Emerging concepts and therapeutic implications of β-adrenergic receptor subtype signaling

Pharmacology & Therapeutics 108 (2005) 257 – 268 www.elsevier.com/locate/pharmthera

Associate editor: P. Molenaar

Emerging concepts and therapeutic implications of h-adrenergic receptor subtype signaling Ming Zhenga,b, Weizhong Zhuc, Qide Hana,b, Rui-Ping Xiaoa,b,c,* a

Institute of Cardiovascular Sciences, Peking University, Beijing 100083, People’s Republic of China b Institute of Molecular Medicine, Peking University, Beijing 100871, People’s Republic of China c Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, NIH, Baltimore, MD 21224, USA

Abstract The stimulation of h-adrenergic receptor (hAR) plays a pivotal role in regulating myocardial function and morphology in the normal and failing heart. Three genetically and pharmacologically distinct hAR subtypes, h1AR, h2AR, and h3AR, are identified in various types of cells. While both h1AR and h2AR, the predominant hAR subtypes expressed in the heart of many mammalian species including human, are coupled to the Gs – adenylyl cyclase – cAMP – PKA pathway, h2AR dually activates pertussis toxin-sensitive Gi proteins. During acute stimulation, h2AR – Gi coupling partially inhibits the Gs-mediated positive contractile and relaxant effects via a Gi – Ghg – phosphoinositide 3kinase (PI3K)-dependent mechanism in adult rodent cardiomyocytes. More importantly, persistent h1AR stimulation evokes a multitude of cardiac toxic effects, including myocyte apoptosis and hypertrophy, via a calmodulin-dependent protein kinase II (CaMKII)-, rather than cAMP – PKA-, dependent mechanism in rodent heart in vivo and cultured cardiomyocytes. In contrast, persistent h2AR activation protects myocardium by a cell survival pathway involving Gi, PI3K, and Akt. In this review, we attempt to highlight the distinct functionalities and signaling mechanisms of these hAR subtypes and discuss how these subtype-specific properties of hARs might affect the pathogenesis of congestive heart failure (CHF) and the therapeutic effectiveness of certain h-blockers in the treatment of congestive heart failure. D 2005 Published by Elsevier Inc. Keywords: G protein-coupled receptors; h-Adrenergic receptor subtypes; h-Adrenergic receptor polymorphism; G proteins; PKA; CaMKII; PI3K; Apoptosis; Hypertrophy; Congestive heart failure; h-Blocker therapy

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subtype-specific functionalities and signaling properties of h-adrenergic receptors in the heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Distinct signaling properties of cardiac h1AR and h2AR revealed from pharmacological and genetic approaches . . . . . . . . . . . . . . . . . 2.2. Dual coupling to Gi and Gs of h2AR but not h1AR . . . . . . . . . . . 2.3. Switch from PKA- to calmodulin-dependent protein kinase II-dependent signaling during sustained h1AR activation . . . . . . . . . . . . . . . 2.4. Persistent h2AR stimulation protects cardiomyocytes by a Gi – phosphoinositide 3-kinase – Akt dependent mechanism . . . . . . . . . 2.5. Heterodimerization of h1AR and h2AR . . . . . . . . . . . . . . . . .

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* Corresponding author. Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA. Tel.: 410 558 8662; fax: 410 558 8150. E-mail address: [email protected] (R.-P. Xiao). 0163-7258/$ - see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.pharmthera.2005.04.006

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3.

Clinical implications of h-adrenergic receptor subtype signaling . . . . . . . . . . 3.1. Possible causal relation between h-adrenergic receptor subtype signaling and the pathogenesis of heart failure . . . . . . . . . . . . . . . . . . . . 3.2. Therapeutic implications of subtype-specific h-adrenergic receptor signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Potential impact of h-adrenergic receptor subtype polymorphisms on the development of CHF . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Enhancing cardiac protective h2AR signaling as a potential therapy for congestive heart failure . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Up-regulation of h3AR in congestive heart failure . . . . . . . . . . . . . 4. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction h-adrenergic receptor (hAR) stimulation by the sympathetic nervous system or circulating catecholamines regulates a wide range of biological processes, from heart pacemaker activity, myocardial contractility, and relaxation to vascular and bronchia smooth muscle tone, to glucose and lipid metabolism. hARs belong to G protein-coupled receptors (GPCRs) characterized by a conserved structural topology of 7-transmembrane domains (7TM) with an extracellular N-terminus and an intracellular C-terminus. In 1967, Lands et al. (1967) classified hARs into h1AR and h2AR as cardiac/lipolytic and vascular/bronchial subtypes, respectively, based on the rank order of potency of a series of structurally related catecholamines, including epinephrine and norepinephrine in different tissues. Over the past 4 decades, the accumulation of a wealth of knowledge of hAR subtype entities and their signaling mechanisms has rendered the hAR as a model system of the GPCR superfamily. Beginning in the early 1980s, Lefkowitz and colleagues developed a series of techniques to purify and reconstitute adrenergic receptors. In 1986, they successfully cloned the gene and cDNA encoding the hamster h2AR (Dixon et al., 1986). This important breakthrough paved the road for the field of GPCR biology. In the next several years, many GPCRs were cloned, including human h1AR (Frielle et al., 1987) and human h3AR (Emorine et al., 1989). The human h1AR and h2AR share 54% homology in their amino acid sequences, while h3AR shares 51% and 46% homology with h1AR and h2AR amino acid sequences, respectively. Although cardiac hAR was initially thought to be h1AR, radioligand binding or pharmacological assays revealed the existence of cardiac h2AR and h3AR. There is now compelling evidence indicating that the predominant hAR subtypes expressed in the heart are h1AR and h2AR in many mammalian species, including human (Brodde, 1988, 1991). These hAR subtypes fulfill different, even opposite, physiological and pathophysiological roles via activating subtype-specific signaling pathways in

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the heart. These characteristics of cardiac hAR subtypes bear important etiological and therapeutic implications for congestive heart failure (CHF).

