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REPORT
MUSCLE CELL BIOLOGY AND CELL MOTILITY
1Department of Oral Biology, College of Dentistry, The Ohio State University, Columbus, Ohio; and 2Department of Physiology, University of Sydney, New South Wales, Australia
Submitted 4 July 2005 ; accepted in final form 6 December 2005
ABSTRACT
The thyroarytenoid muscle, a vocal fold adductor, has important roles in airway protection (e.g., prevention of aspiration) and phonation. Isoform expression of myosin heavy chain (MHC), a major determinant of muscle-shortening velocity, has been reported to be heterogeneous in this muscle in several mammals, differing markedly between the medial and lateral divisions. The objective was to determine the isoform expression patterns of both MHC and myosin light chain (MLC), with the latter having a modulatory role in determining shortening velocity, to further test whether the expression of both myosin subunits differs in multiple specific sites within the divisions of the dog thyroarytenoid muscle, potentially revealing even greater compartmentalization in this muscle. Our results indicate the existence of large gradients in the relative levels of individual MHC isoforms in the craniocaudal axis along the medial layer (i.e., airflow axis), where levels of MHC-I and MHC-IIA are low at both ends of the axis and high in the middle and MHC-IIB has a reciprocal distribution. The lateral layer is more uniform, with high levels of MHC-IIB throughout. The level of MHC-IID is relatively constant along the axis in both layers. Large differences exist in the distribution of MHC isoforms among single fibers isolated from sites along the craniocaudal axis, especially in the lateral layer. Systematic regional variations are apparent in the MLC isoform composition of single fibers as well, including some MLC isoform combinations that are not observed in dog limb muscles. Variations of MHC and MLC isoform expression in the dog thyroarytenoid muscle are greater than previously recognized and suggest an even broader range of contractile properties within this multifunctional muscle.
intrinsic laryngeal muscle; airway protection; vocalization
The thyroarytenoid muscle, along with an antagonist, the cricothyroid muscle, has pivotal roles in pitch control during vocalizations by regulating the tension of the vocal fold. The ability of an individual skeletal muscle to serve specific motor tasks is thought to be due in large part to its myosin isoform composition. Sarcomeric myosin is a hexameric protein composed of two myosin heavy chain (MHC) subunits, two essential myosin light chains (MLCs; MLC1 and MLC3), and two regulatory MLCs (MLC2). Myosin forms cross bridges with actin and hydrolyzes ATP, which provides the energy that drives cyclical cross-bridge interactions that underlie contraction, that is, muscle shortening and/or force production. MHC isoforms are thought to be primarily responsible for differences in muscle-shortening velocity and therefore in power output of vertebrate muscle, with the MLC subunits potentially serving a modulatory role in the determination of contractile properties (7, 30; for review, see Ref. 23). Four MHC isoforms, MHC-I, MHC-IIA, MHC-IID, and MHC-IIB, in order of the increasing muscle-shortening velocity with which they are associated, are expressed in limb skeletal muscles of small mammals. Some larger mammals, including cats, dogs, and humans, appear not to express MHC-IIB in limb skeletal muscle (21, 32, 33). Type I fibers, that is, fibers expressing MHC-I, have the greatest fatigue resistance (9, 18) and are commonly deployed to serve prolonged motor tasks (e.g., posture maintenance), whereas fibers with predominantly MHC-IIB are the least fatigue resistant, yet generate greater power because of their high speed. Fibers expressing MHC-IIA and/or MHC-IID (latter referred to as MHC-IIX in other reports) are intermediate in fatigue resistance and shortening velocity.
