Distribution of myofiber types in the crural musculature of sheep

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1 Okajimas Folia Anat. Jpn., 89(2): 39 45, August, 2012 Distribution of myofiber types in the crural musculature of sheep By Toshihiro KONNO 1 and Kouichi WATANABE Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai , Japan Received for Publication, June 14, 2012 Key Words: crural muscles, myofiber types, postural maintenance, locomotion Summary: In domestic animals, the legs function in both postural maintenance and propulsion. The crural muscles participate in actions of the tarsal and toe joints. Mammalian skeletal muscles consist of myofibers, which are histochemically classified into three myofiber types, slow-twitch/oxidative (SO) or type I, fast-twitch/oxidative/glycolytic (FOG) or type IIA, and fast-twitch/ glycolytic (FG) or type IIB myofibers. The histochemical characteristics of myofiber types reflect an aspect of function that myofibers possess. In the present study, we investigated the composition and average diameter of myofiber types of each muscle in crus of sheep and determined their roles in the movement of tarsal and toe joints. The tibialis cranialis muscle was a flat unipennate muscle and not capable to generate a large tension; however, it could function primarily in posture maintenance and play a cooperative role in adjusting standing posture. The flexor hallucis longus and flexor digitorum superficialis muscles were the major muscles that contributed to posture maintenance in leg musculature. These muscles were capable to generate a large tension and participate primarily in standing posture maintenance. The composition and diameter of myofiber types in ovine crural musculature reflected the role of each muscle in posture maintenance and locomotion. Introduction In mammalian skeletal muscles, the composition of myofiber types differs from muscle to muscle. In hindlimbs, many muscles generating a propulsive force have numerous fast-twitch myofibers (type II), which show a strong alkali-stable myosin ATPase reaction, whereas the deeply situated muscles extending and stabilizing the joints contain many slow-twitch/oxidative (SO) myofibers (type I), which show a weak reactivity for alkali-stable myosin ATPase and a strong activity for NADH dehydrogenase (NADH-D) 1 4). Fast-twitch myofibers have been divided into two types: fast-twitch/ oxidative /glycolytic (FOG) or type IIA and fast-twitch/ glycolytic (FG) or type IIB myofibers, on basis of differences in NADH-D activities 5). Motor units composed of FOG (type IIA) myofibers are activated for moderate force output from walking to running and jumping. In addition, motor units of FG (type IIB) myofibers are recruited for burst contraction necessary for high force output, running and jumping. The tension for maintaining a standing posture is sustained by recruitments of motor units of SO (type I) myofibers, motor units of which are activated from standing to varying locomotion 6 9). Thus the histochemical characteristics of myofiber types appear to reflect an aspect of function that myofibers possess. In domestic animals, the legs functions in both postural maintenance and propulsion. The crural muscles participate in actions of the tarsal and toe joints. In sheep, the tibialis cranialis, peroneus tertius, and peroneus longus muscles flex the tarsal joint, whereas the gastrocnemius and soleus muscles extend the tarsal joint. The extensor digitorum longus and extensor digitorum lateralis muscles are the extensors of toe joints, and the tibialis caudalis, flexor hallucis longus, and flexor digitorum longus muscles are the flexors of toe joints. The flexor digitorum superficialis muscle extends the tarsal joint and flexes the toe joints. In the ovine thigh muscles, type I myofibers are distributed more in the deep portions, and the type IIA myofibers are distributed more in the cranial, caudolateral, Corresponding author: Kouichi Watanabe, Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai , Japan. Phone: ; Fax: ; watakoh@bios.tohoku.ac.jp Grants: a Grant-in-Aid for Scientific Research (C) (No and ) from the Japan Society for the Promotion of Science (JSPS). 1 Current address: Department of Animal Resource Sciences/ Veterinary Medical Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo , Japan.

