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The molecular signaling pathways associated with muscle hypertrophy and atrophy. The binding of IGF-I to its receptor (IGF-IR) causes autophosphorylation of insulin receptor substrate (IRS1). Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that phosphorylates phosphatidylinositol (4,5)-bisphosphate, producing phosphatidylinositol (3,4,5)-trisphosphate, which is a membrane-binding site for phosphoinositide-dependent protein kinase (PDK1). Upon translocation to the sarcolemma, AKT (or protein kinase B, PKB) is phosphorylated by PDK1. Once activated, AKT phosphorylates mammalian target of rapamycin complex 1 (mTORC1) directly and by phosphorylating and inactivating the tuberous sclerosis complexes 1 and 2 (TSC1/2), which otherwise inhibit mTORC1 activation. Following resistance exercise, an influx of calcium ions (Ca2+) via stretch-activated channels (SACs) and the activation of FAK in the costamere can inactivate TSC1/2, thus activating mTORC1. Amino acids entering the muscle fiber cause RagGTPase-dependent translocation of mTORC1 to the lysosome, where it is activated by ras homologous protein enriched in brain (Rheb). mTORC1 subsequently activates 70KDa ribosomal S6 protein kinase (p70S6K), and inhibits 4E-BP (also known as PHAS-1), which is a negative regulator of the eukaryotic translation initiation factor 4E (eIF-4E). Phosphorylated AKT also inhibits glycogen-synthase kinase 3β (GSK3β), a substrate of AKT that blocks protein translation initiated by the eIF-2B protein. All of these actions lead to increased protein synthesis. However, protein degradation can be induced by pro-inflammatory cytokines, such as tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which activate NF-κB via degradation of I-κB, leading to increased transcription of the E3 ubiquitin-ligase, muscle RING-finger protein-1 (MuRF1). Another ligase, Atrogin1 (also known as MAFbx), is up-regulated by mitogen-activated protein kinase (MAPK) p38, while both ligases are up-regulated by forkhead box (FOXO) transcription factors. However, phosphorylated AKT blocks the transcriptional up-regulation of Atrogin1 and MuRF1 by inhibiting FOXO1, while phosphorylated mTORC1 inhibits the up-regulation of Atrogin1 directly. Myostatin also increases protein degradation and decreases protein synthesis by activating MAPKs and the SMAD complex, and by inhibiting PI3K. In addition, myostatin inhibits the myogenic program, thus resulting in a decrease of myoblast proliferation.
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Muscle Growth, Repair and Preservation: A Mechanistic Approach
Robert M. Erskine1,2 and Hans Degens3,4
1School of Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, United
Kingdom; 2Institute of Sport, Exercise and Health, Division of Surgery and Interventional Sciences,
University College London, London, UK; 3School of Healthcare Science, Manchester Metropolitan
University, Manchester, United Kingdom; 4Institute of Sport Science and Innovations, Lithuanian
Sports University, Kaunas, Lithuania.
Address correspondence to: R.M. Erskine, PhD; School of Sport and Exercise
Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF,
United Kingdom; Tel: +44 (0)151 904 6256; Fax: +44 (0)151 904 6284; Email:
Running title: Muscle Growth, Repair and Preservation
Resistance exercise, amino acid ingestion and an anabolic hormone environment all
have the capacity to elevate muscle protein synthesis (MPS), while a catabolic
hormone environment, such as elevated pro-inflammatory cytokines as seen during
disuse, aging, and conditions such as cancer and AIDS, can cause an increase in
muscle protein degradation (MPD). When the rate of MPS exceeds that of MPD there
is a positive net protein balance (NPB) and over a prolonged period of time this
results in accretion of contractile material and muscle growth, or hypertrophy. In
contrast, when NPB is chronically negative, muscle atrophy occurs, i.e. muscle size
decreases. Various signaling pathways within the muscle fiber appear to play a crucial
role in the adaptive processes, and understanding how these pathways can be
modulated will help the design of therapies to prevent or reverse muscle atrophy in a
host of muscle wasting conditions.
Key words: skeletal muscle protein synthesis hypertrophy atrophy IGF-I
mTOR cytokine TNF- interleukin myostatin
Skeletal muscle comprises numerous bundles of long, thin, multinucleated cells called
muscle fibers, each containing a multitude of myofibrils. Each myofibril is composed
of myofilaments (comprising the contractile proteins, actin and myosin) and a variety
of structural proteins, all arranged in a regular configuration throughout the length of
the myofibril, so as to form a series of contractile components, or sarcomeres. The
maximum force that can be generated by a muscle fiber is proportional to the number
of sarcomeres arranged in parallel, or fiber cross-sectional area (CSA), and ultimately
the CSA of the whole muscle (Jones, Rutherford, Parker, 1989). Therefore, there is a
strong relationship between whole muscle CSA and maximum isometric force
measured in vivo (Bamman, Newcomer, Larson-Meyer, Weinsier, Hunter, 2000;
Fukunaga et al., 2001; Kanehisa, Ikegawa, Fukunaga, 1994).
Based on the relationship between muscle size and force generating capacity (Erskine,
Fletcher, Folland, 2014), it is not surprising that an increase in muscle size following
resistance training (RT) is accompanied by an increase in maximal muscle force
(Erskine, Jones, Williams, Stewart, Degens, 2010b; Jones, Rutherford, 1987; Narici et
al., 1996). Not only can this enhance the athletic performance of an individual but it
can also reduce the elevated risk of falling and bone fracture in older people that is
among other factors attributable to sarcopenia (the age-related loss of muscle mass).
The question thus arises as to the mechanisms underlying overload-induced muscle
A multitude of signaling molecules within the muscle fiber are thought to play an
integral role in stimulating muscle protein synthesis (MPS) and degradation (MPD). If
there is a positive net protein balance (NPB), i.e. when the rate of MPS exceeds that
of MPD, the amount of contractile material will increase, enabling the muscle to
hypertrophy and generate more force. Conversely, when NPB is negative, the muscle
will decrease in size, or atrophy, and become weaker. This chapter will explore the
specific signaling pathways involved in MPS and MPD, which help to explain how
skeletal muscle adapts to overload, disuse, ageing and muscle-wasting diseases.
Furthermore, strategies used to preserve or maintain muscle mass during periods of
disuse and wasting, such as RT and nutritional interventions, will be discussed.
In addition to the mechanisms underlying muscle growth and atrophy, there is still
more to be learned about the systems associated with repair following exercise-
induced muscle damage. Several studies have reported that disruption of the
cytoskeletal structure of muscle fibers is accompanied by impairment of muscular
function following damage-inducing exercise (Baumert, Lake, Stewart, Drust,
Erskine, 2016; Friden, Sjostrom, Ekblom, 1983; Lieber, Shah, Friden, 2002). As well
as structural damage to the sarcomere, eccentric exercise can cause raised intracellular
calcium ion (Ca2+) levels (Belcastro, Albisser, Littlejohn, 1996), decreased muscle
force production (Clarkson, Sayers, 1999; Friden et al., 1983), an increase in serum
levels of muscle-specific proteins (Ebbeling, Clarkson, 1989), an increase in muscle
specific inflammation (MacIntyre, Reid, McKenzie, 1995), an increase in proteolytic
enzyme activity (Evans, Cannon, 1991), and a delayed-onset muscle soreness
(MacIntyre et al., 1995). The ultimate repair of the muscle requires the activation of
satellite cells and in this chapter we will consider the various MPD systems and the
role of satellite cells thought to play a major role in muscle damage and repair
following exercise.
While prenatal muscle growth is largely the result of muscle fiber formation, postnatal
maturational muscle growth and that in response to RT is almost entirely attributable
to fiber hypertrophy. Prenatal myogenesis, i.e. the formation of muscle fibers during
embryonic development, involves the proliferation, migration, differentiation and
fusion of muscle precursor cells to form post-mitotic multinucleated myotubes.
