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Clinical Correlates of Increased Levels of Autoantibodies

in Chagas Heart Disease

Andre Talvani, Manoel O.C. Rocha

, Antonio L. Ribeiro

, Enri Borda

,

Leonor Sterin-Borda

& Mauro M Teixeira.

Departamento de Bioqumica e Imunologia, Instituto de Cincias Biolgicas and, Ps-

Graduao em Medicina Tropical, Departamento de Clnica Mdica, Faculdade de Medicina,

UFMG,Belo Horizonte, Brasil andPharmacology Unit, School of Dentistry, University of

Buenos Aires, Buenos Aires, Argentina.

Short title: Anti-M2 autoantibodies in Chagas Heart Disease

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Footnote page:

(1) Informed consent was obtained from all patients and non-infected individuals.

Human experimental guidelines of the Brazilian Ministry of Health were followed in the

conduct of the experiments described here. The authors have not commercial or other

association that may pose a conflict of interest.

(2) This investigation received financial support from FAPEMIG, the UNDP/World

Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR-

WHO A970728) and Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico

(CNPq-Brazil) and National Research Council (CONICET) of Argentina, TDR-WHO

A20771 and University of Buenos Aires (UBACYT), Buenos Aires, Argentina.

(3) Address for correspondence and reprints request

Mauro M Teixeira, M.D.

Departamento de Bioqumica e Imunologia

Instituto de Cincias Biolgicas

Universidade Federal de Minas Gerais

Av. Antnio Carlos, 6627 - Pampulha

31270-901 BELO HORIZONTE MG BRASIL

Phone # 55 31 3499 2651 (direct line)

Fax # 55 31 3441 5963e-mail: mmtex@icb.ufmg.br

Alternative contact:

Dr Leonor Sterin-Borda (leo@farmaco.odon.uba.ar).

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Abstract

Background: Anti-neurotransmitter receptor autoantibodies are found in the serum of

chagasic patients and may play a role in the pathophysiology of the disease. In the present

study, we investigated the presence of autoantibodies and their correlation with the

severity of chronic chagasic cardiomyopathy (CCC). Methods: Anti-M2 cholinergic and

anti-1 adrenergic autoantibodies were measured in sera of non-infected subjects and

chagasic patients with the various clinical forms of the disease using ELISA. Results:

The optical density (OD) and the frequency of anti-M2 and anti-1 antibodies were higher

in chagasic patients than in non-infected subjects. The levels of anti-M2 antibodies were

lower in patients with the indeterminate form than patients with CCC, levels of both

antibodies were similar in patients with CCC, regardless of severity. This is consistent

with lack of correlation between levels of autoantibodies and various parameters of

ventricular dysfunction. Patients with apical lesion, with exercise-induced ventricular

arrhythmias, or those with chronotropic insufficiency had higher levels of anti-M2

autoantibodies. Conclusion: The levels of anti-neurotransmitter autoantibodies in serum

of patients with Chagas heart disease seemed not to correlate with the severity of the left

ventricular dysfunction. Nevertheless, the levels of anti-M2 autoantibodies are increased

in patients in which apical aneurysm, ventricular arrhythmias and chronotropic

incompetence, suggesting that anti-M2 cholinergic autoantibodies may play a role in the

pathogenesis of chagasic cardiac dysautonomia.

Key Words: chagas cardiomyopathy chagas disease neurotransmitter receptor

antibodies anti-adrenergic antibodies anti-cholinergic antibodies

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Introduction

In Latin America, chronic chagasic cardiomyopathy (CCC) affects around 30% of

individuals infected with the protozoan parasite Trypanosoma cruzi (T. cruzi). In a

significant proportion of the latter patients, severe heart disease occurs and is frequently

the cause of death. There are several hypotheses that try to explain the pathogenesis of

severe heart disease in infected individuals, including the role of parasite persistence

(1,2), autoimmune events (3-6) and microvascular dysfunction (7).

Chronic chagasic cardiomyopathy is characterized mainly by a dilated

cardiomyopathy complicated by frequent and complex ventricular arrhythmias and/or

conduction defects (8). Autonomic dysfunction occurs early in the course of the disease

and may associate with worse prognosis (9-12). It has been argued that the blockade of

neurotransmitter receptors by anti-receptor antibodies contributes to the autonomic

dysfunction and worse clinical evolution (12-14). Although a strong association between

circulating antipeptide M2 muscarinic acetylcholine receptor (mAChR) autoantibodies

and the presence of patients' low heart rate variability index, bradycardia and cardiac or

esophageal autonomic dysfunction in chronic chagasic patients was verified (15), it is not

known whether the presence and/or titer of anti-receptor antibodies correlate with CCC

severity.

