Canadian Journal of Cardiology xx (2012) xxx

Review

An “I” on Cardiac Hypertrophic Remodelling: ImidazolineReceptors and Heart Disease

Suhayla Mukaddam-Daher, PhD

Centre Hospitalier de L’Université de Montréal Research Center, and Departments of Medicine and Pharmacology, Université de Montréal,

Montreal, Québec, Canada

ABSTRACTThe centrally-acting sympatholytic imidazoline compound, moxoni-dine, prevents the development of left ventricular hypertrophy andattenuates maladaptive proliferative signalling as well as downstreamapoptotic pathways in spontaneously hypertensive rat and cardiomyo-pathic hamster hearts. The actions are selectively mediated by imid-azoline type-1 receptor (I1-receptor, also named nischarin), nonadren-ergic neurotransmitter receptors mainly found in the brainstemmedulla. We identified cardiac I1-receptors/nischarin and showed thatthey are upregulated in cardiovascular disorders, and are functionalwithout the central nervous system’s contribution. Molecular charac-terization revealed that I1-receptor/nischarin has a unique structurewith multifunctional domains allowing it to perform a number of cellsignalling roles as a scaffolding protein. Nischarin has been associatedwith integrin �5 and inhibition of Rac1 and was shown to interact withinsulin receptor substrates. However, very little is known about cardiac

I1-receptor/nischarin and its role(s) in normal physiology and patho-

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0828-282X/$ – see front matter © 2012 Canadian Cardiovascular Society. Publisheddoi:10.1016/j.cjca.2012.02.007

RÉSUMÉLe composé d’imidazoline sympatholytique d’action centrale, la moxo-nidine, prévient la survenue de l’hypertrophie ventriculaire gauche etatténue la signalisation proliférative maladaptive aussi bien que lesvoies de l’apoptose en aval dans les cœurs de rats spontanémenthypertendus et des hamsters cardiomyopathiques. Les actions sontsélectivement médiées par le récepteur des imidazolines de type 1(récepteur I1, aussi nommé nischarine), les récepteurs des neurotrans-metteurs non adrénergiques principalement trouvés dans la moelle dutronc cérébral. Nous avons identifié les récepteurs I1 cardiaques/nis-charine et montré qu’ils sont régulés à la hausse dans les troublescardiovasculaires, et qu’ils sont fonctionnels sans la contribution dusystème nerveux central. La caractérisation moléculaire a révélé quele récepteur I1/nischarine a une structure unique comportant des do-maines multifonctionnels qui lui permettent d’accomplir plusieursrôles de signalisation cellulaire tout comme une protéine

d’échafaudage. La nischarine a été associée à l’intégrine �5 et à

The hypertensive heart undergoes significant structural cha-nges that occur in response to mechanical stress, induced byhemodynamic overload, and biochemical stress, induced byactivated renin-angiotensin-aldosterone system, sympatheticnervous system (SNS), growth factors, and cytokines. Thesestimuli initiate and maintain left ventricular hypertrophy(LVH) through a complex array of signalling events, withcross-talk and positive and/or negative interaction and contrib-ute to regression to a fetal program of gene expression. Alteredmyocardial growth and composition involve cardiomyocytehypertrophy and loss (by apoptosis or necrosis), cardiac fibro-blast proliferation and conversion to myofibroblasts, increasedsynthesis and deposition of collagen, and progression of inter-stitial and perivascular fibrosis, leading to ventricular stiffen-ing. The initial hypertrophic growth of the left ventricle was

Received for publication January 4, 2012. Accepted February 14, 2012.

Corresponding author: Dr Suhayla Mukaddam-Daher, CRCHUM, 264Rene Levesque East, Pavilion Edouard Asselin (A-301), Montreal, QuébecH2X 1P1, Canada. Tel.: �1-514-890-8000 � 35461; fax: �1-514-412-7377.

