Relationship Between Cardiac β₁-AAB Production And Arrhythmia And The Analysis Of Its Significance

BackgroundG protein-coupled receptor (G protein-coupled receptor, GPCR) is the largest membrane receptor protein family in the body and it is widely distributed in body tissues and organs,β1 adrenergic receptor (β1-AR)located on Myocardial cell membrane is one kind of the GPCR, it plays an important role in regulating cardiac function and activities. In physiological circumstances,β1-AR identify and selectively bind to catecholamines, then active signal transduction pathway in the cell, mediated positive chronotropic, inotropic and dromotropic effect in the heart and participate in energy metabolism of cardimyocyte.β1-AR plays a dominant role in the Nerve - humoral regulation in the function and activation of the heart. Over the past two decades, many scholars have detected antibodies against the second extracellular loop ofβ1-adrenoceptor(β1-AAB)in dilated cardiomyopathy, rheumatic heart disease, Chagas disease and primary cardiac electricity disorders disease and so on. This suggests that this autoantibody may be associated with the pathophysiology mechanisms of the cardiac disease.In 1990, Magnusson et al. discovered that: anti-β1-AR autoantibody (β1-AAB)is able to recognize the functional epitope peptide of the second extracellular loop of theβ1-AR, and it exhibited the agonist-like effect such as positive chronotropic effect. However, there is still some difference to agonist: the stimulation effect of the autoantibody to theβ1-AR receptor is sustained, which showed no desensitization. In 1996, Mijares et al. found thatβ1-AAB can active L-type calcium channel in the membrane of cardimyocytes through cAMP-dependent protein kinase pathway, then enhanced calcium influx and shortened the QT interval. In view of L-type Ca2+ channel current is the main inward currents of the action potential plateau of non-self-regulatory cells and 0 phase depolarization of the self-regulatory, whetherβ1-AAB which has the agonist-like effect is associated with the abnormal electrical activity of heart, then further associated with the occurrence of arrhythmia remains unknown However, in order to confirm this speculation, whether such antibodies existed in sera of patients with arrhythmias should be clarified. Furthermore, clarify whether such autoantibodies are associated with arrhythmia. First-hand information should be provided for conforming thatβ1-AAB is involved in the pathophysiological process of occurrence and development of arrhythmia.Studies have shown that B-lymphocytes, which can produce autoantibodies, existed in healthy organism, and they are usually suppressed or in a low lever status, therefore, they can't induce tissue damage or induce disease. Autoimmunity also often happens in normal human, and the general function is to maintain the physiological homeostasis. In sera from normal human there are a variety of autoantibodies against self-antigen, but the titer is very low, just can assist the body to clear aging self-composition. Therefore, it is known as physiological antibodies. In some pathological conditions, there is a more significant increasing trend of autoantibodies titer than the original physiological antibodies. The autoantibodies which are abnormal in quality and quantity attack self cells and tissues which have target antigen or in turn affect and destroy the immune system, thus produce the pathological changes and dysfunction, then form the autoimmune disease. In immune system, proliferation of lymphocytes is an important event in the immune response to the stimulation of antigen, and results in producing effector lymphocytes. Finally clear the antigen and maintain the internal environment stabilization. In the start-up phase of immune response, T lymphocytes play an essential supplementary role. In addition, there isβ1-AR on the membrane of T lymphocytes. Except the function to the heart ofβ1-AAB which was previously found. Therefore, whetherβ1-AAB can bind to the corresponding receptor on membrane of T lymphocyte, and further affect the immune regulatory function, thereby affect the arrhythmia needs to be further investigated.Our preliminary study has found that antibody against AT1R could promote proliferation of T lymphocytes from rats'spleen. For bothβ1-AR and AT1 receptor are G protein-coupled receptors, andβ1-AR also distributed on T lymphocytes according to previous reports. So we speculate that the immune system may also be one of the targets ofβ1-AAB. If so, what's the function ofβ1-AAB to T lymphocytes? And whether it can affect the function of immune system? All these questions have to be analyzed one by one through experiments.Objective1. To investigate whether there isβ1-AAB in the sera of arrhythmia patients, and if so, to determine the distribution characteristics ofβ1-AAB in various types of arrhythmia patients and its possible clinical significances.2. To observe whetherβ1-AAB can induce the occurrence of arrhythmia in normal rats or heart failure rats, and if so, what are their characteristics?3. To preliminaryly study whetherβ1-AAB can influence the function of immune system, if so, what is the effect ofβ1-AAB on the proliferation of T lymphocytes?Methods1. The synthesized peptides of the second extracellular loop ofβ1-AR (197-222 amino acids) were used as antigen to detectβ1-AAB in the sera of 106 cases arrhythmia patients, 100 cases coronary heart disease patients and 100 cases healthy people by SA-ELISA technology, and analyze the distribution characteristics ofβ1-AAB in various types of arrhythmia patients.2. The synthesized peptides corresponding to the second extracellular loop ofβ1-AR (197-222 amino acids) were used as antigen to immunize rats. During the immunization