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J Am Coll Cardiol, 2007; 49:1684-1692, doi:10.1016/j.jacc.2006.11.051 (Published online 5 April 2007).
© 2007 by the American College of Cardiology Foundation
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CLINICAL RESEARCH: CARDIOMYOPATHY

Fc{gamma} Receptors IIa on Cardiomyocytes and Their Potential Functional Relevance in Dilated Cardiomyopathy

Alexander Staudt, MD*, Petra Eichler, PhD{dagger}, Christiane Trimpert, Msc*, Stephan B. Felix, MD*,* and Andreas Greinacher, MD{dagger}

* Klinik für Innere Medizin B, Ernst-Moritz-Arndt-Universität, Greifswald, Germany
{dagger} Institut für Immunologie und Transfusionsmedizin, Ernst-Moritz-Arndt-Universität, Greifswald, Germany

Manuscript received July 20, 2006; revised manuscript received October 13, 2006, accepted November 6, 2006.

* Reprint requests and correspondence: Dr. Stephan B. Felix, Klinik für Innere Medizin B, Ernst-Moritz-Arndt-Universität, Fr.-Loefflerstr. 23a, 17475 Greifswald, Germany. (Email: felix{at}uni-greifswald.de).


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
Objectives: The purpose of this study was to investigate how cardiac autoantibodies might contribute to cardiac dysfunction in patients suffering from dilated cardiomyopathy (DCM).

Background: In the majority of DCM patients, it is possible to detect antibodies with negative inotropic effect on cardiomyocytes. The manner in which these antibodies impair cardiac function is poorly understood.

Methods: Immunoglobulin (Ig)G was prepared from plasma of 11 DCM patients containing antibodies that induced a negative inotropic effect on cardiomyocytes. We analyzed the effects of antibodies/IgG fragments on calcium transients and on systolic cell shortening of adult rat cardiomyocytes and investigated the dependency of these effects on potential cardiomyocyte Fc receptors.

Results: In contrast to control subjects, intact IgG from DCM patients reduced calcium transients and cell shortening of cardiomyocytes. The F(ab')2 fragments of these antibodies did not induce these effects but inhibited the functional effects of DCM-IgG of the respective patients' IgG. These effects were also inhibited by Fc fragments of normal IgG. Reconstitution of the Fc part by incubation of cardiomyocytes with DCM-F(ab')2 fragments followed by goat-anti-human-F(ab')-IgG again induced reduction of cell shortening and of calcium transients. In rat and human ventricular cardiomyocytes, Fc{gamma} receptors IIa (CD32) were demonstrated by immunofluorescence.

Conclusions: Our findings indicate that DCM-IgG-F(ab')2 bind to their cardiac antigen(s), but the Fc part might trigger the negative inotropic effects via the newly detected Fc{gamma} receptor on cardiomyocytes. These results point to a novel potential mechanism for antibody-induced impairment of cardiac function in DCM patients.

Abbreviations and Acronyms
  DCM = dilated cardiomyopathy
  Ig = immunoglobulin

Dilated cardiomyopathy (DCM) is a frequently occurring myocardial disease with poor prognosis that is characterized by progressive depression of myocardial contractile function and by ventricular dilatation (1,2). Disturbances in humoral immunity play an important role in the cardiac dysfunction of DCM patients (3–9). A number of autoantibodies against various cardiac cell proteins have been identified in DCM: for example, antibodies against mitochondrial proteins, contractile proteins, beta-1-receptors, and muscarinergic receptors (3,10–13). Studies with an animal model for anti–beta-1-receptor antibodies have especially demonstrated the biological relevance of these antibodies and their association to DCM (14). Animal models further indicate that, under certain pathological conditions, DCM can be triggered by autoantibodies. BALB/c mice, deficient for the immune-regulatory program death receptor-1 (PD-1), generate autoantibodies against cardiac troponin I. Antibodies against cardiac troponin I bind to the surface of cardiomyocytes, as shown by immunoelectron microscopy. These autoantibodies induce cardiac dysfunction in animals similar to that seen in human DCM patients (15,16).

