Anabolic and Catabolic Signaling Pathways that Regulate Skeletal Muscle Mass
John J. McCarthy1,2*, Kevin A. Murach1,3
Contact information:
800 South Rose St. 1
Center for Muscle Biology
2
Department of Physiology
3
Department of Rehabilitation Sciences
College of Medicine University of Kentucky Lexington, KY 40536 *Corresponding Author Phone:
(859) 323-4730
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(859) 323-1070
e-mail:
[email protected]
Abstract Skeletal muscle mass is primarily dictated by the balance between the rates of protein synthesis and degradation. Over the last decade, significant progress has been made in defining the anabolic and catabolic signaling pathways that control skeletal muscle mass through the regulation of protein synthesis and degradation. The purpose of this review is to briefly describe known and emerging signaling pathways involved in the regulation of skeletal muscle mass. Two important themes have come to light: 1) the degree of crosstalk between and among anabolic and catabolic signaling pathways involved in the control of skeletal muscle hypertrophy and atrophy, and 2) the balance between anabolism and catabolism that is required to facilitate a proper hypertrophic response. A more in-depth understanding of the anabolic and catabolic signaling pathways that regulate skeletal muscle mass will provide a critical foundation for the development of more effective training programs and nutritional aids to enhance athletic performance.
Key words AMPK, β-adrenergic, β-catenin, anabolic, atrophy, catabolic, FoxO, hypertrophy, MAFbx, mechanotransduction, MuRF-1, myostatin, NF-κB, nitric oxide, PGC-1α4, ribosome biogenesis, TORC1, TRPV1, YAP/TAZ
Introduction Skeletal muscle is one of the abundant tissues of the human body, accounting for roughly 40% of the body mass in men [1]. In addition to its primary function in locomotion, there is a growing recognition that skeletal muscle has an important role in whole-body metabolism and protein homeostasis during aging [2, 3]. The clinical importance of skeletal muscle health is highlighted by the diverse patient population reported to show significant losses in skeletal muscle mass including those suffering from systemic diseases (cancer, sepsis or HIV-AIDS), organ failure (cirrhosis, chronic kidney disease and chronic heart failure) or inactivity (as a result of obesity, rheumatoid arthritis or prolonged bed rest) as well as aging (sarcopenia). Currently, the most effective therapy for maintaining or restoring muscle mass is resistance exercise, which is often not a realistic option for the aforementioned patient populations. Given this state of affairs, there is great interest in defining the anabolic and catabolic signaling pathways that regulate skeletal muscle mass as the basis for developing more effective therapies to prevent or restore the loss of muscle mass associated with systemic disease, bed rest, and aging. Moreover, a better understanding of the signaling pathways that regulate skeletal muscle mass will allow for the design of more effective training and nutritional programs aimed at increasing muscle mass in athletes with the goal of improving performance.
History One of the most remarkable qualities of skeletal muscle, that is often taken for granted, is its ability to specifically alter its physical characteristics (phenotype) in response to a particular type of contractile activity. The most obvious example of this phenotypic plasticity is the significant increase in skeletal muscle mass following a progressive, high-resistance exercise training program. The observation that resistance exercise can increase muscle mass dates back to the to the ancient Greeks when it was reputed that Milo of Crotona achieved his great strength by carrying a calf on his back every day until it was a bull. Current resistance exercise training programs have replaced the bull with barbells and dumbbells, prescribing three sets of 8-12 repetitions for each exercise, performed on alternating days, three times a week [4]. The dramatic increase in muscle
size following resistance exercise is primarily the result of an increase in muscle fiber size (hypertrophy). The contribution from an increase in muscle fiber number (hyperplasia) is likely minor under normal conditions [5-9], but appears to become more prevalent under circumstances of extreme loading and/or hypertrophy in both humans and rodents [10-17].
Skeletal muscle mass is primarily dictated by the balance between the rates of protein synthesis and degradation. Increasing protein synthesis, decreasing protein breakdown, or modulating both factors can result in a net increase in the rate of protein synthesis which leads to muscle hypertrophy [18]. Accordingly, resistance exercise has been shown in both humans and rodents to cause a net increase in the rate of protein synthesis [18-24], and this is primarily dictated by intrinsic cellular processes, and not necessarily by transient alterations in systemic hormones such as testosterone [25]. Recent work in humans has shown that the initial myofibrillar protein synthetic response to resistance exercise in untrained muscle is not necessarily predictive of the hypertrophic response to training [26, 27]. However, once muscle damage characteristic of the first few weeks of a resistance training program has subsided, myofibrillar protein synthesis rates do reflect hypertrophic potential [27].
Baar and Esser (1999) provided the first mechanistic data linking high-force contractions to muscle hypertrophy via the prolonged activation of mTOR (mechanistic target of rapamycin) signaling, the cell’s master regulator of protein synthesis [28-31]. Bodine and colleagues (2001) followed up with the seminal study demonstrating mTOR function was absolutely necessary for skeletal muscle hypertrophy [32]. The importance of mTOR activation was subsequently linked to hypertrophic growth in humans by Mayhew and co-workers (2009), showing that changes in translational signaling following a bout of unaccustomed resistance exercise was predictive of the hypertrophic response after 16 weeks of resistance exercise training [33]. While mTOR signaling is considered to be the primary pathway regulating skeletal muscle mass, other signaling pathways are shown to be capable of regulating skeletal muscle hypertrophy. The focus of this chapter will be to briefly discuss the role of mTOR signaling in muscle
hypertrophy (for an in-depth review of mTOR signaling, refer to the recent review by Saxton and Sabatini [34]), followed by a review of other anabolic signaling pathways involved in the regulation of skeletal muscle mass. The chapter will conclude with a review of catabolic signaling pathways that promote muscle atrophy by inhibiting protein synthesis and/or increasing the rate of protein degradation.
