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RESEARCH REPORT |
Dept. of Orthodontics, Box 100444, JHMHSC, University of Florida, Gainesville, FL 32610-0444;
*corresponding author, widmer{at}dental.ufl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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-cardiac, IIa, and IIb—was determined within the defined masseter partitions by means of Western blot analysis and immunofluorescent localization. Types IIa, IIx, and IIb were the predominant MyHC isoforms observed. Distinct differences in the spatial distribution of these MyHC isoforms were found between muscle regions and varied between sexes. The regionalization of muscle fiber types in the mouse masseter is consistent with the functional compartmentalization of the masseter observed in other species.
KEY WORDS: masseter • mouse • myosin heavy-chain • sexual dimorphism • muscle partition
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The MyHC isoform profile of muscle fibers within the masseter has been shown to be unique. The adult masseter in several species contains a variety of fiber types, including both adult (slow, cardiac, IIa, IIx, and IIb) and developmental (embryonic and neonatal) MyHC isoforms (d'Albis et al., 1986; Soussi-Yanicostas et al., 1990; Monemi et al., 1996; Eason et al., 2000). In limb and trunk muscles, embryonic and neonatal isoforms are replaced after birth by adult slow and fast isoforms, and adult muscle fibers have very little, if any, neonatal MyHC isoform (Lyons et al., 1990; Lu et al., 1999). In addition, MyHC isoform expression within the masticatory muscles is regulated differently from that in the limb, based on recent studies that examined MyHC isoform expression in null mutant IIx mice (Acakpo-Satchivi et al., 1997; Sartorius et al., 1998). Sexual differences in the spatial expression of MyHC isoforms in masticatory muscles have also been reported in several studies (Schiaffino, 1974; Lyons et al., 1986; Talmadge and Roy, 1993; English et al., 1999b). Evidence exists that additional factors such as sex hormones can affect the final phenotype of the masseter in rabbit and mice (English et al., 1999b; Eason et al., 2000).
The mouse is the animal model of choice for the study of masticatory muscle development, due to the availability of transgenic strains targeting genes associated with myosin regulation or expression. However, there is a paucity of information regarding the anatomy and muscle fiber phenotype of the adult mouse masseter. The objective of this study was to refine our understanding of the adult mouse masseter muscle by defining the architecture of this muscle and by examining systematically, in male and female mice, the spatial distribution of MyHC isoforms within anatomical partitions, using Western blot analysis and immunofluorescent localization. The results of this study and those from a previous study of the spatial distribution of MyHC message (unpublished observations) provide insight into the organization and regulation of fiber phenotype within different compartments of the male and female masseter.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Adult Masseter Muscle Architecture
The architecture of the adult mouse masseter was determined by the gross dissection of whole muscle specimens (n = 5). Anatomical partitions were defined by their unique tendons of origin or insertion. For further characterization of muscle compartments, 3 muscles were processed for acetylcholinesterase for the evaluation of endplate distribution according to the method of Karnovsky-Roots, and were visualized with the use of 10% ammonium sulfide.
Tissue
Masseters used in Western blot analysis were obtained from adult male (n = 5, pooled into 2 samples) and female (n = 2) mice. The muscles were separated into 3 anatomical layers (superficial, intermediate, and deep); each layer was then divided into anterior and posterior portions, frozen on dry ice, and stored at -20°C until myosin extraction. Soleus, diaphragm, and ventricle were collected as positive controls. Masseters used in immunofluorescent localization were obtained from adult male (n = 5) and female (n = 5) mice. The muscles were quick-frozen in isopentane that was cooled with dry ice and acetone and stored at -20°C until sectioned. Two masseters of each sex were sectioned at 14 µm in a frontal plane, and 3 were sectioned in a transverse plane. Sections were consecutively mounted on an alternating series of gelatin-coated slides and stored at -20°C until immunofluorescently stained.
