(337), re4. [doi: 10.1126/scisignal.2005096]7Science Signaling Marius Sudol and Junichi Sadoshima (August 5, 2014) Henning Wackerhage, Dominic P. Del Re, Robert N. Judson,muscleThe Hippo signal transduction network in skeletal and cardiac

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R E V I E W

C E L L B I O L O G Y

The Hippo signal transduction network in skeletaland cardiac muscleHenning Wackerhage,1* Dominic P. Del Re,2 Robert N. Judson,1,3

Marius Sudol,4,5 Junichi Sadoshima2

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The discovery of the Hippo pathway can be traced back to two areas of research. Genetic screens infruit flies led to the identification of the Hippo pathway kinases and scaffolding proteins that functiontogether to suppress cell proliferation and tumor growth. Independent research, often in the context ofmuscle biology, described Tead (TEA domain) transcription factors, which bind CATTCC DNA motifsto regulate gene expression. These two research areas were joined by the finding that the Hippo pathwayregulates the activity of Tead transcription factors mainly through phosphorylation of the transcriptionalcoactivators Yap and Taz, which bind to and activate Teads. Additionally, many other signal transductionproteins crosstalk to members of the Hippo pathway forming a Hippo signal transduction network. Wediscuss evidence that the Hippo signal transduction network plays important roles in myogenesis, regen-eration, muscular dystrophy, and rhabdomyosarcoma in skeletal muscle, as well as in myogenesis, organsize control, and regeneration of the heart. Understanding the role of Hippo kinases in skeletal and heartmuscle physiology could have important implications for translational research.

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Introduction

Skeletal and cardiac muscle function to generate body movement and bloodflow. Both skeletal and cardiac muscle cells have sarcomeric motor proteinsthat convert the chemical energy of nutrients into work and heat. Skeletal andcardiac muscles develop from differentiation of the mesoderm, but expressdifferent genes and vary in their morphological and physiological properties.In addition, skeletal muscle has the ability to regenerate after chemical ormechanical damage (1): a property conferred by the activation of residentstem cells known as satellite cells (2–5). In contrast, adult mammalian cardiacmuscle does not effectively regenerate after cardiac injury, such as a heartattack in humans (6). However, neonatal mammalian (7) and adult zebrafishhearts (8) can regenerate, suggesting that the underlying molecular programresponsible for regeneration exists in the heart and may be activated for ther-apeutic purposes. Both heart and skeletal muscle are subject to progressivedegeneration, as seen in aging (9) and muscular dystrophy (10), and skeletal,but not cardiac, muscle can undergo oncogenic transformation, for example,in patients with rhabdomyosarcoma (11–13). Several signal transduction path-ways have been linked to muscle development, regeneration, and disease.Here, we review the evidence for the emerging role of the Hippo signal trans-duction network in these processes.

Discovery of the Hippo signal transduction network

The Hippo pathway is an important signal transduction pathway involved indevelopment, stem cell function, regeneration, and organ size in multiple tissuesin various species, and underlies several human pathologies, including cancer

1School of Medical Sciences, University of Aberdeen, Health SciencesBuilding, Foresterhill, AB25 2ZD Aberdeen, Scotland, UK. 2Department ofCell Biology and Molecular Medicine, New Jersey Medical School, RutgersUniversity, 185 South Orange Avenue, Newark, NJ 07103, USA. 3BiomedicalResearch Centre, University of British Columbia, 317-2194 Health SciencesMall, Vancouver, British Columbia V6T 1Z3, Canada. 4Mechanobiology Insti-tute, National University of Singapore, 5A Engineering Drive 1, Singapore117411, Republic of Singapore. 5Department of Medicine, Mount SinaiSchool of Medicine, One Gustave Levy Place, New York, NY 10029, USA.*Corresponding author. E-mail: h.wackerhage@abdn.ac.uk

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(14–18). Genetic screens designed to identify tumor suppressors in the fruit flyDrosophila melanogaster led to the discovery of the Hippo pathway, includingthe kinases Hippo and Warts, the kinase-binding proteins Salvador and Mob1,and several upstream kinase activators. Consistent with a role as tumor sup-pressors, loss-of-function mutations in the genes encoding these proteinstypically increase cell proliferation and decrease apoptosis (19, 20). Severalgenetic epistasis and protein interaction studies indicate that these proteinsconstitute a distinct, integrated signal transduction pathway (21).

As early as the late 1980s, transcriptional regulators, which are now knownto be part of the Hippo pathway, were studied independently from the work inflies, often in the context of muscle research. The transcription factor Tead1(TEA domain containing 1, also referred to as Tef-1 in early studies) binds toa CATTCC DNA binding motif (22), which is variously referred to as amuscle CAT (MCAT) motif (23), GTIIC motif (22), or Hippo response ele-ment (24). A conserved 66– to 68–amino acid region known as the TEA/ATTS (TEA) domain in Teads (Tead1 to Tead4) binds to MCAT elements(Fig. 1, A and B) (25). The activity of Teads requires binding to transcriptionalcoactivators (26), including Yap (yes-associated protein, encoded by the geneYap1) (27, 28), Taz (transcription coactivator with PDZ binding motif, encodedby the gene Wwtr1) (29–31), and Vglls (Vestigial-like proteins) (26, 32, 33).The discovery that the Hippo pathway inhibits the transcriptional cofactorYorkie (34), which is the fly homolog of Yap and Taz, connected theHippo pathway to the transcriptional regulators Yap, Taz, and Teads.

In recent years, the list of genes and proteins linked to the Hippo path-way has expanded greatly. Research demonstrates that the Hippo pathwayis only one of several signal transduction modules that target the Hippotranscriptional regulators Yap, Taz, Teads, and Vglls. Additionally, the Hippotranscriptional regulators interact with many downstream signaling proteins(35, 36). Therefore, to most appropriately describe the structure of the entiresignaling system, we refer to it as the “Hippo signal transduction network.”