2. Subtype-specific functionalities and signaling properties of B-adrenergic receptors in the heart 2.1. Distinct signaling properties of cardiac b1AR and b2AR revealed from pharmacological and genetic approaches Cardiac hAR signaling and functional properties were traditionally studied using pharmacological assays with selective agonists and antagonists. In particular, using highly selective h1 AR and h2AR antagonists, CGP 20712A (Dooley et al., 1986; Baker, 2005) and ICI 118,551 (O’Donnell & Wanstall, 1980; Baker, 2005), respectively, in conjunction with radioligand binding with [125I]-Cyanopindolol, the densities of these hAR subtypes have been characterized in normal and failing hearts of human and animal models. In human ventricle, the h1AR constitutes 70– 80% of the total hAR complement, while the h2AR constitutes the remaining 20– 30% (Brodde, 1991). In rat ventricular myocytes, the h2AR proportion (30 – 40%) is relatively greater than that in human heart (Xiao et al., 1998). There are a multitude of qualitative and quantitative differences between these hAR subtypes in their functional roles and signaling pathways. For instance, we and others have demonstrated that in rodent and canine hearts, h1ARactivated cAMP signaling increases the phosphorylation of sarcolemmal L-type Ca2+ channels and a multitude of intracellular regulatory proteins, including sarcoplasmic reticulum (SR) protein phospholamban (PLB) and myofilaments (troponin I [TnI] and C protein; Xiao et al., 1994; Kuschel et al., 1999a, 1999b). h2AR-mediated cAMP signaling, however, specifically modulates the Ca2+ channel without affecting the aforementioned intracellular targets in cardiomyocytes of those mammalian species (Xiao et al., 1994; Kuschel et al., 1999a, 1999b). Although h2AR

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1999). These studies on hAR subtype KO mice imply that h1AR plays a predominant role in catecholamine-mediated regulation of heart rate and myocardium contractility, even though h2AR constitutes 20 –30% of the total cardiac hARs. In this regard, hAR-induced positive contractile response in human heart is also predominantly mediated by h1AR stimulation, although h2AR causes a significant positive inotropic effect (Brodde, 1991; Kaumann et al., 1996, 1999). Altogether, pharmacological and genetic manipulations of the h1AR or h2AR system not only reveal distinctly different functional roles of these hAR subtypes in the heart, but also raise a fundamental question as to why h1AR and h2AR are coexpressed in cardiomyocytes if h1AR is sufficient to fulfill h-adrenergic modulation of cardiac performance. 2.2. Dual coupling to Gi and Gs of b2AR but not b1AR The failure of h2AR to produce a proportional contractile response in the heart in rodent and canine hearts is mainly because of a crucial difference between h1AR and h2AR in their G protein coupling. In 1995, we demonstrated that stimulation of h2AR but not h1AR activates Gi proteins in adult rat cardiac myocytes, while both hAR subtypes are able to stimulate the classic Gs – AC –cAMP– PKA signaling pathway (Fig. 1; Xiao et al., 1995). This notion was confirmed in HEK293 cells in 1997 (Daaka et al., 1997), more importantly, in the human heart in 2000 (Kilts et al., 2000). The additional Gi coupling qualitatively and quantitatively modifies the outcome of the Gs signaling, while exhibiting important cardiac protective effects (see below). PTX

β3AR

Gi

Gs

PTX

NOS

NO

?

