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Cardiovascular Research 2003 58(1):55-65; doi:10.1016/S0008-6363(02)00811-8
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Copyright © 2003, European Society of Cardiology

Human chagasic IgGs bind to cardiac muscarinic receptors and impair L-type Ca2+ currents

Ciria Carolina Quintero Hernándeza, Luciane Claudia Barcellosa, Luis Eduardo Díaz Giménezb, Rafael Armando Bonfante Cabarcasc, Simone Garciaa, Roberto Coury Pedrosad, Jose Hamilton Matheus Nascimentoa, Eleonora Kurtenbachb, Masako Oya Masudaa and Antonio Carlos Campos de Carvalhoa,*

aInstituto de Biofísica Carlos Chagas Filho, Universidade do Brasil, Rio de Janeiro, 21949-900 Rj, Brazil
bInstituto de Ciências Biomédicas, Universidade do Brasil, Rio de Janeiro, 21949-900 Rj, Brazil
cUniversidad Centroccidental ‘Lisandro Alvarado’, Barquisimeto, Venezuela
dHospital Universitário Clementino Fraga Filho, Universidade do Brasil, Rio de Janeiro, 21949-900 Rj, Brazil

* Corresponding author. Tel.: +55-21-2280-4399; fax: +55-21-2280-8193. acarlos{at}biof.ufrj.br

Received 10 September 2002; accepted 19 November 2002


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Antibodies against cardiac G protein-coupled receptors have been reported in sera from chronic chagasic patients (CChP) and other non-parasitic cardiomyopathies, but the effects and underlying mechanism of interaction between these antibodies and heart cells are not fully established. To address this point, binding of antibodies purified from sera of CChP patients and normal blood donors (NBD) to cardiac muscarinic acetylcholine receptors (mAChR) and their effect on L-type Ca2+ currents were examined. Methods and Results: Saturation [3H]NMS binding experiments with porcine atrial membranes showed that Bmax in the presence of CChP-immunoglobulin G (IgG) decreased from 280.2±16.08 fmol/mg (control) to 91.00±5.98 fmol/mg, with no apparent change in KD, while NBD-IgG did not significantly alter these parameters. At the single channel level, CChP-IgG decreased both the fast and slow mean open times and Po (from 0.074±0.023 to 0.025±0.007) without changes in single channel conductance. I/V plots of isoproterenol-stimulated whole-cell L-type Ca2+ currents (ICa) from rabbit ventricular cardiomyocytes showed a significant reduction in peak ICa during perfusion with CChP-IgG (at 0 mV: from 10.61±2.97 to 8.45±2.54 pA/pF). NBD-IgGs had no effect on ICa. A CChP-IgG purified against a peptide corresponding to the second extracellular loop of the M2 receptor also impaired L-type Ca2+ currents. All effects of CChP-IgG were blocked by atropine. Conclusions: Our results show that antibodies from CChP bind to mAChR in a non-competitive manner and are able to activate the receptor in an agonist-like form resulting in L-type Ca2+ current inhibition.

KEYWORDS Ca-channel; Cardiomyopathy; Immunology; Receptors; Single channel currents


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Chagas’ disease is caused by a protozoan parasite, Trypanosoma cruzi, that is most commonly found in South and Central America. Chagasic cardiopathy is the most frequent complication of chronic T. cruzi infection. As many as 40% of T. cruzi infected patients will develop chagasic cardiopathy, which can lead to heart failure and sudden death [1]. Several laboratories have reported the presence of antibodies that interact with cardiac G protein-coupled receptors (GPCR) in sera from chronic chagasic patients (CChP). The muscarinic acetylcholine receptors (mAChR) and the β-adrenergic receptors (β-AR) seem to be preferential targets and the antibody receptor interaction elicits agonist-like functional responses that may contribute to the cardiac abnormalities observed in Chagas’ disease [2–6].

