J Muscle Res Cell Motil (2015) 36:405–421

DOI 10.1007/s10974-015-9439-8

REVIEW ARTICLE

Skeletal muscle atrophy: disease-induced mechanisms may mask

disuse atrophy
C. J. Malavaki1,2 • G. K. Sakkas1,2,4 • G. I. Mitrou1 • A. Kalyva1,4 •

I. Stefanidis2 • K. H. Myburgh3 • C. Karatzaferi1,4

Received: 14 July 2015 / Accepted: 8 December 2015 / Published online: 4 January 2016
Ó Springer International Publishing Switzerland 2015

Abstract Disuse atrophy is the loss of skeletal muscle Timing of sampling is crucial as some signalling mecha-
mass due to inactivity or lower than ‘normal’ use. It is not nisms reach their peak early during the atrophy process to
only a furtive component of the ‘modern’ sedentary life- rapidly decline thereafter, while other present high levels
style but also a part of numerous pathologies, where muscle even weeks and months after study initiation. The impor-
loss is linked to disease specific and/or other toxicity fac- tance of such differences lays in future consideration of
tors, eventually leading to wasting (cachexia). Whether appropriate treatment targets. Apart from attempting to
disuse-or-disease induced, muscle loss leads to weakness correct defective genes or negate their effects, technolog-
and metabolic comorbidities with a high societal and ical advances in new rational ways should aim to regulate
financial cost. This review discusses the intricate network specific gene expression at precise time points for the
of interacting signalling pathways including Atrogin-1/ treatment of muscle atrophy in therapeutic protocols
MAFbx, IGF1-Akt, myostatin, glucocorticoids, NF-kB, depending on the origin of atrophy induction.
MAPKs and caspases that seem to regulate disuse atrophy
but also share common activation patterns in other states of Keywords Protein synthesis  Protein degradation 
muscle loss such as sarcopenia or cachexia. Reactive Ubiquitin–proteasome system  Redox status  Mechanical
oxygen species are also important regulators of cell sig- loading  Atrophy countermeasures
nalling pathways that can accelerate proteolysis and
depress protein synthesis. Exercise is an effective coun-
termeasure and antioxidants may show some benefit. We
discuss how the experimental model used can crucially Introduction and background
affect the outcome and hence our understanding of atrophy.
Working definitions

C. J. Malavaki and G. K. Sakkas are equal first authors. The term Disuse Atrophy describes the loss of skeletal
muscle mass due to inactivity (for definitions of muscle
& C. Karatzaferi
[email protected]; [email protected] loss see (A.D.A.M. 2005) and is considered a characteristic
of the modern lifestyle, i.e. it can occur while one is living
1
Muscle Physiology & Mechanics Lab DPESS, University of an otherwise ‘normal’ life. Its health consequences follow a
Thessaly, Karyes, 42100 Trikala, Greece
continuum of severity in body composition disturbances,
2
Division of Nephrology, Department of Medicine, School of from hidden obesity (Sarcopenic Obesity) to evident obe-
Health Sciences, University of Thessaly, BIOPOLIS,
sity in otherwise healthy individuals, to metabolic syn-
Panepistimiou Str, 41500 Larissa, Greece
3
drome (Wannamethee and Atkins 2015), all of which have
Department of Physiological Sciences, Stellenbosch overall increased in frequency.
University, Matieland, Private Bag X1, Stellenbosch 7602,
South Africa Disuse atrophy might occur in a single group of muscles
4 due to long term limb immobilization as in the case of cast
Department of Kinesiology, Institute for Research and
Technology Thessaly, CERTH, 42100 Trikala, Greece application in fracture management (Chen et al. 2007) and

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limb reconstruction approaches, or throughout the whole weakness muscle strength is less than expected given the
body in situations in which bed rest is mandated (Brooks motor unit activation. This appears to be a primary
and Myburgh 2014; Palus et al. 2014). Muscle changes are symptom of a variety of skeletal muscle diseases, including
not limited to loss of total muscle mass or volume, but loss muscular dystrophy and inflammatory myopathy (An-
of myosin content (Campbell et al. 2013) and fibre type nexstad et al. 2014; Yiu and Kornberg 2015). In perceived
transition (slow to fast), may also be evident. Analogous muscle weakness a person feels that extra effort is required
detriments in muscle status, also without loss of fibre to exert a given force but muscle strength is, at least ini-
count, occur with exposure to microgravity (spaceflight tially, normal (Holgate et al. 2011). In the case of a chronic
Talmadge et al. 1996). Without loss of motor units, com- condition (e.g. kidney disease), with a gradual worsening
plete recovery of all aspects of muscle health is expected of systemic effects (including eventual neuropathy), mus-
(Appell 1990), especially in younger individuals (Campbell cle weakness and exercise intolerance are combined to a
et al. 2013). generalized sense of fatigue that is traced to both muscular
Muscle atrophy is also a side effect of numerous and central activation failures (Sakkas and Karatzaferi
pathologies such as cancer, diabetes mellitus, renal failure, 2012). Therefore, divergent types of weakness and fatigue
heart failure and sepsis (Hasselgren and Fischer 1997; do not exactly correspond to distinct causes of atrophy.
Jagoe and Goldberg 2001), where striated muscle is However, irrespectively of origin, muscle atrophy and
directly or indirectly affected by the disease per se (i.e. via weakness, given their implication in metabolic comor-
mechanisms such as systemic toxicity, inflammation and/or bidities and loss of independence, are seen as ‘‘key cost
side effects of treatments such as glucocorticoids, statins, driver(s)’’ (see e.g. Chen et al. 2013), imposing a great
radiation etc.). Nevertheless, at the same time the patient financial burden to national health care systems.
assumes a severely hypokinetic state so that disuse atrophy
becomes an, often masked, component of muscle loss. Modes and models of studying disuse atrophy
Separating the involvement of disuse muscle atrophy from
disease-related muscle loss bears grave significance on The model used to study disuse muscle atrophy and a
diagnosis and treatment strategies. E.g., in the case of the protocol’s duration can crucially affect the experimental
haemodialysis patient, the disease itself causes muscle results and their interpretation. In animals, popular models
atrophy, the administration of the treatment enforces include tail suspension, hind limb suspension, cast immo-
inactivity (Sakkas et al. 2003a) and patients may present bilisation, nerve dissection, hibernation studies and
with malnutrition that has been shown to influence the microgravity exposure. In human subjects microgravity
effectiveness of interventions (Sakkas et al. 2003b). If the may be simulated by bed rest (Gallagher et al. 2005;
catabolic state is left unaddressed, the above and other Trappe et al. 2004). Typically these are longitudinal studies
diseases, eventually lead to Cachexia (Lee and Glass 2011) that vary substantially in duration. In human cross-sec-
a terminal condition which includes both muscle wasting tional studies, characteristics of a specific disease popula-
and fat loss. tion are contrasted to healthy counterparts. Animal models
To further complicate our understanding, aging-related that simulate disease are frequently cross-sectional, con-
muscle loss, termed Sarcopenia also contributes to overall trasting the experimentally-induced disease to control, with
muscle loss in the presence or absence of disease and variable levels of fidelity (Booth et al. 2012).
inactivity. Sarcopenia causes reductions in both fibre size Various methods are used to study manifestations of
and count with a characteristic mosaic change in histo- atrophy in muscle morphology (e.g. MRI, DEXA, fibre CSA,
chemical profile due to loss of the fast fibres (Brooks and immunocytochemistry, etc.), metabolism (from resting
Faulkner 1994). Interestingly some researchers propose energy expenditure to muscle biopsy derived signalling
that repeated bouts of disuse atrophy through the lifespan molecules and protein levels) and function (from whole body
may exacerbate sarcopenia at an older age (Wall et al. or limp testing, to isolated muscle fibre contractile proper-
2013) indicating an overlap and interaction of the under- ties). Regardless of the study methodology, overall duration
lying molecular mechanisms. and timing of sampling are crucial. As it will be seen below,
some signalling mechanisms reach their peak early after
Functional and clinical associations starting the atrophy model, while others maintain high levels
even weeks and months after the initiation.
With muscle loss, comes skeletal muscle weakness, either The following sections of this review aim to report on
true or perceived. In disuse atrophy, strength reduction is current knowledge regarding the molecular signals that
not related to neural activation, in contrast to neurogenic play a role in disuse atrophy. Where possible we separate
atrophy, i.e. when an injury harms the motor nerve that otherwise healthy disuse from disease related atrophy and
activates the muscle (Moresi et al. 2010). In true muscle distinguish differences between animal and human studies.

