| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo 14049-900; 2Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro 21949-900; and 3Laranjeiras National Institute of Cardiology, Rio de Janeiro, Rio de Janeiro 22240-002, Brazil
Submitted 2 January 2004 ; accepted in final form 8 July 2004
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
1-receptor occurred in 50% of them. Electrophysiological experiments using the whole cell configuration of the patch-clamp technique showed that sera from 43% of IDC patients induced a significant decrease (
26%) in isoproterenol-stimulated L-type Ca2+ currents in rabbit ventricular myocytes, whereas the sera from healthy blood donors failed to do so. As expected, IDC sera also decreased the action potential duration (by 10.5%) due to a shortening of the plateau phase. Sera that reduced isoproterenol-stimulated L-type Ca2+ currents did not cause any effect on K+ currents. We conclude that sera from IDC patients have autoantibodies, which interact with muscarinic M2 receptors of rabbit cardiomyocytes, acting in an agonist-like fashion. This action results in changes in electrogenesis, which, as often observed in patients with IDC, could initiate ventricular arrhythmias that lead to sudden death.
calcium channel; electrophysiology; patch clamp; heart failure; autoimmune disease
- and -cardiac myosin heavy chains (3), heart mitochondrial proteins, anti-M7 and ADP/ATP carrier (35), laminin (37), and G protein coupled receptors (GPCR), such as 1-adrenergic (23) and muscarinic M2 receptors (11), have been described. The fact that specific antibodies exist in the sera of patients with IDC and myocarditis (31) and the detection of Coxsackie B virus-specific RNA sequences in myocardial biopsies from both types of patients (1) favors the hypothesis that an immunological disturbance, probably induced by a subclinical viral infection, could lead to the formation of autoantibodies against cardiac proteins.
On the other hand, the hypothesis that primary abnormalities in the immune system could be accounted for one of the possible causes of IDC was recently strengthened by Jahns and coworkers (17). They have shown some evidence that IDC should be considered along with other well-known receptor antibody-mediated diseases, such as Graves disease, myasthenia gravis, or Type B insulin-resistant diabetes. According to Jahns et al. (17), an autoimmune attack against the second extracellular loop of the 1-adrenergic receptor may play an important role in the development of IDC in rats.
Some progress has been achieved concerning the antigenic targets for these auto antibodies, and, in the specific case of the GPCR, they are localized in the second extracellular loop of both adrenergic (1) and muscarinic (M2) receptors (11, 23). Although it is uncertain whether these autoantibodies are the cause or consequence of IDC, understanding their interaction with GPCR is of importance because these proteins constitute one of the chief regulators of cardiac function in health and disease. Interestingly, it has been shown that antibodies against the GPCR are also found in the sera of patients with Chagas disease (10, 26) and hypertrophic cardiomyopathy (27), where they exert agonist-like effects. Antibodies against the third intracellular loop of M2 receptor have also been reported in the sera of Chagasic patients. They were shown in individuals that have a moderate to severe cardiac impairment and seem to be a consequence of intense cell damage. This leads to the exposure of intracellular epitopes that will trigger new autoimmune response (34).
Although autoantibodies against GPCR have been described in patients with IDC and their removal has ameliorated symptoms of the disease (9, 28), the precise mechanisms by which they contribute to mediate cardiac damage have not been clearly defined. Because the importance of GPCR as regulators of cardiac function rests mainly on their ability to modulate ion channels, our purpose was to detect antibodies against M2 and 1-receptors in the sera of patients with IDC and to analyze their electrophysiological effects using freshly isolated left ventricular myocytes from young rabbits.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
-blocking drugs for at least 48 h before the blood sample was collected. The blood was collected and fractioned, and the serum was stored at –20°C until immunological and/or electrophysiological assays were performed. The clinical characterization of the patients is summarized in Table 1.
