The Protective Effect Of Autoantibodies Against The β₃-Adrenoceptor On The Occurring Of Heart Failure In Rat

Background:Chronic heart failure (CHF) is the final clinical manifestation of a variety of human heart diseases, including dilated cardiomyopathy (DCM), coronary artery disease (CAD), hypertensive heart disease (HHD) and rheumatic heart disease (RHD).In China, the prevalence of the heart failure is about 0.9% in adult population and with the aging of population the prevalence of CHF will increase substantially. Although current treatment against some pathogeniesis of heart failure can improve some symptoms, the 5-year survival rate of patients with heart failure remained less than 50%. It indicates that, there may be some other unknown factors involved in the occurrence and development process of heart failure.The pathophysiologic mechanism of heart failure is extremely complex, there are several mechanisms involved in this syndrome. Resent yeas, some autoanbibodies against the G-protein-coupled receptors were detected in patients of heart failure, such as autoantibodies againstβ1 adrenoceptors/α1 adrenoceptors/M2-muscarinic receptors/angiotensinⅡ-1 receptors. Some studies showed that, autoanbibodies against the G-protein-coupled receptors have important pathophysiologic role in the occurrence and development process of heart failure.P3-adrenoceptor is a member of G-protein-coupled receptors. In 2005, our research group found that there is a import autoantibody named autoantibodies against the second extracellular loop ofβ3-adrenoceptor (β3AA) existing in patients of heart failure, and the positive rate ofβ3AA in patients of heart failure is higher than that in normal people. Resent study from our research group showed, there is positive correlation between the titer ofβ3AA and cardiac left ventricular ejection fraction (EF) and negative correlation between the titer ofβ3AA and NYHA (New York Heart Association) function class in patients of heart failure, that is the cardiac function of patients withβ3AA positive is better than patient withβ3AA negative. This result indicates that theβ3AA may have some protect effect on heart and the function is different from the injury effect induced by autoantibodies againstβ1 adrenocepters andα1 adrenoceptors. However, because of the limitation of clinical research, the exact role ofβ3AA in the process of occurrence and development of heart failure is still unknown, so some animal experiments are needed to further confirm the real function ofβ3AA.In addition, Transition from Chronic Compensated to Acute Decompensated Heart Failure is the main reason for hospitalization and death, and myocardial ischemia is one of the main triggers. Then whether the susceptibility to ischemic/reperfusion injury of heart failure rats can be decreased ifβ3AA long term exist in chronic heart failure rats.Therefore, in this study we chosed chronic heart failure rats as starting point, givenβ3AA through intravenous, with cardiac function, heart weight/body weight ratio, serum brain natriuretic peptide and apoptosis of myocardial cells as indicators, from two aspects whetherβ3AA can reduce the degree of heart failure and whetherβ3AA can reduce the susceptibility to ischemic/reperfusion injury of heart failure rats to abserve whetherβ3AA have cardio protective effect to the heart failure rats.Objectives:1.To observe whetherβ3AA can reduce the occurrence of heart failure;2. To observe whetherβ3AA have protect effect on the heart of rats with heart failure undergoing ischemia/reperfusion injury.Methods:1. The preparation ofβ3AA20 healthy Wistar rats (male, initially weighing 140-160g,8 weeks old) were randomly divided into immunize group and control group. Rats were immunized with the second extracellular loops of theβ3AR (176aa～200aa,RVGADAEAQECHSNPRCCSFA S N M P) for 8 weeks. The level ofβ3AA in the sera was detected by ELISA which was improved by our research group.