Heterogeneity of myosin heavy-chain expression in fast-twitch fiber types of mature avian pectoralis muscle1

Transcription

1 Heterogeneity of myosin heavy-chain expression in fast-twitch fiber types of mature avian pectoralis muscle1 B.W.C. Rosser, M. Wick, D.M. Waldbillig, and E. Bandman Abstract: The aims of this study are to investigate the diversity of myosin heavy-chain (MyHC) expression among avian fasttwitch fibers, and to test the hypothesis that dissimilar MyHC isoforms are found in each of the principal avian fast-twitch fiber types. MyHCs within the muscle fibers of the pectoralis of 31 species of bird are characterized using immunocytochemical methods. A library of 11 monoclonal antibodies previously produced against chicken MyHCs is used. The specificity of these antibodies for MyHCs in each of the muscles studied is confirmed by Western blots. The results show that avian fast-twitch glycolytic fibers and fast-twitch oxidative-gylcolytic fibers can contain different MyHCs. Among the species studied, there is also a conspicuous variety of MyHC isoforms expressed. In addition, the results suggest that two epitopes are restricted to chickens and closely allied gallinaceous birds. There are no apparent correlations between MyHC epitope and presupposed contractile properties. However, the presence of different isoforms in different fast-twitch fiber types suggests a correlation between isoform and contractile function. Key words: muscle, fiber, myosin, avian. RbumC : Le but de ce travail Ctait d'ktudier la diversit6 de l'expression de la chaine lourde de myosine dans les fibres rapides d'oiseaux et de verifier l'hypothkse que diffkrentes isoformes de la chaine lourde de myosine se trouvent dans chacun des principaux types de fibres rapides d'oiseaux. Les chaines lourdes de myosine des fibres de muscles pectoraux de 31 espkces d'oiseaux ont CtC caractcris6es par des mtthodes immunocytochimiques. Une banque de 11 anticorps monoclonaux dirigcs contre les chaines lourdes de myosine du poulet a tt6 utiliske. La sp6cificitc de ces anticorps envers les chaines lourdes de myosine dans chacun des muscles CtudiBs a CtC confirmbe par des transferts Western. Les rcsultats indiquent que les fibres rapides B glycolyse anabobie et les fibres rapides B glycolyse acrobie d'oiseaux contiennent diffkrentes chaines lourdes de myosine. 11 y a une grande varictc d'isoformes de chaine lourde de myosine exprimces chez les espbces d'oiseaux CtudiCes. De plus, les rcsultats suggkrent que deux Cpitopes se trouvent seulement chez le poulet et les autres gallinaces trks apparent& au poulet. I1 n'y a pas de corrklation apparente entre un Cpitope de la chaine lourde de myosine et les propribt6s contractiles prtsumces. Cependant, la prcsence de diffkrentes isoformes dans diffkrents types de fibres rapides suggkre qu'il y a une corrtlation entre les isoformes et la capacitt de contraction. Mots clis : muscle, fibre musculaire, myosine, oiseau. [Traduit par la rkdaction] Introduction Myosin, a filamentous protein, is intimately involved in the contraction of all skeletal muscles (Sellers and Goodson 1995; Received August 12, Revised October 16, Accepted October 23, Abbreviations: MyHC, myosin heavy-chain;, fast-twitch oxidative glycolytic; FG, fast-twitch glycolytic; SDS-PAGE, sodium dodecyl sulfate - polyacrylamide gel electrophoresis. B.W.C. ~osse9 and D.M. Waldbillig. University of Saskatchewan, College of Medicine, Department of Anatomy and Cell Biology, Saskatoon, SK S7N 5E5, Canada. M. Wick and E. Bandman. University of California, Department of Food Sciences and Technology, Davis, CA 95616, U.S.A. This paper is dedicated to Dr. John C. George, University of Guelph, in recognition of his pioneering studies of avian muscle fiber types and in honour of his seventy-fifth birthday. Author to whom all correspondence should be addressed. Voet and Voet 1995). In muscle, each myosin molecule is a hexamer consisting of two heavy and four light chains. MyHCs, with molecular weights of approximately , contribute more to skeletal muscle mass than any other myofibrillar protein (Yates and Greaser 1983; Voet and Voet 1995). In vertebrates, MyHCs are encoded by a diverse multigene family (see Bandman et al. 1994) and there are many biochemically distinct but related MyHC isoforms (Bandman et al. 1990,1994). MyHC expression has been studied in an impressive variety of mammalian skeletal muscles and species (see Hoh 1992; Hamalainen and Pette 1993; Schiaffino and Reggiani 1994; Stal et al. 1994). By comparison, there are fewer studies of MyHCs in avian skeletal muscles. Moreover, studies of avian MyHCs have been confined to several muscles of the domestic chicken (Bandman et al. 1990; Williams and Dhoot 1992; Connold et al. 1993; Gauthier and Orofanos 1993; Maier 1994) and their close allies, the turkey (Maruyama et al. 1993; Cherel et al. 1994) and Japanese quail (Merrifield et al. 1989; Alway 1993). Biochem. Cell Biol. 74: (1996). Printed in Canada I Imprimd au Canada

2 Biochem. Cell Biol. Vol. 74, 1996 Muscle fibers, the contractile cells of skeletal muscle, are broadly classified as either fast twitch or slow, depending on their contractile properties (Rosser and George 1986; Williams and Dhoot 1992). Vertebrate skeletal muscles consist of varying proportions of different types of fibers (Peters 1989; Hoh 1992). In the earliest stages of development, fast-twitch and slow muscle fibers contain the same embryonic MyHC isofoms (Bandman et al. 1990, 1994; Russell et al. 1993; Schiaffino and Reggiani 1994). During subsequent stages, each type of fiber may contain a number of different MyHC isofoms (Bandman et al. 1990, 1994; Russell et al. 1993; Schiaffino and Reggiani 1994). In mature vertebrate muscles, fast-twitch and slow fibers contain different MyHC isofoms, although there may be uncommon transitional fiber types containing mixtures of slow and fast isoforms (Bandman et al. 