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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2006.00185.x |
Autoantibody against Cardiac b1-Adrenoceptor Induces
Apoptosis in Cultured Neonatal Rat Cardiomyocytes
Yan GAO1, Hui-Rong LIU1,
Rong-Rui ZHAO1, and Jian-Ming ZHI2*
1 Department of
Cardiovascular Physiology,
Received:
February 21, 2006
Accepted:
April 17, 2006
*Corresponding
author: Tel, 86-21-63846446; Fax, 86-21-63846590 Email, [email protected]
Abstract To clarify whether apoptosis is
involved in the injury processes induced by autoantibody against cardiac b1-adrenoceptor,
we investigated the biological and apoptotic effects of antibodies on cultured
neonatal rat cardiomyocytes. Wistar rats were immunized with peptides
corresponding to the second extracellular loop of the b1-adrenoceptor
to induce the production of anti-b1-adrenoceptor antibodies
in the sera. Immunoglobulin (Ig) G in the sera was detected using synthetic
antigen enzyme-linked immunosorbent assay and purified using the diethylaminoethyl
cellulose ion exchange technique. Apoptosis of cardiomyocytes was evaluated
using agarose gel electrophoresis and flow cytometry. Our results showed that
the positive serum IgG greatly increased the beating rates of cardiomyocytes
and showed an “agonist-like” activity. Furthermore, positive serum
IgG induced cardiomyocyte apoptosis after treatment with b1-adrenoceptor
overstimulation for 48 h. The effects of monoclonal antibody against b1-adrenoceptor
were also found to be similar to those of positive serum IgG. It was suggested
that the autoantibody could induce cardiomyocyte apoptosis by excessive
stimulation of b1-adrenoceptor.
Key words b1-adrenoceptor;
rat cardiomyocyte; autoantibody; apoptosis
In recent years,
autoantibodies against cardiac b1-adrenoceptors have been
found in approximately 30% of patients suffering from dilated cardiomyopathy
(DCM) by enzyme-linked immunosorbent assay [1–3]. The high level of b1-adrenoceptor
autoantibodies is related to poor left ventricular ejection fraction in
patients [4–7]. In
vivo experiments also showed that active immunization of rats stimulated
strong production of anti-b1-adrenoceptor antibodies
in the sera, which had a long-term effect on the heart and induced remarkable
histopathological changes and cardiac dysfunction [8,9]. In addition, it has
been demonstrated that these autoantibodies could specifically recognize the
functional epitope of the second extracellular loop of the corresponding
receptors and display various agonist-like activities without desensitization
[1,2]. All of these results indicated that anti-b1-adrenoceptor
antibodies might be involved in the pathologic processes of heart failure.
It is known that
apoptosis is one of the most common causes of cell loss in humans and animals,
and is characterized by nuclear and cellular fragmentation. There is increasing
evidence that apoptosis is mediated even in a low-grade manner. Moreover,
inhibiting apoptosis can successfully prevent or attenuate heart failure [10].
However, the mechanisms of the pathogenic effects are still unclear.
Previous reports
indicated that overdriving of b1-adrenoceptor could
induce apoptosis in neonatal and adult cardiomyocytes [11]. Furthermore, b1-adrenoceptor
antibodies can produce a positive inotropic response as a partial agonist in
isolated cardiomyocytes [12–14]. The
apoptotic effect of the antibody against b1-adrenoceptor
on cardiomyocytes needs to be investigated.
In this study, we
assessed the biological and apoptotic effects of anti-b1-adrenoceptor
autoantibody on cultured neonatal cardiomyocytes by immunizing rats using a
peptide corresponding to the second extracellular loop of b1-adrenoceptor
as the immunogen.
