Skeletal muscle cross-bridge cycling is dependent on the availability of adenosine triphosphate (ATP). The concentration of ATP in muscle is very low relative to the rate of utilization during exercise (3). Fortunately, the demand for ATP is matched remarkably well by various ATP supply pathways, even during maximal intensity fatiguing exercise (3). However, skeletal muscle has evolved an array of mechanisms to regulate ATP homeostasis that extend beyond the activation of these metabolic pathways. One strategy is to decrease neuromuscular activation during fatigue (10). However, some of the other strategies are more localized, do not depend on central perceptions of fatigue, and will be presented in the following three articles, beginning with the efficiency of the cross-bridges’ ATP utilization (24) and progressing to the activation of two metabolic sensors, and the subsequent upregulation of substrate delivery (see Fig. 1). These two metabolic sensors, whose roles have been delineated very recently, are the intracellular energy sensor, the adenosine monophosphate-activated kinase (AMPK) (15), and the extracellular oxygen sensor, the red blood cell (8).
To maintain intracellular ATP concentrations during exercise, the most obvious solution is to upregulate ATP supply. However, ATP demand may also be downregulated, either by a decline in the number of active skeletal muscle ATPases or by localized slowing of their rates of ATP utilization. It is also important to note that cross-bridge cycling is not only a function of the myosin ATPase enzyme but also contractile protein interactions. Hence, the efficiency of contraction could also be promoted by enhanced protein-protein interaction and force production. In 1985, Cooke and Pate (5) showed how an accumulation of ADP, albeit under experimental conditions, could both slow down cross-bridges’ ATP utilization and enhance their force production. But the first paper in this series cites evidence that the role of ADP accumulation during fatigue is controversial (Table 1 in ref. 24). Nevertheless, its role has been more clearly defined recently (19).
In in vitro experiments using an ADP-analog, researchers (19) could manipulate both ADP and ATP concentrations with much better control than was previously possible. The experimental milieu in these experiments was also manipulated to simulate various levels of fatigue by altering either pH or inorganic phosphate (Pi) contents, or both, in addition to concurrent controlled manipulations of ATP and ADP. One of the main findings of Karatzaferi et al. (19) was that there was competition between ADP and ATP for the nucleotide binding site on myosin which favored ATP under all conditions, even those simulating extreme fatigue. Hence, an effect of ADP is dependent on very low ATP concentrations. However, when force was significantly depressed in the presence of high concentrations of Pi and H+ ions, the relative effect of elevated ADP was more significant.
In the first paper in this series, Myburgh (24) places these new results in the context of previous studies on the efficiency of contraction (for review, see Bangsbo (1)) and current theories and evidence on the extent to which ATP concentrations decline during fatigue. If ATP concentrations do decline to very low levels in micro-environments, as suggested by both theoretical (20) and physiological (18) studies, ADP may indeed have an effect of partial alleviation of the force decrement associated with metabolite accumulation. Myburgh (24) also hypothesizes that, although the elevated Pi during fatigue has a direct negative effect on force production, it could have an indirect positive effect on ATP homeostasis. This hypothesis depends on the presence of a) an intracellular mechanosensor that can feed back the actual force produced (which is reduced at the cross-bridge by Pi) and b) central perception of a mismatch between the expected and the actual force production. Such a role for Pi remains to be proven but could be an integrating factor in the apparently opposing central (10) versus peripheral (11) views of fatigue.
One reason that ADP accumulation during fatigue is not substantial is that it is prevented by the adenylate kinase reaction (3) that produces AMP. However, AMP accumulation during fatigue is also small because it is prevented by the activation of the AMP deaminase enzyme. It is for this reason that authors in the past have overlooked AMP in favor of IMP as a metabolite with potentially important consequences during fatigue (e.g., 11,25). But it is now clear that sufficient AMP can accumulate in muscle to activate the AMP-kinase not only under conditions of severe cellular energy stress (16) but also during submaximal exercise intensities (28,29,30). In the second paper in this series, Hardie (15) presents a fairly chronological perspective of our growing understanding of the multiple important effects of AMPK in the integration of cellular metabolism, particularly during management of a high rate of energy demand.
In skeletal muscle, the activation of AMPK by exercise was first shown in 1996 (29). Its activation is not only dependent on exercise intensity (31) but is also isoform specific (13,29). Hardie (15) illuminates reasons why this kinase is highly sensitive to AMP over the small range of AMP that accumulates intracellularly in skeletal muscle during exercise. AMPK has three subunits (α, β, and γ) and each subunit has different isoforms. Therefore, depending on the various combinations of these isoforms, AMPK could play multiple roles within a cell or different roles in different cell types. In different types of skeletal muscle, different isoforms of the catalytic α-subunit predominate (7), and each is not equally activated by different kinds of exercise (13,28) or in trained versus untrained muscle (7). In his review (15), Hardie explains the significance of these facts for skeletal muscle.
