Abstract
The aim of this study was to determine whether a period of 19 days in hypergravity was long enough to induce changes in the expression of myosin heavy chain (MyHC) isoforms in the muscle spindles. The soleus muscle of 10 male Wistar rats (control: CONT, n=5; hypergravity: HG, n=5) was frozen, cut into serial sections, and labeled with antibodies against MyHCs: I, IIA, IIA + IIX + IIB, slow-tonic, and α-cardiac. Forty CONT and 45 HG spindles were analyzed. The results from HG spindles compared to CONT showed that there was no change in the cross-sectional area of intrafusal fibers. However, along the entire length of B1 fibers, the expression of both MyHC I and α-cardiac was increased significantly, whereas the labeling against MyHC IIA and MyHC slow-tonic was decreased. In B2 fibers, the labeling against MyHC IIA (region A), slow-tonic (region A), and fast myosins (regions A-C) was statistically decreased. In chain fibers, the labeling against both MyHC IIA and fast MyHC was reduced significantly. We conclude that hypergravity has a real impact on the MyHC content in the muscle spindles and induces some inverse changes of those observed in hypogravity for MyHCs I, α-cardiac, and slow-tonic.
M
In extrafusal fibers, the modifications in myosin expression and, more precisely, of myosin heavy chain (MyHC) have been extensively studied. Many studies have established that the MyHC proportions are modified according to environmental changes. Among these factors, gravity changes have been shown to induce marked neuromuscular changes (for review see Edgerton and Roy 1996; Ohira 2000). In skeletal muscles, after a period of microgravity, slow postural muscles have been reported to present an important atrophy and to evolve towards a faster type. Their MyHC content was therefore changed: fast MyHC isoforms were expressed at the expense of slow isoforms. Conversely, when gravity was reinforced (i.e., hypergravity) it has been demonstrated that, in rats born and reared in this environment, slow postural muscles such as the soleus became slower and therefore, in this case, only the slow MyHC isoform was expressed (Picquet et al. 2002).
As for extrafusal fibers, the existence of fiber plasticity was also demontrated in IFs. The expression and the regulation of MyHC isoforms in IFs are very complex and depend on a complex interaction between inductive and suppressive influences of both sensory and motor innervations (for review see Pedrosa-Domellof et al. 1991; Soukup et al. 1995; Walro and Kucera 1999). Consequently, the IF MyHC content was modified as soon as the neural message was changed by deefferentation, deafferentation (Wang et al. 1997), and denervation (Soukup et al. 1995). Moreover, a recent study has shown that MyHC isoforms contained in muscle spindles were also influenced by the gravitational load even though the changes appeared less important than those observed in extrafusal fibers (De-Doncker et al. 2002). Indeed, 14 days of hindlimb unloading (or simulated microgravity) were sufficient to induce both a significant decrease in the slow-twitch MyHC I content and a significant increase in slow-tonic MyHC. Moreover, the α-cardiac MyHC isoform, which is specifically expressed in IFs, was also modified, its proportion being increased along the nuclear bag fibers.
Consequently, the myosin content in the muscle spindles was modified following the level of gravity. During simulated microgravity, the hindlimbs are not in contact with the ground or floor (Riley et al. 1990). Consequently, the postural soleus muscle is often in a shortened position. Therefore, the natural stimulus of spindles, i.e., muscle stretch, is probably reduced. It has been suggested that, in this position, afferent information could be reduced (Falempin and Fodili In-Albon 1999). Because Ia fibers project via interneurons on β and γ fusimotor neurons, which innervate the contractile part of IFs, the lack of afferent activity could in turn influence the myosin content.
On the basis of these results, it was tempting to speculate that, during a period of hypergravity, the soleus muscle could be stretched and the Ia afferent activity could be increased. We therefore suggested that the MyHC content of IFs could be modified during a stay in hypergravity.
Consequently, the aims of our study were to determine whether the expression level of MyHC isoforms was changed after a period of hypergravity and to compare the effects on muscle spindles of hypergravity with those of microgravity.
