J Appl Physiol 102: 2389 –2397, 2007;

doi:10.1152/japplphysiol.01202.2006. Invited Review

HIGHLIGHTED TOPIC Free Radical Biology in Skeletal Muscle

Oxidative stress and disuse muscle atrophy

Scott K. Powers, Andreas N. Kavazis, and Joseph M. McClung
Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, Florida

Powers SK, Kavazis AN, McClung JM. Oxidative stress and disuse muscle atrophy.
J Appl Physiol 102: 2389–2397, 2007; doi:10.1152/japplphysiol.01202.2006.—Skeletal
muscle inactivity is associated with a loss of muscle protein and reduced force-
generating capacity. This disuse-induced muscle atrophy results from both increased
proteolysis and decreased protein synthesis. Investigations of the cell signaling
pathways that regulate disuse muscle atrophy have increased our understanding of
this complex process. Emerging evidence implicates oxidative stress as a key
regulator of cell signaling pathways, leading to increased proteolysis and muscle
atrophy during periods of prolonged disuse. This review will discuss the role of
reactive oxygen species in the regulation of inactivity-induced skeletal muscle
atrophy. The specific objectives of this article are to provide an overview of muscle
proteases, outline intracellular sources of reactive oxygen species, and summarize
the evidence that connects oxidative stress to signaling pathways contributing to
disuse muscle atrophy. Moreover, this review will also discuss the specific role that
oxidative stress plays in signaling pathways responsible for muscle proteolysis and
myonuclear apoptosis and highlight gaps in our knowledge of disuse muscle
atrophy. By presenting unresolved issues and suggesting topics for future research,
it is hoped that this review will serve as a stimulus for the expansion of knowledge
in this exciting field.
oxidants; proteasome; calpain; caspase-3; reactive oxygen species

EXTENDED PERIODS of skeletal muscle inactivity (e.g., limb im-
mobilization, chronic bed rest, or spaceflight) result in a loss of
muscle mass and strength (1, 11, 83). Knowledge of the cell
signaling pathways that regulate disuse muscle atrophy is
important in developing an anti-catabolic strategy to prevent or
retard protein loss and maintain physiological function (12).
Ongoing research in cell signaling has led to an improved
understanding of those factors that contribute to muscle atro-
phy during both disuse and pathologies that promote muscle
wasting. In particular, a large volume of research indicates that
oxidative stress is an important contributor to numerous cellu-
lar signaling pathways that modulate muscle atrophy during
prolonged inactivity. For example, exposure of skeletal muscle
myotubes to oxidative stress (i.e., hydrogen peroxide) in-
creases the expression of important components of the protea-
some proteolytic system (58). Moreover, other signaling path-
ways exist to connect oxidative stress with cellular processes
leading to a loss of cellular protein (44).
The objective of this review is to provide a synopsis of our
present knowledge regarding the link between reactive oxygen
species (ROS) and proteolytic components of disuse skeletal
muscle atrophy. The first segment of this article will introduce
experimental models to study muscle atrophy. We will then
present an overview of the biochemical events leading to the
loss of myonuclei in atrophying muscle followed by a discus-
sion of the principal proteolytic pathways that contribute to
Address for reprint requests and other correspondence: S. K. Powers, Dept.
of Applied Physiology and Kinesiology, PO Box 118205, Univ. of Florida,
Gainesville, FL 32611 (e-mail: spowers@hhp.ufl.edu).
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disuse muscle atrophy. Next, we will outline the major path-
ways involved in skeletal muscle radical production followed
by a discussion of the signaling pathways linking oxidative
stress to increased proteolysis and loss of myonuclei. The final
segment of this treatise will identify voids in our knowledge
about oxidative stress and disuse muscle atrophy in hopes of
stimulating future research in this field.
EXPERIMENTAL MODELS TO INVESTIGATE DISUSE
MUSCLE ATROPHY
Skeletal muscle atrophy can occur due to pathology (e.g.,
cancer, sepsis, or diabetes) and in the absence of disease during
extended durations of inactivity (33, 39). For example, long
periods of bed rest, limb immobilization, spaceflight, or un-
loading the diaphragm via mechanical ventilation is associated
with skeletal muscle atrophy in humans and other animals. Due
to the complexities involved in investigating the mechanisms
responsible for disuse muscle atrophy in humans, numerous
experimental animal models have evolved to simulate unload-
ing conditions that lead to disuse muscle atrophy (Fig. 1). For
example, animal models of limb immobilization (i.e., casting)
have been used to investigate the impact of muscle inactivity
on muscle size and function (11). Moreover, rodent animal
models using a tail suspension technique to unload the hind-
limb locomotor muscles have been used to simulate prolonged
spaceflight or bed rest in humans (1, 83).
