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Journal of Histochemistry and Cytochemistry
Volume 52 (5): 581-590, 2004
Copyright ©The Histochemical Society, Inc.

Fiber Types in Rat Laryngeal Muscles and Their Transformations After Denervation and Reinnervation

Hannah S. Rhee, Christine A. Lucas and Joseph F. Y. Hoh

Department of Physiology and Institute for Biomedical Research, School of Medical Sciences, Faculty of Medicine, University of Sydney, New South Wales, Australia

Correspondence to: Dr. J. F. Y. Hoh, Dept. of Physiology, Bldg F13, University of Sydney, Sydney, NSW 2006, Australia. E-mail: joeh{at}physiol.usyd.edu.au


   Summary
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 Summary
Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
The intrinsic laryngeal muscles cricothyroid (CT) and thyroarythenoid (TA) differ in myosin expression. CT expresses limb myosin heavy chains (MyHCs) and TA expresses an MyHC found in extraocular (EO) muscles, in addition to limb isoforms. We used immunohistochemical (IHC) analyses with highly specific monoclonal antibodies (MAbs) against various MyHCs to study muscle fiber types in rat CT and TA and to investigate whether nerves to laryngeal muscles control MyHC expression. CT was found to have the full complement of limb fiber types. TA had three major fiber types: 2b/eo, co-expressing 2B and EO MyHCs, 2x/2b, co-expressing 2X and 2B MyHCs, and 2x, expressing 2X MyHC. Type 2a and slow fibers were absent. TA consisted of two divisions: the external division (TA-X), which is homogeneously 2b/eo, and the vocalis division (TA-V), composed principally of 2x and 2b/eo fibers with a minority of 2x/2b fibers. TA-V had two compartments that differ in fiber type composition. At 4 weeks after cutting and re-uniting the recurrent laryngeal nerve (RLN), many 2b/eo fibers in the TA-X began to express 2X MyHC, while EO and 2B MyHC expression in these fibers progressively declined. By 12 weeks, up to 16.5% of fibers in the TA-X were of type 2x. These findings suggest that nerve fibers originally innervating 2x fibers in TA-V and other muscles have randomly cross-innervated 2b/eo fibers in the TA-X and converted them into 2x fibers. We conclude that CT and TA are distinct muscle allotypes and that laryngeal muscle fibers are subject to neural regulation.

(J Histochem Cytochem 52:581–590, 2004)

Key Words: muscle fiber • myosin heavy chain • neuronal modulation


   Introduction
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 Summary
 Introduction
Materials and Methods
 Results
 Discussion
 Literature Cited
 
SKELETAL MUSCLE FIBER TYPES have been most extensively studied in limb and trunk muscles that play a role in locomotion and maintenance of posture. The phenotypic characteristics of these fiber types in these muscles have been extensively reviewed (Pette and Staron 1990Go). They can be classified into two broad phenotypes, fast and slow. Slow muscle fibers express slow/ß-cardiac MyHC. The fast fibers can be further divided phenotypically into three fundamental subtypes called 2a, 2x, and 2b, which respectively express 2A, 2X, and 2B MyHC (Lucas et al. 2000Go), generating a range of muscle speeds and power outputs (2a<2x<2b) (Bottinelli et al. 1991Go).

Craniofacial muscles are highly specialized in function, possessing different repertoires for expressing MyHCs, including isoforms not found in normal adult limb muscles. For jaw closers this repertoire includes "superfast" or masticatory MyHC (Qin et al. 2002Go), {alpha}-cardiac MyHC (Hoh et al. 2000Go), and developmental MyHCs (for review see Hoh 2002Go). Laryngeal and EO muscles of rat and rabbit express EO MyHC (Lucas et al. 1995Go; DelGaudio et al. 1995Go; Briggs and Schachat 2000Go), which is associated with high speed of contraction (Close and Luff 1974Go; Asmussen et al. 1994Go) and rapid cross-bridge kinetics (Li et al. 2000Go). EO muscles differ from laryngeal muscles in having the capacity to express developmental, {alpha}-cardiac, and slow-tonic MyHCs. The unique pattern of MyHC gene expression in EO muscles forms part of a broader difference in gene expression pattern (Porter et al. 2001Go) associated with the muscle allotype (Hoh and Hughes 1988Go; Lucas et al. 1995Go), a property related to the developmental origin of the muscle.

