Laboratory of Animal Morphology, Faculty of Agriculture, Tohoku University, Sendai 980

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1 ACTA HISTOCHEM. CYTOCHEM. Vol. 15, No. 3, 1982 HISTOCHMICAL PROPERTIES OF MYOFIBER TYPES IN THIGH MUSCLES OF THE CHICKEN ATSUSHI SUZUKI, TAKESHI TSUCHIYA AND HIDEO TAMATE Laboratory of Animal Morphology, Faculty of Agriculture, Tohoku University, Sendai 980 Received for publication September 21, 1981 and in revised form November 10, 1981 The presence of subtypes of dark and light myofibers distinguished by myosin adenosine triphosphatase (ATPase) reaction after alkaline preincubation was studied in thigh muscles of chickens. The dark myofibers were subclassified into type II and SS, and the light myofibers into type I, SM, and MM myofibers by the ph sensitivity of myosin ATPase. Type I myofibers reacted strongly for acid-stable ATPase and weakly for alkali-stable ATPase. Type II myofibers reacted negatively for acid-stable ATPase and strongly for alkali-stable ATPase. Type SM myofibers reacted strongly for acid-stable ATPase and model ately for alkali-stable ATPase. Type SS myofibers reacted strongly and type MM myofibers reacted moderately for both acid-stable and alkali-stable ATPase. Type II myofibers were subdivided into type IIA with strong activity for NADH: Nitro BT oxidoreductase (NADHOX) and type IIB with weak NADHOX activity. Type IIB myofibers were strong in activity for glycerol-3-phosphate: menadione oxidoreductase (GPOX) whereas many type IIA myofibers were weak. Type IIA myofibers were stained weakly to moderately with 3-hydroxybutyrate: NAD+ oxidoreductase (HBOX) and type IIB myofibers were stained very weakly. Type I, SM, SS, and MM myofibers were stained strongly with NADHOX, weakly to moderately with HBOX, and weakly with GPOX. Myofibers of skeletal muscle have been classified into three to seven types by correlating the relative activity of myosin adenosine triphosphatase (ATPase) and oxidoreductase in chickens (1, 8, 16, 18). In general, the myofibers that show a strong reaction for acid-stable myosin and a weak reaction for alkali-stable myosin ATPase are designated as type I (3, 16) or 9 (1, 5), whereas type II or a myofibers show a weak reaction for acid-stable myosin ATPase and a strong reaction for alkalistable myosin ATPase. In mammals, type I myofibers are also called slow-twitch oxidative myofibers and type II myofibers are termed fast-twitch-oxidative-glycolytic or fast-twitch-glycolytic myofibers (15). These twitch myofibers are contacted by a nerve fiber at a single site. The histochemical reaction for myosin ATPase is related to the contractile properties of myofibers and the activity of oxidoreductases reflects their metabolic aspects (4, 9, 15). This work was supported in part by a grant-in-aid, Ministry of Education, Japan (to A. S.). No , for Scientific Research from the 362

