INTRODUCTION

TOP ABSTRACT INTRODUCTION MHC PLASTICITY IN CARDIAC... MHC PLASTICITY IN LIMB... MHC PLASTICITY IN ADULT... MHC PLASTICITY IN RESPONSE... DOES THE THYROID STATE... CONCLUSIONS AND FUTURE... REFERENCES

MYOSIN IS THE MOST ABUNDANT protein expressed in striated muscle cells, comprising ~25% of the total protein pool. The native myosin protein exists as a complex molecule, composed of two heavy chains (MHCs) and two pairs of light chains. [Although it is recognized that the light chains serve important modulatory roles during muscle contraction, they will not be a focus of this review.] This native protein serves as both a structural and regulatory protein (enzyme) in that 1) it forms the backbone of the sarcomeric structure designated as the contractile apparatus and 2) through its interaction with another sarcomeric protein, actin, it functions as a "motor" and transduces, via its ATPase activity, chemical energy (e.g., ATP) into the mechanical manifestation of force generation and/or sarcomere shortening and hence the muscle's production of mechanical work and power. From both a biological and functional perspective, an important feature concerning muscle structural/functional properties is the existence of the MHC gene family of motor proteins in which specific genes encode MHC protein isoforms. These isoforms have distinctly different ATPase (and shortening velocity) properties, thereby impacting the intrinsic functional properties of the individual myofibers in which they are expressed, which provide the molecular basis of a muscle fiber's functional diversity (94).

At least nine MHC isoforms have been identified in mammalian striated muscles (defined herein as heart and skeletal muscle), and they have been designated as 1) embryonic, 2) neonatal, 3) cardiac alpha (), 4) cardiac beta () or slow type I (as expressed in skeletal muscle), 5) fast type IIa, 6) fast type IIx/IId, 7) fast type IIb, 8) extraocular, and 9) mandibular or masticatory (m-MHC) (94). The embryonic and neonatal isoforms are expressed predominantly in developing skeletal muscles and can be detected in the adult muscle in regenerating fibers (30) as well as in specialized adult muscles such as the masseter and extraocular muscles (94). The - and -MHCs are expressed chiefly in cardiac muscle. -MHC has been detected in certain adult muscles such as the masseter (94). -MHC (type I) expression is not only confined to cardiac muscle but also is found in embryonic skeletal muscle and is the major isoform expressed in adult slow-twitch skeletal muscle (67, 94). In addition to type I MHC, adult skeletal muscles express various proportions of type IIa, IIx, and IIb MHC isoforms. The extraocular MHC isoform is confined chiefly to the eye and laryngeal muscles, whereas the m-MHC is expressed in the mandibular muscles of carnivores (94).

Although it has been long appreciated that the protein expression of these MHC genes is highly plastic in that it can be modulated by a variety of factors, including (but not limited to) fetal/embryonic developmental programs (3, 27, 28, 71, 76), innervation and associated neuronal firing patterns (85, 94), hormonal factors (17), and mechanical activity/inactivity factors (17, 105), the goal of this mini-review is to focus more specifically on the findings to date concerning the role that mechanical activity and inactivity paradigms play in altering MHC expression in both cardiac and limb skeletal muscle of neonatal and adult animal models (including, where feasible, human studies). This will be done in the context of certain hormonal factors such as thyroxin, which has been shown to act either independently of or synergistically with mechanical activity in regulating MHC gene expression. We fully recognize that diaphragm skeletal muscle responds to mechanical intervention and thyroid hormone in a fashion typical of limb skeletal muscle (21, 61, 78, 98, 99); however, this important topic was not covered due to space limitations. Also, several excellent reviews have been reported, the collection of which provide greater depth and breadth on the topic of MHC gene regulation than what can be provided in this mini-review (17, 41, 46, 47, 85, 93, 103, 105, 114).