There are five intrinsic laryngeal muscles: the cricothyroid (CT), thyroarytenoid (TA), lateral cricoarytenoid (LCA), interarytenoid (IA), and posterior cricoarytenoid (PCA). The CT is innervated by the superior laryngeal nerve and all the others are innervated by the recurrent laryngeal nerve (RLN). Intrinsic laryngeal muscles are involved in a number of complex and important functions: airway protection, respiration and phonation. The isometric twitch contraction times of the TA, PCA, and other muscles that control the size of the glottis are in the range for the very fast EO muscles (Martensson and Skoglund 1964Go; Hall–Craggs 1968Go; Hinrichsen and Dulhunty 1982Go). In contrast, contraction time of the CT, which tenses the vocal fold, is two- to fourfold longer, close to values for fast limb muscles of the same species. Earlier work on the rabbit showed that the CT is limb-like in its myosin composition, whereas the TA is EO-like in expressing EO MyHC (Lucas et al. 1995Go). This difference has been confirmed in the rat (Shiotani and Flint 1998bGo). Methods used for the classification of limb muscle fiber types are not applicable to laryngeal muscle fibers (Claassen and Werner 1992Go; DelGaudio et al. 1995Go), and currently a definitive histochemical classification of laryngeal muscle fibers does not exist. Here we used a battery of highly specific monoclonal antibodies (MAbs) to the full range of MyHC isoforms found in laryngeal muscles to study the distribution of MyHCs in rat CT and TA muscle fibers. We established a MyHC-based immunohistochemical (IHC) system for classifying laryngeal muscle fibers.

Since the classical nerve cross-union experiments on limb muscles of Buller et al. (1960)Go, the notion that muscle fibers of the limb allotypes are under the control of the electrical impulse pattern mediated by its nerve supply is very well established (for review see Pette and Staron 1997Go). Whether muscle fibers of craniofacial allotypes are also under neural regulation has received considerably less attention. Transplantation (Hoh and Hughes 1988Go) and electrical stimulation (Hoh et al. 1991Go) studies on cat jaw muscles have shown that the type of innervation or impulse pattern can influence MyHC expression in jaw muscle cells, but only within the phenotypic options defined by the jaw allotype. Studies on laryngeal muscles after denervation (Shiotani and Flint 1998aGo) and reinnervation (Shiotani et al. 2001Go) have shown some changes in the profile of MyHC of whole muscles, but fiber type transformation at the cellular level has not been shown. Rat TA muscle has a vocalis division (TA-V) subjacent to the vocal ligament, and a larger external division (TA-X). Our IHC analysis revealed that all fibers in the rat TA-X co-express 2B and EO MyHCs, whereas the majority of fibers in the TA-V express 2X MyHC. We then made use of this favorable distribution of MyHCs to study neural regulation of TA fibers by transecting and reuniting the RLN, a procedure known to result in random re-innervation of laryngeal muscle fibers (Flint et al. 1991Go). We anticipate that muscle fibers co-expressing 2B/EO MyHCs will be cross-innervated by nerve fibers originally innervating muscle fibers expressing 2X MyHC. The results clearly demonstrated 2X MyHC gene expression in TA-X fibers originally expressing 2B/EO MyHCs, implying that laryngeal muscle fibers are subject to neural regulation.


   Materials and Methods
Top
 Summary
 Introduction
 Materials and Methods
Results
 Discussion
 Literature Cited
 
Surgery and Tissue Preparation
For studies on normal laryngeal muscle fibers, the larynges were removed from four 12-week old female Sprague–Dawley rats sacrificed with an anesthetic overdose. Left RLN transection was performed on 16 10-week old female Sprague–Dawley rats. The animals were anesthetized by IM injection of ketamine HCl 35 mg/kg and xylazine HCl 5 mg/kg. Under a standard dissecting microscope, the larynx was exposed via a midline incision. The left RLN was sectioned close to the larynx and then immediately rejoined end to end with silk sutures. The right RLN was left intact primarily to minimize laryngeal dysfunction in conformity with ethical requirements, but it also served as a control. Animals were allowed to survive for 2, 4, 6, or 12 weeks. At each time interval, four animals were sacrificed by an anesthetic overdose, the RLNs were visually checked for successful reunion, and whole larynges were removed. Excised larynges were mounted on cork with Tissue-Tek (Miles Scientific; Elkhart, IN), frozen in isopentane cooled in liquid nitrogen, then stored at –80C until use. All surgery and handling were performed in accordance with the guidelines of the Animal Research Act and the 1997 NHMRC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the Animal Care and Ethics Committee of the University of Sydney.