E-mail: suhayla.mukaddam-daher@umontreal.ca

considered adaptive, allowing the heart to maintain cardiacoutput in response to the increased workload. However, suchchanges are detrimental, eliciting abnormalities in conductionand contractility, predisposing to coronary artery disease, ar-rhythmias, stroke, sudden death, and heart failure.1-3

Sympathetic outflow to the heart is preferentially activatedin hypertension and heart failure. Excess catecholamines acti-vate the renin-angiotensin system, cytokines, and oxidativestress, accelerate left ventricular remodelling, and worsen myo-cardial function.4-6 Overexpression of �-1 adrenergic receptorsin the heart or chronic activation of cardiac �-adrenergic re-ceptors produces a cardiomyopathic phenotype,7 while genet-ically engineered mice that are unable to synthesize norepi-nephrine exhibit less cardiac hypertrophy and preservedventricular function after aortic banding.8 Deletion of �2A-and �2C-adrenergic receptors leads to cardiac hypertrophy andfailure due to chronically enhanced catecholamine release,9 andsympathectomy prevents cardiac fibrosis in hypertensive ratsindependent of blood pressure.10 Antihypertensive treatmentsthat directly block or oppose the SNS, such as �-blockers andindirectly through renin-angiotensin system blockade, by ang-

iotensin-converting enzyme inhibitors and angiotensin-recep-

by Elsevier Inc. All rights reserved.

2 Canadian Journal of CardiologyVolume xx 2012

physiology, specifically in cardiac remodelling. Our studies have shownthat I1-receptor is expressed in cardiac fibroblasts and myocytes andthat in vitro I1-receptor activation inhibits norepinephrine-induced car-diomyocyte apoptosis and fibroblast proliferation, through differentialeffects on mitogen-activated protein kinases and Akt. Accordingly,apart from centrally-mediated sympatholytic function, I1-receptor inthe heart may control cell growth and death. I1-receptor may be impli-cated in cardiac remodelling and dysfunction, through the inhibition ofapoptotic pathways and/or activation of survival pathways, in a cell-specific manner. Identification of the cardioprotective mechanisms ofcardiac I1-receptor could result in specifically-tailored cell/gene-drivenI1-receptor treatments, and/or treatments that target cardiac I1-recep-tor, which could eventually be important for patients with hypertrophicheart disease.

l’inhibition de Rac1, et a démontré une interaction avec le substrat durécepteur de l’insuline. Cependant, on en connaît très peu sur le ré-cepteur I1 /nischarine cardiaque et son ou ses rôles dans la physiolo-gie normale et la physiopathologie, spécifiquement dans le remode-lage cardiaque. Nos études ont montré que le récepteur I1 est exprimédans les fibroblastes et les myocytes cardiaques, et que l’activation invitro du récepteur I1 inhibe l’apoptose cardiomyocytaire induite par lanorépinéphrine et la prolifération fibroblastique par des effets différen-tiels sur les protéines kinases activées par des agents mitogènes(MAPK: mitogen-activated protein kinase) et l’Akt. En conséquence,indépendamment du fonctionnement sympatholytique d’origine cen-trale, le récepteur I1 dans le cœur peut maîtriser la croissance et lamort cellulaires. Le récepteur I1 peut être impliqué dans le remodelagecardiaque et le dysfonctionnement par l’inhibition des voies apopto-tiques ou l’activation de voies de survie, ou les deux, de manièrespécifique à la cellule. L’identification de mécanismes cardioprotect-eurs du récepteur I1 cardiaque pourrait aboutir à des thérapies adap-tées spécifiquement à la cellule et/ou géniques ciblées sur le durécepteur I1 ou des traitements ciblant le récepteur I1 cardiaque, ou

tor blockers, exert protective effects on the heart, by loweringblood pressure and by preventing or reversing LVH, and isassociated with reduction of cardiovascular risk.3,10 The sec-ond generation centrally-acting antihypertensive compounds,moxonidine and rilmenidine, reduce blood pressure and pre-vent the development of LVH.11-17 Their sympatholytic actionis selectively mediated by imidazoline type-1 receptor (I1-re-ceptor), nonadrenergic neurotransmitter receptor mainlyfound in the central nervous system.18 We have identified car-diac I1-receptors, which may be independently implicated inLVH control. The following sections will focus on the effects ofmoxonidine and I1-receptors in blood pressure and LVH con-trol in heart disease.

