Hypergravity from conception to adult stage: effects on contractile properties and skeletal muscle phenotype
1 Laboratoire de Plasticité Neuromusculaire, UPRES EA 1032, IFR 118,
Bâtiment SN4, Université des Sciences et Technologies de Lille,
59655 Villeneuve d'Ascq cedex, France
2 Laboratoire de Neurobiologie des Restaurations Fonctionnelles, CNRS
Unité Mixte de Recherche 6562, Université de Provence, 52,
Faculté de St Jérôme, 13397 Marseille cedex 20,
France
* Author for correspondence (e-mail: cyelboz{at}hotmail.com)
Accepted 7 May 2004
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Summary |
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Key words: 2G-centrifugation, skinned fiber, rat, soleus, plantaris, contractile and regulatory protein, electrophoresis, RT-PCR
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Introduction |
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Less is known about the adaptation of the muscular system under
hypergravity (HG) conditions, particularly in terms of contractile protein
isoform transitions, and/or Ca2+ activation properties. Most
studies have examined rats born in normal gravity and placed as adults into an
HG environment (Amtmann and Dyama,
1976; Roy et al.,
1996
; Chi et al.,
1998
; Stevens et al.,
2003
). For instance, adult rats exposed for 14 days
(Roy et al., 1996
) or 19 days
in HG (Stevens et al., 2003
)
led to partial slow-to-fast transformation of the slow soleus phenotype
without alteration of the developed forces. The phenotype transformation was
characterized by an increase in fast isoform contents of TnC and TnI in
relation to a higher cooperativity along the thin filament (steeper
Tension/pCa relationship), whereas fast muscle was not (plantaris) or less
(gastrocnemius) modified.
Few experiments have reported the effects of hypergravity on rats reared in
HG. Martin (1980) has shown an
elevation in slow oxidative fibers in soleus and plantaris muscles of 30-day
old rats centrifuged at 2 g for 2 weeks. In the rectus capitis
muscle (responsible for head posture) of rats conceived, born and reared until
the seventh postnatal day at 1.8 g centrifugation, Martrette
et al. (1998
) showed an
increase in slow and perinatal MHC isoform expressions. In a recent study on
rats reared in HG (Picquet et al.,
2002
), we showed that the slow soleus muscle studied in
situ presented an increase in maximal and twitch tensions and was changed
into a slower type, for both contractile parameters and MHC isoform content,
while the fast plantaris was less affected.
Therefore the results obtained under lifelong conditions of hypergravity, when compared to both microgravity and hypergravity applied to adults, appeared opposite in terms of generated forces and MHC isoform transitions. This led us to characterize the effects of HG on Long Evans rats conceived, born and reared in hypergravity (2 g) conditions, in order to understand how a complete life in HG could affect the generated force in relation to the muscle protein transformations. Two muscles were studied: the soleus muscle (a predominantly slow ankle extensor) and its fast agonist, the plantaris.
For this purpose, the different objectives of this work were: (1) analysis,
at the cellular level, of the Ca2+ activation characteristics in
relation to the forces developed by single fibers, in order to complete the
study of Picquet et al.
(2002), who showed, at the
whole muscle level, a reduction of the absolute twitch and maximal tetanic
forces, as well as a decrease in fiber cross sectional area after HG. (2) To
study more deeply phenotype acquisition in muscles continuously exposed to 2
g. The analysis in Picquet et al.
(2002
) was limited to the MHC,
so to obtain a more complete understanding of the isoform transitions
occurring after HG, we studied the three subunits of the troponin molecule
(TnC, TnT and TnI, contributing to the regulation of Ca2+
activation properties) and the set of MLC, especially the regulatory MLC2,
known to modulate the Ca2+ sensitivity of the contractile proteins
by its phosphorylation state (Sweeney and Stull, 1996). Slow-to-slower MHC
changes in contractile properties have already been described in CBR soleus
muscle in situ (Picquet et al.,
2002
), so a study of MLC2 phosphorylation could contribute to our
knowledge of the regulation of this phenotypical modification. Indeed, we have
recently reported an increase of MLC2 phosphorylation associated with
slow-to-fast transitions induced by hindlimb unloading in rat soleus muscle
(Bozzo et al., 2003
). Thus, we
hypothesize that the slow-to-slower characteristics acquired after HG were
associated with a decrease in MLC2 phosphorylation. (3) To estimate by reverse
transcription polymerase chain reaction (RT-PCR) analyses, the gene
regulation of the MHC slow-to-slower transition observed previously
(Picquet et al., 2002
).
