INTRODUCTION

TOP ABSTRACT INTRODUCTION ECC AND THE SR... CURRENT THEORIES IN ECC SR CA2+ RELEASE FROM... BIOPHYSICAL REGULATION OF SR... FAILURE OF SR CA2+... CRITICAL QUESTIONS REFERENCES

PRODIGIOUS EFFORTS TO EXAMINE the relevant mechanisms involved in skeletal muscle fatigue have begun to focus on Ca²⁺ handling within the active muscle cell. It is understood that precise control of intracellular Ca²⁺ is central to optimal performance of skeletal muscle. First and foremost, activator Ca²⁺ released from the sarcoplasmic reticulum (SR) regulates cross-bridge interactions and thus controls force production. Reductions in SR Ca²⁺ release will primarily compromise tension development through reduced Ca²⁺ transients (49, 137, 140). Second, Ca²⁺-activated tension and SR Ca²⁺ accumulation via the SR Ca²⁺-ATPase consume a large fraction of the energy expenditure within the muscle cell (105). Thus regulation of intracellular Ca²⁺ fluxes can greatly influence muscle cell metabolism and may possibly serve as a link between the requirement for force and the metabolic state of the active muscle cell.

Eberstein and Sandow (43) first suggested inhibition of Ca²⁺ release from the SR as a likely and relevant factor in the fatigue process under certain experimental conditions. This has been confirmed time and time again by several different groups in a wide variety of species. Reductions in Ca²⁺ release have been noted in single muscle fibers from mouse (6), single whole (136) and cut fibers from frog (64), and whole bullfrog muscle (10). Moreover, inhibition of SR Ca²⁺ release was noted in the rat after it run to exhaustion (46). (For a more detailed account of the muscle preparations and fatigue protocols please see Refs. 49, 131, 133, and 137.)

Inadequate or suboptimal delivery of Ca²⁺ to the myofilaments via SR Ca²⁺ release could occur under three scenarios: 1) impaired coupling between the voltage sensors in the t-tubule (TT) membrane and the SR Ca²⁺ channel [excitation-contraction coupling (ECC)], 2) reductions in or modification of the SR Ca²⁺ content, or 3) by transient modification of the SR Ca²⁺ channel, reducing its opening probability after activation (64, 137).

It is difficult to assess the contributions of the first hypothesis, considering that the complete mechanism for ECC has yet to be adequately defined. However, Gyorke (64) has suggested that fatigue from tetanic stimulation does not modify function of the t-tubule voltage sensor and is localized to the SR Ca²⁺ channel.

It is possible that SR Ca²⁺ content becomes diminished, considering that the catalytic activity of the SR Ca²⁺-ATPase is inhibited by a single bout of muscle activity (22, 78, 135) or that "releasable" Ca²⁺ may be buffered by luminal phosphate that becomes elevated in skeletal muscle undergoing activity (55, 103, 132). This area will be addressed in BIOPHYSICAL REGULATION OF SR Ca²⁺ release.

Clearly, the most studied and well-supported contention suggests that activity-induced reductions in myoplasmic Ca²⁺ transients are mediated by the SR Ca²⁺ channel and/or its related regulatory proteins. An early observation by Eberstein and Sandow (43) showed that the application of caffeine, a Ca²⁺-channel agonist, reversed the loss of force production in fatigued fibers. This result, confirmed many times (58, 62, 87, 98), pointedly implicates modification of the SR Ca²⁺ channel during fatiguing protocols.

Whereas SR Ca²⁺ channel dysfunction may not be the only cause of fatigue under all conditions of muscle activity, it is considered a viable mechanism for a several different muscle activation patterns. A myriad of studies have demonstrated alterations in myoplasmic Ca²⁺ during and/or after muscle activity. More complete reviews detailing the various fatigue protocols as well as contributions from the myofilaments and the influence of metabolism have been expertly reviewed elsewhere (49, 120, 137, 140). In light of these works, this review seeks to accomplish two related goals: 1) to provide an up-to-date molecular understanding of the Ca²⁺ release process, with considerable attention devoted to the SR Ca²⁺ release channel, its associated proteins, and channel regulation by endogenous compounds; and 2) to examine several putative mechanisms by which cellular alterations resulting from intense and/or prolonged contractile activity will modify SR Ca²⁺ release. This review is not intended to be a complete account of the many studies produced over the past decade. Thus apologies are extended for the exclusion of any work that supports this current undertaking.

