**1. Introduction**

In humans, skeletal muscle is comprises 40-50% of body mass and plays vital roles in locomo‐ tion, heat production during periods of cold stress, and overall metabolism. Skeletal muscles possess a highly plasticity in response to altered activity. Mechanical and metabolic demands elicit marked modifications of gene expression that could lead to gain (hypertrophy) or loss (atrophy) of muscle mass. Indeed, strength training induces marked hypertrophy of exercising muscles. Histochemical analyses clearly show a 10-30% increase in muscle fiber cross-sectional area after 10-12 weeks of resistance training in sedentary subjects [1].

Satellite cells are myogenic stem cells, accounting for 3-9% of the subliminal nuclei associated with adult normal muscle fiber, with the variation widely depending on animal species, age,

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and muscle fiber type [2]. Satellite cells, existing between the basal lamina and the sarcolemma of the fiber, are normally found in a mitotically quiescent in adult muscles. When muscle is injured or mechanically stretched, satellite cells activate to enter the cell cycle. Activated satellite cells have been shown to migrate to the damaged site where they replicate DNA, divide, differentiate, and fuse with the adjacent muscle fiber or form new fibers [3].

It has been reported that satellite cells are activated in compensatory hypertrophy [3, 4], and addition of new nuclei to the growing fiber seems to be required for extreme hypertrophy. Since the myonuclear domain is constant in hypertrophied muscle after mechanical overload‐ ing, many satellite cells must be incorporated adjacent to muscle fibers. In fact, irradiation of satellite cells followed by a loading stimulus results in an attenuated increase in skeletal muscle mass and protein content [5]. Therefore, it is necessary for consecutive processes (the activa‐ tion, proliferation, and differentiation of satellite cells) to elicit muscle hypertrophy in the case of mechanical overloading as well as normal growth. However, several researchers recently suggested satellite cell-independent muscle hypertrophy during mechanical overloading. In addition, some have debated whether the contribution of satellite cells to fiber hypertrophy in adult muscle is minor [6, 7].

In hypertrophied muscle, increasing protein synthesis and decreasing protein degradation are also important events. Phosphatidylinositol-3-kinase (PI3-K)/Akt/ mammalian target of rapamycin (mTOR) signaling has been shown to be crucial to protein synthesis [8, 9]. Me‐ chanical stretching *in vivo* and *in vitro* activates serum response factor (SRF)-dependent signaling in skeletal muscle similar to smooth and cardiac muscles [10, 11]. In contrast, several possible mediators for muscle atrophy have been described. Many negative regulators are proposed to induce muscle atrophy by inhibiting protein synthesis and enhancing protein degradation in skeletal muscle. For example, the ubiquitin proteasome system (UPS) is thought to be a major contributor to many structural proteins [12]. The autophagy-lysosome system has been largely ignored despite the evidence that lysosomal degradation contributes to protein breakdown in atrophying muscles [13]. Recent studies demonstrated that autophagy is an important pathway for appropriate protein degradation in several neuromuscular disorders [14]. The group of Sandri et al. [15, 16] has shown that the autophagy-lysosome and UPS are coordinately regulated during muscle wasting. Furthermore, specific expression of mutant SOD (superoxide dismutase)1G93A in skeletal muscle caused muscle atrophy and weakness mainly via autophagy activation [17]. In this chapter, we summarize possible candidates for proteins that regulate muscle hypertrophy and atrophy. In addition, we describe the possible modulators for switching, proliferation, and differentiation of satellite cells, a possible contributor to muscle hypertrophy.

