I. INTRODUCTION

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The functional units of skeletal muscles are the muscle fibers, long cylindrical multinucleated cells. They vary considerably in their morphological, biochemical, and physiological properties. Different fiber types can be distinguished in each muscle. The fiber type composition, varying from muscle to muscle, is the basis of the well-known structural and functional muscular diversity. The fibers can change their characteristics in response to a large variety of stimuli leading to muscular plasticity. All muscles use Ca²⁺ as their main regulatory and signaling molecule. Therefore, muscle plasticity is closely linked with and highly dependent on the Ca²⁺ handling system.

In 1882 Ringer found that the isolated frog heart contracted when incubated in a solution prepared with London tap water but not in a one prepared with distilled water. This led to the important discovery that the ability of the heart muscle to contract depends on the presence of Ca²⁺ in the external solution (423). Indeed, it has been demonstrated that the function of all muscle types is controlled by Ca²⁺ as a second messenger.

Control of contraction and relaxation by Ca²⁺ in different types of muscle is achieved by three major mechanisms. The first activation mechanism, first discovered and best described, is the troponin-tropomyosin system associated with the actin filaments. It is restricted to skeletal and cardiac muscles. In the second mechanism, found in smooth muscles of vertebrates, Ca²⁺, together with calmodulin (CaM), activates myosin light-chain kinase, which (through phosphorylation of the myosin light chains) initiates muscle contraction. The third mechanism consists of direct binding of Ca²⁺ to myosin which regulates contraction in muscles of certain invertebrates such as scallop. This system depends on the presence of the regulatory light chains of myosin.

The aim of this review is to summarize the present knowledge of muscle plasticity in the context of Ca²⁺ signaling and handling, which is of crucial importance to our understanding of normal muscle function and muscle diseases.

This article focuses mainly on the complexity of the Ca²⁺ handling system in the skeletal muscle of mammals, although reference is made on several occasions to cardiac and smooth muscle and when appropriate to muscles of other vertebrates. Muscle fiber type diversity analyzed at the histological level and functional consequences of fiber type composition are described in some detail (see sect. II) to emphasize the importance of the morphological studies for understanding muscle plasticity in response to a given stimulus and its dependence on the Ca²⁺ handling apparatus.

Speed of muscle contraction and relaxation as well as other physiological parameters are critically dependent on the special composition of components belonging to the Ca²⁺ handling apparatus. Molecular details of the Ca²⁺ cycle as well as muscle fiber-type specific variations are presented and discussed at a structural and functional level. The main players in the Ca²⁺ cycle are indicated in the schematical presentation shown below.
In the resting state of the myofiber, Ca²⁺ concentrations in the cytosol are maintained at ~50 nM. The Ca²⁺ cycle starts with a surface membrane and transverse tubular (T system) depolarization leading to a release of Ca²⁺ from the sarcoplasmic reticulum (SR) via the ryanodine receptor (RyR), which elevates cytosolic Ca²⁺ locally to ~100 fold higher levels. The conversion of an electrical into a chemical signal at the t-tubule membrane is activated through charge-dependent structural changes of the dihydropyridine receptor (DHPR). Because the DHPR in skeletal muscles are not involved to a major degree in the initial increase of myoplasmic Ca²⁺ as Ca²⁺ channels, they are not discussed in detail. In the skeletal muscle Ca²⁺ binds in a fast reaction to one of the troponin subunits [troponin (Tn) C] on the thin filament. Upon Ca²⁺ binding to TnC, contraction is activated.

In addition to triggering muscle contraction through the troponin system, Ca²⁺ may also affect the muscle contraction apparatus through direct interaction with myosin and other motor proteins. In addition, Ca²⁺ controls the energetics of the muscle by regulating the provision of ATP when needed (512). The latter two aspects are not covered by the present review. Because several transcription factors are known to be regulated by Ca²⁺ (reviewed in Ref. 153), another, so far not addressed, possibility for Ca²⁺-dependent regulation of muscular activity would be by transcriptional regulation of genes for proteins important in the Ca²⁺ cycle.

In myofibrils, there is a variety of Ca²⁺-binding proteins (CaM, S100 proteins, calpains, calcineurin, sorcin, and annexins) that are not directly involved in the primary process of muscle contraction and relaxation but that may be important for muscle performance and plasticity. Therefore, some features of these proteins (especially CaM and calpain) with respect to their regulatory effects on the primary Ca²⁺ cycle components are discussed in this review.

Ca²⁺ translocation from the myofibril to the SR is likely to be facilitated in the fast-twitch skeletal muscle by the high-affinity Ca²⁺-binding protein parvalbumin (PV). However, both contraction and relaxation speed decrease as the animal gets bigger. This is mirrored by a decreasing PV content of fast-twitch fibers in larger animals; in humans, these fibers completely lack PV. The energy-dependent Ca²⁺ uptake into the SR is mediated by the SR ATPase, an enzyme that itself is regulated by both Ca²⁺- and CaM-dependent phosphorylation. The Ca²⁺ cycle is completed by binding of Ca²⁺ to the high-capacity, low-affinity Ca²⁺-binding protein calsequestrin.

A major goal of this article is to discuss consequences of malfunctioning of the crucial elements of the Ca²⁺ cycle that may lead to a variety of muscle diseases. Elevated cytoplasmic Ca²⁺ levels can cause activation of certain proteases, lipases, and nucleases. Altered physiological properties of muscle, altered gene transcription and transformation of muscle fibers, necrosis, and apoptosis may be consequences. Well-known Ca²⁺-related diseases described in this article are certain forms of myopathies (related to channel malfunctioning), malignant hyperthermia (related to Ca²⁺ release mechanisms), and dystrophinopathies which involve several Ca²⁺ handling systems and are therefore treated separately in sections IID, IIIB, and V.

Several recent reviews and book articles deal with various aspects of muscle plasticity and single components of the Ca²⁺ cycle apparatus. The reader is referred to just one article on each subtopic which should contain sufficient citations for further reading: muscle plasticity (396), myofibrillar protein isoforms (453), molecular muscle diversity (52), the ryanodine receptor (469), the troponin system (490), PV (390), the Ca²⁺-ATPase (320), calsequestrin (187), dystrophinopathies (499), calcium release channel diseases (351), and muscular channelopathies (296).

	II. SKELETAL MUSCLE AS A DYNAMIC ORGAN

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The fiber types present in a muscle can be distinguished with different morphological, biochemical, or physiological methods. Histochemical methods are based mostly on myofibrillar adenosinetriphosphatase (mATPase) activity or on enzymes of the aerobic and anaerobic energy metabolism (Fig. 1).

View larger version (131K): [in this window] [in a new window]   Fig. 1. Serial cross sections of human deltoid muscle stained for myofibrillar ATPase (mATPase) after preincubation at pH 10.5 or 4.3, cytochrome c oxidase (Cyt C ox), and -glycerophosphatase dehydrogenase (-glyc), respectively. The fiber types are indicated; for IIB (IIX) fibers, see text. They show a largely reciprocal staining pattern after acid and alkaline preincubation, respectively, as well as in the reaction for cytochrome c oxidase and -glycerophosphatase dehydrogenase, respectively. The plate illustrates the establishment of morphological fiber typing. Original magnification, ×95.

