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(Circulation. 2003;108:1633.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Enhanced G_i Signaling Selectively Negates ß₂-Adrenergic Receptor (AR)– but Not ß₁-AR–Mediated Positive Inotropic Effect in Myocytes From Failing Rat Hearts

Rui-Ping Xiao, MD, PhD; Sheng-Jun Zhang, MD; Khalid Chakir, PhD; Pavel Avdonin, PhD; Weizhong Zhu, MD, PhD; Richard A. Bond, PhD; C. William Balke, MD, PhD; Edward G. Lakatta, MD; Heping Cheng, PhD

From the Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, Md (R.X., S.Z., K.C., P.A., W.Z., E.G.L., H.C.); the Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, Tex (R.A.B.); and the Departments of Physiology and Medicine, University of Maryland at Baltimore, and Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, Md (C.W.B.).

Correspondence to Rui-Ping Xiao, MD, PhD, Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, NIH, 5600 Nathan Shock Dr, Baltimore, MD 21224. E-mail xiaor{at}grc.nia.nih.gov

Received January 15, 2003; revision received May 20, 2003; accepted May 21, 2003.

	Abstract

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Background— Myocardial contractile response to ß₁- and ß₂-adrenergic receptor (AR) stimulation is severely impaired in chronic heart failure, in which G_i signaling and the ratio of ß₂/ß₁ are often increased. Because ß₂-AR but not ß₁-AR couples to G_s and G_i with the G_i coupling negating the G_s-mediated contractile response, we determined whether the heart failure–associated augmentation of G_i signaling contributes differentially to the defects of these ß-AR subtypes and, if so, whether inhibition of G_i or selective activation of ß₂-AR/G_s by ligands restores ß₂-AR contractile response in the failing heart.

Methods and Results— Cardiomyocytes were isolated from 18- to 24-month-old failing spontaneously hypertensive (SHR) or age-matched Wistar-Kyoto (WKY) rat hearts. In SHR cardiomyocytes, either ß-AR subtype–mediated inotropic effect was markedly diminished, whereas G_i proteins and the ß₂/ß₁ ratio were increased. Disruption of G_i signaling by pertussis toxin (PTX) enabled ß₂- but not ß₁-AR to induce a full positive inotropic response in SHR myocytes. Furthermore, screening of a panel of ß₂-AR ligands revealed that the contractile response mediated by most ß₂-AR agonists, including zinterol, salbutamol, and procaterol, was potentiated by PTX, indicating concurrent G_s and G_i activation. In contrast, fenoterol, another ß₂-AR agonist, induced a full positive inotropic effect in SHR myocytes even in the absence of PTX.

Conclusions— We conclude that enhanced G_i signaling is selectively involved in the dysfunction of ß₂- but not ß₁-AR in failing SHR hearts and that disruption of G_i signaling by PTX or selective activation of ß₂-AR/G_s signaling by fenoterol restores the blunted ß₂-AR contractile response in the failing heart.

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

	Introduction

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Stimulation of ß-adrenergic receptors (ß-ARs) by catecholamines plays a pivotal role in regulating cardiac function. At least 2 ß-AR subtypes, ß₁-AR and ß₂-AR, are expressed in cardiac myocytes. Chronic heart failure in animal models and in humans is characterized by a severely diminished contractile response to both ß-AR subtypes.^1–3 The heart failure–associated defect of ß-AR contractile response is accompanied by a selective downregulation of ß₁-AR (resulting in a higher ß₂/ß₁ ratio^1–3) and an increase in the amount or activity of pertussis toxin (PTX)–sensitive inhibitory G proteins (G_i) without any abnormality in stimulatory G proteins (G_s).^3–5