### **2. Positive regulators of skeletal muscle mass**

#### **2.1. PI3-K/Akt/mTOR pathway**

The serine/threonine kinase Akt regulates the translational level to be involved to a central pathway of hypertrophy. In muscle, Akt is activated by the upstream PI3-K, induced either by receptor binding or by integrin-mediated activation of focal adhesion kinase (FAK). PI3-K activates Akt, which then has the ability to phosphorylate and change the activity of many signaling molecules. Possible downstream regulators of Akt, mTOR, and glycogen synthase 3-β (GSK-3β) play a crucial role in the regulation of translation. Akt activates mTOR via phosphorylation and inactivation of tuberous sclerosis complex (TSC)-2. Subsequently, mTOR phosphorylates and activates the 70kDa ribosomal protein S6 kinase (p70S6K), which results in increased translation either directly or indirectly by activating initiation and elongations, elongation initiation factor (eIF)-2, eIF-4E [through eukaryotic initiation factor 4E binding protein (4E-BP)], and eEF-2. In addition, Akt also phosphorylates and inactivates GSK-3β, thereby activating translation via the initiation factor eIF-2B [18]. Other functions of Akt include the negative regulation of protein degradation by inhibiting forkhead box O (FOXO) mediated proteasome activity.

#### *2.1.1. Akt*

and muscle fiber type [2]. Satellite cells, existing between the basal lamina and the sarcolemma of the fiber, are normally found in a mitotically quiescent in adult muscles. When muscle is injured or mechanically stretched, satellite cells activate to enter the cell cycle. Activated satellite cells have been shown to migrate to the damaged site where they replicate DNA,

It has been reported that satellite cells are activated in compensatory hypertrophy [3, 4], and addition of new nuclei to the growing fiber seems to be required for extreme hypertrophy. Since the myonuclear domain is constant in hypertrophied muscle after mechanical overload‐ ing, many satellite cells must be incorporated adjacent to muscle fibers. In fact, irradiation of satellite cells followed by a loading stimulus results in an attenuated increase in skeletal muscle mass and protein content [5]. Therefore, it is necessary for consecutive processes (the activa‐ tion, proliferation, and differentiation of satellite cells) to elicit muscle hypertrophy in the case of mechanical overloading as well as normal growth. However, several researchers recently suggested satellite cell-independent muscle hypertrophy during mechanical overloading. In addition, some have debated whether the contribution of satellite cells to fiber hypertrophy in

In hypertrophied muscle, increasing protein synthesis and decreasing protein degradation are also important events. Phosphatidylinositol-3-kinase (PI3-K)/Akt/ mammalian target of rapamycin (mTOR) signaling has been shown to be crucial to protein synthesis [8, 9]. Me‐ chanical stretching *in vivo* and *in vitro* activates serum response factor (SRF)-dependent signaling in skeletal muscle similar to smooth and cardiac muscles [10, 11]. In contrast, several possible mediators for muscle atrophy have been described. Many negative regulators are proposed to induce muscle atrophy by inhibiting protein synthesis and enhancing protein degradation in skeletal muscle. For example, the ubiquitin proteasome system (UPS) is thought to be a major contributor to many structural proteins [12]. The autophagy-lysosome system has been largely ignored despite the evidence that lysosomal degradation contributes to protein breakdown in atrophying muscles [13]. Recent studies demonstrated that autophagy is an important pathway for appropriate protein degradation in several neuromuscular disorders [14]. The group of Sandri et al. [15, 16] has shown that the autophagy-lysosome and UPS are coordinately regulated during muscle wasting. Furthermore, specific expression of mutant SOD (superoxide dismutase)1G93A in skeletal muscle caused muscle atrophy and weakness mainly via autophagy activation [17]. In this chapter, we summarize possible candidates for proteins that regulate muscle hypertrophy and atrophy. In addition, we describe the possible modulators for switching, proliferation, and differentiation of satellite

The serine/threonine kinase Akt regulates the translational level to be involved to a central pathway of hypertrophy. In muscle, Akt is activated by the upstream PI3-K, induced either by

divide, differentiate, and fuse with the adjacent muscle fiber or form new fibers [3].

adult muscle is minor [6, 7].

144 Muscle Cell and Tissue

cells, a possible contributor to muscle hypertrophy.

