Sarcopenia Age-Related Muscle Wasting and Weakness: Mechanisms and Treatments P16 pps

136 R.T. Hepple
1 Introduction
Aging is associated with myriad changes in physiological function. Amongst the
most visible of these changes is a progressive loss of skeletal muscle mass and
function, known as sarcopenia, a process that begins in approximately the 5th to 6th
decade of life (Lexell et al. 1988; Hepple 2003). There is an abundance of studies
examining the involvement of mitochondria in aging, including their role in the
functional and structural deterioration of skeletal muscle with aging. Despite years
of study, the precise involvement of mitochondria in the aging of skeletal muscle
remains to be fully understood. On the other hand, the appeal of a central involve-
ment of mitochondria in age-related changes in skeletal muscle is that this could
provide a unifying explanation for both the loss of skeletal muscle mass (e.g., by
increasing the incidence of apoptosis and increasing ROS-induced activation of the
proteasome) and the decline in skeletal muscle contractile function (e.g., by reduc-
ing muscle aerobic capacity and oxidizing proteins involved in muscle contractile
responses) with aging.
There is a multitude of ways that mitochondria might be involved in sarcopenia.
The potential impact of three important age-related changes in mitochondria will
be considered here: (1) a reduced capacity for generating cellular energy in the
form of adenosine triphosphate (ATP) (Conley et al. 2000; Drew et al. 2003;
Tonkonogi et al. 2003), (2) an increased susceptibility to apoptosis (Chabi et al.
2008; Seo et al. 2008), (3) and an increase in reactive oxygen species (ROS) pro-
duction with aging (Capel et al. 2005; Mansouri et al. 2006; Chabi et al. 2008). In
the context of explaining age-related muscle atrophy, mitochondria have been
implicated in: (i) fiber loss, atrophy and breakage (Lee et al. 1998; Wanagat et al.
2001; Bua et al. 2002); (ii) an increase in apoptosis (Dirks and Leeuwenburgh
2002; Marzetti et al. 2008; Seo et al. 2008); and (iii) activation of protein degrada-
tion pathways via increased reactive oxygen species (ROS) generation (Muller
et al. 2007; Hepple et al. 2008). In the context of explaining impaired muscle con-
tractile function with aging, mitochondria have been implicated in: (i) the decline
of aerobic contractile function secondary to reduced muscle oxidative capacity

(Hepple et al. 2003; Hagen et al. 2004) and reduced muscle ATP generating capac-
ity (Hepple et al. 2004a); (ii) impaired cross-bridge function secondary to oxidative
damage to contractile proteins (Lowe et al. 2001; Prochniewicz et al. 2005;
Thompson et al. 2006); and (iii) impaired Ca
2+
handling secondary to oxidative
damage to the Ca
2+
handling apparatus (Fano et al. 2001; Boncompagni et al. 2006;
Fugere et al. 2006; Thomas et al. 2009).
This chapter will examine the issue of mitochondrial dysfunction in aging
muscles. The first point of examination will be to determine the extent to which
mitochondrial dysfunction occurs (and what is meant by “mitochondrial
dysfunction”). We will then examine why mitochondrial dysfunction occurs, and
the functional consequences of mitochondrial dysfunction in aging muscles.
Finally, we will consider the extent to which the mitochondrial content may be up-
regulated in response to muscle activity as a means of assessing the malleability of
the age-related impairments in mitochondria.
137Alterations in Mitochondria and Their Impact in Aging Skeletal Muscle
2 Age-Related Changes in Mitochondrial Function
Because of the complexity of mitochondrial structural and biochemical organization
and the many roles that mitochondria serve within the cell, the ways in which mito-
chondrial function may be altered, and the consequences thereof, is vast. Underscoring
this point, despite the fact that the mitochondrion was discovered more than a century
ago (Altmann 1890), new insights into the scope of mitochondrial functional altera-
tion, both in a physiological and pathological context, continue to this day. Perhaps
reflecting a limited appreciation of the normal scope of mitochondrial function,
although the term “mitochondrial dysfunction” is used extensively in the literature, the
criteria used in making this qualification are often vague or inaccurate. In the interest
of keeping things simple and given the central importance of mitochondria to cellular

