Fiber Size Distribution in the Flexor Carpi Radialis Muscle of the Cat WILLIAM J. GONYEA Department of Cell Biology, The University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75235
ABSTRACT The pennate flexor carpi radialis muscle of the cat has been shown to have a compartmentalized distribution of muscle fiber types. The possibility exists that the oxidative regions of the FCR are primarily concerned with fine movements including postural adjustments, while fast-twitch glycolytic region is used more for phasic movements. Muscle fiber diameters were measured for all three fiber types to determine if there was a compartmentalization of fiber size that might reflect differences in motor performance between the different regions of the same muscle. This study has demonstrated that the oxidative fiber types are significantly larger in the oxidative compartment when compared with the same fiber types in the glycolytic compartment and that the cross-sectional area of the oxidative region is dominated by oxidative fibers. The glycolytic fibers are significantly larger in the glycolytic compartment when compared to those in the oxidative region of the muscle and they constitute the major fiber type in this region. It has been demonstrated recently that the cat flexor carpi radialis (FCR), a pennate wrist flexor muscle, contains 37%slow-twitch fibers and 63%fast-twitch fibers (Gonyea and Ericson, '77; Gonyea and Bonde-Petersen, '77). The slow-twitch oxidative fibers (SO) were found to be concentrated in the deep region of the muscle located between the origin and insertion tendons. In addition, the fasttwitch glycolytic fibers (FG) were concentrated more peripherally. Muscle spindles were associated with the SO region and were never found in the region containing high concentrations of FG fibers (Gonyea and Ericson, '77). The association of spindles with muscles, or muscle regions, rich in oxidative fibers and the lack of spindles in regions rich in FG fibers has now been demonstrated in a number of muscles from several different mammalian species (Cooper and Daniel, '49; Swett and Eldred, '60; Yellin, '69; Richmond and Abrahams, '75a,b; Maier et al., '76; Gonyea and Ericson, '77). Based on the association between muscle spindles and oxidative muscle fibers and electrophysiological studies which have emphasized that oxidative motor units have low reflex thresholds, Botterman et al. ('78) have postulated that this association ocANAT. REC. (1979) 195: 447-454.
curs when a muscle is involved in vernier contractions including those associated with postural adjustments. Conversely, a high percentage of FG fibers and a low spindle density would be expected in muscles whose use is restricted to relatively coarse and powerful movements. Given that the FCR muscle is compartmentalized into distinct regions, the possibility exists, then, that the slow and fast regions can be called upon to perform quite different tasks. It is known that muscle fibers respond to an increased work load by increasing their size (hypertrophy) (Gonyea and Ericson, '76; Edgerton, '76). In the FCR, i t might be expected that in the oxidative compartment the oxidative fibers (SO and fast-twitch oxidative glycolytic-FOG) would be larger than in other regions of the muscle reflecting their preferential recruitment for postural adjustments and vernier contractions. In the glycolytic compartment, the FG fibers may exhibit differences in functional demand by being larger than those found in the oxidative compartment, for this region of the muscle should be primarily involved in powerful movements. Received Oct. 3. "78. Accepted May 16, '79.
