Specific ␤2AR Blocker ICI 118,551 Actively Decreases
Contraction Through a Gi-Coupled Form of the ␤2AR in
Myocytes From Failing Human Heart
Haibin Gong, PhD; Hong Sun, MD; Walter J. Koch, PhD;
Thomas Rau, MD; Thomas Eschenhagen, PhD; Ursula Ravens, PhD; Jürgen F. Heubach, PhD;
Dawn L. Adamson, MB, BS; Sian E. Harding, PhD
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Background—We have observed direct (noncatecholamine-blocking) negative inotropic effects of the selective ␤2adrenoceptor (AR) antagonist ICI 118,551 in myocytes from failing human ventricle. In this study we characterize the
effect in parallel in human myocytes and in myocytes from animal models where ␤2ARs or Gi proteins are
overexpressed.
Methods and Results—Enzymatically isolated, superfused ventricular myocytes were exposed to ␤AR agonists and
antagonists/inverse agonists, and contraction amplitude was measured. ICI 118,551 decreased contraction in ventricular
myocytes from failing human hearts by 45.3⫾4.1% (n⫽20 hearts/31 myocytes, P⬍0.001) but had little effect in
nonfailing hearts (4.9⫾4%, n⫽5 myocytes/3 hearts). Effects were significantly larger in patients classified as end-stage.
Transgenic mice with high ␤2AR number and increased Gi levels had normal basal contractility but showed a similar
negative inotropic response to ICI 118,551. Overexpression of human ␤2AR in rabbit myocytes using adenovirus
potentiated the negative inotropic effect of ICI 118,551. In human, rabbit, and mouse myocytes, the negative inotropic
effects were blocked after treatment of cells with pertussis toxin to inactivate Gi, and overexpression of Gi␣2 induced
the effect de novo in normal rat myocytes.
Conclusions—We hypothesize that ICI 118,551 binding directs the ␤2AR to a Gi-coupled form and away from the
Gs-coupled form (ligand-directed trafficking). ICI 118,551 effectively acts as an agonist at the Gi-coupled ␤2AR,
producing a direct negative inotropic effect. Conditions where ␤2ARs are present and Gi is raised (failing human heart,
TG␤2 mouse heart) predispose to the appearance of the negative inotropic effect. (Circulation. 2002;105:2495-2501.)
Key Words: myocytes 䡲 receptors, adrenergic, beta 䡲 contractility 䡲 heart failure
egative inotropic effects of ␤2AR antagonists were
first described in the transgenic mouse overexpressing the human ␤2AR, where receptor levels had been
increased 200-fold in the myocardium (TG␤2). Cardiac
contractility and adenylate cyclase activity in these animals was markedly activated in the absence of agonists,
and cAMP production was increased. 1 , 2 Certain
␤-blockers, termed inverse agonists, could reduce basal
contraction by up to 80%, whereas others, termed neutral
antagonists, competitively inhibited the effects of both
agonists and inverse agonists.2 It was hypothesized that the
␤2AR existed in 2 conformations in equilibrium, active
(R*) and inactive (R), and that agonists bound to and
stabilized R*, which then activated adenylate cyclase via
the stimulatory guanine nucleotide– binding protein, Gs. In
TG␤2 mice, overexpression of the ␤2AR had increased
N
proportionately the amount of R* such that cAMP levels
were sufficient to produce maximal contractile activation
even in the absence of agonist. Inverse agonism was
thought to be attributable to binding to R, the inactive
form, shifting the equilibrium away from R*. Basal contraction amplitude decreased as R* levels declined and
adenylate cyclase activity was reduced.
However, negative inotropic effects of ␤2AR blockers have
since been observed under conditions where tonic activation
of contraction through the ␤2AR does not occur. Adaptations
occurred in the TG␤2 mice, with the result that raised Gi
levels reduced both ␤2AR-mediated stimulation of contraction3 and constitutive activation of basal contractility in TG␤2
mice.4 The adaptation was most extreme in the colony used
by our laboratory (TG␤2-NHLI), where basal contraction in
myocytes was decreased to levels equal to, or even below,
Received December 28, 2001; revision received March 14, 2002; accepted March 20, 2002.
From the National Heart and Lung Institute, Imperial College School of Medicine, London, UK; Department of Experimental Surgery (W.J.K.), Duke
University Medical Center, Durham, NC; Institute of Pharmacology (T.R., T.E.), University of Erlangen, Germany; and Institut fur Pharmakologie und
Toxikologie (U.R., J.F.H.), Universitat Carl Gustav Carus, Dresden, Germany.