Although ß₁-AR and ß₂-AR are closely related G protein–coupled receptors, these ß-AR subtypes couple to different G proteins^6–9 and exhibit different effects on phosphorylation of protein kinase A (PKA) target proteins,^10–13 cardiac excitation-contraction coupling,^14,15 and cardiac cell survival and cell death.^16–19 Most importantly, ß₁-AR stimulation activates the G_s-cAMP-PKA signaling cascade, whereas the cardiac ß₂-AR couples to PTX-sensitive G_i proteins, G_i2 and G_i3, in addition to G_s.^{6–9,16–19} The coupling of ß₂-AR to G_i protein functionally confines the ß₂-AR–G_s–stimulated cAMP/PKA signaling to a subsarcolemmal microdomain via the G_i-Gß–phosphatidylinositol 3-kinase pathway^{10–15,20–22} and negates the G_s-mediated protein phosphorylation and positive inotropic effect in ventricular myocytes of many mammalian species (for review, see Reference ²²).

The overall goal of the present study was to determine whether G_i signaling is selectively involved in the heart failure–associated defective contractile response to ß₂-AR stimulation and, if so, to determine whether ß₂-AR/G_s selective activation by certain agonists is able to restore the contractile support by ß-AR stimulation in the failing heart. Specifically, we (1) determined the abundance and function of both ß-AR subtypes (ß₁-AR and ß₂-AR) and G proteins (G_s and G_i) in cardiomyocytes from spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats (WKY), (2) investigated the potential role of G_i signaling in the defects of ß-AR subtype–mediated contractile response in failing SHR cardiomyocytes, (3) and screened a number of ß₂-AR ligands to identify "ß₂-AR/G_s-signaling–selective" agonists and tested the agonists in cardiomyocytes from failing SHR hearts.

	Methods

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Echocardiography and Hemodynamic Studies
Transthoracic echocardiographic studies were performed in rats as previously described.²³ Briefly, a 2D short-axis view of the left ventricle (LV) was obtained at the level of the papillary muscles. M-mode tracings were recorded through the anterior and posterior LV walls. The percentage shortening of the endocardium was calculated as (LVDD-LVSD)/LVDDx100, where LVDD is LV internal diastolic dimension and LVDS is LV internal systolic dimension. The heart rates, blood pressures, and respiratory rates of all animals were monitored at monthly intervals. Blood pressure was measured by the tail-cuff method.

Measurement of Cardiac Myocyte Contraction
Myocytes were isolated from 18- to 24-month-old SHR or WKY rats by use of standard enzymatic techniques.¹⁴ Cells were perfused with a HEPES buffer containing (in mmol/L): 137 NaCl, 5.4 KCl, 1.2 MgCl₂, 1 NaH₂PO₄, 1 CaCl₂, 20 glucose and 20 HEPES (pH 7.4) and electrically stimulated at 0.5 Hz at 23°C. Cell length was monitored by an optical edge-tracking method as previously described.¹⁴ In a subset of experiments, aliquots of cells were incubated with PTX (1.5 µg/mL at 37°C for 3 hours).⁶ An individual myocyte was exposed to only 1 dose of 1 agonist, and measurements were obtained under steady-state conditions after a 10-minute exposure to the designated agonist.

Receptor Radioligand Binding
ß-AR radioligand binding studies were performed in membranes from LVs by use of the nonselective ß-AR antagonist [¹²⁵I]-labeled cyanopindolol (¹²⁵I-CYP), as described previously.¹⁵ Maximal binding (B_max) was measured by use of a saturating amount of ¹²⁵I-CYP on 25 µg of membrane protein from LVs. Inclusion of 10 µmol/L propranolol defined nonspecific binding. For competition isotherms, membranes (25 µg total protein) were incubated with 50 pmol/L ¹²⁵I-CYP and increasing dilutions of ICI118,551 (ICI), a selective ß₂-AR antagonist, as described previously.¹⁵ The percentage of ß₂-AR was calculated by use of GraphPad Prism.

Western Analysis of G_s and G_i Proteins
Cardiac membranes from myocytes were used to determine G_s and G_i protein amounts by Western analysis.¹⁸ The antibodies recognizing the -subunits of G_i2, G_i3, and G_s were obtained from Santa Cruz Biotechnology.

