AJP - Regu AJP citation statistics
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 282: R1687-R1695, 2002; doi:10.1152/ajpregu.00643.2001
0363-6119/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Picquet, F.
Right arrow Articles by Falempin, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Picquet, F.
Right arrow Articles by Falempin, M.
Vol. 282, Issue 6, R1687-R1695, June 2002

Contractile properties and myosin expression in rats born and reared in hypergravity

F. Picquet1, V. Bouet2, M. H. Canu1, L. Stevens1, Y. Mounier1, M. Lacour2, and M. Falempin1

1 Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cedex; and 2 Laboratoire de Neurobiologie des Restaurations Fonctionnelles, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6562, Université de Provence, 52, Faculté de St Jérôme, 13397 Marseille Cedex 20, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of hypergravity (HG) on soleus and plantaris muscles were studied in Long Evans rats aged 100 days, born and reared in 2-g conditions (HG group). The morphological and contractile properties and the myosin heavy chain (MHC) content were examined in whole muscles and compared with terrestrial control (Cont) age-paired rats. The growth of HG rats was slowed compared with Cont rats. A decrease in absolute muscle weight was observed. An increase in fiber cross-sectional area/muscle wet weight was demonstrated, associated with an increase in relative maximal tension. The soleus muscle changed into a slower type both in contractile parameters and in MHC content, since HG soleus contained only the MHC I isoform. The HG plantaris muscle presented a faster contractile behavior. Moreover, the diversity of hybrid fiber types expressing multiple MHC isoforms (including MHC IIB and MHC IIX isoforms) was increased in plantaris muscle after HG. Thus the HG environment appears as an important inductor of muscular plasticity both in slow and fast muscle types.

Long Evans rats; soleus; plantaris; myosin heavy chain; contractile parameters


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MANY STUDIES HAVE INVESTIGATED the remarkable capacity of the muscular tissue to adapt to environmental stimuli. Among many factors, gravity has been shown to induce neuromuscular changes. For instance, skeletal muscles of rats are atrophied under unloading conditions during spaceflight or simulated microgravity, and the degree of the modifications is differential according to the muscle type and muscle function (7, 10). The changes are greater in slow extensor muscles than in fast ones. In the soleus, which is a slow postural muscle, several changes have classically been described in the literature (7) after episodes of microgravity (real or simulated) as follows: 1) a muscular atrophy characterized by decreases in absolute muscle weight and fiber cross-sectional area (CSA); 2) a decrease in absolute and relative strength; and 3) a decrease in twitch contraction time confirmed by changes in histochemical (ATPase staining) and electrophoretic properties [contents in myosin heavy chain (MHC) isoforms; see Ref. 36] during which the soleus muscle changed into a faster type. In the plantaris muscle, which is the fast agonist of the soleus muscle, the following less drastic effects have been reported: 1) a slight muscular atrophy after simulated microgravity (4, 8, 39); 2) no change in the relative tetanic tension (4, 8), and 3) no change (8) or an increase in time to peak (TTP; see Ref. 4).

On the contrary, the impacts of a hypergravity (HG) environment on the muscular system were less substantiated. Until now, studies performed on cockerels or rats (young or adult) during short or long periods of HG exposure have described modifications in the body and muscular masses, protein synthesis rate, and RNA and enzyme activities (3, 6, 15, 21, 31).

In most of the studies performed in HG, the animals were conceived and born in normal gravity and then exposed to HG. Presently, there are no data relative to the morphological, contractile, and biochemical behavior of muscles from rats born and reared in HG. Therefore, the aim of this work was to study the morphological muscle characteristics, contractile properties, and MHC content in two hindlimb extensor muscles [a slow one (the soleus) and its fast agonist (the plantaris)]. According to the function of these two muscles, we predicted that these muscles were more activated during HG and, probably, the soleus muscle became slower and the plantaris muscle became faster.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal groups. Experiments were performed on 55 male Long Evans rats aged 100 days and divided into the following two groups: control rats (Cont, n = 25) and HG rats (n = 30). The 30 HG animals were selected from a population of the first generation derived from couples mated in the chronic centrifuge apparatus. After 15 days, males were taken off, and females stayed until weaning. We studied male rats descending from different littermates. The Cont rats were reared in conditions similar to those of the centrifuge, i.e., the same room, same dark-light cycle (12:12 h), and same temperature inside a standard home cage contained in a gondola. The contractile and morphological properties were determined in right soleus muscles of 11 Cont and 15 HG rats and in right plantaris muscles of 14 Cont and 15 HG rats.

The experiments and the maintenance conditions of the animals were approved by both the Agricultural and Forest Ministry and National Education Ministry (authorization 03805).

Centrifugation apparatus. The apparatus consisted of a velocity-controlled direct-current 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 four extremities of the horizontal arms, 76.5 cm away from the axis of rotation; the gondolas were equipped with an aeration system, lights that reproduced a 12:12-h light-dark cycle, and standard home cages for rats. One gondola was equipped with a video system composed of a wide-angle video camera connected to a video monitor, which allowed knowledge of the exact birth date of litters and to allow control of the movement ability of the animals. During centrifugation, the gondolas were tilted at a constant angle of 60° from vertical, depending on the chosen speed. 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 rat, this angular velocity led to 2-g resultant force. The resultant force was directly measured using a standardized force sensor at the center of the floor of the gondola.

