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RESEARCH REPORT |
Department of Functional Anatomy, Academic Center for Dentistry Amsterdam (ACTA), Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands;
*corresponding author, j.a.korfage{at}amc.uva.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: myosin • single muscle fiber • gel electrophoresis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, which is normally present in the atrium of the heart, and MyHC-fetal, which is normally present in developing muscles. Next to fibers that express only one MyHC isoform, there are also fibers which co-express more than one MyHC isoform, the so-called hybrid fibers. The latter are abundantly present in human jaw-closing muscles (Korfage and Van Eijden, 1999, 2000; Korfage et al., 2000, 2001; Monemi et al., 2000). The occurrence of fibers containing various proportions of different MyHC isoforms can be considered as an important mechanism contributing to the large functional diversity of a particular muscle. Most studies that investigated the distribution of different fiber types in human jaw muscles used either ATPase histochemistry (Eriksson et al., 1982; Rinqvist et al., 1982; Eriksson and Thornell, 1983), immunohistochemistry (Korfage and Van Eijden, 2000; Korfage et al., 2000), gel electrophoresis (Korfage and Van Eijden, 2003), or a combination of these three techniques (Sciote et al., 1994). ATPase histochemistry and immunohistochemistry can distinguish hybrid fibers from pure fibers. However, these methods are not suitable for quantifying the proportions of different MyHC isoforms within single hybrid fibers. A variation in proportion of a particular MyHC isoform would suggest that a continuum of contractile properties exists within the jaw muscles. The aim of the present study was to determine the MyHC contents of single fibers of human jaw muscles and to investigate the proportions of individual MyHC isoforms within the hybrid fibers.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The muscles were obtained within 12 to 36 hrs post mortem. After the muscles were exposed, they were cut from their attachment sites and rapidly frozen in liquid-nitrogen-cooled isopentane. From each frozen muscle, a bundle of fibers (approximately 5 mm long, containing about 200 to 500 fibers) was cut out and freeze-dried overnight. Single fibers were microdissected from these bundles following a procedure similar to that described for human limb muscles (Essén et al., 1975). The single fibers were dissected out by a couple of metal needles under a dissection microscope. During this procedure, great care was taken to ensure that the dissected fiber was not contaminated by parts of other fibers. Each fiber was then divided into two segments for gel electrophoresis and for microscopic examination.
Gel Electrophoresis
The fiber segments destined for gel electrophoresis were diluted in sample buffer (15% [w/v] glycerol, 2% [w/v] DTT, 0.01% [w/v] bromophenol blue, and 1% [w/v] SDS in 62.5 mM Tris/HCl buffer, pH 6.8). The fibers underwent lysis for 2 min at 100°C and were then stored at -80°C until processed for protein separation.
Gel electrophoresis was performed in the presence of SDS in high-glycerol-containing (30%) gels (0.75 mm thickness) with an acrylamide-to-bis-acrylamide ratio of 67:1 in the separating gel (9% total acrylamide, pH 8.8) and of 50:1 in the stacking gel (4% total acrylamide, pH 6.8) (modified from Talmadge and Roy, 1993). The single fiber samples were run at constant current (13.5 mA) for a total of 29 hrs in a custom-built electronic timer device (pulse unit) connected to a power supply that switched the running current on and off (Sant’Ana Pereira et al., 2001). After running was completed, the gels were silverstained and blue-toned (Berson, 1983) for a better quantification of the MyHC isoform bands.
The gels were scanned with an LKB 2202 Ultrascan laser densitometer (LKB, Bromma, Sweden). The MyHC isoforms were identified on the basis of migration as MyHC types I, IIA, and IIX. The total integrated area of the three MyHC peaks was set to 100, and each individual area was expressed as a percentage of the total MyHC.
Microscopic Examination
The fiber segments that were used for microscopic examination were embedded in a double layer of 15% gelatin. Twenty fiber segments were placed in each block of gelatin. This block was frozen in liquid nitrogen and transported to a cryomicrotome (Model HM 500 M, Adamas Instruments BV, Leersum, the Netherlands). Serial transverse sections of 10 µm were cut and collected on slide glasses coated with VectabondTM (Vector Laboratories, Inc., Burlingame, CA, USA). We then examined all fibers under a microscope to check whether they were single or not. Fibers that were found not to be single were omitted from the study.