Mice that have been experimentally infected with T. cruzi develop a chronic cardiopathy similar to that seen in humans [7]. Immunoglobulins purified from sera of these chronic chagasic mice can reduce L-type calcium current in myocytes isolated from control hearts [8]. Monoclonal or polyclonal antibodies generated against the second extracellular loop of the human muscarinic receptor mimic those found in Chagas’ disease and have been used to demonstrate inhibition of L-type calcium currents [9,10].

IgG from asymptomatic chagasic patients’ sera binds to muscarinic receptors from rat atrial membranes or Chinese Hamster Ovary (CHO) cells stably expressing human M2-mAChRs [11,12]. Functionally, these chagasic antibodies activate the mAChRs, decrease cAMP production, increase cGMP and inhibit contractility in the same way as muscarinic agonists [6,13]. We recently showed that CChP antibodies induced disturbances in cardiac electrogenesis and conduction when perfused into isolated rabbit hearts [14]. Given the clear ties between L-type Ca2+ current and cardiac conduction and contractility, we used sera from chronic, symptomatic patients in binding and electrophysiological experiments. We found that CChP-IgG from these symptomatic patients did bind to mAChR, thereby reducing whole cell Ca2+ current by reducing single channel open times and probability.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Human subjects
The present study involved 10 CChP patients (seven from group III and three from group II according to the Los Andes classification) [15] and seven normal blood donors (NBD) referred to as controls. The CChP were selected according to previously described criteria and the sera and IgG from all CChP included in this study had muscarinic-like activity when tested on isolated rabbit hearts [14,16]. The normal blood donors are patients submitted to orthopedic surgery and were otherwise healthy individuals. Written consent was obtained from each patient and World Health Organization and Helsinki Treaty regulations (1963) reviewed in Venice (1983) were followed. The study was approved by the Institutional Committee on Research using Human subjects.

2.2 Purification of human IgG
Total IgG from each patient was individually purified by standard chromatographic procedure on DEAE-Sepharose (Amersham-Pharmacia Biotech, Buckinghamshire, UK) as described previously [14]. Protein concentration in the fractions was determined by the Lowry method [17]. Affinity purified anti-peptide IgGs were obtained by elution of individual human chagasic IgGs on a Sepharose 4B-CNBr-activated column (Amersham-Pharmacia Biotech) covalently linked to a peptide corresponding to residues 168–192 of the second extracellular loop (EL2) of the human M2-mAChR (Research Genetics, Huntsville, AL, USA). The affinity purified IgGs were eluted with 3 mol/l potassium thiocyanate (pH 7.4) followed by immediate extensive dialysis against phosphate buffered saline (PBS).

2.3 Porcine atrium membrane preparation and radioligand binding assay
The binding experiments were performed in membrane preparations from porcine right atrium as described previously [18], substituting imidazole buffer by 25 mmol/l HEPES buffer, pH 7.4. Total protein content was quantified according to Lowry et al. [17] and ranged from 1.0 to 1.7 mg/ml of membrane suspension. Cardiac atrial membrane preparations were incubated at 37°C with [3H]NMS (L-[N-methyl-3H]scopolamine methyl chloride, 80–83 Ci/mmol, Amersham-Pharmacia Biotech) for 120 min and the binding was measured in 2.0 ml of 25 mmol/l HEPES buffer containing 2 mmol/l MgCl2, pH 7.4. Cardiac membranes were collected by rapid vacuum filtration through glass–fiber GF/B filters (Whatman, Maidstone, UK) with a Brandel Cell harvester (Semat, Herts., UK) followed by fast washes with 3x5 ml of ice-cold 10 mmol/l sodium phosphate buffer. The filters were dried, placed in scintillation vials and soaked with 5.0 ml of toluene-based scintillation liquid cocktail. Subsequently, membrane bound radioactivity was determined by liquid scintillation counting (Packard LS counter). Nonspecific binding was defined as tracer binding in the presence of 2 µmol/l atropine and was subtracted in all binding experiments. For saturation assays, [3H]NMS binding was measured at various tracer concentrations (10–1000 pmol/l) to determine the KD and Bmax for ligand binding in the absence or presence of CChP-IgG and NBD-IgG. Competition experiments were performed incubating membranes with a fixed concentration of [3H]NMS (100 pmol/l) and with varying concentrations of IgG. Binding parameters were estimated by least square nonlinear regression analysis curve fitting of experimental data to the rectangular hyperbola binding or sigmoid one site competition models, depending on the particular assay performed, using GraphPad Prism version 3.03 for Windows (GraphPad Software, San Diego, CA, USA).

