Acta Physiol Scand 1990, 140, 175-180
Myosin heavy chain composition of single fibres from m. biceps brachii of male body builders H. K L I T G A A R D , M. Z H O U and E. A. R I C H T E R August Krogh Institute, University of Copenhagen, Denmark
KLITGAARD, H., ZHOU,M. & RICHTER, E. A. 1990. Myosin heavy chain composition of single fibres from m. biceps brachii of male body builders. Acta Physiol Scand 140, 175-180. Received 27 March 1990, accepted 1 1 June 1990. ISSN 0001-6772. August Krogh Institute, University of Copenhagen, Denmark. The myosin heavy chain (MHC) composition of single fibres from m. biceps brachii of young sedentary men (28f0.4 years, mean+SE, n = 4) and male body builders (25 2.0 years, n = 4) was analysed with a sensitive one-dimensional electrophoretic technique. Compared with sedentary men, the body builders had a higher proportion of fibres containing only MHC type IIa ( 3 6 5 4 us 12f2yo; P < 0.05), but a lower proportion of fibres with a coexistence of MHC types I I a and I1 b (16 f3 ‘us 34+ 2%; P < 0.05) and nearly no fibres containing only MHC type I1 b (1 f 1 vs 1 2 5 1 yo;P < 0.05). Myofibrillar ATPase histochemistry only demonstrated a trend towards a higher proportion of type I1 a fibres (3 1 f6 us 25 f6 yo)and a lower proportion of type I1 b fibres (18 5 us 26 6%) within the body builders. These results, therefore, suggest an altered expression of MHC isoforms within histochemical type I1 fibres of human skeletal muscle with body building. Furthermore, in human skeletal muscle differences in expression of MHC isoforms may not always be reflected in the traditional histochemical classification of types I, I1 a, I1 b and I1 c fibres.
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Key words : human skeletal muscle, myofibrillar ATPase histochemistry, myosin isoforms, training.
The classification of mammalian skeletal muscle fibres with myofibrillar ATPase histochemistry into types I, I I a and I I b fibres reflects the expression of distinct myosin heavy chain (MHC) isoforms in these fibres (Billeter et at. 1981, Salviati et al. 1982, Danieli-Betto et al. 1986). Thus, there is evidence to suggest that differences in myosin ATPase activity are related to the M H C isoform composition of the fibres (Wagner 1981). However, a large proportion of human skeletal muscle fibres has recently been demonstrated to contain two or three M H C isoforms in different stoichiometric ratios (Biral et al. 1988, Klitgaard et al. 1990a). Since the dominant M H C isoform ‘sets ’ the histochemical reaction of a fibre (Danieli-Betto et al. 1986), the traditional myofibrillar ATPase staining methCorrespondence : Henrik Klitgaard, August Krogh 13, DK-2100 Institute, Universitetsparken Copenhagen 0, Denmark.
odology is not sensitive enough to reveal subtle changes in the expression of M H C isoforms in human skeletal muscle fibres. The question is therefore whether traditional myofibrillar ATPase histochemistry can give an adequate characterization of fibre type composition in athletes. Using immunohistochemical and electrophoretic techniques it has been reported that endurance training can increase the proportion of fibres with coexistence of slow and fast M H C isoforms (Baumann et al. 1987, Schantz 1986). In a recent cross-sectional study, we also demonstrated a high proportion of fibres with a coexistence of M H C types I and I1 a isoforms in muscles from elite cross-country skiers (Klitgaard et al. 1990b). Endurance training therefore seems to increase the proportion of fibres with coexistence of M H C isoforms. However, to our knowledge no studies have examined M H C isoform expression in single fibres from athletes
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Table 1. Physical characteristics of subjects
Sedentary controls (4) Body builders (4)
Age (years)
Body weight (kg)
Height
28 0.4 25 i2.0
75i2 98 i3*
181 + 4 185+2
*
(4
Values are meansFSE. Numbers in brackets indicate number of subjects in each group. * Statistically significant difference between the groups, P < 0.05. involved in training regimens inducing a muscular hypertrophy. As for other types of exercise, the results from histochemical studies of body builders have been conflicting. Thus, some reports have demonstrated an identical (MacDougall et n l . 1982, Sale et al. 1987) and others a different (Tesch & Larsson 1982) fibre type distribution in skeletal muscles of body builders as compared w-ith control groups. T o extend the biochemical characterization of fibre types in body builders, the present study therefore analysed needle biopsies from a group of young sedentary men and a group of male body builders. Fibre type characteristics were determined using both myofibrillar ATPase histochemistry and electrophoretic analysis of MHC composition in single fibres. Only minor differences were observed in the fibre type distribution with histochemistry. However, the two groups manifested marked differences in MHC composition of single fibres. M,.ZTERIALS A N D METHODS Subjects. Four sedentary 4 oung men and four male bod>- builders participated in this study. Physical characteristics of subjects are given in Table 