Moxonidine in HypertensionMoxonidine is an orally administered moderately lipophilic

antihypertensive imidazoline compound. Its blood pressure-lowering effect equals that of thiazides, calcium antagonists,angiotensin-converting enzyme inhibitors, �- and �-adrener-gic receptor blockers, and the first generation centrally-actingantihypertensive compounds, such as �-methyl dopa and clo-nidine.13,17,19

Moxonidine is a structural homologue of clonidine. It actscentrally to reduce excessive sympathetic tone and peripheralvascular resistance, accompanied by reduced plasma norepi-nephrine and epinephrine levels as well as reduction in renin,angiotensin II, and aldosterone levels.20,21 The effects are se-lectively mediated by imidazoline I1-receptors with lower effectat �2-adrenergic receptors.18 Thus, in comparison with cloni-dine, which binds to both receptor types with equal affinity,moxonidine results in fewer undesirable side effects, such as drymouth, sedation, and mental depression, which have been at-tributed to �2-adrenergic receptors. Also, in contrast to othercentrally acting agents, moxonidine cessation does not result insympathetic overactivity and rebound hypertension. Studies inhypertensive patients show that, in addition to blood pressurelowering, moxonidine offers end-organ protection. Moxoni-dine may be used in treatment of hypertension complicatedwith LVH, coronary artery disease, and ventricular premature

beats.13,15-17,22,23

Moxonidine is well tolerated, safe, and effective in obeseand diabetic patients, in hypertensive patients with metabolicsyndrome, and in postmenopausal women with metabolic syn-drome.24-29 The antihypertensive effect is associated with im-proved glucose tolerance, insulin resistance, and lipid profile aswell as reduction in inflammatory markers, including plasmatumor necrosis factor (TNF)-�,.30,31 In these patients, mox-onidine may be favoured to �-blockers which exert adversemetabolic effects on lipids or insulin sensitivity and can causefurther weight gain.32 The adverse effects may be influenced byunopposed �1-adrenergic stimulation during �-blockade.

Moxonidine reduces exercise-induced sympathetic activa-tion without affecting exercise capacity and also blunts sympa-thetic activation due to mental stress. Thus, it may be consid-ered as an alternative to �-blockers in patients with SNShyperactivity and those bothered by �-blocker’s exercise limi-tation or asthma.13,33 In addition, moxonidine results in a clin-ically relevant reduction in blood pressure and muscle sympa-thetic nerve activity in patients with renal disease, in end-stagerenal disease patients, and patients with resistant hypertension;34-36 and may be an “add-on” treatment in elderly patientswhose hypertension is poorly controlled despite treatment with2 or more antihypertensive agents.35 Moxonidine can be com-bined with peripheral vasodilators and thiazide diuretics,37

which otherwise may further stimulate SNS activity,38 as wellas with angiotensin blockers.28,34 Furthermore, moxonidinepotentiates the antidepressant effects of fluoxetine and parox-etine, at doses inefficient by themselves.39 The anxiolytic andantidepressant effects of moxonidine may be especially impor-tant in cardiovascular diseases.

The cardioprotective mechanisms of moxonidine have beeninvestigated in animal models of essential hypertension andheart failure. In stroke-prone spontaneously hypertensive rats(SHR), moxonidine reduces blood pressure and the degree ofhypertrophy and myocardial fibrosis, capillarization, and re-gressive changes of myocytes.40 We have shown in SHR that4-week treatment with moxonidine reduces blood pressure andheart rate and improves global cardiac performance.41,42 Theeffects are associated with reduced collagen deposition andLVH regression associated with lower ventricular DNA con-

les deux, lesquels pourraient éventuellement être importants chez lespatients ayant une cardiomyopathie hypertrophique.

