(Circulation. 2001;103:1649.)
© 2001 American Heart Association, Inc.
Clinical Investigation and Reports |
Upregulation of ß3-Adrenoceptors and Altered Contractile Response to Inotropic Amines in Human Failing Myocardium
From the Department of Medicine, Unit of Pharmacology and Therapeutics, University of Louvain Medical School (S.M., O.F., J.-L.B.), Brussels, Belgium; INSERM U533, Physiopathologie et Pharmacologie Cellulaires et Moléculaires (J.-N.T., C.G.) and Faculté des Sciences et Techniques (C.G.), Nantes, France; and Department of Pathology, Brigham and Women’s Hospital, and Physiology Program, Harvard School of Public Health (L.K.), Boston, Mass.
Correspondence to Jean-Luc Balligand, Department of Medicine, Unit of Pharmacology and Therapeutics, FATH 5349, University of Louvain Medical School, 53 avenue Mounier, B1200 Brussels, Belgium, e-mail Balligand{at}mint.ucl.ac.be; or Chantal Gauthier, INSERM U533, Physiopathologie et Pharmacologie Cellulaires et Moléculaires, 44093 Nantes, France,
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Abstract |
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Background—Contrary to ß1- and ß2-adrenoceptors, ß3-adrenoceptors mediate a negative inotropic effect in human ventricular muscle. To assess their functional role in heart failure, our purpose was to compare the expression and contractile effect of ß3-adrenoceptors in nonfailing and failing human hearts.
Methods and Results—We
analyzed left ventricular samples from 29 failing (16 ischemic and 13
dilated cardiomyopathic) hearts (ejection fraction 18.6±2%) and 25
nonfailing (including 12 innervated) explanted hearts (ejection
fraction 64.2±3%). ß3-Adrenoceptor proteins
were identified by immunohistochemistry in ventricular cardiomyocytes
from nonfailing and failing hearts. Contrary to
ß1-adrenoceptor mRNA, Western blot analysis of
ß3-adrenoceptor proteins showed a 2- to 3-fold
increase in failing compared with nonfailing hearts. A similar increase
was observed for G
i-2 proteins that couple
ß3-adrenoceptors to their negative inotropic
effect. Contractile tension was measured in electrically stimulated
myocardial samples ex vivo. In failing hearts, the positive inotropic
effect of the nonspecific amine isoprenaline was reduced by 75%
compared with that observed in nonfailing hearts. By contrast, the
negative inotropic effect of ß3-preferential
agonists was only mildly reduced.
Conclusions—Opposite changes occur in ß1- and ß3-adrenoceptor abundance in the failing left ventricle, with an imbalance between their inotropic influences that may underlie the functional degradation of the human failing heart.
Key Words: receptors, adrenergic, beta • heart failure • catecholamines • contractility • nitric oxide synthase
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Introduction |
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Cardiomyocyte ß1- and ß2-adrenoceptors mediate the classic increase in cardiac inotropic state after stimulation by endogenous catecholamines.1 Activation of ß-adrenergic signaling is limited in time by rapid homologous desensitization, mediated by receptor phosphorylation, including through G-protein–coupled receptor kinases (ie, GRK2 or ß-adrenoceptor kinase, ßARK1)2 and in the longer term by agonist-elicited decreases in receptor density.3 In the chronically failing heart, the inotropic response is diminished in the face of elevated circulating catecholamines, in part through decreased ß1-adrenoceptor mRNA and proteins,4 5 6 7 increased abundance and activity of ßARK,8 and increased abundance of the inhibitory G protein, Gi, which couples receptor stimulation to decreases in intracellular production of cAMP, a key mediator for the contractile effects of ß-adrenoceptor stimulation.9 10 11
Transgenic models or gene transfer experiments in failing
cardiomyocytes highlighted the importance of these signaling molecules
in the development of contractile dysfunction and opened new
therapeutic avenues for heart failure. Cardiac-specific overexpression
of ß2-adrenoceptor or
Gs transgenes that positively couple to cAMP
formation resulted in enhanced cardiac contraction and responsiveness
to catecholamines, at least up to 12
weeks.12 13
Adenovirus-mediated transfection of
ß2-adrenoceptors or a ßARK inhibitor in
cardiomyocytes from chronically paced failing rabbit hearts restored
cAMP production in these cells after agonist
stimulation.14 However,
transgenic mice overexpressing
ß1-adrenoceptors that displayed early enhanced