Immunohistochemistry
Serial sections of CT and TA muscles from both sides were cut at 10 µm in a cryostat at –20C. Whole larynges were cut at right angles to the midline, so that reinnervated and control TA muscles appeared in the same section and were treated the same way. Sections from the mid-regions of these muscles that contain the full complement of fibers were stained by IHC. Indirect IHC was performed as previously described (Hoh et al. 1988Go). The primary antibodies used in this study were MAb NOQ7-5-4-D (hereafter referred to as 5-4D) specific to slow/ß-cardiac MyHC (Hoh et al. 1988Go), MAb SC-71 (Schiaffino et al. 1989Go) specific to fast 2A MyHC, MAbs 6H1 and 10F5, specific to fast 2X and 2B MyHCs, respectively (Lucas et al. 2000Go), and MAb 4A6, which is specific to EO MyHC (Lucas et al. 1995Go). The secondary antibodies used were horseradish peroxidase (HRP)-labeled rabbit anti-mouse IG antibody (DAKO; Carpinteria, CA) for MAbs 4A6 and 5-4D and HRP-labeled goat anti-mouse IgM antibody (Sigma; St Louis, MO) for MAbs 6H1 and 10F5.

Quantification of Muscle Fiber Type Distribution
The percentages of fibers expressing slow/ß-cardiac, 2A, 2X, 2B, and EO MyHCs present in the CT, TA-V, and TA-X were calculated based on counts using photomicrographs of tissue sections stained by MyHC IHC. All fibers of the TA (n=943–1102) and CT (n=611–1088) muscles visible in the photomicrographs were counted. Fibers were classified as hybrid if they were clearly stained by more than one anti-MyHC MAb in serial sections. Faintly staining fibers were ignored for the purpose of quantification. Results from four control and four experimental muscles at each time point are presented as mean percentages ± SEM. Statistical comparisons were performed using the unpaired two-tailed Student's t-test and were considered significant at p<0.05.


   Results
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 Summary
 Introduction
 Materials and Methods
 Results
Discussion
 Literature Cited
 
Fiber Types in Normal TA and CT Muscles
Figure 1 shows cross-sections of the CT stained with MAbs to slow (Figure 1A), 2A (Figure 1B), 2X (Figure 1C), 2B (Figure 1D), and EO (Figure 1E) MyHCs. With the exception of anti-EO MyHC, all antibodies stained fibers in this tissue. This is similar to the staining characteristics of limb muscle fibers, which express the slow and three isoforms of fast MyHCs but not the EO MyHC. The percentages of various fiber types based on IHC of four muscles with these MAbs are presented in Table 1. The CT is rich in 2X (61.2%) and slow (19.0%) fibers but relatively poor in 2A (12.5%) and 2B (4.9%) fibers. Hybrid fibers co-expressing two or more MyHCs are present but are less than 3%, prominent among these being fibers co-expressing 2X and 2B MyHCs (2x/2b fibers, 2.1%).



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  		Figure 1

Distribution of MyHC expression in the cricothyroid (CT). Photomicrographs of immunoperoxidase-stained cryostat sections of the rat cricothyroid (CT) muscle using anti-slow MyHC MAb 5-4D (A), anti-2A MyHC MAb SC-71 (B), anti-2X MyHC MAb 6H1 (C), anti-2B MyHC MAb 10F5 (D), and anti-EO MyHC MAb 4A6 (E). (A,C,E) Serial sections in a region poor in 2A and 2B expression. (B,D) Serial sections from a different region of the muscle. Bar = 100 µm.

