Sarcopenia Age-Related Muscle Wasting and Weakness: Mechanisms and Treatments P34 ppt

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Abstract Frailty in the elderly is largely caused by loss of muscle mass and
strength, increased susceptibility to injury, and impaired recovery following
damage, particularly contraction-induced damage. The mechanisms responsible
for the age-related loss of muscle mass and function are unclear although modified
generation of Reactive Oxygen and Nitrogen Species (RONS) have been implicated
in age-related tissue dysfunction. Many studies have provided evidence for the
pivotal role of ROS in signal transduction and recognized these molecules as
second messengers. Aberrant generation of RONS in the mitochondria and cyto-
sol of cells and tissues of old mammals leads to an altered activation of crucial
redox-responsive transcription factors at rest, following acute stress or during the
regenerative process. Data suggest that targeted interventions to suppress altered
mitochondrial ROS generation in muscle of old individuals are necessary to restore
the signal for adaptive responses to contractions. Interventions based on antioxidant
supplementation will suppress ROS signals in both mitochondrial and cytosolic
compartments and hence be ineffective at prevention of age-related loss of muscle
mass and function.
Keywords Skeletal muscle • Ageing • ROS • RONS • HSPs • Adaptive responses
• Mitochondria • Cytosol
A. McArdle (*) and M.J. Jackson
School of Clinical Sciences, University of Liverpool, UK
e-mail:
Reactive Oxygen Species Generation
and Skeletal Muscle Wasting – Implications
for Sarcopenia
Anne McArdle and Malcolm J. Jackson
G.S. Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness,
DOI 10.1007/978-90-481-9713-2_14, © Springer Science+Business Media B.V. 2011
318 A. McArdle and M.J. Jackson
1 Skeletal Muscle Atrophy and Weakness Contribute

to Physical Frailty in the Elderly
Frailty in the elderly (Hadley et al. 1993) is largely caused by loss of muscle
mass and strength, increased susceptibility to injury, and impaired recovery fol-
lowing damage, particularly contraction-induced damage (Faulkner et al. 2007;
Marcell 2003). By the age of 70, the cross-sectional area (CSA) of muscle is
reduced by 25–30% (Porter et al. 1995) associated with a loss in absolute force
generation (Grimby and Saltin 1983) and a decrease in specific force (per unit
CSA) generation (Morse et al. 2005). After 70, strength continues to fall and
power in the lower leg declines at ~3.5% per year (Skelton et al. 1994). These
deficits profoundly impact on the quality of life of even healthy older people,
as many are at, or near thresholds that limit the ability to carry out everyday
tasks (Young and Skelton 1994). This age-related muscle weakness signifi-
cantly increases the risk for elderly falling. Approximately 20% of community-
dwelling elderly fall each year (Prudham and Evans 1981). Many elderly who
fall suffer loss of independence and some never re-enter the community. One
half of the accidental deaths in those over 65 are related to falls. While regular
exercise can modify the rate of muscle deficits, even active elderly people show
significant age-related declines in muscle mass and function (Wiswell et al.
2001). The mechanisms responsible for the age-related loss of muscle mass and
function are unclear although modified generation of Reactive Oxygen and
Nitrogen Species (RONS) have been implicated in age-related tissue dysfunction
(Harman 2003).
2 The Source and Nature of the RONS Generated
by Skeletal Muscle
The source and nature of the RONS generated by muscle of young or adult
mammals during contractions has been studied since the 1980s. Initial studies
demonstrated increased generation of free radicals by contracting skeletal mus-
cles (Davies et al. 1982; Jackson et al. 1983). The main reactive oxygen species
(ROS) produced in the cell are free radical species, such as the superoxide anion
and hydroxyl radical, and non-radical species, such as hydrogen peroxide

