Journal of Gerontology: BIOLOGICAL SCIENCES
2006, Vol. 61A, No. 1, 3–13
Copyright 2006 by The Gerontological Society of America
Regulation of Skeletal Muscle Mitochondrial
Content During Aging
Carrie N. Lyons,1 Odile Mathieu-Costello,2 and Christopher D. Moyes1
1
Department of Biology, Queen’s University, Kingston, Ontario, Canada.
2
Department of Medicine, University of California San Diego.
M
ITOCHONDRIA play a pivotal role in muscle cell
physiology, primarily by providing adenosine triphosphate via oxidative phosphorylation. Variations in
muscle mitochondrial content are observed throughout the
lifetime of an animal. During muscle differentiation,
mitochondrial content increases 5-fold (1). When adult
phenotypes are established, slow-twitch oxidative muscles
contain about 5 times more mitochondria than do fast-twitch
glycolytic muscles, as measured by citrate synthase (CS)
activity levels (2). Mitochondrial content remains plastic in
adult muscle, changing in response to environmental stress
(3) and physiological conditions (4,5). The aging process,
for example, can cause a decline in mitochondrial content,
particularly in muscles that undergo atrophy (6). These
metabolic changes, in combination with declines in
contractile function, lead to a loss of muscle strength and
endurance (7).
Although much is known about the regulatory basis of
distinct contractile phenotypes, determinants of the metabolic phenotype in different fiber types and physiological
states are less clear. Several lines of evidence suggest that
differences in mitochondrial content among muscles of
varied fiber type may be due to a transcriptional network
including the coactivator PGC-1a (peroxisome proliferatoractivated receptor-c coactivator) (8). For example, overexpression of PGC-1a in cultured muscle cells and fat cells
leads to mitochondrial proliferation (9,10), and muscles
rich in mitochondrial enzymes also possess higher levels
of PGC-1a (11). Despite the research focus on these
transcriptional regulators, it is not yet established if steady-
state levels of muscle mitochondria are regulated primarily
by transcriptional controls.
Understanding the mechanistic basis of variations in
mitochondrial content among muscles is complicated by
many morphological differences that influence functional
relationships. For example, the striated muscles differ in
nuclear content. Nuclear content of slow-twitch muscle is
twice that of fast-twitch muscle, based on ultrastructural
analyses (12–16) or biochemical analyses (mg DNA/g
tissue) (17). Changes in the nuclear content of skeletal
muscle are also reported to occur during the aging process
(18–21). Such variations in nuclear content among fiber
types and during aging have important influences on the
genetic control of the muscle phenotype (22). Superimposed
on these morphological differences are regulatory controls at
the level of transcription and translation. Variations in any
or all of these parameters (i.e., nuclear content, transcriptional and translational processes) can influence the
molecular composition of skeletal muscle, e.g., mitochondrial content.
In this study, we compare mitochondrial enzyme and
gene regulatory properties in a spectrum of skeletal muscles
in adult (12 months), old (24 months), and very old (35–36
months) rats. We focus on analysis of CS activity as
a marker for mitochondrial content, but also analyze
activities of other mitochondrial enzymes to assess changes
in enzyme stoichiometries, which often occur in muscle
with age (23–25). We compare CS activity, CS messenger
RNA (mRNA) levels, and DNA content across 10 skeletal
muscles that vary in CS activity over a 5-fold range (2).
3
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Mitochondrial content of skeletal muscle varies among fiber types, and changes in complex ways
during aging. We evaluated the regulatory origins of differences in mitochondrial content among
muscles of varied fiber type in F3443BNF1 rats, and how these regulatory patterns are altered
with aging. In adult (12 month) animals we found that units citrate synthase (CS)/g tissue,
a marker for mitochondrial content, varied ;3-fold among 10 skeletal muscles. Stoichiometric
relationships between CS and isocitrate dehydrogenase, aconitase, and cytochrome c oxidase
were generally preserved across fiber types. Among the 10 muscles of adult rats, CS content
correlated with nuclear content (R2 ¼ 0.36). Muscles differed widely in CS messenger RNA
(mRNA)/DNA (an index of variation in transcriptional regulations) and units CS/CS mRNA (an
index of variation in posttranscriptional regulations). All muscles of aged rats (35 months)
showed an increase in mg DNA/g, suggestive of atrophy. Age-dependent declines in units CS/
DNA were accompanied by reductions in CS mRNA/DNA and/or units CS/CS mRNA,
depending on muscle fiber type. Thus, declines in units CS/DNA with age appeared to be due to
transcriptional as well as translational variations. Differences in mitochondrial content among
muscle fiber types and age groups may arise from variations in nuclear content and
posttranscriptional processes, as well as transcriptional regulation.
4
LYONS ET AL.
Because variations in muscle mitochondrial content are
often thought to arise through transcriptional regulation
(e.g., by PGC-1a and other transcriptional effectors), we
investigate whether CS enzyme content parallels CS mRNA
levels. However, because multiple factors can influence the
molecular composition of skeletal muscle, we also evaluate
the importance of nuclear content and posttranscriptional
processes on mitochondrial abundance across muscles and
during aging. We hypothesize that activity levels of CS, an
enzyme widely held to be transcriptionally regulated (26),
should covary with CS mRNA levels among fiber types
and during aging.
Animal Care
Male F1 hybrid F344 3 Brown Norway rats (Harlan
Sprague Dawley Inc., Indianapolis, IN) were housed 2–3
animals per cage in a temperature-controlled room with
a 12-h light/dark cycle. Animals had access to standard
chow (Harlan Teklad 8604; Madison, WI) and untreated tap
water ad libitum. The study was approved by the University
of California, San Diego, Animal Subjects Committee
and conducted in a pathogen-free facility accredited by the
American Association of Accreditation of Laboratory
Animal Care. The investigation conforms to the Guide for
the Care and Use of Laboratory Animals published by the
U.S. National Institutes of Health (NIH Publication No. 8523, revised 1996).
