Histochemistry 58, 79-87 (1978)
Histochemistry 9 by Springer-Verlag 1978
Subgrouping of Fast Twitch Fibres in Skeletal Muscles of Man A Critical Appraisal Gisela Sjogaard, M.E. Houston*, Else Nygaard and B. Saltin August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark
Summary. Subgroups of fast twitch (FT) muscle fibres were identified by histochemical techniques on muscle samples of m. quadriceps femoris from six male and six female subjects, who had been assigned to three groups; untrained, active and well trained (endurance runners). Slow twitch (ST) and FT fibres were initially identified using the histochemical stain for myofibrillar ATPase, preincubated at pH 10.3 and 4.3. Three people, working independently, then identified the subgroups FTa and FTb on the basis of the staining intensity for only one of the following reactions: ~-glycerophosphate dehydrogenase, c~-GPD; N A D H tetrazolium reductase, N A D H - T R ; and myofibrillar ATPase preincubated at pH 4.6, ATPase (4.6). FTa fibres were clearly distinguished from the darker staining FTb fibres using the ATPase (4.6) reaction. Differences in the staining within the FT fibres using the e-GPD and N A D H - T R reactions were more subtle, and differences between subject groups were evident. The percentage of FTa fibres was overestimated for the untrained and underestimated for the well trained subjects using N A D H - T R . With the c~-GPD stain the percentage of FTa fibres was generally underestimated. When the data for all three stains were compared, only 27% of the FT fibres were placed in the same subgroups. These results demonstrate that the subgrouping of FT fibres in man is more reliable using the differences in pH sensitivity for the myofibrillar ATPase reaction compared to histochemical reactions for oxidative or glycolytic enzymes.
Introduction Skeletal muscle of animal species (man, rat, guinea pig etc.) is composed of two major fibre types as based on contractile properties and histochemically demonstrated myofibrillar ATPase activity (for reviews see Close, 1972; Burke and Edgerton, 1975). These two fibre types have been described as type I or slow twitch (ST) and type II or fast twitch (FT) (Engel, 1962; Gollnick *
Present address : Department of Kinesiology, Universityof WaterIoo, Waterloo, Canada
0301-5564/78/0058/0079/$01.80
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et al., 1972). Within the polulation of type II or FT fibres, there are distinct subgroups of fibres that have been differentiated on the basis of histochemical stains (a) oxidative or glycolytic enzymes (Barnard et al., 1971; Peter et al., 1972; Prince et al., 1976) or (b) using the subtle acid pH sensitivities of the myofibrillar ATPase reaction (Brooke and Kaiser, 1969). For non-human skeletal muscle it has been customary to use the former (a) techniques, resulting in fibre classes that have been described by Peter et al. (1972) as slow twitch oxidative (SO), fast twitch oxidative glycolytic (FOG) and fast twitch glycolytic (FG). Such a classification scheme is also used for human skeletal muscle (Edgerton et al., 1975; Prince et al., 1976), but clinical and recent physiological studies have employed the latter procedure (b) resulting in fibre classes described as Type I, II-A and II-B (Dubowitz and Brooke, 1973; Brooke and Kaiser, 1974), or ST, FTa and FTb (Saltin et al., 1977). A major question that arises is whether subgrouping of the FT fibres using the two aforementioned procedures gives rise to identical fibre subpopulations in man. The purpose of this study was to critically evaluate this question. FT fibres in human skeletal muscle were separately assigned to two subgroups using either a histochemical stain for an oxidative or a glycolytic enzyme as well as myofibrillar ATPase after preincubation at pH 4.6: the results of these three procedures were compared. Materials and Methods 12 healthy subjects volunteered to participate in this study after being fully informed about the nature and risks involved. In order to cover a wide range of muscle characteristics, men and women between the ages of 24-45 years,and having widely different patterns of skeletal muscle activity were assigned to one of three groups: untrained, 4 females; active, 4males; and well trained, 2 male and 2 female endurance runners. Muscle biopsies (20-40 rag) were obtained from the quadriceps femoris muscle (vastus lateralis) 12-16 cm above the patella, with a needle technique (Bergstr6m, 1962). The biopsy was divided into two parts: one was immediately frozen for biochemical analysis of succinate dehydrogenase (SDH) activity (Ess6n et al., 1975), and the other was mounted in an embedding medium for histochemical analysis and frozen in isopentane cooled to the freezing point. The muscle samples were stored at - 8 0 ~ C until analysis. For histochemical analysis, serial cross sections (10 Ixm thick) were cut and stained for myofibrillar ATPase at pH 9.4 (Padykula and Herman, 1955) subsequent to preincubations in media having a pH of 4.3, 4.6 or 10.3 (Brooke and Kaiser, 1969). Reduced nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) was assayed by the method of Novikoff et al. (1961), and alpha glycerophosphate dehydrogenase (~-GPD) by the method of Wattenberg and Leong (1960). Enlarged photomicrographs of muscle cross sections were made for each subject after the removal of periodic acid schiff positive material within the individual fibres (Andersen, 1976). The identity of the individual fibres was marked in these photographs. ST, FT and undifferentiated fibres were identified for each subject, and marked on the photographs by three persons, working independently, and using the myofibrillar ATPase stains preincubated at pH 10.3 and 4.3.The FT fibres were then divided into two groups based on the staining intensity, visualized in the microscope, of only one of the following three histochemical reactions: myofibrillar ATPase (pH 4.6), NADH-TR, or c~-GPD. FT fibres were classified as FTa fibres when the staining intensity was light for myofibrillar ATPase (pH 4.6), dark for NADH-TR or light for c~-GPD. FTb fibres were identified if they were dark, light or dark, respectively, for the above three stains. Finally, a fourth person compared the classification of each fibre from the marked photographs and tabulated the results. Statistics. Only descriptive statistics (mean and range) were employed as this study was exclusively designed for describing the relation between different procedures for the subgrouping of FT-fibres.
