Cardiovascular Research

Cardiovascular Research 2004 63(3):391-402; doi:10.1016/j.cardiores.2004.03.011
© 2004 by European Society of Cardiology

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Copyright © 2004, European Society of Cardiology

Network integration of the adrenergic system in cardiac hypertrophy

Liza Barki-Harrington, Cinzia Perrino and Howard A Rockman^*

Departments of Medicine, Cell Biology and Molecular Genetics, Duke University Medical Center, DUMC 3104 226 CARL Building, Research Drive, Durham, NC 27710 USA

^* Corresponding author. Tel.: +1-919-668-2520; fax: +1-919-668-2524. Email address: h.rockman{at}duke.edu

Received 17 January 2004; revised 4 March 2004; accepted 9 March 2004

	Abstract

Top Abstract 1. Introduction 2. Mechanisms of adrenergic... 3. β Adrenergic signaling... 4. {alpha}AR signaling in... 5. Novel paradigms in... 6. Concluding remarks References

 
Adrenergic receptors play a pivotal role in regulating cardiac function in response to a constantly changing environment. Altered and β adrenergic receptor signaling in vivo is associated with cardiac hypertrophy and failure. This review focuses on the different roles of adrenergic receptors in regulating cardiac function under normal and pathological conditions. Understanding the signaling mechanisms of these receptors in the context of the heart is likely to provide a better therapeutic approach towards treatment of heart failure.

KEYWORDS Adrenergic receptors; Heart failure; Cardiac hypertrophy

	1. Introduction

Top Abstract 1. Introduction 2. Mechanisms of adrenergic... 3. β Adrenergic signaling... 4. {alpha}AR signaling in... 5. Novel paradigms in... 6. Concluding remarks References

 
Activation of the sympathetic nervous system in response to a host of normal or disease-related stimuli is essential to maintain homeostasis in a constantly changing environment. The physiological and metabolic responses to sympathetic activation are mediated through the action of the endogenous catecholamines norepinephrine and epinephrine on adrenergic receptors. Based on their pharmacological properties and molecular structure, adrenergic receptors are divided into three subfamilies: 1 adrenergic receptors (1ARs), which include the subtypes 1_A, 1_B and 1_D; 2Ars, which include 2_A, 2_B and 2_C [1]; and β adrenergic receptors (βARs), which include β1, β2 and β3 [2]. Although they respond to the same catecholamines, adrenergic receptors differ significantly in the types of cellular responses they mediate. It is therefore the repertoire and quantity of different adrenergic receptors that determines the overall response of an organ to the circulating catecholamines [2]. Besides controlling cardiac contractile rate and force, both and βARs play a critical role in regulating blood pressure, airway reactivity and metabolic functions.

The heart expresses all three subtypes of βARs [3], as well as three types of 1ARs: 1_A, 1_B and 1_D ARs [4]. Despite the existence of species-related differences (reviewed in Ref. [5]), βARs are the predominant form of adrenergic receptors expressed in the heart, with a ratio of 80:20 of β1 to β2ARs [6]. Expression of 1ARs is much more variable between different species [3] and altogether seems to comprise only a tenth of total βARs [6]. Adult rat ventricular cardiomyoctes contain a 20:80 ratio of 1_A and 1_B ARs (reviewed in Ref. [4]).

	2. Mechanisms of adrenergic receptor signaling in the heart

Top Abstract 1. Introduction 2. Mechanisms of adrenergic... 3. β Adrenergic signaling... 4. {alpha}AR signaling in... 5. Novel paradigms in... 6. Concluding remarks References

 
Adrenergic receptors belong to the superfamily of G protein-coupled receptors (GPCRs), which contain a conserved structure of seven transmembrane helices linked by three alternating intracellular and extracellular loops [7]. According to the classic paradigm of GPCR signaling, binding of ligand to the receptor induces a sequence of conformational changes that result in its coupling to a heterotrimeric G protein. Activated G proteins then dissociate into G and Gβ subunits, each capable of modulating the activity of a variety of intracellular effector molecules. Thus, receptors that couple to G stimulatory (Gs) or G inhibitory (Gi) G proteins modulate the activity of adenylyl cyclase (AC) to generate the second messenger cAMP and subsequently activate of cAMP-dependent protein kinase (PKA). Gq-coupled receptors activate phospholipase C that in turn generates diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) and activate protein kinase C (PKC). The nature of the intracellular response to catecholamine stimulation therefore depends not only on the type of activated receptor and its expression levels, but also on the G protein it couples to and the intracellular pathways activated by the various second messengers.

