Abstract
Although ligand-free, constitutive β2-adrenergic receptor (AR) signaling has been demonstrated in naive cell lines and in transgenic mice overexpressing cardiac β2-AR, it is unclear whether the dominant cardiac β-AR subtype, β1-AR, shares the ability of spontaneous activation. In the present study, we expressed human β1- or β2-AR via recombinant adenoviral infection in ventricular myocytes isolated from β1β2-AR double knockout mice, creating pure β1-AR and β2-AR systems with variable receptor densities. A contractile response to a nonselective β-AR agonist, isoproterenol, was absent in double knockout mouse myocytes but was fully restored after adenoviral β1-AR or adenoviral β2-AR infection. Increasing the titer of adenoviral vectors (multiplicity of infection 10–1000) led to a dose-dependent expression of β1- or β2-AR with a maximal density of 1207 ± 173 (36-fold over the wild-type control value) and 821 ± 38 fmol/mg protein (69-fold), respectively. Using confocal immunohistochemistry, we directly visualized the cellular distribution of β1-AR and β2-AR and found that both subtypes were distributed on the cell surface membrane and transverse tubules, resulting in a striated pattern. In the absence of ligand, β2-AR expression resulted in graded increases in baseline cAMP and contractility up to 428% and 233% of control, respectively, at the maximal β2-AR density. These effects were specifically reversed by a β2-AR inverse agonist, ICI 118,551 (10−7 M). In contrast, overexpression of β1-AR, even at a greater density, failed to enhance either basal cAMP or contractility; the alleged β1-AR inverse agonist, CGP 20712A (10−6 M), had no significant effect on basal contraction in these cells. Thus, we conclude that acute β2-AR overexpression in cardiac myocytes elicits significant physiological responses due to spontaneous receptor activation; however, this property is β-AR subtype specific because β1-AR does not exhibit agonist-independent spontaneous activation.
G protein-coupled receptors (GPCRs) constitute the largest class of cell surface-signaling molecules, which are widely involved in regulating vital cellular processes. β-Adrenergic receptor (β-AR) is a prototypical GPCR. At least two β-AR subtypes, β1-AR and β2-AR, coexist in the heart of many mammalian species, including human (Xiao and Lakatta, 1993; Xiao et al., 1994; Altschuld et al., 1995; for review see Xiao et al., 1999b). Stimulation of these receptors by catecholamines increases cardiac contractility and heart rate and accelerates cardiac relaxation via a Gs-adenylyl cyclase-cAMP-protein kinase A-signaling cascade. Although there is a high degree of structural and functional similarity between these β-AR subtypes, recent studies have shown that β-AR subtypes play strikingly different functional roles via distinct signaling pathways in the heart. In particular, β2-AR, but not β1-AR, couples to pertussis toxin-sensitive Gi proteins in addition to the well established Gs-signaling pathway (Xiao et al., 1995, 1999a;Kuschel et al., 1999).
A GPCR is proposed to exist in an equilibrium between two conformational states, an inactive form (R) and an active form (R*), that can interact with G proteins (Samama et al., 1993; Bond et al., 1995; Neilan et al. 1999). In addition to ligand-induced activation, a small percentage of receptors are active in the absence of ligand. The ligand-independent activation of β2-AR has been elegantly demonstrated in several systems overexpressing wild-type (WT) β2-AR (Chidiac et al., 1994; Milano et al., 1994; Bond et al., 1995) or expressing the constitutively active mutant of β2-AR (Samama et al., 1993). For example, ligand-free cardiac β2-AR activation is evidenced by significantly increased basal adenylyl cyclase activity, cAMP accumulation, or contractility in the absence of any ligand, and the β2-AR inverse agonist, ICI 118,511 (ICI), reverses these augmentations (Milano et al., 1994; Bond et al., 1995; Xiao et al., 1999a; Zhou et al., 1999a). However, whether β1-AR shares the ability of spontaneous activation is still controversial. In transgenic mice overexpressing β1-AR, the basal adenylyl cyclase activity and cardiac function remain unchanged compared with WT controls (Bertin et al., 1993; Zolk et al., 1998; Engelhardt et al., 1999). Nevertheless, in guinea pig and human cardiomyocytes, in the presence of forskolin, the mixed β1- and β2-AR antagonist, but not β2-AR antagonists, induces a marked decrease in basal L-type Ca2+ current in the absence of ligand (Mewes et al., 1993). More recently, it has been shown that, in canine cardiac myocytes, a β1-AR antagonist, CGP 20712A (CGP), inhibited the basalI Ca by 27% (Nagykaldi et al., 1999). These results were interpreted to indicate spontaneous activation of β1-AR.