β3AA positive IgG and IgG withoutβ3AA were purified, quantified and qualified. SDS-PAGE was used to detect the purity.2. Aortic banding surgeryWistar rats(10 weeks old, weighing 200-220g) were chose and anaesthetized with 10% chloral hydrate solution (30 mg/kg) by peritoneal injection. Below the xiphoid lcm, a small horizon incision was made. Separate the abdomen aortic artery, band the artery with a needle (size7), then draw out the needle and form strictness (approximate 50%).At the same time inject Gentamicin and close the abdomen.3. Preparation ofβ3AA passive immunized modelAdministrate theβ3AA (2μg/g) which was purified to the rats through the tail vein 3 days after the aortic banding surgery. Every 10 days to repeat the immunity and the total period of immunization is 50 days.1 day before the immunization get the blood sample from the rat tail and separate the serum. SA-ELISA is used to detect the level ofβ3AA.In the control group, IgG withβ3AA negative (2μg/g) is administrated to the rats through the tail vein, the immunization process and detect methods is as same as the procedure described above.4. Measurement of the in vivo cardiac functionMS2000, a computerized biological signaling system is used to monitor changes of the cardiac function parameters, such as left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and maximal rate of rise and decline of ventricular pressure (±dp/dtmax).5.Measurement of the Heart weight to body weight ratioThoracotomy, quickly remove the heart and wash the remained blood in the cardiac cavities.Wash intracardiac hematoma s excised with all the cavities being washed clean with normal saline immediately. Remove the blood vessels and tissues around the heart; absorb the water around the heart by filter paper. Weigh the whole heart and calculate the ratio of heart weight to body weight.6. Detect the level of Brain natriuretic peptide (BNP) in the seraELISA kit is used to detect the level of BNP in the sera; the specific operations are carried out in accordance with the instructions.7. The ischemia/reperfusion surgeryLigating the left anterior descending coronary artery for 30 minutes, then loose the ligature for reperfusion 3 hours;in the sham group, suture needle go through the bottom of the left anterior descending coronary artery, but not ligation, other operation is the same as described above.8. Measurement of the myocardial infarct sizeTTC coloration is used to detect the myocardial infarct size ofβ3AA passive immunized heart failure rats after ischemia/reperfusion surgery.9. Caspase 3 ability assayCaspase 3 ability assay kit is used to detect the ability of caspase 3 in rat cardiac tissues.10. Detected Myocardial apoptosis by TdT-mediated dUTP nick end labeling (TUNEL)In Situ Cell Death Detection Kit is used to detect the apoptosis of cardiomyocytes and calculate the ratio.Results:1. Choose the time of transferringβ3 AA to the heart failure rats1.1 The cardiac function changed over time after the aortic banding surgeryAs shown in Table 1,8 weeks after the aortic banding surgery, LVSP and±dp/dtmax were remarkably decreased, LVEDP was significantly increased, the cardiac function begins to decline; 12 weeks and 16 weeks after the aortic banding surgery, the cardiac function progressively deteriorate.1.2β3AA in the sera of the rat changes over time after the aortic banding surgery8 weeks after the aortic banding surgery,β3AA in the sera of rats was remarkably increased (0.31±0.02 vs.0.22±0.05,P<0.05); 12 weeks after the aortic banding surgery, the level ofβ3AA reached the peak; after the 12th week, the level ofβ3AA began to decline (Figure 1).All the data above indicate that, the phrase of 0-8 weeks after the aortic banding surgery is a process of changes of cardiac function from normal to significantly decrease, that means this phrase is very important to the occurrence and development of heart failure; more important, during this phrase theβ3AA was produced and the protective effect ofβ3AA on the rat heart could be singly studied. So in this study, we chose the phrase of 0-8 weeks after the aortic banding surgery to study the protective effect ofβ3AA on the rat heart in the process of occurrence and development of heart failure.2. Successfully prepared heart failure model2.1 cardiac functions decreased in 8 weeks after the aortic banding surgery8 weeks after the aortic banding surgery, LVSP and±dp/dtmax were remarkably decreased, LVEDP was significantly increased. The result showed that the heart failure model was successfully prepared (Figure 3-6).2.2 The ratio of in 8 weeks after the aortic banding surgery8 weeks after the aortic banding surgery, the ratio of heart weight to body weight significant increased compared with the sham group. Suggest that there is reconstruction of heart in heart failure rats (Figure 7).All the data of result 2 showed that there was notably reconstruction of heart and decompensation of cardiac