G Protein Coupled Signaling and Gene Expression Genetic Models and Mechanisms of Transcription in Cardiac Hypertrophy

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1 This Review is part of a thematic series on Gene Expression in Hypertrophy and Stress, which includes the following articles: Gene Expression in Fibroblasts and Fibrosis: Involvement in Cardiac Hypertrophy Roles of Cardiac Transcription Factors in Cardiac Hypertrophy Ras, Akt, and Mechanotransduction in the Cardiac Myocyte G Protein Coupled Signaling and Gene Expression Genetic Models and Mechanisms of Transcription in Cardiac Hypertrophy Ryozo Nagai, Guest Editor Ras, Akt, and Mechanotransduction in the Cardiac Myocyte Downloaded from by guest on April 28, 2018 Peter H. Sugden Abstract The Ras subfamily of 21-kDa ( small ) guanine nucleotide binding proteins [which includes Ha-Ras, Ki(A)-Ras, Ki(B)-Ras, and N-Ras] is universally important in regulating intracellular signaling events in mammalian cells and controls their growth, proliferation, senescence, differentiation, and survival. These Ras isoforms act as membrane-associated biological switches that transduce signals from transmembrane receptors, thus potentially activating a variety of downstream signaling proteins. These include ultimately two Ser/Thr protein kinase families, the extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt (or protein kinase B). Activation of ERK1/2 has been associated with cardiac myocyte hypertrophy (ie, increased cell size and myofibrillogenesis, with concurrent transcriptional changes to a fetal pattern of gene expression), whereas activation of Akt is associated with the increased protein accretion in hypertrophy. Both ERK1/2 and Akt may promote myocyte survival. In the intact heart in vivo and in primary cultures of cardiac myocytes, mechanical strain induces hypertrophy, a process known as mechanotransduction, which may involve Ras, ERK1/2, and Akt. In this study, general and cardiospecific aspects of the regulation of Ras and Akt will be described. The various mechanisms through which mechanical strain might initiate Ras- or Akt-dependent signaling will be discussed. The overall conclusion is that although an involvement of Ras and Akt in mechanotransduction is likely, more work (particularly focusing on mechanoreception) needs to be undertaken before it is unequivocally established. (Circ Res. 2003;93: ) Key Words: mechanical strain small G proteins protein kinases hypertrophy apoptosis The major foci of this review will be the regulation and effects of Ras and Akt activation in the heart, and their potential involvement in workload- or strain-induced myocardial hypertrophy (mechanotransduction). Because of space limitations, description of general aspects regulation of Ras and Akt will be kept to a minimum, although some aspects are discussed in greater depth in the associated online data supplement (available at Normally, only the most recent or comprehensive review articles are cited, but the online data supplement contains details of additional reviews (and articles) that could not be accommodated. Although Ras and Akt were originally identified as viral oncogenes, their wild-type counterparts are vital for regulation of many biological processes in untransformed cells. In the cardiac myocyte, their identified functions include the regulation of growth and survival. The multiple proteins of the Ras subfamily are themselves members of an extensive small guanine nucleotide binding protein (small G protein) superfamily, to which the term molecular switch is often applied. 1 Through their ability ultimately to modulate transcription, Ras proteins generally control cell growth and proliferation, and other facets of cellular biology including Original received August 12, 2002; resubmission received October 28, 2003; accepted October 30, From the National Heart and Lung Institute Division (Cardiac Medicine Section), Faculty of Medicine, Imperial College of Science, Technology and Medicine, London, UK. Correspondence to Peter H. Sugden, DPhil, NHLI Division (Cardiac Medicine Section), Faculty of Medicine, Imperial College London, Flowers Building (4th Floor), Armstrong Road, London SW7 2AZ, UK. p.sugden@imperial.ac.uk 2003 American Heart Association, Inc. Circulation Research is available at DOI: /01.RES F5 1179

2 1180 Circulation Research December 12/26, 2003 Downloaded from by guest on April 28, 2018 senescence/cell cycle arrest, differentiation, and survival. Akt (or protein kinase B, PKB) 2,3 is a Ser/Thr protein kinase that is activated through the phosphoinositide 3-OH kinase (PI3K) pathway, and Ras and Akt signaling may interconnect at this level because of the involvement of Ras in the activation of PI3K. 2,4 Akt regulates cellular processes associated with growth (eg, protein synthesis), survival (eg, apoptosis), and carbohydrate metabolism (eg, glycogenesis, and possibly glycolytic flux and glucose uptake), and it is particularly important in insulin and insulin-like growth factor 1 (IGF1) signaling. Ras and Akt are potentially regulated through several classes of transmembrane receptors, including G protein coupled receptors (GPCRs), receptor protein Tyr kinases (receptor PTKs), and integrins. On agonist engagement, GPCRs signal to membrane-localized heterotrimeric G proteins [ (GDP). in their inactive state, dissociating to the biologically active (GTP) and dimer species on GDP/GTP exchange], whereas receptor PTKs autophosphorylate Tyr residues in their cytoplasmic domains and, in many cases, elicit Tyr phosphorylation of other proteins. In contrast to the unidirectional nature of GPCRs and receptor PTKs, integrin signaling is bidirectional with outside-in signaling involving interaction of integrins with the extracellular matrix (ECM) rather than with soluble ligands. A common feature of Ras and Akt is that both are implicated in hypertrophic growth of the myocardium. 5,6 Although still actively debated, the prevailing view is that adult mammalian ventricular myocytes are terminally differentiated cells which, although capable of limited karyokinesis, are incapable of cytokinesis. In vivo, the myocardium adapts to a requirement for increased contractile power (eg, during increased hemodynamic load) by increasing its muscle mass, a process brought about predominantly by a true hypertrophy (growth in the absence of cell division) of preexisting cardiac myocytes. The coupling process of overload to hypertrophy is known as mechanotransduction, the molecular nature of which remains poorly understood. 7 Myocardial hypertrophy in vivo is characterized by increases in cell size and sarcomerogenesis over and above that which would be predicted for a given stage of development. Although these changes are brought about, at least in part, by increases in expression of constitutive genes, there are also ancillary transcriptional changes [eg, reexpression of atrial natriuretic factor (ANF) and B-type natriuretic factor (BNP); see the online data supplement] that are associated with adaptive hypertrophy but may also be exhibited by the failing heart. Hypertrophy of myocytes in primary culture can be also induced by mechanical strain (see the online data supplement), as well as by (or through) a plethora of agonists including the GqPCR agonism by endothelin-1 (ET-1), angiotensin II (ANGII), or -adrenergic stimulation, and by direct pharmacological activators of protein kinase C (PKC), to which GqPCR signaling is also coupled. 