The overall goal of the present study was to examine whether β-AR subtypes are different in terms of their propensity of spontaneous activation. To avoid complicated interactions between β-AR subtypes, because of the lack of absolutely selective β-AR subtype ligands, we took advantage of a recently developed β1- and β2-AR double knockout (DKO) mouse model (Rohrer et al., 1999) and recent advances in methods to culture adult mouse ventricular myocytes (Zhou et al., 2000). We expressed either β-AR subtype over a wide range of receptor density in cultured β1β2-AR DKO ventricular myocytes and investigated the physiological or biochemical responses in the absence or presence of β-AR subtype inverse agonists. Our results indicate that overexpression of β2-AR is associated with a robust increase in basal cAMP accumulation and contractility, which can be specifically reversed by the β2-AR inverse agonist, ICI. Surprisingly, overexpression of β1-AR to the same or even greater density has no effect on basal cAMP and contractility. These results indicate that β1-AR, unlike β2-AR, is not able to undergo ligand-independent constitutive activation in intact mouse cardiac myocytes.
Experimental Procedures
Myocyte Isolation, Culture, and Adenoviral Infection.
The investigation conforms to National Institutes of Health guiding principles in the care and use of animals. Single mouse cardiac myocytes were isolated from the hearts of 2- to 3-month-old mice with an enzymatic technique and then were cultured and infected with adenoviral vectors, as described previously (Zhou et al., 2000). Before culture, myocytes were washed three times with minimal essential medium (MEM) containing 1.2 mM Ca2+, 2.5% fetal bovine serum (FBS), and 1% penicillin-streptomycin and then plated at 0.5 to ∼1 × 104/cm2 with the same medium in the culture dishes precoated with 10 μg/ml mouse laminin. After 1 h of culture (to achieve attachment), the culture medium was aspirated along with unattached cells. Adenovirus-mediated gene transfer was implemented by adding a minimal volume of the FBS-free MEM containing an appropriate titer of gene-carrying adenovirus. The full volume of FBS-free MEM was supplied after culture for another 1 to 2 h. All experiments were performed after 24 h of adenoviral infection.
Measurement of Cell Contraction.
Cells were placed on the stage of an inverted microscope (Zeiss, model IM-35, Zeiss, Thornwood, NY) and superfused with HEPES-buffered solution consisting of (in mM): CaCl2 1, NaCl 137, KCl 5.4, dextrose 15, MgSO4 1.3, NaH2PO4 1.2, and HEPES 20, pH 7.4 adjusted with NaOH. Each cell was illuminated with red (650–750 nm) light through the normal bright-field path of the microscope and electrically stimulated at 0.5 Hz at 23°C. Cell length was monitored from the bright-field image by an optical edge-tracking method using a photodiode array (model 1024 SAQ, Reticon, Boston, MA) with a 3-ms time resolution (Spurgeon et al., 1990). Tpeak was measured as the time from stimulation to peak shortening; T50 was measured as the time from the peak to 50% relaxation.
Riadioligand-Binding Assay.