2.4 Isolation of ventricular cardiomyocytes
Cells from rabbit ventricles were enzymatically dispersed as previously described [19]. The electrophysiological assays were performed within 6 h of isolation. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication no. 85-23, revised 1996).

2.5 Electrophysiological measurements
Voltage clamp of cardiomyocytes was performed using conventional whole-cell or cell-attached patch-clamp procedures to measure membrane L-type Ca2+ currents (ICa). Isolated myocytes were placed in a bath mounted on the stage of a Nikon inverted microscope (Nikon, Japan). All experiments were performed at room temperature (22–25°C) and all tested drugs and IgGs were added to the external bath solution, which was continuously perfused at 1 ml/min flow rate. Patch electrodes (4–9 M{Omega} when filled either with internal solution or external isotonic Ba2+ solution, described below) were prepared from borosilicate glass pipettes on a two-stage pipette puller (Sutter Instruments, P-97, USA).

2.5.1 Cell-attached configuration
Recordings of single channel currents through L-type Ca2+ channels were performed in isotonic BaCl2 solution in the presence of Bay-K-8644 (RBI, Natick, USA) inside the pipette, to increase single channel conductance, thus improving signal to noise ratio, and to increase mean open time [20], respectively. After gigaohm seal was established and capacitive and series resistance were optimally compensated, the patch was depolarized every 2 s from –90 to –40 mV for 80 ms, to inactivate both fast sodium and T-type Ca2+ currents and then, L-type Ca2+ channels were activated by depolarizing voltage pulses of 300–350 ms duration to 0 mV. Single channel currents carried by Ba2+ were recorded using an Axopatch 200 patch clamp amplifier (Axon Instruments), filtered through an 8-pole Bessel low-pass filter (Frequency Devices, 902LPF1 Haverhill, MA) with 1 kHz cut-off frequency and digitized at the sampling rate of 100 µs per point. Single channel conductance was calculated by linear regression analysis of the current–voltage relationship.

2.5.2 Whole-cell configuration
After achieving whole-cell configuration, capacitance and series resistance were compensated. The series resistance was routinely compensated by 60–80%. Cells were voltage-clamped using a patch-clamp amplifier Model L/M-EPC-7 (List-electronic, Germany). Currents were sampled at a frequency of 2.5 kHz using an analog to digital converter (TL-1, DMA Interface, Axon Instruments) connected to a PC compatible computer. Voltage-clamp protocols were generated by the pCLAMP software v6.03 (Axon Instruments, Foster City, CA). A holding potential of –90 mV was used to minimize run-down of the calcium current [21]. Whole-cell currents through L-type Ca2+ channel were evoked every 3 s from the holding potential to test potentials which varied from –60 to +60 mV for 320 ms after a short prepulse (80 ms) to –40 mV, to inactivate fast Na+ and T-type Ca2+ currents. Replacement of K+ ions with intracellular Cs+ and TEA+ (tetraethylammonium chloride) and extracellular Cs+ blocked K+ currents.

