Effect of exercise on age-related muscle atrophy

NeurobiologyofAging, Vol. 14, pp. 331-335, 1993

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Effect of Exercise on Age-Related Muscle Atrophy AKIHIKO ISHIHARA 1 AND SADAYOSHI TAGUCHI*

Department of Neurochemistry, Faculty of Integrated Human Studies, and *Graduate School of Human and Environmental Studies, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan R e c e i v e d 21 O c t o b e r 1991; R e v i s e d 13 January 1993; A c c e p t e d 1 February 1993 ISHIHARA, A. AND S. TAGUCHI. Effect of exercise on age-related muscle atrophy. NEUROBIOL AGING 14(4) 331-335, 1993.--Fifty-five-week-old male Wistar rats exercised voluntarily in running wheels for 10 weeks. The number and cross-sectional area of fibers in the slow-twitch soleus (SOL) and fast-twitch tibialis anterior (TA) muscles were recorded and compared with those of 20-week-old rats, and of age-matched rats not exercised. Muscle fibers were classified as fast-twitch oxidative glycolytic (FOG), fast-twitch glycolytic (FG), or slow-twitch oxidative (SO). After injection of horseradish peroxidase into the SOL and TA for retrograde neuronal labeling, oxidative enzyme activity of labeled motoneurons in the spinal cord was measured by microspectrophotometry. There were fewer FOG fibers in the SOL, and fewer FG fibers in the TA, at 65 weeks than at 20 weeks of age. The cross-sectional area of FOG and FG fibers in the TA was lower at 65 weeks than at 20 weeks of age. Exercise prevented the atrophy of FOG fibers in the TA. There were no age- or exercise-related differences in the number or oxidative enzyme activity of motoneurons in the SOL or the TA. These findings suggest that exercise can prevent the atrophy of FOG fibers by restoring their decreased metabolic capacity, and by inhibiting the degeneration of neuromuscular junctions. Oxidative enzyme activity Exercise Rat

Spinal motoneuron

Muscle fiber atrophy

AGING is associated with a reduction in the volume of the skeletal muscle. Earlier studies indicated that this reduction is due to decreases in the number (39) and volume (13) of individual muscle fibers. Decreases in the numbers of both slow-twitch (Type I) and fast-twitch (Type II) muscle fibers (38), especially those of Type II fibers (1,4,7,12,35,36,37), have been observed. The decrease in the number of Type 1I fibers may be due to transformation of the Type II fibers into Type I fibers (1,4,7) and to selective loss of Type II fibers (12,35,36,37). Gutmann and Hanzlikov,'i (15) observed that the decrease in the number of muscle fibers in aged rats is the result of degeneration of neuromuscular junctions, which occurs before motoneurons are lost from the spinal cord. However, in older animals there is a continuous decrease in the number of spinal motoneurons (8,17, 18) and of motor nerve fibers (41,42). We found that the decrease in the number of muscle fibers which occurs at a relatively early stage in aged rats is not accompanied by alteration of spinal motoneurons, but the reduction in the number of muscle fibers which occurs later is closely related to a loss of motoneurons (21). Only a few studies have examined the effect of physical activity on age-related muscle fiber atrophy (32,33). If atrophy and loss of muscle fibers are associated with decreased metabolic capacity, and if degeneration of neuromuscular junctions is the result of muscle inactivity alone, then we would expect exercise to delay or reverse these age-related morphological and histochemical

Soleus muscle

Tibialis anterior muscle

changes. Therefore, we examined the effects of exercise on fiber number and on the fiber cross-sectional area in the slow-twitch soleus and fast-twitch tibialis anterior muscles of 55-week-old rats, in which the number of motoneurons is not reduced. We also examined changes in the number of motoneurons in the neuron pool and in motoneuron oxidative enzyme activity. METHOD

Animals and Exercise Ten 55-week-old male Wistar rats were used. Five animals (exercise group) were housed 1 per cage. An exercise wheel was attached to one side of the cage, to which the animal had access for l0 weeks. The running distance was measured each day with a counter attached to the wheel. Another 5 animals (control group at 65 weeks) were also housed one per cage but without a wheel. Food and water were provided ad lib and room temperature was maintained at 22 + 2°C. Body weight was measured every other week. For the study of age-related changes in muscle fibers, five 20-week-old rats (considered to be mature) and five 55-week-old rats were also used as control groups.

