Autoantibodies Against The Second Extracellular Loop Of β₁-Adrenoceptor Could Induce The Injury Of Multiple Organs In Rats And The Possibly Underlying Mechanisms

BACKGROUND AND OBJECTIVEβ-Adrenoceptor (β-AR) is the important member of sympathetic nervous system.β-AR plays an important role on regulating the heart function by mediating the physiological effect of catecholamine.β-AR belongs to G protein–coupled receptor family.β-AR includesβ1,β2 andβ3-AR three types by now. They have the same structural features which are composed with 7 transmembrane domains, 3 intracellular loops and 3 extracellular loops. Among them, the distribution ofβ1-AR in heart is predominant.β1-AR can activate adenyl cyclase (AC) and cyclic adenosine monophosphate (cAMP) system by stimulating G protein (Gs) and displaying positive chronotropic, inotropic and dromotropic actions.In 1987, Wallukat and Wollenberger found the antibodies against the second extracellular loop ofβ1-AR (anti-β1-AR-ECⅡ, Homology of human and rat is 100%) in the sera of patients with idiopathic dilated cardiomyopathy (IDCM). The following studies indicated that the autoantibodies displayed an analogous agonist effect which includes the inhibition of radioligand binding and enhanced signal transduction mediated byβ1-AR. With the further studies, other researches and ours all found that anti-β1-AR-ECⅡnot only existed in the patients with IDCM, but also existed in the patients with Chagasic disease (1994), primarily electrical derangement (1995), hypertensive heart disease and rheumatic heart disease (1999) et al. From the above results, we presumed that there may be a close relationship between anti-β1-AR-ECⅡand many kinds of heart diseases with the changes of cardiac structure and function.In 2000, we observed the formation of anti-β1-AR-ECⅡin the cardiac remodeling models induced by abdominal aorta contraction and Adriamycin. The results indicated that high positive frequencies of anti-β1-AR-ECⅡwere found in abdominal aorta contraction group (87.5%) and Adriamycin group (79.2%). Moreover, with the development of heart failure, the titers of anti-β1-AR-ECⅡdisplayed an increasing, maintaining and decreasing process which suggested that cardiac remodeling or heart failure induced by different reasons all could induce the formation of anti-β1-AR-ECⅡ. In order to study the influence of anti-β1-AR-ECⅡon the cardiac structure and function, we immunized rats with the synthetic peptides according to the sequences of humanβ1-AR-ECⅡfor 18 months. In the late stage of immunization, analogous changes of dilated cardiomyopathy and reduced heart function were observed which suggested that the changes of cardiac structure could induce the formation of anti-β1-AR-ECⅡwhich could aggravate heart injury in return. At last, aggravated heart injury was displayed.The phenomenon of cardiomyocytes lose provoked our attention in the process of 18 months active immunization. Cell lose has two types: apoptosis and necrosis in generally. Apoptosis is an active programmed procedure controlled by genes which is easy to be controlled. There is a research demonstrated that blocking the signal pathway of apoptosis could decrease the area of myocardial infarction significantly and improve heart function. In order to make it clear whether apoptosis existed in the process of DCM induced by anti-β1-AR-ECⅡ, we did an experiment in vitro and found that anti-β1-AR-ECⅡcould induce apoptosis of cultured neonatal cardiomyocytes (2002). In 2003, Staudt et al also demonstrated that the apoptosis of cultured adult cardiomyocytes could be induced by anti-β1-AR-ECⅡ. In 2004, we use active immunization model to demonstrate that anti-β1-AR-ECⅡcould induce apoptosis of cardiomyocytes in vivo. The above results all suggested that apoptosis played an important role in the changes of cardiac structure and function induced by anti-β1-AR-ECⅡ. However, the above researches all used the active immunization model in which we could not exclude the possibly toxic role of antigen peptides. Moreover, the exact mechanism of anti-β1-AR-ECⅡinducing apoptosis was not clear yet. Therefore, making