| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
DEVELOPMENT AND TISSUE PLASTICITY
1Department of Pharmacology, Milton S. Hershey Medical Center, College of Medicine, The Pennsylvania State University, Hershey, Pennsylvania 17033-0850; and2Department of Integrative Physiology, University of Colorado, Boulder, Colorado 80309
Submitted 13 May 2003 ; accepted in final form 5 June 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
1-isoform increased with age in red gastrocnemius (GR),
white gastrocnemius (GW), and extensor digitorum longus (EDL) muscles, and ETr
further increased its levels. Levels of the 2-isoform were
unchanged in GR, had a strong trend for a decrease in GW, and decreased
significantly in EDL. ETr increased expression of the
2-isoform in all three muscle groups. There was no increase
in expression of the 
1-isoform in GR, GW, or EDL with age,
whereas ETr markedly increased its levels in the muscles. There was a marked
decrease with age in expression of the 2-isoform in the
muscle groups that was not reversed by ETr. By contrast,
3-isoform levels increased with age in GR and GW, and ETr was
able to reverse this increase. Na+-K+-ATPase enzyme
activity was unchanged with age in GR and GW but increased in EDL. ETr
increased enzyme activity in GR and GW and did not change in EDL. Myosin heavy
chain isoforms in the muscle groups did not change significantly with age; ETr
caused a general shift toward more oxidative fibers. Thus ETr differentially
modifies age-associated alterations in expression of
Na+-K+-ATPase subunit isoforms, and a mechanism(s) other
than physical inactivity appears to play significant role in some of the
age-associated changes.
-subunit; -subunit; aging; gastrocnemius; extensor digitorum longus; myosin heavy chain
Na+-K+-ATPase consists of a transmembrane catalytic
-subunit and a -subunit. Multiple isoforms of the - and
-subunits have been cloned and sequenced
(29-31,
40,
42). Skeletal muscle of mature
rats expresses the 1- and 2-subunit
isoforms and the three -subunit isoforms
(1-3,
21,
28). Fast and slow
oxidative-rich fibers express more 1- and
1-isoforms than fast glycolytic fibers, whereas the opposite
is true for the 2-isoform
(22,
44). Relative expression of
the 3-isoform in the different fibers has not been
determined.
We demonstrated previously that, in 6- to 30-mo-old Fischer 344 x
Brown Norway rats, advancing age is associated with increased levels of
1- and 1-isoforms and decreased levels of
2- and 2-isoforms in red and white
gastrocnemius (41). Our data
demonstrated, for the first time, dynamic regulation of skeletal muscle
Na+-K+-ATPase isozymes during aging. The mechanism(s)
underlying the age-associated differential expression of
Na+-K+-ATPase subunit isoforms is unclear. One of the
important adaptations associated with aging, common in humans and in animal
models, is reduced spontaneous physical activity
(20,
48). Furthermore, it has been
well established that exercise training alters the abundance of
Na+-K+-ATPase
(11,
17,
33). Therefore, in the present
study, we tested the hypothesis that increased physical activity, by endurance
exercise training, attenuates or reverses age-associated changes in
Na+-K+-ATPase. The corollary to this hypothesis is that
physical inactivity contributes to altered expression of the
Na+-K+-ATPase subunit isoform associated with advancing
age. Our result shows that endurance exercise training differentially modifies
expression of the isoforms in aged skeletal muscle; the training paradigm
reversed the expression of some, but not all, of the subunit isoforms.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
24 ± 4 h after the
last bout of exercise training, the rats were anesthetized with pentobarbital
sodium (35 mg/kg ip) 
15 min after heparin injection (250 U). Skeletal
muscles were dissected after hearts were removed. Thus the three experimental
groups (n = 12-15/group) were as follows: 16-mo-old middle-aged
sedentary (Ms), 29-mo-old senescent sedentary (Ss), and 29-mo-old senescent
exercise-trained (St) rats. All the following measurements were performed
using tissues from a group of five to six animals randomly selected from each
experimental group, except the citrate synthase activity assay, which was
performed using tissues from the entire groups of rats. All animal use
protocols were approved by the institutional animal care committee.
