J Appl Physiol 92: 313–322, 2002;
10.1152/japplphysiol.00832.2001.
Skeletal muscle adaptations in response to voluntary
wheel running in myosin heavy chain null mice
B. C. HARRISON,1 M. L. BELL,1 D. L. ALLEN,2 W. C. BYRNES,1 AND L. A. LEINWAND2
Department of Kinesiology and Applied Physiology, and 2Department of Molecular, Cellular and
Developmental Biology, University of Colorado at Boulder, Boulder, Colorado 80309
1
Received 7 August 2001; accepted in final form 17 September 2001
myosin heavy chain; endurance exercise; muscle plasticity
SKELETAL MUSCLE SHOWS a remarkable adaptive ability,
which is exemplified by alterations in the expression of
a wide range of muscle-specific genes. Paradigms such
as endurance exercise increase muscle activity and
result in changes within the muscle that allow the
elevated metabolic demands to be met more effectively.
Specifically, there are shifts in myosin heavy chain
(MHC) isoform expression that result in a decrease in
the percentage of muscle fibers expressing the faster
MHC IIb isoform and an increase in the percentage of
muscle fibers expressing the slower MHC IIa isoform
(3, 6, 7, 14, 19, 27, 32, 33, 40, 44). These changes in
MHC isoform expression can also be accompanied by
increased levels of key oxidative enzymes (3, 6, 8, 14,
24, 25, 37) and skeletal muscle fiber hypertrophy (3, 14,
27, 44).
Although the plasticity of skeletal muscle in response to endurance exercise is well documented for a
Address for reprint requests and other correspondence: L. A.
Leinwand, Dept. of MCD Biology, Campus Box 347, Univ. of Colorado
at Boulder, Boulder, CO 80309 (E-mail: [email protected]).
http://www.jap.org
number of animal models using both treadmill-based
and voluntary wheel running-based exercise programs,
relatively few studies have combined endurance exercise and a transgenic rodent model (9, 11, 15–18, 21–
23, 26, 29, 31, 34, 37, 38, 42, 43), and none of these has
examined muscle plasticity. Transgenic mouse models
in which a muscle-specific gene has been genetically
altered may shed light on the phenomenon of muscle
plasticity and lead to an increased understanding of
muscle adaptation (41). Given that shifts in MHC isoform expression with endurance exercise seem to follow a defined pattern of change (IIb, IId/x, IIa, I) (32,
33), genetic manipulations within this family of muscle
genes may directly impact the ability of muscle to
adapt to an exercise stimulus.
Two transgenic mouse strains null for an adult fast
skeletal MHC isoform gene have been previously described (1, 4, 36). MHC IIb and MHC IId/x null mice
are viable and exhibit normal myosin-to-actin ratios
via gene compensation within the MHC isoform gene
family (1, 4, 36). Specifically, in the MHC IIb null mice,
there is compensation by the MHC IId/x gene, and in
the MHC IId/x null mice, there is compensation by the
MHC IIa gene (4, 36). Despite these gene compensations, both null strains exhibit specific and distinct
phenotypes. MHC IIb null mice show significant muscle fiber loss in most muscles of the hindlimb combined
with a compensatory hypertrophy of the remaining
skeletal muscle fibers (1, 4). In contrast, MHC IId/x
null mice show a tendency toward maintenance of total
muscle fiber number and muscle fiber hypertrophy
within some fiber populations (36). Both MHC null
strains show regions of muscle pathology in selected
hindlimb muscles that is characterized by small fiber
profiles, increased fibrosis, increased interfiber space,
evidence of muscle fiber degeneration or regeneration,
and evidence of ultrastructural defects including abnormal nuclei, abnormal mitochondria, and myofibrillar disarray (1, 4, 36).
We have previously demonstrated that for the nontransgenic (NTG) C57BL/6 mouse, voluntary wheel
running provides a sufficient endurance exercise stimulus to trigger significant skeletal muscle adaptation
The costs of publication of this article were defrayed in part by the
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8750-7587/02 $5.00 Copyright © 2002 the American Physiological Society
313
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Harrison, B. C., M. L. Bell, D. L. Allen, W. C. Byrnes,
and L. A. Leinwand. Skeletal muscle adaptations in response to voluntary wheel running in myosin heavy chain
null mice. J Appl Physiol 92: 313–322, 2002; 10.1152/
japplphysiol.00832.2001.—To examine the effects of gene
inactivation on the plasticity of skeletal muscle, mice null for
a specific myosin heavy chain (MHC) isoform were subjected
to a voluntary wheel-running paradigm. Despite reduced
running performance compared with nontransgenic C57BL/6
mice (NTG), both MHC IIb and MHC IId/x null animals
exhibited increased muscle fiber size and muscle oxidative
capacity with wheel running. In the MHC IIb null animals,
there was no significant change in the percentage of muscle
fibers expressing a particular MHC isoform with voluntary
wheel running at any time point. In MHC IId/x null mice,
wheel running produced a significant increase in the percentage of fibers expressing MHC IIa and MHC I and a significant decrease in the percentage of fibers expressing MHC IIb.
Muscle pathology was not affected by wheel running for
either MHC null strain. In summary, despite their phenotypes, MHC null mice do engage in voluntary wheel running.
