263
J. Anat. (1992) 180, pp. 263-274, with 7 figures Printed in Great Britain
Myosin heavy chain expression in rabbit masseter muscle during postnatal development JANTINE J. BREDMANI, WIM A. WEIJS', HANS A. M. KORFAGE1, PETER BRUGMAN' AND ANTOON F. M. MOORMAN2
'Department of Functional Anatomy, Academic Centre for Dentistry Amsterdam (ACTA) and 2Department of Anatomy and Embryology, Faculty of Medicine, University of Amsterdam, The Netherlands
(Accepted 12 November 1991)
ABSTRACT
The expression of isoforms of myosin heavy chain (MHC) during postnatal development was studied in the masseter muscle of the rabbit. Evidence is presented that in addition to adult fast and slow myosin, the rabbit masseter contains neonatal and 'cardiac' ac-MHC. During postnatal growth myosin transitions take place from neonatal and fast (IIA, IIA/IIB-referring to a fibre containing both IIA and IIB MHCs) MHC to adult 'cardiac' a-MHC and I/a-MHC. Since there is a temporary population of fibres containing IIA/a-MHC during the first 4 wk of development with a peak in the 3rd to 4th wk, the transition from IIA-MHC to a-MHC may occur in these IIA/a-MHC-containing fibres. The appearance of 'cardiac' a-MHC coincides with the timing of weaning, suggesting that the changes in MHC content, that probably result in a transition to a lower speed of contraction, have functional significance related to weaning. The finding of neonatal MHC in adult rabbits indicates that the masseter develops at a rate and in a way that is distinct from most other skeletal muscles. A spatiotemporal variation in expression of myosin isozymes within the masseter was observed, with many fibres containing more than one myosin type, indicating developmentally regulated spatial differences in function.
INTRODUCTION
The isoform of MHC protein expressed in a skeletal muscle fibre is in part determined by the type of myoblast that forms the fibre. Apart from the well known adult myosin heavy chain (MHC) isoforms, that is, type I (slow contracting), type II A (fast contracting) and type II B (fast contracting), embryonic and neonatal MHC isoforms have been described for developmental stages. The myosin isozymes follow a transition from embryonic either to neonatal or adult slow and from neonatal to adult fast and/or adult slow myosin (Butler-Browne & Whalen, 1984; Butler-Browne et al. 1988; Hoh & Hughes, 1989). It has been shown that masseter muscle occupies a special position among the skeletal muscles. For instance, neonatal MHC often persists in adult rat
and human masseter (d'Albis et al. 1986; ButlerBrowne et al. 1988). In addition, in adult rabbit and human jaw muscles, besides neonatal, slow (type I) and fast (type II A and II B) MHC, a 'cardiac' aMHC is expressed (Bredman et al. 1990b, 1991; d'Albis et al. 1991). Furthermore, single masseter muscle fibres usually express more than one type of MHC (Thornell et al. 1984; Rowlerson, 1990). This might be indicative for a fine gradation of possible contraction speeds in the fibre of the muscle since it has been shown that the speed of maximum velocity of shortening of single muscle fibres (Vmax) is correlated with the MHC composition (Reiser et al. 1985; Eddinger & Moss, 1987; Schiaffino et al. 1988). Previous (immuno-)histochemical studies on masseter muscles have mostly been based on small parts of the muscle, assuming that the whole muscle is similar. In this study special attention was devoted to regional
Correspondence to Dr J. J. Bredman, Department of Functional Anatomy, Academic Centre for Dentistry Amsterdam, University of Amsterdam K2, Meibergdreef 15, 1005 AZ Amsterdam, The Netherlands.
264
Jantine J. Bredman and others
differences within the masseter. Differences in fibre type composition were found when different regions of the muscle were compared. The occurrence of the various MHC isoforms is described in the rabbit masseter muscle during postnatal development. Furthermore, spatiotemporal changes in the MHC expression pattern in the developing masseter are related to postnatal changes in function. MATERIALS AND METHODS
Animals and dissection New Zealand rabbits (4 at each of the following ages) were killed by anaesthetic overdose: newborn (1 d), 7, 14, 21 and 28 d old (weight-range 35-1074 g) and adult (2500-4400 g, 2 males and 2 females). The masseter muscles were dissected from each animal after marks had been placed to indicate the levels and directions of 2 sections. Because the posterior deep portion of the masseter (MPPO compartment; Weijs et al. 1987; Bredman et al. 1990a) has an orientation different from the rest of the masseter it was removed separately. Embryonic hearts were obtained from rabbits at the 15th d of gestation. For the immunohistochemical studies all masseter muscles were fixed in a mixture of methanol: acetone:acetic acid:water (35:35:5:25), dehydrated in a graded series of ethanols, cleared in chloroform and embedded in Paraplast Plus (Monoject, Ireland) (Wessels et al. 1988; Bredman et al. 1990a). For the combined immuno- and enzyme-histochemical (ATPase) study, adult tibialis anterior and soleus muscles were frozen in liquid Freon-22 (monochlorodifluoromethane) cooled with liquid nitrogen and stored at -70 'C. For Western blot analysis, Table 1. Antibody reactivity* MHC Mab no.
a
249-5A4 (anti-a) 219-lDl (anti-I)
+
412-1D5 (anti-a/I/IIB) 333-7H1 (anti-I1 A) 41 1-1D5 (anti-II B) 340-3B5 (anti-IIA/IIB)
+
anti-N
I
IIA
+ +
IIB
N
+ + +
+ + +
* The pattern of reactivity of the 7 anti-MHC Mabs used in this study with the various MHC isozymes, as determined by immuno-
histochemistry, is summarised. +, positive reaction; otherwise no reaction.
tissue specimens were frozen in liquid nitrogen and stored at -70 'C. Production and characterisation of the monoclonal antibodies
The isolation of myosin and the production and characterisation of the monoclonal antibodies (Mabs) 249-5A4, 219-IDI, 412-1D5 and 340-5B3 has been described before (Wessels et al. 1988, 1990 a; de Groot et al. 1989; Bredman et al. 1991). The Mabs 333-7H1 and 411-4F10 were raised against a protein extract from muscle tissue of adult rabbit tibialis anterior and psoas. The antibody against neonatal MHC was kindly provided by Dr G. S. Butler-Browne (INSERM, Paris). The specificity of the antibodies was determined on tibialis anterior and soleus as indicated in Table 1, and has been described in part previously (Bredman et al. 1991 a). Western blot analyses were used to specify the antibodies towards MHC (Wessels et al. 1990 a; Bredman et al. 1991). SDS-glycerol gel electrophoresis of c-MHC was carried out by the method of d'Albis et al. (1991). The specificity of the neonatal Mab has been demonstrated by Butler-Browne et al. (1988).
Immunohistochemistry The embedded masseter muscles (all ages) were cut into 8-10 gim thick serial sections and mounted on microscope slides coated with poly-L-lysine. After deparaffination the sections were pretreated with pronase (0.1 mg/ml, 5-30 min, depending on the age of the animal) to optimise binding of the antibodies with the antigens (Christensen & Strange, 1987). To detect the binding of the specific monoclonal antibodies with the MHC isoforms the indirect unconjugated immunoperoxidase technique (PAP-technique) was applied (Moorman et al. 1984). For the combined immuno-enzyme histochemical study from quench frozen blocks, 8-10 gim thick cryostat-cut serial sections of adult rabbit tibialis anterior and soleus muscles were mounted on microscope slides coated with AAS (3-aminopropyltriethoxysilane) (Henderson, 1989). The frozen sections were fixed overnight in methanol: acetone: acetic acid:water (35:35:5:25) at -20 'C, washed in PBS 3 times for 5 min each, pretreated with pronase (5 min) and allowed to react with the various antibodies according to the PAP-technique (Bredman et al. 1991).
265
Myosin expression in rabbit masseter isnp
180-
0
1 161
0
-111
x
%
E
84 58 -
i
mm
_
36-
_
Wm
26II-
Fig. 1. Lateral view of the masseter (MASS). Lines I, II and III indicate the level of the selected sections. Heavy solid lines indicate the aponeuroses; triangles show the position of the selected sample sites. MPPO, posterior deep masseter; A, anterior; P, posterior; S, superficial; D, deep.
C H F G A D E B Fig. 2. Western blot analyses of rabbit masseter muscles. Proteins of rabbit masseter (B, C, D) and, as a control, tibialis anterior (E, F, G, H, I) were fractionated by 10% SDS-polyacrylamide gel electrophoresis. After electrophoresis the proteins were transferred to nitrocellulose. Lanes A, D and E show parts of the gel that were stained with serva blue prior to transfer to visualise the marker protein. (A), masseter (D) and tibialis anterior (E) proteins. Nitrocellulose strips were incubated with anti-a MHC (B, F), antia/I/IIB MHC (C, G), anti-IIB MHC (H) and anti-IIA MHC (I). mw = molecular weight.
