Agonistic Anti-ß1-Adrenergic Receptor Autoantibodies From Cardiomyopathy Patients Reduce the ß1-Adrenergic Receptor Expression in Neonatal Rat Cardiomyocytes
From the Max Delbrück Center for Molecular Medicine Berlin-Buch (S.P., H.P.L., R.M., G.W.) and the German Heart Institute Berlin (S.P., J.M.), Germany.
Correspondence to Gerd Wallukat, Max Delbrück Center, Department of Molecular Cardiology, Robert Rössle Straße 10, 13125 Berlin, Germany. E-mail molcard{at}orion.rz.mdc-berlin.de
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Abstract |
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Background—Autoantibodies directed against the ß1-adrenergic receptor have been described in patients with dilated cardiomyopathy. These autoantibodies exert an agonistic, chronotropic effect on spontaneously beating cultured neonatal rat cardiomyocytes. We studied the effect of such antibodies on ß1-adrenergic receptor expression.
Methods and Results—Cardiomyocytes were incubated with either the ß-adrenergic agonist isoproterenol or autoantibodies for 72 hours. ß-Adrenergic receptor expression was studied on the mRNA level with semiquantitative reverse transcription–polymerase chain reaction and on the protein level with immunoblotting. Isoproterenol downregulated both mRNA and ß1- and ß2-adrenergic receptor protein subtypes, whereas the anti–ß1-adrenergic receptor autoantibodies decreased only the ß1-adrenergic receptor mRNA and protein. Long-term incubation of cultured cardiomyocytes with isoproterenol or the anti–ß1-adrenergic receptor autoantibodies reduced the acute stimulatory effect of isoproterenol on the myocytes. These effects were prevented by incubating the cells with isoproterenol in the presence of propranolol or with anti–ß1-adrenergic receptor autoantibodies in the presence of bisoprolol. Bisoprolol also abolished the reduction of the ß1-adrenergic receptor expression caused by longer-term incubation with isoproterenol and the autoantibodies.
Conclusions—We conclude that after longer-term treatment with the anti–ß1-adrenergic receptor autoantibodies, the rat cardiomyocytes showed a ß-adrenergic receptor expression similar to that observed in failing hearts from patients with dilated cardiomyopathy.
Key Words: receptors, adrenergic, beta • antibodies • cardiomyopathy • myocytes • heart failure
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Introduction |
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The pathogenesis of dilated cardiomyopathy (DCM) is poorly understood; however, autoimmune mechanisms may be in part responsible. Autoantibodies against various cardiac structures have been detected in DCM patients, including the mitochondrial ADP/ATP carrier,1 laminin,2 myosin heavy chain,3 and the ß1-adrenergic receptor4 5 (ß1-AR). The failing heart is characterized by a reduced responsiveness to ß-adrenergic agonists6 7 due to persistently elevated circulating norepinephrine levels, which desensitize the ß-adrenergic receptor (ß-AR) response8 and foster adrenergic receptor uncoupling to G proteins. Functional impairment to ß-adrenergic stimulation could also be caused by an increase in Gi
activity or
expression.9 There is evidence that alterations
of both ß-AR and Gi processes contribute to
the loss of response. Several investigators showed a downregulation of
the ß1-AR.10 11 12 13 14 In
contrast, the expression of the ß2-AR was
unchanged or only slightly decreased. Primary cultured rat cardiomyocytes are a convenient model to study adrenergic receptors and their signal transduction pathways. Short-term stimulation by the ß-adrenergic agonist isoproterenol causes an increase in the beating frequency of the cardiomyocytes, accompanied by a desensitization and downregulation of the ß-AR after long-term treatment. DCM patient sera containing anti–ß1-AR autoantibodies increase the beating frequency of the cells. The chronotropic activity differs from the effect of isoproterenol in that the ß-AR is not desensitized in short-term experiments.15 We proposed earlier that chronic ß1-AR stimulation by anti–ß1-AR autoantibodies may trigger the development of DCM. Evidence for this hypothesis was supplied by Dörffel et al,16 who removed the anti–ß1-AR autoantibodies in their DCM patients with immunoadsorption. Hemodynamic improvement correlated inversely with the decrease in autoantibody titers.16 After a cardiac assist device was implanted, the autoantibodies disappeared in the DCM patients.17 These results supported the notion that disappearance of anti–ß1-AR autoantibodies is associated with improved cardiac function. To further elucidate the relationship between anti–ß1-AR autoantibodies and ß-AR and their regulation, we studied the long-term effect of these autoantibodies on both the ß1-AR and ß2-AR subtypes.