|
1-receptors (V-R-T-V-E-D-G-E-C-Y-I-Q-F-F-S-N-A-A-V-T-F-G-T-A-I; residues 168–193 for M2 and 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; residues 183–208 for 1, respectively) were commercially synthesized (Research Genetics). Peptide purity was ascertained by mass-spectroscopy analysis. Enzyme immunoassay. ELISA was carried out as previously described (36) with the following modifications: microtiter plates adsorbed with 10 µg/ml of either peptide were saturated with 0.1 M PBS supplemented with 3% (wt/vol) BSA (IgG and protease free; Jackson ImmunoResearch Laboratories; West Grove, PA), and 0.05% (vol/vol) Tween 20 (Merck; Rio de Janeiro, Brazil). Whole sera (dilution ranging from 1:5 to 1:5,120) were used as primary antibody and an affinity-purified goat anti-human IgG peroxidase-conjugated antibody (diluted 1:5,000) was used as the secondary antibody. The bound peroxidase-conjugated antibody was detected after incubation with the chromogenic substrate for peroxidase (o-phenylenediamine). The reaction was stopped with 50 µl of sulfuric acid (2 M; Merck) and optical densities were read at 490 nm (Molecular Devices; Menlo Park, CA). We considered as positive, sera with optical densities higher than the means ± 2SD of HBD sera, at the dilution of 1:40 (36).
Patch-clamp recordings in isolated cardiomyocytes. Isolated left ventricular myocytes were obtained by collagenase type II (Worthington Biochemical) digestion (90 U/mg) of young rabbit hearts (60–70 days), as described elsewhere (25). Rod-shaped cells with well-defined striations and without sarcolemmal blebs were selected for electrophysiological studies under a Nikon TMD inverted microscope equipped with phase contrast. Cells were continuously superfused with external solution at room temperature.
The whole cell configuration of the patch-clamp technique (14) was used to measure both ionic currents and action potentials. An amplifier (model 200B, Axon Instruments; Burlingame, CA) was connected to the preparation via salt bridges (2.5% agar in the pipette solution) and Ag/AgCl electrodes and set in voltage- or current-clamp mode, depending on the experiment. The voltage protocol used to record calcium currents consisted of a holding potential of –90 mV, a prepulse to –40 mV, and a sequence of pulses ranging from –70 to +70 mV, 350 ms in duration. Ca2+ current (ICa) amplitude was measured as the difference between the peak inward current and the current value at the end of the depolarization step, which was previously "baseline corrected." The baseline was set as the average between all the current traces at the very end of the depolarization pulse (350 ms). After that, the baseline value is nearly 0 pA and calcium current was measured as described above.
For K+ currents recordings, the voltage protocol consisted of a holding potential of –40 mV, and 17 square pulses ranging from –120 to +40 mV, 350 ms in duration. Current amplitude was measured as the difference between the steady-state and holding current. Action potentials were recorded by pacing the cells at 0.2 Hz, with suprathreshold current pulses (varying from 15 to 20 pA), 3 ms in duration. Micropipettes were pulled from glass capillaries (model B150F; Sutter Instrument) and had tip resistances in the range of 2–5 M
. Series resistance was 70–80% compensated to give a final value <10 M. The signals coming from the amplifier were filtered at 5 KHz and were sent to a computer through a Digidata 1200 (Axon Instruments) data-acquisition board. The data were sampled at 10 KHz and stored on a computer hard drive for later analysis with PClamp6 software (Axon Instruments). Junction potential varied between 5 and 6 mV and was offset at the beginning of the experiment with the pipette in the bath solution.
Solutions. External solution was composed of (in mM) 150.8 NaCl, 2.7 KCl, 0.5 MgCl2, 6.0 glucose, 10.0 HEPES, and 2.7 CaCl2, pH adjusted to 7.4 with NaOH. To measure Ca2+ currents, pipettes were filled with a solution containing (in mM) 130 CsCl, 10.0 HEPES, 10.0 EGTA, 10.0 tetraethylammonium chloride, 1.0 MgCl2, 5.0 Na2-ATP, and 0.1 Na2-GTP (pH adjusted to 7.3 with CsOH). For K+ currents and action potential measurements, the pipette solution composition was the following (in mM): 150.0 KCl, 10.0 HEPES, 5.0 EGTA, 1.0 MgCl2, 5.0 Na2-ATP, and 0.1 Na2-GTP (pH adjusted to 7.3 with KOH). Where appropriate, cells were exposed to 10–7 M isoproterenol (Iso; Sigma; St. Louis, MO), 5.0 or 10.0 µM 11-{[2-(diethylamino)methyl]–1-piperidyl}acetyl-5–11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepin-6-one (AFDX-116; Tocris Cookson; Bristol, UK), 10.0 µM carbachol (Sigma; St. Louis, MO), and sera from patients with IDC or from HBD, dilutions of 1:50 (vol/vol) in external solution. The bathing solutions were exchanged by gravity perfusion of the whole chamber with the new solution and/or drug. Solutions were prepared with double-distilled water and filtered through 0.22-µm filters (Millipore GSWP 02500) before use.