An interesting and clinically relevant model of inactivity-
induced skeletal muscle atrophy is controlled mechanical ven-
tilation (i.e., ventilator delivers all of the breaths; Fig. 1). In
human medicine, controlled mechanical ventilation is used to
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2390 OXIDATIVE STRESS AND DISUSE MUSCLE ATROPHY
Fig. 1. Human conditions that promote skeletal muscle atrophy and the
corresponding animal models that are used to investigate them.
maintain alveolar ventilation in patients incapable of sustaining
adequate ventilation. Common applications for mechanical
ventilation in medicine include respiratory failure, sepsis, drug
overdose, spinal cord injury, and surgery (34). Numerous
human and animal studies have reported that prolonged me-
chanical ventilation results in a rapid onset of diaphragmatic
fiber atrophy (25, 54, 56, 75).
BIOCHEMICAL ALTERATIONS IN SKELETAL MUSCLE
DURING PROLONGED INACTIVITY
Well-controlled studies, using both the tail suspension and
limb immobilization models of muscle inactivity, indicate that
disuse muscle atrophy occurs due to both an increase in
proteolysis and a decrease in muscle protein synthesis (13, 82).
For example, a study using the hindlimb suspension model of
muscle atrophy reported that the rate of protein synthesis
declines quickly after the onset of muscle unloading (82). This
decrease in muscle protein synthesis reaches a new “lower”
steady-state rate of protein synthesis at ⬃48 h (82). Further-
more, this disuse-induced decrease in muscle protein synthesis
is followed by a large increase in muscle protein degradation.
Hence, reduced activity of skeletal muscle results in a net loss
of total muscle protein due to both a decrease in protein
synthesis and an increase in proteolysis.
Similar to studies using locomotor skeletal muscle, recent
studies indicate that mechanical ventilation-induced diaphrag-
matic atrophy is a result of both elevated proteolysis and
decreased protein synthesis (19, 73, 74). However, during
prolonged mechanical ventilation, the time course of disuse-
induced proteolysis in the diaphragm is more rapid than the
rate of protein breakdown observed in limb muscles exposed to
Fig. 2. Alterations in myonuclear number
during skeletal muscle hypertrophy and atro-
phy. Note that during hypertrophy or disuse
atrophy, myonuclear number is altered in
conjunction with changes in myofibrillar pro-
tein content and myofiber cross-sectional
area (CSA), resulting in a constant ratio of
cytoplasmic area to nuclear number (myo-
nuclear domain).
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periods of unloading. In fact, it would take at least 96 h to
achieve the same level of atrophy in locomotor muscle un-
loaded by hindlimb suspension as observed in the diaphragm
following 12 h of mechanical ventilation (61, 82). In addition,
the rate of mechanical ventilation-induced atrophy also ex-
ceeds that reported in the diaphragm with denervation (26),
spinal cord hemisection (91), and corticosteroid administration
(87). These facts suggest that the signaling associated with
myofiber atrophy during mechanical ventilation may involve
different or accelerated mechanisms compared with other mod-
els of muscle atrophy.
Moreover, it is now clear that muscular dystrophy (72)-,
denervation (14)-, aging (20, 78)-, hindlimb unloading (4, 5,
22)-, and mechanical ventilation (61)-induced disuse muscle
atrophy are associated with a loss of myonuclei, and growing
evidence indicates that the loss is due to a form of apoptosis
called “myonuclear apoptosis” (3, 4, 71). Apoptosis is a highly
regulated form of programmed cell death that is characterized
by specific morphological and biochemical events (55, 66).
Recently it has been reported that a loss of myonuclei via
apoptotic pathways can occur without cell death in multinu-
cleated muscle fibers during disuse muscle atrophy (4, 66, 71).
The loss of nuclei within atrophying muscle fibers appears to
be a strategy to maintain a constant ratio of nuclei per fiber area
(4, 71). It has been postulated that this constant ratio of nuclei
to fiber area (i.e., myonuclear domain) within skeletal muscle
is intended to maintain an optimal nuclear-to-cytoplasm ratio
such that nuclei are added to muscle fibers during growth and
nuclei are lost during atrophy (Fig. 2; Refs. 4, 71).
Identical to locomotor skeletal muscle, our laboratory re-
cently discovered that unloading the diaphragm during pro-
longed mechanical ventilation results in caspase-3 activation
and myonuclear apoptosis (61). The pathways responsible for
this disuse-induced myonuclear apoptosis are a hot topic of
debate and potential candidate pathways will be outlined in the
next segment.
PATHWAYS RESPONSIBLE FOR MYONUCLEAR APOPTOSIS
Myonuclear apoptosis can be induced by three overlapping
pathways: 1) sarcoplasmic (endoplasmic) reticulum (SR), 2)
receptor mediated, or 3) mitochondrial. Each of these apoptotic
pathways can trigger the activation of a unique group of
proteases called “caspases.” To date, over 14 different caspases
have been identified in mammalian cells (66). Collectively,
caspases are endoproteases responsible for the final execution
of cell death or nuclear destruction. In the cell, caspases are
expressed as inactive precursors (i.e., procaspases), which
activate on cleavage, resulting in a series of events leading to
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OXIDATIVE STRESS AND DISUSE MUSCLE ATROPHY
apoptosis. An illustration of the three primary apoptotic path-
ways along with selected caspases and pro/anti-apoptotic pro-
teins is presented in Fig. 3.
