Autoantibodies against M2-muscarinic acetylcholine receptors: new upstream targets in atrial fibrillation in patients with dilated cardiomyopathy

Akiyasu Babaa,*, Tsutomu Yoshikawab, Yukiko Fukudab, Takashi Sugiyamac, Megumi Shimadaa, Makoto Akaishia, Kanji Tsuchimotoa, Satoshi Ogawab and Michael Fud

a Department of Medicine, Kitasato Institute Hospital, Tokyo, Japan
b Department of Medicine, Keio University School of Medicine, Tokyo, Japan
c Center for Clinical Pharmacy and Clinical Sciences, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan
d Wallenberg Laboratory, University of Gothenburg, Gothenburg, Sweden

* Corresponding author. Present address: Department of Cardiology, Kitasato Institute Hospital, 5-9-1 Shirokane Minato-ku, Tokyo, Japan. Tel.: +81-3-3444-6161; fax: +81-3-3448-0553
E-mail address: baba-a{at}kitasato.or.jp

Received 2 December 2003; revised 22 April 2004; accepted 12 May 2004 See page 1091 for the editorial comment on this article1

Abstract

Aim To characterise the clinical significance of M2-muscarinic acetylcholine receptor autoantibodies (M2-AAB) in patients with dilated cardiomyopathy (DCM).

Methods and results Sera from 104 patients with DCM, age-matched with 104 patients with idiopathic atrial fibrillation (Af) and 104 healthy control subjects, were screened for M2-AAB by enzyme-linked immunosorbent assay (ELISA). IgG purified by Protein-A column was also used as a primary antibody in ELISA. In DCM, M2-AAB were detected in 40% of patients using whole sera and in 36% of patients using purified IgG. M2-AAB were also found in several patients with idiopathic Af (23%, 23%), and these frequencies were significantly higher than those in healthy subjects (8%, 8%). Af was more common in AAB-positive than in AAB-negative patients with DCM. Multivariable analysis confirmed that M2-AAB were independent predictors of the presence of Af in such patients. We determined electrophysiological changes by adding patient purified M2-AAB to chick embryos. Purified IgG from both Af and DCM patients exhibited negative chronotropic effects and induced supraventricular arrhythmias.

Conclusion M2-AAB may play a role in mediating the development of Af in patients with DCM.

Key Words: Dilated cardiomyopathy • Autoantibodies • Atrial fibrillation

Introduction

Autoimmunity, viral infection, and genetic predisposition have been proposed as causes of idiopathic dilated cardiomyopathy (DCM). Among them, autoantibodies have become one of the cornerstones in mediating the pathophysiology in such patients. In the programmed cell death-1-deficient mice, in which autoimmune-mediated DCM was induced, autoantibodies against cardiac troponin I was detected, and monoclonal antibody against cardiac troponin I was able to induce cardiac dilatation and dysfunction in wild-type mice.1 Transfer autoantibodies against ß1-adrenoreceptor to healthy rats could induce dilatation and dysfunction of hearts.2 These functional autoantibodies are not limited in the patients with DCM, since anti-myosin autoantibodies were detected in 6 of 10 patients with idiopathic paroxysmal atrial fibrillation (Af) who have no known cardiac abnormalities.3

Af is one of the most common arrhythmias in patients with congestive heart failure (CHF), but the "upstream" target of this arrhythmia remains to be determined.4 Cellular electrical remodelling in Af includes shortening of action potential duration (APD) and effective refractory period that can be explained by concomitant alterations in ion channel activity.5 Data from a dog model suggested that electrical remodelling in the presence CHF did not involve the shortening of APD and the characteristic loss of rate adaptation typically observed by electrical remodelling in the absence of CHF.6

With respect to electrophysiological effects of autoantibodies, anti-ß1-adrenoreceptor autoantibodies exerted positive chronotropic effects7 whereas anti-M2-muscarinic acetylcholine receptor autoantibodies shortened APD.8 Therefore we inferred that anti-M2-muscarinic autoantibodies could be one of the "upstream" targets of Af in patients with DCM. Idiopathic Af may serve as a control as opposed to DCM with permanent Af in which significant cardiac dysfunction is co-existent. We therefore sought to determine the clinical significance of M2-muscarinic autoantibodies in patients with DCM and in those with idiopathic Af. We further evaluated the direct electrophysiological effects of these autoantibodies in chick embryos.