Materials and Methods
Animal Groups
Ten male Wistar rats with initial body weights of 200 g were used for this study. They were randomly divided into two groups: a control (CONT, n = 5) and a 2-g-centrifugated group (HG: n = 5) for a duration of 19 days. The experimentations were approved by both the Ministry of Agriculture and the Ministry of Education in France (Veterinary Service of Health and Animal Protection, authorization 59-00980) and by the Animal Care Committee of the Institute of Biomedical Problems in Moscow, CEI.
Centrifugation Apparatus
The centrifugation was performed in Moscow at the Institute of Biomedical Problems. The apparatus consisted of a velocity-controlled direct-current motor located in the vertical axis of the apparatus and driving two horizontal cross-arms (total length 4 m) at a constant rotation speed. Four free-swinging gondolas were jointed at the four extremities of the horizontal arms. Each gondola contained five rats allowed to maintain a natural tetrapod position. For the centrifugation, the gondolas were tilted at a constant angle of 60° from vertical. The rotations were performed at a constant velocity of 21 rad/min. Taking into account the mass and the inertia of the gondolas and the cage and rat masses, this velocity corresponded to a 2-g resultant force. Each gondola was equipped with an aeration system and with lights that reproduced a circadian rhythm (12-hr light:12-hr dark). The centrifugation was stopped daily for 15 min at 1100 hr for animal care (feeding and cleaning of the cages). Food and tapwater were available ad libitum. For the entire duration of the experiments, the control animals were maintained in the centrifuge room in cages similar to those contained in the gondolas. They were therefore submitted to the same noise, light, and temperature (20 ± 1C).
At the end of the 19 experimental days, both CONT and HG rats were anesthetized with an IP injection of sodium pentobarbital (35 mg.kg−1). The right soleus muscles were excised, weighed, and frozen in isopentane precooled with liquid nitrogen. At the end of the experimental procedure, the animals received a lethal dose of anesthetic (100 mg.kg−1).
Muscle Processing
The soleus muscles of the CONT and HG groups were cut into serial frozen transverse sections (10 μm thick) using a cryostat microtome (Leica CM 1800; Heidelberg, Germany) set at −20C. This specific protocol has already been described by De-Doncker et al. (2002). Briefly, every 280 μm along the muscle, eight sections were made perpendicular to the muscle longitudinal axis. The first section was used as a negative control in the IHC analysis. The next two sections were processed for myofibrillar adenosine 5′-triphosphatase (ATPase) with acid (pH 4.3) and alkaline (pH 10.4) preincubations according to the method of Guth and Samaha (1969). On these sections, we measured the cross-sectional area (CSA) of IFs with an image analyzer (SAMBA 2005; Grenoble, France) in the regions A (equatorial and juxtaequatorial), B (from the end of the periaxial space to the end of the capsule), and C (extracapsular region), these regions being described by Kucera et al. (1978). The remaining sections were labeled with antibodies against myosin heavy chain (MyHC) isoforms.
Antibodies and Labeling
Five primary monoclonal antibodies (MAbs) were used in this study: NCL-MHCs (against MyHC I); SC71 (against MyHC IIA); MY32 (anti-fast MyHC: IIA, IIX, IIB); ALD58 (against slow-tonic MyHC); and F88 (anti-α-cardiac MyHC). The dilutions of these antibodies already reported in De-Doncker et al. (2002) were 1:20, 1:20, 1:2000, 1:5, and 1:1, respectively. NCL-MHCs is commercialized by Tebu-Novocastra (Le Perray en Yvelines, France), SC71 by DSMZ (Braunschweig, Germany), MY32 by Sigma-Aldrich (Saint Quentin Fallavier, France), ALD58 by DSHB (Iowa City, IA), and F88 by Coger (Paris, France). 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 for 2 hr. Serial cross-sections were washed for 5 min in PBS. 