Subgrouping of Fast Twitch Muscle Fibres
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Table 1. Individual subject data and group means for SDH activity, fibre composition and comparison of FT fibre subgroup identifications. ST and undifferentiated fibres were identified using the histochemical reactions for myofibrillar ATPase preincubated at pH 4.3 and 10.3. FTa fibres and FTb fibres (not shown) were identified on the basis of their histochemical staining intensities for myofibrillar ATPase preincubated at pH 4.6 (ATPase 4.6), NADH tetrazolium reductase (NADH-TR) and e-glycer0phosphate dehydrogenase (e-GPD). The percentage of FTb fibres may be determined by subtracting the percentage of ST, undifferentiated and FTa fibres from 100. The asterix (*) indicates that no FTb fibres were apparent based on the e-GPD stain
Subject and subject groups
~ IE .2 E. .= ~ ~ ~
ST Undiffibres ferenti(%) ated fibres (%)
FTa fibres
FT fibres subgrouped identically using
ATPase NADH- c~-GPD ATPase 4.6 TR (%) 4.6 and (%) (%) NADH-TR (%)
ATPase 4.6, NADH-TR, and ~-GPD (%)
Untrained 1
6.1
65
0
2 3 4
2.4 5.3 7.2
51 50 69
0 0 0
20 35 34 27
25 45 47 24
35* 5 14 7
57 72 68 75
43 11 27 15
Mean
5.3
59
0
29
35
15
68
24
Active 5 6 7 8
8.4 7.5 11.5 11.5
62 46 61 69
0 4 2 3
30 44 37 22
31 46 32 18
11 16 4 28*
83 78 88 59
38 25 12 53
Mean
9.7
60
2
33
32
15
77
32
6.6 11.4 11.5 7.5
70 87 64 79
0 0 3 0
25 7 30 20
9 10 13 14
4 13" 3 3
42 63 51 72
22 45 18 17
9.3
75
1
21
12
6
57
26
Well trained 9 10 11 12 Mean
Results
T h e p e r c e n t a g e o f S T fibres ( T a b l e 1) was a p p r o x i m a t e l y t h e s a m e f o r t h e a c t i v e a n d u n t r a i n e d g r o u p s , 6 0 % (46-69), b u t this was 1 5 % h i g h e r in t h e well t r a i n e d subjects, 7 5 % (64-87). F o r all s u b j e c t s t h e S T fibres h a d t h e d a r k e s t s t a i n i n g i n t e n s i t y w i t h t h e N A D H - T R stain, a n d t h e l i g h t e s t s t a i n i n g i n t e n s i t y w i t h t h e ~ - G P D stain. H o w e v e r , t h e level o f s t a i n i n g i n t e n s i t y w a s q u i t e d i f f e r e n t in t h e t h r e e g r o u p s , e s p e c i a l l y w i t h the N A D H - T R stain w h e r e t h e fibres o f t h e well t r a i n e d g r o u p s t a i n e d d a r k e s t a n d the u n t r a i n e d l i g h t e s t (Fig. 1). S D H a c t i v i t y was l o w e s t in t h e u n t r a i n e d g r o u p 5.3 ( 2 . 4 - 7 . 2 ) g m o l x g - i x r a i n - 1 b u t a p p r o x i m a t e l y t h e s a m e for t h e a c t i v e g r o u p a n d well t r a i n e d g r o u p 9.5 ( 6 . 6 - 1 1 . 5 ) g m o l x g - 1 x rain 1. I n e a c h g r o u p t h e r e was a t r e n d t o w a r d a c o r r e l a t i o n b e t w e e n % S T fibres a n d S D H activity. T h e u n d i f f e r e n t i a t e d fibres a v e r a g e d 1 % ( 0 - 4 ) f o r t h e d i f f e r e n t subjects. T h e s e fibres g e n e r a l l y d e m o n s t r a t e d a m e d i u m s t a i n i n g i n t e n s i t y f o r b o t h t h e N A D H - T R a n d t h e e - G P D stains.
Subgrouping of Fast Twitch Muscle Fibres
83
Table 2. Comparisons of the subgrouping of individual FT fibres in h u m a n skeletal muscle using the histochemical reactions for myofibrillar ATPase preincubated at pH 4.6 (ATPase 4.6), N A D H tetrazolium reductase ( N A D H - T R ) and ~-glycerophosphate dehydrogenase (~-GPD). Individual FT fibres identified as F T a and F T b using the ATPase 4.6 stain are compared with the staining intensity they exhibited (dark or light) using the N A D H - T R and c~-GPD stains. Correspondence between comparisons is expressed as per cent. The asterix (*) indicates that no F T b fibres were apparent based on the c~-GPD stain Subjects and subject groups
Untrained 1" 2 3 4
N u m b e r of F T a fibres ATPase 4.6
20 54 61 59
Mean Active 5 6 7 8*
41 48 49 26
Mean Well trained 9 10" 11 12 Mean Overall m e a n Range
37 6 44 35
F T a subgroup correspondance
Number FTb fibres ATPase 4.6
FTb subgroup correspondance NADH-TR light (%)
c~-GPD dark (%)
15 21 27 9
33 i4 7 33
0 95 93 100
22
72
55 0 33
100 86 0
29
62
88 40 91 100
100 0 100 100
NADH-TR dark (%)
c~-GPD light (%)
75 94 95 81
100 13 38 12
86
41
90 90 88 65
37 33 12 100
83
45
35 83 50 71
17 100 9 15
60
35
80
75
76
40
44
70
(9 100)
(0-100)
(0 100)
(35 95)
11 7 0 6
8 5 5 1
The number of FTa fibres classified according to the N A D H - T R stain, as compared to the myofibrillar ATPase stain (pH 4.6), was overestimated in the untrained group by 6%, but underestimated in the well trained by 9%. In the group of active subjects the per cent was approximately the same (Table 1). According to the e-GPD stain, the percentage of FTa fibres was underestimated Fig. 1. Serial cross sections of muscle biopsies from vastus lateralis of an untrained (A, B, C), an active (D, E, F) and a well trained (G, I, H) subject. The fibre types, ST, F T a and F T b have been identified On the histochemical reactions for myofibrillar ATPase, preincubated at pH 4.6 (A, D, G) and are compared with the histochemieal reaction for N A D H tetrazolium reduetase (B, E, I) a n d c~-glycerophosphate dehydrogenase (C, F, H)
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G. Sjogaard et al.