Histochemical Methods The rats were anesthetized with ether. Ten V,1 of 20% horseradish peroxidase (HRP) was injected into the right soleus (SOL)

1 Requests for reprints should be addressed to Dr. A. Ishihara, Department of Neurochemistry, Faculty of Integrated Human Studies, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan. 331

332

ISHIHARA AND TAGUCHI

TABLE l

tions were cut in a cryostat at - 20°C, and were alternately stained for HRP with tetramethyl benzidine and for SDH. Quantitative analysis of SDH staining intensity of HRP-labeled motoneurons was performed by measurement of the density of SDH staining (22,24). Within the cytoplasm of each motoneuron analyzed, staining density was determined within spot areas of 5 to 9 p,m diameter with light of wavelength at 470 nm. The average of values for 2 or 3 different regions a given cell's cytoplasm was calculated. Values were expressed in terms of the optical density in arbitrary units. During the photometric analysis, all lighting conditions, magnifications, and reference points were kept constant. Motoneurons with a perfectly clear nucleus and average cell body diameter greater than 25 Ixm were regarded as alpha motoneurons.

RELATIVE MUSCLE WEIGHTS AT DIFFERENT AGES AND AFTER EXERCISE Group 20 55 65 65

weeks weeks weeks weeks

M. SOL C C C E

45.7 44.5 42.9 51.5

M. TA

-+ 2.34 +- 2.19 +- 2.78 -+ 5.27*

162.3 172.4 155.3 171.9

++-+ +-

5.48 9.05 6.83~: 10.55 +

Relative muscle weight is expressed as mg per 100 g body weight. Values are means +-- SDs for 5 animals. SOL = soleus; TA = tibialis anterior; 20 weeks C = 20-weekold control group; 55 weeks C = 55-week-old control group; 65 weeks C = 65-week-old control group; 65 weeks E = 65-week-old exercise group. *p < 0.05, compared to both 55 weeks C and 65 weeks C groups; tp < 0.05, compared to the 65 wks C group; Sp < 0.01, compared to the 55 weeks C group.

Statistical Analysis

muscle and 20 I~1 of 20% HRP was injected into the left tibialis anterior (TA) muscle. After 24 h they were again anesthetized with pentobarbital sodium. The left SOL and right TA were immediately removed and weighed. The muscle was frozen in liquid nitrogen and 10 ixm transverse sections of the widest point of the muscle belly were cut in a cryostat at - 2 0 ° C . Sections were stained for adenosine triphosphatase after alkaline preincubation (ATPase), and also for succinate dehydrogenase (SDH) and alpha glycerophosphate dehydrogenase (¢x-GPD). ATPase, SDH, and ¢x-GPD were used, respectively to distinguish fast- or slow-twitch type, high- or low-oxidative enzyme activity, and high- or lowglycolytic enzyme activity. Muscle fibers were classified as fasttwitch oxidative glycolytic (FOG), fast-twitch glycolytic (FG), or slow-twitch oxidative (SO) by their histochemical stain intensities. The numbers of muscle fibers of each type were counted directly on photomicrographs of the transverse sections, The crosssectional areas of fibers of each type on the ATPase-stained sections were calculated using a digitizer connected to a personal computer. The lumbosacral enlargement of the spinal cord was removed and frozen in liquid nitrogen. Five Ixm longitudinal sec-

Analysis of variance (ANOVA) was used to assess age-related changes. Student's t test was used to compare independent samples. Data from the exercise group were statistically compared to those from the control group when the animals in both groups were 65 weeks old. RESULTS

Changes With Aging Body weight and muscle weight. Body weights of rats in the control groups at 20, 55, and 65 weeks were 302.8 --- 11.79 g, 396.8 - 14.63 g, and 395.6 -+ 17.98 g, respectively. Although there were no age-related differences in the SOL muscle weight per 100 g body weight, changes in the TA muscle weight per 100 g body weight with aging were significant (ANOVA, p < 0.05). TA muscle weight per 100 g body weight at 65 weeks was significantly lower than that at 55 weeks (Table 1). Muscle fiber number. In the SOL, there were no age-related differences in the total number of muscle fibers. However, changes in the number of FOG fibers with aging were significant (ANOVA, p < 0.05). The number of FOG fibers at 65 weeks was significantly lower than that at 20 weeks. In the TA, changes in the total number of muscle fibers (ANOVA, p < 0.05) and the number of FG fibers (ANOVA, p < 0.05) with aging were significant.

TABLE 2 NUMBERS OF SOLEUS AND TIBIALIS ANTERIOR MUSCLE FIBERS AT DIFFERENT AGES AND AFrER EXERCISE M. SOL Group 20 weeks C Mean SD 55 weeks C Mean SD 65 weeks C Mean SD 65 weeks E Mean SD

M. TA

SO

FOG

Total

SO

FOG

FG

Total

2232 131.8

335 86.9

2566 215.5

54 12.6

4522 458.4

7658 568.5

12233 781.1

2153 134,8

220 79. I

2373 213.1

98t 26.1

4302 371.8

6528t 455.7

10928* 519.4

2166 204.7

138t 92.3

2304 278.8

1265 27.5

4319 429.9

6598t 418.9

11044" 777.7

2110 201.0

171 63.3

2281 260.7

102 36.2

4568 238.0

6148 312.2

10818 411.9

N = 5. SO = slow-twitch oxidative fiber; FOG = fast-twitch oxidative glycolytic fiber; FG = fast-twitch glycolytic fiber. *p < 0.05, tp < 0.01, Sp < 0.001, compared to the 20 weeks C group.

