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Immunocytochemical demonstration of myosin heavy chain expression in human muscle
Journal of the Neurological Sciences, 1989, 91:71-78
71
Elsevier JNS 03153
Immunocytochemical demonstration of myosin heavy chain expression in human muscle Marion Ecob-Prince 2, Mark Hill I and Wendy Brown 3 1Muscular Dystrophy Group Research Laboratories, Newcastle General Hospital, Newcastle upon Tyne NE4 6BE (U.K.), 2Glasgow University Department of Neurology, Institute of Neurological Sciences, Southern General Hospital, Glasgow G51 4TF (U.K.), and 3Department of Surgery, Jefferson Medical College Philadelphia, PA 19107 (U.S.A.)
(Received 28 December, 1988) (Revised, received 9 January, 1989) (Accepted 9 January, 1989)
SUMMARY Three new monoclonal antibodies are shown by immunocytochemical techniques to recognise the adult fast, slow and neonatal myosin heavy chain (MHC) isoforms in adult and fetal human muscle. In fetal muscle of 17-20 weeks of gestation, slow M H C was present only in primary myotubes. Secondary myotubes contained neonatal M H C with different levels of fast and some embryonic MHC. We confirmed the presence of tertiary myotubes in the fetal muscle (Draeger et al. (1987)J. Neurol. Sci., 81: 19-43) and show that these contained fast, neonatal and possibly some embryonic MHC. Fast M H C was therefore present in secondary and tertiary myotubes at least as early as 17 days of gestation.
Key words: Myosin; Human muscle; Development
INTRODUCTION
Myosin is a major muscle-specific protein. It is composed of 2 heavy chains and 4 light chains. The myosin heavy chain (MHC) has many isoforms which are specific for different muscles or fibre types, and some which are developmentally regulated Correspondence to." Dr. Marion Ecob-Prince, Glasgow University, Department of Neurology, Institute of Neurological Sciences, Southern General Hospital, Glasgow G51 4TF, U.K. Telephone: 041-445-2466 ext 3559.
0022-510X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
72 (Rushbrook and Stracher 1979: Hoh and Yeoh 1979; Whalen et al. 1981" Lowey et al. 1983; Lyons et al. 1983: Butler-Browne and Whalen 1984; Mahdavi et al. 1986). The presence of different M H C isoforms correlates well with the histochemical reaction of the myofibrillar ATPase (Billeter et al. t981; Salviati et al. t982; Danieti-Betto et al. 1986). Depending on the acid or alkaline lability of this myofibrillar ATPase (Brooke and Kaiser 1970), human muscle is judged to contain one type of slow-twitch fibre (type 1) and 3 types of fast-twitch fibre (types 2A, 2B, 2C). Further subgroups have now been described (Staron et al. 1983) and are probably attributable to more than one isoform being present in each fibre (Birat et al. 1988). Although biochemical analysis can be done on human muscle homogenates to determine native myosin isoforms (Fitzsimons and Hoh 1981 a,b) or on single human muscle fibres to determine their light and heavy chain composition (Billeter et al. 1981; Biral et al. 1988), the availability of antibodies which recognise different M H C isoforms would facilitate studies especially of development where histochemical typing is unhelpful. Such studies have already been done (Moore et al. 1984; Thornell et al. 1984; Pons et al. 1986; Zhang et al. 1987), the most recent presenting data about quadriceps muscle from a complete range of gestational ages (Draeger et al. 1987). This latter study was the only one to describe the appearance of tertiary myotubes, containing fast myosin, in fetal muscle "after 16 weeks gestation. Their presence, therefore, remains controversial. We now describe the immunocytochemical characterisation of 3 new monoclonal antibodies to rabbit M H C which specifically recognise the homologous isoforms in human muscle. Using these, we have been able to confirm and extend some of the findings described by Draeger et al. (1987).
MATERIALS AND METHODS
Muscle samples Muscle from the quadriceps of patients who were later diagnosed as having no muscle abnormality (on histological, histochemical and electrophysiological grounds) were used as adult normal samples (total of 5, aged 19 years and over). Muscle samples were also collected from the quadriceps muscle of fetuses after either spontaneous (3) or prostaglandin-induced (3) terminations. The fetuses were 17, 18 (2), 19 or 20 (2) weeks of gestation. Muscle samples were frozen in melting isopentane in a bath of liquid nitrogen and were stored in liquid nitrogen.