Hippo pathway

The central Hippo pathway comprises the mammalian STE20-like proteinkinases 1 and 2 (Mst1 and Mst2, encoded by the genes Stk4 and Stk3,orthologs of Hippo in flies) and the large tumor suppressor kinases

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1 and 2 (Lats1 and Lats2, paralogous to Warts in flies) of the NDR (nu-clear dbf2-related) family (Fig. 1A) (37). Multiple proteins in the Hipposignal transduction network interact using WW domains, which aredefined by two highly conserved tryptophans located 20 to 22 amino acidsapart that bind to proline-rich motifs [PPxYor PPxF (PPxY/F)] (38). Mst1or Mst2 (Mst1/2) binds to the auxiliary protein Sav1 (Salvador homolog 1),presumably through interactions of WW domains in Sav1 and noncanon-ical PPxF motifs in Mst1/2 (39, 40), and this complex directly phosphoryl-ates a threonine in the kinase domain of Lats1 or Lats2 (Lats1/2) (Thr1041

of Lats1 or Thr1079 of Lats2) (41). Moreover, Mst1/2 bound to Sav1 bindsto and phosphorylates Mob1a or Mob1b (Mob kinase activator 1), whichthen binds to the autoinhibitory loop of Lats1/2 to promote autophosphor-ylation (Ser909 of Lats1 or Ser872 of Lats2) (41, 42). The PPxY motif ofLats1/2 binds to the WW domains of Yap or Taz (43, 44) (Fig. 1C). Yapis alternatively spliced to produce variants with either one or two WW do-mains (45), and Taz has one WW domain (30). Phosphorylation and ac-tivation of Lats1/2 by Mst1/2 promote the ability of Lats1/2 to phosphorylateYap or Taz (30, 34, 46, 47). Many cell adhesion and cell junction proteins canaffect the activity of Mst1/2 and Lats1/2; however, these proteins primarilyhave been studied in epithelial cells (14, 48), and it is unclear whether theyhave similar functions in skeletal and cardiac muscle cells.

Phosphorylation of Yap or Taz by Lats1/2 occurs on HXRXXS aminoacid motifs (46). Human YAP has five (Ser61, Ser109, Ser127, Ser164, andSer381) and human TAZ has four (Ser66, Ser89, Ser117, and Ser311) HXRXXS

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motifs, all of which are phosphorylated by Lats1/2(49). Phosphorylation of the best-characterized phos-phosite of YAP (Ser127) leads to sequestration in thecytosol by 14-3-3 proteins (50). Mutating YAP Ser127

to alanine prevents phosphorylation and promotesconstitutive activation of its transcriptional function(47, 51). Except in intestinal stem cells (52), expressionof YAP S127A increases cell proliferation and inhibitsapoptosis (47, 51, 53, 54). Ser89 of TAZ is analogous toSer127 of YAP, and the TAZ S89A mutant is also aconstitutive transcriptional activator (30). Yap can beactivated independently of Lats1/2 by phosphorylationby the tyrosine kinase Yes (27, 55). Moreover, the PPxYmotif of the nonreceptor tyrosine phosphatase Ptpn14binds to the WW domain of Yap and inhibits nuclearlocalization of Yap (56–58). The serine and threoninephosphatase PP1 activates Taz by dephosphorylationof Ser89 and Ser311, which induces nuclear localizationand stabilization of Taz (59). Monomethylation of Yapat Lys494 by Setd7 (SET domain containing lysinemethyltransferase 7) leads to cytoplasmic localizationand inhibition of Yap (60). Moreover, murine Yap phos-phorylated at Ser112, which is homologous to Ser127 inhuman YAP, localizes to both the cytoplasm and the nu-cleus of cells grown at low density in culture (61). Thus,Yap and Taz are inhibited and activated by proteins otherthan Lats kinases, suggesting that the phosphorylationstate of YAP Ser127 and TAZ Ser89 may not be sufficientto indicate inhibition of their transcriptional activity.

In the absence of coactivators, Teads repress ex-pression of target genes (25). Yap and Taz bind tothree interfaces on Teads (32) (Fig. 1D) and therebyrelieve repression and promote transcription in a man-ner analogous to transcription coactivators in otherdevelopmental signaling pathways. Chromatin immu-noprecipitation studies in breast epithelial cells show

that Yap and Tead occupy about 80% of the same genomic loci (62). How-ever, Tead-dependent transcription can also occur independently of coacti-vation by Yap or Taz. Like Yap and Taz, binding of some Vgll proteins canactivate Tead-based transcription (32, 33). In contrast, Vgll4 is a repressor ofTeads (26, 63). The Tondu domains of Vgll proteins interact with Teads(32, 33) and may compete for common binding interfaces with Yap andTaz (26) (Fig. 1D). Vgll4 has two Tondu domains, whereas other Vgll para-logs have only one.

Crosstalk with the Hippo pathway

The Hippo pathway extends beyond a simple kinase cascade, leading toinhibition of Yap and Taz. Several proteins have been discovered to eithercompletely or partially bypass the Hippo pathway to target Yap and Taz.Likewise, Yap and Taz can influence the activity of DNA binding transcrip-tion factors other than Teads (Fig. 2).

Cells grown on stiff substrates or at low density form stress fibers(64, 65) composed of filamentous (F)–actin, myosin II, and a-actin (66).Actin polymerization is required for Yap and Taz nuclear localization andactivation by mechanical signals or low-density cell culture (61, 64, 65).Several angiomotin proteins, which are present in skeletal muscle and theheart in addition to other tissues (67), bind to F-actin and Yap and arerequired for inhibition of Yap by mechanical stimuli (68). Disruptionof actin polymerization can activate Lats (69), and expression of a kinase-

P

P

P

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CATTCC

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Tead4

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Interface 2

Interface 3YAP

WW

PPxY/F

Tondu

TEA

Vgll1–3

Vgll4

Tead1–4

Fig. 1. Protein interactions in the Hippo pathway. (A) The Hippo pathway comprises a cassette

of serine and threonine kinases, including Mst1 or Mst2 (Mst1/2) and Lats1 or Lats2 (Lats1/2),that phosphorylates the functionally redundant transcriptional coactivators Yap and Taz (Yap/Taz). The key domains and motifs that enable protein-protein and protein-DNA interactions areWW, Tondu, and Tea domains and PPxY or PPxF motifs (PPxY/F). P, phosphorylation; Yap/Taz,Yap or Taz. (B) Structural model of the TEA domain of Tead (red, yellow) binding to DNA (white).Image adapted with permission from (210). (C) Structural model of the b strands in the WWdomain (green) of YAP in complex with a LATS1 peptide (yellow) containing the PPxY motif.The side-chain moieties of amino acids within the WW domain (red) and the consensus residueswithin the PPxY motif (blue) (assigned P0, P + 1, and Y + 3) are engaged in intermolecularcontacts. Credit: A. Farooq/University of Miami. (D) Structural model of the transactivation domainof Tead4 (red) binding to Yap (orange) at three interfaces or Vgll1 (green) at two interfaces. Imagereproduced with permission from (32).

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dead form of Lats2 can prevent the ability of the actin depolymerizingagents to promote the cytosolic localization of Yap (69). However, knock-down of Lats1 and Lats2 does not rescue Yap and Taz inhibition in cells grownon a soft extracellular matrix (65), suggesting that inhibition of Yap and Tazby mechanical stimuli can function in parallel with or independent of Lats.