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Materials and methods |
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The experiments and the maintenance conditions of the animals were approved by both the Agricultural and Forest Ministry and National Education Ministry (authorization 5900996).
Hypergravity conditions
Hypergravity was obtained in the same centrifugation apparatus as the one
previously used by Picquet et al.
(2002) and described by
Gustave Dit Duflos et al.
(2000
). The centrifuge
consisted of a velocity-controlled DC motor located in the vertical axis of
the apparatus and driving two horizontal cross-arms (length 165 cm) at
constant rotation speed. Four free-swinging gondolas were jointed at the four
extremities of the horizontal arms, 76.5 cm away from the axis of rotation;
the gondolas were equipped with lights that reproduced a 12 h:12 h light:dark
cycle, and standard home cages for rats. One gondola carried a video system
used to observe animal birth and behavior. During centrifugation, the gondolas
were tilted at a constant angle of 60° depending on the centripetal
acceleration for 2 g hypergravity environment.
Counterclockwise rotations were achieved at constant velocities of 3.81 rad
s1. Given the mass and the inertia of the gondolas,
including the home cages and the rats, these angular velocities led to a 2
g resultant force whose direction was always similar to that
exerted in normal gravity (directed dorsoventrally, orthogonal to the
anteroposterior axis of the animal).
Solutions
All reagents were provided by Sigma (St Louis, USA). The composition of all
the solutions was calculated as previously described
(Mounier et al., 1989). Final
ionic strength was 200 mmol l1. Activating solutions
contained MOPS, 10 mmol l1, potassium propionate (KProp, 185
mmol l1), magnesium acetate (MgAc, 2.5 mmol
l1) and various concentrations of free Ca2+ from
CaCO3, buffered with EGTA and added in proportions to obtain
different pCa values (6.84.2, where pCa=log[Ca2+]).
The pSr (pSr=log[Sr2+]) solutions were similar to the pCa
solutions but with free Sr2+ from SrCl2. Relaxing
solution (R) was identical to the skinning solution and composed of 10 mmol
l1 MOPS, 170 mmol l1 KProp, 2.5 mmol
l1 MgAc and 5 mmol l1 EGTA. pH was
adjusted to 7.0 and ATP at 2.5 mmol l1 was added to each
solution. Before applications of the Ca2+ or Sr2+
solutions, each fiber was bathed for 15 min in 2% Brij 58 (polyoxyethylene 20
cetyl ether) under constant stirring. The non-ionic Brij 58 detergent
irreversibly eliminated the ability of the SR of skinned muscles to sequester
and release Ca2+, without altering the actomyosin system.
Force measurement and recording
The experiments were carried out in a thermostatically controlled room
(19±1°C). Fibers were mounted in an experimental chamber and
connected to a strain-gauge (Ford 10, World Precision Instruments, Aston, UK).
A micrometer allowed fiber diameter measurements. The resting sarcomere length
(SL) was determined by diffraction using a Helium/Neon laser (Spectra
Physics, Carlsbad, USA). For soleus as well as for plantaris muscle fibers,
the SL was set to 2.6 µm (120% of resting SL) to allow
optimal isometric tension development upon ionic activation.