2 40 T. Konno and K. Watanabe and caudomedial portions 4). Therefore, in the ovine thigh, type I myofibers are effectively distributed to maintain a standing posture without diminishing the propulsive force of hindlimb. A study of the distribution of myofiber types in the ovine leg musculature is necessary for better understanding of those functions in postural maintenance and propulsive force generation. The present study was undertaken to examine the composition of myofiber types of each crural muscle in sheep and to determine their roles in the movement of tarsal and toe joints. Materials and Methods Muscle samples Six adult female sheep (Corriedale, body weight 47.4 ± 6.3 kg) were used in this study. After slaughter, transverse sections (1 cm thick) were taken from the belly regions of the leg muscles. The animals were handled in accordance with the Guidelines of the Administrative Panel on Laboratory Animal Care of Tohoku University. The muscles sampled are shown in Table 1. The planes of transverse sections in the samples were trimmed specially to be perpendicular to the long axis of myofiber bundles. Samples were frozen in dry ice-cooled acetone and stored at 80 C. Cross sections (10 µm) were cut serially on a cryostat and mounted on microslides. Table 1. Muscle Crural muscles and their action on knee, tarsal, and toe joints Action Tibialis cranialis Flexor of tarsal joint Peroneus tertius Flexor of tarsal joint Peroneus longus Flexor of tarsal joint Extensor digitorum longus Extensor of toe joints Extensor digitorum lateralis Extensor of toe joints Triceps surae Extensors of tarsal joint (antigravity) Gastrocnemius, caput laterale Gastrocnemius, caput mediale Soleus Flexor digitorum superficialis Extensor of tarsal joint (antigravity), Flexor of toe joints (antigravity) Flexor digitorum profundus Flexors of toe joints (antigravity) Tibialis caudalis Flexor hallucis longus Flexor digitorum longus Popliteus Flexor of knee joint Enzyme histochemistry and myofiber typing Fresh cross sections were incubated for the demonstration of myosin ATPase after preincubation at ph 4.3 and at ph ). Serial sections were stained with NADH-D 11 13). The myofibers that reacted strongly to myosin ATPase after acid preincubation and were unreactive or weakly reactive to myosin ATPase after alkaline preincubation were classified as type I myofiber, whereas myofibers that were unreactive to myosin ATPase after acid preincubation and strongly reactive to myosin ATPase after alkaline preincubation were classified as type II myofiber 10 12). The myofibers that reacted moderately to strongly to myosin ATPase after both acid and alkaline preincubation were classified as intermediate type (Type Int.) myofibers. In adult sheep, type II myofibers were subdivided into type IIA myofiber, on the basis of moderate to strong reactivity to NADH-D and type IIB myofiber, on the basis of weak reactivity to NADH-D 5,12). Measurements Photomicrographs of cross sections stained with myosin ATPase after acid or alkaline preincubation were taken from two portions of each muscle sections and used to classify and count myofiber types. The mean percentages of myofiber types were calculated from the two portions of each muscle sections. The diameters of myofiber types were obtained by measuring their smallest dimension with an ocular micrometer. The mean diameters of myofiber types were then calculated from forty myofibers per myofiber types in each muscle. Statistical analyses The mean percentages and diameters of myofiber types were compared by analyses of variance followed by Tukey s contrasts tests. All statistical analysis was performed using R statistical packages ( org). Results Myofiber types Enzyme-histochemical profiles of myofiber types were showed in Fig. 1. Type I myofibers exhibited strong reactivity for acid-stable myosin ATPase and weak or no reactivity for alkali-stable myosin ATPase (Figs. 1a and 1b). Myosin ATPase reactivities of type II myofiber were reversal of type I myofibers (Figs 1a and 1b). In NADH-D activity, type I myofibers were strong, type IIA myofibers were moderate to strong, and type IIB myofibers were weak (Fig. 1c). Type Int. myofibers were reactive for both acid- and alkali-stable myosin ATPase (Fig. 1a, b). Composition of myofiber types The muscles that actioned on tarsal and toe joints as flexor and extensor, respectively, were located cranial to the tibia, whereas the extensor and flexor muscles of tarsal and toe joints, respectively, were located plantar to the tibia (Table 1 and Fig. 2). Among the muscles located cranial to the tibia, the tibialis cranialis muscle possessed a largest population of type I myofibers. In the muscles located plantar to the tibia, the flexor digitorum superficialis muscle contained more than 50% of type I myofibers, suggesting its role in the maintenance of standing