We have recently shown that antibodies from DCM patients induce an acute negative inotropic effect in isolated rat cardiomyocytes through depression of calcium transients. Removal of these antibodies from patients' plasma by therapeutic immunoadsorption improves cardiac function in patients with heart failure due to DCM (8). Only those DCM patients in whom a negative inotropic effect of the antibodies was documented in vitro experienced clinical benefit during the following immunoadsorption (9). A variety of different epitopes on cardiomyocytes might represent potential antigens responsible for the induction of these functional effects in cardiomyocytes. Whereas each of the 2 Fab fragments of an immunoglobulin (Ig)G antibody expresses the hypervariable region that is responsible for specificity of the antibody and by which the antibody binds to its antigen, the Fc part does not differ among antibodies of the same IgG subclass. The Fc part is important for the biological effects of IgG, because it interacts with Fc{gamma} receptors that are expressed on many cells (e.g., leukocytes, endothelial cells, and platelets). Accordingly, we studied the role of the effector part of negative inotropic antibodies. In this context, we also investigated the existence of Fc{gamma} receptors on cardiomyocytes as well as their potential functional relevance. Our findings indicate that Fc{gamma} receptors might play an important pathogenic role in DCM, because cardiac autoantibodies evidently bind with their Fab part to structures on the cardiomyocytes but then crosslink via their Fc part the Fc{gamma} receptors on cardiomyocytes.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Preparation of plasma IgG.   Immunoglobulin G was prepared from plasma of 11 DCM patients with antibodies that induced a negative inotropic effect on cardiomyocytes, as described earlier (9). The negative inotropic effects were defined by a reduction of systolic cell shortening (>10%) caused by a decrease of calcium transients (>10%) (9). The DCM was diagnosed by standard methods (5). Antibodies were purified by an anti-IgG column (PlasmaSelect, Teterow, Germany), dialyzed (molecular weight cutoff 100 kDa) against experimental buffer (117 mmol/l sodium chloride (NaCl), 2.8 mmol/l potassium chloride (KCl), 0.6 mmol/l magnesium chloride, 1.2 mmol/l potassium phosphate monobasic, 1.2 mmol/l potassium dihydrogen phosphate, 20 mmol/l glucose, and 10 mmol/l HEPES; pH 7.3) for 30 h, incubated (56°C, 30 min) for inactivation of complement (8,9), and stored in aliquots at –70°C. Immunoglobulin G from healthy blood donors (control subjects) was prepared identically.

Measurement of intracellular calcium transients and systolic cell shortening.   The isolation of ventricular cardiomyocytes from adult Wistar rats and the measurements of intracellular calcium transients and of systolic cell shortening were performed as described earlier (8,9,17). Briefly, the cardiomyocytes were suspended in experimental buffer and stained with the calcium fluorescent probe Fura 2-AM. Single cardiomyocytes were field-stimulated (1 Hz, 5 ms) and superfused continuously with experimental buffer (2 ml/min) containing DCM patient IgG (300 µg/ml) or the respective F(ab')2 fragments (200 µg/ml), control IgG (300 µg/ml), and anti-Fab antibodies (30 µg/ml). For analysis of the involvement of the specific binding of the F(ab')2 part to its antigen, we pre-incubated the cardiomyocytes with the F(ab')2 fragments (56 µg/ml) of each of the eleven DCM patients for 30 min, rinsed with buffer, and superfused the cardiomyocytes with the intact IgG of the respective patient. To assess the involvement of the Fc part in the functional effects of the autoantibodies, cardiomyocytes were pre-incubated with human Fc fragments (30 µg/ml).

Fluorescence measurements were recorded with a dual-excitation, single-emission fluorescence photomultiplier system (IonOptix, Milton, Massachusetts). Changes in intracellular calcium transients were inferred from the ratio of the fluorescence intensity at 2 wavelengths (9). A video-imaging edge detector system (IonOptix) was used for measurements of cell length, as described elsewhere (9,18). The relative change of calcium transients and systolic cell shortening was expressed as the mean of the experiments, with at least 6 different cardiomyocytes from 6 different cardiomyocyte preparations per IgG preparation (8,9). At baseline, all measured cardiomyocytes showed systolic cell shortening of more than 8% (mean 11.3 ± 0.5%). The experiments were performed in blinded fashion. In all experiments, antibodies with (positive control) and without (negative control) functional activity were additionally tested on the same cardiomyocyte preparation. Each aliquot of cardiomyocytes was used only for a single investigation of the one Ig sample.