G protein (heterotrimeric guanine nucleotide–binding protein)–coupledreceptors (GPCRs) are a large family of seven-transmembrane receptorproteins (70), which can regulate the nuclear translocation of Yap andTaz. Exposing cultured cells to fetal bovine serum or GPCR ligands, in-cluding lysophosphatidic acid or S1P, which activates Ga12/13-, Gaq/11-,and Gai/d-coupled receptors, inhibits phosphorylation of Lats, Yap, andTaz, and promotes nuclear localization of Yap (69). Disruption of the actincytoskeleton by exposing cells to latrunculin B prevents the ability of ly-sophosphatidic acid or fetal bovine serum to activate Yap and Taz (69),suggesting that the cytoskeleton may integrate mechanotransduction andGPCR signaling. In contrast to Ga12/13-, Gaq/11-, and Gai/d-coupled receptors,the activation of GaS-coupled receptors by epinephrine or other ligandsincreases inhibitory phosphorylation and cytosolic localization of Yap (69).GaS increases the production of cyclic AMP (adenosine 5′-monophosphate),which activates protein kinase A (PKA), and exposing cells to forskolin,which increases cyclic AMP, increases phosphorylation of Lats1/2 and Yapand promotes cytosolic localization of Yap (69).

Multiple studies provide evidence for crosstalk between the Wnt–b-catenin and Hippo signaling pathways (35). For example, Yap can form acomplex with b-catenin, Yes, and Tbx5 (T-box transcription factor 5) andis required in colorectal cancer cell lines with high b-catenin activity forproliferation and colony formation in culture and tumor-forming potential

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in orthotopic xenografts in mice (55). Addi-tionally, members of the b-catenin destructioncomplex can promote the cytosolic sequestrationof Yap and Taz and the ubiquitin-dependent deg-radation of Taz and thereby inhibit Yap- and Taz-dependent gene expression (71, 72).

Yap and Taz bind and coactivate transcrip-tion factors other than Teads (36). Of these,Smads (sma gene mothers against decapenta-plegic peptide) and Tbx5 are especially rele-vant to skeletal and cardiac muscle biology.The WW domains of Yap bind to the PPxY mo-tifs of the phosphorylated linker region of thebone morphogenetic protein (BMP) signaling–associated receptor Smad, Smad1, to promoteSmad1-dependent transcription (73). The WWdomains of Yap can also bind to the PPxY motifof Smad7, an inhibitor of transforming growthfactor–b (TGFb) signaling (74). The C termi-nus of Taz binds to complexes of the TGFbsignaling–associated Smads, Smad2 and Smad3,and the DNA binding Smad, Smad4, in the nu-cleus and promotes the expression of Smad2-and Smad3-dependent genes (75). Moreover,Yap and Taz can bind to Tbx5 and promoteTbx5-dependent gene expression (55, 76).

Hippo signaling can influence mTOR (mam-malian target of rapamycin) kinase signaling. Yapdrives the expression of miR-29, which promotesthe degradation of the phosphatase Pten. Pten in-hibits Akt, and Akt indirectly activates mTOR.Knockdown of Lats1 and Lats2 in cells or over-expression of Yap in the skin of mice increases

mTOR activity (77). Mst1/2 can bind in a complex with Akt1, and knock-down of Mst1/2 reduces the activating phosphorylation of Akt1 at Ser473

and reduces phosphorylation of Akt substrates (78). Moreover, Akt phos-phorylates Mst1 at Thr120 and Thr387, leading to inhibition of Mst1 activ-ity (79–81). Because Yap and Taz promote cell proliferation and becausemTOR signaling is required for protein synthesis during cell proliferation,the mechanistic connection between Hippo and mTOR signaling suggeststhat cell proliferation and protein synthesis could be coordinated throughthis crosstalk.

There is evidence that other signaling pathways regulate Hippo sig-naling by altering the transcription of genes encoding proteins in theHippo pathway. For example, exposing cerebellar neuronal precursors tothe secreted glycoprotein Shh (sonic hedgehog) increases Yap1 expression(82). Moreover, the Notch-associated transcription factor Rbpj (recombi-nation signal binding protein for immunoglobulin kappa J) directly inducestranscription of Yap1 and Tead2 in mouse cortical neural stem cells (83). Sox2(sex determining region box 2) directly binds to the Yap1 promoter and in-creases its expression in mesenchymal stem cells (84). Moreover, thepromoter of Yap1 contains several binding sites for the transcription factorGA-binding protein, which also promotes Yap1 expression (85).

The abundance of Yap and Taz protein is controlled by phosphorylation-dependent degradation. Phosphorylation of Ser381 by Lats1 or Lats2 primesYAP for phosphorylation by casein kinases CK1d or CK1e, which createsa phosphodegron motif that leads to Yap degradation by the proteasome(49). Similarly, glycogen synthase kinase 3 of the b-catenin destructioncomplex phosphorylates Taz, creating a phosphodegron motif and target-ing it for degradation (86, 87).

Mechanotransduction

Extracellular

Epinephrine

Gs

G12/13

Gq/11

S1PLPA

Thrombin

Frz Lrp

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Cytoplasm

YapTaz TazYap/Taz Yap

Smad

Set7

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Sav1 Mst1/2

Fig. 2. Schematic of key signaling modules within the Hippo signal transduction network. As detailed inthe main text, evidence suggests that crosstalk between the Hippo pathway and other signaling mod-

ules occurs in parallel to the core kinase cassette. P, phosphorylation; M, methylation; LPA, lysopho-sphatidic acid; S1P, sphingosine 1-phosphate.

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Several other signaling proteins crosstalk with the Hippo pathway.For example, the kinase Lkb1 (also known as serine and threonine proteinkinase 11) (88, 89) and integrin-linked protein kinase (90) interact withHippo transcriptional regulators and may be relevant to skeletal and car-diac muscle biology. Moreover, recent proteomic studies in several celltypes have identified previously uncharacterized protein interactions withmembers of the Hippo signal transduction network. For example, Yorkiecan interact with proteins of lysosomal pathway, and knockdown of theseproteins or neutralization of lysosomal pH increases the activity of a Yorkietranscriptional reporter (91). Likewise, interaction with coiled-coil domain-containing protein 85C regulates the localization of Yap (92). Striatin-interactingphosphatase and kinase (STRIPAK) complexes bind Mst1 and Mst2, but thefunctional implications of this interaction are poorly understood (93, 94). Col-lectively, this diversity of crosstalk supports the notion that the Hippo pathwaydoes not function in isolation but participates in various other pathways as partof a wider Hippo signal transduction network.

