β-Adrenergic Receptor Subtype Signaling in the Heart: from Bench to the Bedside

CHAPTER 9 b-Adrenergic Receptor Subtype Signaling in the Heart: from Bench to the Bedside Weizhong Zhu1, Anthony Yiu-Ho Woo2, Yan Zhang2, Chun-Mei Cao,2 and Rui-Ping Xiao2 1 2

Center for Translational Medicine, Thomas Jefferson University, Philadelphia, PA, USA Institute of Molecular Medicine, Peking University, Beijing, China

I. Overview II. Subtype-Specific Functional Roles of b1AR and b2AR in Regulating Cardiomyocyte Survival and Death A. Prolonged Stimulation of b1AR Triggers Cardiomyocyte Apoptosis and Maladaptive Cardiac Remodeling B. Cardioprotection by b2AR Stimulation III. Mechanisms Underlying b2AR-Coupled Gi Signaling IV. RGS2-Mediated Termination of b2AR-coupled Gi Signaling and Its Potential Pathogenic and Therapeutic Implications V. Ligand-Directed Selective Activation of b2AR-Coupling to Gs or Gi VI. Development of b2AR Agonists into new Drugs for the Treatment of Heart Failure A. Signaling-Selective b2AR Agonists for the Treatment of Heart Failure B. A Combination of b2AR Activation with b1AR Blockade Provides a more Effective Therapy for Heart Failure VII. Future Perspective References

I. OVERVIEW Stimulation of b-adrenergic receptor (bAR), a prototypical member of G protein-coupled receptor (GPCR) superfamily, is broadly involved in metabolic regulation, growth control, muscle contraction, cell survival, and cell death. The major bAR subtypes, b1AR and b2AR, couple to distinct G proteins and differentially regulate cardiac function and remodeling. Three major discoveries have marked the recent research line with respect to bAR subtype-specific Current Topics in Membranes, Volume 67 Copyright 2011, Elsevier Inc. All right reserved.

0065-230X/10 $35.00 DOI: 10.1016/B978-0-12-384921-2.00009-4

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signal transduction. These include: (1) dual coupling of b2AR to Gs and Gi proteins in cardiomyocytes; (2) cardioprotection by b2AR signaling in improving cardiac function and cardiomyocyte viability; (3) PKA-independent, CaMKII-mediated b1AR signaling in triggering myocyte apoptosis and maladaptive cardiac remodeling. These findings indicate that b1AR stimulation is cardiac detrimental, while b2AR stimulation is protective. Heart failure (HF) is a complex clinical syndrome featured by extensive abnormalities in the bAR system, including elevated circulating catecholamine levels, selective downregulation and desensitization of b1AR, and increased b2AR-coupled Gi signaling. In particular, the enhanced Gi signaling negates b1AR- as well as b2AR-mediated contractile response, thus contributing to the pathogenesis of HF. Our recent translational studies support the concept that inhibition of the Gi signaling or selective b2AR-Gs stimulation with fenoterol markedly improves cardiac remodeling and function of the failing heart. In this chapter, we intend to summarize (a) the recent progresses on subtype-specific functional roles of b1AR and b2AR, (b) the mechanisms underlying the activation and termination of the unique b2AR-coupled Gi signaling, and (c) liganddirected selective activation of b2AR-coupled Gs or Gi signaling and their potential therapeutic implications.