	ECC AND THE SR CHANNEL

TOP ABSTRACT INTRODUCTION ECC AND THE SR... CURRENT THEORIES IN ECC SR CA2+ RELEASE FROM... BIOPHYSICAL REGULATION OF SR... FAILURE OF SR CA2+... CRITICAL QUESTIONS REFERENCES

ECC is a sequential process that begins with activation of the muscle cell membrane and concludes with Ca²⁺-activated tension produced by the myofilaments. Several key proteins participate in this process: the dihydropyridine (DHP) receptor, triadin, a 12-kDa FK506binding protein (FKBP12), calsequestrin, calmodulin, junctin, and the SR Ca²⁺-release channel-ryanodine receptor (RYR).

DHP receptor. The skeletal muscle DHP receptor is an oligomeric complex composed of five different subunits (1, 2, , , and ) (27). All five subunits have cytoplasmic domains, yet the 1-subunit appears to be the most important. It contains the binding site for DHPs and other antagonists and also can form an L-type Ca²⁺ channel (26), but Ca²⁺ entry is not required for activation of the SR Ca²⁺-release channel. It is currently believed that the DHP receptors found within the t tubule serve as the voltage sensors associated with asymmetric charge movement, an event consistent with depolarization to the contractile threshold (109). The 1-subunit consists of four repeats with three cytoplasmic loops between each segment (27). The II-III loop has been postulated to have a role in communication between the DHP and RYR receptors, as peptide fragments from the II-III loop of the DHP receptor bound to and activated the purified skeletal RYR Ca²⁺ channel but had no effect on the cardiac receptor (90). Both the 1- and -subunits contain multiple sites for phosphorylation that may be critical during muscle activity. The functions of the other subunits have not been fully defined.

Triadin. Several proteins have been localized in the triadic region and are thought to participate in the interaction between the DHP receptor and the SR Ca²⁺-release channel; among them, triadin, a 95-kDa glycoprotein, has been the most thoroughly studied (19, 20). The most compelling evidence arguing for its participation in ECC is that triadin binds to both the RYR channel and the DHP receptor in protein overlay studies, although no reports of direct in vivo interactions have been published (79). In other studies, triadin has been shown to directly interact with the RYR Ca²⁺ channel via hyperreactive thiols when the channel is in the open configuration (92). Cloning of a protein that appears to be identical to triadin (80) indicates that the protein contains one transmembrane domain and a large luminal component. The luminal region contains a number of positively charged residues and may serve as an anchoring protein for calsequestrin, the moderate affinity-high capacity Ca²⁺-binding protein that has been implicated in modifying SR Ca²⁺ release (69). Continued work should elucidate a more concrete role for triadin.

FKBP12. Four low-molecular-weight binding proteins are associated with each functional RYR channel. These FKBPs function as receptors for the immunosuppressant drugs, FK506 and rapamycin. When these drugs are added to muscle fiber or SR preparations, they induce dissociation of the FKBP12 proteins and appear to reduce or relieve channel inhibition by Mg²⁺ (4), H⁺ (127), and possibly millimolar Ca²⁺ (42). In single-fiber experiments, removal of FKPB12s by rapamycin potentiated Ca²⁺ release evoked by depolarization or caffeine application (86). Interestingly, repeated depolarizations resulted in an irreversible loss of depolarization-induced release but not caffeine-induced activation of the release channel. Although the removal of these proteins appears to affect native channel function, it is not known how the proteins participate in normal channel gating or are modified by extended contractile activity.

Calmodulin. Calmodulin is a cytoplasmic Ca²⁺-dependent protein that shares many similarities with troponin (28). Upon binding up to four Ca²⁺, the Ca²⁺-calmodulin complex can control a large number of calmodulin-dependent enzymes and membrane transport proteins. By binding to the skeletal muscle RYR protein, calmodulin has been shown to modulate channel activity (129). At low Ca²⁺ concentration (submicromolar), calmodulin stimulates Ca²⁺ flux and increases single-channel gating activity (129). At micro- to millimolar concentrations, calmodulin inhibited the RYR severalfold, thus suggesting that calmodulin may play a role in both activation and inhibition of SR Ca²⁺ release (129).

