1521-0081/66/1/308–333$25.00 http://dx.doi.org/10.1124/pr.112.007203 PHARMACOLOGICALREVIEWS PharmacolRev66:308–333,January2014 U.S.GovernmentworknotprotectedbyU.S.copyright ASSOCIATEEDITOR:PAULA.INSEL Cardiac Alpha -Adrenergic Receptors: Novel Aspects of 1 Expression, Signaling Mechanisms, Physiologic Function, and Clinical Importance TimothyD.O’Connell,BrianC.Jensen,AnthonyJ.Baker,andPaulC.Simpson DepartmentofIntegrativeBiologyandPhysiology,UniversityofMinnesota,Minneapolis,Minnesota(T.D.O);CardiologyDivision, UniversityofNorthCarolina,ChapelHill,NorthCarolina(B.C.J.);andCardiologyDivision,VAMedicalCenterandCardiovascular ResearchInstitute,UniversityofCalifornia,SanFrancisco,SanFrancisco,California(A.J.B.,P.C.S.) Abstract.....................................................................................309 I. Introduction.................................................................................310 II. a -Adrenergic Receptor Expression in the Heart .............................................310 1 A. a -Adrenergic Receptor Expression in the Heart in Animal Models .......................310 1 B. Unique Aspects of a -Adrenergic Receptor Expression Profiles in Cardiac Myocytes .......311 1 C. a -Adrenergic Receptor Expression in Human Heart......................................311 1 D D. a1-Adrenergic Receptor Levels Increase Proportionately in Human Heart Failure .........311 ow n E. Conclusions on a1-Adrenergic Receptor Heart Expression.................................311 loa III. a -AR Signaling in Cardiac Myocytes........................................................312 d 1 e d A. Conventional Models of a1-Adrenergic Receptor Signaling ................................312 fro B. New Model for General G-Protein-Coupled Receptor Signaling: G-Protein-Coupled m b Receptors at the Nucleus ................................................................312 y g C. a -Adrenergic Receptors in the Nuclei in Cardiac Myocytes...............................312 u 1 e s 1. Nuclear Localization of a1-Adrenergic Receptors in Cardiac Myocytes .................313 t o 2. Mechanism of a1-Adrenergic Receptor Nuclear Localization...........................314 n N 3. Receptor Orientation in the Inner Nuclear Membrane ................................314 ov e 4. Catecholamine Uptake in Cardiac Myocytes ..........................................314 m b 5. Functional Evidence for Nuclear a1-Adrenergic Receptor Signaling....................315 er 1 6. Localization of Signaling Partners with a -Adrenergic Receptors in Cardiac 6 1 , 2 Myocyte Nuclei ......................................................................316 0 1 7. Nuclear a -Adrenergic Receptor Localization in Vivo..................................316 8 1 8. Pathophysiologic Implications of Nuclear a -Adrenergic Receptor Signaling ...........316 1 9. Summary of a -Adrenergic Receptor Nuclear Localization.............................316 1 IV. a -Adrenergic Receptor Physiologic Function in the Heart....................................317 1 A. a -Adrenergic Receptors Activate Physiologic or Adaptive Hypertrophy ...................317 1 1. a -Adrenergic Receptor-Mediated Hypertrophy in Cell Culture Models ................317 1 2. a -Adrenergic Receptor-Mediated Hypertrophy in Animal Models .....................318 1 3. Summary of a -Adrenergic Receptor in Hypertrophy..................................319 1 B. a -Adrenergic Receptors Prevent Cardiac Myocyte Death.................................319 1 1. a -Adrenergic Receptor-Mediated Myocyte Survival Signaling in Cell Culture Models..319 1 2. a -Adrenergic Receptor-Mediated Myocyte Survival Signaling in Animal Models.......320 1 3. Summary of a -Adrenergic Receptor-Mediated Myocyte Survival Signaling............320 1 ThisworkwassupportedbytheNationalInstitutesofHealthNationalInstituteofGeneralMedicalSciences[GrantsP20-RR017662(to T.D.O.)];NationalInstitutesofHealthNationalHeart,Lung,andBloodInstitute[GrantsK08-HL096836(toB.C.J.)andR01-HL31113(to P.C.S.)];theDepartmentofVeteran’sAffairs[GrantsIO1-BX001078and001970(toP.C.S.),I01BX000740(toA.J.B.)];theAmericanHeart Association, Western States Affiliate (to P.C.S., A.J.B.); the American Heart Association, Greater Midwest Affiliate (to T.D.O.); the GlaxoSmithKlineResearchandEducationFoundationforCardiovascularDisease(toB.C.J.);andtheUniversityofCalifornia,SanFrancisco, FoundationforCardiacResearch(toB.C.J.). Addresscorrespondenceto:Dr.PaulC.Simpson,VAMedicalCenter(111-C-8),4150ClementSt.,SanFrancisco,CA94121.E-mail: [email protected];orDr.TimothyD.O’Connell,E-mail:[email protected]. dx.doi.org/10.1124/pr.112.007203. 308 Cardiaca -AdrenergicReceptors 309 1 C. a -Adrenergic Receptors Augment Contractile Function ..................................321 1 1. a -Adrenergic Receptor Activation of Contraction in In Vitro Models ..................321 1 2. a -Adrenergic Receptor-Mediated Contraction in Transgenic and Gene-Deletion 1 Mouse Models .......................................................................321 3. a -Adrenergic Receptor Activation of Contraction in Humans .........................322 1 4. Summary of a -Adrenergic Receptor Activation of Contraction ........................322 1 D. a -Adrenergic Receptors Induce Ischemic Preconditioning ................................322 1 1. a -Adrenergic Receptor-Mediated Preconditioning in Animal Models ..................322 1 2. a -Adrenergic Receptor-Mediated Preconditioning in Humans.........................322 1 3. Summary of a -Adrenergic Receptor-Mediated Preconditioning........................323 1 E. Conclusions: a -Adrenergic Receptors Are Cardioprotective...............................323 1 1. a -Adrenergic Receptors are Cardioprotective and Prevent Pathologic Remodeling 1 in Heart Failure Unlike Other G -Coupled Receptors .................................323 q 2. a -Adrenergic Receptor-Mediated Cardioprotective Signaling Can Explain the 1 Worsening of Heart Failure with a -Blockers Observed in Clinical Trials..............323 1 3. a -Agonist Therapies Might Improve Heart Failure Outcomes.........................323 1 V. a -Adrenergic Receptors in Human Heart Disease............................................324 1 A. Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial: An a -Adrenergic Receptor Antagonist in Hypertension Increases the Risk of Heart Failure ..324 1 B. Vasodilator-Heart Failure Trial: An a -Adrenergic Receptor Antagonist Does 1 Not Improve Survival in Heart Failure...................................................326 C. a -Adrenergic Receptor Antagonist Therapy in Benign Prostatic Hyperplasia 1 Might Exacerbate Heart Failure .........................................................326 D. Carvedilol: A Nonselective b -Adrenergic Receptor/a -Adrenergic Receptor 1/2 1 Antagonist for Heart Failure.............................................................326 E. Sympatholytics: Reducing Norepinephrine Levels Does Not Improve Heart Failure........327 F. Conclusions and Implications: Are Myocardial a -Adrenergic Receptors 1 Cardioprotective in Humans? ............................................................327 VI. Final Summary .............................................................................327 References ..................................................................................328 Abstract——Adrenergicreceptors(AR)areG-protein- atthemolecularlevel,alpha -ARslocalizetoandsignal 1 coupled receptors (GPCRs) that have a crucial role in at the nucleus in cardiac myocytes, and, unlike most cardiac physiology in health and disease. Alpha -ARs GPCRs, activate “inside-out” signaling to cause 1 signal through Ga , and signaling through G , for cardioprotection. Contrary to past opinion, human q q example,byendothelinandangiotensinreceptors,is cardiac alpha -AR expression is similar to that in 1 thought to be detrimental to the heart. In contrast, the mouse, where alpha -AR effects are seen most 1 cardiac alpha -ARs mediate important protective and convincingly in knockout models. Human clinical 1 adaptivefunctionsintheheart,althoughalpha -ARsare studies show that alpha -blockade worsens heart 1 1 only a minor fraction of total cardiac ARs. Cardiac failure in hypertension and does not improve alpha -ARs activate pleiotropic downstream signaling outcomes in heart failure, implying a cardioprotective 1 to prevent pathologic remodeling in heart failure. roleforhumanalpha -ARs.Insummary,thesefindings 1 Mechanismsdefinedinanimalandcellmodelsinclude identify novel functional and mechanistic aspects of activation of adaptive hypertrophy, prevention of cardiacalpha -ARfunctionandsuggestthatactivation 1 cardiac myocyte death, augmentation of contractility, of cardiac alpha -AR might be a viable therapeutic 1 andinductionofischemicpreconditioning.Surprisingly, strategyinheartfailure. ABBREVIATIONS: a -AR,a -adrenergicreceptor;b-AR,b-adrenergicreceptor;a A-subtype,a A-adrenergicreceptor;a B-subtype, 1 1 1 1 1 a B-adrenergic receptor; a -blocker, a -adrenergic receptor antagonist; a AKO, a A-adrenergic receptor knockout; a BKO, a B- 1 1 1 1 1 1 1 adrenergicreceptorknockout;a ABKO,a AB-adrenergicreceptordoubleknockout;aSkAct,a-skeletalactin;AR,adrenergicreceptor; 1 1 ATR,angiotensinreceptor;ALLHAT,AntihypertensiveandLipid-LoweringTreatmenttoPreventHeartAttackTrial;BPH,benign prostatic hyperplasia; BEST, b-Blocker Evaluation of Survival Trial; cav-3, caveolin-3; CAM, constitutively active mutant; DAG, diacylglycerol;ETR,endothelinreceptor;EMT,extraneuronalmonoaminetransporter;ERK,extracellularsignal–regulatedkinase; GFP, green fluorescent protein; GPCR, G-protein-coupled receptor; HEK, human embryonic kidney; HW, heart weight; IP, inositol phosphate; IP , inositol 1,4,5-trisphosphate; KO, knockout; MEK, mitogen-activated protein kinase kinase; MOXCON, Moxonidine 3 Congestive Heart Failure Trial; MOXSE, Moxonidine Safety and Efficacy Trial; MyHC, myosin heavy chain; NE, norepinephrine; NFAT, nuclear factor of activated T cells; NRVM, neonatal rat ventricular myocyte; NYHA, New York Heart Association; OCT3, organiccationtransporter3;PLCb ,phospholipaseCb ;PKC,proteinkinaseC;PKD,proteinkinaseD;V-HeFT,Vasodilator-Heart 1 1 FailureTrial;WT,wildtype. 310 O’Connelletal. I. Introduction G abundance is 2-fold (Ponicke et al., 1998), and q transgenic mice with 2-fold cardiomyocyte-specific G Adrenergicreceptors(ARs)bindtoandareactivated q overexpression have no discernible cardiac phenotype by the endogenous catecholamine hormones epineph- (Adams et al., 1998; Sakata et al., 1998). rine and norepinephrine (NE). Epinephrine is primar- Furthermore, a -ARs differ from other G -coupled ily produced in and released to the circulation from 1 q receptors in several important ways, including expres- the adrenal gland, whereas NE is synthesized in and sion limited to myocytes within the heart (section II) releasedbysympatheticnerveterminalsintheperiph- andlocalizationandsignalingatthenucleus,asdiscussed eral nervous system and brain. In the heart, the two in section III. main ARs are the b-ARs, which comprise roughly 90% Thus,unlikewhatcanbeseenwithsomeG -coupled ofthetotalcardiacARs,anda -ARs,whichaccountfor q 1 receptors, a -ARs protect the heart by activating an approximately 10% (see section II). 1 adaptive or physiologic hypertrophy, preventing car- In general, acute activation of cardiac b -ARs, the 1 diac myocyte death, augmenting contractile function predominant b-AR subtype(80% ormore of total b-ARs inheartfailureandinducingpreconditioning(section in heart), induces positive inotropic and chronotropic IV). Finally, clinical trials indicate that blockade responses,althoughinheartfailure,wheresympathetic ofa -ARsexacerbatesheartfailure(sectionV),which 1 activation and catecholamine levels are increased, long- could be explained by the cardioprotective functions termactivationofb -ARsexacerbatespathologicremod- 1 of a -ARs identified in cell and animal models. 1 eling (Bristow, 2000; Naga Prasad et al., 2001; Lohse This review summarizes these data, which span et al., 2003). decades, and emphasizes recent findings from our Less is known about cardiac a -ARs, but studies 1 laboratories. from the last thirty years indicate that long-term activationofcardiaca -ARsactivatesbeneficialtrophic 1 II. a -Adrenergic Receptor Expression in signalinginthedevelopingheartandthatthesea -AR- 1 1 the Heart mediated trophic effects in the adult, in many ways, counteract the negative effects of overstimulation of A. a -Adrenergic Receptor Expression in the Heart in 1 b -ARsinheartfailure.Thisreviewwillfocusonthese Animal Models 1 trophic effects of cardiac a -ARs and how activation of 1 In mice and rats, all three a -AR subtype mRNAs, 1 a -ARs might be beneficial in heart failure. 