Skeletal muscle fatigue has been defined as the transient work-induced inability to maintain the expected or required force or power output. The potential mechanisms underlying the impaired muscle function during fatigue are varied and include decreased central nervous system drive, impaired neuromuscular transmission, alterations in the muscle surface and transverse-tubule (t-tubule) membrane action potential, impaired excitation-contraction coupling (ECC), decreased calcium (Ca2+) sensitivity of contraction, and impaired cross-bridge function (for review see (1)). The relative importance of each of these factors depends on the type and duration of the work bout that induced the fatigue, environmental conditions, and the nutritional state and training status of the individual. The characteristics of fatigue, that is, changes in contractile properties, electromyography, force-frequency relationship, and duration of impairment, will vary according to the underlying impairment and therefore provide mechanistic insight.
Low-frequency fatigue (LFF) is characterized by a prolonged recovery and selective loss of force at low frequencies of activation (Fig. 1A) regardless of the protocol used to induce fatigue (1). Indeed, a work-induced decrease in the ratio of force production in response to 20- or 30-Hz stimulation versus 100 Hz is commonly used to define LFF. Although the initial characterization of LFF, which used human subjects, included continuous low-frequency stimulation of a small muscle, the work also described LFF in response to voluntary isometric and dynamic contraction of a large muscle group. It is now clear that LFF occurs in humans in response to voluntary contractions (1).
LFF may be functionally significant, as the motor discharge rate during voluntary muscle activation rarely exceeds 30 Hz (1), an activation frequency at which LFF is manifest. Increased motor drive may compensate for the decreased force generation at low activation frequencies and thereby maintain force. However, the increased central nervous system activation may cause subjects to experience a greater sense of effort and increased fatigability during normal daily activities. Furthermore, LFF of the respiratory muscles occurs after exhaustive respiratory effort and has therefore been suggested to be of clinical significance in respiratory failure (26).
A number of characteristics of LFF give clues as to the underlying mechanisms (1). First, involvement of the central and peripheral nervous system are excluded because of the lack of significant alterations in the electromyogram associated with LFF. Muscle metabolite levels return to near resting levels early in recovery while force remains significantly depressed; thus, a metabolic mechanism is excluded. Second, because this type of fatigue has been induced in isolated muscles and single muscle fibers, the mechanism underlying LFF must lie within the muscle fiber itself. The long-lasting nature of LFF precludes muscle membrane depolarization and alterations in the action potential as probable causes because these derangements recover rapidly upon the cessation of work. The observation that LFF in isolated muscles was completely reversed by the ryanodine receptor (RyR) activator caffeine suggested an impairment in ECC as the underlying mechanism. Finally, the prolonged recovery suggests a protein modification, possibly requiring protein synthesis to restore force.
Evidence to date suggests a functional uncoupling of excitation from contraction related to an elevated Ca2+ concentration in the triadic junction. However, the mechanism and targets of the Ca2+-dependent process are unknown. Likely candidates include the transverse-tubule (t-tubule) voltage sensor (also known as L-type Ca2+ channel and dihydropyridine receptor (DHPR)) and RyR1 Ca2+ release channel in the sarcoplasmic reticulum (SR), yet other recently discovered "minor" triadic proteins may be involved and are currently being characterized. Some of these minor proteins seem to be critical for the structural integrity of the triad and for the functional coupling of the DHPR and RyR and therefore have a potential role in excitation-contraction uncoupling in LFF, eccentric muscle contraction-induced muscle weakness, and aging. Rather than focus on the major triadic proteins, the DHPR and RyR1, which appropriately receive a great deal of attention, this review will focus on the potential role of the minor proteins in LFF. First, the mechanism of skeletal muscle ECC and the role of the DHPR and RyR1 will be reviewed; then, the evidence for excitation-contraction uncoupling in LFF will be described. This will be followed by a discussion of many of the recently characterized triadic proteins, and finally, potential roles these proteins could play in LFF will be suggested.