1990, 1994; Schiaffino et al. 1990; Russell et al. 1993; Staron and Pette 1993). In mature mammalian muscles, three fasttwitch fiber types have been identified and each of these principal fiber types contains a characteristic MyHC isoform (Hoh 1991; Harnalainen and Pette 1993; Bottinelli et al. 1994; Schiaffino and Reggiani 1994). In avian muscle, while several distinct isoforms can also appear sequentially within the same fast-twitch fiber during development (Bandman et al. 1990, 1994), different MyHCs have not been described among the principal fast-twitch fiber types of mature muscle (Menifield 1989; Williams and Dhoot 1992). The purpose of this study is to explore the diversity of MyHC expression among avian fast-twitch fibers and to test the hypothesis that the principal avian fast-twitch fiber types differ from each other in their MyHC content. MyHCs in fibers of the pectoralis muscle of 3 1 species of bird are characterized using a library of monoclonal antibodies previously generated against known chicken MyHCs. The specificity of these antibodies for MyHCs in the muscles studied is verified by Western blots. Our results indicate that there is a pronounced diversity in the MyHC isofoms expressed and that different avian fast-twitch fiber types do contain distinct MyHCs. Materials and methods Fast-twitch fiber nomenclature In mature mammalian muscles, three fast-twitch fiber types have been identified: type 2A, type 2X (also called 2D), and type 2B (Hoh 1991; Hamalainen and Pette 1993; Schiaffino and Reggiani 1994). In this scheme, fiber type determined by MyHC content has been correlated with physiologic performance (Bottinelli et al. 1994). Metabolic criteria, however, have also been incorporated into fast-fiber terminology (see Nemeth 1990) and correlated with physiologic performance (Nemeth et al. 1991). The and FG fibers of the metabolic nomenclature are, respectively, largely synonymous with type 2A and type 2B of the myosin-based scheme (see Nemeth 1990). It has been suggested that past discrepancies between these two schemes were due to the presence of type 2X fibers, of which earlier investigators were unaware (Schiaffino et al. 1990). Avian fast-twitch fiber nomenclature has most often incorporated metabolic criteria (George and Berger 1966; Rosser and George 1986; Kaplan and Goslow 1989; Lundgren and Kiessling 1988; Gaunt et al. 1990; Boesiger 1992; Torrella et al. 1993; Tobalske 1996). Avian type and FG fibers are, respectively, for the most part synonymous with type 2A and type 2B fibers (see Rosser and George 1986). To date, type 2X fibers have not been described in avian muscle. Therefore, in this study, we have chosen to continue to include metabolic criteria to describe fast-twitch fibers as either FG or. Avian and FG fibers are also synonymous with, respectively, red and white fast-twitch fiber types (see George and Berger 1966; Rosser and George 1986). An intermediate fiber type has also been described. These intermediate fibers are between red and white fibers in their metabolic activities and morphologic characteristics (see George and Berger 1966; Rosser and George 1986). In mammalian muscles, a complete spectrum of overlapping metabolic activities has been described among fast-twitch fibers (see Nemeth 1990), and some of these fibers may represent transitional types containing mixtures of different MyHCs (Staron and Pette 1993). The avian fast-twitch intermediate type merits attention because it has been described as a discrete population meeting defined functional demands in flight muscles (Parker and George 1975; Tobalske 1996). However, unless otherwise indicated, in this study for simplicity we refer to both avian intermediate and red fibers as subtypes. Experimental model The pectoralis muscle (M. pectoralis pars thoracicus) of birds extends from the sternum, clavicle, and ribs to the humerus (Raikow 1985). The pectoralis is the most massive muscle in the body of volant species (Hartman 1961). It provides almost all power for the downstroke of the wing in flight and assists in decelerating the wing during the upstroke (Biewener et al. 1992; Dial 1992). The fiber type complement of the pectoralis muscle is generally correlated with a species' mode of flight (George and Berger 1966; Rosser and George 1986; Rosser et al. 1994; Tobalske 1996). We have previously reported the proportion and distribution of fiber types in the pectoralis of some 44 species of birds (Rosser and George 1986; Rosser et al. 1987). Subsequently, additional observations have been published (Edman et al. 1988; Lundgren and Kiessling 1988; Kaplan and Goslow 1989; Gaunt et al. 1990; Boesiger 1992; Evans et al. 1992; Swain 1992; Torrella et al. 1993; Tobalske 1996). Invariably, in those birds that rely upon flapping flight, the pectoralis is composed almost exclusively (99-100%) of fasttwitch fiber types. Slow fibers are found sparingly within the avian pectoralis muscle. They are present within a tiny narrow band, termed the deep red strip, which has been located in the deepest cranial region of the muscle in just four species: chicken, Japanese quail, double-crested cormorant, and red-tailed hawk (Rosser and George 1986; Rosser et al. 1987; Shear et al. 1988). Also, in certain species using soaring flight (such as vultures and pelicans), the pectoralis has an accessory deep belly consisting entirely of slow fibers (Rosser et al. 1994). In addition, flightless forms (such as the kiwi, emu, and ostrich) have comparatively diminutive pectoralis muscles, which contain a high proportion of slow fibers throughout (see Rosser and George 1985). MyHC expression within the pectoralis muscle of the domestic chicken is fairly well understood (Bandman et al. 1990; Gauthier and Orfanos 1993). The muscle consists

