Eiji Tanaka, Department of Orthodontics and Dentofacial Orthopedics, Graduate School of Oral Sciences, The University of Tokushima, 2-5-1 Kuramoto-cho, Tokushima 770-8803, Japan. E: email@example.com
The behavioral differences in muscle use are related to the fiber type composition of the muscles among other variables. The aim of this study was to examine the degree of heterogeneity in the fiber type composition in the rat temporalis muscle. The temporalis muscle was taken from 10-week-old Wistar strain male rats (n = 5). Fiber types were classified by immunohistochemical staining according to their myosin heavy chain content. The anterior temporalis revealed an obvious regional difference of the fiber type distribution, whereas the posterior temporalis was homogeneous. The deep anterior temporalis showed a predominant proportion of type IIA fibers and was the only muscle portion displaying slow type fibers (< 10%). The other two muscle portions, the superficial anterior and posterior temporalis, did not differ significantly from each other and contained mainly type IIB fibers. Moreover, the deep anterior temporalis was the only muscle portion showing slow type fibers (< 10%). In the deep portion, type IIX fibers revealed the largest cross-sectional area (1943.1 ± 613.7 µm2), which was significantly (P < 0.01) larger than those of type IIA and I + IIA fibers. The cross-sectional area of type IIB fibers was the largest in the remaining two muscle portions and was significantly (P < 0.01) larger than that of type IIX fibers. In conclusion, temporalis muscle in rats showed an obvious heterogeneity of fiber type composition and fiber cross-sectional area, which suggests multiple functions of this muscle.
In rodents, the jaw muscles contract symmetrically and a separation in time is present in the forward and backward pulling muscles or muscle portions. The result is a protraction and retraction of the jaw in addition to its depression and elevation (Langenbach & van Eijden, 2001). In rats, the temporalis muscle is anatomically clearly divided into two major portions (anterior and posterior). The orientation of the muscle fibers becomes more horizontal towards the posterior region. This regional difference of fiber orientation may be reflected in a function divergence accompanied by variations in fiber type composition, as described in various other species including human (Korfage et al. 2005a,b).
Skeletal muscles contain various fiber types with different contraction velocities and fatigability characteristics (Bottinelli et al. 1996). These fiber types can be classified by their myosin heavy chain (MyHC) content using immunohistochemistry. Four major fiber types (I, IIA, IIX and IIB), associated with the similarly named myosin isoforms (Korfage et al. 2005a,b), have been identified in adult skeletal muscles of small mammals (Schiaffino & Reggiani, 1996; Pette & Staron, 1997). The MyHC content of muscle fibers is well correlated with their unloaded shortening velocity (Bottinelli et al. 1991), which increases, from slow to fast, in a sequence of fibers expressing MyHC types I, IIA, IIX and IIB (Sciote & Kentish, 1996). The percentage of slow type fibers (type I) has been associated with the duty time of the muscles, i.e. muscles active during a large portion of the day show a higher percentage of slow type fibers. For the jaw system, it has been found that vertically directed muscles have a larger amount of slow type fibers, which is probably related to their effective action line for jaw closure, generation of occlusal forces and continuation of a mandibular posture (Korfage et al. 2005a,b).
The amount of force produced by skeletal muscles depends on the cross-sectional area when the muscle length is constant (Maughan et al. 1983). This area adapts to and increases with the amount of resistance experienced during contraction (McCall et al. 1996). Hence, determination of the fiber type composition and cross-sectional area can be used to characterize the muscle's functional properties and requirements. Previously, we investigated the fiber type composition and cross-sectional area of the masseter and digastric in rats and reported a clear heterogeneity in their fiber characteristics (Sano et al. 2007) consistent with their daily muscle activities recorded by a telemetric electromyogram (EMG) recording system (Kawai et al. 2007). As far as we know, no information is available about the possible regional differences in fiber type composition in the rat temporalis muscle.
The aim of the present study was to examine the degree of heterogeneity in the fiber type composition of the temporalis muscle of the rat. The fiber types are characterized by their content of MyHC isoforms, as identified with monoclonal antibodies (Bredman et al. 1990). We hypothesized that the anterior temporalis region, with its more vertically directed action line, would contain a higher percentage of slow type fibers than the muscle's posterior region (Korfage et al. 2005a,b). Comparison of the current fiber type characteristics in the temporalis with previous data in the masseter and digastric muscles (Hiraiwa, 1978; Tuxen & Kirkeby, 1990; Arai et al. 2006; Sano et al. 2007) could further clarify any difference in function between the rat jaw muscles.
