The Role And Mechanism Of Macrophage Colony-stimulating Factor (M-CSF) In Ventricular Remodeling After Myocardial Infarction In Mice By Regulating Cardiac Macrophage

BackgroundMyocardial infaction（MI）refers to atheromatous（AS）plaque formed on the basis of coronary artery endothelium injury,and then plaque hardening obstructs the artery and causes one or more coronary stenosis and myocardial blood supply insufficiently while the collateral circulation has not been fully established.Then the blood supply to the coronary arteries is drastically reduced or interrupted,which will cause ischemic necrosis of myocardium.A large number of studies have confirmed that the majority of acute MI patients is due to unstable atheroma of the AS plaque in the coronary arteries,followed by the intraluminal hemorrhage and thrombosis,eventrually occluding the arteries.In a few cases,bleeding inside the AS plaque or persistent vasospasm may also result in complete or incomplete occlusion of the coronary artery.Most of the clinical symptoms are persistent symptoms of severe post-sternal pain in the precordial region.Most patients have white blood cells and marked up-regulation of serum myocardial infarction markers,and progressive changes in ECG of myocardial necrosis in the corresponding sites,heart failure（HF）,shock,and various types of VAs can occur.MI has occurred in Europe and the US.About 1.5 million people have AMI every year,and 800 thousands people have recurrent MI.In China,nearly 1,000 thousands people die caused by AMI.Nowadays,with the in-depth study of the clinical symptoms and related mechanisms of MI,the extensive application of thrombolysis and coronary intervention and the rapid improvement of drug treatment,the mortality of MI patients is decreasing yearly.Although most patients with MI have a good prognosis after percutaneous coronary intervention（PCI）,some patients of them still have ventricular remodeling,which is closely related to patients with different degrees of HF and the occurrence of various VAs.Therefore,improving the clinical managements of patients with MI,and inhibiting the structural remodeling and electrical remodeling of the heart after MI,inhibiting and/or reducing of the incidence of VAs in patients with MI and its related mechanisms still play an important role in the management of MI.Lots of researches have confirmed that inflammatory response plays an essential role in the occurrence and development of cardiac structural remodeling and electrical remodeling after MI,and the serum inflammatory factors levels has a significant correlation with the development and prognosis of MI.For example,one study found that interleukin-6（IL-6）is one of the important risk factors for coronary heart disease（CHD）,and the serum IL-6 level is significant higher in patients with MI than those who have no CHD.On the contrary,the reduction of the serum IL-6 level or the inhibition of the serum IL-6 level can make the AS plaques stable in coronary arteries,which can decrease the ratio of cardiovascular events.In addition,another study has found that interleukin 4（IL-4）triggers a downstream signaling cascade through the IL-4 receptor,and then activates the classical JAK-STAT6 signaling pathway,ultimately inhibits the inflammatory responds to improve cardiac remodeling after MI.What’s more,the immune response also has relationship with the ventricular remodeling of the heart.Macrophages are important components of the important natural autoimmune system.They can phagocytose necrotic cardiomyocytes after MI,inhibit inflammation,and directly release cytokines and inflammatory factors to promote pro-inflammatory effects acting as the important regulators of inflammatory responses,thereby promoting structural remodeling and electrical remodeling of the heart after MI.Macrophages are mostly derived from monocytes in the circulational blood,and then enter into the heart tissue to undergo differentiation and finally change to the mature functional macrophages,which will settle down in the heart tissue.Macrophages can exhibit different polarization types when they are in different immune diseases,and different cell stimulating factors.According to the properties or functions,they are mainly divided into two types:The classical activation pathway is polarized to M1 type which is highly expressed i NOS;The second type is the M2 type which is the alternative polarization pathway,and the Arg-1 is highly expressed on the cell surface.The main role of M1 macrophages is to release tumor necrosis factorα（TNF-α）,IL-6 and interleukin 12（IL-12）and other pro-inflammatory factors,which in turn produce a large number of reactive Nitrogen and Oxygen intermediates promoting the process of immune response.In contrast,M2 macrophages secrete inflammatory factors,such as IL-10,transforming growth factor-β（TGF-β）,etc.,which inhibit