Effect of hypothyroidism on myosin heavy chain expression in rat pharyngeal dilator muscles BASIL

J. PETROF,

ALAN

M. KELLY,

NEAL

A. RUBINSTEIN,

AND

ALLAN

I. PACK

Pulmonary and Critical Care Division, Department of Medicine, and Center for Sleep and Respiratory Neurobiology; Department of Pathobiology, School of Veterinary Medicine; and Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 PETROF,BASIL J., ALAN M. KELLY,NEAL A. RUBINSTEIN, ANDALLAN I. PACK. Effect of hypothyroidism on myosin heavy chain expression in rat pharyngeal dilator mu&es. J. Appl. Physiol. 73(1): 179-187, 1992.-Although the association between hypothyroidism and obstructive sleepapneais well established, the effect of thyroid hormone deficiency on contractile proteins in pharyngeal dilator musclesresponsiblefor maintaining upper airway patency is unknown. In the present study, the effects of hypothyroidism on myosin heavy chain (MHC) expression were examined in the sternohyoid, geniohyoid, and genioglossusmusclesof adult rats (n = 20). The relative proportions of MHC isoformspresent were determined using MHC-specific monoclonal antibodies and oligonucleotide probes. All control musclesshoweda paucity of type I MHC fibers, with >90% of fibers containing fast-twitch type II MHCs. In the genioglossus muscle, a population of non-IIa non-IIb fast-twitch type II fibers (putatively identified as type 11xMHC fibers) were detected. Hypothyroidism induced significant changesin MHC expression in all musclesstudied. In the sternohyoid, type I fibers increasedfrom 6.2 to 16.9%, whereastype IIa fibers increasedfrom 25.9 to 30.7%. Type I fibers in the geniohyoid increasedfrom 1.2 to 12.8%,whereastype IIa fibers increased from 34.1to 42.7%.The genioglossusshowedthe smallestrelative increasein type I expressionbut the greatest induction of type IIa MHC. None of the musclesexamined demonstrated reinduction of embryonic or neonatal MHC in responseto thyroid hormone deficiency. In summary, hypothyroidism alters the MHC profile of pharyngeal dilators in a muscle-specific manner. These changesmay play a role in the pathogenesisof obstructive apneain hypothyroid patients. contractile proteins; fiber types; upper airway; obstructive sleepapnea

SEVERAL GROUPShave reported a clinical

association between hypothyroidism and the obstructive sleep apnea syndrome (13,26,28). Although the precise mechanisms by which thyroid hormone deficiency leads to this condition are not known, a number of potential pathophysiological changes have been proposed. These include upper airway narrowing secondary to myxedematous infiltration of pharyngeal structures (26), obesity (26, 28), and abnormalities of ventilatory control (28). In addition, neurological examination has suggested the presence of pharyngeal muscle weakness in some patients with concomitant hypothyroidism and obstructive sleep apnea, leading to the notion that the pharyngeal dilator muscles responsible for maintaining upper airway patency may be affected directly by the lack of thyroid hormone (13).

Thyroid hormone deficiency has been shown to impart a number of morphological and functional changes to skeletal muscle in both animal models (2,14,23) and humans (1, 36). Chief among these alterations are modifications of the myosin heavy chain (MHC) isoform composition of the muscle (10, 14, 18). MHC represents the major structural component of the thick filament of the sarcomere and is encoded by a highly conserved multigene family (24). The MHC also plays the primary role in converting chemical energy into mechanical work in striated muscle through its ability to catalyze ATP hydrolysis. Differences in adenosinetriphosphatase (ATPase) activity, shortening velocity, force generation, and thermodynamic efficiency observed between individual skeletal muscle fibers have been found to correlate directly with the presence of distinct MHC isoforms (5, 8, 34). In mammalian systems, seven sarcomeric MHCs have been identified thus far at both the protein and gene level (24,27). These include 1) embryonic, 2) neonatal, 3) adult fast-twitch type IIa, 4) adult fast-twitch type IIb, 5) adult slow-twitch type I (skeletal) Q-cardiac, 6) a-cardiac, and 7) extraocular muscle-specific MHC. The physiological features of skeletal muscle fibers expressing the adult MHC isoforms are fairly well characterized. In this regard, both shortening velocity and tension development follow a IIb > IIa > I hierarchy (8, 34). Regulation of MHC isoform expression in striated muscle is affected profoundly by manipulations of thyroid hormone status (10, 14, 18). Furthermore, changes in sarcomeric MHC phenotype induced by alterations in thyroid hormone occur in a highly tissue(i.e., muscle) specific manner (18). Little is known about the relative proportions of MHC isoforms normally present in the major pharyngeal dilator muscles. In view of the important functional differences associated with the various MHC isoforms, such information would expand our currently limited knowledge of the contractile properties of these muscles (16, 35). Similarly, nothing is known at present regarding the effects of hypothyroidism on the MHC composition of these muscles. MHC phenotype modifications resulting from thyroid hormone deprivation, if present, could vary considerably among the different upper airway dilator muscles on the basis of previous work demonstrating the muscle specificity of the response. Accordingly, the specific objectives of the present investigation were to 1) determine the normal MHC complement of three major

