Chagas disease (CD) is one of the four leading causes of death from infectious and parasitic diseases in Brazil.^{1 - 3} The elucidation of its pathogenesis was marked by pivotal studies that evaluated cross-reactivity between the ribosomal P protein of Trypanosoma cruzi and human ribosomal proteins (molecular mimicry), as well as by advances in the knowledge of the structure of β-adrenergic receptors, used as antigens to screen for specific anti-β-adrenergic antibodies in chronic Chagas cardiomyopathy (CCC).^{4 , 5}

Köerbele⁶ advocates that CCC is essentially neurogenic, while most authors note it is characterized by irreversible damage caused by the presence of autoantibodies.^{7 , 8} Recent studies have called into question the primary nature of autonomic nervous system abnormalities. Endomyocardial biopsies show continuous and progressive destruction of myocardial fibers with cardiac remodeling, as well as the presence of inflammatory and neuronal mediators in Chagas-related HF and HF of other etiologies.^{9 - 11}

The extent of myocardial damage leads to progressive ventricular dilatation with impairment of cardiac receptors and consequent autonomic dysfunction, which can predate the onset of ventricular dysfunction and occur at all stages of the disease.^{12 - 14}

Anti-β1 and anti-M2 antibodies detected early in the natural history of the disease seem to exert effects on cardiac electrogenesis through an agonistic action similar to that of adrenaline and acetylcholine, respectively. The various forms of Chagas heart disease cannot be distinguished by high serum levels of these antibodies, nor by markers of left ventricular function.^{15 - 19}

In heart failure (HF) of other etiologies, the presence of anti-β1 autoantibodies causes prolonged activation of these cardiac receptors, inducing cell dysfunction, arrhythmias, and apoptosis^{12 , 13 , 18 , 19 , 20} , but in CD, some patients with CCC exhibit low levels of norepinephrine, unlike those with HF in the same functional class but of non-Chagas etiologies.^{21 , 22}

Agonists for receptors coupled pleiotropically to multiple cell signaling pathways may preferentially signal some pathways over others, i.e., display bias towards certain signals. The agonist-like and allosteric effects of autoantibodies and autonomic modulation in CD warrant further investigation.

Within this context, the present study investigated the association of high titers of anti-β1 and anti-M2 antibodies in CCC with autonomic dysfunction as assessed by heart rate variability (HRV) and the behavior of the rate-pressure product (RPP) during cardiopulmonary exercise testing (CPET).

This was a cross-sectional study of 64 patients with CCC recruited from the outpatient cardiomyopathy clinic at the Instituto Nacional de Cardiologia (INC). This convenience sample was followed for a period of 3 years and 8 months, and is part of an extensive database created as part of a doctoral research project.^{23 - 24}

The study sample was approved by the INC Research Ethics Committee, and all patients read and signed an informed consent form.

Inclusion criteria were age over 18 years and positive serology by two methods (hemagglutination and ELISA).²⁵ Patients with pacemakers, on stem cell therapy, with insulin-dependent diabetes mellitus, stage IV hypertension, chronic kidney disease (serum creatinine ≥ 2.5 mg/dL), anemia (hemoglobin < 9.0 mg/dL), thyroid disease, or heart valve disease with hemodynamic compromise were excluded from the sample.

All patients received optimized clinical treatment for HF according to their NYHA functional class. Drugs with β-adrenergic action, vasodilators, diuretics, statins, angiotensin-converting enzyme inhibitors (ACEI), angiotensin receptor blockers (ARB), and antiarrhythmics were prescribed as per routine practice.

Autoimmune reactions directed at G protein-coupled receptors and the production of anti-β1 and anti-M2 autoantibodies acting on these receptors are often associated with changes in HRV, with a consequent increase in morbidity and mortality in patients with HF.^{31 , 32} Decreased HRV is considered an independent risk factor for arrhythmic events and all-cause mortality. Some authors have shown that a lower HRV implies a loss of cardioprotective activity of the parasympathetic system. These changes in HRV occur both in patients without functional LV abnormalities and in those with regional wall motion abnormalities and an EF lower than 50%.^{12 , 13 , 16 , 31 , 33 , 34}