8 Ras and Its Effectors Activation of Ras As well as the four closely related proteins generically known as Ras [Ha-Ras, the two alternatively spliced Ki-Ras species, Figure 1. Ras activation cycle. In its off state, Ras is ligated to GDP. Activation of guanine nucleotide exchange factors (GEFs) stimulates exchange of GTP for GDP and produces biologically active Ras.GTP, which then participates in the activation of c-raf, phosphoinositide 3-OH kinase (PI3K), and Ral.GDS (a GEF for the Ral small G proteins). Innate GTPase activity of Ras, an activity returns Ras.GTP to the off state, the GTPase activity being profoundly stimulated by GTPase-activating protein (GAPs). restrictively expressed Ki(A)-Ras and ubiquitously expressed Ki(B)-Ras, and N-Ras, all with a molecular mass of 21 kda], the Ras subfamily encompasses several other related proteins that will not be discussed further. 1 Ha-Ras and the Ki-Ras species are essentially entirely (plasma) membranebound, 9 although the subcellular localization of N-Ras is less clear. Membrane localization is essential for Ras signaling, and posttranslational lipidation (irreversible farnesylation and methylesterification of Ha-Ras and Ki-Ras, along with reversible palmitoylation of Ha-Ras) contributes significantly to this (see the online data supplement). 1,9 Furthermore, Ha-Ras and Ki-Ras are differentially localized to membrane microdomains (caveolar or noncaveolar lipid rafts, and disordered membrane), and this may account for some of the differential signaling properties of these Ras isoforms. 9 In its biologically inactive state, Ras is ligated to GDP (Figure 1). 1 Exchange of GTP for GDP causes a conformational change, turning the molecular switch to the on position. Exchange is normally very slow but is enhanced by guanine nucleotide exchange factors (GEFs), and the participation of one of these, Sos, in signaling from receptor PTKs such as the epidermal growth factor (EGF) receptor to Ras is well-established (see the online data supplement). 10 Ras.GTP signaling is terminated by its innate GTPase activity, which returns Ras to the GDP-ligated state. 1 Under normal circumstances, the hydrolysis rate is slow, but it is dramatically enhanced by the interaction of Ras.GTP with GTPaseactivating proteins (GAPs) such as p120-ras.gap. Mutated Ras species in which the activation or inactivation processes are altered (see the online data supplement) are either constitutively active (eg, V12Ha-Ras) or inhibitory (dominant-negative) (eg, N17Ha-Ras), and these mutants have been used extensively to examine participation of Ras in biological processes. In contrast to receptor PTK signaling,

3 Sugden Ras, Akt, and Mechanotransduction 1181 Downloaded from by guest on April 28, 2018 the mechanisms involved in activation of Ras by GPCR ligands are less clear, although these ligands are probably of greater importance in Ras regulation in cardiac myocytes. Effectors of Ras.GTP The differential lethality of targeted disruption of each the three Ras genes (the Ki-Ras-null mutation is embryonically lethal in mice 11 but Ha-Ras- or N-Ras-null mice survive as do the double Ha-Ras/N-Ras-null mutants 12 ) suggests that there is significant diversity of function between Ras isoforms, and this may be related to differential localization in plasma membrane subdomains. 9 Three effectors of Ras signaling have been identified with certainty (Figure 1) 1,13 : the protein kinase c-raf (and the A-Raf and B-Raf isoforms), 13 PI3K, 2,4 and Ral.GDS (a GEF for the Ras subfamily member, Ral). 1 More recently, phosphoinositide-phospholipase C has been added to this list. 14,15 Whereas the signaling pathways associated with c-raf or PI3K activation have been studied extensively in the myocardium, those associated with Ral.GDS or phospholipase C have not and these will not be discussed further apart from mentioning that Ral.GDS or related proteins may participate in the hypertrophic response, 16 and that phospholipase C mrna is expressed relatively abundantly in heart. 15 Raf and the Extracellular Signal Regulated Kinase Cascade The Raf family of mitogen-activated protein kinase (MAPK) kinase kinases catalyze the initial step of the 3-membered extracellular signal regulated kinase 1/2 (ERK1/2) MAPK phosphorylation cascade (Figure 2). 13,17 Generally, by modulating transcription factor activity, apoptosis, and other anabolic processes, activation of the ERK1/2 cascade promotes cell growth, division, and survival. The mechanisms that bring about Ras-mediated activation of c-raf are complex and are not entirely clear. 13,17 In outline, c-raf is normally cytoplasmic and does not bind to (membraneassociated) Ras.GDP. However, c-raf has a much higher affinity for Ras.GTP and their interaction translocates c-raf to the membrane where further interactions and modifications (including phosphorylation of a Ser and a Tyr residue) lead to more complete activation (see the online data supplement). 13,17 Counterbalancing the activating phosphorylations, camp-dependent protein kinase and possibly Akt 20 phosphorylate c-raf, reducing its activity and/or ability to interact with Ras.GTP. In contrast to c-raf, B-Raf may be activated by camp, 13,17 although this remains controversial. After its activation, Raf phosphorylates (and activates) the intermediate MAPK kinase (MKK) members of the ERK1/2 cascade on two Ser residues (Figure 2). 21 Two isoforms of MKK are phosphorylated by Raf (MKK1 and MKK2, alternatively known as MEK1 and MEK2, respectively), but whether these differ in their biological functions is unclear. Mutation of these Ser residues to acidic Asp/Glu residues partially mimics phosphorylation and produces a species with greater constitutive activity than the unphosphorylated form. Although this constitutive activity is only a small percentage of the activity of the phosphorylated species, the mutated species is not subject to protein phosphatase mediated de- Figure 2. Gq protein coupled receptor (GqPCR) agonists such as endothelin-1 (ET-1) stimulate the Gq/phospholipase C mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] to diacylglycerols (and inositol 1,4,5- trisphosphate). Membrane-localized diacylglycerols then activate the diacylglycerol-dependent isoforms of protein kinase C (PKC). In a manner that is not understood, a PKC-dependent event probably activates Ras, although it is known that diacylglycerol-dependent guanine nucleotide exchange factors are present in some cells. GTP/GDP exchange on Ras results in the activation of the extracellular signal regulated kinase 1/2 (ERK1/2) cascade through the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) Raf and MAPK kinases 1/2 (MKK1/2). Pharmacological inhibitors PD98059 and U0126 interfere with the activation of the cascade at the MKK1/2 level. ERK1/2 phosphorylate and activate transcription factors (eg, GATA-4, Elk-1), transcriptional coactivators (eg, CBP, p300), and protein kinases [eg, p90-ribosomal protein S6 kinase (p90- RSK)] to induce biological responses. phosphorylation. Finally, MKK1 or MKK2 phosphorylate a Thr and a Tyr residue within a Thr-Glu-Tyr sequence in ERK1 and ERK2 [also known as p44-mapk (or MAPK1) and p42-mapk (or MAPK2), respectively], 21 and thereby activate these species in both the cytoplasmic and nuclear compartments (Figure 3). Nuclear import of ERK1 or ERK2 may require dimerization. 21 As with MKK1/2, it is unclear whether ERK1 and ERK2 differ in their biological roles. ERK1/2 phosphorylate Ser/Thr residues lying N-terminal to Pro in a variety of proteins, although it should be recognized that (Ser/Thr)Pro is a common motif in many proteins and additional factors (eg, possession of an appropriate ERK1/2 docking site) also determine substrate susceptibility. 22 The proteins that ERK1/2 phosphorylates and activates (or, in part, contributes to the activation of) include protein kinases [eg, 90-kDa ribosomal protein S6 kinase (p90rsk) 23 ], transcription factors (eg, Elk-1, 24 which is important in the regulation of c-fos expression), and other signaling proteins (eg, phospholipase A 2, which is involved in prostanoid synthesis 25 ). Effectors of Ras Signaling: PI3K As mentioned, Ras.GTP may be involved in activation of the PI3K pathway. 1,2,4 PI3K phosphorylates membrane 3-OH phos-