During centrifugation, rats were subjected to a gravitoinertial force vector (Z vector) whose direction was always similar to that exerted in normal gravity (parallel to the dorsoventral axis of the animal), and magnitude was two times the normal magnitude exerted on Earth. Consequently, the rats were also subjected to Coriolis accelerations. The amplitude of Coriolis accelerations depended on the speed of animal locomotion within the home cage and led to a deviation of the rat movement trajectory.

For animal care (cleaning and feeding), the centrifugation was stopped for 15 min every week. Food and water were available ad libitum. Food intake was measured. No statistical difference was found between Cont and HG rats except during 7 days after weaning. At this period, the HG rats showed a slight decrease in food intake. However, after acclimation, the HG rats gained body mass at a rate similar to Cont, but their mass remained lower than Cont animals.

Surgical techniques and physiological measurements. The physiological studies were performed 1) in the right soleus of 11 Cont rats and 15 HG rats and 2) in the right plantaris muscles of 14 Cont rats and 15 HG rats. Animals were anesthetized with pentobarbital sodium (30 µg/g). Supplementary doses were injected if necessary (15 µg/g). Under deep anesthesia, assessed by the absence of blink reflexes, all of the muscles of the right thigh and lower limb were denervated, except the studied muscle (either the soleus or the plantaris). 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 muscle (soleus or plantaris) was isolated from surrounding tissues, and its tendon was sutured by a short 3.0 silk suture around a force transducer (FT 10; Grass). Its length was adjusted to produce a maximal twitch tension. The stimulating (Teflon-coated platinum, ø: 125 µm) and reference (Teflon-coated platinum, ø: 250 µm) electrodes were maintained by micromanipulators. Muscle contractions were induced through a stimulating monopolar electrode put under the soleus or the plantaris nerves. The reference electrode was inserted in the adjacent denervated muscle mass. The TTP and the half-relaxation time (HRT) were measured from the maximal twitch tension (Pt) recording. A series of stimulation frequencies, from 20 to 120 Hz, was also applied and permitted to establish the force/frequency relationship from which several parameters could be determined, such as the maximal tetanic tension (P0) obtained at 100 Hz and the ratios between unfused tetanic tension and fused tetanic tension: P20/P0 (tetanic tension at 20 Hz divided by P0) for the soleus muscle and P40/P0 (tetanic tension at 40 Hz divided by P0) for the plantaris muscle, which are indicators of the muscle type (44).

After the measurements of contractile properties, the soleus and plantaris muscles were excised, weighed, and divided into two parts that were frozen in isopentane precooled in liquid nitrogen. One of the two parts was used to determine the MHC isoform composition by SDS-PAGE analysis. On the second part, the muscle typing was established with immunohistochemical methods. After the experiments, animals were killed with a lethal dose of pentobarbital sodium.

Determination of MHC isoforms by SDS-PAGE. The MHC isoforms were determined in five Cont and five HG soleus muscles and in five Cont and five HG plantaris muscles. The first part of the frozen tissues was pulverized under liquid nitrogen in a small steer mortar (30). As already described (40), the MHC composition in plantaris and soleus muscles was determined on a 4.5% glycerol stacking gel and on a 7.5% glycerol separating gel. Electrophoresis was run for 18 h at 12°C (180 volts constant, 13 mA/gel). After the gels were run, the gel slabs were silver stained. The relative proportion of each MHC isoform in each muscle type was determined using a scanning densitometer (Quantiscan Microvial Systems; Biosoft).

Characterization of muscle fiber types by immunohistochemistry. The fiber types were determined in five Cont and five HG soleus and in five Cont and in five HG plantaris muscles by ATPase staining, on which the muscle fiber types were assessed by immunolabeling of MHC isoforms. Frozen serial sections (10 µm thick) were obtained in the second part of soleus and plantaris muscles. The myosin ATPase activity was revealed both with alkaline (pH 10.4) and acid preincubations (pH 4.3 and 4.45) according to the method of Guth and Samaha (13). Additional sections were treated with a double preincubation (32) that differentiated, in a fast muscle, only fibers expressing MHC IIX. The sections were preincubated for 15 min at room temperature in a 20 mM glycine buffer solution containing 20 mM CaCl2 (pH 10.4). They were fixed for 3 min in a 2% methanol-free paraformaldehyde solution containing 0.2 M sodium cacodylate and 75 mM CaCl2 with 11.5% sucrose (pH 7.2) and then further preincubated for 15 min (room temperature) in a solution containing 0.1 M sodium acetate and 0.1 M potassium chloride (pH 4.5, adjusted with acetic acid). Washings with a solution containing 0.1 M Tris and 18 mM CaCl2 (pH 7.2) were made between preincubations and the fixation. Incubation was performed for 25 min at room temperature in a medium containing 40 mM glycine buffer, 20 mM CaCl2, and 2.5 mM ATP (pH 9.4). The sections were then rinsed with 1% CaCl2 and were stained with 2% CoCl2 and 5% (NH4)2S. For the immunohistochemically treated sections, the protocol has already been described in detail (29). The following antibodies were used: NCL-MHCs (Tebu/Novocastra), which recognize MHC I; SC-71 (DSMZ) against MHC IIA; BF-F3 (DSMZ) against MHC IIB; and MY32 (Sigma) against MHC IIA, IIB, and IIX. These antibodies were diluted, respectively, at 1:20, 1:20, 1:10, and 1:2,000. The muscle typing was established on a total of 300 fibers/muscle immunologically identified in serial sections. The CSA of the fibers was also measured on ATPase sections using an image analyzer (Samba, Grenoble, France) on a minimum of 300 fibers/muscle (11 Cont and 15 HG soleus and 14 Cont and 15 HG plantaris).