3 Rosser et al. 717 Table 1. Specificity of antimyhc monoclonal antibodies against chicken MyHCs. Fast Slow Monoclonal antibody* Embryonic* * Neonatal Adult 1 2 Ventricular Apparent specificity NA All myosins AG All fast myosins EB Embryonic and adult fast AB Adult fast 2E Neonatal fast B Embryonic and neonatal fast NA Slow 2 NA Slow 1 and 2 NA Slow 1 NA Slow 2 HVll Ventricular *The preparation and specificity of these monoclonal antibodies against chicken MyHCs (myosin heavy chains) has been detailed elsewhere (Cemy and Bandman 1987; Bandman and Bennett 1988; Bourke et al. 1991). **Embryonic fast refers to multiple isoforms; B103 labels embryonic 1 or 3 but not embryonic 2 (Moore et al. 1992). almost entirely of fast fibers except for the deep red strip that comprises less than 1 % of the muscle and contains about onethird slow fibers (Shear et al. 1988). Throughout the muscle, excepting the red strip, embryonic MyHC isoforms are entirely replaced by a neonatal isoform at about 2 weeks after the birds hatch. This neonatal isoform is in turn wholly replaced by an adult isoform by about 4 months of age. A comparable embryonic to neonatal to adult MyHC transformation also occurs in the pectoralis muscle of the Japanese quail (Merrifield et al. 1989) and the turkey (Maruyama et al. 1993). However, a different sequence of MyHC transformations takes place within the fast-twitch fibers located in the red strip (Shear et al. 1988), as well as within fast-twitch fibers of other muscles of the chicken (Bandman et al. 1990). Species Studied The nomenclature for the 3 1 species studied is, unless otherwise indicated, from Sibley and Munroe (1990). The species studied were as follows: American crow (Corvus brachyrhynchos), American robin (Turdus migratorius), American white pelican (Pelecanus erythrorhynchos), American wigeon (Anas americana), belted kingfisher (Megaceryle alcyon), Boehemian waxwing (Bombycilla garrulus), common or island canary (Serinus canaria), domestic chicken (Gallus gallus; from Crawford 1990), domestic or rouen duck (Anas platyrhynchos; from Crawford 1990), domestic or rock pigeon (Columba livia), great homed owl (Bubo virginianus), grey partridge (Perdix perdix), homed lark (Eremophila alpestris), house wren (Troglodytes aedon), Japanese quail (Coturnix japonica), least sandpiper (Calidris minutilla), long-eared owl (Asio otus), merlin (Falco columbarius), mulga parrot (Psephotus varius), northern flicker (Colaptes auratus), northern harrier (Circus cyaneus), northern shoveller (Anas clypeata), ostrich (Struthio camelus), red-eyed vireo (Vireo olivaceus), red-winged blackbird (Agelaius phoeniceus), rufous-sided towhee (Pipilo erythrophthalmus), short-eared owl (Asiojlammeus), sora rail (Porzana carolina), stilt sandpiper (Micropalama himantopus), Swainson's hawk (Buteo swainsoni), and zebra finch (Taeniopygia guttata). All birds were judged to be mature (adult) individuals, with the exceptions of the ostrich (2 months), house wren (newly fledged), and great homed owl, which were sexually immature (juveniles). Specimens and tissue preparation Blocks of muscle were excised from the more superficial areas of either the right or left pectoralis muscle of each bird. Blocks were removed from a location near the keel of the sternum, about midway between the cranial and caudal edges of the muscle. As in our earlier work (Rosser and George 1986; Rosser et al. 1987), samples were obtained from the sternobrachialis and not the thoracobrachialis portion (Dial 1992) of each muscle. Each block was about 0.6 x 0.6 x 3 cm and was cut so that its long axis ran in the same direction as the muscle fibers. In the smallest birds studied, a block could contain most of the muscle. Blocks of muscle were coated with Tissue-Tek O.C.T. compound (Miles Inc., Elkhart, Ind.), quick frozen in 2-methylbutane cooled to C by liquid nitrogen (Dubowitz 1985), and then stored at -80 C. From tissue blocks representative of each bird, sections of 4-6 p (1 p. = 1 p,m) thickness were cut in a cryostat maintained at -20 C. Two to three sections were picked up on each microscope slide coated with gelatin-chrome alum. An extensive series of serial sections was obtained from each muscle. Histochemistry Avian fast-twitch and slow fibers are readily differentiated on the basis of their myosin ATPase activity (Rosser and George 1986). Avian fast-twitch fibers are further divided into FG and types by the biochemical pathways used to produce the energy for contraction (Rosser and George 1986). A histochemical stain for a mitochondria1 oxidative enzyme is commonly used for this purpose (Rosser and George 1986). Predictably, mainly as a result of the greater density of mitochondria in oxidative fibers, the smaller diameter fibers stain dark and the larger fibers stain light (George and Berger 1966). These fibers are then classified, respectively, as and FG. For the purposes of this study, the fibers can

4 71 8 Biochem. Cell Biol. Vol. 74, 1996 Table 2. Classification of species studied, and monoclonal antibody binding to fiber types found in each avian pectoralis muscle. Order, family, and species Antibody Fiber type NA4 AG6 EB165 AB8 2E9 B103 NA8 NA1 NA2 NA7 HVll Order Struthioniformes Family Struthionidae Ostrich (juvenile) (1) Order Galliformes Family Phasianidae Domestic chicken, red strip" (2) S Domestic chicken, superficiala (2) Japanese quailb (3) Grey partridge FG FG FG FG Order Anseriformes Family Anatidae Rouen (domestic) duck (1) American wigeon (1) Northern shoveler (1) Order Piciformes Family Picidae Northern Flickef (2) Order Coraciiformes Family Cerylidae Belted kingfisher (1) Order Psittaciformes Family Psittacidae Mulga parrot (1) Order Strigiformes Family Strigidae Short-eared owl (1) Long-eared owl (1) Great homed owl (juvenile) (1) Order Columbiformes Family Columbidae Domestic pigeonb (3) Order Gruiformes Family Rallidae Sora rail (1) Order Ciconiiformes Family Scolopacidae Least sandpipef (2) Stilt sandpiper (1) Family Falconidae Merlin (1) Family Accipitridae Northern harrier (1) Swainson's hawk (1) FG FG FG C -t + fd + + P FG FG FG FG

