The Specific Molecular Composition and Structural Arrangement of Eleutherodactylus Coqui Gular Skin Tissue Provide Its High Mechanical Compliance.

A male Eleutherodactylus Coqui (EC, a frog) expands and contracts its gular skin to a great extent during mating calls, displaying its extraordinarily compliant organ. There are striking similarities between frog gular skin and the human bladder as both organs expand and contract significantly. While the high extensibility of the urinary bladder is attributed to the unique helical ultrastructure of collagen type III, the mechanism behind the gular skin of EC is unknown. We therefore aim to understand the structure-property relationship of gular skin tissues of EC. Our findings demonstrate that the male EC gular tissue can elongate up to 400%, with an ultimate tensile strength (UTS) of 1.7 MPa. Species without vocal sacs, Xenopus Laevis (XL) and Xenopus Muelleri (XM), elongate only up to 80% and 350% with UTS~6.3 MPa and ~4.5 MPa, respectively. Transmission electron microscopy (TEM) and histological staining further show that EC tissues' collagen fibers exhibit a layer-by-layer arrangement with an uninterrupted, knot-free, and continuous structure. The collagen bundles alternate between a circular and longitudinal shape, suggesting an out-of-plane zig-zag structure, which likely provides the tissue with greater extensibility. In contrast, control species contain a nearly linear collagen structure interrupted by thicker muscle bundles and mucous glands. Meanwhile, in the rat bladder, the collagen is arranged in a helical structure. The bladder-like high extensibility of EC gular skin tissue arises despite it having eight-fold lesser elastin and five times more collagen than the rat bladder. To our knowledge, this is the first study to report the structural and molecular mechanisms behind the high compliance of EC gular skin. We believe that these findings can lead us to develop more compliant biomaterials for applications in regenerative medicine.

Posterior cricoarytenoid bellies: relationship between their function and histology.

OBJECTIVES/HYPOTHESIS: Complete physiological information about human posterior cricoarytenoid muscle (PCA) is essential and is not only of basic science interest but also could lead directly to understanding phonation and many clinical issues in neurolaryngology. The purpose of the study was to investigate and compare the histochemical and morphological properties to know contractile muscle fiber characteristics of two bellies of the PCA. STUDY DESIGN: Cross-sectional experimental study. METHODS: The PCAs were harvested from the total laryngectomy simples. Serial transverse sections of the two PCA bellies were performed and studied by immunohistochemical analysis. RESULTS: Two separate muscle bellies were always identified within 15 PCA. The following muscle fiber types were observed: I, I-IIA, and IIA. Comparisons of the vertical and horizontal bellies of the PCA reveled differences in the fiber-type composition. CONCLUSION: In our experience, the PCA should be considered as a combination of two functional subunits, which significantly differ in their muscle fiber-type composition.

[The expression of myosin heavy chain isoforms of human normal laryngeal muscles and the difference between the adductor and abductor].

OBJECTIVE: To investigate the expression of myosin heavy chain (MHC) in normal laryngeal muscle and the difference between the adductor and abductor. METHOD: Seven patients with total laryngectomy were enrolled in this study. The adductor muscles were acquired from the lateral cricoarytenoid (LCA) muscle and the abductor muscles were acquired from the posterior cricoarytenoid (PCA) muscle. The expression of myosin heavy chain were detected with fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence staining respectively. RESULT: (1) MHC-II b was expressed in laryngeal muscles at mRNA levels, and not expressed at the protein level; (2) At both mRNA level and protein level, the expression of MHC-I was higher in the PCA muscles than in the LCA muscles while MHC-II level was higher in the LCA muscles than in the PCA muscles. CONCLUSION: (1) MHC-II b protein was not expressed in human laryngeal muscles; (2) Phenotypic differences were significant in laryngeal adductor and abductor muscles based on their different functions. PCA contained larger percentage of MHC-I fibers, while LCA contained more MHC-II fibers.

Fiber types in canine muscles: myosin isoform expression and functional characterization.