Anabolic signaling
Mechanotransduction: translating force to a hypertrophic signal During resistance exercise, muscle tension is a primary impetus for the growth response. As such, the first step in initiating an intracellular signaling cascade that results in hypertrophy is tension sensing. Skeletal muscle fibers perceive tension via a variety of elements within the muscle fiber membrane and intracellular cytoskeleton. Structures linking the muscle fiber membrane to the extracellular matrix, such as integrins, cadherins, and focal adhesion complex proteins, undergo conformational changes when force is applied to them, which affects intracellular behavior. For instance, when the extracellular matrix interacts with membrane-bound integrins, focal adhesion complexes form that mobilize ribosomes and mRNAs which facilitates translation [35]. Muscle cell culture experiments show that disruption of focal adhesion complex proteins blunts intracellular hypertrophic signaling [36]. It is also known that these focal adhesion complexes can directly activate ribosomal proteins to facilitate protein translation [37].
The intracellular actin cytoskeleton is also connected to integrins. Physical interactions between actin and intermediate filaments within skeletal muscle such as desmin and muscle ankryn repeat proteins (MARPs, which are associated with the giant scaffold protein titin) also play mechanosensing roles via these connections. Titin is now recognized as a central mediator of hypertrophic signaling [38, 39], and titin-associated MARPs can migrate away from titin and affect various signaling pathways as well as gene transcription [40]. The Z-disc, which titin and many other myofilaments are anchored to within the sarcomere, is also emerging as a central node for translating
mechanical tension to intracellular signaling [39, 41]. Furthermore, the primary intermediate filament, desmin, deforms myonuclei via direct force transmission from the cell membrane (which can affect transcription) and facilitates intracellular stress signaling [42].
Mechanotransduction in skeletal muscle is a relatively recent area of inquiry, so mechanosensing protein interactions and the consequences are not yet fully elucidated. Despite the relative immaturity of this area, significant progress has been made regarding mechanotransduction and activation of mTOR. Recent evidence indicates that phosphatidic acid (PA), a lipid second messenger, is responsive to mechanical load and directly activates mTOR independent from intermediate signaling [43-45]. This PA is generated from mechanical-sensitive diacylglycerol kinases found in the membrane structures of skeletal muscle [45]. Furthermore, PA supplementation increases muscle protein synthesis in rodents [46], and enhances hypertrophy from resistance exercise training in humans [47]. More work is needed to fully elucidate how mechanical tension is ultimately translated to skeletal muscle growth, but mTOR signaling appears to be a focal point in this process.
mTOR signaling mTOR (mechanistic target of rapamycin) is a member of the phosphoinositide 3-kinase (PI3K)-related kinase family that has been shown to form two separate multi-protein complexes designated mTOR complex 1 (TORC1) and 2 (mTORC2) [48]. mTORC1 is a master regulator of protein synthesis, integrating a number of upstream signals including growth factors (insulin, IGF-1), nutrients (amino acids) and mechanical strain; TORC2 is primarily involved in the regulation of cytoskeleton dynamics and cell proliferation and survival [48, 49].
Although it had been known for some time that insulin-like growth factor-1 is capable of inducing muscle fiber hypertrophy, the underlying mechanism remained unknown until Rommel and colleagues (2001) provided evidence showing that IGF-1 activated TORC1 via the PI3K/AKT pathway [50, 51]. Activation of the PI3K/AKT pathway by IGF-1
increases protein synthesis through AKT-mediated phosphorylation of tuberous sclerosis protein 2 (TSC2) protein, the primary inhibitor of TORC1 activity [52-55]. The phosphorylation of TSC2 by AKT inhibits its activity, resulting in the accumulation of the active form of Rheb (Ras homolog enriched in brain), a potent activator TORC1 [56, 57]. Rheb localizes with the late endosome/lysomal (LEL), along with other key modulators of mTOR such as PA and TSC2, indicating that the LEL is the key site of integration for mTOR signaling during hypertrophy [44]. Indeed, mechanical stimulation increases the association of mTOR with the LEL [58]. Furthermore, amino-acid-mediated stimulation of mTOR is Rheb-dependent, and mTORs association with the LEL is facilitated by amino-acid availability [59]. Once activated by Rheb, TORC1 stimulates protein synthesis by phosphorylating ribosomal S6 kinase (p70S6k1) and eukaryotic initiation factor 4E binding protein (4E-BP1), two factors that enhance the initiation of mRNA translation (thus promoting protein synthesis). In addition to these factors, TORC1 activation has been shown to increase the expression of the ε-subunit of eIF2B holoenzyme, thereby promoting the recruitment of the initiator methionine tRNA to the start codon and increasing protein synthesis [60]. The expression of eIF2Bε was shown to increase following resistance exercise training, with its abundance appearing to be an important determinant of the hypertrophic response [33, 61].