(H2O2) (Palomero and Jackson 2010). The generation of specific RONS, including
superoxide, nitric oxide and hydroxyl radicals by muscle were then described
(Reid et al. 1992a,b; O’Neill et al. 1996; Balon and Nadler 1994; Kobzik et al
1994).
Cells are required to preserve a delicate balance between ROS generation and
elimination to maintain the correct redox status necessary to carry out vital
functions. In excess, ROS can attack cellular structures, such as lipids, proteins, and
DNA, thereby inducing irreversible changes that can lead to the disruption of
319Reactive Oxygen Species Generation and Skeletal Muscle Wasting – Implications
cellular functions and integrity. Under normal physiologic conditions, the reactive
nature of ROS allows their incorporation into the structure of macromolecules in a
reversible fashion. Such reversible oxidative modifications play a critical role in
different signalling pathways that regulate different cellular functions and the fate
of the cell (Sies and Jones 2007). Many studies have provided evidence for the
pivotal role of ROS in signal transduction and recognized these molecules as
second messengers (Powers and Jackson 2008).
Most authors have assumed that the ROS generated by contractions are pre-
dominantly generated by mitochondria due to the increased demand for energy,
but recent data argue against this possibility (for discussion see Jackson 2008).
In order to evaluate the relative magnitude of the increase in ROS activity that
occurs in skeletal muscle fibres in response to contractions, Palomero et al
(2008a) applied a protocol of electrically stimulated, isometric contractions to
single isolated fibres from the mouse Flexor digitorum brevis (FDB) muscle.
Fibres were loaded with 5- (and 6-) chloromethyl-2¢,7¢-dichlorodihydrofluorescein
diacetate (CM-DCFH DA) and measurements of 5- (and 6-) chloromethyl-2¢,7¢-
dichlorofluorescin (CM-DCF) fluorescence from individual fibers were obtained
by microscopy to study ROS in skeletal muscle. This technique has advantages
because of the maturity of the fibres compared with muscle cells in culture and
the analysis of single cells prevents contributions from non-muscle cells. The
contraction protocol used has been shown to (1) to induce release of superoxide

and nitric oxide from muscle cells in culture and muscles of mice in vivo
(McArdle et al. 2001; Pattwell et al. 2004), (2) to lead to a fall in muscle glu-
tathione and protein thiol content (Vasilaki et al. 2006c) and (3) to stimulate
redox-regulated adaptive responses (Vasilaki et al. 2006b) when applied to
intact muscles in vivo. The increase in intracellular DCF fluorescence induced
by the contraction protocol was less than that following exposure of the fibres
to 1 uM hydrogen peroxide. We (Palomero et al. 2008a) calculated that the
likely change in intracellular hydrogen peroxide following addition of 1 uM to
the extracellular medium is ~0.1 uM. Thus it can be inferred that the absolute
level of cytosolic ROS activity in muscle fibres that was achieved following
contractile activity was potentially equivalent to ~0.1 uM hydrogen peroxide.
Such levels of hydrogen peroxide have traditionally been associated with a
signalling role for the oxidant and our recent data indicate that the ROS gener-
ated by contractions are reduced by inhibitors of NADPH oxidase enzymes.
The increase in ROS activity with contractions is also observed where dihydro-
ethidium (DHE) is used as a probe. This probe is predominantly located in the
cytosol, but when DHE is modified to locate within mitochondria (as a probe
called Mito-HE or MitoSox) no increase in mitochondrial fluorescence was
seen during contractions. We conclude that the source of ROS that acts as a
signal for adaptive responses to contractions is not mitochondria, but is associ-
ated with the cytosol. Inhibitor studies indicate that this is likely to be a mem-
brane-located NADPH oxidase that is activated during contractions to generate
superoxide (which is converted to hydrogen peroxide) and these ROS activate
adaptive responses to contractions.
320 A. McArdle and M.J. Jackson
3 Modified ROS Generation Activates Redox-Sensitive
Transcription Factors in Contracting Muscle
Skeletal muscles of adult mice and humans adapt rapidly to contractile activity.
Numerous proteins show adaptive responses to contraction, including the antioxidant
defence enzymes and Heat Shock Proteins (HSPs) that protect against subsequent