Correspondence to Dr Sian E. Harding, National Heart and Lung Institute, Faculty of Medicine, Imperial College School of Science, Technology and
Medicine, Dovehouse St, London SW3 6LY, UK. E-mail [email protected]
© 2002 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000017187.61348.95
2495
2496
Circulation
May 28, 2002
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
those of nontransgenic littermate controls.5 Inhibitory
G-protein (Gi) was found to be increased, and pertussis toxin
treatment, which inactivates Gi, restored both ␤2AR responses
and raised basal myocyte shortening.3,6 Contrary to predictions, we continued to observe inverse agonism in myocytes
from these adapted mice, even though basal activity was not
increased.6
Even more surprisingly, we have seen a similar effect of
ICI 118,551 in myocytes from failing human heart. ␤2ARs are
present in myocytes from failing human heart and can
contribute to increases in contraction after exposure to isoproterenol,7 but they are far from the excess in the TG␤2
mice.8 Inverse agonism attributable to reduction of tonically
activating R*, secondary to excess ␤2ARs, is therefore unlikely. However, failing human heart does have in common
with TG␤2-NHLI mice an increased level of Gi.
In the present study, we have characterized the negative
inotropic effect of ␤AR antagonists in myocytes from failing
human heart, TG␤2-NHLI mice, and other models of ␤2AR or
Gi overexpression. The results lead us to hypothesize that
␤2AR blockers can act as agonists at a Gi-coupled form of the
␤2AR, producing a direct negative inotropic effect without
influencing cAMP levels. This form of stimulus trafficking or
ligand-directed trafficking of receptors between different
G-proteins has been described for ␤2AR and other G-protein
coupled receptors (GPCRs) in several tissues.9
Methods
Contraction Measurements in Myocytes
Tissue was obtained from failing and nonfailing human ventricular
myocardium from TG␤2-NHLI mice and littermate controls, with
transgenic status confirmed as previously described,6 and from
normal rats (Sprague-Dawley; Harlan, Bicester, UK) and rabbits
(New Zealand white; Charles River, Margate, UK). Ethical procedures followed were in accordance with institutional guidelines in all
cases. Isolation of adult myocytes was performed as described for
human,10 mouse,6 rat,11 and rabbit myocardium.11 Myocytes in a
bath volume of 200 ␮L were superfused with Krebs-Henseleit (KH)
solution at 2 mL/min, 37°C. Composition of the KH solution was
(in mmol/L) NaCl 119, KCl 4.7, MgSO4 0.94, KH2PO4 1.2, NaHCO3
25, and glucose 11.5, gassed with 95% O2/5% CO2 to bring the pH
to 7.4. Calcium concentrations were adjusted to give contraction
amplitudes 50% to 75% of maximum (8 mmol/L human, 4 mmol/L
mouse, rat, and rabbit) to increase accuracy of measurements of
negative inotropic effects. Cells were paced at 0.2 Hz (human), 0.5
Hz (rat, rabbit), or 1 Hz (mouse) using field stimulation. Contraction
was measured using a video-motion detector as previously described.6 ICI 118,551 and alprenolol were superfused over stably
contracting myocytes and remained in contact until the decrease in
contraction had reached a steady state, usually after 5 minutes. Only
myocytes showing full reversal of the negative inotropic effect on
washout of ICI 118,551 were used in the analysis.
cAMP Measurement
cAMP was measured using an ELISA kit (Amersham). Freshly
isolated myocytes were incubated for 5 minutes at 37°C in KH, in the
presence and absence of 1 ␮mol/L ICI 118,551. Cells were pelleted
by centrifugation and quickly disrupted using the lysis buffer
Figure 1. Contraction amplitude of myocytes from human or mouse ventricle before, after, and during exposure to 1 ␮mol/L ICI
118,551. A, Myocytes from nonfailing (n⫽5 cells/3 hearts) and failing (n⫽31 cells/20 hearts) human right ventricle. ***P⬍0.001 vs before
or after ICI 118,551. B, Myocytes from littermate (LM, n⫽43 cells/31 hearts) and transgenic (TG␤2-NHLI, n⫽25 cells/17 hearts) mice.