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pharyngeal dilator muscles in a rat model, 2) establish whether thyroid hormone deficiency dictates changes in MHC isoform expression in these muscles, and 3) ascertain whether the effects of hypothyroidism on MHC phenotype in pharyngeal dilator muscles are characterized by a muscle-specific response. METHODS

Adult (6-wk-old) male Sprague-Dawley rats (Taconic Farms, Germantown, NY) were randomly assigned to one of two groups designated normal control (NC) and hypothyroid (HT). All animals were provided with standard rat chow ad libitum and were housed at room temperature with a 1212-h light-dark cycle. Rats assigned to the HT group received water supplemented with 0.05% propylthiouracil, whereas the NC group was maintained on normal drinking water (10, 17). At the end of a 6- to 8-wk period, all animals were weighed, killed with carbon dioxide, and placed immediately on dry ice to prevent muscle catabolism. Blood was collected from each animal and assayed for triiodothyronine (T3) and thyroxine (T,) to confirm thyroidal status with the use of a commercial radioimmunoassay kit (Baxter Healthcare, Cambridge, MA). Because of the quantity of tissue from pharyngeal muscles required for the various analyses (see below), the study was performed on two sets of animals, each with NC and HT cohorts as described above. In the first set of rodents (n = l2), MHC expression was examined by immunohistochemistry and/or enzyme-linked immunosorbent assay (ELISA) to investigate those MHC isoforms (I, IIa, and embryonic) for which a specific monoclonal antibody was available. Because of the lack of a neonatal or IIb MHC-specific antibody and because of previous work indicating that MHC isoform expression is primarily under transcriptional control (27), we employed isoform-specific oligonucleotide probes to study expression of these isoforms (Northern analysis) in a second set of animals (n = 8). The following upper airway dilator muscles were excised and trimmed free of connective tissue: sternohyoid, geniohyoid, and genioglossus. In addition, two hindlimb muscles, the extensor digitorum longus (EDL) and the soleus, were also removed to allow comparisons to well-characterized fast- and slow-twitch muscles, respectively. Muscles were removed in random order and processed within l-2 min of the rat’s death. Rat embryonic @day fetal hindlimb) and neonatal (5 day postnatal hindlimb) muscle samples were also obtained to serve as positive controls in the examination of developmental (embryonic and neonatal) MHC isoform expression. MHC Analysis h72munohistochemistry. Indirect immunofluorescence was performed as previously described (25) on the sternohyoid, geniohyoid, and genioglossus muscles. Briefly, individual muscles were sectioned at midbelly, embedded in mounting medium (Tissue Tek), and then immediately frozen in isopentane precooled with liquid nitrogen. Serial sections (6.pm thick) were cut with a cryostat at -2OOC and air-dried on glass slides. The cryosections were subsequently treated with monoclonal antibodies previously shown to be specific for the slow type I (mAb