447
448
WILLIAM J. GONYEA
This hypothesis assumes that the muscle fiber distribution of a motor unit is restricted, in most cases, to a single compartment. The object of this study was to examine the size distribution of fiber types in the cat FCR muscle to determine if this distribution supports the hypothesis that differences in the SO and FG regions might reflect a compartmentalization of motor performance within the same muscle. If there is no compartmentalization of muscle function or if motor units are not restricted to a single compartment, then i t would be expected that there would be no significant difference in fiber area. Alternatively, fibers located close to the muscle tendons may have a smaller cross-sectional area than fibers located a t the periphery. This could result from muscle fibers having a fusiform shape instead of being cylindrical; hence those fibers close to their origin or insertion would not be cut in the region that would represent their greatest cross-sectional area. MATERIALS AND METHODS
Left and right FCR muscles were obtained from 10 cats of both sexes (body weight = 3.70 2 0.20 kg) after intraperitoneal injection of Nembutal anesthesia (35 mg/kg). All muscles were removed in toto, cleaned of extraneous tissue including fat, connective tissue and all tendon not directly receiving muscle fibers. The muscles were then weighed immediately. As previously described (Gonyea and Ericson, '771, the muscles were prepared for histochemical examination by removing a 1-cm segment containing the greatest girth of the muscle belly. Each segment was mounted in tragacanth gum on a cork disc and rapidly frozen by immersion in Freon that was cooled with liquid nitrogen. Transverse serial sections were cut a t 8 p m on a cryostat ( - 20°C) and mounted on coverslips by thawing and then processed using the following techniques: The methods of alkaline- and acid-stable ATPase (pH 10.4 and 4.3) (Guth and Samaha, '701, reduced nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR; Novikoff et al., '61), succinate dehydrogenase (SDH; modification of Pearse, '72), menadionelinked a-glycerophosphate dehydrogenase (a-GPD) (Wattenberg and Long, '60) and a Gomori trichrome for general muscle morphology. Using these procedures muscle fibers were classified according to Peter et al. ('72) as slow-twitch oxidative (SO), fast-twitch oxi-
dative glycolytic (FOG), or fast-twitch glycolytic (FG). The FCR can be divided into 6 fields based on t h e internal tendonous architecture (Gonyea and Ericson, '77). Muscle fiber diameters were measured according to Brooke ('73) from predetermined areas from each of the 6 fields. Each area was photographed and every muscle fiber in the photograph was typed and measured. Hence, the number of muscle fibers measured in each area for each fiber type was dependent on their size and distribution. Fiber diameters were compared between each of the 6 muscle fields for each fiber type using a nested analysis of variance for unequal sample size. When the F ratio was significant (p < 0.051, the Student-Newman-Keuls (SNK) test was used to determine which sample means differed significantly (Sokal and Rohlf, '69). The estimated proportion of muscle represented by a given fiber type in each field was calculated by multiplying the average diameter of each fiber type by the percentage of fibers of that type within each field and dividing this by the sum of cross-sectional area of all three fiber types (Armstrong et al., '77). RESULTS
The fiber types were not evenly distributed in the FCR, but were found to be concentrated into three regions: fast, intermediate, and slow, each having a significantly different fiber type concentration than the other regions (table 1) and this was similar to that found in our previous study (Gonyea and Ericson, '77). The oxidative fibers (SO and FOG) were found to be concentrated in the slow and intermediate regions and formed an oxidative compartment. The FG fibers were concentrated in the fast region which formed a glycolytic compartment (table l). In this study, i t was felt that the heterogeneity in fiber-type distribution made i t necessary to measure fiber diameters for all six fields to determine if there was a pattern in fiber size distribution for the three fiber types, and if the size distribution corresponded to the three distinct fiber type regions. It can be observed in figure 1 that the fiber diameters do differ from one field to another. In testing the fiber diameters i t was determined, using an analysis of variance, that there were significant differences in fiber diameters for each fiber type when compared between the six fields (F = 466.1 p < 0.001). In addition, the
449
MUSCLE FIBER SIZE TABLE 1
Fiber composition o f t h e FCR Fiber type Compartment Reaon
so
Field
1 Populations, % Proportion of area, % X (N) 3 Populations, % Proportion of area, % X (N) 4 Populations, % Proportion of area, %
Glycolytic
Fast
Oxidative
Intermediate
5 Populations, % Proportion of area, %
Slow
2 Populations, % Proportion of area, %
X (N)
X (N) X (N) 6 Populations, % Proportion of area, % X (N)
FOG
FG
24.1 f 1.92 I 18.5 33.4 (459) 23.2 f 1.92 18.0 34.8 (367) 23.8 f 1.85 18.4 35.9 (407)
30.3 f 2.27 28.0 40.0 (552) 26.6 f 2.24 23.8 40.3 (487) 28.9 f 3.22 25.6 41.0 (518)
45.6 f 2.76 53.5 50.8 (511) 48.2 2 2.39 58.2 54.3 (751) 47.3 f 2.99 56.0 54.8 (766)
37.5 f 1.57 33.3 39.8 (715) 49.4 2 2.08 45.4 38.1 (1076) 52.5 -t 2.16 49.3 37.1 (1212)
31.8 t 2.47 30.4 42.9 (624) 26.5 2 2.84 26.1 40.8 (605) 22.5 f 2.40 21.4 37.5 (565)
30.8 t 2.43 36.3 52.8 (585) 24.1 f 1.86 28.5 49.1 (498) 25.0 f 1.95 29.3 46.4 (636)
' Values are means 2 S.E.
