Can Antioxidants Protect Against Disuse Muscle Atrophy?

Abstract

Long periods of skeletal muscle inactivity (e.g. prolonged bed rest or limb immobilization) results in a loss of muscle protein and fibre atrophy. This disuse-induced muscle atrophy is due to both a decrease in protein synthesis and increased protein breakdown. Although numerous factors contribute to the regulation of the rates of protein breakdown and synthesis in skeletal muscle, it has been established that prolonged muscle inactivity results in increased radical production in the inactive muscle fibres. Further, this increase in radical production plays an important role in the regulation of redox-sensitive signalling pathways that regulate both protein synthesis and proteolysis in skeletal muscle. Indeed, it was suggested over 20 years ago that antioxidant supplementation has the potential to protect skeletal muscles against inactivity-induced fibre atrophy. Since this original proposal, experimental evidence has implied that a few compounds with antioxidant properties are capable of delaying inactivity-induced muscle atrophy. The objective of this review is to discuss the role that radicals play in the regulation of inactivity-induced skeletal muscle atrophy and to provide an analysis of the recent literature indicating that specific antioxidants have the potential to defer disuse muscle atrophy.

Full text

REVIEW ARTICLE
Can Antioxidants Protect Against Disuse Muscle Atrophy?
Scott K. Powers
ÓThe Author(s) 2014. This article is published with open access at Springerlink.com
Abstract Long periods of skeletal muscle inactivity (e.g.
prolonged bed rest or limb immobilization) results in a loss
of muscle protein and fibre atrophy. This disuse-induced
muscle atrophy is due to both a decrease in protein syn-
thesis and increased protein breakdown. Although numer-
ous factors contribute to the regulation of the rates of
protein breakdown and synthesis in skeletal muscle, it has
been established that prolonged muscle inactivity results in
increased radical production in the inactive muscle fibres.
Further, this increase in radical production plays an
important role in the regulation of redox-sensitive signal-
ling pathways that regulate both protein synthesis and
proteolysis in skeletal muscle. Indeed, it was suggested
over 20 years ago that antioxidant supplementation has the
potential to protect skeletal muscles against inactivity-
induced fibre atrophy. Since this original proposal, exper-
imental evidence has implied that a few compounds with
antioxidant properties are capable of delaying inactivity-
induced muscle atrophy. The objective of this review is to
discuss the role that radicals play in the regulation of
inactivity-induced skeletal muscle atrophy and to provide
an analysis of the recent literature indicating that specific
antioxidants have the potential to defer disuse muscle
atrophy.
1 Introduction
Skeletal muscles are essential for both breathing and
locomotion in humans and other animals. Prolonged
periods of skeletal muscle disuse (e.g. chronic bed rest,
limb immobilization or space flight) can lead to muscle
atrophy, impaired contractile performance and overall
muscle weakness. The establishment of a countermeasure
to prevent disuse muscle atrophy requires a mechanistic
understanding of the cellular signalling pathways that
regulate both protein synthesis and protein breakdown in
muscle. In this regard, ongoing research in muscle biol-
ogy has improved our understanding of those factors that
contribute to inactivity-induced muscle atrophy, and evi-
dence indicates that disturbed redox signalling, due to
increased production of reactive oxygen species (ROS)
and decreased antioxidant capacity, is an important reg-
ulator of signalling pathways that control both proteolysis
and protein synthesis in skeletal muscle [1–8]. These
collective results are consistent with the concept that
oxidative stress plays an important regulatory role in
disuse skeletal muscle atrophy, and they raise the question
as to whether antioxidant supplementation is a potential
countermeasure to protect against inactivity-induced
muscle atrophy.
The objectives of this review are twofold: (1) to sum-
marize the current knowledge regarding the mechanistic
links between ROS and muscle atrophy resulting from
prolonged periods of contractile inactivity; and (2) to dis-
cuss the evidence indicating that antioxidant supplemen-
tation can protect skeletal muscles against disuse muscle
atrophy. The discussion begins with a summary of the
experimental models that are used to investigate disuse
muscle atrophy. This is followed by an overview of the
signalling pathways connecting ROS to decreased protein
synthesis and increased proteolysis in skeletal muscle
fibres. The review concludes with a summary of the evi-
dence supporting the concept that specific antioxidant
compounds can delay disuse muscle atrophy.