AC

Gi Gs

β2AR

Giβγ

PI3K

AC

AC

Ca2+

β1AR

Akt

Cell Survival

Contractility, Relaxation, Heart rate

P cAM PKA

Gs

stimulation in the human heart is able to increase cAMP formation and PKA-dependent phosphorylation of intracellular regulatory proteins (PLB, TnI, and C protein), for a given elevation in cAMP production or PKA activation, the h2AR-induced protein phosphorylation or positive inotropic effect is significantly smaller compared with that induced by h1AR stimulation (Kaumann et al., 1996, 1998, 1999). In rat and canine cardiomyocytes, h2AR-mediated augmentation in cAMP is apparently dissociated with its contractile response (Xiao et al., 1994; Altschuld et al., 1995; Kuznetsov et al., 1995; Zhou et al., 1997). More ironically, in mouse cardiomyocytes, h2AR-induced cAMP formation excites a very minor positive inotropic effect (Xiao et al., 1999). Thus, h2AR-stimulated cAMP signaling is functionally compartmentalized, while h1AR-activated cAMP signaling is more diffusible in the cardiac myocytes of many mammalian species, such as human, rat, mouse and dog. Recent advances in transgenic and gene targeted knockout mouse models, together with in vivo and in vitro physiological and pharmacological measurements, have provided well-controlled experimental systems to further dissect the signaling and functional roles of each hAR subtype in regulating myocardial morphology and functional performance in the normal and failing heart. Among many elegant studies, the work by Milano et al. (1994) has attracted considerable attention in the field of cardiovascular biology and medicine. Cardiac-specific overexpression of the human h2AR at 100- to 200-fold in mice markedly enhanced cardiac contractility even in the absence of hagonist, without obvious pathological consequence at the age of 12 months (Milano et al., 1994). This observation was initially interpreted to indicate that the overexpression of hAR might hold great promise to treat heart failure via enhancing the contractility of the failing heart (Milano et al., 1994). Five years later, however, studies in mice with modest (¨ 15-fold) cardiac-specific overexpression of the human h1AR revealed strikingly different phenotypes, including dilated cardiomyopathy, severe cardiac remodeling, and premature death (Engelhardt et al., 1999; Bisognano et al., 2000). These phenotypic characteristics are similar to those in transgenic mice with cardiac-specific overexpression of Gas (5-fold; Iwase et al., 1996), but markedly different from those, discussed above, in the transgenic mice overexpressing cardiac h2AR (Milano et al., 1994). Although studies on hAR transgenic models are fruitful, the overexpression of an individual hAR subtype might cause multiple compensatory alterations in the animal, thus complicating the interpretation. To better dissect the distinct functional and signaling properties of h1AR versus those of h2AR, Kobilka and colleagues developed gene-targeted mice lacking h1AR (h1AR KO) or h2AR (h2AR KO) or both (h1h2 DKO; Rohrer et al., 1996, 1999; Chruscinski et al., 1999). Remarkably, catecholamine stimulation fails to induce any cardiac inotropic or chronotropic response in h1AR KO mice (Rohrer et al., 1996), whereas it produces a full cardiac response in h2AR KO mice (Chruscinski et al.,

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cAMP

PKA CaMKII

Apoptosis, Hypertrophy

Fig. 1. Subtype-specific signaling pathways of cardiac hARs. The dual coupling of h2AR to Gi proteins activates the Gi – Ghg – PI3K – Akt pathway, which, in turn, leads to functional localization and inhibition of the Gs – AC – cAMP – PKA signaling and protects cardiomyocytes against apoptosis. In contrast, h1AR couples exclusively to Gs, which activates the Gs – AC – cAMP – PKA pathway, resulting in positive inotropic and relaxant effects. The newly identified, PKA-independent activation of CaMKII is necessary and sufficient in mediating persistent h1AR stimulation-induced myocyte apoptosis, perhaps cardiac hypertrophy as well. Emerging evidence suggests that cardiac h3AR might also couple to Gi, in addition to Gs, resulting in the activation of the NOS – NO signaling pathway, which negatively regulates cardiac contractility (PTX, pertussis toxin).

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During acute receptor stimulation, the h2AR – Gi coupling activates phosphoinositide 3-kinase (PI3K), which, in turn, mediates compartmentalization of the concurrent Gs-cAMP signaling, thereby negating h2AR-induced PLB phosphorylation and the positive inotropic and lusitropic responses in rat and canine ventricular myocytes (Kuschel et al., 1999a, 1999b; Jo et al., 2002; Fig. 1). During prolonged receptor stimulation, the h2AR –Gi coupling evokes multiple Gsindependent signaling pathways, including the activation of MAPKs (Daaka et al., 1997; Communal et al., 2000; Shizukuda & Buttrick, 2002) and PI3K – Akt (Chesley et al., 2000; Zhu et al., 2001), resulting in cardiac protective effects (Fig. 1). It has been shown that PKA-dependent h2AR phosphorylation switches the receptor G protein coupling from Gs to Gi in HEK 293 cells (Daaka et al., 1997) and rat cardiac myocytes (Zou et al., 1999), which might desensitize the receptor-mediated inotropic response while enhancing its cardiac protective effect during prolonged agonist stimulation. 2.3. Switch from PKA- to calmodulin-dependent protein kinase II-dependent signaling during sustained b1AR activation The traditional view on cardiac hAR signal transduction is that, under physiological conditions, catecholamines induce positive inotropic, lusitropic, and chronotropic responses through the h1AR-mediated activation of the Gs – AC – cAMP – PKA pathway (Fig. 1). Subsequently, PKA leads to the phosphorylation of a panel of key target proteins involved in cardiac excitation – contraction coupling, including sarcolemmal L-type Ca2+ channels, the sarcoplasmic reticulum membrane protein PLB, and some contractile myofilament proteins such as troponin I (TnI) and C protein. Pharmacological studies have suggested that h1AR-induced cardiomyocyte apoptosis is also likely mediated by the cAMP – PKA pathway (Communal et al., 1999; Zaugg et al., 2000). Thus, it was thought that the functional consequences of either acute or persistent h1AR activation might be exclusively mediated by the classic Gs – AC –cAMP –PKA signaling cascade. This perception has been, however, challenged by more recent studies. In particular, persistent h1AR activation augments myocyte contractility and intracellular Ca2+ transients via Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling, independently of the cAMP –PKA pathway in adult rat cardiac myocytes (Fig. 1). This emerging h1AR signaling paradigm is based on following lines of evidence. First, the inhibition of the PKA pathway cannot block sustained h1 AR stimulation-mediated increases in myocyte contraction and Ca2+ transients, whereas the inhibition of CaMKII activation by specific inhibitors or a dominant-negative mutant fully abolishes the effects of sustained h1AR stimulation without affecting those excited by acute h1AR activation (Wang et al., 2004). In contrast, the inhibition of PKA but not CaMKII prevents