Statistical Analysis
Results are expressed as mean±SEM. Significance was determined by use of Student’s t test, with a value of P<0.05 considered to be statistically significant.

	Results

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Chronic Heart Failure Model: SHR
SHRs develop hypertension at 6 to 8 weeks of age. By the age of 18 to 24 months, 50% of these rats develop severe dilated chronic heart failure, many aspects of which have been characterized.²⁴ At the time of study (ie, 18 to 24 months old), all SHRs used in this study had severe physical signs of heart failure (eg, resting tachycardia, tachypnea, pleural and/or pericardial effusions, and ascites). Cardiac dilation in the SHR group was evidenced by echocardiographic measurements (Table 1). Cardiac hypertrophy was confirmed by significant increases in the heart weight/body weight ratio, by echocardiographic measurements of LV posterior wall thickness, and by increased resting cell length (Table 1).

View this table: [in this window] [in a new window]   TABLE 1. Hemodynamic and Echocardiographic Parameters, Heart Weight/Body Weight Ratios, and Resting Cell Length of 18- to 24-Month-Old WKYs and SHRs

Alterations in ß-AR Density and G Protein Abundance in Failing SHR Hearts
We first examined total ß-AR density, ß₁- and ß₂-AR subpopulations, and the abundance of G_s and G_i proteins in myocytes from 18- to 24-month-old SHR and age-matched WKY cardiac myocytes. There was a significant decrease (24%) in the B_max in SHR relative to WKY hearts, without alterations in the binding affinity (Table 2). To define the relative abundance of ß₁-AR and ß₂-AR, competition isotherms were performed by use of the selective ß₂-AR antagonist ICI. The ß₂/ß₁-AR ratio in control animals is 44/56, which is similar to the ratio previously reported.¹⁵ The ß₂/ß₁-AR ratio was significantly increased in SHR relative to WKY hearts (Table 2). This result indicates that the reduction in total ß-AR density is largely accounted for by the loss of ß₁-AR. Thus, a selective downregulation of ß₁-AR leads to a relative increase in the ß₂-AR density in the failing SHR heart, a situation similar to what has been reported in failing human heart.^1–3

View this table: [in this window] [in a new window]   TABLE 2. ß-AR Radioligand Binding Properties and Competition of [125I]ICYP Binding With ß-AR Antagonist ICI in LV Membranes From 18- to 24-Month-Old SHRs and Age- and Sex-Matched WKYs

In addition, both G_i2 and G_i3 but not G_s were elevated by 2-fold in SHR compared with WKY hearts (Figure 1), resulting in an increase in the G_i/G_s ratio, as is the case in human heart failure.^3–5 Taken together, these results suggest that the SHR heart failure model resembles human heart failure with respect to the major changes that occur in the proximal ß-AR signaling system.

View larger version (31K): [in this window] [in a new window]   Figure 1. Gi and Gs protein abundance in SHR and WKY cardiac myocytes. A, Representative Western blots probed with antibodies reacting with -subunits of Gs, Gi3, or Gi2, as indicated. B, Average data of Gs, Gi3, and Gi2 levels in SHR and WKY cardiomyocyte membranes (*P<0.01 vs WKY, n=6 experiments in cells from 12 hearts).

Disruption of G_i Signaling by PTX Selectively Restores ß₂-AR– but Not ß₁-AR–Mediated Positive Inotropic Effect in Cardiomyocytes From Failing SHR Hearts
Although the baseline contraction amplitude in myocytes from failing SHR hearts was not significantly different from that of control animals (see Figure 2 legend), the positive inotropic effect of either ß-AR subtype stimulation was markedly attenuated. Figure 2A shows that myocyte contractile response to ß₁-AR stimulation by norepinephrine (NE) in the presence of ₁-AR and ß₂-AR blockade by prazosin (10^-6 mol/L) and ICI (10^-7 mol/L), respectively, was markedly blunted in SHR myocytes compared with that in WKY cells, consistent with the downregulation of the receptor density. Figure 2B illustrates that ß₂-AR stimulation by zinterol had only a minor positive inotropic effect in SHR ventricular myocytes, whereas it enhanced the contraction amplitude by >2-fold in control cardiomyocytes from age-matched WKYs. The dose-response curve of zinterol to increase contraction amplitude was severely shifted downward in SHR compared with WKY. Thus, myocyte contractile response to either ß₁-AR or ß₂-AR was overtly attenuated in SHR cardiomyocytes relative to WKY myocytes, even though only ß₁-AR density was selectively downregulated, with little change in ß₂-AR density in SHR hearts (Table 2).

