| Background and objectives:Viral myocarditis (VMC) is characterized by myocyte necrosis and degeneration with mononuclear cells infiltration. It is one of the most common acquired susceptible myocardial diseases among children. The clinical manifestation ranges from the asymptomatic state due to the focal inflammation to the fatal congestive heart failure due to diffuse myocarditis. Most patients with VMC can completely recover, while some may develop dilated cardiomyopathy(DCM) and manifest cardiac dilation and heart failure. There is no specifically good effect therapy for it, although patient could survive for some time after immunosuppressive therapy. Performing cardiac transplantation is still the only way at the end stage of the disease. Thereafter, VMC and DCM have become a major cause of morbidity and mortility among children and adolescent.To date, the pathogenesis of VMC developing into DCM remains unclear. Previous researches have revealed that the development of VMC to DMC may result from the direct invasion of virus, the autoimmune reaction and the persistent exist of the virus. Autoimmune injury induced by the infection of virus may play the most important role in the process. So it becomes a worldwide focus. Injection of porcine cardiac myosin into susceptible strains of rats and mice has been shown to cause experimental autoimmune myocarditis(EAM), which resembles myocarditis in humans and can develop into DCM. So EAM is an excellent experimental model for studying the pathogenesis of VMC developing into DCM and the effective therapy. Domestic reports in this field are still relatively rare.Because the pathogenesis of myocarditis remains unclear, treatment is not direct at the diseases themselves but instead of managing the symptoms and complications, such as congestive heart failure, cardiogenic shock, conduction abnormalities and arrhythmia. Recently, because evidences suggest that autoimmune response plays important role in the development of myocarditis, immune therapies are being taken more and more attention for the treatment of myocarditis. However, their effects are still controversial. Autoimmune myocarditis is a T-cell-mediated autoimmune disease. CD4-positive T cells are believed to be the most important for the initiation and mediation of the disease. They are responsible for the production of most of the cytokines that are necessary to stimulate an immune response. Injection of anti-CD4 monoclonal antibody in vivo can disturb the function of CD4 positive T cells through combining with CD4 molecule and induce immune tolerance to the given antigen. It has been reported to be effective in some autoimmune disease. However the effects of anti-CD4 monoclonal antibody have not been studied in EAM.In the present study, cardiac myosin was separated from porcine heart and being successfully purified. Then the EAM rat model was established by injecting cardiac myosin twice. In order to reveal the function of autoimmune injury in the development of EAM, the dynamic changes of inflammation and cardiac function and the relationship between them were observed in detail. In addition, Anti-CD4 monoclonal antibody was used as an immune modulation agent for the first time in the treatment of EAM. It was aimed at evaluating whether anti-CD4 monoclonal antibody could induce immune tolerance to cardiac myosin in EAM and whether the immune tolerance to cardiac myosin could protect rats with EAM from myocardial injury. Thus, the role of autoimmune injury in the development of VMC to DCM was explained in detail. In addition, the present study provide new strategy not only for the treatment of VMC and DCM but also for the treatment of other autoimmune diseases.Methods:1. The preparation, identification and the activity analysis of porcine cardiac myosin(1)The crude myosin was separated from porcine heart by different concentration of potassium buffer, ultra centrifugation, (NH4)2SO4 fraction, et al.(2)The crude cardiac myosin was purified using the method of DEAE-Sephadex A 50 chromatography.(3)Protein concentration was determined by Bradford method.(4)The protein was cursorily identified by polyacrylamide gel electrophoresis (PAGE).(5)The ATPase activity of the protein was measured by the method of Fiske-Subbarow.