Materials and methods
The left temporalis muscle was taken from 10-week-old Wistar strain male rats (n = 5). The animals were killed by an overdose of pentobarbital (300 mg/kg Nembutal, Sanofi Sante, Maassluis, The Netherlands) and muscles were cut from their attachment sites after they had been exposed. The unfixed muscles were rapidly frozen in liquid-nitrogen-cooled isopentane (ca. –120 °C, negating the development of destructive ice crystals) and stored at –80 °C until required for further processing. The experimental procedure was approved by the Animal Ethics Committee of the Medical School of the University of Amsterdam.
Serial transverse sections of 10 µm were cut with a cryomicrotome (CM1850, Leica Microsystems GmbH, Nussloch, Germany). The position of the sections is indicated in Fig. 1A. All of the sections were cut perpendicular to the main orientation of the muscle fibers.
After overnight fixation at –20 °C in a mixture of methanol/acetone/acetic acid/water (35 : 35 : 5 : 25) (Wessels et al. 1988), five consecutive 10-µm sections were incubated with monoclonal antibodies raised against purified myosin (Bredman et al. 1991; Sant’Ana Pereira et al. 1995). Antibody 219-1D1 recognized MyHC-I, antibody 333-7H1 recognized MyHC-IIA, antibody 340-3B5 recognized all fast MyHC isoforms, antibody 332-3D4 recognized MyHC-IIA and MyHC-IIX, and antibody 249-5A4 recognized cardiac-α myosin. This antibody panel could not distinguish hybrid fibers that coexpressed MyHC-IIA, MyHC-IIX and/or MyHC-IIB from pure fibers and therefore we classified them as MyHC-IIA, MyHC-IIX and MyHC-IIB (Korfage et al. 2001). The specificity and characterization of these monoclonal antibodies against MyHC isoforms were demonstrated previously (Wessels et al. 1991; Sant’Ana Pereira & Moorman, 1994; Sant’Ana Pereira et al. 1995). The indirect unconjugated immunoperoxidase technique (peroxydase-anti-peroxidase technique) was applied to detect the specific binding of the different antibodies. Nickel-Diaminobenzidine was used to visualize the staining (Hancock, 1982).
Muscle sampling and muscle section analysis
A clear heterogeneity of the fiber type distribution existed in the anterior temporalis (Fig. 1B). To fully examine this heterogeneity, sample locations were chosen in the superficial and deep portions. The fiber type composition in the posterior temporalis was homogeneous and the sample area was chosen at the centroid of this region. In each sample area, 100–300 fibers (mean 203) were analyzed by photographing the area with a digital camera attached to a microscope. The fiber area outlines were drawn onto a transparent sheet. Fibers were classified by means of the above-mentioned series of five consecutive sections.
The cross-sectional area of the fibers was measured by reading the drawn sheets, together with a grade mark for correction of enlargement, via a flat-bed scanner (Scanjet 4c, Hewlett-Packard) into a personal computer. A custom-made program computed the cross-sectional area of each muscle fiber from the reproduced image.
The proportions and mean cross-sectional areas of the different fiber types were calculated for each muscle portion. Interindividual mean and S.D. values were determined for each portion of the temporalis muscle. All of the data were tested for normality of distribution (Kolmogorov-Smirnov test) and uniformity (Bartelett's test). The differences in the proportion and fiber cross-sectional area between sample sites were tested by one-way analysis of variance with a post-hoc test (Bonferroni test). A probability of less than 0.05 was considered to be significant.
Fiber type composition
A panoramic view of the temporalis muscle incubated with the monoclonal antibody showed clear heterogeneity in the distribution of fiber types (Fig. 1B). The deep portion of the anterior temporalis contained a higher proportion of type IIA fibers and a lower proportion of type IIB fibers (Fig. 2) compared with the rest of the muscle. These differences were significant (P < 0.01, Fig. 3), whereas no significant differences were found between the fiber type compositions of the main part of the superficial anterior and posterior temporalis regions. Moreover, the deep anterior temporalis was the only region to contain pure or hybrid slow type fibers consisting of MyHC-I or MyHC-I/IIA (1.4% and 5.3%, respectively). Although MyHC-IIA was the predominant myosin type in the deep temporalis region (66.0 ± 29.2%), the superficial portion of the anterior temporalis and posterior temporalis showed mostly fibers containing MyHC-IIB (62.5 ± 25.0% and 75.7 ± 8.6%, respectively). In all muscle portions, type IIX was the second most predominant fiber (23.6 ± 18.4% in the superficial anterior, 18.3 ± 19.3% in the deep anterior, 19.3 ± 10.4% in the posterior portion) (Fig. 3). Little cardiac-α myosin was detected in any of the muscle regions.
Fiber cross-sectional areas
In general, the cross-sectional area of fiber types increased significantly in the sequence IIA, IIX and IIB from approximately 1000 µm2 in type IIA to 1990–2850 µm2 in the type IIB fibers (Table 1). These differences were significant (P < 0.01), except for the IIB fibers in the deep anterior temporalis that were equal in size to the type IIX fibers. Of note is that the inter-individual variations in the IIX and IIB fibers of the deep anterior temporalis were larger than in the other muscle regions. The slow fiber types (type I and type I/IIA), found in the deep anterior temporalis, exhibited a smaller (P < 0.05 or P < 0.01) cross-sectional area (635 and 794 µm2, respectively) than the fiber type IIA. The mean fiber cross-sectional area did not differ significantly among the three muscle portions.
