Cardio Troponin I Antibody Induce Cardiac Dysfunction By Combination With Myocardial Cell Membrane α-enolase And Activation Of PTEN/Akt Signaling Pathway

Cardiovascular disease(CVD)is an important disease which seriously endangers human health. Heart failure(HF)is the end stage of various kinds of cardiovascular diseases which main pathophysiological mechanism is ventricular remodeling. In recent years, despite as with the development of early diagnosis and treatment, the incidence of cardiovascular disease is on the decline, the cure rate of acute stage patients also increased significantly, but there are still a considerable number of patients with progressive left ventricular remodeling and chronic congestive heart failure. It is necessary to improve the prevention and cure effect of cardiovascular disease through to delve into the immunological pathogenesis of left ventricular remoding and heart failure.Autoimmune mechanism is the important risk factors of autoimmune myocarditis, myocardial fibrosis and pathological remodeling, but its pathophysiological mechanism is still unclear. Intracellular protein of myocardial rarely contact with immune system during embryo phase which is referred to “covered antigen”, such as myocardial myosin, myocardial actin, myocardial troponin and so on. When myocardial cells are injuried, Intracellular protein of myocardial released into the blood circulation and may stimulate the immune system to produce antibodies against myocardial.We and other researchers found that autoantibodies against cardiac troponin I(c Tn I) have been identified and participate in ongoing myocardial damage that eventually results in heart failure. Researches showed that cardiac troponin I autoantibody(c Tn IAAb) can not only negative interference c Tn I detection system, but also influence the judgement of patients’ condition and may also cause continuous damage of cardiac function. c Tn IAAb may combined with c Tn I expressed on myocardial cell membrane and cause cardiac systolic dysfunction. It is still unknown weather c Tn IAAb is the leading cause of heart dysfunction or only the mark of myocardial damage and how c Tn IAAb combine with cardiac sarcolemmal. The relationship between c Tn IAAb and the prognosis of patients need further research.In 2012, we noticed that acute myocardic infarction(AMI) patients with c Tn IAAb positive have worse prognosis when we study the positive rate of c Tn I autoantibody in AMI patients and the negative interference in c Tn I detection system. One AMI patient with the highest c Tn I autoantibody titer died in hospital because of repeated myocardial infarction and heart failure. On this basis, we screen c Tn IAAb in 131 AMI patients and evaluate the relationship between c Tn IAAb and left ventricular remodeling in this study. Then we choice 10 AMI patients with positive c Tn IAAb and left ventricular remodeling[LVESV( Left ventricular end systolic volume) increased more than 15%]. Using CNBr-activated Sepharose 4b affinity chromatography column coupled with recombinant human c Tn I protein expressed in E.coli, we obtained human c Tn IAAb that mostly cause cardiac injury. We prepared 3 homemade cardiac troponin I monoclonal antibodies(c Tn Im Abs:c Tn Im Ab N1、c Tn Im Ab N2、c Tn Im Ab N3) and explore the pathogenic mechanisms of cardiac dysfunction caused by c Tn IAAb direct contact with the myocardial cell membrane.In this study,we used cardiac troponin T polyclonal antibody(c Tn Tp Ab), cardiac troponin T monoclonal antibody(c Tn Tm Ab) and Immunoglobulin G(Ig G) as control groups. This study consistde of six parts:The first part: One-year follow-up study to evaluate the relationship of c Tn IAAb and left ventricular remodeling in AMI patients. Serum sample was collected from AMI patients with positive c Tn IAAb and left ventricular remodeling which is for further study of c Tn IAAb.This section study included 131 AMI patients at two hospital. Serum samples were obtained. c Tn IAAb were detected by indirect enzyme-linked immunosorbent assay. Echocardiographic assessments were performed at asmission and following about 1 year. The experiment results of this section showed that the positive rates of c Tn IAAb in 131 AMI patients was 17.56%(23/131). 90 of 131 AMI patients tracked the cardiac ultrasound data [19( c Tn IAAb positive) VS 71(c Tn IAAb negative)] and 19 AMI patients appeared left ventricular remodeling. Two classification logistic regression revealed that both peak BNP(OR:1.001, 95%CI:1.000-1.002, p:0.028) and c Tn IAAb(OR:3.552, 95%CI:1.148-10.989, p:0.028) were predictive of left ventricular remodeling. 