Western Blot Analysis
Myosin was extracted from the masseters to be used in Western blot analysis according to a protocol modified from Talmadge and Roy (1993). Myosin heavy-chain isoforms were separated by SDS PAGE according to a protocol modified from Prevost et al. (1995) and Talmadge and Roy (1993). The protein was transferred to an MSI nylon transfer membrane (Fisher Scientific, Pittsburgh, PA, USA) in Tris-glycine buffer at 20 V at 4°C overnight. Western blotting was carried out according to the manufacturer's protocol (BioRad, Hercules, CA, USA). The primary antibodies used were mouse monoclonal antibodies to myosin isoforms developed by Schiaffino and co-workers (Schiaffino et al., 1986,1988): clone BF-45, embryonic MyHC; clone BA-D5, slow MyHC; clone SC-71, IIa MyHC; clone BF-F3, IIb MyHC; clone BF-B6, neonatal MyHC; and clone BA-G5, -cardiac MyHC. In addition, we used MY32 (Sigma Chemical Co., St. Louis, MO, USA) to identify type II MyHC. Using Gel-Pro analyzer software (Media Cybernetics, Silver Spring, MD, USA), we optically scanned Western blots and analyzed them for the relative densities of bands compared with loading and with each other. Based on the integrated optical density (IOD) of each band, the MyHC content of each muscle region was normalized to the band with the greatest IOD and averaged.
Immunofluorescent Staining and Analysis
The sectioned masseters were immunofluorescently stained according to standard protocols (Schiaffino et al., 1989). The same primary antibodies that were used in the Western blots were also used for immunofluorescent staining. Immunostained sections were mounted in glycerol and examined under epifluorescence with a Nikon FXA Photomicroscope (Nikon, Melville, NY, USA).
Images of immunostained sections were captured and analyzed by means of an Optronics 470E digitizing camera (Optronics, Goleta, CA, USA) and Image Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). For the MyHC isoforms, types IIa and IIb, we reconstructed collages of transverse and frontal sections from the acquired images to determine the percent area populated by type IIa or IIb fibers. Regions that contained immunofluorescently stained fibers were segmented from fibers that were not immunostained and the areas of these regions calculated as a percentage of the total section area. A similar sample region of a female and male masseter transverse section was also acquired for the analysis of minimum cross-sectional fiber diameter and the relationship of fiber diameter to the intensity of staining. Significant differences between sex for the MyHC areas and minimum fiber diameters were evaluated by the Mann-Whitney U test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Western Blot Analysis
Based on the Western blot analysis, IIa and IIb MyHC were found to be the major isoforms in the mouse masseter, although small amounts of neonatal, slow, and -cardiac isoforms were also detected. Embryonic MyHC was not identified in the Western blots for either male or female mice. Although Coomassie-blue-stained SDS PAGE gels revealed a band consistent with the IIx isoform in the masseter and diaphragm, the IIx isoform could not directly be evaluated in the Western blots, because there is no available antibody specific to the IIx MyHC isoform. However, MY32 labeled three bands in the masseter Western blot, including a thick band (IIx) located between IIa and IIb bands (Fig. 2A
). Soleus contained slow and IIa isoforms, the diaphragm slow, IIa, IIx and IIb isoforms, and ventricle both slow and -cardiac isoforms. The MyHC isoforms detected in control muscles are the same as previously reported (Schiaffino et al., 1988; Ausoni et al., 1990) and validate the specificity of the antibodies. The IIa and IIb MyHC isoforms were identified in both male and female masseter muscle fibers, and their distributions were found to be localized to specific regions of the muscle. Examination of the normalized IOD representing the IIa or IIb isoform for each of the six regions of the masseter (Fig. 2B) revealed that the IIa isoform was localized to the anterior region of the muscle, while IIb distribution was generally greater in the posterior half of the muscle for both sexes.
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). The density of IIb fibers was greatest in posterior portions of superficial and intermediate layers (Fig. 3C). The female masseter had a higher density of IIa-labeled fibers in the anterior superficial and intermediate layers compared with the male (Fig. 3A). A smaller collection of labeled fibers was also observed within the posterior intermediate layer on the deep side of tendon b. Although this posterior distribution was observed in the male, it was more pronounced in the female. Like the male, the female masseter has a higher density of IIb fibers in the posterior of the muscle (Fig. 3C). Unlike in the male, where some overlap was observed, there was very little overlap in the distribution of IIa and IIb fibers in the female. In both the male and female masseters, there were no fibers that were labeled with both IIa and IIb antibodies. Fibers within central regions of the superficial and intermediate layers, as well as within discrete regions intermingled within the anterior third of the muscle, did not immunostain for either IIa or IIb but were stained only by MY32 antibody in both males and females, suggesting that they were IIx in phenotype. A few fibers were found alongside tendon b that co-labeled with both embryonic and neonatal antibodies. A different group of fibers in this region, also limited in number, was co-labeled with slow and -cardiac MyHC antibodies. This region was the only one identified to have muscle fibers with isoforms other than IIa or IIb and accounted for < 1% of the muscle.