1 a A, a B, and a D, are detected in the heart (Rokosh 1 1 1 Therearethreea -ARsubtypes,thea A,a B,anda D, 1 1 1 1 et al., 1994; Stewart et al., 1994; Cavalli et al., 1997; and all three are expressed in the heart in a cell-type O’Connell et al., 2003). Interestingly, among most specific manner (section II). All three a1-ARs are species, including mouse, guinea pig, rabbit, pig, and G-protein-coupledreceptors(GPCR),andclassica1-AR cow, heart a1-AR levels determined by ligand binding signalingmechanismsinvolvecouplingtotheGq/11(Gaq) are relatively constant (mouse: mean of six studies, familyofG-proteinsandactivationofphospholipaseCb1 ;12 fmol/mg protein) (Steinfath et al., 1992a; Cavalli (PLCb1) at the plasma membrane. Activation of PLCb1 et al., 1997; Yang et al., 1998; Lin et al., 2001; cleaves phosphatidylinositol (PI), increasing inositol tri- O’Connell et al., 2003; Rokosh and Simpson, 2002), sphosphate (IP3) and diacylglycerol (DAG). IP3 binds to with the exception of rat heart, in which a1-AR levels the IP3-receptor to release calcium from intracellular are approximately 10-fold higher (rat: mean of four stores, and DAG activates protein kinase C (PKC). studies,;114fmol/mg)(Steinfathetal.,1992a;Michel Other Gq-coupled GPCRs that signal through Gaq, et al., 1994; Noguchi et al., 1995; Stewart et al., 1994). such as endothelin receptors (ETRs) and angiotensin Determination of cell-type specific expression of receptors (ATRs), are believed to play an important a -ARs in the heart, or any tissue, is hampered by the 1 role in the pathogenesis of heart failure. Hallmarks of lack of validated, subtype-specific a -AR antibodies 1 cardiomyopathy with heart failure include contractile (Jensen et al., 2009c), which is a general problem with dysfunction (both systolic and diastolic), myocyte antibodiesforGPCRs,asreviewed(Micheletal.,2009). hypertrophy, fibrosis, and increased cardiac cell death However,studiesina -ARknockoutmicedemonstrate 1 (Anand and Florea, 2003), which can all be worsened that cardiac myocytes express only the a A- and 1 by G -coupled receptors (Salazar et al., 2007). a B-subtypes, based on lack of [3H]prazosin binding q 1 However, it also needs to be recalled that the view or functional responses in hearts from a AB-double 1 that G -coupled receptor signaling is toxic is based in knockout mice (a ABKO) (McCloskey et al., 2003; q 1 large part on a transgenic mouse model with G O’Connell et al., 2003; Turnbull et al., 2003) as well as q overexpression that markedly exceeds the 2-fold in- lack of binding to a fluorescent a -AR antagonist or 1 crease found in human heart failure (Adams et al., signaling in cardiac myocytes isolated from a ABKO 1 1998; Ponicke et al., 1998; Sakataetal.,1998)andthus hearts (O’Connell et al., 2003; Wright et al., 2008). cannotbeconsideredtosimulatehumanpathophysiology. Ligand binding studies further indicate that the In human heart failure, the maximal increase in a B is predominant, with the a A- and a B-subtypes 1 1 1 Cardiaca -AdrenergicReceptors 311 1 expressedina1:2–4ratioincardiacmyocytes (Rokosh C. a -Adrenergic Receptor Expression in 1 and Simpson, 2002; O’Connell et al., 2003). Despite Human Heart the presence of a D-subtype mRNA, rodent cardiac 1 Inhumanheart,allthreea -ARsubtypemRNAsare 1 myocytes do not appear to express the a D-subtype 1 detected (Jensen et al., 2009a). Furthermore, a -AR protein by binding (O’Connell et al., 2003). However, 1 expressionlevelsinhumanheartdeterminedbyligand the a D might be expressed in the coronary vascula- 1 binding are similar to mouse and most other species ture, based on studies demonstrating a1-AR mediated (human: mean of 6 studies, ;12 fmol/mg protein) reductionsincoronaryflowinisolateda -ARknockout 1 (Bohm et al., 1988; Bristow et al., 1988; Vago et al., hearts (Chalothorn et al., 2003; Turnbull et al., 2003). 1989; Steinfath et al., 1992b; Hwang et al., 1996; This idea is supported by human studies (below). Jensenetal.,2009a).Humanmyocardiumhasthea A- 1 Conversely, rodent cardiac fibroblasts do not express and a B-subtypes, with the a B predominant, similar a -ARs (Stewart et al., 1994; O’Connell et al., 2001), 1 1 1 to other species (Jensen et al., 2009a,b), and the a A 1 and a -agonist infusioninduceshypertrophy without 1 is functional in signaling (R. C. Thomas and P. C. fibrosis (Marino et al., 1991), suggesting that a -AR 1 Simpson, unpublished data). These data suggest that activation does not exacerbate fibrosis associated themouseisamoreappropriatemodeltoapproximate with heart failure. In contrast with a -ARs, most 1 cardiac a -AR function than the rat, which as men- 1 ATRs and ET Rs are in fibroblasts, not cardiac B tioned above, has roughly 10-fold more a -ARs. 1 myocytes(Kimetal.,1995;Grayetal.,1998;Modesti Competition binding experiments do not detect the et al., 1999). a D-subtype in explanted human heart (Jensen et al., 1 Long-term activation of a -ARs and other hypertro- 1 2009a,b). However, the a D-subtype is expressed and 1 phic agonists increases the a A-subtype, without 1 functional in coronary artery smooth muscle cells and desensitizing a -mediated inositol phosphate (IP) 1 might cause vasoconstriction (Jensen et al., 2009b). turnover or growth, while decreasing a B-subtype 1 The a B-subtype is expressed in coronary artery en- 1 mRNA and protein levels in cultured neonatal rat dothelial cells and might induce vasodilation and angio- cardiac myocytes (NRVM) and in rats subjected to genesis (Jensen et al., 2010). aortic banding (Rokosh et al., 1996). Moreover, total a -AR levels are not altered in vivo by hypertrophy or 1 D. a -Adrenergic Receptor Levels Increase 1 heart failure in rats (Rokosh et al., 1996; Sjaastad Proportionately in Human Heart Failure et al., 2003), and a -AR inotropic effects are main- 1 In heart failure, b -ARs are desensitized and down- tained or increased (Wang et al., 2010), in contrast to 1 regulated. In contrast, radioligand-binding studies in- b-ARs that are desensitized and downregulated in dicate that myocardial a -AR levels are slightly heartfailure(Bristowetal.,1982;Bristowetal.,1988). 1 increased in human heart failure (mean of six studies, Partial explanation for the differences in desensiti- increased from ;12 to ;19 fmol/mg protein) (Bohm zation of a -AR and b-ARs might reside in expression 1 et al., 1988; Bristow et al., 1988; Vago et al., 1989; and regulation of G-protein receptor kinases (GRKs). Steinfathetal.,1992b;Hwangetal.,1996;Jensenetal., GRK3 is found exclusively in myocytes, regulates 2009a). This means that a -AR levels in the heart, a -ARs, and is not upregulated in heart failure (Vinge 1 1 which normally represent approximately 11% of the et al., 2001, 2007; Aguero et al., 2012). In contrast, total AR population at baseline (range 2–23%, mean of GRK2 and GRK5 that desensitize b-ARs but not six studies), are proportionately increased to approxi- a -ARs are expressed in many myocardial cell types 1 mately 25% of the total AR population in heart failure and are upregulated in heart failure (Rockman et al., (range 9–41%) (Bohm et al., 1988; Bristow et al., 1988; 1996; Eckhart et al., 2000; Vinge et al., 2001, 2007; Vago et al., 1989; Steinfath et al., 1992b; Hwang et al., Aguero et al., 2012). 