it clear was helpful to determine the pathophysiologic significance of anti-β1-AR-ECⅡand was necessary for the clinical treatment.Some researches demonstrated that anti-β1-AR-ECⅡcould enhance the current of L-type calcium by activating cAMP-PKA pathway. Calcium overload is the important mechanism of apoptosis. In many kinds of cells, calcium can bind with calmodulin (CaM). The compounds of Ca2+/CaM can activate protease depending on Ca2+/CaM. CaMK has four subtypes:Ⅰ,Ⅱ,ⅢandⅣ. Among them, CaMKⅡis a kind of multifunctional protease which is expressed in many important organs. CaMKⅡis composed with four subunits:α,β,γandδ. Among them, the products ofγandδexpress in cardiomyocytes. Up to now, we have found that CaMKⅡδcould induce hypertrophy of cardiomyocytes and also intimately related with apoptosis. So if there is a lot of apoptosis existing in the DCM model induced by anti-β1-AR-ECⅡ, then anti-β1-AR-ECⅡinduce apoptosis by the CaMKⅡδsignal pathway at the dowm stream of calcium?Cardiac remodeling is an important stage in the process of heart failure which may be the possible reason of reduced cardiac function and arrhythmia. Some researches indicated that high level of anti-β1-AR-ECⅡwas detected in the sera of patients with DCM complicating with ventricular arrhythmia and conduction blockade, moreover there was a close relation between anti-β1-AR-ECⅡand arrhythmia. Other researches indicated that anti-β1-AR-ECⅡcould enhance the current of L-type calcium. Then whether anti-β1-AR-ECⅡcan induce arrhythmia directly need further study. The essential mechanisms of arrhythmia are the changes of bioelectricity of cardiomyocytes. In order to investigate the exact mechanism of anti-β1-AR-ECⅡinducing arrhythmia, we will observe the long-term role of anti-β1-AR-ECⅡon resting potential (RP), action potential duration (APD), L-type Calcium current (ICa-L), transient outside potassium current (Ito), delayed-rectifier potassium current (Ik1) and Na+/Ca2+ exchange current (INa-Ca) in cardiomyocytes. Moreover we will observe the acute role of anti-β1AR-ECⅡon bioelectricity of papillary muscles.Researches indicated that the distribution ofβ1-AR is mainly on heart and kidney, and slightly on liver. A lot of researches in the past demonstrated that anti-β1AR-ECⅡcould induce the injury of heart by binding withβ1-AR. Different with agonist; anti-β1-AR-ECⅡhad the character of non-desensitization. Then whether anti-β1-AR-ECⅡexisting in the circulation system could bind withβ1-AR in liver and kidney is not clear. Our previous experiment of long-term active immunization of 18 months with synthetic peptides according to humanβ1-AR-ECⅡon rats, the phenomena of a lot of ascitic fluid and blunt liver edge were observed. In the ascitic fluid, high in protein, more cells and a low glucose level were found by us. Moreover, the isolated ascitic fluid coagulated naturally. In view of the ascitic fluid analysis, the ascitic fluid should be determined as exudate but not transudate. But the reason of ascitic fluid formation was not clear. Considering the phenomenon of blunt liver edge, liver injury may be the possible reason, but we could not exclude the kidney injury. So could anti-β1AR-ECⅡalso damage liver and kidney? In our study, we will do some initial research on the direction.In summary, we will continue the study from two following portions:①observe the changes of intracellular free calcium and CaMKⅡδexpression in the long-term existence of anti-β1-AR-ECⅡ; set up active and passive immunization model and observe whether long-term existence of anti-β1-AR-ECⅡcan induce arrhythmia; moreover analyze the possible mechanism of anti-β1-AR-ECⅡinducing arrhythmia in order to provide the theoretical direction for clinical treatment;②set up active and passive immunization model and observe whether long-term existence of anti-β1-AR-ECⅡcan induce the injury of liver and kidney.I. Anti-β1-AR-ECⅡCould Result in Cardiac Remodeling and its Possible MechanismPART ONE: Long-term Existence of Autoantibodies against the Second Extracellular Loop ofβ1-Adrenoceptor Could Induce the Increase of Calcium/Calmodulin-Dependent Protein