Morphological and functional differences of muscles and muscle fibers (mainly in frog and rabbit) were known for a long time (170, 412, 481). However, systematic fiber typing did not start until the 1950s after Krüger had distinguished in several vertebrates (including humans) fibers with "Fibrillenstruktur" (evenly dispersed myofibrils) and fibers with "Felderstruktur" (myofibrils arranged in bundles) (273, 274, 276). In the following decades several classification systems were proposed that are summarized in Table 1.

Dubowitz and Pearse (106) described in 1960 histochemically two fiber types, termed type I and type II, respectively. They showed that both fiber types displayed "reciprocal" activities of oxidative and glycolytic enzymes (Fig. 1). Using a histochemical assay for mATPase that was earlier introduced by Padykula and Herman (386), Engel (118) reported a low mATPase activity in the type I and a high mATPase activity in the type II fibers in humans. The group of type II fibers was subsequently subdivided into IIA and IIB fibers (Table 1).

Analyzing the rat semitendinosus muscle by electron microscopy, Gauthier (150) also could distinguish three major fiber types (Table 1). In subsequent ultrastructural and morphometric investigations of mammalian muscles, the classification was mainly based on the mitochondrial content and the width of the Z bands (115, 208, 379). With respect to mitochondria, it should be noted that in small mammals (e.g., mouse, rat) the highest amount of mitochondria is seen in type IIA fibers, whereas in larger animals this is the case with type I fibers. Type IIB fibers exhibit the smallest width of the Z band paralleled by the lowest relative volume density of mitochondria. However, human muscle fibers can more reliably be classified by electron microscopy on the basis of the M-band structure (474).

Schiaffino et al. (455) described in 1985 in the rat an additional fast-twitch fiber type that was later termed type 2X. In the late 1980s, Pette and co-workers (18) electrophoretically identified the myosin heavy chain (MHC) IId in rodents. Fibers containing this MHC isoform were termed type IID fibers. It was shown that type IID fibers are identical to type 2X (IIX) fibers and hence these fibers are designated as type IID/X fibers (Table 1).

In different species more than 10 intermediate fiber types have been described histochemically and biochemically in limb and trunk muscles (14, 46, 226, 231, 243, 251, 304, 433, 492, 494). Most of these subtypes or intermediate types have been shown to be hybrid fibers with respect to the coexistence of different types of myosin. In normal mature muscle fibers of humans and rodents, the coexistence of different slow- and fast-type MHC isoforms is frequently observed (30, 38, 94, 287, 395, 446, 447, 476, 493); all three MHC isoforms I, IIa, and IIb can occasionally be coexpressed in a single muscle fiber (447). With increasing age (see below) also the percentage of muscle fibers coexpressing two or three MHC increases (5).

Some muscles of the craniofacial region which are not concerned with locomotion, such as extraocular (e.g., rat, Ref. 548), laryngeal (e.g., rabbit, thyroarytaenoid, Ref. 312), or jaw-closing muscles (e.g., cat, masseter, temporalis, Ref. 434), exhibit "super-fast" fibers. These fiber types contain superfast MHC isoforms and are phenotypically distinct from both fast-twitch oxidative and fast-twitch glycolytic muscle fibers of the body and limbs.

Presently, for practical reasons, the most widely used classification of fiber types is still the one based on the pH lability of the mATPase activity which distinguishes only type I, IIA, and IIB fibers (46). Unfortunately, in many investigations, no distinction is made between type IIB fibers and the recently discovered IIX fibers (164, 452). The fast fiber types showing the lowest activity of the cytochrome c oxidase (or succinate dehydrogenase, SDH) and the highest activity of the -glycerophosphate dehydrogenase are also in humans, analogously to rat and mouse, frequently termed IIB fibers. However, in humans, these fibers express the IIx but not the IIb MHC isoforms (119, 446) and should be correctly designated IIX fibers. Therefore, these fibers are termed in this review IIB when dealing with rat, mouse, or rabbit muscle and IIB(IIX) when dealing with human muscle (Fig. 1). The histochemical staining characteristics of a given fiber type may vary considerably from species to species. The different schemes (Table 1) of classification are not fully interchangeable (79, 370, 488). Only recently simultaneous measurements of mATPase, SDH, and -glycerophosphate dehydrogenase activities and cross-sectional area in MHC-based fiber types have been performed (427). Significant interrelationships between these parameters have been found on a fiber-to-fiber basis.

The fiber types show differences in their oxidative and glycolytic capacities that generally correlate with differences in contractile and other physiological properties (Table 1). However, these correlations are only general ones. In a histochemically defined fiber type, the metabolic profile and the physiological characteristics need to be defined separately.

Twitch contraction (i.e., time to peak) and half-relaxation times differ to a great extent between fast-twitch and slow-twitch muscle fibers, but there is a substantial overlap between these two groups. These properties are critically dependent on the Ca²⁺ sensitivity of the contractile apparatus and on the efficiency of Ca²⁺ uptake into the SR. A third factor is the efficiency of the myosin motor itself, which is composed of different protein isoforms in different muscle fibers. Many events in the Ca²⁺ cycle also contribute to large differences in resistance to fatigue between fast and slow muscles (reviewed in Ref. 497).

Already at the beginning of this century specific regional variations in fiber composition have been noticed in mammalian muscles (96). In the rat, some neck and thoracic (165, 169, 314, 401) and most limb muscles (13, 16, 409, 410, 529) exhibit a predominance of oxidative type I and type IIA fibers in the deep portions and a predominance of glycolytic type IIB fibers in the superficial portions. This is demonstrated with staining for the fast fiber (IIA and IIB) specific PV in the rat extensor digitorum longus muscle (Fig. 2). Lexell et al. (302) found in extensor digitorum longus and tibialis anterior muscle of rabbits an analogous situation. In humans, a similar gradient from deep to superficial has been found in muscles of the upper and lower limb (237, 475), in paravertebral muscles (473), and the masseter muscle (424). Although the trapezius muscle exhibited an increase of type I and IIA fibers at the expense of type IIB (IIX) fibers from cranial to caudal in both genders (305, 306), the temporalis muscle showed an increase of the proportion of pure slow type I fibers in a postero-anterior direction at the expense of hybrid slow/fast fibers (268). Quantitative morphological study of whole vastus lateralis muscle from childhood to old age has revealed a life-long rearrangement of these compartments (475).

View larger version (152K): [in this window] [in a new window]   Fig. 2. Immunohistochemical demonstration of parvalbumin in the superficial and deep portion of rat extensor digitorum longus muscle. Because parvalbumin is involved in the relaxation process, the type IIB fibers that are fast contracting and fast relaxing show throughout a strong staining intensity. Sixty to seventy percent of the type IIA fibers are intermediately stained, whereas the remaining type IIA and the type I fibers that are slow contracting and slow relaxing are nonreactive. The compartmentation of the muscle is clearly seen; the superficial ("white") portion displays a higher relative amount of IIB fibers than the deep ("red:) one. Original magnification, ×60.