energy provision, one criterion that will be considered here in specifying a decline in
mitochondrial function with aging is whether the capacity for energy provision per
unit of mitochondrial volume is reduced. This criterion is distinct from a reduction in
mitochondrial volume per se because a reduced skeletal muscle mitochondrial
volume could occur in response to reduced physical activity with aging and reduce
muscle oxidative capacity without impacting the ability of individual mitochondria to
generate energy.
2.1 Evidence for Reduced Oxidative Capacity Per Mitochondrion
Although many studies have demonstrated a reduced mitochondrial oxidative
capacity with aging at the level of whole muscle (e.g., enzyme assays using whole
muscle homogenates) (Essen-Gustavsson and Borges 1986; Coggan et al. 1992;
Sugiyama et al. 1993), these studies do not reveal the extent to which these
declines might reflect a lower mitochondrial content due to a more sedentary
lifestyle with aging versus changes intrinsic to the aged mitochondria themselves.
Conley and colleagues provided the first in vivo estimation of mitochondrial
function in aging skeletal muscle. Their study showed that there was a greater
decline in the oxidative capacity of human vastus lateralis muscle (inferred from
phosphocreatine recovery following knee extensor exercise) of aged subjects
than could be accounted for by the reduction in mitochondrial volume density
(measured by electron microscopy in muscle cross sections taken from biopsy
samples), revealing a reduced oxidative capacity per volume of mitochondria in
aged human skeletal muscle (Conley et al. 2000) (Fig. 1). Others have examined
the function of mitochondria ex vivo using mitochondria isolated from muscles
of aged individuals or organisms and the results have been mixed, with some
groups finding reduced oxidative capacity or ATP production per unit of
mitochondria in aged rodents (Desai et al. 1996; Drew et al. 2003; Mansouri et al.
2006) and aged humans (Short et al. 2005), and others finding no change in
mitochondria isolated from skeletal muscles of older humans relative to younger
adults (Rasmussen et al. 2003; Hutter et al. 2007). In addition to this, it has been
138 R.T. Hepple

shown that whereas mitochondrial volume density does not decline between
adulthood and senescence in rat fast- or slow-twitch muscles (Mathieu-Costello
et al. 2005) (Fig. 2, panel A), there is a significant reduction in mitochondrial
electron transport chain enzyme activities across this age range (Hagen et al.
2004; Hepple et al. 2006) (Fig. 2, panel B), indicating a reduced oxidative power
per mitochondrion in aged skeletal muscles.
One of the factors suggested to account for inconsistency in some of the findings
is that isolating mitochondria may underestimate the potential for mitochondrial
dysfunction with aging by selectively harvesting the healthiest mitochondria
(Tonkonogi et al. 2003). Although this has not been rigorously tested experimen-
tally, it has been hypothesized that due to increasing fragility of some mitochondria
with aging (Terman and Brunk 2004), this would result in selective harvest of the
healthiest mitochondria in the aged muscles, thereby leading to an underestimate of
the extent of mitochondrial dysfunction in isolated mitochondrial fractions
(Tonkonogi et al. 2003). Furthermore, as mitochondria in skeletal muscle exist in
varying degrees of a reticulum (Bakeeva et al. 1978; Kayar et al. 1988; Ogata and
Yamasaki 1997), experimental isolation of mitochondria would disrupt this struc-
tural arrangement, which could also obscure important changes in mitochondrial
function that would be evident in vivo.
Two other factors relating to the human literature may also contribute to incon-
sistency in observing mitochondrial dysfunction in studies of human subjects.
Firstly, the screening measures required for human studies often results in loss of
the least healthy subjects (Stathokostas et al. 2004), and it would be expected that
this would bias the measures against identifying mitochondrial dysfunction.
Secondly, mitochondrial function measurements in humans have not so far included
subjects who are amongst the oldest of old (>75 years). Since the progression of
sarcopenia exhibits a marked acceleration both in terms of declining muscle mass
(Lexell et al. 1988; Hagen et al. 2004; Baker and Hepple 2006) and impaired
1.5
4

