Molecular regulation of cardiac hypertrophy

The International Journal of Biochemistry & Cell Biology 40 (2008) 2023–2039

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Review

Molecular regulation of cardiac hypertrophy Sean P. Barry a,1 , Sean M. Davidson b,1 , Paul A. Townsend c,∗ a

Medical Molecular Biology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N IEH, United Kingdom The Hatter Cardiovascular Institute, Department of Medicine, Royal Free and University College Medical School, 67 Chenies Mews, University College Hospital, London WC1E 6HX, United Kingdom c Human Genetics Division, MP808, Duthie Building, Southampton General Hospital, Tremona Road, University of Southampton, Southampton SO16 6YD, United Kingdom b

a r t i c l e

i n f o

Article history: Received 30 November 2007 Received in revised form 13 February 2008 Accepted 15 February 2008 Available online 26 February 2008 Keywords: Cardiac Hypertrophy Molecular Signalling Transcription

a b s t r a c t Heart failure is one of the leading causes of mortality in the western world and encompasses a wide spectrum of cardiac pathologies. When the heart experiences extended periods of elevated workload, it undergoes hypertrophic enlargement in response to the increased demand. Cardiovascular disease, such as that caused by myocardial infarction, obesity or drug abuse promotes cardiac myocyte hypertrophy and subsequent heart failure. A number of signalling modulators in the vasculature milieu are known to regulate heart mass including those that influence gene expression, apoptosis, cytokine release and growth factor signalling. Recent evidence using genetic and cellular models of cardiac hypertrophy suggests that pathological hypertrophy can be prevented or reversed and has promoted an enormous drive in drug discovery research aiming to identify novel and specific regulators of hypertrophy. In this review we describe the molecular characteristics of cardiac hypertrophy such as the aberrant re-expression of the fetal gene program. We discuss the various molecular pathways responsible for the co-ordinated control of the hypertrophic program including: natriuretic peptides, the adrenergic system, adhesion and cytoskeletal proteins, IL-6 cytokine family, MEK-ERK1/2 signalling, histone acetylation, calcium-mediated modulation and the exciting recent discovery of the role of microRNAs in controlling cardiac hypertrophy. Characterisation of the signalling pathways leading to cardiac hypertrophy has led to a wealth of knowledge about this condition both physiological and pathological. The challenge will be translating this knowledge into potential pharmacological therapies for the treatment of cardiac pathologies. © 2008 Elsevier Ltd. All rights reserved.

Abbreviations: ACE, angiotensin-converting enzyme; ANF, atrial natriuretic factor; AngII, angiotensin II; ANP, atrial natriuretic peptide; ASK-1, apoptosis signalling kinase-1; AT-1, angiotensin type-1; AR, adrenergic receptors; BNP, B-type natriuretic peptide; CaMKII, calcineurin and Ca2+ /calmodulin-dependent kinase II; CBP, CREB-binding protein; cGMP, guanosine 3 ,5 -cyclic monophosphate; CHF, coronary heart failure; CNP, C-type natriuretic peptide; CT-1, cardiotrophin-1; CVD, cardiovascular disease; DCM, dilated cardiomyopathy; DUSP6, dual-specific phosphatase-6; ECM, extracellular matrix; ERK, extracellular signal receptor-regulated kinase; GPCR, G-protein-coupled receptor; GTP, guanosine triphosphate; HAND, heart and neural crest derivatives; HCM, familial hypertrophic cardiomyopathy; JAK, Janus-activated kinase; LIF-1, leukaemia-inhibitory factor-1; MAPK, mitogen-activated protein kinase; MEF2, myocyte-specific enhancer factor 2; MEK, MAPK/ERK kinase; ␣-/␤-MHC, myosin heavy chain alpha or beta; miRNA, microRNA; NFAT, nuclear factor of activated T cells; NF-␬B, nuclear factor-kappa B; NP, natriuretic peptides; NPR, natriuretic peptide receptor; PDE5A, phosphodiesterase 5A; PE, phenylephrine; PGK-1, protein kinase-1; PI(3)K, phosphatidyl inositol-3 kinase; PKA, protein kinase A; PKC, protein kinase C; SERCA, sarco-/endo-plasmic reticulum Ca2+ -ATPase; SirT, SirT-related protein; SR, sarcoplasmic reticulum; STAT, signal transducer and activator of transcription; TAC, thoracic aortic constriction; THRAP1, thyroid receptor-associated protein 1; VSM, vascular smooth muscle; YFP, yellow fluorescent protein. ∗ Corresponding author. Tel.: +44 23 8079 8692; fax: +44 23 8079 4264. E-mail addresses: [email protected] (S.P. Barry), [email protected] (S.M. Davidson), [email protected] (P.A. Townsend). 1 SPB and SMD contributed equally to this article (surnames listed alphabetically). 1357-2725/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2008.02.020

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Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inherited cardiomyopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The fetal gene program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Myosin heavy chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Natriuretic peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. G-protein-coupled receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Interluekin-6 family of cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Adhesion and cytoskeletal proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Mitogen-activated protein kinase/extracellular signal receptor-regulated kinase signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Histone acetylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Histone acetyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Histone deacetylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. Class III HDACs role in cardiac hypertrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Calcium-mediated pathways towards hypertrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. MicroRNAs—new players in cardiac hypertrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Cardiovascular disease (CVD) is one of the leading causes of death worldwide, accounting for 16.7 million deaths per annum (the World Health Organization— http://www.who.int/dietphysicalactivity/publications/ facts/cvd/en/). CVD encompasses a wide spectrum of cardiac pathologies including 5.3 million people living with heart failure in the USA alone (the American Heart Association—http://www.americanheart.org/presenter. jhtml?identifier=4478). In the various forms of heart failure, excessive cardiac workload leads to an enlargement of the heart in an endeavour to manage the increased hemodynamic demand. This process, known as hypertrophy, is classified as “physiological” hypertrophy when it occurs in healthy individuals following exercise and is not associated with cardiac damage, or “pathological” hypertrophy (Fig. 1). In the case of pathological hypertrophy, although the increased heart size is initially a compensatory mechanism, sustained hypertrophy can ultimately lead to a decline in left ventricular function and thus represents an independent risk factor for heart failure (Levy, Garrison, Savage, Kannel, & Castelli, 1990). The main causes of pathological hypertrophy are hypertension, genetic polymorphisms, and loss of myocytes following ischaemic damage. Altered cardiac metabolism can also be an important component leading to hypertrophy (Rajabi, Kassiotis, Razeghi, & Taegtmeyer, 2007). Indeed, this mechanism is so prevalent in the setting of diabetes that it has led to the description of a specific syndrome of “diabetic cardiomyopathy” (Boudina & Abel, 2007). Hypertrophic growth develops in two ways: concentric hypertrophy is caused by chronic pressure overload and leads to reduced left ventricular volume and increased wall thickness whereas eccentric hypertrophy is caused by volume overload and causes dilation and thinning of the heart wall (Wakatsuki, Schlessinger, & Elson, 2004). Mechanistically, eccentric expansion occurs by addition of sarcomeres in series causing cell elongation; concentric enlargement on the other hand is caused by addition of sarcomeres

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in parallel resulting in increased cell thickness. Accompanying the increase in myocyte size, there is an increase in the number of cardiac fibroblasts causing fibrosis and increased myocardial stiffness (Wakatsuki et al., 2004). This in turn leads to overload and promotes further hypertrophy and cell death, resulting in a detrimental cycle of cardiac enlargement and myocyte loss. Although hypertrophy is ultimately a detrimental process, it is nonetheless highly organized and tailored to the specific needs of the heart. Thus, pressure and volume overload lead to concentric and eccentric hypertrophy via co-ordinated activation of specific intracellular signalling pathways, ultimately altering myocyte shape in a way that is best suited to deal with the specific burden. In this review we will discuss the various molecular pathways that are responsible for the co-ordinated control of the hypertrophic program. 2. Inherited cardiomyopathy The aetiology of cardiomyopathies can be idiopathic, immunological, toxicological or genetic but in general, a defect in normal contractile function is compensated for by hypertrophic remodelling. The two major inherited forms are hypertrophic cardiomyopathy (HCM) characterized by increased wall thickness, reduced ventricular chamber volumes and reduced diastolic function and dilated cardiomyopathy (DCM) characterized by increased chamber volume, defects in systolic function and ventricular tachyarrhythmias. A diverse range of mutations underlie familial hypertrophy, but the great majority are located in genes encoding sarcomeric proteins, and can drastically alter Ca2+ handling and contractility, leading to arrhythmias and sudden cardiac death. These have been thoroughly reviewed previously (Nicol, Frey, & Olson, 2000), but some pertinent examples are given below. The first identified mutation causing a cardiomyopathy was a missense mutation in the ␤-myosin heavy chain (␤-MHC) gene associated with HCM (Geisterfer-Lowrance et al., 1990). The thick filament of the sarcomere con-