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In the discussion section, issues of concern and possible residue, forming a thioester linkage. Then, via a trans-
new directions will be mentioned. thioesterification reaction, activated Ub is transferred from
the E1 to the E2. Following that, an E3 catalyses the for-
mation of a peptide bond between the C-terminal glycine of
Mechanisms of muscle atrophy Ub with a lysine of the target protein. Finally, polyubiq-
uitination condemns the protein to degradation while
At any moment available muscle mass is the result of a ubiquitin molecules are recycled.
balance between muscle anabolic and catabolic processes. In the human genome two E1 enzymes, 20–30 E2
Muscle anabolism is driven by growth hormone (Pupim enzymes and a few hundred substrate-specific E3 enzymes
et al. 2005), insulin, IGF-1 (Hirose et al. 2000; Wang et al. have been identified, with the cascade’s increased com-
2006) and testosterone (Gruenewald and Matsumoto 2003), plexity from conjugation (Jin et al. 2007) to poly-ubiqui-
always dependent upon adequate nutrition (Wall et al. tination (Sadowski and Sarcevic 2010) allowing for diverse
2013), sleep, and mediated by the appropriate exer- substrate-ubiquitin structures directing proteins to different
cise/physical activity stimulus (Sakkas et al. 2008). Muscle fates. Several muscle-specific E3s exist: Muscle Ring
catabolism is mediated by endocrine, inflammatory and Finger 1 (MuRF1) regulates the dissociation and degra-
oxidative stress factors (Powers et al. 2012). Whether dation of myosin light chains 1 and 2, myosin binding
reductions on anabolic or increases in catabolic processes protein C and cardiac Troponin I (Cohen et al. 2009; Kedar
dominate in net muscle loss may be dependent on duration et al. 2004), MuRF3 ligase ubiquitinates soluble myosin
of the disuse. As discussed by Wall et al., early disuse (Fielitz et al. 2007), Ozz ligase (also known as Neuralised-
(\10 days) is characterised by a rise in muscle protein like protein 2) ubiquitinates the embryonic isoform of
breakdown and a decline in synthesis, while in prolonged Myosin Heavy chain (Campos et al. 2010) and Muscle
disuse ([10 days) with protein breakdown rates unchan- Atrophy F-box (MAFbx, also known as Atrogin-1) which
ged, atrophy might be primarily attributed to a decline of ubiquitinates MyoD and the elongation initiation factor 3
post-absorptive and of post-prandial muscle protein syn- (eIF3-f) (Lagirand-Cantaloube et al. 2008, 2009). Particu-
thesis rates (Wall et al. 2013). larly MAFbx and MurF1 have been shown to over-express
There are four known proteolytic systems implicated in in muscle atrophy (Bodine et al. 2001; Brooks and
muscle atrophy resulting from disuse or disease; (A) the Myburgh 2014; Gomes et al. 2001), with the upregulation
calcium-dependent calpain system, (B) the caspase endo- of MAFbx and MurF1 during immobilization being a
protease system, (C) the ubiquitin–proteasome system and consistent finding amongst different study models, in
(D) the lysosomal protease system. The calcium-dependent humans, mice and rats (reviewed in Foletta et al. 2011).
calpain system and the lysosomal protease system are well
described by Powers et al. (2007) and Jackman and Kan- Human Studies of the role of the Atrogin-1/MAFbx
darian (2004). These proteolytic systems intermingle. For and MuRF1 genes and other ligases
example, activation of caspase-3, an essential initial step
for proteolysis initiation yields proteins that are degraded Unilateral lower limb suspension differs from cast immo-
by the ubiquitin–proteasome system (Du et al. 2004). In bilisation. However, the basic tenet is that hypokinesia and
this review, substantial focus will be placed on the ubiq- mechanisms of proteasomal degradation could be expected
uitin–proteasome system, and more specific on the MuRF- to be similar.
1, Atrogin-1/MAFbx networks and their roles in muscle
atrophy. Early disuse (\10 days)

The ubiquitin–proteasome system and the MuRF-1, Already at 3 days of unilateral lower leg unloading,
Atrogin-1/MAFbx networks MuRF1 and Atrogin-1/MAFbx mRNA levels were signif-
icantly upregulated, by threefold and twofold respectively
A polypeptide or a protein destined for proteasomal (Gustafsson et al. 2010). However, at 10 days, De Boer
degradation must be ubiquitinated first. The ubiquitination et al., using the same approach, found an upregulated
process (Fig. 1), involves ubiquitin (Ub)-activating mRNA expression for MuRF-1,by *threefold, but not for
enzymes (E1s), Ub-conjugating enzymes (E2s) and Ub- MAFbx (de Boer et al. 2007).
ligases (E3s) (reviewed in Hershko and Ciechanover 1998;
Hochstrasser 1996). Briefly, in a two-step, ATP-dependent Prolonged disuse ([10 days)
process, E1 binds Ub and catalyses the acyl-adenylation at
the C-terminal of ubiquitin (Chondrogianni et al. 2003), At 2 weeks of limb immobilization, the Atrogin-1/
and the adenylated Ub is transferred to an active site Cys MAFbx gene was found to be upregulated by *2.5 fold

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Fig. 1 Ubiquitination is a
multistep process. Firstly the E1
ubiquitin activating enzyme
binds ubiquitin in an ATP
hydrolysis dependent step. Then
ubiquitin is transferred from the
E1 to the E2 ubiquitin-
conjugating enzyme. Finally the
E3 ubiquitin-protein ligase
enzyme is involved in the
formation of a peptide bond
between a glycine at the
C-terminal of ubiquitin with a
lysine of the target protein. Ub
detachment is mediated by
deubiquitinating enzymes
(DUBs). Ubiquitination has
diverse outcomes.
Polyubiquitination (not shown)
leads to protein degradation. For
a review discussing diverse
forms of ubiquitin modifications
see (Woelk et al. 2007)