Calsequestrin. Calsequestrin is a low-affinity, high-capacity Ca²⁺-binding protein that resides within the SR lumen. It has been shown to be anchored to the junctional SR membrane, thus ensuring a large quantity of Ca²⁺ near the release channel (35). It does not bind to the RYR, but has been shown to bind to triadin (63) and has been implicated in modifying Ca²⁺ release from the SR (39, 69).

Junctin. Junctin was first discovered in canine heart muscle and later in skeletal muscle (76). Amino acid sequencing reveals that, like triadin, it resides on the luminal side of the SR membrane (76). It has recently been shown to bind to triadin, calsequestrin, and the RYR protein (145) and is thus believed to form a luminal complex that regulates SR. It appears that junctin and triadin interact directly in the junctional SR membrane and stabilize a complex that anchors calsequestrin to the RYR. Taken together, these results suggest that junctin, calsequestrin, triadin, and the RYR form a quaternary complex that may be required for normal operation of Ca²⁺ release.

SR RYR Ca²⁺-release channel junctional foot protein. This protein is the critical component in ECC, as Ca²⁺ release is lost in transgenic mice lacking the RYR protein (123). Originally isolated as the junctional foot protein (77), it was later determined to be the SR Ca²⁺-release channel from morphological and pharmacological studies with ryanodine (70, 94, 102). Because the protein has been identified by different methods, it has acquired several names that are frequently used interchangeably. The skeletal muscle RYR is a high-molecular-weight homotetramer containing four subunits. The monomeric protein contains 5,035 amino acids, with an apparent molecular mass of 560 kDa (124). The native protein that has been visualized by electron microscopy appears as a quatrefoil structure (cloverleaf-like) (104). Sequence analysis suggests the presence of cytoplasmic Ca²⁺, calmodulin, and adenine nucleotide binding as well as phosphorylation sites.

	CURRENT THEORIES IN ECC

TOP ABSTRACT INTRODUCTION ECC AND THE SR... CURRENT THEORIES IN ECC SR CA2+ RELEASE FROM... BIOPHYSICAL REGULATION OF SR... FAILURE OF SR CA2+... CRITICAL QUESTIONS REFERENCES

Many cell types are activated by second-messenger systems following receptor-mediated signaling. Smooth muscle and neurons utilize inositol 1,4,5-triphosphate (IP₃) to promote the release of Ca²⁺. Whereas several early studies suggested that IP₃ may modulate SR Ca²⁺ release in skeletal muscle, its inability to rapidly activate skinned fibers argues against IP₃ as the main signaling protein in skeletal muscle (see Ref. 93 for discussion).

The ECC processes are different between mammalian skeletal and cardiac muscle. In heart tissue, the DHP receptor, an L-type Ca²⁺ channel embedded in the t-tubule membrane, mediates the influx of Ca²⁺ following an action potential and voltage-dependent activation. The influx of extracellular Ca²⁺ into the region of the closely opposed SR Ca²⁺ channels then induces a large increase in intracellular Ca²⁺ mediated by the SR channel (138). This process is well defined and has been aptly named calcium-induced calcium release (CICR). In skeletal muscle, Ca²⁺ entry is not required to activate the SR Ca²⁺-release process, as chelation of t-tubule Ca²⁺ does not prevent activation of the RYR channel and SR Ca²⁺ release (8, 41).

In skeletal muscle, a mechanical coupling was proposed by Schneider and Chandler (113), as they suggested that charged molecules located in the t-tubule membrane move during depolarization, and these molecules provide a physical link between the t tubule and the SR Ca²⁺-release process. They observed that voltage-dependent charge movement was closely associated with muscle contraction and could be measured across the t-tubule membrane when all ionic currents were blocked. Subsequent experiments showed that DHPs blocked charge movements and SR Ca²⁺ release with similar voltage and dose dependencies, thus arguing for a critical role of the DHP receptor in the activation of Ca²⁺ release (109). This sequence was later confirmed, as dysgenic myotubes lacking the DHP receptor 1-subunit failed to elicit requisite charge movement and ECC (11). This coupling was restored following transfection of the 1-unit (123, 125).