Methods

Study patients
One hundred and four patients with DCM, who were clinically stable using conventional therapy that included digitalis glycosides, diuretics, angiotensin-converting enzyme inhibitors or beta-blockers, were treated in the Keio Interhospital Cardiology Study (KICS) group and were recruited to participate in the present study between 1998 and 2002. The diagnosis of DCM was based on World Health Organization/International Society and Federation of Cardiology criteria established in 1995.9 A left ventricular ejection fraction (LVEF) of less than 45% was confirmed by radionuclide ventriculography. Coronary angiography was performed in all patients, and myocardial biopsy was performed in 85 patients (82%). Patients with 75% stenosis of the major coronary arteries, myocarditis or one of the other specific cardiomyopathies were excluded.

We also studied 104 idiopathic Af patients and 104 healthy control subjects, who were matched to the DCM patients for gender and age. All cases visited the KICS group in the objective of physical checkup. We defined Af as supraventricular tachyarrhythmia characterised by unco-ordinated atrial activation with consequent deterioration of atrial mechanical function. All "Af" in this study indicate permanent Af according to the classification of the ACC/AHA/ESC Guidelines in 2001.10

Our present study complied with the Declaration of Helsinki. The study protocol was approved by an Institutional Review Committee, and all patients gave their written, informed consent to participate in the present study.

Antibody detection
The autoantibodies of interest were measured in patient sera and purified IgG with an enzyme-linked immunosorbent assay (ELISA), using a synthetic peptide corresponding to the putative sequence of the second extracellular loop of human M2 receptors (amino acid sequence number, 169–193: Val-Arg-Thr-Val-Glu-Asp-Gly-Glu-Cys-Tyr-Ile-Gln-Phe-Phe-Ser-Asn-Ala-Ala-Val-Thr-Phe-Gly-Thr-Ala-Ile).11 The IgG was purified from patients' serum by the protein A–agarose method, using the Affi-Gel Protein-A MAPS II Kit (Bio-Rad Laboratories, Hercules, CA, USA). Using the Peptide Coating Kit (MK100, Takara Bio Inc., Shiga, Japan), these peptides were coated on microtitre plates according to the manufacturer's instructions. The wells were saturated with the blocking solution, and 50 µl of patients' serum or purified IgG dilutions beginning at 1:20 were then added to the coated and saturated microtitre plates. After the wells were washed three times, a peroxidase-conjugated goat anti-human IgG (H+L) antibody (1:1000 dilution, Biosource, Camarillo CA, USA) was added and allowed to react for 1 h. After washing, the bound peroxidase-conjugated antibody was detected by incubating the plates for 1 h with orthophenylenediamine dihydrochloride (Organon Teknika, West Chester, PA, USA). Sulphuric acid (2.5 M) was added to stop the reaction and the optical density was read at 492 nm in a microtitre reader with a positive finding defined as an optical density that was 2.5 times the background density.

Clinical variables
Age, gender, New York Heart Association (NYHA) functional class, LVEF, and other clinical parameters of patients with and without the M2-muscarinic autoantibodies were compared. Clinical variables, including findings from ultrasonic echocardiography and Holter monitoring, were recorded for all patients within 1 month of serum sampling for measurement of autoantibodies. Echocardiography was performed using a commercially available probe and system to assess left ventricular end-diastolic dimensions, end-systolic dimensions, left atrial dimensions, and severity of mitral regurgitation. The Holter electrocardiogram (ECG) was analysed on two standard channels by using the Marquette system (SXP 8000 or MARS 8000) to assess the basal heart rhythm, total number of supraventricular premature contractions (SVPCs) and premature ventricular contractions (PVCs), and occurrence of ventricular tachycardia (VT). PVCs were defined as any beats of ventricular origin faster than the sinus rate. VT was defined as three or more consecutive PVCs with a heart rate 100 beats/min, which was noted in at least one of the tests. Data were averaged from two or more sets of recordings. Blood samples for measurement of serum potassium, plasma noradrenaline, and atrial natriuretic peptide concentrations were collected from a peripheral vein after the patient had rested in the supine position for at least 30 min. Blood samples were placed in ice and spun in a refrigerated centrifuge to separate the plasma, and stored at –80 °C until analysis by appropriate techniques.

