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MUSCLE CELL BIOLOGY AND CELL MOTILITY
1Graduate School of Medicine and 2Frontier Biosciences, Osaka University, Suita City, Osaka; 3Department of Physiology II, Jikei University School of Medicine, Tokyo; and 4Graduate School of Human and Environmental Studies, Kyoto University, Kyoto City, Kyoto, Japan
Submitted 17 June 2005 ; accepted in final form 11 November 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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sarcomere
Muscle satellite cells are myogenic precursor cells that lie between the sarcolemma and the basal lamina of the myofiber (19), and myonuclear accretion occurs through the incorporation of satellite cells into the growing myofibers (23). Satellite cells have been shown to serve as a source of new myonuclei during regeneration after a muscle injury (21, 40, 41) and during functional overload (6, 20, 42). In contrast, hindlimb unloading downregulates the satellite cell mitotic activity (7, 25–27, 39). The mechanisms underlying the modulation of satellite cells in response to va0rying loading conditions are still unclear. Therefore, the present study was performed to investigate the relationship between the distribution of satellite cells and the level of the mechanical load applied to the muscle. In addition, the role of muscle loading on myonuclear number, distribution, size, DNA content, and domain size was investigated.
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METHODS |
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Seventy-five 5-wk-old male Wistar Hannover rats (139 ± 5 g; Nihon CLEA, Tokyo, Japan) were used in the present study. The rats were separated randomly into either a cage-control (n = 40) and a hindlimb-unloaded (n = 35) group. Five control rats were euthanized on the first day of the experiment and served as a preexperimental group. Tail suspension was performed in the hindlimb-unloaded group as previously described (31). Briefly, the tail of the rat was washed and dried. Strips of sticky tape (
5 mm wide and 3 cm long) with good cushion were then placed longitudinally on the dorsal and ventral sides of the midportion of the rat tail. These strips were anchored with tape wrapped loosely around the tail, so that the blood flow in the tail was not impeded. A string was inserted through the gap between the tail and the tape. The string was fastened to the roof of the cage (30 x 30 cm, and 30 cm height) at a height that allowed the forelimbs to support the weight, but prevented the hindlimbs from touching the floor or the walls of the cage. The rats could reach food and water freely using their forelimbs. The control rats were housed individually in identical cages as the unloaded rats. The amount of solid diet (CE-2, Nihon CLEA), which was completely eaten within 12 h, was supplied at 10 AM daily (20 g·day–1·rat–1). The temperature and humidity in the animal room were maintained at 23°C and 55%, respectively. The rats were also maintained under 12:12-h light-dark cycle conditions.
After 16 days, both control and unloaded rats (n = 15 in each group) were euthanized with an overdose of pentobarbital sodium (5 mg/100 g body wt ip). The hindlimb-unloaded rats were anesthetized while the hindlimbs remained unloaded to avoid any effects of acute reloading. The left soleus muscle (n = 5 in each group) was sampled for the single fiber analyses. Ten rats from each group were used for analyses of sarcomere length in the soleus muscle fibers at specific ankle joint angles (see below).
The remaining rats in both the control and unloaded groups were allowed to recover at either 1 G or 2 G (n = 10 in each group). Normal ambulatory cage recovery was allowed for the 1-G group. The rats in the 2-G group were loaded continuously, except 30 min/day for cleaning and feeding, with the use of a four-armed animal centrifuge (1.3 m of radius and 38 rpm of swing speed) powered by a 0.4-kW gear motor and controlled by a general-purpose inverter (43). The 1-G group was housed in the same room equipped with the centrifuge. After 2 or 16 days of recovery, the rats were euthanized (n = 5 per group in each day) and the left soleus muscle was sampled for the single fiber analyses.