S. K. Powers (&)
Department of Applied Physiology and Kinesiology, University
of Florida, PO Box 118205, Gainesville, FL 32611, USA
e-mail: spowers@hhp.ufl.edu
123
Sports Med (2014) 44 (Suppl 2):S155–S165
DOI 10.1007/s40279-014-0255-x

2 Disuse Muscle Atrophy: Experimental Models
Because of both ethical considerations and the com-
plexities associated with studying the mechanisms
responsible for disuse muscle atrophy in humans, animal
models are commonly used to study the cellular mecha-
nisms responsible for muscle atrophy. In this regard,
several animal models are commonly used to simulate
the various types of human disuse muscle atrophy. Spe-
cifically, several conditions can result in disuse muscle
atrophy in humans: (1) prolonged mechanical ventilation,
resulting in inactivity of inspiratory muscles; (2) broken
bone that requires limb immobilization (i.e. casting), and
the resulting muscle inactivity; (3) space flight, resulting
in unloading of skeletal muscles; (4) spinal cord injury;
and (5) prolonged bed rest. For each of these human
conditions that result in muscle atrophy, a corresponding
rodent model exists (Fig. 1). For example, a tail sus-
pension technique to unload the hindlimb muscles of
rodents is frequently utilized to replicate the human
muscle atrophy that occurs during space flight or pro-
longed bed rest. Also, experimental manipulations such
as denervation or spinal cord isolation have been
employed to investigate the impact of prolonged muscle
inactivity on rodent skeletal muscle structure and
function.
In addition to the numerous studies performed on limb
skeletal muscles, a growing number of reports have
examined the impact of reduced contractile activity on
respiratory muscle fibre size and function in both humans
and animals. A common and clinically relevant
experimental model to study inactivity-induced inspira-
tory muscle atrophy is mechanical ventilation. Specifi-
cally, full-support mechanical ventilation is a clinically
important intervention, which can be life-saving in
patients with respiratory failure. However, during full-
support mechanical ventilation, the ventilator delivers all
of the breaths and the patient’s respiratory muscles are
inactive. Both human and animal studies have demon-
strated that prolonged mechanical ventilation results in
extremely rapid inspiratory muscle (i.e. diaphragm) atro-
phy. Indeed, as few as 18 h of mechanical ventilation can
result in significant diaphragmatic atrophy (e.g. [15 %
reduction in fibre cross-sectional area) in both humans
and rodents [9,10]. This rate of disuse muscle atrophy is
unique, as a comparable level of disuse muscle atrophy in
limb skeletal muscles would require at least 96 h of
muscle inactivity to achieve [11,12].
3 Disuse Muscle Atrophy: the Big Picture
The control of skeletal muscle fibre size is determined by
balancing the rates of protein synthesis and degradation
(Fig. 2). Animal studies have established that inactivity-
induced skeletal muscle atrophy occurs because of both
increased proteolysis and decreased muscle protein syn-
thesis [11,13]. For example, muscle protein synthesis
declines within 6 h following the initiation of muscle
inactivity [11,13]. Further, animal studies have revealed
that disuse muscle atrophy is also due to large increases
in muscle protein breakdown [10,11].
Fig. 1 Illustration of several
human conditions that promote
inactivity-induced skeletal
muscle atrophy, along with the
corresponding animal model
that is commonly used to study
each condition
S156 S. K. Powers
123

4 How Do ROS Promote Disuse Muscle Atrophy?
The notion that increased ROS production and disturbances
in redox signalling play a significant role in the promotion
of disuse muscle atrophy was proposed over 20 years ago
[14]. Nonetheless, this view did not receive substantial
scientific investigation until recent years. In the following
sections, the cellular locations responsible for ROS pro-
duction in inactive skeletal muscle are discussed, and
evidence supporting the position that inactivity-induced
oxidative stress plays a key role in the regulation of both
muscle protein synthesis and degradation is presented.
4.1 Prolonged Skeletal Muscle Inactivity Increases
ROS Production
The first evidence that contracting skeletal muscles pro-
duce ROS was reported over 30 years ago by Kelvin
Davies and colleagues [15]. Since that seminal report,
many studies have confirmed that resting skeletal muscles
produce low levels of ROS and that the initiation of con-
tractile activity results in marked increases in ROS pro-
duction in the active fibres [16–20]. Paradoxically, it was
reported in 1991 that prolonged skeletal muscle inactivity
due to immobilization resulted in chronic increases in ROS
production and oxidative damage in the inactive muscle
fibres [14]. This ground-breaking discovery has been con-
firmed by many studies over two decades (reviewed in
references [3–6]).
