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Am J Physiol Regul Integr Comp Physiol 289: R217-R224, 2005. First published March 17, 2005; doi:10.1152/ajpregu.00596.2004
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ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY

Changes in rat soleus muscle phenotype consecutive to a growth in hypergravity followed by normogravity

F. Picquet,1 V. Bouet,2 L. Cochon,1 M. Lacour,2 and M. Falempin1

1Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France; and 2UMR 6149, Neurobiologie Intégrative et Adaptative, Marseille, France

Submitted 1 September 2004 ; accepted in final form 15 March 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It has been demonstrated that a long-term stay in hypergravity (HG: 2G) modified the phenotype and the contractile properties of rat soleus muscle. The ability of this muscle to contract was drastically reduced, which is a sign of anticipated aging. Consequently, our aim was to determine whether rats conceived, born, and reared in hypergravity showed adaptative capacities in normogravity (NG: 1G). This study was performed on rats divided into two series: the first was reared in HG until 100 days and was submitted to normogravity until 115 to 220 postnatal days (HG-NG rats); the second was made up of age paired groups reared in normogravity (NG rats). The contractile, morphological, and phenotypical properties of soleus muscle were studied. Our results showed that the NG rats were characterized by coexpressions of slow and fast myosin, respectively, 76.5 and 23.5% at 115 days. During their postnatal maturation, the fast isoform was gradually replaced by slow myosin. At 220 days, the relative proportions were respectively 91.05% and 8.95%. From 115 to 220 days, the HG-NG rats expressed 100% of slow myosin isoform and they presented a slower contractile behavior compared with their age-matched groups; at 115 days, the whole muscle contraction time was increased by 35%, and by 15%, at 220 days. Our study underlined the importance of gravity in the muscular development and suggested the existence of critical periods in muscle phenotype installation.

muscular properties; myosin transition; gravity change; hindlimb muscle

ALL LIVING ORGANISMS ARE DIRECTLY under the influence of a common and constant factor, gravity. It is well known that the development and the maturation of numerous life systems such as cell division, axonal growth, posture, and body movements are directly regulated by this factor (6, 14, 15, 16).

The effects of real (spaceflight) and simulated (hypodynamia-hypokinesia model, HH) microgravity on the neuromuscular system are well documented. In hindlimb muscles, an exposure to a HH period induces more marked effects in slow extensor muscles such as the soleus than in fast muscles (for a review, see Refs. 23 and 24). Most studies have been carried out in adult rats. Indeed, in soleus muscle from rats submitted to real and simulated microgravity, a muscular atrophy correlated to a decrease both in fiber and muscle cross-sectional area and to a decrease in muscle strength, which has been commonly described (for review, see Ref. 13). A slow-to-fast phenotype transition, characterized by changes in contractile protein isoforms such as myosin heavy and light chains (3, 32) and regulatory proteins (2), has also been reported. Moreover, the proprioceptive information could be disturbed (9), and a cortical reorganization has been demonstrated (5, 10). Furthermore, it has been reported that the effects of microgravity are reversible, as muscle properties are recovered afer reloading (23). Consequently, gravity appears to be a key element necessary to maintain neuromuscular integrity.

Another way to study the importance of gravity was to increase it by centrifugation to obtain hypergravity (HG). Compared with microgravity, this condition offers the advantage of studying long-term changes. It has been established that the GABA immunoreactivity was decreased in the cortex of HG rats, suggesting changes in sensory feedback information from muscle receptors (7). Furthermore, it has been reported that the behavior of rats born and raised in HG was altered compared with control animals (36). In a recent publication, we have established that contractile properties and the phenotype of hindlimb muscles of rats conceived, born, and reared in HG now aged 100 days were modified after a long-term HG period (26). Muscle characteristics appeared reinforced, as slow muscle, such as the soleus, became slower (100% slow myosin) and fast muscle, such as the plantaris, became faster, regarding its kinetic properties. The soleus phenotype was completely unusual, because, in control animals, the appearance of a pure slow phenotype is characteristic of more aged rats (1, 8). Considering these results, it was tempting to determine the adaptive capacities of rats reared in HG and then submitted to normogravity.