SR stress-induced apoptosis. The SR-mediated pathway of
apoptosis is activated via SR stress, resulting in calcium release
leading to activation of caspase-12 (due to either caspase-7 or
calpain activation). Activation of caspase-12 can then lead to
activation of caspase-3 (Fig. 3). Activated caspase-3 then
translocates to the nucleus where it cleaves and deactivates
“inhibitor of caspase-activated DNase” (66). This liberates
caspase-activated DNase, an endonuclease, to fracture genomic
DNA, leading to one of the most important morphological
events of apoptosis (i.e., DNA fragmentation; Ref. 66). This
apoptotic DNA fragmentation can be identified via histological
techniques, including fluorescent labeling of damaged DNA
via probes for single-stranded DNA or terminal deoxynucleo-
tidyl transferase-mediated dUTP nick-end labeling (TUNEL)
or electrophoretic procedures to reveal DNA fragmentation
(i.e., DNA laddering).
Note that increases in cytosolic calcium due to SR stress can
also promote apoptosis via activation of calpain. Calcium-
mediated activation of calpain has been postulated to contrib-
ute to apoptosis in several ways. For example, calpain can
cleave the Bcl2 (B-cell CLL/lymphoma-2) family member Bid
(BH3 interacting domain death agonist), to an active pro-apop-
totic molecule, tBid (truncated Bid). tBid promotes the forma-
tion of the mitochondrial permeability transition pore and
cytochrome c release from the mitochondria, leading to nuclear
apoptosis (15). Moreover, it has been reported that calpain
cleaves a variety of other proteins to promote apoptosis (15).
Hence it is possible that calpain activation is an important
stimulus to promote myonuclear apoptosis in skeletal muscle
during prolonged periods of unloading.
Receptor-mediated apoptosis. A widely studied pathway
leading to apoptosis is the corridor induced by the binding of
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2391
a ligand to one or more extracellular receptors. These ligand-
receptor complexes transmit their signals via a series of pro-
tein-protein interactions (66). The tumor necrosis factor recep-
tor super family is a critical trans-membrane receptor class that
binds both the cytokine tumor necrosis factor-␣ (TNF-␣) and
Fas Ligand (a transmembrane protein on cytotoxic T lympho-
cytes and other immune cells; Ref. 52). Receptor-mediated
apoptotic pathways can be initiated by ligand-induced (e.g.,
TNF-␣) binding, leading to the activation of caspase-8 and -10,
promoting the activation of caspase-3 and subsequent damage
to genomic DNA (66).
Mitochondrial-mediated apoptosis. The mitochondrial path-
way of apoptosis can be initiated via various signals, including
ROS (i.e., oxidative stress) and high levels of cellular calcium.
These factors can induce opening of the mitochondrial perme-
ability transition pore and promote the release of cytochrome c
from the mitochondria into the cytosol (Fig. 3; Ref. 66). In the
cytosol, cytochrome c triggers a signaling pathway leading to
the activation of caspase-9 and subsequently promoting
caspase-3-mediated nuclear DNA fragmentation. Independent
of caspase activation, mitochondrial release of apopto-
sis-inducing factor (AIF) or endonuclease G (Endo G) can also
induce nuclear DNA fragmentation in atrophying skeletal mus-
cle (22, 66).
In general, the release of mitochondrial apoptotic factors
into the cytoplasm is regulated by the Bcl-2 family of apoptotic
regulators (17, 24, 66). Within this family, several pro-apop-
totic (e.g., Bax, Bid, PUMA, etc.) and anti-apoptotic (e.g.,
Bcl-2, Bcl-XL, etc.) members exist (66). For example, Bcl-2
protects the mitochondria against the release of pro-apoptotic
molecules, whereas Bax (Bcl2-associated X-protein) and Bid
facilitate apoptosis by contributing to the opening of the
mitochondrial permeability transition pore, resulting in the
release of cytochrome c and AIF (24, 66). Interestingly, Bcl-2
and Bax influence each other’s function such that the relative
Fig. 3. Pathways involved in myonuclear apop-
tosis in atrophying skeletal muscle. Apoptosis
can be initiated by receptor signaling, sarco-
plasmic reticulum (SR), and/or mitochondrial-
regulated pathways in skeletal muscle. Ligand
receptor (FAS/TNF) binding results in
caspase-3 dependent DNA fragmentation
and/or myofilament release. Reactive oxygen
species (ROS) dysregulation of sarcoplasmic
reticulum calcium (Ca) handling results in
caspase-7 and calpain activation. ROS also
acts on the mitochondria to influence the
opening of the mitochondrial permeability
transition pore to release cytochrome c (Cyt
C). Independent of caspase activity, mito-
chondrial endonuclease G (Endo G), and/or
apoptosis inducing factor (AIF) are also capa-
ble of inducing DNA fragmentation in skeletal
muscle. Note the multiple lines of cross talk
between pathways.