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Fig. 1. Pathological and physiological hypertrophic response to stimuli. When the heart experiences extended periods of elevated workload, it undergoes enlargement in an effort to cope with the increased demand. This process, known as hypertrophy, is classified as “physiological” when it occurs in healthy individuals following exercise or pregnancy and is not associated with cardiac damage. Whereas the increased heart size in “pathological” hypertrophy presents initially as a compensatory mechanism, sustained hypertrophy may ultimately lead to a decline in left ventricular function and as such represents an independent risk factor for heart failure. The main causes of pathological hypertrophy are hypertension, genetic polymorphisms, and loss of myocytes following ischaemic damage. Hypertrophic growth develops in two ways: concentric hypertrophy caused by chronic pressure overload leading to reduced left ventricular volume and increased wall thickness; eccentric hypertrophy due to volume overload and causing dilation and thinning of the heart wall. Eccentric expansion occurs by addition of the contractile sarcomere in series causing cell elongation whereas concentric enlargement is caused by addition of sarcomeres in parallel resulting in increased cell thickness.

sists primarily of myosin, a protein composed of two heavy chains and four light chains which hydrolyses ATP to move actin myofilaments in response to Ca2+ —thereby mediating cardiac contraction. Over 400 mutations in patients with cardiomyopathy have now been identified in 9 separate sarcomeric genes, including troponin, ␣tropomyosin, myosin-binding protein C and actin (Lowey, 2002; Richard et al., 2003) but the majority are localized in the ␤-MHC (MYH7) gene (Richard et al., 2003). Early work on the prominent R403Q mutation in MYH7 showed that it reduced ATPase activity, resulting in an 80% decrease in maximum velocity compared to wildtype myosin (Cuda, Fananapazir, Epstein, & Sellers, 1997), although recent studies have shown the same mutation can actually enhance mechanical performance at the single molecule level (Palmiter et al., 2000). Other ␤-MHC mutations can affect aspects other than contractility, for example the A223T mutation affects thermostability and protein folding, while the S642L mutation may alter the conformation of the actin-binding site (Karkkainen & Peuhkurinen, 2007). In all cases, the end result is compensation for altered workload though hypertrophy. The thin filament of the sarcomere is composed of a double helix of actin bound to tropomyosin and troponin. Troponin is comprised of three subunits: troponin T (TnT), which binds to tropomyosin; troponin C (TnC), the Ca2+ binding subunit and the inhibitory troponin I (TnI)

(Metzger & Westfall, 2004). Mutations have been identified in all three troponin subunits, with mutations in TnT being the most prevalent (Chang & Potter, 2005). Though the mutations responsible for DCM tend to be more diverse and are less well understood than those for HCM, in general it seems that HCM mutations cause increased Ca2+ sensitivity while DCM mutations decrease it. For example the A2V mutation in TnI reduces myocyte contraction and results in DCM (Murphy et al., 2004), while the R21C mutation increases Ca2+ sensitivity and force development and results in HCM (Gomes, Harada, & Potter, 2005). Similarly, TnT mutations from HCM patients tend to increase Ca2+ sensitivity of myofilaments, enhancing contractility, impairing relaxation and causing diastolic abnormalities, whereas many TnT mutations from DCM patients have the opposite effect, i.e. decreased Ca2+ sensitivity. Importantly, in a knock-in mouse model harbouring the TnT K210 mutation which causes DCM (Kamisago et al., 2000; Karkkainen & Peuhkurinen, 2007), increasing Ca2+ sensitivity with the inotropic agent pimobendan ameliorated the cardiac hypertrophy and heart failure, suggesting that restoring Ca2+ sensitivity may be very beneficial in certain DCM patients (Du et al., 2007). These and other examples illustrate that mutations in practically any sarcomeric proteins may eventuate in cardiomyopathy if contractility is affected, thereby initiating the hypertrophic program.

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3. The fetal gene program Following the onset of increased mechanical stress or pressure overload one of the initial molecular changes is reactivation of the so-called fetal gene program—a set of genes that are normally expressed only in the developing heart and are repressed in the adult myocardium. Activation of the fetal gene program allows co-ordinated synthesis of the proteins needed to bring about increased cardiac myocyte size and adjustment to the altered energy demands of these larger cells. Indeed, correspondence analysis between gene expression profiles in normal adult heart, failing heart and fetal heart revealed that the pattern of highly differentially expressed genes is very similar in fetal and failing hearts (Thum et al., 2007). Reinduction of the fetal gene program is associated with pathology—eccentric and concentric hypertrophy induces fetal gene expression, whereas physiological hypertrophy does not. While there are a host of re-expressed fetal genes during hypertrophy, the expression levels of myosin heavy chains, the natriuretic peptides and ␣-actinin, are used most commonly as molecular markers of hypertrophy. 3.1. Myosin heavy chain It has been appreciated for over 20 years that one of the hallmarks of cardiac hypertrophy in patients and in rodent models is an increase in ␤-MHC expression and a decrease in ␣-MHC expression (Izumo et al., 1987; Mahdavi, Lompre, Chambers, & Nadal-Ginard, 1984). Since each isoform has a distinct enzymatic activity, it means that their relative ratios greatly influence cardiac function, thus an increase in ␤-MHC decreases the myosin ATPase enzyme velocity which in turn slows the myocyte contraction rate, a key adaptation to altered workload (Lowes et al., 1997). Reversion of hypertrophy is associated with a return of MHC isoform levels to normal, for example, in patients with dilated cardiomyopathy undergoing ␤-blocker therapy, recovery was associated with increased ␣-MHC and decreased ␤-MHC expression (Lowes et al., 2002). While left ventricular MHC isoform switching has been studied extensively in humans and animal models, one complication is that whereas in adult humans 90% of the total MHC pool is of the ␤-MHC isoform, in adult rodents ␣-MHC is the predominant form, probably because of the much greater heart rate of rodents (LeWinter, 2005). Therefore, while ␤-MHC expression is increased in human heart failure it is not as pronounced as the increase seen in rodents. This has complicated the interpretation of results from animal models. Despite the undeniable genetic link between MHC proteins and hypertrophy, recent work has suggested that increased ␤-MHC expression in an individual cell is not necessarily associated with an increase in hypertrophy (Pandya, Kim, & Smithies, 2006). 3.2. Natriuretic peptides The natriuretic peptides (NP) are a family of hormones that affect the cardiovascular and endocrine systems through their actions in diuresis, natriuresis, vasorelaxation and aldosterone and renin inhibition (Nishikimi, Maeda, &