Data obtained over the last three decades suggest that at least some cases of dilated cardiomyopathy (DCM) could be secondary to an autoimmune disease caused by viruses or protozoa, or even by infections that induce autoimmunity in predisposed individuals.^{13 , 16 , 19}

Autoantibodies that modulate the function of receptors that transduce the regulation of heart rate and contractility by the autonomic nervous system may be implicated in DCM pathogenesis. They are also present in HF of other etiologies and atrial tachyarrhythmias. Whether these autoantibodies can actually cause HF or are just an epiphenomenon in the course of disease, however, remains unknown.³⁵

In the last decade, humoral autoimmunity against β1 adrenergic and cholinergic receptors has come to be considered one of the probable causes of chronic HF. Therapeutic concepts have been directed towards this pathogenic process, and have shown promise in the treatment of end-stage DCM.^{10 , 24}

In CD, evidence suggests that the continuous action of both anti-β1 and anti-M2 autoantibodies is implicated in Chagas cardiomyopathy. Studies in rodents subjected to immunization with peptides representing the sequence of the second extracellular loop of the β1 adrenergic receptor developed left ventricular dilatation and dysfunction, with consistent evidence of chronic stimulation and desensitization of β1 adrenoceptor signaling, effects that were reversible upon removal of the antibodies. These studies support the hypothesis that β1 adrenoceptor G protein-coupled autoantibodies are a decisive cofactor in chronic HF.^{36 , 37}

The present study showed that increased anti-β1 and anti-M2 receptor antibody titers seem to alter nighttime HRV (2:00 to 6:00 a.m.) and exert modulating effects on membrane receptors. A decrease in sympathetic activity was observed during this period in patients with high anti-β1 antibodies, paradoxically to their expected agonist effect. The higher the anti-β1 antibody titer, the lower the SDNN (a marker of sympathetic activity), and the higher the anti-M2 titer, the greater the SDANN, which also reflects sympathetic activity. These changes occurred at night, where an exacerbation of parasympathetic activity could be a relevant trigger in precipitating atrioventricular blocks, symptomatic bradycardia, and syncope.

Considering that both anti-β1 and anti-M2 antibodies behave as allosteric modulators via signal transduction, in settings where sympathetic activity is exacerbated or the parasympathetic activity is blunted, they would act in a modulatory manner to maintain overall balance. The youngest patient in this study was 33 years old and had high titers of anti-β1 antibodies, while the oldest was 87 and had high concentrations of both anti-β1 and anti-M2 antibodies.

These antibodies seem to exert both an inhibitory and a stimulating effect on muscarinic or adrenergic receptors. Whether these effects play any role in a protective response against nighttime tachyarrhythmias and/or bradyarrhythmias through signal transduction remains unclear.

Classically, the stimulation of adrenergic receptors consists of a signaling cascade involving the G_s (stimulatory) protein and activation of adenylyl cyclase, which is accompanied by an increase in cyclic AMP (cAMP) and PKA (cAMP-dependent protein kinase).³⁸

Constant stimulation of β1 adrenergic receptors alters the Ca²⁺-dependent PKA signaling pathway and regulates the Ca²⁺ current by mechanisms involving Ca²⁺/calmodulin-dependent protein kinase II, leading to myocyte apoptosis and changes in cardiac adaptation mechanisms, which in turn lead to ventricular remodeling.^{20 , 23}

Some proteins have allosteric and modulating properties and bind to their substrates non-competitively, with the ability to produce a conformational change in protein structure. Proteins are dynamic compounds, and have multiple functional or conformational states. A signaling molecule interacts with its receptor and activates cellular mechanisms, producing a second signal or change in activity in the cellular protein, altering the metabolic activity of the target cell.^{16 , 38}

In HF of other etiologies, the presence of anti-β1 antibodies is associated with greater morbidity, and some authors believe that these antibodies can be considered predictors of mortality. The CIBIS-ELD study used a novel ELISA approach to investigate the relationship between anti-β1 antibodies in a cohort of HF patients and found that patients with an ejection fraction < 50% and a diastolic diameter > 55 mm had higher heart rates and significantly higher anti-β1 antibody titers.³⁹

Beta-blocker therapy, despite being highly effective, is insufficient to prevent clinical deterioration in the presence of anti-β1 autoantibodies.⁴⁰ In CD, there is evidence showing that patients with CCC have low levels of norepinephrine, unlike their counterparts at the same functional class but with HF of non-Chagas etiologies.²¹

This study also assessed whether anti-β1 and anti-M2 antibodies would have influenced physiological responses during exercise in patients with CD.