In a search for proteins that were targeted for degradation by the muscle-specific ubiquitin ligase Fbxo32 (commonly known as MAFbx or atrogin), Lagirand-Cantaloube and co-workers (2008) identified the eukaryotic initiation factor 3, subunit 5 (eIF3f) [62]. As the largest of the initiation factors, evidence indicates that the eIF3 complex is involved in nearly all aspects of translation initiation; most notable for the current discussion is the finding that eIF3f can serve as a docking site for TORC1 and p70S6k1 [63-65]. Upon activation, TORC1 is recruited to the eIF3f-p70S6K1 complex, leading to the phosphorylation and subsequent release of active p70S6K1, which is then capable of increasing protein synthesis by phosphorylating ribosomal protein S6 (rpS6) and eIF4B [64, 65]. In a series of studies, it was demonstrated that the in vivo and in vitro overexpression of eIF3f induced myotube and muscle fiber hypertrophy, respectively, by stimulating protein synthesis through the phosphorylation of p70S6K1, rpS6 and 4E-BP1
[62, 66]. It will be important to determine if the regulation of muscle hypertrophy by eIF3f is conserved in humans and what role it has in the hypertrophic response following resistance exercise.
Wnt/β-catenin signaling and ribosome biogenesis In their original description of IGF-1 mediated muscle hypertrophy, Rommel and colleagues (2001) also identified glycogen synthase kinase 3β (GSK3β) as a downstream target of PI3K/AKT signaling [51]. In contrast to TORC1, phosphorylation by AKT leads to GSK3β inactivation, which prevents further inhibition via phosphorylation of the translation initiation factor eIF2Bε [51]. The inhibition of GSK3β activity by IGF-1 or lithium (a known inhibitor of GSK3β) treatment, or over-expression of a dominant-negative form of GSK3β, all resulted in a significant myotube hypertrophy, thus confirming the importance of GSK3β inhibition by IGF-1/PI3K/AKT signaling in the regulation of muscle mass [51, 67].
In addition to eIF2Bε, the inactivation of GSK3β by IGF-1 via AKT phosphorylation also results in the stabilization of β-catenin protein [68]. β-catenin is known to exist in two separate intracellular pools; an actin cytoskeleton pool involved in the formation of adherens junctions and a cytoplasmic pool that is the downstream mediator of the Wnt signaling pathway involved in the regulation of gene expression [69]. Under resting conditions, cytoplasmic β-catenin is phosphorylated by GSK3β, targeting it for degradation by the proteasome [70]. Upon GSK3β inhibition, via phosphorylation of Ser9 by AKT or sequestration by Dishevelled following Wnt activation, β-catenin accumulates in the cytoplasm. β-catenin ultimately translocates to the nucleus where it regulates the expression of target genes such as c-Myc and cyclin D [71, 72]. These studies show that β-catenin levels can be regulated by a Wnt-dependent and independent mechanism. Following seven days of mechanical overload induced by synergist ablation, nuclear βcatenin levels increased by over 4-fold in the mouse plantaris muscle through a Wnt-
independent mechanism [73]. In a follow-up study, Armstrong and colleagues (2006) reported that the skeletal muscle-specific inactivation of β-catenin completely prevented muscle fiber hypertrophy, demonstrating the necessity of β-catenin in the regulation of muscle hypertrophy [74]. Associated with the increase in nuclear β-catenin levels during hypertrophy were increased myonuclear c-Myc expression (an established target gene of β-catenin) and a 3-fold increase in total RNA [73]. The increase in the total RNA pool is indicative of ribosome biogenesis given that ~85% of total RNA consists of ribosomal RNA [75]. A greater ribosomal content of the cell results in an increase in protein synthesis as a consequence of greater translational capacity. An increase in the translational capacity of the cell represents an additional mechanism for increasing the rate of protein synthesis in response to a hypertrophic stimulus. While the necessity of enhanced translational efficiency through TORC1 activation is well-established, the importance of increased translational capacity to skeletal muscle hypertrophy is an emerging area of interest [76]. Interestingly, in a review of the literature, Hannan et al., (2003) concluded that increased translational capacity was required for cardiac hypertrophy and that increased translational efficiency alone was not sufficient to promote muscle hypertrophy [77].
Although the exact mechanism(s) involved in the regulation of ribosome biogenesis during muscle hypertrophy have not been fully elucidated, evidence from a number of different studies supports a model in which increased β-catenin expression up-regulates c-Myc expression, which subsequently drives transcription of ribosomal DNA through the regulation of RNA polymerase I and components of the preinitiation complex [73, 78, 79]. Transcription of ribosomal DNA (rDNA) to rRNA by polymerase I is considered the major rate-limiting step in ribosome biogenesis [80]. Both the mitogen-activated protein kinase/extracellular signal-regulated pathway (MAPK/ERK), as well as the mTOR pathway, regulate Pol I formation and activation [81], indicating that these signaling cascades are also mediators of ribosome biogenesis. Pol I regulation via mTORC1 is controlled by upstream binding factor (UBF) [81], which dictates the number of active ribosomal genes (of which there are hundreds in the human genome) [82]. In culture, blocking mTORC1 in myotubes decreases UBF and rRNA abundance, thereby
preventing myotube hypertrophy [83]. Specifically blocking Pol I in muscle cell cultures has the same effect [84]. mTOR also has nuclear localization in muscle, directly interacting with the rDNA promoter and influencing ribosomal gene transcription [85]. The mechanistic evidence suggesting that translational capacity via ribosome biogenesis is a primary mediator of muscle hypertrophic potential is compelling. Observational studies in both rodents and humans also indicate that robust upregulation of ribosome biogenesis is characteristic of, and likely necessary for, successful muscle hypertrophy [84, 86-93]. Future loss- and gain-of-function studies will determine whether ribosome biogenesis and increased translational capacity are indispensable for muscle hypertrophy.
