Changes in myosin heavy chain mRNA and protein

expression in human skeletal muscle with age and

endurance exercise training
Kevin R. Short, Janet L. Vittone, Maureen L. Bigelow, David N. Proctor, Jill M.
Coenen-Schimke, Paul Rys and K. Sreekumaran Nair
Journal of Applied Physiology 99:95-102, 2005. First published Mar 3, 2005;
doi:10.1152/japplphysiol.00129.2005

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Changes in myosin heavy chain mRNA and protein expression in human
skeletal muscle with age and endurance exercise training
Kevin R. Short,1 Janet L. Vittone,2 Maureen L. Bigelow,1 David N. Proctor,3
Jill M. Coenen-Schimke,1 Paul Rys,1 and K. Sreekumaran Nair1
Divisions of 1Endocrinology and 2General Internal Medicine, Department of Internal Medicine,
and 3Department of Anesthesiology, Mayo Clinic School of Medicine, Rochester, Minnesota
Submitted 2 February 2005; accepted in final form 25 February 2005
Short, Kevin R., Janet L. Vittone, Maureen L. Bigelow, David
N. Proctor, Jill M. Coenen-Schimke, Paul Rys, and K. Sreekuma-
ran Nair. Changes in myosin heavy chain mRNA and protein ex-
pression in human skeletal muscle with age and endurance exercise
training. J Appl Physiol 99: 95–102, 2005. First published March 3,
2005; doi:10.1152/japplphysiol.00129.2005.—Aging is associated
with reduced muscle strength and atrophy of type II muscle fibers.
Muscle fiber type and contractile function are primarily determined by
myosin heavy chain (MHC) isoforms. There are few data available on
the effects of aging on MHC isoform expression in humans. In the
present study, we tested the hypothesis that MHC isoform protein
composition and mRNA abundance would favor a fast-to-slow iso-
form shift with aging and in response to endurance exercise training.
Muscle biopsies were obtained from previously sedentary, healthy
men and women, aged 21– 87 yr before (n ⫽ 77) and after (n ⫽ 65)
16 wk of bicycle training (up to 45 min at 80% peak heart rate, 3– 4
days/wk). At baseline, MHC I mRNA was unchanged with age,
whereas IIa and IIx declined by 14 and 10% per decade, respectively
(P ⬍ 0.001). MHC IIa and IIx protein declined by 3 and 1% per
decade with a reciprocal increase in MHC I (P ⬍ 0.05). After training,
MHC I and IIa mRNA increased by 61 and 99%, respectively, and IIx
decreased by 50% (all P ⬍ 0.001). The increase in MHC I mRNA was
positively associated with age, whereas the changes in MHC IIa and
IIx mRNA were similar across age. MHC I protein increased by 6%
and was positively related to age, whereas IIx decreased by 5% and
was inversely related to age. These results suggest that the altered
expression of MHC isoforms with aging is transcriptionally regulated.
In response to endurance exercise, regulation of MHC isoform tran-
scripts remains robust in older muscle, but this did not result in
corresponding changes in MHC protein expression.
muscle contractile proteins; gene expression; isokinetic strength; mus-
cle size
AGING IS ASSOCIATED WITH REDUCED size and strength of human
skeletal muscles (13, 19, 23). With aging, there are also
changes in the size, number, and contractile phenotype of
individual muscle fibers that may impact muscle function.
Classic studies by Larsson et al. (18) and Lexell (22) showed
that the decline in size of the vastus lateralis with normal aging
could be attributed to fewer total muscle fibers and preferential
atrophy of fibers classified as type II (fast twitch) by histo-
chemical staining of myosin ATPase. Those authors also re-
ported that the percentage of each of the three major fiber types
in adult muscle, type I (slow twitch), IIa, and IIb, was largely
unchanged with age. Subsequent investigations by other
groups also showed that the percentage of each fiber type did
Address for reprint requests and other correspondence: K. Sreekumaran
Nair, Endocrinology Research Unit, 5-194 Joseph Mayo Clinic School of
Medicine, 200 First St. SW, Rochester, MN 55905 (E-mail: nair.sree
@mayo.edu).
http://www. jap.org 8750-7587/05 $8.00 Copyright © 2005 the American Physiological Society
J Appl Physiol 99: 95–102, 2005.
First published March 3, 2005; doi:10.1152/japplphysiol.00129.2005.
not appear to change significantly with age in human leg
muscles, but that the size of type II fibers was selectively
reduced in the vastus lateralis and gastrocnemius of older
people (6, 11, 15, 27).
The use of myosin ATPase staining to determine muscle
fiber type is based on the relative expression of isoforms of
myosin heavy chain (MHC), the major contractile protein in
skeletal muscle. Contractile phenotype in adult muscle fibers is
primarily determined by the relative expression of the MHC
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isoforms I, IIa, and IIx (5, 9), which correspond to the histo-
chemically determined fiber types I, IIa, and IIb in humans
(29). Han et al. (9) demonstrated that ATP consumption rate
and tension cost of single human muscle fibers vary with the
expression of MHC isoforms. Whereas most muscle fibers
appear to express a single MHC isoform, isoform coexpression
has been demonstrated in up to ⬃30% of fibers and may vary
with age and exercise training (1, 16, 38). Thus measurement
of fractional MHC isoform composition of muscle provides an
important index of the regulation of contractile phenotype.
The mechanisms that regulate the expression of MHC iso-
forms in aging muscle are not yet clear. It has been reported
that the synthesis rate of MHC protein is reduced with age in
humans (2, 10), but it is not known whether this is a global
change that affects all MHC isoforms or whether there is
isoform-specific regulation. Isoform-specific regulation may
occur at the pretranslational level. Previous work from our
laboratory demonstrated that the mRNA abundance of MHC
IIa and IIx was reduced in vastus lateralis biopsies from
healthy older vs. younger people, whereas MHC I mRNA
content was unchanged (3). This finding is consistent with a
report by Klitgaard et al. (15) in which older men were found
to have smaller type II muscle fibers than young men and a
corresponding decrease in the fractional amount of MHC IIx
protein. In contrast, in studies by Welle and colleagues (36, 37)
and Marx et al. (24), there was no difference detected between
groups of younger and older people (n ⫽ 8 –16 people per
group) in mRNA abundance of MHC isoforms. The study by
Marx et al. also reported that the fractional protein content of
MHC isoforms was also unchanged with age.
Therefore, it is not yet resolved whether expression of MHC
mRNA and protein is altered with age in human skeletal
muscle, and to our knowledge only one study has examined the
age effect on both MHC mRNA and protein (24). Some of the
variation among studies may be due to the use of a small
number of subjects, different methods of sample analysis, or
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
95
96 AGING AND EXERCISE EFFECT ON MHC