in 9 subjects, while in 3 subjects (one from each group) subgrouping was not possible as the staining intensity was virtually the same in all FT fibres (Tables 1 and 2). An analysis of the classification of each single FT-fibre using the myofibrillar ATPase (pH 4.6) and N A D H - T R stains revealed that 67% (42-88) of the fibres were classified identically in both stainings with the discrepancy being largest in the group of well trained subjects and lowest in the active group (Table 1). When taking all stainings (myofibrillar ATPase (pH 4.6), N A D H - T R and e-GDP) into account, only 27% (11-53) of the FT fibres were classified identically for all three stainings. However, only 1% of the fibres classified as FTa fibres according to the myofibrillar ATPase (pH 4.6) stain were classified as FTb fibres according to the N A D H - T R as well as the e-GPD stains. Discussion
Subgrouping of the FT fibres in skeletal muscle of man can be performed with the different methods employed in the present study, but neither the relative number nor the same fibres are assigned to the two subgroups of FT fibres. Several questions then arise: why does a routine stain for myofibrillar ATPase in combination with a stain for the mitochondrial capacity give so distinct a typing pattern in certain species (Peter et al., 1972; Ariano et al., 1973), but not in man; and which combination of stains should be used when typing skeletal muscle of man? In species like the rat and the guinea pig marked differences exist in the metabolic potentials of the different fibres. The red and the white portions of the vastus in the guinea have similar and high myosin ATPase activities and fast times to peak tension (TPT), but the myoglobin content and the activities of different oxidative enzymes are 4-9 times higher in the red as compared to the white vastus (Peter et al., 1972). The soleus on the other hand has a low myosin ATPase activity, a long TPT and barely the same myoglobin and oxidative enzyme content as the red vastus (Peter et al., 1972). These circumstances provide a good basis for a routine stain for myofibrillar ATPase to distinguish between the red vastus and the soleus and a stain for a mitochondrial enzyme to distinguish between the red and the white vastus. In skeletal muscle of man there are also differences between fibres in regard to myosin ATPase and contractile characteristics (Buchthal and Schmalbruch, 1970; Taylor et al., 1974) as well as to the metabolic potentials of the fibres (Ess6n et al., 1975). However, at least for the oxidative and the glycolytic enzymes, the differences are small (Ess6n et al., 1975). Moreover, the training status of the muscle will also influence the metabolic capacity (Henriksson and Reitman, 1976). In contrast to man, where increased physical activity minimizes the differences in the oxidative potentials between ST, FTa and FTb fibres (Henriksson and Reitman, 1976; Jansson and Kaijser, 1977), the same ratio still exists between the fibre types (ST/FOG, ST/FG and F O G / F G ) in rat skeletal muscle after endurance training (Baldwin et al., 1972). Thus, the basis for the use of a metabolic differentiation between fibres in human skeletal muscle is limited.
Subgrouping of Fast Twitch Muscle Fibres
85
Why there is this difference between species in metabolic differentiation is not immediately apparent. Several factors may contribute. One is that skeletal muscles of man contain high proportions of both major fibre types (Johnson et al., 1973), whereas in many species a preponderance of one fibre is frequently found (Ariano et al., 1973). Another important difference is that both FOG fibres and ST fibres of species such as rats (Armstrong et al., 1974), guinea pigs (Edgerton et al., 1970) and lions (Armstrong et al., 1977) appear to be recruited at very low tension developments. In man, under comparable contractile conditions, only ST fibres are depleted of glycogen, suggesting that this fibre type is the only one to be recruited (Gollnick et al., 1973). Thus the specialization in the function of the muscles and the easy recruitment of the F O G fibres in many other species than man may be the explanation for the less distinct metabolic differentiation of FT fibres in skeletal muscle of man. In this connection some recent findings by Spamer and Pette (1977) are of note. They determined enzyme activities in single fibres microdissected from freeze-dried muscle of rabbits and found variability of up to 14 fold in malate dehydrogenase activity, representing a continuum, in FT fibres within the same muscle. This prompted these investigators to conclude that muscle fibre classification according to qualitative enzymological evaluations is unsatisfactory. Whether these results are indicative for other animal species (rats, guinea pigs, lions etc.) is not presently known. The use of the different pH labilities of the myofibrillar ATPase reaction a pH 4.6 for a subdivision of FT fibres gives two distinct populations with an insignificant number of intermediate stained fibres (Table 1; Brooke and Kaiser, 1974). There is however, a drawback with this method. It is not known what the differences in pH lability reflect. Brooke and Kaiser (1970) have suggested that it is related to differences in sulfhydryl groups of the myosin molecule. Furthermore, in an attempt to evaluate possible differences in FTa and FTb fibres, Henriksson and Pette (unpublished observations) have not noted differences in these fibres on the basis of preliminary studies using polyacrylamide gel electrophoresis. These points should not distract from the fact that the subsivision of FT muscle fibres in man according to pH lability appears to have strong physiological importance. Thus the cross sectional fibre area of the FTa fibres is larger than than that of the FTb fibres (Saltin et al., 1977). Further, the FTa and FTb fibres appear to be selectively recruited both in isometric and dynamic exercise as indicated by the glycogen depletion method (Andersen and Sjogaard, 1976; Secher and Nygaard, 1976; Ess6n, 1978). There are also differences in the mean values for oxidative and glycolytic enzymes comparing FTa and FTb fibres, although there is a substantial overlap in enzyme levels (Ess6n et al., 1975) which can be observed by microphotometric evaluation of histochemical stains for N A D H - T R and e-GPD (Halkj~er-Kristensen and Ingemann-Hansen, 1978). These latter findings are in agreement with the previously discussed minor distinction in the metabolic profiles between muscle fibre types, and also reinforces our contention that the histochemical stains for oxidative or glycolytic enzymes provide a poor bais for the subgrouping of FT fibres in man.