A non-canonical Wnt signaling pathway has been described that is also capable of inducing muscle hypertrophy independent of GSK3β or β-catenin [94]. Experiments in myotubes revealed that Wnt7a binding to the frizzled homolog 7 (Fzd7) receptor activated AKT via a PI3K mechanism that did not involve IGF-1 signaling [94]. As expected, AKT activation by Wnt7a resulted in an increase in TORC1 activity, as assessed by rpS6 phosphorylation, and an increase in myotube diameter [94]. Consistent with these findings, over-expression of Wnt7a in skeletal muscle fibers increased cross-sectional area 40-55%; however, whether or not this hypertrophy was caused by TORC1 activation and/or the increase satellite cells associated with Wnt7a over-expression, remains to be determined [94, 95]. β-adrenergic receptor signaling The β-adrenergic receptors (β-AR) are members of the guanine nucleotide-binding Gprotein-coupled receptor family with the β2 subtype being the most abundant in skeletal muscle [96-98]. Signaling through the β2-AR occurs primarily via coupling with the G protein Gαs in skeletal muscle, though there are reports describing signaling events involving Gαi and, more recently, Gβγ [99, 100]. β2-AR coupling to Gαs activates adenylyl cyclase production of cyclic-AMP (cAMP), which in turn activates downstream signaling through protein kinase A (PKA) [101]. Alternatively, β2-AR signaling involving
the Gβγ dimer initiates signaling through a PKA-independent pathway involving PI3K activation of AKT [102-104]. Historically, β-adrenergic receptor agonists (β-agonists) have been used to treat patients suffering from bronchial ailments such as asthma; however, Emery and coworkers (1984) made the fortuitous discovery that administration of the β-agonist clenbuterol caused skeletal muscle hypertrophy in rats and was associated with a 34% increase in muscle protein synthesis [105]. The ability of β-agonists to increase skeletal muscle mass has since been confirmed by numerous studies and shown to be effective in humans as well (reviewed in Lynch GS, 2008) [101]. The increase in muscle mass following β-agonist administration is the result of hypertrophy (an increase in cell size) and not hyperplasia (an increase in cell number) or satellite cell activity [106-108]. The mechanism through which β-agonists exert their anabolic effect has been attributed to enhanced protein synthesis, though studies have reported a decrease in protein degradation as a contributing factor [105, 109-114]. The increase in skeletal muscle mass following 14 days of clenbuterol treatment was completely blocked by coadministration of rapamycin, a potent inhibitor of TORC1 signaling, indicating the anabolic action of β-agonists is the result of an increase in protein synthesis via TORC1 activation [115]. In support of this mechanism, the same authors reported that clenbuterol activated TORC1 signaling, as evidenced by increased phosphorylation of AKT, p70S6K1 and 4E-BP1 [115]. Moreover, these findings suggest that in skeletal muscle, β-agonists function through a β2-AR/ Gβγ complex that activates PI3K/AKT signaling. Interestingly, rapamycin or triciribine (an AKT inhibitor) was unable to block the muscle-sparing effects of clenbuterol following muscle denervation suggesting a different mode of action, possibly involving cAMP/PKA pathway downstream of β2-AR/ Gαs [115, 116]. Upstream in β2-AR signaling, GRK2, a G-protein coupled receptor kinase, plays an integral role in desensitization of adrenergic receptors following stimulation. Eventual desensitization of β2-ARs is of critical importance specifically in heart muscle, since continued activation could lead to pathology and loss of function. In
skeletal muscle, however, loss of GRK2 leads to enhanced hypertrophy following clenbuterol treatment through increased AKT signaling [117]. Worth noting is that β2AR-mediated hypertrophy is not always functional, resulting in reduced normalized strength [117, 118], which is most noticeable in fast-twitch predominant muscles [117, 119]. Impaired relative force production via β2-AR stimulation highlights that successful skeletal muscle hypertrophy is a complex and interrelated process that requires coordination at multiple levels to ultimately facilitate proper function.
In addition to Gαs and Gβγ, lysophosphotidic acid (LPA)-induced muscle hypertrophy was uncovered that involved the Gαi2 isoform [120]. In a series of loss- and gain-offunction experiments, Minetti and colleagues (2011) showed that LPA-induced myotube hypertrophy was mediated by a 40% increase in protein synthesis via TORC1 activation, as indicated by increased phosphorylation of its downstream targets p70S6K1 and rpS6 [120]. TORC1 activation by LPA, however, was independent of AKT but required protein kinase C (PKC) inhibition of GSK3β, though the mechanism linking PKC to TORC1 activation remains to be identified [120]. Furthermore, in vivo upregulation of β2-AR via adeno-associated virus induces hypertrophy independent from mTOR activation, suggesting an alternative pathway can contribute to β2-AR-mediated hypertrophy [121].
Emerging pathways Unlike PI3K/AKT/TORC1 and β2-AR signaling pathways, there are less well-established signaling pathways that have been shown to be capable of regulating muscle hypertrophy. Although it is still too early to know the importance of these “new” pathways in human skeletal muscle hypertrophy, an understanding of how each of these pathways function will provide a more comprehensive knowledge of the signaling pathways that regulate skeletal muscle hypertrophy.