**P⬍0.01,***P⬍0.001 vs before or after ICI 118,551. C, Representative trace from a failing human myocyte. D, Concentration-response
curve for ICI 118,551 in TG␤2-NHLI myocytes (䡩, n⫽6 myocytes/4 hearts) and a failing human myocyte (䢇). Human myocytes were
studied in 8 mmol/L Ca2⫹ and mouse in 4 mmol/L Ca2⫹.
Gong et al
Negative Inotropic Effects of ICI 118,551
2497
supplied with the ELISA kit. Protein was measured using the
Bradford reagent.
Pertussis Toxin Treatment
Freshly isolated myocytes were incubated with pertussis toxin (PTX)
(1.5 ␮g/mL) at 35°C for 2 to 3 hours for mouse and up to 6 hours for
human. For treatment of rabbit myocytes cultured with Adv.␤2AR,
pertussis toxin was added with the virus, and cells were cultured for
an additional 24 hours. After PTX treatment, both PTX-treated and
nontreated cells were kept at room temperature until the time of
experiments.
Infection of Myocytes With Adenovirus
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
E1-deficient–type adenovirus was constructed to express the human
␤2AR (Adv.␤2AR12) or Gi␣2 plus green fluorescent protein
(Adv.Gi␣2.GFP). The control virus, Adv.GFP, was a gift from Drs
Hajjar and del Monte at the Cardiovascular Research Center (Massachusetts General Hospital, Charlestown, Mass). Rat or rabbit
myocytes were cultured as previously described.13 Adenovirus expressing the required protein was initially added to each well,
containing 2⫻104 myocytes in 2 mL medium at 3.5⫻107 PFU for
Adv.Gi␣2.GFP or 107 to 108 for Adv.␤2AR and Adv.GFP. Gi
overexpression was confirmed by immunoblotting techniques as
previously described.14
Materials
Pertussis toxin, (⫺)isoproterenol, (⫺)alprenolol, and CGP 20712A
were obtained from Sigma, and BRL 37344, ICI 118,551, and ICI
215,001 were from Tocris.
Statistical Analysis
Numbers are quoted for myocytes and hearts, but for statistical
purposes, results for several cells from one heart were pooled.
Differences between means were determined using a paired or
unpaired Student’s t test with a level of P⬍0.05 taken to be
statistically significant. For groups of 3 or more, one-way ANOVA
was used with the Fisher test for pair-wise comparison of means.
Results
Negative Inotropic Effects of ␤2AR Antagonists in
Human and Mouse Ventricular Myocytes
Contracting myocytes were exposed to ICI 118,551, a highly
specific antagonist/inverse agonist at ␤2ARs, at a concentration of 1 to 3 ␮mol/L.2 Significant decreases in amplitude
were observed in myocytes from failing human heart and
from TG␤2-NHLI mice (Figure 1). A small decrease was also
observed in myocytes from littermate mice, although this was
significantly less than that in TG␤2-NHLI (P⬍0.001),
whereas no significant effect was seen in myocytes from
nonfailing human heart (Figure 1). In failing human myocytes, effects started to become apparent at concentrations as
low as 3 nmol/L ICI 118,551 (Figure 1). A representative
trace in Figure 1 shows that the negative inotropic effect was
rapidly and completely reversible. Alprenolol had a similar
negative inotropic effect (data not shown).
In failing human heart, ICI 118,551 had significant effects
on beat duration, with time-to-peak contraction and time-to90% relaxation reduced compared with basal contraction
(Figure 2). An expanded trace shows the marked effect on the
second phase of relaxation (Figure 2).
Relation of Inverse Agonist Effect to
Patient Characteristics
Effects were significantly larger in patients classified as
end-stage, NYHA IV (48.8⫾4.9%, n⫽7) than patients in
Figure 2. Beat duration of myocytes from failing human heart in
the presence of 1 ␮mol/L ICI 118,551. A, Expanded trace of
contraction. B, Normalized contraction to show beat duration.
C, Time to peak contraction (TTP), time to 50% relaxation (R50),
and time to 90% relaxation (R90) in the presence (shaded bars)
and absence (open bars) of 1 ␮mol/L ICI 118,551 in 22 myocytes from 13 failing human hearts. Human myocytes were
studied in 8 mmol/L Ca2⫹. Significantly different from basal,
**P⬍0.01, ***P⬍0.001.
NYHA classes II and III (29.9⫾6.4%, n⫽6, P⬍0.05). Dividing by disease etiology did not reveal systematic differences,
with reductions of 42.6⫾6.1% (n⫽8) for idiopathic dilated
cardiomyopathy, 43.8⫾9.9% (n⫽5) for ischemic heart disease, and 40.3⫾8.6% (n⫽3) for congenital heart disease.