Recording ECG of chick embryos
We recorded the ECG of chick embryos in order to determine the acute physiological effects of the M2-muscarinic autoantibodies. As previously described,12 fertile eggs of White Leghorns (Ohmiya Poultry Laboratory, Saitama, Japan) were incubated at 37.5±0.2 °C at a relative humidity of about 65%, and turned automatically every hour (P-1 type, Showa Incubator Laboratory, Ohmiya, Japan). A single injection of carbachol (2, 4, and 8 mg/egg) or purified IgG (1.0 mg/egg) into the air sac of fertile eggs was performed on the 16th day of incubation. The ECG was recorded as bipolar waves between two needles on a thermal array recorder (PTA-1100M, Nihon Koden, Tokyo, Japan). The tracings were recorded from 0 to 60 min for 30-s intervals every 10 min after IgG injection, which was purified from healthy controls without M2-muscarinic autoantibodies , DCM patients with and without M2-muscarinic autoantibodies , and idiopathic Af patients with and without M2-muscarinic autoantibodies , and the heart rate was calculated from the regular R–R intervals. All these purified IgG fractions were confirmed to be free from other anti-myocardial autoantibodies in our previous experiments.13–15 Alterations in heart rate and the occurrence of arrhythmias were recorded every 30 s in all five groups. In order to check the specific effects of purified IgG, we also performed the blocking experiments using atropine (50 µg/egg) or peptides (1.0 mg/egg) corresponding to the epitope of M2-muscarinic autoantibodies.

Statistical analyses
Data are expressed as mean values±SD. Clinical characteristics of patients were compared among the three groups using the Kruskal–Wallis test, and between the two groups by the unpaired two-sided t-test or Mann–Whitney U test. Autoantibody titres of ELISA were evaluated using two-way analysis of variance (ANOVA) and the Bonferroni post hoc test. Logistic regression analysis was used to assess predictors of Af. Four variables, including those with p values 0.10 (univariate analysis), were further assessed by multivariable logistic regression analysis. ECG data of chick embryos were evaluated using the multi-way repeated measures ANOVA, four-way for experiments of carbachol and nine-way for experiments using patients' IgG. Statistical significance was set at . StatView 5.0 software (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses.

Results

Antibody screening
Autoantibodies directed against M2-muscarinic receptors were found in 40% of patients with DCM and 24% of those with idiopathic Af using whole sera as a primary antibody in ELISA. There was a significant difference in the frequency of M2-muscarinic autoantibodies among the three groups. When purified IgG by protein A column was used as a primary antibody, the positive rate was slightly lower than that determined by whole sera in patients with DCM, but there was no difference between the two methods in those with idiopathic Af or control subjects (Table 1). In idiopathic Af and control subjects, all positive cases using purified IgG were also positive by ELISA using whole sera. Autoantibody titres were higher in patients with idiopathic Af and in those with DCM than in control subjects without any relation to the use of primary antibodies of ELISA (Fig. 1). Antibody titres were similar between whole sera and affinity-purified IgG in both healthy control subjects (47.5±28.2 vs. 50.0±26.2) and in patients with idiopathic Af (78.3±30.0 vs. 83.3±31.0). However, in patients with DCM, the titres determined by whole sera were higher than those determined by purified IgG (154.6±71.3 vs. 184.8±89.6).


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Table 1 Clinical characteristics of all subjects

 


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Figure 1 Autoantibody titres against M2-muscarinic receptors in healthy controls, patients with idiopathic atrial fibrillation (Af) and dilated cardiomyopathy (DCM). Autoantibodies determined by protein A-purified IgG (open bars) and autoantibodies determined by whole sera (closed bars). Data are presented as mean values±SD.

 
Clinical profiles and correlation with autoantibodies
There was a significant difference in the cardio-thoracic ratio on the chest X-ray among the three groups. There was no difference in the left atrial dimension and severity of mitral regurgitation as determined by echocardiography between patients with DCM and those with idiopathic Af.

