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Contact author: Lisa B. Thomas, who is now with the Department of Communication Disorders, Marshall University, 1 John Marshall Drive, Huntington, WV 25755. E-mail: thomasl{at}marshall.edu.
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Abstract |
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Method: Larynges from dystrophin-deficient mdx and normal mice were examined histologically.
Results: Results demonstrate that despite the absence of dystrophin in the mdx laryngeal muscles, membrane damage, inflammation, necrosis, and regeneration were not detected in the assays performed.
Conclusions: The authors concluded that these muscles are 1 of only a few muscle groups spared in this model of dystrophin deficiency. The muscles may count on intrinsic and adaptive protective mechanisms to cope with the absence of dystrophin. Identifying these protective mechanisms may improve DMD management. The study also highlights the unique aspects of the selected laryngeal skeletal muscles and their dissimilarity to limb skeletal muscle.
KEY WORDS: laryngeal, muscles, muscular dystrophy, thyroarytenoid, posterior cricoarytenoid
Duchenne muscular dystrophy (DMD) is the most common form of childhood muscular dystrophy, affecting approximately 1 in every 3,500 live male births. Clinical markers of the disease, including mild motor delays and weakness, generally appear by 2 years of age. Notable reductions in motor function and muscle size are present by 6–11 years. The condition generally proves fatal by the end of the 3rd decade of life (Menache & Darras, 2001).
DMD is just one form of a broad array of muscular dystrophies, each with a unique set of presenting features. Although deficits in speech production, expressive language skills, and cognition have been documented in other forms of muscular dystrophy (Kaplan, Osborne, & Elias, 1986; Lacomis, Kupsky, Kuban, & Specht, 1991; Menache & Darras, 2001; Meyerson, Lewis, & Ill, 1984; Mullendore & Stoudt, 1961; Sanders & Perlstein, 1965; Speer et al., 1995; Young & Durant-Jones, 1997), the influence of the Duchenne form of the disease on speech, and particularly voice production, has received little attention in the literature. Only one article investigating the influence of DMD on speech production could be identified in the literature. Mullendore and Stoudt's (1961) article reported moderate to severe articulation errors and the presence of a "dystrophic voice" (p. 256) in children with DMD; however, the exact nature of the voice concerns present in these individuals was not defined.
One mechanism whereby the disease may exert influence upon voice production is via its influence on the diaphragm, the major muscle of respiration. Over time, the disease causes marked changes in the structure and function of the diaphragm (Beck et al., 2006; Stedman et al., 1991). As the diaphragm and its associated muscles of respiration drive the phonatory system, limitations in these muscles can be expected to bring about alterations in voice production. Yet, outside of this indirect respiratory influence on phonation, the impact of DMD on laryngeal aspects of voicing remains to be examined.
The disease is characterized by the loss of dystrophin, a cytoskeletal protein responsible for stabilizing the sarcolemma or plasma membrane of skeletal muscle (see Figure 1). In the absence of dystrophin, the sarcolemma becomes vulnerable to the mechanical forces applied by muscle contraction; focal sarcolemmal tearing often results (Lapidos, Kakkar, & McNally, 2004). The loss of sarcolemmal integrity permits the influx of extracellular calcium (Ca2+) into the muscle fiber and the subsequent activation of protein-destroying enzymes. Over time, the destruction of proteins leads to muscle fiber necrosis (Menache & Darras, 2001).
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The intrinsic muscles of the larynx are skeletal muscles that play a significant role in breathing, swallowing, and voicing. As with the spared extraocular muscles noted above, the laryngeal muscles stand apart from typical limb skeletal muscle in terms of their muscle fiber size, myosin heavy chain isoforms, innervation patterns, regenerative capacity, mitochondrial content, and response to disease (Bendiksen, Dahl, & Teig, 1981; Goding, Al-Sharif, & McLoon, 2005; Konig & von Leden, 1961; Lucas, Rughani, & Hoh, 1995; Merati et al., 1996; Perie, Agbulut, St. Guily, & Butler-Browne, 2000; Perie, St. Guily, Callard, & Sebille, 1997; Rossi & Cortesina, 1965; Sciote, Morris, Brandon, Horton, & Rosen, 2002; Shinners, Goding, & McLoon, 2006; Shiotani & Flint, 1998; Shiotani, Westra, & Flint, 1999). In brief, the above studies show that these specialized muscles display faster peak contraction rates, higher fatigue resistance profiles, smaller motor units, more specialized patterns of remodeling, and stronger resistance to certain neuromuscular diseases than their classic limb muscle counterparts. Among the laryngeal muscles, the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles have been the most widely studied. Given the "special" nature of the laryngeal muscles and their documented similarity to the DMD-spared extraocular muscles (Goding et al., 2005), the response of the laryngeal musculature to DMD is of particular interest.