Much information is available on the biochemical, morphological, and physiological phenotype of specific fiber types and how these fiber types can be transformed. However, little is known on the accompanying changes in the expression of the corresponding genes and the involved control mechanisms. The signals and early cellular events that exert this control are poorly understood. Several major technical problems hamper the analysis of fiber type specific gene expression. Investigations cannot be carried out on cultured cells, since these do not differentiate to the point they do in vivo. Therefore, work on whole animals has to be carried out. Some data are available from transgenic mouse work. For example, the myosin light chain (MLC) 1 fast (1f) promoter (102) or the MLC3f promoter (249) in combination with the enhancer located 3' to the MLC1/3 locus (containing both genes) were found to be expressed in fast type II fibers of animals made transgenic with these regulatory elements coupled to reporter genes. This indicates that the used promotor together with the enhancer contain sufficient information to allow fiber-specific expression. However, correct subtype distribution as found in the normal situation was not achieved. A variety of factors may be responsible for this discrepancy. Maybe not all required DNA elements were present in the constructs. Alternatively, the site of transgene integration into the genome, the chromatin configuration or genomic imprinting during embryonic development might have caused the differences.

The method of direct gene transfer into the muscle has been used to investigate the regulatory sequences for the fiber type-specific expression of the MLC-1 slow/ventricular (MLC-1s/v) gene. Constructs with 5'-flanking regions of this gene showed a preferred expression pattern in the slow fibers (534) as found in the endogenous situation. However, several problems are encountered as well when this method is used. The transgenic DNA is not in a genomic configuration, since it is localized on a extrachromosomal plasmid and expression is relatively low if the muscle is not regenerating. So far, no common sequences present in different genes with the same fiber type specificity are known. It is also not established how the known myogenic transcription factors govern fiber type-specific gene expression. There are some indications that members of the myogenic helix-loop-helix transcription factor family such as MyoD, which is expressed mainly in type II fibers, and myogenin, which is expressed mainly in type I fibers (217, 537), are involved in the differentiation and transformation process. However, there is no clear evidence for a specific causal involvement (271). Differentiation of fast-twitch and slow-twitch fibers can also occur when either the MyoD or the myogenin gene is knocked out. Possibly there exists redundancy in transcription factors for muscle differentiation. Other groups of transcription factors such as MEF-2, M-CAT binding factor, and SRF known to regulate muscle genes have not been investigated for their involvement in fiber type-specific gene expression (453).

Regulation of fiber type-specific gene expression can also be achieved posttranscriptionally. It has been shown, for example, that the pool of transcripts for a muscle gene family producing several isogenes remains constant although levels of the different isogene products may vary greatly during development. This has been demonstrated, for example, for TnC and TnCf isogenes (538).

In conclusion, every muscle within an animal is unique in terms of fiber type composition and distribution pattern within the muscle. In animals the fiber composition of homologous muscles can vary considerably from species to species. Within a species in a given muscle the proportion of type I fibers increases with body size and body weight. In contrast, in humans the interindividual variability of the fiber composition is considerable. Although a given muscle may consist mainly of fast-twitch fibers in one individual, it may be totally made up of slow-twitch fibers in an other individual (237, 527, 544, 545). A muscle can functionally adapt to a broad spectrum of activities. The molecular mechanisms involved in these adaptations and the early molecular and cellular events taking place in these processes are still poorly understood. However, DNA sequences important for the regulation of fiber-specific gene expression as well as transcription factors involved in this process can now be investigated by the use of transgenic animals or by direct gene transfer into the muscle of living animals.

B.  Changes of Fiber Type Composition and Calcium Handling Apparatus During Development and Aging

1.  Development

In vertebrates, most skeletal muscles derive from the paraxial mesodermal tissue that condenses into the segmentally arranged somites. During the further maturation in each somite, the cells are compartmentalized. The dorsolateral compartment is called myotome; it contains two subsets of myogenic precursor cells. The cells of one subset are destined to become the axial musculature, whereas the cells of the other subset migrate into the periphery to form the muscles of the body wall and the limbs (see Refs. 32 and 57 for further references). Myogenic determination occurs independently in somites and limb buds (239). The myogenic precursor cells differentiate to become myoblasts, which later fuse to become three discrete populations of myotubes (first, secondary, tertiary) that then develop into myofibers (104). The later stages of myogenesis are more dependent on the myogenic regulatory factor (MRF) myogenin than early stages (532). Protein and mRNA studies have demonstrated that myosin isozymes follow an embryonic > neonatal > adult transition during mammalian and avian skeletal muscle development (547). In mice, the accumulation of slow MLC in the slow-twitch muscle fibers occurs during prenatal myogenesis, whereas the accumulation of the fast MLC in the fast-twitch muscle fibers is a postnatal phenomenon (541). For further details of embryonic myogenesis and the regulatory pathways underlying the generation of the definitive skeletal muscle diversity, the reader is referred to the following reviews (52, 248).

Studies on chicken muscle have shown that the Ca²⁺ handling system (Ca²⁺ release, storage, and uptake) develops in two stages. A temporary Ca²⁺ regulating system is established at the periphery of the myotubes during myofibrillogenesis [around embryonal day E5.5]. This peripheral system is subsequently replaced by the more highly specialized central system (t tubules/SR) during myotube-to-myofiber transition (between E15 and E16) (510). The avian calsequestrin homolog, a Ca²⁺-binding protein responsible for Ca²⁺ storage in the SR, was detected in limb primordia of chicken embryos as early as E5 (67). Calsequestrin and its mRNA increased ~10-fold before myoblast fusion. Cross-linking studies revealed that, during postnatal development, the oligomerization state of Ca²⁺ regulatory components including the RYR, the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and calsequestrin increased (143). This indicates that protein-protein interactions become more and more complex during development and are important for the correct function of the adult muscle.

The appearance of PV during myogenesis and maturation has been investigated in frog and rat; in Xenopus laevis, PV is first detected at embryonic stages 24-25, when myotomal muscles are differentiating (463). In the rat, PV immunoreactivity appears only postnatally and varies considerably from muscle to muscle. PV can be detected in the tibialis anterior muscle at the fourth postnatal day where it reaches the adult checkerboard pattern 2 days later. In contrast, in the intrinsic muscles of the tongue, in diaphragm, and in intercostal muscles, PV immunoreactivity does not appear until the second week. The fact that differences in PV expression do not correlate in time with the differentiation of fiber types (as judged by myosin ATPase activity) probably suggests that myosin and PV are regulated by different mechanisms (384).

In aging rodents, a progressive loss of muscle fibers paralleled by fiber type conversion (see below) from "fast twitch" to "slow twitch" has been observed (58). This process was later shown to be both muscle and fiber specific (135, 353). In humans too, the changes with age differ from muscle to muscle. For this reason, age changes, mainly the ones in relation to oxidative capacity, are controversially reported in the literature (212), while the selective atrophy of type II (A and B) fibers is well documented (166, 209, 256, 284, 285, 404; see Ref. 515 for further references). Electrophoretic investigation of single fibers of vastus lateralis and biceps brachii muscles of young (23-31 yr old) and elderly men (68-70 yr old) showed, with increasing age, an increasing number of muscle fibers with coexistence of different MHC isoforms (258). Very old subjects (average age, 88 yr) displayed 52.6% muscle fibers coexpressing two or three MHC in the vastus lateralis muscle (5). Thus a separation into slow and fast fibers becomes misleading in very old individuals. In elderly men and old animals, in old age the muscles retain their individual adaptability in response to physical exercise (257, 286, 495, 531). Atrophy of fibers due to aging can be attenuated by training (339).