KinaseⅡδin Rat Cardiomyocytes Objective1. To observe the changes of cardiac structure and function in the process of passive immunization with anti-β1-AR-ECⅡ;2. To observe the changes of intracellular free calcium and the expression of CaMKⅡδin the process of passive immunization with anti-β1AR-ECⅡ.Methods(1) Select healthy adult Wistar rats (180~220g ,body wt, n=60).The Wistar rats used in the present study were obtained from the Animal Center of Shanxi Medical University, P.R. China. Detect anti-β1-AR-ECⅡin the sera by ELISA. The rats with no anti-β1-AR-ECⅡwere divided into the following two groups: (2 rats died for over dose anesthesia; 2 rats died for unknown reasons)①β1AAb group(β1-AR-ECⅡantibody group, n=32):Purify the IgG from sera obtained from active immunized rats. Then quantity the total IgG by BCA method. Inject the purified IgG into vena caudalis of rats at the dose of 0.7μg/g, boosting for every 2 weeks. The experiment lasts 40 weeks.②Negative sera group(n=28):Purify the negative sera, then quantity the total IgG by BCA method. Inject the purified IgG into vena caudalis of rats at the dose of 0.7μg/g, boosting for every 2 weeks. The experiment lasts 40 weeks.(2) Peptides composition: A peptide (HWWRAESDEARRCYNDPKCCDFVTNRA, 197-223 amino acid residues) corresponding to the sequence of the second extracellular loop of humanβ1-AR was synthesized by GL Biochem (Shanghai) Ltd. The purity is 95% (Table. 1). The synthetic peptides were stored at -20℃for use.(3) Set up passive immunization model by injecting anti-β1-AR-ECⅡinto vena caudalis of rats regularly. And detect the level of anti-β1AR-ECⅡin the sera;(4) Use anatomic measurements, Masson staining and heart weight to body weight ratio to reflect the changes of cardiac structure in the process of passive immunization;(5) Monitor Heart rates (HR), left ventricular systolic pressure (LVSP), and Left ventricular diastolic pressure (LVDP) and±dp/dtmax by putting the arterial cannula into left ventricle by right carotid artery;(6) Detect the changes of apoptosis in the process of immunization by TUNEL and detection of caspase-3, 8 and 9 activity in cardiomyocytes;(7) detect the intracellular free calcium in single ventricular myocytes at the late stage of passive immunization labeled with Fluo-3-AM by Confocal microscope;(8) Detect the expression of CaMKⅡδin cardiomyocytes by Western-blot and immunohistochemical method. Results1. Anti-β1-AR-ECⅡcould result in cardiac remodeling of structure1.1 In the process of passive immunization, anti-β1-AR-ECⅡin the sera still maintained a relative permanent levelThe optical density (OD) value of anti-β1-AR-ECⅡin the sera detected by ELISA method ranged from 0.09±0.030 before immunization to 0.21±0.029 at the 4th week after immunization which was higher than 0.08±0.040 (P<0.001) in negative sera group. At the 4th week after immunization, anti-β1-AR-ECⅡkept the high level till to the end of experiment (Fig.1). In the whole immunization process, the level of anti-β1-AR-ECⅡin the sera didn't fluctuate too much. There was no significant difference among different time points of active immunization (P>0.05).1.2 Long-term existence of anti-β1-AR-ECⅡcould result in cardiac remodeling1.2.1 The changes of the ratio of heart weight to body weight in the passive immunizationThe ratio of heart weight to body weight (HW/BW) can reflect whether cardiac remodeling took place. HW/BW began to decrease significantly to 2.10±0.09 at the 24th week after passive immunization which was lower than 2.72±0.04 (P<0.01) in the negative sera group. At the 36th week, the ratio decreased to 2.35±0.05 inβ1AAb group which was still lower than 2.68±0.05 (P<0.01) in the negative sera group (Fig.2C).1.2.2 Anatomic indexAt the end stage of passive immunization, some pathological changes such as enlarged chamber heart and thinning heart wall could be observed (Fig.2B).1.2.3 Masson's trichromic stainingAt the 36th week after passive immunization, a lot of collagen fibers depositing in the stroma of heart could be observed by Masson's t staining (Fig.3).1.3 Long-term existence of anti-β1-AR-ECⅡcould result in the descending