INTRODUCTION

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ADULT MAMMALIAN skeletal muscles are differentiated primarily on the basis of the pattern of fiber types and/or motor units that they express, which can be generally classified into two major groups: slow twitch and fast twitch (28, 29, 34). A characteristic feature spanning the spectrum of muscles supporting antigravity and locomotor activities is the type of myosin heavy chain (MHC) isoform that is expressed across the fiber pool. In adult rodent skeletal muscle, at least four distinct MHC isoforms are expressed: types I, IIa, IIx, and IIb. These MHC isoforms are the products of four distinct MHC genes and can be identified on the basis of their enzymatic and/or functional, immunological, and electrophoretic properties (21, 29, 34). Slow-twitch muscles primarily contain fibers expressing the type I MHC (2, 28, 29, 34), and they form the major population of fibers in the soleus and the vastus intermedius (VI) muscles, which are specialized for antigravity function and postural support (2, 3, 28, 29, 34). In contrast, fast-twitch muscles, such as the plantaris, predominantly contain fibers expressing chiefly the type IIb and IIx MHCs, and these fibers are primarily recruited during activities requiring a high power output (4-6, 11, 12, 29, 33, 34).

In the adult state, skeletal muscle MHC expression is highly plastic, and the phenotype pattern can be influenced by various exogenous factors, including altered functional demand (overuse and decreased use) and thyroid hormone 3,5,3'-triiodothyronine (T₃) (1-7, 11-17, 19, 22, 25, 26, 31, 36). Interestingly, we and others have reported that both T₃ treatment and hindlimb suspension (HS) induce slow-to-fast MHC transformations (7, 11, 12, 16, 17, 19, 22). Despite the similar directional changes in MHC phenotype that occur in response to these contrasting types of intervention, relatively little is known concerning their qualitative and quantitative differences and/or similarities in terms of MHC isoform expression because, until recently, the two interventions have not been examined either individually or in combination with one another under the same experimental conditions.

In an attempt to address this issue, we recently reported (3) that the combination of T₃ treatment plus HS (T₃ + HS) induced a marked remodeling of the MHC phenotype in the rodent slow-twitch soleus muscle, such that it was transformed into essentially a fast-contracting muscle. The characteristic features of the adaptive response included several novel observations. For example, analyses at the single-fiber level revealed that T₃ treatment alone appeared to affect a different fiber pool and distinctly different MHC isoforms compared with that of HS treatment. That is, T₃ induced a greater bias of type IIIa shifts, whereas HS caused a greater bias of IIIx transformation, while neither induced any significant upregulation of the type IIb MHC. However, when the two interventions were combined, the majority of fibers (~70%) in the T₃-suspended soleus muscle simultaneously expressed all four adult MHC proteins, with the type I MHC accounting for only a relatively small portion (<20%) of the MHC pool in the majority of these fibers. In contrast, the type IIb MHC, which is normally not expressed in consistently detectable amounts in the soleus muscle of control animals (11, 14, 17, 22), accounted for as much as 25-35% of the MHC pool in many of these fibers (3). Also, whereas the majority of fibers (~80%) in the control soleus muscle normally express only the type I MHC, essentially none of the fibers in the T₃ + HS soleus muscle expressed only the type I isoform (3). Therefore, due to both the unique pattern and the magnitude of change in MHC expression that occurred in this model, we hypothesize that the combination of T₃ + HS exerts a powerful influence on MHC gene expression that is dominated by pretranslational events. Also, there could be transformations that occur in response to the separate and combined intervention of T₃ treatment and HS that are both temporally regulated and specific to an isoform, depending on the type of muscle affected. Thus, in view of the paucity of data to address these issues, we performed a time course (1, 2, and 4 wk) analysis of MHC expression at both the mRNA and protein levels on soleus, plantaris, and VI muscles by using the combined T₃ + HS model. These analyses were carried out in the context of performing the same analyses on the separate interventions of T₃ treatment and HS. Herein we show that all four MHC isoforms are sensitive pretranslationally to varying degrees as a result of the separate interventions of T₃ treatment and of HS that are both isoform and muscle-type specific. Also, we report for the first time the novel observation that the combination of T₃ + HS induces a rapid, sustained, and marked (80-90%) downregulation of type I MHC gene expression that is mirrored temporally by concomitant marked upregulation of type IIb MHC gene expression that is independent of muscle type.

