J Appl Physiol 102: 1664 –1670, 2007;
doi:10.1152/japplphysiol.01102.2006.
Invited Review
HIGHLIGHTED TOPIC
Free Radical Biology in Skeletal Muscle
The production of reactive oxygen and nitrogen species by skeletal muscle
Malcolm J. Jackson, Deborah Pye, and Jesus Palomero
Division of Metabolic and Cellular Medicine, School of Clinical
Sciences, University of Liverpool, Liverpool, United Kingdom
free radicals; mitochondria; reduced nicotinamide adenine dinucleotide phosphate
oxidase
ALTHOUGH THERE WAS SOME RECOGNITION in the 1950s that skeletal muscle could generate free radical intermediates (10), the
first substantive demonstrations that exercise was associated
with an increase in the formation of the products of the
reactions of free radicals, reactive oxygen species (ROS) or
reactive nitrogen species (RNS), with other biomolecules occurred in the late 1970s (5, 16), and these data were subsequently expanded and confirmed by Kelvin Davies and colleagues in Lester Packer’s laboratory at Berkeley (13, 55).
Many of the assumptions that remain prevalent about free
radicals generated during exercise, such as skeletal muscle
mitochondria being the predominant source for formation of
free radicals and that the species formed were potentially
damaging, appear to have been formulated and expounded in
those early studies.
The techniques used to examine free radical activity in these
early studies utilized the approaches available at that time,
mainly measurements of the products of free radical reaction
with lipids (5, 16), although Davies also utilized electron spin
resonance spectroscopy to examine the relatively stable species
that is seen by direct analysis of tissues (13). Subsequent
studies examined other measures of free radical activity, but
the demonstration of specific species that are generated by
skeletal muscle during contractions occurred only after a further ⬃10 years of research with Reid’s description of superoxide release from the contracting diaphragm (56), the demonstration of nitric oxide (NO) generation by skeletal muscle
(2), and the detection of hydroxyl radical formation by contracting skeletal muscle tissue (51).
Recent studies have clarified a number of the uncertainties
relating to ROS generation in skeletal muscle, with greater
Address for reprint requests and other correspondence: M. J. Jackson, Division
of Metabolic and Cellular Medicine, School of Clinical Sciences, Univ. of
Liverpool, Liverpool L69 3GA, UK (e-mail: [email protected]).
1664
recognition of the nature of the species formed, the identification of multiple sites for their generation, and a further understanding of their potential roles. Our current understanding of
the sites of generation of ROS within skeletal muscle fibers is
shown in schematic form in Fig. 1.
SPECIES GENERATED BY SKELETAL MUSCLE
The primary free radicals generated by skeletal muscle, both
at rest and during activity, are NO and superoxide, the latter of
which dismutates rapidly to form hydrogen peroxide (40).
These substances provide the precursors for generation of
peroxynitrite (9, 53) and hydroxyl radicals in the presence of
catalytic transition metals (53). The limitations of the techniques available have meant that few studies have clearly
identified the nature of the ROS or RNS present within muscle
fibers, and most work has examined species released or formed
on the extracellular surface of muscle cells (53, 56). This
approach is clearly suitable for substances such as hydrogen
peroxide or NO, which can cross the plasma membrane but has
limited application for highly reactive substances, such as the
hydroxyl radical or the superoxide anion.
Some studies have also identified secondary free radical
species that are generated during exercise, such as lipid radicals that may originate in muscle tissue (1). The source of these
species has not been identified, although some data suggest
they may be generated by reaction of hydroxyl radical with
lipids released from muscle tissue or by enzymatic oxidation of
lipid species (52).
To ensure that these species do not inevitably injure skeletal
muscle, the tissue has a well-developed system to regulate
ROS and prevent potentially deleterious effects. These protective systems include both mitochondrial and cytosolic isoforms
of superoxide dismutase (MnSOD and CuZnSOD, respectively), catalase and glutathione peroxidase enzymes, and a
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Jackson MJ, Pye D, Palomero J. The production of reactive oxygen and
nitrogen species by skeletal muscle. J Appl Physiol 102: 1664 –1670, 2007.
doi:10.1152/japplphysiol.01102.2006.—Skeletal muscle has been recognized as a
potential source for generation of reactive oxygen and nitrogen species for more
than 20 years. Initial investigations concentrated on the potential role of mitochondria as a major source for generation of superoxide as a “by-product” of normal
oxidative metabolism, but recent studies have identified multiple subcellular sites,
where superoxide or nitric oxide are generated in regulated and controlled systems
in response to cellular stimuli. Full evaluation of the factors regulating these
processes and the functions of the reactive oxygen species generated are important
in understanding the redox biology of skeletal muscle.
