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J Appl Physiol 91: 183-190, 2001;
8750-7587/01 $5.00
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Vol. 91, Issue 1, 183-190, July 2001

Muscle regeneration during hindlimb unloading results in a reduction in muscle size after reloading

P. E. Mozdziak1, P. M. Pulvermacher2, and E. Schultz2

1 Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27695; and 2 Department of Anatomy, University of Wisconsin Medical School, Madison, Wisconsin 53706


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The hindlimb-unloading model was used to study the ability of muscle injured in a weightless environment to recover after reloading. Satellite cell mitotic activity and DNA unit size were determined in injured and intact soleus muscles from hindlimb-unloaded and age-matched weight-bearing rats at the conclusion of 28 days of hindlimb unloading, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-unloaded rats were significantly (P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb unloading, but they were the same (P > 0.05) as those of weight-bearing rats 2 and 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, number of nuclei per millimeter, and DNA unit size were significantly (P < 0.05) smaller for the injured soleus muscles from hindlimb-unloaded rats than for the soleus muscles from weight-bearing rats at each recovery time. Satellite cell mitotic activity was significantly (P < 0.05) higher in the injured soleus muscles from hindlimb-unloaded rats than from weight-bearing rats 2 wk after reloading, but it was the same (P > 0.05) as in the injured soleus muscles from weight-bearing rats 9 wk after reloading. The injured soleus muscles from hindlimb-unloaded rats failed to achieve weight-bearing muscle size 9 wk after reloading, because incomplete compensation for the decrease in myonuclear accretion and DNA unit size expansion occurred during the unloading period.

notexin; satellite cell; DNA unit; myofiber; atrophy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SKELETAL MUSCLE IS A COMPLEX tissue composed of multinucleate myofibers, the nuclei of which do not divide. Postnatal skeletal muscle growth occurs almost exclusively through a myofiber hypertrophy without myofiber hyperplasia, but concurrent with the myofiber hypertrophy is an increase in DNA content. A mitotically active satellite cell population located between the myofiber basal lamina and sarcolemma (20) donates new myonuclei to enlarging myofibers (22). Satellite cells are not only important in fueling skeletal muscle growth through the addition of new myonuclei but they also play an important role after a muscle injury. Immediately after a wound, myofiber debris is removed, and satellite cells become activated and recapitulate the embryonic sequence of events to form replacement myotubes, which mature into replacement myofibers (5). Recent data suggest that other cell types, such as bone marrow-derived cells, can contribute to muscle regeneration (15), but it is unclear whether the contribution of these other cell types to muscle regeneration is quantitatively meaningful. However, during growth of the replacement myofibers, satellite cells continue to contribute myonuclei. Therefore, satellite cells have a proliferative capacity that is not completely accessed during normal skeletal muscle growth. The individual myonuclei within a myofiber generated by the dividing satellite cells must also have an important role directing skeletal muscle growth, because they direct the transcription of myofibrillar proteins to the myofiber cytoplasm.

The DNA unit is the theoretical amount of cytoplasm supported by each nucleus in a multinucleate myofiber (3, 11, 32). The coordinated expression of muscle-specific proteins within a myofiber must be a complex process, because thousands of nuclei may be found within a myofiber segment, and all myonuclei are not transcriptionally active at one time (28). Similarly, the relationship between myonuclear accretion through the addition of new nuclei and DNA unit size must also be complex because of the different factors regulating satellite cell proliferation, satellite cell fusion, and myofiber size. Although a plethora of growth factors affect satellite cell proliferation and differentiation, it is likely that myofibers play a major role in directing satellite cell proliferation and myofiber fusion (8, 27, 42). Therefore, the interrelationship between the myofiber and the satellite cell population is important in determining ultimate muscle size, because myonuclear accretion is a major determinant of mature muscle size (25).

During normal skeletal muscle growth, there is a developmental program to increase DNA unit size (24-26). However, during an adaptive response in a mature muscle, satellite cell nuclei are added at a sufficient rate to maintain a constant DNA unit size (21). The maintenance of a constant DNA unit size during a myofiber adaptive response in a mature muscle would suggest that myofiber hypertrophy is dependent on myonuclear accretion. It appears that myonuclear accretion does not affect DNA unit size, because irradiation, which does not affect postmitotic nuclei, does not affect the developmental increase in DNA unit size in juvenile muscle (25). The maintenance of a constant DNA unit size during myofiber hypertrophy after an increase in mechanical load (21) suggests a direct relationship between the number of nuclei and the amount of protein per myofiber. Hindlimb unloading affects protein synthesis-degradation pathways (39), interrupts myonuclear accretion (13, 24, 35), and interrupts the developmental program to increase DNA unit size in juvenile animals (24). Therefore, it appears that DNA unit size can be altered under specific experimental conditions that negatively influence ultimate muscle size.