contractility developed cardiac hypertrophy, fibrosis, and heart
failure when studied at a later age (beyond 35
weeks).15 Therefore,
short-term benefits of enhancing
ß1-adrenoceptor signaling must be
distinguished from potential toxic effects of adrenergic stimulation
over longer periods, at least in these animal
models.16 How some or all of
these paradigms apply to human heart failure, however, is unknown. In
particular, the clinical experience that chronic treatment of heart
failure patients with ß-adrenergic blockers slows the progression of
the disease and improves outcomes suggests a more complex regulation of
myocardial inotropism by
catecholamines.17
After molecular cloning of a third ß-adrenoceptor subtype,
the
ß3-adrenoceptor,18
and its detection in human
adipocytes,19 evidence was
provided for its functional role in human ventricular myocardium from
nonfailing, denervated
hearts.20 In contrast to
ß1- and ß2-subtypes,
stimulation of ß3-adrenoceptors with
ß3-preferential agonists or norepinephrine (in
the presence of full blockade with 1-,
ß1-, and
ß2-adrenoceptor antagonists) inhibited cardiac
contractility. This effect implicated the inhibitory G protein
Gi/o, and more recently was shown to involve the
production of nitric oxide (NO) by the endothelial isoform of NO
synthase expressed within the ventricular myocardium, or
eNOS.21 The regulation of
the ß3-adrenoceptor pathway in the human
failing heart, however, is unknown. Here, we first demonstrate the
expression of ß3-adrenoceptors in ventricular
myocytes from nondenervated, nonfailing human hearts and show that
contrary to ß1-adrenoceptors, the abundance of
ß3-adrenoceptors increases in ischemic or
dilated cardiomyopathic hearts. This is paralleled by increased
abundance of Gi-2 coupling proteins in the
same hearts but decreased expression of eNOS. In addition, we show that
these opposite changes in abundance of ß-adrenoceptor subtypes result
in striking impairment of the positive inotropic effect of the
nonspecific ß-adrenoceptor agonist isoprenaline but less attenuation
of the negative inotropic effect of the
ß3-preferential adrenoceptor agonist BRL
37344. These findings substantiate the hypothesis that overstimulation
of the relatively desensitization-resistant
ß3-adrenoceptor22
after increased sympathetic tone and norepinephrine release in the
setting of heart failure may further decrease cardiac inotropy and open
the perspective for correcting the disordered adrenergic regulation of
the failing heart with specific antagonists of the human cardiac
ß3-adrenoceptor.
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Methods |
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Reverse Transcription–Polymerase Chain Reaction Amplification
Because cDNA synthesis is a critical step for reverse transcription–polymerase chain reaction (RT-PCR) quantitation experiments, efforts have been made to optimize reverse transcription for a reproducible maximum efficiency. Total RNA isolated from frozen left ventricular (LV) specimens was reverse transcribed with Superscript II (Gibco BRL) at 45°C for 50 minutes. ß1- and ß2-adrenoceptor cDNA was then amplified by PCR (35 cycles) at the following annealing temperatures: 54°C (ß2-AR), 55.5°C (ß1-AR), or 60°C (36B4) (see sequences of primers in Table 1
). The absence of contamination by genomic
DNA was ascertained in parallel amplifications of RT(-) samples,
which always remained negative. The density of ethidium-stained bands,
analyzed on 3% agarose gels, was quantified with image-analysis
software (ImageJ, NIMH), and data were normalized to the level of
transcripts for the housekeeping gene 36B4 (acidic ribosomal
phosphoprotein
PO).23
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Western Blotting Experiments
Denatured proteins from human ventricular tissues
were separated on 10% SDS-PAGE gels (ß3-AR)
or 12% 6 mol/L urea gels (Gi-2) and
transferred on nitrocellulose. Membranes were incubated for 4 hours
with the primary antibody (ie, anti-eNOS, Transduction Labs;
anti-ß3-adrenoceptors, generously provided by
Dr J. Arch, SmithKline-Beecham Pharmaceuticals, Harlow, UK; or
anti-Gi-2, a gift from Dr M. Böhm,
Universitat zu Köln, Germany), washed in TBST (Tris-buffered saline
containing 0.1% Tween 20), incubated with the secondary antibody at
1:10 000, and revealed by chemiluminescence. Densitometric values for
each sample were expressed as a percentage of the mean value obtained
for the corresponding control (nonfailing) samples analyzed on the same
gel.