An age-related impairment of intrinsic SR function, i.e., the rate of Ca²⁺ uptake and the fractional rate of SR filling, and a decrease in SR volume are the most probable factors underlying the decreased speed of contraction in old fast-twitch motor units (288). Additionally, uncoupling of sarcolemmal excitation and SR Ca²⁺ release have been assumed as a major determinant of weakness and fatigue (89). Indeed, with increasing age, an increase of the number of RYR1 ryanodine receptor uncoupled from DHPR has been found in rat (soleus and extensor digitorum longus muscle; Ref. 418) and human (vastus lateralis muscle; Ref. 89). DHPR-RYR1 uncoupling leads to a significant reduction in the amount of releasable Ca²⁺ in skeletal muscles from old animals and humans. However, the effects of aging considerably vary from muscle to muscle (367).

C.  Fiber Transformations and Modulation of Calcium Signaling and Handling Depending on Altered Neuronal Input, Exercise, and Other Factors

1.  Neural input, cross-reinnervation, and electrical stimulation

Muscle fiber transformations as the basis of muscular plasticity occur in response to a variety of systemic or local stimuli in humans and animals. Investigation of muscle plasticity mainly started after the classical cross-reinnervation experiments of Buller and co-workers in 1960 (50). Since then it has been repeatedly shown that the firing patterns of the innervating motoneurons largely determine the characteristics of muscle fibers (195) (for further references, see Refs. 52, 396, 397, 440). Thus reinnervation by motoneurons with a different firing pattern leads, within a few months, to changed properties of the reinnervated muscles. This is evidenced by an altered fiber type distribution with corresponding changes of the concentration of the fast fiber specific PV (360, 362) and other proteins important for Ca²⁺ handling in the muscle (364). It has been shown that the degree and the time course of the fiber transformation depends on the size ratio of the two muscles which are cross-reinnervated (51, 244). It also depends on the ratio of type IIA and type IIB motoneurons within the reinnervating motor nerve (514).

The effects of cross-reinnervation can, to a large extent, be both reproduced and opposed by long-term electrical stimulation (reviewed in Refs. 396, 399, 400). Artificial stimulation that activates all motor units of the stimulated muscle induces a specific remodeling of the muscle fibers leading to a shift of the fiber type distribution. This remodeling encompasses the major, myofibrillar proteins, membrane-bound and soluble proteins involved in Ca²⁺ dynamics, and mitochondrial and cytosolic enzymes of energy metabolism. Stimulation work has been mainly carried out on fast-to-slow transition by chronic low-frequency stimulation (continuous at 10 Hz) (232, 440) and much less on slow-to-fast transition by phasic high-frequency stimulation [e.g., 60 pulses at 100 Hz every 60 s (309, 310) or 40 pulses at 40 Hz every 5 min (301)].

Both types of conversion show substantial species differences that are still not yet fully understood. They involve, e.g., the replacement of degenerating and de novo formation of regenerated fibers in rabbits (460) and guinea pig (301), whereas they are entirely due to transformation of preexisting fibers in rats (91, 301). When rabbit tibialis anterior muscles were stimulated continuously at 2.5, 5, or 10 Hz for 10 mo, interestingly in muscles that had received 2.5-Hz stimulation, fast myosin isoforms were found to predominate, and the muscles showed the highest levels of oxidative and glycolytic activity (506).

Possible differences in posttranscriptional regulation may result in the transient accumulation of atypical combinations of fast and slow MLC and MHC isoforms, giving rise to the appearance of hybrid fibers (294). Recently, it has been suggested that the drastic depression of the energy state in stimulated muscle fibers could act as an important signal initiating the fast-to-slow transformation process (72). Already after 3 wk of chronic low-frequency stimulation the neuromuscular junctions of the stimulated (fast-twitch) muscles showed a partial transformation toward a morphology characteristic of slow-twitch muscle in rabbits (480). The neuromuscular junctions became smaller, and the secondary postsynaptic folds were more closely spaced. In senescent rats, the fiber shift was significantly less pronounced after low-frequency stimulation (539). This stimulation pattern suppressed the expression of the Ca²⁺-binding protein PV in fast-twitch rabbit muscles (259, 260). In humans, intermittent electromyostimulation could increase endurance without concomitant morphological or biochemical changes (253).

Muscle fiber transformations in consequence of cross-reinnervation or electrical stimulation have been mostly studied in rodents. In humans, the most intensively studied fiber transformations are both the ones following physical exercise and detraining (10, 36, 160). For many years, it has been recognized that endurance training leads to an increase of slow-twitch type I fibers (162, 230, 231). Only much later was it shown conclusively that the fiber distribution can also change in the opposite direction {an increase of fast-twitch type II [A + B(X)] fibers} as a consequence of repeated 30-s "all-out" sprints (120, 121, 229).

Partly comparable with physical exercise and detraining are mechanical overload and hypogravity, respectively. Mechanical overload (induced by stretch or ablation or tenotomy of synergists) leads to hypertrophy. Ultrastructural myofibrillar disruptions, mitochondrial alterations, glycogen pooling, and a significant increase in the number of myonuclei and satellite cells are observed in the early stages (3, 479). Recently, it has been shown that calcineurin plays an important role as a mediator of the Ca²⁺ effect on gene transcription in hypertrophy (111, 366, 468; for more details, see sect. IVE). Additionally, fiber splitting paralleled by a shift of the fiber distribution toward the oxidative type I fibers has been reported (179, 247, 391). Many studies show that in several animal species certain forms of mechanical overload can increase muscle fiber number (10, 247). Overload experiments have shown that active musculature not only produces much of the circulating insulin-like growth factor I (IGF-I) but also utilizes most of the IGF-I produced (see review in Ref. 159). The discovery of the locally produced IGF-I appears to provide the link between the mechanical stimulus and the activation of gene expression.

Exposure to hypogravity decreases muscle strength in humans and animals mostly affecting the postural muscles (108). Zhou et al. (570) showed that fibers expressing only slow (type I) MHC in the vastus lateralis of space craft crew members were significantly reduced after a relatively brief (11 days) exposure to space flight (570). However, it was suggested that adaptive changes subsequent to weightlessness were more dependent on the muscle function (involving mainly postural muscles) than on the fiber type (498). In rats exposed to a 7-day space flight, Riley and co-workers (422) found shrinkage of the majority of the soleus and extensor digitorum longus fibers; in soleus, ~1% of the fibers appeared necrotic. Ca²⁺-activated protease activities of soleus fibers from rats on space craft were significantly increased. Hypogravity conditions induced by walking on crutches (28), bed rest (107, 126, 199) (for further references, see Ref. 139), or hindlimb suspension (11, 216, 382, 443) lead to reduction in muscle mass and strength. The reduction in strength is more pronounced in extensors than in flexors (11, 107, 422), and the muscular changes are species specific (11). In addition to atrophy, fiber in a transitional state (showing a mismatch between MHC isoforms at the mRNA and protein level) and myofibrillar damage have been reported (4, 216, 382, 443). To our knowledge the Ca²⁺-binding proteins have not yet been investigated in muscles exposed to hypogravity.

