Acta Physiol Scand 2001, 171, 225232

Cytokines and oxidative signalling in skeletal muscle

M . B . R E I D and Y . - P . L I
Department of Medicine, Baylor College of Medicine, Houston, TX, USA
ABSTRACT
A growing body of literature indicates that cytokines regulate skeletal muscle function, including gene
expression and adaptive responses. Tumour necrosis factor-a (TNF-a) is the cytokine most
prominently linked to muscle pathophysiology and, therefore, has been studied most extensively in
muscle-based systems. TNF-a is associated with muscle catabolism and loss of muscle function in
human diseases that range from cancer to heart failure, from arthritis to AIDS. Recent advances have
established that TNF-a causes muscle weakness via at least two mechanisms, accelerated protein
loss and contractile dysfunction. Protein loss is a chronic response that occurs over days to weeks.
Changes in gene expression required for TNF-a induced catabolism are regulated by the transcription
factor nuclear factor-jB which is essential for the net loss of muscle protein caused by chronic TNF-a
exposure. Contractile dysfunction is an acute response to TNF-a stimulation, developing over hours
and resulting in decreased force production. Both actions of TNF-a involve a rapid rise in endogenous
oxidants as an essential step in post-receptor signal transduction. These oxidants appear to include
reactive oxygen species derived from mitochondrial electron transport. Such information provides
insight into the cellular and molecular mechanisms of TNF-a action in skeletal muscle and establishes
a scientic basis for continued research into cytokine signalling.
Keywords cachexia, cytokines, free radicals, inammation, muscle contraction, oxidative stress,
skeletal muscle, tumour necrosis factor.
Received 12 December 2000, accepted 24 January 2001

Cytokines are intercellular signalling molecules, typically

proteins or glycoproteins, that mediate various aspects
of cell function, including proliferative and adaptive
responses. Cytokine signalling is essential for a
co-ordinated inammatory response and is fundamental to our understanding of immune cell function. It is
less well-recognized that cytokines also inuence skeletal muscle biology in various settings. Inammatory
myopathies are characterized by leucocyte invasion of
damaged muscle and overt injury of muscle bres,
events that correlate with elevated tissue levels of selected cytokines. Remote inammatory processes
cancer, congestive heart failure, arthritis, etc. often
lead to muscle catabolism and loss of muscle function
that have been attributed to circulating cytokines.
Similar mechanisms have been suggested to cause
cachexia and weakness in systemic diseases such as
sepsis and AIDS. More recently, a physiological role
has been suggested for cytokines in the absence of
disease. Skeletal muscle is capable of synthesizing a
variety of cytokines in response to inammation and

may constitutively express some cytokines under basal

conditions. Exercise is known to alter immunological
function in healthy individuals, a response that may be
linked to altered cytokine levels. And the muscle loss
that often accompanies ageing of otherwise healthy
individuals has been attributed to age-related increases
in circulating cytokine levels.
Despite the potential importance of cytokines as
regulators of skeletal muscle function, relatively little is
known about cytokine effects on muscle or the intracellular events that transduce cytokine signals. Such
information cannot be extrapolated with condence
from data obtained in non-muscle cells because postreceptor signalling mechanisms are often cell typespecic. Thus, primary data from skeletal muscle are
essential. Among the limited information that is available on cytokine actions in skeletal muscle, the most
extensive literature relates to tumour necrosis factor-a
(TNF-a). This review focuses on TNF-a as a prototype
for understanding the cellular and molecular bases of
cytokine action in skeletal muscle.

Correspondence: Michael B. Reid, Pulmonary Medicine, Suite 520B, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
2001 Scandinavian Physiological Society

225

Cytokines, oxidants, and muscle

M B Reid and Y-P Li

R E L E V A N C E O F T N F -a T O M U S C L E
TNF-a is a polypeptide cytokine that promotes
antitumour and immune responses (Schutze et al.
1992). It is primarily released by macrophages although
other cell types, including skeletal muscle (Shindoh
et al. 1995), are capable of synthesizing TNF-a. TNF-a
has long been associated with muscle pathology and
was originally designated `cachectin' in recognition of
its catabolic action (Beutler et al. 1985, Beutler &
Cerami 1986). Experimental animals lose muscle mass
when chronically exposed to exogenous TNF-a (Garcia-Martinez et al. 1993b, Buck & Chojkier 1996) or to
interventions that elevate endogenous TNF-a, e.g.
sepsis (Ahmad et al. 1994) or tumour implantation
(Tessitore et al. 1993). In humans, TNF-a is reported
to stimulate muscle catabolism and contractile dysfunction in inammatory diseases that range from
sepsis to cancer, from congestive heart failure to AIDS
(Espat et al. 1994). Loss of muscle mass leads to
weakness, fatigue, loss of mobility, and decreased
quality of life for individuals with catabolic diseases.
Despite its potential importance in inammatory
disease, the direct effects of TNF-a on skeletal muscle
and the underlying mechanisms remain largely undened.
Studies of non-muscle cell types indicate that TNF-a
receptor binding stimulates a complex cascade of postreceptor signalling events (Hsu et al. 1996, Liu et al.
1996). In selected cell types, these pathways include
reactive oxygen species (ROS) or reactive nitrogen
species (RNS) as mediators of the TNF-a signal
(Schutze et al. 1992). ROS and RNS may inuence a
variety of downstream signalling events including activation of redox-sensitive kinases, alteration of intracellular calcium regulation, transcription factor
activation and gene expression (Hancock 1987). Oxidants are critically important for TNF-a signalling in
some tissues and are largely unimportant in others.
Data obtained over the last few years place skeletal
muscle in the former category (Sen et al. 1997, Li et al.
1998, 1999, 2000, Khanna et al. 1999). Cytosolic oxidant levels are elevated in diaphragm muscle bres of
transgenic mice that overexpress TNF-a and in
wild-type diaphragm bres challenged with exogenous
TNF-a (Li et al. 2000). TNF-a also alters muscle contraction via redox effects. Experimental endotoxaemia
stimulates TNF-a expression, oxidative injury and
contractile dysfunction in rat diaphragm (Shindoh et al.
1995). Cardiac-specic overexpression of TNF-a
depresses diaphragm force production in transgenic
mice, an effect partially reversed by antioxidant treatment (Li et al. 2000). Similarly, antioxidant pre-treatment inhibits the contractile losses caused by
incubating diaphragm of wild-type mice with exogen226

