REVIEW
Glycogen synthase kinase 3(GSK3)in the heart:a point of integration in hypertrophic signalling and a therapeutic target?A critical analysis
PH Sugden,SJ Fuller,SC Weiss and A Clerk
National Heart and Lung Institute (NHLI)Division,Faculty of Medicine,Imperial College London,London,UK
Glycogen synthase kinase 3(GSK3,of which there are two isoforms,GSK3a and GSK3b )was originally characterized in the context of regulation of glycogen metabolism,though it is now known to regulate many other cellular processes.Phosphorylation of GSK3a (Ser21)and GSK3b (Ser9)inhibits their activity.In the heart,emphasis has been placed particularly on GSK3b ,rather than GSK3a .Importantly,catalytically-active GSK3generally restrains gene expression and,in the heart,catalytically-active GSK3has been implicated in anti-hypertrophic signalling.Inhibition of GSK3results in changes in the activities of transcription and translation factors in the heart and promotes hypertrophic responses,and it is generally assumed that signal transduction from hypertrophic stimuli to GSK3passes primarily through protein kinase B/Akt (PKB/Akt).However,recent data suggest that the situation is far more complex.We review evidence pertaining to the role of GSK3in the myocardium and discuss effects of genetic manipulation of GSK3activity in vivo .We also discuss the signalling pathways potentially regulating GSK3activity and propose that,depending on the stimulus,phosphorylation of GSK3is independent of PKB/Akt.Potential GSK3substrates studied in relation to myocardial hypertrophy include nuclear factors of activated T cells,b -catenin,GATA4,myocardin,CREB,and eukaryotic initiation factor 2B e .These and other transcription factor substrates putatively important in the heart are considered.We discuss whether cardiac pathologies could be treated by therapeutic intervention at the GSK3level but conclude that any intervention would be premature without greater understanding of the precise role of GSK3in cardiac processes.
British Journal of Pharmacology (2008)153,S137–S153;doi:10.1038/sj.bjp.0707659;published online 21January 2008
Keywords:cardiac myocytes;transgenic mouse models;myocardial hypertrophy;glycogen synthase kinase 3;protein kinase
B/Akt;mitogen-activated protein kinases;transcription factors;nuclear factor of activated T cells;b -catenin
Abbreviations:APC,adenomatous polyposis coli;BW,body weight;eIF,eukaryotic initiation factor;ERK,extracellular signal-
regulated kinase;GSK3,glycogen synthase kinase 3;HDAC,histone deacetylase;HW,heart weight;IGF 1,insulin-like growth factor 1;LRP,lipoprotein receptor-related protein;MAPK,mitogen-activated protein kinase;MSK,mitogen-and stress-activated protein kinase;NFAT,nuclear factor of activated T cell;PtdIns(4,5)P 2,phosphatidylinositol 4,5-bis phosphate;PtdIns(3,4,5)P 3,phosphatidylinositol 3,4,5-tris phosphate;PI3K,phos-phoinositide 30kinase;PKA,cyclic AMP-dependent protein kinase A;PKB,protein kinase B;PKC,protein kinase C;RSK,p90-ribosomal S6kinase;TAC,transverse aortic constriction;TCF/LEF1,T-cell-specific transcription factor/lymphoid enhancer binding factor 1;TL,tibial length
Introduction
Glycogen synthase kinase 3(GSK3)is emerging as an important therapeutic target in a variety of pathologies (Cohen and Goedert,2004).Here,we will be principally concerned with its role in cardiac growth and heart failure,particularly with that occurring during myocardial hyper-trophy.GSK3may also play an important role in the
cardioprotection afforded by ischaemic preconditioning (Tong et al .,2002),but this is beyond the scope of this review.GSK3was originally identified as a Ser/Thr protein kinase that phosphorylates glycogen synthase on multiple sites in its C-terminal region (Figure 1a)to inhibit its activity (Embi et al .,1980).It is a key signalling protein in the regulation of glycogen synthesis and the activity of GSK3itself is regulated by phosphorylation–dephosphorylation,phosphorylation being inhibitory (Figure 1b).Phosphoryla-tion of GSK3is increased by insulin and this represents a major mechanism by which insulin promotes glycogen accumulation (Cohen,
2006).
Received 26July 2007;revised 16November 2007;accepted 4December 2007;published online 21January 2008
Correspondence:Professor PH Sugden,National Heart and Lung Institute (NHLI)Division,Faculty of Medicine,Imperial College London,Flowers Building (4th Floor),Armstrong Road,London SW72AZ,UK.E-mail:
p.sugden@https://www.doczj.com/doc/de10860588.html,
British Journal of Pharmacology (2008)153,S137–S153
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https://www.doczj.com/doc/de10860588.html,
Since its discovery around 25years ago,GSK3has been implicated in the regulation of many additional biological processes apart from glycogen metabolism.Particular emphasis has been placed on its role in the phosphorylation of transcriptional regulators and translation initiation factors,and the consequent effects on gene and protein expression.It is also notable that GSK3regulates metazoan development by modulating transcriptional activity in the context of the ‘canonical’Wnt pathway (Logan and Nusse,2004;Cadigan and Liu,2006;Gordon and Nusse,2006).The potential role of GSK3in diseases,including diabetes mellitus and neurodegenerative disorders,has led to the proposal that inhibition of GSK3may be therapeutically useful (Cohen and Goedert,2004).However,as discussed by Cohen and Goedert (2004),long-term pharmacological inhibition of GSK3may result in increased malignancies because active GSK3generally restrains gene expression.Analogously,in myocardial hypertrophy (which also involves altered patterns and rate of gene expression),the active forms of GSK3are thought to act as a restraint.Thus,systemic inhibition of GSK3may also promote anomalous hypertrophic growth of the heart possibly leading to heart failure.Conversely,systemic activation of GSK3should reduce hypertrophic growth but might promote diabetes and neurodegeneration.We will return to the topic of GSK3as a point for therapeutic intervention subsequently.
General properties and regulation of GSK3
Mammalian cells express two isoforms of GSK3,GSK3a (molecular mass B 51kDa)and GSK3b (molecular mass B 47kDa).The catalytic domains of GSK3exhibit a high degree of sequence similarity,but the isoforms diverge in their N-and C-terminal sequences (Figure 1c).GSK3a and GSK3b are each phosphorylated on at least two residues (Figure 1c).In terms of their diurnal regulation by hormones,such as insulin,probably the most significant residues are GSK3a (Ser21)and GSK3b (Ser9),phosphorylation of which inhibits their protein kinase activities (Ali et al .,2001;Cohen and Frame,2001;Frame and Cohen,2001;Woodgett,2001;Cohen and Goedert,2004).In addition,‘maturational’or ‘facilitative’phosphorylations of GSK3a (Tyr279)and GSK3b (Tyr216)are required for GSK3activity and may play a structural role (Wang et al .,1994;Cole et al .,2004).They probably involve intramolecular autophosphorylation catalysed by GSK3itself and there is no consistent evidence that changes in the degree of phosphorylation of GSK3a (Tyr279)or GSK3b (Tyr216)play any role in the diurnal regulation of its in vivo activity (Cohen and Goedert,2004;Cole et al .,2004).In other words,GSK3a (Tyr279)and GSK3b (Tyr216)are always stoichiometrically phosphory-lated under physiological conditions.
The protein kinases that phosphorylate and regulate GSK3a (Ser21)and GSK3b (Ser9)are reasonably well estab-lished (Figure 2;Ali et al .,2001;Cohen and Frame,2001;Frame and Cohen,2001;Woodgett,2001;Cohen and Goedert,2004).These sites of phosphorylation lie within an established consensus sequence for cyclic AMP-dependent protein kinase B/Akt (PKB/Akt),namely Arg-Xaa-Arg-Xaa-Xaa-Ser(P)/Thr(P),the optimal site being Arg-Lys-Arg-Xaa-Arg-Thr-Tyr-Ser(P)-Phe-Gly as defined by peptide library screening (Obata et al .,2000).Within Homo sapiens GSK3,the sequences are Ser-Gly-Arg-Ala-Arg-Thr-Ser-Ser(P)-Phe-Ala in GSK3a and Ser-Gly-Arg-Pro-Arg-Thr-Thr-Ser(P)-Phe-Ala in GSK3b .In terms of insulin signalling and glycogen accumulation,PKB/Akt is activated by phosphorylation downstream from phosphoinositide 30kinases (PI3Ks),phos-phatidylinositol 3,4,5-tris phosphate (PtdIns(3,4,5)P 3)and 3-phosphoinositide-dependent kinase 1(Vanhaesebroeck
and
Figure 1Regulation of glycogen metabolism by glycogen synthase kinase 3(GSK3).(a )Casein kinase 2(CK2)phosphorylates a priming Ser residue in the C terminus of glycogen synthase (Ser657in the muscle isoform of Homo sapiens )to initiate a relay of GSK3-catalysed phosphorylations,as described in the text.(b )Glycogen synthase activity is regulated by a phosphorylation–dephosphorylation cycle.In the preprandial state,plasma insulin concentrations are low and GSK3is active.GSK3phosphorylates glycogen synthase and converts it to the less-active (glucose-6-phosphate (G6P)dependent)form,thus inhibiting glycogen synthesis.When plasma insulin concentrations rise after feeding,insulin promotes phosphorylation of GSK3and inhibits GSK3activity.Dephosphorylation of glycogen synthase by protein phosphatases (GS phosphatase)subsequently increases the activity of glycogen synthase and promotes glycogen synthesis.(c )GSK3a and GSK3b exhibit a high degree of homology in their kinase domains,diverging at their N-and C-terminal regions.GSK3a contains an N-terminal Gly-rich domain of unknown function.However,Gly-rich domains are classically associated with binding of nucleotides (Bossemeyer,1994),though this domain is distinct from the ATP-binding site in the kinase domain.Both isoforms are constitutively phosphorylated on a Tyr residue in the kinase domain and this phosphorylation is required for activity.Phosphorylation of a conserved N-terminal Ser residue inhibits the activity of GSK3.
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Alessi,2000;Cohen,2006),and this pathway probably represents the most important regulatory route to inhibition of GSK3by insulin (Cohen,2006).Other kinases also phosphorylate the Arg-Xaa-Arg-Xaa-Xaa-Ser(P)/Thr(P)motif and,for GSK3,the best studied are the p70/p85-S6
kinases (S6Ks)and the p90-ribosomal S6kinase (RSK)family (Fro
¨din and Gammeltoft,1999;Cohen and Frame,2001;Frame and Cohen,2001;Cohen and Goedert,2004).Other kinases including serum/glucocorticoid-regulated kinases (Kobayashi and Cohen,1999)and the RSK-related mitogen-and stress-activated protein kinases (MSKs,Fro
¨din and Gammeltoft,1999)also phosphorylate GSK3.Whereas S6Ks are activated downstream from PKB/Akt,RSKs are activated downstream from the best characterized of the mitogen-activated protein kinases (MAPKs),the extracellular signal-regulated kinases 1/2(ERK1/2).MSKs are activated by both ERK1/2and p38-MAPKs.In addition,cell mem-brane-permeating cyclic AMP analogues,agents which raise cyclic AMP or overexpression of the cyclic AMP-dependent protein kinase A (PKA)catalytic subunit stimulate phosphory-lation of GSK3a (Ser21)and GSK3b (Ser9)(Fang et al .,2000).Furthermore,PKA physically associates with GSK3b in cell cultures ex vivo ,and the PKA catalytic subunit phosphory-lates GSK3a (Ser21)and GSK3b (Ser9)in vitro .The GSK3a (Ser21)and GSK3b (Ser9)phosphorylation sites lie within an ‘adequate’consensus sequence for PKA,the optimum being Arg-Arg-Arg-Arg-Ser(P)-Ile-Hydrophobic-Hydrophobic-Ile as defined by peptide library screening (Songyang et al .,1994),that is basic (Arg-)residues lying N-terminal,hydrophobic residues lying C-terminal to the phosphorylated Ser-residue.Effects of cyclic AMP/PKA on GSK3phosphorylation have been identified sporadically in non-cardiomyocytic cells and tissues (Li et al .,2000;Jensen et al .,2007;Taurin et al .,2007).However,to quote Frame and Cohen (2001)directly,who were admittedly writing some years ago,‘the physiological relevance (of these observations)is not yet clear’.As a major function of cyclic AMP is to promote glycogen breakdown rather than glycogen synthesis,the phosphorylation of GSK3by PKA in the context of glycogen metabolism is somewhat dissonant.
