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Age-related changes in fibre number, fibre size, fibre type composition and adenosine triphosphatase activity in rat soleus muscle
= = = = = = = = = ANNALS Of ANATOMY = = = = = = = = = Age-related changes in fibre number, fibre size, fibre type composition and adenosine triphosphatase activity in rat soleus muscle Satoshi Fujimoto, Jun Watanabe*, Ryokei Ogawa and Shinsuke Kanamura* Departmen t of Anatomy* and Orthopedics, Kansai Medical University, 1 Fumizono-cho, Moriguchi, Osaka 570, Japan
Summary. To study the aging of mu scle fibres in red skeletal muscle, fibre number, fibre diameter and fibre type composition in the soleus mu scle o f male rat s of 3, 12 and 24 months old were examined. The total number of mu scle fibre s remained unchanged, while average diameter increased slightly with increasing age. The staining intensity of myosin adenosine triphosphatase (ATPase) act ivity in the fibres decreased with advancing age. Therefore, observation on the basis of myosin ATPase histochemistry alone is not adequate to study the agin g of mu scle fibres. In the muscles of 24 month-old animals, four fibre types were recognized; 1) many (52%) type l-O fibre s showing weak ATPase and succinate dehydrogenase (SDH) reaction s with slight subsarcolemma l aggregates of diformazan (SAD); 2) some (330/0) type M fibres showing weak ATPa se and intense SDH reactions with marked SAD; 3) a few (12%) type a fibres showing weak ATPase and intense SDH reactions without SAD; and 4) very few (4%) type IIA fibres . Histochemical and morphometric results suggest that type l-O , type M and type a fibres are derived from type I, type I and type IIA fibres, respectively. Furthermore, no transitional fibres from type IIA to type I were observed. The refore , age-related changes in fibre type composition in the muscle cannot be explained by the simple idea that mo st type IIA fibres are transformed into type I fibres. Key words: Skeletal muscl e - Adeno sine triphosphatase Fibre typing - Aging - Rat
Introduction There has been a controversy regarding age-related changes in fibre type composition in muscles. Although Tauchi et al. Correspondence to: S. Kanamur a
Ann Anat (1994) 176: 429-435 Gu stav Fischer Verlag lena
(1971) and Silbermann et al. (1983) showed that type I fibres transformed into type II fibre s in some muscles during aging , Caccia et al. (1979) and Eddinger et al. (1985) reported that type II fibre s transformed into type I fibres during aging. McCarter (1978), Cac cia et al. (1979) and Alnaqeeb and Goldspink (1987) ob served that staining inten sit y of myofibrillar ad enosine triphosphatase (myosin ATPase) acti vity, which is used for fibre typing, decreased during aging. These findings suggest that the controversy concerning the changes in fibre type composition during aging is due to differences in criteria for fibre type classification. In addition, although there was evidence for a loss of muscl e fibre s (Tauchi et al. 1971; Gutmann and Hanzlikova 1976; Ihemelandu 1980; Lexell et al. 1988) and reduction in fibr e size (Lexell and Taylor 1991) during aging, the evidence is not conclusive (Rowe and Goldspink 1969; Eddinger et al. 1985). The present study was therefore undertaken to test the following hypotheses: a) the controversy regarding the changes in fibre type composition during aging is due to a difference in the criteria for fibre type classification, and b) the loss of mu scle fibres and reduction in fibre size occur no t in all muscles but onl y in some particular muscles. In the present study, we mea sured the changes in fibr e type composition in the rat soleu s muscle, a typical antigravity red muscle (Ariano et al. 1973), during aging. For thi s purpose, histochemical methods were used to classify fibre types based on acid- stable myosin ATPase activity and succinate deh ydrogenase (SDH) ac tivity. The age-related cha nges in fibre number and fibre size in the muscle were then measured by morphometry. Furthermore, myosin, in which ATPa se is localized (Miintener and Srihari 1983), was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using anti-myosin polyclonal or monoclonal antibody.
Materials and methods Fifteen male Wistar rats, 3 month-old (young animals), 12 monthold (middle-aged animals), and 24 month-old (old animals) were used. The animals were fed laboratory chow and water ad libitum and housed at 22 ± 1°C and 60 ± 10070 relative humidity under a 12: 12 h light/dark cycle. Under sodium pentobarbital anesthesia, the soleus muscles were quickly removed and frozen on dry-ice. The muscles from right legs were used for morphological and histochemical analysis. Those from left legs were homogenized with 9 volumes of 0.25 M sucrose and used for SDS-PAGE followed by Western blotting.
Classification of fibre types Three serial transverse sections, 10!lm in thickness, were cut at -10°C in a cryostat through the midbelly portion of the muscle. One section was incubated for acid-stable myosin ATPase activity according to the method of Brooke and Kaiser (1970) as described previously (Watanabe et al. 1986; Sakaida et al. 1987). In short, the section was preincubated for 15 min at room temperature in 0.1 M acetate buffer (pH 4.35) containing 0.1 M KCI, washed in distilled water, incubated for 30 min at 37°C in 50 mM glycine-NaOH buffer (pH 9.40) containing 3 mM ATP, 30 mM calcium chloride and 55 mM NaCI, and washed again in distilled water. The section was then immersed for 3 min in 2% aqueous calcium chloride, fixed for 3 min in buffered 4% formalin, washed in distilled water, immersed for 30 sec in 1% ammonium sulfide solution, washed again in distilled water and mounted in glycerol. The second section was incubated for alkali-stable myosin ATPase activity (Sakaida et al. 1987). The section was preincubated for 15 min at room temperature in 0.1 M barbital buffer (pH 10.25), and processed for the detection of myosin ATPase activity as described above. The third section was incubated for succinate dehydrogenase (SDH) activity according to the method of Barka and Anderson (1963). In brief, the section was incubated for 30 min at room temperature in 0.1 M phosphate buffer (pH 7.4) containing 60 mM sodium succinate and 0.1% (w/v) tetranitroblue tetrazolium, fixed in the fixative for 3 min, washed in distilled water and mounted in glycerol. To compare the staining intensity of myosin ATPase or SDH activity between the three age groups, sections cut from the muscles of young, middle-aged and old animals were incubated simultaneously in the same incubation medium. Staining intensity was measured semiquantitatively as the optical density in the centre of each fibre with a microphotometry system (KWSP-l) (Watanabe et al. 1991 b). Readings were made at 430 nm for the myosin ATPase reaction and 535 nm for the SDH reaction with a spot size of 10 urn. In our preliminary experiments, the staining reaction of acid-stable myosin ATPase activity was found to be more reproducible than that of alkali-stable myosin ATPase activity in the muscles of middle-aged and old animals. Therefore, we used acid-stable myosin ATPase activity and SDH activity for fibre typing.