Twenty-four hours after adenoviral infection, cardiac myocytes were harvested in lysis buffer (5 mM Tris-HCl, pH 7.4, with 5 mM EGTA) and homogenized with 15 strokes on ice. Samples were centrifuged at 30,000g for 15 min to pellet membranes. Membranes were resuspended in binding buffer (75 mM Tris-HCl, pH 7.4, 12.5 mM MgCl2, 2 mM EDTA) and stored in aliquots at −80°C. Binding assays were performed on 25 μg of membrane protein using saturating amounts of the β-AR-specific ligand [125I]cyanopindolol (ICYP). Nonspecific binding was determined in the presence of 10 μM propranolol. Reactions were conducted in 250 μl of binding buffer at 37°C for 1 h. The binding reaction was terminated by addition of ice-cold 10 mM Tris-HCl (pH 7.4) to the membrane suspension, followed by rapid vacuum filtration through glass-fiber filters (Whatman GF/C). Each filter was washed three times with an additional 7 ml of ice-cold 10 mM Tris-HCl. The radioactivity of the wet filters was determined in a gamma counter. All assays were performed in duplicate, and receptor density was normalized to milligrams of membrane protein.K d and the maximal number of binding sites (B max) for ICYP were determined by Scatchard analysis of saturation binding isotherms.
Immunocytochemical Staining and Confocal Imaging.
β1β2-DKO cells were infected by either adeno-β1-AR tagged with hemagglutinin (HA) or adeno-β2-AR [multiplicity of infection (m.o.i.) 100] for 24 h. Cells were washed twice with phosphate-buffered saline (PBS) and fixed with cold methanol plus acetone (7:3) for 10 min and rinsed twice with PBS containing 0.2% Triton. Nonspecific binding was reduced by a 30-min incubation with Blotto solution (5% BSA, 2% horse serum, 0.2% Triton, and 0.01% NaN3 in PBS, pH 7.4). Then, cells were incubated for 60 min at room temperature with primary antibodies for HA-tagged β1-AR (anti-HA monoclonal antibody diluted by 1:500) or for β2-AR (β2-AR polyclonal antibodies diluted by 1:100). After the cells were rinsed four times with PBS, including 0.2% Triton, they were stained with Texas Red-conjugated secondary antibodies (1:100, Vector Laboratories, Burlingame, CA) for another 60 min in the dark: horse anti-mouse IgG secondary antibodies were used for β1-AR, whereas goat anti-rabbit IgG secondary antibodies were used for β2-AR staining. As a negative control, cells were incubated with secondary antibodies in the absence of primary antibodies. As an additional negative control, another subset of DKO cells cultured without viral infection was treated with the same protocol. Immunofluorescence was then detected by a laser scanning confocal microscope (LSM-410, Zeiss) with optical section thickness of 1.0 μm.
Measurement of cAMP Accumulation.
After cells were treated with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 1 mM), for 30 min at 37°C in a CO2 incubator, they were incubated with either β-AR agonists or inverse agonists for 10 min. Cells were then harvested, and cAMP levels were assayed as previously described (Xiao et al., 1999a) with minor modifications. Briefly, 10 μl of membrane vesicles (20 μg of total protein) were added to a 40-μl reaction solution to make a final concentration of 4 mM Tris-EDTA and 1 mM IBMX. The reaction was performed at 37°C for 15 min, and 25 μl of supernatant were assayed using a cAMP 3H assay kit obtained from Amersham (Arlington Heights, IL). Protein content was measured using the Bradford method (Bio-Rad, Richmond, CA) with bovine serum albumin as the standard.
Materials.