Negative inotropic effects were also observed for patients
with primary pulmonary hypertension, hypertrophic obstructive cardiomyopathy, and doxorubicin-related cardiomyopathy. Fewer left ventricular samples were studied, but the
average reduction was also significant (33.5⫾3.9%, n⫽7
patients, P⬍0.001). Little difference was observed between
patients who had been treated with ␤-blockers (40.6⫾6.2%
decrease, n⫽6) and those untreated (45.8⫾5.9, n⫽10), although decreases were slightly greater in 2 patients receiving
inotropes1 ␮mol/L (60% in each case).
Subtype Selectivity for Negative Inotropic Effect
of ICI 118,551
Myocytes were challenged with ICI 118,551 in the presence
of the highly selective ␤1AR antagonist CGP 20712A at a
concentration (0.3 ␮mol/L) that would be expected to produce a 3- to 4-log unit shift in the effect of an agent acting at
the ␤1AR (Figure 3). There was no effect of CGP 20712A
itself on basal contraction, and the decrease in amplitude with
ICI 118,551 was unaffected. This indicates that the negative
inotropic effect was not mediated by the ␤1AR subtype.
Isoproterenol, in the continued presence of the ␤1AR antagonist, was able to surmount the effects of ICI 118,551. The
effects of ICI 118,551 were not mimicked by ␤3AR agonists
in either mouse or human myocytes (data not shown).
2498
Circulation
May 28, 2002
Figure 3. ␤1AR blockade with 0.3 ␮mol/L CGP 20712A (CGP) did not prevent negative inotropic effects of ICI 118,551 (ICI, 1 ␮mol/L)
on contraction amplitude (% shortening) of myocytes from failing human heart or TG␤2-NHLI mice. Isoproterenol (Iso, 10 ␮mol/L) in the
presence of ␤1AR blockade reversed negative effects of ICI 118,551. Human myocytes, n⫽7 for ICI/CGP, n⫽5 for Iso; mouse myocytes, n⫽7. Significantly different from control (Con), *P⬍0.05, **P⬍0.01; significantly different from CGP⫹ICI, ##P⬍0.001.
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Negative Inotropic Effect of ICI 118,551 Is Not
cAMP-Related
Involvement of Gi in the Negative Inotropic Effect
of ICI 118,551
cAMP concentrations were measured in myocytes from
TG␤2-NHLI mice (Figure 4). Consistent with our previous
observations in these animals, cAMP levels were not raised
compared with control. Nor did ICI 118,551 significantly
decrease cAMP in either control or TG␤2-NHLI myocytes
(Figure 4). Measurement of cAMP is less reliable in human
myocytes because of the variable number of viable cells. We
therefore tested the effects on contraction of RpcAMPS,
which competes with cAMP for binding to protein kinase A.
RpcAMPS was used at a concentration (100 ␮mol/L) that
could inhibit completely the response of the myocyte to
isoproterenol.6,15 Basal contraction of myocytes from failing
and nonfailing human hearts was unaffected by 40 minutes of
exposure to RpcAMPS, indicating that there was no tonic
support of contraction by cAMP (basal contraction amplitude,
% shortening [8 mmol/L Ca2⫹]: nonfailing, 5.79⫾1.10, ⫹RpCAMPS 5.68⫾1.57, n⫽4; failing, 6.76⫾1.01, ⫹RpcAMPS
5.93⫾0.73, n⫽6). We have previously shown similar results
for TG␤2 mice.6 It is therefore unlikely that the negative
inotropic effects of ICI 118,551 observed in myocytes from
the same animal or patients were secondary to reduction of
cAMP.
For both mouse and human myocytes, PTX treatment to
inactivate Gi abolished the negative inotropic effect of ICI
118,551 (Figure 5). Conversely, overexpression of Gi␣2
induced the effect de novo in rat myocytes, which have a
minor population of ␤2ARs. In untreated rat myocytes, ICI
118,551 at inverse agonist concentrations had little effect on
contraction. After culture for 48 hours with an adenoviral
vector (Adv.Gi␣2.GFP), ICI 118,551 induced a significant
negative response (Figure 6). Myocytes cultured for the same
period without adenovirus or with adenovirus expressing
GFP alone were unaffected.
