Characterization of a monoclonal antibody to myosin specific for mammalian and human type II muscle fibers

Journal of the Neurological Sciences, 1985, 69:247-254

247

Elsevier

Characterization of a Monoclonal Antibody to Myosin Specific for Mammalian and Human Type II Muscle Fibers Judith A. Sawchak, Betty Leung and Saiyid A. Shafiq Department ofNeurology, State Universityof New York,DownstateMedical Center,Brooklyn, NY 11203(U.S.A.) (Received 28 January, 1985) (Accepted 7 March, 1985)

SUMMARY

We have characterized a monoclonal antibody (McAb), ALD-19, generated against slow myosin from chicken anterior latissimus dorsi (ALD) muscle for use in studies of human and animal muscle fiber types. This McAb bound selectively to the 200 kDa myosin heavy chain band in immunoblots against chicken, rat and human myosins and showed selective staining of A bands in the myofibrils. The reactivity of ALD-19 with various myosin types was quantitated by radioimmunoassays. Fiber type analysis revealed unexpected specificity of McAb ALD-19 for type II mammalian muscle fibers. This antibody should, therefore be useful for identification and quantification of normal type II fibers in human muscle biopsy specimens.

Key words: Monoclonal antibody - Muscle fiber types - Myosin heavy chain

INTRODUCTION

Fiber type analysis in normal and diseased human muscle is generally based on the histochemical adenosine triphosphatase (ATPase) technique (Padykula and Hermann 1955) after preincubation at discriminating pH values (Brooke and Kaiser 1970). This work was supported by research grants from the National Institute of Health (NS-18776) and the Muscular Dystrophy Association of America. Address correspondence and reprint requests to: Judith A. Sawchak, M.D., Department of Neurology - Box 1213, State University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, N.Y. 11203, U.S.A. 0022-510X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

248 Although useful as a routine procedure, it is subject to erroneous interpretation (Guth and Samaha 1972; Guth 1973), and is of questionable value for analysis of myosin isoforms among and within fiber types (Billeter et al. 1981; Mabuchi et al. 1982, 1984). In this report we have characterized a monoclonal antibody (McAb) against myosin heavy chain (MHC) which reacts specifically with mammalian and human type II fibers. The potential applications of this and other McAbs to the study of myosin isoforms in human muscle are discussed. MATERIALS AND METHODS The McAb, ALD- 19 described here, was generated against the myosin of the slow anterior latissimus dorsi (ALD) muscle of the chicken using the procedures of Kohler and Milstein (1975). Details of methods of preparation of the slow myosin antigen, production of hybridomas, binding assays, immunoblot autoradiography and immunofluorescence microscopy have been published (Shafiq et al. 1984). Briefly, the hybridoma clones were produced from the fusion of a non-secreting myeloma cell line (Ps NP) and spleen cells from mice immunized with chicken ALD myosin. Solid phase radioimmune assay (RIA) was used for initial screening of hybridoma cultures as well as for determining the specificity of the antibodies produced. The reactivity of the antibody was tested against myosin extracted from chicken breast (fast), heart (ventricular) and gizzard (smooth) muscles as well as against myosins extracted from rat gastrocnemius and human psoas muscles. The epitope recognized by the hybridoma antibody ALD-19 was mapped with respect to myosin heavy and light chains by immunoblots prepared against whole muscle extracts as well as against isolated myosins. To localize antibody binding sites by immunofluorescence microscopy, myofibrils were prepared from chicken ALD, rat gastrocnemius and human psoas muscles, reacted with ALD-19 and visualized by indirect epifluorescence. For analysis of fiber types, frozen sections of chicken ALD and sartorius muscles, rat soleus and gastrocnemius muscles and human vastus lateralis and psoas muscles were reacted for myosin ATPase with preincubation at pH 10.4 and 4.5. Serial sections were then processed for immunofluorescence as described for the myofibrils. RESULTS The binding of ALD-19 antibody with myosins of various muscles of the chicken was examined in solid phase RIAs. Antibody dilution protocols (Fig. la) revealed strong reactivity of McAb ALD-19 with ALD myosin. Under identical conditions cardiac (ventricular) and pectoralis (fast) myosin elicited moderate levels of binding while gizzard (smooth) myosin was unreactive (Fig. la). When a fixed concentration of ALD-19 was reacted with serial dilutions of these myosins, similar results were obtained (Fig. lb). In RIAs, ALD-19 antibody also reacted with myosins isolated from rat gastrocnemius and human psoas muscles as seen in Fig. 2. The binding with mammalian myosins, however, was at a lower level than with chicken ALD myosin which was the immunogen for this antibody.