(Chen et al. 2007). However, in a study of otherwise Mediation by age

healthy volunteers, at 2 weeks upon cast removal, MAFbx
was upregulated by only 62 % (i.e. 0.6 folds), while Interestingly, while an age-related decline in FBXO40,
MuRF1 expression did not change (Jones et al. 2004). another specific F-box protein, was reported, no discernible
Perhaps this discrepancy in magnitude of response can be differences in baseline mRNA and protein levels of atro-
explained by subject selection as, in the former study, the gin-1 and MURF1 have been found between young and old
sample included fracture patients. By 21 days, expression subjects (Stefanetti et al. 2014), prompting these authors to
of MAFbx and MuRF-1 genes may return towards baseline conclude that the ubiquitin proteasome may not be par-
(de Boer et al. 2007). This temporal effect was reiterated ticularly affected by ageing per se.
elsewhere when the obvious reduction of vastus lateralis’
muscle volume at 20 days of lower limb suspension was
not accompanied by obvious changes in MAFbx activity Muscle type differences
(Sakuma et al. 2009).
At 3 days of unloading, only the faster vastus lateralis
Mediation by exercise presented with increased MuRF1 and Atrogin-1/MAFbx
mRNA expression levels (threefold and twofold respec-
At 24 h after cast removal and a first exercise session, tively) but not the slow soleus muscle (Gustafsson et al.
MAFbx and MuRF1 were down-regulated by 39 and 33 %, 2010). As already mentioned, at 20 days of unloading,
respectively, in comparison to levels immediately post- Atrogin-1/MAFbx mRNA levels were not significantly
immobilization, i.e. they returned to levels close to baseline different from baseline in the vastus lateralis muscle, while
(Jones et al. 2004). During 6 weeks of rehabilitation, a muscle volume had significantly decreased (Sakuma et al.
further decline in MuRF1 expression was evident at 1 week, 2009). Moreover, at 60 days of bed rest, MuRF1 protein
while MAFbx expression did not change (Jones et al. 2004). levels were somewhat elevated (by 35 %) only in the
In very prolonged bed rest (60 days), exercise maintained soleus muscle but not in the vastus lateralis muscle (Sala-
muscle type morphometry, composition and ‘‘almost nor- nova et al. 2008). It would appear that the soleus requires
mal’’ levels of MuRF1 in the soleus and vastus lateralis of very prolonged periods of severe hypokinesia to be
otherwise healthy women (Salanova et al. 2008). affected.

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Animal studies of the role of the Atrogin-1/MAFbx Muscle type differences

and MuRF1 genes
In the Nordquist et al. study (Nordquist et al. 2007) men-
The number of studies using rodent models is very large, tioned earlier, MAFbx mRNA and MuRF1 levels increased
thus only a few representative studies of disuse atrophy are in both EDL and soleus muscles (MAFbx somewhat more
therein discussed. Overall time lines are different in animal pronounced in soleus, MuRF1 in EDL). In agreement with
models as their life cycle is shorter than in humans. While human studies, it appears that prolonged bedrest is required
this may seem to be a disantvatage in terms of translation, to see an effect on the soleus similar to what other models
it makes possible to study the rapid signalling responses of atrophy cause on faster muscle.
(minutes or hours), the acute responses (within a few days)
and the long-term responses (weeks or even months) with Mediation by other factors
multiple invasive studies.
Spaceflight not only upregulated MuRF-1, Siah-1A, and
Early disuse Cbl-b, but also appeared to ‘dislocate’ mitochondria, per-
haps by disturbing mitochondria anchoring proteins, with
When rats were unilaterally casted for 1, 3 and 5 days, both an evident upregulation of hypoxia-inducible factor 1b and
MuRF1 and Atrogin-1/MAFbx mRNA levels significantly other genes inducible by oxidative stress (Nikawa et al.
increased (Krawiec et al. 2005). By day 3 upregulation 2004). In that study, oxidative stress in L6 cells appeared to
peaked, while by day 5 mRNA levels returned to day 1 levels selectively induce Cbl-b and Siah-1A but not MuRF1, thus
(still higher than baseline). Interestingly, day 3 was also the the mediators that triggered the expression of ubiquitin
time point where maximum gastrocnemius muscle weight ligase during spaceflight were not clarified.
loss was observed (12 %) indicating an association between
mRNA expression and phenotype very early during KO models
unloading. To further prove the role of the proteasome,
provision of the potent proteasome inhibitor Velcade atten- In a seminal study by Bodine et al., under normal conditions,
uated the 3 day atrophy (by 53 %) (Krawiec et al. 2005). MuRF1 knockout (MuRF1-/-) or MAFbx knockout mice
(MAFbx-/-) appeared phenotypically similar to the wild type
Prolonged disuse (WT) mice. However, at 14 days of denervation, the
MuRF1-/- mice showed a 36 % and the MAFbx-/- mice
In a study mimicking the condition of intensive care patients, showed a 56 % resistance to muscle wasting and atrophy
rats were under mechanical ventilation with blocked neu- compared to WT, showing the critical role of these two ubiq-
romuscular transmission for 1–2 weeks. The *25 % uitin ligases (Bodine et al. 2001). Likewise, Cbl-b-/- mice
reduction in body weight was accompanied by significant subjected to tail suspension showed no changes in force, nor in
increases in mRNA levels of MuRF1 and MAFbx in both their muscle mass compared to WT tail-suspended animals
soleus and extensor digitorum longus (EDL) muscles showed a 20 % decrease in gastrocnemius muscle wet weight
(Nordquist et al. 2007). Exposure of neonatal rats to 16-day (Nakao et al. 2009). The Cbl-b ligase targets the insulin
spaceflight, resulted in a 30 % decrease of gastrocnemius receptor substrate 1 (IRS-1), which it ubiquitinates, to be
muscle wet weight/body weight (ww/bw) with a *40 % degraded then by the proteasome, in both muscle cells and
reduction in fibre CSA accompanied by upregulation of COS7 cells. With tail suspension, WT mice showed increased
MuRF1, and other ligases such as Siah-1A and Cbl-b, the ubiquitination of IRS-1 with a reduction in its protein levels
latter by *eightfold (Nikawa et al. 2004). This indicated that (Nakao et al. 2009). These are very important observations
despite growth, muscle mass did not keep up proportionally. since the IRS-1 is a major substrate protein of the IGF-1
In that same study, at ground level, rats, exposed to 16 days receptor and can therefore influence signalling to the down-
of denervation had a 42 % decrease of gastrocnemius muscle stream PI3K and Akt targets. Hence, the effects of denervation
ww/bw and the most pronounced fibre CSA reduction, with (and possibly other atrophy models in which Cb1-b has not yet
tail suspension giving results in between. Cbl-b, named after been investigated) will clearly also have humoral ramifications.
Casitas B-lineage Lymphoma, is another E3 ligase whose
mRNA levels are dramatically elevated in rats in unloaded
conditions such as spaceflight, tail suspension or denervation Signalling molecules regulating muscle growth
(Nakao et al. 2009; Nikawa et al. 2004). Moreover, protein
levels of Cbl-b in gastrocnemius increased in response to An intricate network of signalling pathways has been studied
both spaceflight and 10 days of tail suspension (Nakao et al. for regulating muscle growth, including positive and negative
2009). regulators. These different pathways crosstalk and modulate