Although this wheel-running activity is lessened compared
with NTG, there is evidence of distinct patterns of muscle
adaptation in both null strains.
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WHEEL RUNNING IN MHC NULL MICE
METHODS
Animals and wheel running. Generation of MHC IIb and
MHC IId/x null mice has been previously described (1). Litters of male MHC IIb and MHC IId/x null mice 6–8 wk of age
were randomly divided into either exercised or nonexercised
(NE) littermate controls. Wild-type C57BL/6 animals served
as NTG controls, and the data for these animals have been
published previously (3). Individual exercise animals were
housed in a cage (47 ⫻ 26 ⫻ 14.5 cm) containing a free wheel
for 1, 2, or 4 wk. Corresponding NE animals were housed in
a standard mouse cage for the same 1-, 2-, or 4-wk period. All
animals were given water and standard hard rodent chow ad
libitum. The exercise wheels used have been previously described (3). Daily exercise values for time and distance run
were recorded for each exercised animal throughout the duration of the particular exercise period (1, 2, or 4 wk). At the
end of the specific exercise period, exercised and NE littermate control animals were killed by cervical dislocation.
Hindlimb muscles of interest (tibialis anterior and gastrocnemius) were dissected, weighed, and frozen in liquid nitrogen-cooled isopentane.
Citrate synthase and NADH-tetrazolium staining. Citrate
synthase (CS) analysis was performed as described by Srere
(39). Briefly, the tibialis anterior and gastrocnemius were
dissected, weighed, and rapidly frozen in liquid nitrogen.
Frozen tissue was then homogenized with a glass homogenizer on ice in 100 mM Tris-HCl. Muscle homogenate protein
concentration was determined by using the Bio-Rad protein
assay (Bio-Rad Laboratories, Hercules, CA). Sample homogenate was then added to a reaction mix of 100 mM Tris-HCl,
1.0 mM dithio-bis(2-nitrobenzoic acid), and 3.9 mM acetyl
coenzyme A. After an addition of 1.0 mM oxaloacetate, absorbance at 412 nm was recorded for a 2-min period. Mean
absorbance change per minute was recorded for each sample,
and CS activity (in ␮mol 䡠 mg protein⫺1 䡠 min⫺1) was then
calculated by using the mercaptide ion extinction coefficient
of 13.6.
For NADH-tetrazolium (NADH-TR) staining, tissue sections were incubated for 30 min at 37°C in a solution of 0.2 M
Trizma base, 1.5 mM NADH, and 1.5 mM nitrotetrazolium
blue. Sections were then serially incubated with acetone
solutions (60, 80, 100, 80, and 60% for 2 min each), and
mounted with Aquamount (Lerner Labs, Pittsburgh, PA).
J Appl Physiol • VOL
NADH-TR staining revealed three distinct fiber profiles: unstained, moderately stained, and intensely stained. Five hundred to 1,000 fibers from approximately five random fields
within the tissue section were counted and grouped into
NADH-TR negative (unstained) and NADH-TR positive
(moderately stained and intensely stained).
Immunohistochemistry. Frozen muscle samples were sectioned into 10-␮m-thick sections with the aid of a Tissue Tek
II microtome/cyrostat (Miles Scientific, Naperville, IL) and
fixed to gelatin-coated slides. Muscle sections were air dried
for 30 min followed by incubation in permeabilizing/blocking
solution (P/BS) (0.12% BSA, 0.12% nonfat dry milk, 0.01%
Triton X-100 in PBS) for 1 h at 4°C. Tissue sections were then
rinsed four times with PBS before primary antibody incubation. Primary antibodies used were NCL-MHCs [Novocastra
Laboratories (NCL), Newcastle on Tyne, UK], which positively labels MHC I-expressing fibers; SC-71, which positively identifies MHC IIa expression; BF-F3, which labels
MHC IIb-expressing fibers; and BF-35, which labels all MHC
isoforms except for MHC IId/x (20). To aid in muscle fiber
identification, MHC isoform-specific antibodies were used in
conjunction with an anti-laminin antibody (1:100 dilution in
P/BS) (Sigma Chemical, St. Louis, MO). NCL-MHC was used
at a 1:20 dilution in P/BS. For all other primary antibodies,
undiluted hybridoma supernatant was used. Primary antibody incubations were carried out for 1 h at room temperature. After primary antibody incubations, tissue sections
were washed three times with P/BS and incubated in P/BS
3 ⫻ 5 min. For MHC isoform-specific antibodies, secondary
antibodies consisted of goat anti-mouse peroxidase conjugates, either IgG (MHCs, SC-71, and BF-35) or IgM (BF-F3).
For the anti-laminin antibody, the secondary antibody used
was a goat anti-rabbit peroxidase conjugate. Secondary antibodies were diluted 1:200 in P/BS, and tissue sections were
incubated for 1 h at room temperature. After secondary
antibody incubation, tissue sections were washed four times
with PBS and then incubated in peroxidase diaminobenzidine (DAB) reaction kit (Vector Labs, Burlinghame, CA)
solution for 5 min. After DAB incubation, tissue sections
were washed four times with distilled H2O, dehydrated with
serial application of 60, 80, and 100% ethanol, and mounted
in Permount (Fisher Scientific, Fair Lawn, NJ).