function in 8 weeks after the aortic banding surgery. So 0-8 weeks after the aortic banding surgery is a important phrase in the process of occurrence and development of heart failure.3. Successfully establishedβ3AA passive immunized heart failure rats3.1 Dose selection of P3AA passive immunization0.5μg,1μg/g,2μg/gβ3AA was respectively used to passive immunize rats after the aortic banding surgery. First day after the immunization, collect the sera and detect the level ofβAA. After 2μg/gβ3AA passive immunized, the average OD value ofβ3AA is 0.67, matched to the clinical level. So we used 2μg/g as the final immunize doze (Figure 8).3.2 Detection the IgG subclass ofβ3AAFigure 9 showed that the main IgG subclass ofβ3AA in the immunized rats is IgG2b subclass, and little belongs to IgG2a subclass. Some studies showed that different IgG subclass have different metabolic half-life. IgG2b has a half-life of 6 days; the average half-life of serum IgG2a is about 8 days. So to determine the main IgG subclass of P3AA can provide a theoretical basis for the cycle of passive immunization (Figure 9).3.3 Determine the elimination time ofβ3AA in the rats after the aortic banding surgeryFirst day after theβ3AA passive immunization, the level ofβ3AA in the sera significantly increased, the average OD value was 0.67, it increased by 0.35 compared with the control group. Approximately 10 days were needed to make sure the OD value ofβ3AA decline by half of the increasing value. So in this study 10 days were chose as the immunizing cycle (Figure 10).3.4 The level of P3AA kept stable in the sera after passive immunized the aortic banding rats2μg/g as the immunizing doze,10 days as the immunizing cycle,β3AA was used to immunize the aortic banding rats. Regular monitoring the level ofβ3AA in the sera showed that the level ofβ3AA could keep stable (Figure 11).4. Cardiac protect effect of P3AA on the heart failure rats4.1β3AA decrease the level of Brain natriuretic peptide (BNP) in the seraSerum BNP levels and clinical severity of heart failure are parallel. Real-time monitoring the level of BNP showed that there was light increase in 2 weeks after theβ3AA was passive immunized to the post-aortic banding rats; 6 weeks later, the level of BNP in theβ3AA passive immunized post-aortic banding rats was lower than the control group in the same time;8 weeks later, the level of BNP was significantly lower. These results indicate that the degree of heart failure inβ3AA passive immunized group is more slight (Figure 12).4.2β3AA improve the cardiac function of heart failure rats8 weeks after the the aortic banding surgery, inβ3AA passive immunized group, reduction ranges of+dp/dtmax and-dp/dtmax were remarkably lower than that in control group which is P3AA negative (Figure 13～16).4.3 P3AA have no effect on the ratio of heart weight to body weight8 weeks after the the aortic banding surgery, in P3AA passive immunized group, heart weight to body weight had no significant changes to the control group (Figure 17).5. Cardiac protect effect of P3AA to the rats with heart failure undergoing ischemia/reperfusion injury5.1β3AA improve the survival situation of the rats with heart failure undergoing ischemia /reperfusion injuryAfter the rats passive immunized byβ3AA underwent the ischemia/reperfusion injury, the survival time could be obviously extended compared with the control group whichβ3AA not passive immunized (Figure 19).5.2β3AA reduce the myocardial infarct size of the rats with heart failure undergoing ischemia/reperfusion injuryAfter the rats passive immunized by P3AA, the myocardial infarct size induced by ischemia/ reperfusion injury was smaller than that in the control group (Figure 20).5.3 P3AA decrease the cardiomyocyte apoptosis of the rats with heart failure undergoing ischemia/reperfusion injuryAfter the rats passive immunized byβ3AA, TUNEL-positive cells and caspase-3 activity in myocardiocytes decreased significantly which were induced by ischemia/reperfusion injury (Figure 21,22).Conclusions:1.P3AA passive immnized the heart failure rats induced by pressrue overload can significantly reduce the degree of deterioration of cardiac function and reduce the level of BNP in the sera;2.β3AA can improve the survival situation of the rats with heart failure undergoing ischemia/reperfusion injury and can also decrease the myocardial infarct size and cardiomyocyte apoptosis induced by ischemia/reperfusion injury.