The aim of the present study was thus to establish whether soleus muscle phenotype from rats conceived, born, and reared in HG was a stilted structure or if it could be remodeled; that is, express other myosin than slow isoforms during a stay in normogravity. For these animals, normogravity was similar to microgravity, compared with the gravity level from their conception.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal groups. The study was performed on a total of 58 male Long-Evans rats divided into two series. The first one was made up of rats conceived, born, and reared in hypergravity until postnatal day 100. The centrifugation apparatus was then stopped and the animals continued their growth in normogravity (HG-NG groups). The HG-NG rats were selected from a population of the first generation derived from couples mated in the centrifugation apparatus. After 15 days, males were removed, whereas females stayed on until weaning. The studied rats descended from different littermates. The second series consisted of terrestrial rats conceived, born, and reared in normogravity (NG groups). The NG rats were reared in conditions similar to those of the centrifuge, that is, same room, same dark-light cycle (12:12 h), and same temperature inside a standard home cage contained in a gondola until postnatal day 100. Both NG and HG-NG rats were then transferred in a thermoregulated room (25°C) with circadian rhythm (12:12 h dark-light) but without centrifugation noise for a maximal duration of 120 days. The HG-NG group comprised four subgroups. HG-NG rats aged 100 days continued their postnatal growth until postcentrifugal days: 1) 15 (group HG-NG-115, n = 6), 2) 30 (group HG-NG-130, n = 6), 3) 60 (group HG-NG-160, n = 8) and 4) 120 (group HG-NG-220, n = 10). Age-paired groups consisted, for NG rats, of NG-115 (n = 6), NG-130 (n = 6), NG-160 (n = 8), and NG-220 (n = 8). Consequently, in any group, the values 115, 130, 160, and 220 indicate the postnatal age of rats, whereas NG and HG-NG represent the growth in normogravity or in hypergravity + normogravity, respectively.

The contractile, morphological, and phenotypical properties were determined in the right soleus muscle of both NG and HG-NG rats. All experimental procedures described below were approved by both the French Agricultural and Forest Ministry and French National Education Ministry (Veterinary Service of Health and Animal Protection, authorization A 59–00980).

Centrifugation apparatus. The apparatus has already been described in detail (26). It consisted of a velocity-controlled DC motor (3.5 kW), located in the vertical axis of the apparatus and driving two horizontal cross-arms (total length: 165 cm) at constant rotation speed. Four free-swinging gondolas were jointed at the 4 extremities of the horizontal arms, 76.5 cm away from the axis of rotation; the gondolas were equipped with standard home cages for rats (30 x 40 cm, each cage containing three rats) with an aeration system and lights that reproduced a 12:12-h light-dark cycle. A video system in the gondolas allowed us to know the exact birth date of litters and to control the movement ability of the animals. During centrifugation, the gondolas were tilted at a constant angle of 60° from the vertical. Counterclockwise rotations were done at a constant velocity of 3.81 rad/s. Given the mass and the inertia of the gondolas, including the home cages and the rats, this angular velocity led to 2 G resultant force. The resultant force was directly measured using a standardized force sensor placed at the center of the floor of the gondola. During centrifugation, rats were subjected to a gravito-inertial force vector whose direction was always similar to that exerted in normal gravity (parallel to the dorso-ventral axis of the animal), and magnitude was twice the normal magnitude exerted on Earth. Consequently, the rats were also subjected to Coriolis force accelerations.

After weaning, female pups and their mothers were removed from the centrifuge. For animal care (cleaning and feeding), the centrifugation was stopped for 15 min every week. Food and water were available ad libitum. The centrifugal rats gained body mass at a rate similar to controls but their mass remained lower than control animals, whereas no statistical difference of food intake was observed between the two populations. After 100 days of centrifugation, the rats were taken off the apparatus and were maintained in normogravity for a maximal duration of 120 days.

In situ isometric contractile properties. The animals were anesthetized with pentobarbital sodium (40 mg/kg ip), and anesthesia was prolonged with further injections of 20 mg/kg, when necessary. The dissection protocol has been described in previous papers originating from our laboratory (12, 19). Briefly, under deep anesthesia, assessed by the absence of blink reflexes, all the muscles of the right thigh and lower limb were denervated, except the soleus muscle. The dissected limb was fixed to maintain the isometric conditions and was immersed in a paraffin oil bath thermostatically controlled (37°C). The limb was stabilized by a combination of pins, clamps, and bars, so that the muscle was maintained in a horizontal position. The soleus muscle was isolated from surrounding tissues, and its tendon was stitched up by a short 3.0 silk suture around a force transducer (Grass, FT 10, USA). Its length was adjusted to produce a maximal twitch tension. The stimulating [Teflon-coated platinum, outside diameter (OD) : 125 µm] and reference (teflon-coated platinum, OD : 250 µm) electrodes were maintained by micromanipulators. Contractions of the soleus muscle were induced by stimulation (Grass model S 8800, Quincy, MA) of the soleus nerve (0.2-ms duration pulses) through monopolar platinum electrodes at twice the minimum voltage required to obtain the maximal twitch response. The reference electrode was inserted into adjacent denervated muscle mass. The soleus muscles were stimulated so as to record the following parameters: 1) a single maximal twitch from which the maximal twitch tension (Pt), the time to peak (TTP), and the half-relaxation time (HRT) were measured; 2) the tension/frequency relationship (for stimulation frequencies ranging from 16 to 120 Hz), which allowed the determination of maximum tetanic tension P0 obtained for a 100-Hz stimulation frequency and of index P20/P0 (ratio of the tetanic tension at 20 Hz to P0); and 3) the fatigue index (FI), previously defined by Winiarski et al. (35).