Postnatal skeletal muscle growth is accompanied by an increase in the number of
myonuclei per muscle fiber (Delhaas, van der Meer, Schaart, Degens, Drost, In Press)
that requires the activation of muscle stem cells, or satellite cells (located at the basal
lamina that surrounds the muscle fiber), which proliferate and fuse with existing
muscle fibers (Jacquemin, Furling, Bigot, Butler-Browne, Mouly, 2004). Once fully
mature, skeletal muscle growth, or hypertrophy, is dependent upon a positive NPB,
i.e. MPS must be greater than MPD (Chesley, MacDougall, Tarnopolsky, Atkinson,
Smith, 1992; Phillips, Tipton, Aarsland, Wolf, Wolfe, 1997), a process that is driven
by an increase in the rate of MPS (Kumar, Atherton, Smith, Rennie, 2009). This leads
to an accretion of myofibrillar proteins and an increase in muscle fiber CSA, which in
turn leads to an increase in the overall CSA of the muscle, thus enabling more force to
be produced. Resistance exercise, i.e. overloading the muscle, has been shown to
increase MPS (Chesley et al., 1992; Phillips et al., 1997), and chronic resistance
exercise, i.e. RT performed over many weeks, is a potent stimulus for skeletal muscle
hypertrophy and strength gains (Erskine et al., 2010b; Jones et al., 1987; Narici et al.,
1996). However, exactly how overloading the muscle leads to a positive NPB and
therefore an increase in muscle size has yet to be fully elucidated. It is thought that the
process necessary for inducing muscle hypertrophy involves a myriad of molecules
within the muscle fiber that form signaling cascades, eventually culminating in
increased MPS and/or decreased MPD. Here we will discuss how insulin-like growth
factor-I (IGF-I), mechanosensors, and amino acids might activate these specific
signaling pathways that lead to MPS and ultimately to muscle growth, or hypertrophy.
The role of IGF-I in muscle growth
IGF-I is produced by the liver and skeletal muscle and thus acts on muscle fibers in an
endocrine and autocrine/paracrine manner (Goldspink, 1999; Stewart, Rotwein,
1996). This growth factor appears to play an integral role in activating a specific
signaling pathway within the muscle fiber that stimulates MPS (Bodine et al., 2001;
Rommel et al., 2001). The local production and release of IGF-I during muscle
contraction (DeVol, Rotwein, Sadow, Novakofski, Bechtel, 1990) activates this
signaling cascade by binding to its receptor, located in the sarcolemma. This causes
autophosphorylation of the insulin receptor substrate (IRS1) and subsequent
phosphorylation of down-stream molecules within this signaling pathway, which
includes phosphatidylinositol-3 kinase (PI3K), protein kinase-B (PKB or Akt), the
mammalian target of rapamycin complex 1 (mTORC1), 70-kDa ribosomal S6 protein
kinase (p70S6K), and eukaryotic initiation factor 4E binding protein (4E-BP) (Fig. 1).
In fact, p70S6K activation is related to gains in skeletal muscle mass following RT,
both in rats (Baar, Esser, 1999) and humans (Terzis et al., 2008), with the increase
occurring mainly in type II fibers (Koopman, Zorenc, Gransier, Cameron-Smith, van
Loon, 2006). Together, these studies implicate mTORC1 and p70S6K as principal
downstream mediators of IGF-I stimulation of skeletal muscle growth.
There is evidence that IGF-I produced in skeletal muscle is more important for
developmental and exercise-induced muscle growth than IGF-I produced by the liver.
This is indicated by the greater muscle mass in transgenic mice over-expressing IGF-I
in skeletal muscle compared to wild-type mice (Coleman et al., 1995; Musaro et al.,
2001) despite normal serum IGF-I levels (Coleman et al., 1995). Furthermore, low
systemic IGF-I levels in liver-specific IGF-I knockout mice does not affect muscle
size (Ohlsson et al., 2009; Yakar et al., 1999). Also in young adult men, elevated
levels of circulating IGF-I do not influence MPS following an acute bout of resistance
exercise (West et al., 2009) or muscle hypertrophy in response to RT (West et al.,
2010). Although in rat skeletal muscle, local IGF-I gene expression increases
proportionately to the progressive increase in external load (DeVol et al., 1990), it is
equivocal whether this occurs in human muscle (Bamman et al., 2001; Bickel et al.,
2005; Bickel, Slade, Haddad, Adams, Dudley, 2003; Hameed, Orrell, Cobbold,
Goldspink, Harridge, 2003; Petrella, Kim, Cross, Kosek, Bamman, 2006; Psilander,
Damsgaard, Pilegaard, 2003). Some of this controversy might be explained by the
elevated expression of two isoforms of the Igf1 gene in animal skeletal muscle in
response to mechanical stimulation (McKoy et al., 1999; Yang, Alnaqeeb, Simpson,
Goldspink, 1996). Thus, at least two IGF-I isoforms exist: (i) IGF-IEa, which is
similar to the hepatic endocrine isoform, and (ii) the less abundant IGF-IEb (in rats)
or IGF-IEc (in humans), otherwise known as mechanical growth factor (MGF).
However, it is not always clear which IGF-I isoform has been measured in the muscle
(Bamman et al., 2001). Furthermore, the age of the participants also influences the
findings, with MGF increasing in young but not old people following an exercise
bout. Interestingly, IGF-IEa does not appear to change in young or older people
following resistance exercise (Hameed et al., 2003), despite its apparent hypertrophic
effect (Musaro et al., 2001).
Skeletal muscle-derived IGF-I does not appear to be the only regulator of adult
muscle mass and function, as unloading induces skeletal muscle atrophy in mice
whose muscles over-express IGF-I (Criswell et al., 1998), and overload induces
hypertrophy even in transgenic mice, which express a dominant negative IGF-I
receptor in skeletal muscle (Spangenburg, Le Roith, Ward, Bodine, 2008). Also in
older people, enhanced muscle strength can be attained following RT without a
significant change in muscle IGF-I gene expression (Taaffe, Jin, Vu, Hoffman,
Marcus, 1996). Thus, it appears that for the development of hypertrophy in adult
muscles, loading is more important than alterations in local and systemic IGF-I levels.
This fits the notion that activation of mTORC1 and p70S6K can also occur
independently of PI3K activation following muscle overload (Hornberger, Sukhija,
Wang, Chien, 2007).
The role of mechanosensors in muscle growth
The process that couples the mechanical forces during a muscle contraction with cell
signaling and ultimately protein synthesis is called mechanotransduction. It has been
shown in rats that passive stretch induces an increase in the expression of myogenic
regulatory factors (Kamikawa, Ikeda, Harada, Ohwatashi, Yoshida, 2013) that may
well underlie the muscle fiber atrophy seen after passive stretching (Coutinho,
DeLuca, Salvini, Vidal, 2006). These responses are probably at least partly mediated
by stretch-activated channels (SACs), which are calcium (Ca2+) and sodium
permeable channels that increase their open probability (the fraction of time spent in
the open state) in response to mechanical loading of the sarcolemma (Franco,
Lansman, 1990a; Franco, Lansman, 1990b; Guharay, Sachs, 1984). It has been
proposed that SACs function as mechanosensors by allowing an influx of Ca2+ into
the muscle fiber (Yeung et al., 2005) following mechanical changes in the
sarcolemma, which activate mTORC1 (Gulati et al., 2008), leading to an increase in
MPS (Kameyama, Etlinger, 1979). Correspondingly, inhibition of SACs by
streptomycin reduces skeletal muscle hypertrophy in response to mechanical overload
(Butterfield, Best, 2009; Spangenburg, McBride, 2006) via attenuation of mTORC1
and p70S6K activation (Spangenburg et al., 2006).
Other mechanosensors might exist in the form of costameres, intra-sarcolemmal
protein complexes that are circumferentially aligned along the length of the muscle
fiber (Pardo, Siliciano, Craig, 1983). Costameres mechanically link peripheral
myofibrils via the Z-disks to the sarcolemma (Fig. 2), thus maintaining the integrity of
the muscle fiber during contraction and relaxation (Pardo et al., 1983). An individual
costamere contains many proteins arranged in a complex structure (Ervasti, 2003;
Patel, Lieber, 1997), which comprises two different laminin receptors, a
dystrophin/glycoprotein complex and an integrin-associated complex, which are
localised in the sarcolemma and bound to intra and extra-cellular structural proteins
(Fig. 2). Thus, the force-producing contractile material is connected to the basal
membrane and ultimately to adjacent muscle fibers (Morris, Fulton, 1994; Patel et al.,
1997; Rybakova, Patel, Ervasti, 2000).
Costameres are receptive to mechanical, electrical and chemical stimuli (Ervasti,
2003). Indeed, mechanical tension is essential in regulating costameric protein
expression, stability and organization, with talin and vinculin, for instance, being up-
regulated in response to muscle contraction (Tidball, Spencer, Wehling, Lavergne,
1999). Regular contractions, as experienced during RT, increase the expression of
costameric proteins, such as desmin (Woolstenhulme, Conlee, Drummond, Stites,
Parcell, 2006), alpha-1-syntrophin and dystrophin (Kosek, Bamman, 2008) in
humans, while focal adhesion kinase (FAK) and paxillin activity are increased in
stretch-induced hypertrophied avian skeletal muscle (Fluck, Carson, Gordon,
Ziemiecki, Booth, 1999). The forces exerted on both the intracellular contractile
proteins and the basal membrane during periods of loading are required to cause
binding of basal membrane laminin to the receptors on the and integrins and on
the dystrophin/glycoprotein complex (Fluck, Ziemiecki, Billeter, Muntener, 2002).
