THE ANATOMICAL RECORD 246185-194 (1996) Myosin Heavy Chain Isoforms in Adult Equine Skeletal Muscle: An lmmunohistochemical and Electrophoretic Study JOSk-LUIS L. RIVERO, ROBERT J. TALMADGE, AND V. REGGIE EDGERTON Department of Comparative Anatomy and Pathological Anatomy, Faculty of Veterinary Science, University of Cordoba, Spain (J.-L.L.R.); and Department of Physiological Science and Brain Research Institute (R.J.T., V.RX.), University of California, Los Angeles, California ABSTRACT B a c k g r o u d The aim of this study was to characterize the myosin heavy chain (MyHC) isoforms present in equine skeletal muscle. Methods: Muscle biopsies were removed from the superficial region of the gluteus medius muscle of five mature horses and analyzed by immunohistochemistry (using a battery of monoclonal antibodies specific for rat MyHC isoforms) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results: Immunohistochemistry allowed subdivision of three different muscle fiber populations containing a single MyHC, one slow and two fast, and two hybrid populations, one containing slow and fast MyHCs and another with both fast-MyHC isoforms. Electrophoresis of MyHC confiimed the existence of three resolvable bands, with an electrophoretic mobility parallel to type I, IIa, and IIx rat MyHCs. The identities of two of these MyHCs were easily comparable with slow type I and fast type IIa MyHCs from rat skeletal muscle. However, a precise identification of the second fast MyHC was not made. Conclusions: These results show the presence of three different MyHC isoforms in mature equine skeletal muscle, whose differential distribution defines three fiber types containing a single MyHC and two hybrid fiber populations containing either both slow and fast type IIa MyHCs or both fast MyHC isoforms. o 19% Wiley-Liss, Inc. Key words: Horse, Gluteus medius, Muscle fiber type, Electrophoresis Myosin is the predominant protein in skeletal muscle, and it makes up the largest portion of the contractile apparatus of muscle fibers. This protein consists of four light chains and two heavy chains. Myosin heavy chain (MyHC) isoforms are encoded by a multigene family (Mahdavi et al., 1987). To date, nine distinct MyHC isoforms have been identified in adult skeletal muscles of a number of species (for reviews, see Pette and Staron, 1990; Schiaffino and Reggiani, 1994). Of these, three MyHCs exist in many species: the p, slow, or type I MyHC and the two fast (IIa and IIb) MyHCs. The differential distribution of these MyHCs defines three main fiber types containing a single MyHC isoform (types I, IIA, and IIB) and a number of hybrid fiber populations containing both I and IIa MyHCs (type C fibers) and IIa and IIb MyHCs (type IIAB fibers). An additional fast MyHC isoform, termed 1Ix or IId, and encoded by a specific mRNA (De Nardi et al., 19931, has been identified in muscles of rat, mouse, guinea pig, and rabbit (Bar and Pette, 1988; Schiaffino et al., 1989; Gorza, 1990; Aigner et al., 1993; Hamalainen and Pette, 1993) by using monoclonal antibodies and gel electrophoretic techniques. The unique expression of this MyHC isoform in a single muscle fiber defines the type IIX or IID muscle fiber type, al0 1996 WILEY-LISS, INC. though its coexpression with other MyHCs also occurs under normal conditions and during fiber type transformation (Termin et al., 1989; Talmadge et al., 1995a). However, in humans the MyHC isoform found in IIB fibers is equivalent t o rat IIx MyHC, not to rat IIb MyHC (Smerdu et al., 1994; Ennion et al., 1995). Several observations have suggested that the differences in MyHC content of single muscle fibers contribute significantly to the differences in maximum shortening velocity (Reiser et al., 1985; Sweeney et al., 1988; Larsson and MOSS,1993; Bottinelli et al., 1991, 1994) and histochemical myofibrillar ATPase activity (Pette and Staron, 1990; Talmadge et al., 1995b), and to the differences in morphological, physiological and biochemical properties among motor units (Larsson et al., 1991). In addition to the four main MyHC isoforms previously described, another slow (slow-tonic MyHC), two fast (types Eom- and IIm-MyHCs), and two specific de- Received October 25, 1995; accepted February 27, 1996. Address reprint requests to JosB-LuisL. Ftivero, Ph.D., Department of Veterinary Anatomy, Faculty of Veterinary Science, University of Cordoba, Medina Azahara 9, 14005 Cordoba, Spain. 