This study was aimed to achieve a definitive and unambiguous identification of fiber types in canine skeletal muscles and of myosin isoforms that are expressed therein. Correspondence of canine myosin isoforms with orthologs in other species as assessed by base sequence comparison was the basis for primer preparation and for expression analysis with RT-PCR. Expression was confirmed at protein level with histochemistry, immunohistochemistry, and SDS-PAGE combined together and showed that limb and trunk muscles of the dog express myosin heavy chain (MHC) type 1, 2A, and 2X isoforms and the so-called "type 2dog" fibers express the MHC-2X isoform. MHC-2A was found to be the most abundant isoform in the trunk and limb muscle. MHC-2X was expressed in most but not all muscles and more frequently in hybrid 2A-2X fibers than in pure 2X fibers. MHC-2B was restricted to specialized extraocular and laryngeal muscles, although 2B mRNA, but not 2B protein, was occasionally detected in the semimembranosus muscle. Isometric tension (P(o)) and maximum shortening velocity (V(o)) were measured in single fibers classified on the basis of their MHC isoform composition. Purified myosin isoforms were extracted from single muscle fibers and characterized by the speed (V(f)) of actin filament sliding on myosin in an in vitro motility assay. A close proportionality between V(o) and V(f) indicated that the diversity in V(o) was due to the different myosin isoform composition. V(o) increased progressively in the order 1/slow < 2A < 2X < 2B, thus confirming the identification of the myosin isoforms and providing their first functional characterization of canine muscle fibers.

Proteomic profiling of rat thyroarytenoid muscle.

PURPOSE: Proteomic methodologies offer promise in elucidating the systemwide cellular and molecular processes that characterize normal and diseased thyroarytenoid (TA) muscle. This study examined methodological issues central to the application of 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE) to the study of the TA muscle proteome using a rat model. METHOD: 2D SDS-PAGE was performed using 4 chemically skinned rat TA muscle samples. Gel images were analyzed and compared. Protein spot detection and matching were performed using computational image analysis algorithms only and computational image analysis followed by visual inspection and manual error correction. A synthetic master gel, constructed to control for uninteresting biological variation and technical artifact due to differences in protein loading and staining, was evaluated against its constituent gels. RESULTS: Manual error correction resulted in a consistent increase in the number of protein spots detected (between 5.8% and 40.9%) and matched (from 25.8% to 70.8%) across all gels. Sensitivity and specificity of the automatic (computational) spot detection procedure, evaluated against the manual correction procedure, were 74.1% and 97.9%, respectively. Evaluation of protein quantitation parameter values revealed statistically significant differences (p < .0001) in optical density, area, and volume for matched protein spots across gels. The synthetic master gel successfully compensated for these intergel differences. CONCLUSIONS: Valid and reliable proteomic data are dependant on well-controlled manageable variability and well-defined unmanageable variability. Manual correction of spot detection and matching errors and the use of a synthetic master gel appear to be useful strategies in addressing these issues. With these issues accounted for, 2D SDS-PAGE may be applied to quantitative experimental comparisons of normal and disease conditions affecting voice, speech, and swallowing function.

Proteomic analysis of rat laryngeal muscle following denervation.

Laryngeal muscle atrophy induced by nerve injury is a major factor contributing to the disabling symptoms associated with laryngeal paralysis. Alterations of global proteins in rat laryngeal muscle following denervation were, therefore, studied using proteomic techniques. Twenty-eight adult Sprague-Dawley rats were divided into normal control and denervated groups. The thyroarytenoid (TA) muscle was excised 60 days after right recurrent laryngeal nerve was resected. Protein separation and identification were preformed using 2-DE and MALDI-MS with database search. Forty-four proteins were found to have significant alteration in expression level after denervation. The majority of these proteins (57%), most of them associated with energy metabolism, cellular proliferation and differentiation, signal transduction and stress reaction, were decreased levels of expression in denervated TA muscle. The remaining 43% of the proteins, most of them involved with protein degradation, immunoreactivity, injury repair, contraction, and microtubular formation, were found to have increased levels of expression. The protein modification sites by phosphorylation were detected in 22% of the identified proteins that presented multiple-spot patterns on 2-D gel. Significant changes in protein expression in denervated laryngeal muscle may provide potential therapeutic strategies for the treatment of laryngeal paralysis.

Effect of deficits in laryngeal sensation on laryngeal muscle biochemistry.