The current hypothetical mechanism suggests a direct interaction between the DHP-receptor tetrads and its closely associated RYR proteins, where voltage-dependent activation of the DHP-receptor proteins triggers Ca²⁺ efflux via the RYR (109). As Ca²⁺ exits the mouth of the channel, it putatively diffuses laterally to the unlinked RYR channels providing a role for Ca²⁺ ion activation or potentiation of the release process (109). Myoplasmic Ca²⁺ rises rapidly from ~50 nmol/l to 1-5 µmol/l, a concentration great enough to saturate the troponin-binding sites allowing interaction between actin and myosin.

The RYR displays a biphasic Ca²⁺-dependent behavior as submicromolar Ca²⁺ potentiates Ca²⁺ channel opening while millimolar Ca²⁺ induces closing of the RYR channel. Ca²⁺ ions in ECC and SR release have received much interest (96). Ca²⁺ has long been proposed as an activator in the Ca²⁺-release process, as the increases in myoplasmic Ca²⁺ after depolarization are thought to produce a positive-feedback CICR. This may be the critical trigger for those channels not directly coupled to t-tubule voltage sensors (109, 143). Inhibition of RYR activity at high concentrations of Ca²⁺ has been suggested to close the RYR after activation (109). However, this hypothetical mechanism does not explain how the SR channel maintains its closed configuration as the myoplasmic or triadic Ca²⁺ concentrations are lowered back to nanomolar level during SR Ca²⁺-ATPase sequestration following cessation of excitation. Although Ca²⁺ ions may not be the direct trigger of SR Ca²⁺ release, it is difficult to rule out the possible contributions they may have to potentiating SR Ca²⁺ release in vivo or under conditions of prolonged contractile activity.

Recently, Stephenson et al. (120) have argued an important role for Mg²⁺ in regulation of SR Ca²⁺ release. Their model suggests that by binding to the low-affinity Ca²⁺-binding site, Mg²⁺ (1 mM) exerts a strong inhibitory effect on channel activity, essentially closing the channel during resting conditions (85). They propose that "coupling" between the DHP receptor and RYR induces dissociation of Mg²⁺, via FKBP12 participation, removing the inhibition allowing Ca²⁺ to exit the channel and thus more fully activating the RYR and its neighboring unlinked RYRs (85). Under their assertion, Ca²⁺ release is controlled by the voltage sensors on the DHP receptor that regulate Mg²⁺ binding and dissociation from the RYR channel.

It has also been proposed that endogenous sulfhydryl (SH) groups present on the RYR protein may be responsible for regulating the gating of the SR Ca²⁺-release channel (1, 111, 112). It has been demonstrated that oxidation of the SH groups stimulates SR Ca²⁺ release in vesicle experiments (2) and muscle fibers (112). Whereas no clear role for the SH regulation has been established, it remains a viable mechanism to modulate channel gating.

Despite considerable effort over the past decade, the mechanism that describes SR Ca²⁺ channel activation is unknown. Although the DHP receptor is voltage regulated, the SR Ca²⁺ channel does not display voltage sensitivity, nor does it appear to be electrically coupled to the DHP receptor. Several hypotheses have suggested that various proteins participate in this process. Roles for triadin and the II-III cytoplasmic loop from the DHP receptor 1-subunit have been proposed but not completely elucidated.

In summary, the following sequence of events is currently thought to explain ECC: 1) sarcolemmal action potential, 2) TT charge movement, 3) coupling of TT charge movement to the SR Ca²⁺-release channel, 4) Ca²⁺ release from the SR, 5) Ca²⁺ binding to troponin, and 6) actomyosin ATP hydrolysis and cross-bridge cycling. As discussed above, because sequestration of Ca²⁺ ultimately restores the Ca²⁺ gradient, thus "loading" the SR membrane, the uptake of Ca²⁺ may be included as an accessory part of the ECC mechanism.

