby inhibiting DNA sensing of cGAS and further inhibiting the activation of its downstream cGAS-STING pathway. Subsequent assays of the phosphorylation levels of TBK1, a key molecule in the cGAS-STING pathway, validated this inference. Further blockage of either G3BP1 or cGAS abolished SHEP1 influence on NFκB, confirming that SHEP1 influences NFκB activation via the G3BP1 forward cGAS-STING pathway. In summary, our findings reveal that during MIRI, SHEP1 expression levels in macrophages infiltrating injured myocardial tissues increased significantly in the early stages and returned to baseline levels within 3 days. It competitively bound to G3BP1, further inhibiting the activation of the RAS-MAPK-ERK pathway to suppress cell migration while inhibiting the activation of the cGAS-STING pathway to reduce the production of pro-inflammatory factors. The specific knockout of SHEP1 in macrophages promoted the infiltration of macrophages and differentiation to a pro-inflammatory phenotype during MIRI, thereby exacerbating myocardial injury and deterioration of cardiac function. Pharmacological inhibition of G3BP1 in IR-injured mice reversed the deterioration of cardiac function due to macrophage SHEP1 deficiency. Given that SHEP1 is only highly expressed at the early stage of MIRI and regulates macrophage migration and pro-inflammatory phenotype, it provides a therapeutic target and research idea for MIRI to suppress excessive inflammation only at this early stage of injury without affecting the later reparative function of macrophages. Declarations Ethics approval: This study and all animal procedures were approved by the animal care and use committee of Fudan University. Consent for publication: Not applicable. Availability of data and materials: The raw data and study materials are available from the corresponding author upon reasonable request. Conflict of Interest statement: The authors declare that there are no conflicts of interest. Funding: This work was supported by a grant from Innovative Research Groups of the National Natural Science Foundation of China (81521001), a key project of the National Natural Science Foundation of China (82130010), and the National Natural Science Foundation of China (81800227). References Bonaventura A, Montecucco F, Dallegri F. Cellular recruitment in myocardial ischaemia/reperfusion injury. Eur J Clin Invest. 2016;46:590-601. Adamo L, Rocha-Resende C, Prabhu SD, Mann DL. Reappraising the role of inflammation in heart failure. Nat Rev Cardiol. 2020;17:269-85. 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A novel and efficient model of coronary artery ligation and myocardial infarction in the mouse. Circ Res. 2010;107:1445-53. Ackers-Johnson M, Li PY, Holmes AP, O'Brien SM, Pavlovic D, Foo RS. A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart. Circ Res. 2016;119:909-20. Parker F, Maurier F, Delumeau I, Duchesne M, Faucher D, Debussche L, et al. A Ras-GTPase-activating protein SH3-domain-binding protein. Mol Cell Biol. 1996;16:2561-9. Omer A, Barrera MC, Moran JL, Lian XJ, Di Marco S, Beausejour C, et al. G3BP1 controls the senescence-associated secretome and its impact on cancer progression. Nat Commun. 2020;11:4979. Liu ZS, Cai H, Xue W, Wang M, Xia T, Li WJ, et al. G3BP1 promotes DNA binding and activation of cGAS. Nat Immunol. 2019;20:18-28. Leuschner F, Rauch PJ, Ueno T, Gorbatov R, Marinelli B, Lee WW, et al. Rapid monocyte kinetics in acute myocardial infarction are sustained by extramedullary monocytopoiesis. J Exp Med. 2012;209:123-37. Cherfils J, Zeghouf M. Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol Rev. 2013;93:269-309. Alam U, Kennedy D. Rasputin a decade on and more promiscuous than ever? A review of G3BPs. Biochim Biophys Acta Mol Cell Res. 2019;1866:360-70. Pazman C, Mayes CA, Fanto M, Haynes SR, Mlodzik M. Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling. Development. 2000;127:1715-25. Mendoza MC, Er EE, Zhang W, Ballif BA, Elliott HL, Danuser G, et al. ERK-MAPK drives lamellipodia protrusion by activating the WAVE2 regulatory complex. Mol Cell. 2011;41:661-71. Tourrière H, Chebli K, Zekri L, Courselaud B, Blanchard JM, Bertrand E, et al. The RasGAP-associated endoribonuclease G3BP assembles stress granules. J Cell Biol. 2003;160:823-31. Sanders DW, Kedersha N, Lee DSW, Strom AR, Drake V, Riback JA, et al. Competing Protein-RNA Interaction Networks Control Multiphase Intracellular Organization. Cell. 2020;181:306-24.e28. Yang P, Mathieu C, Kolaitis RM, Zhang P, Messing J, Yurtsever U, et al. G3BP1 Is a Tunable Switch that Triggers Phase Separation to Assemble Stress Granules. Cell. 2020;181:325-45.e28. Guillén-Boixet J, Kopach A, Holehouse AS, Wittmann S, Jahnel M, Schlüßler R, et al. RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation. Cell. 2020;181:346-61.e17. Cao DJ, Schiattarella GG, Villalobos E, Jiang N, May HI, Li T, et al. Cytosolic DNA Sensing Promotes Macrophage Transformation and Governs Myocardial Ischemic Injury. Circulation. 2018;137:2613-34. Additional Declarations (Not answered) Supplementary Files SupplementalTables.docx SupplementaryFigure1.jpg Supplementary figure 1 Gating strategy for cytometry. The gating strategy for sorting CD11b/CD46 double labeled positive macrophages. Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 12 Apr, 2024 Review # 2 received at journal 11 Apr, 2024 Reviewer # 2 agreed at journal 03 Apr, 2024 Review # 1 received at journal 26 Mar, 2024 Reviewer # 1 agreed at journal 08 Mar, 2024 Reviewers invited by journal 07 Mar, 2024 Submission checks completed at journal 19 Feb, 2024 Editor assigned by journal 17 Feb, 2024 First submitted to journal 17 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3964475","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":277125117,"identity":"870e98ca-13ab-4fef-9a56-8bf31d51ae60","order_by":0,"name":"Junbo 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1","display":"","copyAsset":false,"role":"figure","size":7743259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSHEP1 selectively expressed in macrophages co-cultured with H/R cardiomyocytes fluctuates over time\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003eRepresentative 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, 10μm.\u003cstrong\u003e (b)\u003c/strong\u003eFlow chart of the co-culture system.\u003cstrong\u003e \u003c/strong\u003eSHEP1 mRNA levels\u003cstrong\u003e(c)\u003c/strong\u003e and protein levels \u003cstrong\u003e(d)\u003c/strong\u003e in RAW264.7 co -cultured with hypoxia-reoxygenated cardiomyocytes for different time periods (n = 3 sets of cells). Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3964475/v1/33ab22d536da5e2f89d3142f.jpg"},{"id":52443462,"identity":"e4cb6c20-b1f3-42d4-994f-0cd536fea812","added_by":"auto","created_at":"2024-03-11 17:36:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18779552,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSHEP1 deletion in RAW264.7 co-cultured with H/R cardiomyocytes enhances macrophages migration and inflammation \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e 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. \u003cstrong\u003e(b)\u003c/strong\u003e 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. \u003cstrong\u003e(c)\u003c/strong\u003eRepresentative 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). \u003cstrong\u003e(d) \u003c/strong\u003eRepresentative 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). \u003cstrong\u003e(e) \u003c/strong\u003emRNA levels of IL-1β, MCP-1, 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 24h in our co-culture system described above (n = 3 sets of cells). All data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3964475/v1/99754152b1ed9fb53e9ce09d.jpg"},{"id":52443457,"identity":"5fd872e0-c541-4182-b500-a2eef87fc2ee","added_by":"auto","created_at":"2024-03-11 17:36:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9538469,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSHEP1 regulates macrophages activities primarily through the MAPK signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e RNA-sequencing (RNA-seq) for total RNA extracted from RAW264.7 cells (SHEP1 KO and WT) co-cultured for 0h (SH) or 6h (H6) with H/R primary cardiomyocytes isolated from adult mice. VENN, volcano blots, KEGG enrichment analysis and Heatmap are displayed. \u003cstrong\u003e(b)\u003c/strong\u003e 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 24h (n = 3 sets of cells). All data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3964475/v1/2cc4601a8a56125e302c201d.jpg"},{"id":52443468,"identity":"ffdec6a3-483b-4ee5-9083-bacababf1cce","added_by":"auto","created_at":"2024-03-11 17:36:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16177475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSHEP1 targets G3BP1 to attenuate macrophages migration and inflammation via MAPK signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Mass spectrometry analysis of SHEP1 immunoprecipitates. Selected peptide hits and coverage are shown.\u003cstrong\u003e (b)\u003c/strong\u003e RAW264.7 co-cultured with H/R primary cardiomyocytes or sham were subjected to co-immunoprecipitation (co-IP) respectively with anti-SHEP1, anti-G3BP1 and cGAS antibodies. \u003cstrong\u003e(c)\u003c/strong\u003e SHEP1 knocked-out (KO)/over-expressed (OE) RAW264.7 and their respective negative controls (NC-KO and NC-OE) 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).\u003cstrong\u003e (d)\u003c/strong\u003e 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 24h (n = 3 sets of cells).\u003cstrong\u003e (e)\u003c/strong\u003e 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 24h (n = 3 sets of cells). \u003cstrong\u003e(f)\u003c/strong\u003e 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.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3964475/v1/c1d9629869401e1c4caee5f0.jpg"},{"id":52443461,"identity":"caf50c0a-5f1c-412b-8c82-0a908edbc2c0","added_by":"auto","created_at":"2024-03-11 17:36:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":12359509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of SHEP1 shows sequential characteristic under the condition of MIRI \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003eRepresentative immunohistochemical analyses of SHEP1 at different time points after IR. Scale bar, 500μm. SHEP1 expression were quantified and compared (n=4).\u003cstrong\u003e (b)\u003c/strong\u003e Representative immunohistochemical analyses of SHEP1 in remote area, border area, and infarct area at day 1 after IR. Scale bar, 500μm. SHEP1 expression were quantified and compared (n=4).\u003cstrong\u003e (c)\u003c/strong\u003eS SHEP1 mRNA levels were analyzed by RT-qPCR at different time points after MIRI (n=4). \u003cstrong\u003e(d)\u003c/strong\u003e 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.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3964475/v1/7918769232e47622c737dc09.jpg"},{"id":52443460,"identity":"45efab89-df7a-449f-8167-e38a1826d9f2","added_by":"auto","created_at":"2024-03-11 17:36:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":16887611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMacrophage-specific deficiency of SHEP1 deteriorated cardiac function and cardiac injury under MIRI \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e 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) \u003cstrong\u003e(b)\u003c/strong\u003e Representative heart sections from\u003cem\u003e LysMCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eShep1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eor \u003cem\u003eShep1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e 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).\u003cstrong\u003e (c)\u003c/strong\u003e 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). \u003cstrong\u003e(d)\u003c/strong\u003e Representative Western blot analysis showing the protein levels of Bax and Bcl-2 in \u003cem\u003eLysMCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eShep1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eShep1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice hearts at 1 day after MIRI or sham operation (n=6). \u003cstrong\u003e(e) \u003c/strong\u003eRepresentative Masson trichrome staining of cardiac tissue obtained from \u003cem\u003eLysMCre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eShep1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eShep1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice at day 21 after MIRI. Quantitative analysis of fibrotic areas (n=6). All data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3964475/v1/5067499c28f8df9df8882d99.jpg"},{"id":52443469,"identity":"c6b10f0d-dbc3-4440-9154-7cd3e5c66227","added_by":"auto","created_at":"2024-03-11 17:36:44","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":20315808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMacrophage-specific deficiency of SHEP1 exacerbates cardiac injury through amplifying inflammatory process under MIRI \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e 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\u003csup\u003e+\u003c/sup\u003e (red) cell numbers were counted and compared (n=4; scale bar, 500μm). Flow cytometry analysis for cardiac macrophages \u003cstrong\u003e(b)\u003c/strong\u003e, M1 and M2 \u003cstrong\u003e(c)\u003c/strong\u003e, and Ly6C\u003csup\u003elow\u003c/sup\u003e macrophages \u003cstrong\u003e(d)\u003c/strong\u003e within LV myocardium in\u003cem\u003e LysM Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Shep1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Shep1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice 1 or 3 days after MIRI. n = 5 mice/group pooled from 5 independent experiments. \u003cstrong\u003e(e)\u003c/strong\u003e 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 \u003cem\u003eLysM Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Shep1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003emice and their\u003cem\u003e Shep1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e littermates. All data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3964475/v1/3ff1b64ad55c86400be87cd7.jpg"},{"id":52443458,"identity":"ce7d802f-e258-4227-843c-81a62a8978a7","added_by":"auto","created_at":"2024-03-11 17:36:43","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":11593160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eG3BP1 inhibition mproves cardiac function of myeloid-specific SHEP1 deficiency mice under MIRI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e \u003cem\u003eLysM Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Shep1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice and their \u003cem\u003eShep1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003elittermates 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.