The Coupling of Internal and External Gas Exchange During Exercise

Chapter 10

The Coupling of Internal and External Gas Exchange During Exercise T. Scott Bowen1, Alan P. Benson1 and Harry B. Rossiter1,2 1

Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom, 2Rehabilitation Clinical Trials Center, Division of Pulmonary Critical

Care Physiology and Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, United States

10.1 INTRODUCTION 10.1.1 Introduction to Exercise Bioenergetics Muscular exercise imposes a stress to bodily homeostasis that demands an integrated multiorgan response. The ability to maintain muscular contractions depends on the ability to provide adenosine triphosphate (ATP) at a rate required by the myosin ATPase and the sarco/endoplasmic reticulum Ca21-ATPase (SERCA) for cross-bridge cycling and power production. Skeletal muscle ATP demand can increase from B1 mmol ATP
kg21
min21 at rest to 100 mmol ATP
kg21
min21 or more during exercise in an endurance trained athlete: more than a 100-fold increase. However, ATP is stored at low concentrations in skeletal muscle (B8.2 mmol
kg21). Therefore, sustained exercise demands rapid resynthesis of ATP via phosphocreatine (PCr) breakdown and anaerobic glycolysis forming lactate (substrate level phosphorylation) and oxidative phosphorylation fueled by either carbohydrate or fatty acid derived reducing equivalents. The maximum flux for ATP provision by each of these systems is inversely related to their total capacity, such that, on average, in normal healthy human muscles: (1) the maximum rate at which ATP can be supplied approximately halves between each bioenergetic system; and (2) the maximum capacity for ATP synthesis before depletion of substrate storage increases greater than two fold between each bioenergetic system (Table 10.1). For details see also Chapter 5: Muscle Energetics by Graham Kemp. These properties of muscle bioenergetics allow a wide span of rate and capacity for ATP provision supporting both high-intensity short-duration and moderate-intensity long-duration endurance exercise. In fact, these bioenergetic systems, and the processes that support them, integrate so effectively that [ATP] remains constant during muscle contractions, in all but the most extreme of Muscle and Exercise Physiology. DOI: https://doi.org/10.1016/B978-0-12-814593-7.00010-4 © 2019 Elsevier Inc. All rights reserved.

physiological conductions (Ivy et al., 1987; Rossiter et al., 2002b). This is all the more surprising considering that muscular oxygen storage (in the forms of dissolved O2 and O2 bound to myoglobin), required for oxidative phosphorylation, is also extremely limited. Therefore, the ability to buffer muscular [ATP] during contractions is consequent, in large part, to the exquisite temporal and spatial matching of O2 supply from the atmosphere to the demand in the myocyte to support oxidative phosphorylation. Without this ability, the capacity to sustain highintensity exercise would be limited to only a few seconds. Sustaining exercise in conditions where oxidative phosphorylation is limited, perhaps by gas exchange limitations in the lung or by convective or diffusive limitations in the circulatory system, increases the demand for substrate level phosphorylation, which has a limited capacity (Table 10.1) and results in the intramuscular accumulation of metabolites implicated in muscle fatigue, such as inorganic phosphate (Pi), hydrogen ions (H1), and adenosine diphosphate (ADP) (Poole and Jones, 2005), or in impaired excitationcontraction coupling (Allen et al., 2008; Grassi et al., 2015). Thus, the mechan_ 2m ) and pulisms by which muscle O2 consumption (VO _ 2p ) increase to meet the new monary O2 uptake (VO ̇ 2 kinetcellular demand for oxidative phosphorylation (VO ics), and the mechanisms limiting the maximum rate of O2 delivery and utilization by integrated physiological systems, are each key determinants of endurance exercise tolerance (Murgatroyd et al., 2011). To borrow from the words of Wasserman et al. (2011): “The consistent physiological signal for impending [task failure] during exeṙ 2 to reach a steady state and to cise is the failure of VO meet the [muscle] cellular O2 requirement.” Consequently, the maximum rate of pulmonary O2 _ 2max ) is the typical measure of choice to uptake (VO 217

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TABLE 10.1 Estimated Maximum Rate and Capacity of ATP Provision in Skeletal Muscle of a Healthy Adult

