Muscle fatigue, defined as the fall in maximal force or power production in response to contractile activity, by definition arises at least in part from failure of cross-bridge cycling within the muscle cells. However, this contractile machinery relies on multiple systems to support its work, including the nervous, vascular, and energy systems. Thus, as illustrated in Figure 1, failure at any of the sites upstream from the cross-bridges can and does contribute to the development of fatigue (54). As such, no muscle operates in isolation in vivo. It is well understood at this time that fatigue is complex and task-specific, making the use of both human studies and animal models important. The focus of this review is to provide examples of how integrating a variety of approaches and techniques can provide a more complete understanding of muscle fatigue. Although sometimes results appear contradictory or inconsistent, the wide array of conditions, protocols, and models used to study fatigue contributes to the advancement of this exciting field of inquiry.
Using Fatigue to Probe the Integration of Physiological Systems In Vivo
Integrative approaches to studying muscle fatigue
As so well by implemented by the work of Brenda Bigland-Ritchie and colleagues in the 1970s and 1980s (10,11,91), in vivo studies of skeletal muscle fatigue in humans benefit from an integrated approach that captures information about the neural and contractile properties of muscle during a variety of contraction protocols. For example, using both voluntary and electrically stimulated contractions in a given fatigue protocol allows comparison of the entire pathway of force production with those events occurring distal to the point of stimulation (see Neural activation, below); methods such as transcranial magnetic stimulation and EMG have been essential to advancing our understanding of these neural processes that contribute to muscle fatigue (54). This approach was expanded in the late 1980s and 1990s by the addition of measures of intracellular energy metabolism using in vivo magnetic resonance spectroscopy (MRS) (4,55,67). With addition of this technique, the role of energy production and the inhibition of force production by its by-products (Fig. 1, Bioenergetics) became possible in vivo. Because studies of human muscle fatigue generally involve noninvasive measures, an approach that tackles the question of fatigue from many different directions can be used to infer much about the causes of contraction-induced loss of maximal muscle force or power.
Neural activation of the muscle involves transmission of the signal to contract from the brain to the muscle’s transverse tubules and sarcoplasmic reticulum (SR), with subsequent release of calcium into the cytosol by the SR and initiation of cross-bridge cycling. A combination of voluntary and stimulated muscle contractions can be used to evaluate changes in “central” and “peripheral” activation of muscle, with those distinctions largely established by the location of the stimulation electrodes. For example, a greater decrease in maximal voluntary force compared with the force produced by a separate supramaximal stimulation during a fatiguing contraction protocol provides evidence of “central” fatigue, or fatigue arising from sources proximal to the point of stimulation (10,11,52). An increase in force in response to superimposition of a stimulated contraction during a maximal voluntary contraction can be used to detect incomplete voluntary activation of the muscle (52). Sophisticated techniques including transcranial magnetic stimulation of the brain and spinal cord and complex EMG electrode arrays have also been developed to parse out more specifically the sites of failure in the brain and spinal cord that may contribute to fatigue (54).
Peripheral activation can be evaluated by changes in the m-wave or compound muscle action potential (AP), which is recorded by surface EMG electrodes placed over the muscle. The m-wave is the electrical response to a single, supramaximal stimulation applied to the motor nerve just proximal to its innervation point at the muscle. During fatigue, changes in the amplitude of the compound muscle AP reflect an impairment of propagation at the neuromuscular junction (NMJ) or muscle membrane. Typically, failure at this site is uncommon during voluntary contractions except during very high-intensity, prolonged contraction protocols (52), although high-frequency stimulation protocols can induce this type of peripheral “electrical” fatigue (37). In summary, much attention remains focused on clarifying the complex contributions of neural activation to muscle fatigue in vivo.
Muscular work must be supported by the ready supply of energy for adenosine triphosphate (ATP)ases functioning at the sarcolemma, SR and cross-bridges. The increased energy demand of contraction will result in the accumulation of metabolites known to induce contractile failure, including inorganic phosphate (Pi) and hydrogen ion (H+; (54). Phosphorus MRS can be used to quantify noninvasively and continuously (with ~2–10 s temporal resolution) the changes in these and other energy metabolites in the cytosol of working muscle (4,52,55,62). Various protocols have been used to probe the associations between fatigue and metabolic inhibition of contraction, and the results are consistent with those observed at the cellular (32) and molecular (24) levels. More recently, use of proton MRS has shown that intracellular PO2 is maintained well above critical PO2 during single muscle group contractions (63), and thus oxygen availability does not appear to be a factor per se in fatigue under conditions of unrestricted blood flow (BF).