Although a detailed understanding of why prolonged
inactivity results in increased ROS production in inactive
skeletal muscle remains elusive, a growing number of
studies have provided insight into this interesting
biological phenomenon. Although both xanthine oxidase
and nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase contribute to inactivity-induced ROS
production in skeletal muscle [21,22], mitochondria
appear to be the dominant site of ROS production in
inactive skeletal muscles [23–26]. For example, mito-
chondria isolated from diaphragm muscle of mechanically
ventilated animals released *40 % more ROS during
state 4 respiration than mitochondria obtained from the
diaphragm of control animals [23]. Further, treatment of
animals with a mitochondrial-targeted antioxidant pre-
vented inactivity-induced oxidative stress in the diaphragm
of animals exposed to prolonged mechanical ventilation
[26]. Similar results have been reported in immobilized
limb muscles [24,25]. The mechanisms responsible for this
inactivity-induced increase in mitochondrial ROS produc-
tion remain unknown (see reference [5] for a review).
4.2 Oxidative Stress Can Depress Protein Synthesis
Emerging evidence indicates that exposure of cells to high
levels of ROS can depress protein synthesis. This topic has
been addressed in detail in a previous review [5]; therefore,
only a brief synopsis of these findings is presented here.
Briefly, protein synthesis in cells is accomplished by a
highly structured scheme of signalling pathways, which
culminate in the translation of messenger RNA (mRNA)
into a specific protein. The rate of protein synthesis is
primarily controlled by the efficiency of translation, which
is regulated at the level of initiation [27]. Mounting evi-
dence indicates that oxidants depress protein synthesis by
hindering mRNA translation at the level of initiation
(reviewed in reference [4]). For example, exposure of
Fig. 2 Conservation of skeletal
muscle mass depends on the
balance between the rates of
protein synthesis and
degradation. An increase in the
rate of protein synthesis relative
to the rate of protein breakdown
results in muscle hypertrophy.
Conversely, an increase in the
rate of protein breakdown
relative to the rate of protein
synthesis results in a net loss of
muscle protein, and fibre
atrophy occurs
Antioxidants and Disuse Muscle Atrophy S157
123

cardiac myocytes to oxidative stress [i.e. exposure to
hydrogen peroxide (H
2
O
2
)] inhibits global protein synthe-
sis by *90 % [28]. It follows that the decrease in protein
synthesis that occurs in skeletal muscle exposed to pro-
longed skeletal muscle inactivity could be mechanistically
linked to increased production of ROS within the inactive
muscle fibres.
4.3 Oxidative Stress Increases Proteolysis in Skeletal
Muscle
It has been postulated that increased skeletal muscle
inactivity-induced increases in ROS production and the
resulting oxidative stress accelerate muscle protein break-
down in three different ways. First, oxidative stress pro-
motes the expression of proteins involved in at least three
proteolytic systems, including autophagy, calpain and the
ubiquitin–proteasome system of proteolysis. Second,
inactivity-induced oxidative stress in skeletal muscle
results in the activation of two important proteases, calpain
and caspase-3. Finally, increased ROS production in
muscle fibres can also promote proteolysis by oxidative
modification of myofibrillar proteins, which enhances their
susceptibility to proteolytic processing. A brief summary of
these connections between oxidative stress and proteolysis
is presented in the following sections.
4.3.1 Oxidative Stress Increases Synthesis of Proteolytic
Proteins
The major proteolytic systems found in skeletal muscle can
be categorized into four groups: (1) autophagy (i.e. lyso-
somal proteases); (2) the ubiquitin–proteasome system;
(3) calpains; and (4) caspase-3. Growing evidence reveals
that cellular oxidative stress can increase the expression of
key proteins involved in autophagy and the ubiquitin–
proteasome system, and can increase expression of both
calpain 1 and calpain 2. Specific details of these processes
are highlighted in the following sections.
ROS Increase Expression of Key Autophagy Proteins
Autophagy is a highly regulated proteolytic pathway for
the degradation of non-myofibril cytosolic proteins and
organelles [29]. During autophagy, cytosolic components
(i.e. proteins and organelles) are sequestered into vesicles
called autophagosomes. After formation, these auto-
phagosomes fuse to lysosomes and the cytosolic constitu-
ents are degraded by lysosomal proteases (i.e. cathepsins)
[30]. Although it has been established that several lyso-
somal proteases (i.e. cathepsins B, D and L) are activated
in skeletal muscle undergoing disuse atrophy [31], the role
that the autophagic proteolytic system plays in muscle
atrophy has received limited research attention. Nonethe-
less, emerging studies have revealed that accelerated
autophagy contributes to skeletal muscle atrophy in
response to both fasting and denervation [32,33]. Fur-
thermore, a recent study has demonstrated that auto-
phagosomes are formed in diaphragm muscle during
prolonged mechanical ventilation, suggesting that autoph-
agy contributes to ventilator-induced protein breakdown in
the diaphragm [34].