To determine whether the functional effects are mediated by the F(ab')2 part of the antibodies, F(ab')2 fragments were prepared (pepsin digestion) and purified (fast performance liquid chromatography) from patient IgG and control IgG by the Biotech Company Biogenes (Berlin, Germany). The IgG and the respective F(ab')2 fragments from each patient were used in equimolar concentrations. For assessment of the involvement of the Fc part of antibodies in the functional effects, Fc fragments of normal IgG were used (kindly provided by Behringwerke, Marburg, Germany). For reconstitution of the Fc parts of the IgG-F(ab')2 fragments, cardiomyocytes were pre-incubated with the F(ab')2 fragments of DCM patients (56 µg/ml; 30 min), rinsed, and superfused with goat-anti-human-F(ab') IgG (Sigma-Aldrich Chemie, Taufkirchen, Germany) (30 µg/ml).

Isolation of human cardiomyocytes from endomyocardial biopsies.   Isolation of human cardiomyocytes was performed as described elsewhere (19). From DCM patients (n = 8) we obtained 8 to 10 endomyocardial biopsies from the interventricular septum of the right ventricle (7). Briefly, a part of the endomyocardial biopsies was transported immediately to the laboratory for cell isolation within 5 min, in ice-cold HEPES-buffered salt solution (pH = 7.0) containing the following: 120 mmol/l NaCl, 5.4 mmol/l KCl, 0.5 mmol/l magnesium sulfate, 5.0 mmol/l sodium pyruvate, 20 mmol/l glucose, 10 mmol/l HEPES, and 6 mmol/l nitrilotriacetic acid (Sigma-Aldrich Chemie). The biopsies were placed in a 2-ml tube in a water bath (35°C) for enzymatic digestion. The continuously gassed digestion media consist of the HEPES-buffered solution with a calcium concentration of 50 µmol/l and 1% bovine serum albumin. In the first step the biopsies were incubated with protease type XXIV (4 U/ml) (Sigma-Aldrich Chemie) for 15 min. The biopsies were replaced in a fresh tube containing buffer with a combination of 1 mg/ml collagenase-A (Worthington, Lakewood, New Jersey) and 0.5 mg/ml hyaluronidase (Sigma-Aldrich Chemie). After 20 min the cells were centrifuged (4 min, 240 g), and the supernatant was replaced by a fresh pure collagenase-A solution for additional 20-min incubation. To terminate the digestion process, the cell pellet was washed once in the media without enzyme. Finally, the cell pellet was resuspended in 1.5 ml phosphate buffered saline (PBS) buffer and passed through a filter to remove undigested tissue.

Written consent was obtained from each patient, and the protocol was approved by the Ethics Committee of the University Hospital of Greifswald.

Indirect immunofluorescence.   Isolated cardiomyocytes were allowed to adhere on cover slips (1 h, 37°C); fixed with paraformaldehyde (2% in PBS, 10 min, room temperature, without additional permeabilization); blocked with PBS containing 3% bovine serum albumin/0.05% Tween20 for 15 min; and stained with polyclonal goat antibodies against Fc{gamma} receptor I (CD 64), Fc{gamma} receptor II (CD 32), Fc{gamma} receptor IIa, Fc{gamma} receptor IIb, Fc{gamma} receptor IIb/c, and Fc{gamma} receptor III (CD 16) (4 µg/ml, 2 h, room temperature). Goat IgG was used as negative control, and a polyclonal goat antibody against troponin I served as positive control. Furthermore, rat leukocytes were employed as an additional positive control for detection of Fc{gamma} receptors IIa to demonstrate that the antibody, which recognizes human Fc{gamma} receptors IIa, also reacts with the rat Fc{gamma} receptors IIa. Human fibroblasts served as additional negative control. For detection, cells were incubated with rhodamine-conjugated bovine anti-goat IgG antibodies (30 min, room temperature, 1:400). The cellular nuclei were stained with DAPI (4',6-Diamidino-2-phenyindole, dilactate) (Sigma-Aldrich Chemie) (1 µg/ml in PBS) for 2 min. Before immunofluorescence analysis of a high-power microscopic field, the identity of cells was additionally verified by light microscopy.

Isolated human cardiomyocytes were treated according to the same immunofluorescence protocol as rat cardiomyocytes but with antibody treatment carried out in solution. All primary and secondary antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Cover slips were mounted on slides with GelMount (Sigma-Aldrich Chemie) and examined with a fluorescence microscope (Leica DMLB, Benzheim, Germany). A constant exposure time of 10 s was used for rhodamin-conjugated antibodies, and 50 ms was used for DAPI staining.