The same stimulated contractions that are used to quantify changes in peripheral activation of the muscle can be used to indirectly evaluate events such as excitation–contraction coupling (ECC) and calcium kinetics within the muscle during fatigue. Decreases in force in response to supramaximal stimulation of the motor nerve following a contraction protocol indicate the magnitude of peripheral fatigue, or that arising from changes at the NMJ, muscle membrane or within the myocytes themselves (67). Further, a decrease in stimulated twitch or tetanic force in the absence of a decrease in the amplitude of the m-wave suggests that the failure of force production is occurring distal to the sarcolemma (52). Changes during stimulated contractions in the rates of force development and, more commonly, relaxation also support the interpretation of contraction-induced changes in calcium kinetics or cross-bridge cycling (11).
In the next three sections, we describe three studies that applied different combinations of the measures described above to address distinct scientific questions. In the first case, muscle fatigue was used to perturb homeostasis in individuals with multiple sclerosis (MS), to evaluate the cardiovascular response to contractions in this population (70). In another example of the utility of an integrative approach to evaluating fatigue mechanisms, an in silico study of fatigue that built upon studies in vivo by manipulating known factors in fatigue is described (16). Finally, our third example illustrates how understanding the bioenergetic contributions to fatigue can explain the fatigue resistance of older muscle under isometric conditions in vivo, and reveal new information about age-related changes in muscle metabolism.
Fatigue as a means of understanding multiple physiological systems: MS
Fatigue protocols can also be used to stress the intact organism to evaluate the integration of various physiological systems in pathological conditions. For example, in response to a report that persons with MS had an inadequate blood pressure response to isometric forearm contractions (76), a fatigue study was conducted during which simultaneous measures of isometric force, beat-by-beat heart rate and blood pressure, intracellular energy metabolites and pH, neural activation and ratings of perceived exertion were obtained (70). The hypothesis tested was that the blunted pressor response in MS observed by Pepin et al. (76) was in fact appropriate to a blunted metabolic response to the contraction protocol, as had been observed previously in this population (57). Although endurance time, and changes in heart rate, perceived exertion and neural activation were similar in MS and controls during the sustained submaximal isometric contraction, the pressor response and changes in [Pi] and pH were blunted in MS compared with controls. Further, the changes in mean arterial pressure were associated with the changes in both [Pi] and pH. Notably, the MS group had a normal response to the cold pressor test and Valsalva maneuver, both nonexercise tests of autonomic function. Thus, this integrative fatigue paradigm revealed that the blunted pressor response in MS was appropriate to the smaller metabolic perturbation during fatigue, and not a result of cardiovascular dysautonomia in this cohort.
Integrative, in silico study of fatigue
Due to the complexity and interrelationships between systems that support muscle force production, the study of fatigue in vivo is somewhat limited to indirect measures. Recently, a computational model was developed to address this limitation (16,17). Information in the literature from in vivo studies of neural activation, contractile characteristics, and energy metabolism were combined with de novo experimental observations to develop the model. This relatively comprehensive model used an integrated approach, including some preexisting components (33), to evaluate the sites of failure during fatigue of human skeletal muscle. This approach allows manipulation of specific variables representing explicit physiological functions to determine their independent effects on fatigue; once developed, the model was applied to the question of age-related fatigue resistance.
The approach involved development of distinct modules reflecting relevant physiological processes including motor unit behavior, bioenergetics, contractile properties, calcium and force-generation kinetics, and biomechanical properties (Fig. 2) (16,17). The modules were populated with data from the literature, and once the forward dynamics simulations were initiated, no further input was given to the model. The output from the model agreed well with reports from in vivo studies (62), both in young muscle and when adjusted to reflect age-related changes in neuromuscular properties (16). Changes in phosphocreatine and pH reflected changes in experimental data, including age-related differences. Fatigue was well correlated with the concentration of diprotonated Pi, in both age groups. Analysis of the model output in response to various manipulations of the modules indicated that the predominant effect of age was that of a decreased reliance on glycolytic ATP production in the older muscle; a result that agrees well with available in vivo data (62). Thus, a modular, computational approach to the study of fatigue is feasible and allows independent manipulation of various mechanisms of fatigue. Future applications in aging and pathological conditions should prove useful in augmenting in vivo studies and our overall understanding of the mechanisms of fatigue.
Age-related differences in acidosis during muscle contractions
On balance, the literature is clear in showing that older muscle fatigues less than young during isometric contraction protocols (19). Under these conditions, the primary source of this difference in fatigue appears to be the fact that older muscle develops markedly less intracellular acidosis than young muscle during a variety of contraction protocols (20,56,62). Concomitantly, muscle in healthy older adults relies more on oxidative metabolism to meet the ATP needs of the cell during contraction (20,62). To determine whether this difference in energetic response was related to an inability of older muscle to increase glycolytic flux to the same extent as young muscle, acidosis and ATP production from the creatine kinase reaction, glycolysis and oxidative phosphorylation were determined in young and older adults during contraction protocols in which BF to the lower leg was left intact or occluded (62). The results of this study showed that, although older muscle relied relatively less on glycolytic ATP production during free-flow conditions compared with young, this difference was abolished during ischemia, indicating no impairment on the part of the older muscle to produce energy from glycolysis when necessary. Notably, the age-related difference in fatigue observed during free-flow conditions were also found during ischemic contractions; thus differences in fatigue were not attributable to differences in oxygen availability. In a follow-up study, the availability of glycogen and intracellular oxygen as substrates for energy metabolism was examined using carbon-13 and proton spectroscopy, respectively (Tonson et al, personal communication). Once again, age-related differences in acidosis and fatigue during intermittent maximal contractions were observed, despite similar [glycogen] at baseline and rapid equilibration of cytosolic PO2 well above critical PO2 (63) in both age groups during the contraction protocol. Thus, by combining approaches, it was demonstrated that age-related differences in intracellular acidosis were not a result of differences in substrate availability, at least in the form of glycogen and oxygen.