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Methods |
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Neonatal Rat Cardiomyocytes
Neonatal rat cardiomyocytes were prepared as described earlier.4 15 18 About half the isolated cells were myocytes, and the rest were fibroblasts and other cells. Briefly, single cells were dissociated from the minced ventricles of 1- to 2-day-old Wistar rats by use of a 0.25% crude trypsin solution and were cultured in Halle SM20-I medium containing 2 µmol/L fluorodeoxyuridine,18 19 which prevents proliferation of any nonmyocyte cells. Longer-term treatment with drugs or with anti–ß1-AR autoantibodies was performed for 72 hours. During this time, we observed no dedifferentiation of the cultured cells. The control cells were timed controls. Isoproterenol, sodium deoxycholate, and aprotinin were purchased from Sigma. Propranolol was acquired from Isis-Porem Pharma. Bisoprolol was a gift from E. Merck.
Immunoglobulin Fraction Preparation
Serum was obtained from patients with idiopathic DCM. The
diagnosis was verified by cardiac catheterization of
all patients. Biopsies confirmed the diagnosis and ruled out other
heart diseases. The serum immunoglobulin fraction was isolated by
ammonium sulfate precipitation at a saturation of 40% and washed
twice. The samples were dialyzed against 1 L dialyzed buffer (10
mmol/L mono- and dibasic sodium phosphate, 154 mmol/L NaCl
solution, pH 7.2) at 4°C for 30 hours. The antibodies were taken up
in dialyzed buffer, stored at -20°C, and used in a dilution of
1:40.
RNA Preparation
Cardiomyocytes (
6x106 per flask) were
transferred from flasks to reaction tubes and centrifuged for 3
minutes at 3000g at 4°C. After the medium was removed,
total RNA was isolated as described by Chomczynski and
Sacchi.20
DNAse Treatment
Isolated RNA (18-µL) samples were mixed with 2.2 µL
10xDNase buffer (40 mmol/L Tris-HCl, 6 mmol/L
MgCl2, pH 7.5)/2 µL DNase I (Pharmacia Biotech)
and heated at 37°C for 10 minutes. The RNA purification was performed
with the RNeasy Kit (Qiagen GmbH). The RNA concentration was determined
by UV absorption. The ratio of optical densities at 260 and 280 nm was
between 1.8 and 2 in all cases.
Reverse Transcription
For reverse transcription (RT) into cDNA, 1 µg of total RNA
was used. First, RNA was denatured together with 25 pmol random
hexamers for 5 minutes at 70°C and then reverse transcribed by
incubation for 60 minutes at 42°C in the presence of 0.5 mmol/L
dNTP, 0.01 mmol/L dithiothreitol, 1 U Superscript reverse
transcriptase, 1 U RNase H (both Gibco-BRL Life Technologies GmbH) in
50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, and 3 mmol/L
MgCl2. The final volume was 20 µL.