Preparation of tissue homogenates. Total tissue homogenates were
prepared as described previously
(41). Briefly, skeletal
muscles (200-300 mg) were pulverized and homogenized with a Polytron
(Brinkmann Instruments, Westbury, NY) at a speed of 6.5 (11.0 full scale) for
three 20-s periods at 4°C in a buffer containing 10 mM Tris · HCl
(pH 7.5), 1 mM EDTA, protease inhibitors (500 µM phenylmethylsulfonyl
fluoride), 1 µM leupeptin, 1 µM pepstatin, and 10 µM E-64. Protein
concentrations were determined by a protein assay (Bio-Rad, Melville, NY).
Western blot. Na+-K+-ATPase subunits were
resolved by SDS-PAGE and immunoblotted as previously described
(41). For analysis of the
-subunits, equal amounts of homogenates (80 µg) were first
deglycosylated with N-glycosidase F (Glyko, Novato, CA) for 18-20 h
at 37°C according to the manufacturer's instructions. Antibodies for
1- and 2-isoforms were kindly provided by
K. Sweadner (Harvard University). Antibodies for 1 (SpEtB1)-
and 2 (GP50)-isoforms were kindly provided by P.
Martin-Vasallo (Tenerife, Spain) and P. Beesley (Royal Holloway and Bedform
New College, Egham, Surrey, UK), respectively, and antibody for the
3-isoform was purchased from Upstate Biotechnology (Lake
Placid, NY). Bound monoclonal antibodies were detected with rabbit anti-mouse
IgG antibody followed by 125I-labeled protein A (ICN, Costa Mesa,
CA), whereas bound polyclonal antibodies were detected with
125I-labeled protein A alone. The blots were subjected to
autoradiography for the purpose of displaying the images. Subsequently, band
signal intensities were quantitated by a PhosphorImager (Molecular Dynamics,
Sunnyvale, CA). Because of the relatively low abundance of the -subunit
isoforms, a chemiluminescent detection method was used (SuperSignal West Pico,
Pierce, Rockford, IL). Blots were exposed to multiple films to ensure that
signals were within the linear range of the film, and relative intensity of
the bands was quantitated by densitometry (Molecular Dynamics laser
densitometer). Because the anti--subunit antibodies generate a
significant number of nonspecific bands, we verified the bands of interest by
running skeletal muscle controls to observe band shifts at appropriate
molecular sizes before and after N-glycosidase F treatment, as
demonstrated previously (37)
(data not shown). An N-glycosidase F-digested brain microsomal
preparation, in which levels of the -subunits are much higher than in
skeletal muscle, was run in the reference lane of the blots to identify the
appropriate bands (data not shown). The apparent molecular size of all core
-subunit isoforms is 35 kDa as revealed by molecular mass markers.
The transferred gels and portions of some of the membranes were stained with
Coomassie blue to verify efficient transfer of proteins and equal loading,
respectively (data not shown).
Enzyme activity. Citrate synthase activity in plantaris muscles
was determined as described previously
(34) in the entire group of
exercise-trained and control rats. Ouabain-sensitive, Na+- and
K+-stimulated hydrolysis of [
-32P]ATP was
determined as described by Feschenko and Sweadner
(14) with slight
modifications. Briefly, tissue homogenates were diluted in 10 mM Tris with 1
mM EDTA (pH 6.8, room temperature) to 150 µg/40 µl. After 10 freeze-thaw
cycles in dry ice-acetone, the homogenates were added to a buffer (0.16 ml
total) containing (in mM) 100 NaCl, 15 KCl, 5 MgCl2, 5
NaN3, 50 Tris (pH 7.8, room temperature), and 1 EGTA. After a 5-min
preincubation, the reaction was started by the addition of 5.5 mM
[-32P]ATP. Nonspecific enzyme activity was assayed in the
presence of 3 mM ouabain. The reaction was allowed to proceed for 25 min and
stopped with 0.5 ml of quenching solution (1 N sulfuric acid and 0.5% ammonium
molybdate). After 1 ml of isobutanol was added, the phosphomolybdate complex
was extracted into the organic phase by vigorous vortexing and separated by
centrifugation, and 0.5 ml of the extract was counted with a scintillation
counter. For each sample, the assay was performed in triplicate. Specific
activity is calculated as the difference between total and nonspecific
activity.
