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ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY
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
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ABSTRACT |
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 TOP ABSTRACT MATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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muscular properties; myosin transition; gravity change; hindlimb muscle
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.
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MATERIAL AND METHODS |
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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.
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RESULTS |
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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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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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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 |
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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 
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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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GRANTS |
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ACKNOWLEDGMENTS |
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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. Section 1734 solely to indicate this fact.
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, type IIA fiber; a thin solid arrow, fiber expressing the antibodies MHC I + MHC IIA; and a thick arrow, type I fiber.
