S100A12 Aggravates Acute Myocardial Infarction Injury Through Excessive Neutrophil Extracellular Traps Formation

Background and Objective:Since the late 1950s,the incidence of cardiovascular system diseases has been increasing year by year,which are often accompanied by many dangerous complications that seriously threaten the quality of life and the health of patients,and are a major public health problem that needs to be addressed in the field of medical research worldwide^[1][2].Acute myocardial infarction is one of the most critical cardiovascular diseases due to its vicious course,high mortality rate,and often accompanied by multiple complications.Due to the wide range of causes and complex molecular mechanisms of acute myocardial infarction,it poses great difficulties in its prevention,early diagnosis and treatment^[3][4].Myocardial infarction occurs as a gradual evolutionary process from myocardial necrosis to scar repair,and the heart undergoes several phases of acute hypoxia,cellular necrosis,cellular proliferation,and scar formation^[5].The increase in the number of neutrophils in the circulatory system or their volume in patients with acute infarction is positively correlated with the size of the infarct area,left ventricular function and clinical outcome^[6].Once infarction has occurred,neutrophils are rapidly recruited to the damaged area and are responsible for maintaining the initial acute proinflammatory response.Neutrophils have been shown to facilitate the healing phase after acute myocardial infarction by helping to shift macrophages toward a repair phenotype^[7].Therefore,the treatment of acute myocardial infarction should take into account the dual pro-inflammatory and anti-inflammatory effects of neutrophils.S100A12 is a newly discovered calmodulin that is not expressed in mice^[8]and is mainly distributed in human myeloid cells^[9]（especially most abundant in neutrophils.）The effects of S100A12 include enhancing chemotactic activity,inducing axonal growth,and promoting cytokine production^[10].Many studies have been reported on S100A12 and immune system diseases,respiratory and digestive tract inflammation,while its role and mechanisms in cardiovascular diseases have only been published in recent years^[11].Previous studies in our laboratory identified S100A12 as a possible new biomarker molecule for the early diagnosis of acute ST-segment elevation myocardial infarction^[2].Therefore,this study attempted to elucidate the role of S100A12 in the occurrence and progression of acute myocardial infarction,and its regulatory mechanisms,to provide a new experimental basis and intervention targets for the prevention of myocardial cell necrosis,myocardial cell fibrosis and the occurrence of ventricular remodeling after acute infarction.Methods and Results:（1）Preparation of S100A12 specific transgenic mice and its effect on the progression of acute myocardial infarction in mice1.Construction of transgenic miceMethods:Nanjing University Nanjing Model Animal Center was commissioned to prepare and breed the model of neutrophil-specific Lysozyme M promoter S100A12-TG transgenic mice required for this project,and continue to feed and reproduce in our laboratory.2.Establishment of a mouse model of acute myocardial infarctionMethods:Male mice at about 10 weeks（w）were selected and the left anterior descending branch of the coronary artery was ligated and modeled using Lysozyme M promoter S100A12-TG transgenic mice（TG）as the experimental group and their littermates wild type（WT）mice as the control group.The sham operation group（sham）was performed with the same procedure as the infarct operation group,except that the heart was exposed and the vessels were not ligated.Cardiac function was measured by ultrasound in small animals at 1 day（d）,3 d,7 d,14 d,and 28 d after infarction,respectively,and the expression levels of S100A12 at different time points were detected,and the level of ventricular remodeling after 28 d was observed by staining,and the level of myocardial tissue apoptosis in mice was detected.Results:The expression of S100A12 was significantly up-regulated in the hearts of TG mice after coronary ligation,and the highest expression was observed at 1 d.Compared with the WT group,mice in the TG group had more severe cardiac functional impairment,larger myocardial necrosis area,and heavier myocardial tissue apoptosis levels.3.Detection of neutrophil extracellular traps（NETs）in myocardial tissue after acute myocardial infarctionMethods:Western blotting,immunohistochemistry and immunofluorescence staining were applied to detect the production of NETs in cardiac tissues of TG and WT mice at different time points after infarction surgery.Results:After