Fibre counting and measurement of fibre diameter Photographs were taken from SDH- or ATPase-stained sections at a magnification of x 40 and enlarged to a final magnification of x 140. The photographs taken from SDH- and ATPase-stained sections were separately reconstructed to cover the entire muscle profile. Since the sections were cut in the midbelly region of the muscle, almost all the fibres were present in each reconstructed photograph (Watanabe et al. 1986; Alnaqeeb and Goldspink 1987). This reduced the risk of area sampling errors arising from the
uneven distribution of the various fibre types (Alnaqeeb and Goldspink 1987). The total fibre number in each muscle was measured by counting all the fibres in the reconstructed photograph of the SDH-stained section. Then, every fibre in the reconstructed photograph was identified as one of the fibre types, using reconstructed photographs of ATPase-stained sections, and the number of each fibre type was counted. Fibre number counting showed that all fibre types were distributed homogeneously in the muscles of young, middle-aged or old animals. Therefore, the average fibre diameter in each muscle was determined in randomly-selected areas in the reconstructed photograph of SDH-stained section after the identification of fibre type as mentioned above. About 300 fibres in the selected areas were analyzed per animal. No morphological abnormalities were seen in the muscles from young, middle-aged and old animals. No morphological abnormalities were found in the heart, lung, liver, kidney, spleen, small and large intestine, pancreas or testis in these animals.
Biochemical methods Proteins in the homogenate from soleus muscles were measured by the method of Lowry et al. (1951), and then, analyzed by SDS-PAGE according to the method of Laemmli (1970). Some gels were then stained with Coomassie Brilliant Blue. The remaining gels were subjected to Western blot analysis as described previously (Watanabe et al. 1991 a) by the use of anti-myosin polyclonal antibody (Cappel, West Chester, PA), or anti-myosin heavy chain monoclonal antibody from clone MY-32 (BioMakor, Rehovot, Israel).
Statistical analysis Data were subjected to the multiple x2-test or analysis of variance (ANOVA) followed by Duncan's multiple range test. All statistical comparisons were made above the 95% level of confidence.
Results Body weights of young, middle-aged and old animals were 310 ± to.5g, 716 ± 35.1 g and 616 ± 46.9g(means ± S.E. for five animals) respectively. The values in middle-aged and old animals were greater than the value in young animals (P < 0.05; Duncan's multiple range test), and the value in old animals was greater than that in middle-aged animals (P < 0.05). The soleus muscle weights of young, middle-aged and old animals were 152 ± 7.5 mg, 213 ± 37.5 mg and 185 ± 12.6 mg respectively. The values in middle-aged and old animals were greater than the value in young animals (P < 0.05), whereas no significant difference was seen between the value in middle-aged animals and that in the old animals (ANOVA). Fibre type composition Muscle fibres were classified according to the acid-stable myosin ATPase activity, sarcoplasmic SDH activity for interfibrillar mitochondria, and subsarcolemmal SDH activity for subplasmalemmal mitochondria. The fibres from young, middle-aged, and old animals were categorized as shown in Table 1.
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Table 1. Staining properties of muscle fibres seen in the soleus muscle of young, middle-aged and old rats Acid-stable adenosine triphosphatase (ATPase) activity
Succinate dehydrogenase (SDH) activity
Staining intensity")
Staining intensity b)
SAD')
Weak Strong Weak Strong Weak
Slight Marked Slight Marked Slight
Strong Strong
Marked Marked
Strong
Negligible
Acid-stable
Alkali-stable
Strong Strong Moderate Moderate Weak
Negative Negative Negative Negative Negative - Weak Strong Negative - Weak Negative - Weak
Fibre type (I) (IA) (I-M) (IA-M) (1-0)
IIA M
Negative Weak
0
Weak
") Staining intensity was classified into four classes on the basis of the results of microphotometry; negative (optical density of 0-0.04), weak (0.05-0.15), moderate (0.16-0.30) and strong (0.31- ). b) Staining intensity measured in the centre of each fiber was classified into four classes on the basis of the results of microphotometry; negative (optical density of 0-0.04), weak (0.05-0.10), moderate (0.11 -0.20) and strong (0.21 -). C) Subsarcolemmal aggregates of diformazan. The magnitude of SAD was defined as "negligible", "slight" or "marked" on the basis of visual inspection.
Table 2. Age-related changes in fibre type composition in rat soleus muscle ") Fibre type composition (070) Young (3 months old)
Middle-aged (12 months old)
Old (24 months old)
74.9 ± 1.27 3.7 ± 0.45 0 0 0
0 0 79.6 ± 4.94 2.7 ± 0.50 0
0 0 0 0 52.0 ± 0.63
21.4 ± 0.94
15.4 ± 3.79
3.7 ± 0.64
Type (I) (lA) (I-M) (IA-M) (1-0)
IIA M
0
2.3 ± 0.74
32.6 ± 3.81
0
0
0
11.7 ± 2.55
") Values are means ± S.E. for three animals.
The soleus muscles of young animals contained 1) many typical type I (slow twitch-oxidative) fibres, 2) a few type IIA (fast twitch-oxidative, glycolytic) fibres, and 3) very few atypical type I (IA) fibres (Figs. 1A and 1B) (Table2). There were no type lIB fibres in the muscle. In the muscle of middle-aged animals, four fibre types were recognized; 1) many type I (I-M) fibres, 2) very few atypical type I (I-AM) fibres, 3) very few type M fibres showing weak ATPase reaction and intense SDH reaction with marked subsarcolemmal aggregates of diformazan, and 4) a few type IIA fibres (Figs. 1C and 10). The staining intensity of the ATPase reaction was apparently lower in type I-M and type IA-M fibres than in type I and type IA fibres, as determined by simultaneous incubation of sections from young and middle-aged animals in the same incubation medium. In the muscles of old animals, four fibre types were recognized; 1) numerous type I (1-0) fibres, 2) a few type 0 fibres showing a weak ATPase reaction and an intense SDH reaction without subsarcolemmal aggregates of diformazan, 3) some type M fibres and 4) very few type IIA fibres (Figs. 1E and iF). The staining intensity of the ATPase reaction is apparently lower in type 1-0 fibres than in type I or type I-M fibres. Fibre number
The total number of fibres in the soleus muscle was statistically unchanged during senescence (ANOVA) (Table3). The number of type IIA fibres was unchanged from young to middle age, but decreased markedly from middle to old age (Duncan's multiple range test). The number of type I or type IA fibres in young animals was similar to that of type I-M or type IA-M fibres in middle-aged animals. The number of type 1-0 fibres in old animals was smaller than that of type I fibres in young animals or that of type I-M fibres in middle-aged animals. On the other hand, type M fibres appeared in middle-aged animals and increased markedly from middle to old age. Type 0 fibres appeared in old animals. Fibre size
The averaged diameter of total muscle fibres in the soleus muscle increased slightly with increasing age (Duncan's multiple range test) (Table4). In the muscles of young animals, the diameter was greater in type I or type IA fibres than in type IIA fibres. There was no difference in the values between type I and type IA fibres. In middle-aged animals, the value in type I-M, type IA-M or type M fibres was greater than type IIA fibres. TypeI-M, type IA-M and type M fibres were approximately equal in size. In old animals, the value in 1-0 fibres was greatest, followed in order by type M fibres, type 0 fibres and type IIA fibres. The value in type IIA fibres was unchanged from young to middle age, but decreased markedly from middle to old age. The value in type I-M or type IA-M fibres in middle-aged animals was similar to that in type I or type IA fibres in 431
Figs. 1A - t F. Serial transverse sections of the soleus muscles incubated for acid-stable adenosine triphosphatase activit y (A, C, and E) or incubated for succinate dehydrogenase activity (B, D, and F). x 270. Figs. t A and t B. Young (3 month-old) animal s. I, typical type I fibre; lA , atypical type I fibre; IIA , type llA fibre. Figs. t C and t D. Middle-aged (12 month-old) anim als. I, type I-M fibre; lA, type IA-M fibre; IIA, type IIA fibre; M, type M fibre. Figs. t E and t F. Old (24 month-old) an imals. I, type 1-0 fibre; HA, type llA fibre; M, type M fibre; 0 , type 0 fibre.