In total, 92 single fibers were selected at random for immunohistochemical examination. Therefore, the sections of these fibers were incubated with monoclonal antibodies raised against purified myosin (Bredman et al., 1991), namely, antibodies 219-1D1, 333-7H1, 332-3D4, and 249-5A4, recognizing MyHC-I, MyHC-IIA, MyHC-IIA and MyHC-IIX, and MyHC-cardiac , respectively, and antibody NCL-MHCn (Novocastra Laboratories Ltd, Newcastle Upon Tyne, UK), recognizing MyHC-fetal. The indirect unconjugated immunoperoxidase technique (PAP-technique) was applied for detection of the specific binding of the different antibodies. Nickel-DAB was used for visualization of the immunolabeling.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Three distinct MyHC isoforms could be identified by migration, namely, MyHC type I, IIA, and IIX (Fig. 1). In contrast to the electrophoretic separation of MyHC isoforms, the present antibody panel was unable to distinguish hybrid fibers that co-expressed MyHC-IIA and -IIX from pure fibers that express only MyHC-IIA (compare fibers 1 and 3 with fiber 5 in Fig. 1). An extra band corresponding to MyHC-fetal was not observed. Also, among the 92 single fiber sections, no immunolabeling was seen with the MyHC-fetal antibody. In 3 fibers, an extra band was seen which migrated closely above the MyHC-I band which might correspond to MyHC-cardiac , since the sections of these fibers were immunolabeled with the antibody against MyHC-cardiac . However, this band was difficult to separate by the laser densitometer. Therefore, MyCH-cardiac was not considered further in the evaluation of the present study.
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). The temporalis had more pure slow fibers than pure fast fibers; the reverse was true for the digastric. The temporalis contained more hybrid than pure fibers, while the digastric showed the opposite. Between the two bellies of the digastric, a difference was seen in the proportions of the fast MyHC isoforms, i.e., the anterior belly had more fibers expressing MyHC-IIX, while the posterior belly had more fibers expressing MyHC-IIA.
In both the temporalis and digastric muscles, the predominant hybrid fibers were fibers co-expressing MyHC-IIA and -IIX (Table). A smaller number of hybrid fibers co-expressed either MyHC-I and -IIA, MyHC-I and -IIX, or MyHC-I, -IIA, and -IIX. The proportions of the MyHC isoforms within the hybrid fibers varied largely (Fig. 2). For instance, the sample of hybrid MyHC-IIA/IIX fibers consisted, on the one extreme, of fibers with a relative small proportion (< 10%) of MyHC-IIA and a large proportion (>90%) of MyHC-IIX, while, on the other extreme, there were fibers with a large proportion of MyHC-IIA and a small proportion of MyHC-IIX. Note that in the MyHC-I/IIX fibers, the proportions were not normally distributed, and that about 50% of the fibers contained about 90% MyHC-IIX. In general, in all observed combinations of MyHC isoforms, there was a predominance for the fastest isoform: The mean proportion of MyHC-IIA in hybrid MyHC-I/IIA fibers was 58.6%, of MyHC-IIX in MyHC-I/IIX fibers 71.0%, of MyHC-IIX in MyHC-IIA/IIX fibers 62.2%, and of MyHC-IIX in MyHC-I/IIA/IIX fibers 63.0%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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With the antibody panel we used, we were unable to distinguish hybrid MyHC-IIA/IIX fibers from pure MyHC-IIA fibers (Fig. 1). Therefore, in our earlier work, hybrid MyHC-IIA/IIX fibers were classified as pure MyHC-IIA fibers (Korfage and Van Eijden, 1999; Korfage et al., 2000, 2001). By using gel electrophoresis, we were now able to separate MyHC-IIA from MyHC-IIA/IIX fibers and noticed that about 40% of the fibers in our sample from the temporalis and about 20% of the fibers from the digastric muscle sample were actually hybrid fibers that co-expressed both MyHC-IIA and -IIX. Thus, in our earlier studies, the amounts of pure MyHC-IIA fibers were overestimated (about 6 times in the temporalis and about 1.8 times in the digastric).