'X (Nl
=
mean (sample size)
fiber diameters differed significantly when compared between the three fiber types (F = 7.962 p < 0.005). The mean fiber diameters were then compared for all six fields (table 1). The slow-twitch region was found to contain 75%oxidative fibers (SO and FOG) while the intermediate region contained 68%oxidative fibers. However, the fast-twitch region of the FCR contained only 50%oxidative fibers. Muscle fibers were measured in areas adjacent to the origin and insertion tendons in fields 6 and 2 respectively. Instead of these fields containing the smallest SO fibers they contained some of the largest (table 1). The largest SO muscle fibers (X = 39.8 f 0 . 3 0 7 ~ )were located in the compartments that have a high oxidative fiber ratio (fields 5 , 2 and 6). The SO fibers located in the high oxidative regions were significantly larger than the SO fibers located in the fast-twitch region. The smallest SO fibers (X = 33.4 f 0 . 3 7 2 ~ )were not located near one of the tendons but a t the periphery of the muscle in field No. l. In fact, the SO fibers located around the periphery of the muscle (fast-twitchregion) were significantly smaller than those located in the oxidative compartment (table 1). The largest FOG fibers (field No. 5 , %= 42.9 f 0.366 p ) were located in the oxidative compartment. However, the smallest FOG fibers
were also located in this compartment (field , were closely No. 6, X = 37.5 f 0 . 3 1 4 ~ 1but associated with the origin tendon. The FOG fibers located in the glycolytic compartment exhibited little size variation between fields. The largest FG fibers were located in the fast-twitch region in field No. 4 (X = 54.8 f 0 . 4 5 0 ~ )The . FG fibers located in the fasttwitch region were significantly larger than those found in the slow-twitch region (table 1). The FG fibers located in the intermediate region were also found to be significantly larger than those in the slow-twitch region even though these fibers were in the oxidative compartment. However, these fibers were generally smaller than those found in the fasttwitch region. The distribution of fiber types in each field tended to overestimate the importance of SO and FOG fibers and underestimate the contribution of FG fibers to the cross-sectional area of each field (table 1). This was due to the FG fibers having the largest cross-sectional area of all three fiber types (table 1). Nevertheless, the slow and intermediate regions of the FCR were dominated, based on fiber proportions, by oxidative fibers. In these two regions, the FG fibers constitute only 30.8% (intermediate) and 28.8% (slow) of the estimated cross-sectional area, while the cross-sectional area of
450
WILLIAM J. GONYEA
the fast region was dominated by the FG fibers (55.9%). DISCUSSION AND SUMMARY
Although the FCR is a mixed muscle in terms of fiber types, the distribution of the fiber types was found not to be uniform (Gonyea and Ericson, '77). In fact, the SO fibers were found to be concentrated near one surface of the muscle while the FG fibers were concentrated near the other surface. It has also been shown that the muscle spindles were always associated with the regions high in oxidative fibers (in the slow and intermediate regions) and were never associated with the region containing a high concentration of FG fibers (glycolytic compartment). We originally postulated that the association of muscle spindles with oxidative fibers and the differential distribution of muscle fibers into oxidative and glycolytic compartments might allow these regions to function independently of one another when called upon to perform different tasks (Gonyea and Ericson, '77). Botterman et al. ('78) have suggested that the compartmentalization of fiber types in mixed muscles and the association of muscle spindles with those regions rich in oxidative fibers reflect a strong structure-function relationship. In this regard, it has been postulated that the region of a mixed muscle with a high oxidative index and spindle density would be involved in vernier contractions including those associated with postural adjustments. Conversely, regions with a high percentage of FG fibers and a low spindle density would be restricted to relatively coarse and powerful movements (Botterman et al., '78). This study has demonstrated that in the FCR