The impact of dystrophin deficiency on the laryngeal muscles was recently considered by Marques, Ferretti, Vomero, Minatel, and Neto (2007). Using the dystrophin-deficient mdx mouse model of DMD (Bulfield, Siller, Wight, & Moore, 1984; Hoffman, Brown, & Kunkel, 1987), the authors examined the effects of the disease on the lateral cricoarytenoid (LCA), PCA, lateral thyroarytenoid (LTA), medial thyroarytenoid (MTA), and cricothyroid (CT) muscles in adult (4 months of age) and aged (18 months of age) animals. Results demonstrate that despite the absence of dystrophin in these muscles, there was no histological evidence of the disease process in the LCAs, PCAs, LTAs, and MTAs. Interestingly, the CT muscle showed mild evidence of pathology. Levels of pathological markers in the CT, although notable for their deviation from the other intrinsic muscles, did not approach the levels of pathology observed in the tibialis anterior: a stereotypically affected limb muscle examined in the study. The authors concluded that, with the exception of the CT, the intrinsic laryngeal muscles are protected from the effects of dystrophin deficiency, but potential sparing mechanisms were not studied.
The purposes of the current investigation were to (a) examine the effects of dystrophin deficiency on the PCA and TA muscles of the mdx mouse and (b) initiate the search for a cause of laryngeal muscle sparing. The PCA and TA muscles were selected for study for two primary reasons. First, the TA and PCA muscles offer vital contributions to laryngeal function. Knowledge of their involvement or sparing in DMD will offer a significant clinical contribution and enhance the field's understanding of potential speech and/or swallowing concerns in patients with DMD. Second, a vast body of literature demonstrates the special features of these two muscles and their deviation of classic limb skeletal muscle (Bendiksen et al., 1981; Goding et al., 2005; Konig & von Leden, 1961; Lucas et al., 1995; Merati et al., 1996; Perie et al., 1997, 2000; Rossi & Cortesina, 1965; Sciote et al., 2002; Shinners et al., 2006; Shiotani & Flint, 1998; Shiotani et al., 1999). Knowledge of their ability to function amid the loss of typical scaffolding would contribute to this growing body of literature and offer insight into the mechanical properties of the muscles. We hypothesized that the PCA and TA muscles would be spared from the pathological effects of dystrophin deficiency and that constitutive properties of the muscles, not adaptive mechanisms, would explain the preferential sparing.
Materials and Method
Animals
Use of experimental animals was approved by the Institutional Animal Care and Use Committee at the University of Kentucky. Twelve 8-week old male C57BL (control) and 12 mdx mice were obtained from the Jackson Laboratory (Bar Harbor, ME). The mdx mouse strain is the result of spontaneous X-linked mutation of the C57BL/10ScSn strain (Bulfield et al., 1984), resulting in the impaired expression of full-length dystrophin in skeletal muscle (Hoffman et al., 1987). The strain is considered to be a standard animal model for the study of human DMD (Gillis, 1999). The wild type C57BL/10ScSn was used as the control.
The mice were euthanized by CO2 asphyxia. Whole larynges and orbits as well as the diaphragm and gastrocnemius/soleus muscles (typical limb skeletal muscles)1 were quickly dissected, embedded in an optimal cutting temperature medium, and frozen in 2-methylbutane cooled to its freezing temperature with liquid nitrogen. The extraocular muscles (included in the orbit) were selected for study because of their well-documented status as a spared muscle group and their structural and functional similarity to laryngeal muscle. The diaphragm and gastrocnemius/soleus are recognized as three muscles displaying the classic pathological cascade of DMD. The inclusion of these three muscles permitted the comparison of the laryngeal muscles with stereotypically affected muscle groups.