1996; Jensen et al., 2009a). Given that sympathetic B. Unique Aspects of a -Adrenergic Receptor drive and catecholamine levels are increased in heart 1 Expression Profiles in Cardiac Myocytes failure(Cohnetal.,1984),thiscouldimplythata -ARs 1 sustain adrenergic function when b -ARs are down- Recent studies provide unique information on the 1 regulated. In fact, a -AR-induced positive inotropy, expression and distribution of a -ARs in cardiac 1 1 which at baseline is minimal, can be equal to b-AR- myocytes. First, both the a A- and a B-subtypes 1 1 mediated inotropy in ventricular muscle strips isolated localize to and signal at the nuclear membrane, but from human heart failure patients (Skomedal et al., not the plasma membrane, in adult mouse cardiac 1997), as reviewed in more detail below. myocytes(Huangetal.,2007;Wrightetal.,2008;Wright et al., 2012), as reviewed in section III. Second, a A- 1 E. Conclusions on a -Adrenergic Receptor subtype expression and function are graded in adult 1 Heart Expression cardiac myocytes, from high levels to none, whereas the a B-subtype is expressed in all cardiac myocytes (un- In summary, a -ARs constitute a minority of the 1 1 published data). totalcardiacARpopulationinhumansatbaseline,and 312 O’Connelletal. thisseemstoholdacrossspecies,withtheexceptionof elsewhere (Hein and Michel, 2007; Cotecchia, 2010; rats where a -ARs levelsare;10-fold higherthan any Jensen et al., 2011). To date, over 70 downstream sig- 1 otherspecies.Thisshouldbeconsideredwheninterpret- naling molecules have been implicated in cardiac a -AR 1 ing results from studies of a -ARs in rats, particularly signaling, using the NRVM model of a -AR-stimulated 1 1 in cultured NRVMs, the most common cardiac myocyte cardiac myocyte hypertrophy (Jensen et al., 2011). Some culture model. data suggest interactions with b-arrestin (Pediani et al., Cardiac myocytes of all species have all three a -AR 2005;Stanasilaetal.,2008;Hennenbergetal.,2011)and 1 subtype mRNAs, but only the a A- and a B-subtype Gbg (Vettel et al., 2012). 1 1 receptor proteins are detected. In humans, the a D- 1 B.NewModelforGeneralG-Protein-CoupledReceptor subtype is present in coronary smooth muscle and Signaling: G-Protein-Coupled Receptors at might regulate coronary vasoconstriction, whereas the the Nucleus a B-subtype is in coronary endothelial cells and might 1 regulate vasodilation and angiogenesis. In contrast, It is now clear that several GPCRs localize to and a -ARs are not expressed by cardiac fibroblasts. signalatthenucleus,or“inside-out”signaling.Nuclear 1 In heart failure, a -ARs are not downregulated as signaling is seen in several cell types, including 1 are b -ARs and thus become a greater share (25%) of neurons, hepatocytes, and cardiac myocytes, as 1 ARsintheheart.Thisincreaseina -ARscouldsuggest reviewed previously (Gobeil et al., 2006; Boivin et al., 1 that a -ARs have a compensatory or adaptive role in 2008;Bkailyetal.,2009;Tadevosyanetal.,2012).The 1 heart failure, as suggested by studies showing that GPCRs include receptors for prostaglandin E in the 2 a -mediated inotropy can be similar to b-AR-mediated brain (Gobeil et al., 2002), angiotensin II (AT1R) in 1 inotropy in heart failure (Skomedal et al., 1997). The thebrainandinHEKandChinesehamsterovarycells idea that a -ARs might have an adaptive and pro- (Lu et al., 1998; Chen et al., 2000; Lee et al., 2004), 1 tective role in the heart is a central theme of this plateletactivatingfactorintheliverandbrain(Marrache review and is discussed in following sections. et al., 2002), apelin in the brain (Lee et al., 2004), bradykinininHEKcells(Leeetal.,2004),andglutamate in neurons (O’Malley et al., 2003). III. a -AR Signaling in Cardiac Myocytes 1 Several recent studies show that GPCRs localize Thefollowingsectionsreviewtheconventionalmodels to nuclei in binucleate adult cardiac myocytes, as of a -AR localization and signaling at the plasma reviewed previously (Tadevosyan et al., 2012). Specif- 1 membrane, or “outside-in” signaling, and evidence for ically, ETRs are detected on nuclei isolated from adult novelmodels,suggestingthata -ARsandotherGPCRs cardiac myocytes, and endothelin stimulates nuclear 1 signalfromthecardiacmyocytenucleus,or“inside-out” calciumtransients(Boivinetal.,2003).ATRsandb-ARs signaling. are also detected on nuclei isolated from adult cardiac myocytes and mediate increased RNA synthesis A. Conventional Models of a -Adrenergic 1 (Boivin et al., 2006; Tadevosyan et al., 2010; Vaniotis Receptor Signaling et al., 2011). These findings indicate that GPCR local- Conventional models of GPCR signaling describe ization to the nucleus could regulate important phys- receptoractivationattheplasmamembraneleadingto iologic functions in adult cardiac myocytes. However, initiation of downstream signaling within the cell, themajorityoftheseotherreceptorscanlocalizealsoto commonly referred to as “outside-in” signaling. Fur- the myocyte plasma membrane; for example, 95% of thermore, classic models of GPCR function suggest ETRsareonthesarcolemma(Boivinetal.,2003;Wright that GPCRs are expressed on the cell membrane and etal.,2012),sothattherelativefunctionalsignificance are only internalized after receptor phosphorylation of nuclear versus surface localization is uncertain. and subsequent desensitization (Drake et al., 2006). Despite thedatareviewed above, theprevalentview a -ARs signal through the G /11 class of G-proteins, isthatGPCRs,includinga -ARs,arelocalizedprimar- 1 q 1 leading to activation of PLCb and increases in ily to the plasma membrane in heart and myocytes. 1 IP /calcium signaling and activation of PKC (Graham Thisimpressionisbasedpredominantlyonradioligand 3 etal.,1996;PiascikandPerez,2001).Thea B-subtype bindingtomembranefractions,bindingassaysinwhole 1 might also signal through G (Hu and Nattel, 1995; cells (Filipeanu et al., 2006) and studies with a -AR i 1 Steinbergetal.,1985;Akhteretal.,1997;Melienetal., antibodies. Difficulties with these approaches are dis- 2000; Snabaitis et al., 2005). In NRVM, historically cussed in the next section. the primary cell model used to study cardiac a -AR 1 C. a -Adrenergic Receptors in the Nuclei in signaling, a -AR-induced increases in IP are readily 1 1 3 Cardiac Myocytes observed, but in adult cardiac myocytes this is controversial. The general consensus is that a -ARs Cellular localization of signaling molecules deter- 1 signal through the G /PLCb -IP /PKC pathway, but mines function, emphasizing the importance of a -AR q 1 3 1 downstreamsignalingpathwaysarediverse,asreviewed subcellularlocalizationincardiacmyocytes.Thefollowing Cardiaca -AdrenergicReceptors 313 1 sections review the limitations and advantages of discarded normally (Simpson, 2006). Whole-cell bind- differentapproachesto detecta -ARsubcellularlocaliza- ing assays are limited by the lack of radioligands that 1 tion and the evidence that a -ARs are in the cardiac do not enter the cell (Filipeanu et al., 2006). 