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3964475/v1/d0d225f0c16a124b53239587.jpg"},{"id":71856114,"identity":"cbe0fd11-8c81-4a62-bf9a-e6c77f7a5e9e","added_by":"auto","created_at":"2024-12-19 08:11:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":114401981,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3964475/v1/0c985a68-b3ab-421e-a1c1-8d4277abd569.pdf"},{"id":52443455,"identity":"ad020542-df63-4efc-bb3e-68194cc6c80b","added_by":"auto","created_at":"2024-03-11 17:36:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18982,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-3964475/v1/05abf75a4a53a2a256060a58.docx"},{"id":52443456,"identity":"c26864a7-f430-40b3-aa05-bdf71121a43d","added_by":"auto","created_at":"2024-03-11 17:36:42","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7448252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 1 Gating strategy for cytometry.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe gating strategy for sorting CD11b/CD46 double labeled positive macrophages.\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3964475/v1/79d17820002e6f6ced9b16b8.jpg"}],"financialInterests":"(Not answered)","formattedTitle":"SHEP1 alleviates cardiac ischemia reperfusion injury via targeting G3BP1 to regulate macrophage infiltration and inflammation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute myocardial infarction (AMI) is a major threat to human health worldwide. Revascularization therapy significantly improves the prognosis of AMI, but death rates due to secondary reperfusion injury remain persistently high. Accumulating evidence suggests that inflammatory mediators released from the innate and adaptive immune responses contribute to the progression of myocardial ischemia-reperfusion injury (MIRI) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. MIRI induces a vigorous immune response, augmented by the generation and release of various inflammatory cytokines and chemokines that accelerate peripheral monocyte mobilization and infiltration to exacerbate myocardial injury [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This ultimately leads to adverse outcomes including infarct expansion, left ventricular (LV) systolic dysfunction, and LV dilation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Macrophages involved in MIRI exhibit functional heterogeneity, with pro-inflammatory macrophages infiltrating initially to clear the irreversibly damaged tissues, followed by a reparative phase of anti-inflammatory macrophages that drives the resolution of inflammation, tissue repair, and coronary angiogenesis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Both migration and differentiation play vital roles in inflammatory processes. However, little is known about the initial mechanisms by which monocytes are recruited to the sites of injury, the diversity of monocyte-derived macrophages, or the instructive cues that specify their fate.\u003c/p\u003e \u003cp\u003eTo explore the regulators that modulate monocyte recruitment and fate specification during MIRI, we previously sequenced transcriptomes of peripheral blood mononuclear cells derived from 10 patients assigned to percutaneous coronary intervention due to acute ST-segment elevation myocardial infarction at successive timepoints launching around the intervention. The results demonstrated that the mRNA levels of SHEP1 were significantly associated with revascularization [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], suggesting that it may play a role in MIRI. SHEP1, encoded by the \u003cem\u003eSH2D3C\u003c/em\u003e gene, is an Src homology 2 (SH2) -containing NSP scaffold that mediates protein-protein interactions through conserved modular domains [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Its architectures include an amino-terminal SH2 domain that binds to phosphorylated tyrosine residues, followed by a proline/serine-rich region, and a conserved carboxyl-terminal domain with homology to CDC25-related guanine nucleotide exchange factors (GEFs), which promote guanine nucleotide exchange on Ras family GTPases. Although the GEF domain of SHEP1 binds to several Ras proteins, it appears to lack enzymatic activity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It has been shown that NSP family members affect the migration and adhesion of T cells and B cells by regulation of cell signaling [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, they act as essential regulators of monocyte migration during atherosclerosis development [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, whether and how SHEP1 participates in macrophage function during MIRI remains unclear.\u003c/p\u003e \u003cp\u003eTo ascertain the role of SHEP1 in macrophages during MIRI, we examined cardiac repair in mice genetically engineered to lack SHEP1 in macrophages. Our findings reveal that a deficiency of SHEP1 in macrophages antagonizes optimal cardiac rehabilitation by promoting macrophage infiltration while fostering pro-inflammatory responses, thus providing an attractive new approach to preventing adverse events in MIRI.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and culture of adult mouse primary cardiomyocytes\u003c/h2\u003e \u003cp\u003eMouse cardiomyocytes were isolated from WT mice using Liberase enzymatic digestion as previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell co-culturing\u003c/h2\u003e \u003cp\u003eThe cell co-culturing system was performed using Corning Transwell chambers (0.4 \u0026micro;m pores, 6.5 mm diameter) where primary cardiomyocytes from adult wild-type C57 mouse were hypoxia-injured (in low-glucose DMEM without FBS, incubated in a 37\u0026deg;C air-tight incubator with 0.1%O2, 5% CO2 balanced by N2 for 6 hours) in the upper well, subsequently reoxygenated (back to high-glucose DMEM with 10% FBS), and concurrently inserted into lower well laid with RAW264.7 followed by incremental periods of co-culturing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and quantitative PCR (qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted with TRIZOL reagent (93289, Sigma) according to the manufacturer\u0026rsquo;s protocol and quantified using a Nanodrop 2000. Total RNAs (1000ng) were used to perform the reverse transcription with the PrimeScript RT reagent kit (Takara, DRR037A). To determine relative mRNA abundance, qPCR was performed with SYBR Green Master Mix (Yeason) on an ABI StepOnePlus system according to the manufacturer\u0026rsquo;s protocol. Data were analyzed with StepOnePlus software. The primers were synthesized from Tsingke Biotechnology and sequence information is provided in Supplementary Table\u0026nbsp;1. Mouse 18S was used for normalization. The experiment was repeated three times.\u003c/p\u003e \u003cp\u003eFor RNA-seq, after RNA extraction and quantification, Oliago(dT)-attached magnetic beads were used to purify mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at an appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by second-strand cDNA synthesis. Afterward, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from the previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double-stranded PCR products from the previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single-strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make a DNA nanoball (DNB) which had more than 300 copies of one molecular. DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on the BGIseq500 platform (BGI-Shenzhen, China). The sequencing data was filtered with SOAPnuke (v1.5.2) by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterward clean reads were obtained and stored in FASTQ format. The clean reads were mapped to the reference genome using HISAT2 (v2.0.4). Bowtie2 (v2.2.5) was applied to align the clean reads to the reference coding gene set, then the expression level of the gene was calculated by RSEM (v1.2.12). The heatmap was drawn by heatmap (v1.0.8) according to the gene expression in different samples. Essentially, differential expression analysis was performed using the DESeq2 (v1.4.5) [with Qvalue\u0026thinsp;\u0026le;\u0026thinsp;0. 