GSK3is also regulated through the so-called ‘canonical’Wnt pathway (Figure 3a),which is important in metazoan development (Figure 3a;Logan and Nusse,2004;Cadigan and Liu,2006;Gordon and Nusse,2006;see also https://www.doczj.com/doc/de10860588.html,/~rnusse/wntwindow.html).Wnts are a family of extracellular palmitoylated glycoproteins encoded by 19genes in H.sapiens .It is still not entirely clear how Wnts are presented to their target cells,two possibilities being presentation by a ‘donor’cell in the form of cell surface-tethered molecules or presentation in a diffusible form.On presentation to its target cell,Wnt interacts with heptahelical (seven transmembrane spanning)receptors known collectively as Frizzled (10genes in H.sapiens ),and with single transmembrane-pass co-receptors known as low-density lipoprotein receptor-related proteins (LRPs).In the absence of Wnt,GSK3is catalytically active in a complex with axin and the adenomatous polyposis coli (APC)protein and phosphorylates b -catenin.The pool of b -catenin,which is phosphorylated by GSK3,seems to be specifically involved in regulating gene expression,and a separate additional pool is involved in cell–cell interactions at adherens junctions (Perez-Moreno and Fuchs,2006).Phosphorylation of b -catenin by GSK3is a signal for its ubiquitination and degradation by the ubiquitin–proteasome system.In the presence of Wnt,the intracellular scaffolding
protein
Figure 2Signalling pathways leading to the phosphorylation and inhibition of glycogen synthase kinase 3(GSK3).The following scheme is a simplification of the events and only the salient features of the pathways are outlined.Receptor protein tyrosine kinase (RPTK)agonists (for example,insulin)stimulate the activity of phosphoinositide 30kinase (PI3K),which phosphorylates the membrane phospholipid phosphatidylinositol 4,5-bis phosphate (PtdIns(4,5)P 2),to produce phosphatidylinositol 3,4,5-tris phosphate (PtdInsP 3).PtdInsP 3remains in the plane of the membrane,activating 3-phosphoinositide-dependent kinase 1(PDK1)and also causing translocation of protein kinase B/Akt (PKB/Akt)to the membrane where it is phosphorylated on Thr308and partially activated by PDK1.Phosphorylation of PKB/Akt(Ser473)by the mammalian target-of-rapamycin complex 2(not shown)fully activates the kinase.PKB/Akt subsequently phosphorylates the two GSK3isoforms on a Ser-residue in each of their N-terminal domains to inhibit their activities (see Figure 1c).Gq-protein-coupled receptor (GqPCR)agonists (for example,endothelin-1(ET-1))stimulate phospholipase C b which hydrolyses PtdIns(4,5)P 2to produce diacylglycerol and inositol 1,4,5-tris phosphate.The former is the physiological activator of the diacylglycerol-regulated protein kinase C isoforms (which can also be activated by the tumour-promoting phorbol esters),whereas the latter is important in the regulation of intracellular Ca 2tmovements.Protein kinase C activates the extracellular signal-regulated kinases 1/2(ERK1/2)cascade,which involves the small guanine nucleotide-binding protein Ras.Conver-sion of Ras.GDP to its biologically active form,Ras.GTP,leads to the hierarchical activation of three levels of protein kinases.Raf phosphorylates and activates mitogen-activated protein kinase kinases 1/2(MKK1/2),which then phosphorylate and activate ERK1/2.The signalling pathway then diverges.One consequence of activation of ERK1/2is their phosphorylation and activation of the p90-ribosomal subunit S6kinases (RSKs),which phosphorylate and inhibit GSK3.Some RPTK agonists (for example,epidermal growth factor,EGF),in addition to activating PKB/Akt (Clerk et al .,2006),activate the ERK1/2cascade by stimulating GTP/GDP exchange on Ras via the Grb2/Sos guanine nucleotide exchange factor system,and thus activate Ras.In addition,PKB/Akt also activates the mammalian target-of-rapamycin complex 1(mTOR).This leads to phosphorylation and activation of p70/p85-ribosomal subunit S6kinases (S6Ks),thence to phosphorylation and inhibition of GSK3.Agents that raise cyclic AMP concentrations and activate cyclic AMP-dependent protein kinase A (PKA)may bring about phosphorylation and inhibition of GSK3,though the physiological significance of this mode of regulation is somewhat obscure.
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Dishevelled associates with the transmembrane Wnt–Frizzled–LRP complex (Figure 3a).The axin–APC–GSK3complex is then recruited,with coordinate phosphorylation of Dishevelled and of the intracellular domain of LRP.In this context,GSK3is unable to phosphorylate b -catenin,dephospho-b -catenin accumulates and subsequently migrates into the nucleus.In the nucleus,T-cell-specific transcription factor/lymphoid enhancer binding factor 1(TCF/LEF1)is normally maintained in a repressed state by association with Groucho.b -Catenin displaces Groucho and,with TCF/LEF1,forms a complex that activates transcription of TCF/LEF1-regulated genes.
Substrates of GSK3:general points
GSK3preferentially phosphorylates substrates on Ser/Thr residues in a ‘relay’fashion.A ‘priming’phosphorylation on a Ser/Thr residue is catalysed by a protein kinase distinct from GSK3.This increases the rate of phosphorylation by GSK3at a Ser/Thr residue à4to the priming phosphorylation (that is,the fourth residue lying N-terminal to the priming phosphorylation,for example,Ser -Xaa-Xaa-Xaa-Ser(P )).The first GSK3-mediated phosphorylation may serve to prime a second phosphorylation at any Ser/Thr residue lying à4relative to this.The extent of such relays may be limited and consist of only two residues as in the transcription factor c-Jun (residues 239and 243for H.sapiens c-Jun,the former being the GSK3site and the latter being the priming site;Cohen and Goedert,2004).In contrast,a relay system of up to five Ser residues between residues 641and 657(the latter representing the priming site;Figure 1a)potentially operates in the N terminus of H.sapiens muscle glycogen synthase.Individual priming phosphorylations are catalysed by one of a number of different protein kinases,including casein kinases (for example,casein kinase 2catalyses the priming phosphorylation in glycogen synthase),PKA,cyclin-depen-dent protein kinases,MSKs and dual specificity tyrosine phosphorylated and regulated kinases (Cohen and Goedert,2004).The crystal structure of GSK3b accounts for the requirement for a priming phosphorylation and for the inhibitory effect of phosphorylation of Ser9(Dajani et al .,2001;Frame et al .,2001).Thus,GSK3b (Arg96)interacts with the priming phosphate in a substrate,orientating it for phosphorylation at the à4position.Phosphorylation of GSK3b (Ser9)causes an intramolecular interaction with Arg96,and the N terminus of the enzyme then serves as a pseudosubstrate to block both the binding site for the priming phosphate and access of any substrate to the catalytic pocket.
A brief overview of myocardial/cardiac myocyte hypertrophy
In this review,we use the term ‘hypertrophy’to indicate an increase in heart or cardiac myocyte size above that which would normally be achieved at any given stage of postnatal mammalian growth (Dorn et al .,2003;Dorn,2007),though the boundaries are not absolute as discussed later.Cardiac
Figure 3Regulation of the transcriptional co-regulator b -catenin.(a )Glycogen synthase kinase 3(GSK3)participates in the regulation of the canonical Wnt pathway.In the absence of Wnt,a pool of GSK3is present in a complex with axin and the adenomatous polyposis coli (APC)protein.In this complex,GSK3is catalytically active and it phosphorylates previously ‘primed’b -catenin (that is,phosphorylated by casein kinase 1on Ser45)at three further sites:Thr41,Ser37and Ser33.This polyphospho-b -catenin is then degraded through the ubiquitin–proteasome system.When Wnts engage to the extracellular domain of their transmembrane receptors (Frizzled)and to the lipoprotein receptor-related protein (LRP)co-receptor,the axin–APC–GSK3complex in association with the scaffold protein Dishevelled (Dsh)binds to the intracellular domains of Frizzled and LRP,and phosphorylations occur.GSK3is now unable to phosphorylate b -catenin,which accumulates and enters the nucleus.Here,it displaces the co-repressor Groucho from its complex with T-cell-specific transcription factor/lymphoid enhancer binding factor 1(TCF/LEF1),initiating gene expression.(b )It is suggested (Shevtsov et al .,2006)that a similar scheme may operate when endothelin-1(ET-1)binds to its receptor in cardiac myocytes (the ET A receptor seems to be the principal endothelin-1receptor).This leads to phosphorylation and activation of protein kinase B/Akt (PKB/Akt)(Figure 2)which then phosphorylates and inhibits GSK3promoting b -catenin accumulation.It is however unclear which pool(s)of GSK3is/are involved in this particular phosphorylation of b -catenin.
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myocytes terminally differentiate during the perinatal period and are unable to undergo any further complete cycles of cell division.They therefore grow as the organism grows,a process that we call‘eutrophy’(Dorn et al.,2003).They are additionally capable of considerable growth on suitable stimulation(hypertrophy).In vivo,myocyte hypertrophy is an important adaptational response that allows the heart to maintain an adequate cardiac output with improved cardiac contractility in a variety of physiological conditions.‘Physiological’,‘compensated’or‘adaptive’(all are synony-mous)hypertrophy such as is seen in pregnancy(Eghbali et al.,2005),endurance exercise,for example,voluntary running(Allen et al.,2001)or post-prandially in Burmese pythons(Andersen et al.,2005)is probably beneficial(or at least not disadvantageous)and is reversible upon removal of the stimulus.Post-prandial hypertrophy is perhaps an area which is perhaps semantically difficult to differentiate from eutrophy,because,in Burmese pythons,it represents normal behaviour.Even in‘pathological’conditions(for example,hypertension or following a survived myocardial infarction),the heart may initially adapt beneficially.However, such a beneficial consequence may ultimately prove detri-mental,leading to heart failure(Opie et al.,2006).This is the stage when cardiac hypertrophy becomes symptomatic,thus in a clinical context the term is usually associated with heart failure and the hypertrophy is‘pathological’,‘decompen-sated’or‘maladaptive’,possibly because of ancillary changes (for example,fibrosis,reduced contractile performance and chamber dilatation/wall thinning).It is not clear whether, in pathological conditions,the compensated phase is a compulsory waypoint on the path to decompensation or whether it is dispensable.
A number of extracellular stimuli and receptors are implicated in the development of myocyte hypertrophy (Sugden and Clerk,1998;Heineke and Molkentin,2006). These include particularly Gq-protein-coupled receptor (GqPCR)agonists(for example,endothelin-1,a-adrenergic stimuli),peptide growth factors that bind to receptor protein tyrosine kinases(for example,epidermal growth factor, platelet-derived growth factor,fibroblast growth factor)and cytokines binding to gp130family receptors(for example, cardiotrophin-1,leukemia inhibitory factor).b-Adrenergic receptors may also promote hypertrophy,though whether this is through classical coupling to GsPCRs or GiPCRs is less clear.The signalling pathways activated by the various stimuli and the role(s)of each pathway in the hypertrophic response have been subject to considerable debate over recent years(Sugden and Clerk,1998;Heineke and Molkentin,2006).These pathways frequently operate through phosphorylation and/or dephosphorylation events, and a number of protein kinases and phosphatases may be involved.In the context of hypertrophy,much emphasis has been placed on cyclic AMP-dependent protein kinase C (PKC),the four principal MAPK cascades(ERK1/2,ERK5, p38-MAPKs and c-Jun N-terminal kinases),calcineurin (protein phosphatase2B)and PKB/Akt.Other protein kinases may be involved in a specific context.For example,Janus kinases are predominantly activated by cytokine receptors (Haan et al.,2006),whereas activation of Ca2t/calmodulin-dependent kinases may be a particularly important response to increased nuclear Ca2tconcentrations(Wu et al.,2006). The dogma is that activation of intracellular signalling pathways leads to changes in the phosphorylation status of transcription and translation factors,and these modulate cardiac myocyte gene and protein expression.Such changes lead to and are ultimately responsible for the hypertrophic state.
In vivo or ex vivo,hypertrophy is characterized by increased myocyte size and sarcomerogenesis,and changes in the expression of characteristic index genes(for example, increased expression of atrial natriuretic factor,B-type natriuretic peptide,b-myosin heavy chain),though there is discussion about how closely these are linked to hypertrophy as opposed to heart failure(Dorn et al.,2003).Other variables are assessed frequently in whole animals,for example,heart (ventricular)weight to body weight ratio(HW/BW).In cases of specific regional hypertrophy(left or right ventricular), these weights are used as numerators.Sometimes,tibial length(TL)is used as a denominator as it is less sensitive to dietary state than BW.In vivo,the extent of fibrosis can also be assessed(unchanged in compensated hypertrophy, increased in decompensated hypertrophy)as can the con-tractile properties of the heart(increased contractility in compensated hypertrophy,decreased contractility in decom-pensated hypertrophy).Indeed,the decreased contractility may be linked to the increased fibrosis.Ex vivo,increases in the rate of protein synthesis(difficult to measure in vivo) are often used.In many publications,authors choose to examine only a limited number of aspects of the hyper-trophic response to delineate‘hypertrophy’(for example, increased atrial natriuretic factor expression or increased rates of protein synthesis).Whether a single index is sufficient to define hypertrophy is a different matter,but here we use the term‘hypertrophy’without any attempt at more detailed definition.
GSK3and cardiac myocyte hypertrophy
As outlined above,the canonical Wnt pathway(which involves GSK3)is important in developmental growth of the heart(Eisenberg and Eisenberg,2007).In addition,a number of studies suggest that GSK3(but GSK3b in particular)plays a pivotal role in cardiac hypertrophy(Hardt and Sadoshima,2002;Kerkela¨et al.,2007).In essence,active GSK3prevents hypertrophic growth of cardiac myocytes, probably through phosphorylation and inhibition of transcriptional regulators including b-catenin,nuclear factor of activated T cells(NFATs),GATA4,etc.,and the transla-tional regulator eukaryotic initiation factor2B e(eIF2B e),as described below.However,in H.sapiens,although inhibition of GSK3b is seen in end-stage heart failure resulting from coronary artery disease or idiopathic dilated cardiomyopathy, it is not observed in chronic hypertrophy(Haq et al., 2001).This may simply reflect the late stage at which samples were examined.We will now review the data on GSK3and cardiac hypertrophy(some of which is very recent)and also highlight specific questions which remain.
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Inhibition of GSK3in cardiac myocyte hypertrophy Hypertrophic agonists such as endothelin-1,or a-or b-adrenoceptor ligands increase phosphorylation of GSK3b(Ser9),resulting in a40–60%inhibition of GSK3b activity(Haq et al.,2000;Morisco et al.,2000).It is notable that these particular studies do not provide any data for phosphorylation or activity of GSK3a.However,GSK3a is expressed in the heart and,at least with respect to the regulation of glycogen synthase,plays a similar though less prominent role to GSK3b(Mora et al.,2005).In neonatal rat cardiac myocytes,we detect both GSK3a and GSK3b in approximately equal abundance(though the former appears less active)and we also observe phosphorylation of both GSK3a(Ser21)and GSK3b(Ser9)in response to insulin or endothelin-1(Markou et al.,2008).This suggests that GSK3a should not be ignored and,indeed,a very recent publication (Zhai et al.,2007)has gone some way in rectifying this omission.
Inhibition of GSK3activity with Lit(as LiCl)promotes features of the hypertrophic response(Haq et al.,2000)and is likely to affect both isoforms.It is also of note that LiCl is not a particularly effective inhibitor of GSK3.The IC50is only B1–2m M and,at the concentration normally used(10m M), inhibition is only about50%(Stambolic et al.,1996;Davies et al.,2000).Furthermore,specificity may be a problem as casein kinase2and two protein kinases activated by p38-MAPK are also at least partially inhibited by Lit(Davies et al.,2000).This is pertinent as p38-MAPK is activated by endothelin-1or a-adrenergic agonism in cardiac myocytes (Clerk et al.,1998),and casein kinase2sometimes catalyses the priming phosphorylation of GSK3substrates(for example, glycogen synthase;Picton et al.,1982;Cohen and Goedert2004).Thus,any effects of Litin isolated cells or tissues may result from inhibition of both casein kinase2 and GSK3.However,as discussed in depth by Davies et al. (2000),Litmay be a more selective inhibitor of GSK3(as opposed to casein kinase2)at physiological Ktconcentra-tions and at the lower physiological Mg2tconcentrations than those that were used in vitro.Even at these Ktand Litconcentrations,the IC50of Litfor GSK3is only B2m M, suggesting that Litshould be used at10m M.Litalso powerfully inhibits inositol monophosphatase(Hallcher and Sherman,1980),and this may influence inositol phospho-inositide signalling.