Tissue Harvest
Tissues were harvested from animals, ages 12 (adult), 24
(old), and 35–36 (very old) months. Because the maximal
life span of these rats is 41–42 months (27), the chosen age
groups allow for an analysis of the aging process over the
last third of the life span, which is the period in humans
when the greatest losses in muscle structure and function
occur (7). Animals were weighed, anesthetized with 40–70
mg of sodium pentobarbital, and the following muscles were
excised as quickly as possible from both sides of the body:
adductor longus (Add-Lon), biceps femoris (Bic-Fem),
gluteus superficialis (Glu-Sup), plantaris (Plan), extensor
digitorum longus (EDL), lateral gastrocnemius (Lat-Gas),
medial gastrocnemius (Med-Gas), soleus (Sol), tibialis
anterior (Tib-Ant), and diaphragm (Dia). All muscles were
harvested within 1.5 hours of administration of anesthetic.
Whole muscle samples were weighed immediately, flashfrozen in liquid nitrogen (N2), and stored in a cryovial at
808C for biochemical and molecular assays. For Lat-Gas,
only the deep (fast-twitch oxidative) portion of the muscle
was frozen for further analysis. Similarly, only the
superficial (fast-twitch glycolytic) portions of the Med-Gas
and Tib-Ant were retained.
Enzyme Assays
Frozen muscle samples were powdered using a mortar
and pestle under liquid N2 and stored at 808C until further
use. For enzyme assays, powdered tissue was extracted in
20 volumes of a buffer composed of 20 mM HEPES, 1 mM
Tissue Protein, DNA, and RNA Content
Protein content (mg/g) of muscle homogenates was
measured using a commercial protein assay kit (Bio-Rad,
Hercules, CA), based on the Bradford method for protein
quantification (29).
DNA content was assayed using DNA-specific fluorescent
dyes. Aliquots of muscle homogenate from enzyme assays
were reserved and stored at 208C for assay of mg DNA/g
tissue. These measurements are a good estimate of nuclear
DNA content because mitochondrial DNA comprises less
than 2% of the total DNA content of skeletal muscle (30).
Thawed homogenate was diluted 1:6 in DNA digestion buffer
and incubated overnight at 558C. Digestion buffer contained
100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH
8.0), 0.5% sodium dodecyl sulfate, and proteinase K at 0.2
mg/ml. Digested homogenate (2 ll) and DNA standards (1.0–
60 ng of purified DNA) were loaded onto a 96-well black
plate. DNA concentration was quantified according to the
manufacturer’s instructions with Pico-Green (Molecular
Probes, Eugene, OR), a reagent that fluoresces when bound
to double-stranded DNA. Fluorescence was detected (excitation 480 nm, emission 520 nm) using a Spectramax Gemini
fluorometer (Molecular Devices).
Differences in mg RNA/g tissue of muscles of varied fiber
type (17,31,32) must be considered when comparing levels
of specific mRNA transcripts across muscles (31). Thus, we
measured the total RNA content of all samples to report CS
mRNA/g tissue. Frozen powdered tissue was quickly
weighed, diluted 10- to 40-fold in GTC solution (4 M
guanidium thiocyanate [GTC], 25 mM sodium citrate, 0.5%
sarkosyl, 1.0% b-mercaptoethanol, pH 7.0), and homogenized using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). Total RNA was extracted from the
homogenate using a standard phenol-chloroform procedure
according to the methods of Chomczynski and Sacchi (33).
After the first phase separation, a small volume (,5%) of
the aqueous top layer was reserved for quantification of
RNA using the fluorescent reagent Ribo-Green (Molecular
Probes). The remainder of the aqueous layer was precipitated and further purified to isolate RNA suitable for
northern blot analysis. To quantify mg RNA/g tissue,
samples and standards (10–200 ng of purified transfer RNA)
were loaded onto a 96-well black plate and measured
(excitation 480 nm, emission 536 nm). To account for
possible effects of the RNA extraction procedure (i.e., GTC,
phenol, chloroform) on fluorescence of Ribo-Green, an
equal volume (1 ll) of a mock extraction (i.e., no tissue) was
added to each well of the RNA standards.
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METHODS
EDTA, 0.1% Triton X-100 (pH 7.2) and homogenized with
a glass homogenizer. Specific activities of isocitrate dehydrogenase (IDH), aconitase (ACON), cytochrome c
oxidase (COX), and CS were measured on a SpectraMAX
Plus spectrophotometer (Molecular Devices, Sunnyvale,
CA). IDH, CS, and ACON were assayed according to
previously published protocols (1,28). COX was assayed at
550 nm with 50 mM Tris-HCl (pH 8.0) containing 50 lM
reduced cytochrome c (1). IDH and ACON activities were
measured immediately after homogenization, as activity was
found to decline after a 30-minute incubation on ice.
MITOCHONDRIAL CONTENT IN AGING MUSCLE
Statistics
Data are presented as mean 6 standard error of the mean
(SEM). A two-way analysis of variance (ANOVA) was used
to determine significant main effects ( p , .05) of age and
muscle fiber type on variations in enzyme activity and
enzyme stoichiometries. A one-way ANOVA followed by
the Tukey-Kramer test was used to detect significant
differences ( p , .05) within one muscle fiber type across
three age groups (adult, old, very old). An independent t test
was used to detect differences ( p , .05) within one muscle
type between the adult and very old age groups. Regression
analysis was performed to determine linear relationships
between two variables ( p , .05). All statistical analyses
were performed with JMPIN (Thomson Learning, Florence,
KY) statistical software.