186 J.-L.L. RIVER0 E T AL. velopmental MyHCs, the embryonic and neonatal isoforms, are expressed in the extraocular musculature, in muscles derived from the first branchial arch, in developing muscles, and under certain pathological conditions (Pette and Staron, 1990). Some studies have investigated the composition of MyHC isoforms in equine skeletal muscle by using immunohistochemistry (Snow et al., 1981; Sinha et al., 1992) or gel electrophoresis (Billeter et al., 1987; Sosnicki et al., 1989; Yamaguchi et al., 1993; Barrey et al., 1995) or a combination of both methods (Hermanson et al., 1991; Cobb et al., 1994). The content of MyHCs of five equine muscles has also been investigated by using enzyme-linked immunosorbent assay (Barrey et al., 1995).All of these investigations distinguished two different MyHCs: slow and fast. However, none of them was able to separate either different fast MyHCs or different fast-twitch muscle fiber types according to difference in MyHC content. Recently, the gluteus medius of the horse has been found to contain two fast MyHCs with differing electrophoretic mobilities as determined by the protocol for sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) separation of MyHC proposed by LaFramboise et al. (1990; Serrano et al., 1996). Nevertheless, no clear dichotomy was found between the two bands. A recent improved SDSPAGE technique has provided high-resolution separation of MyHC isoforms (Talmadge and Roy, 1993).Similarly, over the past few years a large number of monoclonal antibodies to specific MyHCs in rat skeletal muscles has been produced (Ecob-Prince et al., 1989; Schiaffino et al., 1989; Hughes et al., 1993), and these have proved useful for identifying different types of MyHC in several species. In this study we examined muscle biopsies from the equine gluteus medius to characterize the MyHC content of single muscle fibers by using a battery of monoclonal antibodies and homogenates of these biopsy specimens by a sensitive 8% SDS-PAGE technique. This study provides an immunohistochemical evaluation of MyHC isoforms and a gel electrophoretic separation of MyHCs. These results provide a basis for comparisons of skeletal muscle fiber types in horses and other several mammalian species. In the present study we compared properties of MyHCs in equine skeletal muscle with those previously described for the rat (Schiaffno et al., 1989). selected (2 cm below the gluteal fascia) to obtain a high percentage of type I1 fibers in biopsy specimens. After collection, muscle samples were mounted on cork blocks using OCT embedding medium and oriented so that myofibers could be cut transversely (Dubowitz, 1985). Specimens were systematically frozen by immersion in isopentane (30 sec) and kept at freezing point in liquid nitrogen a t -160°C (Dubowitz, 1985). Muscle samples were transported in dry ice for 48 hr and stored at -80°C until analyzed. lmmunohistochemistry Samples from the rat medial gastrocnemius muscle were also included as a control in the immunohistochemical analysis. Transverse serial sections of 10 Fm thickness were obtained on a cryostat microtome (Reichert Jung 2800 Frigocut E) at -20°C and mounted on gelatin-coated glass slides. The serial sections were reacted with a series of 14 different monoclonal antibodies (primary antibodies, MAbs) specific to rat MyHCs. The specificity of these MAbs against MyHC isoforms in rat skeletal muscle is presented in Table 1. The avidin-biotin peroxidase complex (ABC) immunohistochemical procedure was used for the localization of primary antibody binding following instructions for kits PK-6102 and AK-5010 (Vector Laboratories, Burlingame, CA, USA). Phosphate buffered saline (PBS) was used as a buffer for all IgG class primary antibodies and Tris buffered saline (TBS) for all IgM class primary antibodies. Tissue sections were allowed to warm to room temperature for 10-20 min and then rehydrated with buffer (either PBS or TBS) for 10-20 min. Samples were subsequently preincubated in a 1.5% blocking solution of either stock horse or goat serum in PBS or TBS, respectively, at room