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European Journal of Heart Failure 2006 8(5):460-467; doi:10.1016/j.ejheart.2005.10.019
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© 2006 European Society of Cardiology

Effects of autoantibodies removed by immunoadsorption from patients with dilated cardiomyopathy on neonatal rat cardiomyocytes

Jie Chena,b, Lisa Larssona, Espen Haugena, Olga Fedorkovaa, Eva Angwalda, Finn Waagsteina,c and Michael Fua,d,*

a Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital Göteborg, Sweden
b Department of Cardiology, Xinhua Hospital, Shanghai Second Medical University Shanghai, PR China
c Department of Cardiology, Sahlgrenska University Hospital Göteborg, Sweden
d Department of Medicine, Sahlgrenska University Hospital Göteborg, Sweden

* Corresponding author. Heart Failure Center, Department of Medicine c/o Wallenberg Laboratory, Sahlgrenska University Hospital, SE 41345 Göteborg, Sweden. Tel.: +46 31 3422923; fax: +46 31 823762.Email address: Michael.fu{at}wlab.gu.se


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Introduction: Immunoadsorption has been shown to improve cardiac performance and reduce mortality in patients with dilated cardiomyopathy. In this study, the underlying mechanism for these beneficial effects was investigated in cultured rat cardiomyocytes.

Methods and results: Immunoadsorption was performed in patients with dilated cardiomyopathy (n=7). Antibody-induced complement-dependent cytotoxicity was investigated by colorimetric MTT. Autoantibodies against the β1-adrenoceptor were detected by ELISA and purified. Column eluent from six patients exhibited a cytotoxic effect, three patients were positive for the β1-adrenoceptor autoantibodies. The purified autoantibodies were able to visualize the β1-adrenoceptors by immunocytofluorescence on rat cardiomyocytes, and also displayed partial agonist properties and induced a positive chronotropic effect, which were blocked by the β1-selective antagonist bisoprolol and the peptide corresponding to the β1-adrenoceptor. Column eluent from one patient induced apoptosis in nick end labelling test (8.1±1.7% vs. 2.9±1.2% in control, p<0.05).

Conclusion: Autoantibodies removed by immunoadsorption from patients with dilated cardiomyopathy have a pathophysiological role, as shown by the complement-dependent cytotoxicity and chronotropic action on rat cardiomyocytes. This implies that removal of circulating autoantibodies might be part of the underlying mechanism for improved cardiac function.

Key Words: Autoantibodies • β1-Adrenoceptor • Cytotoxicity • Dilated cardiomyopathy • Immunoadsorption

Received May 18, 2005; Revised July 30, 2005; Accepted October 31, 2005


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Dilated cardiomyopathy (DCM) is a chronic myocardial muscle disease characterized by decreased myocardial contractility, dilation of the heart and progressive heart failure. Patients with DCM are usually significantly younger than those with ischaemic cardiomyopathy. The prognosis of advanced DCM is dismal, there are 1.99 hospitalizations per patient per year, the 1-year mortality rate is 30.5% [1] and DCM is also one of the main reasons for heart transplantation, but the aetiology of DCM is still unknown. Disturbances in humoral and cellular immunity have been described in patients with DCM [2]. It has been shown that DCM is often related to elevated levels of autoantibodies against cardiac functional or structural proteins, including mitochondrial, β1-adrenoceptor, M2 muscarinic receptor, and myosin heavy chain [3-5]. Active immunization of rabbits with a peptide corresponding to the second extracellular loop of human β1-adrenoceptor has been shown to induce similar morphological changes in the heart to those found in DCM patients [6].

DCM can develop congestive heart failure refractory to conventional treatment with angiotensin converting enzyme (ACE) inhibitors, digitalis, diuretics and β-adrenoceptor blockade. Emerging therapy using immunoadsorption is receiving increased attention. A number of case studies and two controlled clinical trials have shown that the removal of cardiac autoantibodies, belonging to the immunoglobulin G (IgG) fraction which are extractable by immunoadsorption (IA), results in a significant improvement in haemodynamic parameters, in these patients [7,8]. Follow-up evaluations of these studies suggest a reduced morbidity in the patient group treated with IA compared to the control group who received conventional therapy only [9].