Matsuoka, 2006). The NPs are potent endogenous inhibitors of hypertrophy. There are three members: atrial natriuretic peptide (ANP) also known atrial natriuretic factor (ANF), brain or B-type natriuretic peptide (BNP) and C-type (CNP). In the heart, ANP expression is confined to the atria, BNP is expressed in both the atria and ventricles and the extent of CNP expression in the heart is unclear. Following their secretion, the N-terminals of NPs are cleaved to release the biologically active C-terminal derived peptide and exert their effects by binding to the natriuretic peptide receptors (NPRs), also known as guanylyl cyclase receptors. Binding to the receptor induces guanylyl cyclase activity and subsequent conversion of guanosine triphosphate (GTP) to the second messenger guanosine 3 ,5 -cyclic monophosphate (cGMP) (Gardner, Chen, Glenn, & Grigsby, 2007). NP signalling is terminated either though binding to NPR-C which internalises and degrades NPs thus removing them from the circulation or through cleavage by neutral endoperoxidase (Gardner, 2003; Richards, 2007). A feedback loop also exists where ANP inhibits the expression of its own receptor by cGMP-mediated transcriptional repression (Hum et al., 2004). ANP and BNP are found at high levels during embryonic development and in early neonates but are absent in healthy adults (Gardner, 2003). Hypertrophic stimuli dramatically increase the expression of ANP and BNP through the transcription factor GATA-4 and their main function in the myocardium is to inhibit the hypertrophic response (Gardner et al., 2007; Richards, 2007). Deletion of ANP in mice causes hypertension and hypertrophy under resting conditions, while volume and pressure overload both activate the hypertrophic response in the mutant mice to a greater extent than in wild-type mice (Mori et al., 2004; Wang et al., 2003). When wild-type and ANP−/− mice were fed a low-salt diet, differences in blood pressure disappeared, however ANP−/− mice still had more extensive hypertrophy, demonstrating that the anti-hypertrophic effects of ANP are independent of increased blood pressure (Feng et al., 2003). ANP-deficient mice also had enhanced expression of extracellular matrix (ECM) proteins following hypertrophy, suggesting that ANP may reduce hypertrophic remodelling through inhibition of ECM deposition (Wang et al., 2003). Deletion of BNP, unlike ANP, did not result in hypertrophy or hypertension but caused an increase in interstitial fibrosis in the ventricles which was exacerbated in response to ventricular overload (Tamura et al., 2000). Further evidence for the anti-hypertrophic activity of ANP and BNP comes from studies in NPR-A-deficient mice. NPR-A is the sole receptor for ANP and BNP and deletion of the receptor abolishes their functionality in the cardiovascular system (Lopez, Garbers, & Kuhn, 1997). NPR-A−/− mice suffer from hypertension, interstitial fibrosis, ventricular hypertrophy and heart failure-like symptoms (Kishimoto, Rossi, & Garbers, 2001; Knowles et al., 2001; Oliver et al., 1997). As with the ANP-deficient mice, controlling hypertension through drugs fails to ameliorate the hypertrophic phenotype, again demonstrating that hypertrophy is functionally distinct from elevated blood pressure in the context of reduced natriuretic signalling (Knowles et al., 2001). Specific deletion of NPR-A in the heart using an ␣-MHC-Cre driver definitively showed that natriuretic peptides have

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a direct effect on the myocardium (Holtwick et al., 2003). These mice exhibited a mild increase in hypertrophy associated with upregulation of ANP, ␤-MHC and ␣-skeletal actin, and hypertrophy following transverse aortic constriction was exacerbated when compared to wild-type controls (Holtwick et al., 2003). Again, these effects were not attributable to differences in BP since the mice were found to be slightly hypotensive. Interestingly, in humans, certain ANP- and BNP-receptor polymorphisms have been associated with left ventricular mass in essential hypertension (Rubattu et al., 2006). In contrast to ANP and BNP, less is known about the role of CNP in hypertrophy. CNP is predominately produced in vascular and endothelial cells where it is important in the regulation of vascular tone, blood flow and pressure (Ahluwalia & Hobbs, 2005). CNP has also been shown to have anti-hypertrophic effects in vitro and at the same concentration appears to be equally as potent as ANP and BNP (Rosenkranz, Woods, Dusting, & Ritchie, 2003; Tokudome et al., 2004). Moreover, CNP administration in vivo inhibits both fibrosis and hypertrophy following coronary artery ligation (Soeki et al., 2005). This result has been confirmed in transgenic mice overexpressing CNP and in transgenic rats expressing a dominant-negative NPR-B, the sole receptor for CNP (Langenickel et al., 2006; Wang et al., 2007). CNP has been shown to be produced by the heart, where levels increase during CHF and indeed CNP may have some prognostic value in terms of disease severity (Del Ry, Passino, Maltinti, Emdin, & Giannessi, 2005; Kalra et al., 2003). The signalling pathways responsible for NP-mediated inhibition of hypertrophy are not entirely established. It is clear that the mitogen-activated protein kinase (MAPK) pathway is involved; ANP was shown to inhibit p38 MAPK and ERK activity in smooth muscle cells in response to angiotensin II and NPR-A-deficient mice have increased basal phosphorylation of p38, ERK and Akt in the heart (Kilic et al., 2005; Sharma et al., 2002). All three NP hormones induce cGMP production which appears to be the common mediator of NPs in hypertrophy. Stimulating adult cardiac myocytes with a cGMP analogue has the same anti-hypertrophic effect as treating with the NPs themselves (Calderone, Thaik, Takahashi, Chang, & Colucci, 1998; Rosenkranz et al., 2003). Similarly, nitric oxide, which induces cGMP via the soluble GC receptor, can block hypertrophy (Calderone et al., 1998). Precisely how increased cGMP production prevents cardiac hypertrophy has not been entirely elucidated but it likely involves cGMPdependent protein kinase-1 (PGK-1) a serine/threonine kinase activated by cGMP. PGK-1 inhibits l-type Ca2+ channels thus reducing Ca2+ transient amplitude and blocking calcineurin-mediated activation of NFAT, a transcription factor that is obligatory for hypertrophy (Fiedler et al., 2002) (Fig. 2). Indeed, pharmacological inhibition of PGK-1 abrogates the effects of BNP and CNP in cardiac myocytes (Calderone et al., 1998). cGMP is normally broken down by phosphodiesterase-5A (PDE5A) and Takimoto et al. recently showed that the PDE5A inhibitor sildenafil (commonly known as Viagra) reduces hypertrophy and improves heart function in response to transverse aortic constriction (TAC) in mice, demonstrating that increased bioavailablility of cGMP has beneficial effects in hypertrophy (Takimoto et

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al., 2005). Importantly, sildenafil was able to reverse preestablished hypertrophy, and this was accompanied by activation of PKG-1. Another facet to NP signalling is their effect on intracellular Ca2+ concentrations (Fig. 2). NPR-A-deficient mice have increased activity of the cardiac Na+ /H+ exchanger NHE-1, which increases intracellular pH and [Ca2+ ] and leads in turn to induction of calcineurin and Ca2+ /calmodulin-dependent kinase II (CaMKII) (Kilic et al., 2005). Moreover, pharmacological inhibition of NHE1 reverses the hypertrophic phenotype of NPR-A−/− mice. NPs are also known to inhibit the renin-angiotensin system (RAS), for example pharmacological inhibition of the angiotensin type 1 (AT1) receptor with losartan reverses the left ventricular hypertrophy of NPR-A−/− mice. Upon crossing NPR-A−/− mice onto an AT1-receptor-deficient background, fibrosis is no longer present. However, in these mice, hypertrophy and survival in response to coronary artery ligation remains the same, indicating that NP-mediated RAS inhibition may play a prominent role in causing fibrosis during remodelling (Nakanishi et al., 2005). These studies therefore all clearly demonstrate that ANP, BNP and CNP reduce blood pressure, hypertrophy and fibrosis through multiple pathways. Why then does hypertrophy still occur in patients with high-endogenous levels of antihypertrophic NPs? Possibly they serve to slow down the progression of hypertrophy for a time but are insufficient to halt progression in the setting of prolonged pressure or volume overload. Interestingly, a study from Kuhn’s group suggests that the anti-hypertrophic effect of NPs may be impaired during heart failure. Patients with congestive heart failure were found to have elevated levels of the NP metabolizing NPR-C and produced meagre amounts of cGMP in response to ANP, suggesting hypo responsiveness of NRP-A (Kuhn et al., 2004). When hemodynamic stress was eased with a left ventricular assist device, NPR-A activity was restored and NPR-C levels returned to normal. A recent study has also suggested that NPR-B may have a heretofore-unappreciated role in heart failure. In failing hearts from mice which underwent TAC, CNP induced twice as much guanylyl cyclase activity as ANP, suggesting that NPR-B activity is unaltered in heart failure while NPR-A activity is attenuated (Dickey et al., 2007). The anti-hypertrophic, vasodilatory and diuretic properties of NPs have generated interest in the potential clinical use of NPs. Indeed, human BNP (nesiritide) has been approved by the US FDA in the treatment of heart failure, although a recent meta-analysis suggested that there may be some unappreciated safety issues (Aaronson & SacknerBernstein, 2006; Colucci et al., 2000; Mentzer et al., 2007). Since BNP is highly upregulated during hypertrophy, there has been intense interest in its use as a biomarker for the diagnosis of heart failure. In a multi-centre study of 1586 patients, measuring serum levels of BNP proved very effective—the predictive accuracy was 83.4% using a cut off of 100 pg/ml and using a cut off point of 50 pg/ml, the negative predictive value was 96% (McCullough et al., 2002; Moe, 2006). Plasma BNP levels are also predictive of cardiovascular mortality and heart failure hospitalisation and using BNP levels as a guide in clinical decision making may