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NOQ7.5.4D), fast type IIa (mAb SC-71), and embryonic (mAb 2B6) MHC isoforms (11,25,32). The genioglossus muscle was also immunostained with a commercially available (Sigma Chemical, St. Louis, MO) polyclonal antibody (MY-32) that recognizes all fast isomyosins (15). All antibodies were diluted in standard salt solution (1.2 M KCl, 0.5 M Na,HPO,, 0.5 M KH,PO,, 1.0 M MgCl,) as follows: NOQ7.5.4D (1:30), SC-71 (l:lOO), 2B6 (1:20), and MY-32 (1:200). After a l-h incubation at 37OC with the aforementioned antibodies, sections were rinsed in standard salt and then incubated for an additional hour at 37OC with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (IgG, 1:20 in standard salt) secondary antibody (Cappel Laboratories, Cochranville, PA). After further rinsing, the sections were mounted in 50% glycerol-50% standard salt solution. Sections receiving only the secondary antibody served as a control for background activity and demonstrated no reaction. Serially stained sections of the sternohyoid and geniohyoid muscles were examined under fluorescence microscopy, and the total number of positively and negatively staining fibers in each section were counted; note that this type of quantitative analysis could not be performed on the genioglossus due to the multiplanar orientation of its fibers that precludes accurate fiber counts. EUSA. Actomyosin was extracted from both pharyngeal and hindlimb muscle samples as described by Narusawa et al. (25). Whole muscles were homogenized in a solution (3 ml/g tissue) containing 0.6 M KCl, 0.015 M Tris, 0.01 M dithiothreitol, 0.01 M NaPPi, and 2 mM MgCl,. The following protease inhibitors were also included in the above solution: leupeptin (0.5 pi/ml), aprotinin (0.5 pi/ml), and phenylmethylsulfonyl fluoride (1 pi/ml). The homogenate was shaken gently on ice for 1 h and then centrifuged at 19,000 rpm (Sorvall SS-34 rotor, New-town, CT) for 20 min at 4OC. The resulting supernatant was diluted with 5 volumes of a 2-mM MgSO, solution containing protease inhibitors in the concentrations stated above. This mixture was subsequently allowed to shake slowly on ice for 15-30 min until a precipitate was formed. After a further centrifugation at 8,000 rpm (Sorvall HB4 rotor) for 10 min at 4”C, the resulting pellet was suspended in a buffer consisting of 50% glycerol and 20 mM NaPPi. This final suspension was stored at -2OOC. Quantitative protein assays were carried out on the actomyosin preparations according to the method of Lowry et al. (22) with the use of bovine serum albumin (BSA) as a protein standard. Quantitation of MHC isoforms in whole muscle samples by ELISA was performed as previously described (19). Briefly, 1 pg of actomyosin in 50 ~1 of Tris-buffered saline (TBS) was loaded in triplicate into polyvinyl microtiter plate wells. After 1 h at room temperature, unbound antigen was removed by flooding the wells with TBS. To prevent nonspecific binding of antibody, the wells were then filled with blocking solution (1% BSATBS) and incubated for 30 min before the plate was washed with TBS-0.05% Tween-20 (TTBS). The MHCspecific antibodies diluted in 1% BSA-0.05% Tween-20, i.e., NOQ7.5.4D (1:25), SC-71 (1:250), and 2B6 (1:50), were then added at 50 ~1 per well. After 1 h, the plate was

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washed again with TTBS and then incubated with 50 ~1 per well of alkaline phosphatase-labeled goat anti-mouse IgG diluted 1:2,000 in 1% BSA-0.05% Tween-20. Unbound secondary antibody was washed off with TTBS 1 h later, and color development was initiated by adding 50 pi/well of alkaline phosphatase substrate solution. The reaction was stopped after 2 h by adding 0.5 M NaOH (50 ~1 per well), and the plates were read at 405 nm (model 2550 EIA Reader, Bio-Rad Laboratories, Richmond, CA). Northern analysis. Pharyngeal and hindlimb muscle samples were stored at -7OOC. The frozen tissues were homogenized, and total cellular RNA was subsequently isolated by use of the guanidinium thiocyanate-phenolchloroform extraction method (4). Concentrations were determined spectrophotometrically, and RNA (3 pg per sample) was added to sample buffer (22.5 ~1 dimethyl sulfoxide, 4.5 ~1 0.1 M NaH,PO,, 6.6 ~1 glyoxal). The samples were denatured by heating for 30 min at 55OC and then placed immediately on ice. RNA samples were size fractionated on a 1% agarose gel containing 10 mM NaH,PO, and transferred to nylon filters (GeneScreen, Biotechnology Systems, NEN Research Products, Boston, MA) according to standard procedures. The filters were hybridized with isoform-specific synthetic oligonucleotide probes uniquely complementary to 20 bases of the 3’-untranslated region of neonatal and type IIb MHC mRNA transcripts; the sequences of the probes used in this study have been reported earlier (14,24). The probes were labeled at the 5’ end with gamma [32P]ATP using T4 polynucleotide kinase. Filters were prehybridized overnight at 61°C (neonatal MHC) or 53OC (IIb MHC) in a solution containing 10% dextran sulfate, 10X Denhardt’s solution (0.2% BSA, 0.2% polyvinylpyrollidone, 0.2% Fitoll), 0.1% sodium dodecyl sulfate, 6X NET (900 mM NaCl, 90 mM Tris-HCl, pH 8.3,6 mM EDTA), and 100 pg/ml denatured salmon sperm DNA. Labeled oligonucleotide probe (250,000 cpm/ml) was then added. After hybridization at the same temperature for 16-24 h, the filters were washed three times at room temperature and once at the hybridization temperature for 5 min with 6X standard saline citrate (0.9 M NaCl, 0.09 M sodium citrate, pH 7.0): 0.1% sodium dodecyl sulfate. Autoradiography was performed at -7OOC for 48 h with two intensifying screens, and mRNA levels were quantitated by scanning densitometry. Statistical

Analysis

All data are reported as means t SE and were analyzed with a NWA STATPAK (version 4.1) statistical package (Northwest Analytical, Portland, OR). Differences between groups (NC vs. HT) and within a group (muscle type) were tested using a two-way analysis of variance. For variables identified as significant by analysis of variance, comparisons between and within groups were performed with Student’s two-tailed t test for independent and dependent samples, respectively. The NewmanKeuls post hoc test was also used to determine differences between pairs of means in the case of multiple comparisons. Statistical significance was defined as P < 0.05.