Emerging evidence indicates that oxidative stress
increases the expression of autophagy genes in skeletal
muscles. Indeed, a recent study has revealed that increased
cellular ROS production promotes the expression of the
autophagy-related beclin 1 and cathepsin L genes in cul-
tured cells [35]. Importantly, another report has indicated
that inactivity-induced oxidative stress also promotes the
expression of autophagy-related proteins in human skeletal
muscle [34]. These findings have been confirmed in rodent
locomotor muscles exposed to prolonged immobilization
[24]. Specifically, prevention of inactivity-induced increa-
ses in mitochondrial ROS emission in hindlimb muscles
prevents the expression of cathepsin L [24]. Together,
these results suggest that oxidative stress increases gene
expression of selected autophagy-related genes, which
have the potential to increase the rate of autophagy-medi-
ated protein breakdown in cells.
Oxidative Stress Increases Expression of Proteins
Required for the Ubiquitin–Proteasome System The ubiq-
uitin–proteasome system comprises the total proteasome
complex (26S), which includes a core proteasome subunit
(20S) combined with a regulatory complex (19S) attached
at the end of the 20S proteasome core [36,37]. The 26S
proteasome degrades ubiquitinated proteins only. Hence,
the 26S proteasome degradation pathway is active when
ubiquitin binds to protein substrates and labels these mol-
ecules for breakdown. The binding of ubiquitin to protein
substrates is a three-step process, which requires the par-
ticipation of three families of ubiquitin-activating enzymes
[4]. In this regard, evidence indicates that the ubiquitin-
conjugating enzyme E2
14k
is an important regulator of
ubiquitin–protein conjugation in skeletal muscle [1]. Fur-
ther, several skeletal muscle-specific ubiquitin E3 ligases
(e.g. atrogin-1 and muscle ring finger-1) exist, and these
proteins play important roles in skeletal muscle atrophy
[38,39].
Abundant evidence confirms that oxidative stress pro-
motes increased gene expression of proteins involved in the
ubiquitin–proteasome system. For instance, in vitro
experiments have revealed that exposure of C2C12 myo-
tubes to H
2
O
2
increases the expression of specific ubiqui-
tin-activating enzymes that contribute to muscle protein
breakdown, including E2
14k
, atrogin-1 and muscle ring
finger-1 [1,40]. Similarly, in vivo experiments have
revealed that oxidative stress augments the expression of
atrogin-1 and muscle ring finger-1 in rodent skeletal
S158 S. K. Powers
123

muscles [24,26]. Together, these results verify that ROS-
induced oxidative stress promotes the expression of key
components of the ubiquitin–proteasome system of prote-
olysis in skeletal muscle.
Oxidative Stress Increases Calpain Expression Calpains
are Ca
2?
-dependent cysteine proteases and are located in
all mammalian cells [41]. While several calpain isoforms
exist, the two best characterized calpains located in skeletal
muscle are calpains 1 and 2 [41]. Active calpains promote
the release of sarcomeric proteins by cleaving cytoskeletal
proteins (e.g. titin and nebulin) that anchor contractile
elements [41–43]. Further, calpain can break down selected
kinases and phosphatases, and can also degrade oxidized
contractile proteins, such as actin and myosin [41,44].
Several reports have indicated that oxidative stress
increases the expression of calpains in both C2C12 myo-
tubes and human myoblasts. For example, exposure of
C2C12 myotubes to H
2
O
2
increases calpain 1 mRNA
levels [40]. Further, exposure of human myoblasts to H
2
O
2
promotes the expression of both calpain 1 and calpain 2
[45]. Together, these investigations suggest that oxidative
stress increases calpain expression in cultured muscle cells.
At present, it is unknown if oxidative stress can increase
the expression of calpain in skeletal muscle fibres in vivo.
4.3.2 Oxidative Stress Increases Protease Activation
As discussed in the previous sections, oxidative stress
increases the expression of several important proteolytic
proteins. This section presents robust evidence demonstrating
that oxidative stress can promote the activation of selected
proteases (e.g. calpain and caspase-3) in skeletal muscles.
Elevated Cellular ROS Production Activates Calpain
Numerous studies have concluded that oxidative stress
increases calpain activity in muscle cells. For example,
treatment of C2C12 myotubes with H
2
O
2
activates cal-
pain 1 and stimulates myotube atrophy [40]. Similarly,
exposure of human myoblasts to H
2
O
2
increases the
activities of both calpain 1 and calpain 2 [45]. Moreover,
prevention of oxidative stress via antioxidants can prevent
calpain activation in inactive diaphragm muscle in vivo
[26,46]. Similarly, mitochondrial-targeted antioxidants can
prevent calpain activation in immobilized limb muscles
[24]. Collectively, these studies have confirmed that
increased production of ROS in skeletal muscle promotes
the activation of calpain.