Overexpression of ␤2AR in Rabbit Myocytes
Enhances Negative Inotropic Effects of ICI 118,551
Rabbit myocytes were transfected with Adv.␤2AR, and the
total ␤AR number was increased from 44.5⫾8.6 fmol/mg
protein (mean⫾SEM, n⫽7) to 284.8⫾55.6 fmol/mg protein
(P⬍0.002). In control cells, the percentage of ␤1ARs was
85.5⫾3.4%, and in ␤2AR-overexpressing cells, 16.3⫾5.6%
(P⬍0.001). Unlike rat, normal untransfected rabbit myocytes
showed a small depression of contraction in response to ICI
118,551 (Figure 7), which was similar in freshly isolated or
cultured cells. ␤2AR-overexpressing myocytes had enhanced
negative inotropic responses to ICI 118,551 (48.5⫾4.7%
[n⫽19] decrease in contraction versus 18.5⫾2.2% [n⫽23] in
cultured myocytes not exposed to Adv.␤2AR [P⬍0.001]).
Pertussis toxin treatment abolished the increase in response to
ICI 118,551 brought about by ␤2AR overexpression (Figure 7).
Discussion
Figure 4. cAMP levels (fmol/mg protein) in preparations of myocytes from control (littermate, n⫽3 preparations) and TG␤2-NHLI
(n⫽4) mice in the presence and absence of 1 ␮mol/L ICI
118,551. Each preparation was tested in triplicate.
We have observed a substantial direct negative inotropic
effect of the classical ␤2AR-selective antagonist/inverse agonist, ICI 118,551, on myocytes from failing human heart. In
many aspects, this effect resembled that observed in myocytes from transgenic mice overexpressing the human ␤2AR,
which we have characterized in parallel. Various trivial
explanations for the negative inotropic effect can be excluded; first, block of endogenous catecholamines is unlikely,
because these will not be present in superfused single
Gong et al
Negative Inotropic Effects of ICI 118,551
2499
Figure 5. Pertussis toxin treatment abolished the negative inotropic effect of ICI 118,551. Paired experiments are shown, with
responses to ICI 118,551 in myocytes from the same hearts before and after pertussis toxin treatment (human, n⫽3 hearts with 4 myocytes before and 5 myocytes after PTX; mouse, n⫽5 hearts/myocytes). Human myocytes were studied in 8 mmol/L Ca2⫹ and mouse in
4 mmol/L Ca2⫹. Significantly different from control, *P⬍0.05, **P⬍0.01. Pertussis toxin treatment had no significant effect on basal contractility in either human (P⫽0.17) or mouse (P⫽0.28) myocytes.
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
myocytes. Second, nonspecific or membrane-stabilizing effects, where the lipophilic nature of some ␤-blockers is
thought to depress contractility by an effect on sarcolemmal
ion channels, would be expected in normal mouse and rat or
in nonfailing human myocytes also. Third, general fatigue or
rundown of myocyte contraction is excluded, because only
reversible negative inotropic effects were analyzed.
Several lines of evidence suggest that the effect is mainly
associated with the ␤2AR. It occurred in TG␤2 and failing
human heart, which have in common the strong contribution
of ␤2ARs in ventricular myocardium. Negative inotropic
effects of ICI 118,551 were not prevented by ␤1AR blockade
and could be reversed by isoproterenol in the presence of the
␤1AR blocker. Overexpression of the ␤2AR in rabbit myocytes markedly enhanced the negative response in this species. Importantly, stimulation of the ␤3AR, which has previ-
ously been reported to depress myocardial contraction, did
not mimic the response to ICI 118,551.
However, it is clear that the negative inotropic effect is not
related to a reduction of the constitutively active form of the
␤2AR, R*. In neither the TG␤2-NHLI myocytes nor those
from failing human heart was basal contraction raised above
control values. cAMP did not tonically support basal contraction in mouse myocytes or myocytes from failing or nonfailing human heart. The original hypothesis for inverse agonism, that preferential binding of ICI 118,551 to R (the
inactive form of the ␤2AR) shifts the equilibrium away from
R* and so reduces adenylate cyclase activation, cannot
provide an explanation for these results. Evidence from this
study implicates Gi in the negative inotropic effect of ICI
118,551. Overexpression of Gi␣2 induced the negative inotropic of ICI 118,551 de novo in rat myocyte, whereas
Figure 6. Overexpression of Gi␣2 using an adenoviral vector induced the negative inotropic effect of 1 ␮mol/L ICI 118,551 in normal rat
myocytes. Bar graph, contraction amplitude (% shortening, 4 mmol/L Ca2⫹) of myocytes from 5 animals cultured for 48 hours either
with (13 myocytes) or without (9 myocytes) Adv.Gi␣2.GFP. Infected cells were identified by green fluorescence. ***P⬍0.001 vs control.