Histology and Immunocytochemistry
Eight mdx and 8 control mice were used for histological and immunocytochemical investigations. Serial 10-µm thick coronal and transverse sections of whole larynges were collected to permit cross-sectional examination of the TA (encompassing vocalis and muscularis portions) and PCA, respectively. Similarly, serial 10-µm thick cross-sections of whole orbits were obtained to reveal the superior, inferior, lateral, and medial rectus muscles, and the superior oblique muscles. Finally, 10-µm thick cross-sections of whole gastrocnemius/soleus and diaphragm muscles were collected. For each histological or immunocytochemical assay, slides from each of the above muscle groups were processed concurrently to allow for the comparison across muscles.
Histology. For overall morphology and central nuclei counts, 15 sections of each muscle (control and mdx TA, PCA, extraocular, and gastrocnemius/soleus) were randomly selected and stained with hematoxylin and eosin (Sheehan & Hrapchack, 1980). After staining, slides were dehydrated in an ethanol series, cleared with xylene, mounted in Permount, and viewed with a Nikon E600 microscope (Nikon Inc., Melville, NY). Images were captured with a Spot RT digital camera (Diagnostic Instruments Inc., Sterling Heights, MI) and a PowerMac G4 computer (Apple Computer Inc., Cupertino, CA) equipped with Spot RT software, Version 4.0 (Diagnostic Instruments Inc.). To determine the percentage of nuclei in the central position, a marker of fiber regeneration, the total number of muscle fibers and the number of fibers with centrally positioned nuclei were counted in control and mdx TA (full muscle, all fields), PCA (full muscle, all fields), extraocular (full muscle, all fields) and gastrocnemius/soleus muscles (three to five random fields per muscle). The quantitative analysis was performed by trained personnel who were blinded to the experimental conditions. Training was provided by a muscle physiologist (Francisco H. Andrade) with 18 years of experience. A one-way analysis of variance with Games–Howell post hoc testing was applied to the data to determine the significance of findings.
Immunocytochemistry. The dystrophin–glycoprotein complex (DGC; dystrophin, dystroglycans, sarcoglycans, syntrophins, dystrobrevin, and laminin-2) links the muscle's cytoskeleton to the extracellular matrix and, thereby, stabilizes the sarcolemma during muscle contraction (see Figure 1). Dystrophin is recognized as the pivotal protein within the complex (Cohn & Campbell, 2000; Lapidos et al., 2004; Rando, 2001). The absence of dystrophin disrupts the integrity of the entire DGC; the link between cytoskeletal and extracellular structures is lost. To examine the presence or absence of various components of the DGC and of the dystrophin homolog, utrophin, we used monoclonal antibodies specific for dystrophin (NCL-DYS2), β-dystroglycan (NCL-b-DG), and utrophin (NCL-DRP2) purchased from Novocastra Laboratories (Burlingame, CA). Briefly, 10-µm thick frozen sections of control and mdx muscles were fixed with 2% paraformaldehyde, blocked with phosphate-buffered saline (PBS) with 0.1% bovine serum albumin, and incubated with primary antibody in a humid chamber at 4 °C overnight. After washing with PBS, immunoreactivity was visualized by incubation for 1 hr in Alexa-Fluor 488-conjugated secondary antibody (Molecular Probes Inc., Eugene, OR). Sections were rinsed in PBS, mounted with Immu-Mount (Shandon Lipshaw, Pittsburgh, PA), and visualized by fluorescence microscopy.
Sarcolemmal Integrity
Live cells are capable of controlling the uptake and distribution of injected dyes (Horobin, 1988). Consequently a vital dye can be injected into a living animal and its distribution can be assessed to determine the integrity of cell membranes. In this study, the vital dye Evans blue (Sigma Chemical Corp., St. Louis, MO; 50 mg/kg body weight, intraperitoneal) was injected into 4 mdx and 4 control mice to assess sarcolemmal integrity (Matsuda, Nishikawa, & Tanaka, 1995; Straub, Rafael, Chamberlain, & Campbell, 1997). Mice were euthanized approximately 18 hr after injection, and the muscles were quickly dissected, embedded, and frozen as described above. Unfixed muscle sections were mounted with Immu-Mount and examined by fluorescence microscopy to determine the extent of intracellular dye incorporation, a sign of sarcolemmal damage.