1. The sedentary young men did not participate in any regular sports activity and had not done so for the last 7 years (range 3-11 years) before the time of examination. The MHC composition of single fibres from m. biceps brachii of these subjects has been published elsewhere (Klitgaard et al. 1990a). The body builders had trained 2-5 h per day, 3-6 times a week, for the last 6 years (range 2-12 years) and two of them were in the national elite. None of them had taken anabolic steroids during the last 2 years before the time of examination. The study was approved by the Copenhagen Ethics Committee. Biopsies. Needle biopsies were taken by suction from m. biceps brachii to secure 100-150 mg samples. One part of the sample was frozen immediately in liquid nitrogen and used for dissection of 2- to 4-mmlong fibres for analysis of MHC composition. The
other part of the sample was trimmed, mounted and frozen in isopentane, cooled with liquid nitrogen and used for histochemical analysis. M H C composition of singlefibres. Single fibres from the muscle specimens were dissected and prepared for electrophoretic analysis of MHC composition as previously described (Salviati et al. 1982). Electrophoresis of the fibres was performed in 616 SDSPAGE gels with 37.504 glycerol (w/v) in the separating gel (Klitgaard et al. 1990b). Gels were run at 70 V overnight. This electrophoretic system has previously demonstrated a clear resolution of MHC types I, I1 a and I1 b, as verified by immunoblotting with monoclonal antibodies, in both human m. vastus lateralis and m. biceps brachii (Biral ef til. 1988, Klitgaard et al., 1990b,c). On average, 50-60 fibres were analysed in each subject from both groups. A total of 224 fibres was analysed in the sedentary control group and 217 fibres in the group of body builders. Historhemistry. Serial transverse sections (10 pm) were cut in a cryotome at 20 "C. The sections were mounted on coverslips and stained for myofibrillar ATPase at pH 9.4 after both alkaline (pH 10.3) and acid (pH 4.3 and 4.6) preincubations (Klitgaard et al. 1990~).Fibres were characterized as I, IIa, I I b and I I c fibres. On average, 382 (range 294-640) fibres were classified in each sample. The areas of a minimum of 50 type I, 30 type IIa and 20 type I I b fibres were measured in each sample with a digitizer. Statistics. The non-parametric Mann-Whitney test was applied for statistical comparison between the groups. The 0.05 confidence level was chosen to indicate statistical significance. ~
RESULTS Fibre typing from myofibrillar ATPase histochemistry (Fig. 1) revealed a similar proportion of type I fibres in the sedentary controls and the body builders (48 2 us 5 1 i3 yo, mean F SE). I n contrast, there was a trend towards a larger proportion of type I1 a fibres (3 1 6 us 25 6%) and a lower proportion of type I1 b fibres (18 & 5 1'5 26f6Y0) in the body builders, but these
Myosin expression in muscle of body builders
177
group contained all three M H C isoforms (Klitgaard et al. 1990a). In the body builders 41 f 4 % (range 33-45) of the fibres contained only MHC type I, and 6 4 3 yo(2-14) showed coexistence of M H C types I and I I a (Fig. 3). This was similar f to the 36+5Y0 (24-43) and 6 + 2 % (3-ll), respectively, found within the sedentary controls. However, the body builders had a larger proportion of fibres containing only M H C type I1 a (36+4 [28-451 us 1 2 f 2 % [6-171; P < 0.05); as compared with the sedentary controls, but a lower proportion of fibres with a coexistence of M H C types I I a and I I b ( 1 6 f 3 [lo-221 us 34 2 yo[29-401; P < 0.05) and nearly no fibres containing only M H C type I1 b (1 f 1 [&3] 'us 12+1'30 [10-15]; P < 0.05). Type I TypeIIa Type I1 b The two different systems for classification of Fig. 1. Fibre type distribution (meansfSE), de- fibre types are compared in Fig. 4, and the termined by myofibrillar ATPase histochemistry, in arrows illustrate their relationship. Nearly all of m. biceps brachii of young sedentary men ( 0 2 8 0.4 the fibres showing coexistence of MHC types I years, n = 4)and male body builders ( 0 2 5 i2 years, and I I a had a clear majority of M H C type I n = 4). On average, 382 (range 294-640) fibres were (Fig. 2, lane v). These fibres would react as typed in each sample. histochemical type I fibres. M H C determination of single fibres and myofibrillar ATPase histochemistry therefore gives a similar type I fibre population in both the sedentary controls (42 us 48%) and in the body builders (47 us 51 yo). As regards the type I1 fibre population, the two classification systems did not give similar Fig. 2. Identification of human myosin heavy chain results. From comparison of the percentage isoforms in single fibres of m. biceps brachii after fibres typed histochemically as types I1 a and I1 b SDS-PAGE in 6% gels with high glycerol content and silver staining. Only the MHC region is shown. with the percentage distribution of single fibres Lane (i), purified myosin from human m. biceps containing M H C types I1a and I1 b it appears brachii; lanes (ii) to (vi), single fibres from m. biceps that in sedentary controls a large proportion of brachii containing