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2392 OXIDATIVE STRESS AND DISUSE MUSCLE ATROPHY
concentration of these two proteins may determine whether the
cell undergoes apoptosis when faced with stimuli such as
oxidative stress or calcium overload (24, 66).
PROTEOLYTIC PATHWAYS IN SKELETAL MUSCLE
Numerous proteolytic systems contribute to the degradation
of muscle proteins. The principal proteases in skeletal muscle
can be classified into three major categories: 1) lysosomal
proteases, 2) Ca2⫹-activated proteases (i.e., calpain), and 3) the
proteasome system. Furthermore, recent evidence reveals that
another protease, caspase-3, may also contribute to select
forms of muscle atrophy (21, 61). A brief overview of each of
these major proteolytic systems follows.
Lysosomal proteases. Lysosomes are a component of the
degradative machinery in many mammalian cells. Lysosomes
are membrane-bound vesicles containing acid hydrolases that
include proteases, glycosidases, lipases, and phosphatases (8).
Therefore, lysosomes are intracellular compartments dedicated
to the degradation of macromolecules.
The four major lysosomal proteases are cathepsins L, B, D,
and H (8, 79). Although these cathepsins are ubiquitously
expressed in all tissues, the concentrations of cathepsins in
cells vary, with the greatest levels found in tissues containing
inherently high rates of protein turnover (i.e., kidney, liver, and
spleen), whereas tissues with slow protein turnover (i.e., skel-
etal muscle) possess much lower levels (8). Moreover, cathep-
sin levels differ between skeletal muscle fiber types. Type I
(slow, oxidative) fibers exhibit higher levels of cathepsins
compared with type IIb (fast, glycolytic) fibers (8).
Although some debate continues, it is generally agreed that
lysosomal protease activity increases in disuse muscle atrophy
(8, 27, 79, 85). For instance, work by Taillandier et al. (79)
indicates that 9 days of hindlimb suspension promoted a 2.5-fold
increase in lysosomal protease activity in rat soleus muscle. In an
effort to determine the quantitative contribution of cathepsins to
muscle atrophy, these investigators inhibited cathepsin activity
during periods of muscle disuse and evaluated the rate of muscle
atrophy. Their results revealed that inhibition of cathepsin activity
resulted in only a small decrease (⬍18%) of the loss of muscle
protein associated with disuse muscle atrophy. Furthermore, be-
cause lysosomal proteases are not involved in the degradation of
myofibrillar proteins, it seems unlikely that cathepsins play a
dominant role in the proteolysis associated with inactivity in
skeletal muscles (79).
Calpain-mediated proteolysis. Calpains are Ca2⫹-dependent
cysteine proteases that are found in all vertebrate cells (28).
Although numerous members of the calpain family of pro-
teases exist, the two best-characterized calpains found in skel-
etal muscle are termed ␮-calpain and m-calpain and refer to
micromolar and millimolar amounts of calcium required to
activate each respective calpain isoform (28). Although
calpains are not at present known to directly degrade the
contractile proteins actin and myosin, they participate in
sarcomeric protein release by cleaving cytoskeletal proteins
(e.g., titin, nebulin) that anchor contractile elements (43,
67). Moreover, calpain is known to degrade several kinases
and phosphatases, including calcium/calmodulin-dependent
kinase (CaM kinase II), protein kinase C (PKC-␣, PKC-␤I,
PKC-␤II, and PKC-␥), and calcineurin (28, 32, 81).
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In vivo calpain activity is regulated by several factors
including cytosolic calcium levels and the concentration of the
endogenous calpain inhibitor, calpastatin (28). More specifi-
cally, calpain activity is increased by a sustained elevation in
free calcium in the cytosol and/or a decrease in cytosolic
calpastatin levels (28). In regard to calpain’s contribution to
disuse muscle atrophy, it is clear that skeletal muscle inactivity
is associated with an increase in both cytosolic calcium levels
and calpain activity (51). Although the mechanism responsible
for this inactivity-mediated calcium overload is unknown, it is
possible that intracellular production of ROS could play a key
role in disturbances in calcium homeostasis (42). A potential
biochemical mechanism to link oxidative stress with calcium
overload is that ROS-mediated formation of reactive aldehydes
(i.e., 4-hydroxy-2,3-trans-nonenal) can inhibit plasma mem-
brane Ca2⫹-ATPase activity (77). Hence, an oxidative stress-
induced decrease in membrane Ca2⫹-ATPase activity would
impede Ca2⫹ removal from the cell and promote intracellular
Ca2⫹ accumulation. Nonetheless, it is currently unknown as to
whether this mechanism is the single explanation for inactivity-
mediated calcium overload in muscle.
