Myosin heavy chain expression in human laryngeal muscles

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August 1996

Scientific Sessions- - Tuesday

8:00 AM to 12:00 NOON Rooms 20 and 21, WCC RESEARCH FORUM: Sponsored by the Committee for Research in Otolaryngology of the American Academy of Otolaryngology-Head and Neck Surgery Foundation, Inc., and the Association for Research in Otolaryngology THOMAS R. PASIC, MD, and DIANNE DURHAM, PhD (co-chairpersons), Madison, Wis., and Kansas City, Kan. SESSION A: Laryngeal Disorders (Room 20 - WCC) LISA ORLOFF, MD, DONALD WEED, MD, and DIANNE DURHAM, PhD (moderators), San Diego, Calif., Nashville, Tenn., and Kansas City, Kan. 8:00 AM

New Data Regarding Muscle Fiber Types of Canine Posterior Crlcoarytenold Muscle: The Single Fiber Gel Electrophoresls Technique YA ZHEN WU, MD (presenter), MICHAEL J. BAKER, ROGER L. CRUMLEY, MD, ROBERT H.I. BLANKS, PhD, and VINCENT J. CAIOZZO, PhD, Irvine, Calif.

Previous studies have shown that some single muscle fibers may simultaneously coexpress more than one type of myosin heavy chain (MHC) isoform, but little work has been done to characterize such a phenomenon in laryngeal muscle. Hence the objective of this study was to analyze the MHC content of single fibers in the canine posterior cricoarytenoid (PCA) muscle (n = 5). Forty single fibers were microdissected from each of the three distinct regions (horizontal, oblique, vertical) of the PCA muscle. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the content of the four MHC isoforms (type I, type IIA, type IIX, type liB) within single fibers. Results showed that the PCA is heterogeneous for MHC composition: single fibers from the horizontal region showed a larger percentage (48%) of slow-twitch type I MHC than in the fibers of the oblique (33%) or vertical (31%) regions (p < 0.001). Similarly, more fast-twitch type IIA MHC was found in the single fibers of the oblique region (42%) than in fibers of the horizontal region (29%) (/9 < 0.01). Additionally, more fast-twitch type IIX MHC was expressed in fibers of the vertical region (27%) than in those of the horizontal region (17%) (p < 0.001). With respect to polymorphic expression, 22.6% of all PCA fibers sampled coexpressed more than one MHC, and a majority of these fibers contained both type IIA and type IIX. This percentage is in sharp contrast to the results of previous work, which indicated that the lat-

eral cricoarytenoid (LCA) muscle contains a much higher percentage (41.1%) of polymorphic fibers. Because MHC isoforms are largely responsible for the mechanical properties of a muscle, these results suggest that the three regions of the PCA may have distinct functions during phonation and inspiration. Further electromyographic analysis may confirm the speculation that the horizontal region is responsible for phonation, whereas the oblique and vertical regions hold abductive/inspiratory functions. 8:15 AM

A Comparison of Succinate Dehydrogenase Activity in Rat Laryngeal and Hind Limb Muscles ALBERT L MERATI, MD, (presenter), WILLIAM J,D, TURNER, MD, SUE BODINE-FOWLER, PhD, and DAVID J. PIEROTII, PhD, San Diego and La Jolla, Calif.

Succinate dehydrogenase (SDH) activity is often used as a marker of a muscle's oxidative capacity and relative fatigue resistance. In hind limb muscles, the fastest muscle fibers (i.e., the liB fibers) have a very low oxidative capacity. Immunohistochemical and in situ histochemistry done in our laboratory has revealed that the thyroarytenoid (TA) muscle is composed of exclusively fast fibers expressing type IIX, liB, or IlL myosin heavy chains. Interestingly, qualitative SDH staining has suggested that the TA muscle is highly oxidative. In this study we measured SDH activity of individual muscle fibers in the TA muscle using a quantitative histochemical assay developed by Martin et al. (J Histochem Cytochem 1985;33:1053-9). The larynx of four adult male Sprague-Dawley rats was removed, frozen, and sectioned. The TA muscle was identified in cross-section, and SDH activity, fiber type, and fiber size were determined for a sample of muscle fibers (-40 fibers/muscle). The mean SDH rate for the TA muscle was 0.072 optical density units/minute (• This rate is 1.5 times higher than the SDH rates measured in the soleus, a predominantly slow, highly oxidative hind limb muscle. The TA and other head and neck muscles are unusual in that they contain primarily fast fibers with very high oxidative capacity. In the TA muscle this may represent adaptation to the demands of airway regulation and protection. This is an underexplored area in laryngeal function and disease. This work was funded, in part, by a grant from the American Laryngological Association. 8:30 AM

Myosln Heavy Chain Expression in Human Laryngeal Muscles WILLIAM J.D. TURNER, MD, (presenter), and SUE C. BODINEFOWLER, PhD, San Diego and La Jolla, Calif.