Swallowing deficits in elderly people are significant clinical problems and may be associated with impaired pharyngolaryngeal sensation. However, the extent to which sensory innervation affects the motor system is unclear. Our purpose was to examine differences in biochemical properties of laryngeal muscles following sensory nerve ablation. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to evaluate laryngeal muscles of young and old Fischer 344/Brown Norway rats, and rats that underwent sensory ablation via bilateral section of the superior laryngeal nerve, internal branch (SLNi), or mixed sensory-motor nerve ablation via left-sided recurrent laryngeal nerve (RLN) section. In lateral thyroarytenoid muscle, a reduction was found in the proportion of the most rapidly contracting myosin heavy chain isoform (type 2B) with SLNi section, RLN section, and aging. Section of the SLNi did not alter the proportion of any myosin heavy chain isoform within the lateral cricoarytenoid or posterior cricoarytenoid muscles, but RLN section resulted in a reduction in the proportion of type 2B. Accordingly, alteration in biochemical properties of the lateral thyroarytenoid muscle alone was demonstrated following sensory ablation. We conclude that sensory changes may affect properties of laryngeal muscles, and may thus have an impact on motor control during critical functions, such as airway protection during swallowing.

Age-related alterations in myosin heavy chain isoforms in rat intrinsic laryngeal muscles.

Deficits in voice and swallowing are found in the elderly, but the underlying neuromuscular mechanisms are unclear. A potential mechanism may be denervation-induced muscle fiber transformation to a slower-contracting type of muscle fiber. This study examined young, old, and denervated rat laryngeal muscles (lateral thyroarytenoid, lateral cricoarytenoid, and posterior cricoarytenoid) to examine differences in myosin heavy chain (MHC) composition. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that all muscles were composed predominately of type IIB MHC. With aging and denervation, type IIB was reduced and type IIX, a slower-contracting isoform, was increased in the lateral thyroarytenoid and lateral cricoarytenoid muscles. In the posterior cricoarytenoid muscle, the MHC composition was relatively unchanged. These findings suggest that aging may affect laryngeal adductory function by altering muscle fiber type composition to a slower-contracting isoform, in a manner similar to that observed with denervation.

Unloaded shortening velocity and myosin heavy chain variations in human laryngeal muscle fibers.

Myosin description in human laryngeal muscles is incomplete, but evidence suggests the presence of type I, IIA, IIX, and tonic myosin heavy chain (MHC) fibers. This study describes the unloaded shortening velocity (V0) of chemically skinned laryngeal muscle fibers measured by the slack test method in relation to MHC content. Skeletal fibers from human laryngeal and limb muscle biopsy specimens were obtained for determination of V0, and subsequently, glycerol-sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to determine the MHC isoform content. The fibers from human limb muscle had shortening speeds similar to those in previous reports on human skeletal fibers. Type I, IIA, and IIX fibers of laryngeal muscle had shortening speeds similar to those of fibers from limb muscle, but laryngeal fibers with heterogeneous MHC expression had a wide range of shortening speeds, some being nearly twice as fast as limb fibers. In addition, MHC isoform bands from human extraocular muscle comigrated with some bands from laryngeal muscle--a finding suggesting that extraocular myosin may also be expressed.

Laryngeal reinnervation with the hypoglossal nerve. I. Physiology, histochemistry, electromyography, and retrograde labeling in a canine model.

This study was performed to determine whether the hypoglossal nerve (cranial nerve XI [XII]) would serve as a useful donor for laryngeal reinnervation by anastomosis to the recurrent laryngeal nerve (RLN). Twenty hemilarynges in 10 dogs were studied prospectively after XII-RLN anastomosis (group A; n = 5), split XII-RLN anastomosis (group B; n = 3), XII-RLN anastomosis with a 2-cm interposition graft (group C; n = 2), no treatment (group D; n = 5), RLN section (group E; n = 2), or ansa cervicalis-RLN anastomosis (group F; n = 3). Spontaneous activity was observed monthly by infraglottic examination through permanent tracheostomies and was recorded by electromyography. Laryngeal adductory pressure and induced phonation were obtained by stimulating the RLN while passing a pressure transducer balloon or humidified air through the glottis. At sacrifice, the laryngeal muscles were stained for adenosine triphosphatase to determine the ratio of type I to type II fibers. Retrograde labeling of the brain stem was performed with horseradish peroxidase. Infraglottic examination at 6 months showed a full range of adductory motion in groups A and B during the swallow reflex, comparable with that in group D. Groups C and F showed good bulk and tone, but little spontaneous motion. Group E remained paralyzed. Stimulation of the transferred nerves caused more activity in groups A and B than in the other groups; groups C and F partially adducted at high levels. The laryngeal adductory pressure responses of groups A and B were similar to those of group D. The XII-reinnervated larynges were capable of producing normal induced phonation. Retrograde labeling of the RLN showed that the reinnervating axons originated only in the hypoglossal nucleus. Electromyography of the reinnervated adductor muscles confirmed spontaneous activity in the dogs (awake). Histochemical analysis confirmed slow-to-fast transformation of both the posterior and lateral cricoarytenoid muscles, indicating that significant reinnervation occurred. We conclude that the hypoglossal nerve functions well as a donor for adductory reinnervation of the larynx.