Statistical analysis. All results are expressed as means ± SE. For the two muscles, after a one-way ANOVA, the intergroup comparisons (soleus HG vs. Cont, plantaris HG vs. Cont) were performed using Student's t-test with a significance level of P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Morphological data. The morphological parameters are reported in Table 1. The values of body weights (BW), absolute [muscle wet weight (MWW)], and relative (MWW/BW) muscle weights showed that, after 100 days of HG, the rats presented BW (-40%) and MWW (soleus -42%, plantaris -46%) significantly smaller than their age-paired Cont groups. However, when the results were expressed in terms of MWW/BW, the ratios were not significantly different compared with Cont data. The CSA of the fibers were measured on a minimum of 300 fibers/muscle: on slow fibers (expressing only the slow MHC I isoform) and on intermediate (int) + fast fibers (expressing a combination of slow MHC I isoform with one or more fast MHC isoforms). With the aim to clarify the data and to perform a statistical analysis, these populations were expressed in terms of "slow type" and "int + fast types." The absolute CSA of slow and int + fast fiber types decreased significantly in each HG muscle when compared with paired Cont muscles. In the soleus muscle, the decrease reached 30% for the CSA of slow type fibers, whereas in plantaris muscle this decrease was 41 and 17% for the CSA of slow and int + fast type fibers, respectively. On the contrary, when normalized to MWW, the CSA were increased significantly by 20% for the slow fibers in the HG soleus muscle and by 54% for int + fast fibers in the HG plantaris muscle.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Mean values of morphological parameters

Contractile characteristics. The contractile parameters of Cont and HG soleus and Cont and HG plantaris are reported in Table 2. After HG, the soleus muscles showed significantly increased values in kinetic parameters (TTP: +51%, HRT: +76%, P20/P0: +19%). The absolute twitch and maximal tetanic forces were reduced significantly (-23 and -31%, respectively). On the contrary, when normalized to MWW, twitch and maximal tetanic forces increased (+44 and +9%, respectively). The plantaris muscle was differently affected by an HG period, since TTP, HRT, and P40/P0 were decreased (by -10, -42, and -20%, respectively). The absolute twitch and maximal tetanic forces were decreased (-28 and -44%), whereas normalized maximal twitch force increased significantly by 40% and maximal tetanic force remained unchanged.

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Mean values of studied contractile parameters for the soleus and plantaris muscles in cont and HG groups

Electrophoretic determination of MHC isoforms. An example of electrophoretic migration profiles is given in Fig. 1A. Four MHC isoforms were determined according to their increasing order of electrophoretic mobility (MHC IIA, IIX, IIB, and I). In a previous study (30), we established the level of migration of these isoforms in our gels. Two MHC isoforms were detected in the Cont soleus [the predominant slow MHC I and the fast MHC IIA isoform (Fig. 1, lane 1)]. After HG, the MHC IIA band disappeared (Fig. 1, lane 2). Figure 1, lanes 3 and 4, show plantaris muscles for Cont and HG groups, respectively. The four MHC isoforms were expressed both in the Cont and HG plantaris muscles.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 1.   A: electrophoretic determination of myosin heavy chain (MHC) isoforms. The MHC isoforms were identified in increasing order of their electrophoretic mobility. Lane 1, control (Cont) soleus; lane 2, hypergravity (HG) soleus; lane 3, Cont plantaris; lane 4, HG plantaris. B: densitometric analysis of the identified MHC isoforms. MHC isoform composition, measured by densitometry, was determined for soleus and plantaris muscles. Results are expressed as means ± SE. * Statistical difference between HG and Cont paired groups.

A densitometer analysis, presented in Fig. 1B, permitted determination of the relative proportion of each MHC isoform. The Cont soleus was composed of 63.8% MHC I and 36.2% MHC IIA. The HG situation induced the disappearance of MHC IIA, with HG soleus muscles containing only the MHC I isoform (100%). The Cont and HG plantaris muscles expressed the four MHC isoforms (IIA, IIX, IIB, and I) in proportions that were not significantly different between Cont and HG groups.

Immunological identification of muscle fiber types by their MHC content. The identification of muscle fiber types was performed by histochemistry associated with immunological staining. On serial sections, the fiber MHC isoform expression was determined in a total of 300 fibers/muscle. Figure 2 shows the antibody reactivity in Cont muscles [soleus (Fig. 2, 2 and 3) and plantaris (Fig. 2, 7-10)] and in the HG muscles [soleus (Fig. 2, 4 and 5) and plantaris (Fig. 2, 12-15)]. Positive fibers were characterized by a dark color; negative fibers showed no color, except for the antibody against MHC IIB in which negative fibers were slightly colored. The ATPase method, which allowed differentiation of fibers containing the MHC IIX isoform, is shown in Fig. 2, 1 (Cont soleus), 6 (Cont plantaris), and 11 (HG plantaris). The fibers that were weakly colored (light staining) did not express the MHC IIX isoform, whereas the dark fibers contained the isoform.