Results
Immunocytochemistry was used to determine the presence or absence of dystrophin and to examine the integrity of the DGC in control and mdx muscles. As anticipated, the dystrophin protein was identified in the laryngeal muscles of control mice but was absent in the corresponding muscles of mdx mice (see Figure 2). Further, β-dystroglycan, an associated transmembrane protein of the DGC responsible for linking the extracellular matrix to the cytoskeleton, was identified along the perimeter of the cell membrane in control muscles; it was not expressed in the mdx model. These results confirm the absence of the dystrophin protein and the disruption of the DGC in mdx laryngeal muscles.
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Comparison of the current study with the recent study by Marques et al. (2007) offers valuable information related to the effect of the disease on the larynx. Both studies found no evidence of sarcolemmal disruption in mdx TA and PCA, as shown by a clear resistance to Evans blue penetration, and no evidence of muscle fiber degeneration (i.e., fibrosis, necrosis, inflammation) or regeneration (i.e., central nucleation). Marques et al. found similar results in the LCA muscle. Interestingly, however, they did identify moderate myopathic changes (i.e., elevated levels of central nucleation) in the CT. Although the pathological findings in the CT were clearly not of the same magnitude as those of the limb muscle examined in the study, they did point to a potentially greater susceptibility to the effects of dystrophin deficiency in this muscle. Marques et al.'s work joins other recent studies showing the CT muscle to be more similar to limb skeletal muscle than other craniofacial muscles (Hyodo, Kawakita, & Desaki, 2001; Rhee, Lucas, & Hoh, 2004).
Some differences between the current study and the original work by Marques et al. (2007) are of note. First, the two studies examined mice at different ages. The current study used young adult (8-week-old) mdx and control mice. In the mdx model, the dystrophic phenotype is fully expressed in the majority of skeletal muscles by this point in the lifespan (Porter et al., 2002). We found no disease effects in the laryngeal muscles in the 8-week-old animals; however, by that same time disease effects were clearly demonstrated in diaphragm and limb muscle. Marques et al. examined older mice: adult (4 months) and aged (18 months). They found no changes in central nucleation or morphology in spared muscles throughout the lifespan. These findings suggest that the sparing identified in this study was not a factor of lack of maturation or lack of disease progression but was, instead, a true marker of disease resistance.
The current study extends the work of Marques et al. (2007) by considering the potential role of utrophin compensation in laryngeal muscle sparing. Utrophin is a protein found near the neuromuscular and myotendinous junctions of mature muscle fibers and the sarcolemma of regenerating and developing fibers that shares many structural similarities with dystrophin (Khurana et al., 1991; Rando, 2001). As with its sister protein dystrophin, utrophin binds the muscle fiber's F-actin filament to the sarcolemma's β-dystroglycan and provides structural support to the muscle. Because of its similarity to dystrophin, some have implicated utrophin as a compensatory mechanism in DMD. Studies examining the potential role of utrophin in dystrophin deficiency have yielded conflicting results (Dowling et al., 2002; Kleopa, Drousiotou, Mavrikiou, Ormiston, & Kyriakides, 2006; Mizuno et al., 1993; Porter et al., 1998; Shim & Kim, 2003; Tinsley et al., 1998). Results of the investigation do not, however, support the utrophin compensation theory as a mechanism of sparing; mdx PCA and TA muscles showed no evidence of widespread utrophin expression near the sarcolemmal boundary, suggesting that utrophin is not acting as a dystrophin substitute in these muscles.
Study Limitations
Several limitations of the current study are of note. First, the study examined mice at only the 8-week point in the lifespan. Use of an alternative time course method, examining mice from 8 weeks of age through 2 years of age (death), would have permitted the determination of the laryngeal muscles' sparing over the disease's full course. Similar time course studies in extraocular muscles have demonstrated sparing throughout the course of the lifespan in the primary extraocular muscles and a mild, delayed response to dystrophin deficiency in accessory ocular muscles (Porter et al., 1998). Further, the recent study of laryngeal muscles by Marques et al. (2007) examined the mdx mouse at two points in the lifespan: 4 months (adult) and 18 months (aged). Muscles identified as spared in their study (LCA, PCA, LTA, and MTA) remained resistant to the disease throughout the life course. The single laryngeal muscle showing a mild response to the disease—the CT—did evidence an increase in central nucleation with advancing age. Although the above studies on extraocular and laryngeal muscles point to lifelong sparing in muscles showing sparing at the 8-week point, documentation of sparing in late life would be appropriate.
