Ya Zhen Wu

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.

The Effect of Denervation and Reinnervation on PCA Cell Cycle Gene Expression

Problem: While it is known that denervation of skeletal muscle can induce atrophy, the effect of denervation on muscle cell cycle genes is unclear. Further, while attempted reinnervation of laryngeal muscle has thus far been unsuccessful in restoring function, cell cycle gene expression might be utilized to predict reinnervation success. The objective of this study was to examine the effects of denervation and reinnervation of the posterior cricoarytenoid (PCA) muscle on key genes involved in the cell cycle. Methods: Female Sprague-Dawley rats were assigned to 1 of 3 groups: control (CON, n = 24); denervation (DEN, n = 24); or reinnervation (REIN, n = 24). DEN and REIN animals had their left recurrent laryngeal nerves transected and ligated at the fourth tracheal ring while REIN animals subsequently had their nerves repaired using 10–0 suture. Animals were then euthanized after 7, 14, and 30 days, and mRNA expression examined using RT-PCR. If significant group differences were seen using one-way ANOVA (P < 0.05), Tukey post-hoc tests were employed. Results: Expression of p21, p53, GADD45 alpha, GADD45 beta, GADD45 gamma, and cyclin D1 genes were compared. At all time points, cyclin D1 and p53 gene expressions were unchanged with DEN or REIN. However, DEN muscles were found to express significantly higher levels of p21, GADD45 alpha, and GADD45 beta than CON at all time points. Conclusion: Interestingly, while REIN p21, GADD45 alpha, and GADD45 beta expressions were found to be elevated at 7 and 14 days, GADD45 alpha and GADD45 beta levels at 30 days were not significantly different from CON, threreby suggesting a recovery of REIN muscle gene expression at later time points. Significance: Paradoxically, while the restoration of some cell cycle genes to normal levels in REIN muscles might be seen as an indication of successful reinnervation of the PCA, no significant functional recovery was associated. Support: None reported.

Thyroid hormone level and the Cn-NFAT pathway in laryngeal muscle

but resistance showed no changes. Conclusion: In the Reinke’s edema group, the voice intensity was controlled by laryngeal adjustment, but a greater expiratory effort was needed because of a greater increase in glottal resistance. In the RNP group, aerodynamic expiratory power was extremely high, and airway resistance did not increase even with a high-intensity voice. Intensity control by expiratory effort was considered to be unavoidable in this group because of limited laryngeal adjustment. Significance: The results suggest that if there is sufficient ability for laryngeal adjustment, the vocal intensity is controlled first by laryngeal adjustment and by expiratory adjustment in response to increases in glottal resistance but that controlling intensity is considered to be by expiratory effort when laryngeal adjustment ability is poor. Support: None reported.

Does MyoD control type IIB MHC expression in denervated laryngeal muscle

Problem: The myogenic helix-loop-helix transcription protein MyoD is thought to regulate Type IIB myosin heavy chain (MHC) gene expression in response to alterations in skeletal muscle. The purpose of this study was to test whether such a regulation also exists within muscles of larynx. Larynges were denervated, and MyoD and Type IIB mRNA levels in posterior cricoarytenoid (PCA) and thyroarytenoid (TA) muscles were analyzed. Methods: Female rats were randomly assigned either to control (CON, n 7) or denervation (DEN, n 7) groups. DEN animals had their left recurrent laryngeal nerves surgically transsected at the level of the fourth tracheal ring. After 30 days, the left PCA and TA muscles were then excised from each animal and frozen. mRNA transcripts were isolated, and reverse transcription was performed on 1 g of purified mRNA. RT-PCR products were probed with primer sets for MHC (embryonic, neonatal, Type I, Type IIA, Type IIX, Type IIB) and for MyoD using rat sequence data. PCR products were visualized using Syber Green–stained gels scanned and analyzed using ImageQuant software. Means for each muscle were used for t tests (P 0.05). Results: DEN TAs were found to express significantly more Type IIX MHC mRNA than CON TAs, while DEN PCAs were found to express significantly more embryonic, neonatal, and Type IIX MHC than CON. DEN PCAs were found to express significantly less Type IIB MHC mRNA than CON. Interestingly MyoD mRNA levels in DEN TAs were unchanged from CON, while DEN PCAs actually expressed a significantly higher MyoD mRNA level than CON. Conclusion: Denervation of laryngeal muscles resulted in a shift in myosin heavy chain (MHC) isoform expression. Yet while DEN muscles exhibited Type IIB MHC mRNA level decreases, MyoD mRNA levels were not correspondingly reduced. Significance: Alterations in laryngeal muscle MHC expression following denervation not related to MyoD expression may hold a functional significance clinically. Support: None reported.