Hippo signaling and skeletal muscle myogenesis

Several studies implicate the Hippo signal transduction network inmuscle development, regeneration, and disease. Skeletal muscle myogen-esis begins around embryonic day 8 (E8) in mice, when Pax3 (paired boxprotein-3)–positive cells in the dermomyotome begin to undergo epithelial-to-mesenchymal transitions, delaminate, and migrate (95). Migratory Pax3-positive cells begin to express the myogenic regulatory transcriptionfactors Myf5 and MyoD (MyoD is expressed later than Myf5) (96–98),which promote the specification of Pax3-positive progenitor cells intomononucleated myoblasts (98). Overexpression of Myf5 or MyoD innon-muscle cells is sufficient to initiate myogenesis in vivo (99), suggest-ing that these factors function redundantly (100, 101). MyoD induces theexpression of the transcription factors myogenin and Mrf4, causing myoblaststo undergo terminal differentiation, in which they fuse into multinucleatedmyotubes that mature into striated muscle fibers (98). MyoD has been re-ported to bind more than 20,000 DNA loci in myoblasts and multinucleatedmyotubes (102). Although some of these loci might be detected due to non-specific MyoD binding (103), the large number of sites is consistent with theidea that MyoD acts as a “pioneer” transcription factor. Pioneer transcriptionfactors stably bind and “preselect” a set of genes to be expressed in a given

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cell lineage (104). Consistent with this model,MyoD recruits histone methyltransferases and ace-tyltransferases to the enhancers and promotersof myoblast-associated genes, which prepares thechromatin for active transcription (105, 106).

There is both direct and indirect evidence thatthe Hippo signal transduction network is involvedin the regulation of myogenic differentiation incultured myoblasts in vitro and during embryonicmyogenesis in vivo. Studies suggest that Yap isactive in myoblasts and inhibits terminal differ-entiation into myotubes (107, 108). In Xenopuslaevis embryos, overexpression of constitutivelyactive Yap increases proliferation of neural pro-genitor cells and reduces the expression of mark-ers of somatic muscle differentiation, includingMyoD (109). The evolutionarily conserved ECR111enhancer, which contains an MCAT motif thatbinds Teads, is located ~111 kilo–base pairsupstream of the Myf5 gene and is required forthe expression of Myf5 in ventral somatic com-partments (110). Consistent with a potential role

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of Hippo signaling in activating ECR111, overexpression of YAP S127Ain C2C12 myoblasts increasesMyf5 expression (107). It is unknown whetherYap1 is required for Myf5 expression during developmental myogenesis invivo because mice with global knockout of Yap1 die around E8.5 (111).Global knockout of Wwtr1, the gene encoding Taz (112), does not resultin an obvious skeletal muscle phenotype, and in-depth analysis of this tissuewas not the focus of that study.

Additional circumstantial evidence suggests that inhibition of Yap isessential for myoblast differentiation into multinucleated myotubes. Cellculture conditions optimal for differentiating myoblasts into myotubesare similar to those that inhibit Yap activity in other cell types. Myoblastsgrown to a high degree of confluence maximize cell-cell contact, which isknown to inhibit Yap in other cell types (46, 65) and enhances myoblastdifferentiation (113). Similarly, myoblast differentiation is enhanced whencells are cultured on soft hydrogel substrates with a stiffness of about 12 kPasimilar to the stiffness of muscle, rather than directly on plastic, which is muchstiffer (114). Reducing the concentration of serum in the medium, whichshould inhibit Yap by reducing activation of GPCR signaling (69), alsopromotes terminal differentiation of C2C12 myoblasts (113). Together, theseresults imply that inhibition of Yap may be a key event for the terminaldifferentiation of myoblasts into myotubes.

In contrast to the observation that active Yap prevents the differentia-tion in myoblasts (107) and activated satellite cells (108), some genes withMCAT motifs are expressed in differentiated muscle. In zebrafish, activa-tion of a transgenic reporter for Tead-dependent transcription is highlyabundant in differentiated trunk muscles at 2 and 3 days after fertiliza-tion (Fig. 3A) (115). Similarly, the expression of genes that encode proteinscharacteristic of differentiated muscle, such as cardiac troponin T (Tnnt2)(116) and a-actin (117) in mouse, relies at least partially on MCAT responseelements (118). One possible explanation is that different Tead, Yap, Taz,and Vgll complexes may selectively target myoblast or differentiated mus-cle genes, leading to differential expression. Similar target specificity occursin flies where the homologs of Vgll and Teads homologs form complexesthat target different genes than complexes of Yorkie and Tead homologs(26, 119). In mammals, Yap1 is highly expressed in myoblasts and inhibitsdifferentiation (107, 108), whereasWwtr1 [Taz (120–122)], Vgll2 (123–126),and Tead4 (127) are more highly expressed in differentiated muscle and pro-mote differentiation. A second possibility is that different Tead, Yap, Taz, and

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Plasmalemma

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Fig. 3. The Hippo signal transduction network and skeletal muscle. (A) Green fluorescent protein

(GFP) fluorescence in a 4xGTIIC:dGFP (MCAT reporter) zebrafish at 2 days after fertilization. Notethe intense GFP signal in skeletal muscle (arrowheads). Image reproduced with permission from(115). (B) Transmission electron micrograph of a satellite cell (sc) between the plasmalemma andbasal lamina. Image reproduced with permission from (4). (C) Immunofluorescence of ex vivomuscle fibers showing that Yap abundance (red) is higher in activated (48 hours, MyoD+) than inquiescent (0 hours, Pax7+) satellite cells. Image reproduced with permission from (108).

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Vgll complexes target the same genes but exert different effects. During neu-rogenesis, the transcription factors Sox2, Sox3, and Sox11 are expressed atdifferent stages of neuronal differentiation and bind to identical DNA loci;however, the function of the different isoforms can be either to preselect genesfor expression or to actively promote their expression (128). A third possibilityis that Yap or Taz could not only coactivate MCATelements but also bind andcoactivate additional transcription factors such as Smads (75, 129) or Tbx5(55, 76) to drive myoblast or myotube and differentiated muscle-specificgene expression.

The Hippo pathway in satellite cells and skeletalmuscle regeneration

In skeletal muscle, nuclei within differentiated muscle fibers do not divideand, thus, do not contribute to regeneration. Resident Pax7-expressing stemcells, called satellite cells (4), proliferate and differentiate in response to injuryto give rise to new muscle (130). Satellite cells reside between the basal lam-ina and the plasma membrane of differentiated muscle cells (4, 5, 131) (Fig.3B). The nuclei of satellite cells account for between 1.4 and 7.3% of allnuclei within an adult human muscle (132). Satellite cells undergo self-renewing cell divisions and, when stimulated, can differentiate into musclefibers (2). Satellite cells are essential for regeneration (3), but not for short-term hypertrophy after overload (133).

