========= 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 skeletalmuscle, fibre number, fibre diameter and fibre type com­position in the soleus muscle o f male rat s of 3, 12 and24 months old were examined. The total number of musclefibre s remained unchanged, while average diameter increas­ed slightly with increasing age. The staining intensity ofmyosin adenosine triphosphatase (ATPase) act ivity in thefibres decreased with advancing age. Therefore, observationon the basis of myosin ATPase histochemistry alone is notadequate to study the agin g of mu scle fibres. In the musclesof 24 month-old animals, four fibre types were recognized;1) many (52%) type l-O fibre s showing weak ATPase andsuccinate dehydrogenase (SDH) reactions with slight subsar­colemmal 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 andmorphometric results suggest that type l-O, type M and typea fibres are derived from type I, type I and type IIA fibres,respectively. Furthermore, no transitional fibres from typeIIA to type I were observed. The refore , age-related changesin fibre type composition in the muscle cannot be explainedby the simple idea that most type IIA fibres are transformedinto type I fibres.

Key words: Skeletal muscl e - Adeno sine triphosphatase ­Fibre typing - Aging - Rat

Introduction

There has been a controversy regarding age-related changesin fibre type composition in muscles. Although Tauchi et al.

Correspondence to: S. Kanamura

Ann Anat (1994) 176: 429-435

Gustav Fischer Verlag lena

(1971) and Silbermann et al. (1983) showed that type I fibrestransformed into type II fibre s in some muscles duringaging , Caccia et al. (1979) and Eddinger et al. (1985)reported that type II fibre s transformed into type I fibresduring aging. McCarter (1978), Caccia et al. (1979) andAlnaqeeb and Goldspink (1987) observed that staining in­tensity of myofibrillar adenosine triphosphatase (myosinATPase) acti vity, which is used for fibre typing, decreasedduring aging. These findings suggest that the controversyconcerning the changes in fibre type composition duringaging is due to differences in criteria for fibre type classifica­tion. In addition, although there was evidence for a loss ofmuscl e fibre s (Tauchi et al. 1971; Gutmann and Hanzlikova1976; Ihemelandu 1980; Lexell et al. 1988) and reduction infibr e size (Lexell and Taylor 1991) during aging, the evidenceis not conclusive (Rowe and Goldspink 1969; Eddinger et al.1985).

The present study was therefore undertaken to test thefollowing hypotheses: a) the controversy regarding thechanges in fibre type composition during aging is due to adifference in the criteria for fibre type classification, and b)the loss of mu scle fibres and reduction in fibre size occurnot in all muscles but onl y in some particular muscles.

In the present study, we measured the changes in fibr etype composition in the rat soleu s muscle, a typical anti­gravity red muscle (Ariano et al. 1973), during aging. Forthi s purpose, histochemical methods were used to classifyfibre types based on acid- stable myosin ATPase activity andsuccinate dehydrogenase (SDH) activity. The age-relatedchanges in fibre number and fibre size in the muscle werethen measured by morphometry. Furthermore, myosin, inwhich ATPase is localized (Miintener and Srihari 1983), wasanalyzed by sodium dodecyl sulfate-polyacrylamide gel elec­trophoresis (SDS-PAGE) followed by Western blotting usinganti-myosin polyclonal or monoclonal antibody.

Materials and methods

Fifteen male Wistar rats, 3 month-old (young animals), 12month­old (middle-aged animals), and 24 month-old (old animals) wereused. The animals were fed laboratory chow and water ad libitumand housed at 22 ± 1°C and 60 ± 10070 relative humidity under a12: 12h light/dark cycle. Under sodium pentobarbital anesthesia,the soleus muscles werequickly removed and frozen on dry-ice. Themuscles from right legs were used for morphological andhistochemical analysis. Those from left legs were homogenized with9 volumes of 0.25 M sucrose and used for SDS-PAGE followed byWestern 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 activityaccording to the method of Brooke and Kaiser (1970) as describedpreviously (Watanabe et al. 1986; Sakaida et al. 1987). In short, thesection was preincubated for 15 min at room temperature in 0.1 Macetate buffer (pH 4.35) containing 0.1 M KCI, washed in distilledwater, incubated for 30 min at 37°C in 50 mM glycine-NaOH buf­fer (pH 9.40) containing 3 mM ATP, 30 mM calcium chloride and55 mM NaCI, and washed again in distilled water. The section wasthen immersed for 3 min in 2% aqueous calcium chloride, fixed for3 min in buffered 4% formalin, washed in distilled water, immersedfor 30 sec in 1% ammonium sulfide solution, washed again indistilled 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 15min at room tem­perature in 0.1 M barbital buffer (pH 10.25), and processed forthe detection of myosin ATPase activity as described above. Thethird section was incubated for succinate dehydrogenase (SDH) ac­tivity according to the method of Barka and Anderson (1963). Inbrief, the section was incubated for 30 min at room temperature in0.1 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 ofyoung, middle-aged and old animals were incubated simultaneous­ly in the same incubation medium. Staining intensity was measuredsemiquantitatively as the optical density in the centre of each fibrewith a microphotometry system (KWSP-l) (Watanabe et al. 1991 b).Readings were made at 430 nm for the myosin ATPase reaction and535 nm for the SDH reaction with a spot size of 10 urn. In ourpreliminary experiments, the staining reaction of acid-stablemyosin ATPase activity was found to be more reproducible thanthat of alkali-stable myosin ATPase activity in the muscles of mid­dle-aged and old animals. Therefore, we used acid-stable myosinATPase activity and SDH activity for fibre typing.

