Numerous environmental paracrine factors are associated to the regulation of skeletal myogenesis, thus, for example, Notch signaling plays a role in cell fate determination in the lateral dermomyotome [147], in the amplification of Pax3+/Pax7+ progenitors cells and in the myoblastic proliferation [148].

Overall, Wnt protein family has been determined along with *Shh* to be the main molecules required to activate myogenesis [149,150,151,152] (Figure 2).

In response to *Shh* and Wnt signals from the notochord and neural tube, somites delaminate to form the sclerotome and dermomyotome. The notochord and the floor plate of the neural tube secrete ventralizing signals, including *Shh* whereas the surface ectoderm and the dorsal neural tube secrete Wnt proteins [153]. These two morphogens will induce the induction of MRFs leading to the delamination, specification and myoblast differentiation.

In a remarkable study realized from chicken cultures of presomitic mesoderm cells, A.E Munsterber et al. have shown that Wnts could mimic the inducer effect of the dorsal neural tube and that *Shh* could mimic the effect of the floor plate and the notochord [25,154]. These results indicated that Wnt1, Wnt3, Wnt4 and *Shh* could replace the inducer role of the dorsal neural tube and the floor plate/Notochord structures. Ectopically implanted Wnt1, Wnt3a and Wnt4 expressing cells in chicken alter the process of somite compartmentalization *in vivo*, resulting in an enhanced recruitment of somitic cells into the myogenic lineage [155]. In the same study, authors reported no difference on somite development between neural tube (expressing Wnt1 and 3a) and surface ectoderm (expressing Wnt4), suggesting that surface ectoderm promote myogenesis by Wnt4 secretion [155].

In mouse, similar experiments indicated that the expression of Myf5 is induced by a signal emanating from neural tube and that the expression of MyoD is depending of a signal coming from surface ectoderm [156]. Wnt7a secreted by the surface ectoderm and Wnt1/Wnt3a secreted by dorsal neural tube regulate MyoD and Myf5 expression, respectively whereas Wnt4 and Wnt5a regulate both MyoD and Myf5 expression [132]. Furthermore, it has been shown that Wnt1, Wnt3a and Wnt4 are expressed in the dorsal half of the neural tube when epaxial myogenesis is initiated [153]. Regulation of hypaxial muscle specification is less understood but Tajbakhsh et al, 1998 have shown that Wnt7a and to a lesser extent Wnt4 and Wnt5a can activate myogenesis in mouse paraxial mesoderm explants [132]. Conversely, sFRPs, inhibitor of Wnt signaling, markedly reduce myogenesis by antagonizing Wnts [157,158].

80 Skeletal Muscle – From Myogenesis to Clinical Relations

called satellite cell population [145,146].

required to activate myogenesis [149,150,151,152] (Figure 2).

ectoderm promote myogenesis by Wnt4 secretion [155].

*3.1.2. Extrinsic pathway*

myoblastic proliferation [148].

cells from the central dermomyotome to the myotome [128,133,137,138,139,140,141]. These cells enter the myogenic program after activating of the myogenic regulatory factors (MRFs): Myf5 and Mrf4 [2,142]. Double mutant Pax3/Pax7 mouse embryos suffer from a major skeletal muscle deficit where only the early myotome is formed, but the cells in these structures do not activate Myf5 or MyoD and consequently fail to enter the myogenic program consequently, they die or migrate towards a non myogenic fate [143,144]. Pax3 and Myf5/Mrf4 control the activation of MyoD, another MRF family member and MyoD positive myogenic precursors differentiate into myofibers or remain as a proliferating population

Numerous environmental paracrine factors are associated to the regulation of skeletal myogenesis, thus, for example, Notch signaling plays a role in cell fate determination in the lateral dermomyotome [147], in the amplification of Pax3+/Pax7+ progenitors cells and in the

Overall, Wnt protein family has been determined along with *Shh* to be the main molecules

In response to *Shh* and Wnt signals from the notochord and neural tube, somites delaminate to form the sclerotome and dermomyotome. The notochord and the floor plate of the neural tube secrete ventralizing signals, including *Shh* whereas the surface ectoderm and the dorsal neural tube secrete Wnt proteins [153]. These two morphogens will induce the induction of

In a remarkable study realized from chicken cultures of presomitic mesoderm cells, A.E Munsterber et al. have shown that Wnts could mimic the inducer effect of the dorsal neural tube and that *Shh* could mimic the effect of the floor plate and the notochord [25,154]. These results indicated that Wnt1, Wnt3, Wnt4 and *Shh* could replace the inducer role of the dorsal neural tube and the floor plate/Notochord structures. Ectopically implanted Wnt1, Wnt3a and Wnt4 expressing cells in chicken alter the process of somite compartmentalization *in vivo*, resulting in an enhanced recruitment of somitic cells into the myogenic lineage [155]. In the same study, authors reported no difference on somite development between neural tube (expressing Wnt1 and 3a) and surface ectoderm (expressing Wnt4), suggesting that surface

In mouse, similar experiments indicated that the expression of Myf5 is induced by a signal emanating from neural tube and that the expression of MyoD is depending of a signal coming from surface ectoderm [156]. Wnt7a secreted by the surface ectoderm and Wnt1/Wnt3a secreted by dorsal neural tube regulate MyoD and Myf5 expression, respectively whereas Wnt4 and Wnt5a regulate both MyoD and Myf5 expression [132]. Furthermore, it has been shown that Wnt1, Wnt3a and Wnt4 are expressed in the dorsal half of the neural tube when epaxial myogenesis is initiated [153]. Regulation of hypaxial muscle specification is less understood but Tajbakhsh et al, 1998 have shown that Wnt7a and to a

MRFs leading to the delamination, specification and myoblast differentiation.

Use of somites from transgenic mice expressing lacZ reporter gene under the control of Myf5 epaxial promoter in presence of a constitutive form of -catenin or in presence of the neural tube lead to a strong activation of the reporter, indicating that the Wnts secreted by the neural tube act mainly through canonical pathway [159]. In the surface ectoderm, signaling pathway initiated by Wnt7a differs from that by Wnt1. Wnt7a binds to Frz7 and signals through Protein kinase C, a β-catenin independent pathway [122,149]. Recently, the Wnt PCP pathway has been implicated in regulating the orientation of myocyte growth in the developing myotome [160]. As reported during osteogenic differentiation or during recruitment, maintenance, and differentiation of human bone marrow mesenchymal stem cells a cross-talk between canonical and non-canonical Wnt signaling exists and may lead to functional antagonism [161,162].

In chick embryos, Nohno's group analyzed the myogenic effects of Wnt4 overexpression in the limb bud in order to evaluate the significance of this factor in skeletal muscle formation during embryogenesis. They found that Wnt4 treatment induced an increase in the muscle mass, particularly in fast-type muscle size. Furthermore, ectopic Wnt4 induces muscle satellite markers Pax7 and MyoD expression [60].

As extrinsic factors, myostatin (Mstn), a member of the TGF- superfamily, has been proposed as a regulator of embryonic myogenesis [30,32,34,163,164,165,166]. Myostatin, also called GDF8 (growth differentiation factor 8), is a secreted growth factor that belongs to the transforming growth factor- (TGF-) superfamily of growth and differentiation factors [30]. In mice, myostatin is predominantly expressed in skeletal muscle tissues from the period of embryogenesis to adulthood suggesting a role for this factor in the control of muscle development and function [30,166]. The role of myostatin in muscle comes from the phenotype of myostatin-deficient animals. Myostatin was first found to regulate muscle mass in mice from which the gene encoding myostatin has been knocked-out. The resulting "mighty mice" displayed muscle overgrowth due to both hyperplasia (increased number of muscle fibers) and hypertrophy (increased size of individual muscle fibers). These effects on muscle mass are persistent throughout the life of the animals. The phenotype of these mice suggested that myostatin functions as a negative regulator of muscle growth. Interestingly, the function of myostatin appears to have been conserved across diverse species. Natural mutations in the myostatin gene have been identified in double-muscled animals such as the Belgian blue cattle [29,167,168]. The recent identification of a hypermuscular child with a loss-of-function mutation in the myostatin gene suggests that the function of myostatin is similarly conserved in humans [169]. In support of this, myostatin sequence has been highly conserved through evolution, among species ranging from zebrafish to humans [30]. At early embryonic stages, myostatin expression is restricted to the myotome compartment of the developing somites, and myostatin has been proposed to play an essential role in skeletal muscle growth and development [30,165].

It is of note that many others autocrine or paracrine factors, such as Notch, Tgf-, Bmp, Noggin, mi-RNA…, play a subtle but crucial role in orchestrating the regulation of embryonic myogenesis [170,171,172,173,174,175,176]. Undoubtedly, the complete decoding of this intricate network will take several years to be complete.

Role and Function of Wnts in the Regulation of Myogenesis: When Wnt Meets Myostatin 83

which in adults contain approximately 6–8 times more nuclei than in neonates [177]. In adult muscle, satellite cells are mitotically quiescent and do not express myogenic regulatory factors (MRFs) [178,179]. Their role changes to one of providing myonuclei for homeostasis and hypertrophy or in response to the more sporadic demands for myofiber repair and regeneration [180]. In the latter context, satellite cell activation is dramatically enhanced; they reenter the cell cycle and proliferate [181,182], at this stage they are often referred to as either myogenic precursor cells or myoblasts [182,183]. Some resulting myoblasts differentiate and fuse to form replacement myofibers, whereas some remain as continual dividing cells to replenish the pool of satellite cells in anticipation of future rounds of regeneration [182,184].

Pax7 is expressed in quiescent as well as activated satellite cells, and it is down regulated when satellite cells commit to muscle differentiation [177,185,186]. Compared to embryonic satellite cells, quiescent adult satellite cells do not require Pax7 for self-renewal and regeneration [187]. The progression of activated satellite cells toward myogenic differentiation is mainly controlled by Myf5 and MyoD [188] and is followed by fusion into regenerating fibers. Satellite cells undergo different fates, giving rise to a few Pax7+MyoD– cells, which return to quiescence (satellite stem cell population), and many Pax7+MyoD+ cells, which differentiate [189]. These asymmetric divisions are mainly regulated by Notch signaling [190,191,192]. In addition, satellite stem cells which represent approximately 10% of Pax7+ mouse satellite can undergo planar symmetric cell divisions to drive expansion of their population [190]. By a subtractive hybridization approach, Legrand et al. found that Wnt7a is expressed during muscle regeneration and acts through Fzd7 receptor to induce symmetric satellite stem cell expansion and thus enhance muscle regeneration. Wnt7a signaling though Frz7 receptor requires association with Vangl2, a membrane protein involved in the regulation of PCP suggesting that Wnt7a utilizes this non-canonical pathway to control the orientation of satellite cell division [62]. They suggest that Wnt7a regulates the homeostatic maintenance of the satellite stem cell pool by modulating the increase in satellite stem cell expansion during regenerative myogenesis and that basal levels of PCP

signaling are insufficient to maintain the satellite cell pool at normal levels [62].

phenotype is consistent with the findings of Liu et al. [198].

Aging is accompanied by a decline in muscle mass and strength, a phenomenon referred as to sarcopenia [193]. It is clear that fitness is greater at any age in individuals who exercise regularly versus those who do not and that sarcopenia is reduced in physically active elderly people. A decline in the number of satellite cells, their proliferative capacities, or both, may contribute to sarcopenia. One of the mechanisms responsible for the reduced regenerative potential of old muscle seems to be the decline in Notch signaling [194]. Aging of skeletal muscle is characterized by an increase in fibrous connective tissue and adipose tissue and by an impairment of muscle regenerative potential [195,196]. This decrease of aged-muscle regeneration can be enhanced by direct activation of the Notch pathway [194] or by exposure to a youthful systemic environment [197]. Very interestingly, these agerelated effects are associated with increased canonical Wnt signaling in the satellite cell population, possibly resulting from increased amounts of Wnt or Wnt-like molecules in the serum of aged animals [24]. This generalized role of Wnt signaling in promoting an aging

From Bentzinger et al. Cold Spring Harb Perspect Biol 2012

**Figure 2.** Embryonic myogenesis. Illustration of the morphogen gradients along the rostral–caudal axis of the embryo. (C) Schematic of transverse sections through the embryo at early (i) and late (ii) stages of somitogenesis. (Ci) Morphogens secreted from various domains in the embryo specify the early somite to form the sclerotome (SC) and dermomyotome (DM). Wnts secreted from the dorsal neural tube (NT) and surface ectoderm (SE) along with bone morphogenetic protein (BMP) from the lateral plate mesoderm maintain the undifferentiated state of the somite, whereas Sonic hedgehog (Shh) signals from the neural tube floor plate and notochord (NC) induce the formation of the sclerotome. (Cii) As the sclerotome segregates, muscle progenitor cells (MPCs) from the dorsomedial (DML) and ventrolateral (VLL) lips of the dermomyotome mature give rise to the myotome (MY). At the level of the limb bud, Pax3-dependent migrating MPCs delaminate from the ventrolateral lips to later give rise to limb muscles.

### **3.2. Postnatal myogenesis**

### *3.2.1. Satellite cells*

The main role of satellite cells during the early postnatal period is to provide myonuclei for skeletal muscle growth. They contribute to the postnatal growth of syncital muscle cells, which in adults contain approximately 6–8 times more nuclei than in neonates [177]. In adult muscle, satellite cells are mitotically quiescent and do not express myogenic regulatory factors (MRFs) [178,179]. Their role changes to one of providing myonuclei for homeostasis and hypertrophy or in response to the more sporadic demands for myofiber repair and regeneration [180]. In the latter context, satellite cell activation is dramatically enhanced; they reenter the cell cycle and proliferate [181,182], at this stage they are often referred to as either myogenic precursor cells or myoblasts [182,183]. Some resulting myoblasts differentiate and fuse to form replacement myofibers, whereas some remain as continual dividing cells to replenish the pool of satellite cells in anticipation of future rounds of regeneration [182,184].

82 Skeletal Muscle – From Myogenesis to Clinical Relations

From Bentzinger et al. Cold Spring Harb Perspect Biol 2012

**3.2. Postnatal myogenesis**

*3.2.1. Satellite cells*

of this intricate network will take several years to be complete.