Fiber-type percentage, fiber cross-sectional area, and total
fiber number. Fiber-type percentage, fiber cross-sectional
area (CSA), and total fiber number were assessed using
National Institutes of Health (NIH) Image 6.1 image analysis
software (NIH, Bethesda, MD) in conjunction with a light
microscope attached to a microcomputer (Power Computing,
Austin, TX). The percentage of muscle fibers expressing a
particular MHC isoform was determined via counts of 500–
1,000 fibers in four to six regions from both the deep and
superficial portion of the muscle. For the gastrocnemius, in
which there are distinct regional differences in MHC isoform
expression (MHC I and MHC IIa are mostly restricted to the
deep portions of the muscle, and MHC IIb is mostly restricted
to the superficial portion of the muscle), fiber counts were
weighted to account for the relative CSAs of these distinct
muscle regions. Mean CSA for fibers expressing a particular
MHC isoform was determined by tracing the circumference of
50 fibers chosen from 5–10 random fields within the muscle.
It has been previously demonstrated that 50 fibers provide an
adequate sample size for this analysis (30), and this finding
has been confirmed in our hands for both NTG and MHC null
tissue (data not shown). CSA measurements were performed
on tissue sections stained for the MHC isoform of interest.
For both fiber-type percentage and CSA measurements, unstained fibers were counted as negative for the MHC isoform
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(3). The specific aim of the current study was threefold:
first, to determine whether MHC null mice would undergo voluntary exercise to a sufficient extent to induce
muscle adaptation; second, to determine the plasticity
of MHC IIb and MHC IId/x null mouse skeletal muscle
in response to voluntary wheel running; and finally, to
determine whether engaging in voluntary exercise
would affect the muscle pathology exhibited by MHC
null mice.
We demonstrate that, despite the phenotypic
changes observed in the MHC null strains, both MHC
IIb and MHC IId/x null mice engage in a significant
level of voluntary activity on a cage wheel. We also find
that this level of wheel-running activity is sufficient to
induce similar skeletal muscle adaptations as seen in a
NTG mouse, although the specifics and extent of the
adaptation differ between the two MHC null strains.
Finally, we show that voluntary wheel running does
not appear to affect the localized muscle pathology
observed in either MHC null strain.
WHEEL RUNNING IN MHC NULL MICE
RESULTS
Wheel-running activity. MHC IIb null animals ran
an average of 4.4 ⫾ 0.3 h/night for an average distance
J Appl Physiol • VOL
of 4.7 ⫾ 0.4 km/night with an average speed of 17.3 ⫾
0.9 m/min (Fig. 1). Although this running duration was
not significantly different from NTG, both running
distance and average speed were significantly reduced
compared with NTG values reported previously (3). In
contrast, MHC IId/x null mice ran an average of 2.8 ⫾
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of interest, and fibers showing either intermediate or dark
staining were counted as positive for the MHC isoform of
interest.
Total fiber number was calculated by using the formula
total fiber number ⫽ (muscle CSA-interfiber space)/mean
fiber CSA. Muscle CSA was determined by circumscribing
the entire muscle with the image analysis software described
above. Interfiber space was determined by measuring fiber
area for all fibers within five separate muscle regions and
then subtracting this value from the total screen area for
each region. For the MHC IIb null animals, mean fiber CSA
was determined by using the formula (MHC I/100 ⫻ mean
MHC I fiber CSA) ⫹ (percent MHC IIa/100 ⫻ mean MHC IIa
fiber CSA) ⫹ (MHC IId/x/100 ⫻ mean MHC IId/x fiber CSA).
For the MHC IId/x null animals, mean fiber CSA was determined by using the formula (MHC I/100 ⫻ mean MHC I fiber
CSA) ⫹ (MHC IIa/100 ⫻ mean MHC IIa fiber CSA) ⫹ (MHC
IIb/100 ⫻ mean MHC IIb fiber CSA).
Muscle pathology. In the MHC IIb null mice, there is
evidence of muscle pathology in the superficial portions of the
gastrocnemius and quadriceps that is characterized by abnormal fiber profiles, evidence of muscle fiber degeneration
and/or regeneration, and ultrastructural abnormalities (4).
To investigate the effects of wheel running on this pathology,
the area of the pathological region was quantified for the
gastrocnemius by using the image analysis system described
previously and expressed as a percentage of the total gastrocnemius CSA. This procedure was performed on tissue sections stained with the anti-laminin antibody as the pathological region is easily identified with this staining (see Ref. 4;
Fig. 4).
In the MHC IId/x null strain, there is diffuse pathology
throughout the tibialis anterior muscle that is characterized
by small fiber profiles, increased interstitial fibrosis, and
myofibrillar disarray (1, 36). Because this pathology is not
restricted to a particular muscle region, we were unable to
quantify pathological area using the procedure used for the
MHC IIb null animals. Instead, we used ␣-bungarotoxin to
identify denervated muscle fibers as we and others have observed a tight correlation between ␣-bungarotoxin staining and
pathological appearance (small fiber profiles, increased fibrosis,
and increased interfiber space) and feel that this stain provides
a valid tool for the quantification of the muscle pathology seen
in these animals (5, 10, 28, 35). Sections were air dried briefly,
incubated in a 1:100 dilution of the ␣-bungarotoxin, biotin-XX
conjugate antibody (Molecular Probes, Eugene, OR) for 1 h at
room temperature, followed by an avidin-horseradish peroxidase conjugate at 1:200 dilution, and then visualized with the
DAB reaction kit described previously.