Here, the presence and levels of anti-adrenergic (1) and anti-muscarinic (M2)

receptor antibodies were evaluated in serum of a group of 58 individuals. We also

investigated the correlation between the titers of autoantibodies and the following aspects

of clinical CCC manifestations: left ventricular systolic function and response to effort.

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Methods

Study population

We performed this initial study with 6 healthy individuals and 52 chagasic

patients with different clinical forms of the disease. All individuals were recruited at the

Referral Center for Training on Infectious and Parasitic Diseases (CTR-DIP) at Hospital

das Clinicas, Universidade Federal de Minas Gerais (UFMG) and underwent a complete

clinical examination and performed the following laboratory workup: full blood count,

free T4, TSH, glucose, potassium, creatinine, blood urea nitrogen, electrocardiogram

(ECG), chest X-Ray, a 24 hour Holter examination, echoDopplercardiography (ECHO)

and a treadmill exercise test. Patients with hypertension, diabetes, thyroid or renal

disturbances or any other cardiac or systemic diseases and those using steroidal drugs

were excluded from this study, as these conditions could prevent adequate interpretation

of cardiac disease severity on immune parameters. The study received ethical clearance

from the Ethics Review Board of Universidade Federal de Minas Gerais.

Chagasic patients were also categorized into groups according to the degree of

heart dysfunction, as previously described (8). Briefly, patients with indeterminate form

(IND) (n=8) or chronic chagasic cardiomyopathy grade I (CCC I) (n=8) were those with

normal ECG and radiological studies or with only minor alterations in their ECHO (e.g.

regional contraction defects), respectively. Patients classified as CCC II/III (n=7) were

those with minor or moderate ECG alterations, including block of the anterosuperior

division of the left branch, right bundle branch block or uniform ventricular premature

contractions. Patients classified as CCC IV (n=15) were those manifesting severe

conduction defects (e.g. left bundle branch block, left anterior divisional block with right

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bundle branch block or total atrioventricular block) or complex ventricular arrhythmias

(complex ventricular premature beats, non-sustained or sustained ventricular

tachycardia). Finally, patients classified as CCC V were those with ventricular

enlargement, as observed on the ECHO, irrespective of the presence or not of

arrhythmias or conduction defects (8). The control group was formed by non-infected

(NI) healthy individuals (Table 1).

A maximal stress test was performed according to the standard Bruce protocol.

Chronotropic insufficiency was arbitrarily defined as the inability to achieve at least 85%

of the predicted heart rate according to Astrands formula (220-age) at peak exercise (16).

Patients with exercise-induced arrhythmias were those who presented non-sustained

ventricular tachycardia or increase ventricular premature beats number (NVPB) or

complexity (polimorphic or repetitive forms) clearly related to the exercise. Patients

underwent ECHO with color flow using an ATL Philips HDI 5000 apparatus operated by

an experienced echocardiographer, blinded to the clinical status of the patients.

Segmental contractility was evaluated according to the method described by American

Society of Echocardiography (17) and typical apical lesions in the left ventricle wall were

recognized. The left ventricular ejection fraction (LVEF) was obtained by Simpson's

method using the software provided with the equipment.

Measurement of antibodies against anti-M2 cholinergic and anti-1 adrenergic receptors

Serum samples were obtained by conventional venipuncture, centrifuged and

stored at -80oC until used in an immunoassay (ELISA) with M2 synthetic cholinergic

peptide (18) and 1 adrenergic synthetic peptide (14) as coating antigens, as previously

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described (19). The samples were assayed in parallel at a 1/50 dilution and optical

density (OD) values were measured with an ELISA reader (Uniskan Laboratory System).

This antibody dilution was found to be optimal to separate chronic chagasic

cardiomyopathy patients from control indeterminate form of Chagas heart disease.