Maximum flux Capacity

mmol ATP
kg21 s21 21

mmol ATP
kg

ATP

PCr

Anaerobic Glycolysis

Carbohydrate Oxidation

Fatty Acid Oxidation

2.6a

2.3

1.1

0.6

0.3

8.2

32

75

3500

b

Estimates are based on a glycogen replete 70 kg active male with a maximum oxygen uptake of 4 L
min21 and an active muscle mass of 20 kg. Estimates are based on data from magnetic resonance spectroscopy and muscle biopsy (Sahlin et al., 1998; Kemp et al., 2015). Values are expressed as mmol ATP
kg wet weight s21. a Maximum rate of ATP provision from stored ATP is determined by the rate of the specific ATPase. b Given typical adipose storage within the human body, the maximum capacity of fatty acid oxidation is also effectively unlimited, at least over the durations typical of common physical activities.

FIGURE 10.1 The interaction of physiological systems coupling internal and external gas exchange based on the 1967 conceptualization of Wasserman et al. (1967). The ability to fuel muscular contractions is dependent on the functioning of a number of linked physiological systems, each of which is subject to deterioration with aging or chronic disease. V0 O2, O2 uptake; V0 /Q0 , ventilation/perfusion ratio; V0 CO2, carbon dioxide output; Pyr., pyruvate; lact., lactate; util., utilization. Redrawn from Wasserman, K., et al., 1967. J. Appl. Physiol. 22, 7185 with permission.

estimate the physiological limits to endurance exercise. This is precisely because muscle O2 storage is low, meaning that O2 exchange across the pulmonary alveolarcapillary interface (external respiration) must be closely matched to that at the muscle mitochondria (internal respiration). If not, the limited O2 stores of the body would become rapidly depleted and tolerance for endurance exercise would be extremely limited. However, _ 2max, while Wasserman’s statement reminds us that VO important, is far from the sole physiological measure of efficacy for exercise endurance. Indeed, during the activities of daily living most humans do not sustain exercise _ 2max . The dynamics (or kinetics) by at, or even close to, VO which the systems for oxidative phosphorylation can respond to alterations in exercise demand will determine “metabolic stability.” This is the concept that the ability to make a rapid adjustment in oxidative metabolism during exercise, allows the energetic demands of the task to be met with only minor derangements in muscle metabolism (e.g., breakdown of PCr and accumulation of Pi):

metabolic stability means that strain on muscle metabolism can be met in a steady state. However, slow adjustments in oxidative metabolism cause large intramuscular metabolic strain and exceed the threshold for stability, resulting in non-steady-state physiology, muscle fatigue, and exercise intolerance. Thus, bioenergetics kinetics are as key to understanding mechanisms limiting endurance _ 2max exercise tolerance as more familiar indices such as VO (Murgatroyd et al., 2011; Zoladz and Grassi, 2011; Grassi et al., 2015). These concepts are succinctly encapsulated in “Wasserman’s gears,” which depicts the systemic integration of physiological mechanisms underlying the bodily responses to exercise (Fig. 10.1). Transiting from muscle to lung in Fig. 10.1, the physiological systems that are each required to respond rapidly to alterations in exercise demands include: (1) mechanical to metabolic coupling in the muscle; (2) gas transport between muscle capillary and mitochondrion; (3) regional matching of O2 delivery to its requirement in heterogeneous muscles; (4) O2 and

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CO2 carrying capacity of the blood; (5) cardiac output; (6) pulmonary vascular function; (7) O2 and CO2 transport between pulmonary capillary and alveolus; (8) ventilation; and (9) pulmonary mechanics and respiratory muscle function (Roman et al., 2016). This chapter will explore the coupling of internal to external O2 exchange during dynamic exercise. It will discuss how pulmonary gas exchange can be used to shed light on: the mechanisms controlling, and limiting, muscular oxidative phosphorylation in skeletal muscle; how these are altered by training, aging, or chronic disease; and how they contribute to determining exercise tolerance.