5 Rosser et al. 719 Table 2 (concluded). Antibody Fiber Order, family, and species type NA4 AG6 EB165 AB8 2E9 B103 NA8 NA1 NA2 NA7 HVll Family Pelecanidae American white pelicana (2) Order Passeriformes Family Vireonidae Red-eyed vireo (1) Family Corvidae American crow (1) Family Bombycillidae Bohemian waxwing (1) Family Muscicapidae American robin (1) Family Certhiidae House wren (juvenile) (1) Family Alaudidae Homed lark (1) Family Fringillidae Common canary (1) Zebra finch (1) Red-winged blackbird" (2) Rufous-sided towhee (1) FG FG Note: The muscle studied is the pectoralis (M. pectoralis pars thoracicus). Unless otherwise indicated, all birds are mature (adult) individuals. Classification follows Sibley and Munroe (1990) and Sibley and Ahlquist (1990). Numbers in brackets = number (n) of animals studied. Immunofluorescence: +, positive reaction; -, negative reaction; f some fibers positive, others negative; blank space, not done. S, slow. "The red strip is located in the deepest cranial edge of the belly of the chicken pectoralis. In two adult chickens, approximately 29 and 37% of the that react with NA8 also react with NA7. No fiber reacts with NA7 alone. %103 labels ends of muscle fibers in the Anseriformes. 'Approximately 4 and 40%, respectively, of the FG and the fibers react positively with B103. dapproximately 30 and 25%, respectively, of the fibers in only the deep half of the muscle react positively with AG6. 'The occasional cell is faintly stained for B103. Gxtremely rare occurrence of FG and fibers reacting with HVI 1 but not with other antibodies against slow myosins. gapproximately 13% of the FG fibers are labeled by B103. happroximately 87% of the fibers throughout the muscle are labeled by B labeled one fiber in one red-winged blackbird. fibers consist of both small diameter red and slightly larger intermediate subtypes (see George and Berger 1966; Rosser and George 1986). In a muscle containing both fast-twitch and slow fibers, one need perform the histochemical reaction for myosin ATPase to differentiate these fiber types. However, if it is known that no slow fibers are present, as in the superficial regions of the avian pectoralis, FG and fibers can be differentiated solely on the basis of their mitochondria1 enzymes. Therefore, in the present study, two sections from each series were stained by histochemical methods for the demonstration of NADH activity (Dubowitz 1985). NADH, aproduct of the citric acid cycle and fatty acid oxidation (Voet and Voet 1995), is prevalent in mitochondria (Dubowitz 1985). MyHC isoforms and primary antibodies At least eight MyHC isoforms occur in chicken muscles, as shown by immunocytochemical techniques (see Bandman et al. 1990, 1994). These include three embryonic, one neonatal, and one adult isoform found within the fast-twitch fibers of the pectoralis and other muscles; two isoforms found in the slow fibers of the anterior latissimus dorsi muscle (also termed latissimus dorsi pars cranialis) and the deep red strip of the pectoralis; and a ventricular isoform found in the heart. A battery of monoclonal antibodies have been used to differentiate these isoforms. The antibodies used in this paper and their reactivity with known chicken MyHCs are summarized in Table 1. Immunocytochemical techniques Immunocytochemical techniques, described by Shear et al. 1988, were used to demonstrate MyHC isoforms. Briefly, sections from each series were first blocked in a solution consisting of 5% horse serum, 1% bovine serum albumin, and 5 mm EDTA in PBS (0.02 M sodium phosphate buffer, 0.15 M sodium chloride, ph 7.2). Sections were then incubated for 75 min at room temperature with a primary antibody diluted in blocking solution. Primary antibodies NA4, AG6, EB165, AB8, NA8, NA1, NA2, NA7, and HVl1 were each used at a dilution of 1:5000. However, 2E9 and B103 were used at a

6 720 Biochem. Cell Biol. Vol Figs Transverse serial sections of the pectoralis muscle of the Japanese quail. Each scale bar = 50 p (1 p = 1 pm) Large arrowheads indicate FG fibers, and small arrows indicate fibers. Fig. 1, histochemical demonstration of NADH activity, demonstrating FG and fibers. Figs. 2-9 respectively, immunocytochemical demonstration of labeling by antimyhc primary antibodies NA4, AG6, EB165, AB8,2E9, B103, NA8, and NA2. The same FG and fibers are indicated by the large arrow, head and smaller arrow respectively, throughout this series of figures. While all FG and fibers react with NA4 (Fig. 2), AG6 (Fig. 3), EB165 (Fig. 4), and B103 (Fig. 7), only fibers react with AB8 (Fig. 5) and only FG fibers react with 2E9 (Fig. 6). Neither FG nor fibers react with NA8 (Fig. 8) or NA2 (Fig. 9).

7 Rosser et al Figs. 1&15. Transverse serial sections of grey partridge pectoralis. Each scale bar = 50 p. Large arrowheads indicate FG fibers, and small arrows indicate fibers. Fig. 10, histochemical demonstration of NADH activity, revealing FG and fibers. Figs , respectively, immunocytochemical demonstration of labeling by primary antibodies AG6, AB8, 2E9, B103, and NA2. Throughout this series of figures the same FG and fiber are indicated by the large arrowhead and smaller arrow, respectively. While only FG fibers react with AG6 (Fig. 11) and AB8 (Fig. 12), only fibers react with B103 (Fig. 14). Fibers react with neither 2E9 (Fig. 13) nor NA2 (Fig. 15). Figs Transverse serial sections of sora rail pectoralis. Each scale bar = 50 p. Large arrowheads indicate FG fibers, and small arrows indicate fibers. Fig. 16, histochemical demonstration of NADH activity, showing FG and fibers. Figs. 17 and 18, respectively, immunocytochem~cal demonstration of labeling by primary antibodies EB 165 and B103. The same FG and fibers are shown throughout this series of figures by the large arrowhead and small arrow, respectively. While only fibers react with EB165 (Fig. 17), only FG fibers are labeled by B103 (Fig. 18).