In patients with DCM, the presence of Af was more common in autoantibody-positive than in autoantibody-negative patients. However, there was no difference between the two groups with regard to age, gender, NYHA functional class, concomitant medications, echocardiographic parameters, LVEF, serum potassium, plasma noradrenaline or atrial natriuretic peptide levels, or other arrhythmias on Holter ECG monitoring (Table 2).


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Table 2 Clinical characteristics of DCM group

 
Univariate analysis of each variable showed that M2-muscarinic autoantibodies, plasma atrial natriuretic peptide level, and use of digitalis and beta-blockers were candidates for multivariable analysis. It was shown that the presence of M2-muscarinic autoantibodies was the sole independent predictor of Af in patients with DCM (Table 3).


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Table 3 Predictors of Af in DCM Group

 
Electrophysiological effects of purified M2-muscarinic autoantibodies
In the chick embryo system, carbachol decreased heart rate in a dose–response manner, and atropine was able to block these effects (Fig. 2(a)). Any purified IgG from patients without M2-muscarinic autoantibodies was unable to affect heart rate until 60 min after injection (Fig. 2(b)). In contrast, heart rate decreased 30–60 min after injection of purified IgG from patients with DCM and also from those with idiopathic Af who were positive for M2-muscarinic autoantibodies. There was no difference in the negative chronotropic effects of M2-muscarinic autoantibodies between the two groups (Fig. 2(c)). Moreover, atrial arrhythmias actually occurred during the observation period when purified IgG from patients with M2-muscarinic autoantibodies were added (Fig. 3, Table 4). Pre-administration of atropine or peptides corresponding to the epitope of autoantibodies completely abolished these effects (Fig. 2(c), Table 4).



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Figure 2 Heart rate change after the injection on chick embryos. (a) Effects of carbachol, (b) effects of purified IgG from patients without M2-muscarinic autoantibodies, and (c) effects of purified IgG from patients with M2-muscarinic autoantibodies. Atropine or epitope peptides were used for blocking experiments. Af, atrial fibrillation, DCM, dilated cardiomyopathy.

 


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Figure 3 Supraventricular arrhythmias which was induced by purified IgG from patients with autoantibodies in chick embryos. Atrial arrhythmia indicated by arrows. N, normal sinus beats.

 

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Table 4 Occurrence of supraventricular premature contractions by adding autoantibodies on chick embryos

 
Discussion

We studied the clinical features of a large sample of DCM patients, and demonstrated that the presence of autoantibodies directed against the second extracellular loop of M2-muscarinic receptors was associated with the occurrence of Af. Such autoantibodies were also found in patients with idiopathic Af. It was demonstrated that atrial arrhythmias occurred when purified autoantibodies were added to chick embryos in vitro.

Detection of autoantibodies directed against M2-muscarinic receptors
A number of studies have shown that autoantibodies directed against M2-muscarinic receptors can be detected in approximately 40% of patients having such conditions as DCM, Chagasic disease, and idiopathic VT.16 A recent report showed that autoantibodies are also found in some patients with ischaemic cardiomyopathy or hypertensive heart disease,17 although the prevalence was lower than in patients with DCM. In previous studies, patients' whole sera have commonly been used as a primary antibody for ELISA. However, we could not establish the role of IgG subclasses of autoantibodies using ELISA with whole sera. The IgG subclass 3 has a more powerful effect in terms of complement activation than other IgG subclasses. In myocardial biopsy samples from DCM patients, not only the deposits of IgG on the cell membrane but also the expression of the terminal factor of complement, C5b-9 complex, were observed.18 A recent report of immuno-adsorption therapy for DCM indicated that autoantibodies belonging to IgG subclass 3 could play an important role in cardiac dysfunction among patients with DCM.19 Therefore, in the present study we performed additional experiments using IgG purified by Protein A column. There was no difference in the frequency and titres between these two methods in patients with idiopathic Af and control subjects. However, both the frequency and titres as determined by whole sera were higher than those determined by purified IgG in patients with DCM. Presence of IgG subclass 3, which cannot bind Protein A, may be responsible for the differences in the frequency and titres for such patients.