2 HISTOCHEMISTRY OF CHICKEN MYOFIBER TYPES 363 Avian muscles have slow tonic myofibers contacted by several nerve endings along their surface in addition to the fast twitch myofibers (7). The latissimus dorsi cranialis (anterior) muscle of chickens is a tonic muscle; it comprises multipleinnervated myofibers (7). These myofibers are divided into two types : dark or type II and light or type I by the reactivity for myosin ATPase after alkaline preincubation (2, 13). In the two myofiber types the reaction for myosin ATPase remains unchanged after acid or alkaline preincubation. Two types of tonic myofibers have been recently reported to exist in the biventer cervicis muscle of the chick (23). These indicate that the staining profile for myosin ATPase varies among slow tonic myofbers and that the ph sensitivity of myosin ATPase in the tonic myofibers differs from that of mammalian type I and II myofibers. The pubo-ischio-femoralis (adductor) muscle of chickens is considered to be a postural muscle which possesses tonic myofibers (6). It possesses many myofibers corresponding to type I myofibers in chickens (1) and Japanese quails (19), but it is not clear whether the ph sensitivity of myosin ATPase in those myofibers is similar to that of the tonic myofiber. The purpose of this study was to verify the presence of subtypes of two myofiber types classified by the reaction for myosin ATPase at ph 9.4 in thigh muscles of chickens. The ph sensitivity of myosin ATPase and the activity of oxidoreductases in myofibers were examined to clarify the histochemical properties of myofiber types. MATERIALS AND METHODS Six male chickens of broiler type (Ross 1 strain) at 8 to 10 weeks of age were used in this study. They were killed by cervical dislocation and bleeding. Muscle samples were removed from the middle part or belly of m. femorotibialis medius, m. flexor cruris medialis, m. flexor cruris lateralis pars pelvica, m. iliofibularis, and m. pubo-ischio-fermoralis pars medialis and pars lateralis. The nomenclature for muscle follows that of Nomina Anatomica Avium (24). The samples were rapidly frozen in a mixture of dry ice and acetone. Serial cross sections (10µm thigh) were cut on a cryostat. Fresh sections were incubated for myosin ATPase (EC ) (14) after acid and alkaline preincubation (3, 20). The ph of the acid preincubation solution was 4.3, 4.5, 4.7, and 5.0. The ph of the alkaline preincubation solution was 9.4, 10.55, and The alkaline solution was changed three times and the acid solution was changed once during the time period of preincubation. The time of preincubation was 7 min for the acid solution and 27 min for the alkaline solution. Both preincubation and incubation for the ATPase reaction was carried out at room temperature (18-19 C). Other sections were stained with NADH : Nitro BT oxidoreductase (EC , NADHOX), with sn-glycerol-3-phosphate : menadione oxidoreductase (EC , GPOX) and with 3-hydroxybutyrate : NAD+ oxidoreductase (EC , HBOX) (10). The myofibers that showed a strong (-N{- to -~}}}-) reaction for acid-stable myosin ATPase and a weak (+ to +) reaction for alkali-stable myosin ATPase were denoted as type I according to the nomenclature of Brooke and Kaiser (3), whereas the myofibers that showed a negative (-) reaction for acid-stable myosin ATPase and a strong (I-N- to +) reaction for alkali-stable myosin ATPase were denoted as type II myofibers. The myofibers that reacted strongly for both acid-stable myosin ATPase and alkali-stable myosin ATPase were denoted as type SS according to the ter-

3 364 SUZUKI ET AL. minology of Suzuki and Gassens (20). The myofibers that reacted strongly for acid-stable myosin ATPase and moderately (-H-) for alkali-stable myosin ATPase were denoted as type SM (20). Type II myofibers were subdivided into type IIA and JIB myofibers by differences in activity for NADHOX as shown by Shafiq et al. (16). RESULTS Type I myofibers reacted strongly for ATPase after preincubation at ph 4.3 and 4.5 and moderately for ATPase after preincubation at ph 4.7 and 5.0 (Fig. 1). They reacted weakly for ATPase after preincubation at ph and negatively or very weakly after preincubation at ph Type II myofibers reacted negatively for ATPase after preincubation at ph below 4.5 but strongly for ATPase after preincubation at ph (Figs. 1, 2). The myofibers that showed a moderate to strong reaction for ATPase after preincubation at ph and after preincubation at ph were subdivided into three types (SM, SS, and MM) by differences in the reactivity for ATPase after preincubation at these phs (Figs. 1, 2). In the designation (SM, SS, and MM), the former letter was used to express the intensity of reaction for acid-stable myosin ATPase and the latter one to express the intensity of reaction for alkali-stable myosin ATPase. The myofibers that reacted strongly (S) for ATPase after preincubation at ph below 5.0 and moderately (M) after preincubation at ph were denoted as type SM myofibers (Fig. 1). The myofibers that reacted strongly (S) for ATPase after preincubation at ph and strongly (S) for ATPase after preincubation at ph were denoted as type SS myofibers (Fig. 2). The type SM and SS myofibers were stained slightly more strongly with ATPase after preincubation at ph 10.7 than after preincubation at ph 9.4 or (Figs. lc-d, 2c-d), but the increase in the intensity of staining was obscure in the other types. The myofibers that reacted moderately (M) for ATPase after preincubation at ph and moderately (M) for ATPase after preincubation at ph were named type MM so that they could be distinguished from type SS and SM myofibers Table 1, ph sensitivity of myosin ATPase of myofibers in thigh muscle of chickens FIGS. 1 and 2. Myosin ATPase activity of iliofibularis muscle (Fig. 1) and pubo-ischio-femoralis muscle pars medialis (Fig. 2) from chickens. a) preincubation at ph 4.3. b) preincubation at ph 4.7. c) preincubation at ph 9.4, d) preincubation at ph Myofiber types are labeled I, II, SM, SS, and MM. x 180

