Sarcopenia Age-Related Muscle Wasting and Weakness: Mechanisms and Treatments P17 docx

146 R.T. Hepple
burdens of mtDNA damage at the whole muscle level and very much higher fractions
of muscle fibers exhibiting complex IV enzyme activity deficiency, and yet in these
patients neither individual muscle fibers lacking complex IV activity (Fig. 6) nor
their muscles as a whole are grossly atrophied relative to healthy individuals of the
same age (Jacobs 2003). As such, the degree to which this phenomenon might
contribute to sarcopenia remains an important area of investigation.
As suggested above, one specific manner in which mitochondria are proposed to
be involved in sarcopenia involves apoptosis (Pollack and Leeuwenburgh 2001;
Chabi et al. 2008; Seo et al. 2008). Mitochondria play a key role in regulating
apoptosis, via the mitochondrial permeability transition pore (mPTP) which regu-
lates the release of cytochrome c into the cytoplasm. A variety of stimuli, such as
high Ca
2+
and high ROS exposure, can lead to opening of the mPTP, allowing cyto-
chrome c to leak out of the mitochondria and into the cytoplasm. Once released into
the cytoplasm, cytochrome c binds with Apaf-1 and caspase 9, leading to the
formation of an apoptosome, activation of caspase 9 and subsequent commitment
of the apoptotic pathway via activation of caspase 3. In support of a role for
apoptosis in age-related muscle atrophy, many studies have reported an increase in
pro-apoptotic signaling in aged muscles (Alway et al. 2002; Dirks and Leeuwenburgh
2002; Giresi et al. 2005; Baker and Hepple 2006; Rice and Blough 2006; Chabi
et al. 2008). On the other hand, differences in the degree of muscle atrophy between
Fig. 6 Succinate dehydrogenase and complex IV doubly-stained cross-section of muscle from a
patient with heteroplasmic mtDNA mutation. Note that the complex IV deficient fibers (blue
fibers) are no different in size than fibers with normal complex IV activity (brown-orange fibers)
(Reproduced from Taivassalo and Haller [2005], with permission from The American College of
Sports Medicine)
147Alterations in Mitochondria and Their Impact in Aging Skeletal Muscle
individuals in senescent animals do not track well with differences in expression of
pro-apoptotic transcripts (Baker and Hepple 2006). In addition, although the pro-

gression of muscle atrophy with aging correlates generally with an increase in
number of apoptotic nuclei in both fast-twitch and slow-twitch muscles, it is strik-
ing that there are markedly more apoptotic nuclei in the slow-twitch soleus muscle
than the fast-twitch extensor digitorum longus muscle, despite very similar amounts
of atrophy (Fig. 7; data taken from (Rice and Blough 2006)). This difference may
relate to the fact that muscle fibers are multi-nucleated and, therefore, apoptotic
loss of a nucleus within a given myocyte does not need to result in loss of the myo-
cyte entirely. As such, a difference in the incidence of apoptotic nuclei between
muscles having the same amount of atrophy could reflect differences in the ability
of these muscles to regenerate and repair, e.g., via recruitment of satellite cells.
Whether this or another explanation applies awaits further investigation.
Notwithstanding some uncertainty about the degree to which apoptosis directly
explains the degree of muscle atrophy with aging, recent data suggests that accu-
mulation of non-heme iron in skeletal muscle mitochondria may be one mechanism
leading to an increased incidence of mitochondrial-mediated apoptosis in aged
skeletal muscle. Specifically, accumulation of non-heme iron with aging is hypoth-
esized to exacerbate mitochondrial ROS generation (and thus oxidative damage)
via the Fenton reaction, wherein the increased mitochondrial damage leads to an
increased probability of mPTP opening (Seo et al. 2008). This notion is consistent
with the aforementioned increase in mitochondrial ROS generation in aged skeletal
muscles (Section 2.2), and observations indicating greater accumulation of non-
heme iron in mitochondria isolated from aged skeletal muscle (Seo et al. 2008). In
addition, mitochondria from aged muscles exhibit a greater release of cytochrome
Fig. 7 Muscle mass in the fast-twitch extensor digitorum longus (EDL) muscle and slow-twitch
soleus muscle (Sol) versus the density of TUNEL-positive nuclei (a marker of apoptotic nuclei)
as sarcopenia progresses with aging (Data reproduced from Rice et al. [2006])
148 R.T. Hepple
c in response to ROS-induced stress (Chabi et al. 2008), which may in part explain
the increased susceptibility to mitochondrial-driven apopotosis in aging muscle.
Thus, collectively, there is substantial evidence that apoptosis increases in aged