Hippo signaling is likely to play a role in the proliferation and differ-entiation of satellite cells. The expression of Yap1 increases about threefoldduring activation of mouse satellite cell grown in mitogen-rich medium(108) (Fig. 3C), and overexpression of human YAP S127A in activated sat-ellite cells increases proliferation and inhibits differentiation (108),consistent with the activation and function of Yap in other stem and progen-itor cells (51, 53, 54). Additional evidence suggests that Yap activity insatellite cells may be regulated by pathways that crosstalk to the Hippopathway. S1P, which activates Yap through GPCR signaling (69, 134), pro-motes the proliferation of satellite cells (135). Notch, which can increase theexpression of Yap1 in cortical neural stem cells (83), promotes satellite cellproliferation (136). Recent reports suggest that Notch can also promotesatellite cell self-renewal (137) and niche colonization (138). Wnt sig-naling regulates myogenesis during development and differentiation ofsatellite cells (139). Yap activates the expression of Bmp4 in satellite cells(108), and Bmp4 protein promotes proliferation and inhibits differentia-tion of satellite cells (140). Moreover, overexpression of YAP S127A inmuscle satellite cells increases the expression of genes encoding proteinsangiomotin-like 2 and Frmd6 (also known as Willin) and decreases theexpression of genes that encode GPCRs (107). Because these changes ingene expression should result in inhibition of Yap activity (68, 141, 142),this observation suggests that negative feedback mechanisms may serveto limit Yap-dependent cell proliferation. Thus, these data suggest that Yapmay be an important regulator of the proliferation of activated satellite cells,and future studies should test the role of Yap on skeletal muscle regenera-tion in vivo.

The Hippo pathway in terminally differentiated skeletal muscle

In adult humans, muscle fibers can be up to ~20 cm long (143), and asingle fiber may contain several tens of thousands of nuclei (144). Thehuman vastus lateralis of young males comprises ~400,000 to 900,000muscle fibers, and the number of fibers decreases during aging (145).Fibers can be distinguished into slow type I, intermediate type IIa, andfast type IIx and IIb fibers based on the presence of myosin heavychain isoforms and on the abundance and isoforms of other motor,metabolic, and mitochondrial proteins (146, 147). The number of fi-

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bers and the percentage of different fiber types vary greatly both withinand between differing muscles of the body and among individuals(145, 148, 149). Muscle fibers hypertrophy in response to overload(for example, as a result of resistance training), and increase mitochon-drial biogenesis and change the concentrations and isoforms of motorand metabolic proteins in response to endurance training (147). Thus,differentiated skeletal muscle has a high degree of plasticity, enabling itto change its force production and metabolic capacity in response tovarious types of stimuli.

Hippo signaling may regulate gene expression in differentiated skeletalmuscle. Genes and reporter genes with MCAT elements are activelyexpressed in differentiated skeletal muscle (115–117, 150). Acute resist-ance exercise increases expression of the genes encoding cysteine-rich an-giogenic inducer 61 and connective tissue growth factor (Ctgf) (151), thelatter of which has three MCAT elements in its proximal promoter (62).These genes are frequently used as marker genes for Yap and/or Taz ac-tivity [for example (152)], suggesting that Yap or Taz may be activated byacute resistance exercise. Teads have been shown to regulate a-actin pro-moter activity in a model of stretch overload–induced hypertrophy inchicken (117). Overexpression of Tead1 in muscle fibers in mice causesa fast-to-slow fiber type transition, but not hypertrophy (153). Moreover,denervation of fast, but not slow, skeletal muscle induces atrophy and in-creases the expression ofMst1 (154). Mst1 can phosphorylate Ser207 of theforkhead transcription factor Foxo3a, promoting muscle atrophy in mice(154) and thereby increasing the expression of genes encoding skeletalmuscle atrophy–regulating factors known as atrogins (155). Future studiesare needed to clarify the role of the Hippo pathway in overload-inducedhypertrophy, the regulation of muscle fiber type–specific gene expression,muscle atrophy, and other related phenomena.

Hippo pathway and myopathies

Perturbation of Hippo signaling may contribute to the pathology of differ-ent myopathies, including muscular dystrophies (10). We recently foundthat the expression of human YAP S127A in muscle fibers in adult micecauses a “fulminant” myopathy characterized by atrophy, signs of centro-nuclear myopathy, deterioration, and, after several weeks, death of the mice(156). The WW domain–containing protein Bag3 (Bag family molecularchaperone regulator 3) interacts directly with the Hippo pathway proteinsangiomotin 1, angiomotin 2, and Lats1, and is a positive regulator of Yapand Taz and of Ctgf expression (157). Bag3 knockout causes a fulminantmuscular dystrophy in mice (158), and in humans, loss-of-function muta-tions of Bag3 are associated with severe childhood muscular dystrophy(159). Loss-of-function mutations in the WW domain–containing proteindystrophin (DMD) causes either Duchenne’s (severe) or Becker’s (mild)muscular dystrophy in humans (10, 160). It is unknown whether DMD,or the related protein utrophin, uses WW domains to interact with theHippo pathway and whether mutations in these proteins perturb Hipposignaling and thereby contribute to the pathology of muscular dystrophies.However, considering the relatively small number of human proteins withWW domains (39), it is intriguing that mutations in these proteins causehuman muscular dystrophy.

Other studies further support a role for perturbation of Hippo signalingin myopathies. The Hippo pathway target gene CTGF is highly expressedin muscles of patients with muscular dystrophy (161). Overexpression ofCtgf in mouse skeletal muscle is sufficient to cause muscular dystrophy(162), whereas knockout of Ctgf in a mouse model of muscular dystrophypartially ameliorates the pathology (163). Laminopathies, which can resultin muscular dystrophy, are diseases that result from mutation in LMNA,which encodes the nuclear lamina protein lamin A/C (164). Human

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myoblasts with LMNA mutations have impaired ability to produce appropri-ate cytoskeletal rearrangements in response to mechanical stimuli, such asthe changes in substrate stiffness (165). Consistent with the role of Hippo inmechanotransduction (64, 65), YAP is more active, and CTGF expression ishigher in LMNA mutant compared to wild-type myoblasts cultured on sub-strates with the same stiffness (165). Collectively, these results suggest thatdysregulation of Hippo signaling in skeletal muscle due to the mutations inDMD, BAG3, or LMNAmay result in changes in expression of Hippo targetgenes such as CTGF and thereby contribute to pathology.

