1 Faculty of Biology, University of Konstanz, D-78457 Konstanz, Germany; and 2 Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, France
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ABSTRACT |
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Time-dependent changes in myosin heavy chain
(MHC) isoform expression were investigated in rat soleus muscle
unloaded by hindlimb suspension. Changes at the mRNA level were
measured by RT-PCR and correlated with changes in the pattern of MHC
protein isoforms. Protein analyses of whole muscle revealed that MHCI
decreased after 7 days, when MHCIIa had increased, reaching a transient maximum by 15 days. Longer periods led to inductions and progressive increases of MHCIId(x) and MHCIIb. mRNA analyses of whole muscle showed
that MHCIId(x) displayed the steepest increase after 4 days and
continued to rise until 28 days, the longest time period investigated.
MHCIIb mRNA followed a similar time course, although at lower levels.
MHCI mRNA, present at extremely low levels in control soleus, peaked
after 4 days, stayed elevated until 15 days, and then decayed.
Immunohistochemistry of 15-day unloaded muscles revealed that MHCI
was present in muscle spindles but at low amounts also in extrafusal
fibers. The slow-to-fast transitions thus seem to proceed in the order
MHCI
MHCIIa
MHCIId(x)
MHCIIb. Our
findings indicate that MHCI
is transiently upregulated in some
fibers as an intermediate step during the transition from MHCI
to MHCIIa.
hindlimb suspension; messenger ribonucleic acid; myosin heavy chain isoforms; reverse transcriptase-polymerase chain reaction; slow-to-fast transition
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INTRODUCTION |
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SKELETAL MUSCLE has the capacity of changing its
phenotype in response to altered functional demands. This
"plasticity" has been documented in numerous studies, using
various experimental protocols of increased and decreased mechanical
loading, as well as enhanced or reduced contractile activity. Among
these protocols, chronic low-frequency stimulation (CLFS) of
fast-twitch muscle and unloading of slow-twitch muscle by hindlimb
suspension represent extremes, leading to muscle fiber type transitions
in opposite directions. CLFS induces fast-to-slow transitions, whereas
unloading evokes slow-to-fast transitions (for reviews, see Refs. 1, 22, 30). The latter have become a challenging topic of muscle research
in view of prolonged exposition to weightlessness under the conditions
of spaceflight. At the level of myofibrillar proteins, these adaptive
fast-to-slow and slow-to-fast responses encompass exchanges of fast
with slow and slow with fast protein isoforms, respectively. As shown
by whole muscle and single fiber analyses, these changes are clearly
reflected by sequential transitions in myosin heavy chain (MHC) isoform
expression (17, 23). In low-frequency stimulated fast rat muscle, MHC
isoform transitions generally follow the order of MHCIIb MHCIId(x)
MHCIIa
MHCI
(16). In rabbit, the MHCIIa
MHCI
transition includes a transient upregulation of the
MHCI
isoform (23, 24). In rat, however, the MHCIIa
MHCI
transition is difficult to attain. It occurs only after very long
stimulation periods (15, 35), and this may explain that, under our
experimental conditions, an upregulation of MHCI
has as yet not been
observed (26). The difficulty to attain in rat muscle the MHCIIa
MHCI
transition, the ultimate step of the fast-to-slow
transformation, appears to be in contrast to the facility to elicit the
opposite change, namely the MHCI
MHCIIa transition by
unloading rat slow muscle (3, 6, 19, 30). Studies at the mRNA and
protein levels suggest that, under these conditions, the switch from
MHCI
to MHCIIa is followed by further shifts toward other fast MHC
isoforms, i.e., MHCIIa
MHCIId(x)
MHCIIb (12, 14). To
our knowledge, the time course of transitions in the expression of MHC
mRNA isoforms has been followed in unloaded rat soleus muscle only in a
single study based on Northern blot hybridization (12). However, the
question of MHC
upregulation was not addressed.
The present study was undertaken to investigate in more detail the time
course and complete MHC transition profile when a slow muscle turns
fast. In this context, we were interested whether MHCI is a member
of the transition profile as in the rabbit. We quantitatively assessed
changes in mRNA and protein levels of the various MHC isoforms in rat
soleus muscle unloaded for 4, 7, 15, and 28 days by hindlimb
suspension. Quantitative changes in the transcript levels of MHCI
,
MHCI
, MHCIIa, MHCIId(x), and MHCIIb were assessed by highly
sensitive RT-PCR (16, 26) and correlated with quantitative changes in
the pattern of MHC protein isoforms.