8 Biochem. Cell Biol. Vol. 74, 1996 Figs Transverse serial sections of long-eared owl pectoralis. Each scale bar = 50 k. Small arrows indicate fibers. Fig. 19, histochemical demonstration of NADH activity, revealing fibers only. Fig. 20, immunocytochemical demonstration of labeling by primary antibody AG6. The same two fibers are indicated by the small arrows in these figures. While the fiber on the right is labeled by AG6, the fiber on the left is not. Only a minority of the fibers react with AG6. Fig. 21. Transverse section of great homed owl (juvenile) pectoralis muscle. Scale bar = 50 p. All of these fibers (determined by NADH histochemistry, not shown) are labeled by the primary antibody AG6. dilution of 1 :2000. Primary antibody dilutions were determined empirically and on the basis of their known reactivity with appropriate control muscles. Sections were then rinsed twice, 5 min for each rinse, with PBS. Binding of the primary antibodies was then visualized by incubating the sections for 30 min at room temperature with a fluoresceinated horse antimouse IgG secondary antibody (Vector Laboratories, Burlingame, Calif.) used at a dilution of 1:64 in PBS. Sections were then rinsed twice, 5 min for each rinse, in PBS and subsequently fixed for 3 min in 4% buffered formalin. Sections were again rinsed twice, 5 min for each rinse, in PBS and then mounted in citifluor mountant (Marivac Ltd., N.S.). Western blots SDS-PAGE and Western blot analyses were done to determine whether the antibody reactivity demonstrated by our immunocytochemical techniques was, in fact, a result of antibody affinity for MyHCs. For each species, a piece of pectoralis muscle (-0.5 cm3) was excised from the frozen tissue blocks embedded in O.C.T. compound. Excess O.C.T. compound was cut away at -20 C using arazor blade, and myosin was extracted from each piece of muscle (described by Bandman et al. 1982). The myosin samples were analyzed by SDS- PAGE on 10% polyacrylamide gels and electrophoretically transferred to nitrocellulose (see Bandman 1985). The nitrocellulose blots were then incubated with 5% nonfat milk powder - 3% Tween-20 in PBS (PBS - TM) for 30 min at 37 C. Subsequently, for 60 min at room temperature with agitation, each blot was incubated with one of the following monoclonal antibodies: NA4, EB165, AG6, or B103. The myosin extracted from each pectoralis was normalized to give relatively similar appearances when reacted with NA4 at 1 : The antibodies were diluted in PBS-TM as follows: NA4 (1 : 100 OOO), EB 165 (1 : 25 OOO), AG6 (1 : ) and B103 (1 : ). In addition, myosin from the gallinaceous birds (chicken, Japanese quail and grey partridge) was also similarly blotted and incubated with AB8 (1:5000) or 2E9 (1:2500). Other antibodies were not done because either our immunocytochemical results demonstrated that they did not react with the pectoralis samples or their reactivity with chicken muscle had been published elsewhere. The blots were then washed three times in PBS-TM over 15 min and subsequently incubated with horseradish-peroxidase-conjugated goat antimouse IgG (Cappel Laboratories, Pa., 1:1000 in PBS-TM) for 60 rnin at room temperature with agitation. The blots were then washed three times in a 0.1 M Tris buffer (ph 7.4) over 15 min. The blots were developed in 0.1 M Tris (ph 7.4) containing diaminobenzidine (0.2 mg/ml) - H,O, (0.024%). Results Histochemistry and immunocytochemistry The histochemical and immunocytochemical results for the 3 1 species studied are summarized in Table 2, where the birds are arranged phylogenetically according to Sibley and Ahlquist (1990) and Sibley and Munroe (1990). Figures 1-21 illustrate the histochemical and irnmunocytochemical profiles of those pectoralis muscles representative of the overall results. Fast-twitch fibers were readily differentiated as either FG or by their staining for NADH activity. In concurrence with earlier works (George and Berger 1966; Rosser and George 1986), fibers had comparatively smaller diameters and were darkly stained, and FG fibers had larger diameters and were lightly stained (Figs. 1, 10, 16, and 19). Unambiguous FG and fiber types were found in the pectoralis muscle of 11 species, including Japanese quail, grey partridge, rouen (domestic) duck, American wigeon, northern shoveler, domestic pigeon, sora rail, least sandpiper, northern harrier, American robin, and the red-winged blackbird. The pectoralis of the ostrich and the chicken each consisted chiefly of FG fibers (see following paragraph). The remaining 18 species had a pectoralis containing fibers only. The fibers could be discerned as red and intermediate subtypes in 9

9 Rosser et al. 723 Fig. 22. Western blot analyses of myosin extracted from avian pectoralis muscle. Myosin extracted from the pectoralis muscle of each species studied was subjected to SDS-PAGE and electrophoretically transferred to nitrocellulose for immunoblot analysis. Arrows indicate the mobility of molecular mass standards. The prominent band at 200 kda is the MyHC. The gels were organized as follows: Lane 1, ostrich; lane 2, domestic chicken; lane 3, Japanese quail; lane 4, grey partridge; lane 5, rouen (domestic) duck; lane 6, American wigeon; lane 7, northern shoveler; lane 8, northern flicker; lane 9, belted kingfisher; lane 10, mulga parrot; lane 11, short-eared owl; lane 12, great homed owl; lane 13, long-eared owl; lane 14, domestic pigeon; lane 15, sora rail; lane 16, merlin; lane 17, northern harrier; lane 18, Swainson's hawk; lane 19, American white pelican; lane 20, red-eyed vireo; lane 21, American crow; lane 22, Bohemian waxwing; lane 23, American robin; lane 24, house wren; lane 25, homed lark; lane 26, common canary; lane 27, zebra finch; lane 28, red-winged blackbird; lane 29, rufous-sided towhee. From each gel, a series of Western blots was prepared, which were then individually incubated with the NA4 antibody, the AG6 antibody, the EB165 antibody, or the B103 antibody. Results indicate that NA4 and EB165 reacted with MyHCs from all species studied. AG6 labeled MyHCs from all species except the short-eared (lane 11) and the long-eared (lane 13) owls. B103 reacted with MyHCs from Japanese quail (lane 3), sora rail (lane 15), American crow (lane 