only MHC type I (ii), type IIa (iii), both the histochemical types I I a and I1 b fibres type I1 b (iv) and with a coexistence of MHC types I manifest coexistence of M H C types I1 a and I1 b and IIa (v) and types IIa and IIb (vi). (Fig. 4). On the other hand, within the body builders the histochemical type I1a fibres seem to contain only M H C type I1 a, whereas nearly all differences were not significant. Almost no I I c the histochemical type I1 b fibres contain both fibres were found in both groups. Fibre areas of M H C types I I a and I I b . type I (6825f955 us 5519&533pm2) but especially type I1a (10,959 f1035 'us 7286 241 DISCUSSION pm2;P < 0.05) and type I1 b fibres (9837 & 1282 'us 621 1 f294 ,urn2; P < 0.05) were larger in the Using a sensitive electrophoretic technique the body builders as compared with the sedentary present study demonstrated a different pattern of M H C expression in single fibres from m. controls. T h e electrophoretic analysis identified single biceps brachii of body builders, as compared fibres containing only M H C type I (Fig. 2, lane with a group of sedentary controls. In contrast, ii), M H C type I I a (lane iii)), M H C type I I b traditional myofibrillar ATPase histochemistry (lane iv) and fibres with coexistence of M H C only revealed minor differences. By comparing types I and I1 a (lane v) and M H C types I1a and the M H C composition and the myofibrillar I I b (lane vi). Also, one fibre in the sedentary ATPase reaction of single fibres from rat skeletal
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MHC 1
MHCI/lla
K
MHC Ila MHCllalllb MHCllb
Fig. 3. Fibre tj-pe composition, determined by MHC composition of single fibres, in m. biceps brachii of young sedentary men ( 0 2 8 + 0 . 4 years, n = 4) and male body builders ( a 2 5 f 2 . 0 years, n = 4). 224 fibres were analysed in the sedentary control group and 217 in the group of body builders. Values are means SE.
muscles (Danieli-Betto et a / . 1986), it has been shown that fibres containing only M H C types I, I1 a and I1 b will react as histochemical types I, I I a and I I b fibres respectively. Fibres with a coexistence of two M H C isoforms will react histochemically as the dominant isoform. Only with nearly equal fractions of MHC types I and I1 a will the fibre react histochemically as a type I I c fibre. The major part of the relativelj large proportion of fibres showing coexistence of two M H C isoforms will therefore not be adequately characterized by traditional myofibrillar ATPase histochemistry, as also demonstrated for human skeletal muscle in the present study. This has functional implications, since the M H C content of a fibre is related to its shortening velocity (Reiser et nl. 1985, Fitts et ul. 1989) and 'contraction economy' (energy utilization per unit force; Crow & Kushmerick, 1982). This study did not employ histochemical typing of single fibres because it is debatable whether such fibre typing is able adequately to distinguish between fibre types (Staron & Pette 1987). Only major fibre types can be given reliable classification since the histochemical reaction is influenced by the sample thickness and since classification is done without the aid of surrounding fibres (Staron & Pette 1987). We
therefore preferred to compare, within the same sample, a population of fibres classified in a cross-section of muscle stained for myofibrillar ATPase histochemistry with a population of single fibres classified by M H C composition. As demonstrated in Fig. 4, it is possible to compare the two classification systems even though myofibrillar ATPase histochemistry is done on a tissue slice and M H C composition determined in single fibres. This is possible because of knowledge of the staining properties of the different M H C isoforms mentioned above (Danieli-Betto et a/. 1986). It is still debated whether usage of muscles can change the expression of myosin isoforms (Saltin & Gollnick 1983). Recent longitudinal training studies of both rats (Green et al. 1983) and humans (Schantz 1986) suggest that this is possible. The almost complete absence of fibres containing only M H C type IT b within the body builders, together with the low proportion of fibres showing coexistence of M H C types I I a and I I b and the large proportion of fibres containing only M H C type IIa, suggests an increased expression of M H C type I I a and a decreased expression of M H C type I I b with body building. Of course, the cross-sectional design of the present study provides only a
Myosin expression in muscle of body builders
179
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Fig. 4. Comparison of the fibre type composition, determined by single-fibre analysis of MHC composition (MHC) and myofibrillar ATPase histochemistry (histochemistry), in m. biceps brachii of young sedentary men (2810.4 years, n = 4) and male body builders (25k2.0 years, n = 4). The arrows indicate the relationship between the two fibre type classification systems. The dashed arrow illustrates the possibility that some of the body builders’ histochemical type I1 a fibres might exhibit coexistence of MHC types IIa and I1 b (see ‘Results’).