temperature. Following preincubation, excess blocking solution was removed, and the primary antibody was applied and allowed to incubate overnight in a humid chamber a t 4°C. The sections were washed in buffer for 10 min and then reacted with a biotinylated second antibody for 60 min at room temperature. Sections were again washed with buffer for 10 min and reacted in ABC reagent for 60 min at room temperature. Diaminobenzidine tetrahydrochloride (DAB, kit SK-4100, Vector Laboratories) was used as chromogen to localize peroxidase in all IgG class primary antibodies (2-4 min at MATERIAL AND METHODS room temperature), and a premixed BCIP/NBT soluMuscle Samples tion (Sigma B-6404) containing levamisole (Vector Percutaneous needle muscle biopsies were obtained Laboratories, SP-5000) was used to reveal the reaction from the right gluteus medius muscle of five clinically in all IgM class primary antibodies. After staining, healthy adult horses (three 3-year-old active thorough- slides were soaked for 10 min in distilled water, dehybred mares and two inactive Andalusian stallions, one drated in graded ethanol series, and coverslipped with 13 years old and one 17 years old) according to the permount. The same region of biopsy specimens was analyzed technique outlined by Lindholm and Piehl(l974). The gluteus medius was selected because it is the muscle for all 14 different MAbs by using a computer-enmost frequently sampled when studying the effects of hanced image processing system connnected to a vidgrowth, training, and performance in the equine ath- eoprinter. This system includes an image normalizing lete because it is a major propulsive muscle active in procedure based on gray levels to allow a more objeclocomotion and is easily accessible (Lindholm and tive categorization of the staining intensity of each Piehl, 1974). The histochemical fiber type distribution muscle fiber. Thus, the reactivity of -125 fibers per of this muscle varies extensively as a function of sam- biopsy specimen against the different MAbs was studpling depth and probably reflects different functional ied and correlated. In addition, stained cross sections demands on this muscle (L6pez-Rivero et al., 1992). In were photographed on an Olympus BH-2 microscope this study, a relatively superficial sampling site was with a Nikon camera attachment. 187 MYOSIN HEAVY CHAIN IN HORSES TABLE 1. Specificity of the monoclonal antibodies (MAbs) against rat skeletal muscle myosin heavy chain (MsHC) used in this studv' MyHC Isoforms IIb Emb MAb I IIa IIX BF-B6" - - - - ++ ++- + + + + - + ++ +- - t N ~ O ~ - BA-G!j2 - BF-452 F5g4 slow5 Fast5 SC-712 BF-F3' A4.743 RT-D~~ - N2.2613 BF-352 BF-G62 + +- - + +- + +- - - - - - - - - * + - + + ~ - Neo a + - - - +- +? + + + +- - - - - - - - - - - - + + + - - - Reference Schiaffino et al. (1988) Ecob-Prince et al. (1989) Kucera et al. (1992) Schiaffino et al. (1988) Miller et al. (1985) Ecob-Prince et al. (1989) Ecob-Prince et al. (1989) Schiaffino et al. (1989) Schiaffino et al. (1989) Hughes et al. (1993) Schiaffino et al. (1989) Hughes et al. (1993) Schiaffino et al. (1989) Schiaffino et al. (1988) 'Each MAb bound to specific MyHC isoforms as determined by the references listed and suppliers' instructions. + , Positive reaction for that MAb with that specific MyHC isoform; -, no reaction between MAb and MyHC isoform. Antibody BF-G6 reacted primarily with embryonic (Emb) MyHC; however, this MAb also bound type Ilb MyHC at a lower intensity (5). All MAbs used were type immunoglobulin (Ig) G, except MAbs BF-F3 and RT-D9, which were IgM. Neo, neonatal; a,a-cardiac. 2From Dr. S. Schiafino (University of Padova, Italy). 3From the Developmental Studies Hybridoma Bank. 4From Dr. Stockdale (Stanford University, CAI. 