Polymerase Chain Reaction
Polymerase chain reaction (PCR) was performed with 2 µL (3
µL for ß1-AR) cDNA in a final reaction volume
of 50 µL. The assay mix contained 50 mmol/L KCl, 10 mmol/L
Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2,
0.2 mmol/L dNTP, 1 mmol/L of the respective primers, and 1.5
U of Taq DNA polymerase (Gibco-BRL Life Technologies GmbH)
(Table
). After
initial denaturation at 95°C for 3 minutes and further denaturation
for 1 minute, primer annealing was carried out at 60°C for 1 minute
and extension at 72°C for 1 minute. The number of cycles was 27 for
GAPDH, 33 for ß1-AR, and 31 for
ß2-AR. The PCR products were
analyzed after 2% agarose gel electrophoresis. We could not
coamplify GAPDH with either ß1-AR or
ß2-AR because the amplification rates of the
products were not the same. During coamplification of any 2
sequences, competition occurs for available Taq polymerase,
nucleotides, and magnesium. Consequently, the reaction
condition may differ from PCR to PCR. We therefore carried out the PCR
with aliquots of the same cDNA in separate tubes with GAPDH as an
external standard. Because the ß1-signal was
weaker in preliminary experiments, a greater quantity of
ß1-mRNA was used in the reaction than
ß2-mRNA or GAPDH. DNA sequencing of both
strands from the ß1-AR and
ß2-AR PCR products was done by InViTek. To
quantify the PCR products, agarose gels were filmed with a video
camera, and the optical density was measured by densitometry (Raytest).
The amount of the PCR products was normalized to the signal of the
external standard GAPDH.
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Protein Isolation
Cardiomyocytes were washed with PBS buffer (mmol/L: dibasic
sodium phosphate 9.1, monobasic sodium phosphate 1.7, NaCl 150,
pH 7.4). The cells were then scratched off with a rubber policeman,
added to 0.1 mL RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate,
0.1% SDS) with freshly added inhibitors (µL/mL: 10 mg/mL
PMSF 10, aprotinin 30, 100 mmol/L sodium orthovanadate 10) and
homogenized by passage through a 21-gauge needle. The
lysate was incubated on ice for 1 hour and centrifuged at
maximal speed for 30 minutes at 4°C. The protein concentration was
measured according to the method of Lowry et
al.21
Electrophoresis and Western Blotting
Electrophoresis was performed with 7.5%
SDS–polyacrylamide gels. Before they were loaded onto the gel,
the samples were heated for 1 minute at 95°C. Thereafter, the
separated proteins were blotted on nitrocellulose. The blots were then
incubated with blocking buffer (5% skim-milk powder and TBST: 10
mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05% Tween-20, pH 8.0) for 1
hour at room temperature, followed by incubation with 1 µg/mL
anti–ß1-AR or
anti–ß2-AR polyclonal rabbit antibodies (Santa
Cruz Biotechnology Inc) in blocking buffer overnight at 4°C. The
antibodies were directed against an 18-mer
(ß1-AR, 446 to 464) or 19-mer
(ß2-AR, 399 to 418) peptide of the carboxy
terminus of the ß-AR. After 2 washes for 7 minutes with TBST
incubation with horseradish peroxidase–conjugated anti-rabbit
antibodies (Sigma), was carried out at a dilution of 1:12 000 for 1
hour at room temperature. The blots were then washed 3 times with TBST
and once with TBS for 5 minutes. Detection of the proteins was
performed with an ECL Kit (Amersham Buchler GmbH & Co KG). The
immunostaining intensity was measured
densitometrically. To check the signal specificity,
ß1-AR or ß2-AR
polyclonal antibodies were preincubated with the corresponding peptides
(5 µg/mL for ß1-AR; 20 µg/mL for
ß2-AR) for 5 hours at room temperature.
Immunoprecipitation
We used IgG fractions from 3 patients with DCM previously
applied to the stimulatory test. These IgG fractions contained
autoantibodies against the ß1-AR. These
autoantibody-containing IgG fractions had been collected by
immunoadsorption, and their specificity had been tested in a functional
test as described below. The autoantibodies were specifically directed
against amino acids 197 to 222 of the second extracellular loop of the
ß1-AR. First,
anti–ß1-AR autoantibodies were linked to
Fc-specific anti-human IgG and conjugated with magnetic beads by
overnight incubation at 4°C on a rotator. After 3 washes with PBS,
solubilized membrane proteins were incubated with the joined antibodies
for 2 hours at 4°C and washed 3 times with RIPA buffer. The
precipitated proteins were eluted with 70 µL SDS sample buffer by
heating at 95°C for 3 minutes. Precipitated proteins were separated
by 7.5% SDS-PAGE and blotted on nitrocellulose. We used a commercial
peptide antibody for detection raised against the
ß1-AR C terminus (Santa Cruz).