Antibodies Antibodies to rabbit myosin (WBMHC f, s or n) were produced by inoculation of Balb/c mice with native adult fast myosin (rabbit psoas) or adult slow myosin (rabbit soleus) or SDS-denatured neonatal myosin (3-day-old rabbit leg muscle). Hybridomas were established using the NS-1 cellline and techniques o f Milstein (Galfre and Milstein 1981). The specificity of the antibodies to myosin heavy chain was shown on Western blots. Antibodies from ascites fluid, diluted 1 : 250 in phosphate-buffered saline, were used throughout the study.
73 Antibodies to human embryonic (NOQ 23.6.2C) or adult fast (NOQ 7.5.2B) myosin were described by Draeger et al. (1987) and were kindly supplied by Dr. Annette Draeger and Dr. Robin Fitzsimons. Hybridoma culture supernatants were used without dilution. All antibodies were adsorbed to the cryostat sections for 1 h at 37 °C. Sections were washed in PBS and the second antibody (1:40 in PBS) of rhodamine-conjugated rabbit anti-mouse immunoglobulin (Dakopatts) was adsorbed for 40 min at 37 ° C.
RESULTS
The monoclonal antibodies to rabbit myosin heavy chains were used to stain serial cryostat sections of adult human muscle. Histochemical typing of individual fibres on adjacent serial sections was done by the methods of Brooke and Kaiser (1970) for myofibrillar ATPase (Fig. 1D, E, F). In this adult muscle, antibody WBMHC-n stained
Fig. 1. Serial cryostat sections of an adult h u m a n muscle which have been stained with antibodies to neonatal (A), fast (B), or slow (C) myosin heavy chain, or which have been used to demonstrate myofibrillar ATPase after alkaline preincubation (D) or acid preincubation at pH 4.3 (E) or pH 4.5 (F). The single type 2C fibre, seen in A, contains neonatal and fast with a little slow myosin heavy chain, and stains darkly at all pH values. Other fibre types are indicated in panel F. Bar = 20 #m.
74 no fibres except an occasional type 2Cfibre (Fig. IA) which was possibly aregenerating fibre. The antibody WBMHC-f stained all type 2A and 2B (fast-twitch) fibres, and the antibody WBMHC-s stained only type 1 (slow-twitch fibres) as shown in Fig, 1B and C, respectively. Monoclonal antibodies which recognised embryonic (NOQ 23.6.2C) or adult fast (NOQ 7.5.2B) human myosin (Draeger et al. 1987) were similarly used to stain sections of adult human muscle samples. Antibody NOQ 23.6.2C failed to stain any fibres. Antibody NOQ 23.6.2C stained all type 2A fibres and about 95~o of type 2B fibres. In muscle of fetuses aged 17-19 weeks, the staining pattern of the antibodies to rabbit M H C was different to that found in adult human muscle. The antibody WBMHC-n (Fig. 2A) which recognised neonatal rabbit MHC, stained all the fibres brightly except for some scattered large-diameter fibres which it stained only weakly. These larger diameter fibres were negative or pale when stained with antibody W B M H C - f (Fig. 2B) to fast M H C but were all stained brightly with antibody
Fig. 2. Serial cryostat sectionsof a 17-weekfetal human muscle which have been stained with antibodies to neonatal (A), fast (B), or slow (C) myosin heavy chain, or with antibodies to embryonic(D) or fast (E) human myosinheavychain. Smalldiametertertiary myotubescontain both neonatal and fast myosinheavy chain (arrows). Bar = 20/~m.
75 WBMHC-s (Fig. 2C) to slow MHC: they were probably Wohlfart type B fibres (Fenichel 1966). Antibody WBMHC-f stained brightly a population about 21 ~o of the fibres, of very small diameter fibres (arrow in Fig. 2B) which also contained neonatal myosin (arrow in Fig. 2A): it stained all other fibres either brightly or at an intermediate level. Antibody WBMHC-s stained only the putative Wohlfart type B fibres (Fig. 2C). The antibody NOQ 23.6.2C to embryonic human myosin (Fig. 2D) stained all fibres in 17-19 week old fetal muscle with equal intensity except for a slightly brighter staining of some fibres with a very small diameter. Staining with the antibody NOQ 7.5.2B to fast MHC-h was generally weaker than with our antibody (Fig. 2E vs. 2B, respectively), but the same variations in staining intensity between individual fibres were evident with both antibodies. However, we found that the intensity of staining with NOQ 7.5.2B also varied between fascicles of the same muscle and between different muscles within a leg. In muscle from fetuses aged 20 weeks, the Wohlfart type B fibres were either negative or very weakly stained with antibody WBMHC-n (Fig. 3A), were negative with antibody WBMHC-f (Fig. 3B) but were strongly stained with antibody WBMHC-s (Fig. 3C). The remaining fibres were all stained strongly with antibody WBMHC-n (Fig. 3B) but were stained with various intensities by WBMHC-f (Fig. 3C). Although there were fibres with a less-than-average diameter (Fig. 3B), the small-diameter fibres seen in earlier samples were now less evident (about 5 ~o of the total fibre number). No fibres stained with antibody NOQ 23.6.2C to embryonic human MHC (not shown).