Single fibers were first immersed in a pCa 4.2 solution to measure the
initial force. Next, they were checked for their strontium reactivity by
successive exposures to pSr solutions: submaximal steady state tensions
obtained in pSr 5.8, 5.4, 5.0, 4.6, were normalized to maximal Sr2+
activated tension (pSr 3.4), in order to deduce the half maximal activation
(Sr2+-affinity or pSr50) by strontium from the linear
part of the TensionpSr (TpSr relationship). Fibers were then
activated with various pCa (from 6.8 to 4.2 for all fibers). The steady state
submaximal tensions P were expressed as a percentage of the maximal
tension P0 (induced by the saturating pCa 4.2 solution),
and reported as Tension-pCa (TpCa) relationships. Fiber type was
determined, using the difference between Ca2+ and Sr2+
activation characteristics for fast and slow fibers, the fast muscle fibers
being less sensitive to Sr2+ than slow fibers
(Kerrick et al., 1980). Fiber
type was therefore determined by establishing the
value or difference
between the respective Ca2+ and Sr2+-affinity criteria,
pCa50 and pSr50 (Ca2+ and Sr2+ concentration needed to
elicit 50% of P0). Typically, the
value of slow
fiber is <0.30 pCa units, and
1.00 pCa units in fast fibers. Two other
important parameters were deduced from the TpCa relationships: the
threshold for activation by Ca2+, reflecting the sensitivity of the
contractile system, and the steepness of the TpCa curve, corresponding
to the cooperativity between the different regulatory proteins within the thin
filament. Fast type fibers could also be distinguished from slow type ones by
a higher Ca2+ threshold (lower pCa value) and a steeper TpCa
curve, pCa50 not usually being significantly different
(Stevens et al., 1993
). The
steepness of the TpCa curve was determined by the Hill coefficient
nH, calculated according to the following equation:
P/P0=([Ca2+]/K)nH/{1+([Ca2+]/K)nH},
where P/P0 is the normalized tension and
K is the apparent dissociation constant
(pK=logK=pCa50).
Mono-dimensional electrophoresis for troponin and MHC analyses
Muscles stored at 80°C were pulverized under liquid
N2. Half the powder was dissolved in a buffer consisting of
Tris-HCl 125 mmol l1, pH 6.8, 2% SDS, 5%
ß-mercaptoethanol and 10% glycerol with 0.02% Bromophenol Blue. As
previously described (Kischel et al., 2001), the different isoforms of the
three troponin subunits were separated on a one-dimensional 10%20%
gradient gel and identified by immunoblotting (see next section).
As already described (Toursel et al.,
2000), the MHC composition was determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4.5% stacking gel
and on a 7.5% separating gel. Electrophoresis was for 20 h at 12°C (180 V
constant, 13 mA/gel). After the gel run, the MHC gel slabs were silver
stained. The relative proportions of MHC isoforms in each sample was
determined by integrating densitometry software (GS-700 Imaging Densitometer,
Biorad, Ivry s/Seine, France). At least two independent measurements were
performed on each sample. They were always quite similar and the mean value is
reported.
Immunoblotting
Electrotransfer was performed on a 0.2 µm nitrocellulose sheet (Advantec
MFS, Pleasanton, CA, USA). The membranes were blocked with a
phosphate-buffered saline solution (PBS, pH 7.4) containing 5% non-fat dry
milk and 0.2% sodium azide. All the membranes were incubated overnight with
each primary antibody. As previously described
(Stevens et al., 2002), a
monoclonal antibody (5C5 from Sigma, specific to
sarcomeric actin)
allowed actin signal recognition, serving as internal control. For TnT, the
fast isoforms were identified with the JLT-12 monoclonal antibody from Sigma;
the slow TnT isoforms were detected using a polyclonal antibody previously
characterized and provided by Pette and collaborators
(Härtner et al., 1989
).
For TnC, both slow and fast isoforms (5050% recognition) were
identified by another polyclonal antibody provided by Pette and collaborators
(Härtner and Pette,
1990
). For TnI, slow and fast isoforms were identified by two
separate polyclonal antibodies also provided by Härtner and Pette
(1990
). Previous studies from
our team (Stevens et al.,
2003
), however, have shown that each TnI antibody (slow or fast)
recognized each TnI isoform (slow or fast), respectively, and with the same
affinity. The primary antigenantibody complexes were detected by
extravidin peroxidase and biotinylated conjugate antibodies against mouse or
guinea pig IgGs (Sigma). The signals were visualized by an enhanced
chemiluminescence (ECL) kit (Amersham, Bucks, UK). Signal intensities were
evaluated by integrating densitometry. At least two independent measurements
were performed on each sample (averaged value reported).
The bound antibodies could be removed by an incubation at 50°C for 40 min with occasional stirring in a stripping buffer (100 mmol l1 2-mercaptoethanol, 2% SDS, 62.5 mmol l1 Tris-HCl, pH 6.7). The membrane was washed twice in PBS at room temperature and the success of the stripping was tested by incubating the membrane with the secondary antibodies corresponding to the previously tested antibodies and ECL detection. Immunodetection of the other proteins was then performed as described above. To ensure that there was no muscle protein loss during incubation of the nitrocellulose membrane in the stripping buffer, the membrane was reincubated at the end of the experiments with the first used antibody and the intensities of the signals compared. No significant difference between the signals was measured.