young animals. The value in type 1-0 fibres in old animals was slightly greater than that in type I fibres in young animals or the value in type I-M fibres in middle-aged animals . Fibre size distribution of each fibre type is summariz ed in Figure 2. Biochemical results
Protein contents (mg/g wet tissue) in the soleus muscle of young , middle-aged and old animals were 61 ± 4.2,
52 ± 7.7 and 42 ± 2.7 (means ±S.E. for five animals), respectively.
When the homogenate was applied to SDS-PAGE (Fig. 3 A) followed by Western blotting using the polyclonal antibody (Fig. 3 B), four bands at 220 kD (myosin heavy chain) , 37 kD (a fragment of digested myosin heavy chain), 18 kD (myosin light chain fragment 2; LCF 2) and 15 kD (myosin light chain fragment 3; LCF 3) were detected. The staining intensities of bands at 220, 37, and 15 kD were
432
Table 3. Age-related changes in fibre number in rat soleus muscles
Total (I) (IA) (I-M) (IA-M) (1-0)
IIA
Young
Middle-aged
Old
1920 ± 114.9
2020 ± 71.8
1797 ± 75.9
1438 ± 24.4 71 ± 8.6
NF NF
NF NF NF
1608 ± 99.8 55 ± 10.1
NF NF NF NF
NF
411±18.0
M
NF
0
NF
Discussion
934 ± 11.3
311 ± 76.6
67 ± 11.5
47 ± 14.9
586 ± 68.5
NF
210 ± 45.8
Values are means ± S.E. for three animals. NF, not found. Table 4. Age-related changes in fibre size in rat soleus muscles Fibre diameter (urn) Middle-aged Old
Young Total
56.5 ± 0.27
60.6 ± 0.16
68.6 ± 0.37
I IA I-M IA-M
57.7 ± 0.76 59.3 ± 0.30
NF NF 61.5 ± 0.24 61.7 ± 0.27
1-0
NF NF NF
NF NF NF NF
NF
76.1 ± 0.79
IIA
52.9 ± 0.43
57.6 ± 0.27
31.0 ± 1.59
M
NF
62.0 ± 0.31
71.4 ± 0.91
0
NF
NF
39. t ± 1.59
Values are means ± S.E. for three animals. NF, not found. YOUNG
I
IA
I I M NONE
M
>U .. Z
w
~
ow
a:
u.
0
0 NONE
1
L
OLD
1(1-0 )
...
10
IIANONE
IIA
o M
04080006010
FIBER
"'
0
NONE
o
40
decreased with increasing age, whereas that of the band at 18 kD was similar in all age groups. MY-32 anti-myosin heavy chain monoclonal antibody recognized myosin heavy chain (Fig. 3 C). The staining intensity of the band at 220 decreased slightly with increasing age.
eo
DIAMETER ( pm )
Fig. 2. Frequency distribution of each fibre type in the soleus muscles from young, middle-aged, and old rats. NONE, could not be found.
As shown in the present study, the total number of muscle fibres in the soleus muscle remained unchanged during senescence. It is therefore probable that the decrease in total fibre number, reported in previous studies (Tauchi et al. 1971; Gutmann and Hanzlfkova 1976; Ihemelandu 1980; Lexell et al. 1988), is not a universal aging phenomenon. Moreover, the average diameter of total fibres in the muscle increased gradually with increasing age. These findings support the tested hypothesis that the loss of muscle fibres and reduction in fibre size do not occur in all muscles but only in some particular muscles. The staining intensity of myosin ATPase activity in fibres of the soleus muscle decreased gradually with advancing age. Furthermore, histochemical and biochemical results suggest that quantitative and/or conformational change in myosin molecules occur during senescence. Therefore, observation on the basis of ATPase histochemistry alone is probably inadequate to examine the muscle aging. Eddinger et al. (1985) showed that the percentage of type I fibres in the soleus muscle in Fisher 344 rats increased, but that of type IIA fibres decreased from middle to old age. However, Alnaqeeb and Goldspink (1987) found that the percentages of type I and IIA fibres in the muscles of CFY SpragueDawley rats remained unchanged from middle to old age. When type M and type 0 fibres are classified into type I and type IIA, respectively, the present results are in agreement with those of Alnaqeeb and Goldspink (1987). However, if fibres showing weak ATPase reaction (types 1-0, M and 0) are simply classified into type I, the results are compatible with those of Eddinger et al. (1985). The controversy regarding the age-related changes in fibre type composition found between the results of previous studies is probably due to differences in the criteria of fibre type identification. In type M fibres, the staining intensity of the ATPase reaction was intermediate between type I (IA-M or I-M) and type IIA fibres. However, both the staining properties of SDH activity and fibre size distribution in type M fibres resembled those in type I (lA-M) fibres. Furthermore, decrease in type I fibres from middle to old age (type I-M 1608 to type 1-0 934) can be explained by the transformation of some (about 550) type I-M fibres into type M fibres. Therefore, type M fibres are probably derived from type I fibres (Fig. 4). Although the ATPase staining reaction was weak in type 0 fibres, the staining properties of SDH activity in type 0 fibres were similar to those in type IIA fibres except for the absence of subsarcolemmal aggregates of difor-
433
1 2 3
4 5 6
123
1 2 3
4 5 6
4 5 6
220-
220-
67-
67-
-
45-
45-37
=11
2514 -
_18 -15
C
8
A
- 37
Figs. 3A - 3 C. SOS-PAGE and Western blot ana lysis of solubilized homo genates from the soleus muscle of young (lanes 1 and 4), middleaged (lanes 2 and 5), and old (lanes 3 and 6) rats. Numbers at the left or right side indicate molecular mass in kilo-Daltons. Fig. 3A. SOS-PAGE. Solubilized homogenate (lanes 1- 3, 5 mg wet tissue/ lane; lanes 4 - 6, 9 - 11 ~g protein/lane) was applied onto gradient (4 - 20%) polyacrylamide gel. Stained with Coo massie Brilliant Blue. Fig. 3 B. Western blot anal ysis. Solubilized homogenate (lanes I - 3, 3 mg wet tissue/lane; lanes 4 - 6, 5 ug protein/lane) was subjected to SOS-PAGE, transferred onto a nitrocellulose membrane, and stained with an anti-myosin polyclonal antibody. Fig. 3 C. Western blot analysis. Solubilized homogenate (lanes 1- 3. 5 mg wet tissue/ lane; lanes 4 - 6, 20 ug protein/l ane) was subjected to SOS-PAGE, transferred, and stained with an anti-myosin heavy chain monoclon al antibody from clone MY-32.