3 Functional involvement of ovine crural muscles 41 Fig. 1. Myofiber types in the peroneus longus muscle of sheep. a: Myosin ATPase reactivity after acid preincubation (ph 4.3). b: Myosin ATPase reactivity after alkaline preincubation (ph 10.5). c: NADH dehydrogenase activity. I: Type I myofiber. A: Type IIA myofiber. B: Type IIB myofiber. *: Type Int. myofiber. Bars = 20 μm. Table 2. Distribution of myofiber types in ovine crural muscles Muscle Percentages of myofiber types (%) I IIA IIB Int. Tibialis cranialis 54.1 ± 14.1 a 27.7 ± 11.9 b 17.6 ± 5.8 b 0.6 ± 1.3 c Peroneus tertius 27.0 ± 5.1 b 44.2 ± 8.5 a 28.4 ± 10.6 b 0.4 ± 0.7 c Peroneus longus 22.6 ± 6.1 b 44.7 ± 7.8 a 28.6 ± 6.3 b 4.1 ± 5.0 c Extensor digitorum longus 25.5 ± 6.4 b 46.6 ± 8.2 a 23.6 ± 6.2 b 4.3 ± 8.0 c Extensor digitorum lateralis 18.8 ± 3.9 b 44.1 ± 3.9 a 36.2 ± 3.4 a 0.9 ± 1.3 c Triceps surae Gastrocnemius, caput laterale 29.7 ± 3.8 b 40.6 ± 5.7 a 28.0 ± 3.8 b 1.6 ± 4.0 c Gastrocnemius, caput mediale 31.2 ± 9.7 b 46.0 ± 9.3 a 22.4 ± 6.7 b 0.4 ± 0.8 c Soleus ± ± ± ± 0.0 Flexor digitorum superficialis Superficial portion 51.2 ± 15.8 a 21.7 ± 11.8 b 25.4 ± 5.7 b 1.6 ± 3.9 c Deep portion 57.8 ± 5.6 a 20.5 ± 8.0 b 20.1 ± 7.1 b 1.5 ± 3.8 c Flexor digitorum profundus Tibialis caudalis 7.5 ± 2.4 c 59.7 ± 3.4 a 32.6 ± 4.2 b 0.2 ± 0.4 d Flexor hallucis longus 41.6 ± 5.0 a 36.3 ± 7.3 a 21.7 ± 8.3 b 0.4 ± 1.1 c Flexor digitorum longus 21.5 ± 7.2 b 47.6 ± 5.3 a 30.5 ± 6.3 b 0.4 ± 1.0 c Popliteus 19.2 ± 6.5 c 48.2 ± 3.3 a 32.6 ± 6.6 b 0.1 ± 0.2 d Mean ± S.D., a > b > c > d within a muscle, P < 0.05, n = 6 posture. The flexor hallusis longus muscle also exhibited a large population (>40%) of type I myofibers (Table 2 and Fig. 2A). The muscle with the lowest population of type I myofibers was the tibialis caudalis muscle, which exhibited the highest type IIA population among the crural muscles (Table 2). Muscles with lower type I populations tended to have a large population of type IIA myofibers (Table 2). The myofibers possessing high oxidative activity (type I and IIA) accounted for more than 60% of myofibers in all crural muscles (Fig. 2B). All crural muscles expect for the soleus muscle possessed small population of type Int. myofibers ( %; Table 2 and Fig. 2A). The soleus muscle was consisted of only type I myofibers (Tabel 2 and Fig. 2A). Diameters of myofiber types Although the populations of type I and IIA myofibers were high in the crural muscles, the diameters of type IIB myofibers were larger than that of type I and IIA myofibers in all crural muscles except for the flexor hallucis

4 42 T. Konno and K. Watanabe Fig. 2. Schematic representations of distribution of myofiber types in sheep crural musculature. (A) Percentages of type I myofibers in each muscle was presented on a cross-sectioned diagram of sheep crus. (B) Percentages of oxidative myofibers (type I and type IIA myofibers) were presented on a cross-sectioned diagram of sheep crus. longus muscle, suggesting that the crural muscles participate in the burst contraction (Table 3). The flexor hallucis longus muscle had the largest type I myofibers among the crural muscles (Table 3). The flexor muscles of toe joints (the flexor digitorum superficialis, tibialis caudalis, flexor hal lucis longus, and flexor digitorum longus muscles) had larger type I myofibers than the extensor muscles of toe joints (the extensor digitorum longus and extensor digitorum lateralis muscles), consistent with their antigravity actions on toe joints (Table 3 and Fig. 3A). Within the muscles that act on tarsal joint, the peroneus longus muscle with the smallest type I population exhibited the smallest diameter of type I myofibers; however, no correlations were found between the diameters of type I myofibers and the actions (flexor or extensor) on the tarsal joint (Table 3 and Fig. 3B).