RESULTS
Regulation of Mitochondrial Content in Different
Muscles of Adult Animals
We found that mg DNA/g tissue and CS activity correlate
across 10 muscles of varied fiber-type characteristics
(Tables 1 and 2) in adult animals, with almost 40% of the
variance in mitochondrial content explained by differences
in DNA content (Figure 1A, R2 ¼ 0.36, p , .0001). The
most parsimonious explanation for this linear relationship is
that CS gene expression is regulated primarily by constitutive pathways, and that differences arise from variations in
DNA content. If so, then DNA content should also predict
CS mRNA levels, which should likewise predict CS enzyme
content. CS mRNA/g tissue (Figure 1C) was quantified
using paired measurements of CS mRNA/total RNA (via
northern blot) and total RNA/g tissue (via Ribo-Green). We
found no relationship between CS mRNA/g tissue and mg
DNA/g tissue among the muscles (Figure 1C), suggesting
that the tissues vary in degree of transcriptional control over
CS gene expression. The potential impact of contaminating
sources of nonmuscle nuclei in our measurements of mg
DNA/g tissue should be considered, but it is expected that
these sources also contribute to measurements of mRNA
levels and enzyme activity.
Next, we compared CS activity levels to CS mRNA
content across muscles, an index of posttranscriptional
variation among tissues. Because CS is not known to be
allosterically or covalently regulated, catalytic activity is
a good reflection of enzyme content (36). If CS enzyme
levels were determined primarily by transcriptional regulation, we would predict a linear relationship between CS
mRNA/g tissue and CS activity/g tissue. The lack of
relationship between these parameters (Figure 1E) suggests
an influence of posttranscriptional controls on CS content in
different muscles.
As a result of this analysis, it is clear that some tissues
have more CS protein than would be expected based on CS
mRNA, and other tissues have more CS mRNA than would
be expected based on DNA content. For example, deep LatGas maintains one of the highest levels of CS activity/g
tissue with one of the lowest quantities of CS mRNA/g
tissue (Figure 1E). Thus, the linear relationship between
units CS/g tissue and mg DNA/g tissue is not a simple
relationship between constitutive gene expression and
nuclear DNA content.
Changes in Muscle Composition With Age
To investigate how the regulation of mitochondrial
content is altered with age, we evaluated the same set of
muscles in adult (12 months), old (24 months), and very old
(35–36 months) rats. In Table 1, aging-related compositional changes are reported for each muscle. With the
exception of Add-Lon, considerable atrophy occurred in all
tissues, as indicated by an 18%–51% loss in absolute muscle
mass. As body mass changed very little with age (;10%
higher in old animals, see legend of Table 1), similar
declines (17%–49%) in muscle-specific mass were observed. Although there was a trend for decreased protein
(mg/g tissue), no significant declines were observed with
age except in the superficial Tib-Ant (18%), Bic-Fem
(21%), and Dia (13%). DNA levels/g muscle significantly
increased in all of the fast-twitch muscles of very old
animals (by 33%–75%); no differences were observed in the
old age group relative to adults. RNA content (mg/g tissue)
also significantly increased in many of the fast-twitch
muscles (up to 2.3-fold), except in the superficial Med-Gas
and superficial Tib-Ant. No significant changes in DNA or
RNA occurred in slow-twitch and mixed muscles, with the
exception of a 49% increase in DNA in the deep Lat-Gas.
Regulation of Mitochondrial Content in
Different Muscles of Aged Animals
The linear relationship between DNA and CS activity
across muscles in adult animals (R2 ¼ 0.36) is preserved in
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Northern Blot Analysis
Purified total RNA was denatured and fractionated (5–20
lg/lane) with a standard 1% agarose-formaldehyde gel
system. RNA was transferred overnight onto Duralon-UV
membrane (Stratagene, La Jolla, CA) and then cross-linked
with a UV Crosslinker (Fisher Scientific, Pittsburgh, PA).
A complementary DNA (cDNA) probe for COX IV was
constructed as previously described (34). CS (GenBank
accession no. AF461496) was amplified from rat gastrocnemius cDNA template at 57.58C using the following primers:
59-gaaacatcrgttcttgatcc-39 and 59-gtgtattccagatgtagtcwcg
taa-39. The resulting 765-bp product was used as a cDNA
probe for CS mRNA. Cytochrome c (CYT-C) (GenBank
accession no. NM012839) was amplified from rat skeletal
muscle cDNA template at 528C using 59-cgggacgtctccc
taaga-39 and 59-gctattaggtctgccctttc-39, yielding a 351-bp
product that was used as a cDNA probe. Hybridization and
phosphorimaging conditions were as described previously
(35). Signal intensities for all genes were corrected for
loading differences by probing for 18S RNA.
Separate blots were used for every tissue, with each blot
containing adult and very old samples. For comparison
among tissues, a normalizing blot was generated using
samples that appeared on each tissue blot. After probing the
normalizing blot with CS, the relative signal strength of
CS mRNA across tissues was calculated and used to compare blots.
5
LYONS ET AL.