05. To take an insight into the change of phenotype, GO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.geneontology.org/\u003c/span\u003e\u003cspan address=\"http://www.geneontology.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) and KEGG(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.kegg.jp/\u003c/span\u003e\u003cspan address=\"https://www.kegg.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) enrichment analysis of annotated different expressed genes was performed by Phyper (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://en.wikipedia.org/wiki/Hypergeometric_distribution\u003c/span\u003e\u003cspan address=\"https://en.wikipedia.org/wiki/Hypergeometric_distribution\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) based on Hypergeometric test. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold (Qvalue\u0026thinsp;\u0026le;\u0026thinsp;0. 05) by Bonferroni.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eProtein sample preparation, IP-MS, and Western blotting assay\u003c/h2\u003e \u003cp\u003eTotal proteins were extracted in RIPA lysis buffer (Beyotime, China) supplemented with a Complete Mini protease inhibitor cocktail (Roche, 11836153001). The protein concentration was determined by the BCA protein assay kit (Bio-Rad, 5000006JA).\u003c/p\u003e \u003cp\u003eFor immunoprecipitation (IP), the Pierce Classic IP Kit (Thermo Scientific\u0026trade;, 26146) was utilized according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003cp\u003eFor identification of shep1-interacting proteins, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed. Immunoprecipitates were separated with SDS\u0026ndash;PAGE and the gel was stained with Coomassie brilliant blue for protein band visualizing. The entire lane was cut into 1cm gel slices and subjected to in-gel digestion. The mass spectrometry analysis was conducted by PTM-BIO (China). The ranking of the identified proteins was based on the peptide coverage of each identification. The related proteins were selected and subjected to further validation by immunoprecipitation and immunoblot. This study focused on G3BP1.\u003c/p\u003e \u003cp\u003eAfter quantified, protein samples (10\u0026ndash;20 \u0026micro;g) were loaded on SDS-polyacrylamide gels for electrophoresis and then transferred to PVDF membranes. After blocking with 5% BSA for 1 h, the membranes were incubated with primary antibodies overnight at 4\u0026deg;C. After the subsequent washes, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Weiao Biological Company, China) at room temperature for 1 h. The blots were visualized and detected by Chemiluminescence Reaction (LuminataTM Forte, Millipore, USA) and ChemiDocTM Imaging System (Bio-Rad, CA, US). The density of the protein blots was analyzed by ImageJ (Version 1.53c, NIH, USA). Detailed information on the primary antibodies used in immunoblotting, including source, catalog number, and dilution, is listed in Supplementary Table\u0026nbsp;2. We also provide full scans of all the blots as Supplementary Dataset.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMigration wound healing\u003c/h2\u003e \u003cp\u003eMigration assays were performed using Corning Transwell chambers (8 \u0026micro;m pores, 6.5 mm diameter, USA). Primary cardiomyocytes were isolated from adult wild-type C57 mice, laid in the lower wells, and exposed to H/R injury. After reoxygenation, RAW264.7 cells were seeded in the upper chambers containing FBS-free DMEM. After 24 h of co-culture, cells that traversed through the membrane were fixed with 4% PFA for 30 min and stained with 0.05% crystal violet solution (Solarbio, China) for 20 min.\u003c/p\u003e \u003cp\u003eA wound-healing assay was conducted in 6-well plates by scratching the cell monolayer with a 200 \u0026micro;l pipette tip. Cells were washed with PBS twice and cultured in conditional media of H/R cardiomyocytes. Images were collected at the beginning and 24 h after scratching. The wound-healing speed was calculated as the ratio of the healed areas at 24 h to the original injured areas.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA interference\u003c/h2\u003e \u003cp\u003eRAW264.7 cells were transfected with siRNAs by Lipofectamine\u0026trade; 3000 transfection reagent (Invitrogen\u0026trade;, L3000150) for 48h before stimulation according to the manufacturer\u0026rsquo;s instruction. The siRNAs used were as follows: si-cGAS (5'-CGGCAGCUAUUAUGAACAU-3'), si-G3BP1(5\u0026prime;-CCCTATGGAAATCATTCCT-3\u0026prime;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eMice were fed with a standard rodent chow diet and housed in micro isolator cages in a special pathogen-free facility. The number of animals in each experimental group is indicated in the tables or figure legends. This study and all animal procedures conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH publication NO. 85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1996), and were approved by the animal care and use committee of Fudan University.\u003c/p\u003e \u003cp\u003eWild-type (WT) C57BL6/J mice (male, 14\u0026ndash;16 weeks old; SLRC Laboratory Animal, Shanghai, China) were used in this study.\u003c/p\u003e \u003cp\u003eMyeloid-specific SHEP1-deficient mice were generated by crossing SHEP1\u003csup\u003efl/fl\u003c/sup\u003e mice with LysM-Cre mice and are designated as LysM Cre\u003csup\u003e+\u003c/sup\u003e SHEP1\u003csup\u003efl/fl\u003c/sup\u003e (CKO) in this paper (constructed by Cyagen Biosciences Inc. China). For experiments described in this paper, adult male mice (14 to 16 weeks of age) were used.\u003c/p\u003e \u003cp\u003eEGCG administration: Mice were intraperitoneally injected with EGCG (40mg/kg) right after reperfusion, and afterward once a day for 3 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCardiac I/R injury model\u003c/h2\u003e \u003cp\u003eMice underwent surgical cardiac I/R injury as described previously [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Briefly, the mice were anesthetized with 2% isoflurane inhalation using an isoflurane delivery system. Subsequently, a small skin cut (1.2 cm) was made over the left chest, and the left anterior descending coronary artery (LAD) was ligated via a slipknot using 6\u0026ndash;0 silk for 45 min before the slipknot was gently loosened to induce reperfusion injury. The suture was passed 2\u0026ndash;3 mm below the tip of the left auricle. Sham-operated animals underwent the same procedure without tying the slipknot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTriphenyltetrazolium chloride (TTC) staining\u003c/h2\u003e \u003cp\u003eTTC staining was conducted for evaluation of cardiac infarction size. After the mice were anesthetized with 2% isoflurane, the slipknot was tied again, and 1% Evans blue (w/v, Sigma, USA) was injected into the aortic root to perfuse the left ventricle. Then, the heart was rapidly excised, cut into 1 mm slices, and incubated with 1% TTC solution (Sigma, USA) at 37\u0026deg;C for 10 min. Consecutive slices were scanned by a white light scanner (Canon, Japan). LV area, AAR, and infarct area were determined by computerized planimetry and comprehensively analyzed in serial sections of each mouse) using ImageJ software. The infarction degree was calculated as the ratio of the infarction area to the AAR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTransthoracic echocardiography\u003c/h2\u003e \u003cp\u003eCardiac function was assessed with a Vevo2100 Ultrasound system (VisualSonics, Toronto, Canada). Mice