(6)The accurate identification of the protein was performed by mass spectrum analysis.2. The establishment of Lewis rat EAM model and treatment with anti-CD4 monoclonal antibody104 Lewis rats were randomly divided into six groups which include normal group, control group, myosin-immunized group, saline-treated group, IgG-treated group and anti-CD4 monoclonal antibody treated group. On days 0 and 7, a total volume of 0.2ml emulsion containing 1mg cardiac myosin was injected subcutaneously in each rear footpad of rats in myosin-immunized group, saline-treated group, IgG-treated group and anti-CD4 monoclonal antibody treated group. The same volume of emulsion without cardiac myosin was injected to rats in the control group at the same manner as a control. Immune tolerance was induced by injection of 4mg/kg anti-CD4 monoclonal antibody on days -2, -1, 0, 1. Anti-CD4 monoclonal antibody was replaced by IgG or saline in IgG-treated group and saline-treated group respectively as control.3. Observation and sample collectionRat serum samples were obtained before immunization from each group through the vein of tail. After immunization, clinical symptoms were observed every day. Then, on days 18, 30 and 49 after the first immunization, echocardiography was performed. After echocardiography examination, serum samples were obtained through vena cava inferior. Then rats were sacrificed and hearts were obtained immediately in asepsis, weighted and stored for further use. Spleens were also obtained on day 18 after the first immunization and used to perform lymphocyte proliferation assay immediately.4. Sample examination:(1)Echocardiography was performed to evaluate the cardiac structure and cardiac function of the rats.(2)Heart samples were stained with H-E and Masson's trichrome. Inflammation and fibrosis was examined by light scope. Pathologic score was calculated.(3)The dynamic changes of the serum titer of anti-cardiac myosin autoantibody were tested by Enzyme-linked immunosorbent assay (ELISA).(4)Lymphocyte proliferation to cardiac myosin was observed by lymphocyte proliferation assay in vitro.(5)The serum concentrations of Th1-typing cytokines including IFN-γand IL-2 and Th2-typing cytokines including IL-6 and IL-10 were analyzed by ELISA(6)The mRNA expression of Th1-typing cytokines including IFN-γand IL-2 and Th2-typing cytokines including IL-4 and IL-10 in the hearts were examined by reverse transcription polymerase chain reaction (RT-PCR).Results:1. The yield of crude cardiac myosin was 15mg/g, while the yield of purified cardiac myosin was 10mg/g.2. The specific activity of the Ca2+-activiated ATPase of the crude protein was 0.19Piμmol/(mg.min), while its activity was 0.22Piμmol/(mg.min) after purification.3. The purity of the protein was analyzed by SDS-PAGE throughout the process of preparation. Results showed that cardiac myosin was gradually separated from other proteins. The SDS-PAGE chart of the crude protein showed three bands. Their molecular weights were the same as the light and heavy chains of standard cardiac myosin. The SDS-PAGE chart of the purified myosin has no difference with the crude protein.4. The ratio of A280/A260nm of crude myosin was 0.52, while it became 1.45 after purification, which meaned that the content of nuclear acid significantly decreased.5. In the present study, native-PAGE was used to identify the protein further. Results showed that there was one band in the native-PAGE raceway and three bands in the SDS-PAGE raceway, which were suggested to be representative of the non-degenerated and the degenerated myosin respectively.6. When the standard myosin and prepared myosin were estimated in one SDS-PAGE, results showed that their bands are approximately the same. However, there were impurity protein in the raceway of the standard myosin, and there was scarcely any contamination with impurity protein in the raceway of prepared myosin.7. The band in the raceway of the active-PAGE was identified further by mass spectrum. Results showed that it was cardiac myosin.8. Changes of heart structure and cardiac function of EAM rats: On day 18 after the first immunization, all rats in EAM group showed massive pericardial effusion, both IVS and LVPW were significantly increased, while LVDd and LVFS were significantly decreased, relative to the values in normal and control group, which indicated an acute stage. On day 30 after the first immunization, pericardial