View larger version (21K): [in this window] [in a new window]   Figure 2. PTX selectively restores ß2- but not ß1-AR–mediated positive inotropic effect in SHR cardiomyocytes. A, Dose-response of ß1-AR stimulation by NE (plus 1-AR and ß2-AR blockade); B, dose-response of ß2-AR stimulation by zinterol to increase myocyte contraction in presence or absence of PTX. Baseline contraction amplitudes are 5.60±0.23% (n=126 cells from 10 hearts) and 4.93±0.33% (n=76 cells from at least 10 hearts) of resting cell length for WKY and SHR, respectively. Data are expressed as % of control (n=6 to 10 cells from 6 to 10 hearts). PTX does not alter basal contraction (4.88±0.14% and 5.52±0.34% of resting cell length for SHR and WKY cells, respectively; n=28 cells from 6 hearts for each group).

To test the hypothesis that exaggerated G_i signaling might be differentially involved in the impairment of ß₂-AR versus that of ß₁-AR in the failing heart, we treated myocytes with PTX to disrupt G_i function. Remarkably, PTX not only enhanced the contractile response to ß₂-AR stimulation in both failing SHR and control myocytes but also abolished the difference between the 2 groups (Figure 2B). In contrast, PTX did not affect the whole dose-response of ß₁-AR stimulation by NE in either WKY or SHR myocytes (Figure 2A). These results indicate that enhanced G_i signaling is specifically involved in the defect of ß₂-AR contractile response but not in the dysfunction of ß₁-AR signaling in the SHR heart failure model. These results also suggest that ß₂-AR–coupled G_s signaling remains largely intact in the failing heart.

Screening for Ligands Bypassing ß₂-AR/G_i Pathway
To determine whether disruption or avoidance of the ß₂-AR–coupled G_i signaling pathway is able to restore ß₂-AR–mediated contractile support in the failing SHR heart, we searched for ligands that activate ß₂-AR–coupled G_s signaling but not the G_i pathway. Six known ß₂-AR–selective agonists and 2 nonselective ß-AR agonists were tested in the presence of the ß₁-AR antagonist CGP20712A (CGP; 3x10^-7 mol/L). Zinterol had been well characterized in previous studies as a selective ß₂-AR agonist.^{6–9,10–15} The specificity of other tested agonists, including salbutamol, procaterol, and fenoterol, for ß₂-AR stimulation was verified by the fact that the highly selective ß₁-AR antagonist CGP (3x10^-7 mol/L) did not block their contractile responses, whereas it fully abolished the effect induced by the ß₁-AR agonist NE (10^-7 mol/L) (Figure 3). Cell contractile response to ß-AR stimulation and its PTX sensitivity were then measured to represent the overall readout of G_s (in PTX-treated cells) or combined G_s-G_i signaling (in non–PTX-treated cells).

View larger version (20K): [in this window] [in a new window]   Figure 3. Specificity of ß2-AR agonists fenoterol (FEN), procaterol (PROC), and salbutamol (SALB). ß1-AR selective antagonist CGP (3x10-7 mol/L) blocks contractile response induced by NE (10-7 mol/L) plus 1-AR and ß2-AR blockade but not equipotent positive inotropic effects induced by fenoterol, procaterol, or salbutamol [n=6 to 8 cells from 6 hearts for each group, *P<0.001 vs NE (-CGP)].