acute h1AR signaling-mediated positive inotropic and lusitropic effects (Wang et al., 2004). Second, tracking cAMP production and CaMKII activation over an extended time course (24 hr) reveals that a progressive and persistent CaMKII activation is accompanied by a rapid desensitization of the cAMP – PKA signaling, consistent with the previous notion (Hausdorff et al., 1990) indicating that h1AR signaling undergoes a time-dependent switch from the PKA-dominant pathway to the CaMKII-dominant pathway after receptor stimulation (Wang et al., 2004; Fig. 1). Likewise, sustained h1AR stimulation markedly increases cardiomyocyte apoptosis via a CaMKII-, rather than PKA-, dependent mechanism (Fig. 1). In 2 genetically welldefined h1AR experimental settings, that is, myocytes from h2AR KO mice or those from h1h2 DKO in conjunction with adenoviral gene transfer of h1AR, the apoptotic effects of persistent h1AR stimulation are fully prevented by inhibiting CaMKII activity, but not by blocking PKA activation (Zhu et al., 2003). Furthermore, the overexpression of a cardiac CaMKII isoform, CaMKII-yC, markedly aggravates the h1AR-induced apoptotic death of cardiomyocytes (Zhu et al., 2003). Our recent studies have shown that enhanced CaMKII activation is both necessary and sufficient for h1AR-mediated apoptotic heart cell death. Moreover, h1AR-activated CaMKII signaling, instead of the PKA pathway, is essentially involved in CHF-associated fetal gene expression (Bristow et al., personal communication) and catecholamine-induced cardiomyocyte hypertrophy (Morisco et al., 2000). Thus, the time-dependent h1AR signaling switch from the PKA-dominant pathway to the CaMKII-dominant pathway underscores that CaMKII constitutes a determinant of clinically important heart disease phenotypes, and that CaMKII inhibition might offer a promising approach for targeting adverse myocardial remodeling in the context of enhanced h1AR signaling, as is the case for CHF. This perception has been recently validated by the fact that CaMKII inhibition via cardiacspecific overexpression of a CaMKII peptide inhibitor in transgenic mice significantly prevents maladaptive remodeling and cardiac contractile dysfunction from excessive hAR stimulation or myocardial infarction (Zhang et al., 2005). The notion that enhanced CaMKII activation mediates the detrimental effects of chronically elevated hAR signaling is consistent with the phenotypes of heart failure and premature death in transgenic mice overexpressing cardiac CaMKII-yC (Zhang et al., 2003) and is supported by a central role of CaMKII signaling pathway in insulting factor-induced apoptosis in several naive cell lines (Wright et al., 1997; Fladmark et al., 2002). In supporting the cardiac toxic effects of persistent h1AR signaling, levels of plasma norepinephrine, an endogenous h1AR agonist, and an active anti-h1AR antibody (Limas et al., 1989) have been directly associated with CHF mortality in humans (Hagar & Rahimtoola, 1995). Experimentally elevating the active anti-h1AR antibody is sufficient to trigger cardiomyopathy and CHF in animal models (Jahns et al.,

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2004). Despite many unanswered questions with respect to the exact signaling pathways responsible for h1AR-mediated cardiac toxic effects, emerging evidence points to h1ARactivated CaMKII as a major player in the progress of cardiac pathological remodeling, particularly myocyte hypertrophy and apoptosis, and ultimately contributing to myocardial contractile dysfunction and the progression of CHF. It remains highly controversial as to whether chronic activation of the cAMP – PKA pathway causes cardiac detrimental consequences. For instance, enhanced signaling of the AC – cAMP – PKA pathway by overexpressing adenylyl cyclase type V or VI does not produce CHF, but unexpectedly alleviates CHF in the Gaq-overexpressing mouse (Gao et al., 1999; Tepe et al., 1999; Roth et al., 2002). More ironically, enhanced h2AR stimulation by agonists or modest overexpression of the receptor, while marked elevating cAMP –PKA signaling, exhibits beneficial effects in several CHF models (Dorn et al., 1999; Liggett et al., 2000; Ahmet et al., 2004). Nevertheless, the cardiacspecific overexpression of PKA catalytic subunit by as modest as 2.4-fold or the overexpression of Gas by 5-fold leads to CHF phenotype in mice (Antos et al., 2001; Vatner et al., 2000). These paradoxical observations challenge the conventional wisdom that the cAMP – PKA pathway is solely responsible for h1AR-induced cardiac detrimental effects. Resolving these paradoxes should reveal valuable etiological insights and novel therapeutic targets for CHF. 2.4. Persistent b2AR stimulation protects cardiomyocytes by a Gi – phosphoinositide 3-kinase – Akt dependent mechanism The aforementioned studies have provided compelling evidence that persistent h1AR activation is toxic to cardiomyocytes in vivo and in vitro. However, the scenario of persistent h2AR stimulation is strikingly different. In particular, the stimulation of h2AR in adult mouse cardiomyocytes lacking native h1AR protects myocytes against catecholamine-induced myocyte apoptosis in vivo (Zhu et al., 2001; Patterson et al., 2004). These studies performed in the ‘‘genetically pure h2AR systems’’ are, in principle, consistent with earlier pharmacological studies that the blockade of h1AR but not h2AR prevents catecholamine-, hypoxia-, or reactive oxygen species (ROS)-induced apoptotic death in both neonatal and adult rat cardiomyocytes (Communal et al., 1999; Chesley et al., 2000; Zaugg et al., 2000). The cardiac beneficial effects of persistent h2AR signaling have been further confirmed in recent in vivo studies: Selective activation of h2AR by fenoterol for 8 weeks exerts a clear antiapoptotic effect and improves cardiac performance in an ischemic rat heart failure model (Ahmet et al., 2004). The opposite effects of h1AR and h2AR on the fate of cardiomyocytes might contribute, at least in part, to the markedly different outcomes of genetic manipulations of these hAR subtypes in mouse genetic models (Milano et al., 1994; Dorn et al., 1999; Engelhardt et al., 1999; Liggett et al., 2000; Bisognano et al., 2000).