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Fig. 2. Molecular pathways controlling transcription factor activation during hypertrophy. Binding of IL-6 family cytokines (IL-6, CT-1 and LIF) to the heterodimeric gp130 receptor allows recruitment of STAT3 and Gab1. STAT3 translocates to the nucleus while Gab1 binds to SHP2, promoting downstream activity of the Raf/MEK/ERK, PI(3)K/Akt and MEK5/ERK5 pathways and subsequent activation of the NFAT and GATA-4 transcription factors. Negative control of hypertrophy is brought about by binding of ANP and BNP to the NPR-A receptor. This receptor contains guanylyl cyclase activity which promotes accumulation of cGMP as does nitric oxide (NO). cGMP-mediated activation of PKG-1 abrogates calcineurin-mediated NFAT nuclear localisation and also affects Ca2+ levels by inhibiting the cardiac Na+ /H+ exchanger NHE-1.

lead to improved patient outcome (de Denus, Pharand, & Williamson, 2004; Koglin et al., 2001; Moe, 2006). 4. G-protein-coupled receptors G-protein-coupled receptors (GPCRs) are crucial in normal cardiovascular function and in mediating the “fight or flight” response to endogenous catecholamines such as adrenaline, noradrenaline and angiotensin (see Table 1). The adrenergic receptors (ARs) are GPCRs that are highly abundant in the major arteries of the body; including the aorta, pulmonary and coronary arteries and are instrumental in orchestrating the hypertrophic response through control of contractility, vascular tone and metabolism. Like all GPCRs, adrenergic receptors couple to heterodimeric G-proteins which consist of G␣ and G␤␥ subunits (see Fig. 3). ␤-Adrenergic receptors couple to

G␣s (or simply Gs), inducing adenylyl cyclase activity, accumulation of cAMP and consequently activation of protein kinase A (PKA). PKA phosphorylates several proteins involved in cardiac contraction, including l-type calcium channels, ryanodine receptors, phospholamban and troponin (Marian, 2006), increasing their activity and hence cardiac contraction. ␣-ARs couple to Gq and activate phospholipase C (PLC), resulting in hydrolysis of phosphoinositol 4,5-bisphosphate. The products of this reaction are inositol 1,4,5-triphosphate (IP3 ) and diacyl glycerol (DAG). DAG in turn activates protein kinase C (PKC), an important step in the development of concentric hypertrophy—indeed, inhibition of PKC abrogates GPCR-mediated hypertrophy in mice (Arimoto et al., 2006; Takeishi et al., 2000). Patients with heart failure have elevated circulating catecholamines and increased adrenergic drive which

Table 1 The major distinguishing characteristics of the pathways activated by ␣- and ␤-adrenoreceptor types, and common agonists and antagonists used to distinguish between them Receptor type

Agonists

Antagonists

Coupled G-protein type

Downstream signalling pathways

␣-Adrenergic

Serotonin Angiotensin II Phenylephrine

Phentolamine Prazosin

Gq (G␣q/␣11 )

InsP3 DAG PKC, MAPK

␤-Adrenergic

Adrenaline Noradrenaline Isoproterenol

Atenalol Propranalol

Gs

Adenyl cyclase PKA

The many different sub-types of ␣- and ␤-receptors are not indicated.

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Fig. 3. Adrenergic signalling in hypertrophy. Activation of the ␣1 -adrenergic receptor allows coupling of the G-protein Gq and activation of phospholipase C (PLC). PLC promotes hydrolysis of phosphoinositol 4,5-bisphosphate and the products of this reaction are inositol 1,4,5-triphosphate (IP3 ) and diacyl glycerol (DAG). DAG in turn activates protein kinase C (PKC) and MAPK which are involved in hypertrophy and vasoconstriction. ␤-Adrenergic receptors on the other hand couple to Gs, induce adenylyl cyclase and accumulation of the second messenger cAMP. The resulting PKA activity affects several aspects of the hypertrophy program, including phosphorylation and inactivation of the SERCA inhibitor phospholamban. This has the effect of increasing Ca2+ influx into the cytoplasm and increasing Ca2+ re-uptake into the sarcoplasmic reticulum, causing myocyte contraction and vasodilation.

inversely correlate with survival (Lohse, Engelhardt, & Eschenhagen, 2003). This chronic adrenergic drive is one of the hallmarks of hypertrophy. The development of ␤blockers by Sir James Black revolutionized the treatment of heart failure, reducing the overall risk of death by over 30% (Black, Crowther, Shanks, Smith, & Dornhorst, 1964). Treatment with ␤-blockers halts left ventricular remodelling, improves left ventricular ejection fraction and has a positive effect on mortality outcomes (Sabbah, 2004). In patients with DCM, hemodynamic recovery subsequent to ␤-blocker treatment is associated with a restoration of normal ␣-MHC/␤-MHC balance, reduced ANP expression and increased sarco-/endo-plasmic reticulum Ca2+ -ATPase (SERCA) expression (Lowes et al., 1997). In heart failure, the increased adrenergic drive is initially beneficial through increased contractility and heart rate, however chronic adrenergic stimulation is thought to be ultimately detrimental (Bristow, 2000). This is borne out in studies of transgenic mice overexpressing ␤1 -ARs, which have increased cardiac contractility when young, but pronounced cardiac hypertrophy and fibrosis when older, which eventually leads to heart failure (Engelhardt, Hein, Wiesmann, & Lohse, 1999). This suggests that short-term ␤1 -ARs may indeed improve

cardiac function but prolonged hyper-adrenergic drive eventually leads to pathology. Thus, in humans a gain of function ␤1 -receptor polymorphism leads to greater risk of cardiomyopathies (Mason, Moore, Green, & Liggett, 1999). Despite the increased levels of circulating catecholamines accompanying hypertrophy, heart failure is associated with attenuated ␤-AR signalling as a consequence of receptor desensitisation (Sabbah, 2004). This is brought about by receptor phosphorylation by PKA, PKC or G-protein-coupled receptor kinases (GRKs), leading to receptor internalisation, uncoupling from G-proteins or downregulation of AR mRNA expression (Lohse et al., 2003). While ␤-blockers are undoubtedly the most efficacious class of drugs in the treatment of heart failure, there is still some debate as to the exact nature of their action. They may either combat the negative effects of prolonged ␤-adrenergic stimulation or cause re-sensitization of the ␤-AR system (Lohse et al., 2003). Overexpression of GRK2 in the vasculature reduces sensitivity to ␤-AR vasorelaxation and induces hypertension and hypertrophy in the myocardium (Eckhart, Ozaki, Tevaearai, Rockman, & Koch, 2002), while inhibition of GRK2 can restore normal GPCR signalling and lead to improved outcomes in a mouse heart failure model (Rockman et al., 1998).

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A plethora of animal studies confirm that adrenergic signalling is a potent hypertrophic stimulus. Adrenergic agonists such as PE, endothelin-1 and angiotensin II have long been recognized as potent inducers of hypertrophy and transgenic overexpression of ␤1 -ARs, Gs or Gq leads to hypertrophic remodelling and systolic dysfunction (D’Angelo et al., 1997; Engelhardt et al., 1999; Iwase et al., 1996). Likewise, overexpression of angiotensin II (AngII) is sufficient to induce hypertrophy and heart failure in transgenic mice (Paradis, Dali-Youcef, Paradis, Thibault, & Nemer, 2000). Indeed angiotensin-converting enzyme (ACE) inhibitors are comparable to ␤-blockers in treating hypertrophy in the clinic. Intriguingly though, AngII receptor-deficient mice still undergo hypertrophy in response to mechanical stretch, suggesting that other factors are involved (Harada et al., 1998). Several studies have used mice overexpressing a Gq inhibitor peptide (GqI) in cardiomyocytes to demonstrate that TAC-mediated pressure overload requires Gq to induce hypertrophy although the mice suffered fibrosis and apoptosis to the same extent as wild-type mice (Akhter et al., 1998; Wettschureck et al., 2001). Interestingly, these mice remain susceptible to hypertrophy in response to chronic PE, angiotensin II and serotonin (Keys, Greene, Koch, & Eckhart, 2002). In contrast, mice in which expression of GqI was restricted to vascular smooth muscle (VSM) under the control of the SM22␣ promoter were resistant to these forms of hypertrophy (Keys et al., 2002). This may indicate that chronic Gq-mediated signalling induces hypertrophy through its hypertensive effects rather than by directly affecting the myocardium (Keys et al., 2002). This study suggests that it is peripheral resistance and catecholamine production in hypertensive individuals that is largely responsible for cardiac hypertrophy. Interestingly, mechanisms other than hypertrophy can contribute to improved cardiac function following transverse aortic constriction. Mice overexpressing GqI show better recovery from TAC than wild-type controls even though they cannot increase left ventricular size to compensate (Esposito et al., 2002). 5. Interluekin-6 family of cytokines Along with adrenergic agonists, cytokines play a major role in inducing hypertrophy. The main hypertrophic cytokines are all members of the IL-6 family and include IL-6 itself, leukaemia-inhibitory factor (LIF) and cardiotrophin-1 (CT-1). All IL-6 cytokines utilise a common receptor unit glycoprotein 130 (gp130) in combination with ligand-specific receptors and mediate their effects by the JAK/STAT, MAPK and PI(3)K pathways (Fig. 2). In the early nineties, a search for novel mediators of hypertrophy using an expression cloning approach lead to the identification of CT-1 (Pennica et al., 1995). Soon after, CT-1 was shown to promote cardiac myocyte survival and induce an eccentric form of hypertrophy both in vitro and in vivo (Jin et al., 1996; Sheng, Pennica, Wood, & Chien, 1996; Wollert et al., 1996). CT-1 signals through the heterodimeric LIF-R/gp130 receptor and it seems that separate downstream pathways are responsible for the hypertrophic and anti-apoptotic effects of this cytokine. In cardiac myocytes, CT-1 induces the activity of several signalling mediators