2.5.3 Solutions and reagents
Control Tyrode solution contained (mmol/l): 150.8 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 11.0 D-glucose, 10.0 HEPES, pH 7.4 adjusted with NaOH. Ca2+-free Tyrode has no added CaCl2. Whole-cell L-type Ca2+ currents were recorded with an internal pipette solution containing (in mmol/l): 120.0 CsCl, 20.0 NaCl, 2.0 MgCl2, 0.5 CaCl2, 20.0 TEA-Cl, 11.0 EGTA, 10.0 HEPES, pH adjusted to 7.2 with CsOH and pCa of 9.0 (calculated using the WinMaxc software v2.05, Stanford, CA, USA). External solution was control Tyrode. Single channel Ba2+ currents through L-type Ca2+ channels were recorded with the pipette filled with an isotonic Ba2+ solution containing (in mmol/l): 96.0 BaCl2, 10.0 HEPES, 0.01 Bay-K-8644, pH adjusted to 7.4 with CsOH. External solution in this case was a K-aspartate depolarizing solution containing (in mmol/l): 140.0 aspartic acid, 140.0 KOH, 10.0 HEPES, 5.0 D-glucose, 1.0 EDTA, 5.0 MgCl2, pH adjusted to 7.4 with KOH. All reagents whose origin was not specified were of the highest grade available and were purchased from Sigma–Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany).

2.6 Statistical analysis
The statistical tests used are specified in the Results for each case.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 [3H]NMS equilibrium binding experiments: effect of chagasic antibodies
To determine whether CChP-IgG interacted directly with the ligand binding site of M2-mAChRs, we performed saturation binding experiments in porcine atrial membranes. Fig. 1A shows the saturation isotherm of [3H]NMS binding to porcine right atrium membrane preparations in the absence (control, open triangles) and in the presence of individually purified NBD-IgG (open circles) or CChP-IgG (filled circles). Maximal [3H]NMS binding to cardiac mAChR was decreased in the presence of 250 µg/ml CChP-IgG, compared to NBD-IgG and control. Scatchard plots for all conditions were linear indicating that the radioligand bound to a homogeneous population of muscarinic receptors (Fig. 1A, inset).


Figure 1
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  		Fig. 1 Effect of chagasic antibodies on the equilibrium of [3H]NMS binding to M2-mAChR. In binding isotherm curves (A), membranes from porcine heart containing the M2-mAChR (0.060 mg of protein) were incubated in quadruplicate with [3H]NMS 10 to 1000 pmol/l for 120 min at 37°C in the absence (open triangle) and presence of 250 µg/ml of CChP-IgG (filled circles) or NBD-IgG (open circles). In the displacement curves (B), membranes (0.060 mg of protein) were incubated in quadruplicate with 100 pmol/l [3H]NMS for 120 min at 37°C in the absence and presence of increasing concentrations of CChP-IgG (filled circles) or NBD-IgG (open circles). Data points represent mean±S.E.M. of three to five independent experiments. Inset in (A) shows Scatchard plots for control (open triangles), NBD-IgG (open circles) and CChP-IgG (filled circles). IgGs from six CChP and four NBD were used in distinct experiments.

 
We also analyzed the effect of CChP-IgG on [3H]NMS displacement assays. Fig. 1B shows the binding of [3H]NMS in the presence of varying concentrations of IgGs. The inhibition curves showed monophasic behavior indicating that chagasic and non-chagasic IgGs recognize single sites on the muscarinic receptor. The CChP-IgG was able to inhibit the [3H]NMS binding to a maximal extent of 80% (closed circles), while NBD-IgG inhibited about 40% of the specific radioligand binding (open circles) when IgG concentration was 750 µg/ml.