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The cardiac protective effect of persistent h2AR signaling is largely mediated by the h2AR – Gi coupling, which, in turn, activates a cell survival pathway sequentially involving Gihg, PI3K, and Akt (Fig. 1). This perception is corroborated by following lines of evidence. First, h2AR, but not h1AR, activates a Gi –Ghg – PI3K – Akt cell survival-signaling pathway, and the inhibition of this pathway converts h2AR signaling from survival to apoptotic (Zhu et al., 2001). Second, in cultured adult rat myocytes, h2AR blockade enhances the apoptotic effect of norepinephrine in a PTX-sensitive manner, suggesting that the h2AR protective effect is Gi-dependent (Communal et al., 1999). Third, the effect of h2AR stimulation in protecting cardiomyocytes from hypoxia and ROS is also attributable to the Gi –PI3K –Akt pathway (Chesley et al., 2000). Thus, the h2AR – Gi – Ghg –PI3K –Akt signaling cascade counteracts h1AR-mediated apoptosis and protects myocytes against a wide range of apoptotic insults. The extracellular signal-regulated protein kinases (ERK1/ 2) and p38 MAPK are also implicated in h2AR –Gi-mediated antiapoptotic signaling (Communal et al., 2000; Shizukuda & Buttrick, 2002). We and others have, however, provided evidence that argues against any essential role p38 MAPK in regulating myocyte apoptosis. The activation of p38 MAPKs by the transgenic overexpression of activated mutants of upstream kinases MKK3bE and MKK6bE neither induces nor suppresses cardiomyocyte apoptosis (Liao et al., 2001). Given the fact that p38 MAPK plays an essential role in cardiac hypoxia- and ischemia-induced cardiomyocyte injury and apoptosis (Bogoyevitch et al., 1996; Sugden & Clerk, 1998; Nakano et al., 2000; Zheng et al., 2005), it is unlikely that h2AR-mediated antiapoptotic effect is attributed to p38 MAPK signaling. 2.5. Heterodimerization of b1AR and b2AR Increasing evidence suggests that GPCRs may exist as both homodimers or heterodimers (Hebert et al., 1996; Jones et al., 1998). The physical interaction of GPCRs within or among different families creates new entities endowed with distinctly different ligand-binding and signaling properties. For example, h1AR and h2AR subtypes form heterodimers in HEK 293 cells (Lavoie et al., 2002; Mercier et al., 2002), and the heterodimerization of these hAR subtypes inhibits h2AR internalization and its ability to activate ERK1/2 MAPK signaling (Lavoie et al., 2002). The coexpression of both h1AR and h2AR in HEK293 cells also reduces the high affinity binding of subtype-selective ligands, as compared with the expression of a single subtype in those cells (Lavoie & Hebert, 2003). Because the heterodimerization of h1AR and h2AR alters both the functional and signaling properties of these receptors, the dimeric h1AR/h2AR should be considered as pharmacologically and functionally distinct population of hARs. Thus, many well-established paradigms for hAR signaling and functionality need to be revisited in the

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context of the coexistence of multiple receptor subtypes and their homo- and heterodimers. In addition, it remains to be explored whether h1AR and h2AR undergo heterodimerization in cardiomyocytes and, if so, to determine the potential physiological or pathological relevance of their heterodimerization.