including ERK1/2, p38 MAPK, STAT3, PI(3)K/Akt and NF␬B (Craig, Wagner, McCardle, Craig, & Glembotski, 2001). Blocking the MAPKs, Akt or NF-␬B abrogated the protective effect of CT-1 against hypoxia, while inhibiting STAT3 blocked CT-1-mediated hypertrophy, clearly delineating a separate signalling pathway in each process (Brar, Stephanou, Pennica, & Latchman, 2001; Craig et al., 2001; Sheng et al., 1997). While the downstream pathways that control CT-1mediated hypertrophy were being deciphered, several studies found that CT-1 levels are increased in animal models of hypertrophy (Ishikawa et al., 1996; Pan et al., 1998) and in human patients (Lopez et al., 2005; Talwar, Squire, Downie, Davies, & Ng, 2000; Talwar, Squire, Downie, O’Brien, et al., 2000; Talwar et al., 2002; Tsutamoto et al., 2001), and in most cases high-CT-1 levels correlate with disease severity. Currently, BNP is one of the most widely used markers of heart failure, however a recent study suggests that combining CT-1 and BNP measurements may be a greater predictor of mortality in patients suffering from congestive heart failure than either marker alone (Tsutamoto et al., 2007). Interestingly, in patients with DCM, ischaemic cardiomyopathy (ICM) or hypertensive heart failure, augmented CT-1 expression is accompanied by downregulation of gp130, suggesting a compensatory mechanism may control excessive IL-6 family cytokine-mediated hypertrophy (Zolk, Ng, O’Brien, Weyand, & Eschenhagen, 2002). CT-1 may reduce gp130 levels by promoting its ubiquitination and degradation (Gonzalez et al., 2007). CT-1 can be induced by adrenergic stimulation via a cAMP response element in the CT-1 promoter (Funamoto et al., 2000) but whether CT-1 contributes to hypertrophic remodelling induced by adrenergic drive is unknown. An interesting experiment therefore would be to examine the extent of adrenergic-induced hypertrophy in mice with a cardiac-specific knockout of CT-1. As well as crosstalk with the adrenergic system, CT-1 may have a role in hypertrophy induced by other GPCRs. Angiotensin II (AngII) treatment induces CT-1 and LIF release from cardiac fibroblasts, while pharmacological inhibition of AngII blocks CT-1 and LIF-mediated hypertrophy (Fukuzawa et al., 2000; Sano et al., 2000). Like CT-1, LIF induces hypertrophy in cultured cardiac cells and leads to rapid tyrosine phosphorylation of gp130, JAK1, STAT1, STAT3 and ERK (Kodama et al., 1997; Kunisada et al., 1996; Villegas, Villarreal, & Dillmann, 2000a). LIF and CT-1 are the only factors known to induce serial assembly of sarcomeres resulting in myocyte elongation, and several downstream pathways are postulated to be involved in this effect. Expression of the dominant-negative MEK5 blocks LIF-induced elongation of cardiac myocytes in culture, and hearts of MEK5 transgenic mice have a similar appearance to hearts of mice treated with LIF or CT-1 (Nicol et al., 2001). As well as promoting addition of sarcomeres in series, LIF/MEK5/ERK5 may also interfere with addition of sarcomeres in parallel and thus may serve as a focal point in the decision of myocytes to undergo eccentric or concentric hypertrophy (Nicol et al., 2001). It is important to keep in mind that chemical inhibitors commonly used to determine the involvement of ERK1/2 in signalling pathways also inhibit MEK5/ERK5 (Nishimoto & Nishida, 2006).

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LIF is also involved in the altered Ca2+ signalling that accompanies hypertrophy through downregulation of SERCA (Villegas, Villarreal, & Dillmann, 2000b) and phosphorylation of the l-type Ca2+ channel Ca(v)1.2 in cardiac myocytes leading to increases in Ca2+ currents (Takahashi et al., 2004). Ca(v)1.2 phosphorylation is dependent on ERK and results in downstream activation of CaMKII, CaMKIV and calcineurin. The resulting increase in Ca2+ signalling may play a prominent role in LIF-mediated hypertrophy since inhibitors of CaMKII and CaMKIV block LIF-dependent fetal gene activation and cell-size increase (Kato et al., 2000; Takahashi et al., 2004). In addition to ERK1, 2 and 5, LIF promotes STAT3 phosphorylation and inhibition of STAT3 with a dominant-negative construct in cardiac myocytes blocks LIF-mediated hypertrophy (Kunisada et al., 1998). Like CT-1, LIF is induced in response to hemodynamic overload and is upregulated in patients with CHF (Eiken et al., 2001; Tsutamoto et al., 2001). Mice with a cardiac-specific deletion of gp130 are overtly normal but whereas wild-type mice undergo compensatory hypertrophy following TAC, gp130−/− mice experience dilated cardiomyopathy and increased myocyte apoptosis (Hirota et al., 1999). This maladaption to mechanical stress causes all the gp130−/− to die within 7 days of TAC and suggests that the gp130 system controls a critical juncture in the transition from compensatory hypertrophy to heart failure (Hirota et al., 1999). The JAK/STAT pathway (particularly STAT3) appears to be centrally responsible for the biological effects of gp130 stimulation, and overexpression of the JAK inhibitor SOCS3 abolishes gp130 signalling in cardiac myocytes (Yasukawa et al., 2001). Transgenic overexpression of STAT3 in the heart is sufficient to induce hypertrophy (Kunisada et al., 2000); crossing IL-6 transgenic mice with IL-6 receptor transgenic mice showed that chronic activation of IL6/gp130 and STAT3 is sufficient to induce hypertrophy (Hirota, Yoshida, Kishimoto, & Taga, 1995). It seems most plausible that each of the JAK/STAT, PI(3)K and MAPK pathways are necessary for a fully fledged gp130-dependent hypertrophic response but that each pathway controls primarily a separate facet. There are also likely several points of crosstalk and it will be important to delineate precise signalling pathways in order to better tailor pharmaceutical interventions. Since there is evidence that gp130 signalling can have a protective effect in addition to its role in hypertrophy (Berry et al., 2004; Zou et al., 2003), the challenge will lie in dissecting out the protective facets of gp130 signalling from those that are responsible for maladaption to hypertrophic stress.