Functional Test
Four-day-old neonatal cardiomyocyte cultures were
used to determine the stimulatory effect of isoproterenol on the
spontaneously beating rate as described
elsewhere.4 15 17 18
Statistical Analysis
Values are given as mean±SEM. We used Student's t
test for paired and unpaired groups or 1-way ANOVA with the Bonferroni
post hoc test for selected pairs.
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Results |
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RT-PCR
RT-PCR resulted in single, specific products of the expected size (Figure 1
, top). Sequence
analysis was carried out with the corresponding known sequences
of both receptor subtypes.22 23 For
quantification, we optimized the reactions. The cycle number was
titrated for sufficient but exponential amplification (Figure 1, bottom). Furthermore, RT was done with different amounts of RNA. The
amplification of the ß1-AR and the
ß2-AR was proportional to the amount of RNA
(0.5 to 1.25 µg). For all further experiments, 1 µg of total RNA
was used for the RT. GAPDH expression was unchanged in differently
treated cells. We were therefore able to use GAPDH as an external
standard.
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Four-day-old neonatal rat cardiomyocytes were treated for
72 hours with isoproterenol. Both the ß1-AR and
ß2-AR mRNAs were significantly reduced after
treatment with isoproterenol (Figure 2),
to the same level, compared with untreated cells
(ß1-AR, 71.6%; ß2-AR,
73±7%). Incubation with anti–ß1-AR
autoantibodies had different effects on the mRNA expression of the
receptor subtypes (Figure 3). After 72
hours of treatment, the expression of ß1-AR
mRNA was significantly decreased to 66±5% of control and corresponded
to the isoproterenol-treated cells. In contrast, the
ß2-AR mRNA was unchanged by the
anti–ß1-AR autoantibody incubation (94±9%).
To check whether or not the resulting downregulation of the
ß1-AR mRNA was a specific
anti–ß1-AR autoantibody–mediated effect,
cells were treated with IgG fractions from healthy controls (negative
IgG fraction). Figure 3 shows that the ß1-AR
was expressed equally in untreated cells and in cells incubated with
negative IgG fractions (110%). In comparison, the expression was
markedly reduced in anti–ß1-AR
autoantibody–treated cells.
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Western Blotting
ß1-AR and
ß2-AR immunodetection was carried out with
polyclonal antibodies raised against a peptide of the carboxy terminus.
For the ß1-AR, a 42-kDa protein was detected,
and for the ß2-AR, a 65-kDa protein was
detected. To check the specificity of the signals, antibodies were
neutralized with the respective peptides before immunodetection (Figure 4). Furthermore, immunoprecipitation was
performed for the ß1-AR using
anti–ß1-AR autoantibodies of DCM patients. The
precipitated proteins were electrophoretically separated, transferred
to nitrocellulose, and detected by the Santa Cruz
ß1-AR antibodies. Figure 4b shows that the
anti–ß1-AR autoantibodies directed against the
second extracellular loop precipitated a 42-kDa protein that was
detected by the second ß1-AR antibodies
directed against the carboxy terminus. The left lane revealed that
additional signals at 55 kDa were due to cross-reactions with the
second polyclonal anti-rabbit antibodies. The quantification ranges for
both receptor subtypes were determined by protein dependency
experiments (Figure 4). The signals increased proportionally with the
protein concentrations. For semiquantitative analysis of the
ß1-AR, 20 µg of protein was sufficient,
whereas 40 µg of protein was loaded for the
ß2-AR experiments.
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Isoproterenol treatment (Figure 5)
led to a significant reduction of both ß-AR protein levels
(ß1-AR, 50±12%;
ß2-AR, 79±5%). Incubation with
anti–ß1-AR autoantibodies (Figure 6) decreased only the
ß1-AR protein level (65±13%), but not the
ß2-AR (117±5%). This subtype-specific effect
of the anti–ß1-AR autoantibodies confirmed the
mRNA expression data (Figure 3
) shown earlier. In contrast,
non–anti–ß1-AR autoantibody–containing IgG
fractions did not influence the ß1-AR protein
expression. Because isoproterenol and the
anti–ß1-AR autoantibodies reduced the
ß1-AR, we tested whether or not a specific
ß1-adrenergic blocker would abolish the
ß1-AR downregulation. Cardiomyocytes were
treated with isoproterenol and bisoprolol simultaneously
for 72 hours. We applied anti–ß1-AR
autoantibodies and bisoprolol to the cells in the same manner and found
that bisoprolol partly prevented the isoproterenol-induced
ß1-AR reduction (Figure 7). The effect of the
anti–ß1-AR autoantibodies was completely
blocked by bisoprolol.