**2.1. PI3-K/Akt/mTOR pathway**

**2. Positive regulators of skeletal muscle mass**

Disruption of the Akt1 gene causes growth retardation and apoptosis [19], whereas deletion of Akt2 causes defects in glucose metabolism but not altered growth [20]. The striking effect of Akt1 on muscle size was demonstrated by the transient transfection of a constitutively active inducible Akt1 transgene in skeletal muscle *in vivo* [15, 21]. Downstream mediators (p70S6K, S6) of protein synthesis were activated, but satellite cells were not incorporated [21]. Akt1 transgenic muscles showed increased strength, showing that a functional hypertrophy was elicited [21]. Moreover, muscle mass was completely preserved in denervated transgenic Akt mice [22]. The effects of Akt on muscle mass regulation can be mediated by several different downstream effectors, including GSK-3β, mTOR, and FOXO.

#### *2.1.2. mTOR and mTOR signaling complex (mTORC) 1*

mTOR exists in two functionally distinct multi-protein signaling complexes, mTORC1 and mTORC2. In general, only signaling by mTORC1 is inhibited by rapamycin, and thus the growth regulatory effects of rapamycin are primarily exerted through the mTORC1 complex [23]. mTORC1 regulates several anabolic processes including protein synthesis, ribosome biogenesis, and mitochondrial biogenesis, as well as catabolic processes such as autophagy [23]. Two of the most studied mTORC1 targets are the 4E-BP1 and p70S6K, which both play important roles in the initiation of mRNA translation.

mTORC1 is activated in response to hypertrophic stimuli such as increased mechanical loading, feeding, and growth factors [24, 25]. In fact, hypertrophy induced by mechanical loading, insulin-like growth factor (IGF)-I, and clenbuterol is significantly, if not completely, blocked by rapamycin [25]. In addition, overexpression of constitutively active Akt activates mTORC1 signaling and induces hypertrophy through a rapamycin-sensitive mechanism [26]. These findings support the hypothesis that mTORC1 is sufficient to induce hypertrophy, however, the hypertrophic stimuli employed in these studies also induce signaling through PI3-K and Akt. Signaling through PI3-K/Akt can regulate mTOR-independent growth regulatory molecules [GSK-3β, tuberin (TSC-2), and FOXO]. However, it was not clear if signaling by mTORC1 was sufficient, or simply permissive, for the induction of hypertrophy. To address this issue, overexpression of Ras homolog enriched in the brain (Rheb) was recently used as a means to induce a PI3-K/Akt-independent activation of mTORC1 [27]. Marked increases in protein synthesis and hypertrophy have been recognized in several muscles of Rheb abundant mice [27]. Stretch-induced activation of mTOR signaling was not abolished in the skeletal muscle of Akt1-/- mice [28]. Therefore, mechanically induced signaling through mTOR is not dependent on Akt. Furthermore, Akt-independent stimulation of mTOR may be regulated by phosphorylation of TSC-2. For instance, TSC-2 is inhibited by FAK in 293T cells [29] indicating that up-regulated FAK with increased loading could stimulate protein synthesis via TSC-2 inhibition. All these regulatory influences may explain the rise in the level of phosphorylated p70S6K. These results suggested that the activation of mTORC1 is indeed sufficient to induce hypertrophy, at least in part by increasing protein synthesis.

Although one of the most well-characterized upstream triggers of mTOR signaling in skeletal muscle is IGF-I, mechanical loading has been shown to activate mTOR by an IGF-I independent pathway involving PLD via its metabolite phosphatidic acid. More recently, Hornberger et al. [30] extended these initial findings by showing mTOR activation following eccentric contrac‐ tions via PLD synthesis but not PI3-K-Akt activity. It has been shown that PLD1, but not PLD2, was a downstream effector of Rheb's activation of mTOR. In contrast, Vps34 is a class III PI3K previously shown to mediate amino acid activation of p70S6K by mTOR. In skeletal muscle, MacKenzie et al. [31] reported that high-resistance contractions increased Vps34 activity possibly in response to increased intramuscular leucine levels. In addition to Vps34, two groups reported the exciting discovery that the Rag family of GTPases was necessary and sufficient for amino acid activation of the mTOR pathway [32]. Therefore, mTOR is currently thought to be the major hub for the integration of an array of upstream signaling pathways which, when activated, ultimately result in increased translational efficiency [8]. Figure 1 summarizes the anabolic pathway (PI3-K/Akt/mTOR and SRF-dependent) regulating skeletal muscle mass.