10.1.2 Definitions We will first define a few key terms that are commonly used, but occasionally misused, in exercise physiology. Firstly, we want to distinguish between fatigue and intolerance. The term “fatigue” is here reserved for the specific condition of muscle fatigue, which is defined as the reduction in the maximum evocable power that a muscle can produce, and which is rapidly reversible by rest. Fatigue is an ongoing process during sustained exercise, which in extremis may contribute to exercise intolerance (Froyd et al., 2013; Coelho et al., 2015; Ferguson et al., 2016). “Exercise intolerance” is the inability to continue an exercise task, and defines the point at which a human slows or stops exercise, despite the encouragement or desire to continue. Exercise intolerance is synonymous with the term “task failure.” Fatigue may bring about intolerance should the maximum evocable power fall below the demands of the task (Bigland-Ritchie et al., 1986). This may be one mechanism by which exercise intolerance occurs in humans (Ferguson et al., 2016; Keir et al., 2016), but it is certainly not exclusive. Exercise intolerance can also be determined by central nervous system constraints on muscle activation or triggered factors such as by pain, dyspnea, or fear or anticipation of impending symptoms (Cannon et al., 2016). We also wish to disambiguate the terms exercise intensity and power output. Power output (named watts, and measured in joules per second) is here defined as the external mechanical manifestation of the conversion of chemical to mechanical power in the skeletal muscles, commonly measured at the flywheel during cycling or calculated during treadmill exercise. This combines two components: 1. “Work efficiency” which is the fraction of intramuscular energy used in addition to that required for basal metabolism that ultimately produces power. This includes energy lost during two key steps: mitochondrial coupling (ATP/molecular oxygen ratio; P/O) and

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contractile coupling (ATP/power output ratio; P/W); resulting in a work efficiency of B25% in healthy human muscle. The remaining B75% is lost as heat. 2. “Economy” which is the product of work efficiency and the fraction of muscle power production that is converted to “useful” external mechanics. This step includes loss of muscle force or power in the application of biomechanics, such as the stretching of the muscle cytoskeleton, muscle-tendon complex function, and the accuracy of movements or “skill.” In exercise physiology the term work rate is often used synonymously with power output, although power is the appropriate term according to the convention of syste`me international d’unite´s (SI unit). Intensity, on the other hand, has several meanings. We believe that the important concept to grasp is that intensity and power output are not interchangeable, linear functions of one another. A 10% increase in power output does not, by necessity, beget a 10% increase in intensity. One meaning of intensity is the subjective rating of perceived exertion. The “Borg CR-10” rating scale during exercise increases approximately curvilinearly with power, with dynamics dependent upon fitness and primary pathological states (Jones and Killian, 2000). Other definitions of intensity use a fraction of peak exercise responses, such as percentage of maximum heart rate (HR), HR reserve (i.e., a fraction of the range _ 2max, or multiples of a between resting and peak HR) or VO standardized resting metabolic rate (metabolic equiva_ 2max is widelents). Although the use of percentage VO spread, it is a suboptimal approach (Rossiter, 2011; Poole and Jones, 2012). This is because different individuals demonstrate markedly different kinetics of gas exchange, ventilation, metabolism, and acidbase balance during _ 2max work rates derived from the same percentage VO (Roston et al., 1987). Here we will use a definition of exercise intensity that is based upon bioenergetics (Whipp, 1996; Davies et al., 2017), which clusters a range of power outputs depending upon the individual’s ability to meet the energetic demands of exercise by wholly aerobic means (the term “wholly aerobic” will be discussed in more detail later), and whether or not a steady state is achieved.

10.2 GAS EXCHANGE DURING EXERCISE 10.2.1 Exercise Intensity Domains The predominant energy system recruited during exercise is highly dependent on the intensity of the exercise being performed. As such, the ability to accurately characterize exercise intensity, for example, for a relevant basis comparison among individuals’ exercise responses, or to

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inform an accurate basis for exercise prescription, is a central tenet of exercise physiology. Here we assign exercise intensity based on an individual’s response kinetics in gas exchange and metabolic acidosis. Four intensity domains are identified: moderate, heavy, very-heavy, and severe intensity. Intensity domains are separated by threshold events in metabolic rate: lactate threshold _ 2max (Whipp, 1996) (LT), critical power (CP), and VO (Fig. 10.2). While intensity domains are typically expressed in units of external power (these being

FIGURE 10.2 Schematic of the exercise intensity domains which ̇ 2 ,LT); heavy (VO ̇ 2 between LT and CP); veryinclude: moderate (VO _ 2max ); and severe (VO ̇ 2 .VO _ 2max , and without ̇ 2 .CP and ,VO heavy (VO ̇ 2 slow component). The VO ̇ 2 response (A), power duration (B), and a VO blood lactate profile (C) are dependent on the exercise intensity that is performed. The dotted line in (A) represents the expected increase in ̇ 2 predicted from sub-LT work rates in the absence of the VO ̇ 2 slow VO component. From Rossiter, H.B., 2011. 1, 203244 with permission. Copyright r 2011 American Physiological Society. All rights reserved.

