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1 Department of Kinesiology and Applied Physiology, and 2 Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado 80309
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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.
myosin heavy chain; endurance exercise; muscle plasticity
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 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 (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.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 litter-mate 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 µmol · mg
protein
1 · min1) was then
calculated by using the mercaptide ion extinction coefficient of 13.6.
Immunohistochemistry. Frozen muscle samples were sectioned into 10-µm-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 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.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Wheel-running activity.
MHC IIb null animals ran an average of 4.4 ± 0.3 h/night for
an average distance 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 ± 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.
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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).
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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 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).
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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 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).
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Muscle fiber-type percentage.
For the MHC IIb null mice, the percentage of fibers expressing a
particular MHC isoform was not affected by voluntary wheel-running 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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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 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).
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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
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.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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, 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 cage-mounted 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 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 time-to-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), 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 wheel-running activity of these animals was reduced compared with both NTG and MHC IIb null, we did observe shifts in the percentages of muscle fibers 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, voluntary wheel running did not affect the percentage of-bungarotoxin-positive muscle fibers in the tibialis anterior.
Overall, these results provide indirect evidence that the increased
activity of wheel running did not affect the extent of the muscle
pathology seen in either MHC null strain. Further analysis of the
pathological regions, however, would be required before a final
conclusion is reached on this issue.
In summary, we demonstrate that, despite their phenotypes, MHC IIb and
MHC IId/x null mice show significant levels of voluntary wheel-running
activity when presented with a cage wheel. The extent of this
wheel-running activity, however, is reduced compared with that seen in
NTG control animals (3). Despite this reduced exercise
stimulus, there were significant adaptations in both MHC null strains
(Table 3). The distinct responses
of these transgenic mouse strains to the endurance exercise paradigm
supports the evidence that the individual MHC isoforms have unique
functional roles in determining the physiological properties of
skeletal muscle and indicates that targeted inactivation of a
particular MHC isoform has specific effects on the adaptational
response of skeletal muscle.
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ACKNOWLEDGEMENTS |
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The authors thank Deborah Whitney for help with the CS assay.
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FOOTNOTES |
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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.
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: leslie.leinwand{at}colorado.edu).
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.
Received 7 August 2001; accepted in final form 17 September 2001.
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TOP
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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