were mildly anesthetized with 0.5% isoflurane and placed on a heated ECG platform. The left ventricle and the aortic outflow tract were observed under the two-dimensional B-Mode, and the sample line was placed at the maximum cross-section of the left ventricle to guide the recording of serial M-Mode echocardiographic images. The left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), fractional shortening (FS), left ventricular end-systolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD) were measured from at least three distinct frames for each mouse, and the average was used for data analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIHC and immunofluorescence staining\u003c/h2\u003e \u003cp\u003eAnalysis was performed using paraffin-embedded sections of the heart tissue samples fixed with 4% paraformaldehyde. All tissue samples were characterized using hematoxylin and eosin (H\u0026amp;E) staining before further immunostaining. After the inhibition of endogenous peroxidase activity, the sections were incubated with primary anti-SHEP1 (sc-100792, Santa Cruz) at 4\u0026deg;C overnight. Following the incubation, a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) was used according to the manufacturer\u0026rsquo;s instructions. After the visualization with 3,3\u0026rsquo;-diaminobenzidine, the sections were counterstained with hematoxylin. As for immunofluorescence analysis, after deparaffinization, heat-mediated antigen retrieval in citrate buffer pH 6.0, and blocking with 5% horse serum at room temperature for 30 min, samples were incubated with primary antibodies overnight at 4\u0026deg;C. The primary antibodies anti-SHEP1 (Santa Cruz, 1:100, sc-100792) and anti-F4/80 (AbD Serotec, 1:200, MCA497GA) were utilized for these experiments. Signals were visualized with Alexa fluorescence-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). TUNEL staining was performed with an In Situ Apoptosis Detection kit (Yeasen, Shanghai, China), according to the manufacturer\u0026rsquo;s instructions. Cell nuclei were stained with DAPI. Five fields or more in \u0026gt;\u0026thinsp;3 different sections/animals were examined by a technician who was blinded to the treatment groups. Images were obtained using an Olympus microscope and quantified using Image J. For each heart sample, at least five random fields were measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCell extract, flow cytometry, and magnetic beads sorting\u003c/h2\u003e \u003cp\u003eThe hearts were dissected, carefully cut into small pieces with fine scissors, and enzymatically digested with type II collagenase (1.5 mg mL-1, Worthington Biochemical, Lakewood, NJ, USA), elastase (0.25 mg mL-1, Worthington Biochemical), and DNase I (0.5 mg mL-1, Worthington Biochemical) for 1 h at 37\u0026deg;C. Following digestion, the tissues were passed through 70-\u0026micro;m cell strainers, washed, and stained with antibodies for fluorescence-activated cell sorter (FACS) analysis. To block the non-specific binding of antibodies to Fcγ receptors, isolated cells were incubated first with anti-CD16/32 antibody (BD Biosciences, #553142) at 4\u0026deg;C for 5 min. The primary antibodies against the following were obtained from BD Biosciences (Franklin Lakes, NJ, USA): CD45.2 (#564616), CD11b (#552850), Ly-6G (#560599), Ly6C (#563011), CD64 (Biolegend, #139309), CD206 (Thermo, 17-2061-82). The obtained results are expressed as cell number/\u0026micro;g tissue. Flow cytometric analysis was performed using a FACSAria\u0026trade; flow cytometer (BD Biosciences), and the obtained data were analyzed with FlowJo 7.6.1 software (Tree Star, Ashland, OR, USA).\u003c/p\u003e \u003cp\u003eMagnetic cell sorting was performed using QuadroMACS (Miltenyi Biotec, 130-091-051) according to the manufacturer\u0026rsquo;s instructions, anti-F4/80 MicroBeads (Miltenyi Biotec, 130-110-443) were utilized for separating macrophages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were collected and expressed as average\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). T-test were conducted when comparing 2 independent groups with the normalized distribution of values. For n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4 per group, Mann-Whitney tests were used. ANOVA-test or Kruskal-Wallis (with low numbers) were carried out when comparing more than 2 groups. For two groups and multiple time points, a two-way ANOVA was used. Statistical analysis was conducted using GraphPad Prism 8.0 (GraphPad Prism Software Inc., San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSHEP1 selectively expressed in macrophages co-cultured with H/R cardiomyocytes fluctuates over time\u003c/h2\u003e \u003cp\u003eWe first co-stained heart tissue slices from wild-type C57BL6 mice that had undergone cardiac IR operation with fluorescent-labeled antibodies for macrophages (F4/80, red) and SHEP1 (green) to investigate whether SHEP1 selectively expressed in macrophages. And DAPI (blue) was used to stain the nuclei. The results demonstrated that SHEP1 was specifically localized in the cytoplasm of macrophages in merged confocal images (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo mimics microenvironment in vivo under the condition of MIRI, we established co-culture systems with transwells between macrophages and hypoxia-reoxygenated (H/R) cardiomyocytes (flow chart shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) to investigate the evolution of SHEP1 expression in RAW264.7 cells over time, with the results that SHEP1 mRNA levels peaked at 3 hours and protein levels peaked at 6 hours after co-culture, then both gradually declined to baseline level within 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec \u003cb\u003e\u0026amp; d\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSHEP1 deletion in RAW264.7 co-cultured with H/R cardiomyocytes enhances macrophages migration and inflammation\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo observe the effect of SHEP1 on the biological function of monocyte \u003cem\u003ein vitro\u003c/em\u003e, we thereupon constructed SHEP1 knock-out (KO) and over-expressed (OE) RAW264.7 monoclonal cell lines for verification by qPCR and western-blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea \u003cb\u003e\u0026amp; b)\u003c/b\u003e. By the above transwell co-culture systems, the migration of SHEP1-KO RAW264.7 group was significantly promoted than that of NC-KO group, whereas it showed the opposite trend between the SHEP1-OE group and NC-OE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). And another result with the same biological characteristics was confirmed by wound healing \u003cem\u003ein vitro\u003c/em\u003e among these 4 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimultaneously, the effect of SHEP1 regulating inflammation in macrophage was also investigated through using the co-culture system described above. qRT-PCR showed that the pro-inflammatory cytokine IL-1β, chemokine MCP-1 and CCR2 were obviously upregulated in the SHEP1-KO RAW264.7 group (lower-chamber), while down-regulated in the SHEP1-OE group, compared with their corresponding control groups. Unlike that CCR2 showed significant differences at the baseline level, the differences of IL-1β and MCP-1 were gradually occurred after co-culture stimulation, which might mainly be due to that IL-1β and MCP-1 are inflammatory factors produced by macrophages in response to the stimulation of an inflammatory environment, whereas CCR2 represents the susceptibility of macrophages to chemokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSHEP1 regulates macrophages