Interaction between integrins and the extra-cellular matrix causes rapid
phosphorylation of FAK (Cary, Guan, 1999), which subsequently activates p70S6K
independently of Akt (Durieux et al., 2009; Klossner, Durieux, Freyssenet, Flueck,
2009). This probably occurs via the phosphorylation and, thus, inactivation of
tuberous sclerosis complex 2 (Gan, Yoo, Guan, 2006; Malik, Parsons, 1996), thus
activating mTORC1 as shown in Fig. 1.
The role of amino acids in muscle growth
Both resistance exercise (Biolo, Maggi, Williams, Tipton, Wolfe, 1995; Phillips et al.,
1997) and amino acid/protein ingestion (Tang, Moore, Kujbida, Tarnopolsky, Phillips,
2009; Yang et al., 2012b) stimulate MPS independently, while a combination of the
two augments MPS even further (Biolo, Tipton, Klein, Wolfe, 1997; Tipton,
Ferrando, Phillips, Doyle, Wolfe, 1999). Both stimuli cause an increase in mTORC1
activation (Apro, Blomstrand, 2010; Moore, Atherton, Rennie, Tarnopolsky, Phillips,
2011) but it is unclear whether they stimulate MPS via different signaling pathways,
or whether the combination of the two stimulates the same pathway more than either
stimulant on its own. It is thought that amino acids cause Rag GTPases to interact
with raptor (a regulatory protein associated with mTORC1), leading to the
translocation of mTORC1 to the lysosomal membrane, where Rheb (a Ras GTPase)
activates mTORC1 (Kim, Guan, 2009; Sancak et al., 2010; Sancak et al., 2008), as
shown in Fig. 1. Of the essential amino acids (EAAs), the branched-chain amino acids
(BCAAs: isoleucine, leucine, and valine), and particularly leucine, are the most potent
stimulators of the mTORC1 signaling pathway (Anthony et al., 2000). Leucine
supplementation stimulates muscle protein accretion in cultured cells (Haegens,
Schols, van Essen, van Loon, Langen, 2012) and can also reduce MPD in healthy men
(Nair, Schwartz, Welle, 1992), and it is possible that the action of leucine occurs via
its metabolite, β-hydroxy-β-methylbutyrate (HMB) (Aversa et al., 2011; Pimentel et
al., 2011).
The timing of amino acid ingestion appears crucial for an optimal anabolic response
to a single bout of resistance exercise: ingesting amino acids immediately before an
exercise bout promotes a greater increase in MPS compared to ingestion immediately
after the bout (Tipton et al., 2001). This effect was attributed to an increased blood
flow during exercise, and therefore an increased delivery of amino acids to the active
muscle when they were ingested prior to exercise (Tipton et al., 2001). As well as the
timing, the amount of protein ingested is integral in producing an optimal anabolic
environment following resistance exercise (Moore et al., 2009; Witard et al., 2014;
Yang et al., 2012a). For example, the MPS dose response to ingested protein after a
single bout of resistance exercise in healthy young men is saturated at 20 g protein,
and any additional ingested protein is simply oxidized (Moore et al., 2009; Witard et
al., 2014). This suggests that if the rate of protein ingestion after resistance exercise
exceeds the rate at which it can be incorporated into the muscle, the excess protein is
not used for MPS. This dose-response relationship seems to be altered with age, as in
older men increased rates of MPS were found when participants ingested 40 g protein
following a resistance exercise bout (Yang et al., 2012a). Therefore, older muscle
appears to be less sensitive to amino acids, which has been termed ‘anabolic
resistance’, something that may be attributable to impaired ribosome genesis in older
muscle (Chaillou, 2017). Finally, the type and quality of the ingested protein appears
to be important when it comes to MPS (Tang et al., 2009; Yang et al., 2012b).
Following a single bout of resistance exercise and the ingestion of whey, soy or
casein, each containing 10 g EAA, larger increases in blood EAA, BCAA, and leucine
concentrations were found following the ingestion of whey compared to either soy or
casein (Tang et al., 2009), suggesting a greater availability of these amino acids for
protein synthesis following whey protein ingestion. This may be a reflection of the
different rate of protein digestion and absorption of amino acids between the protein
types (Boirie et al., 1997; Dangin et al., 2001; Dangin et al., 2003) and explain why
MPS was greater following ingestion of whey compared to casein, both at rest and
after exercise (Tang et al., 2009). In older muscle, the rate of MPS appears to be
greater with whey than soy protein ingestion following resistance exercise (Yang et
al., 2012b), which could be due to the ~28% greater leucine content in whey versus
soy protein (Drummond, Rasmussen, 2008) as well as differences in digestion and
absorption rate.
There is therefore striking evidence to support the acute effects of amino acid
ingestion and resistance exercise on MPS via their independent and complementary
effects on mTORC1 activation. It is also well known that repeated bouts of resistance
exercise over a prolonged period of time, i.e. a RT program, leads to gains in both
muscle size and strength (Erskine et al., 2010b; Jones et al., 1987; Narici et al., 1996).
Therefore, the amplification of the anabolic environment within the muscle seen with
the combination of both amino acid/protein ingestion and resistance exercise (Biolo et
al., 1997; Tipton et al., 1999) suggests that RT with protein supplementation should
confer greater gains in skeletal muscle size and strength than RT alone. However, the
evidence for protein supplementation enhancing the increases in muscle size and
strength following longer term RT programs in young (Erskine, Fletcher, Hanson,
Folland, 2012; Hartman et al., 2007) and older (Candow et al., 2008; Verdijk et al.,
2009b) individuals is equivocal. The controversy surrounding the longer-term RT
studies could be due to methodological differences/limitations between studies. For
example, considerable inter-individual variability exists in the response to RT
(Erskine, Jones, Williams, Stewart, Degens, 2010a; Hubal et al., 2005) and yet many
studies have used small sample sizes (Godard, Williamson, Trappe, 2002; Hulmi et
al., 2009; Willoughby, Stout, Wilborn, 2007) that may have limited the statistical
power required to detect an influence of protein supplementation. Different measures
of muscle hypertrophy may also compound this discrepancy. For example, some
studies have determined muscle thickness using ultrasonography (Candow et al.,
2008; Vieillevoye, Poortmans, Duchateau, Carpentier, 2010) or whole body fat-free
mass assessed via dual-energy X-ray absorptiometry (Hartman et al., 2007), while
others have used magnetic resonance imaging to provide a more precise assessment of
muscle size, but still found no effect of protein supplementation on muscle
hypertrophy following RT (Coburn et al., 2006; Erskine et al., 2012; Holm et al.,
2008; Hulmi et al., 2009). There are, however, circumstances where protein
supplementation may have a beneficial effect on muscle hypertrophy and strength
gains. For example, whole body RT (incorporating multiple muscle groups rather than
an individual muscle) could create a requirement for an increase in exogenous protein
(due to a greater absolute MPD) that might not be satisfied by habitual protein intake
alone. This might be particularly beneficial in the early phase of a RT program when
MPS and MPD are likely to be higher than towards the end (Hartman, Moore,
Phillips, 2006; Phillips, Tipton, Ferrando, Wolfe, 1999). In addition, older individuals
need to ingest at least twice as much protein (40 g) to maximally stimulate MPS
(Moore et al., 2015; Yang et al., 2012a) compared to the 20 g dose in younger people
(Moore et al., 2009; Witard et al., 2014), which may reflect an ‘anabolic resistance’ in
old age. Therefore, previous studies that have not shown a beneficial effect of protein
supplementation on muscle size and strength gains in older people may have been
administering suboptimal amounts and types of protein per RT session. In frail elderly
individuals, whose protein requirements are probably higher than in old, healthy
people, more recent evidence supports the combination of 60 g/day milk protein
supplementation and resistance exercise (twice weekly for six months) in increasing
total leg lean mass composition over resistance training alone (Tieland et al., 2012).