DISCUSSION The monoclonal antibodies to rabbit MHC all cross-reacted specifically with the homologous human isoforms. Antibody WBMHC-f recognised all type 2A and type 2B
Fig. 3. Serial cryostat sections of a 20-weekfetal human muscle which have been stained with antibodies to neonatal (A), fast (B) or slow (C) myosin heavy chain. The Wohlfart B fibres which stain brightly for slow myosin heavy chain (C) lack fast and have only residual neonatal myosin. Bar = 20/~m.
76 fast-twitch fibres in adult muscle. In serial frozen sections of fetal human muscle, it stained fibres with several levels of intensity ranging from negative to strong although each fibre contained similar levels of embryonic myosin as shown by antibody NOQ 23.6.2C. Therefore, antibody WBMHC-f did not cross-react with embryonic human myosin. (Such cross-reactivity could not be checked on Western blots except by using pyrophosphate gels (Fitzsimons and Hoh 1981) which were not available to us.} Antibody WBMHC-f similarly gave a different pattern of staining to that of WBMHC-n which recognised neonatal MHC. and so this antibody to fast MHC also did not cross-react with neonatal myosin. By similar arguments the specificity of WBMHC-n to neonatal and WBMHC-s to slow human myosin heavy chain could be established. Thus 3 newly-described antibodies are now available to study myosin expression in human muscle. We have used these antibodies already to look at myosin expression in human muscle which has regenerated in culture in the presence or absence of neurones (Ecob-Prince et al. 1989). These antibodies also recognised several distinct populations of fibres in fetal human muscle of 17-20 weeks gestation. Using a much larger number of fetal human muscle samples, Draeger et al. (1987) have already described myosin expression in developing muscle from 8 to 40 weeks of gestation. Our results in no way compare to such a comprehensive study but we can nevertheless consider myosin expression over a limited period of gestation. We confirm their and others (Moore et al. 1984: Pons et al. 1986; Zhang et al. 1987) finding that slow myosin is present only in primary, large diameter (Wohlfart type B) fibres at 17-20 weeks of gestation. A second phase of slow myosin expression was found in secondary and tertiary fibres after 29 weeks of gestation (Draeger et al. 1987). In addition, the use of serial sections allowed us to conclude that these primary fibres also contained low levels of neonatal MHC. sometimes also associated with low levels of fast MHC. especially in the 17-19-week-old samples. By 20 weeks of gestauon, these fibres contained almost exclusively slow MHC. We also confirm the finding of very small diameter fibres, described as "tertiary" by Draeger et al. (1987), which stained brightly with antibodies to adult fast myosin. In addition, we were also able to show that most of these also contained neonatal M H C and that a few possibly contained both neonatal MHC and embryonic myosin in addition to the fast MHC. Our results differed from those of Draeger et al. (1987) in that we found expression of adult fast M H C in fibres other than tertiary myotubes in all of our san~les from 17-20 weeks of gestation, whereas they found it only after about 23 weeks o f gestation. There was, therefore, the possibility that our antibody to fast M H C was cross-reacting with either embryonic or neonatal myosin and that it was this cross-reactivity which was giving a staining of the fetal muscle in other than tertiary fibres. We have Shown that this was not the case. However, in adult human muscle, our antibody t o fast M H C (WBMHC-f) gave slightly different results to that (NOQ 7.5.28) used by Draeger et al. (1987), suggesting that they were recognising different epitopes on MHC. Antibody W B M H C - f stained all type 2A and all type 2B fibres. Antibody NOQ 7.5.2B stained all type 2A but not all type 2B fibres. This antibody also recognises only type 2A fibres in rat. cat and mouse (Hoh et al. 1988; our own observations). It has therefore been
77 suggested that N O Q 7.5.2B may recognise a type of 2A M H C which is present in all type 2A fibres and in varying amounts in type 2B fibres in human skeletal muscles (Draeger et al. 1987). If this is so, then this type of M H C is not expressed in developing human muscle in fibres other than tertiary myotubes until after 23 weeks of gestation. On the other hand, our results show that a (different?) MHC, present in all adult type 2A and type 2B fibres, is present at least as early as 17 weeks gestation. In other studies, the presence of a type 2B M H C was detected at 14-16 weeks gestation (Pons et al. 1986) and a heterologous pattern of staining for fast MHC, similar to that shown by this study, has been seen at 19 weeks gestation (Zhang et al. 1987). In conclusion, we have described the immunocytochemical characterisation of 3 monoclonal antibodies which specifically recognise human muscle M H C isoforms. We also confirm the earlier work of Draeger et al. (1987) but, in using serial sections, have been able to extend some of their observations about the myosin content of individual fibres. We found tertiary myotubes but were able to show that these contained a mixture of M H C rather than pure fast myosin. We have also described the appearance of a fast M H C in fibres of other than of tertiary origin as early as 17 weeks of gestation.