Statistical analysis. All data are reported as means ⫾ SE.
Statistical significance was determined for all measures with
a one-way ANOVA combined with the Fisher’s least-squares
differences post hoc test. Regression analysis was performed
for each dependent variable and total running duration,
distance, and volume (time ⫻ distance). Because the observed correlation patterns were the same for duration, distance, and volume, only significant R values for total running
duration vs. dependent variable are reported. Statistical significance was set at P ⬍ 0.05. No significant differences were
observed for any variable among the NE control animals (1-,
2-, or 4-wk litters), so these animals were pooled into a single,
NE group for each genotype.
315
Fig. 1. Overall and weekly wheel-running values for myosin heavy
chain (MHC) IIb, MHC IId/x null, and nontransgenic control (NTG)
mice. A: mean running duration (h/night). For the MHC IIb null
mice, running duration was not significantly different from NTG. For
the MHC IId/x null animals, running duration was significantly
reduced compared with both NTG and MHC IIb null overall and for
weeks 1 and 2 (§P ⬍ 0.01). B: mean running distance (km/night).
MHC IIb null running distance was significantly reduced compared
with NTG overall and for weeks 1, 2, and 4 (*P ⬍ 0.01). MHC IId/x
running distance was significantly reduced compared with NTG for
week 4 (**P ⬍ 0.001) and significantly reduced compared with both
NTG and MHC IIb null overall and for weeks 1 and 2 (§P ⬍ 0.01). C:
average running speed (m/min). Average speed for both MHC null
strains was significantly reduced compared with NTG overall and for
weeks 1, 2, and 4 (*P ⬍ 0.01 and **P ⬍ 0.001 for MHC IIb null and
MHC IId/x null, respectively). Values are means ⫾ SE for n ⫽ 12
animals for overall and week 1, 8 animals for week 2, and 4 animals
for week 4 per genotype.
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WHEEL RUNNING IN MHC NULL MICE
Table 1. Body and skeletal muscle mass in MHC-IIb
null and MHC-IId/x null with and without
wheel running
Exercise
Body Wt,
g
Tibialis Anterior,
mg
Gastrocnemius,
mg
MHC-IIb null
NE
1 wk
2 wk
4 wk
21.4 ⫾ 0.7
19.9 ⫾ 0.9
22.9 ⫾ 1.1
22.6 ⫾ 0.6
NE
1 wk
2 wk
4 wk
23.2 ⫾ 1.8
23.3 ⫾ 2.1
22.9 ⫾ 1.6
24.8 ⫾ 2.7
26.7 ⫾ 1.3*
21.9 ⫾ 1.8*
25.9 ⫾ 0.5*
27.5 ⫾ 1.0*
54.2 ⫾ 3.0*
46.9 ⫾ 8.3*
67.6 ⫾ 4.0*
54.8 ⫾ 4.4*
MHC-IId/x null
41.0 ⫾ 2.3
42.0 ⫾ 3.5
33.7 ⫾ 0.8
40.8 ⫾ 4.7
140.1 ⫾ 6.1
138.9 ⫾ 10.4
124.2 ⫾ 4.6
146.6 ⫾ 13.2
Values are means ⫾ SE; n ⫽ 4 animals per condition. MHC,
myosin heavy chain; NE, no exercise. * Significantly reduced compared with corresponding MHC-IId/x value (P ⬍ 0.01).
J Appl Physiol • VOL
Fig. 2. Citrate synthase (CS) activity in ␮mol 䡠 mg protein⫺1 䡠 min⫺1
with 4 wk of wheel running compared with nonexercised (NE) control
in NTG, MHC IIb null, and MHC IId/x null mice for the tibialis
anterior (A) and gastrocnemius (B). In the NE condition, CS activity
was significantly elevated in the MHC IIb null animals compared
with NTG and MHC IId/x null for both muscles (*P ⬍ 0.0001). After
4 wk of wheel running, CS activity was significantly increased over
baseline NE values in all strains for both muscles (**P ⬍ 0.05).
Values are means ⫾ SE for n ⫽ 8 animals per condition.
and the gastrocnemius (Fig. 2). After 4 wk of voluntary
wheel running, CS activity was significantly elevated
in NTG and both MHC null strains (Fig. 2). In the
MHC IIb null tibialis anterior, there was a significant
correlation between total running duration and CS
activity (R ⫽ 0.914, P ⫽ 0.029).
NADH-TR analysis revealed that in the NE condition, MHC IIb null animals showed a significantly
greater percentage of NADH-TR positive fibers in both
the tibialis anterior and the gastrocnemius compared
with either NTG or MHC IId/x null. In fact, in the
MHC IIb null tissue samples, 100% of all fibers were
moderately or darkly stained with the NADH-TR stain
(Fig. 3). In the MHC IIb null animals, voluntary wheel
running did not alter the percentage of NADH-TR positive fibers. In the MHC IId/x null animals, however, a
significant increase in the percentage of NADH-TR positive fibers was observed in gastrocnemius with 2 and 4
wk of wheel running (46.5 ⫾ 2.9% NE vs. 56.0 ⫾ 0.7%
2 wk of exercise and 60.2 ⫾ 1.3% 4 wk of exercise;
P ⬍ 0.05).