ECC occurs at the interface of two membrane systems, the invaginations of the surface membrane known as t-tubules and the intracellular SR (Fig. 2A). The SR is a specialized form of endoplasmic reticulum (ER), dedicated to the storage and release of Ca2+ for the regulation of muscle contraction. The SR can be divided into two domains, the longitudinal SR and the junctional SR. The longitudinal SR is made up of a network of interconnected tubules enriched in proteins involved in uptake of Ca2+ into the SR such as the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). As the longitudinal SR approaches the t-tubule, the tubules coalesce into an enlarged sac known as the terminal cisterna. The region of the terminal cisterna facing the t-tubule is the junctional face membrane. At the junction of A and I bands in mammalian skeletal muscle, two terminal cisternae and one t-tubule make up the triad. Numerous proteins, many of them uncharacterized, are found in triad preparations (Fig. 2B). Of primary importance are the L-type Ca2+ channel in the t-tubule membrane and the RyR in the junctional face membrane. Depolarization of the t-tubule membrane induces a conformational change in the L-channel, which, in turn, opens RyR1, allowing Ca2+ efflux from the SR (21).
In mammals, there are three RyR isoforms (for review, see (13)). RyR1 and RyR2 are the predominant isoforms in skeletal and cardiac muscle, respectively. RyR3 has a wide tissue distribution and is expressed at very low levels in skeletal muscle. RyRs are homotetrameric channels. Each monomer consists of greater than 5000 residues and has a molecular mass of approximately 565 kDa. RyRs are arranged in lattice-like arrays in the junctional face membrane where they contact their neighbors at the corners, and every other RyR1 is coupled to a DHPR tetrad. The carboxy-terminal third of the channel spans the SR (Fig. 3). The amino two-thirds of the protein is cytoplasmic, makes critical interactions with the DHPR, and provides a scaffolding to support numerous accessory proteins. Some of the accessory proteins are involved in the phosphorylation/dephosphorylation of the channel, including cAMP-dependent protein kinase (PKA) and phosphodiesterase 4D3 (PDE4D3), Ca/calmodulin-dependent protein kinase (CaMKII), and protein phosphatase 1 (PP1). The EF-hand Ca2+-binding proteins, calmodulin (CaM) and S100A1 modulate Ca2+ activation of RyR1 and may compete for the same binding site on the channel. In vitro, both these proteins enhance channel opening in nM Ca2+ concentrations (near resting intracellular Ca2+ concentrations) (13,32). Although genetic ablation of CaM is lethal, S100A1 knockout animals are viable and exhibit reduced voltage-activated SR Ca2+ release (27). Thus, disrupting the interaction between CaM or S100A1 and RyR1 would be predicted to depress SR Ca2+ release. Furthermore, oxidation of CaM (4), or oxidation, nitrosylation, or glutathionylation of RyR1 (3) reduces CaM regulation of the channel. Similar work has not been performed with S100A1. The FK506-binding protein FKBP12 is thought to stabilize the tetrameric channel and aid in coordinated gating. The function of the glycolytic enzyme aldolase is unclear, but it may serve as an anchor for the neutral Ca2+-activated protease calpain-3. A soluble form of the chloride intracellular channel (CLIC-2) binds RyR1 and stabilizes the closed state of the channel (22). The channel also interacts with the SR membrane proteins triadin and junction, which, in turn, interact with calsequestrin and histidine-rich calcium-binding protein (HRC). Calsequestrin and HRC are low-affinity, high-capacity Ca2+-binding proteins that act to buffer the free Ca2+ concentration within the SR lumen. Homer1 is a scaffolding protein that has been reported to enhance RyR1 activation (12). However, more recently, Homer1 has been localized to the z-disk where its putative function is to link mechanical stretch to the activation of mechanosensitive channels (31).