Acta Physiol Scand 2001, 171, 225232

ous TNF-a (Li et al. 2000). At least two oxidantdependent mechanisms exist by which TNF-a may
compromise muscle function.
First, TNF-a promotes loss of muscle protein.
Recent studies have shown that TNF-a can directly
stimulate protein loss in differentiated muscle cells
(Li et al. 1998, 2000), refuting previous conclusions
that TNF-a acts only via indirect mechanisms (Rofe
et al. 1987, Goldberg et al. 1988, Goodman 1991,
Garcia-Martinez et al. 1993b). Supporting a role for
oxidants in this process, Buck & Chojkier (1996)
have demonstrated that either antioxidants or nitric
oxide (NO) synthase inhibitors can inhibit muscle
wasting and dedifferentiation in a mouse model of
TNF-a-induced cachexia. A primary pathway by
which TNF-a may induce protein loss is via oxidative
activation of nuclear factor-jB (NF-jB), a redoxsensitive transcription factor. Sen et al. (1997) demonstrated TNF-a/NF-jB signalling in undifferentiated myoblasts and determined that this pathway is
regulated by the glutathione cycle. As detailed below,
the TNF-a/NF-jB pathway is regulated by mitochondrial ROS and is a primary mediator of TNF-ainduced protein loss.
Secondly, TNF-a may disrupt contractile function
without overt loss of muscle protein. Weakness and
fatigue are common symptoms of infection (Friman &
Ilback 1998), cancer chemotherapy (Simon & Zittoun
1999), overtraining (Budgett 1998) and other clinical
conditions in which gross cachexia may be absent. Loss
of muscle function is associated with the cell-mediated
immune response and circulating cytokines have been
implicated as possible regulators (Chang & Bistrian
1998). Recent data indicate that muscle function may
be compromised by chronic elevation of circulating
TNF-a with no change in muscle mass or ultrastructure
(Li et al. 2000). Systemic TNF-a administration has
been shown to acutely diminish force production of
muscle in anaesthetized animals (Wilcox et al. 1994).
In vitro experiments indicate that TNF-a can act directly
on isolated muscle preparations to decrease force
(Wilcox et al. 1996), although such decrements are not
observed uniformly (Diaz et al. 1993). Contractile losses induced by TNF-a can be prevented or reversed by
antioxidant treatment (Li et al. 2000), suggesting that
force decrements are caused by a rise in intracellular
oxidant levels.
Integrating the information outlined above, we have
developed an experimental model of the intracellular
mechanisms by which TNF-a may compromise skeletal
muscle function. This model is depicted in Figure 1.
The current article reviews the scientic basis of this
model and the evidence on which it rests. Each of the
sections below addresses a major component of this
model.
2001 Scandinavian Physiological Society

Acta Physiol Scand 2001, 171, 225232

Figure 1 Mechanistic model of TNF-a-induced weakness. During

inammatory processes, TNF-a is synthesized and released into the

extracellular milieu by monocytes, macrophages and other nonimmune cell types. The resultant rise in circulating TNF-a levels leads
to increased ligand binding to TNF-a receptors on the skeletal muscle
sarcolemma. Receptor activation stimulates intracellular production of
mitochondrial ROS which promote muscle weakness via two parallel
pathways. One action of ROS alters myolament function, diminishing force production. A second action enhances expression of
proteins in the ubiquitinproteasome pathway, leading to degradation
and loss of contractile proteins.

T N F - a R E C E PT O R S I N S KE L E T A L
M U S CL E
Cellular responses to TNF-a may be mediated by either
of two distinct receptor populations located on the cell
surface, the 55 kDa TNF-receptor 1 (TNFR1) and the
75 kDa TNF-receptor 2 (TNFR2) (Tartaglia & Goeddel 1992). Skeletal muscle appears to express both
TNFR1 and TNFR2. In homogenates of mouse diaphragm, the two receptor subtypes are detectable in
roughly equal amounts (Li et al. 2000). Several lines of
evidence suggest that TNFR1 mediates the catabolic
response to TNF-a. In differentiated myotubes, TNF-a
induces protein loss by activating NF-jB (Li & Reid
2000), a pathway regulated by TNFR1 (Liu et al. 1996).
In a mouse model of cancer cachexia, muscle wasting
can be partially inhibited by knocking out the TNFR1
gene (Llovera et al. 1998c). Alternatively, muscle catabolism can be blunted in these animals by inducing
overexpression of the soluble TNFR1 receptor (Llovera
et al. 1998b); elevated receptor levels in the circulation
selectively chelates circulating TNF-a thereby inhibiting
cytokine activity. Pilot data from our research further
suggest that TNFR1 is most likely to mediate contractile dysfunction (unpublished results).
O XI D A N T S I N T N F - a S I G N A L L I N G
Little is known about the post-receptor signalling events
by which TNF-a stimulates oxidant production.
Inammation is generally reported to increase oxidant
2001 Scandinavian Physiological Society