19 AMI patients with left ventricular remodeling included 10 c Tn IAAb positive AMI patients and 9 c Tn IAAb negative AMI patients. Serum samples from 10 AMI patients with c Tn IAAb positive and left ventricular remodeling can be used for follow-up studies.The second part: c Tn I affinity chromatography was used to purify c Tn IAAb. The purified protein was obtained from serum samples from 10 AMI patients with c Tn IAAb positive and left ventricular remodeling. IF, IHC and Western Blotting were used to prove the purified protein can specificity combine with c Tn I. The experiment results of this section showed that 2.24 mg purification protein(0.8ml, 2.8mg/ml) was obtained from 36 ml serum samples. The purified protein was separated by SDS-PAGE.There are two bands at 20 KD and 50 KD stained by Coomassie Brilliant Blue which match with the bands of immunoglobulin. Using IF, IHC and Western Blotting tests, we proved that the purified protein can not only bind with c Tn I of human, rat and mice but also bind to cardiac sarcolemmal. Since the purified protein was obtained from 10 AMI patients with c Tn IAAb positive and left ventricular remodeling using c Tn I affinity chromatography, we speculated that the c Tn IAAb may play a pathological role in left ventricular remodeling according to other literatures reported that c Tn IAAb associated with cardiac dysfunction and poor prognosis after AMI. Howere, we need more laboratory evidences to validate this scientific hypothesis.The third part: Study the hypertrophy, apoptosis and autophagy of neonatal rat cardiac myocytes(NRVM) co-cultured with c Tn IAAb in virto. The experiment results of this section showed that autophagy-related genes Atg5 and Becn1 m RNA and t autophagy-related proteins LC3 II and Beclin-1 were significantly upregulated when NRVM co-cultured with c Tn IAAb in virto after 12 h, then significantly downregulated after 24 h. Following this, apoptosis related proteins Bax and Bcl2 changed after 24 h,then Bax and Bcl2 changed more obvious after 48 h. With the co-cultured time passing by, pro-apoptotic protein Bax gradually upregulated whereas anti-apoptosis protein Bcl2 gradually downregulated in the group of NRVM co-cultured with c Tn IAAb. During the whole process, NRVM co-cultured with c Tn IAAb appeared reciprocal balance between autophagy and apoptosis whereas with batch equilibrium experiments, the same dose of c Tn Tp Ab control and human Ig G control had no these changes. Howere we didn’t observe NRVM hypertrophy in this section study. The experiment result of this section shows that c Tn IAAb was a kind of harmful stimuli to NRVM. On the basis of these experimental results, here came several new questions. Firstly, what is the myocardial cell membrane protein which combined with c Tn IAAb? Is it c Tn I? Secondly, what is the mainly antigen epitopes of c Tn AAb which induced autophagy and apoptosis in NRVM? We focused on these new questions in next step research.The fourth part: Choose a c Tn Im Ab for follow-up study which can replace c Tn IAAb. Analysis the antigen epitopes of 3 homemade c Tn Im Abs(c Tn Im Ab N1, c Tn Im Ab N2, c Tn Im Ab N3) and find out the best c Tn Im Ab that can combine with the membrane protein of myocardial cell and some cell lines.Then purify and identify the cell membrane protein which can combine with c Tn Im Ab. The experiment results of this section firstly showed that the purified human c Tn IAAb mainly bind with cardiac sarcolemmal at a.a.r.26-49 of c Tn I. The antibody c Tn Im Ab N1 can reduce the binding rate of c Tn IAAb with the membrane protein of myocardial cell and BCG823 cell line. Then we firstly confirmed that the target antigen binding with c Tn Im Ab N1 is α-enolase(ENO1) but not c Tn I using c Tn Im Ab N1 affinity chromatography, flight mass spectrometry, immunoprecipitation and Western Blotting. At next section experiments, we will focus on the biological changes of NRVM cocultured with c Tn Im Ab N1 in virto and the relationship between ENO1 and the apopotosis of NRVM. These scientific problems shoule be solved at next section experiments.The fifth part: Study whether the pathological changes of NRVM caused by c Tn Im Ab N1 are the same as c Tn IAAb. What kind of changes did ENO expression and pathological changes appear in NRVM since c Tn Im Ab N1 can combine with ENO1 on cell membrane? The experiment results of this section showed that we successfully used c Tn Im Ab N1 instead of c Tn IAAb and induced time- and dose-dependent apoptosis. Meanwhile, ENO1 expression significantly upregulated consistent with the increasing apoptosis when NRVM co-cultured with c Tn Im Ab N1 whereas ENO1 gene silence