Enzyme-histochemistry
Cryostat-cut serial sections of quench-frozen adult rabbit tibialis anterior and soleus muscles were mounted on glass slides coated with AAS. The sections were incubated for Ca2' activated adenosine triphosphatase (ATPase) at pH 9.4 (Staron et al. 1983). Preincubations (pH 4.4, 4.6, 10.5) were used to distinguish the several histochemical fibre types (Staron & Pette, 1986; Bredman et al. 1990 a).
the belly of the masseter (line II), about 9 mm above this level (line I) and halfway through the belly of the posterior deep masseter (MPPO compartment) (line III) (Fig. 1). The exact position of the lines was described previously (Bredman et al. 1990a). In the different sections 6 sample sites were selected to represent best the anatomical compartments (Weijs et al. 1987; Bredman et al. 1990 a) (see Fig. 1). Sample 1 is situated in the anterior deep masseter (MPAN compartment) (line I), sample 2 is situated in the
Morphometric techniques The sections used for the immunohistochemical studies were taken at 3 different sites: halfway through
Table 2. Fibre types present in postnatal and adult masseter muscle* Distinct fibre types
Immunohistochemistry anti-a MHX anti-I MHC anti-a/I/II B MHC anti-IIA MHC anti-IIB MHC anti-IIA/IIB MHC MHC composition Enzyme histochemistry
pH 4.4; 4.6; 10.5**
7
8
9
-
-
-
+ + IIA
+ + + + II A/II B
+ + + II B
+ + + + + I/II A/a
IIA
IIAB
IIB
n.t.
1
2
3
4
5
6
-
+ + + I/a
+ + -
+ + -
(I)ac
a
+ + + + IIA/a
_-
+ + I I
I
I
I
IIC
+/-
-
* The fibre types were classified by their MHC composition as determined by immunohistochemistry. Neonatal Mab is omitted. I, intermediate staining of anti-I MHC. ** The fibre types were previously determined by enzyme histochemistry (ATPase reactivity) (Bredman et al. 1990a, b). n.t., not tested by enzyme histochemistry since this fibre type is not present in adult masseter muscle.
Jantine J. Bredman and others
266
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Fig. 4. Immunohistochemical analysis of serial sections in the MSS3 compartment of newborn (A, F, I, M, Q), 7 d (B, F J, N R), 28 d (C, G, K, 0, S) and adult rabbit masseter muscle (D, H, L, P, T). The sections were stained immunohistochemically with anti- a MHC (A-D), anti-I MHC (F-H), anti-11A MHC (I, K, L), anti-11IA/11IB MHC (J), anti-ez/I/II B MHC (M), anti-TI B MHC (N-P) and anti-N MHC (Q-T). I-MHC fibre (1), I/cz-MHC fibre (2), (1)/cz-MHC fibre (3), cc-MHC fibre (4), IIA/ax-MHC fibre (5), IIA-MHC fibre (6), IIA/IIBMHC fibre (7) and II B-MHC fibre (8) are indicated. S, muscle spindle. The scale bar at bottom right represents 21 gim in Q, 15 gim in R, 41
gm in S and 52 gm in _T.
269
Myosin expression in rabbit masseter fibre type appears, containing exclusively a-MHC (designated as oa-MHC fibre) (Figs 4C, 5). During this developmental period the number of IIA/I1BMHC fibres is gradually decreasing while the number of a-MHC and I/f-MHC fibres is increasing (Fig. 5). During these weeks the number of I/IIA/fx-MHC fibres remains the same as at the age of 7 d (about 3 %). A small number of fibres contains exclusively I-MHC. The small number of II B-MHC fibres (1.5 %) are found only in a restricted region (MPPO compartment) of the muscle. The change in the number of II A/a-MHC fibres follows a unique pattern: they are absent in newborn rabbits, reach a maximum number at 21-28 d and then gradually disappear (Figs 5, 6D). This strongly suggests that this fibre type is a transitional form.
Adult masseter muscle. The different fibre types present in the adult rabbit masseter are shown in Tables 2 and 3 and Figure 4(D, H, L, P, T). The I/IIA/(x-MHC fibres have disappeared at adult age. Apart from these, the same fibre types as in the 28 d masseter are found. The expression of neonatal myosin During the first 4 wk of development, N-MHC is present in 90 % of the muscle fibres; however, a decrease in immunoreactivity was seen. All II AMHC, II A/II B-MHC, II A/oc-MHC, I/II A/at-MHC and a-MHC fibres contain N-MHC. Furthermore, N-MHC is present in widely varying concentrations in the I-MHC fibres (0-20 % during the first 4 wk) and in the I/a-MHC fibres (0%O at d 1, 56-62 % from d 7 until d 28). As described above, the number of JIBMHC fibres drops steeply in the first 4 wk. The II BMHC fibre lose their N-MHC; in the first 3 wk 90 % of the II B-MHC fibres contain N-MHC, in the 4th wk only 3 % contain N-MHC. In adult animals only 20 % of the total fibre population contains N-MHC. N-MHC is only found in a part of the IIA-MHC, IIA/IIB-MHC and aMHC fibre types, all other fibre types contain no NMHC. However, the fact that the oldest of the investigated animals showed the least number of NMHC containing fibres suggests that the neonatal myosin might disappear completely.
Regional differences between sample sites (compartments) Postnatal changes in fibre type distribution in the 6 sample sites (compartments) are shown in Figure 6.
I/a
IIA/Il B
10 "
IIA/a 1
7
14
21
28
A
Age Fig. 5. Postnatal changes in fibre type distribution in the rabbit masseter muscle. The percentages of 6 different fibre types are shown per age. Other fibre types are omitted as they were present only in small amounts.
For each age heterogeneity between the compartments is shown; however, during development, trends of regional difference do change. For example, 1 wk rabbits show differences between compartments in their percentage of IIA-MHC fibres. The MSSl(d) contains 72% whilst the MPPO contains only 33 %. At the adult stage in the deep MSSl compartment the amount of IIA-MHC fibres had decreased to 25 % but in the MPPO compartment the amount had increased during development to 49 %. For II A/II BMHC fibres at 7 d great differences are found between the MPPO (60%) and the MSS3 (30 %) compartments. At the adult stage the amount of II A/IT BMHC had decreased in the MPPO compartment to 13 % and in the MSS3 compartment to 0 %. Comparison of 7 d and adult masseter shows that a fibre type with the highest (or lowest) concentration in a certain compartment at 7 d, does not usually show the highest (or lowest) concentration in the same compartment at adult age. This means that between ages the fibre composition can be very different in the same compartment. All in all our results show that directly after birth the masseter is heterogeneous, but the different compartments develop in a different way so that adult heterogeneity seems to originate during postnatal development. Distribution of.fibre types within muscle compartments
Despite differences in fibre composition between sample sites most of the Mabs used show a regular pattern of distribution of MHC within compartments. 18-2
270
Jantine J. Bredman and others
However, there are 3 Mabs (anti-I MHC, anti-II B MHC, anti-N MHC) sometimes showing, also within compartments, considerable heterogeneity in distribution. Variation of anti-I MHC at the newborn stage is shown in Figure 7A. In the MSS4 compartment the anterior/superficial part contains no I-MHC. The deep part of the middle masseter (MSM 12 compartment, Weijs et al. 1987; Bredman et al. 1990a) also shows no I-MHC. Examples of the heterogeneity of anti-IIB MHC and anti-N MHC in the adult MSS4 compartment are shown in Figure 7 B and C. The IIB-MHC is only present in a small restricted part of the compartment whilst the N-MHC appears along the tendon planes.
80
DISCUSSION
Postnatal expression of 'cardiac' a myosin
'Cardiac' c-MHC is present only in a few extrafusal fibres (type I/a-MHC) in the newborn rabbit masseter. At the age of 7 d this myosin appears in II AMHC and I/IIA-MHC fibres, and at 14 d exclusively ct-MHC containing fibres appear. The I/ct-MHC and ax-MHC fibres show a gradual increase in number until the adult stage. d'Albis et al. (1991) reported the appearance of a-MHC in rabbit masseter during the 3rd wk after birth. Apparently the amount of ac-MHC was too low to determine its existence with SDSglycerol gel electrophoresis before 14 d. Simul-
A
70
% 60 t 50 40
I
1
7
14
21
28
Adult 1 Age (days)
7
14
21
28
Adult
D
Fig. 6. Postnatal changes in fibre type distribution in the 6 sample sites (compartments) of the masseter muscle. (A) IIA-MHC fibres; (B) IIA/IIB-MHC fibres; (C) I/a-MHC fibres; (D) IIA/ca-MHC fibres; (E) a-MHC fibres; (F) I-MHC fibres. The vertical axis gives the percentage frequency of the different fibre types in a compartment.