	MHC PLASTICITY IN CARDIAC MUSCLE

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Developmental impact. In both humans and small animals, during the fetal developmental period of the heart, -MHC is the principal isoform initially expressed in the ventricles (66); -MHC, however, is the principal isoform expressed in atria of small and large neonatal and adult animals (66). However, in rodents shortly after birth, when thyroid hormone (T₃, 3,5,3'-triiodothyronine) titers begin to increase progressively, expression of the -MHC becomes rapidly decreased and is replaced by the -MHC such that, by ~3 wk of age, the -MHC is exclusively expressed in the ventricles of small mammals (but not in large mammals such as humans). As the rodent heart continues to mature to adulthood, the -MHC becomes reexpressed, accounting for ~10-15% of the total MHC pool. This regulation of the -MHC is thought to be due to an increase in the mechanical stress gradient that occurs across the wall of the myocardium as the chamber enlarges during maturation to adulthood (103, 120). In humans, throughout the growth and development phase of the heart, it appears that the -MHC remains as the dominant isoform that is expressed in the ventricles under normal conditions, although there have been reports that low levels of -MHC can be detected (79). This residual pool of -MHC appears to be absent in patients under hemodynamic overload, suggesting there is some degree of cardiac MHC plasticity in the human heart. This is further illustrated by the increased expression of -MHC in the atria of human hearts under pressure overload (13, 93).

MHC plasticity in the adult rodent heart. As described above, the adult heart of small animals such as rodents and mice chiefly expresses the -MHC throughout the adult state. This MHC pattern can change toward increased -MHC expression under several pathophysiological conditions, including 1) chronic pressure overload (induced hypertension) (53, 68, 77, 80), 2) hypothyroidism or thyroid deficiency (TD) (17, 36, 52), 3) diabetes (33, 35, 54), 4) chronic energy (caloric) deprivation (55, 100), and 5) chronic endurance-type running (42, 69, 70). In the models of hypertension, caloric restriction, and exercise, the heart can increase expression of the -MHC approximately two- to fourfold above control levels. -MHC expression, however, can become exclusively expressed with TD and diabetes (56). Of the five interventions, TD appears to be the most potent. T₃ has been shown to exert its biological action via binding to its nuclear receptors, the thyroid hormone receptors (31, 91). The thyroid hormone receptors are ligand-dependent transcription factors that modulate transcriptional activity via interaction with specific DNA recognition sequences known as thyroid-responsive elements (TREs) located in the promoter region of the target genes (14, 39, 45). Several TREs are thought to be strategically located in the promoter region of the both the - and -MHC genes (38, 51, 60, 72, 89). T₃ regulates - and -MHC gene transcription in an antithetical fashion, increasing the expression of -MHC while simultaneously decreasing that of -MHC (89, 119). Interestingly, there is some evidence to suggest that the interventions of diabetes and chronic energy deprivation may mediate their effects either directly or indirectly through the actions of T₃ (56, 100). However, the mechanism(s) of overload-induced upregulation of -MHC gene transcription appears to be more complex and involves a different set of trans-acting factors and cis-regulatory elements in the -MHC promoter.

	MHC PLASTICITY IN LIMB SKELETAL MUSCLE

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Mammalian skeletal muscles can be generally classified into two major groups, slow-twitch and fast-twitch, based on their intrinsic contractile properties, which are, in part, determined by their MHC expression profiles (85, 94). Slow muscles of mammals predominantly express the slow type I MHC isoform with some varied proportion of type IIa, the slowest of the fast MHCs (11, 40, 41, 88, 94, 105). This profile is exemplified in muscles such as the soleus and vastus intermedius, which play a strong role in antigravity function. Fast muscles of small mammals such as the gastrocnemius-plantaris complex, the vastus, the extensor digitorum longus, and the tibialis anterior predominantly express the two fast isoforms, IIx and IIb, with variable proportions, depending on the muscle, the region of the muscle, or the specific animal (11, 32, 40, 41). It is interesting to note that, although the IIb MHC gene has been identified in the human genome (115, 116), evidence for its expression at the protein level has not been reported (84, 105). Thus, in human muscle, there appears to be only two fast isoforms that are expressed (IIa and IIx) in addition to the slow type I MHC. MHC analysis of human soleus muscle shows that this muscle expresses an approximately equal mix of type I and IIa isoforms, but the type IIx MHC is not expressed (58). In contrast, human fast muscles such as the vastus lateralis express a mix of all three types of MHC isoforms at variable proportions (58, 64), depending on the physical fitness and activity of the subject. For example, in moderately active humans (e.g., not undergoing rigorous endurance or resistance-type training exercise), the MHC profile of the quadriceps (vastus) muscle is ~50% slow type I, 40% type IIa, and 10% type IIx (5, 9, 62). On the other hand, world-class marathon runners and ultra-endurance athletes have been reported to have remarkably high type I fibers in their major muscle groups (as much as 95%) (9, 86), whereas muscles of sprinters and power weight lifters predominantly consist of the IIa/IIx fibers (7, 9). Whether these extreme patterns of MHC gene expression are due exclusively to genetics, specificity of training, or some combination of factors is uncertain.