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one another however not always in a clear way (Fig. 2). Here, which is involved in protein synthesis and skeletal muscle
seven important pathways and factors will be presented: IGF1- growth has been well characterized over the last years
Akt, FOXO, myostatin, glucocorticoids, NF-kB, MAPKs and (Schiaffino and Mammucari 2011). This pathway is initi-
ROS-induced signalling, mostly based on animal studies. ated by binding of the Insulin like Growth Factor-1 (IGF-1)
Human studies are few as they require multiple invasive to its specific IGF-1 receptor (IGF-1R) stimulating a
monitoring, while animal data are more feasible and also allow cytoplasmatic signaling cascade. The IGF-1 binding stim-
for more definitive answers on selected factors via ‘non-phys- ulates the IGF-1R tyrosine kinase activity and therefore the
iological’ models, including knocking out a single molecule or phosphorylation of several IGF-1R protein substrates (IRS)
applying inhibitors of molecules affecting whole pathways. is initiated. Subsequently the phosphorylated receptor’s
substrate becomes the binding site for the phosphatidyl
IGF1-PI3K-Akt signalling insitol 3 kinase (PI3K), which binds via its p38 regulatory
subunit. The p38 subunit releases the catalytic activity of
The IGF-1-Akt axis has unique regulatory functions in p110 subunit of PI3K, and therefore the PI3K lipid kinase
controlling both protein synthesis and protein degradation activity is stimulated. The membrane phospholipid-phos-
(Fig. 2). The IGF-1/PI3K/Akt/mTOR signaling pathway phatidylinositol-4,5-bisphosphate (PIP2), is phosphorylated

Fig. 2 The IGF-1-Akt axis controls both protein synthesis and (p70S6KK kinase) leading in translation initiation. It also controls
protein degradation. Binding of IGF1 to its receptor leads to muscle growth by inactivating the glycogen synthase kinase b
phosphorylation of insulin receptor substrate (IRS). Phosphorylated (GSK3b) which inhibits protein synthesis via the eukaryotic initiation
IRS recruits and activates phosphatidylinositol-3-kinase (PI3K), not factor 2B (eIF2B), and repressing FOXO signalling. FOXO tran-
shown, which in turn recruits phosphoinositidedependent kinase 1 scription factors regulate the expression of genes encoding MuRF1
(PDK1). PDK1 activates Akt by phosphorylation at serine 308. The and Arogin-1/MAFbx ligases that lead to muscle atrophy. Moreover,
activated Akt (translocated to the membrane) indirectly phosphory- MyoD family proteins have been implicated as nuclear downstream
lates and activates mTOR kinase. Activated mTOR has two targets of P13K/Akt for myogenin induction (Xu and Wu 2000). The
downstream targets which are involved in protein synthesis. It can IGF-Akt pathway is controlled by various feedback pathways (not
either phosphorylate the eukaryotic translation initiation factor shown in this simplified scheme), for a discussion see (Schiaffino and
4Ebinding protein 1 or it can phosphorylate and activate the S6K1 Mammucari 2011)

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by PI3K into phosphatidylinositol-3,4,5- triphosphate myopathy, loss of contractile muscle mass, muscle weak-
(PIP3). PIP3 is a binding substrate for the serine/threonine ness and premature death (Risson et al. 2009). Further-
kinase Akt. more, when regenerating denervated rat muscles were
The activated Akt in turn phosphorylates and activates a transfected with a plasmid encoding for constitutively
second kinase namely the mammalian target of rapamycin active Akt the denervation induced atrophy was mitigated
or mTOR kinase. The activated mTOR has two downstream and fibre hypertrophy was induced during regeneration.
targets which are involved in protein synthesis. It can either However, the increase in muscle fibre size was repressed
phosphorylate the eukaryotic translation initation facton 4E- when the drug rapamycin was injected, which is a known
binding protein 1 or it can phosphorylate and activate the inhibitor of the mTOR protein. Rapamycin injection
p70S6K kinase. The translation initiation factor eIF4F is a resulted in complete blocking of the phosphorylation of
protein complex composed of three subunits, the eIF4E, the p70S6K and the target ribosomal protein S6 (Pallafacchina
eIF4A and the eIF4G. All three subunits are necessary for et al. 2002) confirming the downstream pathway affected.
the formation of the complex and the initiation of translation In humans, at 48 h of lower leg immobilization, Akt
in eukaryotes. The subunit eIF4E can only be available for phosphorylation decreases by 25 and 10 % in phosphory-
complex formation when it is not bound to another partner lation at Ser473 and Thr308 respectively, were indicative
protein i.e. the 4E-binding protein 1. The activated mTOR of a decrease in the protein synthesis pathway (Urso et al.
kinase phosphorylates the 4E-BP1 and therefore dissociates 2006). Therefore, alterations of the Akt/mTOR pathway
it from the eIF4E subunit. The dissociated free eIF4E may occur in humans as well, but it is again crucial at
becomes then available for the eIF4F complex formation which time frame the samples are collected since it is
and the subsequent initiation of translation and protein possible that this step takes place only in the very begin-
synthesis (Carrera 2004). On the other hand, p70S6K, a ning of the atrophy process. In any case the well charac-
70 kDa serine/threonine kinase phosphorylates and acti- terized and consistent results that come from the rat model
vates the 40S ribosomal protein S6 (S6RP). Which increases studies cannot always be representative of the human
the translation of mRNA containing a 50 terminal oligopy- condition.
rimidine transcripts (Aloni et al. 1992). These transcripts
further promote protein synthesis as they encode key com-
ponents of the translational machinery such as ribosomal FOXO
proteins and elongation factors. Therefore, if in the above
IGF-1/PI3K/Akt/mTOR pathway any of the steps is inhib- The IGF-1/PI3K/Akt pathway also affects FOXO sig-
ited or any of the factors involved is down-regulated, it is nalling (Zhao et al. 2007). The forkhead box O (FOXO)
obvious that protein synthesis will be seriously affected. proteins belong to the Forkhead family of transcription
factors, and are linked to muscle atrophy, since upon
Early and later disuse activation they regulate the transcription of apoptosis, cell
cycle inhibition and cellular metabolism genes (Accili and
As early as 6 h after immobilization, in rats, the fractional Arden 2004; Carlsson and Mahlapuu 2002; Tran et al.
rate of protein synthesis was reduced by 37 % compared to 2003) (Engelbrecht et al. 2010). FOXO proteins can be
the control (Booth and Seider 1979; Thomason and Booth found either in the cytoplasm or in the nucleus with
1990). Moreover, reduction in body weight after 7 days dephosphorylated FOXO considered to be the active form
immobilization was accompanied by reductions in soleus since then it can be located in the nucleus and regulate the
fibre cross-sectional area, glycogen content and an increase transcription of the target genes. Phosphorylation of FOXO
in connective tissue density, while treatment with IGF by the Akt kinase, translocates the FOXO proteins from the
largely increased glycogen content, blunted fibre atrophy nucleus to the cytoplasm where they are unable to involve
and ameliorated the increase in connective tissue density, in the transcription process (Brunet et al. 1999). So when
without salvaging soleus weight (da Silva et al. 2011). there is inactivation of the IGF-1/PI3K/Akt pathway, Akt
Such data suggest that interventions to the IGF-signaling kinase fails to phosphorylate the FOXO proteins, which
pathways could be applied early after induction of atrophy, then promotes the transcription in the nucleus of several
but could also be effective at later stages. atrophy-related genes, particularly the ‘‘atrogenes’’
encoding the E3 ligases discussed earlier (Stitt et al. 2004).
KO models It has been shown that FOXO regulates the expression
of genes encoding MuRF1 and Atrogin-1/MAFbx ligases.
Downstream of IGF itself, when mTOR signalling is Human patients on mechanical ventilation who develop
inhibited, muscle atrophy was observed. In muscle specific severe fibre atrophy and decreased diaphragm force gen-
mTOR knockout mice, the animals showed severe erating capacity, showed decreased phosphorylation of Akt