Role of Glycogen in Skeletal Muscle ECC and Function
As noted in section 2, mechanistic studies of contraction-induced loss of maximal muscle force or power in skeletal muscle have benefited from a whole-body, integrative approach. However, this approach does not tackle the question of fatigue at the isolated whole muscle or cellular levels, which can help explain processes observed in vivo. Indeed, single fiber studies have provided invaluable knowledge about fatigue mechanisms in the complex systems that support muscle force, for example, myosin–actin interactions, regulation of cytosolic Ca2+ by the SR, the force–Ca2+ relationship and the role of cellular metabolism (1,72,89). In the following sections, we discuss how one of these important systems, cellular glycogen metabolism, affects muscle fatigue.
Compartmentalized energy utilization and production in the muscle cell
Skeletal muscle is faced with challenging problems related to metabolic regulation during exercise, where energy turnover nearly instantly can increase several hundred-fold during high intensity exercise and more than 20-fold during more prolonged aerobic exercise (85). This is achieved without substantial and potentially deleterious decreases in global myocellular ATP concentration, demonstrating a remarkable precision of adjusting the rate of ATP generating processes to the energy requirements (44). However, the muscle cell forms many microenvironments with restricted diffusional access of metabolites and high ATPase activity during muscle contraction, which may result in some locations within the cell where the metabolic environment differs from the measured global energy status.
The sequence of events leading to muscle contraction is known as ECC (Fig. 3) and a number of the complex steps in ECC and relaxation are either directly or indirectly dependent on the energy level of the muscle fiber. Both the ion pumping at the transverse tubular (t)-system and sarcolemma (Na+/K+-ATPase) as well as the myosin ATPases at the contractile apparatus and the SR Ca2+-ATPase are directly dependent on ATP (Fig. 3, steps 1, 5, and 6) and are affected by either low (ATP) or high by-products of metabolism (e.g., ADP, Pi). Further, some of the ion channels involved in muscle activation and relaxation are indirectly regulated by the energy status of the cell, including the SR Ca2+ release channel (25). The highly organized muscle cell forms many compartments and thereby may have microenvironments with high ATPase activity and restricted diffusional access of metabolites. Thus, skeletal muscle cells represent an example of spatial organization where energy production from energy stores and energy-dependent steps in ECC are not located in close proximity. Still, within this organization, glycogen particles are distributed in local depositions and thereby serve as an efficient energy store for the different steps in ECC.
Glycogen is the carbohydrate energy store for ATP production and located primarily within muscle and liver cells. Glycogen can be observed by electron microscopy as spherical particles, widely distributed within the cell in close proximity to the sites of energy consumption, creating optimal conditions for efficient regulation (72,75). Conventionally, three distinct subcellular localizations of glycogen have been defined: i) intermyofibrillar glycogen, which is located between the myofibrils in close proximity to SR and mitochondria; ii) intramyofibrillar glycogen, which is located within the myofibrils interspersed among the contractile filaments most often in the I-band of the sarcomere; and iii) subsarcolemmal glycogen, which is clustered just beneath the sarcolemma primarily next to mitochondria, lipids, and nuclei (72). In relative terms, intermyofibrillar glycogen is the major site of glycogen deposition, constituting approximately 75% of the cell’s total store of glycogen, whereas intramyofibrillar and subsarcolemmal glycogen each account for 5%–15% of total glycogen. The glycogen particles are connected with proteins involved in glycogen metabolism and form a dynamic carbohydrate–protein complex comprising a subcellular compartment that has the ability to respond to the metabolic requirements of the cell (29,88). Moreover, it has been demonstrated that these glycogen particles can be associated physically with the SR (29,88).