Table 1 summarizes the effect of CChP-IgG and NBD-IgG on NMS binding. Mean values for Bmax were significantly reduced in the presence of CChP-IgG (91.00±5.98 fmol/mg of protein, P<0.001) when compared to those obtained in the presence of NBD-IgG or in the absence of IgG (233.1±25.58 and 280.2±16.08 fmol/mg of protein, respectively). The affinity of [3H]NMS to the receptor in the presence of CChP-IgG was not altered (KD of 150.1±26.15 in control and 116.1±27.30 pmol/l in CChP-IgG). The affinity of [3H]NMS in the presence of the same concentration of NBD-IgGs, showed a tendency to increase (KD 342.4±88.20 pmol/l) but this was not significant. The calculated Bmax and the equilibrium dissociation constant (KD) of [3H]NMS binding to the porcine right atrium under control conditions were respectively 280.2±16.08 fmol/mg of protein and 150.1±26.15 pmol/l and these values are in agreement with those reported in the literature [22] for NMS binding to M2-mAChR. These data suggest that CChP-IgGs induce an apparent non-competitive reduction in the maximal number of binding sites and do not alter the affinity of the receptor to the ligand.


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  		Table 1 Saturation isotherm kinetic parameters of [3H]N-methyl scopolamine binding to porcine right atrium mAChRs: effect of chagasic IgG

 
3.2 Effects of chagasic antibodies on single channel L-type Ca2+ currents
Because muscarinic receptors are known to affect L-type Ca2+ channels, Ca2+ currents at the single channel level were measured. CChP-IgG (Fig. 2A and B) or NBD-IgG (Fig. 2C and D) were perfused in the bath solution to compare channel gating behavior using the cell-attached patch clamp method. Identical protocols (Fig. 2A, top) were used before and during exposure to the IgGs. The presence of CChP-IgG in the perfusate had a striking effect in the overall channel activity during the test pulse; compare Fig. 2A (in control solution) to Fig. 2B (in the presence of CChP-IgG, 80 µg/ml). Ensemble average currents (Fig. 2A and B, bottom traces) clearly demonstrate the inhibition of ICa by CChP-IgG. In contrast, the presence of NBD-IgG (compare Fig. 2C in control solution to 2D in the presence of NBD-IgG, 80 µg/ml) did not affect single channel activity during the test pulse. The similarity of ensemble currents (Fig. 2C and D, bottom traces) confirms the lack of NBD-IgG-mediated effect.


Figure 2
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  		Fig. 2 Decreased activity of single L-type Ca2+ channels in cardiomyocytes treated with chagasic antibodies. Upper panels, single channel currents recorded from the same cell-attached patch in the absence (A) and presence (B) of a CChP-IgG (80 µg/ml) added to the external bath. Lower panels, cell-attached single channel activity recorded before (C) and during (D) perfusion with NBD-IgG (80 µg/ml). Pulse protocol in all single channel recordings consisted of 300-ms steps to 0 mV following an 80-ms duration prepulse to –40 mV; holding potential was kept constant throughout the experiment at –90 mV. The bottom traces in each panel represent the ensemble averages calculated from 300 sweeps before and after antibody addition. Current and time calibrations are shown at the lower right corner.

 
Table 2 shows a quantitative description of the single channel kinetics parameters under control conditions and in the presence of CChP-IgG and NBD-IgG. In line with the decrease in single channel activity during CChP-IgG exposure, the channel mean open time, which was best fitted by a double decaying exponential function, significantly decreased, from 5.36±0.63 ms (n = 15) to 2.34±0.45 ms (n = 9, P = 0.002) for the first exponential, and from 35.39±4.72 ms to 22.43±1.45 ms (P = 0.04) for the second exponential. In contrast, there was no significant change in the mean closed time. Concomitant with the decrease in single channel mean open times, the channel open probability (Po) was significantly reduced, from 0.074±0.023 to 0.025±0.007 (P = 0.04). Channel availability and single channel conductance were unaffected by the presence of CChP-IgG (values for slope conductances were similar: 24.4±7.9 pS under control conditions versus 23.99±6.45 pS in the presence of CChP-IgG, n = 3). Additionally, none of the single channel properties evaluated was affected by NBD-IgGs.