Myosin expression in rabbit masseter
271
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Fig. 7. Heterogeneous distribution of Mabs in newborn and adult masseter muscle. (A) Transverse section of aI1 d masseter muscle at line II, stained with anti-I MHC. The whole section is shown. (1) Mid-part of the M554 compartment; (2) mid-part of the MSM 12 (Weijs et al. 1987; Bredman et al. 1990a) compartment. (B, C) Serial transverse sections of an adult masseter muscle showing the MSS4 compartment at line II, stained with anti-I1 B MHC (B) and anti-N MHC (C). Scale bar represents 350 gim in A and 260 gim in C.
taneously with the increase of a-MHC expression a decrease of II A-MHC fibres and especially II A/IIBMHC fibres is evident. This implies that during postnatal growth a myosin isozyme transition takes place in fibres from fast (II A, II A/IIB and N) MHC to relatively slow a-MHC and/or I/a-MHC. This hypothetical transition of fibre types is reminiscent of transitions described in the literature from fast to slow MHC expression in presumptive slow muscles (Kugelberg, 1976; Pelloni-Mueller et al. 1976; Butler-Browne & Whalen, 1984; Hoh et al. 1988; d'Albis et al. 1989). This hypothesis is supported by the unique pattern of transitional postnatal appearance of II A/a-MHC fibres. These fibres are present in largest numbers at the age of 21-28 d (Figs 5, 6D). The IIA/a-MHC fibres and/or I/IIA/oc-MHC fibres contain 2 or more myosin isozymes and might therefore be in the process of transition. IIA/oc-MHC might be replaced by aMHC and I/IIA/a-MHC by I/a-MHC. In view of the great numbers in which the a-MHC fibres and I/aMHC fibres appear and the disappearance of large
numbers of II A/II B-MHC fibres in the adult masseter it is quite possible that this latter fibre type somehow changes into c-MHC or I/a-MHC fibres, possibly via IIA-MHC fibres. The peak of IIA/a-MHC fibres at the age of 21-28 d corresponds with the time that the mammalian feeding apparatus undergoes a switch in function from suckling to chewing (Maeda et al. 1981; Herring, 1985). At the age of 3-4 wk rabbits have just started to feed independently on solid food (Yardin, 1974; Sorensen et al. 1968). Since the speed of contraction of a single skeletal muscle fibre is largely determined by its MHC content, Vna- of fast MHC (3.1 muscle length/s) is about 4 times the Vmax of slow MHC (0.7 muscle length/s) and fl-MHC (0.8 muscle length/s) (Marston & Taylor, 1980; Reiser et al. 1985; Rome et al. 1990) and the ATPase activity (we have no information on Vkax of 'cardiac' a-MHC) in heart atrium is 2 to 3 times higher than in slow (ventricular ,/) MHC (Pope et al. 1980; McNally et al. 1989) so it seems likely that the muscle fibres containing ax-MHC
272
Jantine J. Bredman and others
will show contraction speeds intermediate between fibres containing I-MHC and IIA-MHC. Thus conversion of IIA-MHC and IIB-MHC to a-MHC may imply a conversion to a slower muscle. This should be established by physiological studies. The notion that the masseter might reveal a developmentally-regulated decrease in contraction speed underlines a similar idea predicted by the muscle architecture (Weijs et al. 1987; Langenbach & Weijs, 1990). Weijs et al. (1987) and Langenbach & Weijs (1990) demonstrated that the masseter muscle of young rabbits contains relatively long fibres, meaning that their absolute speed of shortening must be high, relative to adult animals. The authors concluded that in the course of postnatal development a decrease of speed of contraction might be related to changing functional requirements. In contrast to the above named hypothesis, Close (1964) reported that the speed of contraction of the rat soleus remains the same during postnatal development though a transition from fast to slow MHC expression takes place (Butler-Browne & Whalen, 1984; d'Albis et al. 1989). Our finding of 'cardiac' a-MHC in the rabbit masseter muscle fibres supports the idea that individual muscle fibres might, by varying their MHC content, adapt their contraction speed to the conditions in the muscle during postnatal growth. However, not only masticatory muscles contain cardiac aMHC: we (Bredman et al. 1990b) showed the existence of 'cardiac' oc-MHC containing muscles in the cranial area of adult rabbit. This led us to suggest that embryonic neural crest cells might play a role in inducing the expression of specific MHCs in skeletal muscle, so that the expression of MHC would be determined by the developmental history of the tissue.
Expression of neonatal and slow myosin
Until the fourth postnatal week about 90 % of the fibres contain N-MHC. Thereafter a decrease in number of N-MHC containing fibres takes place so that in adult masseter N-MHC is expressed in only 20 % of the fibres (the oldest rabbits contain the least N-MHC containing fibres). The finding of N-MHC in the adult rabbit masseter distinguishes it from limb muscles. In normal adult limb muscles N-MHC is never seen (Guth & Samaha, 1972; Butler-Browne & Whalen, 1984; Butler-Browne et al. 1988). But in masseter muscle of adult human (Butler-Browne et al. 1988) and 3 month old rat (d'Albis et al. 1989) the N-MHC is still expressed. Soussi-Yanicostas et al. (1990) demonstrated, in the
human, that this late maturation is also apparent from biochemical data with respect to the presence of slow tropomyosin, MLCIs, MLC2s, slow MHC and fast MHC in the masseter unlike in the quadriceps. It is tempting to speculate about a functional meaning for this late expression of N-MHC. d'Albis et al. (1986) and Soussi-Yanicostas et al. (1990) proposed that the masseter develops at a different rate and/or a different way compared with other skeletal muscles. The expression of N-MHC extends the number of different MHCs already present in masticatory muscles. According to Rowlerson (1990) the mixed composition of MHC in the rabbit masseter muscle might reflect their rather rapid masticatory movements compared with herbivores. Furthermore, it might be an adaptation to cope with a diet of variable texture. The N-MHC was absent or present only in very small amounts in the fibres containing type I-MHC. During the first weeks the latter fibres are topographically evenly distributed throughout the muscle (Fig. 7A) (though they are absent in some compartments). The relatively large diameter of these I-MHC fibres, at the newborn stage, points according to Hoh & Hughes (1989) to a primary myotube origin. According to the literature the slow primary myotubes synthesise embryonic myosin (until about 2 wk in rat soleus and 11 d in cat masseter; Butler-Browne & Whalen, 1984; Hoh & Hughes, 1989). We could not check this in the rabbit masseter because no monoclonal antibody against embryonic MHC was available. The development of heterogeneity in relation to muscle architecture All ages show heterogeneity in MHC content between the different sample sites. This heterogeneity varies with age. For example, the 1 wk masseter shows the highest percentage of II A-MHC in the deep MSS 1(d) compartment while the adult masseter shows the highest percentage of IIA-MHC in the superficial MSS4 compartment. The muscle starts with a mixture of I-MHC, II A-MHC and II B-MHC containing fibres and from this, a mixture of I-MHC, II A-MHC, IIB-MHC and a-MHC containing fibres develops, leading to quite different heterogeneity in the adult. It is possible that the changes have a functional meaning, with different pre- and postweaning roles for different regions, but also that different regions have different developmental speeds. Within compartments heterogeneity in distribution of fibre types can appear. In some compartments a
fibre type can be absent, restricted to a small part (see
Myosin expression in rabbit masseter
for example Fig. 7) or appear homogeneously. Heterogeneity in distribution of one fibre type can be the result of motor unit distribution. All muscle fibres belonging to a single motor unit belong to the same immunohistochemical category (Edstr6m & Kugelberg, 1968). Furthermore, Stalberg & Eriksson (1987) implied that motor units are restricted to certain regions or compartments. When only a single motor unit, consisting of a fibre type that is relatively rare, is present in a compartment a picture like Figure 7B might appear, but when 3 or more units of this fibre type are present the whole compartment will show the specific fibre type. This could explain interindividual variation present in rabbit masseter muscle compartments. Despite the individual age variations in fibre percentages, the pattern of appearance and disappearance of specific MHC types is always similar. ACKNOWLEDGEMENTS
We thank Dr G. S. Butler-Browne, INSERM, Paris, France for providing the neonatal antibody. We also thank Cars Gravemeyer, Ans van Horssen and Ad van Horssen for their photographic and art work and Sabina Tesink-Takema and Carol Verhoek-Pocock for their expert technical assistance.