View larger version (184K):
[in this window]
[in a new window]
  		Fig. 2.   Muscle typing determined by immunohisto- and histochemical methods. 1-3 and 4-5, Cont and HG soleus muscles, respectively; 6-10 and 11-15, Cont and HG plantaris muscles, respectively. Reactivity of the antibodies is presented as follows: anti-MHC I (2, 4, 7, and 12), anti-MHC IIA (3, 8, and 13), anti-MHC IIB (9 and 14, on which positive fibers are indicated by black arrows), and anti MHC II (5, 10, and 15); 1, 6, and 11 show the histochemical method that differentiates, by a dark color, fibers expressing MHC IIX. Scale bar = 140 µm.

The Cont soleus muscles contained fibers that expressed the MHC I and IIA isoforms, whereas, after HG, the slow MHC I was only expressed. In these two groups, no type IIX was detected. In the Cont and HG plantaris muscles, the four MHC isoforms were expressed, since positive fibers were identified in all sections. Indeed, some fibers of the plantaris muscles showed a dark staining indicating the presence of MHC IIX.

Using the combination of enzyme histochemistry and immunochemistry, we were able to distinguish several MHC isoform coexpressions (Fig. 3). The fiber types were classified according to their MHC expressions in 1) pure types, corresponding to the expression of only one MHC isoform, slow (I) or fast (IIA, IIX, or IIB), 2) hybrid types presenting two, three, or four MHC isoforms according to a coexpression order based on the "next-neighbor rule" i.e., Iright-arrowIIAright-arrowIIXright-arrowIIB, and 3) atypic hybrid types, corresponding to a coexpression of two, three, or four MHC isoforms with gaps in the transition order.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3.   Repartition of fiber types within each muscle. Fibers were classified according to their MHC isoform coexpressions. Results are expressed as means ± SE. * Statistical difference between HG (A) and Cont (B) paired group.

In the Cont soleus muscle, the following three fiber populations could be detected: two pure fiber types, the predominant type I (68%) and the type IIA (27%); and one hybrid population (5%) with MHC I and MHC IIA being coexpressed. After HG, only the pure slow type (100% MHC I) was identified in the soleus muscle.

The plantaris muscle showed multiple MHC isoform combinations. In the Cont plantaris, we distinguished four pure populations of fibers expressing either the MHC I isoform (11%), the MHC IIA isoform (32%), the MHC IIX isoform (2.6%), or the MHC IIB isoform (2.7%). Five hybrid types coexpressed multiple MHC isoforms according to the next-neighbor rule as follows: Iright-arrowIIAright-arrowIIXright-arrowIIB, i.e., I + IIAright-arrow2%, IIA + IIXright-arrow29%, IIX + IIBright-arrow6%, I + IIA + IIXright-arrow0.25%, and IIA + IIX + IIBright-arrow12.5%. Three atypic hybrid types were also detected as follows: I + IIXright-arrow0.25%, IIA + IIBright-arrow1.5%, and I + IIA + IIBright-arrow0.2%.

In the HG plantaris, the pure fiber types represented a similar content as in Cont muscles (~48%). However, the fibers containing MHC I and MHC IIA isoforms were decreased by 5.2 and 17.7%, respectively, whereas the fibers containing MHC IIX or MHC IIB were overexpressed by 12.6 and 11.5%, respectively. Concerning the hybrid types, a slight redistribution between the proportion in the different fiber types and an increase in the number of hybrid groups appeared. Moreover, the atypic hybrid fiber types appeared increased, and the proportion of such fibers reached 5% after HG.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study presents, for the first time, the impacts of a long stay in an HG environment on rats submitted from their conception until 100 days postbirth to HG conditions. We chose to compare the effects of this long HG duration on two ankle extensor muscles [a slow one (the soleus) and its fast agonist (the plantaris muscle)]. Thus morphological, contractile properties and MHC contents of these muscles were analyzed. The primary findings showed that 1) the animal growth was slowed down, with associated decreases in the two muscle weights being observed, 2) the relative maximal tensions of the two muscles were increased after HG, and 3) the contractile properties of each muscle were modified according to changes in their phenotypes. Thus, after HG, the slow soleus became slower and the fast plantaris presented a higher proportion of pure fibers that expressed the fastest MHC isoforms (IIX and IIB). These main results were discussed and compared with those obtained in a real or simulated microgravity situations.

