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Cardiovascular Research 1999 41(1):116-125; doi:10.1016/S0008-6363(98)00225-9
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Copyright © 1999, European Society of Cardiology

Alterations in cardiac beta-adrenergic receptors in chagasic mice and their association with circulating beta-adrenoceptor-related autoantibodies

Leonor Sterin-Borda*, Gabriela Gorelik, Miriam Postan, Stella Gonzalez Cappa and Enri Borda

Centro de Estudios Farmacológicos y Botánicos (CEFYBO), Consejo Nacional de Investigaciones Cientiéficas y Técnicas de la República Argentina (CONICET) and School of Medicine and Dentistry, University of Buenos Aires, Buenos Aires, Argentina

* Corresponding author. CEFYBO-CONICET, Serrano 669-5to. Piso, 1414 Buenos Aires, Argentina. Tel.: +54-1-855-7194; fax: +54-1-856-2751.

Received 17 February 1998; accepted 2 June 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objetive: Cardiac tissue from chagasic mice was studied to evaluate the expression and biological activity of β-adrenoceptors in association with circulating β-adrenoceptor-related autoantibodies. Methods: BALB/c inbred mice that were either treated or not treated with atenolol (2.5 mg/kg) and infected or not infected with 1x104 trypomastigotes (CA-1 strain) were sacrificed weekly up to week nine. Morphological, binding and contractility studies were performed on the four different groups of animals. The effect of their serum antibodies was also assayed in binding and contractility studies on normal heart preparations. Results: Hearts from chagasic myocarditis mice showed a β-adrenoceptor-related dysfunction, with a decrease in heart contractility, impaired response to exogenous β-adrenoceptor agonist and a significant reduction in β-adrenergic binding sites. Those effects were maximum at eight–nine weeks post-infection and were improved by treating infected mice with atenolol. In addition, serum or IgG from chagasic myocarditis mice was capable of interacting with cardiac β-adrenoceptors, reducing the number of binding sites and inhibiting the contractile response to exogenous norepinephrine. IgG effects that were observed in normal myocardium, were highest in sera from mice eight–nine weeks post-infection and correlate with the degree of myocarditis. Moreover, chagasic autoantibodies from infected mice recognized a peptide corresponding to the sequence of the second extracellular loop of the human β1-adrenoceptor. Conclusions: (1) The development of alterations in β-adrenergic receptors, related to cardiac dysfunction, may be associated with the presence of circulating antibodies against these receptors and (2) it is possible that the chronic deposits of these autoantibodies in cardiac β-adrenoceptors could lead to a progressive blockade with sympathetic denervation, a phenomenon that has been described in the course of chagasic myocarditis.

KEYWORDS Chagasic myocarditis; β-Adrenoceptors; Antiheart antibodies; Cardiac dysfunction; Anti-adrenoceptor antibodies


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Chagas’ disease, one of the most important determinants of congestive heart failure and sudden death in the world, is caused by a parasite, Trypanosoma cruzi (T. cruzi). The paradoxical severe involvement of the heart in the absence of any intracellular forms of the parasite during chronic disease has prompted many investigators to propose the involvement of autoimmune mechanisms in the pathogenesis of this cardiomyopathy [1]. Evidence is accumulating to support the autoimmune hypothesis, including the presence of myocardial lymphomononuclear cell infiltration [2, 3], abnormalities in circulating lymphocyte subpopulations [4, 5]and the presence of antimyocardial antibodies [6]. Among the antimyocardial antibodies, we have identified autoantibodies against neurotransmitter receptors in human and murine chagasic sera. These autoantibodies were not only able to bind to the β1-adrenoceptor but they also displayed an agonist-like activity, triggering neurotransmitter receptor-mediated biological effects [7–10]. These effects produce a variety of functional consequences in myocardial behaviours, such as alterations in the physiology and biochemistry of the myocardium [9].