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G. Sjogaard et aI.
Acknowledgement. This study was performed with economical support from the Danish Natural Science Foundation.
References Andersen, P. : Capillary density in skeletal muscle of man. Acta physiol, scand. 95, 203-205 (1975) Andersen, P., Sjogaard, G.: Selective glycogen depletion in the subgroups of Type II muscle fibres during intense submaximal exercise in man. Acta physiol, scand. 96, 26A (1976) Ariano, M,A., Armstrong, R.B., Edgerton, V.R. : Hindlimb muscle fiber populations of five mammals. J. Histochem. Cytochem. 21, 51-55 (1973) Armstrong, R.B., Saubert IV, C.W., Sembrovich, W.L., Shepherd, R.E., Gollnick, P.D. : Glycogen depletion in rat skeletal muscle fibers at different intensities and durations of exercise. Pfliigers Arch. 352, 243-256 (1974) Armstrong, RB., Marum, P., Saubert IV, C.W., Seeherman, H.J., Taylor, C.R.: Muscle fiber activity as a function of speed and gait. J. Appl. Physiol. 43, 672~77 (1977) Baldwin, K.M., Klinkerfuss, G.H., Terjung, R.L., Mol& P.A., Holloszy, J.O. : Respiratory capacity of white, red and intermediate muscle: Adaptive response to exercise. Am. J. Physiol. 222, 373-378 (1972) Barnard, R.J., Edgerton, V.R., Furukawa, T., Peter, J.B. : Histochemical, biochemical, and contractile properties of red, white and intermediate fibers. Am. J. Physiol. 220, 410-414 (1971) Bergstr6m, J. : Muscle electrolytes in man. Scand. J. clin. Lab. Invest. Suppl. 68 (1962) Brooke, M.H., Kaiser, K.K.: Some comments on the histochemical characterization of muscle adenosine triphosphatase. J. Histochem. Cytochem. 17, 431432 (1969) Brooke, M.H., Kaiser, K.K.: Three "myosin ATPase" systems: The nature of their pH lability and sulfhydryl dependance. J. Histochem. Cytochem. 18, 670~572 (1970) Brooke, M.H., Kaiser, K.K.: The use and abuse of muscle histochemistry. Ann. N.Y. Acad. Sci. 228, 121 144 (1974) Buchthal, F., Schmalbruch, H.: Contraction times and fibre types in intact human muscle. Acta physiol, scand 79, 435-452 (1970) Burke, R.E., Edgerton, V.R.: Motor unit properties and selective involvement in movement. In: Exercise and sport sciences reviews. J.H. Wilmore, J.F. Keough (ed.), Vol. 3, pp. 31 81. New York: Academic Press 1975 Close, R.I.: Dynamic properties of mammalian skeletal muscles. Physiol. Rev. 52, 129-197 (1972) Dubowitz, V., Brooke, M.H. : Muscle biopsy: A modern approach. In: Major problems in neurology, Vol. 2 London and New York: W.B. Saunders 1973 Edgerton, V.R., Simpson, D.R., Barnard, R.J., Peter, J.B.: Phosphorylase activity in acutely exercised muscles. Nature 225, 866-867 (1970) Edgerton, V.R., Smith, J.L., Simpson, D.R. : Muscle fiber type populations of human leg muscles. Histochem. J. 7, 259-266 (1975) Engel, W.K. : The essentiality of histo- and cytochemical studies of skeletal muscle in the investigation of neuromuscular disease. Neurology 12, 778-784 (1962) Ess6n, B., Jansson, E., Henriksson, J., Taylor, A.W. Saltin, B.: Metabolic characteristics of fiber types in human skeletal muscle. Acta physiol, scand. 95, 153-165 (1975). Ess6n, B. : Glycogen depletion of different fibre types in human skeletal muscle during intermittent and continuous exercise. Submitted for publication (1978). Gollnick, P.D., Armstrong, R.B., Saubert IV, C.W., Piehl, K., Saltin, B.: Enzyme activity and fiber composition in skeletal muscle of untrained and trained men. J. Appl. Physiol. 33, 312-319 (1972) Gollnick, P.D., Armstrong, R.B., Saubert IV, C.W., Sembrovich, W.L., Shepherd, R.E. : Glycogen depletion patterns in human skeletal muscle fibers during prolonged work. Pfl/igers Arch. 244, 1-12 (1973) Halkj~er-Kristensen, J., Ingemann-Hansen, T.: Quantitative histochemistry of single fibers in the human quadriceps muscle. Histochem. J. 10, 497-504 (1978) Henriksson, J., Reitman, J.S.: Quantitative measures of enzyme activities in type I and type II muscle fibres of man after training. Acta physiol, scand. 97, 392 397 (1976) Jansson, E., Kaijser, L.: Muscle adaptation to extreme endurance training in man. Acta physiol. scand. 100, 315-324 (1977)