limited basis for such a statement, but it would which a higher content of M H C type I I a and a be consistent with the fact that body building lower content of MHC type I I b was demontraining involves intense repetitive contractions strated, as compared with an inactive, agecharacterized by demands on not only strength matched control group (Klitgaard et af. 1990~). In conclusion, this study demonstrates a but muscular endurance as well. This has been proposed to result in an increased proportion of different expression of M H C isoforms in m. histochemical type I fibres, resembling the biceps brachii of body builders compared with histochemical profile of skeletal muscles from sedentary controls. This may suggest that the endurance athletes (Tesch & Larsson 1982). training increased the expression of M H C type However, other histochemical studies of body I I a at the expense of expression of M H C type builders have reported a fibre type distribution I1 b. identical to sedentary subjects (MacDougall et af. 1982; Sale et al. 1987). This is in agreement with This study was supported by grants from the Danish the present study which suggests that an adapResearch Academy, the Danish Research Council for tation occurs only in the M H C expression of the Natural Sciences, the Obel Family Foundation, the histochemical type I1 fibre population. Our the Knud Hejgaard Foundation and the John Meyer results are also consistent with a recent study on Foundation. H. Klitgaard was funded by the Faculty muscle biopsies from m. vastus lateralis and m. of Natural Sciences, University of Copenhagen, biceps brachii of strength-trained elderly men in Denmark. We are grateful to Mrs Lise Riis-Jacobsen 9
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and hlrs Marianne Hemmingsen for their skilful technical assistawe and to Mrs Lis H. Christensen for typing of the manuscript.
1990b. Co-existence of myosin heavy chain I and
I1 a isoforms in human skeletal musck fibres with
endurance training. P’ugers Archiv (in press). H., MANTONI,M., SCHIAFFINO, S., KLITGAARD, ALSONI, S., GORZA,L., LAURENT-WINTER, C., SCHNOHR,P. & SALTIN,B. 1990c. Function, morphology and protein expression of ageing REFERENCES skeletal muscle: a cross sectional study of elderly men with different training backgrounds. Acta BAUMILXN, H., J~ZGGI,M., SOLAUD, F., HOWALD, H. & Physiol Scand 140, 11-54. SCHACB,M.C. 1987. Exercise training induces MACDOUGALL, J.D., SALE,D.G., ELDER,G.C.B. & transitions of myosin isoform subunits within SUTTON,J.R. 1982. Muscle ultrastructural charachistochemically typed human muscle fibres. PJugers teristics of elite powerlifters and bodybuilders. Eur .4rrh 409, 349-360. 3 Appl Physiol48, 117-126. BILLLTER,R., HEIZMASU,C.W., HOWALD,H. & REISER, G.G. & GREASER, P.J., Moss, R.L., GUILLIAN, , E. 1981. Analysis of myosin light and heavy M.L. 1985. Shortening velocity in single fibres types in single human skeletal muscle fibres. from adult rabbit soleus muscles is correlated with Eur 3 Biochem 116, 389-395. myosin heavy chain composition. 3 B i d Chem 260, BIRAL, D., BETTO,R., DANIELI-BETTO, D. & SALVIATI, 9077-9080. G , 1988. Myosin heavy chain composition of single SALE, D.G., MACDOUGALL, J.D., ALWAY,S.E. & fibres from normal human muscle. Biochem 3 250, SUTTON, J.R. 1987. Voluntary strength and muscle 307-308. characteristics in untrained men and women and CROW, 1f.T. & KUSHMERICK, M.J. 1982. Chemical male bodybuilders. 3 Appl Physiol 62, 17861793. energetics of slow- and fast-twitch muscles of the SALTIN, B. & GOLLNICK,P. 1983. Skeletal muscle mouse. 