Elsevier

Clinical Immunology

Volume 212, March 2020, 108346
Clinical Immunology

Impairment of agonist-induced M2 muscarinic receptor activation by autoantibodies from chagasic patients with cardiovascular dysautonomia

https://doi.org/10.1016/j.clim.2020.108346Get rights and content

Highlights

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    The role of serum autoantibodies in Chagas disease-related dysautonomia was examined.

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    M2 muscarinic receptor antibodies inhibit agonist-induced Gi protein activation.

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    These antibodies also inhibit agonist-mediated arrestin-2 recruitment.

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    Anti-M2 receptor autoantibodies could function as negative allosteric modulators.

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    These autoantibodies could play a pathogenic role in chagasic dysautonomia.

Abstract

Previous studies showed that circulating autoantibodies against M2 muscarinic receptors (anti-M2R Ab) are associated with decreased cardiac parasympathetic modulation in patients with chronic Chagas disease (CD). Here we investigated whether the exposure of M2R to such antibodies could impair agonist-induced receptor activation, leading to the inhibition of associated signaling pathways. Preincubation of M2R-expressing HEK 293T cells with serum IgG fractions from chagasic patients with cardiovascular dysautonomia, followed by the addition of carbachol, resulted in the attenuation of agonist-induced Gi protein activation and arrestin-2 recruitment. These effects were not mimicked by the corresponding Fab fractions, suggesting that they occur through receptor crosslinking. IgG autoantibodies did not enhance M2R/arrestin interaction or promote M2R internalization, suggesting that their inhibitory effects are not likely a result of short-term receptor regulation. Rather, these immunoglobulins could function as negative allosteric modulators of acetylcholine-mediated responses, thereby contributing to the development of parasympathetic dysfunction in patients with CD.

Introduction

Patients with chronic Chagas disease (CD) can exhibit alterations in cardiac autonomic regulation. Both inflammatory and degenerative lesions of the intrinsic autonomic innervation of the heart – particularly at the parasympathetic branch- as well as abnormal autonomic regulation of heart rate and blood pressure (cardiovascular dysautonomia) have been reported in chronic patients with cardiac, digestive or combined forms of CD [[1], [2], [3]]. Early autonomic involvement has also been demonstrated in patients with the indeterminate or apparently asymptomatic form of T. cruzi infection [4,5].

The identification and pharmacological characterization of functional circulating autoantibodies against autonomic neurotransmitter receptors (β1- and β2-adrenergic receptors, and M2 muscarinic acetylcholine receptors) (anti-β1AR Ab, anti-β2AR Ab and anti-M2R Ab) in patients with chronic CD suggested that the interaction between those antibodies and their target receptors could be involved in the development of clinical manifestations. Actually, different studies have shown a high prevalence of anti-β1AR Ab in chronic chagasic patients with ventricular arrhythmias [6] as well as high levels of anti-M2R Ab in those with sinus node dysfunction [6], increased ventricular repolarization heterogeneity [7], esophageal achalasia [8] and megacolon [9,10]. Interestingly, a high prevalence (98%) of anti-β1AR Ab combined with anti-M2R Ab in CD patients with cardiomyopathy vs 34% in those asymptomatic suggests that the latter are at risk of developing cardiomyopathy in the future [10]. Among these studies, a remarkably strong association between anti-M2R Ab and cardiovascular dysautonomia was found in both symptomatic and asymptomatic chronic infected patients [[11], [12], [13]], suggesting that this antibody fraction could serve as a marker for cardiac autonomic dysfunction in chronic CD. Moreover, further studies demonstrated a significant negative correlation between serum levels of anti-M2R Ab and the heart rate variability (HRV) high-frequency power (HF), which suggests an inhibitory effect of these antibodies on vagal modulation [5].

The general aim of this study was to elucidate the immunological and pharmacological bases for the inhibitory role of anti-M2R Ab on cardiac parasympathetic modulation in CD. Most previous studies have focused on pathogenic mechanisms for anti-M2R Ab on the basis of their ability to activate their target receptor (agonist-like activity) [6,13,14], but only a few reports have actually envisioned their potential to impair agonist-induced M2R activation, leading to a decreased parasympathetic activity [11,12,15,16]. Because previous studies had found that anti-M2R Ab could enhance M2 muscarinic receptor/receptor (M2R/M2R) interaction by receptor crosslinking [17], we hypothesized that a crosslinking-mediated conformational change in the M2R elicited by these antibodies would promote impairment of agonist-induced M2R activation, leading to the attenuation of M2R-associated signal transduction pathways. According to this view, we investigated whether the exposure of M2R to anti-M2R Ab from chagasic patients with dysautonomia could inhibit carbachol-induced Gi protein activation and arrestin-2 (Arr-2) recruitment in a crosslinking-dependent manner. In addition, we examined the potential involvement of short-term M2R regulation and allosteric inhibition in these modulatory effects.

Section snippets

Patients

Patients with chronic CD and healthy noninfected volunteers were recruited at DF Santojanni Hospital (Buenos Aires, Argentina). Diagnosis of CD was made on the basis of two positive out of three standard serological reactions against T. cruzi [17]. All individuals were subjected to clinical and cardiological assessment, including standard 12-lead ECG, 24-h Holter ECG and 2-D echocardiography. Those ones with chronic systemic diseases, any infectious diseases other than CD or previous treatment

Clinical and serological characterization of patients

The overall patient population (n = 45) had a median age of 55 (20) years and two-thirds were women. Age, gender, and hemodynamic data were similar across the three groups (Table 1). Both frequency and serum reactivity for anti-M2R Ab were higher in DCD patients (n = 15) than in NDCD (n = 15) or control subjects (n = 15), but no statistical differences were found between the two latter groups (Table 1, and Fig. 1A). A strong association between anti-M2R Ab and dysautonomia in chagasic patients

Discussion

The clinical and prognostic value of cardiovascular dysautonomia in Chagas heart disease is still a matter of debate [3,26]. However, this syndrome – particularly parasympathetic dysfunction – has been associated with arrhythmias and sudden death in other clinical conditions [27]. These findings, together with the early appearance of cardiac autonomic abnormalities suggest that cardiovascular dysautonomia could be associated with increased morbidity and mortality in CD [3,27]. Given that serum

Conclusion

This study proposes a novel inhibitory role for anti-M2R Ab in cardiac parasympathetic dysautonomia secondary to CD (Fig. 7). These antibodies interact with and crosslink M2R – most probably engaged in homooligomers – thereby promoting a conformational change in the receptor molecule, which generates an impaired activation state upon agonist binding. As a result, anti-M2R Ab elicit inhibition of both agonist-mediated Gi protein activation and Arr-2 recruitment, most likely through an allosteric

Source of funding support

This work was funded by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (PIP grants No 0816 to JCG and No 0377 to CIW) and Fundación Alberto J. Roemmers (grant for medical research to SPB). SPB was supported by a CONICET fellowship. CIW and JCG are established researchers from CONICET.

Declaration of Competing Interest

None of the authors has any conflict of interests with the subject matter or materials discussed in this manuscript.

Acknowlegdements

We would like to thank J. Miranda Dávalos, A.C. Santos and C.A. Gou for their valuable technical assistance.

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