Materials and Methods
Synthetic peptide and
anti-b1-adrenoceptor
monoclonal antibody
The free peptide
(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), corresponding to the
sequence of the second extracellular loop of the human b1-adrenoceptor,
was synthesized by the Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai,
China). The peptide was analyzed by high
performance liquid chromatography on a Vydac C-18 column, and amino acids
were analyzed by an automated amino acid analyzer (Beckman,
was a kind gift from Prof. Yvonne MAGNUSSON (Wallenberg Laboratory,
Immunization
Twelve healthy Wistar
rats (180–
G was prepared from collected sera by graded salt precipitation in 35%–
Enzyme-linked
immunosorbent assay
The synthesized peptide
was dissolved in a
Culture of neonatal
cardiomyocytes
Primary cultures of neonatal
rat cardiomyocytes were prepared according to the method originally described
by Simpin and Savion [16] with minor modifications. Positive serum IgG for
autoantibodies against b1-adrenoceptors, negative
control IgG, monoclonal antibody for b1-adrenoceptor, or
isoprenaline was used to treat the cells. After 30 min treatment, the number of
beating single cardiomyocytes or clusters of synchronously contracting
cardiomyocytes was counted and compared with that of pre-treatment. The basal
beating rate was 110±20 beats per minute (b.p.m.). After 48 h of antibody or
isoprenaline treatment, the cardiomyocytes were collected and cell apoptosis
was detected.
DNA fragmentation (DNA
ladder) assay
The protocol of Moore
and Matlashewski [17] was used to isolate fragmented DNA. The experiment was
normalized using equal volumes of cultures for each group. In brief,
cardiomyocytes (approximately 3.0´106) from each treated
sample were washed with PBS and the pellets were homogenized with 1 ml of lysis
buffer (
Flow cytometry assay
Cell apoptosis was
estimated by flow cytometry assay as described previously [18]. Briefly, 1.0´106 cells were harvested, washed twice with cold
PBS (pH 7.4) and fixed in 70% ice-cold ethanol. After centrifugation, washing
with PBS and re-centrifugation, the cells were suspended in 1 ml of DNA
staining solution (PBS containing 10 mg/ml RNase A
and 50 mg/ml
propidium iodide) and incubated for 30 min at room temperature in the dark.
Then at least 1.0´105 cells from each sample
were examined, and the percentage of apoptotic cells was calculated using
CellQuest and modifit software packages (Becton Dickinson, San Jose, USA).
Statistical analysis
Results are expressed as
the mean±standard deviation. The average of the antibody titer is expressed as
the geometric mean value. Student’s paired or non-paired t-test and
ANOVA were done with SPSS version
10.0 software (SPSS,
Results
Antibody
production and titers
Fourteen days after the
first immunization, the titer in the sera was less than 1:10. However, the antibody
titer in the immunized rats was increased to 1:(142.5±6.0) (P<0.01
compared with the titer lower than 1:
Chronotropic effects on
cultured cardiomyocytes
Spontaneously beating rates
of rat neonatal cardiomyocytes were used to assess the chronotropic effects
of the antibodies. The data showed that the negative serum IgG control had no
effect on beating rates, but the positive serum IgG for b1-adrenoceptor,
monoclonal antibody for b1-adrenoceptor and b1-adrenoceptor
agonist isoprenaline significantly increased the beating rates of
cardiomyocytes. The positive serum IgG at the concentrations of 0.3, 0.6 and
1.2 mM increased the rates to
DNA ladder production by
autoantibodies against b1-adrenoceptor
and isoprenaline
The positive serum IgG,
monoclonal antibody and isoprenaline were added into cardiomyocyte culture media. After incubation for
48 h, all of the cultured neonatal cardiomyocytes showed characteristic DNA
fragmentation, with a ladder of the
internucleosomal DNA bands representing
integer multiples of the internucleosomal DNA length (approximately 180
bp), but DNA ladders were not detected in the negative serum IgG group or the
control group without any treatment (Figs. 2–4). This suggested that the overdriving of b1-adrenoceptor
induced cardiomyocyte apoptosis.
Apoptosis detection by
flow cytometry
The effects of positive
serum IgG against b1-adrenoceptor (Figs.
5 and 6) and monoclonal antibody (Figs. 7 and 8) were
examined at various concentrations by flow cytometry. Treatment with the
antibodies resulted in a concentration-dependent increase in the apoptotic
populations (Table 3). The apoptotic effects of the positive serum IgG
group were increased significantly to 11.5%±2.8%, 14.1%±2.7% and 21.6%±4.2% at
the concentrations of 0.3 mM, 0.6 mM and 1.2 mM, respectively. In the monoclonal antibody group, the
percentages of the apoptotic cells were 14.3%±2.7% at 10 nM, 17.6%±2.3% at 50
nM and 30.4%±1.8% at 100 nM. All of these data were significantly different
compared with that of the corresponding control (P<0.01).