It is of note that many others autocrine or paracrine factors, such as Notch, Tgf-, Bmp, Noggin, mi-RNA…, play a subtle but crucial role in orchestrating the regulation of embryonic myogenesis [170,171,172,173,174,175,176]. Undoubtedly, the complete decoding

**Figure 2.** Embryonic myogenesis. Illustration of the morphogen gradients along the rostral–caudal axis of the embryo. (C) Schematic of transverse sections through the embryo at early (i) and late (ii) stages of somitogenesis. (Ci) Morphogens secreted from various domains in the embryo specify the early somite to form the sclerotome (SC) and dermomyotome (DM). Wnts secreted from the dorsal neural tube (NT) and surface ectoderm (SE) along with bone morphogenetic protein (BMP) from the lateral plate mesoderm maintain the undifferentiated state of the somite, whereas Sonic hedgehog (Shh) signals from the neural tube floor plate and notochord (NC) induce the formation of the sclerotome. (Cii) As the sclerotome segregates, muscle progenitor cells (MPCs) from the dorsomedial (DML) and ventrolateral (VLL) lips of the dermomyotome mature give rise to the myotome (MY). At the level of the limb bud, Pax3-dependent

The main role of satellite cells during the early postnatal period is to provide myonuclei for skeletal muscle growth. They contribute to the postnatal growth of syncital muscle cells,

migrating MPCs delaminate from the ventrolateral lips to later give rise to limb muscles.

Pax7 is expressed in quiescent as well as activated satellite cells, and it is down regulated when satellite cells commit to muscle differentiation [177,185,186]. Compared to embryonic satellite cells, quiescent adult satellite cells do not require Pax7 for self-renewal and regeneration [187]. The progression of activated satellite cells toward myogenic differentiation is mainly controlled by Myf5 and MyoD [188] and is followed by fusion into regenerating fibers. Satellite cells undergo different fates, giving rise to a few Pax7+MyoD– cells, which return to quiescence (satellite stem cell population), and many Pax7+MyoD+ cells, which differentiate [189]. These asymmetric divisions are mainly regulated by Notch signaling [190,191,192]. In addition, satellite stem cells which represent approximately 10% of Pax7+ mouse satellite can undergo planar symmetric cell divisions to drive expansion of their population [190]. By a subtractive hybridization approach, Legrand et al. found that Wnt7a is expressed during muscle regeneration and acts through Fzd7 receptor to induce symmetric satellite stem cell expansion and thus enhance muscle regeneration. Wnt7a signaling though Frz7 receptor requires association with Vangl2, a membrane protein involved in the regulation of PCP suggesting that Wnt7a utilizes this non-canonical pathway to control the orientation of satellite cell division [62]. They suggest that Wnt7a regulates the homeostatic maintenance of the satellite stem cell pool by modulating the increase in satellite stem cell expansion during regenerative myogenesis and that basal levels of PCP signaling are insufficient to maintain the satellite cell pool at normal levels [62].

Aging is accompanied by a decline in muscle mass and strength, a phenomenon referred as to sarcopenia [193]. It is clear that fitness is greater at any age in individuals who exercise regularly versus those who do not and that sarcopenia is reduced in physically active elderly people. A decline in the number of satellite cells, their proliferative capacities, or both, may contribute to sarcopenia. One of the mechanisms responsible for the reduced regenerative potential of old muscle seems to be the decline in Notch signaling [194]. Aging of skeletal muscle is characterized by an increase in fibrous connective tissue and adipose tissue and by an impairment of muscle regenerative potential [195,196]. This decrease of aged-muscle regeneration can be enhanced by direct activation of the Notch pathway [194] or by exposure to a youthful systemic environment [197]. Very interestingly, these agerelated effects are associated with increased canonical Wnt signaling in the satellite cell population, possibly resulting from increased amounts of Wnt or Wnt-like molecules in the serum of aged animals [24]. This generalized role of Wnt signaling in promoting an aging phenotype is consistent with the findings of Liu et al. [198].

### *3.2.2. Non satellite cells*

Interestingly, a variety of non-satellite cell, located outside the basal lamina can also participate in skeletal muscle regeneration in the adult. These include pericytes, endothelial cells, mesoangioblasts [177,199,200,201,202], and interstitial cells (PICs) [203], as well as other cell types that are not fully characterized [26,204,205]. Polesskaya et al. found that a side population of cells harboring surface markers CD45 and Sca1 (CD45+:Sca1+) isolated from regenerating muscle readily underwent myogenic differentiation *in vitro* [26]. As quiescent satellite cells, activated satellite cells and satellite cell derived myoblasts do not express CD45 and satellite cell lineage do not express Sca1, CD45+:Sca1+ cells constitute a cellular pool distinct of muscle satellite cells. The fact that (i) activation of the canonical Wnt pathway by LiCl in isolated CD45+:Sca1+ cells was sufficient to induce muscle specification, (ii) myogenic commitment of CD45+:Sca1+ cells were induced by co-culture with cells ectopically expressing Wnt proteins and, (iii) the number of CD45+:Sca1+ cells was decreased in injured muscle treated with sFRPs, demonstrated clearly a functional requirement for canonical Wnt in the myogenic specification of CD45+:Sca1+ cells. These exciting results establish that myogenic progenitors derived from non-satellite cell can have a physiological role in muscle regeneration.

Role and Function of Wnts in the Regulation of Myogenesis: When Wnt Meets Myostatin 85

Takata et al. reported that Wnt4 exhibited a low but significant myogenic activity with an increase of proliferation rate in myoblast cells [60]. Conversely, Otto and co-workers found, using co-culture of Wnt4-producing NIH-3T3 cell line on single muscle fiber culture, a 24% reduction of satellite-cell proliferation [207]. These conflicting results could be attributed either to the different specificities of the cellular models used or to the fact that effective concentration and physiological activity of ectopicWnt4 can be eminently variable, depending on the nature and activity of produced Wnt4. Taken together, these data are in agreement with a modest effect of Wnt4 on proliferation, suggesting a role of this factor in enhancing cell fusion rather than controlling the number of nuclei available for fusion.

Knockdown of Wnt4 expression in C2C12 and satellite cells inhibits their differentiation and further confirms that Wnt4 acts as an effector of myogenesis (Figure 3). However, addition of siWnt4 after switching to differentiation medium or 24 h before did not decrease significantly the differentiation rate. This confirms that Wnt4 acts as a positive regulator of

**Figure 3.** Effects of Wnt4 overexpression and Wnt4 silencing on C2C12 myoblasts and satellite cells differentiation. Stably transfected polyclonal C2C12 myoblasts with empty (Ctrl) or Wnt4-containing

Myoblastic differentiation involves two major steps, their reversible withdrawal from cell cycle and the subsequent expression and activation of myogenic factors. Myf5 and MyoD are expressed in early myogenesis. As cells progress toward a differentiated phenotype,

(Wnt4) expression vector were grown 2 days in proliferation medium followed by 4 days in differentiation medium. C2C12 myoblasts and satellite cells transfected with siRNA specific to Wnt4 (siWnt4) and negative control siLuc were grown 2 days in proliferation medium followed by 4 days in differentiation medium. Fluorescent images of C2C12 myoblasts were obtained by immunostaining

with a monoclonal anti-myosin antibody. Nuclei were labeled by DAPI staining.

myogenic differentiation only if it is expressed in the proliferative phase.

From Bernardi et al. Am J Physiol Cell Physiol-2011

### *3.2.3. Wnt-related regulation of myoblast proliferation and differentiation*

In addition to the crucial role of Wnt proteins in satellite cells fate and maintenance, they play an essential role in the control of proliferation and differentiation in various cellular types.

In order to better characterize the role of Wnt proteins in the control of muscle cell differentiation, Bernardi et al. established Wnt expression pattern on C2C12 myoblasts and satellite cells [37]. They found that, among the 19 existing Wnts, only the expression of Wnt4 was strongly activated from the early steps of differentiation. Interestingly, by modulating Wnt4 expression level with overexpression or silencing approaches, they reported a strong differentiation promoting activity of this factor (Figure 3). The relative increase in myotube size being comparable 48, 72, and 96 h after switching to differentiation medium indicates that Wnt4 signaling controls myotube size at the early steps of differentiation during myotube formation. The lack of Wnt4-mediated hypertrophic effect observed when cells were transfected the day of differentiation or later indicates that this factor has to be expressed during the proliferative stage to be effective.

Fusion consists of two distinct phases: myoblast/myoblast fusion to form nascent myotubes and subsequent myoblast/myotubes fusion, resulting in a rapid accretion of size. The efficiency of the first phase can be evaluated by measuring the fusion index, which represents the proportion of the total cell population that has fused. After being switched to differentiation medium, Wnt4 overexpression in C2C12 and satellite cells gave a 62% and a 67% fusion index increase, respectively, suggesting that Wnt4 signaling control nascent myotube formation. Recently, Tanaka et al. confirms that Wnt4 overexpression increased troponin-T positive cells in proliferation medium and enhanced myotube formation of differentiated C2C12 cells [206].

Takata et al. reported that Wnt4 exhibited a low but significant myogenic activity with an increase of proliferation rate in myoblast cells [60]. Conversely, Otto and co-workers found, using co-culture of Wnt4-producing NIH-3T3 cell line on single muscle fiber culture, a 24% reduction of satellite-cell proliferation [207]. These conflicting results could be attributed either to the different specificities of the cellular models used or to the fact that effective concentration and physiological activity of ectopicWnt4 can be eminently variable, depending on the nature and activity of produced Wnt4. Taken together, these data are in agreement with a modest effect of Wnt4 on proliferation, suggesting a role of this factor in enhancing cell fusion rather than controlling the number of nuclei available for fusion.

Knockdown of Wnt4 expression in C2C12 and satellite cells inhibits their differentiation and further confirms that Wnt4 acts as an effector of myogenesis (Figure 3). However, addition of siWnt4 after switching to differentiation medium or 24 h before did not decrease significantly the differentiation rate. This confirms that Wnt4 acts as a positive regulator of myogenic differentiation only if it is expressed in the proliferative phase.

From Bernardi et al. Am J Physiol Cell Physiol-2011

84 Skeletal Muscle – From Myogenesis to Clinical Relations

a physiological role in muscle regeneration.

expressed during the proliferative stage to be effective.

differentiated C2C12 cells [206].

*3.2.3. Wnt-related regulation of myoblast proliferation and differentiation*

In addition to the crucial role of Wnt proteins in satellite cells fate and maintenance, they play an essential role in the control of proliferation and differentiation in various cellular types.

In order to better characterize the role of Wnt proteins in the control of muscle cell differentiation, Bernardi et al. established Wnt expression pattern on C2C12 myoblasts and satellite cells [37]. They found that, among the 19 existing Wnts, only the expression of Wnt4 was strongly activated from the early steps of differentiation. Interestingly, by modulating Wnt4 expression level with overexpression or silencing approaches, they reported a strong differentiation promoting activity of this factor (Figure 3). The relative increase in myotube size being comparable 48, 72, and 96 h after switching to differentiation medium indicates that Wnt4 signaling controls myotube size at the early steps of differentiation during myotube formation. The lack of Wnt4-mediated hypertrophic effect observed when cells were transfected the day of differentiation or later indicates that this factor has to be

Fusion consists of two distinct phases: myoblast/myoblast fusion to form nascent myotubes and subsequent myoblast/myotubes fusion, resulting in a rapid accretion of size. The efficiency of the first phase can be evaluated by measuring the fusion index, which represents the proportion of the total cell population that has fused. After being switched to differentiation medium, Wnt4 overexpression in C2C12 and satellite cells gave a 62% and a 67% fusion index increase, respectively, suggesting that Wnt4 signaling control nascent myotube formation. Recently, Tanaka et al. confirms that Wnt4 overexpression increased troponin-T positive cells in proliferation medium and enhanced myotube formation of

Interestingly, a variety of non-satellite cell, located outside the basal lamina can also participate in skeletal muscle regeneration in the adult. These include pericytes, endothelial cells, mesoangioblasts [177,199,200,201,202], and interstitial cells (PICs) [203], as well as other cell types that are not fully characterized [26,204,205]. Polesskaya et al. found that a side population of cells harboring surface markers CD45 and Sca1 (CD45+:Sca1+) isolated from regenerating muscle readily underwent myogenic differentiation *in vitro* [26]. As quiescent satellite cells, activated satellite cells and satellite cell derived myoblasts do not express CD45 and satellite cell lineage do not express Sca1, CD45+:Sca1+ cells constitute a cellular pool distinct of muscle satellite cells. The fact that (i) activation of the canonical Wnt pathway by LiCl in isolated CD45+:Sca1+ cells was sufficient to induce muscle specification, (ii) myogenic commitment of CD45+:Sca1+ cells were induced by co-culture with cells ectopically expressing Wnt proteins and, (iii) the number of CD45+:Sca1+ cells was decreased in injured muscle treated with sFRPs, demonstrated clearly a functional requirement for canonical Wnt in the myogenic specification of CD45+:Sca1+ cells. These exciting results establish that myogenic progenitors derived from non-satellite cell can have

*3.2.2. Non satellite cells*

**Figure 3.** Effects of Wnt4 overexpression and Wnt4 silencing on C2C12 myoblasts and satellite cells differentiation. Stably transfected polyclonal C2C12 myoblasts with empty (Ctrl) or Wnt4-containing (Wnt4) expression vector were grown 2 days in proliferation medium followed by 4 days in differentiation medium. C2C12 myoblasts and satellite cells transfected with siRNA specific to Wnt4 (siWnt4) and negative control siLuc were grown 2 days in proliferation medium followed by 4 days in differentiation medium. Fluorescent images of C2C12 myoblasts were obtained by immunostaining with a monoclonal anti-myosin antibody. Nuclei were labeled by DAPI staining.

Myoblastic differentiation involves two major steps, their reversible withdrawal from cell cycle and the subsequent expression and activation of myogenic factors. Myf5 and MyoD are expressed in early myogenesis. As cells progress toward a differentiated phenotype, myogenin and MRF4 are induced and cooperatively establish the irreversible commitment to terminal differentiation. Myf5 is a factor reported to be high in the Go phase, where cells were arrested to differentiate [208]. Borello et al. have previously showed a direct regulation of Myf5 expression by canonical Wnt signaling during somitogenesis [2]. Bernardi et al. found that the expression of Myf5 was activated by Wnt4 overexpression and strongly inhibited by Wnt4 silencing during C2C12 myoblast and satellite cells differentiation, suggesting that myogenic activity of Wnt4 is related to an increase of Myf5 expression.