 

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  		Table 1

Distribution of fiber types in normal rat laryngeal musclesa

 
Figure 2 shows low-power views of cross-sections of the rat TA stained with the same battery of MAbs. There are two major subdivisions of the TA, the internal or vocalis division (TA-V) lying subjacent to the vocal ligament (upper part of each panel in Figure 2), and the external (TA-X) divisions forming the major portion of the muscle (lower part of each panel in Figure 2). TA-V in the rat consists of two clearly distinct rostral (upper left corner of each panel) and caudal compartments. The MAb 5-4D that stains limb slow fibers failed to react with any of the fibers present in the TA (Figure 2A). MAb SC-71, which stains 2a fibers, also failed to stain fibers in TA except for scattered fibers at an intensity level barely above background (Figure 2B). Interestingly, MAb 6H1, which stains fast 2x fibers, strongly stained a population of fibers restricted to the TA-V (Figure 2C), although faint staining of scattered TA-X fibers was also seen. In contrast, the MAbs 10F5 and 4A6, which react with fast 2B and EO MyHC, respectively, revealed a major new fiber type characterized by the co-expression of these two MyHCs (Figures 2D and 2E, respectively). The whole of the TA-X division is composed of fibers strongly co-expressing these MyHCs (2b/eo fibers). No fibers stained strongly for 2A or 2X MyHC. Therefore, except for possible expression of trace amounts of other MyHCs, TA-X is homogeneously 2b/eo. These fibers are also present, but at lower abundance, in the TA-V.



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  		Figure 2

Distribution of MyHC expression in the thyroarytenoid (TA). Low-power photomicrographs of immunoperoxidase-stained semi-serial sections of the rat thyroarytenoid (TA) muscle using anti-slow MyHC MAb 5-4D (A), anti-2A MyHC MAb SC-71 (B), anti-2X MyHC MAb 6H1 (C), anti-2B MyHC MAb 10F5 (D), and anti-EO MyHC MAb 4A6 (E). The external division (TA-X) forms the bulk of the muscle occupying the central and lower part of each panel, and the rostral and caudal compartments of the vocalis division (TA) are located in the upper left and right regions, respectively. Bar = 100 µm.

 
The percentages of various fiber types in the normal TA based on IHC with these MAbs are presented in Table 1. The rostral and caudal portions of TA-V differ in fiber type composition. The rostral portion consists of a large proportion of hybrid 2b/eo fibers (62.0%), some pure 2x fibers (27.7%), and a few hybrid 2x/2b fibers (4.1%). The caudal portion consists of a large portion of pure 2x fibers (73.2%), some hybrid 2b/eo fibers (11.9%), and a few hybrid 2x/2b fibers (7.3%). Low amounts of pure 2b and eo fibers were also found in both rostral and caudal TA-V but not in the TA-X. A few fibers (<1%) clearly expressed three MyHCs (2x/2b/eo) in TA-V but not in TA-X. The virtual homogeneity of 2b/eo fibers in rat TA-X was confirmed in all four rats studied. Such homogeneity of the 2b/eo fibers in the TA-X serves as an ideal background for the detection of changes in fiber type after experimental manipulation.

Fiber Types in Reinnervated TA Muscles
Figure 3 shows serial sections of representative TA-X muscles stained with anti-2X, anti-2B, and anti-EO MAbs at various times after RLN section and reunion. At 2 weeks there was no apparent change in pattern of staining of the TA-X with these mAbs (Figures 3A–3C). After 4 weeks, however, there was emergence of a population of fibers in the TA-X that are strongly positive for fast 2X MyHC. These fibers continued to stain for 2B and EO MyHCs (Figures 3D–3F), and are therefore examples of fibers that strongly express three MyHC isoforms significantly. By 6 weeks there was a further increase in the proportion of 2X MyHC-positive fibers. At this time, fibers staining for 2X MyHC started to show a substantial decrease in staining for 2B MyHC, whereas staining for EO MyHC remained strong (Figures 3G–3I). By 12 weeks, fibers staining for 2X MyHC were no longer positive for 2B and EO MyHC (Figures 3J–3L). IHC of the intact right TA-X in all operated animals was essentially identical to that in unoperated animals (data not shown).



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  		Figure 3

Changes in MyHC expression in the TA-X after recurrent laryngeal nerve section and reunion. Photomicrographs of immunoperoxidase-stained semi-serial sections of the external division of the thyroarytenoid (TA-X) at 2 weeks (A–C), 4 weeks (D–F), 6 weeks (G–I), and 12 weeks (J–L) after recurrent laryngeal nerve section and reunion. The sections are stained with anti-2X MyHC MAb (A,D,G,J), with anti-2B MyHC MAb (B,E,H,K), and with anti-EO MyHC MAb (C,F,I,L). Bar = 100 µm.