REFERENCES
D'ALBIS A, JANMOT C, BECHET JJ (1986) Comparison of myosins from the masseter muscle of adult rat, mouse and guinea-pig. European Journal of Biochemistry 156, 291-296. D'ALBIS A, COUTEAUX R, JANMOT C, ROULET A (1989) Specific programs of myosin expression in the postnatal development of rat muscles. European Journal of Biochemistry 183, 583-590. D'ALBIS A, JANMOT C, MIRA JC, COUTEAUX R (1991) Characterization of a ventricular VI myosin isoform in rabbit masticatory muscles. Developmental and neural regulation. Basic and Applied Myology 1, 23-34. BREDMAN JJ, WEIJS WA, MOORMAN AFM, BRUGMAN P (1990a) Histochemical and functional fibre typing of the rabbit masseter muscle. Journal of Anatomy 168, 31-47. BREDMAN JJ, WEIJS WA, MOORMAN AFM (1990b) Expression of 'cardiac-specific' myosin heavy chain in rabbit cranial muscles. In Muscle and Motility 2; Proceedings of XIXth European Conference (ed. G. Mar6chal & U. Carraro), pp. 329-335. Brussels: Intercept. BREDMAN JJ, WESSELs A, WEIJS WA, KORFAGE JAM, SOFFERS CAS, MOORMAN AFM (1991) Demonstration of 'cardiac-specific' myosin heavy chain in masticatory muscles of human and rabbit. Histochemical Journal 23, 160-170. BUTLER-BROWNE GS, WHALEN RG (1984) Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle. Developmental Biology 102, 324-334. BUTLER-BROWNE GS, ERIKSSON PO, LAURENT C, THORNELL LE (1988) Adult human masseter muscle fibers express myosin isozyme characteristics of development. Muscle and Nerve 11, 610-620. CHRISTENSEN L, STRANGE L (1987) Universal immunoperoxidase
273 staining protocol to optimize the use of polyclonal and monoclonal antibodies. Journal of Histotechnology 10, 11-15. CLOSE R (1964) Dynamic properties of fast and slow skeletal muscles of the rat during development. Journal of Physiology 173, 74-95. DE GROOT IJM, LAMERS WH, MOORMAN AFM (1989) Isomyosin expression patterns during rat heart morphogenesis: an immunohistochemical study. Anatomical Record 224, 365-373. EDDINGER TJ, Moss RL (1987) Mechanical properties of skinned single fibers of identified types from rat diaphragm. American Journal of Physiology 253, c210-c218. EDSTROM L, KUGELBERG E (1968) Histochemical composition, distribution of fibres and fatiguability of single motor units. Journal of Neurology, Neurosurgery and Psychiatry 31, 424-433. GUTH L, SAMAHA FJ (1972) Erroneous interpretations which may result from application of the 'myofibrillar ATPase' histochemical procedure to developing muscle. Experimental Neurology 34, 465-475. HENDERSON C (1989) Aminoalkylsilane: an inexpensive, simple preparation for slide adhesion. Journal of Histotechnology 12, 123-124. HERRING SW (1985) The ontogeny of mammalian mastication. American Zoology 25, 339-349. HOH JFY, HUGHES S, HALE PT, FITZSIMONs RB (1988) Immunocytochemical and electrophoretic analyses of changes in myosin gene expression in cat limb fast and slow muscles during postnatal development. Journal of Muscle Research and Cell Motility 9, 30-47. HOH JFY, HUGHES S (1989) Immunocytochemical analysis of the perinatal development of cat masseter muscle using anti-myosin antibodies. Journal of Muscle Research and Cell Motility 10, 312-325. KUGELBERG E (1976) Adaptive transformation of rat soleus motor units during growth. Journal of the Neurological Sciences 27, 269-289. LANGENBACH GEJ, WEIJS WA (1990) Growth patterns of the rabbit masticatory muscles. Journal of Dental Research 69, 20-25. MAEDA N, HANAI H, KUMEGAWA M (1981) Postnatal development of masticatory organs in rats. III Effect of mastication on the postnatal development of the m. masseter superficialis. Anatomischer Anzeiger (Jena) 150, 424-427. MARSTON SB, TAYLOR EW (1980) Comparison of the myosin and actomyosin ATPase mechanisms of the four types of vertebrate muscles. Journal of Molecular Biology 139, 573-600. MCNALLY EM, KRAFT R, BRAVO M, TAYLOR DA, LEINWAND LA (1989) Full-length rat alpha and beta 'cardiac' myosin heavy chain sequences. Journal of Molecular Biology 210, 665-671. MOORMAN AFM, DE BOER PAJ, LINDERS MTH, CHARLES R (1984) The histone H5 variant in Xenopus laevis. Cell Differentiation 14, 113-123. PEDROSA F, SOUKUP T, THORNELL LE (1990) Expression of an alpha cardiac-like myosin heavy chain in muscle spindle fibres. Histochemistry 95, 105-113. PELLONI-MUELLER G, ERMINI M, JENNY E (1976) Myosin light chains of developing fast and slow rabbit skeletal muscle. FEBS Letters 67, 68-74. POPE B, HOH JFY, WEEDS A (1980) The ATPase activities of rat cardiac myosin isozymes. FEBS Letters 118, 205-208. REISER PJ, Moss RL, GIULIAN GG, GREASER ML (1985) Shortening velocity in single fibers from adult rabbit soleus muscles is correlated with myosin heavy chain composition. Journal of Biology and Chemistry 260, 9077-9080. ROME LC, SOSNICKI SS, GOBLE DO (1990) Maximum velocity of shortening of three fibre types from horse soleus muscle: implications for scaling with body size. Journal ofPhysiology 431, 173-185. ROWLERSON AM (1990) Specialization of mammalian jaw muscles:
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fibre type compositions and the distribution of muscle spindles. In Neurophlvsiologv of the Jaws and Teeth (ed A. Taylor). pp. 1 51. London: Macmillan. SCHIAFFINO S, AUSONI S, GORZA L, SAGGIN L, GUNDERSEN K (1988) Myosin heavy chain isoforms and velocity of shortening of type 2 skeletal muscle fibres. Acta PhYsiologica Scandinavica 134, 575-576. SORENSEN MF, ROGERS JP, BASKETT TS (1968) Reproduction and development in confined swamp rabbits. Journal of Wildlife Management 32, 520-531. STALBERG E, ERIKSSON PO (1987) A scanning electromyographic study of the topography of human masseter single motor units. Archives of Oral Biologi' 32, 793-797. STARON RS, HIKIDA RS, HAGERMAN FC (1983) Reevaluation of human muscle fast-twitch subtypes: evidence for a continuum. Histochemistry 78, 33-39. STARON RS, PETTE D (1986) Correlation between myofibrillar ATPase activity and myosin heavy chain composition in rabbit muscle fibers. Histochemistry 86, 19-23. SOUSSI-YANICOSTAS N, BARBET JP, LAURENT-WINTER C, BARTON P, BUTLER-BROWNE GS (1990) Transition of myosin isozymes during development of human masseter muscle. Development 108, 239-249. THORNELL LE, BILLETER R, ERIKSSON PO, RINGQVIST M (1984) Heterogeneous distribution of myosin in human masticatory
muscle fibres as shown by immunocytochemistry. Archives of Oral Biology 29, 1-5.
WEIJS WA, BRUGMAN P, KLOK EM (1987) The growth of the skull and jaw muscles of the rabbit and its functional consequences in the New Zealand rabbit (Oryctolagus cuniculus). Journal of Morphology 194, 143-161. WESSELs A, VERMEULEN JLM, MOORMAN AFM, BECKER AE (1988) Immunohistochemical detection of myosin heavy chain isoforms in large sections of whole human hearts. In Sarcomeric and Nonsarcomeric Muscles: Basic and Applied Research Prospects for the 90s, (ed. U. Carraro), pp. 311-316. Padova: Unipress. WESSELs A, VERMEULEN JLM, VIRAiGH Sz, KA~LMAN F, LAMERS WH, MOORMAN AFM (1990 a) Spatial distribution of 'tissue specific' antigens in the developing human heart and skeletal muscle: II, an immunohistochemical analysis of myosin heavy chain isoform expression patterns in the embryonic heart. Anatomical Record 229, 355-368. WESSELs A, SOFFERS CAS, VERMEULEN JLM, MYINDESS TA, BREDMAN JJ, MOORMAN AFM (1990b) Expression of a 'cardiacspecific' myosin heavy chain in intrafusal fibres of the developing human spindle. In Muscle and Motility 2; Proceedings of XIXth European Conference (ed. G. Marechal & U. Carraro), pp. 71-77. Brussels: Intercept. YARDIN M (1974) Sur l'ontogenese de la mastication chez le lapin (Oryctolagus cuniculus). Mammalia 38, 737-747.