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Johnson, M.A., Polgar, J., Weightman, D., Appleton, D.: Data on the distribution of fibre types in thirty-six human muscles. An autopsy study. J. Neurol. Sci. 18, 111 129 (1973) Novikoff, A.B., Shin, W., Drucker, J.: Mitochondrial localization of oxidation enzymes: Staining results with two tetrazolium salts. J. Biophys. Biochem. Cytol. 9, 47 61 (1961) Padykula, H.A., Herman, E. : The specificity of the histochemical method for adenosine triphosphotase. J. Histochem. Cytochem. 3, 170-195 (1955) Peter, J.B., Barnard, R.J., Edgerton, V.R., Gillespie, C.A., Stempel, K.E.: Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits. Biochemistry 11, 2627-2633 (1972) Prince, F.P., Hikida, R.S., Hagerman, F.C.: Human muscle fibre types in power lifters, distance runners and untrained subjects. Pflfigers Arch. 363, 19 26 (1976) Saltin, B., Henriksson, J., Nyggard, E., Andersen, P.: Fiber types and metabolic potentials of skeletal muscles in sedentary man and endurance runners. Ann. N.Y. Acad. Sci. 301, 3-29 (1977) Secher, N., Nygaard-Jensen, E. : Glycogen depletion pattern in types I, IIA and IIB muscle fibres during maximal voluntary static and dynamic exercise. Acta physiol, scand. Suppl. 440, 287 (1976) Spamer, C., Pette, D. : Activity patterns of phosphofructokinase, glyceraldehyde phosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle. Histochemistry 52, 201-216 (1977) Taylor, A.W., Ess~n, B., Saltin, B.: Myosin ATPase in skeletal muscle of healthy man. Acta physiol, scand. 91, 568 570 (1974) Wattenberg, L.W., Leong, J.L. : Effects of coenzyme Q 10 and menadione on succinate dehydrogenase activity as measured by tetrazolium salt reduction. J. Histochem. Cytochem. 8, 296-303 (1960) Received June 13, 1978
Histochemistry 9 by Springer-Verlag 1978
Subgrouping of Fast Twitch Fibres in Skeletal Muscles of Man A Critical Appraisal Gisela Sjogaard, M.E. Houston*, Else Nygaard and B. Saltin August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark
Summary. Subgroups of fast twitch (FT) muscle fibres were identified by histochemical techniques on muscle samples of m. quadriceps femoris from six male and six female subjects, who had been assigned to three groups; untrained, active and well trained (endurance runners). Slow twitch (ST) and FT fibres were initially identified using the histochemical stain for myofibrillar ATPase, preincubated at pH 10.3 and 4.3. Three people, working independently, then identified the subgroups FTa and FTb on the basis of the staining intensity for only one of the following reactions: ~-glycerophosphate dehydrogenase, c~-GPD; N A D H tetrazolium reductase, N A D H - T R ; and myofibrillar ATPase preincubated at pH 4.6, ATPase (4.6). FTa fibres were clearly distinguished from the darker staining FTb fibres using the ATPase (4.6) reaction. Differences in the staining within the FT fibres using the e-GPD and N A D H - T R reactions were more subtle, and differences between subject groups were evident. The percentage of FTa fibres was overestimated for the untrained and underestimated for the well trained subjects using N A D H - T R . With the c~-GPD stain the percentage of FTa fibres was generally underestimated. When the data for all three stains were compared, only 27% of the FT fibres were placed in the same subgroups. These results demonstrate that the subgrouping of FT fibres in man is more reliable using the differences in pH sensitivity for the myofibrillar ATPase reaction compared to histochemical reactions for oxidative or glycolytic enzymes.
Introduction Skeletal muscle of animal species (man, rat, guinea pig etc.) is composed of two major fibre types as based on contractile properties and histochemically demonstrated myofibrillar ATPase activity (for reviews see Close, 1972; Burke and Edgerton, 1975). These two fibre types have been described as type I or slow twitch (ST) and type II or fast twitch (FT) (Engel, 1962; Gollnick *
Present address : Department of Kinesiology, Universityof WaterIoo, Waterloo, Canada