of heart functionWith the time of passive immunization, heart function cut down gradually (Fig.4). In the whole immunization, heart rates didn't change obviously. Only at the 36th week of immunization, heart rates decreased to 311±20.3 times/min which was lower than 360±23.0 times/min (P<0.05) in negative sera group (Fig.4A). +dp/dtmax significantly descended to 213±31.1 Kpa/s at the 24th week of immunization which was lower than 510±29.0 Kpa/s (P<0.01) in negative sera group. At the 36th week, it was 289±39.6 Kpa/s inβ1AAb group which was lower than 505±30.0 Kpa/s (P<0.01) in negative sera group (Fig.4B). -dp/dtmax began to decrease obviously at the 24th week of immunization. At that time, -dp/dtmax was -233±34.7 Kpa/s which was lower than -415±31.0 Kpa/s (P<0.01) in negative sera group. At the 36th week, -dp/dtmax was -283±29.6 Kpa/s which was also lower than -421±29.0 Kpa/s (P<0.01) in negative sera group (Fig.4C). There was no significant difference of LVSP and LVDP betweenβ1AAb group and negative sera group.Above results suggested that the long-tem passive immunization with anti-β1-AR-ECⅡcould result in the formation of cardiac remodeling and decreased heart function. But the possible mechanisms of these changes were not clear which need further study.2. The possible mechanisms of cardiac remodeling induced by anti-β1-AR-ECⅡ2.1 Long-term existence of anti-β1-AR-ECⅡcould induce the increase of apoptosis in cardiac myocytes2.1.1 Long-term existence of anti-β1-AR-ECⅡcould induce the increase of caspase-3,8 and 9 activities of cardiomyocytesIn the passive immunization, fluorescent quantitation was used to detect the activity of caspase3, 8 and 9. Use AFC standard to make the standard curve (Fig.5). The slope rate of the straight line was 0.998 by fitting every point. The best incubation time of caspase3, 8 and 9 was determined in Fig.6. When incubate sample and fluorescence substrate for 30min, the fluorescence intensity of negative control was near to the peak, whereas the fluorescence intensity of samples were in low level. When incubating them for 60min, the fluorescence intensity of negative control was near to the bottom of the curve, whereas the fluorescence intensity of samples was increasing. Incubating 90min, the fluorescence intensity of negative control was still at the bottom, whereas the fluorescence intensity of samples was still increasing to a high level. Incubating 120min, the fluorescence intensity of negative control began to increase, whereas the fluorescence intensity of samples was still increasing. According to the standard, we select the time point with high fluorescence intensity of sample and low fluorescence intensity of negative control. So the incubation time was determined at 90min.In the process of passive immunization, the activity of caspase-3, 8 and 9 increased with the time of immunization (Fig.7). The activity of caspase-8 began to increase at the 16th week. At that time, the activity of caspase-8 was 13.41±6.14 pmol/h/mg which was higher than 3.45±0.97 pmol/h/mg (P<0.01) in the negative sera group. After that, the activity of caspase-8 kept increasing. At the 36th week, it reached the peak (34.1±10.09 pmol/h/mg) which was significantly higher than 4.15±1.09 pmol/h/mg (P<0.01) in the negative sera group (Fig.7A). Caspase-9 had the same variation regularity. At the 16th week, it increased to 7.47±0.94 pmol/h/mg which was higher than 1.27±0.18 pmol/h/mg (P<0.01) in the negative sera group. It kept rising. At the 36th week, it reached the peak 16.24±3.31 pmol/h/mg which was higher than 1.38±0.37 pmol/h/mg (P<0.01) in the negative sera group (Fig.7B). The variation regularity of caspase-3 was the same. At the 16th week, it increased to 18.84±7.23 pmol/h/mg which was significantly higher than 1.49±0.18 pmol/h/mg (P<0.01) in the negative sera group. At the 36th week, it reached the peak (25.06±6.80 pmol/h/mg) which was higher than 1.83±0.60 pmol/h/mg (P<0.01) in the negative sera group (Fig.7C).2.1.2 Anti-β1-AR-ECⅡexisting for a long time in the sera could induce the increase of apoptosis in cardiomyocytesAt the 36th week after immunization, there