Two-dimensional electrophoresis for MLC analysis
The other half of the muscle powder was used to extract myofibrillar
protein for MLC analysis by 2-D gel electrophoresis. Myofibrillar proteins
were extracted from 710 mg of dry muscle powder as described previously
(Toursel et al., 2000), washed
first with a solution containing 6.3 mmol l1 EDTA (pH 7),
pepstatin 0.1%, phenylmethylsulfonylfluoride (PMSF) 1%, and then with a second
solution containing KCl 50 mmol l1, pepstatin 0.1%, PMSF 1%.
The myofibrillar proteins were resuspended in 500 µl of milliQ-filtered
water and their concentration determined by a protein assay kit (Dc Protein
Assay, Bio-Rad) to prepare samples having a final quantity of 50 µg. Then,
the proteins were precipitated for 2 h with acetone (8 v/v), followed by
centrifugation for 1 h at 13 000 g. The pellet was dissolved
in Laemmli solution (Laemmli,
1970
) for SDS-PAGE or in rehydration buffer for 2-D gel
electrophoresis.
Proteins were separated by two-dimensional (2-D) gel electrophoresis using
a procedure similar to those previously described by Morano et al.
(1988), Gonzalez et al.
(2002
) and Bozzo et al.
(2003
). For the first dimension
or isoelectric focusing (IEF), proteins were solubilized in 8 mol
l1 urea, 2% Chaps, 0.01 mol l1
dithitreitol (DTT) and a 2% carrier ampholites (Amersham) buffer, and then
separated using the Ettan IPGphor Isoelectric Focusing System (Amersham) on
3.5% acrylamide strips with immobilized pH gradients (47) (Amersham). Strips
were rehydrated at 50 V for 12 h andproteins focused under the following
voltage conditions: 500 V for 1 h, 5001000 V for 1 h, 8000100
000 V h1. Temperature was kept constant at 20°C. After
reduction with 6 mol l1 urea, 30% glycerol, Tris-HCl 0.375
mol l1, pH 8.8, 2% DTT and alkylation using the same buffer
with additional 2.5% iodoacetamide, the strips were embedded in 4%
polyacrylamide stacking gel and the proteins separated by SDS-PAGE on a 12%
polyacrylamide gel for 8 h at 150 V at low temperature (4°C). Following
electrophoresis, gels were silver stained. The positions of slow and fast
isoforms of MLC on 2-D gels were determined according to their isoelectric
point in the first IEF dimension, and to markers of appropriate molecular mass
in the second dimension (Bozzo et al.,
2003
). All two-dimensional gels were digitized with an Epson 1650
scanner at a resolution of 200 dpi. The spots were analyzed densitometrically
determining BAP (Brightness Area Product) with a constant threshold after
black/white inversion using Adobe Photoshop Software
(Bozzo et al., 2003
).
Statistical analysis
All the data are reported as means ± S.E.M. The
statistical significance of the difference between means was determined using
the Student's t-test. Differences at or above 95% confidence level
were considered significant (P<0.05).
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Results |
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Ca2+ activation properties of isolated skinned fibers
The TpCa curves and their derived parameters are illustrated for
soleus and plantaris in Fig. 1.
We chose to test only slow fibers in both CONT and CBR soleus muscles
(Fig. 1Ai,Bi). Slow fibers of
CBR rat soleus exhibited a mean TpCa curve shifted to lower calcium
concentrations when compared to CONT. The Ca2+ affinity parameter
(pCa50) was increased by 0.14 pCa units and the steepness
(nH value) of the curve was elevated by 55% in CBR fibers.
The calcium sensitivity (pCa threshold) was not affected.
|
Ca2+ activation properties of fast plantaris fibers were not altered after hypergravity conditions (Fig. 1Aii,Bii, bottom). All characteristics of CBR fibers from plantaris presented values identical to those of CONT ones.
Protein expression of troponin isoforms
The expression patterns of the different isoforms of the three subunits of
troponin are illustrated in Fig.