Separation of myosin isoforms. The method for separation of myosin isoforms is basically identical to that described by Yu et al. (49), with only slight modifications. Briefly, total tissue homogenates (0.075 µg/15 µl) were separated on 6% SDS gel (0.75 mm thick) at 15°C. Electrophoresis was performed at a constant voltage of 120 V for 24 h, and the gels were stained with silver stain (Amersham Pharmacia Biotech, Piscataway, NJ) immediately after electrophoresis. Relative intensity of the bands was quantitated by densitometry (Molecular Dynamics laser densitometer).
Statistical analysis of data. Values are means ± SE. Oneway ANOVA was used to compare group means, and Duncan's test was used for post hoc analysis. Data were examined at P < 0.05 and P < 0.10 to indicate statistical significance and trends, respectively.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
Effect of exercise training on subunit isoform expression.
Expression of the subunit isoforms was examined in red gastrocnemius, white
gastrocnemius, and extensor digitorum longus (EDL) muscles. Total tissue
homogenates were used in the present study to avoid unintentional selection of
subcellular pools. In red gastrocnemius muscle, levels of the
1-isoform increased in Ss rats compared with Ms rats
(Fig. 1). Expression of the
1-isoform in senescent rats further increased after exercise
training: 1.00 ± 0.06, 1.25 ± 0.08, and 1.44 ± 0.06
arbitrary densitometry units in Ms, Ss, and St rats, respectively. With age,
there was a statistically insignificant decrease in expression of the
2-isoform. Exercise training increased expression of the
2-isoform in St rats to levels significantly higher than
that in Ms and Ss rats: 1.00 ± 0.05, 0.88 ± 0.06, and 1.53
± 0.07 arbitrary densitometry units in Ms, Ss, and St rats,
respectively. Similar changes were observed in white gastrocnemius muscle
(Fig. 1), except there was a
strong trend for a decrease in the 2-isoform in Ss rats, and
exercise training failed to further increase the already elevated levels of
the 1-isoform in Ss rats (1: 1.00 ±
0.11, 1.77 ± 0.15, 1.83 ± 0.26 arbitrary densitometry units in
Ms, Ss, and St rats, respectively; 2: 1.00 ± 0.12,
0.74 ± 0.07, and 1.40 ± 0.05 arbitrary densitometry units in Ms,
Ss, and St rats, respectively). In EDL muscle, the changes are also similar to
that in the gastrocnemius muscle: there was a strong trend for an increase in
the 1-isoform in Ss rats, and exercise training
significantly increased the levels (1.00 ± 0.26, 2.01 ± 0.39,
and 2.70 ± 0.36 arbitrary densitometry units in Ms, Ss, and St rats,
respectively); there was a small, but significant, decrease in the
2-isoform in Ss rats, and exercise training reversed that
decrease (1.00 ± 0.06, 0.78 ± 0.05, and 0.98 ± 0.06
arbitrary densitometry units in Ms, Ss, and St rats, respectively).
|
Levels of the 1-isoform in red gastrocnemius muscle were
slightly increased in Ss rats compared with Ms rats, although the difference
was not statistically significant (Fig.