muscle there is a significant difference in muscle fiber diameters when compared between the different compartments. The smallest SO fibers were located in the glycolytic compartment while the largest SO fibers were located in the oxidative compartment. The largest FOG fibers were also located in the oxidative compartment while those that were located in the glycolytic compartment were generally smaller in size. The largest FG fibers were found in the glycolytic compartment and the smallest FG fibers were found in the oxidative compartment. One possibility for the significant differences in fiber size distribution for each fiber type may be that muscle units, especially SO and FG units, are restricted to either the oxi-
dative or glycolytic compartment. However, a t present there is no physiological evidence to support this hypothesis, and the definitive studies of the function of this muscle must await further analysis. This study has also suggested that the structure-function relationship between oxidative fibers and muscle spindles may also be extended to include skeletal muscle fiber size. ACKNOWLEDGMENTS
I extend my appreciation to Ms. Sharon Marushia and Mary Banks for their technical assistance and to Ms. Billie Price for typing the manuscript. I would also like t o extend my appreciation to Dr. Carl Saubert for his suggestion to use muscle fiber contributions to cross-sectional area. This work was supported by Grant 2-R01-AM1761504from the National Institute of Arthritis, Metabolism, and Digestive Diseases. LITERATURE CITED Armstrong, R. B., P. Marum, C. W. Saubert, IV, H. J. Seeherman and C. R. Taylor 1977 Muscle fiber activity as a function of speed and gait. J. Appl. Physiol., 43: 672-677. Botterman, B. R., M. D. Binder and D. G. Stuart 1978 Functional anatomy of the association between motor units and muscle receptors. Am. Zool., 18: 135-152. Brooke, M. H. 1973 The pathologic interpretationof muscle histochemistry. In: The Striated Muscle. C. M. Pearson and K. F. Mostofi, eds. Baltimore, Maryland, Williams and Wilkins, pp. 86-122. Cooper, S.,and P. M. Daniel 1949 Muscle spindles in human extrinsic eye muscles. Brain, 72: 1-24. Edgerton, V. R. 1976 Neuromuscular adaptation to power and endurance work. Can. J. Appl. Sport Sci., 1: 49-58. Gonyea, W., and F. Bonde-Petersen 1977 Contraction properties and fiber types of some fore- and hind limb muscles in the cat. Exp. Neurol., 57: 637-644. Gonyea, W. J., and G. C. Ericson 1976 An experimental model for the study of exercised-induced skeletal muscle hypertrophy. J. Appl. Physiol., 40(4): 630-633. 1977 Morphological and histochemical organization of t h e flexor carpi radialis muscle in the cat. Am. J. Anat., 148: 329-344. Guth, L., and F. J. Samaha 1970 Procedure for the histochemical demonstration of actomyosin ATPase. Exp. Neurol., 28: 365-367. Maier, A,, D. R. Simpson and V. R. Edgerton 1976 Histological and histochemical comparisons of muscle spindles in three hind limb muscles of the guinea pig. J. Morph., 148: 185-192. Novikoff, A. B., W. Y. Shin and J. Drucker 1961 Mitochondrial localization of oxidative enzymes: Staining results with two tetrazolium salts. J. Biophys. Biochem. Cytol., 9: 47-61. Pearse, E. 1972 Histochemistry. Williams and Wilkins Company, Baltimore. Peter, J. B., R. J. Barnard, V. R. Edgerton, C. A. Gillespie and K. F. Stempel 1972 Metabolic profiles of three fibre types of skeletal muscle in guinea pigs and rabbits. Biochemistry, 11: 2627-2633.
MUSCLE FIBER SIZE Richmond, F. J. R., and V. C. Abrahams 1975a Morphology and enzyme histochemistry of dorsal muscles of the cat neck. J. Neurophysiol., 38: 1312-1321. 19751, Morphology and distribution of muscle spindles in dorsal muscles of the cat neck. J. Neurophysiol., 38: 1322-1339. Sokal. R. R., and F. J. Rohlf 1969 Biometry. W. H. Freeman & Co., San Francisco, 776 pp. Swett, J. E., and E. Eldred 1960 Distribution and number of
451
stretch receptors in medial gastrocnemius and soleus muscles. Anat. Rec., 137: 453-460. Wattenberg, L. W., and J. L. Long 1960 Effects of coenzyme Q 10 and menadione on succinate dehydrogenase activity as measured by tetrazolium salt reaction. J. Histochem. Cytochem., 8: 296-303. Yellin, H. 1969 A histochemical study of muscle spindles and their relationship to extrafusal fiber types in the rat. Am. J. Anat., 125: 31-46.