0301-5564/78/0058/0079/$01.80
80
G. Sjogaard et al.
et al., 1972). Within the polulation of type II or FT fibres, there are distinct subgroups of fibres that have been differentiated on the basis of histochemical stains (a) oxidative or glycolytic enzymes (Barnard et al., 1971; Peter et al., 1972; Prince et al., 1976) or (b) using the subtle acid pH sensitivities of the myofibrillar ATPase reaction (Brooke and Kaiser, 1969). For non-human skeletal muscle it has been customary to use the former (a) techniques, resulting in fibre classes that have been described by Peter et al. (1972) as slow twitch oxidative (SO), fast twitch oxidative glycolytic (FOG) and fast twitch glycolytic (FG). Such a classification scheme is also used for human skeletal muscle (Edgerton et al., 1975; Prince et al., 1976), but clinical and recent physiological studies have employed the latter procedure (b) resulting in fibre classes described as Type I, II-A and II-B (Dubowitz and Brooke, 1973; Brooke and Kaiser, 1974), or ST, FTa and FTb (Saltin et al., 1977). A major question that arises is whether subgrouping of the FT fibres using the two aforementioned procedures gives rise to identical fibre subpopulations in man. The purpose of this study was to critically evaluate this question. FT fibres in human skeletal muscle were separately assigned to two subgroups using either a histochemical stain for an oxidative or a glycolytic enzyme as well as myofibrillar ATPase after preincubation at pH 4.6: the results of these three procedures were compared. Materials and Methods 12 healthy subjects volunteered to participate in this study after being fully informed about the nature and risks involved. In order to cover a wide range of muscle characteristics, men and women between the ages of 24-45 years,and having widely different patterns of skeletal muscle activity were assigned to one of three groups: untrained, 4 females; active, 4males; and well trained, 2 male and 2 female endurance runners. Muscle biopsies (20-40 rag) were obtained from the quadriceps femoris muscle (vastus lateralis) 12-16 cm above the patella, with a needle technique (Bergstr6m, 1962). The biopsy was divided into two parts: one was immediately frozen for biochemical analysis of succinate dehydrogenase (SDH) activity (Ess6n et al., 1975), and the other was mounted in an embedding medium for histochemical analysis and frozen in isopentane cooled to the freezing point. The muscle samples were stored at - 8 0 ~ C until analysis. For histochemical analysis, serial cross sections (10 Ixm thick) were cut and stained for myofibrillar ATPase at pH 9.4 (Padykula and Herman, 1955) subsequent to preincubations in media having a pH of 4.3, 4.6 or 10.3 (Brooke and Kaiser, 1969). Reduced nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) was assayed by the method of Novikoff et al. (1961), and alpha glycerophosphate dehydrogenase (~-GPD) by the method of Wattenberg and Leong (1960). Enlarged photomicrographs of muscle cross sections were made for each subject after the removal of periodic acid schiff positive material within the individual fibres (Andersen, 1976). The identity of the individual fibres was marked in these photographs. ST, FT and undifferentiated fibres were identified for each subject, and marked on the photographs by three persons, working independently, and using the myofibrillar ATPase stains preincubated at pH 10.3 and 4.3.The FT fibres were then divided into two groups based on the staining intensity, visualized in the microscope, of only one of the following three histochemical reactions: myofibrillar ATPase (pH 4.6), NADH-TR, or c~-GPD. FT fibres were classified as FTa fibres when the staining intensity was light for myofibrillar ATPase (pH 4.6), dark for NADH-TR or light for c~-GPD. FTb fibres were identified if they were dark, light or dark, respectively, for the above three stains. Finally, a fourth person compared the classification of each fibre from the marked photographs and tabulated the results. Statistics. Only descriptive statistics (mean and range) were employed as this study was exclusively designed for describing the relation between different procedures for the subgrouping of FT-fibres.
Subgrouping of Fast Twitch Muscle Fibres
81
Table 1. Individual subject data and group means for SDH activity, fibre composition and comparison of FT fibre subgroup identifications. ST and undifferentiated fibres were identified using the histochemical reactions for myofibrillar ATPase preincubated at pH 4.3 and 10.3. FTa fibres and FTb fibres (not shown) were identified on the basis of their histochemical staining intensities for myofibrillar ATPase preincubated at pH 4.6 (ATPase 4.6), NADH tetrazolium reductase (NADH-TR) and e-glycer0phosphate dehydrogenase (e-GPD). The percentage of FTb fibres may be determined by subtracting the percentage of ST, undifferentiated and FTa fibres from 100. The asterix (*) indicates that no FTb fibres were apparent based on the e-GPD stain
Subject and subject groups
~ IE .2 E. .= ~ ~ ~
ST Undiffibres ferenti(%) ated fibres (%)
FTa fibres
FT fibres subgrouped identically using
ATPase NADH- c~-GPD ATPase 4.6 TR (%) 4.6 and (%) (%) NADH-TR (%)
ATPase 4.6, NADH-TR, and ~-GPD (%)
Untrained 1
6.1
65
0
2 3 4
2.4 5.3 7.2
51 50 69
0 0 0
20 35 34 27
25 45 47 24
35* 5 14 7
57 72 68 75
43 11 27 15
Mean
5.3
59
0
29
35
15
68
24
Active 5 6 7 8
8.4 7.5 11.5 11.5
62 46 61 69
0 4 2 3
30 44 37 22
31 46 32 18
11 16 4 28*
83 78 88 59
38 25 12 53
Mean
9.7
60
2
33
32
15
77
32
6.6 11.4 11.5 7.5
70 87 64 79
0 0 3 0
25 7 30 20
9 10 13 14
4 13" 3 3
42 63 51 72
22 45 18 17
9.3
75
1
21
12
6
57
26
Well trained 9 10 11 12 Mean
Results
T h e p e r c e n t a g e o f S T fibres ( T a b l e 1) was a p p r o x i m a t e l y t h e s a m e f o r t h e a c t i v e a n d u n t r a i n e d g r o u p s , 6 0 % (46-69), b u t this was 1 5 % h i g h e r in t h e well t r a i n e d subjects, 7 5 % (64-87). F o r all s u b j e c t s t h e S T fibres h a d t h e d a r k e s t s t a i n i n g i n t e n s i t y w i t h t h e N A D H - T R stain, a n d t h e l i g h t e s t s t a i n i n g i n t e n s i t y w i t h t h e ~ - G P D stain. H o w e v e r , t h e level o f s t a i n i n g i n t e n s i t y w a s q u i t e d i f f e r e n t in t h e t h r e e g r o u p s , e s p e c i a l l y w i t h the N A D H - T R stain w h e r e t h e fibres o f t h e well t r a i n e d g r o u p s t a i n e d d a r k e s t a n d the u n t r a i n e d l i g h t e s t (Fig. 1). S D H a c t i v i t y was l o w e s t in t h e u n t r a i n e d g r o u p 5.3 ( 2 . 4 - 7 . 2 ) g m o l x g - i x r a i n - 1 b u t a p p r o x i m a t e l y t h e s a m e for t h e a c t i v e g r o u p a n d well t r a i n e d g r o u p 9.5 ( 6 . 6 - 1 1 . 5 ) g m o l x g - 1 x rain 1. I n e a c h g r o u p t h e r e was a t r e n d t o w a r d a c o r r e l a t i o n b e t w e e n % S T fibres a n d S D H activity. T h e u n d i f f e r e n t i a t e d fibres a v e r a g e d 1 % ( 0 - 4 ) f o r t h e d i f f e r e n t subjects. T h e s e fibres g e n e r a l l y d e m o n s t r a t e d a m e d i u m s t a i n i n g i n t e n s i t y f o r b o t h t h e N A D H - T R a n d t h e e - G P D stains.