2.1. β Adrenergic receptor signaling
Regulation of cardiac function in response to catecholamine stimulation is controlled primarily through the actions of βARs. Both β1 and β2AR subtypes couple to Gs and activate AC, resulting in elevated cAMP levels and subsequent activation of PKA (reviewed in Ref. [8]). PKA activation is a critical step in the mediation of contractility through phosphorylation of L-type calcium channels and phospholamban to regulate calcium influx and reuptake (reviewed in Refs. [5,6]). Despite the dominant role of the Gs/AC/cAMP pathway in βAR signaling (particularly that of β1ARs), different subtypes of βARs are capable of coupling to other G proteins, thereby activating more than one intracellular signaling pathway. In addition to Gs, β2ARs couple to Gi (pertussis-toxin sensitive pathway) both in vitro [9] and in the heart [10–13]. Studies in neonatal cardiomyocytes of β1AR knockout mice show that stimulation of β2ARs is characterized by a biphasic effect on contraction rate with an initial PKA-independent increase in rate, followed by a PTX-sensitive decrease in rate of contraction [14]. Switching of β2AR from Gs to Gi-coupling was found not only to inhibit AC activity but also to initiate signaling of mitogen-activated protein kinase (MAPK) by the Gβ subunits of Gi, in a process that is regulated by PKA-mediated phosphorylation of the receptor [15]. More recent studies show that βAR/Gi coupling can also activate the cytosolic effector molecule phosholipase A2 (cPLA2) in the heart, in a cascade that triggers positive enhancement of calcium signaling and contraction, and is independent of cAMP production [16]. Moreover, β2AR regulation of cPLA2 was found to depend upon the status of βAR/AC coupling: while efficient βAR/AC coupling exerts a negative constraint on the cPLA2 pathway, decreased βAR/AC coupling promotes signaling through the βAR/cPLA2 cascade [17]. Together, these data indicate that β2ARs have the capacity to signal through multiple pathways and that they are able to utilize alternative compensatory mechanisms under conditions of altered or defective βAR/Gs/AC coupling.

As opposed to the relatively well-understood role of β1ARs and β2ARs in regulating cardiac function, the role of β3ARs in the heart remains unclear. Studies in human ventricular biopsies [18] and in β3 knockout mice [19] have suggested a role for these receptors in reduction of cardiac contractility through coupling to Gi and inhibition of AC. However, cardiac specific overexpression of human β3ARs in mice was shown to induce a significant, agonist-dependent increase in AC activity and cardiac contractility [20]. Moreover, specific β3ARs overexpression was accompanied by a significant reduction in β1ARs, a similar picture to that observed in human heart failure [21].

The ability of adrenergic receptors to activate multiple pathways through different G proteins and second messengers often imparts diversity to adrenergic receptor responsiveness under different conditions. Many factors, among them distribution, type of species [22] and age [23] determine the effect of a certain receptor on cardiac response. Comparative studies of the role of species in βAR responsiveness reveal that whereas β1- and β2AR-induced contractility is mediated through a cAMP-dependent pathway in rats and humans, in mice only β1AR-induced contractility uses the Gs/AC pathway myocytes, while β2ARs enhances the contractile function through a pathway that is not associated with a significant elevation of cAMP and is not modulated by PTX-sensitive G proteins [22]. Developmental stage is another critical factor in determining the overall function of adrenergic receptors as illustrated by the finding that activation of β2ARs by low agonist concentrations leads to a positive cAMP-dependent inotropic response in neonatal but not adult ventricular myocytes [24].

2.2. Adrenergic receptor signaling
AR signaling in the heart is diverse and involves activation of multiple signaling pathways that regulate cardiac output as well as cellular growth responses. The first point of regulation that determines which pathway is activated occurs at the receptor level. All three subtypes of 1ARs 1_A, 1_B and 1_D are expressed in the heart, the main subtype being 1_B [4,25,26]. A recent study in two lines of transgenic mice, a constitutively active 1_B-overexpressing model and 1_D knockout model, showed that the 1_BAR is involved in regulation of contractile function and cardiac growth, whereas the 1_D AR does not participate in contractile function and is involved more in regulating arterial blood pressure [27]. The effect of AR-mediated stimulation on contractile function also depends on the developmental state of the animals: while stimulation of 1ARs leads to an increase in rate of contraction in neonatal cardiomyocytes, it decreases both chronotropy [28] and inotropy [29] in adult myocytes. More recently, it has been found that both 1_A/C and 1_BARs are required for normal development of physiological cardiac hypertrophy in male but not female mice [30], suggesting that gender differences also play an important role in AR-mediated signaling.