YOUNG
MIDDLE-AGED
OLD
I(I+IA) -I(I-M+IA-M) -1(1 -0)
-...
IIA
..........
-- ---
-... M - -
--
IIA-
M
a IIA
Fig. 4. Probable pathways of fibre aging in the rat soleus muscle. I, type I fibre including typical type I, type lA, type I-M, type IA-M and type 1-0 fibres; M, type M fibre; 0 , type 0 fibre; IIA, type IIA fibre.
mazan in type 0 fibres . Furthermore, the fibre size distribution of type 0 fibres was similar to that of type IIA fibre s. Moreover, the decrease in the number of type IIA fibre s from middle to old age can be explained by the change of type IIA fibres into type 0 fibre s. The se strongly suggest that type 0 fibre s are derived from type IIA fibres, although the possibility that origins of typ e 0 fibr es are type M fibr es or myosatellite cells can not be ruled out (Fig. 4). The size of type IIA fibr es in old animals was a pparently smaller than that in young or middle-aged animals. The small-sized type IIA fibres are neither newly differentiated type IIA fibre s deri ved from myosatellite cells nor split fibres, because transitional fibres between myosatellite cells and type IIA fibres (Caccia et a1. 1979) coult not be found
in the mu scles of old an imals, and the nu mber of type IIA fibres decreased markedly from middle to old age. The small-sized type IIA fibre s are probably degenerating type IIA fibre s. There are several anti-myosin monoclonal antibodies for fibre typing (Marini et al. 1989; Gorza 1990; Fiichtbauer eta1. 1991). In our preliminary experiments, commercially available anti -myosin monoclonal antibodies were helpful for fibre type identification in muscle sections from young animals. Ho wever, these antibodies were inadequate for the fibre type identification in sections from old animals, becau se all soleus fibr es were homogeneously stained with an an ti-slow myosin an tibody. Although the present biochemical result s suggest the occurrence of conformational changes in certain epitopes of myosin molecules, the epitope specificity of the fibr e type-specific monoclonal antibodies has not yet been elucidated. Caccia et al. (1979) and Eddinger et al. (1985) suspected that most type IIA fibres in the soleu s muscles were transformed into type I fibres during aging . Caccia et al. (1979) assum ed the presence of "transitional or tru e intermediate" fibres bet ween type IIA to type I in the muscles of old animals. The transitional fibres exhibited high mitochondrial oxidative enzyme acti vity and ATPase activity intermed iate between those of type I and type IIA fibr es, and increased from middle (0.3 %) to old age (15.8% ) (Caccia et a1. 1979). These cha racteristics are coincident with those of type M fibres found in the present study. However,
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the origin of type M fibres is not type IIA fibres but probably type I fibres as described above. There may be no true transitional fibres between type I and type IIA fibres in the soleus muscle of old animals.
References Alnaqeeb MA, Goldspink G (1987) Changes in fiber type, number and diameter in developing and aging skeletal muscle. J Anat 153: 31-45 Ariano MA, Armstrong RB, Edgerton VR (1973) Hindlimb muscle fiber populations of five mammals. J Histochem Cytochern 21: 51-55 Barka T, Anderson PJ (1963) Histochemistry. Harper and Row, New York, p 314. Brooke MH, Kaiser KK (1970) Muscle fiber types: how many and what kind? Arch Neurol 23: 369 - 379 Caccia MR, Harris JB, Johnson MA (1979) Morphology and physiology of skeletal muscle in aging rodents. Muscle Nerve 2: 202-212 EddingerTJ, Moss RL, Cassens RG (1985) Fiber number and type composition in extensor digitorum longu s, soleus, and diaphragm muscles with aging in Fisher 344 rats. J Histochem Cytochem 33: 1033-1041 Fuchtbauer E-M, Rowlerson AM, Got z K, Friedrich G, Mabuchi K, Gergely J, JockuschH (1991) Direct correlation of parvalbumin levels with myosin isoforms and succinate dehydrogena se activity on frozen sections of rodent muscle. J Histochem Cytochem 39: 355 - 361 Gorza L (1990) Identification of a nobel type 2 fiber population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies. J Histochem Cytochem 38: 257 - 263 Gutmann E, Hanzlikova V (1976) Fast and slow motor units in aging. Gerontology 22: 280 - 300 Ihemelandu EC (1980) Decrease in fiber number of dog pectineus muscle with age. J Anat 130: 69 - 73 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680 - 685
Lexell J, Taylor CC (1991) Variability in muscle fibre areas in whole human quadriceps muscle: effects of increasing age. J Anat 174: 239-249 LexellJ, 'Iaylor CC, Sjostrom M (1988) What is the cause of the aging atrophy? Total number, size and proportion of different fiber types studied in whole vastus lateral is muscle from 15- to 83-year-old men. J Neurol Sci 84: 275 - 294 Lowry OH, Rosebrough AL, Farr AL , Randall RJ (1951) Protein measurement with the Folin phenol reagent. J BioI Chern 193: 265-275 Marini J-F, Pons F, Anoal M, Leger J. Leger JJ (1989) Anti -myosin heavy chain monoclonal antibodies reveal two IB (fast) fiber subtypes. J Histochem Cytochem 37: 1721-1729 McCarter R (1978) Effects of age on contraction of mammalian skeletal muscle. Aging 6: 1-21 Muntener M, Srihari T (1983) Changes of myosin and its ATPase in experimentally induced fiber transformation in the rat. Exp Neurol 80: 471-478 Rowe RWD, Goldspink G (1969) Muscle fibre growth in five different muscles in both sexes of mice. I Normal mice. J Anat 104: 519-530 Sakaida M. Watanabe J, Kanamura S, Tokunaga H, Ogawa R (1987) Physiological role of skeletal muscle glycogen in starved mice. Anat Rec 218: 267 - 274 Silbermann M, Finkelbrand S, Weiss A. Gershon D, Reznick A (1983) Morphometric analysis of aging skeletal muscle following endurance training. Muscle Nerve 6: 136- 142 Tauchi H, Yoshioka T, Kobayashi H (1971) Age changes of skeletal muscles of rats. Gerontologia 17: 219 - 227 Watanabe J, Kanamura S. Kanai K, Shugyo Y (1986) Cytochemical and biochemical glucose 6-phosphatase activity in skeletal mu scle cells of mice. Anat Rec 214: 25 - 31 WatanabeJ, Kanai K, KanamuraS (1991a) Measurement of NADPH-ferrihemoprotein reductase content in sections of liver. J Histochem Cytochem 39: 1635-1643 Watanabe J, Kanamura R (1991 b) An improved microphotometry system for measurement of cytochrome P-450 in hepatocyte cytoplasm. J Histochem Cytochem 39: 689-694
Accepted August 24, 1993