3 Gen Physiol 79, 147-166. adaptability : significance for metabolism and pefE. & BETTO,R. 1986. D.4XIELI-BETT0, D., ZERBATO, formance. I n : Handbook of Physiology. Section 10, Type 1, 2A and 2B myosin heavy chain electrcSkeletal Muscle, Chapter 19, pp. 555-631. American phoretic analysis of rat muscle fibres. Biochem Physiological Society, Williams & Wilkins Biophys Res Commun 138, 981-987. Company, Baltimore, MO. FITTS, R.H., COSTILL,D.L. & GARDETTO, P.R. 1989. SALVIATI, G., BETTO,R. & DANIELI-BETTO, D. 1982. Effects of swim exercise training on human muscle Polymorphism of myofibrillar proteins of rabbit fibre function. 3 Appl Physiol 66, 465475. skeletal-muscle fibres. Biochem 3 207, 261-272. GREES,H.J., KLCG,G.,4., REICHMA SCHANTZ,P.G. 1986. Plasticity of human skeletal U., WIEHRER,W. & PETTE, D. 1983. Exercisemuscle. Acta Physiol Scand 128, Suppl. 558. induced fibre type transition with regard to myosin, STARON, R.S. & PETTE,D. 1987. The multiplicity of parvalbumin, and sarcoplasmic reticulum in combinations of myosin light chains and heavy muscles of the rat. PJUgers .4rrh 400, 432138. chains in histochemically typed single fibres. SCHIAFFINO, I., s., BETTO, Biochem 3 243, 687-693. KLITGAARD, H., ZHOU, % R., SALVIATI, G. & SALTIN, B. 1990a. Aging alters TESCH, P.A. & LARSSON, L. 1982. Muscle hypertrophy the myosin heavy chain composition of single fibres in bodybuilders. Eur 3 Appl Physiol49, 301-306. P.D. 1981. Formation and characterization from human skeletal muscle. .4rfa Physiol Scand WAGNER, 140, 55-62. of myosin hybrids containing essential light chains KLITGAARD, H., BERGMAN, O., BETTO,R., SALVIATI, and heavy chains from different muscle myosin. 3 Biol Chem 256, 2493--2498. G., SCHIAFFINO, s., CLAUSEN, T. & SALTIN,B.
Myosin heavy chain composition of single fibres from m. biceps brachii of male body builders H. K L I T G A A R D , M. Z H O U and E. A. R I C H T E R August Krogh Institute, University of Copenhagen, Denmark
KLITGAARD, H., ZHOU,M. & RICHTER, E. A. 1990. Myosin heavy chain composition of single fibres from m. biceps brachii of male body builders. Acta Physiol Scand 140, 175-180. Received 27 March 1990, accepted 1 1 June 1990. ISSN 0001-6772. August Krogh Institute, University of Copenhagen, Denmark. The myosin heavy chain (MHC) composition of single fibres from m. biceps brachii of young sedentary men (28f0.4 years, mean+SE, n = 4) and male body builders (25 2.0 years, n = 4) was analysed with a sensitive one-dimensional electrophoretic technique. Compared with sedentary men, the body builders had a higher proportion of fibres containing only MHC type IIa ( 3 6 5 4 us 12f2yo; P < 0.05), but a lower proportion of fibres with a coexistence of MHC types I I a and I1 b (16 f3 ‘us 34+ 2%; P < 0.05) and nearly no fibres containing only MHC type I1 b (1 f 1 vs 1 2 5 1 yo;P < 0.05). Myofibrillar ATPase histochemistry only demonstrated a trend towards a higher proportion of type I1 a fibres (3 1 f6 us 25 f6 yo)and a lower proportion of type I1 b fibres (18 5 us 26 6%) within the body builders. These results, therefore, suggest an altered expression of MHC isoforms within histochemical type I1 fibres of human skeletal muscle with body building. Furthermore, in human skeletal muscle differences in expression of MHC isoforms may not always be reflected in the traditional histochemical classification of types I, I1 a, I1 b and I1 c fibres.
+
+
Key words : human skeletal muscle, myofibrillar ATPase histochemistry, myosin isoforms, training.
