Complement-coagulation System Interaction Is Involved In Basic And Clinical Translational Research On The Occurrence And Development Of Arrhythmogenic Right Ventricular Cardiomyopathy

Part 1:Activation expression of myocardial complement system in patients with arrhythmogenic right ventricular cardiomyopathy(ARVC)Background:Arrhythmogenic right ventricular cardiomyopathy(ARVC)is an inherited cardiomyopathy characterized by malignant ventricular arrhythmia and sudden cardiac death(SCD),and it is one of the important causes of SCD in young people under 35 years old.The featured pathological change of ARVC is the diffuse or segmental fibro-fatty replacement of functional myocardial tissue,with or without inflammatory cell infiltration.The main objective of clinical treatment of ARVC is the prevention of arrhythmia and SCD.However,there is a lack of treatment targeted at important molecules involved in the progression of ARVC.Our team has reported that significant infiltration of inflammatory cells exists in more than 70%of AC heart tissues in the Chinese population.Complement is an important active component in inflammation,and the intervention of the complement system with small-molecule drugs or genetic methods can inhibit the inflammatory response and pathological remodeling of cardiac tissue in the mice model of ARVC(Des-/-).However,there has been no systematic study on complement activation in myocardial tissue of ARVC patients.Objective:To explore the specific activation status of the complement system in ARVC patients.Methods:The complement system-related proteins were quantified in right ventricular tissues from four patients with ARVC,four patients with dilated cardiomyopathy(DCM),and four normal discarded donor hearts,using a high-resolution quantitative proteomics technique called Tandem Mass Tags(TMT).Then we used several conventional experimental techniques,including immunoblotting,quantitative real-time polymerase chain reaction(RT-qPCR),and immunohistochemical staining,to verify the results of TMT in another cohort which included sixteen patients with ARVC,sixteen patients with DCM,and sixteen normal discarded donor hearts.Results:Using TMT technique,we found that several complement system-related proteins were significantly activated in right ventricular tissues from ARVC patients compared with normal controls(C3,Fold change=1.74,P=0.008;C6,Fold change=1.52,P=0.001;C7,Fold change=1.57,P=0.001;C8,Fold change=1.53,P=0.005;C9,Fold change=1.78,P=0.0001).The DCM patients that we enrolled could be considered as heart failure controls because they are accompanied with lower left ventricular eject fraction compared with ARVC patients(25.00±2.45%VS.47.53±4.87%).To exclude the possibility that complement activation was a common feature of myocardial tissues from heart failure patients,we compared the complement system-related proteins expression in myocardial tissues from ARVC patients and DCM patients,and we found significantly higher expression of complement system-related proteins in ARVC patients(C3,1.74±0.33 VS.1.09±0.11,P=0.018;C6,1.52±0.14 VS.1.20±0.17,P=0.041;C7,1.57±0.18 VS.0.98±0.06,P=0.002;C8A,146±0.10 VS.0.97±0.24,P=0.019;C9,1.78±0.15 VS.1.48±0.12,P=0.034),suggesting that complement system-related proteins were specifically activated in right ventricular tissues form ARVC patients.In the validation cohort,we identified several specifically activated complement system-related proteins in ARVC patients using immunoblotting,including Factor B(Fold change=1.795,P<0.001),ASP(Fold Change=4.194,P<0.05),C5b-9(Fold change=1.585,P<0.001),C5aR(Fold change=4.167,P<0.05).By RT-qPCR,we revealed elevated expression of complement-related receptor genes,such as C5aR and C5L2,in the ARVC group.Besides,immunohistochemical staining results showed increased deposition of complements(C5aR,C5b9,and Factor B)in myocardial tissues from ARVC patients compared with normal controls,and the complements deposit was more likely to be observed around areas with obvious fibro-fatty replacement.Conclusion:The complement system is specifically activated in myocardial tissues from ARVC patients.Part 2:Complement-coagulation system crosstalk promotes ARVC disease progressionBackground:Arrhythmogenic right ventricular cardiomyopathy(ARVC)is an inherited cardiomyopathy,characterized by malignant arrhythmia and right ventricular(partial left ventricular involved)dysfunction.Through quantitative proteomics,we found the complement system(C3,C6,C7,C8 and C9)was significantly activated in the cardiac tissue of ARVC.The complement system plays a role mainly through three activation pathways(classical-,alternative-and