Polymorphism of MHC isoform expression in single myofibers. Before examining how various forms of activity/inactivity induce skeletal muscle transformations related to development as well as adult muscle plasticity, it is important to point out that single fibers of both developing and adult skeletal muscle exist as hybrids, i.e., fibers coexpressing more than one MHC isoform in various combinations (37, 96, 109, 110). This is best illustrated by single-fiber analyses of the plantaris muscle during development at 5-, 20-, and 40-day postpartum (37) (Fig. 1). This study (37) showed that, at 5 days of age, the muscle contains a large population of fibers that is polymorphic, with a predominance of fibers coexpressing the embryonic and neonatal isoforms (~60%). The other 40% of the fibers coexpressed various combinations of developmental and adult isoforms consisting mainly of embryonic/neonatal/I- and embryonic/neonatal/IIb-expressing fibers (37). By 20 days of age, ~10% of the fibers expressed pure type IIb or type I isoforms, and the rest of the fiber population coexpressed various combinations of adult fast MHC isoforms with some low proportion of neonatal MHC (37). By the adult state, i.e., 40 days of age, only a small pool of fibers (~15%) expressed one MHC isoform (I or IIb), whereas the rest of the population consisted of hybrid fibers coexpressing adult fast MHC isoforms with ~35% coexpressing IIx and IIb (37). This polymorphic nature of single fibers was also demonstrated by Caiozzo et al. (18) in the adult plantaris. Among the adult skeletal muscles, the soleus seems to consist of the most homogeneous fiber-type population. In fact, single-fiber analyses of adult rat soleus muscle revealed that over 70% of the total fibers express only type I MHC isoform, 5-10% of the fibers express type IIa isoform, and the remaining are type I/IIa hybrid fibers coexpressing type I and IIa MHC isoforms in different relative proportions (15) (see Fig. 3). The significance of MHC polymorphism in single myofibers is not clear; perhaps, it could lead to a more efficient MHC transition in a muscle under altered functional demands. Given the pattern of MHC expression in rodent muscles, it would appear that the phenotype remodeling resulting in the isoform shifts largely involves changes in MHC isoform content among the many fibers that normally express a combination of isoforms such as IIx/IIa and IIx/IIb and type I/IIa (15, 16, 18, 37). Therefore, this polymorphism could be a characteristic of fibers with high adaptive potential, i.e., hybrid fibers are more suitable to switch phenotype to meet new functional demands.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Myosin heavy chain (MHC) isoform polymorphism in single fibers of the plantaris muscle in normal developing rats at 5, 20, and 40 days of age. The x-axis represents the fiber types based on different combinations of coexpressed MHC isoforms. The y-axis represents the %fiber type relative to the total fiber population. The fill of each bar represents a specific MHC isoform (see bar legend). The percentage of a given MHC isoform within each fiber type can be determined by comparing height of bar for a given isoform to total height of the corresponding bar. Note that other fiber types are also found, but they represent only a minor proportion (<2%); therefore, they are not shown on the graph for simplification. E and Emb, embryonic; N and Neo, neonatal; I, type I; A, type IIa; X, type IIx; B, type IIb. [Data adapted from di Maso et al. (37).]