Nitric oxide (NO) signaling Inhibition of nitric oxide synthase (NOS) by NG-nitro-L-arginine methyl ester (L-NAME) administration significantly blunted muscle hypertrophy induced by mechanical overload
of the rat plantaris muscle [122, 123]. Building on these early studies, Ito and colleagues (2013) uncovered a signaling pathway by which neuronal NOS (nNOS) production of nitric oxide mediates muscle hypertrophy [124]. Upon mechanical loading, nNOS derived nitric oxide reacts with superoxide to generate peroxynitrite. Peroxynitrite then activates the TRPV1 (transient receptor potential cation channel, subfamily V, member 1) channel causing an increase in intracellular Ca2+ levels via release from the sarcoplasmic reticulum. Elevation of intracellular Ca2+ causes an increase in protein synthesis through TORC1 activation by an unknown mechanism; though speculative at this time, the authors proposed a similar Ca2+/calmodulin mechanism as that used by amino acids to stimulate TORC1 activity [124, 125]. In agreement with these findings, the administration of the TRPV1 agonist capsaicin activated TORC1 signaling to a comparable level as that observed with mechanical overload [124]. Germline deletion of NOS production elicits a mitochondrial unfolded protein response as well as an upregulation in protein degradative pathways, resulting in impaired muscle growth during development [126]. These findings allude to the important but often overlooked relationship between mitochondrial function (which provide ATP for biosynthetic processes like protein synthesis and ribosome biogenesis) and hypertrophic potential. NOS scavenges reactive oxygen species (ROS) such as superoxides, which cause oxidative stress and can be damaging to the cell. However, a physiological balance of ROS seem to promote hypertrophy [127]. More work is needed to fully elucidate the mechanisms whereby NOS, either directly or indirectly, contributes to hypertrophy. PGC-α4 signaling PGC-1α (peroxisome proliferative activated receptor, gamma, coactivator 1 alpha) was originally identified as a coactivator of PPARγ in brown adipose tissue, but has since been shown to have an important role in skeletal muscle adaptation to exercise [128, 129]. Although the over-expression of PGC-1α in skeletal muscle was found to ameliorate the loss of muscle mass caused by denervation, there was no evidence to suggest PGC-1α might have a role in regulating skeletal muscle hypertrophy [130]. Recently, Ruas et al., (2012) identified a splice variant of PGC-1α, PGC-1α4, capable of inducing muscle fiber hypertrophy when over-expressed both in vitro and in vivo [131].
The mechanism through which PGC-1α4 promotes hypertrophic growth appears to be through the down-regulation of myostatin expression while simultaneously increasing IGF-1 expression [131]. Importantly, in humans, PGC-1α4 expression was found to be dramatically increased in response to a 8-week training program consisting of both resistance and endurance exercises [131]. Moreover, leg press performance (number of repetitions) following the training program correlated with the increase in PGC-1α4 expression, suggesting PGC-1α4 expression might serve as a readout for optimizing a strength training program [131]. Further investigation showed that PGC-1α4 transcription is dependent on G protein-coupled receptor 56 (GPR56); over-expression of this receptor induces myotube hypertrophy while knockdown attenuates hypertrophy in mice, and receptor regulation is enhanced during hypertrophy in mice and humans [132]. Since the original work showing a connection between PGC-1α4 and hypertrophy with resistance exercise, others have shown that this PGC-1α splice variant is similarly up-regulated with endurance exercise [133, 134] and hypoxia [135]. More work is needed to determine whether PGC-1α4 drives hypertrophy, or is part of the global remodeling response to exercise in humans.
microRNAs MicroRNAs (miRs) are small (~ 22 nucleotides), noncoding RNAs that regulate gene expression through a post-transcriptional mechanism [136]. A small family of musclespecific miRs, referred to as myomiRs, have been shown to have an important role in muscle development and disease [137]. In response to mechanical overload in mice, myomiR expression is drastically altered, suggesting a regulatory role in the hypertrophic process [138]. For instance, miR-1 and -133a are known to target growth factor transcripts for degradation (specifically IGF1); these two myomiRs are downregulated during overload, which may allow for up-regulation of growth factor synthesis in skeletal muscle to promote hypertrophy [138]. The expression of myomiRs has also been found to change in response to both resistance and endurance exercise following a single bout or with training in humans [139]. Davidsen and co-workers (2011) identified a small group of miRs that were differentially expressed between low- and high-responders following a 12-week resistance exercise program [140]. In particular,
the change in miR-378 expression in response to training was positively correlated with gains in skeletal muscle mass [140]. Most recently, it has been reported that myomiRs are stabilized via packaging into extracellular vesicles (e.g. exosomes) or via chaperone proteins and effluxed from skeletal muscle under various conditions [141, 142]. Trafficking of miRNAs appears to be a method of intercellular regulation between and amongst tissues. For example, muscle stem cells (known as satellite cells) release miR206 into the microenvironment which specifically regulates proper extracellular matrix (ECM) deposition during hypertrophy [143]. ECM remodeling is necessary for coordinating the hypertrophic process, but excessive ECM can inhibit muscle hypertrophy [143, 144]. Release of miRNAs from muscle fibers into the extracellular space could also serve to clear specific myoMirs from muscle in order to facilitate growth processes [145]. To have a better understanding of the molecular mechanisms through which myomiRs regulate skeletal muscle hypertrophy, future studies will need to focus on identifying target genes and their connection to anabolic signaling.