M B Reid and Y-P Li Cytokines, oxidants, and muscle

levels by mechanisms that may include the sphingosine/

ceramide pathway (Quillet-Mary et al. 1997), phospholipase A2 signalling (Nethery et al. 1999), arachidonic acid
metabolism (Oe et al. 1994) and increased intracellular
calcium (Chakraborti et al. 1999). Possible sources of
oxidants within muscle cells include ROS derived from
mitochondrial electron transport (Bejima & Ji 1999,
McArdle et al. 1999) or from enzymatic sources, e.g.
NADPH-like oxidases (Bejima & Ji 1999), xanthine
oxidase (Stofan et al. 2000) or cyclooxygenase (Okabe
et al. 1985). Inammatory mediators also can stimulate
NO production by muscle (Williams et al. 1994), thereby
inuencing redox-sensitive processes.
The importance of endogenous oxidants in TNF-a
signal transduction is highly cell type-specic (Sen &
Packer 1996). In skeletal muscle, oxidant signalling
appears to be critical. The most direct evidence has been
obtained using 2,7-dichlorouorescin, a uorochrome
probe that detects cytosolic oxidants in skeletal muscle
bres (Murrant et al. 1999). A recent study (Li et al.
2000) evaluated oxidant levels in diaphragms of transgenic mice that constitutively overexpress a cardiacspecic transgene for secreted TNF-a. In muscles of
transgenic animals, sarcoplasmic oxidants were elevated
relative to muscles of littermate controls. Similarly, acute
exposure to exogenous TNF-a increased oxidant levels
in diaphragms from wild-type mice. Functional data
provide further support for oxidant involvement in
TNF-a signalling. In differentiated myotubes, ROS
appear to modulate the TNF-a/NF-jB pathway. The
TNF-a activation of NF-jB is partially inhibited by
treating myotubes with catalase (Li et al. 1998), an
antioxidant enzyme that dehydrates hydrogen peroxide.
Conversely, exogenous hydrogen peroxide can directly
activate NF-jB in the absence of TNF-a (Li et al. 1998).
In excised diaphragm, TNF-a effects on contractile
function also appear to be oxidant-mediated. Contractile
losses are reversed or inhibited by N-acetylcysteine (Li
et al. 2000), a reduced thiol-donor that supports glutathione resynthesis and has antioxidant properties.
The ROS that mediate TNF-a/NF-jB signalling are
most likely to derive from electron transfer reactions at
ubiquinone, the primary site of ROS synthesis in
mitochondrial electron transport (Chance et al. 1979).
The TNF-a activation of NF-jB is prevented by
complex I inhibitors, i.e. rotenone and amytal, that
block electron transport upstream of ubiquinone.
Complex I inhibitors were effective in differentiated
myotubes derived both from the C2C12 cell line and
from rat primary myocytes (Li et al. 1999). Antimycin A
blocks downstream of ubiquinone, increasing ROS
production, and was found to exaggerate NF-jB
activation (Li et al. 1999). Thus, the available data suggest that mitochondria-derived ROS are a primary
source of oxidants induced by TNF-a.
227

Cytokines, oxidants, and muscle

M B Reid and Y-P Li

In principle, endogenous NO gives rise to a cascade

of RNS that also could mediate TNF/NF-jB signalling.
Skeletal muscle constitutively expresses NO synthase
and continually produces RNS at low rates (Reid et al.
1998). The TNF can stimulate RNS production. In turn,
RNS can elevate intracellular oxidant levels in muscle
(Murrant et al. 1999) as occurs with TNF-a stimulation
(Li et al. 2000). In a mouse model of TNF-a-induced
cachexia, muscle wasting was partially inhibited by systemic administration of a NO synthase inhibitor (Buck
& Chojkier 1996). However, data from cultured myotubes argue against a direct effect of RNS in TNF/NFjB signalling (Li et al. 1999). The pathway is not affected
by NO synthase inhibitors nor is NF-jB activated by
direct application of exogenous NO donors. Thus, the
importance of RNS as mediators of the TNF/NF-jB
pathway remains unclear in skeletal muscle.
T H E C A TA B O L I C RE S P O N S E T O T N F -a
A primary cause of weakness and fatigue in inammatory disease is loss of muscle mass (Moldawer &
Sattler 1998, Anker & Rauchaus 1999, Tisdale 1999,
Farber & Mannix 2000). This effect is strongly linked
to TNF-a (Espat et al. 1994) which stimulates net
nitrogen loss and catabolism of muscle protein when
administered to experimental animals (Oliff et al. 1987,
Garcia-Martinez et al. 1993b, Tayek 1996, Tracey et al.
1998). Administration of TNF-a also produces complex humoral and behavioural effects that could contribute to loss of muscle mass in vivo (Tessitore et al.
1993). Investigators therefore sought to determine
whether muscle catabolism was a direct effect of
TNF-a by testing excised muscles in vitro. Pharmacological concentrations of TNF-a failed to evoke
detectable protein loss over periods of 14 h, leading
to the conclusion that TNF-a induces muscle catabolism by an indirect systemic effect (Rofe et al. 1987,
Goldberg et al. 1988, Goodman 1991, Garcia-Martinez
et al. 1993b).
Cachexia does not develop over several hours,
however. In inammatory disease, muscle wasting
occurs over weeks to months to years. It, therefore,
seemed possible that a longer-term study might be
necessary to resolve the catabolic action of TNF-a.
Subsequent studies using cell culture techniques
achieved this goal. Using differentiated myotubes, it has
been established that protein loss is accelerated by
TNF-a concentrations within the range measured in
patients (Li et al. 1998). This process does not involve
accelerated death of muscle cells, either by apoptosis or
necrosis. Rather, individual myotubes grow smaller as
total protein and muscle-specic protein contents
progressively decline. This process resembles the bre
atrophy that is typical of human cachexia.
228