We proposed the possibility that the anti-neurotransmitter receptor antibodies may play a role in the pathogenesis of chagasic chronic cardiomyopathy. In fact, chronic Chagas’ heart disease is a cardioneuromyopathy in which the sympathetic and parasympathetic nervous systems are affected [10]. The dysautonomic syndrome is seen before the development of symptoms and signs of Chagas’ cardiomyopathy and could be explained by a slow and progressive blockade of cardiac neurotransmitter receptors by autoantibodies [9]. In fact, the presence of chagasic IgG with autonomic activity was higher in sera from seropositive patients with dysautonomia than from those seropositive patients without dysautonomia [9]. However, the origin of myocardial injuries and dysfunction during the chronic state of the disease has been a controversial issue.

Although evidence is accumulating concerning the presence of circulating β-adrenoceptors autoantibodies and their ability to trigger in vitro neurotransmitter-receptor-mediated biological responses in normal heart, little is known about the functional implication of their in vivo fixation to myocardium during T. cruzi infection. Here, we performed studies to correlate the functionality of myocardium from T. cruzi-infected mice with the expression and activity of β-adrenergic receptors. In addition, the relationship between the onset of cardiac β-adrenoceptor dysfunction and the presence of circulating autoantibodies displaying β-adrenergic activity has been investigated.

Results demonstrated, for the first time, changes in myocardial β-adrenergic receptors in T. cruzi-infected mice that are related to circulating autoantibodies against β1-adrenoceptors. We confirmed that such autoantibodies arise as a result of the infection, and that myocardial contractility as well as β-adrenoceptor expression decrease as a consequence of autoantibody fixation to chagasic heart. We also show that treatment of infected mice with atenolol can prevent these effects.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animals and schedule of infection
BALB/c (H-2d) inbred male mice, which were three months old and weighed between 38–40 g, were allowed food ad libitum. They were kept in a temperature- and light-controlled room (photoperiod: 14 h light and 10 h darkness). Mice were infected intraperitoneally (i.p.) with 1x104 trypomastigotes CA-1, which is a non-lethal strain. The chosen dose was the one reported to induce the most efficient antibody response against the bloodstream forms of the parasite [11]. Animals were killed weekly from one to eight–nine weeks post-infection. Parasitemia was determined by the microhaematocrit technique [12]and specific antibodies were detected by direct agglutination and an indirect immunofluorescence test [11]. The animals were classified as being positive or negative for myocarditis. Myocarditis was assessed by histological studies on heart slices from infected animals; 98% of animals infected with T. cruzi exhibited myocarditis and myocardial parasitosis. Uninfected mice, matched in age, body weight and sex, were used as controls (normal mice). Animals were divided into four groups: I, normal mice injected with phosphate buffer solution (PBS); II, normal mice treated with atenolol; III, infected mice and IV, infected mice treated with atenolol. Seven to ten mice from each group were sacrificed weekly and morphological, binding and contractile assays were performed simultaneously. Additionally, the serum and IgG from those animals were used to carried out binding and contractile experiments on normal heart. All studies were done following a double blind experimental protocol. The investigation conformed with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1985).

2.2 Morphological studies
The hearts were fixed in Bouin–Hollande's fixative for 48 h, cut into two halves, each containing the four heart cavities and embedded in paraffin. Sections (5 µm) were obtained, stained with hematoxilin and eosin and Giemsa or Masson's trichrome stains, as required. The histological sections were evaluated using a light microscope on a single blind basis, as described previously [13]. Briefly, eight areas of the heart (the two atria, the upper and lower halves of the right and left ventricular walls, and the septum) were observed separately with respect to the severity and extent of inflammation and evaluated as 1+ for a single inflammatory focus, 2+ for multiple non-confluent inflammatory foci, 3+ for diffuse inflammation with partial involvement of the wall (subendocardial or subepicardial) and 4+ for inflammation that extended through the wall (from epicardium to endocardium or from endocardium to endocardium in the case of the septum). A number was obtained for each section by adding the values of all areas and the heart inflammation index was derived from the mean of the values obtained from the two sections. Myocardial parasite nests were identified on atria and ventricles. Parasite nests were found in the heart after the first week post-infection. The number of myocardial parasite nests found at weeks two–three post-infection was three to five; at weeks five–six post-infection, five to nine were found, and at weeks eight–nine post-infection, the number was more than ten.