At the end of the fatigue index, the right soleus muscles of the experimental animals were removed, weighed (determination of the soleus muscle wet weight, MWW), frozen in isopentane precooled to its freezing point by liquid N2, and stored at –80°C. The muscles were divided into two parts to perform both immunohistochemical and electrophoretical determinations of myosin heavy-chain isoforms on the same muscle.

Characterization of muscle fiber types by immunohistochemistry. At the midbelly, the soleus muscles of the NG and HG-NG groups were cut into serial 10-µm-thick cross sections in cryostat microtome at –20°C. The protocol relative to immunohistochemically treated sections has been described in detail (25). The following antibodies were used: NCL-MHCs (Tebu-Novocastra), which recognizes MHC I, SC-71 (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ) against MHC IIA, BF-F3 (DSMZ) against MHC IIB and MY32 (Sigma) against MHC IIA, IIB, and IIX. These antibodies were diluted at 1/20, 1/20, 1/10 and 1/2,000, respectively. The binding of the primary antibodies was localized by an immunoperoxidase reaction using the Novostain Universal Quick Kit (Tebu/Novocastra). Serial cross sections were incubated in prediluted blocking serum (normal horse serum) for about 10 min. The excess serum was blotted, and sections were incubated with primary antibodies diluted in PBS solution for 2 h at room temperature. Serial cross sections were washed for 5 min in PBS solution. Then, they were incubated for 30 min in prediluted biotinylated universal secondary antibody. At the end of the 30 min, sections were washed with PBS for 5 min and incubated in ready-to-use streptavidin/peroxidase complex reagent for 30 min. To label the serial cross sections, the peroxidase substrate solution (diaminobenzidine, DAB; Sigma-Aldrich) was added after the sections had been washed for 5 min with PBS. Finally, after dehydration with alcohol and toluene, the slides were mounted in Eukitt resin. Positive fibers were characterized by a brown color, while negative fibers remained unlabeled. Muscle typing was established from a total of 300 fibers per muscle, immunologically identified in serial sections. In our laboratory, we have previously verified that such a sample is representative of all muscle fiber types contained in the muscle. This approximation is conceivable because Kugelberg (17), in a previous paper, has reported that, in soleus rat muscle, the fibers are more evenly distributed throughout the muscle cross section than in the other hindlimb muscles. Indeed, the muscle fiber distribution in the soleus muscle is homogenous.

Determination of MHC isoforms by SDS PAGE. The MHC isoforms were studied in NG and HG-NG soleus muscles. The first part of the frozen tissues was pulverized under liquid nitrogen in a small steel mortar (30). As already described (33), the MHC composition was determined by polyacrylamide gel electrophoresis using a 4.5% glycerol stacking gel and a 7.5% glycerol separating gel. Electrophoresis was run for 18 h at 12°C (180 V constant, 13 mA per gel). After the gels were run, the gel slabs were silver-stained. Briefly, they were postfixed for 20 min in a solution containing 50% methanol and 5% acetic acid and for 10 min in a 50% methanol solution. Between each fixation, the gels were rinsed with distilled water. They were thus silver-stained in a mix of 0.25% silver nitrate and 0.02% of formaldehyde (from a 35% stock solution). They were developed with 0.04% formaldehyde (35%) and 2% sodium carbonate. The staining was stopped with 5% acetic acid. The relative proportion of each MHC isoform in each muscle type was established using a scanning densitometer (Multi analyst, Biorad).

Statistical analysis. All of the results are presented as means ± SE. After a one-way ANOVA was performed, a Bonferroni test was used to establish the intergroup difference within an identical experimental condition (i.e., between NG rats, from 115 to 220 days and between HG-NG rats from 115 to 220 days). This test allowed us to compare the temporal evolution of NG and HG-NG rats during their adult maturation. To compare the degree of muscle modifications after growth in NG and HG-NG rats, a Student's t-test was used between age-paired groups. Statistical significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Morphological data. The evolution of body weights (BW), MWW, and normalized MWW to body weight (MWW/BW) are reported in Table 1. In NG rats, as a function of age, from 115 to 220 days, the results showed a gradual increase both in BW (+37% ) and in MWW (+43%), whereas the ratio MWW/BW remained unchanged. During the same period, the HG-NG group presented increases in BW (+80%) and MWW (+55%), whereas MWW/BW was decreased (–13%). Consequently, the difference between values of NG and HG-NG groups decreased from 115 to 220 days. Indeed, at 115 days, the BW of HG-NG animals represented 64% of BW in NG rats (vs. 71% for MWW), whereas at 220 days, these differences were 84% for BW and 77% for MWW, respectively, compared with age-paired groups. The comparison between NG and HG-NG age-paired groups revealed that the BW and MWW of HG-NG rats were significantly smaller than those of NG rats. The MWW/BW ratios were also significantly lower, except for rats aged 115 days, in which the tendency was reversed.