Glycogen and muscle function
In line with the glycogen particle distribution, studies from the single fiber to the whole body level collectively indicate that steps in muscle ECC and relaxation are affected by glycogen levels, which may link low glycogen levels with a decreased muscle function (18,73,75). Thus, there is now compelling evidence that low muscle glycogen and/or glycolytic-derived energy are associated with SR Ca2+ release and reuptake, and Na+/K+-pump function (Fig. 3, steps 1, 3, 4 and 6; for review see (72,75). This may also explain the importance of glycogen as a fuel during exercise, which is a fundamental concept in exercise physiology. Some of the first studies using the needle biopsy technique, in the late 1960s, demonstrated that there is a strong correlation between muscle glycogen content and endurance capacity during prolonged cycling exercise (9), and an inability to continue exercise when glycogen stores are limited (42). These observations have subsequently been confirmed numerous times and it is now well established that glycogen oxidation is of major importance for ATP regeneration during both prolonged exercise (>1 h), and also during high-intensity intermittent exercise (39). In this context, it is noteworthy that endurance and high-intensity exercise training not only improve performance, but also increase muscle glycogen content (39). However, the link between glycogen depletion and the development of fatigue, as well as the precise mechanism(s) whereby muscle glycogen affects the series of events that ultimately result in fatigue, are not fully understood.
Muscle glycogen can delay fatigue by maintaining energy transduction during aerobic exercise, as the maximum rate of ATP production is much higher for muscle glycogen than for blood glucose or fat oxidation (85). Further, glycogen may be important by producing tricarboxylic acid cycle intermediates permitting the maintenance of oxidative metabolism (85). Low glycogen per se may limit glycogen phosphorylase activity and hence the overall glycolytic rate. Although the in vitro Km values of phosphorylase for glycogen are <3 mM glucosyl units and the cellular concentration is usually >100 mM glucosyl units, the Km of phosphorylase for glycogen is substantially higher in vivo than in vitro (51). Interestingly, depletion of glycogen particles may be localization-dependent, with a higher relative utilization of intramyofibrillar glycogen content (75).
The important role of muscle glycogen is also apparent when studying its effect on the steps of ECC, which is consistent with the understanding of muscle glycogen as having rapid mobilization, being the precursor of glycolytic derived ATP, having a high ATP production rate and that glycogen and the glycolytic enzymes are widely distributed within the cell in close proximity to the energy consumption locations.
Muscle glycogen and SR function
Studies from the levels of SR vesicles, mechanically skinned and intact rodent single fibers, and human studies have pointed to a modulating role of glycogen availability on SR Ca2+ handling (18,26,73,74). A reduction in [glycogen] within the myofibrils is correlated with impaired SR Ca2+ release both in human muscle after prolonged exercise and in skinned rat muscle fibers after repeated tetanic stimulation (73,74). Furthermore, it was recently shown that intact single mouse muscle fibers undergoing repeated tetanic stimulations exhibit a steep decrease in tetanic cytosolic [Ca2+] when intramyofibrillar glycogen within the myofibrils reaches low levels (71). Thus, the results of several recent studies support a link between glycogen depletion within myofibrils and decreased SR Ca2+ release. At present, little is known about the precise mechanism(s) that link low glycogen levels in the muscle with an impaired SR Ca2+ release rate.
Possible effects of glycogen on the ryanodine receptor
In skeletal muscle, Ca2+ is released from the SR Ca2+ stores via specific Ca2+ channels (RyR1 isoform in skeletal muscle, Fig. 3, step 3). The RyR1 channels are located at the junctional SR of the SR-t tubule triad, which ensures efficient Ca2+ release to the contractile proteins. The RyR channels are modulated by numerous factors and in intact muscle, RyR1 interacts with multiple molecules and metabolites and is regulated by various cellular processes (e.g., phosphorylation and oxidation). During exercise and with glycogen depletion, the main physiological modulators of RyR are conceivably: 1) Ca2+ (both cytosolic and SR luminal), 2) protein phosphorylation, 3) the redox state, and 4) the cellular energy status (for review, see Dhar-Chowdhury et al. (25).
With respect to the role of glycogen affecting the SR Ca2+ release, low glycogen may lead to local changes in metabolic status of the compartmentalized cell, especially in the triad region with RyR localization. This may lead to increased free [Mg2+] and decreased free [ATP], which within the physiological range of changes in these metabolites, are strong regulators of the RyR1 (13). Further, clear evidence has accumulated demonstrating that glycolysis preferentially regulates membrane ion transport mechanisms. In line with this concept, physiological data directly support the concept of a regulatory role of glycolysis on SR membrane proteins, as glycolytic enzymes are associated with these membranes (38). Indeed, it was found that especially the glycolytic intermediate, fructose 1,6-bisphosphate, increased the opening probability of RyR channels (38). However, low glycogen has also been demonstrated to modulate the Ca2+ release rate in isolated vesicles without restricted metabolic space and during resting metabolic conditions. This result may indicate a crucial role of the metabolic machinery associated with the SR in maintaining endogenous metabolism. In line with this, analysis of the RyR protein sequence reveals that it contains many phosphorylation sites. The capacity of protein kinase A (PKA) and Ca2+-calmodulin-dependent kinase II (CaMKII, fast twitch fibers) to activate the RyR1 channel has been reported (31), although studies in skinned and intact skeletal muscle fibers have not supported this idea (12). However, the impact of phosphorylation and/or dephosphorylation on single RyR channel behavior and the role of glycogen and energy status is at present not fully unraveled.