1 myocyte nucleus, derived from studies of localization, Immunochemical detection,eitherbyimmunoblotor agonist uptake, and signaling. Physiologic implications cell/tissue staining, is another commonly used tech- of nuclear a -ARs are also suggested. This novel nuclear nique to detect a -ARs. However, none of 10 commer- 1 1 a -AR signaling paradigm in cardiac myocytes is illus- ciala -ARantibodiesarespecificfora -ARsingeneral 1 1 1 trated in Fig. 1. or for any subtype, as documented by the fact that no 1.NuclearLocalizationofa -AdrenergicReceptorsin antibody detects a band in wild-type (WT) tissue that 1 Cardiac Myocytes. Limitations with the techniques is absent in tissue from a -AR knockout (KO) mice 1 used to detect a -ARs, radioligand binding and a -AR (Jensen et al., 2009c). This nonspecificity of anti- 1 1 antibodies, might explain the conventional view that GPCR antibodies is a general problem, reviewed a -ARs localize mainly to the plasma membrane. recently, emphasizing that a -AR antibodies need to 1 1 Ligand binding assays typically involve homogeniza- be validated using KO tissue (Michel et al., 2009). tionofhearttissueorculturedcellsfollowedbyahigh- Nonspecificity of a -AR antibodies calls into question 1 speed ultracentrifugation to isolate total membrane previous reports with these reagents, for example, fractions. This high-speed ultracentrifugation pulls work suggesting a -AR localization to the plasma 1 down all membranes, and if subcellular markers are membrane and t-tubules in adult rat cardiac myocytes not used, this technique does not distinguish between (O-Uchi et al., 2008) or a study using immunoprecip- plasma, sarcoplasmic, and nuclear membranes (Lin itation of a -ARs with potential signaling partners 1 et al., 2001; Rokosh and Simpson, 2002; O’Connell (Fujita et al., 2001). et al., 2003). Furthermore, most purified membrane Anantibodytothe1D4epitopetagattheCterminus preparations exclude over 65 to 85% of total heart of the a A detects surface membrane expression in 1 a -ARsthatarefoundin“debris”andlow-speedpellets heart sections of a transgenic mouse (Lin et al., 2001). 1 Fig.1. Modelfora -ARsignalingatthenuclearmembrane.Inadultcardiacmyocytes,catecholaminea -ARagonists(NE/PE)areactivelytransported 1 1 intothemyocyteviaorganiccationtransporter3(OCT),whichcanbeinhibitedbycorticosterone.Themembrane-permeablea -ARantagonistprazosin 1 (andsimilarderivatives)cancrosstheplasmamembranetoinhibitsignaling,whereasthemembraneimpermeablea -ARantagonistCGP12177Afails 1 toinhibitsignaling.Themodelsuggeststhatactivea -ARslocalizetotheinnernuclearmembranewiththeligand-bindingdomainfacingthespace 1 betweentheouterandinnernuclearmembranes(ONMandINM,respectively).Onthebasisofthisorientation,bindingofagonisttoa -ARsinduces 1 signalinginsidethenucleus,possibly throughGa ,althoughdownstreamintranuclear signalingpathwaysremaintobedefined.Weproposethat q activationofnucleara -ARscaninduceintranuclearhypertrophicsignalingaswellasextranuclearsignaling,includingactivationofERKincaveolae 1 andsurvivalsignalingorphosphorylationofcardiactroponinIatthesarcomereandcontractilefunction.HDAC,histonedeacetylase;CaCh,calcium channel;RYR,ryanodinereceptor;PTP,mitochondrialpermeabilitytransitionpore;ER/SR,endoplasmic/sarcoplasmicreticulum;NR,nucleoplasmic reticulum;NPC,nuclearporecomplex. 314 O’Connelletal. However, it is problematic whether receptor localiza- localization sequencesare recognized bya class ofpro- tion with 170-fold overexpression simulates that of teins known as importins that bind these sequences endogenous a -ARs (Lin et al., 2001). and facilitate transport of the target protein to the 1 A few studiesusemembrane fractionation combined nucleus. This importin-mediated nuclear localiza- with the caveolar marker caveolin-3 (cav-3) to detect tion not only occurs for proteins that target to the a -AR binding in caveolae in NRVMs (Fujita et al., nucleoplasm but for proteins that target the inner 1 2001; Lanzafame et al., 2006). In NRVMs, a caveolar nuclear membrane as well (King et al., 2006; Cook fraction defined in this way contains most or all et al., 2007; Lusk et al., 2007). Importin-mediated a -mediated IP turnover (Morris et al., 2006) and 27% nuclear localization was previously described for the 1 of total a -AR binding, both a A and a B (Lanzafame type 1 parathyroid hormone receptor (Pickard et al., 1 1 1 et al., 2006). The value of 27% a -AR binding in 2006, 2007) and more recently for the gonadotropin- 1 caveolae in NRVMs agrees well with a more recent releasing hormone type 1 receptor (Re et al., 2010). study finding 20% of total a -ARs in adult myocyte Recent experiments identify nuclear localization se- 1 membranes defined by high levels of cav-3 (Wright quencesinthea A-anda B-subtypes,andmutationof 1 1 et al., 2008). these sequences results in loss of nuclear localization Thecontrarynotionthata -ARslocalizeprimarilyto for each subtype in adult mouse cardiac myocytes 1 the nucleus arises from three main lines of evidence. (Wright et al., 2012). First, 80% of total a -AR binding in adult mouse 3. Receptor Orientation in the Inner Nuclear 1 cardiac myocytes is found in nuclear membranes Membrane. As described above, nuclear membrane defined by the marker LAP2 (Wright et al., 2008, proteins are targeted to the inner nuclear membrane 2012). In NRVMs, nuclear a -AR binding is also through nuclear localization sequences, similar to 1 observed (Buu et al., 1993), and 73% of total a -ARs proteins in the nucleoplasm. Also important is the 1 are in noncaveolar membranes that might be nuclear orientationofinnernuclearmembraneproteins,which (Lanzafame et al., 2006), in good agreement with the could affect how they signal. For GPCRs, such as results in adult myocytes. a -ARs,iftheligand-bindingdomainfacestheinsideof 1 Second, BODIPY-prazosin is a fluorescent analog of thenucleus,theligandwouldhavetoenterthenucleus the a -AR antagonist prazosin that binds all three and signaling would be initiated on the cytoplasmic 1 a -AR subtypes with equal affinity and fluoresces only side in the space between the inner and outer nuclear 1 when bound to receptor (Daly et al., 1998; Mackenzie membranes. Conversely, if the ligand-binding domain et al., 2000; Pediani et al., 2005). BODIPY-prazosin faces the space between the outer and inner nuclear staining of living adult cardiac myocytes identifies membranes, then signaling would be activated inside endogenousa -ARsonthenuclearmembranebutdoes the nucleus. 