ACKNOWLEDGEMENTS We would like to thank the clinicians involved in providing muscle samples under recognised ethical guidelines; T. Walls, D. Roberts and D. Turnbull. The antibodies to rabbit myosin were produced during the doctoral studies of one of us (W. B.) in the laboratory of Professor S. Salmons. Drs. Robin Fitzsimons and Annette Draeger generously provided us with antibodies N O Q 23.6.2C and N O Q 7.5.2B. This work was supported by grants from the Muscular Dystrophy Group of Great Britain and Northern Ireland and the Medical Research Council. We would also like to thank Mrs. M. McColl for her skillful preparation of the manuscript. REFERENCES Billeter, R., C. W. Heizmann,H. Howald and E. Jenny ( 1981)Analysisof myosinlight and heavy chain types in single human skeletal muscle fibers. Eur. J. Biochem., 116: 389-395. Biral, D., R. Betto, D. Danieli-Bettoand A. Salviati(1988) Myosinheavy chain compositionof single fibres from normal human muscle. Biochem. J., 250: 307-308. Brooke, M.H. and K.K. Kaiser (1970) Muscle fiber types: how many and what kind? Arch. Neurol., 23: 369-379. Butler-Browne, G. S. and R.G. Whalen (1984) Myosin isozyme transitions occurring during the postnatal development of the rat soleus, Dev. Biol., 102: 324-334. Danieli-Betto, D., E. Zerbato and R. Betto (1986) Type 1, 2A and 2B myosinheavy chain electrophoretic analysis of rat muscle fibers, Biochem. Biophys. Res. Commun., 138: 981-987. Draeger, A., A.G. Weeds and R.B. Fitzsimons (1987) Primary, secondary and tertiary myotubes in developing skeletal muscle: a new approach to the analysis of human myogenesis.J. Neurol. Sci., 81: 19-43. Ecob-Prince, M., M. Hill and W. Brown (1989) Myosinheavy chain expressionin human musclecocultured with mouse spinal cord. J. Neurol. Sci., 90: 167-177. Fenichel, G.M. (1966) A histochemical study of developing human skeletal muscle. Neurology (Minneap.), 16: 741-745.
78 Fitzsimons, R. B and J. F. Y. Hoh (198 la) Embryonic and foetal myosins in human skeletal muscle. The presence of fetal myosins in Duchenne muscular dystrophy and infantile spinal muscular atrophy. J. Neurol. Sci.. 52: 367-384. Fitzsimons, R.B. and J.F.Y. Hoh (1981b) Isomyosins in human type 1 and type 2 skeletal muscle fibres. Biochem. J.. 193: 229-233. Galfre, G. and C. Milstein (1981 ) Preparation ofmonoclonal antibodies: strategies and procedures. Methods Enzymol., 23: 1-45. Hob, J.F.Y. and G.P.S. Yeoh (1979) Rabbit skeletal myosin isoenzymes from fetal, fast-twitch and slow-twitch muscles. Nature, 280: 321-323. Hoh, J. F. Y., S. Hughes, P.T. Hale and R.B. Fitzsimons (1988) Immunocytochemical and electrophoretic analyses of changes in myosin gene expression in cat limb fast and slow muscles during postnatal development. J. Musc. Res. Cell Motil., 9: 30-47. Lowey, S., P. A. Benfield, D.D.I.~blanc and G. S. Waller (1983) Myosin isozymes in avian skeletal muscles. I. Sequential expression of myosin isozymes in developing chicken pectoralis muscles. J. Musc. Res. Cell Motil., 4: 695-716. Lyons, G. E.. J. Haselgrove, A. M. Kelly and N. Rubinstein (1983) Myosin transitions in developing fast and slow muscles of the rat hind limb. Differentiation, 25: 168-175. Mahdavi, V., E.E Strehler, M. Periasamy, D.F. Wieczorek, S. Izumo and B. Nadal-Ginard (1986) Sarcomere myosin heavy chain gane family: organization and pattern of