variable control for physical activity status. Thus the first Table 1. Subject characteristics
purpose of the present study was to test the hypothesis that
Younger Middle-aged Older
expression of MHC II mRNA and protein is reduced with age Variable Gender (21–37 yr) (40 –58 yr) (60 – 87 yr)
in human skeletal muscle. To address this question, muscle
biopsies of the vastus lateralis were obtained from 77 healthy, No. of participants W 10 12 17
M 12 11 15
non-exercise-trained men and women between the ages of 21 Weight, kg W 68.7⫾1.8 69.1⫾2.2 69.5⫾2.0
and 80 yr. We also examined the strength of the association M 85.7⫾2.7 90.4⫾2.8 86.0⫾2.8
between MHC expression and muscle strength in these people. BMI, kg/m2 W 24.2⫾0.5 25.1⫾0.6 26.3⫾0.6
Larsson et al. (18) reported that the total cross-sectional area of M 25.8⫾0.6 28.1⫾0.6 27.6⫾0.7
type II fibers was significantly related to muscle strength in Body fat, % W 34.1⫾1.2 37.2⫾1.1 39.3⫾1.1
M 23.9⫾1.0 25.8⫾1.7 27.1⫾1.3
younger and older people. Klitgaard et al. (15), however, did Fat-free mass, kg W 41.4⫾0.8 39.3⫾0.9 38.7⫾0.9
not find a significant relationship between muscle strength and M 60.4⫾1.7 60.9⫾2.6 56.6⫾1.4
fiber area or MHC isoform content in young and older un- V̇O2peak, l/min W 1.95⫾0.05 1.66⫾0.06 1.26⫾0.05
trained men, but this negative finding may be due to the use of M 3.15⫾0.11 2.73⫾0.19 1.96⫾0.12
a small number of subjects (7– 8 per group). Values are means ⫾ SE. W, women; M, men; BMI, body mass index;
The second purpose of the study was to determine whether V̇O2peak, peak oxygen uptake. Statistically significant differences between men
an endurance exercise training program would alter MHC and women were present for each variable. There was not a statistically
isoform expression and whether the effect would vary with significant change in BMI with age. Body fat increased (r ⫽ 0.40), whereas
fat-free mass (r ⫽ ⫺0.46), and V̇O2peak (r ⫽ ⫺0.81) decreased with age, P ⬍
age. We are aware of only one study that prospectively exam- 0.01.
ined the effect of endurance exercise training on MHC mRNA