Role and Function of Wnts in the Regulation of Myogenesis: When Wnt Meets Myostatin 87

this discrepancy and suggest that data emanating from established cell lines should be considered with care [206]. Moreover, the analysis of cellular response during myoblast differentiation can be made more complex by the implication of other signaling pathways

Recently, an elegant work from Rudnicki's group showed that Wnt signaling is also implicated in the control of muscular hypertrophy [27]. Skeletal muscle mass depends upon a dynamic balance between anabolic and catabolic processes. Muscle hypertrophy is characterized by an increase of the diameter of muscle fibers and increased protein synthesis, mainly by activation of the IGF1/Akt/mTORC1 pathway. They observed that Wnt7a binding to Fzd7 receptor directly activates the Akt/mTOR growth pathway, thereby inducing myofiber hypertrophy. Besides, they found that anabolic activity of Wnt7a is related to the

Myostatin acts through the receptor-associated proteins Smad2 and Smad3 [33,212,213]. Phosphorylated Smad2 and Smad3 form heterodimeric complex with the common mediator Smad4. These activated Smad proteins function as the key intracellular mediators of signaling for myostatin as they translocate into the nucleus, and activate the transcription of the target genes through interaction with DNA and other nuclear factors [214,215]. Takata et al. reported that Wnt4 had no effect on Smad2 phosphorylation, but it antagonized Smad2 phosphorylation induced by Mstn in differentiated C2C12 myoblasts [60]. They suggest that Wnt4 exert its myogenic effect by acting against Mstn. By using CAGA reporter assay, Bernardi et al. confirmed the inhibition of Mstn-activated Smad signaling pathway by Wnt4 [37]. Contrary to differentiated myoblasts, Wnt4 decreased the CAGA reporter activity in proliferating myoblasts below the basal level meaning that Wnt4 has a stronger anti-Mstn activity during proliferation than during differentiation. Furthermore, the fact that the addition of Mstn did not reverse the Wnt4-induced CAGA reporter inhibition in myoblasts and satellite cells suggests that Mstn acts upstream of Wnt4 or that Wnt4 regulates the Mstn/Smad pathway independently of Mstn. The inhibition of the Mstn pathway by Wnt4 can be associated with the inhibition of Mstn expression and/or inhibition of the Mstn/Smad transduction pathway. By using sqRT-PCR and promoter reporter experiments, authors showed that Wnt4 acted as an inhibitor of Mstn expression. Moreover, study with LiCl, strongly suggests that Wnt4 regulates negatively Mstn expression through activation of the canonical -catenin pathway. However, while Wnt4 down-regulates Mstn expression in proliferative and differentiated cells, the activation of canonical signaling by LiCl inhibits Mstn expression only when cells are in proliferative or early differentiated states. Thus, in function of the differentiation state, various signaling pathways can be implicated in the inhibition of Mstn expression by Wnt4 in myogenic cells. A biphasic mode of action of Wnt4 can be proposed: in proliferative and early differentiated stages, Wnt4 acts by inducing canonical signaling, and later in the differentiation, canonical pathways is relaxed and another pathway maintains Mstn at low level. However, the fact that Wnt4 displayed the strongest CAGA reporter inhibiting activity in proliferative myoblasts, whereas Wnt4 had

activation of a new non-canonical pathway implicating stimulatory G protein, Gαs.

like the non-canonical Wnt/calcium signaling.

*3.2.4. Relation between Wnt4 and Myostatin*

These authors by measuring Tcf/LEF gene canonical reporter activity and axin2 expression (a protein induced by canonical Wnt pathway), in the presence or absence of canonical Wnt pathway stimulators (LiCl or BIO), reported canonical Wnt pathway activation by Wnt4. Although this factor was originally described as a non-canonical Wnt, it has also been implicated in the activation or inhibition of the canonical Wnt pathway [55,56,57,58,59,60,61]. Numerous reports showed that Wnt-related myogenic activity is mediated mainly through a stimulation of canonical signaling. Thus, Takata et al. found that myogenic activity of Wnt4 is linked to an increase of -catenin signaling [60]. Likewise, Armstrong et al. have reported that the expression of -catenin is necessary for physiological growth of skeletal muscle and that Wnt signaling pathway induces -catenin activation of growth-control genes during overload induced skeletal muscle hypertrophy [209,210]. By using C2C12 and satellite cells, Han et al. reported recently that activation of canonical pathway by R-spondin2, promote myogenic differentiation and hypertrophic myofiber formation [211]. When LiCl was added to the culture medium, the area of myotubes and index fusion were increased in both C2C12 and satellite cells, reaching values similar to those observed after Wnt4-induced hypertrophy [37]. On the other hand, the fact that LiCl exerts a higher Tcf/LEF activation than Wnt4 treatment, whereas the extent of differentiation is the same suggests that this process is dependent on other signaling pathways. In this respect, work of Otto et al. (61) on isolated muscle fibers showed that Wnt4 inhibited satellite cell proliferation but that canonical Wnts such as Wnt1, Wnt3a, and Wnt5a induced a greater degree of proliferation than control cells, confirming the implication of other pathways in Wnt4 signaling [207]. The stronger activity in terms of Tcf/LEF activation of LiCl compared with Wnt4 (+100% on C2C12 myoblasts, +250% on satellite cells) treatments could explain the discrepancy between proliferative activities of Wnt4 and LiCl. Indeed, as described above, Wnt4 shows a weak effect on mitogenicity, whereas 10 mM LiCl has a strong anti-proliferative activity (-15% on satellite cells, -48% on satellite cells), suggesting that low levels of Tcf/LEF activation regulate myogenic differentiation, whereas higher levels regulate preferentially the proliferation. A study reported by Anakwe et al. is also in agreement with a relation between the level of Tcf/LEF activity and the type of cellular response elicited [209]. They reported an increase in the number of terminally differentiated cells in Wnt4 transfected myogenic cells, whereas the overexpression of canonical Wnt3a decreases the number of terminally differentiated myogenic cells. Making more complex the Wnt4-related signaling in muscle, Tanaka et al. showed very recently that Wnt4 promotes myogenesis and suppresses canonical Wnt signaling, culture conditions and C2C12 myoblasts characteristics may be at the origin of this discrepancy and suggest that data emanating from established cell lines should be considered with care [206]. Moreover, the analysis of cellular response during myoblast differentiation can be made more complex by the implication of other signaling pathways like the non-canonical Wnt/calcium signaling.

Recently, an elegant work from Rudnicki's group showed that Wnt signaling is also implicated in the control of muscular hypertrophy [27]. Skeletal muscle mass depends upon a dynamic balance between anabolic and catabolic processes. Muscle hypertrophy is characterized by an increase of the diameter of muscle fibers and increased protein synthesis, mainly by activation of the IGF1/Akt/mTORC1 pathway. They observed that Wnt7a binding to Fzd7 receptor directly activates the Akt/mTOR growth pathway, thereby inducing myofiber hypertrophy. Besides, they found that anabolic activity of Wnt7a is related to the activation of a new non-canonical pathway implicating stimulatory G protein, Gαs.

### *3.2.4. Relation between Wnt4 and Myostatin*

86 Skeletal Muscle – From Myogenesis to Clinical Relations

myogenin and MRF4 are induced and cooperatively establish the irreversible commitment to terminal differentiation. Myf5 is a factor reported to be high in the Go phase, where cells were arrested to differentiate [208]. Borello et al. have previously showed a direct regulation of Myf5 expression by canonical Wnt signaling during somitogenesis [2]. Bernardi et al. found that the expression of Myf5 was activated by Wnt4 overexpression and strongly inhibited by Wnt4 silencing during C2C12 myoblast and satellite cells differentiation, suggesting that myogenic activity of Wnt4 is related to an increase of Myf5 expression.

These authors by measuring Tcf/LEF gene canonical reporter activity and axin2 expression (a protein induced by canonical Wnt pathway), in the presence or absence of canonical Wnt pathway stimulators (LiCl or BIO), reported canonical Wnt pathway activation by Wnt4. Although this factor was originally described as a non-canonical Wnt, it has also been implicated in the activation or inhibition of the canonical Wnt pathway [55,56,57,58,59,60,61]. Numerous reports showed that Wnt-related myogenic activity is mediated mainly through a stimulation of canonical signaling. Thus, Takata et al. found that myogenic activity of Wnt4 is linked to an increase of -catenin signaling [60]. Likewise, Armstrong et al. have reported that the expression of -catenin is necessary for physiological growth of skeletal muscle and that Wnt signaling pathway induces -catenin activation of growth-control genes during overload induced skeletal muscle hypertrophy [209,210]. By using C2C12 and satellite cells, Han et al. reported recently that activation of canonical pathway by R-spondin2, promote myogenic differentiation and hypertrophic myofiber formation [211]. When LiCl was added to the culture medium, the area of myotubes and index fusion were increased in both C2C12 and satellite cells, reaching values similar to those observed after Wnt4-induced hypertrophy [37]. On the other hand, the fact that LiCl exerts a higher Tcf/LEF activation than Wnt4 treatment, whereas the extent of differentiation is the same suggests that this process is dependent on other signaling pathways. In this respect, work of Otto et al. (61) on isolated muscle fibers showed that Wnt4 inhibited satellite cell proliferation but that canonical Wnts such as Wnt1, Wnt3a, and Wnt5a induced a greater degree of proliferation than control cells, confirming the implication of other pathways in Wnt4 signaling [207]. The stronger activity in terms of Tcf/LEF activation of LiCl compared with Wnt4 (+100% on C2C12 myoblasts, +250% on satellite cells) treatments could explain the discrepancy between proliferative activities of Wnt4 and LiCl. Indeed, as described above, Wnt4 shows a weak effect on mitogenicity, whereas 10 mM LiCl has a strong anti-proliferative activity (-15% on satellite cells, -48% on satellite cells), suggesting that low levels of Tcf/LEF activation regulate myogenic differentiation, whereas higher levels regulate preferentially the proliferation. A study reported by Anakwe et al. is also in agreement with a relation between the level of Tcf/LEF activity and the type of cellular response elicited [209]. They reported an increase in the number of terminally differentiated cells in Wnt4 transfected myogenic cells, whereas the overexpression of canonical Wnt3a decreases the number of terminally differentiated myogenic cells. Making more complex the Wnt4-related signaling in muscle, Tanaka et al. showed very recently that Wnt4 promotes myogenesis and suppresses canonical Wnt signaling, culture conditions and C2C12 myoblasts characteristics may be at the origin of

Myostatin acts through the receptor-associated proteins Smad2 and Smad3 [33,212,213]. Phosphorylated Smad2 and Smad3 form heterodimeric complex with the common mediator Smad4. These activated Smad proteins function as the key intracellular mediators of signaling for myostatin as they translocate into the nucleus, and activate the transcription of the target genes through interaction with DNA and other nuclear factors [214,215]. Takata et al. reported that Wnt4 had no effect on Smad2 phosphorylation, but it antagonized Smad2 phosphorylation induced by Mstn in differentiated C2C12 myoblasts [60]. They suggest that Wnt4 exert its myogenic effect by acting against Mstn. By using CAGA reporter assay, Bernardi et al. confirmed the inhibition of Mstn-activated Smad signaling pathway by Wnt4 [37]. Contrary to differentiated myoblasts, Wnt4 decreased the CAGA reporter activity in proliferating myoblasts below the basal level meaning that Wnt4 has a stronger anti-Mstn activity during proliferation than during differentiation. Furthermore, the fact that the addition of Mstn did not reverse the Wnt4-induced CAGA reporter inhibition in myoblasts and satellite cells suggests that Mstn acts upstream of Wnt4 or that Wnt4 regulates the Mstn/Smad pathway independently of Mstn. The inhibition of the Mstn pathway by Wnt4 can be associated with the inhibition of Mstn expression and/or inhibition of the Mstn/Smad transduction pathway. By using sqRT-PCR and promoter reporter experiments, authors showed that Wnt4 acted as an inhibitor of Mstn expression. Moreover, study with LiCl, strongly suggests that Wnt4 regulates negatively Mstn expression through activation of the canonical -catenin pathway. However, while Wnt4 down-regulates Mstn expression in proliferative and differentiated cells, the activation of canonical signaling by LiCl inhibits Mstn expression only when cells are in proliferative or early differentiated states. Thus, in function of the differentiation state, various signaling pathways can be implicated in the inhibition of Mstn expression by Wnt4 in myogenic cells. A biphasic mode of action of Wnt4 can be proposed: in proliferative and early differentiated stages, Wnt4 acts by inducing canonical signaling, and later in the differentiation, canonical pathways is relaxed and another pathway maintains Mstn at low level. However, the fact that Wnt4 displayed the strongest CAGA reporter inhibiting activity in proliferative myoblasts, whereas Wnt4 had

the strongest Mstn expression inhibiting activity in differentiated myoblasts, implies that Wnt4 regulates both Mstn expression and Mstn/Smad signaling pathway. In silico analysis in the Mstn promoter revealed the presence of three putative Tcf/LEF binding sites within a 2-kb sequence upstream of the *Mstn* gene at position -1889, -1028, and -743 confirming the observed regulation of the Mstn expression by Wnt/-catenin pathway. Analyze of reciprocal regulation of the both signaling pathways showed that whereas Mstn inhibits the accumulation of nuclear -catenin in differentiated C2C12 [36], Mstn was not able to inhibit Tcf/LEF activity induced by Wnt4 overexpression in proliferative and differentiated C2C12 myoblasts [37]. Thus, contrary to adipogenic differentiation of human bone marrow-derived mesenchymal stem cells in which there is a cross-communication of the Mstn signal with Wnt/-catenin pathway, Wnt4/-catenin signaling pathway is not affected by Mstn.

Role and Function of Wnts in the Regulation of Myogenesis: When Wnt Meets Myostatin 89

Yann Fedon, Anne Bonnieu, Barbara Vernus, Francis Bacou and Henri Bernardi\*

[1] Kusserow A, Pang K, Sturm C, Hrouda M, Lentfer J, et al. (2005) Unexpected complexity

[2] Borello U, Berarducci B, Murphy P, Bajard L, Buffa V, et al. (2006) The Wnt/beta-catenin pathway regulates Gli-mediated Myf5 expression during somitogenesis. Development

[3] Moon RT, Bowerman B, Boutros M, Perrimon N (2002) The promise and perils of Wnt

[4] Cadigan KM, Nusse R (1997) Wnt signaling: a common theme in animal development.

[5] Church VL, Francis-West P (2002) Wnt signalling during limb development. Int J Dev

[6] Veeman MT, Axelrod JD, Moon RT (2003) A second canon. Functions and mechanisms

[7] Logan CY, Nusse R (2004) The Wnt signaling pathway in development and disease.