Skeletal muscle atrophy is known to occur in response to disuse and numerous
chronic conditions, such as cancer, AIDS, and senile sarcopenia (the age-related loss
of muscle mass). Here we will focus on the mechanisms underlying sarcopenia, which
is the major cause of muscle weakness in older individuals (Evans, 1995). Although
the causes of sarcopenia are not fully understood, disuse, chronic systemic
inflammation and neuropathic changes leading to motoneuron death are thought to
play an integral role (Degens, 2010). Motoneuron death results in denervation of
muscle fibers, and ultimately the loss of muscle fibers (hypoplasia). Selective atrophy
of type II fibers (Lexell, Taylor, Sjostrom, 1988) and a decrease in the proportion of
type II fibers (Jakobsson, Borg, Edstrom, Grimby, 1988; Larsson, 1983; Larsson,
Sjodin, Karlsson, 1978) are thought to be caused by denervation accompanied by
reinnervation of these fibers by axonal sprouting from adjacent slow-twitch motor
units (Brooks, Faulkner, 1994; Faulkner, Larkin, Claflin, Brooks, 2007).
Chronic low-grade inflammation and sarcopenia
Physiological aging is associated with chronic low-grade inflammation, a condition
that has been termed inflammaging (Franceschi et al., 2000). Inflammaging is
characterized by elevated serum levels of pro-inflammatory cytokines such as
interleukin 1 (IL-1), IL-6 and tumor necrosis factor- (TNF-), as well as acute
phase proteins such as C-reactive protein, or CRP (Bartlett et al., 2012; Erskine et al.,
2017; Franceschi et al., 2000), and increased circulating levels are associated with
lower muscle mass and weakness in old age (Erskine et al., 2017; Visser et al., 2002).
Furthermore, the levels of cytokines that counteract the inflammatory state, such as
IL-10, are reduced with age (Bartlett et al., 2012; Lio et al., 2002). The pro-
inflammatory cytokines, IL-1, IL-6 and TNF- are produced by both skeletal muscle
fibers and adipose cells, and are therefore also members of the adipokine family. As
we age, we accumulate more adipose tissue, which is deposited in the subcutaneous,
visceral and intramuscular regions, and it is particularly the visceral fat that appears to
contribute to the inflammatory environment (Pedersen, 2009).
TNF- induces the production of reactive oxygen species (ROS), altering vascular
permeability, which leads to leukocyte infiltration of the muscle fiber (Evans et al.,
1991) and further ROS generation by the leucocytes. This release of ROS also
activates NF-κB via degradation of I-κB (Fig. 1), which results in the increased
expression of key enzymes of the ubiquitin-proteasome MPD system (Li, Schwartz,
Waddell, Holloway, Reid, 1998). In addition, TNF- interferes with satellite cell
differentiation and therefore muscle growth and regeneration in old age by reducing
the expression of myogenic regulatory factors (MRFs) (Degens, 2010). The MRFs are
a family of muscle-specific transcription factors (MyoD, myogenin, MRF4 and myf-
5) that regulate the transition from proliferation to differentiation of the satellite cell
(Langen et al., 2004; Szalay, Razga, Duda, 1997). The TNF--induced activation of
NF-κB results in a loss of MyoD mRNA (Guttridge, Mayo, Madrid, Wang, Baldwin,
2000) and, via activation of the ubiquitinproteasome pathway (UPP) (Reid, Li, 2001;
Saini, Al-Shanti, Faulkner, Stewart, 2008), breakdown of MyoD and myogenin. Thus,
part of the attenuated hypertrophic response in elderly compared to younger muscle
(Welle, Totterman, Thornton, 1996) could be due to a decrease in MRFs, as seen in
overload-induced hypertrophy in older rats (Alway, Degens, Krishnamurthy, Smith,
2002a). TNF- also stimulates the release of proteolytic enzymes, such as lysozymes
(e.g. cathepsin-B) from neutrophils (Farges et al., 2002), which are thought to
contribute to the MPD process (Friden, Kjorell, Thornell, 1984; Kasperek, Snider,
1985), but the main action of the pro-inflammatory cytokines in muscle atrophy is
thought to occur via the UPP.
It has been suggested that the UPP cannot breakdown intact sarcomeres, so additional
mechanisms are proposed to be involved (Solomon, Baracos, Sarraf, Goldberg, 1998).
For example, the activation of caspase-3 is thought to lead to cleavage of the
myofilaments, actin and myosin, which are then degraded by the UPP (Du et al.,
2004; Lee et al., 2004; Ottenheijm et al., 2006). The UPP involves numerous
enzymes, or ligases, that regulate the ubiquitination (the coupling of ubiquitin to
protein substrates), so that the ‘tagged’ protein fragments can be identified by the
proteasome for the final step of MPD (DeMartino, Ordway, 1998). These ubiquitin
ligases may also degrade MyoD and inhibit subsequent satellite cell activation and
differentiation (see above), thus exacerbating the effects of MPD on muscle size by
impairing satellite cell associated muscle growth. Expression of two genes that encode
the E3 ubiquitin ligases, muscle-specific atrophy F box (MAFbx, also known as
Atrogin-1) and muscle RING Finger 1 (MuRF1), has been shown to increase during
different types of muscle atrophy (Gomes, Lecker, Jagoe, Navon, Goldberg, 2001;
Lecker et al., 2004). The structure and function of the UPP will be discussed in more
detail, below, with regard to muscle damage and repair following exercise.
The role of myostatin in muscle atrophy
Myostatin, otherwise known as growth differentiation factor-8 (GDF-8), is part of the
transforming growth factor β superfamily and is produced in skeletal muscle. The role
of myostatin as a negative regulator of muscle mass has been demonstrated by
knocking out the gdf-8 gene in mice, which leads to a 2-3 fold increase in skeletal
muscle mass (McPherron, Lawler, Lee, 1997). Conversely, administering myostatin to
wild-type mice induces substantial muscle wasting (Zimmers et al., 2002). Further
examples of myostatin’s regulatory effect can be seen in bovine (McPherron, Lee,
1997) and human (Schuelke et al., 2004) cases, where mutation of the gdf -8 gene
leads to a reduction in myostatin production and considerably enlarged skeletal
Once bound to the activin IIB receptor (ActRIIB), a signaling cascade is activated that
leads to MPD. Key signaling proteins in this pathway include SMAD 2 and 3 (Sartori
et al., 2009), which form a complex with SMAD 4 that then translocates to the
nucleus where it targets genes encoding MRFs (Rodino-Klapac et al., 2009), and
inhibits differentiation via the reduction of MyoD expression (Langley et al., 2002)
(Fig. 1). In addition, myostatin reduces Akt/ mTORC1/p70S6K signaling (Amirouche
et al., 2009; Trendelenburg et al., 2009) (Fig. 1) and is associated with smaller
myotube size (Trendelenburg et al., 2009). Accordingly, the inhibition of myostatin in
mature mice leads to increased activation of p70S6K, ribosomal protein S6 and skeletal
muscle MPS (Welle, Burgess, Mehta, 2009). Myostatin appears to not only inhibit
satellite cell differentiation and MPS, but also to induce the expression of atrogenes
(genes associated with muscle atrophy) via activation of the p38 mitogen-activated
protein (MAP) kinase, Erk1/2, Wnt and c-Jun N-terminal kinase (JNK) signaling
pathways (Huang et al., 2007; Philip, Lu, Gao, 2005; Yang et al., 2006) (Fig. 1). This
is in accord with the observation that myostatin induces cachexia by activating the
UPP, i.e. phosphorylating the ubiquitin E3 ligases MAFbx and MuRF1, via FOXO1
activation rather than via the NF-κB pathway (McFarlane et al., 2006). However,
myostatin-induced atrophy persists despite inhibiting the expression of the two E3
ligases (Trendelenburg et al., 2009). Therefore, it is likely that myostatin negatively
regulates muscle growth via multiple pathways (Fig. 1). A lower expression of
myostatin may therefore help to maintain muscle mass at old age, a situation reflected
by the attenuated loss of muscle mass and regenerative capacity in old myostatin-null
mice compared to age-matched wild-type mice (Siriett et al., 2006).
Combating sarcopenia
RT has been shown to increase muscle size and strength in old (Reeves, Narici,
Maganaris, 2004), very old (Harridge, Kryger, Stensgaard, 1999) and frail (Fiatarone
et al., 1994) individuals. This beneficial effect of RT is attributable to an increase in
MPS in the atrophied muscle (Kimball, O'Malley, Anthony, Crozier, Jefferson, 2004)
and a reduction in MPD as a result of a reduced MAFbx and MuRF-1 gene expression
(Jones et al., 2004; Mascher et al., 2008). A reduction in atrogene expression can be
realized by the ability of phosphorylated Akt to block FoxO1, which would suppress
the transcription of MuRF1 and MAFbx (Sandri et al., 2004), as shown in Fig. 1. In
addition to the Akt/FoxO-1 pathway, mTORC1 also blocks MuRF1 and MAFbx
transcription (Sandri et al., 2004). Therefore, while a degree of MPD is required for
muscle remodeling, RT appears to reverse atrophy via the inhibiting effect of
Akt/mTORC1 on MPD and the positive effect of mTORC1 activation on MPS (Apro
et al., 2010; Moore et al., 2011), thus resulting in net protein synthesis (albeit to a
lesser extent in elderly compared to younger muscle).
