Báo cáo Y học: Mitochondrial DNA deletion mutations A causal role in sarcopenia docx

MINIREVIEW
Mitochondrial DNA deletion mutations
A causal role in sarcopenia
Debbie McKenzie, Entela Bua, Susan McKiernan, Zhengjin Cao, Jonathan Wanagat and Judd M. Aiken
Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, WI, USA
Mitochondrial DNA (mtDNA) deletion mutations accu-
mulate with age in tissues of a variety of species. Although
the relatively low calculated abundance of these deletion
mutations in whole tissue homogenates led some investiga-
tors to suggest that these mutations do not have any phy-
siological impact, their focal and segmental accumulation
suggests that they can, and do, accumulate to levels sufficient
to affect the metabolism of a tissue. This phenomenon is
most clearly demonstrated in skeletal muscle, where the
accumulation of mtDNA deletion mutations remove critical
subunits that encode for the electron transport system (ETS).
In this review, we detail and provide evidence for a molecular
basis of muscle fiber loss with age. Our data suggest that the
mtDNA deletion mutations, which are generated in tissues
with age, cause muscle fiber loss. Within a fiber, the process
begins with a mtDNA replication error, an error that results
in a loss of 25–80% of the mitochondrial genome. This
smaller genome is replicated and, through a process not well
understood, eventually comprises the majority of mtDNA
within the small affected region of the muscle fiber. The
preponderance of the smaller genomes results in a dysfunc-
tional ETS in the affected area. As a consequence of both the
decline in energy production and the increase in oxidative
damage in the region, the fiber is no longer capable of self-
maintenance, resulting in the observed intrafiber atrophy
and fiber breakage. We are therefore proposing that a pro-

cess contained within a very small region of a muscle fiber
can result in breakage and loss of muscle fiber from the tissue.
Keywords: muscle loss; intrafiber atrophy; aging.
INTRODUCTION
Adenosine triphosphate is the carrier of free energy in most
living cells and is generated by the process of oxidative
phosphorylation that occurs in the mitochondrion. This free
energy is required for mechanical work, active transport of
molecules and ions and the synthesis of biomolecules.
Disturbances in energy production, due to mitochondrial
DNA (mtDNA) mutations, have been shown, in mito-
chondrial myopathies and cellular myopathy models, to
have a negative impact on the function of cells, specific
tissues and, ultimately, the whole animal. These mutations
include missense mutations, protein synthesis mutations,
copy number mutations and insertion-deletion mutations
[1]. Mitochondrial deletion mutations present as human
disease states in a number of mitochondrial myopathies.
The evolving, progressive nature of mtDNA mutations has
led researchers to focus on the contribution of mtDNA
mutations to the aging process. Sarcopenia is a clinically
recognized manifestation of the aging process that presents
as muscle mass and function loss over time. We used young,
middle-aged and old muscles from rodents and primates to
test whether mtDNA deletion mutations are associated with
the negative physiological impact of sarcopenia.
SARCOPENIA
An age-related loss of muscle mass and function occurs in
skeletal muscle of a variety of mammalian species; this
process is referred to as sarcopenia. In humans, specific

skeletal muscles undergo a  40% decline in muscle mass
between the ages of 20 and 80 years [2]. The public health
ramifications of this large decline are evident in the clinical
presentation, which includes decreased mobility, energy
intake and respiratory function. These declines affect both
the nutrition and the ability of elderly people to live
independently.
Progressive muscle wasting has also been demonstrated in
rodents and nonhuman primates. These sarcopenic changes
are evidenced by a significant reduction in muscle cross-
sectional area, muscle mass loss and fiber number loss over
time. In the Fischer 344 · Brown Norway (FBN) hybrid
rat, the difference between the rectus femoris muscles of
18- and 38-month-old animals is striking. Muscle cross-
sectional area is reduced by 30% in the older animals and
the muscle composition is more heterogeneous including an
increase in fibrotic tissue. A significant reduction in muscle
mass (45%) is observed between 18- and 36/38-months of
age as well as a significant (27%) loss of muscle fibers
counted at the midbelly (Fig. 1).
Although the molecular events responsible for sarcopenia
are unknown, the muscle mass loss is due to fiber atrophy
[2–4] and fiber loss [2,5,6]. A variety of mechanisms
Correspondence to J. M. Aiken, 1656 Linden Drive, Madison, WI
53706, USA.
Fax: + 1 608 262 7420, Tel.: + 1 608 262 7362,
E-mail:
Abbreviations: mtDNA, mitochondrial DNA; ETS, electron transport
system; FBN, Fischer 344 · Brown Norway rat; MERRF, myoclonic
epilepsy and ragged red fiber; CPEO, chronic progressive external