#### **2.2. Serum Response Factor (SRF)**

SRF is an ubiquitously expressed member of the MADS (MCM1, Agamous, Deficiens, SRF) box transcription factor family, which binds the core sequence of SRF/CArG boxes [CC (A/T)6 GG] as homodimers. Functional CArG boxes have been found in several promoter regions of muscle-specific genes such as the skeletal α-actin and myosin light chain 1/3 genes. SRFdependent signaling plays a major role in a variety of physiological processes, including cell growth, migration, and cytoskeletal organization [33]. Previous results obtained with specific SRF knock-out models by the Cre-LoxP system emphasize a crucial role for SRF in postnatal skeletal muscle growth and regeneration by modulating interleukin (IL)-4 and IGF-I mRNA expression [34]. More recently, Mokalled et al. [35] demonstrated that members of the myocardin family of transcriptional coactivators, MASTR and myocardin-related transcription factor (MRTF)-A, are up-regulated in satellite cells during muscle regeneration. In addition, skeletal muscle regeneration exhibited the impairment in mouse possessing double-knockout satellite cells (MASTR and MRTF-A). As proposed by Mokalled et al. [35], the promoting role on muscle regeneration seems to be attributable to both MASTR/myocyte enhancer factor 2 and/or MRTF-A/SRF complexes.

To address this issue, overexpression of Ras homolog enriched in the brain (Rheb) was recently used as a means to induce a PI3-K/Akt-independent activation of mTORC1 [27]. Marked increases in protein synthesis and hypertrophy have been recognized in several muscles of Rheb abundant mice [27]. Stretch-induced activation of mTOR signaling was not abolished in the skeletal muscle of Akt1-/- mice [28]. Therefore, mechanically induced signaling through mTOR is not dependent on Akt. Furthermore, Akt-independent stimulation of mTOR may be regulated by phosphorylation of TSC-2. For instance, TSC-2 is inhibited by FAK in 293T cells [29] indicating that up-regulated FAK with increased loading could stimulate protein synthesis via TSC-2 inhibition. All these regulatory influences may explain the rise in the level of phosphorylated p70S6K. These results suggested that the activation of mTORC1 is indeed

Although one of the most well-characterized upstream triggers of mTOR signaling in skeletal muscle is IGF-I, mechanical loading has been shown to activate mTOR by an IGF-I independent pathway involving PLD via its metabolite phosphatidic acid. More recently, Hornberger et al. [30] extended these initial findings by showing mTOR activation following eccentric contrac‐ tions via PLD synthesis but not PI3-K-Akt activity. It has been shown that PLD1, but not PLD2, was a downstream effector of Rheb's activation of mTOR. In contrast, Vps34 is a class III PI3K previously shown to mediate amino acid activation of p70S6K by mTOR. In skeletal muscle, MacKenzie et al. [31] reported that high-resistance contractions increased Vps34 activity possibly in response to increased intramuscular leucine levels. In addition to Vps34, two groups reported the exciting discovery that the Rag family of GTPases was necessary and sufficient for amino acid activation of the mTOR pathway [32]. Therefore, mTOR is currently thought to be the major hub for the integration of an array of upstream signaling pathways which, when activated, ultimately result in increased translational efficiency [8]. Figure 1 summarizes the anabolic pathway (PI3-K/Akt/mTOR and SRF-dependent) regulating skeletal