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RESULTS

Mean T, levels in the HT group of animals were significantly reduced compared with the NC rodents (25.8 t 2.1 vs. 91.0 t 12.4 ng/dl; P < O.OOl), thus confirming the hypothyroid status of the HT animals. Similar decreases in T, values were also found; mean T, levels were 0.30 t 0.06 and 5.23 t 0.42 pgldl in the HT and NC groups, respectively (P < 0.001). Body weight was significantly lower in HT animals relative to the NC group (226.2 t 7.8 vs. 453.7 t 24.5 g; P < O.OOl), a finding consistent with previous studies of thyroid hormone-deficient rats (14,23). Hyoid Muscles Figures 1 and 2 show indirect immunofluorescence of representative serial sections of the sternohyoid and geniohyoid muscles from both NC and HT animals. For both muscles, there were increases in the proportion of fibers staining for types I and IIa MHC in the HT group. In addition, in the HT animals the two muscles contained fibers (indicated in Figs. 1 and 2 by asterisks) that costained for both MHC isoforms. These results are confirmed and expanded by quantitative fiber counts performed on the sternohyoid and geniohyoid muscles in all rodents from both experimental groups (summarized in Table I). For both muscles studied and in both groups of animals, the number of fibers counted in tissue sections was not significantly different. In control animals, there was a paucity of fibers expressing type I MHC in both upper airway dilator muscles. However, the sternohyoid contained an approximately fivefold greater proportion of these fibers than did the geniohyoid (P < 0.001). Conversely, the geniohyoid in controls demonstrated a larger percentage of IIa fibers, with approximately one-third of its fibers expressing IIa MHC compared with roughly one-quarter in the sternohyoid (P < 0.001). The imposition of hypothyroidism led to substantial alterations in the percentages of fibers expressing types I and IIa MHC in both muscles. Type I fiber proportion increased from 6.2 t 0.6 to 16.9 t 1.2% in the sternohyoid muscle (P < 0.0001). This was accompanied by an increase in IIa fibers from 25.9 t 0.6 to 30.7 t 1.4% (P < 0.01). The effects of hypothyroidism on the geniohyoid muscle were even more marked. In this muscle, there was a lo-fold increase in the proportion of type I fibers, increasing from 1.2 t 0.1 to 12.8 t 1.7% (P < 0.0001). The proportion of IIa fibers also increased in the geniohyoid, from 34.1 3- 1.3 to 42.7 t 1.3% (P < 0.001). The ELISA determinations of type I and IIa MHC expression in whole muscle homogenates (shown in Figs. 3 and 4) were in general accordance with these results. In control animals, the sternohyoid contained a greater amount of type I MHC (P < O.Ol), whereas the geniohyoid exhibited a higher level of IIa MHC expression (P < 0.05). Hypothyroidism induced a significant increase in type I MHC expression in both the sternohyoid (P < 0.0001) and geniohyoid (P < 0.001) muscles. Thyroid hormone deprivation also led to increased IIa MHC expression in the geniohyoid (P < 0.05), whereas the increase in IIa MHC in the sternohyoid did not quite reach statistical significance (P = 0.07). A representative Northern blot demonstrating expres-

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FIG. 1. Type I and IIa fibers in sternohyoid muscle of normal control (NC) and hypothyroid (HT) animals. Serial transve rse sections were stained with monoclonal antibodies against types I and IIa myosin heavy chain (MHC) and were vi:sualized by indirect immunofluorescence: NC stained for type I MHC (A); NC stained for type IIa MHC (B); HT stained for type I MHC (C); HT stained for type IIa MHC (D). Note increased proportion of fibers expressing types I and IIa MHC in HT muscle. In addition, HT sections demonstrate a number of transitional fibers coexpressing both types I and IIa MHC (*). Scale bar, 50 pm.

sion of the IIb MHC isoform in these muscles is shown in Fig. 5. The specificity of the probe was confirmed by the strong signal observed for RNA samples obtained from the EDL and the lack of detectable signal in the soleus muscle (9, 14, 18). For the group as a whole, there was a trend toward reduced IIb MHC expression in the sternohyoid and geniohyoid muscles of HT rats (Fig. 6), although this did not achieve statistical significance. Finally, for both the NC and HT groups, the embryonic and neonatal MHC isoforms were not detected in either of the hyoid muscles. GenioglossusMuscle