Subgrouping of Fast Twitch Muscle Fibres
83
Table 2. Comparisons of the subgrouping of individual FT fibres in h u m a n skeletal muscle using the histochemical reactions for myofibrillar ATPase preincubated at pH 4.6 (ATPase 4.6), N A D H tetrazolium reductase ( N A D H - T R ) and ~-glycerophosphate dehydrogenase (~-GPD). Individual FT fibres identified as F T a and F T b using the ATPase 4.6 stain are compared with the staining intensity they exhibited (dark or light) using the N A D H - T R and c~-GPD stains. Correspondence between comparisons is expressed as per cent. The asterix (*) indicates that no F T b fibres were apparent based on the c~-GPD stain Subjects and subject groups
Untrained 1" 2 3 4
N u m b e r of F T a fibres ATPase 4.6
20 54 61 59
Mean Active 5 6 7 8*
41 48 49 26
Mean Well trained 9 10" 11 12 Mean Overall m e a n Range
37 6 44 35
F T a subgroup correspondance
Number FTb fibres ATPase 4.6
FTb subgroup correspondance NADH-TR light (%)
c~-GPD dark (%)
15 21 27 9
33 i4 7 33
0 95 93 100
22
72
55 0 33
100 86 0
29
62
88 40 91 100
100 0 100 100
NADH-TR dark (%)
c~-GPD light (%)
75 94 95 81
100 13 38 12
86
41
90 90 88 65
37 33 12 100
83
45
35 83 50 71
17 100 9 15
60
35
80
75
76
40
44
70
(9 100)
(0-100)
(0 100)
(35 95)
11 7 0 6
8 5 5 1
The number of FTa fibres classified according to the N A D H - T R stain, as compared to the myofibrillar ATPase stain (pH 4.6), was overestimated in the untrained group by 6%, but underestimated in the well trained by 9%. In the group of active subjects the per cent was approximately the same (Table 1). According to the e-GPD stain, the percentage of FTa fibres was underestimated Fig. 1. Serial cross sections of muscle biopsies from vastus lateralis of an untrained (A, B, C), an active (D, E, F) and a well trained (G, I, H) subject. The fibre types, ST, F T a and F T b have been identified On the histochemical reactions for myofibrillar ATPase, preincubated at pH 4.6 (A, D, G) and are compared with the histochemieal reaction for N A D H tetrazolium reduetase (B, E, I) a n d c~-glycerophosphate dehydrogenase (C, F, H)
84
G. Sjogaard et al.
in 9 subjects, while in 3 subjects (one from each group) subgrouping was not possible as the staining intensity was virtually the same in all FT fibres (Tables 1 and 2). An analysis of the classification of each single FT-fibre using the myofibrillar ATPase (pH 4.6) and N A D H - T R stains revealed that 67% (42-88) of the fibres were classified identically in both stainings with the discrepancy being largest in the group of well trained subjects and lowest in the active group (Table 1). When taking all stainings (myofibrillar ATPase (pH 4.6), N A D H - T R and e-GDP) into account, only 27% (11-53) of the FT fibres were classified identically for all three stainings. However, only 1% of the fibres classified as FTa fibres according to the myofibrillar ATPase (pH 4.6) stain were classified as FTb fibres according to the N A D H - T R as well as the e-GPD stains. Discussion
Subgrouping of the FT fibres in skeletal muscle of man can be performed with the different methods employed in the present study, but neither the relative number nor the same fibres are assigned to the two subgroups of FT fibres. Several questions then arise: why does a routine stain for myofibrillar ATPase in combination with a stain for the mitochondrial capacity give so distinct a typing pattern in certain species (Peter et al., 1972; Ariano et al., 1973), but not in man; and which combination of stains should be used when typing skeletal muscle of man? In species like the rat and the guinea pig marked differences exist in the metabolic potentials of the different fibres. The red and the white portions of the vastus in the guinea have similar and high myosin ATPase activities and fast times to peak tension (TPT), but the myoglobin content and the activities of different oxidative enzymes are 4-9 times higher in the red as compared to the white vastus (Peter et al., 1972). The soleus on the other hand has a low myosin ATPase activity, a long TPT and barely the same myoglobin and oxidative enzyme content as the red vastus (Peter et al., 1972). These circumstances provide a good basis for a routine stain for myofibrillar ATPase to distinguish between the red vastus and the soleus and a stain for a mitochondrial enzyme to distinguish between the red and the white vastus. In skeletal muscle of man there are also differences between fibres in regard to myosin ATPase and contractile characteristics (Buchthal and Schmalbruch, 1970; Taylor et al., 1974) as well as to the metabolic potentials of the fibres (Ess6n et al., 1975). However, at least for the oxidative and the glycolytic enzymes, the differences are small (Ess6n et al., 1975). Moreover, the training status of the muscle will also influence the metabolic capacity (Henriksson and Reitman, 1976). In contrast to man, where increased physical activity minimizes the differences in the oxidative potentials between ST, FTa and FTb fibres (Henriksson and Reitman, 1976; Jansson and Kaijser, 1977), the same ratio still exists between the fibre types (ST/FOG, ST/FG and F O G / F G ) in rat skeletal muscle after endurance training (Baldwin et al., 1972). Thus, the basis for the use of a metabolic differentiation between fibres in human skeletal muscle is limited.