The hindlimb-unloading paradigm is an excellent model for the muscular atrophy and myosin heavy chain isoform transitions that occur during spaceflight (38), and it is an excellent model for muscle inactivity. The majority of studies employing the hindlimb-unloading model aimed at gaining insight into satellite cells and DNA unit size have employed mature animals to understand the adaptive response of a mature muscle to different levels of functional activity. However, hindlimb unloading is also an effective model to gain insight into the effects of weightlessness on juvenile muscle. It is known that hindlimb unloading/weightlessness induces apoptosis (1), decreases myonuclear number in adult rats (2, 4, 17), reduces DNA unit size (19), and induces muscular atrophy in mature skeletal muscle. Second, it is known that hindlimb unloading suppresses satellite cell mitotic activity in juvenile skeletal muscle and inhibits skeletal muscle growth (13, 24, 35). Third, it is known that hindlimb unloading does not inhibit myofiber formation after a muscle injury but reduces growth of the newly formed replacement myofibers (27). However, the effect of a muscle injury in conjunction with hindlimb unloading on muscle recovery after the resumption of weight bearing has been a relatively unexplored area of study. The objective of these studies was to examine whether a muscle that was injured and repaired in a weightless environment would attain normal myofiber size, satellite cell mitotic activity, and DNA unit size after the resumption of weight bearing. The rationale was to examine the possibility that the impaired development after injury induced by a weightless environment would be recovered after the resumption of weight bearing.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. All experimental procedures involving animals were approved by the University of Wisconsin Animal Care Committee. The myotoxic snake venom notexin was used to kill myofibers and activate satellite cells for replacement of the injured myofibers (27, 41) in the right soleus muscle of 48-day-old male Sprague-Dawley rats (~200 g, n = 35). Each rat was anesthetized (90 mg/kg body wt ketamine and 9 mg/kg body wt xylazine), the soleus muscle was exposed, and a single injection of 0.3 ml of notexin (20 µg/ml in 0.9% NaCl) was delivered through a 27-gauge needle (41). After recovery from anesthesia, the rats were randomly assigned to a hindlimb-unloading (HU, n = 18) or a weight-bearing control (WB, n = 17) group. The HU rats were immediately placed into a hindlimb-unloading apparatus (24, 27, 30). Briefly, the rats were attached by their tail to a trolley system that allowed only the forelimbs to touch the cage floor, but the rats had free movement to obtain food and water ad libitum. The remaining WB rats were maintained in the same room as the HU rats during the hindlimb-unloading period, with food and water provided ad libitum. The HU rats were hindlimb unloaded for 28 days. Near the end of the suspension period, the rats were randomly assigned to three groups for different post-hindlimb-unloading recovery times. The first group (group 1) of HU (n = 6) and WB (n = 5) rats was killed immediately after 28 days of hindlimb unloading. The second group (group 2) of HU (n = 6) and WB (n = 6) rats was killed 2 wk after removal from the hindlimb-unloading apparatus. The third group (group 3) of HU (n = 6) and WB (n = 6) rats was killed 9 wk after removal from the hindlimb-unloading apparatus. All rats were killed by an overdose of Beuthanasia-D (Schering-Plough Animal Health, Kenilworth, NJ; 0.25 ml/kg body wt). The post-hindlimb-unloading recovery times were based on a previous study (24).

Nuclear labeling. The rats in group 1 were given a single injection (100 mg/kg body wt ip) of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) on days 26, 27, and 28 of hindlimb unloading. The rats in group 2 were implanted subcutaneously with miniosmotic pumps (Alzet model 2ML2, Alza, Palo Alto, CA) containing BrdU while they were under general anesthesia (90 mg/kg body wt ketamine and 9 mg/kg body wt xylazine) immediately after they had been removed from the hindlimb-unloading apparatus. The rats in group 3 were implanted with miniosmotic pumps containing BrdU 7 wk after the conclusion of hindlimb unloading. In groups 2 and 3, the miniosmotic pumps were implanted to deliver BrdU to the rats over the 2 wk preceding their death. The miniosmotic pumps were designed to deliver BrdU at 250 µg/h to label all cells that entered the S phase of the cell cycle over the 2 wk after implantation (25).

Immunohistochemistry and image analysis. Myofiber segment preparation and analysis were similar to procedures employed by Mozdziak et al. (23). Soleus muscles were weighed, tied to sticks, and immersed in Carnoy's solution (60% ethanol, 30% chloroform, 10% acetic acid) for 24 h. Soleus muscles were hydrated to 70% ethanol, mechanically teased apart with fine tweezers, hydrated through an ethanol series, and digested with collagenase (480 U/ml PBS; catalog no. C-2139, Sigma Chemical, St. Louis, MO) at 37°C for 4 h. Subsequently, DNA was denatured with 2 N HCl for 1 h. Myofiber segments were washed four times with PBS and incubated overnight with the primary monoclonal antibody anti-BrdU (Becton-Dickinson, Mountain View, CA) diluted 1:10 with PBS containing 0.5% Tween 20 and 0.5% bovine serum albumin. BrdU-labeled nuclei were detected with goat anti-mouse IgG conjugated to fluorescein isothiocyanate (ICN Biomedicals, Irvine, CA) diluted 1:50 with PBS containing 0.5% Tween 20 and 10% goat serum. Myofiber nuclei (satellite cell nuclei + myonuclei) were counterstained with propidium iodide (50 µg/ml PBS). Myofiber segments were resuspended in mounting medium [75% (vol/vol) glycerol, 75 mM KCl, 10 mM tris(hydroxymethyl)aminomethane, 2 mM MgCl2, 2 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mM NaN3, pH 8.5, 1 mg/ml p-phenylenediamine] and mounted on glass slides.