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MATERIALS AND METHODS |
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Animals and muscles. Adult male Wistar
rats (initial body wt 280 g) were randomly divided into two groups:
control and hindlimb suspension unloading groups. The animal
experiments, as well as the animal maintenance conditions, were
approved by the French Ministries of Agriculture and Education
(veterinary service of health and animal protection, authorization no.
03805). Hindlimb suspension was performed as previously described (28).
After various periods of hindlimb suspension (4, 7, 15, and 28 days), the animals (5-7 for each time point) were anesthetized by
intraperitoneal injection of ethylcarbamate and killed by
exsanguination, and soleus muscles from both control and
hindlimb-suspended rats were removed. The muscles were weighed (Table
1), frozen in liquid N2, and stored at 70°C
until analyzed. Frozen muscle tissue was pulverized under liquid
N2 in a small steel mortar. One
part of the muscle powder was used for MHC mRNA analysis, and the other was used for MHC protein analysis.
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Preparation of total RNA. Muscle powder was homogenized (1:10, wt/vol) in cold TRI reagent (Molecular Research Center, Cincinnati, OH). Total RNA was isolated according to the producer's instructions for RNA preparation using the following modifications: 1) after homogenization, proteins and insoluble material were removed by 10 min centrifugation at 12,000 g at 4°C; 2) phase separation was performed using 1-bromo-3-chloropropane (Fluka, Buchs, Switzerland); and 3) isopropanol (0.125 ml) and 0.125 ml of a solution containing 1.2 M sodium citrate and 0.8 M NaCl were used for RNA precipitation. Pellets were resuspended in 30 µl of diethyl pyrocarbonate-treated water. RNA concentration (µg/µl) was assessed spectrophotometrically.
Oligonucleotide primers.
Oligonucleotide primers specific to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) and -skeletal actin, MHCIIb, MHCIId(x),
MHCIIa, MHCI
(16), and MHCI
(26) were derived from published cDNA
sequences. The 5'-ends of sense primers were labeled with
digoxigenin to allow chemiluminescence detection of the amplified products.
RT-PCR. Total RNA stock solution (2 µg) was reverse-transcribed (25) in a 20-µl volume using the
following assay mixture: RT buffer (50 mM Tris · HCl,
pH 8.5, 8 mM MgCl2, and 30 mM
KCl), 20 units avian myoblastosis virus RT, 40 units RNase inhibitor (Roche), 2.5 µM primer p(dT)15
(Roche), and each 0.625 mM deoxynucleoside triphosphate (dNTP;
Pharmacia). Incubation was performed for 60 min at 42°C. Two
microliters of the 1:5 diluted RT assay were amplified separately for
each of the seven sequences and transferred in 23 µl of the following
PCR incubation mixture: PCR buffer [50 mM
Tris · HCl and 15 mM
(NH4)2SO4],
specific antisense and sense primers (0.2 µM), dNTPs (0.25 mM), and
0.63 units Expand High-Fidelity polymerase (Roche). The optimized
MgCl2 concentrations were 2 mM for
GAPDH, -skeletal actin, MHCI
, and MHCIIa and 2.5 mM for MHCI
,
MHCIId(x), and MHCIIb. For amplification, the following conditions were chosen: denaturation at 94°C, annealing at
59°C, and synthesis at 72°C. The number of cycles was adjusted
to allow product detection in the exponential range of amplification.
Cycle numbers were 13 for
-skeletal actin, 18 for GAPDH, and varied according to their expression levels from 17 to 26 for the different MHC sequences. To allow quantitative analysis of gene expression, the
amplification reactions were monitored using cDNA standards. For this
purpose, PCR fragments were purified from primers, nucleotides, and
nonspecific reaction products using the QIAEX DNA gel extraction procedure (QIAGEN) after electrophoretic separation on 1.5% agarose gels (25). The purified DNA was spectrophotometrically quantified, and
known amounts of each sequence were amplified in parallel to the
samples. The calculated first-strand copy numbers of standard cDNA
fragments were 105, 4 × 105, and
106 molecules for GAPDH and
-skeletal actin; 103, 4 × 103, and
104 molecules for MHCI
; 4 × 105,
106, and
107 molecules for MHCI
;
104,
105, and
106 molecules for MHCIIa; and
103,
104, and
105 molecules for MHCIIb and MHCIId(x).