It will be interesting to determine whether more selective inhibitors of GSK3,which have been recently developed (Cohen and Goedert,2004),have the same effect on cardiac myocyte hypertrophy as Lit.As a first step here,we have shown that a relatively high concentration(10m M)of the GSK3inhibitor,1-azakenpaullone(Kunick et al.,2004), increases myocyte area but produces an‘elongated’pheno-type compared with the a-adrenergic agonist,phenylephrine (Markou et al.,2008).Like all work with inhibitors,we should add the caveat that1-azakenpaullone may not be specific.It does also inhibit cyclin-dependent kinase1/cyclin B and cyclin-dependent kinase5/p25,but the IC50is some two orders of magnitude greater(Kunick et al.,2004).Results with Litwere less convincing in our hands.Furthermore, the acute changes in transcript abundances induced by 1-azakenpaullone differ from those induced by endothelin-1.A more detailed examination of the‘pro-hypertrophic’effects of1-azakenpaullone and other GSK3inhibitors is probably justified.Interestingly,another GSK3inhibitor, 6-bromoindirubin-30-oxime,promotes proliferation(hyper-plasia)of adult or neonatal rat cardiac myocytes,with loss of phenotype in the case of the adult cell(Tseng et al.,2006). This is somewhat confusing because the mammalian cardiac myocyte is thought to undergo terminal differentiation and withdraw from the proliferative cell cycle during the perinatal period.However,the results are perhaps consonant with the ability of6-bromoindirubin-30-oxime to maintain pluripotency of embryonic stem cells through activation of the‘canonical’Wnt pathway(Sato et al.,2004). Phosphorylation of GSK3in ex vivo cardiac myocyte hypertrophy and in vivo cardiac hypertrophy
In all studies relating to phosphorylation and inhibition of GSK3by physiological means in the heart,the presumption seems to be that the signal is propagated through PKB/Akt. For insulin or insulin-like growth factor1(IGF1),which potently activates PKB/Akt but not ERK1/2in cardiac myocytes(Clerk et al.,2006),this is indeed likely to be the case(Markou et al.,2008).However,as discussed above, GSK3may be phosphorylated by other protein kinases.In relation to phosphorylation of GSK3by b-adrenergic agonism,Morisco et al.(2000)demonstrated that PKB/Akt is phosphorylated/activated in response to b-adrenoceptor stimulation,but the link from PKB/Akt to phosphorylation of GSK3b was not made.Indeed,the time course for activation of PKB/Akt is delayed relative to phosphorylation of GSK3b(Ser9),suggesting that other pathways are involved. An input from Ca2t/calmodulin-dependent kinase into GSK3phosphorylation was also detected,though any possible direct input from PKA was not considered.With respect to endothelin-1,it is interesting that Haq et al.(2000) inhibited the increase in phosphorylation of GSK3b(Ser9) with the PI3K inhibitors,LY294002or wortmannin,indicating that endothelin-1probably signals to GSK3b by activating PKB/Akt.In our hands,endothelin-1does not substantially activate PKB/Akt(Clerk et al.,2006).Further-more,the increases in phosphorylation of GSK3b(Ser9)and GSK3a(Ser21)induced by endothelin-1in cardiac myocytes are unaffected by LY294002but are suppressed by inhibitors of the ERK1/2cascade(Markou et al.,2008).This suggests that ERK1/2and thus possibly RSKs(which lie downstream from and are activated by ERK1/2)are required in this context rather than PI3K–PKB/Akt.In vivo,pro-hypertrophic manipulations such as increasing left ventricular pressure by transverse aortic constriction(TAC)rapidly(within10min) increase phosphorylation of GSK3(Brancaccio et al.,2003), but the signalling pathways are ill defined.By4weeks,these differences are no longer detectable,c.f.in man(Haq et al., 2001),and the abundance and activity of GSK3a,but not GSK3b,may even be increased(Zhai et al.,2007).
Some comments on insulin and IGF1in relation to cardiac hypertrophy and GSK3
Although perhaps tangential to the main topic of this review, we thought that it might be useful to include some
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comments on the effects of insulin(and IGF1)on the growth of the myocardium,given that insulin is probably the agonist most effective in inducing GSK3phosphoryla-tion.It has been known for many years that insulin stimulates cardiac protein synthesis(Morgan et al.,1971; Smith et al.,1986)and inhibits cardiac protein degradation (Rannels et al.,1975;Sugden and Smith,1982).Indeed,it is one of the most powerful modulators of these processes yet identified,and decreased plasma insulin concentrations probably contribute to the loss of cardiac protein mass that occurs in starvation(Preedy et al.,1984).IGF1stimulates cardiac myocyte protein synthesis to the same extent as insulin and inhibits protein degradation(Fuller et al.,1992; Decker et al.,1995).With either agonist,there is a net accumulation of protein in cardiac myocytes ex vivo(Decker et al.,1995),suggesting that they participate in the regula-tion of cardiac mass.Inducible,conditional targeted deletion of the cardiac myocyte insulin receptor gene in mice decreases heart size,but increases expression of the supposed hypertrophic index gene,b-myosin heavy chain(Belke et al., 2002),though it should be added that others have failed to detect significant differences in the expression of this gene in wild-type and insulin receptor-null mice(Hu et al.,2003). Although HW and HW/TL were decreased in the insulin receptor-null mice,they exhibited the same absolute extent of hypertrophy(HW/TL)as wild-type animals following TAC and the expression of two hypertrophic index genes was increased.However,after TAC,there was a greater degree of dilatation and fibrosis in the insulin receptor-null mice, and contractility was also decreased,suggesting that they were decompensating(Hu et al.,2003).Overall,though, insulin signalling seemed dispensable for TAC-induced hypertrophy.
In addition to the study of Decker et al.(1995),the effects of IGF1on cardiac myocyte hypertrophy(as defined earlier) have been studied both ex vivo and in vivo.Thus,Ito et al. (1993)treated myocytes with IGF1and showed that the myocyte cell area was twofold greater than controls.What is not clear is to what extent this difference was a reflection of ‘shrinkage’of control cells(this criticism can be made of most experiments of this type,including our own).There was also evidence of transcriptional changes that were consistent with a hypertrophic response.Duerr et al.(1995) infused IGF1in vivo and showed increases in left ventricular weight/TL at‘medium’or‘high’rates of infusion and in right ventricular weight/TL at‘high’rates.McMullen et al.(2004) cardiospecifically overexpressed the IGF1receptor and showed that HW/BW and cardiac myocyte size were increased,with some evidence of increased hypertrophic ‘index’gene expression.There is thus evidence that IGF1is a bona fide inducer of hypertrophy(as defined above).
At least part of the stimulation of cardiac myocyte protein synthesis by insulin is mediated through PI3K(Pham et al., 2000).Furthermore,insulin and IGF1are the most powerful activators of PKB/Akt in cardiac myocytes yet identified (Pham et al.,2000;Wang et al.,2000;Clerk et al.,2006). Insulin induces inhibition and phosphorylation of GSK3a (Ser21)and GSK3b(Ser9),an effect that in cardiac myocytes is mediated largely through the PI3K pathway(Wang et al., 2000;Markou et al.,2008).As summarized in a recent review (Clerk et al.,2007),cardiospecific activation of PI3K or PKB/ Akt signalling in transgenic mice generally promotes cardiac growth and cardioprotection(though the resulting pheno-type differs significantly between laboratories,possibly because a number of different methodologies have been used to activate PI3K or PKB/Akt signalling).Equally,there is an element of growth reversal in mice expressing‘dominant-negative’(inhibitory)transgenes.
In contrast to the studies of others(for example,Ito et al., 1993),we have never been able to detect any increases in surface area or sarcomerogenesis in primary cultures of neonatal rat cardiac myocytes treated with insulin or IGF1 (Clerk et al.,2006),nor have we been able to establish that IGF1greatly increased activities of promoters for hyper-trophic‘index’genes using luciferase reporter assays as compared with more established hypertrophic agonism,for example,a-adrenergic agonists(SJ Fuller and PH Sugden, unpublished observations).Thus,while some of the experi-ments with insulin or IGF1are consistent with a primary or exclusive role for GSK3in cardiac myocyte hypertrophy,the simple interpretation of our own experiments is not.Of course,it is possible that other PI3K-or PKB/Akt-dependent pathways override the input from GSK3inhibition or that signalling inputs additional to GSK3may be required,but those are imponderables.Indeed,inhibition of GSK3by insulin or IGF1may contribute to their antiapoptotic effects,as discussed elsewhere in this review.Our view coincides closely with that of Shiojima and Walsh(2006),as expressed in their eloquent review.Thus,insulin couples cardiac protein mass to nutritional status through its pancreatic secretion,whereas IGF1controls maturational eutrophy through the hypothalamopituitary axis(Shiojima and Walsh,2006;Catalucci et al.,2008).
Effects of genetic manipulation of GSK3signalling on cardiac myocyte hypertrophy ex vivo
Further evidence to support a role for GSK3b in cardiac myocyte hypertrophy derives from overexpression of wild-type GSK3b or expression of a constitutively active mutant form of GSK3b,GSK3b(Ser9Ala),which renders GSK3b resistant to the inhibitory phosphorylation.These approaches prevent or reduce the development of the hypertrophic phenotype induced by endothelin-1or b-adrenergic agonism in isolated cardiac myocytes(Haq et al.,2000;Morisco et al.,2000).Compensated hypertrophy is usually associated with increased myocyte survival(in part through activation of the ERK1/2cascade and PKB/Akt)and an enzymically inactive‘dominant-negative’inhibitor of GSK3,GSK3b(Lys85Met/Lys86Ile),inhibits tumour necrosis factor a/cycloheximide-induced apoptosis(Hirotani et al., 2007).A recent publication by Zhai et al.(2007)likewise concluded that overexpression of GSK3a reduces cardiac myocyte size and decreases the hypertrophic response to phenylephrine but increases apoptosis,whereas‘knock-down’of GSK3a with shRNA has the opposite effects (increased cell size and reduced apoptosis).These studies suggest that inhibition of GSK3is pro-hypertrophic but, importantly,is also pro-survival whereas GSK3activity is antihypertrophic and antisurvival.
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Effects of genetic manipulation of GSK3signalling on myocardial hypertrophy in vivo
A number of studies have also been performed in genetically manipulated mice in which transgene expression is placed under the control of the a-myosin heavy chain promoter to ensure a high degree of cardiospecific expression(Palermo et al.,1995).In the ventricles of these animals,the transgene is expressed in myocytes largely from the perinatal period through to adulthood.Overexpression of wild-type GSK3b or expression of GSK3b(Ser9Ala)reduces maturational growth of the heart(Antos et al.,2002;Michael et al.,2004),and reduces the cardiac hypertrophy induced by isoprenaline infusion,TAC or overexpression of constitutively activated calcineurin(Antos et al.,2002).Furthermore,inducible expression of GSK3b(Ser9Ala)in the adult heart reduces the extent of established hypertrophy produced by TAC,as assessed by left ventricular weight/BW(Sanbe et al.,2003).In a mouse model of familial hypertrophic cardiomyopathy resulting from expression of a mutant b-myosin heavy chain transgene(simulating the inheritable human disease to a greater or lesser extent),the increase in cardiac mass was reduced by co-expression of GSK3b(Ser9Ala),although gender and diet were complicating factors(Luckey et al., 2007).These studies suggest that activation of GSK3may be therapeutically valuable.However,to complicate matters, overexpression of wild-type GSK3b also induces diastolic dysfunction because of disturbances in Ca2tmovements (Michael et al.,2004).The heart becomes unable to regulate its diastolic Ca2tconcentration because of reduced Ca2treuptake into the sarcoplasmic reticulum through inhibition of the sarcoplasmic(endoplasmic)reticulum Ca2tATPase pump.
Two very recent publications from the group of Junichi Sadoshima have extended these observations.In the first of these,Hirotani et al.(2007)generated mice cardiospecifically expressing‘dominant-negative’GSK3b(Lys85Met/Lys86Ile). These mice displayed cardiac hypertrophy(as expected)with improved cardiac contractility(adaptive hypertrophy)in the absence of further interventions.Following TAC that was adequate to induce decompensation,there were no differ-ences in terms of relative(to sham-operated controls) hypertrophy of GSK3b(Lys85Met/Lys86Ile)mice compared with non-transgenic mice,but the GSK3b(Lys85Met/Lys86Ile) mice displayed greater cardiac contractility with less fibrosis or apoptosis.Conversely,inducible postnatal cardio-specific overexpression of GSK3b increased GSK3activity and resulted in the development of left ventricular dysfunc-tion,increased HW/TL,fibrosis and apoptosis,and induced early death.(Quite why cardiomyocyte-specific overexpression of a transgene should lead to fibrosis is unclear.Cardiac myocytes are not normally thought to participate in synthesis of the extracellular matrix.Explanations could include secretion of pro-fibrotic paracrine factors(Kemp et al.,2004)or compulsory coupling of changes in contrac-tility to fibrosis.)The conclusions from these experiments are that inhibition of GSK3leads to a compensated cardiac phenotype,but perversely activation of GSK3leads to decompensation(c.f.Michael et al.,2004).In the second publication(Zhai et al.,2007),the focus was GSK3a which,as summarized by Zhai et al.(2007),may differ to some extent from GSK3b in terms of the biological responses which they each provoke.Indeed,TAC induced expression of GSK3a and caused an overall increase in its activity in the absence of changes in GSK3b(Zhai et al.,2007).Cardiospecific over-expression of wild-type GSK3a reduced cardiac growth(c.f. GSK3b;Michael et al.,2004;Hirotani et al.,2007),and reciprocally reduced expression and activity of GSK b.After TAC,the authors conclude that left ventricular hypertrophy is inhibited in the GSK3a transgenic mice.This is true in absolute terms,but the differences are less clear in relative terms because the GSK3a transgenic mice have lower HW/BW or HW/TL ratios at the outset.This shows the type of consideration that can bedevil such work.Be that as it may, contractile function in the control and GSK3a transgenic mice did not differ at baseline,but was worse in the GSK3a transgenic after TAC,possibly because of increased apoptosis and fibrosis.In other words,the‘antihypertrophic’effects of GSK3activation could simply result from increased myocyte loss.On the basis of experiments with inhibitors and GSK3 constructs,the authors devise what,to our minds,is a rather fanciful signalling pathway to account for their findings. They suggest that increased expression/activity of GSK3a in transgenic mice leads to inhibition of ERK1/2signalling by inhibiting PKC d and PKC e.Assuming that the central roles of ERK1/2as being pro-hypertrophic and pro-survival are accepted,inhibition of ERK1/2should decrease hypertrophy and increase apoptosis,but the proposed signalling pathway could also effectively constitute a positive feedback loop, whereby inhibition of ERK1/2would promote further activation of GSK3through inhibiting phosphorylation of GSK3a(Ser21)and GSK3b(Ser9),presumably increasing myocyte losses.