expression. Med. Sci. Sports Exercise, 18: 299-308. Moore, S.E.. O. Hurko and F. Walsh (1984) Immunocytochemical analysis of fibre type differentiation in developing skeletal muscle. J. Neuroimmunol., 7: 137-149. Pons, F.. J. O.C. Leger, M. Chevallay, F. M. S. Tome, M. Fardeau and J.J. Leger t 1986) Immunocytochemical analysis of myosin heavy chains in human fetal skeletal muscles. J. Neurol. Sci., 76: 151-163. Rushbrook. J.I. and A. Stracher (1979) Comparison of adult, embryonic and dystrophic myosin heavy chains from chicken muscle by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and peptide mapping. Proe. Natl. Acad. Sci. USA, 76: 4331-4334. Salviati, G.. R. Betto and D. Danieli-Betto (1982) Polymorphism of myofibrillar proteins of rabbit skeletal muscle fibers: an electrophoretic study of single fibers. Biochem. J., 207: 261-272. Staron. R.S., R.S. Hikida and F.C. Hageman (1983) Myofibrillar ATPase activity in human muscle fast-twitch subtypes. Histochemistry, 78: 405-408. Thornell, L.E.. R. Billeter, G.S. Butter-Browne, P.-O. Erickson, M. Ringqvist and R.G. Whalen (t984) Development of fiber types in human fetal muscle: an immunocytochemieal study. J. Neurol. Sci.. 66: 107-115. Whalen. R.G.. S.M. Sell, G.S. Butler-Browne, K. Schwartz, P. Bouveret and I. Pinset-Harstrom (1981) Three myosin heavy-chain isozymes appear sequentially in rat muscle development. Nature. 292: 805-809. Zhang, Y.. J.H. Sher, B. Leung and S.A. Shafiq (1987) An immunocytochemical study- of type l muscle fibres in developing human skeletal muscles. J. Neurol. Sci., 80: 1-12.
71
Elsevier JNS 03153
Immunocytochemical demonstration of myosin heavy chain expression in human muscle Marion Ecob-Prince 2, Mark Hill I and Wendy Brown 3 1Muscular Dystrophy Group Research Laboratories, Newcastle General Hospital, Newcastle upon Tyne NE4 6BE (U.K.), 2Glasgow University Department of Neurology, Institute of Neurological Sciences, Southern General Hospital, Glasgow G51 4TF (U.K.), and 3Department of Surgery, Jefferson Medical College Philadelphia, PA 19107 (U.S.A.)
(Received 28 December, 1988) (Revised, received 9 January, 1989) (Accepted 9 January, 1989)
SUMMARY Three new monoclonal antibodies are shown by immunocytochemical techniques to recognise the adult fast, slow and neonatal myosin heavy chain (MHC) isoforms in adult and fetal human muscle. In fetal muscle of 17-20 weeks of gestation, slow M H C was present only in primary myotubes. Secondary myotubes contained neonatal M H C with different levels of fast and some embryonic MHC. We confirmed the presence of tertiary myotubes in the fetal muscle (Draeger et al. (1987)J. Neurol. Sci., 81: 19-43) and show that these contained fast, neonatal and possibly some embryonic MHC. Fast M H C was therefore present in secondary and tertiary myotubes at least as early as 17 days of gestation.
Key words: Myosin; Human muscle; Development
INTRODUCTION
Myosin is a major muscle-specific protein. It is composed of 2 heavy chains and 4 light chains. The myosin heavy chain (MHC) has many isoforms which are specific for different muscles or fibre types, and some which are developmentally regulated Correspondence to." Dr. Marion Ecob-Prince, Glasgow University, Department of Neurology, Institute of Neurological Sciences, Southern General Hospital, Glasgow G51 4TF, U.K. Telephone: 041-445-2466 ext 3559.