II. SUBTYPE-SPECIFIC FUNCTIONAL ROLES OF b1AR AND b2 AR IN REGULATING CARDIOMYOCYTE SURVIVAL AND DEATH A. Prolonged Stimulation of b1AR Triggers Cardiomyocyte Apoptosis and Maladaptive Cardiac Remodeling The persistent stimulation of b1AR and b2AR exhibits distinct outcomes under certain pathological circumstances such as HF. Specifically, persistent stimulation of b1AR in mouse cardiomyocytes lacking b2AR (b2AR knockout or b1b2 double knockout) in conjunction with adenoviral gene transfer of b1AR triggers cardiomyocyte apoptosis by a CaMKII-dependent mechanism that is independent of PKA signaling (Zhu et al., 2003). Furthermore, b1AR-activated CaMKII signaling, but not the PKA pathway, is involved in catecholamineinduced neonatal rat cardiomyocyte pathological growth (hypertrophy) (Sucharov, Mariner, Nunley, Long, Leinwand, & Bristow, 2006). These in vitro observations have been corroborated by in vivo studies using genetic manipulation of either b1AR or CaMKII signaling components. In particular, cardiacspecific overexpression of the human b1AR at a modest level (15-fold) leads to maladaptive cardiac remodeling, severe dilated cardiomyopathy, and premature death of transgenic mice (Bisognano et al., 2000; Engelhardt, Hein, Wiesmann, & Lohse, 1999). This is further supported by the severe HF and

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premature death of transgenic mice with cardiac-specific overexpression of CaMKII-dC (Zhang et al., 2003). In contrast, the cardiac-specific expression of a peptide inhibitor of CaMKII prevents myocyte apoptosis, maladaptive remodeling, and cardiac contractile dysfunction following excessive bAR stimulation or myocardial infarction (MI; Zhang et al., 2005). In clinical settings, elevations in plasma norepinephrine, an endogenous b1AR agonist, and a stimulatory anti-b1AR antibody are directly associated with HF mortality in humans and animal models (Hebert, 2007). Thus, the b1AR-evoked persistent activation of CaMKII might be a primary etiological mechanism that underlies b1AR-induced adverse myocardial remodeling and resultant cardiomyopathy. In contrast to the detrimental effects of persistent CaMKII activation, the consequence of sustained PKA activation in the heart remains controversial. Enhanced AC-cAMP-PKA signaling by overexpressing AC type V or VI does not produce HF and, instead, alleviates HF in some genetic mouse models (Lai et al., 2008; Roth et al., 1999, 2002; Tang, Gao, Roth, Guo, & Hammond, 2004). Likewise, enhanced cAMP-PKA signaling by b2AR agonist stimulation or by overexpression of the receptor (by  30-fold over its endogenous level) protects cardiomyocytes against apoptosis and exerts beneficial effects in several HF models (Liggett et al., 2000). These findings differ from the HF phenotype of transgenic mice overexpressing either the Gas subunit or the PKA catalytic subunit (Antos et al., 2001; Iwase et al., 1997). Although the exact mechanism underlying the discrepancy is unknown, it might be attributable to a b2ARmediated antiapoptotic effect and the different spatial profile of the cAMP-PKA signaling induced by b2AR compared with that induced by the overexpression of Gas or the PKA catalytic subunit (Antos et al., 2001; Iwase et al., 1997). Nevertheless, these paradoxical observations challenge the conventional wisdom that the cAMP-PKA pathway is solely responsible for b1AR-induced cardiac detrimental effects. Resolving these paradoxes should reveal valuable etiological insights and novel therapeutic targets for the treatment of HF.

B. Cardioprotection by b2AR Stimulation In contrast to the cardiotoxic effects of persistent b1AR activation in vivo and ex vivo, persistent b2AR stimulation is cardioprotective. In mice lacking the native b2AR, the stimulation of endogenous b1AR with isoproterenol (ISO) triggers more severe myocardial apoptosis in vivo compared with wild type control mice (Patterson et al., 2004). This is consistent with earlier pharmacological studies showing that the activation of b2AR attenuates catecholamine-, hypoxia-, or reactive oxygen species (ROS)-induced apoptosis in both neonatal and adult rat cardiomyocytes (Chesley et al., 2000; Zhu, Zheng, Koch, Lefkowitz, Kobilka, & Xiao, 2001). The cardiac beneficial effects of persistent

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b2AR signaling have been further manifested in recent in vivo studies; the selective activation of b2AR by fenoterol exerts a profound antiapoptotic effect and improves cardiac performance in an ischemic rat HF model (Ahmet et al., 2008). However, further investigation is merited for understanding the exact signaling events that underlie b2AR-mediated cardiac protection (see following sections).