It has been shown that column eluent from immunoadsorption containing the eliminated antibodies decreases cell contraction in adult rat cardiomyocytes by depression of Ca2+ transients [10]. Purified autoantibodies against the β1-adrenoceptor from column eluent increased L-type Ca2+ current and prolonged action potential duration [11]. The functional significance of these removed autoantibodies remains to be further elucidated. In the present study, the effects of autoantibodies removed by immunoadsorption from DCM patients on complement-dependent cytotoxicity (CDC), chronotropic action and apoptosis in cultured neonatal rat cardiomyocytes were investigated.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1. Study patients and Immunoadsorption
Seven DCM patients (age 54.7±2.4, left ventricular ejection fraction [LVEF]<40%) were included. Coronary heart disease was excluded by angiography. Acute myocarditis was excluded according to the Dallas criteria [12]. Patients were excluded if they had active infectious disease, cancer, chronic alcoholism or heart failure due to other known origins. All patients were treated with optimal conventional treatment for at least 3 months. Immunoadsorption was performed with protein A columns (Immunosorba, Fresenius, Germany). The first two patients underwent three IA courses, at 1-month intervals, each course consisted of three sessions on 3 days. The other five patients underwent one IA course, with five sessions on five consecutive days and received 0.5 g/kg polyclonal IgG (OCTAPHARMA, Schweiz) intravenously to reduce the risk of infection after the last session. Invasive haemodynamic measurements using a Swan Ganz thermodilution catheter were carried out in these five patients. Written informed consent was obtained from each patient, and the protocol was approved by the Ethics Committee at the University of Göteborg.

2.2. Collection column eluent and purification of β1-AAB
Column eluent (CE) from IA columns containing the eliminated IgG was collected and neutralized (0.18 M KH2PO4, 1.92 M K2HPO4, pH 8.0) during the first IA session, followed by extensive dialyzation against phosphate buffer saline (PBS). Micro BCATM protein assay kit (Pierce, IL) was used to determine the protein concentration of antibodies. Aliquots of CE were stored at –20 °C until use. In addition, polyclonal IgG (Octapharma), which is prepared from pooled IgG from more than 3500 healthy blood donors and usually used as immunoglobulin substitution after IA in DCM patients, was used for control purposes.

H26R peptide corresponding to the second extracellular loop (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) (197-222) of the human β1-adrenoceptor was synthesized using the solid-phase method and verified by mass spectral analysis (LSUHSC Core Labs, USA). β1-AAB was purified by affinity chromatography using a column of H26R peptide coupled to CNBr-activated sepharose (Amersham, Sweden) as previously described [13]. The anti-peptide antibodies adsorbed on the affinity column in PBS were eluted with 0.2 M glycine, pH 2.8. Immediately after elution, the antibodies were neutralized in 100 µl 1 M Tris-HCl, pH 9.0. The purified β1-AAB was dialyzed extensively against PBS, and stored at –20 °C until use. The concentration of β1-AAB was measured by optical density at 280 nm.

2.3. Cell culture
Primary cultures of cardiomyocytes were prepared from the ventricles of neonatal Wistar rats by the method of Simpson and Savion with some modifications [14]. Briefly, hearts from 1-3 days old neonatal rats were removed aseptically. The atria and aorta were discarded, and the ventricles were minced and trypsinized at 37 °C with gentle stirring in G-solution (in mM: 137 NaCl, 5.4 KCl, 1.47 KH2PO4, 1.08 Na2HPO4, 6.1 glucose, pH 7.4) containing 0.1% trypsin (Gibco) for 10 min. The supernatant was collected; fresh enzyme solution was added to the tissue and digested for another 10 min. This step was repeated until the tissue was completely digested. The first 2 supernatants were discarded. The supernatant from each digestion was rendered inactive by adding fetal calf serum (FCS, Gibco) to the cell-enzyme mixture, centrifuged and resuspended in Dulbecco's modified Eagle medium (DMEM, Gibco) containing 10% heat-inactived FCS, 100 U/ml penicillin and 100 mg/ml streptomycin. After preplaced at 37 °C for 2 h, the unattached cardiomyocytes were seeded at a density of 1x105 cells/cm2 in 35 mm dishes (Corning, USA) for beating test. BrdU (5-Bromo-2'-deoxyuridine, Sigma) at a final concentration of 0.1 mM was added to the culture to inhibit the proliferation of non-myocytes.