Invited Review
FREE RADICAL GENERATION BY MUSCLE
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Fig. 1. Schematic representation of potential mechanisms for generation of reactive oxygen species (ROS) in skeletal muscle. CAT, catalase; CuZnSOD, copper,
zinc superoxide dismutase (SOD1); ecSOD, extracelluar superoxide dismutase (SOD3); GPx, glutathione peroxidase 1; eNOS, endothelial nitric oxide synthase
(NOS3); NO, nitric oxide; MnSOD, manganese superoxide dismutase (SOD2); nNOS, neuronal nitric oxide synthase (NOS1); PM oxido-reductase, plasma
membrane-located oxido-reductase; PLA2, phospholipase A2; VDAC, voltage-dependent anion channel; GPx, glutathione peroxidase. [Modified from Ref. 28.]
J Appl Physiol • VOL
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Invited Review
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FREE RADICAL GENERATION BY MUSCLE
number of direct scavengers of ROS, including glutathione,
vitamin E, and ascorbate. In general, slow-twitch, mitochondria-rich (type I) fibers have an increased content of protective
systems compared with fast (type II) fibers.
ENDOGENOUS SITES FOR ROS GENERATION IN
SKELETAL MUSCLE
Superoxide
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Mitochondria. Standard texts cite mitochondria as the major
site of superoxide generation in tissues (22), and many authors
have reiterated early reports that 2–5% of the total oxygen
consumed by mitochondria may undergo one electron reduction with the generation of superoxide (4, 38). There has been
considerable debate about the site(s) of superoxide generation
within mitochondria, but most data now indicate that complexes I and III of the electron transport chain are the main sites
of mitochondrial superoxide production (3, 48). In complex I,
the main site of electron leakage to oxygen appears to be the
iron-sulphur clusters, and in complex III it appears to be the Qo
semiquinone (48). Recent data also indicate that complex III
releases superoxide to both sides of the inner mitochondrial
membrane, providing a potential source for cytosolic superoxide generation, although CuZnSOD has also been reported to
be partially located in the mitochondrial intermembrane space
where it may minimize this possibility (48). When related to
exercise, many researchers have assumed that the increased
ROS generation that occurs during contractile activity is directly related to the elevated oxygen consumption that occurs
with increased mitochondrial activity, implying potentially a
50- or 100-fold increase in superoxide generation by skeletal
muscle during aerobic contractions (e.g., see Refs. 31, 64).
A number of recent findings have argued against a major
formation of superoxide within mitochondria. Brand and colleagues have reassessed the rate of production of ROS by
mitochondria and indicated that the upper estimate of the
proportion of the electron flow giving rise to ROS was
⬃0.15%, or ⬍10% of the original minimum estimate (60), and
it is becoming increasingly clear that even this low rate of
production may be further reduced by intrinsic control mechanisms. Thus this same research group has highlighted the
potential role of uncoupling proteins (specifically uncoupling
protein-3 in skeletal muscle) as regulators of mitochondrial
production of ROS (6, 7), acting to protect mitochondria
against oxidative damage. In addition, there has been considerable debate about the effect of changes in the stage of
mitochondrial respiration on ROS generation by mitochondria
(17, 25, 35), and some evidence indicates that, during aerobic
contractile activity, skeletal muscle mitochondria are predominantly in state III, and this limits their generation of ROS
during contractions (17, 25, 35).
Measurements of intracellular ROS generation by contracting skeletal muscle have only been rarely undertaken, but
where these are available for skeletal muscle cells, the data
have indicated that intracellular ROS activity only increased by
a modest two- to fourfold during contractions (43, 65), which
appears to support the more recent assessments of the likely
magnitude of mitochondrial ROS generation. As a final twist to
this evolving picture of the role of mitochondria in ROS
generation, Kozlov and colleagues (35) have recently used new
spin probe approaches to examine ROS generation by skeletal
muscle mitochondria and concluded that no ROS were released
from isolated mitochondria of young rats (although a significant release was detected from isolated mitochondria from the
tissues of old rats).