'From Novocastra. nemius muscle according to their MyHC content (Fig. 1 In addition to cross sections for immunohistochemis- and Table 2). No fibers were labeled with MAb BF-B6, try, 30 cross sections of 20 pm thickness were obtained indicating that no embryonic or neonatal MyHCs were from each biopsy sample on the cryostat, placed in pre- present in this control muscle. The fibers that were cooled (-20°C) microcentrifuge tubes, and stored at labeled with the MAb that reacts with slow MyHC and -70°C in preparation for myofibrillar protein isolation. unlabeled with MAb fast were identified as type I (fiIsolated myofibrils were prepared from these cross sec- bers containing type I MyHC only, e.g., fiber labeled 1 tions according to Thomason et al. (1986). Briefly, myo- in Fig. 1).A few fibers reacted positively with the antifibrils were extracted from minced cross sections in slow and anti-fast MAbs and with MAb SC-71, but not small aliquots (200 pl) of an ice-cold homogenization with RT-D9 and BF-F3; these fibers (e.g., fiber labeled buffer [250 mM sucrose, 100 mM KC1, 5 mM EDTA, 2 in Fig. 1)contained type I and IIa MyHCs and correand 20 mM tris (hydroxymethyl) aminomethane (Tris), spond with classical type IIC fibers. Some fibers were pH 6.81. The samples were subsequently homogenized labeled with the MAb directed against all fast MyHCs by hand with a micropestle. Extracts were then centri- and with MAb SC-71 and BF-35, but not with the other fuged at 1,000 rpm for 10 min at room temperature. MAbs, demonstrating that they contained type IIa The supernatant was discarded, and sediment was re- MyHC only (type IIA, e.g., fiber labeled 3 in Fig. 1).In suspended in the same volume of a n ice-cold premixed addition, a few fibers in the control rat muscle stained resuspension solution (150 mM KC1 and 20 mM Tris, positively with MAbs fast, SC-71, and RT-D9, but not pH 7.0). The protein concentration of the final myo- with slow and BF-F3; thus, these fibers (type IIAX, fibrillar suspension was assayed after Bradford (1976). e.g., fiber 4 in Fig. 1)contained types IIa and IIx MyMyofibrillar protein was then boiled in sample buffer HCs. Many fibers were labeled with MAb RT-D9 but (Laemmli, 1970) for 2 min a t a final concentration of unlabeled with MAbs BF-35 and BF-F3, so these fibers (type IIX, e.g., fiber 5 in Fig. 1) contained type IIx 0.250 mg of proteidml of sample buffer, MyHC exclusively. A very low proportion of fibers Myosin heavy chain electrophoresis was performed following the protocol for SDS-PAGE separation de- (termed type IIXB, not shown) were demonstrated to scribed in detail by Talmadge and Roy (1993). Accord- coexpress type IIx and IIb MyHCs; these fibers were ingly, 20-30-pl aliquots of diluted myofibrillar protein unreacted with MAb BF-35 but reacted positively with were electrophoresed in a large-gel apparatus (CBS MAbs RT-D9 and BF-F3. Finally, some fibers were laScientific SG-200) placed in a Styrofoam box containing beled with the MAb that reacted with IIb MyHC exclucooling packs to maintain the temperature below 10°C. sively (BF-F3) and with MAbs BF-35 (specific to all Separating gels were stained with Coomasie blue, MyHCs except 11x1, RT-D9 (directed against type IIx dried, and scanned with an Alpha Innotech IS-1,000 and IIb MyHCs), and BF-G6 (that also bound type IIb videoscanning densitometric system, and photo- MHC at a lower intensity); therefore, these fibers (type IIB; e.g., fiber labeled 7 in Fig. 1)contained unequivographed. cally type IIb MyHC only. Electrophoresis RESULTS lmmunohistochemistry Rat Eight of the 14 MAbs used in this study allowed us to characterize seven groups of fibers in the rat gastroc- Horse The reactivity patterns of all MAbs to MyHC isoforms in horse muscle fibers are summarized in Table 3. The MAbs that proved most efficient for the discrim- Fig. 1. Serial cross sections of rat control medial gastrocnemius muscle stained with some monoclonal antibodies directed against specific MyHC isoforms (see Table 1 for specificities). A BF-B6. B Slow. C: Fast. D: SC-71. E BF-35. F BF-G6. G RT-D9. H BF-F3. The fibers labeled 1, 3, 5, and 7 contain type I, IIa, IIx, and 1% MyHCs, respectively; fiber 2 contains type I and IIa MyHCs; and fiber 4 contains type IIa and IIx MyHCs. Bar = 100 pm. 189 MYOSIN HEAVY CHAIN IN HORSES TABLE 2. Immunohistochemical identification of muscle fiber types in the rat according to their myosin heavy chain (MvHC) content' MAb BF-B6 Slow Fast SC-71 BF-35 BF-G6 RT-D9 BF-F3 1. I (I) 2. IIC (I+IIa) 3. IIA (IIa) Fiber types 4. IIAX 5. IIX (IIa+IIx) (11~) 6. IIXB (IIx + IIb) 7. IIB (IIb) 'MAb, monoclonal antibody;the number of each fiber type (1-7) corresponds to those in Figure 1, except fiber 6 (not shown). + , -, and 2: positive, negative, and moderate