	SR CA2+ RELEASE FROM AN EXPERIMENTAL PERSPECTIVE

TOP ABSTRACT INTRODUCTION ECC AND THE SR... CURRENT THEORIES IN ECC SR CA2+ RELEASE FROM... BIOPHYSICAL REGULATION OF SR... FAILURE OF SR CA2+... CRITICAL QUESTIONS REFERENCES

When evaluating SR Ca²⁺ channel function, it is important to understand and evaluate the techniques from which the data were derived. Much of our current understanding regarding channel function was elucidated through the application of three different techniques (discussed below). Concurrent with the development and application of techniques used to study channel function was the identification of a variety of chemical modulators that can regulate the RYR Ca²⁺ channel activity. The list of activators of SR Ca²⁺ release includes anthraquinones, halothane, fatty acid derivatives, ryanodine, Ca²⁺, adenine nucleotides, caffeine, oxidizing reagents such as hydrogen peroxide, and sulfhydryl reagents (see Table 1 for specific concentrations). Paralleling the list of activators was the identification of compounds that inhibit Ca²⁺ release. Among them are the polycationic dye ruthenium red; lactate, both Ca²⁺ and Mg²⁺ in the millimolar range; and reducing agents, such as dithiothreitol and glutathione. Whereas many of these compounds are not physiological, several have proved extremely useful in describing channel regulation following physiological modification. In particular, experiments with caffeine and ryanodine have led to descriptions of channel behavior that is much like a "fingerprint," indicative of a certain type of channel dysfunction. As these reagents become more widely utilized, our ability to detect important, and yet subtle, changes in channel function will be advanced.

Vesicle flux assays. The majority of the early studies examined SR Ca²⁺ release by using isolated SR vesicle preparations (2, 94, 111). These vesicles could be actively loaded in the presence of Ca²⁺ and ATP, developing a 3,000-fold Ca²⁺ gradient across the SR vesicle. Once loaded, a wide variety of reagents can be used to trigger Ca²⁺ efflux. SR vesicle flux experiments are advantageous because they closely parallel in vivo Ca²⁺ release, as a large gradient provides the driving force for release. Triad preparations can also demonstrate activation by t-tubule depolarization following ionic substitution (7). The greatest advantage of this assay is that it yields representative data by averaging the kinetic behavior of a considerable number of channels contained by the vesicles. However, several drawbacks detract from the global utility of this assay. SR isolation is time consuming, it is possible that key SR proteins are lost as a result of the lengthy procedure, and, last, these assays do require significant amounts of protein, usually requiring larger muscle samples (~1 g muscle needed to isolate SR, the yield is ~1 mg/g muscle). Furthermore, because the assays are performed under "active" loading conditions where extracellular Ca²⁺ is monitored, it is possible that reagents that affect Ca²⁺-ATPase activity or modify membrane permeability can influence Ca²⁺ movements independent of SR Ca²⁺ release. For example, it is very difficult to perform SR Ca²⁺ release assays at different H⁺ concentrations, as Ca²⁺ loading is significantly inhibited at lower pH values, thus reducing the driving force for SR release.

Ryanodine-binding assays. Our greatest understanding of release channel regulation was advanced by the development of the ligand-binding assay using tritiated ryanodine (50, 94, 102). Ryanodine is a neutral plant alkaloid and a naturally occurring insecticide (72) that binds with nanomolar affinity to open Ca²⁺ channels. The greatest benefit of this assay is that ryanodine binds specifically to its receptor (the RYR-release channel) and to no other protein in the SR. With few exceptions, compounds that activate the SR Ca²⁺ channel and stimulate Ca²⁺ release also enhance the binding of ryanodine. The opposite is true for Mg²⁺, as it reduces SR Ca²⁺ release in a concentration-dependent manner and inhibits the binding of the ryanodine to its receptor. These macroscopic assays are not technically difficult, and they provide ample opportunity to manipulate a wide variety of conditions that would be difficult to carry out in flux assays. However, the binding is quite slow (typically 3 h) and thus it does not provide temporal data consistent with SR Ca²⁺ release in single-fiber or vesicle experiments.

