- Muscle fatigue from intense contractile activity is thought to result from accumulating metabolic by-products directly inhibiting contractile proteins. However, the magnitude of the decrease in contractile function caused by these metabolic by-products is smaller than the decrease in force and velocity, suggesting other factors contribute to fatigue.
- Intracellular calcium concentration ([Ca++]i), initiates contraction, also decreases during fatiguing contractions; however, the magnitude of the change in Ca++ alone also is too small to account for the observed decrease in force.
- The emerging view is that elevated levels of metabolic by-products act synergistically with the decrease in [Ca++]i to inhibit contractile function during fatiguing contractions by decreasing the Ca++-sensitivity of the myofilaments. Indeed, preliminary modeling efforts suggest that much of the loss in muscle force can be predicted accurately based on this information.
Intense contractile activity of skeletal muscle lasting even a few minutes results in a rapid decline in its ability to generate force, velocity, and power — a phenomenon that characterizes fatigue (1). In an exercising human, the causes of fatigue are multifactorial, ranging from changes in the impulse to move in the brain to decreased release of Ca++ from the sarcoplasmic reticulum (SR) in the muscle cell to inhibition of myosin, the molecular motor of myosin (3). The crucial questions now are, which factors play the most prominent role in reducing contractile function and what are the molecular mechanisms underlying these effects? Prevailing hypotheses stem from two key observations: 1) elevated levels of metabolites (most notably hydrogen ions [H+] and phosphate [Pi]) directly inhibit the function of the contractile proteins (11) and 2) myoplasmic Ca++ levels ([Ca++]i) decrease during fatiguing contractions leading to less activation of the myofilaments (3). Based on our work and other related experiments, we hypothesize that the accumulation of metabolic by-products and compromised release of Ca++ from the SR act synergistically to cause much of the loss in muscle function during fatiguing contractions from intense contractile activity (Fig. 1). This short article focuses on highlighting recent data that have led to this hypothesis, with a specific focus on how elevated metabolites act to decrease the ability of Ca++ to activate the myofilaments.
Intracellular Ca++ Concentration Compromised During Fatiguing Contractions
The increase in Ca++ inside the muscle cell in response to stimulation from the motor neuron is the trigger of contraction, allowing myosin to bind to its molecular track, actin, and generate force and motion. Ca++ regulates contraction through the action of the regulatory proteins troponin (Tn) and tropomyosin (Tm) that are bound to the actin filament. Significant advances in our understanding of the molecular basis of this activation process have occurred in the last 20 yr (21) and demonstrate that Ca++ binds to the C subunit of troponin (TnC) and induces the c-terminal end of the inhibitory subunit (TnI) to dissociate from actin and bind to a hydrophobic patch on TnC (41). These events release the constraints on tropomyosin that hold it in a position that prevents myosin from binding strongly to actin. This work demonstrates just how crucial Ca++ is to contraction and the potential impact on contractile function if compromised during fatiguing contractions.
The relation between Ca++ and contractile properties has been well characterized as sigmoidal, with both muscle force and velocity increasing steeply before plateauing at a saturating [Ca++] (Fig. 1). The shape of this relation is indicative of the highly cooperative nature of myosin's binding to actin (22) and also highlights that the impact of reduced [Ca++]i, caused by sustained activation, will be highly dependent on its initial concentration and the magnitude of the decrease. For example, a small decrease in Ca++ could cause a very large decrease in force if the change occurs near the steep portion of the relation. Based on this relation and the importance of Ca++ to contraction, several researchers have sought to determine the effect of fatiguing stimulation on myoplasmic Ca++.
In what is now considered a landmark study, Lee et al. (25) examined the effect of repeated stimulation on [Ca++]i and isometric force in an isolated, intact single muscle fiber preparation. Within the first minute of stimulation, isometric force had declined by roughly 10% but [Ca++]i increased slightly. However, as stimulation continued and the frequency increased, force dropped by greater than 70%, and [Ca++]i began to decrease, ultimately being reduced to approximately 50% of the initial value. In a subsequent experiment, a chemical agent (caffeine) was used to open fully the Ca++-release channels in the SR and almost completely restored both [Ca++]i and force to initial values. This suggests that the decrease in [Ca++]i played a causative role in the decrements in isometric force through a mechanism involving compromised release of Ca++ from the SR. Similar observations were made in a follow-up study focused on the recovery from the sustained contraction (44), which demonstrated that after a similar amount of contractile activity in the same preparation, much of the force recovered in parallel with the return of [Ca++]i. More recently, a similar finding was reported using a whole mouse tibialis anterior muscle and using more sophisticated methods for quantifying changes in [Ca++]i (2). Because of a constant high stimulation frequency (100 Hz) in this study, the force generated by the isolated muscle decreased steeply from the first contraction, whereas the [Ca++]i declined concomitant with the force decrements; as with the earlier study (44), force recovered in parallel with [Ca++]i (2). Indeed, because of the higher stimulation frequency (100 Hz) in this study, the force generated by the isolated muscle decreased steeply from the first contraction (2), which suggests that at these high stimulation rates, the loss of [Ca++]i levels could contribute to the loss of force even in the early stages of the sustained contraction. These observations provide compelling evidence to support the notion that the decrease in [Ca++]i contributes to the loss in the force-generating capacity of muscle.