Acta Physiol Scand 2001, 171, 225232

The catabolic action of TNF-a undoubtedly reects

altered gene expression in muscle cells. Transcriptional
regulation of this response is mediated in part by NF-jB
(Liu et al. 1996). As reviewed elsewhere (Baeuerle &
Baltimore 1996, Barnes & Karin 1997), this transcription factor exists in the cytoplasm of muscle cells as an
inactive complex bound by the inhibitory protein I-jBa.
TNF-a stimulates ubiquitin conjugation of I-jBa and
subsequent degradation of I-jBa by the 26S proteasome. Nuclear factor-jB is thereby activated and
translocates to the myotube nucleus. As noted above,
this process is mediated by endogenous ROS that
appear to derive from mitochondrial electron transport.
This process also appears to be essential for TNFa-induced catabolism. A recent study (Li & Reid 2000)
has established a causeeffect relationship using musclederived transdominant negative cell lines that overexpress mutant variants of I-jBa. These variants lacked
the Ser-32 and Ser-36 phosphorylation sites necessary
for ubiquitin conjugation. Accordingly, the mutant
I-jBa could not be targeted for proteasomal degradation and NF-jB could not be activated. This prevented
TNF-a-induced catabolism, identifying an essential role
for NF-jB in the regulation of protein loss.
The genes regulated by NF-jB remain to be established. However, regulatory proteins of the ubiquitin/
proteasome pathway are probable sites of transcriptional regulation in this setting. Expression of genes for
ubiquitin and for subunits of the 26S-proteasome
are upregulated in muscles of cancer patients
(Williams et al. 1999) and slim AIDS patients (Llovera
et al. 1998a). Acute, intravenous injection of TNF-a
causes time-dependent increases in free ubiquitin
(Garcia-Martinez et al. 1993a), conjugated ubiquitin
(Garcia-Martinez et al. 1993a), and ubiquitin
mRNA (Garcia-Martinez et al. 1994) in the limb muscles of intact rats. Ubiquitin mRNA is increased in
muscle by experimental sepsis (Garcia-Martinez et al.
1995) or tumour implantation (Llovera et al. 1997), and
proteasome inhibitors suppress protein breakdown in
rodent muscle after experimental sepsis (Hochstrasser
1995, Mitch & Goldberg 1996, Tawa et al. 1997). Such
studies establish the physiological relevance of ubiquitin/proteasome activity in regulating TNF-a effects on
muscle. But mechanistic questions remain: does TNF-a
act directly on muscle cells to upregulate the pathway or
is this a secondary effect of systemic stimuli? Which
regulatory components of the pathway are induced?
What transacting elements are involved at the promoter
level to increase gene expression?
C O N T R AC T IL E D Y S F U N C T I O N
Muscle weakness often occurs in inammatory diseases
without overt loss of muscle protein (Budgett 1998,
2001 Scandinavian Physiological Society

Acta Physiol Scand 2001, 171, 225232

Friman & Ilback 1998, Simon & Zittoun 1999). Prior

studies indicate that cytokines in general, and TNF-a in
particular, can cause contractile dysfunction (Wilcox
et al. 1992, 1994, 1996, Hopkins 1996). Recent observations in our laboratory support the relevance of such
effects in vivo (Li et al. 2000). We studied muscle function in a strain of transgenic mice that overexpresses a
constitutively active TNF-a transgene in cardiac
myocytes. Exaggerated synthesis of TNF-a stimulates
marked changes in myocardial structure and function
that closely resemble the cardiac pathophysiology of
congestive heart failure. As in human disease, the
genetically altered heart also sheds TNF-a and functions as an endocrine organ to increase circulating
TNF-a levels. This humoural stimulus appears to affect
remote organs e.g. lung, liver and spleen weights are
increased but there is no evidence of muscle wasting.
Overall appearance, body weight and spontaneous
behaviour of transgenic animals are not different from
littermate controls. Nor is there evidence of peripheral
muscle dysfunction. Limb muscle weights do not differ
from control and the contractile properties of selected
limb muscles (extensor digitorum longus, soleus) are
normal.
In contrast, cardiac-specic overexpression of TNFa in transgenic animals is associated with profound
weakening of the diaphragm (Li et al. 2000). Force
production is 50% of control across the entire range of
activation frequencies. This weakness does not have an
obvious structural basis. Diaphragm weight is normal
and there is no evidence of ultrastructural damage or
apoptosis. Cytosolic oxidant levels are elevated in
muscle bres of the diaphragm, suggesting oxidant
production is exaggerated. This oxidative stimulus
appears to mediate the contractile dysfunction. Acute
incubation with N-acetylcysteine, a reduced thiol donor
with antioxidant properties, reverses much of the force
decit seen in the diaphragm of transgenic animals.
Changes that occur in the diaphragm appear to be stimulated by cardiac-derived TNF-a as similar changes
can be induced in the diaphragm of wild-type animals
by acute exposure to exogenous TNF-a in vitro. TNF-a
stimulates a rise in cytosolic oxidants within wild-type
muscle bres and diaphragm force production falls.
The latter response is inhibited by N-acetylcysteine.
How does cardiac overexpression of TNF-a weaken
the diaphragm without affecting limb muscles?
Retention of normal function may reect a less intense
TNF-a stimulus as limb muscles have lower blood ow
than the diaphragm and are not exposed to pericardial
uid. Alternatively, limb muscles could be insensitive to
this action of TNF-a. The intracellular mechanism,
whereby TNF-a disrupts contractile function of the
diaphragm, also remains enigmatic. In non-muscle cell
types, TNF-a has been shown to alter cytosolic calcium
2001 Scandinavian Physiological Society

M B Reid and Y-P Li Cytokines, oxidants, and muscle

via effects on intracellular stores (Chakraborti et al.