In vivo, RyR1 is activated via the voltage-driven conformational change in the DHPR; however, in vitro, RyRs can be activated in a biphasic manner by Ca2+ (13). In the absence of other channel regulators, RyR1 is closed at resting cytoplasmic Ca2+ levels (approximately 50 nM). As the Ca2+ concentrations on the cytoplasmic face of the channel increases into the μM range, channel opening increases and reaches a peak at a Ca2+ concentration near 0.1 mM. Further increases in Ca2+ progressively close the channel. This biphasic Ca2+ dependence is modulated by numerous endogenous ligands including adenine nucleotides and magnesium, which enhance and inhibit channel opening, respectively (13).
L-Type Ca2+ Channel (DHPR)
In skeletal muscle, the L-type Ca2+ channel is located primarily in the t-tubule and is composed of five subunits, a pore-forming α1 (Cav1.1), an intracellular β, a membrane spanning γ, and an extracellular α2, which is linked via a disulfide bond to a membrane spanning δ subunit. The α1 subunit is composed of four repeating motifs (I-IV) of six transmembrane α-helices (S1-S6). The S4 α-helix of each repeating motifs is highly positively charged and imparts voltage-sensitivity to the channel. A hairpin-loop between S5 and S6 forms part of the channel pore. DHPRs in the t-tubule oligomerize into tetrads with each of the four DHPRs making multiple contacts with RyR1 (5). These contacts involve the intracellular loop between the repeat motifs II and III of α1, the α1 carboxy-terminal tail, and the β-subunit (Fig. 2B). The contacts transduce the depolarization-induced conformational change in the DHPR to RyR1. Thus, weakening these interactions would decrease the efficiency of ECC and impair force development.
In vitro, two electrical signals originating from the DHPR can be recorded; the t-tubular charge movement and the L-type Ca2+ current (for details, see (21)). The t-tubular charge movement originates in the voltage-driven translocation of charges, predominately within the four S4 α-helices, across the electrical field of the t-tubular membrane. The L-current is carried by the influx of Ca2+ from the t-tubular lumen into the triadic junction. The charge movement is an absolute requirement for skeletal muscle ECC. The function of the L-current is unclear as extracellular Ca2+ can be removed without abolishing contraction.
Ca2+ Fluxes Across the Transverse-Tubule Membrane
A number of pathways for Ca2+ influx across the surface membrane and t-tubules have recently been identified (for details, see (10)). In nonexcitable cells, depletion of intracellular Ca2+ stores triggers the influx of Ca2+ across the plasma membrane to refill the depleted store, that is, store-operated Ca2+-entry (SOCE). Recently, SOCE has been clearly demonstrated in skeletal muscle. The two primary components underlying SOCE-stromal interaction molecule 1 (Stim1), the Ca2+ store sensor, and Orai1, a component of the SOCE channel-are highly expressed in skeletal muscle. Stim1 colocalizes with RyR1. SOCE in skeletal muscle occurs across the t-tubule membrane, is controlled by the Ca2+ content in the region of SR adjacent to each section of t-tubule, and can be activated by individual Ca2+ release events before exhaustion of the SR Ca2+ store.
A role for SOCE in muscle fatigue comes from observation that muscle with reduced Stim1 expression generated lower maximal tetanic tension and fatigued more rapidly than muscle with normal Stim1 content. The increase susceptibility to fatigue was associated with an inability to maintain SR Ca2+ stores. Thus, SOCE is required to sustain SR Ca2+ stores during repeated contractions (10).
Excitation-coupled Ca2+ entry (ECCE) is a store-independent Ca2+ entry pathway that is activated upon prolonged or repetitive depolarization (10). The molecular entities underlying this current are not well defined. The current requires functionally coupled RyRs and DHPRs and is independent of Stim1 and Ori1. It may be mediated by the L-type Ca2+ channel or an as of yet unidentified Ca2+-permeable channel within the t-tubule and coupled to the RyR-DHPR complex.
