This Article Abstract Full Text (PDF) All Versions of this Article: 105/21/2497    most recent 01.CIR.0000017187.61348.95v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gong, H. Articles by Harding, S. E. Search for Related Content PubMed PubMed Citation Articles by Gong, H. Articles by Harding, S. E. Related Collections Receptor pharmacology Animal models of human disease Cell signalling/signal transduction Genetically altered mice Heart failure - basic studies

(Circulation. 2002;105:2497.)
© 2002 American Heart Association, Inc.

Clinical Investigation and Reports

Specific ß₂AR Blocker ICI 118,551 Actively Decreases Contraction Through a G_i-Coupled Form of the ß₂AR 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

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 sian.harding{at}ic.ac.uk

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— We have observed direct (noncatecholamine-blocking) negative inotropic effects of the selective ß₂-adrenoceptor (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 ß₂ARs or G_i proteins are overexpressed.

Methods and Results— Enzymatically isolated, superfused ventricular myocytes were exposed to ßAR 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 ß₂AR number and increased G_i levels had normal basal contractility but showed a similar negative inotropic response to ICI 118,551. Overexpression of human ß₂AR 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 G_i, and overexpression of G_i2 induced the effect de novo in normal rat myocytes.

Conclusions— We hypothesize that ICI 118,551 binding directs the ß₂AR to a G_i-coupled form and away from the G_s-coupled form (ligand-directed trafficking). ICI 118,551 effectively acts as an agonist at the G_i-coupled ß₂AR, producing a direct negative inotropic effect. Conditions where ß₂ARs are present and G_i is raised (failing human heart, TGß₂ mouse heart) predispose to the appearance of the negative inotropic effect.

Key Words: myocytes • receptors, adrenergic, beta • contractility • heart failure

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Negative inotropic effects of ß₂AR antagonists were first described in the transgenic mouse overexpressing the human ß₂AR, where receptor levels had been increased 200-fold in the myocardium (TGß₂). 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 ß -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 ß₂AR 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, G_s. In TGß₂ mice, overexpression of the ß₂AR had increased 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 ß₂AR blockers have since been observed under conditions where tonic activation of contraction through the ß₂AR does not occur. Adaptations occurred in the TGß₂ mice, with the result that raised G_i levels reduced both ß₂AR-mediated stimulation of contraction³ and constitutive activation of basal contractility in TGß₂ mice.⁴ The adaptation was most extreme in the colony used by our laboratory (TGß₂-NHLI), where basal contraction in myocytes was decreased to levels equal to, or even below, those of nontransgenic littermate controls.⁵ Inhibitory G-protein (G_i) was found to be increased, and pertussis toxin treatment, which inactivates G_i, restored both ß₂AR 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. ß₂ARs 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 TGß₂ mice.⁸ Inverse agonism attributable to reduction of tonically activating R*, secondary to excess ß₂ARs, is therefore unlikely. However, failing human heart does have in common with TGß₂-NHLI mice an increased level of G_i.

In the present study, we have characterized the negative inotropic effect of ßAR antagonists in myocytes from failing human heart, TGß₂-NHLI mice, and other models of ß ₂AR or G_i overexpression. The results lead us to hypothesize that ß₂AR blockers can act as agonists at a G_i-coupled form of the ß₂AR, 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 ß₂AR and other G-protein coupled receptors (GPCRs) in several tissues.⁹

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Contraction Measurements in Myocytes
Tissue was obtained from failing and nonfailing human ventricular myocardium from TGß₂-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 µL 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 µmol/L ICI 118,551. Cells were pelleted by centrifugation and quickly disrupted using the lysis buffer 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 µg/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.ß₂AR, 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
E₁-deficient–type adenovirus was constructed to express the human ß₂AR (Adv.ß₂AR¹²) or G_i2 plus green fluorescent protein (Adv.G_i2.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 2x10⁴ myocytes in 2 mL medium at 3.5x 10⁷ PFU for Adv.G_i2.GFP or 10⁷ to 10⁸ for Adv.ß₂AR and Adv.GFP. G_i 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

Top Abstract Introduction Methods Results Discussion References

 
Negative Inotropic Effects of ß₂AR Antagonists in Human and Mouse Ventricular Myocytes
Contracting myocytes were exposed to ICI 118,551, a highly specific antagonist/inverse agonist at ß₂ARs, at a concentration of 1 to 3 µmol/L.² Significant decreases in amplitude were observed in myocytes from failing human heart and from TGß₂-NHLI mice (Figure 1). A small decrease was also observed in myocytes from littermate mice, although this was significantly less than that in TGß₂-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).