EFFECT OF EXERCISE ON MUSCLE ATROPHY

333

TABLE 5 OXIDATIVE ENZYME ACTIVITY OF ALPHA MOTONEURONS IN THE SOLEUS AND TIBIALIS ANTERIOR NEURON POOLS AT DIFFERENT AGES AND AFTER EXERCISE

TABLE 3 CROSS-SECTIONAL AREAS OF SOLEUS AND TIBIALIS ANTERIOR MUSCLE FIBERS AT DIFFERENT AGES AND AFTER EXERCISE M. SOL Group 20 weeks Mean SD 55 weeks Mean SD 65 weeks Mean SD 65 weeks Mean SD

M. TA

SO

FOG

SO

FOG

FG

4110 210

3970 224

2550 110

2560 140

2780 150

3910 180

3700 240

2450 230

2270* 240

2450? 140

3840 220

3680 290

2510 110

2240* 170

2420? 160

4010 190

3870 200

2520 120

25005 150

2550 120

C

C

C

Group 20 55 65 65

weeks weeks weeks weeks

SOL Neuron Pool C C C E

0.63 0.62 0.61 0.59

± ± ± ±

TA Neuron Pool

0.036 0.052 0.055 0.055

0.49 0.49 0.47 0.46

± 0.076 ± 0.081 ±- 0.105 - 0.106

Oxidative enzyme activity is expressed as optical density (arbitrary units). Motoneurons with an average cell body diameter greater than 25 Ixrn were regarded as alpha motoneurons. Values are mean ± SDs for 5 animals.

E

Fiber area is expressed as p,m2, and was measured from serial crosssections stained for adenosine triphosphatase after alkaline preincubation. n = 5. *p < 0.05, %p < 0.01, compared to the 20 weeks C group; Sp < 0.05, compared to the 65 weeks C group.

The total numbers of muscle fibers and the numbers of FG fibers at 55 weeks and 65 weeks were significantly lower than those at 20 weeks. There was a significant increase in the number of SO fibers in the TA associated with aging (ANOVA, p < 0.01) (Table 2). Fiber cross-sectional area. In the SOL, there were no agerelated differences in the cross-sectional area of the muscle fibers. In the TA, changes in the cross-sectional areas of FOG (ANOVA, p < 0.05) and FG (ANOVA, p < 0.01) fibers with aging were significant. The cross-sectional areas of FOG and FG fibers at 55 and 65 weeks were significantly lower than those at 20 weeks (Table 3). Motoneuron number and oxidative enzyme activity. There were no age-related differences in the total number or the oxidative

TABLE 4 NUMBERS OF HRP-LABELED MOTONEURONS IN THE SOLEUS AND TIBIALIS ANTERIOR NEURON POOLS AT DIFFERENT AGES AND AFTER EXERCISE SOL Neuron Pool Group

Alpha

Gamma

enzyme activity of motoneurons in the SOL or the TA (Tables 4 and 5). There were no histopathological abnormalities in the muscle or spinal cord.

Effects of Exercise Running distance. The running distance increased until the 7th week of exercise (Fig. 1). The mean daily running distance for the 10 weeks of exercise was 3,718 meters. Body weight and muscle weight. Body weights of the exercise group were significantly lower than those of the control group from the 4th to 10th week of exercise (Fig. 2). The SOL muscle weight per 100 g body weight in the exercise group was significantly greater than that in the control groups at 55 weeks and at 65 weeks. TA muscle weight per 100 g body weight in the exercise group was significantly greater than that in the control group at 65 weeks (Table 1). Muscle fiber number. The SOL and TA showed no exerciserelated differences in the total number of muscle fibers or in the number of fibers of each type (Table 2). Fiber cross-sectional area. In the SOL, there were no exerciserelated differences in the cross-sectional area of muscle fibers. In the TA, the cross-sectional area of FOG fibers in the exercise group was significantly greater than that in the control group at 65 weeks (Table 3). Motoneuron number and oxidative enzyme activity. There were no exercise-related differences in the total number or the oxidative

TA Neuron Pool Total

Alpha

Gamma

Total

>, "0

20 weeks C Mean SD 55 weeks C Mean SD 65 weeks C Mean SD 65 weeksE Mean SD

28 3.5

10 1.6

38 4.7

74 9.7

31 5.8

105 13.9

28 5.2

9 2.6

37 6.9

70 9.0

30 4.8

101 13.1

v

t-.