tent and synthesis as well as a transient apoptotic effect in left

Suhayla Mukaddam-DaherImidazoline Receptors and Heart Disease

3

ventricles that subsided by 4 weeks of treatment, evidenced byboth decreased, mitochondrial apoptotic Bax to anti-apoptoticBcl2 ratio and caspase3 activation.42 These effects coincidewith an early rise in the levels of atrial natriuretic peptide(ANP),42 a cardiac diuretic, natriuretic, and vasodilatory hor-mone, with antiproliferative, antifibrotic, and anti-inflamma-tory properties.43 Then, left ventricular natriuretic peptides arenormalized after 4 weeks of treatment, indicating treatmentefficacy.43 Similar results have been observed in obese SHR, amodel of metabolic syndrome seen in humans, where moxoni-dine improved the metabolic profile, reduced cardiac collagendeposition, and normalized left ventricular natriuretic peptidesynthesis.44,45

In addition, moxonidine treatment attenuated circulatingand cardiac inflammatory cytokines, TNF-�, interleukin (IL)-1�, and IL-6, and decreased phosphorylation of Akt and p38mitogen-activated protein kinase (MAPK) in SHR left ventri-cles.41,46 Importantly, the cytokines, IL-1�, IL-6, and TNF-�are implicated in the pathogenesis of cardiac hypertrophy andcardiomyocyte apoptosis, in vivo and in vitro,47-49 and in stim-ulation of cardiac fibroblast proliferation and differentiationinto activated myofibroblasts, which produce large amounts ofcollagens.50 Activation of p38 in cardiomyocytes leads to arapid onset of lethal cardiomyopathy associated with cardio-myocyte hypertrophy, interstitial fibrosis, and contractile dys-function.51 p38 is activated by cytokines and, in turn, it isinvolved in the release of proinflammatory cytokines,52 amechanism by which p38 contributes to myocytes cell death.48

Akt (or protein kinase B), a serine/threonine kinase with potentanti-apoptotic action in vitro and in vivo, is a downstreamtarget of phosphoinositide 3-kinase (PI3-K) and is linked tohyperplasia and hypertrophy of cardiomyocytes and increasedproliferation and collagen synthesis in fibroblasts.53 Akt acti-vation reduces cardiomyocyte death and protects against isch-emia-reperfusion injury,54 but sustained activation of Akt sig-nalling results in progression from adaptive to maladaptivehypertrophy and fibrosis.55

Clearly, the benefits of moxonidine on the hypertensiveheart involve anti-inflammatory effects. However, it is not clearwhether attenuation of p38 MAPK and Akt by moxonidine issubsequent to reduced cytokine levels, and/or interference withcytokine signalling in the heart. Alternatively, it may be due toreduced hemodynamic stress and subsequent effects of integ-rins, a family of transmembrane receptors that function asmechanotransducers, converting mechanical forces to bio-chemical signals. Integrin engagement with ligands activatesfocal adhesion kinase (FAK) and integrin-linked kinase. Thesekinases assemble onto the integrin scaffold to activate tyrosinekinases, such as Src, which induce oxidative stress and cardio-myocyte hypertrophy and apoptosis,56 through the activationof multiple signal transduction pathways. FAK/Src complexactivates extracellular signal-regulated kinase (ERK)1/2, c-JunN-terminal kinase (JNK), PI3-K/Akt/the mammalian target ofrapamycin (mTOR), and PI3-K/Rac1/p38 MAPK cascades;and integrin-linked kinase signals through Akt-nuclear factor(NF)-�B, Akt-mTOR, and Rac1.57,58

However, it is important to note that the benefits in SHRhearts which could be attributed to reduction of blood pressurewere also observed at subhypotensive doses.41,42,46 Also, addi-tion of a subhypotensive dose of moxonidine to an angiotensin-

receptor blocker, eprosartan, caused a larger reduction in left

ventricular mass than either treatment alone, despite the ab-sence of fall in blood pressure in response to moxonidine.46

These studies suggest an additional pressure-independent ef-fect of moxonidine on LVH control. Further studies are re-quired to investigate the mechanisms through which moxoni-dine exerts its cardiac benefit.