Catabolic signaling As dramatic as the increase in skeletal muscle mass can be as the result of resistance exercise training, so the loss of skeletal muscle mass can be following periods of disuse. Muscle atrophy can also occur as the consequence of certain systemic diseases (cancer, sepsis or HIV-AIDS), organ failure (cirrhosis, chronic kidney disease and chronic heart failure), inactivity (as a result of rheumatoid arthritis or prolonged bed rest) and aging. In contrast to muscle hypertrophy, muscle atrophy comes about when the rate of protein degradation exceeds the rate of protein synthesis, either through an increased degradation and/or decreased protein synthesis.
In an effort to determine if a common mechanism might underlie muscle atrophy brought about by differing catabolic conditions, Bodine and colleagues (2001) searched for genes that shared a similar pattern of expression in response to denervation, immobilization, and hind limb unloading [146]. Two genes, MAFbx (Muscle Atrophy Fbox) and MuRF-1 (Muscle RING Finger 1), were identified that were significantly upregulated in all three models of muscle atrophy [146]. Concurrently, Gomes and co-
workers reported the identification of atrogin-1 (also known as MAFbx) in atrophying muscle caused by food deprivation, diabetes, cancer and renal failure [147]. Further analysis revealed that both MAFbx and MuRF-1 are skeletal muscle-specific ubiquitin ligases involved in protein degradation through the ubiquitin-proteosome (UPP) system [146, 147]. The central importance of these two genes to the skeletal muscle atrophy process was confirmed by experiments showing that inactivation of either MAFbx or MuRF-1 resulted in a blunted atrophic response under catabolic conditions [146]. Recently, UPP activation independent from MAFBx and MuRF-1 has been observed during skeletal muscle remodeling [148]. Additional ubiquitin ligases, such as MUSA1 [149] and Nedd4-1 [150, 151], have also been implicated in the UPP system and could emerge as important regulators of muscle catabolism.
Shifting protein balance via UPP activation to favor breakdown generally results in atrophy, but it is important to highlight that a certain degree of breakdown is important for maintaining protein integrity and an optimal hypertrophic response [152, 153]. For instance, muscle-specific knockout of a protein that is essential for proper proteasome activity impairs muscle growth and function during post-natal development [154]. Studies in humans also report acute up-regulation of MAFBx and MuRF-1 after acute resistance exercise [155], which highlights that muscle protein breakdown is a component of the hypertrophic process. A controlled level of breakdown likely supports amino acid-reallocation during times of growth, and also prevents the aggregation of mis-folded and non-functional proteins. More work is needed to understand the complex interplay between protein breakdown and synthesis in mediating muscle fiber size.
AKT/Foxo signaling The ability of IGF-1 to block MAFbx expression under catabolic conditions induced by the synthetic glucocorticoid dexamethasone indicated, paradoxically, that the PI3K/AKT signaling pathway was involved in the regulation of muscle atrophy [156]. A downstream target of PI3K/AKT signaling through which IGF-1 could inhibit MAFbx expression was the Forkhead box O (FoxO) class of transcription factors; phosphorylation by AKT causes the Foxo protein to remain sequestered in the cytoplasm, unable to activate
transcription of target genes such as MAFbx and MuRF-1 [157, 158]. Subsequent studies confirmed such a mechanism by providing evidence demonstrating that IGF-1 activation of AKT leads to repression of MAFbx and MuRF-1 expression through FoxO phosphorylation [159, 160]. Further, over-expression of a constitutively active form of FoxO3 isoform caused a significant increase in MAFbx expression and reduced myotube diameter by 50% [159]. Conversely, Southgate and colleagues (2007) reported that FoxO1 was also able to promote muscle atrophy by inhibiting TORC1 signaling through the up-regulation of 4E-BP1 expression [161]. In addition to describing a key signaling pathway regulating muscle atrophy, these studies reveal the degree of crosstalk between anabolic and catabolic pathways in skeletal muscle.
Further evidence of the degree of functional overlap between anabolic and catabolic signaling is highlighted by the importance of FoxO3 in mediating autophagy. Separate from the UPP system, autophagy is another mechanism of protein breakdown that utilizes autophagosomes and lysosomes to facilitate systematic degradation and recycling of cellular components. In skeletal muscle, FoxO3 directly controls autophagy through the transcription of autophagy-related genes [162]. Although autophagy is a protein degradative process, and excessive autophagy contributes to atrophy in various catabolic conditions [163], inhibition of autophagy also results in atrophy and myopathy and is required to maintain muscle mass [162, 164]. This again highlights that a fine balance between protein synthesis and breakdown is required in order to undergo optimal muscle growth.
Given that the UPP is the major contributor to muscle protein degradation, and both MAFbx and MuRF-1 are E3 ubiquitin ligases, identifying the proteins each one targets for degradation is important for understanding their respective catabolic action and how it promotes muscle atrophy. MAFbx had been shown to target eIF3f and MyoD for degradation whereas thick myofilament proteins, such as myosin-binding protein C, myosin light chains 1 and 2 and myosin heavy chain, are targeted for degradation by MuRF-1 [62, 165, 166]. These findings suggest MAFbx and MuRF-1 have distinct functions in muscle atrophy; MAFbx modulates the expression level of genes involved in
regulating protein synthesis while MuRF-1 influences the level of protein degradation [167].