Forskolin, isoproterenol hydrochloride, propranolol, alprenolol, IBMX, and minimal essential medium were purchased from Sigma (St. Louis, MO). ICI 118,551 was kindly supplied by ICI Pharmaceutic Group (Wilmington, DE). CGP 20712A was kindly supplied by CIBA-GEIGY Corp. (East Hanover, NJ). Fetal bovine serum, penicillin-streptomycin, and mouse laminin were purchased from Life Technologies (Gaithersburg, MD). The cAMP assay kit was purchased from Amersham. [125I]Cyanopindolol was purchased from NEN Life Science Products, Inc. (Boston, MA). Anti-HA monoclonal antibody and β2-AR polyclonal antibody were purchased from Berkeley Antibody Co. (Berkeley, CA) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. The secondary antibodies were purchased from Vector Laboratories.
Data Analysis.
Data are reported as means ± S.E.M. Student's t test, paired t test, or ANOVA were used, when appropriate, to test for differences among the means. A value of P < .05 was considered to be statistically significant.
Results
Characterization of β1β2-AR DKO Mouse Ventricular Myocytes.
Previous in vivo studies in the β1β2-AR DKO mouse model have shown that elimination of both β1- and β2-AR has little impact on resting cardiovascular function but completely abolishes the cardiac response to β-AR agonist stimulation (Rohrer et al., 1999). In this study, we further characterized cardiac morphological and physiological properties of the DKO mouse at the single cell level. Ventricular myocytes from adult DKO mice were almost identical with those from WT mice with respect to cell length (Table1) and membrane capacitance (153 ± 8.3 pF, n = 7, for DKO cells, versus 158 ± 15.3 pF, n = 25, for WT controls; P > .05). All of the basal contractile properties, including twitch amplitude (TA), time to peak (Tpeak), and time to 50% relaxation (T50), were unaltered in DKO as compared with WT cells (Table 1). TA of the first beat after rest, which reflects the maximal contractile reserve, was also similar in DKO (9.47 ± 0.52% of rest cell length, n = 23) and WT cells (9.19 ± 0.53% of rest cell length, n = 28). After 24 h of culture, steady-state TA in DKO cells was largely preserved, whereas the kinetics of contraction, including T50 and Tpeak, were slowed by 30 to ∼40%, as was the case with WT cells (Table 1; also see Zhou et al., 2000).
Consistent with the genotype, there were no detectable β1- or β2-receptors in DKO cells as assayed by radioligand-binding assay and confocal immunocytochemical staining (data not shown). The disruption of both β1- and β2-AR signaling was confirmed in single myocytes by the lack of a contractile response to the β-AR agonist, isoproterenol (ISO, 10−6M), which activates all known β-AR subtypes (Fig.1A). This observation is in contrast to a robust inotropic effect of ISO in WT cells (Fig. 1C). To determine whether the downstream β-AR-signaling pathway is adaptively altered in DKO cells, adenylyl cyclase was directly activated by forskolin. As shown in Fig. 1, B and C, forskolin (Fsk, 10−6M) induced a marked increase in contraction amplitude in DKO cells, comparable with that in WT cells. These results indicate that β1- and β2-AR are the major functional β-AR subtypes in cardiac myocytes and that DKO cells provide a virtually null β-AR background with intact cAMP-signaling pathway.
Expression of β1-AR or β2-AR Subtype in DKO Myocytes.
To investigate β1- and β2-AR subtype signaling individually, we expressed either β-AR subtype in cultured DKO mouse myocytes using adenovirus-mediated gene transfer. The exact level of receptor protein expression was measured by radioligand-binding assay using ICYP. Figure2 shows that the expression level of either β1-AR or β2-AR depended on the titer (m.o.i., 0–1000) of adeno-β1-AR or adeno-β2-AR. The average maximal receptor density was 1207 ± 173 fmol/mg protein for β1-AR, and 821 ± 38 fmol/mg protein for β2-AR at m.o.i. 1000. Because β1-AR is the predominant β-AR subtype (β1-AR: 33.5 ± 1.2; β2-AR: 11.9 ± 1.7 fmol/mg,n = 3) and constitutes ∼70% of the total β-ARs in WT cells, the relative increase in β2-AR over WT control (∼69-fold) was greater than that of β1-AR (∼36-fold). In addition, there was no significant change in β-AR affinity for ICYP at different expression levels (Fig. 2). There was no detectable radioligand-binding signal in DKO cells infected with a marker transgene β-galactosidase, as expected.