Wu and co-workers (35, 37) provided the first descriptions of MHC isoform expression in canine laryngeal muscles, including the thyroarytenoid muscle. Their results and those of several other studies have revealed differences in MHC isoform expression and/or muscle fiber types between the medial division, commonly referred to as the vocalis region, and the lateral (also referred to as external in some reports) division of the canine (37), feline (16), and human thyroarytenoid muscle (13, 19, 36). Furthermore, Yokoyama et al. (38) and Imamura et al. (16) reported that heterogeneity in muscle fiber-type distribution exists within the lateral (external) division of feline thyroarytenoid muscle. It is therefore reasonable to expect that even more extensive regional differences in MHC isoform expression exist between specific compartments in canine thyroarytenoid muscle in addition to those between the vocalis and lateral divisions reported previously (37). We therefore undertook a more thorough examination of the regional distribution of MHC isoforms in dog thyroarytenoid muscle by consistently sampling multiple specific sites along the medial and lateral layers and along a mediolateral continuum across the thickest part of the muscle. The MHC isoform composition of single muscle fibers isolated from different regions of the thyroarytenoid muscle were also examined. We recently reported the existence of fiber types in canine extraocular muscles, identified on the basis of MLC isoform expression patterns, that had not been described previously (3) and in limb muscles of several mammalian species (4). There has been only one report of an examination of the MLC isoform composition of mammalian (human) laryngeal muscles (11). We therefore also examined the MLC isoform composition of the same thyroarytenoid homogenates and single fibers to obtain a more complete understanding of the regional distribution of myosin subunit isoforms in this muscle. The results indicate an even greater complexity than previously reported in the isoform expression patterns of MHCs and MLCs in the dog thyroarytenoid muscle that far exceeds the variability found among fibers in dog limb muscles.
MATERIALS AND METHODS
Sample preparation.
The care and use of animals in this study were conducted in accordance with institutionally (Ohio State University) approved IACUC protocols. Thyroarytenoid (TA), tibialis cranialis (TC, frequently referred to as tibialis anterior in other species), and deep portion of the lateral gastrocnemius (DG) muscles from adult beagles were harvested immediately after euthanasia. A schematic of the dog thyroarytenoid muscle is shown in Fig. 1. The larynx was pinned to a wax-lined tray and cut along its length to expose the lumina and to visualize the vocal folds. The mucosa and underlying nonmuscular elements of the vocal fold were carefully removed from the left and right thyroarytenoid muscle. Samples, cut parallel to the longitudinal axis of the muscle fibers, 
1 mm thick (mediolateral axis) and 1 mm wide (craniocaudal axis), and 90% of the length of the muscle fibers, were obtained from the medial layer and from the lateral layer of either the right or left thyroarytenoid muscle from eight dogs. Five samples from different regions were obtained from both layers, beginning with the most cranial aspect (referred to as M1 and L1 in the medial layer and lateral layer, respectively) and ending with the most caudal aspect of the muscle (M5 and L5). The cross section of the dog thyroarytenoid approximates a triangle, with the portion labeled M3 in this report corresponding to the apex of the triangle and the lateral layer corresponding to the base. In addition, nine samples that were similarly shaped and also cut parallel to the longitudinal axis of the muscle fibers, except for the thickness being 0.5 mm, were then obtained across the thickest part of the contralateral thyroarytenoid muscle in three of the same dogs, beginning at the medial layer (ML1, corresponding to M3 of the thyroarytenoid muscle on the first side sampled) and ending on the lateral layer (ML9, corresponding to L3 on the other side). The direction of sampling for the nine ML samples was along an imaginary line connecting the apex and the middle of the base of the triangle-shaped cross section of the muscle. All of the samples were dissected using a stereomicroscope (magnification, x10–x30) to ensure consistency in sampling sites among all dogs. During the dissections, the muscle was kept moist with relaxing solution (2.0 mM EGTA, 4.0 mM MgATP, 1.0 mM free Mg2+, 10.0 mM imidazole, and sufficient KCl to achieve an ionic strength of 180 mM; pH 7.0) (27). The dissected samples were gently blotted onto filter paper to remove excess relaxing solution, weighed and homogenized in gel sample buffer (6), and further prepared for gel electrophoresis as described by Bicer and Reiser (3). The samples were then stored at –40°C until being loaded onto gels. Bundles of muscle fibers were dissected along the longitudinal axis of the fibers (1 mm wide and 1 mm thick) from several regions of the thyroarytenoid as well as the TC and DG muscles from four additional dogs and stored in glycerinating