Product analysis and quantitative evaluation. The digoxigenin-labeled DNAs were electrophoretically separated on 6% polyacrylamide gels and visualized after electroblotting (Hybond N; Amersham) by an antibody-linked assay followed by a peroxidase-catalyzed chemiluminescence reaction (Roche). The signals were photographically documented (Hyperfilm ECL; Amersham) and evaluated by integrating densitometry. At least two measurements were performed on each sample (animal and time points).
Immunohistochemistry. A monoclonal
antibody directed against MHCI (clone F-88 12F8,1) was from Biocytex
(Marseille, France). The following monoclonal antibodies directed
against adult MHC isoforms were also used: 7HCS-15 (specific to MHCI)
and SC-71 (specific to MHCIIa). Biotinylated horse anti-mouse IgG
(rat-absorbed, affinity-purified) was from Vector Laboratories
(Burlingame, CA). Freshly cut, 9-µm-thick, frozen sections were
air-dried at room temperature for 2 h. Sections were washed in PBS
containing 0.1% Tween 20 and in PBS alone. They were then incubated
for 15 min in 3%
H2O2
in methanol, washed, and incubated for 2 h in a blocking solution (1%
BSA, 10% horse serum, and 0.1% Tween 20 in PBS, pH 7.4). The primary
monoclonal antibody was applied overnight at 4°C. Primary mouse IgG
monoclonal antibodies were used as undiluted culture supernatants
(F-88) or were diluted in blocking solution (7HCS-15, 1:40; SC-71,
1:1,000). Control sections were processed in parallel incubations in
which the primary antibody was substituted with nonspecific control
mouse IgG. After 30 min of incubation with biotinylated horse
anti-mouse IgG (diluted 1:200 in blocking solution), sections were
washed and incubated for 30 min with biotin-avidin horseradish
peroxidase complex (Vectastatin Elite; Vector Laboratories). Staining
solution (0.07% diaminobenzidine, 0.05%
H2O2,
and 0.03% NiCl2 in 50 mM
Tris · HCl, pH 7.5 ) was applied for 4 min.
MHC protein electrophoresis. MHC isoforms were analyzed as previously described (13). Gels were silver-stained, and relative concentrations of MHC isoforms were evaluated by integrating densitometry. At least two independent measurements were performed on each sample.
Statistical analyses. Data are presented as means ± SD. All data were analyzed using Student's t-test to determine differences between values from control and unweighted muscles. The acceptable level of significance was set at P < 0.05.
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RESULTS |
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Changes in MHC protein
isoforms. Unloading of soleus muscle up to 28 days led
to progressive loss of weight (Table 1) and changes in the pattern of
the electrophoretically separated MHC isoforms (Figs.
1 and 2). MHCI,
the predominant isoform in normal soleus muscle (~95%), decreased
within the first week of unloading, reaching a relative concentration
of ~75% at day 7. MHCIIa, which represented 5% of total MHC isoforms in control soleus, was elevated by day 4 and reached a maximum of
~25% after 1 wk. With longer periods of unloading, when MHCIId(x)
and also MHCIIb started to rise, MHCIIa decayed. Thus 28-day
unloaded soleus muscle exhibited a thoroughly rearranged MHC isoform
pattern in which all fast isoforms were elevated at the expense of
MHCI. Even though MHCI remained the predominant isoform (~70%) in
28-day unloaded soleus muscle, MHCIId(x) now represented the major fast
isoform (~18%). MHCIIa and MHCIIb amounted to ~8 and 3%,
respectively.
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Under the applied conditions of electrophoresis, only a single MHCI
band was separated, such that MHCI and MHCI
could not be
distinguished. Therefore, changes in their proportions due to unloading
were not detected. An interesting observation was that MHCIa, an
additional slow MHC isoform recently described and electrophoretically
characterized by a mobility higher than MHCI
(8, 9), seemed to be
present at elevated levels in the 15- and 28-day unloaded muscles (Fig.
1). However, this band was not separated well enough in all samples to
allow reproducible densitometric evaluation. Probably, MHCIa was only
separated when the MHCI band was less bulky in the unloaded muscles.
Changes in MHC mRNA isoforms. To
assess mRNA concentrations by molecule number, RT-PCR were performed
using specific primers for the various MHC isoforms, as well as for
-skeletal actin and GAPDH. Known amounts of purified PCR fragments
were used as external DNA standards for quantitative evaluations of
molecule numbers (25).
-Skeletal actin and GAPDH mRNAs served as
controls to assess the validity of the method.