Fibre counting and measurement of fibre diameter

Photographs were taken from SDH- or ATPase-stained sections ata magnification of x 40 and enlarged to a final magnification ofx 140. The photographs taken from SDH- and ATPase-stained sec­tions were separately reconstructed to cover the entire muscleprofile. Since the sections were cut in the midbelly region of themuscle, almost all the fibres were present in each reconstructedphotograph (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 andGoldspink 1987).

The total fibre number in each muscle was measured by countingall the fibres in the reconstructed photograph of the SDH-stainedsection. Then, every fibre in the reconstructed photograph wasidentified as one of the fibre types, using reconstructedphotographs of ATPase-stained sections, and the number of eachfibre type was counted.

Fibre number counting showed that all fibre types weredistributed homogeneously in the muscles of young, middle-agedor old animals. Therefore, the average fibre diameter in each musclewas determined in randomly-selected areas in the reconstructedphotograph of SDH-stained section after the identification of fibretype as mentioned above. About 300 fibres in the selected areaswere analyzed per animal.

No morphological abnormalities were seen in the muscles fromyoung, middle-aged and old animals. No morphological abnor­malities were found in the heart, lung, liver, kidney, spleen, smalland large intestine, pancreas or testis in these animals.

Biochemical methods

Proteins in the homogenate from soleus muscles were measuredby the method of Lowry et al. (1951), and then, analyzed bySDS-PAGE according to the method of Laemmli (1970). Some gelswere then stained with Coomassie Brilliant Blue. The remaininggels 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 chainmonoclonal 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 statisticalcomparisons were made above the 95% level of confidence.

Results

Body weights of young, middle-aged and old animals were310 ± to.5g, 716 ± 35.1 g and 616 ± 46.9g(means ± S.E.for five animals) respectively. The values in middle-aged andold animals were greater than the value in young animals(P < 0.05; Duncan's multiple range test), and the value inold 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 and185 ± 12.6mg respectively. The values in middle-aged andold 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 oldanimals (ANOVA).

Fibre type composition

Muscle fibres were classified according to the acid-stablemyosin ATPase activity, sarcoplasmic SDH activity for in­terfibrillar mitochondria, and subsarcolemmal SDH activityfor subplasmalemmal mitochondria. The fibres fromyoung, middle-aged, and old animals were categorized asshown in Table 1.

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Fibre type composition (070)

Table 2. Age-related changes in fibre type composition in rat soleusmuscle ")

Table 1. Staining properties of muscle fibres seen in the soleusmuscle of young, middle-aged and old rats

") Staining intensity was classified into four classes on the basis ofthe results of microphotometry; negative (optical density of0-0.04), weak (0.05-0.15), moderate (0.16-0.30) and strong(0.31- ).b) Staining intensity measured in the centre of each fiber wasclassified into four classes on the basis of the results ofmicrophotometry; 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 ofSAD was defined as "negligible", "slight" or "marked" on thebasis of visual inspection.

The total number of fibres in the soleus muscle wasstatistically unchanged during senescence (ANOVA)(Table3). The number of type IIA fibres was unchangedfrom young to middle age, but decreased markedly frommiddle to old age (Duncan's multiple range test). Thenumber of type I or type IA fibres in young animals wassimilar to that of type I-M or type IA-M fibres in middle-ag­ed animals. The number of type 1-0 fibres in old animalswas smaller than that of type I fibres in young animals orthat of type I-M fibres in middle-aged animals. On the otherhand, type M fibres appeared in middle-aged animals andincreased markedly from middle to old age. Type 0 fibresappeared in old animals.

The soleus muscles of young animals contained 1) manytypical type I (slow twitch-oxidative) fibres, 2) a fewtype IIA (fast twitch-oxidative, glycolytic) fibres, and 3) veryfew 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 typeswere recognized; 1) many type I (I-M) fibres, 2) very fewatypical type I (I-AM) fibres, 3) very few type M fibresshowing weak ATPase reaction and intense SDH reactionwith marked subsarcolemmal aggregates of diformazan,and 4) a few type IIA fibres (Figs. 1C and 10). The stainingintensity of the ATPase reaction was apparently lower intype I-M and type IA-M fibres than in type I and type IAfibres, as determined by simultaneous incubation of sectionsfrom young and middle-aged animals in the same incuba­tion medium.