Developmental impact. Although the formation of the skeletal muscle system begins during the latter stages of fetal development, it is important to recognize that, in terms of MHC gene expression, both slow and fast muscles are still in an undifferentiated state at the time of birth. For example, in the rodent soleus muscle, which is destined to be composed chiefly of slow, type I fibers, both the embryonic and neonatal isoforms are abundantly expressed shortly after birth, accounting for ~50% of the total MHC pool with the remaining protein comprised of the slow type I MHC (3, 12, 27, 29). In fast muscles, the degree of undifferentiation is even greater, as the embryonic and neonatal MHC isoforms account for ~90% of the total MHC pool shortly after birth (3, 37). During the first 3-4 wk of neonatal development, both slow and fast muscles rapidly grow and differentiate into their adult MHC phenotype (3, 29, 37). Although this process requires an intact nerve (3), the two muscle types appear to differ substantially in the primary factors that drive this transformation process. On the basis of recent studies comparing muscle development of neonatal rats raised in a microgravity environment of spaceflight vs. rodents raised on Earth, it appears that slow muscle types are heavily dependent on weight-bearing activity (which begins during the second week following birth) for both normal growth and the optimal expression of type I MHC (1). In the absence of the weight-bearing state, muscle growth is dramatically reduced, and the MHC pool becomes biased to fast MHC expression, with the type IIa MHC becoming the most predominant isoform that is expressed (1). In contrast, it appears that weight-bearing activity is not essential for the fast-type muscles to achieve the adult fast MHC phenotype, which, as mentioned above, normally consists chiefly of the fast IIx and IIb isoforms (1). Instead, an intact thyroid state (e.g., circulating T₃ levels) appears to be necessary to downregulate the neonatal isoform and replace its expression with the IIb isoform (1, 37). If insufficient T₃ is available, the fast muscle remains undifferentiated for a longer duration; whether this undifferentiated state remains permanent with severe TD remains to be determined (37). Interestingly, TD in the neonatal state actually induces greater expression of the slow type I MHC in both slow and fast skeletal muscles, and this occurs independently of gravity (1). Thus it appears that circulating T₃ is necessary for the normal growth axis of the body as well as the skeletal muscle system to occur. However, weight-bearing activity is necessary for the ability of skeletal muscle to fully express the type I () MHC gene in antigravity muscles.

	MHC PLASTICITY IN ADULT SKELETAL MUSCLE IN RESPONSE TO INCREASED ACTIVITY PARADIGMS

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In this review, we will focus on three models of increased mechanical activity: 1) the continuous functional overloading of targeted muscles by the surgical removal of their synergists, 2) the intermittent periodic loading of muscles through heavy resistance-training paradigms (e.g., high loading but relatively low contraction frequencies), and 3) endurance exercise such as running (i.e., relatively low force output but high contractile frequencies during each training session). The functional overload model has been primarily used on small animals to establish the upper limits of muscle fiber remodeling in response to a chronic level of overload stress. This perturbation has been shown to cause marked fiber enlargement (without concrete evidence of hyperplasia; Ref. 108) along with significant changes in the contractile protein phenotype (11, 18, 101, 102, 113). The resistance-training paradigms have been shown to induce modest degrees of muscle fiber enlargement, and some evidence of hyperplasia under extreme conditions involving certain human and animals subjects has also been shown (48). Endurance exercise is not associated with significant increases in muscle fiber enlargement; rather, the adaptations are confined chiefly to increases in mitochondrial expression and moderate fast-to-slower MHC isoform shifts (41).

Functional overload. Studies on the functional overload model clearly show that, in fast muscles such as the plantaris, the relative expression of the fast type IIb and IIx MHCs decreases at both the protein and mRNA levels, whereas both the fast IIa and slow type I are increased in expression at both the mRNA and protein level (14, 18, 90, 101, 104). If an endurance-exercise stimulus is imposed on muscles that are functionally overloaded, the exercise stimulus enhances the repression of the fast IIb MHC while further augmenting the expression of the fast IIa MHC (97). Thus the combined intervention of overload and endurance exercise further biases MHC expression in a direction toward a slower phenotype. Recent studies using transgenic mice (74, 112, 118) and rodent muscles transfected with the type I MHC promoter (43) showed that functionally overloaded plantaris muscles increase type I MHC gene expression by a tight coupling of transcriptional and translational events. The control of transcription appears to be mediated by factors that interact with regions in the type I MHC gene promoter that reside in the first 300 base pairs upstream of the initiation start site (43, 74, 75, 112, 117, 118).

Resistance training. Studies using resistance-training paradigms have used both small animals, such as rats, and humans as subjects. In the rodent model, contraction paradigms of either the concentric, eccentric, or isometric type, in which the targeted muscle group is electrically stimulated to contract against a computer-driven ergometer system (14, 19), have been shown to induce significant decreases in type IIb expression (protein and mRNA) and concomitant increases in the expression of the fast type IIx MHC. In humans, resistance training has been shown to produce a similar response with the exception that it is the fast type IIx MHC that is downregulated and the fast IIa MHC that is upregulated (2, 7, 22, 63). If the subjects are detrained for several weeks, it appears that this process is reversed and the reexpression of the fast type IIx appears to be even greater than the level that was originally expressed in the pretraining period (5). Furthermore, although it appears that the level of type IIa expression in human subjects is associated to a greater extent in individuals involved in resistance training, it is uncertain whether the MHC profiling exerts a strong influence on strength adaptations, especially in the initial stages of training (22).