0022-510X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
72 (Rushbrook and Stracher 1979: Hoh and Yeoh 1979; Whalen et al. 1981" Lowey et al. 1983; Lyons et al. 1983: Butler-Browne and Whalen 1984; Mahdavi et al. 1986). The presence of different M H C isoforms correlates well with the histochemical reaction of the myofibrillar ATPase (Billeter et al. t981; Salviati et al. t982; Danieti-Betto et al. 1986). Depending on the acid or alkaline lability of this myofibrillar ATPase (Brooke and Kaiser 1970), human muscle is judged to contain one type of slow-twitch fibre (type 1) and 3 types of fast-twitch fibre (types 2A, 2B, 2C). Further subgroups have now been described (Staron et al. 1983) and are probably attributable to more than one isoform being present in each fibre (Birat et al. 1988). Although biochemical analysis can be done on human muscle homogenates to determine native myosin isoforms (Fitzsimons and Hoh 1981 a,b) or on single human muscle fibres to determine their light and heavy chain composition (Billeter et al. 1981; Biral et al. 1988), the availability of antibodies which recognise different M H C isoforms would facilitate studies especially of development where histochemical typing is unhelpful. Such studies have already been done (Moore et al. 1984; Thornell et al. 1984; Pons et al. 1986; Zhang et al. 1987), the most recent presenting data about quadriceps muscle from a complete range of gestational ages (Draeger et al. 1987). This latter study was the only one to describe the appearance of tertiary myotubes, containing fast myosin, in fetal muscle "after 16 weeks gestation. Their presence, therefore, remains controversial. We now describe the immunocytochemical characterisation of 3 new monoclonal antibodies to rabbit M H C which specifically recognise the homologous isoforms in human muscle. Using these, we have been able to confirm and extend some of the findings described by Draeger et al. (1987).
MATERIALS AND METHODS
Muscle samples Muscle from the quadriceps of patients who were later diagnosed as having no muscle abnormality (on histological, histochemical and electrophysiological grounds) were used as adult normal samples (total of 5, aged 19 years and over). Muscle samples were also collected from the quadriceps muscle of fetuses after either spontaneous (3) or prostaglandin-induced (3) terminations. The fetuses were 17, 18 (2), 19 or 20 (2) weeks of gestation. Muscle samples were frozen in melting isopentane in a bath of liquid nitrogen and were stored in liquid nitrogen.
Antibodies Antibodies to rabbit myosin (WBMHC f, s or n) were produced by inoculation of Balb/c mice with native adult fast myosin (rabbit psoas) or adult slow myosin (rabbit soleus) or SDS-denatured neonatal myosin (3-day-old rabbit leg muscle). Hybridomas were established using the NS-1 cellline and techniques o f Milstein (Galfre and Milstein 1981). The specificity of the antibodies to myosin heavy chain was shown on Western blots. Antibodies from ascites fluid, diluted 1 : 250 in phosphate-buffered saline, were used throughout the study.
73 Antibodies to human embryonic (NOQ 23.6.2C) or adult fast (NOQ 7.5.2B) myosin were described by Draeger et al. (1987) and were kindly supplied by Dr. Annette Draeger and Dr. Robin Fitzsimons. Hybridoma culture supernatants were used without dilution. All antibodies were adsorbed to the cryostat sections for 1 h at 37 °C. Sections were washed in PBS and the second antibody (1:40 in PBS) of rhodamine-conjugated rabbit anti-mouse immunoglobulin (Dakopatts) was adsorbed for 40 min at 37 ° C.
RESULTS
The monoclonal antibodies to rabbit myosin heavy chains were used to stain serial cryostat sections of adult human muscle. Histochemical typing of individual fibres on adjacent serial sections was done by the methods of Brooke and Kaiser (1970) for myofibrillar ATPase (Fig. 1D, E, F). In this adult muscle, antibody WBMHC-n stained
Fig. 1. Serial cryostat sections of an adult h u m a n muscle which have been stained with antibodies to neonatal (A), fast (B), or slow (C) myosin heavy chain, or which have been used to demonstrate myofibrillar ATPase after alkaline preincubation (D) or acid preincubation at pH 4.3 (E) or pH 4.5 (F). The single type 2C fibre, seen in A, contains neonatal and fast with a little slow myosin heavy chain, and stains darkly at all pH values. Other fibre types are indicated in panel F. Bar = 20 #m.
74 no fibres except an occasional type 2Cfibre (Fig. IA) which was possibly aregenerating fibre. The antibody WBMHC-f stained all type 2A and 2B (fast-twitch) fibres, and the antibody WBMHC-s stained only type 1 (slow-twitch fibres) as shown in Fig, 1B and C, respectively. Monoclonal antibodies which recognised embryonic (NOQ 23.6.2C) or adult fast (NOQ 7.5.2B) human myosin (Draeger et al. 1987) were similarly used to stain sections of adult human muscle samples. Antibody NOQ 23.6.2C failed to stain any fibres. Antibody NOQ 23.6.2C stained all type 2A fibres and about 95~o of type 2B fibres. In muscle of fetuses aged 17-19 weeks, the staining pattern of the antibodies to rabbit M H C was different to that found in adult human muscle. The antibody WBMHC-n (Fig. 2A) which recognised neonatal rabbit MHC, stained all the fibres brightly except for some scattered large-diameter fibres which it stained only weakly. These larger diameter fibres were negative or pale when stained with antibody W B M H C - f (Fig. 2B) to fast M H C but were all stained brightly with antibody
Fig. 2. Serial cryostat sectionsof a 17-weekfetal human muscle which have been stained with antibodies to neonatal (A), fast (B), or slow (C) myosin heavy chain, or with antibodies to embryonic(D) or fast (E) human myosinheavychain. Smalldiametertertiary myotubescontain both neonatal and fast myosinheavy chain (arrows). Bar = 20/~m.