1 not detect receptors at the plasma membrane (Wright Recent studies with nuclear GPCRs detect signaling etal.,2008).Nucleiisolatedfromadultcardiacmyocytes in isolated nuclei, implying that nuclear receptors are confirm positive BODIPY-prazosin staining of endoge- likely oriented with the ligand-binding domain facing nous nuclear a -ARs (Wright et al., 2012). outward and the C terminus facing the nucleoplasm. 1 Third, a reconstitution system, in which a -AR-GFP ETRsinducecalciumtransientsinisolatednuclei(Boivin 1 fluorescent fusion proteins are expressed in cultured et al., 2003), and b-ARs and ATRs induce transcrip- adult a ABKO cardiac myocytes, recapitulates the tional responses in isolated nuclei (Tadevosyan et al., 1 nuclear localization of the endogenous a -ARs (Huang 2010; Vaniotis et al., 2011). These studies suggest 1 et al., 2007; Wright et al., 2008, 2012). that nuclear GPCR signaling is activated inside the Studies in other cells provide some support for the nucleus, thereby indicating an orientation in the results in cardiac myocytes. In recombinant cells inner nuclear membrane similar to GPCRs at the expressing a -ARs, for example, HEK293 cells, all plasma membrane where the C terminus faces the 1 a -AR subtypes show some intracellular localization cytoplasm. 1 (Daly et al., 1998; Mackenzie et al., 2000; Chalothorn 4. Catecholamine Uptake in Cardiac Myocytes. etal.,2002).Inprimaryculturesofsmoothmusclecells, A prerequisite for nuclear a -AR signaling is that NE 1 endogenous a -ARs are also found on both the plasma and other a -AR ligands must traverse the plasma 1 1 membraneandintracellular,usingafluorescentligand, membrane, transit to the nucleus, and bind to and BODIPY-FL prazosin (Mackenzie et al., 2000). activate receptors in a time course consistent with 2. Mechanism of a -Adrenergic Receptor Nuclear signaling. In nonneuronal cells, this process is known 1 Localization. Themechanismfornucleartargetingin- as NE “uptake-2” (Obst et al., 1996) and is facilitated volves nuclear localization sequences embedded in the by extraneuronal monoamine transporter/organic cat- protein. These nuclear localization sequences typically ion transporter 3 (EMT/OCT3) (Zwart et al., 2001; consist of mono- or bi-partite basic residues, usually Schomig et al., 2006). EMT/OCT3 is expressed most lysinesandargininesorglycine-argininerepeats(Dono abundantly in heart (Zwart et al., 2001), where it is etal.,1998;Hock etal.,1998; Luetal.,1998).Nuclear presentonboththeplasmaandnuclearmembranesin Cardiaca -AdrenergicReceptors 315 1 adultcardiacmyocytes(Wrightetal.,2008).Inneonatal Terzic et al., 1992; Gambassi et al., 1998; Zhang et al., myocytes, uptake of [3H]NE is observed, but the time 1998; Woo and Lee, 1999; Ross et al., 2003; O-Uchi scale of nearly an hour before NE is detected in the et al., 2005; Luo et al., 2007; Ichishima et al., 2010), nucleus is not sufficiently rapid to account for a -AR ratherthansecondsaswouldbeexpectedforareceptor 1 signaling (Buu et al., 1993). at the sarcolemma. However, a more sensitive fluorescent-based cate- Finally, there is functional evidence that agonist cholamineuptakeassayshowsthatcatecholaminesare uptake is required for a -AR signaling. Inhibition of 1 taken up very rapidly in cultured adult mouse cardiac EMT/OCT3-mediated catecholamine uptake with cor- myocytes.Inthissystem,catecholamineuptakebegins ticosterone,anEMT/OCT3antagonist,preventsa -AR 1 within seconds, is clearly increased by 5 minutes, activation of ERK in cultured adult mouse cardiac peaksat30minutes,andisantagonizedbyadditionof myocytes (Wright et al., 2008). unlabeled NE 15 minutes prior to catecholamine up- Insummary,thekineticsofagonistuptakeinmyocytes, takemeasurement,indicatingspecificity(Wrightetal., EMT/OCT3 biochemistry and biology, including kinet- 2008). ics, heart expression, and inhibition by corticosterone, Further consistent with rapid uptake, the intrinsic and the latency of a -AR physiologic responses are 1 uptakekineticsofOCT3,whichistherateatwhichone all consistent with agonist uptake and activation of transporter moves a cation, show that OCT3-mediated nuclear a -ARs. 1 cation transport is in the time frame of seconds. Thus, 5. Functional Evidence for Nuclear a -Adrenergic 1 the uptake kinetics of catecholamines by recombinant Receptor Signaling. a -ARs have numerous signaling OCT3 expressed in HEK293 cells is a V ;30,000 1 max effectsinthecytosol,asreviewedlater.Thus,ifa -ARs pmol/mg protein/min and a K ;900 mM for NE, and 1 m signal in the nucleus, then that signal must be aV ;13,000pmol/mgprotein/minandaK ;500mM max m transduced out of the nucleus to reach these cytosolic for epinephrine (Duan and Wang, 2010). Catechol- targets,defininganinside-out(nuclear-to-cytoplasmic) amine uptake is observed in seconds, with half- signaling mechanism. Three sets of data support the maximum response seen in ;2 minute (Duan and idea that a -signaling is initiated in the nucleus. Wang, 2010). It is likely that the kinetic properties of 1 First, CGP-12177A [4-[3-[(1,1-dimethylethyl)amino]2- the transporter are relatively consistent from cell to hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one cell and that expression level will dictate the absolute hydrochloride], an a -antagonist that does not cross amount of uptake and OCT3 expression is highest in 1 membranes (Staehelin et al., 1983; Levin et al., 2002; heart (Zwart et al., 2001). Brahmadevara et al., 2003, 2004), does not block In mice, OCT3-mediated heart uptake of the neuro- a -AR-ERK signaling in cultured adult mouse cardiac toxin cation methyl-4-phenylpyridinium acetate is ob- 1 myocytes, whereas the prototypical a -AR antagonist served within minutes of infusion (;4000 ng/g tissue 1 prazosin, which freely crosses the plasma membrane, 5 minutes after infusion), and this uptake is inhibited does block a -AR-ERK signaling (Wright et al., 2008). by 75% in OCT3KO mice (Zwart et al., 2001), indi- 1 Second, mislocalization mutants of both the a A- and cating a rapid and robust uptake system. The pheno- 1 a B-subtype, in which the nuclear localization sequen- type of OCT3KO mice is further interesting. Thus, 1 