6. Adhesion and cytoskeletal proteins In addition to the cytoskeletal proteins being primary end-targets of the hypertrophic response, they co-operate with adhesion molecules to sense the initial stress and transmit the hypertrophic signal inside the cells. Overload of the myocardium causes mechanical stress to the myocytes. This cellular stretch signal is transduced by integrins at focal adhesion sites on the cell surface and can result in direct rearrangement of

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the actin cytoskeleton. Overload also causes kinases such as the Src tyrosine kinase (Kuppuswamy et al., 1997) and the integrin-linked kinase, ILK-1, to associate with the cytoskeleton and become activated. Overexpression of ILK-1 in mouse myocardium results in a compensated ventricular hypertrophic phenotype whereas mice with cardiomyocyte-restricted expression of a kinase-inactive ILK are apparently resistant to the development of cardiac hypertrophy (Lu et al., 2006). In response to Src activation or ILK-1 overexpression (Kuppuswamy et al., 1997; Lu et al., 2006), the induction of hypertrophy is mediated by certain members of the Ras family of small GTPases. Overexpression of some of these small GTPases is sufficient to induce hypertrophy. For example, overexpression of the prototypical Ras protein in transgenic mice causes development of cardiac hypertrophy and diastolic dysfunction (Hunter, Tanaka, Rockman, Ross, & Chien, 1995). Similarly, cardiac overexpression of V12Rac1 produces profound cardiac hypertrophy and ventricular dilation (Sussman et al., 2000). On the other hand, cardiac overexpression of RhoA leads to atrial enlargement and conduction defects but not ventricular hypertrophy (Sah et al., 1999). Thus, a network of small GTPases act downstream of the cytoskeleton to control the activation of pro-hypertrophic signalling pathways including the JAK/STAT, MAPK, and calcineurin pathways (reviewed in Ruwhof & van der, 2000) described below. 7. Mitogen-activated protein kinase/extracellular signal receptor-regulated kinase signalling A large body of evidence supports a role for MEK and its downstream kinases ERK1/2 (extracellular signalregulated kinases) in the development of hypertrophy. For instance, ERK1/2 are activated in response to practically every known hypertrophic agonist (Bueno & Molkentin, 2002) and transgenic expression of constitutively active MEK1 induces concentric hypertrophy in vivo (Bueno & Molkentin, 2002; Bueno et al., 2000; Clerk, Fuller, Michael, & Sugden, 1998). Similarly, inhibition of the MEK1-ERK1/2 pathway by drugs or adenoviral expression of dominantnegative constructs reduces the hypertrophic response of cardiac myocytes in culture (Bueno et al., 2000; Bueno & Molkentin, 2002; Clerk et al., 1998). Surprisingly however, it was recently shown that the hypertrophic response to either pressure overload stimulation, neuroendocrine agonist infusion or exercise is unaffected in ERK1−/− ERK2+/− mice (Purcell et al., 2007). In order to eliminate the possibility that the remaining ERK2 expression in these mice was sufficient to account for this response, the experiments were repeated in mice in which ERK1/2 was completely inhibited by overexpression of dual-specificity phosphatase-6 (DUSP6) in the heart. Again, there was no effect on the development of hypertrophy (Purcell et al., 2007). It seems that a possible explanation for this observation is that DUSP6 overexpression was driven by an inducible ␣-MHC promoter and was therefore restricted to cardiomyocytes. As explained previously, emerging evidence suggests that GPCR pathways in other cell types such as vascular smooth muscle can also be central to the development of hypertrophy. Furthermore, by analogy with

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experiments with mice expressing GqI in cardiac myocytes, some hypertrophic stimuli may act via induction of hypertension and/or secondary release of ␤-adrenergic stimuli, which would then act via signalling pathways not requiring MEK-ERK1/2 (see Fig. 3). Interestingly, after long-term transverse aortic constriction, DUSP6 transgenic and ERK1−/− ERK2+/− , mice suffer from cardiac decompression and signs of heart failure associated with enhanced myocyte cell death, indicating that this pathway does play an important role in myocyte survival. Mutations in upstream activators of the ERK pathway are associated with cardiomyopathies in humans. For example, of the patients with Noonan or LEOPARD syndrome with mutations in two mutation hotspots in the serine/threonine kinase RAF1 gene, 95% suffered from hypertrophic cardiomyopathy (Pandit et al., 2007). These mutations impair binding of the inhibitory 14-3-3 protein to RAF1, resulting in insufficient auto-inhibition and thus increased ERK activity. Interestingly, despite the evidence cited above that cardiac ERK activity is not required for the development of hypertrophy, expression of cardiac-specific dominantnegative Raf-1 in mice attenuates hypertrophy after pressure overload (Hunter et al., 1995). As has been pointed out however, Raf-1 directly complexes with many other signalling proteins, including Ras, apoptosis signal-regulating kinase-1 (ASK-1), Rok-␣, cdc25, casein kinase-2, retinoblastoma protein, and MEKK1, any of which could also affect the cardiac hypertrophic response independently of ERK1/2 (Purcell et al., 2007). For example, ASK-1 is also involved in myocyte survival by way of regulating apoptosis, and mice with cardiomyocyte-specific disruption of Raf-1 demonstrate cardiomyocyte-specific apoptosis, left ventricular systolic dysfunction and heart dilatation, all of which are ASK-1 dependent (Yamaguchi et al., 2004). 8. Histone acetylation 8.1. Histone acetyltransferases Histone acetyltransferases (HATs) such as p300 and CREB-binding protein (CBP) are transcriptional coactivators that cause the relaxation of chromatin structure and promote gene activation. Overexpression of CBP/p300 is sufficient to induce hypertrophy and left ventricular remodelling in transgenic mice (Gusterson, Jazrawi, Adcock, & Latchman, 2003; Yanazume, Hasegawa, et al., 2003). Interestingly, cardiac remodelling after myocardial infarction, which involves hypertrophy of the remaining cardiomyocytes, also requires p300 HAT activity (Miyamoto et al., 2006). Acetylation of histones alters gene expression in cardiac cells in response to pharmacologically induced hypertrophy and simulated ischaemia and reperfusion (Davidson et al., 2005; Davis, Pillai, Gupta, & Gupta, 2005; McKinsey & Olson, 2004b)—see Table 2. Treatment of primary neonatal cardiomyocytes with PE causes them to undergo hypertrophy, as demonstrated by an increase in ANP production, protein-to-DNA ratio, mean cell area and length, and sarcomeric organization as well as an increase in the activity of p300 (Davidson et al., 2005; Yanazume, Hasegawa, et al., 2003). A major mechanism of

PE-mediated cardiac hypertrophy therefore appears to be enhancement of p300/CBP HAT activity. Proteins with HAT activity such as p300 and CBP tend to be large, and act as scaffolding proteins that can bridge between basal transcription factors and various transactivators. Thus, it seems likely that CBP/p300 is an essential co-factor for a distinct subset of genes necessary to initiate the hypertrophic response. Acetylation of the GATA-4 transcription factor by CBP/p300 results in eccentric left ventricular hypertrophy (Molkentin & Olson, 1997; Yanazume, Morimoto, Wada, Kawamura, & Hasegawa, 2003). In fact, GATA-4-mediated hypertrophy is an interesting example of how different post-translational modifications on the same protein can have distinct effects on cardiac remodelling, since in contrast to the above, activation of the MEK-ERK pathway by hypertrophic stimuli leads to phosphorylation of GATA-4 at serine 105 and development of concentric hypertrophy. Activation of p300/CBP in response to PE occurs via the downstream protein kinase pathways downstream of Gq that were described above, specifically ERK, p38, MSK1 and PKA (Davidson et al., 2005; Markou, HadzopoulouCladaras, & Lazou, 2004). 8.2. Histone deacetylases Hypertrophic growth involves control of cardiomyocyte gene expression at multiple molecular levels. In addition to their interaction with HATs, master transcriptional regulators of gene expression in cardiac myocytes such as myoD, MEF2, and GATA-4/-5/-6 (Chanalaris et al., 2005; Kolodziejczyk et al., 1999; Liang & Molkentin, 2002) recruit histone deacetylases (HDACs) to remodel chromatin as part of the mechanism by which they control gene expression (Davis et al., 2005; Lu, McKinsey, Nicol, & Olson, 2000; Townsend et al., 2007). In direct opposition to the HATs described above, HDACs mediate the removal of acetyl groups. There are more than a dozen individual HDAC enzymes which can be divided into three main classes—class I HDACs (HDACs 1, 2, 3, and 8), class II HDACs (HDACs 4, 5, 6, 7, 9, and 10) and class III which are discussed in the following section. HDACs have been shown to be important endpoint targets of cell-signalling pathways involved in the induction of altered gene expression in cardiac hypertrophy and ischaemia/reperfusion injury (Frey, Katus, Olson, & Hill, 2004) and are currently undergoing intense investigation as possible therapeutic regulators of hypertrophy-associated cardiac disease. In adult cardiac myocytes activation of the MEF2 transcription factor in response to stress signalling activates a pro-hypertrophic gene expression profile. Stress signalling activates MEF2 by causing the nuclear export of class II HDACs, possibly regulated through protein kinase D (Vega et al., 2004), which would normally associate with MEF2 and suppress its activity in normal cells. Thus, class II HDACs play a key role in suppression of hypertrophy (Kolodziejczyk et al., 1999; Lu et al., 2000; Miska et al., 1999). Consistent with this notion, mice lacking HDAC9 or HDAC 5, class II HDACs, are supersensitive to stress signals and both mouse models showed enhanced hypertrophy in response to pathological stimuli (Chang et al., 2004; Zhang