Sarcoplasmic reticulum. Studies have identified NAD(P)H
oxidase enzymes associated with the sarcoplasmic reticulum
(SR) of cardiac (8) and skeletal muscle (67). The superoxide
generated by these enzymes appears to influence calcium
release by the SR through oxidation of the ryanodyne receptor
(8). The skeletal muscle enzyme described appears to preferentially use NADH as substrate (67). Some inhibitor studies
have indicated that extracellular superoxide release from stimulated myotubes was reduced by treatment with diphenyleneiodonium, a nonspecific inhibitor of NAD(P)H oxidases (53),
although the NADH oxidase described by Xia et al. (67) is
localized to the SR and hence seems unlikely to contribute to
the extracellular release.
Plasma membrane. Numerous studies have indicated that
skeletal muscle cells release superoxide into the extracellular
space (e.g., see Refs. 41, 53, 56, 69). All cells contain plasma
membrane redox systems capable of undertaking electron
transfer across the plasma membrane. Only the NADPH oxidase system of phagocytic and nonphagocytic cells has been
extensively described. An NAD(P)H oxidase complex has
been reported to be constitutively expressed in diaphragm and
limb muscles of the rat and localized to the region of the
plasma membrane (30). The enzyme contains four of the
subunits that are found in the enzyme in phagocytic cells
(gp91phox, p22phox, p47phox, and p67phox), all of which were
associated with the cell membranes (37). Whether this complex
predominantly releases superoxide to the inside or the outside
of the plasma membrane cannot be ascertained from the experiments reported (30).
Other plasma membrane redox systems are less well characterized but are capable of transferring electrons from intracellular reductants to appropriate extracellular electron acceptors. No such system has been described in skeletal muscle, but
other cell types contain networks of enzymes that perform this
function (59). Morré (47) has described external NADH oxidase (ECTO-NOX) proteins that exhibit a hydroquinone
(NADH) oxidase activity and a protein disulphide-thiol exchange activity. The current understanding of these systems is
that they accept electrons from the hydroquinones of the
plasma membrane, such as reduced coenzyme Q10 (45), and
can reduce a number of nonphysiological (e.g.. ferricyanide
and WST-1) and physiological (e.g., protein thiols or oxygen)
electron acceptors outside the cell, although oxygen is likely to
be a major acceptor in vivo (15). Transfer of electrons from
cytosolic NAD(P)H to the plasma membranes has been proposed to occur through either NADH-cytochrome b5 oxidoreductase or NAD(P)H quinone oxidoreductase (15). Thus,
through a series of linked steps, intracellular NAD(P)H can act
as substrate for superoxide generation on the cell surface. de
Grey (14) and Morré et al. (46) have hypothesized that such
systems may generate extracellular superoxide to help maintain
NADH homeostasis in glycolytic tissues and to help maintain
cell viability where mitochondria become defective during
ageing of tissues, such as skeletal muscle.
The relevance of these processes to skeletal muscle contractions has not been established, but it is feasible that such
systems are activated during contractile activity or that the
Invited Review
FREE RADICAL GENERATION BY MUSCLE
NO
NO is generated continuously by skeletal muscle, a production that is increased by contractions (2). Skeletal muscle
normally expresses the neuronal (type I or nNOS) and the
endothelial (type III or eNOS) isoforms of NO synthase
(NOS). nNOS is strongly expressed in fast-twitch muscle fibers
and localized to the muscle sarcolemma, where it is associated
with the dystrophin-glycoprotein complex. eNOS is localized
to the muscle mitochondria (34). Inducible NOS (type II) is
also expressed in skeletal muscle in some inflammatory conditions, but it does not play a significant role in normal muscle
(61). Release of NO was originally demonstrated from isolated
muscles in vitro (2), although the cellular source of the NO
released was unclear. Our recent analysis of myotubes in
culture has confirmed that skeletal muscle cells per se release
increased amounts of NO during contractile activity (53), a
release that was greatly reduced by the NOS inhibitor N Gnitro-L-arginine methyl ester. nNOS appears to be the prime
source of the NO released from skeletal muscle (26). Passive
stretching of muscle has also been shown to increase NO
release from rat skeletal muscle in vitro (63), and nNOS
expression is increased by repeated exposure of muscle to
contractile activity or passive stretching (56, 63).
POTENTIAL NONMUSCLE SITES FOR GENERATION OF ROS
THAT INFLUENCE SKELETAL MUSCLE
REDOX HOMEOSTASIS
Nonmuscle sources may play a major role in modifying
muscle redox state under certain conditions. Most notable
appears to be the role played by phagocytic white cells following insult to skeletal muscle. It is clear that substantial injury to
muscle fibers is accompanied by invasion of the area with
macrophages and other phagocytic cells (e.g., see Ref. 42).
