Transcriptional mechanisms regulating skeletal muscle ...zider.free.fr/papers/Paper (2).pdf ·...

Movement is a defining feature of all animals, even if it is sometimes restricted to specific developmental stages. Larval migration, feeding, flight, reproduction and blood circulation all rely on the directional and coordinated movement that is afforded by muscles. The evolutionary advantages of efficient locomotion led to several solutions for the construction of the motile organs (the muscles) in all animal phyla. In all muscle cells, myosin II motor proteins and actin filaments generate force and movement. In the striated muscles that are used for locomotion, acto myosin contraction is amplified in serial and parallel arrangements of numerous contractile units, called sarcomeres. These are made up of actin and myosin filaments arranged in highly ordered, almost crystalline arrangements, as well as hundreds of regulatory proteins such as the troponin–tropomyosin complex, and scaffolding and cytoskeletal crosslinking proteins such as αactinin, myomesi n and the kinase titin1,2 (FIG. 1).

In vertebrates, striated muscle cells are found in two tissues: skeletal and heart muscle (FIG. 1). Although they both have highly ordered myofibril structures, they have distinct embryonic origins and are tailored for particular purposes by different genetic programmes. Furthermore, in vertebrates specialized skeletal muscles with different contractile (slowtwitch or fasttwitch) and metabolic properties coexist3,4.

Such distinctions are determined by the activity of specific transcription factors, the myogenic regulatory factors (MRFs), which act together with pleiotropic transcription

factors and epigenetic regulatory mechanisms to control muscle development and postnatal remodelling. During embryonic development, these genetic programmes determine the primary differentiation and also the future metabolic and contractile properties of tissues in specific anatomical compartments, thus controlling the commit ment of future slowtwitch or fasttwitch muscles. Postnatally, the degree of muscle use and the specifi c innervation patterns determine the composition and turnover rates of muscle contractile proteins, as well as of the supporting metabolic enzymes, ion channels and signal transduction proteins. This remodelling of differentiated muscle determines the contractile properties and the preferred sources of energy. It is governed by signalling cascades (modulated by growth factors and cytokines, steroid hormones and mechanical activity) that control the transcriptional activit y of musclespecific and pleiotropic transcription factors. These genetic programmes also regulate changes in muscle mass. During embryonic development, muscle mass increases predominantly by proliferative growth of myoblasts. Postnatally, the contribution of cell proliferation decreases, and hypertrophic growth and remodelling of preexisting muscle fibres dominates; the resident stem cells (the satellite cells) are then mostly engaged in damage repair5–7.

Recent research has shed important light on the basic mechanisms that lead to the commitment of precursor cells to the muscle lineage (myogenesis). As a result, we now have a better understanding of how the

*Max-Planck-Institute for Heart and Lung Research, Department for Cardiac Development and Remodelling, Benekestrasse 2, 61231 Bad Nauheim, Germany.‡King’s College London, Randall Division for Cell and Molecular Biophysics and Cardiovascular Division, Muscle Signalling and Development Section, New Hunt’s House, Guy’s Campus, London SE1 1UL, UK.e-mails: thomas.braun@ mpi-bn.mpg.de; [email protected]:10.1038/nrm3118

MyofibrilThe structural unit of striated muscle fibres, which is formed from longitudinally joined sarcomeres. Several myofibrils form each fibre.

MyoblastsEmbryonic cells that will become a muscle cell or part of a muscle cell.

Transcriptional mechanisms regulating skeletal muscle differentiation, growth and homeostasisThomas Braun* and Mathias Gautel‡

Abstract | Skeletal muscle is the dominant organ system in locomotion and energy metabolism. Postnatal muscle grows and adapts largely by remodelling pre-existing fibres, whereas embryonic muscle grows by the proliferation of myogenic cells. Recently, the genetic hierarchies of the myogenic transcription factors that control vertebrate muscle development — by myoblast proliferation, migration, fusion and functional adaptation into fast-twitch and slow-twitch fibres — have become clearer. The transcriptional mechanisms controlling postnatal hypertrophic growth, remodelling and functional differentiation redeploy myogenic factors in concert with serum response factor (SRF), JUNB and forkhead box protein O3A (FOXO3A). It has also emerged that there is extensive post-transcriptional regulation by microRNAs in development and postnatal remodelling.

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α

Paraxial mesodermThe mesodermal areas that form directly lateral to the neural tube.

Rostrocaudal axisA description of anatomical location in animals. Rostral (from the latin rostrum meaning beak) refers to the anterior (‘nose-end’) of the animal and caudal (from the latin caudum meaning tail) refers to the posterior (‘tail or feet end’).

SomitesMesodermal structures found on either side of the neural tube in vertebrate embryos that eventually give rise to muscle, skin and vertebrae.

Delamination A process in embryology in which cells from a single layer separate to form two different layers, or laminae.

functional diversity of different striated muscles evolves on the develop mental level, and how physiological stimuli are translated into changes in muscle gene expression patterns, metabolic flow and protein turnover. This recent research has also revealed how postnatal muscle

remodelling uses myogenic transcription factors that are also active during early development. In this Review, we outline how tight developmental regulation, mechanical functions and the homeostasis of the organism are interlinked during development and postnatal muscle adaptation. We focus on recent insights into the transcriptional and posttranscriptiona l control that commits myogenic cells to proliferation or differentiation, determines their lineage fate as slowtwitch or fasttwitch striated muscles and regulates the postnatal adaptive remodelling of committed muscle cells. Where appropriate, we consider relevant similarities between skeletal and cardiac striated muscles.

Transcriptional control of myogenesisSkeletal muscle cells of higher vertebrates arise during midgestation (in mice between embryonic day 9 (E9) and E12) from three different locations within the middle layer of cells in the primitive embryo: the segmented somitic paraxial mesoderm, the unsegmented cranial paraxial mesoderm and the prechordal mesoderm; these represent different parts of the mesoderm along the rostrocauda l axis (reviewed in REF. 8). The skeletal muscles of the trunk and limbs are derived from cells of the segmented paraxial mesoderm (known as somites), which form on either side of the neural tube in vertebrate embryos. Somites further differentiate into a dorsal part, called the dermomyotome (which retains an epithelial structure) and a ventra l part, called the sclerotome (which breaks up into the mesenchyme that contributes to the axial skeleto n of the embryo). The first muscle cells are formed in the myotome, which is located directly underneath the dermomyotome and is formed by cells that separate by delamination from the dermomyotome (reviewed in REF. 9) (BOX 1).

In all the anatomical sites where skeletal muscle forms, determination and terminal differentiation of muscle cells are governed by a network of four MRFs: myogenic

Figure 1 | Striated muscle structure. The contractile machinery of skeletal muscle syncytial myotubes (left) and single cardiomyocytes (right) is formed from long arrays of sarcomere units, which are joined into myofibrils. The sarcomere (bottom) is constructed from interdigitating, antiparallel filaments of actin and myosin, the elastic titin filaments and the crosslinker proteins for actin — α-actinin, myosin and myomesin. Sarcomeres contain many other accessory components, including proteins involved in transcriptional regulation and turnover control. The transcription factor CLOCK, the transcriptional cofactors muscle LIM protein (MLP), muscle ankyrin-repeat proteins (MARPs) and LIM domain-binding protein 3 (LDB3) are found at the Z-disk and/or the I-band. Multifunctional components of the protein turnover machinery include sequestosome 1 (SQSTM1), NBR1 and the muscle-upregulated RING finger proteins (MURFs). MYOZs, myozenins.