2.3 In vivo treatment with atenolol
Atenolol (2.5 mg/kg) was administered i.p. daily as a single dose. Mice received the first dose one week before T. cruzi infection and were treated until sacrifice; parasitemia was evaluated in the same period. Mice infected with T. cruzi and injected with PBS were used as controls (untreated); untreated mice were injected once a day using the protocol employed for atenolol administration. In order to evaluate drug toxicity, groups of normal mice were inoculated with drug using the same schedule as before. Mice treated with atenolol did not show lethality and the parasitemia levels were similar to those of non-treated infected mice.

2.4 Purification of IgG
Total IgG was isolated from the sera of T. cruzi-infected mice and normal mice, which was either treated or untreated by precipitation with 50% ammonium sulphate [14], followed by two or three washes and reprecipitation with 33% (NH4)2SO4. Total IgG was dialyzed overnight against the elution buffer and then passed through columns of DEAE–cellulose (Sigma, St. Louis, MO, USA) that had been equilibrated with the elution buffer (10 mM phosphate, pH 8) [15]. The eluted peaks were concentrated by ultrafiltration (Minicon B15 concentrator, Amicon, USA) to about 9–10 mg of protein per ml. The degree of IgG purification was tested by microimmunoelectrophoresis, using goat anti-mouse total serum protein (Cappel Laboratories, USA) and goat anti-mouse total Ig (Cappel Laboratories). Only one line of precipitation was obtained in all cases. Additionally, IgG from the same sera cited above was also purified on protein A agarose columns (Pierce, USA).

2.5 Preparation of membranes
Heart membrane from different groups (treated or untreated, infected or noninfected) was prepared essentially as described by Wei and Sulake [15]. Hearts were removed and placed on a glass plate containing a modified Krebs–Ringer bicarbonate (KRB) buffer [8], gassed with 5% CO2 in oxygen, pH 7.4, where fat, large vessels, connective tissue and blood were eliminated. Homogenization was accomplished at 4°C by homogenizing the tissue twice in four volumes of cold buffer containing 0.25 M sucrose, 50 mM Tris–HCl, pH 7.4, and 10 mM MgCl2 using a Polytron PT-20 at a setting of three for 15 s. The homogenates were filtered through four layers of gauze and centrifuged at 700 g for 15 min, 10 000 g for 15 min and 40 000 g for 30 min. The pellet was resuspended in 2.5 ml of 50 mM Tris–HCl, pH 7.4, and 10 mM MgCl2. Spleen cell membranes were prepared from the normal mouse spleen that was removed and homogenized in RPMI 1640 medium (Gibco, NY, USA) in a teflon glass homogenizer. Cell suspensions were depleted of red blood cells by water lysis and then washed with 5% fetal calf serum-supplemented medium. The cells were then centrifuged and the pellet was resuspended in 1:4 volumes of 50 mM Tris–HCl, pH 8, and 10 mM MgCl2. The suspensions were homogenized twice using a Polytron PT-20 at a setting of three for 15 s. The homogenates were filtered and centrifuged at 1000 g for 15 min and at 40 000 g for 30 min. The pellets were resuspended in 2 ml of 50 mM Tris–HCl, pH 7.4, and 10 mM MgCl2.