SRF is an ubiquitously expressed member of the MADS (MCM1, Agamous, Deficiens, SRF) box transcription factor family, which binds the core sequence of SRF/CArG boxes [CC (A/T)6 GG] as homodimers. Functional CArG boxes have been found in several promoter regions of muscle-specific genes such as the skeletal α-actin and myosin light chain 1/3 genes. SRFdependent signaling plays a major role in a variety of physiological processes, including cell growth, migration, and cytoskeletal organization [33]. Previous results obtained with specific SRF knock-out models by the Cre-LoxP system emphasize a crucial role for SRF in postnatal skeletal muscle growth and regeneration by modulating interleukin (IL)-4 and IGF-I mRNA expression [34]. More recently, Mokalled et al. [35] demonstrated that members of the myocardin family of transcriptional coactivators, MASTR and myocardin-related transcription factor (MRTF)-A, are up-regulated in satellite cells during muscle regeneration. In addition, skeletal muscle regeneration exhibited the impairment in mouse possessing double-knockout satellite cells (MASTR and MRTF-A). As proposed by Mokalled et al. [35], the promoting role on muscle regeneration seems to be attributable to both MASTR/myocyte enhancer factor 2

sufficient to induce hypertrophy, at least in part by increasing protein synthesis.

muscle mass.

146 Muscle Cell and Tissue

**2.2. Serum Response Factor (SRF)**

and/or MRTF-A/SRF complexes.

**Figure 1.** Anabolic pathway regulating skeletal muscle mass. The major anabolic pathway regulating protein synthesis in skeletal muscle is mTOR/TORC1 signaling. Upstream triggers (IGF-I, mechanical loading, amino acids) activate mTOR signaling through a number of different intermediary proteins such as Rheb, phospholipase D1 and its metabo‐ lite PA, and Vps34. Although myostatin signals through the ActRIIB-ALK4/5 heterodimer activate Smad2/3, reduced stimulation of myostatin in the presence of IGF-I and mechanical loading cannot block the functional role of Akt. Myo‐ sin-actin interaction by mechanical loading activates STARS /MRTF-A/SRF signaling. The accumulation of MuRF in muscle tissue during inactivity (hindlimb suspension, immobilization, etc.) is known to inhibit SRF-dependent tran‐ scription of muscle-specific genes. However, the functional role of SRF is not abrogated under such conditions, which lower MuRF expression because of marked inhibition of FOXO by abundant Akt. ActRIIB: activin receptor IIB; ALK4/5: activin-like kinase 4/5; eIF: eukaryotic initiation factor; FOXO: Forkhead box O; IGF-I: insulin-like growth fac‐ tor-I; IRS-1: insulin receptor substrate-1; MRTF-A: myocardin-related transcription factor-A; mTOR: mammalian target of rapamycin; MuRF: muscle ring-finger protein; PA: phosphatidic acid; PI3-K: phosphatidylinositol 3-kinase; p70S6K: 70 kDa ribosomal protein S6 kinase; Rheb: Ras homolog enriched in brain; SRF: serum response factor; STARS: striated muscle activators of Rho signaling; TORC1: a component of TOR signaling complex 1; TSC: tuberous sclerosis com‐ plex. Data from Sakuma et al. [130]

It is proposed that the transcriptional activity of SRF is regulated by muscle ring finger (MuRF)-2 [36] and striated muscle activators of Rho signaling (STARS) [37]. At the M-band, the mechanically modulated kinase domain of titin interacts with a complex of the protein products of the atrogenes NBR1, p62/sequestosome 1 (SQSTM1), and MuRFs [36]. This complex dissociates under mechanical arrest, and MuRF-1 and MuRF-2 translocate to the cytoplasm and the nucleus [36]. One of the probable nuclear targets of MuRFs is SRF [36], suggesting that the MuRF-induced nuclear export and transcriptional repression of SRF may contribute to amplifying the transcriptional atrophy program. Thus, it is possible that MuRF-2 abrogates the synergistic transactivation of SRF and SRF-linked molecules *in vivo*. On the other hand, SRF activity is sensitive to the state of actin polymerization. G-actin monomers inhibit SRF activity, whereas polymerization of actin occurs in response to serum stimulation and RhoA signaling. Thus, signal inputs lower the ratio of globular actin to fibrillar actin liberating the binding of MRTF-A to globular actin resulting in the nuclear accumulation of MRTF-A and subsequent SRF-dependent gene expression [38]. It has been well established that overexpres‐ sion of STARS contributes to the nuclear translocation of MRTF-A and MRTF-B [37, 39], and these factors activate SRF transcription.