Muscle preparation and fiber dissection. Immediately after removal, the left soleus muscle was cleaned of excess fat and connective tissues and weighed (wet weight). The muscle was carefully torn longitudinally while being examined under a microscope, and the middle (longest) segment was stored in a cellbanker (Nihon Zenyaku, Tokyo, Japan) at –80°C until analyzed. The muscle fiber segments stored in the cellbanker were thawed instantly at 35°C. The fibers were placed in Dulbecco's modified Eagle's medium (Invitrogen) containing 20 µM 5'-bromo-2'-deoxyuridine (BrdU; Becton Dickinson, Mountain View, CA), 0.2% type I collagenase, 1% antibiotics, and 10% newborn calf serum for 4 h at 35°C to digest the collagens. The collagenase-treated segments were fixed in 4% buffered formaldehyde for 10 min. Entire single muscle fibers, sampled from tendon to tendon, were isolated with the use of fine needles. The fibers were collected carefully using pipettes to avoid any damage. The collected fibers were separated into three tubes (30 fibers in each tube; tubes 1–3) and immersed in Dulbecco's modified Eagle's medium containing 10% newborn calf serum. The working solution of type I collagenase was gel purified to remove the clostripain, which supposedly strips the basal lamina of the fibers (3).
Immunohistochemistry and nuclear labeling. Immunohistochemistry was performed to label BrdU and M-cadherin in the single fibers as described previously (24, 25). The collected single fibers in the tubes were permeabilized with 1% Triton X-100 diluted with phosphate-buffered saline (PBS) for 10 min. Subsequently, the single fibers in the tubes were blocked with 10% normal goat serum diluted with PBS for 15 min.
Tube 1 was incubated overnight with primary polyclonal antibody specific to M-cadherin (Santa Cruz Biolotechnology) diluted at 1:20 with PBS containing 0.5% Tween 20 and 0.5% bovine serum albumin (BSA). Tube 2 was incubated overnight with primary monoclonal antibody specific to BrdU diluted at 1:20 with PBS containing 0.5% Tween 20 and 0.5% BSA. The fibers in tube 3 were incubated overnight with both antibodies specific to M-cadherin and BrdU. The M-cadherin-positive (quiescent) satellite cells were detected with goat anti-rabbit IgG conjugated to either fluorescein (tube 1, Chemicon International) or rhodamine (tube 3, Chemicon International) diluted at 1:50 with PBS containing 0.5% Tween 20 and 0.5% BSA, respectively. The BrdU-positive nuclei (mitotic active) satellite cells were detected with goat anti-mouse IgG conjugated to fluorescein isothiocyanate (tubes 2 and 3; Jackson ImmunoResearch) diluted at 1:50 with PBS containing 0.5% Tween 20 and 0.5% BSA. Furthermore, the fibers in tubes 1 and 2 were rinsed with 0.1% Triton X-100, and were stained by propidium iodide (PI; 25 µg/ml PBS) for 5 min. After being stained, the single fibers were rinsed with PBS and stored in PBS at 4°C until the time of analysis. Immediately before the analyses were made with the use of a confocal microscope, the fibers were mounted in glycerol on coverslips with "struts" of hardened nail polish on the corners to minimize fiber compression.
Confocal microscopy. A FV-300 confocal microscope with an argon laser (488 nm of peak wavelength) and a He-Ne laser (543 nm of peak wavelength) (Olympus) was used to analyze the fiber length and cross-sectional area (CSA), number and maximum CSA of the myonuclei, DNA concentration in each myonucleus, sarcomere number, and distribution of quiescent and mitotically active satellite cells.