In the muscles of old animals, four fibre types wererecognized; 1) numerous type I (1-0) fibres, 2) a few type 0fibres showing a weak ATPase reaction and an intense SDHreaction 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 ortype I-M fibres.

Fibre number

Staining SAD')intensity b)

Succinate dehydrogenase(SDH) activity

Acid-stable Alkali-stable

Staining intensity")

Acid-stable adenosinetriphosphatase (ATPase)activity

Fibre type

(I) Strong Negative Weak Slight(IA) Strong Negative Strong Marked(I-M) Moderate Negative Weak Slight(IA-M) Moderate Negative Strong Marked(1-0) Weak Negative Weak Slight

- WeakIIA Negative Strong Strong MarkedM Weak Negative Strong Marked

- Weak0 Weak Negative Strong Negligible

- Weak

Young Middle-aged Old(3 months (12 months (24 monthsold) old) old)

Type

(I) 74.9 ± 1.27 0 0(lA) 3.7 ± 0.45 0 0(I-M) 0 79.6 ± 4.94 0(IA-M) 0 2.7 ± 0.50 0(1-0) 0 0 52.0 ± 0.63

IIA 21.4 ± 0.94 15.4 ± 3.79 3.7 ± 0.64

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.

Fibre size

The averaged diameter of total muscle fibres in the soleusmuscle increased slightly with increasing age (Duncan'smultiple range test) (Table4). In the muscles of younganimals, the diameter was greater in type I or type IA fibresthan in type IIA fibres. There was no difference in the valuesbetween type I and type IA fibres. In middle-aged animals,the value in type I-M, type IA-M or type M fibres wasgreater than type IIA fibres. TypeI-M, type IA-M andtype M fibres were approximately equal in size. In oldanimals, the value in 1-0 fibres was greatest, followed inorder by type M fibres, type 0 fibres and type IIA fibres.The value in type IIA fibres was unchanged from young tomiddle age, but decreased markedly from middle to old age.The value in type I-M or type IA-M fibres in middle-agedanimals 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 activity (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) animals. 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) animals. 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 animalswas slightly greater than that in type I fibres in younganimals or the value in type I-M fibres in middle-agedanimals . Fibre size distribution of each fibre type is sum­marized in Figure 2.

Biochemical results

Protein contents (mg/g wet tissue) in the soleus muscle ofyoung, 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 polyclonalantibody (Fig. 3 B), four bands at 220 kD (myosin heavychain) , 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. Thestaining intensities of bands at 220, 37, and 15 kD were

432

Discussion

decreased with increasing age, whereas that of the band at18 kD was similar in all age groups.

MY-32 anti-myosin heavy chain monoclonal antibodyrecognized myosin heavy chain (Fig. 3C). The staining in­tensity of the band at 220 decreased slightly with increasingage.

As shown in the present study, the total number of musclefibres in the soleus muscle remained unchanged duringsenescence. It is therefore probable that the decrease in totalfibre 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 muscleincreased gradually with increasing age. These findings sup­port the tested hypothesis that the loss of muscle fibres andreduction in fibre size do not occur in all muscles but onlyin some particular muscles.

The staining intensity of myosin ATPase activity in fibresof the soleus muscle decreased gradually with advancingage. Furthermore, histochemical and biochemical resultssuggest that quantitative and/or conformational changein myosin molecules occur during senescence. Therefore,observation on the basis of ATPase histochemistry alone isprobably inadequate to examine the muscle aging. Eddingeret al. (1985) showed that the percentage of type I fibres inthe soleus muscle in Fisher 344 rats increased, but that oftype IIA fibres decreased from middle to old age. However,Alnaqeeb and Goldspink (1987) found that the percentagesof 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 andtype IIA, respectively, the present results are in agreementwith those of Alnaqeeb and Goldspink (1987). However, iffibres showing weak ATPase reaction (types 1-0, M and 0)are simply classified into type I, the results are compatiblewith those of Eddinger et al. (1985). The controversy regard­ing the age-related changes in fibre type composition foundbetween the results of previous studies is probably due todifferences 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) andtype IIA fibres. However, both the staining properties ofSDH activity and fibre size distribution in type M fibresresembled those in type I (lA-M) fibres. Furthermore,decrease in type I fibres from middle to old age (type I-M1608 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 Ifibres (Fig. 4).