muscles and that age-related changes in mitochondria are likely to be involved.
4.2 Involvement of Mitochondria in Age-Related Muscle
Dysfunction
In addition to the potential involvement of mitochondria in the age-related loss of
muscle mass, there is considerable support for the involvement of mitochondria in
impaired muscle function with aging. For example, there is a progressive decline in
skeletal muscle aerobic function with aging that is not due to loss of capillaries
(Hepple and Vogell 2004; Mathieu-Costello et al. 2005), but rather correlates with
a progressive loss of mitochondrial oxidative capacity in aging muscles (Hagen
et al. 2004) (Fig. 8). As noted in Section 3, a decline in muscle mitochondrial oxi-
dative capacity may be caused by a reduction in the expression of PGC-1a in aged
muscles (Baker et al. 2006; Chabi et al. 2008). In this context, it is important to note
that aged muscles, particularly in senescence, are characterized by an accumulation
of very small muscle fibers. Although this area requires further study, it seems
likely that a large proportion of these small fibers are denervated (Hepple et al.
2004b) and that a sub-fraction of these may be attempting to regenerate. The reason
this is relevant here is that these small fibers have lower levels of markers of
Fig. 8 Muscle maximal oxygen uptake (VO
2max
) in pump-perfused rat hindlimb versus the flux
capacity of complex I–III in homogenates of gastrocnemius muscle. The figure shows that
the age-related decline in VO
2max
parallels the decline in flux capacity through a key part of the
mitochondrial electron transport chain (Reproduced from Hagen et al. [2004], with permission
from The Gerontological Society of America)
149Alterations in Mitochondria and Their Impact in Aging Skeletal Muscle
mitochondrial content (e.g., complex IV activity) (Fig. 9), and because of this they
contribute significantly to the lower muscle oxidative capacity. Furthermore, den-
ervation, or perhaps failure to reinnervate, may be constraining the mitochondrial

content of these fibers, secondary to the aforementioned reduction in drive on mito-
chondrial biogenesis that occurs in denervated muscle (Adhihetty et al. 2007)
(Section 3.1). Thus, the reduction of muscle mitochondrial oxidative capacity with
aging may have an important neurological involvement. This point needs further
consideration in the experimental literature.
As noted in Section 3.2, aged muscles are also characterized by mitochondria
that emit higher levels of ROS. This increase in mitochondrial ROS generation in
aging skeletal muscles can exacerbate oxidative damage to proteins, which has
been shown to inhibit the biological activity of enzymes, particularly those contain-
ing iron-sulfur centers (Bota et al. 2002; Ma et al. 2009). In addition, several pro-
teins involved in muscle contraction are known to be specifically targeted by
oxidative stress, and thus, likely contribute to the impairment in muscle contractile
function with aging. Prochniewicz et al. (2005) previously showed using in vitro
motility assays that although actin function was unaltered with aging, the catalyti-
cally active portion of myosin (heavy meromyosin) was impaired in muscles of
aged versus young adult rats. In addition, this difference in actin versus myosin
function with aging corresponded to differences in the susceptibility of actin versus
Fig. 9 Senescent rat gastrocnemius muscle cross-section stained for complex IV activity.
Note that the very small fibers have a lower complex IV activity than the larger fibers, showing
that the accumulation of these very small fibers in aged muscle, particularly in senescence,
contributes to the overall decline in muscle oxidative capacity with aging (R.T. Hepple
[unpublished])
150 R.T. Hepple
myosin to accumulate oxidative damage to cysteine molecules (Prochniewicz et al.
2005). Similarly, there is an increase in oxidative damage, particularly nitrotyrosine
damage, to the sarcoplasmic reticulum ATPase in aged muscles (Fugere et al. 2006;
Thomas et al. 2009), and this is thought to contribute to decreases in maximal
SERCA activity in aged muscle (Thomas et al. 2009). As such, the collective evi-
dence suggests that oxidative damage to various proteins within skeletal muscle,
and the mitochondria therein, can lead to functional deterioration in aging skeletal

muscle.
5 Plasticity of Mitochondria in Aging Muscles
Given the above evidence of reduced mitochondrial oxidative capacity and
increased ROS generation with aging, both of which have been attributed in part to
accumulation of damaged mitochondria secondary to reduced mitochondrial
renewal, an obvious question is whether aged muscle simply loses the capacity to
increase its mitochondrial content. The majority of what we know about this ques-
tion has been obtained from experiments examining changes in muscle mitochon-
drial oxidative capacity in response to exercise training or chronic electrical
stimulation. Significantly, an emerging concept is that the capacity for mitochon-
drial biogenesis in response to muscle activation, while relatively preserved in the
younger of the old, becomes severely impaired in the oldest old.
There are many studies showing that aged muscles can respond favorably by
increasing markers of mitochondrial content in response to endurance exercise
training in both the human (Orlander and Aniansson 1980; Hagberg et al. 1989;
Meredith et al. 1989; Short et al. 2003) and animal model (Cartee and Farrar 1987;
Rossiter et al. 2005; Betik et al. 2008) literature. However, it is important to realize
that these prior studies have not considered potential differences in the endurance
training responses between late middle age versus the senescent period (i.e., when
survival rates drop below 50%), and it is the senescent period when the consequences
of aging for skeletal muscle become most severe. To address this issue, we recently
examined the effect of aging on the responses of the skeletal muscle aerobic
machinery to endurance training in rat skeletal muscles. Interestingly, whereas
skeletal muscle aerobic function (in situ maximal oxygen consumption) and
mitochondrial enzyme activities increased significantly when endurance exercise
training was imposed in late middle age and continued for 7 weeks (Betik et al.
2008) (Fig. 10), the skeletal muscles completely lost this positive adaptation when
the training was continued for 7 months into the senescent period (Betik et al. 2009)
(Fig. 11). Further to this, the normally robust response of PGC-1a expression to
endurance exercise training seen in studies of rodents (Baar et al. 2002; Terada