Myofiber segments were observed with a Nikon (Biophot) microscope equipped with epifluorescence illumination. BrdU-labeled nuclei were visualized with a fluorescein isothiocyanate filter set, and myofiber nuclei were visualized with a Texas red filter set. Images of nuclei visualized with the fluorescein isothiocyanate filters and nuclei visualized with the Texas red filters were acquired using a Spot-2 charge coupled device camera (Diagnostic Instruments, Sterling Heights, MI). Mean myofiber segment diameter, myofiber segment length, number of BrdU-labeled nuclei, and number of propidium iodide-labeled nuclei were obtained for each myofiber segment using Metamorph software (Universal Imaging, West Chester, PA). Care was taken to analyze only connective tissue-free myofiber segments to avoid including any nonmyofiber nuclei in the counts of BrdU- or propidium iodide-labeled nuclei. The criterion for concluding analysis was counting >= 1,000 myofiber nuclei (satellite cell nuclei + myonuclei) for each muscle (23-26).

Calculations. An index of satellite cell mitotic activity was expressed as the number of BrdU-labeled nuclei per 1,000 total myofiber nuclei (satellite cell nuclei + myonuclei). The cytoplasmic volume-to-nucleus ratio (DNA unit size) was estimated after estimation of myofiber segment volume from myofiber segment diameter and length estimates [DNA unit size = pi (myofiber segment diameter/2)2 × (myofiber segment length)/myofiber nuclei].

Statistical analysis. Body weight, muscle weight, myofiber diameter, DNA unit size, the number of myofiber nuclei per millimeter, and the BrdU-labeling data for rats killed 2 and 9 wk after the conclusion of hindlimb unloading were analyzed using the General Linear Models procedure of SAS (34) to examine the effect of hindlimb unloading, muscle injury, and recovery time on each parameter. The data were analyzed using a split-plot design. Animal was the whole plot treatment, and leg was the subplot treatment. Least squares means were compared using least significant differences (29). If population variances were unequal, then a logarithmic transformation was performed on the data before analysis. The BrdU-labeling data at the conclusion of hindlimb unloading were analyzed using the General Linear Models procedure of SAS to examine the effect of muscle injury and hindlimb unloading on satellite cell mitotic activity. Least squares means were separated using least significant differences (29).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Growth after reloading. Body weights were higher (P < 0.05) for HU and WB rats 9 wk after reloading than at the conclusion of hindlimb unloading, indicating that the rats were growing during the post-hindlimb-unloading period. However, the body weights of the WB rats did not significantly (P > 0.05) change between the conclusion of hindlimb unloading and 2 wk after reloading (Table 1). The HU rats weighed significantly (P < 0.05) less than the WB rats at the conclusion of hindlimb unloading, but the body weights of HU rats were not significantly (P > 0.05) different from those of WB rats 2 and 9 wk after reloading.

                              
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  		Table 1.   Soleus muscle weight, body weight, and soleus muscle weight relative to body weight for intact and injured HU and WB rats

Myofiber diameters of uninjured soleus muscles from HU rats were smaller (P < 0.05) than those of uninjured soleus muscles from WB rats. Similarly, myofiber diameters of soleus muscles from injured HU rats were smaller (P < 0.05) than those of injured soleus muscles from WB rats (Fig. 1). Myofiber diameters of the injured muscles from HU and WB rats were also smaller than those of the uninjured muscles (Fig. 1). Myofiber diameters increased (P < 0.05) between each post-hindlimb-unloading recovery time for soleus muscles from HU and WB rats, indicating that the myofibers were growing. Similarly, hindlimb unloading in combination with muscle injury resulted in a smaller myofiber diameter, muscle weight, and muscle weight-to-body weight ratio than in uninjured soleus muscles from HU rats or all muscles from WB rats.


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  		Fig. 1.   Myofiber diameters after hindlimb unloading. Data are from rats killed at the conclusion of the unloading period (0 wk recovery), rats killed after 2 wk of reloaded recovery (2 wk recovery), and rats killed after 9 wk of reloaded recovery (9 wk recovery). WB, soleus muscles from weight-bearing rats; WB + IN, injured soleus muscles from weight-bearing rats; HU, soleus muscles from hindlimb-unloaded rats; HU + IN, injured soleus muscles from hindlimb-unloaded rats. Values are means ± SE. Values with different superscripts (a-g) are significantly different (P < 0.05).