cellular damage (Hollander et al. 2003; McArdle et al. 2004, 2005). ROS have
become increasingly recognised to mediate some adaptive responses of skeletal
muscle to contractile activity through activation of redox-sensitive transcription fac-
tors (Jackson et al. 2002; McArdle et al. 2004; Jackson 2005; Ji et al. 2006; Gomez-
Cabrera et al. 2008; Ristow et al. 2009). Nuclear factor kappa B (NFkB) is one such
factor, along with Activator Protein-1 (AP-1) and Heat Shock Factor 1 (Cotto and
Morimoto 1999). These transcription factors are involved in remodelling, production
of other cytoprotective proteins and production of inflammatory cytokines. ROS are
principal regulators of NFkB activation in many situations (Moran et al. 2001).
NFkB family members expressed in skeletal muscle play critical roles in modulating
the specificity of NFkB (Bar-Shai et al. 2005; Hayden and Ghosh 2008). In skeletal
muscle, NFkB modulates expression of genes associated with myogenesis (Bakkar
et al. 2008; Dahlman et al. 2009), catabolism-related genes (Bar-Shai et al. 2005;
Peterson and Guttridge 2008; Van Gammeren et al. 2009) and cytoprotective pro-
teins during adaptation to contractile activity (Vasilaki et al. 2006b). Moreover,
skeletal muscle has been identified as an endocrine organ producing cytokines via
NFkB activation following stresses such as systemic inflammation or physical strain
(Lee et al. 2007). The specificity of the responses of skeletal muscle cells to NFkB
activation is likely to be largely due to subtle differences in NFkB activation such as
B binding sequences and NFkB dimer formation that regulate expression of specific
genes (Bakkar et al. 2008). Activation of NFkB by ROS involves oxidation of key
cysteine residues in upstream activators of NFkB and the process can be inhibited
by antioxidants or reducing agents (Hansen et al. 2006) and more recently by HSPs
(Chen and Currie 2006).
Evidence from our laboratory and others have demonstrated that the HSP
content of skeletal muscles increases rapidly following a demanding but non-
damaging period of isometric contractions and this is termed the stress response
(McArdle et al. 2001; Vasilaki et al. 2006b) and this increased HSP content is part
of a more widespread adaptive response in transcription of cellular proteins
(McArdle F et al. 2004). Data also demonstrated that this was associated with sig-

nificant protection against subsequent damage (McArdle F et al. 2004). Definitive
data demonstrating a functional role of HSPs in protection against damage and
rapid recovery from damage was provided by a study using HSP70 overexpressor
mice whereby muscles of these mice were protected against the secondary deficit
characteristic of lengthening contraction – induced damage in mice and resulted in
a more rapid recovery of maximum force generation (McArdle et al. 2004).
The signal for increased HSP production following exercise has been a topic of
interest for some time and oxidative stress, hyperthermia and modified energy
321Reactive Oxygen Species Generation and Skeletal Muscle Wasting – Implications
supplies have all been proposed to play a role. Data from our laboratory has
provided evidence that the primary signal for activation of transcription of HSPs in
skeletal muscle in both rodents and humans following isometric contractions is an
increased production of reactive oxygen species (ROS). Studies in mice have dem-
onstrated that increased HSP production is associated with increased detection of
ROS in the muscle extracellular space (McArdle et al. 2001) and a transient fall in
protein sulphydryl groups and this occurred in the absence of any significant
change in muscle temperature. Supplementation of human subjects with nutritional
antioxidants abolished the exercise-induced increase in muscle HSP content
(Khassaf et al. 2001). Further studies in humans have demonstrated that although
the production of HSPs is dependent upon the intensity of exercise, exercise condi-
tions and the training status of the individuals (Morton et al. 2008; Palomero et al.
2008b), an equivalent rise in muscle temperature without exercise did not result in
increased muscle content of HSPs (Morton et al. 2007) although the cumulative
effect of heat and ROS production may result in a reduction in threshold for ROS-
induced HSP production.
Changes in HSP content of muscle can play a direct role in modification of ROS
production and thus feedback to modify the activation of the stress response.
Neuronal nitric oxide synthase (nNOS) produces nitric oxide but also produces
superoxide at low levels of L-arginine (Heinzel et al. 1992; Pou et al. 1992). nNOS
is localised to the plasma membrane of muscle cells, associated with the dystrophin