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Fig. 1. a: Titration curves ofMcAb ALD-19 with myosins extracted from chicken ALD, cardiac, pectoralis and gizzard muscles. One/~g of myosin from each source was reacted with serial dilutions of the McAb. b: Titration curves of the McAb ALD-19 (1 : 9 dilution) with serial dilutions of myosin from chicken ALD, cardiac, pectoralis and gizzard muscles. Both sets of curves (a and b) were obtained by indirect solid phase RIA using 125I-labeled goat anti-mouse IgG ([~2SI]GAM) as the second antibody.

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Fig. 2. Indirect RIA ofMcAb ALD-19 with myosin from rat and human muscle. One/~g of myosin from each source was reacted with serial dilutions of the antibody followed by [125I]GAM.

250 To localize the epitope for the McAb ALD-19, myosin extracted from the chicken A L D as well as the extract of fresh, chicken whole A L D muscle were tested by immunoblot autoradiography procedure (Towbin et al. 1979; Shafiq etal. 1984) (Figs. 3a,b). In the immunoblot against purified A L D myosin, McAb ALD-19 reacted with the 200 k D a peptide band on the gels corresponding to the myosin heavy chain and not with the light chains (Fig. 3b). In immunoblots against the whole A L D muscle homogenate which contained numerous peptide bands, again only one reactive band corresponding to the myosin heavy chain was detected (Fig. 3a). Similarly, in the immunoautoradiographs of rat and human myosins (Figs. 4a,b) the reacting band corresponded to the 200 k D a M H C band; however, minor bands migrating in front of the M H C band also showed some reactivity; these minor bands presumably represented proteolytic fragments produced during purification and storage of myosin. By indirect immunofluorescent staining, myofibrils isolated from the chicken ALD as well as those isolated from rat gastrocnemius and human psoas muscles gave a strong reaction with ALD- 19. In most fibrils the A band stained in a uniform manner, although in some it exhibited a thin central zone of weaker fluorescence (Figure not shown). The selective A band staining by immunofluorescence confirmed the results of RIA and of immunoblotting on the antimyosin specificity of the ALD-19.

3 Fig. 3. Immunoautoradiographsof whole chicken ALD muscle homogenate (a) and myosin from chicken ALD muscle (b) treated with McAb ALD-19. The muscle or myosin preparations were run on sodium dodecyl sulfate--polyacrylamidegel electrophoresis (SDS-PAGE), transferred to nitrocellulose paper, treated with ALD-19 followed by [125I]GAM,and developed autoradiographically. Proteins displayed by SDS-PAGE and stained with Coomassieblue are on the left and the immunoautoradiographsare on the fight in each panel. In each case, McAb ALD-19 reacted only with the 200 kDa peptide band (arrowheads) on the gels corresponding to the myosin heavy chain.

251

4 Fig. 4. Immunoautoradiographsof myosin extracted from rat gastrocnemius (a) and human psoas (b) musclesand treated withMcAbALD-19.The myosinswere treated and displayedas describedin Figure3. As with chick myosins(Fig.3), ALD-19 antibody reacted specificallywith mammalian200 kDa MHC bands (arrowheads). F o r fiber type analysis, cryosections of various muscles were reacted for myosin ATPase technique and compared to serial sections of the same muscles treated for immunofluoreseence with the ALD-19 antibody. In the tonic chicken ALD muscle all fibers were equally reactive with ALD-19. In the chicken sartorius muscle, this McAb reacted with both type I and II fibers, although less intensely with the latter. By contrast, in rat gastroenemius and soleus muscles and in normal human vastus lateralis and psoas muscles, type II fibers were strongly reactive while type I fibers were uniformly nonreactive (Figs. 5a,b). Comparison of rat and human muscle sections treated for immunofluorescence with McAb ALD-19 with adjacent sections stained for ATPase with acid preincubation revealed that both type IIA and IIB fibers were consistently and equally reactive with this antibody. This pattern of immunofluorescence was not altered by pretreatment of muscle sections with acetone or formalin prior to antibody application. DISCUSSION We have demonstrated that a McAb, ALD-19, raised against the slow-tonic myosin of chicken ALD muscle, displays an unusual specificity. In chicken skeletal muscles, this antibody reacted strongly with both slow-tonic (i.e. type III) (Barnard et al. 1982) and slow-twitch (type I) fibers but weakly with fast (type II) fibers. Conversely, in mammalian muscles it reacted only with fast (type II) fibers. These results were unexpected since previous immunocytochemical studies utilizing conventional