75 WBMHC-s (Fig. 2C) to slow MHC: they were probably Wohlfart type B fibres (Fenichel 1966). Antibody WBMHC-f stained brightly a population about 21 ~o of the fibres, of very small diameter fibres (arrow in Fig. 2B) which also contained neonatal myosin (arrow in Fig. 2A): it stained all other fibres either brightly or at an intermediate level. Antibody WBMHC-s stained only the putative Wohlfart type B fibres (Fig. 2C). The antibody NOQ 23.6.2C to embryonic human myosin (Fig. 2D) stained all fibres in 17-19 week old fetal muscle with equal intensity except for a slightly brighter staining of some fibres with a very small diameter. Staining with the antibody NOQ 7.5.2B to fast MHC-h was generally weaker than with our antibody (Fig. 2E vs. 2B, respectively), but the same variations in staining intensity between individual fibres were evident with both antibodies. However, we found that the intensity of staining with NOQ 7.5.2B also varied between fascicles of the same muscle and between different muscles within a leg. In muscle from fetuses aged 20 weeks, the Wohlfart type B fibres were either negative or very weakly stained with antibody WBMHC-n (Fig. 3A), were negative with antibody WBMHC-f (Fig. 3B) but were strongly stained with antibody WBMHC-s (Fig. 3C). The remaining fibres were all stained strongly with antibody WBMHC-n (Fig. 3B) but were stained with various intensities by WBMHC-f (Fig. 3C). Although there were fibres with a less-than-average diameter (Fig. 3B), the small-diameter fibres seen in earlier samples were now less evident (about 5 ~o of the total fibre number). No fibres stained with antibody NOQ 23.6.2C to embryonic human MHC (not shown).
DISCUSSION The monoclonal antibodies to rabbit MHC all cross-reacted specifically with the homologous human isoforms. Antibody WBMHC-f recognised all type 2A and type 2B
Fig. 3. Serial cryostat sections of a 20-weekfetal human muscle which have been stained with antibodies to neonatal (A), fast (B) or slow (C) myosin heavy chain. The Wohlfart B fibres which stain brightly for slow myosin heavy chain (C) lack fast and have only residual neonatal myosin. Bar = 20/~m.
76 fast-twitch fibres in adult muscle. In serial frozen sections of fetal human muscle, it stained fibres with several levels of intensity ranging from negative to strong although each fibre contained similar levels of embryonic myosin as shown by antibody NOQ 23.6.2C. Therefore, antibody WBMHC-f did not cross-react with embryonic human myosin. (Such cross-reactivity could not be checked on Western blots except by using pyrophosphate gels (Fitzsimons and Hoh 1981) which were not available to us.} Antibody WBMHC-f similarly gave a different pattern of staining to that of WBMHC-n which recognised neonatal MHC. and so this antibody to fast MHC also did not cross-react with neonatal myosin. By similar arguments the specificity of WBMHC-n to neonatal and WBMHC-s to slow human myosin heavy chain could be established. Thus 3 newly-described antibodies are now available to study myosin expression in human muscle. We have used these antibodies already to look at myosin expression in human muscle which has regenerated in culture in the presence or absence of neurones (Ecob-Prince et al. 1989). These antibodies also recognised several distinct populations of fibres in fetal human muscle of 17-20 weeks gestation. Using a much larger number of fetal human muscle samples, Draeger et al. (1987) have already described myosin expression in developing muscle from 8 to 40 weeks of gestation. Our results in no way compare to such a comprehensive study but we can nevertheless consider myosin expression over a limited period of gestation. We confirm their and others (Moore et al. 1984: Pons et al. 1986; Zhang et al. 1987) finding that slow myosin is present only in primary, large diameter (Wohlfart type B) fibres at 17-20 weeks of gestation. A second phase of slow myosin expression was found in secondary and tertiary fibres after 29 weeks of gestation (Draeger et al. 1987). In addition, the use of serial sections allowed us to conclude that these primary fibres also contained low levels of neonatal MHC. sometimes also associated with low levels of fast MHC. especially in the 17-19-week-old samples. By 20 weeks of gestauon, these fibres contained almost exclusively slow MHC. We also confirm the finding of very small diameter fibres, described as "tertiary" by Draeger et al. (1987), which stained brightly with antibodies to adult fast myosin. In addition, we were