1 Laboratory of Exercise Biochemistry, Institute of Health and Sport Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8574; 2 Japan Institute of Sports Sciences, Kita-ku, Tokyo 115-0056; and 3 Graduate School of Integrated Science and Art, University of East Asia, Shimonoseki, Yamaguchi 751-5803, Japan
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ABSTRACT |
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Age-related but not artificially induced muscle fiber atrophy has been shown to occur without any decrease in myonuclear number, although these results remain controversial. The present study was carried out to clarify whether age difference affects the degree of decrease in myonuclear number occurring with denervation-induced fiber atrophy. After denervation of 3-wk-old (young) and 4-mo-old (mature) mice, single myofibers were isolated from the plantaris muscles by alkali maceration, and their fiber cross-sectional area (CSA), myonuclear number, and cytoplasm-to-myonucleus (C/N) ratios were analyzed. Fiber CSA in both young and mature mice decreased with denervation. Myonuclear number decreased in young mice 5 and 10 days after denervation but was unchanged in mature mice 10 and 120 days after denervation. C/N ratio decreased in mature mice but was unchanged in denervated young mice. These results suggest that age differences affect the degree of decrease of myonuclear number with denervation and that fiber cytoplasmic atrophy may occur without decrease in myonuclear number.
age difference; change in myonuclear number; fiber atrophy; single myofiber; alkali maceration
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INTRODUCTION |
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MYOFIBERS, which are typical multinucleated cells, can modify their cytoplasmic mass as a result of adaptation to functional demand. Although the mechanism of change in fiber cytoplasmic mass is still unclear, changes in myonuclear number may play an important role. Recently, Allen et al. (2) reviewed the relationship between myonuclear number and fiber cytoplasmic mass. They mentioned that, in many cases, changes in myonuclear number appear to be synchronized with changes in fiber cytoplasmic mass. In short, myofibers undergo cytoplasmic hypertrophy and atrophy accompanied by an increase and a decrease in myonuclear number, respectively, under physiological or experimental conditions (1, 4, 6, 7, 10).
However, Manta et al. (9) showed that myofibers obtained from subjects ages 60 yr and above had smaller fiber size, but their myonuclear numbers were similar to those obtained from 17- to 30-year-old subjects. This observation not only indicates that myofibers can undergo cytoplasmic atrophy without decrease in myonuclear number but also suggests that aged muscle subjected to artificially induced fiber atrophy may not show any decrease in myonuclear number. If this is true, the degree of decrease in myonuclear number should be different between young and mature subjects when fiber atrophy is experimentally induced. However, little is yet known about whether age difference affects the degree of decrease in myonuclear number caused by artificially induced fiber atrophy.
Denervation induces marked fiber atrophy and decrease in myonuclear number (11, 13). Therefore, the use of denervated muscle is one feasible method for investigating the effects of age difference on the change in myonuclear number associated with fiber atrophy. The present study aims to clarify whether the effect of denervation on decreased myonuclear number is different in young and mature mice and whether mature muscle undergoes cytoplasmic atrophy without any decrease in myonuclear number. In the present study, we have shown that after denervation, myonuclear number decreases in young mice but remains unchanged in mature mice. Our results suggest that fiber cytoplasmic atrophy can occur without decrease in myonuclear number.
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MATERIALS AND METHODS |
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Animals and surgical procedures. The present study was approved by the Animal Experimental Committee of the University of Tsukuba. Three-week-old (young) and four-month-old (mature) male ICR mice were used in this study. Animals were housed in an air- and humidity-controlled room. Food and water were provided ad libitum.
The sciatic nerve was dissected from animals under pentobarbital sodium or diethyl ether anesthesia. The skin of the proximal region of the hindlimb was opened so as not to cut any veins, and the sciatic nerve was carefully exposed, after which the nerve was cut to the maximum extent from the proximal to distal end. Denervation was carried out on mice at the ages of 3 wk and 4 mo. The plantaris muscles were removed at 0, 5, and 10 days after denervation of young mice, and at 0, 10, and 120 days after denervation of mature mice.Preparation of single myofibers. To obtain a large number of fine single myofibers, we developed a new fiber isolation method. Plantaris muscles were fixed with calcium-chelated 4% paraformaldehyde for more than 2 days. Fixed muscles were divided into several bundles by pulling the tendon with tweezers, and nonmuscle tissue was removed to the maximum degree possible under a binocular microscope. The muscle bundles were macerated in 40% NaOH solution for 3 h at room temperature and then shaken for 8 min to separate the bundle into single myofibers. Isolated myofibers were rinsed twice with phosphate-buffered saline (pH 7.2) for neutralization. They were then mounted on gelatin-coated glass slides and air-dried. Prepared single myofibers were stained with hematoxilin so that the myonuclei could be seen. Myofibers were also isolated from glycerol-extracted muscle: hindlimb muscles still attached to the bone were soaked overnight in 50% glycerol diluted with calcium-chelated saline at 4°C, and the myofibers were then mechanically isolated from the muscle tissue with sharpened fine tweezers under a binocular microscope. These mechanically isolated single myofibers were also fixed with calcium-chelated 4% paraformaldehyde before being mounted on a glass slide.