This process appears to be essential for preparation of the
tissue to allow effective regeneration to occur, but involves the
release of substantial amounts of ROS from the phagocytic
cells (22, 39). The magnitude of this release can be such that
damage to previously undamaged muscle cells may result (68).
Fig. 2. Effect of NG-nitro-L-arginine methyl ester (LNAME) treatment on microdialysate measurements of
extracellular superoxide release from the resting gastrocnemius muscle of mice. Data from groups of
untreated (solid bars) and L-NAME-treated (open
bars) mice are shown. The additional superoxide detected following inhibition of NO synthesis appears to
be due the lack of formation of peroxynitrite. *P ⬍
0.05 compared with untreated mice. [Data redrawn
from Close et al. (9).]
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substrate level rises to increase electron transfer across the
membrane through these systems. During the initial contractions of muscle and where muscle contractions are maintained
at a high intensity, NADH levels are reported to rise acutely to
levels ⬎200 ␮M (24, 58), and it may be that this stimulates
activity of the putative NADH-dependent plasma membrane
oxido-reductase system, allowing regeneration of intracellular
NAD⫹, with the resulting one electron reduction of molecular
oxygen on the outside of the plasma membrane. The characteristics of the release of superoxide from skeletal muscle are
compatible with the involvement of such a system.
Other endogenous generation systems. A number of other
potential systems have been described within skeletal muscle.
Reid and colleagues have recently described phospholipase
(PL) A2-dependent, intracellular superoxide generation (21).
The enzyme involved was calcium independent and appeared
distinct to the calcium-dependent PLA2 that was also reported
to stimulate ROS production by Nethery et al. (49) and Zuo et
al. (69). Reid and colleagues rationalized these apparently
disparate findings by hypothesizing that the calcium-independent PLA2 was a major determinant of ROS activity under
resting conditions, whereas, during contractions, heat stress, or
other processes elevating intracellular calcium, the calciumdependent PLA2 was activated and stimulates ROS production
at supranormal rates. The subcellular location of these processes appears relatively undefined, although the calciumdependent PLA2 has been reported to contribute both to
mitochondrial ROS formation (50) and to the extracellular
release of superoxide through a lipoxygenase-dependent
system (70).
There has also been considerable speculation concerning
a role for muscle-associated xanthine oxidase in superoxide
generation by skeletal muscle, data that are primarily based
on the effects of the xanthine oxidase inhibitors, allopurinol
or oxypurinol (e.g., see Ref. 20). Skeletal muscle cells per se
have been reported not to contain significant amounts of
xanthine dehydrogenase or oxidase (23), although these
enzymes will inevitably be present in associated endothelial
cells.
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FREE RADICAL GENERATION BY MUSCLE
INTERACTIONS AND CROSS TALK BETWEEN ROS FORMED
IN SKELETAL MUSCLE
Interactions of NO and Superoxide
Transmembrane Signaling of ROS and RNS Activities
A number of pieces of evidence indicate that changes in the
intracellular ROS generation or redox environment in skeletal
muscle can influence ROS activities outside of the muscle cell
and vice versa. While this process partially reflects the ability
of skeletal muscle to generate relatively long-lived species that
can cross membranes such as hydrogen peroxide, there is also
evidence that more subtle effects occur. Thus Reid and coworkers (56) reported that the addition of scavenger enzymes
J Appl Physiol • VOL
CONCLUSIONS
Skeletal muscle clearly produces a variety of ROS and RNS
in controlled and regulated processes, both at rest and during
contractile activity. Although initial data suggested the major
generation of superoxide occurred “by accident” or as a “byproduct,” due to leakage from the mitochondrial electron
transport chain, recent data have cast considerable doubt on
this theory. In contrast, evidence is emerging of a closely
controlled generation of ROS and RNS at multiple subcellular
sites in response to a variety of mechanical and metabolic
stimuli. Understanding the factors regulating this production,
the processes underlying it, and the functions of the species
generated may potentially provide a means of optimizing
skeletal muscle function in a variety of disparate physiological
and disease states.
ACKNOWLEDGMENTS
The authors acknowledge the collaboration of many colleagues.
GRANTS
We thank the BBSRC, European Commission, Wellcome Trust, Research
into Ageing, and the US National Institute on Aging for financial support of
this work.
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