Accumulation of these mtDNA deletion mutations would cause a decline in the energy production of the affected cells, result in abnormal electron transport system ETS enzyme phenotypes, a
Trang 1M I N I R E V I E W
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
I N T R O D U C T I O N
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
S A R C O P E N I A
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: aiken@ahabs.wisc.edu
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)
Trang 2have 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 quadricepmuscle, 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
A C C U M U L A T I O N O F m t D N A D E L E T I O N
M U T A T I O N S W I T H A G E
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
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, mtDNA4977in 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]
D E L E T I O N M U T A T I O N S A C C U M U L A T E
F O C A L L Y
Initial studies utilizing radioactive PCR methods calculated the abundance of the specific deletion mutation, mtDNA4977, 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).
Trang 3in 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
E T S A B N O R M A L I T I E S A C C R U E
W I T H A G E
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 lm to 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
E T S A B N O R M A L F I B E R S A T R O P H Y
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++).
Trang 4quantitate 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–/SDHnormalphenotype 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–/SDHnormal regions are shorter than
COX–/SDH++ regions and their CSA slightly
decrea-ses The COX–/SDH++ regions are larger than the
COX–/ SDHnormal 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–/SDHnormal 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
E T S A B N O R M A L F I B E R S C O N T A I N
m t D N A D E L E T I O N M U T A T I O N S
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 replicareplica-tive 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
a
Each fiber was examined through 1000 lm.bEstimates 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.
Trang 5out-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)
A C K N O W L E D G E M E N T S
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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