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or protein expression in human skeletal muscle. O’Neill and
colleagues (25) found that the mRNA abundance for MHC IIx
was reduced in vastus lateralis muscle in young men after
performing 7 consecutive days of bicycle training. This ap-
pears to be in agreement with previous studies that have
reported that endurance training results in a decrease in the
percentage of fibers classified as type IIb by myosin ATPase
histochemistry (7, 12). These changes in fiber type percents
were observed after 6 wk of bicycle training in young men (12)
or after a 9- to 12-mo walking and jogging program in older
men and women (7). To our knowledge, however, the effects
of endurance exercise training on MHC mRNA or protein
expression have not been compared in younger and older
people. Therefore, subjects in the present study were tested
before and after completion of a 16-wk program of standard-
ized bicycle exercise training to determine whether the hypoth-
esized shift from fast to slow MHC isoform expression would
vary with age.
METHODS
Participants. Healthy men and women who exercised ⬍30 min on
two or fewer occasions per week during the previous 9 mo were
recruited from the local community. Physical activity levels were
confirmed by questionnaire (33). Health status was assessed by
medical history, physical examination, blood chemistries (complete
blood count and comprehensive chemistry panel, including liver
enzymes, creatinine, electrolytes, and glucose), urinalysis, and resting
electrocardiogram. Exclusion criteria included body mass index ⬎32
kg/m2, tobacco use, use of ␤-blockers, diabetes or other metabolic or
endocrine disorders, history of alcohol or substance abuse, and debil-
itating chronic illness. Thirty-nine women and 38 men between the
ages of 21 and 87 yr met these criteria and were enrolled after
providing written and oral consent. Consent was obtained before any
tests were performed. The Mayo Foundation Institutional Review
Board approved the study. Physical characteristics of the men and
women in each decade have been published elsewhere (30, 31). A
summary version of those data is provided in Table 1, in which the
participants were grouped as young (21–37 yr), middle aged (40 –56
yr), or older (60 – 87 yr).
Study protocol. Participants were randomized to either a 16-wk
endurance exercise or control program. A similar 5-day protocol was
completed at baseline and again within 1 wk of completion of the
training or control phases. During each study period, a weight-
maintaining diet (55% carbohydrate, 30% fat, and 15% protein) was
provided for the first 4 days. On the morning of day 4, subjects were
admitted to the General Clinical Research Center (GCRC). The
following morning (day 5), muscle biopsy samples from the vastus
lateralis (33) were obtained.
The exercise program was performed on a stationary bicycle.
Training started with three sessions per week, lasting 20 min each, at
an intensity eliciting 70% of the peak heart rate. Peak heart rate during
cycling was measured during an aerobic capacity test as described
below. Intensity, duration, and number of sessions were gradually
increased so that the final month of training consisted of four sessions
per week at 80% of maximal heart rate for 40 min. Exercise specialists
supervised each session and recorded heart rates. Compliance with the
target workloads and number of sessions was ⬎90%. The exercise
protocol was completed by 41 of 47 participants originally assigned.
The control group was taught a series of flexibility exercises and
encouraged to perform them at home while maintaining their regular
lifestyle. Follow-up tests were available for 37 of the 43 people in the
control group. Subsequently, 24 control group members opted to
complete the exercise program, yielding a total of 65 people studied
before and after exercise training.
Because the goal of the study was to examine effects of the exercise
program, participants were instructed to maintain body weight.
Weight was recorded weekly, and the GCRC dietary staff provided
further guidance if weight changed ⬎2%. Only one person in the
exercise group discontinued the study because of excessive weight
loss.
Body composition and aerobic capacity. Fat and fat-free mass were
determined by dual-energy X-ray absorptiometry (DPX-L, Lunar,
Madison, WI). Thigh muscle area was measured by using a single-
slice (6-mm thickness) computed tomography scan (model C-150,
Imatron, San Francisco, CA) at the midpoint between the knee and
hip. Areas of muscle, fat, and bone were estimated by manual
planimetry using custom software (21).
A standard treadmill stress test was performed initially to ensure
cardiovascular health and was followed on another day with measure-
ment of peak oxygen uptake (V̇O2 peak) on a bicycle ergometer (26).
The volume and composition of expired gases using breath-by-breath
analysis, heart rate by 12-lead electrocardiogram, and blood pressure
were continuously monitored throughout the tests (26). The posttrain-
ing assessment was made within 3 days of the completion of the last
training bout.