In keeping with the theme that ATP can be defended directly or indirectly by metabolites, it is worth noting that the AMP-activated kinase plays several roles in this regard ranging from activating mechanisms to improve ATP supply, to inhibiting mechanisms that may utilize, for other purposes, the substrates needed to produce ATP. For example, what has only recently come to light is that AMPK inhibits glycogen synthase and that glycogen status itself influences AMPK activation during exercise (32). AMPK also upregulates glucose uptake by muscle (21), thus providing a source of carbohydrate for ATP supply that is not dependent on the finite intracellular stores. But, as Hardie explains (15), the role of AMPK in metabolism is more complex than was at first suspected: it can also increase fatty acid uptake into the mitochondria (23), thus providing even more substrate for ATP supply through fat oxidation.
The activation of AMPK also has longer-term benefits for ATP protection during exercise. For example, AMPK is involved in stimulating mitochondrial biogenesis by activating gene transcription (30). It is likely that AMPK can also inhibit energy-expensive anabolic pathways by inhibiting gene transcription of anabolic enzymes (33). Hardie concludes his review (15) by emphasizing that AMPK plays many roles, both short- and long-term, but that during exercise it plays a role in mechanisms that adjust the supply of ATP to more effectively balance the demand.
Up to this point in the symposium, most of the focus was directed toward intracellular actions of metabolites of ATP hydrolysis or downstream effects of the metabolite AMP. However, muscle tissue cannot work long in isolation: occlusion of blood flow causes a rapid onset of fatigue, whereas exercise improves perfusion of the active muscle (3). One mechanism for regulation of this enhanced perfusion is the focus of the third paper in this series (8), particularly, the role of a mobile oxygen sensor that can act peripherally, rapidly and specifically.
It is well known that oxidative phosphorylation has the capacity to supply skeletal muscle with more ATP than other pathways. However, it depends on the supply of oxygen. Therefore, a sensor (or sensors) is required that can quickly determine a change in oxygen demand. However, a mechanism must also exist to distribute the oxygen efficiently to the precise region where it is required. As is often the case when multiple physiological tissues and systems need to interact, there may be multiple controllers, some of which are more sensitive than others. Ellsworth (8) describes the evolution of the idea that the red blood cells, the carriers of oxygen, could themselves be the mobile sensors referred to above.
Although it has been know since 1969 that ATP is released into the venous circulation of the working muscle during sustained contractions (12), more recent evidence shows that red blood cells contain reasonably high levels of ATP, have the capacity to generate more ATP glycolytically, and actually release ATP under hypoxic, hypercapnic conditions (2). For those of us who approach fatigue from an intramuscular perspective, it may still not be evident why ATP release into plasma may facilitate the maintenance of ATP supply within the muscle. However, it is also crucial to understand that the endothelium contains purinergic receptors that can bind ATP and that this event results in vasodilation (26). Ellsworth delineates several experimental studies showing unequivocally that there is a graded response in red cell ATP release in response to decreased oxygenation (6,9). Further studies, both in vitro (17) and in human subjects (14), have indicated that the crucial effect of hypoxia that results in the red cell ATP release, is a decrease in hemoglobin oxygen saturation.
Although ATP is a metabolic product rather than a metabolite, it serves as an example of an important regulatory mechanism that is active peripherally during exercise to delay or alleviate fatigue by improving supply of a crucial substrate for intracellular ATP homeostasis. However, purely localized vasodilation at the site of ATP release would be less effective in enhancing oxygen delivery than would be a localized response that is also propagated upstream from the hypoxic region (27). Ellsworth (8) also explains how and why communication across a capillary bed can greatly enhance blood flow and that this can be affected by physiological amounts of ATP released into either arteriolar lumens or collecting venules (4,22). In the latter case, the conducted effect on the upstream arterioles was even greater than in the former. This paper also highlights those issues that remain to be properly explained such as the signals within the red cells that link desaturated hemoglobin and ATP release, and the mechanisms of conducting the vasodilatory signal upstream from ATP release or across capillary beds.
In summary, the main messages of this series of papers (8,15,24) are that metabolites are not necessarily “bad” for skeletal muscle but play an important positive role in ATP homeostasis by modulating the demand-supply equilibrium, either for ATP itself or for substrates of ATP resynthesis. There are multiple mechanisms that not only modulate supply but can also modulate demand. These mechanisms are operative peripherally within the exercising skeletal muscle, as well as in the surrounding vasculature without the requirement for any central intervention.
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