Morphometric analysis of single myofibers. The thickness of single myofibers did not permit all myonuclei to be seen in focus, so we synthesized several different focused images into a single image by using computer software (NIH Image, National Institutes of Health) to enable all the myonuclei within the fiber segment to be counted. Fiber cross-sectional area (CSA) and cytoplasmic volume were calculated by using the following formulas: fiber CSA = 3.14 · (w/2)2 and cytoplasmic volume = fiber CSA · l, where w and l are the width and length of the myofiber measured on the image, respectively. In this study, average nuclear domain size, or cytoplasmic volume per myonucleus, is represented as cytoplasm-to-myonucleus (C/N) ratio. Differences among each group were determined by using one-way ANOVA followed by Scheffé's post hoc test, with P < 0.01 regarded as significant for individual comparisons.
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RESULTS |
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Single myofibers.
In this study, we isolated single myofibers from paraformaldehyde-fixed
muscles by alkali maceration for morphometric analysis. Our alkali
maceration technique separated muscle tissue into single myofibers
without the need for a mechanical isolation process (Fig.
1A). These myofibers possessed
obvious cross-striations and multinuclei with distinct nucleoli and
showed no structural damage. All nuclei in single myofibers were round,
and their long axes were in line with the direction of the single
myofiber (Fig. 1, B-D). When single myofibers were
mechanically isolated from paraformaldehyde-fixed muscle without alkali
maceration, many single myofibers contained spindle-shaped nuclei that
were not in line with the myofiber. We have confirmed that capillary,
which is the most significant nonmuscle cell around myofibers, has a spindle-shaped nucleus. Therefore, we believe that our alkali maceration method isolates single myofibers without nonmuscle cells. Although fine myofibers could be isolated from
glycerol-extracted muscle by means of mechanical isolation (Fig.
1E), some of these myofibers contained nonmuscle cells or
were in a partly contracted state (Fig. 1, F and
G).
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Effects of denervation.
After denervation, none of the mice regained any movement in their
hindlimbs in the period between denervation and dissection, indicating
that no innervation occurred. Changes in fiber CSA, myonuclear number
per millimeter of fiber length, and C/N ratio after denervation in
3-wk-old and 4-mo-old mice are presented in Fig.
2. There are two points of interest.
First, myonuclear number in 4-mo-old mice was unchanged even after 120 days of denervation: myonuclear number in mature mice at both 10 and
120 days after denervation showed no significant difference from
day 0. However, a relatively short-term denervation
period (5 and 10 days) induced a decrease in myonuclear number in
3-wk-old mice. Second, C/N ratio was unchanged in 3-wk-old mice: C/N
ratio at both 5 and 10 days after denervation in 3-wk-old mice showed
no significant differences from day 0. However, C/N
ratio decreased with the elapse of time in 4-mo-old mice after
denervation. We carried out similar denervation experiments twice, and
the results of both the first and second trials were in close
agreement. In this study, the results of one trial are presented.
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DISCUSSION |
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Changes in fiber cytoplasmic mass and myonuclear number are not necessary synchronized. To gain an understanding of the relationship between them, it is necessary to find a factor that affects the degree of change of myonuclear number. The present study was carried out to clarify whether age differences affect the extent of decrease in myonuclear number resulting from denervation-induced fiber atrophy. Morphometric analysis of single myofibers isolated from denervated plantaris muscles of 3-wk-old and 4-mo-old mice showed that myonuclear number is unchanged at 10 and 120 days after denervation in 4-mo-old mice but that it decreases at 5 and 10 days after denervation in 3-wk-old mice. These results suggest that age difference affects the degree of decrease in myonuclear number and that fiber cytoplasmic atrophy can occur without a decrease in myonuclear number.