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Summary. To study the aging of mu scle fibres in red skeletal muscle, fibre number, fibre diameter and fibre type composition in the soleus mu scle o f male rat s of 3, 12 and 24 months old were examined. The total number of mu scle fibre s remained unchanged, while average diameter increased slightly with increasing age. The staining intensity of myosin adenosine triphosphatase (ATPase) act ivity in the fibres decreased with advancing age. Therefore, observation on the basis of myosin ATPase histochemistry alone is not adequate to study the agin g of mu scle fibres. In the muscles of 24 month-old animals, four fibre types were recognized; 1) many (52%) type l-O fibre s showing weak ATPase and succinate dehydrogenase (SDH) reaction s with slight subsarcolemma l aggregates of diformazan (SAD); 2) some (330/0) type M fibres showing weak ATPa se and intense SDH reactions with marked SAD; 3) a few (12%) type a fibres showing weak ATPase and intense SDH reactions without SAD; and 4) very few (4%) type IIA fibres . Histochemical and morphometric results suggest that type l-O , type M and type a fibres are derived from type I, type I and type IIA fibres, respectively. Furthermore, no transitional fibres from type IIA to type I were observed. The refore , age-related changes in fibre type composition in the muscle cannot be explained by the simple idea that mo st type IIA fibres are transformed into type I fibres. Key words: Skeletal muscl e - Adeno sine triphosphatase Fibre typing - Aging - Rat
Introduction There has been a controversy regarding age-related changes in fibre type composition in muscles. Although Tauchi et al. Correspondence to: S. Kanamur a
Ann Anat (1994) 176: 429-435 Gu stav Fischer Verlag lena
(1971) and Silbermann et al. (1983) showed that type I fibres transformed into type II fibre s in some muscles during aging , Caccia et al. (1979) and Eddinger et al. (1985) reported that type II fibre s transformed into type I fibres during aging. McCarter (1978), Cac cia et al. (1979) and Alnaqeeb and Goldspink (1987) ob served that staining inten sit y of myofibrillar ad enosine triphosphatase (myosin ATPase) acti vity, which is used for fibre typing, decreased during aging. These findings suggest that the controversy concerning the changes in fibre type composition during aging is due to differences in criteria for fibre type classification. In addition, although there was evidence for a loss of muscl e fibre s (Tauchi et al. 1971; Gutmann and Hanzlikova 1976; Ihemelandu 1980; Lexell et al. 1988) and reduction in fibr e size (Lexell and Taylor 1991) during aging, the evidence is not conclusive (Rowe and Goldspink 1969; Eddinger et al. 1985). The present study was therefore undertaken to test the following hypotheses: a) the controversy regarding the changes in fibre type composition during aging is due to a difference in the criteria for fibre type classification, and b) the loss of mu scle fibres and reduction in fibre size occur no t in all muscles but onl y in some particular muscles. In the present study, we mea sured the changes in fibr e type composition in the rat soleu s muscle, a typical antigravity red muscle (Ariano et al. 1973), during aging. For thi s purpose, histochemical methods were used to classify fibre types based on acid- stable myosin ATPase activity and succinate deh ydrogenase (SDH) ac tivity. The age-related cha nges in fibre number and fibre size in the muscle were then measured by morphometry. Furthermore, myosin, in which ATPa se is localized (Miintener and Srihari 1983), was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using anti-myosin polyclonal or monoclonal antibody.
Materials and methods Fifteen male Wistar rats, 3 month-old (young animals), 12 monthold (middle-aged animals), and 24 month-old (old animals) were used. The animals were fed laboratory chow and water ad libitum and housed at 22 ± 1°C and 60 ± 10070 relative humidity under a 12: 12 h light/dark cycle. Under sodium pentobarbital anesthesia, the soleus muscles were quickly removed and frozen on dry-ice. The muscles from right legs were used for morphological and histochemical analysis. Those from left legs were homogenized with 9 volumes of 0.25 M sucrose and used for SDS-PAGE followed by Western blotting.
Classification of fibre types Three serial transverse sections, 10!lm in thickness, were cut at -10°C in a cryostat through the midbelly portion of the muscle. One section was incubated for acid-stable myosin ATPase activity according to the method of Brooke and Kaiser (1970) as described previously (Watanabe et al. 1986; Sakaida et al. 1987). In short, the section was preincubated for 15 min at room temperature in 0.1 M acetate buffer (pH 4.35) containing 0.1 M KCI, washed in distilled water, incubated for 30 min at 37°C in 50 mM glycine-NaOH buffer (pH 9.40) containing 3 mM ATP, 30 mM calcium chloride and 55 mM NaCI, and washed again in distilled water. The section was then immersed for 3 min in 2% aqueous calcium chloride, fixed for 3 min in buffered 4% formalin, washed in distilled water, immersed for 30 sec in 1% ammonium sulfide solution, washed again in distilled water and mounted in glycerol. The second section was incubated for alkali-stable myosin ATPase activity (Sakaida et al. 1987). The section was preincubated for 15 min at room temperature in 0.1 M barbital buffer (pH 10.25), and processed for the detection of myosin ATPase activity as described above. The third section was incubated for succinate dehydrogenase (SDH) activity according to the method of Barka and Anderson (1963). In brief, the section was incubated for 30 min at room temperature in 0.1 M phosphate buffer (pH 7.4) containing 60 mM sodium succinate and 0.1% (w/v) tetranitroblue tetrazolium, fixed in the fixative for 3 min, washed in distilled water and mounted in glycerol. To compare the staining intensity of myosin ATPase or SDH activity between the three age groups, sections cut from the muscles of young, middle-aged and old animals were incubated simultaneously in the same incubation medium. Staining intensity was measured semiquantitatively as the optical density in the centre of each fibre with a microphotometry system (KWSP-l) (Watanabe et al. 1991 b). Readings were made at 430 nm for the myosin ATPase reaction and 535 nm for the SDH reaction with a spot size of 10 urn. In our preliminary experiments, the staining reaction of acid-stable myosin ATPase activity was found to be more reproducible than that of alkali-stable myosin ATPase activity in the muscles of middle-aged and old animals. Therefore, we used acid-stable myosin ATPase activity and SDH activity for fibre typing.