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  		Table 1. Distribution in body weights, muscle wet weights, and ratio of rats reared in normogravity and of rats reared in hypergravity for 100 days followed by normogravity

 
Contractile properties. The contractile properties in both NG and HG-NG rats are summarized in Table 2. The muscle maturation between days 115 and 220 in NG groups revealed that both TTP and HRT parameters increased and stabilized after 130 days. The ratio P20/P0 did not significantly change except at 160 days when its value surprisingly became lower than those obtained for both NG-115 and NG-130 groups. The normalized maximal tetanic force presented identical values through all studied groups. Similar results were observed regarding the normalized twitch strength data, except in NG-160 rats, which presented an increased mean value compared with NG-115 group.


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  		Table 2. Evolution of contractile parameters in NG and HG-NG groups

 
The growth in normogravity of HG rats (HG-NG groups) showed that both TTP and HRT parameters remained unchanged between 115 and 220 days. The ratio P20/P0 only showed a decreased value in HG-NG-220 rats when compared with HG-NG-160 animals. The normalized muscle force decreased from 115 to 220 days, the values becoming significantly lower after 160 days for the twitch strength and after 220 days for the maximal tetanic force.

The comparison between age-paired groups in NG and HG-NG rats revealed that at 115 days, HG-NG rats presented significantly higher kinetic (TTP and HRT) values. Furthermore, they showed an increase in muscle force both in normalized twitch and maximal tetanic strengths. At 130 days, the soleus muscle in HG-NG rats remained significantly slower (except for HRT parameter) and stronger than in NG rats. At 160 days, the muscle showed only higher values of TTP and P20/P0, the muscle force being similar to control values. At 220 days, only the kinetic TTP parameter was significantly greater in HG-NG rats. For both NG and HG-NG series of rats, the FI was unchanged.

Identification of muscle fiber type by immunohistochemistry. Figure 1 shows representative serial cross sections from NG and HG-NG rats, in which muscle fiber types were identified by immunoreactivity. The MHC expressions were determined on a total of 300 fibers per muscle. Figure 1 illustrates the antibody reactivity in NG (Fig. 1: parts 1, 2, 5, 6, 9, 10, 13, 14) and in HG-NG (Fig. 1: parts 3, 4, 7, 8, 11, 12, 15, 16) rats. We have chosen to present the staining with NCL-MHCs (Fig. 1: parts 1, 3, 5, 7, 9, 11, 13, 15) and SC-71 (Fig. 1: parts 2, 4, 6, 8, 10, 12, 16) antibodies, respectively, directed against MHC I and MHC IIA isoforms. In Fig. 1, the positive fibers were characterized by a dark color, whereas negative fibers remained uncolored. In NG rats, from 115 to 220 days, fibers expressing MHC I and/or MHC IIA could be identified, the number of type I fibers being largely predominant. In HG-NG rats at any age, the soleus muscle presented only fibers containing MHC I isoform, as all sections incubated with SC-71 antibody remained uncolored. For both NG and HG-NG rats, the reactivity with MY32 antibody was strictly identical to that observed with SC-71. Moreover, in any of the NG and HG-NG groups, sections treated with BF-F3 antibody remained negative, indicating that soleus muscle did not express MHC IIB isoform.



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  		Fig 1 Serial sections illustrating muscle typing determined by immunohistochemistry in normogravity (NG) and hypergravity-normogravity (HG-NG) rats. The experimental conditions NG or HG-NG and the antibodies used are indicated at the top of the figure. The age of the animals is reported on the left of the photos: 115 days, 1–4; 130 days, 5–8; 160 days, 9–12; and 220 days, 13–16. Examples of fiber types are indicated by {blacktriangleup}, type IIA fiber; a thin solid arrow, fiber expressing the antibodies MHC I + MHC IIA; and a thick arrow, type I fiber.

 
The relative proportions of fiber types within each group are reported in Fig. 2. The quantitative data revealed that the soleus muscle of NG-115 rats contained ~76% of type I fibers, ~14% of fast type IIA fibers, and ~10% of fibers coexpressing the two MHC isoforms, MHC I + MHC IIA. During muscle maturation in normogravity, the amount of fibers expressing MHC I significantly increased between 115 and 130 days, remained stable until 160 days, and showed again a significant increase at 220 days, when data were compared with those obtained at 115 days. Hybrid fibers containing both MHC I + MHC IIA were decreased 1) between 115 and 130 days (respectively from 10% to 2.8%) and 2) at 220 days (~5%). They then showed an unexpected increase at 160 days. The fast IIA fiber did not show any variation along the studied period. The muscle fiber types of HG-NG rats remained identical from 115 to 220 days; the entire muscle presented only type I fibers. The examination of muscle fiber types in HG-NG groups revealed that the soleus muscle only presented slow-type fibers whose composition remained unchanged along the studied period.



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  		Fig 2 Repartition of fiber types within each muscle group. The fibers were classified according to their MHC isoform coexpressions.The results were expressed as means ± SE. *Statistical difference between age-paired groups using the Student's t-test. Letters a and b, indicate statistically significant difference between NG-115 and NG-130, respectively, by using the Bonferroni test. For all statistical tests, statistical significance was accepted at P < 0.05.