	METHODS

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Animal care and experimental design. Because of the large number of rats used in the present study and the limited facilities to perform HS simultaneously on >20 rats at a time, the study was conducted in three separate phases designated as phase I (4 wk), phase II (2 wk), and phase III (1 wk) on the basis of the duration of the experiment. For a given phase, 32 adult female Sprague-Dawley rats (weight, 220-260 g; Taconic Farms, Germantown, NY) were randomly assigned to one of four experimental groups (n = 8 in each): 1) normal control (Con); 2) normal HS (HS); 3) T₃-treated control (T₃); and 4) T₃ plus HS (T₃ + HS). Animals designated to be suspended (HS and T₃ + HS) were treated with a noninvasive tail-casting technique, as described previously (11). This technique utilized a swivel-harness system that was incorporated into the casting materials; the swivel harness was attached to a hook on the top of the cage. The hook was adjusted to allow only the front legs of the animal to reach the floor. Suspended animals were free to move about the cage by using their front legs. Animals designated for T₃ treatment (T₃ and T₃ + HS) were given daily intraperitoneal injections of T₃ (150 µg · kg¹ · day¹) throughout the experimental period of each phase. This protocol has been shown to 1) elevate plasma T₃ levels, 2) induce marked cardiac hypertrophy and cardiac isomyosin shifts, and 3) elevate resting metabolic rate. These three conditions collectively are the hallmarks of a hyperthyroid state (3, 7, 20, 31). Animals were housed in light- and temperature-controlled quarters and were provided with food and water ad libitum. The protocols were approved by our Institutional Animal Care Committee.

Tissue processing and biochemical analyses. At the designated time point, subgroups of the animals were sedated with a lethal injection of Nembutal (50 mg/kg). The chest was opened to obtain the heart, which was rapidly removed, trimmed of the aorta and veins, weighed, and frozen. From the right hindlimb, the fast-twitch plantaris muscle, the slow-twitch soleus muscle, and the predominantly slow-twitch VI muscle were rapidly removed, grossly trimmed of connective tissue, quickly frozen on dry ice, and stored at 70°C for RNA and protein coextraction for MHC analysis. On the left side, only the soleus muscle was removed and processed for MHC analyses in single fibers and immunohistochemistry, as reported in the companion paper (1). The present study focused on these particular muscles because they collectively express divergent MHC profiles (see Table 3) and they show various degrees of responsiveness to both altered thyroid state and weightlessness (4-6, 11, 12, 15, 17, 19, 31). Furthermore, although the soleus muscle is considered to be the prototype antigravity muscle due to its predominance of slow, type I MHC expression, this study also included analyses of another slow muscle prototype, the VI. This muscle is unique in that it expresses a predominance of the slower isoforms, as seen in the soleus, but unlike the soleus, it also typically expresses detectable amounts of two additional fast isoforms (types IIb and IIx) which predominate in a fast muscle (see Table 3). Thus it was of interest to contrast the response of the VI to the slow (soleus) and fast (plantaris) prototypes.

Total RNA and protein isolation. Total cellular RNA and total muscle proteins were simultaneously coextracted from frozen muscle samples by using the TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the company's protocol, which is based on the method described by Chomczynski (8). Total proteins were separated in the organic phase and subsequently precipitated with isopropanol, washed with guanidine hydrochloride and ethanol, and suspended in 1% SDS (8). Protein concentration was determined by the Bio-Rad protein assay kit, using gamma globulin as standard. Samples were adjusted to protein concentration of 1 mg/ml with 1% SDS and were stored at 20°C until they were later analyzed for MHC distribution pattern by using SDS-PAGE (33). Total RNA was precipitated from the aqueous phase with isopropanol, and, after it was washed with ethanol, it was dried and suspended in a small volume of 0.5% SDS in Tris-EDTA buffer containing 10 mM Tris, pH 8.0, and 1 mM EDTA. This method of RNA extraction can yield high-quality undegraded RNA, free of protein and DNA [on the basis of an optical density at 260 nm/optical density at 280 nm (OD₂₆₀/OD₂₈₀) of ~2] and, as tested by agarose gel electrophoresis with ethidium bromide staining, showing both 28S and 18S ribosomal RNA with an intensity ratio of ~2:1 (9). The RNA concentration was determined by OD at 260 nm (using an OD₂₆₀ unit equivalent to 40 µg/ml). The RNA samples were stored frozen at 70°C until they were subsequently analyzed, by Northern hybridization, for MHC mRNA expression.