3. Clinical implications of B-adrenergic receptor subtype signaling 3.1. Possible causal relation between b-adrenergic receptor subtype signaling and the pathogenesis of heart failure A possible causal relation between hAR stimulation and the pathogenesis of CHF have evolved progressively during the past 3 decades. Because a hallmark of CHF is the diminished cardiac contractile performance, the early prevalent view was that the reduced hAR contractile support is a causal factor for the progression of heart failure (Braunwald & Bristow, 2000). However, clinical administration of hAR agonists unexpectedly increased the mortality of patients with CHF, despite the short-term gain of inotropic support. Paradoxically, some hAR antagonists were able to improve cardiac function and decrease the mortality of patients with CHF (Bristow, 2000). Furthermore, over the past couple of decades, physician-scientists have appreciated that CHF is, in fact, associated with elevated circulating catecholamine levels and hyperadrenergic drive. There is a close positive relation between levels of circulating catecholamines (especially norepinephrine) and adverse clinical outcomes of CHF patients (Cohn et al., 1984). The infusion of catecholamines causes cardiomyopathy and remodeling, ultimately leading to heart failure in various animal models (Braunwald & Bristow, 2000). The current perception is that hyperadrenergic drive initially activates a multitude of compensatory mechanisms to maintain normal cardiac output; persistent receptor stimulation then causes desensitization and downregulation of hARs, particularly h1AR subtype, and eventually, cardiac pathological remodeling and heart failure (Lohse et al., 2003). This conceptual breakthrough forms the rationale for h-blocker therapy for CHF. As discussed above, the essence of cardiac hAR signaling is subtype specific and time dependent. In this regard, there is a large body of evidence that the cardiac toxic effects of catecholamines are mediated mainly, if not exclusively, by persistent h1AR stimulation. First, the administration of isoproterenol, a hAR agonist, exhibits protective, rather than detrimental, consequences in in vivo or in cultured cardiomyocytes from mice lacking native h1AR (Zhu et al., 2001; Patterson et al., 2004). Second, enhanced h1AR signaling by either receptor overexpression or agonist stimulation consistently evokes cardiac detrimental effects, as manifested by myocyte apoptosis, overall cardiac hypertrophy and fibrosis, and heart failure (Communal et al., 1999; Engelhardt et al.,

1999; Bisognano et al., 2000; Zhu et al., 2003). Third, clinical and basic bench studies have demonstrated that autoimmune antibodies that react and activate h1AR are sufficient to induce cardiomyopathy and CHF in humans and animal models (Limas et al., 1989; Jahns et al., 2004). Furthermore, h1AR selective blockers such as metoprolol and bisoprolol exhibit clear-cut beneficial effects in improving the cardiac function and survival in CHF patients (Bristow et al., 2004; Greenberg, 2004) and animal models (Harding et al., 2001; Morita et al., 2002). Thus, the current consensus is that sustained h1AR stimulation serves as a causal factor of CHF, while h2AR activation may actually be protective for the failing heart. 3.2. Therapeutic implications of subtype-specific b-adrenergic receptor signaling Chronologically, hAR blocking agents were categorized into 3 generations: nonselective hAR blocking agents, for example, propranolol and nadolol; h1AR-selective blocking agents, for example, metoprolol and bisoprolol; and nonselective (a1 and h) adrenergic receptor blocking agents, for example, carvedilol and bucindolol Bristow (Bristow, 2000). Over the last several years, a host of clinical trials have been set out to compare the clinical outcomes of h1AR-selective such as metoprolol and bisoprolol versus a nonselective hAR blocker, carvedilol. Placebo-controlled mortality trials with carvedilol (Metra et al., 1994; Krum et al., 1995; Olsen et al., 1995), bisoprolol (CIBIS-II Investigators and Committees, 1999), and metoprolol (MERITHF Study Group, 1999; Hjalmarson et al., 2000) have demonstrated a long-term reduction in total mortality, cardiovascular mortality, sudden cardiac death, and death due to the progression of CHF in patients with NYHA class II –IV. Interestingly, both h1AR-selective and nonselective h-blockers substantially reduce morbidity and mortality in patients with CHF to a similar extent (Metra et al., 2000; Poole-Wilson et al., 2003). Because h1AR-selective blockers are equally effective in treating CHF, it is plausible that h-blocker therapy for CHF might be mainly mediated by antagonizing h1AR cardiac toxic effects. However, a recently published COMET study has shown a 17% advantage of carvedilol (25 mg twice daily) over immediate-release metoprolol (50 mg twice daily) in reducing the mortality in CHF patients (Poole-Wilson et al., 2003). Despite the long-term and large scope (3029 patients) nature of the COMET trial, this study was inappropriately designed in the choice of dose and dosage regimen, thus significantly compromising its weight in evaluating the effectiveness of h1AR-selective versus that of nonselective h-blockers (Bristow et al., 2004). Multiple mechanisms might be involved in the cardiac beneficial effects of h-blockers in the failing heart. Among these, the delay or amelioration of h1AR-mediated cardiac toxic effects, including myocyte hypertrophy and apoptosis, may play a central role. Other mechanisms have also been