3
2
1
0
0.4
0.3
0.2
0.1
0
1.0
Oxidative capacity
(mM ATP s
−1
)
0.5
0.0
Oxidative capacity/
n
v
(mt,f)
(mM ATP (s %)
−1
)
n
v
(mt,f) (%)
Fig. 1 The rate of muscle phosphocreatine resynthesis following muscle contractions, used as a
surrogate of muscle oxidative capacity, was slower in muscle of elderly human adults (black bar)
versus young adults (open bar) (left panel). Although the mitochondrial volume density
(Vv[mt,f]%) was also lower in the elderly subjects (middle panel), taking the quotient of oxidative

capacity and Vv[mt,f]% revealed a lower oxidative capacity per mitochondrion in the muscle of
elderly humans (right panel) (Figure reproduced from Conley et al. [2000], with permission from
The Physiological Society)
139Alterations in Mitochondria and Their Impact in Aging Skeletal Muscle
muscle function (Hagen et al. 2004; Hepple et al. 2004a) between late middle age
and senescence, study of very old human subjects may reveal changes in mitochon-
drial function that have not been previously identified. These important issues
remain to be adequately addressed in the literature.
Fig. 2 Whereas aging is not associated with a reduction in mitochondrial volume density in either
the slow-twitch soleus (Sol) muscle or the fast-twitch extensor digitorum longus (EDL) muscle
between adulthood (12 month) and senescence (35 month old) in rats (top panel), there is a sig-
nificant reduction in complex IV activity of the electron transport chain in the slow-twitch soleus
muscle and mixed fast-twitch muscles like the red region of gastrocnemius (Gr), the mixed region
of gastrocnemius (Gmix) and the plantaris (Plan) muscle (The top panel was adapted with permis-
sion from the American Physiological Society from data provided in Mathieu-Costello [2005].
The bottom panel was adapted with permission from Mary Ann Liebert, Inc. from a figure appear-
ing in Hepple et al. [2006])
140 R.T. Hepple
One means by which the oxidative capacity per mitochondrion could be
reduced with aging is via a selective loss of electron transport chain function. As
the activities of different mitochondrial enzymes normally scale proportionally
across a wide range of muscle oxidative capacity (Davies et al. 1981), this kind of
alteration could be revealed by examining the activity of electron transport chain
enzymes relative to other mitochondrial enzyme pathways. In this context,
complex IV of the electron transport chain often exhibits a disproportionately
lower activity with aging relative to other mitochondrial enzymes (Navarro and
Boveris 2007). This has also been seen in aged skeletal muscles (Hepple et al.
2005, 2006). The reasons for a greater decline in complex IV activity remain to be
agreed upon, but strong candidates include the accumulation of oxidative damage
(Navarro and Boveris 2007; Choksi et al. 2008) and/or incorrect assembly of the

subunit proteins.
2.2 Aged Mitochondria Exhibit Greater ROS Generation
Another indication of impaired mitochondrial function with aging is an increase in
mitochondrial ROS generation. Although some ROS production is a normal part of
mitochondrial physiology (Droge 2002) and is considered essential to facilitate
adaptations in skeletal muscle (Gomez-Cabrera et al. 2005), excessive ROS pro-
duction can lead to adverse consequences for skeletal myocytes. There are several
studies showing that mitochondria isolated from skeletal muscles of aged humans
(Capel et al. 2005) or rodent models (Bejma and Ji 1999; Capel et al. 2004;
Mansouri et al. 2006; Vasilaki et al. 2006; Chabi et al. 2008) emit higher levels of
ROS. On the basis of experiments using rotenone to inhibit complex I, it was sug-
gested that the majority of the increase in mitochondrial ROS emission with aging
was from complex I due to reverse electron transfer between complex II and com-
plex I (Capel et al. 2005) (Fig. 3).
In summarizing age-related changes in mitochondrial function, although the
findings are not uniformly in agreement, several lines of evidence suggest that
aging is associated with a reduction in skeletal muscle mitochondrial oxidative
capacity which exceeds that explainable by a reduction in muscle mitochondrial
content. Furthermore, mitochondria from aged muscles pump out higher levels of
ROS, which contributes to the greater accumulation of oxidative damage with
aging, and likely plays a key role in impaired muscle function with aging and its
greater vulnerability to apoptosis and excessive protein degradation. Therefore,
while physical inactivity may be contributing to a declining muscle oxidative
capacity with aging, the basis of mitochondrial functional alterations with aging
likely includes aging-specific changes that are not reversible by restoring physical
activity alone.
141Alterations in Mitochondria and Their Impact in Aging Skeletal Muscle
3 Factors Accounting for Mitochondrial Dysfunction
in Aged Muscles
Given the aforementioned evidence for mitochondrial dysfunction in aged