2A and their relative expression reported in
Fig. 2B. All the troponin
subunit identifications were performed using specific antibodies against slow
and fast Tn isoforms (see Materials and methods). As described in
Fig. 2A,B (top), TnT protein is
composed, in control soleus from Long Evans rats, of four fast isoforms:
TnT1f, 2f, 3f and 4f and three slow isoforms: TnT1s, 2s and 3s. In CBR rats,
the relative distribution of fast isoforms was modified with an upregulation
of TnT3f (20% increase) and a downregulation of TnT1f (50% decrease) when
compared to CONT soleus. However, no change was observed for TnTs isoforms. In
CBR soleus muscles (Fig. 2A,B),
the relative percentages of slow (fast) TnI isoforms were increased
(decreased) by 20%. The same effects (amount and direction) were observed
for TnC isoforms.
|
No variation in Tn (T, I and C) isoform expression in the plantaris was seen in hypergravity conditions.
Protein expression of MLC isoforms and MLC2 phosphorylation
Since no difference was found for the three Tn subunits in plantaris, the
effects of hypergravity on MLC expression and MLC2 phosphorylation were tested
only in soleus muscles. The CONT soleus expressed predominantly the slow
isoforms of MLC, slow MLC1 (84%) and slow MLC2 (92%), when compared to their
respective fast counterparts, fast MLC1 (16%) and fast MLC2 (8%)
(Table 2). The MLC3 was
detected in the 2-D gels but at very low levels. In CBR soleus, the
transitions could be seen only for MLC1 isoform distribution and were
characterized by a 12% increase in slow MLC1 isoform, with a concomitant
decrease in fast MLC1, which remained slightly persistent (4%). No change was
observed in MLC2 or MLC3 expression.
|
Analysis of MLC2 phosphorylation after hypergravity is also shown in
Table 2 and in
Fig. 3, which illustrates a 2-D
gel and indicates the position of the different MLC2 spots (s, s1 and f). As
described previously (Bozzo et al.,
2003), the positions of slow and fast MLC2 isoforms were confirmed
by immunoblotting (data not shown). In the CONT soleus, slow MLC2 was the
predominant regulatory light chain and appeared, in fact, to be separated into
two spots: MLC2s (as described above) and another spot with a more acidic
isoelectric point, MLC2s1. MLC2s and MLC2s1 have previously been identified by
alkaline phosphatase experiments (Bozzo et
al., 2003
) as unphosphorylated and phosphorylated spots,
respectively. Hypergravity conditions did not modify the extent of
phosphorylation of the MLC2s1 isoform in soleus muscles since there was no
change in MLC2 spot unphosphorylated/phosphorylated distribution in CBR soleus
compared to control muscles (Fig.
3). For the fast MLC2 isoforms, both CONT and CBR soleus presented
one single spot, the unphosphorylated MLC2f.
|
Protein and mRNA expressions of MHC isoforms
The changes in MHC isoform composition are described in
Fig. 4. These data were
obtained from the same animals as Picquet et al.
(2002). HG conditions had
important and significant effects on the MHC isoform repartition in soleus
muscle. At the protein level (Fig.
4B), two MHC isoforms were expressed in the CONT soleus: the
predominant slow MHCI isoform (64%) and the fast MHCIIa isoform (36%). As
previously reported (Picquet et al.,
2002
), the slow MHCI was significantly increased in CBR muscles
since its expression reached 100% of total MHC content, while MHCIIa was no
longer expressed. RTPCR analysis of MHC mRNA isoform content of the SOL
muscle demonstrated that the four MHC mRNA isoforms (MHCI, IIa, IId/x and IIb)
were present in CONT and CBR soleus muscles
(Fig. 4C). The slow MHCI
isoform was increased compared to CONT (
78% versus 58%) while
fast MHCIIa and IId/x mRNA isoforms were decreased by 50% in CBR rats. MHCIIb
mRNA isoform, present at very low amounts in CONT SOL (
3%), was not
affected.
|
At both protein and mRNA levels, the CONT plantaris muscle expressed the
four MHC isoforms previously described, but with a predominance of the fast
isoforms in the order MHCIId/x MHCIIb >MHCIIa >MHCI. The protein and
mRNA isoform distributions were not modified in CBR plantaris muscles.