III. MECHANISMS UNDERLYING b2AR-COUPLED G i SIGNALING The classic view on bAR-coupled Gs signaling involves an agonist-induced change in the receptor conformation that causes the activation of the Gs protein, leading the formation of the second messenger, cAMP, which activates PKA and downstream signaling. The termination of this cascade occurs when GPCR kinases (GRKs) and the second messenger kinase, PKA, phosphorylate the activated receptor and promote the binding of b-arrestins which sterically block the coupling of Gs to the receptor. It is widely accepted that prolonged exposure of bAR to an agonist leads to reduced Gs-mediated responses such as cAMP production and positive inotropic effect. When this occurs in the context of diminished responsiveness to a diverse array of other agonists (heterologous desensitization), it generally results from a negative feedback regulation by PKA-mediated phosphorylation of bARs. Exposure to an agonist also leads to GRKs-dependent, agonist-specific or homologous desensitization, which proceeds the PKA-dependent phosphorylation and constitutes the most efficient means to desensitize GPCRs. HF is characterized by defective bAR system, including increased circulating catecholamine levels, reduced b1AR density and signaling efficiency, and enhanced b2AR-coupled Gi signaling (Rockman, Koch, & Lefkowitz, 2002; Ungerer, Bohm, Elce, Erdmann, & Lohse, 1993). GRK2 (also known as bARK1), the best characterized member of the GRK family, plays a predominant role in desensitizing bARs and has been implicated as a cause factor of HF (Rengo, Lymperopoulos, Leosco, & Koch, 2011). Emerging evidence suggests that activation of GRK2 as well as PKA is essentially involved in the activation of the b2AR-coupled Gi signaling in mammalian cells. First, early work has shown that b2AR-induced activation of ERK1/2 in HEK293 cells is mediated by a Gi-dependent mechanism, and that phosphorylation of b2AR by PKA is a prerequisite for the switch of the receptor coupling from Gs to Gi (Daaka, Luttrell, & Lefkowitz, 1997). Second, our recent studies have demonstrated that, in addition to PKA-mediated phosphorylation, elevated b2AR phosphorylation by GRK2 acerbates the Gi signaling, whereas inhibition of GRK2 activity profoundly suppresses the b2AR-Gi coupling (Zhu et al., unpublished data). Since GRK2 upregulation occurs prior to the onset