However, in both the intact fiber and whole-muscle preparation, several other variables in addition to [Ca++]i likely are changed, making it difficult to ascribe all of the force loss to decreased [Ca++]i. Whereas in some preparations and stimulation rates, [Ca++]i can be observed to decline in tandem with the loss of force (2), in other preparations during the early stages of the sustained contraction, [Ca++]i can be unaffected or even increase slightly at a time when isometric force is depressed by approximately 10%–15% (25). This observation demonstrates that factors other than [Ca++]i must cause the decline in force at this early stage of the protocol. Furthermore, although studies clearly demonstrate that [Ca++]i is decreased during fatiguing contractions, the magnitude of the change in concentration has still not been determined definitively. Indeed, some estimates suggest that the change might be quite small relative to the range of [Ca++]i values reached during normal contraction (5,7). Quantification of the decrease in the absolute concentration is difficult because the fluorescent indicator dyes used to measure changes in [Ca++]i offer only an indirect measure of the actual concentration. In an attempt to quantify the absolute values, Allen and Trajanovska (5) used prior estimates in rested skeletal muscle (7) to predict values during sustained activation using data in which the protocol induced a 33% reduction in the Ca++ signal (2). Using this method, they estimated that the [Ca++]i decreased from an initial value of 1.26 μM (pCa 5.9) to 0.83 μM (pCa 6.1) at the end of the protocol. If these estimates are correct, they would represent a relatively small change in [Ca++]i based on the full force-calcium relation and potentially have a limited impact on the force-generating capacity of the myofilaments because of the sigmoidal shape of the relation (Fig. 1). Indeed, examination of the force-pCa curve from a skinned single-muscle fiber suggests that, absent any other changes in the intracellular milieu, this magnitude of a decrease in [Ca++]i would cause only an approximately 5% decrease in isometric force (Fig. 1), which cannot account for the 50%–70% decrease in force observed in the latter stages of a sustained contraction (1). Thus, it is becoming clear that this decrease in [Ca++]i alone cannot account fully for the magnitude of the force decrements during these latter stages of in vivo sustained contractions (47) or the rapid and large changes observed at higher stimulation rates in isolated muscle (2). To account for both the initial decline in force despite increasing [Ca++]i in the earlier stages of these protocols and the more pronounced decrease in force than in [Ca++]i in the latter stages, several authors have suggested that elevations in metabolic by-products (most notably Pi and H+) play a stronger role in causing the decline in force (1).
Elevated Metabolites Cannot Account Fully for Losses in Contractile Function
Elevated levels of metabolic by-products undoubtedly are the most cited putative agents of the decline in force during sustained contractions (8,34). The list of these metabolic by-products includes H+, which when elevated decreases the intracellular pH (i.e., acidosis) and Pi, which accumulates during intense exercise because the cell uses creatine phosphate to maintain the adenosine triphosphate (ATP) concentration in the cell (8). Indeed, the intracellular pH in the myoplasm can decrease from a neutral value (7.0–7.2) at rest to less than 6.4 during intense contractile activity, whereas Pi can increase from 1–5 μM to more than 30 μM (8). Other factors that change within the cell during fatiguing contractions include adenosine diphosphate (ADP) and reactive nitrogen and oxygen species (ROS/RNS) (45); however, the increase in ADP does not reach a level that could significantly impact contractile function (9), and the concentrations of ROS/RNS are notoriously hard to quantify, making it hard to identify their contribution to the decline in force (45).