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and FOXO1 and an increase in transcription of both MuRf1 unloaded soleus muscles but had no effect in the weight-
and MAFbx ubiquitin ligases (Levine et al. 2011). bearing muscles (Senf et al. 2010). Moreover the increase
In myoblast culture it is possible to do complex studies of nuclear FOXO3 that occurred during immobilization
to define the importance of these observations. Stitt et al. was prevented in the muscles injected with Hsp70. On the
reported that if in dexamethasone treated myotube cultures other hand Hsp70 did not alter the amount of FOXO3
that show elevated MuRF1 and MAFbx levels, IGF-1 is mRNA or protein levels (Senf et al. 2010). Also when
overexpressed, then IGF-1 blocks the dexamethasone-in- soleus muscle was injected with an Atrogin-1/MAFbx
duced atrophy (Stitt et al. 2004). This was achieved via the promoter reporter together with a FOXO3 plasmid, FOXO3
activation of the IGF-1/PI3K/Akt pathway and resulted in caused a twofold induction of the atrogin-1 promoter, while
the almost complete blockage of the MuRF1 and MAFbx when the Hsp70 plasmid was also injected, FOXO3 was
genes upregulation which was stimulated by dexametha- inhibited from inducing the expression of Atrogin-1.
sone. Furthermore it was shown that the FOXO1 and Interestingly when the same experiment was carried out
FOXO3 transcription factors were the link between the using a MuRF1 promoter-reporter, it was found that Hsp70
IGF/PI3K/Akt pathway and the ubiquitin ligases network was unable to block the FOXO3 transcriptional activity
(Sandri et al. 2004; Stitt et al. 2004). IGF-1 treatment in (Senf et al. 2010). When a mutant of FOXO3 unable to
denervated mice spared the muscle mass and markedly bind DNA was injected, together with either the MuRF1
decreased the extent of upregulation of the two atrophy reporter or the MAFbx reporter, it was shown to signifi-
genes. Moreover Stitt et al. showed that WT FOXO1, cantly activate the MurF1 but not the MAFbx promoter.
which could be phosphorylated by Akt, was found almost This indicates that FOXO3 regulates MAFbx transcription
entirely in the cytoplasm, while a mutant FOXO1 in which through a DNA-binding manner but uses a different
the Thr were replaced by Ala residues, and thus it could not mechanism for the regulation of the MuRF1 gene tran-
be phosphorylated, was found localized in the nucleus (Stitt scription through a DNA binding independent manner,
et al. 2004). Inhibition of the PI3K/Akt resulted in the perhaps needing a second transcription factor for inducing
dephosphorylation and the translocation of the WT FOXO1 MuRF1 expression (Senf et al. 2010). In support to this
into the nucleus. hypothesis, Waddelle et al. showed that in cultured myo-
FOXO1 alone was not sufficient to induce the expres- tubes the co-transfection of both FOXO1 and the Gluco-
sion of the MuRF1 and MAFbx ligases as shown by its corticoid receptor synergistically activated the MuRf1 gene
overexpression in cultured myotubes (Stitt et al. 2004). (Waddell et al. 2008). Importantly, the MuRf-1 promoter
However it was necessary to mediate the IGF-1 inhibition presents with both a glucocorticoid response element
of the dexamethasone induced MuRF1 and MAFbx (GRE) and a directly adjacent FOXO binding element
upregulation. This was concluded since in myotube cul- (Waddell et al. 2008).
tures expressing the unphosphorylatable mutant FOXO1,
IGF-1 could not inhibit the ligases overexpression, while in Relation to autophagy
myotubes expressing the WT FOXO1 the IGF-1 mediated
blockage of MuRF1 and MAFbx overexpression was The FOXO family also regulates the expression of genes
achieved (Stitt et al. 2004). involved in autophagy pathways and lysosomal degrada-
However, FOXO3 has also been shown to be both suf- tion (Mammucari et al. 2008; Zhao et al. 2008), for an
ficient and necessary to induce muscle atrophy. Sandri astute discussion see (Sandri 2013). In cultured myotubes
et al. used a WT FOXO3 and a mutant FOXO3 where all Zhao et al. showed that the FOXO3-mediated protein loss
Thr were replaced by Ala, (i.e. a constitutively active was achieved by the activation of both the ubiquitin/pro-
unphosphorylatable FOXO3) (Sandri et al. 2004). In cul- teasome pathway and lysosomal proteolysis (Zhao et al.
tured myotubes both FOXO3 forms resulted in increased 2007). The protein breakdown was estimated to be *50 %
MAFbx mRNA levels. After treatment with IGF-1, myo- proteasomal and *40 % lysosomal, but after treatment
tubes expressing WT FOXO3 sharply reduced MAFbx with constitutively active FOXO3 the overall degradation
mRNA levels while those expressing the mutant FOXO3 increased by 66 %, with 20 % proteasomal and 70 %
showed no reduction in MAFbx (Sandri et al. 2004). lysosomal degradation (Zhao et al. 2007). Moreover, fast
Heat shock protein 70 (Hsp70) appears to have an muscle fibres transfected with constitutively active FOXO3
important role in the regulation of atrophy signalling were filled with a large number of autophagic vacuoles
pathways, as injection with Hsp70 was shown to abolish (Mammucari et al. 2007) while many autophagy related
soleus muscle atrophy, in bilaterally immobilised rats (Senf genes were upregulated along with the MuRF1 and MAFbx
et al. 2008). Again in rats, either under weight-bearing genes in atrophic muscles after denervation or in myotube
conditions or under hind limb unloading, Hsp70 could cultures expressing constitutively active FOXO3. Autop-
decrease the phosphorylation of FOXO3 by at least 50 % in hagy genes (LC3b, Atg12 l, Atg4b, Beclin1, Ulk2,