was about 7.86±0.43% cardiomyocytes took place apoptosis which was higher than 0.86±0.29 % (P<0.01) in the negative sera group at the same time (Fig.8G). The representative figures were shown in Fig.8A-F. Among them, Fig.8A-C belonged to negative sera group; Fig.8D-F belonged toβ1AAb group.The above results suggested that anti-β1-AR-ECⅡcould increase apoptosis in cardiomyocytes, but the mechanism of augmented apoptosis need further study.2.2 Anti-β1-AR-ECⅡexisting for a long time in the sera could induce the increase of intracellular free calcium in rats'ventricular myocytesAt the end stage of passive immunization, the intracellular free calcium in rats'ventricular myocytes was detected by confocal microscope. The data showed that the level of intracellular free calcium was 456.34±35.47 in anti-β1-AR-ECⅡgroup which was significantly higher than 51.96±1.18 (P<0.001) in negative sera group (Fig.9).The above results suggested that long-term role of anti-β1-AR-ECⅡcould result in the increase of intracellular free calcium. But how the elevated calcium resulted in apoptosis was not clear. In another word, the downstream signal pathway of calcium inducing apoptosis need further study.2.3 Long-term existence of anti-β1-AR-ECⅡcould result in the increasing expression of CaMKⅡδin cardiomyocytes2.3.1 Western blot resultIn the passive immunization with anti-β1-AR-ECⅡ, the expression of CaMKⅡδincreased with the time (Fig.10). At the 16th week after immunization, the expression of CaMKⅡδincreased to 1.19±0.38 which was higher than 0.39±0.22 (P<0.05) in the negative sera group. After that, the expression of CaMKⅡδcontinued to increase. At the 36th week after immunization, it reached the peak (1.94±0.77) which was higher than 0.46±0.27 (P<0.05) in the negative sera group.2.3.2 Immunohistochemical resultIn the process of passive immunization, the expression of CaMKⅡδincreased gradually. At the 36th week, the expression of CaMKⅡδsignificantly increased which was higher than that in the negative sera group (Fig.11).The above results suggested that the elevated expression of CaMKⅡδin cardiomyocytes may be related with the increased apoptosis of cardiomyocytes in the passive immunization.Summary1. Long-term existence of anti-β1-AR-ECⅡcould result in cardiac remodeling;2. Heart function decreased gradually in the long-term passive immunization with anti-β1-AR-ECⅡ;3. Long-term existence of anti-β1-AR-ECⅡcould result in the increase of apoptosis;4. Long-term existence of anti-β1-AR-ECⅡcould result in the increase of intracellular free calcium;5. Long-term existence of anti-β1-AR-ECⅡcould result in the increasing expression of CaMKⅡδin cardiomyocytes.PART TWO: The Autoantibodies against the Second Extracellular Loop ofβ1-Adrenoceptor Could Induce Arrhythmia in vivo and in vitro and the possibly Underlying MechanismObjective1. To observe whether anti-β1-AR-ECⅡcould induce arrhythmia directly;2. On papillary muscle level and cell level, using microelectrode recording AP and patch clamp to study the possible mechanism of anti-β1-AR-ECⅡinducing arrhythmia which is helpful for the clinical treatment of patients with high level of anti-β1-AR-ECⅡin the sera.Methods(1) Select healthy adult Wistar rats (180~220g, body wt,n=172).The Wistar rats used in the present study were obtained from the Animal Center of Shanxi Medical University, P.R. China. Detect anti-β1-AR-ECⅡin the sera by ELISA. The rats with no anti-β1-AR-ECⅡwere divided into the following four groups: (5 rats died for over dose anesthesia; 4 rats died in nubibus reason)①β1AR group(n=64):inject the mixture of antigen peptides and immunoadjuvant into the dermis of back at the dose of 0.4μg/g, boosting for every 2 weeks. The experiment lasts 40 weeks.②Vehicle group(n=48):inject the mixture of saline and immunoadjuvant into the dermis of back boosting for every 2 weeks. The experiment lasts 40 weeks.③β1AAb group(n=32):Purify the IgG from sera obtained from active immunized rats. Then quantity the total IgG by BCA method. Inject the purified IgG into vena caudalis of rats at the dose of 0.7μg/g boosting for every 2 weeks. The experiment lasts 40 weeks.