Furthermore, we examined intracellular distribution of either β-AR subtype expressed in the DKO mouse myocytes using adenoviral gene transfer (m.o.i. 100). Figure 3 shows that, in the absence of agonist stimulation, specific immunofluorescence for β1-AR or β2-AR is largely concentrated on cell surface membranes, including transverse tubules, with little staining of the cytosol, resulting in a clear striated pattern.
To determine whether the expressed receptors couple to downstream signaling pathway and modulate contractility, we examined effects of the nonselective β-AR agonist, ISO, on cellular cAMP accumulation and contraction. ISO (10−6 M) significantly elevated cAMP accumulation in DKO cells infected with either β1-AR (at m.o.i. 100, from 10.1 ± 2.4 to 33.8 ± 6.3 pmol/mg protein, n = 4,P < .05) or β2-AR (at m.o.i. 100, from 23.2 ± 7.4 to 37.89 ± 8.42 pmol/mg protein,n = 4, P < .05). Concomitantly, stimulation of either β-AR subtype by ISO (10−6 M) markedly augmented TA with a comparable maximal response (11.8 ± 1.1% of rest cell length for adeno-β1-AR at m.o.i. 100, n = 10, P < .01; 11.9 ± 1.0% of rest cell length for Adeno-β2-AR at m.o.i. 100,n = 11, P < .01), which was selectively blocked by specific β-AR antagonists, CGP or ICI, respectively (Fig. 4). These results indicate that expression of β1-AR or β2-AR in DKO myocytes fully restores the functionality of β-AR, thus providing pure β1-AR and β2-AR experimental systems.
Receptor Density and Spontaneous β-AR Subtype Signaling.
Next, we examined spontaneous activation of β-AR subtypes by expressing β1-AR or β2-AR over a wide range of receptor density in the null background of DKO myocytes. The possible spontaneous receptor activation was determined using cellular cAMP and contractility as the biochemical and physiological readouts. In DKO cells expressing β2-AR at m.o.i. ≥100, the baseline cAMP was significantly elevated in the absence of ligand (Fig.5A). Similarly, baseline contraction amplitude was also markedly increased in these cells (Fig. 5B and Table1). These results are consistent with previous observations in a β2-AR overexpression transgenic model (Milano et al., 1994; Xiao et al., 1999a). In sharp contrast, overexpression β1-AR to the same or even higher levels had no effect on either baseline cAMP or contractility (Fig. 5 and Table 1). Furthermore, neither T50 nor Tpeak of contraction was altered in cells overexpressing β1-AR, but both were abbreviated in cells infected with adeno-β2-AR at m.o.i. 100 or 1000 (Table 1). Taken together, these results indicate that ligand-free β-AR signaling is subtype specific, i.e., it is evident for β2-AR but not for β1-AR.
Effects of β-AR Inverse Agonists.
To further characterize ligand-independent β-AR signaling, β-AR inverse agonists were used to inhibit spontaneous receptor activation. A number of β-AR ligands, including ICI (Milano et al., 1994; Bond et al., 1995), CGP (Nagykaldi et al., 1999), and propranolol (Mewes et al., 1993), have been reported as β-AR inverse agonists based on their effects on unliganded receptors. However, the effect and potency of any given ligand are variable depending on the experimental conditions (Chidiac et al., 1996; Guerrero and Minneman, 1999). Taking advantage of the null background of uninfected DKO cells, we first examined possible nonspecific effects of a β2-AR inverse agonist, ICI, a β1-AR inverse agonist, CGP, and the nonselective antagonist, propranolol. Neither the selective antagonists, ICI (10−7 M) and CGP (10−6 M), nor the nonselective antagonist, propranolol, had any significant effect on baseline contraction amplitude in the absence of adenoviral infection (−5.6 ± 4.5, 9.4 ± 6.6, and 12.6 ± 6.4% of control, respectively,n = 7 for ICI and CGP, n = 12 for propranolol).