ophthalmoplegia; KSS, Kearns–Sayre syndrome; COX, cytochrome c
oxidase; SDH, succinate dehydrogenase; LCM, laser capture micro-
dissection; CSA, cross-sectional area.
(Received 28 November 2001, accepted 15 February 2002)
Eur. J. Biochem. 269, 2010–2015 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02867.x
have been proposed for fiber loss. These include contrac-
tion-induced injury, deficient satellite cell recruitment,
denervation/renervation, endocrine changes, oxidative
stress and mitochondrial DNA damage.
We propose that the latter two mechanisms, in concert,
contribute to the progressive age-related loss of muscle
mass. Our working hypothesis is based on the idea that
oxidative damage to the mitochondrial genome has the
potential to trigger a deletion event. Accumulation of these
mtDNA deletion mutations would cause a decline in the
energy production of the affected cell(s), result in abnormal
electron transport system (ETS) enzyme phenotypes, atro-
phy and would, ultimately, lead to fiber loss.
Working with an animal model known to undergo
sarcopenia, we addressed the question of whether mtDNA
deletion mutations initiate the events that lead to sarcope-
nia. We examined a quadricep muscle, rectus femoris, from
three different age groups of FBN rats (5-, 18-, and 36/38-
month-old). Fiber number at the midbelly, individual fiber
cross-sectional area, electron transport system abnormalities
and mtDNA deletion mutations were analyzed. In the
remainder of this review, we will present data that support
the hypothesis that mitochondrial DNA deletion mutations
play a critical role in sarcopenia.
ACCUMULATION OF mtDNA DELETION

MUTATIONS WITH AGE
Mitochondria generate most of the energy in cells. They
contain their own genomes (2–10 per mitochondria) that
replicate independently of the nuclear genome [7]. The
mitochondrial genome, in mammals, is  16 kb in length
and encodes 22 tRNA, 13 subunits of the electron transport
system and its own 16S and 26S ribosomal RNAs [8]. The
mitochondrial transcripts are synthesized as long polycis-
tronic messages and are transcribed from both strands.
The mtDNA genome is thought to be a major target of
oxidative damage for several reasons. First, the mtDNA
genome is located adjacent to the primary source of reactive
oxygen species, the electron transport system (reviewed in
Cadenas & Davies [9]). The lack of histone cognates [10] and
the minimal repair systems [11] in the mitochondria (as
compared to the nucleus) increase the likelihood of
oxidative damage occurring and being maintained in the
mitochondrial genome. The levels of oxidatively damaged
bases in mtDNA are 10- to 20-fold higher than that
observed in nuclear DNA [12,13]. Also, the contiguous,
compact nature of the mitochondrial coding region (all but
the displacement loop region encode either mRNAs or
tRNAs) increases the chance that a mutation event will
affect a gene product.
MtDNA mutations have been shown, in humans, to
result in a number of diseases including myopathies and
encephalopathies, a broad class of conditions characterized
by muscle weakness and central nervous system dysfunc-
tion. Myopathies can be divided into two major groups: (a)
those caused by a single mtDNA base substitution, such as

Leber’s hereditary optic nerve atrophy and myoclonic
epilepsy and ragged red fiber (MERRF) [14,15]; and (b)
diseases caused by large mtDNA deletion mutations such as
chronic progressive external ophthalmoplegia (CPEO) and
Kearns–Sayre Syndrome (KSS) [16–18]. In myopathy
patients, the levels of the mutated mtDNA genomes are very
high, 73–98% mutated mtDNA in symptomatic MERRF
patients [19]. Deletion mutations are generally present as
20–80% of all mtDNA genomes in KSS patients [20].
The first age-associated mtDNA alterations identified
where those that were also detected in the mitochondrial
myopathies (reviewed by Wallace [1]). Initial studies
focussed on the ÔcommonÕ deletion, mtDNA
4977
in humans.
Although this common deletion was not detected in normal
aged individuals by Southern blot analysis, it was detectable
using the more sensitive PCR. This particular deletion
mutation was found to accumulate, with age, in a variety of
human tissues [21,22]. The highest levels were detected in
nerve and muscle tissue [23], the same tissues in which
mitochondrial enzyme activities were observed to decline
with age. Later studies demonstrated the presence of
different deletion mutations that accrued within and
between different human tissues [21,24–28]. Subsequent
studies identified multiple mtDNA deletion mutations in
a variety of species including rhesus monkeys [29], mice
[30–33] and rats [34–36].
Using PCR, we analyzed tissue homogenates prepared
from specific skeletal muscles of rhesus monkey, mice and