The rat temporalis muscle can be divided anatomically into three parts: a superficial and deep part in the anterior region and a posterior region (Weijs, 1973). Anatomically, each of these parts is separated from the other parts by loose connective tissue. Furthermore, in the frontal plane, the anterior part shows a heterogeneous fiber orientation, as the deep part has a more oblique orientation. The directions of the fibers in the posterior part are more homogeneous (Weijs, 1973). As the fiber type composition is clearly different in the anterior temporalis, the rat temporalis is divided into three parts according to the classification of Weijs (1973). The superficial portion of the anterior temporalis shows a fiber composition similar to that of the posterior temporalis, in all probability indicating a similar function. The deep portion of the anterior temporalis shows, compared with the rest of the muscle, an increased amount of IIA fibers, whereas the amount of IIB-fibers is greatly decreased. Moreover, slow type fibers (containing MyHC-I or MyHC-I/IIA) are found only in this deep part of the muscle.
In general, type I (slow) and type IIA fibers possess many mitochondria and exhibit high oxidative enzyme activity and low phosphorylase activity, reflecting their well-developed aerobic metabolism and resistance to fatigue (Ten Cate, 1995; Anderson & Neufer, 2006). In contrast, the faster muscle fibers (types IIX and IIB) rely more on anaerobic (glycolytic) activity and consequently fatigue more easily (Anderson & Neufer, 2006). The posterior temporalis and the superficial portion of the anterior temporalis contain a dominant proportion of type IIB fibers, which also have the largest cross-sectional area. This implies that these muscle portions work as power producers during, for instance, chewing and biting. The deep portion of the anterior temporalis contains a significant amount of type I fibers in addition to the predominant amount of type IIA fibers (also, to some extent, a fatigue-resistant fiber type). This indicates that this portion might have a function involving prolonged muscle activity, for instance during a postural function keeping the mandible at a rest position. Moreover, the deep anterior temporalis shows the longest activity burst during chewing (Weijs & Dantuma, 1975).
As in humans, the rat temporalis muscle is a jaw-closing muscle. However, unlike humans, lateral excursion of the mandible is not present in rats (Creanor & Noble, 1995). In rodents, all of the muscles contract symmetrically and a separation in time is present in forward and backward pulling muscles, resulting in a retraction and protraction of the jaw added to the hinge motion (Langenbach & van Eijden, 2001). Our recent study using a telemetry system revealed that the superficial portion of the anterior superficial temporalis in rats contributes to the chewing power stroke (data not shown). In addition, because the deep portion of the anterior temporalis has a more oblique orientation (Weijs, 1973) than the superficial portion, the deep portion is assumed to play an important role in mandibular posture and in controlling anteroposterior mandibular movements. Our results suggest that the superficial and deep portions of the anterior temporalis muscle in rats play a similar function to the corresponding anterior and posterior temporalis muscles in humans.
Behavioral differences in muscle use, as expressed by their EMG, are largely determined by differences in fiber type composition. Muscles with a significant amount of slow type fibers (MyHC-I) show a longer daily duty time (Hensbergen & Kernell, 1997) and a larger daily number of bursts (van Wessel et al. 2005). We recently examined daily jaw muscle activity in the adult rat by use of a radio-telemetry system in relation to their fiber type composition. In support of our results, the anterior belly of the digastric muscle, containing ca. 5–8% of slow type fibers, shows a longer duty time and larger daily number of bursts and average burst length for activities exceeding 5% of peak EMGs (Kawai et al. 2007) than the masseter muscle, which contains no slow type fibers at all. Compared with the superficial masseter muscle, the anterior temporalis muscle shows a longer duty time for activities exceeding 2% of the peak EMGs and a relatively shorter duty time for activities exceeding 40–50% of the peak EMGs. The low level of EMG activity is likely to occur at rest and the higher levels during mastication (Kawai et al. 2007, 2008). The heterogeneity in fiber type composition combined with findings concerning the daily jaw muscle activity suggests that the various regions of the rat temporalis muscle are involved in a wide range of functions differing in contraction amplitude, incidence and duration, such as chewing, grooming and keeping a mandibular rest position.
The authors gratefully thank Prof. Theo van Eijden, who unfortunately died on February 28, 2007, for his constructive initiatives during the design of the study. We are grateful to Jan Harm Koolstra for his constructive criticism. This research was supported in part by a grant (no. 18592235) for Science Research from the Ministry of Education, Science and Culture, Japan.