Oxygen and Muscle Fatigue
The previous two sections have described how an integrative approach can elucidate the sources of fatigue in vivo, and how muscle glycogen metabolism is essential to supporting contractile function at the cellular level. In this section, we turn to the role of oxygen in the development of fatigue. Although numerous factors play into the fatigue process that occurs in skeletal muscle during exercise (highly dependent on the type of exercise), one of the more studied factors has been that of oxygen availability to the working muscle. It has been well appreciated that reduced oxygen availability to exercising muscle has profound consequences on muscle fatigue. However, it remains uncertain as to the precise mechanisms by which O2 availability may affect the fatigue process. Certainly, when the exercise is of a very high intensity, the oxygen availability to the respiring mitochondria may be insufficient for the demand for ATP, resulting in an instability of the cellular metabolic homeostasis and leading to changes that induce fatigue. However, at submaximal exercise intensities, the role of O2 availability in the fatigue process becomes a bit more curious. Increases in oxygen uptake and ATP utilization rise in unison until V˙O2max is reached, and at that point the demand for more ATP cannot be met by increases in mitochondrial respiration or oxygen delivery. This tight coupling of ATP demand by the working muscle to the ATP supply from the mitochondria only becomes uncoupled at the highest exercise intensities (see review by Sahlin, (84). The manner in which O2 availability may affect the fatigue process at less than the highest work intensities remains puzzling, and may have important consequences for human health, for example as in the hypoxia that develops with living at altitude, in some lung and heart diseases, and during the process of human aging.
Since the early work of Krogh (61), researchers have attempted to measure and model the diffusion of oxygen to the interior of cells. Although the importance of O2 for maintaining energy balance within cells became appreciated with the delineation of oxidative phosphorylation, it has been postulated that the metabolism and function of many cell types can be affected by intracellular O2 tensions at levels well above those considered rate limiting for mitochondrial function (83). This field of research has been difficult to reconcile due to the problems involved in the precise measurement of intracellular PO2. Whereas some of the methods for studying intracellular oxygenation and its effects on cell function have had methodological constraints, appropriate models to study these questions have also been a difficulty. There are limited data in skeletal muscle attempting to measure intracellular PO2 values. For example, it is exceedingly difficult to remove the variability and heterogeneity of the microcirculation and fiber population from estimates or measurements of intracellular PO2. Whole muscle measurements of myoglobin saturation, using MRS technology (79) are able to at least measure the mean PO2 from a large number of cells. The consensus from this work suggests that while intracellular PO2 drops to very low levels during moderate- to high-intensity exercise, the PO2 remains above that which limits mitochondrial respiration. And although knowing the PO2 within a working muscle fiber is important for understanding mitochondrial function, it has become just recently appreciated that the mitochondria can form a reticulum throughout the myofiber and the utilization of O2 in one area of the myofiber may transfer the energy charge to another area of the fiber closer to the ATP demand (35).
Rumsey and colleagues (83) suggested that there is not simply a minimal “critical” value for [O2] within the cell below which oxidative phosphorylation becomes compromised, but rather a range of O2 values that influence both the metabolic and respiratory state of the cell. The regulation of tissue respiration and metabolism has generally been considered to be controlled by the levels of the substrates needed to rephosphorylate ADP through oxidative phosphorylation, principally [ADP], [Pi], the [ATP]/[ADP][Pi] ratio, and the redox ratio of NADH/NAD (see review by (5). It has been demonstrated (45) that the levels of some of these proposed regulators may be influenced by the amount of O2 available for tissue respiration, even when the O2 available is above that considered “critical” for tissue respiration. It was demonstrated (40,46), using 31P-MRS, that the factors thought to regulate tissue respiration and metabolism during work (ADP, Pi, phosphocreatine, etc.) may be modulated by an interaction with tissue oxygenation levels. It was shown that, depending on the degree of tissue oxygenation, quite different amounts of these proposed regulators were required to attain similar levels of V˙O2, suggesting that the mitochondrial sensitivity to these proposed regulators of respiration and metabolism could be altered by tissue oxygenation levels. Additionally, Hogan and Welch (47) demonstrated that at similar V˙O2 during low arterial PO2 versus normal arterial PO2 conditions, there is a greater intracellular muscle [lactate] and muscle lactate efflux in the hypoxemic condition. It has been postulated that this is a result of an increase in the rate of glycolysis brought on in part by the changes (due to PO2) in the concentration of some of those substrates listed above (ADP, Pi, Ca2+) that regulate glycolysis, and that these changes can alter the development of fatigue. This would occur independent of any O2 limiting situation.