Statistics.   Results are expressed as mean ± SEM. The analyses included comparisons between the groups (control subjects vs. DCM) and within the groups. Analyses were performed by Mann-Whitney U tests and Wilcoxon signed rank tests. Significance was assessed at the p < 0.05 level.


   Results
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 Abstract
 Methods
 Results
Discussion
 References
 
Effects of intact IgG and F(ab')2 fragments of DCM patients.   The intact IgG fractions (300 µg/ml) of plasma from 11 DCM patients with negative inotropic antibodies (mean age = 46 ± 3 years; 10 male/1 female patient; ejection fraction <30%, New York Heart Association functional class III to IV) immediately reduced calcium transients by 14 ± 1% and systolic cell shortening of rat cardiomyocytes by 19 ± 1% (p < 0.001 vs. control IgG). Control IgG had no effect (Fig. 1A). The F(ab')2 fragments of patient IgG and control subjects showed no functional effects (p < 0.001 vs. DCM IgG), which indicates that binding of F(ab')2 fragments to its antigen alone is not sufficient to induce negative inotropic effects (Fig. 1B).


Figure 1
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  		Figure 1 Effects of Intact IgG/Antibody Fragments on Isolated, Field-Stimulated Rat Cardiomyocytes

Changes of calcium transients and systolic cell shortening during superfusion (% changes from baseline; mean ± SEM; n = 6 experiments for each patient's/control sample) with: intact immunoglobulin (Ig)G from plasma of healthy blood donors (control IgG) and intact IgG from dilated cardiomyopathy (DCM) patients (DCM-IgG); F(ab')2 fragments of control IgG and of respective DCM-IgG; intact DCM-IgG after pre-incubation with the F(ab')2 fragments of control subjects and of respective DCM patients; intact DCM-IgG after pre-incubation of cardiomyocytes with human Fc fragments; and goat-anti-human-F(ab')-IgG after pre-incubation of cardiomyocytes with F(ab')2 fragments of control IgG and of respective DCM patients. +++p < 0.001 significantly different from control IgG; ***p < 0.001 significantly different from DCM-IgG.

 
When we pre-incubated cardiomyocytes with the F(ab')2 fragments (56 µg/ml) of each of the 11 DCM patients for 30 min, rinsed with buffer, and superfused the cardiomyocytes with the intact IgG of the respective patient, the F(ab')2 fragments blocked the negative inotropic effect of the intact patient's IgG (p < 0.001 vs. DCM IgG) (Fig. 1B). The F(ab')2 fragments of control IgG (n = 6), in contrast, did not inhibit the effects of intact IgG of DCM patients (Fig. 1B). This indicates that the DCM F(ab')2 fragments inhibit binding of intact antibodies to their respective antigen: a process evidently crucial for inducing the observed functional effects of DCM IgG on cardiomyocytes (Fig. 2).


Figure 2
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  		Figure 2 Summary of Functional Findings

Intact immunoglobulin (Ig)G of dilated cardiomyopathy (DCM) patients induced negative inotropic effects by binding via their Fab part to the antigenic epitope on cardiomyocytes and then via their Fc part to the Fc{gamma} receptor IIa (Fc{gamma}R) (A). The F(ab')2 fragments of these antibodies inhibited the effect of intact DCM IgG (B), as did Fc fragments of normal IgG (C). Reconstitution of Fc parts by sequential incubation of cardiomyocytes with DCM F(ab')2 fragments and goat-anti-human F(ab') IgG induced a negative inotropic effect (D) comparable to findings for intact DCM IgG. A detailed description of Fc receptor cross linking is in the Discussion section. AG = antigen.

 
To assess whether the antibodies in different DCM patients bind to the same or to different antigen-binding sites, cardiomyocytes were pre-incubated with F(ab')2 fragments of 1 patient, rinsed, and then superfused with the IgG antibodies of another patient. Three different patients' F(ab')2 fragments were tested in this way against antibodies of 3 other DCM patients. As an indication for the involvement of different cardiac antigens in 8 of 9 blocking experiments, the DCM F(ab')2 fragments did not inhibit the effects of functionally active, intact antibodies from other DCM patients (data not shown).