2.6 Binding assay
Binding assays were carried out under two different conditions: (a) on cardiac membrane from noninfected or infected mice and (b) on normal cardiac membrane and spleen cells in the presence of normal and chagasic serum or the corresponding IgG [17–19]. In the former, sera or IgG from infected mice at different stages were preincubated with cardiac or spleen membrane (3–5 mg/ml protein for hearts and 0.5–1 mg/ml protein for spleen cell membranes) with different dilutions of normal and chagasic serum or IgG for 30 min at 30°C in 50 mM Tris–HCl, pH 7.4, and 10 mM MgCl2. The membranes were washed twice by centrifugation and resuspended. For [3H]dihydroalprenolol (3H-DHA) binding, 100 µl of membrane suspension and different concentrations of 3H-DHA (Dupont/New England Nuclear, Boston, MA, USA; specific activity, 81.4 Ci/mmol) were incubated, with shaking, for 15 min at 37°C in a total volume of 150 µl of 50 mM Tris–HCl, pH 8, and 10 mM MgCl2. In the other series of experiments, when β-adrenoceptor binding parameters of cardiac membranes from different groups were calculated, each membrane suspension was incubated in a final volume of 150 µl with 0.1–6 nM in the same buffer and under the same conditions as above. In both cases, binding was stopped by the addition of ice-cold buffer and, after filtration under mild pressure, Whatman GF/c filters were rinsed and counting in a Beckman spectrometer. Non-specific binding was determined by filtering aliquots of membranes incubated in the presence of 10–6 M propranolol, which did not exceed 25% of the specific binding. Results are expressed as femtomoles and 3H-DHA values specifically bound per milligram of protein (fmol/mg prot).

2.7 Enzyme-linked immunosorbent assay (ELISA)
A 50-µl volume of the peptide (H–W–W–R–A–E–S–D–E–A–R–R–C–Y–N–D–P–K–C–C–D–F–V–T–N–R–C) (20 µg/ml), corresponding to the second extracellular loop of the human β1-adrenergic receptor in 0.1 M Na2CO3 buffer, pH 9.6, was used to coat microtiter plates (Costar) at 4°C overnight. After blocking the wells with 1% bovine serum albumin in PBS for 1 h at 37°C, different dilutions of sera or purified IgG from normal or infected mice (50 µl) were allowed to react with peptide for 2 h at 37°C [9]. The wells were then thoroughly washed with 0.05% Tween 20 in PBS. A 50-µl volume of goat anti-mouse IgG alkaline phosphatase conjugate (1:5000, v/v; Sigma) was added and incubated for 1 h at 37°C. After several washing steps, p-nitrophenyl phosphate (1 mg/ml) was added as substrate and the reaction was stopped at 30 min. Optical density (O.D.) values were measured with an ELISA reader (Uniskan Laboratory System).

2.8 Purification of anti-peptide antibodies by affinity chromatography
The IgG fraction from eight chagasic mice (nine weeks post-infection) affinity chromatographed on the synthesized peptide, which was covalently linked to AffiGel 15 gel (Bio-Rad, Richmond, CA, USA). The IgG fraction was loaded on the affinity column, which had been equilibrated with PBS, and the non-anti-peptide fraction was first eluted with the same buffer. Specific anti-peptide autoantibodies were then eluted with 3 M KSCN, 1 M NaCl, followed by immediate extensive dialysis against PBS. The IgG concentrations of both non-anti-peptide antibodies and specific anti-β1-adrenergic receptor peptide antibodies were determined by radial immunodiffusion assay and their immunological reactivities against the β1 receptor peptide were evaluated by ELISA [9].

2.9 Adrenoceptor functional studies
Normal or infected animals (nine weeks post-infection) that were either treated with atenolol or untreated were decapitated and the atria were removed quickly and placed in a glass chamber containing KRB solution that was gassed with 5% CO2 in oxygen at 30°C, pH 7.4. After a stabilization period of 30 min, control values for tension were recorded using a force transducer coupled to an ink writing oscillograph, as previously described [6]. The preparations were paced by means of a bipolar electrode using a SK4 Grass stimulator, with a stimuli duration of 2 ms and a voltage that was 10% above threshold. The constant resting tension applied to the atria (preload tension) was 350 mg. Inotropic effects (dF/dt) were assessed by recording the maximum rate of tension development during electrical stimulation at a frequency of 300 beats/min. To obtain the maximum serum or IgG effect, different concentrations of sera or IgG were added to normal mouse hearts every 15 min. The weights of the atria were not significantly different between the experimental groups (10.3±1.1 mg).

2.10 Drugs
Freshly prepared solutions of atenolol and norepinephrine (Sigma) were used. All concentrations quoted in the text represent the final values in the bath solution.