Essentially, all three subtypes of 1ARs that are expressed in the heart couple to their signal transduction machinery primarily through the PTX-insensitive Gq/11 family, thereby leading to intracellular calcium mobilization through a Gq/PLC/PKC pathway [31]. However, several lines of evidence indicate that in addition to Gq/11, cardiac 1ARs are associated with PTX-sensitive Gi [28,32,33]. The negative chronotropic effect induced by 1AR activation in adult rat hearts [34] was found to be a result of 1AR coupling to Gi proteins in the course of development [35]. Data from transgenic mice show that cardiac-specific wild-type 1_BAR overexpression results in increased PLC activity together with attenuation of basal and agonist-stimulated AC activity, which is reversed by PTX [29]. 1ARs also have the capacity to couple to alternative effector molecules such as phosholipase D (PLD) [36] and to activate a number of calcium/calmodulin sensitive kinases [37]. Additionally, these receptors couple to numerous intracellular calcium mobilization pathways via voltage-dependent and independent calcium channels [38].

2.3. Mechanisms of adrenergic receptor desensitization and internalization
Repeated or sustained exposure to agonists often results in rapid attenuation of receptor responsiveness in a process termed desensitization. Lack of responsiveness is usually the result of a combination of different mechanisms during which receptors are rapidly phosphorylated, internalized into intracellular compartments or downregulated due to reduced protein synthesis/degradation of existing receptors (Reviewed in [39]). Stimulation of and β adrenergic receptors by catecholamines leads to their phosphorylation by second messenger-regulated kinases (PKA, PKC), or by G protein-coupled receptor kinases (GRKs) [40–42]. βAR-mediated activation of PKA or AR-mediated activation of PKC triggers indiscriminative phosphorylation of both agonist-bound and non-agonist bound receptors in a process of heterologous desensitization [31,39]. Conversely, GRK-mediated phosphorylation is limited only to the agonist-bound receptor and it promotes the recruitment of arrestin molecules to uncouple the receptor from the G protein [39].

An important consequence of agonist-mediated receptor phosphorylation is subsequent endocytosis of agonist-bound receptors into intracellular compartments [43]. Internalization of GPCRs can occur via two distinct pathways, namely clathrin-coated pits and caveolae, which serve as microdomains for integrating the transport machinery. Internalization of the β2AR in response to agonist occurs through a clathrin-coated pit pathway involving GRK and β arrestin [44], although data in myocyte membranes show that co-localization of β2ARs in caveolae is required for their physiologic signaling [45]. More recently, it was demonstrated that β1ARs can utilize both internalization pathways, depending on the kinase that phosphorylates the receptor [46]. The preferred internalization pathway is GRK/β arrestin-mediated and proceeds via clathrin coated pits. Alternatively, β1ARs can also internalize via caveolae through a PKA-dependent cascade [46].

Association of βARs with different downstream scaffolding proteins is critical for their internalization and recycling. One of the general mechanisms to recruit GPCR into signaling complexes is by its carboxyl terminal PDZ domain: a protein interaction module of 80–100 residues that recognizes and binds the C-terminal of four residues on their target protein [47]. β2ARs were shown to interact with the Na⁺/H⁺ exchanger regulatory factor through the PDZ binding motif in their carboxyl terminus in a process that is critical for receptor recycling after sequestration [48]. Additionally, studies in neonatal cardiomyocytes infected with tagged β1ARs or β2ARs showed differences in the internalization properties of these receptors that were dependent upon PDZ domain interactions. While β2ARs underwent endocytosis in response to the non-selective β agonist isoproterenol, β1ARs remained on the cell surface due to association of their PDZ binding domain with proteins that prevented receptor/Gi interactions [49]. Mutation in the PDZ domain resulted in a β2AR-like response of a biphasic effect on myocyte contractility suggesting that although β1ARs have the machinery to couple to Gi, it is prevented from doing so in cardiac myocytes [49]. In addition to the PDZ domain, other regions of the carboxyl tail region of the β2AR were also recently shown to affect β2AR recycling after agonist mediated internalization [50].

While βAR endocytosis has been studied extensively, mechanisms of AR internalization are less defined. Evidence suggest that activation of PKC plays an important role in 1_BAR internalization in response to adrenergic agonists and PKC activators (reviewed in Ref. [31]). 1_BARs also undergo rapid endocytosis following exposure to agonist through a clathrin-coated pit mechanism [51] in a partially arrestin-dependent pathway [52].