 
Muscle myosin heavy-chain composition. The different MHC isoforms were determined in soleus muscle by electrophoresis. The gel migrations are presented in Fig. 3. The MHC isoforms contained in the soleus muscles were identified by comparing their electrophoretic migration with that of a mix of fast (extensor digitorum longus) and slow (soleus) muscles (Fig. 3: lane 5). We were able to determine four MHC isoforms already described in our previous studies (26, 27). According to their increasing order of electrophoretic mobility, in lane 5, fast MHC isoforms were identified as being MHC IIA, MHC IIX, MHC IIB, and slow MHC isoform as MHC I. In Fig. 3, lanes 1 and 2 present examples of soleus muscle electrophoretic migration at 115 days (lane 1: NG rat, lane 2: HG-NG rat). Examples of profiles at 220 days are illustrated in lane 3 (NG rats) and in lane 4 (HG-NG rats). In NG rats, we identified two different MHC isoforms (MHC I, which was predominant, and MHC IIA) between 115 and 220 days. During this period, MHC I gradually increased at the expense of MHC IIA. In HG-NG rats at both 115 and 220 days, only the slow MHC I isoform could be determined (lanes 3 and 4, respectively).



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  		Fig 3 A: electrophoretic migration of myosin heavy-chain isoforms. The MHC isoforms were identified according to their electrophoretic mobility. Lane 1, NG-115 soleus muscle; lane 2, HG-NG-115 soleus muscle; lane 3, NG-220 soleus muscle; lane 4, HG-NG-220 soleus muscle; lane 5, mix of soleus and extensor digitorum longus muscles. B: densitometric determination of MHC isoforms expressed in NG and HG-NG rats. The results were reported as means ± SE. *Statistical difference between age-paired groups using the Student's t-test. Letter a indicates statistical difference with NG-115, by using the Bonferroni test. For all statistical tests, statistical significance was accepted at P < 0.05.

 
The total amount of MHC isoforms was then obtained by densitometry. The results are summarized in Fig. 3B. In NG groups, two MHC isoforms were identified, MHC I and MHC IIA. The densitometric measurements revealed the marked predominance of MHC I isoform at the expense of MHC IIA. During muscle maturation in normogravity, MHC I became significantly more expressed: ~76% at 115 days to ~90% at 220 days contrary to MHC IIA that declined (from ~24% at 115 days to 10% at 220 days). In HG-NG groups, the MHC expression was significantly modified, as only MHC I isoform was detected. Consequently, it could be deduced that the soleus muscle was made up of 100% MHC I from 115 to 220 days.


   DISCUSSION
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study presents for the first time on a timescale of 120 days from the 100th postnatal day the evolution of phenotypical and contractile muscle behavior in terrestrial rats and in rats conceived, born, and reared in HG, and then submitted to normogravity. In a previous paper, we have demonstrated that soleus muscle from rats conceived, born, and reared in HG presented a complete slow phenotype (26). Consequently, our aim was to determine whether these animals were able to express fast myosin in a situation of normogravity corresponding for these animals to hypogravity or whether their protein expression potential was durably modified by hypergravity. Our results proved that soleus muscle appeared unable to express fast myosin, although some muscle contractile properties changed with increasing to age.

Morphological data and muscle strength. Our data showed that for both NG and HG-NG rats, body and muscle weights increased with age. The comparison between the two experimental series suggested a differential maturation. The ratio MWW/BW remained lower in HG-NG rats compared with age-paired controls, this latter result indicating that the growth of BW was proportionately bigger than the growth of MWW. Nevertheless, BW and MWW in HG-NG rats always remained lower than their age-matched controls.

Consequently, two relevant findings could be deduced from our results: first, the growth of HG-NG rats remained slow even after a long stay in normogravity and second, from the MWW/BW data, the growth of soleus muscles of these animals was delayed compared with NG rats. This latter point could be explained by a decrease in food intake of rats reared in HG. However, no statistical difference was found between HG rats and their age-matched group, except during 7 days postweaning. Only at that time, the HG rats showed a slight decrease in food intake (26). After acclimation, the HG rats gained body mass at a rate similar to controls, but their mass remained lower than control animals. Consequently, after a long stay in HG, the rats could be considered acclimated to HG environment. A durable modification in the balance of anabolism/catabolism proteins could also explain the mass differences between NG and HG-NG rats caused by a decrease in protein synthesis due to the hypergravity environment. Indeed, previous studies have demonstrated that hypergravity induced, in growing rats, a marked decrease in the weight and muscular protein content (22). A final hypothesis was that normogravity must have been perceived as hypogravity by the HG-NG rats, since the soleus muscles were involved in an antigravity function. In such a case, the soleus muscles would be less solicited and muscular growth could be slowed.