Many hormones (e.g., growth hormone, insulin, thyroid hormones, sex hormones) exert a strong systemic influence on skeletal muscles during development as well as in the adult stage (for further references, see Refs. 134, 136). The hormonal effect on muscles is also mirrored in the widespread use and misuse of hormone analogs, e.g., in sports or meat production.

For the thyroid hormones, it has been shown in rats that both hypo- and hyperthyroidism were paralleled by modifications in the fiber type composition. 3,3',5-Triiodothyronine (T₃) induces terminal muscle differentiation and regulates fiber type composition via direct activation of the muscle-specific myoD gene family (103). Gender- and muscle-specific differences were observed in regulation of myosin heavy chain isoforms by thyroid hormones (289). PV distribution and concentration were largely unaffected in all thyroid states. This indicates that the muscular alterations are likely caused by a direct action of the thyroid hormone on muscle fibers, and not via their nervous input (363). The sexually dimorphic muscles (e.g., perineal, masticatory, laryngeal), also under strong hormonal control, will not be further considered.

In addition to these specific and/or systemic stimuli, also local and unspecific stimuli can elicit muscular reactions. As an example, fiber transformations of fast- into slow-twitch fibers, and vice versa, have been observed in neck muscles of the rat after an incision of the overlying skin (359).

In mammalian muscles, fiber transformations probably occur according to the following scheme (modified from Refs. 30, 164, 229, 231, 359)
Endurance exercise or overload, for example, lead to a transformation in the direction from right to left, whereas detraining or hypogravity leads to a transformation in the opposite direction. The fiber type at right is IIB in rodents and IIX in humans (see above). The "intermediate" fiber types are intermediate with respect to metabolic profile and myosin composition. In normal muscles, these fiber types are found in only small amounts. However, in muscles undergoing a transformation, their percentages are increased independently of the direction of the fiber transformation. Such an increase has been shown for type IB and IIC fibers (between type I and IIA in the scheme) in rats after unspecific stimulation (359) and for IIC fibers in healthy humans after physical training (231) or in patients with cervical dysfunctions (527, 545). Subjects with mandibular prognatism and deficient occlusion have revealed in their masseter muscle an increased frequency of intermediate IM fibers (between type I and IIA) (425, 516).

Muscle fiber transformations paralleled by an altered fiber composition are also encountered as secondary effects in muscle diseases as hereditary myotonias and periodic paralyses, which are disorders of skeletal muscle excitability. Myotonia is caused by runs of nerve independent action potentials at the sarcolemma (Fig. 3, myotonic response). Incomplete muscle relaxation and transient muscle stiffness are the consequences of this hyperexcitability (234, 435). Paralysis is brought about by strong membrane depolarization and following inexcitability of the sarcolemma. Before 1990, the underlying genetic defects were not known in any of these diseases in humans and animals (mouse, goat, and horse). Since that time, most of the disorders have been recognized as mutations in genes coding for voltage-dependent ion channels. Recently, the diseases, called muscular channelopathies, were reclassified and grouped as either sodium channel (SkM1) disorders, chloride channel (ClC-1) disorders (Fig. 3, Table 2), or Ca²⁺ channel disorders. This subject has been extensively reviewed (203, 295, 296). The most frequent disease of this group of disorders is probably the recessive chloride channel myotonia (affected between 1:23,000 to 1:50,000). The aim of this section is to discuss the secondary consequences of increased muscle excitability and activity on muscle structure, function, and fiber type composition in the different affected species.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Myotonia and muscle fiber type changes. Mutations in Na+ (1) or Cl (2) channels or the lack of the sarcolemmal chloride channel (ClC-1; 2) can lead to overexcitability of the sarcolemma (myotonia). Normal muscle responds with single action potentials upon single stimuli, whereas myotonic muscle often responds with runs of action potentials. Increased membrane excitation can cause protein kinase C (PKC) activation in the nucleus and changes in the pattern of myogenic regulating factors (MRF). The myogenic factors control gene transcription and therewith couple membrane excitation to the muscle fiber type. A second signaling pathway involves cytoplasmic Ca2+. The propagation of action potentials into the transverse tubule system (TT) activates the L-type Ca2+ channel and stimulates Ca2+ release from the sarcoplasmic reticulum (SR) via the ryanodine receptor (RyR). A Ca2+ signaling pathway into the nucleus is suggested.

The hereditary Na⁺ and Cl channelopathies are not accompanied by muscle fiber necrosis, regeneration, or persistent weakness. In some cases of the dominant chloride channel disorder myotonia congenita (Thomsen) muscle fiber hypertrophy, an increased number of central nuclei, and type I fiber atrophy were observed (45), whereas other cases were normal. In recessive myotonia (Becker), also caused by ClC-1 gene mutations, three-quarters of the patients show muscle hypertrophy. A slight increase of serum creatine kinase (CK) was found in some cases. Occasionally, muscle biopsies from patients with paramyotonia congenita, a dominant Na⁺ channel disorder, showed focal myofibrillar damage (149). Myotonia of mouse and goat are caused by chloride channel (ClC-1) defects; however, the phenotype shows differences between the species. In murine myotonia, the degree of muscle stiffness and the frequency of the myotonic discharges are much more pronounced compared with myotonia in humans and goats. Thus myotonic mouse muscle is a biological model of a chronically and extensively stimulated muscle (267, 336, 550). In aged myotonic mice, elongation of tendons and bone deformations have been observed (193). This points to a considerable increase of force development of myotonic muscle in vivo.

In mice and to a minor extent in the myotonic goat and humans, muscle histological, immunohistochemical, and biochemical investigations revealed secondary changes of myotonia. Reduced glycolytic enzyme activity (482) and changes in the muscular lipid composition (414) were reported. Both findings are consistent with increased amounts of mitochondria in myotonic muscles. In 1984, Jockusch and co-workers (501) showed that in myotonic adr muscle the content of PV was drastically reduced (adr, arrested development of righting response). They suggested that the impaired muscle relaxation seen in adr mice could be a consequence of the PV deficiency. After clarifying that electrically induced myotonia is responsible for the aftercontractions of adr muscle (336), the biochemical changes in myotonic muscle were reinterpreted as fiber type transformations in response to the different stimulation pattern. The most drastic changes occur in predominantly fast-twitch muscle in the myotonic mouse. Electrophoretic and histochemical analysis (234) revealed a shift from IIb to IIa myosin heavy chain expression in the predominantly fast-twitch tibialis anterior and gastrocnemius muscles. The slow-twitch soleus muscle, consisting of ~70% type I fibers in the normal mouse, shows a composition of only 50% type I and 50% type IIA fibers in the myotonic mouse. This may be explained by the different pattern of electrical and mechanical activity of myotonic soleus muscle. Alternatively, consequences of the smaller size of myotonic mice (about one-half of the body weight of controls) can contribute to the reduction of type I fiber number. The RNA for the type IIB specific protein PV was found much decreased in myotonic muscle, whereas the mRNA for a slow-twitch muscle specific protein p19/6.8 was increased (261). The chronic application of tocainide, a drug which normalizes membrane excitability, reverted the fiber type transformations of myotonic muscle (235, 416). These results indicate that the fiber type abnormalities of myotonic muscle are secondary adaptations to the different pattern of electrical stimulation.