5 Functional involvement of ovine crural muscles 43 Table 3. Diameters of myofiber types in ovine crural muscles Muscle Diameter of myofiber types (µm) I IIA IIB Tibialis cranilais 43.7 ± 4.6 b 43.2 ± 3.9 b 53.0 ± 7.0 a Peroneus tertius 43.2 ± 3.1 b 43.3 ± 2.5 b 49.0 ± 3.4 a Peroneus longus 34.7 ± 2.4 b 36.3 ± 2.1 b 48.0 ± 1.9 a Extensor digitorum longus 37.2 ± 2.3 b 36.8 ± 2.5 b 53.7 ± 6.2 a Extensor digitorum lateralis 36.1 ± 2.9 c 39.7 ± 2.3 b 50.2 ± 2.7 a Triceps surae Gastrocnemius, caput laterale 46.8 ± 2.1 b 40.6 ± 0.7 c 53.9 ± 4.2 a Gastrocnemius, caput mediale 39.6 ± 3.8 c 45.8 ± 3.2 b 51.2 ± 2.2 a Soleus 49.1 ± 2.1 N/A N/A Flexor digitorum superficialis Superficial portion 44.4 ± 2.0 b 47.8 ± 2.9 b 49.7 ± 1.5 a Deep portion 42.3 ± 2.0 c 52.2 ± 3.3 b 54.7 ± 3.4 a Flexor digitorum profundus Tibialis caudalis 40.8 ± 3.6 b 42.5 ± 3.9 b 50.9 ± 3.3 a Flexor hallucis longus 57.5 ± 3.4 a 50.8 ± 1.1 b 54.5 ± 2.2 a Flexor digitorum longus 43.8 ± 2.4 b 45.1 ± 3.8 b 52.1 ± 4.1 a Popliteus 49.6 ± 1.2 b 49.0 ± 4.4 b 59.0 ± 4.4 a Mean ± S.D., a > b > c within a muscle, P < 0.05, n = 6 Discussion The composition of myofiber types differs from muscle to muscle and reflects the function of each muscle, such as maintenance of standing posture, locomotion, and generation of burst force. In the hip and thigh musculature of sheep, the gluteus medius, gluteus accessorius, gluteus profundus, and vastus intermedius muscles are known to have a large population of type I myofibers 4). Because of the slow contractility and high antifatiguability of type I myofiber, these muscles are considered as to participate largely in the maintenance of standing posture. In the crural muscles, the tibialis cranialis, soleus, and flexor digitorum superficials muscles possessed a large population of type I myofibers, indicating their roles in the maintenance of standing posture. In the ovine hip and thigh musculature, type II myofibers account for a large population of myofibers in the semitendinosus, semimembranosus, adductor, and gluteobiceps muscles, and these muscles participate in the generation of propulsive force 4). In the crus of sheep, the extensor digitorum lateralis and tibialis caudalis muscles were found to have a large population of type II myofiber, indicating that these muscles participate largely in locomotion. In crural musculature of dogs, craniolateral muscles tend to have less type I myofibers than plantar muscles 2). Also in rats 3), the leg muscles with high type I myofibers population are located plantar to tibia. These previous observations indicate that the plantar muscles play a role in the maintenance of standing posture in digitigrades and plantigrade animals. In sheep, however, the tibialis cranialis muscle was found to possess a large population of type I myofibers, suggesting that a tension generated by craniolateral crural muscles may participate in maintaining a standing posture of unguligrade animals. The tibialis cranialis muscle is a flat shaped unipennate muscle with a relatively long myofiber length, and its architecture is therefore suitable for a rapid contraction rather than a generation of large and constant tension 14). While keeping a standing posture, plantar antigravity muscles of crus extend the tarsal joint. The tibialis cranialis muscle, which is a flexor muscle of the tarsal joint, may play a cooperative role in adjusting a standing posture. In guinea pigs and cats, the soleus muscle consists of 100% type I myofibers 1). The soleus muscle of humans and rats contains predominantly type I myofibers 3,15), suggesting its important role in maintaining a standing posture. However, not all mammals have the soleus muscle; it is diminutive in horse, and dogs lack this muscle 16,17). In sheep, the soleus muscle consisted of 100% type I myofibers. However, the soleus muscle of sheep is a thin and filamentous muscle with long myofiber length, indicating that the tension from this muscle during posture maintenance is small 14). The flexor digitorum superficialis muscle is an antigravity muscle of tarsal and toe joints. The architecture of the flexor digitorum superficialis muscle with a multipennate arrangement of short myofibers indicates its ability to generate large tension 14). The flexor digitorum superficialis muscle contained a large population of type I myofibers, suggesting its role in posture maintenance. The high population of type I myofibers in the flexor digitorum superficialis muscle has also reported in dogs 2). In rats, however, type I myofiber population in the flexor digitorum

6 44 T. Konno and K. Watanabe Fig. 3. Comparisons of type I myofiber diameter between the extensor and flexor muscles of tarsal and toe joints. (A) Comparison of diameter of type I myofiber between the extensor and flexor muscles of toe joints. (B) Comparison of diameter of type I myofiber diameter between the flexor and extensor muscles of tarsal joint. Error bars: standard error of means; *P < 0.05 (analysis of variances followed by Tukey s contrasts test), n = 6. Note: The flexor superficialis muscle was a flexor of toe joints (A) and extensor of tarsal joint (B).