As in the ex vivo experiments,these in vivo experiments are consistent with an antihypertrophic role for active GSK3,but also with a potentially undesirable pro-apoptotic role with concurrent reductions in contractility.Thus,the question becomes whether the antihypertrophic action is simply a reflection of the pro-apoptotic effects?Leaving this aside for the time being,it is worth considering the potential effects of GSK3activation on the best-established substrate for GSK3, glycogen synthase,and on glycogen metabolism generally. Active(dephosphorylated)GSK3phosphorylates glycogen synthase to inhibit glycogen synthesis,and inhibition (phosphorylation)of GSK3a(Ser21)/GSK3b(Ser9)increases the rate of glycogen synthesis(Figures1a and b).Over-expression of GSK3or expression of the GSK3b(Ser9Ala) mutant might therefore be expected to diminish glycogen stores.In the non-stressed heart,changes in GSK3activity as a consequence of phosphorylation appear to play a minimal role in regulating cardiac glycogen levels,as transgenic mice in which wild-type GSK3a/b genes are replaced by transgenes encoding Ser21Ala/Ser9Ala-mutated forms of GSK3a/b(that is,a GSK3a(Ser21Ala)/GSK3b(Ser9Ala)‘knock-in’)exhibit similar levels of cardiac glycogen as their wild-type litter-mates(Mora et al.,2005).However,during pathophysiolo-gical cardiac stresses(for example,TAC),it is possible that altered glycogen metabolism resulting from GSK3activation may affect cardiac metabolism and mass and,ultimately,the development of cardiac pathologies.Furthermore,the con-centration of glycogen and the activity of the‘intracellular
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fuel gauge’,AMP-activated protein kinase,are reciprocally related,the link possibly being the glycogen-binding b subunit of AMP-activated protein kinase (Hardie,2007).A decrease in cardiac glycogen should activate AMP-activated protein kinase,which would lead to inhibition of anabolic processes such as protein accumulation (Hardie et al .,2003;Wojtaszewski et al .,2003;Hardie,2007).Although such a scheme is hypothetical,it suggests that the consequences of GSK3activation or inhibition might not simply rely directly on its own ability to regulate transcription and translation (see below)directly.Perhaps unexpectedly,the GSK3a (Ser21-Ala)/GSK3b (Ser9Ala)‘knock-in’mice have significantly high-er HW/BW ratios than the wild-type animals in the absence of any significant differences in absolute HW or BW (K Sakamoto,personal communication),though it is not clear that this is attributable to increased myocytic protein mass.Obviously,further experimentation is needed.
Potential downstream targets of GSK3in cardiac myocyte hypertrophy
As mentioned,GSK3requires a priming phosphorylation of its substrates to direct it to Ser/Thr residues in the à4position,but the identities of kinases responsible for the
priming phosphorylations are not always clear (Cohen and Goedert,2004).GSK3phosphorylates a range of substrates and,generally,inhibits their biological activities (Cohen and Goedert,2004).Here,we will focus on substrates for which there is evidence of a potential role in hypertrophy.It is entirely possible that several or even all of the protein enumerated may participate in the hypertrophic response.Although most of the substrates identified are transcriptional regulators,the specific changes in transcription associated with changes in GSK3activity have not been thoroughly characterized.
Nuclear factors of activated T cells
The role of NFATs,which are encoded by five genes (NFATc1–NFATc4,NFAT5)producing multiple alternatively spliced transcripts,is best defined in T-cell activation (Crabtree and Olson,2002;Gallo et al .,2006).NFATs are phosphorylated on Ser-(and maybe Thr-)residues in their N-terminal regions.These phosphorylations mask nuclear localization signals and retain NFATs in the cytoplasm possibly by binding of 14-3-3proteins,inhibition of which promotes cardiac myocyte hypertrophy (Liao et al .,2005).T-cell receptor engagement increases intracellular Ca 2t,which binds to the Ca 2tsensor protein calmodulin,and the Ca 2t–calmodulin complex binds to and
activates
Figure 4Potential glycogen synthase kinase (GSK3)Ser/Thr-directed phosphorylation relays in nuclear factor of activated T cells (NFATs).(a )Sequences for Homo sapiens NFATs were aligned by CLUSTAL W (1.83).Accession numbers are NFATc1,NP_006153.2(825residues);NFATc2,NP_036472.2(921residues);NFATc3,NP_004546.1(1068residues)and NFATc4,NP_004545.2(902residues).Alignment of the ‘atypical’NFAT,NFAT5,has been omitted.The extended and highly conserved Ser/Thr relays (potentially phosphorylated by GSK3)present in the N-terminal regions of NFATc1,NFATc2,NFATc3and NFATc4are in underlined,bold italicized type.In NFATc1,these sites are thought to be phosphorylated by GSK3.Other more limited relays are present in individual NFATs and all contain a Ser-rich domain N-terminal to the GSK3consensus sequences that is phosphorylated by casein kinase 1.(b )GSK3relays (underlined,bold italicized type)present in GATA4(1–217).(c )GSK3relays (underlined,bold italicized type)in mouse myocardin (residues 454–466and 624–636)as identified by Badorff et al .(2005).Identical sequences are present in H.sapiens myocardin.There are also ‘out-of-phase’relays in these regions (shaded,bold and italicized),which are not as strongly conserved in H.sapiens myocardin.
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calcineurin(protein phosphatase2B).Calcineurin dephos-phorylates cytoplasmically localized NFATs,allowing them to migrate into the nucleus to promote gene expression. GSK3phosphorylates PKA-primed NFATc1on relays of Ser residues(Figure4a;Beals et al.,1997;Neal and Clipstone, 2001;Sheridan et al.,2002),and thus inhibits NFATc1-dependent gene expression.Whether PKA is the physiologi-cal priming kinase for NFATs is unclear.A‘systems biology’approach has suggested that dual specificity tyrosine phos-phorylated and regulated kinases may catalyse the priming phosphorylation of at least NFATc2(Gwack et al.,2006). Although these relays are conserved in other NFATs (Figure4a),the individual identities of the sites phosphory-lated are not as well characterized as for NFATc1.However, GSK3is thought to be involved in the phosphorylation of other NFATs,viz NFATc2(Okamura et al.,2004),NFATc3 (Nilsson et al.,2006)and NFATc4(Graef et al.,1999).Equally, it is important to note that GSK3is not the only kinase that phosphorylates and regulates the localization of NFATs. Casein kinase1phosphorylates NFATc2in a Ser-rich region more N-terminal than the GSK3sites and in the region of the nuclear localization signal(Okamura et al.,2004).This retains NFATc2in the cytoplasm and the region is conserved in other NFATs.
Some years ago,the calcineurin/NFAT system was identi-fied as an important signalling component in cardiac hypertrophy,with NFATc3(rather than the NFATc4species studied in the original report)ultimately emerging as a particularly important mediator of the response(Molkentin et al.,1998;Wilkins et al.,2002;Wilkins and Molkentin, 2004).The NFAT isoforms expressed in the heart are not fully defined,partly because of alternative splicing and partly because of the limited specificity of the commercially available antibodies previously available.However,at the mRNA level,NFATc2transcripts appear to be expressed in considerably greater abundance than the others(LJ De Windt,personal communication).In cardiac myocytes, endothelin-1reportedly promotes translocation of both GSK3and NFATs to the nucleus,and the translocation of NFATs is opposed by expression of GSK3b(Ser9Ala)(Haq et al.,2000).Furthermore,in transgenic mice,cardiospecific expression of GSK3b(Ser9Ala)attenuates the increase in nuclear NFAT induced by constitutively activated calcineur-in(Antos et al.,2002).These two studies suggest that NFATs may be a target for GSK3in the heart,and phosphorylation of NFATs by GSK3may constitute one aspect of the antihypertrophic effect of GSK3.However,the NFAT isoforms involved and the physiological conditions which inhibit GSK3and promote dephosphorylation of NFATs remain to be fully defined.
b-Catenin
In the canonical Wnt pathway(described above),the Wnt–Frizzled–LRP complex in association with Dishevelled prevents GSK3from phosphorylating b-catenin and thus promotes b-catenin accumulation(Logan and Nusse,2004; Cadigan and Liu,2006;Gordon and Nusse,2006).b-Catenin migrates to the nucleus to promote gene expression via TCF/ LEF1.Although this pathway is important in cardiac development(Eisenberg and Eisenberg,2007),the influence of GSK3signalling through b-catenin in the context of myocardial hypertrophy is less clear.It has been proposed that hypertrophic G-protein-coupled receptor agonists pro-mote accumulation of b-catenin in cardiac myocytes in a Wnt-independent manner,but which requires phosphoryla-tion and inhibition of GSK3by PKB/Akt(Figure3b;Haq et al.,2003;Shevtsov et al.,2006).Consistent with this,the pro-hypertrophic inhibition of GSK3in the GSK3b(Lys85-Met/Lys86Ile)transgenic mouse promotes accumulation of b-catenin(Hirotani et al.,2007).Furthermore,hearts from Dishevelled-1null mice(three Dishevelled genes are expressed in mice)or from mice with cardiospecific deletion of b-catenin exhibit a reduced hypertrophic response to TAC (Chen et al.,2006;van der Schans et al.,2007).However,in another setting,cardiospecific deletion of b-catenin causes mild cardiac hypertrophy and does not inhibit the cardiac hypertrophy induced by infusion of angiotensin II(Baurand et al.,2007).Indeed,stabilization(and therefore increased protein expression)of b-catenin impairs adaptive hyper-trophic growth response to angiotensin II(Baurand et al., 2007;Zelarayan et al.,2007).It would seem that further studies are required to clarify the role of b-catenin in cardiac myocyte hypertrophy and any influence of GSK3 upon this.
GATA4and myocardin
GATA4(Liang and Molkentin,2002)and myocardin(Liu and Olson,2006;Parmacek,2007)are two more transcriptional regulators which may be involved in hypertrophy and which are potentially phosphorylated by GSK3to inhibit their effects on gene expression.The transcriptional transactiva-ting activity of GATA4is,at least in part,regulated by nucleocytoplasmic shuttling.Thus,GATA4contains a‘non-classical’nuclear localization signal lying within residues 270–324,and a nuclear export signal lying within residues 49–54(Philips et al.,2007).In a transgenic mouse over-expressing constitutively active PKB/Akt,the nuclear pool of GATA4was increased,though whether this resulted from any GSK3-mediated phosphorylation was not readily amen-able to investigation(Condorelli et al.,2002).In the context of stimulation of cardiac myocytes by b-adrenergic receptor agonists,GSK3phosphorylates GATA4to promote its export from the nucleus through the exportin,Crm1(Morisco et al., 2001).Circumstantial evidence from in vitro experiments suggests that the site of phosphorylation lies within the N terminus(2–116)when this region is primed by PKA,but the site(s)has not been clearly defined in vivo(Morisco et al., 2001).There are three potential GSK3phosphorylation relay systems within GATA4(2–116)(Figure4b).Although Morisco et al.(2001)do not exclude the possibility that other sites exist in GATA4(117–205),there are no Ser/Thr‘relays’in this region(Figure4b).PKA requires an Arg residue(s)within four residues N-terminal to the phosphorylation site and a hydrophobic residue att1(Songyang et al.,1994).The most likely site for GSK3-mediated phosphorylation is thus Ser105with the priming site lying at Ser109.It is not clear whether such a scheme applies in vivo.However,Ser105can also be phosphorylated by ERK1/2and,in this instance,
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phosphorylation of GATA4increases its transcriptional transactivating activity(Liang et al.,2001).The location of the GSK3phosphorylation sites in GATA4awaits resolution. Myocardin was identified as a muscle-specific transcrip-tional co-regulator,which modulates gene expression in association with serum response factor(Wang et al.,2001a). It may be phosphorylated by GSK3on two relays of Ser residues,Ser454–Ser466and Ser624–Ser636,(Figure4c; Badorff et al.,2005).Although Ser466and Ser636presumably serve as priming phosphorylations,the kinases responsible have not been identified.GSK3catalyses phosphorylation of myocardin and inhibits its effects on cardiac myocyte gene expression and hypertrophy,but the biochemical mecha-nisms have not been explored further(Badorff et al.,2005). The Jun family,CREB and c-Myc
Other transcriptional regulators that are likely to be important in cardiac hypertrophy are phosphorylated by GSK3in other systems.These include CREB,Jun family proteins(c-Jun,JunB,JunD)and c-Myc(Clerk et al.,2007). Phosphorylation of CREB presents something of a conundrum.Phosphorylation of CREB catalysed by PKA (and possibly a variety of other kinases,for example, especially the RSK-related MSKs,see Deak et al.,1998)at Ser133or Ser119in the H.sapiens‘long’or‘short’CREB isoform,respectively,was thought originally to increase its transactivating activity,though it now deemed‘necessary, but not sufficient’(Johannessen and Moens,2007).The site (Ser129-Arg-Arg-Pro-Ser133(P)-Tyr)is actually closer to a PKA consensus sequence than to one for RSK-related kinases, though GqPCR-coupled hypertrophic agonists certainly induce phosphorylation of CREB(Ser133)in an ERK1/2 cascade-dependent(RSK-dependent?)manner in cardiac myocytes(Harrison et al.,2004).However,phosphorylation of CREB by GSK3at Ser129in the‘long’CREB isoform with a PKA priming phosphorylation at Ser133decreases its DNA-binding activity(Bullock and Habener,1998).It would therefore seem that phosphorylation of Ser129is dependent on phosphorylation of Ser133,but their effects on transcrip-tion could be opposite.It has been proposed that reduction in the GSK3-mediated phosphorylation of CREB(Ser129) plays a role in the cardiac myocyte hypertrophy induced by hypoxia/reoxygenation and the inhibition of GSK3may involve the ERK1/2cascade,PKB/Akt and/or PKA(El Jamali et al.,2004).Further study of the role of the various CREB phosphorylation sites in relation to cardiac myocyte hyper-trophy is warranted.
