| BackgroundRecent studies have showed that autoantibodies against the second extracellular loop ofβ1-adrenoceptor (β1-AA) and the autoimmune mechanisms were involved in the occurrence of many heart diseases such as idiopathic dilated cardiomyopathy, Chagasic disease, hypertensive heart disease, and so on. The pathophysiological role ofβ1-AA in the occurrence and the development of heart disease have been paid much attention among researchers. However, these former studies obtained the results by active immunizing animal models which used the synthetic peptide corresponding to the epitope peptide of the second extracellular loop of humanβ1-adrenoceptor (β1-AR-ECⅡ) to immune the rats. But there are two major deficiencies in this model. Firstly, it is difficult to control the levelβ1-AA in the process of active immunization, which suggested that the serum level ofβ1-AA was much higher than the actual level in clinical patients. Secondly, the model could not exclude the possible toxic role of antigenic peptides itself. Therefore, we should establish a much better model to mimic clinical condition, which showed a pathophysiological significance in the study of myocardial dysfunction disease related toβ1-AA.Further studies found thatβ1-AA had an"agonist-like" activity, which could lead to an increasing of intracellular free calcium in isolated cardiomyocytes of normal mice. However, it is not clear whether the long-term existence ofβ1-AA would affect the level of intracellular free calcium in cardiomyocytes. It is related to the basic point whether we could use Ca2+ as the key in the treatment of cardiac dysfunction induced byβ1-AA.Objectives1. To set up aβ1-AA rat model of passive immunity, so as to keepβ1-AA serum levels in the rats the same as that in clinical patients.2. To observe long-term effects ofβ1-AA on intracellular free calcium in in rats' cardiomyocytes by using the above model. Materials and Methods1. Preparation of anti-humanβ1-adrenergic receptor autoantibodies (β1-AA)1.1 Synthesis peptides of anti-humanβ1-AA:According to epitope peptides of humanβ1-AR-ECⅡ(197-223, H-W-W-R-A-E-S-D-E-A-R-R-C-Y-N-D-P-K-C-C-D-F-V-T-N-R-A), the sequence of the second extracellular loop of humanβ1-AR was synthesized by GL Biochem (Shanghai) Ltd. The purity was>95%. The synthetic peptides were stored at-20℃for the future qualitation and quantitation of the serum antibodies, and even their functional examination.1.2 Preparation ofβ1-AA:Using such antigenic peptides to the Wistar rats (8 weeks, weight 160-180g, n=20) for a period of 8 weeks of active immunization and preparing anti-β1-AA serum, detecting its valency by Streptavidin-enzyme linked immunosorbent assay (SA-ELISA). Extracting and purifyingβ1-AA serum by MAb Trap Kit. The purity of the antibody solution was detected by polyacrylamide gel electrophoresis (PAGE) and then the protein quantification of the extract was detected with the BCA Kit.2. Establishment of passive immunization model2.1 Passive immunization:According to the results of the pre-experiment, injected 0.7μg/g of the purifiedβ1-AA into the normal rats via the tail vein, every 2 weeks to enhance immunity, and the boosting dose should correspond to the level ofβ1-AA in the immune rats, for a whole period of 40 weeks of immunization. Rats in the control group were injected 0.7μg/g of negative IgG via tail vein, with the same immunization procedures and test methods.2.2 Examination of cardiac function of the rats:After intraperitoneal injection of urethane (1g/kg, i.p) anesthesia according to their weights, rats of passive immunization for 40 weeks were taken to adopt the left ventricular catheterization through the right common carotid artery, till the emergence of the left ventricular feature waveform (Figure 1), and after it stabled for 15 or 20 minutes, using BL-410 biological signal analysis system to analyze and record the cardiac function parameters, including left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVDP), left ventricular maximum rate of pressure rise (+dp/dtmax) and left ventricular maximum rate of pressure decline (-dp/dtmax).3. Determination of intracellular free calcium through confocal laser scanning microscopy in ventricular myocytes of passive immunization rats in late phase 3.1 Isolation of myocardial cells:Rats of passive immunization for 40 weeks were anesthetized. The hearts were quickly removed from the chest and retrogradely aortic perfused with the Langendorff perfusion device and enzymaticly digested into single cardiomyocytes, which were placed into high potassium KB solution, and put it aside for 2-4 hours for the later experiment.3.2 Measurement of the level of intracellular free calcium in ventricular myocytes by Laser scanning confocal microscope (LSCM):After recalcification of the isolated cardiac cells suspension by adding Tyrode's solution, loaded the fluorescent indicator Fluo-3/AM, put to a special confocal dish, and observed with an inverted microscope. Selected 10 fields of vision for each rat and chose rod-shaped, with integrated cell membrane and well-shaped cells for detection. Analyzed the results with the image analysis software LSM510.4. Determination of intracellular free calcium in normal rats' ventricular myocytes with ion imaging systemSingle myocardial cells were obtained from healthy Wistar rats by the above method. Loaded Fura-2/AM fluorescent indicator, put to the perfusion