Caspase-3 and muscle atrophy. Recent evidence suggests
that the protease caspase-3 contributes to protein degradation
and muscle atrophy (21, 61). In this regard, caspase-3 activa-
tion promotes degradation of actomyosin complexes, and in-
hibition of caspase-3 activity suppresses the overall rate of
proteolysis in diabetes-mediated cachexia and myofiber atro-
phy in mechanical ventilation disuse (21, 61).
As discussed previously, control of caspase-3 activity in the
cell is complex and involves numerous interconnected signal-
ing pathways. In the case of diabetes-induced muscle atrophy,
it seems possible that caspase-3 is activated by caspase-12 (via
a calcium release pathway) and/or activation of caspase-9 (via
a mitochondrial pathway). A potential interaction between
these caspase-3 activation pathways is that both of these can be
activated by ROS (Fig. 3; Ref. 66).
Moreover, note that calpastatin is a substrate for both
caspase-3 and calpain. It follows that increases in caspase-3 or
calpain activity lower calpastatin levels in cells and promote
calpain activation (21, 28, 88). Furthermore, increased calpain
activity can lead to the activation of caspase-3 (15). Therefore,
cross-talk between the calpain and caspase-3 proteolytic sys-
tems could play a role in the regulation of myofilament release
in skeletal muscle during periods of disuse.
The proteolytic release of myofilaments during disuse mus-
cle atrophy is important because the bulk of muscle proteins
(50 –70%) exist in actomyosin complexes (84). Although the
proteasome system can degrade monomeric contractile pro-
teins (i.e., actin and myosin), it is unable to break down intact
actomyosin complexes (28). Hence, myofilaments must be
released from the sarcomere as monomeric proteins prior to
degradation by the proteasome system (84, 89). This fact
dictates that the release of myofilaments is potentially the
rate-limiting step in muscle protein degradation. Again, evi-
dence reveals that both calpain and caspase-3 are capable of
producing actomyosin disassociation (21, 28, 84). Hence, ac-
tivation of one or both of these proteases is a requirement for
proteolytic degradation of myofilaments during conditions that
result in muscle atrophy.
Proteasome-mediated proteolysis. The total proteasome
complex (26S) is comprised of a core proteasome subunit
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OXIDATIVE STRESS AND DISUSE MUSCLE ATROPHY
(20S) coupled with a regulatory complex (19S) connected to
each end of the 20S core (30, 31, 33, 84). Interestingly,
proteins can be degraded by either the 26S proteasome or the
20S protease core.
The 26S proteasome degrades ubiquitinated proteins. It
follows that the 26S proteasome degradation pathway is only
active after ubiquitin covalently binds to protein substrates and
marks them for degradation. The binding of ubiquitin to
protein substrates is often a three-step process that initially
requires the ubiquitin-activating enzyme (E1). Following acti-
vation, the ubiquitination of specific proteins is provided by
one of a variety of E2s and by specialized E3s that recognize
specific protein substrates. In this regard, numerous studies
reveal that the ubiquitin-conjugating enzyme E214k is an im-
portant regulator of skeletal muscle ubiquitin-protein conjuga-
tion (58, 70, 80). Furthermore, E214k interacts with a specific
E3 ligase (i.e., E3␣) to promote muscle protein degradation in
catabolic states. In addition, other unique ubiquitin E3 ligases
(e.g., atrogin1/muscle atrophy F-box and muscle ring finger-1)
exist in skeletal muscle, and these ligases play essential roles in
skeletal muscle atrophy (10, 29, 70, 80).
The process of ubiquinated protein degradation requires a
coordinated effort of both the 19S regulatory complex and the
20S proteasome core. The 19S regulatory complex possesses
inherent ATPase activity and is a required participant in ATP-
dependent degradation of ubiquitinated proteins (18). The
ubiquitinated protein is recognized and bound by the 19S
regulators of the 26S proteasome, and energy from ATP
hydrolysis removes the polyubiquitin chain and unfolds the
substrate protein. The unfolded protein then enters the 20S core
proteasome and is degraded in a process that does not require
energy from ATP (31, 70). Also, note that evidence reveals that
the 20S core proteasome can degrade oxidized proteins without
ubiquitination (30, 31). Therefore, ROS-mediated oxidation of
proteins can promote muscle protein breakdown via 20S core
proteasome alone.
PRODUCTION OF ROS IN QUIESCENT SKELETAL MUSCLES
It is now clear that ROS are produced in both inactive and
contracting skeletal muscles (44, 69). When ROS production in
cells exceeds the antioxidant capacity to eliminate these oxi-
dants, oxidative stress occurs. A pro-oxidant state in cells can
alter the structure and function of proteins, lipids, and nucleic
acids, resulting in cellular injury and, in extreme circum-
stances, cell death.