To determine if elevated H+ and Pi play a causative role, researchers exposed permeablized single muscle fibers from rested muscle to the concentrations of H+ and Pi reached during fatiguing contractions (10,14,24,34). Initial experiments performed below physiological temperatures (10°C–15°C) suggested that exposing muscle fibers isolated from rested muscle to fatiguing levels of Pi or H+ can decrease maximal isometric force by 50% or more (34). However, more recent work has demonstrated that closer to physiological temperatures (30°C), the same increase in [Pi] still significantly reduces isometric force but by only 5%–20% (14). Similarly, the depressive effects of acidosis on maximal isometric force are significant but less pronounced at physiological temperatures, with a fatiguing level of acidosis (pH, 6.2 vs 7.0) decreasing force by only approximately 10% at 30°C (24,38). The most recent work has shown that when both of these metabolites (Pi and H+) are elevated, as is the case during fatiguing contractions, the effects on isometric force seem to be additive, causing the depressive effect on isometric force to reach approximately 33% (32). However, the magnitude of the decrease in isometric force in these single-fiber experiments often is less than is observed at the end of fatiguing contractions when the intracellular concentrations of Pi and H+ might be equivalent to those used in fiber experiments (43). Therefore, it is difficult to attribute the large reductions in isometric force, observed during fatiguing contractions, solely to these biochemical changes and suggests that some other single factor or combination of factors must contribute to this loss in force.
Pi and H+ Decrease Myofilament Sensitivity to Ca++
In addition to directly affecting maximal isometric force, elevated levels of Pi and H+ also seem to decrease isometric force at subsaturating Ca++ levels (19,30). This effect has been most carefully characterized in skinned muscle fibers where the concentration of Ca++ as well as Pi and H+ can be controlled directly. Early work demonstrated that fatiguing levels of acidosis (high H+) and Pi can decrease isometric force at any given level of Ca++ (19,30). The sigmoidal relation between Ca++ and muscle force (22) means that, at intermediate levels of Ca++, force can be reduced by a much larger amount than at saturating levels when operating near the steep portion of this relationship (Fig. 1). This effect would be amplified given that elevated levels of Pi and H+ decrease the sensitivity of the myofilaments to Ca++, meaning that, when on the steep portion of the force-calcium relation, small reductions in myoplasmic Ca++ result in large decreases in force (Fig. 1). In other words, elevated Pi and H+ can cause a rightward shift in the force-calcium relation that exacerbates the effects of reduced Ca++. Consistent with this notion, the [Ca++]i required to elicit 50% of the maximal isometric force (pCa50, a measure of Ca++-sensitivity) is increased by twofold in the presence of a fatiguing level of Pi (16). Unlike the effect of Pi on force at saturating [Ca++] (pCa 4), the effects at subsaturating [Ca++] seem to be more pronounced near physiological temperatures (16); indeed, 30 μM Pi causes the pCa50 to increase more than fourfold at 30°C in mammalian muscle fibers (16). A similar observation was made for the effect of acidosis, where decreasing pH from 7.0 to 6.2 increased the [Ca++] required to elicit 50% of the maximal force by greater than 10-fold at physiological temperatures (33). Furthermore, when both H+ and Pi are elevated simultaneously, the reductions are even more pronounced resulting from a more pronounced rightward shift (i.e., decreased Ca++ sensitivity) near physiological temperatures (33).
These findings imply that elevated levels of Pi and H+ act in concert with reduced [Ca++]i to cause the large reductions in isometric force observed during sustained contractions. Strong support for this hypothesis comes from recent simulations of muscular force during fatigue protocols that incorporate both fatigue-induced changes in [Ca++]i and Pi (5). In this investigation, the authors used data from skinned single-fiber experiments that examined the effect of Pi on the force-calcium relation at 30°C (16) and combined information with measures of the decrement in [Ca++]i during fatiguing contractions of an isolated mouse tibialis anterior muscle (2). Using this information, the authors generated a series of force-calcium curves at increasing levels of Pi, and, using measured values of Pi and Ca++ at different time points during the fatigue protocol as inputs into the model, they were able to closely match the decay in isometric force (5). This very powerful approach suggests that elevated levels of Pi and decrements in [Ca++]i act in combination to reduce isometric force resulting from intense contractile activity.
Although profound, this finding is somewhat limited in that it applied only to a specific experimental result; however, if the levels of Ca++ and Pi were known from other experiments, with different stimulation protocols, it is likely that the model would generate similar fits where different patterns of stimulation led to the decline in force. A more important limitation is that the results have been applied so far only to predict the drop in isometric force and not yet used to predict the loss of contraction velocity or power, both of which are reduced during fatiguing contractions (43). Importantly, our laboratory has demonstrated that elevated levels of Pi have no effect on actin filament velocity in the motility assay (17) (a measure analogous to unloaded shortening velocity in muscle fibers). Therefore, the changes in velocity observed in response to fatiguing stimulation are unlikely to be attributed to elevations in Pi.