Subgrouping of Fast Twitch Muscle Fibres
85
Why there is this difference between species in metabolic differentiation is not immediately apparent. Several factors may contribute. One is that skeletal muscles of man contain high proportions of both major fibre types (Johnson et al., 1973), whereas in many species a preponderance of one fibre is frequently found (Ariano et al., 1973). Another important difference is that both FOG fibres and ST fibres of species such as rats (Armstrong et al., 1974), guinea pigs (Edgerton et al., 1970) and lions (Armstrong et al., 1977) appear to be recruited at very low tension developments. In man, under comparable contractile conditions, only ST fibres are depleted of glycogen, suggesting that this fibre type is the only one to be recruited (Gollnick et al., 1973). Thus the specialization in the function of the muscles and the easy recruitment of the F O G fibres in many other species than man may be the explanation for the less distinct metabolic differentiation of FT fibres in skeletal muscle of man. In this connection some recent findings by Spamer and Pette (1977) are of note. They determined enzyme activities in single fibres microdissected from freeze-dried muscle of rabbits and found variability of up to 14 fold in malate dehydrogenase activity, representing a continuum, in FT fibres within the same muscle. This prompted these investigators to conclude that muscle fibre classification according to qualitative enzymological evaluations is unsatisfactory. Whether these results are indicative for other animal species (rats, guinea pigs, lions etc.) is not presently known. The use of the different pH labilities of the myofibrillar ATPase reaction a pH 4.6 for a subdivision of FT fibres gives two distinct populations with an insignificant number of intermediate stained fibres (Table 1; Brooke and Kaiser, 1974). There is however, a drawback with this method. It is not known what the differences in pH lability reflect. Brooke and Kaiser (1970) have suggested that it is related to differences in sulfhydryl groups of the myosin molecule. Furthermore, in an attempt to evaluate possible differences in FTa and FTb fibres, Henriksson and Pette (unpublished observations) have not noted differences in these fibres on the basis of preliminary studies using polyacrylamide gel electrophoresis. These points should not distract from the fact that the subsivision of FT muscle fibres in man according to pH lability appears to have strong physiological importance. Thus the cross sectional fibre area of the FTa fibres is larger than than that of the FTb fibres (Saltin et al., 1977). Further, the FTa and FTb fibres appear to be selectively recruited both in isometric and dynamic exercise as indicated by the glycogen depletion method (Andersen and Sjogaard, 1976; Secher and Nygaard, 1976; Ess6n, 1978). There are also differences in the mean values for oxidative and glycolytic enzymes comparing FTa and FTb fibres, although there is a substantial overlap in enzyme levels (Ess6n et al., 1975) which can be observed by microphotometric evaluation of histochemical stains for N A D H - T R and e-GPD (Halkj~er-Kristensen and Ingemann-Hansen, 1978). These latter findings are in agreement with the previously discussed minor distinction in the metabolic profiles between muscle fibre types, and also reinforces our contention that the histochemical stains for oxidative or glycolytic enzymes provide a poor bais for the subgrouping of FT fibres in man.
86
G. Sjogaard et aI.
Acknowledgement. This study was performed with economical support from the Danish Natural Science Foundation.
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Johnson, M.A., Polgar, J., Weightman, D., Appleton, D.: Data on the distribution of fibre types in thirty-six human muscles. An autopsy study. J. Neurol. Sci. 18, 111 129 (1973) Novikoff, A.B., Shin, W., Drucker, J.: Mitochondrial localization of oxidation enzymes: Staining results with two tetrazolium salts. J. Biophys. Biochem. Cytol. 9, 47 61 (1961) Padykula, H.A., Herman, E. : The specificity of the histochemical method for adenosine triphosphotase. J. Histochem. Cytochem. 3, 170-195 (1955) Peter, J.B., Barnard, R.J., Edgerton, V.R., Gillespie, C.A., Stempel, K.E.: Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits. Biochemistry 11, 2627-2633 (1972) Prince, F.P., Hikida, R.S., Hagerman, F.C.: Human muscle fibre types in power lifters, distance runners and untrained subjects. Pflfigers Arch. 363, 19 26 (1976) Saltin, B., Henriksson, J., Nyggard, E., Andersen, P.: Fiber types and metabolic potentials of skeletal muscles in sedentary man and endurance runners. Ann. N.Y. Acad. Sci. 301, 3-29 (1977) Secher, N., Nygaard-Jensen, E. : Glycogen depletion pattern in types I, IIA and IIB muscle fibres during maximal voluntary static and dynamic exercise. Acta physiol, scand. Suppl. 440, 287 (1976) Spamer, C., Pette, D. : Activity patterns of phosphofructokinase, glyceraldehyde phosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle. Histochemistry 52, 201-216 (1977) Taylor, A.W., Ess~n, B., Saltin, B.: Myosin ATPase in skeletal muscle of healthy man. Acta physiol, scand. 91, 568 570 (1974) Wattenberg, L.W., Leong, J.L. : Effects of coenzyme Q 10 and menadione on succinate dehydrogenase activity as measured by tetrazolium salt reduction. J. Histochem. Cytochem. 8, 296-303 (1960) Received June 13, 1978