MHC mRNA analysis by Northern blot. Because four different probes needed to be tested (types I, IIa, IIx, and IIb), each sample was loaded on four separate gels; ~5 µg of each sample were loaded on each gel. Gel and transfer conditions were as explained previously (5, 6, 17, 31). Oligonucleotides complementary to the 3' untranslated sequences of the MHC mRNAs were purchased from Chemgene (Waltham, MA). The sequences for the oligonucleotides used for MHC types I, IIa, and IIb were as reported by Gustafson et al. (15). For MHC type IIx, we used a 20-base probe complementary to the 3' nontranslated sequence of a cDNA clone isolated by De Nardi et al. (10) from rat diaphragm muscle of the following 5'3' sequence: GGTCA CTTTC CTGCT TTGGA. The 5' ends of the probes were labeled to 1-2 × 10⁹ counts · min¹ · µg DNA¹ (cpm/µg) with [-³²P]ATP by using polynucleotide kinase. The blots were prehybridized and then hybridized overnight, washed, and exposed to autoradiographic film with an intensifying screen, as described in detail previously (5, 6, 12, 17, 31).

Skeletal MHC isoform analyses. Skeletal MHCs were separated by using a SDS-PAGE technique (33). The gels were run at 275 V for ~22 h under refrigeration. After electrophoresis, the gels were stained for 1 h with Brilliant blue G-250 (Sigma Chemical, St. Louis, MO) and destained with 25% methanol and 10% acetic acid. The separated MHC bands were scanned and quantified by using a Molecular Dynamics Densitometer. The peaks of interest representing the distinct MHC isoforms were identified in the digitized densitometric data sets. The area of each peak was determined by integration (Image Quant Software, Molecular Dynamics). The MHC identity on the resulting banding pattern was verified on the basis of Western blotting techniques by using a battery of MHC antibodies as obtained from the laboratory of Dr. S. Schiaffino (data not shown).

Statistical analyses and data presentation. All data are reported as means ± SE. For the body and muscle weight data presented in Table 1, the data were analyzed by using one-way ANOVA and a Newman-Keuls post hoc test for multigroup comparisons (37). The data for a given muscle MHC protein or mRNA isoform were analyzed by using a three-way ANOVA. The three factors were time (1, 2, and 4 wk), T₃ status (euthyroid, T₃), and HS (no HS, HS). Differences among groups at any given time point were determined by using a Newman-Keuls post hoc test. All statistical analyses were performed by using a computer software package (Systat, Evanston, IL). Statistical significance was set at P < 0.05. Data for MHC protein and mRNA analyses were presented in two ways. First, the absolute response for each isoform in a given muscle was presented in tabular form (Tables 2 and 3). Second, in keeping with previous studies involving the interaction of mechanical activity and thyroid state on MHC expression (12, 31), directional changes across muscles and as a function of time were plotted relative to the control response (see Figs. 2-4). In this way, one can more easily simultaneously examine and compare the directional changes for both MHC protein and mRNA in a given muscle over the time course of the experiment (12, 31).