Fig. 5. Serial frozen sections of human psoas muscle treated with histochemical myosin ATPase reaction with alkaline (pH 10.4) preincubation (a) and indirect immunotluorescence with McAb ALD-19 followed by FITC-GAM (b). All type II fibers in this mixed human muscle are strongly fluorescent ( × 600).

253 antisera against chicken ALD myosin for fiber typing of mammalian muscles yielded different results. Gauthier and Lowey (1979) examined the reactivity of rat skeletal muscles with an antibody raised against chicken ALD myosin. Their antibody reacted specifically with type I fibers except that in the soleus, the type II fibers were found to be reactive with antibodies against both slow (ALD) and fast (pectoralis) myosins of the chicken. Pierobon-Bormioli et al. (Sartore et al. 1978; Pierobon-Bormioli et al. 1979, 1980) on the other hand, found that their anti-chick ALD antisera were specific for chicken muscles and did not cross-react with mammalian and human muscle fibers except for a proportion of fibers in extraocular muscles and muscle spindles. Since cross-reactivity with a McAb represents the sharing of a single antigenic determinant (Kohler and Milstein 1975) our findings suggest an antigenic site common to both chick slow-tonic and mammalian fast-twitch myosins which is apparently absent in the mammalian slow-twitch myosin. Such a determinant has not been detected previously, possibly because of a relatively low titer of antibody against such an epitope in polyclonal antisera. Whether this unexpected finding has an evolutionary significance must await detailed epitope mapping analysis of chicken and mammalian myosin heavy chains. Recent two-dimensional electrophoretic studies of proteins in single human skeletal fibers have indicated a heterogenous mixture of myosin isoforms both among fiber types as well as in fibers of the same histochemical type (Billeter et al. 1981); there is, thus, a wider spectrum of fiber "types" than that revealed by routine histochemical methods. McAbs offer distinct advantages in the study of various myofibriUar proteins in that McAbs to specific determinants can be produced in large quantities and serve as highly selective molecular probes. In particular, immunocytochemical procedures employing McAbs are well suited to studies of human fiber type heterogeneity with respect to protein isoforms. The McAb, ALD-19, described in this report should be useful for identification and quantification of the full set of normal mature type II (A and B) muscle fibers in human biopsy specimens. Currently there is evidence which points to changes in human myosin isoforms during development (Fitzsimmons and Hoh 1981; Strohman et al. 1983; Sher et al. 1984) and in neuromuscular disorders, e.g., Duchenne's muscular dystrophy (Pennington 1971; Giometti et al. 1980; Fitzsimmons and Hoh 1981), spinal muscular atrophy (Fitzsimmons and Hoh 1979), and nemaline myopathy (Streter et al. 1976; Dalla Libera et al. 1978; Giometti et al. 1980; Stuhlfauth et al. 1983). ACKNOWLEDGEMENTS

The authors are grateful to Dr. Khalid M.H. Butt for aid in obtaining normal human muscle, to Dr. Joanna H. Sher who provided normal human biopsy specimens, and to Ms. Helen R. Watson for secretarial assistance.