The apparent ‘anabolic resistance’ to RT in older compared to young muscle (Kimball
et al., 2004; Welle et al., 1996) may not be due to an age-related reduction in the
mechanosensitivity of the mTORC1 signaling pathway (Hornberger, Mateja, Chin,
Andrews, Esser, 2005), although others do see a reduction in the translational
signaling during overload (Thomson, Gordon, 2006). It could therefore be that the
rates of transcription and translation are reduced during overload, which may be a
consequence of impaired ribosome biogenesis in older muscle (Chaillou, 2017).
Another factor that might be considered is the proposed requirement of satellite cell
recruitment for the development of hypertrophy. An impaired satellite cell recruitment
would then result in impaired hypertrophy and part of the problem might be a decline
in satellite cell number (Shefer, Van de Mark, Richardson, Yablonka-Reuveni, 2006),
particularly in type II muscle fibers of older people (Verdijk et al., 2007). Yet,
paradoxically, older muscle appears to have an increased regenerative drive and
protein synthesis that is more pronounced the more severe the sarcopenia. For
instance, muscle mass is lower despite higher p70S6K activation and MPS in old
compared to young adult rats (Kimball et al., 2004). IGF-I appears to play a key role
in the activation and proliferation of satellite cells (Scime, Rudnicki, 2006), while
differentiation is regulated by MRFs (Langen et al., 2004; Szalay et al., 1997).
However, despite an increased regenerative drive as reflected by elevated IGF-I
expression (Edstrom, Ulfhake, 2005) and MRF mRNA expression (Alway, Degens,
Lowe, Krishnamurthy, 2002b) in old rat muscle, MRF protein levels are reduced
(Alway et al., 2002b). This reduction might be caused by a concomitant increase in Id
protein expression (Alway et al., 2002b), which inhibits MRF expression and DNA
binding capacity. The result is that during an hypertrophic stimulus, satellite cell
activation and proliferation can occur in older rat skeletal muscle but with limited
differentiation (Edstrom et al., 2005). In elderly human skeletal muscle, however, the
capacity for satellite cells to proliferate and differentiate in response to RT does not
appear to be diminished (Mackey et al., 2007; Verdijk et al., 2009a). It should be
noted, however, that the relative age in the human studies was less than that in the rat
studies and it may be that beyond a given age in humans, the differentiation of
satellite cells may also be diminished, particularly when associated with chronic low-
grade systemic inflammation.
Although satellite cells are generally considered to play an important role in muscle
hypertrophy, the conditional ablation of satellite cells by tamoxifen in Pax7-DTA
mice did not attenuate the development of 100% hypertrophy induced by overload
(McCarthy et al., 2011). It may well be that the microcirculation is crucial for the
development of hypertrophy as it has been found that in genetically modified mice
with muscle hypertrophy (due to myostatin knock out) and high-oxidative fibers
(overexpression of oestrogen-related receptor gamma) the regenerative capacity was
normal despite a lower satellite cell content, but higher capillary density (Omairi et
al., 2016). Also in aged mice, the blunted hypertrophic response was associated not
only associated with a lower satellite cell content (Ballak et al., 2015), but also with
impaired angiogenesis (Ballak et al., 2016). These data suggest that impaired
angiogenesis and/or reductions in the capillarization, that would result in an impaired
delivery of substrates, of the muscle may underlie the impaired regenerative capacity
and anabolic resistance in old age
Extra stimulation of the mTORC1 pathway may overcome the anabolic blunting. As
discussed above, older people need to ingest more protein than younger individuals to
stimulate maximal MPS (Moore et al., 2015; Moore et al., 2009; Volpi, Mittendorfer,
Rasmussen, Wolfe, 2000; Yang et al., 2012a). The greater activation of the mTORC1
pathway when combining RT with protein/amino acid ingestion may inhibit MPD and
augment MPS, thereby improving the hypertrophic response. The observation that
BCAA administration attenuates the loss of body mass in mice bearing a cachexia-
inducing tumor (Eley, Russell, Tisdale, 2007) is promising and suggests that it may
also enhance the hypertrophic response in this condition. Furthermore, HMB has been
shown to attenuate the reduction in MPS in rodents following the administration of a
cachectic stimulant (Aversa et al., 2011; Eley, Russell, Baxter, Mukerji, Tisdale,
In addition to RT and amino acid supplementation, various pharmaceutical therapies
have been proposed to combat sarcopenia. Supplementation of the anabolic steroid,
testosterone, augments muscle mass in older men, healthy hypogonadal men, older
men with low testosterone levels, and men with chronic illness and low testosterone
levels (Bhasin et al., 2006). It is thought that testosterone can reverse sarcopenia by
suppressing skeletal muscle myostatin expression, while simultaneously stimulating
the Akt pathway (Kovacheva, Hikim, Shen, Sinha, Sinha-Hikim, 2010) to increase
MPS and decrease MPD. Furthermore, administration of a myostatin antagonist has
led to satellite cell activation, increased MyoD protein expression, and greater muscle
regeneration after injury in old murine skeletal muscle (Siriett et al., 2007).
In normal skeletal muscle, cytoskeletal proteins act as a framework that keeps the
myofibrils aligned in a lateral position by connecting the Z-disks to one another and to
the sarcolemma (Friden et al., 1984; Friden et al., 1983). Following eccentric exercise,
Z-disk streaming (disturbance of Z-disk configuration) and misalignment of the
myofibrils is a common characteristic (Friden et al., 1984). Eccentric contractions are
defined as contractions where muscles lengthen as they exert force and generally
result in more muscle damage than concentric contractions (Clarkson, Hubal, 2002). It
has been suggested that this is due to fewer motor units being recruited during
eccentric exercise, leading to a smaller CSA of muscle being activated than during a
concentric contraction at the same load (Enoka, 1996). It has also been demonstrated
that the extent of muscle damage is due to strain (the change in length) rather than the
amount of force generated by the muscle (Lieber, Friden, 1993; Lieber, Friden, 1999).
Eccentric exercise not only causes alterations in the cytoskeletal structure, but also
increases in the activity of proteolytic enzymes (Arthur, Booker, Belcastro, 1999;
Kasperek et al., 1985; Stupka, Tarnopolsky, Yardley, Phillips, 2001). The positive
correlation between the proteolytic enzyme activity and a rise in serum concentrations
of muscle-specific proteins, e.g. creatine kinase (CK), post exercise (Arthur et al.,
1999; Kasperek et al., 1985; Stupka et al., 2001), suggests that the degree of
activation of the proteolytic machinery is related to the degree of muscle damage.
Therefore, it is feasible that the activity of these proteolytic enzymes may be required
for the remodeling of skeletal muscle in response to exercise, where the regulated
degradation of cellular proteins (Ordway, Neufer, Chin, DeMartino, 2000) may be a
pre-requisite for subsequent adaptive repair and growth.
There are three main systems that contribute to the controlled MPD following muscle
damage; 1) the release of calpain, a non-lysosomal, Ca2+-dependent neutral protease
that mediates the dismantling of myofibrils (Belcastro, Shewchuk, Raj, 1998), 2) the
inflammatory response, which includes lysosomal proteolysis (Farges et al., 2002),
and 3) the ATP-dependent UPP, which coordinates the demolition of protein
fragments liberated by the aforementioned degradation systems (DeMartino et al.,
1998). Recent findings suggest that myostatin is also implicated in the MPD process
following damaging muscle contractions (Ochi et al., 2010).