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Discussion |
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Body and muscle mass, diameter and maximal tension of muscle fibers
As already described and discussed by Picquet et al.
(2002), CBR rats exhibited
body and muscle masses lower than those of control muscles. These authors
concluded that the decrease in muscle mass was correlated with the decrease in
body mass, which could, in turn, be related to a slowing down in the growth of
the rats conceived and reared in hypergravity. They also observed a
significant decline in the absolute CSA of the slow soleus fibers (30%)
and the fast plantaris (17%) ones. These observations were confirmed by
our measurements on skinned fiber diameters, the difference in the amplitude
(10% in soleus and 6% in plantaris fiber diameters) of the
decreases being possibly explained by the different techniques of analysis and
muscle fiber treatments (skinning) used.
Our results showed that the maximal tensions (kN m2) were
increased by 34% in CBR soleus fibers. This also corresponded with the results
of Picquet et al. (2002),
obtained at the whole soleus muscle level. Here, the increase in specific
maximal force could be linked either to an increased number of cross-bridges
or to an increase in force output per cross-bridge. The first hypothesis would
suppose an elevated synthesis/degradation ratio of proteins in CBR soleus
fibers, a fact that was not supported by the observed decrease in fiber
diameter. Some studies have suggested that hypergravity did not induce changes
in protein synthesis (Almurshed and
Grunewald, 2000
), but had a general sparing effect on muscle
proteins in rats that were not gaining body mass, thus permitting them to
maintain muscle protein levels (Roy et
al., 1996
). Another explanation for the increase in the number of
cross-bridges would be a modification in the myofilament lattice spacing,
already proposed for microgravity conditions
(Fitts et al., 2000
). The
second hypothesis, i.e. the elevated maximal tension P0
(kN m2) explained by an increase in force per cross-bridge,
was in good agreement with the observed increase in Ca2+ affinity
in CBR soleus fibers (see below).
For the plantaris muscle, the absence of alteration in specific force P0 (kN m2) in hypergravity, agreed with the lack of changes in calcium activation characteristics (especially in Ca2+ affinity parameter).
Contractile properties and troponin isoform transitions
This is the first time that changes in body and muscle growth of rats
conceived, born and reared in hypergravity, have been shown to be accompanied
by modifications in calcium activation characteristics of soleus but not
plantaris muscle fibers. These changes consisted principally of a higher
Ca2+ affinity and a higher cooperativity between proteins of the
thin filament, indicating that CBR soleus fibers were able to produce more
efficient contractions. These effects were specific to CBR animals (including
animal gestation, birth and development), since only slight modifications (and
in the opposite direction) have been reported for adult rats placed in
hypergravity for a period of 19 days
(Stevens et al., 2003). In our
study, the electrophoretic analyses showed an increase in the relative
proportions of slow isoforms of troponin subunits. It is well known that the
isoform type of the three Tn subunits present in the muscle fibers can
influence the shape of the TpCa curve
(Schiaffino and Reggiani,
1996
). Here, the isoform composition of the three subunits (T, C
and I) was affected. Thus, upregulation of TnC and TnI slow isoforms
versus downregulation of the fast ones could explain very well the
higher Ca2+ affinity observed in CBR soleus fibers. Indeed, slow
fibers generally exhibit higher Ca2+ affinity than fast ones
(Mounier et al., 1989
;
Schiaffino and Reggiani,
1996
). Changes at the TnT level were less marked: the slow TnT
content was not modified and a rearrangement within the fast TnT isoforms
occurred, consisting of an increased relative level of TnT3f at the expense of
TnT1f, one of the two isoforms (with TnT4f) more representative of fast
muscles. Thus, we suggested that the higher cooperativity observed in CBR
soleus fibers might be linked to the rearrangement in TnT fast isoforms.
Tropomyosin transformations can also be envisaged to explain the modifications
in Ca2+ affinity (Schachat et
al., 1987
).
MLC and MLC2 isoform transitions
In our study, MLC slow-to-slower changes were seen only for the slow (fast)
MLC1 isoform, which was significantly increased (decreased) in hypergravity.
Thus, transitions at the total MLC level were less marked than for MHC (see
below) and troponin isoforms. Such lesser effects of environmental conditions
on MLC changes have already been described elsewhere
(Ingalls et al., 1996;
Stevens et al., 2000
).