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Table 2 p300/CBP histone acetyltransferase acts as a scaffold factor, bridging transcription factors and cofactors, by bringing a number of proteins into a ternary transcriptional complexes which lead to cardiac hypertrophy (a selection of hypertrophic stimuli, acting through p300/CBP and their corresponding transcription cofactors are shown) Hypertrophy agent

Transcription (co)factor

Hypertrophic target protein

Reference

PE, MAPK activation

MyoD MEF2D SRF AP-1 GATA-4, -5, -6 NFAT Nished NF-␬B MEF2 GATA-4 MEF2 SRF dHAND Nkx2.5 GATA-5, -6 (NFAT)

ANP MHC Desmin

Yanazume, Hasegawa, et al. (2003), Davidson et al. (2005)

MLC-2v

Mathew et al. (2004)

Cardiac actin ANP ET-1

Molinari et al. (2004) Molkentin and Olson (1997), Yanazume, Morimoto, et al. (2003)

AngII Isoproterenol infusion Emb1 overexpression Pressure overload

(SERCA)

et al., 2002). In contrast, class I HDACs are considered to play a pro-hypertrophic role although the relevant gene targets are less well described (McKinsey & Olson, 2004a; Townsend et al., 2007). Taken together, class I and II HDACs play opposing roles in control of hypertrophy and there is a clear benefit to the development of class I HDAC-specific inhibitors as therapeutic agents which would not interfere with the class II-dependent inhibition of pro-hypertrophic pathways (Fig. 4). It is anticipated that the clinical use of HDAC inhibitors would allow the control of key hypertrophic genes, and would provide a novel molecular and therapeutic approach. The development of HDAC inhibitors as experimental therapeutic agents for the treatment of cancer has progressed well, although most of these agents are not isoform specific. By contrast, the bicyclic tetrapeptide HDAC inhibitors FK228 and Spiruchostatin A appear to possess selectivity towards class I HDACs (unpublished data; YurekGeorge et al., 2007), an activity profile which may make

Fig. 4. The role of histone acetylation in regulating cardiac hypertrophy. Histones play a key role in packaging DNA in chromatin and the acetylation of lysine in histone protein subunits modulates the “open” or “closed” status of the DNA. Chromatin “opening”, or relaxation, permits the access of transcriptional machinery to potential hypertrophy-regulating genes, such as the anti-hypertrophic GSK3␤ gene or pro-hypertrophic ANP gene. The regulation of lysine acetylation is governed by histone deacetylases (HDACs) and histone acetylases (HATs). Class I and II HDACs play key opposing roles in modulating cardiac hypertrophy. Class II HDACs are thought to repress whereas class I HDACs induce pro-hypertrophic genes. A novel molecular therapy might aim to target and inhibit class I HDACs thereby regulating cardiac hypertrophy.

them particularly attractive as starting points to develop novel therapies for cardiovascular disease. Indeed, it has already been shown that Spiruchostatin A is a highly potent inhibitor (∼pM) of HDACs in cardiac myocytes and effectively interferes with the pro-hypertrophic effects of phenylephrine and urocortin (Davis et al., 2005).

8.3. Class III HDACs role in cardiac hypertrophy Class III HDACs, sirtuins, have an impact on a range of cellular processes, which include gene silencing, cellular ageing and genotoxic/DNA damage repair (Longo & Kennedy, 2006). Class III HDACs are unique in that they require nicotinamide adenine dinucleotide (NAD) for catalytic activity, as such they are thought to function as rheostats of metabolism and accordingly stress. Recently, class III HDAC activity has been associated with inhibition of cardiac hypertrophy and enhanced cardiac myocyte survival. Sir2␣ is expressed in the nuclei of cardiac myocytes and its overexpression has been shown to reduce apoptosis in response to serum starvation and also to significantly increase the size of cardiac myocytes (Alcendor, Kirshenbaum, Imai, Vatner, & Sadoshima, 2004). Conversely, inhibition of Sir2␣ with nicotinamide, sirtinol, or by expression of dominant-negative Sir2␣ induced myocyte apoptosis. An in vivo cardioprotective role for Sir2 expression was seen in dogs where levels were increased significantly in hearts with heart failure induced by rapid pacing superimposed on stable, severe hypertrophy. A direct role for Sir2␣ in cardiac myocyte hypertrophy is suggested by experiments demonstrating that it modifies chromatin structure by deacetylating histone protein H2A.z (Chen I.Y. et al., 2006). Recent animal studies using Sirt1, the mammalian homologue of yeast Sir2␣, showed that its overexpression induces ␣-MHC expression. Cardiac Sirt1 levels also increase during cardiac hypertrophy following pressure overload (Alcendor et al., 2004; Alcendor et al., 2007; Pillai et al., 2008). Strikingly, however, moderate overex-

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pression of Sirt1 attenuated age-dependent increases in cardiac hypertrophy, apoptosis, cardiac dysfunction, and expression of senescence markers, and therefore seems to cause a paradoxical cardioprotective response. However, high-level expression of Sirt1 promotes apoptosis, hypertrophy and decreased cardiac function, thereby stimulating the development of cardiomyopathy. These observations suggest that Sirt1 can impede aging and confer stress resistance to the heart in vivo, yet these beneficial effects are dose/expression dependent. Moreover, a transcriptional influence of Sirt1 in a model of ventricular remodelling in type 2 diabetes, a disorder which predisposes to fatal coronary heart disease, has shown a role for forkhead transcription factors (Vahtola et al., 2008). In Goto-Kakizaki rats Sirt1 modulation of the proapoptotic forkhead class O transcription factor 3a (FOXO3a) regulates the cardiac myocyte lifespan and hypertrophy. Clearly the sirtuins, have a remarkable role in the heart which is yet to be fully understood. These genetic studies demonstrate that sirtuins, if carefully regulated, can repress cardiac hypertrophy, reducing the hearts sensitivity to stress, and promote longevity. 9. Calcium-mediated pathways towards hypertrophy Calcium increase is another potential trigger of the translocation of pro-hypertrophic transcription factors to the nucleus. Nuclear factor of activated T cells (NFAT) was originally discovered as a transcription factor involved in the development of T cells. However it is also involved in the development of the heart, as well as skeletal muscle and the nervous system. As per many transcription factors involved in cardiac development, it is also activated during hypertrophy. In this case it is normally retained in the cytosol, but when dephosphorylated by calcineurin it translocates to the nucleus (Fig. 2). Calcineurin is a serine-threonine protein phosphatase that is activated in the presence of calcium, and can also be affected by the redox state of the cell (reviewed in Wilkins & Molkentin, 2004). Since calcineurin is a target of the immunosuppressive drugs ciclosporin A and FK506, these drugs have been used to evaluate the role of the calcineurin/NFAT pathway in rodent models of cardiac hypertrophy (Wilkins & Molkentin, 2004). However, since these drugs also affect NFAT, more specific inhibition of calcineurin by cardiac-specific overexpression of endogenous inhibitors Cain/Cabin-1 or A-kinase anchoring protein 79 has been used to more specifically demonstrate the involvement of calcineurin in hypertrophy (De Windt et al., 2001). Furthermore, genetic deletion of specific isoforms of calcineurin or NFAT attenuates development of hypertrophy. NFAT appears to serve as an integrator of many different signalling pathways, some of which (GSK3␤, JNK, p38, PKA, MEKK1, CK1) cause its nuclear export, and others of which (AP1, MEF-2, GATA-4) interact with and modulate its transcriptional activity as co-factors (reviewed in Crabtree & Olson, 2002). A link between calcium and the regulation of hypertrophic transcriptional pathways by acetylation has recently been discovered. In this case, it appears to be the nuclear export of HDAC4 (Backs, Song, Bezprozvannaya,