Although the ATPase staining reaction was weak intype 0 fibres, the staining properties of SDH activity intype 0 fibres were similar to those in type IIA fibres exceptfor the absence of subsarcolemmal aggregates of difor-

o

o 40 eo

M

IMNONE I0 NONE "'

10

NONE IIA

L

IA

FIBER DIAMETER ( pm )

I IANONE M

04080006010... 10

YOUNGI

OLD1(1-0 )

Table 3. Age-related changes in fibre number in rat soleus muscles

Young Middle-aged Old

Total 1920 ± 114.9 2020 ± 71.8 1797 ± 75.9

(I) 1438 ± 24.4 NF NF(IA) 71 ± 8.6 NF NF(I-M) NF 1608 ± 99.8 NF(IA-M) NF 55 ± 10.1 NF(1-0) NF NF 934 ± 11.3

IIA 411±18.0 311 ± 76.6 67 ± 11.5

M NF 47 ± 14.9 586 ± 68.5

0 NF 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)Young Middle-aged Old

Total 56.5 ± 0.27 60.6 ± 0.16 68.6 ± 0.37

I 57.7 ± 0.76 NF NFIA 59.3 ± 0.30 NF NFI-M NF 61.5 ± 0.24 NFIA-M NF 61.7 ± 0.27 NF1-0 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.

Fig.2. Frequency distribution of each fibre type in the soleusmuscles from young, middle-aged, and old rats. NONE, could notbe found.

>-U ..Zw~ 0owa:u.

433

1 2 3 4 5 61 2 3 4 5 6

67-

45-

-37 - 37

=1125-

_1814 - -15

C

4 5 6123

-

8

220-

67-

45-

A

220-

Figs. 3A - 3C. SOS-PAGE and Western blot ana lysis of solubilized homogenates from the soleus muscle of young (lanes 1and 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 Coomassie Brilliant Blue.Fig. 3B. Western blot analysis. Solubilized homogenate (lanes I - 3, 3 mg wet tissue/lane; lanes 4 - 6, 5 ug protein/lane) was subjected toSOS-PAGE, transferred onto a nitrocellulose membrane, and stained with an anti-myosin polyclonal antibody.Fig. 3C. Western blot analysis. Solubilized homogenate (lanes 1- 3. 5 mg wet tissue/ lane; lanes 4 - 6, 20 ug protein/l ane) was subjectedto SOS-PAGE, transferred, and stained with an anti-myosin heavy chain monoclon al antibody from clone MY-32.

in the muscles of old animals, and the number of type IIAfibres decreased markedly from middle to old age. Thesmall-sized type IIA fibre s are probably degeneratingtype IIA fibre s.

There are several anti-myosin monoclonal antibodies forfibre typing (Marini et al. 1989; Gorza 1990; Fiichtbauereta1. 1991). In our preliminary experiments, commerciallyavailable anti -myosin monoclonal antibodies were helpfulfor fibre type identification in muscle sections from younganimals. However, these antibodies were inadequate for thefibre type identification in sections from old animals,becau se all soleus fibr es were homogeneously stained withan an ti-slow myosin an tibody. Although the present bio­chemical result s suggest the occurrence of conformationalchanges in certain epitopes of myosin molecules, the epitopespecificity of the fibr e type-specific monoclonal antibodieshas not yet been elucidated.

Caccia et al. (1979) and Eddinger et al. (1985) suspectedthat most type IIA fibres in the soleus muscles weretransformed into type I fibres during aging . Caccia et al.(1979) assumed the presence of "transitional or tru e in­termediate" fibres between type IIA to type I in the musclesof old animals. The transitional fibres exhibited highmitochondrial oxidative enzyme activity and ATPase activi­ty intermediate between those of type I and type IIA fibres,and increased from middle (0.3%) to old age (15.8% ) (Cac­cia et a1. 1979). These cha racteristics are coincident withthose of type M fibres found in the present study. However,

M

aIIA

OLD

----­IIA-

MIDDLE-AGED

--IIA

I(I+IA) -I(I-M+IA-M) -1(1-0)-... ..........-... M--

YOUNG

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 sfrom middle to old age can be explained by the change oftype IIA fibres into type 0 fibre s. The se strongly suggestthat type 0 fibre s are derived from type IIA fibres, althoughthe possibility that origins of type 0 fibres are type M fibresor myosatellite cells can not be ruled out (Fig. 4).

The size of type IIA fibres in old animals was apparentlysmaller than that in young or middle-aged animals. Thesmall-sized type IIA fibres are neither newly differentiatedtype IIA fibre s derived from myosatellite cells nor splitfibres, because transitional fibres between myosatellite cellsand type IIA fibres (Caccia et a1. 1979) coult not be found

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-Mand type 1-0 fibres; M, type M fibre; 0 , type 0 fibre; IIA, type IIAfibre.

434

the origin of type M fibres is not type IIA fibres but prob­ably type I fibres as described above. There may be no truetransitional fibres between type I and type IIA fibres in thesoleus muscle of old animals.

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Accepted August 24, 1993

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