Another means of regulation, the phosphorylation of the MLC2 isoform, has
previously been described as positively correlated with a slow-to-fast
phenotype transformation in slow muscles
(Bozzo et al., 2003). Here, the
slow-to-slower phenotype transitions induced in CBR soleus were not
accompanied by variations in MLC2 phosphorylation. One could have expected a
decrease in phosphorylation. A possible explanation for these latter results
could be the low level of MLC2 phosphorylation existing in the slow soleus
muscle. Moreover, decreases (increases) in MLC2 phosphorylation have generally
been associated with declined (elevated) Ca2+ affinities
(Sweeney and Stull, 1986
).
This was not the case in this study, since we did not demonstrate any
change in phosphorylation paralleling the decrease in Ca2+ affinity
in CBR soleus. As previously described by Bozzo et al.
(2003), in unloaded soleus,
MLC2 phosphorylation was increased alongside a decrease in Ca2+
affinity (Gardetto et al.,
1989
; Stevens et al.,
1993
). These observations led us to suggest that modifications in
Ca2+ affinity induced by changes in the gravity factor could not be
directly explained by variations in MLC2 phosphorylation.
MHC isoform transitions
The most important biochemical changes in CBR soleus reported here were
characterized by slow-to-slower transitions, observed at the MHC level. Our
results are in agreement with those previously described by Martin
(1980) on Sprague-Dawley rats
submitted to centrifugation at 30 days of age, but not with studies on rats
placed in hypergravity for 2 weeks as adults
(Roy et al., 1996
;
Stevens et al., 2003
). This
suggested that a nervous factor could be participating in the observed
transformations. Indeed, Krasnov et al.
(1992
) have demonstrated in
soleus from rats grown in hypergravity that the volumes of the bodies, nucleus
and nucleolus in motoneurons from the spinal cord at the level of lumbar
enlargement were increased. These authors thus suggested a higher functional
motoneuron activity, associated with an elevated content of slow and
intermediate muscle fibers.
In our conditions and as previously described
(Picquet et al., 2002), the
CBR soleus muscle only expressed the slow MHCI isoform. A total disappearance
of all fast isoform expression, more precisely of MHCIIa, was observed. These
changes were in agreement with those occurring at the mRNA level, i.e. a shift
from MHCIIa mRNA (
30% in CONT/
15% in CBR) to MHCI mRNA (
60% in
CONT/
80% in CBR). However, MHCIIa mRNA was still present in CBR muscles
even though the protein was no longer expressed, suggesting altered
translational and/or post-translational regulation during hypergravity.
In plantaris muscle, we also found no changes in maximal tension or
contractile characteristics in MHC and troponin isoform compositions. The lack
of changes in MHC isoforms described in this study does not conflict with the
increased number of hybrid fibers observed by Picquet et al.
(2002). Indeed, a
rearrangement in the MHC isoform distribution among the different fibers would
not necessary lead to a change in MHC content at the whole muscle level.
Therefore, as was the case in real or simulated microgravity conditions,
hypergravity at 2 g preferentially affected the phenotype of
slow hindlimb extensor muscles.
In conclusion, the present study performed on rats conceived, born and
reared in hypergravity, reported effects on soleus muscle that are contrary to
those observed in adult rats exposed to hypergravity. Indeed, in the latter
(Stevens et al., 2003), only a
few modifications were shown, consisting of a decrease in Ca2+
affinity, and a slight slow-to-fast transition of TnC and I isoforms; MHC and
TnT isoforms were unaffected. In contrast, the present study on CBR rats
highlights an increase in Ca2+ affinity and important changes in
the expression of myofibrillar proteins: troponins present slow-to-slower
transitions and MHC isoform pattern are reduced to the single expression of
the slow MHCI isoform. In this context, other periods of hypergravity
initiation could be further examined with the aim of understanding which
critical steps of rat gestation, birth and/or development influence the
transformations induced by changes in gravity factor. Indeed, since the
muscles in our study were removed at the adult stage, we could not determine
whether or not the MHC plasticity under centrifugation went through
remodelling steps including modified early-expressed myosin isoforms, like
embryonic or neonatal MHC (Martrette et
al., 1998
).
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Acknowledgments |
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References |
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