Launikonis et al. (19) described a small action-potential-mediated Ca2+ influx across the t-tubule membrane that could not be ascribed to any known skeletal muscle ion channel. The current rapidly inactivates and therefore raises the Ca2+ concentration in the triadic junction only during the initial action potential of a train. The authors speculate that this Ca2+ influx primes the RyRs to activation by the DHPR.
EXCITATION-CONTRACTON UNCOUPLING IN LFF
In general, three different preparations have been used to study the cellular mechanisms underlying LFF, in vivo exercise in conjunction with skeletal muscle biopsies, isolated whole muscles or individual muscle fibers, and mechanically peeled muscle fibers. After dynamic knee extensor exercise, which reduced evoked isometric tension at stimulation frequencies of 10 and 20 Hz stimulation but not at 50 or 100 Hz, Hill et al. (14) obtained biopsies of the vastus lateralis. In this preparation, SR function from fresh and fatigued muscle was studied under identical conditions eliminating any metabolic or ionic influences. LFF resulted in a reduced rate of SR Ca2+ release and a reduced rate of Ca2+ uptake back into the SR. Despite the depressed rate of Ca2+ uptake, there was no difference in the rate of adenosine-5'-triphosphate (ATP) hydrolysis by SERCA, suggesting there was either an uncoupling of Ca2+ transport from ATP hydrolysis by the pump or there was an increased leak of Ca2+ out of the SR. Furthermore, there was a significant correlation between the decreased ratio of torque generated at 20:50 Hz and the rate of SR Ca2+ release, but no relationship was found between the rate of Ca2+ uptake and muscle relaxation or rate of torque generation. These authors concluded that modification of the RyR protein contributed to LFF. An obvious limitation to this type of work is that SR Ca2+ release could not be triggered via the normal DHPR-RyR coupling.
To circumvent this limitation, intact mouse muscle fibers were intermittently stimulated, and isometric tension and intracellular Ca2+ were simultaneously recorded (1). The induction of LFF was exemplified by a long-lasting reduction in isometric tension at low stimulation frequencies. There was no change in Ca2+ sensitivity of contraction or maximal tension. Conduction of the action potential into the depths of the t-tubule was not impaired as the reduction in tetanic Ca2+ across the width of the muscle fibers was uniform. In addition, direct activation of RyR1 with caffeine restored tension in the fatigued muscle, demonstrating that SR Ca2+ stores were not depleted. Thus, the main cause of LFF in these intact fibers was a reduction in SR Ca2+ release, which in conjunction with the sigmoid Ca2+ dependence of contraction, caused the loss of force at low stimulation frequencies.
As illustrated in Figure 1B, because of the sigmoid force-Ca2+ relationship, a decrease in the intracellular Ca2+ transient could give rise to LFF. In fresh muscle (solid arrows), high-frequency stimulation elicits a Ca2+ transient that falls on the plateau of the force-Ca2+ curve. Although fatigue may reduce the Ca2+ transient in response to high-frequency stimulation, it would still fall near the plateau of the force-Ca2+ curve, and tension would be maintained (dashed arrow). In contrast, the Ca2+ transient elicited by low-frequency stimulation falls on the steep section of the curve. A reduction in the Ca2+ transient here would cause significant reduction in force (Fig. 1B, low-frequency solid vs dashed arrow).
An advantage of intact muscle fibers is that the physiological ECC mechanism remains intact; however, a limitation is the inability to manipulate the intracellular environment to study the mechanisms underlying LFF. Peeled muscle fibers maintain the DHPR-RyR coupling mechanism and allow manipulation of the cellular environment. In this preparation, a segment of muscle fiber is isolated, and the surface membrane is mechanically peeled from the fiber. When the surface membrane tears away from the t-tubules, the t-tubule membrane seals over and traps the extracellular solution in the t-tubule lumen. The t-tubules can then be polarized and depolarized via ion substitution in the bath media, and because the link between the DHPR and RyR remains intact, SR Ca2+ release can be induced via the physiological ECC pathway.