Muscle fiber CSA. For both MHC null strains, wheel
running resulted in a significant increase in the mean
CSA of selected skeletal muscle fiber populations. Specifically, in the MHC IIb null animals, there was a
significant increase in the CSA of fibers expressing
MHC I in the gastrocnemius with 2 and 4 wk of wheel
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0.2 h/night for an average distance of 2.4 ⫾ 0.2 km/
night at an average speed of 14.3 ⫾ 0.6 m/min (Fig. 1).
All MHC IId/x null values were significantly lower
than those previously recorded for NTG (3), and the
running duration and distance values were significantly reduced compared with MHC IIb null.
Comparison of wheel-running values across time indicated that the wheel-running activity patterns of
both null strains were altered compared with NTG
control mice (Fig. 1). Running durations were similar
for NTG and MHC IIb null at all time points, whereas
MHC IId/x null animals showed significantly reduced
running durations compared with both NTG and MHC
IIb null for weeks 1 and 2 and a tendency for reduced
running duration compared with NTG for week 4 (P ⫽
0.07; Fig. 1A). For running distance and average speed,
MHC IId/x null values were significantly reduced compared with NTG at all time points (Fig. 1, B and C).
MHC IId/x null running distance values for weeks 1
and 2 were also significantly lower than the corresponding MHC IIb null values at these time points
(Fig. 1B). For the MHC IIb null mice, running distance
and average speed were significantly reduced compared with NTG at all time points (Fig. 1, B and C).
Body and muscle mass. As previously noted, body
mass of both MHC null strains was significantly reduced compared with NTG (NTG data not shown), and
MHC IIb null muscle mass was significantly reduced
compared with both NTG and MHC IId/x muscle mass
(Table 1) (1, 4, 32). For both MHC null strains, body
mass and skeletal muscle mass were unaffected by
voluntary wheel running at any time point (Table 1).
Muscle oxidative capacity. It is well established that
endurance exercise leads to an increase in muscle oxidative capacity (3, 6, 8, 14, 24, 25). In the MHC null
animals, muscle oxidative capacity was addressed via
analysis of muscle CS activity and of NADH-TR staining patterns in both NE and exercised animals. In the
NE condition, CS activity was significantly elevated in
the MHC IIb null animals compared with either NTG
or MHC IId/x null mice for both the tibialis anterior
WHEEL RUNNING IN MHC NULL MICE
317
Fig. 3. NADH-tetrazolium (NADH-TR)
staining of the medial gastrocnemius of
NTG, MHC IIb null, and MHC IId/x
null with (4 wk) and without (NE)
wheel running. For both NTG and
MHC IId/x null, a gradient of fiber
staining is visible, from unstained to
darkly stained. In the MHC IIb null
samples, all fibers are moderately or
darkly stained. Wheel running was
found to increase the percentage of
NADH-TR-positive fibers for both NTG
and MHC IId/x null but not for MHC
IIb null.
J Appl Physiol • VOL
In the tibialis anterior of the MHC IId/x null animals, there was a significant increase in the percentage of fibers expressing MHC IIa combined with a
significant decrease in the percentage of fibers expressing MHC IIb with 4 wk of wheel running (Fig. 5B). For
this muscle, there was a significant correlation between the percentage of muscle fibers expressing MHC
IIa and total wheel-running duration (R ⫽ 0.695, P ⫽
0.012). In the MHC IId/x null gastrocnemius, the percentage of fibers expressing MHC I increased from
1.6 ⫾ 0.2% in the NE condition to 2.7 ⫾ 0.4% with 4 wk
of wheel running (P ⬍ 0.05; Fig. 5D) and there was a
significant correlation between the percentage of muscle fibers expressing MHC I and total wheel running
duration (R ⫽ 0.609, P ⫽ 0.035). The MHC IId/x null
gastrocnemius also showed a general trend of a decrease in the percentage of fibers expressing MHC IIb
(86.7 ⫾ 1.8% NE vs. 80.7 ⫾ 3.6% 4 wk of exercise, P ⫽
0.125) and an increase in the percentage of fibers
expressing MHC IIa (12.4 ⫾ 1.8% NE vs. 15.6 ⫾ 2.5%
4 wk of exercise, P ⫽ 0.208).
Given that there is a gap in the MHC isoform continuum (IIb, IIa, I) in the MHC IId/x null animals, we
used combined fluorescence immunohistochemistry
(monoclonal antibody SC-71 plus monoclonal antibody
BF-F3) to determine whether there were fibers coexpressing MHC IIa and MHC IIb. Analysis of NE and
exercised MHC IId/x null muscle sections with this
technique, however, did not reveal any hybrid IIa/IIb
fibers, indicating that the muscle’s ability to coexpress
these two isoforms may be limited (data not shown).