The mechanism(s) responsible for ROS-mediated cal-
pain activation appear(s) to be linked to oxidative stress-
induced disturbances in calcium homeostasis. The two key
factors that regulate calpain activity in cells are cytosolic
calcium levels and the concentration of the endogenous
calpain inhibitor, calpastatin [41]. Specifically, calpain can
be activated by a sustained elevation in cytosolic free
calcium and/or a decrease in cytosolic levels of calpastatin
[41]. During prolonged periods of muscle inactivity, it is
established that muscle inactivity is accompanied by ele-
vated cytosolic calcium levels and increased calpain acti-
vation [47]. Although the mechanism responsible for this
disuse-induced increase in cellular calcium remains under
investigation, it is possible that increased ROS production
plays a key role in this event [48]. A potential connection
between oxidative stress and increased cytosolic calcium is
ROS-driven formation of reactive aldehydes (i.e.
4-hydroxy-2,3-trans-nonenal), which can impede plasma
membrane Ca
?2
ATPase activity [49]. Logically, a decline
in membrane Ca
?2
ATPase activity would hinder Ca
?2
removal from the cell, resulting in increased cytosolic
Ca
?2
. It is also possible that oxidation of the ryanodine
receptor can increase Ca
?2
leakage from the sarcoplasmic
reticulum, resulting in increased cytosolic Ca
?2
levels [50].
Nonetheless, it is not clear which of these mechanisms is
responsible for inactivity-mediated calcium overload
within skeletal muscle, and this subject remains an active
area of research.
Oxidative Stress Promotes Caspase-3 Activation
Research has revealed that the activation of caspase-3
contributes to skeletal muscle protein degradation and fibre
atrophy [51–53]. Specifically, active caspase-3 results in
the degradation of actomyosin complexes, and inhibition of
caspase-3 activity suppresses the overall rate of proteolysis
in diabetes-mediated cachexia and disuse-induced muscle
atrophy [51–53].
Numerous studies have confirmed that oxidative stress
activates caspase-3 in skeletal muscle. For instance,
exposing C2C12 myotubes to H
2
O
2
activates caspase-3
[54]. Further, several studies have confirmed that preven-
tion of inactivity-induced oxidative stress in skeletal mus-
cles prevents caspase-3 activation [24,26,46].
Control of caspase-3 activity in cells is complex and
involves numerous signalling pathways. Inactivity-induced
caspase-3 activation in skeletal muscle can occur by acti-
vation of caspase-12 via a calcium release pathway and/or
activation of caspase-9 via a mitochondrial signalling
pathway [7]. A potential interaction between these caspase-
3 activation pathways is that both signalling pathways can
be activated by ROS [3,55]. Finally, note that caspase-3
can also be activated by calpain activation via a cross-talk
mechanism between these two proteases [51,56].
Regardless of which pathway is responsible for inactivity-
induced caspase-3 activation in skeletal muscle, it is
apparent that ROS can promote caspase-3 activation.
4.3.3 Protein Oxidation Accelerates Proteolysis
Another link between oxidative stress and increased pro-
tein turnover is that oxidation of skeletal muscle proteins
Antioxidants and Disuse Muscle Atrophy S159
123

increases their vulnerability to proteolytic breakdown.
Indeed, Davies and Goldberg were the first to demonstrate
that ROS accelerate proteolysis [57]. This early work has
been confirmed, and it is now clear that oxidized proteins
are swiftly degraded by several proteases, including the
ubiquitin–proteasome system [36,37,44]. Further, evi-
dence has revealed that oxidation increases the suscepti-
bility of skeletal muscle myofibrillar proteins to
degradation by both calpains and caspase-3. Indeed, oxi-
dation of sarcomeric proteins (e.g. myosin heavy chain, a-
actinin, actin and troponin I) increases their breakdown by
both calpain and caspase-3 in a dose-dependent manner
[44].
The connection between high cellular levels of ROS and
accelerated protein breakdown is due, in part, to the fact
that oxidation of muscle proteins results in unfolding of the
affected proteins, resulting in enhanced susceptibility to
proteolysis [57]—that is, oxidative modification of a pro-
tein results in a change in the molecular structure such that
the formerly protected peptide bonds are now exposed to
enzymatic breakdown [15,44].
4.4 Summary: How Do ROS Promote Muscle
Atrophy?