6
Table 1. Changes in Muscle Composition With Age
Age
Absolute
Mass (g)
Specific Mass
(mg/g Body Weight)
Protein
(mg/g Tissue)
Sol
Adult
Old
Very Old
0.20 6 0.009
0.21 6 0.007
0.13 6 0.006*y (13)
0.37 6 0.013
0.37 6 0.008
0.26 6 0.011*y (13)
136 6 4
127 6 6
114 6 12
0.85 6 0.011
0.82 6 0.039
0.98 6 0.069
0.85 6 0.034
—
0.77 6 0.038
Add-Lon
Adult
Old
Very Old
0.11 6 0.009 (8)
0.09 6 0.004 (6)
0.08 6 0.008 (13)
0.20 6 0.014 (8)
0.16 6 0.011 (6)
0.17 6 0.014 (13)
177 6 8
174 6 11
170 6 10
0.61 6 0.087
0.56 6 0.077
0.71 6 0.049
0.17 6 0.013
—
0.17 6 0.011 (6)
Dia
Adult
Old
Very Old
1.30 6 0.079 (9)
1.22 6 0.061
1.06 6 0.049* (13)
2.50 6 0.13 (9)
2.13 6 0.11
2.07 6 0.07* (13)
173 6 8
194 6 6
y
169 6 4
0.82 6 0.048
0.86 6 0.044
0.97 6 0.034 (4)
0.52 6 0.059
—
0.49 6 0.053 (6)
Lat-Gas (deep)
Adult
Old
Very Old
1.31 6 0.042
1.20 6 0.040
0.64 6 0.048*y (13)
2.49 6 0.06
2.08 6 0.07*
1.27 6 0.08*y (13)
188 6 10
186 6 14
165 6 11
0.69 6 0.020
0.74 6 0.031
0.99 6 0.054*y
0.26 6 0.011
—
0.24 6 0.018 (6)
Med-Gas (superficial)
Adult
Old
Very Old
1.11 6 0.061
0.90 6 0.019*
0.64 6 0.046*y (13)
2.11 6 0.11
1.56 6 0.03*
1.24 6 0.08*y (13)
192 6 7
178 6 8
165 6 7
0.56 6 0.032
0.60 6 0.037
0.79 6 0.013*y
0.25 6 0.035 (6)
—
0.26 6 0.016 (9)
EDL
Adult
Old
Very Old
0.20 6 0.005
0.20 6 0.004
0.14 6 0.007*y (13)
0.38 6 0.008
0.35 6 0.007
0.27 6 0.009*y (13)
152 6 11
175 6 7
151 6 8
0.52 6 0.006
0.58 6 0.005*
0.75 6 0.021*y
0.54 6 0.018
—
0.75 6 0.036*
Glu-Sup
Adult
Old
Very Old
1.43 6 0.074
1.43 6 0.062
0.98 6 0.053*y (13)
2.72 6 0.11
2.48 6 0.10
1.88 6 0.09*y (13)
189 6 2
187 6 15
148 6 12
0.48 6 0.009
0.48 6 0.015
0.67 6 0.023*y
0.38 6 0.051
—
0.53 6 0.021*
Plan
Adult
Old
Very Old
0.48 6 0.022
0.42 6 0.009
0.30 6 0.020*y (13)
0.92 6 0.044
0.73 6 0.019*
0.59 6 0.038*y (13)
213 6 8
213 6 6
190 6 14
0.59 6 0.019
0.67 6 0.033
1.03 6 0.11*y
0.35 6 0.065 (8)
—
0.81 6 0.033*
Tib-Ant (superficial)
Adult
Old
Very Old
0.85 6 0.018
0.80 6 0.021
0.48 6 0.026*y (13)
1.62 6 0.02
1.39 6 0.04*
0.94 6 0.04*y (13)
207 6 5
188 6 6
169 6 11*
0.48 6 0.020
0.54 6 0.009
0.78 6 0.038*y
0.29 6 0.035 (8)
—
0.43 6 0.073 (7)
Bic-Fem
Adult
Old
Very Old
3.90 6 0.170
3.52 6 0.079
2.82 6 0.103*y (13)
7.43 6 0.27
6.14 6 0.20*
5.49 6 0.16* (13)
194 6 3
181 6 6
152 6 12*
0.44 6 0.015
0.48 6 0.011
0.58 6 0.029*y
0.45 6 0.013
—
0.59 6 0.021*
Muscle
DNA
(mg/g Tissue)
RNA
(mg/g Tissue)
Slow-Twitch
Mixed
Notes: Mass of bilateral muscles is the average of both muscles; all other measurements were performed on tissue from one side (random selection) of each
individual. N ¼ 10 for muscle mass, unless otherwise indicated in parentheses. For Lat-Gas, Med-Gas, and Tib-Ant, mass is reported for entire muscle; other
measurements were performed on indicated portions. N ¼ 5 for protein, DNA, and RNA, unless otherwise indicated. RNA was not measured in old animals. Fiber-type
classification for Sol, Plan, EDL, superficial Tib-Ant, deep Lat-Gas, and superficial Med-Gas is based on analysis performed on other individuals from this cohort
of animals (39). Classification for other muscles is based on published fiber-type data (2). Mean body mass (in g) for adult, old, and very old animals was 525 6 13,
577 6 15, and 519 6 10, respectively.
* and y indicate significantly different than adult and old, respectively (one-way analysis of variance, p , .05).
Add-Lon ¼ adductor longus; Bic-Fem ¼ biceps femoris; Dia ¼ diaphragm; EDL ¼ extensor digitorum longus; Glu-Sup ¼ gluteus superficialis; Lat-Gas ¼ lateral
gastrocnemius; Med-Gas ¼ medial gastrocnemius; Plan ¼ plantaris; Sol ¼ soleus; Tib-Ant ¼ tibialis anterior.
the old age group (R2 ¼ 0.48, p , .001, data not shown).
Conversely, in very old animals, only 10% of the variation
in CS activity across muscles is attributed to differences in
DNA content (Figure 1B, R2 ¼ 0.10, p ¼ .03). Because of the
similarity between adult and old animals, the effects of aging
on CS mRNA expression were evaluated only in the very old
age group. As with the adults, there was no simple relationship between either DNA content and CS mRNA levels
(Figure 1D) or CS mRNA levels and CS activity (Figure 1F).
In analyzing specific changes within each muscle with
age, we found that units CS/g tissue varied little (Table 2),
but units CS/DNA significantly declined (by 19%–50%) in
almost all tissues (Figure 2A). This decline signifies major
changes in the regulation of nucleus-encoded mitochondrial
gene expression. In some tissues that we classified as Group
I (superficial Tib-Ant, superficial Med-Gas, deep Lat-Gas,
Dia), declines in units CS/DNA are paralleled by declines in
CS mRNA/DNA (Figure 2, A and B). These changes
suggest that declines in transcription may occur in these
muscles. In other tissues that we classified as Group II (Plan,
Sol, Bic-Fem, Glu-Sup), decreases in units CS/DNA are
paralleled by declines in units CS/CS mRNA (Figure 2, A
and C), indicating reductions in posttranscriptional activity.
Finally, in two muscles that we classified as Group III,
declines in both CS mRNA/DNA and units CS/CS mRNA
accompany the decreases in units CS/DNA (Figure 2, A–C).
Overall, our results reveal muscle-specific mechanisms for
maintaining mitochondrial enzyme levels in aging tissue,
with changes occurring at the transcriptional and/or posttranscriptional levels.