solution (same ionic composition as relaxing solution, but with one-half the water substituted with glycerol) to be able to isolate single fibers by dissection at a later time. Single fibers were dissected from these bundles in a petri dish containing cold relaxing solution, and their volumes were estimated for gel-loading purposes by measuring their length with a submersed ruler and comparing their diameters with the thickness of submersed nylon black monofilaments of known thickness. Each isolated single fiber was treated with 1% Triton X-100 (Sigma Chemical, St. Louis, MO) in relaxing solution for 5 min to wash away any soluble proteins that might have been retained during storage in the glycerinating solution. Fibers were then rinsed in relaxing solution and transferred into individual microcentrifuge tubes. Sarcomeric proteins in these fibers were then extracted directly in gel sample buffer (2 µl sample buffer/nl fiber volume). The single-fiber samples were allowed to dissolve in the sample buffer for 30 min at room temperature, heated at 65°C for 2 min, immediately chilled on ice for 5 min, and stored at –40°C until being loaded onto gels. All of the homogenates and single-fiber samples were thawed at room temperature, briefly vortexed, and centrifuged for 15 s before being loaded onto gels. Single fibers from the TC and DG were run as limb fast and slow fibers to determine the fast and slow MLC isoforms of thyroarytenoid muscles.
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MALDI-TOF-MS and LC-MS/MS.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (1, 2, 5) were conducted in the Campus Chemical Instrument Center at The Ohio State University to assist with the identification of the proteins postulated to be MHC isoforms. The gel used to generate the bands to be analyzed was fixed with 50% ethanol and 10% acetic acid for 1 h, washed with 50% methanol and 10% acetic acid overnight at room temperature, and stained with Coomassie blue (0.1% Coomassie Brilliant Blue R-250 in 20% methanol and 10% acetic acid) for 2 h. The gel was destained with 50% methanol and 10% acetic acid. The bands of interest were excised and stored in 5% acetic acid solution at 4°C until MALDI-TOF-MS and LC-MS/MS. The gel slices were trimmed to minimize background polyacrylamide material and washed in 50% methanol-5% acetic acid for several hours, dried with acetonitrile (ACN), and reconstituted with DTT to reduce the cysteines. Iodoacetamide was added to alkylate the cysteines, and the gel was washed again with cycles of ACN and ammonium bicarbonate. The gel slices were digested at room temperature overnight with sequencing grade trypsin from Promega using the Montage In-Gel Digestion kit from Millipore (Billerica, MA) according to the manufacturer's recommended protocol. The peptides were extracted from the polyacrylamide with 50% ACN and 5% formic acid several times and then pooled. The extracted pools were concentrated in a speed vacuum to 25 µl. Capillary nano-LC MS/MS was performed on a Micromass hybrid quadrupole time-of-flight Q-TOF II mass spectrometer (Micromass, Wythenshawe, UK) equipped with an orthogonal nanospray source (New Objective, Woburn, MA) operated in positive ion mode. The capillary LC system was a Dionex UltiMate system (Dionex, Sunnyvale, CA). Solvent A was composed of water containing 50 mM acetic acid, and solvent B was composed of ACN. A 5-cm, 75-µm inner diameter BioBasic C18 column (New Objective) packed directly into the nanospray tip was used to perform chromatographic separation. Aliquots (5.0 µl) of each sample were injected into the column for analysis. Peptides were eluted directly from the column into the Q-TOF system using a gradient of 2–80% solvent B for 48 min, with a flow rate of 300 nl/min. Total run time was 55 min. The nanospray capillary voltage was set at 3.0 kV, and the cone voltage was set at 40 V. The source temperature was maintained at 100°C. Mass spectra were acquired from a 400–2,000 mass-to-charge ratio (m/z) every 0.9 s with a resolution of 8,000 full-width half-maximum and recorded using MassLynx 4.0 with automatic switching functions. When the desired peak was detected at a minimum of 15 ion counts, the mass spectrometer was automatically switched to acquire the collision-induced dissociation MS/MS spectrum of the individual peptides and the mass spectra were acquired from a 75/2,000 m/z to detect NH4+. Collision energy was set and was dependent on charge state recognition properties. Sequence information from the MS/MS data was processed using Mascot Distiller software (Matrix Science, Boston, MA) with standard data processing parameters. Database searches were performed using the Mascot software programs (25). The following parameters were used for the Mascot search: carboxyamidomethyl-modified cysteine residues, two missed cleavages, oxidation of methionine residues, and peptide mass tolerance of ±0.6 Da for precursor and ±1.2 Da for fragment ions. Protein identification was checked manually, and proteins with a probability-based molecular weight search (MOWSE) score (24) of 56 or higher with at least one peptide having a y or b ion sequence tag of three residues or more were accepted.