Early specialization of the superfast myosin in extraocular and laryngeal muscles.

Extraocular muscle (EOM) exhibits high-velocity, low-tension contractions compared with other vertebrate striated muscles. These distinctive properties have been associated with a novel myosin heavy chain (MyHC) isoform, MyHC-EO. An atypical MyHC, MyHC IIL, has also been identified in laryngeal muscles that have similarly fast contractile properties. It co-migrates with MyHC-EO on high-resolution SDS gels, but appeared to be encoded by a different mRNA. We combined CNBr peptide maps and full-length cDNA sequences to show that rabbit muscle EO and IIL MyHCs are identical. Analysis of the 5; untranslated region (5;UTR) of the mRNAs identified three variants that result from a combination of alternative splicing and multiple transcription initiation sites. This complex pattern of 5;UTRs has not been reported previously for MyHC genes. We identified the human homologue of the MyHC-EO gene in GenBank, and analyzed the 5; upstream region, which revealed a paucity of muscle-specific transcription factor binding sites compared with the other MyHC genes. These features are likely to be critical to the unique regulation and tissue-specific expression of the MyHC-EO/IIL gene. Phylogenetic analysis indicates that MyHC-EO/IIL diverged from an ancestral MyHC gene to generate the first specialized fast myosin. The catalytic S1 head domain is more closely related to the fast MyHCs, while the rod is more closely related to the slow/cardiac MyHCs. The exon boundaries of the MyHC-EO are identical to those of the embryonic MyHC gene and virtually identical to those of the &agr; and (&bgr;) cardiac genes. This implies that most of the current exon boundaries were present in the ancestral gene, predating the duplications that generated the family of skeletal and cardiac myosin genes.

Are hybrid fibers a common motif of canine laryngeal muscles? Single-fiber analyses of myosin heavy-chain isoform composition.

BACKGROUND: The canine lateral cricoarytenoid muscle contains a large proportion of muscle fibers that coexpress various combinations of myosin heavy-chain isoforms (ie, so-called hybrid fibers). OBJECTIVE: To test the hypothesis that hybrid fibers are a common motif throughout laryngeal muscles. DESIGN: The posterior cricoarytenoid, canine cricothyroid, and thyroarytenoid muscles were removed from 5 beagle dogs. The posterior cricoarytenoid and canine cricothyroid muscles were each dissected into horizontal, oblique, and rectus regions. The thyroarytenoid was separated into medial and lateral regions. Approximately 40 single fibers were microdissected from each region ( approximately 1800 total fibers were sampled) and placed into a denaturing sample buffer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was then used to separate the individual myosin heavy-chain isoforms. RESULTS: Each laryngeal muscle contained hybrid fibers; however, the types and proportions of hybrid fibers were clearly muscle specific. Within a given muscle, there were relatively minor regional differences in the types and proportions of hybrid fibers. CONCLUSION: If the myosin heavy-chain isoform composition of a single fiber can be used as a "physiological marker," then the extent of hybridism may reveal the diversity of activity required of a given laryngeal muscle.

Single-fiber myosin heavy-chain isoform composition of rodent laryngeal muscle: modulation by thyroid hormone.