Laryngeal muscle serves a unique function in phonation, respiration, and airway protection. Understanding the basic biochemical and morphologic characteristics of these muscles is important in understanding their function. No study presently exists that accurately characterizes the

Otolaryngology Head and Neck Surgery

Scientific Sessions- - Tuesday

Volume 115 Number 2

muscle fiber types present in human laryngeal muscles. Previous studies have relied on biochemical markers which can distinguish only between fast and slow fibers and cannot differentiate between fast myosin heavy chain (MHC) isoforms or hybrid fibers which express multiple isoforms. We examined the thyroarytenoid (TA), lateral cricoarytenoid (LCA), posterior cricoarytenoid (PCA), interarytenoid (IA), and cricothyroid (CT) muscles obtained from two organ donors and five cadavers with normal larynges using immunohistochemistry with MHC-specific antibodies and in situ hybridization using specific riboprobes to human slow, IIA, and IIX MHC. Muscle fiber type distribution and fiber size were determined for six of each muscle using MHC antibodies. A total of 300 to 600 fibers per muscle were analyzed. The specific fiber type distributions for each muscle were as follows: TA, 23% slow, 50% IIA, 27% IIX; LCA, 25% slow, 62% IIA, 12% IIX; PCA, 52% slow, 37% IIA, 11% IIX; IA, 32% slow, 62% IIA, 6% IIX; and CT, 40% slow, 57% IIA, 1.5% IIX. Average fiber size ranged from 750 to 1650 Hm2, which is one half to one third the size of limb skeletal muscle fibers. In all muscles slow fibers were significantly smaller than type IIA or IIX fibers. In situ hybridization revealed that fiber typing obtained via antibodies could not distinguish hybrid fibers, and depending on the muscle, 20% to 90% of those fibers typed as IIA using antibodies expressed both IIA and IIX MHC messenger RNA. No fibers expressed both slow and fast isoforms. All fibers hybridized with at least one of the three isoforms. In situ hybridization revealed that those fibers previously typed as liB actually expressed the IIX isoform. This study provides previously unavailable basic information about the MHC composition of normal laryngeal muscles, which is intimately linked with their function. This work was supported by a grant from the American Laryngologic Association. 8:45 AM

A Rat Model of Wound Repair after Laryngotracheal Injury DAVID G. DILLARD, MD (presenter), IAN N. JACOBS, MD, and ANTHONY A. GAL, MD, Atlanta, Ga,

Acquired subglottic stenosis occurs in a small percentage of premature infants who require chronic ventilation. Endotracheal tube injury to the mucosal surface of the airway may result in cartilage exposure and subsequent chondritis. This leads to cartilage remodeling and collagen deposition. The new scar tissue may narrow the subglottic airway, resuiting in airway compromise. Wound repair after injury is a complex process that involves epidermal cells, monocytes, fibroblasts, cytokines, and extracellular matrix proteins. To better understand airway wound healing we investigated sequential changes in wound repair and scar tissue formation after posterior cricoid injury in rats. Twenty-eight male adult rats underwent a midline laryngofissure to expose the posterior wall of the cricoid. A rectangular area (1 x 3 mm) of mucosa, submucosa, and

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perichondrium was removed under the microscope. The underlying cricoid cartilage was exposed. The laryngofissure site was then closed. Four rats were sacrificed at each time period (0, 1, 2, 4, 7, 14, and 21 days). Their larynges were harvested, and 5-p.m thick sections were stained with hematoxylin and eosin. Additional sections underwent immunehistochemical staining for fibronectin, cellular fibronectin, procollagen, and tenascin. Optical density measurements of staining intensity were performed on computer-aided microscopy equipment. The wound sites reepithelialized in 4 days by migration and proliferation of basal epithelial cells. Epithelial maturation was complete by 7 days. Fibroblasts infiltrated into the submucosa by 2 days. Fibronectin staining intensity peaked between two and four and returned to normal by 21 days after injury. Procollagen appears later and peaks at 7 days. Tenascin appears between 2 and 4 days at the periphery of the injury site. The expression of these extracellular matrix proteins and cellular elements results in the scar tissue in the airway. This immunohistochemical model of wound repair will serve as a basis for our future studies on the effect of growth factors in the etiology and prevention of subgiottic stenosis. 9:00 AM

Tracheal Reconstruction: Long-term Growth Potential of Auricular Perichondrium and Utility of a Tubed Myoperiosteal Free Flap PAUL T. HOFF, MD (presenter), a n d RAMON M. ESCLAMADO, MD, Ann Arbor, Mich., and Cleveland, Ohio

Reconstruction of extensive laryngotracheal stenoses continues to pose a significant challenge. Previous work from our laboratory has shown the utility of vascularized perichondrium for airway reconstruction. Preliminary work by other investigators suggests that vascularized periosteum may also provide suitable grafting material. We propose in this study to address two different questions using separate animal models. In the first study we propose to examine long-term cartilage growth, epithelial growth, and luminal patency in our previously described rabbit model. Second, we propose to use a canine model to evaluate the efficacy of vascularized periosteum as a donor material to reconstruct a segmental tracheal defect so that this may soon be applied in the clinical setting. 9:15 AM

The Effect of Platelet Derived Growth Factor in Laryngotracheal Surgery Utilizing a Rabbit Model DAVID L. WALNER, MD (presenter), ROBIN T. COTTON, MD, DEAN M. TORIUMI, MD, a n d J. PAUL WILLGING, MD, Cincinnati, Ohio, a n d Chicago, III.

Growth factors are proteins that help regulate the inflammatory response and wound healing in tissues. Multiple growth factors including platelet-derived growth factor (PDGF) have been identified as important cell mediators