rats for the presence of age-associated mtDNA deletion
mutations [29,30,35]. Deletion mutations were observed in
all three species. In rhesus monkey skeletal muscle, there
was a significant increase in the number and frequency of
mtDNA deletion mutations with age [29]. The number of
deletion mutations increased most dramatically in rhesus
monkeys greater than 20 years of age. Some of the deletion
products were common among the rhesus monkeys while
others were unique. The rodent studies yielded similar
information, multiple mtDNA deletion mutations accumu-
lated with age. Unlike rhesus monkeys, however, common
deletion events were rare in both rats and mice suggesting
that they might initiate or accumulate differently in these
animals [33,36,37].
DELETION MUTATIONS ACCUMULATE
FOCALLY
Initial studies utilizing radioactive PCR methods calculated
the abundance of the specific deletion mutation,
mtDNA
4977
, to be < 0.1% of the total mtDNA present
Fig. 1. Analysis of FBN rat rectus femoris muscles from three different
age groups. Muscle weight is represented by the black bars using the
left axis, fiber number is represented by the white bars using the right
axis, muscle cross-sectional area is represented by the width of the
black bars using the x-axis. Different letters associated with like bars
indicate a significant difference (P <0.05).
Ó FEBS 2002 mtDNA deletions and sarcopenia (Eur. J. Biochem. 269) 2011
in the tissue homogenate [22,23]. The low abundance of this
deletion led to the speculation that mtDNA deletion

mutations are of minor physiological significance. All of
the initial quantitative analyses, however, were performed
using cellular homogenates, in which thousands of cells were
present. Estimating the abundance of mtDNA deletion
mutations from homogenates assumed an equal cellular
distribution of the deletion-containing genomes. In situ
hybridization analyses of aged muscle tissue provided the
first evidence of a focal accumulation of mtDNA deletion
mutations. The initial in situ hybridization studies of age-
associated mtDNA deletion mutations were performed on
human skeletal muscle and were focussed on the common
deletions [38,39]. Using mtDNA probes located either
within or outside the deleted regions, high levels of mtDNA
deletion mutations were localized to individual cells. This
suggests that deletion mutations accumulate focally and
that neither their abundance nor distribution can be
accurately assessed in cellular homogenates.
Further evidence for the focal and mosaic distribution of
the mtDNA deletion mutations came from muscle fiber
bundle analyses [40]. In these studies, defined numbers of
skeletal muscle fibers were dissected from old rhesus
monkeys. Two classes of samples were analyzed for
mtDNA deletion products, those containing either: (a)
several thousand individual muscle fibers or (b) defined
groups of 75 or 10 fibers per bundle. We found that the
number of amplification products decreased with the
reduction in the number of muscle fibers analyzed, but that
there was a significant increase in the abundance of specific
deletion-containing genomes in the samples containing
fewer fibers [40]. These experiments demonstrated that

mtDNA deletion mutations are not distributed evenly
throughout a muscle group, but rather focally accumulate
to high levels in a subset of fibers.
ETS ABNORMALITIES ACCRUE
WITH AGE
Dramatic changes in the activities of specific ETS enzymes
were observed in myopathy patients [41–44] and were later
demonstrated to occur in humans with age [45,46]. We
examined the muscle tissue of young and old rhesus
monkeys and rats, histologically, for changes in the activity
of two ETS enzymes with age, cytochrome c oxidase (COX)
and succinate dehydrogenase (SDH). Several of the COX
subunits are encoded by the mitochondrial genome and,
thus, the absence of COX activity would be indicative of
changes in the mtDNA. Although SDH is entirely encoded
by the nuclear genome, decreases in mitochondrial energy
output result in a compensatory up-regulation of mito-
chondrial synthesis and of nuclear-encoded transcripts. For
the rat studies, entire muscles were dissected, cut at the
midbelly and embedded. Muscle biopsy samples (vastus
lateralis) were embedded for the rhesus monkey studies.
Sections were then obtained using a cryostat and, depending
on the study, 100–200 serial, 8–10-lm thick sections were
produced. Samples were analyzed by staining every seventh
slide (i.e. the first, seventh, 14th, etc.) for COX activity and
every eighth slide (i.e. the second, eighth, 15th, etc.) for SDH
activity. Fibers containing ETS abnormalities would be
expected to show a negative reaction for COX and often,
but not always, hyper-reactive for SDH (Fig. 2). All stained
slides were analyzed by light microscopy and all COX