Two-way ANOVA followed by a two-tailed Student t-test was performed to assess the statistical significance of differences between mean percentages of each MHC isoform at different sites within the thyroarytenoid muscle. P < 0.05 was accepted as statistically significant.
RESULTS
Identification of electrophoretically separated MHC isoforms.
Four prominent MHC electrophoretic bands were detected in samples of dog thyroarytenoid muscle. The specific MHC isoforms in the individual bands were identified from the results of a series of immunoblots, dot blots, MALDI-TOF-MS, and LC-MS/MS (Fig. 2). Three MAbs, 2F7 (anti-MHC-IIA), 6H1 (anti-MHC-IID), and 10F5 (anti-MHC-IIB), were used to probe nitrocellulose blots onto which electrophoretically separated MHC isoforms in homogenates of dog thyroarytenoid and rat diaphragm (latter used as positive control) were transferred. Antibodies 10F5 and 6H1 recognized the second- and third-fastest migrating bands in rat diaphragm, identifying these as MHC-IIB and MHC-IID, respectively. The 2F7 antibody did not react with high specificity to any of the rat bands that were transferred from denaturing gels. Therefore, myosin was extracted from four individual skinned fibers isolated from a rat lateral gastrocnemius muscle, and the nondenatured myosin from these fibers was probed with 2F7 on a dot blot (Fig. 2A). This antibody recognized the myosin from only one of these fibers, and this fiber was the only one of the four that expressed the slowest migrating MHC isoform. This isoform was therefore identified as MHC-IIA. The fastest migrating band in the rat diaphragm comigrated with MHC-
in adult rat ventricle (data not shown), with the latter identified as in previous studies (26, 28) and was therefore identified as MHC-I.
The fastest migrating band was the least abundant in the dog thyroarytenoid muscle sample used for immunoblot analysis (see Fig. 2H, in which gel was stained for total protein). This MHC band comigrates with the -isoform of cardiac MHC in dogs (4) and is therefore identified as MHC-I. Others have demonstrated that MHC-I and the predominant ventricular MHC isoform (i.e., MHC-) in large mammals are the same protein (10, 20). The specificity of the same antibodies for individual dog MHC isoforms was generally lower that that for rat MHC isoforms. Antibody 2F7 recognized the slowest migrating band (MHC-IIA in rat) in dog thyroarytenoid but also exhibited, on average, moderate reactivity with the other three MHC bands (Fig. 2E). Antibody 10F5 exhibited the greatest reactivity with the second-fastest migrating band (MHC-IIB in rat) in dog thyroarytenoid but also reacted with the other MHC bands (Fig. 2G). Antibody 6H1 did not recognize any of the dog MHC isoforms. The reactivity and specificity of these antibodies were not improved after nondenatured myosin was extracted from dog single fibers (data not shown). Because of the remaining uncertainty in the identification of the dog MHC isoforms, all four MHC bands from a dog thyroarytenoid sample were analyzed using MALDI-TOF-MS and LC-MS/MS (Table 1). The probability-based MOWSE score (ranging from 2,203 to 4,363) for identification of each of the MHC bands greatly exceeded the minimum score (i.e., 56) required for statistical significance to exclude the possibility of nonrandom matches. The identification of MHC isoforms in dog thyroarytenoid muscle (Fig. 2I) is thus based on a combination of results from Western blot analysis, MALDI-TOF-MS, and LC-MS/MS. The band identified as MHC-IIB was never observed in any sample of dog fast or slow limb muscle in this study or in another recent study (4). Snow et al. (33) reported that dog limb skeletal muscle does not contain IIB fibers, consistent with the lack of expression of the MHC-IIB isoform in limb muscle in this study. We cannot exclude the possibility that any