As with the factors that regulate muscle metabolism, the factors that cause reduced tension development and muscle fatigue interact in a complex fashion (2,89). It is likely that various changes in the intracellular milieu can directly affect cross-bridge function or inhibit SR Ca2+ kinetics, both of which will reduce tension production. Accumulation of H+ and Pi has both been implicated in these processes, although the role of changes in pH remains controversial. Increases in [Pi] have been linked to decreased contractility in isolated muscle fibers (15) and decreases in SR Ca2+ release (6). Figure 4 (from Hogan et al. (46); using 31P-MRS) shows the effect of different PO2 on one of these critical modulators of cellular function (including fatigue)—intracellular Pi. It can be seen that at the same submaximal work intensity (rates of ATP utilization and mitochondrial respiration will be the same for any of the varied PO2), the intracellular Pi is increased with decreasing PO2, and this may have influenced the earlier onset of fatigue in the low PO2 conditions. In addition, recent evidence has pointed to the role of intracellular [Ca2+] as a significant factor in the fatigue process, and there is good evidence that release of Ca2+ from the SR complex may be inhibited by changes in the other intracellular constituents mentioned previously (including Mg2+) as the ATP requirements become excessive (1).
Finally, numerous investigations have also implicated oxygen radical formation during intense muscle contractions as a causative agent in the fatigue process (86). However, there remains a paucity of information related to the manner in which intracellular oxygen tension affects reactive oxygen species (ROS) production during contractile activities. It has been known from the early 1980s that ROS are generated by working skeletal muscle (23). ROS are generated in low amounts within resting skeletal muscle, and it is generally agreed that ROS production increases with contractile activity. Since ROS generation is dependent on the metabolism of O2, processes related to intracellular oxygenation are of critical importance to this field of research. However, the oxygen dependence of ROS generation is poorly understood (21), and it has only recently become evident (22) that hypoxia can paradoxically induce ROS generation—thereby potentially affecting the process of fatigue. Ferreira and Reid (30) have postulated that muscle force production is sensitive to the muscle redox status, with inhibition of force at either a very reduced or a very oxidized muscle redox state.
At this time, after years of investigation, we still know very little about intracellular O2 levels in muscle at rest or during the conditions of increased mitochondrial respiration caused by contractile activity and how this modulates ROS formation and the other factors that can induce fatigue. In terms of contractile function, it should be clear that the changes in the intracellular metabolic state induced by altered oxygenation states can have a profound impact on cellular function. Increases in Pi, ADP, H+, ROS and other metabolites that occur with altered oxygenation state, independent of respiratory rate, can seriously impair the activity of the contractile and ion pump processes. In this way, cell function can be compromised by the altered metabolic state of the cell that results from altered O2 availability, even when there is enough O2 available for the required respiratory rate.
Is There a Disconnect between Human and Animal Fatigue Models?
In this final section of our review, we focus on the role of BF in muscle fatigue, to provide some historical perspective on how studies of both humans and animal models have contributed to our understanding of fatigue over the years. Almost without exception, experimental evidence gathered in animal species proves valuable to furthering our knowledge of human physiology and the basis for relieving the burden of human disease. Comparative sequence analysis has revealed genetic homologies across phenotypically diverse species and, as the genes and diseases are the same, so is the therapeutic treatment. Insertion of a human disease gene into the mouse genome causes that disease in the mouse which then becomes indispensable as a model for helping to understand and treat the disease in humans. It should be noted that engineered animal models of disease that produce a phenotype similar to that found in humans may not faithfully reproduce the mechanistic bases for that disease. As a result, therapeutic interventions developed from such animal models often fail in human trials because the models are too narrowly focused on a single pathological pathway. This overengineered approach, combined with a lack of diversity of animal models (i.e., reliance on mice versus larger animals), has slowed drug discovery.
Given the above, it is difficult to understand a defensible scientific approach to solving any physiological or medical problem that does not garner relevant evidence from both humans and animal species. This section highlights the sequence of discovery across species for several major scientific advances as they relate directly to our present knowledge of muscle fatigue and especially the role of O2 delivery and metabolism in those processes.
The alleviation of fatigue and exercise intolerance in health and particularly in endemic chronic diseases such as heart failure and diabetes remains a cornerstone principle of modern medicine and National Institutes of Health funding strategies. For rhythmic contractions, muscle performance is inextricably connected to the sustained and adequate supply of oxygen. Consequently, any discussion of fatigue and fatigue mechanisms is not complete without consideration of muscle BF and O2 supply and their potential to impact contractile performance.
In 1987, the ACSM Annual Meeting provided a forum (87) for luminaries in the field to pose sentinel questions regarding what was then known regarding skeletal muscle(s) BF during exercise. One impetus for this symposium was the, then recent, article by Andersen and Saltin (3), documenting BF in the human quadriceps approximately 2.5 L·min−1·kg−1. This value was far greater than found previously by indicator washout/dilution, venous occlusion plethysmography, or bolus infusion of cold saline in humans (3,49,58) and raised major concerns regarding control of the exercise hyperemia and muscle metabolism itself. Cross-species comparisons featured in that symposium included the rat and dog as well as human studies and highlighted the primary role played by research on animal muscle(s) in past and future scientific discovery.