The classification of mammalian skeletal muscle fibres with myofibrillar ATPase histochemistry into types I, I I a and I I b fibres reflects the expression of distinct myosin heavy chain (MHC) isoforms in these fibres (Billeter et at. 1981, Salviati et al. 1982, Danieli-Betto et al. 1986). Thus, there is evidence to suggest that differences in myosin ATPase activity are related to the M H C isoform composition of the fibres (Wagner 1981). However, a large proportion of human skeletal muscle fibres has recently been demonstrated to contain two or three M H C isoforms in different stoichiometric ratios (Biral et al. 1988, Klitgaard et al. 1990a). Since the dominant M H C isoform ‘sets ’ the histochemical reaction of a fibre (Danieli-Betto et al. 1986), the traditional myofibrillar ATPase staining methCorrespondence : Henrik Klitgaard, August Krogh 13, DK-2100 Institute, Universitetsparken Copenhagen 0, Denmark.
odology is not sensitive enough to reveal subtle changes in the expression of M H C isoforms in human skeletal muscle fibres. The question is therefore whether traditional myofibrillar ATPase histochemistry can give an adequate characterization of fibre type composition in athletes. Using immunohistochemical and electrophoretic techniques it has been reported that endurance training can increase the proportion of fibres with coexistence of slow and fast M H C isoforms (Baumann et al. 1987, Schantz 1986). In a recent cross-sectional study, we also demonstrated a high proportion of fibres with a coexistence of M H C types I and I1 a isoforms in muscles from elite cross-country skiers (Klitgaard et al. 1990b). Endurance training therefore seems to increase the proportion of fibres with coexistence of M H C isoforms. However, to our knowledge no studies have examined M H C isoform expression in single fibres from athletes
175
176
H . Klitgaard et al.
Table 1. Physical characteristics of subjects
Sedentary controls (4) Body builders (4)
Age (years)
Body weight (kg)
Height
28 0.4 25 i2.0
75i2 98 i3*
181 + 4 185+2
*
(4
Values are meansFSE. Numbers in brackets indicate number of subjects in each group. * Statistically significant difference between the groups, P < 0.05. involved in training regimens inducing a muscular hypertrophy. As for other types of exercise, the results from histochemical studies of body builders have been conflicting. Thus, some reports have demonstrated an identical (MacDougall et n l . 1982, Sale et al. 1987) and others a different (Tesch & Larsson 1982) fibre type distribution in skeletal muscles of body builders as compared w-ith control groups. T o extend the biochemical characterization of fibre types in body builders, the present study therefore analysed needle biopsies from a group of young sedentary men and a group of male body builders. Fibre type characteristics were determined using both myofibrillar ATPase histochemistry and electrophoretic analysis of MHC composition in single fibres. Only minor differences were observed in the fibre type distribution with histochemistry. However, the two groups manifested marked differences in MHC composition of single fibres. M,.ZTERIALS A N D METHODS Subjects. Four sedentary 4 oung men and four male bod>- builders participated in this study. Physical characteristics of subjects are given in Table 1. The sedentary young men did not participate in any regular sports activity and had not done so for the last 7 years (range 3-11 years) before the time of examination. The MHC composition of single fibres from m. biceps brachii of these subjects has been published elsewhere (Klitgaard et al. 1990a). The body builders had trained 2-5 h per day, 3-6 times a week, for the last 6 years (range 2-12 years) and two of them were in the national elite. None of them had taken anabolic steroids during the last 2 years before the time of examination. The study was approved by the Copenhagen Ethics Committee. Biopsies. Needle biopsies were taken by suction from m. biceps brachii to secure 100-150 mg samples. One part of the sample was frozen immediately in liquid nitrogen and used for dissection of 2- to 4-mmlong fibres for analysis of MHC composition. The
other part of the sample was trimmed, mounted and frozen in isopentane, cooled with liquid nitrogen and used for histochemical analysis. M H C composition of singlefibres. Single fibres from the muscle specimens were dissected and prepared for electrophoretic analysis of MHC composition as previously described (Salviati et al. 1982). Electrophoresis of the fibres was performed in 616 SDSPAGE gels with 37.504 glycerol (w/v) in the separating gel (Klitgaard et al. 1990b). Gels were run at 70 V overnight. This electrophoretic system has previously demonstrated a clear resolution of MHC types I, I1 a and I1 b, as verified by immunoblotting with monoclonal antibodies, in both human m. vastus lateralis and m. biceps brachii (Biral ef til. 1988, Klitgaard et al., 1990b,c). On average, 50-60 fibres were analysed in each subject from both groups. A total of 224 fibres was analysed in the sedentary control group and 217 fibres in the group of body builders. Historhemistry. Serial transverse sections (10 pm) were cut in a cryotome at 20 "C. The sections were mounted on coverslips and stained for myofibrillar ATPase at pH 9.4 after both alkaline (pH 10.3) and acid (pH 4.3 and 4.6) preincubations (Klitgaard et al. 1990~).Fibres were characterized as I, IIa, I I b and I I c fibres. On average, 382 (range 294-640) fibres were classified in each sample. The areas of a minimum of 50 type I, 30 type IIa and 20 type I I b fibres were measured in each sample with a digitizer. Statistics. The non-parametric Mann-Whitney test was applied for statistical comparison between the groups. The 0.05 confidence level was chosen to indicate statistical significance. ~