course we collected the peripheral blood from tail vein to detect the ratios of CD4+ and CD8+ T lymphocytes by flow cytometry technology. By the end of immunization, the positive and negative IgGs of seraβ1-AAB were purified respectively by affinity chromatography.3. The heart failure models of rats were established by constricting the abdominal aorta. After the models formation, theβ1-AAB negative rats were selected by SA-ELISA technology, and were injected acutely withβ1-AAB through tail vein. Then we observed the effects ofβ1-AAB on ECG in heart failure rats, sham rats and healthy rats.4. In cultured lymphocytes from mesenteric lymph nodes, we observed the effects ofβ1-AAB positive IgGs on T lymphocytes proliferation induced by Con A by CCK-8 Detection Kit.Results1. The high positive rate ofβ1-AAB in the sera of patients with arrhythmias.In 106 cases of patients with arrhythmia, 58 cases were positive forβ1-AAB, so the positive rate was 52.8%, which was significantly higher than that in the normal subjects (5.0%) or in coronary heart disease groups (24.0%), (P<0.01) (see Table 1-1,1-2). This result indicates the positive rate ofβ1-AAB in patients with arrhythmia is higher than that in patients of coronary heart disease patients or normal subjects.2. The distribution characteristics ofβ1-AAB in the sera of patients with different types of arrhythmia.By analyzing the contents ofβ1-AAB based on the sera OD values, we found in patients with ventricular arrhythmia, atrial arrhythmia and conduction abnormalities arrhythmia, the OD values were 0.35±0.02,0.20±0.01 and 0.22±0.02 respectively which were all higher than that of the normal control group (0.13±0.01, P<0.01). In addition, compared with the atrial arrhythmia and conduction abnormalities arrhythmia, the OD values of ventricular arrhythmia were significantly different (P<0.01) (see Figure 1-1). These results indicate that the distribution ofβ1-AAB is higher in patients of arrhythmia than that in the normal controls, and maybeβ1-AAB is more relevant with ventricular arrhythmias.3. Establishment of the active immunization rats models and purification ofβ1-AAB.After active immunization with antigen peptides of the second extracellular loop ofβ1-AR in rats, theβ1-AAB contents in sera increased significantly at 4 weeks, and reached the peak at 10 weeks (at 1:20 dilution the OD value was 3.43±0.72, P<0.01, vs. the control group at the same period) (see Figure 2-1). The anti-serum of rats was purified to obtainβ1-AAB IgGs.4. Heart failure models of rats were successfully established.After the heart failure models were set up for 12 weeks, the left ventricular systolic pressure (LVSP) and the maximum rate of ventricular pressure rise and fall (±dp / dtmax) were lower in the heart failure group than that in the sham group (P<0.05), while the end-diastolic pressure of left ventricular (LVEDP) was higher than that in the sham group (P<0.05) (see Table 2-1). The ratios of heart weight and body weight in the heart failure group was significantly higher than that in the sham group at the same period (P<0.01) (see table 2-2). These results above indicated that the rat models of heart failure were established successfully.5. The effects ofβ1-AAB on ECG of normal rats and heart failure ratsWhenβ1-AAB IgGs were infused from the tail vein of rats acutely, varying degrees and different types of arrhythmia were monitored in the normal rats and heart failure rats. and the ventricular arrhythmia was one of the most common (see Figure 2-2). In addition, the frequency of arrhythmia in the heart failure group was significantly higher than that in the normal rats (see Figure 2-3).6. Changes of T lymphocyte subtypes in the peripheral blood of immunized ratsThe ratios of CD4+ and CD8+ T lymphocyte began to increase in theβ1-AR antigen peptide immunization group and control group after treatment for 24 hours. And it reached a higher level in theβ1-AR antigen peptide immunization group at the 7th day (3.26±0.32), then increased gradually during the study period. However, the ratios of CD4+ and CD8+ T lymphocyte achieved peak at the third day (2.72±0.47), then decreased gradually (see Table 3-1). These results indicate that there is the emergence of significant immune hyperfunction in theβ1-AR antigen peptide immunization group compared with the control group.7. The effects ofβ1-AAB on T lymphocytes proliferationThe results detected by CCK-8 kits showed that three concentrations ofβ1-AAB (0.01, 0.1, 1 mol/L) promoted proliferation of T lymphocytes induced by Con A in a dose-dependent manner (OD values were 0.49±0.05, 0.69±0.08, 0.82±0.07), which was similar to the role ofβ1-AR agonist isoprenaline (see Figure 3-1). In addition, the effects ofβ1-AAB could be neutralized by theβ1-AR-specific blockers Metoprolol or the antigen peptides corresponding to the second extracellular loop ofβ1-AR, but simple Metoprolol orβ1-AR antigen peptides had no significant effect on proliferation of T lymphocytes (see Figure 3-2).Conclusions1. In the sera of patients with arrhythmia, the positive rate ofβ1-AAB was significantly higher than that of patients with coronary heart disease and healthy subjects, and theβ1-AAB might be more relevant with ventricular arrhythmias.2.β1-AAB had a direct-induced arrhythmia effects in normal rats and heart failure rats, and the ventricular arrhythmia was most common. In addition, the susceptibility to arrhythmia increased markedly in heart failure rats, indicating that the existence of high level ofβ1-AA in sera of patients with heart failure may have more considerable pathophysiologic significance.3.β1-AAB were reported for the first time to promote the proliferation of T lymphocytes by activating ofβ1-AR, and affect the immune regulation function of the organism.