J. Anat. (1992) 180, pp. 263-274, with 7 figures Printed in Great Britain
Myosin heavy chain expression in rabbit masseter muscle during postnatal development JANTINE J. BREDMANI, WIM A. WEIJS', HANS A. M. KORFAGE1, PETER BRUGMAN' AND ANTOON F. M. MOORMAN2
'Department of Functional Anatomy, Academic Centre for Dentistry Amsterdam (ACTA) and 2Department of Anatomy and Embryology, Faculty of Medicine, University of Amsterdam, The Netherlands
(Accepted 12 November 1991)
ABSTRACT
The expression of isoforms of myosin heavy chain (MHC) during postnatal development was studied in the masseter muscle of the rabbit. Evidence is presented that in addition to adult fast and slow myosin, the rabbit masseter contains neonatal and 'cardiac' ac-MHC. During postnatal growth myosin transitions take place from neonatal and fast (IIA, IIA/IIB-referring to a fibre containing both IIA and IIB MHCs) MHC to adult 'cardiac' a-MHC and I/a-MHC. Since there is a temporary population of fibres containing IIA/a-MHC during the first 4 wk of development with a peak in the 3rd to 4th wk, the transition from IIA-MHC to a-MHC may occur in these IIA/a-MHC-containing fibres. The appearance of 'cardiac' a-MHC coincides with the timing of weaning, suggesting that the changes in MHC content, that probably result in a transition to a lower speed of contraction, have functional significance related to weaning. The finding of neonatal MHC in adult rabbits indicates that the masseter develops at a rate and in a way that is distinct from most other skeletal muscles. A spatiotemporal variation in expression of myosin isozymes within the masseter was observed, with many fibres containing more than one myosin type, indicating developmentally regulated spatial differences in function.
INTRODUCTION
The isoform of MHC protein expressed in a skeletal muscle fibre is in part determined by the type of myoblast that forms the fibre. Apart from the well known adult myosin heavy chain (MHC) isoforms, that is, type I (slow contracting), type II A (fast contracting) and type II B (fast contracting), embryonic and neonatal MHC isoforms have been described for developmental stages. The myosin isozymes follow a transition from embryonic either to neonatal or adult slow and from neonatal to adult fast and/or adult slow myosin (Butler-Browne & Whalen, 1984; Butler-Browne et al. 1988; Hoh & Hughes, 1989). It has been shown that masseter muscle occupies a special position among the skeletal muscles. For instance, neonatal MHC often persists in adult rat
and human masseter (d'Albis et al. 1986; ButlerBrowne et al. 1988). In addition, in adult rabbit and human jaw muscles, besides neonatal, slow (type I) and fast (type II A and II B) MHC, a 'cardiac' aMHC is expressed (Bredman et al. 1990b, 1991; d'Albis et al. 1991). Furthermore, single masseter muscle fibres usually express more than one type of MHC (Thornell et al. 1984; Rowlerson, 1990). This might be indicative for a fine gradation of possible contraction speeds in the fibre of the muscle since it has been shown that the speed of maximum velocity of shortening of single muscle fibres (Vmax) is correlated with the MHC composition (Reiser et al. 1985; Eddinger & Moss, 1987; Schiaffino et al. 1988). Previous (immuno-)histochemical studies on masseter muscles have mostly been based on small parts of the muscle, assuming that the whole muscle is similar. In this study special attention was devoted to regional
Correspondence to Dr J. J. Bredman, Department of Functional Anatomy, Academic Centre for Dentistry Amsterdam, University of Amsterdam K2, Meibergdreef 15, 1005 AZ Amsterdam, The Netherlands.
264
Jantine J. Bredman and others
differences within the masseter. Differences in fibre type composition were found when different regions of the muscle were compared. The occurrence of the various MHC isoforms is described in the rabbit masseter muscle during postnatal development. Furthermore, spatiotemporal changes in the MHC expression pattern in the developing masseter are related to postnatal changes in function. MATERIALS AND METHODS
Animals and dissection New Zealand rabbits (4 at each of the following ages) were killed by anaesthetic overdose: newborn (1 d), 7, 14, 21 and 28 d old (weight-range 35-1074 g) and adult (2500-4400 g, 2 males and 2 females). The masseter muscles were dissected from each animal after marks had been placed to indicate the levels and directions of 2 sections. Because the posterior deep portion of the masseter (MPPO compartment; Weijs et al. 1987; Bredman et al. 1990a) has an orientation different from the rest of the masseter it was removed separately. Embryonic hearts were obtained from rabbits at the 15th d of gestation. For the immunohistochemical studies all masseter muscles were fixed in a mixture of methanol: acetone:acetic acid:water (35:35:5:25), dehydrated in a graded series of ethanols, cleared in chloroform and embedded in Paraplast Plus (Monoject, Ireland) (Wessels et al. 1988; Bredman et al. 1990a). For the combined immuno- and enzyme-histochemical (ATPase) study, adult tibialis anterior and soleus muscles were frozen in liquid Freon-22 (monochlorodifluoromethane) cooled with liquid nitrogen and stored at -70 'C. For Western blot analysis, Table 1. Antibody reactivity* MHC Mab no.
a
249-5A4 (anti-a) 219-lDl (anti-I)
+
412-1D5 (anti-a/I/IIB) 333-7H1 (anti-I1 A) 41 1-1D5 (anti-II B) 340-3B5 (anti-IIA/IIB)
+
anti-N
I
IIA
+ +
IIB
N
+ + +
+ + +
* The pattern of reactivity of the 7 anti-MHC Mabs used in this study with the various MHC isozymes, as determined by immuno-
histochemistry, is summarised. +, positive reaction; otherwise no reaction.
tissue specimens were frozen in liquid nitrogen and stored at -70 'C. Production and characterisation of the monoclonal antibodies
The isolation of myosin and the production and characterisation of the monoclonal antibodies (Mabs) 249-5A4, 219-IDI, 412-1D5 and 340-5B3 has been described before (Wessels et al. 1988, 1990 a; de Groot et al. 1989; Bredman et al. 1991). The Mabs 333-7H1 and 411-4F10 were raised against a protein extract from muscle tissue of adult rabbit tibialis anterior and psoas. The antibody against neonatal MHC was kindly provided by Dr G. S. Butler-Browne (INSERM, Paris). The specificity of the antibodies was determined on tibialis anterior and soleus as indicated in Table 1, and has been described in part previously (Bredman et al. 1991 a). Western blot analyses were used to specify the antibodies towards MHC (Wessels et al. 1990 a; Bredman et al. 1991). SDS-glycerol gel electrophoresis of c-MHC was carried out by the method of d'Albis et al. (1991). The specificity of the neonatal Mab has been demonstrated by Butler-Browne et al. (1988).
Immunohistochemistry The embedded masseter muscles (all ages) were cut into 8-10 gim thick serial sections and mounted on microscope slides coated with poly-L-lysine. After deparaffination the sections were pretreated with pronase (0.1 mg/ml, 5-30 min, depending on the age of the animal) to optimise binding of the antibodies with the antigens (Christensen & Strange, 1987). To detect the binding of the specific monoclonal antibodies with the MHC isoforms the indirect unconjugated immunoperoxidase technique (PAP-technique) was applied (Moorman et al. 1984). For the combined immuno-enzyme histochemical study from quench frozen blocks, 8-10 gim thick cryostat-cut serial sections of adult rabbit tibialis anterior and soleus muscles were mounted on microscope slides coated with AAS (3-aminopropyltriethoxysilane) (Henderson, 1989). The frozen sections were fixed overnight in methanol: acetone: acetic acid:water (35:35:5:25) at -20 'C, washed in PBS 3 times for 5 min each, pretreated with pronase (5 min) and allowed to react with the various antibodies according to the PAP-technique (Bredman et al. 1991).
265
Myosin expression in rabbit masseter isnp
180-
0
1 161
0
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%
E
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i
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_
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_
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Fig. 1. Lateral view of the masseter (MASS). Lines I, II and III indicate the level of the selected sections. Heavy solid lines indicate the aponeuroses; triangles show the position of the selected sample sites. MPPO, posterior deep masseter; A, anterior; P, posterior; S, superficial; D, deep.
C H F G A D E B Fig. 2. Western blot analyses of rabbit masseter muscles. Proteins of rabbit masseter (B, C, D) and, as a control, tibialis anterior (E, F, G, H, I) were fractionated by 10% SDS-polyacrylamide gel electrophoresis. After electrophoresis the proteins were transferred to nitrocellulose. Lanes A, D and E show parts of the gel that were stained with serva blue prior to transfer to visualise the marker protein. (A), masseter (D) and tibialis anterior (E) proteins. Nitrocellulose strips were incubated with anti-a MHC (B, F), antia/I/IIB MHC (C, G), anti-IIB MHC (H) and anti-IIA MHC (I). mw = molecular weight.