Effects of HG on morphological properties and muscle strength. The body weight of growing rats was significantly lower (-40%) under 2-g loading, and the mean absolute masses for soleus and plantaris muscles were also decreased by 42 and 46%, respectively. The effects of a 2-g environment on growing rats could appear relatively surprising. In fact, the physiological function of these two agonist muscles is an antigravity function (20), and one may expect a hypergrowth under an HG situation. Our results showed a decrease in muscle weights. This may be the consequence of a decrease in protein synthesis resulting from the HG environment. Indeed, previous studies have demonstrated that HG induced, in growing rats, a marked decrease in the weight and muscular protein content (21). However, our data showed that the ratio MWW/BW was unchanged after HG both in soleus and plantaris muscles. This result supported the hypothesis that HG induced a slowing down in the growth of the entire animal. This might be a consequence of a nutritional deficiency already described in HG conditions (11, 41) and/or a decrease in the muscle body fat reserves (11, 14). Moreover, a transient elevation of circulating corticosterone that returns to control levels after 5 days was demonstrated during HG (27). This increase in corticosteone triggered the lipolysis (for review, see Ref. 24). Taking into account other data previously published, it also appeared that HG may have differential effects, depending on the species, age, and sex of the animals (rats) and the duration of HG. Indeed, Roy et al. (31) described in adult male Wistar rats submitted 2 wk at 2 g 1) no change in the absolute soleus and medial gastrocnemius muscle mass and 2) a significant increase of the relative muscle mass. On the contrary, Amtmann and Oyama (3) described, in female Sprague-Dawley rats, a decrease in body mass after a 810-day exposition at 2.76 g. These latter results are in agreement with our data.

Our results showed that, after HG, absolute values of CSA were significantly lower than age-matched Cont. Indeed, the absolute CSA of the slow fibers decreased in the soleus by 30%. In the plantaris, the CSA of slow and fast fibers decreased by 41 and 17%, respectively. However, the analysis of the normalized CSA (CSA/MWW) revealed, for each fiber type, an increase by 20% in the soleus (100% slow fibers) and by 54% in the fast fibers of the plantaris (95% of the total fiber content) after HG. Therefore, the fiber atrophy was exceeded by total body weight loss (and MWW) so that the ratio was increased. This could indicate that protein in the muscle was slightly spared compared with protein in other tissues. Moreover, it is not clear whether the water content was changed by 2-g conditions. The relative CSA would increase if muscle water was reduced, thus inducing an MWW decrease. Unfortunately, we have no measurement of the water content in our conditions. However, it has been reported that the water balance in rats exposed to a chronic centrifugation was not modified after 12 days of HG (26). Finally, it should be pointed out that HG affected slow and fast fibers, whereas, in microgravity, atrophy was more developed in slow than in fast fibers (7, 10).

Another aim of this study was to determine the effects of HG on the force level developed by the soleus and the plantaris muscles. Our results showed that absolute Pt and P0 significantly decreased in the soleus (-23 and -31%, respectively) and in the plantaris (-28 and -44%, respectively). When normalized to MWW, the maximal twitch forces increased in both muscles, and the maximal tetanic tension increased significantly only in the soleus. Previous studies (23) have suggested that cross-bridge formation involves intermediate steps in which the cross-bridge transforms from a low- to a high-force state. In microgravity, a decline in force per CSA could reflect a drop in the force level per cross-bridge and/or a reduction in the number of cross-bridges consecutive to a loss of myofibrillar protein (22). Taking into account the increase in force after HG, we suggested that the force developed per cross-bridge was reinforced in HG conditions. However, we cannot rule out a supplementary hypothesis, i.e., a possible increase in the number of cross-bridges, perhaps because of an increased intracellular Ca2+ concentration or because of a link to a modification in the filament lattice spacing, an hypothesis already proposed for microgravity (22). These results thus clearly demonstrate the reverse of observations in microgravity, which first demonstrate either losses of forces (soleus muscle; see Ref. 7) or no change (plantaris; see Ref. 4) and, second, a much larger effect in slow than in fast muscles (44).

Effects of HG on kinetics and fiber typing. In the soleus muscle, our results showed that the kinetic parameters were modified after a 100-day period of HG. TTP, HRT, and the P20-to-P0 ratio significantly increased (+51, +77, and +19%, respectively) and changed into those of a slower muscle type. The acquisition of slower contractile parameters was confirmed by the electrophoretic and histochemical profiles. Indeed, after this 2-g period, only the MHC I isoform was detected in all of the studied fibers. MHC IIA and type IIA fibers identified by SDS-PAGE and ATPase staining, respectively, and normally expressed in the Cont rats had disappeared in the HG soleus muscle. These results were in agreement with those of Martin (19) who observed that HG resulted in a progressive increase in the proportion of slow oxidative fibers, up to 100% in developing Sprague-Dawley rats, when the centrifugation was initiated at 30 days of age. On the contrary, using 60-day-old rats, Roy et al. (31) demonstrated that, after 2 wk at 2 g, the soleus of centrifuged rats had a slightly lower percentage of fibers expressing the MHC I isoform and a higher percentage of fibers (15 vs. 10%) that coexpressed type I and IIA MHC isoforms. Therefore, these differential effects might again be linked to the animals and to their developmental state when HG was applied. It has been demonstrated that, in the soleus muscle, the MHC isoforms and their mRNA expression were not modified during postnatal undernutrition (43).