4 HISTOCHEMISTRY OF CHICKEN MYOFIBER TYPES 36.5

5 366 SUZUKI ET AL. in this study (Fig. 2). Type MM myofibers existed numerously in the m. puboischio-femoralis pars medialis. The type MM and SS myofibers were unchanged in reaction for ATPase after acid or alkaline preincubation, differing from the type I, II, and SM myofibers that possessed a myosin ATPase inhibited either by acid preincubation or by alkaline preincubation (Table 1). Type I, SS, SM, and MM myofibers were stained strongly with NADHOX and weakly with GPOX (Fig. 3, Table 2). Type I, SM, and MM myofibers were weak to moderate in HBOX activity and type SS myofibers weak (Figs. 4, 5, Table 2). Type II myofibers with strong activity for NADHOX were denoted as type IIA and those with weak activity for NADHOX as type IIB myofibers (Figs. 4, 5). Type IIB myofibers were stained very weakly with HBOX and strongly with GPOX (Figs. 4c-d, 5c-d). Type IIA myofibers were stained weakly to moderately with HBOX and weakly to strongly with GPOX (Figs. 4c-d, 5c-d). In general, the intensity of staining of GPOX activity was inversely related to that of HBOX activity in type II myofibers (Table 2). The m. pubo-ischio-femoralis pars medialis had type IIA myofibers with weak Fin. 3. NADHOX activity of iliofibularis muscle (a) and pubo-ischio-femoralis muscle pars medialis (b) from chickens. Figures 3a and 3b indicate the same fields as in Figures 1 and 2 respectively. Myofiber types are labeled I, II, SM, SS, and MM. X 180 TABLE2. Histochemicalactivityfor oxidoreductasein myofibertypesin thigh muscleof chickens FIGS. 4 and 5. Histochemical properties of m. flexor cruris medialis (Fig. 4) and lateralis pars pelvica (Fig. 5) from chickens. a) Myosin ATPase activity after preincubation at ph b) NADHOX activity. c) GPOX activity. d) HBOX activity. Myofiber types are labeled 1, IIA, 1113,and SM. x 180

6 HISTOCHEMISTRY OF CHICKEN MYOFIBER TYPES 367

7 368 SUZUK I ET AL. activity of GPOX but no type IIA myofibers with strong activity of GPOX. In general, type IIA myofibers with weak activity of GPOX were greater in number than type IIA myofibers with strong activity of GPOX in the other muscles. The m. pubo-ischio-femoralis pars medialis possessed type IIA, SS, and MM myofibers; type IIA and SS myofibers were located at the region near the femur and fewer than type MM myofibers. The m, pubo-lschlo-femoralis pars lateralis possessed numerous type IIA myofibers and very few type SM myofibers. The flexor cruris medialis, iliofibularis, and femorotibialis medius muscle consisted of type I, IIA, JIB, and SM myofibers; they possessed more type II (IIA plus IIB) myofibers than type I and SM myofibers. The m. flexor cruris lateralis pars pelvica had type IIA and IIB myofibers. The myofiber types were distributed unevenly in the muscles. DISCUSSION The myofibers reacting strongly for myosin ATPase after alkaline preincubation were divided into type II and SS myofibers by the ph sensitivity of myosin ATPase, whereas the myofibers with weak or moderate reaction for myosin ATPase were classified into type I, SM, and MM myofibers in the thigh muscles of chickens. Talesara and Goldspink (22) have reported that type I myofibers were stained strongly with ATPase after preincubation at ph 4.35 and weakly with ATPase after preincubation at 4.6 and 5.0. The type I myofibers of thigh muscles reacted moderately for myosin ATPase at ph The ph sensitivity of myosin ATPase in type II myofibers of the thigh muscles resembles that of type II myofibers of avian pectoralis and gastrocnemius muscle (22) and of mammalian type IIA myofibers (3, 20). Toutant et at. (23) have shown that slow tonic /9 myofibers are divided into /9i and /92 myofibers on the basis of the ph sensitivity of myosin ATPase in the biventer cervicis muscle. The ph sensitivity of /31 myofibers resembles that of type I myofibers of the thigh muscles, whereas the /92 myofibers appear to correspond to type SS myofibers. The activity of myosin ATPase in type SS and MM myofibers was not affected by preincubation at ph The staining profile for myosin ATPase in type MM and SS myofibers of the m. pubo-ischio-femoralis pars medialis is similar to that of two types of tonic myofibers in the latissimus dorsi cranialis (2, 13). Type MM and SS myofibers were considered to be a tonic myofiber. The type SM myofibers showing the staining profile intermediate between type SS and I myofibers seem to possess a characteristic of tonic myofibers. The presence of type I, SM, SS, and MM myofibers indicates that tonic myofibers vary in histochemical properties. A similar diversification of myofiber types has been observed in the hind limb muscle of the copper pheasant (11). The type JIB myofibers of mammals react positively for myosin ATPase after preincubation at ph 4.5 and negatively after preincubation at ph below 4.3 (3, 20). All type II myofibers were unstained with myosin ATPase at ph 4.5 in the thigh muscles of chickens; they were divided into type IIA and JIB myofibers by differences in the intensity of NADHOX activity. Chicken type JIB myofibers differ slightly from mammalian type JIB myofibers in the ph sensitivity of myosin ATPase. Type I myofibers of chickens resemble mammalian type I myofibers in the ph sensitivity of myosin ATPase (3, 20).