1999). This represents a feasible target for TNFa-induced oxidants as oxidative modication alters the
function of proteins in the sarcoplasmic reticulum,
including the ryanodine-sensitive calcium release
channel (Hamilton & Reid 2000) and the calcium-sensitive ATPase (Klebl et al. 1998). Alternatively, force
may be inhibited via an effect of TNF-a-induced oxidants on the myolaments. Prior studies of intact single
bres have established that exogenous ROS reversibly
inhibit myolament function in mouse skeletal muscle
(Andrade et al. 1998); TNF-a-induced ROS might exert
a similar effect.
POTENTIAL INVOLVEMENT
IN MYOGENESIS AND MUSCLE
A D AP T A TI O N
TNF-a has growth factor-like effects that stimulate
proliferation and differentiation in various cell types
(Tracey & Cerami 1993). There is circumstantial evidence to suggest similar effects on immature skeletal
muscle myocytes. TNF-a rapidly activates NF-jB (see
above). In turn, NF-jB stimulates myoblast cell cycle
progression by regulating the cyclin D1 gene (Guttridge
et al. 1999) and enhances the activity of serum response
factor (SRF), a transcription factor essential for myoblast differentiation and proliferation (Franzoso et al.
1996, Perona et al. 1997). NF-jB may also be required
for myoblast fusion (Lee et al. 1997) and the expression
of genes for muscle-specic proteins including myosin
heavy chains, caveolin 3, and GLUT4 (Kaliman et al.
1999). By activating NF-jB, TNF-a may function as a
myogenic regulator. Recent evidence is consistent with
a physiological role for this cytokine. TNF-a is constitutively synthesized at low levels by skeletal muscles
of healthy humans (Saghizadeh et al. 1996). Moreover,
TNF-a expression is upregulated in humans following
strenuous exercise (Camus et al. 1998, Ostrowski et al.
1999). This suggests a functional role for TNF-a in the
post-exercise state, perhaps as an activator of satellite
cell differentiation.
R O L E S F O R O T H E R CY T O K I N E S
An understanding of TNF-a signalling in isolation is
not sufcient. In the intact organism, TNF-a never
functions as a solitary stimulus. Inammatory processes
that elevate TNF-a levels also increase circulating levels
of interleukin-1 (IL-1), interleukin-6 (IL-6), interferon-c
(IFN-c) and other catabolic cytokines (Argiles &
Lopez-Soriano 1998, Chang & Bistrian 1998). Thus,
TNF-a signalling is invariably modulated by other
receptor-mediated stimuli. It is likely that cytokine
co-stimulation is synergistic, exaggerating TNF-a
229

Cytokines, oxidants, and muscle

M B Reid and Y-P Li

effects on muscle. For example, IL-1 might well be

synergistic. Chronic IL-1 administration is associated
with negative nitrogen balance, loss of lean body mass,
and decrements in muscle weight and protein content
(Cooney et al. 1999). IL-1 stimulates oxidant production ROS, RNS or both in cardiac myocytes (Cheng
et al. 1999), vascular smooth muscle (Privat et al. 1999),
endothelial cells (Tampo et al. 1999) and other cell types
(Tolias et al. 1999). In non-muscle cells, IL-1 also has
been shown to activate NF-jB and increase expression
of NF-jB-dependent genes (Das et al. 1995). In differentiated muscle, it is conceivable that IL-1 could
exaggerate TNF-a effects on muscle protein loss,
contractile function and the upstream events that
regulate these responses. One possible source of IL-1
and other co-stimulatory cytokines is the muscle itself.
Skeletal muscle has the capacity to express catabolic
cytokines including TNF-a (De Bleeker et al. 1999), IL1 (Belec et al. 1997) and IL-6 (Bartoccioni et al. 1994).
This capacity raises the theoretical possibility of positive feedback. TNF-a is a stereotypical stimulus for
cytokine expression by non-muscle cells. If TNF-a
were to similarly stimulate synthesis and release of
cytokines by muscle bres, this would provide a
mechanism for amplifying the inammatory response
within muscle tissue.
Our research in this area has been supported by NIH grant
#HL59878 and by a grant-in-aid from the American Heart Association/Texas Chapter.

R E F E RE N C E S
Ahmad, S., Karlstad, M.D., Choudhry, M.A. & Sayeed, M.M.
1994. Sepsis-induced myobrillar protein catabolism in rat
skeletal muscle. Life Sci 55, 13831391.
Andrade, F.H., Reid, M.B., Allen, D.G. & Westerblad, H.
1998. Effect of hydrogen peroxide and dithiothreitol on
contractile function of single skeletal muscle bres from
mouse. J Physiol 509, 565575.
Anker, S.D. & Rauchaus, M. 1999. Insights into the
pathogenesis of chronic heart failure: immune activation
and cachexia. Curr Opin Cardiol 14, 211216.
Argiles, J.M. & Lopez-Soriano, F.J. 1998. Catabolic
proinammatory cytokines. Curr Opin Clin Nutr Metab Care
1, 245251.
Baeuerle, P.A. & Baltimore, D. 1996. NF-kappa B: ten years
after. Cell 87, 1320.
Barnes, P.J. & Karin, M. 1997. Nuclear factor-KB a pivotal
transcription factor in chronic inammatory disease. New
Engl J Med 336, 10661071.
Bartoccioni, E., Michaelis, D. & Hohlfeld, R. 1994.
Constitutive and cytokine-induced production of
interleukin-6 by human myoblasts. Immunol Lett 42,
135138.
Bejima, J. & Ji, L.L. 1999. Aging and acute exercise enhance
free radical generation in rat skeletal muscle. J Appl Physiol
87, 465470.
230