Statistics. Where indicated, data are presented as means ± SE and their statistical significance tested with Student's paired t-test or nonpaired test with P < 0.05 or P < 0.01.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
1-receptors with the use of ELISA. Figure 1A shows typical optical density curves read at 490 nm against sera dilutions of both groups, IDC (squares) and HBD (circles), for the M2 (open symbols) and 1 (closed symbols) peptides, respectively. The wide range of dilutions was used to determine the point where the interaction between peptide and antibody was optimal. For the M2 and 1 peptides, the differences in optical density magnitudes between IDC and HBD groups were statistically significant at all sera dilutions (P < 0.01 and P < 0.05, respectively). In Fig. 1B, optical density values measured at a dilution of 1:40 were plotted for each individual of the two groups (squares for IDC patients and circles for HBD individuals) for both M2 and 1-peptides. All sera from patients with IDC and none of the HBD group recognized the M2 peptide at 1:40 dilution. Accordingly, the mean values for these two groups were statistically different (P < 0.01). In contrast, only 50% of the sera from IDC patients interacted with the 1-peptide, whereas no recognition was detected for the HBD group. Again, there was a significant difference between the averages for the two groups at P < 0.05. Repetition of the enzyme immunoassay (twice) during the evolution of the disease in the patient and the control group did not show changes in antibody titers (sera collected at 24-mo interval).
|
-adrenergic pathway was activated with 10–7 M Iso and the Iso-stimulated L-type ICa (ICa,L) were recorded. Each serum was tested before and after the stimulation of the -adrenergic pathway and repeated at least three times. The addition of serum from IDC patient (03R) did not affect ICa (Fig. 2A), but when the -adrenergic pathway was activated, application of the same serum reduced Iso-stimulated ICa,L by 19% (Fig. 2B). This effect was completely reversed after a 10- to 15-min washout with control solution. In contrast, addition of HBD sera (n = 04) did not cause a significant change in either ICa or Iso-stimulated ICa,L (Fig. 2, C and D), although a slight increase in current amplitude could be recorded in both situations. Six of the fourteen IDC patients' sera tested were able to decrease ICa under Iso stimulation. The average decrease in Iso-stimulated ICa,L induced by these six sera was 26.9 ± 4.3% (Fig. 2, E and F). None of the sera affected the activation curve of the ICa,L (data not shown).
|
-adrenergic pathway was fully stimulated with 10–7 M Iso, the addition of the sera did not cause any further effect (Fig. 3B). This was observed with sera from three IDC patients and the average increase in ICa was 21.5 ± 9.13%. We also performed experiments to analyze the possible modulation of K+ currents with special attention to the sera, which triggered activation of the muscarinic pathway. The addition of sera, which were capable of reducing Iso-stimulated ICa,L, had no significant effect on the current-voltage relationship for K+ currents either before or after the stimulation of the -adrenergic pathway (Fig. 3C), indicating that the serum-induced muscarinic activation did not affect K+ channels in the isolated rabbit ventricular myocytes. Effects on K+ currents from sera with "adrenergic-like" behavior were also analyzed and again no significant effect was observed (not shown).
|
|
|
To further characterize the electrophysiological effects of the autoantibodies present in the sera of IDC patients, we measured action potentials in single ventricular myocytes using the current-clamp mode of the patch-clamp technique. The cells were paced at 0.2 Hz, and the effect of sera were analyzed in the absence and presence of Iso (10–7 M) in the bath solution. As expected, the effects were only observed after the stimulation of the -adrenergic pathway. Representative tracings are shown in Fig. 6 and show that the serum from an IDC patient (20R) reduced the time course of the action potential (Fig. 6A), in a manner similar to 10 µM carbachol (Fig. 6B), a well-known muscarinic agonist. Both effects were observed in the same cell and were completely reversed after washout. The effects of the serum were also prevented by the addition of AFDX-116 (Fig. 6C). Four of the six sera that induced a decrease in Iso-stimulated ICa,L also induced a significant shortening on the action potential duration by 10.5% (Fig. 6D) (P < 0.01). In searching for a correlation between the presence of functionally active anti-M2 antibodies in the sera of IDC patients and the clinical stage of the disease, we plotted the sera-induced reduction of Iso-stimulated ICa,L versus the LVEF (Fig. 7A) and the presence of atrial fibrillation in the patients (Fig. 7B). A good correlation (r = –0.79) was seen between the reduction of Iso-stimulated ICa,L and LVEF, whereas a marked reduction (>20%) of ICa,L was strongly correlated with the presence of atrial fibrillation.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
1-receptors to screen for the presence of antibodies against these receptors in the sera of patients with IDC. The sequences were selected based on previous studies (11, 23) that consider both peptides to be involved in an autoimmune recognition of the two receptor types. Our results show that the patients with IDC studied have antibodies against both muscarinic M2 (100%) and adrenergic 1 (50%)-receptors.