effusion disappeared. Although IVS and LVPW in EAM rats were reduced on day 30 after the first immunization, they were still higher than those of controls. LVFS in EAM rats increased, although they were still significantly lower than those of controls, which indicated a subacute stage. On day 49 after the first immunization, the wall thickness in EAM rats including IVS and LVPW was rather thin and the ventricle was then dilated. LVFS was significantly decreased in comparison to control group after a transient improving in subacute stage, which indicated a chronic stage.9. Changes of histopathology in EAM rats: On day 18 after the first immunization, all rats in EAM group developed typical severe autoimmune myocarditis. H-E staining showed that large numbers of inflammatory cells infiltrated all layers of ventricular wall, and degeneration and necrosis were also evident. On day 30 after the first immunization, the number of inflammatory cells was reduced. On day 49, there were few inflammatory cells found in myocardia. Masson's trichrome staining showed that there were dense collagen fibers surrounding the surviving myocytes on days 18, 30 and 49 after the first immunization.10. Changes of the serum titer of anti-cardiac myosin autoantibody in EAM rats: On days 18 and 30 after the first immunization, high titers of the autoantibody were tested, while on day 49 after the first immunization, the titer of the antibody was significantly decreased.11. Changes of the serum concentrations of Th1-typing cytokines including IFN-γand IL-2 and Th2-typing cytokines including IL-6 and IL-10 in EAM rats: Th1-typing cytokines were significantly elevated in EAM rats throughout the disease, showing their peak on day 18, and decreased on days 30 and 49. Interestingly, the serum level of Th2-typing cytokine IL-6 and IL-10 showed their peak on day 30 after the first immunization.12. Changes of mRNA expression of Th1-typing cytokines including IFN-γand IL-2 and Th2-typing cytokines including IL-4 and IL-10 in hearts of EAM rats: Compared to control group, the mRNA expressions of IFN-γand IL-2 were significantly increased in myocardia of rats with EAM. Their maximal mRNA expressions were examined on day 18 after the first immunization, and decreased along with the prolongation of the time after EAM. The expressions of IL-4 and IL-10 mRNA in myocardia of rats with EAM had been increased on day 18 after the first immunization and reached their peak on day 30 after the first immunization.13. The correlations between LVFS and cytokines: LVFS was negative correlated to Th1-typing cytokines and positive correlated to Th2-typing cytokines on days 18 and 30 after the first immunization (P<0.01). However, on day 49, there were no correlations between LVFS and serum levels of Thl and Th2-typing cytokines(IFN-γ: r=-0.082, IL-2: r=-0.117, P>0.05; IL-6: r=0.436, IL-10: r=-0.031, P>0.05). Also, the correlations between LVFS and mRNA expression of Thl and Th2- typing cytokines in the hearts of EAM rats had no statistic significance(IFN-γ: r=0.456, IL-2: r=-0.459, P>0.05; IL-6: r=0.229, IL-10: r=-0.008, P>0.05). Interestingly, on day 49 after the first immunization, the serum level of Th1-typing cytokine IFN-γwas negative correlated to fibrosis scores (r=-0.831, P<0.05), while Th2-typing cytokine IL-10 was positive correlated to fibrosis scores (r=0.851, P<0.01). As far as mRNA expressions of the cytokines in the hearts were concerned, Th1-typing cytokine IFN-γwas also negative correlated to fibrosis scores (r=-0.751, P<0.05), while Th2-typing cytokines IL-4 and IL-10 were positive correlated to fibrosis scores (IL-4: r=0.876, P<0.01 IL-10: r=0.726, P<0.05)14. Immune tolerance induced by anti-CD4 monoclonal antibody on day 18 after the first immunization:(1)Suppression of lymphocyte proliferation by anti-CD4 monoclonal antibody in vitro: Lymphocytes obtained from rats in saline-treated group and IgG-treated group had remarkable proliferative response to porcine cardiac myosin. In anti-CD4 monoclonal antibody treated group, however, lymphocytes showed no significant proliferation to this antigen. When stimulated with non-specific mitrogen, Con A, Lymphocytes obtained from control group, saline-treated group, IgG-treated group and McAb-treated group displayed similar thymidine incorporation.