4 1182 Circulation Research December 12/26, 2003 Downloaded from by guest on April 28, 2018 Figure 3. Activated ERK1/2 rapidly appear in the nucleus after exposure of cardiac myocytes to PMA. Myocytes were stained with an antibody to phospho-erk1/2. A, Control. Staining is largely confined to the cytoplasm with negligible staining of nuclei (two of the nuclei indicated by the arrows). B, After exposure to 1 mol/l PMA for 8 minutes, both cytoplasmic and nuclear staining were increased (two of the nuclei indicated by the arrows). Adapted from Chiloeches A, Paterson HF, Marais RM, Clerk A, Marshall CJ, Sugden PH. Regulation of Ras.GTP loading and Ras-Raf association in neonatal rat ventricular myocytes by G protein-coupled receptor agonists and phorbol esters: activation of the ERK cascade by phorbol esters is mediated by Ras. J Biol Chem. 1999;274: , by permission of the American Society for Biochemistry and Molecular Biology phoinositides, producing the membrane-localized second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] when PtdIns(4,5)P 2 is substrate (Figure 4). 4 PI3K is a heterodimer consisting of a catalytic subunit (molecular mass 110 kda) and a regulatory subunit (see the online data supplement). 4 There are three species of catalytic subunit (p110, p110, and p110 ) in the class 1 A PI3Ks, and these heterodimerize with one of the three species of regulatory subunit (p85, p85, or p55). For class 1 B PI3K, the only catalytic subunit so far identified is p110, and the only regulatory subunit is p101. All PI3K catalytic subunits possess a lipid kinase domain, which may also display a limited ability to phosphorylate Ser/Thr residues in proteins, and a Ras interaction domain. Interactions of PI3K regulatory subunits with membrane proteins following receptor stimulation place PI3Ks in the plane of the membrane. For class 1 A PI3Ks, these interactions are with autophosphorylated phospho-tyr residues in receptor PTKs and their associated docking proteins; for class 1 B PI3K, these interactions are with heterotrimeric G protein dimers. Thus, PI3K is placed in proximity to the Ras.GTP formed in a receptor PTK- or GPCR-dependent manner and its membrane substrates. Many of the signaling functions of PtdIns(3,4,5)P 3 depend on its recognition by and binding of proteins containing pleckstrin homology (PH) domains. Other PH domain proteins may bind selectively to other phosphoinositides such as PtdIns(3,4)P 2 or PtdIns(4,5)P 2. PtdIns(3,4,5)P 3 -dependent signaling is terminated by hydrolysis either to reform PtdIns(4,5)P 2 by 3 -lipid phosphatases such as PTEN (a tumor suppressor, mutation of which can predispose to malignancies), or to PtdIns(3,4)P 2 by 5 -lipid phosphatases such as the SHIPs. Regulation of Ras Activation in the Heart As reviewed earlier, 5 previous work in isolated myocytes or in transgenic mice has suggested that V12Ha-Ras induces a hypertrophic phenotype in the myocardium, although some inconsistencies in the in vivo response in particular have been Figure 4. Phosphoinositide 3-OH kinase (PI3K) is activated by receptor protein tyrosine kinases (eg, the insulin or EGF receptor) and phosphorylates the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ], to phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ]. PI3K is inhibited by wortmannin or LY Formation of PtdIns(3,4,5)P 3 induces membrane-binding of Akt, placing it in the vicinity of the PtdIns(3)P-dependent kinase, PDK1. Part of the cell s complement of PDK1 is constitutively localized at the membrane because of its higher affinity for PtdIns(3,4,5)P 3 and because it also binds to PtdIns(3,4)P 2, some of which is formed by hydrolysis of PtdIns(3,4,5)P 3 by 5 -lipid phosphatases. Recently identified 3-deoxy-phosphatidyl-myo-inositols, SH5 and SH6, 55,56 prevent membrane localization of Akt by competing with PtdIns(3,4,5)P 3 -binding to the Akt pleckstrin homology (PH) domain. Akt is phosphorylated and activated by PDK1, and it then phosphorylates or instigates the phosphorylation of a range of substrates to induce biological responses. mtor indicates mammalian target-of-rapamycin; p70s6k, 70-kDa ribosomal protein S6 kinase; 4E-BP1, eukaryotic initiation factor 4E-binding protein; GSK3, glycogen synthase kinase 3; FOXO, Forkhead box transcription factors of class O; 6-PF-2-K, 6-phosphofructo-2-kinase; AMPK, AMP-activated protein kinase; and enos, endothelial nitric oxide synthase. Activation of p70s6k is complex and involves multiple phosphorylations (including phosphorylation indirectly instigated by Akt and direct phosphorylation by PDK1). detected. Conversely, inhibitory N17Ha-Ras reduces agoniststimulated ANP expression in isolated myocytes. As might be predicted given the importance of membrane localization of Ras, 9 farnesyl transferase inhibitors attenuate some of the morphological and transcriptional changes associated with norepinephrine- or ET-1 induced myocyte hypertrophy. 26 Inhibition of hydroxymethylglutaryl CoA reductase, the ratecontrolling step for polyprenoid and cholesterol biosynthesis, by the statins is a less-specific way of reducing Ras farnesylation, but statins reduce stimulation of protein accumulation by ANGII in cardiac myocytes, and importantly, the inhibition can be prevented by mevalonate. 27 However, polyprenoids are involved in the modification of other small G proteins other than Ras, so these results must be interpreted with caution. In isolated myocytes, ET-1, the pharmacological -adrenergic agonist phenylephrine (PE), and phorbol 12- myristate 13-acetate (PMA), a phorbol ester that directly activates certain PKC isoforms, 28 each maximally activate Ras rapidly (30 seconds to 1 minute), 29 as does ANGII and

5 Sugden Ras, Akt, and Mechanotransduction 1183 Downloaded from by guest on April 28, 2018 some other agonists to a lesser extent. 29,30 In the case of ET-1, PE, and PMA, this leads to the formation of complexes of Ras.GTP and c-raf, which, with PMA or ET-1, possess MKK1-activating activity. 29 After exposure to PMA, activated ERK1/2 appear in the nucleus (Figure 3), and this is inhibited by microinjection of neutralizing Ras antibodies, 29 demonstrating that Ras is essential for activation of ERK1 by PMA. GqPCR-mediated activation of Ras is thus potentially able to regulate nuclear events in cardiac myocytes. Activation of Ras by GqPCR agonists presumably involves stimulation of a Ras.GEF or inhibition of a Ras.GAP, the former being the more likely to produce the rapid activation of Ras occurring in the cardiac myocyte. 29 Probably the best-characterized Ras.GEF is Sos, 10 and Sos protein is expressed in cardiac myocytes. 30 What is less clear is whether Sos is involved in the activation of Ras by ET-1, etc, in these cells. By binding to their GqPCRs, ET-1 and PE stimulate the activity of Gq protein regulated phosphoinositide phospholipase C and hydrolysis of PtdIns(4,5)P 2 to (membranelocalized) diacylglycerol (DAG), 31 the physiological activator of conventional and novel PKC isoforms (Figure 3), 28 whose action is mimicked pharmacologically by suitable phorbol esters. In addition to activating Ras, another feature shared by PMA, ET-1, and PE is that they rapidly ( 30 seconds) translocate PKC from the soluble to the particulate fraction (interpreted as indicating PKC activation) in cardiac myocytes, 32,33 and there is some evidence that PKCs participate in the activation of Ras in cardiac myocytes. 29 Because of the rapidity of the response, it seems likely that, if PKC signals to Ras, the interconnection between them cannot be complex, although the mechanism is not understood. Interestingly, the existence of a signaling cassette complex between PKC, N-Ras, and c-raf has been demonstrated in fibroblasts, 34 and there is evidence that PKC -containing signaling cassettes are present in the cardiac myocyte. 