2). Exercise training markedly increased its expression in St rats
compared with Ms and Ss rats: 1.00 ± 0.22, 1.51 ± 0.19, and 4.69
± 0.45 arbitrary densitometry units in Ms, Ss, and St rats,
respectively. Expression of the 2-isoform, by contrast, was
markedly decreased in Ss rats compared with Ms rats, and exercise training
failed to reverse the decrease: 1.00 ± 0.10, 0.23 ± 0.07, and
0.23 ± 0.05 arbitrary densitometry units in Ms, Ss, and St rats,
respectively. Expression of the 3-isoform is very different
from expression of the 1- and 2-isoforms:
its levels significantly increased in Ss rats, and exercise training reversed
that increase (1.00 ± 0.16, 2.16 ± 0.17, and 1.44 ± 0.17
arbitrary densitometry units in Ms, Ss, and St rats, respectively), although
not completely.
|
In white gastrocnemius muscle, expression of -subunit isoforms is
almost identical to that in red gastrocnemius muscle
(Fig. 2): 1.00 ± 0.35,
1.04 ± 0.36, and 3.22 ± 0.37 arbitrary densitometry units for
the 1-isoform in Ms, Ss, and St rats, respectively; 1.00
± 0.19, 0.39 ± 0.18, and 0.25 ± 0.08 arbitrary
densitometry units for the 2-isoform in Ms, Ss, and St rats,
respectively; and 1.00 ± 0.30, 3.30 ± 0.29, and 1.63 ±
0.51 arbitrary densitometry units for the 3-isoform in Ms,
Ss, and St rats, respectively. In EDL muscle, expression of the
1- and 2-isoforms is also similar to that in
gastrocnemius muscle: 1.00 ± 0.18, 1.34 ± 0.30, and 3.51
± 0.55 arbitrary densitometry units for the 1-isoform
in Ms, Ss, and St rats, respectively, and 1.00 ± 0.16, 0.41 ±
0.13, and 0.43 ± 0.15 arbitrary densitometry units for the
2-isoform in Ms, Ss, and St rats, respectively. Because of an
insufficient amount of tissue samples, expression of the
3-isoform in EDL muscle was not examined.
Table 1 summarizes the above
data regarding expression of the subunit isoforms.
|
Na+-K+-ATPase enzyme activity. Ouabain-sensitive, Na+- and K+-stimulated ATPase enzyme activity was determined in total tissue homogenates. In red and white gastrocnemius muscle, there were no detectable changes in enzyme activity between Ms and Ss rats; however, enzyme activity increased significantly after exercise training (Fig. 3; red gastrocnemius: 21.63 ± 0.97, 22.51 ± 1.52, and 29.13 ± 0.37 µmol · mg protein-1 · h-1 in Ms, Ss, and St rats, respectively; white gastrocnemius: 44.93 ± 4.21, 45.14 ± 3.16, and 57.50 ± 3.30 µmol · mg protein-1 · h-1 in Ms, Ss, and St rats, respectively). In EDL muscle, enzyme activity was significantly higher in Ss than in Ms rats, and exercise training failed to further increase this elevated level of enzyme activity: 46.07 ± 2.41, 55.35 ± 3.04, and 58.45 ± 1.36 µmol · mg protein-1 · h-1 in Ms, Ss, and St rats, respectively.