Some CPET variables are markers of severity, including HR reduction in the first minute of recovery and a decline in blood pressure during exercise. However, these parameters were not associated with the presence of anti-β1 or anti-M2 antibodies. It is well known that initial exertion-induced tachycardia depends on suppression of vagal activity, while further increases in HR depend on autonomic (specifically, sympathetic) increments according to exercise intensity.³⁰

In this study, a normal chronotropic response and RPP were found to be associated with high anti-β1 titers. Patients with a normal inotropic response had high anti-M2 titers. The agonistic action of these antibodies on β adrenergic receptors explains the increase in HR and blood pressure. The increase in RPP was attributed to the additional positive allosteric modulating action of the anti-β1 adrenergic antibody during exercise, acting as a ligand that modifies the action of an orthosteric agonist, endogenous activator, or antagonist by binding to the allosteric site of the receptor.³⁸

Higher anti-M2 titers reflect a modulating action or interaction with other signaling pathways (signal transduction), which stimulate sympathetic action in the heart and other systems that participate in the ANS signaling cascade.³⁸ Anti-M2 antibodies appear to exert both negative and positive allosteric modulating actions. Both anti-β1 and anti-M2 antibodies modulate the cascade of adaptive ANS responses during exercise.^{41 , 42}

Considering the findings of previous studies on autoantibodies,³⁷ at least three distinct effects of these antibodies on their respective receptors must be considered: they modulate binding of true ligands; they activate various effector pathways; and they modulate the disposition of the receptor and simultaneous binding to the receptor. However, how autoantibodies cause changes in receptor activity and how this relates to the pathogenesis and severity of CCC remains unclear.

Whether the presence of circulating antibodies has any role in the clinical outcome of patients with CCC is still an open matter; future clinical trials are needed to assess the prognostic nature of these antibodies. Some authors have shown that immune cross-reactivity is not always harmful, and a balanced parasite/host relationship may exist, allowing the disease to remain clinically silent for many years.^{11 , 19}

In Chagas’ heart disease, myocardial remodeling seems to be related to etiopathogenic mechanisms linked to neuronal activation. The prognosis is worse compared to that of other cardiomyopathies, with a disturbingly high incidence of sudden death secondary to arrhythmias, and current therapeutic approaches are based on results obtained in heart diseases of another etiologies.^{12 , 13 , 16}

Including measurement of anti-β1 and anti-M2 autoantibody in the routine workup of patients with CD, plus exercise testing and 24-hour Holter monitoring, would create a panel for assessment of the whole spectrum of HRV behavior and its relationship with autoantibodies in the long-term follow-up of these patients.

This study has some limitations, including: 1) the absence of a control group for comparison of findings; 2) the fact that the sample was not separated into groups by NYHA functional class for analysis of HRV and autoantibody behavior according to the degree of cardiac remodeling; 3) the fact that autoantibody titers were not compared with those measured in cardiomyopathies of other etiologies; 4) the lack of functional testing of anti-β1 and anti-M2 antibodies (in a Langendorff preparation) from the serum of these patients for comparison with HRV behavior and measurement of a potential active role of these antibodies in ANS modulation, since functionally active antibodies can modulate vagal action even in the absence of structural heart disease.^{43 - 45} Finally, since autoantibodies are part of the immune background of heart disease, 5) the lack of tools and diagnostic criteria specific for CCC makes it difficult to interpret the data and monitor the natural history of these patients.

The data presented herein show that anti-β1 and anti-M2 autoantibodies seem to exert a modulating effect on the autonomic nervous system, altering nighttime measures of HRV (SDNN and SDANN). There was an inverse association between anti-β1 antibodies and sympathetic activity, and a direct association between anti-M2 and sympathetic activity during this period.

The rate-pressure product correlated significantly with anti-β1 antibody titers, although as an independent variable (5%), the association was weak.