Right, Western blot of infected myocyte preparation. Con indicates cultured 48 hours without virus; Adv.Gi, cultured 48 hours with
Adv.Gi␣2.GFP; and Gi␣1, Gi␣2, Gi␣3, standards.
2500
Circulation
May 28, 2002
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Figure 7. Enhancement of the negative inotropic effect of 1
␮mol/L ICI 118,551 after overexpression of the human ␤2AR
using an adenoviral vector in rabbit myocytes and its reversal by
pertussis toxin. Contraction amplitude (% shortening) of rabbit
myocytes cultured for 24 hours without additions (control, 23
myocytes/11 hearts), with Adv.␤2AR (␤2, 19 myocytes/10 hearts),
or with Adv.␤2AR plus pertussis toxin (␤2⫹PTX, 10 myocytes, 3
hearts). Basal, 4 mmol/L Ca2⫹; ICI 118,551, 1 ␮mol/L. Significantly different from respective basal, **P⬍0.01, ***P⬍0.001;
significantly different from control or ␤2⫹PTX, ##P⬍0.01.
pertussis toxin treatment to inactivate Gi prevented the effect
in mouse, rabbit, or human myocytes.
To reconcile these conflicting results, we propose a modification to the original hypothesis for inverse agonism. In the
new scheme, ICI 118,551 binds to and stabilizes an additional
active isoform (R#) of the ␤2AR that couples through Gi to a
pathway with a direct negative inotropic effect in the ventricular myocyte. ICI 118,551 is, in effect, an agonist at R#. This
occurs in parallel to the normal R*-Gs coupling that activates
adenylyl cyclase. R* and R# are in equilibrium, possibly with
an intermediate inactive form (R). In normal mouse, rat, or
human, the basal contractile state is not influenced by ␤2ARs
through either Gs or Gi, as evidenced by the lack of effect of
pertussis toxin or RpcAMPS on contraction amplitude. Agonists bind to R* and activate the adenylate cyclase pathway,
thereby increasing contraction. Inverse agonists bind to R# but
have little effect on basal contraction when Gi is in the normal
range.
In TG␤2 mice (original phenotype1), total ␤2AR levels are
massively increased, although the equilibrium between isoforms is not necessarily altered. The excess R* is sufficient to
activate the adenylate cyclase pathway to produce levels of
cAMP that will stimulate contraction above basal. As in the
original hypothesis, inverse agonists have negative inotropic
effects mainly by decreasing levels of R* via a shift in
equilibrium toward R#, reducing Gs activation of adenylyl
cyclase and the raised basal contraction.
In TG␤2-NHLI mice, Gi has upregulated. If the ␤2AR (R#)
binds directly to Gi, the excess Gi will shift the equilibrium
toward R# and away from R*. This accounts for the decrease
in basal contraction relative to the original phenotype. In our
TG␤2-NHLI myocytes, the level of R* has dropped below
that which can activate basal contraction. Inverse agonists
bind to R#, and the increased amount of R# and Gi is now
sufficient to mediate a negative inotropic response. A similar
situation occurs in myocytes from failing human ventricle; ie,
␤2ARs are active, Gi is increased, and there is no basal
activation of adenylyl cyclase through Gs. These special
circumstances reveal a direct negative inotropic effect of
␤-blockers mediated through the ␤2ARs.
Evidence for ligand-specific receptor active states, or
stimulus-trafficking of receptors, has been obtained previously for several G-protein– coupled receptors, including the
␤2AR.9 When receptors can activate distinct subcellular
pathways through 2 different G-proteins, the rank order of
potency of agonists often differs for the 2 effects. Furthermore, mutations of the receptor can preferentially affect
responses through one pathway but not the other.9 Evidence
for a ␤2AR/Gi link has been accumulating for some time, with
Gi tonically inhibiting the positive inotropic and apoptotic
effects of ␤2ARs in normal rat myocytes.16 –18 PKAdependent phosphorylation of the ␤2AR was shown to switch
the ␤2AR from Gs to Gi coupling in HEK29 cells.19 In human
atria, immunoprecipitation studies have shown stimulation of
Gi through the ␤2AR, with inhibition by pertussis toxin
increasing activation though Gs/adenylate cyclase.20 The
present study is the first demonstration of such a link in
human ventricle, although it differs from previous reports in
that an inverse, rather than conventional, agonist produces the
effect.