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 and negative fibers remained unlabeled. To estimate the coloration intensities, we have developed a densitometric approach previously described in detail (De-Doncker et al. 2002). The IHC-labeled slides were examined with an image analyzer (SAMBA 2005). To avoid a disparity in the labeling, some experimental precautions were taken. For each antibody, all the sections were processed with the same diluted solution. Moreover, the slides were incubated in a common DAB solution. The zero of the densitometer was adjusted on the negative fibers in a given section; the slight variations of labeling between each slide were therefore discarded. The IHC-labeling slides were analyzed with the image analyzer, which was made up of a digital camera fixed to a standard optical light microscope. The camera was coupled to a computer with video monitor and image acquisition and storage modules. The measures were quantified to 256 gray levels (level 1 corresponding to black color and to 0% of light transmission, and level 256 to white color and 100% of light transmission), which were then converted automatically into optical density, taking into account the studied area. The densitometric values were expressed in percentage (OD = log10(1/light transmission). According to Nibbering et al. (1986), the formation of the reaction product after DAB/H2O2 staining for immunoper-oxidase-labeled cells indicated a linear relationship between the amount of enzyme-coupled antibodies bound to cells and the amount of enzyme reaction product. This densitometric analysis has already been used as a semiquantitative approach in rat muscle fibers (Sant'Ana Pereira et al. 1995). The immunoreactivity in muscle spindle IHC is usually scored as absent (-), weak to moderate (+), and strong (+ +), which does not allow detection of fine modifications in the expression level of myosin isoforms. Consequently, we intentionally chose to express our results as values obtained using densitometric measurements.
As previously described by Kucera (1981), in the most nucleated equatorial region of IFs, the labeling was localized around the IF. In this case, the densitometric measurement was performed only on the colored part. The IF center, which was not stained, was discarded.
Identification of IF Types
The muscle spindles were identified in both CONT and HG soleus muscle groups as encapsulated structures containing small-diameter fibers and presenting three regions (A,B, and C) classically described (Kucera et al. 1978). IFs were classified as B1 (nuclear bag1), B2 (nuclear bag2), and chain (nuclear chain) fibers according to their histochemical labeling (Ovalle and Smith 1972; Kucera et al. 1978) and IHC profiles, as previously reported (Pedrosa-Domellof et al. 1991; Kucera et al. 1992; Soukup et al. 1995; Walro and Kucera 1999). Rat muscle spindles usually contain four IFs: one B1, one B2, and two chain fibers (Kucera and Walro 1988b). The diameter of these IFs decreases in the following order: nuclear B2 > B1 > chain fibers. Nuclear B1 fibers exhibit low acid and alkaline ATPase activity along their intracapsular region but have high acid ATPase activity at their poles. Nuclear B2 fibers have an acid-stable ATPase activity along their length and an intracapsular alkaline-stable ATPase activity, which is lost beyond the capsular sleeve. Nuclear chain fibers have low acid and high-alkaline ATPase activities along their entire length (Kucera et al. 1978). The nuclear B1 and B2 fibers were labeled with both ALD58 antibody, specific for the slow-tonic MyHC isoform, and F88 antibody, specific for the α-cardiac MyHC isoform. Nuclear chain fibers did not express these two MyHC isoforms except in a very short equatorial length where the slow-tonic isoform was detected (Pedrosa et al. 1989; Pedrosa-Domellof et al. 1991).
Statistical Analysis
All results were expressed as means ± SD. After an ANOVA, Student's t-test was used to establish the intergroup comparisons and statistical significance was accepted at p<0.05. ∗Indicates a significant difference with the A region, †with the B region, and §with the control group.
Mean values of morphological parameters a
Values are expressed as means ± SD. Morphological parameters are reported as follows: body weight (BW), muscle wet weight (MWW), and ratio MWW:BW of CONT and HG groups, the sample appearing in brackets.
Results
Morphological Characteristics
Table 1 reports the body weight (BW), the muscle wet weight (MWW), and the ratio MWW:BW after 19 days of experiment for CONT and HG groups. The values of BW remained similar, whereas both the MWW and the ratio MWW:BW were significantly increased (+ 21% and +27%, respectively).
