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Molecular Insights into Muscle Homeostasis, Atrophy and Wasting

Muscle homeostasis is guaranteed by a delicate balance between synthesis and degradation of cell proteins and its alteration leads to muscle wasting and diseases. In this review, we describe the major anabolic pathways that are involved in muscle growth and homeostasis and the proteolytic systems that are over-activated in muscle pathologies. Modulation of these pathways comprises an attractive target for drug intervention.


INTRODUCTION
Skeletal muscle represents one of the most plastic tissues of our body. The presence of heterogeneous population of myofibers confers to skeletal muscle a high degree of plasticity necessary to modulate its morpho-functional properties in response to different external stimuli, such as exercise, variation in hormone levels, oxygen and nutrient supply, and activity of motor neurons. However, this important and critical feature of skeletal muscle is impaired in senescent muscle and severely compromised in different diseases, leading to alteration in the functional performance and in the regenerative capacity of skeletal muscle.

MUSCLE HOMEOSTASIS: A MATTER OF BAL-ANCE BETWEEN PROTEIN SYNTHESIS AND DEG-RADATION
The morpho-functional properties of skeletal muscle are guaranteed by a dynamic turnover between net protein synthesis and degradation and by cell turnover.
The prevalence of protein synthesis leads to muscle growth and hypertrophy, whereas the reduced protein synthesis rates associated with an increase in protein degradation results in muscle atrophy and, in certain pathologic conditions, in muscle wasting. Muscle cell turnover, which is a guarantee by the activity of satellite cells, the muscle stem cells compartment, and by other precursor cells, is also another important homeostatic process of the adult skeletal muscle.
The signaling pathway that regulates protein turnover is mediated by the Insulin-like Growth Factor 1 (IGF1) and by a cascade of intracellular effectors, including Akt, which has the ability to control both protein synthesis, by activating the kinase mTOR, and protein degradation by repressing the transcription factor FoxO [4, 5].