reaction, respectively. ination of fiber types in horses are illustrated in Figure 2. No fibers were labeled with MAbs BF-B6 (Fig. 2A), Neo (not shown in Fig. 21, or BA-G5 (not shown), indicating that no embryonic, neonatal, or a-cardiac MyHCs were present in the gluteus medius muscle of adult horses. Conversely, all fibers were labeled with anti-myosin MAb F59 used in this study (Fig. 2B). The fibers that reacted with the MAb Slow (Fig. 2C) directed against slow MyHC (e.g., fiber labeled 1 in Fig. 2) were identified as type I fibers. Many fibers were labeled with MAb fast (Fig. 2D), reacting with all fast (type 11) MyHCs in rats; these fibers were identified as type I1 fibers. A high proportion of these fibers were also positive for MAbs SC-71 (Fig. 2E), A474 (Fig. 2F), N2.261 (Fig. 2G), and BF-35 (Fig. 2H). Because all these MAbs bind to IIa-MyHC in rat, these fibers were designated as containing type IIa MyHC only (e.g., fiber labeled 2 in Fig. 2) and were identified as type IIA fibers. Another high proportion of fibers reacted positively with MAb against fast MyHCs but were negative with MAbs SC-71, A4.74, N2.261, and BF-35 (e.g., fiber labeled 3 in Fig. 2). These fibers were considered as containing a MyHC isotype other than IIa MyHC (IIb or 11x1 and were identified as type IIB fibers because they correspond mainly to fibers typed by myofibrillar ATPase as IIB (River0 et al., 1996). A few fibers (- 10-15%) that stained positively to all fastMyHCs were demonstrated to coexpress the two fast MyHCs (e.g., fiber labeled 4 in Fig. 2). These fibers reacted inconsistently (positive or negative) with MAbs SC-71, BF-35, and BF-G6 but were always positive to MAb N2.261 and negative to MAb A4.74. They were identified as type IIAB fibers. In addition, a few fibers (<1-2%) reacted positively with anti-slow and anti-fast MyHCs MAbs and with MAbs SC-71, A4.74, N2.261, BF-35, and BF-G6. These fibers (Fig. 3) contain both type I and IIa MyHCs and were identified as type IIC. In summary, MAbs allowed to identify five groups of fibers in horse skeletal muscle (Table 3 and Fig. 4). Surprisingly, the anti-IIb and antiembryonic MyHC BF-G6 MAb in rat muscle reacted with equine muscle fibers containing type I or IIa MyHC isofoms (Fig. 21). Similarly, the antiembryonic MyHC BF-45 MAb for rat muscle also reacted positively in horse muscle fibers containing type I-MyHC isoform (Fig. W). No specificity was found for RT-D9 and BF-F3 MAbs in horse muscle (Fig. 2K,L). No fibers were labeled with the BF-F3 MAb, and all fibers containing fast MyHCs were labeled with RT-D9, although the intensity of staining in fibers containing IIa-MyHC only was weaker than in those fibers containing only the other fast MyHC. Electrophoresis Electrophoresis of MyHC from gluteus medius muscle biopsies of adult horses by using 8% SDS-PAGE produced a total of three resolvable bands (Fig. 5). The identity of the MyHCs in each of the three bands was compared with the mobility of MyHCs in rat skeletal muscle (Talmadge and Roy, 1993) in order of mobility I > IIb or IIx > IIa. Type I MyHC was the fastest-migrating band (lower band), and type IIa MyHC was the slowest-migrating band (upper band). The second-fastest MyHC migrated to approximately the same level as IIx MyHC of rat muscle (Talmadge and Roy, 1993). DISCUSSION By using different MAbs, it was possible to identify four major MyHC isoforms in rat muscle: the slow or type I MyHC and the three fast IIa, IIx, and IIb MyHC. The differential distribution of these MyHCs defines four major fiber types containing a single MyHC isoform (I, IIA, IIX, and IIB) and three hybrid fiber populations containing type I and IIa MyHC (fibers type IIC), IIa and IIx MyHC (type IIAX), and type IIx and In3 (type IIXB). These results are consistent with previous studies in rats (De Nardi et al., 1993; Schiaffino and Reggiani, 1994; Talmadge et al., 1995a). No fibers containing all three fast MyHC isoforms (IIa + IIx + IIb) were observed in the present study (Bottinelli et al., 1994; Talmadge et al., 1995a). Likewise, we could not distinguish between type IIXB (fibers containing a larger amount of IIx than of IIb MyHC) and IIBX (fibers containing a larger amount of IIb and a smaller amount of IIx MyHC fibers; Bottinelli et al., 1994). Conversely, only three different MyHC isoforms in horse skeletal muscle were identified by using immunohistochemical and electrophoretic techniques: one slow and two fast isoforms. With multiple MAbs against MyHC isoforms, immunohistochemstry allowed subdivision of the fibers in equine skeletal muscle into five major types on the basis of their MyHC composition. No previous immunohistochemical studies have reported two different fast MyHC isoforms in equine skeletal muscle. Snow et al. (19811, using typespecific anti-rabbit myosin sera, observed that type I fibers (identified histochemically) contained only slow 190 J.