In DKO cells infected with adeno-β2-AR (m.o.i. 100), ICI significantly decreased the baseline contraction amplitude to 58.4 ± 2.7% of control (n = 7, P< .01, Fig. 6), confirming the existence of spontaneous β2-AR activation. Furthermore, ICI fully reversed the enhanced basal cAMP level in DKO cells overexpressing β2-AR at m.o.i. 100 (Fig. 6C). However, CGP failed to reveal any spontaneous β1-AR activity, because it had no discernible effect on the baseline contraction amplitude (Fig. 6) in DKO cells infected with adeno-β1-AR at m.o.i. 100. The nonselective β-AR antagonist, propranolol (10−6 M), fully reversed the enhancement of contraction amplitude induced by β2-AR spontaneous activation without altering the baseline contractility of cells expressing β1-AR (Fig. 6B). These results reinforce the conclusion that β-AR subtypes exhibit distinctly different spontaneous activity.
Discussion
Subtype-Specific β-AR Spontaneous Activation.
In the present study, we combined the β1β2 DKO mouse model with the adenoviral gene expression technique to create a pure experimental setting to individually study β-AR subtypes in mouse cardiac myocytes over a wide range of receptor density. The primary finding of this study is that overexpression of β2-AR increases the basal cAMP accumulation and contraction amplitude in a receptor density-dependent manner and that the β2-AR inverse agonist, ICI, can fully reverse these changes, with little nonspecific effect in the uninfected DKO myocytes. The observations in cardiac myocytes acutely overexpressing β2-AR are, therefore, in agreement with previous findings in the transgenic model chronically overexpressing β2-AR (Milano et al., 1994; Xiao et al., 1999a). Thus, both in vivo and in vitro, acute or chronic overexpression of β2-AR reveal constitutive activity of the receptor in mouse ventricular myocytes. Because basal cAMP level and contractility are concomitantly enhanced, the positive inotropic effect induced by spontaneous β2-AR activation is likely mediated by the cAMP-signaling pathway, as is the case in TG4 mice (Xiao et al., 1999a).
To our surprise, the property of spontaneous activation is not shared by β1-AR, the dominant β-AR subtype in the heart. Overexpression of β1-AR to similar or even greater levels has virtually no effect on basal cAMP, contraction amplitude, or contractile kinetics (Table 1). Furthermore, the alleged β1-AR inverse agonist, CGP, does not affect the basal cAMP level or contractility in myocytes overexpressing β1-AR (Fig. 6). These observations are consistent with the results from transgenic mice overexpressing β1-AR, although the β1-AR level (5–15 fold over WT) in the transgenic model (Engelhardt et al., 1999) was considerably lower compared with that in the present study. Because the present experimental setting avoids the potential nonspecificity of β-AR agonists or antagonists, the complicated interactions between β1- and β2-AR subtypes, and the possible compensatory changes in transgenic models, the present results provide evidence that the β1-AR, unlike β2-AR, does not undergo spontaneous activation in mouse cardiac myocytes. Similar findings were observed in cultured (wild-type) rat ventricular myocytes overexpressing β1-AR or β2-AR (R.-P. Xiao and S.-J. Zhang, unpublished observations).