IGF-1
IGF-1 exists in different isoforms, due to alternative splicing of the initial transcript. The different isoforms can exert different roles and can act as a systemic growth factor or alternatively in a autocrine/paracrine manner [6][7][8].
At molecular level, human and rodent IGF-1 gene contains six exons, separated by five introns. In rodent, Exons 1 and 2 represent leader exons and encode distinct 5´UTRs, as well as different parts of the signal peptide. Exon 3 encodes part of the signal peptide as well as part of the mature IGF-1 peptide. Exon 4 encodes the rest of the mature peptide and part of the amino-terminal region of the E-peptide. Exons 5 and 6 encode two distinct carboxy-terminal E-peptides, namely Ea and Eb, and the 3´UTR.
In humans, similarly to rodent IGF-1 gene, Exons 1 and 2 represent leader exons; however, differently to murine IGF-1 gene, three mRNA variants with alternatively spliced-end have been identified [6]. Splicing of human exon 4 to exon 6 yields an mRNA sequence which encodes the Ea-peptide, shares 91% homology with the mouse Ea-peptide. The second mRNA derives from an exon 4-5 splice variant, which encodes an extension peptide termed Eb-peptide. The third mRNA splice variant, termed as Ec-peptide, contains exon 4, part of exon 5 and exon 6. This human Ec-peptide shares 73% homology with rat Eb-peptide and is considered to be its counterpart [9].
Whether different IGF-1 isoforms exert different biological functions is still an open question.
Gene expression analysis performed in muscle of both human and wild type mice revealed that IGF-1Ea results are more highly expressed as compared to both IGF-1Eb and IGF-1Ec isoforms, while IGF-1Eb appears to be more expressed than IGF-1Ec isoform [6]. IGF-1Ea promotes satellites cell differentiation and provides most of the mature IGF-1 for stimulating protein synthesis. IGF-1Eb isoform can be responsible for satellite cells activation and proliferation and can be induced by physical exercise and stretch [10].
Previous work demonstrated that muscle restriction of IGF-1Ea (mIGF-1) transgene (MLC/mIGF-1) expression sustained muscle hypertrophy and regeneration in young and senescent skeletal muscle [11], enhanced the recruitment of circulating stem cells in injured muscle [ Additional work is necessary to define the role of the different IGF-1 isoforms in the physiopathology of skeletal muscle and to characterize the specific signal transduction pathways that might be activated by each isoform.
The Mitogen-Activated Protein (MAP) kinase pathway basically mediates the proliferative response of muscle cells to the activity of IGF-1, whereas the kinase Akt pathway stimulates muscle differentiation and might trigger muscle hypertrophy. The activity of Akt involves the disruption of the tuberous sclerosis complex-1 and -2 proteins (Tsc1 and Tsc2) and the subsequent phosphorylation and activation of mTOR (Fig. 1). Then, mTOR phosphorylates and activates the 70-kDa ribosomal protein S6 kinase (p70S6K) (Fig. 1) and inhibits 4E-BP, a negative regulator of the protein initiation factor eIF-4E, increasing translation.
In particular, mTOR exerts its role forming two functionally distinct multiprotein signaling complexes, known as mTOR Complex 1 (mTORC1) and 2 (mTORC2). At structural level, mTORC1 is formed by three components: mTOR, regulatory protein associated with mTOR (Raptor), and mammalian lethal with Sec13 protein 8 (mLST8) [26-28] (Fig. 1). Like mTORC1, mTORC2 contains mTOR and mLST8, but differently to mTORC1, it contains Rictor (rapamycin insensitive companion of mTOR), [ 1). At functional level, mTORC1 is inhibited by rapamycin, whereas mTORC2 is insensitive to the activity of rapamycin. Fig. (1). A schematic representation of the effects of the Insulin-like Growth Factor 1 (IGF-1) on muscle growth and homeostasis. IGF-1 stimulates mTORC1 activity and inhibits FoXO expression through the PI3K-Akt pathway, promoting protein synthesis and counteracting protein degradation. IGF-1 induces also mTORC2 activity, along with other signal pathways, to stimulate proliferation, survival, and cell migration. mTORC1 plays a central role in regulating several anabolic processes including protein and lipid synthesis, and ribosome biogenesis while also suppressing catabolic pathways [38]. mTORC1 directly phosphorylates S6K1 that in turn activates several substrates that promote mRNA translation initiation [39-42] (Fig. 1). Conversely to mTORC1, which regulates cell growth and metabolism, mTORC2 controls proliferation and survival, inducing the phosphorylation and activation of Akt [43], which phosphorylates and inhibits several substrates, including the FoxO1/3a transcription factors, the metabolic regulator GSK3b, and the mTORC1 inhibitor TSC2 (Fig. 1). Moreover, mTORC2 phosphorylates several members of the PKC family [29, 43-46], regulating cytoskeletal remodeling and cell migration.
Additional pathways that are modulated by IGF-1 are ROCK2, p90RSK and the components of the kinase cascade p42/p44 MAPK, which act in concert to promote muscle differentiation and maturation [47-50].

THE PROTEOLYTIC SYSTEMS OF MUSCLE WASTING
A general feature associated with several pathologic conditions such as both genetic diseases and acquired disorders (i.e. muscle disuse, cachexia, heart failure, sarcopenia) is the lose of adaptability of skeletal muscle leading to wasting, a process in which the delicate balance between anabolic and catabolic process is impaired [51,52]. Nevertheless, the mechanisms underlying muscle atrophy and wasting associated with different conditions might be different. For example, PI3K/Akt/mTOR pathway is not modulated in human sarcopenic sedentary subject [6], neither physical activity, normally associated with an increased activity of Akt/mTOR/p70S6K in young individuals, is able to stimulate this anabolic pathway in sarcopenic animal models [53]. In contrast, cancer cachexia significantly reduces the rates of muscle protein synthesis.
Among proteolytic systems ubiquitin-,calpain-, caspase-, and autophagy-mediated protein and organelles degradation are the principal pathways activated in several pathologies, leading to myofiber degeneration, wasting and impaired muscle regeneration [54].