Total fiber number. Wheel running did not affect
total fiber number in either the gastrocnemius or the
tibialis anterior for NTG, MHC IIb null, and MHC
IId/x null (Table 2). Overall, the results for total fiber
number reinforced the previous findings, indicating
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running (Fig. 4C) and a significant increase in the CSA
of fibers expressing MHC IIa in the gastrocnemius and
tibialis anterior with 1, 2, and 4 wk of wheel running
(Fig. 4, A and C). In the MHC IId/x null mice, the mean
CSA of fibers expressing MHC IIa was increased in the
gastrocnemius and tibialis anterior with 2 and 4 wk of
wheel running (Fig. 4, B and D). For the MHC IId/x
null gastrocnemius, there was a significant correlation
between total wheel-running duration and the mean
CSA of MHC IIa expressing fibers (R ⫽ 0.720, P ⫽
0.008). Wheel running also produced a significant increase in the mean CSA of MHC IId/x expressing
muscle fibers in the MHC IIb null tibialis anterior (Fig.
4A) and of MHC IIb-expressing muscle fibers in the
MHC IId/x null tibialis anterior and gastrocnemius
(Fig. 4, B and D).
Muscle fiber-type percentage. For the MHC IIb null
mice, the percentage of fibers expressing a particular
MHC isoform was not affected by voluntary wheelrunning activity for either the tibialis anterior or the
gastrocnemius (Fig. 5, A and C). In the MHC IIb null
gastrocnemius, there was a trend toward an increase
in the percentage of MHC IIa expressing fibers (52.2 ⫾
5.8% NE vs. 58.8 ⫾ 10.4% 4 wk of exercise) combined
with a decrease in the percentage of MHC IId/x expressing fibers (37.2 ⫾ 5.4% NE vs. 30.53 ⫾ 8.3% 4 wk
of exercise), but these changes were not significant
(P ⬎ 0.1; Fig. 5C). This lack of significant change is in
contrast to the NTG response, in which significant
alterations in fiber-type percentage have been reported
with voluntary wheel running for both of these hindlimb muscles (3). Regression analysis of MHC isoform
composition in the MHC IIb null animals revealed no
significant correlation between the percentage of muscle fibers expressing a particular MHC isoform and
total wheel-running duration.
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WHEEL RUNNING IN MHC NULL MICE
that the MHC IIb null strain was more affected in
terms of total fiber number than the MHC IId/x null
strain (1, 4, 36). Specifically, in both analyzed muscles,
total fiber number was significantly reduced in the
MHC IIb null mice compared with NTG, and, in the
gastrocnemius, MHC IIb null total fiber number was
significantly reduced compared with both NTG and
MHC IId/x null (Table 2).
Muscle pathology. We have previously reported that
both MHC null strains exhibit regions of muscle pathology as a consequence of elimination of a particular
MHC isoform gene (1, 4, 36). We therefore wished to
test whether increased muscle activation due to wheel
running would exacerbate this muscle pathology.
Quantification of the muscle pathology in the superficial gastrocnemius of the MHC IIb null animals revealed no difference between the NE and 4-wk exercised animals (17.9 ⫾ 2.7% of total gastrocnemius CSA
vs. 18.8 ⫾ 3.8% of total gastrocnemius CSA, respectively). In contrast to the MHC IIb null phenotype, in
which the pathology is localized to the superficial
portions of the gastrocnemius and quadriceps (4), the
pathology in the MHC IId/x null animals is restricted
to the tibialis anterior and is more diffuse in nature
(36). For these animals, the extent of the muscle
J Appl Physiol • VOL
pathology was quantified through the use of ␣-bungarotoxin staining to identify denervated muscle fibers. Analysis of these results revealed no difference
in the percentage of ␣-bungarotoxin-positive fibers in
the tibialis anterior of both NE and 4-wk exercised
animals (39.4 ⫾ 6.3% vs. 45.7 ⫾ 7.2%, respectively).
Overall, these data provide indirect evidence that
wheel running did not result in increased muscle
pathology in either of the MHC null strains.
DISCUSSION
Given that the shifts in MHC isoform expression in
response to an endurance exercise stimulus are
thought to progress through a specific pattern of
change (IIb, IId/x, IIa, I) (32, 33), genetic perturbations
within the MHC isoform gene family might therefore
disrupt the adaptive ability of skeletal muscle and
could perhaps provide insight into the phenomenon of
muscle plasticity. As discussed previously, both MHC
IIb and MHC IId/x null mice show distinct phenotypes
and patterns of gene compensation within the family of
MHC isoform genes. Despite this gene compensation,
MHC null mice exhibit significant loss of skeletal muscle mass, alterations in total muscle fiber number,
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Fig. 4. Mean muscle fiber cross-sectional area (CSA) with and without wheel running in MHC IIb (A and C) and
MHC IId/x null mice (B and D) for the tibialis anterior and gastrocnemius. A: for the MHC IIb null tibialis anterior,
there was a significant increase in the mean CSA of muscle fibers expressing MHC IIa with 1, 2, and 4 wk of wheel
running (**P ⬍ 0.001) and of MHC IId/x-expressing fibers with 2 and 4 wk of wheel running (***P ⬍ 0.01). B: for
the MHC IId/x null tibialis anterior, there was a significant increase in the mean CSA of MHC IIa-expressing fibers
with 2 and 4 wk of wheel running (**P ⬍ 0.001) and of MHC IIb-expressing fibers with 1 and 4 wk of wheel running
(***P ⬍ 0.01). C: for the MHC IIb null gastrocnemius, there was a significant increase in the mean CSA of MHC
I-expressing fibers with 2 and 4 wk of wheel running (*P ⬍ 0.001) and of MHC IIa-expressing fibers with 1, 2, and
4 wk of wheel running (**P ⬍ 0.001). D: for the MHC IId/x null gastrocnemius, there was a significant increase in
the mean CSA of MHC IIa-expressing fibers with 2 and 4 wk of wheel running (**P ⬍ 0.001) and of MHC
IIb-expressing fibers with 1 and 4 wk of wheel running (***P ⬍ 0.01). Values are means ⫾ SE for n ⫽ 4 animals
per condition.