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Fast-Twitch
MITOCHONDRIAL CONTENT IN AGING MUSCLE
7
Table 2. Changes in Specific Activities of Mitochondrial Enzymes With Age
Muscle
Age
CS (Units/g Tissue)
IDH (Units/g Tissue)
ACON (Units/g Tissue)
COX (Units/g Tissue)
Sol
Adult
Old
Very Old
14.1 6 1.4
14.6 6 0.5
11.7 6 1.2
0.62 6 0.21 (3)
0.59 6 0.09 (3)
0.79 6 0.28 (3)
1.56 6 0.19
1.60 6 0.30
1.10 6 0.32
19.6 6 1.3
18.5 6 1.6
15.1 6 2.3
Add-Lon
Adult
Old
Very Old
16.7 6 2.4
17.5 6 3.1
13.4 6 1.0
1.07 6 0.27
1.49 6 0.41
1.05 6 0.06
1.80 6 0.44
2.27 6 0.67
1.41 6 0.08
25.3 6 3.8
27.7 6 5.9
20.3 6 1.4
Dia
Adult
Old
Very Old
33.0 6 1.8
35.3 6 1.2
31.2 6 0.8
1.28 6 0.05
1.27 6 0.09
1.24 6 0.06
3.11 6 0.24
3.07 6 0.17
3.34 6 0.17
36.6 6 1.6
32.4 6 1.0
33.5 6 1.5
Lat-Gas (deep)
Adult
Old
Very Old
24.2 6 0.9
25.6 6 1.8
19.3 6 1.3y
1.65 6 0.08
1.70 6 0.08
1.19 6 0.08*y
3.10 6 0.23
3.27 6 0.13
2.24 6 0.15*y
31.0 6 2.2
31.2 6 1.5
22.4 6 1.0*y
Med-Gas (superficial)
Adult
Old
Very Old
20.9 6 3.1
19.4 6 1.6
20.1 6 1.2
1.49 6 0.25
1.53 6 0.18
1.46 6 0.09
1.86 6 0.38
1.86 6 0.21
1.82 6 0.12
23.3 6 3.1
21.9 6 1.9
23.0 6 2.4
EDL
Adult
Old
Very Old
16.2 6 0.5
19.6 6 0.8
18.5 6 1.3
1.79 6 0.11
1.88 6 0.06
1.70 6 0.14
2.15 6 0.11
2.68 6 0.12*
2.31 6 0.15
28.9 6 1.7
31.2 6 0.6
27.1 6 1.6
Glu-Sup
Adult
Old
Very Old
10.1 6 0.4
10.9 6 0.7
11.5 6 0.6
0.72 6 0.09
0.61 6 0.04
0.69 6 0.06
0.84 6 0.06
0.95 6 0.09
1.26 6 0.16*
15.9 6 1.2
16.1 6 0.8
16.3 6 2.5
Plan
Adult
Old
Very Old
19.6 6 0.5
19.0 6 1.3
16.5 6 1.3
1.75 6 0.18
1.50 6 0.07
1.16 6 0.18*
2.56 6 0.12
2.32 6 0.18
2.06 6 0.28
30.4 6 0.8
25.3 6 1.5*
24.2 6 1.6*
Tib-Ant (superficial)
Adult
Old
Very Old
12.7 6 0.8
14.2 6 0.9
16.1 6 1.3
1.37 6 0.13
1.36 6 0.07
1.48 6 0.08
1.63 6 0.16
1.71 6 0.10
1.95 6 0.18
20.2 6 0.2
20.9 6 1.2
23.2 6 0.9
Bic-Fem
Adult
Old
Very Old
13.2 6 0.7
13.8 6 0.4
13.7 6 1.2
0.81 6 0.05
0.72 6 0.03
0.83 6 0.11
1.21 6 0.07
1.29 6 0.07
1.19 6 0.08
20.3 6 0.8
21.6 6 0.6
18.3 6 2.8
Slow-Twitch
Mixed
Notes: N ¼ 5, unless otherwise indicated in parentheses.
*Indicates significantly different than adult (one-way analysis of variance, p , .05).
y
Indicates significantly different than old ( p , .05).
CS ¼ citrate synthase; IDH ¼ isocitrate dehydrogenase; ACON ¼ aconitase; COX ¼ cytochrome c oxidase; Add-Lon ¼ adductor longus; Bic-Fem ¼ biceps femoris;
Dia ¼ diaphragm; EDL ¼ extensor digitorum longus; Glu-Sup ¼ gluteus superficialis; Lat-Gas ¼ lateral gastrocnemius; Med-Gas ¼ medial gastrocnemius; Plan ¼
plantaris; Sol ¼ soleus; Tib-Ant ¼ tibialis anterior.
Changes in Transcript Levels of Other
Nucleus-Encoded Mitochondrial Genes
To further investigate muscle-specific changes in transcriptional regulation of bioenergetic gene expression, we
also evaluated transcript levels for CYT-C and COX IV, two
other nucleus-encoded mitochondrial genes. Consistent with
Figure 2B, Group I tissues with decreased CS mRNA/DNA
showed lower levels of CS mRNA relative to total RNA
(Figure 3A), providing further evidence for a downregulation of CS transcriptional activity in these muscles.
However, there were no declines in transcripts for CYT-C or
COX IV (Figure 3, B and C); in fact, a trend for increased
expression is observed in the deep Lat- and superficial MedGas. Group II tissues, which showed no reduction in CS
mRNA/DNA with age (Figure 2B), likewise exhibited no
decrease in CS mRNA relative to total RNA (Figure 3A).
Moreover, Sol and Glu-Sup displayed significant or modest
increases in CS, CYT-C, and COX IV transcripts with age
(Figure 3, A–C). Two exceptional tissues (EDL, Plan)
demonstrated significant declines in CYT-C expression (by
19%–34%, respectively), with no changes in other transcripts. The lack of a consistent trend in mRNA levels for all
three nuclear genes indicates that complex regulation by
transcriptional activators/suppressors underlies differential
expression profiles within and across different muscles.