THE UBIQUITIN-PROTEASOME SYSTEM
The ubiquitin-proteasome system is one of the signal transduction pathways that mediates the turnover of muscle protein. It has been demonstrated that several pathologic conditions, leading to muscle wasting (e.g. glucocorticoid treatment, sepsis, fasting, cancer, and acidosis) [55][56][57][58] or associated with muscle atrophy [59-61] over-activate a program of gene expression in which the ubiquitin-proteasome pathway represents the major player.
The pathway involves an enzymatic cascade starting with the conjugation of protein substrates with ubiquitin and terminating with the degradation of targeted protein to small peptides and amino acids (Fig. 2). E1 enzymes activate the ubiquitin proteins, which move from E1 to the E2 ubiquitinconjugating enzyme. The last step of the reaction involves the ubiquitin-protein ligases E3, which recognize the protein that will be ubiquitinated (Fig. 2). The polyubiquitineted proteins are transferred, in an ATP-dependent reaction, in the 26S proteasome complex where the proteins are degraded (Fig. 2). The ubiquitin itself is then released and reused for a new enzymatic reaction. , whereas atrogin-1 has been shown to target both the muscle regulatory factor MyoD and the eukaryotic initiation factor of protein synthesis eIF-e [68, 69]. MuRF1, but not Atrogin-1, can be also transcriptionally activated by NF-kB, a critical player activated by several catabolic stimuli, including oxidative and inflammatory cytokines, promoting muscle atrophy. Of note, other factors implicated in the regulation of muscle Fig. (2). A schematic representation of the ubiquitin-mediated protein degradation. The ubiquitin-activating enzyme-E1 activates ubiquitin, which is then transferred to a ubiquitin-conjugating enzyme-E2. The ubiquitin-protein ligase-E3 binds to E2 and to protein substrate which becomes polyubiquitinated and subsequently degraded by the proteasome, generating short peptides and free ubiquitin that can be further reused. . It has been also demonstrated that FoxO3 regulates another proteolytic pathways, namely autophagy (discussed below), coordinating two proteolytic systems, namely the proteasome, which is involved in removal of proteins, and the autophagy, involved in organelles clearance.
Several factors and stimuli including glucocorticoids, oxidative stress, and inflammatory cytokines might activate the ubiquitin-proteasome system [57]. Activation of the ubiquitin-proteasome system has been reported in both experimental model of cancer cachexia and human patients with advanced cancer. It has been reported that the decreased activity of signaling pathway mediated by IGF-1, associated with different model of cancer cahexia, can lead to muscle atrophy [62], via the activation of the "atrophy program". The reduction in IGF-1 expression and activity results in the inhibition of the AKT [73,74]. Under anabolic conditions, AKT blocks, by phosphorylation and subsequent sequestration in the cytoplasm, the function of the FoxO transcription factors family [75]. Reduction in anabolic factors, including IGF-1, causes the inhibition of the PI3K/AKT pathway. In such conditions, the dephosphorylated active form of FoxO factors entering the nucleus which transactivate the expression of atrogin-1 and MuRF1 [64]. The ubiquitin-proteasome system can be also activated, although there are conflicting findings, in aging-related muscle wasting, a state known as sarcopenia [76,77]. It has been demonstrated that the decline in muscle mass during aging is associated with a reduction in IGF-1/AKT pathway with a consequent activation of atrogin-1 and MuRF1 [7, 11, 78-82]. Nevertheless, due to different discrepancies among different studies [78,83], the involvement of MuRF1 and atrogin-1 in aging related muscle loss require additional study. Other chronic diseases such as Chronic Obstructive Pulmonary Disease (COPD), diabetes, Chronic Kidney Disease (CKD), and Congestive Heart Failure (CHF) impinge the IGF-1 signaling and activate the ubiquitin-proteasome system [61, 63, [84][85][86][87]. Of note, a common feature of these complex diseases is the increase in angiotensin II (Ang II), which promotes muscle wasting through multiple mechan [87]. AngII can: i) reduce IGF-1 expression and increase cytokine signaling such as glucocorticoid and IL-6, resulting in increased protein breakdown; ii) increase oxidative stress; iii) inhibit AMPK, impinging energy balance; iv) inhibit satellite cell function and muscle regeneration.