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Gabarapl1 and PI3 KIII) were all induced by the expres- however FOXO activation is blocked, atrophy was pre-
sion of caFOXO3 in myotubes, but were also found ele- vented in myotubes (Sandri et al. 2004), further supporting
vated in mouse muscles after denervation-induced atrophy the role of FOXO in glucocorticoid induced muscle
and fasting (Zhao et al. 2007). Likewise, Mammucari et al., atrophy.
reported that 1 day of fasting induced a number of genes Myostatin, a member of the TGF-b family, is a negative
related to autophagy and autophagy regulation, while with regulator of muscle growth and its overexpression in male
3 days denervation a similar profile appeared (with the transgenic mice was associated with lower muscle mass
additional induction of Beclin1 and Atg12), while Akt (Reisz-Porszasz et al. 2003). It is thought to contribute to
activation prevented FOXO3 activation and subsequent the pathophysiology of muscle wasting in many patho-
autophagy (Mammucari et al. 2007). Of course, autophagy logical conditions e.g. in HIV infection, where serum
is crucially implicated in muscle health, including exercise/ myostatin protein concentrations were found to increase in
mechanical loading responses and adaptations, as by HIV- infected men with weight loss (Gonzalez-Cadavid
removing abnormal or damaged organelles it maintains the et al. 1998). Myostatin causes muscle cell atrophy by
functional status of the muscle cell, (as discussed by Sandri inhibiting Akt phosphorylation and reversing the IGF-I/Akt
2013). hypertrophy pathway (McFarlane et al. 2006), while
Therefore, FOXO3 is shown to control degradation myostatin KO mice do not show reduction on muscle mass
through either, ubiquitin–proteasome or the lysosomal-au- or fibre CSA in response to a synthetic glucocorticoid,
tophagy pathways. At the same time, the IGF-1/PI3K/Akt dexamethasone (Gilson et al. 2007).
pathway determines whether FOXO factors will be inac-
tive/phosphorylated. Thus, the downregulation of the IGF- Early disuse
1/PI3K/Akt pathway, not only inhibits protein synthesis via
mTOR pathway, but also promotes FOXO function since The endogenous expression of myostatin increases by
its dephosphorylated form will be enhanced. The above and glucocorticoids, notably mostly in myotubes expressing
other data point to the intricate interplay between the fast myosin heavy chain (MyHC) isoform (Artaza et al.
mechanisms promoting the synthesis or the degradation of 2002). Myostatin mRNA has been found to peak at day 3
intracellular muscle components. after denervation of mouse gastrocnemius, neatly accom-
panying reductions in muscle w/w and fibre CSA, while
Glucocorticoids and myostatin myostatin protein levels peaked at 7 days (Shao et al.
2007). Phospho-Smad2 protein had a similar temporal
Various disease conditions characterized by muscle atro- profile to that of myostatin mRNA also peaking at 3 days
phy have been associated with increase in circulating glu- (Shao et al. 2007). In a squirrel hibernation model, myo-
cocorticoids levels, implicating these hormones in muscle statin protein levels, in mixed hindlimb muscles, were
atrophy, specifically in fast muscle types, Palus et al. similar to pre-hibernation values however total and phos-
(2014). hpo-Smad2 values increased dramatically (Brooks et al.
The synthetic glucocorticoid dexamethasone is used to 2011).
induce muscle proteolysis in cell culture (Thompson et al.
1999) or in vivo (Hasselgren 1999). In skeletal muscle, Late disuse
glucocorticoids decrease the rate of protein synthesis and
increase the rate of protein breakdown (Lofberg et al. The denervation study by Shao et al. (Shao et al. 2007)
2002) contributing significantly to the level of muscle extended to 56 days of observation. Maximum decline of
atrophy. Glucocorticoids can cause muscle atrophy by fibre CSA was observed at day 14 which gradually reached
altering the production of growth factors that control a size *20 % of control, by day 56. After peaking at
locally the muscle mass development (Schakman et al. 3 days, myostatin mRNA levels declined to a new ‘plateau’
2008). As described above, IGF-I stimulates the develop- between days 28–56 (still significantly higher than control).
ment of muscle mass by increasing protein synthesis and While levels of myostatin protein remained throughout
myogenesis while decreasing proteolysis and apoptosis. significantly higher than control, they showed a steady
Since studies have shown that glucocorticoids inhibit the decline. Phospho-Smad2 protein continued on a similar
production of IGF-I (Gayan-Ramirez et al. 1999), then temporal profile to that of myostatin mRNA, remaining
IGF-I has been proposed to be a key molecule in the glu- higher than control at 56 days (Shao et al. 2007). In a
cocorticoid-induced muscle atrophy pathway. On the other squirrel hibernation model, myostatin protein levels during
hand, glucocorticoid treatment induces atrogin-1 and the early and late arousal phases greatly exceeded pre-
MuRF1 expression and muscle wasting in cell culture and hibernation and hibernation values with phospho-Smad2
in vivo (Sandri et al. 2004; Schakman et al. 2008). When significantly increased as well, while total Smad protein

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was back to baseline (Brooks et al. 2011). Interestingly, the Studies indicating the involvement of NF-kB in
disparate temporal patterns of Smad7, a negative regulator cachexia have been reported in cell culture and in animal
of myostatin signalling, and follistatin, a negative regulator models. In cell culture, TNF-a upregulates NF-kB,
of Myostatin (inhibiting its binding to its receptor), indi- increases myofibrillar proteolysis (Li and Reid 2000), and
cating an alternative control of myostatin signalling suppresses myosin synthesis (Guttridge et al. 2000). In
(Brooks et al. 2011), which seems to be independent of animal experiments, it has been shown that injection of
temperature (Nowell et al. 2011). Thus further work, taking NF-kB decoy oligonucleotides into cachectic mice with
advantage of the unique myostatin response in hibernation, adenocarcinoma tumours attenuates muscle wasting sig-
could help clarify the complexities of TGFb related nificantly (Kawamura et al. 1999). To decipher mecha-
signalling. nisms of cancer cachexia, Wyke and Tisdale, using a
mutant NF-kB inhibitory protein I-kBa in muscle cell
cultures, showed that the expression of a cancer cachectic
Muscle type differences
factor, proteolysis-inducing factor (PIF), was also depen-
dent on initiation of transcription by NF-kB, similar to
Myostatin plays a role in specification of muscle types,
TNF-a (Wyke and Tisdale 2005). As those authors astutely
inhibiting fast MyHC and facilitating slow MyHC
commented, the above mechanisms oppose that of gluco-
expression during myogenic differentiation (Wang et al.
corticoids, pointing to temporal variations of NF-kB
2012). It is impressive that slow muscles are spared from
bearing significance in mechanism interpretation.
atrophy even after 7 months of hibernation in ground
squirrels, with diaphragm myostatin mRNA declining by
Muscle type considerations
400 % in early hibernation (by 50 % in soleus later on),
while myostatin mRNA does not significantly change in
In the Hunter and Kandarian study (Hunter and Kandarian
faster muscles, exhibiting 25 % atrophy (Nowell et al.
2004), after 10 days of unloading the expected atrophy in
2011). Moreover, in a study with a clinical relevance to
fast fibres was attenuated by *65 % in both KO strains.
sarcopenia, antibody-directed myostatin inhibition miti-
However, NF-kB-KO mice showed less sparing of soleus
gated loss of fast muscle mass and function in ageing mice
slow fibre CSA vs fast fibre CSA, while soleus atrophy was
(Murphy et al. 2010). In that study, 14 weeks of treatment
abolished in the Bcl3-KO. Moreover, the usual slow-to fast
not only statistically reduced myostatin mRNA, but also
MyHC transition with unloading did not occur in these
mRNA levels of Smad3 and Atrogin1, with a tendency for
KOs.
reduced MuRF1 in the fast muscle studied (Murphy et al.
2010). Such findings corroborate earlier suggestions that
MAPKs and TNFa
phosphorylation of Smad proteins in myostatin signalling
may be a common step in the signal transduction pathway
Mitogen–activated protein kinases (MAPKs) include
for the TGFb superfamily members (Shao et al. 2007).
growth factor-regulated extracellular signal related kinases
1 and 2 (ERK 1/2) and the stress-activated MAPKs, c-jun
NF-kB NH2-terminal kinase (JNK) and p38 MAPK (Flach and
Bennett 2010). MAPK pathways regulate diverse processes
NF-kB represents a family of five transcription factors ranging from proliferation and differentiation to apoptosis
(p65, c-Rel, RelB, p52 and p50) expressed in skeletal (Qi and Elion 2005).
muscle (Hunter et al. 2002), which play a key role in Several MAPKs have been associated with disuse atro-
activation of immune and inflammatory responses (Zhang phy. JNK activity was significantly elevated in atrophic
et al. 2007) and mediate the effect of inflammatory soleus of rats following an extended period of immobi-
cytokines, particularly tumor necrosis factor-a (TNF-a) lization (Hilder et al. 2003). Childs et al., found that
during the process of muscle wasting and cachexia Atrogin-1/MAFbx gene is a downstream target of p38
(Peterson et al. 2011; van de Vyver and Myburgh 2012). MAPK signalling and exposure of rats to hindlimb sus-
Activation of NF-kB often occurs by the ubiquitination and pension or immobilization, resulted in elevated p38 activity
degradation of the inhibitory protein IkB (Jackman and in soleus muscle (Childs et al. 2003). This suggested that
Kandarian 2004), in response to TNF-a (Peterson et al. p38 is involved in triggering the up-regulation of MAFbx
2011). Interestingly, knocking out the genes encoding p50 in unloading-induced atrophy. Li et al., found that exposing
(Nfkb1-/-), or the NF-kB co-trans activator, Bcl-3 myotubes to TNF-a promoted the activation of the p38,
(Bcl3-/-) inhibited muscle atrophy, showing that these ERK 1/2 and JNK. The observed TNF-a induced increase
two factors are involved in disuse atrophy (Hunter and in atrogin-1/MAFbx gene expression was independent by
Kandarian 2004). ERK or JNK inhibitors but dependent on p38 signalling