coronary artery ligation,the expression of NETs related protein was the highest at the first day of myocardial infarction,and then decreased gradually with time.And the above proteins were more expressed in the TG group mice compared with the WT group.4.Intraperitoneal injection of NETs inhibitor Dnase I to observe whether Dnase I has a therapeutic effect on S100A12-induced infarct injuryMethods:Male mice at about 10 w were randomly divided into four groups:WT+Saline group,WT+Dnase I group,TG+Saline group and TG+Dnase I group.Mice in each group were injected intraperitoneally with Saline or Dnase I（10 mg/kg/d）every other day starting one day before coronary ligation until 28 d postoperatively.Cardiac function,ventricular remodeling and myocardial tissue apoptosis were measured at 3 and 28 d post-infarction,respectively.Results:Compared with the Saline group,the mice in the Dnase I group showed reduced production of NETs and apoptosis at 1 d of infarction,and the WT+Dnase I and TG+Dnase I groups decreased to similar levels.28 d after infarction,the mice in the TG+Dnase I group recovered their cardiac function and slightly reduced the area of left ventricular fibrosis.（2）Study on the regulatory role of S100A12 expression on the biological function of neutrophils1.To investigate the effect of S100A12 on primary neutrophils in miceMethods:Bone marrow-derived primary neutrophils were extracted from WT and TG mice,respectively.Transwell assay was performed to observe the migration ability of the primary neutrophils,Western blotting to detect the changes of NETs-related protein expression,and fluorescence staining to observe the formation of NETs and intracellular reactive oxygen content.Results:Compared with WT group,primary neutrophils from TG group were more susceptible to apoptosis,had stronger cell migration ability,expressed more NETs-related proteins,and had higher intracellular reactive oxygen species content,and immunofluorescence staining and Sytox green staining showed a significant increase in NETs production.2.To investigate whether S100A12 has a role in regulating NETs at the ex vivo levelMethods:A differential interference contrast microscope（DIC）was used to capture the real-time dynamic process of S100A12 promoting NETs production in neutrophils.Western blotting and q PCR methods were used to examine the effects of S100A12knockdown and overexpression on NETs-related protein production.Results:Compared with controls,neutrophils immediately after S100A12 stimulation showed flattening,increased cell adhesion,earlier intracellular vacuolization,enhanced plasma membrane permeability,and more rapid release of depolymerized chromatin.Inhibition of S100A12 expression caused a decrease in NETs production;overexpression of S100A12 promoted increased NETs formation.3.Effect of S100A12 expression in neutrophils on the level of apoptosis of co-cultured cardiomyocytesMethods:After neutrophils were transfected with small interfering RNA and overexpression plasmid of S100A12,they were co-cultured with HL-1 cardiomyocytes.The apoptosis of co-cultured cardiomyocytes was detected by TUNEL staining and Western blotting.Results:Compared with the control group,the apoptosis level of cardiomyocytes co-cultured with neutrophils knocking down S100A12 decreased,and the apoptosis level of cardiomyocytes co-cultured with neutrophils overexpressing S100A12 increased.When neutrophils overexpressing S100A12 were treated with Dnase I,the apoptosis level of cardiomyocytes co cultured with them decreased to normal.（3）Study on the molecular mechanism of S100A12 regulation of NETs formation1.To explore the role and mechanism of hypoxia induced increase in S100A12 expression in neutrophilsMethods:The expression of hypoxia inducible factor-1α（HIF1α）,Lysozyme,S100A12and NETs-related proteins were detected by Western blotting in neutrophils after different times of hypoxia.Dual luciferase reporter gene assay was performed to detect the transcriptional regulation of S100A12 gene by HIF1α.The changes in protein levels and m RNA of S100A12 after knockdown of HIF1αwere compared.Results:The expression of HIF1α,Lysozyme,S100A12 and NETs-related proteins in neutrophils all gradually increased with the prolongation of hypoxia;HIF1αwas an important transcription factor of S100A12.2.Search for receptor molecules in neutrophils that mediate increased expression of S100A12-regulated NETsMethods:Primary neutrophils from WT and TG mice were extracted,and the expression differences of Toll like receptor 4（TLR4）and the receptor of advanced glycation end products（RAGE）between the two groups were detected.Transfection of overexpression plasmid and small interference RNA were used to detect whether the