In non-myocytes,c-Jun,JunB and JunD are phosphory-lated by GSK3close to their DNA-binding domains to inhibit DNA binding and prevent Jun-directed gene expression (Nikolakaki et al.,1993).Phosphorylation of c-Jun by GSK3 also promotes its ubiquitination and degradation(Wei et al., 2005).Similarly,phosphorylation of c-Myc by GSK3may prime it for proteolysis by the ubiquitin–proteasome system (Henriksson et al.,1993;Sears et al.,2000;Gregory et al., 2003).We have recently shown that1-azakenpaullone or insulin rapidly increases Jun/Fos binding to the AP-1 consensus sequence(TGAGTCA)in cardiac myocytes,but to a lesser extent than endothelin-1(Markou et al.,2008).The transcription factor most clearly associated with the AP-1sequence is JunB.Consistent with this,binding of JunB to the AP-1sequence is negatively regulated by GSK3,thus inhibition of GSK3should increase JunB binding to the AP-1 sequence(Nikolakaki et al.,1993).H.sapiens JunB contains a GSK3relay close to the DNA-binding site between residues 251and259(248–256in rat)with Ser259(Ser256in rat) representing the priming site.Interestingly,expression of JunB protein is also increased by1-azakenpaullone or insulin,a finding which complicates interpretation of AP-1-binding data because increased binding could simply represent a mass action effect rather than a dephosphoryla-tion event(Markou et al.,2008).
Eukaryotic initiation factor2B
In addition to changes in gene expression,cardiac myocyte hypertrophy is associated with increases in the rate of protein synthesis.One point of translational regulation involves eIF2(Proud,2007).During translational initiation, eIF2.GTP brings the initiator Met-tRNA i to the40S ribosomal subunit.GTP is hydrolysed during the initiation process, generating eIF2.GDP which is released and must be recharged with GTP before being recycled.eIF2B is the guanine nucleotide exchange factor for eIF2.Of the five eIF2B subunits,the e subunit binds to eIF2and enhances GTP/GDP exchange.eIF2B e is phosphorylated on at least four sites,including a GSK3site at Ser535in rat eIF2B e (Ser540in H.sapiens;Ser536in mouse)with a priming site at Ser539(Ser544in H.sapiens;Ser540in mouse)(Wang et al., 2001b).Phosphorylation of rat eIF2B e(Ser535)by GSK3 inhibits its activity,preventing recycling of eIF2and inhibiting translational initiation.Conversely,inhibition of GSK3reduces phosphorylation of eIF2B e(Ser535),increases the recycling of eIF2and hence increases translational initiation.Although dephosphorylation of eIF2B e(Ser535) has the potential to increase the rate of protein synthesis, it is not clearly established that this is the case.Thus, in Chinese hamster ovary cells,dephosphorylation of ‘eIF2B e(Ser535)’(residue numbering for the hamster unknown) occurs in response to insulin(a powerful stimulator of protein synthesis generally and the initiation step in particular)via a PI3K-dependent pathway(Wang et al., 2002).Although GSK3inhibitors(Litand two pharmacolo-gical inhibitors)also cause the dephosphorylation of eIF2B e (Ser535),they do not affect overall eIF2B activity(Wang et al.,2002).Thus,the significance of eIF2B e(Ser535) phosphorylation in translational initiation is difficult to assess but again,the GSK3inhibitors have‘additional effects’on the protein synthesis machinery,suggesting that the results should be interpreted cautiously.
In rat cardiac myocytes,b-adrenergic receptor agonism decreases phosphorylation of eIF2B e(phospho-Ser535),prob-ably through inhibition of GSK3(Hardt et al.,2004).(Note that there is some confusion about the residue numbering because the commercial suppliers state that the anti-phospho-eIF2B e antibody is raised against H.sapiens eIF2B e (phospho-Ser539),which is the correct numbering for the GSK3site if the N-terminal Met-residue is not included.) In the GSK3b(Lys85Met/Lys86Ile)transgenic mouse, Glycogen synthase kinase3and cardiac hypertrophy
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pro-hypertrophic inhibition of GSK3promotes dephosphory lation of the GSK3phosphorylation site(eIF2B e(phospho-Ser540))(Hirotani et al.,2007).Thus,eIF2B e may represent a relevant site of regulation in the hypertrophic response. Relationship of GSK3signalling to class I histone deacetylases in the heart
Histone acetylation and deacetylation regulate gene expres-sion in eukaryotic cells and tissues,including the heart (Backs and Olson,2006).Whereas histone acetyltransferases stimulate gene expression by disrupting chromatin,histone deacetylases(HDACs)repress gene expression.However, because individual genes may encode positive or negative regulators of cell processes,histone acetylases and HDACs can each exert individual positive or negative effects. Mammalian HDACs fall into three classes based on their similarity to yeast orthologues.Generally,class II HDACs repress cardiac gene expression and myocyte growth but the situation regarding the classes I and III HDACs is less clear.It has been proposed that class I HDACs might repress antihypertrophic pathways and thus promote the hyper-trophic response(Kook et al.,2003).Hence,pharmacologi-cally or genetically induced deficiency in class I HDAC activity leads to an inhibition of the hypertrophic response. Recently,an interesting connection between the class I HDAC,HDAC2,and GSK3has been revealed(Trivedi et al., 2007).This does not involve positioning of HDACs down-stream from GSK3,but vice versa.The pathway proposes that HDAC2normally represses expression of the gene encoding inositol polyphosphate-5-phosphatase f.This enzyme (Minagawa et al.,2001)hydrolyses the50phosphate in PtdIns(3,4,5)P3(Figure5),the phospholipid signalling molecule controlling the activation of PKB/Akt(Figure2; Vanhaesebroeck and Alessi,2000).The proposal(Trivedi et al.,2007)is that a decrease in HDAC2increases expression of inositol polyphosphate-5-phosphatase f,which decreases the plasma membrane pools of PtdIns(3,4,5)P3.This prevents activation of PKB/Akt and the subsequent inhibition of GSK3,ultimately preventing the development of hyper-trophy induced by,for example,TAC.The evidence for involvement of HDAC2in hypertrophy via GSK3is primarily genetic or morphological,though the phosphorylation of PKB/Akt and GSK3is decreased in HDAC2-null mice (Trivedi et al.,2007).
It should be noted that inositol polyphosphate-5-phos-phatase f will not only deplete the plasma membrane of PtdIns(3,4,5)P3but also of any5-phosphoinositides,includ-ing phosphatidylinositol4,5-bis phosphate(PtdIns(4,5)P2) (Minagawa et al.,2001).In an analogous paradigm,depletion of PtdIns(4,5)P2(induced by overexpression of constitutively active G a q and hence supranormal activation of phospho-lipase C b(Adams et al.,1998)has profound effects in cardiac myocytes and promotes apoptosis(Howes et al.,2003),a phenomenon not examined in detail by Trivedi et al.(2007). The pro-apoptotic effects of PtdIns(4,5)P2depletion observed by Howes et al.(2003)were attributed to resultant decreases in PtdIns(3,4,5)P3and repression of the PKB/Akt-dependent pro-survival and-growth pathways.The low level of
tonic Figure5Relevant aspects of phosphoinositide metabolism.Phosphatidylinositol4,5-bis phosphate(PtdIns(4,5)P2)is an important phospholipid in cell signalling producing three separate‘second messenger’molecules.It is hydrolysed by a variety of phospholipase C (PLC)isoforms to produce diacylglycerol and inositol1,4,5-tris phosphate.The former is the physiological activator of the diacylglycerol-regulated protein kinase C isoforms(see Figure2),whereas the latter is important in the regulation of intracellular Ca2tmovements.In cardiac myocytes,the phospholipase C b isoforms are best studied in this regard.In a second signalling pathway,PtdIns(4,5)P2is phosphorylated by phosphoinositide30kinase(PI3K)to form phosphatidylinositol3,4,5-tris phosphate(PtdIns(3,4,5)P3).This regulates the activity of the 3-phosphoinositide-dependent kinase/protein kinase B(Akt)signalling pathway(see Figure2).Phosphoinositides are dephosphorylated (hydrolysed)by a number of lipid phosphatases.One such phosphatase which hydrolyses the5-phosphate of phosphatidylinositol4,5-bis phosphate and PtdIns(3,4,5)P3is inositol polyphosphate-5-phosphatase f,which produces phosphatidylinositol4-phosphate and phosphatidylinositol3,4-bis phosphate,respectively.
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activity exhibited by PKB/Akt in cardiac myocytes that can be reduced by PI3K inhibitors(Pham et al.,2000)may be relevant because this may be important in diurnal growth and survival.However,it seems unlikely that PKB/Akt and GSK3would be the sole signalling proteins affected by inositol polyphosphate-5-phosphatase f expression(or by supranormal phospholipase C b activation).Depletion of PtdIns(4,5)P2would ultimately be expected to interfere with GqPCR-mediated activation of phospholipase C b,PKC and the ERK1/2cascade(and maybe with phosphorylation of GSK3via the ERK1/2cascade)with consequent repression of hypertrophy,but we are not aware of any studies in myocardial preparations.This is important because GqPCR/ G a q signalling is involved in in vivo hypertrophy induced, for example,by TAC(Akhter et al.,1998).Further examina-tion of paradigms involving modulation of HDCA2expres-sion(for example,further biochemical evidence from measurement of phosphoinositide concentrations and sig-nalling protein activities)is needed.
GSK3and apoptosis
GSK3activity has been associated with both apoptosis and cell survival,though it is perhaps the former as is seen in neuroblastoma cells(Bijur et al.,2000)and cardiac myocytes (Menon et al.,2007)that has attracted more attention(Jope and Johnson,2004).A pro-apoptotic role of GSK3in the heart is consistent with the findings of Hirotani et al.(2007) in the GSK3b(Lys85Met/Lys86Ile)-expressing transgenic mouse and in cardiac myocytes,as discussed earlier.Hirotani et al.(2007)also show that expression of the antiapoptotic and rapidly turning-over Bcl2family member,Mcl-1 (Yang-Yen,2006),is increased in GSK3b(Lys85Met/Lys86Ile)-expressing mice,though whether increased expression of Mcl-1is a prime determinant of the improved myocyte survival is unclear.As suggested,a pro-apoptotic role of GSK3is important because promotion of apoptosis in experimental situations could be equated with inhibition of hypertrophy. GSK3research in relation to myocardium
hypertrophy:some unanswered questions
Although a number of studies support the notion of an antihypertrophic function for active GSK3(particularly GSK3b),it is our view that the information is incomplete and a number of issues remain to be addressed.First,there seems to be confusion over likely signalling events leading to GSK3phosphorylation and inhibition.The presumption is that PKB/Akt(through the PI3K pathway)exclusively regulates GSK3,but other kinases clearly phosphorylate the same inhibitory sites.Second,although a very recent publication(Zhai et al.,2007)has helped to redress the balance,too much emphasis may have been placed on GSK3b as opposed to GSK3a.Our results(Markou et al.,2008) clearly demonstrate phosphorylation of both isoforms and others have identified a role for GSK3a in the regulation of glycogen synthesis in the heart(Mora et al.,2005).Third, although Litions are widely used as inhibitors of GSK3,their use is compromised by their low efficacy and specifi-city.More selective small molecule inhibitors(Cohen and Goedert,2004)are becoming increasingly available and should probably be used in preference to Lit.In our experiments(Markou et al.,2008),we have made use of 1-azakenpaullone,as described above.Fourth,if GSK3is involved with the export of transcription factors from the nucleus or in the regulation of nuclear-localized transcrip-tion factors,it should be present in that compartment,as we have observed(Markou et al.,2008).However,glycogen synthesis is primarily a cytoplasmic process.The implication is that at least two pools of GSK3must exist.There is some evidence of this in skeletal muscle(Jensen et al.,2007),but there is very little information on subcellular compartmen-talization of GSK3signalling within cardiac myocytes.Fifth, even though some transcription factors are already impli-cated in GSK3signalling,the integration of transcription factor activities and the genes that they regulate is still unknown.Sixth,the studies in transgenic mice are confu-sing.Several studies using overexpression of wild-type GSK3b or GSK3a,or expression of GSK3b(Ser9Ala)suggest that catalytically active forms of GSK3are antihypertrophic in the heart in vivo.However,other studies have suggested that GSK3activity,although antihypertrophic,is also pro-fibrotic and pro-apoptotic and that it decreases contractile function, with inhibition of GSK3by transgenesis having the opposite effect(Michael et al.,2004;Hirotani et al.,2007;Zhai et al., 2007).All these studies involve transgene overexpression that is difficult to control(for example,differences in copy number and genetic background between lines can be problematical),and the effects can be difficult to interpret. Further studies with GSK3a(Ser21Ala)/GSK3b(Ser9Ala)‘knock-in’mice in which single copies of the transgenes are under the control of their endogenous promoters may be more informative,but here,preliminary experiments suggest that HW/BW is increased in the‘knock-in’mice(K Sakamoto, personal communication).Overall,we consider that the precise molecular mechanisms through which GSK3partici-pates in the hypertrophic response still need to be determined.
Is manipulation of GSK3activity a viable myocardial therapy?