The total number of fiber nuclei, labeled by PI, as well as the number of nuclei in the proximal, central, and distal portions of the fiber, were counted under a microscope (tubes 1 and 2). The numbers of M-cadherin-labeled and BrdU-labeled nuclei also were counted throughout the fiber length to determine the distribution of quiescent and mitotically active satellite cells, respectively. The relative distance of each satellite cell from the center (0%) to the end of fiber (100%) was measured using the laser-scanned image with calibrated measurement software (Olympus). A maximum-intensity projection rotated orthogonally to the long axis of the fiber was produced from the stack, and the fiber CSA was measured at three nonoverlapping proximal, central, and distal regions (tubes 1 and 2). The fiber length and the length of 10 consecutive sarcomeres from each of the three regions along the fiber were measured by Nomarski optic scanning techniques. The overall mean sarcomere length from the three regions was calculated, and based on the fiber length, and the total number of sarcomeres was estimated. Overall myonuclear domain size (5, 14) was calculated as (average fiber CSA x fiber length)/number of myonuclei per fiber. Myonuclear domain size also was estimated for the proximal, central, and distal regions where myonuclear number and fiber size were analyzed. Furthermore, at least 30 myonuclei were laser scanned at a given intensity of the laser with a proper filter set for PI. The mean emission intensity (EI) of PI was measured at a given sensitivity to estimate the myonuclear DNA concentration (EI/pixel, where 1 pixel = 0.06 µm2) and the total DNA content (EI/pixel x CSA x 103). The EI of fluorescence was quantified as 4,096 levels of scale. The maximum CSA of a myonucleus was determined by serial cross-sectional scanning.
Relationship between sarcomere length and ankle joint angle.
The mean length of sarcomere length in single fibers during unloading and at rest on the floor was estimated in the soleus muscle fixed at specific ankle joint angles in unloaded (immediately after the 16-day unloading period) and of the age-matched control rats (n = 10 in each group) as reported previously (16). The entire hindlimbs were isolated bilaterally and submerged in 4% buffered formaldehyde, which fixed the anterior angle of the ankle joint at either 30, 120, 140, or 160° (n = 5 for each angle). The soleus muscles were removed after 30 min of fixation. Subsequently, each muscle was divided into three regions, and single muscle fibers were isolated from the proximal (n = 30), central (n = 30), and distal (n = 30) regions of the muscle with the use of fine forceps under the microscope. The fibers were mounted on a slide glass with coverslip with struts of hardened nail polish on the corners to minimize fiber compression. The length of 10 consecutive sarcomeres was measured in each region. The average of a single sarcomere length in each region was calculated.
Statistical analyses. All values are expressed as means ± SE. Significant differences were examined by two-way ANOVA, followed by Scheffé's post hoc test. Differences were considered significant at P < 0.05.
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RESULTS |
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15% vs. age-matched 1-G controls, P < 0.05; Fig. 1A). However, body weight was normalized after the 16-day recovery period at 1 G (P < 0.05). Acute 2-day exposure to 2 G resulted in a significant reduction of body weight in both control and unloaded rats (P < 0.05). The body weight of the unloaded group was not increased after 16 days of recovery at 2 G and was significantly lower than the body weight of the other age-matched groups (P < 0.05). These results may be related to stress due to the hypergravity environment created by using animal centrifuge, even though the daily food consumption was identical. Significant hypertrophy of the adrenals (50% and 25% vs. the 1-G control group) and atrophy of the thymus (–32% and –29%) were noted 2 days after 2-G exposure of the cage control and the previously unloaded groups, respectively (data not shown). After 16 days of recovery at 2 G, the adrenals of these groups were 14% and 35% larger and the thymus was 32% and 25% smaller than in the age-matched controls, respectively (P < 0.05).
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170% within 16 days of recovery at either a 1-G or 2-G environment, but was still lower than in the age-matched control group (P < 0.05). The mean relative weights showed a similar response (Fig. 1C), although the mean relative soleus weight in the group recovering at 2 G was similar to the age-matched control due to a lower mean body weight (Fig. 1A).
Muscle fiber size.
The mean CSA was generally similar throughout the fiber length in both the control and unloaded groups (Fig. 2). Compared with the preexperimental values, the mean fiber sizes in the control group were increased by 58% and 85% after 16 or 32 days of growth, respectively (P < 0.05). Mean fiber sizes were 46% and 66% smaller in the unloaded group than in the preexperimental and the age-matched controls, respectively (P < 0.05). Compared with the unloaded group, the mean fiber sizes were 114% and 95% larger after 16 days of recovery at 1 G or 2 G, respectively. The fiber sizes, however, were still 40% and 46% smaller than in the age-matched controls (P < 0.05). Two days of reloading had no effect on fiber size.