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  		Table 2 Single channel kinetic parameters of L-type Ca2+ channels under control conditions and after addition of chagasic and normal blood donor antibodies

 
3.3 Effects of chagasic antibodies on the β-adrenergic stimulated L-type Ca2+ currents
The aim of these experiments was to evaluate if the depressing effect of CChP-IgGs on Ca2+ currents was maintained when the β-adrenergic pathway is activated. Thus in all whole-cell experiments we followed the classical procedure [23,24] of first enhancing ICa through activation of the β-AR by isoproterenol (1 nmol/l), and then adding the IgG of interest. Fig. 3 shows that CChP-IgG (250 µg/ml) depressed Ca2+ current through L-type channels (Fig. 3A), reducing the mean peak current density at 0 mV from 10.61±2.97 to 8.45±2.54 pA/pF (n = 7, P<0.01). IgG from NBD had no effect (Fig. 3B); peak calcium current density at 0 mV was 12.37±1.91 pA/pF under control conditions and 11.84±1.73 pA/pF in the presence of NBD-IgG (250 µg/ml, n = 4).


Figure 3
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  		Fig. 3 Effect of IgG on L-type Ca2+ currents in the whole-cell configuration. Left, I–V relations for mean peak Ca2+ current densities. Right, current–time recordings at 0 mV. (A) Effect of CChP-IgG: control Tyrode solution plus isoproterenol (ISO), 1 nmol/l, open circles, and in the presence of ISO+250 µg/ml CChP-IgG, filled circles, n = 7. (B) Effect of NBD-IgG: control, same as (A) (open circles) and ISO+NBD-IgG (250 µg/ml), filled circles, n = 4. In all cases, each data point represents mean±S.E.M. On the right side, representative current–time records at 0 mV under control condition (a) and 5 min after addition of 250 µg/ml IgG are shown. In all experiments the voltage protocol described in Section 2 was repeated in the same cell in the presence and absence of the IgG of interest. IgGs from four CChP and four NBD individual patients were used for each experiment. *Significantly different with respect to control, P<0.05 and {dagger}P<0.01, paired Student's t-test.

 
3.4 Blocking the effect of chagasic antibodies by the use of muscarinic antagonists
Because CChP-IgG act as M2-mAChR agonists, inhibition of M2-mAChR should modulate the effect of chagasic antibodies on L-type Ca2+ current. Therefore, Ca2+ currents were recorded in the presence of atropine sulfate, a muscarinic antagonist. Fig. 4 shows the unitary current records obtained in the absence (Fig. 4A) and in the presence (Fig. 4B) of CChP-IgG (80 µg/ml). Single channel activity is unaffected by CChP-IgG when atropine is present in the bath solution (Po=0.079±0.022 in control and 0.096±0.026 in the presence of CChP-IgG, n = 4). Ensemble average currents (Fig. 4A and B, bottom traces) as expected, do not change, as well as mean open or mean closed times (not shown). At the whole-cell level, Fig. 4C shows that atropine sulfate (1.0 µmol/l) is able to completely block the depressing effect of the CChP-IgG on the L-type Ca2+ current: peak calcium current densities of 10.67±3.02 in control compared to 10.47±3.03 pA/pF in IgG+atropine, at 0 mV.


Figure 4
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  		Fig. 4 Effect of chagasic antibodies on L-type Ca2+ currents in the presence of muscarinic antagonist. Upper panels, single channel recordings from the same patch on a rabbit ventricular cardiomyocyte, before (A) and during (B) perfusion with CChP-IgG (80 µg/ml) in the presence of 1 µmol/l atropine sulfate. Pulse protocol consisted of 350 ms steps from –40 mV to 0 mV throughout experiment, from a holding potential of –90 mV. The bottom traces in each panel represent the ensemble averages calculated from 100 sweeps before and 100 sweeps after antibody addition in the presence of atropine sulfate. Lower panels: left, I–V relations for mean peak calcium current densities and, right, current–time recordings at 0 mV. (C) Atropine blocks the effect of CChP-IgG in the whole-cell configuration: control, ISO (1 nmol/l)+atropine sulfate (1 µmol/l), open circles, and 5 min after addition of 250 µg/ml CChP-IgG, filled circles, n = 4. Each data point represents mean±S.E.M. On the right side, representative current records at 0 mV under control condition (a) and 5 min after addition of 250 µg/ml IgG are shown.