Enzyme-histochemistry
Cryostat-cut serial sections of quench-frozen adult rabbit tibialis anterior and soleus muscles were mounted on glass slides coated with AAS. The sections were incubated for Ca2' activated adenosine triphosphatase (ATPase) at pH 9.4 (Staron et al. 1983). Preincubations (pH 4.4, 4.6, 10.5) were used to distinguish the several histochemical fibre types (Staron & Pette, 1986; Bredman et al. 1990 a).
the belly of the masseter (line II), about 9 mm above this level (line I) and halfway through the belly of the posterior deep masseter (MPPO compartment) (line III) (Fig. 1). The exact position of the lines was described previously (Bredman et al. 1990a). In the different sections 6 sample sites were selected to represent best the anatomical compartments (Weijs et al. 1987; Bredman et al. 1990 a) (see Fig. 1). Sample 1 is situated in the anterior deep masseter (MPAN compartment) (line I), sample 2 is situated in the
Morphometric techniques The sections used for the immunohistochemical studies were taken at 3 different sites: halfway through
Table 2. Fibre types present in postnatal and adult masseter muscle* Distinct fibre types
Immunohistochemistry anti-a MHX anti-I MHC anti-a/I/II B MHC anti-IIA MHC anti-IIB MHC anti-IIA/IIB MHC MHC composition Enzyme histochemistry
pH 4.4; 4.6; 10.5**
7
8
9
-
-
-
+ + IIA
+ + + + II A/II B
+ + + II B
+ + + + + I/II A/a
IIA
IIAB
IIB
n.t.
1
2
3
4
5
6
-
+ + + I/a
+ + -
+ + -
(I)ac
a
+ + + + IIA/a
_-
+ + I I
I
I
I
IIC
+/-
-
* The fibre types were classified by their MHC composition as determined by immunohistochemistry. Neonatal Mab is omitted. I, intermediate staining of anti-I MHC. ** The fibre types were previously determined by enzyme histochemistry (ATPase reactivity) (Bredman et al. 1990a, b). n.t., not tested by enzyme histochemistry since this fibre type is not present in adult masseter muscle.
Jantine J. Bredman and others
266
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Fig. 4. Immunohistochemical analysis of serial sections in the MSS3 compartment of newborn (A, F, I, M, Q), 7 d (B, F J, N R), 28 d (C, G, K, 0, S) and adult rabbit masseter muscle (D, H, L, P, T). The sections were stained immunohistochemically with anti- a MHC (A-D), anti-I MHC (F-H), anti-11A MHC (I, K, L), anti-11IA/11IB MHC (J), anti-ez/I/II B MHC (M), anti-TI B MHC (N-P) and anti-N MHC (Q-T). I-MHC fibre (1), I/cz-MHC fibre (2), (1)/cz-MHC fibre (3), cc-MHC fibre (4), IIA/ax-MHC fibre (5), IIA-MHC fibre (6), IIA/IIBMHC fibre (7) and II B-MHC fibre (8) are indicated. S, muscle spindle. The scale bar at bottom right represents 21 gim in Q, 15 gim in R, 41
gm in S and 52 gm in _T.
269
Myosin expression in rabbit masseter fibre type appears, containing exclusively a-MHC (designated as oa-MHC fibre) (Figs 4C, 5). During this developmental period the number of IIA/I1BMHC fibres is gradually decreasing while the number of a-MHC and I/f-MHC fibres is increasing (Fig. 5). During these weeks the number of I/IIA/fx-MHC fibres remains the same as at the age of 7 d (about 3 %). A small number of fibres contains exclusively I-MHC. The small number of II B-MHC fibres (1.5 %) are found only in a restricted region (MPPO compartment) of the muscle. The change in the number of II A/a-MHC fibres follows a unique pattern: they are absent in newborn rabbits, reach a maximum number at 21-28 d and then gradually disappear (Figs 5, 6D). This strongly suggests that this fibre type is a transitional form.
Adult masseter muscle. The different fibre types present in the adult rabbit masseter are shown in Tables 2 and 3 and Figure 4(D, H, L, P, T). The I/IIA/(x-MHC fibres have disappeared at adult age. Apart from these, the same fibre types as in the 28 d masseter are found. The expression of neonatal myosin During the first 4 wk of development, N-MHC is present in 90 % of the muscle fibres; however, a decrease in immunoreactivity was seen. All II AMHC, II A/II B-MHC, II A/oc-MHC, I/II A/at-MHC and a-MHC fibres contain N-MHC. Furthermore, N-MHC is present in widely varying concentrations in the I-MHC fibres (0-20 % during the first 4 wk) and in the I/a-MHC fibres (0%O at d 1, 56-62 % from d 7 until d 28). As described above, the number of JIBMHC fibres drops steeply in the first 4 wk. The II BMHC fibre lose their N-MHC; in the first 3 wk 90 % of the II B-MHC fibres contain N-MHC, in the 4th wk only 3 % contain N-MHC. In adult animals only 20 % of the total fibre population contains N-MHC. N-MHC is only found in a part of the IIA-MHC, IIA/IIB-MHC and aMHC fibre types, all other fibre types contain no NMHC. However, the fact that the oldest of the investigated animals showed the least number of NMHC containing fibres suggests that the neonatal myosin might disappear completely.
Regional differences between sample sites (compartments) Postnatal changes in fibre type distribution in the 6 sample sites (compartments) are shown in Figure 6.
I/a
IIA/Il B
10 "
IIA/a 1
7
14
21
28
A
Age Fig. 5. Postnatal changes in fibre type distribution in the rabbit masseter muscle. The percentages of 6 different fibre types are shown per age. Other fibre types are omitted as they were present only in small amounts.
For each age heterogeneity between the compartments is shown; however, during development, trends of regional difference do change. For example, 1 wk rabbits show differences between compartments in their percentage of IIA-MHC fibres. The MSSl(d) contains 72% whilst the MPPO contains only 33 %. At the adult stage in the deep MSSl compartment the amount of IIA-MHC fibres had decreased to 25 % but in the MPPO compartment the amount had increased during development to 49 %. For II A/II BMHC fibres at 7 d great differences are found between the MPPO (60%) and the MSS3 (30 %) compartments. At the adult stage the amount of II A/IT BMHC had decreased in the MPPO compartment to 13 % and in the MSS3 compartment to 0 %. Comparison of 7 d and adult masseter shows that a fibre type with the highest (or lowest) concentration in a certain compartment at 7 d, does not usually show the highest (or lowest) concentration in the same compartment at adult age. This means that between ages the fibre composition can be very different in the same compartment. All in all our results show that directly after birth the masseter is heterogeneous, but the different compartments develop in a different way so that adult heterogeneity seems to originate during postnatal development. Distribution of.fibre types within muscle compartments
Despite differences in fibre composition between sample sites most of the Mabs used show a regular pattern of distribution of MHC within compartments. 18-2
270
Jantine J. Bredman and others
However, there are 3 Mabs (anti-I MHC, anti-II B MHC, anti-N MHC) sometimes showing, also within compartments, considerable heterogeneity in distribution. Variation of anti-I MHC at the newborn stage is shown in Figure 7A. In the MSS4 compartment the anterior/superficial part contains no I-MHC. The deep part of the middle masseter (MSM 12 compartment, Weijs et al. 1987; Bredman et al. 1990a) also shows no I-MHC. Examples of the heterogeneity of anti-IIB MHC and anti-N MHC in the adult MSS4 compartment are shown in Figure 7 B and C. The IIB-MHC is only present in a small restricted part of the compartment whilst the N-MHC appears along the tendon planes.
80
DISCUSSION
Postnatal expression of 'cardiac' a myosin
'Cardiac' c-MHC is present only in a few extrafusal fibres (type I/a-MHC) in the newborn rabbit masseter. At the age of 7 d this myosin appears in II AMHC and I/IIA-MHC fibres, and at 14 d exclusively ct-MHC containing fibres appear. The I/ct-MHC and ax-MHC fibres show a gradual increase in number until the adult stage. d'Albis et al. (1991) reported the appearance of a-MHC in rabbit masseter during the 3rd wk after birth. Apparently the amount of ac-MHC was too low to determine its existence with SDSglycerol gel electrophoresis before 14 d. Simul-
A
70
% 60 t 50 40
I
1
7
14
21
28
Adult 1 Age (days)
7
14
21
28
Adult
D
Fig. 6. Postnatal changes in fibre type distribution in the 6 sample sites (compartments) of the masseter muscle. (A) IIA-MHC fibres; (B) IIA/IIB-MHC fibres; (C) I/a-MHC fibres; (D) IIA/ca-MHC fibres; (E) a-MHC fibres; (F) I-MHC fibres. The vertical axis gives the percentage frequency of the different fibre types in a compartment.