activities primarily through the MAPK signaling pathway\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular mechanisms of SHEP1 in various cell activities, we carried out RNA-sequencing (RNA-seq) for total RNA extracted from RAW264.7 cells (SHEP1-KO group and control group, respectively) co-cultured for 0 h or 6 h with H/R primary cardiomyocytes isolated from adult wild-type C57 mice. The transcriptome-wide role of SHEP1 in RAW264.7 cells was evaluated in the co-culture system to identify the key pathways that may be affected by the loss of SHEP1. In total, 720 differential genes were obtained from a Venn diagram analysis of different cell groups for the same co-culture duration and different co-culture durations from the same cell group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Subsequently, KEGG pathways analysis among these differential genes indicated that the primary RAS-MAPK-ERK pathway was identified from top 20 pathways and partially involved in cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo illuminate the regulatory role of SHEP1 in RAS-MAPK-ERK signaling pathways, western blotting was used to detect the expression of ERK phosphorylation and its downstream protein WAVE2. The results showed that it was a negative regulation of this pathway by SHEP1, with increased expression of ERK phosphorylation and WAVE2 in SHEP1-KO RAW264.7 cells, while decreased expression in the SHEP1-OE group, when they co-cultured with H/R cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSHEP1 targets G3BP1 to attenuate macrophages migration and inflammation via MAPK signaling pathway\u003c/h2\u003e \u003cp\u003eTo further explore interactive proteins targeted by SHEP1, Co-IP mass spectrometry and competition assay were used to elucidate these potential molecular cluster in RAW264.7 cells and their possible regulation of the RAS-MAPK-ERK pathway. Firstly, differentially expressed proteins binding to SHEP1 in RAW264.7 cells between co-cultured with H/R cardiomyocytes for 9 hours and the normoxia group was analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Among these differential proteins, Ras-GTPase-activating protein SH3 domain binding protein1(G3BP1) was an optimal candidate since it could bind to the SH3 domain of Ras-GTPase-activating protein (RasGAP), which might regulate RAS-MAPK-ERK signaling pathway[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Subsequently, the direct interaction between SHEP1 and G3BP1 was confirmed through Co-IP experiment when RAW264.7 cells were co-cultured with H/R cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSecondly, via gene regulation of targeting protein G3BP1, the effect of SHEP1 on expression of its downstream proteins in MAPK signaling pathway was also observed. The results indicated that siRNA-assisted knockdown of G3BP1 abrogated increased expression of WAVE2 in SHEP1-KO RAW264.7 group and decreased expression of WAVE2 in the SHEP1-OE group, which showed that through G3BP1, SHEP1 had a regulatory effect on the RAS-MAPK-ERK pathway, thereby affecting cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eThirdly, G3BP1 has been revealed to promote the activation of cGAS via DNA binding, and subsequently to activate NF-κB in previous studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. To clarify that SHEP1 suppresses the expression of inflammatory factors by competitive binding with G3BP1 to interrupt the G3BP1-cGAS interaction, western blotting was applied to detect the expression of TBK1 phosphorylation and proteins of NF-κB pathway. The following experiments demonstrated that the binding of SHEP1 and G3BP1 was enhanced in macrophages co-culturing with H/R cardiomyocytes, whereas the binding of G3BP1 and cGAS was synchronously abated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Simultaneously, SHEP1 was also found to suppress the activation of cGAS, with evidence of the repression of TBK1 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee) and the activation of proteins of NF-κB pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef) in SHEP1-KO RAW264.7 group, while an opposite trend was observed in the SHEP1-OE group. Further, blockage of either G3BP1 or cGAS could abolish these effects of SHEP1 on proteins related to NF-κB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe expression of SHEP1 shows sequential characteristic under the condition of MIRI\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eImmunohistochemistry showed that SHEP1 was expressed mainly in the infarct and border areas after MIRI, with low expression in the non-infarct areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Through continuous observation of expression of SHEP1 in histological sections for 7 days, SHEP1 area was found to reach its peak at 24 hours after MIRI and afterward experienced a gradual decline (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). To elucidate the expression of SHEP1 in cardiac tissue under the condition of MIRI at different reperfusion time points, we checked the gene expression levels and protein abundance of SHEP1 in the myocardium. The results uncovered that mRNA level of SHEP1 increased as early as 3 hours after MIRI and peaked at 6 hours, while protein accumulation progressively increased from 9 hours to 24 hours and subsequently declined (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). These data in vivo showed the similar tendency with the western blot results of cells in vitro.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMacrophage-specific deficiency of SHEP1 deteriorated cardiac function and cardiac injury under MIRI\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the crucial biological role of SHEP1 on regulation of cardiac function and cardiac injury under the condition of MIRI in vivo, LysM Cre\u003csup\u003e+\u003c/sup\u003e SHEP1\u003csup\u003efl/fl\u003c/sup\u003e (CKO) and SHEP1\u003csup\u003efl/fl\u003c/sup\u003e (flox) littermates were generated by using gene-editing technology and then underwent the operation of IR. Echocardiography was performed to observe the difference among various groups. The data showed that despite similar baseline echocardiographic parameters, macrophage-specific deficiency of SHEP1 group resulted in worse cardiac function both 4 days and 21 days after MIRI, with a significant reduction in the ejection fraction (EF%) and fractional shortening (FS%), compared with the control group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Subsequently, the pathologic character of myocardial injury was evaluated, according to infarct area/ areas at risk (AAR) ratio and myocardial apoptosis markers. The findings displayed that AAR ratio in CKO mice with SHEP1 deficiency in macrophages was obviously higher than that in control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Similar results were confirmed by further evidence that the increased TUNEL\u003csup\u003e+\u003c/sup\u003e cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec) in CKO mice group were more obvious, concurrently, higher expression of pro-apoptotic molecule BAX and lower expression of anti-apoptosis molecule Bcl-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed) in LV tissue samples could be observed in CKO mice group 24 hours after MIRI, compared with the control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, myocardial fibrosis post myocardial infarction is secondary pathological changes, which could be evaluated by Masson\u0026rsquo;s trichrome staining. The results showed that myocardial fibrosis 21 days after MIRI increased significantly in the CKO group, compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Although the increase in SHEP1 expression in macrophages after MIRI returned to baseline levels within 3 days, the reason why the difference between various groups was still significant might be due to the reflection of the integrated effect of the lack of SHEP1, the early inflammatory and myocardial injury.