THE CALPAIN PATHWAY
Calpains, which have been identified in many organisms [88], are calcium-activated cysteine proteases characterized by the presence of two subunits, a large subunit of 80kDa that contains protease activity and a smaller subunit of 30kDa, functioning as a regulator of calpain activity [89]. Based on Ca 2+ concentration dependence, two ubiquitous calpain isoforms, µ and m, are well characterized, and are commonly referred as the ubiquitous/conventional/typical calpains. Among the different subtypes, calpain 3 is predominantly expressed in skeletal muscle and it maintains proteolytic activity at physiological Ca 2+ level [89]. More recently, it has been demonstrated that calmodulin (CaM), a known transducer of the calcium signal, binds and facilitates Calpain 3 autolytic activation in skeletal muscle [90]. At physiological levels, calpains participate in many cellular processes, including cytoskeletal remodeling [91], cell mobility [92], myofibril maintenance [93], signal transduction [94], cell cycle progression [95], regulation of gene expression [96] apoptosis [88], and long term potentiation [97].
Calpains are generally localized at the level of the Z disk of the sarcomere [105] where they initiate the proteolytic cleavage of muscle proteins that anchor the sarcomere, including titin, nebulin, desmin, filamin, troponin and tropomyosin, [106,107]. Another molecular target of calpains is fodrin, the non-erythroid spectrin protein, whose alteration impinges muscle physiology and myofibers survival [106][107][108].
The activity of calpains is regulated by the endogenous inhibitor calpastatin, which prevents both enzyme activation and expression of catalytic activity, by modulating intracellular Ca 2+ concentration [109,110] (Fig. 3).
Of note calpains, conversely to other proteolytic systems, cleave target proteins at specific sites, leaving large polypeptide fragments with altered physiological properties. Moreover, the calpains target proteins can be "marked" for degradation by post-translational modifications, such as phosphorylation [111].
The activity of calpains can play also a role in muscle injury under physiological, such as exercise, [112] or pathologic, such as Duchenne and Becker muscular dystrophies (DMD/BMD), conditions [113]. Of interest is the observation that calpain3 is associated with limb-girdle muscular dystrophy type 2A [114], and might control myoblast fusion and maturation, suggesting a role in muscle function [115]. More recently, two sarcomeric tropomodulin (Tmod) isoforms, Tmod1 and Tmod4, have been characterized, which represent novel proteolytic targets of m-calpain [116]. The levels of m-calpain resulted elevated in dystrophic soleus muscle of mdx mice and were associated with loss of Tmod1 from the thin filament pointed ends, suggesting that Tmod proteolysis, by m-calpain, represents a novel mechanisms that may contribute to DMD pathology [116]. In other reports, the catabolic activity of calpains, along with the ubiquitin-proteasome pathway, has been associated with muscle atrophy induced by chronic hypobaric hypoxia and with sarcopenia [117]. Sarcopenia is a pathologic consequence of aging, associated with loss of muscle mass and function. It has been demonstrated that muscle-specific overexpression of calpastatin greatly reduces sarcopenia [118].
Other reports provided additional insights into the pathogenic role of calpain in sarcopenia. One of the leading mechanistic theories for aging is the oxidative damage hypothesis, based also on the evidences that age-related changes accelerate with elevated oxidative stress [119,120]. It has been demonstrated that calpains activate the degradation of oxidated myofibrillar proteins [121], suggesting a mechanistic link between oxidative stress and proteolysis [121,122]. In addition, it has been reported that the reduced calpain activity, by forced expression of a calpastatin transgene, attenuates muscle wasting and prevents the shift from slow to fast fiber type in muscle unloading [123]. Another potential target of calpain is Akt via the chaperone heat shock protein 90 (HSP90), which under physiological conditions protects phosphorylated active form of Akt. It has been demonstrated that calpain activation reduces HSP90-client proteins binding, thus reducing Akt activation [124]. Calpain activation might also regulate other factors and pathways, such as STAT, NF-kB, PKC, calcineurin, Cdc42/RhoA leading to muscle atrophy and wasting [125][126][127][128][129][130]. More recently, it has been demonstrated that calpain activity increased, while calpastatin expression is reduced in the muscle of experimental models of cancer cachexia, namely AH-130 hepatoma and the C26 colon carcinoma [131]. Nevertheless, a specific inhibition of Ca 2+ -dependent proteases did not block muscle wasting [131], suggesting that i) loss of myofibrillar proteins in muscle wasting requires the synergistic action of at least two proteolytic systems and ii) targeting the catabolic stimuli rather than the proteolytic activity of a single pathway would likely be the most efficient therapeutic approach for complex diseases as cancer cachexia.
Collectively these studies indicate that calpains exert critical role in both muscle homeostasis and diseases and represent potential therapeutic targets.