The mechanism of the negative inotropic effect is not yet
known, and we can only speculate at this point. The abbreviation of the second phase of relaxation is consistent with a
decrease in APD and resembles our previous findings with
the IkATP channel opener Lemakalim in human myocytes.21
The rapidity of the effect is also more consistent with a direct
action on a membrane channel rather than a second
messenger-mediated mechanism.
Are these pharmacological effects likely to be relevant to
the clinical use of ␤-blockers? Clearly, the unopposed ␤2AR
responses and high Gi levels in heart failure predispose to the
demonstration of negative inotropic effects of ␤-blockers. It
is known that ␤-blockers must be titrated carefully in patients
with heart failure, and it has been assumed that the initial
decrease in cardiac output22 is a consequence of withdrawal
of tonic sympathetic support. The present study suggests that
direct negative inotropic effects of ␤-blockers might also
contribute to this initial decline in contractility. ICI 118,551 is
an experimental compound, not used in patients with heart
failure. However, previous studies23 have shown negative
inotropic effects of carvedilol and metoprolol in muscle strips
from failing human heart at clinically relevant concentrations.
The ␤2AR/Gi-mediated negative inotropic effect of
␤-blockers may also be the tip of the iceberg. There are many
potential Gi-activated pathways that would not have immediate contractile effects but could modify the response of
ventricular muscle to apoptotic stimuli17,24 or hypertrophic
agents (MAP kinase19). The fact that a ␤-blocker can act
directly through ␤2ARs and Gi allows the possibility that
these pathways are active during ␤-blockade and could
contribute to the recovery of ventricular function produced by
these agents in failing human heart.
Acknowledgments
This work was supported by Wellcome Trust (Dr Gong and Dr Sun),
the National Heart Research Fund (D. Adamson), and Deutsche
Forschungsgemeinschaft, Germany (Dr Ravens and Dr Heubach).
Gong et al
We are grateful to Peter O’Gara for preparation of the myocytes and
would like to thank the Immunology Department of Harefield
Hospital for the help with tissue samples.
References
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
1. Milano CA, Allen LF, Rockman HA, et al. Enhanced myocardial function
in transgenic mice overexpressing the ␤2-adrenergic receptor. Science.
1994;264:582–586.
2. Bond RA, Leff P, Johnson TD, et al. Physiological effects of inverse
agonists in transgenic mice with myocardial overexpression of the
␤2-adrenoceptor. Nature. 1995;374:272–276.
3. Xiao RP, Avdonin P, Zhou YY, et al. Coupling of ␤2-adrenoceptor to Gi
proteins and its physiological relevance in murine cardiac myocytes. Circ
Res. 1999;84:43–52.
4. Du XJ, Vincan E, Woodcock DM, et al. Response to cardiac sympathetic
activation in transgenic mice overexpressing ␤2-adrenergic receptor. Am J
Physiol. 1996;271:H630 –H636.
5. Heubach JF, Trebess I, Wettwer E, et al. L-type calcium current and
contractility in ventricular myocytes from mice overexpressing the
cardiac ␤2-adrenoceptor. Cardiovasc Res. 1999;42:173–182.
6. Gong H, Adamson DL, Ranu HK, et al. The effect of Gi-protein inactivation on basal, ␤1- and ␤2AR-stimulated contraction of myocytes from
transgenic mice overexpressing the ␤2-adrenoceptor. Br J Pharmacol.
2000;131:594 – 600.
7. del Monte F, Kaumann AJ, Poole-Wilson PA, et al. Coexistence of
functioning ␤1- and ␤2-adrenoceptors in single myocytes from human
ventricle. Circulation. 1993;88:854 – 863.
8. Bristow MR, Ginsburg R, Umans V, et al. B1-and B2-adrenergic receptor
subpopulations in nonfailing and failing human ventricular myocardium:
coupling of both receptor subtypes to muscle contraction and selective
B1-receptor down-regulation in heart failure. Circ Res. 1986;59:297–309.
9. Kenakin T. Inverse, protean, and ligand-selective agonism: matters of
receptor conformation. FASEB J. 2001;15:598 – 611.