(2)Inhibitory effect of McAb on serum anti-cardiac myosin autoantibody: Rats in saline-treated group and IgG-treated group produced high titer of anti-cardiac myosin autoantibody and it was much lower in anti-CD4 monoclonal antibody treated group than in saline-treated group or IgG-treated group.15. The effects of anti-CD4 monoclonal antibody on EAM.(1)Prevention of heart failure and ventricular remodeling: A significant reduction in IVS and LVPW and a significant increase in LVFS were found in anti-CD4 monoclonal antibody treated group, compared to saline-treated group and IgG-treated group.(2)Inhibitory effects of anti-CD4 monoclonal antibody on inflammation and fibrosis: Gross analysis of rats' hearts in anti-CD4 monoclonal antibody treated group revealed a significant decrease in the typical signs of myocarditis: hypertrophy, pericardial effusion and pallor. Microscopically, very little infiltration of inflammatory cells and fibrosis in myocardia were observed in anti-CD4 monoclonal antibody treated rats. Pathological scores of rats in anti-CD4 monoclonal antibody treated group were significantly lower than those of rats in saline-treated group and IgG-treated group.16. The effects of anti-CD4 monoclonal antibody on Th1 and Th2-typing cytokines on day 18 after the first immunization: Th1-typing cytokines were significantly higher in saline-treated group and IgG-treated group compared to control group and treatment with anti-CD4 monoclonal antibody significantly reduced them. Moreover, treatment with anti-CD4 monoclonal antibody significantly up-regulated the serum level of Th2-typing cytokines of EAM rats.Conclusions:1. In the present study, high purity and activity cardiac myosin was separated by biochemical methods.2. EAM model was established by injection of cardiac myosin separated in the first part.3. The development of EAM included acute, subacute and chronic stages.4. The unbalance of CD4 positive T cell subsets contributed to the initiation and development of the disease and directly leads to the cardiac dysfunction and ventricular remodeling at acute and subacute stage, while on the chronic stage, Thl and Th2-typing cytokines undirectly contributed to the cardiac function and ventricular remodeling through intervening in the fibrosis.5. Immune tolerance to cardiac myosin in EAM rats was successfully induced by anti-CD4 monoclonal antibody. Through the induction of immune tolerance to cardiac myosin, the development of EAM was successfully prevented. So it is proved further that CD4 positive T cells play a key role in the development of EAM.6. Treatment with anti-CD4 monoclonal antibody changed the Thl/Th2 balance from Thl to Th2, which maybe one of the mechanisms of the induction of immune tolerance.Innovation and significance1. In the present study, the traditional method of preparing cardiac myosin was improved on. In order to avoid the disadvantage of Western blot, native-PAGE and mass spectrum analysis were used in the present study to identify the separated protein. The yield of the protein separated in the present study was higher than everbefore. Therefore, the present study laid the foundation for clinical and research application of cardiac myosin. In addition, the method of mass spectrum analysis used in the present study provides evidence for the application of this technology in the field of medicine, as well as in the field of large protein analysis.2. EAM was successfully induced by immunizing cardiac myosin separated in the present study. The dynamic detection of the development of EAM is performed. At the same time, the relationship between cardiac function and CD4 positive T cell was analyzed dynamically. It revealed the function and mechanism of autoimmune injury leading to cardiac dynamics and ventricular remodeling in more detail.3. Anti-CD4 monoclonal antibody was used for the first time in the treatment of EAM rats. Immune tolerance to cardiac myosin was successfully induced by injection of this antibody in vivo. The induction of immune tolerance prevented the development of EAM. It is revealed that anti-CD4 monoclonal antibody had great potential for the treatment of viral myocarditis and dilated cardiomyopathy in clinic. In addition, the present study provided important evidence for the application of this antibody in other autoimmune diseases.
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