muscles, an important question is why this occurs. Many different ideas are
currently being explored, with some gaining experimental support. These include
a reduced mitochondrial turnover, which leads to accumulation of poorly
functioning mitochondria, and denervation which by some mechanism yet to be
fully identified, leads to increased ROS generation and also low mitochondrial
content in afflicted fibers.
3.1 Evidence for Decreased Mitochondrial Turnover with Aging
Mitochondrial protein exhibits a continual turnover, with the enzymes having a
half-life of approximately 7 days (Booth and Holloszy 1977), although recent evidence
from murine liver suggests mitochondrial turnover may be much more rapid, on the
order of 2 days (Miwa et al. 2008). One reason for this high rate of turnover is that
2.5
Young
Elderly
*
1.5
Mitochondrial H
2
O
2
Release
0.5
2
1
0
Basal
#
#
+Rotenone
Fig. 3 Reactive oxygen species (ROS) production, as determined by hydrogen peroxide (H

2
O
2
)
release, increases with aging in mitochondria isolated from human skeletal muscle. Furthermore,
inhibition of Complex I ROS generation with Rotenone markedly reduces the elevated ROS
generation with aging, suggesting that most of the increase in ROS with aging is due to reverse
electron transfer from complex II to complex I (Figure reproduced from Capel et al. [2005], with
permission from Elsevier)
142 R.T. Hepple
mitochondria normally produce some ROS, which even at physiological levels may
oxidatively damage the mitochondrial proteins and mitochondrial DNA, leading to
impaired enzyme function. This impaired enzyme function, particularly if it were
to occur in the electron transport chain, could elevate ROS production and lead to
a downward spiral in mitochondrial function. Thus, continual renewal of mitochon-
drial proteins is thought to be essential to the proper function of the mitochondria.
It follows that changes in the rate at which mitochondria are turned over with aging
can contribute to age-related cellular impairment.
Consistent with the idea that accumulation of oxidative damage can impair mito-
chondrial enzyme activity, elevating oxidative stress in aging muscle can reduce
aconitase enzyme activity without reducing its protein content (Bota et al. 2002).
The significance of this observation is that aconitase has an iron-sulfur center,
which renders it particularly susceptible to oxidative damage, and thus it provides
a useful biomarker of oxidative damage in mitochondria. In accounting for impaired
mitochondrial function in aged skeletal muscles it is relevant that a major enzyme
involved in the degradation of oxidatively damaged mitochondrial proteins (Lon
protease) declines with aging (Bota et al. 2002), and mitochondrial protein synthe-
sis rate declines in aged muscle (Rooyackers et al. 1996). Further to this latter point,
there is evidence that the reduced mitochondrial protein synthesis may occur sec-
ondary to a reduced drive on mitochondrial biogenesis, based upon the decreased