35 However, the DAG-dependent PKCs may not represent the only intracellular DAG receptors that are able to activate Ras. Thus, Ras.GEFs that bind DAG and phorbol esters (eg, Ca 2 - and phorbol ester binding Ras.GEFs such as Ras.GRP) have been identified, 36 although it should be pointed out that Ras.GRP appears to be primarily neuronal and may not be of significance in the myocardium. In terms of transcriptional regulation in the cardiac myocyte, it is worth mentioning that ERK1/2 instigate phosphorylation and activation of the transcription factors including Elk-1 37 and GATA-4 38 (Figure 2), and that Ras activates the NF-AT3 (NF-ATc4) transcription factor in an ERK1/2 cascade-dependent manner. 39 In addition, the transcriptional coactivators CBP and p300 are activated through ERK1/2. 40 All these transcription factors have been implicated in the regulation of gene expression in cardiac myocyte hypertrophy. Elk-1 has been identified as an important factor in transcription of the immediate early gene c-fos, 24 which has long been known to be rapidly expressed on hypertrophic stimulation, whereas GATA-4 regulates transcription of hypertrophic index genes including ANF and BNP genes. 41 In addition to stimulating transcription in their own right, NF-ATs cooperate with GATA-4 to increase expression of the BNP gene. 42 We and others have proposed that the ERK1/2 cascade represents an important signaling pathway in myocyte hypertrophy. 8,43 Convincing evidence of an in vivo role has recently been presented, 44 with transgenic mice expressing constitutively activated MKK1 in a cardiac myocyte-directed manner exhibiting a largely adaptive hypertrophy in which the hearts enlarge, fetal genes are reexpressed (see the online data supplement), and dp/dt max is increased (although there is a typical negative lusitropic effect). Additionally, myocytes isolated from these hearts show increased resistance to apoptosis. However, ERK1/2 activities are not increased in the hypertrophied hearts of transgenic mice cardiospecifically expressing V12Ha-Ras, although the activities of c-jun N-terminal kinase, a stress-responsive MAPK, are. 45 Although this finding has been interpreted as implicating JNKs, and not ERK1/2, in promotion of Ras-mediated hypertrophy, an alternative view is that the activation of JNKs is an effect of the ensuing Ras-induced cardiac failure rather than an initiator of adaptive hypertrophy. Much attention has been focused recently on the triplemembrane-passing signal pathway of Ras-dependent signaling. 46 In this pathway, GqPCR stimulation causes phosphorylation and transactivation of the EGF receptor, with subsequent activation of the ERK1/2 cascade and increased c-fos expression. ET-1 and other GqPCR agonists transactivate the EGF receptor in cardiac myocytes, and this may play an important role in myocardial hypertrophy The mechanism involves matrix metalloproteinases, cleavage of extracellular surface-bound pro heparin-binding EGF and heparin-binding-egf shedding, followed by activation of the EGF receptor. 47,49,50 The pathway(s) connecting GqPCR activation to metalloproteinase stimulation is obscure and may variously involve PKC, Ca 2, reactive oxygen species (ROS), and nonreceptor PTKs. 50 Other possible pathways of ERK1/2 cascade activation include the endocytotic -arrestin dependent pathway (see the online data supplement), although this has been studied mainly in connection with GsPCR/GiPCR signaling; however, it has also been implicated in signaling from GqPCRs such as the ET A - and AT1a- receptors, although its relevance to the cardiac myocyte has not been fully explored. Regulation of Akt (PKB) Research into Akt is very active currently and has been summarized in recent excellent reviews, 2,3 (see also the online data supplement) and only basic salient features are summarized here. Like Ras, Akt is encoded by three genes in mammals, with Akt1/PKB being the most widely expressed and best studied. The differing phenotypes displayed by mice with targeted genomic disruption of Akt1 or Akt 2 (both of which are viable) not only imply that Akt1 and Akt2 may fulfill different roles in vivo, but also that there may be a limited ability of Akt1 to substitute for Akt2, and vice versa. 51,52 As described earlier, activation of PI3K increases the membrane content of PtdIns(3,4,5)P 3, and this leads to activation of Akt (Figure 4). There are two strands to this. Akt contains an N-terminal PH domain that binds to PtdIns(3,4,5)P 3, and formation of PtdIns(3,4,5)P 3 translocates inactive Akt from the cytoplasm to the membrane. However,

6 1184 Circulation Research December 12/26, 2003 Downloaded from by guest on April 28, 2018 in spite of some reports to the contrary, the general view is that binding of Akt to PtdIns(3,4,5)P 3 is not sufficient to activate the kinase. Another PH domain kinase, PtdIns(3)Pdependent kinase 1 (PDK1), 2 has greater affinity for PtdIns(3,4,5)P 3 than Akt, and additionally binds to PtdIns(3,4)P 2, some of which is produced by SHIP-mediated hydrolysis of PtdIns(3,4,5)P 3. Thus, a proportion of the cell s complement of PDK1 is constitutively bound to the membrane, and, in contrast to Akt, binding of PDK1 to these 3-phosphoinositides does activate the kinase. The translocation of Akt to the membrane thus juxtaposes the two kinases, and Akt is phosphorylated on Thr 308 in Akt1 (or an equivalent Thr residue in Akt2 and Akt3). However, full activation of Akt1 also requires phosphorylation of Ser 473. The kinase responsible for Akt1(Ser 473 ) phosphorylation is not clear, 2 although experiments using a PDK1 / embryonic stem cell line suggest that it is not PDK1. 53 It has been suggested that Akt1(phospho-Thr 308 ) autophosphorylates or transphosphorylates Akt1(Ser 473 ), although recently a distinct PDK2 activity has been described. 54 Importantly, Akt is not the only substrate of PDK1, which is directly involved in the phosphorylation of other AGC family kinases such as the 70-kDa ribosomal protein S6 kinases (p70s6ks). 2 In some cases, these kinases may mediate some of the effects originally ascribed to Akt because an overlap in recognition of substrate oligopeptide motifs (for details of Akt specificity, see the online data supplement). The problem of overlapping substrate specificities has been compounded by the fact that, until recently, 55,56 there were no small molecule inhibitors of Akt, only of PI3K (wortmannin, LY294002). Biological Roles of Akt A multiplicity of biological effects have been ascribed to Akt (Figure 4). By inhibiting apoptosis at multiple points, Akt promotes cell survival. The regulation of apoptosis and its significance in the cardiac myocyte has recently been reviewed in depth and will not be described in detail. 57 Akt modulates carbohydrate metabolism through phosphorylation and inhibition of glycogen synthase kinase 3 (GSK3), although the inhibition of GSK3 has wider consequences in the heart. 58 It may also regulate fuel metabolism in heart by activating 6-phosphofructo-2-kinase, which produces the allosteric activator of the 6-phospho-1-fructokinase step of glycolysis, fructose 2,6-bisphosphate, 59 and through (a probably indirect) inhibition of AMP-regulated protein kinase. 59,60 The role of Akt in cardiac fuel metabolism will not be discussed further, but it has been reviewed recently. 59 Akt regulates protein synthesis through mechanisms involving the activation of the mammalian target-of-rapamycin, eukaryotic initiation factor 4E-binding proteins (4E-BPs), and p70s6k (see the online data supplement). 