2.11 Statistical analysis
Statistical significance was determined by the two-tailed t-test for independent populations. Analysis of variance and Student–Newman–Keuls test was employed when multiple comparisons were necessary. Differences between means were considered significant if P was equal to or less than 0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Distribution of autoantibodies against β-adrenoceptors in the course of murine T. cruzi infection
The distribution of anti-β-adrenoceptor activity in the course of T. cruzi infection was studied. The inhibitory effect of 1/50 to 1/100 dilutions of sera from chagasic mice on 3H-DHA binding to cardiac or lymphocyte membranes is shown in Fig. 1. Positive effects were defined as >20% inhibition of 3H-DHA binding compared to the same dilutions of normal IgG. Inhibition by less than 20% was not found to be significantly different from the results found with normal sera. It can be seen that serum anti-β2-adrenergic activity in lymphocyte membranes appeared in the first week post-infection, peaked at week three and drastically decreased thereafter. In contrast, the interference by serum of β1-adrenergic radioligand binding to cardiac membranes appeared at the second week post-infection, drastically decreased at four weeks and then increased proportionally with the time post-infection. Sera from non-infected mice (control), which was used at the same concentrations at different weeks, as indicated for chagasic sera, had no effect on either cardiac or lymphocyte membranes (data not shown).


Figure 1
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  		Fig. 1 Distribution of the inhibitory effect of sera from T. cruzi-infected mice on 3H-DHA binding to normal cardiac membrane ({circ}) and normal lymphocyte membranes (bullet) related to the evolution period of experimental chagasic infection. Cardiac (3 mg/ml) or lymphocyte (1 mg/ml) membranes were incubated for 30 min at 30°C with 1/50–1/100 dilutions of serum from mice infected with T. cruzi CA-1 strain. Then, they were incubated with 3H-DHA (3 and 1.2 nM for cardiac and lymphocyte membranes, respectively), as described in Section 2. Ten chagasic sera in each week to interact with cardiac and lymphocyte membranes was studied. Cardiac Bmax values (fmol/mg protein) for normal sera=72.4±7.3; chagasic sera were considered positive when Bmax was <55.3±5.1. Lymphocyte Bmax=26.4±2.2; chagasic sera were considered positive when Bmax was <19.2±1.4.

 
3.2 Relation between the degree of myocarditis and β-adrenergic activity of serum from T. cruzi-infected mice
Fig. 2 shows that both the ability of serum to interact with myocardial β1-adrenoceptors (A) and the degree of myocarditis (B) increased with time after infection; the myocardial parasitosis increased in parallel with the inflammation index and the degree of myocardial β-adrenergic antibody activity. Thus, the number of myocardial parasite nests at weeks two–three post-infection was less than five, at weeks five–six post-infection was five to nine and at weeks eight–nine post-infection was more than ten; nests were identified on both atria and ventricular tissues [16]. Parasite nests were found in the heart after the first week post-infection. Inflammation was found to be associated with parasitized cells and in areas where parasites were not detected. Inflammation consisted mainly of mononucleated cell infiltrates (lymphocytes, macrophages, plasma cells), with fewer polymorphonuclear cells and eosinophils. Inflammatory cells were found focally, infiltrating necrotic myocardial fibres or in the interstitium surrounding necrotic or histologically normal myocardial fibres. Diffuse inflammatory cell infiltration was found infrequently.


Figure 2
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  		Fig. 2 Parallel study between the degree of myocarditis and β-adrenergic activity of serum from T. cruzi-infected mice. (A) Bmax values (fmol/mg protein) of 3H-DHA binding to cardiac membrane in the presence of chagasic serum ({square}) and normal serum ({image}); 1/50 dilutions. (B) Inflammation index in mice at different weeks post-infection with T. cruzi ({square}). Data for normal mice ({image}) are also shown. Data represent the mean±SEM of eight animals in each group. *P<0.05 versus one week post-infection.

 
To ascertain whether the onset of circulating β-adrenergic antibody could be associated with cardiac β-adrenergic receptor dysfunction, competitive binding and contractile assays with IgG from mice sacrificed nine weeks after infection were performed.