In the plantaris muscle, HG conditions induced decreases in TTP (-11%), HRT (-42%), and the P40-to-P0 ratio (-20%). This acquisition of faster contractile properties could be related to the reorganization in the proportions of pure fibers, especially the increase in fibers expressing the fastest MHC isoforms (IIX and IIB). The HRT parameter has classically been described as dependent on the relative expression of the different sarco(endo)plasmic reticulum Ca2+ (SERCA) isoforms (18). The changes observed in HRT values, i.e., increase in the soleus muscle and decrease in the plantaris muscle, suggested that, after HG, the levels in the slow SERCA 2a isoform and the fast SERCA 1a isoform were overexpressed in the two muscles.

Consequently, both slow and fast muscles were affected by HG, whereas, in simulated or real microgravity, fast muscles were less modified than slow muscles. Indeed, in soleus muscles, a decrease in normalized force concomitant with a decrease in slow MHC and with an increase in fast MHC isoforms has been commonly described after microgravity (7, 10, 44). In the unloaded soleus muscle, the decreases in TTP, HRT, and force were correlated with changes in SERCA kinetics and with a de novo appearance of fibers coexpressing MHC I and/or various MHC II isoforms (for review, see Ref. 25). On the contrary, normalized tensions of fast muscles such as gastrocnemius, tibialis anterior, or extensor digitorum longus were maintained after microgravity (for review, see Ref. 7). Moreover, the myosin distribution was not modified in the medial portion of the gastrocnemius and tibialis anterior (25). Thus it appears that HG had a more global impact than microgravity on the muscular system.

Effects of HG on the MHC repartition and their regulation. Our results demonstrated that the HG environment had a real impact on MHC isoform transitions. After HG, the soleus muscle became slower, after the transition MHC IIAright-arrowMHC I. Indeed, only the pure fiber type remained, expressing the MHC I isoform. On the contrary, in plantaris muscles, HG induced a subtle reorganization of the MHC expression within the pure and hybrid fibers without modification of the total content in each MHC isoform. We suggested that generally the transitions followed the next-neighbor rule as proposed by Pette and Staron (28). Interestingly, in plantaris muscles submitted to HG, a larger amount of fibers (5% in HG vs. 1.5% in Cont conditions) presented atypic MHC expressions. The increase in fibers that coexpressed multiple MHC isoforms was characteristic of a muscle presenting a state of transition, as already described in spaceflight (2, 38), after hindlimb unloading (9, 38) and electrical stimulation (34). Thus all of the results indicated that HG induced changes in the expression of the MHC isoforms, since it was also described in microgravity. However, the results were the opposite. In fact, in real or simulated microgravity, the soleus muscle acquired fast MHC isoforms (36), and few or no changes appeared in fast muscles (35). Conversely, it is interesting to note that HG induced changes in the fast plantaris. The changes in MHC isoforms resulting from microgravity have been proven to be related to their different mRNA expression (36). Therefore, we can suggest that the alterations in the MHC isoform content after HG could result from a modification in mRNA expression levels.

It has been well established that mechanical properties (17) and the myosin expression pattern (5) were dependent on motor innervation. It has been demonstrated that, in rat soleus muscle, the suppression of motor innervation induces an increase in fiber diversity (37). Moreover, a neonatal denervation blunted the upregulation of MHC IIB in the plantaris muscle and of MHC I in the soleus muscle and shifted the pattern to greater expression of MHC IIA and IIX in both muscles (1). In our conditions, the new myosin repartition after HG, in soleus and plantaris muscles, should be a direct consequence of a modification and/or an increase in the motor nervous message during HG. To support this hypothesis, it has been previously demonstrated that, during the two HG phases of parabolic flight, the electromyographic activity of the rat soleus muscle was immediately strongly increased (16). This increase originated from the recruitment of new motor neurons, with a shift toward higher-amplitude units being observed. It can therefore be argued that, during the 100-day period of the 2-g environment, an increase in the recruitment of soleus motor neurons was achieved and influenced the myosin expression during development. Moreover, in terrestrial conditions, the extensor muscle activity of hindlimb is regulated by information originating from vestibular receptors. It has been demonstrated that, during spaceflight, this information is reduced (42). Inversely, it is tempting to suppose that, during HG, the vestibular information was increased and therefore hindlimb muscles received an increased motor discharge. Moreover, it has been demonstrated in the rat, by using the Fos protein expression, that the otolith-vestibulo-olivar pathways are strongly activated by an increase of the gravitoinertial forces (12).

Conclusion

Our results show that HG produced some inverse effects to those observed in real or simulated microgravity. Indeed, both soleus and plantaris muscles were affected by HG. The soleus muscle presented an increase in relative maximal tension and a change to a slower type, as a function of both kinetic parameters and MHC isoform expression. The fast plantaris became a faster muscle (kinetic parameters increased). Moreover, a new diversified repartition of the MHC isoforms was revealed in this fast muscle. Our results thus demonstrated that the slow and fast extensor muscles presented a specific adaptation to a chronic HG. Finally, in the soleus muscle, the changes seemed to be the result of a qualitative continuity from micro- to hypergravitational effects; for instance, microgravity induces decreases in force, TTP, and MHC I expression; additionally, 1 g represents normality and 2 g induces increases in force, TTP, and MHC I expression. In the plantaris muscle, this continuity rule did not fully apply since this fast muscle was slightly affected by microgravity.