Concerning the muscle spindle sample, we studied 40 spindles in the CONT group and 45 in the HG group. The rat spindles usually contained one B1, one B2, and two chain fibers. However, some spindles showed three chain fibers in both CONT and HG groups. This was the case for three of the 40 CONT spindles and two of the 45 HG spindles. Consequently, in the CONT group the total number of studied fibers was for B1 40, for B2 40, and for chain 83. In the HG group, these samples were, respectively, 45, 45, and 92. The CSA of IFs were examined in CONT and HG rats, from A to C regions. There was no statistical difference between the two experimental groups. The ATPase labeling revealed no difference between CONT and HG groups.
Transverse sections of CONT and HG soleus muscles (10-μm thick) containing a spindle with one B1 (thin arrow), one B2 (thick arrow), and two chain fibers (triangle). The illustrated sections were processed with the following antibodies: NCL-MHCs (anti-MyHC I), SC71 (anti-MyHC IIA), and MY32 (anti-fast MyHC: IIA + IIX + IIB) from
Identification of MyHC Isoforms in IFs
The MyHC content was identified by antibodies along the IF length. The results illustrated in Figure 1 and 2 as follows.
NCL-MHCs Antibody (Against MyHC I Isoform). In the CONT group, B2 fibers were labeled and expressed the MyHC I isoform from A to C regions, whereas B1 fibers were labeled only at the end of the B region and in the C region, and chain fibers were negative (Figures 1A1-1A3 for the CONT group). In the HG group (Figures 1A4-1A6), B2 and chain labeling remained similar, whereas B1 fibers appeared reactive against the NCL-MHCs antibody from the A to the C region.
SC71 Antibody (Against MyHC IIA Isoform). B1 fibers of CONT and HG rats were labeled in the A, B, and C regions, B2 and chain fibers being stained only in the A region (Figures 1A7, 1B8, and 1C9, CONT spindles; HG spindles Figures 1A10, 1B11, and 1C12).
MY32 Antibody (Against MyHC IIA + IIX + IIB Isoforms). In both experimental groups, B2 and chain fibers were reactive against this antibody from A to C regions, whereas B1 fibers were never labeled (Figure 1: CONT Figures 1A13, 1B14, and 1B15; HG Figures 1A16, 1B17, and 1C19).
ALD58 Antibody (Against Slow-tonic MyHC Isoform). In both CONT and HG groups, only B1 and B2 fibers contained the slow tonic MyHC isoform in A, B and C regions, and only in the A and inner B regions, respectively (Figure 2: Figures 2A1, 2B2, and 2C3 for CONT rats; Figures 2A4, 2B5, and 2C6 for HG rats).
F88 Antibody (Against α-cardiac MyHC Isoform). In CONT rats, the α-cardiac MyHC was expressed in B2 fibers from A to C regions, the chain fibers being absolutely negative. However, B1 fibers were reactive against F88 only from outer B to C regions (Figures 2A7, 2B8, and 2B9). For HG rats, B2 and chain labeling remained identical, but the α-cardiac MyHC was now expressed from A to C regions in B1 fibers (Figures 2A10, 2B11, and 2C12).
Densitometric Data
Because slight variations in MyHC content after HG were not clearly visible with a single antibody labeling, we refined the data using densitometric measurements. The densitometric values are reported in Table 2 (CONT group) and Table 3 (HG group). The results are expressed as percentage of optical density (OD) with OD = log (1/light transmission) considering that 0% of light transmission corresponded to black color and 100% of light transmission to white color. Consequently, the more elevated the OD value, the more important the myosin content. However, the results were not directly quantitative but rather were semiquantitative.
In the CONT group, the densitometric data showed that, for B1 fibers, labeling against NCL-MHCs (anti-MyHC I), SC71 (anti-MyHC IIA), ALD58 (anti-slow-tonic MyHC), and F88 (anti-α-cardiac MyHC) antibodies was significantly decreased in the C region compared to the A region (NCL-MHCs and F88) and compared to both A and B regions (SC71 and ALD58). In B2 fibers, labeling against MY32 (anti-MyHC IIA + IIX + IIB) and F88 was significantly reduced in the C region vs the A and B regions.
In the HG group, B1 fibers exhibited a significantly increased labeling against both NCL-MHCs and F88 antibodies in the B and C regions compared with the A region. Moreover, the densitometric results revealed marked changes in labeling amounts. After HG, these fibers became reactive from A to C regions against NCL-MHCs and F88 antibodies (p<0.05), the labeling being increased. Conversely, the binding intensity was statistically decreased for SC71 in the A and B regions and for ALD58 in the A to C regions.