PLATE I EXPLANATION OF FIGURES
1 Cross sections of the FCR processed histochemically to demonstrate the differences in fiber type distribution and fiber cross-sectional area in the different compartments of this muscle. x 270. Oxidative compartment A ATPase, alkaline preincubation C NADH-TR activity
452
Glycolytic compartment B ATPase, alkaline preincubation D NADH-TR activity
MUSCLE FIBER SIZE William J. Gonyea
PLATE 1
453
ABSTRACT The pennate flexor carpi radialis muscle of the cat has been shown to have a compartmentalized distribution of muscle fiber types. The possibility exists that the oxidative regions of the FCR are primarily concerned with fine movements including postural adjustments, while fast-twitch glycolytic region is used more for phasic movements. Muscle fiber diameters were measured for all three fiber types to determine if there was a compartmentalization of fiber size that might reflect differences in motor performance between the different regions of the same muscle. This study has demonstrated that the oxidative fiber types are significantly larger in the oxidative compartment when compared with the same fiber types in the glycolytic compartment and that the cross-sectional area of the oxidative region is dominated by oxidative fibers. The glycolytic fibers are significantly larger in the glycolytic compartment when compared to those in the oxidative region of the muscle and they constitute the major fiber type in this region. It has been demonstrated recently that the cat flexor carpi radialis (FCR), a pennate wrist flexor muscle, contains 37%slow-twitch fibers and 63%fast-twitch fibers (Gonyea and Ericson, '77; Gonyea and Bonde-Petersen, '77). The slow-twitch oxidative fibers (SO) were found to be concentrated in the deep region of the muscle located between the origin and insertion tendons. In addition, the fasttwitch glycolytic fibers (FG) were concentrated more peripherally. Muscle spindles were associated with the SO region and were never found in the region containing high concentrations of FG fibers (Gonyea and Ericson, '77). The association of spindles with muscles, or muscle regions, rich in oxidative fibers and the lack of spindles in regions rich in FG fibers has now been demonstrated in a number of muscles from several different mammalian species (Cooper and Daniel, '49; Swett and Eldred, '60; Yellin, '69; Richmond and Abrahams, '75a,b; Maier et al., '76; Gonyea and Ericson, '77). Based on the association between muscle spindles and oxidative muscle fibers and electrophysiological studies which have emphasized that oxidative motor units have low reflex thresholds, Botterman et al. ('78) have postulated that this association ocANAT. REC. (1979) 195: 447-454.
curs when a muscle is involved in vernier contractions including those associated with postural adjustments. Conversely, a high percentage of FG fibers and a low spindle density would be expected in muscles whose use is restricted to relatively coarse and powerful movements. Given that the FCR muscle is compartmentalized into distinct regions, the possibility exists, then, that the slow and fast regions can be called upon to perform quite different tasks. It is known that muscle fibers respond to an increased work load by increasing their size (hypertrophy) (Gonyea and Ericson, '76; Edgerton, '76). In the FCR, i t might be expected that in the oxidative compartment the oxidative fibers (SO and fast-twitch oxidative glycolytic-FOG) would be larger than in other regions of the muscle reflecting their preferential recruitment for postural adjustments and vernier contractions. In the glycolytic compartment, the FG fibers may exhibit differences in functional demand by being larger than those found in the oxidative compartment, for this region of the muscle should be primarily involved in powerful movements. Received Oct. 3. "78. Accepted May 16, '79.
447
448
WILLIAM J. GONYEA
This hypothesis assumes that the muscle fiber distribution of a motor unit is restricted, in most cases, to a single compartment. The object of this study was to examine the size distribution of fiber types in the cat FCR muscle to determine if this distribution supports the hypothesis that differences in the SO and FG regions might reflect a compartmentalization of motor performance within the same muscle. If there is no compartmentalization of muscle function or if motor units are not restricted to a single compartment, then i t would be expected that there would be no significant difference in fiber area. Alternatively, fibers located close to the muscle tendons may have a smaller cross-sectional area than fibers located a t the periphery. This could result from muscle fibers having a fusiform shape instead of being cylindrical; hence those fibers close to their origin or insertion would not be cut in the region that would represent their greatest cross-sectional area. MATERIALS AND METHODS