Immunostains of the genioglossus muscle revealed, as in the case of the hyoid muscles, a notable lack of type I MHC fibers along with a larger number of fibers containing type IIa MHC. For this muscle with varying fiber orientations, the ELISA analysis (Figs. 3 and 4) allowed a more quantitative assessment of type I and IIa MHC isoform expression than could be made with the immunohistochemical approach. In the NC group, the genioglossus contained a lower level of type I MHC than the sternohyoid (P < O.Ol), whereas its type I MHC content was not significantly different from that of the geniohyoid. Type IIa MHC expression in the genioglossus of controls was substantially greater than that found in either the sternohyoid (P < 0.01) or geniohyoid (P < 0.01). Hypo-

thyroidism resulted in an increase in type I MHC (P < 0.05) as well as type IIa MHC (P < 0.0001) expression in the genioglossus muscle. Compared with the sternohyoid and geniohyoid muscles, these changes represented the smallest relative increase in type I MHC (P < 0.05) and the greatest relative induction of type IIa MHC expression (P < 0.05). Northern analysis revealed that type IIb MHC expression was detectable at low levels in the genioglossus of control rats. This is of particular interest in view of the fact that there appeared to be a substantial number of fibers in this muscle that contained a fast isomyosin (as indicated by a lack of reactivity with the slow-twitch type I MHC-specific antibody and confirmatory positive staining with the generic fast-twitch type II MHC-directed antibody) that did not immunostain positively for IIa MHC (see Fig. 7). Collectively, the above data suggest that the genioglossus contains a population of fibers expressing a non-IIa non-IIb fast-twitch type II MHC isoform (19, 32). As was the case for the hyoid muscles, there was a trend toward reduced IIb MHC expression in this muscle under hypothyroid conditions (Fig. 6). In addition, neither developmental MHC isoform was detected in the genioglossus muscles of NC and HT rats. Hindlimb Muscles

Results of the ELISA analysis indicated that both hindlimb muscles in the NC group had greater amounts

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FIG. 2. Type I and IIa fibers in geniohyoid muscle of NC and HT animals. A: NC stained for type I MHC; B: NC stained for type IIa MHC; C: HT stained for type I MHC; D: HT stained for type IIa MHC. HT muscle sections show ed percentage of fibers expressing types I and IIa MHC as well as fibers that costain for 2 MHCs (*). Scale bar, 50 pm.

of slow-twitch type I MHC than was observed in any of the control pharyngeal muscles (P < 0.01). Type IIa MHC expression in control hindlimb muscles was not significantly different from that seen in the sternohyoid and geniohyoid. However, both the EDL and soleus contained a lower level of IIa MHC than the genioglossus (P < 0.01). Thyroid hormone deficiency led to increased type I MHC expression in both hindlimb muscles (P < 0.05), as well as an increase in the level of IIa MHC expression in the EDL (P < 0.001). In contrast, the soleus demonstrated reduced levels of IIa MHC expression in the HT animals (P < 0.0001). Northern analysis showed substantial IIb MHC expression in the EDL, whereas there was little or no detectable IIb MHC signal in the 1. MHC expression in fibers of the sternohyoid and geniohyoid muscles in normal control and hypothyroid animals TABLE

Control

Type I MHC fibers, % Type IIa MHC fibers, % Total fibers counted

Hypothyroid

Sternohyoid

Geniohyoid

6.2rt0.6

1.2rto.1*

16.9+1.2t

123+1.7t

25.9kO.6

34.1+1.3*

30.7+1.4t

42.7*1.3t

4,766+271

4,514+170

4,731+279

4,960*298

Sternohyoid

Geniohyoid

Values are means + SE. MHC, myosin heavy chain; NC, normal control; HT, hypothyroid. * P < 0.01, sternohyoid vs. geniohyoid (NC group). t P < 0.01, NC vs. HT group for a given muscle.

soleus. Hypothyroidism did not result in significant changes in IIb expression in these muscles. Developmental MHCs were not found to be present in any of the adult hindlimb muscles examined. DISCUSSION

Considerable attention has been devoted during the past decade to the study of the pharyngeal dilator muscles. Such interest has arisen from the recognition that the coordinated action of these muscles is critical for the maintenance of upper airway patency (29,35). Although the possibility of a myopathy involving the upper airway has been recently suggested as a potential etiologic factor in obstructive sleep apnea associated with hypothyroidism (13), this is the first study to demonstrate that thyroid hormone deficiency does in fact lead to alterations in contractile protein gene expression in the muscles responsible for dilating the upper airway. The present study establishes that hypothyroidism results in increased expression of types I (slow-twitch) and IIa MHC in rat pharyngeal dilator muscles and that these changes occur in a muscle-specific manner. Although the response to hypothyroidism was qualitatively similar (i.e., increased expression of types I and IIa MHC) among the three pharyngeal dilator muscles as well as a fast-twitch hindlimb muscle (EDL), there were important quantitative differences in the effects on these muscles. For example, the genioglossus exhibited the greatest induction of IIa MHC expression while also

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NC

* P < 0.05 **P < 0.001

0.5

IN PHARYNGEAL

HT 12345 ,./ +S?* 4 \,

0.4

8 2

_\\ ‘_ _\ \ \\__\. \ .’