Box 1 | Skeletal muscle cell specification and differentiation during embryogenesis

Formation of trunk musclesThe morphogenetic events that are involved in the formation of the myotome have been extensively studied. Cell tracing methods have shown that a first wave of myogenic factor 5 (MYF5)-expressing muscle progenitor cells, which progressively withdraw from the cell cycle, delaminate and migrate towards the rostral somite, making up the medial region of epithelial somites. Myofibres differentiate in rostrocaudal (head to tail) and mediolateral (centre outwards) directions (reviewed in REF. 133). This initial wave of myogenesis is followed by a second wave of myoblasts. These cells come from all four lips of the dermomyotome, which is the epithelial cell layer that comprises all of the mesodermal somites (but not the sclerotome) and that gives rise to the axial skeleton. The dorso–medial lip is initially the sole contributor to myogenesis, followed by the posterior lip, until eventually the anterior and lateral borders begin to contribute to myogenesis134. It should be emphasized that the myotome that is formed by the epithelial borders of the dermomyotome is composed of postmitotic cells, which do not contribute to further muscle growth. Only the continuous addition of proliferating muscle progenitor cells allows the primary muscle compartment to expand.

Formation of head musclesThe formation of head muscles differs significantly from the formation of their counterparts in the trunk and limbs. The head musculature originates from the cranial paraxial mesoderm (CPM), which is located anterior to the somites; additional input to head muscles comes from the lateral splanchnic mesoderm (SpM). Cells from the CPM migrate to the proximal part of the central region of the branchial arches, and SpM-derived cells contribute to the distal region. Later in development, CPM-derived myogenic cells of the first branchial arch will form the masseter muscle, whereas SpM- derived myogenic cells generate the lower jaw muscles (reviewed in REF. 135). Formation of head muscles is also strongly influenced by cranial neural crest cells136. Deletion of neural crest cells leads to severe reduction of jaw muscles after the onset of muscle specification and differentiation135,137, and this is probably due to interactions between muscles and neural crest-derived tendons138.

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Neural crestA group of embryonic cells that separate from the embryonic neural plate and migrate, giving rise to the spinal and autonomic ganglia, peripheral glia, chromaffin cells, melanocytes and some haematopoietic cells.

Hypaxial musclesMuscles that usually lie ventral to the vertebrae and are innervated by the ventral ramus of the spinal nerves.

Epaxial muscles Muscles that usually lie dorsal to the vertebrae (in fish and amphibiae they lie dorsal to the septum). They are innervated by the dorsal ramus of the spinal nerves.

factor 5 (MYF5), musclespecific regulatory factor 4 (MRF4; also known as MYF6), myoblast determination protein (MYOD) and myogenin. MRFs are transcription factors that activate many downstream genes to initiate muscle cell differentiation. MYOD and MYF5 are muscle specific transcription factors and constitute a crossregulator y transcriptional network that is at the core of muscle cell determination and differentiation (FIG. 2); disruption of this network completely abrogates skeletal muscle formation. MYF5 and MYOD are generally thought to act as determination genes, whereas myogenin is essential for the terminal differentiation of committed myoblasts (reviewed in REF. 10). MRF4 seems to have a dual role: it is thought to be a differentiation gene acting in postmitotic maturating cells, but it is also expressed by undifferentiated proliferating cells in which it might act as a determination gene11. The upstream signals (transcription factors and extracellular signals (BOX 2)) that activate MRFs differ significantly at various anatomical locations (FIG. 2), although some molecular cues are shared.

Regulation of trunk skeletal muscle formation. Numerous transcription factors, such as paired box, homeobox and Tbox proteins, have been identified as binding upstream of MRF genes. None of these factors is exclusively expressed in the muscle progenitor cells that give rise to differentiated muscl e cells, which suggests that these trans cription factor s prepare the stage for additional actors (such as MRFs) to initiate myogenesis. Alternatively, they may act together with other transcription factors to allow the activation of MRFs. In both cases, instructive or permissive inductional cues are necessary to achieve stable selfsustained activation of MRFs in muscle progenitor cells and muscle cell formation.

The differentiation of hypaxial muscles and epaxial muscles seems to differ with regard to the transcriptional network that activates MRFs. Indeed, the target genes of paired box protein 3 (PAX3), which is upstream of the MRFs, differ in hypaxial and epaxial muscles.

In mouse epaxial muscles, PAX3 seems to activate MYF5 by controlling the expression of Dmrt2, which in turn activates the epaxial enhancer of Myf5 in somites12. Furthermore, analysis of mice depleted of PAX3 and MYF5 showed almost complete loss of trunk muscles and a loss of MyoD expression; this indicates that MyoD expression depends on either PAX3 or MYF5 (REF. 13). At present, it is not known precisely how PAX3 activates MyoD expression, although experiments in presomitic explant cultures suggest that WNT signalling regulates MyoD expression in a PAX3dependent manner14. Taken together, current evidence suggests that PAX3 mediates the activation of MYOD and MYF5 in a tight interplay with muscleinducing signals. Early experiments showed that ectopic expression of PAX3 is sufficient to induce the expression of MYOD, MYF5 and myogenin in the absence of inducing tissues (that is, neural tube and paraxial surface epithelium) in both the paraxial and latera l plate mesoderm of chicken embryo, and in the neural tube15,16; however, directed expression of PAX3 in vivo did not induce a stable myogenic fate. Similarly, expression of LBX1, a PAX3 target gene, resulted in the

activation of myogenesis in vitro and transient induction of myogenic determination genes in vivo in chicken embryos13,16. Interestingly, mutants of the Pax3 paralogue Pax7 do not show overt muscle defects during mouse development17, and the combined loss of PAX3 and PAX7 yields a phenotype that is similar to PAX3 mutants, with the initial formation of skeletal muscle occurring in the myotome until E10.5. This indicates that the initial formation of skeletal muscle cells in the myotome occurring up to E10.5 is not directly under the control of PAX3 and PAX7. As compound PAX3 and PAX7 mutants show a severe disruption of muscle development at later stages, with very few differentiated muscle cells18, it is possible

Figure 2 | Different ways to activate the genetic programme of muscle differentiation. All muscle cells express a core set of myogenic factors (for example, myogenic factor 5 (MYF5), muscle-specific regulatory factor 4 (MRF4), myoblast determination protein (MYOD) and myogenin), which are required for myogenic differentiation. Other transcription factors reflect lineage-specific differences and are necessary for the activation of myogenic factors and/or proliferation and survival of muscle progenitor cells. Muscle groups from the head, including extraocular, tongue and laryngeal muscles, and branchial arches, are derived from occipital somites, cranial paraxial mesoderm, splanchnic mesoderm and prechordal mesoderm. In these cells, pituitary homeobox 2 (PITX2) predominantly controls the myogenic hierarchy, leading to the activation of MYF5 and MYOD and eventually terminal differentiation induced by myogenin. By contrast, muscles from the limbs and the trunk are all derived from trunk somites. In limb muscles, sine oculis homeobox homologue (SIX) and eyes absent (EYA) proteins regulate paired box protein 3 (PAX3), which in turn controls the proliferative myogenic cell pool, the differentiation of which is induced by a cascade involving MYF5, MRF4, MYOD and myogenin. In trunk muscles, MYF5 or MRF4 can show parallel activation of MYOD and myogenin, whereas PAX3 acts upstream of MYOD. Solid lines represent direct control and dashed lines represent indirect control. TBX1, T-box transcription factor. Figure modified, with permission, from REF. 150 © (2009) Elsevier.