The abundance of hybrid MyHC-IIA/IIX fibers found in the present study is in agreement with the results of an immunological study of the digastric muscle in elderly people (Monemi et al., 2000), where an antibody panel was used that could distinguish hybrid MyHC-IIA/IIX fibers from pure MyHC-IIA fibers. Similarly, many of these hybrid fibers were found in human laryngeal muscles (Wu et al., 2000). The overall difference in fiber type composition (pure and hybrid) between the temporalis and the digastric muscles, and between both bellies of this muscle (Table), was also noticed in earlier ATPase histochemical (Eriksson et al., 1982; Eriksson and Thornell, 1983) and immunohistochemical (Korfage and Van Eijden, 1999, 2000; Korfage et al., 2000, 2001; Monemi et al., 2000) studies.
In the present study, MyHC-fetal could not be distinguished by electrophoresis. In 3 fibers, an extra band was seen in the gels which might correspond with MyHC-cardiac , because sections of these fibers were immunolabeled with the MyHC-cardiac antibody. In an immunohistochemical study on sections of the temporalis (Korfage and Van Eijden, 1999) we found that MyHC-cardiac and MyHC-fetal were expressed in, respectively, about 5% and 9% of the fibers in the most posterior muscle portion. In the present sample, a lower percentage (about 3%) of the fibers expressed MyHC-cardiac , and none of the fibers expressed MyHC-fetal. We have no explanation for the absence of fibers expressing MyHC-fetal. Maybe the proportion of this MyHC isoform is too low for detection, or its band co-migrates with another MyHC band. Further analysis should be made to investigate the MyHC contents of these particular MyHC isoforms in muscle fibers.
The shortening velocity as determined by the slack test (V0) showed that, in human limb muscles, pure MyHC-I fibers are approximately four times slower than MyHC-IIA fibers, and approximately nine times slower than MyHC-IIX fibers (Bottinelli et al., 1996). The shortening velocity of hybrid fibers is said to lie between the individual MyHC isoforms they express (Larsson and Moss, 1993; Bottinelli et al., 1996). The various hybrid fiber types that are found in the jaw muscles would, therefore, result in a more smooth transition of contractile properties (Butler-Browne et al., 1988). This is needed to produce a controlled positioning and force induction of the mandible during complex tasks like speaking and chewing. We found that the proportions of individual MyHC isoforms in hybrid fibers are highly variable. Such a continuum within hybrid fibers was also found in human limb (Larsson and Moss, 1993) and laryngeal muscles (Wu et al., 2000), and in rabbit limb muscles (Peuker and Pette, 1997). This large variability suggests that, also in the jaw muscles, a large range of contractile properties exists among fibers containing a particular combination of MyHC isoforms. This diversity in contractile fiber properties contributes to the heterogeneous function of the jaw muscles and is consistent with earlier reports on the heterogeneity of contractile properties of masticatory motor units (Kwa et al., 1995; Turkawski and Van Eijden, 2001; Van Eijden and Turkawski, 2001). Interestingly, the hybrid fibers in our sample had a tendency for the expression of the faster MyHC isoform.
We also found that approximately 5% of the fibers co-expressed MyHC-I and -IIX but not MyHC-IIA. These hybrid fibers were also found in the soleus of the rat which was affected by hyperthyroidism and reduced stress (Caiozzo et al., 1998). Apparently, transitions of MyHC isoforms do not always follow the orderly transition of MyHC-I 
-IIA -IIX as suggested by Schiaffino and Reggiani (1994). Further investigations are needed to elucidate these particular hybrid fiber types in human jaw muscles.
It should be noted that MyHC is not the sole protein that determines contractile properties (Bottinelli, 2001), and that a large variation in properties exists within fibers that express the same MyHC isoform. A recent study (Morris et al., 2001) noticed a large variability in unloaded shortening velocities of pure single MyHC type I fibers of the human anterior superficial masseter. The reason for the variation among these pure MyHC type I fibers might be that the MyHC-I isoform consists of different subforms, each with a slightly different physiological property. This hypothesis is supported by the finding that, in the adult rabbit masseter, 4 different subforms of MyHC-I are expressed (English et al., 1998). Furthermore, a recent study in humans identified two novel MyHC genes which are structurally close to the MyHC type I isoform (Desjardins et al., 2002). Whether these isoforms are expressed in the human jaw muscles is still unclear.
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ACKNOWLEDGMENTS |
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Received October 28, 2002; Last revision February 12, 2003; Accepted February 28, 2003
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REFERENCES |
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