 
The proportions of fibers expressing 2X, 2B, and EO MyHCs in the four reinnervated TA-X muscles at each time point after nerve reanastomosis are shown in Figure 4 . This shows that between 2–4 weeks a rapid increase in fibers expressing 2X MyHC occurred, reaching 14.8% at 4 weeks, during which time fibers expressing 2B (91.8%) and EO (96.0%) MyHC were significantly reduced. By 6 weeks, 2X MyHC expression had reached a sustained average of 17% while 2B (82.8%) and EO (91.5%) MyHC expression further dropped significantly, the decrease of EO MyHC expression lagging significantly behind that of 2B MyHC. By 12 weeks a stable transformation had been reached, with 16.5% of fibers expressing 2X MyHC and a matching fall in 2B (84.0%) and EO (83.0%) MyHC expression.



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  		Figure 4

Changes in MyHC distribution in TA-X fibers after recurrent laryngeal nerve section and reunion. Histograms show the percentages (±SEM, n=4) of fibers expressing 2X (black), 2B (white), EO (striped) MyHCs in the reinnervated TA-X at 0, 2, 4, 6, or 12 weeks after reinnervation. Symbols (*p<0.05; §p<0.005; {ddagger}p< 0.001; {dagger}p<0.0005) indicate statistical significance of changes in MyHC expression compared with respective controls (t-tests). The difference between values for 2B and EO MyHC expression at 6 weeks is also significantly different (p<0.05, t-test).

 
IHC changes also occurred in the vocalis region after RLN denervation and reinnervation, but they took place against a more complex background of fiber type distributions, and were not investigated in detail except for muscles 12 weeks after operation. In the rostral TA-V, which normally has a relatively low proportion of 2x fibers (27.7%), there was a small increase (to 32.4%) in the proportion of 2x fibers accompanied by a decrease in the proportion of 2b/eo fibers. In the caudal TA-V, which normally has a high abundance of 2x fibers (73.2%), there was a small decrease (to 67.6%), with a similar corresponding increase in the proportion of 2b/eo hybrid fibers.


   Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
Classification of Rat Laryngeal Muscle Fiber Types
The present work is the first to classify muscle fiber types in the CT and TA using a panel of highly specific antibodies to all MyHCs found in laryngeal muscles. The results clearly show that the rat CT has four fundamental fiber types, slow, 2a, 2x, and 2b, identical to those in limb fast muscle (Lucas et al. 2000Go). This is consistent with the MyHC composition of whole rat CT studied by SDS-PAGE (Shiotani and Flint 1998bGo). The absence of EO MyHC expression in the CT is consistent with rabbit (Lucas et al. 1995Go), dog (Wu et al. 2000cGo), and human (Wu et al. 2000bGo) CT.

Fiber types of the other intrinsic laryngeal muscles innervated by the RLN have been studied in various species by classical myosin ATPase histochemistry. Both fast and slow fibers were found, but further classification into subtypes of fast fibers on histochemical grounds was problematic (Claassen and Werner 1992Go; DelGaudio et al. 1995Go). For example, no fibers from the rat TA could be classified according to the standard fiber type categories established for limb muscles (DelGaudio et al. 1995Go). Two recent investigations have shed much light on this problem. The first was the discovery that TA in the rabbit expresses EO MyHC in addition to limb fast MyHCs (Lucas et al. 1995Go). This finding was confirmed in the rat, in which the apparently laryngeal muscle-specific MyHC was initially called 2L MyHC (DelGaudio et al. 1995Go) but was later shown to be identical to EO MyHC (Briggs and Schachat 2000Go). The second finding that helps to explain anomalous histochemical behavior of laryngeal muscle fibers is the fact that they contain a high proportion of fibers that co-express multiple MyHCs (hybrid fibers). Wu and co-workers, using SDS-PAGE analysis of single fibers, showed that 20–40% of laryngeal fibers in dog, human, and rat co-express two or more MyHCs (Wu et al. 1998Go; Wu et al. 2000aGo–c).