et al. 2002) and young adult humans (Norrbom et al. 2004) was abolished in
senescent rat skeletal muscles following 7 months of endurance exercise training in
both the slow-twitch soleus muscle and the fast-twitch plantaris muscle (Fig. 12)
(Betik et al. 2009). On the basis of these results, therefore, it appears that senescent
151Alterations in Mitochondria and Their Impact in Aging Skeletal Muscle
muscle in particular has a markedly diminished capacity to increase mitochondrial
biogenesis in response to an endurance training stimulus, and that this is due in part
to an impaired ability to up-regulate PGC-1a. This finding of reduced adaptability
with endurance training in senescence is consistent with studies demonstrating that
skeletal muscle from the oldest old also has a diminished plasticity in response to
resistance exercise training (Slivka et al. 2008; Raue et al. 2009) and functional
overload (Blough and Linderman 2000).
The aforementioned results indicate that senescent skeletal muscle loses its abil-
ity to generate new mitochondria in advanced age, suggesting that the reduced
Fig. 10 Muscle oxygen uptake during incremental muscle contractions in distal rat hindlimb
muscles pump-perfused in situ (top) and the activity of complex IV in homogenates of plantaris
(Plan) and gastrocnemius (Gas) muscle (bottom) in sedentary late middle aged rats and late middle
aged rats exercise-trained for 7 weeks (Reproduced from Betik et al. [2008], with permission from
The Physiological Society [London])
152 R.T. Hepple
mitochondrial turnover rate with aging is secondary to this diminished capacity to
make new mitochondria. However, an important question remains: is it that senes-
cent muscle loses its adaptive plasticity per se, or is the limitation the result of the
much lower exercise stimulus that can be sustained in very old age. To help address
this issue, a recent study examined the response of young adult versus senescent
skeletal muscle to an acute bout of low frequency electrical stimulation.
Interestingly, these experiments revealed that whereas the cell signaling pathway,
including molecules involved in driving mitochondrial biogenesis (e.g., adenosine
monophosphate protein kinase [AMPK] activation), was relatively intact in the
highly oxidative region of the tibialis anterior muscle, there was a blunted response

Fig. 11 Muscle maximal oxygen uptake (VO
2
max) during incremental muscle contractions in
distal rat hindlimb muscles pump-perfused in situ (top) and the activity of complex IV in homo-
genates of plantaris (Plan) and Soleus (Sol) muscle (bottom) in sedentary senescent rats and
senescent rats trained for 7 months beginning in late middle age (Data reproduced from Betik
et al. [2009])
153Alterations in Mitochondria and Their Impact in Aging Skeletal Muscle
in the highly glycolytic region of this muscle in senescence (Ljubicic and Hood
2009). These data are generally consistent with another study showing that AMPK
activation is markedly blunted in aged muscles following either pharamacological
stimuli or an acute exercise bout (Reznick et al. 2007). What is not yet clear, how-
ever, is the degree to which an attenuated mitochondrial biogenesis response is a
general property of all muscle fibers in an aged muscle, versus there being an
increasing proportion of muscle fibers which cannot contribute to the whole muscle
mitochondrial biogenesis response (e.g., those that have become denervated and/or
which are undergoing regeneration). Irrespective of this point, the growing consen-
sus is that aging muscle, particularly in senescence, displays an impaired ability to
up-regulate mitochondrial biogenesis and this in turn plays an important role in the
attenuated benefits of endurance exercise training for skeletal muscle aerobic
capacity in senescence. Future studies need to address whether this loss of adaptive
plasticity is an immutable consequence of aging, or if other interventions yet to be
identified can help restore the adaptive response to increased muscle use.
6 Conclusions
Mitochondrial changes in aging skeletal muscles, and the implications these have for
the decline in both muscle mass and its function with aging, have constituted an
intensive area of study. The aforementioned chapter provides some context for the
current knowledge in this area and areas that will be refined through further study.
Given the central importance of mitochondrial biology to so many facets of normal
Fig. 12 PGC-1 protein expression in plantaris (Plan) and soleus (Sol) muscles of sedentary senes-

cent rats and senescent rats trained for 7 months beginning in late middle age. *P < 0.05 versus
Sedentary group (Data reproduced from Betik et al. [2009])
154 R.T. Hepple
cell function, particularly in tissues with a wide metabolic scope like skeletal muscle,
new discoveries about the significance of changes in mitochondria for aging skeletal
muscles, and their potential remedy through lifestyle modification (e.g., exercise
training, diet) and/or medical intervention (e.g., pharmaceuticals, gene therapy), will
remain at the forefront of our quest to promote healthy aging.
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