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Distribution of satellite cells. In general, both mitotically active and quiescent satellite cells were distributed evenly along the fiber length in control muscle fibers, although the number of mitotically active cells was slightly less at the fiber ends (Fig. 5A). Unloading resulted in a decrease in both mitotically active and quiescent satellite cells in the central region of the fiber, i.e., the mean number of these cells in each 10% interval of fiber length immediately after the termination of the 16-day unloading period was only 0.01 and 0.05 vs. 0.37 and 0.46 in the age-matched control, respectively. In contrast, the number of satellite cells in the regions close to the fiber ends were unaffected by unloading. The distribution of satellite cells was normalized by 16 (Fig. 5C), but not two (Fig. 5B), days of reloading. In addition, there was no effect of G level on satellite cell distribution in either the control or unloaded groups throughout the experimental period.
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30°, 120°, 140°, and 160° of ankle joint angle in control rats were 2.8, 2.0, 1.9, and 1.9 µm, respectively. The greater the degree of dorsiflexion, the longer the sarcomere length was. However, the values at 140° and 160° were identical, suggesting that the plantarflexion-related shortening reached a plateau level. The mean sarcomere length at a given ankle joint angle was increased after 16 days of hindlimb unloading. It was also noted that the sarcomere length at both proximal and distal regions of fiber was greater than that at the central region of fiber with ankle joint angle at 30° and 120° in both unloaded and control rats (Fig. 10B). The mean sarcomere length at proximal and distal regions of fiber was 2.9 µm, and that of the central region was 2.6 µm when the ankle joint angle was maintained at 30° on the floor in control rats (P < 0.05). The sarcomere lengths at the fiber ends and the central region were reduced to 2.02 and 1.98 µm, respectively, when the ankle joint was passively extended acutely by hindlimb unloading. However, these levels were slightly increased to 2.3 and 2.1 µm after 16 days of unloading.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Effects of unloading. Hindlimb unloading-related atrophy of the soleus muscle fibers has been reported in previous studies (29, 44–46). In the present study, fiber atrophy was closely associated with a decrease in the number of myonuclei, which were evenly distributed throughout the fiber length. This loss of myonuclei most likely results in a decrease in protein synthesis and, therefore, may be a major factor in the observed reduction of fiber CSA with unloading. Myonuclear domain size, i.e., the volume of cytoplasm that may be regulated by a single myonucleus (5, 14), also decreased with chronic unloading. Because domain size would be unaffected if both the fiber CSA and myonuclear number were changed in parallel, the data indicate that the amount of fiber atrophy was greater than the loss of myonuclei.
The mean size of the myonuclei was increased after unloading, whereas the total DNA content in a single myonucleus was unchanged. Therefore, the DNA concentration in a myonucleus was lower in the muscle fibers of unloaded than control rats and this may impact nuclear function. The data obtained in the present study suggest that the cause of muscle fiber atrophy was closely related to the reduction in both the number and function of myonuclei that, in turn, may result in an imbalance between protein synthesis and degradation (4, 8, 11, 12). The loss of myonuclei may be related to apoptotic mechanism as suggested by Allen et al. (1) who reported an 15-fold increase in the number of nuclei staining for terminal deoxynucleotidyl transferase, an indicator of apoptosis, after 14 days of hindlimb unloading.
The electromyogram (EMG) activity of the soleus muscle also decreases in response to gravitational unloading, such as during hindlimb suspension and/or exposure to actual µ-G created by parabolic flight of a jet airplane (17, 30, 34, 36). Generally, hindlimb suspension and µ-G exposure results in plantarflexion of the ankle joints (90–160°), which then results in passive shortening of the ankle plantarflexors, such as the soleus (17, 31, 36). Tension development in the soleus is low when the ankle joint is plantarflexed, and no tension is detected if the anterior angle exceeds 120° (16). This inhibition in electromechanical activity most likely plays a key role in the reduction in soleus muscle fiber size associated with unloading.