④Negative sera group(n=28):Purify the negative sera, then quantity the total IgG by BCA method. Inject the purified IgG into vena caudalis of rats at the dose of 0.7μg/g, boosting for every 2 weeks. The experiment lasts 40 weeks.(2) Observe whether arrhythmia can be induced by anti-β1-AR-ECⅡby the measurement of cardiac function and in vivo;(3) Observe whether triggered activity can be induced by adding anti-β1-AR-ECⅡto the isolated papillary muscle of guinea pig and analyze the changes of action potential duration (APD) ;(4) Observe whether arrhythmia can be induced by long-term existence of anti-β1-AR-ECⅡin the active and passive immunization model and analyze the changes of QT interval;(5) Analyze the possible mechanisms of anti-β1-AR-ECⅡinducing arrhythmia by patch clamp and fluorescent assay of intracellular calcium.Results1. Anti-β1-AR-ECⅡcould result in cardiac remodeling of electricity1.1 Anti-β1-AR-ECⅡcould induce arrhythmia on normal rats1.1.1 Anti-β1-AR-ECⅡcould directly induce arrhythmia on normal rats in vivo68μM anti-β1-AR-ECⅡcould induce arrhythmia in 66.7% (6/9) normal rats (weight: 180-220g). The percent was higher than 12.5% (1/8) in negative sera group and 11.1% (1/9) in saline group. Premature ventricular contraction (PVC) was the major type of arrhythmia (Fig.12A). We also could see considerable premature supraventricular contraction (PSVC, Fig.12B). After injecting anti-β1-AR-ECⅡinto artery, we observed the frequency of arrhythmia per hour. We find that the frequency of arrhythmia is 16±10.28 times/hour in anti-β1AR-ECⅡgroup which was higher than 4±1.00 times/hour (P<0.01) in saline group and 5±0.82 times/hour (P<0.01) in the negative sera group (Fig.12C).1.1.2 Anti-β1-AR-ECⅡcould induce triggered activity on the papillary muscles of guinea pigsAdding 0.1μM anti-β1-AR-ECⅡto the papillary muscle of guinea pig, almost all isolated papillary muscles took place triggered activity inducing by train (Fig.13B). Moreover its role didn't attenuate with time (the longest observation time is 2 hours). Its role was similar to Isoprenaline (ISO, 1μM, Fig.13A). But the papillary muscle displayed more sensitive in anti-β1-AR-ECⅡgroup than ISO. The triggered activity induced by anti-β1-AR-ECⅡand ISO could be blocked by metoprolol (MET) which was a selectiveβ1-AR antagonist (Fig.13C). Moreover, anti-β1-AR-ECⅡcould prolong the APD of papillary muscle of guinea pig to 355.5±32.98 ms which was higher than 306±18ms (P<0.05) in saline group and 277.2±27.30 ms (P<0.01) in negative sera group. MET could block the role of anti-β1-AR-ECⅡprolonging APD (P<0.001, Fig.13D).1.1.3 Anti-β1-AR-ECⅡcould induce after-depolarization and triggered activity on the papillary muscles of ratsAdding 1μM anti-β1-AR-ECⅡto the papillary muscles of rats, most isolated papillary muscles (60%, 9/15) took place triggered activity inducing by train (Fig.14A). EAD could be observed on some other papillary muscles (46.7%, 7/15, Fig.14B). Moreover DAD could be observed on some papillary muscles (26.7%, 4/15, Fig.14C). EAD, DAD and triggered activity could not be observed in ISO (1μM) group. MET (10μM) could inhibit EAD, DAD and triggered activity induced by anti-β1-AR-ECⅡ(Fig.14D).The above results suggested that anti-β1-AR-ECⅡcould induce arrhythmia by inducing triggered activity and after depolarization on normal cardiomyocytes. Because anti-β1-AR-ECⅡexited in our body for a long time, so what were the exact effects of anti-β1-AR-ECⅡon electrocardio-activity need further study.1.2 Long-term existence of anti-β1-AR-ECⅡcould induce arrhythmia1.2.1 In the process of immunization with peptides according to the sequence ofβ1-AR-ECⅡ, different kinds and degrees arrhythmia could be observed in rats; moreover Q-T interval was prolonged obviously1.2.1.1 In the process of active immunization, the level of anti-β1-AR-ECⅡshowed a naturally generated and extinctive processAt the 4th week after first immunization, the level of anti-β1-AR-ECⅡwas 0.4±0.21 which was higher than 0.10±0.05 (P<0.05) in the vehicle group. After that, the level of anti-β1AR-ECⅡincreased more and more. At the 8th week, it reached the peak.