|
Expression of the myosin heavy chain isoforms. Previous studies suggested a fiber type-dependent differential expression of the Na+-K+-ATPase subunit isoforms (22, 44). To examine whether the observed alterations in expression of subunit isoforms with age and exercise training can be correlated with muscle fiber type changes, relative abundance of myosin heavy chain isoforms was determined by SDS-PAGE. Overall, there was no statistically significant change in the level of the myosin heavy chain isoforms in red gastrocnemius, white gastrocnemius, and EDL muscles between Ms and Ss animals (Fig. 4). After exercise training, there was an increase in myosin isoforms I and IIX in red gastrocnemius muscle and a statistically insignificant decrease in IIB in St rats compared with Ss rats. In white gastrocnemius muscle, exercise training increased levels of myosin isoform IIX in St rats, with no significant change in IIB. In EDL muscle, myosin isoforms IIA and IIX appear to migrate close together and could not be resolved in the gel. Exercise training caused a significant decrease in the level of myosin isoform IIB, with no detectable change in the IIA/X in St rats.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
Our study shows that senescent Fischer 344 x Brown Norway rats can be trained to undergo a fairly vigorous endurance running paradigm. Because of the physical condition of the senescent rats, the animals were not trained at a higher intensity to avoid unreasonable loss of animals due to injury. It is important to note that the paradigm significantly increased citrate synthase enzyme activity in plantaris muscle of the senescent rats. In recruited skeletal muscle, citrate synthase activity is known to increase with endurance training. Therefore, increases in this activity provide a peripheral marker of skeletal muscle adaptation to exercise. Furthermore, our result showed a significant shift toward slower myosin isoforms in red and white gastrocnemius muscle after exercise training. These data strongly suggest that the senescent rats have received exercise training of sufficient intensity. Along with significant changes in expression of the Na+-K+-ATPase isoforms in white gastrocnemius muscle, our results suggest recruitment of this skeletal muscle under our training paradigm. This is somewhat unexpected, because previous studies suggested that recruitment of white gastrocnemius muscle occurs only when exercise training is at very high intensity (35, 36). Whether the advanced age of the rats is responsible for this apparent difference is unclear. On the other hand, we cannot completely eliminate the possibility that some general training effect was producing the training-induced changes. To limit the number of exercise-trained animals to a more manageable size, we chose not to include exercise-trained young or matured rats in this study. We believe that the simpler experimental design is justified, because in this study we specifically are interested in whether exercise training reverses the changes that occur during senescence, not during growth or maturation of the rats. Some important questions, such as whether exercise training has similar effects in young vs. old rats, cannot be answered by the present study.
In the present study, red gastrocnemius, white gastrocnemius, and EDL
muscles were examined, because they represent different muscle types. In the
rat, red gastrocnemius muscle consists of predominantly fast oxidative
glycolytic fibers mixed with oxidative fibers, white gastrocnemius muscle
consists of predominantly fast glycolytic fibers, and EDL muscle consists of
roughly equal amounts of fast oxidative glycolytic and fast glycolytic fibers.
We have detected qualitatively very similar, but less dramatic, age-associated
changes in expression of Na+-K+-ATPase than we reported
previously (41). In addition,
the previously observed age-associated increase in enzyme activity between
middle-aged and senescent rats is not apparent in this study, at least in red
and white gastrocnemius muscle (a significant increase was detected in EDL
muscle). The reason for these apparent differences is not clear. A careful
review of the history of the rat colonies reveals that our previous
experiments were performed using animals before the 1999 rederivation of the
Harlan colony. We speculate that the difference could be due to subtle
variations between the colonies. Lifespan data for the present colony are not
available (National Institute on Aging Office of Biological Resources and
Resource Development, personal communication). It is possible that a greater
magnitude of changes may be observed if older rats were used. In addition,
because the rats were housed and trained in Boulder, CO (altitude 1,650
m), an unexpected influence of high altitude on expression and activity of the
subunit isoforms cannot be excluded. Interestingly, Green et al.
(16) showed that
Na+-K+-ATPase in human skeletal muscle decreased after a
21-day expedition at high altitude. It must be emphasized, however,
qualitatively, that data from the present study and our previous study are in
excellent agreement. Furthermore, the small differences in no way obscure the
conclusions of this study.
As mentioned above, ouabain-sensitive Na+- and K+-stimulated ATPase enzyme activity in red and white gastrocnemius muscle was unchanged between Ms and Ss rats. By contrast, enzyme activity in EDL muscle increased with age, similar to our previously reported results. Exercise training increased Na+-K+-ATPase activity in red and white gastrocnemius muscle. Thus it seems unlikely that physical inactivity plays an important role in the increased enzyme activity that was detected in the present and previous studies.
Expression of the two -subunit isoforms in skeletal muscle responded
very differently to aging and exercise training. Because increasing physical
activity did not reverse the increased levels of the
1-isoform, we conclude that the age-associated increase in
the 1-isoform is unlikely to be due to physical inactivity.