THE CASPASE PROTEOLYTIC SYSTEM
Another proteolytic system dysregulated in several diseases, is represented by Caspases [132,133] which comprise two major groups, namely the initiator caspases and the effector caspases.
Caspase-2, -8, -9 and -10 belong to the initiator caspases and as such initiate the proteolitic cascade, whereas caspases-3, -6 and -7 are members of the effector caspases, which are activated by initiator caspases and are responsible for the proteolytic cleavage of a broad spectrum of cellular targets, which ultimately lead to cell death [132] (Fig. 4).
Caspases can be activated by either extrinsic death stimuli, in which specific ligands triggers a cascade of events leading first to the activation of initiator caspases, or alternatively by intrinsic death stimuli inducing mitochondrial release of cytochrome c (Fig. 4), which binding to Apaf1 leads to the formation of the apoptosome [132,133].
A key role of caspases is to inactivate proteins that protect living cells from apoptosis. Caspases can stimulate the caspase-activated deoxyribonuclease (CAD), leading to DNA fragmentation [134,135]. Caspases can also cleave structural proteins such as lamins, promoting nuclear lamina destruction and chromatin condensation [136,137], desmin, generating a small product (N-desmin) that activates apoptotic pathways [138], gelsolin, altering cell motility and morphology, β -catenin, altering cell-cell contacts and cellmatrix focal adhesions, DNA repair, mRNA splicing, and DNA replication [133,139,140]. The activation of apoptotic pathway by Caspase activity is also associated with different muscular diseases, including Duchenne and facioscapulo-humeral dystrophies [141]. One of the proposed mechanisms that mediates the activation of caspase pathway in muscular dystrophy is the Endoplasmic Reticulum (ER) stress [142][143][144][145] that activates the initiator caspases-12 and-4 and in turn the caspase-9 and 3, leading to apoptosis [146][147][148][149][150].
Activated caspase-12 can also carry out apoptotic events directly into the nucleus [151], whereas deleting caspase-12 preserves muscle function in the mdx dystrophic mouse model, resulting in improved force generation [152].
Caspases are also involved in cancer-associated cachexia and in the inhibition of muscle differentiation [153].
Of note, caspases activity has been also associated to different cell functions, which are independent of inducing apoptosis [171]. Caspase activity is involved, along with the ubiquitin protesome system, in the modulation of satellite cell activity during muscle growth and regeneration, promoting Pax7 degradation; thus guaranteeing the acquisition of a differentiation competent state [172,173]. The opposite and mutually exclusive effects exerted by caspases, namely death and non-death caspase function, depend on the duration of the signaling cascade, where cell death is characterized by sustained caspase activity, whereas non-death function is guaranteed by a transient activity responses [172,[174][175][176].
Moreover, increased proteolysis can be achieved by synergic activity of different proteolytic system, such as caspase and ubiquitin-proteasome [177].