21), and zebra finch (lane 27). of these 18 species, including northern flicker, belted kingfisher, mulga parrot, long-eared owl, Swainson's hawk, American crow, American robin, homed lark, and red-winged blackbird. Intermediate fiber types can pose a problem when metabolic criteria are incorporated into fiber typing (see Rosser and George 1986). In the present study, the only slight difficulties that we encountered in this regard were with ostrich and domestic chicken pectoralis. While these muscles have been reported to contain minor populations of smaller-diameter moderately oxidative fibers (Rosser and George 1985, 1986; Edman 1988), the chicken pectoralis is usually considered to be a fairly homogeneous mass of FG fibers (see Bandman et al. 1990). For simplicity, in this study, we refer to all of the fast- twitch fibers in the chicken and the ostrich pectoralis as FG. The antibody NA4 reacted with every fiber, fast twitch and slow, in all samples (Fig. 2). This antibody appears to be a general marker for all sarcomeric myosin in that it also labels all MyHCs from developing fast-twitch chicken fibers (Moore et al. 1992) and all fiber types in rat extensor digitorum muscle (B.W.C. Rosser, unpublished results). AG6 reacted with almost all of the fast-twitch fibers in the pectoralis muscles that we studied (Figs. 3 and 21). The only exceptions were the grey partridge (Fig. 11) and two species of owl belonging to the genus Asio (Fig. 20). In the grey partridge, although all FG fibers reacted with AG6, none of the fibers did. In short-eared and long-eared owls, approxi-

10 Biochem. Cell Biol. Vol. 74, 1996 Fig. 23. Western blot analyses of myosin extracted from pectoralis muscle of galliforms. Myosin extracted was subjected to SDS-PAGE and electrophoretically transferred as in Fig. 22. The gels were organized as follows: lane 2, domestic chicken, lane 3, Japanese quail; and lane 4, grey partridge. From each gel, Western blots were incubated with either the AB8 antibody or the 2E9 antibody. Results indicate that AB8 reacted with MyHCs from the pectoralis of each of the three galliforms studied. 2E9 labelled MyHCs from Japanese quail pectoralis only. mately 30 and 25%, respectively, of the fibers in only the deep half of the pectoralis reacted with AG6. This antibody did not label any of the slow fibers that we studied. Similarly, EB165 reacted with almost all of the fast-twitch fibers that we studied (Fig. 4). The only exceptions were the FG fibers in the pectoralis muscles of both sora rail (Fig. 17) and pigeon. Nevertheless, the fibers of these two species were labeled by this antibody (Fig. 17). AB8 and 2E9 were confined in their reactivity to the order Galliformes. Throughout the bulk of the chicken pectoralis, all fast-twitch fibers are labeled with only AB8 in the adult and by only 2E9 in the neonate (Cerny and Bandman 1987). In the red strip, however, the fast-twitch fibers of the adult react with neither 2E9 nor AB8 (Shear et al. 1988). In Japanese quail and grey partridge, the muscle contained both FG and fibers (Figs. 1 and 10). In mature Japanese quail, the fibers were labeled by AB8 and the FG fibers by 2E9 (Figs. 5 and 6). In adult grey partridge, however, only the FG fibers were labeled by AB8 and neither fiber type reacted with 2E9 (Figs. 12 and 13). B103 reacted with the pectoralis of several phylogenetically diverse species. Previous work had shown that this antibody recognizes embryonic and neonatal MyHC isoforms in developing chicken pectoralis (Bandman and Bennett 1988). In this study, we also observed B103 reacting with the fasttwitch fibers in the red strip of mature chickens. In the other Galliforms, we found that B 103 labeled both the and the FG fibers in the adult Japanese quail pectoralis (Fig. 7) but only the and not the FG fibers in the grey partridge (Fig. 14). In addition, respectively 4 and 40% of the FG and fibers of the northern shoveler pectoralis were labeled by B103. This antibody also reacted with the occasional fiber () in the fledgling great horned owl; all FG but no fibers of both the sora rail (Fig. 18) and the least sandpiper; 13% of the FG fibers in the northern harrier; all fibers () in Swainson's hawk, American crow, and zebra finch; 87% of the fibers () in the juvenile house wren; and a single fiber in one of two red-winged blackbirds. B 103 also labeled the ends of muscle fibers in the pectoralis of the Anseriforms studied. In these birds, this antibody normally labels the pectoralis of neonates (E. Bandman, unpublished observations). Similarly, the tapered ends of the fast-twitch fibers in the chicken pectoralis were reported to contain both neonatal and adult MyHCs (Rosser et al. 1995). Antibodies against slow MyHCs (NA1, NA2, NA7, and NA8) reacted with only two of our samples: ostrich pectoralis and the deep red strip of the chicken pectoralis. ATPase fiber typing has shown that the ostrich pectoralis contains from 13 to 39% slow muscle fibers (Rosser and George 1985). Similarly, ATPase and immunocytochernical techniques have shown that the red strip of the chicken pectoralis normally contains about one third slow fibers (see Shear et al. 1988). Antibodies against slow MyHCs did not react with pectoralis from any other species that we studied (Figs. 8, 9, and 15). Conversely, of those antibodies labeling fast-twitch fibers, only NA4 was colocalized with the antibodies against slow fibers. HV11, which normally reacts with ventricular MyHC, also reacts with certain embryonic and regenerating avian skeletal muscle fibers (Hartley et al. 1991). It is expressed in all differentiating avian fibers and is considered to be a definitive marker for the activation of the myogenic program (Bourke et al. 1991). In the present study, its reactivity was restricted to just several FG and fibers in the pectoralis of the least sandpiper. Western blots Western blots for 29 of the 31 species studied are presented in Figs. 22 and 23. Westerns were not done for the two sandpipers. These blots not only confirmed our immunocytochemical findings, but also showed that the chicken monoclonal antibodies were recognizing MyHCs in the pectoralis of each of the other species studied. Almost all myosin extracts yielded a well-defined MyHC band, which indicated that the MyHCs had not degraded. The most noteworthy exception to this was that of the Bohemian waxwing pectoralis, in which there appeared to be some degradation of the MyHCs, as shown by the presence of several bands on each gel. The monoclonal antibodies NA4 and EB165 each labeled MyHCs from all species studied (Fig. 22). AG6 labeled MyHCs from all species except the long- and the