What are the upper limits to skeletal BF during exercise?
As demonstrated for the heart (left ventricle, >6 L·min−1·kg−1) (65) and skin (2–3 L·min−1·kg−1) (81), it was appreciated that organ BF could reach extraordinarily high levels. However, despite the existence of similar values in individual rodent muscles, skepticism regarding the radioactive microsphere technique precluded mainstream acceptance of these values. Specifically, in rats running at 75 m·min−1, several muscles including the red gastrocnemius and vastus intermedius reached BF in excess of 3 L·min−1·kg−1 (64). Furthermore, Musch and colleagues (69) recorded similarly BF in the locomotor muscles of exercise trained foxhounds running maximally on the treadmill. In 1993, Richardson and colleagues (80) considered that the prolonged duration of the Andersen and Saltin (3) knee extension protocol may have constrained both work rate at fatigue and maximal BF. Using the same techniques as Andersen and Saltin during a more rapidly work rate-incremented knee extensor exercise test in trained cyclists, Richardson et al. (80) found that BF increased to approximately 4 L·min−1·kg−1, the highest measured to date in human muscle(s). These values in humans led to widespread acceptance of the very high animal BF gathered using microspheres. Today, the microsphere technique has measured some of the greatest BF in skeletal muscle (rats running at 96 m·min−1, vastus intermedius, 6.8 L·min−1·kg−1) and also the diaphragm (costal diaphragm, 5.5 L·min−1·kg−1) (78). As well as establishing the extraordinary vascular capacity of skeletal muscle(s), Barclay and Stainsby’s (7) experiments using their classic canine gastrocnemius-plantaris model made it evident that, during maximal contractions, skeletal muscle oxidative capacity was limited by O2 delivery and not mitochondrial metabolism as was subsequently proven for human muscle by Richardson et al. (79).
How heterogeneous is BF among/within muscles during exercise?
As discussed in Poole et al. (77), animal muscle may be more highly stratified with respect to fiber type(s) than their human counterparts. This property means that animals may be ideal models for investigating skeletal muscle functional vascular and metabolic control during exercise as related to distinct fiber types and their recruitment during different exercise intensities and durations. Thus, Laughlin and Armstrong (64) determined that, in the resting rat, the soleus muscle supported a BF some sixfold that of the white vastus lateralis and 2.5-fold that of the red gastrocnemius. However, during high speed running at 75 m·min−1 the red gastrocnemius BF increased to 1.4- and 7.5-fold that of the soleus and white vastus lateralis, respectively. Although many of the highly oxidative muscles were recruited at relatively slow running speeds their low oxidative counterparts (such as the white gastrocnemius) required far greater speeds. These investigations also dispelled the notion that muscles comprised of slow oxidative (type I) fibers sustained higher peak BF (and metabolic rates) than fast twitch oxidative (type IIa) muscles. Human muscles may possess very different fiber types both across muscles (e.g., %type I; soleus 88%, rectus femoris [RF], 36%) and also, to a lesser extent, with respect to depth from the skin (deeper muscle has more type I fibers; (48). In 2000, Kalliokoski and colleagues (50) using H2O15 positron emission tomography (PET) identified a distinct heterogeneity of BF among the thigh extensors during submaximal exercise. Specifically, BF increased from the vastus lateralis and RF (most superficial) toward the deeper vastus medialis and vastus intermedius (~2-fold that of the VL). This is the precise pattern expected based upon Laughlin and Armstrong’s (64) data obtained in rats almost two decades earlier!
Rat muscles composed of type I fibers sustain a higher O2 delivery/O2 utilization ratio during contractions compared with type IIs, such that their microvascular PO2 (which constitutes the O2 pressure driving blood–myocyte O2 flux) is greater and falls more slowly after the onset of contractions (8,66). Based on these data and that of Kalliokoski et al. (50), Koga and colleagues (59) hypothesized that the deeper thigh muscles should deoxygenate more slowly after exercise onset. Using high-power Time Resolved Spectroscopy–near-infrared spectroscopy the mean response time for the deeper RF deoxygenation was indeed substantially slower than its superficial region (i.e., superficial RF, 37 s versus deep RF, 65 s, P < 0.05). This finding calls into question the interpretation of more superficial NIRS measurements as representative of the muscle as a whole.
What is the role of nitric oxide in the exercise hyperemia?
Early human studies investigating the role of nitric oxide (NO) in regulating the skeletal muscle BF response to exercise produced conflicting results (49). Whereas some investigators (27,34) found a significant role for NO in the hyperemic response through NO synthase (NOS) blockade, others (28,90) did not. The reasons for these conflicting results remain unclear, but they may have been associated with differences in experimental design and methodologies used to measure BF (i.e., venous occlusion plethysmography, Doppler ultrasound, constant-infusion thermodilution technique) along with varied exercise paradigms (i.e., arm versus leg exercise; exercising at different work intensities) used in these investigations. In addition, unlike research using animals it is challenging to achieve full NOS blockade in humans.