In the introduction to this article,we briefly assessed the possible utility of GSK3activation as an antihypertrophic therapy.This was the most obvious clinical application of the earlier findings(Haq et al.,2000;Antos et al.,2002),but perhaps the utility of such manoeuvres should be recon-sidered in the light of more recent work that has shown increased fibrosis,apoptosis and decreased contractility in mice in which GSK3is activated(Michael et al.,2004; Hirotani et al.,2007;Zhai et al.,2007).Indeed,inhibition may be a better course of action.If activation of GSK3is beneficial,it is difficult to see how this could be achieved directly using small molecules.In contrast,if inhibition is beneficial,small molecule inhibitors are available and ex vivo experiments have shown that inhibition may represent the philosopher’s stone of myocardial therapeutics,namely Glycogen synthase kinase3and cardiac hypertrophy
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myocyte regeneration(Tseng et al.,2006).However,as pointed out by Cohen and Goedert(2004),there are fears about the potential oncogenicity of pleiotropic GSK3 inhibition.Currently,the only feasible method which we can envisage whereby therapeutic modulation of GSK3in the heart could be achieved cardiospecifically is through a ‘gene therapy’approach.To summarize,we just do not know whether to inhibit or activate GSK3,or simply not to interfere.What might be useful would be the further study of a transgenic mouse model involving inducible expression of GSK3a(Ser21Ala)or GSK3b(Ser9Ala)at a stage that would be most clinically relevant,that is,when the heart is already failing.Admittedly,Sanbe et al.(2003)carried out such a study in mice with established TAC-induced left ventricular hypertrophy,but did not examine the contractile function of the hearts.Perhaps this is one model that should be revisited. Conflict of interest
The authors state no conflict of interest.
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Hippo信号通路 一、Hippo信号通路概述 Hippo 信号通路，也称为Salvador / Warts / Hippo（SWH）通路，命名主要源于果蝇中的蛋白激酶Hippo（Hpo），是通路中的关键调控因子。该通路由一系列保守激酶组成，主要是通过调控细胞增殖和凋亡来控制器官大小。 Hippo信号通路是一条抑制细胞生长的通路。哺乳动物中，Hippo信号通路上游膜蛋白受体作为胞外生长抑制信号的感受器，一旦感受到胞外生长抑制信号，就会激活一系列激酶级联磷酸化反应，最终磷酸化下游效应因子YAP和TAZ。而细胞骨架蛋白会与磷酸化后的YAP和TAZ结合，使它滞留在细胞质内，降低其细胞核活性，从而实现对器官大小和体积的调控。 二、Hippo信号通路家族成员 虽然Hippo信号通路在各个物种中保守性很高，但是相同功能的调控因子或效应因子在不同物种间还是存在着差异，下表中我们对比了果蝇与哺乳动物中Hippo信号通路相同功能的关键因子[1]。
Expanded(Ex) FRMD6/Willin 含有FERM结构域的蛋白，能与Kibra及Mer结合，调控Hippo信号通路的上游信号 Dachs(Dachs) 肌浆球蛋白myosin的一种，能结合Wts 并促进其降解 Kibra(Kibra) WWC1 含有WW结构域的蛋白，能与Ex及Mer 结合，调控Hippo信号通路的上游信号 Merlin(Mer) NF2 含有FERM结构域的蛋白，能与Kibra及Ex结合，调控Hippo信号通路的上游信号 Hippo(Hpo) MST1,MST2 Sterile-20-样激酶，磷酸化并激活Wts Salvador(Sav) WW45(SAV1) 含有WW结构域的蛋白，能起到一个脚手架蛋白的作用，易化Hippo对Warts的磷酸化 Warts（Wts）LATS1,LATS2 核内DBF-2相关激酶，能磷酸化Yki并使之失活 Mob as tumor suppressor(Mats) MOBKL1A,MOBKL1B 能与Wts结合的激酶，与Wts结合后能 促进Wts的催化活性 Yorkie(YKi) YAP,TAZ 转录共激活因子，能在非磷酸化的激活状态下与转录因子Sd结合，并激活下游靶基因的转录。这些受调控的下游靶基因主要参与了细胞的增殖、生长并抑制凋亡的发生 Scalloped(Sd) TEAD1,TEAD2,TEAD3, TEAD4 能与Yki结合的转录因子，与Yki共同 作用，调控靶基因的转录 三、Hippo信号通路的功能 近十年相关研究结果表明，无论是果蝇还是哺乳动物，Hippo信号通路都可以通过调节细胞增殖、凋亡和干细胞自我更新能力实现对器官大小的调控。Hippo信号通路异常会导致大量组织过度生长。此外，大量研究证实，Hippo信号通路在癌症发生、组织再生以及干细胞功能调控上发挥着重要功能[2][3][4]。 a.Hippo信号通路在器官大小控制中的作用 起初，关于Hippo信号通路的研究主要集中在器官大小的调控。大量研究表明，Hippo 途径主要通过抑制细胞增殖并促进细胞凋亡，继而实现对器官大小的调控。激酶级联反应是该信号传导的关键。Mst1/2激酶与SA V1形成复合物，然后磷酸化LATS1/2；活化后的LATS1/2激酶随即磷酸化Hippo信号通路下游关键效应分子——Y AP和TAZ，同时抑制了
第3次作业 一、作文题（本大题共60分，共 2 小题，每小题 30 分） 1. For this part, you are allowed thirty minutes to write a composition on the topic Work and Leisure in no less than 100 words. You may begin your composition with “People generally believe that a life without leisure is really terrible.” Remember to write clearly. 2. Instructions:建议你在30分钟内，根据下面所给的题目和提纲用英语写出一篇不少于80词的短文。 1.你在英语学习中遇到什么困难； 2.你如何克服这些困难。 二、填空题（本大题共40分，共 10 小题，每小题 4 分） 1. Negative student''s ______ (respond) to online learning are also due to time factors, particularly for students with part-time or full-time jobs. 2. The meeting began ______ (punctuality) at nine o’clock. 3. It’s Friday again. What about going to the pub to have some ______ (entertain)? 4. She felt quite ______ (loose) after taking a trip to the seaside. 5. In the United States, all children between the ages of 5 and 18 must ______ (attendance) school. 6. The reporter checked with a variety of sources to guard against ______ reports. (accurate ) 7. The criminal was sent to a mental hospital for ______ by doctors. (assess) 8. Only a few of us could ______ his statement, because it was too profound. (comprehension) 9. ______ , he failed in the examination again though he tried many times.( fortunate) 10. At the Olympic Games our representatives were in ______ with the best swimmers from all parts of the world. (compete) 答案： 一、作文题（60分，共 2 题，每小题 30 分） 1. 参考答案： Work and Leisure People generally believe that life without leisure is really terrible. A person who works day and night will gradually feel rather tired. This tiredness, if continues for quite a long time,
一种新型防窃电装置的设计和应用 赵兵吕英杰邹和平 （中国电力科学研究院，北京市，100192） The Design and Application of a New Anti-stealing Electricity Device ZHAO Bing，LV Yingjie, ZOU Heping (China Electric Power Research Institute, Beijing, 100192) ABSTRACT: Even though effectively interdicting the action of stealing electricity is significant, reliable and practical means of anti-stealing electricity is lacking. Now, a new anti-stealing electricity device is introduced in this article. The electro- information is directly collected from high-tension side and which is acted as the criterion of stealing Electricity. Further more, the design method and the function of this device is explained, especially the power module. In the end an ensample is given to explain the feasible and reliable. KEYWORDS:Anti-stealing Electricity, Data collection Device, Power Supply 摘要：有效遏制窃电行为具有重大意义，但目前缺乏可靠实用的防窃电手段。为此本文介绍了一种新型防窃电装置，该装置直接采集用户一次侧用电信息，作为判断窃电的依据，本文对该装置的功能及设计方法进行了说明，并详细说明介绍了从高压侧获取电源的方法。最后给出了防窃电实例证明该装置的实用性。 关键词：防窃电，采集器，高压侧获取电源 1 引言 随着市场经济的发展和人民生活水平的提高。用电需求日益扩大。为节省用电开支，追求高额利润。许多不法个人或单位采取各种方式窃电。使国家蒙受巨大的经济损失。据统计，北京市供电企业的被窃电量1996年时为1.36亿千瓦时，折合人民币4030万元；2001年，北京地区被窃电量2亿千瓦时，折合人民币9050万元；到2002年，被窃电量已经上升为2.22亿千瓦时，折合人民币1.02亿元。吉林省年售电量230亿千瓦时，窃电造成的线损如果为一个百分点，就丢失电量2亿多千瓦时，折合人民币1亿多元。湖北省用电量859亿千瓦时,被窃的电占线损比例高达21．3％，窃电量约为15亿千瓦时,折合人民币7亿多元，居全国第一。因此，完善防窃电技术，有效遏制窃电行为具有重大意义。 2 现有窃电方式及防窃电技术 计量的电能取决于电压、电流、功率因数三者与时间的乘积，改变以上任一要素，都可达到窃电 的目的。现存的窃电手法种类很多，归纳起来主要分为以下几种方式[1]：(1)电压法窃电。即将接人电能计量设备的电压回路断开或松掉，导致电压回路失压或欠压。(2)电流法窃电。即对接入电能计量 设备的电流回路(计量电流互感器——CT，current transformer一二次侧)实施短路或开路，造成流入计量设备的电流减少。(3)错相法窃电，也称移相 法窃电或相角法窃电。即改变电能计量设备电压回路或电流回路的接线。从而改变计量设备正确的电压或电流相位关系。使电能计量不准。(4)扩差法 窃电。从物理上破坏电能计量设备的计量机理，扩 大电能计量设备误差。(5)表前分流窃电。在电能 计量设备前私自接线用电，使一部分电流没有流经计量设备，从而使电量少计。 对于电压法及错相法窃电可采用高速交流采 样芯片采集相电压、电流信号，通过与阈值电压相 比较可判断是否存在失压或欠压；同时，采用傅立叶算法可计算电流与电压间的相量角，从而判断是否存在错相窃电。对于扩差法窃电及表前分流窃电，主要采取硬件防范措施，如采用专用计量箱或 电表箱、封闭变压器低压出线端至计量设备的导体、采用防撬铅封等，均可取得一定的效果。但是，
1、对模拟信号（一维信号，是时间的函数）进行采样后，就是 离散 信号，再进行幅度量化后就是 数字信号。 2、若线性时不变系统是有因果性，则该系统的单位取样响应序列h(n)应满足的充分必要条件是 当n<0时，h(n)＝0 。 3、序列)(n x 的N 点DFT 是)(n x 的Z 变换在 单位圆 的N 点等间隔采样。 4、)()(5241n R x n R x ==，只有当循环卷积长度L ≥8 时，二者的循环卷积等于线性 卷积。 5、已知系统的单位抽样响应为h(n)，则系统稳定的充要条件是 ()n h n ∞ =-∞ <∞∑ 6、用来计算N ＝16点DFT ，直接计算需要（N 2）16*16＝256_次复乘法，采用基2FFT 算法，需要__(N/2 )×log 2N ＝8×4＝32 次复乘法。 7、无限长单位冲激响应（IIR ）滤波器的基本结构有直接Ⅰ型，直接Ⅱ型，_级联型_和 并联型_四种。 8、IIR 系统的系统函数为)(z H ，分别用直接型，级联型，并联型结构实现，其中 并联型的运算速度最高。 9、数字信号处理的三种基本运算是：延时、乘法、加法 10、两个有限长序列 和 长度分别是 和 ，在做线性卷积后结果长度是 __N 1＋N 2－1_。 11、N=2M 点基2FFT ，共有 M 列蝶形，每列有N/2 个蝶形。 12、线性相位FIR 滤波器的零点分布特点是 互为倒数的共轭对 13、数字信号处理的三种基本运算是： 延时、乘法、加法 14、在利用窗函数法设计FIR 滤波器时，窗函数的窗谱性能指标中最重要的是___过渡带宽___与__阻带最小衰减__。 16、_脉冲响应不变法_设计IIR 滤波器不会产生畸变。 17、用窗口法设计FIR 滤波器时影响滤波器幅频特性质量的主要原因是主瓣使数字滤波器存在过渡带，旁瓣使数字滤波器存在波动，减少阻带衰减。 18、单位脉冲响应分别为 和 的两线性系统相串联，其等效系统函数时域及频域表 达式分别是h(n)=h1(n)*h2(n), =H1(ej ω)×H2(ej ω)。 19、稳定系统的系统函数H(z)的收敛域包括 单位圆 。 20、对于M 点的有限长序列x(n)，频域采样不失真的条件是 频域采样点数N 要大于时域采样点数M 。
