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Muscle Growth, Repair and Preservation: A Mechanistic Approach

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Muscle Growth, Repair and Preservation: A Mechanistic Approach

Robert M. Erskine1,2 and Hans Degens3,4

1
School 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:

R.M.Erskine@ljmu.ac.uk

Running title: Muscle Growth, Repair and Preservation

 2

SUMMARY

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

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INTRODUCTION

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

hypertrophy.

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
 4

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.
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MUSCLE GROWTH

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

 6

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.

[FIGURE 1 NEAR HERE]

 7

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
 8

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
 9

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).

[FIGURE 2 NEAR HERE]

 10

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,
 11

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;
 12

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.
 13

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
 14

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).

MUSCLE ATROPHY

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
 15

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
 16

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 ubiquitin–proteasome 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.

 17

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,

 18

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

muscles.

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
 19

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
 20

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
 21

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
 22

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,

2007).

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).

MUSCLE DAMAGE AND REPAIR

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
 23

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.,

 24

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): -calpain

(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.

 25

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

 26

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.,

1985).
 27

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
 28

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).

[FIGURE 3 NEAR HERE]

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
 29

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).

 30

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).

SUMMARY

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
 31

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.

 32

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FIGURE LEGENDS

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 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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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.

 51

Figure 1
 52

Figure 2A

Figure 2B
 53

Figure 3

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