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implicated in h-blocker therapy. These include the restoration of cardiac excitation –contraction coupling and Ca2+ handling machineries (Freeman et al., 2001; Reiken et al., 2001, 2003), anti-ischemic action of h-blockers by reducing heart rate and improving cardiac perfusion and metabolism, and antiarrhythmic effects, thus reducing the incidence of sudden death in CHF patients (Lohse et al., 2003). Nevertheless, the exact mechanism underlying the beneficial effects of h-blockers in improving the function of the failing heart merits further basic and clinical investigations. 3.3. Potential impact of b-adrenergic receptor subtype polymorphisms on the development of CHF Recent progress in human population genetics have revealed an important linkage between certain polymorphisms (occurrence with a frequency >1% in a population) of hAR genes and clinical phenotypes in terms of predisposition, prognosis, and response to h-blocker therapy in patients with CHF. Two common polymorphisms in the h1AR, Ser49Gly and Arg389Gly, and 3 in the h2AR, Arg16Gly, Gln27Glu, and Thr164Ile, have been identified and functionally characterized (Small et al., 2003). In vitro and in vivo studies have shown that most of the polymorphic hARs exhibit either enhanced or reduced signaling efficiency relative to their allelic counterparts. For instance, when overexpressed, the h1AR-Arg389 (a glycine in transgenic mice is substituted by an arginine at amino acid position 389) variant leads to sensitized agonistmediated receptor stimulation in fibroblast cell lines (Mason et al., 1999). Moreover, cardiac-targeted transgenic expression of h1AR-Arg389 in mice exhibits reduced receptor – agonist signaling in activating adenylyl cyclase, abnormal expression of fetal and hypertrophy genes, and decreased cardiac contractility compared with h1AR-Gly389 hearts (Kass, 2003; Mialet Perez et al., 2003). This phenotype is, to a large extent, recapitulated in homozygous, end-stage failing human hearts. Thus, it is speculated that the human h1AR-Arg389 variant predisposes to heart failure because of the hyperactive signaling of the receptor, leading to depressed receptor coupling and ventricular dysfunction. Among the aforementioned 3 h2AR polymorphisms, the Ile164 variant displays markedly blunted signaling efficiency in response to agonist stimulation in vitro (Green et al., 1993) and in the overexpression transgenic mouse model (Turki et al., 1996). It is noteworthy that the prognosis of CHF patients with h2AR-Ile164 is much worse than that of patients with the wild-type h2AR-Thr164 (Liggett et al., 1998), consistent with the general notion that h2AR activation is cardiac protective (Ahmet et al., 2004; Patterson et al., 2004). In light of the opposing functional roles of h1AR and h2AR in regulating cardiomyocyte fate and morphology, it is not surprising to find that the particular case of ‘‘gain-of-function’’ of the h1AR-Arg389 variant, as well as the ‘‘loss-of-function’’ of h2AR-Thr164 variant, is more likely associated with an increased risk of

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progression and worse prognosis of CHF patients. However, it is noteworthy that this is not always the case for all identified h1AR or h2AR polymorphisms (see Kirstein & Insel, 2004; Leineweber et al., 2004, for recent reviews). 3.4. Enhancing cardiac protective b2AR signaling as a potential therapy for congestive heart failure In many types of CHF, the reduction in h1AR and h2AR inotropic responses is often accompanied by an upregulation of Gi (Eschenhagen et al., 1992; Bohm et al., 1994; Xiao et al., 2003) and a selective down-regulation of h1AR (Bristow et al., 1986; Kiuchi et al., 1993; Xiao et al., 2003). Based on above discussions, it is reasonable to assume that the selective down-regulation of h1AR and the up-regulation of h2AR – Gi signaling in the failing heart from humans (Gong et al., 2002; Harding & Gong, 2004) and animal models (Xiao et al., 2003) may represent cardiac adaptive mechanisms to protect myocytes against apoptosis and consequently ameliorate cardiomyopathy and contractile dysfunction. Several studies have provided supporting evidence for this perception. Moderate overexpression of cardiac h2AR (¨ 30-fold) restores ventricular function and cardiac morphology in CHF mice with cardiac-specific overexpression of Gaq (Dorn et al., 1999). Moreover, h2AR stimulation by fenoterol restores the contractile response of myocytes from spontaneous hypertensive rat failing hearts (Xiao et al., 2003) and markedly protects myocardium from ischemia-induced hypertrophy and apoptosis and improves the performance of the ischemic failing heart (Ahmet et al., 2004). However, the exact mechanism underlying fenoterolmediated beneficial effects in the failing heart merits future investigation. In this regard, our preliminary studies have shown that fenoterol, the apparent h2AR – Gs-selective agonist, not only restores the diminished h2AR positive inotropic effect but also exhibits a potent antiapoptotic effect in culture mouse and rat cardiomyocytes in a PTXinsensitive manner (Zhu et al., unpublished data), as is the case in vivo in the rat ischemic heart failure model (Ahmet et al., unpublished data), suggesting that fenoterol protects cardiomyocytes against apoptosis via a Gi-independent mechanism. Furthermore, the cardiac protective effects of chronic h2AR stimulation with fenoterol have synergy with h1AR blockade by metoprolol in the ischemic rat heart failure model (Ahmet et al., unpublished data; also see below). Nevertheless, future studies are required to delineate the mechanism responsible for fenoterol-mediated, Giindependent cardiac protection. It is widely accepted that prolonged agonist exposure leads to a decrease in hAR responsiveness, that is, desensitization. hARK1 (also known as GPCR kinase 2, GRK2) plays a central role in agonist-induced desensitization of both h1AR and h2AR and is crucially involved in the development of various etiologies of heart failure (Williams et al., 2004; Tachibana et al., 2005). Importantly, inhibiting