Quantitative evaluations of the data from several animals at different
time points of hindlimb suspension are summarized in Figs.
3 and 4.
Over the time period under study, the level of GAPDH mRNA remained
stable (Fig. 3). Conversely, the amount of the -skeletal actin mRNA
decreased in muscles unloaded for longer than 7 days. MHCI mRNA, the
predominant isoform in control soleus (2.42 ± 0.26 × 108 molecules/µg total RNA),
tended to decline in the unloaded muscle, especially during the second
week, although the decreases attained at 15 and 28 days were not
significant. Very low concentrations of MHCI
mRNA (0.5 ± 0.2 × 105 molecules/µg total
RNA) were detected in control soleus, but, after 4 days of unloading,
MHCI
mRNA was markedly elevated (2.7 ± 1.1 × 106 molecules/µg total RNA). It
thus attained a similar level as MHCIIb mRNA in the 28-day unloaded
muscle (Fig. 4). However, the rise of MHCI
mRNA was transitory, and
it started to decay after 15 days.
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The three fast MHC mRNA isoforms were already elevated in the 4-day unloaded muscle. MHCIIa mRNA, present at 1.8 ± 0.2 × 107 molecules/µg total RNA in the control, had increased threefold by 15 days (Fig. 3). With prolonged unloading (28 days), however, MHCIIa mRNA decayed, attaining its basal level. Among all MHC isoforms, MHCIId(x) mRNA displayed the steepest rise within the first 4 days (Fig. 4). The low amount in control soleus (2.5 ± 0.26 × 105 molecules/µg total RNA) had increased ~40-fold after 4 days. Thereafter, it continued to rise, but at a lower rate, being ~60-fold elevated (1.5 ± 0.08 × 107 molecules/µg total RNA) over control in 28-day unloaded soleus muscle. Elevated transcript levels were also observed for MHCIIb (Fig. 4). Its basal level in control soleus (2.0 × 105 molecules/µg total RNA) was in the same range as that of MHCIId(x) mRNA. It was elevated after 4 days and continued to rise, reaching an ~13-fold increase in 28-day unloaded soleus muscle (2.7 ± 0.3 × 106 molecules/µg total RNA).
A summary of the major alterations in MHC isoform expression at the
level of relative MHC mRNA concentrations is given in Fig.
5. The temporal pattern of the changes in
relative magnitudes resembles that shown for the proteins in Fig. 2.
The only exception is MHCIIa peaks by 15 days, at a time when its
protein has started to decay.
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Immunohistochemical findings. To
investigate the histological distribution of MHCI expression, we
performed immunohistochemical stainings with an antibody previously
proven to be highly specific to MHCI
(24). As expected, MHCI
expression was restricted in control soleus muscle to the muscle
spindles (results not shown). However, 15-day unweighted soleus muscles
displayed an additional weak reaction in extrafusal fibers. In
comparison with MHCIIa and MHCI
, however, the staining intensity for
MHCI
was very low, even with the highly sensitive method of
enhancement used. Nevertheless, not all fibers were equally reactive.
The predominant MHC complement of those fibers displaying weak MHCI
reactivity was determined by immunohistochemical stainings of serial
sections. As can be seen, MHCI
expression was observed in
MHCIIa-positive fibers (Fig. 6,
fiber 1) and in MHCI
-positive
fibers (Fig. 6, fiber 2).
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DISCUSSION |
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As first shown by Vrbová (33) in 1963, unloading of rabbit soleus by tenotomy turns this slow muscle into a faster-contracting muscle. Numerous studies on rats have since confirmed that mechanical unloading of soleus muscle induces slow-to-fast transitions (22, 30). Besides alterations in contractile properties (28), these transitions are best illustrated by changes in the pattern of MHC isoforms (4, 6-8, 18-20, 27, 29). Collectively, these studies have demonstrated increases in the relative concentration of MHCIIa and an induction of MHCIId(x) that is normally not expressed in soleus muscle of adult rats. A few studies even reported the additional appearance of low amounts of MHCIIb under these conditions (2, 5, 8).