glycoprotein complex (Vranić et al. 2002) and HSP90 is also associated with
nNOS. HSP90 is thought to modify the action of nNOS since the presence of
HSP90 dose-dependently inhibits the superoxide anion radical generation from
nNOS. At lower levels of L-arginine where marked superoxide anion radical
generation occurred, HSP90 caused a more dramatic enhancement of NO synthesis
from nNOS as compared to that under normal L-arginine (Song et al. 2002). The
balance of production of NO and/or superoxide anion radical by nNOS may also be
linked to the cellular localisation of nNOS since it has also been proposed that, in
certain pathological conditions including Duchenne muscular dystrophy, deloca-
lised nNOS produces altered patterns of NO/superoxide although the role of HSP90
in this production is unknown. The interaction between HSPs and other ROS
generating systems is yet to be determined.
4 HSPs Interact with and Mediate Activation
of Transcription Factors
The dependence of a stress response in muscles following non-damaging exercise
on the initial level of HSPs in the quiescent muscle seems to be due to a feedback
mechanism by which increased cellular HSPs deactivate Heat Shock Factor 1
(HSF1), the main transcription factor thought to be responsible for the acute stress
response (Pirkkala et al. 2001). It is also possible that other adaptations to exercise
322 A. McArdle and M.J. Jackson
may play a role in this lack of response, such as an increase in ROS defences
(McArdle et al. 2001) which would also reduce the ROS signal. Thus, the threshold
for activation of the stress response changes in muscles with altered HSP content
or altered oxidant/antioxidant status (termed redox status).
Data have demonstrated interactions between cellular HSPs and the activation of
other transcription factors, particularly NFkB and AP-1, which are involved in
remodelling, production of other cytoprotective proteins and production of inflam-
matory cytokines. These studies have concentrated on the protective role of HSPs in
ameliorating the activation of the pro-inflammatory pathways of NFkB whereby
high levels of HSP70 and HSP27 have been shown to suppress the pro-inflammatory

pathway of NFkB (Chen and Currie 2006). Heat shock treatment suppresses NFkB
activation in mucosal cells of endotoxin treated mice by inhibiting the phosphoryla-
tion and degradation of the NFkB inhibitor, IkB-a and prior heat shock treatment
also inhibits IkB kinase (IKK) activation and results in a decreased cytoplasmic level
of IKK-a and IKK complex insolublisation (Pritts et al. 2000; Yoo et al. 2000; Chen
et al. 2004). In non-muscle cells, HSP70 and HSP27 have been found to interact
directly with NFkB, IkB-a, IKK-a, and IKK-b in suppress, resulting in the suppres-
sion of NFkB (Shimizu et al. 2002; Guzhova et al. 1997; Park et al. 2003).
It is entirely feasible that a similar interaction is present in skeletal muscle
cells and that this interaction not only modulates cytokine production by skel-
etal muscle, but other pathways in which NFkB and AP-1 may be involved. The
pattern and time course of HSP production in skeletal muscles to different
forms of exercise and other stresses differs and our data have shown that differ-
ent HSPs provide specific protection to various aspects of damage and regen-
eration. It is likely that there is some specificity in these interactions with
specific HSPs modulating different aspects of transcription factor activation or
inhibitor degradation and the induction of the stress response in skeletal muscle
may act as a shut-down mechanism of NFkB - mediated cytokine production by
muscle cells.
The interaction of HSPs with AP-1 and NFkB is further complicated since sev-
eral HSPs are known to contain promoters which can be regulated by both NFkB
and AP-1. For example, HSP90 contains a promoter which is regulated by NFkB
and downregulation of the p65 component of NFkB resulted in reduced constitutive
expression of HSP90 (Ammirante et al. 2008). HSPs can also contain an AP-1
promoter (e.g. Hosokawa et al. 1993).
5 Changes in and HSP Content and Redox Status of Muscles
Facilitate Successful Myogenesis and Rapid Regeneration
Following Damage
Controlled changes in transcription factor activation and deactivation are crucial to
successful myogenesis and regeneration. During myoblast proliferation and fusion,