[8] Abiola M, Favier M, Christodoulou-Vafeiadou E, Pichard AL, Martelly I, et al. (2009) Activation of Wnt/beta-catenin signaling increases insulin sensitivity through a reciprocal regulation of Wnt10b and SREBP-1c in skeletal muscle cells. PLoS One 4:

[9] Brisken C, Heineman A, Chavarria T, Elenbaas B, Tan J, et al. (2000) Essential function of Wnt-4 in mammary gland development downstream of progesterone signaling. Genes

[10] Kim Y, Kobayashi A, Sekido R, DiNapoli L, Brennan J, et al. (2006) Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination. PLoS Biol 4: e187. [11] Nishizuka M, Koyanagi A, Osada S, Imagawa M (2008) Wnt4 and Wnt5a promote

[12] Park JS, Valerius MT, McMahon AP (2007) Wnt/beta-catenin signaling regulates nephron induction during mouse kidney development. Development 134: 2533-2539. [13] Bryja V, Schambony A, Cajanek L, Dominguez I, Arenas E, et al. (2008) Beta-arrestin and casein kinase 1/2 define distinct branches of non-canonical WNT signalling

*INRA, UMR866 Dynamique Musculaire Et Métabolisme, Montpellier, France*

of the Wnt gene family in a sea anemone. Nature 433: 156-160.

of beta-catenin-independent Wnt signaling. Dev Cell 5: 367-377.

signaling through beta-catenin. Science 296: 1644-1646.

adipocyte differentiation. FEBS Lett 582: 3201-3205.

**Author details**

Stéphanie Gay

**5. References**

133: 3723-3732.

Biol 46: 927-936.

e8509.

 \*

Corresponding Author

Dev 14: 650-654.

Genes Dev 11: 3286-3305.

Annu Rev Cell Dev Biol 20: 781-810.

pathways. EMBO Rep 9: 1244-1250.

*INRA, LPG Campus de Baulieu, Rennes France*

Mstn has been described as an inhibitor of myotube formation in C2C12 and satellite cells [32,33,34,35]. The reversion of Wnt4 activity by recombinant Mstn and the absence of hypertrophic activity of this factor in Mstn-/- mice demonstrated clearly that Wnt4 requires the presence of Mstn to elicit its differentiation promoting activity [37]. Thus the negative regulation of Mstn by Wnt4 is a crucial step for the myotube formation and hypertrophy. The fact that the addition of Mstn reverses the hypertrophic activity of Wnt4 but does not reverse the Wnt4-induced CAGA reporter inhibition implies that *1*) Wnt4 acts upstream of Mstn and that *2*) Wnt4 inhibits CAGA reporter by another pathway than Mstn/Smad signaling. Furthermore, the demonstration that Wnt4 can dominantly overcome the expression and activity of atrophic Mstn opens possibilities of the manipulation of Wnt4 or -catenin levels as a likely target for therapeutic design.

## **4. Conclusion**

Development, growth and maintenance of muscle mass are critical for long-term health and quality of life. In this regard, activation of satellite cells and regulation of anabolic/catabolic muscular pathways play key roles. Production of new myofibrils and degradation of existing proteins is a delicate equilibrium, which, depending on the condition, can promote muscle growth or loss. In this matter, skeletal muscle has the capacity to continuously regulate its size in response to a variety of external cues and serves as the most significant repository for protein in the body of healthy adult humans (~50% total protein content). To achieve a correct development and to maintain homeostasis, the biological response of the skeletal muscle implies a multitude of regulatory signaling pathways that orchestrates myogenesis in embryonic development and enables adult muscle repair. There is much interest in understanding the cellular and molecular mechanisms underlying skeletal muscle homeostasis and regeneration in different contexts because such knowledge might help in the development of cell therapies for diseases characterized by skeletal muscle degeneration. As described in this review, it is clear that Wnt play multiple and essential actions both in developmental and postnatal muscles. Depending not only of the Wnt signaling pathways implicated but also of the other interacting signaling pathways, Wnt proteins can act to promote or inhibit myogenesis. Further study will be necessary to elucidate the complex network that regulates embryonic and adult myogenesis.

### **Author details**

88 Skeletal Muscle – From Myogenesis to Clinical Relations

the strongest Mstn expression inhibiting activity in differentiated myoblasts, implies that Wnt4 regulates both Mstn expression and Mstn/Smad signaling pathway. In silico analysis in the Mstn promoter revealed the presence of three putative Tcf/LEF binding sites within a 2-kb sequence upstream of the *Mstn* gene at position -1889, -1028, and -743 confirming the observed regulation of the Mstn expression by Wnt/-catenin pathway. Analyze of reciprocal regulation of the both signaling pathways showed that whereas Mstn inhibits the accumulation of nuclear -catenin in differentiated C2C12 [36], Mstn was not able to inhibit Tcf/LEF activity induced by Wnt4 overexpression in proliferative and differentiated C2C12 myoblasts [37]. Thus, contrary to adipogenic differentiation of human bone marrow-derived mesenchymal stem cells in which there is a cross-communication of the Mstn signal with

Wnt/-catenin pathway, Wnt4/-catenin signaling pathway is not affected by Mstn.

network that regulates embryonic and adult myogenesis.

**4. Conclusion**

Mstn has been described as an inhibitor of myotube formation in C2C12 and satellite cells [32,33,34,35]. The reversion of Wnt4 activity by recombinant Mstn and the absence of hypertrophic activity of this factor in Mstn-/- mice demonstrated clearly that Wnt4 requires the presence of Mstn to elicit its differentiation promoting activity [37]. Thus the negative regulation of Mstn by Wnt4 is a crucial step for the myotube formation and hypertrophy. The fact that the addition of Mstn reverses the hypertrophic activity of Wnt4 but does not reverse the Wnt4-induced CAGA reporter inhibition implies that *1*) Wnt4 acts upstream of Mstn and that *2*) Wnt4 inhibits CAGA reporter by another pathway than Mstn/Smad signaling. Furthermore, the demonstration that Wnt4 can dominantly overcome the expression and activity of atrophic Mstn opens possibilities of the manipulation of Wnt4 or

Development, growth and maintenance of muscle mass are critical for long-term health and quality of life. In this regard, activation of satellite cells and regulation of anabolic/catabolic muscular pathways play key roles. Production of new myofibrils and degradation of existing proteins is a delicate equilibrium, which, depending on the condition, can promote muscle growth or loss. In this matter, skeletal muscle has the capacity to continuously regulate its size in response to a variety of external cues and serves as the most significant repository for protein in the body of healthy adult humans (~50% total protein content). To achieve a correct development and to maintain homeostasis, the biological response of the skeletal muscle implies a multitude of regulatory signaling pathways that orchestrates myogenesis in embryonic development and enables adult muscle repair. There is much interest in understanding the cellular and molecular mechanisms underlying skeletal muscle homeostasis and regeneration in different contexts because such knowledge might help in the development of cell therapies for diseases characterized by skeletal muscle degeneration. As described in this review, it is clear that Wnt play multiple and essential actions both in developmental and postnatal muscles. Depending not only of the Wnt signaling pathways implicated but also of the other interacting signaling pathways, Wnt proteins can act to promote or inhibit myogenesis. Further study will be necessary to elucidate the complex Yann Fedon, Anne Bonnieu, Barbara Vernus, Francis Bacou and Henri Bernardi\* *INRA, UMR866 Dynamique Musculaire Et Métabolisme, Montpellier, France*

Stéphanie Gay *INRA, LPG Campus de Baulieu, Rennes France*

### **5. References**

<sup>\*</sup> Corresponding Author

	- [14] Kohn AD, Moon RT (2005) Wnt and calcium signaling: beta-catenin-independent pathways. Cell Calcium 38: 439-446.

Role and Function of Wnts in the Regulation of Myogenesis: When Wnt Meets Myostatin 91

[31] Williams MS (2004) Myostatin mutation associated with gross muscle hypertrophy in a

[32] Joulia D, Bernardi H, Garandel V, Rabenoelina F, Vernus B, et al. (2003) Mechanisms involved in the inhibition of myoblast proliferation and differentiation by myostatin.

[33] Langley B, Thomas M, Bishop A, Sharma M, Gilmour S, et al. (2002) Myostatin inhibits myoblast differentiation by down-regulating MyoD expression. J Biol Chem 277: 49831-

[34] Rios R, Carneiro I, Arce VM, Devesa J (2002) Myostatin is an inhibitor of myogenic

[35] Trendelenburg AU, Meyer A, Rohner D, Boyle J, Hatakeyama S, et al. (2009) Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube

[36] Guo W, Flanagan J, Jasuja R, Kirkland J, Jiang L, et al. (2008) The effects of myostatin on adipogenic differentiation of human bone marrow-derived mesenchymal stem cells are mediated through cross-communication between Smad3 and Wnt/beta-catenin

[37] Bernardi H, Gay S, Fedon Y, Vernus B, Bonnieu A, et al. (2011) Wnt4 activates the canonical beta-catenin pathway and regulates negatively myostatin: functional

[38] Wodarz A, Nusse R (1998) Mechanisms of Wnt signaling in development. Annu Rev

[39] Fradkin LG, Noordermeer JN, Nusse R (1995) The Drosophila Wnt protein DWnt-3 is a secreted glycoprotein localized on the axon tracts of the embryonic CNS. Dev Biol 168:

[41] Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, et al. (2003) Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423: 448-452. [42] Mason JO, Kitajewski J, Varmus HE (1992) Mutational analysis of mouse Wnt-1 identifies two temperature-sensitive alleles and attributes of Wnt-1 protein essential for

[43] Zakin LD, Mazan S, Maury M, Martin N, Guenet JL, et al. (1998) Structure and expression of Wnt13, a novel mouse Wnt2 related gene. Mech Dev 73: 107-116. [44] Binari RC, Staveley BE, Johnson WA, Godavarti R, Sasisekharan R, et al. (1997) Genetic evidence that heparin-like glycosaminoglycans are involved in wingless signaling.

[45] Reichsman F, Smith L, Cumberledge S (1996) Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction. J Cell

[46] Hausmann G, Banziger C, Basler K (2007) Helping Wingless take flight: how WNT

implication in myogenesis. Am J Physiol Cell Physiol 300: C1122-1138.

child. N Engl J Med 351: 1030-1031; author reply 1030-1031.

differentiation. Am J Physiol Cell Physiol 282: C993-999.

size. Am J Physiol Cell Physiol 296: C1258-1270.

signaling pathways. J Biol Chem 283: 9136-9145.

[40] Miller JR (2002) The Wnts. Genome Biol 3: REVIEWS3001.

proteins are secreted. Nat Rev Mol Cell Biol 8: 331-336.

transformation of a mammary cell line. Mol Biol Cell 3: 521-533.

Exp Cell Res 286: 263-275.

Cell Dev Biol 14: 59-88.

Development 124: 2623-2632.

Biol 135: 819-827.

202-213.

49840.

[31] Williams MS (2004) Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med 351: 1030-1031; author reply 1030-1031.

90 Skeletal Muscle – From Myogenesis to Clinical Relations

pathways. Cell Calcium 38: 439-446.

human diseases. Dev Dyn 239: 1-15.

morphogen gradient. Cell 87: 833-844.

and hypertrophy. Faseb J 20: 580-582.

a new TGF-beta superfamily member. Nature 387: 83-90.

Dev Cell 12: 113-127.

Biol 10: 293-300.

205: 1292-1294.

215-235.

841-852.

186-191.

to cardiomyogenic cells. J Biol Chem 280: 16838-16842.

ventral axis of the Drosophila wing. Development 124: 871-880.

mature muscle fibers isolated in vitro. Dev Biol 45: 260-275.

[14] Kohn AD, Moon RT (2005) Wnt and calcium signaling: beta-catenin-independent

[15] Koyanagi M, Haendeler J, Badorff C, Brandes RP, Hoffmann J, et al. (2005) Noncanonical Wnt signaling enhances differentiation of human circulating progenitor cells

[16] Minami Y, Oishi I, Endo M, Nishita M (2010) Ror-family receptor tyrosine kinases in noncanonical Wnt signaling: their implications in developmental morphogenesis and

[17] Tu X, Joeng KS, Nakayama KI, Nakayama K, Rajagopal J, et al. (2007) Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation.

[18] Neumann CJ, Cohen SM (1997) Long-range action of Wingless organizes the dorsal-

[19] Strigini M, Cohen SM (2000) Wingless gradient formation in the Drosophila wing. Curr

[20] Zecca M, Basler K, Struhl G (1996) Direct and long-range action of a wingless

[21] Bischoff R (1975) Regeneration of single skeletal muscle fibers in vitro. Anat Rec 182:

[22] Konigsberg UR, Lipton BH, Konigsberg IR (1975) The regenerative response of single

[23] Lipton BH, Schultz E (1979) Developmental fate of skeletal muscle satellite cells. Science

[24] Brack AS, Conboy MJ, Roy S, Lee M, Kuo CJ, et al. (2007) Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis. Science 317: 807-810. [25] Munsterberg AE, Kitajewski J, Bumcrot DA, McMahon AP, Lassar AB (1995) Combinatorial signaling by Sonic hedgehog and Wnt family members induces

[26] Polesskaya A, Seale P, Rudnicki MA (2003) Wnt signaling induces the myogenic specification of resident CD45+ adult stem cells during muscle regeneration. Cell 113:

[27] von Maltzahn J, Bentzinger CF, Rudnicki MA (2012) Wnt7a-Fzd7 signalling directly activates the Akt/mTOR anabolic growth pathway in skeletal muscle. Nat Cell Biol 14:

[28] Steelman CA, Recknor JC, Nettleton D, Reecy JM (2006) Transcriptional profiling of myostatin-knockout mice implicates Wnt signaling in postnatal skeletal muscle growth

[29] Kambadur R, Sharma M, Smith TP, Bass JJ (1997) Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle. Genome Res 7: 910-916. [30] McPherron AC, Lawler AM, Lee SJ (1997) Regulation of skeletal muscle mass in mice by

myogenic bHLH gene expression in the somite. Genes Dev 9: 2911-2922.

	- [47] Neumann S, Coudreuse DY, van der Westhuyzen DR, Eckhardt ER, Korswagen HC, et al. (2009) Mammalian Wnt3a is released on lipoprotein particles. Traffic 10: 334-343.