Statistical analysis

Data are expressed as means SEM or median and interquartile range. Analysis

was performed using the computer program GraphPrism (GraphPad, San Diego, CA,

USA). Comparison between groups carried was out by using Analysis of Variance

(ANOVA) followed by Student-Newman-Keulss post test (parametric distribution) or

Kruskal Wallis followed by Dunns post test (non-parametric distribution). For

association between variables, data were analyzed using linear regression analysis and

Spearmans rank correlation test. Probability values were considered significant when

p

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Results

There was no significant difference in the age distribution of non-infected

individuals and chagasic patients (Table 1). In agreement with the clinical parameters

used to classify the group, patients with CCC V had lower left ventricle ejection fraction

and greater diastolic diameter in comparison with patients with the other degrees of CCC.

There was great variation in the number of ventricular premature beats in 24 hour and

patients with CCC II/III or worse had greater number of premature beats than those with

IND form or CCC 1 (Table 1).

The distribution of anti-M2 cholinergic and anti-1 adrenergic autoantibodies

detected by ELISA is shown in Table 2. It can be seen that the frequency of anti-M 2

cholinergic autoantibody was higher in CCC patients (mean values: 86.3%) than in the

IND form (mean values 37.5%) of Chagas disease. The non-infected individuals were

negative in the study system. When the distribution of anti-1 adrenergic autoantibodies

was evaluated no differences in the frequency between CCC patients (mean value 67.2%)

and IND (mean value 77.1%) was observed. Table 2 also shows the distribution of both

autoantibodies in the different degrees of CCC and it can be appreciated that no

differences existed between the different degrees of CCC.

Figure 1 shows the levels of cholinergic (M2) and adrenergic (1) autoantibodies

in serum from non-chagasic and chagasic individuals. It can be seen that chagasic

patients presented greater levels of both autoantibodies. However, when chagasic patients

were grouped according to disease severity (8), there was no difference in the OD values

of both M2 and 1 autoantibodies among the different clinical groups (Figure 1). The lack

of correlation between levels of autoantibodies and disease severity is further re-enforced

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when the levels of antibodies and clinical parameters of ventricular dysfunction were

compared (Table 3).

Interestingly, patients presenting apical lesion in the left ventricle had higher

levels of anti-M2 antibodies (Figure 2), but not anti-1 antibodies than patients without

apical lesion (Figure 2). Patients that presented ventricular arrhythmias during or

immediately after exercise had higher levels of anti-M2 antibodies, but not anti-1

antibodies than patients who did not present exercise-induced arrhythmias (Figure 3A).

Moreover, the levels of anti-M2 antibodies were higher in patients with than those

without chronotropic insufficiency (Figure 3B). The levels of anti-1 antibodies were

similar in patients with or without chronotropic incompetence (Figure 3B).

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Discussion

Global systolic left ventricular dysfunction is the strongest predictor of morbidity

and mortality during Chagas heart disease (20,21). It has been argued that an

autoimmune response against antigens present in heart tissue may favor the development

of the more severe forms of Chagas cardiomyopathy. Antibodies against adrenergic and

cholinergic receptors are among the many autoantibodies that have been described in

Chagas disease. For example, antibodies against 1-adrenoceptores and M2 mAChR have

been found in the sera of patients and experimental animals with Chagas disease

(5,12,14). These antibodies may induce acute functional alterations of isolated hearts

from experimental animals (e.g. enhance or decrease contractility) and may also interact

with the respective receptor and induce sequestration and endocytosis of the receptor (22-

25). Thus, it is clear that chagasic patients have anti-M2 or anti-1 receptor antibodies and

that the binding of these autoantibodies to the receptors may have functional

consequences. However, it is unclear whether the autoantibodies have a role in the

pathogenesis of Chagas disease and/or reflect structural damage to the heart. In regard to

the latter possibility, anti-M2 and anti-1 antibodies are also found in serum of patients

with other forms of heart disease (12,26-28).

Our results showed that individuals with chronic chagasic cardiomyopathy had

elevated levels of both adrenergic and cholinergic autoantibodies when compared with

non-infected controls. The frequency of anti-M2 autoantibody was higher in CCC than in

IND form of the disease, suggesting that the anti-M2 autoantibody could be used as an

early marker of evolution in Chagas cardiomyopathy. However, levels of this

autoantibody were not able to differentiate the various forms of Chagas heart disease.

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This was reflected in the lack of correlation between levels of autoantibodies and the left

ventricular ejection fraction or the left ventricular end-diastolic diameter, both important

parameters of left ventricular dysfunction. Thus, although the presence of antibodies is

associated with the presence of Chagas heart disease, there seems to be no association

between the levels of antibodies and the degree of left ventricular function. The latter

results suggest that left ventricular dysfunction appears not to be the cause of augmented

serum levels of anti-M2 and anti-1 antibodies in Chagas disease and, on the other hand,

the antibodies may not have a direct role in the pathogenesis of the left ventricular

dysfunction that accompanies the most severe cases of Chagas disease. Alternatively, it is

possible that the titers of circulating autoantibodies do not reflect the amount of those

fixed on myocardium neurotransmitter receptors.