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that PAX3 and/or PAX7 are responsible for the enlargement of muscle precursor cell populations. This would increase the bias towards myogenic differentiation and thus enable myogenic cells to respond to environmental cues16. This view is further supported by the observation that PAX3 directly regulates components of the fibroblast growth factor (FGF) signalling pathway, which have a role in the expansion of the myogenic progenitor cell pool19.

In hypaxial muscle cells, PAX3 seems to directly activate MYF5 but not MYOD. Similarly, a study in muscle stem cellderived myoblasts showed that expression of MYF5 is regulated by PAX7 (REF. 20). However, despite having a role in the activation of MYF5 and the morpho genesis of the hypaxial dermomyotome, PAX3 does not seem to be essential for the development of hypaxial muscles, as the lateral halves of somites from E9.25 PAX3‑mutant embryos transplanted into the limb bud of chicken host embryos differentiated normally21. Furthermore, recent studies using a PAX3–engrailed fusion protein, which acts as a transcriptional repressor, suggested that PAX3 directly regulates Myf5 in the hypaxial somite and its limb muscle derivatives. In these mice, the expression of Myf5, but not of MyoD, was compromised in hypaxial muscles. The authors postulated that the regulation of Myf5 by PAX3 in hypaxial muscles was achieved by a PAX3 consensus site located in a 145 bp regulatory element –57.5 kb from Myf5. Mutation of the PAX3binding site in the context of the 145 bp element abolished all expression in transgenic mouse embryos22. Similarly, sequences have been described that contain a predicted homeobox adjacent to

a putative paired domainbinding site within the –58 to –56 kb distal Myf5 enhancer, which directs Myf5 expression in myogenic progenitor cells in limbs. Both PAX3 and mesenchyme homeobox gene 2 (MEOX2) transcription factors could bind these consensus sites in vitro, so they are potential regulators23. However, there is no valid genetic evidence showing that MEOX2 has a significant effect on Myf5 expression in the limb, despite some early reports claiming a reduced expression of MYF5 in MEOX2mutant limbs. It seems more likely that sine oculis homeobox homologue 1 (SIX1) and SIX4 (see below) occupy the homeoboxbinding site in the 145 bp element. In fact, it was recently shown that SIX1 and SIX4 regulate the transcription of MYF5 in the limb together with PAX3, by binding to the 145 bp element –57.5 kb from Myf5. An additional regulatory element seems to contribute to the expression of Myf5 in the hindlimbs, and this element might be responsible for some of the remaining expressio n of Myf5 in the hindlimb buds of SIX1 mutants24.

SIX proteins are also involved in the regulation of myogenesis in the limb. SIX proteins act, at least in part, upstream of myogenic regulatory factors and PAX proteins (FIG. 2). The human and mouse genome contains 6 SIX genes (SIX1–SIX6), 3 of which (SIX1, SIX4 and SIX5) are expressed from E8 in overlapping expression patterns in somites, limb buds, dorsal root ganglia and branchial arches. SIX proteins form complexes with their transcriptional coactivators, eyes absent (EYA) proteins, to stimulate transcription synergistically25. SIX1 mutants die at birth and show selective muscle hypoplasia in the diaphragm, forelimb, distal ventral hindlimb and abdomen26. The muscle phenotype is aggravated in compound mutants of SIX1 and SIX4, which do not have myogenic progenitor cells in their limb buds, resulting in legs with no muscles27. Furthermore, neither double SIX1 and SIX4 mutants nor double EYA1 and EYA mutants express Pax3 in the hypaxial dermomyotome, indicating that Six and Eya lie upstream of Pax3 in the genetic hierarchy of hypaxial myogenesis. SIX1 and SIX4 double mutants also show a reduced expression of MyoD, myogenin and other myotomal markers, although the early activation of MYF5 in the epaxial somite is un affected27. However, later in development the expression of SIX genes seem to depend on MRFs, indicating a dual role for SIX proteins both upstream and downstream of MRFs during myogenesis28. The role of SIX proteins in the differentiation of muscle fibres intro slowtwitch or fasttwitch is discussed below.

MRFs are also assisted by many other factors such as PBX and MEIS proteins29, which function as heterodimers and act as cofactors with basic helix–loop–helix (bHLH) proteins such as MRFs and various homeobox transcription factors. PBX and MEIS proteins participate in the feedforward mechanisms that enable MRFs to activate ‘early’ genes of the muscle differentiation programme immediately, whereas genes activated later on in the differentiation programme need both MRFs and the additional participation of one of the earlier MRF targets29 (reviewed in REF.10). Myocyte enhancer factor 2 (MEF2) proteins are also important members of this regulatory circuit. MEF2 proteins interact directly with MYOD

Box 2 | Extracellular signals directing muscle development

The activation of the network of transcription factors that controls skeletal muscle development depends on paracrine factors that are released by adjacent tissues, such as the neural tube, notochord, surface ectoderm and lateral mesoderm. Several secreted factors have been identified that determine the spatial and temporal onset of myogenesis. Surprisingly, no consensus has been reached as to whether these molecules instruct naive cells (instructive induction), amplify a pool of committed progenitors and/ or enable a default differentiation pathway (permissive induction) or primarily prevent programmed cell death of muscle progenitor cells (reviewed in REF. 139). From numerous studies, it is clear that sonic hedgehog (SHH) and WNT signalling have pivotal roles in the induction of myogenesis. Moreover, other signalling molecules, such as Noggin and bone morphogenetic proteins (BMPs) — which inactivate and activate receptors of the transforming growth factor-β (TGFβ) superfamily, respectively — play an important part in orchestrating the activation of myogenesis140.

A study in mice showed that the canonical β-catenin-mediated WNT signalling pathway acts co-operatively with SHH, its receptor Patched and its downstream target, GLI (a zinc-finger transcription factor), to regulate the expression of myogenic factor 5 (Myf5). According to this model, the SHH–Patched–GLI pathway is not sufficient to induce myogenesis if the local concentration of β-catenin does not suffice to support full transcriptional activity of lymphoid enhancer-bracking factor 1 LEF1 or other transcription factors, which facilitate the localization of β-catenin to specific cis-regulatory elements. The transcription of the first myogenic transcription factor, MYF5, is activated in the epaxial domain only when both SHH and canonical WNT signalling pathways are activated at the onset of somitogenesis141. Despite compelling evidence for the decisive role of canonical β-catenin-mediated WNT signalling in the induction of myogenesis, other authors reported that adenylyl cyclase signalling through protein kinase A (PKA) and its target transcription factor, cAMP-responsive element-binding protein (CREB), are required for WNT-directed myogenic gene expression142. It is possible, however, that the WNT signalling pathway that involves PKA and CREB acts in parallel to canonical β-catenin-mediated WNT signalling and becomes limiting only under specific conditions.

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Masseter musclesA specific subset of branchiomeric muscles that are derived from the first branchial arch and are involved in mastication.

Pharyngeal muscleA subgroup of head muscles acting on the pharynx to control swallowing.

in vitro and synergistically activate reporter genes that contain Eboxes and MEF2binding sites30. Eboxes and MEF2binding sites are often located close to one another within the promoters and enhancers of musclespecific genes, suggesting a model in which MRFs and MEF2 bind DNA and activate transcription in a cooperative manner. Three of the four MEF2 proteins (MEF2A, MEF2C and MEF2D) are expressed in skeletal muscle, and expression and splicing of these isoforms is altered in response to MYOD, which further supports a role for MYOD in the feedforward mechanism that is driven by MRFs29.