In HG spindles, B2 fibers showed a decreased reactivity against SC71 (A region) and MY32 and ALD58 (A and B regions) antibodies (p<0.05). In chain fibers, the labeling intensity was significantly decreased for SC71 and MY32 antibodies in the A region and in the A and B regions, respectively.
Discussion
The aim of the present study was to determine by IHC the effects of a hypergravity environment on MyHC isoform expressions among the three types of IFs in rat soleus muscle.
Soleus sections of CONT and HG groups showing a spindle from the
For the CONT group, the depth and the regional variation of labeling with NCL-MHCs, MY32, ALD58, and F88 antibodies were in agreement with other studies (Pedrosa et al. 1990; Pedrosa-Domellof et al. 1991; Kucera et al. 1992; McWhorter et al. 1995; Soukup et al. 1995; De-Doncker et al. 2002). However, some differences were observed with SC71 antibody labeling: a low level of labeling was seen in the B2 fibers. This point has been discussed in a previous article (De-Doncker et al. 2002) and was attributed to a different dilution used by other authors (Kucera et al. 1992). A discrepancy in labeling was observed in the nuclear B1 fibers with MY32 and SC71 antibodies. Nuclear B1 fibers were labeled by SC71 along their entire length, whereas MY32 antibody never labeled these fibers. This was surprising because the MY32 antibody is supposed to react with all fast-twitch MyHC isoforms (Schiaffino et al. 1989). SC71 antibody is specific for MyHC IIA isoform and never crossreacts with other MyHC isoforms. Therefore, the fast-twitch MyHC isoform, recognized in nuclear B1 fibers by the SC71 antibody but not by MY32, could be a specific muscle spindle MyHC isoform whose epitope resembles that recognized by SC71 on the MyHC IIA isoform. This possible existence of other specific muscle spindle MyHC isoforms, not expressed in extrafusal fibers, has previously been suggested by other authors (Kucera et al. 1992; Pedrosa-Domellof et al. 1993). Kucera et al. (1992) suggested that the labeling intensity and the regional variation with MY32 were higher and broader than those with SC71 (MyHC IIA) and BF-F3 (MyHC IIB) antibodies, indicating the possible existence of a specific fast-twitch MyHC isoform expressed by the IFs but not by the extrafusal fibers.
Densitometric analysis of the IHC labeling in intrafusal fibers of the control group a
The densitometric analysis is presented as a percentage of B1, B2, and chain fibers in the CONT group. Values are given for each antibody in the three different muscle spindle regions (A,B,C).
Significant difference of labeling with the A region and
with the B region. NL, no labeling.
After 19 days of hypergravity, labeling with NCL-MHCs, SC71, MY32, ALD58, and F88 antibodies was modified. The expression of both MyHC I and α-cardiac appeared in the A region and increased in B and C regions of B1 fibers. The novel expression of MyHC I and MyHC α-cardiac in the A region of B1 fibers was not the result of a crossreaction between NCL-MHCs and F88 antibodies. Indeed, the epitope detected by the F88 in the MyHC α-cardiac was not present in embryonic, neonatal, slow-twitch, fast-twitch, and slow-tonic isoforms (Pedrosa et al. 1990). Labeling against SC71 and ALD58 antibodies was decreased along the full length of B1 fibers and in the A region of B2 fibers. For chain fibers, a significant decrease in SC71 labeling was also observed in their A region. Moreover, MY32 labeling was decreased along the entire length of B2 fibers and in the A and B regions of chain fibers.
Two major points are discussed here: (a) the impact of neural regulation on expression of the MyHC isoform in IFs in both developing and adult rats, and (b) the influence of the gravitational load on expression of these myosin isoforms.
Densitometric analysis of the IHC labeling in IFs of the HG group a
Densitometric analysis is presented as a percentage of B1, B2, and chain fibers in the HG group. Values are given for each antibody in the A, B, C muscle spindle regions.