RESULTS Fibre typing from myofibrillar ATPase histochemistry (Fig. 1) revealed a similar proportion of type I fibres in the sedentary controls and the body builders (48 2 us 5 1 i3 yo, mean F SE). I n contrast, there was a trend towards a larger proportion of type I1 a fibres (3 1 6 us 25 6%) and a lower proportion of type I1 b fibres (18 & 5 1'5 26f6Y0) in the body builders, but these
Myosin expression in muscle of body builders
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group contained all three M H C isoforms (Klitgaard et al. 1990a). In the body builders 41 f 4 % (range 33-45) of the fibres contained only MHC type I, and 6 4 3 yo(2-14) showed coexistence of M H C types I and I I a (Fig. 3). This was similar f to the 36+5Y0 (24-43) and 6 + 2 % (3-ll), respectively, found within the sedentary controls. However, the body builders had a larger proportion of fibres containing only M H C type I1 a (36+4 [28-451 us 1 2 f 2 % [6-171; P < 0.05); as compared with the sedentary controls, but a lower proportion of fibres with a coexistence of M H C types I I a and I I b ( 1 6 f 3 [lo-221 us 34 2 yo[29-401; P < 0.05) and nearly no fibres containing only M H C type I1 b (1 f 1 [&3] 'us 12+1'30 [10-15]; P < 0.05). Type I TypeIIa Type I1 b The two different systems for classification of Fig. 1. Fibre type distribution (meansfSE), de- fibre types are compared in Fig. 4, and the termined by myofibrillar ATPase histochemistry, in arrows illustrate their relationship. Nearly all of m. biceps brachii of young sedentary men ( 0 2 8 0.4 the fibres showing coexistence of MHC types I years, n = 4)and male body builders ( 0 2 5 i2 years, and I I a had a clear majority of M H C type I n = 4). On average, 382 (range 294-640) fibres were (Fig. 2, lane v). These fibres would react as typed in each sample. histochemical type I fibres. M H C determination of single fibres and myofibrillar ATPase histochemistry therefore gives a similar type I fibre population in both the sedentary controls (42 us 48%) and in the body builders (47 us 51 yo). As regards the type I1 fibre population, the two classification systems did not give similar Fig. 2. Identification of human myosin heavy chain results. From comparison of the percentage isoforms in single fibres of m. biceps brachii after fibres typed histochemically as types I1 a and I1 b SDS-PAGE in 6% gels with high glycerol content and silver staining. Only the MHC region is shown. with the percentage distribution of single fibres Lane (i), purified myosin from human m. biceps containing M H C types I1a and I1 b it appears brachii; lanes (ii) to (vi), single fibres from m. biceps that in sedentary controls a large proportion of brachii containing only MHC type I (ii), type IIa (iii), both the histochemical types I I a and I1 b fibres type I1 b (iv) and with a coexistence of MHC types I manifest coexistence of M H C types I1 a and I1 b and IIa (v) and types IIa and IIb (vi). (Fig. 4). On the other hand, within the body builders the histochemical type I1a fibres seem to contain only M H C type I1 a, whereas nearly all differences were not significant. Almost no I I c the histochemical type I1 b fibres contain both fibres were found in both groups. Fibre areas of M H C types I I a and I I b . type I (6825f955 us 5519&533pm2) but especially type I1a (10,959 f1035 'us 7286 241 DISCUSSION pm2;P < 0.05) and type I1 b fibres (9837 & 1282 'us 621 1 f294 ,urn2; P < 0.05) were larger in the Using a sensitive electrophoretic technique the body builders as compared with the sedentary present study demonstrated a different pattern of M H C expression in single fibres from m. controls. T h e electrophoretic analysis identified single biceps brachii of body builders, as compared fibres containing only M H C type I (Fig. 2, lane with a group of sedentary controls. In contrast, ii), M H C type I I a (lane iii)), M H C type I I b traditional myofibrillar ATPase histochemistry (lane iv) and fibres with coexistence of M H C only revealed minor differences. By comparing types I and I1 a (lane v) and M H C types I1a and the M H C composition and the myofibrillar I I b (lane vi). Also, one fibre in the sedentary ATPase reaction of single fibres from rat skeletal
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Fig. 3. Fibre tj-pe composition, determined by MHC composition of single fibres, in m. biceps brachii of young sedentary men ( 0 2 8 + 0 . 4 years, n = 4) and male body builders ( a 2 5 f 2 . 0 years, n = 4). 224 fibres were analysed in the sedentary control group and 217 in the group of body builders. Values are means SE.