Myosin-based classification of muscle fiber types has been widely accepted for limb muscles. This system has the advantage of using unique molecular markers that indicate the relative contractile property of each fiber type (Bottinelli et al. 1991Go). The present study shows that an unambiguous classification of rat TA muscle fibers based on MyHC composition that takes into account hybrid fibers is possible (Table 1). Rat TA has three predominant fiber types, 2x, 2x/2b, and 2b/eo. The most abundant fibers are the 2b/eo, followed by 2x and 2x/2b fibers. There are some pure eo and 2b fibers. Fibers clearly showing all three MyHCs (2x/2b/eo) were rare, on average less than 1%. These observations are in general agreement with the findings based on single-fiber SDS-PAGE (Wu et al. 2000aGo), except that Wu et al. reported a high abundance (about 20%) of 2x/2b/eo fibers. The data of Wu et al. can be reconciled with ours on the assumption that the 2X component in their 2x/2b/eo fibers is almost uniformly very low. They would thus have included the 2b/eo fibers that stained faintly with anti-2X MAb, thereby greatly increasing the abundance of 2x/2b/eo fibers. Because a small amount of a third MyHC is unlikely to affect fiber function significantly, ignoring the faint staining greatly simplifies classification without sacrificing functional significance.

Slow and 2a fibers are conspicuously absent in the rat TA, in agreement with Wu et al. (2000a)Go, but are present in dog (Wu et al. 2000cGo) and human TA (Wu et al. 2000bGo). There are also interesting species differences in the expression of the other MyHCs in the TA. EO MyHC is absent in dog TA (Wu et al. 2000bGo), and 2B MyHC is absent in human TA (Wu et al. 2000bGo). A myosin-based classification of laryngeal muscle fiber types is able to cope with all these species variations.

Functional Implications of the Fiber Type Distribution in Rat TA and CT Muscles
Although isotonic studies on laryngeal muscles of experimental animals have not been reported, the TA is expected to have a much higher speed of shortening than the CT in view of its MyHC composition. It is the properties of myosin in a muscle fiber that control the speed of shortening, power and economy of the fiber (Schiaffino and Reggiani 1996Go). The maximal speed of shortening (Vmax) of a fiber is directly proportional to the myosin ATPase activity of its myosin (Bárány 1967Go). Rabbit TA myosin, which contains a significant proportion of EO MyHC (Lucas et al. 1995Go), has an ATPase that is 25–55% higher than that of CT or limb fast muscle myosin (Syrovy and Gutmann 1971Go). Extraocular muscles (EOMs) also express EO MyHC, in addition to limb and other MyHCs. Mechanical analysis of rabbit EOM single fibers in which fmin, the dynamic stiffness parameter indicative of the cross-bridge cycling rate, was measured, revealed the presence of fibers faster than limb fast fibers (Li et al. 2000Go). These observations suggest that EO MyHC is associated with fast fiber mechanics. TA expresses EO MyHC and two of the fastest limb MyHC isoforms, 2B and 2X, while the CT has abundant slow MyHC and relatively little 2B MyHC. Therefore, rat TA is expected to have a much higher Vmax than rat CT. The importance of rapid glottic movements for the survival of the animal might have been responsible for the evolution of the expression of the EO MyHC in laryngeal muscles.

Our IHC analysis revealed that TA-X, rostral TA-V, and caudal TA-V contain decreasing proportions of 2b/eo fibers and are expected to be the fastest. Therefore, their relative speeds of contraction would be in the order TA-X > rostral TA-V > caudal TA-V. TA-X and TA-V are known to have distinct functions. The former, together with LCA and IA, functions in adducting the vocal folds. This action closes the glottis in opposition to the action of the PCA, which abducts the vocal folds and opens the glottis. The presence of a virtually homogeneous population of 2b/eo fibers in the TA-X ensures a high speed of vocal fold adduction, which is clearly very important as a defence against aspiration pneumonia and choking by foreign bodies. Contraction of TA-V increases the tension of the non-ligamentous portion of the vocal fold. This is thought to be important in phonation (Hirano et al. 1969Go). The functional significance of two separate compartments of the vocalis is obscure. The fiber type composition of the rostral TA-V suggests that it should be faster than the caudal TA-V and that these compartments may play different roles during vocalization.