"o

c t-

28 5.3

10 2.0

38 6.8

70 6.8

29 5.3

99 12.0

I

8

2

0

L. 1

26 3.7

9 3.0

35 6.6

68 6.4

28 4.5

96 10.6

Motoneurons with an average cell body diameter greater than 25 N,m were regarded as alpha motoneurons (n = 5).

II

i II I I

I

I

I

I

2

3

4

5

I

I

I

I

7

8

9

10

wks of exercise

FIG. 1. Voluntary running distances of exercised rats. Values are means + SEs for 5 animals.

334

ISHIHARA AND TAGUCHI 500 E

400 0 a3

300

~ I

55

I 57

I 59

I 61

1 63

I 65

Age (weeks)

FIG. 2. Body weights of the control (11) and exercised (Q) rats. Exercise began when the animals were 55 weeks old and continued for 10 weeks. Values are means +- SDs for 5 animals. *p < 0.05. enzyme activity of motoneurons in the SOL or the TA (Tables 4 and 5). DISCUSSION Selective atrophy of Type I1 fibers occurs in skeletal muscles during aging (14,30,31,34,35). Age-related preferential degeneration of spinal motoneurons (17,18), especially those innervating Type II fibers, has also been observed (8,12,26,27). In this study, we found that atrophy of FOG and FG fibers in the TA occurred at 65 weeks of age. However, neither the number nor the metabolic capacity of TA motoneurons changed. These findings are consistent with those obtained in a previous study of the rat extensor digitorum longus muscle (23). To study the effect of exercise on muscle fiber atrophy we used 55- to 65-week-old rats because muscle fiber atrophy is not related to degeneration of the corresponding motoneurons or motor nerve fibers. Therefore, muscle fiber atrophy at this age may be largely due to muscle inactivity. If muscle fiber atrophy at this age depends on reduced physical activity, physical exercise may result in age-related structural and functional changes in muscle fibers. In this study, a 10-week period of exercise was found to prevent atrophy of FOG fibers in the TA. Although both FOG and FG fibers in the TA were atrophied in the control groups, exercise affected only the FOG fibers. The kind of voluntary running exercise used in this study was aerobic, high-intensity, and known to selectively recruit FOG muscle units (25). In another study (29), high-intensity strength training prevented age-related atrophy of

fibers in the rat soleus and plantaris muscles, while low-intensity, endurance swim training had no such effect. Thus, selective prevention of FOG fiber atrophy is an adaptive response and depends on muscle unit type and the nature of the exercise. The mechanisms by which exercise prevents the atrophy of FOG fibers are not clearly understood but probably involve different factors operating at various levels of the neuromuscular system. One possibility is that exercise results in changes in the neuromuscular junctions. Degeneration of neuromuscular junctions and an accompanying reduction in the capacity for maintenance of synaptic function were observed in animals of about the same age as those exercised in the present study (28). Andonian and Fahim (3) studied old mice and found that endurance exercise results in functional adaptation and prevents age-related nerve terminal degeneration in fibers of fast-twitch muscles, although the effect depended on the age of the animal. Exercise has been shown to prevent functional age-related changes in the neuromuscular junctions of the mouse fast-twitch muscle and are associated with muscle activity patterns during aging (2). Another explanation for the effect of exercise on muscle fiber atrophy is that exercise may restore a previously-reduced metabolic capacity to adapt to the functional demands of exercise. Enzyme activities of skeletal muscle normally decline with age (16,40), but several biochemical studies (5,6,11,29,32) indicate that endurance exercise can raise the levels of metabolic enzymes in muscles. Despite the age- and exercise-related changes in TA muscle fibers, there were no such changes in the fiber cross-sectional area in the SOL. This suggests that these age-related changes depend on muscle activity, and, therefore, that tonic slow-twitch SOL fibers maintain their pre-existing recruitment pattern and activity conditions while TA fibers become less active with aging. In very old animals, there was a loss of spinal motoneurons, in addition to loss and atrophy of muscle fibers (21,23). Furthermore, remodeling of neuromuscular systems is considered to occur at this age (9,10,20) although the capacity for such remodeling is less in older animals (19). It is important to determine whether or not age-related muscle fiber atrophy in very old animals can also be prevented by exercise. To answer this question, exercise training using rats older than 120 weeks will be required. ACKNOWLEDGEMENTS

This research was supported by a grant from the Meiji Life Foundation of Health and Welfare in Japan.

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