BACKGROUND: Studies have shown that canine laryngeal muscle contains a large number of muscle fibers that coexpress varying combinations of myosin heavy-chain (MyHC) isoforms. Currently, it is not clear whether this phenomenon is unique to canine laryngeal muscle or occurs in all mammals. OBJECTIVES: To examine the single-fiber MyHC isoform composition of rodent laryngeal muscle and to examine the plasticity of single-fiber MyHC isoform composition via manipulation of thyroid state. RESULTS: (1) Findings of single-fiber electrophoretic analyses clearly demonstrate that most fibers in both the posterior cricoarytenoid and thyroarytenoid muscles exhibit MyHC polymorphism. However, the proportions and patterns of polymorphism appear to be muscle specific. (2) Although the fast type IIL isoform was observed in fibers from both muscles, it was always coexpressed in combination with other MyHC isoforms (ie, no pure type IIL fibers were found), and always represented a minor proportion of the total MyHC pool. (3) Altering the thyroid state proved a useful tool for exploring the scope of MyHC isoform expression in these muscles. While the posterior cricoarytenoid muscle seemed more sensitive to the thyroid state, transitions in both muscles were primarily confined to the fast type IIX and IIB MyHC isoforms. CONCLUSION: The findings of this study support the concept that single-fiber MyHC polymorphism occurs commonly in mammalian laryngeal muscle.

Myosin heavy chain expression in human laryngeal muscle fibers. A biochemical study.

Since the intrinsic laryngeal muscles in humans are involved in specialized functions, one may suppose that this would be associated with the expression of specific myosin heavy chain (MHC) isoforms, as has been reported for the rat, dog, and rabbit. In order to determine which MHCs are expressed in the human laryngeal muscles, biochemical analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed. Thyroarytenoid and posterior cricoarytenoid muscles were obtained from a 7-month-old infant and 4 adults. In the adult human laryngeal muscles, 3 bands were resolved identical to those previously described in the human limb muscles (I, IIA, and IIB MHCs). In contrast, muscles from the infant also expressed fetal MHC and a novel MHC not observed in other human skeletal muscles. This novel band migrated at the same level as the laryngeal MHC previously described in the rat. Since these 2 isoforms disappear in the adult, the persistence in the infant could be correlated with the immature development of laryngeal functions and, in particular, phonation.

Myosin heavy chain composition in human laryngeal muscles.

OBJECTIVES: Myosin heavy chain (MHC) composition of human thyroarytenoid (TA), lateral cricoarytenoid (LCA), interarytenoid (IA), vocalis, posterior cricoarytenoid (PCA), and cricothyroid muscles were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western bolt techniques. The presence of superfast MHC was also assessed using antibodies directed against the extraocular MHC. STUDY DESIGN: MHC protein was analyzed using fresh human laryngeal muscles. METHODS: Laryngeal muscles excised from cadavers were processed for SDS-PAGE. The composition of MHC isoforms was determined by densitometry. Western blot was carried out to identify specific bands. RESULTS: MHC types IIA and IIB are the predominant MHC components in human laryngeal muscles. The adductor muscles--TA, LCA, and IA--have a higher percentage of type IIB MHC and a lower percentage of type I when compared with the abductor--PCA. The rank file order for type IIB MHC composition (TA > LCA > or = IA > PCA) is the same in all specimens. A band migrating between type IIA and type I was observed in several specimens. Although similar to type IIL in rats, this atypical band did not react with anti-extraocular MHC antibody on Western blot. CONCLUSION: Characterization of laryngeal muscles determined by the composition of MHC is correlated with function and neural input. Human laryngeal muscle is characterized by a predominance of fast-type MHCs in laryngeal closing muscle and mixed fast-slow type MHCs in respiratory and phonatory muscle groups. Although an atypical myosin band similar to type IIL (superfast) MHC in rat was identified, it did not react with anti-extraocular MHC antibody.

Neurochemical substances in neurons of the canine intrinsic laryngeal muscles.

The localization of vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), catecholamines (CA) and carbon monoxide (CO) in neurons of the intrinsic laryngeal muscles of the dog was investigated using immunohistochemistry. Cells with VIP-like immunoreactivity were found between the striated muscle fibres of the cricothyroid and posterior cricoarytenoid muscles, some aggregated into a ganglion and some solitary. Neurons with immunoreactivity to haem oxygenase-2 (HO-2), an immunohistochemical marker for CO, were also seen. VIP-or HO-2-negative cells also existed in various numbers in the intramuscular ganglia. No CGRP-like immunoreactive neurons were found although CGRP-like immunoreactive varicose fibres were seen in the intramuscular ganglia. No neurons or fibres immunoreactive to tyrosine hydroxylase (TH), the key enzyme in synthesis of CA, were detected. The exact function of these intramuscular ganglia is yet to be clarified, but they may be sensory neurons.

Postnatal development of myosin heavy chain isoforms in rat laryngeal muscles.

