(Circulation. 1999;99:65-72.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Activation of ß2-Adrenergic Receptors Hastens Relaxation and Mediates Phosphorylation of Phospholamban, Troponin I, and C-Protein in Ventricular Myocardium From Patients With Terminal Heart Failure
From the Babraham Institute, Cambridge, UK (A.K., L.S.); Department of Pharmacology, University of Melbourne, Victoria, Australia (P.M., K.B.); and Max-Delbrück Centre of Molecular Medicine, Cardiology, Berlin, Germany (S.B., D.V., P.H., P.K., E.-G.K.).
Correspondence to Alberto Kaumann, MD, PhD, Babraham Institute, Cambridge CB2 4AT, United Kingdom. E-mail alberto.kaumann{at}bbsrc.ac.uk
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Abstract |
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Background—Catecholamines hasten cardiac relaxation through ß-adrenergic receptors, presumably by phosphorylation of several proteins, but it is unknown which receptor subtypes are involved in human ventricle. We assessed the role of ß1- and ß2-adrenergic receptors in phosphorylating proteins implicated in ventricular relaxation.
Methods and Results—Right ventricular trabeculae, obtained from freshly explanted hearts of patients with dilated cardiomyopathy (n=5) or ischemic cardiomyopathy (n=5), were paced at 60 bpm. After measurement of the contractile and relaxant effects of epinephrine (10 µmol/L) or zinterol (10 µmol/L), mediated through ß2-adrenergic receptors, and of norepinephrine (10 µmol/L), mediated through ß1-adrenergic receptors, tissues were freeze clamped. We assessed phosphorylation of phospholamban, troponin I, and C-protein, as well as specific phosphorylation of phospholamban at serine 16 and threonine 17. Data did not differ between the 2 disease groups and were therefore pooled. Epinephrine, zinterol, and norepinephrine increased contractile force to approximately the same extent, hastened the onset of relaxation by 15±3%, 5±2%, and 20±3%, respectively, and reduced the time to half-relaxation by 26±3%, 21±3%, and 37±3%. These effects of epinephrine, zinterol, and norepinephrine were associated with phosphorylation (pmol phosphate/mg protein) of phospholamban 14±3, 12±4, and 12±3; troponin I 40±7, 33±7, and 31±6; and C-protein 7.2±1.9, 9.3±1.4, and 7.5±2.0. Phosphorylation of phospholamban occurred at both Ser16 and Thr17 residues through both ß1- and ß2-adrenergic receptors.
Conclusions—Norepinephrine and epinephrine hasten human ventricular relaxation and promote phosphorylation of implicated proteins through both ß1- and ß2-adrenergic receptors, thereby potentially improving diastolic function.
Key Words: heart failure • receptors, adrenergic, beta 2 • catecholamines • phosphoproteins • contractility, diastole
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Activation of cardiac ß-adrenergic receptors by catecholamines augments contractility and hastens relaxation. Catecholamines are thought to promote relaxation (ie, cause lusitropic effects)1 by inducing the phosphorylation, catalyzed by cAMP-dependent protein kinase A (PKA), of at least 2 proteins, phospholamban1 2 3 and troponin I,4 and perhaps of C-protein.5 6 Phospholamban can also be phosphorylated by a Ca2+/calmodulin–dependent protein kinase7 (CaM kinase). Nonphosphorylated phospholamban exerts a braking effect on the Ca2+ ATPase that pumps Ca2+ into the sarcoplasmic reticulum.3 Phospholamban phosphorylation results in liberation of Ca2+ ATPase activity, which accelerates its pumping activity, thereby decreasing Ca2+ concentrations at the contractile proteins and hastening myocyte relaxation.1 2 3 Phosphorylation of troponin I desensitizes contractile proteins to Ca2+, thus providing another mechanism for relaxation.4 8 9
Diastolic function deteriorates in congestive heart failure,10 even when systolic function is unimpaired.11 Relaxation of ventricular myocardium is retarded in heart failure and correlates with prolongation of calcium transients, suggesting a decreased capacity to restore low basal Ca2+ levels.12 One factor that correlates with the prolongation of calcium transients is deficient generation of cAMP, which is observed in heart failure.13 Although it has been reported that phosphorylation of phospholamban, troponin I, and C-protein is decreased in failing human ventricle compared with normal myocardium,14 positive lusitropic responses to the catecholamine dobutamine are still observed in patients with heart failure.15 The affinity16 and agonist potency17 of dobutamine are similar at both ß1- and ß2-adrenergic receptors, and it is unknown which subtype is involved. We therefore sought to determine whether both ß1- and ß2-adrenergic receptors can mediate positive lusitropic effects and whether implicated proteins are phosphorylated. This is not the case in a variety of animal models in which ß1- but not ß2-adrenergic receptors mediate hastening of ventricular relaxation.18 19 20 21 22 23 However, there is evidence in human atrium24 25 and human ventricular myocytes from normal and failing hearts26 that both ß1- and ß2-adrenergic receptors mediate not only inotropic but also lusitropic effects. We now confirm this in ventricular trabeculae from failing ischemic and cardiomyopathic hearts and show it to be associated with phosphorylation of phospholamban, troponin I, and C-protein for both ß1- and ß2-adrenergic receptors.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Patients
Informed consent was obtained from all patients (Table 1
). All patients had terminal
heart failure and underwent cardiac transplant surgery. This study was
approved by the ethics committees of the Papworth Hospital,
Cambridgeshire (United Kingdom), and Alfred Hospital, Melbourne
(Australia).