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 AGING AND EXERCISE EFFECT ON MHC
Fig. 1. Representative electrophoretic gel separation of myosin heavy chain
(MHC) isoforms in human skeletal muscle by SDS-PAGE. Results from
duplicate analysis from 2 participants (samples 1 and 2) are shown.
Muscle strength. Isokinetic knee extensor strength was measured
on a Cybex II dynamometer (Ronkonkoma, NY). Subjects were
seated with the hip joint angle between 90 and 100° of flexion and had
stabilizing straps over the chest, hips, and thigh. The knee joint was
aligned with the axis of the lever arm, and the leg was attached to the
lever arm just above the ankle. Each subject was given a familiariza-
tion and brief warm-up before testing. Testing was performed at a
speed of 180°/s (3.14 rad/s). From a starting point of 90° of knee
flexion, subjects were instructed to perform a maximal force voluntary
97
5°C using a Hoefer SE600 electrophoresis unit (Amersham Bio-
sciences, Piscataway, NJ). The upper running buffer contained 0.08%
vol/vol 2-mercaptoethanol as recommended by Blough et al. (4) for
improved separation of MHC isoforms.
Gels were fixed overnight in 50% vol/vol methanol and 10%
vol/vol acetic acid. A silver staining protocol was used to visualize
protein bands (Fig. 1). The following incubation steps were performed
after the gels were washed briefly in deionized water: 1) 5 min in 8
mM Na2S2O3 and then wash three times for 30 s in deionized water;
2) 25 min in 12 mM AgNO3 and then wash three times for 1 min in
deionized water; 3) 10 min in 280 mM Na2CO3, 0.16 mM Na2S2O3,
and 0.2% vol/vol formaldehyde; and 4) 10 min in 37.2 mM Na2-
EDTA and then wash in deionized water. Stained gels were dried
between cellophane sheets and digitally imaged on a Kodak Image
Station 1000 (Kodak Scientific, Rochester, NY). The relative density
of each myosin isoform band was measured using Kodak Image
Analysis Software 3.5 (Kodak Scientific) and expressed as the fraction
of the total MHC density in each gel lane. Therefore, with this
procedure, it is possible to determine whether there is a change in the

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knee extension through the range of motion. A series of five consec-
utive contractions was performed with each leg separately. The
highest torque production recorded from the five attempts using the
self-reported dominant leg was used for data analysis. Calibration of
the machine was performed before testing by hanging standard
weights from the lever arm.
Quantification of MHC mRNA. A real-time quantitative polymerase
chain reaction (PCR) system (ABI Prism 7700, PE Biosystems, Foster
City, CA) was used to measure the abundance of mRNAs in muscle
tissue (3). RNA was extracted from 25 mg of skeletal muscle from
individual subjects by TRIzol method (Life Technologies, Gaithers-
burg, MD), treated with DNase (Life Technologies), and then reverse
transcribed using the TaqMan Reverse Transcription Reagents (PE
Biosystems). The primer and probe sequences used for human MHC
I, MHC IIa, MHC IIx, and 28S ribosomal RNA (used for normaliza-
tion because it does not change with age in our hands) were previously
described (3). Samples were run in triplicate with coamplification of
the target gene and 28S rRNA within each well and quantified by
normalizing the target signal for the 28S rRNA signal. Finally, for
each gene, the average transcript level of the young group was
assigned a value of 1.0, and all individual values were linearly
transformed relative to this value and expressed in arbitrary units
(AU).
Quantification of MHC protein. The relative protein abundance of
MHC isoforms was measured using SDS-PAGE. Muscle samples
(150 mg) from each participant were homogenized in 250 mM
sucrose, 2 mM EDTA, and 10 mM Tris-EDTA, pH 7.4, and then
centrifuged at 1,000 g as previously described (28). The resulting
pellet, containing the myofibrillar fraction, was rehomogenized in 176
mM KCl, 10 mM Tris 䡠 HCl, and 2 mM EDTA, pH 7.2. After
determination of the protein concentration (DC Protein Assay, Bio-
Rad, Hercules, CA), an aliquot was adjusted to a concentration of 3
mg/ml and then diluted fourfold in sample loading buffer (66 mM
Tris, 19 mM EDTA, 1.05% vol/vol SDS, 0.008% vol/vol bromophe-
nol blue, 810 mM 2-mercaptoethanol, and 40% vol/vol glycerol,
pH 6.7).
Samples were heated to 95°C for 4 min, cooled to room tempera-
ture, centrifuged briefly, and then loaded on 0.75-mm-thick polyacryl-
amide gels for protein separation. The discontinuous gel recipe of
Talmadge and Roy (32) was used, consisting of an 8% separation gel,
Fig. 2. Decline in muscle size and strength with age. A: single-leg peak
a 4% stacker gel, and 30% glycerol in the gel matrix. Samples were isokinetic knee extensor torque performed at 180°/s (3.14 rad/s). B: cross-
run in duplicate with pre- and poststudy samples in adjacent lanes. A sectional area of muscle at the midthigh. C: peak isokinetic torque after
semipurified MHC preparation from rabbit muscle (Sigma Chemical, covariate adjustment (Adj) for thigh muscle area. A single regression line is
St. Louis, MO) was loaded on each gel as a molecular weight standard shown in C because men and women did not differ after adjustment for muscle
and for quality control monitoring. Gels were run for 24 h at 275 V at area.