Role of the Fc part of the antibodies in the functional effects.   The F(ab')2 fragments lack the Fc part of intact IgG. In contrast to the specific antigen binding sequence of the F(ab')2 part, the Fc parts of various antibodies of the same IgG subclass do not differ in their structure. To assess the involvement of the Fc part in the functional effects of the autoantibodies, we pre-incubated cardiomyocytes with human Fc fragments (30 µg/ml) for 30 min, rinsed, and superfused the cardiomyocytes with the IgG antibodies of a DCM patient (n = 6), and analyzed the inotropic effects. Whereas Fc fragments alone demonstrated no effect (data not shown), pre-incubation of cardiomyocytes with Fc fragments of normal IgG completely inhibited the functional effects of intact DCM IgG (p < 0.001 vs. DCM IgG) (Fig. 1C).

To verify that both the Fab part of DCM IgG and the Fc part are required for the functional effects of the antibodies, we reconstituted the Fc part of the F(ab')2 fragments of DCM IgG antibodies by sequential incubation of rat cardiomyocytes with the F(ab')2 fragments of DCM IgG, followed by an intact goat-anti-human-F(ab') IgG. This resulted again in reduction of systolic cell shortening (–20 ± 2%) and of calcium transients (–14 ± 2%) comparable to intact DCM patient IgG (p < 0.001 vs. control IgG) (Fig. 1C). Sequential incubation of cardiomyocytes with F(ab')2 fragments of control IgG and goat-anti-human-F(ab') IgG demonstrated no functional effects (Fig. 1C). This is further evidence that DCM IgG induces functional effects on cardiomyocytes only if it can bind with its Fab part and its Fc part to cardiomyocytes (Fig. 2). This view of a potential mechanism is supported by the fact that, after removal of immune complexes by ultracentrifugation, the negative inotropic effects of intact DCM patient IgG remain detectable. Potential direct anti-Fc{gamma} receptor-IIa activity of DCM IgG was excluded by an established system using platelets (20). Preincubation of platelets with DCM sera (n = 6) did not cause platelet activation; nor did it inhibit platelet activation by sera (n = 2) of patients with heparin-induced thrombocytopenia that still activated platelets via the Fc{gamma} receptor IIa.

Detection of Fc{gamma} receptors on rat and human cardiomyocytes.   Binding of the IgG Fc part to cells occurs via Fc{gamma} receptors. By immunofluorescence we detected Fc{gamma} receptors II (CD32) on rat cardiomyocytes (Fig. 3), which stained positive with anti-Fc{gamma} receptor-IIa antibodies but not with anti-Fc{gamma} receptor IIb, anti-Fc{gamma} receptor IIb/anti-Fc{gamma} receptor IIc, anti-Fc{gamma} receptor III, or anti-Fc{gamma} receptor I antibodies (Figs. 3 and 4).Go Human cardiomyocytes isolated from endomyocardial biopsies of DCM patients (n = 8) also stained positive for the Fc{gamma} receptor IIa but not for Fc{gamma} receptor IIb or Fc{gamma} receptor IIc (Fig. 5). However, it cannot be excluded that the other Fc{gamma} receptors are expressed below the detection limit of the immunofluorescence method employed. Goat-normal IgG and human fibroblasts served as negative control subjects. Antibodies against troponin I and rat leukocytes served as positive control subjects (Figs. 3, 4, and 5). Counterstaining with DAPI demonstrates that the positive immunofluorescence staining is located on cardiomyocytes. However, in negative control subjects (goat IgG) the cardiomyocytes produced no positive immunofluorescence staining (Fig. 4). In all immunofluorescence experiments, the majority of the cells (>90%) displayed the typical staining as shown in the figures.


Figure 3
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  		Figure 3 Fc{gamma} Receptor on Rat Cardiomyocytes

Immunofluorescent staining of isolated rat cardiomyocytes with a rhodamine-conjugated bovine anti-goat secondary antibody after pre-incubation with polyclonal goat primary antibodies against Fc{gamma} receptor I (CD64), Fc{gamma} receptor II (CD32), or Fc{gamma} receptor III (CD16); goat immunoglobulin (Ig)G and antibodies against troponin I, serving as negative and positive control subjects, respectively (counterstaining with 4',6-Diamidino-2-phenyindole, dilactate [DAPI]; representative figures of n = 6 experiments for each antibody).