Fig. 3 shows that IgG from T. cruzi-infected mice inhibited the binding of the β-adrenoceptor radioligand to its receptors on cardiac membranes in a concentration-dependent manner, having a maximal inhibitory effect at 1x10–7–1x10–6 M. Control IgG from non-infected mice had no effect. Scatchard analysis of the data showed that IgG from T. cruzi post-infected mice (week nine) decreased the number of binding sites (Bmax) without causing changes in the equilibrium dissociation constant (Kd) compared with normal IgG, thus indicating an irreversible interaction (see insert in Fig. 3).


Figure 3
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  		Fig. 3 Inhibition of 3H-DHA binding on normal cardiac membrane by normal IgG ({circ}) and murine chagasic IgG (bullet) obtained at nine weeks post-infection. Cardiac membranes (3 mg/ml) were incubated for 30 min at 30°C with different concentrations of normal and chagasic IgG and then with 3 nM 3H-DHA. Values are expressed as mean±SEM of IgG from eight chagasic and seven normal mice. The insert shows the Scatchard analysis of the saturation curve for the data. *P<0.001 versus normal IgG.

 
Moreover, sera or IgG from infected mice were able to react with a synthetic peptide corresponding to the sequence of the second extracellular loop of human β1-adrenergic receptor. Fig. 4A shows the presence of anti-peptide antibody in the sera and the corresponding IgG from mice nine weeks post-infection by an enzyme immunoassay. O.D. values were always more than two standard deviations (SD) higher than those from normal mice.


Figure 4
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  		Fig. 4 (A) Immunoreactivity of anti-β1-adrenergic antibodies of normal sera (bullet) or IgG ({blacksquare}), and of chagasic sera ({circ}) or chagasic IgG ({square}) from mice at nine weeks post-infection, tested by ELISA. Values are expressed as the mean±SD of eight infected and twenty normal mice. (B) Concentration–response curve of affinity chromatography purified anti-peptide IgG upon normal atria contractility (dF/dt). Atria were incubated for 10 min with each anti-peptide IgG concentration, either alone (– {triangleup} –) or in the presence of peptide (- - {triangleup} - -) or atenolol (– {blacktriangleup} –). Changes in dF/dt were expressed in g/s. Values are expressed as the mean±SEM of six experiments in each group. *P<0.001 versus normal serum or IgG, respectively.

 
To reveal the β1-adrenergic activity of murine anti-peptide chagasic IgG, its functional effect upon murine atria contractility was studied. It can be seen in Fig. 4B that the affinity purified anti-peptide IgG increased normal atria dF/dt in a concentration-dependent manner. This IgG effect could be neutralized after preincubating the antibodies with the β1 peptide (5x10–5 M). Atenolol also abrogated the action of affinity purified anti-peptide IgG.

Chagasic IgG from mice nine weeks post-infection was also able to inhibit the inotropic positive concentration–response curve of exogenous norepinephrine (NE) (Fig. 5A). It is shown that the stimulatory effects of different NE concentrations were inhibited by chagasic IgG, while IgG from non-infected mice did not alter NE's positive inotropic response. When the activity of the β-adrenoceptor system was studied on atria from mice nine weeks after infection, a decrease in their response to exogenous NE was also detected. It can be seen in Fig. 5B that atria from T. cruzi-infected mice showed a concentration-dependent impairment in their response to exogenous NE, in comparison with atria from control non-infected mice.


Figure 5
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  		Fig. 5 (A) Inhibitory effect of chagasic IgG obtained nine weeks post-infection on the concentration–response curve of exogenous norepinephrine (NE) upon atria contractility (dF/dt). Normal mice atria were exposed to NE either alone ({triangleup}) or in the presence of 1x10–8 M normal IgG ({circ}) or chagasic IgG (bullet). After 30 min of equilibration, atria were incubated for 30 min with IgG, then, NE was added. Each concentration of NE reacted with atria for 2 min. The Lineweaver–Burke plots [34]of the concentration–response curves of NE show typical non-competitive inhibition between both NE and chagasic IgG. Values represent the mean±SEM of six chagasic and six normal mice. (B) Concentration–response curve of norepinephrine (NE) upon dF/dt of isolated atria from normal ({circ}) and chagasic (bullet) mice nine weeks after infection with T. cruzi. Values are expressed as the mean±SEM of eight mice in each group. *P<0.001 versus normal IgG; **P<0.001 versus normal atria.