警告标志 (警告车辆、行人注意危险地点的标志） 十字交叉Ｔ形交叉Ｔ形交叉Ｔ形交叉Ｙ形交叉 环形交叉向左急弯路向右急弯路反向弯路连续弯路 上陡坡下陡坡两侧变窄右侧变窄左侧变窄 窄桥双向交通注意行人注意儿童注意牲畜 注意信号灯注意落石注意落石注意横风易滑 傍山险路傍山险路堤坝路堤坝路村庄
隧道渡口驼峰桥路面不平 过水路面 有人看守铁路道 口 无人看守铁路道口注意非机动车事故易发路段 慢行 左右绕行左侧绕行右侧绕行施工 注意危险 叉形符号(表示多股铁道与道路交叉) 禁令标志 （禁止或限制车辆、行人交通行为的标志） 禁止通行禁止驶入禁止机动车通行禁止载货汽车通 行禁止三轮机动车 通行
禁止大型客车通 行禁止小型客车通 行 禁止汽车拖、挂车 通行 禁止拖拉机通行禁止农用运输车 通行 禁止二轮摩托车 通行禁止某两种车通 行 禁止非机动车通 行 禁止畜力车通行禁止人力货运三 轮车通行 禁止人力客运三轮车通行禁止人力车通行禁止骑自行车下 坡 禁止骑自行车上 坡 禁止行人通行 禁止向左转弯禁止向右转弯禁止直行禁止向左向右转 弯禁止直行和向左 转弯 禁止直行和向右 转弯禁止掉头禁止超车解除禁止超车禁止车辆临时或 长时停放
禁止车辆长时停 放 禁止鸣喇叭限制宽度限制高度 限制质量 限制轴重限制速度解除限制速度停车检查停车让行 减速让行会车让行 指示标志 （指示车辆、行人行进的标志） 直行向左转弯向右转弯直行和向左转弯 直行和向右转弯 向左和向右转弯靠右侧道路行驶靠左侧道路行驶立交直行和左转 弯行驶 立交直行和右转 弯行驶 环岛行驶 单行路(向左或向 右) 单行路(直行) 步行鸣喇叭
数字信号处理的应用与发展趋势 作者：王欢 天津大学信息学院电信三班 摘要： 数字信号处理是应用于广泛领域的新兴学科，也是电子工业领域发展最为迅速的技术之一。本文就数字信号处理的方法、发展历史、优缺点、现代社会的应用领域以及发展前景五个方面进行了简明扼要的阐述。 关键词： 数字信号处理发展历史灵活稳定应用广泛发展前景 数字信号处理的简介 1.1、什么是数字信号处理 数字信号处理简称DSP，英文全名是Digital Signal Processing。 数字信号处理是利用计算机或专用处理设备以数字的形式对信号进行采集、变换、滤波、估值、增强、压缩、识别等处理，以得到符合人们需要的信号形式。 DSP系统的基本模型如下： 数字信号处理是一门涉及许多学科且广泛应用于许多领域的新兴学科。它以众多的学科为理论基础，所涉及范围及其广泛。例如，在数学领域、微积分、概率统计、随即过程、数值分析等都是数字信号处理的基本工具；同时与网络理论、信号与系统、控制论、通信理论、故障诊断等学科也密切相关。近年来的一些新兴学科，如人工智能、模式识别、神经网络等，都是与数字信号处理密不可分的。数字信号处理可以说许多经典的理论体系作为自己的理论基础，同时又使自己成为一门新兴学科的理论基础。 1.2、数字信号系统的发展过程 数字信号处理技术的发展经历了三个阶段。 70 年代DSP 是基于数字滤波和快速傅里叶变换的经典数字信号处理, 其系统由分立的小规模集成电路组成, 或在通用计算机上编程来实现DSP 处理功能, 当时受到计算机速度和存储量的限制,一般只能脱机处理, 主要在医疗电子、生物电子、应用地球物理等低频信号处理方面获得应用。 80 年代DSP 有了快速发展, 理论和技术进入到以快速傅里叶变换(FFT) 为主体的现代信号处理阶段, 出现了有可编程能力的通用数字信号处理芯片, 例如美国德州仪器公司(TI公司) 的TMS32010 芯片, 在全世界推广应用, 在雷达、语音通信、地震等领域获得应用, 但芯片价格较贵, 还不能进 入消费领域应用。 90 年代DSP 技术的飞速发展十分惊人, 理论和技术发展到以非线性谱估计为代表的更先进的信号处理阶段, 能够用高速的DSP 处理技术提取更深层的信息, 硬件采用更高速的DSP 芯片, 能实时地完成巨大的计算量, 以TI 公司推出的TMS320C6X 芯片为例, 片内有两个高速乘法器、6 个加法器, 能以200MHZ 频率完成8 段32 位指令操作, 每秒可以完成16 亿次操作, 并且利用成熟的微电子工艺批量生产,使单个芯片成本得以降低。并推出了C2X 、C3X 、C5X 、C6X不同应用范围的系列, 新一代的DSP 芯片在移动通信、数字电视和消费电子领域得到广泛应用, 数字化的产品性能价 格比得到很大提高, 占有巨大的市场。 1.3、数字信号处理的特点
第三次在线作业 单选题 (共40道题) 收起 1.（ 2.5分） – Can I help you? --__________. ? A 、I want that blue dress. ? B 、 OK. ? C 、That’s OK. ? D 、I can help you. 我的答案：A 此题得分：2.5分 2.（2.5分） – What else do you want? -- ___________. ? A 、Nothing else. ? B 、I’m fine. ? C 、What do you want? ? D 、It doesn’t matter. 我的答案：A 此题得分：2.5分 3.（2.5分） – What kind of room do you want? -- _____. ? A 、Room 1. ? B 、I have no idea. ? C 、I want a single room. ? D 、I’m sorry. 我的答案：C 此题得分：2.5分 4.（2.5 分） – Have you made a reservati on? -- ____________. ? A 、Yes, I have. ? B 、I did. ? C 、Yes, I will. ? D 、Thank you. 我的答案：A 此题得分：2.5分 5.（2.5分） – Excuse me, where can I che ck out? -- ____________. ? A 、At the reception desk. ? B 、You’re welcome. ? C 、Nice to see you. ? D 、When you are ready. 我的答案：A 此题得分：2.5分 6.（2.5分） – I really enjoyed the concert. -- _______. ? A 、What will you do tomorrow? ? B 、I’m glad to hear that. ? C 、It’s my pleasure. ? D 、I’m sorry to hear that. 我的答案：B 此题得分：2.5分 7.（2.5分） – What do you think of this concert? -- ________. ? A 、I don’t think so.
2、对一个带限为3f kHz ≤的连续时间信号采样构成一离散信号，为了保证从此离散信号中能恢复出原信号，每秒钟理论上的最小采样数为多少？如将此离散信号恢复为原信号，则所用的增益为1，延迟为0的理想低通滤波器的截止频率该为多少？ 答：由奈奎斯特采样定理，采样频率必须大于两倍的信号最高频率，236s f kHz kHz >?=每秒钟理论上得最小采样数为6000。如将此离散信号恢复为原信号，为避免混淆，理想低通滤波器的截止频率为采样频率的一半，即 32 s kHz Ω=。 3、有限频带信号11()52cos(2)cos(4)f t f t f t ππ=++，式中，11f kHz =。用5s f kHz =的冲激函数序列()T t δ进行 取样。 （1）画出()f t 及采样信号()s f t 在频率区间(10,10)kHz kHz -的频谱图。 （2）若由()s f t 恢复原信号，理想低通滤波器的截止频率c f 。 解：（1）()f t 在频率区间(10,10)kHz kHz -的频谱图 /kHz -10 0 1 2 10 ()s f t 在频率区间(10,10)kHz kHz -的频0谱图 （2）25002 s c f f Hz ≥ = 4、有一连续正弦信号cos(2)ft π?+，其中20f Hz =，6 π ?=。 （1）求其周期0T ； （2）在t nT =时刻对其采样，0.02T s =，写出采样序列()x n 的表达式； （3）求()x n 的周期N 。 解：（1）011 0.0520 T s f = == （2）在t nT =时刻，4()cos(2)cos(2200.02)cos()6 5 6 x n f nT n n π π π?ππ=+=?+=+ （3） 25 425 ππ=，所以5N =。
资深PI最新文章解析信号通路 ------------------------------------------------------------------------------------------------------------------------------------ 摘要：来自新加坡分子与细胞生物学研究院，癌症与发育细胞生物学部的研究人员获得了YAP－TEAD4复合物在YAP因子N端结构域相互作用，以及在TEAD4 C端结构域与YAP相互作用的晶体结构，从中研究人员认为YAP中的PXXΦP片段是与TEAD4相互作用的关键结构，这为研究Hippo信号通路提供了重要的分子机理线索。这一研究成果公布在《Genes Development》杂志上。 生物通报道：来自新加坡分子与细胞生物学研究院，癌症与发育细胞生物学部的研究人员获得了YAP－TEAD4复合物在YAP因子N端结构域相互作用，以及在TEAD4 C端结构域与YAP相互作用的晶体结构，从中研究人员认为YAP中的PXXΦP片段是与TEAD4相互作用的关键结构，这为研究Hippo信号通路提供了重要的分子机理线索。这一研究成果公布在《Genes Development》杂志上。 领导这一研究的是新加坡分子与细胞生物学研究院宋海卫博士，其早年毕业于河南大学化学系，之后进入中科院生物物理研究院进行分子生物学方面的学习，1998年获得利兹大学（The University of Leeds）分子生物学专业博士学位。目前任新加坡分子与细胞生物学研究所资深研究员。 Hippo信号转导通路是几年前发现的一个信号转导通路。研究发现Hippo信号通路是参与调控器官大小发育的关键信号通路，这一观点首先在果蝇中被发现，后来的研究发现在哺乳动物的发育过程中Hippo有相同的功能。06年Cell发表的一篇文章证实Hippo 是一种细胞分裂和死亡的控制开关。Hippo信号转导通路通过促进细胞调亡和限制细胞
数字信号处理是将信号以数字方式表示并处理的理论和技术。数字信号处理与模拟信号处理是信号处理的子集。 简介 简单地说，数字信号处理就是用数值计算的方式对信号进行加工的理论和技术，它的英文原名叫digital signal processing，简称DSP。另外DSP也是digital signal processor的简称，即数字信号处理器，它是集成专用计算机的一种芯片，只有一枚硬币那么大。有时人们也将DSP看作是一门应用技术，称为DSP 技术与应用。 《数字信号处理》这门课介绍的是：将事物的运动变化转变为一串数字，并用计算的方法从中提取有用的信息，以满足我们实际应用的需求。 本定义来自《数字信号处理》杨毅明著，由机械工业出版社2012年发行。 特征和分类 信号(signal)是信息的物理体现形式，或是传递信息的函数，而信息则是信号的具体内容。 模拟信号(analog signal)：指时间连续、幅度连续的信号。 数字信号(digital signal)：时间和幅度上都是离散(量化)的信号。 数字信号可用一序列的数表示，而每个数又可表示为二制码的形式，适合计算机处理。 一维(1-D)信号: 一个自变量的函数。 二维(2-D)信号: 两个自变量的函数。 多维(M-D)信号: 多个自变量的函数。 系统：处理信号的物理设备。或者说，凡是能将信号加以变换以达到人们要求的各种设备。模拟系统与数字系统。 信号处理的内容：滤波、变换、检测、谱分析、估计、压缩、识别等一系列的加工处理。 多数科学和工程中遇到的是模拟信号。以前都是研究模拟信号处理的理论和实现。 模拟信号处理缺点：难以做到高精度，受环境影响较大，可靠性差，且不灵活等。数字系统的优点：体积小、功耗低、精度高、可靠性高、灵活性大、易于大规模集成、可进行二维与多维处理 随着大规模集成电路以及数字计算机的飞速发展，加之从60年代末以来数字信号处理理论和技术的成熟和完善，用数字方法来处理信号，即数字信号处理，已逐渐取代模拟信号处理。 随着信息时代、数字世界的到来，数字信号处理已成为一门极其重要的学科和技术领域。 数字信号处理器 DSP芯片，也称数字信号处理器，是一种特别适合于进行数字信号处理运算的微处理器，其主要应用是实时快速地实现各种数字信号处理算法。根据数字信号处理的要求，DSP芯片一般具有如下主要特点： （1）在一个指令周期内可完成一次乘法和一次加法；
本次作业是本门课程本学期的第3次作业，注释如下：大学英语II 第3单元作业题 一、单项选择题(只有一个选项正确，共40道小题) 1. –What time is it? –____________________. (A) My watch keeps good time (B) My watch is five minutes fast (C) My watch says three o’clock (D) I say three o’clock 你选择的答案：[前面作业中已经做正确] [正确] 正确答案：C 解答参考：[第三单元] 本题问句是“现在几点钟”，C项“我的表是三点钟”。A项“我的表走时准确”，B项“我的表快五分钟”，D项“我说是三点钟”都不符合习惯表达法。 2. –Could you tell me the time, please? –____________________. (A) Yes, I can (B) No, I can’t (C) It’s four thirty (D) It’s Friday 你选择的答案：[前面作业中已经做正确] [正确] 正确答案：C 解答参考：[第三单元]C 本题仍是问时间，回答应该直接告诉对方时间，故C项正确。 3. –What day is tomorrow? –Today is Tuesday. So it’s________. (A) Monday (B) Wednesday (C) Thursday (D) Saturday 正确答案：B 解答参考：[第三单元]B 本题问“明天是星期几”，回答“今天是星期二，所以明天是星期三”。 4. –When is your birthday? –____________________. (A) It’s April 18th (B) It’s Sunday (C) It’s August (D) I was born in 1988
1.序列a{n}为{1,2,4},序列b(n)为{4,2,1},求线性卷积a(n)*b(n) 答：a(n)*b(n)={4,10,21,10,4} 2.序列x1(n)的长度为N1，序列x2(n)的长度为N2，则他们线性卷积长度为多少? 答：N1+N2-1 第二次 1.画出模拟信号数字化处理框图，并简要说明框图中每一部分的功能作用。 第三次 1.简述时域取样定理的基本内容。 第四次 1.δ(n)的Z变换是？ 答：Z(δ(n))=1 2.LTI系统，输入x（n）时，输出y（n）；输入为3x（n-2），输出为？ 答：3y(n-2 第五次 1、已知序列Z变换的收敛域为｜z｜>2，则该序列为什么序列？ 答：因果序列加右边序列
∑ x(n)e^(-jwn)而 Z 变换为 X （z ）= ∑ x(n)Z^(-n) ∑ x(n)e^(-jwn)= ∑ x(n)e^-j(w + 2mπn) ∑x (n )e ^(-j 2πkn /N )∑ [δ(n) + 2δ(n - 5)e ^(-jwkn /5) (2) y(k)=e^(j2k2π/10)x(k)=W 10 x(k) 1. 相同的 z 变换表达式一定对应相同的时间序列吗？ 答：不一定，因为虽然 z 变换的表答式相同，但未给定收敛域，即存在因果序列和反因果 序列两种情况。 2．抽样序列在单位圆上的 z 变换，等于其理想抽样信号的傅立叶变换？ 答：相等，傅里叶变换 X （e^jw ）= +∞ -∞ +∞ -∞ 令 Z=e^(-jw)即 X(z)|z=e^jw=X(e^jw)此时正是对应在单位圆上 3．试说明离散傅立叶变换和 z 变换之间的关系。 答： 抽样序列在单位圆上的 z 变换，等于其理想抽样信号的傅立叶变换。 第七次 1. 序列的傅里叶变换是频率 w 的周期函数，周期是 2π 吗？ 答：是，X(e^jw)= +∞ -∞ +∞ -∞ (m 为整数) 2. x(n)=sinw(n)所代表的序列不一定是周期的吗? 答：不一定，在于 w （n ）是否被 2π 整除。 1.一个有限长为 x （n ）（1）计算序列 x （n ）的 10 点 DFT 变换 （2）前序列 y （n ）的 DFT 为 y （k ）=e^(j2k2π/10)x(k),式中 x(k)是 x(n)10 点离散傅里叶变 换，求序列 y(n) 答: (1) X(k)= = N -1 n =0 9 n =0 =1+2e^(-j πk) =1+2(-1)^k (k=0,1,2,3……9) -2k
视窗 × loading... 大学英语三第三次在线作业 单选题 (共40道题) 展开 收起 1.（ 2.5分）– Can I help you? --__________. ?A、I want that blue dress. ?B、OK. ?C、That’s OK. ?D 、I can help you. 我的答案：A此题得分：2.5分 2.（2.5分）– What else do you want? -- ___________. ?A、Nothing else. ?B、I’m fine. ?C、What do you want? ?D、It doesn’t matter. 我的答案：A此题得分：2.5分 3.（2.5分）– What kind of room do you want? -- _____.