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Functional Tests
Isoproterenol stimulation was measured in untreated cells
and in cells preincubated with 10 µmol/L isoproterenol or
anti–ß1-AR autoantibodies for 72 hours. A
significant reduction in the stimulatory effect (Figure 8) was observed with both isoproterenol
and anti–ß1-AR autoantibodies
(P<0.001). When isoproterenol was applied together with the
nonselective ß-adrenergic antagonist
propranolol for 72 hours, the decrease in number of beats
was completely blocked. Similar results were found after long-term
treatment of anti–ß1-AR autoantibodies
together with the ß1-specific
antagonist bisoprolol.
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Discussion |
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We showed that nonselective isoproterenol downregulated both ß1- and ß2-AR mRNA and protein in neonatal rat cardiomyocytes, whereas the selective anti–ß1-AR autoantibodies downregulated only ß1-AR mRNA and ß1-AR protein. These findings underscore the specific action of the anti–ß1-AR autoantibodies. Our observations were confirmed by experiments in which we incubated the cells with isoproterenol or anti–ß1-AR autoantibodies in the presence of bisoprolol. Bisoprolol, which occupied the ß1-AR, inhibited the receptor downregulation from both isoproterenol and anti–ß1-AR autoantibodies. Finally, our functional studies support our conclusions in that propranolol blocked the desensitization afforded by preincubation with isoproterenol and bisoprolol blocked the desensitization afforded by preincubation with ß1-agonistic anti–ß1-AR autoantibodies.
Immunoblotting revealed bands at 42 kDa and 65 kDa
for the ß1-AR and
ß2-AR, respectively. Our molecular weight
estimates for the ß1-AR differ from those of
other investigations.23 24 25 Hebert et
al26 performed immunoblotting
experiments and showed that the human ß2-AR
expressed in Sf9 cells had a monomeric (43 to 50 kDa) and a dimeric (85
to 95 kDa) form. We demonstrated that our
immunoblotting signal represented the
ß1-AR by showing that the 42-kDa protein was
recognized by 2 different antibodies. One of these antibodies (from the
patients) precipitates the 42-kDa protein, and the other (Santa Cruz)
was used for the detection. Because ß-ARs are
glycoproteins,27 their migration
characteristics in SDS-PAGE depend on their glycosylation state. We
therefore treated the cardiomyocytes with 2 µg/mL
tunicamycin for 72 hours to inhibit the protein
N-glycosylation (data not shown).
Immunoblotting revealed no differences between
untreated cells and tunicamycin-treated cells in terms of the
ß1-AR molecular weight signal. We conclude that
some of the ß1-AR in cultured
cardiomyocytes may not be glycosylated. Such an effect
could possibly explain the uncommon migration characteristics in
SDS-PAGE. However, we cannot exclude the possibility that this protein
is a proteolytic degradation product.
Our data showed that prolonged isoproterenol treatment reduced both the ß1-AR and ß2-AR mRNA and protein levels, which is in accord with the observation that isoproterenol causes a downregulation of ß1-AR and ß2-AR mRNA levels in DDT1-MF2 hamster cells, C6 glioma cells, and H9c2 cells.28 29 30 Anti–ß1-AR autoantibodies had an exclusive effect on the ß1-AR gene and protein expression. In contrast, the ß2-AR expression was nearly unchanged. The modulation of the ß1-AR expression was abolished by the ß1-adrenergic blocker bisoprolol. The IgG fractions from healthy controls did not influence the ß1-AR expression. Thus, the downregulation was indeed a specific effect of the anti–ß1-AR autoantibodies.