cesaremutated,donotactivateERKinculturedadult OCT3KO mice have a trend toward reduced heart size mouse cardiac myocytes (Wright et al., 2012). These in males [WT heart weight (HW) 160 mg, OCT3KO mislocalization mutants are not redirected to the HW145mg,n=7,P=0.138,a10%reduction](Zwart plasmamembrane,whichwouldprovideamorecrucial et al., 2001), reminiscent of the small heart phenotype testoftherequirementfornuclearlocalization,butthe seeninmalea ABKO mice (WTHW 147mg,a ABKO 1 1 122 mg, n = 33–27, P , 0.05, a 17% reduction) mutants do show that nuclear localization is required (O’Connell et al., 2003), but the number of OCT3KO for a1-AR signaling in adult cardiac myocytes. Finally, mice analyzed was small (n = 7) (Zwart et al., 2001). activation of nuclear a1-ARs leads to the activation of Thus, the kinetics of catecholamine uptake in myo- ERK in caveolae at the plasma membrane (Wright cytes (Wright et al., 2008) and the biochemistry and etal.,2008),andthenuclearexportinhibitorleptomycin biology of OCT3 (Zwart et al., 2001; Duan and Wang, Bblocks a1-AR-mediatedactivationofERK,suggesting 2010) are consistent with a1-AR responses initiated by thata1-ARsignalingtoERKatcaveolaemustoriginate agonist activation of nuclear receptors. in the nucleus (Wright et al., 2012). How signals are Inagreementwith theideathata -AR agonistmust transported from the nucleus to cytosolic targets is 1 be transported into the myocyte for signaling, there uncertain. However, a -ARs activate PKC, a molecule 1 is a long latency of a -AR responses after agonist known to translocate upon activation, suggesting 1 addition, in contrast to the rapid onset for b-AR a possible mechanism to transmit a signal out of the agonism. Specifically, the latency for contractile or nucleus. Taken together, these studies provide func- calcium responses to a -agonism in isolated myocytes tional evidence for a -AR signaling initiated in the 1 1 is 2 to 5 minutes in nine studies (Tohse et al., 1990; nucleus. 316 O’Connelletal. 6. Localization of Signaling Partners with a -Adren- localization of a -ARs in vivo. As mentioned, overex- 1 1 ergic Receptors in Cardiac Myocyte Nuclei. To effect presseda A-ARsintransgenicmicelocalizetotheplasma 1 nuclear a -AR signaling in cardiac myocytes, a -ARs membrane based on immunohistochemical staining for 1 1 mustcolocalizewithdownstreamsignalingpartnersin an epitope tag (Lin et al., 2001). However, very high thenuclearmembrane.However,theiridentitiessofar receptor levels, about 170-fold over basal, might cause remain unclear. artifactual localization, and the lack of validated a -AR 1 A fraction of Gaq colocalizes with a1-ARs at the antibodies (Jensen et al., 2009c) make conventional nucleus, based on immunocytochemistry in a1ABKO immunohistochemical approaches problematic. cardiac myocytes expressing a1-AR-GFP fluorescent Conversely, in a different a1A-subtype transgenic fusion proteins, and on subcellular fractionation of model, in which an a A-subtype GFP fusion protein is 1 WT adult cardiac myocytes (Wright et al., 2008). In expressed at a much lower level, approximately 5-fold NRVMs, approximately 56% of Gaq is in caveolar over basal, a1-ARs are detected at the nuclei in ven- membranes defined by cav-3, and the remainder is in tricular tissue sections with a GFP antibody (Wright noncaveolar membranes, some of which might be nu- et al., 2008). This result with the a A-GFP transgenic 1 clear (Morris et al., 2006). micesuggeststhata -ARnuclearlocalizationobserved 1 AroleinnuclearsignalingisuncertainforPLCb ,the 1 in cultured cardiac myocytes can represent a -AR lo- 1 classic a -coupled PLC. PLCb is detected in the nuclei 1 1 calization in vivo. ofadultcardiacmyocytesusingtheG12PLCb antibody 1 8. Pathophysiologic Implications of Nuclear from Santa Cruz Biotechnology (Santa Cruz, CA) a -Adrenergic Receptor Signaling. ETRs, ATRs, and 1 (Wright et al., 2008), but this and other commercial b-ARs signal in isolated nuclei from adult cardiac PLCb antibodies are not proven specific for PLCb 1 1 myocytes (Boivin et al., 2003; Tadevosyan et al., 2010; using KO tissues. In NRVMs,91% of PLCb is detected 1 Vaniotis et al., 2011). However, it is difficult to assign in a caveolar fraction using a Santa Cruz antibody a functional significance to nuclear signaling by these (Lanzafame et al., 2006), and forced expression of GPCRs in cardiac myocytes, because the majority of PLCb b with an N-terminal enhanced GFP tag detects 1 ETRs, ATRs, and b-ARs localize to the plasma localization on the sarcolemma but not the nucleus membrane (although quantitative ligand binding in (Grubb et al., 2008). Also in NRVMs, expression of subcellularfractionsforATRsisnotpossibleduetolow a C-terminal peptide from PLCb b blocks a - and 1 1 level of expression). Conversely, approximately 80% of G -mediated IP turnover and aspects of hypertrophy q a -ARs localize to the nuclei in adult mouse cardiac (Grubb et al., 2008; Filtz et al., 2009). Finally, the 1 myocytes. Interestingly, in pathologic settings, a -AR substrate for PLCb , phosphatidylinositol 4,5-bisphos- 1 1 signaling is clearly protective (sections IV and V), phate, is not detected in nuclear membranes (Zhang whereas ETR and ATR signaling can exacerbate path- et al., 2013). Taken together, these data suggest that ologic remodeling (Harada et al., 1999; Yang et al., PLCb mightnotbeinvolvedina -ARnuclearsignaling. 1 1 2004). This raises the possibility that differences in An interesting alternate mediator of nuclear a -AR 1 receptor localization might lead todifferencesbetween signaling is PLC«, which might be regulated by small physiologic and pathologic signaling. In other words, GTPases (Rho, Ras, Rap) and Gbg subunits, but not nuclear receptors, like a -ARs, might be protective, byGa (Lopezetal.,2001).PLC«inNRVMsandheart 1 q whereas ETRs and ATRs at the plasma membrane isscaffoldedtomuscle-specificAkinase-anchoringpro- mightinducepathologicsignaling(Wrightetal.,2012). tein at the nuclear envelope with PKD, one key hyper- Although these ideas remain to be tested, differential trophic signaling molecule (Zhang et al., 2011, 2013). localization of G -coupled receptors could have signif- Knockdown of PLC« inhibits a -and Ga -stimulated q 1 q hypertrophy in NRVMs, but has no effect on IP turn- icant implications for their physiologic functions and over, and expression of PLC« causes hypertrophy, an for therapeutic targeting of Gq-coupled receptors in effect that requires PLC« catalytic activity (Zhang heart disease. et al., 2011, 2013). Myocyte-specific PLC« KO also in- 9. Summary of a1-Adrenergic Receptor Nuclear hibits hypertrophy with pressure overload in vivo Localization. Overall, the majority of current data (Zhang et al., 2013). Extensive evidence suggests that supports the idea that a1-ARs localize to and signal PLC« mediates hypertrophy by hydrolysis of phos- fromthenucleiinadultcardiacmyocytesinvitroand phatidylinositol 4-phosphate (PI4P) at the nuclear in vivo. Identification of functional nuclear localiza- envelope, with generation of DAG and activation of tion sequences in each a1-subtype provides a mecha- PKD, but upstream mechanisms are uncertain (Zhang nistic basis to support nuclear a -AR localization, 1 et al., 2013). Clearly, definition of nuclear a -AR sig- orientedwiththeC-terminaltailinthenucleoplasm. 