CCN2 Derived From NLRX1-deficient Cardiomyocytes Regulates Macrophage Conversion To Phenotype Involved In Post-infarction Repair

Background:Myocardial infarction and its associated complications are the leading causes of death with high morbidity and mortality,posing a huge threat to human health.How to reduce myocardial ischemia injury and improve infarct healing is of great scientific significance in current cardiovascular research filed.Following myocardial infarction,injured cardiomyocytes release damage-associated molecular pattern which is recognized by immune cells such as macrophages,triggering an immune response.Macrophages play an important role in inflammatory response,infarct healing and repair,and ventricular remodeling following myocardial infarction.Macrophages exhibit phenotypic diversity and functional plasticity.Reshaping the phenotype of macrophage by enhancing its reparative function is expected to promote the healing of infarcted hearts.NLRX1,a recently identified regulatory pattern recognition receptor belonging to the NLR family,is highly expressed in the heart and is significantly increased after myocardial infarction,however,the role of NLRX1 in post-infarction inflammation and healing is poorly understood.This thesis proposes to use NLRX1 knockdown/overexpression and myocardial infarction models to observe the role and possible mechanisms of NLRX1 in inflammatory response and tissue repair after myocardial infarction.Objectives:1.To investigate the role of NLRX1 in the inflammation and infarct healing following infarction.2.To determine the effect of NLRX1 knockdown/overexpression on the primary cardiomyocytes upon hypoxic injury.3.To dissect the molecular mechanism underlying the cross-talk between the cardiomyocytes and macrophages in the infarcted heart.Methods:1.Mine and analyze The Human Protein Atlas database to clarify the NLRX1expression abundance in various tissues.Tissues of heart,liver,spleen and lung were extracted from C57BL/6J wild-type mice and the expression of NLRX1 was verified by Western blotting.Background gene modification was applied to C57BL/6J mice to construct NLRX1 knockout model.The experimental animals were divided into four groups:(1)C57BL/6J mice sham-operated group(WT-Sham);(2)C57BL/6J mice operated group(WT-MI);(3)NLRX1 KO mice sham-operated group(NLRX1 KO-Sham);(4)NLRX1 KO mice operated group(NLRX1 KO-MI).First,a myocardial infarction model was prepared,the control group was not ligated,and the rest of the operations were performed as in the surgical group.Western blotting was performed to detect the expression abundance of NLRX1 before and after myocardial infarction;The survival rate of WT and NLRX1 KO mice after MI was observed and echocardiography was used to analyze cardiac function;Masson staining and Sirius red staining were used to detect the extent of fibrosis in cardiac tissue;Single cell sequencing was performed to analyze the effect of NLRX1 knockdown on the phenotype and function of macrophages in infarcted hearts;Flow cytometry was performed to detect the presence and expression abundance of CD11b~+CD9~+macrophages in the infarcted hearts and to sort CD11b~+CD9~+macrophages;transcriptome sequencing was performed to analyze the gene expression characteristics and functional properties of CD11b~+CD9~+macrophages.2.1-3 day SD rat primary cardiomyocytes were extracted;Knockdown of NLRX1 in cardiomyocytes using si RNA;Overexpression of NLRX1 in cardiomyocytes infected with adenovirus;Cell viability was detected by different time oxyglucose deprivation and by CCK8 assay;Cardiomyocyte apoptosis was detected by Annexin V/PI assay.3.Transcriptome sequencing analysis to screen for possible differential expression and related genes in NLRX1 knockdown/overexpressing primary cardiomyocytes after hypoxic injury;Western blotting assay was performed to compare the expression levels of NLRX1 in the cardiomyocytes and macrophages and the expression content of CCN2protein in the cardiomyocytes and infarcted heart tissues after hypoxic injury;q PCR was performed to detect the effect of knockdown of NLRX1 in the cardiomyocytes with hypoxic injury cell supernatant on the expression of typical tissue repair-related genes and inflammation-related genes in bone marrow-derived macrophages and expression of typical tissue repair genes after stimulation of human homologous recombinant CCN2protein in bone marrow-derived macrophages.ELISA assay for CCN2 in cardiomyocyte supernatant after hypoxic injury.Results:1.Mined of The Human Protein Atlas database and Western blotting reveal that NLRX1 is highly expressed in the heart and cardiomyocytes.NLRX1 expression is significantly increased after myocardial infarction.2.Compared with WT mice,NLRX1 KO mice exhibit improved cardiac function,increased collagen content in the infarct zone and improved healing,and increased survival following myocardial infarction.Single-cell RNA sequencing analysis showed that infarct macrophages are clustered into seven subpopulations,with cluster 5 of CD9~+macrophages predominantly present in the infarcted hearts of NLRX1 KO mice.CD9~+macrophages are characterized by high expression of wound healing associated genes,and downregulation of proinflammatory cytokines and chemokines.CD9,Il1rn,Gpnmb and SPP1.3.Flow cytometry analysis showed markedly increased number of CD9~+macrophages and higher mean fluorescence intensity of CD9 in NLRX1 KO mice.RNA-seq analysis revealed that CD9~+macrophages exhibit a reparative and anti-inflammatory phenotype.4.At the cellular level,NLRX1 knockdown attenuated hypoxia-induced apoptosis in cardiomyocytes,and NLRX1 overexpression exacerbated hypoxia-induced apoptosis in cardiomyocytes.5.Verification experiments revealed that NLRX1 is significantly highly expressed in primary cardiomyocytes and H9C2 cardiomyocyte cell line.A small amount of NLRX1expression is detected in the RAW264.7 macrophage cell line,and NLRX1 expression is lowest in bone marrow-derived macrophages.6.Supernatants from NLRX1 deficient cardiomyocytes upon hypoxia stimulation skewed macrophage polarization towards a reparative and inflammation-suppressed phenotype,with upregulation of anti-inflammatory repair-related genes like SPP1,Fn1,and Il1rn,and downregulation of inflammation-related genes such as Irf7,Ifit3,and Isg15.7.Transcriptome sequencing results suggested that CCN2 expression level are negatively correlated with NLRX1 expression in cardiomyocytes upon hypoxia:CCN2 is significantly upregulated in NLRX1 knockdown cardiomyocytes,whereas,CCN2 is remarkably downregulated in NLRX1-overexpressing cardiomyocytes in response to hypoxia stimulation.8.In vitro,both protein expression of CCN2 and secreted CCN2 in the supernatant are increased in NLRX1 knockdown cardiomyocytes upon hypoxia stimulation.In vivo,protein expression of CCN2 was significantly elevated in the infarcted hearts of NLRX1KO mice.9.Exogenous CCN2 stimulation mediated macrophage activation towards a reparative phenotype.Conclusion:1.Single cell RNA-sequencing identifies a novel subset of CD9~+reparative macrophages,which are predominantly present in the infarcted hearts of NLRX1 KO mice.2.CCN2,whose expression are significantly increased in NLRX1 deficient cardiomyocytes upon hypoxia and markedly elevated in NLRX1 KO infarcted hearts,is identified as a cardiomyocytes-derived mediator modulating macrophage towards a reparative phenotype,highlighting the cross-talk between cardiomyocytes-derived mediators and macrophages in the infarcted hearts.