Myostatin signaling Myostatin is a member of the transforming growth factor-β (TGF-β) superfamily that acts as a potent negative regulator of skeletal muscle mass [168]. Myostatin is a “myokine”, or a hormone-like protein (cytokine) released from muscle, that can act in an autocrine or paracrine fashion. Myostatin binds the activin type IIB receptor, which in turn activates activin receptor-like kinase 4 (ALK4) or ALK5 receptor and initiates downstream signaling through the Smad2/Smad3 transcription factor complex [169]. While the evidence is clear that inactivation of myostatin expression or activity promotes skeletal muscle hypertrophy, the mechanism by which myostatin induces muscle atrophy remains to be fully defined [168, 170, 171]. Studies have shown that myostatin is capable of inhibiting AKT/TORC1 signaling through a Smad2/3-dependent mechanism [170, 172, 173]. In vivo, Smad3 activation induces MAFbx, inhibits mTOR and protein synthesis, and promotes atrophy [174]. Recent muscle cell culture evidence indicates that Smad 3 synergistically increases FoxO-mediated expression of MAFbx and MuRF-1 via DNA binding at promoter regions for these genes [175], suggesting myostatin may function via this mechanism. Regardless, these findings provide yet another example of the crosstalk between the signaling pathways that regulate skeletal muscle mass.
AMPK signaling The AMP-activated protein kinase (AMPK) is a multi-protein kinase composed of a catalytic subunit (α) and two regulatory subunits (βγ) [176]. In response to cellular stress that causes a decrease in cellular energy levels (as sensed by an increase in the AMP:ATP ratio), AMPK is activated and functions to restore the energy status of the cell by turning on catabolic pathways and turning off ATP-consuming anabolic pathways such as protein synthesis [176]. As such, AMPK is called the “energy sensor” of the cell. Chronic activation of AMPK ultimately results in mitochondrial biogenesis [177], making AMPK a primary mediator of endurance exercise adaptations. The central role that
AMPK plays in the regulation of cell metabolism, and protein synthesis in particular, suggest that it could also be involved in regulating skeletal muscle mass.
Inoki and colleagues (2003) provided evidence that AMPK was able to inhibit protein synthesis through phosphorylation of TSC2, a potent inhibitor of TORC1 activity [178]. Various in vivo experiments in rodents subsequently showed that AMPK and mTOR are inversely regulated with hypertrophic stimuli [179-181]. In AMPK knockout mice, muscle mass and fiber size was increased as a result of TORC1 activation [182]. Further, AMPK inactivation accelerated the rate of muscle hypertrophy induced by mechanical overload, thus confirming the idea that AMPK acts to limit skeletal muscle growth [183]. AMPK down-regulation also attenuates atrophy during muscle disuse [184].
In addition to inhibiting TORC1 through TSC2 activation, AMPK has also been shown to restrict muscle growth by inducing expression of FoxO transcription factors and expression of target genes MAFbx and MuRF-1 [185]. In myotubes, activation of AMPK by the AMP analog AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) caused enhanced myofibrillar protein degradation that was likely the result of FoxO mediated up-regulation of MAFbx and MuRF-1 expression [185]. Accordingly, when AMPK activation is suppressed in the presence of AICAR, MuRF-1 is not up-regulated [186].
The reciprocal relationship between AMPK and mTOR forms the basis of the “molecular interference” phenomenon, which stipulates that optimal hypertrophic adaptation from resistance training (i.e. maximal mTOR activation) cannot be realized in circumstances when endurance training adaptations are occurring simultaneously (i.e. AMPK will interfere with mTOR when training “concurrently”). While chronic and excessive AMPK activation will likely inhibit maximal hypertrophic potential, it is too simplistic to reduce endurance and resistance exercise adaptations to two distinct and competing pathways. For instance, resistance exercise increases AMPK activation [187-192] while aerobic exercise stimulates mTOR [193-195] in humans. In fact, mechanical strain has recently been linked directly to AMPK activation and energy homeostasis [196]. It follows that the evidence for molecular interference inhibiting hypertrophy in humans is quite limited
[197]. There is likely a balance between AMPK and mTOR activation that still allows for maximal hypertrophic adaptation in vivo, but more work is needed to elucidate the limits of this interplay.
NF-κB signaling NF-κB is a dimeric transcription factor involved in a range of biological processes including inflammatory and immune responses, and is rapidly activated by inflammatory cytokines such as TNF-α [198]. In skeletal muscle, however, NF-κB expression was reported to dramatically increase during muscle atrophy that was independent of TNF-α [199]. Subsequent studies showed that NF-κB signaling was in fact necessary and sufficient for mediating the hypertrophic response [200, 201]. The mechanism through which NF-κB signaling promoted atrophy remained unknown until recently when Jackman and colleagues (2012) showed that NF-κB directly regulates the expression of MuRF-1 through a Bcl-3 dependent mechanism [202]. Furthermore, NF-κB sites are required for MuRF-1 activation during disuse atrophy [203]. Alternatively, a member of the TNF family of inflammatory cytokines, TWEAK (TNF-like weak inducer of apoptosis), has been shown to induce skeletal muscle atrophy through up-regulation of MuRF-1 via NF-κB activation [204]. Indeed, high levels of TWEAK promote muscle atrophy in vivo via activation of the “canonical”, or traditional NF-κB signaling pathway [205, 206]. However, through the TWEAK receptor fibroblast growth factor-inducible 14 (Fn14), TWEAK (or potentially some other ligand) can also signal through the “noncanonical” NF-κB pathway which involves synthesis of NF-κB inducible kinase (NIK). Skeletal muscle hypertrophy in humans is associated with Fn14 up-regulation [207, 208] and resistance exercise causes non-canonical NF-κB activation without TWEAK induction [209], suggesting a complex regulation of this pathway that is likely dependent on TWEAK levels and the nature of the stimulus [210].