2.4. Complement-dependent cytotoxicity
CE-induced CDC effect was determined by MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] assay as previously described [15] with some modifications. Primary culture cardiomyocytes were seeded in 96 well flat bottom cell culture plates (TPP, Switzerland) at a concentration of 2.5x104 cells per well and cultured in DMEM containing 10% FCS. On the third day the cells were incubated for 2 h in fresh DMEM containing 1% FCS. Test CE or control IgG were diluted in the same medium, and sterilized by filtration through 0.22 µm membrane filters. 100 µl of each sample was added in quadruple wells. The plate was incubated at 37 °C for 3 h in CO2 incubator. After 2 washes with DMEM, freshly reconstituted baby rabbit complements (Cedarlane, Canada) diluted 1:20 with DMEM were added to each well. The plate was incubated as above for 2 h. After 2 washes, cell viability was measured by MTT cell proliferation kit (ATCC, Manassas, USA) in accordance with the manufacturer's protocol. 100 µl DMEM and 10 µl of MTT reagent were added to each well. The plate was incubated at 37 °C for 4 h in CO2 incubator. 100 µl of detergent was added in all the wells. The plate was gently swirled in the dark overnight at room temperature. The plate was read at 590 nm in microplate spectrophotometer system (Molecular Devices, USA). The reference wavelength 650 nm was used. The percentage of cytotoxicity for each sample was calculated using the formula: % Cytotoxicity=(Maximum uptake–sample uptake)/Maximum uptakex100. The cutoff value of the cytotoxicity of test CE was taken as mean value and two times the standard deviation (S.D.) of the cytotoxicity obtained from healthy control IgG. This procedure was repeated twice in different cultures.

2.5. Fluorescence microscope immunocytochemistry and ELISA
Cultivated cardiomyocytes on coverslips were fixed in 2% paraformaldehyde for 20 min, washed with cold PBS, and blocked for 10 min with 10% goat serum (Zymed, USA). Subsequently, cells were incubated with affinity purified β1-AAB (67 nM, diluted with 1% goat serum) overnight at 4 °C. After washing three times with PBS, the slides were incubated with FITC conjugated goat anti-human antibody (Jackson Immunoresearch, USA), diluted 1:100 with 1% goat serum for 1 h at room temperature. After washing and mounting, slides were examined by a Zeiss Axioplan fluorescence microscope. Enzyme-linked immunosorbent assay (ELISA) with peptide H26R was performed as previously described in order to detect β1-AAB from column eluent [13].

2.6. Cellular physiological studies
Chronotropic effect was measured as previously described [4]. Briefly, on the third or fourth day the cells were incubated for 2 h in 1 ml of fresh serum-containing medium. Thereafter, the dishes were transferred to the heated stage of an inverted microscope (Olympus, Japan), where 10 small circular fields of the cell layer were observed at 37 °C through the perforations of a metal template, equilibrated with 5% CO2 and 95% air. The data represent observations on 30 cells or cell clusters of synchronously beating myocytes in three different cultures. The basal beating rate on the third and fourth day averaged 136±15 (S.D.) beats per minute (bpm). Immunoglobulin or β-adrenoceptor agonist (–)-isoproterenol (Sigma) was added after a stabilizing period of 15 min. The cell motion were introduced into video camera (HAMAMATSU, Japan) and videotaped by a VHS recorder. An increase in beating rate more than mean value and two times S.D. from healthy control IgG was considered to be positive.