Discussion
Increasing evidence
suggests that autoimmune response is involved in the pathogenesis of a number of
cardiovascular diseases, including DCM. Among them, the biological, functional
and pathogenic properties of anticardiac receptor antibodies have been
extensively investigated. It was recently reported that autoantibody against
cardiac troponin I could cause DCM in normal mice [19]. Jahns et al.
also revealed these antibodies against various synthetic receptor peptides in
51% of patients suffering from DCM, but only the subgroup against the second
extracellular loop could bind and stimulate human b1-adrenoceptor
of the cell membrane [4]. Approximately 31% of patients with DCM develop
autoantibody against this epitope. Much research has indicated that
autoantibody against b1-adrenoceptor also
played an important role in the pathogenesis of DCM, such as cardiac
morphologic changes, deposition of collagen and obvious functional impairment
[7,20,21]. An earlier study found that active immunization with the synthetic
peptide corresponding to the second extracellular loop of the human cardiac b1-adrenoceptor
in rabbits or rats could induce a remarkable production of anti-receptor
peptide antibody, which had the same biological and immunological properties as
those of autoantibody in the sera of patients with DCM [13]. Therefore, in the
present study, we used a synthetic peptide corresponding to the second
extracellular loop of human b1-adrenoceptor as the
antigen to immunize rats to induce the generation of autoantibody, and then
investigate the role of anti-b1-adrenoceptor antibody
on cultured neonatal cardiomyocytes.
The apoptosis in hearts
of patients with heart failure has increased the possibility that apoptosis
contributes to the pathophysiology of myocardial failure. Pharmacologic studies
of cardiomyocytes in vitro demonstrate that b1-adrenoceptor
can stimulate apoptosis. It was reported that overexpression of b1-adrenoceptor
was associated with myocyte apoptosis and the development of DCM in transgenic
mice [22,23]. Here, we used cultured neonatal cardiomyocytes as a model to
study whether overstimulation of b1-adrenoceptor by
autoantibody could induce apoptosis. We chose neonatal cardiomyocytes as a
model because the cultured technique of neonatal rat cardiomyocytes is well
developed and accepted, and reliable. This study showed that positive serum IgG
against the second extracellular loop of b1-adrenoceptor
could clearly increase the beating rates and induce apoptosis in cultured
neonatal cardiomyocytes in a dose-dependent manner.
It is known that
positive serum IgG is a polyclonal antibody, which recognizes different
independent epitopes on the antigen; monoclonal antibody is identical, but
recognizes only one specific epitope and has a defined specificity for the
antigen. To verify that the agonist-like activity and the apoptotic effects of
the positive serum IgG were produced by the second extracellular loop of b1-adrenoceptor,
we also made a comparative study of the above effects between positive serum
IgG and the monoclonal antibody group. Similarly, the monoclonal antibody could
also increase the beating rates of cardiomyocytes, which was consistent with
the previous report by Staudt et al. [14]. Furthermore, we demonstrated
that the monoclonal antibody was able to induce the cardiomyocyte apoptosis
dose-dependently. It also indicated that it is possible for autoantibody
against b1-adrenoceptor to induce
myocardial injury through the apoptotic pathway.
Much evidence suggests
that autoantibody against b1-adrenoceptor displays
the “agonist-like” activity without desensitization, which is very
different from the b1-adrenoceptor agonist,
isoprenaline. It is known that excessive stimulation using isoprenaline leads
to apoptosis [24]. Therefore, to further confirm that overstimulation of b1-adrenoceptor
by autoantibody might exactly induce myocardial apoptosis, we made another
comparative study with isoprenaline. Our results showed that isoprenaline
significantly increased the beating rate of cultured cardiomyocytes and
definitely induced cardiomyocyte apoptosis. According to previous research, the
isoprenaline-induced apoptosis is mediated by activation of the cAMP/PKA
pathways and voltage-dependent calcium influx, which induces cardiac
hypertrophy and subsequent DCM [24]. Thus, it can be speculated that the
signaling transduction pathway of autoantibody-induced apoptosis might be
related to isoprenaline.
In conclusion, our
results indicated that anti-b1-adrenoceptor
autoantibody could produce chronotropic effects and induce the apoptosis of
cultured cardiomyocytes by overstimulation of b1-adrenoceptors,
which might be responsible for antibody-induced myocardial injury. All of these
results provide promise for the development of potential clinical approaches in
which Ig-adsorption therapy might substitute heart transplantation and become
the standard therapy for DCM.
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