The effect of anti–ß1-AR autoantibodies
on ß-ARs in neonatal rat cardiomyocytes displays
similarities to effects observed in failing human hearts. Several
studies show that only the ß1-AR mRNA levels
are reduced in failing hearts.11 12 14 The
expression of the ß2-AR mRNA is unchanged or
only slightly decreased.13 The downregulation has
been attributed to the effects of norepinephrine, which
differs from isoproterenol in terms of ß-AR affinity.
Norepinephrine is a potent ß1-AR
and 1-AR agonist and has little action on
ß2-AR, whereas isoproterenol has a powerful
action on all ß-AR.31
Similarly, radioligand binding studies reveal that only the ß1-AR but not the ß2-AR density is lower in hearts from DCM patients,12 13 a condition in which circulating ß1-AR antibodies have been described.15 16 17 Our functional data corresponded with the results on mRNA and protein levels. Longer-term treatment with isoproterenol and anti–ß1-AR autoantibodies led to a loss of cardiomyocyte isoproterenol responsiveness. In failing hearts, the reduction of the ß1-AR number is in accord with the decreased positive inotropic effect of isoproterenol.7 Our findings elucidate the role of circulating ß1-AR autoantibodies in DCM patients. We suggest that, like norepinephrine in any form of heart failure, the autoantibodies stimulate, downregulate, and reduce the responsiveness of contractile mechanisms.
ß-Blockers offer a further medical approach to heart failure therapy in DCM patients.32 We showed that ß1-AR downregulation with either isoproterenol or anti–ß1-AR autoantibodies was prevented by ß-adrenergic blockers. In Western blotting experiments, bisoprolol partly abolished the isoproterenol-induced ß1-AR downregulation and blocked the anti–ß1-AR autoantibody–induced downregulation completely. Similarly, in the functional test, the chronotropic response was restored by propranolol and bisoprolol. Not only norepinephrine but also anti–ß1-AR autoantibodies could be involved in the decrease of ß1-AR number and ß-adrenergic responses in DCM patients. The possibility that anti–ß1-AR autoantibodies could play a role in the pathogenesis of DCM is underscored by the association between the autoantibodies and cardiac function.15 16 17 In these patients, the removal of anti–ß1-AR autoantibodies with immunoadsorption led to improved cardiac function. Randomized trials will be necessary to confirm these results; however, our present data may provide a mechanism by which these autoantibodies lead to and maintain DCM.
ß1-AR downregulation probably depends on microtubules, because colchicine pretreatment inhibited ß1-AR but not ß2-AR downregulation in a glioma cell line.33 The microtubule stabilizer taxol inhibited the effects of colchicine on isoproterenol-induced ß1-AR downregulation. The effect appears to occur distal to the receptor at the level of Gs protein and/or via cAMP generation.34 Hori et al35 demonstrated that ß-adrenergic stimulation with norepinephrine induced microtubular disassembly via the ß1-AR. The process was accompanied by increased calcium influx, which may have inhibited tubulin polymerization, thereby disrupting cellular integrity. The ß2-AR may be regulated by alternative mechanisms. For instance, in rat pulmonary cells, isoproterenol leads to downregulation of both ß1- and ß2-AR; however, dexamethasone selectively inhibits the downregulation of the ß2- but not the ß1-AR.36 This process involves the transcription factor, cAMP response element binding protein (CREB). We do not know to what extent altered regulation via microtubular function or differential regulation of the cAMP response element by CREB may have contributed to the phenomena we observed. Further investigations of ß1- and ß2-adrenergic signaling pathways and their regulation in cardiomyocytes will be necessary to elucidate these issues.
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Acknowledgments |
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This work was financially supported by a Biomed-2 European Project (BMH4-CT95–1008) and by the Sparkassen Stiftung Medizin. We are grateful to Prof A. Wollenberger for his scientific suggestions. We thank Prof F.C. Luft for helping us with the manuscript. We thank Holle Schmidt, Monika Wegener, and Karin Karczewski for excellent technical assistance.
Received January 29, 1998; revision received May 22, 1998; accepted June 18, 1998.
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