In order to determine the specific action of the autoantibodies, we performed the experiments with the administration of the β1-selective adrenoceptor antagonist bisoprolol (Tocris), the protein kinase A inhibitor Rp-Adenosine-3',5'-cyclic monophosphorothioate triethylammonium salt (RpcAMPS) (Calbiochem) or peptide H26R.

2.7. TUNEL assay
To purify cardiomyocytes and reduce fibroblasts and cell debris, discontinuous percoll (Amershan) gradient (densities 40.5%, 58.5% and 75%) separation was performed as previously described with slight modifications [16]. The cell suspension was layered onto the top of the gradients. The discontinuous gradient was centrifuged at 900xg for 30 min. Two bands at the interface of 40.5-58.5% percoll and at the interface of 58.5-75% percoll were collected and used as cardiomyocytes. The cardiomyocytes were washed and seeded on type I collagen-coated coverslip (BD Biosciences) at a concentration of 5x104 cells per coverslip in DMEM containing 10% FCS. After 2 days in culture, the medium was replaced with DMEM containing 0.5% FCS. Treating with column eluent or control IgG pool on cardiomyocytes was started 24 h after placing in DMEM with 0.5% FCS. In situ cell death was detected in cultured cardiomyocytes by using Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labelling (TUNEL) assay kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. The cells were washed with cold phosphate-buffered saline and fixed with 2% paraformaldehyde for 1 h at room temperature. After washing with PBS, the cells were incubated in permeabilisation solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. The cells were then rinsed with PBS and incubated with the TUNEL reaction mixture for 1 h at 37 °C in a humidified chamber. After washing with PBS, samples were counterstained with mounting medium (Vector, USA) containing 1.5 µg/ml 4',6-diamidino-2-phenylindole (DAPI) and observed under a fluorescence microscope. At least 5 random fields were analysed for apoptotic myocytes in each slide of six independent experiments (n=6). The proportion of TUNEL-positive cardiomyocytes was expressed as a percentage of the total cells counted.

2.8. Statistical analysis
The data are given as means±S.E.M. of independent experiments based on different cell cultures. Significant differences in chronotropic effect was analysed with Student's t-test. For comparison of apoptotic rate, the Mann-Whitney U-tests were used. A probability level of P<0.05 was chosen as the least significant difference.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1. Clinical findings
After the first IA session, the plasma IgG level in DCM patients was decreased by 81%, from 11.9±1.0 g/l to 2.4±0.8 g/l. Comparing cardiac performance at baseline and after the last IA session by invasive hemodynamic measurements (n=5), cardiac output (CO) increased from 4.8±0.3 to 5.5±0.5 l/min, pulmonary capillary wedge pressure (PCWP) decreased from 15.2±2.7 to 11±3.5 mm Hg, and mean pulmonary arterial blood pressure (PAP) decreased from 27.0±3.4 to 20.0±4.0 mm Hg. By echocardiographic assessment (n=7), LVEF increased from 26.7±2.3% to 29.0±3.6%.

3.2. Effect of CE-induced complement-dependent cytotoxicity
Using the mean value and 2S.D. of the cytotoxicity obtained from healthy control IgG as a cut-off, CE from six of the seven patients exhibited a positive CDC effect on neonatal rat cardiomyocytes at a protein concentration of 6.7 µM. Patient 7 had the most intensive effect (60.3±1.8%), while patient 4 had no effect in comparison with control IgG (Table 1). This cytotoxic effect was CE-induced and complement-dependent. Neither CE (6.7 µM) nor complements (1:20) alone had a cytotoxic effect on cardiomyocytes within 2-3 h incubation. Furthermore, if complements were heat-inactived, the cytotoxic effect on the cardiomyocytes induced by CE disappeared. Cell destruction was also directly visualized under a phase-contrast microscope before MTT assay (Fig. 1). Target cardiomyocytes were swollen and lost their pseudopods, they finally died as defined by a granular cytoplasm and pyknotic nucleus.