Furthermore, endurance exercise training appears to elicit tissue-specific
differential effects in expression of the 1-isoform. The
mechanism(s) underlying this tissue-specific regulation of the
1-isoform is not clear. It is worth noting that the
age-associated changes occurred without significant switches in myosin
isoforms and that, despite a switch toward slower fibers in both tissues,
exercise training affects red and white gastrocnemius muscle differently. Thus
the tissue-specific effect of exercise training does not appear to be
primarily due to fiber type changes.
With regard to the 2-isoform, its levels in red and white
gastrocnemius muscle were not significantly different between Ms and Ss rats,
in accordance with our previously reported observations, even though a trend
toward decreased levels is evident. Indeed, the decrease in the
2-isoform in EDL muscle of Ss rats reached statistical
significance. Previously we observed decreased expression of the
2-isoform between young (6-mo-old) and middle-aged
(18-mo-old) rats (41). Thus,
collectively, advancing age is associated with decreased levels of the
2-isoform. Exercise training increased levels of the
2-isoform in senescent rats in all three muscle groups
examined. The reversal could be due to a switch of fiber types, because the
present study shows that exercise training increases the relative amount of
oxidative fiber and that expression of the 2-isoform is
higher in this fiber type. However, it appears unlikely that this is the
primary, or sole, reason, because data from our previous study and the present
study (2-isoform in EDL muscle) showed that decreased
expression of the 2-isoform with advancing age is not
dependent on fiber type changes.
How these changes in the Na+-K+-ATPase
-subunit isoforms ultimately affect function of the skeletal muscle in
aging and after endurance exercise training remains to be elucidated. The
-subunit isoforms appear to have different affinities for
Na+ and K+
(6,
13,
24), and the
1-isozyme is a better substrate for phosphorylation by
kinase (4,
14). Insulin and exercise
appear to selectively translocate the 2-isozyme from the
intracellular site to the plasma membrane
(21,
25). Importantly, He and
co-workers (18) recently
demonstrated that skeletal muscle from a mouse lacking one copy of the
1-isoform gene showed lower force than skeletal muscle from
a wild-type mouse. Conversely, mouse skeletal muscle lacking one copy of the
2-isoform gene showed greater force. Although it is
difficult to extrapolate data from a genetically manipulated animal model, it
may be speculated that an increase in 1-isoform level and a
decrease in 2-isoform level in skeletal muscle with
advancing age may result in altered contractile function.
Similar to the -subunits, the three -subunit isoforms also
exhibited unique adaptations in their expression during aging and in response
to exercise training. Our data demonstrated that expression of the
1-isoform is highly sensitive to exercise training. Whether
the increase is primarily due to an increase in oxidative fiber after exercise
training cannot be definitively determined in the present study, although our
previous study demonstrated that increased expression of the
1-isoform in aged skeletal muscle is not associated with a
significant switch in expression of myosin isoforms.
Levels of the 2-isoform decreased dramatically with age,
and exercise training failed to reverse or modify the decrease, making it the
only subunit isoform examined in the present study in which expression is not
modified by exercise training. The data suggest that factors other than
physical inactivity are important for its altered expression with age or that,
in aged skeletal muscle, expression of the 2-isoform lost its
responsiveness to increased physical activity. Levels of the
3-isoform increased in aged skeletal muscles, and exercise
training substantially reversed that increase. Thus physical inactivity may be
responsible, at least in part, for increased expression of the
3-isoform with aging. Expression of the
3-isoform in skeletal muscle is among the highest in the
various tissues examined (1).
Whether the 3-isoform performs a special function in skeletal
muscle is an interesting question to be explored in future studies. It remains
to be determined in skeletal muscle whether the -subunit isoforms play
different roles in the assembly of the -complex or during
differentiation of muscle cells. The age-associated changes in levels of
2- and 3-isoforms may be a compensatory
response to aging of skeletal muscle or may signify a pathological
maladaptation process. However, lower levels of the 3-isoform
in younger rats and reversal of increased levels of the
3-isoform in aged skeletal muscle by exercise training seem
to suggest that, in rat skeletal muscle, a low level of the
3-isoform is preferred.
