The Exploreation On Role And Mechanism Of Macrophage In Sympathetic Hyperinnervation Post MI

BackgroundSudden cardiac death is one of the most common complication of myocardial infarction(MI).0.46-3.7 in 100 thousand people died per year.In clinical practice,patients tended to suffer ventricular tachycardia(VT)and ventricular fibrillation(VF)frequently,also known as "sympathetic electrical storm",which is correlated with sympathetic hyperinnervation and activation.In spite of high dose of anti-arrhythmia drug administration、ICD transplantation and ablation are widely used in the electrical storm post-MI,the arrhythmia could not be terminated effectively.In canines and rodents,sympathetic targeted left Stellate Ganglion Block,renal sympathetic ablation as well as para-sympathetic stimulation showed beneficial effects on preventing ventricular arrhythmia from happening.However,the small clinical sample and clinical evidence is lacking.For now,cardiac autonomic nerve remodeling characterized by sympathetic hyperinnervation is rising to be important element for VT and VF.Therefore,searching for the molecular machenism for sympathetic hyperinnervation may provide novel theoretical basis and therapeutic target for clinical malignant arrhythmia post MI.Studies have shown that inflammation response played an important role in the pathogenesis of peripheral nerve damage and innervation.This founding unveiled the mist of the pathogenesis of autonomic innervation.Inflammation-dominated sympathetic sprouting adjacent to the necrotic region following myocardial infarction(MI)has been implicated in theetiology of arrhythmias resulting in sudden cardiac death.Studies have shown that through the producing nerve growth factor(NGF),the infiltrated macrophages in the infarcted heart build the connection between inflammatory reaction and sympathetic innervation,with the underlying mechanism unclear.Macrophages are mainly divided into two phenotypes:the inflammatory M1 subtype and anti-inflammatory M2 subtype.Studies relating the myocardial remodeling found that the phenotype and function of macrophageare changing consequently in a certain time course.In the early inflammatory stage of MI,M1 macrophages are infiltrated massively and peaked at day 3 post-MI;at day 5 post-MI,M2 macrophages increased gradually and peaked at 7 days post-MI.Then,macrophages began to disappear.Therefore,further exploration of the specific molecular that could regulate macrophage phenotype and function could provide novel therapeutic target for sympathetic hyperinnervation and ventricular arrhythmia.The latest founding by Xu et al.published in Nature Communication showed that Notch signal involved in the shifting of macrophage phenotype and macrophage dominated inflammatory reaction regulation,while the role of the Notch in the process of sympathetic hyperinnervation post-MI has yet to be explored.Therefore,we speculated that the activation of Notch signaling was involved in the macrophage phenotype shifting post-MI,and this process could affect NGF synthesis and potentially involve in sympathetic hyperinnervation post-MI.ObjectiveWe investigated whether Notch regulates macrophage responses to inflammation and affects cardiac sympathetic reinnervation in rats undergoing MI.MethodsProtocols 1The experimental rats were randomly assigned to the following groups:I:Sham +DMSO group:coronary arterywere threading without ligation,while DMSO was injected by tail vein injection,50ul per time,starting from 30 minutes before MI and then daily until sacrifice;II:Sham + DAPT group:The manner of model establishment and DAPT injection were consistent with I,while the dose of DAPT was 10mg/kg dissolved in 50ul DMSO;III:MI + DMSO group:The left anterior descending coronary artery was ligated permanently to establish MI model,while the dose and the injection manner of DMSO were consistent with I.IV:MI + DAPT group:The manner of model establishment were the same as III,while the dose and injection manner of DAPT were the same as II.At 3 days post-MI,we isolated infiltrated macrophage in the infarcted myocardium,infarcted myocardium,infarcted border myocardium,remote myocardium to define mRNA level Notch signaling related protein(NICD1、Hesl)by RT-PCR.We double stained CD68 and CD163,CD68 and Argl,as well as conducted RT-PCR to define macrophage subtype.Elisa was applied to measure the plasma TNF-a and IL-1β level.At 7 days post-MI,we measuredsemi-quantitative sympathetic innervation withimmunofluorescence staining of tyrosine hydroxylase(TH)and growth-associated protein(GAP43);we applied the volume-pressure system to measure the hemodynamic indexes;in addition,we applied the programmed electrical stimulation to measure the ventricular arrhythmia susceptibility.Protocols2To determine the effect of DAPT to NGF production,we divided the infarcted rats into the following groups:I:MI group;Ⅱ:MI + DAPT low dose group(5mg/kg);Ⅲ:MI + DAPT high dose group(10 mg/kg).7 days later,rats were sacrificed.We determine the NGF mRNA and protein level in the infarcted zone and infarcted border by RT-PCR and WestemBlot.In addition,we double stained CD68 and NGF to determine the level of macrophage mediated NGF production.Protocols3In