319
WHEEL RUNNING IN MHC NULL MICE
Fig. 5. Fiber-type percentages with and without wheel
running in MHC IIb null (A and C) and MHC IId/x null
(B and D) mice for the tibialis anterior and gastrocnemius. A and C: in the MHC IIb null animals, no significant differences in the percentage of muscle fibers
expressing a particular MHC isoform were observed
with any duration of wheel running compared with NE.
B: in the MHC IId/x null tibialis anterior, the percentage of fibers expressing MHC IIa was significantly
increased with 4 wk of wheel running compared with
NE, whereas the percentage of fibers expressing MHC
IIb was significantly reduced (**P ⬍ 0.05 and ***P ⬍
0.05, respectively). D: for the MHC IId/x null gastrocnemius, the percentage of fibers expressing MHC I was
significantly increased compared with NE with 4 wk of
wheel running (*P ⬍ 0.05). Values are means ⫾ SE for
n ⫽ 4 animals per condition.
J Appl Physiol • VOL
mouse, the fiber-type composition of this muscle is
⬃45% MHC IIa-expressing fibers whereas in the MHC
IId/x null animals, 82% of the muscle fibers express
MHC IIa (36). The increased relative content of the
slower contracting MHC IIa may adversely impact the
function of this muscle, and it has been previously
demonstrated that both time-to-peak tension and timeto-relaxation of the MHC IId/x null diaphragm are
significantly increased compared with NTG (1). It is
therefore possible that the reduced running capacity of
the MHC IId/x null animals is due, at least in part, to
a reduced ventilatory capacity in these animals and to
an inability to increase the rate of ventilation appropriately in the face of an exercise stimulus. It is also
important to note that the mouse diaphragm does not
normally contain MHC IIb expressing fibers (36),
Table 2. Total fiber number in the tibialis anterior
and gastrocnemius of NTG, MHC-IIb null, and
MHC-IId/x null with and without wheel running
Exercise
Tibialis Anterior
Gastrocnemius
NTG
NE
4 wk
2,971 ⫾ 253
2,548 ⫾ 334
NE
4 wk
1,847 ⫾ 66*
1,805 ⫾ 92*
6,069 ⫾ 350
7,196 ⫾ 578
MHC-IIb null
1,933 ⫾ 108†
2,345 ⫾ 181†
MHC-IId/x null
2,265 ⫾ 361
2,287 ⫾ 334
NE
4 wk
5,738 ⫾ 453
5,871 ⫾ 309*
Values are means ⫾ SE; n ⫽ 4 animals per condition. NTG,
nontransgenic. * Significantly reduced compared with corresponding
NTG value (P ⬍ 0.05). † Significantly reduced compared with corresponding NTG and MHC-IId/x null values (P ⬍ 0.001).
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evidence of compensatory hypertrophy, and regions of
muscle pathology (1, 4, 36).
Wheel-running activity. Given the phenotypes of the
MHC null strains, an initial question was whether
these animals would choose to exercise on a cagemounted free wheel to the same extent as a NTG
mouse. It might have been predicted that the MHC IIb
null strain would exhibit impaired wheel performance
compared with the MHC IId/x null strain, as the MHC
IIb null phenotype is in many ways more severe than
that of the MHC IId/x null strain (more significant and
systematic reductions in muscle mass combined with
greater alterations in muscle fiber number and size) (1,
4, 36). Surprisingly enough, when the wheel-running
activities of these strains were compared, it was the
MHC IId/x null strain that exhibited the lowest level of
wheel-running activity compared with either NTG or
MHC IIb null strains.
For the MHC IIb null mice, compensatory shifts in
MHC expression may contribute to the reduced running speed compared with NTG. The fast-contracting
MHC IIb isoform is missing; there is a marked increase
in the level of MHC IId/x expression, and the relative
content of the slower contracting MHC IIa and MHC I
isoforms is increased (4). This pattern of wheel-running activity is also consistent with previous data that
demonstrated a reduction in maximum contractile
force in isolated MHC IIb null skeletal muscle (1). In
addition to the functional effects of altered MHC isoform expression, decreased muscle mass and muscle
fiber loss would most likely also contribute to decreased force production and therefore reduced running velocity in the MHC IIb null animals.