Role and Function of Wnts in the Regulation of Myogenesis: When Wnt Meets Myostatin 93

[63] Kengaku M, Capdevila J, Rodriguez-Esteban C, De La Pena J, Johnson RL, et al. (1998) Distinct WNT pathways regulating AER formation and dorsoventral polarity in the

[64] Strutt DI, Weber U, Mlodzik M (1997) The role of RhoA in tissue polarity and Frizzled

[65] Boutros M, Mlodzik M (1999) Dishevelled: at the crossroads of divergent intracellular

[66] Habas R, Kato Y, He X (2001) Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1. Cell 107: 843-854. [67] Winter CG, Wang B, Ballew A, Royou A, Karess R, et al. (2001) Drosophila Rhoassociated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the

[68] Fanto M, McNeill H (2004) Planar polarity from flies to vertebrates. J Cell Sci 117: 527-

[69] Bhanot P, Brink M, Samos CH, Hsieh JC, Wang Y, et al. (1996) A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 382: 225-230. [70] Bhat KM (1998) frizzled and frizzled 2 play a partially redundant role in wingless signaling and have similar requirements to wingless in neurogenesis. Cell 95: 1027-1036. [71] Hsieh JC, Rattner A, Smallwood PM, Nathans J (1999) Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein.

[72] Chen CM, Strapps W, Tomlinson A, Struhl G (2004) Evidence that the cysteine-rich domain of Drosophila Frizzled family receptors is dispensable for transducing

[73] Zhang J, Carthew RW (1998) Interactions between Wingless and DFz2 during

[74] Dann CE, Hsieh JC, Rattner A, Sharma D, Nathans J, et al. (2001) Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains.

[77] Nusse R, Rulifson E, Fish M, Harryman-Samos C, Brink M, et al. (2000) Interactions between wingless and frizzled molecules in Drosophila. Ernst Schering Res Found

[78] Cadigan KM, Liu YI (2006) Wnt signaling: complexity at the surface. J Cell Sci 119: 395-

[79] He X, Semenov M, Tamai K, Zeng X (2004) LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way. Development 131: 1663-1677. [80] Tamai K, Semenov M, Kato Y, Spokony R, Liu C, et al. (2000) LDL-receptor-related

[75] Hsieh JC (2004) Specificity of WNT-receptor interactions. Front Biosci 9: 1333-1338. [76] Rulifson EJ, Wu CH, Nusse R (2000) Pathway specificity by the bifunctional receptor

chick limb bud. Science 280: 1274-1277.

signaling pathways. Mech Dev 83: 27-37.

signalling. Nature 387: 292-295.

actin cytoskeleton. Cell 105: 81-91.

Proc Natl Acad Sci U S A 96: 3546-3551.

Nature 412: 86-90.

Workshop: 1-11.

402.

Wingless. Proc Natl Acad Sci U S A 101: 15961-15966.

Drosophila wing development. Development 125: 3075-3085.

frizzled is determined by affinity for wingless. Mol Cell 6: 117-126.

proteins in Wnt signal transduction. Nature 407: 530-535.

533.

[63] Kengaku M, Capdevila J, Rodriguez-Esteban C, De La Pena J, Johnson RL, et al. (1998) Distinct WNT pathways regulating AER formation and dorsoventral polarity in the chick limb bud. Science 280: 1274-1277.

92 Skeletal Muscle – From Myogenesis to Clinical Relations

phosphorylation. J Neurochem 92: 1550-1553.

transduction pathways. Genome Biol 5: 234.

epithelial cells by Wnt genes. Mol Cell Biol 14: 6278-6286.

Oncogene 25: 7461-7468.

1349-1358.

167-177.

369-387.

236: 2800-2807.

Cell 4: 535-547.

Physiol Renal Physiol 282: F431-441.

rats. J Am Soc Nephrol 14: 1223-1233.

[47] Neumann S, Coudreuse DY, van der Westhuyzen DR, Eckhardt ER, Korswagen HC, et al. (2009) Mammalian Wnt3a is released on lipoprotein particles. Traffic 10: 334-343. [48] Panakova D, Sprong H, Marois E, Thiele C, Eaton S (2005) Lipoprotein particles are

[49] Schulte G, Bryja V, Rawal N, Castelo-Branco G, Sousa KM, et al. (2005) Purified Wnt-5a increases differentiation of midbrain dopaminergic cells and dishevelled

[50] Yang-Snyder J, Miller JR, Brown JD, Lai CJ, Moon RT (1996) A frizzled homolog

[51] Huang HC, Klein PS (2004) The Frizzled family: receptors for multiple signal

[52] Mikels AJ, Nusse R (2006) Wnts as ligands: processing, secretion and reception.

[53] Wong GT, Gavin BJ, McMahon AP (1994) Differential transformation of mammary

[54] Shimizu H, Julius MA, Giarre M, Zheng Z, Brown AM, et al. (1997) Transformation by Wnt family proteins correlates with regulation of beta-catenin. Cell Growth Differ 8:

[55] Bernard P, Fleming A, Lacombe A, Harley VR, Vilain E (2008) Wnt4 inhibits betacatenin/TCF signalling by redirecting beta-catenin to the cell membrane. Biol Cell 100:

[56] Jordan BK, Shen JH, Olaso R, Ingraham HA, Vilain E (2003) Wnt4 overexpression disrupts normal testicular vasculature and inhibits testosterone synthesis by repressing steroidogenic factor 1/beta-catenin synergy. Proc Natl Acad Sci U S A 100: 10866-10871. [57] Liu CF, Parker K, Yao HH (2010) WNT4/beta-catenin pathway maintains female germ cell survival by inhibiting activin betaB in the mouse fetal ovary. PLoS One 5: e10382. [58] Lyons JP, Mueller UW, Ji H, Everett C, Fang X, et al. (2004) Wnt-4 activates the canonical beta-catenin-mediated Wnt pathway and binds Frizzled-6 CRD: functional implications of Wnt/beta-catenin activity in kidney epithelial cells. Exp Cell Res 298:

[59] Surendran K, McCaul SP, Simon TC (2002) A role for Wnt-4 in renal fibrosis. Am J

[60] Takata H, Terada K, Oka H, Sunada Y, Moriguchi T, et al. (2007) Involvement of Wnt4 signaling during myogenic proliferation and differentiation of skeletal muscle. Dev Dyn

[61] Terada Y, Tanaka H, Okado T, Shimamura H, Inoshita S, et al. (2003) Expression and function of the developmental gene Wnt-4 during experimental acute renal failure in

[62] Le Grand F, Jones AE, Seale V, Scime A, Rudnicki MA (2009) Wnt7a activates the planar cell polarity pathway to drive the symmetric expansion of satellite stem cells. Cell Stem

required for Hedgehog and Wingless signalling. Nature 435: 58-65.

functions in a vertebrate Wnt signaling pathway. Curr Biol 6: 1302-1306.

	- [81] Kato M, Patel MS, Levasseur R, Lobov I, Chang BH, et al. (2002) Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J Cell Biol 157: 303-314.

Role and Function of Wnts in the Regulation of Myogenesis: When Wnt Meets Myostatin 95

[97] Miller JR, Hocking AM, Brown JD, Moon RT (1999) Mechanism and function of signal transduction by the Wnt/beta-catenin and Wnt/Ca2+ pathways. Oncogene 18: 7860-

[98] Wallingford JB, Fraser SE, Harland RM (2002) Convergent extension: the molecular control of polarized cell movement during embryonic development. Dev Cell 2: 695-

[99] Marlow F, Topczewski J, Sepich D, Solnica-Krezel L (2002) Zebrafish Rho kinase 2 acts downstream of Wnt11 to mediate cell polarity and effective convergence and extension

[100] Franco CA, Liebner S, Gerhardt H (2009) Vascular morphogenesis: a Wnt for every

[101] Miller JR, Moon RT (1996) Signal transduction through beta-catenin and specification

[102] Willert K, Nusse R (1998) Beta-catenin: a key mediator of Wnt signaling. Curr Opin

[103] Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, et al. (1996) Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science 272:

[104] Rubinfeld B, Souza B, Albert I, Muller O, Chamberlain SH, et al. (1993) Association of

[105] Su LK, Vogelstein B, Kinzler KW (1993) Association of the APC tumor suppressor

[106] Yost C, Farr GH, 3rd, Pierce SB, Ferkey DM, Chen MM, et al. (1998) GBP, an inhibitor of GSK-3, is implicated in Xenopus development and oncogenesis. Cell 93: 1031-1041. [107] Zeng L, Fagotto F, Zhang T, Hsu W, Vasicek TJ, et al. (1997) The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis

[108] Yost C, Torres M, Miller JR, Huang E, Kimelman D, et al. (1996) The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus

[109] Aberle H, Bauer A, Stappert J, Kispert A, Kemler R (1997) beta-catenin is a target for

[110] Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, et al. (1997) Activation of betacatenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275:

[111] Rubinfeld B, Robbins P, El-Gamil M, Albert I, Porfiri E, et al. (1997) Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 275: 1790-1792. [112] Itoh K, Brott BK, Bae GU, Ratcliffe MJ, Sokol SY (2005) Nuclear localization is required

[113] Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, et al. (1996) Functional interaction of beta-catenin with the transcription factor LEF-1. Nature 382: 638-642.

7872.

706.

movements. Curr Biol 12: 876-884.

Genet Dev 8: 95-102.

formation. Cell 90: 181-192.

1023-1026.

1787-1790.

vessel? Curr Opin Genet Dev 19: 476-483.

protein with catenins. Science 262: 1734-1737.

of cell fate during embryogenesis. Genes Dev 10: 2527-2539.

the APC gene product with beta-catenin. Science 262: 1731-1734.

embryos by glycogen synthase kinase 3. Genes Dev 10: 1443-1454.

for Dishevelled function in Wnt/beta-catenin signaling. J Biol 4: 3.

the ubiquitin-proteasome pathway. EMBO J 16: 3797-3804.

[97] Miller JR, Hocking AM, Brown JD, Moon RT (1999) Mechanism and function of signal transduction by the Wnt/beta-catenin and Wnt/Ca2+ pathways. Oncogene 18: 7860- 7872.

94 Skeletal Muscle – From Myogenesis to Clinical Relations

Frizzled and LRP. Development 131: 5103-5115.

and patterns. Bioessays 24: 811-820.

signaling. J Biol Chem 275: 4374-4382.

Proc Natl Acad Sci U S A 94: 11196-11200.

391: 357-362.

13636-13641.

Biol 3: 683-686.

Sci 116: 2627-2634.

protein mediates Wnt signalling in mice. Nature 407: 535-538.

frizzled receptors. Proc Natl Acad Sci U S A 94: 2859-2863.

tissue inhibitors of metalloproteases. Protein Sci 8: 1636-1642.

their significance to Wnt signal transduction. Dev Biol 253: 1-17.

[81] Kato M, Patel MS, Levasseur R, Lobov I, Chang BH, et al. (2002) Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J Cell Biol 157: 303-314. [82] Liu G, Bafico A, Harris VK, Aaronson SA (2003) A novel mechanism for Wnt activation of canonical signaling through the LRP6 receptor. Mol Cell Biol 23: 5825-5835. [83] Cong F, Schweizer L, Varmus H (2004) Wnt signals across the plasma membrane to activate the beta-catenin pathway by forming oligomers containing its receptors,

[84] Pinson KI, Brennan J, Monkley S, Avery BJ, Skarnes WC (2000) An LDL-receptor-related

[85] Wehrli M, Dougan ST, Caldwell K, O'Keefe L, Schwartz S, et al. (2000) arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature 407: 527-530. [86] Jones SE, Jomary C (2002) Secreted Frizzled-related proteins: searching for relationships

[87] Glinka A, Wu W, Delius H, Monaghan AP, Blumenstock C, et al. (1998) Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature

[88] Krupnik VE, Sharp JD, Jiang C, Robison K, Chickering TW, et al. (1999) Functional and

[89] Kawano Y, Kypta R (2003) Secreted antagonists of the Wnt signalling pathway. J Cell

[90] Rattner A, Hsieh JC, Smallwood PM, Gilbert DJ, Copeland NG, et al. (1997) A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of

[91] Melkonyan HS, Chang WC, Shapiro JP, Mahadevappa M, Fitzpatrick PA, et al. (1997) SARPs: a family of secreted apoptosis-related proteins. Proc Natl Acad Sci U S A 94:

[92] Uren A, Reichsman F, Anest V, Taylor WG, Muraiso K, et al. (2000) Secreted frizzledrelated protein-1 binds directly to Wingless and is a biphasic modulator of Wnt

[93] Lin K, Wang S, Julius MA, Kitajewski J, Moos M, Jr., et al. (1997) The cysteine-rich frizzled domain of Frzb-1 is required and sufficient for modulation of Wnt signaling.

[94] Banyai L, Patthy L (1999) The NTR module: domains of netrins, secreted frizzled related proteins, and type I procollagen C-proteinase enhancer protein are homologous with

[95] Bafico A, Liu G, Yaniv A, Gazit A, Aaronson SA (2001) Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow. Nat Cell

[96] Wharton KA, Jr. (2003) Runnin' with the Dvl: proteins that associate with Dsh/Dvl and

structural diversity of the human Dickkopf gene family. Gene 238: 301-313.

	- [114] Molenaar M, van de Wetering M, Oosterwegel M, Peterson-Maduro J, Godsave S, et al. (1996) XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86: 391-399.

Role and Function of Wnts in the Regulation of Myogenesis: When Wnt Meets Myostatin 97

[132] Tajbakhsh S, Borello U, Vivarelli E, Kelly R, Papkoff J, et al. (1998) Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5. Development

[133] Hollway G, Currie P (2005) Vertebrate myotome development. Birth Defects Res C

[134] Brent AE, Tabin CJ (2002) Developmental regulation of somite derivatives: muscle,

[135] Ordahl CP, Le Douarin NM (1992) Two myogenic lineages within the developing

[136] Kassar-Duchossoy L, Giacone E, Gayraud-Morel B, Jory A, Gomes D, et al. (2005) Pax3/Pax7 mark a novel population of primitive myogenic cells during development.

[137] Braun T, Rudnicki MA, Arnold HH, Jaenisch R (1992) Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death.

[138] Tajbakhsh S, Rocancourt D, Buckingham M (1996) Muscle progenitor cells failing to respond to positional cues adopt non-myogenic fates in myf-5 null mice. Nature 384:

[139] Lagha M, Sato T, Bajard L, Daubas P, Esner M, et al. (2008) Regulation of skeletal muscle stem cell behavior by Pax3 and Pax7. Cold Spring Harb Symp Quant Biol 73:

[140] Francis-West PH, Antoni L, Anakwe K (2003) Regulation of myogenic differentiation

[141] Buckingham M (2006) Myogenic progenitor cells and skeletal myogenesis in

[142] Tajbakhsh S, Rocancourt D, Cossu G, Buckingham M (1997) Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD.