vitro study:Bone marrow macrophages(BMM)were extracted and cultured in M-CSF for 3 days,then the cultured cells are divided into the following treatment group:Control group,LPS(10 μg/ml)+IFN-y(20 ng/ml)group,LPS(10μg/ml)+IFN-y(20 ng/ml)+ DAPT group(10 μg/ml);Control group,IL-4(10 ng/ml)group,IL-4+DAPT group.Results(1)The animal study showed that,in accordance with the macrophage polarization time course,Notch signaling-associated protein NICD1 and Hesl were significantly activated in day 3 post-MI,and fell back to normal level at day 7 post-MI.RT-PCR and immunofluorescence double staining quantified and localized the activation of Notch signaling.The data showed that Notch signaling was remarkably activated in infiltrated macrophages and mainly co-stained with CD68 positive macrophages,instead of other cell type as myocyte and myofibroblast.(2)Notch inhibitor N-N-(3,5-difluorophenacetyl-1-alanyl)-S-phenylglycine-t-butyl ester(DAPT)(i.v.)decreased the number of macrophages,decreases in the plasma levels of TNF-α and IL-1β and significantly increased the M2 macrophage activation profile in the early stages as well as attenuated the expression of nerve growth factor(NGF)in a dose-dependent manner.Eventually,NGF-induced sympathetic hyperinnervation was blunted,as assessed by the immunofluorescence of tyrosine hydroxylase(TH)and growth associated protein 43(GAP-43).At 7 days post-MI,the arrhythmia score of programmed electric stimulation in the vehicle-treated infarcted rats was higher than that in rats treated with DAPT.Further However,our data showed unexpected deterioration in cardiac function and enlargement of infarcted area despite of the improved ventricular arrhythmia susceptibility.(3)Our in vitro study on bone marrow macrophage(BMM)pointed out that DAPT could shift the M1 macrophage phenotype towards M2 phenotype,and reduce the NGF production of LPS/IFN-γ inducedM1 macrophage in a Notch-dependent manner.In the opposite,Notch signaling played no role in NGF production in M2 macrophages.ConclusionsIn the setting of rat MI model,we foundNotch signalingwas activated in infiltrated macrophage post-MI and dominated the macrophage subtype shifting process post MI.Notch inhibition at early stage could shift macrophage towards M2 subtype during the acute inflammatory response phase and downregulated NGF expression,probably through a macrophage-dependent pathway,thus eventually preventing the process of sympathetic hyperinnervation.Our study demonstrated that Notch signaling may involve in the sympathetic reinnervation in macrophages dependent manner,thus providing a novel therapeutic target for ventricular arrhythmia post-MI.BackgroundsFor now,the role of inflammation on the process of neural remodeling has been broadly verified.In animal experiment of rodents and canine,multiple anti-inflammatory drugs,and the inhibitory regants targeting NF-kB、PPAR-γpresented beneficial role in ameliorating sympathetic remodeling,while the connection between "inflammation response" and "sympathetic innervation"remained unclear for a long time.Until 2009,Wernli G et al.discovered that through NGF production,macrophage presented to be the "bridge" between "inflammation"and "sympathetic innervation":they depleted the macrophage through injection of liposome and found that the macrophages infiltrated in the infarcted heart was the main source of the nerve growth factor(NGF).We regulated macrophage phenotype through interfering Notch signaling.Consequently,we found that by inhibiting Notch signaling,macrophages were shifted to M2 direction,NGF expression was decreased,then sympathetic nerve remodeling was improved.The above data indirectly proved that Ml macrophages were involved in the sympathetic nerve post MI.However,it may be premature to block the Notch signal to polarize the macrophages to the M2 direction,which leads to the improvement of the heart function of MI 7.Confirming the role of M1 macrophage on NGF production in the first part,we then searched for the upstream molecular target for the regulation of M1 macrophage function.M1 macrophage is characterized by increased IL-1βexpression.Pro-inflammatory cytokines like IL-1β was reported to be involved in diabetes peripheral neuropathy and retina neuropathy through regulating the production of nerve nutritional factor.Previous study demonstrated that the mature and secretion of IL-1β in tissue macrophage is completely dependent on P2X7R signaling mediated caspase-1 activation.Under the condition of hypoxia、cell damage and mechanical stress,massive ATP was released outside of the cell membrane,then activated P2X7R,alerting cell necrosis/damage immune status.P2X7R is a highly unusual ATP-gated,non-selective cation channel(K+、Na+、Ca2+,and so on)expressed primarily on cells of haematopoietic origin such as macrophages and microglia.It mainly functions through producing IL-1β in M1 macrophage in inflammatory response,while the function in M2 macrophages is quite slim.Mounting evidence supports the hypothesis that inflammation mediated growth factor and cytokines modulate sympathetic spouting after myocardial infarction(MI).MyeloidP2X7 signal has been shown to activateNOD like-3 protein(NLRP3)inflammasome,a master regulator