Endurance exercise. The effects of endurance exercise, such as running, on the MHC profile appear to be both muscle specific and dose dependent. For example, in rodents, when the animals are trained to run at moderate to high intensity (~30 m/min; ~20% incline at ~75% maximal O₂ uptake) for several weeks, the running effects on the MHC profile of the soleus are manifest only when running lasted for longer durations (60 and 90 min/day), with the overall profile maintained with a slow, type I MHC predominance (32). In mixed fast muscles and in portions of fast muscles (red vastus and gastrocnemius), which have an intrinsic bias to type IIx and IIa MHC expression, both the type IIa and IIx MHCs are upregulated relative to the sedentary state, whereas the IIb MHC is significantly downregulated (32). If the running is extended for more extreme durations, it is possible to induce increased expression of the type I MHC (32, 49). An exception to these high-intensity exercise-induced MHC shifts to a slower muscle phenotype appears to occur when rodents are trained at moderate to high running intensity without the added stress of working against a steep incline. Under these conditions, the exercise induces upregulation of the IIb MHC, whereas that of the IIx MHC is decreased (97). To our knowledge, this is the first report that a specific activity paradigm can enhance the expression of a fast isoform (e.g., IIb) in an already fast-twitch muscle. This type of regulation has been primarily associated with inactivity paradigms as described below.

	MHC PLASTICITY IN RESPONSE TO DECREASED WEIGHT-BEARING ACTIVITY PARADIGMS

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A variety of approaches have been used to examine muscle fiber plasticity in response to reduced weight-bearing activity, and this review will summarize results on four key models: 1) spaceflight, in which the lower limb muscles remain active but are unloaded (i.e., they produce low force output); 2) hindlimb suspension, in which only the lower limbs are prevented from weight bearing; 3) spinalization (spinal transection, ST), in which the cord has been either injured or severed; and 4) spinal isolation (SI), in which the cord is severed in two places and afferent (sensory) input is eliminated between the two lesions. It is important to point out that in the first two models, although the lower limbs (leg muscles) become relatively unloaded in terms of their force-generating requirements, the extensor muscles remain electrically active, based on the electromyographic patterns that are elicited under these conditions (105). However, in spinalization or spinal injury, electrical activity of the motoneuron pools can be activated by sensory input, but the force-generation outputs of the muscles are dramatically reduced (105). In the case of the unique model of SI, the motoneurons connected to the muscle remain intact, but the neurons are electrically silent due to their inactivation (50). Thus the key feature of this latter model is that it establishes a true inactivity baseline in that the muscles are electrically silent and unloaded, but they still can be influenced by trophic factors released from the neurons. In each of these experimental settings, the extensor muscles of the legs undergo marked degrees of atrophy, and the MHC profiles can be significantly altered to reflect slow to fast transitions in MHC phenotype, especially in the antigravity muscles (105).

Spaceflight and hindlimb suspension. Exposure of rodents to the microgravity environment of spaceflight of varying duration (4-16 days) induces, in addition to the atrophy response, significant transitions in the MHC isoforms. These effects are most prominent in slow muscle types involved in antigravity function. These adaptations chiefly involve downregulation of the slow type I MHC with the concomitant de novo expression of the fast type IIx MHC (1, 10, 11, 20). These transitions appear to consist of hybrid fibers that express both the slow type I and fast type IIx MHC (20, 105). Additional findings show that these transformations are regulated by pretranslational events, since the mRNA profiles are generally consistent with those observed at the protein level (1, 20). Similar patterns are observed with the hindlimb suspension model (15, 16, 57, 111), with the exception that, over time, the slow to fast transitions become more exaggerated because the unloaded conditions can be extended for longer durations, i.e., up to 8 wk (111). Although human studies involving spaceflight and ground-based models of unloading have not been extensive, the available information suggests that human muscle undergoes type I to type II MHC transitions similar to those reported for rodents; these transformations occur on a relatively similar time scale as those reported above for rodents (105).