Most ligands tested, including salbutamol, procaterol, and zinterol, enhanced the contraction amplitude of WKY myocytes in a dose-dependent manner, with a maximal increase of 2-fold (Figure 4A through 4C). Inhibition of G_i function by PTX enabled the ß₂-AR agonists to induce a full contractile response, with the maximal response being a 3-fold increase (Figure 4A through 4C). In other words, with respect to their inotropic effects, inhibition of G_i signaling converted these partial ß₂-AR agonists to full agonists. These results are consistent with previous findings that ß₂-AR can simultaneously activate G_s and G_i.^6–9 A similar potentiating effect of PTX was observed for ß₂-AR stimulation induced by other known ß₂-AR agonists such as terbutaline and celenbuterol, or by nonselective agonists, epinephrine and isoproterenol, plus the ß₁-AR blocker CGP (data not shown).

View larger version (25K): [in this window] [in a new window]   Figure 4. Effects of PTX treatment on contractile responses to ß2-AR agonists. Dose-response of contraction amplitude in myocytes subjected to salbutamol (A), procaterol (B), zinterol (C), and fenoterol (D) in presence and absence of PTX. Data are expressed as % of control (% C; n=6 to 8 cells from 6 hearts for each data point). Baseline contractions are 5.29±0.13% (n=146 cells from 24 hearts) and 5.57±0.13% (n=166 cells from 24 hearts) of resting cell length for PTX-treated and nontreated group, respectively.

Interestingly, another selective ß₂-AR agonist, fenoterol,²⁵ evoked a full contractile response that was insensitive to PTX treatment (Figure 4D). It appears that fenoterol selectively stimulates the ß₂-AR/G_s signaling pathway, whereas other ß₂-AR agonists activate both G_s and G_i proteins in terms of their effects on cardiac myocyte contractility.

Rescue of the Depressed ß₂-AR Contractile Response by Fenoterol in Myocytes From Failing SHR Hearts
We next determined whether the apparent ß₂-AR/G_s signaling–specific agonist fenoterol could rescue the diminished contractile response to ß₂-AR stimulation in myocytes from failing SHR hearts. Figure 5A shows typical continuous recordings of myocyte contractile responses to zinterol or fenoterol. Fenoterol induced a robust increase in contraction amplitude in a representative SHR cell. This effect was completely reversed by the ß₂-AR antagonist ICI (10^-7 mol/L), indicating that the positive inotropic effect of fenoterol is mediated by selective ß₂-AR stimulation. In contrast, zinterol had a very minor effect in another cell from the same heart. Figure 5B shows the average dose-response of contraction amplitude to fenoterol in cardiomyocytes from SHR and age-matched WKY rats. The 2 dose-response curves virtually overlapped each other, in sharp contrast to the markedly suppressed contractile response to zinterol in failing SHR heart cells (Figure 2B). Thus, fenoterol is able to restore a full contractile response to ß₂-AR stimulation in SHR myocytes even in the absence of PTX, thereby abolishing the difference between SHR and WKY.

View larger version (13K): [in this window] [in a new window]   Figure 5. Fenoterol restores contractile response of failing SHR myocytes to ß2-AR stimulation. A, Examples of cell contractile response to zinterol (ZINT, 10-5 mol/L) and fenoterol (FEN, 10-6 mol/L) in representative SHR myocytes. B, Dose-response of fenoterol in myocytes from failing SHR and nonfailing WKY hearts. See legend of Fig. 2 for baseline contraction. Data are presented as % of control (% C; n=10 to 18 cells from 5 hearts for each data point).