-L.L. RIVER0 ET AL. Fig. 2. Serial cross sections of the gluteus medius muscle of one horse stained with a number of monoclonal antibodies against specific MyHC isoforms. A BF-B6. B: F59. C: Slow. D: Fast. E: SC-71. F: A4.74. G N2.261. H BF-35. I: BF-G6. J BF-45. K: RT-D9. L BF-F3. The fibers labeled 1 , 2 , 3 , and 4 contain type I MyHC, type IIa MyHC, type IIb or IIx MyHCs, and type IIa and IIb or IIx MyHCs, respectively. Bar = 250 pm. myosin, whereas fibers typed by myofibrillar ATPase histochemical staining into IIA and IIB fibers contained only a single fast myosin isoform. In a more recent immunocytochemical study using anti-slow (54D), anti-fast (lAlO), and anti-fast red (5-2B) MAbs with cross reactivity for type I, all type I1 and type IIa MyHCs, respectively, in a number of species, did not enable subclassification of type I1 MyHCs in equine gluteus medius muscle (Sinha et al., 1992). Similarly, a clear discrimination between fibers containing either 191 MYOSIN HEAVY CHAIN I N HORSES TABLE 3. Pattern of reactivity of the various monoclonal antibodies against MyHC in horse muscle fiber tmes MAb BF-B6 Neo BA-G5 F59 Slow BF-45 Fast SC-71 A4.74 N2.261 BF-35 BF-G6 RT-D9 BF-F3 Dilution 1:5.000 150 1:lOO 1:5 1:lOO 1:lO 1:200 1:100,000 1:lOO 1:lOO 1:20,000 1:5,000 1:1,000 1:lO 1. I (1) - IIC* (I + IIa) - Fiber Types (MyHC content) 2. IIA 3. IIAB (IIa) (IIa + IIb/IIx) - - 4. IIB (IIb/IIx) - - + + + + + + + + +- MAb, monoclonal antibody.The number of each fiber type (1-4)corresponds t o those in Figure 2. The fiber type IIC (*) is shown in Figure 3. +, -, 2,positive, negative, and intermediate reaction, respectively. slow or fast MyHCs has been obtained by immunohistochemistry in both horse diaphragm (Cobb et al., 1994) and gluteus medius muscle (Serrano et al., 1996), but subdivision of type I1 fibers was not possible. Nevertheless, only MAbs directed against slow and all fast MyHC isoforms were tested in these latter studies. Analysis of MyHC gels confirmed the existence of three MyHC isoforms in the superficial region of the equine gluteus medius muscle (Fig. 5). Using one- and two-dimensional gel electrophoresis of proteins from lyophilized microdissected single equine muscle fibers, Billeter et al. (1987) reported distinct band patterns for only one slow and one fast MyHC isoforms. Later, electrophoretic methods similar to those used in the present study were applied on samples from equine biceps brachii (Hermanson et al., 1991) and diaphragm (Cobb et al., 19941, but only a single fast MyHC was observed. The difference between these two studies and the present results might be explained by the fact that horse biceps brachii (Hermanson et al., 1991) and diaphragm (Cobb et al., 1994) muscles are exclusively composed of type I and type IIA muscle fibers. Sosnicki et al. (1989) identified fast and slow fiber types in horse skeletal muscle by differences in the mobility of their MyHCs. In that study, there was also an indication of a slight difference in mobility between fast fiber subtypes, but this difference was inconsistent. SDS-PAGE analysis of histochemically pretyped single fibers was used in that study. Similar observations were reported in two more recent studies of equine skeletal muscles by using a 5-8% SDS-PAGE separating gel with 2540% glycerol (Yamaguchi et al., 1993; Barrey et al., 1995). In the latter study, it was concluded that an enzyme-linked immunoassay method made it possible to measure a wide range of MyHC contents in equine muscles, but the technique only used two complementary monoclonal antibodies specific against slow and all fast MyHC isoforms. In a recent study, two