Myosin expression in rabbit masseter
271
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Fig. 7. Heterogeneous distribution of Mabs in newborn and adult masseter muscle. (A) Transverse section of aI1 d masseter muscle at line II, stained with anti-I MHC. The whole section is shown. (1) Mid-part of the M554 compartment; (2) mid-part of the MSM 12 (Weijs et al. 1987; Bredman et al. 1990a) compartment. (B, C) Serial transverse sections of an adult masseter muscle showing the MSS4 compartment at line II, stained with anti-I1 B MHC (B) and anti-N MHC (C). Scale bar represents 350 gim in A and 260 gim in C.
taneously with the increase of a-MHC expression a decrease of II A-MHC fibres and especially II A/IIBMHC fibres is evident. This implies that during postnatal growth a myosin isozyme transition takes place in fibres from fast (II A, II A/IIB and N) MHC to relatively slow a-MHC and/or I/a-MHC. This hypothetical transition of fibre types is reminiscent of transitions described in the literature from fast to slow MHC expression in presumptive slow muscles (Kugelberg, 1976; Pelloni-Mueller et al. 1976; Butler-Browne & Whalen, 1984; Hoh et al. 1988; d'Albis et al. 1989). This hypothesis is supported by the unique pattern of transitional postnatal appearance of II A/a-MHC fibres. These fibres are present in largest numbers at the age of 21-28 d (Figs 5, 6D). The IIA/a-MHC fibres and/or I/IIA/oc-MHC fibres contain 2 or more myosin isozymes and might therefore be in the process of transition. IIA/oc-MHC might be replaced by aMHC and I/IIA/a-MHC by I/a-MHC. In view of the great numbers in which the a-MHC fibres and I/aMHC fibres appear and the disappearance of large
numbers of II A/II B-MHC fibres in the adult masseter it is quite possible that this latter fibre type somehow changes into c-MHC or I/a-MHC fibres, possibly via IIA-MHC fibres. The peak of IIA/a-MHC fibres at the age of 21-28 d corresponds with the time that the mammalian feeding apparatus undergoes a switch in function from suckling to chewing (Maeda et al. 1981; Herring, 1985). At the age of 3-4 wk rabbits have just started to feed independently on solid food (Yardin, 1974; Sorensen et al. 1968). Since the speed of contraction of a single skeletal muscle fibre is largely determined by its MHC content, Vna- of fast MHC (3.1 muscle length/s) is about 4 times the Vmax of slow MHC (0.7 muscle length/s) and fl-MHC (0.8 muscle length/s) (Marston & Taylor, 1980; Reiser et al. 1985; Rome et al. 1990) and the ATPase activity (we have no information on Vkax of 'cardiac' a-MHC) in heart atrium is 2 to 3 times higher than in slow (ventricular ,/) MHC (Pope et al. 1980; McNally et al. 1989) so it seems likely that the muscle fibres containing ax-MHC
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will show contraction speeds intermediate between fibres containing I-MHC and IIA-MHC. Thus conversion of IIA-MHC and IIB-MHC to a-MHC may imply a conversion to a slower muscle. This should be established by physiological studies. The notion that the masseter might reveal a developmentally-regulated decrease in contraction speed underlines a similar idea predicted by the muscle architecture (Weijs et al. 1987; Langenbach & Weijs, 1990). Weijs et al. (1987) and Langenbach & Weijs (1990) demonstrated that the masseter muscle of young rabbits contains relatively long fibres, meaning that their absolute speed of shortening must be high, relative to adult animals. The authors concluded that in the course of postnatal development a decrease of speed of contraction might be related to changing functional requirements. In contrast to the above named hypothesis, Close (1964) reported that the speed of contraction of the rat soleus remains the same during postnatal development though a transition from fast to slow MHC expression takes place (Butler-Browne & Whalen, 1984; d'Albis et al. 1989). Our finding of 'cardiac' a-MHC in the rabbit masseter muscle fibres supports the idea that individual muscle fibres might, by varying their MHC content, adapt their contraction speed to the conditions in the muscle during postnatal growth. However, not only masticatory muscles contain cardiac aMHC: we (Bredman et al. 1990b) showed the existence of 'cardiac' oc-MHC containing muscles in the cranial area of adult rabbit. This led us to suggest that embryonic neural crest cells might play a role in inducing the expression of specific MHCs in skeletal muscle, so that the expression of MHC would be determined by the developmental history of the tissue.
Expression of neonatal and slow myosin
Until the fourth postnatal week about 90 % of the fibres contain N-MHC. Thereafter a decrease in number of N-MHC containing fibres takes place so that in adult masseter N-MHC is expressed in only 20 % of the fibres (the oldest rabbits contain the least N-MHC containing fibres). The finding of N-MHC in the adult rabbit masseter distinguishes it from limb muscles. In normal adult limb muscles N-MHC is never seen (Guth & Samaha, 1972; Butler-Browne & Whalen, 1984; Butler-Browne et al. 1988). But in masseter muscle of adult human (Butler-Browne et al. 1988) and 3 month old rat (d'Albis et al. 1989) the N-MHC is still expressed. Soussi-Yanicostas et al. (1990) demonstrated, in the
human, that this late maturation is also apparent from biochemical data with respect to the presence of slow tropomyosin, MLCIs, MLC2s, slow MHC and fast MHC in the masseter unlike in the quadriceps. It is tempting to speculate about a functional meaning for this late expression of N-MHC. d'Albis et al. (1986) and Soussi-Yanicostas et al. (1990) proposed that the masseter develops at a different rate and/or a different way compared with other skeletal muscles. The expression of N-MHC extends the number of different MHCs already present in masticatory muscles. According to Rowlerson (1990) the mixed composition of MHC in the rabbit masseter muscle might reflect their rather rapid masticatory movements compared with herbivores. Furthermore, it might be an adaptation to cope with a diet of variable texture. The N-MHC was absent or present only in very small amounts in the fibres containing type I-MHC. During the first weeks the latter fibres are topographically evenly distributed throughout the muscle (Fig. 7A) (though they are absent in some compartments). The relatively large diameter of these I-MHC fibres, at the newborn stage, points according to Hoh & Hughes (1989) to a primary myotube origin. According to the literature the slow primary myotubes synthesise embryonic myosin (until about 2 wk in rat soleus and 11 d in cat masseter; Butler-Browne & Whalen, 1984; Hoh & Hughes, 1989). We could not check this in the rabbit masseter because no monoclonal antibody against embryonic MHC was available. The development of heterogeneity in relation to muscle architecture All ages show heterogeneity in MHC content between the different sample sites. This heterogeneity varies with age. For example, the 1 wk masseter shows the highest percentage of II A-MHC in the deep MSS 1(d) compartment while the adult masseter shows the highest percentage of IIA-MHC in the superficial MSS4 compartment. The muscle starts with a mixture of I-MHC, II A-MHC and II B-MHC containing fibres and from this, a mixture of I-MHC, II A-MHC, IIB-MHC and a-MHC containing fibres develops, leading to quite different heterogeneity in the adult. It is possible that the changes have a functional meaning, with different pre- and postweaning roles for different regions, but also that different regions have different developmental speeds. Within compartments heterogeneity in distribution of fibre types can appear. In some compartments a
fibre type can be absent, restricted to a small part (see
Myosin expression in rabbit masseter
for example Fig. 7) or appear homogeneously. Heterogeneity in distribution of one fibre type can be the result of motor unit distribution. All muscle fibres belonging to a single motor unit belong to the same immunohistochemical category (Edstr6m & Kugelberg, 1968). Furthermore, Stalberg & Eriksson (1987) implied that motor units are restricted to certain regions or compartments. When only a single motor unit, consisting of a fibre type that is relatively rare, is present in a compartment a picture like Figure 7B might appear, but when 3 or more units of this fibre type are present the whole compartment will show the specific fibre type. This could explain interindividual variation present in rabbit masseter muscle compartments. Despite the individual age variations in fibre percentages, the pattern of appearance and disappearance of specific MHC types is always similar. ACKNOWLEDGEMENTS
We thank Dr G. S. Butler-Browne, INSERM, Paris, France for providing the neonatal antibody. We also thank Cars Gravemeyer, Ans van Horssen and Ad van Horssen for their photographic and art work and Sabina Tesink-Takema and Carol Verhoek-Pocock for their expert technical assistance.