Myosin heavy chain isoform expression in laryngeal muscle in response to thyroid hormone level

Problem: Recent experience in the field of human solid organ transplantation indicates that orthotopic transplantation is possible when major histocompatibility complex (MHC) mismatches are present. Due to a relative shortage of organ donors compared with potential organ recipients, reliance on ABO blood type matching alleviates some of the difficulties associated with organ procurement. We undertook this study to investigate the feasibility of canine orthotopic laryngeal transplantation performed solely on the basis of a blood type match between the donor and recipient animals. Methods: Canine laryngeal transplantation was performed between 2 pairs of blood type-matched adult male beagles. Selective motor reinnervation of laryngeal adductor and abductor muscles using distal branches of the recurrent laryngeal nerve and superior laryngeal nerve sensory reinnervation was performed in the transplant recipients. Subsequent histocompatibility testing revealed DLA-DRB sequences with nonidentity at 14 positions and DLA-DQB sequences with nonidentity at 13 positions, indicating an MHC mismatch. Immunosuppression was achieved using FK-506 and steroids. Results: Two canines survived for 76 and 180 days postoperatively. Recovery of laryngeal function was seen in one animal, with return of a strong bark, normal deglutition, airway protection, and good exercise tolerance. Electrophysiologic testing at 6 months demonstrated physiologic laryngeal motion of both abductors and adductors with laryngeal stimulation and tube occlusion. In the second animal, 2 episodes of acute rejection were successfully treated with steroid pulses, although this animal subsequently developed ankylosis of the cricoarytenoid joint secondary to inflammation. Conclusion: These results indicate that laryngeal transplantation in the setting of blood type match with MHC mismatch is possible using FK-506 and steroid immunosuppression. However, MHC matching is recommended, given that episodes of acute rejection appear to compromise laryngeal function. Clinical Significance: This research will help refine and improve the surgical techniques and immunosuppressive regimen that will be used in human orthotopic laryngeal transplantation. (Supported by a VA Merit Review Grant.) Poster 76

Analysis of myosin heavy chain isoforms in canine lateral cricoarytenoid muscle using a novel gel electrophoresis technique

It is well known that the larynx has three functions: phonation, lower airway protection, and respiration. Many studies have explored the mechanisms of phonatory function and lower airway protection of the larynx, whereas there have been few studies dealing with laryngeal respiratory function. Recently it was revealed that the sensory afferents from the larynx transmit to the medullary respiratory neurons and affect respiratory rhythmogenesis. Furthermore, some studies have reported an interaction between the medullary respiratory neurons and laryngeal motoneurons located in the nucleus ambiguus. From these findings it was suggested that the larynx participates in respiration actively and influences respiratory regulation. Therefore we studied the changes in the activity of the intrinsic laryngeal muscles during hypercapnia in the decerebrated cat. The electromyographic activity of the posterior cricoarytenoid and lateral cricoarytenoid muscles were recorded simultaneously with an electromyogram of the diaphragm, intratracheal pressure, and endotracheal partial concentration of 02 and CO 2. Hypercapnia was induced by inhalation of CO 2 gas, and the end-tidal CO 2 was maintained at 8% to 10%. The activity of the intrinsic laryngeal muscles during hypercapnia was analyzed in comparison with that during eucapnia. In hypercapnia, both the posterior cricoarytenoid and lateral cricoarytenoid muscles increased their electromyographic activities, and the intratracheal pressure during expiration was elevated to a higher level than that in eucapnia. These findings suggested a further widening of the glottis during inspiration to decrease inspiratory resistance and a further narrowing of the glottis during expiration to prevent alveolar collapse. Thus it may be concluded that the larynx actively participates in respiratory regulation under the control of the brain stem.