The developmental transitions of myosin heavy chain (MHC) isoforms of rat posterior cricoarytenoid (PCA), thyroarytenoid (TA), cricothyroid (CT), and lateral cricoarytenoid (LCA) muscles were examined by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot techniques. The muscles were microscopically dissected from animals on postnatal days 0, 3, 7, 10, 14, 21, 28, 35, 45, and 55 and from adult animals. Silver-stained SDS-PAGE gels of each muscle were analyzed densitometrically to measure the composition of MHC isoforms, and Western blot was carried out to identify specific bands. Characterizations of the internal laryngeal muscles determined by the composition of MHCs were correlated with their function in the adult. Temporally, differentiation reflects onset of function. Differentiation of isoforms and transition to adult forms occur first in the TA muscle, followed by the PCA, LCA, and CT muscles. Expression of type IIL was observed only in muscles innervated by the recurrent laryngeal nerve. Postnatally observed developmental differences of myosin phenotypes suggest that regulation of MHC expression is influenced by neural activity or other environmental factors.

Expression of extraocular-superfast-myosin heavy chain in rat laryngeal muscles.

Superfast myosin heavy chain (MHC), which is found in jaw-closing muscle and extraocular muscle (EOM), may also be found in rat laryngeal muscles. Immunostaining and Western blot using anti-EOM antibody were performed to identify and localize EOM MHC in laryngeal muscles. Specific reactivity of laryngeal IIL MHC was confirmed by Western blot and on immunostaining, all fibers in the lateral part of thyroarytenoid muscle reacted with EOM antibody. A scattered pattern of positive fibers was observed in the medial part of the thyroarytenoid, the posterior cricoarytenoid and the lateral cricoarytenoid muscles. EOM MHC was not detected in the cricothyroid muscle. The expression of EOM MHC in rat laryngeal muscle is consistent with the functional demands of the airway protection reflex.

Age- and gender-related elastin distribution changes in human vocal folds.

The composition of the lamina propria in human vocal folds has been shown to affect vocal performance. Elastin plays a significant role in the biomechanical effects of the lamina propria. We obtained 19 larynges from the state medical examiner from subjects whose cause of death was unrelated to the trachea and laryngeal regions. The sample contained male and female subjects in the infant, adult, and geriatric age groups. We stained the vocal folds for elastin with Verhoeff's elastic tissue stain and studied them with use of an image analysis system configured for light microscopy. Distributions of elastin were measured from superficial to deep within the lamina propria (from epithelium to vocal muscle). These elastin distributions were then compared with the use of statistical software. The data showed that there was an increase in elastin content from the infant through geriatric stages. No gender-related differences were found. Infant folds had about 23% of the elastin found in adults, and geriatric subjects had about 879% of the elastin found in adults. Both of these results were statistically significant (p < 0.05). The distributions were consistent with previous observations that the lamina propria is a layered structure with most of the elastin present in the intermediate layer. This layer was larger in geriatric subjects than in adult and pediatric subjects. We observed that the fiber diameter appeared to be larger in geriatric subjects (this observation is currently being verified with electron microscopy) whereas smaller, spiraled fibers appeared in pediatric subjects.

A new concept in laryngeal muscle: multiple myosin isoform types in single muscle fibers of the lateral cricoarytenoid.

This report describes the first known investigation of canine laryngeal muscle in which single fibers were dissected and their myosin heavy chain (MHC) isoform content was analyzed. Both SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot techniques were used. The data from single fiber SDS-PAGE indicate that the lateral cricoarytenoid (LCA) is predominantly a fast muscle composed of the following MHC isoforms: Type I, 16.3%; Type IIA, 71.3%; Type IIX, 10.4%; and Type IIB, 2.0%. The results reveal a phenomenon that, to our knowledge, has not been previously described for laryngeal muscle: the presence of two or more MHC isoforms in a single canine LCA muscle fiber. A large number (41%) of muscle fibers coexpressed two or more MHC isoforms. The three most common patterns of coexpression were Type IIA/IIX (72%), Type IIA/I (16%), and Type IIA/IIX/I (8%). Interestingly, the fast Type IIX MHC isoform was typically present with other isoforms and rarely found by itself in individual fibers. Additional experiments are underway to determine whether other laryngeal muscles exhibit such an unusually high ratio of MHC isoform polymorphism.