View larger version (35K): [in this window] [in a new window]   Figure 1. Contraction amplitude of myocytes from human or mouse ventricle before, after, and during exposure to 1 µmol/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 (TGß2-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 TGß2-NHLI myocytes (, n=6 myocytes/4 hearts) and a failing human myocyte (). Human myocytes were studied in 8 mmol/L Ca2+ and mouse in 4 mmol/L Ca2+.

In failing human heart, ICI 118,551 had significant effects on beat duration, with time-to-peak contraction and time-to-90% relaxation reduced compared with basal contraction (Figure 2). An expanded trace shows the marked effect on the second phase of relaxation (Figure 2).

View larger version (26K): [in this window] [in a new window]   Figure 2. Beat duration of myocytes from failing human heart in the presence of 1 µmol/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 µmol/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.

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 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 ß-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 µmol/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 ß₁AR antagonist CGP 20712A at a concentration (0.3 µmol/L) that would be expected to produce a 3- to 4-log unit shift in the effect of an agent acting at the ß₁AR (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 ß₁AR subtype. Isoproterenol, in the continued presence of the ß₁AR antagonist, was able to surmount the effects of ICI 118,551. The effects of ICI 118,551 were not mimicked by ß₃AR agonists in either mouse or human myocytes (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 3. ß1AR blockade with 0.3 µmol/L CGP 20712A (CGP) did not prevent negative inotropic effects of ICI 118,551 (ICI, 1 µmol/L) on contraction amplitude (% shortening) of myocytes from failing human heart or TGß2-NHLI mice. Isoproterenol (Iso, 10 µmol/L) in the presence of ß1AR 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.

Negative Inotropic Effect of ICI 118,551 Is Not cAMP-Related
cAMP concentrations were measured in myocytes from TGß 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 TGß₂-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 µ 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 TGß₂ 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.

View larger version (31K): [in this window] [in a new window]   Figure 4. cAMP levels (fmol/mg protein) in preparations of myocytes from control (littermate, n=3 preparations) and TGß2-NHLI (n=4) mice in the presence and absence of 1 µmol/L ICI 118,551. Each preparation was tested in triplicate.

Involvement of G_i in the Negative Inotropic Effect of ICI 118,551
For both mouse and human myocytes, PTX treatment to inactivate G_i abolished the negative inotropic effect of ICI 118,551 (Figure 5). Conversely, overexpression of G_i2 induced the effect de novo in rat myocytes, which have a minor population of ß₂ARs. 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.G_i2.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.

View larger version (16K): [in this window] [in a new window]   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.

View larger version (19K): [in this window] [in a new window]   Figure 6. Overexpression of Gi2 using an adenoviral vector induced the negative inotropic effect of 1 µmol/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.Gi2.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.Gi2.GFP; and Gi1, Gi2, Gi3, standards.

Overexpression of ß₂AR in Rabbit Myocytes Enhances Negative Inotropic Effects of ICI 118,551
Rabbit myocytes were transfected with Adv.ß₂AR, and the total ßAR 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 ß₁ARs was 85.5±3.4%, and in ß₂AR-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. ß₂AR-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.ß₂AR [P< 0.001]). Pertussis toxin treatment abolished the increase in response to ICI 118,551 brought about by ß₂AR overexpression (Figure 7).

View larger version (16K): [in this window] [in a new window]   Figure 7. Enhancement of the negative inotropic effect of 1 µmol/L ICI 118,551 after overexpression of the human ß2AR 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.ß2AR (ß2, 19 myocytes/10 hearts), or with Adv.ß2AR plus pertussis toxin (ß2+PTX, 10 myocytes, 3 hearts). Basal, 4 mmol/L Ca2+; ICI 118,551, 1 µmol/L. Significantly different from respective basal, **P<0.01, ***P<0.001; significantly different from control or ß2+PTX, ##P<0.01.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have observed a substantial direct negative inotropic effect of the classical ß₂AR-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 ß₂AR, 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 myocytes. Second, nonspecific or membrane-stabilizing effects, where the lipophilic nature of some ß-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 ß₂AR. It occurred in TGß₂ and failing human heart, which have in common the strong contribution of ß₂ARs in ventricular myocardium. Negative inotropic effects of ICI 118,551 were not prevented by ß₁AR blockade and could be reversed by isoproterenol in the presence of the ß₁AR blocker. Overexpression of the ß₂AR in rabbit myocytes markedly enhanced the negative response in this species. Importantly, stimulation of the ß₃AR, which has previously 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 ß₂AR, R*. In neither the TGß 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 ß₂AR) shifts the equilibrium away from R* and so reduces adenylate cyclase activation, cannot provide an explanation for these results. Evidence from this study implicates G_i in the negative inotropic effect of ICI 118,551. Overexpression of G_i2 induced the negative inotropic of ICI 118,551 de novo in rat myocyte, whereas pertussis toxin treatment to inactivate G_i 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 ß₂AR that couples through G_i 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*-G_s 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 ß₂ARs through either G_s or G_i, 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 G_i is in the normal range.

In TGß₂ mice (original phenotype¹), total ß₂AR 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 G_s activation of adenylyl cyclase and the raised basal contraction.

In TGß₂-NHLI mice, G_i has upregulated. If the ß₂AR (R^#) binds directly to G_i, the excess G_i 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 TGß₂-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 G_i is now sufficient to mediate a negative inotropic response. A similar situation occurs in myocytes from failing human ventricle; ie, ß₂ARs are active, G_i is increased, and there is no basal activation of adenylyl cyclase through G_s. These special circumstances reveal a direct negative inotropic effect of ß-blockers mediated through the ß₂ARs.

Evidence for ligand-specific receptor active states, or stimulus-trafficking of receptors, has been obtained previously for several G-protein–coupled receptors, including the ß₂AR.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 ß₂AR/G_i link has been accumulating for some time, with G_i tonically inhibiting the positive inotropic and apoptotic effects of ß₂ARs in normal rat myocytes.^16–18 PKA-dependent phosphorylation of the ß₂AR was shown to switch the ß₂AR from G_s to G_i coupling in HEK29 cells.¹⁹ In human atria, immunoprecipitation studies have shown stimulation of G_i through the ß₂AR, with inhibition by pertussis toxin increasing activation though G_s/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 Ik_ATP 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 ß-blockers? Clearly, the unopposed ß₂AR responses and high G_i levels in heart failure predispose to the demonstration of negative inotropic effects of ß-blockers. It is known that ß-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 ß-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 ß₂AR/G_i-mediated negative inotropic effect of ß-blockers may also be the tip of the iceberg. There are many potential G_i-activated pathways that would not have immediate contractile effects but could modify the response of ventricular muscle to apoptotic stimuli^17,24 or hypertrophic agents (MAP kinase¹⁹). The fact that a ß-blocker can act directly through ß₂ARs and G_i allows the possibility that these pathways are active during ß-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). 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.

Received December 28, 2001; revision received March 14, 2002; accepted March 20, 2002.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Milano CA, Allen LF, Rockman HA, et al. Enhanced myocardial function in transgenic mice overexpressing the ß₂-adrenergic receptor. Science. 1994; 264: 582–586.[Abstract/Free Full Text]
   
2. Bond RA, Leff P, Johnson TD, et al. Physiological effects of inverse agonists in transgenic mice with myocardial overexpression of the ß₂-adrenoceptor. Nature. 1995; 374: 272–276.[CrossRef][Medline] [Order article via Infotrieve]
   
3. Xiao RP, Avdonin P, Zhou YY, et al. Coupling of ß₂-adrenoceptor to G_i proteins and its physiological relevance in murine cardiac myocytes. Circ Res. 1999; 84: 43–52.[Abstract/Free Full Text]
   
4. Du XJ, Vincan E, Woodcock DM, et al. Response to cardiac sympathetic activation in transgenic mice overexpressing ß₂-adrenergic receptor. Am J Physiol. 1996; 271: H630–H636.[Abstract/Free Full Text]
   
5. Heubach JF, Trebess I, Wettwer E, et al. L-type calcium current and contractility in ventricular myocytes from mice overexpressing the cardiac ß₂-adrenoceptor. Cardiovasc Res. 1999; 42: 173–182.[Abstract/Free Full Text]
   
6. Gong H, Adamson DL, Ranu HK, et al. The effect of G_i-protein inactivation on basal, ß₁- and ß₂AR-stimulated contraction of myocytes from transgenic mice overexpressing the ß₂-adrenoceptor. Br J Pharmacol. 2000; 131: 594–600.[CrossRef][Medline] [Order article via Infotrieve]
   
7. del Monte F, Kaumann AJ, Poole-Wilson PA, et al. Coexistence of functioning ß₁- and ß₂-adrenoceptors in single myocytes from human ventricle. Circulation. 1993; 88: 854–863.[Abstract/Free Full Text]
   
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.[Abstract/Free Full Text]
   
9. Kenakin T. Inverse, protean, and ligand-selective agonism: matters of receptor conformation. FASEB J. 2001; 15: 598–611.[Abstract/Free Full Text]
   
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.[CrossRef][Medline] [Order article via Infotrieve]
    
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.[CrossRef][Medline] [Order article via Infotrieve]
    
12. Akhter SA, Skaer CA, Kypson AP, et al. Restoration of ß-adrenergic signaling in failing cardiac ventricular myocytes via adenoviral-mediated gene transfer. Proc Natl Acad Sci U S A. 1997; 94: 12100–12105.[Abstract/Free Full Text]
    
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.[Abstract/Free Full Text]
    
14. Gierschik P, Codina J, Simons C, et al. Antisera against a guanine nucleotide binding protein from retina cross-react with the ß-subunit of the adenylyl cyclase-associated guanine nucleotide binding protein, N_s and N_i. Proc Natl Acad Sci U S A. 1985; 82: 727–731.[Abstract/Free Full Text]
    
15. Adamson DL, Money-Kyrle AR, Harding SE. Functional evidence for a cyclic-AMP related mechanism of action of the ß₂-adrenoceptor in human ventricular myocytes. J Mol Cell Cardiol. 2000; 32: 1353–1360.[CrossRef][Medline] [Order article via Infotrieve]
    
16. Zhou YY, Cheng H, Bogdanov KY, et al. Localized cAMP-dependent signaling mediates ß₂-adrenergic modulation of cardiac excitation-contraction coupling. Am J Physiol. 1997; 273: H1611–H1618.[Abstract/Free Full Text]
    
17. Chesley A, Lundberg MS, Asai T, et al. The ß₂-adrenergic receptor delivers an antiapoptotic signal to cardiac myocytes through G_i-dependent coupling to phosphatidylinositol 3'-kinase. Circ Res. 2000; 87: 1172–1179.[Abstract/Free Full Text]
    
18. Communal C, Singh K, Sawyer DB, et al. Opposing effects of ß₁- and ß₂-adrenergic receptors on cardiac myocyte apoptosis: role of a pertussis toxin-sensitive G protein. Circulation. 1999; 100: 2210–2212.[Abstract/Free Full Text]
    
19. Daaka Y, Luttrell LM, Lefkowitz RJ. Switching of the coupling of the ß₂-adrenergic receptor to different G proteins by protein kinase A. Nature. 1997; 390: 88–91.[CrossRef][Medline] [Order article via Infotrieve]
    
20. Kilts JD, Gerhardt MA, Richardson MD, et al. ß₂-adrenergic and several other G protein-coupled receptors in human atrial membranes activate both G_s and G_i. Circ Res. 2001; 87: 705–709.[Abstract/Free Full Text]
    
21. Tweedie D, O’Gara P, Harding SE, et al. The effect of alterations to action potential duration on ß-adrenoceptor-mediated after-contractions in human and guinea-pig ventricular myocytes. J Mol Cell Cardiol. 1997; 29: 1457–1467.[CrossRef][Medline] [Order article via Infotrieve]
    
22. Waagstein F, Caidahl K, Wallentin I, et al. Long-term ß-blockade in dilated cardiomyopathy: effects of short- and long-term metoprolol treatment followed by withdrawal and readministration of metoprolol. Circulation. 1989; 80: 551–563.[Abstract/Free Full Text]
    
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.[CrossRef][Medline] [Order article via Infotrieve]
    
24. Communal C, Colucci WS, Singh K. P³⁸ mitogen-activated protein kinase pathway protects adult rat ventricular myocytes against ß-adrenergic receptor-stimulated apoptosis: evidence for G_i-dependent activation. J Biol Chem. 2000; 275: 19395–19400.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				G. Ding, R. F. Wiegerinck, M. Shen, A. Cojoc, C. M. Zeidenweber, and M. B. Wagner Dopamine increases L-type calcium current more in newborn than adult rabbit cardiomyocytes via D1 and {beta}2 receptors Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2327 - H2335. [Abstract] [Full Text] [PDF]

				L. P. Collis, S. Srivastava, W. A. Coetzee, and M. Artman beta2-Adrenergic receptor agonists stimulate L-type calcium current independent of PKA in newborn rabbit ventricular myocytes Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2826 - H2835. [Abstract] [Full Text] [PDF]

				X.-H. Zhang, G.-R. Li, and J.-P. Bourreau The effect of adrenomedullin on the L-type calcium current in myocytes from septic shock rats: signaling pathway Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2888 - H2893. [Abstract] [Full Text] [PDF]

				V. O. Nikolaev, M. Bunemann, E. Schmitteckert, M. J. Lohse, and S. Engelhardt Cyclic AMP Imaging in Adult Cardiac Myocytes Reveals Far-Reaching {beta}1-Adrenergic but Locally Confined {beta}2-Adrenergic Receptor-Mediated Signaling Circ. Res., November 10, 2006; 99(10): 1084 - 1091. [Abstract] [Full Text] [PDF]

				E. J. Birks, P. D. Tansley, J. Hardy, R. S. George, C. T. Bowles, M. Burke, N. R. Banner, A. Khaghani, and M. H. Yacoub Left Ventricular Assist Device and Drug Therapy for the Reversal of Heart Failure N. Engl. J. Med., November 2, 2006; 355(18): 1873 - 1884. [Abstract] [Full Text] [PDF]

				S. Mittra and J.-P. Bourreau Gs and Gi coupling of adrenomedullin in adult rat ventricular myocytes Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1842 - H1847. [Abstract] [Full Text] [PDF]

				M. Metra, C. Torp-Pedersen, K. Swedberg, J. G.F. Cleland, A. Di Lenarda, M. Komajda, W. J. Remme, B. Lutiger, A. Scherhag, M. A. Lukas, et al. Influence of heart rate, blood pressure, and beta-blocker dose on outcome and the differences in outcome between carvedilol and metoprolol tartrate in patients with chronic heart failure: results from the COMET trial Eur. Heart J., November 1, 2005; 26(21): 2259 - 2268. [Abstract] [Full Text] [PDF]

				S. Maudsley, B. Martin, and L. M. Luttrell The Origins of Diversity and Specificity in G Protein-Coupled Receptor Signaling J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 485 - 494. [Abstract] [Full Text] [PDF]

				S. Maudsley, L. Davidson, A. J. Pawson, R. Chan, R. L. de Maturana, and R. P. Millar Gonadotropin-Releasing Hormone (GnRH) Antagonists Promote Proapoptotic Signaling in Peripheral Reproductive Tumor Cells by Activating a G{alpha}i-Coupling State of the Type I GnRH Receptor Cancer Res., October 15, 2004; 64(20): 7533 - 7544. [Abstract] [Full Text] [PDF]

				S. Pepe, O. W.V van den Brink, E. G Lakatta, and R.-P. Xiao Cross-talk of opioid peptide receptor and {beta}-adrenergic receptor signalling in the heart Cardiovasc Res, August 15, 2004; 63(3): 414 - 422. [Abstract] [Full Text] [PDF]

				M. Zaugg and M. C. Schaub Cellular mechanisms in sympatho-modulation of the heart Br. J. Anaesth., July 1, 2004; 93(1): 34 - 52. [Abstract] [Full Text] [PDF]

				A. El-Armouche, O. Zolk, T. Rau, and T. Eschenhagen Inhibitory G-proteins and their role in desensitization of the adenylyl cyclase pathway in heart failure Cardiovasc Res, December 1, 2003; 60(3): 478 - 487. [Abstract] [Full Text] [PDF]

				K. Chakir, Y. Xiang, D. Yang, S.-J. Zhang, H. Cheng, B. K. Kobilka, and R.-P. Xiao The Third Intracellular Loop and the Carboxyl Terminus of {beta}2-Adrenergic Receptor Confer Spontaneous Activity of the Receptor Mol. Pharmacol., November 1, 2003; 64(5): 1048 - 1058. [Abstract] [Full Text] [PDF]

				R.-P. Xiao, S.-J. Zhang, K. Chakir, P. Avdonin, W. Zhu, R. A. Bond, C. W. Balke, E. G. Lakatta, and H. Cheng Enhanced Gi Signaling Selectively Negates {beta}2-Adrenergic Receptor (AR)- but Not {beta}1-AR-Mediated Positive Inotropic Effect in Myocytes From Failing Rat Hearts Circulation, September 30, 2003; 108(13): 1633 - 1639. [Abstract] [Full Text] [PDF]

				J.-C. Peter, P. Eftekhari, P. Billiald, G. Wallukat, and J. Hoebeke scFv Single Chain Antibody Variable Fragment as Inverse Agonist of the {beta}2-Adrenergic Receptor J. Biol. Chem., September 19, 2003; 278(38): 36740 - 36747. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 105/21/2497    most recent 01.CIR.0000017187.61348.95v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gong, H. Articles by Harding, S. E. Search for Related Content PubMed