–
and/
or SDH
++
fibers were noted. ETS abnormal (COX
–
and/
or SDH
++
) were followed along the 1000–2000 lmto
determine the length of the ETS abnormal region. Using
these two enzymatic stains, we demonstrated the age-
associated increase of ETS abnormalities in several different
muscles and animal models [36,47]. For example, in the
rectus femoris of FBN rats, no COX
–
/SDH
++
fibers were
observed in the 5-month-old animals and only one was
found among the nine 18-month-old rats. At 36/38-months
of age, however, a total of 184 COX
–
/SDH
++
regions were
observed in 11 rats (Table 1). As only 1 mm of tissue was
examined ( 3% of the length of the rectus femoris),
extrapolation was used to determine that  7% of the
muscle fibers of aged rectus femoris muscles contained ETS

abnormal regions.
ETS ABNORMAL FIBERS ATROPHY
While following these abnormal fibers for 1000–2000
microns, we noted that many of the ETS abnormal fibers
displayed an overt decline in cross-sectional area (intrafiber
atrophy) within the ETS abnormal region of the fiber. To
Fig. 2. Electron transport system abnormalities. Consecutive, serial cross-sections of the rectus femoris from a 36-month-old FBN rat identifying a
fiber (indicated by the arrow) that stains negative for cytochrome c oxidase (COX
–
) and hyperreactive for succinate dehydrogenase (SDH
++
).
2012 D. McKenzie et al. (Eur. J. Biochem. 269) Ó FEBS 2002
quantitate the extent of atrophy in these ETS abnormal
regions, the cross-sectional area (CSA) of both ETS
normal and abnormal regions was measured along the
length of the 1000 lm. The smallest (minimum CSA)
value of the fiber CSA in the ETS abnormal region was
divided by the average value of the fiber CSA in the ETS
normal region within the same fiber [36,52]. In the ETS
normal fibers, the ratio between minimum CSA and
average CSA was determined. The cross-sectional area
ratio of the ragged red phenotype is significantly smaller
than that observed in either normal fibers or fibers that
display a COX
–
/SDH
normal
phenotype. These data clearly
demonstrate that ETS abnormalities have a localized

physiological impact on the cell and can result in fiber
atrophy. Subsequent longitudinal analysis of atrophied
fibers also showed that some decrease in CSA until they
are no longer observable by light microscopy, suggesting
that they are broken. The fiber can often be found again
several sections later.
Our analysis of the cross-sectional area of ETS abnormal
fibers also demonstrates that longer ETS abnormal regions
are more likely to atrophy than shorter ETS abnormal
regions. COX
–
/SDH
normal
regions are shorter than
COX
–
/SDH
++
regions and their CSA slightly decrea-
ses. The COX
–
/SDH
++
regions are larger than the
COX
–
/SDH
normal
regions and the CSA declines to a
greater extent. These studies were performed in the rat and

suggest a process in the rat quadricep muscle in which the
initial phenotype is COX
–
/SDH
normal
. The phenotype
progresses with time to the COX
–
/SDH
++
phenotype,
associated with fiber atrophy and fiber breakage. A similar
process was observed in the rhesus monkey skeletal muscle.
ETSABNORMALFIBERSCONTAIN
mtDNA DELETION MUTATIONS
We have recently defined the mtDNA genotype associated
with the abnormal ETS phenotypes in aged muscle fibers.
Using laser capture microdissection (LCM), we have
amplified the mtDNA from both abnormal and normal
regions of fibers. As described by Cao et al.[37],the
mtDNA deletion mutations were concomitant with the
COX
–
/SDH
++
regions of affected muscle fibers. Single
fiber sections, 10-lm thick, of normal and abnormal
regions of rat rectus femoris muscle fibers were isolated
using LCM. When total DNA from each fiber section was
subjected to whole mitochondrial genome PCR, smaller