0.3

18S9

g

\,

_’

+*

9

0.2

0.1

0.0 SH

GH

CC

EDL

FIG. 3. Enzyme-linked immunosorbent assay (ELISA) determination of type I MHC expression in sternobyoid (SH), geniohyoid (GH), genioglossus (GG), and extensor digitorum longus (EDL) muscles of NC (0) and HT (@) animals. Type I MHC expression increased as result of the hypothyroid state in all muscles examined (soleus not shown because values exceeded this scale at antibody dilution employed here). All values are expressed as group mean (*SE) absorbance per microgram of actomyosin.

showing the smallest relative increase in type I MHC. Furthermore, the effect of thyroid hormone deficiency on the above fast-twitch muscles was qualitatively different from that seen in a slow-twitch muscle (soleus), which was the only muscle to demonstrate reduced IIa MHC expression in the hypothyroid state. The results of the present study are therefore consistent with an earlier report of the tissue or muscle specificity of thyroid hormone action (18). This could occur through a number of 0.6 * P < 0.05 **P < 0.001 0.6

FIG. 5. Northern blot analysis of type IIb MHC expression. Sternohyoid (lane I), geniohyoid (lane 2), genioglossus (hne 3), soleus (lane 4), and extensor digitorum long-us (lane 5) muscles of NC and HT animals are shown. Autoradiogram depicted is representative of experiments performed in 8 animals. There was a relative lack of IIb MHC mRNA transcripts in genioglossus and soleus muscles of both NC and HT samples, whereas strong IIb MHC signal was obtained for the 2 hyoid muscles and EDL.

potential mechanisms. Different levels of hormone delivery due to variations in local blood flow or metabolism are possible. It appears more likely, however, that these tissue-specific responses are a result of differences in tram and cis regulatory factors. In regard to the former possibility, recent work has identified the existence of several T, receptor isoforms encoded by the c-erbA protooncogene, which are themselves differentially regulated by thyroid hormone in a highly complex and tissuespecific manner (17). Hence, qualitative as well as quantitative differences in nuclear T, receptors may exist among the pharyngeal muscles. In addition, muscle-specific variations in the thyroid-responsive element and other cis acting elements of the gene may also play an important role. In the present study, hypothyroidism was associated with a significant reduction in body mass, as has been described previously in rats (10, 14, 23). This raises the question of whether weight loss per se may have affected MHC expression in the muscles studied. Previous investigators have reported that weight loss resulting from food restriction leads to selective reductions in the crosssectional area of type II fibers, with little or no change in the number or proportions of different fiber types (12,

z i% 8 0.4 s

0.2

0.0 SH

GH

GG

EDL

SOL

4. ELISA determination of type IIa MHC expression in SH, GH, GG, EDL, and soleus (SOL) muscles of NC (Cl) and HT (M) rats. Hypothyroidism induced significant increases in type IIa MHC expression in GH, GG, and EDL. However, type Ha expression was decreased in hypothyroid SOL. All values are group mean (&SE) absorbance per microgram of actomyosin. FIG.

SH

GH

cc

EDL

SOL

6. Densitometric quantitation of IIb MHC expression in SH, GH, GG, EDL, and SOL muscles of NC (Cl) and HT @) rats. All values are expressed as group mean (*SE) in arbitrary units. FIG.

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FIG. 7. Genioglossus muscle sections from NC animal. A: section immunostained with monoclonal antibody (mAb SC-71) directed against type IIa MHC. B: section stained with polyclonal antibody (MY-32) reacting with all fast-twitch type II MHC fibers. Many fibers do not stain positively for IIa MHC in A, whereas all fibers do contain fast isomyosins, as revealed in B. See text for further interpretation. Scale bar, 50 pm.

20). It is therefore unlikely that body weight reductions induced the highly significant changes in MHC expression observed with hypothyroidism in the present study. In addition, the increased type IIa MHC protein levels determined in whole muscle homogenates of hypothyroid pharyngeal muscles are not in keeping with the reported atrophic response of type II fibers to malnutrition. Hence, the alterations in MHC expression observed in the present study are likely to have resulted primarily from the hypothyroid state per se rather than associated reductions in body weight. D’Albis and co-workers (6) showed that developmental isomyosins are eliminated from the rat tongue by - 1 mo after birth. The results of the present study, in which embryonic MHC was undetectable in all of the pharyngeal muscles examined at -12 wk of age in euthyroid animals, are in keeping with this finding. In the adult, reexpression of both embryonic and neonatal MHCs at the mRNA transcript level has been reported in certain muscles as a result of thyroid hormone deficiency (18). However, in the present study we found no evidence of a recapitulation of the developmental program of MHC expression in any of the muscles examined.