Gathered together with data obtained in microgravity conditions, our results underline the importance of the gravity factor in muscle physiology.


    ACKNOWLEDGEMENTS

We thank Dr. G. S. Butler-Browne for the growing of hybridomas producing SC-71 and BF-F3 antibodies initially developed by Schiaffino et al. (33) and Laetitia Cochon for technical assistance. The hybridomas are commercialized by Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany.


    FOOTNOTES

The Laboratoire de Plasticité Neuromusculaire was supported by grants from the Centre National d'Etudes Spatiales (3194), the Conseil Régional du Nord-Pas-de-Calais, and the Fonds Européen de Développement Régional (F 007), and the Laboratoire de Neurobiologie des Restaurations Fonctionnelles was supported by UMR 6562 Centre National de la Recherche Scientifique/Université de Provence and by grants from the Centre National d'Etudes Spatiales.

Address for reprint requests and other correspondence: F. Picquet, Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille, Bâtiment 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.

10.1152/ajpregu.00643.2001

Received 31 October 2001; accepted in final form 22 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adams, GR, McCue SA, Zeng M, and Baldwin KM. Time course of myosin heavy chain transitions in neonatal rats: importance of innervation and thyroid state. Am J Physiol Regulatory Integrative Comp Physiol 276: R954-R961, 1999[Abstract/Free Full Text].

2.   Allen, DL, Yasui W, Tanaka T, Ohira Y, Nagaoka S, Sekiguchi C, Hinds WE, Roy RR, and Edgerton VR. Myonuclear number and myosin heavy chain expression in rat soleus single muscle fibers after spaceflight. J Appl Physiol 81: 145-154, 1996[Abstract/Free Full Text].

3.   Amtmann, E, and Oyama J. Effect of chronic centrifugation on the structural development of the musculoskeletal system of the rat. Anat Embryol (Berl) 149: 47-70, 1976[Medline].

4.   Brown, M, and Hasser EM. Differential effects of reduced muscle use (hindlimb unweighting) on skeletal muscle with aging. Aging Clin Exp Res 8: 99-105, 1996[Medline].

5.   Butler-Browne, GS, and Whalen RG. Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle. Dev Biol 102: 324-334, 1984[ISI][Medline].

6.   Chi, MMY, Manchester JK, and Lowry OH. Effect of centrifugation at 2G for 14 days on metabolic enzymes of the tibialis anterior and soleus muscles. Aviat Space Environ Med 69: A9-A11, 1998[Medline].

7.   Edgerton, VR, and Roy RR. Neuromuscular adaptations to actual and simulated spaceflight. In: Handbook of Physiology, Environmental Physiology. Bethesda, MD: Am. Physiol. Soc, 1996, vol. 1, p. 721-763.

8.   Elder, GCB Effect of hindlimb suspension on the development of dystrophic hamster muscle. Exp Neurol 99: 187-200, 1988[Medline].

9.   Fauteck, SP, and Kandarian SC. Sensitive detection of myosin heavy chain composition in skeletal muscle under different loading conditions. Am J Physiol Cell Physiol 268: C419-C424, 1995[Abstract/Free Full Text].

10.   Fitts, RH, Riley DR, and Widrick JJ. Physiology of a microgravity environment. J Appl Physiol 89: 823-839, 2000[Abstract/Free Full Text].

11.   Fuller, C. History of chronic acceleration: what we learned from rodent studies. In: Ann Meet Int Gravitational Physiol 22nd Budapest, Hungary, 2001.

12.   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].

13.   Guth, L, and Samaha FJ. Qualitative differences between actomyosin ATPase of slow and fast mammalian muscles. Exp Neurol 25: 138-152, 1969[ISI][Medline].

14.   Keil, LC. Changes in growth and body composition of mice exposed to chronic centrifugation. Growth 33: 83-88, 1969[ISI][Medline].

15.   Kita, F, Koike K, Metori K, and Takahashi S. Protein synthesis in fast and slow muscles of developing cockerels loaded with 2 G for 3 weeks. Aviat Space Environ Med 69: 145-148, 1998[Medline].

16.   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].

17.   Lømo, T. Long-term effects of altered activity on skeletal muscle. Biomed Biochem Acta 48: S432-S444, 1989[Medline].

18.   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 2: 889-897, 1998.

19.   Martin, WD. Effects of chronic centrifugation on skeletal muscle fibers in young developing rats. Aviat Space Environ Med 51: 473-479, 1980[Medline].

20.   Martin, WD, and Romond EH. Effects of chronic rotation and hypergravity on muscle fibers of soleus and plantaris muscles of the rat. Exp Neurol 49: 758-771, 1975[ISI][Medline].

21.   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].

22.   McDonald, KS, and Fitts RH. Effect of hindlimb unloading on rat soleus fiber force, stiffness, and calcium sensitivity. J Appl Physiol 79: 1796-1802, 1995[Abstract/Free Full Text].

23.   Metzger, JM, and Moss RL. pH modulation of the kinetics of Ca2+ sensitive cross-bridge state transition in mammalian single skeletal muscle fibers. J Physiol 428: 751-764, 1990[Abstract/Free Full Text].

24.   Moran, MM, Stein TP, and Wade CE. Hormonal modulation of food intake in response to low peptin levels induced by hypergravity. Exp Biol Med 226: 740-745, 2001[Abstract/Free Full Text].