A variety of techniques has been used to show the presence of antibodies against many proteins in IDC, and their overall prevalence varies according to the technique and stringency of the criteria used to discriminate between positive and negative sera. However, one of the most interesting features of the antibodies against GPCRs is their ability not only to recognize those receptors, but to bind to the allosteric site on the second extracellular loop and lead to activation of the downstream signal transduction cascade (19). This is especially important because Liu et al. (22) reported that normal individuals also have antibodies against M2 and 1-adrenergic receptors (11% and 10% of the population studied, respectively), but did not report functional effects for these autoantibodies.
Previous work with M2 and 1-autoantibodies from patients with IDC, as well as antibodies raised against the M2 and 1-peptide sequences, have shown that they are capable of inhibiting the binding of specific 1-adrenergic (18, 23) and M2 (11) agonists on cardiac cell membranes. They have also been shown to modulate the chronotropic response of spontaneously beating cultured neonatal rat heart myocytes (8, 24).
In this study, we show functional effects of the M2 autoantibodies from patients with IDC at the ion channel level. To our knowledge, this is the first time that antibodies from IDC patients have been shown to negatively modulate ICa,L in isolated cardiomyocytes.
Our results show that sera from 43% of IDC patients decreased the action potential duration by 10.5% and reduced the Iso-stimulated ICa,L by 26%. The former effect seems to be a direct consequence of the latter, because it is characterized essentially by a reduction of the plateau phase. Nonetheless, we did not neglect that part of the shortening observed in the action potential duration could be due to activation of K+ currents that are sensitive to ACh. This is a controversial point because the proteins Kir3.1 and Kir3.4, which form the muscarinic K+ channel, are not always found in ventricular cells. However, Koumi et al. (20) have shown that ACh reversibly shortens (by 74%) the action potential duration and increases the whole cell K+ currents in human ventricular myocytes. More recently, Dobrzynski et al. (7) demonstrated the presence of the GIRK1/GIRK4 heteromultimer in ferret ventricular cells and concluded that the ACh-induced shortening of ventricular action potential also observed in these cells was primarily the result of the activation of ACh-stimulated K+ channel. Surprisingly, we observed no effect of the IDC sera on the K+ currents, indicating that, at least in this disease model, the sera-activated muscarinic pathway did not trigger the activity of ACh-stimulated K+ channel.
Whereas all sera were positive for the M2 receptor in the ELISA assays, just 43% of them inhibited the ICa,L through activation of the cardiac muscarinic receptor. Sera from seven patients (02R, 03R, 04R, 07R, 10R, 19R, and 22R) displayed positive autoantibody titers for both M2 and 1-receptors. Two of them (03R and 10R) showed the muscarinic agonistic-like effects reported above, whereas other three (02R, 07R, and 22R) had an adrenergic-like effect represented by an increase of the basal ICa (Fig. 3A). In the remaining two sera, the ratio between M2 and 1-autoantibodies seems to be functionally "balanced," leading to a null net effect on either pathway.
We have also shown that the muscarinic-like effects induced by the sera from IDC patients could be blocked either by the selective M2 receptor antagonist AFDX-116 or by preincubation of the sera with the peptide corresponding to the second extracellular loop of the M2 receptor. Therefore, the results shown here cannot be attributed to an antagonistic effect of the 1-antibodies that are present in the sera of some of these patients or to other, as-yet-unidentified, antibodies that could be present in the sera of the remaining patients.
We tried to evaluate the mechanisms by which these autoantibodies were acting downstream of the M2 receptor. Our results suggest the involvement of the classic PKA pathway. This finding is in contrast with the results reported by Nascimento et al. (29), who have shown that the decrease in ICa induced by monoclonal antibodies against the M2 receptor occurs via the PKG pathway. We believe that such difference can be attributed to the fact that sera from patients have polyclonal antibodies and polyclonality can induce pleiotropy of response.