?A、Room 1. ?B、I have no idea. ?C、I want a single room. ?D、I’m sorry. 我的答案：C此题得分：2.5分 4.（2.5分）– Have you made a reservation? -- ____________. ?A、Yes, I have. ?B、I did. ?C、Yes, I will. ?D、Thank you. 我的答案：A此题得分：2.5分 5.（2.5分）–Excuse me, where can I check out? -- ____________. ?A、At the reception desk. ?B、You’re welcome. ?C、Nice to see you. ?D、When you are ready. 我的答案：A此题得分：2.5分 6.（2.5分）– I really enjoyed the concert. -- _______. ?A、What will you do tomorrow? ?B、I’m glad to hear that.
信号注入法小电流接地故障保护中注入信号检测技术研究 李盼盼，潘贞存，陈 青,丛 伟，施啸寒 （山东大学电气工程学院，济南 250061） 摘 要：针对基于信号注入法的小电流接地故障保护中注入信号由于幅值较小、干扰严重而导致检测困难的现状，研究了一种模拟滤波和数字滤波相结合的注入信号检测方法，分别设计了模拟滤波器和数字滤波器，分析了各自的传递函数特性，采用PSPICE和MATLAB分别对模拟滤波器和数字滤波器的工作特性进行了仿真，仿真结果表明，本文所研究的注入信号检测方法具有较高的灵敏度和可靠性，完全能够满足现场应用的要求。 关键词：注入信号小电流接地系统信号检测滤波器 0 引言 通过电压互感器的二次侧注入特殊信号进行小电流接地故障选线原理自上世纪90年代初被提出以来，已获得了较为广泛的应用。理论上该方法具有较高的选线准确率，并能够进行故障定位。但由于注入信号微弱，加之现场运行环境恶劣，难于准确提取出注入信号，致使容易出现误选或漏选等选线失败的现象，特别是过渡电阻较大且系统对地分布电容也较大时，对选线的准确率的影响也进一步增大。 与配网自身的高电压、大电流相比，注入信号不仅量值很小、而且能量很低，因此，能否准确、可靠的检测出注入信号，是保证选线准确率的根本。本文设计了一种模拟滤波与数字滤波相结合的信号处理方法，通过设计不同特性的滤波器，能够可靠的将弱信号与强信号分离，并通过仿真试验验证了信号检测的效果。 1 信号注入法基本原理与注入信号的选取 信号注入法是在发生单相接地故障后向系统注入特殊频率的信号，通过跟踪、检测注入信号的路径和特征来实现故障选线。系统正常运行时，三相对称，系统中没有零序分量；线路发生金属性单相接地故障后，故障相对地电压降为零，两非故障相电压升高为线电压，零序电压升高为相电压。此时TV的故障相一次绕组被短接，其二次绕组中也无感应电压，使TV的故障相在故障期间处于“闲置”状态。利用这一特点，可以通过TV 的故障相二次侧将信号电流注入到故障系统中[1]。注入信号仅在故障线路中流通，且越过接地点后注入信号不再存在，只要检测各出线中有无注入信号电流，便可找出故障线路[2]，这就是信号注入法选线的基本原理。 从TV二次侧注入的信号电流感应到一次系统中，实质上是人为增加了故障系统的零序电流，因此需保证在不影响配电系统的正常运行的前提下合理的选择注入信号的幅值和频率，并分析不同幅值频率对检测环节的影响。 1.1注入信号幅值的选取 对注入信号幅值的选取首先要不影响配电系统的正常运行，其次要考虑到TV的容量有限，注入信号过大容易烧毁二次保险，另外由于信号源本身经济性的制约，注入信号源宜向系统提供幅值相对较小的信号。具体幅值的大小还要考虑过渡电阻、系统分布电容、系统中性点接地方式、系统电压等级等诸多因素的影响，确保在极端的故障情况下也能可靠检测到注入信号电流[3]。 假设系统电压等级为10kV、TV变比为100：1，注入信号装置输出的电流为5A，则耦合到一次系统后注入信号的大小为0.05A，再经过零序TA耦合进入保护，设TA变比为50：1，最终进入保护的电流大小为1mA，此电流是未考虑其他影响因素分流前的结果。如此微弱的信号对检测技术提出了更高的要求，如何从相差千倍以上的工频电流中提取微弱的注入信号成为了应用该原理需要解决的关键问题。 1.2注入信号频率的选取 注入信号频率的选取主要取决于以下几个因素：（1）注入信号的频率必须与电网的固有频率相区分；（2）注入信号的频率不能太低，否则易受电力系统中工频和直流分量的影响，检测误差增加；注入信号的频率也不能太高，否则会使得系统容抗变大，降低检测灵敏度[4]；（3）为了满足数字滤波器的设计要求，在工频信号的一个采样周期内，对注入信号的采样点数也应为整数，且满足采样定理的要求。 在许多文献中提出了应用20Hz[4]，60Hz[5]、220Hz[6]等频率的信号。选用20Hz主要是为了减
题目1 正确 获得1.0分中的1.0分 标记题目 题干 is considered to be ________ the other students in her class. 选择一项： a. the most intelligent b. intelligent as well c. less intelligent d. as intelligent as 题目2 正确 获得1.0分中的1.0分 标记题目 题干 — Did the medicine make you feel better? — No. The more_________, _________ I feel. 选择一项： a. medicine I take; and the worse b. I take medicine; worse c. medicine I take; the worse d. I take medicine; the worse 题目3 正确 获得1.0分中的1.0分
标记题目 题干 This is the ______ photo I have ever taken. 选择一项： a. worse b. best c. most worst d. better 题目4 正确 获得1.0分中的1.0分 标记题目 题干 - Hi, Jane. Would you like to go to the ball this evening? - Sorry, Frank. ____ tomorrow's lessons, I have no time to go out with you. 选择一项： a. Not preparing b. Not to prepare c. Being not prepared d. Not having prepared 题目5 正确 获得1.0分中的1.0分
一、 填空题 1、 对模拟信号（一维信号，是时间的函数）进行采样后，就是 离散 信号，再进行幅度量化后就是 数字 信号。 2、若线性时不变系统是有因果性，则该系统的单位取样响应序列h(n)应满足的充分必要条件是 当n<0时，h(n)＝0 。 3、序列)(n x 的N 点DFT 是)(n x 的Z 变换在 单位圆 的N 点等间隔采样。 4、)()(5241 n R x n R x ==，只有当循环卷积长度L ≥8 时，二者的循环卷积等于线性卷积。 5、已知系统的单位抽样响应为h(n)，则系统稳定的充要条件是 ()n h n ∞ =-∞ <∞∑ 6、巴特沃思低通滤波器的幅频特性与阶次N 有关，当N 越大时，通带内越_平坦______，过渡带越_窄___。 7、用来计算N ＝16点DFT ，直接计算需要__（N 2 ）16*16＝256_ __次复乘法，采用基2FFT 算法，需要__(N/2 )×log 2N ＝8×4＝32_____ 次复乘法。 8、无限长单位冲激响应（IIR ）滤波器的基本结构有直接Ⅰ型，直接Ⅱ型，_级联型____和 _并联型__四种。 9、IIR 系统的系统函数为)(z H ，分别用直接型，级联型，并联型结构实现，其中 并联型 的运算速度最高。 10、数字信号处理的三种基本运算是： 延时、乘法、加法 11、两个有限长序列 和 长度分别是 和 ，在做线性卷积后结果长度是__N 1＋N 2－1_____。 12、N=2M 点基2FFT ，共有__ M 列蝶形，每列有__ N/2 个蝶形。 13、线性相位FIR 滤波器的零点分布特点是 互为倒数的共轭对 14、数字信号处理的三种基本运算是： 延时、乘法、加法 15、在利用窗函数法设计FIR 滤波器时，窗函数的窗谱性能指标中最重要的是___过渡带宽___与__阻带最小衰减__。 16、_脉冲响应不变法_设计IIR 滤波器不会产生畸变。 17、用窗口法设计FIR 滤波器时影响滤波器幅频特性质量的主要原因是主瓣使数字滤波器存在过渡带，旁瓣使数字滤波器存在波动，减少阻带衰减。 18、单位脉冲响应分别为 和 的两线性系统相串联，其等效系统函数时域及频域表达式分别是h(n)=h 1(n)*h 2(n), =H 1(e j ω )×H 2(e j ω )。 19、稳定系统的系统函数H(z)的收敛域包括 单位圆 。 20、对于M 点的有限长序列x(n)，频域采样不失真的条件是 频域采样点数N 要大于时域采样点数M 。 二、 选择题 1、下列系统（其中y(n)为输出序列，x(n)为输入序列）中哪个属于线性系统？( D ) A. y(n)=x 3(n) B. y(n)=x(n)x(n+2)
收稿日期:2008 07 01 作者简介:刘顺会(1971 ),男,湖北荆州市人,博士,主要从事生物信息学研究。 基金项目:国家自然科学基金(30572124)、广东省科技厅(2004B31201001)、教育部科学技术研究重点项目(205116)、广东省自然科学基金(5002855)联合资助。 *广东药学院临床医学院 文章编号:1004 4337(2008)06 0641 06 中图分类号:R392 4 文献标识码:A 医学数学模型探讨 T 细胞受体信号转导通路的动力学分析 刘顺会 肖兰凤 * 黄树林 (广东药学院生命科学与生物制药学院 广州510006) 摘 要: 目的:建立T 细胞受体信号转导途径的动力学模型,通过模型仿真揭示T 细胞受体信号途径各分子间的动态调控过程,简要分析模型的动力学特性。方法:根据数据库KEGG 及相关中英文文献,提取T 细胞受体信号转导各条通路相关分子作用的方式及数量关系,利用M atlab 7.0的S imulin k 工具箱构建信号途径的动力学模型并仿真。结果:模型仿真结果与文献符合得较好,能够从数量上反映T 细胞受体信号转导途径中各分子间复杂的调控关系,并能通过模型仿真发现和验证该信号途径中的关键节点分子。结论:模型基本反映了T 细胞受体信号转导途径的动力学特征,可以作为后续的精确定量关系研究的基础。 关键词: T 细胞受体; 信号转导; 动力学模型 T 细胞特异性抗原或T CR/CD3的特异性抗体可引起T 细胞跨膜受体以及膜附近的其它信号分子的活化,并引起T 细胞形态改变、细胞因子分泌、细胞粘附性改变等免疫应答,从而调节T 细胞的增殖、分化或凋亡,该过程涉及一系列下游信号转导和基因表达调控。T 细胞活化时信号传递由T 细胞表面抗原识别受体(T cell receptor ,T CR)介导,外来信号经受体及相关蛋白传递给胞内的蛋白酪氨酸激酶,此后启动三条下游信号途径:一为磷脂酶C 1(Phospholipase C 1,PL C 1)介导的信号途径,二为Ras M A PK 途径,三为共刺激分子介导的磷酸肌醇激酶 3(PI3K)辅助信号途径。同时,为保持免疫应答的平衡,避免过度活化,T 细胞的活化过程还受到抑制性信号通路的调节,这些通路彼此交织,构成一个十分复杂的T 细胞活化调控网络[1]。 随着各种复杂的信号转导网络中各个分子通道的确定,如何从系统水平上定量地分析各信号转导网络的动态特征就成为当前系统生物学的研究重点。除各种并行、高通量的实验技术外,数学建模和仿真是另外一种研究复杂生化网络的强有力手段,比如在细胞代谢研究领域已经很广泛地利用数学模型分析具有多个调控环的代谢网络[2]。实践表明,通过建立生化网络的数学模型并进行计算机仿真能够拟合现有的实验数据,解释实验中观测到的现象,预测一些不能通过当前实验手段获得的结果,减少实验的强度和数量,并验证实验提出的可能机制。 本研究建立了T 细胞受体信号转导途径的动力学模型,通过模型仿真揭示了T 细胞受体信号途径各分子间的动态调控过程,并对模型的动力学特性进行了简要分析。1 材料与方法 1 1 T 细胞受体信号转导途径 T 细胞受体信号转导途径(图1)摘自K EG G 数据库(ht tp://ww w.g eno me.jp/keg g/)。本研究根据相关中英文文献,对图中涉及的信号转导相关分子之间的作用方式及数量关系进行了详细研究和确证,并据此定义整个信号转导途径的变量(包括自变量和因变量)和变量之间的关系。1 2 动力学建模 本研究采用S 系统方程(S system equations )[2]来描述信号转导的生化级联反应过程。对于含有n 个因变量X 1,X 2,!,X n (其数量随时间而变化)和m 个自变量X n +1 ,X n +2,!, X n +m (一般设定为某些不变常量)的生化级联反应系统,其动 力学时变方程可以表达为: d X i /d t =V +i -V -i i =1,2,!,n (1) 式中X i 的生产函数V i +=V i +(X 1,!,X n ,X n +1,!,X n +m )和消耗函数V i -=V i -(X 1,!,X n ,X n +1,!,X n +m )是所有变量的函数,式(1)用S 系统方程可表达为: d X i /d t = i n +m j =1 X g ij j - i n +m j =1 X h ij j i =1,2,!,n (2) 式(2)中 i 和 i ( i 、 i >0)分别表示生产函数和消耗函数的速率常数,g ij 和h ij 分别表示变量X j 在因变量X i 的生产和消耗过程中的动力学阶,其中g ij 或h ij >0表示X j 在X i 的生产或消耗过程中起"正"调控作用,反之,如果g ij 或h ij <0则表示X j 在X i 的生产或消耗过程中起?负#调控作用,而当g ij 或h ij =0时则表示X j 在X i 的生产或消耗过程中不起任何调控作用。 641