Despite the apparent lack of association between left ventricular function and

level of autoantibodies, there were significant associations between the level of anti-M2

antibodies and the presence of apical lesion, exercise-induced arrhythmia and the

chronotropic response to exercise. None of the latter parameters were associated with the

level of anti-1 receptor antibodies. Segmental changes of myocardial contractility,

especially of the apex and the postero-inferior wall of the left ventricle, are common in

Chagas heart disease (29). The apical aneurysm is the most peculiar finding in Chagas

disease, although also observed in other types of heart disease, with thinning and deficit

preferentially localized on the apex of the left ventricle, with or without thrombus

(30,31). In the endemic area, aneurysms of the left ventricle were diagnosed in 18.8%

patients and were related to symptoms, specially palpitations, ECG abnormalities, such as

ventricular extra-systoles and conduction abnormalities, and impairment of the

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ventricular function (32). Moreover, left ventricular aneurysms are associated with the

development of malignant sustained ventricular arrhythmias (33) and are independent

predictors of death (34). The pathogenetic hypothesis for the development of the left

ventricular aneurysms includes mechanical, electrical, inflammatory, ischemic and

autonomic mechanisms. Our results showed that patients with apical aneurysm had

higher levels of anti-M2 receptor antibodies than patients without the lesion, although the

exact meaning of this finding remains unknown.

The occurrence of ventricular arrhythmia during exercise testing is related with

increased mortality due to Chagas disease (35). Here, we found that there was an

association between the levels of anti-M2 receptor antibodies and the presence of effort-

induced ventricular arrhythmias, as demonstrated by exercise testing. Experimental

studies have shown that activation of muscarinic receptors by anti-receptor antibodies

may induce their internalization and, consequently, loss of parasympathetic function (12).

The loss of parasympathetic tonus could then potentially facilitate the occurrence of

arrhythmias, especially during exercise.

Indeed, the level of anti-M2, but not anti-1, receptor antibodies was greater in

patients in whom the presence of chronotropic incompetence during exercise testing was

detected. Preserved autonomic modulation is a major determinant of physiological heart

rate response to exercise. Chronotropic incompetence, which is independent of left

ventricular dysfunction in Chagas disease, may be considered a manifestation of

autonomic dysfunction (11).The latter findings may be secondary to chronic muscarinic

receptor stimulation by the antibodies and resulting receptor desensitization and

impairment of parasympathetic function. This is consistent with our previous results

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demonstrating the presence of parasympathetic impairment in the absence of heart failure

in Chagas disease (11).

In conclusion, we showed that the levels of anti-M2 or anti-1 receptors in serum

of patients with Chagas disease seemed not to correlate with the severity of the left

ventricular dysfunction. Nevertheless, the levels of anti-M2 antibodies were greater in

patients with apical aneurysm, in those with exercise-induced ventricular arrhythmias and

were related to the presence of chronotropic incompetence. Overall, these results point to

an important role of anti-M2 receptor antibodies in the pathogenesis of the dysautonomia

frequently observed in Chagas disease.

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Acknowledgements

This investigation received financial support from FAPEMIG, the UNDP/World

Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR-

WHO A970728 and A20771) and Conselho Nacional de Desenvolvimento Cientfico e

Tecnolgico (CNPq-Brazil) and National Research Council (CONICET) of Argentina,

and University of Buenos Aires (UBACYT), Buenos Aires, Argentina.

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Legends of Figures

Figure 1. Scattergram showing immunoreactivity of serum anti-M2 cholinergic (A) and

anti-1 adrenergic (B) autoantibodies tested by ELISA. Dots represent the individual

optical density (OD) values for each serum sample at 1/50 dilution from 6 non-infected

(NI), 8 infected patients in indeterminate form (IND) and 52 chronic chagasic

cardiomyopathy (CCC). Dotted line shows cutoff values (mean OD values 2 SD from

NI group): anti-M2 cholinergic, 0.200, and anti-1 adrenergic, 0.100. Horizontal lines

show median OD values. *p < 0.0005 versus NI.