The calpain protein degradation system
Calpain is a multidomain protein composed of two subunits, a catalytic 80-kDa
subunit and a regulatory 30-kDa subunit (Suzuki, Sorimachi, Yoshizawa, Kinbara,
Ishiura, 1995). In skeletal muscle, three homologous isozymes of calpain with
different Ca2+ sensitivities have been identified (DeMartino et al., 1998):
(active at micromolar Ca2+ concentrations), m-calpain (active at millimolar Ca2+
concentrations), and n-calpain (requiring very high Ca2+ concentrations). It appears
that, although the
- and m-calpain 80-kDa subunits are quite different, both have
similar binding domains; the proteolytic site of a cysteine proteinase, the calpastatin
(an endogenous inhibitor of calpain activity) binding domain, and the Ca2+-binding
domain (Belcastro et al., 1996). The 30-kDa subunit is extremely hydrophobic, which
may help to act as an anchor to the membrane proteins (Belcastro et al., 1996). While
there is evidence to suggest calpain is localized and activated at or around the
sarcolemma, thus targeting the membrane-associated proteins (Belcastro et al., 1998),
others have demonstrated that calpain also targets Z-disk proteins, such as desmin and
-actinin (Goll, Dayton, Singh, Robson, 1991). The action of other proteolytic
complexes, including lysosomal enzymes and the UPP, may have a part to play in
MPD immediately after damaging eccentric muscle contractions, but as their activity
does not peak until later in the muscle damage/repair process (Belcastro et al., 1998;
Kasperek et al., 1985), it is more likely that calpain and/or mechanical stress is the
initial effector of cytoskeletal protein breakdown.
Calpain is activated by raised intracellular [Ca2+] (Belcastro et al., 1998). Initial
mechanical damage to the sarcoplasmic reticulum (SR) and muscle plasma membrane
caused by eccentric muscle contractions could lead to SR vacuolization and an
increase in intracellular [Ca2+] (Clarkson et al., 2002; Warren, Hayes, Lowe,
Armstrong, 1993). The intracellular [Ca2+] could rise further following an increased
open probability of SACs as a consequence of increased strain on the skeletal muscle
fibers during forced lengthening (Lieber et al., 1993). It is thought that the activation
of calpain is pivotal in the breakdown of cytoskeletal proteins, including desmin and
-actinin, rather than mechanical stress applied to the “over-stretched” sarcomeres
during eccentric contractions per se (Belcastro et al., 1998). The activity of calpain is
not only dependent on the intracellular [Ca2+] but also on the concentration of its
inhibitor, calpastatin, and condition of degradable substrates, i.e. the ultrastructural
proteins. To become fully active, calpain undergoes autolysis into its subunits. It is
likely that the influx of excess Ca2+ into the muscle fiber (via the SACs, SR calcium
channels and sarcolemmal lesions) binds to the specific domain on the 80-kDa calpain
subunit, thereby inhibiting calpastatin. Once calpain is free of calpastatin, it may
begin autolysis and/or bind to its substrate (with the help of Ca2+) and begin the
process of MPD (Belcastro et al., 1996). A positive relationship between calpain
activity and neutrophil accumulation within skeletal muscle after exercise suggests
that the calpain-degraded protein fragments act as chemoattractants, thus localizing
leukocytes to the site of muscle damage (Belcastro et al., 1998; Raj, Booker,
Belcastro, 1998).
The inflammatory response
Exercise-induced damage to muscle fibers elicits an inflammatory response that
results in movement of fluid, plasma proteins and leukocytes to the site of injury
(Clarkson et al., 2002). Leukocytes have the ability to break down intracellular
proteins with the aid of lysosomal enzymes (Friden et al., 1984), but exactly how the
inflammatory response regulates MPD and muscle repair following eccentric exercise
is not entirely clear. However, the purpose of the post-exercise-induced inflammatory
response is to promote clearance of damaged muscle tissue and prepare the muscle for
repair (MacIntyre et al., 1995), a process that is sub-classified into acute and
secondary inflammation.
The acute phase response in skeletal muscle begins with the complement system
when fragments from the damaged fiber(s) serve as chemoattractants, luring
leukocytes to the injured area (Belcastro et al., 1998; Evans et al., 1991). As a
consequence there is an accumulation of neutrophils, the histological hallmark of
acute inflammation (MacIntyre et al., 1995), in and around the site of injury, which
peaks around 4 hours after exercise-induced damage has occurred (Evans et al.,
1991). The accumulation of neutrophils has been reported to be more significant after
eccentric than concentric exercise, and is most likely related to the degree of damage
incurred (Evans et al., 1991). Pro-inflammatory cytokines, such as IL-1, IL-6, TNF-,
and acute phase proteins, e.g. CRP, act as mediators of inflammatory reactions. TNF-
induces the production of ROS, altering vascular permeability, which leads to
leukocyte infiltration into the muscle fiber (Evans et al., 1991). TNF- also stimulates
the release of cytoxic factors from neutrophils, such as lysozymes and ROS (Friden et
al., 1984; MacIntyre et al., 1995), which are responsible for at least a part of the MPD
process following exercise-induced damage (Friden et al., 1984; Kasperek et al.,
It may take up to seven days to see a significant infiltration of monocytes (precursors
to macrophages) within the damaged muscle fiber (Evans et al., 1991), which carry
out further phagocytic activity inside the muscle fiber. Furthermore, considerable
increases in the quantity of lipofuscin granules (generally considered to be the
indigestible residue of lysosomal degradation) in sore muscles three days after
exercise suggests that lysozyme activity plays a major part in the secondary
inflammatory process (Farges et al., 2002; Friden, 1984). The role of the
inflammatory response in muscle regeneration is therefore thought to be the further
breakdown of damaged muscle proteins via lysozymes, the engulfing of protein
fragments by macrophages and the activation of the UPP by the pro-inflammatory
cytokines released from the neutrophils. These cytokines simultaneously stimulate the
proliferation of satellite cells, crucial for the regeneration of the damaged area (Chen,
Jin, Li, 2007).
The ubiquitin-proteasome pathway
This pathway is recognized as the major non-lysosomal complex responsible for the
degradation of cellular proteins. The UPP has received much attention due to its
involvement in cellular processes, where protein degradation is a key regulatory or
adaptive event (Attaix et al., 1998; Attaix, Combaret, Pouch, Taillandier, 2001;
Ciechanover, 1994). There are two types of ubiquitin in human skeletal muscle: free
and conjugated (Thompson, Scordilis, 1994). In its free state ubiquitin is a normal
component of the non-stressed muscle fiber but it also forms complexes, or
conjugations, with abnormal proteins and then returns to its free state (Fig. 3). The
conjugation of ubiquitin with denatured proteins within the muscle fiber “tags” these
proteins for recognition by a non-lysosomal protease to be subsequently degraded in a
process that requires ATP (Attaix et al., 1998). The UPP, therefore, consists of two
major components that represent the system’s functionally distinct parts. Ubiquitin is
the element that covalently binds to the protein due to be broken down, while the 26S
proteasome, a large protease complex, catalyses the degradation of the ubiquitin-
tagged proteins (DeMartino et al., 1998).
The cellular proteins are selected for degradation by the attachment of multiple
molecules of ubiquitin, or a polyubiquitin chain, which is built by repeated cycles of
conjugation via the action of E1, E2, and E3 conjugating enzymes (Fig. 3). Ubiquitin
is initially activated in the presence of ATP by the ubiquitin-activating enzyme, E1,
which then transfers ubiquitin to E2, one of the ubiquitin-conjugating enzymes. E2
then binds the ubiquitin molecule to the protein substrate, which is selected for
tagging by E3 (Attaix et al., 2001). The 26S proteasome is able to discriminate
between ubiquitinated and non-ubiquitinated proteins, and rapidly degrades the
polyubiquitinated proteins, deriving the energy for this process from ATP hydrolysis
(Hershko, Ciechanover, 1998).
The 26S proteasome is composed of a 20S proteasome and two 19S (PA700)
regulatory modules (Attaix et al., 2001; Ciechanover, 1994; DeMartino et al., 1998).
The 20S proteasome is the proteolytic core, containing multiple catalytic sites. PA700
binds to each end of the proteasome cylinder and elicits ATPase activity in order to
unfold and/or translocate the ubiquitinated proteins to the catalytic sites within the
20S proteasome (Fig. 3). PA700 is also thought to be responsible for disassembling
the polyubiquitinated chain, a process requiring its isopeptidase activity.
The total amount of ubiquitin found in skeletal muscle is muscle fiber-type specific,
with a greater abundance of ubiquitin found in type I fibers (Riley et al., 1992).
Furthermore, a three to seven times higher density of conjugated ubiquitin was found
at the Z-discs than anywhere else in the muscle fiber, which suggests that, like
calpain, ubiquitin targets the cytoskeletal proteins of muscle fibers. One main
difference between the two systems, however, is that calpain is activated a lot earlier
than the ubiquitin-proteasome pathway (Feasson et al., 2002; Stupka et al., 2001).
Furthermore, the action of the UPP may be prolonged post-exercise in order to
increase the intracellular concentration of amino acids (Tipton, Wolfe, 1998), which
would stimulate MPS via mTORC1 activation.