Acta Physiol Scand 2001, 171, 225232

Belec, L., Authier, F.J., Chazaud, B., Piedouillet, C., BarlovatzMeimon, G. & Gherardi, R.K. 1997. Interleukin (IL)-1
beta and IL-1 beta mRNA expression in normal and diseased
skeletal muscle assessed by immunocytochemistry,
immunoblotting, and reverse transcriptase-nested polymerase chain reaction. J Neuropathol Exp Neurol 56, 651663.
Beutler, B. & Cerami, A. 1986. Cachectin and tumor necrosis
factor as two sides of the same biological coin. Nature 320,
584588.
Beutler, B., Mahoney, J., LeTrang, N., Pekala, P. & Cerami, A.
1985. Purication of cachectin, a lipoprotein lipasesuppressing hormone secreted by endotoxin-induced
Raw264.7 cells. J Exp Med 161, 984995.
Buck, M. & Chojkier, M. 1996. Muscle wasting and
dedifferentiation induced by oxidative stress in a murine
model of cachexia is prevented by inhibitors of nitric oxide
synthesis and antioxidants. EMBO J 15, 17531765.
Budgett, R. 1998. Fatigue and underperformance in athletes:
the overtraining syndrome. Br J Sports Med 32, 107110.
Camus, G., Nys, M., Poortmans, J.R. et al. 1998.
Endotoxaemia, production of tumour necrosis factor alpha
and polymorphonuclear neutrophil activation following
strenuous exercise in humans. Eur J Appl Physiol 79, 6268.
Chakraborti, T., Das, S., Mondal, M., Roychoudhury, S. &
Chakraborti, S. 1999. Oxidants, mitochondria, and calcium:
an overview. Cell Signal 11, 7785.
Chance, B., Sies, H. & Boveris, A. 1979. Hydroperoxide
metabolism in mammalian organs. Physiol Rev 59, 527605.
Chang, H.R. & Bistrian, B. 1998. The role of cytokines in the
catabolic consequences of infection and injury. J Parenter
Enteral Nutr 22, 156166.
Cheng, X.S., Shimokawa, H., Momii, H. et al. 1999. Role of
superoxide anion in the pathogenesis of cytokine-induced
myocardial dysfunction in dogs in vivo. Cardiovasc Med 42,
651659.
Cooney, R.N., Maish, G.O. III, Gilpin, T., Shumate, M.L.,
Lang, C.H. & Vary, T.C. 1999. Mechanism of IL-1 induced
inhibition of protein synthesis in skeletal muscle. Shock 11,
235241.
Das, K.C., Lewis-Molock, Y. & White, C.W. 1995. Thiol
modulation of TNF-alpha and IL-1 induced MnSOD gene
expression and activation of NF-kappa B. Mol Cell Biochem
148, 4557.
De Bleeker, J.L., Meire, V.I., Declerq, W. & Van Akan, E.H.
1999. Immunolocalization of tumor necrosis factor-alpha
and its receptors in inammatory myopathies. Neuromuscul
Disord 9, 239246.
Diaz, P.T., Julian, M.W., Wewers, M.D. & Clanton, T.L.
1993. Tumor necrosis factor and endotoxin do not
directly affect in vitro diaphragm function. Am Rev Resp
Dis 148, 281287.
Espat, N.J., Copeland, E.M. & Moldawer, L.L. 1994. Tumor
necrosis factor and cachexia: a current perspective. Surg
Oncol 3, 255262.
Farber, M.O. & Mannix, E.T. 2000. Tissue wasting in patients
with chronic obstructive pulmonary disease. Neurol Clin 18,
245262.
Franzoso, G., Carlson, L., Brown, K., Daucher, M.B.,
Bressler, P. & Siebenlist, U. 1996. Activation of the

2001 Scandinavian Physiological Society

Acta Physiol Scand 2001, 171, 225232

serum response factor by p65/NF-kappaB. EMBO J 15,

3412.
Friman, G. & Ilback, N.G. 1998. Acute infection: metabolic
responses, effects on performance, interaction with
exercise, and myocarditis. Int J Sports Med 19 (Suppl. 3),
S172S182.
Garcia-Martinez, C., Agell, N., Llovera, M., Lopez-Soriano,
F.J. & Argiles, J.M. 1993a. Tumour necrosis factor-alpha
increases the ubiquitinization of rat skeletal muscle
proteins. FEBS Lett 323, 211214.
Garcia-Martinez, C., Lopez-Soriano, F.J. & Argiles, J.M.
1993b. Acute treatment with tumour necrosis factor-alpha
induces changes in protein metabolism in rat skeletal
muscle. Mol Cell Biochem 125, 1118.
Garcia-Martinez, C., Llovera, M., Agell, N., Lopez-Soriano,
F.J. & Argiles, J.M. 1994. Ubiquitin gene expression in
skeletal muscle is increased by tumour necrosis factoralpha. Biochem Biophys Res Commun 201, 682686.
Garcia-Martinez, C., Llovera, M., Agell, N., Lopez-Soriano,
F.J. & Argiles, J.M. 1995. Ubiquitin gene expession in
skeletal muscle is increased during sepsis: involvement of
TNF-alpha but not IL-1. Biochem Biophys Res Comm 217,
839844.
Goldberg, A.L., Kettelhut, I.C., Furuno, K., Fagen, J.M. &
Barascos, V. 1988. Activation of protein breakdown and
prostaglandin E2 production in rat skeletal muscle in fever
is signaled by a macrophage product distinct from
interleukin 1 or other known monokines. J Clin Invest 81,
13781383.
Goodman, M.N. 1991. Tumor necrosis factor induces skeletal
muscle protein breakdown in rats. Am J Physiol 260,
E727E730.
Guttridge, D.C., Albanese, C., Reuther, J.Y., Pestell, R.G. &
Baldwin, A.S.J. 1999. NF-kappaB controls cell growth and
differentiation through transcriptional regulation of cyclin
D1. Mol Cell Biol 19, 57855799.
Hamilton, S. & Reid, M.B. 2000. RyR1 modulation by
oxidation and calmodulin. Antiox Redox Signal 2, 4145.
Hancock, J.T. 1987. Superoxide, hydrogen peroxide and nitric
oxide as signalling molecules: their production and role in
disease. Br J Biomed Sci 54, 3846.
Hochstrasser, M. 1995. Ubiquitin, proteasomes, and the
regulation of intracellular protein degradation. Curr Opin
Cell Biol 7, 215223.
Hopkins, P.M. 1996. Human recombinant TNF-alpha affects
rat diaphragm muscle in vitro. Intensive Care Med 22, 359362.
Hsu, H., Shu, H.-B., Pan, M.-G. & Goeddel, D.V. 1996.
TRADDTRAF2 and TRADDFADD interactions dene
two distinct TNF receptor 1 signal transduction pathways.
Cell 84, 299308.
Kaliman, P., Canicio, J., Testar, X., Palacin, M. & Zorzano, A.
1999. Insulin-like growth factor-II, phosphatidylinositol 3kinase, nuclear factor-kappaB and inducible nitric oxide
synthase dene a common myogenic signaling pathway.
J Biol Chem 274, 1743717444.
Khanna, S., Roy, S., Packer, L. & Sen, C.K. 1999. Cytokineinduced glucose uptake in skeletal muscle: redox regulation
and the role of alpha-lipoic acid. Am J Physiol 276,
R1327R1333.