Left and right FCR muscles were obtained from 10 cats of both sexes (body weight = 3.70 2 0.20 kg) after intraperitoneal injection of Nembutal anesthesia (35 mg/kg). All muscles were removed in toto, cleaned of extraneous tissue including fat, connective tissue and all tendon not directly receiving muscle fibers. The muscles were then weighed immediately. As previously described (Gonyea and Ericson, '771, the muscles were prepared for histochemical examination by removing a 1-cm segment containing the greatest girth of the muscle belly. Each segment was mounted in tragacanth gum on a cork disc and rapidly frozen by immersion in Freon that was cooled with liquid nitrogen. Transverse serial sections were cut a t 8 p m on a cryostat ( - 20°C) and mounted on coverslips by thawing and then processed using the following techniques: The methods of alkaline- and acid-stable ATPase (pH 10.4 and 4.3) (Guth and Samaha, '701, reduced nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR; Novikoff et al., '61), succinate dehydrogenase (SDH; modification of Pearse, '72), menadionelinked a-glycerophosphate dehydrogenase (a-GPD) (Wattenberg and Long, '60) and a Gomori trichrome for general muscle morphology. Using these procedures muscle fibers were classified according to Peter et al. ('72) as slow-twitch oxidative (SO), fast-twitch oxi-
dative glycolytic (FOG), or fast-twitch glycolytic (FG). The FCR can be divided into 6 fields based on t h e internal tendonous architecture (Gonyea and Ericson, '77). Muscle fiber diameters were measured according to Brooke ('73) from predetermined areas from each of the 6 fields. Each area was photographed and every muscle fiber in the photograph was typed and measured. Hence, the number of muscle fibers measured in each area for each fiber type was dependent on their size and distribution. Fiber diameters were compared between each of the 6 muscle fields for each fiber type using a nested analysis of variance for unequal sample size. When the F ratio was significant (p < 0.051, the Student-Newman-Keuls (SNK) test was used to determine which sample means differed significantly (Sokal and Rohlf, '69). The estimated proportion of muscle represented by a given fiber type in each field was calculated by multiplying the average diameter of each fiber type by the percentage of fibers of that type within each field and dividing this by the sum of cross-sectional area of all three fiber types (Armstrong et al., '77). RESULTS
The fiber types were not evenly distributed in the FCR, but were found to be concentrated into three regions: fast, intermediate, and slow, each having a significantly different fiber type concentration than the other regions (table 1) and this was similar to that found in our previous study (Gonyea and Ericson, '77). The oxidative fibers (SO and FOG) were found to be concentrated in the slow and intermediate regions and formed an oxidative compartment. The FG fibers were concentrated in the fast region which formed a glycolytic compartment (table l). In this study, i t was felt that the heterogeneity in fiber-type distribution made i t necessary to measure fiber diameters for all six fields to determine if there was a pattern in fiber size distribution for the three fiber types, and if the size distribution corresponded to the three distinct fiber type regions. It can be observed in figure 1 that the fiber diameters do differ from one field to another. In testing the fiber diameters i t was determined, using an analysis of variance, that there were significant differences in fiber diameters for each fiber type when compared between the six fields (F = 466.1 p < 0.001). In addition, the
449
MUSCLE FIBER SIZE TABLE 1
Fiber composition o f t h e FCR Fiber type Compartment Reaon
so
Field
1 Populations, % Proportion of area, % X (N) 3 Populations, % Proportion of area, % X (N) 4 Populations, % Proportion of area, %
Glycolytic
Fast
Oxidative
Intermediate
5 Populations, % Proportion of area, %
Slow
2 Populations, % Proportion of area, %
X (N)
X (N) X (N) 6 Populations, % Proportion of area, % X (N)
FOG
FG
24.1 f 1.92 I 18.5 33.4 (459) 23.2 f 1.92 18.0 34.8 (367) 23.8 f 1.85 18.4 35.9 (407)
30.3 f 2.27 28.0 40.0 (552) 26.6 f 2.24 23.8 40.3 (487) 28.9 f 3.22 25.6 41.0 (518)
45.6 f 2.76 53.5 50.8 (511) 48.2 2 2.39 58.2 54.3 (751) 47.3 f 2.99 56.0 54.8 (766)
37.5 f 1.57 33.3 39.8 (715) 49.4 2 2.08 45.4 38.1 (1076) 52.5 -t 2.16 49.3 37.1 (1212)
31.8 t 2.47 30.4 42.9 (624) 26.5 2 2.84 26.1 40.8 (605) 22.5 f 2.40 21.4 37.5 (565)
30.8 t 2.43 36.3 52.8 (585) 24.1 f 1.86 28.5 49.1 (498) 25.0 f 1.95 29.3 46.4 (636)
' Values are means 2 S.E.