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98 AGING AND EXERCISE EFFECT ON MHC

proportional content of each of the three MHC isoforms, but it was not
possible to determine whether the total MHC concentration varied
with age or exercise training.
Statistical analysis. Summary data are reported as means ⫾ SE.
Differences between men and women and between pre- and posttest-
ing within groups were analyzed by using unpaired and paired t-tests,
respectively. Pearson correlation coefficients were used to measure
association among selected variables. Multiple regression analysis
was used to determine the contributions of leg muscle size and MHC
protein content to variance in knee extensor torque. Covariate adjust-
ment of knee extensor torque for muscle cross-sectional area was
performed (34) so that the residual effect of age could be graphically
represented. A P value ⬍ 0.05 was considered statistically significant
for all tests.
RESULTS
Subject characteristics at baseline are shown in Table 1. The
data are summarized into three age groups to simplify the
presentation, but analysis of age effects was performed by
linear regression. As expected, there were significant differ-
ences between men and women for most variables. With the
Values for muscle MHC isoform mRNA and protein content
at baseline are shown in Fig. 3. At the mRNA level, MHC I
abundance did not vary with age, whereas both MHC IIa and
IIx transcripts declined by 14 and 10% per decade, respec-
tively. At the protein level, the relative content of MHC I
increased with age by 3.5% per decade, whereas there was a
decline of 3% per decade for MHC IIa and 1% per decade for
MHC IIx. There were no differences between men and women
in average MHC mRNA abundance or fractional protein con-
tent nor any significant differences in the changes in MHC
expression with age.
The association between the fractional MHC protein content
and peak knee extensor torque is shown in Fig. 4. Peak torque
was negatively related to the fraction of MHC I protein and
positively related to the fraction of MHC IIa protein. After
covariate adjustment of peak torque values for differences in
thigh muscle cross-sectional area, however, the fraction of
MHC protein isoforms was no longer significantly correlated
with peak torque (data not shown). Multivariate regression
confirmed that MHC protein did not make a significant con-

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tribution to the explanation of the variance in knee extensor
exception of body weight and body mass index, there were torque once thigh muscle area was entered into the model.
significant age-related changes in body composition, reflecting
increasing adiposity and decreasing fat-free mass. The peak
aerobic capacity (V̇O2 peak) declined with age by 8% per decade
in both men and women.
There were no statistically significant changes in the phys-
iological characteristics of the control group between measure-
ments made at baseline and 16 wk later. In exercisers, there
were small but statistically significant (P ⬍ 0.001) reductions
in body weight (0.6 ⫾ 0.2 kg) and body mass index (0.2 ⫾ 0.1
kg/m2) but no change in dual-energy X-ray absorptiometry-
determined body fat or fat-free mass. V̇O2 peak during cycling
improved by 9.5% on average (P ⬍ 0.001). The magnitude of
posttraining changes in body mass, body mass index, and
maximal oxygen uptake in the exercise participants was not
statistically different between men and women and did not vary
with age of the participant. These physiological characteristic
data have been presented previously (30, 31).
Peak isokinetic knee extensor torque was on average 48%
higher in men than women (P ⬍ 0.001) and declined with age
at a rate of 13% per decade in men and 8% per decade in
women (Fig. 2A). Cross-sectional area of the midthigh muscle
was on average 32% higher in men than women (P ⬍ 0.001)
and declined 5% per decade in men and 4% per decade in
women (Fig. 2B). Peak torque was strongly associated with
thigh muscle area in men and women combined (r ⫽ 0.88, P ⬍
0.001). After covariate adjustment of peak torque for differ-
ences in muscle area, differences in knee extensor torque
between men and women were eliminated, but there was still a
significant decline in peak torque with age of 5% per decade
(Fig. 2C). In the control group, there was no change from
baseline to follow-up in muscle size or strength (data not
shown). The exercise group had an average 6% increase in
peak isokinetic knee extensor torque (75.7 ⫾ 4.8 N䡠 m pre- Fig. 3. Age effect on expression of MHC mRNA and protein isoforms. The
training, 78.5 ⫾ 5.2 N䡠 m posttraining; P ⬍ 0.01) after training, relative abundance of mRNA transcripts for each isoform (left) are shown in
but the change was similar in young (7%), middle-aged, (4%), arbitrary units (AU) after normalization for 28S rRNA. Protein expression of
and older (6%) groups. Leg muscle area did not change each isoform (right) is shown as the fraction of each isoform relative to the
total MHC in each gel lane. There were no statistical differences in expression
significantly in response to training in the exercise group between women and men, so a single regression line is shown in each panel
(overall average, 129 ⫾ 5 cm2 pretraining, 133 ⫾ 4 cm2 except for MHC I mRNA, where there was not a statistically significant
posttraining; P ⫽ 0.17). association with age.