11 Rosser et al. short-eared owls (Fig. 22). B103 reacted with MyHCs from Japanese quail, sora rail, American crow, and zebra finch (Fig. 22). AB8 labeled MyHCs in the pectoralis of the domestic chicken (see also Cerny and Bandman 1987), Japanese quail, and grey partridge (Fig. 23). 2E9 reacted with MyHCs from the Japanese quail pectoralis (Fig. 23), as well as neonatal chicken pectoralis (not shown; see Cerny and Bandman 1987). Certain of our immunocytochemical results were not verified by the Westerns: AG6 in the long- and the short-eared owls and B 103 in grey partridge, northern shoveler, and northern harrier. In each of these instances, however, our immunocytochemical results indicated that the antibody reacted with a very minor proportion of the total fiber population. It seems reasonable to suggest, therefore, that the Western blots were not sensitive enough to detect the very low concentrations of labeled MyHCs in each of these muscles. Discussion Fiber type complement The belly of the pectoralis muscle consists almost entirely (99-100%) of fast-twitch fibers in those birds relying mainly on flapping flight (see Experimental model in Materials and methods). This observation is corroborated by our results showing that antibodies against slow MyHCs labeled only known slow fibers in the ostrich pectoralis and chicken red strip. These antibodies also labeled the slow fibers in the accessory deep belly of the pectoralis, found in soaring birds (Rosser et al. 1994). fibers are capable of sustained rapid contraction, and FG fibers of a more powerful but fast-fatiguing contraction (Peters 1989). fibers predominate in the pectoralis of all birds capable of migratory flight, and FG fibers are in the majority in the pectoralis of those relatively few species capable of only short-duration explosive flapping flight (see Rosser and George 1986). Although most smaller bodied migratory birds (such as sparrows, warblers, and hummingbirds) have a pectoralis consisting entirely of fibers, the pectoralis of larger bodied birds (such as pigeons, ducks, and herons) usually contains a minority of FG fibers (see Rosser and George 1986). Presumably these FG fibers assist in providing an increase in power during certain activities such as take-off (Rosser and George 1986; Welsford et al. 1991). In certain species, the intermediate subtype of the fibers may perform functions not unlike those of the FG fibers of larger species (Parker and George 1975; Tobalske 1996). The fiber type complement of the pectoralis muscle in the birds of this study largely concurs with published observations on 14 species, including ostrich, domestic chicken, Japanese quail, domestic duck, northern flicker, belted kingfisher, great homed owl, domestic pigeon, least sandpiper, northern harrier, white pelican, American crow, American robin, and redwinged blackbird (George and Berger 1966; Rosser and George 1985, 1986; Rosser et al. 1987, 1994; Tobalske 1996). For the remaining 17 species studied, the fiber type complement of the pectoralis has not been previously reported. For these species, however, a comparable fiber type complement has been shown in at least one related species in the same genus, family, or order. Differences in MyHC content between avian FG and fibers Differences in MyHC content were not found between FG and fibers in leg muscles of adult chickens (Williams and Dhoot 1992; Maier 1994). Similarly, earlier works did not demonstrate any difference between these two fiber types in the pectoralis muscles of Japanese quail (Memfield et al. 1989) or other Galliformes (Bandman et al. 1990; Maruyama et al. 1993). Fast-twitch fibers in the latissimus dorsi muscles of adult Japanese quail (Alway 1993) and turkey (Cherel et al. 1994) were also reported to be homogeneous in their MyHC content. However, Bandman and Bennett (1988) used a battery of monoclonal antibodies against chicken MyHCs to show that adult chicken gastrocnemius and posterior latissimus dorsi muscles contained, respectively, three and two different populations of fast-twitch fibers. Nevertheless, whether these fibers were either type FG or was not determined. We find clear differences in MyHC content between avian FG and fibers. This was the case in 5 of the 11 species having both fiber types, including Japanese quail, grey partridge, domestic pigeon, sora rail, and least sandpiper. Using monoclonal antibodies generated against chicken MyHCs, in each of these species we were able to differentiate all FG and fibers as typed by their NADH activity. A small proportion (13%) of FG fibers in a sixth species, northern harrier, could be differentiated from fibers in the same muscle. We could not discern any differences in MyHC content between FG and fibers in the remaining 5 species, including domestic duck, American wigeon, northern shoveler, American robin, and red-winged blackbird. This does not necessarily mean that in each of these species the MyHCs contained in the FG and the fibers were indistinguishable. Rather, it could indicate that the antibodies that we used were unable to detect any differences. In addition, we did not identify differences in MyHC content between the red and intermediate subtypes in nine of the species studied. Epitopes and isoforms The monoclonal antibodies used in this study were originally generated against specific MyHC isoforms from various developmental stages and muscles of the chicken, and each antibody has been shown to recognize one or more isoforms (see Table 1). In fact, each monoclonal antibody recognizes only a small specific epitope (amino acid sequence) at a precise location on each MyHC molecule (Moore et al. 1992). The same epitope may appear on several different MyHC isoforms and, thus, the antibody against that epitope will recognize each isoform containing the epitope. None of the antibodies that we use has been shown to recognize more than one epitope. The question then arises: is a monoclonal antibody that recognizes a specific epitope in chicken recognizing the identical amino acid sequence when it labels fibers from another species? The answer is that we do not know. However, for the purposes of this paper, this is not crucial. We identify pronounced heterogeneity of MyHCs among the fast-twitch fibers of the avian pectoralis muscle, and any interpretation of our data will support this. For example, the antibody AB8 labels (see Table 2) almost all fast-twitch fibers in adult chicken pectoralis, only FG but not fibers in grey par-