Prolonged muscle inactivity results in increased mito-
chondrial ROS production and, consequently, disturbed
redox signalling (i.e. oxidative stress) in the inactive
skeletal muscle fibres. This inactivity-induced oxidative
stress results in increased proteolysis in muscle fibres,
which is due to increased expression of key proteolytic
proteins, augmented protease activation and increased
oxidation of skeletal muscle proteins. Further, oxidant
stress has the potential to depress muscle protein synthesis.
Collectively, the ROS-induced increase in proteolysis and
decrease in protein synthesis results in a net loss of muscle
protein (Fig. 3).
5 Can Treatment with Antioxidants Prevent Disuse
Muscle Atrophy?
The previous sections discussed the factors that explain the
biological connection between increased ROS production
and inactivity-induced skeletal muscle atrophy. A frequent
experimental approach to determine cause and effect
between increased ROS production and disuse muscle
atrophy is the treatment of animals with antioxidants to
prevent inactivity-induced oxidative stress in the inactive
muscles. Using this approach, evidence exists both for and
against the notion that antioxidants can prevent disuse
muscle atrophy. Most of these studies have used an indi-
vidual antioxidant. In the following sections, the efficacy of
specific antioxidants to protect against disuse muscle
atrophy is examined. The discussion focuses on those
antioxidants that have received significant experimental
attention.
5.1 Vitamin E/Vitamin E Analogues and Disuse
Muscle Atrophy
Both vitamin E and vitamin E analogues have been widely
investigated as antioxidant interventions to protect against
disuse muscle atrophy [12,14,46,58–60]. Vitamin E is
one of the most widely distributed antioxidants in nature
and is the major antioxidant found in cell membranes. The
generic term ‘vitamin E’ refers to eight structural isomers
of tocopherols and tocotrienols [61]. Among these mole-
cules, a-tocopherol (the all-rac form) is the best known and
possesses the highest antioxidant capacity [61]. In addition
to its antioxidant activity, vitamin E is known to promote
gene expression of selected muscle proteins [61].
In regard to vitamin E and muscle atrophy, Kondo et al.
[14] provided the first investigation demonstrating that
administration of vitamin E to rats protected against
immobilization-induced hindlimb muscle atrophy. Since
that early study, several other reports have concluded that
vitamin E can completely or partially protect rodents
against muscle atrophy induced by hindlimb unloading,
immobilization or denervation [59,60,62,63].
Although studies have reported that vitamin E can blunt
disuse muscle atrophy, the mechanisms behind this pro-
tection remain unclear. However, a recent study has sug-
gested that the protective effect of vitamin E against disuse
muscle atrophy could be due to modulation of muscle
proteolysis-related genes, rather than its antioxidant func-
tion [60]. For example, treatment of animals with high
levels of vitamin E (60 mg/kg body mass, twice weekly)
increased the expression of heat shock protein 72 and
decreased the expression of several proteases, including
calpain and caspase-3, in inactive skeletal muscles [60]. In
theory, both of these changes in gene expression could
provide protection against disuse muscle atrophy [56,64–
66]. Therefore, it is possible that although vitamin E pro-
tects against disuse muscle atrophy, this protection is
achieved by alterations in gene expression and not neces-
sarily the antioxidant function of vitamin E [60]. Clearly,
additional research is required to determine the precise
mechanism(s) responsible for vitamin E-mediated protec-
tion against disuse muscle atrophy.
Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-car-
boxylic acid) is a cell-permeable and water-soluble ana-
logue of vitamin E [67]. Like vitamin E, Trolox has
antioxidant properties that are derived from direct scav-
enging of H
2
O
2
and other ROS [67]. Several investigations
have demonstrated that treatment of animals with Trolox
S160 S. K. Powers
123

(i.e. an intravenous infusion of 4 mg/kg/h) protects the
diaphragm against ventilator-induced diaphragmatic atro-
phy [2,12,46,58]. The mechanism responsible for this
protection appears to be, at least in part, related to the fact
that oxidative stress is required to activate both calpain and
caspase-3 in the diaphragm during prolonged mechanical
ventilation [26,46].
In contrast to the findings that Trolox protects against
ventilator-induced diaphragmatic atrophy, two studies have
concluded that Trolox supplementation does not protect
against inactivity-induced limb muscle wasting. Specifi-
cally, these studies demonstrated that treatment of mice
with Trolox did not protect against hindlimb unloading-
induced muscle atrophy [68,69]. The reason(s) for these
divergent findings across the Trolox studies are unclear but
could be related to the duration of muscle inactivity and/or
the dosage of Trolox that was used in the experiments.