than wild-type amplifications were observed in all of the
COX
–
/SDH
++
regions demonstrating the association of
deletion mutations with the ETS abnormal phenotype.
These deletion-containing genomes were the only mtDNA
genomes detected in the ETS abnormal regions while only
wild-type genomes were found in the ETS normal regions.
LCM coupled with PCR analysis has clearly demonstrated
that age-associated mtDNA deletion mutations are locali-
zed to specific cells identified as abnormal by histo-
chemical analysis. When the same ETS abnormal region
was sampled in two different places from the same fiber
(i.e. 70 lm apart), the same deletion product was obtained.
The accumulation of the same deletion product in both
COX
–
/SDH
++
regions of skeletal muscle [37] and indi-
vidual cardiomyocytes [48] suggests that mtDNA deletion
mutations are clonal events.
Two hypotheses have been proposed to account for this
clonal expansion phenomenon. De Grey [49] proposed that
damaged mitochondria degrade more slowly than intact
mitochondria. The abnormal mitochondrial would there-
fore accumulate in the cell by the Ôsurvival of the slowestÕ.
Due to the low proton gradient present in defective

mitochondria, the production of free radicals would be
decreased and, hence, less damage to the mitochondrial
membranes. The second hypothesis, based on the size of the
mtDNA deletion mutations observed, presumes that the
smaller deletion-containing genomes would have a replica-
tive advantage [50]. Support for the replicative advantage of
the smaller genomes is provided by re-population kinetic
studies using several mtDNA forms after severe mtDNA
depletion by ethidium bromide. These studies demonstrated
that the replication and maintenance of mtDNA in human
cells is highly dependent on molecular features, as partially
deleted mtDNA molecules re-populated cells significantly
faster than full-length molecules [51].
This work, in total, has led us to propose the following
mechanism for the role of mitochondrial DNA deletion
mutations in sarcopenia. A portion of the mtDNA genome
is deleted by an as yet unknown mechanism, possibly as the
result of oxidative damage. As mtDNA genomes carrying a
deletion would be smaller, they would have a replicative
advantage over wild-type genomes. As the ratio of deletion-
containing genomes increased, the mitochondria would
become deficient in major subunits of the ETS (i.e. the
COX
–
phenotype) and an energy deficiency would occur
(Fig. 3B). Concurrent with the energy deficiency would be
increased oxidative damage [36]. Signals from the nuclear
genome would then trigger mitochondrial amplification in
an effort to overcome the energy deficiencies. This increased
synthesis of mitochondria would subsequently result in an

increase in the production of the nuclear subunits of the
ETS system (i.e. the COX
–
/SDH
++
phenotype) (Fig. 3C).
However, as the deletion mutations would continue to
Table 1. Ragged red fiber content of rat rectus femoris muscles.
Age
No.
of animals
Fibers examined
a
(mean±SD)
Ragged red fibers
(mean±SD)
Estimated no. of RRF
per muscle (%)
b
5 Months 6 10 426 ± 714 0
c
0
18-Months 9 10 075 ± 707 0.11 ± 0.33
c
4 (0.04%)
36/38-Months 11 7606 ± 1257 17.0 ± 14
d
567 (7.5%)
a
Each fiber was examined through 1000 lm.

b
Estimates were determined by dividing the mean number of ETS abnormalities found in
1 mm of tissue by 0.03 (3% of the approximate 3.0 cm length of rectus femoris), percentages were determined by dividing the resulting value
by the mean number of fibers.
c,d
Values with different superscripts were significantly different.
Ó FEBS 2002 mtDNA deletions and sarcopenia (Eur. J. Biochem. 269) 2013
out-replicate the wild-type mtDNA genomes, both energy
deficiencies and oxidative damage would continue to accrue.
In response, the fiber atrophies (Fig. 3D) and, eventually,
breaks (Fig. 3E).
ACKNOWLEDGEMENTS
Research in our laboratory is supported by the National Institutes of
Health Grant Nos. RO1 AG11604, AG17543, P01 AG11915 and T32
AG00213.
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Fig. 3. Model of mtDNA deletion mutations and ETS abnormalities in
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abnormal region. a, COX
normal
/SDH
normal
;b,COX
–
/SDH
normal
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c–e,COX
–
/SDH
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.
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