Until the early 1990s it was widely believed that ROS
production was limited in noncontracting skeletal muscle and
that oxidative injury does not occur in inactive muscles. How-
ever, many studies indicate that oxidative injury occurs during
periods of disuse in locomotor skeletal muscles (45, 47–50, 53)
and in the unloaded diaphragm during prolonged mechanical
ventilation (23, 75, 90). It is currently unknown which ROS
producing pathways are dominant contributors to inactivity-
induced oxidative injury in skeletal muscles. Indeed, it is
possible that oxidative stress in inactive skeletal muscle may be
due to the interaction of several major oxidant production
pathways, including xanthine oxidase production of superox-
ide, nitric oxide synthase production of nitric oxide, NADPH
oxidase-mediated production of superoxide, and mitochondrial
production of superoxide (7, 23, 37, 38, 50, 86; Fig. 4).
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Fig. 4. Simplified diagram illustrating pathways capable of producing super-
oxide (O䡠2) and nitric oxide (NO䡠) in skeletal muscle during periods of disuse.
Candidates for the production of reactive oxygen include NADPH oxidase,
xanthine oxidase, and muscle mitochondria. Nitric oxide is likely produced by
skeletal muscle nitric oxide synthase.
WHAT INTRACELLULAR SIGNALING PATHWAYS LINK
OXIDATIVE STRESS TO MUSCLE ATROPHY?
Recent studies reveal that ROS species can serve as second
messengers in cellular signal transduction pathways that regu-
late both normal physiological signaling and pathological sig-
naling leading to proteolysis and cell death via apoptosis. This
seemingly contradictory signaling function of ROS is presum-
ably linked to the level of ROS and the overall redox state in
the cell. That is, low levels of ROS promote cell adaptation and
survival, whereas high levels of ROS modulate signaling
pathways that lead to proteolysis and cell death.
Abundant research demonstrates that oxidative stress con-
tributes to disuse muscle atrophy. The first evidence that
oxidants played a key signaling role in the regulation of disuse
muscle atrophy was provided by Kondo et al. (46). The
pioneering work of Kondo and colleagues revealed that immo-
bilization of skeletal muscles is associated with increased
radical production, resulting in oxidative injury in inactive
muscle fibers (46, 47, 50). Importantly, this work also demon-
strated that disuse muscle atrophy could be delayed by exog-
enous antioxidants. These early observations have subse-
quently been confirmed by others (6, 9, 60).
How does inactivity-induced oxidative stress in skeletal
muscle contribute to muscle atrophy? At present, a complete
answer to this question is not available. Nonetheless, it appears
that oxidative stress could contribute to disuse muscle atrophy
by influencing one or more of the following cell signaling
pathways: 1) regulation of cytosolic calcium levels and subse-
quent activation of calpain (as previously discussed); 2) control
of mitogen activated protein kinase (MAPK) signaling; and
3) activation of the nuclear factor (NF)-␬B pathway. A brief
discussion of these possibilities follows.
OXIDATIVE STRESS ACTIVATES MAPK SIGNALING
The redox regulation of MAPK provides another potential
link between oxidative stress and skeletal muscle atrophy. It is
well established that MAPKs play a key role in cell signaling
because control of numerous cellular signaling pathways is
achieved via activation or deactivation of regulatory proteins
through phosphorylation (16). Indeed, the highly conserved
MAPK family is one of the major kinase families that regulate
the conversion of cell signals into cellular responses. These
protein kinases contribute to the regulation of life and death
decisions made in response to various stress signals (e.g.,
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2394 OXIDATIVE STRESS AND DISUSE MUSCLE ATROPHY
oxidative stress), as the actions of both pro- and antiapoptotic
factors are often regulated by the phosphorylation status of key
elements in the execution of apoptosis or survival (59).
All eurkaryotic cells possess multiple MAPK pathways. The
three best characterized MAPK subfamilies are c-Jun NH2-
terminal kinase (JNK), p38 MAPK, and extracellular signal-
regulated kinase (ERK; Ref. 64). All three of these MAPK
pathways are structurally similar, but functionally distinct.
Importantly, ERK, JNK, and p38 have all been shown to be
activated by oxidative stress and could potentially participate
in pathways influencing muscle protein breakdown or myo-
nuclear apoptosis. The classic MAPK activation cascade con-
sists of three sequential intracellular protein kinase activation
steps leading to the activation of numerous protein kinases,
nuclear proteins, and transcription factors, resulting in down-
stream signal transduction (16, 64). A brief overview of the
functions of ERK, p38, and JNK follows.