THE AUTOPHAGIC PATHWAY
Autophagy is an important homeostatic mechanism for all eukaryotic organisms and modulates different processes such as proliferation, cell death, differentiation during both embryogenesis and postnatal development [178]. The activation of autophagy involves several distinct steps, namely i) induction, ii) phagophore formation, involving donor doublemembrane structures, iii) autophagosome formation and sequestration of cellular components, iv) autophagosomelysosome fusion, v) degradation of sequestered components (Fig. 5).
Basal level of autophagy is essential for physiological turnover of old and damaged organelles, whereas the activation of autophagy beyond a certain threshold promote cell alterations and muscle atrophy [183][184][185][186][187][188][189][190]. For example, exercise promotes beneficial effects on muscle by improving overall mitochondrial quality through the selective removal of damaged or dysfunctional mitochondria [191,192]. Indeed, long-life regular exercise in humans has been shown to preserve functional autophagy, guaranteeing better muscle mass and strength in senior sportsmen than elderly sedentary subjects [193]. Danon Disease (DD) and Glycogen Storage Disease type II (GSDII) represent two autophagic vacuolar myopathies, in which an autophagy block correlates with the severity of the disease [194]. Activity-dependent regulation of autophagy is also important in stabilizing the neuromuscular junction. It has been reported that muscle-specific loss of Atg7, a principal component of the autophagy machinery leads to neuromuscular junctions fragmentation, precocious synaptic dysfunction, and partial denervation [193]. Of note, basal autophagy is also essential to maintain the satellite cells quiescent state in mice [195].
Interestingly, it has been demonstrated that activated autophagy, by low-protein diet, is able to counteract myofiber apoptosis and to improve mitochondrial function in patients affected by COL6/collagen VI-related myopathies [196]. Fig. (4). Schematic representation of the caspase-mediated apoptosis. Caspases can be activated by either extrinsic or intrinsic death stimuli. Extrinsic death signals activate the initiator caspases, which in turn induce the effector caspases, promoting the proteolytic cleavage of cellular targets. Intrinsic death stimuli induce mitochondrial damage, resulting in the cytosolic release of cytochrome c that binding to Apaf1, resulting in the activation of initiator caspases and then to the induction of effector caspases and subsequently to cell death.
Recently, a set of guidelines for the examination of macroautophagy and related processes has been reported [207].

ROLE OF EXERCISE AND PROTEIN INTAKE AS EFFECTIVE COUNTERMEASURES TO THE DELE-TERIOUS CONDITION OF MUSCLE ATROPHY AND WASTING
Several interventions have been suggested to counteract muscle atrophy and wasting.
Physical exercise can counteract muscle atrophy through multiple metabolic and transcriptional adaptations. Exercise acts on signaling pathways involved in muscle mass maintenance, such as the IGF-1/Akt/mTOR axis and on catabolic pathways, such as those mediated by FoxOs transcriptional factors [208][209][210].
Physical exercise can also attenuate muscle atrophy through the modulation of muscle catabolic pathways. Interestingly, it has been demonstrated that 12 weeks of resistance training in young women are sufficient to modulate the basal expression level of two molecular marker of the ubiquitin proteasome dependent proteolysis, MuRF-1 and FOXO3A [211]. Moreover, it has been demonstrated that exercise training enhances the cellular autophagic flux, inducing protein turnover and cellular organelles quality control processes [212,213]. All these data demonstrate that physical exercise can counteract muscle atrophy favorably modulating the catabolic pathway.
Of note, nutrition also plays an important role in muscle homeostasis. It has been demonstrated that food intake can regulate muscle anabolic stimuli. In particular, the Essential Amino Acids (EAAs) have been described as the most important nutritional input for synthesis of new proteins; indeed the EAA Leucine is considered as the major nutritional regulator of protein anabolism for its ability to activate mTOR pathway and to inhibit the proteasome ubiquitin one [214].
Moreover, an antioxidant cocktail of rutin, vitamin E, vitamin A, zinc, and selenium is able to restore the defective leucine stimulation of protein synthesis and function of aged rats [215]. Taurine (2-aminoethane-sulfonic acid), a sulfurcontaining semi-essential aminoacid has been demonstrated to regulate ubiquitin-proteasome system (UPS) and autophagy [216], promoting also the maintenance of cellular integrity and helping to overcome the resistance to anabolic stimuli and loss of muscle mass in elderly [217].
Recent literature has highlighted that the combination of exercise with increased protein intake can represent a plausible strategy to counteract muscle wasting. In this regard, it has been demonstrated that protein supplementation combined with both aerobic and resistance training is able to increase muscle mass, strength, and physical performance in elderly participants [218].

CONCLUSION
Muscle homeostasis is the result of a delicate balance between anabolic and catabolic processes, which if altered can lead to debilitating conditions. Thus, modulation of the anabolic and catabolic pathways represents an attractive therapeutic intervention to counteract muscle wasting and to guarantee the quality control of cellular components.

CONSENT FOR PUBLICATION
Not applicable.

CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.