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of HF and contributes to the development of HF (Choi, Koch, Hunter, & Rockman, 1997; Iaccarino, Tomhave, Lefkowitz, & Koch, 1998; Perrino, Naga Prasad, Schroder, Hata, Milano, & Rockman, 2005; White, Hata, Shah, Glower, Lefkowitz, & Koch, 2000) enhanced GRK2 activation may play an important role in the exacerbated b2AR-coupled Gi signaling in the failing heart. Indeed, disruption of Gi signaling with PTX or inhibition of GRK2 with a peptide inhibitor, bARK-ct, can restore cardiac contractile response to bAR stimulation in multiple HF models (Chakir et al., 2009; Koch et al., 1995; Tachibana, Naga Prasad, Lefkowitz, Koch, & Rockman, 2005; Xiao et al., 2003). It is also noteworthy that, while the b1AR subtype cannot couple to Gi under normal conditions, b1AR contractile response is cross-inhibited by enhanced b2AR-Gi signaling in the failing heart. Thus, the enhanced b2AR-Gi signaling contributes to the dysfunction of both b1AR and b2AR in the failing heart (Lokuta et al., 2005; Sato, Gong, Terracciano, Ranu, & Harding, 2004; Xiao & Balke, 2004; Zhu et al., 2005). Taken together, these recent studies have defined GRK2 as an important etiological link between enhanced b2AR-Gi signaling and the development of HF. These studies also suggest that the previously reported beneficial effects of bARK-ct in improving the function of the failing heart (Choi et al., 1997; Iaccarino et al., 1998; Perrino et al., 2005; White et al., 2000) is mediated, at least in part, by attenuating GRK2-dependent b2AR-Gi signaling. IV. RGS2-MEDIATED TERMINATION OF b2 AR-COUPLED G i SIGNALING AND ITS POTENTIAL PATHOGENIC AND THERAPEUTIC IMPLICATIONS As discussed above, in contrast to the bAR-Gs signaling, the b2AR-Gi signaling is enhanced by PKA- and GRK2-mediated phosphorylation of the receptor (Daaka et al., 1997; Hausdorff, Lohse, Bouvier, Liggett, Caron, & Lefkowitz, 1990; Liu, Ramani, Soto, De Arcangelis, & Xiang, 2009). The next fundamental question is what is the mechanism underlying the termination of the b2AR-Gi signaling. In this regard, it has been shown that upon GPCR activation, GDP is exchanged for GTP on the Ga subunit, resulting in dissociation of the Ga from Gbg subunits and the activation of downstream effectors. The intrinsic GTPase activity of the a subunit of G proteins serves as a molecular clock, turning down GPCR signaling via returning G proteins to the GDPbond heterotrimeric form. Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) which accelerate GTPase-mediated hydrolysis of GTP to GDP on Ga, thus reconstituting the heterotrimeric G protein complex and terminating G protein signaling (Hollinger & Hepler, 2002; Ross & Wilkie, 2000).

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The small RGS B/R4 subfamily members, RGS2-5, are the predominant RGS proteins expressed in the cardiovascular system (Hao, Michalek, Zhang, Zhu, Xu, & Mende, 2006; Riddle, Schwartzman, Bond, & Insel, 2005). In the heart of mammalian species, RGS2-5 are abundantly expressed (Riddle et al., 2005). RGS3-5 equally regulate both Gai/o and Gaq/11(Hao et al., 2006), whereas cardiomyocyte RGS2 displays selectivity for Gaq/11 (Hao et al., 2006; Heximer, Watson, Linder, Blumer, & Hepler, 1997; Zhang, Watson, Zahner, Rottman, Blumer, & Muslin, 1998). In particular, RGS2 can negatively regulate the signaling of Gq-coupled receptors, including a1A-adrenergic receptor, angiotensin II receptor 1A, and interleukin receptor (Hao et al., 2006; Zou, Roy, Zhao, Kirshenbaum, Karmazyn, & Chidiac, 2006). Deregulation of RGS2 has been implicated in the pathogenesis of cardiac hypertrophy and hypertension (Wieland, Lutz, & Chidiac, 2007). It has been recently reported that RGS2 can physically interact with b2AR (Roy, Lemberg, & Chidiac, 2003), in addition to Gq-coupled GPCRs such as M1 muscarinic receptor (Bernstein et al., 2004) and a1AAR (Hague, Bernstein, Ramineni, Chen, Minneman, & Hepler, 2005). More importantly, our recent studies have provided multiple lines of evidence to demonstrate that RGS2 is a primary terminator of the b2AR-Gi signaling. These include (a) prolonged absence of agonist stimulation for 24 h impairs the b2ARGi signaling, resulting in enhanced b2AR- but not b1AR-mediated contractile response in cultured adult mouse cardiomyocytes; (b) increased b2AR contractile response is accompanied by a selective upregulation of RGS2 in the absence of alterations in other major cardiac RGS proteins (RGS3-5) or Gs, Gi or bAR subtypes; (c) administration of a bAR agonist, ISO, prevents RGS2 upregulation and restores the b2AR-Gi signaling in cultured cells; (d) RGS2 ablation, similar to bAR agonist stimulation, sustains the b2AR-Gi signaling in cultured cells, whereas adenoviral overexpression of RGS2 suppresses agonist-activated b2AR-Gi signaling in cardiomyocytes and HEK293 cells (Chakir et al., 2011). Thus, RGS2 functions as a novel negative regulator of the b2AR-Gi signaling. Since RGS2 constitutively binds to b2AR (Roy et al., 2003), it is reasonable to assume that the b2AR-coupled Gi signaling is constitutively suppressed by RGS2 under physiological conditions, leading to apparent Gs-predominant b2AR signaling in cardiac myocytes of most mammalian species except mouse (Xiao, Cheng, Zhou, Kuschel, & Lakatta, 1999; Xiao et al., 2006). These findings have revealed a novel negative regulation of b2AR-activated Gi signaling by RGS2, and suggest that bAR agonist-induced switch of b2AR signaling from Gs to Gi pathway is mediated, at least in part, by agonist-induced downregulation of RGS2 protein. The selective upregulation of RGS2 and the concurrent augmentation of b2AR contractile response induced by the lack of bAR stimulation in cultured myocytes are intriguing given the fact that b-blocker therapy can resensitize bAR-mediated contractile support and improve cardiac function in patients with