In the myotonic goat, the proportion of fast myosin isoforms was found to be increased in all muscles tested (326), in contrast to the results with myotonic mouse (234). The difference may be explained by the fact that the muscle fiber type composition of bigger mammals differs from that of small rodents by a higher proportion of type I fibers at the expense of type IIA and IIB fibers. In human myotonic muscle, a lack of fast-twitch glycolytic type IIB (IIX) fibers was reported (77). This is consistent with the reduction of type IIB fibers in mice and the reduction of the MHCIIb isoform.

In addition to the fiber type changes, muscle hypertrophy was observed in some human disorders with myotonia, independent of whether they are based on Cl channel (45) or on Na⁺ channel mutations. Our favored interpretation for this finding is that the myotonia is equivalent to exercise and stimulates protein synthesis in muscle. In contrast to human myotonia, mouse myotonia is accompanied by reduced body weight and reduced muscle mass of the affected animals (542). This seems first surprising, but as mentioned above, mouse myotonia is more intense than human and goat myotonia. A reduced opportunity of food intake, insufficient respiration, and other handicaps may be responsible for the growth restriction of myotonic mice.

Fiber type transformation in myotonic muscle and in chronically stimulated muscle has been described at the protein and mRNA levels for many years. However, it is still not clarified how altered muscle membrane and contractile activity influences muscular gene transcription. In the last few years two signal transduction pathways have been discovered that seem to be important for the coupling of membrane excitation and altered gene transcription. First, it was shown by Huang et al. (215) that protein kinase C (PKC) couples membrane excitation to acetylcholine receptor inactivation (Fig. 3). After electrical stimulation of denervated chicken muscle the activity of PKC in the nucleus was found 100-fold increased, and this increase was correlated with the inactivation of AChR subunit genes. Later the same group showed that the myogenin gene, coding for a transcription factor belonging to a family of myogenic factors (48), declined in transcriptional activity after electrical stimulation comparable to the rate of AChR gene inactivation (213). It has been reported that phosphorylation by PKC inactivates myogenin (303). Compared with controls, myotonic adr muscle is characterized by increased levels of the myogenic factors myogenin and herculin (or MRF4) and a reduction of the MyoD level. The differences in mRNA levels of MHCIIb, MHCIIa, MHCIIx, and MHCI genes (158) were attributed to the different pattern of myogenic factors.

The second signal transduction cascade that came into question involves intracellular Ca²⁺ (Fig. 3). Huang and Schmidt (214) showed that electrical stimulation, via an increase of Ca²⁺, causes AChR -subunit gene inactivation. It has not been clarified whether this mechanism also involves myogenic factors. Recently, it was shown that calcineurin, a calcium-dependent phosphatase, is a possible mediator of fiber type conversion in response to electrical stimulation. The overexpression of calcineurin in cultured muscle caused slow-fiber-specific gene expression, whereas calcineurin inhibition led to a slow to fast conversion of rat soleus muscle in vivo (66) (for further discussion, see sect. IVE). In other cell systems (neurons, glial and liver cells, and T lymphocytes) CaM and CaM-binding proteins have been detected in the nucleus (15). It has further been shown (in nonmuscle cells) that CaM, via the activation of CaM-dependent kinases II or IV, can phosphorylate transcription factors, and it was suggested that CaM may have a general role in RNA processing or splicing (15). Calcium, in most cases together with CaM, can use different routes to signal through ras to modulate survival, differentiation, and plasticity in neurons (130).

	III. PLASTICITY OF THE CALCIUM HANDLING APPARATUS

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A.  Calcium release from the SR

1.  Structural and functional considerations

The RyR to which the plant alkaloid ryanodine specifically binds is the major channel for Ca²⁺ release from intracellular stores in skeletal muscle; it mediates the t-tubular depolarization-induced Ca²⁺ release from the SR (Fig. 4). Several review articles exist on the structure and function of the RyR (74, 131, 324, 458). In skeletal muscle, activation of Ca²⁺ release from the SR is controlled by a voltage sensor in the transverse tubular (tt) membrane (459). Elementary Ca²⁺ signal events have recently been subcellularly localized in the skeletal muscle. These signals represent openings of individual RyR in the SR membrane and have been termed Ca²⁺ sparks and Ca²⁺ quarks, respectively (reviewed in Ref. 371). The initial Ca²⁺ release activates additional Ca²⁺ sparks by Ca²⁺-induced Ca²⁺ release from the SR. It is believed today that the signal transmission from the DHPR to the RyR is achieved by mechanical coupling. This is fully compatible with the original hypothesis of Schneider and Chandler (459) that charged components in the sarcolemma and t tubules move in response to depolarization, and this is coupled to a charged component in the SR. That asymmetric charge movement is related to the excitation-contraction coupling could be demonstrated by many studies. For example, it has been shown that in soleus muscle of paraplegic rats (after spinal cord transsection) the voltage dependence of contraction (twitches and K⁺ contractures) and charge movements changed in parallel (109) compared with normal animals. Another study shows that T₃, which shifts the soleus muscles toward fast physiology, also increases the amount of charge movement and both the voltage dependence of charge movement and tension shifted to more positive potentials (110). Voltage-dependent depolarization-induced activation is independent of a Ca²⁺ inward current (reviewed in Ref. 64). However, the maintenance of the function of the voltage sensor depends on external Ca²⁺. It seems that Ca²⁺ has a stabilizing effect that supports excitation-contraction (EC) coupling (reviewed in Refs. 340, 458). In contrast, the heart muscle RyR (RyR2) is activated during EC coupling by Ca²⁺ influx through the DHPR, a phenomenon referred to as Ca²⁺-induced Ca²⁺ release (reviewed in Ref. 123). Because Ca²⁺ influx through the Ca²⁺ sensor is of secondary importance for skeletal muscle physiology, this mechanism is not discussed in this article.

View larger version (35K): [in this window] [in a new window]   Fig. 4. The ryanodine receptor and its function in Ca2+ release. Proposed arrangement of proteins in the SR and target proteins of Ca2+ in the cytoplasm. The transverse tubular membrane is part of the plasma membrane of the muscle fiber. The interaction of the -subunit of the Ca2+ channel, also known as dihydropyridine receptor (DHPR), and the Ca2+ release channel of the SR called ryanodine receptor (RyR1) connects both membranes, tubular and SR membranes. This connection is responsible for electromechanical coupling. Several cytoplasmic and SR proteins are associated with the DHP/RyR complex (triadin, calsequestrin, FK506 binding protein, and calmodulin). Calcium release from the SR via the RyR1 triggers muscle contraction and multiple cellular effects by binding of Ca2+ to a variety of other target proteins. Reuptake of Ca2+ from the cytoplasm into the SR is carried out by the SR calcium pump.