Myopathies resulting from nongenetic causes may also involve Hipposignaling. Statins are widely prescribed drugs that act on the mevalonatepathway, the activation of which increases Yap and Taz transcriptional activ-ity (166, 167). Statins induce myopathies with symptoms ranging from mus-cle weakness to rhabdomyolysis in as many as 1.5 million patients per year(168). Thus, investigating whether the inhibition of Yap and Taz contributesto statin-induced myopathies may have important clinical ramifications.

Hippo and rhabdomyosarcoma

Rhabdomyosarcomas, diagnosed on the basis of the presence of rhabdo-myoblasts, are the most common soft tissue sarcomas in children and ado-lescents (11). Rhabdomyosarcomas are classified as embryonal (ERMS),alveolar (ARMS), and pleomorphic (anaplastic). ERMS occur in infantsand young children, ARMS occur in adolescents and young adults andhave a poorer prognosis than ERMS, and pleomorphic rhabdomyosarcomasoccur in adults and are relatively rare (11). About 70 to 80% of ARMS ex-press PAX3-FOXO1 or PAX7-FOXO1 fusion genes, which indicates a poorerprognosis than other ARMS (169). Expression of PAX3-FOXO1 in mice withhomozygous loss of the gene encoding tumor protein 53 (p53) or homozygousloss of the gene encoding cyclin-dependent kinase inhibitor 2A (CDKN2A,also known as p16/Ink4A) gives rise to tumors that resemble human ARMS(170), supporting the tissue-specific, oncogenic potential of this fusion gene.

Given that YAP S127A overexpression drives proliferation and inhibitsdifferentiation of C2C12 myoblasts (107) and activated satellite cells (108),deregulation of Hippo signaling may contribute to the pathogenesis of rhabdo-myosarcoma. The Ras association (RalGDS/AF-6) domain family (Rassf )of proteins contain SARAH (Salvador, Rassf, and Hippo) domains that bindto and inhibit Mst1 and Mst2 (171). The expression of the tumor suppressorRASSF4 is increased in PAX3-FOXO1–positive ARMS (172). The PAX3-FOXO1 fusion protein directly binds to the 5′ enhancer of RASSF4, andknockdown of RASSF4 in cultured ARMS cell lines reduces cell prolifera-tion and the survival of mice with ARMS xenotransplants (172). Similar toother Rassf proteins, RASSF4 binds and inhibits MST1 in human myoblastand ARMS cell lines (172); however, a functional connection betweenRASSF4 and YAP in ARMS has not been identified.

Hippo signaling may also be important in the development of ERMS.YAP is more abundant in the nucleus of ERMS compared to ARMS pa-tient samples, which, in some cases, may be explained by increased copynumber of the YAP1 locus (173). Overexpression of YAP S127A in acti-vated, but not quiescent, satellite cells causes ERMS-like tumors in micewith high penetrance and a short latency to tumor onset. Cessation of YAPS127A transgene expression in YAP S127A–driven ERMS-like tumors inmice or a knockdown of YAP in human ERMS cells causes differentiationof tumor cells into myosin heavy chain–expressing muscle fibers. Knock-down of YAP reduces proliferation and anchorage-independent growth inhuman ERMS cells in culture and decreases the tumor burden in micewith human ERMS xenotransplants. Combined analyses of Yap and Tead1genome-wide chromatin immunoprecipitation and quantitative reversetranscription polymerase chain reaction (RT-qPCR) studies and cDNA mi-croarrays of YAP S127A–driven ERMS-like tumors suggest that in mouse

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ERMS myoblasts, Yap and Tead1 bind to and increase the expression ofgenes that regulate cell proliferation (Ccnd1 and Cdc6), as well as oncogenesand cancer-related genes (Met, Myc, and Birc5), and conversely repress theexpression of genes typically expressed in terminally differentiated skel-etal muscle (Myl4,Myh2,Mybph, and Tnnc2) (173). Other studies provideadditional evidence for a connection between Hippo signaling and rhabdo-myosarcoma. One case of a spindle cell variant of ERMS contained aTEAD1-NCOA2 fusion gene (174), and knockdown of the tyrosine kinaseYes, which binds and phosphorylates Yap (27, 55), reduces proliferation ofARMS and ERMS cell lines (175). Collectively, these studies suggest thatHippo pathway dysregulation causes or contributes to rhabdomyosarcoma,identifying the Hippo pathway as a treatment target for these cancers.

The Hippo pathway in heart development

The heart is a heterogeneous organ comprising cardiomyocytes, endocar-dial cells, valvular components, connective tissues, cells of the electricalconduction system, as well as the smooth muscle and endothelial cells ofthe coronary arteries and veins (176). It develops from mesodermal pro-genitor cells located in the anterior region of the primitive streak and theproepicardium (176). These cardiac progenitor populations migrate awayfrom the primitive streak and give rise to the primary and secondary heartfields. Cells in the primary heart field differentiate and form a linear hearttube that begins to beat. The heart tube grows unevenly, using cells fromthe primary heart field to form the lower bulk of the heart. Cells from thesecondary heart field migrate to the heart tube to form the outflow tract.Heart looping enables the developing heart tube to fold within the peri-cardial cavity (6, 176). The growth of the heart during embryonic devel-opment is driven by cardiomyocyte and precursor cell proliferation, butjust after birth, cardiomyocyte proliferation stops and heart growth occursprimarily by cardiomyocyte hypertrophy (176). These processes are regu-lated through complex intrinsic and extrinsic signaling events among cellsin both heart fields.

Several lines of evidence suggest that Hippo signaling is involved inheart development. Pathways that crosstalk with the Hippo pathway,including BMP, Wnt, Notch, and Shh (51, 54, 71, 177, 178), regulatetranscription factors critical for heart development. The genes encodingthe transcription factors Gata4 and Nkx2.5 are expressed in the primaryand secondary heart fields, along with the gene encoding Tbx5, whichis expressed only in the primary heart field, and these proteins work inconcert to promote cardiomyocyte differentiation and heart maturation(179). Tbx5, which can be coactivated by both Yap and Taz (55, 76), isa critical mediator of embryonic heart development (180), as mutationsin TBX5 cause Holt-Oram syndrome, which is characterized by heartand limb abnormalities (181). Conditional deletion of Sav1, Lats2, orMst1 and Mst2 in Nkx2.5-positive cardiomyocytes in mice increasesproliferation, leading to cardiomegaly and perinatal lethality (Fig. 4A)(182). Crossing Sav1 conditional knockout mice to those with heterozy-gous deletion of Ctnnb1, which encodes b-catenin, rescues cardiomyocytehyperproliferation, indicating that Wnt–b-catenin signaling acts down-stream of Hippo signaling (182). Cardiomyocytes from Sav1 conditionalknockout mice have increased expression of Wnt–b-catenin target genes,including the transcription factors Sox2 and Snail2. Because Sox2 in-creases the expression of Yap (84), this could represent a positive feed-back mechanism. Moreover, using either Nkx2.5- or Tnnt2-Cre–mediatedexcision to create conditional deletion of Yap1 in cardiomyocytes during em-bryonic development results in lethality between E10.5 and E16.5 (183, 184).Hearts of these mice have normal cardiac looping and chamber formation,but reduced cardiomyocyte proliferation and smaller, thinner ventricles,indicating that Yap is an important regulator of embryonic cardiac growth.