Immunohistochemistry
Immunohistochemical localization of the
ß3-AR was performed on cryostat sections of
human endomyocardial biopsy specimens from failing and nonfailing
hearts. The same rat monoclonal
anti-ß3-adrenoceptor primary antibody was
used, followed by detection with an avidin-biotin complex
immunoperoxidase method and diaminobenzidine as chromogen. Control
experiments were run in parallel with normal rat IgG as primary
antibody.
Contractility Measurements
Ventricular samples were electrically
stimulated (0.6 Hz) in a tissue chamber perfused with oxygenated
Tyrode’s solution (2.7 mmol/L CaCl2;
37±0.5°C) and tensions recorded at steady state by a mechanoelectric
force transducer (Akers, AE 801; SensoNor), as described
previously.24 The cumulative
concentration-response curves of isoprenaline and the
ß3-preferential agonist BRL 37344 were
determined by superfusion with increasing concentrations of the drugs.
Changes in tension were expressed as percent increase (or decrease) of
baseline (each sample being its own control). To determine agonist
potencies from the concentration-response curves, the concentrations
producing 50% of maximum effect (EC50) were determined by fitting
curves with the Bolzmann equation. pD2 values were calculated according
to the equation pD2 =-log(EC50).
Drugs
Isoprenaline was obtained from Sigma Chemical Co and
BRL 37344 (4-[-[2-hydroxy-(3-chlorophenyl)ethyl-amino]propyl]
phenoxyacetate) from Research Biochemicals Int.
Statistical Analysis
Data are presented as mean±SEM. Statistical
significance of the drug effect was assessed by 1-way ANOVA followed by
a Dunnett test. Comparison of the concentration-response curves was
performed by 2-way ANOVA. Statistically significant differences between
groups in terms of mRNA or protein expression were calculated by a
Student’s t test for unpaired
data.
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Results |
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Patient Characteristics
Nonfailing heart tissues were obtained from 3 sources, with the approval of local ethics committees. Thirteen samples of denervated hearts were collected from endomyocardial biopsy specimens in transplanted patients (mean ejection fraction 64.3±6%). None of these patients showed any histological sign of rejection in biopsy specimens taken in parallel. All received immunosuppressive therapy, including azathioprine, prednisolone, and cyclosporin, but were free of any cardiovascular-specific medications. Eight samples of normal, innervated myocardium were obtained from donor hearts that were not transplanted for technical reasons (mean ejection fraction 64.1±2%). Six of 8 donors had received synthetic inotropic amines in the 48 hours preceding explantation. Four additional nonfailing hearts were explanted from patients with cystic fibrosis.
Failing myocardial tissue was obtained from explanted hearts
of 29 recipients at transplantation (mean ejection fraction 18.6±2%).
Sixteen patients had ischemic cardiomyopathy and 13 had dilated
cardiomyopathy. Details regarding their drug regimens are shown in
Table 2.
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Expression of ß-Adrenoceptor Subtypes in
Normal and Failing Hearts
Cardiomyocytes are the predominant cell type within
normal human ventricle to express ß1- and, to
a much lesser extent, ß2-adrenoceptors. The
abundance of these 2 subtypes is known to be altered in the failing
heart, with a reduction of ß1-adrenoceptor
proteins,5 whereas
ß2-adrenoceptors are primarily
unchanged.3 Accordingly, we
found that the abundance of mRNAs for
ß1-adrenoceptors was reduced by 48.1±8.5% in
failing hearts (n=14 patients; 9 ischemic and 5 dilated
cardiomyopathies) compared with levels measured in extracts from
nonfailing patients (1 denervated and 4 nondenervated hearts;
P<0.05). In the same extracts,
the abundance of ß2-adrenoceptor mRNAs was
unchanged (P=0.08).
Using a monoclonal antibody specific for the human
ß3-adrenoceptor in extracts of nonfailing
hearts, we found a single immunodetected band of a size compatible with
a glycosylated form of the receptor, as observed in other human
tissues.25 Of note, the
abundance of ß3-adrenoceptor proteins was
higher in nonfailing, nondenervated cardiac tissues than in tissues
from nonfailing but denervated transplanted hearts
(Figure 1B). Moreover, compared with levels observed in
nonfailing, nondenervated hearts,
ß3-adrenoceptors were significantly increased
in failing cardiac tissues from either ischemic or dilated
cardiomyopathic hearts
(Figure 1A and 1B).