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Isolated Right Ventricular Trabecula Carnae
Explanted hearts were obtained immediately (<1 minute) after
removal from the patient. The endocardial layer of the right
ventricular free wall was rapidly dissected in ice-cold,
preoxygenated (95% O2-5%
CO2) modified Krebs' solution containing
(mmol/L) Na+ 125, K+ 5,
Ca2+ 2.25, Mg2+ 0.5,
Cl- 98.5,
SO42- 0.5,
HCO3- 29,
HPO42- 1, and EDTA 0.04 at the
surgical theater. Trabeculae (width usually <1 and not
>1.3 mm) were dissected, set up at optimum length, and paced to
contract isometrically at 60 bpm at 37°C in a bath containing the
above solution supplemented with (mmol/L) Na+ 15,
fumarate 5, pyruvate 5, L-glutamate 5, and glucose 10 as
described.27
In all experiments, contractile force and its first derivative were recorded simultaneously. Cross-sectional area of trabeculae that were not snap-frozen was determined from the length and weight of the muscle at the end of the experiment, assuming a density of 1.063. Rapid freezing of trabeculae prevented measurements of length and weight under conditions of contraction.
Specific Activation of ß1- and
ß2-Adrenergic Receptors
To irreversibly block tissue uptake of
catecholamines and 
-adrenergic receptors,
trabeculae were incubated for 90 minutes with
phenoxybenzamine followed by washout.28
To establish conditions for selective activation of ß1- and ß2-adrenergic receptors, experiments were carried out as described previously on ventricular preparations from hearts without advanced failure27 and ventricular myocytes from a donor heart and hearts in terminal failure.26 To determine ß1-selective activation, inotropic concentration-effect curves to (-)-norepinephrine in the presence of ICI118551 (50 nmol/L) (to selectively block ß2-adrenergic receptors) were determined in the absence and presence of the ß1-selective blocker CGP20712A.24 25 26 27 For ß2-selective activation, inotropic concentration-effect curves to (-)-epinephrine in the presence of CGP20712A (300 nmol/L) were determined in the absence and presence of ICI118551 (50 nmol/L).24 25 26 27 In these studies, only a single concentration-effect curve was determined on each trabeculum. After equilibrium effects were reached with the highest catecholamine concentration, (-)-isoproterenol was added at a concentration (200 µmol/L) that surmounts27 the blockade caused by CGP20712A and ICI118551. The experiments were terminated by increasing the CaCl2 concentration to 9.25 mmol/L in the presence of catecholamines. In 4 additional patients, the effects of 9.25 mmol/L CaCl2 were also investigated in the absence of catecholamines but in the presence of CGP20712A.
To determine relaxation and protein phosphorylation mediated by ß1-adrenergic receptors, trabeculae were exposed for 2 hours to ICI118551 (50 nmol/L) followed by incubation for 5 minutes with 10 µmol/L (-)-norepinephrine. To assess ß2-adrenergic receptor–mediated effects for tissues from the same patient, trabeculae exposed for 2 hours to CGP20712A (300 nmol/L) were incubated for 5 minutes with 10 µmol/L (-)-epinephrine or 10 µmol/L zinterol. To assess maximal effects mediated through both ß1- and ß2-adrenergic receptors, CGP20712A-treated trabeculae were exposed for 5 minutes to 200 µmol/L (-)-isoproterenol. Basal protein phosphorylation was determined in trabeculae from the same patient incubated with either CGP20712A or ICI118551 but not agonist.