Moxonidine in Heart FailureAnimal studies have shown beneficial effects of moxonidine

in heart failure. After myocardial infarction in rats, chronicmoxonidine treatment suppressed sympathetic activity, pre-vented cardiac hypertrophy, and restored interstitial collagencontent to sham values.59 In heart failure BIO 14.6 hamsters, agenetic model of dilated cardiomyopathy, moxonidine im-proved diastolic and systolic cardiac functions and exerted dif-ferential inflammatory/anti-inflammatory responses that cul-minated in attenuated cardiac apoptosis and fibrosis andaltered ventricular protein expression of collagen types. Al-though observed in moderate as well as advanced heart failurehamsters, the benefits were more evident in the moderate heartfailure group.60 These studies confirm and contrast studies inhumans. Dickstein et al.61 and Swedberg et al.62 reported thatin patients with congestive heart failure, chronic treatmentwith moxonidine effectively attenuated sympathetic tone andimproved the hemodynamic profile. However, the phase IIItrial, the Moxonidine Congestive Heart Failure (MOXCON)trial in patients with NYHA class II-IV heart failure and leftventricular ejection fraction � 35%, reported more deaths inthe moxonidine-treated group,63,64 which led to its abrupt ter-mination. Several reasons could be hypothesized for the failingtrial: patients with very low baseline plasma norepinephrinewould not benefit from sympathetic suppression; force-titra-tion of very high doses (5-10 times higher than the recom-mended antihypertensive doses) could lead to strong suppres-sion of much needed residual sympathetic activity to the heart.Also lack of patient compliance leading to rebound effect of thedrug, or increased myocardial metabolism,61,62,65,66 may un-derlie the findings of the trial. Keeping in mind that the use of�-blockers is recommended in heart failure, these studiesclearly demonstrate that we do not properly understand themechanism of action of sympatholytic compounds, specificallycentrally acting compounds, which may have alternative drugtargets or different target distribution, regulation, cell signal-ling pathways, and interaction with other mechanisms stimu-lated in hypertension and heart failure.

Imidazoline I1-receptor: discovery, distribution, andmechanisms of action

Blood pressure reduction by centrally acting compounds,moxonidine and rilmenidine, is dependent on inhibiting re-lease of the neurotransmitter norepinephrine, in the brainstemrostral ventrolateral medulla (RVLM), 1 of the vasomotor cen-tres that regulate peripheral SNS activity. Szabo et al.67 showedin pithed rabbits with electrically stimulated sympathetic out-flow that rilmenidine and moxonidine, injected intravenously,dose-dependently lowered blood pressure and plasma norepi-nephrine concentration and inhibited stimulation-evoked car-dioacceleration. Inhibition of norepinephrine release by mox-

onidine has also been demonstrated in pithed SHR.68

4 Canadian Journal of CardiologyVolume xx 2012

The actions of centrally acting compounds are mediated by2 functionally and genetically distinct receptors: �2-adrenergicreceptors and nonadrenergic imidazoline I1-receptors, withhigher affinity at imidazoline I1-receptors.

The existence of nonadrenergic imidazoline I1-receptorsimplicated in central regulation of the circulation was first pro-posed by Bousquet and coworkers69 in 1984, based on func-tional studies. Later, binding studies identified I1-receptors inbrainstem medulla, adrenal chromaffin cells, platelets, liver,and kidneys.70 Imidazoline I1-receptor signalling has beenmostly investigated in vitro in rat neuronal pheochromocy-toma PC12 cells, because these cells constitutively express I1-receptors but not �2-adrenergic receptors, as confirmed withradioligand binding and molecular approaches. In these cells,stimulation by imidazolines activates phosphatidylcholine-phospholipase C (PC-PLC), followed by increased formationof diacylglycerol (DAG) and release of phosphocholine. Theaccumulation of the lipid second messenger DAG augmentsarachidonic acid release through DAG lipase. Subsequent sig-nalling steps include activation of protein kinase C (PKC) anddownstream MAPKs.70-73 Other studies revealed that I1-recep-tor activation decreases forskolin-stimulated cyclic adenosinemonophosphate (cAMP).74 In rat superior cervical ganglionneurons, I1-receptor activation by moxonidine inhibits volt-age-dependent N-type Ca2� current.75