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The Hippo pathway in cardiac regeneration and growth

The potential for cardiomyocyte proliferation in adult mammals is lowand declines with age (185–187). Therefore, after injury, the adult mam-malian myocardium replaces lost cardiomyocytes with fibrotic scar tissue,and the functional output of the heart is reduced. In contrast, the neonatalmouse heart can regenerate after partial resection or ischemia within thefirst week after birth (7). In these animals, mature cardiomyocytes, ratherthan distinct cardiac stem or progenitor cells, undergo proliferation to pro-mote regeneration (188). In neonatal mice, conditional deletion of Yap1 incardiomyocytes, using aMHC-Cre, impairs heart regeneration after ische-mia (189). In contrast, cardiomyocyte-specific overexpression of constitu-tively active (S112A) murine Yap (homologous to human YAP S127A)reduces fibrosis and promotes cardiac regeneration beyond postnatalday 7 (Fig. 4B) (189). This is consistent with the observation that over-expression of active Yap promotes cardiomyocyte proliferation both in vivoand in cultured cardiomyocytes (183, 184, 190). In response to myocardialinfarction, Yap can be found in the nuclei of cardiomyocytes surroundingthe site of injury (190), suggesting increased activation of Yap at the borderof the infarcted area. Moreover, adult mice with conditional Yap1 knockoutusing aMHC-Cre display greater injury, increased apoptosis, and reducedproliferation of cardiomyocytes after myocardial infarction (190). However,it is unknown whether Yap is involved in cardiac remodeling and myocar-dial regeneration after myocardial infarction, and if so, which cells expressYap1, how are they activated, and how do they contribute to the functionalrecovery of the heart. Conditional deletion of Sav1, or Lats1 and Lats2, incardiomyocytes using Nkx2.5-Cre stimulates proliferation in uninjuredhearts and increases proliferation, reduces fibrosis, and improves cardiacfunction in response to partial heart resection at postnatal day 8 in mice,as well as myocardial infarction in adult mice (191). Thus, inhibition ofHippo signaling promotes heart regeneration through increased cardio-myocyte proliferation.

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Whether Hippo signaling also plays a role incardiac hypertrophy is less clear. Conditional de-letion of Yap1 in cardiomyocytes in Tnnt2-Cremice during development does not affect the sizeof cardiomyocytes (184). Likewise, postnatal retro-orbital injection (192) of adenovirus encoding GFPand Tnnt2-Cre into mice with heterozygous floxedalleles of Yap1 results in targeted deletion of Yap1in a small percentage of cardiomyocytes. GFP-positive (Yap1-deleted) cardiomyocytes have normalsize at baseline and after pressure overload stress(184). Likewise, aMHC promoter–driven YAP S127Aor S112A in the heart of postnatal mice does notalter cardiomyocyte size (184, 189). In contrast,conditional overexpression of wild-type Yap in neo-natal cardiomyocytes increases size and inducesexpression of genetic markers of the hypertrophicfetal cardiomyocytes, including genes that encodeatrial natriuretic factor, brain natriuretic peptide,and b-myosin heavy chain (190). Exposing culturedcardiomyocytes to the GPCR agonist phenylephrineinduces hypertrophy and increases expression ofatrial natriuretic factor mRNA, and expression ofshort hairpin RNAs targeting endogenous Yap1 at-tenuates the effects of phenylephrine in other celltypes (69), suggesting that phenylephrine may in-hibit Hippo signaling in these cells. Moreover, adultmice with conditional deletion of Yap1 with aMHC-

Cre have reduced cardiomyocyte hypertrophy in response to myocardialischemia (190). Thus, Yap can either promote or inhibit cardiomyocytehypertrophy, a discrepancy that may be explained by the method of genetargeting, the timing or duration of Yap depletion, the type of myocardialstress, or the mutational status of Yap1. Nevertheless, these studies revealthat Yap, and potentially Hippo signaling, can influence cardiomyocytehypertrophy.

Mst1 is activated by oxidative stress and promotes cardiomyocytedeath (193, 194). Overexpression of Mst1 promotes apoptosis of culturedcardiomyocytes. In contrast, the inhibition of endogenous Mst1 by expres-sion of a kinase-inactive (K59R) variant, which functions as a dominantnegative, reduces cardiomyocyte apoptosis induced by pharmacologicalinhibition of protein kinase C or protein phosphatases (195). Furthermore,cardiomyocyte-specific overexpression of Mst1 causes a dose-dependentincrease in cardiomyocyte apoptosis: mice with low amounts of Mst1overexpression have a modest increase in basal apoptosis, whereas micewith higher expression have a robust increase in apoptosis and rapidlyprogress to dilated cardiomyopathy, heart failure, and premature death(195). The SARAH domain of Rassf1a binds to Mst1 and promotesits activation in response to pressure overload in mouse hearts (196).Cardiomyocyte-specific overexpression of Rassf1a increases Mst1 activa-tion and exacerbates cardiac dysfunction induced by pressure overload. Incontrast, overexpression of Rassf1a with a mutation in the SARAH do-main (L308P), which cannot bind Mst1, acts as a dominant negativeand protects against cardiac injury in this context. Furthermore, cardiomyo-cyte-specific deletion of Rassf1A attenuates Mst1 activation and is protectiveagainst injury and heart failure due to pressure overload. Knockout ofRassf1A in mice does not protect against fibrosis and cardiac hypertrophyinduced by tumor necrosis factor–a (196), indicating cell type–specific effectsof Rassf1A in heart.