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMacrophage-specific deficiency of SHEP1 exacerbates cardiac injury through amplifying inflammatory process under MIRI\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAS an important source for inflammatory cytokines, macrophages play a critical role in the regulation of inflammation caused by myocardial injury. To investigate the role of SHEP1 in macrophage mediated inflammatory response, the operation of IR was conducted in both CKO mice and flox littermates. The results indicated that by the comparison of their differences at timepoints of 1 and 3 days after the initiation of reperfusion, immunofluorescence staining of the myocardium in CKO mice appeared obviously enhanced infiltration of macrophages into the myocardium, compared with that in flox littermates (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnd the number of CD11b/CD46 double labeled positive macrophages in CKO mice hearts were significantly higher than that of control group whether 1 day or 3 days after MIRI (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb and \u003cb\u003esupplementary Fig.\u0026nbsp;1\u003c/b\u003e). Simultaneously, the ratio of M1/M2 of macrophages in CKO mice hearts by flow cytometry was found to be higher than that of hearts in flox littermates 1 day after MIRI, without difference 3 days after MIRI (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Besides, the proportion of Ly6c\u003csup\u003elow\u003c/sup\u003e/macrophages among various groups showed no remarkable difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). These above flow cytometry results implied the positive role of SHEP1-deficiency improving macrophage differentiation under MIRI.\u003c/p\u003e \u003cp\u003eInflammatory cytokines are important regulators of inflammatory reaction under the condition of various injuries. We further conducted magnetic cell sorting for F4/80\u003csup\u003e+\u003c/sup\u003e cells from total cardiac cells of different groups of mice at 1 and 3 days after MIRI and then applied RNA analysis of the isolated cells to quantify their pro-inflammatory phenotype. The results showed that compared to the control group, the pro-inflammatory factors IL-1β and MCP-1 were significantly enriched in the CKO mice group at 1 and 3 days after MIRI. And the similar expression trend was consistent with C-C motif chemokine receptor 2 (CCR2), whereas inconsistent with TGF-β and IFN-β (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEpigallocatechin gallate, an inhibitor of G3BP1, improves cardiac function of myeloid-specific SHEP1 deficiency mice under MIRI\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo verify whether SHEP1 also competitively bound to G3BP1 to improve heart function \u003cem\u003ein vivo\u003c/em\u003e, we first selected epigallocatechin gallate (EGCG), an inhibitor of G3BP1 extracted from green tea, as targeting reagent, and then administered it to the MIRI model mice once daily, intraperitoneally for 3 days. The results indicated that a significant improvement in cardiac function in CKO mice group receiving EGCG occurred, with increased ejection fraction (EF%) and fractional shortening (FS%), compared with that in CKO mice group receiving PBS. What\u0026rsquo;s more, the increasing extent in the above two parameters in CKO mice group was significantly higher than those in Flox mice group receiving EGCG and PBS (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the course of myocardial ischemia-reperfusion injury, large quantities of monocytes invade the heart within 24 h as the dominant immune cells obliterate debris [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]; however, excessive inflammation is thought to exacerbate adverse remodeling and heart failure pathogenesis. This highlights the need for an appropriate extent of anti-inflammatory approaches within the desired time window to effectively suppress injurious processes without negatively interfering with the reparative response. However, little is known about the variety of monocytes and monocyte-derived macrophages recruited to the heart during MIRI, or the mechanisms that modulate monocyte recruitment and fate determination. In our model of clinically relevant MIRI, we observed an increased level of SHEP1 in the myocardium at early time points after injury, with subtotal expression in F4/80\u003csup\u003e+\u003c/sup\u003e macrophages. Using myeloid cell-specific SHEP1 knockout mice, we demonstrated for the first time that genetic depletion of SHEP1 in macrophages exacerbated MIRI through enhanced infiltration and pro-inflammatory differentiation of monocytes, revealing a role for SHEP1 in macrophages during MIRI, implying that targeting SHEP1 on macrophages may constitute a therapeutic approach for the prevention of IR injury.\u003c/p\u003e \u003cp\u003eAs mentioned above, SHEP1 was only highly expressed in the early stage of MIRI and decreased to baseline levels within 3 days. The early expression of SHEP1 could counteract excessive macrophage infiltration and inflammatory factor secretion only at the early stages of MIRI to further reduce myocardial tissue damage and cardiac function deterioration caused by an exaggerated inflammatory response, without directly impeding the reparative function of macrophages in the middle and late stages of injury. This makes it an ideal target and research direction for MIRI therapy.\u003c/p\u003e \u003cp\u003eSpecialized receptors on the cell surface transduce extracellular stimuli and initiate signaling cascades that culminate in diverse functional outcomes. This organization is achieved through specific high-affinity protein-protein interactions and multiprotein complex formation facilitated by conserved modular domains. Src homology region 2 (SH2) is one of the most prevalent conserved modular domains [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Accordingly, we assumed that SHEP1 manipulates macrophage functionality through protein-protein interactions, and subsequently conducted protein mass spectrometry in the immunoprecipitates of SHEP1. G3BP1 was screened from those identified proteins because it can link SHEP1 to both the migration and inflammation pathways.\u003c/p\u003e \u003cp\u003eG3BP1 was initially discovered as a binding protein of the Ras-GTPase-activating protein (RAS-GAP), which is an inactivator of RAS GTPase via hydrolysis of the Ras-bound GTP molecule to GDP [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. With no enzymatic activity, G3BP1 acts on the RAS signaling pathway by interacting with GAP [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Here, we demonstrated the combination of SHEP1 and G3BP1 to a greater extent when co-cultured with hypoxia-reoxygenated cardiomyocytes, indicating a potential role of SHEP1 in RAS signaling pathway manipulation. This was confirmed by RNA-sequencing of RAW264.7, co-cultured with hypoxia-reoxygenated cardiomyocytes, and further confirmed by protein analysis, in which SHEP1 suppressed phosphorylation of ERK1/2 as well as expression of WAVE2, an activator of the Arp2/3 actin nucleator essential for cell migration through actin assembly [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Further blocking of G3BP1 by siRNA abolished SHEP1 influence on WAVE2, confirming that SHEP1 acts on the RAS-MAPK-ERK pathway through G3BP1.