	3. β Adrenergic signaling in cardiac hypertrophy and failure

Top Abstract 1. Introduction 2. Mechanisms of adrenergic... 3. β Adrenergic signaling... 4. {alpha}AR signaling in... 5. Novel paradigms in... 6. Concluding remarks References

 
A prolonged increase in cardiac workload due to neurohormonal stimulation or acute damage causes the myocardium to undergo a hypertrophic response that may or may not be protective against deteriorating ventricular function [53–56]. Indeed, sustained cardiac hypertrophy is a leading predictor for progressive deterioration into heart failure [57,58]. Interestingly, both cardiac hypertrophy and heart failure are characterized by marked alterations in components of the adrenergic system.

3.1. β Adrenergic receptor levels in cardiac hypertrophy and failure
One of the most prominent characteristics of the failing heart is a marked desensitization and downregulation of βARs [59]. Evidence to support a role for β1ARs in heart failure comes from several studies using transgenic technology to overexpress β1ARs in mouse myocardium. These studies showed that overexpression of β1ARs in mice causes hypertrophy and interstitial fibrosis in young animals, which proceeds to cardiac dysfunction as the animals age [60,61]. Hemodynamic studies in these mice showed that although maximal contractility and rate of isovolemic relaxation were initially increased in young animals, systolic and diastolic functions were already impaired and progressed to a steady decline of left ventricular contractility and relaxation [62]. Although overexpression of wild-type (WT) β1AR in the healthy myocardium is clearly deleterious, it remains to be determined whether it is advantageous under conditions of chronic downregulation. Additionally, the effect of β1AR knockout on the outcome of hypertrophy and heart failure is not known at this time.

As opposed to a specific marked downregulation of β1ARs, there is no significant change in the level of desensitized β2ARs in the failing heart [59]. The consequent resulting change in the ratio of β1/β2ARs may indicate a prominent role for β2ARs under conditions where β1AR signaling is suppressed. Indeed, overexpression of human β2ARs (60–100-fold over endogenous levels) was shown to enhance cardiac function without deterioration into heart failure [63,64]. Mice overexpressing the β2AR show augmented baseline cardiac function in a ligand-independent manner and have greater contraction amplitudes, larger calcium transients and faster relaxation times compared to their non transgenic littermates [65]. Intracoronary adenoviral-mediated overexpression of β2ARs in rabbit hearts also increased in vivo hemodynamic function compared to control rabbits [66]. In contrast, high levels of β2ARs in the myocardium (200–350-fold over endogenous levels) resulted in age-dependent progression to cardiomyopathy [64,67].

Several studies addressed the issue of whether β2AR overexpression is advantageous or deleterious during cardiac hypertrophy and failure. Induction of chronic pressure overload using the transverse aortic constriction (TAC) method [68] in high level β2AR overexpressing mice resulted in worse heart failure compared to sham-operated mice [69]. Transgene overexpression was reduced by 70% and β2AR binding—by 65%, 8 weeks after TAC [70]. Isolated cardiomyocytes from these mice showed presence of hypertrophy under basal conditions and the same extent of increase in cell size after 9 weeks of TAC as the WT littermates [71]. Contrary to the deleterious actions of β2ARs during pressure overload, β2AR overexpression preserved left ventricular contractility in a mouse model of myocardial infarction (MI) where despite presenting similar cardiac hypertrophy and LV chamber size to WT animals, transgenic animals had a significantly higher level of left ventricular contractility [69]. In another system, adenoviral-mediated transfer of human β2ARs to ventricular myocytes from chronically paced rabbits led to restoration of βAR signaling in vitro [72]. Hence, expression of β2ARs may be advantageous in some conditions and deleterious in others, depending on the types of signals that are initiated.

Besides differences in βAR subtypes, it is becoming evident that genetic heterogeneity in the structure of both β1 and β2ARs has a remarkable effect on heart failure predisposition. Human β1ARs were found to be polymorphic at amino acid residue 389 (substitute of Arg to Gly). Overexpression of the β1Arg389 in mice was found to induce an initial increase in receptor function and contractility, followed by a marked deterioration in both parameters, suggesting predisposition of β1Ang389 to heart failure [73]. Together with the findings in β1AR overexpressing mice [60,61], these results suggest that initial hyper stimulation of the β1AR system may culminate in depressed receptor function and ventricular dysfunction. Predisposition to heart failure was also identified in individuals who were homozygous for β1Ang389 and 2CDel322-325 [74].

Like β1ARs, a unique polymorphism at amino acid residue 164 (Thr to Ile substitute), that contributes to the outcome of heart failure was identified in the human β2AR [75,76]. The β2Ile164 variation displayed decreased basal and agonist-mediated AC activity [76], and was related to significantly worse survival in individuals with heart failure [77].