61 This aspect of Akt signaling is perhaps particularly important in considering the outcome of expressing constitutively active Akt in the cardiac myocytes of transgenic mice, but like cardiac protein synthesis generally, its importance is underestimated. Finally, Akt phosphorylates and activates endothelial nitric oxide synthase, 62 which is important in regulation of cardiac function and is present in both cardiac myocytes and cardiac endothelial cells. Akt Enhances Cell Survival by Inhibition of Apoptosis Akt increases cell survival through transcription-independent and -dependent mechanisms. 63 Bcl-2 proteins play critical positive and negative roles in the regulation of outer mitochondrial membrane integrity, which is crucial for cell survival. 57 Bcl-2 itself and Bcl-X L are antiapoptotic proteins associated with this compartment and probably to prevent proapoptotic Bcl-2 family proteins such as Bax and Bak from forming poorly characterized pores through which cytochrome c and other proteins are released into the cytoplasm, thereby promoting apoptosome formation. Membrane integrity is maintained by formation of heterodimers of Bax or Bak with Bcl-2 or Bcl-X L. BH3-only Bcl-2 family proteins such as Bad, Bid, and Bim interact with Bcl-2/Bcl-X L through their BH3 domains and, by removing the antiapoptotic influence of Bcl-2/Bcl-X L on Bax and Bak, promote pore formation. However, phosphorylation of Bad(Ser 136 ) and its consequent sequestration by proteins (which recognize phospho- Ser residues in sequence-specific contexts) tethers Bad in the cytoplasm, thus diminishing its proapoptotic potential. 57 Although this phosphorylation was originally attributed to Akt, more recent evidence has indicated that it is p70s6k, which recognizes the same consensus sequence as Akt, that phosphorylates Bad(Ser 136 ), 64 although this is perhaps a minor detail because Akt is intimately involved in p70s6k activation. 2,3 Equally, a second Ser lying in an apparent Akt consensus sequence [Bad(Ser 112 )] is probably phosphorylated in an ERK1/2-dependent manner by p90rsks (which also recognize the Akt consensus sequence), and this may also promote sequestration of Bad. 65,66 The phosphorylation of Bad is complex and is described in more detail in the online data supplement. Another transcription-independent antiapoptotic action of Akt is the phosphorylation and inhibition of caspase 9, an initiator caspase of the mitochondrial pathway of apoptosis present in the apoptosome. 57 Phosphorylation has been demonstrated for the human enzyme, 67 but the site is not conserved in other species. 68 Furthermore, the putative phosphorylation site represents a relatively unfavored consensus sequence for phosphorylation by Akt, and together, these findings cast doubt about the wider significance of caspase 9 phosphorylation. One of the best established transcription-dependent antiapoptotic Akt-dependent pathways involves phosphorylation of Forkhead box transcription factors of class O subfamily (FOXOs) (see the online data supplement). 69 In humans, FOXOs contain three consensus sequences for phosphorylation by Akt, and phosphorylation of these promotes localization to the cytoplasm, a process that is partly dependent on proteins, although the situation is more complex. 69,70 FOXOs promote transcription of proapoptotic paracrine factors and/or their receptors, although confusingly, FOXOs may protect cells from (potentially proapoptotic) oxidative stress under some circumstances by induction of Mn 2 - dependent superoxide dismutase, 71 an enzyme that is known to be cardioprotective. 72 The proapoptotic Fas ligand (FasL) gene, which encodes the ligand for the cell surface Fas receptor, contains three FOXO consensus binding sequences

7 Sugden Ras, Akt, and Mechanotransduction 1185 Downloaded from by guest on April 28, 2018 in its promoter region and its transcription is upregulated by the (dephosphorylated) FOXO, FKHRL1. 69,70 In addition, FOXOs may play a role in promoting the expression of proapoptotic Bim while reducing expression of antiapoptotic Bcl-2 family genes. 69,70 There are at least three less well-characterized transcription-dependent pathways for Akt-mediated cell survival, namely those dependent on the NF- B, CREB, or p53 transcription factors. Nuclear translocation of NF- B, which is normally retained in the cytoplasm in unstimulated cells through sequestration by I B, increases expression of genes encoding inhibitor of apoptosis proteins 73 and (as first shown by Zong et al 74 ) antiapoptotic Bcl-2 family members. Akt-mediated activation of NF- B may involve stimulation of pathways leading to I B phosphorylation (which results in proteasomal I B degradation) or more direct effects on NF- B itself. 63 The transcription factor CREB is a substrate for camp-dependent protein kinase and other protein kinases, and its transactivating activity is increased by phosphorylation of Ser 133. This residue is also phosphorylated by Akt, 75 and the Aktdependent upregulation of expression of Bcl-2 may be mediated through phosphorylation of CREB. 76 Akt may also regulate the transcription-dependent proapoptotic activity of p53 by promoting the translocation of Mdm2, a p53 ubiquitin ligase, to the nucleus, leading to proteasomal degradation of p53 and increased cell survival. 77 Glycogen Synthase Kinase 3: An Important Effector of Akt Signaling GSK3 was originally identified as a Ser/Thr protein kinase that phosphorylates and inhibits glycogen synthase, the enzyme that catalyzes the rate-controlling step in glycogen synthesis, although the influence of GSK3 is now recognized to be considerably wider. The regulation of GSK3 has been reviewed recently (see also the online data supplement), 78 and will only described in outline in this review. GSK3 represents a major substrate of Akt, with phosphorylation of Ser 21 in GSK3 or Ser 9 in GSK3 being inhibitory (this is one pathway through which insulin promotes glycogen accumulation), and mutation of these residues to Ala- produces constitutively active kinases. These Ser residues are also phosphorylated by other protein kinases such as p90rsks and p70s6ks and thus represent intersections allowing crosstalk between signaling pathways. In addition, GSK3 is regulated through the Akt-independent, developmentally important Wnt pathway, although the insulin-sensitive pool of GSK3 and Wnt pathway associated pool of GSK3 appear to be separate. 78 The Wnt pathway regulates the stability of -catenin, a protein involved directly in both transcriptional regulation and cell adhesion, and Wnt-mediated inhibition of GSK3 causes accumulation of unphosphorylated -catenin, which is less susceptible to degradation than the phosphorylated species. Akt and GSK3 in the Myocardium: Regulation of Cell Survival and Growth Regulation of Akt in the Myocardium The potential therapeutic benefit of activation of the PI3K/ Akt pathway in the heart is manifest. 6 However, there have been relatively few studies of the regulation of Akt and most have used only phosphorylation of the Akt1(Ser 473 )-like site as the sole criterion of activation. In cardiac myocytes, insulin and H 2 O 2 induce the greatest degree of Akt(Ser 473 ) phosphorylation; serum is a moderately powerful agonist, but PMA, ET-1, and PE are at best only weak agonists The differing potencies of insulin or PE in stimulating Akt(Ser 473 ) phosphorylation are also reflected in enzymic activity of Akt. 81 Ras.GTP loading on its own is clearly insufficient to activate the PI3K-Akt pathway in cardiac myocytes (because Ras is strongly activated by PMA, ET-1 and PE in cardiac myocytes 29 ), and additional signals (eg, receptor PTK phosphorylation) are presumably required. Cytokine signaling [the interleukin-6 related cytokines, leukemia inhibitory factor, 82 and cardiotrophin-1 (CT-1), 83 or tumor necrosis factor 84 ] also stimulates the phosphorylation/activity of Akt (and PI3K activity and p70s6k phosphorylation) in cardiac myocytes, and erythropoietin has recently been identified as another factor that activates Akt. 85 The cytoprotective properties of insulin and cytokines are discussed in greater detail in the following section. The activation of Akt by H 2 O 2 in cardiac myocytes 79 is something of an anomaly, although it has been detected in other cells. H 2 O 2 may be insulin-mimetic in this regard through increasing Tyr phosphorylation of the insulin receptor or other receptor PTKs by inhibition of protein tyrosine phosphatases. 