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pathway, as atrogin-1/MAFbx up-regulation and the asso- production, especially when exercise-induced muscle
ciated increase in ubiquitin conjugating activity were both damage is involved, however, exercise per se induces
blunted by p38 inhibitors such as curcumin (Li et al. 2005). antioxidant mechanisms as well (Paschalis et al. 2007).
Inactivity also increases mitochondrial ROS production
(Zhao et al. 2008). When a deficiency of enzymatic and
Relation to insulin resistance
non-enzymatic antioxidants is coupled with an overpro-
duction of ROS/RNS then oxidative/nitrosative stress
Atrophy is often accompanied by insulin resistance in the
occurs. Muscle atrophy caused by immobilization (Kondo
fast muscles, e.g. in rat tibialis anterior immobilisation
et al. 1991) or unloading (Lawler et al. 2003) have both
(Hirose et al. 2000). In atrophic soleus, insulin resistance
been associated with elevated ROS levels and inhibited
correlated with JNK activity (Hilder et al. 2003). Elevated
antioxidant mechanisms. Abundant research demonstrates
JNK activity in atrophic soleus muscle may contribute to
that redox stress contributes to disuse muscle atrophy via
the increase of insulin resistance often seen in atrophic
the following cell signalling pathways: (1) activation of
muscles and not only to the reduction of muscle mass
calcium-dependent calpain system, (2) activation of
(Zhang et al. 2007). Observations in non-obese, normo-
nuclear factor (NF-kB) pathway and (3) control of mitogen
glycemic human subjects, where insulin resistance was
activated protein kinase (MAPK) signalling (for extensive
associated with increased phosphoryation of the JNK in the
reviews see (Pellegrino et al. 2011) and (Powers et al.
vastus lateralis biopsy, linked to increased intramyocellular
2011). Moreover, all of the above pathways can be
lipids and higher total body and abdominal adipose stores,
entwined with disease mechanisms, as already discussed
i.e. an overall metabolic stress state (Masharani et al. 2011)
here or elsewhere, (e.g. see Kaltsatou et al. 2015) thus, as
further corroborate the cell and animal findings.
judiciously commented by Pellegrino et al. (2011) the
‘‘mere coexistence’’ of atrophy and redox imbalance indi-
Redox stress and disuse muscle atrophy ces does not automatically attribute a causative role to
redox stress in muscle atrophy.
There are many sources of oxygen (ROS and nitrogen In the 90s it was clearly shown that TNF-a stimulates
(RNS) reactive species (Fig. 3), which can play either ROS production (Goossens et al. 1995). Moreover, ROS
beneficial (see (Finkel 2001)) or harmful roles (see Powers was shown to modulate TNF-a/NF-kB signalling, in
et al. 2012) in skeletal muscle. Mitochondria are a main mouse-derived C2C12 muscle cell lines and rat muscle
site for the production of ROS like superoxide (O2-), by primary cultures, presenting hydrogen peroxide and its
its dismutation, hydrogen peroxide (H2O2) and other derivatives as important components of the pathway via
derivatives, (Murphy 2009). Exercise can increase their promotion of I-kBa degradation (Li et al. 1998). Research

Fig. 3 The major endogenous and exogenous sources of reactive (MAPK) signalling. Reactive nitrogen species (RNS, not shown)
oxygen species (ROS). Redox stress contributes to disuse muscle appear to cause inhibition of the antioxidant defences. The combi-
atrophy via the following cell signalling pathways: (1) activation of nation of increased ROS and increased RNS can affect a double blow
calcium-dependent calpain system, (2) activation of nuclear factor on muscle redox status, by promoting protein damage/proteolysis and
(NF-kB) pathway and (3) control of mitogen activated protein kinase reducing antioxidant defences