[143] Relaix F, Rocancourt D, Mansouri A, Buckingham M (2005) A Pax3/Pax7-dependent

[144] Buckingham M (2007) Skeletal muscle progenitor cells and the role of Pax genes. C R

[145] Gros J, Manceau M, Thome V, Marcelle C (2005) A common somitic origin for

[146] Schultz E (1996) Satellite cell proliferative compartments in growing skeletal muscles.

[147] Ben-Yair R, Kalcheim C (2008) Notch and bone morphogenetic protein differentially act on dermomyotome cells to generate endothelium, smooth, and striated muscle. J

population of skeletal muscle progenitor cells. Nature 435: 948-953.

embryonic muscle progenitors and satellite cells. Nature 435: 954-958.

cartilage and tendon. Curr Opin Genet Dev 12: 548-557.

125: 4155-4162.

Embryo Today 75: 172-179.

Genes Dev 19: 1426-1431.

Cell 71: 369-382.

Cell 89: 127-138.

Biol 330: 530-533.

Dev Biol 175: 84-94.

Cell Biol 180: 607-618.

266-270.

307-315.

somite. Development 114: 339-353.

in the developing limb bud. J Anat 202: 69-81.

vertebrates. Curr Opin Genet Dev 16: 525-532.

[132] Tajbakhsh S, Borello U, Vivarelli E, Kelly R, Papkoff J, et al. (1998) Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5. Development 125: 4155-4162.

96 Skeletal Muscle – From Myogenesis to Clinical Relations

Xenopus embryos. Cell 86: 391-399.

polarity gene dTCF. Cell 88: 789-799.

Science 303: 1483-1487.

413.

14: 1583-1588.

2330-2336.

Biol 47: 467-477.

crossed. Dev Cell 11: 601-612.

Wnt target gene expression. EMBO Rep 1: 24-28.

fates in Xenopus. J Biol Chem 275: 12701-12711.

hair production. Development 135: 3103-3111.

cell's compass. Cold Spring Harb Perspect Biol 1: a002964.

of skeletal muscle: from somite to limb. J Anat 202: 59-68.

Frizzled functions. Science 300: 1529-1530.

[114] Molenaar M, van de Wetering M, Oosterwegel M, Peterson-Maduro J, Godsave S, et al. (1996) XTcf-3 transcription factor mediates beta-catenin-induced axis formation in

[115] van de Wetering M, Cavallo R, Dooijes D, van Beest M, van Es J, et al. (1997) Armadillo coactivates transcription driven by the product of the Drosophila segment

[116] Brannon M, Brown JD, Bates R, Kimelman D, Moon RT (1999) XCtBP is a XTcf-3 corepressor with roles throughout Xenopus development. Development 126: 3159-3170. [117] Nelson WJ, Nusse R (2004) Convergence of Wnt, beta-catenin, and cadherin pathways.

[118] Perez-Moreno M, Fuchs E (2006) Catenins: keeping cells from getting their signals

[119] Hecht A, Kemler R (2000) Curbing the nuclear activities of beta-catenin. Control over

[120] Wang HY, Malbon CC (2003) Wnt signaling, Ca2+, and cyclic GMP: visualizing

[121] Kuhl M, Sheldahl LC, Park M, Miller JR, Moon RT (2000) The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet 16: 279-283. [122] Kuhl M, Sheldahl LC, Malbon CC, Moon RT (2000) Ca(2+)/calmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell

[123] Slusarski DC, Corces VG, Moon RT (1997) Interaction of Wnt and a Frizzled homologue triggers G-protein-linked phosphatidylinositol signalling. Nature 390: 410-

[125] Vladar EK, Antic D, Axelrod JD (2009) Planar cell polarity signaling: the developing

[126] Strutt D, Warrington SJ (2008) Planar polarity genes in the Drosophila wing regulate the localisation of the FH3-domain protein Multiple Wing Hairs to control the site of

[127] Katoh M (2005) WNT/PCP signaling pathway and human cancer (review). Oncol Rep

[128] Buckingham M, Bajard L, Chang T, Daubas P, Hadchouel J, et al. (2003) The formation

[129] Parker MH, Seale P, Rudnicki MA (2003) Looking back to the embryo: defining

[130] Gridley T (2006) The long and short of it: somite formation in mice. Dev Dyn 235:

[131] Gilbert SF (2003) The morphogenesis of evolutionary developmental biology. Int J Dev

transcriptional networks in adult myogenesis. Nat Rev Genet 4: 497-507.

[124] Nusse R (1999) WNT targets. Repression and activation. Trends Genet 15: 1-3.

[148] Vasyutina E, Lenhard DC, Birchmeier C (2007) Notch function in myogenesis. Cell Cycle 6: 1451-1454.

Role and Function of Wnts in the Regulation of Myogenesis: When Wnt Meets Myostatin 99

[163] Thomas M, Langley B, Berry C, Sharma M, Kirk S, et al. (2000) Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. J Biol Chem

[164] Taylor WE, Bhasin S, Artaza J, Byhower F, Azam M, et al. (2001) Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells. Am J Physiol Endocrinol

[165] Manceau M, Gros J, Savage K, Thome V, McPherron A, et al. (2008) Myostatin promotes the terminal differentiation of embryonic muscle progenitors. Genes Dev 22:

[166] Kollias HD, McDermott JC (2008) Transforming growth factor-beta and myostatin

[167] Grobet L, Martin LJ, Poncelet D, Pirottin D, Brouwers B, et al. (1997) A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat Genet 17:

[168] McPherron AC, Lee SJ (1997) Double muscling in cattle due to mutations in the

[169] Schuelke M, Wagner KR, Stolz LE, Hubner C, Riebel T, et al. (2004) Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med 350: 2682-

[170] Duprez D, Bell EJ, Richardson MK, Archer CW, Wolpert L, et al. (1996) Overexpression of BMP-2 and BMP-4 alters the size and shape of developing skeletal

[171] Sela-Donenfeld D, Kalcheim C (2002) Localized BMP4-noggin interactions generate the

[172] Amthor H, Christ B, Rashid-Doubell F, Kemp CF, Lang E, et al. (2002) Follistatin regulates bone morphogenetic protein-7 (BMP-7) activity to stimulate embryonic

[173] Souza TA, Chen X, Guo Y, Sava P, Zhang J, et al. (2008) Proteomic identification and functional validation of activins and bone morphogenetic protein 11 as candidate novel

[174] McCarthy JJ (2008) MicroRNA-206: the skeletal muscle-specific myomiR. Biochim

[175] Chen JF, Mandel EM, Thomson JM, Wu Q, Callis TE, et al. (2006) The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation.

[176] Sweetman D, Goljanek K, Rathjen T, Oustanina S, Braun T, et al. (2008) Specific requirements of MRFs for the expression of muscle specific microRNAs, miR-1, miR-206

[177] Tedesco FS, Dellavalle A, Diaz-Manera J, Messina G, Cossu G (2010) Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells. J Clin Invest 120: 11-19.

dynamic patterning of noggin expression in somites. Dev Biol 246: 311-328.

signaling in skeletal muscle. J Appl Physiol 104: 579-587.

myostatin gene. Proc Natl Acad Sci U S A 94: 12457-12461.

elements in the chick limb. Mech Dev 57: 145-157.

muscle mass regulators. Mol Endocrinol 22: 2689-2702.

muscle growth. Dev Biol 243: 115-127.

Biophys Acta 1779: 682-691.

and miR-133. Dev Biol 321: 491-499.

Nat Genet 38: 228-233.

275: 40235-40243.

Metab 280: E221-228.

668-681.

71-74.

2688.

[163] Thomas M, Langley B, Berry C, Sharma M, Kirk S, et al. (2000) Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. J Biol Chem 275: 40235-40243.

98 Skeletal Muscle – From Myogenesis to Clinical Relations

mammals. Embo J 18: 6867-6872.

Development 125: 2019-2030.

Development 121: 651-660.

of myogenesis. Cold Spring Harb Perspect Biol 4.

Cycle 6: 1451-1454.

495-505.

228: 86-94.

4247-4255.

Genes Dev 16: 114-126.

expression. Development 135: 237-247.

cells. J Cell Physiol 212: 817-826.

elongation of early muscle fibres. Nature 457: 589-593.

differentiation. J Cell Biochem 101: 1109-1124.

[148] Vasyutina E, Lenhard DC, Birchmeier C (2007) Notch function in myogenesis. Cell

[149] Cossu G, Borello U (1999) Wnt signaling and the activation of myogenesis in

[150] Teillet M, Watanabe Y, Jeffs P, Duprez D, Lapointe F, et al. (1998) Sonic hedgehog is required for survival of both myogenic and chondrogenic somitic lineages.

[151] Bentzinger CF, Wang YX, Rudnicki MA (2012) Building muscle: molecular regulation

[152] Duprez D, Fournier-Thibault C, Le Douarin N (1998) Sonic Hedgehog induces proliferation of committed skeletal muscle cells in the chick limb. Development 125:

[153] Christ B, Brand-Saberi B (2002) Limb muscle development. Int J Dev Biol 46: 905-914. [154] Munsterberg AE, Lassar AB (1995) Combinatorial signals from the neural tube, floor plate and notochord induce myogenic bHLH gene expression in the somite.

[155] Wagner J, Schmidt C, Nikowits W, Jr., Christ B (2000) Compartmentalization of the somite and myogenesis in chick embryos are influenced by wnt expression. Dev Biol

[156] Cossu G, Kelly R, Tajbakhsh S, Di Donna S, Vivarelli E, et al. (1996) Activation of different myogenic pathways: myf-5 is induced by the neural tube and MyoD by the

[157] Borello U, Coletta M, Tajbakhsh S, Leyns L, De Robertis EM, et al. (1999) Transplacental delivery of the Wnt antagonist Frzb1 inhibits development of caudal paraxial mesoderm and skeletal myogenesis in mouse embryos. Development 126:

[158] Gustafsson MK, Pan H, Pinney DF, Liu Y, Lewandowski A, et al. (2002) Myf5 is a direct target of long-range Shh signaling and Gli regulation for muscle specification.

[159] Alvarez-Medina R, Cayuso J, Okubo T, Takada S, Marti E (2008) Wnt canonical pathway restricts graded Shh/Gli patterning activity through the regulation of Gli3

[160] Gros J, Serralbo O, Marcelle C (2009) WNT11 acts as a directional cue to organize the

[161] Baksh D, Boland GM, Tuan RS (2007) Cross-talk between Wnt signaling pathways in human mesenchymal stem cells leads to functional antagonism during osteogenic

[162] Baksh D, Tuan RS (2007) Canonical and non-canonical Wnts differentially affect the development potential of primary isolate of human bone marrow mesenchymal stem

dorsal ectoderm in mouse paraxial mesoderm. Development 122: 429-437.

[178] Cornelison DD, Wold BJ (1997) Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 191: 270-283.

Role and Function of Wnts in the Regulation of Myogenesis: When Wnt Meets Myostatin 101

[195] Goldspink G, Fernandes K, Williams PE, Wells DJ (1994) Age-related changes in collagen gene expression in the muscles of mdx dystrophic and normal mice.

[196] Conboy IM, Rando TA (2005) Aging, stem cells and tissue regeneration: lessons from

[197] Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, et al. (2005) Rejuvenation of aged progenitor cells by exposure to a young systemic environment.

[198] Liu H, Fergusson MM, Castilho RM, Liu J, Cao L, et al. (2007) Augmented Wnt

[199] Dellavalle A, Sampaolesi M, Tonlorenzi R, Tagliafico E, Sacchetti B, et al. (2007) Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells.

[200] Sampaolesi M, Blot S, D'Antona G, Granger N, Tonlorenzi R, et al. (2006) Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature 444:

[201] Galvez BG, Sampaolesi M, Brunelli S, Covarello D, Gavina M, et al. (2006) Complete repair of dystrophic skeletal muscle by mesoangioblasts with enhanced migration

[202] Sampaolesi M, Torrente Y, Innocenzi A, Tonlorenzi R, D'Antona G, et al. (2003) Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of

[203] Mitchell KJ, Pannerec A, Cadot B, Parlakian A, Besson V, et al. (2010) Identification and characterization of a non-satellite cell muscle resident progenitor during postnatal

[204] Gussoni E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, et al. (1999) Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401: 390-394. [205] LaBarge MA, Blau HM (2002) Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury. Cell

[206] Tanaka S, Terada K, Nohno T (2011) Canonical Wnt signaling is involved in switching from cell proliferation to myogenic differentiation of mouse myoblast cells. J Mol Signal

[207] Otto A, Schmidt C, Luke G, Allen S, Valasek P, et al. (2008) Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration. J Cell Sci

[208] Lathrop B, Olson E, Glaser L (1985) Control by fibroblast growth factor of

[209] Anakwe K, Robson L, Hadley J, Buxton P, Church V, et al. (2003) Wnt signalling regulates myogenic differentiation in the developing avian wing. Development 130:

differentiation in the BC3H1 muscle cell line. J Cell Biol 100: 1540-1547.

signaling in a mammalian model of accelerated aging. Science 317: 803-806.

Neuromuscul Disord 4: 183-191.

muscle. Cell Cycle 4: 407-410.

Nature 433: 760-764.

Nat Cell Biol 9: 255-267.

ability. J Cell Biol 174: 231-243.

mesoangioblasts. Science 301: 487-492.

development. Nat Cell Biol 12: 257-266.

574-579.

111: 589-601.

121: 2939-2950.

3503-3514.

6: 12.

[195] Goldspink G, Fernandes K, Williams PE, Wells DJ (1994) Age-related changes in collagen gene expression in the muscles of mdx dystrophic and normal mice. Neuromuscul Disord 4: 183-191.

100 Skeletal Muscle – From Myogenesis to Clinical Relations

others? J Cell Sci 121: 2975-2982.

Semin Cell Dev Biol 18: 870-882.

regeneration. Physiol Rev 84: 209-238.

dystrophy. Biochim Biophys Acta 1772: 272-283.

therapeutic potential. Trends Mol Med 14: 82-91.

Tissue Res 267: 99-104.

15: 666-673.

134.

357.

677-687.

Nature 431: 466-471.

Sci Med Sci 58: M911-916.