	Discussion

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Enhanced G_i Signaling Selectively Contributes to the Diminution of ß₂-AR– but Not ß₁-AR–Mediated Contractile Response in Myocytes From Failing SHR Hearts
Although previous studies have demonstrated that PTX treatment partially restores the diminished contractile response to mixed ß-AR stimulation in a rat myocardial infarction heart failure model²⁶ and in myocytes from failing human hearts,²⁷ the present study provides the first documentation that enhanced G_i signaling is, in fact, selectively involved in heart failure–associated attenuation in ß₂-AR– but not ß₁-AR–mediated increase in myocyte contractility. This is because inhibition of ß₂-AR/G_i signaling by PTX treatment enables ß₂-AR stimulation to induce a full contractile response, thus abolishing the difference between the failing and normal hearts, whereas it does affect ß₁-AR–induced inotropic effect. These findings are consistent with our previous notion that ß₂-AR but not ß₁-AR dually couples to G_s and G_i and that the G_i coupling exhibits an inhibitory effect on the G_s signaling.^6–9

Although the heart failure–associated defect of ß₂-AR contractile response is largely attributable to enhanced ß₂-AR/G_i signaling, multiple mechanisms might be involved in ß₁-AR dysfunction, including receptor downregulation^1–3 (Table 2) and receptor desensitization resulting from upregulated G protein–coupled receptor kinases. In this regard, it has been shown that the level and enzymatic activity of ß-AR kinase 1 (ßARK1), a prototypic G protein–coupled receptor kinase,²⁸ are significantly elevated in human²⁹ and SHR failing hearts.³⁰ The increased ßARK1 abundance/activity may contribute to the reduced ß₁-AR signaling in chronically failing hearts. Further studies are needed to determine the relative contributions of enhanced G_i function and ßARK1 to subtype-specific defects of ß-AR in the context of heart failure.

Selective Downregulation of ß₁-AR May Serve as a Protective Mechanism in Heart Failure
It has been highly controversial as to whether enhancing ß-AR signaling is beneficial or deleterious for the failing heart. The prevalent view is that chronically increasing nonselective ß-AR stimulation is toxic to the heart, because there is an inverse relationship between the plasma level of catecholamines and survival in heart failure patients³¹ and because ß-AR blockade reduces both the morbidity and mortality in heart failure patients.³² More recent studies have demonstrated that ß₁-AR induces apoptosis in cultured rodent cardiomyocytes.^16–19 Similarly, cardiac transgenic overexpression of ß₁-AR in mice leads to marked myocyte hypertrophy, apoptosis, and heart failure.^33,34 Thus, chronic ß₁-AR stimulation is detrimental to the myocardium.

In contrast, selective enhancement of ß₂-AR signaling might be beneficial for the failing heart. This hypothesis is supported by the fact that ß₂-AR stimulation protects cardiac myocytes against a wide array of apoptotic insults.^16–19 In addition, overexpression of cardiac ß₂-AR by 100- to 200-fold does not induce hypertrophy or heart failure,³⁵ at least up to the age of 1 year. In fact, crossing transgenic mice overexpressing cardiac ß₂-AR at appropriate levels (30-fold) with transgenic mice overexpressing G_q not only improves cardiac performance but also prevents hypertrophy in the G_q overexpression heart failure model,³⁶ although extremely high levels of ß₂-AR overexpression induce pathological phenotypes^36,37 and fail to rescue the genetic mouse heart failure model.^36,37 Furthermore, the beneficial effect of ß₂-AR stimulation in the context of heart failure is clearly supported by the analysis of polymorphisms of ß₂-AR in chronic heart failure patients. The prognosis of heart failure patients with Ile164 polymorphism (a Thr-to-Ile switch at amino acid 164 with reduced ß₂-AR signaling)³⁸ relative to patients without the ß₂-AR variant is much worse, and they are significantly more likely to receive earlier aggressive interventions or cardiac transplantation.³⁹

In the present study, we found that in failing SHR hearts, ß₁-AR is selectively downregulated with little or no loss of ß₂-AR, similar to the situation of chronic human heart failure.^1–3 In light of evidence for the opposing effects of ß₁-AR and ß₂-AR stimulation on cardiac myocyte apoptosis^16–19 and the distinct phenotypes of transgenic overexpression of ß₁-AR versus ß₂-AR,^33–35 the selective downregulation of ß₁-AR may represent a cardiac protective mechanism to slow the progression of cardiomyopathy and contractile dysfunction.