bath of ion imaging system, using the inverted microscope to measure them in groups:(1) Normal control group (Tyrode, n=6), in normal Tyrode's solution; (2) Negative IgG group (negative IgG, n=6), giving negative IgG; (3) Isoprenaline group (Isoprenaline, n=6), giving 1μmol/L of Isoprenaline; (4)β1-AR autoantibodies group (β1-AA, n=6), giving 0.1μmol/Lβ1-AA; (5)β1-AA plusβ1-AR antagonist group (β1-AA+Bisoprolol, n=6), giving 0.1μmol/Lβ1-AA and 1μmol/L Bisoprolol; (6) Bisoprolol group (Bisoprolol, n=6), giving 1μmol/L Bisoprolol; (7)β1-AA plusβ1-AR-ECⅡgroup (β-AA+β1-AR-ECⅡ, n=6), giving 0.1μmol/Lβ1-AA and 1μmol/Lβ1-AR-ECⅡ; (8)β1-AR-ECⅡgroup (β1-AR-ECⅡ, n=6), giving 1μmol/Lβ-AR-ECⅡ. Observing the changes of the intracellular resting calcium in ventricular myocytes of the rats and comparing the results with that from Isoprenaline.Results1. Preparation ofβ1-AA through active immunization with functional epitope peptides ofβ1-AR-ECⅡ1.1 The level ofβ1-AA in rats increased during active immunization:Two weeks after initial immunization,β1-AA appeared in the serum of the rats of the immune group, in which the antibody increased quickly and reached a peak at the 8th week (OD value 2.99±0.14 vs. 0.43±0.12, P<0.05, vs. control group, Figure 2). At the same time, there was noβ1-AA in the control group, indicating the success of active immunization. 1.2 The qualitation and quantitation determination ofβ1-AA protein:The results of PAGE showed that only one band appeared, with a molecular weight of about 160KDa, consistent with the IgG standard (Figure 3), indicating satisfactory results of the purification. Meanwhile, the protein concentration of the extract was determined by the BCA kit (Table 1).2. Established passive immunization rat models by purifiedβ1-AA2.1 The level ofβ1-AA in rats during the process of passive immunization matched that in patients:Afterβ1-AA was passively transferred to normal rats, the serum levels of this antibody were gradually increased, reaching a peak at the 16th week of passive immunization and then declining slowly. There were no statistical differences among those groups of 8,16,32,36,40 weeks, but if it was compared with the control group, there were significant differences (P<0.001, P<0.05, Figure 5). In the whole process of passive immunization, the levels ofβ1-AA in sera of immune rats remained 0.19±0.01-0.22±0.02, matching to that of patients with heart disease.2.2 The cardiac function in vivo decreased in the final phase of passive immunity:At the 40th week of passive immunization byβ1-AA, the cardiac function in vivo of the rats in immune group decreased (Figure 6). In the passive immunization group,+dp/dtmax and-dp/dtmax were significantly lower than those in the control group (P<0.001, P<0.01). LVDP was drastically higher (P<0.001) than that in the control. LVSP did not change obviously, and there were no obvious differences between the two groups or in themselves (P>0.05). This indicated that the long-term existence ofβ1-AA had led to the decline of cardiac function in rats.3. The levels of intracellular free calcium in ventricular myocytes increased in long-term existence ofβ1-AA in rats3.1 The levels of intracellular free calcium in myocytes in rats increased at the final phase of passive immunization:At the 40th week of passive immunization byβ1-AA, LSCM, a high-resolution imaging system, was used to make an accurate quantitative analysis to the fluorescence of intracellular probe. We found that the level of intracellular calcium in single ventricular myocytes in rats of the immune group was 10 times higher than that of the control group (P<0.001, Figure7A,7B), indicating that the Long-term existence ofβ1-AA could increase the levels of intracellular free calcium in rats.3.2β1-AA made the level of intracellular free calcium in myocytes in normal rats continue rising, but this effect could be antagonized by the selective antagonist ofβ1-AR:In this experiment, as we had to monitor the level of the intracellular free calcium of the ventricular myocardial cells for a long period, we used the ion-imaging system, and used different oxygen saturated liquids to perfuse continuously to make the cells survive in good condition, so as to ensure the reliability of the experimental results. The results showed thatβ1-AA could significantly increase the level of intracellular free calcium in myocardial cells in normal rats (Figure 9A, B), which was corresponding to the effect ofβ1-AR agonist Isoprenaline (Figure 8A, B), but the action ofβ1-AA was continuous and non-desensitizing (Figure 8A,9A). It was almost possible to wholly antagonize the increasing of intracellular free calcium caused byβ1-AA after using 1μmol/L of Bisoprolol, the selective antagonist of theβ1-AR (Figure 10A). The action ofβ1-AA to increase the intracellular free calcium could be reversed by humanβ1-AR-ECⅡfunctional epitope peptides (Figure 10B).Conclusions1. It was possible to establish cardiac insufficiency models corresponding to the levels of antibody in clinical patients through long-term passive immunization with purifiedβ1-AA.2.β1-AA could increase the concentration of myocardial intracellular free calcium throughβ1-adrenergic receptor (β1-AR), which was similar with the role of isoprenaline. But the difference betweenβ1-AA and isoprenaline was that the role ofβ1-AA might not desensitize.
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