#### *2.2.1. The functional role of SRF during muscle hypertrophy*

In adults, SRF activity could be important for the control of skeletal muscle mass. In fact, SRF also enhances the hypertrophic process in muscle fibers after mechanical overloading [40-42]. For example, Flück et al. [40] utilized a stretch-induced hypertrophic model, in which a weight equal to 10% of body weight was attached to the left wing of a rooster to induce enlargement of the anterior latissimus dorsi muscle. Gordon et al. [10] also indicated a significant increase in SRF protein in the soleus and plantaris muscles after 8 days of functional overload caused by surgical ablation of the gastrocnemius muscle in rats. In humans, 8 weeks of resistance training (leg presses, squats, and leg extensions) induced increases in SRF mRNA (3-fold) and nuclear protein (1.25-fold) in human muscle [41]. The same training also increases in the mRNA levels of several SRF-targeted molecules {alpha-actin, myosin heavy chain (MHC) IIa, and IGF-I [34]}. They proposed the induction of these molecules by SRF in human hypertrophied muscle, though they did not provide any direct evidence such as transcriptional activation by increased binding of SRF to the promoter region of alpha-actin, MHC IIa, and IGF-I. Although SRF would regulate proliferation and differentiation using different pathways, it would mainly activate the differentiation of satellite cells during muscle hypertrophy. Indeed, it was shown that, in mechanically overloaded muscles of rats, the SRF protein co-localized with MyoD and myogenin in myoblast-like cells during the active differentiation phase [42]. In this study, abundant SRF protein at 2 days was failed to be detected after mechanical overloading, when many proliferating satellite cells and/or myoblasts are expected to exist. In addition, the location of the SRF protein did not correspond with that of BrdU-positive satellite cells or ED-1 positive macrophages in the hypertrophied plantaris muscle [42].

As indicated earlier, by reducing the cytoplasmic concentration of monomeric G-actin, STARS promotes the nuclear translocation of SRF transcriptional co-activator-A and -B (MRTF-A and MRTF-B), resulting in an increase in SRF-mediated gene transcription [37]. A real-time PCR analysis conducted by Lamon et al. [41] demonstrated that increased mechanical loading from resistance training in humans caused significant increases in the upstream modulators of SRF (STARS mRNA; 3.4-fold, MRTF-A mRNA; 2.5-fold, MRTF-B mRNA; 3.6-fold, and RhoA protein; 2-fold). SRF seems to regulate the transcriptional facilitation of the alpha-actin promoter by the androgen receptor (AR) during muscle hypertrophy. Using male adult Sprague-Dawley rats, Lee et al. [43] showed an increase in AR protein of 106% and 279% after 7 and 21 days in mechanically overloaded plantaris muscles by surgical ablation of two synergistic muscles. Co-overexpression of either SRF or active RhoA with AR indicated a synergistic 36- and 28-fold increase in the skeletal alpha-actin promoter activity. In contrast, cotransfection of AR, SRF, and active RhoA induced a 180-fold increase in skeletal a-actin promoter activity. Therefore, it is possible that intimate linkages among these three modulators induce alpha-actin expression in hypertrophied muscle *in vivo*. Intriguingly, experiments using C2C12 cells indicated that this AR coactivation for the alpha-actin promoter requires a co-expressed full-length SRF and SRF-binding site but not AR's direct binding to GRE sites [44].