Subsequent analysis of extracts from epicardial, midmyocardial, and
subendocardial layers from both ventricles showed no transmural
difference in ß3-adrenoceptor abundance (not
shown).
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Immunolocalization of
ß3-Adrenoceptors in the Human
Ventricle
To identify the specific cell type(s) expressing
ß3-adrenoceptors within the human ventricle,
the same antibody was used in immunohistochemical experiments on
similar tissue samples
(Figure 2). No staining was detectable above background when
the primary antibody was omitted or nonspecific rat IgG was used.
However, with the specific antibody, a strong signal was detected in
cardiac myocytes from nonfailing, nondenervated ventricular tissue
(Figure 2A). A light staining was also present in vessels.
Consistent with immunoblotted signals as described above, the intensity
of cardiomyocyte labeling appeared to be higher in sections of
ventricular tissue from dilated or ischemic cardiomyopathic hearts
(Figure 2C and 2E).
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Changes in Abundance of
Gi-2 and eNOS Proteins
Our previous
studies21 had demonstrated
that the contractile effect of ß3-adrenoceptor
agonists is mediated, at least in part, through a pertussis
toxin–sensitive stimulation of NO production. To assess potential
changes in the expression of these signaling molecules in the failing
heart, we compared the abundance of Gi-2
proteins, the main Gi isoform in human
ventricle,11 and eNOS, the
NO synthase constitutively expressed in human
cardiomyocytes,21 in failing
and nonfailing hearts
(Figure 1C
and 1D).
In agreement with previous
studies,9 10 11
we found that the level of immunoblotted Gi-2
proteins was increased in failing myocardium from ischemic (n=11
patients) and dilated (n=8 patients) cardiomyopathic hearts compared
with nonfailing hearts (n=5 patients: 3 nondenervated, 2 denervated)
(Figure 1D). By contrast, the abundance of eNOS proteins was
decreased in ischemic (n=16 patients) and dilated (n=8 patients)
cardiomyopathic hearts compared with nonfailing hearts (n=10 patients:
6 nondenervated, 4 denervated)
(Figure 1C). Again, no transmural difference in eNOS
abundance was observed between the various layers of myocardium from
both ventricles (not shown).
Contractile Responsiveness to
ß-Adrenergic Agonists
To assess the functional consequences of these
expressional changes in ß-adrenergic signaling molecules, we analyzed
the contractile response of human ventricular biopsy specimens both to
isoprenaline, a nonspecific ß-adrenergic agonist, and to BRL 37344, a
ß3-preferential adrenergic agonist, ex vivo.
Basal tension values in nondenervated, nonfailing hearts were
537±204.3 µN in dissected trabeculae (n=4) and 63.8±21.1 µN in
biopsy specimens (n=6), and 675±157.6 µN and 847±139.4 µN in
ischemic (n=15) and dilated (n=16) cardiomyopathic trabeculae,
respectively. Variations between trabeculae and biopsy specimens are
explained by anatomic differences in sarcomere
orientation24 . In LV samples
from nonfailing, nondenervated hearts, isoprenaline produced an
increase in absolute contractile tension with a pD2 of 7.52±0.06 and
an effect at 1 µmol/L amounting to 370±69% over basal contractile
tone
(Figure 3A), and BRL 37344 produced a decrease in absolute
tension with a pD2 of 9.08±0.21 and a maximum reduction of 44±4%
below basal contractile tension at 1 µmol/L
(Figure 3B). This negative inotropic effect was attenuated by
the specific antagonist of the human
ß3-adrenoceptor, L-748,337 (with 0.1 µmol/L
L-748,337: -23±6.0% [n=6]; without L-748,337: -50.3±1.8%
[n=6] of basal tone;
P<0.01). In trabeculae from
failing hearts, however, the positive inotropic effect of 1 µmol/L
isoprenaline was greatly reduced to 94±24% and 12±14% over basal
tension in ischemic and dilated cardiomyopathic hearts, respectively
(Figure 3A) (pD2 7.34±0.21 and 7.64±0.15), and the negative
inotropic effect of BRL 37344 was attenuated to 32±8% and 18±6%
below basal tension, respectively
(Figure 3B) (pD2 8.3±0.20 and 8.3±0.23). Therefore, the
failing hearts exhibited a loss of responsiveness to both agonists that
was more prominent for isoproterenol than for the
ß3-preferential adrenergic agonist BRL 37344
(Figure 3C).