A Ca2+-induced uncoupling of ECC was demonstrated in mechanically peeled muscle fibers (1). The uncoupling was Ca2+ and time dependent and could be induced upon a 60-s exposure to 2.5 μM Ca2+, a physiologically relevant Ca2+ concentration. Furthermore, the temperature and pH dependence of the uncoupling suggested an enzymatic process. However, Ca2+-dependent phosphorylation or dephosphorylation were not involved in the process as neither removal of ATP to prevent phosphorylation, nor phosphatase inhibition prevented the Ca2+-induced uncoupling. Similarly, oxidation was excluded as a mechanism because the reducing agent dithiothreitol (DTT) did not significantly alter the process. Later work suggested a role for the Ca2+-dependent proteases known as calpains (34).
Simultaneous recordings of force and intracellular Ca2+ in single muscle fibers supported the hypothesis of Ca2+-induced ECC uncoupling in LFF (1). A threshold Ca2+-time integral required to induce ECC uncoupling was defined by varying the duty cycle of the stimulation protocol and including caffeine in some of the bath solutions. Stimulation protocols that exceeded the threshold induced LFF.
Although Bruton et al. (6) confirmed the role of reduced Ca2+ release in LFF in mouse fibers that generate little reactive oxygen species (ROS) during fatiguing stimulation, they also showed that in rat fibers, which do generate ROS, there was a reduction in the Ca2+ sensitivity of contraction. In addition, the reduced Ca2+ sensitivity but not the depressed SR Ca2+ release could be partially reversed by reducing agents. Thus, the work-induced decrease in Ca2+ sensitivity was attributed to ROS, but the mechanism underlying the reduced Ca2+ release remained unclear.
Taken together, this work suggests that depressed SR Ca2+ release and possibly a reduced Ca2+ sensitivity of contraction make a significant contribution to the development of LFF. Although there may be a small reduction in the rate of SR Ca2+ uptake, it is not sufficient to reduce SR Ca2+ load to the point where release is significantly impaired. Because LFF can be overcome by a strong activating stimulus, for example, high-frequency stimulation or caffeine, the impaired SR Ca2+ release can be attributed to a decrease in the strength of the functional coupling between the DHPR and the RyR. Furthermore, the uncoupling seems to be a Ca2+-mediated process. Before addressing mechanisms by which elevated triadic Ca2+ might impair ECC, the newly characterized triadic proteins that might be involved in maintaining efficacy of ECC will be reviewed.
MINOR TRIADIC PROTEINS
In myotubes devoid of RyR1 or DPHR, the SR and t-tubules still arrange into triads. Conversely, when RyR1 and the DHPR are heterologously coexpressed triads that are not formed, the proteins remain uncoupled and depolarization-induced Ca2+ release is not reconstituted. These results catalyzed the search for proteins required for triad formation. Although the DHPR and RyR are the best characterized, numerous other proteins are present in the skeletal muscle triad. A number of these proteins have been characterized, and their role in ECC also has been investigated (33). Some of them have been associated with impaired muscle function associated with aging or eccentric contractions. The location of many of these proteins is illustrated in Figure 2B, and their putative functions are described below.
Junctophilin is a single transmembrane domain SR protein. The carboxy-terminal is located within the SR, whereas the amino terminal interacts with the cytoplasmic face of the t-tubule membrane via a 14 residue MORN (membrane occupation and recognition nexus) motif (33). There are four isoforms, JP-1,-2,-3, and -4. JP-1 is expressed exclusively in skeletal muscle. JP-2 is expressed in cardiac, skeletal, and smooth muscles. Suppression of JP-1 and -2 in mature skeletal muscle resulted in deformed triads, reduced SOCE, and decreased SR Ca2+ stores (16). Thus, JP-1 and -2 are critical for the formation and maintenance of the skeletal muscle triad. Recently, we have shown that JP-1 and JP-2 content is reduced in adult skeletal muscle following a bout of eccentric muscle contractions, at a time when tetanic tension was significantly reduced. Furthermore, there was a significant correlation between the magnitude of the strength deficit and the reduction in JP-1 and JP-2 content (8). Although the aim of that work was to induce muscle damage rather than LFF, the two phenomena may share an underlying impairment, namely excitation-contraction uncoupling.