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 AGING AND EXERCISE EFFECT ON MHC 99
exercise effects on MHC mRNA and protein. Therefore, exer-
cise-induced changes in MHC mRNA and protein were pooled
by age group and are shown in Fig. 6. All of the changes in
mRNA were statistically significant within the young, middle-
aged, and older groups, but statistically significant changes in
protein were only present in the young group (increase in MHC
I, decrease in MHC IIx) and the middle- aged group (decrease
in MHC IIx).
DISCUSSION

The major findings from the present study were the age- and
exercise-related changes in expression of MHC isoform
mRNA and protein in human skeletal muscle. In a relatively
large group of healthy men and women, we found that both
MHC mRNA and protein expression of MHC II isoforms
declined with age, in support of our hypothesis. In response to
a 4-mo program of moderate-intensity endurance exercise
training, MHC I and IIa mRNA increased and IIx decreased,
shifting transcript expression from fast to slow isoforms in all
age groups. However, a training effect on MHC isoform

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protein expression in this same direction was detected in
younger people, but there was no change in the composition of
MHC isoform protein in older people. This suggests that some
adaptations to exercise remain robust in older muscles, i.e.,

Fig. 4. Association between fractional protein expression of each MHC

isoform and peak isokinetic knee extensor torque. Separate regression lines are
shown for men (E, dotted lines) and women (F, solid lines) for MHC I and IIa.
Regression lines are not shown for MHC IIx protein because there was not a
significant relationship to knee extensor torque.

There were no statistically significant changes in either

MHC mRNA or protein expression in the control group. In the
exercise group, there were changes in both MHC mRNA and
protein (Fig. 5). On average, mRNA abundance was increased
by 63% for MHC I (1.07 ⫾ 0.09 AU pretraining, 1.74 ⫾ 0.15
AU posttraining), increased 99% for MHC IIa (0.72 ⫾ 0.08
AU pretraining, 1.43 ⫾ 0.14 AU posttraining), and decreased
50% for MHC IIx (0.78 ⫾ 0.09 AU pretraining, 0.39 ⫾ 0.05
AU posttraining) (all P ⬍ 0.001). The change in MHC I
mRNA abundance showed a modest positive correlation with
age (Fig. 5), but changes in MHC IIa and IIx mRNA did not
vary with age. Changes in MHC protein content were smaller
than the mRNA responses. On average, the fractional content
of MHC I protein increased by 6% (0.37 ⫾ 0.02 pretraining,
0.43 ⫾ 0.02 posttraining; P ⫽ 0.004), whereas MHC IIx
protein decreased by 5% (0.26 ⫾ 0.01 pretraining, 0.21 ⫾ 0.02
posttraining; P ⫽ 0.001), and MHC IIa protein was not
significantly changed (0.37 ⫾ 0.02 pretraining, 0.36 ⫾ 0.02 Fig. 5. Changes in MHC mRNA and protein isoforms after the 16-wk
posttraining; P ⫽ 0.39). The change in MHC I protein was endurance exercise training program. Values shown represent the change from
pre- to posttraining, with zero representing no change. Units of measurement
inversely correlated with age, whereas the change in MHC IIx are the same as in Fig. 3. There were no statistical differences in expression
protein was positively correlated with age, as shown in Fig. 5. between women and men, so a single regression line is shown in each panel in
There were no differences between men and women in the which there was a statistically significant association with age.