However, it might be argued that β1-AR desensitization due to spontaneous activation might account for the lack of changes in basal cAMP and contractile parameters in myocytes overexpressing β1-AR and the apparent absence of β1-AR spontaneous activity. However, several lines of evidence argue against this hypothesis. First, β1-AR desensitization is not evident under our experimental conditions, because ISO stimulation augmented contractility (Fig. 4), with an EC50 in the nanomolar range (data not shown). Second, the ligand-binding results indicate that the K d of ICYP for β1-AR is similar to that for β2-AR (Fig. 2, legend), further suggesting that the affinity of β1-AR to ligand remains intact in our experimental system. Because receptor internalization is an important desensitization mechanism, we also examined whether overexpressing β1-AR causes receptor internalization. The confocal imaging data further exclude the possibility of receptor desensitization, because most of β1-ARs as well as β2-ARs are retained on the sarcolemmal and transverse tubule membranes in the absence of agonist stimulation (Fig.3).
Thus, the capacity to manifest spontaneous activity may not exist in all GPCRs. In agreement with our findings, the highly conserved gonadotropin receptors, luteinizing hormone receptor, and follicle-stimulating hormone receptor, have markedly different constitutive activity (Kudo et al., 1996; Schulz et al., 1999). In addition, dopamine receptor subtypes 1A and 1B also exhibit strikingly different constitutive activity, and a point mutation in the third intracellular loop completely abolishes this difference (Tiberi and Caron, 1994; Charpentier et al., 1996). In fact, the amino acid sequence of the third intracellular loop of β1-AR also markedly differs from that of β2-AR (Green and Liggett, 1994). The importance of this difference in determining the constitutive activation of β-AR subtypes merits further investigations. It is interestingly to note that the propensity of a receptor to undergo spontaneous activation is not necessarily related to the capacity to create a constitutively active mutant of the receptor. For example, both β1-AR and β2-AR can be readily mutated to generate constitutive activity (Samama et al., 1993;Lattion et al., 1999), even though they exhibit markedly different magnitudes of spontaneous activity, as evidenced by the present results.
Physiological Relevance of Spontaneous β2-AR Signaling.
As shown in Table 1 and Fig. 5, the baseline contractility was proportionally enhanced with increasing β2-AR density. For example, at the maximal receptor density (adeno-β2-AR, 1000), the basal contraction amplitude is ∼66% of ISO-induced maximal contraction amplitude in WT cells (Fig. 1C; Fig. 5B). Because a relatively high receptor density of β2-AR (69-fold over WT) is required in this case, it is inferred that only a small fraction of β2-ARs exhibits spontaneous activity at any given time. Alternatively, the spontaneously activated receptors may have a lower intrinsic efficacy than the ligand-stimulated receptors. Although ligand-free β2-AR signal exerts little effect on cardiac contractility under physiological conditions, an overexpression of β2-AR may still provide a potential therapeutic strategy to provide contractile support for the failing heart. Recent studies have demonstrated that cardiac-specific overexpression of β2-AR markedly augments cardiac function in an agonist-independent manner, without causing cellular or cardiac hypertrophy (Milano et al., 1994; Bond et al., 1995; Xiao et al., 1999a). Moreover, a 30-fold overexpression of β2-AR not only rescues cardiac function but also reverses cardiac hypertrophy induced by Gqα overexpression (Dorn et al., 1999). In contrast, increasing evidence has demonstrated that there is an inverse relationship between plasma norepinephrine levels and survival in patients with chronic heart failure (Cohn et al., 1984) and that β-AR blockade provides salutary effects on morbidity and mortality in patients with heart failure (Eichhorn and Bristow, 1996). In addition, cardiac transgenic overexpression of β1-AR by 5- to 15-fold in mice leads to marked myocyte hypertrophy, accompanied by fibrosis within a few weeks after birth and heart failure within a few months (Engelhardt et al., 1999). These studies support the idea that spontaneous β2-AR activity may have important implications in gene therapy of chronic heart failure (Drazner et al., 1997; Maurice et al., 1999). In fact, our recent studies have shown that spontaneous β2-AR activation provides the heart contractile support via anI Ca-independent signaling pathway (Zhou et al., 1999b). This unique property may also contribute to the normal phenotype of β2-AR overexpression transgenic models (Milano et al., 1994; Bond et al., 1995; Xiao et al., 1999a).