 
In addition, the mechanical activity developed by atria from mice nine weeks post-infection, registered after a stabilization period (30 min), was lower than that of normal atria and significantly decreased in a time-dependent manner (Fig. 6). Furthermore, when mice were treated with atenolol during the course of infection, the contractility of isolated atria was normalized, reaching similar values of dF/dt to those obtained with atenolol-treated non-infected mice (Fig. 6). Moreover, atenolol treatment prolonged survival, decreasing the mortality rate of mice at nine weeks after infection from 22% in untreated infected mice to 4% in atenolol-treated infected mice.


Figure 6
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  		Fig. 6 Time course of dF/dt (g/s) developed by atria isolated from normal (– {circ} –) and chagasic (– • –) mice. The contractility of atria from normal (- - {circ} - -) or chagasic (- - bullet - -) mice treated in vivo with atenolol is also shown. Values are expressed as the mean±SEM of eight mice in each group. dF/dt at time zero corresponds to that registered after the stabilization period (30 min). *P<0.001 versus normal atria.

 
To confirm that impairment of atrial mechanical activity could be related to an alteration in the cardiac β-adrenoceptor population, cardiac tissue from mice nine weeks post-infection with T. cruzi and from normal mice were used to characterize receptor expression. Table 1 shows the binding parameters calculated from the normal (non-infected) and experimental (infected) groups that either were or were not treated in vivo with atenolol. Heart membranes from T. cruzi-infected animals showed a decrease in β-adrenergic receptor binding sites, without having significant changes in Kd values in comparison with control groups. The number of binding sites increased in atenolol-treated T. cruzi-infected mice, reaching values of Bmax similar to those of atenolol-treated non-infected mice (Table 1).


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  		Table 1 Characterization of 3H-DHA binding to cardiac membranes from chagasic and normal mice

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Among the immunologic factors that seemed to be involved in the pathophysiologic mechanisms of Chagas’ disease, we can mention the presence of autoantibodies (IgG fraction) against β-adrenoceptors and muscarinic cholinoceptors in the sera of human [7, 9]and experimental model [5, 6, 17]of Chagas’ disease.

We have already reported the existence of circulating IgG in chagasic patients that reacts with β1- and β2-adrenoceptor-rich tissues [18, 19]. Here, we confirmed this issue in mice infected with the non-lethal T. cruzi CA-1 strain using a synthetic peptide corresponding to the second extracellular loop of human β1-adrenoceptors. Knowing that myocardial and spleen cell membranes are tissues that are rich in β1- and β2-adrenoceptors, respectively [20], we observed that the distribution of antibodies against both β1- and β2-adrenoceptors in the course of experimental Chagas disease varied as a function of time post-infection. Thus, an inverse correlation was observed between β1- and β2-adrenergic activity of sera from T. cruzi-infected mice, showing that β1-adrenergic activity increased, while β2-adrenergic activity decreased during the course of T. cruzi infection. Similarly, in the course of human infection, we described that the prevalence of anti-β1-adrenergic antibody is low in the acute stage, and increases with infection time, while anti-β2-adrenergic antibody appears during the acute stage and decreases thereafter [19]. In view of the complete homology between the mouse and human β-adrenoceptors [21, 22]and the similarity in the distribution of both β2- and β1-adrenergic autoantibodies in the course of human and murine infection, it may be assumed that these autoantibodies have the same origin in humans as in mice. It has already been shown that the second extracellular loop of cardiovascular G protein-coupled receptors is an antigenic target for the generation of autoantibodies in patients with cardiomyopathies [23]. The fact that murine chagasic IgG was able to react with a synthetic peptide from the second extracellular loop of human β1-adrenoceptors and that the affinity purified anti-peptide IgG modified the contractility of normal murine atria confirms this possibility. Cross-reactivity epitopes between the parasite and the β1-adrenoceptor could occur [24, 25]. Different results were reported in a study of the evolution of the autoimmune anti-receptor response in the acute and chronic phase of T. cruzi Tehuantepec infection in mice, in which, chronically infected mice failed to recognize β1-adrenoceptor peptide [22]. The discrepancy in the results could be related to the genesis of the immune response triggered by different parasite strains inducing modifications in the course of the disease.