254 REFERENCES Barnard, E.A., J. M. Lyles and J. A. Pizzen (1982) Fibre types in chicken skeletal muscles and their changes in muscular dystrophy, J. Physiol. (Lond.), 331: 333-354. Billeter, R., C.W. Heismann and E. Jenny (1981) Analysis of myosin light and heavy chain types in single human skeletal muscle fibers, Europ. J. Biochem., 116: 389-395. Brooke, M. H. and K.K. Kaiser (1970) Muscle fiber types - - How many and what kind? Arch. NeuroL, 23: 369-379. Dalla Libera, L., A. Margreth, I. Mussini, C. Cerri and S. Scarlato (1978) Myosin polymorphism in human skeletal muscles, Muscle & Nerve, 1: 280-291. Fitzsimmons, R.B. and J. F. Y. Hoh (1981 ) Embryonic and foetal myosins in human skeletal muscle - - The presence of foetal myosins in Duchenne muscular dystrophy and infantile spinal muscular atrophy, jr. Neurol. Sci., 52: 367-384. Gautheir, G. and S. Lowey (1979) Distribution of myosin isoenzymes among skeletal muscle fiber types, J. Cell Biol., 81: 10-25. Giometti, C.S., M. Barany, M.J. Danon and N.G. Anderson (1980) Muscle protein analysis, Part2 (Two-dimensional electrophoresis of normal and diseased human skeletal muscle), Clin. Chem., 26: 1152-1155. Guth, L. (1973) Fact and artifact in the histochemical procedure for myofibrillar ATPase, Exp. Neurol., 41: 440-450. Guth, L. and F.J. Samaha (1972) Erroneous interpretations which may result from application of the "myofibrillar ATPase" histochemical procedure to developing muscle, Exp. Neurol., 34: 465-475. Kohler, G. and C. Milstein (1975) Continuous cultures of fused cells secreting antibody of predefined specificity, Nature (Lond.), 256: 495-497. Mabuchi, K., D. Szvetko, K. Pinter and F.A. Streter (1982) Type 2B to 2A fiber transformation intermittently stimulated rabbit muscles, Amer. J. Physiol., 242: C372-C381. Mabuchi, K., K. Pinter, Y. Mabuchi, F. Streter and J. Gergely (1984) Characterization of rabbit masseter muscle fibers, Muscle & Nerve, 7: 431-438. Padykula, H. A. and E. Hermann (1955) Factors affecting the activity of adenosine triphosphatase and other phosphatases as measured by histoehemical techniques, J. Histochem. Cytochem., 3: 161-169. Pennington, R.J. (1971) In: O. Bodansky and A. L. Latner (Eds.), Advances in Clinical Chemistry, Academic Press, New York, London, pp. 409-442. Pierobon-Bormioli, S., P. Torresan, S. Sartore, B. Moschini and S. Schiaffino (1979) Immunohistochemical identification of slow-tonic fibers in human extrinsic eye muscles, Invest. Ophthalmol. Visual Sci., 18: 303-306. Pierobon-Bormioli, S., S. Satore, M. Vitadello and S. Schiaffino (1980) "Slow" myosins in vertebrate skeletal muscle - - An immunofluorescence study, ,/. Cell Biol., 85: 672-681. Sartore, S., S. Pierobon-Bormioli and S. Schiaffino (1978) Immunohistochemical evidence for myosin polymorphism in the chicken heart, Nature (Lond.), 274: 82. Shafiq, S. A., T. Shimizu and D.A. Fischman (1984) Heterogeneity of type 1 skeletal muscle fibers revealed by monoclonal antibody to slow myosin, Muscle & Nerve, 7: 380-387. Sher, J. H., B. Leung, Y. Zhang and S. A. Shafiq (1984) Recognition of human fetal and regenerating muscle fibers by the same myosin specific monoclonal antibodies, J. Neuropath. Exp. Neurol., 43: 318. Streter, F.A., Karl-Erik Astrom, F. C. A. Romanul, R.R. Young and H. Royden Jones Jr. (1976) Characteristics of myosin in nemaline myopathy, J. Neurol. Sci., 27:99-116. Strohman, R.C., J. Micou-Eastwood, C. A. Glass and R. Matsuda (1983) Human fetal muscle and cultured myotubes derived from it contain a fetal-specific myosin light chain, Science, 221: 955-957. Stuhlfauth, I., F.G.I. Jenekens, J. Willemse and B.M. Jockusch (1983) Congenital nemaline myopathy, Part 2 (Quantitative changes in alpha actinin and myosin in skeletal muscle), Muscle & Nerve, 6: 69-74. Towbin, H., T. Staehelin and J. Gordon (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets - - Procedure and some applications, Proc. Nat. Acad. Sci. (U.S.A.), 76: 4350-4354.