Figure 2. Distribution of anti-M2 cholinergic and anti-1 adrenergic cholinergic

autoantibodies in sera of chagasic patients with apical lesion. Dots represent the

individual optical density (OD) values for each serum sample at 1/50 dilution from

infected patients in the absence (30 patients) or in the presence (22 patients) of left

ventricular apical lesions. Horizontal lines show median OD values.

Figure 3. Distribution of anti-M2 cholinergic and anti-1 adrenergic autoantibodies in

sera of chagasic patients with extra systolic beats and chronotropic incompetence.

Chagasic patients were submitted to a treadmill exercise and the presence or absence of

(A) exercise-dependent ventricular arrhythmia or (B) chronotropic defect evaluated. Dots

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represent the individual optical density (OD) values for each serum sample at 1/50

dilution. Horizontal lines show median OD values.

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Table 1. Clinical characteristics of non-infected individuals (NI) and patients classified with

different levels of chronic chagasic cardiomyopathy (CCC).

________________________________________________________________________

NI (n=6) IND (n=8) I (n=8) II/III (n=7) IV (n=15) V (n=14)

Age 38 6.9 47 3.4 48 8.2 47 4.5 44 2.0 43 2.3

Gender 67 38 50 29 47 79

(% male)

LVEF (%) 68 6 64 4 66 2 60 3 61 2 45 3#

LVDD 48 1 48 1 49 1 47 2 50 1 62 1#(mm)

NVPB ND 1 3 725 86 840

in 24 h [0-5] [0-258] [399-5676] [18-2863] [192-3002]

Values are shown as mean SD, except for NVPB in 24h that are shown as median [25%-75% percentile].

NI: non-infected. IND: indeterminate form. LVEF: left ventricle ejection fraction. LVDD: left ventricle

diastolic diameter. NVPB: number of ventricular premature beats in 24 h. # P < 0.01 when compared to

non-infected and furthermore chagasic individuals. Non-normally distributed data were transformed before

performing ANOVA and means comparisons.

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Table 2. Distribution of anti-M2 cholinergic and anti-1 adrenergic autoantibodies in

non-infectd individuals and infected patients

anti-M2 cholinergic anti-1 adrenergic

Groups number positive/total percentage number positive/total percentage

NI 0/6 0 0/6 0

IND 3/8 37.5* 6/8 77.1

CCC I 7/8 87.5 5/8 62.5

CCC II/III 5/7 71.4 4/7 57.1

CCC IV 14/15 93.3 13/15 86.6

CCC V 12/14 85.7 9/14 64.3

______________________________________________________________________________

Microtitre wells were coacted with 1 g peptides (anti-M2 and anti-1) and ELISA was carried

out in the presence of sera from non-infected individuals (NI), indeterminate form (IND) and

different degree of chronic chagasic cardiomyopathy (CCC I to V) infected patients. Optical

density (OD) values more than 2 SD about normal mean were taken as positive. Cutoff values

for anti-M2 cholinergic 0.200 and for anti-1 adrenergic 0.100. Prevalence values of anti-M2

cholinergic and anti-1 adrenergic autoantibodies differ with *p < 0.0005 versus CCC I to V.

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Table 3. Lack of correlation between parameters of ventricular dysfunction and levels of

anti-adrenergic or anti-cholinergic receptor antibodies in patients with chronic chagasic

cardiomypathy (CCC).

LVEF (%) LVEDD (mm) NVPB (in 24 h)

Anti-adrenergic

antibodies

R= 0.053

p= 0.725

R= 0.052

p= 0.733

R= 0.027

p= 0.858

Anti-cholinergic

antibodies

R=0.005

p= 0.973

R= 0.120

p= 0.406

R= 0.167

p= 0.270

LVEF: left ventricle ejection fraction; LVEDD: left ventricle end-diastolic diameter; NVPB:

number of ventricular premature beats.

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Figure 1

NI IND I II/III IV V

0.0

0.5

1.0

1.5

Chronic Chagasiccardiomyopathy

All forms

OD

(405nm)

NI IND I II/III IV V0.00

0.25

0.50

0.75

Chronic Chagasiccardiomyopathy

All forms

OD

(405n

m)

A

B

anti-M2

anti-1

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Figure 2:

Absence Presence Absence Presence0.0

0.5

1.0

1.5

Left ventricle apical lesion

p

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Figure 3:

Absence Presence Absence Presence0.0

0.5

1.0

1.5

Exercise-induced ventricular arrthymia

p