In a previous study (16), we showed that a passive shortening of the sarcomere length in the soleus muscle fibers was induced in response to plantarflexion of the ankle joint. The present data indicate that the sarcomeres of soleus muscle fibers are stretched when the rats maintain a quadrupedal posture on the floor. The degree of sarcomere stretch was greater at the distal and proximal regions than in the central region of the fibers. Hindlimb unloading, on the other hand, resulted in passive shortening of the sarcomeres, especially in the central region. Although the mean sarcomere length was increased slightly toward the control level after 16 days of unloading due to a reorganization of the sarcomeres (16), the mean length in the central region of the fiber was still only 2.1 µm. We previously demonstrated that both the inherent (passive) tension of a relaxed muscle with the rat anesthetized and the in vivo (active) tension developed by conscious rats were not detectable when the sarcomere length was <2.1 µm (16). Therefore, these data suggest that the tension production and/or the mechanical load applied to the central region of the soleus muscle fibers were lower than normal throughout the 16 days of unloading in the present study.
Satellite cells are closely associated with postnatal muscle growth (23, 38) and regeneration from muscle injury (13, 21, 40, 41). In the present study, unloading resulted in a dramatic loss of satellite cells, mainly at the central region of the muscle fibers. The data suggest that the loss of satellite cells was closely related to a decrease in the mechanical load applied to the muscle fibers due to the plantarflexion-related passive shortening of sarcomeres. These results suggest that passive stretch of muscle fibers may be necessary to maintain both the activation and proliferation of satellite cells. This view is consistent with the report of Jejurikar et al. (15), who reported that chronic denervation of soleus and gastrocnemius muscles of rats increases the susceptibility of satellite cells to apoptosis. Combined, these data suggest that the suicide-related pathways for the removal of satellite cells may be induced in response to chronic unloading. It is not clear, however, why the patterns and degrees of unloading-related decreases were similar between the mitotically active and quiescent satellite cells. Neither is it known why the unloading effects on the distribution of satellite cells and myonuclei and the fiber CSA were different along the fiber length.
Effects of reloading. The loss of satellite cells after an unloading period was associated with a decrease in mean sarcomere length (see above). Ambulation on the floor results in the stretching of the soleus muscle fibers and, thus, increases both the sarcomere length (Fig. 10) and the load applied to the fibers (16). Therefore, we hypothesized that satellite cell number would be increased in response to acute reloading. In the present study, however, the numbers of both mitotically active and quiescent satellite cells were not increased by 2 days of reloading, although they were increased after 16 days after.
In contrast, Mozdziak et al. (25) reported that satellite cell mitotic activity, i.e., the number of BrdU-labeled nuclei per 1,000 total myofiber nuclei (satellite cell nuclei + myonuclei), was increased in rat soleus muscle 2 wk after recovery from unloading. The reasons for such an apparent discrepancy are unclear. However, one possibility may be due to the methodological differences. Mozdziak et al. (25) implanted miniosmotic pumps and 250 µg BrdU/h was delivered continuously in vivo for 2 wk to label all cells that entered the S phase of the cell cycle. In contrast, in the present study we first isolated muscle fiber segments and then labeled with BrdU for 4 h in vitro. We then counted the number of satellite cells, excluding myonuclei. In a pretest, we also compared the number of BrdU-positive satellite cells in one group, in which BrdU was injected intraperitoneally 2 days before sampling and in one group, in which the fibers were labeled in vitro as in the current study. The results were similar for the two groups. Other factors that may explain the different results in these two studies may be the differences in the species studied, i.e., Sprague-Dawley (25) vs. Wistar Hannover (present study) rats, and the different durations of unloading, i.e., 28 (25) vs. 16 (present study) days.
Previous studies (9, 10, 40) have reported an early activation of satellite cells in response to muscle injury. Schultz et al. (40) indicated that activation of satellite cells at the damaged region of a muscle fiber reached to a peak within 24–48 h after the injury. Furthermore, a large increase in satellite cell number in soleus muscle fibers of normal control rats was noted within one day after the intramuscular transplantation of satellite cells, and this may be related to muscle damage from the injection (Matsuoka Y, Kawano F, Wang XD, Terada M, Ogura A, and Ohira Y, unpublished observations). However, transplantation-related increase was not observed in the soleus muscle fibers of unloaded rats. Together, these results suggest that the responses of satellite cells to injury of normal fibers and to reloading of previously unloaded fibers are different. Chronic unloading of a muscle appears to downregulate both number and activation of satellite cells. Furthermore, myonuclear number and fiber CSA, which are regulated by satellite cell function, remained unchanged after 2 days of ambulation in the present study.