Processing of Trabeculae for Protein
Phosphorylation and Immunodetection
Freeze-clamped tissue derived from contracting
trabeculae was homogenized in a histidine
buffer containing NaF 25 mmol/L and phenylmethanesulfonyl
fluoride 100 µmol/L as described.29 The
homogenates were centrifuged at
100 000g; the pellet contained phospholamban, and the
supernatant contained troponin I and C-protein.
Protein Phosphorylation
The method used, back-phosphorylation, has
previously been described29 and adapted to human cardiac
tissue.14 25 The phosphorylation reaction
was started by the addition of [
-32P]ATP and
the catalytic subunit of cAMP-dependent protein kinase. We used 30 µg
of protein per assay. After 5 minutes of incubation, the reaction was
terminated with trichloroacetic acid, the resulting pellets were
solubilized and boiled, and proteins were separated by
PAGE.29 Radioactive bands corresponding to phospholamban,
troponin I, and C-protein were identified according to molecular mass
and by immunodetection.14 The calculated difference in
32P incorporation between control and
agonist-treated trabeculae was considered to reflect
agonist-induced endogenous phosphate incorporation into the
intact trabeculae and is expressed as picomoles of
phosphate per 1 mg protein. Protein was determined30 with
BSA as the standard.
Site-Specific Western Blot Analysis of
Phospholamban
Specific antibodies against phospholamban phosphoserine 16 and
phospholamban phosphothreonine 17 were used as reported.31
Crude membrane fractions were solubilized at room temperature in a
lysis buffer containing SDS. Blots were incubated with antibodies
raised against a synthetic oligopeptide sequence of phospholamban with
either a phosphorylated serine 16 or threonine 17
residue31 and visualized with an enhanced
chemiluminescence-based detection system. To demonstrate the
specificity of the immunological reaction, the procedures were also
performed in the presence of 0.1 µmol/L of the corresponding
11–amino-acid-residue oligopeptides of phospholamban,
phosphorylated at either Ser16 or Thr17.
Statistical Analysis
Data are expressed as sample mean±SE. Student's paired
t test or 1-way ANOVA followed by the Bonferroni method was
used for multiple comparisons by use of InStat (GraphPad software
version 2.0). We used P<0.05 as the limit for statistical
significance.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Positive Inotropic and Lusitropic Effects Mediated Through ß1- and ß2-Adrenergic Receptors
(-)-Norepinephrine caused positive inotropic effects in the presence of the ß2-selective blocker ICI118551 that were antagonized with high potency by the ß1-selective blocker CGP20712A. The threshold concentrations of (-)-norepinephrine to increase contractile force were 300 nmol/L and 60 µmol/L in the absence and presence of CGP20712A (300 nmol/L), respectively, and were therefore mostly mediated through ß1-adrenergic receptors. (-)-Epinephrine caused positive inotropic effects in the presence of the ß1-selective blocker CGP20712A that were antagonized by the ß2-selective ICI118551. The threshold concentrations of (-)-epinephrine were 60 nmol/L and 20 µmol/L in the absence and presence of ICI118551 (50 nmol/L), respectively, consistent with mediation through ß2-adrenergic receptors. These results from hearts in terminal failure (data from 22 trabeculae of patients 1 through 3 of Table 1
; not shown) agree with previous
data from ventricular tissue from hearts in mild
failure27 and ventricular
myocytes.26
To obtain robust phosphorylation signals, we selected a
relatively high catecholamine concentration, 10
µmol/L, which, however, under the conditions of the present and
previous studies,26 27 is receptor subtype–specific and
produces nearly maximal effects through ß1- or
ß2-adrenergic receptors. Marked positive
inotropic and lusitropic effects of (-)-norepinephrine and
(-)-epinephrine, mediated through ß1-
and ß2-adrenergic receptors, respectively, were
similar to those of (-)-isoproterenol (Figures 1 and 2).