lectin pathway),but at present,none of the above-mentioned pathway interventions could improve clinical outcomes in clinical trials.In 2006,huer-lang M et al.found that the complement system can also be activated by the coagulation system:in C3 knockout mice,the coagulation factor thrombin can cleave C5,resulting in subsequent cascade activation of C5-9 complement factors.However,the detailed mechanism of complement-coagulation system interaction in ARVC myocardial tissue has not been fully revealed.Objective:This study aims to explore the crosstalk between the coagulation system and the complement system,as well as its role in tissue inflammation and pathological remodeling in ARVC.And,we wanted to confirm thrombin was a specific intervention target for the pathological progress of ARVC disease.Methods:Firstly,the proteomics(Part 1)data was used to analyze the expression levels of coagulation factors(Tissue factor,Factor VIII,Factor IX,Factor X,Factor XII,Fibrinogen,Thrombin,etc.)in the myocardial tissue of ARVC.In the validation cohort 16 ARVC,16 DCM and 16 Normal donors),molecular experiments,such as western blotting and immunostaining,confirmed the specific expression activation of the coagulation system in cardiac tissue of ARVC.Then,the disease phenotype of ARVC mouse model(Desmin knockout,Des-/-)was identified by cardiac ultrasound,electrocardiogram,HE/Masson pathological stain and immunostaining.The Des-/-mice were crossed with the purchased C3 gene knockout mice to obtain Des-/-C3-/-double knockout mice(inhibiting the three traditional activation pathways of complement system).The mice(Des-/-C3-/-,Des-/-,WT)were raised for a long time,and their survival conditions were observed and recorded.The cardiac function and pathological injury were evaluated at 4 months and 12 months respectively.Immunohistochemistry(IHC)and enzyme-linked immunosorption assay(ELISA)were used to analyze the levels of complement expression in cardiac tissue(C5)and peripheral blood(C5a)of Des-/-C3-/-mice,confirming the existence of other complement activation pathways in ARVC myocardial tissue.Furthermore,IHC were used to investigate the expression characteristics of coagulation factor(fibrinogen,thrombin)in different genotypes of mice.Finally,thrombin-specific small molecule inhibitor,lepirudin,was intraperitoneally injected into mice of various genotypes,and the expression levels of complement in the myocardial tissue of the mice and the changes of the myocardial pathological damage were analyzed.Results:After adjustment for protein expression level of normal,quantitative proteomics suggested that specific expression activation of coagulation factor in cardiac tissue of ARVC patients(ARVC VS.DCM:Factor XIII A,Fold change 1.27±0.26 VS.0.79±0.10,P=0.026;Fibrinogen,Fold change 1.77 ± 0.23 VS.1.44 ± 0.38,P=0.026;Thrombin,Fold change 1.69 ± 0.004 VS.1.09±0.21,P=0.004).Western Blot was also used to confirm the activated expression of fibrinogen and thrombin in ARVC myocardial tissue in the validation cohort(16 ARVC,16 DCM,16 normal donors)(Fibrinogen Fold change=2.201 ± 0.21,P<0.001;Thrombin Fold change=1.664 ±0.24,P<0.05).Compared with WT mice,the left ventricle of Des-/-mice is significantly expanded(WT VS.Des-/-,LVESD(mm),1.842±0.1114 VS.2.833±0.3436,P=0.0208),and cardiac contractility significantly decreased(EF(%),82.18±2.685 VS.59.14 ± 8.418,P=0.0262)).The myocardium of Des-/-mice showed obvious fibrosis,with a activation of lipid deposition and lipogenesis related genes.The deposition of cardiac complement increased in ARVC mice(Des-/-),mainly concentrated in the areas with severe lesion.C3 gene knockout in Des-/-mice resulted in the deterioration of disease phenotype:the mortality rate was significantly increased(C3-/-Des-/-VS Des-/-,16.6%(13/78)VS 4.3%(3/70),P=0.0041),the left ventricular systolic function(LVFS,%)was significantly decreased(23.38±1.97 VS 27.39±0.98,P<0.05)during the 8-13 month.And the degree of pathological damage of myocardial tissue increased by 18%.IHC staining of C5 was performed on the myocardial tissue of C3-/-des-/-mice,and the deposition was significantly increased.ELISA was used to detect the concentration of C5a/C5adesArg,and it was found that the plasma expression level of C3-/-des-/-was significantly higher than that of WT(5.5±0.82 nM,n=4 vs.0.56±0.12 nM,n=6),indicating that there were other complement activation pathways with different traditional three activation pathways in ARVC myocardial tissue.By intraperitoneal injection of lepirudin in Des-/-mice(PBS was used in the control group),the pathological features(replacement index)of myocardial tissue