5.2 N-Acetylcysteine and Disuse Muscle Atrophy
N-Acetylcysteine is a small molecule comprising cysteine
with an acetyl group attached to the nitrogen atom. N-
acetylcysteine is widely used clinically as a mucolytic
agent and as a treatment for paracetamol (acetaminophen)
overdose. Further, N-acetylcysteine is also sold as a
nutritional supplement, is a direct scavenger of ROS [70]
and can provide cysteine for glutathione synthesis (i.e.
glutathione is an important non-enzymatic antioxidant)
[71]. To date, only two studies have investigated the ability
of N-acetylcysteine to protect against disuse muscle
atrophy. The first study concluded that although treatment
of animals with N-acetylcysteine in the diet prevented the
increase in nuclear factor (NF)-kappaB activity induced by
hindlimb suspension, N-acetylcysteine treatment did not
protect against inactivity-induced muscle atrophy [72]. In
contrast, another investigation demonstrated that treatment
of animals with N-acetylcysteine (150 mg/kg intrave-
nously) prevented mechanical ventilator-induced oxidative
stress, averted the activation of both calpain and caspase-3,
and protected the diaphragm against disuse-induced dia-
phragm fibre atrophy [70]. It is difficult to determine if
these divergent results are due to a disparity between
studies in the plasma levels of N-acetylcysteine resulting
from the different routes of drug delivery (i.e. dietary
intake versus intravenous infusion), and additional research
is required to determine whether N-acetylcysteine has the
potential to be an effective agent against disuse-induced
muscle atrophy.
5.3 Mitochondrial-Targeted Antioxidants and Disuse
Muscle Atrophy
As discussed earlier, mitochondria appear to be the domi-
nant site of ROS production in skeletal muscles during
prolonged periods of inactivity [23–26]. Therefore, in
theory, a mitochondrial-targeted antioxidant could prevent
inactivity-induced oxidative stress and protect muscles
against disuse atrophy. In this regard, three reports have
concluded that a mitochondrial-targeted antioxidant can
protect both limb and respiratory muscles against
Fig. 3 Steps leading from
oxidative stress to muscle fibre
atrophy. Inactivity-induced
oxidative stress can promote
muscle protein breakdown in
three major ways: (1) oxidative
stress increases gene expression
of key proteolytic proteins;
(2) cellular oxidative stress can
activate selected proteases
(i.e. calpain and caspase-3); and
(3) oxidants can oxidize
myofibrillar proteins and
enhance their susceptibility to
proteolytic processing. Further,
oxidative stress can depress
muscle protein synthesis.
Collectively, this increased
proteolysis and decreased
muscle protein synthesis result
in a net loss of muscle protein
and, consequently, fibre
atrophy. ROS reactive oxygen
species
Antioxidants and Disuse Muscle Atrophy S161
123

inactivity-induced atrophy. Specifically, SS-31 (D-Arg-
2060dimethylTyr-Lys-Phe-NH
2
) is a synthetic aromatic
cationic tetrapeptide that selectively targets and concen-
trates in the inner mitochondrial membrane [73]. Impor-
tantly, the mitochondrial targeting of SS-31 provides
selective scavenging of mitochondrial ROS. Numerous
studies in isolated mitochondria, cultured cells and animal
models have shown that SS-31 can selectively scavenge
mitochondrial ROS and protect mitochondrial function
during periods of increased ROS production [25,26,74,
75]. The first report documenting the ability of a mito-
chondrial-targeted antioxidant to protect against disuse
muscle atrophy revealed that treatment of animals with SS-
31 protects the rat diaphragm against the disuse muscle
atrophy that occurs during prolonged mechanical ventila-
tion [26]. Subsequently, two additional investigations have
concluded that treatment of both rats and mice with SS-31
protects against immobilization-induced atrophy in hind-
limb muscles [24,25]. The mechanism of protection
against atrophy by this specific mitochondrial-targeted
antioxidant appears to be protection against inactivity-
induced oxidative stress and prevention of protease acti-
vation (e.g. caspase-3 and calpain) in the muscle [24,26].