25.   Ohira, Y. Neuromuscular adaptation to microgravity environment. Jpn J Physiol 50: 303-314, 2000[ISI][Medline].

26.   Ortiz, RM, and Wade CE. Water balance in rats exposed to chronic centrifugation. J Appl Physiol 89: 56-60, 2000[Abstract/Free Full Text].

27.   Ortiz, RM, Wang TJ, and Wade CE. Influence of centrifugation and hindlimb suspension on testosterone and corticosterone excretion in rats. Aviat Space Environ Med 70: 499-504, 1999[Medline].

28.   Pette, D, and Staron RS. Mammalian skeletal muscle fiber type transitions. Int Rev Cytol 170: 143-223, 1997[Medline].

29.   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[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.   Roy, RR, Roy RE, Talmadge RJ, Mendoza R, Grindeland RE, and Vasques M. Size and myosin heavy chain profiles of rat hindlimb extensor muscle fibers after 2 weeks at 2 G. Aviat Space Environ Med 67: 854-858, 1996[Medline].

32.   Sant'ana Pereira, JA, De Haan A, Wessels A, Moorman AF, and Sargeant AJ. The mATPase histochemical profile of rat type IIX fibres: correlation with myosin heavy chain immunolabelling. Histochem J 27: 715-722, 1995[ISI][Medline].

33.   Schiaffino, S, Gorza L, Sartore S, Saggin L, Ausoni S, Vianello M, Gunderson K, and Lømo T. Three myosin heavy chain isoforms in type 2 skeletal muscle fibres. J Muscle Res Cell Motil 10: 197-205, 1989[ISI][Medline].

34.   Staron, RS, Gohlsch B, and Pette D. Myosin polymorphism in single fibers of chronically stimulated rabbit fast-twitch muscle. Pflügers Arch 408: 444-450, 1987[ISI][Medline].

35.   Stevens, L, Firinga C, Gohlsch B, Bastide B, Mounier Y, and Pette D. Effects of unweighting and clenbuterol on myosin light and heavy chains in fast and slow muscles of rat. Am J Physiol Cell Physiol 279: C1558-C1563, 2000[Abstract/Free Full Text].

36.   Stevens, L, Sultan KR, Peuker H, Gohlsch B, Mounier Y, and Pette D. Time-dependent changes in myosin heavy chain mRNA and protein isoforms in unloaded soleus muscle of rat. Am J Physiol Cell Physiol 277: C1044-C1049, 1999[Abstract/Free Full Text].

37.   Stevens, L, Toursel T, Lenfant AM, Falempin M, and Mounier Y. Suppression of motor innervation induces fiber diversity in rat soleus muscle. Basic Appl Myol 10: 181-190, 2000.

38.   Talmadge, RJ, Roy RR, and Edgerton VR. Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers. J Appl Physiol 81: 2540-2546, 1996[Abstract/Free Full Text].

39.   Thomason, DB, Herrick RE, Surdyka D, and Baldwin KM. Time course of soleus muscle myosin expression during hindlimb suspension and recovery. J Appl Physiol 63: 130-137, 1987[Abstract/Free Full Text].

40.   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[ISI][Medline].

41.   Wade, CE. Modulation of metabolism in relation to body weight in altered gravity. In: Ann Meet Int Gravitational Physiol 22nd Budapest, Hungary, 2001.

42.   Watt, DG, Money KE, and Tomi LM. MIT/Canadian vestibular experiments on the Space lab-1 mission. III. Effects of prolonged weightlessness on a human otolith-spinal reflex. Exp Brain Res 64: 308-315, 1986[Medline].

43.   White, P, Cattaneo D, and Dauncey MJ. Postnatal regulation of myosin heavy chain isoform expression and metabolic enzyme activity by nutrition. Br J Nutr 84: 185-194, 2000[ISI][Medline].

44.   Winiarski, AM, Roy RR, Alford EK, Chiang PC, and Edgerton VR. Mechanical properties of rat skeletal muscle after hindlimb suspension. Exp Neurol 96: 650-660, 1987[ISI][Medline].

Am J Physiol Regul Integr Comp Physiol 282(6):R1687-R1695
0363-6119/02 $5.00 Copyright © 2002 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
M. F. Patterson, G. M. M. Stephenson, and D. G. Stephenson
Denervation produces different single fiber phenotypes in fast- and slow-twitch hindlimb muscles of the rat
Am J Physiol Cell Physiol, September 1, 2006; 291(3): 518 - 528.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
F. Picquet, V. Bouet, L. Cochon, M. Lacour, and M. Falempin
Changes in rat soleus muscle phenotype consecutive to a growth in hypergravity followed by normogravity
Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2005; 289(1): R217 - R224.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
L. Stevens, C. Bozzo, T. Nemirovskaya, V. Montel, M. Falempin, and Y. Mounier
Contractile properties of rat single muscle fibers and myosin and troponin isoform expression after hypergravity
J Appl Physiol, June 1, 2003; 94(6): 2398 - 2405.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Picquet, F.
Right arrow Articles by Falempin, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Picquet, F.
Right arrow Articles by Falempin, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online