In vivo studies confirmed that the vasodepressor effect ofimidazoline I1-receptor activation occurs through PC-PLCpathway,70,71 and includes ERK activation, as well as nitricoxide (NO) release from inducible NO synthase.76-78 In SHR,the vasodepressor response to intravenous moxonidine wascompletely prevented by microinjection of a PC-PLC inhibi-tor, D609, into the brainstem.71 Also, the central blockade ofNO synthase by local injections of NG-nitro-L-argininemethyl ester (L-NAME) abolished the hypotension and vaso-dilatation elicited by a central injection of moxonidine77; and aspecific inducible NO synthase inhibitor prevented the de-crease in blood pressure and renal sympathetic nerve activityevoked by intravenous or central injections of moxonidine.78

Knockdown of central imidazoline I1-receptors by antisenseoligodeoxynucleotide abrogated rilmenidine-evoked hypoten-sion and ERK1/2 activation in the RVLM.76 Intravenous mox-onidine increases the release of gamma-aminobutyric acid(GABA) in the RVLM and central infusion of moxonidineupregulates the protein expression of GABA receptors in theRVLM, demonstarting that GABAergic mechanisms in theRVLM are responsible for the hypotension and sympathoinhi-bition of moxonidine.79

Our studies in normotensive and hypertensive rats revealedthat acute intravenous injections of moxonidine lower bloodpressure and increase sodium and water excretion, by mecha-nisms that include natriuretic peptide, ANP, and B-type natri-uretic peptide (BNP).80-82 Thus, in addition to inhibition ofrenal nerve activity, stimulation of natriuretic peptides by mox-onidine contributes to acute and long-term control of bloodpressure.

Moxonidine stimulation of natriuretic peptides, which areprimarily of cardiac origin, led to the investigation of the effectof imidazolines on ANP release from the heart. Perfusion ofisolated rat hearts with imidazoline compounds, includingmoxonidine, stimulated ANP release.83,84 These studies in de-

centralized hearts indicated for the first time, a direct cardiac

target for moxonidine (I1-receptor) that was functional with-out the contribution of the central nervous system.

Binding and immunohistochemical studies confirmed thepresence of I1-receptors in heart atria and ventricles,85 andrevealed I1-receptor upregulation in rat hypertension and ham-ster and human heart failure.60,85 Upregulated I1-receptors inSHR hearts were normalized by chronic in vivo treatment withmoxonidine.44,86 In contrast, heart I1-receptors were furtherupregulated by in vivo moxonidine treatment in hamster heartfailure.60 The regulation of cardiac I1-receptors in cardiovascu-lar diseases and in response to exogenous pharmacological treat-ment implies that cardiac I1-receptors are responsive to the cardio-vascular environment; and, more importantly, that their functionappears to be more complex than that ascribed to their sympatho-lytic actions. These studies formed the basis of our interest inI1-receptors in cardiac cell growth and death rather than in bloodpressure regulation by moxonidine. A closer look at I1-receptorstructure and signalling would lead to a better understanding ofcardiac I1-receptor function.

Molecular characterization and cellular functions

Imidazoline I1-receptors have been independently clonedby 2 groups. Piletz and colleagues87 cloned an I1-receptor can-didate protein, from a human hippocampal cDNA expressionlibrary by the use of 2 separate antibodies recognizing imidaz-oline binding sites. Further characterization led to the identi-fication of an imidazoline receptor antisera-selected (IRAS)167 kD protein encoding 1504 amino acid residues. Overex-pression of IRAS in Chinese hamster ovary or PC12 cell lineswere found to induce high affinity, specific imidazoline bind-ing capacity.88

On the other hand, during their search for proteins thatbind to integrin tail regions, to identify downstream effectors,Alahari and coworkers89,90 found a novel protein that interactspreferentially with the �5 cytoplasmic domain of the �5�1fibronectin receptor, which, upon overexpression in fibro-blasts, evokes changes in cytoskeletal organization and pro-found inhibition of cell migration through inhibition of Rac1and subsequent NF-�B pathway.89,90 Alahari called it nis-charin, a Sanskrit word that connotes slow motion. Interest-ingly, upon searching the mouse genome sequence with nis-charin, the newly-identified protein appeared to be the mousehomologue of IRAS. Studies with overexpression or suppres-sion with antisense confirmed that IRAS/nischarin is a func-tional I1-receptor.91-94