Fibre counting and measurement of fibre diameter Photographs were taken from SDH- or ATPase-stained sections at a magnification of x 40 and enlarged to a final magnification of x 140. The photographs taken from SDH- and ATPase-stained sections were separately reconstructed to cover the entire muscle profile. Since the sections were cut in the midbelly region of the muscle, almost all the fibres were present in each reconstructed photograph (Watanabe et al. 1986; Alnaqeeb and Goldspink 1987). This reduced the risk of area sampling errors arising from the
uneven distribution of the various fibre types (Alnaqeeb and Goldspink 1987). The total fibre number in each muscle was measured by counting all the fibres in the reconstructed photograph of the SDH-stained section. Then, every fibre in the reconstructed photograph was identified as one of the fibre types, using reconstructed photographs of ATPase-stained sections, and the number of each fibre type was counted. Fibre number counting showed that all fibre types were distributed homogeneously in the muscles of young, middle-aged or old animals. Therefore, the average fibre diameter in each muscle was determined in randomly-selected areas in the reconstructed photograph of SDH-stained section after the identification of fibre type as mentioned above. About 300 fibres in the selected areas were analyzed per animal. No morphological abnormalities were seen in the muscles from young, middle-aged and old animals. No morphological abnormalities were found in the heart, lung, liver, kidney, spleen, small and large intestine, pancreas or testis in these animals.
Biochemical methods Proteins in the homogenate from soleus muscles were measured by the method of Lowry et al. (1951), and then, analyzed by SDS-PAGE according to the method of Laemmli (1970). Some gels were then stained with Coomassie Brilliant Blue. The remaining gels were subjected to Western blot analysis as described previously (Watanabe et al. 1991 a) by the use of anti-myosin polyclonal antibody (Cappel, West Chester, PA), or anti-myosin heavy chain monoclonal antibody from clone MY-32 (BioMakor, Rehovot, Israel).
Statistical analysis Data were subjected to the multiple x2-test or analysis of variance (ANOVA) followed by Duncan's multiple range test. All statistical comparisons were made above the 95% level of confidence.
Results Body weights of young, middle-aged and old animals were 310 ± to.5g, 716 ± 35.1 g and 616 ± 46.9g(means ± S.E. for five animals) respectively. The values in middle-aged and old animals were greater than the value in young animals (P < 0.05; Duncan's multiple range test), and the value in old animals was greater than that in middle-aged animals (P < 0.05). The soleus muscle weights of young, middle-aged and old animals were 152 ± 7.5 mg, 213 ± 37.5 mg and 185 ± 12.6 mg respectively. The values in middle-aged and old animals were greater than the value in young animals (P < 0.05), whereas no significant difference was seen between the value in middle-aged animals and that in the old animals (ANOVA). Fibre type composition Muscle fibres were classified according to the acid-stable myosin ATPase activity, sarcoplasmic SDH activity for interfibrillar mitochondria, and subsarcolemmal SDH activity for subplasmalemmal mitochondria. The fibres from young, middle-aged, and old animals were categorized as shown in Table 1.
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Table 1. Staining properties of muscle fibres seen in the soleus muscle of young, middle-aged and old rats Acid-stable adenosine triphosphatase (ATPase) activity
Succinate dehydrogenase (SDH) activity
Staining intensity")
Staining intensity b)
SAD')
Weak Strong Weak Strong Weak
Slight Marked Slight Marked Slight
Strong Strong
Marked Marked
Strong
Negligible
Acid-stable
Alkali-stable
Strong Strong Moderate Moderate Weak
Negative Negative Negative Negative Negative - Weak Strong Negative - Weak Negative - Weak
Fibre type (I) (IA) (I-M) (IA-M) (1-0)
IIA M
Negative Weak
0
Weak
") Staining intensity was classified into four classes on the basis of the results of microphotometry; negative (optical density of 0-0.04), weak (0.05-0.15), moderate (0.16-0.30) and strong (0.31- ). b) Staining intensity measured in the centre of each fiber was classified into four classes on the basis of the results of microphotometry; negative (optical density of 0-0.04), weak (0.05-0.10), moderate (0.11 -0.20) and strong (0.21 -). C) Subsarcolemmal aggregates of diformazan. The magnitude of SAD was defined as "negligible", "slight" or "marked" on the basis of visual inspection.
Table 2. Age-related changes in fibre type composition in rat soleus muscle ") Fibre type composition (070) Young (3 months old)
Middle-aged (12 months old)
Old (24 months old)
74.9 ± 1.27 3.7 ± 0.45 0 0 0
0 0 79.6 ± 4.94 2.7 ± 0.50 0
0 0 0 0 52.0 ± 0.63
21.4 ± 0.94
15.4 ± 3.79
3.7 ± 0.64
Type (I) (lA) (I-M) (IA-M) (1-0)
IIA M
0
2.3 ± 0.74
32.6 ± 3.81
0
0
0
11.7 ± 2.55
") Values are means ± S.E. for three animals.
The soleus muscles of young animals contained 1) many typical type I (slow twitch-oxidative) fibres, 2) a few type IIA (fast twitch-oxidative, glycolytic) fibres, and 3) very few atypical type I (IA) fibres (Figs. 1A and 1B) (Table2). There were no type lIB fibres in the muscle. In the muscle of middle-aged animals, four fibre types were recognized; 1) many type I (I-M) fibres, 2) very few atypical type I (I-AM) fibres, 3) very few type M fibres showing weak ATPase reaction and intense SDH reaction with marked subsarcolemmal aggregates of diformazan, and 4) a few type IIA fibres (Figs. 1C and 10). The staining intensity of the ATPase reaction was apparently lower in type I-M and type IA-M fibres than in type I and type IA fibres, as determined by simultaneous incubation of sections from young and middle-aged animals in the same incubation medium. In the muscles of old animals, four fibre types were recognized; 1) numerous type I (1-0) fibres, 2) a few type 0 fibres showing a weak ATPase reaction and an intense SDH reaction without subsarcolemmal aggregates of diformazan, 3) some type M fibres and 4) very few type IIA fibres (Figs. 1E and iF). The staining intensity of the ATPase reaction is apparently lower in type 1-0 fibres than in type I or type I-M fibres. Fibre number
The total number of fibres in the soleus muscle was statistically unchanged during senescence (ANOVA) (Table3). The number of type IIA fibres was unchanged from young to middle age, but decreased markedly from middle to old age (Duncan's multiple range test). The number of type I or type IA fibres in young animals was similar to that of type I-M or type IA-M fibres in middle-aged animals. The number of type 1-0 fibres in old animals was smaller than that of type I fibres in young animals or that of type I-M fibres in middle-aged animals. On the other hand, type M fibres appeared in middle-aged animals and increased markedly from middle to old age. Type 0 fibres appeared in old animals. Fibre size
The averaged diameter of total muscle fibres in the soleus muscle increased slightly with increasing age (Duncan's multiple range test) (Table4). In the muscles of young animals, the diameter was greater in type I or type IA fibres than in type IIA fibres. There was no difference in the values between type I and type IA fibres. In middle-aged animals, the value in type I-M, type IA-M or type M fibres was greater than type IIA fibres. TypeI-M, type IA-M and type M fibres were approximately equal in size. In old animals, the value in 1-0 fibres was greatest, followed in order by type M fibres, type 0 fibres and type IIA fibres. The value in type IIA fibres was unchanged from young to middle age, but decreased markedly from middle to old age. The value in type I-M or type IA-M fibres in middle-aged animals was similar to that in type I or type IA fibres in 431
Figs. 1A - t F. Serial transverse sections of the soleus muscles incubated for acid-stable adenosine triphosphatase activit y (A, C, and E) or incubated for succinate dehydrogenase activity (B, D, and F). x 270. Figs. t A and t B. Young (3 month-old) animal s. I, typical type I fibre; lA , atypical type I fibre; IIA , type llA fibre. Figs. t C and t D. Middle-aged (12 month-old) anim als. I, type I-M fibre; lA, type IA-M fibre; IIA, type IIA fibre; M, type M fibre. Figs. t E and t F. Old (24 month-old) an imals. I, type 1-0 fibre; HA, type llA fibre; M, type M fibre; 0 , type 0 fibre.