receptor RAGE affected the production of NETs.Results:There was no significant difference in the expression of TLR4 receptor between the two groups of primary neutrophils.And the protein and m RNA expression levels of RAGE receptor in S100A12 overexpression group were significantly higher than those in the control group.NETs production was significantly decreased after interfering with RAGE expression;MPO,cit H3 and PAD4 expression were significantly increased upon S100A12 stimulation after overexpression of RAGE.3.Screening for new receptor protein molecules interacting with S100A12Methods:Tandem Mass Tags（TMT）quantitative proteomics was applied to compare differentially expressed proteins between WT and TG groups,and the expression of target proteins was verified in primary neutrophils from both groups of mice.Laser confocal scanning microscopy was used to detect the co-localization relationship between the target protein and S100A12 in cells,and the interaction between the target protein and S100A12was verified by immunoprecipitation（Co-IP）technique.After transfection of S100A12overexpression plasmid and small interfering RNA,we examined whether S100A12affected the expression of the target protein.Results:Combined with mass spectrometry analysis and literature reports,annexin a5（ANXA5）was screened as a possible target protein associated with S100A12 and validated.The expression of ANXA5 was also significantly increased under hypoxic conditions.Co-localization of ANXA5 with S100A12 was observed under confocal microscopy,and Co-IP experiments verified the interaction between ANXA5 and S100A12.Both protein and m RNA expression of ANXA5 decreased when S100A12 expression was interfered;after overexpression of S100A12,the expression of ANXA5 increased significantly.4.To investigate whether S100A12 regulates the expression of RAGE receptor through ANXA5 and affects the production of NETsMethods:Overexpression plasmids of ANXA5 and small interfering RNA were transfected to probe whether ANXA5 affects the expression of RAGE and NETs-related proteins.Next,RAGE overexpression plasmid was used to transfect neutrophils low in ANXA5 to see if overexpression of RAGE could restore the effects of knockdown of ANXA5.Results:The protein and m RNA expression of RAGE,MPO and cit H3 were decreased when ANXA5 was overexpressed,and the expression levels of these proteins were increased when ANXA5 was overexpressed.And overexpression of RAGE was able to restore the effect of knockdown of ANXA5.5.To investigate whether S100A12 regulates the expression of ANXA5 receptor through RAGE,forming a positive feedback regulationMethods:After transfection of neutrophils with RAGE overexpression plasmid and small interfering RNA,Western blotting and q PCR experiments were used to verify whether RAGE affected the expression level of ANXA5.Results:Both protein and m RNA expression of ANXA5 decreased with low expression of RAGE;after overexpression of RAGE,the expression of ANXA5 increased.6.To find and validate the transcription factors mediating the positive feedback regulation of ANXA5 and RAGEMethods:The promoter region of RAGE was searched from the NCBI website,and the transcription factors of RAGE were jointly predicted by PROMO,JASPAR,Gene Cards and UCSC websites and verified in neutrophils overexpressing ANXA5.Dual luciferase reporter gene assays were performed to test the activation of the RAGE promoter by the transcription factor.Neutrophils were transfected with interfering RNA of this transcription factor and altered levels of RAGE were examined after transfection.The transcription factor for ANXA5 was found in the same way as above.Results:Overexpression of ANXA5 increased the expression of transcription factor STAT3,which can improve the transcriptional activity of RAGE,and increased expression of transcription factor GATA1,which can improve the transcriptional activity of ANXA5.Conclusion:1.After acute myocardial infarction,the expression level of S100A12 increased significantly.2.S100A12 aggravates the myocardial injury after acute myocardial infarction,and its mechanism may be related to the increase of NETs production caused by S100A12.Intraperitoneal injection of NETs inhibitor Dnase I can significantly reduce the myocardial infarction injury of mice in S100A12 overexpression group.3.In vivo,transcription factor HIF1αincreased S100A12 transcriptional activity under hypoxia;Overexpression of S100A12 can accelerate the formation of NETs,which is mediated by RAGE.4.In vivo,overexpression of S100A12 increased the expression of ANXA5 through transcription factor GATA1,and ANXA5 increased the expression of RAGE through transcription factor STAT3,which promoted the production of NETs.