Physiopathological effects such as depression of cardiac electrogenesis and conduction were observed by others when studying sera from Chagasic patients (5) or from mothers with children affected by the neonatal lupus syndrome (6). In Chagas disease, the autoimmune response, characterized by the presence of autoantibodies against GPCRs, seems to result from a molecular mimicry that exists between Trypanosoma cruzi antigens and functional epitope targets in the mammalian M2 and 1-receptors. In that regard, Masuda et al. (26) have shown that the muscarinic-like action of Chagasic antibodies could be blocked by the incubation of sera from the patients with peptides derived from the P2 family of T. cruzi ribosomal protein, carrying a stretch of negative charges at the carboxyl terminal end. In neonatal lupus, a well-known autoimmune syndrome that is associated with autoantibodies reactive to intracellular ribonucleoproteins (SSA/Ro and SSB/La), antibodies present in the mother's serum also have the ability to induce cardiac disturbances that are related to ICa,L activity (12, 33). However, in this case, direct interaction of the antibodies with the pore-forming subunit (-1C) of the L-type Ca2+ channel occurs, leading to a strong inhibition (50%) of the current flowing through this channel (38). In a recent paper, Christ and coworkers (5) reported a positive chronotropic effect on cardiomyocytes in culture and a significant increase in the time course of the action potential and ICa,L as well as cell shortening induced by antibodies against the 1-receptor from patients with IDC.
Many authors have tried to correlate the presence and/or titers of autoantibodies detected by ELISA with their functional effects (18) and with clinical features of the patients, such as electrocardiographical abnormalities (16), New York Heart Association functional class (11, 16), and with the evolution of the disease (2). The results have been extremely variable.
Our studies allowed us to evaluate sera from IDC patients and determine which had significant effects on ICa,L channels. We compared the degree of Ca2+ channel inhibition to the extent of LVEF decrease for IDC patients who had functionally active antibodies. A correlation coefficient of –0.79 was found, which can be taken as significant, given all other variables involved in such a complex disease.
We therefore speculate that the presence of functional antibodies might be taken into consideration to establish the severity of the disease and the prognosis for the patient, but certainly additional studies will be necessary to confirm this.
More importantly, five of the six patients with functionally active muscarinic antibodies presented atrial fibrillation (Fig. 7B) and two of them died as a consequence of congestive cardiomyopathy. This clinical finding points toward the likely contribution of the M2 autoantibodies to the pathogenesis of IDC because the high prevalence of atrial fibrillation in these patients could be a direct consequence of the marked decrease of ICa,L and therefore reduction of action potential duration induced by the anti-M2 antibodies. Chiale et al. (4) also reported a high prevalence of circulating anti-M2 antibodies in patients with sinus node dysfunction due to either primary or Chagasic etiology. According to Nattel (30), a decrease in inward currents and an increase in outward currents is one of the possible mechanisms that promote atrial fibrillation, because this leads to a reduction of the refractory period of the action potential. It has been shown that decreases in ICa that are large enough to lead to a decrease in the action potential duration, and promote the induction and maintenance of atrial fibrillation by multiple reentry circuits (13). In addition, a reduction of 30% in the ICa,L in experimental models of congestive heart failure has also been observed (21).
Overall, our results indicate that functionally active muscarinic antibodies in the sera of IDC patients could be playing a role in the cardiac imbalance commonly observed in this illness.
The relevance of autoantibodies against GPCRs to the clinical status of the patients with IDC is further substantiated by immunoadsorption experiments. There was great hemodynamic improvement observed after the removal of GPCR circulating antibodies from the sera of IDC patients (9, 28), making this technique a promising therapeutic approach for this particular type of cardiac disease.
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  B. Olshansky, H. N. Sabbah, P. J. Hauptman, and W. S. Colucci Parasympathetic Nervous System and Heart Failure: Pathophysiology and Potential Implications for Therapy Circulation, August 19, 2008; 118(8): 863 - 871. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
) did not induce any reduction in the basal Ca2+ current (ICa; n = 03 cells). B: the same serum caused a marked reduction on isoproterenol (Iso)-induced L-type ICa (ICa-Iso; 
) increased the basal ICa by 17%. B: the same serum fail to cause a further increase in ICa-Iso (
) and after (
). B: the same cell was again exposed to serum but now in the presence of 10 µM AFDX-116 (