young animals. The value in type 1-0 fibres in old animals was slightly greater than that in type I fibres in young animals or the value in type I-M fibres in middle-aged animals . Fibre size distribution of each fibre type is summariz ed in Figure 2. Biochemical results
Protein contents (mg/g wet tissue) in the soleus muscle of young , middle-aged and old animals were 61 ± 4.2,
52 ± 7.7 and 42 ± 2.7 (means ±S.E. for five animals), respectively.
When the homogenate was applied to SDS-PAGE (Fig. 3 A) followed by Western blotting using the polyclonal antibody (Fig. 3 B), four bands at 220 kD (myosin heavy chain) , 37 kD (a fragment of digested myosin heavy chain), 18 kD (myosin light chain fragment 2; LCF 2) and 15 kD (myosin light chain fragment 3; LCF 3) were detected. The staining intensities of bands at 220, 37, and 15 kD were
432
Table 3. Age-related changes in fibre number in rat soleus muscles
Total (I) (IA) (I-M) (IA-M) (1-0)
IIA
Young
Middle-aged
Old
1920 ± 114.9
2020 ± 71.8
1797 ± 75.9
1438 ± 24.4 71 ± 8.6
NF NF
NF NF NF
1608 ± 99.8 55 ± 10.1
NF NF NF NF
NF
411±18.0
M
NF
0
NF
Discussion
934 ± 11.3
311 ± 76.6
67 ± 11.5
47 ± 14.9
586 ± 68.5
NF
210 ± 45.8
Values are means ± S.E. for three animals. NF, not found. Table 4. Age-related changes in fibre size in rat soleus muscles Fibre diameter (urn) Middle-aged Old
Young Total
56.5 ± 0.27
60.6 ± 0.16
68.6 ± 0.37
I IA I-M IA-M
57.7 ± 0.76 59.3 ± 0.30
NF NF 61.5 ± 0.24 61.7 ± 0.27
1-0
NF NF NF
NF NF NF NF
NF
76.1 ± 0.79
IIA
52.9 ± 0.43
57.6 ± 0.27
31.0 ± 1.59
M
NF
62.0 ± 0.31
71.4 ± 0.91
0
NF
NF
39. t ± 1.59
Values are means ± S.E. for three animals. NF, not found. YOUNG
I
IA
I I M NONE
M
>U .. Z
w
~
ow
a:
u.
0
0 NONE
1
L
OLD
1(1-0 )
...
10
IIANONE
IIA
o M
04080006010
FIBER
"'
0
NONE
o
40
decreased with increasing age, whereas that of the band at 18 kD was similar in all age groups. MY-32 anti-myosin heavy chain monoclonal antibody recognized myosin heavy chain (Fig. 3 C). The staining intensity of the band at 220 decreased slightly with increasing age.
eo
DIAMETER ( pm )
Fig. 2. Frequency distribution of each fibre type in the soleus muscles from young, middle-aged, and old rats. NONE, could not be found.
As shown in the present study, the total number of muscle fibres in the soleus muscle remained unchanged during senescence. It is therefore probable that the decrease in total fibre number, reported in previous studies (Tauchi et al. 1971; Gutmann and Hanzlfkova 1976; Ihemelandu 1980; Lexell et al. 1988), is not a universal aging phenomenon. Moreover, the average diameter of total fibres in the muscle increased gradually with increasing age. These findings support the tested hypothesis that the loss of muscle fibres and reduction in fibre size do not occur in all muscles but only in some particular muscles. The staining intensity of myosin ATPase activity in fibres of the soleus muscle decreased gradually with advancing age. Furthermore, histochemical and biochemical results suggest that quantitative and/or conformational change in myosin molecules occur during senescence. Therefore, observation on the basis of ATPase histochemistry alone is probably inadequate to examine the muscle aging. Eddinger et al. (1985) showed that the percentage of type I fibres in the soleus muscle in Fisher 344 rats increased, but that of type IIA fibres decreased from middle to old age. However, Alnaqeeb and Goldspink (1987) found that the percentages of type I and IIA fibres in the muscles of CFY SpragueDawley rats remained unchanged from middle to old age. When type M and type 0 fibres are classified into type I and type IIA, respectively, the present results are in agreement with those of Alnaqeeb and Goldspink (1987). However, if fibres showing weak ATPase reaction (types 1-0, M and 0) are simply classified into type I, the results are compatible with those of Eddinger et al. (1985). The controversy regarding the age-related changes in fibre type composition found between the results of previous studies is probably due to differences in the criteria of fibre type identification. In type M fibres, the staining intensity of the ATPase reaction was intermediate between type I (IA-M or I-M) and type IIA fibres. However, both the staining properties of SDH activity and fibre size distribution in type M fibres resembled those in type I (lA-M) fibres. Furthermore, decrease in type I fibres from middle to old age (type I-M 1608 to type 1-0 934) can be explained by the transformation of some (about 550) type I-M fibres into type M fibres. Therefore, type M fibres are probably derived from type I fibres (Fig. 4). Although the ATPase staining reaction was weak in type 0 fibres, the staining properties of SDH activity in type 0 fibres were similar to those in type IIA fibres except for the absence of subsarcolemmal aggregates of difor-
433
1 2 3
4 5 6
123
1 2 3
4 5 6
4 5 6
220-
220-
67-
67-
-
45-
45-37
=11
2514 -
_18 -15
C
8
A
- 37
Figs. 3A - 3 C. SOS-PAGE and Western blot ana lysis of solubilized homo genates from the soleus muscle of young (lanes 1 and 4), middleaged (lanes 2 and 5), and old (lanes 3 and 6) rats. Numbers at the left or right side indicate molecular mass in kilo-Daltons. Fig. 3A. SOS-PAGE. Solubilized homogenate (lanes 1- 3, 5 mg wet tissue/ lane; lanes 4 - 6, 9 - 11 ~g protein/lane) was applied onto gradient (4 - 20%) polyacrylamide gel. Stained with Coo massie Brilliant Blue. Fig. 3 B. Western blot anal ysis. Solubilized homogenate (lanes I - 3, 3 mg wet tissue/lane; lanes 4 - 6, 5 ug protein/lane) was subjected to SOS-PAGE, transferred onto a nitrocellulose membrane, and stained with an anti-myosin polyclonal antibody. Fig. 3 C. Western blot analysis. Solubilized homogenate (lanes 1- 3. 5 mg wet tissue/ lane; lanes 4 - 6, 20 ug protein/l ane) was subjected to SOS-PAGE, transferred, and stained with an anti-myosin heavy chain monoclon al antibody from clone MY-32.