The levels of M-cadherin, an adhesion factor for satellite cells (24), expression in the central region of rat soleus muscle were decreased after 16 days of hindlimb unloading (Matsuoka Y, Kawano F, Wang XD, Terada M, Ogura A, and Ohira Y, unpublished observations). And the M-cadherin levels were gradually increased during ambulation recovery. The unloading-related inhibition of M-cadherin expression was prevented, if the muscle was kept at a stretched position (ankle dorsiflexed) using a plaster cast. Furthermore, the unloading-related decrease in the number of satellite cells is prevented when the soleus muscle is stretched at a dorsiflexed position (Wang XD, Matsuoka Y, Kawano F, Terada M, and Ohira Y, unpublished observations). Chronic stretching of the soleus muscle during hindlimb unloading also prevents atrophy (33). These results suggest that unloading-associated decrease of satellite cell number may be closely related to the lowered expression of M-cadherin, which is influenced by passive shortening of fibers.
The mean fiber CSA was significantly larger in the 16-day reloaded group than in the unloaded group (Fig. 2). The numbers of myonuclei (Figs. 6 and 7) and of both mitotically active and quiescent satellite cells per fiber (Figs. 4 and 5) also were higher in the reloaded than unloaded group. In addition, the number of satellite cells in the central region of the fiber was normalized in the reloaded group (Fig. 5C). These results suggest that the increase in the number and/or function of satellite cells in the reloaded rats played a key role in the recovery of myonuclear number and function, which, in turn, resulted in the recovery of fiber size.
Effects of G levels.
The unloading-related detrimental effects on fiber size, myonuclear properties, and sarcomere number recovered to the preexperimental control levels after 16 days of ambulation at 1 G. Recovery at 2 G had similar effects. We (17) previously reported that the EMG activity in the rat soleus muscle was increased during the ascending (hypergravity) phase of parabolic flight of a jet airplane. Thus, it appears that there is an increase of motoneuron activation in response to an acute elevation of the G level during parabolic flight. In contrast, the EMG level in the soleus was unaffected when rats were exposed chronically to a 2-G environment in an animal centrifuge (unpublished observation). The soleus muscle was maintained at a similar length at 2 G as at 1 G, i.e., the ankle joints at 30° position, once the rats acclimated to the new 2-G environment. Thus there was no extra loading of the muscle due to stretch under these conditions. Our conclusion is that the recovery of muscle fiber properties was not enhanced by an increase in G level during the recovery period, because the degree of mechanical stretch applied to muscle fibers was similar at both G levels used.
In conclusion, the effects of mechanical unloading and reloading on the properties of rat soleus muscle fibers were investigated in male Wistar Hannover rats. Myonuclear number and domain size, fiber CSA, and the total number of mitotically active and quiescent satellite cells of whole muscle fibers were lower than control after 16 days of unloading. These values then returned to control values after 16 days of reloading. A passive shortening of the soleus muscle fibers (and sarcomeres) was induced due to the plantarflexion of the ankle joints during hindlimb suspension of rats. The shortening of the sarcomeres was most evident in the central region of the muscle fibers, the region that showed the greatest loss of satellite cells. Thus there appears to be a close link between a decrease in tension development due to short sarcomere lengths and the loss of satellite cells that results in a decrease in fiber size during unloading. The responses of total number of both mitotic active and quiescent satellite cells and myonuclei per whole fiber, as well as fiber CSA, to unloading or reloading were similar, suggesting that the function and/or number of satellite cells play an important role in the load-dependent regulation of muscle fiber properties.
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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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P < 0.05 vs. Pre, R+0, and the age-matched control, respectively.