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HF. The beneficial effects of b-blockers might be mediated, in part, by increasing RGS2 expression. In contrast, adrenergic overdriving, as is the case in hypertension and cardiac hypertrophy in different animal models, is accompanied by a selective downregulation of RGS2 (Heximer et al., 2003; Wang et al., 2008; Zhang et al., 2006). In this regard, recent studies have shown that RGS2 gene silencing blocks a1AR-induced cardiac myocyte hypertrophy (Hao et al., 2006), and that pressure overload by trans-aortic constriction results in enhanced Gq signaling, exacerbated cardiac hypertrophy, HF, and premature death in RGS2-deficient mice as compared to wild type counterparts (Takimoto et al., 2009), implying that RGS2 may play a central role in protecting the heart against stress-induced maladaptive remodeling. Thus, under various pathological circumstances, downregulation or malfunction of RGS2 is expected to enhance Gq- and Gi-mediated signaling, constituting a pathogenic element for the development of HF in addition to its known role in the pathogenesis of hypertension (Tsang, Woo, Zhu, & Xiao, 2010).

V. LIGAND-DIRECTED SELECTIVE ACTIVATION OF b2 AR-COUPLING TO G s OR G i It is now well established that any given ligand for a GPCR does not simply possess a single defined efficacy. Rather, a ligand possesses multiple efficacies, depending on the specific down-stream signal transduction pathway analyzed. This diversity reflects ligand-specific GPCR conformations and is often referred to as ‘‘Functional Selectivity.’’ It has been known for a century that stereoisomers of catecholamines differ in their potency and efficacy. However, the molecular basis for differences in efficacy of GPCR ligand stereoisomers has remained poorly defined till now. b2AR couples dually to Gs and PTX-sensitive Gi proteins, resulting in functionally opposing effects on cardiomyocyte contractility. A fundamental question regarding receptor–G protein interaction is, therefore, whether different agonists can traffic a receptor to different intracellular signaling pathways. Recent studies have demonstrated, while most b2AR agonists activate both Gs and Gi, fenoterol selectively activates Gs (Xiao et al., 2003). Furthermore, we have synthesized all fenoterol stereoisomers, including R,R-, R,S-, S,R-, and S,S-forms, and found that the R,R-fenoterol fails to activate Gi signaling, as evidenced by the absence of PTX-sensitivity of its contractile response and its inability to activate Gi-dependent ERK1/2 signaling, but S,Rfenoterol exhibits a robust PTX-sensitivity in these responses, suggesting that the S,R-isomer enables b2AR to activate both Gs and Gi (Woo et al., 2009). The same conclusion holds true for some fenoterol derivatives. For instance, S,Rmethoxy-fenoterol, but not R,R-methoxy-fenoterol, activates b2AR-coupled Gi signaling in cardiomyocytes (Woo et al., 2009). Thus, in addition to b2AR

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phosphorylation, stereoisomers of an agonist can direct b2AR to different G protein(s). This finding is important because it is the first account to show that even the subtle chemical differences within a ligand stereoisomer pair are sufficient to stabilize GPCR conformations with distinct G-protein coupling properties, highlighting how important it is to carefully examine both the ‘‘active’’ and the ‘‘inactive’’ stereoisomer to understand the exact mechanism of action and cellular effects of a GPCR ligand. This finding may also have broad reaching implications in GPCR biology and signaling pathway-targeted drug development (Seifert & Dove, 2009).