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Recent studies point to the existence of a previously unrecognized MHC isoform (referred to as 11x or IId) that is present in a number of rodent muscles, particularly the diaphragm (3, 19,32). Although previous work, in which fiber types were characterized by acid and alkaline preincubation myosin ATPase staining, has indicated that approximately one-third of the fibers in the rat diaphragm are type IIb (9), it now appears that a large proportion of these fibers are in fact 11x/d (19,32). Type 11x/d fibers exhibit high succinate dehydrogenase activity and might also tend to be classified as fast oxidative glycolytic fibers (frequently equated with IIa fibers) in studies employing oxidative enzyme histochemical staining methods to determine fiber type (32). These findings illustrate the potential pitfalls involved in making inferences regarding muscle fiber MHC composition on the basis of indirect histochemical techniques. Combined immunochemical and Northern analysis in the present study has identified a group of fibers in the genioglossus muscle that contain a non-IIa non-IIb fasttwitch MHC isoform. This conclusion is based on the fact that, although they do react strongly with a generic type II MHC-directed antibody, these fibers do not immunostain positively with either the type I (slow-twitch) or type IIa specific monoclonal antibodies. Nor is it likely that the majority of these fibers contain the type IIb MHC isoform, because there was a relative lack of IIb MHC mRNA transcripts in this muscle. It is reasonable to speculate, therefore, that these fibers may actually contain the 11x/d isoform (19, 32); there is indirect evidence in support of this contention. First, Hellstrand (16) demonstrated in physiological studies that the tongue displays a fast contraction profile while remaining relatively resistant to muscle fatigue, characteristics consistent with the 11x/d fiber (32). Second, Bar and Pette (3) reported the presence of 11x/d myosin in the rat tongue on the basis of protein electrophoresis experiments. In addition, although the two other pharyngeal dilator muscles examined in this study did contain substantial amounts of IIb MHC transcript, we cannot exclude the presence of the 11x/d MHC isoform within these muscles as well. Indeed, van Lunteren and colleagues (35) found that the cat geniohyoid muscle also exhibits enhanced fatigue resistance despite fast contraction kinetics. The myosin isoform profiles of pharyngeal muscles in the euthyroid state and their modification by hypothyroidism have important implications for muscle function. Experiments on single muscle fibers have established that there are significant functional differences among fibers expressing types I, IIa, and IIb MHC. The maximum shortening velocity (V,,) of type IIb fibers is approximately three to four times that of type I fibers, with the V,,,,, of type IIa fibers intermediate between the two (8,34). Previous work in vitro has shown that maximum muscle efficiency occurs when the velocity of shortening during muscle contraction is -30% of V,,, (21). Hence, it may be bioenergetically advantageous for a muscle that is called on to operate at higher shortening velocities to utilize a larger proportion of fast type II fibers. Indeed, Rome and colleagues (30) provided evidence that the recruitment pattern for different muscle

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fiber types adopted in vivo is in fact a strategy designed, at least in part, to achieve optimal mechanochemical efficiency for the particular task being demanded of the muscle. In addition, certain actions by their very nature cannot be performed if muscle fibers are unable to shorten with sufficient rapidity. The paucity of type I MHC fibers in the pharyngeal dilator muscles examined in the present study is in keeping with previous histochemical studies (myosin ATPase activity) of these muscles in the cat (7), as well as in humans (33), and is consistent with the multiple functions of these muscles (mastication, swallowing, speech, respiration) in which relatively high shortening velocities might be required. The increase in slow-twitch type I fibers observed in the hypothyroid pharyngeal muscles could theoretically reduce muscle efficiency at high shortening velocities. On the other hand, an increase in slow-twitch type I fibers is normally accompanied by elevated levels of oxidative enzymes, with an attendant increase in resistance to muscle fatigue. However, existing evidence indicates that this is not necessarily the case in hypothyroidism. Several authors have shown that mitochondrial components and associated oxidative enzymes are actually reduced in thyroid-deficient muscle despite an increase in type I fiber proportion (1,2). Hence hypothyroidism appears to result in an uncoupling of the customary relationship between MHC composition and the oxidative capacity of the muscle. There are conflicting data concerning the susceptibility of hypothyroid muscle to fatigue, with studies suggesting fatigue resistance to be increased (36), decreased (l), and unchanged (23). These inconsistencies likely are related to differences in experimental conditions (isometric vs. isotonic, variations in oxygen delivery) utilized in these studies. Although pharyngeal muscle fatigue can be shown to occur in normal humans in the experimental setting (31), it is currently unknown whether fatigue is induced as a consequence of the repeated pharyngeal muscle activation required to terminate episodes of upper airway occlusion that occur throughout the night in patients with sleep apnea. Furthermore, as alluded to earlier, the anticipated effects of hypothyroidism on pharyngeal muscle fatigability are currently unclear. In addition to the energetics considerations outlined above, alterations in MHC isoform composition also have important implications for the absolute level of force that a muscle is capable of producing. Under isometric conditions, IIb fibers generate substantially greater tension than type I and IIa fibers, based to a large degree on the greater cross-sectional area of IIb fibers (8, 9). Specific tension (tension/cross-sectional area) also appears to be 20-30% greater in fast type II than in slow type I fibers (8). Consistent with these observations, McAllister and co-workers (23) recently reported that the fast-twitch plantaris muscle of hypothyroid rats demonstrated a significant increase over controls in the proportion of type I fibers (similar in magnitude to the changes observed in the present study) and that these changes were associated with a 15% decrease in isometric force development. The divergence in force-generating capacity among fiber types is even greater under more physiologically isotonic conditions, with the entire forcevelocity relationship shifted in favor of the faster fibers.