Index of satellite cell mitotic activity. The index of satellite cell mitotic activity at the conclusion of hindlimb unloading was lower (P < 0.05) in the HU than in the WB rats (Table 2), confirming previous studies showing that hindlimb unloading suppressed satellite cell mitotic activity in the rat soleus (13, 35). The index of satellite cell mitotic activity was higher (P < 0.05) in the injured soleus muscles from WB rats than in the uninjured soleus muscles from WB rats at all experimental times (Table 2, Fig. 2). However, there were no differences (P > 0.05) between the injured and uninjured soleus muscles from the HU rats at all experimental times (Table 2, Fig. 2). Satellite cell mitotic activity was significantly (P < 0.05) higher in soleus muscles from HU rats than in soleus muscles from WB rats 2 wk after reloading, but satellite cell mitotic activity had fallen to nearly the same level as in soleus muscles from WB rats at 9 wk after reloading. There was no significant (P > 0.05) difference in satellite cell mitotic activity between the injured soleus muscles from HU and WB rats 9 wk after reloading.

                              
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  		Table 2.   Satellite mitotic activity at the conclusion of hindlimb unloading



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  		Fig. 2.   Index of satellite cell mitotic activity after hindlimb unloading. Values are means ± SE. Values with different superscripts (a-e) are significantly different (P < 0.05).

DNA unit size. DNA unit size increased between all ages, confirming previous results showing that DNA unit size expansion is a component of normal skeletal muscle growth (24-26). The injured muscles from the WB and HU rats exhibited a smaller (P < 0.05) DNA unit size than the contralateral control muscles at all times (Fig. 3). Hindlimb unloading resulted in a smaller DNA unit size than in soleus muscles from WB rats at all times when the intact soleus muscles from HU rats are compared with the intact soleus muscles from WB rats and the injured soleus muscles from HU rats are compared with the injured soleus muscles from WB rats.


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  		Fig. 3.   DNA unit size (cytoplasmic volume-to-nucleus ratio) after hindlimb unloading. Values are means ± SE. Values with different superscripts (a-h) are significantly different (P < 0.05).

Nuclei per millimeter. The number of nuclei per millimeter was the same in the injured muscles from WB rats and the contralateral control muscles at 0 and 2 wk of recovery, but it was significantly lower than in intact muscles from WB rats 9 wk after reloading (Fig. 4). Similarly, the number of nuclei per millimeter in the injured soleus muscles was lower than in the uninjured soleus muscles at all times examined for the HU rats. The number of nuclei per millimeter was lower for the intact soleus muscles from HU rats than for the intact soleus muscles from WB rats and for the injured soleus muscles from HU rats than the injured soleus muscles from WB rats at all times.


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  		Fig. 4.   Number of nuclei per millimeter after hindlimb unloading. Values are means ± SE. Values with different superscripts (a-g) are significantly different (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that muscle injury combined with muscle inactivity results in a long-term reduction in muscle/myofiber size after the resumption of weight bearing compared with injury in a weight-bearing environment or muscle inactivity without injury. Previous reports have shown that muscular inactivity reduces satellite cell mitotic activity in growing rats (13, 35), reduces DNA unit size in mature rats (19), and disrupts DNA unit size expansion in growing rats (24). Hindlimb unloading does not affect myogenic cell proliferation during the early stages of regeneration, despite the reduction in myofiber growth in the later stages of myofiber regeneration (27). Therefore, injured soleus muscles from HU rats consist of functional myofibers that are smaller in size than muscles subjected to all other treatments at the conclusion of the hindlimb-unloading period. The present study extends previous findings by studying the effect of muscle injury in a weightless environment on muscle recovery after the resumption of weight bearing.

Hindlimb unloading and recovery. Hindlimb unloading disrupts skeletal muscle growth by suppressing satellite cell mitotic activity, and the atrophy associated with hindlimb unloading induces apoptosis (1) of preexisting myonuclei. Therefore, hindlimb unloading has a detrimental effect on nuclear accretion-nuclear maintenance pathways in skeletal muscle. Previous studies have shown that a reduction in myonuclear accretion early in muscle development results in the long-term reduction in muscle size (24, 25), and a reduction in myonuclear accretion prevents the hypertrophic response after an increase in functional load (33). Myofiber diameters remained significantly smaller in soleus muscles from HU rats than in soleus muscles from WB rats throughout the end of the experiment. The lower muscle weights and myofiber diameters from the HU rats support the idea that the soleus muscles from HU rats remained significantly smaller than the soleus muscles from WB rats after reloading.