'X (Nl
=
mean (sample size)
fiber diameters differed significantly when compared between the three fiber types (F = 7.962 p < 0.005). The mean fiber diameters were then compared for all six fields (table 1). The slow-twitch region was found to contain 75%oxidative fibers (SO and FOG) while the intermediate region contained 68%oxidative fibers. However, the fast-twitch region of the FCR contained only 50%oxidative fibers. Muscle fibers were measured in areas adjacent to the origin and insertion tendons in fields 6 and 2 respectively. Instead of these fields containing the smallest SO fibers they contained some of the largest (table 1). The largest SO muscle fibers (X = 39.8 f 0 . 3 0 7 ~ )were located in the compartments that have a high oxidative fiber ratio (fields 5 , 2 and 6). The SO fibers located in the high oxidative regions were significantly larger than the SO fibers located in the fast-twitch region. The smallest SO fibers (X = 33.4 f 0 . 3 7 2 ~ )were not located near one of the tendons but a t the periphery of the muscle in field No. l. In fact, the SO fibers located around the periphery of the muscle (fast-twitchregion) were significantly smaller than those located in the oxidative compartment (table 1). The largest FOG fibers (field No. 5 , %= 42.9 f 0.366 p ) were located in the oxidative compartment. However, the smallest FOG fibers
were also located in this compartment (field , were closely No. 6, X = 37.5 f 0 . 3 1 4 ~ 1but associated with the origin tendon. The FOG fibers located in the glycolytic compartment exhibited little size variation between fields. The largest FG fibers were located in the fast-twitch region in field No. 4 (X = 54.8 f 0 . 4 5 0 ~ )The . FG fibers located in the fasttwitch region were significantly larger than those found in the slow-twitch region (table 1). The FG fibers located in the intermediate region were also found to be significantly larger than those in the slow-twitch region even though these fibers were in the oxidative compartment. However, these fibers were generally smaller than those found in the fasttwitch region. The distribution of fiber types in each field tended to overestimate the importance of SO and FOG fibers and underestimate the contribution of FG fibers to the cross-sectional area of each field (table 1). This was due to the FG fibers having the largest cross-sectional area of all three fiber types (table 1). Nevertheless, the slow and intermediate regions of the FCR were dominated, based on fiber proportions, by oxidative fibers. In these two regions, the FG fibers constitute only 30.8% (intermediate) and 28.8% (slow) of the estimated cross-sectional area, while the cross-sectional area of
450
WILLIAM J. GONYEA
the fast region was dominated by the FG fibers (55.9%). DISCUSSION AND SUMMARY
Although the FCR is a mixed muscle in terms of fiber types, the distribution of the fiber types was found not to be uniform (Gonyea and Ericson, '77). In fact, the SO fibers were found to be concentrated near one surface of the muscle while the FG fibers were concentrated near the other surface. It has also been shown that the muscle spindles were always associated with the regions high in oxidative fibers (in the slow and intermediate regions) and were never associated with the region containing a high concentration of FG fibers (glycolytic compartment). We originally postulated that the association of muscle spindles with oxidative fibers and the differential distribution of muscle fibers into oxidative and glycolytic compartments might allow these regions to function independently of one another when called upon to perform different tasks (Gonyea and Ericson, '77). Botterman et al. ('78) have suggested that the compartmentalization of fiber types in mixed muscles and the association of muscle spindles with those regions rich in oxidative fibers reflect a strong structure-function relationship. In this regard, it has been postulated that the region of a mixed muscle with a high oxidative index and spindle density would be involved in vernier contractions including those associated with postural adjustments. Conversely, regions with a high percentage of FG fibers and a low spindle density would be restricted to relatively coarse and powerful movements (Botterman et al., '78). This study has demonstrated that in the FCR muscle there is a significant difference in muscle fiber diameters when compared between the different compartments. The smallest SO fibers were located in the glycolytic compartment while the largest SO fibers were located in the oxidative compartment. The largest FOG fibers were also located in the oxidative compartment while those that were located in the glycolytic compartment were generally smaller in size. The largest FG fibers were found in the glycolytic compartment and the smallest FG fibers were found in the oxidative compartment. One possibility for the significant differences in fiber size distribution for each fiber type may be that muscle units, especially SO and FG units, are restricted to either the oxi-