In the mouse BC₃H1 cell line, which serves as a model for muscle differentiation, it was found that in the proliferative state of the cells the predominant release channel was inositol 1,4,5-trisphosphate (IP₃) sensitive and therefore identified as the endoplasmic reticulum Ca²⁺ channel, whereas after differentiation, the Ca²⁺ mobilization potential was mostly caffeine sensitive, indicative of the RyR (SR Ca²⁺ channel) (96b). This suggests that differentiation of the BC₃H1 myoblast phenotype induces the expression of RyR and reduces IP₃ receptor activity, a process which might also take place in muscle development in vivo. Investigations on the expression of RyR isoforms and IP₃ receptors during development of skeletal muscle or in the specialized adult muscle indicate that various combinations of Ca²⁺ release channels could contribute to the fine tuning of Ca²⁺ regulation in the skeletal muscle (reviewed in Ref. 486).

Because the RyR has a very central position in the context of Ca²⁺ handling in muscle physiology and plasticity, it is not surprising that it is also a molecular switch that is highly complex and a target of many regulatory pathways. We therefore discuss its structure and regulation in some detail and summarize the knowledge on putative interacting molecules that could contribute to its performance.

The RyR is a homotetramer (see Ref. 324), and 50% of all these complexes are located in close proximity to the DHPR (131, 140-142, 408). In addition, it has been shown that RyR channels are highly clustered square structures arranged in regular rows and that the corners of adjacent channels contact each other (438). There is ~66% amino acid sequence identity among the skeletal, cardiac, and brain isoforms (324). Fast and slow skeletal muscle fibers contain predominantly one RyR isoform (RyR1), but the RyR density is higher in fast fibers (81). Some of the biochemical features of the RyR and other molecules discussed in this review article are listed in Table 3.

Ca²⁺ dependence of the RyR activity is achieved by several different mechanisms. The Ca²⁺ release properties of isolated triad preparations (composed of the terminal cisternae of the SR, the RyR, and the transverse tubular membrane) could be shown to be influenced in a dual mode by Ca²⁺ (561). The channel is activated by low Ca²⁺ concentration (50% activation at 0.5 µM) and inhibited at higher Ca²⁺ concentration (50% inhibition at 0.15 mM), suggesting that there are two classes of Ca²⁺-binding sites involved in channel regulation. This leads to a situation of positive- and negative-feedback regulation of Ca²⁺ release by Ca²⁺ which is reflected in a bell-shaped curve of Ca²⁺-dependent Ca²⁺ release (Fig. 5). Single RyR channel measurements in a lipid bilayer experiment showed that increasing the luminal (SR) Ca²⁺ concentration from 0.1 to 250 µM increased channel activity at negative holding potentials at the cytosolic side. Increase of Ca²⁺ concentrations from 1 to 10 mM in the "luminal" chamber resulted in a decrease of channel activity at negative holding potentials and increased activities at positive holding potentials. This suggests that luminal Ca²⁺ flux through the RyR regulates channel activity by allowing Ca²⁺ to have access to activation and inactivation sites that are on the cytoplasmic domain of the RyR (196). It is highly likely that different modes of Ca²⁺ handling in different fiber types or at different stages during development affect the activity of the RyR differentially. When the RyR is activated by t-tubule depolarization, the released Ca²⁺ may cause further increase in the rate of Ca²⁺ release, and this is followed by a reduction in the rate of Ca²⁺ release (96b).

View larger version (18K): [in this window] [in a new window]   Fig. 5. Ca2+ dependence of Ca2+ release of SR vesicles. Effect of Ca2+ and calmodulin on 45Ca2+ efflux from SR vesicles. Relative Ca2+ efflux rates are first-order efflux constants of 45Ca2+ determined in the absence () or presence () of calmodulin at the indicated concentrations of free Ca2+ in the efflux media. For experimental details, see Reference 522.

Nitric oxide (NO) was found to inhibit the RyR in skeletal (343) and heart muscle (567). Both the rate of Ca²⁺ release from the SR and the open probability were affected. This inhibition causes depression of contractile force, and because the major form of the NO synthase in muscle is of the Ca²⁺/CaM-dependent type, this regulation would represent another feedback loop in Ca²⁺ signaling. Ca²⁺ would activate NO synthase through CaM, and NO would reduce Ca²⁺ release from intracellular stores. In a recent article by Xu et al. (558), direct action of NO on the cardiac RyR was demonstrated through S-nitrosylation of thiol groups.

Free Mg²⁺ is present in the muscle at millimolar concentrations. At this concentration this ion inhibits RyR channel activity (281, 290). Mg²⁺ could bind either to the activating high-affinity Ca²⁺-binding site in a competitive fashion or to the low-affinity inhibitory Ca²⁺-binding site (338). A third possibility is binding to another site that would block Ca²⁺ conduction (477). An explanation why Mg²⁺ cannot permanently inhibit RyR is provided by Hain et al. (178) in that Mg²⁺ can only block the nonphosphorylated state of the RyR which can be phosphorylated by protein kinase A or CaM kinase. The effect of Mg²⁺ is also mediated through phosphatase 2C, which is activated by Mg²⁺ (reviewed in Ref. 469).

An important question concerning intracellular Ca²⁺ storage organelles is how they get refilled after Ca²⁺ is depleted. It is known for many cell types that depletion of intracellular Ca²⁺ stores results in the activation of a store-operated Ca²⁺ channel at the plasma membrane that enables the reloading of internal Ca²⁺ stores. This Ca²⁺ current, the Ca²⁺ release-activated current (I_CRAC), may be responsible for long-term Ca²⁺ effects and oscillations. The signals involved in transmitting the information of Ca²⁺ depletion as well as the channel themselves are not well characterized. By single-cell patch-clamp analysis in cultured skeletal muscle cells, Hopf et al. (206) found Ca²⁺ leak channels that were sensitive to two new dihydropyridine compounds, as well as to manganese influx and an inhibitor of tyrosine kinase. Thus the Ca²⁺ leak channel might have an important function for filling the intracellular Ca²⁺ stores in normal contractile activity besides the voltage-dependent Ca²⁺ channel, which also has been shown to mediate the store refilling. However, it was shown that continuous muscle activity in mammalian skeletal muscle causes a dramatic increase of total muscle Ca²⁺ (122). However, Ca²⁺ concentrations in store organelles were not measured directly in these studies.

Taken together, the importance of the RyR for Ca²⁺-regulated muscle function can hardly be overemphazised. This large protein complex provides on the one hand the entry site for activating signals coming through surface membrane depolarization and DHPR activation. On the other hand, it releases Ca²⁺ from the SR, a process which is tightly controlled by the concentration of Ca²⁺ in the SR as well as by many other factors playing regulatory roles with mostly unknown molecular mechanisms.

In skeletal muscle, direct interaction of the DHPR in the t tubules of the plasma membrane with the RyR in the SR is believed to be responsible for EC coupling via a structural change in the DHPR that induces a structural alteration in the RyR, which finally triggers the opening of Ca²⁺ release channels (426) (see Fig. 4). Biochemical evidence for a link between the two receptors has been reported by Marty et al. (327). RyR interact with a variety of accessory proteins believed to modulate the activity of these Ca²⁺ channels (reviewed in Ref. 317). The following proteins have been shown to bind directly to the RyR or affect the gating properties of the RyR: glyceraldehyde-3'-phosphate dehydrogenase (41), aldolase (41), annexin VI (97), 170-kDa low-density lipoprotein binding protein (80), S100 protein (124, 325), CaM, 60-kDa CaM-dependent protein kinase (82), calsequestrin (365, 560), FK506 binding protein (233), triadin (173), and junctin (238). Whether some of these proteins use common docking sites on the RyR is presently not known. Because this review focuses on Ca²⁺-related issues, we discuss in more detail calsequestrin and CaM. They are both potentially involved in Ca²⁺-governed RyR function and regulation. In addition, new literature on FK506 binding protein is reviewed since this protein seems to have a major function for RyR regulation.