Selective Activation of ß₂-AR/G_s Signaling in Failing SHR Hearts
Because heart failure in many instances is related to a selective loss of ß₁-AR and because chronic stimulation of ß₂-AR exhibits no known detrimental effects, as discussed above,³⁵ ß₂-AR would appear to be a potentially important therapeutic target for the treatment of chronic heart failure. However, most of the commonly used nonselective ß-AR agonists (eg, isoproterenol) cannot discriminate ß₂-AR from ß₁-AR. In addition, most ß₂-AR agonists cannot discriminate ß₂-AR–coupled G_s signaling from G_i signaling. The situation is even worse in chronically failing hearts with elevated G_i signaling, such that ß₂-AR stimulation by zinterol elicits only a minor contractile response (Figure 2). To unmask the ß₂-AR/G_s signaling from the inhibition by G_i, we identified fenoterol as an apparent ß₂-AR/G_s–selective agonist, as evidenced by the lack of PTX sensitivity of its positive inotropic effect (Figure 3). In cardiomyocytes from failing SHR hearts, fenoterol elicits a contractile response that is comparable to that in cells from nonfailing WKY heart cells (Figure 4). It is also noteworthy that there may be some advantages to use of fenoterol instead of ß₁-AR agonists to provide contractile support in the failing heart. Our preliminary experiments have shown that, unlike ß₁-AR stimulation, ß₂-AR stimulation by fenoterol does not induce cardiac myocyte apoptosis (Zhu et al, unpublished data), although it increases cAMP/PKA signaling, perhaps because of distinct compartmentation of cAMP signal in response to different ß-AR subtype activation.^{11–13,40,41}

Regarding receptor theory, the G_s-specific ß₂-AR stimulation, as demonstrated here for native ß₂-AR in intact cardiomyocytes, provides new evidence to support the idea that there are multiple active conformational states for a given receptor^42–45 and that ligands may lead the receptor to a subset of downstream signaling pathways (ligand-directed receptor trafficking).⁴⁵ In contrast to fenoterol, a well-characterized ß₂-AR inverse agonist, ICI, can direct ß₂-AR to a G_i-coupled form and result in a negative inotropic effect in cardiomyocytes from failing human hearts or rabbit and mouse myocytes in which ß₂-AR and/or G_i proteins are overexpressed.⁴⁴

In summary, the present study reveals 3 major findings. First, enhanced G_i signaling is selectively involved in the dysfunction of ß₂-AR but not of ß₁-AR. Inhibition of G_i signaling with PTX enables ß₂-AR stimulation to induce a full contractile response in myocytes from failing SHR hearts without affecting ß₁-AR contractile response. Second, ß₂-AR–coupled G_s and G_i pathways appear to be activated in an agonist-dependent manner. Most agonists tested stimulate both pathways, whereas fenoterol predominantly activates G_s signaling, as manifested by the lack of PTX sensitivity of its positive inotropic effect. Third, the apparent ß₂-AR/G_s–selective agonist fenoterol fully restores ß₂-AR contractile response in failing SHR hearts. Therefore, we conclude that enhanced G_i signaling is specifically involved in the dysfunction of ß₂-AR but not of ß₁-AR in regulating myocyte contractility in the failing heart and that an apparent selective activation of ß₂-AR–coupled G_s, bypassing the G_i pathway, provides a novel means to restore the diminished ß₂-AR responsiveness in the SHR heart failure model. Thus, signaling-selective receptor stimulation by agonists may offer a wide range of novel therapeutic opportunities by enhancing the desired effects while avoiding the adverse side effects of therapeutic agents.

	Acknowledgments

 
This work was supported by the National Institutes of Health (NIH) intramural research programs (Drs Xiao, Lakatta, and Cheng), NIH extramural grants (Drs Bond and Balke), and the American Heart Association (Dr Balke).

	Footnotes

 
All authors except Drs Balke and Bond are employees of the US Government.

	References

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