ST and SI. ST of both cats and rats has been shown to cause transformations in MHC expression that are qualitatively similar in fashion to those reported for spaceflight/hindlimb suspension experiments (105). In the cat, the type I MHC is downregulated and the fast IIx MHC (the fastest MHC isoform identified in cat muscle; there is little evidence that type IIb MHC is expressed) becomes predominantly expressed (105). However, rodents show a higher degree of MHC isoform transformation after ST in that there is greater downregulation of the type I MHC with accompanying increases in the relative expression of all three fast MHC isoforms (105, 107). Interestingly, the type IIx MHC, which is normally not expressed in the soleus muscle, increases extensively in relative expression and can account for almost 50% of the total MHC pool (105, 107). Furthermore, there is also de novo expression of the IIb isoform, but this isoform only accounts for ~10% of the MHC pool. Clearly, the slow muscle of ST rats is essentially converted to a fast muscle, based on the MHC profile and shortening velocities seen in these muscles after 3 and 6 mo of ST (105). Importantly, in humans suffering spinal cord injuries with lower extremity paralysis and the inability to weight bear, there appears to be a considerable delay in the transformation of slow to fast MHC profiles compared with that seen in animal models (8, 23, 24, 105). For example, although there is little evidence for these transitions occurring during the first ~10 mo following the initial trauma, patients with long-term spinal cord injuries show significant type I to IIa and IIx transitions after many months, and the type IIa and IIx MHCs isoforms have been reported to account for as much as 90% of the MHC protein pool in some of these patients (8). The reason for such a slow transition process is unknown. Also, these changes in MHC gene expression seem to be uncoupled from the rapid muscle atrophy that takes place as the muscles lose their weight-bearing capacity.

	DOES THE THYROID STATE MODULATE ACTIVITY/INACTIVITY-INDUCED CHANGES IN SKELETAL MHC EXPRESSION?

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In the past two decades, it has become increasingly apparent that thyroid hormone (T₃) exerts a profound effect on the plasticity of striated muscle given its strong influence on the expression of the MHC gene family of motor proteins (17, 26, 65, 81, 121). The altered T₃ state as well as the altered loading state (mechanical activity) induce similar shifts in MHC gene expression in striated muscles. For example, under conditions of either hypothyroidism (TD) or increased loading state, all muscles are transformed to a slower phenotype. The magnitude and specific isoform shifts in this transformation vary with the muscle type. For example, IIa/IIx-to-type I MHC transformations are observed in slow muscles, whereas IIb/IIx-to-IIa/type I shifts are observed in fast, mixed muscles (17). In contrast, it has been shown that either hyperthyroidism (+T₃) or unloading induces muscle transformation to a faster phenotype involving repression of type I () MHC expression and enhancement of fast IIx/IIb MHC isoforms (17). In these latter transformations, the impact of +T₃ on the type II MHCs appears to differ from that of unloading-induced stimuli, depending on the muscle in question. In soleus muscle, the type IIa MHC is upregulated in response to +T₃ treatment, whereas the type IIx MHC is increased in response to unloading (15, 16). Also, in the vastus intermedius muscle, which is normally biased to expressing the slow type I MHC but also expresses all three fast type II MHCs, T₃ treatment induces the upregulation of all three type II MHCs, whereas unloading primarily upregulates expression of the IIx MHC, as is the case for the soleus. Consequently, although the mechanical loading state and the T₃ state demonstrate similar effects on mainly the type I MHC gene, subtle differences, which appear to be muscle specific, are seen in terms of type II MHC expression.