\u003c/p\u003e \u003cp\u003eAdditionally, recent studies have revealed that G3BP1 abrogates cGAS stimulation, subsequently activating NF-κB by enhancing cGAS-DNA association independent of SGs [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], despite its essential role in promoting the formation of SGs [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. As a cytosolic DNA sensor, cGAS is activated robustly in myocardial infarction, triggering downstream events mediated by the STING cascade. This, in turn, promotes macrophage transformation toward the M1-like subtype [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our study confirmed the G3BP1-cGAS combination, ulteriorly disclosing that it was compromised when co-cultured with H/R cardiomyocytes, consistent with the dissociation of G3BP1 from cGAS upon stimulation by DNA as revealed in previous studies. In contrast, the SHEP1-G3BP1 combination escalated resulting from an increase in SHEP1 expression. Together with a lack of direct combination detected between SHEP1 and cGAS, we concluded that SHEP1 might compete with cGAS in binding with G3BP1 to suppress cGAS activation. This competitive binding relationship needs to be confirmed by further molecular protein experiments. Therefore, SHEP1 reduces the binding of cGAS to G3BP1 by binding to G3BP1, thereby inhibiting DNA sensing of cGAS and further inhibiting the activation of its downstream cGAS-STING pathway. Subsequent assays of the phosphorylation levels of TBK1, a key molecule in the cGAS-STING pathway, validated this inference. Further blockage of either G3BP1 or cGAS abolished SHEP1 influence on NFκB, confirming that SHEP1 influences NFκB activation via the G3BP1 forward cGAS-STING pathway.\u003c/p\u003e \u003cp\u003eIn summary, our findings reveal that during MIRI, SHEP1 expression levels in macrophages infiltrating injured myocardial tissues increased significantly in the early stages and returned to baseline levels within 3 days. It competitively bound to G3BP1, further inhibiting the activation of the RAS-MAPK-ERK pathway to suppress cell migration while inhibiting the activation of the cGAS-STING pathway to reduce the production of pro-inflammatory factors. The specific knockout of SHEP1 in macrophages promoted the infiltration of macrophages and differentiation to a pro-inflammatory phenotype during MIRI, thereby exacerbating myocardial injury and deterioration of cardiac function. Pharmacological inhibition of G3BP1 in IR-injured mice reversed the deterioration of cardiac function due to macrophage SHEP1 deficiency. Given that SHEP1 is only highly expressed at the early stage of MIRI and regulates macrophage migration and pro-inflammatory phenotype, it provides a therapeutic target and research idea for MIRI to suppress excessive inflammation only at this early stage of injury without affecting the later reparative function of macrophages.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eThis study and all animal procedures were approved by the animal care and use committee of Fudan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe raw data and study materials are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest statement:\u003c/strong\u003e The authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by a grant from Innovative Research Groups of the National Natural Science Foundation of China (81521001), a key project of the National Natural Science Foundation of China (82130010), and the National Natural Science Foundation of China (81800227).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBonaventura A, Montecucco F, Dallegri F. 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Circulation. 2018;137:2613-34.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Myocardial ischemia-reperfusion injury, macrophage, SHEP1, G3BP1, cGAS","lastPublishedDoi":"10.21203/rs.3.rs-3964475/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3964475/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The macrophage-associated inflammation response plays an important role in myocardial ischemia-reperfusion injury (MIRI). During MIRI, the role and molecular mechanism of SHEP1 regulating macrophage remains unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e By co-cultured with hypoxia reoxygenation cardiomyocytes \u003cem\u003ein vitro\u003c/em\u003e, macrophages with SHEP1 knockout or overexpression were detected cell migration ability and related proinflammatory factors; and the molecular network regulated by SHEP1 was identified through transcriptome-wide analysis; then its target molecules were verified by co-immunoprecipitation method. \u003cem\u003eIn vivo\u003c/em\u003e, an ischemia-reperfusion heart model was established to observe the changes in cardiac function, cardiac tissue injury and inflammation of macrophage-specific deficiency of SHEP1 mice, and to analyze the improvement of cardiac function by administrating inhibitors for targeted molecules of SHEP1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFindings:\u003c/strong\u003e 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\u003cem\u003e in vivo\u003c/em\u003e. RNA-sequencing and co-immunoprecipitation mass spectrometry showed that macrophage-derived SHEP1 competitively bound to G3BP1 to suppress inflammation via the MAPK pathway. And administrating inhibitor of G3BP1 could improve cardiac function in macrophage-specific deficiency of SHEP1 mice under MIRI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e SHEP1 targeted G3BP1 to antagonize cardiac ischemia-reperfusion injury by inhibiting infiltration and proinflammatory responses of macrophages, which provided a potential and clinically significant therapeutic target for MIRI.\u003c/p\u003e","manuscriptTitle":"SHEP1 alleviates cardiac ischemia reperfusion injury via targeting G3BP1 to regulate macrophage infiltration and inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-11 17:36:08","doi":"10.21203/rs.3.rs-3964475/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-04-12T14:19:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-04-11T12:32:23+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-04-03T10:50:01+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-03-26T14:19:04+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-03-08T10:17:04+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-03-07T20:05:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-19T11:33:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-17T15:29:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2024-02-17T15:29:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"697a8fcf-9796-4e2a-9320-710ce757c87d","owner":[],"postedDate":"March 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29208000,"name":"Biological sciences/Cell biology/Cell migration/Integrin signalling"},{"id":29208001,"name":"Biological sciences/Immunology/Cell death and immune response"}],"tags":[],"updatedAt":"2024-12-19T08:10:21+00:00","versionOfRecord":{"articleIdentity":"rs-3964475","link":"https://doi.org/10.1038/s41419-024-07282-5","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2024-12-18 05:00:00","publishedOnDateReadable":"December 18th, 2024"},"versionCreatedAt":"2024-03-11 17:36:08","video":"","vorDoi":"10.1038/s41419-024-07282-5","vorDoiUrl":"https://doi.org/10.1038/s41419-024-07282-5","workflowStages":[]},"version":"v1","identity":"rs-3964475","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3964475","identity":"rs-3964475","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}