5.4 Miscellaneous Antioxidants/Antioxidant Cocktails
and Disuse Muscle Atrophy
While vitamin E, Trolox and mitochondrial-targeted anti-
oxidants have received significant investigative attention, a
limited number of studies have examined the impact of
other antioxidants on disuse muscle atrophy. For example,
only two studies have investigated the effect of curcumin
(an antioxidant found in the spice turmeric) on disuse
muscle atrophy. Unfortunately, these studies arrived at
divergent conclusions regarding the ability of curcumin to
protect against disuse muscle atrophy. Specifically, one
report concluded that curcumin (a 1 % dose in the diet) did
not protect against hindlimb unloading-induced muscle
atrophy [72]. In contrast, the second report concluded that
treatment with curcumin (600 mg/kg given intraperitone-
ally three times daily) protected the diaphragm against
ventilator-induced muscle wasting [76]. Unfortunately, it is
unclear if these divergent findings are due to the varying
routes of curcumin administration (i.e. dietary intake ver-
sus intraperitoneal injection) resulting in markedly differ-
ent levels of circulating curcumin between the two
investigations. Clearly, additional experiments are required
to resolve whether curcumin has the potential to protect
against disuse muscle atrophy.
Further, a new report has suggested that treatment of
mice with beta-carotene (a lipid soluble antioxidant) can
protect against denervation-induced muscle atrophy [77].
This is an interesting new finding, and additional studies
are warranted to determine if beta-carotene is protective
against other forms of disuse muscle atrophy.
Finally, one study has investigated the effects of a
complex antioxidant cocktail on protection against hind-
limb unloading-induced muscle atrophy in rodents [78].
This experiment treated animals with an antioxidant
cocktail (including vitamin E, vitamin C and beta-caro-
tene) contained in the diet. Although this form of antioxi-
dant supplementation resulted in increased antioxidant
capacity within the hindlimb muscles of the treated ani-
mals, it did not protect against hindlimb unloading-induced
muscle atrophy [78]. These results clearly illustrate the
concept that not all antioxidant treatments are successful in
preventing disuse muscle atrophy.
6 Summary and Conclusions
It is well known that disuse skeletal muscle atrophy
occurs during prolonged bed rest, during limb immobili-
zation and as a result of prolonged mechanical ventilation.
Several lines of evidence couple increased production of
ROS to disuse muscle atrophy via ROS-mediated
increases in proteolysis. Further, it is also possible that
oxidative stress can depress muscle protein synthesis,
which is another contributory factor to the loss of muscle
protein and fibre atrophy that occurs during prolonged
Fig. 4 Illustration of the potential role that antioxidants can play in
protection against disuse muscle atrophy. Specifically, prolonged
skeletal muscle inactivity leads to increased mitochondrial reactive
oxygen species (ROS) production and oxidative stress in the inactive
muscle fibres. This increased ROS production and oxidative stress can
promote decreased protein synthesis and promote proteolysis in the
muscle, leading to skeletal muscle atrophy. In theory, treatment with
selected antioxidants can block disuse-induced oxidative stress and
protect muscle fibres against disuse muscle atrophy
S162 S. K. Powers
123

inactivity. Developing an effective countermeasure to
protect against inactivity-induced muscle atrophy is
important. In this regard, prevention of inactivity-induced
muscle atrophy requires an intervention that can maintain
protein synthesis and/or decrease proteolysis in muscles
exposed to prolonged periods of inactivity. Therefore,
since antioxidants can prevent inactivity-induced oxida-
tive stress in skeletal muscles, treatment of animals with
antioxidants could potentially maintain protein synthesis
and prevent accelerated proteolysis (Fig. 4). Indeed, sev-
eral studies have suggested that selected antioxidants (e.g.
vitamin E, Trolox and mitochondrial-targeted antioxi-
dants) have the potential to decrease inactivity-induced
muscle atrophy of both limb and respiratory muscles.
Nonetheless, at present, the use of antioxidants as a
therapeutic intervention to protect athletes and patient
populations against disuse muscle atrophy is not widely
accepted. Therefore, additional research is required to
completely establish that antioxidant treatments are both
safe and effective in protecting against inactivity-induced
muscle atrophy.
Acknowledgments This article was published in a supplement
supported by the Gatorade Sports Science Institute (GSSI), a division
of PepsiCo, Inc. The supplement was guest edited by Lawrence L.
Spriet, who attended a meeting of the GSSI Expert Panel in February
2013 and received honoraria from the GSSI for his meeting partici-
pation and the writing of his manuscript. He received no honoraria for
guest editing the supplement. L.L.S. selected peer reviewers for each
paper and managed the process. Scott K. Powers, PhD, also attended
the meeting of the GSSI Expert Panel in February 2013 and received
honoraria from the GSSI for his meeting participation and the writing
of this manuscript. The views expressed in this manuscript are those
of the author and do not necessarily reflect the position or policy of
PepsiCo, Inc. The author’s work in this research area has been sup-
ported by National Heart, and Lung Institute grants R01 HL-072789,
R01HL-087839, R01AR064189 and R21AR063805 awarded to Scott
K. Powers.
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
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