Single-channel analysis. By fusing isolated SR vesicles to lipid bilayer membranes, the SR Ca²⁺ gating characteristics can be evaluated (117). Much of the pharmacological and biochemical data generated from the two macroscopic assays have been confirmed on the single-channel level by using isolated proteins. The fused protein remains sensitive to almost all of the known activators and inhibitors of Ca²⁺ release and ryanodine binding. The channel is permeable to both mono- and divalent cations, but because the channel is almost three times more permeable to Cs⁺ than Ca²⁺, Cs⁺ is frequently used as the charge carrier. Following fusion and incorporation of the Ca²⁺-release channel into the bilayer membrane, both the cytoplasmic and luminal sides of the channel protein can be manipulated. This technique is technically demanding, but direct access to a single-gating channel can provide important and detailed information on a molecular level. However, this technique may be self-limiting where macroscopic assays might provide more conclusive data. For example, it was previously determined that SR Ca²⁺ release was reduced by 30% after a run to exhaustion in rats (46). It is believed that as few as one RYR channel exists per vesicle (some vesicles contain no channels), and if only 30% of the channels are dysfunctional, it is possible that 70% of the fusion events will contain unmodified RYR proteins. Thus many assays would have to be conducted to ensure that the stated effect was observable at the single-channel level. Still, this technique was used to determine the Ca²⁺ sensitivity of gating RYR channels isolated from denervated rat muscle (38). In the native state, Ca²⁺ channel experiments are performed as many as six to ten times to confirm channel behavior.

The physiological relevance of the above biochemical and pharmacological characterizations have been advanced and clarified in single-fiber studies. From these studies, it appears as if the Ca²⁺-release process may be dually regulated (85). DHP receptor-mediated activation exerts the most important control over the SR Ca²⁺-release process. However, modulation of SR Ca²⁺ release can be accomplished through a variety of reagents that are known to stimulate or inhibit RYR activity. Considering this evidence, it is important to evaluate reagents typically used in both biochemical and various fiber preparations. Some, but not all, ligands that modulate RYR channel activity in SR vesicle experiments perform similarly at the level of a single muscle fiber. Lamb and Stephenson (85), using skinned fibers with intact TT, have shown that after a normal depolarization Ca²⁺ release and muscle contraction could be completely inhibited by high Mg²⁺ concentrations, a result consistent with SR vesicle studies (95). They demonstrate that the function of the SR Ca²⁺ channel is controlled by the TT under resting conditions but it is also sensitive to the changing intracellular milieu. On the other hand, in a different study, Lamb et al. (84) demonstrated that reductions in intracellular pH have little effect on ECC in preparations where Ca²⁺ release is controlled by native voltage activation mediated by the DHP receptor. These two studies should serve as a caveat when attempting to support results from single-fiber experiments with data obtained from SR vesicle or single-channel preparations.

	BIOPHYSICAL REGULATION OF SR CA2+ RELEASE

TOP ABSTRACT INTRODUCTION ECC AND THE SR... CURRENT THEORIES IN ECC SR CA2+ RELEASE FROM... BIOPHYSICAL REGULATION OF SR... FAILURE OF SR CA2+... CRITICAL QUESTIONS REFERENCES

In addition to the chemical/mechanical/electrical mechanisms that govern ECC, release of Ca²⁺ from the SR lumen is dependent on the physical constraints of the ion channel and luminal ion concentration. A reduction in Ca²⁺ ion flux through the channel may be affected by altering the gating characteristics, modification of the channel conductance, and/or by a reduction in the SR lumen/cytoplasm Ca²⁺ gradient that determines the driving force for release.

The SR channel gates in a two-state (open or closed) model. The two primary gating characteristics that determine ion flow are the probability that the channel is in the open state (P_o) and mean open time, the average time the channel remains in the open configuration once open. On the single-channel level, compounds that close the channel generally do so by reducing the P_o. This may be accomplished by reducing the time spent in the open configuration or reducing the frequency of opening. This behavior has been observed by several laboratories, with many of the compounds listed in Table 1. For example, Smith et al. (117) demonstrated this by using Mg²⁺ to inhibit the SR release channel.