inflammatory responses.In addition,M2 macrophages can regulate the immune,and promote the regeneration of new blood vessels and the remodeling and repair of myocardial tissue.In summary,macrophages are a“double-edged sword”in the pathophysiological process of heart after MI,which plays an important role in the ventricular remodeling and the occurrence and development of VAs.Macrophage colony-stimulating factor（M-CSF）is a polypeptide hormone-like hematopoietic growth factor released from damaged endothelial cells,which can promote the proliferation and differentiation of hematopoietic progenitor cells into mononuclear macrophages,and it is really essential in the growth,proliferation or differentiation of macrophages,and is a factor for inflammatory diseases.Because M-CSF has the function of promoting macrophages maturation,it has chemotactic characteristics on monocytes.Therefore,this study intends to use mice as experimental animals to establish a MI model to observe the role of M-CSF and related macrophages in cardiac remodeling after MI,and to investigate the effects of macropahges on the myocardial remodeling and ventricular arrhythmias after MI and related mechanisms regulating by M-CSF.We hope to provide some new ideas and experimental basis for clinical treatment of MI and improvement of prognosis after MI.Part I The expressions of M-CSF and macrophages in myocardium following MIObjectives:To investigate the levels and role of M-CSF and macrophages at different time points in myocardium after MI.Methods:90 male C57BL/6J wild type mice（WT）weighing 20-25 g and 8-10weeks old were selected,and a mouse protocol of MI was made by ligation of the left anterior descending（LAD）.All mice were obtained and fed two weeks after recovery from splenectomy（not only to eliminate spleen-derived stem cells,but also to prevent the homing of bone marrow-derived stem cells）.The mice were divided into a sham operated group（Sham group,n=30）,a 3-day group after MI（MI/3d,n=30）,and a7-day group after MI（MI/7d,n=30）.The mice in the Sham group were given a ligature through the LAD without ligation,the MI/3d group and MI/7d group were ligated to the LAD to create a MI model,and were sacrificed at the third day and the7^thday after MI.The levels of M-CSF of 3 groups were detected by RT-PCR and Western-blot.The levels of M1 and M2 macrophages in myocardium were detected by immunofluorescence.The expressions of NF-κB p65,STAT3 and STAT6 in heart tissues after MI were measured by Western-blot.Results:The results of RT-PCR and Western-blot indicated that the levels of M-CSF significantly increased in the MI/3d group and the MI/7d group than in the Sham group,and the level of M-CSF in MI/3d group was also significantly higher than in the MI/7d group（all P<0.05）.Immunofluorescence assay indicated that the levels of M1 macrophages increased significantly at the 3rd day（P<0.05）and then decreased,while the levels of M2 macrophages gradually increased at the 3rd day and the levels of M2 macrophages reached a peak at the 7th day after MI（all P<0.05）.The results of Western-blot showed that the expressions of NF-κB p65,STAT3 and STAT6in the MI/3d group increased significantly than in the MI/7d group,and the levels of above proteins in this two groups were also higher than the Sham group（all P<0.05）.Conclusion:The expressions of M-CSF,NF-κB p65,STAT3 and STAT6 in myocardial tissues following MI significantly increased in the early time points of MI.The M2 macrophage increased continuously after MI,while the M1 macrophage increased significantly in the acute phase after MI,then decreased timely,from which we speculated that M-CSF and macrophages play an important role in myocardial remodeling after MI.Part II Effects and mechanisms of M-CSF on inflammation and cell apoptosis after MI in mice by regulating cardiac macrophages Objectives: To observe the effects and related mechanisms of M-CSF on inflammation and cell apotosis after MI in mice by regulating cardiac macrophages.Methods: 110 male C57BL/6J WT mice weighing 20-25 g and 8-10 weeks old were selected.All mice were recovered for 2 weeks after splenectomy.The MI model was made by ligation of the LAD.All mice were divided into 3 groups.The sham-operated mice were given a ligation line through the LAD without ligation（Sham group,n=30）,and the MI model was prepared by ligation of the LAD.The M-CSF（500 ug/kg/d）was given intraperitoneal injection for the first 5 days after surgery,which was the MM group（n=40）.And after MI model was completed,intraperitoneal injection of normal 5 consecutive days（MC group,n=40）.All mice in the 3 groups were fed in a barrier environment for 1 week.At the end of the study,allmice were sacrificed.The expression of M1 and M2 macrophages in cardiac tissues were detected by immunofluorescence assay,and inflammatory factors such as IL-4,IL-6,IL-10,TNF-α,interferon