View larger version (93K): [in this window] [in a new window]   View larger version (23K): [in this window] [in a new window]   Fig. 1.   A: representative myosin heavy chain (MHC) mRNA signals (types I, IIa, IIx, and IIb) in both soleus (top) and plantaris (bottom) muscles in 4 experimental groups for 3 time points after Northern blot hybridization with specific MHC oligoprobes. Also shown is signal after hybridization with 18S ribosomal RNA probe. Con, normal control; HS, normal plus hindlimb suspension; T3, 3,5,3'-triiodothyronine (T3) treatment; T3 + HS, T3 treatment + HS. B: representative MHC protein for both soleus and plantaris muscles, as separated by SDS-PAGE. Vastus intermedius (VI) muscle is not shown to reduce amount of material presented.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Shifts () in %MHC protein (types I, IIa, IIx, and IIb) and mRNA signal from normal Con value in 3 experimental groups at 3 time points (1, 2, 4 wk) in soleus muscle.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Shifts in %MHC protein (types I, IIa, IIx, and IIb) and mRNA signal from normal Con value in 3 experimental groups at 3 time points (1, 2, 4 wk) in VI muscle.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Shifts in %MHC protein (types I, IIa, IIx, and IIb) and mRNA signal from normal Con value in 3 experimental groups at 3 time points (1, 2, 4 wk) in plantaris muscle.

	RESULTS

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Body weight and muscle weight. Relative to the Con group, both the absolute and relative muscle weights were significantly different in both the soleus and plantaris muscles after 2 wk of intervention involving the two groups exposed to suspension (Table 1). In view of the fact that there were some differences in body weight across the various experimental groups, due to the large number of animals processed in the present study, we used the normalized muscle mass for comparing the responses for a given muscle. This relative reduction in normalized muscle mass generally was similar between the 2- and 4-wk time points. T₃ treatment also induced small reductions in mass for the soleus and plantaris muscles. These observations are consistent with previous findings which indicate that both muscle unloading and T₃ treatment causes a reduction in the normalized muscle mass (2, 6, 7, 12, 17). In addition, analyses of heart weight revealed that for all T₃-treated animals, the heart-to-body weight ratios were significantly higher than those of the non-T₃-treated groups, the increase ranged from 30 to 60%, and the increase was significant at each time point (data not shown). These increases in relative heart weight verify that the T₃-injection protocols were effective in inducing a hyperthyroid state, which is characterized by cardiac enlargement (20).

Type I MHC mRNA and protein expression. There was a general pattern which indicated that type I MHC mRNA abundance for all three muscles was reduced relative to that expressed in the Con group under the three experimental manipulations imposed (Table 2 and Fig. 1). The results also suggest that the magnitude of the response was T₃ + HS > T₃ > HS at any given time point. The impact of the combined T₃ + HS manipulation also was readily apparent after only 1 wk of treatment. These findings suggest that the relative level of type I MHC mRNA expression is compromised by both the state of unloading and by T₃ treatment, and the response becomes augmented further when the two manipulations are combined. These responses, seen at the message level in each of the muscles, were essentially mirrored at the protein level of analysis (Figs. 2-4). However, the peak shifts in type I MHC protein were seen after 4 wk of manipulation, whereas the changes seen in the message signal often were of greater magnitude for the HS and T₃ + HS groups at the earlier time points, i.e., 2 wk.

Type IIa MHC mRNA and protein expression. In contrast to the response seen for type I MHC mRNA, changes in the type IIa MHC mRNA profile were much more varied across the three muscles, particularly during the early stages of a given manipulation (Tables 2 and 3, Figs. 2-4). For example, in the soleus muscle, although the changes were somewhat variable during the first two time points across the manipulations, all three experimental groups showed an increase in type IIa MHC mRNA relative abundance after 4 wk of treatment compared with Con values (Table 2 and Fig. 3). The combined intervention (T₃ + HS) induced a greater relative increase than either manipulation alone (T₃ or HS). In the VI muscle, this pattern of increase appeared to only hold true for the T₃ treatment alone, and no clear cut response was apparent when T₃ treatment and suspension were combined. On the other hand, in the plantaris muscle, the overall pattern of change appeared to be different than that seen for the two slow muscles, i.e., there was a net reduction in type IIa MHC mRNA relative abundance compared with the control state in response to both T₃ and T₃ + HS treatments (Fig. 4). These observations suggest that the type IIa MHC gene may be differentially regulated by various interventions in fast vs. slow types of skeletal muscle. The changes seen at the protein level for the type IIa MHC also were similar in direction to that seen at the mRNA level for the various treatments at the later time points (Figs. 2-4). Although there were some exceptions to these parallel responses, they were attributed to the relatively small changes seen for this particular isoform. Clearly the regulation of type IIa MHC is complex and is differentially affected by all three interventions (including their interactive effects) in both a temporal and muscle-specific fashion.