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100 AGING AND EXERCISE EFFECT ON MHC
Fig. 6. Changes in MHC mRNA and protein in response to endurance exercise
training summarized by age group. Units of measurement are the same as in
Fig. 3. *Statistically significant change within age group from pre- to postex-
ercise training, P ⬍ 0.05.
gene expression at the mRNA level, whereas other adaptations
may be either prevented or delayed, i.e., alteration in the
expression of specific proteins.
Changes in MHC expression with age. The present data
demonstrate that with age there is a decrease in the fractional
content of MHC IIa and IIx protein in human leg muscle and
a corresponding increase in the proportion of MHC I. This
finding is consistent with previous reports that the cross-
sectional area occupied by histochemically typed fast-twitch
fibers is reduced in muscle from older people (6, 11, 15, 18, 22,
27). Those studies also suggested that the decline in fast-twitch
fiber area is primarily due to a selective reduction in the size of
type II fibers because most studies report that the percentage of
each fiber type appears not to change significantly with age (6,
11, 15, 18, 22, 27). We are aware of only two other reports in
which the effect of age on MHC protein expression in human
muscle has been presented, but those results of those studies
were contradictory, and both included a smaller number of
subjects (all men) than the present study. In agreement with our
results, Klitgaard et al. (15) found that the fraction of type II
MHC was reduced and MHC I was increased in vastus lateralis
biopsies obtained from 8 older (68 yr old) compared with 7
young (28 yr old) untrained men. A similar, but nonsignificant,
trend was observed in the biceps brachii of these same men. In
contrast, Marx et al. (24) studied a larger number (n ⫽ 16 per
group) of younger (22 yr old) and older (74 yr old) untrained
men, but they did not detect any differences in MHC protein
isoforms. Other studies have examined the MHC isoform
expression within single muscle fibers and found an increase
with age in the number of fibers that coexpress multiple
isoforms (1, 16, 20). Such information is useful to understand
how MHC expression is controlled within single fibers, but the
J Appl Physiol • VOL
technical demands of such work have meant that most of these
studies have analyzed muscle samples from a small number of
people. A strength of the present investigation was the inclu-
sion of a large number of subjects (n ⫽ 77) across most of the
adult life span, thus demonstrating that the effect of age on
MHC expression is a continuous process after linear growth
potential is achieved.
The present study provided some indication that age-related
changes in muscle fiber type are controlled at the pretransla-
tional level. We found that the abundance of mRNA transcripts
for MHC IIa and IIx declined with age, whereas MHC I mRNA
content was unchanged. This selective alteration in transcript
availability for protein synthesis may be one means through
which altered protein expression pattern is regulated. These
alterations in MHC isoform mRNAs represent either an altered
rate of gene transcription or altered stability of the transcripts.
The global fractional synthetic rate of MHC protein (a measure
of translational rate) has been shown to decline with age in
human muscle (2, 10). Further work is need to determine
whether translational regulation of specific MHC isoform tran-
Downloaded from jap.physiology.org on June 23, 2005
scripts is affected by aging because such mechanisms could
also contribute to protein expression pattern. The changes in
MHC mRNA we observed are in agreement with a previous
report from our laboratory in which muscle biopsies of the
vastus lateralis from young (⬃25 yr old, n ⫽ 7), middle-aged
(⬃50 yr old, n ⫽ 12), and older (⬃71 yr old, n ⫽ 14) men and
women were analyzed by using the same real-time PCR
method used in the present study (3). In contrast, in studies by
Marx et al. (24) and Welle et al. (36, 37), there were no
detectable differences in abundance of mRNA of MHC I, IIa,
or IIx in comparisons between younger and older people. The
reason for the discrepant findings among these studies is not
apparent because it appears that healthy, non-exercise-trained
subjects were examined in all of these studies and that biopsy
samples were obtained from the vastus lateralis muscle after
2–3 days of diet and physical activity control.
Changes in MHC expression in response to endurance
exercise. In response to the endurance exercise program, there
was a general shift from fast to slow MHC isoform expression.
The increase in MHC I mRNA was modestly higher in older
people compared with younger people, whereas the increase in
MHC IIa mRNA and decrease in MHC IIx mRNA were similar
in young, middle-aged, and older people. The intriguing find-
ing was that changes in MHC protein were age dependent, with
a significant increase in MHC I and decrease in MHC IIx in
young people, whereas there were no significant differences in
the older group, and intermediate changes occurred in middle-
aged people. There are two plausible explanations for these
results. First, older muscles at baseline already had a smaller
fraction of MHC IIx protein compared with young, so there is
a smaller margin for further suppression in response to train-
ing. Thus a greater stimulus, in the form of a more vigorous or
longer training program, may be required to cause a detectable
shift in the MHC isoform protein composition in older people.
Second, older muscles may be slower to respond to the exer-
cise stimulus at the level of protein expression. The synthesis
rate of MHC protein declines with age in untrained people (2,
10), so it may take longer to upregulate translational machinery
and produce a measurable change in concentration of specific
proteins in older muscles. It was previously shown that syn-
thesis rate of total MHC protein is increased in response to a
99 • JULY 2005 • www.jap.org
 AGING AND EXERCISE EFFECT ON MHC
12-wk resistance training program in older people (2), but
MHC isoform distribution was not measured, so it is unclear
whether there is selective translational regulation of specific
isoforms. Our laboratory has reported elsewhere that the sub-
jects in the present study responded to the endurance training
with increased mRNA abundance for genes encoding mito-
chondrial proteins, nuclear transcription factors, and glucose
