Studies On Macrophage Heterogeneity In Non-ischemic Cardiac Adverse Remodeling

Object:Cardiovascular diseases have the highest mortality in the world.In the course of the disease,the inflammation can aggravate myocardial hypertrophy,fibrosis,apoptosis and other adverse remodeling,which leads to heart failure.Various factors can cause the adverse remodeling of the heart,in which systemic inflammation is an important pathogenic mechanism.Cardiac macrophages exhibit heterogeneity in the source and function in homeostasis and disease settings.This study is to investigate the heterogeneity of macrophages in obesity and doxorubicin-induced cardiac adverse remodeling,to clarify the regulatory mechanism of macrophage proliferation in non-ischemic cardiac adverse remodeling.Methods:Obese mouse models were constructed by giving 6-week-old mice with high-fat diet(HFD)or Western diet(WD)on C57BL6/j background.Echocardiography and MRI(Magnetic resonance imaging)were used to detect cardiac function in mice.After modeling,the plasma and heart were collected,and the expression of inflammatory factors were detected by RT-PCR,Western blot and ELISA.The number and polarization of macrophages in the heart of obese mice were detected by flow cytometry and immunofluorescence.Flow cytometry was used to detect the proportion,polarization and proliferation of macrophages from different sources in the heart of Lyz2^cre/+R26^tdTOMATO/+ and Cx3cr1^creER/+R26^tdTOMATO/+HFD mice.CD45⁺CD11B⁺F4/80⁺macrophages analyzed by single-cell sequencing via 10x Genomics were sorted by flow cytometry from the hearts of CD and HFD 24-week mice.Based on the differences in gene expression in cells,macrophages are divided into different clusters,using functional enrichment to analyze the biological functions of different clusters,and using Monocle and Mpath to simulate the trajectorys between clusters.The function of Ccr2 cluster was verified by Ccr2 inhibitors in vivo.Immunofluorescence experiments of immune cells were performed on the heart tissues of patients with dilated cardiomyopathy(DCM).Six-week-old mice with C57BL6/j background were intraperitoneally injected with doxorubicin(Dox)to construct a model of myocarditis(DiCM).Flow cytometry was used to detect proportion,polarization and proliferation ability of resident and bone marrow-derived macrophages(BMDMs)in the heart of Cx3cr1^creER/+R26^tdTOMATO/+DiCM mice.SR-A1^f/fand SR-A1^cko mice were given Dox,and their cardiac function,morphology,inflammatory factors and apoptosis were detected.In vitro,Western blot was used to detect the signaling pathway of c-Myc regulated by Scavenger receptor A1(SR-A1)in BMDMs.In vivo,macrophage c-Myc knocked down or overexpressing lentiviruses were injected by tail vein into mice,then cardiac function was detected to verify the effect of c-Myc in macrophages on DiCM progression.Results:After HFD or WD,the left ventricular ejection fraction(LVEF)and left ventricular shortening fraction(LVFS)of the obese mice were significantly decreased.Meanwhile,the levels of inflammatory factors Tnf?,Il-1?,and Il-6 in the heart increased,and the levels of Il-6 in the plasma increased.Immunofluorescence and flow cytometry analysis also found that the number of macrophages in the heart of HFD mice increased significantly,mainly pro-inflammatory.In the Lyz2^cre/+R26^tdTOMATO/+and Cx3cr1^creER/+R26^tdTOMATO/+obese hearts,flow cytometry analysis showed that the number of Lyz2⁺bone marrow-derived macrophages increased significantly,and mainly polarized toward Ly6C⁺pro-inflammatory.However,Cx3cr1⁺resident macrophages maintain their quantity through proliferation,and are mainly polarized toward CD206⁺reparative type.Single-cell sequencing analysis found that cardiac macrophages were divided into9 clusters at steady state,and their functions were related to antigen presentation,proliferation,and cardiovascular development.While obese mouse cardiac macrophages were divided into 10 clusters,in which,Ccl and other inflammation-related clusters and Mki67 cluster increased significantly.Among the three origins of cardiac macrophages,the resident Folr2 cluster accounts for 29%of the steady-state heart,and was not replaced by bone marrow-derived macrophages after HFD.The embryonic liver-derived Fabp4 cluster accounts for 22%of the steady-state mouse heart,which is significantly reduced after HFD.After inhibiting Ccr2 activity,the cardiac function of WD mice was significantly improved.The number of macrophages in the heart of DCM patients increased.Flow cytometry analysis showed that during the development of DiCM mice,Cx3cr1^-bone marrow-derived macrophages were dominant and pro-inflammatory polarized,but gradually decreased during the development of DiCM,while Cx3cr1⁺resident macrophages proliferated after the disease,gradually restored the proportion,and polarized towards the reparative type.In DiCM,SR-A1^cko mice had more severely impaired cardiac function than SR-A1^f/f mice,and knockout of SR-A1 on macrophages increased the inflammatory and cardiomyocyte apoptosis.Western blot results showed that SR-A1 on macrophages regulated proliferation through the TAK1-MKK4-P38-c-Myc-SIRT1 signaling pathway.Then,the impact of c-Myc on macrophages was confirmed by knockdown and overexpression lentivirus of c-Myc in vivo that it played a protecting role in DiCM.Conclusion:The results of this study by two non-ischemic cardiac adverse remodeling induced by obesity and Dox respectively show that macrophages from different sources have different effects.During the course of the disease,BMDMs,such as the Ccr2 cluster,increase significantly and play a pro-inflammatory role,thereby aggravating the damage to the heart.While resident macrophages have increased proliferative capacity,mainly polarizing towards reparative type,thus expressing myocardium protective effects.The SR-A1-c-Myc axis in macrophages may mediate cell proliferation.Our study provides a new theoretical basis and an important target for clinical treatment of cardiac ischemic adverse remodeling.