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hARK1-mediated desensitization of hAR by hARK-ct restores the diminished hAR responsiveness and largely reverses the impaired myocardial contractile performance in mouse heart failure models (Rockman et al., 1998; Harding et al., 2001). Similar results were observed in cultured failing human cardiomyocytes (Williams et al., 2004). The beneficial effects of the inhibition of hARK1 by hARK-ct is largely mediated by enhancing h2AR signaling, because h1AR blockade markedly augments, rather than compromises, the beneficial effects of hARK-ct in improving survival and cardiac performance (Harding et al., 2001). Conversely, the up-regulation of hARK1 precedes and is thought to participate in the pathogenesis of CHF (Ungerer et al., 1994; Anderson et al., 1999; Cho et al., 1999). Consistent with this perception, transgenic mice overexpressing cardiac hARK1 manifest many pathological alterations observed in CHF, including dysfunction of hAR signaling and cardiac hypertrophy (Koch et al., 1995; Korzick et al., 1997). Based on these studies, we envision that a combination of h1AR blockade with h2AR stimulation might open a more effective therapeutic avenue for the treatment of CHF. However, h2AR – Gi signaling offsets h2AR – Gs-induced contractile response in the normal heart (Jo et al., 2002) and negates both h1AR- and h2AR-mediated positive inotropic effects in the failing heart (Sato et al., 2004). Interestingly, the inhibition of Gi signaling or the disruption of the association of hARs with PI3K restores hAR inotropic response in a variety of CHF models (Brown & Harding, 1992; Kompa et al., 1999; Xiao et al., 2003) and markedly ameliorates the development of CHF (Nienaber et al., 2003). Thus, exaggerated h2AR – Gi – PI3K signaling might contribute to the contractile dysfunction of the failing heart, despite its antiapoptotic effect. For the enhancement of h2AR signaling to serve as a potential therapy for CHF, the current challenge is how to retain h2AR – Gi-mediated antiapoptotic effect, while avoiding its negative inotropic effect. Recent studies have demonstrated that CHF-associated increase in h2AR –Gi signaling is responsible for the blunted h1AR- as well as h2AR-mediated contractile support and the overall contractile dysfunction, mainly via activating sarcolemmal Na+/Ca2+ exchanger (NCX) and subsequently depleting sarcoplasmic reticulum Ca2+ load (Sato et al., 2004; Xiao & Balke, 2004). Thus, NCX might represent a more specific downstream target of h2AR signaling for new therapies in the treatment of CHF, via enhancing contractile support without compromising the Gi-PI3K-mediated antiapoptotic effect (Fig. 1). 3.5. Up-regulation of b3AR in congestive heart failure While h3AR is primarily expressed in adipose tissues, it is also expressed in cardiomyocytes. Unlike the stimulation of h1AR and h2AR, the activation of h3AR leads to negative regulation of cardiac contractility via the receptorcoupled Gi signaling (Gauthier et al., 1996), which increases NO production via the activation of endothelial constitutive

NO synthase (NOS) in human cardiac myocytes (Gauthier et al., 1998; Fig. 1). Notably, the expression of h3AR is increased by 2- to 3-fold in CHF (Moniotte et al., 2001; Morimoto et al., 2004). It is presently unknown whether the increased h3AR expression exerts an antiapoptotic effect, similar to that of h2AR, thus protecting the failing heart. On the other hand, the up-regulation of h3AR is expected to cause a greater cardiac contractile depression, thereby contributing to the contractile dysfunction in CHF. The potential pathophysiological relevance of the CHF-associated up-regulation of h3ARs awaits further study.

4. Concluding remarks Recent progress in cardiac hAR signaling is marked by discoveries on subtype-specific and time-dependent signaling mechanisms, receptor heterodimerization, dual G protein coupling, and several new intracellular signaling pathways. These advances have greatly deepened our understanding of the causal relation between hAR stimulation and the pathogenesis of CHF. The distinct h1AR and h2AR biology and signaling properties also shed new light on the clinical variation in the effectiveness of h-blockers and the markedly different impact of hAR subtype polymorphisms on the progression of CHF. The opposing functional roles of h1AR and h2AR in the progression of CHF provide the rationale for a combination of h1AR blockade with h2AR activation as a new prevention and intervention strategy for the treatment of CHF. Acknowledgments The authors would like to thank Dr. H. Cheng for critical comments and discussions. This work is supported by the Chinese National Natural Science Foundation (30100215), Peking University 985 Project, Chinese National Key Project 973 (G2000056906), and Chinese Young Investigator Award (30225036), and, in part, by NIH intramural research grant (WZW and RPX). References Ahmet, I., Krawczyk, M., Heller, P., Moon, C., Lakatta, E. G., & Talan, M. I. (2004). Beneficial effects of chronic pharmacological manipulation of h-adrenoreceptor subtype signaling in rodent dilated ischemic cardiomyopathy. Circulation 110, 1083 – 1090. Altschuld, R. A., Starling, R. C., Hamlin, R. L., Hensley, J., Castillo, L., Fertel, R. H., et al. (1995). Response of failing canine and human heart cells to h2-adrenergic stimulation. Circulation 92, 1612 – 1618. Anderson, K. M., Eckhart, A. D., Willette, R. M., & Koch, W. J. (1999). The myocardial beta-adrenergic system in spontaneously hypertensive heart failure (SHHF) rats. Hypertension 33, 402 – 407. 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, 997 – 1004.

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