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; n=4 patients) and failing ischemic (
; n=6 patients) and dilated (
; n=7 patients) cardiomyopathic heart tissues. P<0.001 for ischemic and dilated cardiomyopathic hearts vs nonfailing hearts by 2-way ANOVA. B, Concentration-dependent negative inotropic effect of ß3-preferential agonist, BRL 37344, in nondenervated nonfailing ( |
Discussion |
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We demonstrated that in addition to ß1- and ß2-adrenoceptors, ß3-adrenoceptors are expressed in human nonfailing ventricular myocardium. This substantiates our previous observation of a functional response to catecholamines in the human heart through a ß-adrenergic receptor that is distinct from the ß1- and ß2-subtypes. Indeed, contrary to ß1- and ß2-adrenoceptor pathways, activation of this receptor by norepinephrine in the presence of
1- and
ß1-ß2-antagonists, as
well as by ß3-preferential adrenoceptor
agonists such as BRL
3734420 21 and
CGP 12177 (which also combines antagonistic properties for
ß1-ß2-adrenoceptors),26
decreases contractile force. Of note, the order of potency of these
agonists differs for their lipolytic and inotropic effects, a
phenomenon likely due to differences in their specificity for the
ß3-receptor across species and even tissues
(for a review, see Gauthier et
al27 ). In addition to
specific transcripts,20 we
now identified ß3-adrenoceptor proteins in
human ventricular muscle as observed in other human tissues with the
same antibody
(Figure 1A). The specificity of the antibody for the
ß3-subtype, as opposed to human
ß1- and
ß2-adrenoceptors, was previously ascertained
against recombinant human
ß1-ß2-ß3-subtypes
heterologously expressed in Chinese hamster ovary (CHO) cells, with no
cross-reactivity to
ß1-ß2-subtypes.25
Of interest, we found a robust expression of the
ß3-adrenoceptor in nonfailing hearts from
normal donors, further extending the potential physiological role of
this pathway in the innervated myocardium as opposed to denervated,
transplanted hearts. In the latter, we also found a downregulation of
ß1-adrenoceptor mRNA (data not shown),
although this may be at variance with previous functional
results.28 Moreover, we
showed that the ß3-adrenoceptor is expressed
predominantly in human cardiomyocytes
(Figure 2), where downstream signaling molecules such as
Gi-2 and eNOS are colocalized, suggesting a
direct rather than paracrine regulation of cardiomyocyte contractility
by ß3-adrenoceptor stimulation. The functional
significance of the fainter staining observed in vessels awaits the
results of additional studies of human vascular reactivity.
A downregulation of
ß1-adrenoceptors is well established in the
failing human heart,5 whereas
ß2-adrenoceptors are primarily
unchanged.3 Accordingly, we
found that the abundance of ß1- (but not
ß2-) adrenoceptor mRNAs was significantly
decreased in failing myocardium. Aside from this downregulation, the
desensitization of failing myocardium to the positive inotropic effect
of catecholamines has been attributed in part to the upregulation of G
proteins of the i isoform that couple to
inhibition of adenylyl
cyclase.10 In the present
study, as in previous
studies,9 10 11
the abundance of Gi-2 proteins was increased
in failing ventricular myocardium
(Figure 1D). Because
ß1-adrenoceptors are the predominant
positively inotropic ß-adrenoceptors in cardiac muscle, their
downregulation is thought to account for a significant part of the loss
of inotropic response to catecholamines in the failing heart.
Accordingly, we observed >75% attenuation of the positive inotropic
effect of isoprenaline in ventricular biopsy specimens from ischemic
and dilated cardiomyopathic hearts. Moreover, we now show that contrary
to ß1-adrenoceptors, the abundance of the
negatively inotropic ß3-adrenoceptors
increases in the failing myocardium. This was apparent both by
immunohistochemistry and Western blotting in whole-muscle extracts
across the different ventricular layers
(Figures 1 and 2). That these increased
ß3-adrenoceptors are functional was
ascertained from the observation that stimulation of failing heart
tissue with ß3-preferential agonists did
produce a negative inotropic effect ex vivo
(Figure 3).