Zinterol, a ß2-selective partial agonist
effective in human ventricle32 and atrium,25
also caused positive inotropic and lusitropic effects, but the
lusitropic effects tended to be smaller than those of the
catecholamines (Figures 1 and 2) and
the onset of response tended to be slower than with
catecholamines (Figure 1).
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Comparison of Inotropic and Lusitropic Potencies of
(-)-Norepinephrine and (-)-Epinephrine Through
ß1- and ß2-Adrenergic Receptors
To compare the inotropic and lusitropic potency of
(-)-norepinephrine through
ß1-adrenergic receptors with the corresponding
potencies of (-)-epinephrine through
ß2-adrenergic receptors, we determined
concentration-effect curves for the 2 catecholamines under
receptor-selective conditions. The lusitropic potencies of both
(-)-norepinephrine and (-)-epinephrine were
significantly greater than the corresponding inotropic potencies
(Figure 3 and Table 2).
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shows basal levels; 
, effects of
(-)-norepinephrine or (-)-epinephrine; 
,
maximal stimulation of ß1- and
ß2-adrenergic receptors with 200 µmol/L
(-)-isoproterenol; and 
, effect of raising extracellular
Ca2+ to 9.25 mmol/L. Values given are sample mean±SE
from 11 patients. Basal values for force and t50 were not
different between tissues used for (-)-norepinephrine and
(-)-epinephrine (P>0.2). Basal time to peak
force values were shorter in tissues used for
(-)-norepinephrine concentration-response curves
(P=0.01), possibly because of stimulation of
ß1-adrenergic receptors by endogenous
(-)-norepinephrine. Errors not shown when smaller than
symbol.
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High Ca2+ Does Not Hasten Relaxation
High extracellular calcium concentration has been shown to
abbreviate the duration of Ca2+ transients in
canine cardiomyocytes23 and to hasten
relaxation in guinea pig cardiomyocytes.33 To
examine the effects of a high Ca2+ concentration,
we compared the effects of 9.25 mmol/L CaCl2
with those of basal conditions (2.25 mmol/L) in
trabeculae from patients 8 and 15 through 17 in Table 1. Increasing extracellular Ca2+ to
9.25 mmol/L failed to hasten relaxation, whereas
(-)-norepinephrine, administered as a positive control,
hastened relaxation (Figure 4). In 11
trabeculae from 4 patients, Ca2+
9.25 mmol/L increased contractile force from 1.2±0.4 (SE from 4
patients) to 6.6±2.0 mN/mm2 but did not change
relaxation parameters. The t50 and
time to peak values were 127±7 and 126±5 ms, and 179±15 and 186±7
ms at 2.25 and 9.25 mmol/L CaCl2,
respectively.
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Phosphorylation of Phospholamban, Troponin I,
and C-Protein
Both ß1- and
ß2-adrenergic agonists reduced the capacity of
tissues to incorporate 32P-phosphate into
phospholamban, troponin I, and C-protein in vitro, as assessed after
back-phosphorylation and shown
representatively in Figure 5. This result indicates a higher
endogenous phosphorylation state of each
protein in the intact tissue after 5 minutes of exposure to the
agonists compared with that of non–agonist-exposed tissues. The amount
of protein phosphorylation in the intact
ventricular trabeculae caused by the agonists
was calculated from the back-phosphorylation reaction.
(-)-Norepinephrine, (-)-epinephrine, and
(-)-isoproterenol caused similar phosphorylation of
phospholamban, troponin I, and C-protein through
ß1-, ß2-, or
ß1- plus ß2-adrenergic
receptors, respectively (Figure 6).
Zinterol also induced phosphorylation of the 3 proteins
through ß2-adrenergic receptors (Figure 6).
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Site-Specific Phosphorylation of
Phospholamban
Both (-)-norepinephrine and (-)-epinephrine
induced site-specific phosphorylation of Ser16 and
Thr17 residues of phospholamban through ß1- (2
patients) and ß2-adrenergic receptors (5
patients), respectively (Figure 7), as
demonstrated in competition assays with synthetic phospho-oligopeptides
(Figure 7). The (-)-epinephrine-evoked
phosphorylation of phospholamban at both Ser16 and
Thr17 was prevented by ICI118551 (50 nmol/L) (Western blots from 1
patient; not shown).