 

Figure 4
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  		Figure 4 Fc{gamma} Receptor Type IIa on Rat Cardiomyocytes

Immunofluorescent staining of isolated rat cardiomyocytes with a rhodamine-conjugated bovine anti-goat secondary antibody after preincubation with polyclonal goat primary antibodies against Fc{gamma} receptor IIa, Fc{gamma} receptor IIb, or Fc{gamma} receptor IIb/c. Immunofluorescence of fibroblasts and rat leukocytes with a rhodamine-conjugated bovine anti-goat secondary antibody after preincubation with polyclonal goat primary antibodies against Fc{gamma} receptor IIa served as negative and positive control subjects, respectively (counterstaining with 4',6-Diamidino-2-phenyindole, dilactate [DAPI]; representative figures of n = 6 experiments for each antibody).

 

Figure 5
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  		Figure 5 Fc{gamma} Receptor Type IIa on Human Cardiomyocytes

Immunofluorescent staining of human cardiomyocytes isolated from endomyocardial biopsies with a rhodamine-conjugated bovine anti-goat secondary antibody after preincubation with polyclonal goat primary antibodies against Fc{gamma} receptor IIa, Fc{gamma} receptor IIb, or Fc{gamma} receptor IIc; goat immunoglobulin G and antibodies against troponin I, serving as negative and positive control subjects, respectively (representative figures of n = 6 experiments for each antibody).

 
The polyclonal anti-Fc{gamma} receptor II antibody (8 µg/ml) alone induced negative inotropic effects (cell shortening: –18.1 ± 3% and calcium transient: –11.3 ± 2%); anti-Fc{gamma} receptor IIa antibodies induced a moderate cardiodepressant effect (cell shortening: –11.4 ± 2% and calcium transient: –7.9 ± 1%). For this reason, we did not perform preincubation of cardiomyocytes with these antibodies for inhibition of the functional effects induced by DCM IgG. Preincubation of cardiomyocytes with a tyrosine kinase blocker (PP2 [4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], 1 µmol/l)—a crucial enzyme of the signal cascade of Fc{gamma} receptor IIa—blocked the effects of intact DCM IgG (cell shortening: –1.3 ± 1% and calcium transient: –0.9 ± 1%).


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Cardiac Fc{gamma} receptors IIa and their potential functional relevance.   Disturbances of the humoral immune system play an important role in the pathogenesis of DCM. The present study suggests that DCM IgGs, although binding to their respective cardiac epitopes via their Fab parts, induce their negative inotropic effects via their Fc part by binding to the newly detected Fc{gamma} receptor IIa on cardiomyocytes. The Fc{gamma} receptors IIa can induce an activating signal via its cytoplasmic domains, thereby possibly triggering the negative inotropic effect. The proposed model of Fc{gamma} receptor-dependent activation of cardiomyocytes by DCM autoantibodies provides an explanation of why antibodies directed against different antigens on cardiomyocytes can induce the same functional effects. However, we can merely speculate about the potential role of the interaction between antibodies and the Fc{gamma} receptor on the pathogenesis of DCM.

Figure 2 summarizes our major findings: whereas intact IgG of DCM patients induced negative inotropic effects (Fig. 2A), their respective F(ab')2 fragments did not. However, these F(ab')2 fragments inhibited the effect of intact DCM IgG (Fig. 2B), as did Fc fragments of normal IgG (Fig 2C). When we reconstituted the Fc part of the human-DCM-F(ab')2 fragments by adding an anti-human-F(ab') antibody, the resulting "extra-long" IgG molecule induced a pronounced negative inotropic effect comparable to findings for intact DCM IgG (Fig. 2D). Interestingly, we observed in previous studies that DCM antibodies of the IgG-3 subclass, which are the longest IgG molecules, play an important role in the negative inotropic effects of DCM antibodies on cardiomyocytes in vitro (21). In accordance with these in vitro experiments, we were able to measure the improvement in cardiac function of DCM patients during immunoadsorption therapy only if antibodies belonging to the IgG-3 subclass were also effectively depleted—but not if IgG-1, IgG-2, and IgG-4 antibodies alone were removed from patient plasma (21,22). This might indicate that the Fc part of IgG-3 has enhanced affinity for the involved Fc receptor, as is the case for the Fc{gamma} receptor IIa (23), or it might indicate a large spatial distance between the antigens involved in DCM and the Fc{gamma} receptor that could be bridged only by the longer Fc part of IgG-3. Biologically, this might represent a protection mechanism that prevents all antibodies against cardiomyocytes from inducing Fc{gamma} receptor-dependent cell activation, with potential, consequent, severe cell damage. The DCM antibodies do not bind with their Fab part to the Fc{gamma} receptor but to other antigens on the cardiomyocyte. We assume that the antigen(s) allows binding of several antibodies in close proximity to each other: these clustered antibodies then crosslink the Fc{gamma} receptor IIa via their Fc-part, thereby causing clustering and activation. As in other cells, Fc{gamma} receptors IIa require clustering before they are activated; this might also be the case for activation of Fc{gamma} receptor IIa in cardiomyocytes. Monomeric IgG or Fc parts that do not perform crosslinking can inhibit this activation (24). Furthermore, as in other cells, it is possible that a polymorphism of cardiac Fc{gamma} receptor IIa might influence the response of the receptor (25). However, this point should be elucidated in a larger study population.