ERK1/2. The first identified and best studied MAPK cascade
is the ERK pathway (16). ERK is composed of two isoforms,
ERK1 and ERK2, and are collectively referred to as ERK1/2
(16, 64). ERK1/2 can be activated by a number of mitogens,
including epidermal growth factor, platelet-derived growth
factor, transforming growth factor, and insulin. In addition,
ERK1/2 can be activated by endotoxin and oxidative stress
(16). The activation of ERK1/2 by oxidative stress is consistent
with the hypothesis that low and adequate levels of ROS are
mitogenic (64, 68). Activation of ERK1/2 regulates the tran-
scriptional activity of AP-1, NF-␬B, c-Myc, and the cell
survival protein Bcl-2 (68). Although it has been reported that
ERK1/2 can promote apoptosis, ERK1/2 can also function as
an anti-apoptotic factor following oxidative stress (64). To
date, however, there is little evidence to link ERK1/2 activation
with disuse muscle atrophy.
p38. p38 is an important MAPK member that is activated in
response to various physiological stresses such as osmotic
Fig. 5. Simplified overview of ROS signaling
pathways leading to activation of mitogen ac-
tivated protein kinases in skeletal muscle.
Skeletal muscle cytosolic or mitochondrial
ROS activates apoptosis-stimulating kinase 1
(ASK1) stimulating phosphorylation (p) of jun
NH2-terminal kinase (JNK) and/or p38 regula-
tion of myonuclear apoptosis-associated tran-
scription factors (AP1, p53, c-Myc, NF␬B).
JNK/p38 activation by ASK1 can lead to cy-
tochrome c release and caspase-3 activation. In
contrast, low levels of ROS (dashed lines) can
result in the phosphorylation of the extracellu-
lar regulated kinases 1 and 2 (ERK 1/2), which
functions in cellular survival. mtPTP, mito-
chondrial permeability transition pore.
J Appl Physiol • VOL
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stress, endotoxins, and oxidative stress (64). The link between
oxidative stress and p38 activation occurs via apoptosis stim-
ulating kinase 1 (ASK1; Ref. 59). ASK1 is a ubiquitously
expressed member of the MAPK family that activates both p38
and JNK by phosphorylating and activating respective MAPK
kinases (Fig. 5). Five isoforms of p38 have been identified:
p38␣, p38␤, p38␤2, p38␥, and p38␦ and expression of these
isoforms varies among tissues (16). For example, p38␣ is
highly expressed in bone morrow and leukocytes, p38␤ is
expressed in heart and brain, and p38␥ is predominantly
expressed in skeletal muscle (16).
Among the phosphorylation targets of p38 are several im-
portant transcription factors, including p53, NF-␬B, and ATF2.
Of particular importance to apoptosis is the fact that activation
of the tumor suppressor protein p53 results in the expression of
the pro-apoptosis protein Bax. Moreover, p38 signaling has
been shown to promote the expression of atrogin 1 in myotubes
(57). Collectively, these data suggest a potential role for
oxidative stress-induced activation of p38 in disuse muscle
atrophy.
JNK. JNK (also known as stress-induced kinase) has three
isoforms (JNK1, JNK2, JNK3) that are encoded by three different
genes. JNK1 and JNK2 are ubiquitously expressed, whereas
JNK3 is only expressed in brain, heart, and testis (16). JNK can be
activated in response to many of the same stimuli that activate
p38, such as osmotic stress, endotoxins, and oxidative stress.
Moreover, similar to p38, oxidative stress-induced activation of
JNK occurs via the ASK1 pathway (Fig. 5; Ref. 76).
The specific molecular targets of JNK include the transcrip-
tional factors AP-1, p53, and c-Myc and many other nontran-
scriptional factors such as Bcl-2 family members. In this
regard, there is growing evidence that JNK plays an important
role in oxidative stress-mediated apoptosis. Indeed, because
ROS themselves are not able to activate caspases, ROS-
mediated apoptosis requires another death-signaling pathway
102 • JUNE 2007 • www.jap.org

OXIDATIVE STRESS AND DISUSE MUSCLE ATROPHY
such as JNK (59, 76). Preliminary evidence implicating JNK as
a mediator of ROS-induced apoptosis can be summarized in
three observations. First, JNK is readily activated in cells by
exposure to physiological levels of ROS (59, 76). Secondly, in
ASK1⫺/⫺ cells, JNK is not activated in response to ROS and
ASK⫺/⫺ cells are resistant to ROS-mediated apoptosis, al-
though the role of p38 could not be eliminated (76). Finally,
overexpression of a dominant negative mutant of JNK provides
some resistance to ROS-induced apoptosis (76). Collectively,
these findings support a link between ROS, JNK, and apopto-
sis. However, it is currently unknown if JNK activation is
responsible for the myonuclear apoptosis that occurs during
disuse muscle atrophy.
MAPK cross talk. While each member of the MAPK super
family is considered unique, each member forms only a part of the
complex and interactive network of phosphorylation signaling
pathways (16). Indeed, each member of the MAPK cascade shares
communication with a number of upstream and downstream
kinases along with transcription factors that interact and integrate
these different pathways. Therefore, this redundancy results in a
complex cross-talk and signal convergence among MAPK family
members. Moreover, each MAPK family member has feedback
loops that impact their activation and regulate the activation of
other MAPK family members (16).