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  		Table 1 Effects of column eluent from DCM patients on cytotoxicity and chronotropic response

 


Figure 1
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  		Fig. 1 Direct visualization of cardiomyocyte destruction by column eluent (CE)-induced complement-dependent cytotoxicity. After incubation with (A) CE from patient 7 (B) healthy control IgG for 3 h, complements (1:20) were added to cardiomyocytes and incubated for additional 2 h. Target cardiomyocytes were swollen and lost their pseudopods, finally they died as defined by a granular cytoplasm and pyknotic nucleus (arrows). Bar=50 µM.

 
3.3. Positive chronotropic effect of CE and affinity purified β1-AAB
CE from 5 patients had a positive chronotropic effect on spontaneous beating neonatal rat cardiomyocytes (Table 1). At a concentration of 13.4 µM, CE from patient 1 increased beating frequency by 27.5±2.1 bpm, whereas CE from patient 7 only increased beating by 7.7±1.1 bpm. At a concentration of 0.3 µM, CE from patient 1 increased beating frequency by 10.3±1.0 bpm, but CE from patient 7 did not increase beating frequency. CE from patients 4 and 6 did not show a positive chronotropic effect in comparison with control IgG, even at a concentration at 26.8 µM.

CEs from the first three patients were positive when ELISA was applied (Table 1). β1-AABs were purified from these three patients and they were able to visualize β1-adrenoceptor by fluorescence immunocytochemistry staining (Fig. 2). Addition to the cultured neonatal rat cardiomyocytes of affinity purified β1-AABs led in all instances to an increase in beating frequency. This effect was concentration dependent (Fig. 3).


Figure 2
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  		Fig. 2 Indirect immunofluorescence after incubation with (A) purified autoantibodies against β1-adrenoceptor (β1-AAB) from column eluent, (B) healthy control IgG on culture cardiomyocytes. Intense dot-like fluorescence over the cardiomyocytes is observed with β1-AAB incubation. Bar=20 µM.

 


Figure 3
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  		Fig. 3 Plot of concentration dependency of the chronotropic effect induced by autoantibodies against β1-adrenoceptor. Basal rate was 136±15 beats per minute. Increase in beating frequency is expressed as mean±S.E.M. of 30 observed cells or cell clusters.

 
The effects of β1-AAB are similar to the properties of a "partial agonist". Addition of 10 µM isoproterenol increased beating frequency by 50.3±2.8 bpm, whereas addition of 67 nM β1-AABs from patients 1, 2 and 3 increased beating frequency by 22.3±1.7 bpm, 16.7±1.7 bpm, and 20.7±2.3 bpm, respectively. The positive chronotropic effect induced by purified β1-AABs was completely abolished by addition of the β1-selective adrenoceptor antagonist bisoprolol (1 µM). Preincubation of purified β1-AABs with the H26R peptide (1:30), before addition to the cultured cardiomyocytes, annihilated their positive chronotropic effect. The peptide had no inhibitory effect on the cardiomyocytes by itself. In addition, 30 min after addition of RpcAMPS (50 µM), a specific inhibitor of the cAMP-dependent protein kinase A, the agonistic effect of β1-AAB was blocked (Fig. 4).


Figure 4
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  		Fig. 4 Inhibition of the positive chronotropic effect of autoantibodies against β1-adrenoceptor (β1-AAB, 67 nM) by β1-adrenoceptor antagonist bisoprolol (Biso, 1 µM), cAMP-dependent protein kinase A inhibitor RpcAMPS (50 µM) and preincubation with antigen (peptide H26R, 2 µM) respectively. ***p<0.001 vs. β1-AAB.

 
Furthermore, in some cell cultures where the cardiomyocytes were overgrown by the fibroblasts, addition of β1-AAB sometimes induced arrhythmia, such as "bigeminy or trigeminy".