Type IIx MHC mRNA and protein expression. In the soleus muscle, the relative abundance of the type IIx MHC mRNA signal was very low, and type IIx MHC protein was inconsistently detected in the control muscles. However, type IIx MHC mRNA expression was increased significantly with all treatments (compared with the normal Con condition), with the T₃ + HS group showing a greater responsiveness relative to that of the individual treatments (Tables 2-3, and Figs. 1-4). Also, this pattern of mRNA response in the HS and T₃ + HS groups appeared to peak at the 2-wk time point relative to either the 1- or 4-wk values. A similar pattern occurred in the VI muscle, with the maximum increase in signal occurring at 4 wk in both the HS and T₃ + HS groups (Table 2, Fig. 3). In contrast, the opposite pattern of response occurred in the plantaris muscle in that the type IIx mRNA signal was reduced across all treatments and time points, except that the HS group showed little change at the earlier time points (Fig. 4). This overall pattern of response in the plantaris is similar to that observed for the type IIa MHC and further suggests that there is a differential level of regulation of the fast type IIa and IIx isoforms in fast vs. slow types of muscle. The directional changes in the type IIx mRNA responses for each of the three muscles were also seen at the protein level, and these changes were significant by the 2-wk time point. Peak changes, however, were not seen until 4 wk of treatment (Table 3 and Figs. 2-4).

Type IIb MHC mRNA and protein expression. In the soleus muscle, which normally does not express any significant (e.g., consistently detectable) amounts of IIb MHC mRNA and protein under normal control conditions, both the HS and T₃ + HS groups (but not the T₃ group alone) demonstrated a significant increase in the IIb MHC mRNA signal for each time point (Table 2, Figs. 1 and 2). A similar pattern occurred in the VI muscle (Fig. 3), including also a great response to T₃ after 2 and 4 wk of treatment. This pattern also was mimicked in the plantaris muscles (Figs. 1 and 4). Consequently, the general pattern of adaptive change for the IIb MHC gene is directly opposite to that of the type I MHC gene in response to the perturbations of either T₃ treatment or muscle unloading.

	DISCUSSION

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Previous studies have investigated the separate effects of T₃ treatment (4, 7, 15, 19, 22) and of states of unloading and/or spaceflight (4, 6, 11, 17) on MHC isoform expression (including some analyses involving MHC isoform mRNA). However, to our knowledge, no study has systematically examined, especially in a controlled, time-related experiment, either the separate or combined interventions of T₃ treatment and of muscle unloading on MHC expression, particularly by using approaches involving a primary focus on mRNA analyses to assess the role of pretranslational events in this adaptive process. Thus we undertook the present study in which we report several novel observations concerning MHC plasticity that could not be predicted on the basis of previous reports.

Overall, the combination of T₃ + HS exerted a more pronounced and rapid transformation in MHC gene expression that was, in part, both qualitatively and quantitatively different than that of either intervention alone. Moreover, the transformations which occurred in response to both the separate and combined interventions were both muscle type (fast vs. slow) and MHC isoform specific. The key features of the adaptive response for each MHC isoform are delineated below.

Type I MHC responses. The combination of T₃ + HS caused a marked decrease (by 80-90%) in type I mRNA levels that essentially was optimized after only 1 wk in each of the three muscles examined. This downregulation of type I MHC mRNA most likely contributes significantly to the transformation of MHC phenotype that we have reported to occur elsewhere (3) and in a companion study (1) in the soleus muscle, such that individual fibers normally expressing only the type I MHC become fully repressed (3). These fibers become rapidly converted into an assortment of hybrid fibers that express different combinations of multiple isoforms, including a predominance that expresses all four MHC isoforms, with the faster isoforms (types IIx and IIb) that account for the major component of the total MHC pool (1, 3). Furthermore, the marked impact of T₃ + HS on pretranslational processes reported herein is in contrast to the adaptive process seen in response to short-term (6-14 days) spaceflight in the soleus muscle, in which translational and posttranslational processes appear to more strongly contribute to the reduced level of type I MHC content that is maintained in the unloaded state (2, 6, 17). Thus the unique paradigm of elevated circulating T₃ coupled with muscle unloading rapidly creates an environment that strongly impacts either the transcriptional activity of the type I MHC gene or the inherent stability of the type I mRNA pool. This response, in turn, contributes to the progressive downregulation of type I MHC protein expression that occurs throughout the time course of the study in each of the muscles (Figs. 2-4).