Significant difference of labeling with control group,
with the A region, and
with the B region. NL, no labeling.
Neural Regulation of IF MyHC Isoform Expression in Developing Rat
Several studies have shown that sensory innervation is required for the development of muscle spindles and for the expression of spindle-specific MyHC isoforms. The motor innervation contributed to the regional variation of the different MyHC isoforms along the length of the nuclear bag fibers (Kucera and Walro 1988a; Zelena 1994; Soukup et al. 1995). The regulation of MyHC isoform expression along IFs was very complex and reflected interactions between inductive and/or suppressive effects of both motor and sensory innervation, as well as the intrinsic properties of specific myoblast lineages (Kucera and Walro 1990a,c; Pedrosa-Domellof et al. 1991; Soukup et al. 1995; Walro and Kucera 1999). Moreover, the afferent influence diminished and motor influence increased with increasing distance from the equator of IFs (Pedrosa et al. 1989; Kucera et al. 1992).
Sensory innervation has a specific modulatory key role in slow-tonic MyHC isoform expression. Fetal rat denervation using bungarotoxin (a non-selective neurotoxin) (Kucera and Walro 1990b), neonatal denervation (Thornell et al. 1989; Soukup et al. 1994), and deafferentation (Kucera and Walro 1988a) prevented the expression of the slow-tonic MyHC isoform in nuclear bag fibers. However, after neonatal de-efferentation, the diversity in slow-tonic MyHC distribution along the intrafusal B2 fibers was modified and this MyHC isoform was expressed more intensely and over most of the B2 fiber length (Soukup et al. 1990a; Pedrosa-Domellof et al. 1991). Conversely, efferents rather than afferents might induce and maintain expression of the α-cardiac MyHC isoform (Pedrosa et al. 1990; Soukup et al. 1999). On the one hand, during normal development the expression of the α-cardiac MyHC isoform appeared in nuclear bag fibers 1 day after the arrival of γ motor innervation (Pedrosa et al. 1990; Pedrosa and Thornell 1990). On the other hand, neonatal de-efferentation induced a decrease in the expression of this MyHC isoform in B2 fibers and its removal in B1 fibers (Pedrosa et al. 1990; Soukup et al. 1999).
Neural Regulation of IF MyHC Isoform Expression in Adult Rat
Perturbations in spindle sensory and motor innervations of adult rat produced less severe alterations in MyHC isoform expression than did similar lesions in developing IFs (Wang et al. 1997). Contrary to fetal and neonatal rat, adult rat deafferentation never induced degeneration of muscle spindles, but altered the stereotypical MyHC profiles of intrafusal fibers (Wang et al. 1997). Indeed, IFs in adult chronically deafferented muscles upregulated fast MyHC isoforms, particularly MyHC IIA, and to a much lesser degree the MyHC I isoform at the expense of developmental MyHC isoforms. The activity imposed on nuclear bag fibers by efferents or muscle stretch might modify the MyHC I expression along these IFs (Kucera et al. 1992), as is the case for extrafusal fibers deprived of motor innervation (Harris et al. 1989). In the presence of motor innervation and in the absence of sensory innervation, the MyHC content of IFs assumed more extrafusal-like features (Kucera and Walro 1988a,b; Walro et al. 1989). Therefore, as suggested by Wang et al. (1997) and Walro et al. (1997), the upregulation of MyHC isoforms expressed by extrafusal fibers reflected a de-repression of these isoforms. After adult de-efferentation of the rat extensor digitorum longus muscle, Wang et al. (1997) have shown that B1 fibers that normally expressed the α-cardiac MyHC isoform moderately in the outer B region ceased to express this MyHC isoform. Moreover, B2 fibers continued to express this isoform but less intensely than normal, and this MyHC expression was limited to a shorter region of B2 fibers, as described in the studies of Pedrosa et al. (1990) and Wang et al. (1997).
Finally, according to these data, it appeared that MyHC expression in muscle spindles was directly under neural influence, both sensory and motor. However, the impact of the gravitational environment on neural activity is now understood, and recent data have demonstrated that this environment may also regulate the MyHC expression in muscle spindles.