The transcriptional regulation of head muscles. Recent evidence suggests that head muscle development follows a distinct programme that does not require the action of PAX3 and PAX7. Initial specification of mouse massete r muscles depends on the bHLH genes MyoR and capsulin31. Additional players are pituitary homeobox 2 (Pitx2) and Tbox transcription factor 1 (Tbx1), which collaborate with the core myogenic programme to gener ate head muscles. Both Pitx2 and Tbx1 are expressed widely in the developing mouse embryo and play an important part in the formation of head muscles (FIG. 2). Indeed, PITX2 mutants do not properly develop the muscles that are derived from the first branchial arch32 and they lack extraocular muscles33. TBX1 mutants also suffer from impaired myogenesis in the first branchial arch and lack the muscles that normally originate from other arches owing to severe malformations of these structures34,35. It has recently been shown that TBX1 and MYF5 act syner gistically to govern myogenesis in pharyngeal muscle progenitor s, thereby acting as a complementary pathway to that involving PAX3 and MYF5 in the body36. It is possible that PITX2 and TBX1 regulate the quiescence and selfrenewal of muscle progenitors in the head similarl y to PAX3 and PAX7 in trunk skeletal muscles.

Post-transcriptional control by microRNAsThe human genome contains thousands of noncoding RNAs, the beststudied class of which are microRNAs (miRNAs) (reviewed in REF. 37), which regulate gene expression at the transcriptional and posttranscriptiona l levels. miRNAs suppress gene expression through their complementarity to the sequence of one or more mRNAs, usually at a site in the 3′ untranslated region. The formation of an miRNA–target complex results either in inhibition of protein translation or in degradation of the mRNA transcript through a process similar to RNA interference38. There is no doubt that the formation, maintenance, and physiological and pathophysiological responses of skeletal muscles, with all their complex regulatory circuits, are subject to regulation by noncoding RNAs. In fact, the increase of complexity provided by the extent of genomic noncoding sequences provides a satisfying explanation for the intricate layers of regulation found in skeletal muscle cells.

Many miRNAs are expressed in skeletal and cardiac muscle. Some of them are found specifically in skeletal and/or cardiac muscle, or at least are highly enriched in these tissues, suggesting specific roles in myogenesis39. The expression of the musclespecific miRNAs

miR1, miR133, miR206 and miR208 seems to be under the control of a core muscle transcriptional network, which involves the pleiotropic serum response factor (SRF), MYOD, and the bHLH transcription factor Twist in co operation with MEF2 (REFS 40–43). Chromatin immuno precipitation followed by microarray (ChIP–chip) analysis indicated that MYOD and myogenin bind sequences upstream of miR1 and miR133 (REF. 41). Furthermore, MEF2 has a crucial role in the control of an intragenic enhancer located in the miR‑1–2 locus (which contains miR1 and miR2)44. Recently, analysis of mice deficient in MYF5 and MYOD revealed a surprisingly specific requirement of MYF5 for miR1 and miR206 expression. At early developmental stages, the expression of both miR1 and miR206 was almost entirely absent in the somites of MYF5mutant embryos, whereas mouse embryos lacking MYOD showed an apparently normal expression of both miRNAs45. Whereas miR133 and miR206 are expressed as independent transcriptional units, miR208 is encoded by an intron of its host gene, αmyosin heavy chain (αMHC)46. Both miR208 and

αMHC are heart specific and concurrently expressed during development, suggesting that their expression is controlled by a common regulatory element46.

Some experiments suggest that miRNAs act as modulators of myogenic differentiation, in particular because some miRNAs, such as miR1 and miR133 (FIG. 3a), are absent from undifferentiated myoblasts and are strongly upregulated upon differentiation47. An increase of miR1 expression in tissue culture accelerates myoblast differentiation by downregulating histone deacetylase 4 (HDAC4), a repressor of muscle differentiation, and knockdown of miR1 impedes myogenic differentiation40. Although a similar role for miR1 in facilitating differentiation has been shown in the developing mouse heart, where a tissuespecific overexpression of miR1 induces premature differentiation of cardiomyocytes43, conclusive evidence for an indispensable role of miR1 in the regulation of myogenic differentiation is still missing. Recent studies, however, suggest that reexpression of miR206 in human rhabdomyosarcoma cells promotes myogenic differentiation and blocks tumour growth in xenografted mice by switching the global mRNA expression profile to one that resembles mature muscle48. This finding supports an important modulatory role for miR206 in the control of myogenesis (FIG. 3b), although miR206knockout mice show no major arrest of myogeni c differentiation49.

The modulatory role of miRNAs on muscle differentiation is underscored by several observations on the role of miR1, miR27, miR206 and miR486 in the regulation of PAX3 and PAX7 (FIG. 3c). miR1, miR27 and miR206 were found to inhibit PAX3 and to thereby release the inhibitory effect of PAX3 on terminal muscle differentiation50,51; an analogous role was described for the regu lation of PAX7, which is repressed by miR–1 and miR206 (REFS 52,53). Therefore, the feedback loop amplifying term inal MYODinduced muscle differentiation involves the transcriptional switch towards late factors such as MEF2C, but also the epigenetic downregulation of muscle stem cell factors such as PAX3 and PAX7.

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AntagomirsSynthetic or genetically engineered oligonucleotides used to silence endogenous microRNAs.

Soleus muscleA muscle in the calf of the leg that flexes the ankle; in rodents it is predominantly composed of slow-twitch fibres.

The regulation of myogenesis by miR1 and miR133 is unclear, as the human and mouse genomes each contain two bicistronic loci, which both express miR1 and miR133. It has been reported that the selective disruption of the miR‑1–2 locus in mice results in a severe cardiac phenotype, including 50% embryonic lethality with frequent ventricular septum defects54, which is surprising given that the sequences of miR1 and miR133 are identical and that both loci are expressed in skeletal and cardiac muscle. Combined knockouts of the miR12–miR133a1 and miR11–miR133a2 clusters are still pending and will provide more definitive answers. Nevertheless, it seems likely that miR1 and miR133 are required to delineate the phenotype of differentiated muscl e cells, which is consistent with the conclusions drawn from the miR1 overexpression experiment55.

Currently views on miRNA functions remain disparate and are complicated by some underlying technical problems. For example, massive overexpression might not necessarily reflect a physiological function of an individual miRNA. Instead, the nonphysiological presence of large amounts of an exogenous miRNA might cause numerous offtarget effects, as also demonstrated for small interfering RNAs, and could lead to wrong conclusions56. Similarly, knockdown approaches using antagomir s, although highly appealing because of their relative ease of use and wide applicability, might hold some pitfalls that need to be explored57.

