Fiber Types in Rat Laryngeal Muscles and Their Transformations After Denervation and Reinnervation
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
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Summary |
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(J Histochem Cytochem 52:581590, 2004)
Key Words: muscle fiber myosin heavy chain neuronal modulation
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Introduction |
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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. 2002),
-cardiac MyHC (Hoh et al. 2000
), and developmental MyHCs (for review see Hoh 2002
). Laryngeal and EO muscles of rat and rabbit express EO MyHC (Lucas et al. 1995
; DelGaudio et al. 1995
; Briggs and Schachat 2000
), which is associated with high speed of contraction (Close and Luff 1974
; Asmussen et al. 1994
) and rapid cross-bridge kinetics (Li et al. 2000
). EO muscles differ from laryngeal muscles in having the capacity to express developmental,
-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. 2001
) associated with the muscle allotype (Hoh and Hughes 1988
; Lucas et al. 1995
), 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 1964; HallCraggs 1968
; Hinrichsen and Dulhunty 1982
). 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. 1995
). This difference has been confirmed in the rat (Shiotani and Flint 1998b
). Methods used for the classification of limb muscle fiber types are not applicable to laryngeal muscle fibers (Claassen and Werner 1992
; DelGaudio et al. 1995
), 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), 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 1997
). Whether muscle fibers of craniofacial allotypes are also under neural regulation has received considerably less attention. Transplantation (Hoh and Hughes 1988
) and electrical stimulation (Hoh et al. 1991
) 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 1998a
) and reinnervation (Shiotani et al. 2001
) 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. 1991
). 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.
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Materials and Methods |
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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. 1988). 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. 1988
), MAb SC-71 (Schiaffino et al. 1989
) specific to fast 2A MyHC, MAbs 6H1 and 10F5, specific to fast 2X and 2B MyHCs, respectively (Lucas et al. 2000
), and MAb 4A6, which is specific to EO MyHC (Lucas et al. 1995
). 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=9431102) and CT (n=6111088) 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.
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Results |
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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 3A3C). 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 3D3F), 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 3G3I). By 12 weeks, fibers staining for 2X MyHC were no longer positive for 2B and EO MyHC (Figures 3J3L). 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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Discussion |
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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 1992; DelGaudio et al. 1995
). 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. 1995
). 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. 1995
). This finding was confirmed in the rat, in which the apparently laryngeal muscle-specific MyHC was initially called 2L MyHC (DelGaudio et al. 1995
) but was later shown to be identical to EO MyHC (Briggs and Schachat 2000
). 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 2040% of laryngeal fibers in dog, human, and rat co-express two or more MyHCs (Wu et al. 1998
; Wu et al. 2000a
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. 1991). 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. 2000a
), 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), but are present in dog (Wu et al. 2000c
) and human TA (Wu et al. 2000b
). 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. 2000b
), and 2B MyHC is absent in human TA (Wu et al. 2000b
). 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 1996). The maximal speed of shortening (Vmax) of a fiber is directly proportional to the myosin ATPase activity of its myosin (Bárány 1967
). Rabbit TA myosin, which contains a significant proportion of EO MyHC (Lucas et al. 1995
), has an ATPase that is 2555% higher than that of CT or limb fast muscle myosin (Syrovy and Gutmann 1971
). 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. 2000
). 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. 1969). 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 1998a) and reinnervation (Shiotani et al. 2001
) 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. 2001). 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. 1991), 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. 1998). 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 1985
), 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. 2000a). 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. 2000
; Caiozzo et al. 2003
). 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 1997
; Stephenson 2001
). The number of hybrid fibers may increase very significantly during transitional states brought about by neural and hormonal perturbations (Pette and Staron 1997
). 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. 1995; Wu et al. 2000a
), 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 1989). 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 1962
). 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. 2001
).
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. 1995). The fact that TA and CT differ in developmental origin, being derived from the sixth and fourth branchial arches, respectively (Sperber 1989
), 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 2000). 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.
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Acknowledgments |
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Footnotes |
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