 
3.5 Effect of the affinity purified anti-muscarinic chagasic antibodies on L-type Ca2+ channels
To further support the hypothesis that chagasic IgGs exert their action through activation of the M2-mAChR, CChP-IgG from one patient was purified against a peptide corresponding to the sequence of the EL2 of the human M2-mAChR, named V25I, and assayed. L-type Ca2+ currents were recorded under the same conditions described above but now perfusing the affinity purified anti-muscarinic CChP-IgG (20 µg/ml, Fig. 5). Reduction of single channel activity could be seen 2 min (Fig. 5B) after IgG perfusion reaching maximal inhibition at 8 min (Fig. 5C). Ensemble currents (Fig. 5, bottom) further illustrate the time-dependent inhibition. The same blocking effect on L-type Ca2+ channel is seen at whole-cell level, as shown in Fig. 5D, where Ca2+ currents were obtained in the presence of isoproterenol (1 nmol/l).


Figure 5
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  		Fig. 5 Decreased activity of L-type Ca2+ channel by affinity purified anti-muscarinic chagasic antibodies. Upper panels, consecutive traces from one representative experiment in cell-attached mode. (A) Control; (B) 2 min; (C) 8 min after starting perfusion with the affinity purified anti-muscarinic CChP-IgG (20 µg/ml) from one patient. The bottom traces in each panel represent the ensemble averages calculated from 100 sweeps, before and after antibody addition. (D) Left, whole cell peak current–voltage curve for L-type Ca2+ current. Control (open circles) was recorded in Tyrode solution containing 1 nmol/l isoproterenol. Five minutes after addition of the affinity purified CChP-IgG (20 µg/ml) the current was depressed (filled circle); partial reversion of the effect was seen after a 5-min wash-out (open square). Right, current–time records at 0 mV, showing calcium currents under control (a), 5 min exposure to the antibody (b) and after 5-min wash-out (c).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We demonstrated that CChP-IgG binds to cardiac mAChRs and that this binding results in functional activation of muscarinic receptors and the consequent inhibition of L-type Ca2+ channels. For binding experiments we used porcine atrium membrane preparations known to be rich in muscarinic receptors. To assay for the effects of muscarinic receptors on ion currents through L-type Ca2+ channels, we used isolated rabbit ventricular myocytes, which under isoproterenol pre-stimulation have been extensively used to demonstrate activation of the M2-mAChR, given the technical difficulties involved in isolating viable atrium cells. The functional epitope recognized by the antibodies is probably located in the second extracellular loop (EL2) on the M2-mAChR which is highly conserved between the species used in this study [22,25].

This is the first report of Ca2+ current inhibition by IgGs derived from clinically well-defined CChP patients. In fact, sera used in this study were obtained from chronic chagasic patients with symptoms and signs of cardiopathy, belonging to groups II and III of Los Andes classification. Goin et al. [6] showed that anti-M2 antibodies from asymptomatic chagasic patients (groups Ia and Ib) were able to recognize the mAChR decreasing contractility in rat atria, and proposed that the presence of these antibodies might serve as an early marker of cardiac autonomic dysfunction. We have not used sera from asymptomatic patients in the present study but our results using the whole rabbit heart Langendorff preparation show that some of these sera are indeed able to reduce heart rate and/or induce AV conduction blockage [16]. Thus, the presence of functionally active anti-M2 antibodies in chagasic patients’ sera may serve as an early marker of cardiac dysautonomia but is also indicative of disease progression.