REDOX REGULATION OF NF-␬B SIGNALING
Another potential link between oxidative and muscle disuse
atrophy involves the redox regulation of the NF-␬B family of
transcriptional activators. NF-␬B comprises a family of five
transcription factors (p65, Rel B, c-rel, p52, and p50) and,
when activated, can promote a wide range of cellular outcomes
depending on the cell type (reviewed in Refs. 36, 41, 42). All
five of the NF-␬B family members are expressed in skeletal
muscle and Hunter et al. (35) first reported that skeletal muscle
inactivity leads to increased NF-␬B transcriptional activity.
Since this discovery in 2002, accumulating evidence indicates
that a specific NF-␬B pathway is required for disuse muscle
atrophy (reviewed in Refs. 36, 41). Nonetheless, although
NF-␬B regulates the expression of over 100 genes, a detailed
understanding of the specific NF-␬B targets required for disuse
muscle atrophy remains incomplete.
Although it has long been believed that NF-␬B activation is
under redox control, how ROS regulate NF-␬B transcriptional
activity remains contentious. For example, ROS are known to
enhance the signal transduction pathways (e.g., activation of
kinases) that promote both NF-␬B activation in the cytoplasm
and translocation to the nucleus (40, 62). This observation is
consistent with the concept that ROS can promote NF-␬B
activation and subsequent gene expression. In contrast, the
DNA binding activity of oxidized NF-␬B is diminished, sug-
gesting that ROS may also inhibit NF-␬B transcriptional ac-
tivity (40). Therefore, although NF-␬B was once considered to
be a prototypic redox sensitive transcription factor, the fact that
ROS can both promote and inhibit NF-␬B transcriptional
activation has lead to considerable debate regarding the redox
control of NF-␬B signaling (62). Clearly, future research will
be required to unravel the uncertainties about the redox regu-
lation of NF-␬B in physiologically relevant settings in skeletal
muscle during prolonged periods of inactivity.
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Invited Review
2395
CONCLUSIONS AND UNANSWERED QUESTIONS
Inactivity-induced skeletal muscle atrophy can occur due to
a variety of conditions, including prolonged bed rest, limb
immobilization, and spaceflight. Several lines of evidence
directly link ROS to disuse muscle atrophy via ROS-mediated
regulation of proteolysis. Importantly, several studies suggest
that antioxidants can serve as therapeutic agents in delaying the
rate of disuse muscle atrophy.
Although it is clear that ROS-mediated signaling contributes
to disuse muscle atrophy, numerous unanswered questions
remain. For example, does the role that ROS play in muscle
atrophy differ between different models of skeletal muscle
inactivity (e.g., immobilization vs. microgravity)? Another key
question is “which ROS pathways are active in unloaded
skeletal muscle”? Moreover, “if multiple oxidant production
pathways are active, what is the relative contribution of each
pathway to the regulation of cell signaling pathways involved
in disuse muscle atrophy”? Resolution of these issues will
provide the information needed to develop therapeutic strate-
gies to prevent oxidant production or scavenge ROS to prevent
oxidative injury in the muscle fiber during prolonged periods of
inactivity.
Is the production of ROS a requirement for disuse muscle
atrophy or does oxidative stress merely regulate the rate of
muscle atrophy? A related question is, “do ROS simply act as
second messengers to control muscle atrophy or is ROS-
mediated oxidative injury a requirement for oxidant regulation
of muscle atrophy”? Both of these questions are important and
unresolved.
Another unresolved question is “does oxidative stress nega-
tively influence the rate of protein synthesis in skeletal muscle
during periods of inactivity”? To date, the majority of previous
research related to ROS and muscle atrophy has focused on
proteolysis, and it is unclear if ROS contribute to the downregu-
lation of protein synthesis that occurs during disuse in skeletal
muscle; hence, this is an interesting area for future work. In this
regard, emerging evidence reveals that oxidative stress can inhibit
protein synthesis in several cell types (2, 63, 65).
A final area for future research relates to the pathways
responsible for myonuclear apoptosis in skeletal muscle. As
discussed earlier, disuse muscle atrophy is associated with a
loss of myonuclei from muscle fibers due to myonuclear
apoptosis (3, 71). Although it is well known that ROS can
contribute to signaling pathways, leading to apoptosis, it is
unknown if ROS represent the primary signal to promote
myonuclear apoptosis in atrophying skeletal muscle (55, 66).
Hopefully, questions outlined in this review stimulate muscle
biologists to pursue research in the area of ROS and skeletal
muscle atrophy. Future scientific advances in cell signaling
will provide the tools required to answer these important
questions that will ultimately result in therapeutic approaches
to prevent or diminish disuse muscle atrophy.
GRANTS
Our work in this research area has been supported by National Heart, Lung,
and Blood Institute Grants R01-HL-62361 and R01-HL-072789.
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