expression of peroxisome proliferator activated receptor coactivator gamma 1
alpha (PGC-1a) in aged skeletal muscle (Baker et al. 2006; Chabi et al. 2008).
Finally, mitochondrial autophagy, whereby whole organelles are engulfed and
enzymatically degraded in lysosomes, is thought to be impaired in aging muscles
(Terman and Brunk 2004). Collectively, these changes lead to a reduced mitochon-
drial protein turnover with aging, due to the combined effects of reduced mitochon-
drial protein synthesis, impaired removal of oxidatively damaged mitochondrial
proteins, and reduced mitochondrial autophagy. As implied above, the expected
impact of this reduced mitochondrial turnover would not only be manifest as a
reduced oxidative capacity per unit of mitochondrial volume because the longer
mitochondrial protein dwell-time would exacerbate accumulation of oxidative
damage, but also an increase in mitochondrial ROS generation secondary to, for
example, a relatively greater reduction in complex IV activity (by allowing oxygen
to accumulate to higher levels this favors production of ROS). This is consistent
with the above-mentioned increase in mitochondrial ROS generation with aging in
both rodent (Mansouri et al. 2006) and human (Capel et al. 2005) skeletal muscles.
3.2 Role of Denervation in Mitochondrial Dysfunction
The mechanistic basis for an increase in mitochondrial ROS production with aging
may be due in part to a decreased mitochondrial renewal and resultant accumulation
of ‘aged’ mitochondria, as described above (Section 3.1). In addition to this, recent
evidence suggests that denervation may also be a predisposing factor. For example,
143Alterations in Mitochondria and Their Impact in Aging Skeletal Muscle
skeletal mitochondrial ROS generation was shown to increase following surgical
denervation (Adhihetty et al. 2007; Muller et al. 2007), and in disease models
where there is loss of skeletal muscle a-motor neurons (Muller et al. 2007).
Denervation is thought to affect muscle fibers and the mitochondria therein in sev-
eral important ways. Perhaps the most important is the removal of neurotrophic
influences that affect the drive on mitochondrial biogenesis. This is consistent with
evidence showing a decreased expression of factors involved in driving mitochon-
drial biogenesis (e.g., PGC-1a, PGC-1b, mitochondrial transcription factor A) in

skeletal muscle following denervation (Raffaello et al. 2006; Adhihetty et al. 2007;
Sacheck et al. 2007) and that the pattern of their decline mirrors a decline in mito-
chondrial enzyme activities (Adhihetty et al. 2007). In addition, denervation is
thought to increase phospholipase A signaling, resulting in hydrolysis of the mito-
chondrial membrane phospholipids and subsequent release of mitochondrial
membrane-derived hydroperoxides (Bhattacharya et al. 2009). Finally, denervation
also leads to an increase in pro-apoptotic factors, particularly those involving
mitochondrial-driven apoptosis (Adhihetty et al. 2007). Collectively, therefore,
denervation can have several important effects on mitochondria that may contribute
to the increase in ROS generation observed in aging muscles.
4 Role of Mitochondria in Age-Related Muscle Deterioration
As noted in the Introduction, the appeal of a role for mitochondria in sarcopenia is
that it may provide a unifying explanation for both the reduction of muscle mass
and the impairment in contractile function in aging muscles. To this end, the
following section will address the evidence that mitochondria are involved in both
the mass and functional declines in aging skeletal muscles.
4.1 Involvement of Mitochondria in Age-Related Muscle Atrophy
As noted above, mitochondria in aging skeletal muscle exhibit numerous changes
and several of these could have important implications in the context of age-related
muscle atrophy. Firstly, the age-related increase in mitochodrial ROS generation is
thought to induce protein degradation via NF-kB-induced activation of the
proteasome (Jackman and Kandarian 2004; Powers et al. 2005). Although direct
evidence of how this might be involved in sarcopenia remains to be provided, this
idea is consistent with evidence that proteasome activity increases in aging skeletal
muscle in a manner that is similar to the trajectory of age-related muscle atrophy
(Hepple et al. 2008) (Fig. 4). This view is also consistent with observations in mice
showing that muscle atrophy with (i) aging, (ii) superoxide dismutase 1 knockout,
and (iii) experimental models of amyotrophic lateral sclerosis, correlates with the
amount of muscle mitochondrial ROS production (Muller et al. 2007). Furthermore,
144 R.T. Hepple