Despite the smaller size of soleus muscles from HU rats than of soleus muscles from WB rats, satellite cell mitotic activity was higher in soleus muscles from HU rats than in soleus muscles from WB rats immediately after reloading, suggesting that satellite cells were producing myonuclei for the compensatory increase in muscle size. However, the satellite cell compensatory response was transient and, consequently, insufficient to bring the myonuclear compliment to control levels by the end of the recovery period. A number of factors may have contributed to the elevated level of satellite cell mitotic activity immediately after the resumption of weight bearing. For example, the resumption of weight bearing reestablishes normal levels of electrical activity in the soleus (9), which was suppressed during hindlimb unloading, and reestablishment of normal levels of electrical activity may play a role in stimulating satellite cell mitotic activity (31). Lastly, the low-level myofiber damage without wholesale myofiber destruction (18, 37, 40) that has been reported after the resumption of weight bearing may play a role in stimulating satellite cell mitotic activity after reloading. It was also likely that any myofiber repair process was complete by the 7th wk after reloading, when BrdU infusion began for the last group of rats, because myotubes appear 3-4 days after notexin injection, and regeneration is considered complete 21-28 days after notexin injection (41). Therefore, any beneficial effects of low-level myofiber damage to stimulate satellite cell proliferation likely were not present during the last BrdU infusion period. Despite the fact that satellite cell mitotic activity increased after reloading, the transient nature of the compensation in the satellite cell population was insufficient for the muscle to attain normal muscle size and number of nuclei per millimeter 9 wk after reloading.

The injured muscle may not fully recover after the resumption of weight bearing, because the weight-bearing load did not create conditions that sufficiently stimulated satellite cells to divide for long enough to produce a sufficient number of nuclei for the enlarging myofibers to reach normal size. A second possibility is that conditions created during hindlimb unloading changed the satellite cells in the muscle to the extent that they were unable to respond to the stimulatory conditions created by the changes in electrical activity or the low level of myofiber damage after the reinstatement of weight bearing, because the in vivo environment has been shown to alter the subsequent in vitro behavior of the satellite cells (10). The failure of the muscle to fully recover after the resumption of weight bearing may be related to a decrease in satellite cell proliferation potential induced by the hindlimb-unloading-induced reduction in functional activity (10). Insulin-like growth factor-I (IGF-I) restores satellite cell in vitro proliferation potential after a hindlimb immobilization-induced reduction in in vitro proliferation potential, and overexpression of IGF-I protects muscle against age-related muscular atrophy (6). However, overexpression of IGF-I does not protect the muscle from hindlimb-unloading atrophy (12). It is possible that IGF-I administration after the resumption of weight bearing may aid in muscle recovery by restoring satellite cell proliferation potential in vivo as it does in vitro, because a minimum level of mechanical loading may be required for IGF-I to affect the satellite cell population in vivo to aid muscle recovery.

Satellite cell mitotic activity was lower in soleus muscles from WB rats than in soleus muscles from HU rats at the last recovery time, suggesting that the muscle could have been compensating for the hindlimb-unloading-induced reduction in mitotic activity. A previous study (24) showed that satellite cell mitotic activity was the same in the soleus muscles from HU and WB rats at 9 wk after hindlimb unloading. A potential reason for the discrepancy between studies may be related to differences in the age of the animals used. The rats employed in the previous study (24) were subjected to hindlimb unloading at a young age. Therefore, after reloading at the younger age, DNA unit size may have been capable of making a larger expansion, allowing for a greater amount of growth in diameter independent of myonuclear accretion. At the older age employed in the present study, DNA unit size may not have been capable of the same level of increase, independent of myonuclear accretion, at reloading. However, satellite cell mitotic activity was at such a low level at 9 wk after hindlimb unloading that any potentially remaining compensatory response was likely insignificant.

A second mechanism involved in changing muscle size is increasing DNA unit size. Previous studies have shown that a constant DNA unit size is maintained after an increase in functional load in mature muscle, suggesting that myonuclear accretion is the rate-limiting step of the hypertrophic response (21). Mature muscle may maintain a constant DNA unit size during hypertrophy, because the quantity of protein within a myofiber may be most easily changed by adding more of the necessary machinery to synthesize protein (DNA units) without changing the average quantity of protein produced per nucleus. During normal skeletal muscle growth or during muscle regeneration, the relationship between myonuclear accretion and DNA unit size expansion is more complicated than an increase in mature muscle mass in response to an increase in functional load, because changes in DNA unit size (i.e., changes in the average amount of protein produced per myonucleus) occur concurrently with myonuclear accretion. During normal skeletal muscle growth, DNA units are added to the myofiber during the early postnatal period. The addition of nuclei to the myofiber early in development may prepare the myofiber for the subsequent growth that occurs through DNA unit size expansion in the absence of the addition of new nuclei (26). It appears that a major effect of hindlimb unloading is a disruption in the normal developmental program for DNA unit size expansion (24) (Fig. 3). Therefore, the reduction in satellite cell mitotic activity combined with the disruption in DNA unit size expansion during the hindlimb-unloading period is responsible for the smaller soleus muscles from HU rats than from WB rats 9 wk after reloading.