Although mostly investigated in cell motility and cancerresearch, the inhibition of Rac1 by nischarin, and henceI1-receptor, is attractive to cardiovascular research. Rac1, asmall guanosine triphosphatase catalytic subunit of nicoti-namide adenine dinucleotide phosphate oxidase, is a keysignalling protein at which hypertension-associated hemo-dynamic and biochemical stresses converge to induce cellproliferation, hypertrophy, and apoptosis mediated by in-creasing intracellular reactive oxygen species (ROS) andsubsequent NF-�B/p38, JNK, or PI3-K/Akt pathways.95-97

The Rac1-ROS signalling pathway is a crucial mediator ofcardiovascular disease,96 and targeting inhibition of Rac1may be a therapeutic approach for pressure-overload in-duced cardiovascular disease.

I -receptor/nischarin is not a classic transmembrane recep-

1tor; it is rather a protein localized to the cytosol, anchored on

es.

Suhayla Mukaddam-DaherImidazoline Receptors and Heart Disease

5

the inner layer of plasma membranes; and it has multiple do-mains that allow it to bind several proteins to regulate differentbiological functions.94 As mentioned earlier, nischarin, and byinference I1-receptor, influences cytoskeletal organizationthrough binding to the �5�1 integrin, the principal cellularreceptor for fibronectin. It associates with Lin-11/Isl-1/Mec-3kinases (LIMK) to regulate breast and colon epithelial cell in-vasion by negative modulation of the LIMK/cofilin pathway.98

I1-receptor also associates with insulin receptor substrates inHEK293 cells and enhances insulin receptor substrate-4-de-pendent insulin activation of ERK1/2,99 mechanisms that mayexplain the benefits of moxonidine in patients with metabolicsyndrome. PC12 and COS7 cells stably transfected with I1-receptor exhibit prolonged cell survival when challenged withproapoptotic stimuli, suggesting an antiapoptotic effect of I1-receptor.100,101 Activation of I1-receptor also protects againstnaphthazarin-induced oxidative toxicity and astrocyte celldeath,102 and protects against glutamate-induced neurotoxic-ity in neuronal cell culture experiments.103

We have shown that I1-receptor is expressed in cardiac myo-cytes and fibroblasts. Activation of I1-receptor by moxonidineis cell-selective: in cardiomyocytes, it protects against cell death

Figure 1. Schematic diagram showing hypothetical mechanisms onischarin. I1-receptor/nischarin is a cytosolic protein anchored onphosphatidyl choline phospholipase C (PC-PLC) with subsequent act(MAPK) pathway.70-73 I1-receptor associates with integrin �5 subuniMAPK pathways.89,90 Also, I1-receptor associates with insulin recsignalling.99 Activation of I1-receptor inhibits cytokines,41 through ainteraction of intracellular MAPKs, phosphoinositide 3-kinase (PI-3K)/Dotted arrows indicate inhibition. Solid arrows indicate stimulatireceptors; JNK, c-Jun N-terminal kinase; ROS, reactive oxygen speci

by mechanisms that include inhibition of norepinephrine-

stimulated p38 MAPK phosphorylation while maintainingAkt phosphorylation.41 In fibroblasts, moxonidine results incell death, accompanied by inhibition of norepinephrine-in-duced p38 MAPK and Akt phosphorylation.41 The signallingpathways leading to differential effects of I1-receptor activationin cardiomyocyte and fibroblasts is under current investigationand may include cell-specific differences in integrin-dependentsignal transduction pathways.

Importantly, changes in fibroblast proliferation and apop-tosis quantitatively and qualitatively influence changes in theextracellular matrix, including formation and remodelling.Thus, these in vitro studies point to a role of I1-receptor as aprotein involved in many cellular effects, including cell survi-val/death and extracellular matrix dynamics, and raise interestin this protein beyond its central actions on blood pressurecontrol. However, very little is known about cardiac I1-recep-tor and its role(s) in normal physiology and pathophysiology,specifically in cardiac remodelling.