the HSP content of cells is relatively high and this is primarily due to the expression
323Reactive Oxygen Species Generation and Skeletal Muscle Wasting – Implications
and activation of the developmental Heat Shock Factor 2 (HSF2). HSP content then
falls gradually with maturation of the cells, along with expression of HSF2
(McArdle et al. 2006). Expression of HSF1 increases at the later stages of myogen-
esis once myotubes have been formed (McArdle et al. 2006) such that these cells
are now stress responsive. Data from our laboratory examining NFkB activation
in vivo following muscle damage have demonstrated a secondary and relatively late
phase of NFkB activation at 14 and 28 days post-damage, a time associated with a
secondary phase of remodelling, maturation and reinnervation of skeletal muscle
fibres.
Myogenesis and regeneration are dependent on changes in ROS generation since
muscle cells with altered ROS production demonstrate a failure of successful myo-
genesis in culture. This may be associated with aberrant activation of redox-respon-
sive transcription factors. For example, primary myoblasts from glutathione
peroxidase 1 null mice do not fuse to form multinuclear myotubes in culture (Lee
et al. 2006).
Thus, it is clear that RONS generation plays a major role in determining tran-
scriptional activation in skeletal muscle during contraction-induced adaptive
responses and alteration of such generation results in adaptive and functional
deficits.
6 Modified Generation of Reactive Oxygen Species (ROS)
Have Been Implicated in Age-Related Skeletal Muscle
Dysfunction
The mechanisms responsible for the age-related loss of muscle mass and function
are unclear. Initial studies implicated an increase in oxidative damage in all tissues,
including skeletal muscle, in the functional decline of those tissues (Sastre et al.
2003; Drew et al. 2003; Vasilaki et al. 2006b,c).
Detrimental roles of ROS in tissues have been widely studied and a chronic
increase in the production of ROS has been implicated in a number of pathological

conditions such as cancer and ageing (Jackson et al. 2002). In contrast, it is now
accepted that acute changes in ROS generation are essential for physiological sig-
nalling processes. These include ROS acting as short-lived messengers in signal
transduction pathways such as those involved in cellular differentiation, prolifera-
tion, maturation and programmed cell death via activation of redox-responsive
transcription factors (Jackson et al. 2002). However, these processes are still poorly
defined and in particular there is a lack of information on the magnitude, time
course and localisation of such redox changes in tissues. A chronic accumulation
of oxidative damage has been postulated as a major component of the ageing
process for over 50 years (Harman 1956). Mitochondria have been claimed to be
the major site of reactive oxygen species (ROS) generation that contributes to
increased oxidative damage during ageing (see Sanz et al. 2006 for a review) and
isolated skeletal muscle mitochondria from old organisms release a greater amount
324 A. McArdle and M.J. Jackson
of hydrogen peroxide that is attributable to increased superoxide generation by
electron transport chain complexes (Lass et al. 1998; Mansouri et al. 2006; Vasilaki
et al. 2006c). Studies of the mutations in mitochondrial DNA in a number of cell
types have shown that these accumulate with age (Shah et al. 2009; Taylor et al.
2003). Mutations in mitochondrial DNA can theoretically disrupt the function of
the respiratory chain thereby compromising the production of ATP from oxidative
phosphorylation. Although much of the current data has concentrated on mitochon-
dria as a predominant site for ROS generation during ageing, alternative cellular
sites for ROS generation are receiving increasing attention. For example, copper,
zinc superoxide dismutase (SOD1) is normally located in the cytosol and mitochon-
drial intermembrane space and mice lacking SOD1 show a shortened lifespan and
an acceleration of the normal age-related changes in structure and function of several
tissues (Muller et al. 2007). It must be noted however that, although the oxidative
stress theory of ageing is by far the most popular theory on ageing, data in support
of this theory in mammalian systems is sparce (Pérez et al. 2009).
We have undertaken a number of studies to define the site of the defect in adap-