Junctional Face Protein 45 kDa (JFP-45) (not to be confused with junctophilins) also is a single transmembrane protein linking the SR and t-tubules. It interacts, via its C-terminal, with calsequestrin in the lumen of the SR and with the DHPR via its N-terminal (2). JFP-45 knockout mice are viable but exhibit reduced voluntary wheel running and muscle weakness. The weakness was associated with decreased Cav1.1 expression, reduced t-tubular charge movement, and reduced depolarization-induced Ca2+ release but normal RyR expression (9). Thus, JFP-45 is thought to target Cav1.1 to the t-tubule and to maintain the DHPR complex.
Mitsugumin-29 (Mg-29; mitsugumi means triad junction in Japanese) is a 29 kDa four transmembrane domain t-tubular protein (33). In micrographs of the triad, the t-tubules seem elongated to provide a greater interface with the junctional face membrane of the SR. Deletion of Mg-29 resulted in swollen t-tubules, vacuolated SR, and misaligned triads; thus, Mg-29 is thought to maintain the t-tubules in the elongated conformation that allows triad formation (23). Deletion of Mg-29 significantly impaired SOCE in myotubes and increased susceptibility to fatigue in mature muscle (25).
Triadin (TRISK) was initially thought to be a skeletal muscle-specific protein, it is now understood that triadin is expressed in numerous tissues, and the triadin gene can undergo multiple splicings to generate at least four isoforms, Trisk95 (Triadin Skeletal 95 kDa; the initial triadin identified), Trisk51, Trisk49, and Trisk32 (20). All four isoforms have a single transmembrane domain, a short cytoplasmic domain, and a long SR luminal tail. Trisk95 and -51 are localized to the terminal cisternae, whereas trisk32 is located throughout the SR. When located in the terminal cisternae, trisks are associated with RyR1 and may bind calsequestrin.
Two groups (24,30) generated pan-triadin knockout animals and reported similar results. In fast- but not slow-twitch skeletal muscle, there were subtle alterations in the architecture of triads in which the t-tubules were oriented longitudinally or obliquely rather than transversal. Although calsequestrin content was reduced, it was properly localized to the terminal cisternae. As a consequence of the reduce calsequestrin content, the SR Ca2+ stores were reduced. Interestingly, the mice generated by Oddoux et al. (24) but not those of Shen et al. (30) exhibited muscle weakness, performing worse than wild-type animals on both in vivo and in vitro tests of strength. Both groups concluded that triadins are required for the proper arrangement of the triad and for normal expression of calsequestrin. Furthermore, if triadins are involved in anchoring calsequestrin at the terminal cisternae, an additional protein, possibly junctin, also must be involved.
Alternative splicing of the aspartyl-β-hydroxylase gene results in one of four single transmembrane domain proteins, aspartyl-β-hydroxylase, humbug, junctate, or junctin. Junctin and junctate are localized to the skeletal muscle triad (33). Junctin has a short cytoplasmic domain, a long SR luminal tail, and binds RyR1 and calsequestrin. It may communicate the extent of SR Ca2+ load to the RyR1. Junctate is a 33-kDa single transmembrane domain SR protein. The carboxy-terminal tail, within the SR lumen, has a moderate affinity and high capacity for Ca2+ binding. Overexpression in skeletal muscle increased SR Ca2+ load and enhanced RyR1-mediated Ca2+ release.