transporter 4, increased activity of mitochondrial enzymes, and
increased synthesis rate of mixed (total) muscle proteins, all of
which were changed similarly in younger and older people (30,
31). A notable exception was that insulin sensitivity was
increased in young but not older people (31), so, despite
evidence that many of the adaptive processes remain intact in
older people, there are some variables that may take a rela-
tively longer period to respond to the exercise program. Further
studies on the time course of adaptations are needed to address
this issue.
To our knowledge, this is the first study to directly compare
the effects of endurance exercise training on MHC expression
in younger and older people. In fact, there is limited informa-
tion available on the effects of exercise training on MHC
isoform expression in human muscle, especially at the mRNA
level. In a cross-sectional comparison between sedentary and
running-trained older men (n ⫽ 8 and 5 per group, respec-
tively), there were no differences in fractional MHC isoform
protein composition, although the runners did have a higher
percentage of type I fibers by histochemical analysis (15).
Another study showed that, in response to a short-term (1 h per
day for 7 consecutive days) bicycle training program, MHC IIx
mRNA abundance decreased in the muscles of young men, but
there were no changes in either MHC I or IIa mRNA (25).
Considering that we also observed a decline in MHC IIx
mRNA in addition to increases in MHC I and IIa mRNA after
16 wk of training, it is possible that the time course of
transcriptional regulation varies among individual MHC iso-
forms. In agreement with our results, other studies that used the
histochemical fiber-typing approach found a decrease in the
percentage of fibers expressing type II myosin ATPase and a
corresponding increase in type I fibers in a comparison of
sedentary vs. chronically endurance-trained men (27), after a
6-wk bicycle training program in young men (12), and after a
9- to 12-mo walking and jogging program in older men and
women (7). The finding by Coggan et al. (7) that fiber type was
changed after a longer training program than used in the
present study further supports the possibility that some adap-
tations to exercise may be slower in older people.
Relation of MHC expression and muscle strength. Thigh
muscle cross-sectional area and peak isokinetic knee extensor
torque declined with age in both men and women, in agreement
with previous observations (13–15, 19, 23). Muscle size is
clearly a major determinant of muscle strength, but the decline
in knee extensor torque with age persisted after covariate
adjustment for thigh muscle cross-sectional area, an index of
specific force. This indication that muscle quality declines with
age is consistent with previous reports in which knee extensor
torque was normalized for thigh muscle size in younger and
older people (14, 15, 23). We assessed whether the age-related
shift in MHC expression contributed to the decline in muscle
quality because MHC isoforms are the major determinant of
contractile speed, force, and energy cost as reported in single
muscle fibers (5, 9). In multiple regression analysis, Larsson
J Appl Physiol • VOL
101
and Karlsson (19) found that the percent and the proportional
cross-sectional area occupied by type II fibers were significant
determinants of muscle strength and endurance. In the present
study, although the fractional content of MHC I and IIa
proteins was modestly correlated with knee extensor peak
torque, this relationship was eliminated after adjusting peak
torque for differences in muscle size. Klitgaard and colleagues
(15) also did not find a significant correlation between MHC
isoforms and specific force in young and old men, although
MHC protein isoform composition was related to muscle speed
of movement. Similarly, Jubrias et al. (14) did not detect an
association between MHC protein isoforms and specific force,
but they examined a smaller age range, from 65 to 80 yr. Thus
the decline in muscle-specific force with age appears to be
related to changes in properties other than MHC expression.
Contractile studies of single muscle fibers from older people
have demonstrated reduced speed and force characteristics
compared with young controls that persist after controlling for
the smaller size of older fibers (8, 17, 20), although a more
recent study did not reach this same conclusion (35).
Downloaded from jap.physiology.org on June 23, 2005
In conclusion, the present study demonstrates that, in healthy
untrained men and women, there is a shift with age in the
fraction of MHC protein from fast (decrease in MHC IIx) to
slow (increase in MHC I) isoforms. The mRNA abundance of
MHC isoforms shifts with age in the same direction, suggest-
ing a role for transcriptional regulation of fiber type. Although
there is an association between fractional MHC protein expres-
sion and isokinetic knee extensor strength, MHC protein iso-
forms do not explain the decline in specific force with age. We
also found that, in response to a standardized 4-mo program of
moderate-intensity endurance exercise, MHC isoform mRNA
profile shifted in favor of fast to slow expression in younger
and older people but that this change was evident at the
protein-expression level in younger but not older people. These
findings suggest that older muscles may require a longer period
in translating adaptive responses at the gene-transcript level
into changes in the expression of MHC protein isoforms. This
may represent reduced translational rate (fractional synthesis
rate) in response to stimuli such as endurance exercise.
ACKNOWLEDGMENTS
The authors are grateful for the valuable technical support provided by
Becca Kurup, Jane Kahl, and Dawn Morse. We also thank the GCRC nurses,
dieticians, and support staff, and the staff of the Healthy Living Center for help
with completion of the studies and training sessions, as well as the volunteers
who completed the protocol.
GRANTS
This study was funded by National Institutes of Health Grants MO1-RR-
00585 (for the Mayo Clinic General Clinical Research Center) and RO1-AG-
09531 (to K. S. Nair). Additional support was provided by the Mayo Foun-
dation and the Dole-Murdock Professorship (to K. S. Nair) and by National
Research Service Award T32-DK-07352 and the Mayo-Thompson Fellowship
(to K. R. Short).
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