Nevertheless, given the general similarities between the effects of mechanical loading and effects of the thyroid state on MHC gene regulation and the fact that both stimuli impact transcriptional events (44, 74, 75, 87), the question arises as to whether the effects of mechanical loading on, for example, type I MHC gene expression are mediated via the action of the thyroid hormone, a potent modulator of type I MHC transcriptional activity (87, 89, 119). In an attempt to address this issue, our group has used several different experimental paradigms in which thyroid hormone (T₃) either 1) was competed against altered loading states (i.e., +T₃ vs. functional overload; TD vs. hindlimb suspension; Refs. 34, 101) or 2) was used in conjunction with an altered loading state (+T₃ plus hindlimb suspension or TD plus functional overload; Refs. 15, 18, 57) to test possible interactions between the two stimuli on MHC gene expression. We reasoned that, if thyroid hormone and mechanical interventions acted through different processes (signaling pathways and transcriptional control mechanisms), one intervention would override the other when they were competed, whereas the two would act synergistically when they were allowed to interact. The findings suggest the following. 1) When +T₃ was competed against functional overload and hypothyroidism was competed against hindlimb suspension, the thyroid state effectively neutralized the mechanical intervention altering type I MHC expression (34, 101). These results suggested that the two stimuli could be acting via either a common pathway or two independent pathways; in the latter case, they would neutralized one another. 2) When the +T₃ stimulus was administered in conjunction with hindlimb suspension and TD was used in conjunction with functional overload, their combined effects were synergistic (more than additive) in terms of affecting type I MHC gene expression (15, 18, 57). These latter findings suggest that T₃ and the mechanical loading state exert their regulation through different pathways and/or mechanisms. In support of this conclusion, we have also demonstrated that, in the T₃ plus hindlimb suspension model and in the model of TD plus functional overload, thyroid treatment by itself, mechanical intervention by itself, and their combined interactions exert their respective effects by inducing a MHC gene distribution profile that is clearly different from one experimental condition to another as manifest across the spectrum of individual fibers comprising the test muscles (15). This conclusion was based on observations that there is a uniquely different polymorphic distribution (two or more MHC isoforms become expressed in a different fiber) pattern of the various MHCs that comprise the individual fibers making up the muscles of the above-mentioned experimental groups when the data are plotted in a MHC histogram distribution profile (see Figs. 2 and 3). The idea that a given fiber expresses only a single MHC isoform appears to hold true only in the case of the slow MHC fibers that are expressed in the soleus muscle of normal animals (Fig. 3). In normal, fast muscles and in both fast and slow muscles that undergo alterations in MHC gene expression as reviewed herein, the pattern of MHC expression becomes more polymorphic such that multiple isoforms are contributing to the biochemical composition of the individual fibers and the polymorphic pattern differs significantly with different perturbations (15, 18, 57).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Single-fiber MHC protein isoform composition of plantaris muscles after 6 wk of experimental manipulations. All possible combinations of adult MHC isoform expression are shown along the x-axis. Each bar represents the proportion of a given population relative to the total population of fibers examined for that experimental group. Solid, dark gray, light gray, and open bars represent relative amounts of slow type I, fast type IIa, fast type IIx, and fast type IIb MHC protein isoforms, respectively, found within any given fiber type. Note that the control (CON) plantaris muscles contained a large number of polymorphic fibers, with the predominant population coexpressing the fast type IIx and IIb MHC protein isoforms. The combined treatment of thyroid deficiency and overloading (T3+OV; T3 = 3,5,3'-triiodothyronine) produced a significant reduction in the proportion of population of IIx to IIb fibers and a large increase in fibers coexpressing all 4 MHC protein isoforms. [Adapted from Caiozzo et al. (18).]

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Single-fiber MHC protein isoform composition of soleus muscles after 4 wk of experimental manipulations. All possible combinations of adult MHC isoform expression are shown along the x-axis. Each bar represents the proportion of a given population relative to the total population of fibers examined for that group. Solid, dark gray, light gray, and open bars represent relative amounts of slow type I, fast type IIa, fast type IIx, and fast type IIb MHC protein isoforms, respectively, found within any given fiber type. Note that the control soleus muscles predominantly contained fibers expressing only type I MHC isoform (>70%). T3 is associated with reduction in homogeneous type I fibers to <10% and an increase in polymorphic fibers coexpressing I/IIa and I/IIa/IIx. Similarly, hindlimb suspension (HS) is associated with a decrease in pure type I fibers to ~40%, with the remaining fibers expressing at least three (I, IIa, IIx) or all 4 MHC isoforms. Interestingly, the combined treatment (T3+HS) transformed the normally homogeneous soleus fiber population into one possessing a most heterogeneous profile. In fact, fibers expressing all 4 MHC isoforms were predominant. N = number of analyzed fibers per group. [Data adapted from Caiozzo et al. (15).]