As judged from whole muscle analyses, the slow-to-fast conversion in response to unloading seems to encompass a similar sequential order in MHC isoform exchanges, although in the opposite direction, as observed during the fast-to-slow transition. However, the changes during unloading are less pronounced than those during enhanced neuromuscular activity (16). This may be due to the fact that two processes occur in parallel during hindlimb suspension: 1) altered gene expression and 2) muscle atrophy. Especially in the case of isoforms that are upregulated in the unloaded muscle, their increases at both the mRNA and protein levels are attenuated or obscured by the atrophy of the muscle. The pronounced atrophy (Table 1) could explain the observation that MHCIIa protein starts to decrease at a time when its mRNA is still rising. Another difference between the two models is that MHCIIa is persistently upregulated in muscles undergoing fast-to-slow transition, whereas it is only transiently elevated during slow-to-fast transition. Thus fast-to-slow conversion leads to an accumulation of MHCIIa after downregulation of MHCIIb and MHCIId(x) (16), whereas expression of MHCIIa in the unloaded muscle represents an early stage of the transformation process.
As judged from the time course of the increases in MHCIIa and MHCIId(x) mRNAs, it appears that the upregulation of these two mRNA isoforms occurs in synchrony rather than sequentially. A similar temporal relationship between the changes in MHCIId(x) and MHCIIa mRNAs has been observed during fast-to-slow conversion in rat muscle (16) and also in rabbit muscle, where single fiber analyses identified numerous fibers coexpressing up to four MHC mRNA isoforms. The observation that increases in MHC isoforms, which seem to occur in parallel at the mRNA level, differ at the protein level could point to posttranscriptional regulation. However, one also must consider that the changes at the protein level are given as relative concentrations. Thus the relative increase in MHCIIa protein (Fig. 2) may not be due to enhanced translation but obviously results from the decrease in MHCI protein.
The parallel increases in MHCIIa and MHCIId(x) mRNAs raise the question as to their upregulation within the same or in different fibers. The possibility exists that analyses of whole muscle RNA preparations reveal parallel MHCIIa and MHCIId(x) mRNA increases because type I fibers start to express MHCIIa at the same time when preexisting type IIA fibers start to upregulate MHCIId(x). Therefore, single fiber analyses are necessary to further investigate this question. Such studies would answer the question as to the homogeneity or heterogeneity of myonuclear expression patterns within individual fibers.
The increases in MHCIId(x) and MHCI are the most conspicuous changes
in the MHC mRNA pattern. MHCIId(x) mRNA exhibits the steepest
increase, ultimately even exceeding the transcript level of MHCIIa in
control soleus. Contrary to findings based on Northern blot
hybridizations (12), the rise in MHCIId(x) is not transitory but
persists under our experimental conditions. Similarly, MHCIIb mRNA
continues to rise during the whole time period under study.
The detection of the MHCI transcript in total RNA preparations from
unweighted soleus muscle raises the question as to the site of its
enhanced expression. MHCI
is normally expressed only in intrafusal
fibers of limb muscles, especially the
bag2 type (21). This is consistent
with the very low amounts of MHCI
mRNA in normal soleus muscle and
the finding that the immunohistochemical reaction for MHCI
is
restricted to spindles. The immunohistochemical detection of low
amounts of MHCI
in some type I and type IIA fibers of 15-day
unloaded soleus muscle, however, shows that this isoform is expressed
in some fibers of transforming rat muscle, although not as a major MHC
isoform compared with the rabbit (24).
Different experimental models such as microgravity, hindlimb suspension, tenotomy, and denervation elicit in soleus muscle comparable slow-to-fast transitions (1, 22, 30). Their common denominator could be the reduction of the tonic impulse pattern normally delivered to the soleus muscle by its nerve (32). Contractile activity and specific neural impulse patterns are regarded as major regulatory factors in the control of MHC isoform expression and other phenotype properties in adult skeletal muscle (10, 11, 33, 34).
Taken together, the changes in expression levels of MHC mRNA and
protein isoforms in unloaded rat soleus muscle reflect slow-to-fast transitions in the order MHCI
(MHCI
)
MHCIIa
MHCIId(x)
MHCIIb. Contrary to the rabbit, the
upregulation of MHCI
does not seem to be a major stage of the
transformation process in rats, and probably, the intermediate
expression of MHCI
does not occur in all fibers.
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ACKNOWLEDGEMENTS |
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This study was supported by the Deutsche Forschungsgemeinschaft, grant Pe 62/25-3. L. Stevens is the recipient of a stipend from the Alexander von Humboldt Foundation.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. Pette, Faculty of Biology, Univ. of Konstanz, D-78457 Konstanz, Germany (E-mail: dirk.pette{at}uni-konstanz.de).
Received 14 May 1999; accepted in final form 5 August 1999.
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