For the MHC IId/x mice, one factor that may influence the wheel-running performance of these animals
is the function of the diaphragm muscle. In the NTG
320
WHEEL RUNNING IN MHC NULL MICE
Table 3. Summary of muscle adaptations in response to voluntary wheel running in NTG, MHC-IIb null, and
MHC-IId/x null mice
NTG
MHC isoforms
Wheel performance
Muscle and body mass
Muscle oxidative capacity
Muscle fiber CSA
Total fiber number
Muscle pathology
MHC isoform shifts
MHC-IIb Null
R
IIb 3 IId/x 3 IIa 3 I
MHC-IId/x Null
R
IIb3 IId/x 3 IIa 3 I
IIb 3 IId/x 3 IIa 3 I
medium
7
1
1
7
7
7
low
7
1
1
7
7
1 I 1 IIa and 2 IIb
high
7
1
1
7
NA
1 IIa and 2 IIb
CSA, cross-sectional area; 7, no change; 1, increased; 2, decreased; NA, not applicable; R, unavailable.
J Appl Physiol • VOL
expressing MHC I, IIa, and IIb. This finding is especially intriguing given that there is a gap in the typical
progression of MHC isoform change due to the absence
of the MHC IId/x isoform (IIb, IIa, I). These results
might indicate that, in the MHC IId/x null context,
MHC isoform expression was able to “jump” from MHC
IIb to MHC IIa. If this were the case, then we might
have expected to see hybrid fibers coexpressing both
MHC IIa and MHC IIb. With the use of immunohistochemistry, however, we did not see any evidence of
such fibers. This result suggests that there was not
significant coexpression of these two MHC isoforms but
does not rule out the possibility of trace amounts of
coexpression that was below the level of detection by
immunofluorescence. There is certainly evidence that,
under some conditions, muscle fibers may coexpress
many unique and novel MHC isoform combinations
including those isoforms that are not neighbors in the
above scheme (12, 13). Further analysis is needed to
investigate how MHC isoform transformations occur in
the MHC IId/x null context.
Muscle pathology. In the mdx mouse, a transgenic
model with significant muscle degeneration/regeneration and muscle pathology, there is conflicting evidence
regarding the effects of exercise. Specifically, voluntary
wheel running has been shown to increase muscle force
output and decrease muscle fatigability (16, 23),
whereas eccentric exercise has been shown to be significantly more damaging to mdx muscle than to control muscle (11). An additional question regarding the
MHC null mice, therefore, was whether wheel-running
activity would affect the muscle pathology observed in
these animals.
For the MHC IIb null animals, we have previously
indicated that the first evidence of this muscle pathology appears in these animals by ⬃20 days of age (4).
The lack of visible muscle pathology before this time
indicates a possible connection between the development of the pathology and the elevated activity of the
animals as they mature and begin locomotion. If the
development of muscle pathology is linked to muscle
activity, wheel running might therefore increase the
extent of the pathology in these animals. Quantification of the relative area of the pathological region,
however, showed no difference between exercise and
NE animals. For the MHC IId/x null animals, volun-
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which indicates that the function of this muscle should
not be impaired in the MHC IIb null animals and thus
would not contribute to the decreased running performance in these animals.
Muscle adaptation. Given that both strains did
choose to run on the cage wheel, the next logical question was how would the MHC null muscle adapt to the
exercise stimulus, and how this would adaptation be
different from the typical NTG control response. As
seen with wheel running in NTG control animals (3),
wheel running resulted in an increase in muscle oxidative capacity and muscle fiber hypertrophy in both
MHC null strains. Contrary to the NTG control response, the percentage of muscle fibers expressing a
particular MHC isoform was not significantly altered
with wheel running in the MHC IIb null animals.
Overall, the MHC IIb null data suggest that, in the NE
condition, these animals are already adapted to a more
oxidative state. MHC isoform expression is shifted
toward the slower isoforms, as the MHC IIb isoform is
not present, and there is gene compensation by the
MHC IId/x gene. As previously reported, this gene
inactivation combined with gene compensation results
in an increase in the relative percentages of MHC I,
MHC IIa, and MHC IId/x expressing fibers (4). In the
present study, we report that this shift in MHC isoform
expression is accompanied by an elevation in CS activity and an increased percentage of NADH-TR-positive
fibers. It is possible that the increased oxidative capacity of unexercised MHC IIb null skeletal muscle is of
sufficient magnitude to reduce the need for further
MHC isoform adaptation in the face of this specific
wheel-running stimulus. The lack of significant MHC
isoform transformations in the MHC IIb null animals,
however, should not be taken as evidence that MHC
isoform transformations cannot occur in these animals.
There were nonsignificant alterations in the fiber-type
composition of the MHC IIb null gastrocnemius with
wheel running, and it is certainly possible that we
would see significant MHC isoform shifts with either a
longer exercise period or an exercise stimulus of
greater intensity.
For the MHC IId/x null strain, although the wheelrunning activity of these animals was reduced compared with both NTG and MHC IIb null, we did observe shifts in the percentages of muscle fibers
WHEEL RUNNING IN MHC NULL MICE
The authors thank Deborah Whitney for help with the CS assay.
This project was supported by a grant from the American College
of Sports Medicine to B. C. Harrison and by National Institute of
General Medical Sciences Grant GM-29090 to L. A. Leinwand. D. L.
Allen is supported by a research grant from the Muscular Dystrophy
Association.
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