Postnatal control of muscle phenotypeSkeletal muscle has a remarkable ability to rapidly adjust to changes in physiological requirements, by changes in excitability, contractile characteristics, metabolism, and fibre size and mass (see below). These sweeping changes involve the concerted control of transcription regulation, protein synthesis, protein degradation and metabolic flow, the details of which have only recently emerged. During postnatal adaptation of muscle, or muscle remodelling, aspects of early developmental programmes can be reactivated and can cooperate with specific factors to determine fibretype characteristics58; MEF2, nuclear factor of activated T cells (NFAT), myogenin, JUNB and SRF have prominent roles in these processes59–64. Posttranslational modifiers of histones (histone acetylases and HDACs)64,65 and of transcription factors (protein kinases and phosphatases, and ubiquitin and SUMO transferases) cooperate in the complex interactions between chromatin structure and transcriptional machinery. Many of these responses are modulated by muscle activity, either by sensing neuronal activity though intra cellular Ca2+ levels or directly by mechanical strain. In addition, various hormonal and cytokine signalling mechanisms feed into the same pathways.

The adult skeletal musculature contains highly differentiated fibre types: one slowtwitch, oxidative fibre type (type I) and three fasttwitch, glycolytic fibre types (type 2)4. Fasttwitch fibre development follows defined patterns, involving control by SIX1 and SIX4 (see above)66, which also play an important part postnatally in the adaptation to the fast muscle phenotype25. By contrast, slowtwitch fibres are determined by the transcriptional repressor BLIMP1 (REF. 67), which suppresses the transcription factor SOX6 (REFS 68,69). The distinct origin of fasttwitch and slowtwitch fibres is also highlighted by the observation that the developmental lineages and the regeneration of slow and fasttwitch muscles stems from the recruitment of intrinsically different myogenic precursors70 or satellite cells71. Postnatally, SIX1 co operates with EYA1 in fibre type differentiation. Indeed, forced coexpression of SIX1 and EYA1 in the slowtwitch fibres of soleus muscle induced a transition to a fasttwitch fibre type, with the replacement of the slower myosin heavy chain isoforms I and IIA by the faster IIB and/or IIX, accompanied by the activation of other fasttwitch fibrespecific genes and a switch to glycolyti c metabolism.

For physiological, activitydependent adaptation of existing fibres, the type of neuronal activity acting on a fibre is probably the most important factor determining fibre type. The firing pattern of neurons innervating fasttwitch and slowtwitch muscle fibres has markedly different frequencies and temporal patterns, leading to different membrane potentials and ultimately to net differences in intracellular Ca2+ levels. These changes can be sensed by the Ca2+activated Ser phosphatase calcineurin (also known as PP2B), which dephosphorylates and thereby activates NFAT. This can then act as a calcineurindependent sensor and can translate nerve activity in skeletal muscle into altered fibretypespecific gene expression programmes (FIG. 4). The nuclear version

Figure 3 | Regulation of major myogenic pathways by microRNAs. a | Regulation of myogenic differentiation by miR-1 and miR-133. miR-1 and miR-133 are downstream of the myogenic transcription factors myogenic factor 5 (MYF5) and myocyte enhancer factor 2C (MEF2C) and control the expression of differentiation genes: first, through the pleiotropic transcription factor serum response factor (SRF); second, by inhibiting the assembly of splicing-regulatory protein complexes by neural polypyrimidine tract-binding protein 2 (nPTB2), which controls the splicing of many mRNAs in muscles; and third, by inhibiting histone deacetylase 4 (HDAC4), which blocks myogenic differentiation. b | miR-206 is downstream of the myogenic master transcription factor myoblast determination protein (MYOD) and feeds back on MyoD expression by inhibiting follistatin and bone morphogenetic proteins (BMPs), ultimately determining the expression of differentiation markers such as utrophin. c | Regulation of proliferation and differentiation by the myoblast transcription factors paired box protein 3 (PAX3) and PAX7 is controlled upstream by miR-1, miR-27, miR-206 and miR-486, which repress PAX3 and PAX7 and thus promote the terminal differentiation of myoblasts downstream of MYOD.

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Tibialis anterior muscle A muscle in the front muscle compartment of the lower leg that helps to extend the ankle; in rodents it is predominantly composed of fast-twitch fibres.

MicrogravityGravity below the 1Gal experienced on Earth; for example, during space flight.

Ubiquitin–proteasome system A system of selective, ATP-dependent protein degradation, in which target proteins that have been conjugated by ubiquitin are degraded by the 26S proteasome. The ubiquitin conjugation step requires the activity of highly specific ubiquitin ligases.

AutophagyA catabolic process involving the engulfment of (usually damaged) organelles and long-lived proteins or protein aggregates by double-membrane vesicles (autophagosomes) that fuse with lysosomes to form autolysosomes, in which their contents are degraded by acidic lysosomal hydrolases.

of NFAT (NFATC1) cooperates with numerous musclespecific cofactors such as MYF5 (REF. 72) to modulate musclespecific gene expression59, thus controlling the expression of fast and slow myosin isoforms73. The activit ydependent nuclear import and export of NFATC1 is a rapid event, occurring within minutes of the appropriate slowtype stimulation pattern74. Its activity is counteracted by the calcineurin inhibitor CAIN (also known as CABIN1), which promotes nuclear export of NFATC1 both in soleus and stimulated tibialis anterior muscle.

Further regulation of calcineurin activity is mediated by a family of sarcomereassociated proteins known as myozenins (MOYZs; also known as FATZs and calsarcins), which are encoded by the three MYOZ genes. MYOZ2 is expressed in adult cardiac and slowtwitch skeletal muscle, whereas MYOZ1 is restricted to fasttwitch skeletal muscle. MYOZs interact with calcineurin and the PDZ domain and LIM domaincontaining protein LIM domainbinding protein 3 (LDB3; also known as ZASP, cypher and oracle) (reviewed in REF. 2). MYOZ2knockout mice show increased calcineurin signalling in striated muscles, suggesting that MYOZ2 acts as a brake on calcineurin activity75. As a result of the abnormal calcineurin activation, MYOZ2knockout mice have an excess of slowtwitch skeletal muscle fibres, which is consistent with the established roles of calcineurin and NFAT in activitydependent regulation of fibre types59. Similarly, MYOZ2knockout mice activate a cardiac hypertrophic gene programme in the absence of hypertrophy and show enhanced cardiac growth in response to pressure overload75.

Owing to the probable function of the sarcomere Zdisk (FIG. 1) as a sensor for muscle mechanical strain (reviewed in REF. 76), calcineurin activity might also be mechanically modulated by the MYOZs in the Zdisk. The interplay of transcriptional regulation by calcineurin, SIX1 and EYA, and MRFs, which seems possible from these observations, and the control of SIX1 activity in fibretype transition is currently unclear.

Postnatal control of muscle massThe adaptive changes of muscle in response to changes in activity not only determine the contractile phenotype but also importantly affect muscle protein turnover and thus muscle mass. Muscle mass increases by hypertrophy (increased cellular protein content) and is controlled by anabolic and catabolic mechanisms, which regulate the increased synthesis of muscle proteins or their degradation, respectively. The atrophic loss of muscle mass often responds to inverse stimuli and can be triggered by disuse, microgravity, catabolic steroids such as glucocorticoids, cytokines such as tumour necrosis factor (TNF), genetic factors, acidosis and catabolic nutritional states. Hypertrophy and atrophy are associated with changes in sarcomeric protein composition (BOX 3), metabolic enzymes and contractile phenotype, and atroph y especially can be regarded as an extreme endstage of activityregulated adaptation. In fact, disuse d muscle loss is associated with a shift towards a fasttwitch phenotype and the reactivation of developmental transcription

factors4. The coordinated changes of transcriptional and splice mechanisms, protein turnover and cell fate integrates signalling pathways from hormone and cytokine receptors, as well as the sarcomere itself.