12 Biochern. Cell Biol. Vol. 74, 1996 Table 3. Epitopes contained in fast-twitch MyHC isofonns in chicken vectoralis muscle. Isofom Embryonic 1 and 3 Embryonic 2 Neonatal Adult Epitopes Note: Each epitope is named for the monoclonal antibody that labels it. Table 4. Four new MyCHs in the avian pectoralis muscle and their epitopes. MyHC Owl () Partridgelowl () Pigeon (FG) Rail (FG) Epitopes tridge, and only fibers but not FG fibers in the Japanese quail. The simplest interpretation of these observations is that there are two different MyHC isoforms; one with and one without the AB8 epitope. Thus, we demonstrate heterogeneity of MyHCs. On the other hand, if the antibody is recognizing more than one epitope, at least three MyHC isoforms would be present: that with the AB8-1 epitope, that with the AB8-2 epitope, and that with neither epitope. Using this interpretation, we demonstrate an even greater heterogeneity. Therefore, for the purposes of this paper, whether each antibody is recognizing the same epitope in each species does not alter our overall conclusion of MyHC heterogeneity. How many avian fast-twitch MyHCs? Bandman and colleagues (1990, 1994) have identified at least three embryonic, one neonatal, and one adult fast-twitch MyHC isoform in chicken pectoralis muscle (Table 3). The most conservative interpretation of our results is that in which each antibody recognizes the identical epitope in each species, and it is possible for all mature avian pectoralis fibers of a particular type to contain more than one MyHC isoform. Even with this cautious interpretation, we can discern at least four new MyHCs in the avian pectoralis muscle (Table 4). It would be an overstatement to claim that we have shown four new avian isoforms because, the same isofom can presumably vary slightly from species to species. We have, however, demonstrated at least four new structural varieties of avian MyHCs. Evolution of MyHCs The first birds appeared some 150 x lo6 years ago (Sibley and Ahlquist 1990; Feduccia 1995), and the radiation of birds into the modem orders occurred about 65 x lo6 years ago (Feduccia 1995). It is thought that the chicken myosin heavy-chain gene family has expanded within the last 70 x lo6 years (Moore et al. 1993). In this study, the monoclonal antibodies 2E9 and AB8 labeled MyHCs in only those birds belonging to the order Galliformes, including chicken, Japanese quail, and grey partridge. These antibodies also labeled MyHCs in the turkey (Mamyama et al. 1993). All four of these species are close relatives within the family Phasianidae. Within class Aves, Anserifoms (ducks and geese) diverged from the same stock as Galliforms (Sibley and Ahlquist 1990). Yet, Anseriform MyHCs are labeled by neither 2E9 nor AB8. It seems reasonable to postulate, therefore, that these antibodies are labeling epitopes that appeared since the emergence of the order Galliformes. Studies of other Galliform families could provide further insight into the evolution of avian MyHCs. Functional considerations Different MyHC isoforms have each been correlated with contractile properties of fast-twitch fibers in the avian pectoralis (Reiser et al. 1988) and mammalian muscle (Bottinelli et al. 1994). Other factors, however, such as myosin light-chain composition (Bottinelli et al. 1994; VanBuren et al. 1994) and metabolic criteria (Nemeth et al. 1991) have also been correlated with physiologic properties of muscle fibers. While FG and fibers each have distinctive contractile properties, in our study there appears to be no general correlations between monoclonal antibody labeling and fiber type. There are several possible explanations for this apparent lack of correlation. The particular epitope labeled by an antibody may not be crucial in determining the contractile properties of the MyHC molecule. Conversely, the antibody could be recognizing different epitopes in different species. Also, as indicated in the preceding, there are several other determinants of muscle fiber function. It has been suggested that the diversity of contractile function in birds may be less dependent on MyHC isoform composition than that in mammals (Bandman et al. 1994). However, the presence of different MyHCs in avian and FG fiber types does suggests a correlation between MyHC content and contractile function. Acknowledgements The authors thank the many associates and colleagues in Saskatoon and at the University of Saskatchewan who provided the specimens. The following individuals are acknowledged for their cooperation and generosity: Ms. Claire Bullaro and Mr. Ken King of Saskatoon; Drs. Gary Bortolotti and Karen Wiebe of the Department of Biology; Dr. Helene Philibert of the Department of Veterinary Pathology; Drs. Jocelyn Hudon and Lynn W. Oliphant and Mr. Pat Thompson of the Department of Veterinary Anatomy; Dr. Hank Classen and Mr. Robert Gonda of the Department of Animal and Poultry Science; and Dr. Cheri L. Gratto-Trevor of the Canadian Wildlife Service. This study was supported by operating grants awarded to B.W.C.R. by the Saskatchewan Health Research Board and by the Natural Sciences and Engineering Research Council of Canada. References Alway, S.E Stretch induces non-uniform isomyosin expression in the quail anterior latissimus dorsi muscle. Anat. Rec. 237: 1-7. Bandman, E Continued expression of neonatal myosin heavy chain in adult dystrophic skeletal muscle. Science (Washington, D.C.), 227: Bandman, E., and Bennett, T Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep

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