3.2. Receptor–G protein coupling during cardiac hypertrophy and failure
Another major characteristic of the failing heart is that chronic exposure to high levels of circulating catecholamines results in a marked impairment in the ability of both β1ARs and β2ARs to couple to their respective G proteins [59]. Studies in transgenic animals overexpressing Gs confirm that enhanced signaling in the βAR/Gs/AC pathway initially leads to increased cardiac function, but is detrimental to the heart in the long run [78]. Another important observation is the significant elevation in the levels of Gi in failing hearts [79]. Since β1/β2AR ratios are significantly altered in heart failure, coupling of β2ARs to Gi is likely to play a role in heart failure pathology. Indeed, disruption of Gi signaling by PTX was found to enhance the contractile response in rat ventricular myocytes [10], suggesting that β2AR/Gi coupling contributes to adequate regulation of contractility by putting the break on Gs/AC pathways. In support of this possibility are recent findings made in crosses of β2AR overexpressing transgenic mice with mice expressing an inactivated Gi2 subunit. Gi2 knockout/β2AR overexpressing mice developed a more pronounced cardiac hypertrophy and earlier heart failure compared to the β2AR overexpressing animals, indicating an essential protective role for Gi2 during chronic β2AR signaling [80].

β2AR/Gi coupling can initiate signaling in a number of cascades that may have a tremendous effect on the outcome of hypertrophy and heart failure. For example, it has been demonstrated that while activation of β1ARs in mouse cardiac myocytes leads to cellular apoptosis, β2AR stimulation results in activation of a Gi-Gβ/PI3K mediated survival pathway [81,82]. Therefore, it has been suggested that β2AR/Gi coupling may act to activate a protective anti-apoptotic pathway during enhanced stimulation of β1ARs by excess of catecholamines [49]. Other pathways that are regulated by switching of cardiac β2AR coupling from Gs to Gi in myocytes are L-type calcium channels (reviewed in Refs. [5,83]), and activation of MAP kinase pathways [34,84–87]. Recent studies also indicate that besides myocytes, chronic β2AR stimulation increases DNA synthesis and proliferation of human cardiac fibroblasts [88].

3.3. βAR desensitization: role of phosphoinositide 3-kinase
Desensitization of adrenergic receptors in patients with heart failure is attributed in part to a significant increase in the levels of β adrenergic receptor kinase 1 (βARK 1, also known as GRK2) [89]. The role of βARK in myocardial contractility and its potential therapeutic effects have been studied extensively and will not be discussed here (for reviews, see Refs. [6,90,91]). Binding of cytosolic βARK to liberated Gβ subunit of the heterotrimeric G protein facilitates its translocation to the plasma membrane where it then phosphorylates the agonist-bound receptor (reviewed in Ref. [92]). It has recently been shown that βARK forms a stable cytosolic complex with phosphoinositide 3-kinase (PI3K) in the heart and that translocation of βARK to the receptor facilitates agonist-mediated translocation of PI3K to the plasma membrane [93]. Overexpression of the catalytically inactive PI3K, or the use of selective PI3K inhibitors was found to severely attenuate the ability of β2ARs to internalize following agonist stimulation [93,94]. The region of PI3K that specifically associates βARK was identified as the phosphoinositide kinase (PIK) domain of the enzyme [94]. Moreover, overexpression of PIK domain led to competitive displacement of endogenous PI3K from βARK and to a marked attenuation in agonist-induced internalization of β2ARs [94].

Several lines of evidence point to the involvement of PI3K in cardiac hypertrophy [95]. Cardiac-specific expression of a constitutively active p110 subunit of PI3K resulted in a significant increase in heart size, whereas expression of the inactive form of p110 reduced it [96]. Furthermore, cardiac-specific overexpression of constitutively active Akt, the downstream effector of PI3K, also led to an increase in heart size [82]. To directly assess the role of PI3K in hypertrophy, its activity was measured in response to pressure overload-induced hypertrophy in the mouse [97]. These experiments showed that short term TAC results in a specific activation of p110PI3K (PI3K) without a change in the levels of PI3K [97]. Activation of PI3K upon pressure overload was completely absent in mice overexpressing the C-terminus of βARK (βARKct), which binds and sequesters Gβ subunits [98], indicating that PI3K activation in pressure overload is Gβ-dependent.

Long-term pressure overload in transgenic mice was used as a model to study the role of PI3K in the transition from hypertrophy to heart failure. Mice overexpressing the Gq inhibitor peptide [99], and mice lacking endogenous epinephrine and norepinephrine, underwent an 8-week TAC protocol. The results showed that while WT animals displayed marked hypertrophy along with significant increases in PI3K and Akt activities, the transgenic animals presented a significantly attenuated hypertrophic phenotype and had no increase in PI3K and Akt activities [55]. Importantly, mice with attenuated hypertrophy and reduced PI3K activation showed significantly improved cardiac function compared to WT mice [55].