This decrease in shortening velocity and a larger portion of the loss of power is more likely attributable to changes in intracellular pH rather than Pi. For example, our laboratory has demonstrated recently that fatiguing levels of acidosis (i.e., low pH) decrease both maximal velocity and sensitivity of the thin filament to Ca++ in a motility assay (Fig. 2), suggesting this change contributes to changes in contraction velocity (13,15,17). We contend that much of this decrease in velocity can be attributed to the slowing of a specific step in myosin's cross-bridge cycle, specifically the release of ADP from actomyosin (13). However, we also have demonstrated that the depressive effect of acidosis on actin filament velocity is more pronounced in the presence of regulatory proteins than with actin in isolation (15), suggesting that that the muscle regulatory proteins, Tn and Tm, help to mediate the depressive effects of acidosis during fatiguing contractions. Therefore, we hypothesize that adding the effects of acidosis, both on the velocity-calcium (15) and force-velocity relation (24), to a model such as that of Allen and Trajanovska (5) may enable it also to explain changes in other contractile properties such as shortening velocity and the power-generating capacity (43).
Based on these data, the decline in muscle force response to sustained intense contractile activity, results, in large part, from the combined effects of increased metabolites and reduced [Ca++]i. This implies that the decrease in force observed in human muscle, in response to intense contractile activity (e.g., (8), results because the muscle becomes less responsive to stimulation by the central nervous system; in other words, central stimulation may be maintained but, inside the muscle, the myofilaments are less responsive to activation by Ca++. This notion is consistent with the seminal observations of Merton (28) and others that assert that the cause of such reductions in muscle force are distal to the alpha-motor neuron (1,20). Given this knowledge, a deeper understanding of the adjustments that occur during fatiguing contractions will require us to determine the molecular mechanisms underlying the depression in Ca++ sensitivity of the myofilaments by Pi and H+.
What Are the Mechanisms Underlying Decreased Ca++ Sensitivity?
The data from skinned single muscle fibers previously detailed provide strong evidence that fatiguing levels of Pi and H+ depress the Ca++-sensitivity of the myofilaments; however, these data provide little information about the molecular structures or mechanisms underlying these effects. To gain a more complete understanding of how these changes contribute to the decline in force, it is important to elucidate these mechanisms; several researchers have begun to tackle this question.
Fabiato and Fabiato (19) originally suggested that when elevated, H+ might bind to the Ca++-binding site of TnC thereby preventing Ca++ from binding and activating the thin filament. Parsons et al. (37) directly tested this hypothesis by incorporating a modified construct of TnC that fluoresces on binding to Ca++. They observed that the acidosis-induced force depression paralleled a decrease in the fluorescence signal from their modified TnC, suggesting that a drop in the muscle's intracellular pH (from 7.0 to 6.5) decreases the affinity of Ca++ for its binding sites on TnC. Indeed, this observation in muscle fibers was consistent with prior observations determined in solution using isolated Tn complexes (36). This decrease in affinity could be due to Ca++ attaching to its two binding sites more slowly or releasing from its binding sites more quickly (i.e., a slower on-rate or a faster off-rate). To test this question, Longyear et al. (27) recently examined the effect of a TnC mutation (V43Q) that putatively prolongs the Ca++-bound configuration of TnC, effectively slowing calcium's off-rate from TnC (42). Consistent with predictions, this mutation increased Ca++ sensitivity under normal pH (7.4); however, the size of the acidosis-induced shift in the velocity-calcium relation was nearly identical to that of wild-type TnC (27), suggesting that during fatiguing contractions, acidosis slows the on-rate of Ca++ binding to TnC and likely has no effect on the off-rate. The next step in this line of research will be to determine if acidosis might decrease the ability of Ca++ to bind to TnC by exploring the possibility that the increased [H+] will change the total charge of TnC, reducing its affinity for Ca++ (26,37). Alternatively, acidosis might affect TnC's important interactions with TnI and, in doing so, indirectly reduce its affinity for Ca++(6).