VI. DEVELOPMENT OF b2AR AGONISTS INTO NEW DRUGS FOR THE TREATMENT OF HEART FAILURE A hallmark of HF is the desensitization of bAR signaling, characterized by downregulation of bAR, reduced signaling efficient of remaining receptors, increased Gi signaling, and elevated circulating catecholamine levels. However, HF-associated loss of bAR is selective for b1AR, with little change in b2AR. Previous studies have demonstrated that (a) b2AR dually couples to the Gi and the Gs signaling pathways in the heart with the Gi coupling negating the Gs-mediated contractile response, whereas b1AR couples solely to the Gs signaling cascade (Kilts et al., 2000; Xiao, 2001; Xiao et al., 1994, 2003); (b) b1AR and b2AR stimulation oppositely regulate cardiomyocyte viability and myocardial remodeling with b1AR detrimental and b2AR protective (Bisognano et al., 2000; Engelhardt et al., 1999; Liggett et al., 2000; Patterson et al., 2004; Zhu et al., 2001); and (c) b1AR blockade exhibits salutary effects on patients with HF, thus becoming a major therapy in the treatment of HF (Bristow, 2000; Sabbah, 1999). Our recent research focus is to translate bAR subtype signaling principles into drug development and novel therapies to improve the structure and function of the failing heart. Specifically, we have tested the hypothesis that Gs-selective activation of b2AR alone or in combination with clinically used b1AR blockers might provide a potential novel therapy with greater efficacy and fewer side effects for HF.

A. Signaling-Selective b2AR Agonists for the Treatment of Heart Failure In HF, impaired bAR response is often associated with increased Gi signaling and selective downregulation of b1AR (higher b2/b1 ratio) (Bristow et al., 1986; Eschenhagen et al., 1992). We have demonstrated that inhibition of Gi with PTX restores the markedly depressed b2AR contractile response in myocytes from the failing heart of the rat (Xiao et al., 2003). Further, we have identified a

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unique b2AR agonist, fenoterol, which selectively activates b2AR-Gs signaling, bypassing the Gi pathway, fully restores the diminished b2AR inotropic effect in myocytes from failing spontaneously hypertensive rat (SHR) hearts in the absence of PTX (Xiao et al., 2003). This suggests that selective activation of the b2AR-Gs signaling may provide a useful therapeutic target for the treatment of HF. Follow-up in vivo studies have further demonstrated that prolonged use of fenoterol not only improves cardiac function, but also retards cardiac maladaptive remodeling, and that the overall beneficial effects of fenoterol are greater than the salutary effects of b1AR blockade in a rat HF model (Ahmet, Krawczyk, Heller, Moon, Lakatta, & Talan, 2004; Ahmet, Lakatta, & Talan, 2005). Specifically, the effectiveness of the b2AR agonist in attenuating left ventricle (LV) dilatation, functional decline, and myocyte apoptosis significantly exceeds that of the clinically sued b1AR blocker, metoprolol (Ahmet et al., 2004). Since fenoterol and its derivatives can increase myocardial contractility in vivo and delay the development of HF in the rat ischemic HF model, we envision some of the promising new Gs-signaling selective b2AR agonists may be developed into drugs to improve the structure and function of the failing heart. The therapeutic effect of b2AR stimulation has been recently confirmed in a rat model of autoimmune myocarditis (Nishii et al., 2006).