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Hence, to the extent that thyroid hormone deprivation resulted in increased expression of types I and IIa MHC in the pharyngeal muscles examined in this study, one would anticipate a reduction in their ability to exert force and thereby dilate the upper airway. Moreover, given the fact that these muscles act in a collective fashion to maintain upper airway patency, adverse changes in the function of individual muscles could conceivably be additive in their detrimental effects on upper airway caliber. In summary, the imposition of hypothyroidism in rats leads to increased expression of types I and IIa MHC in the fibers of pharyngeal dilator muscles, with the magnitude of the response characterized by tissue or muscle specificity. In view of the anticipated reduction in forcegenerating capacity associated with the observed alterations in muscle fiber phenotype, as well as potential alterations in fatigue resistance, these changes may play a role in the pathogenesis of obstructive sleep apnea in hypothyroid patients. Further studies are needed to determine the physiological impact of these findings. The authors thank Dr. Robin Fitzsimons for the gift of antibody NOQ7.5.4D, Dr. Stefano Schiaffino for the use of antibody SC-71, Zs. Paltzman and John Leferovitch for expert technical assistance, and Kathleen Haeber for invaluable help in preparing the manuscript. This investigation was supported by a Clinician-Scientist Award from the Medical Research Council of Canada (B. J. Petrof), National Heart, Lung, and Blood Institute (NHLBI) Grant HL-15835 to the Pennsylvania Muscle Institute (A. M. Kelly and N. A. Rubinstein), a grant from the Muscular Dystrophy Association of America (A. M. Kelly), and NHLBI Specialized Center of Research Grant HL-42236 (A. I. Pack). Address for reprint requests: B. J. Petrof, Center for Sleep and Respiratory Neurobiology, Hospital of the University of Pennsylvania, 991 Maloney Bldg., 3600 Spruce St., Philadelphia, PA 19104. Received 18 June 1991; accepted in final form 29 January 1992. REFERENCES 1. ARGOV, Z., P. F. RENSHAW, B. BODEN, A. WINOKUR, AND W. J. BANK. Effects of thyroid hormones on skeletal muscle bioenergetics. J. Clin. Inuest. 81: 1695-1701, 1988. 2. BALDWIN, K. M., A. M. HOOKER, R. E. HERRICK, AND L. F. SCHFUDER. Respiratory capacity and glycogen depletion in thyroid-deficient muscle. J. Appl. Physiol. 49: 102-106, 1980. 3. BAR, A., AND D. PETTE. Three fast myosin heavy chains in adult rat skeletal muscle. FEBS Lett. 235: 153-155, 1988. 4. CHOMCZYNSKI, P., AND N. SACCHI. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987. 5. CROW, M. T., AND M. J. KUSHMERICK. Chemical energetics of slow- and fast-twitch muscles of the mouse. J. Gen. Physiol. 79: 147-166,1982. 6. D’ALBIS, A., R. COUTEAIJX, C. JANMOT, AND A. ROULET. Specific programs of myosin expression in the postnatal development of rat muscles. Eur. J. Biochm. 183: 583-590, 1989. 7. DICK, T. E., AND E. VAN LIJNTEREN. Fiber subtype distribution of pharyngeal dilator muscles and diaphragm in the cat. J. Appl. Physiol. 68: 2237-2240, 1990. 8. EDDINGER, T. J., AND R. L. MOSS. Mechanical properties of skinned single fibers of identified types from rat diaphragm. Am. J. Physiol. 253 (CeZl Physiol. 22): C210-C218, 1987. 9. EDDINGER, T. J., R. L. Moss, AND R. G. CASSENS. Fiber number and type composition in extensor digitorum longus, soleus, and diaphragm muscles with aging in Fisher 344 rats. J. H&o&em. Cytochem. 33: 1033-1041,1985. 10. GAMBKE, B., G. E. LYONS, J. HASELGROVE, A. M. KELLY, AND N. A. RUBINSTEIN. Thyroidal and neural control of myosin transitions during development of rat fast and slow muscles. FEBS Lett. 156: 335-339,1983. 11. GAMBKE, B., AND N. A. RUBINSTEIN. A monoclonal antibody to the

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