Repair following exercise-induced muscle damage
Following the orderly demolition of damaged/cleaved muscle proteins (via the
aforementioned MPD systems) in response to eccentric contractions, the damaged
muscle fibers need to undergo repair. The activation, proliferation and fusion of
satellite cells with damaged muscle fibers, and the subsequent differentiation into
myoblasts, are crucial for this repair process (McCarthy et al., 2011; Petrella et al.,
2006). In fact, it has been shown that while the hypertrophic response maybe
maintained in the absence of satellite cell recruitment, recovery from damage was
severely impaired under these conditions (McCarthy et al., 2011).
As previously discussed, IGF-I and MRFs are integral in the activation and
differentiation of satellite cells (Langen et al., 2004; Scime et al., 2006; Szalay et al.,
1997), which probably explains why skeletal muscle IGF-I and MRF expression are
increased following stretch-induced damage (Bickel et al., 2005; Petrella et al., 2006;
Yang, Alnaqeeb, Simpson, Goldspink, 1997; Yang, Creer, Jemiolo, Trappe, 2005). To
repair the muscle, satellite cells fuse with damaged fibers and differentiate into
myonuclei, but may even form new fibers in the case of complete fiber necrosis. The
orderly proliferation and subsequent differentiation is crucial for optimal repair. For
the initial proliferation of satellite cells the inflammatory environment is beneficial,
but this inflammation must be transient to allow the cells to differentiate (Pelosi et al.,
2007). Therefore, it may be that chronic low-grade systemic inflammation, e.g. during
ageing, may underlie the delay in muscle regeneration (Langen et al., 2006).
Skeletal muscle is able to hypertrophy in response to a variety of anabolic stimuli,
which include resistance exercise, amino acid ingestion, and an increase in IGF-I
expression. All these stimuli are able to activate the mTORC1 signaling pathway,
which stimulates MPS and inhibits MPD. When the rate of MPS exceeds MPD, there
is a positive net protein balance (NPB) and an accretion of contractile material occurs,
leading to muscle hypertrophy and an increase in strength. Inducing muscle
hypertrophy can have beneficial effects on individuals suffering from cachectic
conditions, such as cancer, AIDS, and sarcopenia, where muscle atrophy can have
devastating effects on an individual’s quality of life. Muscle atrophy occurs when
there is a negative NPB, i.e. when the rate of MPD is greater than MPS. There are a
number of stimuli that have been associated with muscle atrophy, including
chronically elevated levels of pro-inflammatory cytokines (e.g. IL-1, IL-6 and TNF-
), a reduction in IGF-I and increased expression of myostatin. Furthermore,
strenuous unaccustomed exercise can cause mechanical damage to the muscle, which
activates MPD systems, including calpain, inflammation and the ubiquitin-proteasome
protein degradation pathway. Damaged proteins within the muscle fiber are broken
down, resulting in an increased intracellular amino acid concentration, which in turn
activates mTORC1 and increases MPS, thus helping to repair the muscle. Elevated
local IGF-I and MRF expression facilitates the repair process by activating satellite
cells and enabling fusion with existing fibers. Many of the molecular signaling
pathways associated with muscle hypertrophy, atrophy and repair have been
identified. However, there is still much to be learned about these pathways, and
understanding them may help us to prevent or reverse muscle atrophy associated with
a host of muscle wasting conditions.
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Figure 1. The molecular signaling pathways associated with muscle hypertrophy and atrophy.
The binding of IGF-I to its receptor (IGF-IR) causes autophosphorylation of insulin receptor substrate
(IRS1). Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that phosphorylates phosphatidylinositol
(4,5)-bisphosphate, producing phosphatidylinositol (3,4,5)-trisphosphate, which is a membrane-binding
site for phosphoinositide-dependent protein kinase (PDK1). Upon translocation to the sarcolemma,
AKT (or protein kinase B, PKB) is phosphorylated by PDK1. Once activated, AKT phosphorylates
mammalian target of rapamycin complex 1 (mTORC1) directly and by phosphorylating and
inactivating the tuberous sclerosis complexes 1 and 2 (TSC1/2), which otherwise inhibit mTORC1
activation. Following resistance exercise, an influx of calcium ions (Ca2+) via stretch-activated
channels (SACs) and the activation of FAK in the costamere can inactivate TSC1/2, thus activating
mTORC1. Amino acids entering the muscle fiber cause RagGTPase-dependent translocation of
mTORC1 to the lysosome, where it is activated by ras homologous protein enriched in brain (Rheb).
mTORC1 subsequently activates 70KDa ribosomal S6 protein kinase (p70S6K), and inhibits 4E-BP
(also known as PHAS-1), which is a negative regulator of the eukaryotic translation initiation factor 4E
(eIF-4E). Phosphorylated AKT also inhibits glycogen-synthase kinase (GSK3β), a substrate of AKT
that blocks protein translation initiated by the eIF-2B protein. All of these actions lead to increased
protein synthesis. However, protein degradation can be induced by pro-inflammatory cytokines, such
as tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which activate NF-κB via degradation of
I-κB, leading to increased transcription of the E3 ubiquitin-ligase, muscle RING-finger protein-1
(MuRF1). Another ligase, Atrogin1 (also known as MAFbx), is up-regulated by mitogen-activated
protein kinase (MAPK) p38, while both ligases are up-regulated by forkhead box (FOXO) transcription
factors. However, phosphorylated AKT blocks the transcriptional up-regulation of Atrogin1 and
MuRF1 by inhibiting FOXO1, while phosphorylated mTORC1 inhibits the up-regulation of Atrogin1
directly. Myostatin also increases protein degradation and decreases protein synthesis by activating
MAPKs and the SMAD complex, and by inhibiting PI3K. In addition, myostatin inhibits the myogenic
program, thus resulting in a decrease of myoblast proliferation.
Figure 2. Schematic representation of (A) the location of costameres within a skeletal muscle
fiber and (B) the proteins that constitute the costamere. Costameres are protein complexes
circumferentially aligned along the length of the muscle fiber that connect peripheral myofibrils at the
Z-disks to the sarcolemma and beyond to the extra-cellular matrix (ECM). The costamere comprises a
dystrophin/glycoprotein complex and a focal adhesion complex (FAC), which includes the integrin-
associated tyrosine kinase focal adhesion kinase (FAK). Fig. 2B modified from (Fluck et al., 2002)
with permission.
Figure 3. Breakdown of the protein fragment via the ubiquitin proteasome pathway. Multiple
ubiquitin (Ub) molecules form a polyubiquitin chain in a process involving the Ub-activating (E1), Ub-
conjugating (E2), and Ub-ligating (E3) enzymes. The targeted protein fragment is then selected for
degradation, or “tagged”, via the covalent attachment of the polyubiquitin chain. The tagging enables
the 19S (PA700) module to recognize the protein fragment, so that it can be further degraded by the
20S core into oligopeptides after it has been de-ubiquitinated and the Ub molecules released recycled.
Adapted from (Milacic, Dou, 2009) with permission.
Figure 1
Figure 2A
Figure 2B
Figure 3
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Background/objectives: The purpose of this study was to determine whether circulating pro-inflammatory cytokines, elevated with increased fat mass and ageing, were associated with muscle properties in young and older people with variable adiposity. Subjects/methods: Seventy-five young (18-49 yrs) and 67 older (50-80 yrs) healthy, untrained men and women (BMI: 17-49 kg/m(2)) performed isometric and isokinetic plantar flexor maximum voluntary contractions (MVCs). Volume (Vm), fascicle pennation angle (FPA), and physiological cross-sectional area (PCSA) of the gastrocnemius medialis (GM) muscle were measured using ultrasonography. Voluntary muscle activation (VA) was assessed using electrical stimulation. GM specific force was calculated as GM fascicle force/PCSA. Percentage body fat (BF%), body fat mass (BFM), and lean mass (BLM) were assessed using dual-energy X-ray absorptiometry. Serum concentration of 12 cytokines was measured using multiplex luminometry. Results: Despite greater Vm, FPA, and PCSA (P<0.05), young individuals with BF% ⩾40 exhibited 37% less GM specific force compared to young BF% <40 (P<0.05). Older adults with BF% ⩾40 showed greater isokinetic MVC compared to older BF% <40 (P=0.019) but this was reversed when normalised to body mass (P<0.001). IL-6 correlated inversely with VA in young (r=-0.376; P=0.022) but not older adults (P>0.05), while IL-8 correlated with VA in older but not young adults (r⩾0.378, P⩽0.027). TNF-alpha correlated with MVC, lean mass, GM FPA and maximum force in older adults (r⩾0.458; P⩽0.048). Conclusions: The age- and adiposity-dependent relationships found here provide evidence that circulating pro-inflammatory cytokines may play different roles in muscle remodelling according to the age and adiposity of the individual.International Journal of Obesity accepted article preview online, 29 August 2016. doi:10.1038/ijo.2016.151.