The current study examined a single theory of muscle sparing in DMD: utrophin replacement. A number of other means of sparing have been proposed that were not investigated in this initial study. Some have considered the compensatory overexpression of other associated proteins (e.g., the integrins), the mechanical advantage offered by a smaller fiber size, the presence of a superior mechanism of calcium ion (Ca2+) handling, and/or the presence of advanced fiber regeneration processes (Andrade et al., 2000; Fischer et al., 2005; Karpati & Carpenter, 1986; Karpati, Carpenter, & Prescott, 1988; Khurana et al., 1995; McLoon, Rowe, Wirtschafter, & McCormick, 2004; Porter et al., 2003). Future work with the laryngeal muscles may consider other potential mechanisms of laryngeal muscles sparing in response to the disease process.
Finally, a double knock-out mouse, lacking both dystrophin and utrophin, is available (Porter et al., 1998). Studies in extraocular muscles have shown a loss of muscle sparing in mice lacking both proteins (Porter et al., 1998). The inclusion of the utrophin–dystrophin deficient mouse as an additional model in the current study would have permitted an additional level of investigating the utrophin replacement theory.
Implications
Laryngeal biophysiology. The investigation supported previous research suggesting that the laryngeal muscles are not "typical" skeletal muscles (Bendiksen et al., 1981; Goding et al., 2005; Konig & von Leden, 1961; Lucas et al., 1995; McMullen & Andrade, 2006; Merati et al., 1996; Perie et al., 1997, 2000; Rossi & Cortesina, 1965; Sciote et al., 2002; Shinners et al., 2006; Shiotani & Flint, 1998; Shiotani et al., 1999). Rather, laryngeal muscles appear to be among only a few specialized muscle groups that make notable departures from typical skeletal muscle structure and function. Differences in contractile protein profiles, mitochondrial density, and innervation patterns have been documented in these specialized muscles, as have variations in response to neuromuscular disease. Thus, the laryngeal muscles appear to express a unique phenotype that may play a role in protecting them from disease.
Clinical and translational implications. Studies regarding diseases and disorders of the human laryngeal muscles are often based on observations of visual and auditory perceptual voice characteristics and indirect acoustic and aerodynamic measures of vocal function. Although these functional observations are important to the overall understanding of voice production, they often do not provide additional information about the underlying biology and physiology of the voice producing mechanism. The results of this study provide new information about the biophysiology of two of the laryngeal muscles. First and foremost, these laryngeal muscles retain normal structure and function in the absence of a vital cytoskeletal protein, a finding that suggests the presence of refined adaptive abilities, specialized constitutive features, and/or unique biomechanics of the laryngeal muscles. Second, utrophin does not replace dystrophin in the dystrophin-deficient laryngeal muscles, suggesting that other mechanisms of sparing are at play within this muscle group. Finally, the preferential sparing of the TA and PCA muscles in this study offers another layer of evidence suggesting that these muscles differ from limb skeletal muscles in the mouse. We acknowledge that this may not extrapolate to humans. However, if future results demonstrate human consistency in these findings, it would suggest significant implications for voice therapy. For example, several voice therapy approaches suggested in the literature (Deem & Miller, 2000; Froeschels, 1944; Froeschels, Kastein, & Weiss, 1955; Hicks & Bless, 2000; Stemple, 1993; Stemple, Lee, D'Amico, & Pickup, 1994) are based on principles of exercise physiology in classic skeletal muscle. Evidence from this study and many others strongly suggests that the laryngeal muscles differ from classic limb skeletal muscle. As a result, the appropriateness of freely generalizing the limb muscle strengthening literature to the laryngeal musculature may be in question. Further research to differentiate the biophysiology of the various intrinsic laryngeal muscles and their response to activity may help shed light on the actual processes of improvement with therapy or lead to new, more effective voice therapies.
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Footnotes |
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Received November 28, 2006
Accepted August 29, 2007
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