improved the apoptosis. On the other hand, the batch equilibrium experiments, the same dose of c Tn Tp Ab control and human Ig G control had no these changes. The experiment results of this section showed that myocardial apoptosis caused by c Tn IAAb not only associated with the dominant epitope binding to c Tn IAAb on cardiac sarcolemmal, but also the concentration and duration of c Tn IAAb. These experiment results may explain why there was a clear cause-and-effect relationship between c Tn IAAb and heart function damage in animal model, but the pathogenetic role of c Tn IAAb in dilated cardiomyopathy(DCM) and ischemic cardiomyopathy(IHD) had always been controversial. In this section. c Tn Im Ab N1 reappear the biological effect of c Tn IAAb in virto through ENO1 up regulation in NRVM. But it is still unknow whether c Tn Im Ab can induce heart damage in animal model through inflammatory infiltration or apoptosis. What was the relationship between pathologic changes and ENO1 expression in vivo? Which signaling pathway did mediate these pathological changes? These scientific problems shoule be solved at next section experiments.The sixth part: Induce myocardial injury model in Balb/c mice by intraperitoneal injection c Tn Im Ab N1 instead of c Tn IAAb. Observe the changes of heart tissue and preliminary study the signal pathways and molecular mechanisms in order to explain the relationship and pathogenetic mechanism between c Tn Im Ab/c Tn IAAb and heart injury.We hope these experiment results can provide a further evidence for the involvement of autoimmunity heart disease and help to find new targets and strategies for the prevention and treatment of ventricular remodeling after AMI. The experiment results of this section, using echocardiography and cardiac catheter, we confirmed that cardiac systolic dysfunction can be successfully induced in Balb/c model mice by injection c Tn Im Ab N1. Using HE,masson and Tunel stain, we found calcified plaque composited in epicardium, myocardial collagen deposition and myocardial cell apoptosis increased whereas heart shape and myocardial cell size were not hypertrophy.These results were in accordance with the third part experiment results. Then using IHC, we found the function and pathological changes of group I mice injected c Tn Im Ab N1 were compensatory and non-specific inflammatory because there were not CD3,CD8,CD4,CD20,CD68 lymph mononuclear cell infiltrated in heart tissue. Using Path Scan Akt Signaling Antibody Array and Western Blotting, we found c Tn Im Ab N1 activated PTEN- Akt signaling pathways by PTEN Ser380 increased and Akt Thr308 decreased accordance with high expressed of α-enolase in vivo and in vitro. As we knew that inactivation of PTEN results in hypertrophy, PTEN Ser380 up regulation in our study consist with the results in third and sixth parts which we didn’t find myocardial cell hypertrophy in group I.When we silence gene in myocardial cell by si-RNA targeted to α-enolase, PTENAkt signaling pathway is not actived and myocardial cells apoptosis decreased.In conclusion, in this study we give the solid evidence that c Tn AAb is one of the independent risk factor in left ventricular remodeling after AMI. The c Tn Im Ab against c Tn I N-terminal antigen sites 26-49(c Tn Im Ab N1)is the main part of c Tn IAAb. c Tn Im Ab N1 activated PTEN- Akt signaling pathways by PTEN Ser380 increased and Akt Thr308 decreased with high expressed of α-enolase through c Tn Im Ab N1 cross react with α-enolase expressed on myocardial cell membrane in vivo and in vitro. c Tn Im Ab N1 cause time- and does-dependent myocardial apoptosis but not myocardial hypertrophy whereas silence α-enolase of myocardial cell can improve the myocardial apoptosis in virto. Then c Tn Im Ab N1 successfully induced the Balb/c mice model character as heart systolic dysfunction, increased myocardial collagen deposition, calcified plaque formation and increased myocardial cell apoptosis. When we silence gene in myocardial cell by si-RNA targeted to α-enolase, PTEN-Akt signaling pathway activation and myocardial cells apoptosis were decreased. In conclusion, This study is an important supplement to pathogenesis of autoimmune heart disease and provides strategy to the clinical intervention autoimmune heart disease and autoimmune injury after myocardial injury.In this study, the patients’ serum and organizations were wastes from clinical laboratory and approved by Changhai Hospital Ethics Committee and The People’s Liberation Army 94 Hospital Medical Ethics Committee. Experimental animals were approved by The Second Military Medical University Animal Care and Use Committee...