Whereas it is theoretically possible that the channel conductance may be modified, this scenario is unlikely during fatigue. There are very few examples in which conductance has been modified by anything other than nonbiological organic molecules. Ryanodine can induce subconductance states in single-channel experiments (68, 82). Furthermore, subconductance behavior was also recently reported after the removal of FKBP12 by rapamycin (4), but this was also accompanied by an increase in the P_o, making it difficult to extrapolate these results to conditions of muscle fatigue.

Recent work indicates that the SR lumen/cytoplasmic Ca²⁺ gradient may become diminished as a result of either inadequate refilling following Ca²⁺ release or by reducing the amount of free releasable Ca²⁺ stored within the lumen.

The total SR Ca²⁺ content has been determined to be 1-2 mM per fiber volume (51, 55) and represents the driving force for Ca²⁺ release into the myoplasm. Fryer et al. (56) have indicated that SR Ca²⁺ content is different between fast and slow type muscle. Luminal Ca²⁺ content is dependent on several factors including, but not limited to, function of the SR Ca²⁺ ATPase, leakiness of the SR membrane and/or RYR channel, and the total amount of Ca²⁺ released that is a function of duration and frequency of the contractile protocol. Inhibition of the SR ATPase (22, 91) and reductions in Ca²⁺ accumulation by SR have been demonstrated after exhaustive running in rats (22, 78), in single-fiber experiments (135, 141), and in exercising humans (17). Evidence suggests that with prolonged stimulation resting intracellular Ca²⁺ concentrations are significantly elevated (89). However, one early report suggested that SR Ca²⁺ content is not modified (61).

Reductions in the amount of releasable SR Ca²⁺ due to the formation of a Ca²⁺-P_i precipitate would also decrease SR Ca²⁺ release, as has been suggested by Fryer et al. (55). P_i can rise to 30-40 mM in the active muscle cell (23), a concentration sufficiently high enough for P_i to enter the SR and complex with Ca²⁺ forming a precipitate (57). Recent work from Fryer et al. (55, 57) and Westerblad and Allen (132) indicates that physiological concentrations of P_i will reduce force by decreasing SR Ca²⁺ release, an event whose time course of recovery is consistent with the resolubilization of the Ca²⁺-P_i precipitate (103). Continued work in this area should reveal more evidence detailing the implications of reduced SR Ca²⁺ content and/or the effects of P_i on SR Ca²⁺ concentrations during muscle fatigue. Further study should help to clarify the effect of prolonged contractile activity on SR Ca²⁺ load and loading parameters.

	FAILURE OF SR CA2+ RELEASE AND MUSCLE FATIGUE

TOP ABSTRACT INTRODUCTION ECC AND THE SR... CURRENT THEORIES IN ECC SR CA2+ RELEASE FROM... BIOPHYSICAL REGULATION OF SR... FAILURE OF SR CA2+... CRITICAL QUESTIONS REFERENCES

Intense or prolonged contractile activity can reduce muscle force output that recovers after the cessation of activity. This sequential pattern of events is typically referred to as muscle fatigue. In part, the length of the recovery period is one of several characteristics that distinguish different types of fatigue, in particular, low-frequency fatigue (LFF) and high-frequency fatigue (HFF) (74, 75). HFF is characterized by a rapid loss of force that recovers very quickly once muscle activation is reduced or stopped (75). It has been suggested that HFF is most likely due to disturbances in muscle excitation related to ionic imbalances that are readily reversible (24, 75). It has been questioned whether this type of fatigue is normal in healthy muscle (75). Conversely, LFF, first described by Edwards et al. (44), is characterized by a loss of force primarily at lower stimulation frequencies, from which recovery is slow and may persist for hours or days, despite the absence of any gross disturbances in the muscle (75). It is important to point out that this type of fatigue can be caused by a variety of forms of activity, but loss of force is noticeable at lower stimulation frequencies. It has been suggested that LFF may be due to a wider variety of mechanisms including, but not limited to, metabolic, mitochondrial, and/or SR disruptions. Despite the fact that the etiology of muscle fatigue is very dependent on that stimulation frequency or muscle activation protocol, along with other considerations such as fiber type, state of training, etc., SR Ca²⁺ release remains a focal point.