2001 Scandinavian Physiological Society

M B Reid and Y-P Li Cytokines, oxidants, and muscle

Klebl, B.M., Ayoub, A.T. & Pette, D. 1998. Protein oxidation,

tyrosine nitration, and inactivation of sarcoplasmic
reticulum Ca2+-ATPase in low-frequency stimulated rabbit
muscle. FEBS Lett 422, 381384.
Lee, K.H., Kim, D.G., Shin, N.Y. et al. 1997. NF-kappaBdependent expression of nitric oxide synthase is required
for membrane fusion of chick embryonic myoblasts.
Biochem J 324, 237242.
Li, Y.-P. & Reid, M.B. 2000. NF-kB mediates the protein loss
induced by TNF-a in differentiated skeletal muscle
myotubes. Am J Physiol 279, R1165R1170.
Li, Y.-P., Schwartz, R.J., Waddell, I.D., Holloway, B.R. &
Reid, M.B. 1998. Skeletal muscle myocytes undergo
protein loss and reactive oxygen-mediated NF-kB
activation in response to tumor necrosis factor a. FASEB
J 12, 871880.
Li, Y.-P., Atkins, C.M., Sweatt, J.D. & Reid, M.B. 1999.
Mitochondria mediate tumor necrosis factor-a/NF-kB
signaling in skeletal muscle myotubes. Antiox Redox Signal 1,
97104.
Li, X., Moody, M.R., Engel, D. et al. 2000. Cardiac-specic
overexpression of TNF-a causes oxidative stress and
contractile dysfunction in mouse diaphragm. Circulation
102, 16901696.
Liu, Z.-G., Hsu, H., Goeddel, D.V. & Karin, M. 1996.
Dissection of TNF receptor 1 effector functions: JNK
activation is not linked to apoptosis while NF-kB activation
prevents cell death. Cell 87, 565576.
Llovera, M., Garcia-Martinez, C., Lopez-Soriano, F.J. &
Argiles, J.M. 1997. TNF can directly induce the expression
of ubiquitin-dependent proteolytic system in rat soleus
muscles. Biochem Biophys Res Comm 230, 238241.
Llovera, M., Garcia-Martinez, C., Agell, N. et al. 1998a.
Ubiquitin and proteasome gene expression is increased in
skeletal muscle of slim AIDS patients. Int J Mol Med 2,
6973.
Llovera, M., Garcia-Martinez, C., Lopez-Soriano, J. et al.
1998b. Protein turnover in skeletal muscle of tumourbearing mice overexpressing the soluble TNF receptor-1.
Cancer Lett 130, 1927.
Llovera, M., Garcia-Martinez, C., Lopez-Soriano, J. et al.
1998c. Role of TNF receptor 1 in protein turnover during
cancer cachexia using gene knockout mice. Mol Cell
Endocrinol 142, 183189.
McArdle, A., van der Meulen, H.H., Catapano, M., Symons,
M.C., Faulkner, J.A. & Jackson, M.J. 1999. Free radical
activity following contraction-induced injury to the
extensor digitorum longus muscles of rats. Free Radic Biol
Med 26, 10851091.
Mitch, W.E. & Goldberg, A.L. 1996. Mechanisms of muscle
wasting; the role of the ubiquitinproteasome pathway.
New Engl J Med 335, 18971905.
Moldawer, L.L. & Sattler, F.R. 1998. Human immunodifcency virus-associated wasting and mechanisms
associated with inammation. Semin Oncol 25, 7381.
Murrant, C.L., Andrade, F.H. & Reid, M.B. 1999. Exogenous
reactive oxygen and nitric oxide alter intracellular oxidant
status of skeletal muscle bres. Acta Physiol Scand 166,
111121.