fast MyHC isoforms were identified in samples of equine gluteus medius muscle by using a 6% SDS-PAGE technique (Serrano et al., 19961, but a clear differentiation of the two bands was not evident, and quantification by densitometric analysis of MyHC bands was not possible. In the present study, the electrophoretic method resulted in a consistent differentation of two type I1 horse MyHC isoforms (Fig. 5). The fastest-migrating band observed in the present study had an electrophoretic mobility identical to that of type 1 MyHC in rat muscle (Talmadge and Roy, 1993), whereas the slowest-migrating MyHC band comigrated with rat IIa MyHC. Moreover, all MAbs that bound to I and IIa MyHC isotypes in rat skeletal muscle showed a clear consistency in reactivity with different fiber types in horses. In consequence, on the basis of MyHC analysis, it seems clear that type I and type IIa MyHC isoforms exist in the equine gluteus medius. By contrast, the identity of the second fast MyHC (middle band in Fig. 5) was difficult to determine. We have adopted the terminology used in other mammalian species such as rat, mouse, guinea pig, and rabbit to design the fast MyHC isoforms in horse muscle: IIa, IIx, and IIb (Schiaffino et al., 1989). This adoption is based on the assumption that the structure of each MyHC isotype is generally conserved between species, whereas greater sequence divergence may be found between different isotypes within the same species (Schiaffho and Reggiani, 1994). However, some important differences in the primary structure of the second fast MyHC isoform identified in horse muscle with regard to those of IIx and IIb MyHCs in rat were evident from the different staining pattern of some MAbs in both species. Thus, the unequal reactivity of MAbs BF-G6, RT-D9, and BF-F3 between rat and horse muscles (Tables 2, 3) clearly show that epitopes recognized by these MAbs in type IIb MyHC isoform of the rat are not contained in the fast MyHC isoform of the horse other than the IIa isotype. Moreover, this fast MyHC was not recognized by the MAb BF-35, which binds to all MyHCs in rat except the IIx isotype (Schiaffino et al., 1989). Because its electrophoretic mobility was closer t o type IIx than to type IIb 192 J.-L.L. RIVER0 ET AL. Fig. 3. Serial cross sections of the gluteus medius muscle of one horse stained with slow (A), fast (B), and SC-71 (C) monoclonal antibodies. The arrow indicates a fiber coexpressing type I and IIa MyHC isoforms. Bar = 100 p m . MyHC in rat muscles, a possible explanation could be that this equine MyHC is a type IIb MyHC but with a higher molecular weight than rat type IIb-MyHC. Another plausible explanation, more in agreement with our immunohistochemical and electrophoretic findings, might be that this equine MyHC could be more closely related to rat type IIx MyHC than to a type IIb MyHC. Two human skeletal MyHC genes have been identified for fast IIa and IIx MyHCs based on pattern of expression and sequence homology with corresponding rat genes (Smerdu et al., 1994; Ennion et al., 1995). The distribution of these IIa and IIx MyHC transcripts defines two major fast muscle fiber types expressing a single MyHC mRNA, i.e., either IIa or IIx MyHC RNA. Fiber typing by ATPase histochemistry showed that IIa MyHC transcripts are more abundant in histochemical type IIA fibers, whereas IIx MyHC transcripts are more abundant in type IIB fibers (Smerdu et al., 1994). This observation strongly suggests that the so-called human IIB fibers actually express a MyHC isoform equivalent to the rat IIx MyHC isoform and not to the rat IIb isoform and would therefore be more accurately classified as IIX fibers (Ennion et al., 1995). Further studies are required to confirm if a similar situation occurs with those equine muscle fibers that contain a fast MyHC isoform other than IIa MyHC isoform. Our immunohistochemical results also show a cross reactivity of some MAbs between rat and horse MyHC isoforms. Thus, type I and IIa MyHC isoforms in the horse have an epitope common to type IIb MyHC in rat (recognized by MAb BF-GG), and type I MyHC of horses has another epitope common to embryonic MyHC of the rat (recognized by MAbs BF-G6 and BF-45). The present results also indicate that, even under normal