1 naling partners is a promising area for the future. Ligand uptake into the cell via EMT/OCT3 pro- 7. Nuclear a -Adrenergic Receptor Localization in vides a mechanism for receptor activation. The sig- 1 Vivo. Itispossiblethatlocalizationobservedinisolated naling mechanisms of nuclear a -ARs remain unclear, 1 or cultured cardiac myocytes does not reflect the true as do the physiologic implications of nuclear versus Cardiaca -AdrenergicReceptors 317 1 sarcolemmalsignalingbya -ARsandotherG -coupled were involved, as in other types of cells. These papers 1 q receptors. were the first demonstration that catecholamines induce cardiac myocyte hypertrophy directly. This finding was later confirmed in cultured adult rat and IV.a -AdrenergicReceptorPhysiologicFunction 1 cat cardiac myocytes (Simpson, 1988; Fuller et al., in the Heart 1990; Ikeda et al., 1991; Volz et al., 1991; Clark et al., In the heart, AR physiology is largely focused on 1993). acuteb-ARmediatedregulationofcontractilefunction, Cardiac hypertrophyis clearly linked to induction of whereas chronic b-AR signaling is maladaptive and gene transcription, and in NRVMs, a -ARs induce 1 b-AR antagonists are now standard therapy in heart a pattern of hypertrophic gene transcription charac- failure. Short-term a -AR signaling can increase terized by re-expression of genes normally expressed 1 contractility, as reviewed below, but this has not been only in the fetal heart (Simpson et al., 1989). Early studied in detail in vivo. On the other hand, many studiesidentifiedagroupofthese“fetalgenes”induced studies now indicate that chronic a -AR signaling is by a -ARs, including c-myc (Starksen et al., 1986), 1 1 adaptive, protecting the heart from pathologic stress atrial natriuretic factor (Knowlton et al., 1991, 1993), throughactivationofphysiologichypertrophy,survival a-skeletal actin (aSkAct) (Bishopric et al., 1987; Long signaling, augmentation of contractility, and ischemic et al., 1989; Karns et al., 1995), and b-myosin heavy preconditioning. These data are described below. chain(bMyHC)(Waspeetal.,1990;Kariyaetal.,1993, 1994). A. a -Adrenergic Receptors Activate Physiologic or 1 Acrucialstudyofendogenoustranscriptioninintact Adaptive Hypertrophy NRVMs proved that a -ARs stimulate transcription 1 Cardiac myocyte hypertrophy is the most common not only of a fetal gene (aSkAct), but also all RNA cellular response in the heart to pathologic stress, but species,includingthemRNAforanadultgene(cardiac hypertrophy is not always maladaptive (Frey and actin), and total RNA (ribosomal and transfer) (Long Olson, 2003). Cardiac hypertrophy occurs during et al., 1989). Several subsequent studies focusing on normal physiologic development and in response to a -AR-mediated transcriptional regulation delineated 1 exerciseandalsoasanadaptiveresponsetopathologic a host of transcriptional factors and modifiers acti- stress. Physiologic or adaptive hypertrophy is charac- vated by a -ARs in cardiac myocytes, including TEF-1 1 terized by an increase in heart and cardiac myocyte (Kariya et al., 1993, 1994; Karns et al., 1995; McLean size without fibrosis and an overall improvement in et al., 2003), GATA-4 (Morimoto et al., 2000; Liang function. etal.,2001a,b),Egr-1(Jinetal.,2000),Elk1(McWhinney In contrast, pathologic or maladaptive hypertrophy et al., 2000), Vgl-4 (Chen et al., 2004), Rlf (Post et al., is characterized by an increase in heart and cardiac 2002),CREB(Markouetal.,2004),Zfp260(Debrusetal., myocyte size accompanied by combinations of cardiac 2005),andclass2 histone deacetylase(Vega etal.,2004; cell death, fibrosis, vessel loss, reduced innervation, Liu et al., 2009). and, most importantly, declining function. Clinically, Mechanistically, the a A-subtype is implicated in 1 cardiac hypertrophy in Framingham adults is corre- a -AR mediated hypertrophy in NRVMs through the 1 latedwithasignificantlyincreasedriskofheartfailure use of a -AR subtype-specific pharmacologic agents 1 and sudden death (Levy et al., 1990). Thirty years of (Autelitanoand Woodcock, 1998).Severalmechanisms researchfromcellculturetogeneticallymodifiedmice, for a -AR-mediated hypertrophic signaling are pro- 1 summarized below, demonstrates clearly that a -ARs posed, and a multitude of signal transducers are 1 can mediate a physiologic or adaptive form of cardiac implicated (Jensen et al., 2011). Certain molecules, hypertrophythatoffsetspathologicremodelinginheart based on frequency in the literature, might be failure. considered essential or “core” molecules required for 1. a -Adrenergic Receptor-Mediated Hypertrophy in a -AR-mediatedhypertrophicsignaling,includingPLC 1 1 Cell Culture Models. Primary cultures of NRVMs are (Filtzetal.,2009;Zhangetal.,2013),PKC(a,d,and«, the most common cell culture model used to examine threemainisotypesactivatedbya -ARs)(Henrichand 1 signaling in cardiac myocytes. The original and now Simpson, 1988; Kariya et al., 1991, 1993, 1994; Karns classic experiments (Glembotski, 2013) demonstrated et al., 1995; Haworth et al., 2000; Rohde et al., 2000; that catecholamines acting through a -ARs produce Braz et al., 2002, 2004; Vega et al., 2004; Carnegie 1 a direct trophic response in NRVMs (Simpson et al., et al., 2008), PKD (Haworth et al., 2000; Vega et al., 1982; Simpson, 1983, 1985). At the time, there was 2004; Harrison et al., 2006; Avkiran et al., 2008; debate as to whether catecholamines induced hyper- Bossuyt et al., 2008, 2011; Carnegie et al., 2008; Liu trophy through increasing blood pressure or through etal.,2009),ERK(Buenoetal.,2000;Xiaoetal.,2001; a direct action on cardiac myocytes, that is, whether Barron et al., 2003; O’Connell et al., 2003), and class myocyte hypertrophy was regulated in some way only 2 histone deacetylase (Vega et al., 2004; Backs et al., by“load”orwhethergrowthfactorsandtheirreceptors 2006, 2008; Harrison et al., 2006; Liu et al., 2009).