band includes other MHC isoforms. The gel format used in this study to separate MHC isoforms is almost identical to that used in a study by Wu et al. (35), wherein different antibodies were used in Western blot analysis to identify the MHC isoforms expressed in canine laryngeal muscles, including the thyroarytenoid. The results of our MHC isoform band identification (including relative gel migration order) are identical to those of Wu et al. (35). Furthermore, the order of migration of the four MHC isoforms in adult rat muscle (identified using Western blot analysis and dot blots) is identical to the migration order of dog MHC isoforms (determined using Western blot analysis, MALDI-TOF-MS, and LC-MS/MS), providing additional support for the identification of MHC isoforms in this study.
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15% total MHC) is at the most medially prominent part (M3) of the vocal fold (referred to by others as vocalis division of thyroarytenoid; see DISCUSSION). All three of the fast-type MHC isoforms are abundantly expressed in the canine thyroarytenoid muscle, but their distributions along the medial and lateral layers are strikingly different. The pattern of MHC-IIA, the isoform that has been associated with the slowest rates of muscle shortening in mammalian limb muscles among the fast-type isoforms (see DISCUSSION) mirrors the pattern of MHC-I expression; that is, MHC-IIA is lower on the lateral layer and peaks at the same point on the medial layer. The expression pattern of the physiologically fastest isoform of the four isoforms detected (MHC-IIB; see DISCUSSION) was opposite that of MHC-I and MHC-IIA: Generally, higher levels (60–70%) were found on the lateral layer, and the lowest level (2%) was found at the most medially-prominent part of the vocal fold (vocalis). Therefore, the physiologically slowest and fastest MHC isoforms, I and IIB, respectively, have reciprocal patterns of expression along the medial layer of the canine thyroarytenoid muscle. At each sampled site along the lateral layer, MHC-IIB is the predominant isoform and MHC-I is the least abundant. The parallel relationship between the levels of MHC-I and MHC-IIA and the reciprocal relationship between the levels of MHC-I and MHC-IIB are shown in Fig. 4. The expression level of MHC-IID, the fast-type isoform associated with intermediate shortening velocities in previous studies (see DISCUSSION), is expressed at a relatively consistent level (30–40%) throughout both layers of this muscle. Therefore, the isoform expression pattern along the entire lateral layer for all MHC isoforms is relatively constant, in contrast to the varied pattern along the medial layer.
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70% of total MHC at site ML1 (same site as M3 in previous section), both of these isoforms are virtually absent at site ML9 (same site as L3 in previous section). In addition, whereas MHC-IIB is just detectable at ML1, it comprises nearly 60% of total MHC at ML9. The level of MHC-IID is relatively constant (30–40%) across the thickest part of the thyroarytenoid.
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3% of all the fibers along the medial layer expressed exclusively MHC-IIB. Results from individual portions along the medial and lateral layers indicate that differences exist within the layers as well. For clarity, differences within layers are not represented in Table 2. Although analysis of homogenates along the lateral layer indicates that relatively high levels of MHC-IIB exist along the entire layer, the distribution among single fibers differed markedly between L1 and L5. Coexpression of MHC-IID and MHC-IIB was the most common pattern (40 of 61 fibers) in the L1 region, whereas exclusive expression of MHC-IIB was the predominant (21 of 34 fibers) pattern in the L5 region.