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A trabeculum of 1 patient (patient 8 in Table 1) was
exposed to high Ca2+ (9.25 mmol/L), which
increased contractile force (basal, Ca2+
2.25 mmol/L) from 1.4 to 10.5 mN but did not shorten the
t50 of relaxation, shorten the time to the onset
of relaxation, or produce phosphorylation at Ser16 or
Thr17 of phospholamban (not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Both ß1- and ß2-Adrenergic Receptors Hasten Relaxation Through cAMP Pathways
Our results show that both the ß1- and ß2-adrenergic receptors mediate hastening of relaxation of human ventricular myocardium, consistent with an involvement of cAMP-dependent pathways for both receptors. (-)-Norepinephrine and (-)-epinephrine had similar lusitropic potencies through ß1- and ß2-adrenergic receptors, respectively, and the lusitropic intrinsic activity of (-)-epinephrine was nearly that of (-)-norepinephrine. As expected from cAMP pathways, the PKA substrates implicated in relaxation,1 2 3 4 5 6 Ser16 phospholamban, troponin I, and C-protein were phosphorylated by (-)-norepinephrine through ß1-adrenergic receptors and by (-)-epinephrine and zinterol through ß2-adrenergic receptors. These findings are at variance with evidence in all other species studied so far (cat, sheep, rat, dog),18 19 20 21 22 23 in which ß2-adrenergic receptors, unlike ß1-adrenergic receptors, appear reluctant to couple to the cAMP pathway that hastens relaxation, and stress the importance of direct work on human myocardium.
Species-dependent differences between the functions of
ß1- and ß2-adrenergic
receptors may possibly be due to differences in coupling to the
Gs-protein/cAMP pathway and differential coupling
to Gs and Gi, guanine
nucleotide–sensitive transducer proteins that cause
activation and inhibition of the cAMP pathway, respectively. In cat
heart, stimulation of the Gs/cAMP pathway through
ß1- and ß2-adrenergic
receptors is proportional to the corresponding receptor
densities,34 suggesting similar coupling of these 2
receptors, but only ß1-adrenergic receptors
hasten relaxation.18 In contrast, human cardiac
ß2-adrenergic receptors are more tightly
coupled to the Gs/cAMP
pathway27 28 35 36 than are
ß1-adrenergic receptors, and this was later
confirmed with human recombinant receptors.37 38 The
selective coupling of human ß2-adrenergic
receptors probably contributes to the marked lusitropic effects of
(-)-epinephrine.36 In ventricular
myocytes from nonfailing rat hearts, the
ß2-adrenergic–Gs/cAMP
pathway leading to relaxation can be demonstrated only in the presence
of pertussis toxin,22 presumably after inactivation of
functional Gi. This suggests that coupling of the
rat cardiomyocyte ß2-adrenergic
receptor is tighter to Gi than to
Gs. The tighter coupling of human
ß2-adrenergic receptors to the
Gs/cAMP pathway compared with
ß1-adrenergic receptors may explain why, even
in failing human ventricle (this work) in which
Gi function and mRNA levels
(Gia2) are increased,39 40 41
ß2-adrenergic receptor–mediated relaxation can
still occur through the Gs/cAMP pathway. It is
thus possible that in nonfailing hearts with unaltered
Gi function,
ß2-adrenergic receptors may mediate even more
marked relaxant effects than in failing hearts.
One limitation of this study is that the magnitude of lusitropic
effects did not correlate (not shown) with the degree of protein
phosphorylation in the same ventricular
preparation. Protein phosphorylation peaks may have
preceded the observed lusitropic response, an issue that can be
clarified only with future kinetic experiments. Our findings of
significant PKA-dependent phosphorylation of
phospholamban, troponin I, and C-protein through both
ß1- and ß2-adrenergic
receptors agree, however, with an earlier report of PKA
stimulation42 suggesting a causal relationship. Unlike the
situation in many other species,18 19 20 21 22 23 human
ventricular (this work) and atrial25
ß2-adrenergic receptors appear to function
mainly through a PKA-dependent pathway. Support for this concept has
also recently been provided for the
ß2-adrenergic receptor–mediated increases in
L-type Ca2+ current with an obligatory
involvement of PKA in human atrial myocytes.43 Zinterol
increased the L-type Ca2+ current and appeared to
dissociate slowly from the ß2-adrenergic
receptors.43 Correspondingly, we attribute the relatively
slow inotropic and lusitropic onset observed in human
ventricular trabeculae (Figure 1) to
slow equilibration of zinterol with
ß2-adrenergic receptors.