Are transitions in MHC gene expression obligated to follow a prescribed sequence? Previous reports proposed that, in adult muscles, the normal transition in MHC expression, especially in response to a powerful model of chronic electrical stimulation (not covered in this review), proceeds in an ordered sequence in the direction IIb  IIx  IIa  I and vice versa, depending on the type of perturbation imposed on the target muscle (85, 95, 105, 109). In this scheme, it is proposed that, if a muscle initially expressed only the type I MHC and was subjected to an unloading stimulus, the muscle would be expected to proceed by first expressing the IIa MHC; from this point, if the IIx were to be upregulated, the transition would proceed via the coupled downregulation of the IIa MHC and so forth. In other words, the muscle fiber would be obligated to express the type IIa before being programmed to express the IIx and vice versa. However, in several of the rodent models examined in this review (e.g., SI, hindlimb suspension, hindlimb suspension plus T₃ treatment, TD plus functional overload) unique patterns of MHC isoform expression were observed as a result of the quantitative analyses of MHC expression that occurred at the single-fiber level in these collective experiments (15, 18, 106) (see Figs. 2 and 3). These observations revealed that a significant percentage of the fibers simultaneously expressed either type I/IIx hybrids, type I/IIb hybrids, or type I/IIx/IIb hybrids, i.e., patterns of MHC gene transition that do not conform to the model of ordered sequence of transition presented above. Interestingly, in the +T₃ plus suspension model (15), after as little as 1 wk of intervention, ~15% of the fiber pool of the soleus muscle simultaneously and abundantly expressed all four adult MHCs (type I, IIa, IIx, and IIb). This same pattern was observed in the plantaris muscle after 6 wk of TD plus functional overload (18). Furthermore, it is noteworthy to mention that this exception in single-fiber polymorphism was also observed in human muscle fibers when subjects were on bed rest. A small proportion of fibers was found to coexpress type I and IIx in the absence of IIa (6). These fibers were termed jump fibers (6) in that they do not conform to proposed sequential transition of the continuum theory of MHC isoform expression (95). Collectively, these findings suggest that 1) individual muscle fibers comprising both fast and slow muscles are not obligated to follow a fixed sequence of transitioning MHC gene expression, and 2) the majority of fibers comprising both fast and slow muscles have the genetic machinery to express all the adult MHC isoforms known to be expressed in the limb muscles of rodents. These findings suggest that there is a tremendous degree of plasticity in MHC gene expression that can be induced in striated muscle, depending on the environmental conditions imposed on a given muscle.

	CONCLUSIONS AND FUTURE DIRECTIONS

TOP ABSTRACT INTRODUCTION MHC PLASTICITY IN CARDIAC... MHC PLASTICITY IN LIMB... MHC PLASTICITY IN ADULT... MHC PLASTICITY IN RESPONSE... DOES THE THYROID STATE... CONCLUSIONS AND FUTURE... REFERENCES

The findings reported in this brief review clearly show that both cardiac and skeletal muscles of experimental animals are initially undifferentiated at the time of birth and that these tissues undergo a marked level of growth and differentiation in attaining the adult MHC phenotype. In rodents, both an intact thyroid axis and weight-bearing activity appear to be needed for normal muscle growth and the MHC differentiation process to occur in both antigravity and locomotor muscles. The cardiac muscle of small mammals is highly sensitive to TD, diabetes, energy deprivation, and hypertension, and each of these interventions induces either downregulation or maintained expression of the fast -MHC, which is coupled to marked upregulation of the -MHC to increase the expression of an isoform that functions to economize myocardial contractile function. In skeletal muscle, interventions that unload or reduce the weight-bearing activity of the muscle cause slow-to-fast MHC conversions, whereas fast-to-slow conversions are seen when the muscles become either chronically overloaded or subjected to intermittent loading, as occurs during resistance training and endurance exercise. The regulation of MHC gene expression appears to be strongly regulated by transcriptional events, based on recent findings on transgenic models and animals transfected with promoter-reporter constructs. Findings also show that the thyroid state exerts equally profound transitions in the MHC phenotype in skeletal muscles, which appear to mimic that seen in the mechanical-loading paradigms. However, the mechanisms by which T₃ and mechanical stimuli exert their control on transcriptional processes appear to be different. Additional findings show that individual skeletal muscle fibers have the genetic machinery to express simultaneously all of the adult MHCs in unique combinations under certain experimental conditions. This degree of heterogeneity among the individual fibers would ensure a large functional diversity in performing complex movement patterns. Future studies must now focus on the signaling pathways and the underlying mechanisms of the transcriptional/translational machinery that controls these marked degrees of plasticity; these studies may help us understand the morphological organization and functional implications of the muscle fiber's capacity to express such a diversity of motor proteins.