an increase in mitochondrial ROS generation following surgical denervation in
skeletal muscle precedes muscle atrophy by several days (Muller et al. 2007).
Despite the appeal of denervation being a cause of muscle atrophy in aged muscle,
it is important to note that it is currently not known whether death of a-motor
neurons is the cause versus the effect of myofiber atrophy and/or death in aged
muscles. Interestingly, recent experiments in transgenic mice have examined a
muscle-specific over-expression of uncoupling protein 1 (the isoform normally
found in brown adipose tissue) by using a muscle creatine kinase promotor to limit
expression to the myocytes, and these mice exhibit deterioration of neuromuscular
junctions and retrograde a-motor neuron degeneration (Dupuis et al. 2009),
showing that mitochondrial dysfunction within myocytes can be a cause of dener-
vation. In addition, these animals exhibited a progressive loss of muscle mass
(Dupuis et al. 2009). As such, these latter experiments show that abnormalities in
mitochondrial metabolism within skeletal muscle fibers can be an initiating event
in denervation. Therefore, the extent to which denervation is the initiating event in
muscle atrophy with aging versus denervation occurring secondary to mitochon-
drial dysfunction in aging myocytes requires further study.
Some of the most compelling data examining the role of mitochondrial dysfunc-
tion in age-related muscle atrophy has been the studies examining the co-localiza-
tion of mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage with
focal regions of fiber atrophy and breakage along the length of individual muscle
fibers in aged muscle (Lee et al. 1998; Wanagat et al. 2001; Bua et al. 2002). The
hypothesis most frequently cited to explain the significance of the aforementioned
co-localization phenomenon is that mtDNA damage occurs segmentally along the
length of individual muscle fibers (due to the accumulated effects of ROS) and that
Fig. 4 Plantaris muscle mass versus the chymotrypsin-like activity of the proteasome in young
adult (8 month old), late middle aged (30 month old) and senescent (35 month old) rats (Figure
reproduced from Hepple et al. [2008], with permission from The American Physiological Society)
145Alterations in Mitochondria and Their Impact in Aging Skeletal Muscle
this damage is propagated by clonal expansion of damaged/mutated mtDNA within

this region, leading to synthesis of mitochondria containing faulty electron trans-
port chain enzymes (specifically those containing mtDNA-encoded subunits),
which in turn is eventually manifest as a complex IV deficient fiber segment. This
focal mitochondrial dysfunction is thought to have numerous consequences, includ-
ing insufficient ATP supply, impaired protein synthesis, increased susceptibility to
apoptosis, and increased mitochondrial ROS production, all of which may contrib-
ute to fiber atrophy and/or death (Wanagat et al. 2001).
Despite the elegance of experiments supporting this hypothesis, and the logical
appeal of the explanation, the significance of this phenomenon for sarcopenia
should be carefully scrutinized. Firstly, the only study to have examined this
phenomenon in skeletal muscles from aging humans (Bua et al. 2006) found that
although muscle fiber segments exhibiting complex IV deficiency co-localized
with regions having a large burden of mtDNA damage, these fiber segments were
not atrophied relative to regions with normal complex IV activity (Bua et al. 2006)
(Fig. 5). Secondly, patients with so-called mtDNA disease exhibit much higher
Slide 11
a
b
100
80
60
40
Percentage of deleted mtDNA
20
0
51118253239
Slide number
46 53 60 67 74 81 88 95 100
C
Slide 25 Slide 46 Slide 74

Fig. 5 Serial cross-sections of human skeletal muscle doubly-stained for succinate dehydrogenase
and complex IV activity (top panels) depicting a fiber with a lack of complex IV activity (blue fiber
indicated by arrow). Although fiber segments with complex IV deficiency (depicted as the blue
region in the reconstructed fiber, bottom panel) exhibited high levels of deleted mitochondrial DNA
(middle panel), these regions did not exhibit atrophy relative to fibers with normal complex IV
activity (depicted as orange regions in the reconstructed fiber, bottom panel) (Reproduced from
Bua et al. [2006], with permission from The American Society of Human Genetics)