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by that group, (Li et al. 2003) and others, showed that peroxide release from mitochondria in soleus but partially
hydrogen peroxide stimulates ubiquitin-conjugating activ- in plantaris muscle fibres (Min et al. 2011).
ity revealing that NF- kB activation by TNF-a is a redox- It is perhaps counterintuitive for a role of redox stress in
sensitive process (Li et al. 2003). atrophy, that while slow muscles present with more
RNS on the other hand appear to cause inhibition of the developed mitochondrial networks than fast muscle, fast
antioxidant defences. As reported by Lawler and Son- muscles present with more atrophy in various disease states
g,(Lawler and Song 2002) using RNS donors on diaphragm (or as in above, may not fully respond to ‘anti-ROS’
rat muscle caused a high, dose- dependent inhibition of agents). Perhaps this has to do with mitochondrial ROS
antioxidant enzymes, with catalase, the enzyme that neu- leaking which appears to be more pronounced in fast
tralizes hydrogen peroxide, appearing most affected. Of muscle fibres than slow (Anderson and Neufer 2006). Such
note was the observation of donor- dependent effect. Thus fibre type differences are not yet fully explored, especially
for example a pure NO donor (DEANO) inhibited glu- in disease states (see for a discussion of the issue, Kaltsatou
tathione peroxidase more, while the mitochondria super- et al. 2015).
oxide dismutase (Mn-SOD) was most affected by SNP than
other RNS donor compounds (Lawler and Song 2002).
From the above and previous observations it becomes Countermeasures and future research
evident that the combination of increased ROS and
increased RNS can affect a double blow on muscle redox This review focused on the mechanisms and the signalling
status, by promoting protein damage/proteolysis and pathways involved in protein loss during muscle atrophy
reducing antioxidant defenses. and highlighted mechanisms that could be targeted in order
As mentioned, the human body strives to increase its to assist with maintaining/restoring muscle function.
overall antioxidant capacity in times of need (Paschalis As described earlier, the IGF-1-Akt axis controls both
et al. 2007). Naturally antioxidants are seen as possible protein synthesis and protein degradation (Fig. 2). IGF1
therapeutic agents to protect against of correct muscle analogues might prove really useful for counteracting
atrophy (Powers 2014). Known antioxidants such as vita- muscle loss and weakness, as suggested previously (Bon-
min E have already attracted a lot of attention. Treatment aldo and Sandri 2013). However, anabolic factors alone
with vitamin E (60 mg/kg body mass, twice weekly) par- (such as growth hormone and IGF-1) cannot prevent
tially protected whole soleus muscle against atrophy muscle loss, while addition of intermittent exercise can
induced by immobilization or denervation, via downregu- attenuate the atrophic effect of bed rest (Brooks et al. 2010)
lation of calpains, caspases-3, -9, and -12, and ubiquitin and hind limb suspension (see Barton and Morris for a
ligases MuRF-1 and MAFbx. (Servais et al. 2007). Sparing review on strategies to counteract muscle atrophy (Barton
effects on muscle fibre CSA was similar for both type I and and Morris 2003)). PI3K-Akt activity has been shown to
IIa soleus fibres. While supplementation somehow protect have a critical role in cell survival and proliferation.
against thiobarbituric acid-reactive substances (TBARS), Therefore, besides the inefficacy of IGF1 as treatment
reducing overall muscle lipid peroxidation, all in all vita- without exercise, the fact that Akt can promote tumorige-
min E did not modify other oxidative stress markers nesis is a factor to consider prior to widespread use of IGF1
(Servais et al. 2007). Treatment with a vitamin E analogue for anabolic purposes (Malavaki et al. 2013).
(Trolox) blunted diaphragm atrophy in mechanically ven- MuRF1 and MAFbx/Atrogin-1 are both over-expressed
tilated rats, again via blocking the activation of both cal- in muscle atrophy resulting in increased proteasomal
pain and caspase-3, proteolytic pathways (Whidden et al. degradation of the skeletal muscle proteins (Bodine et al.
2010). Likewise, beta-carotene, a lipid soluble antioxidant 2001; Brooks and Myburgh 2014; Gomes et al. 2001).
has been also found to attenuate soleus muscle loss, in a Upregulation may be counteracted and almost negated with
denervation-induced muscle atrophy mouse model by only one bout of exercise, as found by Jones et al. (2004), a
repressing the expression of Atrogin-1 and MuRF-1 at the finding which helps to highlight the potency of mechanical
early stage of atrophy (Ogawa et al. 2013). loading in fighting muscle atrophy.
Moreover, there is continuous development of and Myostatin is a key molecule in glucocorticoid-induced
studies on the efficacy of synthetic antioxidant compounds. muscle atrophy. It is a known skeletal muscle anti-growth
In a 14-day immobilisation study in mice, both soleus and factor and causes muscle cell atrophy by inhibiting Akt
plantaris muscles were spared from atrophy with the phosphorylation and reversing the IGF-I/Akt hypertrophy
administration of a novel mitochondrial-targeted antioxi- pathway (McFarlane et al. 2006). Therefore, myostatin
dant (SS-31) via inhibition of the calpain and caspase-3 blockade has been considered as a potential treatment for
system (Min et al. 2011). Some muscle type specificity was muscle wasting syndromes and, despite early disappointing
observed, as SS-31 fully blocked the increase in hydrogen results, more recent Phase 2 trials aim to prove that it can

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provide meaningful, functional, benefit (Smith and Lin ‘anabolic’ effect of muscle contraction per se in halting
2013). It should be noted that resistance exercise and disuse induced signalling needs to be further addressed, at
essential amino acid supplementation during bed rest sup- least in the cases of non-dystrophic muscle.
pressed myostatin gene expression, while remobilization In summary, this review discussed current knowledge of
after bed rest was sufficient to increase IGF-I mRNA, mechanisms underpinning muscle atrophy and highlighted
irrespective of countermeasures taken during bed rest issues that may lead to new therapies. Skeletal muscle
(Brooks et al. 2010). weakness is associated with a large assortment of condi-
Muscle atrophy caused by inflammation, immobilization tions that involve muscle loss, ranging from disuse-induced
(Kondo et al. 1991) or unloading (Lawler et al. 2003) has atrophy to age-related atrophy, to disease-induced muscle
been associated with elevated ROS and RNS levels. While loss. It is evident that a great deal of mechanistic infor-
RNS appear to inhibit the antioxidants mechanisms mation is available and that there are common/shared steps
(Lawler and Song 2002) ROS modulate TNF-a/NF-kB at the molecular level in the complex pathways that result
signalling (Li et al. 1998) with NF-kB activation by TNF-a in skeletal muscle atrophy, whether in the context of dis-
being shown to be a redox-sensitive process (Li et al. 2003) ease or in non-disease situations. Future research could
leading to increased proteolysis through the ubiquitin– lead to identification of biological targets, in the NF-kB
proteasome pathway. The interplay of ROS/RNS and dis- and other pathways, where appropriately designed decoy
ease mechanisms is still under exploration as the coexis- oligonucleotides would intervene to prevent or diminish
tence of increased levels of reactive species with atrophy is disuse atrophy. Apart from attempting to correct defective
observed does not in itself constitute a cause -and-effect genes or negate their effects, technological advances in
(Pellegrino et al. 2011). new rational ways should aim to regulate specific gene
Excitingly, the advent of decoy oligonucleotides appears expression at specific time points, allowing for personal-
to provide a means to selectively regulate gene expression ized novel therapeutic protocols dependent on the origin of
(Ahmad et al. 2013). In animal experiments, injection of the atrophy induction, comorbidities and projected
NF-kB decoy oligonucleotides into cachectic mice with outcome.
adenocarcinoma tumours attenuated muscle wasting sig-
nificantly (Kawamura et al. 1999). Therefore, agents that Acknowledgments G.K.S, G.M, I.S., and C.K., acknowledge the
support of the European Union (European Social Fund—ESF) and
inhibit NF-kB, should be capable to inhibit muscle protein Greek national funds through the Operational Program ‘‘Educational
degradation when cancer cachexia factors are involved and Lifelong Learning’’ of the National Strategic Reference Frame-
(Wyke and Tisdale 2005). Such interventions are likely to work (NSRF)—Research Funding Program: Thales Investing in
take a long time to become viable treatments and may also knowledge society through the European Social Fund, (MuscleFun
Project-MIS 377260). K.H.M. acknowledges H2020 MCAS-RISE-
be used only in severe cases of atrophy. 645648-Muscle Stress Relief. C.M. was supported by ‘‘IKY FEL-
The benefit of existing natural (plant) or synthetic LOWSHIPS OF EXCELLENCE FOR POSTGRADUATE STUDIES
agents, such as antioxidants has not been yet fully IN GREECE-SIEMENS PROGRAM’’.
explored. As discussed earlier, treatment with Vitamin E,
could, at least partially, protect against muscle atrophy
induced by immobilization by dampening muscle proteol- References
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