The macroscopic Ca2+ current modulation by serum and antibodies raised against the second extracellular loop of the M2-mAChR has been previously described. Mijares et al. [8] demonstrated that sera from T. cruzi infected mice decreased the basal macroscopic Ca2+ current in guinea pig ventricular myocytes via muscarinic stimulation. Similar results were observed on basal and isoproterenol-stimulated macroscopic Ca2+ currents using rabbit polyclonal antibodies to a synthetic peptide corresponding to the second extracellular loop of the M2-mAChR [9]. Moreover, isoproterenol-stimulated macroscopic Ca2+ currents were reduced in the presence of monoclonal antibodies against the M2-mAChR in isolated ventricular myocytes [10]. Our results at the single channel level demonstrate that the shortening of the mean open time and the reduction of open probability of the cardiac L-type Ca2+ channel explain the inhibition of the macroscopic Ca2+ current produced by antibody interaction. Additionally, the reduction in L-type Ca2+ current correlates well with the atrioventricular conduction blockade reported by our laboratory [14] in isolated rabbit hearts perfused with CChP-IgGs by the Langendorff technique, since the action potential propagation in the AV node is essentially dependent on L-type Ca2+ currents.

Our results further support the notion that the second extracellular loop of the M2-mAChR is a preferential target of the functionally active antibodies present in chagasic patients’ sera. Purified antibodies obtained by affinity chromatography through a column coupled to the V25I peptide (corresponding to EL2 of the M2-mAChR), retained the capacity of inhibiting single and macroscopic Ca2+ channel currents (see Fig. 5), whereas the flow-through lost this ability (data not shown). Given that anti-muscarinic antibodies seem to interact with the second extracellular loop of the receptor, a region outside the agonist binding pocket, a non-competitive inhibition by the chagasic IgGs, is expected. In the present report we used NMS as ligand and porcine atria or transfected CHO cell membranes (results not shown) as M2-mAChR source. Our results clearly show that IgGs from symptomatic CChP patients inhibited the binding of [3H]NMS to muscarinic receptors without changing the affinity of the receptor to the ligand, suggesting a non-competitive interaction. Similar results were reported by Goin et al. [11] who showed that IgGs from sera of asymptomatic chagasic patients with autonomic dysfunction were able to displace [3H]QNB from rat atrial membranes. It should however be pointed out that while the IgGs used in this study displaced the ligand binding almost completely, Goin et al. [11] showed that IgGs from asymptomatic chagasic patients inhibited QNB binding to a much lesser extent (about 30%). This level of inhibition is similar to that obtained using normal blood donor IgGs in the present study.

Liu et al. [26], using ELISA, showed the presence of serum antibodies against M2-mAChR in 11% of 408 healthy subjects. The antibodies were present in low titers and their frequency increased with age. In that study, functional effects of these anti-M2 antibodies were not tested. Another study by Jahns et al. [27] involving 108 normal subjects reported the presence of antibodies against β1-AR detected by ELISA, but when these antibodies were assayed by cAMP measurements in Chinese hamster fibroblast cells expressing human β-AR, only one serum was able to functionally activate the receptor. Importantly, our results show that although NBD-IgGs may bind to the M2-mAChR, they fail to activate them, based upon the measurements of Ca2+ currents in the present work and also in the effect in whole heart preparations [14].

Preliminary results using CHO cells stably transfected with the human M2-mAChR show that the IgGs used in the present study are able to activate potassium channels sensitive to carbachol present in these cells. Thus, the presence of antibodies against M2-mAChRs with agonist-like actions may explain the bradycardia (through activation of IkACh) and the atria-ventricular conduction blockage (through reduction in ICa), frequently associated to chronic chagasic cardiopathy in patients with Chagas’ disease.

Time for primary review 22 days.


   Acknowledgements
 
This study was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (PRONEX-CNPq), Fundação Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES) and Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).


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

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