YOUNG
MIDDLE-AGED
OLD
I(I+IA) -I(I-M+IA-M) -1(1 -0)
-...
IIA
..........
-- ---
-... M - -
--
IIA-
M
a IIA
Fig. 4. Probable pathways of fibre aging in the rat soleus muscle. I, type I fibre including typical type I, type lA, type I-M, type IA-M and type 1-0 fibres; M, type M fibre; 0 , type 0 fibre; IIA, type IIA fibre.
mazan in type 0 fibres . Furthermore, the fibre size distribution of type 0 fibres was similar to that of type IIA fibre s. Moreover, the decrease in the number of type IIA fibre s from middle to old age can be explained by the change of type IIA fibres into type 0 fibre s. The se strongly suggest that type 0 fibre s are derived from type IIA fibres, although the possibility that origins of typ e 0 fibr es are type M fibr es or myosatellite cells can not be ruled out (Fig. 4). The size of type IIA fibr es in old animals was a pparently smaller than that in young or middle-aged animals. The small-sized type IIA fibres are neither newly differentiated type IIA fibre s deri ved from myosatellite cells nor split fibres, because transitional fibres between myosatellite cells and type IIA fibres (Caccia et a1. 1979) coult not be found
in the mu scles of old an imals, and the nu mber of type IIA fibres decreased markedly from middle to old age. The small-sized type IIA fibre s are probably degenerating type IIA fibre s. There are several anti-myosin monoclonal antibodies for fibre typing (Marini et al. 1989; Gorza 1990; Fiichtbauer eta1. 1991). In our preliminary experiments, commercially available anti -myosin monoclonal antibodies were helpful for fibre type identification in muscle sections from young animals. Ho wever, these antibodies were inadequate for the fibre type identification in sections from old animals, becau se all soleus fibr es were homogeneously stained with an an ti-slow myosin an tibody. Although the present biochemical result s suggest the occurrence of conformational changes in certain epitopes of myosin molecules, the epitope specificity of the fibr e type-specific monoclonal antibodies has not yet been elucidated. Caccia et al. (1979) and Eddinger et al. (1985) suspected that most type IIA fibres in the soleu s muscles were transformed into type I fibres during aging . Caccia et al. (1979) assum ed the presence of "transitional or tru e intermediate" fibres bet ween type IIA to type I in the muscles of old animals. The transitional fibres exhibited high mitochondrial oxidative enzyme acti vity and ATPase activity intermed iate between those of type I and type IIA fibr es, and increased from middle (0.3 %) to old age (15.8% ) (Caccia et a1. 1979). These cha racteristics are coincident with those of type M fibres found in the present study. However,
434
the origin of type M fibres is not type IIA fibres but probably type I fibres as described above. There may be no true transitional fibres between type I and type IIA fibres in the soleus muscle of old animals.
References Alnaqeeb MA, Goldspink G (1987) Changes in fiber type, number and diameter in developing and aging skeletal muscle. J Anat 153: 31-45 Ariano MA, Armstrong RB, Edgerton VR (1973) Hindlimb muscle fiber populations of five mammals. J Histochem Cytochern 21: 51-55 Barka T, Anderson PJ (1963) Histochemistry. Harper and Row, New York, p 314. Brooke MH, Kaiser KK (1970) Muscle fiber types: how many and what kind? Arch Neurol 23: 369 - 379 Caccia MR, Harris JB, Johnson MA (1979) Morphology and physiology of skeletal muscle in aging rodents. Muscle Nerve 2: 202-212 EddingerTJ, Moss RL, Cassens RG (1985) Fiber number and type composition in extensor digitorum longu s, soleus, and diaphragm muscles with aging in Fisher 344 rats. J Histochem Cytochem 33: 1033-1041 Fuchtbauer E-M, Rowlerson AM, Got z K, Friedrich G, Mabuchi K, Gergely J, JockuschH (1991) Direct correlation of parvalbumin levels with myosin isoforms and succinate dehydrogena se activity on frozen sections of rodent muscle. J Histochem Cytochem 39: 355 - 361 Gorza L (1990) Identification of a nobel type 2 fiber population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies. J Histochem Cytochem 38: 257 - 263 Gutmann E, Hanzlikova V (1976) Fast and slow motor units in aging. Gerontology 22: 280 - 300 Ihemelandu EC (1980) Decrease in fiber number of dog pectineus muscle with age. J Anat 130: 69 - 73 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680 - 685
Lexell J, Taylor CC (1991) Variability in muscle fibre areas in whole human quadriceps muscle: effects of increasing age. J Anat 174: 239-249 LexellJ, 'Iaylor CC, Sjostrom M (1988) What is the cause of the aging atrophy? Total number, size and proportion of different fiber types studied in whole vastus lateral is muscle from 15- to 83-year-old men. J Neurol Sci 84: 275 - 294 Lowry OH, Rosebrough AL, Farr AL , Randall RJ (1951) Protein measurement with the Folin phenol reagent. J BioI Chern 193: 265-275 Marini J-F, Pons F, Anoal M, Leger J. Leger JJ (1989) Anti -myosin heavy chain monoclonal antibodies reveal two IB (fast) fiber subtypes. J Histochem Cytochem 37: 1721-1729 McCarter R (1978) Effects of age on contraction of mammalian skeletal muscle. Aging 6: 1-21 Muntener M, Srihari T (1983) Changes of myosin and its ATPase in experimentally induced fiber transformation in the rat. Exp Neurol 80: 471-478 Rowe RWD, Goldspink G (1969) Muscle fibre growth in five different muscles in both sexes of mice. I Normal mice. J Anat 104: 519-530 Sakaida M. Watanabe J, Kanamura S, Tokunaga H, Ogawa R (1987) Physiological role of skeletal muscle glycogen in starved mice. Anat Rec 218: 267 - 274 Silbermann M, Finkelbrand S, Weiss A. Gershon D, Reznick A (1983) Morphometric analysis of aging skeletal muscle following endurance training. Muscle Nerve 6: 136- 142 Tauchi H, Yoshioka T, Kobayashi H (1971) Age changes of skeletal muscles of rats. Gerontologia 17: 219 - 227 Watanabe J, Kanamura S. Kanai K, Shugyo Y (1986) Cytochemical and biochemical glucose 6-phosphatase activity in skeletal mu scle cells of mice. Anat Rec 214: 25 - 31 WatanabeJ, Kanai K, KanamuraS (1991a) Measurement of NADPH-ferrihemoprotein reductase content in sections of liver. J Histochem Cytochem 39: 1635-1643 Watanabe J, Kanamura R (1991 b) An improved microphotometry system for measurement of cytochrome P-450 in hepatocyte cytoplasm. J Histochem Cytochem 39: 689-694
Accepted August 24, 1993
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