α（IFN-α）and monocyte chemotactic protein-1（MCP-1）were detected by ELISA.The levels of NF-κB p65,STAT3 and STAT6 were measured by Western-blot and also the levels of Bax,Caspase 3,C-Casp 3 and Bcl-2.TUNEL staining was used to observe the apoptosis of cardiomyocytes in the 3groups after MI.Results: The levels of M1 and M2 type macrophages in heart tissues showed that,the level of M1 macrophages in the MC group was significantly higher than that in the Sham group and MM group（all P<0.05）while the M1 macrophage in the MM group was also higher than in the Sham group,but there was no significant differences（P>0.05）.When compared with the Sham and MC groups,the level of M2 macrophage in the MM group significantly increased（all P< 0.05）,while the level of M2 macrophage in the MC group was higher than that in the Sham group（P>0.05）.The serum and tissue levels of IL-6 and TNF-α increased in the MC and the MM groups than in the Sham group（all P<0.05）,and the levels of IL-6 and TNF-αsignificantly decreased in the MM group when compared with the MC group（all P<0.05）.In contrast,the expressions of IL-4 or IL-10 in the MM group increased differently than in MC group.The serum levels of IFN-α in the MC group also decreased than that in the MC group（all P<0.05）.When compared with Sham group,the expressions of NF-κB p65,STAT3 and STAT6 in myocardial tissues in MC and MM groups increased,and the levels of STAT3 and STAT6 were significantly higher in the MM group than in the MC group at the end of the protocol（all P<0.05）.The levels of STAT6 in the infarcted area in the MM group and MC group were all higher than that in the non-infarcted area（all P<0.05）.The results of TUNEL staining showed that the apoptosis rate of cardiomyocytes in the MC and MM groups were higher than in Sham group and the MM group was significantly lower than in the MC group（all P<0.05）.Western-blot assay showed that the expressions of Bax,Caspase 3and C-Casp 3 in myocardial tissue in the MC group increased than in the Sham group（all P<0.05）while the Bcl-2 protein decreased than in the Sham group（P<0.05）.Compared with the MC group,the levels of Bax,Caspase 3 and C-Casp 3 in the MM group were significantly lower than that in MC group（all P<0.05）.Conclusion: M-CSF can significantly increase the expression of M2 macrophages and reduce the level of M1 macrophages,then inhibit the inflammatory response and decrease the apoptosis of myocardial cells in mice,and NF-κB/STAT signaling pathway has an essential role in this process.Part III Effects and mechanisms of M-CSF on ventricular structural remodeling after MI in mice by regulating cardiac macrophagesObjectives: To investigate the effects and mechanisms of M-CSF on ventricular structural remodeling after MI in mice by regulating cardiac macrophages.Methods: 110 male C57BL/6J WT mice weighing 20-25 g and 8-10 weeks old were selected.All mice were recovered for 2 weeks after splenectomy.The MI model was made by ligation of the LAD.All mice were divied into 3 groups.The sham-operated mice were given a ligation line through the LAD without ligation（Sham group,n=30）,and the MI model was prepared by ligation of the LAD.The M-CSF（500 ug/kg/d）was given intraperitoneal injection for the first 5 days after surgery,which was the MM group（n=40）.And after MI model was completed,intraperitoneal injection of normal saline for 5 days（MC group,n=40）.All mice in the 3 groups were fed in a barrier environment for 1 week.After the model was completed,the survival status of the 3 groups of mice was observed every day,and the death ratio was calculated,the survival curve was drawn after complete the experiment.Echocardiography was used to evaluate cardiac function in the three groups of mice at the end of the protocol.Body weight（BW）,heart weight（HW）,lung weight（LW）,and tibia length（TL）were measured.The myocardial infarction size and the myocardial interstitial collagen deposition and collagen deposition volume fraction was measured by the Sirius Red Staining.The levels of atrial natriuretic peptide（ANP）,brain natriuretic peptide（BNP）,β-myosin heavy chain（β-MHC）and fibrosis related indicators（Collage I,Collage III）were detected by ELISA after MI in myocardial tissue of infarcted area.The expression of CD31 was detected by immunohistochemistry in the peri-infarct area of mice one week after MI.Results: The survival rate in the MC group and the MM group decreased than in the Sham group,and the live rate of the MC group decreased than that in the MM group.The cardiac function in the MC group and the MM group significantly decreased than in Sham group,and the left ventricular end-diastolic diametion（LVEDd）,left ventricular end-systolic diametion（LVSDd）,left ventricular end-diastolic posterior wall（LVVd）,left ventricular end-systolic posterior wall（LVVs）,the systolic interventricular septum（IVSs）and the diastolic interventricular