Clinical profile of autoantibody-positive patients with DCM
We previously reported that Af had been noted in some younger patients with DCM who had no serious mitral regurgitation. The occurrence of Af in such patients could not be predicted by neurohumoral factors, LV systolic function, or NYHA functional class.20 Therefore, we decided to perform multivariable logistic analysis including various confounding clinical factors to determine the clinical significance of the presence of anti-M2-muscarinic autoantibodies. We believe that this study is the first to show that the presence of M2-muscarinic autoantibodies is the sole independent predictor of Af in such patients. However, it was still unclear whether or not the autoantibodies per se induced Af, since the autoantibodies might be responsible for the co-existent cardiac dysfunction seen in such patients. Therefore, we also examined autoantibodies in patients with idiopathic Af who have almost normal cardiac function. We found that M2-muscarinic autoantibodies were positive in certain patients with idiopathic Af. Thus, the presence of such autoantibodies was associated with Af in the absence of underlying left ventricular dysfunction.

Physiological action of autoantibodies
In the present report, we studied the direct physiological effects of autoantibodies using chick embryos, which provided high reproducibility with minimal inter-individual variability. We performed the experiments under the administration of atropine or human epitope peptides in order to determine the specific action of autoantibodies. The antigen epitope of the M2-muscarinic autoantibodies exists in the 25 amino acid sequences over the putative second extracellular loop of the receptors. These sequences have 80% homologies between chicken and human. The homology is 94% in the 17 amino acids within the C-terminal residue.

Our present experiments using chick embryos yielded twofold results on the effects of M2-muscarinic autoantibodies. The first was the negative chronotropic actions of the autoantibodies, suggesting that M2-muscarinic autoantibodies could affect the sinus node function. A previous study also showed that M2-muscarinic autoantibodies which were obtained from patients with DCM decreased beating rate without inducing desensitisation in cultured rat cardiac myocytes.21 In addition, these autoantibodies inhibited radioligand binding to M2-muscarinic receptors, and enhanced muscarinic receptor-mediated signalling as evidenced by a decrease in isoproterenol-stimulated cyclic-adenosine monophosphate levels in a single guinea pig ventricular cell.22

The second result of our present experiment was the proarrhythmic effect of patients' purified immunoglobulin G, which was confirmed by the occurrence of atrial premature contraction in both patients with idiopathic Af and with DCM. Short-term exposure which lacks of ion channel remodelling was supposed as the reason why our present experiments could not induce the development of the atrial fibrillation. It is also noteworthy that nothing except for the immunoglobulin G was included in patients' sample, which was purified by Protein-A column. This is the first direct evidence, so far as we know, that M2-muscarinic autoantibodies have the pro-arrhythmic effect.

Therapeutic implication
As in atrial remodelling, downregulation of some cardiac ion channels occurs in chronic Af. Several studies have shown that both the mRNA and protein level of Kir3.1–3.4 and current density of IK,Ach were reduced in atrial tissue obtained from patients with chronic Af.23–25 On the other hand, the activity of the acetylcholine esterase, which was responsible for the degradation of acetylcholine, was reduced in chronic Af,26 suggesting that the reduced IK,ACh density might be partly compensated by the increased level of acetylcholine in the synaptic cleft.

Even during chronic Af, the activation of the decreased IK,Ach current could contribute to the initiation and perpetuation of atrial fibrillation. Recent findings have suggested that the IK,Ach is constitutively active in patients with chronic Af.27 Kovoor et al.28 also showed that induction of Af was completely abolished in mice deficient in the IK,ACh channel. It was also well known that shortening of APD, mediated by activation of the IK,Ach can also induce Af.

In this connection, autoantibodies against M2-muscarinic receptors increased outward potassium current, resulting in a shortening of effective refractory period in guinea pig ventricular myocytes,8 although activity of atrial IK,ACh by patients with DCM is also reduced.29 Our present data suggest that the M2-muscarinic autoantibodies could provide an electrophysiological basis of arrhythmia in both patients with idiopathic Af and in those with DCM. In this regard, immuno-modulatory therapy including immuno-suppressive agents and immuno-adsorption technique could be a new therapeutic target in such patients.

Acknowledgments

This study was supported in part by Grant-in-Aid for Young Scientists (No. 14770342, 2002) from the Japan Society for the Promotion of Science, and by Grant-in-Aid for studying abroad (2001) from Mitsukoshi Health and Welfare Foundation.

Footnotes

1 doi: 10.1016/j.ehj.2004.05.001. Back

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