The evidence that PI3K is activated during the hypertrophic process and that it may play a role in the transition to heart failure led to the investigation of the hypothesis that downregulation of βARs under conditions of chronic catecholamine stimulation can be prevented through interference with PI3K interaction with the receptor complex [100]. Exposure of mice with cardiac-specific overexpression of a catalytically inactive mutant of PI3K to a chronic administration of catecholamines prevented βAR downregulation and desensitization. Moreover, development of heart failure in response to pressure overload was significantly ameliorated in these animals (Fig. 1) [100]. However, direct knockout of PI3K was insufficient to prevent receptor downregulation, probably indicating the importance of recruitment of other PI3K isoforms to the activated receptor [100]. These data establish a novel role for receptor localized-PI3K in regulation of βAR turnover in vivo and show that a strategy that blocks βARK-mediated recruitment of PI3K may provide a novel therapeutic approach to restore normal βAR signaling and preserve cardiac function.

View larger version (77K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 PI3Kinact overexpression delays development of cardiac failure following chronic pressure overload induced by TAC. (A) Representative serial echocardiography in conscious WT and PI3Kinact mice with chronic pressure overload. (B) βARK1-associated PI3K activity is decreased in membrane fractions from hearts PI3Kinact mice compared to WT animals. Adapted with permission from Ref. [100].

 

	4. AR signaling in myocardial hypertrophy and failure

Top Abstract 1. Introduction 2. Mechanisms of adrenergic... 3. β Adrenergic signaling... 4. {alpha}AR signaling in... 5. Novel paradigms in... 6. Concluding remarks References

 
4.1. Adrenergic receptor levels in hypertrophy
ARs are the principal mediators of the hypertensive response in the cardiovascular system. However, evidence in recent years point to an additional major role for these receptors in mediating cardiac hypertrophy. Initial studies in neonatal cardiomyocytes have shown that stimulation of 1_AARs induces a hypertrophic phenotype, including increase in cell size and altered gene expression [101,102]. Additionally, mRNA of 1_CAR was found to be elevated under conditions of chronic catecholamine infusion and pressure overload [103]. Surprisingly however, investigation of the role of 1_AARs in vivo yielded a different result. Cardiac-specific overexpression of the 1_AAR subtype in transgenic mice resulted in a marked enhancement of cardiac contractility that was reversible by 1_AAR blockade, but despite these changes transgenic animals failed to display any evidence of hypertrophy [104]. Two important observations arise from these data: (1) 1_AARs possess an inotropic function that may be beneficial under conditions of desensitized βAR signaling, and (2) The AR-mediated hypertrophic response in vivo is mediated by more than one subtype of the 1ARs. This conclusion is supported by the recent findings that physiological hypertrophy such that is seen during normal development is dependent upon the presence of both 1_A/C and 1_BAR subtypes [30]. Male mice with a double knockout of 1_A/C and 1_BARs failed to develop physiological hypertrophy during development and showed smaller myocyte and heart size, reduced basal and phenylephrine stimulated ERK MAP kinase activation and decreased exercise tolerance (Fig. 2) [30]. Importantly, double knockout mice that were exposed to pressure overload showed decreased survival compared to their WT littermates suggesting that both 1AR subtypes are required for an adaptive response to cardiac stress [30].

View larger version (74K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 1A and 1B adrenergic receptors are required for physiological hypertrophy. (A) Whole heart cross sections from WT and 1A/1B double knockout mice showing a small heart phenotype in male double knockout mice compared to WT littermates. (B) Hypertrophic signaling in 1A/1B double knockout mice: Phenylephrine-mediated activation of ERK is selectively attenuated in 1A/1B double knockout mice compared to WT suggesting the involvement of this pathway in the small heart phenotype. Adapted with permission from Ref. [30].

 
Elucidation of the role of 1_BARs in cardiac hypertrophy has been complicated by several observations made in transgenic mice. Knockout of the 1_BAR was shown to prevent the development of hypertrophy induced by a chronic infusion of norepinephrine, suggesting that 1_BAR stimulation is involved in the hypertrophic response [105]. However, studies of the effect of WT 1_BAR overexpression on cardiac hypertrophy and heart failure yield slightly different results. Indeed, myocyte-specific overexpression of 1_B is deleterious to the heart and results in severe left ventricular dysfunction and dilated cardiomyopathy, however, as in the case of the 1_AAR overexpression it does not show any signs of spontaneous hypertrophy [29,106,107]. Marked hypertrophy was demonstrated only in transgenic mice that were chronically exposed to phenylephrine [108]. In contrast to these findings, recent studies conducted in mice with systemic overexpression of 1_BARs showed development of hypertrophy [109,110], suggesting that ARs in other cardiac cells such as fibroblasts as well as ARs in the central nervous system may be involved in the hypertrophic response.