Calsequestrin is the main Ca²⁺-binding protein of the SR, with high capacity and low affinity for Ca²⁺. The biochemical features of this protein are discussed in section IVD (see also Table 3). Calsequestrin contains no transmembrane segments and is therefore believed to be located within the lumen of the SR (132, 387). A region of the protein (amino acid 86-191) was shown to bind to the junctional face membrane of the SR (71). Experiments with ryanodine receptor Ca²⁺ release agonists, such as polylysine or caffeine, suggest that the RyR, when activated by such agents, induces a release of Ca²⁺ from calsequestrin (219, 464). Experiments using fluorescent probes that were bound to SR proteins point in the same direction. In the presence of calsequestrin, the fluorescence intensity of the probe increased with luminal Ca²⁺ but not in its absence (220). Therefore, it was concluded that the Ca²⁺-dependent conformational change of calsequestrin causes a change in the shape of SR membrane proteins, including the RyR. A recent study shows that calsequestrin controls the RyR channel in a phosphorylation state-dependent fashion. Calsequestrin, exclusively when phosphorylated, enhanced the open probability of the RyR fivefold and increased the open time twofold (509). It is possible that calsequestrin interacts with the 95-kDa protein triadin, a protein thought to bind to both the RyR and the DHPR (63). Because triadin contains a region of basic amino acids in the luminal domain, it was suggested that it could interact with the acidic protein calsequestrin and provide a functional connection to the RyR (335). Recently, it was found that the luminal domain of triadin interacts with calsequestrin in a Ca²⁺-dependent manner and therefore triadin might anchor calsequestrin to the junctional region of the SR and might thus be important for functional coupling in the Ca²⁺ cycle (172). Purified triadin inhibits [³H]ryanodine binding to the solubilized heavy fraction of the SR of rabbit skeletal muscle and reduces the opening of the RyR (380). The same study shows that calsequestrin potentiates the Ca²⁺-dependent [³H]ryanodine binding and that this effect was reduced by triadin. That triadin functionally interacts with the skeletal RyR was also shown by antibodies directed against the COOH-terminal part of triadin, which induces a decrease in the rate of Ca²⁺ release from SR vesicles as well as a decrease of the open probability of the RyR Ca²⁺ channel incorporated in lipid bilayers (167). Junctin, another calsequestrin binding protein of 26 kDa with sequence homology to triadin, was found abundantly in junctional membranes and could therefore as well serve to bring calsequestrin in proximity to the RyR (559). In a recent study, the close proximity of calsequestrin and the RyR in fast- and slow-twitch rabbit skeletal muscle was demonstrated by immunoblot analysis of chemically cross-linked membrane vesicles enriched in triad junctions (365).

CaM binds to the RyR and affects its function in a complex positive and negative way (357, 465). CaM is a ubiquitous intracellular Ca²⁺ receptor containing typical EF-hand structural elements that bind Ca²⁺ in a specific way (see Fig. 9 and Ref. 311). The term EF-hand refers to the two COOH-terminal -helical sequence stretches in PV which are oriented in a perpendicular way and connected by a loop that contains amino acid residues serving as Ca²⁺ ligands. Proteins containing such structural elements are named EF-hand proteins (272). Generally, at submicromolar Ca²⁺ concentrations (10⁷ to 10⁹ M), CaM activates the channel by increasing the open probability in a dose-dependent fashion, and at high free Ca²⁺ (10 µM), CaM (0.1-1 µM) inhibits channel activity (53). It has been shown that CaM inhibits Ca²⁺ release from skeletal SR vesicles by a factor of 2-3 (337). Half-maximal inhibition was found between 0.1 and 0.2 µM and maximal inhibition at 1-5 µM CaM. The bell-shaped Ca²⁺ dependence of Ca²⁺ release (see Fig. 5) between 0.1 and 100 µM was not shifted by CaM. Only its amplitude was altered. In the absence of ATP, CaM decreased the mean open time of the skeletal RyR by ~40% (478), and nanomolar CaM concentrations inhibited ryanodine binding to the purified brain RyR (334). In a recent study using mutant mice expressing only type 1 or 3 RyR, it was shown that CaM regulates the Ca²⁺-induced Ca²⁺ release of skeletal muscle in a RyR isoform-specific fashion (221).

Tripathy et al. (522) found that activation of the Ca²⁺ release channel by CaM occurs at <0.2 µM Ca²⁺, whereas at micro- to millimolar Ca²⁺ concentration, CaM was inhibitory. Binding kinetics revealed on and off rates of 50 and 30 s¹, respectively, indicating that CaM exerts its two opposing effects on channel activity without dissociation from the RyR. Another independent investigation (53) comes to the same conclusion concerning the modulatory activity of CaM.

CaM-dependent protein kinase was found to be tightly associated with several junctional terminal cisternae (JTC) proteins of which several were phosphorylated in a Ca²⁺-dependent way, indicating that this system is involved in regulation of functions linked to these structures (68).

A 60-kDa CaM-dependent protein kinase in the junctional cisternae of the SR of rabbit fast muscle inhibits RyR function, although it does not directly phosphorylate the Ca²⁺ channel (82). However, this kinase phosphorylates triadin and a histidine-rich Ca²⁺-binding protein (204, 421, 470), possibly explaining the regulatory function of this enzyme.

In addition to being involved in regulation of the RyR, CaM is a key signal transmitter in a broad variety of other important muscle activities such as metabolism. Furthermore, CaM might have modulatory functions as an activator of CaM kinases and phosphatase and by this means indirectly affects the Ca²⁺ cycle. Examples of CaM targets with known catalytic or regulatory functions in the skeletal muscle are phosphorylase kinase (188), glycogen synthase kinase (552), CaM kinase II (250), calcineurin (171) (see also sect. IVE), NO synthase (42), and dystrophin (6). For biochemical features, see Table 3. A more complete list of CaM targets is presented in Reference 432. Recently, Pyk2, a stress-related kinase that signals through mitogen-activated protein kinase and promotes apoptosis, has been proposed to be activated by CaM (90). Another newly discovered group of enzymes shown to be Ca²⁺/CaM dependent are DAPK, serine/threonine kinases, involved in apoptotic Ca²⁺ signaling and shown to be present in the skeletal muscle (70, 245). It is not clear whether all these mentioned pathways are important for gene regulation in skeletal muscle, but all the necessary components have been shown to exist in muscle cells.

The tetrameric RyR channel binds four molecules of the FK506 binding protein (233), which has a stabilizing effect on the RyR and coordinates its activity (43). In a recent article it has been postulated that FK506 binding protein also coordinates the opening of several adjacent channels to release Ca²⁺ in a simultaneous way (328). Such a coupled gating would allow the regulation of channels that are not associated with vol