Neural Control of Laryngeal Muscle Fiber Types
Earlier studies on rat laryngeal muscles after denervation (Shiotani and Flint 1998aGo) and reinnervation (Shiotani et al. 2001Go) have shown that innervation does influence the profile of MyHCs resolved on SDS gels. These studies do not rigorously argue in favor of neural regulation of laryngeal muscle fibers because the observed MyHC profile changes may be the result of differential changes in fiber diameters of different types of muscle fibers. The present work using IHC has shown fiber type transformation at the cellular level during the course of laryngeal muscle reinnervation, thus providing cogent evidence that laryngeal muscle fibers are subject to neural regulation.

In this study we used highly specific MAbs to detect changes in MyHC expression of TA muscle fibers at the cellular level after denervation and reinnervation. We observed that a significant proportion of the 2b/eo fibers in the TA-X underwent a progressive transformation from expressing 2B/EO MyHC to expressing 2X MyHC exclusively. At 4 weeks postoperatively, 2b/eo fibers began to co-express 2X MyHC, and in the course of subsequent weeks 2B and EO MyHCs were progressively co-repressed, 2B MyHC more rapidly than EO MyHC, so that by 12 weeks these two MyHCs were completely replaced by 2X MyHC in 16.5% of fibers. This degree of transformation agrees well with the increase in 2X MyHC observed by SDS-PAGE (Shiotani et al. 2001Go). The sequence of changes in MyHC expression can be summarized as follows:

2B/EO -> 2X/2B/EO -> 2X/EO -> 2X

Given that reinnervation of laryngeal muscles by the regenerating nerve fibers occurs randomly (Flint et al. 1991Go), some 2b/eo muscle fibers in the TA are expected to be reinnervated by nerve fibers originally innervating 2x muscle fibers in TA-V or some other laryngeal muscle. The transformation of 2b/eo fibers into 2x fibers in the reinnervated TA-X can be accounted for by the assumption that neural impulse patterns along nerve fibers innervating 2x and 2b/eo muscle fibers in the RLN are qualitatively different, each type being able to induce the expression of the appropriate MyHC in the muscle fibers they innervate. We propose that the muscle fiber type transformation observed is the consequence of the cross-innervation between two subtypes of fast muscle fibers, 2x and 2b/eo. This type of cross-innervation is different from and more refined than the classical cross-innervation, which is between nerves to whole fast and slow muscles.

To our knowledge, cross-innervation studies between subtypes of fast muscle fibers have not been described thus far. In classical experiments, subtypes of limb fast muscle fibers were reinnervated by nerve fibers to slow muscle fibers, while slow muscle fibers were reinnervated by nerve fibers to fast muscle fibers. Nerves to slow muscle fibers carry tonic, low frequency impulses that bring about fast-to-slow fiber transformation via Ca2+ activation of the phosphatase calcineurin acting on the nuclear factor NFAT (Chin et al. 1998Go). A refinement of the impulse pattern hypothesis would suggest that the three subtypes of limb fast fibers are controlled by three distinct patterns of firing in their respective nerve fibers. In vivo recording of electrical impulses of motor units from rat limb fast muscle does show a wide range of patterns of activity (Hennig and Lomo 1985Go), but the relationship between impulse pattern and fast fiber subtypes has not been defined. Furthermore, little is currently known about the molecular mechanisms involved in regulating fast fiber subtypes.

Significance of Hybrid Fibers
A striking feature of the TA is the preponderance of fibers co-expressing 2 MyHCs, particularly 2b/eo fibers. This is in agreement with the findings of SDS-PAGE analysis of single fibers in rat TA (Wu et al. 2000aGo). Hybrid fibers expressing 2B MyHCs are also found in limb muscles, but these occur generally as a relatively minor component of the total fiber population in white portions of fast muscles, although those co-expressing 2A/2X may be constitute a substantial proportion (up to 40%) of red portions of fast muscles (Lucas et al. 2000Go; Caiozzo et al. 2003Go). Fibers in normal limb muscles are known to be in a dynamic state, undergoing fiber type transitions and generating hybrid fibers in the process (Pette and Staron 1997Go; Stephenson 2001Go). The number of hybrid fibers may increase very significantly during transitional states brought about by neural and hormonal perturbations (Pette and Staron 1997Go). Our finding of transient expression of 2X MyHC in 2b/eo fibers during reinnervation supports this view.