3.4. Effect of CE induced apoptosis
After 24 h incubation with CE from DCM patients, the apoptotic rate of cardiomyocytes was measured by TUNEL assay. Only CE from patient 1 demonstrated a significant increase in apoptotic rate (Fig. 5), the others did not show significant effect on apoptosis in comparison with control IgG pool. This CE induced apoptosis is dose-dependent (Fig. 6). The apoptotic rate induced by CE from patient 1 and healthy control IgG pool at concentration of 13.4 µM was 8.1±1.7% and 2.9±1.2% respectively (p<0.05).


Figure 5
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  		Fig. 5 Column eluent (CE) increased frequency of DNA strand breaks as assessed by TUNEL assay and DAPI staining on cultured cardiomyocytes. Cells were treated with CE from patient 1 or healthy control IgG. Arrows indicated fragmented chromatin in nuclei of apoptotic cells. Bar=20 µM.

 


Figure 6
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  		Fig. 6 Effects of various dilutions of column eluent from patient 1/healthy control IgG (control) on cultured neonatal rat cardiomyocytes. The values (% apoptotic cells) are mean±S.E.M. for 6 independent experiments. *p<0.05 vs. control at same concentration.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
A variety of autoantibodies directed against different cardiac antigens have been detected in patients with DCM [3-5]. These autoantibodies can recognize antigens on the surface of cardiomyocytes and activate complement system through "classical pathway", finally form membrane attack complex and lyse the target cells by perforin. Immune cytolysis induced by CDC reduces cell viability. The reduction of tetrazolium salts (MTT assay) is now widely accepted as a reliable way to examine cell viability. In the present study, CE from six of the seven patients (85.7%) exhibited positive CDC effect on neonatal rat cardiomyocytes by MTT assay. Patient 7 exhibited the most intensive CDC effect among these patients. The intensity of CDC induced by CE was not only dependent on the quantity of the autoantibodies in the CE, but also on their affinity of binding to antigens on the surface of the cardiomyocytes. Since IgG-3 is the most active complement-fixing IgG subclass [17], the subclass of the autoantibodies should be taken into consideration. Recent findings indicate that IgG-3 might play a significant role in the cardiac dysfunction of DCM. The removal of antibodies of the IgG-3 subclass might represent an essential mechanism of IA in DCM [18].

Since autoantibodies in the sera of healthy subjects have a low frequency and low titre, and the frequency increases with age [19], this may explain why in the present study IgG from healthy subjects exhibited a 6.6% cytotoxic effect on cultured cardiomyocytes. Of course, we could not exclude the preformed natural antibodies (PNAb) between human and rats. However, this CDC effect induced by healthy control IgG was low level and served as background.

The results of the present study clearly demonstrate that the affinity-purified β1-AAB not only recognized H26R peptide in ELISA and denatured β1-adrenergic receptor in immunocytochemistry, but also played a functional role on cardiomyocytes. The inhibition of protein kinase A by RpcAMPS suppressed the "partial agonist" effect produced by β1-AAB, indicating that the adenylate cyclase/protein kinase A system constituted the pathway by which β1-AAB displayed its positive chronotropic effect. The characteristics of the purified β1-AAB in our cultures were similar to those described by Wallukat et al. [20].

The functional significance of cardioreactive autoantibodies in DCM remains to be elucidated. It is possible that they serve simply as a marker of myocardial injury, in which case they should be assessed as an epiphenomenon. On the other hand, there is a growing body of evidence to suggest that cardioreactive autoantibodies are pathogenetically relevant to DCM. Autoantibodies against cardiac troponin I (cTNI) are responsible for DCM in programmed cell death-1 (PD-1) receptor-deficient mice. Administration of monoclonal antibodies against cTNI induced dilatation and dysfunction of hearts in wild-type mice [21]. Currently, the most investigated autoantibody is β1-AAB. Immunization with synthetic peptides or fusion-proteins corresponding to the second extracellular loop of the β1-adrenoceptor induced morphological changes similar to those observed in human DCM in the hearts of rabbits [6] and rats [22]. If cardioreactive autoantibodies only serve as an epiphenomenon in DCM patients, it is difficult to explain the improvement of cardiac performance and reduction of morbidity with 3-year follow-up after IA [9,23]. Our in vitro data indicate that β1-AAB purified from CE do not simply reflect an epiphenomenon associated with DCM; rather, they play a functional role as a β1-adrenoceptor partial agonist.