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). Type IIa fibers were concentrated in the more ventral regions of the male and female masseter muscle, while IIb fibers were concentrated in the dorsal half of the muscle. This dorsal-ventral organization was verified in the frontal sections. The percentage of total masseter cross-sectional area expressing a specific MyHC isoform was found to differ between males and females. The area covered by type IIa was significantly greater in the female than in the male (29 ± 3% vs. 16 ± 4%; p < 0.01), while type IIb was significantly greater in the male (mean ± SD) (32 ± 5% vs. 18 ± 2%; p < 0.01). Other MyHC isoforms that were examined by immunostaining accounted for only 1% of the fibers. The remaining unstained fibers, putative IIx fibers, comprised almost half the masseter and were not statistically different (mean ± SD) (male, 52 ± 2%; female, 53 ± 4%; p = 0.6).
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). Examination of the IIa fiber size in the male found a unimodal distribution of size. Type IIb fiber diameter was found to be significantly larger than that of type IIa fibers in both sexes (Fig. 3D). In both sexes, there was a bimodal distribution of immunofluorescent intensity representing very intense and moderately intense staining, but there was no correlation of the two intensity groups with fiber diameter. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Previous reports have also confirmed the presence of IIa and IIb fibers in the mouse masseter muscle, although these studies relied only on selective sampling of the muscle rather than on a survey of the entire muscle (d'Albis et al., 1986; Acakpo-Satchivi et al., 1997; Sartorius et al., 1998; Eason et al., 2000). Sex differences have also recently been reported in mice (Eason et al., 2000), and the results of this previous study were similar to what was reported in this study. In both, the male was shown to possess more IIb fibers than the female; conversely, the female had more IIa fibers. However, the spatial distribution of IIa and IIb fibers in males and females differed between studies. For example, Eason et al. (2000) observed a greater number of IIa fibers in the posterior region of the male, while we observed a higher density of IIa fibers in the anterior region. Differences in the results of the two studies may be due to differences in the age of the mice used. Young, two- to four-month-old adult mice were used in this study; Eason et al. (2000) used 10-month-old mice. Differences may also be due to the sampling methods used. Eason et al. (2000) evaluated only a single section through the masseter, sampling 6 randomly selected 250-µm2 areas in 4 defined regions. Immunolabeled fibers were expressed as a percentage of total fibers in the sampled area. In contrast, we evaluated entire muscle cross-sections at 4 levels in the dorsoventral direction as well as the anteroposterior direction.
In the study by Eason et al. (2000), castration was shown to alter the phenotype of the male mouse masseter, increasing IIa expression; IIb expression was not affected. Ovariectomy had no effect on either IIa or IIb expression. These observations indicate that androgen levels may influence the sexual dimorphism observed in the masseter by affecting a shift in phenotype to type IIb fibers while minimizing the type IIa phenotype. For the IIb phenotype to be achieved in limb muscle, a transition of IIa 
IIx IIb is believed to be necessary (Talmadge and Roy, 1993; Pette and Staron, 1997). In null mutant IIx knockout mice, IIb fibers were not observed within the masseter, although they were still present in limb muscle (Acakpo-Satchivi et al., 1997; Sartorius et al., 1998), indicating that the expression of MyHC may be regulated differently in the masseter than in the limb. Although no IIb protein was detected in the masseter in IIx null mutants, message could be detected, albeit in diminished amounts. In a companion study, the spatial distribution of MyHC isoform message was examined within masseter compartments (unpublished observations). When the observed spatial distribution of message is compared with that of MyHC protein, they often do not correlate. One possible explanation for this protein-message mismatch in the masseter may be that certain messages may exist in excess, to allow for more rapid transition to a new fiber phenotype in response to changes in muscle fiber activity patterns or from interactions with androgens or other factors. Alternatively, the excess message may itself perform a regulatory function. Similar protein and message mismatches in other muscles have been observed to occur in response to events leading to transitions from one isoform to another (Peuker et al., 1998; Andersen et al., 1999; Stevens et al., 1999).
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ACKNOWLEDGMENTS |
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Received June 4, 2001; Last revision October 15, 2001; Accepted November 27, 2001
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REFERENCES |
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