DISCUSSION
The results of this study reveal three important features of the dog thyroarytenoid muscle: 1) the extent of regional differences in MHC isoform expression, especially within the medial and lateral layers, is much greater than that reported in earlier studies; 2) the difference in sarcomeric protein isoform expression, as determined on the basis of analysis of homogenates of small regions of the thyroarytenoid, appears to be restricted primarily to MHC isoforms, with small regional differences in the relative amount of slow-type MLC2; and 3) there are several fast or slow fiber types as determined on the basis of analysis of the MLC isoform composition of single fibers in the dog thyroarytenoid that are not typically observed in dog limb skeletal muscle (4). Our results are consistent with a study by Wu et al. (37) in which the expression patterns of MHC isoforms were compared between the medial and lateral portions of the dog thyroarytenoid muscle, with relatively more fast myosin in the lateral portion. However, our results reveal even more complex expression patterns of not only MHC isoforms but also MLC isoforms in multiple regions of the medial and lateral portions and across the thickest portion of this muscle from the medial apex to the lateral base. Although fast fibers predominate in the dog thyroarytenoid muscle (this study; see also Ref. 37), some of the specialization of this muscle is found among slow fibers, with only one of the slow fibers being a conventional slow fiber expressing exclusively slow-type MHC isoforms. That is, most of the slow fibers in this muscle differ from conventional slow limb fibers with respect to MLC isoform expression of fast-type MLC1 (S1F fibers) or atrial MLC1 (S1E/A fibers). Our analysis was limited to one-dimensional (1D) gel electrophoresis. It is possible that additional differences in sarcomeric protein isoform expression in specific regions could be detected using 2D gel electrophoresis of single fibers. Furthermore, we cannot exclude the possibility that more than four MHC isoforms, some of which might comigrate with the identified isoforms, might be expressed in dog thyroarytenoid muscle. Concerning the latter point, however, the total number and identification of MHC isoforms that we observed is identical to that reported by Wu and colleagues (35, 37) in canine cricothyroid, cricoarytenoid, and thyroarytenoid muscles. Although it has been reported that cat (21), dog (33), and human (32) limb skeletal muscles do not express MHC-IIB, unlike smaller mammals, it is clear that this isoform is expressed in canine laryngeal muscles and, at least in some regions of the thyroarytenoid, is the predominant isoform. Because MHC-IIB is associated with the fastest velocities of muscle shortening compared with the other three major MHC isoforms (MHC-I, MHC-IIA, MHC-IID) expressed in mammalian limb skeletal muscles (8), the presence of MHC-IIB in at least some laryngeal muscles is consistent with these muscles having contractile properties that are markedly faster than those of most limb skeletal muscles in the same species (12, 14, 22).
Our results reveal the presence of 21 fiber types on the basis of combinations of MHC and MLC isoforms in single fibers of the dog thyroarytenoid muscle. This finding far exceeds the six fiber types observed on the basis of the same criteria in a recent study involving analysis of myosin subunit isoforms in fibers from 10 dog limb muscles and diaphragm (4). Therefore, the repertoire of contractile properties is potentially extremely large among fibers comprising the dog thyroarytenoid muscle, given the correlations between MHC isoform expression and contractile properties of human laryngeal muscles as demonstrated by others (11, 31). Furthermore, given the marked regional differences in the distribution of the fiber types, the contractile properties of specific compartments are likely to be highly divergent and to drive diverse motor functions. The dog thyroarytenoid muscle is not unique among laryngeal muscles, because coexpression of MHC isoforms in single muscle fibers is a consistent finding among studies of rat, dog, and human cricoarytenoid and thyroarytenoid muscles (11, 29, 35–37).
It is generally thought that motor functions of the thyroarytenoid related to vocalization are served by the medial (vocalis) division. This is the portion of the dog thyroarytenoid in which we and Wu et al. (37) have reported the highest level of MHC-I. It is noteworthy that many of the fiber types in dog thyroarytenoid muscle described in the present report that are different from the typical fast- and slow-type fibers of mammalian limb muscles express MHC-I as well as slow-type MLC2 and should therefore be considered slow fibers. Although these atypical fibers were found in several regions of the thyroarytenoid muscle, it is possible that they have roles in vocalization or perhaps in closures of the airway more prolonged than could be performed by fast-type fibers that generally have lower fatigue resistance.