It was previously shown [18, 26]that chagasic IgG interacts with lymphocyte β2 adrenoceptors that specifically bind to T helper cells (CD4+), increasing cAMP production. So, it could be supposed that this antibody would induce an early immunosuppressive response, preventing the host immunological rejection, allowing parasite survival and contributing to the chronic course of the disease. Moreover, during the four weeks post-infection, this immunosuppressive state would lead to lowering anti-β-adrenergic activity, allowing the highest degrees of parasitemia. However, it should be stated that the inhibition of the immune response must be transient: after week four post-infection, an increase in anti-β1-adrenergic receptor activity, probably related to the increment in blood stream flagellates [25], was observed. Supporting this, a high degree of parasitemia has been related to the absence of synthesis of specific antibodies and of any cytokines that are able to kill intracellular parasites [27]. Also, the role of T cells in controlling parasitemia and susceptibility to T. cruzi infection has been demonstrated [28–30].

On the other hand, parasite invasion of myocardium could induce myocardial injury, exposing cardiac tissue antigens that, in turn, could trigger the production of autoantibodies, i.e. anti-β1-adrenoceptor antibodies. This is in agreement with our results that demonstrate a strong relationship between the ability of serum to interact with myocardium β1-adrenoceptors, the inflammation index and the amount of myocardial parasitosis. Autoantibodies against β1-adrenoceptors that are present from the first phase of infection might be partly responsible not only for the chronic classic Chagas cardiomyopathy but also for the early phase heart failure. Thus, even if these autoantibodies behaved as β1-adrenergic agonists, they diminished the reactivity of myocardium to exogenous neurotransmitter, suggesting that, while in an early phase they were able to activate the β1-adrenergic receptors, later on, they might bind irreversibly to those receptors, inducing their down-regulation.

To address this, we found a cardiac functional disorder in infected mice. It was characterized by a decrease in atria contractility accompanied by a diminished response to exogenous NE with a simultaneous loss in the number of β-adrenoceptors. Both alterations were normalized by treating chagasic mice with the specific β1-receptor antagonist drug, atenolol. These results suggest that atenolol could act by preventing antibody fixation to cardiac receptor and support the role of anti-β1 antibodies in the desensitization process. β-Adrenergic down-regulation is a nearly universal finding in heart failure and might be secondary to an increase in circulating catecholamines. However, serum NE levels are not elevated in patients with chronic chagasic cardiomyopathy [31]. Thus, down-regulation may have been induced by circulating antibodies that in turn interact with the heart's β1-adrenergic signalling system and trigger its metabolic regulatory pathways. The mechanism of antibody-mediated desensitization is not clear, but it could involve receptor phosphorylation with the subsequent loss of functional activity. Similar findings of human chagasic autoantibodies with muscarinic cholinergic activity [32]and circulating antibodies associated with other disorders [33, 34]support our hypothesis.

Taken together, these observations point to a potential role of these autoantibodies in the pathogenesis of chronic chagasic cardioneuromyopathy. It is possible that the chronic deposition of these autoantibodies to cardiac β-adrenoceptors could induce desensitization, internalization and/or intracellular degradation of the receptors, leading to progressive blockade with sympathetic denervation, a phenomenon that has been described in the course of chagasic myocarditis.

Time for primary review 23 days.


   Acknowledgements
 
This work was supported by a Grant from the CONICET and UBACYT, Argentina. We thank Ms. Elvita Vannucchi for skilful technical assistance.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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