B. A Combination of b2AR Activation with b1AR Blockade Provides A More Effective Therapy for Heart Failure In the final analysis a main measure of therapeutic efficacy of the treatment for HF is its effect on mortality. In recent in vivo studies, we have compared the efficacy of a combined therapy with a b1AR blocker and a b2AR agonist (fenoterol plus metoprolol) with that of a single therapy of either the b1AR blocker or the b2AR agonist, in terms of animal mortality as a primary outcome. We have found that the beneficial effects of the combined therapy on animal survival, cardiac remodeling and function exceed the effects of either single therapy (Ahmet et al., 2005, 2008). Since standard therapy for advanced HF includes RAS inhibition, we have also compared the combined (fenoterol plus metoprolol) therapy with a combination of b1AR blocker and angiotensin-converting enzyme inhibitor, and found that the combined therapy of the b1AR blocker and the b2AR agonist is equally effective compared to the standard therapy for HF with respect to mortality and exceeds the latter with respect to cardiac remodeling and myocardial infarction (MI) expansion (Ahmet et al., 2008). Altogether, recent in vivo studies have revealed that in the rat HF model, b2AR activation alone or in combination with a b1AR blocker or a RAS

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inhibitor is superior to b1AR blockade alone in preventing MI expansion, improving animal survival, and attenuating LV maladaptive remodeling, contractile dysfunction, myocyte apoptosis, and arrhythmia (Ahmet et al., 2008). These in vivo studies have provided a proof of principle for the combined therapy of b2AR agonists with the clinically used b1AR blockers or RAS inhibitors for the treatment of HF.

VII. FUTURE PERSPECTIVE Studies over the past decade have greatly enriched our understanding of bAR subtype-specific signal transduction and biological functions in normal and disease conditions, but also raised many perplexing questions regarding b2AR signaling and the potential interaction between bAR subtypes in the failing heart. First, if the b2AR-coupled Gi signaling is inactive during b2AR stimulation with R,R-isomers of fenoterol and its derivatives, what is the mechanism underlying their prosurvival effects in cardiomyocytes and the beneficial effects of fenoterol in vivo? Second, what is the mechanism by which enhanced b2AR-Gi signaling cross-inhibits b1AR-mediated contractile support in the failing heart? In other words, how do b1AR and b2AR interact with each other in the failing heart? To address these important questions, it is necessary to systematically characterize bAR subtype signaling complexes and dissect their signaling circuitries at the gene, protein, and cellular levels using interdisciplinary approaches, and integrate what we have learned into better understanding of how they function in the whole animal level. These mechanistic studies will greatly expand our understanding of subtype-specific bAR signaling in special and GPCR biology in general. As another frontline of future directions, extensive efforts should be continuously invested into translating bench discoveries into clinic use. References Ahmet, I., Krawczyk, M., Heller, P., Moon, C., Lakatta, E. G., & Talan, M. I. (2004). Beneficial effects of chronic pharmacological manipulation of b-adrenoreceptor subtype signaling in rodent dilated ischemic cardiomyopathy. Circulation, 110(9), 1083–1090. Ahmet, I., Krawczyk, M., Zhu, W., Woo, A. Y., Morrell, C., & Poosala, S., et al., (2008). Cardioprotective and survival benefits of long-term combined therapy with b2-adrenoreceptor (AR) agonist and beta1 AR blocker in dilated cardiomyopathy postmyocardial infarction. J Pharmacol Exp Ther, 325(2), 491–499. Ahmet, I., Lakatta, E. G., & Talan, M. I. (2005). Pharmacological stimulation of b2-adrenergic receptors (b2AR) enhances therapeutic effectiveness of b1AR blockade in rodent dilated ischemic cardiomyopathy. Heart Fail Rev, 10(4), 289–296. Antos, C. L., Frey, N., Marx, S. O., Reiken, S., Gaburjakova, M., & Richardson, J. A., et al., (2001). Dilated cardiomyopathy and sudden death resulting from constitutive activation of protein kinase a. Circ Res, 89(11), 997–1004.

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