also able to show that most of these also contained neonatal M H C and that a few possibly contained both neonatal MHC and embryonic myosin in addition to the fast MHC. Our results differed from those of Draeger et al. (1987) in that we found expression of adult fast M H C in fibres other than tertiary myotubes in all of our san~les from 17-20 weeks of gestation, whereas they found it only after about 23 weeks o f gestation. There was, therefore, the possibility that our antibody to fast M H C was cross-reacting with either embryonic or neonatal myosin and that it was this cross-reactivity which was giving a staining of the fetal muscle in other than tertiary fibres. We have Shown that this was not the case. However, in adult human muscle, our antibody t o fast M H C (WBMHC-f) gave slightly different results to that (NOQ 7.5.28) used by Draeger et al. (1987), suggesting that they were recognising different epitopes on MHC. Antibody W B M H C - f stained all type 2A and all type 2B fibres. Antibody NOQ 7.5.2B stained all type 2A but not all type 2B fibres. This antibody also recognises only type 2A fibres in rat. cat and mouse (Hoh et al. 1988; our own observations). It has therefore been
77 suggested that N O Q 7.5.2B may recognise a type of 2A M H C which is present in all type 2A fibres and in varying amounts in type 2B fibres in human skeletal muscles (Draeger et al. 1987). If this is so, then this type of M H C is not expressed in developing human muscle in fibres other than tertiary myotubes until after 23 weeks of gestation. On the other hand, our results show that a (different?) MHC, present in all adult type 2A and type 2B fibres, is present at least as early as 17 weeks gestation. In other studies, the presence of a type 2B M H C was detected at 14-16 weeks gestation (Pons et al. 1986) and a heterologous pattern of staining for fast MHC, similar to that shown by this study, has been seen at 19 weeks gestation (Zhang et al. 1987). In conclusion, we have described the immunocytochemical characterisation of 3 monoclonal antibodies which specifically recognise human muscle M H C isoforms. We also confirm the earlier work of Draeger et al. (1987) but, in using serial sections, have been able to extend some of their observations about the myosin content of individual fibres. We found tertiary myotubes but were able to show that these contained a mixture of M H C rather than pure fast myosin. We have also described the appearance of a fast M H C in fibres of other than of tertiary origin as early as 17 weeks of gestation.
ACKNOWLEDGEMENTS We would like to thank the clinicians involved in providing muscle samples under recognised ethical guidelines; T. Walls, D. Roberts and D. Turnbull. The antibodies to rabbit myosin were produced during the doctoral studies of one of us (W. B.) in the laboratory of Professor S. Salmons. Drs. Robin Fitzsimons and Annette Draeger generously provided us with antibodies N O Q 23.6.2C and N O Q 7.5.2B. This work was supported by grants from the Muscular Dystrophy Group of Great Britain and Northern Ireland and the Medical Research Council. We would also like to thank Mrs. M. McColl for her skillful preparation of the manuscript. REFERENCES Billeter, R., C. W. Heizmann,H. Howald and E. Jenny ( 1981)Analysisof myosinlight and heavy chain types in single human skeletal muscle fibers. Eur. J. Biochem., 116: 389-395. Biral, D., R. Betto, D. Danieli-Bettoand A. Salviati(1988) Myosinheavy chain compositionof single fibres from normal human muscle. Biochem. J., 250: 307-308. Brooke, M.H. and K.K. Kaiser (1970) Muscle fiber types: how many and what kind? Arch. Neurol., 23: 369-379. Butler-Browne, G. S. and R.G. Whalen (1984) Myosin isozyme transitions occurring during the postnatal development of the rat soleus, Dev. Biol., 102: 324-334. Danieli-Betto, D., E. Zerbato and R. Betto (1986) Type 1, 2A and 2B myosinheavy chain electrophoretic analysis of rat muscle fibers, Biochem. Biophys. Res. Commun., 138: 981-987. Draeger, A., A.G. Weeds and R.B. Fitzsimons (1987) Primary, secondary and tertiary myotubes in developing skeletal muscle: a new approach to the analysis of human myogenesis.J. Neurol. Sci., 81: 19-43. Ecob-Prince, M., M. Hill and W. Brown (1989) Myosinheavy chain expressionin human musclecocultured with mouse spinal cord. J. Neurol. Sci., 90: 167-177. Fenichel, G.M. (1966) A histochemical study of developing human skeletal muscle. Neurology (Minneap.), 16: 741-745.
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