Diversity of β-AR Subtype Signaling.
The present finding that β1-AR and β2-AR differ in their ability to undergo spontaneous activation provides another line of evidence for the diversity of β-AR subtype signaling. Our previous studies have shown that ligand-activated β2-AR, but not β1-AR, stimulates Gi proteins in addition to Gs (Xiao et al., 1995, 1999a), resulting in opposing effects on cardiac contractility. The efficiency of the coupling of β2-AR to Gi, to a large extent, appears to underlie the species-dependent differences in cardiac β2-AR functional roles (for review see Xiao et al., 1999b). For example, in WT mouse cardiac myocytes, β2-AR stimulation elicits a robust contractile response only after Gi function is inhibited via pertussis toxin (PTX)-mediated rebosylation, whereas in rat and canine cardiac myocytes, β2-AR contractile responses are present but can be further enhanced by PTX treatment. In this regard, it is surprising to find that, unlike native WT mouse β2-AR or the chronically overexpressed human β2-AR in transgenic mice (TG4 mice) (Xiao et al., 1999b), stimulation of the acutely expressed β2-AR in mouse ventricular myocytes with ISO elicits a robust positive inotropic response in the absence of PTX treatment (Fig. 4). It is also noteworthy that β2-AR in noninnervated rat neonatal cardiomyocytes exhibits a much lower PTX sensitivity compared with that in innervated adult rat cardiomyocytes (Steinberg et al. 1999). The reduced β2-AR sensitivity to PTX treatment in both acutely expressed receptor in vitro and the noninnervated neonatal myocytes suggests that innervation or chronic receptor stimulation may play an essential role in promoting the receptor-Gi coupling. Additionally, the acutely expressed and chronically expressed β2-ARs may have different intracellular distributions with different accessibility to other signaling components, such as G proteins. Thus, the diversity of receptor signaling not only depends on the receptor subtypes and conformational states but also relates to cell types and cell conditions.
In summary, the present results reveal another facet of the intrinsic difference between β1- and β2-AR, in addition to their distinct ligand-induced activation: the β2-AR, but not β1-AR, exhibits spontaneous activation in a receptor density-dependent manner. This finding provides new insights into the signaling diversity of these closely related receptor subtypes in the physiological context of a cardiac myocyte.
Acknowledgments
We thank Dr. Harold Spurgeon and Bruce Ziman, for excellent technical assistance, and Dr. Robert Leftkowitz, for generously providing the β2-AR adenoviral vectors.
Footnotes
- Received March 24, 2000.
- Accepted July 18, 2000.
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Send reprint requests to: Rui-Ping Xiao, M.D., Ph.D., Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Dr., Baltimore, MD 21224. E-mail:xiaor{at}grc.nia.nih.gov
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↵1 Present address: Pediatric Cardiology, New York University Medical Center, New York, NY 10016.
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This work was supported by National Institutes of Health intramural (E.G.L., H.C., R.-P.X.) and extramural grants (B.K.K.), and by a grant from the National Science Foundation for Outstanding Youth of China (H.C.).
Abbreviations
- GPCR
- G protein-coupled receptor
- β-AR
- β-adrenergic receptor
- WT
- wild type
- ICI
- ICI 118,551
- CGP
- CGP 20712A
- DKO
- double knockout
- ISO
- isoproterenol
- FBS
- fetal bovine serum
- MEM
- minimal essential medium
- Tpeak
- the time from stimulation to peak shortening
- T50
- the time from the peak to 50% relaxation
- TA
- cell twitch amplitude
- ICYP
- [125I]cyanopindolol
- m.o.i.
- multiplicity of infection
- PBS
- phosphate-buffered saline
- HA
- hemagglutinin
- IBMX
- 3-isobutyl-1-methylxanthine
- PTX
- pertussis toxin
- The American Society for Pharmacology and Experimental Therapeutics