fibrosis,calcification deposition,and inflammatory cell infiltration in Des-/-mice decreased by 25.5%.The expression level of C5 protein also decreased significantly(P<0.01).This indicates that in ARVC myocardial tissue,the coagulation system can promote the activation of the complement system and lead to the pathological progression of the disease.Conclusions:The complement system and coagulation system is activated in cardiac tissue of ARVC.And the coagulation factor,thrombin,can activate complement system to mediate myocardial pathologic injury in ARVC myocardial tissue.The crosstalk between coagulation and complement system may be one of the therapeutic targets of ARVC.Part 3:Activation of complement and coagulation system predicts poor prognosis in patients with ARVCBackground:ARVC is a type of hereditary cardiomyopathy with malignant ventricular arrhythmia and heart failure as the main clinical manifestations.At present,clinical risk classification of ARVC patients is mainly based on cardiac magnetic resonance imaging and cardiac ultrasound.There is no blood biomarker that can effectively reflect the disease process and predict the risk of malignant cardiovascular events.Based on proteomics and molecular biology experiments,the first two parts confirmed the activation of complement-coagulation system in ARVC myocardial tissue and may be involved in ARVC disease progressionObjective:To explore the correlation between plasma complement-coagulation factors and the risk of disease progression and clinical prognosis risk in patients with ARVCMethods:Plasma of 87 patients with ARVC,24 patients with DCM and 63 healthy volunteers were collected,and their clinical data(including baseline data,blood test,echocardiography,electrocardiogram,etc.)were collected at the same time.The concentrations of plasma complement and coagulation factors were detected by enzyme-linked immunosorbent assay(ELISA).Spearman’s test was used to analyze the correlation between plasma complement-coagulation factor concentration and clinical characteristics of ARVC patients.Cardiovascular adverse events during the follow-up period of ARVC patients were defined as death,heart transplantation and in the waiting list for heart transplantation.The patients were grouped according to the occurrence of cardiovascular adverse events during the follow-up period,and the plasma complement expression levels between the groups were compared.ROC diagnostic test was used to find the optimal cut-off value,and Kaplan-Meier curve was further used to analyze the difference in the incidence of cardiovascular adverse events between the groups during the follow-up period(Log rank test was used to calculate the significance of the difference between the groups).Results:The age of ARVC patients matched the normal control(38.59±1.62 VS.42.57 ±1.72),sC5b9(314.28 ± 19.11 VS.244.92±10.97 VS.221.43±33.96,P=0.004),thrombin 301.1±86.47 vs.15.62±4.95 vs.87.13±21.55,P=0.001)in plasma of ARVC patients was significantly higher than normal controls and DCM patients.DCM serves as a positive control for heart failure,indicating that complement and coagulation factors in plasma are specifically increased in ARVC patients.Based on Spearman’s correlation test,the concentration of plasma sC5b9 was significantly correlated with the baseline major arrhythmia event(MACE)and the incidence of atrial fibrillation in ARVC patients(sC5b9～MACE at baseline,P=0.033;sC5b9～AF(p=0.457,P=0.001)).The sC5b9 concentration showed a negative correlation with left ventricular ejection fraction(LVEF,p=-0.306,P=0.004)and right ventricular ejection fraction(RVEF,p=-0.300,P=0.045),and sC5b9 increased with the right ventricle inner diameter expansion(RV ESV,p=0.336,P=0.024;RVID,p=0.427,P<0.001),indicating that sC5b9 was associated with ventricular systolic function and tissue remodeling in ARVC patients.During an average of 17.30 ± 9.51 months(Mean±SD)of clinical follow-up,21 patients with ARVC underwent adverse events,including 15 heart transplants,5 deaths,and 1 in the waiting list for heart transplants.The plasma complement factor concentration of ARVC patients with adverse events was significantly higher than that of patients without adverse events:sC5b9(434.55±32.17 vs.276.01±21.02,P<0.001).The patients were grouped based on cutoff value found by ROC,and Kaplan-Meier curve analysis was used to find significant differences between the groups(sC5b9=356 ng/ml,Log Rank P=0.0036).Conclusions:Plasma complement can be used as a biomarker for the severity of clinical symptoms(arrhythmia,ventricular contractile function,and structural remodeling)and clinical prognosis risk in ARVC patients.