tive responses following contractile activity in muscle from aged mice. We exam-
ined the effect of contractile activity on various indicators of ROS activity in
muscle from old compared with adult mice. A protocol of contractile activity
caused a significant fall in the total glutathione content of contracting muscles from
adult mice, but less of a fall in muscles from old animals and this was associated
with a diminished release of extracellular superoxide from the muscles of old mice
(Vasilaki et al. 2006c). Vasilaki et al. (2007) also reported a contraction-induced
increase in the 3-nitrotyrosine content of muscle from adult mice that was not seen
in the muscle from old mice. These data all suggest that the contraction-induced
increase in ROS activities is reduced in muscle from old mice compared with that
from muscle of adult mice.
The chronic increase in the activities of regulatory enzymes for ROS (SOD1 and
SOD2 and catalase) and HSP content seen in muscle from old mice (Kayani et al.
2008b) appears to reflect an attempt to adapt to a chronic increase in ROS activities.
Despite this attempted adaptation, increased muscle oxidation remains evident in
the muscle from old mice (Broome et al. 2006). The effects of these changes on the
ROS signals that normally stimulate adaptations to contractions are unknown.
7 The Altered Generation of ROS in Muscles of Old Mice
is Associated with an Inability of Muscles of Old Individuals
to Adapt to Stress
Activation of redox-responsive transcription factors in response to an acute stress
such as exercise is aberrant in muscles of old humans and mice. These muscles
demonstrate both chronic constitutive activation of redox-sensitive transcription
factors (Vasilaki et al. 2006b; Cuthbertson et al. 2005) and an inability to further
activate these transcription factors following an acute non-damaging contraction
325Reactive Oxygen Species Generation and Skeletal Muscle Wasting – Implications
protocol (Vasilaki et al. 2006b). The chronic activation of transcription factors such
as NFkB in muscles of old mice is associated with chronic increases in the expres-
sion of a number of genes. For example, increased content and activities of antioxi-
dant defence enzymes such as the superoxide dismutases and catalase (Broome

et al. 2006), increased content of HSPs (Vasilaki et al. 2006b; Kayani et al. 2008b)
and increased production of cytokines and chemokines by muscle cells (Febbraio
and Pedersen 2005).
The inability to further activate NFkB in response to an acute contraction
protocol is associated with severe attenuation of normal changes in expression of
cytoprotective genes (Demirel et al. 2003; Heydari et al. 2000; Locke and Tanguay
1996; Muramatsu et al. 1996; Rao et al. 1999; Vasilaki et al. 2006b). We have
shown that the increases in HSP content and antioxidant enzyme activities stimu-
lated by isometric contractions in muscles of adult rodents were abolished in
muscles of old rodents (Vasilaki et al. 2002, 2006b). These severely blunted
adaptive responses to acute contractions in muscles from old rodents contribute to
age-related muscle dysfunction (McArdle et al. 2004a; Broome et al. 2006) and can
be overcome by activation of the transcription factor through alternative, pharma-
cological routes (Kayani et al. 2008a). Transgenic overexpression of HSP70 in
skeletal muscle throughout life partially preserved muscle function in old mice and
prevented the age-related chronic activation of transcription factors and changes in
muscle content of cytoprotective proteins at rest (McArdle et al. 2004b; Broome
et al. 2006). The mechanisms by which an increased muscle content of HSP70
exerts these effects on NFkB are unclear although overexpression of HSP70
throughout life also prevented the accumulation of markers of oxidative damage in
muscle from old mice (Broome et al. 2006).
A diminished ability to respond to the stress of contractions plays an important
role in other age-related defects in muscle function and adaptation. Ljubicic and
Hood (2008) reported a severe attenuation of the signalling pathways involved in
mitochondrial biogenesis in type II muscle fibres of old rats following contractions
compared with that seen in fibres from young rats. ROS play an important role in
the activation of these signalling cascades (Irrcher et al. 2009). These authors sug-
gest that ROS affect mitochondrial biogenesis via the upregulation of transcrip-
tional regulators as peroxisome proliferator-activated receptor-gamma coactivator-1
protein-alpha (PGC-1alpha), suggesting that an aberrant activation of ROS genera-

tion following contractions may be responsible for the diminished mitochondrial
biogenesis in muscles of old rats. This blunted or absent adaptation to stress in
muscle of old humans and mice is not limited to the exercise response. Skeletal
muscle of healthy elderly humans demonstrates a reduction in anabolic sensitivity
and responsiveness of muscle protein synthesis pathways. Cuthbertson et al. (2005)
demonstrated a reduction in the phosphorylation of mTOR and downstream
translational regulators in response to essential amino acid (EAA) ingestion when
compared with the young despite a greater plasma EAA availability in elderly
subjects. The authors concluded that the nutrient signal was not transduced as well
by old as by young muscle, resulting in a lower protein synthesis response to the
same stimulus.