Hippo pathway and YAP/TAZ signaling The Hippo pathway got its name when a group of researchers noticed that a mutation of one of the key kinase components (Hpo) resulted in tissue overgrowth in the fruit fly,
specifically around the head, that was reminiscent of how a hippopotamus looked [211]. The Hippo pathway has since been recognized as a key mediator of organ size control [212]. In brief, Hippo signaling involves phosphorylation of MST1 and MST2, which acts on the downstream kinases large tumor suppressor kinases 1 and 2 (LATS1 and LATS2), which in turn phosphorylates and represses the transcriptional cofactor Yesassociated protein (YAP) and/or its mammalian paralogue TAZ (transcriptional coactivator with PDZ-binding motif). When YAP/TAZ is de-phosphorylated, it acts as a transcriptional co-activator and affects gene transcription. In various tissues, a high degree of cross-talk is also apparent between the Hippo pathway and defined muscle mass-regulating pathways, such as Akt/mTOR, Myostatin/SMAD, and AMPK, and the Hippo pathway may be activated by a variety of receptors [213-215]. However, the mechanisms of Hippo regulation on muscle fiber size were not explored until recently. YAP was first shown to promote hypertrophy via TEAD transcription factors in skeletal muscle, independent from mTOR activity or MAFbx and MuRF-1 transcription [216]. Indeed, YAP expression increases with a different time course than mTORC1 activation during mechanical overload, and may also regulate muscle mass via ribosome biogenesis and/or altered SMAD2/3 activity [217]. The Hippo pathway and YAP/TAZ regulation are exciting new targets for muscle mass regulation and merit further interrogation.
Summary There have been significant advancements during the last decade in our understanding of the anabolic and catabolic signaling pathways involved in the regulation of skeletal muscle mass. These advances have uncovered the mechanistic details of such signaling pathways and demonstrated the complex crosstalk that occurs between pathways. Future studies will certainly continue to enhance our understanding of the aforementioned pathways as well as better define the importance of emerging pathways not often considered in the field of skeletal muscle plasticity. While there still remains much to be learned, one conclusion that is clear is that the regulation of skeletal muscle mass represents the orchestrated output of multiple anabolic and catabolic signaling pathways.
Figure legend Figure 1. Anabolic and catabolic signaling pathways that regulate skeletal muscle mass. Clenbuterol, IGF-1 and Wnt 7a are able to activate PI3K/AKT signaling through binding to β-AR, IGFR-1 and Fzd7 receptor, respectively. AKT inhibits TSC2 activity which results in Rheb activation of TORC1, localized at the late endosome-lysosome (LEL). TORC1 increases protein synthesis through phosphorylation of S6K1 (p70S6K1) and 4EBP1. AKT is also able to increase protein synthesis through inhibition of GSK3β. Inactivation of GSK3β leads to stabilization of β-catenin resulting in c-Myc induction of ribosome biogenesis. Protein synthesis can be inhibited through AMPK activation of TSC2 or mTOR. AMPK can also increase protein degradation through FoxO regulation of MAFbx and MuRF-1 expression. MAFbx and MuRF-1 are muscle-specific ubiquitin ligases that target proteins for degradation, such as eIF3f and myosin, respectively. FoxO up-regulation of MAFbx and MuRF-1 expression can also be mediated through myostatin binding to the ActIIRβ receptor with the downstream activation of Smad 2 and Smad 3 transcription factors. Canonical activation of NF-κB through an unknown mechanism, that is independent of inflammatory cytokines such as TNF-α, increases MuRF-1 expression through activation of Bcl-3 and facilitates protein degradation. Noncanonical activation of NF-κB via Fn14 may result in muscle hypertrophy. YAP/TAZ is regulated via the Hippo pathway, of which the core components are MST1 and 2 and LATS 1 and 2. Activated YAP/TAZ can promote myogenic gene transcription via TEADs or augment protein synthesis via other mechanisms. Arrow indicates activation where as a crossbar indicates inhibition.
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clenbuterol
IGF-1
β-AR
IGFR
Wnt7a
myostatin
Fzd7
sarcolemma
?
ActIIRβ
DGK
Fn14
PA
PI3K
canonical NF-𝜅B
AKT
Smad 2/3
GSK3β
Bcl-3
non-canonical
NIK
︎ AMP:ATP ratio
TSC2 β-catenin Rheb
AMPK
mTOR
FoxO
LEL
protein synthesis and/or ➡︎ protein degradation?
c-Myc
ribosome biogenesis
MST 1/2 Myonucleus
eIF3F S6K1
4E-BP1
LATS 1/2 YAP/TAZ
MuRF-1
MAFbx
TEADs
YAP/TAZ ︎ translational capacity
︎ translational efficiency protein synthesis
myogenic gene transcription? protein degradation