86 However, H 2 O 2 -mediated Akt phosphorylation, unlike insulin, does not result in the anticipated phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) in myocytes, 79 a phosphorylation that promotes protein synthesis. In fact, unlike insulin, relatively low concentrations of H 2 O 2 strongly inhibit the overall rates of protein synthesis 79 and are cytotoxic to myocytes. 87 In spite of these findings, ROS such as H 2 O 2 have been implicated in the promotion of growth and the stimulation of expression of some genes in cardiac myocytes. 88,89 The biological reasons for the ability of H 2 O 2 to activate Akt are unclear. They may genuinely relate to growth promotion or may represent an endogenous attempt to increase survival. Akt and Myocardial Protection Apoptotic cells have been detected in the myocardium in a variety of pathological situations, although estimates of the frequency differ dramatically. Although there has been disagreement about the significance of apoptosis in myocardial pathologies, the elegant experiments of Kitsis and coworkers have demonstrated that even relatively low levels of initiator caspase 8 activity induce myocardial failure. 90 That activation of Akt is directly protective in whole heart and isolated cardiac myocytes is now well-documented (see review 6 and online data supplement for details of the original articles), the suggestion strongly being that this is due to a reduction in apoptosis. This is likely to be one facet of the cardioprotective and/or antiapoptotic effects of insulin and IGF1 (see, for example, Aikawa et al 91 ; see the online data supplement for further references). In addition, Akt and its effectors have been implicated in the protection mediated by ischemic preconditioning Overexpression of constitutively activated G q activates phospholipase C and this results in

8 1186 Circulation Research December 12/26, 2003 Downloaded from by guest on April 28, 2018 PtdIns(4,5)P 2 hydrolysis. In the myocardium, although this potentially activates the PKC-Ras-ERK1/2 pathway, it also depletes the membrane of the PI3K substrate and reduces Akt activation. 95 This is an interesting result, because it may provide an explanation for some inconsistencies. The balance between activation of signaling pathways and between the overall biological result could be shifted depending on the membrane PtdIns(4,5)P 2 content. It could even explain why some investigators (including ourselves 79 ) have failed to see activation of Akt with powerful GqPCR agonists such as ET-1. Cytokine-based signaling may also involve Akt. CT-1 is both hypertrophic and is a potent survival factor in cardiac myocytes. 96 It stimulates phosphorylation of Bad(Ser 136 ) through a PI3K-dependent pathway (presumably through Akt or p70s6k), and increases cardiac myocyte survival after serum starvation. 82 These antiapoptotic effects of CT-1 are also in part dependent on the ERK1/2 cascade, which is activated by this ligand, 97 and NF- B. 98 Leukemia inhibitory factor is also cytoprotective in the cardiac myocyte, inhibiting doxorubicin-induced apoptosis and promoting phosphorylation of Bad through PI3K. 99 The Bad phosphorylation site was not specified in this study, 99 but was presumably Bad(Ser 136 ). Bad(Ser 112 ) undergoes antiapoptotic phosphorylation in a manner that appears to be largely dependent on the ERK1/2 cascade and the p90rsks, 65,66 and such a pathway exists in cardiac myocytes. 81 Thus, inhibition of Badmediated apoptosis may be regulated through both PI3K/Akt and the ERK1/2 cascade. This agrees with work showing that, although the protective effects of IGF1 against hypoxia in myocytes are dependent in part on PI3K (and Akt?), ERK1/2 (and CREB) are also involved, 78 although in this instance, the conclusion was that the predominant cytoprotective influence was attributable to increased expression of anti-apoptotic Bcl Signaling from IL-6 related cytokines (CT-1, leukemia inhibitory factor) is mediated through their individual receptors and a common signal transducer, glycoprotein130 (gp130). The importance of IL-6 related cytokine signaling in cardiac survival in vivo is evident from experiments with transgenic mice in which the gp130 gene has been targeted for disruption in cardiac myocytes. 100 Though these mice do not show an overt phenotype when the heart is unstressed, they display a greatly increased incidence of dilated cardiomyopathy, cardiac apoptosis, and mortality in response to the stress of pressure-overload. 100 The expression of mrna and protein for CT-1 and gp130 is also increased in the transition of adaptive hypertrophy to failure. 101 This fits with the increased phosphorylation of Akt(Ser 473 ) and inhibition of GSK3 detected in hearts from cardiomyopathic patients, 102 and can rationalized in terms of an endogenous attempt on the part of the heart to survive. Very recently, activation of cardiac Akt by erythropoietin has been demonstrated and may contribute to the hematocrit-independent cardioprotection instigated by this factor. 85,103 Finally, it has been suggested that Akt mediates genderrelated susceptibility to cardiac disease. 17 -Estradiol reduces staurosporine-induced apoptosis in cardiac myocytes. 104 Because Akt is activated by estrogens and phytoestrogens, gender-related (and possibly nutritional) differences in susceptibility to cardiac disease may be due to Akt activation and inhibition of the transactivating activity of FOXOs. 105 GSK3 and Myocardial Hypertrophy and Protection Inhibition of GSK3 by phosphorylation of Ser 9 through the PI3K/Akt pathway promotes ET-1 or -adrenergic mediated hypertrophy of cultured cardiac myocytes. 106,107 Furthermore, transfection or adenoviral transfer of GSK3 (Ser 9 3Ala) inhibits ET-1 or isoproterenol-induced hypertrophy as assessed by sarcomerogenesis or ANF protein expression. 106,107 Inhibition was blocked by Li, which, at concentrations of 10 mmol/l, inhibit GSK3 (but probably not many other protein kinases) by about 40%. 106,107 GSK3 is also inhibited in cardioprotection by ischemic preconditioning through a PI3K-dependent pathway, 92,93 and inhibitors of GSK3 mimic the protective effects of preconditioning. 93 It should be noted that the GSK3 signaling pathways thus far identified in relation to hypertrophy are an example of negative control with active GSK3 maintaining phosphorylation and inactivation of, eg, transcription factors in the basal state. Only when GSK3 is inhibited do they become activated, but this is dependent on the appropriate protein Ser/Thr phosphatases dephosphorylating the proteins concerned. The pathways through which inhibition of GSK3 promotes myocyte growth and ischemic preconditioning are not clear. With respect to growth, ET-1 or PE-mediated activation of Akt promotes dephosphorylation and stabilization of -catenin through (Wnt-independent) inhibition of GSK Mutated stabilized -catenin (GSK3 phosphorylation sites deleted) promotes an increase in cell surface area and protein synthesis, although it does not promote sarcomerogenesis or ANF expression, 108 which are presumably regulated by additional pathways. Of ancillary interest is the observation that Wnt signaling itself induces aggregation and adhesion of isolated neonatal rat cardiac myocytes by increasing formation of cadherin- -catenin complexes, 109 presumably through GSK3. Wnt-independent stabilization of -catenin could be relevant to hypertrophy (at least in cultured cells) in terms of the promotion of intercellular contacts. GSK3 phosphorylates other transcription factors in addition to -catenin. These include the c-jun DNA binding domain (inhibiting DNA binding and attenuating c-jun dependent transcription), 110,111 and NF-AT2 (also known as NF-ATc or NF-ATc1). 112 Phosphorylation of NF-ATs promotes their exclusion from the nucleus, and accordingly, ET-1 promotes nuclear translocation of NF-AT (possibly NF-AT2) in myocytes in a manner that is inhibited by expression of GSK3 (Ser 9 3Ala). 106 Somewhat confusingly, ET-1 also translocates (presumably inactivated?) GSK3 to the nucleus, 106 the function of which is unclear. The GSK3- mediated stimulation of ANF expression by -adrenergic agonism may be mediated by the cardiac myocyte-restricted transcription factor GATA Activated GSK3 promotes nuclear export of GATA-4 in cardiac myocytes through the nuclear exportin, Crm1, 107 although it is not clear whether GSK3 phosphorylates GATA-4 directly. Of ancillary interest is the finding that -adrenergic agonism promotes the association of NF-AT2 and GATA-4 in cardiac myocytes. 113 The