Influence of the Gravitational Load on Muscle Spindle MyHC Isoforms
De-Doncker et al. (2002) have reported that expression of some MyHC isoforms along intrafusal fibers of rat soleus muscle was modified after a 14-day period of hypogravity. These authors observed that, whereas histochemical profiles of IFs were maintained, there was a decrease in slow type 1 and an increase in slow-tonic MyHC isoform expressions in B and C regions of B1 fibers. Moreover, the α-cardiac MyHC expression was significantly decreased along the entire length of B2 fibers and in the B and C regions of B1 fibers. Furthermore, some chain fibers expressed the slow type I and α-cardiac MyHC isoforms over a short distance of the A region, although these isoforms were never expressed in the control intrafusal fibers. De-Doncker et al. (2002) suggested that the decrease in α-cardiac and the increase in slow-tonic MyHC isoforms in nuclear bag fibers probably reflected a decrease in the activity pattern of the motor nerves during a hypogravity period, considering the results of several studies on denervation or de-efferentation experiments (Pedrosa et al. 1990; Soukup et al. 1990a,1999; Wang et al. 1997). The structure of rat muscle spindle was not modified by a hypogravity period. Indeed, IFs were not atrophied and their number per muscle spindle was not changed by hypogravity conditions, as previously described by Soukup et al. (1990b) and De-Doncker et al. (2002). These authors concluded that IFs were more resistant to myogenic atrophy than extrafusal fibers, as suggested earlier by Yellin and Eldred (1970) and Maier et al. (1972). In a previous report (De-Doncker et al. 2002), we hypothesized that during a 14-day period of hypogravity (HH), muscle spindles were little or not at all stimulated, considering the shortened position of the rat soleus muscle. Therefore, muscle spindle afferent activities were probably reduced (Ohira et al. 1992). An indirect proof of this statement was provided by the fact that the reactivation of Ia fibers by tendinous vibrations constituted an effective countermeasure to prevent muscle atrophy developed during hindlimb unloading conditions (Falempin and Fodili In-Albon 1999). It is known that Ia afferents project in the spinal cord onto α-skeletomotor and β-skeletofusimotor neurons, and onto interneurons which, in turn, project onto γ-fusimotor neurons (Bernstein and Goldberg 1995). We suggest that this reduction in muscle afferent activity could modify MyHC isoform expression by altering the pattern discharge of β- and γ-fusimotor neurons that innervate the contractile portion of the IF. Another factor should also be implied in the modifications of motor neuron activity. It has been demonstrated that simulated weightlessness induced a decrease in acetylcholinesterase activity in neuromuscular junctions of both extrafusal and intrafusal fibers, which might be linked with a decrease in α- and γ-motor neuron activities (Tang et al. 2002). This study refuted our first work (De-Doncker et al. 2002), which suggested that changes in MyHC expressions of intrafusal fibers after 14 days of weightlessness could be due to a decreased influence of γ- and/or β-motor innervation during the simulated microgravity environment. To complete the preceding results, we studied the effects of a hypergravity environment on MyHC isoform expression along the three types of IFs of rat soleus muscle. However, contrary to what occurred in a hypogravity environment, the soleus muscle in hypergravity was probably stretched and, consequently, the muscle spindle afferent activities were increased. By a reflex pathway, considering the study of Bernstein and Goldberg (1995), the muscle spindle motor activity was also increased. The MyHC α-cardiac and MyHC I increases in B1 fibers and MyHC slow-tonic decrease in nuclear bag fibers reflected a reinforced motor innervation influence during a hypergravity period, contrary to the hypogravity situation (De-Doncker et al. 2002). Two hypotheses might explain the discrepancy between hypogravity and hypergravity concerning F88 labeling in B2 fibers: the duration of exposure to a modified gravitational load (14 vs. 19 days) and/or different myogenic lineages (Kucera and Walro 1990a; Walro and Kucera 1999). Moreover, after a hypergravity situation, it has been demonstrated that the proportion of slow-type extrafusal fibers expressing the MyHC I isoform was increased up to 100% (Martin and Romond 1975; Picquet et al. 2002). After adult rat deafferentation (Wang et al. 1997), MyHC I isoform was slightly overexpressed in muscle