dative or glycolytic compartment. However, a t present there is no physiological evidence to support this hypothesis, and the definitive studies of the function of this muscle must await further analysis. This study has also suggested that the structure-function relationship between oxidative fibers and muscle spindles may also be extended to include skeletal muscle fiber size. ACKNOWLEDGMENTS
I extend my appreciation to Ms. Sharon Marushia and Mary Banks for their technical assistance and to Ms. Billie Price for typing the manuscript. I would also like t o extend my appreciation to Dr. Carl Saubert for his suggestion to use muscle fiber contributions to cross-sectional area. This work was supported by Grant 2-R01-AM1761504from the National Institute of Arthritis, Metabolism, and Digestive Diseases. LITERATURE CITED Armstrong, R. B., P. Marum, C. W. Saubert, IV, H. J. Seeherman and C. R. Taylor 1977 Muscle fiber activity as a function of speed and gait. J. Appl. Physiol., 43: 672-677. Botterman, B. R., M. D. Binder and D. G. Stuart 1978 Functional anatomy of the association between motor units and muscle receptors. Am. Zool., 18: 135-152. Brooke, M. H. 1973 The pathologic interpretationof muscle histochemistry. In: The Striated Muscle. C. M. Pearson and K. F. Mostofi, eds. Baltimore, Maryland, Williams and Wilkins, pp. 86-122. Cooper, S.,and P. M. Daniel 1949 Muscle spindles in human extrinsic eye muscles. Brain, 72: 1-24. Edgerton, V. R. 1976 Neuromuscular adaptation to power and endurance work. Can. J. Appl. Sport Sci., 1: 49-58. Gonyea, W., and F. Bonde-Petersen 1977 Contraction properties and fiber types of some fore- and hind limb muscles in the cat. Exp. Neurol., 57: 637-644. Gonyea, W. J., and G. C. Ericson 1976 An experimental model for the study of exercised-induced skeletal muscle hypertrophy. J. Appl. Physiol., 40(4): 630-633. 1977 Morphological and histochemical organization of t h e flexor carpi radialis muscle in the cat. Am. J. Anat., 148: 329-344. Guth, L., and F. J. Samaha 1970 Procedure for the histochemical demonstration of actomyosin ATPase. Exp. Neurol., 28: 365-367. Maier, A,, D. R. Simpson and V. R. Edgerton 1976 Histological and histochemical comparisons of muscle spindles in three hind limb muscles of the guinea pig. J. Morph., 148: 185-192. Novikoff, A. B., W. Y. Shin and J. Drucker 1961 Mitochondrial localization of oxidative enzymes: Staining results with two tetrazolium salts. J. Biophys. Biochem. Cytol., 9: 47-61. Pearse, E. 1972 Histochemistry. Williams and Wilkins Company, Baltimore. Peter, J. B., R. J. Barnard, V. R. Edgerton, C. A. Gillespie and K. F. Stempel 1972 Metabolic profiles of three fibre types of skeletal muscle in guinea pigs and rabbits. Biochemistry, 11: 2627-2633.
MUSCLE FIBER SIZE Richmond, F. J. R., and V. C. Abrahams 1975a Morphology and enzyme histochemistry of dorsal muscles of the cat neck. J. Neurophysiol., 38: 1312-1321. 19751, Morphology and distribution of muscle spindles in dorsal muscles of the cat neck. J. Neurophysiol., 38: 1322-1339. Sokal. R. R., and F. J. Rohlf 1969 Biometry. W. H. Freeman & Co., San Francisco, 776 pp. Swett, J. E., and E. Eldred 1960 Distribution and number of
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stretch receptors in medial gastrocnemius and soleus muscles. Anat. Rec., 137: 453-460. Wattenberg, L. W., and J. L. Long 1960 Effects of coenzyme Q 10 and menadione on succinate dehydrogenase activity as measured by tetrazolium salt reaction. J. Histochem. Cytochem., 8: 296-303. Yellin, H. 1969 A histochemical study of muscle spindles and their relationship to extrafusal fiber types in the rat. Am. J. Anat., 125: 31-46.
PLATE I EXPLANATION OF FIGURES
1 Cross sections of the FCR processed histochemically to demonstrate the differences in fiber type distribution and fiber cross-sectional area in the different compartments of this muscle. x 270. Oxidative compartment A ATPase, alkaline preincubation C NADH-TR activity
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Glycolytic compartment B ATPase, alkaline preincubation D NADH-TR activity
MUSCLE FIBER SIZE William J. Gonyea
PLATE 1
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