In skeletal muscle of exercise-trained senescent rats, as the result of a
marked increase in the 1-isoform, a decrease in the
3-isoform, and unchanged levels of the
2-isoform, a significant shift toward more
1-isozyme may be expected, if the elevated levels of
-subunits indeed combine with -subunits to form functional units.
Interestingly, Klip and co-workers demonstrated that intracellular membranes
of red skeletal muscles contain primarily 2- and
1-isoforms
(28) and that insulin
stimulates their translocation from the internal membrane to the plasma
membrane (21). Thus one may
speculate that levels of the 21-type
Na+ pump in the internal membrane may be elevated in aged skeletal
muscle after exercise training. Such an elevated pool of intracellular
Na+ pump may boost availability of the plasma membrane
Na+ pump when there is acute demand. In addition, it appears that
isozymes with the 2-isoform have lower apparent affinity for
K+ (12) and higher
affinity for Na+ (5,
7). Thus the age-associated
decrease in the 2-isoform could significantly affect the
kinetics of ion transport in aging skeletal muscle.
Helwig et al. (19)
demonstrated exercise-induced tissue- and isoform-specific alterations in
expression of the Na+-K+-ATPase subunit isoforms in rats
with myocardial infarction. In their study, exercise training failed to alter
expression of all the subunit isoforms examined, except the
2-isoform. The reason(s) for these apparent differences
between their study and ours is not clear but could be due to a difference in
the exercise-training paradigm or, perhaps more interestingly, could suggest
some fundamental differences in responses of the subunit isoforms to exercise
training between aged rats and rats with heart failure.
Cellular mechanisms responsible for the exercise-induced altered expression
of the subunit isoforms in senescent rats remain unclear. Previous study in
young rats demonstrated that an acute bout of exercise increased mRNA content
of 1- and 2-isoforms, but not
2- and 1-isoforms
(45). It was speculated that
an increase in intracellular Na+ content could have played a role.
Indeed, we and others reported that elevation of intracellular Na+
in various cell types in culture increased expression of the subunit isoforms
and/or pump units (26,
43,
47). Nevertheless, it is
interesting to note that, in our study using the skeletal muscle
C2C12 cell line, increased Na+ transport is
associated with increased expression of the 2-isoform but
not the 1-isoform. These data suggest that the response of
skeletal muscle cells in culture to the change in Na+ is different
from that of intact skeletal muscle or that a mechanism(s) other than changes
in intracellular Na+ plays an important role in expression of the
subunit isoforms. Cellular mechanisms responsible for the divergent responses
of the three -subunit isoforms clearly cannot be explained by changes in
intracellular Na+ alone and remain to be elucidated.
In conclusion, aging of skeletal muscle is associated with distinct patterns of alterations in expression of the Na+-K+-ATPase subunit isoforms, and exercise training differentially modifies these age-associated alterations. Because endurance exercise training was able to reverse only some of the age-associated changes, physical inactivity appears to play, at most, only a partial role in the age-associated changes. Future investigations will explore the role of other well-known aging-related factors, including hormonal changes and oxidative stress, in altering expression of the Na+-K+-ATPase subunit isoforms during aging.
|
DISCLOSURES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
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.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
3 subunit: the presence of 3 in lung and
liver addresses the problem of the missing subunit. J Biol
Chem 272:
22405-22408, 1997.2-but not
1-isoform abundance in heart, muscle, and brain.
Am J Physiol Cell Physiol 260:
C958-C964, 1991.2- and 2-isoforms. Am J Physiol
Cell Physiol 265:
C680-C687, 1993.1 but not 2
or 3 isoforms of Na,K-ATPase are efficiently phosphorylated
in a novel protein kinase C motif. Biochemistry
35: 14098-14108,
1996.[Medline]
21 and
22 isozymes of the Na,K-ATPase.