Type IIb MHC responses. In contrast to the response of the type I MHC noted above, T₃ + HS induced a remarkable increase in the expression of the type IIb MHC in both the soleus and VI muscles (e.g., antigravity muscles). Such a response could not be achieved (or predicted) on the basis of responses seen with either T₃ treatment or HS alone. Whereas previous studies have reported some evidence for augmenting trace amounts of type IIb MHC expression on the basis of both protein (14) and mRNA (6, 11, 17) analyses, we are not aware of any study that has reported either an activity- or hormone-induced paradigm with the effect of increasing type IIb MHC expression in soleus muscle as reported in the present study. The impact of such a response involving type IIb MHC expression in the soleus occurred to such an extent that this isoform (which is the fastest MHC isoform in striated limb muscle) likely became sufficiently expressed in the majority of fibers in the T₃ + HS soleus to chiefly contribute to its transformation into a faster contracting muscle, based on its maximal velocity of shortening (3). The events augmenting type IIb MHC expression appear to be largely driven by pretranslational processes (and, we postulate, by transcriptional processes as well). Consequently, it appears that the unique combination of T₃ + HS treatment creates a novel environment in the regulation of type IIb MHC gene expression in a fiber domain that is normally incapable of expressing quantitative amounts of this gene. Clearly, more research is needed to elucidate the mechanism of this underlying unique type of regulation (see Type IIa MHC responses).

Type IIx MHC responses. Previous studies suggest that in rat skeletal muscles, the type IIx MHC is upregulated at both the protein and mRNA level in response to HS and/or spaceflight (6, 17). The present results not only build on this information but show that the increased expression of the type IIx MHC in antigravity skeletal muscles (soleus and VI) can be augmented to a similar extent at the protein level by both HS and T₃ treatment. Interestingly, although both interventions contribute to the augmentation of type IIx expression that occurs in response to T₃ + HS, HS in and of itself appears to generate a disproportionately greater mRNA signal relative to the level of MHC protein that is induced (Figs. 2-4); this could be caused by an impaired translation efficiency of the type IIx MHC product in the unloaded state. Additionally, in contrast to the responses seen in the soleus and VI muscles, in the plantaris muscle the type IIx MHC was downregulated in response to all three interventions. This pattern of response essentially refutes the logical premise that muscles inherently biased to expressing an abundance of the type II MHC isoforms would be highly responsive to upregulation of the type IIx MHC via stimuli normally inducing slow-to-fast MHC transitions. This assumption is based on findings that suggest that the type IIx MHC isoform is normally expressed in all fast-twitch muscles (10, 21, 28, 29, 34). Moreover, our present findings extend the classical findings of Izumo et al. (19) that show that altered T₃ states regulate MHC expression in a tissue-specific manner; we further suggest that mechanical loading may also exert a similar tissue-specific effect on MHC regulation.

Type IIa MHC responses. Of the four adult MHC isoforms, the findings of the present study suggest that the type IIa MHC, in relative terms, is both the least abundantly expressed and the most complexly regulated isoform. This is illustrated by the fact that 1) type IIa is either not appreciably affected or is downregulated by unloading in all the muscles investigated, which is consistent with previous observations on soleus muscle in response to spaceflight (6, 19); 2) type IIa is upregulated in response to T₃ treatment in slow muscle types but is downregulated in fast muscle types, respectively; and 3) type IIa expression is increased in the soleus but decreased in both the VI and plantaris in response to T₃ + HS. Part of the complexity in both the analyses of and the interpretation of adaptations for this particular isoform is that, as mentioned above and elsewhere (29, 34), type IIa is the least expressed of all the adult MHC isoforms in the spectrum of extensor muscles comprising the hindlimb of rodents, and the plasticity of expression is relatively small compared with the other isoforms studied in the experimental paradigms examined herein.