spindles. In our study, we suggested that afferent activity from muscle spindles was increased. Consequently, MyHC I isoform expression should be decreased. However, the expression of this MyHC isoform was increased along the full length of B1 fibers after a hypergravity period. We therefore supposed that the excitatory motor influence prevailed on the inhibitory afferent influence, at least for the MyHC I isoform regulation. Wang et al. (1997) have also demonstrated that IFs upregulated fast MyHC isoforms after adult rat deafferentation. They concluded that excitatory motor influence was de-repressed by the lack of afferent activity. In our study we considered that both afferent and motor innervation were similarly increased after hypergravity. Therefore, given the conclusions of Wang et al. (1997), the increase in motor innervation influence should upregulate the fast MyHC isoforms. However, our results showed a decrease in fast MyHC isoform expression along IFs. Consequently, contrary to MyHC I regulation, the inhibitory influence prevailed on the excitatory motor influence for fast MyHC isoform expression. At least for these MyHC isoforms (slow and fast), the hypergravity induced some inverse changes of those observed in a hypogravity environment. Conversely, this was not the case for fast MyHC isoform expression. Indeed, the fast MyHC isoforms were not changed after a hypogravity period, whereas they were downregulated in IFs after hypergravity. It therefore appears that IFs were more sensitive to a hypergravity environment. Because chronically deafferented adult muscle spindles overexpressed fast MyHC isoforms along IFs (Walro et al. 1997; Wang et al. 1997) and because these MyHC isoforms appeared down-regulated in our study, we can therefore suggest that muscle spindle afferent activities were increased. It is well known that the γ-motor innervation (dynamic and static) of IFs play a major role in control of the muscle spindle sensitivity during different positions and activities (for reviews see Hulliger 1984; Prochazka et al. 1988; Banks 1994). After 14 days of HH or 19 days of HG, it is possible that subtle modifications in MyHC isoform expression along IFs would result in very little functional adaptation. However, these modifications would change the contraction of IF extremities and consequently would affect the control of the muscle spindle sensitivity. Moreover, fast extrafusal type II fibers containing MyHC II isoforms are less stiff than the slow extrafusal type I fibers containing the MyHC I isoform (Petit et al. 1990). Therefore, changes in MyHC isoform expression by IFs would modify the viscoelastic properties of IFs, which would affect the detection of the muscle stretching by muscle spindles. Consequently, the discharge of muscle spindle afferents would be modified after 19 days of HG, as has been demonstrated after 14 days of HH (De-Doncker et al. 2003), and muscle proprioception would be disrupted.
Comparison between hypergravity (HG) and hypogravity (HH) conditions vs control rats on the immunolabeling of IFs a
Effects of gravity changes on intrafusal MyHC isoforms are synthetically reported. The decrease, the increase, or the maintenance of MyHC content are respectively indicated by -, +, and NC (no change). A de novo expression is indicated by NE (novel expression). NL, no labeling.
Comparison between hypergravity (HG) and hypogravity (HH) conditions vs control rats on the immunolabeling of IFs a
Effects of gravity changes on intrafusal MyHC isoforms are synthetically reported. The decrease, the increase, or the maintenance of MyHC content are respectively indicated by −, +, and NC (no change). A de novo expression is indicated by NE (novel expression). NL, no labeling.
Our conclusion is that IFs are more resistant than extrafusal fibers to changes in the gravitational load. Moreover, the balance between afferent and motor innervation influences was probably modified during both hypogravity and hypergravity (see Table 4 and 5). This imbalance differently regulated the MyHC isoform expression along the three types of IFs. Contrary to Kucera et al. (1993) and Walro et al. (1997), who attributed the mechanism of afferent influence only to neurotrophically mediated substances, our data suggest that afferent nerve activity and an intact reflex arc are also required to determine levels and regional variations of MyHC isoform expression along intrafusal fibers.
Footnotes
Acknowledgements
Supported by grants from the Centre National d'Etudes Spatiales (8411) and the Conseil Régional du Nord-Pas-de-Calais.