Trimeric intracellular cation selective channels (TRIC) are ubiquitously expressed homotetrameric proteins localized to the SR, endoplasmic reticulum, and nuclear membranes. TRIC-B is the common form. TRIC-A (also know as SRP-27 and mitsugumin-33) is specific to excitable tissue and is expressed at higher levels in fast-twitch than in slow-twitch muscle. In planar lipid bilayer experiments, TRIC-A is selective for monovalent cations and has single channel characteristics similar to those described for the SR K+ channel. TRIC-A may mediate cationic countercurrent required to prevent the development of a negative SR membrane potential upon the release of Ca2+ from the SR (35).
Glycolytic and gluconeogenic enzymes are associated with SR membranes and are thought to maintain the local concentration of ATP at a level sufficient for optimal SERCA activity. Some of these enzymes also may play a structural role in the maintenance of the triad. Aldolase, for example, is physically associated with RyR1 (19).
Calpain-3 is a member of the calpain family of neutral Ca2+-dependent proteases. A fraction of the muscle calpain-3 localizes to the triad and associates with aldolase and the RyR. Aldolase, RyR1 content, and SR Ca2+ release are reduced in skeletal muscle from calpain-3-deficient animals suggesting calpain-3 may play a role in the structural stability of the triad (19).
POTENTIAL TARGETS AND MECHANISMS
The characteristics of LFF, in particular the apparent lack of effect of LFF on SR Ca2+ stores, provide some insight into potential targets among the minor triadic proteins. The relatively minor effect of LFF on force production at high stimulation frequencies and the ability of caffeine to restore force during LFF demonstrate that there is sufficient Ca2+ within the SR to fully activate contraction. Thus, modification of components of the store-operated, excitation-coupled, and action-potential activated Ca2+ entry pathways do not significantly contribute to LFF. Similarly, the counter current required for SR Ca2+ release (which may be mediated in part by TRIC-A) does not seem to be impaired because activation of RyR1, rather than flux through the open channel, is impaired. Therefore, the minor triadic proteins thought to play a role in maintaining the structural integrity of triads, such as junctophilin-1 and -2 (16,17), JFP-45 (9), and Mg-29 (23), are the likely candidates to be involved in LFF-induced excitation-contraction uncoupling (Fig. 4). The components of the triad that maintain the DHPR and RyR1 in the proper position and conformation for the functional coupling of these two channels are unknown. However, it is clear from heterologous coexpression experiments that the two channels alone are insufficient. The junctophilins seem to function as a bridge between the t-tubule and SR and maintain the proper distance between the DHPR and RyR1. By maintaining the elongated structure of the t-tubules, Mg-29 also contributes to the proper alignment of the DHPR and RyR1. Thus, alterations in any of these proteins could significantly alter the architecture of the triad and decrease the efficacy of ECC (Fig. 4B and C). Indeed, our work has shown that the eccentric contraction-induced force decline correlated with the loss of JP-1 and JP-2. In addition, muscle from animals lacking Mg-29 exhibits a force-frequency relationship that is reminiscent of LFF. Similarly, given that JFP-45 spans the SR membrane and maintains the DHPR complex, disruption of JFP-45 may decrease the efficacy of coupling by altering the conformation or alignment of the DHPR (Fig. 4C). The ill-defined modifications that disrupt the function of these proteins could be alterations in the proteins themselves, altered protein-protein interactions or, particularly in the case of the junctophilins, altered protein-lipid interaction. Whichever modification occurs, it seems to be initiated by a rise in triadic Ca2+. Elevated triadic Ca2+ could initiate a number of pathways leading to reduced efficacy of ECC. Likely candidates include activation of calpains, phospholipase A, protein kinase C, and increased mitochondrial ROS production.