Maintenance of Mitochondrial Enzyme Stoichiometries
Although aging caused significant declines in enzyme
activities relative to DNA content, regulatory changes in
enzyme synthesis seemed to match reductions in muscle
size. Thus, specific activity levels (per g tissue) of CS, IDH,
ACON, and COX remained constant with age in most
tissues. Of these enzymes, all are encoded entirely by
nuclear DNA, except for COX, which is partially encoded
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Fast-Twitch
8
LYONS ET AL.
Figure 1. Regulation of mitochondrial content in different muscles of adult
and very old animals. Individual paired measurements of citrate synthase (CS)
activity and DNA content are compared across 10 skeletal muscles in adult (A)
and very old (B) animals. Mean CS messenger RNA (mRNA) content and DNA
content of each muscle are compared in adult (C) and very old (D) animals.
Mean CS activity and CS mRNA content of each muscle are compared in adult
(E) and very old (F) animals. For C–F, each muscle is represented by a unique
symbol: adductor longus (black circle), biceps femoris (black triangle),
diaphragm (black diamond), extensor digitorum longus (gray triangle), gluteus
superficialis (black square), deep lateral gastrocnemius (gray square), superficial
medial gastrocnemius (gray circle), plantaris (open triangle), soleus (open
circle), and superficial tibialis anterior (open square). N ¼ 4–5 for all
measurements. R2 and significance level were determined by linear regression.
Standard error of the mean bars that cannot be seen are occluded by the symbol.
by mtDNA. A two-way ANOVA was used to determine
main effects of age on enzyme activity. No significant effect
of age was found on IDH (F ¼ 1.54, p ¼ .2184) or ACON
(F ¼ 2.62, p ¼ .077) activity. A significant main effect of age
was found on CS (F ¼ 4.34, p ¼ .015) and COX (F ¼ 5.44,
p ¼ .006) activity. However, a Tukey post hoc analysis of
Age 3 Muscle did not reveal any significant differences
across age groups for specific muscles. To more closely
evaluate the effects of aging on enzyme activity within each
muscle, a one-way ANOVA was used (see Table 2). This
analysis revealed that a few tissues displayed significant
DISCUSSION
Many aspects of the muscle phenotype change in response
to development and aging, including cellular morphology
(e.g., myonuclear domain), contractile protein profiles (e.g.,
myosin isoforms), and the bioenergetic machinery (e.g.,
mitochondrial content). Although these aspects of the
remodeling process have been studied in isolation, the extent to which they are co-regulated remains largely unknown.
There is a general relationship between these aspects of fiber
type, but it is not clear if this relationship is due to shared
regulatory pathways or parallel but independent pathways. In
discussing these distinct features that contribute to a fibertype phenotype, it is important to recognize that a change in
one parameter (e.g., myosin profile) does not cause a change
in another parameter (e.g., myonuclear domain), although
they may change in parallel.
Determining the mechanistic basis of differences in
mitochondrial content between tissues and physiological
states is difficult because of complex controls on transcriptional, translational, and posttranslational processes. For
example, differences in DNA content and general turnover
rates of RNA, protein, and organelles have major regulatory
influences on mitochondrial content. However, recent
studies have implicated PGC-1a, acting through a network
of transcription factors, as a master controller of mitochondrial biogenesis. These transcriptional regulators (along with
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changes in some enzymes with age. Significant losses
(20%–34%) occurred in all enzymes in the deep Lat-Gas of
very old animals. In Plan, COX and IDH were 20%–30%
lower, and although not significant, CS and ACON levels
appeared modestly depressed. Although Sol and Add-Lon
showed a trend for lower activities of most enzymes with
age, the changes were not statistically significant. Interestingly, ACON levels were significantly higher (by 50%) in
the Glu-Sup and EDL (by 25%). Although the differences
were not statistically significant, other enzymes appeared to
be elevated in the Glu-Sup, EDL, and superficial Tib-Ant.
With respect to enzyme stoichiometries, ratios of IDH/
CS, ACON/CS, and COX/CS did not significantly change
with age in most muscles. We found no main effect of age
on IDH/CS (F ¼ 1.95, p ¼ .147) or ACON/CS (F ¼ 0.185,
p ¼ .8318). There was a significant main effect of age on
COX/CS (F ¼ 5.02, p ¼ .008); however, a Tukey post hoc
analysis of Age 3 Muscle revealed no significant differences
among age groups for specific muscles. To more closely
evaluate the effects of aging on enzyme ratios within each
muscle, a one-way ANOVA was performed. This analysis
revealed no significant differences in enzyme ratios among
age groups in any muscle, with the exception of a 17% decline in COX/CS in EDL (very old vs adult; Figure 4, A–C).
Though enzyme stoichiometries were not largely affected
by aging, there was a significant main effect of muscle type
on IDH/CS (F ¼ 33.5, p , .0001), ACON/CS (F ¼ 9.25,
p , .0001), and COX/CS (F ¼ 12.87, p , .0001). Despite
this variation among some muscles (Figure 4), it is important to note that COX/CS and ACON/CS ratios are
similar among the majority of the muscles (IDH/CS is more
variable).
MITOCHONDRIAL CONTENT IN AGING MUSCLE
9
other general transcriptional machinery) could account for
patterns of mitochondrial gene expression among muscles
and during aging by two separate regulatory mechanisms.
First, constitutive expression of nucleus-encoded mitochondrial genes in combination with differences in nuclear DNA
content might explain a significant fraction of the variation
in mitochondrial content between muscles or physiological
states. Thus, a slow oxidative muscle might have more CS
enzyme/g tissue simply because it has more nuclear DNA/g
tissue (12). Second, superimposed on this constitutive
background are tissue-specific variations in transcriptional
regulation—that is, myonuclei in oxidative muscles may
express more mitochondrial enzyme because of a relatively
high activity of transcriptional activators, such as PGC-1a,
on mitochondrial genes. Both of these mechanisms can
affect the relationship between DNA, mRNA, and enzyme
levels within a tissue.