However, changes in contractile response did not strictly
parallel those in ß3-receptor abundance.
Despite increased ß3-adrenoceptors, the
negative inotropic effect was blunted in cardiomyopathic tissue
compared with responses observed in nonfailing cardiac tissue. This may
be explained by concurrent alterations in postreceptor coupling
mechanisms in the failing heart. Although intracellular pathways
coupling ß3-adrenoceptor stimulation are still
incompletely characterized, we had shown that they involved the
production of NO in the human
ventricle.21 Our observation
of decreased eNOS expression in failing hearts provides one explanation
for the attenuated ß3-response. However, when
compared with responses measured in nonfailing heart, the negative
inotropic effect of ß3-adrenoceptor agonists
was more preserved in failing hearts than the
ß1-ß2 positive
inotropic effect of isoprenaline, thereby potentially producing an
imbalance between these inotropically opposed pathways
(Figure 3C). Aside from the increased receptor abundance, the
relative persistence of the ß3-adrenoceptor
response may be related to the resistance of this subtype to homologous
desensitization because it lacks the consensus sequences for
phosphorylation by GRKs,22
or to the parallel upregulation of Gi-2
proteins, because the coupling of
ß3-adrenoceptors to their functional effect
was shown to be sensitive to pertussis
toxin.20 Therefore, the
attenuated contractile effect of isoprenaline, a nonspecific
ß1-ß2-ß3-agonist,
in failing hearts may integrate a persistent
ß3-adrenoceptor, negatively inotropic effect
superimposed on a sharply downregulated ß1 response. A similar
imbalance is even more likely to occur in vivo in the face of increased
local and circulating catecholamines, eg, norepinephrine, which has a
relatively high affinity for the
ß3-adrenoceptor (unlike the
ß2-subtype).18
Nevertheless, because they were mostly based on mechanical experiments
in vitro, these paradigms will need further rigorous validation in
patients.
The concurrent activation of inverse inotropic pathways,
mediated by distinct ß-adrenoceptor subtypes, helps to rationalize
previous observations on the regulation of catecholamine responsiveness
of the heart in vivo. After our demonstration of the countervailing,
negatively inotropic influence of eNOS on the ß-adrenergic response
in isolated
cardiomyocytes,29 others
observed that inhibition of cardiac NOS or eNOS gene disruption results
in a potentiation of the positive inotropic effect of isoprenaline or
dobutamine in dogs,30
humans,31 and eNOS-deficient
mice.32 Of note, this
potentiation was only apparent in patients with heart
failure,33 where we would
anticipate a prevailing influence of the ß3/NO
pathway over
ß1-ß2-signaling,
according to our present results. Likewise, a recent study showed that
the potentiation of the positive inotropic effect of isoprenaline by
NOS inhibition is abolished in mice homozygously deficient for the
ß3-adrenoceptor,34
confirming the involvement of this subtype in the NO-dependent
regulation of catecholamine responsiveness in vivo. The
ß3-adrenoceptor pathway may subserve different
physiological functions in the heart
(Figure 4). In the normal heart, it may exert a negative
counterregulation against excessive positive inotropic stimulation,
thereby moderating oxygen consumption, preventing calcium overload and
ultimately cardiomyocyte toxicity, as exemplified by the phenotype of
ß1-adrenoceptor–overexpressing
mice.15 At early stages of
cardiac dysfunction, endogenous NO production may, in addition to
attenuating
ß1-ß2-adrenergic
inotropic responses, improve diastolic
relaxation,35 thereby
compensating systolic dysfunction by increasing diastolic reserve. In
terminally failing hearts, the residual negative inotropic effect may
become maladaptive and aggravate systolic dysfunction
(Figure 4, right). More insights into these potential roles
and their impact on the clinical use of ß-blockers should be gained
from linear studies of animal models and patients with progressive
heart failure as new specific antagonists for the human
ß3-adrenoceptor become
available.
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Acknowledgments |
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This work was supported by grants from the INSERM and Fondation de France (Dr Gauthier), the NIH (Dr Kobzik), and ARC and FNRS-FRSM grants (Dr Balligand). We thank Karine Laurent and Mortéza Erfanian for technical assistance and the Cardiovascular Surgery Division, Saint-Luc University Hospital (Brussels) and Laënnec Hospital (Nantes), for providing human tissues.
Received September 29, 2000; revision received December 4, 2000; accepted December 7, 2000.
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