The catecholamine (-)-isoproterenol can induce
phosphorylation of phospholamban at both Ser16 (through
PKA) and Thr17 (through CaM kinase) in rodent
ventricle.7 44 The CaM kinase–catalyzed
phosphorylation of phospholamban can contribute to
increased contractility and hastened relaxation,
provided dephosphorylation is
negligible.44 Type 1 phosphatase catalyzes this
dephosphorylation, and the activity of type 1
phosphatase can, in turn, be inhibited by isoproterenol through
PKA-catalyzed phosphorylation of protein phosphatase
inhibitor-1 in guinea pig ventricular
myocytes.45 Our results show for the first time that Thr17
phosphorylation of phospholamban occurs in failing
human ventricle through both ß1- and
ß2-adrenergic receptors. We suggest that the
CaM kinase–catalyzed phosphorylation of Thr17 of
phospholamban in human ventricle can be demonstrated because its
dephosphorylation is retarded by
simultaneous inhibition of type 1 phosphatase by the
ß1- or ß2-adrenergic
receptor–mediated phosphorylation of protein
phosphatase inhibitor-1. High extracellular
Ca2+ concentration does not appear to result in
activation of sarcoplasmic reticulum CaM kinase because, in contrast to
the catecholamines, it does not hasten relaxation in
trabeculae from nonfailing12 or failing hearts
(Figure 4) and can actually prolong contractions and
Ca2+ transients of trabeculae from
failing human ventricle.12 In agreement with this, we have
seen in the trabeculae of patient 8 in Table 1 that
high Ca2+ concentration did not hasten relaxation
and did not induce phosphorylation at Thr17 or Ser16 of
phospholamban, whereas (-)-epinephrine caused these effects
through ß2-adrenergic receptors, in line with
an indirect role of PKA but not necessarily of high
Ca2+ concentration per se. In contrast,
concentrations of (-)-norepinephrine (through
ß1-adrenergic receptors) and
(-)-epinephrine (through ß2-adrenergic
receptors) and Ca2+, which cause matching
increases in contractile force, are associated with marked hastening of
relaxation with the catecholamines only (even in the
presence of high Ca2+ concentration; Figure 3) but not with Ca2+ alone.
Possible Clinical Relevance
We have conclusively shown that (-)-epinephrine, acting
through ß2-adrenergic receptors, and
(-)-norepinephrine, acting through
ß1-adrenergic receptors, hasten relaxation with
similar potency and efficacy and cause phosphorylation
of proteins implicated in the relaxation process. These results require
verification in myocardium from normal hearts of donors not
treated with drugs, such as ACE inhibitors. It seems
reasonable, however, to suggest that diastolic function of
failing heart may be improved by the action of endogenous
catecholamines, mediated through both
ß1- and ß2-adrenergic
receptors. For example, it is conceivable that during stress
endogenous plasma epinephrine surges elicit not
only tachycardia but also beneficial hastening of
ventricular relaxation, mediated at least partly through
ß2-adrenergic receptors, thus producing a
relative lengthening of diastole.
Because ß2-adrenergic receptors hasten relaxation in failing human ventricular myocardium, it could be clinically desirable to selectively improve diastolic function under conditions in which ß1-adrenergic receptors are blocked. This may happen in patients with chronic heart failure undergoing treatment with ß1-selective blockers.46 It is plausible that in these patients endogenous epinephrine may actually contribute to an improvement in diastolic function via ß2-adrenergic receptors. The likelihood of this occurring is enhanced by the observation that at least in human atrium ß2-adrenergic receptor function is increased by long-term ß1-adrenergic receptor blockade.24
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Acknowledgments |
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Dr Kaumann is grateful to the British Heart Foundation and Dr Molenaar to the NHMRC (Australia) for support. Petra Hempel was supported by the Sonnenfeld-Stiftung, Berlin, Germany. We thank Dr David Kaye for patient details and the surgeons of Papworth-Everard Hospital (Cambridgeshire, United Kingdom) and Alfred Hospital (Melbourne, Australia) for explanted hearts.
Received May 26, 1998; revision received August 19, 1998; accepted September 22, 1998.
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