Myofiber regeneration. The myotoxic snake venom notexin effectively kills all myofibers in the soleus (41). After administration of notexin, myofibers degenerate and phagocytoze, and satellite cells recapitulate the embryonic sequence of events to completely rebuild the damaged muscles. As expected, the myofibers were rebuilt by the resident satellite cell population after injury, and myofiber diameters remained smaller in the injured than in the intact muscle at all times. Satellite cell mitotic activity was higher in the injured muscles from WB rats at the conclusion of the hindlimb-unloading period and after 2 wk of recovery, suggesting that the satellite cell population was contributing nuclei to the enlarging regenerated myofibers after the injury. Despite the elevated levels of satellite cell mitotic activity at 2 wk after reloading, the number of nuclei per millimeter was lower in the injured than in the intact muscle 9 wk after reloading, which may suggest that satellite cell proliferation potential in vivo may have been reduced after injury, because muscle injury reduces satellite cell proliferation potential in vitro (36). Although the regenerated myofibers underwent a postinjury developmental program for DNA unit size expansion, the DNA unit size remained smaller in the injured than in the intact soleus muscles from WB rats. Overall, it appears that the regenerated muscles remained smaller than the intact muscles because of a smaller number of myonuclei and a reduced DNA unit size.

Reloading of injured muscle. The focus of the present study was to examine the effect of a muscle injury in a weightless environment on muscle size after the resumption of weight bearing. It has been previously shown that injury during hindlimb unloading does not affect myogenic cell proliferation but affects growth of the newly formed myofibers (27). Therefore, it was important to examine the possibility that the reduction in myofiber growth observed in the soleus muscles from HU rats would remain a permanent feature of the muscle after reloading. Muscle injury in a weightless environment clearly had a very detrimental long-term effect on ultimate skeletal muscle size after reloading, because the soleus muscle weight-to-body weight ratios and myofiber diameters remained smaller in the injured soleus muscles from HU rats than in all other soleus muscles through 9 wk after the resumption of weight bearing. Hindlimb unloading impedes the rate and the degree of skeletal muscle maturation after an injury, because expression of the normal component of myosin heavy chain isoforms is delayed in HU rats compared with WB rats (7, 14), and there is an interruption in myonuclear accretion. It appears that a minimal level of activity is required for a regenerating muscle to follow the normal maturational process (7, 14), whereas increasing functional activity does not alter regenerating myofiber maturation (16). Muscle regeneration is a recapitulation of the embryonic sequence of events leading to myofiber formation. Therefore, hindlimb unloading inhibits muscle maturation at the earliest possible time after myofiber formation. Injured soleus muscles from HU rats were smaller than intact soleus muscles from HU rats, and the injured muscles had a smaller DNA unit size at reloading. After reloading, satellite cell mitotic activity was similar in injured and intact soleus muscles from HU rats, whereas DNA unit size remained smaller in injured soleus muscles from HU rats than in intact soleus muscles from HU rats, suggesting that muscle injury in a weightless environment had a more detrimental effect than hindlimb unloading on muscle size after reloading.

At all times examined, satellite cell mitotic activity was the same in the injured and uninjured soleus muscles from HU rats. It is likely that the reloading induces a response in the satellite cell population that masks any residual effect of the muscle injury on satellite cell proliferation. Second, it is possible that the proliferative capacity of the satellite cell population is reduced by injury, because muscle injury (36) and immobilization (10) result in a reduction in in vitro proliferation potential. The net effect of the potential reduction in in vivo satellite cell proliferation potential was a significantly lower number of nuclei per millimeter in the injured soleus muscles from HU rats than in muscles subjected to all other treatments 9 wk after reloading, suggesting that hindlimb unloading in combination with muscle injury resulted in a reduction in the number of myonuclei, with each myonucleus controlling a smaller volume of cytoplasm.

In summary, the factors controlling skeletal muscle recovery after reloading of a previously injured muscle appear to be remarkably similar to the changes observed after the reloading of juvenile skeletal muscle. Interestingly, a common feature in all the small muscles is a reduced DNA unit size. A feature of normal postnatal muscle development is an increase in DNA unit size, but DNA unit size is resistant to change in mature muscle (21). The pattern of change in DNA unit size observed in this study suggests that DNA unit size is intimately involved in establishing ultimate myofiber size. It appears that the major factor impacting on muscle recovery after reloading is myofiber size at reloading. The injured muscles from HU rats were smaller and had a smaller DNA unit size than intact skeletal muscles at reloading, and the injured muscles remained smaller than muscles subjected to all other treatments throughout the 9-wk recovery period. Taken together, the observations from this study suggest that the hindlimb-unloaded environment affected the soleus muscle during a window of development when ultimate myofiber dimensions are determined. It appears that ultimate myofiber dimensions after injury are not modulated by the stresses associated with normal weight bearing, although they can be modulated by the excessive functional demands that induce myofiber hypertrophy (21). Similarly, the response of the satellite cell population after reloading of the injured muscle is the same as that of intact muscle, suggesting that the satellite cell population in injured muscle is responding to the same cues as hindlimb-unloaded intact soleus muscles. Response to these cues may be modulated by DNA unit size within the myofibers. The injured muscles from HU rats have undergone a twofold challenge to ultimate muscle size: 1) hindlimb unloading resulted in the muscle missing a window of opportunity for myonuclear accretion and DNA unit size expansion, and 2) muscle injury caused the resident satellite cell population to completely rebuild the muscle in a weightless environment, providing the opportunity for weightlessness to perturb muscle development. The perturbation of muscle development by muscle injury had the long-term effect of reducing muscle size after reloading.