septum（IVSd）all significantly increased（all P<0.05）while the fractional shorting（FS）and the left ventricular ejection fraction（LVEF）were significantly lower than that in the Sham group（all P<0.05）.The ventricular function significantly improved in the MM group than in MC group,and the LVEDd,LVSDd,LVPWd,LVPWs,IVSs,and IVSd all significantly reduced while the FS and LVEF increased than in the MC group（all P<0.05）.The results of HE staining showed that the myocardial infarct size in the MM group was significantly lower than that in the MC group（P<0.05）.The levels of collagen deposition in infarction tissue in the MC group increased significantly than in Sham group after MI while the collagen deposition in the MM group was differently lower than that in the MC group（all P<0.05）.Compared with the sham group,the expressions of ANP,BNP and β-MHC in heart tissues in the MC group significantly increased（all P<0.05）while the expressions of above factors were lower in the MM group than in the MC group（all P<0.05）.The results of ELISA showed that the levels of Collage I and Collage III in the MC group increased than in the Sham group,and the levels of Collage I and Collage III in the MM group were significantly lower than in the MC group（all P<0.05）.The expression of CD31 in the infarction area in the MM group significantly increased than in the MC group after MI（P<0.05）.Conclusion: M-CSF can significantly improve the survival rate of mice after MI by regulating cardiac macrophage,and can increase the live rate of mice,the cardiac function and LVEF after MI,and reduce the infarction size of mice,promote the regeneration of new blood vessels while reducing myocardial hypertrophy and improving myocardial fibrosis.Part IV Effects and mechanisms of M-CSF on ventricular electrical remodeling after MI in mice by regulating macrophagesObjectives: To investigate the effects and mechanisms of M-CSF on ventricular structural remodeling after MI in mice by regulating macrophages.Methods: 110 male C57BL/6J WT mice weighing 20-25 g and 8-10 weeks old were selected.All mice were recovered for 2 weeks after splenectomy.The MI model was established by ligation of the LAD.All mice were divided into 3 groups.The sham-operated mice were given a ligation line through the LAD without ligation（Sham group,n=30）,and the MI model was prepared by ligation of the LAD.The M-CSF（500 ug/kg/d）was given intraperitoneal injection for the first five days after surgery,which was the MM group（n=40）.And after MI model was completed,intraperitoneal injection of normal saline for 5 consecutive days（MC group,n=40）.All mice in the 3 groups were fed in a barrier environment for 1 week.Langendorff electrophysiological monitoring technique was used to record the electrocardiogram（ECG）of mice after MI,and the ventricular effective refractory period（ERP）of the 3groups was measured by S1S2 programmed stimulation and the action potential duration（APD）,action potential electrical alternation,and various ventricular arrhythmias（VAs）were measured by S1S1 programmed stimulation.The expression of symrosine hydroxylase（TH）in infarction tissues was detected by immunofluorescence.The expression of connexin 43（Cx43）in heart tissues of the three groups were detected by Western-blot.Results: The ECG results of the 3 group showed that there was no statistically significant difference in the PR interval and RR interval（all P<0.05）.The QRS interval,the QT interval,and the corrected QT interval（QTc）in the MC group and MM group significantly prolonged than in the Sham group,and above indicators in MC group decreased than in the MM group（all P < 0.05）.The ERP in the MC group and MM group significantly shortened than in the Sham group,and the ERP in MM group increased than in the MC group（all P < 0.05）.The APD90 prolonged in the MC group and MM group than in the Sham group while it shortened in MM group than in the MC group（all P<0.05）.The thresholds of APD alternation in the MC group and the MM group shortened than in the Sham group（all P<0.05）,and the electrical alternating thresholds in the MM group were higher than in the MC group（P<0.05）.The incidence of VAs increased in the MC group than in the Sham group（P<0.05）while the ratio of VAs in the MM group decreased than in the MC group（P<0.05）.The TH levels in MC and MM groups increased than in the Sham group while the TH level in the MC group increased than in the MM group（all P<0.05）.The level of Cx43 in myocardial tissue in the MC group decreased than in the Sham group（P<0.05）.The level of Cx43 in myocardial tissue in the MM group increased highly than in the MC group（P<0.05）.Conclusion: M-CSF can significantly increase ERP and decrease the VAs ratio after MI By regulating cardiac macrophage.It may be related to the decrease of sympathetic hyperdistribution,inhibition of Cx43 degradation,and reduction of myocardial cell uncoupling.