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Prolonged unaccustomed exercise involving muscle lengthening (eccentric) actions can result in ultrastructural muscle disruption, impaired excitation-contraction coupling, inflammation and muscle protein degradation. This process is associated with delayed onset muscle soreness and is referred to as exercise-induced muscle damage. Although a certain amount of muscle damage may be necessary for adaptation to occur, excessive damage or inadequate recovery from exercise-induced muscle damage can increase injury risk, particularly in older individuals, who experience more damage and require longer to recover from muscle damaging exercise than younger adults. Furthermore, it is apparent that inter-individual variation exists in the response to exercise-induced muscle damage, and there is evidence that genetic variability may play a key role. Although this area of research is in its infancy, certain gene variations, or polymorphisms have been associated with exercise-induced muscle damage (i.e. individuals with certain genotypes experience greater muscle damage, and require longer recovery, following strenuous exercise). These polymorphisms include ACTN3 (R577X, rs1815739), TNF (-308 G>A, rs1800629), IL6 (-174 G>C, rs1800795), and IGF2 (ApaI, 17200 G>A, rs680). Knowing how someone is likely to respond to a particular type of exercise could help coaches/practitioners individualise the exercise training of their athletes/patients, thus maximising recovery and adaptation, while reducing overload-associated injury risk. The purpose of this review is to provide a critical analysis of the literature concerning gene polymorphisms associated with exercise-induced muscle damage, both in young and older individuals, and to highlight the potential mechanisms underpinning these associations, thus providing a better understanding of exercise-induced muscle damage.
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We hypothesize that the attenuated hypertrophic response in old mouse muscle is (1) partly due to a reduced capillarization and angiogenesis, which is (2) accompanied by a reduced oxidative capacity and fatigue resistance in old control and overloaded muscles, that (3) can be rescued by the antioxidant resveratrol. To investigate this, the hypertrophic response, capillarization, oxidative capacity, and fatigue resistance of m. plantaris were compared in 9- and 25-month-old non-treated and 25-month-old resveratrol-treated mice. Overload increased the local capillary-to-fiber ratio less in old (15 %) than in adult (59 %) muscle (P < 0.05). Although muscles of old mice had a higher succinate dehydrogenase (SDH) activity (P < 0.05) and a slower fiber type profile (P < 0.05), the isometric fatigue resistance was similar in 9- and 25-month-old mice. In both age groups, the fatigue resistance was increased to the same extent after overload (P < 0.01), without a significant change in SDH activity, but an increased capillary density (P < 0.05). Attenuated angiogenesis during overload may contribute to the attenuated hypertrophic response in old age. Neither was rescued by resveratrol supplementation. Changes in fatigue resistance with overload and aging were dissociated from changes in SDH activity, but paralleled those in capillarization. This suggests that capillarization plays a more important role in fatigue resistance than oxidative capacity.
p>A central tenet of skeletal muscle biology is the existence of an inverse relationship between the oxidative fibre capacity and its size. However, robustness of this relationship is unknown. We show that superimposition of Estrogen-related receptor gamma (Errγ) on the myostatin (Mtn) mouse null background (Mtn(-/-)/Errγ(Tg/+)) results in hypertrophic muscle with a high oxidative capacity thus violating the inverse relationship between fibre size and oxidative capacity. We also examined the canonical view that oxidative muscle phenotype positively correlate with Satellite cell number, the resident stem cells of skeletal muscle. Surprisingly, hypertrophic fibres from Mtn(-/-)/Errγ(Tg/+) mouse showed satellite cell deficit which unexpectedly did not affect muscle regeneration. These observations 1) challenge the concept of a constraint between fibre size and oxidative capacity and 2) indicate the important role of the microcirculation in the regenerative capacity of a muscle even when satellite cell numbers are reduced.</p
We examined the involvement of focal adhesion kinase (FAK) in mechano-regulated signalling to protein synthesis by combining muscle-targeted transgenesis with a physiological model for un- and reloading of hindlimbs. Transfections of mouse tibialis anterior muscle with a FAK expression construct increased FAK protein 1.6-fold versus empty transfection in the contralateral leg and elevated FAK concentration at the sarcolemma. Altered activation status of phosphotransfer enzymes and downstream translation factors showed that FAK overexpression was functionally important. FAK auto-phosphorylation on Y397 was enhanced between 1 and 6 h of reloading and preceded the activation of p70S6K after 24 h of reloading. Akt and translation initiation factors 4E-BP1 and 2A, which reside up- or downstream of p70S6K, respectively, showed no FAK-modulated regulation. The findings identify FAK as an upstream element of the mechano-sensory pathway of p70S6K activation whose Akt-independent regulation intervenes in control of muscle mass by mechanical stimuli in humans.
Vastus lateralis muscle samples were obtained by needle biopsy from 18 healthy elderly men (65-82 yr) participating in a double blind, placebo (PL)-controlled trial of recombinant human GH (rhGH) and exercise and assessed for muscle morphology and skeletal muscle tissue expression of GH and insulin-like growth factors (IGFs). Subjects initially underwent progressive resistance training for 14 weeks and were then randomized to receive either rhGH (0.02 mg/kg, sc) or PL while undertaking a further 10 weeks of training. Muscle samples were obtained at baseline and at 14 and 24 weeks. The mean (+/- SEM) cross-sectional areas of type I and II fibers were similar (type I, 3891 +/- 167 microns2; type II, 3985 +/- 200 microns2) at baseline and increased (P < 0.01) by 16.2 +/- 4.1% and 11.8 +/- 3.8%, respectively, after the initial 14-week training period. After treatment (weeks 14-24), two-way repeated measures ANOVA revealed a main effect of time for type I (P < 0.01) and type II fibers (P < 0.05), but no...
Interaction of the cell surface integrin receptors with extracellular matrix proteins results in the activation of intracellular signaling pathways, including activation of the p42/p44 mitogen-activated protein kinases. The protein tyrosine kinase focal adhesion kinase, or FAK, is linked to integrin signaling and interacts with several molecules involved in signal transduction, Here we report that exposure of fibroblast cells to extracellular matrix proteins activates the p70/p85 ribosomal S6 kinase (S6K) pathway in a ligand dependent manner. Treatment of cells with inhibitors of phosphatidylinositol 3-kinase, or FRAP (FKBP 12/rapamycin-associated protein) blocks integrin-mediated activation of S6K. In contrast to the integrin-directed activation of the mitogen-activated protein kinases, cytochalasin D treatment does not inhibit S6K activation, Treatment with the protein tyrosine kinase inhibitors herbimycin A and genistein completely blocks S6K activation, indicating a requirement for tyrosine kinase activity. Overexpression of the COOH-terminal noncatalytic domain of FAK, FRNK (FAK-related non-kinase) in chick embryo cells results in a significant reduction in the integrin-mediated activation of S6K and a concomitant reduction in FAK tyrosine phosphorylation, These results indicate at least a partial requirement for FAK in the S6K activation pathway.
1. Increases in strength and size of the quadriceps muscle have been compared during 12 weeks of either isometric or dynamic strength training. 2. Isometric training of one leg resulted in a significant increase in force (35 +/- 19%, mean +/- S.D., n = 6) with no change in the contralateral untrained control leg. 3. Quadriceps cross-sectional area was measured from mid-thigh X-ray computerized tomography (c.t.) scans before and after training. The increase in area (5 +/- 4.6%, mean +/- S.D., n = 6) was smaller than, and not correlated with, the increase in strength. 4. The possibility that the stimulus for gain in strength is the high force developed in the muscle was examined by comparing two training regimes, one where the muscle shortened (concentric) and the other where the muscle was stretched (eccentric) during the training exercise. Forces generated during eccentric training were 45% higher than during concentric training. 5. Similar changes in strength and muscle cross-sectional area were found after the two forms of exercise. Eccentric exercise increased isometric force by 11 +/- 3.6% (mean +/- S.D., n = 6), and concentric training by 15 +/- 8.0% (mean +/- S.D., n = 6). In both cases there was an approximate 5% increase in cross-sectional area. 6. It is concluded that as a result of strength training the main change in the first 12 weeks is an increase in the force generated per unit cross-sectional area of muscle. The stimulus for this is unknown but comparison of the effects of eccentric and concentric training suggest it is unlikely to be solely mechanical stress or metabolic fluxes in the muscle.