8 HISTOCHEMISTRY OF CHICKEN MYOFIBER TYPES 369 The reactivity for myosin ATPase in type SM and SS myofibers of chickens differs slightly from that in type SM and SS myofibers of pigs (20), since the intensity of staining in myosin ATPase of chicken SM and SS myofibers increased somewhat after preincubation at ph Such increases in the intensity of staining of myosin ATPase after preincubation at ph have been shown in avian pectoralis, gastrocnemius (22) and biventer cervicis muscles (23). The myofibers that react positively for both acid-stable and alkali-stable myosin ATPase are designated as type TIC (3) or type II intermediate (9). Similar intermediate types are subdivided into three types (SM, SS, and MS) by the ph sensitivity of myosin ATPase in growing pigs; they represent a transitional form in the process of transformation of type II into type I myofibers (20, 21). The type SS myofibers indicate a middle stage in the process of transformation and the type SM myofibers represent the last stage. If type SS myofibers were transformed progressively into type SM myofibers in the m. pubo-ischio-femoralis pars medialis, type SM myofibers would be found near the type SS myofibers as observed in growing pigs (20, 21). However, type SM myofibers were not found in this muscle. The type SS myofibers may represent a static state in the muscle of chickens at 8 to 10 weeks of age. In pigs the proportion of the intermediate types decreases after 4 weeks of age (21). Khan (8) has classified type II red myofibers into two subtypes and type II white myofibers into three subtypes by the reactivity for myosin ATPase after preincubation of a cold solution of K2-EDTA. In addition, type I red myofibers were subdivided into type IRA and IRB myofibers. The type I myofibers in thigh muscles seem to correspond to type IRA myofibers and type SM myofibers to type IRB myofibers. The type IIA myofibers fall under the category of type II red myofibers and the type JIB myofibers under the category of type II white myofibers. In the previous study (18, 19), the type A and B myofibers correspond to type IIA and JIB myofibers respectively, whereas the type C myofibers are subdivided into type I, SM, and MM myofibers in this study. Myofibers with strong reaction for alkali-stable myosin ATPase are considered to be fast contracting, whereas myofibers with weak reaction for alkali-stable myosin ATPase are slow contracting (4, 9, 15). The activity of NADHOX represents a capacity for oxidative metabolism and the activity of GPOX reflects a capacity for glycolytic metabolism (15). The myofibers with strong reaction for alkali-stable myosin ATPase show a strong activity for GPOX in guinea pigs and rabbits (15). Type IIA myofibers with strong activity for GPOX correspond to fast-twitch-oxidative-glycolytic myofibers of Peter et al. (15). On the other hand, type IIA myofibers with weak activity for GPOX are considered to be oxidative in metabolic TABLE 3. Functional and metabolic profiles of nyofiber types in thigh muscles of chickens