muscles (Danieli-Betto et a / . 1986), it has been shown that fibres containing only M H C types I, I1 a and I1 b will react as histochemical types I, I I a and I I b fibres respectively. Fibres with a coexistence of two M H C isoforms will react histochemically as the dominant isoform. Only with nearly equal fractions of MHC types I and I1 a will the fibre react histochemically as a type I I c fibre. The major part of the relativelj large proportion of fibres showing coexistence of two M H C isoforms will therefore not be adequately characterized by traditional myofibrillar ATPase histochemistry, as also demonstrated for human skeletal muscle in the present study. This has functional implications, since the M H C content of a fibre is related to its shortening velocity (Reiser et nl. 1985, Fitts et ul. 1989) and 'contraction economy' (energy utilization per unit force; Crow & Kushmerick, 1982). This study did not employ histochemical typing of single fibres because it is debatable whether such fibre typing is able adequately to distinguish between fibre types (Staron & Pette 1987). Only major fibre types can be given reliable classification since the histochemical reaction is influenced by the sample thickness and since classification is done without the aid of surrounding fibres (Staron & Pette 1987). We
therefore preferred to compare, within the same sample, a population of fibres classified in a cross-section of muscle stained for myofibrillar ATPase histochemistry with a population of single fibres classified by M H C composition. As demonstrated in Fig. 4, it is possible to compare the two classification systems even though myofibrillar ATPase histochemistry is done on a tissue slice and M H C composition determined in single fibres. This is possible because of knowledge of the staining properties of the different M H C isoforms mentioned above (Danieli-Betto et a/. 1986). It is still debated whether usage of muscles can change the expression of myosin isoforms (Saltin & Gollnick 1983). Recent longitudinal training studies of both rats (Green et al. 1983) and humans (Schantz 1986) suggest that this is possible. The almost complete absence of fibres containing only M H C type IT b within the body builders, together with the low proportion of fibres showing coexistence of M H C types I I a and I I b and the large proportion of fibres containing only M H C type IIa, suggests an increased expression of M H C type I I a and a decreased expression of M H C type I I b with body building. Of course, the cross-sectional design of the present study provides only a
Myosin expression in muscle of body builders
179
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Fig. 4. Comparison of the fibre type composition, determined by single-fibre analysis of MHC composition (MHC) and myofibrillar ATPase histochemistry (histochemistry), in m. biceps brachii of young sedentary men (2810.4 years, n = 4) and male body builders (25k2.0 years, n = 4). The arrows indicate the relationship between the two fibre type classification systems. The dashed arrow illustrates the possibility that some of the body builders’ histochemical type I1 a fibres might exhibit coexistence of MHC types IIa and I1 b (see ‘Results’).
limited basis for such a statement, but it would which a higher content of M H C type I I a and a be consistent with the fact that body building lower content of MHC type I I b was demontraining involves intense repetitive contractions strated, as compared with an inactive, agecharacterized by demands on not only strength matched control group (Klitgaard et af. 1990~). In conclusion, this study demonstrates a but muscular endurance as well. This has been proposed to result in an increased proportion of different expression of M H C isoforms in m. histochemical type I fibres, resembling the biceps brachii of body builders compared with histochemical profile of skeletal muscles from sedentary controls. This may suggest that the endurance athletes (Tesch & Larsson 1982). training increased the expression of M H C type However, other histochemical studies of body I I a at the expense of expression of M H C type builders have reported a fibre type distribution I1 b. identical to sedentary subjects (MacDougall et af. 1982; Sale et al. 1987). This is in agreement with This study was supported by grants from the Danish the present study which suggests that an adapResearch Academy, the Danish Research Council for tation occurs only in the M H C expression of the Natural Sciences, the Obel Family Foundation, the histochemical type I1 fibre population. Our the Knud Hejgaard Foundation and the John Meyer results are also consistent with a recent study on Foundation. H. Klitgaard was funded by the Faculty muscle biopsies from m. vastus lateralis and m. of Natural Sciences, University of Copenhagen, biceps brachii of strength-trained elderly men in Denmark. We are grateful to Mrs Lise Riis-Jacobsen 9
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and hlrs Marianne Hemmingsen for their skilful technical assistawe and to Mrs Lis H. Christensen for typing of the manuscript.
1990b. Co-existence of myosin heavy chain I and
I1 a isoforms in human skeletal musck fibres with
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