Role of the IgG Fab.   In our study, F(ab')2 fragments of a particular patient did not block the effects of the IgG of other DCM patients. This finding strongly indicates that different antibody binding sites are involved in different patients. We cannot differentiate whether these antigens are exposed on the same protein or on different proteins. After binding of the DCM IgG to their respective antigens via the Fab part, their Fc parts bind to Fc{gamma} receptors (Fig. 2).

Although the F(ab')2 fragments we assessed had no short-term functional activity, some DCM autoantibodies might very well also induce specific effects caused by binding to their epitope via the Fab part alone. This might be the case for antibodies directed against the beta-1-receptor, which induce a positive inotropic effect and an increase of cyclic adenosine monophosphate in cardiomyocytes (26,27) or for antibodies directed against cardiac troponin I, which induce enhancement of calcium current in cardiomyocytes (16). However, because all these experiments were performed with total IgG antibodies, the role of the Fc part of these antibodies remains to be elucidated. In our experiments, effects induced by cross-linking of the antigen on cardiomyocytes are unlikely, because the F(ab')2 fragments of the antibodies had no functional effects on cardiomyocytes, although they should logically be able to crosslink the proteins to which they bind as effectively, as does total IgG (Fig. 1). We also addressed the issue of inhibitory anti-idiotype antibodies by our experiments. Anti-idiotype F(ab')2 fragments would be able to inhibit DCM idiotype antibodies and could be an explanation for the inhibitory effect of F(ab')2 fragments. However, in our experiments, the F(ab')2 fragments and the DCM IgG were incubated sequentially (not at the same time; i.e., we first incubated with F(ab')2, washed the cells, and then incubated with intact DCM IgG). It is therefore highly unlikely that anti-idiotype antibodies are the reason for the inhibitory effect of some F(ab')2 fragments.

A recent study disclosed that intact IgG (but not F(ab')2 fragments) markedly ameliorates experimental giant cell myocarditis (28). The authors primarily explained this effect by suppression of dendritic cells. Furthermore, Gullestad et al. (29) suggested that IgG treatment might induce beneficial effects in patients with heart failure. On the basis of the present study, some of the effects might have been induced by functional blockade of cardiac Fc{gamma} receptors by high-dose IgG.

Our experiments imply that IgG binding to cardiomyocyte surface antigens as well as Fc{gamma} receptors IIa accomplishes cross-linking of the Fc{gamma} receptors IIa and thereby initiates signaling. This binding initiates a process that inhibits acute cardiomyocyte contractility by suppression of calcium transients. In hematopoietic cells, activation of Fc{gamma} receptor IIa induces an increase of intracellular calcium levels via tyrosine kinase pathways (30,31). Inhibition of the cardiosuppressant effects of DCM antibodies by the tyrosin kinase inhibitor PP2 provides further evidence for an important role of Fc{gamma} receptors IIa in DCM.

The present study has parallel implications with respect to another severe immune-mediated disorder in cardiovascular medicine: heparin-induced thrombocytopenia (32). In heparin-induced thrombocytopenia, antibodies are generated against the self-protein, platelet factor 4, when platelet factor 4 forms multimolecular complexes with heparin (33). This results in immune complexes, which activate platelets by cross-linking their Fc{gamma} receptors IIa (20). Intravascular activation of platelets results in enhanced thrombin generation and can lead to catastrophic thromboembolic complications (34). We hypothesize that a similar mechanism is basically involved in DCM (i.e., autoantibodies bind to their antigen via the Fab part and then induce cardiac dysfunction by activating Fc{gamma} receptors IIa on cardiomyocytes). This model might also be potentially applied for other antibody-mediated degenerative autoimmune disorders.


   Footnotes
 
This study was supported in part by a grant from the Deutsche Forschungsgemeinschaft (SFB-TR 19) and by the Department of Cardiovascular Medicine.


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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