The comparison of the strength data between NG and HG-NG groups revealed that HG-NG rats were stronger until 30 days in normogravity. Afterward, there was no difference in strength between NG and HG-NG rats. Since results were expressed in terms of normalized muscle force, they indicated that the stay in normogravity had induced in HG-NG rats an increase in muscle mass coupled with an increase in muscle force. These increases might also be due to a rise in the proportion of contractile myofibrillar proteins in relation with motor activity. Indeed, it has been previously demonstrated that in HG, the locomotor activity is reduced compared with control animals (34). HG rats, which are suddenly submitted to normogravity, present an increased sensorimotor activity (15). This locomotor activity could be more intense during the first days in normogravity, the animals later adapting to their new environment and thus decreasing their exploratory capacity. Basically, this increased locomotor activity could be the cause of 1) an increase only within muscle mass (in the 15 days after being placed into normogravity), and 2) an increase in muscle force (from 30 days in normogravity), later coupled with an increase in muscle mass resulting from natural growth.

Myosin expression and kinetic parameters. In NG rats, basically, parameters such as TTP, HRT, and to a lower extent P20/P0 revealed increased values from 115 to 130 days. Afterward, the values stabilized until 220 days. These data were in relation with the percentages of fiber expression since from 130 days, the relative amounts of fiber containing slow MHC I and both MHC I and MHC IIA were increased and decreased, respectively, before they stabilized at 220 days. A previous study focusing on the myosin changes during postnatal life has suggested that the mature muscle phenotype was achieved from the 125th day in the soleus muscle (18). However, the changes do not usually take on that importance. Moreover, in this study, the rats that were used did not belong to Long-Evans species. One hypothesis is the existence of a key period in the motor message maturation. It is possible that there was an increase in the electromyographical activity (EMG) activity, which became more tonic and reinforced the MHC I expression. The increase in slow MHC could be sufficient to induce an increase in contractile parameters. Unfortunately, there is no data in the literature relative to an EMG maturation, specific to Long-Evans rats.

In a previous paper (26), we have already reported that, surprisingly, our control rat soleus presented contractile parameters (TTP, HRT, and P20/P0) less slow than those usually observed in Wistar control rats, although the experimental setup such as as force transducer was strictly identical. Similar results have already been reported by Elder and Vasallo (11), in young Long-Evans rats. Our explanation is, thus, the existence of interspecies variability.

The comparison of HRT values between NG and HG-NG rats demonstrated that they were significantly lower in NG-115 rats than in the age-matched animals. From day 130, HRT values of HG-NG rats became similar to those of NG rats. This parameter has been proved to be dependent on calcium ATPase isoform in the sarcoplasmic reticulum (21). Consequently, we postulate that ATPase isoforms became identical in NG and HG-NG rats, from 130 days.

In a previous paper (26), we have established that the soleus muscle of rats born and reared in HG became slow. In our study, when applied to such animals, normogravity seemed unable to transform the muscle phenotype. In hypogravity, simulated or real, a slow-to-fast transition for contractile protein is commonly described, which was not the case in our experiments. In fact, HG-NG rats could be considered in "hypogravity" from the very moment they were installed in normogravity. Their disability to express fast myosin remains unclear. It could be the result of a change in the nervous motor message during HG. Indeed, it has been demonstrated that, during HG, slow muscles became slower. Then, we hypothetized that the nervous message was reinforced (from tonic to more tonic) and was sufficient to alter the muscle phenotype. Unfortunately, it is difficult to perform the measurement of EMG in the centrifugation apparatus, but we have some data on animal behavior. It has been reported that during HG, the locomotor activity of the rats is reduced (7, 15). Animals exposed to 2 G walked more slowly than off-centrifuge control animals. Moreover, they adopted a different form of locomotion that resulted from a four-footed stance which increased the postural stability (7). These authors therefore suggested that, in HG, the locomotion is differently controlled, compared with normogravity.

As the body weight was increased by a factor of 2 during HG, we argue that, to support this increased body charge, the EMG is increased too. Unfortunately, we have no information concerning the future of EMG during prolonged stay in HG. Nevertheless, a previous study has demonstrated that during 2 G phases of parabolic flight, this activity was transiently increased (20). According to our hypothesis, the EMG activity could, in turn, modify the afferent message coming from muscle receptor, such as neuromuscular spindles. Indeed, it has been reported that 19 days in HG are sufficient to induce some intrafusal myosin changes (28). Considering that afferent fibers coming from intrafusal fiber are projected onto {alpha} and {gamma} motoneurons, they could also modify, by reflex pathway, the EMG activity during HG.

HG rats which are submitted to normogravity present an increased locomotor activity (15), whereas they perceive normogravity as hypogravity. We postulate that the existence of a contact of hindlimb plantar soles with the ground together with locomotor movements, which are usually nonexistent in microgravity, could contribute to the maintenance of slow myosin despite gravity change.