More recently, Guerci et al. [45] investigated the functional role of SRF in adult mammalian muscle using SRFflox/flox: HAS-Cre-ERT2 mice injected with tamoxifen. During the compensatory hypertrophy phase, growth was completely slow in the SRF-deleted plantaris muscle, demonstrating that SRF is necessary for overloaded-induced myofiber hypertrophy. Intrigu‐ ingly, Guerci et al. [45] showed that the lack of SRF in myofibers affected satellite cell prolif‐ eration and fusion to the growing fibers. In their genetic mouse model, Cre recombinase is expressed only in myofibers but not in satellite cells. Furthermore, Guerci et al. [45] identified the secreted molecules mediating these paracrine effects and whose expression is under the control of SRF by using a global transcriptomic approach allowing the identification of genes activated by SRF. In SRF-deleted muscles, the overexpression of IL-6 is sufficient to restore satellite cell proliferation, but not satellite cell fusion and overall growth. Cox2/IL-4 overex‐ pression rescues satellite cell recruitment and muscle growth without affecting satellite cell proliferation, identifying altered fusion as the limiting cellular events precluding the hyper‐ trophic growth of SRF-deleted muscles. Guerci's excellent finding was further supported by Bruusgaard et al. [46], showing that the addition of nuclei precedes increased fiber size during compensatory hypertrophy and that this constituted the major cause of hypertrophy. How‐ ever, the contribution of satellite cells to muscle hypertrophy has been a controversial issue [6, 47]. In fact, McCarthy et al. [48] suggested that satellite cell-depleted skeletal muscle undergoes extensive fiber hypertrophy after mechanical overloading. Therefore, further examination of SRF's role in muscle hypertrophy is needed.

#### *2.2.2. Defects of SRF signaling with muscle wasting*

### **Sarcopenia**

abrogates the synergistic transactivation of SRF and SRF-linked molecules *in vivo*. On the other hand, SRF activity is sensitive to the state of actin polymerization. G-actin monomers inhibit SRF activity, whereas polymerization of actin occurs in response to serum stimulation and RhoA signaling. Thus, signal inputs lower the ratio of globular actin to fibrillar actin liberating the binding of MRTF-A to globular actin resulting in the nuclear accumulation of MRTF-A and subsequent SRF-dependent gene expression [38]. It has been well established that overexpres‐ sion of STARS contributes to the nuclear translocation of MRTF-A and MRTF-B [37, 39], and

In adults, SRF activity could be important for the control of skeletal muscle mass. In fact, SRF also enhances the hypertrophic process in muscle fibers after mechanical overloading [40-42]. For example, Flück et al. [40] utilized a stretch-induced hypertrophic model, in which a weight equal to 10% of body weight was attached to the left wing of a rooster to induce enlargement of the anterior latissimus dorsi muscle. Gordon et al. [10] also indicated a significant increase in SRF protein in the soleus and plantaris muscles after 8 days of functional overload caused by surgical ablation of the gastrocnemius muscle in rats. In humans, 8 weeks of resistance training (leg presses, squats, and leg extensions) induced increases in SRF mRNA (3-fold) and nuclear protein (1.25-fold) in human muscle [41]. The same training also increases in the mRNA levels of several SRF-targeted molecules {alpha-actin, myosin heavy chain (MHC) IIa, and IGF-I [34]}. They proposed the induction of these molecules by SRF in human hypertrophied muscle, though they did not provide any direct evidence such as transcriptional activation by increased binding of SRF to the promoter region of alpha-actin, MHC IIa, and IGF-I. Although SRF would regulate proliferation and differentiation using different pathways, it would mainly activate the differentiation of satellite cells during muscle hypertrophy. Indeed, it was shown that, in mechanically overloaded muscles of rats, the SRF protein co-localized with MyoD and myogenin in myoblast-like cells during the active differentiation phase [42]. In this study, abundant SRF protein at 2 days was failed to be detected after mechanical overloading, when many proliferating satellite cells and/or myoblasts are expected to exist. In addition, the location of the SRF protein did not correspond with that of BrdU-positive satellite cells or ED-1

As indicated earlier, by reducing the cytoplasmic concentration of monomeric G-actin, STARS promotes the nuclear translocation of SRF transcriptional co-activator-A and -B (MRTF-A and MRTF-B), resulting in an increase in SRF-mediated gene transcription [37]. A real-time PCR analysis conducted by Lamon et al. [41] demonstrated that increased mechanical loading from resistance training in humans caused significant increases in the upstream modulators of SRF (STARS mRNA; 3.4-fold, MRTF-A mRNA; 2.5-fold, MRTF-B mRNA; 3.6-fold, and RhoA protein; 2-fold). SRF seems to regulate the transcriptional facilitation of the alpha-actin promoter by the androgen receptor (AR) during muscle hypertrophy. Using male adult Sprague-Dawley rats, Lee et al. [43] showed an increase in AR protein of 106% and 279% after 7 and 21 days in mechanically overloaded plantaris muscles by surgical ablation of two synergistic muscles. Co-overexpression of either SRF or active RhoA with AR indicated a

these factors activate SRF transcription.