of inflammationand mediate the cleaved mature and secretion of IL-1β in M1 subtype macrophage.However,the role of P2X7 in NGF secretion and cardiac nerve remodeling remains unexplored.ObjectiveWe assessed whether A-740003,a P2X7R antagonist,alerts inflammatory meditators and attenuates sympathetic reinnervation after MI,and whether lnterleukin-1β(IL-1β)is involved in the protection.MethodProtocols 1To investigate the link betweenP2X7R and IL-1β in vivo,Twenty-five rats were randomly divided into 4 groups:I:Naive group;ⅡI:LPS group:the subclinical dose of 2 mg/kg in normal saline were injected intraperitoneally;III:LPS+ BzATP group:The injection dose and manner of LPS was the same as I;BzATP,a P2X7R agonist,BzATP(50μg/5μL per rat)were administrated3 hours after LPS injection;IV:LPS +BzATP + NLRP3 inhibitor group:BzATP and NLRP3 inhibitor16673-34-0 were given to lipopolysaccharide(LPS)primed naive rats.The dose and injection manner of BzATP and LPS were the same as Ⅲ,while 16673-34-0 was injected 30 min before LPS injection.In LPS-primed rats,BzATP induced caspase-1 activationand mature IL-1β release was neutralized by NLRP3 inhibitor.Six hours after LPS injection,the temporal left ventricular expression of NLRP3,pro-caspase-1,caspase-1,P2X7R and matureIL-1β were detected by Western blot.Protocols 2To investigate the time-course activation of P2X7R and post-MI,twenty-seven rats were divided into 6 groups(MI after 0,0.5,1,3,5,and 7days).The temporal expression of NLRP3,P2X7R and matureIL-1β were detected by Western blot.Protocols 3To investigate the role of P2X7R on the process of sympathetic hyperinnervation,one hundred and thirty nine rats were randomly divided into the following groups:I:Sham surgery(n =15);Ⅱ:Ligation surgery(n = 28);Ⅲ:Ligation surgery +A-740003(n = 28):A-740003 was dissolved in distilled water and administered intraperitoneally using a previously validated dose of 50 mg/kg given daily for 7 d starting one day prior surgery;IV:Ligation surgery +Anakinra(n = 30):2 mg/kg rat recombinant IL-1R antagonist(Anakinra)(R&D Systems)was dissolved in 0.67ml of sodium chloride 0.9%and given subcutaneously immediately and then daily after surgery;V:MI +A-740003+Anakinra(n = 28):The dose and injection manner of A-740003 and Anakinra were the same as previously described.At 3 and 7 days post-MI,rats were sacrificed.At 3 days post-MI,the expression of P2X7R,NLRP3,caspase-1 and IL-1β were confirmed by Western blot.At 7 days post-MI,before sacrificing,we applied the volume-pressure system to measure the hemodynamic indexes;in addition,we applied the programmed electrical stimulation to measure the ventricular arrhythmia susceptibility;then after sacrificing,we measured macrophage infiltration by immunochemistry staining of CD68;in addition,we double stained CD68 and NGF to determine the level of macrophage mediated NGF production and measured the sum protein level of NGF in the infarcted border zone by Western blot.At last,we measuredsemi-quantitative sympathetic innervation withimmunofluorescence staining of tyrosine hydroxylase(TH)and growth-associated protein(GAP43).Results(1)In the heart tissue of lipopolysaccharide(LPS)-primed naive rats,we observed that LPS priming alone significantly increased NLRP3 levels in naive rats but failed to induce the robust production of cleaved caspase-1 and mature IL-1β.3’-O-(4-benzoyl)benzoyl adenosine 5’-triphosphate(BzATP),a P2X7R agonist,induced caspase-1 activation and mature IL-1β release,which was further neutralized by a NLRP3 inhibitor(16673-34-0).(2)Our data of immunofluorescence double staining showed that in the infarcted myocardium tissue,the activatedP2X7 was mainly co-localized with infiltrated macrophages.Western blot analysis showed thatthe MI model was associated with a marked activation of P2X7-NLRP3 inflammasome and IL-1β in the left ventricle,while administration of A-740003(P2X7R antagonist)significantly prevented the increase of NLRP3/IL-1β.Immunohistochemistry data showed that A-740003 and/or Anakinra(IL-1 receptor antagonist)significantly reduced macrophage infiltration and NGF positive macrophages and NGF protein levels.Eventually,immunofluorescence semi quantitative of TH and GAP43 showed that sympathetic hyperinnervation was blunted to a similar degree by A-740003 and A-740003 +Anakinra,moreover,usage of Anakinra partly attenuated sympathetic spouting,suggesting that efficacy of P2X7 on neural remodeling was mediated,at least partly by IL-1 p.(3)In in-vitro studies,immunofluorescence double staining revealed that P2X7R was co-localized with both M1 and M2 macrophages.Western blot analysis revealed that BzATP stimulation could further increase NGF and IL-1β expression in M1 macrophages,which was blunted by IL-1β inhibitor Anakinra.In contrast,the activation of P2X7R could not increase the expression of either NGF and IL-1β.ConclusionTogether,these data implicate thatBzATP upregulated the surface expression of NGF in M1 macrophages via IL-1β.Pharmacological inhibition of P2X7R protect ventricular arrhythmia by attenuating sympathetic innervation via NLRP3/IL-1βpathway following MI.Therapeutic interventions targeting P2X7 pathway may constitutea novel approach to prevent infarction injury.