Skeletal muscle expresses two ubiquitous calpains, m-calpain and µ-calpain, and the muscle specific calpain, calpain-3. Calpain-3 and µ-calpain are activated in the physiological Ca2+ concentration range. However, Ca2+-dependent uncoupling was induced in mouse muscle fibers lacking calpain-3 (34), and the calpain inhibitor, calpeptin, did not prevent LFF in intact muscle fibers (1). Although in vivo exercise increased m- and μ-calpain activity and in vitro activation of endogenous calpain activity proteolyzed RyR1 and an unidentified 88-kDa junctional SR protein, proteolysis of RyR1 did not alter SR Ca2+ flux or SR [3H]ryanodine binding, a sensitive indicator of channel activity (1). Furthermore, Ca2+-induced ECC uncoupling was not associated with an increase in RyR, DPHR, or triadin proteolysis. Thus, if calpain-mediated proteolysis is in involved in ECC uncoupling, the target(s) are unknown.
Phospholipase A2 is a family of 20 distinct phospholipases clustered into multiple groups including the following: secreted (sPLA2), cytosolic (cPLA2), and Ca2+-independent (iPLA2). Skeletal muscle expresses all three of these types of PLA2s, and in vivo exercise significantly increased PLA2 activity (11). PLA2 activation leads to hydrolysis of the fatty acid from the second carbon of the glycerol backbone of phospholipids (sn-2 position) liberating arachidonic acid. The effects of arachidonic acid production are difficult to predict because it can bind a number of ion channels, activate nitric oxide synthase and phosphokinases A and C, and alter membrane fluidity. Furthermore, arachidonic acid is a precursor for the generation of various eicosanoids and related bioactive lipid mediators. In addition, the remaining free fatty acids and lysophospholipids can destabilize membrane lipid-lipid and lipid-protein interactions (7). Thus, the destabilizing effect of PLA2 products could significantly alter the structural interactions required to maintain the functional integrity of the triad.
Inositol phosphates also have been shown to modulate skeletal muscle ECC. Shen et al. (29) demonstrated that the accumulation of phosphatidyl inositol-3,5-phosphate (PidIns-3,5-P2), a component of the SR, activates RyR1 and, in excess, impairs SOCE. Accumulation of PidIns-3,5-P2 in the SR because of the genetic ablation of a newly identified muscle-specific inositol phosphatase resulted in significantly decreased run time to exhaustion, severe contractile impairment, and rapid fatigue in both the soleus and extensor digitorum longus (EDL) muscles.
An increase in intracellular Ca2+ causes protein kinase C (PKC) to associate with the plasma membrane where it can be activated by diacylglycerol. All of the minor triadic proteins discussed above, with the exception of junctate, have predicted PKC phosphorylation sites and therefore may be modified upon PKC activation. Phosphorylation can alter protein-protein interactions and could thereby disrupt interactions critical for the maintenance of the triad. However, Ca2+-induced ECC uncoupling is not mediated by protein phosphorylation as the uncoupling occurred in the absence of ATP and protein phosphorylation would likely be reversed during the prolonged time course of LFF.
The role of ROS in skeletal muscle fatigue has recently been reviewed in detail by Reid (28). Both increased mitochondrial Ca2+ and arachidonic acid enhance mitochondrial ROS production, which can in turn damage proteins and membranes and reduce muscle performance. However, the role of ROS in LFF is questionable, as the reducing agent DTT did not prevent Ca2+-induced excitation-contraction uncoupling, and neither DTT nor the antioxidant N-acetylcysteine prevented fatigue induced decrease in Ca2+ release in intact fibers (6).
An uncoupling of t-tubule excitation from SR Ca2+ release contributes to LFF. It is clear that the interactions between the DHPR and RyR1 are insufficient to maintain the critical coupling between the two channels and that additional components of the triad must contribute. Of the numerous minor triadic proteins, the junctophilins and JFP-45 that span the gap between the SR and t-tubule, and mitsugumin-29, which maintains the conformation of the t-tubule, seem to play a critical role in maintaining the architectural integrity of the triad. Thus, modification of these proteins could decrease the efficacy of ECC and may contribute to LFF (15,18).
This work was supported by research grants from the National Institutes of Health (NIH AG023902) and the American Heart Association (AHA 0160407Z).
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