148 Muscle Cell and Tissue

*2.2.1. The functional role of SRF during muscle hypertrophy*

positive macrophages in the hypertrophied plantaris muscle [42].

Aging is associated with progressive declines in muscle mass, quality, and strength, a condi‐ tion known as sarcopenia. Lean muscle mass generally contributes to ~50% of total body weight in young adults, but this value declines with aging, to just 25% at 75-80 years of age [49]. At the muscle fiber level, sarcopenia is characterized with specific type II fiber atrophy and fiber loss. Although the specific contribution of each is unknown, several possible signaling factors (Akt-mTOR, RhoA-SRF, and autophagy) have been proposed to contribute to age-related muscle atrophy [50, 51]. In fact, using crude and fractionated homogenates, our recent study has clearly demonstrated a blunted expression of SRF protein in the quadriceps and triceps brachii in aged mice [52]. Immunofluorescence microscopy also indicated the selective loss of SRF protein in the cell cytosol but not in satellite cells in sarcopenic mice. In addition, our data showed a decrease in MRTF-A mRNA (50-70%) and protein (76%) levels in only the nuclear fraction with age. Furthermore, we observed 40-60% decreases in the amount of STARS mRNA in the quadriceps and triceps brachii of 24-month-old mice [52].

A decrease of SRF expression achieved using a transgenic approach was found to accelerate the atrophic process in muscle fibers with age [53]. These SRF-deleted mice showed marked deposition of intermuscle lipid with aging. One morphologic aspect of sarcopenia is the infiltration of muscle tissue components by lipids, because of the increased frequency of adipocyte or lipid deposition [54] within muscle fibers. As with precursor cells in bone marrow, liver, and kidney, muscle satellite cells expressing the adipocytic phenotype increased with age [55], although this process is still relatively poorly understood in terms of its extent and spatial distribution. Lipid deposition may result from a net buildup of lipids due to the reduced oxidative capacity of muscle fibers with aging [54]. These lines of evidence clearly showed a defect of SRF-signaling in aged mammalian muscle.

#### **2.3. Muscular disorder**

SRF appears to be linked to the degenerative process during muscular dystrophy. Significant reduction in the amount of SRF has been observed [56], 40-50% and 50-65% at 2 and 12 weeks of age, respectively, in merosin-deficient congenital muscular dystrophy. Our immunohisto‐ chemical analysis demonstrated that mature normal mice exhibited an abundant SRF protein in the cytoplasm of several muscle fibers, while the *dy* mice did not. There is no direct evidence of a link between SRF disorders and the pathogenesis of disease in the skeletal muscle.

However, Lange et al. [36] observed that a mutation in the TK domain of titin, a possible modulator of SRF, disrupted Nbr1 binding and led to hereditary myopathy with early respiratory failure (HMERF). HMERF patient muscle biopsies revealed a Nbr1 diffusible localization, cytoplasmic p62/SQSTM1 aggregates, and the selective accumulation of MuRF-2 in centralized nuclei. Unfortunately, the localization of SRF has not been determined in the muscle of HMERF patients. In contrast, a natural dominant-negative form of SRF was dem‐ onstrated to be elevated in human heart failure [57]. The dominant negative SRF isoform potently inhibited SRF-dependent function, showing the biochemical phenotype seen in SRFnull mice [57]. In addition, a subsequent human heart failure study showed decreases in fulllength SRF and elevated expression of a caspase-3-cleaved product of SRF [58]. A more recent review [59] proposed various disorders to be linked with the SRF mutation as shown by many reliable studies using cell-specific SRF-knockout phenotypes.