Satellite cells play important parts in skeletal muscle repair7,77,78 and thus, in the longterm, homeostasis of muscl e tissue. They are also thought to be responsible for early postnatal muscle growth. By contrast, their role in eliciting hypertrophic growth is disputed, as hypertrophic growth in adults seems to occur without satellite cell activation79. Owing to the large number of recent studies on satellite cells, which would require a separate review, and their unclear role in regulation of postnatal muscle mass, we do not discuss satellite cells further.

Transcriptional control of muscle protein turnover. Recently, the expression of catabolic genes was found to be regulated by the transcription factor forkhead box O1 (FOXO1) and FOXO3A80,81. The activity of FOXO famil y proteins is predominantly regulated by phosphorylation: dephosphorylated FOXOs translocate to the nucleus and are transcriptionally active, whereas phosphorylated FOXOs are sequestered in the cytoplasm. Signalling through several receptor Tyr kinases activates the phospho inositide 3kinase (PI3K)–AKT pathway, a key regulator of muscle mass and metabolism that directly stimulates protein synthesis by activating mammalian target of rapamycin (mTOR) and its downstream targets82 (FIG. 4). Apart from its anabolic effects on protein synthesis and carbohydrate metabolism, AKTmediated phosphoryl ation also blocks FOXO1 and FOXO3A nuclear import and transcriptional activity.

Genes depending on FOXO proteins for their expression in muscle include a range of atrophyrelated genes known as atrogenes, such as atrogin 1 (also known as FBXO32 and MAFBX1) and the muscleupregulated RING finger (MURF) ubiquitin ligases82. The protein products of atrogenes target myofibrillar, metabolic and transcriptional proteins for degradation by both the ubiquiti n–proteasome system and the autophagy–lysosomal system83. FOXO3A, which seems to be the dominant FOXO protein in muscles, also controls the transcription of ubiquitous autophagyrelated genes (which can also be regarded as atrogenes), such as those genes encodin g microtubuleassociated protein 1 light chain 3 (LC3), sequestosome 1 (SQSTM1) and probably the related protein NBR1, and BNIP3 — BNIP3 seems to mediate the effect of FOXO3 on autophagy84,85. Therefore, repressing the transcription of atrogenes augments the AKTmediated increase of protein translation by shutting down protein breakdown, thus increasing muscle mass.

Apart from AKT signalling, many pathways with input from both catabolic and anabolic signals affect FOXOmediated transcription and therefore atrogene expression (FIG. 4). A direct amplification seems to be provided by atrogin 1, which triggers the degradation of calcineurin86, thus inhibiting the anabolic transcriptional signals mediated by the calcineurin–NFAT pathway (see above). Furthermore, although the transfer of Lys48linked polyubiquitin chains generally targets proteins for proteasomal degradation, atrogin 1 in cardiac muscle mediates

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α

β

βκ

Lys63linked polyubiquitylation of FOXO1 and FOXO3A, which leads to their activation independently of AKT and provides an atrogin 1dependen t positive feedback mechanism for atrophic gene expression87. However,

although these mechanisms have been linked mainly to muscle atrophy, it is now emerging that autophagy has an important role not only under the extreme conditions of atrophic muscle remodelling but also in muscle

Figure 4 | Control of postnatal muscle transcription and protein turnover. a | Protein synthesis is transcriptionally regulated by anabolic transcription factors (shown in green), including myogenic regulatory factors, transcription factors such as serum response factor (SRF), JUNB, CLOCK and nuclear factor of activated T cells (NFAT), and transcriptional cofactors and modifiers such as muscle LIM protein (MLP) and the muscle ankyrin repeat proteins (MARPs). Several of these are multi-compartment proteins shuttling to the nucleus from the sarcomere, where they mostly associate with the Z-disk or I-band and can thus respond to mechanical strain-induced conformational changes in the sarcomere. Protein degradation is controlled by a set of catabolic transcription factors (shown in magenta) that include forkhead box protein O1 (FOXO1), FOXO3A, myogenin (MYOG), KLF14, nuclear factor-κB (NF-κB), SMAD2 and SMAD3. FOXO proteins and NF-κB regulate the transcription of atrogenes (shown in orange): atrogin 1, muscle-upregulated RING finger (MURF) ubiquitin ligases, and components of the autophagy pathway such as microtubule-associated protein 1 light chain 3 (LC3), BNIP3, sequestosome 1 (SQSTM1) and possibly NBR1. In addition, the atrogenes MURF1, MURF2 and SQSTM1 translocate to the nucleus and repress SRF activity, presumably by MURF ubiquitin ligase activity. The anabolic regulation of protein synthesis by the phosphoinositide 3-kinase (PI3K)–AKT cascade represses the degradation signals mediated by FOXO proteins. The postnatal pro-atrophic action of myogenin is downstream and redundant with histone deacetylase 4 (HDAC4) and HDAC5. Several amplifying feedback mechanisms exist that either repress or augment anabolic or catabolic signals, including SRF-mediated expression of miR-486, which represses FOXO proteins in hypertrophy, and FOXO protein- mediated expression of miR-1, which represses insulin-like growth factor 1 (IGF1) in atrophy. Additional inputs exist through the activity of hormones and cytokines, especially anabolic signalling through IGF1, leptin and androgen receptor (AR), and catabolic signals through tumour necrosis factor receptor (TNFR), myostatin and glucocorticoid receptor (GR). b | Fibre-type remodelling depends largely on the sensing of Ca2+ levels by calcineurin and the resultant modulation of NFAT and myogenic factor 5 (MYF5) activity; this is antagonized by the myozenins (MYOZs), which are sarcomeric calcineurin inhibitors. c | Ca2+, the activator of muscle contraction, also indirectly regulates the mechanical feedback systems embedded in the sarcomere. Titin has emerged as a sarcomeric strain-sensor at the Z-disk, I-band and M-band that feeds into the activity and localization of transcription factors and atrogenes. Solid lines represent direct actions and dashed lines represent indirect actions. Bold dashed arrows represent receptor-linked signals. CBFβ, core-binding factor β; IKK, inhibitor of NF-κB kinase; MEF2C, myocyte enhancer factor 2C; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homologue.

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maintenance, by removing defective organelles and contributing to energy homeostasis88. The expression of some atrogenes is also regulated by nuclear factorκB (NFκB) (reviewed in REF. 89), thereby linking catabolic signalling and cytokine signalling (for example, through TNF) (FIG. 4). How FOXO proteins and NFκB signalling are coordinated is currently unclear — the deletion of the upstream activating kinase of NFκB, inhibitor of NFκΒ kinase B (IKKB), protects muscle from atroph y90, whereas constitutively active FOXO protein s are sufficien t to induce atrophy80,81.