10. Harding SE, Jones SM, O’Gara P, et al. Isolated ventricular myocytes from
failing and non-failing human heart; the relation of age and clinical status of
patients to isoproterenol response. J Mol Cell Cardiol. 1992;24:549–564.
11. Harding SE, Vescovo G, Kirby M, et al. Contractile responses of isolated
rat and rabbit myocytes to isoproterenol and calcium. J Mol Cell Cardiol.
1988;20:635– 647.
12. Akhter SA, Skaer CA, Kypson AP, et al. Restoration of ␤-adrenergic
signaling in failing cardiac ventricular myocytes via adenoviral-mediated
gene transfer. Proc Natl Acad Sci U S A. 1997;94:12100 –12105.
Negative Inotropic Effects of ICI 118,551
2501
13. Davia K, Hajjar RJ, Terracciano CMN, et al. Functional alterations in
adult rat myocytes after overexpression of phospholamban using adenovirus. Physiol Genomics. 1999;1:41–50.
14. Gierschik P, Codina J, Simons C, et al. Antisera against a guanine
nucleotide binding protein from retina cross-react with the ␤-subunit of
the adenylyl cyclase-associated guanine nucleotide binding protein, Ns
and Ni. Proc Natl Acad Sci U S A. 1985;82:727–731.
15. Adamson DL, Money-Kyrle AR, Harding SE. Functional evidence for a
cyclic-AMP related mechanism of action of the ␤2-adrenoceptor in human
ventricular myocytes. J Mol Cell Cardiol. 2000;32:1353–1360.
16. Zhou YY, Cheng H, Bogdanov KY, et al. Localized cAMP-dependent
signaling mediates ␤2-adrenergic modulation of cardiac excitationcontraction coupling. Am J Physiol. 1997;273:H1611–H1618.
17. Chesley A, Lundberg MS, Asai T, et al. The ␤2-adrenergic receptor
delivers an antiapoptotic signal to cardiac myocytes through Gi-dependent
coupling to phosphatidylinositol 3⬘-kinase. Circ Res. 2000;87:
1172–1179.
18. Communal C, Singh K, Sawyer DB, et al. Opposing effects of ␤1- and
␤2-adrenergic receptors on cardiac myocyte apoptosis: role of a pertussis
toxin-sensitive G protein. Circulation. 1999;100:2210 –2212.
19. Daaka Y, Luttrell LM, Lefkowitz RJ. Switching of the coupling of the
␤2-adrenergic receptor to different G proteins by protein kinase A.
Nature. 1997;390:88 –91.
20. Kilts JD, Gerhardt MA, Richardson MD, et al. ␤2-adrenergic and several
other G protein-coupled receptors in human atrial membranes activate
both Gs and Gi. Circ Res. 2001;87:705–709.
21. Tweedie D, O’Gara P, Harding SE, et al. The effect of alterations to
action potential duration on ␤-adrenoceptor-mediated after-contractions
in human and guinea-pig ventricular myocytes. J Mol Cell Cardiol.
1997;29:1457–1467.
22. Waagstein F, Caidahl K, Wallentin I, et al. Long-term ␤-blockade in
dilated cardiomyopathy: effects of short- and long-term metoprolol
treatment followed by withdrawal and readministration of metoprolol.
Circulation. 1989;80:551–563.
23. Maack C, Cremers B, Flesch M, et al. Different intrinsic activities of
bucindolol, carvedilol and metoprolol in human failing myocardium. Br J
Pharmacol. 2000;130:1131–1139.
24. Communal C, Colucci WS, Singh K. P38 mitogen-activated protein kinase
pathway protects adult rat ventricular myocytes against ␤-adrenergic
receptor-stimulated apoptosis: evidence for Gi-dependent activation.
J Biol Chem. 2000;275:19395–19400.
Specific ß2AR Blocker ICI 118,551 Actively Decreases Contraction Through a Gi-Coupled
Form of the ß 2AR in Myocytes From Failing Human Heart
Haibin Gong, Hong Sun, Walter J. Koch, Thomas Rau, Thomas Eschenhagen, Ursula Ravens,
Jürgen F. Heubach, Dawn L. Adamson and Sian E. Harding
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Circulation. published online May 6, 2002;
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2002 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circ.ahajournals.org/content/early/2002/05/06/01.CIR.0000017187.61348.95.citation
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located,
click Request Permissions in the middle column of the Web page under Services. Further information about
this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation is online at:
http://circ.ahajournals.org//subscriptions/