9 Sugden Ras, Akt, and Mechanotransduction 1187 Downloaded from by guest on April 28, 2018 overall conclusion is that inhibition of GSK3 should increase binding of c-jun to its consensus sequences, and promote nuclear entry/retention of NF-AT2 and GATA-4, thus stimulating gene expression from promoters sensitive to these transcription factors. Manipulation of Akt and GSK3 Activities in Transgenic Mice Akt can be constitutively activated by membrane-targeting (eg, introduction of a c-src myristoylation motif), or by mutation of Thr 308 and Ser 473 to Asp, and three groups have reported the phenotypes of mice expressing such species in a cardiac myocyte directed manner The prediction that the phenotype should resemble that for mice expressing a constitutively activated PI3K 117 is largely borne out. The hearts are larger as a result of an increase cardiac myocyte mean size, and there is resistance to ischemia/reperfusion injury. 116 There were some changes in contractile properties, although these were somewhat variable between the mouse lines. However, where detected, any enhancement of cardiac contraction-relaxation may be the result of changes in Ca 2 movements mediated by upregulation of SERCA2a. 118 From the signal transduction standpoint, p70s6k was phosphorylated as predicted but, surprisingly, GSK3 phosphorylation was not universally detected, although this could relate to time of sampling. When GSK3 phosphorylation was detected, nuclear translocation of GATA-4 was also observed. 114 The mammalian target-of-rapamycin (see the online data supplement) was necessary for the expression of the Akt1(Thr Asp,Ser Asp) phenotype and p70s6k activation. 116 Gene expression profiling by DNA microarrays of one Akt transgenic line did not reveal any changes in the expression of hypertrophic marker genes, although expression of genes that might contribute to the effects of Akt on cardiomyocyte survival, metabolism, and growth was affected. 119 Overall, the phenotypes of the activated Akt transgenics suggest that Akt has two roles: (1) to coordinate the myocardial mass with maturational and nutritional signals 120 and (2) to cytoprotect. 6 Transgenic mice have also been used to study the role of GSK3 in the heart. 121 Cardiac myocyte directed expression of GSK3 (Ser 9 3Ala) alone has little effect on heart weight/ body weight ratio, but in combination with expression of constitutively activated calcineurin, GSK3 (Ser 9 3Ala) reduced calcineurin-induced increases in heart weight/body weight ratio and reduced accumulation of NF-ATs in the nucleus. 121 Similarly, GSK3 (Ser 9 3Ala) reduces the increase in relative heart weight induced by -adrenergic stimulation or aortic constriction. From the point of view of transcriptional criteria of hypertrophy, results were confusing. Although the calcineurin-induced increase in -myosin heavy chain expression was reduced by GSK3 (Ser 9 3Ala), the expression of ANF or BNP was increased. 121 The outcome of activation of the receptor-mediated [Fas ligand (FasL)/Fas] apoptosis pathway in myocytes is ambiguous, 57 but may be rationalized to some extent by its actions on GSK3. Although FasL is reportedly proapoptotic in cultured myocytes, 122 the phenotype of mice expressing FasL in a cardiac myocyte directed manner is one of cardiac inflammation and mild hypertrophy, rather than frank apoptosis. 123 This result is somewhat unexpected (forced FasL expression would be predicted to be proapoptotic), although it has recently been suggested that FasL inhibits GSK3 in isolated neonatal cardiac myocytes through the PI3K pathway and thus promotes their hypertrophy. 124 In vivo, mice lacking a functional Fas receptor display a cardiomyopathic rather than a hypertrophic response after aortic constriction and GSK3 was not inhibited (unlike the wild-type situation). 124 However, mice lacking functional FasL did mount a hypertrophic response after aortic constriction, possibly because Fas receptor transactivation from other receptors is a feature of the hypertrophic response. 124 Involvement of Ras and Akt in Cardiac Mechanotransduction The topic of mechanotransduction in the heart has been fully reviewed (see also the online data supplement), 7 and the focus here will primarily relate to involvement of Ras and Akt in this process. The commonly used experimental models (see the online data supplement) ex vivo are cyclical or static strain of myocytes attached a deformable matrix (which demonstrably induces hypertrophy) and perfused hearts. In vivo, constriction of the (thoracic) aorta is probably the most frequently used intervention. Given the established functions of Ras and Akt, it is inherently likely that they, at least in part, mediate mechanotransductional growth, although direct evidence is relatively exiguous. Likewise, direct evidence that they are activated during mechanotransduction is relatively sparse, and it is necessary largely to rely on surrogate indicators, such as activation of the ERK1/2 cascade for Ras, and phosphorylation of p70s6k or GSK3 for Akt. An alternative strategy is to establish the participation in mechanotransduction of factors known to activate Ras or Akt, thus peptide growth factors, ROS, and ECM signaling will be discussed. Mechanosensitive ion channels will not be discussed as there is currently no evidence of which the author is aware that their activity affects Ras or Akt. With respect to direct evidence of Ras and Akt activation in mechanotransduction, there is only one report to my knowledge that strain increases Ras.GTP loading (in statically stretched myocytes). 125 The activation of Ras in vivo has not been studied. Phosphorylation of Akt (and of enos) in an LY inhibited manner in matrix-embedded stretched myocytes has been demonstrated, 126 and increased phosphorylation of Akt (and of GSK3 ) has been detected in acute (10-minute) pressure-overload in mouse hearts. 127 Furthermore, work in the early 1990s showed that strain activates a multiplicity of signaling molecules that are known to lie either upstream or downstream of Ras, including phospholipases, PKC, ERK1/2, and p90rsk. 125 In vivo, acute or chronic pressure overload activates ERK1/2 and p90rsk, and consistent with a role for Gq/11PCRs in hypertrophy, conditional targeted genomic disruption in the heart of the subunits of the heterotrimeric G proteins, Gq and G11, 130 or cardiac myocyte-specific expression of a peptide that interferes with interaction of GqPCRs with Gq, 131 reduces pressure-overload hypertrophy. Disruption of the G protein