[178] Cornelison DD, Wold BJ (1997) Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 191: 270-283. [179] Grounds MD, Garrett KL, Lai MC, Wright WE, Beilharz MW (1992) Identification of skeletal muscle precursor cells in vivo by use of MyoD1 and myogenin probes. Cell

[180] Zammit PS (2008) All muscle satellite cells are equal, but are some more equal than

[181] Charge SB, Rudnicki MA (2004) Cellular and molecular regulation of muscle

[182] Dhawan J, Rando TA (2005) Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation and replenishment. Trends Cell Biol

[183] Price FD, Kuroda K, Rudnicki MA (2007) Stem cell based therapies to treat muscular

[184] Fukada S, Uezumi A, Ikemoto M, Masuda S, Segawa M, et al. (2007) Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells 25: 2448-2459. [185]  Cell Stem Cell 5: 132-

[188] Tajbakhsh S, Bober E, Babinet C, Pournin S, Arnold H, et al. (1996) Gene targeting the myf-5 locus with nlacZ reveals expression of this myogenic factor in mature skeletal

[189] Zammit PS, Golding JP, Nagata Y, Hudon V, Partridge TA, et al. (2004) Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J Cell Biol 166: 347-

[190] Kuang S, Kuroda K, Le Grand F, Rudnicki MA (2007) Asymmetric self-renewal and

[191] Kassar-Duchossoy L, Gayraud-Morel B, Gomes D, Rocancourt D, Buckingham M, et al. (2004) Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice.

[192] Shinin V, Gayraud-Morel B, Gomes D, Tajbakhsh S (2006) Asymmetric division and cosegregation of template DNA strands in adult muscle satellite cells. Nat Cell Biol 8:

[193] Marcell TJ (2003) Sarcopenia: causes, consequences, and preventions. J Gerontol A Biol

[194] Conboy IM, Conboy MJ, Smythe GM, Rando TA (2003) Notch-mediated restoration of

muscle fibres as well as early embryonic muscle. Dev Dyn 206: 291-300.

commitment of satellite stem cells in muscle. Cell 129: 999-1010.

regenerative potential to aged muscle. Science 302: 1575-1577.

	- [210] Armstrong DD, Wong VL, Esser KA (2006) Expression of beta-catenin is necessary for physiological growth of adult skeletal muscle. Am J Physiol Cell Physiol 291: C185-188.

**Section 2**

**Molecular Biology**

## **Molecular Biology**

102 Skeletal Muscle – From Myogenesis to Clinical Relations

adipogenesis. Mol Cell Biol 23: 7230-7242.

responses. Cell 95: 737-740.

system. Embo J 19: 1745-1754.

10649-10659.

421.

[210] Armstrong DD, Wong VL, Esser KA (2006) Expression of beta-catenin is necessary for physiological growth of adult skeletal muscle. Am J Physiol Cell Physiol 291: C185-188. [211] Han XH, Jin YR, Seto M, Yoon JK (2011) A WNT/beta-catenin signaling activator, Rspondin, plays positive regulatory roles during skeletal myogenesis. J Biol Chem 286:

[212] Bogdanovich S, Krag TO, Barton ER, Morris LD, Whittemore LA, et al. (2002) Functional improvement of dystrophic muscle by myostatin blockade. Nature 420: 418-

[213] Rebbapragada A, Benchabane H, Wrana JL, Celeste AJ, Attisano L (2003) Myostatin signals through a transforming growth factor beta-like signaling pathway to block

[214] Derynck R, Zhang Y, Feng XH (1998) Smads: transcriptional activators of TGF-beta

[215] Massague J, Wotton D (2000) Transcriptional control by the TGF-beta/Smad signaling

**Chapter 5**

© 2012 Heck et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0], which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Role of Heat Shock Proteins in Skeletal Muscle**

The ability of the human organism to respond with rapid and appropriate modification during physiological challenges is an essential feature for its own survival. These modifications are inseparable from a satisfactory adjustment of the physiological processes of the whole body, where physiological systems tend to maintain relatively constant composition of the internal environment (milieu intérieur), despite the constant challenges

The homeostatic regulation of any physiological variable depends on cooperative mechanisms which are activated simultaneously or in succession. Thus, intense or critical challenges to life require numerous and complex mechanisms to restore or to maintain the homeostasis. In this way, challenges generate a stress situation to the body and, depending on the severity, can cause harmful effects. Moreover, moderate challenges, which are also caused by stress, results in profitable physiological adjustments [3]. In this context, stress response may be considered a nonspecific response of the body to any demand. A stressor is an agent that produces stress at any time by different ways. The physiological adaptations of the body represent the chronologic development of the response to stressors when their

Since the muscle represents about ¾ of the body mass, a healthy muscular system is associated with the status of the other system of the body. Sick muscle system has harmful effects on human healthy and to its the capacity of interaction in this world. Skeletal muscle is a high plastic tissue that may be modified by use or disuse. Muscle composition, at chemical and structural levels, can be altered and these modification are related to the specific type of exercise that the organism is submitted. For example, the well know effect of strenght training is the hypertrophy of the muscle, which is associated to an increase in force production capacity. Strenght training may induce hypertrophy (muscle fiber enlargement)

and reproduction in any medium, provided the original work is properly cited.

that the body is submitted daily to, which is known as homeostatic control [1, 2].

Thiago Gomes Heck, Sofia Pizzato Scomazzon,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/47815

**1. Introduction**

action is prolonged [4].

Mirna Stela Ludwig and Paulo Ivo Homem de Bittencourt Jr.

## **Role of Heat Shock Proteins in Skeletal Muscle**

Thiago Gomes Heck, Sofia Pizzato Scomazzon, Mirna Stela Ludwig and Paulo Ivo Homem de Bittencourt Jr.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/47815

### **1. Introduction**

The ability of the human organism to respond with rapid and appropriate modification during physiological challenges is an essential feature for its own survival. These modifications are inseparable from a satisfactory adjustment of the physiological processes of the whole body, where physiological systems tend to maintain relatively constant composition of the internal environment (milieu intérieur), despite the constant challenges that the body is submitted daily to, which is known as homeostatic control [1, 2].

The homeostatic regulation of any physiological variable depends on cooperative mechanisms which are activated simultaneously or in succession. Thus, intense or critical challenges to life require numerous and complex mechanisms to restore or to maintain the homeostasis. In this way, challenges generate a stress situation to the body and, depending on the severity, can cause harmful effects. Moreover, moderate challenges, which are also caused by stress, results in profitable physiological adjustments [3]. In this context, stress response may be considered a nonspecific response of the body to any demand. A stressor is an agent that produces stress at any time by different ways. The physiological adaptations of the body represent the chronologic development of the response to stressors when their action is prolonged [4].

Since the muscle represents about ¾ of the body mass, a healthy muscular system is associated with the status of the other system of the body. Sick muscle system has harmful effects on human healthy and to its the capacity of interaction in this world. Skeletal muscle is a high plastic tissue that may be modified by use or disuse. Muscle composition, at chemical and structural levels, can be altered and these modification are related to the specific type of exercise that the organism is submitted. For example, the well know effect of strenght training is the hypertrophy of the muscle, which is associated to an increase in force production capacity. Strenght training may induce hypertrophy (muscle fiber enlargement)

© 2012 Heck et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0], which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and hyperplasia (increase in muscle fiber number) while endurance training promotes elevated muscle oxidative capacity (increase in mitochondrial number). All these adaptations are related to the health status of the body. On the other hand, the absence of exercise stimulus results in atrophy process and loss of functional capacity, marked by both impaired force production and metabolism of the muscle [5].

Role of Heat Shock Proteins in Skeletal Muscle 107

are barely detectable under non-stressful conditions) could be promptly synthesized under a condition of 'homeostatic stress', this being any 'homeostasis threatening' condition, such as heat, glucose deprivation, lack of growth factors and so forth. Habitually, research groups indistinctly use HSP70 as a unified term for both constitutive and inducible form. However, HSP70 is the preferable form to be used when one refers to the inducible HSP72 protein

In rodents, the Hsp70 family consists of at least nine members that differ from each other by the intracellular localization and expression pattern. Two of them, Grp78 and Grp75, are localized in the lumen of the endoplasmic reticulum and in the mitochondrial matrix, respectively, whereas the remaining seven HSP70s reside mostly in the cytosol. The only cytosolic HSP70 abundantly and constitutively expressed in all cells is Hspa8 (related to Human constitutive form HSP73). The related form for HSP72 in rodents are two proteins encoded by almost identical genes, *Hspa1a* and *Hspa1b*, termed collectively Hsp70i. As in humans, the expression of Hsp70i in rodents is low or undetectable in most "resting" normal cells and tissues, but it increases rapidly in a variety of stress conditions [14-16].

The heat shock response is regulated by high conserved cis-acting regions of the DNA (heat shock elements – HSE) and also by high conserved DNA associated trans-acting proteins named Heat Shock Transcription Factors (HSF) (Morimoto et al., 1992). While more simple organisms as insects (*Drosophila melanogaster)* and yeasts (*Saccharomyces cerevisiae)* have only one HSF, rodents have at least 2 HSF (HSF1 and HSF2) and humans have 3 isoforms of HSF (HSF1, HSF2 e HSF4) [17-20]. Possibly, more complex organisms could have used multiple HSF against different challenges during evolution. Comparing the structure of each isoform of HSF in one single species, the sequence of amino acids may be 40% identical, as HSF1 and HSF2 in mice [21, 22]. Comparing among species, the homology may be as great as 92%, as seen between HSF1 of human and rodents, or in HSF2 (95% homology between humans and rodents). Interestingly, HSF1 and HSF2 may be activated by distinct physiological phenomenon (Sistonen et al., 1992). While HSF1 and HSF2 are found in all kinds of cells, HSF4 is specifically for cells such as neurons or cardiac and skeletal muscle cells. It has also inhibitory function in heat shock response as negative regulator of HSPs expression [23, 24]. Additionally, in experimental models and cell culture procedures, is possible to identify differences in the activation of HSF1 and HSF2. The former is activated seconds after stress and this response is attenuated quickly, while the latter appears to present a latency period between the event and the response, but it remains activated for 72 h. This HSF different latency for activation suggest a cooperative role of HSF isoforms in cell protection [19] and

In unstressed cells, HSP70 may bind to the regulatory protein HSF for prevention of the trimmer formation of HSF that is required for HSPs transcription. Under stressful conditions, the free HSP70 captures the denatured proteins and then dissociates from the HSP70-HSF complex allowing the formation of HSF trimmer, thus triggering a HSP70 production. Once synthesized, new HSP70 molecules may be involved in a variety of

encoded by HSPA1A gene [12, 13]

that these genes are high conserved in nature [7].

cellular processes and exert different functions [25, 26].

In this chapter, we focus on cell physiology and molecular biology of the muscle cells from a special point of view concerning to the stress response: the role of heat shock proteins (HSPs) in muscle.

## **2. Classical roles of heat shock proteins expression**

Living organisms respond at cellular level to unfavorable conditions such as heat shock, and other stressful situations of many different origins, by a rapid, vigorous and transient acceleration in the rate of expression of specific genes: the heat shock genes. The products of these genes are commonly referred to as stress proteins or heat shock proteins (HSPs). Besides activation of heat shock genes, the expression of most other genes is inhibit as a result of stress. Thus, the stress intensity and duration leads to a perturbation of normal gene expression, which, if prolonged, can have drastic consequences for cells and system homeostasis [6, 7].

HSPs are highly conserved proteins in both eukaryotic and prokaryotic organisms and are expressed in many cell types including striated skeletal muscle. The first report about HSPs was documented by Ritossa [8, 9], after a serendipitous heat shock in salivary gland cells of *Drosophila buskii*, but heat shock proteins were only characterized later in 1974 [10]. Actually, HSPs are categorized in families according to their molecular sizes and include HSP110, HSP100, HSP90, HSP70, HSP60, HSP30 and HSP10 subclasses. In this chapter, the role of HSPs in muscle will be discussed in terms of the most studied (due to its evident high expression in mammalian cells under stress conditions) and conserved: the 70-kDa family (HSP70), which comprises a number of related proteins whose molecular weights range from 66 to 78 kDa. Many studies in human, rat and mice will be listed throughout the text, thus it is necessary to learn about the HSP70 isoforms that are encoded by a multigene family in each mammalian that will be listed below.

In humans, there are at least 13 distinct genes so far studied [11]. For the rationalization of the current nomenclature, human HSP70 genes (rat and mouse, also) have given the locus symbol HSPAx, where A defines members of HSP70 family and X designates the individual loci. In this sense, HSPA8 is the human gene that encodes a 73-kDa constitutive form of HSP70 (HSP73 or HSC70, the cognate form), while HSPA1A gene, located at the major histocompatibility complex (MHC) III region, encodes an inducible form (HSP72 or simply HSP70). In humans, but not in the rat or the mouse, there is an even higher inducible form (HSP70B') encoded by HSPA6 gene. Other representative members, besides mitochondrial (HSP75) and endoplasmic reticulum (HSP78) members of HSP70 family, are found in the intracellular space. While the constitutive form is expressed in a wide variety of cell types at basal levels (being only moderately inducible), the so-called inducible HSP70 forms (which are barely detectable under non-stressful conditions) could be promptly synthesized under a condition of 'homeostatic stress', this being any 'homeostasis threatening' condition, such as heat, glucose deprivation, lack of growth factors and so forth. Habitually, research groups indistinctly use HSP70 as a unified term for both constitutive and inducible form. However, HSP70 is the preferable form to be used when one refers to the inducible HSP72 protein encoded by HSPA1A gene [12, 13]

106 Skeletal Muscle – From Myogenesis to Clinical Relations

(HSPs) in muscle.

impaired force production and metabolism of the muscle [5].

**2. Classical roles of heat shock proteins expression**

family in each mammalian that will be listed below.

and hyperplasia (increase in muscle fiber number) while endurance training promotes elevated muscle oxidative capacity (increase in mitochondrial number). All these adaptations are related to the health status of the body. On the other hand, the absence of exercise stimulus results in atrophy process and loss of functional capacity, marked by both

In this chapter, we focus on cell physiology and molecular biology of the muscle cells from a special point of view concerning to the stress response: the role of heat shock proteins

Living organisms respond at cellular level to unfavorable conditions such as heat shock, and other stressful situations of many different origins, by a rapid, vigorous and transient acceleration in the rate of expression of specific genes: the heat shock genes. The products of these genes are commonly referred to as stress proteins or heat shock proteins (HSPs). Besides activation of heat shock genes, the expression of most other genes is inhibit as a result of stress. Thus, the stress intensity and duration leads to a perturbation of normal gene expression, which, if prolonged, can have drastic consequences for cells and system homeostasis [6, 7].