To sum up, when gathering the data, the soleus muscle appears deeply modified by a long stay in HG. Our study underlines the importance of gravity in the maintenance of neuromuscular integrity. However, at present, several questions remain unanswered. Further studies could be undertaken to clarify these issues. Moreover, by using combinations of different ages in relation with different alternating periods of HG and normogravity, critical periods for gene or protein expressions both in the embryonic and postnatal developments could be determined.


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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by grants from the Centre National d'Etudes Spatiales (8411) and the Conseil Régional du Nord-Pas-de-Calais.


   ACKNOWLEDGMENTS
 
The authors wish to thank Dr. G. S. Butler-Browne for the growing of hybridomas producing SC 71 and BF F3 antibodies initially developed by Dr. Schiaffino (31). The hybridomas were produced by Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Picquet, Laboratoire de Plasticité Neuromusculaire, UPRES EA 1032, IFR 118, Université des Sciences et Technologies de Lille, Bat SN4, 59655 Villeneuve d'Ascq cedex, FRANCE (E-mail: florence.picquet{at}univ-lille1.fr)

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
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ansved T. Effects of immobilization on the rat soleus muscle in relation to age. Acta Physiol Scand 154: 291–302, 1995.[ISI][Medline]
  2. Bastide B, Kischel P, Puterflam J, Stevens L, Pette D, Jin JP, and Mounier Y. Expression and functional implications of troponin T isoforms in soleus muscle fibers of rat after unloading. Pflügers Arch 444: 345–352, 2002.[CrossRef][ISI][Medline]
  3. Bozzo C, Stevens L, Toniolo L, Mounier Y, and Reggiani C. Increased phosphorylation of myosin light chain associated with slow-to-fast transition in rat soleus. Am J Physiol Cell Physiol 285: C575–C583, 2003.[Abstract/Free Full Text]
  4. Brocard F, Clarac F, and Vinay L. Gravity influences the development of inputs from the brain to lumbar motoneurons in the rat. Neuroreport 14: 1697–1700, 2003.[CrossRef][Medline]
  5. Canu MH, Langlet C, Dupont E, and Falempin M. Effects of hypodynamia-hypokinesia on somatosensory evoked potentials in the rat. Brain Res 978: 162–168, 2003.[Medline]
  6. Chabbert C, Brugeaud A, Lennan G, Lehouelleur J, and Sans A. Electrophysiological properties of the utricular primary transducer are modified during development under hypergravity. Eur J Neurosci 17: 2497–2500, 2003.[Medline]
  7. D'Amelio F, Wu LC, Fox RA, Daunton NG, Corcoran ML, and Polyakov I. Hypergravity exposure decreases gamma-aminobutyric acid immunoreactivity in axon terminals contacting pyramidal cells in the rat somatosensory cortex: a quantitative immunocytochemical image analysis. J Neurosci Res 53: 135–142, 1998.[Medline]
  8. Danieli-Betto D, Betto R, Megighian A, Midrio M, Salviati G, and Larsson L. Effects of age on sarcoplasmic reticulum properties and histochemical composition of fast- and slow-twitch rat muscles. Acta Physiol Scand 154: 59–64, 1995.[Medline]
  9. De Doncker L, Picquet F, Petit J, and Falempin M. Effects of hypodynamia-hypokinesia on the muscle spindle discharges of rat soleus muscle. J Neurophysiol 89: 3000–3007, 2003.[Abstract/Free Full Text]
  10. Dupont E, Canu MH, and Falempin M. A 14-day period of hindpaw sensory deprivation enhances the responsiveness of rat cortical neurons. Neuroscience 121: 433–439, 2003.[CrossRef][ISI][Medline]
  11. Elder G and Vasallo F. Histochemical and contractile properties of soleus muscle trained during development. Pflügers Arch 407: 166–169, 1986.[Medline]
  12. Falempin M, and In-Albon SF. Influence of brief daily tendon vibration on rat soleus muscle in non-weight-bearing situation. J Appl Physiol 87: 3–9, 1999.[Abstract/Free Full Text]
  13. Fitts RH, Riley DR, and Widrick JJ. Physiology of a microgravity environment invited review: microgravity and skeletal muscle. J Appl Physiol 89: 823–839, 2000.[Abstract/Free Full Text]
  14. Gharib C and Custaud MA. Orthostatic tolerance after spaceflight or simulated weightlessness by head-down bed-rest. Bull Acad Natl Med 186: 733–746, 2002.[Medline]
  15. Gustave Dit Duflo S, Gestreau C, and Lacour M Fos expression in the rat brain after exposure to gravito-inertial force changes. Brain Res 861: 333–344, 2000.[Medline]