231

Cytokines, oxidants, and muscle

M B Reid and Y-P Li

Nethery, D., Stofan, D., Callahan, L., Dimarco, A. & Supinski,

G. 1999. Formation of reactive oxygen species by the
contracting diaphragm is PLA(2) dependent. J Appl Physiol
87, 792800.
Oe, H., Kuzuya, T., Hoshida, S. et al. 1994. Calcium overload
and cardiac myocyte cell damage induced by arachidonate
lipoxygenation. Am J Physiol 267, H1396H1402.
Okabe, E., Kato, Y., Kohno, H., Hess, M.L. & Ito, H. 1985.
Inhibition by free radical scavengers and by cyclooxygenase inhibitors of the effect of acidosis on calcium
transport by masseter muscle sarcoplasmic reticulum.
Biochem Pharmacol 34, 961968.
Oliff, A.D., Defeo-Jones, M., Boyer, M. et al. 1987. Tumors
secreting human TNF/cachectin induce cachexia in mice.
Cell 50, 555563.
Ostrowski, K., Rohde, T., Asp S., Schjerling, P. & Pedersen,
B.K. 1999. Pro- and anti-inammatory cytokine balance in
strenuous exercise in humans. J Physiol 515, 287291.
Perona, R., Montaner, S., Saniger, L., Sanchez-Perez, I.,
Bravo, R. & Lacal, J.C. 1997. Activation of the nuclear
factor-kappaB by Rho, CDC42, and Rac-1 proteins. Genes
Dev 11, 463475.
Privat, C., Stepien, O., David-Dulho, M. et al. 1999.
Superoxide release from interleukin-1B-stimulated human
vascular cells: in situ electrochemical measurement. Free
Radic Biol Med 27, 554559.
Quillet-Mary, A., Jaffrezou, J.-P., Mansat, V., Bordier, C.,
Naval, J. & Laurent, G. 1997. Implication of mitochondrial
hydrogen peroxide generation in ceramide-induced
apoptosis. J Biol Chem 272, 2138821395.
Reid, M.B., Kobzik, L., Bredt, D.S. & Stamler, J.S. 1998.
Nitric oxide modulates excitationcontraction coupling in
the diaphragm. Comp Biochem Physiol 119, 211218.
Rofe, A.M., Conyers, R.A.J., Bais, R., Gamble, J.R. & Vadas,
M.A. 1987. The effects of recombinant tumour necrosis
factor (cachectin) on metabolism in isolated rat adipocyte,
hepatocyte and muscle preparations. Biochem J 247, 789792.
Saghizadeh, M., Ong, W.T., Garvey, W.T., Henry, R.R. &
Kern, P.A. 1996. The expression of TNF-alpha by human
muscle. Relationship to insulin resistance. J Clin Invest 97,
11111116.
Schutze, S., Machleidt, T. & Kronke, M. 1992. Mechanisms of
tumor necrosis factor action. Sem Oncol 2, 1624.
Sen, C.K. & Packer, L. 1996. Antioxidant and redox
regulation of gene transcription. FASEB J 10, 112.
Sen, C.K., Khanna, S., Resznick, A.Z., Roy, S. & Packer, L.
1997. Glutathione regulation of tumor necrosis factor-ainduced NF-kB activation in skeletal muscle-derived L6
cells. Biochem Biophys Res Comm 237, 645649.
Shindoh, C., Hida, W., Ohkawara, Y. et al. 1995. TNF-alpha
mRNA expression in diaphragm muscle after endotoxin
administration. Am J Respir Crit Care Med 152, 16901691.
Simon, A.M. & Zittoun, R. 1999. Fatigue in cancer patients.
Curr Opin Oncol 11, 244249.

232

Acta Physiol Scand 2001, 171, 225232

Stofan, D., Callahan, L.A., DiMarco, A.F., Nethery, D.E. &

Supinski, G.S. 2000. Modulation of release of reactive
oxygen species by the contracting diaphragm. Am J Respir
Crit Care Med 161, 891898.
Subramaniam, A., Jones, W.K., Gulick, J., Wert, S., Neumann,
J. & Robbins, J. 1991. Tissue-specic regulation of the amyosin heavy chain gene promoter in transgenic mice. J
Biol Chem 266, 2461324620.
Tampo, Y., Tsukamoto, M. & Yonaha, M. 1999. Superoxide
production from paraquat evoked by exogenous NADPH
in pulmonary endothelial cells. Free Radic Biol Med 27,
588595.
Tartaglia, L.A. & Goeddel, D.V. 1992. Two TNF receptors.
Immunol Today 13, 151153.
Tawa, N.E., Odessey, R. & Goldberg, A.L. 1997. Inhibitors of
the proteasome reduce the accelerated proteolysis in
atrophying rat skeletal muscle. J Clin Invest 100, 197203.
Tayek, J.A. 1996. Effects of tumor necrosis factor-a on
skeletal muscle amino acid metabolism studied in vivo.
J Am Coll Nutr 15, 164168.
Tessitore, L., Costelli, P. & Baccino, F.M. 1993. Humoral
mediation for cachexia in tumour-bearing rats. Br J Cancer
67, 1523.
Tisdale, M.J. 1999. Wasting in cancer. J Nutr 129, 243S246S.
Tolias, C.M., McNeil, C.J., Kazlauskaite, J. & Hillhouse, E.W.
1999. Astrocytes rather than neurones mediate interleukin1beta dependent nitric oxide and superoxide radical release
in primary hypothalamic rat cell cultures. Neurosci Lett 273,
5760.
Tracey, K.J. & Cerami, A. 1993. Tumor necrosis factor, other
cytokines and disease. Annu Rev Cell Biol 9, 317343.
Tracey, K.J., Wei, H., Manogue, K.R. et al. 1998. Cachectin/
tumor necrosis factor-a induces cachexia, anemia, and
inammation. J Exp Med 167, 12111227.
Wilcox, P., Osborne, S. & Bressler, B. 1992. Monocyte
inammatory mediators impair in vitro hamster diaphragm
contractility. Am Rev Resp Dis 146, 462466.
Wilcox, P.G., Wakai, Y., Walley, K.R., Cooper, D.J. & Road,
J. 1994. Tumor necrosis factor alpha decreases in vivo
diaphragm contractility in dogs. Am J Respir Crit Care Med
150, 13681373.
Wilcox, P., Milliken, C. & Bressler, B. 1996. High-dose
tumor necrosis factor alpha produces an impairment of
hamster diaphragm contractility. Attenuation with a
prostaglandin inhibitor. Am J Respir Crit Care Med 153,
16111615.
Williams, G., Brown, T., Becker, M., Prager, M. & Giroir, B.P.
1994. Cytokine-induced expression of nitric oxide synthase
in C2C12 skeletal muscle myocytes. Am J Physiol 267,
R1020R1025.
Williams, A., Sun, X., Fischer, J.E. & Hasselgren, P.O. 1999.
The expression of genes in the ubiquitinproteasome
proteolytic pathway is increased in skeletal muscle from
patients with cancer. Surgery 126, 744749.

2001 Scandinavian Physiological Society