    ACKNOWLEDGEMENTS

The authors thank Fei Zou (University of Wisconsin) for assistance with statistical methods.


    FOOTNOTES

Support was provided by US Department of Agriculture/National Research Initiative for Competitive Grants Program Grant 96-35206-3524 (P. E. Mozdziak) and National Aeronautics and Space Administration Grant NAG2-1220 (E. Schultz) and, in part, by funds provided under North Carolina State University Project NC06590.

Address for reprint requests and other correspondence: P. E. Mozdziak, Dept. of Poultry Science, Scott Hall/Campus Box 7608, North Carolina State University, Raleigh, NC 27695 (E-mail: pemozdzi{at}unity.ncsu.edu).

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.

Received 9 January 2001; accepted in final form 26 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allen, DL, Linderman JK, Roy RR, Bigbee AJ, Grindeland RE, Mukku V, and Edgerton VR. Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting. Am J Physiol Cell Physiol 273: C579-C587, 1997[Abstract/Free Full Text].

2.   Allen, DL, Linderman JK, Roy RR, Grindeland RE, Mukku V, and Edgerton VR. Growth hormone/IGF-I and/or resistive exercise maintains myonuclear number in hindlimb-unweighted muscles. J Appl Physiol 83: 1857-1861, 1997[Abstract/Free Full Text].

3.   Allen, DL, Roy RR, and Edgerton VR. Myonuclear domains in muscle adaptation and disease. Muscle Nerve 22: 1350-1360, 1999[ISI][Medline].

4.   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: 145-151, 1996[Abstract/Free Full Text].

5.   Anderson, JE. Murray L. Barr Award Lecture. Studies of the dynamics of skeletal muscle regeneration: the mouse came back! Biochem Cell Biol 76: 13-26, 1998[ISI][Medline].

6.   Barton-Davis, ER, Shoturma DI, Musaro A, Rosenthal N, and Sweeney HL. Viral-mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function. Proc Natl Acad Sci USA 95: 15603-15607, 1998[Abstract/Free Full Text].

7.   Bigard, XA, Merino D, Serrurier B, Lienhard F, Guezennec YC, Bockhold KJ, and Whalen RG. Role of weight-bearing function on expression of myosin isoforms during regeneration of rat soleus muscles. Am J Physiol Cell Physiol 270: C763-C771, 1996[Abstract/Free Full Text].

8.   Bischoff, R. Interaction between satellite cells and skeletal muscle fibers. Development 109: 943-952, 1990[Abstract/Free Full Text].

9.   Blewett, C, and Elder GC. Quantitative EMG analysis in soleus and plantaris during hindlimb suspension and recovery. J Appl Physiol 74: 2057-2066, 1993[Abstract/Free Full Text].

10.   Chakravarthy, MV, Davis BS, and Booth FW. IGF-I restores satellite cell proliferative potential in immobilized old skeletal muscle. J Appl Physiol 89: 1365-1379, 2000[Abstract/Free Full Text].

11.   Cheek, DB. The control of cell mass and replication. The DNA unit---a personal 20-year study. Early Hum Dev 12: 211-239, 1985[ISI][Medline].

12.   Criswell, DS, Booth FW, DeMayo F, Schwartz RJ, Gordon SE, and Fiorotto ML. Overexpression of IGF-I in skeletal muscle of transgenic mice does not prevent unloading-induced atrophy. Am J Physiol Endocrinol Metab 275: E373-E379, 1998[Abstract/Free Full Text].

13.   Darr, KC, and Schultz E. Hindlimb suspension suppresses muscle growth and satellite cell proliferation. J Appl Physiol 67: 1827-1834, 1989[Abstract/Free Full Text].

14.   Esser, KA, and White TP. Mechanical load affects growth and maturation of skeletal muscle grafts. J Appl Physiol 78: 30-37, 1995[Abstract/Free Full Text].

15.   Ferrari, G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, and Mavilio F. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279: 1528-1530, 1998[Abstract/Free Full Text].

16.   Ferry, A, Noirez P, Salah IB, Le Page C, Wahrmann JP, and Rieu M. Effect of increased physical activity on growth and differentiation of regenerating rat soleus muscle. Eur J Appl Physiol 76: 270-276, 1997.

17.   Hikida, RS, Van Nostran S, Murray JD, Staron RS, Gordon SE, and Kraemer WJ. Myonuclear loss in atrophied soleus muscle fibers. Anat Rec 247: 350-354, 1997[Medline].

18.   Kasper, CE. Sarcolemmal disruption in reloaded atrophic skeletal muscle. J Appl Physiol 79: 607-614, 1995[Abstract/Free Full Text].