9 370 SUZUKI ET AL. profiles; they fall under the category of fast-twitch-oxidative myofibers found in the rat (12) and mouse (17). Type JIB myofibers correspond to fast-twitch-glycolytic myofibers, whereas type I, SM, SS, and MM myofibers possessing histochemical properties of tonic myofibers fall under the category of slow oxidative myofibers (Table 3). The dissimilarity in the ph sensitivity of myosin ATPase between the type I, SM, SS, and MM myofibers implies that a little difference in contractile properties and function exists among the tonic myofibers. The m. latissimus dorsi cranialis stops the wing from drooping. The m. biventer cervicis elevates the head and prevents it from sinking. The slow tonic myofibers of these two muscles are considered to act to hold the wing and the head or neck in their normal position. The type MM myofibers that exist numerously in the m. pubo-ischio-femoralis pars medialis, a postural muscle, are thought to function in maintenance of posture. Type I, SM, and SS myofibers seem to be involved in postural function, although they may differ in postural activity. The type IIA and JIB myofibers of the m. pubo-ischio-femoralis pars lateralis, m. femorotibialis medius, m. flexor cruris medialis, m. flexor cruris lateralis pars pelvica, and m. iliofibularis are used for movements requiring power such as standing, walking or running. The standing position developed by the type IIA and JIB myofibers of these muscles is presumed to be maintained by the type I and SM myofibers of them and by the type MM and SS myofibers of the m. pubo-ischio-femoralis pars medialis. The distribution pattern of six myofiber types in the thigh muscles differed greatly with muscles or with different regions of muscles. An examination of the composition of myofiber types and their localization in the thigh muscles is in progress. REFERENCES 1. Ashmore, C. R, and Doerr, L.: Postnatal development of fiber types in normal and dystrophic skeletal muscle of the chick. Exp. Neurol. 30; 431, Asiedu, S. and Shafiq, S. A.: Actomvosin ATPase activity of the anterior latissimus dorsi muscle of the chicken. Exp. Neurol. 35; 211, Brooke, M. H. and Kaiser, K. K.: Muscle fiber types: How many and what kind? Arch. Neurol. 23; 369, Burke, R. E., Levine, D. N., Zajac, F. E. III, Tsairis, P, and Engel, W. K.: Mammalian motor units : Physiological-histochemical correlation in three types in cat gastrocnemius. Science 174; 709, Guth, L., Samaha, F. J. and Wayne Albers, R.: The neural regulation of some phenotypic differences between the fiber types of mammalian skeletal muscle. Exp. Neurol. 26; 126, Helmi, C. and Cracraft, J.: The growth patterns of three hind limb muscles of the chicken. 7. Hess, A.: Structural differences of fast and slow extrafusal muscle fibres and their nerve endings in chickens. J. Physiol.157; Khan, M. A.: Histochemical sub-types of three fibre-types of avian skeletal muscle. Histochemistry 50; 9, Kugelberg, E.: Adaptive transformation of rat soleus motor units during growth. J. Neurol. Sci. 27; 269, Lojda, Z., Cossrau, R. and Schiebler, T. H.: Enzyme Histochemistry: A Laboratory Manual. Springer-Verlag, Berlin, Heidelberg, New York, 1979, p Manabe, N., Sato, E. and Ishibashi, T.: Histochemical analysis of skeletal muscle fibers in the copper pheasant Phasianus soemmerringii. Tori 29; 109, 1981 (in Japanese; English summary). 12. Nemeth, P., Hofer, H.-W, and Pette, D.: Metabolic heterogeneity of muscle fibers classified

10 HISTOCHEMISTRY OF CHICKEN MYOFIBER TYPES 371 by myosin ATPase. Histochemistry 63;191, Ovalle, W. K. Jr.: Histochemical dichotomy of extrafusal and intrafusal fibers in an avian slow muscle. Amer. J. Anat. 152; 587, Padykula, H. A. and Herman, E.: The specificity of the histochemical method for adenosine triphosphatase. J. Histochem. Cytochem. 3; 170, Peter, J. B., Barnard, R. J., Edgerton, V. R., Gillespie, C. A. and Stempel, K. E.: Metabolic profiles of three types of skeletal muscle in guinea pigs and rabbits. Biochemistry 11; 2627, Shafiq, S. A., Askanas, V. and Wilhorat, A. T.: Fiber types and preclinical changes in chicken muscular dystrophy. Arch. Neurol. 25; 560, Spurway, N. C.: Objective characterization of cells in terms of microscopical parameters: an example from muscle histochemistry. Histochem. J. 13; 269, Suzuki, A.: Histochemistry of the chicken skeletal muscle. I. Classification of individual muscle fibers. Tohoku J. Agr. Res. 23; 45, Suzuki, A. and Tamate, H.: Histochemical properties and fiber type composition of the pectoral and thigh muscles of the Japanese quail. Acta histochem, cytochem. 12; 69, Suzuki, A. and Cassens, R. G.: ph sensitivity of myosin adenosine triphosphatase and subtypes of myofibres in porcine muscle. Histochem. J. 12; 687, Suzuki, A. and Cassens, R. G.: A histochemical study of myofiber types in muscle of the growing pig. J. Anim. Sci. 51;1449, Talesara, G. L, and Goldspink, G.: A combined histochemical and biochemical study of myofibrillar ATPase in pectoral, leg and cardiac muscle of several species of bird. Histochem. J. 10; 695, Toutant, J. P., Rouaud, T. and Le.Douarin, G. H.: Histochemical properties of the biventer cervicis muscle of the chick: a relationship between multiple innervation and slow-tonic fibre types. Histochem. J. 13; 481, Vanden Berge, J. C.: Myologia. In Nomina Anatomica Avium. An Annotated Anatomical Dictionary of Birds, ed. by J. J. Baumel, A. S. King, A. M. Lucas, J. E. Breazile and H. E. Evans, Academic Press, London, 1979, p. 175.