In the present study, CE from 5 patients had a positive chronotropic effect on cardiomyocytes, but two of these were negative in ELISA. A possible explanation is that these two patients might have had autoantibodies against the first extracellular loop of the β1-adrenoceptor. It has been shown that of 24 DCM patients with distinct positive chronotropic effect, 58% of the cases had autoantibodies that recognized the first extracellular loop and 42% recognized the second extracellular loop of the β1-adrenoceptor [4]. Another explanation is that these patients might have other antibodies that induced positive chronotropic effect, such as autoantibodies against β2-adrenoceptor, angiotensin II AT1 receptor [24].

The present study found that CE from patient 1 induced apoptosis on cultured rat cardiomyocytes. Since adult cardiac myocytes have strictly limited ability to regenerate, sustained programmed cell death is likely to contribute to the development and progression of heart failure in DCM. Experiments with cultured adult cardiomyocytes have demonstrated that apoptosis could be induced in vitro by β1-adrenoceptor monoclonal autoantibody [25]. Although heat-shock protein 60 (Hsp60) is generally considered to be an intracellular chaperone polypeptide, it expresses at the surface of endothelial cells. Autoantibody against Hsp60 in systemic lupus erythematosus (SLE) induced apoptosis on cultured endothelial cells [26]. Numerous antibodies against cardiac structures have been detected in DCM patient, such as autoantibodies against the β1-adrenoceptor and the M2 muscarinic receptor [4,5]. On the other hand, some autoantibodies against cardiac functional or structural proteins are currently undetectable. We cannot exclude that CE from patient 1 might contain unknown autoantibodies, which can recognize and activate the Fas receptor or other similar proteins, inducing apoptosis.

IA represents an additional therapeutic approach, which hitherto has been shown to induce haemodynamic improvement and reduce mortality in DCM patients. Results of 3-year follow-up after IA in DCM patients reported recently by two research groups [9,23] showed that IA significantly reduced hospitalisations and improved haemodynamics. Although the hemodynamic improvements (e.g. LVEF) in our study seem to be less than in other reports [7,8], which might be due to different protocols and different immunoadsorbent devices, as well as DCM patients with different levels of heart failure, our in vitro results indicated that autoantibodies in DCM patients had pathophysiological effects on cardiomyocytes. Taken together with in vitro studies from other groups [10,11], the evidence suggests that removal of these autoantibodies improved cardiac performance in DCM patients. Thus IA is an important emerging therapy in the management of severe heart failure and may serve as a "bridge to transplant". However, it is important to identify the subgroups of DCM patients who will benefit the most from IA by further combined clinical and pre-clinical studies.

4.1. Study limitations
Although two different IA protocols were used in these seven patients, the CEs that we used in the cardiomyocytes were all prepared during first session of IA. Because the same type of protein A columns were used in these seven patients and 81% of IgG in serum was removed by IA after the first session, the effects on cultured cardiomyocytes were comparable. CE from larger, randomized and controlled clinical studies is required to confirm our findings.

Although neonatal rat cardiomyocytes constitute a valid tool to study in vitro effects and mechanisms of autoantibodies from patients with DCM, extrapolation to humans should only be made with caution and extrapolation of these in vitro findings to the human in vivo situation should be reconsidered.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
Autoantibodies removed by immunoadsorption from patients with DCM have a pathophysiological role as shown by their complement-dependent cytotoxicity and chronotropic action on cultured neonatal rat cardiomyocytes.


   Acknowledgements
 
The authors thank Dr. Reza Mobini for helpful discussions and Mrs. Helen Svensson for her skilful technical assistance. This work was supported by the Swedish Heart Lung Foundation.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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