HSPs are highly conserved proteins in both eukaryotic and prokaryotic organisms and are expressed in many cell types including striated skeletal muscle. The first report about HSPs was documented by Ritossa [8, 9], after a serendipitous heat shock in salivary gland cells of *Drosophila buskii*, but heat shock proteins were only characterized later in 1974 [10]. Actually, HSPs are categorized in families according to their molecular sizes and include HSP110, HSP100, HSP90, HSP70, HSP60, HSP30 and HSP10 subclasses. In this chapter, the role of HSPs in muscle will be discussed in terms of the most studied (due to its evident high expression in mammalian cells under stress conditions) and conserved: the 70-kDa family (HSP70), which comprises a number of related proteins whose molecular weights range from 66 to 78 kDa. Many studies in human, rat and mice will be listed throughout the text, thus it is necessary to learn about the HSP70 isoforms that are encoded by a multigene

In humans, there are at least 13 distinct genes so far studied [11]. For the rationalization of the current nomenclature, human HSP70 genes (rat and mouse, also) have given the locus symbol HSPAx, where A defines members of HSP70 family and X designates the individual loci. In this sense, HSPA8 is the human gene that encodes a 73-kDa constitutive form of HSP70 (HSP73 or HSC70, the cognate form), while HSPA1A gene, located at the major histocompatibility complex (MHC) III region, encodes an inducible form (HSP72 or simply HSP70). In humans, but not in the rat or the mouse, there is an even higher inducible form (HSP70B') encoded by HSPA6 gene. Other representative members, besides mitochondrial (HSP75) and endoplasmic reticulum (HSP78) members of HSP70 family, are found in the intracellular space. While the constitutive form is expressed in a wide variety of cell types at basal levels (being only moderately inducible), the so-called inducible HSP70 forms (which In rodents, the Hsp70 family consists of at least nine members that differ from each other by the intracellular localization and expression pattern. Two of them, Grp78 and Grp75, are localized in the lumen of the endoplasmic reticulum and in the mitochondrial matrix, respectively, whereas the remaining seven HSP70s reside mostly in the cytosol. The only cytosolic HSP70 abundantly and constitutively expressed in all cells is Hspa8 (related to Human constitutive form HSP73). The related form for HSP72 in rodents are two proteins encoded by almost identical genes, *Hspa1a* and *Hspa1b*, termed collectively Hsp70i. As in humans, the expression of Hsp70i in rodents is low or undetectable in most "resting" normal cells and tissues, but it increases rapidly in a variety of stress conditions [14-16].

The heat shock response is regulated by high conserved cis-acting regions of the DNA (heat shock elements – HSE) and also by high conserved DNA associated trans-acting proteins named Heat Shock Transcription Factors (HSF) (Morimoto et al., 1992). While more simple organisms as insects (*Drosophila melanogaster)* and yeasts (*Saccharomyces cerevisiae)* have only one HSF, rodents have at least 2 HSF (HSF1 and HSF2) and humans have 3 isoforms of HSF (HSF1, HSF2 e HSF4) [17-20]. Possibly, more complex organisms could have used multiple HSF against different challenges during evolution. Comparing the structure of each isoform of HSF in one single species, the sequence of amino acids may be 40% identical, as HSF1 and HSF2 in mice [21, 22]. Comparing among species, the homology may be as great as 92%, as seen between HSF1 of human and rodents, or in HSF2 (95% homology between humans and rodents). Interestingly, HSF1 and HSF2 may be activated by distinct physiological phenomenon (Sistonen et al., 1992). While HSF1 and HSF2 are found in all kinds of cells, HSF4 is specifically for cells such as neurons or cardiac and skeletal muscle cells. It has also inhibitory function in heat shock response as negative regulator of HSPs expression [23, 24]. Additionally, in experimental models and cell culture procedures, is possible to identify differences in the activation of HSF1 and HSF2. The former is activated seconds after stress and this response is attenuated quickly, while the latter appears to present a latency period between the event and the response, but it remains activated for 72 h. This HSF different latency for activation suggest a cooperative role of HSF isoforms in cell protection [19] and that these genes are high conserved in nature [7].

In unstressed cells, HSP70 may bind to the regulatory protein HSF for prevention of the trimmer formation of HSF that is required for HSPs transcription. Under stressful conditions, the free HSP70 captures the denatured proteins and then dissociates from the HSP70-HSF complex allowing the formation of HSF trimmer, thus triggering a HSP70 production. Once synthesized, new HSP70 molecules may be involved in a variety of cellular processes and exert different functions [25, 26].

HSP70s are known to function as intracellular molecular chaperones that facilitate protein transport, prevent protein aggregation during folding and protect newly synthesized polypeptide chains against misfolding and protein denaturation. The molecular chaperones properties of such a protein allow them to assist the non-covalent assembly/disassembly of other macromolecular structures without being permanent components of such structures. Additionally, molecular chaperones assist the unfolded protein to achieve its single correct three-dimensional configuration (by still unknown mechanism it has evolved to generate this folded state), without becoming a constituent of the final folded protein [25, 26].

Role of Heat Shock Proteins in Skeletal Muscle 109

**Table 1.**

Characteristics and functions of HSP70

Most proteins destined for cell organelles are synthesized in cytosol and must cross one or more organelle membranes to reach their functional destination. For example, in the mitochondria, 95% of the proteins are made as precursor proteins in the cytosol and are mainly post translationally imported into the mitochondrial sub-compartments. In this situation, cytosolic HSP70 play an important role for maintenance of a transport-competent conformation of precursor proteins. The precursor protein is translocated in an unfolded state and are refolded later, sorted to their final destination and assembled into functional complexes [27].

The chaperone function of HSP70 includes the inhibition of the formation of nascent polypeptides. This inhibition is dose-dependent over a range of 0.1-0.4 nmoles of HSP70 and this effect is greater for the larger polypeptides. All these data suggest that high concentration of HSP70 can perturb the normal folding of nascent proteins, block cell growth and impair the cell viability. Then, these are reasons that may explain why the cells of human body have to carefully auto-regulate the levels of HSP70. Many characteristics and functions of HSFP70 are listed in table 1 and are discussed in this text.

Since the skeletal muscle is one of the most adaptable tissues of the body, every structural aspect of the muscle that can change in response to the stimulus challenge (or to its lack) may require the chaperon action of HSP70 at the molecular level. For example, major adaptations to (dis)use muscle, such as fiber type distribution, fiber diameter, myosin heavy chain profile and mitochondrial distribution, are factors that are associated to the up- or down-regulation of HSP70.

### **3. HSP70 function in the muscle**

The ability of muscle cells to express HSPs (mainly HSP70s) represents a cytoprotection mechanism because HSP70 proteins share the same overall structure. They are composed of an actin-like N-terminal nucleotide binding/ATPase domain of 45 kDa, a substrate-binding domain (SBD) of approximately 15 kDa and a C-terminal domain of approximately 10 kDa that is involved in co-chaperone binding (HU et al, 2006). It is of note that N- and C-terminal domains have expressive relevance to antigen presentation, an important way by which HSP70s participate in immune responses. With this structure, HSP70 may act as a molecular chaperone inside the muscle cell: they facilitate protein transport; prevent protein aggregation during folding; protect newly synthesized polypeptide chains against misfolding and protein denaturation [28, 29].

HSP70s are known to function as intracellular molecular chaperones that facilitate protein transport, prevent protein aggregation during folding and protect newly synthesized polypeptide chains against misfolding and protein denaturation. The molecular chaperones properties of such a protein allow them to assist the non-covalent assembly/disassembly of other macromolecular structures without being permanent components of such structures. Additionally, molecular chaperones assist the unfolded protein to achieve its single correct three-dimensional configuration (by still unknown mechanism it has evolved to generate

this folded state), without becoming a constituent of the final folded protein [25, 26].

later, sorted to their final destination and assembled into functional complexes [27].

functions of HSFP70 are listed in table 1 and are discussed in this text.

down-regulation of HSP70.

**3. HSP70 function in the muscle**

misfolding and protein denaturation [28, 29].

Most proteins destined for cell organelles are synthesized in cytosol and must cross one or more organelle membranes to reach their functional destination. For example, in the mitochondria, 95% of the proteins are made as precursor proteins in the cytosol and are mainly post translationally imported into the mitochondrial sub-compartments. In this situation, cytosolic HSP70 play an important role for maintenance of a transport-competent conformation of precursor proteins. The precursor protein is translocated in an unfolded state and are refolded

The chaperone function of HSP70 includes the inhibition of the formation of nascent polypeptides. This inhibition is dose-dependent over a range of 0.1-0.4 nmoles of HSP70 and this effect is greater for the larger polypeptides. All these data suggest that high concentration of HSP70 can perturb the normal folding of nascent proteins, block cell growth and impair the cell viability. Then, these are reasons that may explain why the cells of human body have to carefully auto-regulate the levels of HSP70. Many characteristics and

Since the skeletal muscle is one of the most adaptable tissues of the body, every structural aspect of the muscle that can change in response to the stimulus challenge (or to its lack) may require the chaperon action of HSP70 at the molecular level. For example, major adaptations to (dis)use muscle, such as fiber type distribution, fiber diameter, myosin heavy chain profile and mitochondrial distribution, are factors that are associated to the up- or

The ability of muscle cells to express HSPs (mainly HSP70s) represents a cytoprotection mechanism because HSP70 proteins share the same overall structure. They are composed of an actin-like N-terminal nucleotide binding/ATPase domain of 45 kDa, a substrate-binding domain (SBD) of approximately 15 kDa and a C-terminal domain of approximately 10 kDa that is involved in co-chaperone binding (HU et al, 2006). It is of note that N- and C-terminal domains have expressive relevance to antigen presentation, an important way by which HSP70s participate in immune responses. With this structure, HSP70 may act as a molecular chaperone inside the muscle cell: they facilitate protein transport; prevent protein aggregation during folding; protect newly synthesized polypeptide chains against **Table 1.** Characteristics and functions of HSP70

Historically, studies about the effects of exercise on heat shock protein expression have been dedicated to HSP70 analysis in cardiac or skeletal muscle after exhaustive animal protocols (for review see Noble *et al.*, 2008) [30]. It has been demonstrated that 30 minutes after an exercise bout there is an increase in mRNA expression of muscle inducible form of HSP70 (HSP72) and that is a later heat shock response related to mRNA of constitutive form of HSP70 (HSP73) [31]. Similar response may be observed in humans, and are related to glycogen depletion and the muscle heating [32]. This acute heat shock response (intracellular HSP70 content) remained increased 24 hours after an acute exercise session, according to exercise load [33].

Role of Heat Shock Proteins in Skeletal Muscle 111

The chaperone function of HSP70 is more than microscopic laboratory measurements research field. Muscle disuse results in muscular atrophy that is represented by decrease in muscle mass, fiber cross sectional area and total myofibrillar protein content. In this situation contractile protein breakdown exceeds protein synthesis. Moreover, in atrophied muscle there occurs an increase in the proportion of fibers containing the fast myosin heavy chain by transformation from the slow myosin heavy chain (MyHC-I/β) to the fast myosin heavy chain (MyHC-IId/x). As early as 18 h after muscle disuse and persisting for as long as for 18 days, it is possible it is possible to measure a decrease in HSP72 in soleus muscle [39]. Interestingly, previous heat treatment is a strategy to induce HSP70 expression in muscle and this molecular adaptation results in maintenance of muscle mass during 7 days period of immobilization [40]. In this way, HSP70 expression appears to have, not a full protective effect on muscle mass, fiber cross sectional area and total myofibrillar protein content, but a preventive effect on the decrease in MyHC-I/β and the increase in MyHC-IId/x induced during the atrophy process [41]. These evidences suggest that HSP70 can inhibit a key

signaling pathway for atrophy in muscle cell preventing the muscular atrophy.

the effects of estrogen modulation on heat shock response [42].

expression and regulating cell cycle progression [46].

Heat treatment has also been tested in humans. Short wave diathermy therapy is a clinical strategy that means to increase deep heating of tissues with higher water content. This strategy may promote a 58%-increase in HSP70 expression in *vastus lateralis* [42]*.* It is possible that the previous heat treatment cannot reduce markers of muscle damage but it is able to reduce muscular pain, to preserve strength and to improve range of motion following eccentric contractions. Curiously, there is a gender difference in heat shock response in both basal and exercise-induced HSP70 levels, with men showing lower pre-exercise levels and an attenuated HSP70 response as compared to women's values. The gender difference may be explained by

If muscle disuse is a trouble, the reuse of the musculature may represent many stages of soreness. After immobilization, the reload process to the muscle implies in newest molecular adaptations. If a less-required muscle is submitted to a challenge, the HSP70 expression increases greatly (~200%) in the first two weeks of reload process and return to basal levels (above disuse levels) as early as in 8 weeks [39]. This effect is accompanied by increase in percentage of slow type I MyHC fibers (MyHC-I/β). Although many factors appear to be related to the down- and up-regulation of HSP70 function, the expression of this protein is

Although initially the HSP70s have been described essentially in studies that addressed molecular chaperone action of such proteins, HSP70s have also been studied as limiting of protein aggregation, facilitating protein refolding and maintaining structural function of proteins [43]. Intracellular HSP70s have further been demonstrated to be anti-inflammatory [44, 45], providing cytoprotection through anti-apoptotic mechanisms, inhibiting gene

Besides the now classical molecular chaperone action, the most remarkable intracellular effect of HSP70 is the inhibition of nuclear factor κB (NF-κB) activation, which has profound implications for immunity, inflammation, cell survival and apoptosis. Indeed, HSP70 blocks

closely related with the morphological and functional changes of muscle cells.

In humans, repeated bouts of eccentric exercise showed an impressive result in terms of HSP70 expression. Sets of Eccentric contractions contractions of the elbow flexors promote ~2-fold increase in HSP70 expression in biceps brachii. Four weeks later, the basal levels of HSP70 was reduced but the muscle still presents the heat shock response to exercise in the same magnitude but in less HSP70 content [34, 35]. Accompanying this effect, higher levels of both serum creatine kinase, soreness, lower levels of both relaxed arm angle and measured torque (indicators of muscle damage) occurs after the first bout, but the levels of these parameters are lower in the second bout. This may represents an association between muscular adaptations and the phenomenon called "acquired thermo tolerance" in terms of HSP70 expression. This study suggests that equivalent HSP70 response imply equivalent levels of stress in each bout and that may be an indicative that the heat shock response is a marker of muscle adaptation. Moreover, basal modification of HSP70 levels remains four weeks after the first bout of exercise and suggests that a single exercise challenge can promote deeper molecular adaptations in muscle cells [34].