19.   Kasper, CE, and Xun L. Cytoplasm-to-myonucleus ratios in plantaris and soleus muscle fibres following hindlimb suspension. J Muscle Res Cell Motil 17: 603-610, 1996[ISI][Medline].

20.   Mauro, A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9: 493-498, 1961[Free Full Text].

21.   McCall, GE, Allen DL, Linderman JK, Grindeland RE, Roy RR, Mukku VR, and Edgerton VR. Maintenance of myonuclear domain size in rat soleus after overload and growth hormone/IGF-I treatment. J Appl Physiol 84: 1407-1412, 1998[Abstract/Free Full Text].

22.   Moss, FP, and Leblond CP. Satellite cells as the source of nuclei in muscles of growing rats. Anat Rec 170: 421-436, 1971[Medline].

23.   Mozdziak, PE, Fassel T, Gregory R, Schultz E, Greaser ML, and Cassens RG. Quantitation of satellite cell proliferation in vivo using image analysis. Biotech Histochem 69: 249-252, 1994[ISI][Medline].

24.   Mozdziak, PE, Pulvermacher PM, and Schultz E. Unloading of juvenile muscle results in a reduced muscle size 9 wk after reloading. J Appl Physiol 88: 158-164, 2000[Abstract/Free Full Text].

25.   Mozdziak, PE, Schultz E, and Cassens RG. Myonuclear accretion is a major determinant of avian skeletal muscle growth. Am J Physiol Cell Physiol 272: C565-C571, 1997[Abstract/Free Full Text].

26.   Mozdziak, PE, Schultz E, and Cassens RG. Satellite cell mitotic activity in posthatch turkey skeletal muscle growth. Poult Sci 73: 547-555, 1994[ISI][Medline].

27.   Mozdziak, PE, Truong Q, Macius A, and Schultz E. Hindlimb suspension reduces muscle regeneration. Eur J Appl Physiol 78: 136-140, 1998.

28.   Newlands, S, Levitt LK, Robinson CS, Karpf AB, Hodgson VR, Wade RP, and Hardeman EC. Transcription occurs in pulses in muscle fibers. Genes Dev 12: 2748-2758, 1998[Abstract/Free Full Text].

29.   Ott, L. An Introduction to Statistical Methods and Data Analysis. Belmont, CA: Duxbury, 1993.

30.   Park, E, and Schultz E. A simple hindlimb suspension apparatus. Aviat Space Environ Med 64: 401-404, 1993[Medline].

31.   Putman, CT, Dusterhoft S, and Pette D. Satellite cell proliferation in low-frequency-stimulated fast muscle of hypothyroid rat. Am J Physiol Cell Physiol 279: C682-C690, 2000[Abstract/Free Full Text].

32.   Ralston, E, and Hall ZW. Restricted distribution of mRNA produced from a single nucleus in hybrid myotubes. J Cell Biol 119: 1063-1068, 1992[Abstract/Free Full Text].

33.   Rosenblatt, JD, and Parry DJ. Adaptation of rat extensor digitorum longus muscle to gamma irradiation and overload. Pflügers Arch 423: 255-264, 1993[ISI][Medline].

34.   SAS Institute. SAS User's Guide: Statistics, version 5. Cary, NC: SAS Institute, 1985.

35.   Schultz, E, Darr KC, and Macius A. Acute effects of hindlimb unweighting on satellite cells of growing skeletal muscle. J Appl Physiol 76: 266-270, 1994[Abstract/Free Full Text].

36.   Schultz, E, and Jaryszak DL. Effects of skeletal muscle regeneration on the proliferation potential of satellite cells. Mech Ageing Dev 30: 63-72, 1985[ISI][Medline].

37.   St. Pierre, BA, and Tidball JG. Differential response of macrophage subpopulations to soleus muscle reloading after rat hindlimb suspension. J Appl Physiol 77: 290-297, 1994[Abstract/Free Full Text].

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, Biggs RB, and Booth FW. Protein metabolism and beta -myosin heavy-chain mRNA in unweighted soleus muscle. Am J Physiol Regulatory Integrative Comp Physiol 257: R300-R305, 1989[Abstract/Free Full Text].

40.   Vijayan, K, Thompson JL, and Riley DA. Sarcomere lesion damage occurs mainly in slow fibers of reloaded rat adductor longus muscles. J Appl Physiol 85: 1017-1023, 1998[Abstract/Free Full Text].

41.   Whalen, RG, Harris JB, Butler-Browne GS, and Sesodia S. Expression of myosin isoforms during notexin-induced regeneration of rat soleus muscles. Dev Biol 141: 24-40, 1990[ISI][Medline].

42.   Yablonka-Reuveni, Z, Seger R, and Rivera AJ. Fibroblast growth factor promotes recruitment of skeletal muscle satellite cells in young and old rats. J Histochem Cytochem 47: 23-42, 1999[Abstract/Free Full Text].

J APPL PHYSIOL 91(1):183-190
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