Myogenin, which as discussed above is a key regu lator of muscle development, also has a key role in muscle atrophy by directly regulating the transcription of MURF1 (also known as TRIM63) and atrogin 1 (REFS 64,91). Myogenin is rapidly upregulated in denervated muscle, and its trans criptional actions on atrogenes are apparently controlled by HDAC4 and HDAC5 (REF. 64). This provides a bridge between early muscle development and postnatal adaptation of muscle. The catabolic programmes controlled by FOXO proteins, myogenin and NFκB are in balance with a network of musclespecific and pleiotropic transcription factors that cooperate in only partly understood ways to control the maintenance

of the differentiated pheno typ e (FIG. 4). Of the pleiotropic factors, SRF is required for postnatal hypertrophy, and JUNB promotes muscle growth partly by directly interacting with and repressing FOXO proteins 63. Given the extensive involvement of miRNAs in feedback regulation during development, it is not surprising that miRNAs also have roles in muscle maintenance and remodellin g. In fact, miR486, which induces myoblast differentiation by inhibiting PAX7 (see above), promotes PI3K–AKT signalling by downregulating phosphatase and tensin homologue (PTEN; which inhibits PI3K) and directly inhibits FOXO1A itself 92. miR1 also amplifies the atrophic response; it is trans criptionally regu lated by FOXO proteins and suppresses insulin growth factor 1 (IGF1) expression, thus inhibitin g AKT signalling93 (FIG. 4). These pathways are extensively modulated by mechanical stress and cytokine and hormona l signals (see below).

Mechanical control of protein turnover. Slow and fasttwitch fibres show distinct mechanical behaviour, but the mechanisms regulating their differentiation and adaption are also partly sensitive to mechanical stress. The adaptive changes of muscle to changes in mechanical load and activity not only affect the contractile phenotype but also have an impact on muscle mass.

Recent progress shows that hypertrophic and atrophic signalling pathways communicate with several ‘hubs’ within the sarcomere. Mechanical forces seem to have important roles in modulating the conformation and thus the activity of protein complexes94,95. Several transcription factors and transcriptional modifiers communicate with mechanosensors embedded in the sarcomere. The Zdisk coordinates several direct links to mechanically modulated transcriptional regulation. MOYZ2, a negative regulator of calcineurin activity, seems to be mechanically sensitive75. The transcriptional coregulator muscle LIM protein (MLP; also known as CRP3) weakly associates with the sarcomeric cytoskeleton, but translocates to the nucleus in response to mechanical strain, suggesting that it acts as a transducer of mechanical strain, although whether it is a direct mechanosensor is still controversial96–98. CLOCK, another transcription factor that is also located at the Zdisk, has also been implicated in the mechanically modulated interplay of circadian rhythm, mechanical activity and energy demand regulation99 (FIG. 4). Similarly to MLP, CLOCK seems to shuttle between Zdisks and the nucleus in response to mechanical strain and to contribute to MYODdependent gene expression in the daily cycling maintenance of adult skeletal muscle100. This activity may be important to coordinate the actual disuse atrophy programmes with circadian rhythms, to prevent muscle atrophy initiation during sleep. The recent identification of core binding factorβ (CBFβ), a key element of JUNBmediated gene expression, at the Zdisk101 emphasizes the fact that links between cytoskeletal mechanical stress and anabolic gene expression seem to be a prominent feature in postnatal muscle growth and maintenance control. Thus, several mechanically responsive direct transcriptional links exist between the sarcomeric Zdisk and the nucleus.

Box 3 | Transcriptional control of sarcomere assembly

The concerted interplay of transcription factors and the modulation of their action on the transcript level by microRNAs orchestrate the final functional differentiation of muscle cells: the formation of contractile structures in sarcomeres and myofibrils (FIG. 1). The assembly (and disassembly) of these multiprotein complexes (sarcomere assembly or sarcomerogenesis) follows ordered pathways143, regulated on the transcriptional, translational and post-translational levels. Furthermore, myofibril assembly involves the participation of transient scaffolds and adaptors, notably the microtubule network, which seems to be necessary both for the directional dispersion of protein intermediates and for that of mRNAs (reviewed in REF. 1). The highly ordered assembly of sarcomeres also requires the giant molecular ruler protein titin, which has several functions: it forms a giant scaffold that interacts with almost all other sarcomeric proteins; it acts as an elastic spring; and it is a signal transduction protein acting through its carboxy-terminal protein kinase domain94,144. Therefore, it is unsurprising that titin depletion or truncation disrupts sarcomere assembly (reviewed in REF. 145).

The appearance of sarcomeric proteins, notably the contractile proteins actin and myosin and the structural proteins of the Z-disk and M-line (myomesin and α-actinin, respectively) follows a strictly ordered programme of sequential transcription, translation and incorporation into nascent cytoskeletal structures, with many components only transiently associating with intermediates of the assembly process. The actin filament system and actin-associated cytoskeletal proteins — such as α-actinin and the Z-disk portion of titin — assemble first, followed by the formation of an M-line scaffold of titin and myomesin and the integration of myosin filaments (reviewed in REF. 1). This ordered sequence of the appearance and incorporation of sarcomeric proteins seems to be due to both transcriptional and translational mechanisms (which regulate the sequential appearance of sarcomeric proteins) as well as post-translational control mechanisms that coordinate their ordered incorporation into assembling sarcomeres. Recently, it emerged that the expression of genes for different parts of the sarcomere is regulated by different transcription factors.

Although early myogenic differentiation requires, among other factors, myocyte enhancer factor 2A (MEF2A), the expression of myosin filament and M-line proteins is under the control of the late factor MEF2C146,147. The actin filament proteins are regulated independently and require serum response factor (SRF), which is necessary for de novo sarcomere assembly in the embryo because it mediates the transcription of other of key sarcomeric proteins such as troponin C148. SRF also has a pivotal role in postnatal hypertrophic growth of differentiated cardiac and skeletal muscle61,62,149.

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CachexiaA syndrome of muscle loss that is usually caused by increased catabolic metabolism.

E3 ubiquitin ligases A group of proteins that mediate the transfer of ubiquitin, often by linking the catalytic activity of an E2 transferase to recognition and binding of the specific substrates.

Mechanosensitive signal complexes are also found at the Iband (FIG 1). Ankyrinrepeat domain 2 (ANKRD2), cardiac ankyrin repeat protein (CARP) and diabetesassociated ankyrin repeat protein (DARP) — collectively known as the muscle ankyrin repeat proteins (MARPs) — can form a complex with the Iband proteins titin, myopalladin and calpain 3 (REF. 102). This complex is sensitive to stretch and muscle injury and links to the transcriptional pathways that control cell survival and muscle gene expression103,104 (FIG. 4). Owing to the very elastic nature of the Iband region of titin, it is perhaps not surprising to find mechanically modulated transcriptional links in this region, which may predominantl y sense the passive strain on the sarcomere.

At the Mband, the mechanically modulated kinase domain of titin interacts with a complex of the protein products of the atrogenes NBR1, SQSTM1 and the MURFs105,106. This complex dissociates under mechanica l arrest, and MURF1 and MURF2 (MURF3 has not yet been analysed) translocate to the cytoplasm and the nucleus105,107. SQSTM1 was also recently shown to translocate together with MURF1 and MURF2 to the nucleus in mechanically arrested skeletal muscle s107. One of the probable nuclear targets of MURFs is SRF105,108,109, suggesting that the MURFinduced nuclear export and transcriptional repression of SRF may contribute to amplifying the transcriptional atrophy programme105,107,110; for example, by suppressing SRFdependent miR486 expression (FIG. 4). The multiple sarcomeric and nuclear localizations of MURFs suggest their intracellular localization may also be important in coordinating their diverse functions in protein turnover regulation. Titin might act as an activitydependent brake on protein degradation by suppressing the expression or stability of atrogenes (FIG. 4). Both NBR1 and SQSTM1 promote the autophagy of poly ubiquitylated proteins by interacting with LC3 (REFS 111–114), but the substrates they recruit for degradation remain to be identified. In addition, these proteins interact with several protein kinases apart from titin and seem to have major signalling functions115,116. MURF1 also seems to be required for TNFinduced reduction in skeletal muscle force development117, suggesting that MURF signalling might be involved in the NFκBmediated atrophy response89 that is a hallmark of cachexia118. Direct links to hormone and cytokine receptors, such as those between the atrogene SQSTM1 and TNF receptor signalling, suggest a complex interplay of mechanical and cell surface receptorlinked signalling116. Thus, the differentiated sarcomere serves as a complex feedback device for the adaptive remodelling of the contractile machinery and its supporting energy metabolism in response to mechanical load, and several mechanically active regions feed back to distinct transcriptional programmes. It seems likely that further such mechano–transcriptional links will emerge.