REFERENCES
D'ALBIS A, JANMOT C, BECHET JJ (1986) Comparison of myosins from the masseter muscle of adult rat, mouse and guinea-pig. European Journal of Biochemistry 156, 291-296. D'ALBIS A, COUTEAUX R, JANMOT C, ROULET A (1989) Specific programs of myosin expression in the postnatal development of rat muscles. European Journal of Biochemistry 183, 583-590. D'ALBIS A, JANMOT C, MIRA JC, COUTEAUX R (1991) Characterization of a ventricular VI myosin isoform in rabbit masticatory muscles. Developmental and neural regulation. Basic and Applied Myology 1, 23-34. BREDMAN JJ, WEIJS WA, MOORMAN AFM, BRUGMAN P (1990a) Histochemical and functional fibre typing of the rabbit masseter muscle. Journal of Anatomy 168, 31-47. BREDMAN JJ, WEIJS WA, MOORMAN AFM (1990b) Expression of 'cardiac-specific' myosin heavy chain in rabbit cranial muscles. In Muscle and Motility 2; Proceedings of XIXth European Conference (ed. G. Mar6chal & U. Carraro), pp. 329-335. Brussels: Intercept. BREDMAN JJ, WESSELs A, WEIJS WA, KORFAGE JAM, SOFFERS CAS, MOORMAN AFM (1991) Demonstration of 'cardiac-specific' myosin heavy chain in masticatory muscles of human and rabbit. Histochemical Journal 23, 160-170. BUTLER-BROWNE GS, WHALEN RG (1984) Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle. Developmental Biology 102, 324-334. BUTLER-BROWNE GS, ERIKSSON PO, LAURENT C, THORNELL LE (1988) Adult human masseter muscle fibers express myosin isozyme characteristics of development. Muscle and Nerve 11, 610-620. CHRISTENSEN L, STRANGE L (1987) Universal immunoperoxidase
273 staining protocol to optimize the use of polyclonal and monoclonal antibodies. Journal of Histotechnology 10, 11-15. CLOSE R (1964) Dynamic properties of fast and slow skeletal muscles of the rat during development. Journal of Physiology 173, 74-95. DE GROOT IJM, LAMERS WH, MOORMAN AFM (1989) Isomyosin expression patterns during rat heart morphogenesis: an immunohistochemical study. Anatomical Record 224, 365-373. EDDINGER TJ, Moss RL (1987) Mechanical properties of skinned single fibers of identified types from rat diaphragm. American Journal of Physiology 253, c210-c218. EDSTROM L, KUGELBERG E (1968) Histochemical composition, distribution of fibres and fatiguability of single motor units. Journal of Neurology, Neurosurgery and Psychiatry 31, 424-433. GUTH L, SAMAHA FJ (1972) Erroneous interpretations which may result from application of the 'myofibrillar ATPase' histochemical procedure to developing muscle. Experimental Neurology 34, 465-475. HENDERSON C (1989) Aminoalkylsilane: an inexpensive, simple preparation for slide adhesion. Journal of Histotechnology 12, 123-124. HERRING SW (1985) The ontogeny of mammalian mastication. American Zoology 25, 339-349. HOH JFY, HUGHES S, HALE PT, FITZSIMONs RB (1988) Immunocytochemical and electrophoretic analyses of changes in myosin gene expression in cat limb fast and slow muscles during postnatal development. Journal of Muscle Research and Cell Motility 9, 30-47. HOH JFY, HUGHES S (1989) Immunocytochemical analysis of the perinatal development of cat masseter muscle using anti-myosin antibodies. Journal of Muscle Research and Cell Motility 10, 312-325. KUGELBERG E (1976) Adaptive transformation of rat soleus motor units during growth. Journal of the Neurological Sciences 27, 269-289. LANGENBACH GEJ, WEIJS WA (1990) Growth patterns of the rabbit masticatory muscles. Journal of Dental Research 69, 20-25. MAEDA N, HANAI H, KUMEGAWA M (1981) Postnatal development of masticatory organs in rats. III Effect of mastication on the postnatal development of the m. masseter superficialis. Anatomischer Anzeiger (Jena) 150, 424-427. MARSTON SB, TAYLOR EW (1980) Comparison of the myosin and actomyosin ATPase mechanisms of the four types of vertebrate muscles. Journal of Molecular Biology 139, 573-600. MCNALLY EM, KRAFT R, BRAVO M, TAYLOR DA, LEINWAND LA (1989) Full-length rat alpha and beta 'cardiac' myosin heavy chain sequences. Journal of Molecular Biology 210, 665-671. MOORMAN AFM, DE BOER PAJ, LINDERS MTH, CHARLES R (1984) The histone H5 variant in Xenopus laevis. Cell Differentiation 14, 113-123. PEDROSA F, SOUKUP T, THORNELL LE (1990) Expression of an alpha cardiac-like myosin heavy chain in muscle spindle fibres. Histochemistry 95, 105-113. PELLONI-MUELLER G, ERMINI M, JENNY E (1976) Myosin light chains of developing fast and slow rabbit skeletal muscle. FEBS Letters 67, 68-74. POPE B, HOH JFY, WEEDS A (1980) The ATPase activities of rat cardiac myosin isozymes. FEBS Letters 118, 205-208. REISER PJ, Moss RL, GIULIAN GG, GREASER ML (1985) Shortening velocity in single fibers from adult rabbit soleus muscles is correlated with myosin heavy chain composition. Journal of Biology and Chemistry 260, 9077-9080. ROME LC, SOSNICKI SS, GOBLE DO (1990) Maximum velocity of shortening of three fibre types from horse soleus muscle: implications for scaling with body size. Journal ofPhysiology 431, 173-185. ROWLERSON AM (1990) Specialization of mammalian jaw muscles:
274
Jantine J. Bredman and others
fibre type compositions and the distribution of muscle spindles. In Neurophlvsiologv of the Jaws and Teeth (ed A. Taylor). pp. 1 51. London: Macmillan. SCHIAFFINO S, AUSONI S, GORZA L, SAGGIN L, GUNDERSEN K (1988) Myosin heavy chain isoforms and velocity of shortening of type 2 skeletal muscle fibres. Acta PhYsiologica Scandinavica 134, 575-576. SORENSEN MF, ROGERS JP, BASKETT TS (1968) Reproduction and development in confined swamp rabbits. Journal of Wildlife Management 32, 520-531. STALBERG E, ERIKSSON PO (1987) A scanning electromyographic study of the topography of human masseter single motor units. Archives of Oral Biologi' 32, 793-797. STARON RS, HIKIDA RS, HAGERMAN FC (1983) Reevaluation of human muscle fast-twitch subtypes: evidence for a continuum. Histochemistry 78, 33-39. STARON RS, PETTE D (1986) Correlation between myofibrillar ATPase activity and myosin heavy chain composition in rabbit muscle fibers. Histochemistry 86, 19-23. SOUSSI-YANICOSTAS N, BARBET JP, LAURENT-WINTER C, BARTON P, BUTLER-BROWNE GS (1990) Transition of myosin isozymes during development of human masseter muscle. Development 108, 239-249. THORNELL LE, BILLETER R, ERIKSSON PO, RINGQVIST M (1984) Heterogeneous distribution of myosin in human masticatory
muscle fibres as shown by immunocytochemistry. Archives of Oral Biology 29, 1-5.
WEIJS WA, BRUGMAN P, KLOK EM (1987) The growth of the skull and jaw muscles of the rabbit and its functional consequences in the New Zealand rabbit (Oryctolagus cuniculus). Journal of Morphology 194, 143-161. WESSELs A, VERMEULEN JLM, MOORMAN AFM, BECKER AE (1988) Immunohistochemical detection of myosin heavy chain isoforms in large sections of whole human hearts. In Sarcomeric and Nonsarcomeric Muscles: Basic and Applied Research Prospects for the 90s, (ed. U. Carraro), pp. 311-316. Padova: Unipress. WESSELs A, VERMEULEN JLM, VIRAiGH Sz, KA~LMAN F, LAMERS WH, MOORMAN AFM (1990 a) Spatial distribution of 'tissue specific' antigens in the developing human heart and skeletal muscle: II, an immunohistochemical analysis of myosin heavy chain isoform expression patterns in the embryonic heart. Anatomical Record 229, 355-368. WESSELs A, SOFFERS CAS, VERMEULEN JLM, MYINDESS TA, BREDMAN JJ, MOORMAN AFM (1990b) Expression of a 'cardiacspecific' myosin heavy chain in intrafusal fibres of the developing human spindle. In Muscle and Motility 2; Proceedings of XIXth European Conference (ed. G. Marechal & U. Carraro), pp. 71-77. Brussels: Intercept. YARDIN M (1974) Sur l'ontogenese de la mastication chez le lapin (Oryctolagus cuniculus). Mammalia 38, 737-747.