10 1188 Circulation Research December 12/26, 2003 Downloaded from by guest on April 28, 2018 subunit genes would be expected to prevent agonistdependent activation of Ras. 29 Local Release of Peptides and Other Species That Activate Ras or Akt Conditioned medium from mechanically strained cardiac myocytes stimulates hypertrophic responses in naïve myocytes, 125 suggesting that local release of hypertrophic factors is important. A variety of approaches both ex vivo and in vivo (see the online data supplement) support a role for (local) release of ANGII or endothelins. As mentioned above, both ET-1 and ANGII activate Ras in cardiac myocytes, 29,30 although there is little evidence that they are effective activators of Akt in these cells. 79 Metalloproteinase-mediated shedding of HB-EGF may be important in both pressureoverload hypertrophy and in GqPCR agonist-induced hypertrophy. 47 As mentioned earlier, activation of the EGF receptor potentially stimulates Ras.GTP loading and Akt activation. All of these correlative data certainly implicate Ras and possibly implicate Akt. Less explored is any involvement of gp130-related signaling. Although gp130-linked signaling is implicated in cardiac hypertrophy ex vivo, 96 possibly through the involvement of ERK1/2 96 and Akt, 82,83 results of experiments in which expression of CT-1 or leukemia inhibitory factor has been examined in vivo under overload conditions have been equivocal. 101,132 However, cardiomyocytic overexpression of a dominant-negative gp130 reduced the magnitude of pressure-overload hypertrophy in transgenic mice. 129 As mentioned, pressure-overload of hearts of transgenic mice in which the gp130 gene had been cardiomyocyte-specifically disrupted produced a dilated cardiomyopathy rather than an adaptive hypertrophy, 100 raising the possibility that an adaptive response would have manifested itself had the gp130 gene not been disrupted. Although ROS are cytotoxic at higher concentrations, 87 they may directly promote growth of cardiac myocytes under more benign circumstances. 88 From the signaling standpoint, H 2 O 2 activates MAPK cascades and the PI3K/Akt pathway in cardiac myocytes, 79,133,134 although it is not known whether Ras is involved. As described in the online data supplement, there is evidence that mechanical strain increases ROS production in cardiac myocytes. Extracellular Matrix Based Signaling More general details of ECM-based and focal adhesion based signaling are given in the online data supplement. In vivo, the adult cardiac myocyte interacts both with other myocytes and with the ECM. 135 The attachment of the myocyte to the ECM and any subsequent stretching of the myocyte during hemodynamic overload may stimulate growth. One cardiac myocyte specific locus for interaction with the ECM is the costamere. Costameres are protein complexes that lie adjacent to the Z lines of subsarcolemmal myofibrils and contain cytoskeletal proteins such as vinculin, 1 -integrin, and other proteins, 135 including the recently identified 1 -integrin interacting protein, melusin. 136 Interactions between integrins and the ECM are important in myocyte growth and hypertrophy. 137 Targeted disruption the 1 -integrin gene renders mice intolerant to pressure-overload and leads, in the longer term, to dilated cardiomyopathy even in the unloaded situation. 138 Disruption of the melusin gene prevents pressure-overload hypertrophy in vivo, but does not prevent hypertrophy induced by subpressor doses of ANGII or PE. 127 Parenthetically, the phosphorylation of Akt or GSK3 was decreased in melusin-null mice compared with wild-type after short- (10 minutes) or long-term (7 days) pressure overload. 127 Interaction between laminins of the ECM, the dystrophin-dystroglycan-sarcoglycan complex, and myofibrillar structures represents another example of myocyte-ecm interaction in vivo, 139 and the interaction may influence nuclear events, possibly being transmitted by the cytoskeletal/intermediate filament protein, desmin. 140 Like many cells, cardiac myocytes in culture form focal adhesions where they attach to an ECM substrate such as fibronectin. 135 The simple attachment of myocytes to a fibronectin matrix induces activation of focal adhesion related signaling pathways, 141 and myocyte growth and hypertrophy. 141,142 However, the relationship of focal adhesions in cultured myocytes to myocyte-ecm interactions in vivo are not understood in detail. 135 Focal adhesion signaling proteins such as the focal adhesion kinase (FAK), the docking protein p130cas, and the adaptor/scaffold protein paxillin are located in the region of the Z line in isolated neonatal cardiac myocytes and may play a role in the maintenance of sarcomere structure. 143,144 The concept is emerging that FAK is not so much a protein kinase as a self-regulating docking protein providing a scaffold that activates other signaling molecules. Myocyte strain or pressure overload ex vivo activates focal adhesion signaling (eg, increased Tyr phosphorylation of FAK), and activation of focal adhesion signaling is known to lead to stimulation of multiple signaling pathways including the Ras-ERK1/2 and PI3K/Akt pathways in other cells. 148 Integrin/focal complex based signaling has also been implicated in the pressure-overloaded ventricle in vivo, 149,150 although, in this situation, the cellular heterogeneity of the heart is a complicating factor and it is not clear whether this signaling pathway is sufficient on its own to induce the hypertrophic response. Interestingly, the pattern of Tyr phosphorylation of FAK was not consistent with a role in pressure-overload induced LV hypertrophy in vivo, although the pattern of expression and Tyr phosphorylation of the p125fak-related protein kinase, PYK2, was more so. 151 The overall conclusion is that ECM-based signaling, possibly by acting through focal adhesions, is a facet of strain-induced hypertrophy. Do Ras and Akt Participate in Overload-Induced Hypertrophy In Vivo? Overall Conclusions Although it seems likely that Ras and Akt participate in cardiac mechanotransduction, this still remains to be definitively established. Probably the best direct test would be to use transgenic mice in which the various Ras or Akt genes are inactivated or cardioselectively express suitable dominantnegative Ras or Akt mutants. Targeted genomic disruption of Ha-Ras and/or N-Ras genes, or of the Akt1 or Akt2 genes is not lethal in mice, although, phenotypically, the mice may differ from their wild-type littermates. 12,51,52 Mice that selectively express dominant-negative Akt in their cardiac myo-

11 Sugden Ras, Akt, and Mechanotransduction 1189 Downloaded from by guest on April 28, 2018 cytes have also been derived. 116 These animals should be suitable for direct experimentation on the effects of hemodynamic overload, although no experiments of this nature have been reported. Acknowledgments The author s scientific work was supported principally by the British Heart Foundation, with some additional funding from the Wellcome Trust. References 1. Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins. Physiol Rev. 2001;81: Vanhaesebroeck B, Alessi DR. The PI3K-PDK1 connection: more than just a road to PKB. Biochem J. 2000;346: Brazil DP, Hemmings BA. Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem Sci. 2001;26: Vanhaesebroeck B, Leevers SJ, Ahmadi K, Timms J, Katso R, Driscoll PC, Woscholski R, Parker PJ, Waterfield MD. Synthesis and function of 3-phosphorylated inositol lipids. Annu Rev Biochem. 2001;70: Clerk A, Sugden PH. 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15 Ras, Akt, and Mechanotransduction in the Cardiac Myocyte Peter H. Sugden Downloaded from by guest on April 28, 2018 Circ Res. 2003;93: doi: /01.RES F5 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 2003 American Heart Association, Inc. All rights reserved. Print ISSN: Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: Data Supplement (unedited) at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Circulation Research is online at: