Specific β2AR Blocker ICI 118,551 Actively Decreases Contraction Through a Gi-Coupled Form of the β2AR in Myocytes From Failing Human Heart
Background— We have observed direct (noncatecholamine-blocking) negative inotropic effects of the selective β2-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 β2ARs or Gi proteins are overexpressed.
Methods and Results— Enzymatically isolated, superfused ventricular myocytes were exposed to βAR 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 β2AR number and increased Gi levels had normal basal contractility but showed a similar negative inotropic response to ICI 118,551. Overexpression of human β2AR 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 Giα2 induced the effect de novo in normal rat myocytes.
Conclusions— We hypothesize that ICI 118,551 binding directs the β2AR 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 β2AR, producing a direct negative inotropic effect. Conditions where β2ARs are present and Gi is raised (failing human heart, TGβ2 mouse heart) predispose to the appearance of the negative inotropic effect.
Received December 28, 2001; revision received March 14, 2002; accepted March 20, 2002.
Negative inotropic effects of β2AR antagonists were first described in the transgenic mouse overexpressing the human β2AR, where receptor levels had been increased 200-fold in the myocardium (TGβ2). 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 β -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 β2AR 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 TGβ2 mice, overexpression of the β2AR 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 β2AR blockers have since been observed under conditions where tonic activation of contraction through the β2AR does not occur. Adaptations occurred in the TGβ2 mice, with the result that raised Gi levels reduced both β2AR-mediated stimulation of contraction3 and constitutive activation of basal contractility in TGβ2 mice.4 The adaptation was most extreme in the colony used by our laboratory (TGβ2-NHLI), where basal contraction in myocytes was decreased to levels equal to, or even below, those of nontransgenic littermate controls.5 Inhibitory G-protein (Gi) was found to be increased, and pertussis toxin treatment, which inactivates Gi, restored both β2AR 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. β2ARs 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 TGβ2 mice.8 Inverse agonism attributable to reduction of tonically activating R*, secondary to excess β2ARs, is therefore unlikely. However, failing human heart does have in common with TGβ2-NHLI mice an increased level of Gi.
In the present study, we have characterized the negative inotropic effect of βAR antagonists in myocytes from failing human heart, TGβ2-NHLI mice, and other models of β 2AR or Gi overexpression. The results lead us to hypothesize that β2AR blockers can act as agonists at a Gi-coupled form of the β2AR, 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 β2AR and other G-protein coupled receptors (GPCRs) in several tissues.9
Contraction Measurements in Myocytes
Tissue was obtained from failing and nonfailing human ventricular myocardium from TGβ2-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 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.β2AR, 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 β2AR (Adv.β2AR12) or Giα2 plus green fluorescent protein (Adv.Giα2.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.Giα2.GFP or 107 to 108 for Adv.β2AR and Adv.GFP. Gi overexpression was confirmed by immunoblotting techniques as previously described.14
Pertussis toxin, (−)isoproterenol, (−)alprenolol, and CGP 20712A were obtained from Sigma, and BRL 37344, ICI 118,551, and ICI 215,001 were from Tocris.
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.
Negative Inotropic Effects of β2AR Antagonists in Human and Mouse Ventricular Myocytes
Contracting myocytes were exposed to ICI 118,551, a highly specific antagonist/inverse agonist at β2ARs, at a concentration of 1 to 3 μmol/L.2 Significant decreases in amplitude were observed in myocytes from failing human heart and from TGβ2-NHLI mice (Figure 1). A small decrease was also observed in myocytes from littermate mice, although this was significantly less than that in TGβ2-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-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).
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 β-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 β1AR 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 β1AR (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 β1AR subtype. Isoproterenol, in the continued presence of the β1AR antagonist, was able to surmount the effects of ICI 118,551. The effects of ICI 118,551 were not mimicked by β3AR agonists in either mouse or human myocytes (data not shown).
Negative Inotropic Effect of ICI 118,551 Is Not cAMP-Related
cAMP concentrations were measured in myocytes from TGβ 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 TGβ2-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 TGβ2 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.
Involvement of Gi in the Negative Inotropic Effect of ICI 118,551
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 Giα2 induced the effect de novo in rat myocytes, which have a minor population of β2ARs. 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.Giα2.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 β2AR in Rabbit Myocytes Enhances Negative Inotropic Effects of ICI 118,551
Rabbit myocytes were transfected with Adv.β2AR, and the total βAR 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 β1ARs was 85.5±3.4%, and in β2AR-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. β2AR-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.β2AR [P< 0.001]). Pertussis toxin treatment abolished the increase in response to ICI 118,551 brought about by β2AR overexpression (Figure 7).
We have observed a substantial direct negative inotropic effect of the classical β2AR-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 β2AR, 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 β-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 β2AR. It occurred in TGβ2 and failing human heart, which have in common the strong contribution of β2ARs in ventricular myocardium. Negative inotropic effects of ICI 118,551 were not prevented by β1AR blockade and could be reversed by isoproterenol in the presence of the β1AR blocker. Overexpression of the β2AR in rabbit myocytes markedly enhanced the negative response in this species. Importantly, stimulation of the β3AR, 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 β2AR, R*. In neither the TGβ 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 β2AR) 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 Giα2 induced the negative inotropic of ICI 118,551 de novo in rat myocyte, whereas 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 β2AR 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 β2ARs 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 TGβ2 mice (original phenotype1), total β2AR 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 TGβ2-NHLI mice, Gi has upregulated. If the β2AR (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 TGβ2-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, β2ARs 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 β-blockers mediated through the β2ARs.
Evidence for ligand-specific receptor active states, or stimulus-trafficking of receptors, has been obtained previously for several G-protein–coupled receptors, including the β2AR.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 β2AR/Gi link has been accumulating for some time, with Gi tonically inhibiting the positive inotropic and apoptotic effects of β2ARs in normal rat myocytes.16–18⇓⇓ PKA-dependent phosphorylation of the β2AR was shown to switch the β2AR from Gs to Gi coupling in HEK29 cells.19 In human atria, immunoprecipitation studies have shown stimulation of Gi through the β2AR, 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 β-blockers? Clearly, the unopposed β2AR responses and high Gi levels in heart failure predispose to the demonstration of negative inotropic effects of β-blockers. It is known that β-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 β-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 β2AR/Gi-mediated negative inotropic effect of β-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 β-blocker can act directly through β2ARs and Gi allows the possibility that these pathways are active during β-blockade and could contribute to the recovery of ventricular function produced by these agents in failing human heart.
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.
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- ↵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.
- ↵Gierschik P, Codina J, Simons C, et al. Antisera against a guanine nucleotide binding protein from retina cross-react with the β-subunit of the adenylyl cyclase-associated guanine nucleotide binding protein, Ns and Ni. Proc Natl Acad Sci U S A. 1985; 82: 727–731.
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