Boushel et al. (14) have demonstrated that the hyperemic responses to incremental knee extension exercise in both the vastus lateralis and vastus medialis are reduced in individuals subjected to both NOS and cyclooxygenase blockade. Similarly, Heinonen et al. (41) using PET scanning showed that BF was reduced in the working quadriceps femoris muscle by 13% during NOS and cyclooxygenase blockade and if one examines closely the representative cross-sectional PET BF images produced in that study it is evident that the reductions in BF occurred in the muscles that were located very close to the femur (the more highly oxidative muscles). These studies provided compelling evidence that NO bioavailability plays a significant role in the hyperemic response to leg exercise in humans. They also suggest that NO production and bioavailability make a greater contribution to the hyperemic response of the deep (and more oxidative) muscles found in the human thigh.
Interestingly, the results of Heinonen et al. (41) are very similar to those produced by Hirai et al. (43) nearly 2 decades ago. Specifically, Hirai et al. measured BF with the radioactive microsphere technique in individual muscles or muscle parts of the hindlimb during moderate treadmill exercise both before and after NOS and cyclooxygenase blockade. Results demonstrated that BF was reduced in 16 of the 28 muscles examined and that the reductions in BF were highly correlated with the oxidative capacity of the individual muscles. In agreement with Heinonen et al. (41), the muscles demonstrating the largest NOS-blockade-induced reductions in BF in the thigh region of the rat were those located deep and close to the femur.
Can animal studies help unravel the complexities of vascular control in age muscle?
Understanding how aging affects vascular control and the exercise hyperemia in skeletal muscle is central to maintaining health, exercise tolerance and quality of life. However, without defining the healthy aging response the interactions between aging and age-related diseases (i.e., chronic heart failure, diabetes, chronic obstructive pulmonary disease) cannot be defined and the most effective countermeasures to exercise intolerance undertaken.
Animals studies have consistently demonstrated that working muscle BF is not different (either at submaximal or maximal treadmill running speeds) in young (2–3 yr) versus old (10–14 yr) beagles (36) or young adult (12 months) and senescent (24 months) rats (60). In addition, Musch et al. (68) found that although total hindlimb muscle BF might be unaffected by age per se during submaximal treadmill exercise in mature (6–8 months) and senescent (27–29 months) rats, there was a profound redistribution within and among the 28 different muscles or muscle parts examined. Specifically, in the old rats BF was increased in eight highly glycolytic muscles but reduced in six highly oxidative muscles.
Moving ahead 10 yr, PET scanning measurements of exercising knee extensor BF revealed no difference in total muscle flow in young (26 ± 6 yr) and old (77 ± 6 yr) men. However, as found in rats, BF to both the vastus intermedius and vastus medialis were significantly higher in the old men (82). In addition, in the old subjects, there was a markedly greater heterogeneity of intramuscular flow found within each of the muscles. These findings, initially in rats and subsequently in humans, reveal a complex effect of aging on skeletal muscle vascular control and provide irrevocable evidence that animal studies are indispensable to unravelling the impact of aging on muscle vascular control and hence age-related exercise intolerance.
Perusal of the Nobel prize-winning science over more than a century reveals an effective symbiosis between animal and human research leading to major advances in physiology and medicine (Fig. 5). This is nowhere more true than for progress in understanding the fundamental mechanisms of fatigue and the role of muscle(s) BF and O2 delivery in facilitating or limiting exercise tolerance. Animal experimentation has yielded basic scientific information, unraveled vasomotor control pathways and framed key questions that can then be addressed ethically in humans. As dealt with above, this symbiosis has been especially effective for discovering: 1) The upper limits of muscle perfusion. 2) The extraordinary degree of BF heterogeneity among and within muscles that permits exquisite matching of O2 delivery to V˙O2 during exercise. 3) The essential role of NO in vasomotor control. 4) How aging impacts the exercise hyperemia. These provide just a few of a multitude of examples where investigative science (and knowledge of fatigue mechanisms) has advanced through judicious use of both animal and human models.
Integrative approaches to the study of fatigue can provide unique information about the interaction of physiological systems during stresses such as fatigue. Information about multiple systems informs the overall interpretation of fatigue data, and provides the opportunity to develop novel hypotheses about how systems function under a variety of conditions. Solving the complex mystery of fatigue requires both a combination of approaches within a given study, as well the use of varied models across studies; a full understanding of fatigue will best be served by communication among researchers working at all levels. In this review, we have provided examples of successful approaches to the study of skeletal muscle fatigue, including a variety of applications to understanding fatigue in aging. Future work that expands upon the approaches described here and other approaches in use today will continue to illuminate from the molecular scale to whole-body function the complex factors involved in the development of muscle fatigue.
The authors report no conflicts of interest. There are no funding sources to acknowledge for this review. The results of the present study do not constitute endorsement by ACSM.
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