Clinical PerspectiveMoxonidine is an imidazoline compound used in hyperten-

c hypertrophy control by imidazoline type-1 receptor (I1-receptor)/er layer of plasma membrane. Activation of I1-receptor increasesof protein kinase C (PKC) and the mitogen-activated protein kinasehibits Rac1 activation and subsequent nuclear factor-�B (NF-�B) orubstrates (IRS)1-4 and leads to subsequent Akt/Rac1 and MAPKidentified mechanism. The cross-talk and positive and/or negatived Rac1 determines the fate of the cardiac myocyte and fibroblast.41

K, extracellular signal-regulated kinase; GPCR, G-protein coupled

f cardiathe innivationt and ineptor syet unAkt, anon. ER

sion treatment in Europe, Brazil, Middle East, Far East, and

6 Canadian Journal of CardiologyVolume xx 2012

South Africa. Its use is contraindicated in heart failure treat-ment, which probably influenced its absence from the NorthAmerican markets. Our studies revealed cardiac benefits ofmoxonidine in hypertensive and heart failure animals. The ac-tions of moxonidine, which are primarily mediated by centralI1-receptors may also include direct actions on cardiac I1-recep-tors. However, the direct implication of cardiac I1-receptors inmoxonidine’s in vivo cardioprotective effects and the mecha-nisms mediating them have not been demonstrated. Systemicadministration of moxonidine cannot dissociate the relativecontribution of brain receptors with subsequent blood pres-sure-lowering from the direct consequences of cardiac receptoractivation on LVH regression and improved cardiac function,although a pressure-independent outcome 41,42,46 suggests alocal cardiac effect.

Recent studies by Alahari’s group104 show higher expressionof nischarin mRNA in normal human breast tissue than intumour tissue, associated with decreased �5 integrin expres-sion, FAK phosphorylation, and Rac activation. They alsoshow that nischarin suppression leads to faster cell growth.Nischarin may be a novel tumour suppressor that limits breastcancer progression by regulating �5 integrin expression andsubsequently �5 integrin-, FAK-, and Rac-mediated signal-ling.104 Therefore, based on the intrinsic antiproliferativeaction of nischarin and our studies showing upregulatedI1-receptors/nischarin in SHR hearts,85 it is tempting tospeculate that I1-receptor upregulation is an adaptive re-sponse of the myocardium to pressure overload and thatinhibition of Rac1 through upregulated I1-receptor couldcontribute to LVH control. Accordingly I1-receptor overex-pression may be a novel approach for the treatment of car-diac hypertrophy and prevention of heart failure. Alterna-tively, cardiac I1-receptor suppression in the presence ofLVH may be deleterious. Studies with genetic modificationof cardiac I1-receptors will validate this hypothesis and carrythe research into the clinical direction.

To this end, current studies aim at determining whethercardiac I1-receptor knockout/knockin modulates myocar-dial oxidative stress and the development of cardiac hyper-trophy in response to a chronic hemodynamic load, associ-ated with increased pressure and neurohormonal/growthpromoting stimuli (Fig. 1). These studies will delineatedownstream signalling pathways that are both unique andcommon to the SNS/norepinephrine and imidazoline I1-receptors, including natriuretic peptides, inflammatory cy-tokines, and oxidative stress through Rac1-ROS pathway,with the intent of better understanding the important ho-meostatic, as well as the deleterious interactions that occurbetween these systems. The elucidation of possible interac-tions of these systems on cardiac survival and/or apoptosismay be of major interest to prevent cardiac pathologies pro-gression from compensation to decompensation. Finally,while the antihypertensive action of moxonidine is appreci-ated, a cardiac-selective drug with cell-specific and/or path-way-selective properties may confer additional benefit. Newdrugs or procedures specifically targeting the heart to in-crease I1-receptor levels and/or activity may prevent the de-velopment of cardiac remodelling and heart failure and

would be a good addition to treatment regimens.

Funding SourcesSMD’s studies have been supported by grants from the Ca-

nadian Institutes of Health Research and the Heart and StrokeFoundation of Canada.

DisclosuresThe authors have no conflicts of interest to disclose.

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