Hormonal control of muscle growth. Muscle mass can also be regulated by hormones. One of the most prominent known muscle growth factors is IGF1, which is secreted by myocytes in an autocrine manner in response to mechanical strain as a musclespecific splice

variant119,120, or by the liver. Signalling through IGF1 receptors activates the PI3K–AKT signalling pathway (FIG. 4) and thus represses FOXO protein activity, promoting muscle growth. The activity of IGF1 on the AKT pathway is antagonized by myostatin (also known as GDF8) (reviewed in REF. 121) primarily in differentiated fibres122–124. The importance of myostatin as a negative regulator of muscle mass is highlighted by its increased serum levels in patients with heart failure, which leads to skeletal muscle cachexia that is completely blocked by genetic deletion of the gene encoding myostatin in heart tissue125,126. The myostatinactivated transcription factors SMAD2 and SMAD3 relay downstream myostatin signalling into an atrophy programme that also depends on FOXO proteins 123 and that is amplified by the inhibition of AKT and mTOR signalling122. These observations suggest that the myostatin and AKT pathways crosstalk at the cell signalling and transcriptional levels (FIG. 4). Intriguingly, FOXO proteindependent atrophy through SMAD2 and SMAD3 is independent of MURF1 and atrogin 1, but also leads to a decrease in the levels of these atrogenes122,123. Whether atrophy is then promoted by the activation of other E3 ubiquitin ligases or by the repression of prohypertrophic pathway s remains unclear.

Another hormone that affects muscle mass is leptin, a major regulator of energy intake and expenditure. Leptin was shown to positively regulate muscle mass by suppressing the activity of FOXO3A, further demonstrating how muscle and fat tissue metabolism are interlinked127 (FIG. 4).

A further hormonal input feeding into the AKT–FOXO protein nexus occurs by signalling through the cytoplasmic steroid receptors, the anabolic androgen receptor and the catabolic glucocorticoid receptor. The muscle anabolic effects of androgens have long been known; however, it has only recently been shown, using musclespecific androgen receptorknockout mice, that the myocytic androgen receptor is required for the production of the androgeninduced IGF1 isoform IGF1EA128 and can thus regulate autocrine or paracrine activation of the muscle AKT signalling pathway (FIG. 4). Conversely, the glucocorticoid receptor acts upstream of FOXO proteins and is itself regulated in a feedback loop by the IGF1–AKTactivated mTOR129, thus providing a crosstalk loop between anabolic and catabolic hormonal signals. This feedback loop also involves the transcription factor KLF15, which is implicated in several skeletal muscle metabolic processes. Exploiting such crossconnection s to suppress the unwanted atrophic effects of corticoid treatment might be an interesting practical application of this observation.

Conclusions and perspectivesThe versatility and plasticity of striated muscles is due to finely tuned networks of transcription factors and their regulation by extracellular and intracellular cytoskeletal signals. The embryonic origin and the functional remodelling of slowtwitch oxidative or fasttwitch glyco lytic muscle fibres has become mechanistically clear. Similarly, the control of muscle mass in response

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SarcopeniaThe degenerative loss of skeletal muscle mass and strength associated with ageing and pathological processes.

to activity has become clearer by the identification of the major pathways controlling atrogene expression and the cytokine and mechanical stimuli that modulate their activity on the gene and protein levels. Model organisms from nematodes to vertebrates have proved invaluable in outlining these molecular mechanisms. It will now be important to identify how the mechanisms specifying embryonic fibre fates are postnatally modulated by activityregulate d pathways. The redeployment of embryonic factors in postnatal muscle remodelling is an exciting recent development, both for our mechanistic understanding of postnatal adaptation and for the identification of pathways amenable to pharmacological intervention. It is still an open question how muscl e stem cells are specified, and how this population is maintained to ensure proper muscle homeostasis under different physio logical conditions. Understanding these mechanisms in more detail may lead to the identification of molecular targets that could be exploited to direct cellular therapies for muscular dystrophies, which are partly manifested by depletion of the satellite cell pool. Similarly, preventing dysfunction or decline of the number of satellite cells during ageing could ameliorate sarcopenia, a major cause of agerelated disability.

Many of the pathways controlling postnatal muscle adaptation and remodelling also operate in the heart. Indeed, research on skeletal muscle has spearheaded the research on muscle remodelling and brought the molecular analysis of cardiac atrophy and remodelling to a new level. However, we still need to understan d the complex interplay of hypertrophic and atrophic factors, the nuclear roles of atrogenes and the intriguing role of

circadian rhythm transcription factors in coordinating muscle growth. This information will be increasingl y important for the development of new muscle therapeutics.

The emerging pivotal role of miRNAs in muscle development and postnatal remodelling adds additional layers of complexity, if not even complications, to the analysis and understanding of the molecular networks of these processes. However, exploiting miRNAs in pathological states — for example, miR206 in rhabdomyo sarcoma or miR1 and miR486 in muscle atrophy — could open the door to a whole new class of therapeutics with a wide range of applications. The technical problems of implementing nucleic acids as therapeutic agents are by no means trivial, and the promise of miRNAs in therapy will therefore probably face practical challenges. It seems logical and tempting to target the downstream effectors of atrophic pathways, the protein products of the atrogenes, in the pursuit of smallmolecule drugs against atrophy. Indeed, a smallmolecule inhibitor of MURF1 that is active in cultured muscle cells was recently reported, suggesting that MURFs might indeed be accessible pharmacological targets130. However, targeting MURFs may lead to unexpected physiological effects, as MURFs are expressed early at the onset of muscle differentiation109,131, show high homology and have a probable role in protein homeostasis. Furthermore, a recent report showed that mutations in MURF1 cause cardiac disease132. Harnessing the past research on muscle development and signalling for therapeutic purposes is therefore likely to depend on further insights into the basic mechanisms of these complex regulatory networks.

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AcknowledgementsWe acknowledge the contributions that have also advanced the field by those authors whose work we could not cite owing to space constraints. Work in the authors’ laboratories was funded by: the UK Medical Research Council; the British Heart Foundation; The Excellence Cluster Cardio-Pulmonary System (ECCPS) of the Justus-Liebig-University, Giessen, Germany, the Goethe University, Frankfurt, Germany and the Max-Planck-Institute for Heart and Lung Research (DFG) Germany; and the University of Giessen and the University of Marburg Lung Center (UGMLC), funded by the government of the state of Hessen, Germany.

Competing interests statementThe authors declare no competing financial interests.

FURTHER INFORMATIONThomas Braun’s homepage: http://www.mpg.de/369653/herz_lungenforschung_wissM10Matthias Gautel’s homepage: http://www.kcl.ac.uk/schools/biohealth/research/randall/res-sections/musclesig/gautel/mgautel

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