In contrast to the low abundance of hybrid fibers in the white regions of normal limb fast muscle, we observe that virtually 100% of fibers in the normal TA-X are of this 2b/eo type and that pure eo and 2b fibers are rare in the rest of this muscle. The tight coupling of EO and 2B MyHC expression in the TA does not reflect a common regulatory pathway for these isoforms, because rat PCA expresses a disproportionately high level of 2B MyHC relative to EO MyHC (DelGaudio et al. 1995Go; Wu et al. 2000aGo), suggesting that these isoforms are regulated by separate mechanisms. Therefore, it is likely that co-expression of 2B and EO MyHCs in the normal TA results from the convergence two types of neural signals because of the complex functional demands on this muscle. There may be three types of neural signals regulating the expression of the EO, 2B, and 2X MyHCs, analogous to those controlling the limb 2b, 2x and 2a fibers. Minor populations of hybrid fibers in the TA may represent fibers in transition just as those in limb fibers.

Allotypic Differences Between CT, TA and Other Muscles
Our results demonstrate that CT and limb muscles have identical phenotypic options for MyHC expression, whereas the TA shows a great difference in phenotype compared with CT and limb muscles. The CT has the full range of slow, 2a, 2x, and 2b fibers found in fast limb muscle, suggesting that they may be allotypically the same. However, these muscles differ in developmental origin. The cricothyroid develops from the fourth branchial arch, whereas limb muscles are derived from somitic mesoderm (Sperber 1989Go). In the bat, the CT is very different from limb muscles, being highly specialized for generating ultrasonic pulses used in echolocation. It has ultrastructural features of a very fast muscle, with extremely well-developed sarcoplasmic reticulum and mitochondria (Revel 1962Go). This fact serves to caution us against assuming that CT and limb muscles are allotypically identical. The expression of a broader spectrum of muscle genes in rat CT and limb fast muscles should be compared by using sensitive methods of screening gene expression patterns such as cDNA microarrays, the usefulness of which has been demonstrated in defining the different pattern of gene expression between limb and EO muscles (Porter et al. 2001Go).

It has been proposed that the TA and CT are allotypically distinct on the basis that the former in the rabbit has the capacity to express EO MyHC (Lucas et al. 1995Go). The fact that TA and CT differ in developmental origin, being derived from the sixth and fourth branchial arches, respectively (Sperber 1989Go), lends further support to this view. An alternative explanation for the fact that CT does not express EO MyHC is the absence of an appropriate neural signal to induce it. This possibility can be tested by cross-innervating the CT with the RLN.

The major phenotypic options of the rat TA fibers are to express 2B/EO or 2X MyHCs. In contrast to the CT, there are very few pure 2b fibers and no 2a and slow fibers in the TA. Absence of pure 2b fibers in the TA has been suggested above to be due to the convergence of neural signals for EO and 2B MyHC. At present it is not clear whether the absence of 2a and slow fibers in the TA is intrinsic, i.e., part of the allotypic property, or extrinsic, i.e., the absence of the appropriate neural impulse traffic to induce these isoforms. Chronic stimulation of the RLN or cross-innervation of TA by the superior laryngeal nerve innervating the CT should resolve this question.

Although both TA and CT have 2x fibers, these 2x fibers belong to distinct muscle allotypes and are potentially different in phenotype in some respects. It would therefore be very interesting to further explore differences in gene expression pattern in these fibers more broadly using microchemical methods for detecting myofibrillar proteins and cDNA microarray analysis with identified 2x fibers from these muscles.

The co-expression of 2B and EO MyHCs in a laryngeal muscle raises the question of whether these isoforms are always co-expressed in other muscles expressing these isoforms. Rat EO muscle also expresses 2B and EO MyHCs at the whole-muscle level, but these MyHCs are not co-expressed in the same fiber (Rubinstein and Hoh 2000Go). EO MyHC is principally localized in the endplate zone of orbital singly innervated fibers that express embryonic MyHC elsewhere along the length of the fiber, whereas 2B MyHC is expressed in a subpopulation of global fibers that do not coexpress EO MyHC. Therefore, the patterns of expression, and probably the molecular signaling pathways for the induction of EO and 2B MyHCs in the two different types of craniofacial muscles in the rat, are distinct, reflecting their difference in allotype.


   Acknowledgments
 
Supported by a grant from the National Health and Medical Research Council of Australia.


   Footnotes
 
Received for publication September 15, 2003; accepted December 17, 2003


   Literature Cited
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 Literature Cited
 

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