Our results also reveal a smooth continuum in MHC isoform expression pattern along the mediolateral axis, with the greatest relative amount of the physiologically slowest isoform, MHC-I, found in the medial layer and the least amount of this isoform found in the lateral layer. The physiologically fastest isoform in mammalian limb skeletal muscles, MHC-IIB (see, e.g., Ref. 8), has the opposite pattern of expression, being expressed at the lowest level on the medial layer and at the highest level on the lateral layer. However, there is no abrupt change in the isoform expression pattern across the muscle. This fact argues against the idea that the thyroarytenoid, at least in dogs, is composed of two functionally separate divisions, with one being the medial (vocalis) division and the other being the lateral division as suggested by others for the cat thyroarytenoid (16). It is possible that this continuum in MHC isoform expression allows for graded contractions of the thyroarytenoid in some motor functions such as pitch control, which, even in dogs, spans a broad range from low frequencies during growling to high frequencies in response to painful stimuli.
Wu et al. (37) reported that MHC-IID and MHC-IIB are the predominant isoforms in the medial and lateral regions, respectively, of the dog thyroarytenoid muscle. However, our results derived from analysis of specific compartments within both regions show that MHC-IIA dominates in the middle of the medial region, MHC-IIB dominates at the cranial and caudal edges of the lateral region, and the level of MHC-IID is relatively invariant (30–40% of total MHC) across both regions. Our results indicate that the heterogeneity in MHC isoform expression in dog thyroarytenoid muscle is greater than previously recognized.
The F2S fiber type that we found in the dog thyroarytenoid muscle, expressing fast-type isoforms of MHC and MLC along with MLC2s and a small amount of MHC-I, was not detected in any of the 10 dog limb muscles and diaphragm that we examined in a recent study involving analysis of 292 fibers (4). This fiber type might therefore have a function that is unique to the control of vocal fold tension or movement. Given that it is a fiber type with fast and slow myosin isoforms, it might be involved in fine-tuning vocal fold mechanical properties by having contractile properties that are not shared with other fast or slow fibers. The slow fibers with the embryonic/atrial MLC1 isoform that we observed in this study are not identical to the prevalent slow fibers in the orbital layer of dog rectus muscles, which also express embryonic/atrial MLC1, because the latter did not appear to express slow-type MLC1 (3).
In summary, the dog thyroarytenoid muscle is extremely complex with respect to MHC and MLC isoform expression patterns and the highly compartmentalized distribution of fibers expressing different combinations of these isoforms. A considerable effort is required to fully understand the fundamental contractile properties of the different types of fibers comprising the thyroarytenoid muscle. This information is essential to the understanding of how this muscle is able to subserve the diverse motor functions in which it is involved. A better understanding of the properties of the thyroarytenoid muscle not only would advance knowledge of a highly specialized muscle but also could allow researchers to gain insight into the etiology of voice disorders in which the thyroarytenoid muscle has a role (e.g., Ref. 17).
GRANTS
This project was supported by National Science Foundation Grant IOB 0133613.
ACKNOWLEDGMENTS
We are grateful to Martin Spencer for extensive and extremely insightful discussions regarding the anatomy, especially the anatomical compartmentalization and functions, of the mammalian thyroarytenoid muscle. Drs. Kari Green-Church and Liwen Zhang provided valuable advice on sample preparation and interpretation of data related to MALDI-TOF-MS and LC-MS/MS analyses.
FOOTNOTES
Address for reprint requests and other correspondence: P. J. Reiser, Dept. of Oral Biology, College of Dentistry, The Ohio State Univ., 305 W. 12th Ave., Columbus, OH 43210 (e-mail: reiser.17{at}osu.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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