Biochemistry 34:
319-325, 1995.[Medline]
3-1 and
3-2 isozymes.
Biochemistry 34:
9897-9903, 1995.[Medline]
1- and 2-isoforms of Na-K-ATPase play
different roles in skeletal muscle contractility. Am J Physiol
Regul Integr Comp Physiol 281:
R917-R925, 2001.2 and 1 subunits of
the Na+/K+-ATPase from intracellular compartments to the
plasma membrane in mammalian skeletal muscle. J Biol
Chem 267:
5040-5043, 1992. subunit
isoforms of the Na+,K+-ATPase is muscle type-specific.
FEBS Lett 328:
253-258, 1993.[Web of Science][Medline]
1, 2, and 3
isoforms expressed in HeLa cells. J Biol Chem
266: 16925-16930,
1991.-subunit isoforms in
C2C12 skeletal muscle cell. Mol Cell
Biochem 211:
79-84, 2000.[Web of Science][Medline]
and
subunits of the Na-K-ATPase differ in distinct subcellular membranes
from rat skeletal muscle. Biochemistry
36: 7726-7732,
1997.[Medline]
3 subunit. J Biol
Chem 271:
22754-22758, 1996.-subunit gene is expressed in testis. Proc Natl Acad Sci
USA 91:
12952-12956, 1995.
and subunit
protein levels respond to hypokalemic challenge with isoform and muscle type
specificity. J Biol Chem 271:
32653-32658, 1996.This article has been cited by other articles:
|
|
 |
|
  A. C. Betik, D. J. Baker, D. J. Krause, M. J. McConkey, and R. T. Hepple Exercise training in late middle-aged male Fischer 344 x Brown Norway F1-hybrid rats improves skeletal muscle aerobic function Exp Physiol, July 1, 2008; 93(7): 863 - 871. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. J. Green, T. A. Duhamel, R. D. Stewart, A. R. Tupling, and J. Ouyang Dissociation between changes in muscle Na+-K+-ATPase isoform abundance and activity with consecutive days of exercise and recovery Am J Physiol Endocrinol Metab, April 1, 2008; 294(4): E761 - E767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. J. Green, T. A. Duhamel, G. P. Holloway, J. W. Moule, J. Ouyang, D. Ranney, and A. R. Tupling Muscle Na+-K+-ATPase response during 16 h of heavy intermittent cycle exercise Am J Physiol Endocrinol Metab, August 1, 2007; 293(2): E523 - E530. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Li, L. I. Sinoway, and Y.-C. Ng Aging augments interstitial K+ concentrations in active muscle of rats J Appl Physiol, April 1, 2006; 100(4): 1158 - 1163. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Reis, L. Zhang, S. Cala, K. N. Jew, L. C. Mace, L. Chung, R. L. Moore, and Y.-C. Ng Expression of phospholemman and its association with Na+-K+-ATPase in skeletal muscle: effects of aging and exercise training J Appl Physiol, October 1, 2005; 99(4): 1508 - 1515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Barr, H. J. Green, D. S. Lounsbury, J. W. E. Rush, and J. Ouyang Na+-K+-ATPase properties in rat heart and skeletal muscle 3 mo after coronary artery ligation J Appl Physiol, August 1, 2005; 99(2): 656 - 664. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. J. Green, D. J. Barr, J. R. Fowles, S. D. Sandiford, and J. Ouyang Malleability of human skeletal muscle Na+-K+-ATPase pump with short-term training J Appl Physiol, July 1, 2004; 97(1): 143 - 148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C. Mace, B. M. Palmer, D. A. Brown, K. N. Jew, J. M. Lynch, J. M. Glunt, T. A. Parsons, J. Y. Cheung, and R. L. Moore Influence of age and run training on cardiac Na+/Ca2+ exchange J Appl Physiol, November 1, 2003; 95(5): 1994 - 2003. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Clausen Effects of age and exercise training on Na+-K+ pumps in skeletal muscle Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2003; 285(4): R720 - R721. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