We have developed a novel single-myofiber isolation method (see MATERIALS AND METHODS). This isolation method has two advantages in the morphometric analysis of single myofibers. First, a large number of single fibers can be obtained without artificial selection in sampling because alkali maceration almost completely separates muscle tissue into single myofibers. This is essential for statistical analysis. Second, structurally intact single myofibers can be isolated. Some myofibers isolated by using the conventional mechanical isolation method contained nonmuscle cells or were in a partly contracted state (Fig. 1, F and G). Moreover, mechanical isolation is likely to cause myonuclear loss. When myofibers were mechanically isolated from 10-day denervated muscles, a smaller number of myonuclei was observed compared with those isolated by means of alkali maceration (Table 1). It is possible that the previously reported denervation-induced decrease in myonuclear number is partly due to artificially induced myonuclear loss. If this is the case, our alkali maceration method represents an improved method for isolating single myofibers for morphometric analysis. However, we have no evidence that the denervation-induced decrease in myonuclear number observed in 3-wk-old mice was not due to artificial nuclear loss.
Fast-type fiber has a smaller number of myonuclei than slow-type fiber (3). Therefore, a denervation-induced fiber type shift from slow to fast type may have caused the observed decrease in myonuclear number. However, we had already confirmed that the plantaris muscle of normal adult ICR mice comprises exclusively the fast type by examination of the myosin heavy chain isoform by means of immunohistological and electrophoretic techniques (data not shown). Thus the possibility that a denervation-induced fiber type shift causes the observed decrease in myonuclear number can be ruled out.
We found that myonuclear number remains unchanged with 10- and 120-day denervation in 4-mo-old mice but decreases with 5- and 10-day denervation in 3-wk-old mice. Our finding of no decrease in myonuclear number with long-term denervation is not wholly in agreement with the results of previous studies reporting that mean myonuclear number per millimeter of fiber length in rat extensor digitorum longus muscle decreased by 65, 47, and 32% compared with controls 2, 4, and 7 mo after denervation, respectively (13), and that of rat soleus muscle fibers decreased by 43% with 10 wk of denervation (11).
Although the reason for the lack of change in myonuclear number in mature mice after denervation is unclear, it may be due to age-related changes in the myofiber. In normal growth, the myofibers of ICR mice show increased myonuclear number over the first 5 wk of life (our unpublished data), indicating that myofibers in 3-wk-old murine muscle contain newly formed myonuclei but those of 4-mo-old mice muscles do not. If some nerve derivatives are specifically needed for newly formed myonuclei to survive, it is possible that only a proportion of myonuclei in young muscle enter the apoptotic process to disappear when the supply of nerve derivatives is arrested by denervation. The neuregulins, or ErbB ligands (5, 8) provided from neuron to myofiber, might be needed for newly formed myonuclei to survive. Trachtenberg (12) demonstrated that myonuclear apoptosis occurring in normal neonatal, botulinum toxin-paralyzed, and denervated muscle could be prevented by the application of exogenous neuregulin.
The mechanism of denervation-induced fiber atrophy seems to be different in young and mature mice because of the age-related difference in myofibers. The C/N ratio, or average nuclear domain size, decreased in 4-mo-old mice but remained unchanged in 3-wk-old mice after denervation, suggesting that denervation-induced fiber atrophy is associated with a decrease in myonuclear number in young mice, whereas it is associated with a decrease in nuclear domain size in mature mice. However, denervation-induced fiber atrophy in 3-wk-old mice also would be associated with a decrease in nuclear domain size. In normal growth, the C/N ratio continues to increase after 3 wk; mean C/N ratio of plantaris muscle is ~15,000 and 4,000 in 4-mo-old and 3-wk-old mice, respectively (Fig. 2). Therefore, it is thought that the lack of change in C/N ratio after denervation observed in 3-wk-old mice is the result of some decrease in C/N ratio. Previous studies reporting a decrease in myonuclear number associated with fiber atrophy showed a decrease in C/N ratio, too (7, 13). Thus it is possible that the decrease in nuclear domain size rather than that of nuclear number is important for fiber cytoplasmic atrophy.
In summary, we have developed a novel and practical single-myofiber isolation method, which led us to the following observations. 1) Denervation induces fiber cytoplasmic atrophy in both 3-wk-old and 4-mo-old mice. 2) However, myonuclear number decreases with denervation in 3-wk-old mice but remains unchanged in 4-mo-old mice. These results suggest that age differences are likely to affect the degree of decrease in myonuclear number resulting from denervation and that fiber atrophy can occur without a decrease in myonuclear number.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. Soya, Institute of Health and Sport Sciences, Univ. of Tsukuba, Tsukuba, Ibaraki 305-8574, Japan (E-mail: soya{at}taiiku.tsukuba.ac.jp).
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.
April 3, 2002;10.1152/ajpcell.00025.2002
Received 18 January 2002; accepted in final form 31 March 2002.
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