It also is possible that the acidosis-induced decrease in Ca++-sensitivity is mediated through alternations in the function of TnI. Indeed, prior fiber experiments have shown that the force-pCa relation in fast and slow muscle fibers display different sensitivities to acidosis (31); because fast and slow skeletal muscles express the same TnC, this suggests that the differential response can be attributed to the different isoforms of TnI. Longyear et al. (27) have begun to probe the mechanism of a putative TnI-mediated effect using a construct in which the c-terminal end of TnI is truncated. This region of TnI is thought to play a central role in the regulation of myosin's binding to actin (41) and, therefore, represents an interesting region with which to probe the role of TnI. Using the in vitro motility assay, it was found that partial truncation of the C-terminal end of TnI caused the actin filaments to be less affected by a fatiguing level of acidosis (27). Thus, this suggests that, in addition to a role for TnC, TnI also might play a role in the reduced Ca++-sensitivity that contributes to the decline in muscle force.
The putative mechanism by which elevated Pi reduces Ca++-sensitivity is thought to relate to its direct effects on myosin (12,16,30); a notion supported by findings that Pi does not impede Ca++ ability to bind to TnC in contrast to acidosis (36). In addition, Pi does not affect the velocity-calcium relation (15), again in contrast to the effects of acidosis on velocity (17). Instead, elevated levels of Pi are thought to directly reverse the force-generating steps of myosin as it binds to actin (18,40). This is important to muscle activation because the strong-binding of myosin to actin also activates the thin filament through a Ca++-independent mechanism (22). Therefore, when Pi is high, such as during fatiguing contractions, it likely reduces the number of strongly bound heads at any given level of Ca++, which, in turn, decreases the level of thin filament activation (16).
Additional insight into these mechanisms has been gained by researchers who have investigated whether the depressive effects of Pi and H+ on Ca++-sensitivity can be reversed. These findings also would have relevance to identifying novel therapies that could attenuate the decline in muscle force observed in clinical populations, where it can greatly limit physical function (23). One compound that may hold considerable promise for reversing the effects of acidosis and Pi is the ATP analog 2-deoxy-ATP (dATP). This compound has been shown to enhance the force-generating capacity and unloaded velocity in vitro (39). In addition, more recently it has been shown to improve cardiac function in models of heart failure (35). Encouraged by these findings, Longyear et al. (27) determined the effect of an ATP analog on the acidosis-induced depression of the velocity-calcium relation in the motility assay using skeletal muscle myosin (Fig. 2). We found that substituting dATP for ATP in our assays attenuated much of the acidosis-induced depression in filament velocity. Indeed, at pH 6.5, most of the filaments remained stationary at low pH with normal ATP but recovered to move at more than 50% of control velocity. Interestingly, this improvement in actin filament velocity was evident only under the acidic conditions experienced during fatiguing contractions (Fig. 2). This finding is exciting for two reasons: first, it offers promise for the development of treatments for the decline in muscle force during fatiguing contractions in clinical populations and, second, may provide new insights into the responsible for the decrease in force because the effects of dATP on specific steps in the cross-bridge cycle have been characterized previously (39). For example, dATP is thought to accelerate both the rate at which myosin attaches strongly to actin and the rate at which it detaches from actin by accelerating the rate of ADP release from actomyosin (39), and both of these steps are believed to be slowed by acidosis (13,29). Therefore, the finding that dATP reverses the acidosis-induced depression in velocity provides additional support for the hypothesis that acidosis slows both myosin's weak-to-strong binding transition and the rate of ADP release. Thus, dATP may be an ideal compound to attenuate the depressive effects of acidosis during fatiguing contractions.
Although the accelerated ADP release might be expected to reduce myosin's duty ratio and force, this effect could likely be offset by dATP's ability to accelerate the weak-to-strong binding transition (39). These findings suggest that both steps in the cross-bridge cycle are inhibited by a fatiguing level of acidosis. Thus, this compound could potentially increase contraction velocity under the acidic conditions without compromising force generation and therefore represents a promising approach to attenuate force loss during fatiguing contractions in clinical populations.
During intense contractile activity, metabolic by-products, most notably Pi and H+, accumulate inside the muscle cells. These agents act to directly inhibit the force- and motion-generating capacity of muscle's molecular motor, myosin, but alone cannot account for the magnitude of the decrements in muscle function. The amount of Ca++ released from the SR also is compromised during intense contractile activity, which in and of itself likely has only a small impact on muscle force. However, taking into account that Pi and H+ act to decrease the Ca++-sensitivity of the myofilaments, it is possible to account for much of the loss in contraction force and velocity. Although there are likely other factors that contribute to the reduction in contractile function, given the magnitude of the depression in muscle function that can be attributed to decreased Ca++-sensitivity, it is critically important to gain a deeper understanding of the structures and mechanisms that underlie this decreased Ca++-sensitivity.
E.P. Debold was supported by a Grant-in-Aid from the American Heart Association (14GRNT20450002).
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