As opposed to the observation that cardiac overexpression of the WT 1_BAR does not alter heart size, overexpression of a constitutively active form of 1_BAR was found to induce a marked hypertrophic phenotype including increases in heart/body weight ratio, cross-sectional area and hypertrophic markers [111]. Enhanced 1_BAR activity in the heart was particularly detrimental after TAC-induced pressure overload, causing rapid progression into heart failure [112]. Furthermore, hearts of these animals showed a significant increase in the activities of ERK and JNK MAP kinases and a reduced response to isoproterenol compared to non transgenic animals [27]. The disparity between the WT and constitutively active overexpression of 1_BAR in mediating the hypertrophic response may stem from activation of different signaling pathways by each receptor: Whereas signaling of the WT receptor is largely dependent upon catecholamine stimulation, the constitutively active receptor is characterized by persistent agonist-independent signaling that may continuously activate growth related cascades.

4.2. AR signaling pathways in hypertrophy
A number of studies have established that stimulation of ARs in neonatal ventricular myocytes induces a hypertrophic response that is characterized by activation of immediate early genes, upregulation of contractile protein genes [113,114] and reactivation of embryonic genes [101,115]. Two signaling pathways have been indicated in recent years to mediate the AR-induced cardiac response: (1) A Gq-mediated pathway and (2) A Gi/G12/13-Rho-mediated pathway. Gq presents the convergence point for several receptors such as ARs, angiotensin and endothelin receptors that have all been implicated in mediating the hypertrophic response [6,116]. Indeed, overexpression of a Gq inhibitor peptide, which blocks all Gq-mediated pathways, was found to significantly attenuate activation of MAP kinase pathways and development of hypertrophy in vivo [99,117]. Downstream of Gq-coupling AR activation was found to result in an increase in ANF expression through a Ras and MEK kinase pathway that lead to activation of JNK MAP kinase [118]. Additional support for the role of JNK in 1AR-mediated hypertrophy comes from experiments where expression of a specific JNK inhibitor blocks AR mediated protein synthesis and reorganization of actin cytoskeleton [119]. PI3K [120] and P70^6s kinase [4] were also implicated in AR-mediated hypertrophy in a cascade that does not involve activation of ERK or p38 MAP kinases [4].

Besides coupling to the Gq/PKC/Ras pathway, ARs also effectively couple to Gi G proteins in adult rat myocytes, thereby inducing a negative chronotropic effect [35]. Recent evidence indicate that the 1AR-mediated hypertrophic response is partly mediated by coupling to G12/13 and activation of the Rho family of low molecular weight GTP binding proteins that lead to JNK activation [119]. Activation of the Rho family was shown to occur by Gq coupling as well [121]. Together, these data suggest that ARs can mediate the hypertrophic response via at least three different pathways: (1) Gq/PKC/Ras/JNK kinase, (2) Gq/Rho and (3) G12/13/Rho/JNK.

A possible role for 2ARs in cardiac hypertrophy comes from studies in mice with a double knockout of 2_A/2_C which display higher plasma levels of catecholamine and develop cardiac hypertrophy and reduced cardiac contractility [122]. Induction of pressure overload in 2_A/2_C knockout mice decreased survival dramatically compared to wild-type and 2_B KO mice [123]. The excess in mortality rates in these groups was attributed to increased cardiac hypertrophy with heart failure, fibrosis and elevated catecholamine levels [123].

	5. Novel paradigms in GPCR signaling: receptor dimerization

Top Abstract 1. Introduction 2. Mechanisms of adrenergic... 3. β Adrenergic signaling... 4. {alpha}AR signaling in... 5. Novel paradigms in... 6. Concluding remarks References

 
The accepted paradigm for signaling of GPCRs is that these receptors function as single units (monomers), independently capable of coupling to a G protein and activating/inhibiting effector molecules. However, a rapidly growing body of evidence shows that GPCRs exist as homo- and heterodimers (reviewed in Ref. [124]). Heterodimerization between GPCRs often present characteristics that differ from homogeneous populations thereby generating previously unrealized diversity of function.