Impact of Myonuclear Content on
CS Activity Across Fiber Type
The initial data of this study, which revealed a significant
correlation (R2 ¼ 0.36) between units CS/g tissue and mg
DNA/g tissue in adult tissues (Figure 1A), suggested that
constitutive expression in combination with differences in
DNA content could be a primary explanation for variation
in CS activity among muscles of different fiber type. On
the basis of this observation, we hypothesized that each
myonucleus would produce the same amount of transcript,
which in turn would generate the same amount of protein.
However, we found that neither CS mRNA/DNA stoichiometries nor units CS/CS mRNA stoichiometries were
preserved across tissues (Figure 1, C and E). We conclude
that no simple regulatory pattern or process is responsible
for the tissue-specific differences in CS enzyme levels. The
relative importance of transcriptional and posttranscrip-
tional processes in regulating these changes is specific for
each muscle.
Although CS is traditionally considered to be transcriptionally regulated (26), our data in conjunction with other
reports (37,38) suggests that differences in CS content
among muscles arise through other mechanisms. Consider
the patterns in Sol versus deep Lat-Gas of adult animals.
Compared to deep Lat-Gas, Sol has 6 times more CS
mRNA/DNA (0.436 vs 0.08, Figure 1C), 40% fewer units
CS/g tissue (14.1 vs 24.2, Table 2), and 92% fewer units
CS/CS mRNA (38 vs 439, Figure 1E). This comparison
offers several insights into the regulation of CS enzyme
synthesis. First, the difference in CS mRNA/DNA ratios
indicates that Sol may have higher rates of transcription for
CS gene expression than does deep Lat-Gas, possibly due to
greater activity of transcriptional regulators for mitochondrial gene expression. Second, variation in CS enzyme
content between the tissues cannot be easily attributed to
transcriptional mechanisms, as CS mRNA levels do not
predict CS enzyme levels. Finally, different ratios of units
CS/CS mRNA between the muscles suggest that deep LatGas has higher rates of posttranscriptional activity for CS
enzyme synthesis compared to Sol. Although the mechanism remains to be elucidated, our data provide evidence
that posttranscriptional pathways are involved in the
regulation of CS enzyme content in different muscles.
Influence of Aging on Myonuclear Content
In our study, muscles of adult and old animals were
similar in most respects. With respect to nuclear content,
changes in mg DNA/g were observed only in the very old
(35–36 months) age group. The same age-dependent pattern
is seen in human studies (18–21), where increases in DNA
are not seen until after the age of 60. Increases in mg DNA/g
tissue with age occurred preferentially in fast-twitch muscles,
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Figure 2. Muscle-specific changes in the regulation of mitochondrial content with age. Measurements of citrate synthase (CS) activity, DNA content, and CS
messenger RNA (mRNA) content in each tissue are expressed as (A) units CS/DNA (B) CS mRNA/DNA, and (C) units CS/CS mRNA. All values are mean 6
standard error of the mean, expressed relative to adult within a tissue (open bars, adult; solid bars, very old). Muscles are grouped according to their pattern of change
with age (Groups I–III). N ¼ 5 unless otherwise indicated in parentheses. *Indicates significantly different than adult ( p , .05). Add-Lon ¼ adductor longus;
Bic-Fem ¼ biceps femoris; Dia ¼ diaphragm; EDL ¼ extensor digitorum longus; Glu-Sup ¼ gluteus superficialis; Lat-Gas ¼ lateral gastrocnemius; Med-Gas ¼
medial gastrocnemius; Plan ¼ plantaris; Sol ¼ soleus; Tib-Ant ¼ tibialis anterior.
10
LYONS ET AL.
as is seen in human aging (18–21). It is likely that the
increase in DNA can be attributed to a reduction in fiber
size, which may arise two ways. First, the changes in
muscle morphology may arise from atrophy, where an
increase in nuclear content per gram of muscle coincides
with a loss of contractile machinery. Second, an increase
in nuclear content may also occur when a muscle actively
converts a fiber from Type II to Type I, a transition that is
known to occur in some models of aging (6,24). The
magnitude of the change in myonuclear content associated
with a change in fiber type depends on the relative
proportion of Type I versus Type II fibers. Because Type I
fibers typically have the highest nuclear content (12), the
impact of fiber-type switching would be greatest when
a predominantly Type II muscle becomes a predominantly
Type I muscle. In these animals, it has been shown that
aging does not affect the proportion of Type I fibers in Sol,
EDL, or Lat-Gas (39); therefore, changes we observed in
nuclear content in these muscles are not associated with
a change in fiber type. The proportion of Type I fibers
does increase in Plan, Med-Gas, and Tib-Ant (39);
however, the Type I fibers comprise less than 20% of
the total in each of these muscles, even in very old
animals. Thus, an increase in proportion of Type I fibers in
these muscles (which are predominantly Type II) may
contribute to, but does not account for, the observed
increase in muscle nuclear content.
Overall, our data add to a growing body of evidence that
aging results in a higher DNA content of muscle tissue,
which is expected to have profound effects on gene
expression. Interestingly, mg RNA/g tissue paralleled mg
DNA/g tissue in most muscles (Add-Lon, EDL, Glu-Sup,
Plan, superficial Tib-Ant, and Bic-Fem). However, in other
muscles, mg RNA/g tissue did not change despite increases
in mg DNA/g tissue; this finding may reflect changes in
global transcription or RNA stability with age.