Chang, & Olson, 2006) that is increased after phosphorylation by calcium/calmodulin kinase II. Thus, an increase in cellular calcium results in the balance tipping in favour of histone acetylation, and therefore the loosening of inhibitory nucleosomal structure. Impaired calcium homeostasis is a prominent feature in the transition from compensatory hypertrophy to heart failure and causes contractile dysfunction and development of arrhythmias. It is now well appreciated that downregulation of the SERCA is a major cause of dysregulated Ca2+ signalling and impaired potentiation of contractile force in heart failure (Frank, Bolck, Erdmann, ¨ & Schwinger, 2003; Kogler et al., 2006; Meyer et al., 1995). Several studies have demonstrated that SERCA expression and hence Ca2+ SR re-uptake is reduced in overloadinduced hypertrophy and heart failure (Frank et al., 2003; Mercadier et al., 1990; Schultz et al., 2004). Treating neonatal cardiac myocytes with an ␣1 -adrenergic agonist induces a hypertrophic response in parallel with a reduction in SERCA2 levels (Prasad et al., 2007). This is accompanied by a reduction in SERCA activity and downregulation of Ca2+ signalling. SERCA-deficient mice are not viable but the effect of deleting a single allele shows that SERCA is necessary for adaptation to pressure overload. SERCA2+/− mice have reduced levels of SR Ca2+ uptake and release and have a much greater propensity towards developing heart failure (Schultz et al., 2004). They also exhibit greater hypertrophy and more extensive fibrosis following 10 weeks of TAC-induced pressure overload (Schultz et al., 2004). Likewise, inhibition of phospholamban blocks hypertrophy and improves cardiac function in rodents and has been suggested as a therapeutic strategy to combat hypertrophy in patients (Hoshijima et al., 2002; Minamisawa et al., 1999; Sato et al., 2001). However, loss of a functional phospholamban allele in humans actually results in dilated cardiomyopathy, suggesting that increasing SR Ca2+ uptake may not always be a positive goal in treating hypertrophy (Haghighi et al., 2003). Using right ventricular myocar¨ dial muscle preparations, Kogler et al. showed that SERCA2 is downregulated in unloaded conditions but upregulated by prolonged preload, which is an adaptive response to ¨ increased demand (Kogler et al., 2006). While the desirability of targeting SERCA for therapy is still under debate it is clear that altered levels of SERCA affects the heart’s ability to adapt to chronic pressure overload and studies consistently show that SERCA has a prominent role to play in the progression to heart failure. 10. MicroRNAs—new players in cardiac hypertrophy MicroRNAs (miRNA) are non-coding RNAs 18–25 nucleotides in length whose function is to functionally silence-specific mRNA transcripts. There are over 400 known miRNAs in the human genome, a number which is increasing weekly (Kim, 2005). In the past 12 months, a series of papers have placed miRNAs at the centre stage in regulating the hypertrophic program. Several studies have employed a microarray approach to detect miRNAs differentially expressed during hypertrophy. Van Rooij and colleagues identified 28 differentially expressed miRNAs common to TAC and calcineurin A-mediated hypertrophy

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and found that many of these were also overexpressed in failing human hearts (van Rooij et al., 2006). Moreover, several of these miRNAs have the ability to induce hypertrophy when expressed in cardiac myocytes in culture. A transgenic approach revealed that myocardial overexpression of miR-195 in mice causes an increase in left ventricular wall size, upregulation of ANP, BNP and ␤-MHC and reduced cardiac output (van Rooij et al., 2006). Three separate groups analysed miRNA expression at various times after aortic banding in mice and found dozens of differentially expressed miRNAs (Cheng et al., 2007; Sayed, Hong, Chen, Lypowy, & Abdellatif, 2007; Tatsuguchi et al., 2007). Thum et al. analysed the miRNA expression profiles from heart failure patients and found a high degree of similarity to fetally expressed miRNAs (Thum et al., 2007). Care` et al. focused their studies on miR-1 and miR-133 which had previously been shown to have roles in skeletal myoblast differentiation and proliferation (Chen J.F. et al., 2006). They utilized three separate models of hypertrophy: TAC, transgenic expression of constitutively active Akt and exercise. In all three models both miR-1 and miR-133 were downregulated, a finding which was recapitulated in hearts from patients with DCM (Care` et al., 2007). Functional studies revealed that in culture, either miRNA could block the induction of hypertrophy, while in vivo, inhibition of miR-133 using an oligonucleotide “antagomir” had the opposite effect. The authors highlight possible targets of miR-133 such as RhoA, Cdc42 and NELFA/Whsc2 which have previously been shown to have roles in hypertrophic remodelling. Expression of miR-1 in culture inhibits serum-induced hypertrophy and is associated with inhibition of RasGAP, Cdk9 and fibronectin expression (Sayed et al., 2007). Elegant work from the Olson lab demonstrated that in mice, co-ordinate regulation of ␣- and ␤-MHC is controlled by miR-208 located in intron 27 of the ␣-MHC gene (van Rooij et al., 2007). Specific deletion of miR208 resulted in compromised cardiac function and sarcomeric abnormalities. When these mice were subjected to TAC there was little sign of increased myocyte size or fibrosis. Moreover the miR-208-deficient mice failed to upregulate ␤-MHC but instead increased ␣-MHC expression to compensate. In contrast, ANP and BNP were upregulated as in wild-type mice, demonstrating that miR-208 is needed for ␤-MHC upregulation and cardiac remodelling but not for induction of other stress response factors. Intriguingly, miR-208’s role in hypertrophy may involve silencing of thyroid receptorassociated protein 1 (THRAP1), a previously unappreciated target of miR-208 (van Rooij et al., 2007). This silencing of THRAP1 modulates the activity of thyroid hormone and in turn expression of ␤-MHC. While understanding the role of each miRNA in hypertrophy is a few years away, these early studies have clearly shown that miRNAs can control myocyte hypertrophy in vitro and in vivo. Although some reports have corroborated the role of a specific miRNA – for example two groups independently reported that miR-1 blocked hypertrophy – other reports conflict. For example, both pro- and antihypertrophic effects have been claimed for miR-21 (Cheng et al., 2007; Tatsuguchi et al., 2007). As is often the case in an emerging field of study, there is initially much conflict-

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ing data and controversy. Nonetheless, exciting times are ahead in understanding the role of microRNAs in cardiac pathology. Taken together, these recent reports show that miRNA play a powerful role in co-ordinating the response to cardiac stress and are sure to open the door to previously unappreciated medical therapies. 11. Conclusion By extension of the general observation that cardiac “hypertrophy recapitulates ontogeny”, one might anticipate that other cardiac developmental signalling pathways would be integral to hypertrophy. Indeed, emerging evidence suggests that the Wnt/␤-catenin pathway is involved in hypertrophy, although, its role appears to more complicated than simply that of an on/off activator/repressor. Again, this appears to reflect its multi-faceted roles in cardiac development, during the early stages of which activation is required for commitment to the cardiac lineage, while downregulation is required for later stages of differentiation (reviewed in Zelarayan, Gehrke, & Bergmann, 2007). Dissection and characterisation of the signalling pathways leading to cardiac hypertrophy has led to a wealth of knowledge about this condition both physiological and pathological (Heineke & Molkentin, 2006). Although these pathways have still to be defined further and there are undoubtedly future pathways and players to be discovered; the challenge will be to translate this scientific knowledge and understanding into potential pharmacological therapies for the treatment of pathological cardiac hypertrophy. Acknowledgements The authors would like to thank all of the laboratories cited within this review for their insightful and exceptional research investigating cardiac hypertrophy—we would also like to apologise to those researchers whose data we could not discuss due to space constraints. Research mentioned from the authors own laboratories was funded by the Biotechnology and Biological Sciences Research Council (PAT), the British Heart Foundation (PAT), the Gerald Kerkut Charitable Trust (PAT) and the Medical Research Council (SMD). SPB is funded by a British Heart Foundation PhD studentship. References Aaronson KD, Sackner-Bernstein J. Risk of death associated with nesiritide in patients with acutely decompensated heart failure. J Am Med Assoc 2006;296:1465–6. Ahluwalia A, Hobbs AJ. Endothelium-derived C-type natriuretic peptide: More than just a hyperpolarizing factor. Trends Pharmacol Sci 2005;26:162–7. Akhter SA, Luttrell LM, Rockman HA, Iaccarino G, Lefkowitz RJ, Koch WJ. Targeting the receptor-Gq interface to inhibit in vivo pressure overload myocardial hypertrophy. Science 1998;280:574– 7. Alcendor RR, Gao SM, Zhai PY, Zablocki D, Holle E, Yu XZ, et al. Sirt1 regulates aging and resistance to oxidative stress in the heart. Circ Res 2007;100:1512–21. Alcendor RR, Kirshenbaum LA, Imai S, Vatner SF, Sadoshima J. Silent information regulator 2 alpha, a longevity factor and class III histone deacetylase, is an essential endogenous apoptosis inhibitor in cardiac myocytes. Circ Res 2004;95:971–80.

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