Evidence for the existence of direct interactions among adrenergic receptors, and between adrenergic receptors and other GPCR families began with identification of cross talk between these receptors. Overexpression of β2ARs in cells that express β1/β2ARs at a ratio of 80:20 resulted in a reduction of the maximal response to agonist [125]. Furthermore, blockade of each receptor subtype alone caused a small reduction in response to norepinephrine; however, when present simultaneously, both antagonists completely abolished the agonist effect [125]. These results were among the first to indicate that βAR activation results in a complex and sometime synergistic interaction between β1 and β2ARs. In a later study, cardiac specific overexpression of β2ARs was shown to attenuate β1AR response and to cause cardio-depression at high concentrations [126,127]. Direct assessment of the ability of β1AR and β2AR to engage oligomeric interactions revealed similar interaction propensity, suggesting that under equivalent expression levels the two receptor subtypes are expected to form an equal proportion of homo and heterodimers [128]. This study also indicated that the majority of the expressed receptors are in dimer form and that the type of interactions formed is determined by relative levels of expression [128]. A subsequent study found that although dimerization did not affect AC production, β1/β2AR dimers had a profound effect on trafficking and downstream signaling properties of the receptors [129]. While agonist stimulation of β2ARs overexpressing cells led to internalization and activation of ERK MAP kinases, co expression of β1ARs together with β2ARs blocked β2AR internalization and ERK activation [129]. Together, these data suggest that the interactions between β1- and β2ARs in the heart may be an additional critical event in the overall response of the heart to stimuli. Thus, it is possible that in a healthy heart the high β1AR expression prevents growth-related signaling of β2ARs, whereas downregulation of βARs in response to excess of catecholamines may allow a previously blocked β2AR signaling pathways to proceed.

Besides their interactions between same family members, adrenergic receptors were also found to form oligomers with GPCRs from different GPCR families including opioid [130] and angiotensin II type 1 receptors [131]. An interaction between 2AR and β2AR has been suggested almost 25 years ago [132] and it was only recently demonstrated that two close relative of these receptors, 2AR and β1ARs form dimers that lead to alterations of their signaling properties [133]. Inter-family interactions between endogenous βARs and AT1Rs were recently demonstrated to take place in the heart [131]. A very important consequence of this interaction was that blockade of one of the two receptors in complex was enough to inhibit signaling and trafficking of both receptors (Fig. 3). Thus, treatment of murine myocytes with a β blocker completely obliterated Angiotensin receptor/Gg coupling and contractility, and treatment of mice with a selective angiotensin receptor blocker attenuated heart rate response to a β agonist [131]. Together, accumulating data regarding oligomer formation by adrenergic indicate a much more complex role for each receptor in regulating cardiac function.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 (A) β blockers attenuate angiotensin II-mediated myocyte contractility. Summary of % cellular shortening (%CS) and rate of cell shortening (–dL/dt) from 5 individual hearts (10–15 myocytes from each heart) at baseline and following stimulation with the different agents. Remarkably, co-administration of the β blockers propranolol or metoprolol together with Ang II abolishes the Ang II mediated myocyte contractility (B) The angiotensin receptor blocker valsartan decreases ISO-stimulated elevation in heart rate. In vivo assessment of change of intact, vagotomized, wild-type mice in heart rate in response to increasing doses of the β agonist isoproterenol ISO () following acute administration of 250 µg valsartan () or 1 µg propranolol (). Increasing doses of ISO yielded a marked elevation in heart rate. Pretreatment with a single dose of propranolol resulted in a marked shift of the ISO response curve to the right, as expected from a classical competitive β antagonist. In contrast, a single dose of valsartan resulted in a significant 25% reduction in the maximal heart rate, without a rightward shift indicating that valsartan-mediated attenuation of the ISO response is not through competitive antagonism of the βAR. Adapted with permission from Ref. [131].

 

	6. Concluding remarks

Top Abstract 1. Introduction 2. Mechanisms of adrenergic... 3. β Adrenergic signaling... 4. {alpha}AR signaling in... 5. Novel paradigms in... 6. Concluding remarks References

 
Adrenergic receptors play a pivotal role in regulating cardiac function under both normal and pathologic conditions. In vivo studies of cardiac adrenergic signaling show that these receptors can initiate a variety of signaling cascades depending on their developmental stage, species and level of expression. It is also noteworthy to recognize that adrenergic receptors do not function in isolation but rather they form complex interactions among themselves as well as with other GPCRs, making expression levels and ratios between different receptors an important factor in the overall response to agonists and antagonists. Therefore, appreciation of the overall consequence of adrenergic receptor function in cardiac hypertrophy and failure requires both a complete understanding of the unique signaling properties of each adrenergic receptor, as well as its interactions with other receptors in the heart.

	Notes

 
Time for primary review 16 days

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Top Abstract 1. Introduction 2. Mechanisms of adrenergic... 3. β Adrenergic signaling... 4. {alpha}AR signaling in... 5. Novel paradigms in... 6. Concluding remarks References

 

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