Regulation of Enzyme Content in Aging Muscles
It is important to consider how changes in fiber type
might influence the patterns in mitochondrial gene expression. As we discussed previously in the context of
myonuclear content, remodeling associated with fiber-type
shifts could potentially contribute to the metabolic profile of
muscles with age. As discussed previously, three of the
muscles in our aging model are known to experience an
increase in the proportion of Type I fibers: Plan, Med-Gas,
and Tib-Ant (39). As Type II fibers are remodeled to Type I
fibers, it may be predicted that the mitochondrial content
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Figure 3. Muscle-specific changes in nucleus-encoded mitochondrial transcripts with age. Expression of citrate synthase (CS) (A), cytochrome c (CYT-C) (B), and
cytochrome c oxidase IV (COX IV) (C) messenger RNA (mRNA) were evaluated by northern blot analyses. All values are the mean hybridization signal for each gene,
corrected for loading by 18S and expressed relative to adult within a tissue (open bars, adult; solid bars, very old). Muscles are grouped as in Figure 2. Add-Lon ¼
adductor longus; Bic-Fem ¼ biceps femoris; Dia ¼ diaphragm; EDL ¼ extensor digitorum longus; Glu-Sup ¼ gluteus superficialis; Lat-Gas ¼ lateral gastrocnemius;
Med-Gas ¼ medial gastrocnemius; Plan ¼ plantaris; Sol ¼ soleus; Tib-Ant ¼ tibialis anterior. N ¼ 5 unless otherwise indicated in parentheses. *Indicates significantly
different than adult ( p , .05). D, Representative northern blot (Glu-Sup tissue).
MITOCHONDRIAL CONTENT IN AGING MUSCLE
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would increase. However, there was no detectible change
in mitochondrial content (i.e., units CS/g) in these muscles
as a result of aging. As with myonuclei, the fiber-type conversions do not appear to play a major role in determining
mitochondrial content.
Although units CS/g tissue remained relatively stable,
significant declines in units CS/DNA occurred in almost all
muscles (Figure 2A), which signifies major changes in the
regulation of CS enzyme synthesis. In Group I muscles
(superficial Tib-Ant, superficial Med-Gas, deep Lat-Gas,
Dia), these declines may be linked to changes in
transcriptional activity, as CS mRNA/DNA ratios declined
in older animals (Figure 2B). In the deep Lat-Gas, relatively
low levels of CS mRNA/DNA (Figure 1C) in adult animals
may predispose this tissue to declines in transcriptional
control of CS gene expression with age. The trend of lower
CS mRNA/DNA in Group I muscles is paralleled by
a decline in specific CS mRNA expression relative to total
RNA (Figure 3A). With respect to other nucleus-encoded
mitochondrial genes, CYT-C mRNA/total RNA and COX
IV mRNA/total RNA did not significantly change (Figure 3,
B and C). This finding suggests that gene-specific regulatory
mechanisms are differentially affected by aging. In Group II
tissues (Plan, Sol, Bic-Fem, Glu-Sup), declines in units CS/
DNA may be linked to decreases in posttranscriptional
activity, as indicated by decreases in units CS/CS mRNA
(Figure 2C). It is unknown whether the declines in units
CS/DNA that we observed in Group II muscles with age are
linked to changes in elongation, or other points of
posttranscriptional control. It is interesting to note that the
basis of assignment of muscles to Groups I, II, and III
(Figures 2 and 3) does not appear to be linked to the fibertype profile, as each group is composed of muscles of
different fiber type.
Despite much research over the last few decades, it
remains equivocal as to whether specific activity levels of
mitochondrial respiratory and/or matrix enzymes are
affected during aging (40–42). Discrepancies among reports
are often due to differences in experimental design including
muscle type, species, strain, age group comparisons, and
tissue preparation (tissue homogenates vs isolated mitochondria). Several groups have shown that changes in
enzyme activity are dependent on muscle fiber type
(6,43,44). Our study is one of the few to evaluate multiple
mitochondrial enzymes across a broad spectrum of skeletal
muscles in the context of aging. We found a significant main
effect of age on CS and COX activity with all muscle groups
pooled together. However, analysis of individual muscle
comparisons across age groups revealed that most enzyme
activities were unaltered with age, with a few exceptions.
Most notably, the deep Lat-Gas exhibited significant losses
(20%–30%) in CS, ACON, IDH, and COX activities/g
tissue. A decline in mitochondrial enzyme activity specifically in the deep Lat-Gas (compared to other muscles) has
been previously reported (43); however, the mechanism
behind this muscle-specific pattern is unknown. Other
tissues that showed significant changes in enzyme activity
were the Glu-Sup and EDL (each exhibiting a 50% and 25%
increase in ACON, respectively) and Plan (34% decline in
IDH and 20% decline in COX). In a study of similar design
11
Figure 4. Mitochondrial enzyme stoichiometries do not change with age.
Paired measurements of cytochrome c oxidase (COX), isocitrate dehydrogenase
(IDH), aconitase (ACON), and citrate synthase (CS) activities were analyzed in
homogenates of 10 muscles from adult (open bars), old (gray bars), and very old
(black bars) animals. Enzyme stoichiometries are expressed as ratios of IDH/CS
(A), ACON/CS (B), and COX/CS (C). Muscles grouped under the same
horizontal line are not significantly different from each other (two-way analysis
of variance (ANOVA), main effect of muscle type, p , .05). Add-Lon ¼ adductor
longus; Bic-Fem ¼ biceps femoris; Dia ¼ diaphragm; EDL ¼ extensor digitorum
longus; Glu-Sup ¼ gluteus superficialis; Lat-Gas ¼ lateral gastrocnemius; MedGas ¼ medial gastrocnemius; Plan ¼ plantaris; Sol ¼ soleus; Tib-Ant ¼ tibialis
anterior. *Indicates significantly different than adult (one-way ANOVA, p ,
.05). N ¼ 5 for all enzymes, except IDH in Sol (N ¼ 3).
12
LYONS ET AL.
ACKNOWLEDGMENTS
Funding for this research was provided by grants from the Natural
Sciences and Engineering Research Council of Canada (to C.D.M.), the
American Federation of Aging Research (to C.N.L.), and the National
Institutes of Health (to O.M.C.).
We thank Dr. Li Cui, Dr. Yan Ju, and Margarita Trejo-Morales for
assistance with tissue harvesting.
Address correspondence to Chris Moyes, PhD, Department of Biology,
Queen’s University, Kingston, Ontario, Canada, K7L 3N6. E-mail:
[email protected]
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