  16. Ilyin EA and Oganov VS. Microgravity and musculoskeletal system of mammals. Adv Space Res 9: 11–19, 1989.[Medline]
  17. Kugelberg E. Histochemical composition, contraction speed and fatiguability of rat soleus motor units. J Neurol Sci 20: 177–198, 1973.[CrossRef][ISI][Medline]
  18. LaFramboise WA, Daood MJ, Guthrie RD, Butler-Browne GS, Whalen RG, and Ontell M. Myosin isoforms in neonatal rat extensor digitorum longus, diaphragm, and soleus muscles. Am J Physiol Lung Cell Mol Physiol 259: L116–L122, 1990.[Abstract/Free Full Text]
  19. Leterme D and Falempin M. Compensatory effects of chronic electrostimulation on unweighted rat soleus muscle. Pflügers Arch 426: 155–160, 1994.[CrossRef][ISI][Medline]
  20. Leterme D and Falempin M. EMG activity of three rat hindlimb muscles during microgravity and hypergravity phase of parabolic flight. Aviat Space Environ Med 69: 1065–1070, 1998.[Medline]
  21. Loukianov E, Ji Y, Grupp IL, Kirkpatrick DL, Baker DL, Loukianova T, Grupp G, Lytton J, Walsh RA, and Periasamy M. Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase. Circ Res 83: 889–897, 1998.[Abstract/Free Full Text]
  22. Martrette JM, Hartmann N, Vonau S, and Westphal A. Effects of pre- and perinatal exposure to hypergravity on muscular structure development in rat. J Muscle Res Cell Motil 19: 689–694, 1998.[Medline]
  23. Ohira M, Hanada H, Kawano F, Ishihara A, Nonaka I, and Ohira Y. Regulation of the properties of rat hind limb muscles following gravitational unloading. Jpn J Physiol 52: 235–245, 2002.[CrossRef][ISI][Medline]
  24. Ohira Y, Tanaka T, Yoshinaga T, Kawano F, Nomura T, Nonaka I, Allen DL, Roy RR, and Edgerton VR. Ontogenetic, gravity-dependent development of rat soleus muscle. Am J Physiol Cell Physiol 280: C1008–C1016, 2001.[Abstract/Free Full Text]
  25. Picquet F, Stevens L, Butler-Browne GS, and Mounier Y. Differential effects of a six-day immobilization on newborn rat soleus muscles at two developmental stages. J Muscle Res Cell Motil 19: 743–755, 1998.[CrossRef][Medline]
  26. Picquet F, Bouet V, Canu MH, Stevens L, Mounier Y, Lacour M, and Falempin M. Contractile properties and myosin expression in rats born and reared in hypergravity. Am J Physiol Regul Integr Comp Physiol 282: R1687–R1695, 2002.[Abstract/Free Full Text]
  27. Picquet F and Falempin M. Compared effects of hindlimb unloading vs. terrestrial deafferentation on muscular properties of the rat soleus. Exp Neurol 182: 186–194, 2003.[CrossRef][ISI][Medline]
  28. Picquet F, De-Doncker L, and Falempin M. Expression of myosin heavy chain isoforms in rat soleus muscle spindles after 19 days of hypergravity. J Histochem Cytochem 51: 1–11, 2003.[Abstract/Free Full Text]
  29. Ribotta M, Sandillon F, and Privat A. Influence of hypergravity on the development of monoaminergic systems in the rat spinal cord. Brain Res Dev Brain Res 111: 147–157, 1998.[Medline]
  30. Ricart-Firinga C, Stevens L, Canu MH, Nemirovskaya TL, and Mounier Y. Effects of beta(2)-agonist clenbuterol on biochemical and contractile properties of unloaded soleus fibers of rat. Am J Physiol Cell Physiol 278: C582–C588, 2000.[Abstract/Free Full Text]
  31. Schiaffino S, Gorza L, Sartore S, Saggin L, Ausoni S, Vianello M, Gundersen K, and Lomo T. Three myosin heavy-chain isoforms in type 2 skeletal muscle fibres. J Muscle Res Cell Motil 10: 197–205, 1989.[CrossRef][ISI][Medline]
  32. Stevens L, Bastide B, Kischel P, Pette D, and Mounier Y. Time-dependent changes in expression of troponin subunit isoforms in unloaded rat soleus muscle. Am J Physiol Cell Physiol 282: C1025–C1030, 2002.[Abstract/Free Full Text]
  33. Toursel T, Bastide B, Stevens L, Rieger F, and Mounier Y. Alterations in contractile properties and expression of myofibrillar proteins in wobbler mouse muscles. Exp Neurol 162: 311–320, 2000.[CrossRef][ISI][Medline]
  34. Walton K, Heffernan C, Sulica D, and Benavides L. Changes in gravity influence rat postnatal motor system development: from simulation to space flight. Gravit Space Biol Bull 10: 111–118, 1997.[Medline]
  35. Winiarski AM, Roy RR, Alford EK, Chiang PC, and Edgerton VR. Mechanical properties of rat skeletal muscle after hind limb suspension. Exp Neurol 96: 650–660, 1987.[CrossRef][ISI][Medline]
  36. Wubbels RJ and de Jong HA. Vestibular-induced behaviour of rats born and raised in hypergravity. Brain Res Bull 52: 349–356, 2000.[CrossRef][Medline]



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