Yap is also important for heart homeostasis in adult mice. Homozygousdeletion of Yap1 in cardiomyocytes of postnatal mice using aMHC-Cre

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Fig. 4. The Hippo signal transduction network and the heart. (A) Conditional cardiomyocyte-

specific knockout of Sav1 (Salv) results in cardiac hypertrophy (cardiomegaly) associated withincreased cardiomyocyte proliferation. ra, right atrium; la, left atrium; rv, right ventricle; lv, leftventricle. Image reproduced with permission from (182). (B) Serial sections of Masson’s trichrome–stained wild-type (WT) and aMHC promoter–driven Yap S112A transgenic (Tg1 and Tg2) heartsshowing scar tissue (blue) 21 days after transient ligation of the left anterior descending artery atpostnatal day 7. Image reproduced with permission from (189).

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results in a rapidly developing dilated cardiomyopathy, and these mice die ofheart failure by 12 weeks of age (190). Conditional knockout of Yap1 in theheart leads to robust increases in cardiomyocyte apoptosis and fibrosis withsuppressed cardiac function, possibly due to suppression of the prosurvivalkinase Akt. Activation of Hippo signaling may contribute to arrhythmogeniccardiomyopathy. Neurofibromin 2 (Nf2), a cytoskeletal protein that modu-lates Yap activity through physical interaction with the Hippo pathwaymembers Ww45 and Kibra (197), and Mst1 are activated in a mouse modelof arrhythmogenic cardiomyopathy and in human patients (198). The in-activation of Yap in these contexts may contribute to increased adipogenesis,which is a hallmark and contributing factor in arrhythmogenic cardio-myopathy. Thus, Yap is likely a mediator of cardiomyocyte survival, prolif-eration, and signal transduction in the adult mammalian heart.

The myocardin family of coactivators bind to the transcription factor Srf(serum response factor) and may affect both Yap and Srf signaling in musclecells (199). Myocardins inhibit skeletal muscle differentiation (200) and drivethe expression of contractile genes in smooth muscle and cardiac cells (201).Yap physically interacts with myocardin and inhibits its ability to elicit expres-sion of contractile genes through interaction with Srf (199). The presence of aPPxY motif in myocardin suggests that it could bind to the WW domains ofYap, although this has not been empirically tested.

The Hippo signal transduction network as a drug target

Given the importance of Hippo signaling in skeletal and cardiac musclebiology, drugs that target this pathway may be relevant for therapeuticpurposes. Verteporfin, which is used as a photosensitizer in photodynamictherapy in the eye (202), can disrupt the interaction between Yap and Teadin human embryonic kidney (HEK) 293 cells, inhibit activation of Teadtarget genes, and reverse Yap-induced hepatomegaly in vivo (203). Therefore,verteporfin may be useful in treating hyperproliferative muscle disorders, suchas rhabdomyosarcoma. In addition, drugs that target GPCRs, such as b-blockers(69, 204–206), or the mevalonate pathway, such as statins (166, 167), po-tentially could be used to target the Hippo pathway in muscle disease or topromote regeneration. Statins and GPCR-targeting drugs are among the mostwidely prescribed drugs, and it has been estimated that 30 to 50% of allmedications exert their effect via GPCRs (207). Although not all statins andGPCR-targeted drugs necessarily affect Hippo signaling, this suggests that theeffects and side effects of these drugs should be considered in light of po-tential impact on Hippo signaling. The availability of approved drugs thatmay target the Hippo pathway provides an exciting opportunity to testwhether they can be used to treat skeletal muscle or heart diseases.

Conclusions and outlook

Since the late 1980s, proteins in the Hippo signal transduction networkhave been identified as regulators of skeletal and cardiac muscle gene ex-pression, development, organ growth, stem cell function, regeneration, anddisease. Additionally, in nonmuscle cells, proteins in the Hippo pathwayinteract with proteins involved in skeletal and cardiac muscle biology, sug-gesting that these mechanisms may exist in muscle and the heart. Howev-er, our knowledge of the Hippo signal transduction network in skeletal andcardiac muscle is modest compared to that of other signal transductionpathways in these tissues. Thus, there are still many unanswered questionsin this field, for example:

• Can information from the ENCODE project (208) and other genome-wide analyses that have fundamentally changed our understanding ofchromatin and gene regulation lead to better understanding of the regula-tion of gene expression by the Hippo pathway? Genome-wide chromatinimmunoprecipitation analysis shows that Yorkie DNA binding correlates

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with the activating chromatin mark, trimethylation of Lys4 of histone H3.The same study also shows that Yorkie directly binds to chromatin-remodelingcomplexes (209). Thus, do Yap and Taz have similar functions to Yorkie?How do the Hippo transcriptional regulators interact with chromatin andchromatin-remodeling proteins throughout the mammalian genome, es-pecially in skeletal muscle and heart cells, and during tumorigenesis inrhabdomyosarcoma?

• Given that Yap and Taz typically promote cell proliferation, which isassociated with progressive shortening of telomeres, is there a relationshipbetween the Hippo pathway and aging in skeletal muscle and the heart?

• Is the Hippo pathway involved in regulating the skeletal and heartmuscle adaptation to endurance and resistance exercise training or in me-diating other forms of muscle plasticity?

• Given that several approved drugs target Hippo signaling, can thesedrugs be used to treat skeletal and heart muscle diseases, including mus-cular dystrophy, cellular damage after myocardial infarction, and rhabdo-myosarcoma? Are there other molecules that can target Hippo signalingspecifically through Hippo pathway proteins that are only present inmuscle?

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Acknowledgments: We would like to thank A. Farooq, B. Link, J. Miesfeld, W. J. Hong,A. Pobbati, S. Veeraraghavan, T. Heallen, and J. F. Martin for sending high-resolutionfigures of their work. Funding: Research in the Aberdeen lab has been supported by aMedical Research Council project grant (99477; H.W., P. S. Zammit, and C. De Bari), SarcomaUK grant (H.W., G. Murray, R. Urcia, and M. Jaspars), Friends of Anchor grant (H.W. andC. De Bari), and a grant by Tenovus Scotland (G11/05; C. De Bari and H.W.). Research inthe Sudol lab was supported by the Mechanobiology Institute of the National University ofSingapore. Research in the Sadoshima lab has been supported by NIH grants HL112330,HL102738 (J.S.), and HL122669 (D.P.D.R.) and American Heart Association ScientistDevelopment grant 11SDG7240066 (D.P.D.R.). Competing interests: The authors de-clare that they have no competing financial interests.

Submitted 22 January 2014Accepted 2 July 2014Final Publication 5 August 201410.1126/scisignal.2005096Citation: H. Wackerhage, D. P. Del Re, R. N. Judson, M. Sudol, J. Sadoshima, The Hipposignal transduction network in skeletal and cardiac muscle. Sci. Signal. 7, re4 (2014).

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