7 Functional involvement of ovine crural muscles 45 superficialis muscle is less than 10% 3), suggesting that the flexor digitorum superficialis muscle in plantigrade animals may participate primarily in locomotion. Composition of myofiber types in a muscle reflects a function of each muscle. The maximum tension generated by each muscle relies on predominantly a number of myofibers within a muscle, and a tension of each myofiber differs and depends on the cross-sectional area of individual myofibers. The architecture of the tibialis cranialis and soleus muscles indicates that these muscles are not suitable to generate a large tension 14). However, the average diameters of their type I myofibers indicated that the individual myofibers in these muscles generate a tension and participate in posture maintenance. The flexor hallucis longus muscle is an antigravity muscle of the toe joints and possesses a bipennate arrangement of short myofibers 14). The average diameter of its type I myofibers was the largest in the crural muscles, reflecting a magnitude of its contribution to posture maintenance. Collectively, the composition and average diameter of myofiber types in ovine crural musculature reflected the role of each muscle in posture maintenance, locomotion, and burst contraction. The tibialis cranialis muscle was not capable to generate a large tension; however, it could function primarily in posture maintenance and play a cooperative role in adjusting standing posture. The flexor hallucis longus and flexor digitorum superficial muscles were the major muscles that contributed to posture maintenance in crural musculature. These muscles are capable to generate a large tension and participate primarily in standing posture maintenance. Acknowledgement Authors thank Drs. Atsushi Suzuki and Shyuichi Ohwada for their advices in pursuing this study. This study was partly supported by a Grant-in-Aid for Scientific Research (C) (No and ) from the Japan Society for the Promotion of Science (JSPS). References 1) Ariano MA, Armstrong RB, Edgerton VR: Hindlimb muscle fiber populations of five mammals. J Histochem Cytochem 1973; 21: ) Armstrong RB, Saubert CW 4th, Seeherman HJ, Taylor CR: Distribution of fiber types in locomotory muscles of dogs. Am J Anat 1982; 163: ) Armstrong RB, Phelps RO: Muscle fiber type composition of the rat hindlimb. Am J Anat 1984; 171: ) Suzuki A, Tamate H: Distribution of myofiber types in the hip and thigh musculature of sheep. Anat Rec 1988; 221: ) Peter JB, Barnard RJ, Edgerton VR, Gillespie CA, Stempel KE: Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits. Biochemistry 1972; 11: ) Smith JL, Edgerton VR, Betts B, Collatos TC: EMG of slow and fast ankle extensors of cat during posture, locomotion, and jumping. J Neurophysiol 1977; 40: ) Walmsley B, Hodgson JA, Burke RE: Forces produced by medial gastrocnemius and soleus muscles during locomotion in freely moving cats. J Neurophysiol 1978; 41: ) Dum RP, Kennedy TT: Physiological and histochemical characteristics of motor units in cat tibialis anterior and extensor digitorum longus muscles. J Neurophysiol 1980; 43: ) Dum RP, Burke RE, O Donovan MJ, Toop J, Hodgson JA: Motorunit organization in flexor digitorum longus muscle of the cat. J Neurophysiol 1982; 47: ) Brooke MH, Kaiser KK: Muscle fiber types: how many and what kind? Arch Neurol. 1970; 23: ) Suzuki A: A comparative histochemical study of the masseter muscle of the cattle, sheep, swine, dog, guinea pig, and rat. Histochemistry 1977; 51: ) Suzuki A, Cassens RG: A histochemical study of myofiber types in the serratus ventralis thoracis muscle of sheep during growth. J Anim Sci 1983; 56: ) Suzuki A, Watanabe K, Konno T, Ohwada S: Distribution of myofiber types in the hip and thigh musculature of pigs. Anim Sci J 1999; 70: ) Konno T, Suzuki A: Myofiber length and myofiber arrangement in the antebrachial and leg muscles of sheep. Okajimas Folia Anat Jpn 2000; 77: ) Edgerton VR, Smith JL, Simpson DR: Muscle fibre type populations of human leg muscles. Histochem J 1975; 7: ) Spoor CF, Badoux DM: The M. soleus in the domestic dog (Canis familiaris). Anat Histol Embryol 1989; 18: ) Meyers RA, Hermanson JW: Horse soleus muscle: postural sensor or vestigial structure? Anat Rec A Discov Mol Cell Evol Biol 2006; 288:

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