The type of stimulation protocol that is most like prolonged submaximal exercise is LFF. Fatigue induced by low-frequency stimulation tends to follow a stereotypical pattern of force decline (133). During the initial phase, force declines rapidly to ~80% of control. During the second phase, a more stable force production pattern predominates, with a slight additional reduction in force to 70%. This is followed by a rapid decline of force to ~30-40% and is referred to as "phase three." It has long been suggested that reductions in SR Ca²⁺ release were involved in loss of force observed at low stimulation frequencies (44) and are suggested to be the primary factor in phase-three fatigue (133). Currently, several mechanisms are likely candidates to explain the reductions in SR Ca²⁺ channel activity following contractile activity. They include, but are not limited to, elevated Ca²⁺ concentrations, alterations in metabolic homeostasis within the "microcompartmentalized" triadic space, and modification by reactive oxygen species (ROS).

Elevated Ca²⁺. Westerblad et al. (135) have suggested that reductions in force, particularly LFF, can be explained by a reduction in the SR Ca²⁺ release. Consistent with a reduction in Ca²⁺ release is a general loss of Ca²⁺ homeostasis where intracellular Ca²⁺ is elevated significantly. Lamb et al. (83) have shown that raised intracellular Ca²⁺ in skinned fibers abolishes ECC and alters the structure of the t-tubule SR triad. Reductions in Ca²⁺ release after exposure to 100 µM Ca²⁺ were noted to be time dependent (longer exposures induce greater uncoupling), temperature dependent (uncoupling 13-fold slower at 3°C compared with 23°C), and pH dependent (slower at lower pH values). Interestingly, Mg²⁺ elevated to 10 mM did not prevent or induce uncoupling at high Ca²⁺. Whereas these studies focus on ECC as a whole, they do not pinpoint the critical step that is modified by elevated Ca²⁺. This, however, was examined by Williams (139), who recently demonstrated that in skinned frog fibers SR Ca²⁺ release is directly affected by elevated Ca²⁺. A single 5-min exposure to 500 nM Ca²⁺, a concentration noted to accompany fatiguing stimulation (135), reduced caffeine-stimulated SR Ca²⁺ release. Chin and Allen (29) and Chin et al. (32) have further characterized this phenomenon. Their work suggests that the duration of exposure must be considered along with the level to which intracellular Ca²⁺ concentration ([Ca²⁺]_i) is raised, arguing that the "Ca²⁺-time integral" is most critical in predicting the deficit in SR Ca²⁺ release induced by raise in intracellular Ca²⁺ (32).

	CRITICAL QUESTIONS

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Several areas need to be considered to provide a clearer understanding of any potential mechanism that involves reduced SR Ca²⁺ release.

1) Proteins. Continued evaluation of all proteins, existing and emerging, needs to focus on more precise anatomic placement, leading to a more advanced understanding of individual or complex function. A more accurate map of the triadic space should help determine the accessibility of diffusable mediators that may modify SR Ca²⁺ channel function.

2) Multiple regulators. Many important metabolites have been shown to alter SR Ca²⁺ channel function, but they are often evaluated independently. Our current knowledge would be greatly extended if experiments were designed to assess how multiple regulators interact with each other and with key triadic proteins.

3) Techniques. What new and emerging techniques will shed light on events that have yet to be described during or following intense prolonged muscle activity? For example, utilization of single-channel analysis or Ca²⁺ sparks techniques that may provide molecular insights into channel dysfunction.

Conclusions. In light of the fact that the detailed mechanism that describes ECC and SR Ca²⁺ release in muscle fibers is currently unresolved, our common understanding suggests that a variety of contractile patterns that elicit muscle fatigue appear to involve alterations in ECC and reduced SR Ca²⁺ release. Several putative mechanisms have been evaluated that represent collective ideas forwarded by many different investigators. Much work has significantly focused the discussion and areas of experimentation regarding SR Ca²⁺ release and muscle fatigue. A continued synthesis of experimental techniques, from isolated protein experiments involving single-channel analysis to controlled experiments using fiber bundles activated by an intact nerve, should provide further understanding of muscle performance observed during animal or human activity.