conditions, multiple MyHCs can coexist in single equine muscle fibers as in rat (Talmadge et al., 1995a1,humans (Klitgaard et al., 19901, and other species (Aigner et al., 1993). The rarely occurring muscle fiber type containing both fast and slow MyHC substantiates the results reported by Snow et al. (1981) and Sinha et al. (1992). These fibers correspond to the type IIC muscle fiber identified histochemically, a muscle fiber type abundant in newborn foals but extremely scarce in mature horses (Snow and Valberg, 1994). In addition, a high percentage of fibers coexpressed both fast MyHCs, even in inactive animals. The different relative quantities of these two fast MyHCs within a given muscle fiber might explain the inconsistency observed in the staining intensity for a number of MAbs used in this study to label these fibers (SC-71, BF-35, BF-G6; Fig. 4). This inconsistency also could result from differences in myosin-associated proteins (i.e., myosin light chains) in the different fibers by blocking or allowing the MAb to bind (Schiaffino and Reggiani, 1994). Nevertheless, the MAb N2.261 was sensitive enough for the type IIa MyHC because it always labeled these hybrid fibers. In conclusion, the present study clearly shows the existence of three MyHC isoforms in adult equine skeletal muscle: one slow and two fast. The differential distribution of these MyHCs defines three major fiber types containing a single MyHC and two intermediate hybrid fiber populations containing both slow and fast IIa-MyHCs and the two fast MyHCs. Monoclonal antibodies specific for rat MyHC isoforms used in this study permit a clear separation of two fast MyHC isoforms in horse skeletal muscle. Whereas the identity of one of these two fast MyHC isoforms seems to be clearly a type IIa-MyHC isoform, the present results are not conclusive regarding the second fast MyHC isoform. Further molecular analyses are necessary to clarify this point. In contrast with other small species (i.e., rat, mouse, rabbit, etc.) in which three fast MyHC isoforms (IIa, IIx, and IIb) exist, the presence of only two fast MyHC isoforms in horses may be characteristic of 193 MYOSIN HEAVY CHAIN IN HORSES Slow Fast SC-71 A 4 . 7 4 N2.261 BF-35 BF-G6 1 1 1 . 0 D O ~ 0 0 0 0 o 0 o 0 o a a B O a o o a la0 0 0 0 0 j7jo a a o 0 Fig. 4. Schematic illustration of the five fiber types than can be delineated in the superficial region of the equine gluteus medius muscle by using monoclonal antibodies against specific MyHC isoforms. MHC Content Fiber Type 0 0 0 I I a !+Ha IIc a o 0 o 0 0 o Ila Ila Ha + Ilb/llx llab a o As indicated, the fiber population coexpressing both fast MyHCs spans a diversity of reactions against SC-71, BF-35, and BF-G6, but fiber 5 was the most common fiber to coexpress these two HyHCs. - AB-X I- Fig. 5. Eight-percent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel of normal horse gluteus medius muscle of five horses (lanes 1-5) myosin heavy chains (MyHCs).This gel shows the separation profile of the three MyHCs in adult horse muscle. Ths isoforms are identified as types I (I), IIa (A), and IIb or IIx (B/X). larger animals that have slower intrinsic velocities of shortening than smaller mammals (Rome et al., 1990). sity of Padova, Italy) and Dr. F. Stockdale (Standford University, CAI for the generous gift of MAbs. The MAbs developed by Dr. H. Blau (A4.74 and N2.261) were obtained from the Developmental Studies Hybridoma Bank, maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, and the Department of Biology, University of Iowa, Iowa City, IA, under contract N01-HD-2-3144 from the NICHD. ACKNOWLEDGMENTS This study was completed while Jose-Luis L. Rivero was working at the Department of Physiological Sciences, University of California at Los Angeles, USA, and his work supported by scholarships from the Spanish D.G.C.Y.T. (Ref: PR94-202) and the University of Cordoba, Spain. We thank Drs. S. Schiaffino (Univer- 194 J.-L.L. RIVER0 ET AL, LlTERATUR E CITED Aigner, S., B. Gohlsch, N. Hamalainen, R.S. Staron, A. Uber, U. Wehrle, and D. Pette 1993 Fast myosin heavy chain diversity in skeletal muscles of the rabbit: heavy chain IId, not IIb, predominates. Eur. J . Biochem., 211t367-372. Bar, A., and D. 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