relatively simple to measure), they more accurately reflect thresholds in metabolism (often expressed in units of ̇ 2). Power and VO ̇ 2 are linearly related up to LT, makVO ing the need for a distinction somewhat moot. However, ̇ 2 are no longer linearly above LT power output and VO related, meaning that it is important to recognize that the expression of intensity domains in units of external power is an (over) simplification and can lead to misinterpretation of exercise responses. Moderate-intensity exercise constitutes the range of ̇ 2 that reside below LT, and is characterized by the VO absence of a sustained metabolic acidosis and the attainment of a steady state in pulmonary gas exchange (as well as ventilation and HR) typically within B23 min. ̇ 2 between LT and Heavy-intensity exercise, that is, VO CP, is characterized by a sustained, but stable, arterial acidosis (increased [H1] and reduced [HCO32]), increased arterial lactate concentration ([L2]), and a delayed attainment of a steady state in pulmonary gas exchange (delayed as long as 15 min in some cases). This steady state comes at the expense of an inefficiency of energy conversion, which is manifest as an increase in the “func_ 2p : the VO _ 2p cost of power output tional gain” of VO _ (ΔVO2p /ΔW, measured in units of mL
min21
W21). ̇ 2 slow component,” This inefficiency is termed the “VO because its appearance is delayed relative to the start of exercise and, once manifest, it is slow to develop (Poole et al., 1994; Grassi et al., 2015). The asymptote of the hyperbolic relationship between power output and tolerable duration, CP (which is, itself, a metabolic power; Barker et al., 2006), delineates the upper limit of heavyintensity exercise. Metabolic rates that exceed CP are within the veryheavy-intensity domain. CP represents the greatest metabolic rate for which a steady state in pulmonary gas exchange, muscle metabolism, and blood acidbase status can be achieved, and above which muscle fatigue is first observed. Metabolic rates above this threshold cannot ̇ 2 inexorably increases during be met in a steady state: VO very-heavy-intensity exercise, and, if exercise is contin_ 2max. In the very-heavẏ 2 will eventually reach VO ued, VO intensity domain arterial [L2] and [H1] rise inexorably and [HCO32] continues to fall until cessation, or the limit of tolerance. Severe-intensity exercise includes all metabolic rates _ 2max from the start of exercise, and cause that exceed VO _ 2max before a VO ̇ 2 slow intolerance or attainment of VO component can be expressed.

10.2.2 Ramp-Incremental Exercise By far the most common assessment of aerobic function in humans is the ramp-incremental (RI) exercise test (Whipp et al., 1981). During this clinical assessment, the

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power output increases as a smooth function of time until volitional intolerance. By providing a gradually increasing power demand, using a treadmill or cycle ergometer, this protocol spans exercise intensity domains. It provides several variables of aerobic function that are clinically and prognostically significant. Here we will focus on four variables that are closely related to muscle metabolic _ 2max, functional gain (related to work function: LT, VO efficiency), and kinetics (mean response time; how ̇ 2 responds to meet energetic demands; quickly VO Whipp et al., 1981).

10.2.2.1 Lactate Threshold The LT, originally termed the anaerobic threshold, is the metabolic rate at which lactate production in the active musculature exceeds the rate of systemic lactate clearance. Using infusions of lactate containing radiolabeled carbon, Stanley et al. (1985) demonstrated that, at low power outputs, the rate of systemic lactate appearance was essentially matched by its rate of disappearance. At high power outputs, however, lactate appearance exceeded clearance, and hence lactate accumulated in the arterial blood (Stanley et al., 1985). Thus, during a rampincremental exercise test arterial lactate concentration increases at a metabolic rate where blood lactate appearance exceeds clearance: this metabolic rate is termed the LT. Variables that influence the rate of lactate appearance during incremental exercise are many, include phosphofructokinase activity (related to flux through anaerobic glycolysis), the cytosolic redox potential (greater NADH/ NAD ratio favors lactate formation), the relative activity of lactate dehydrogenase (high activity favoring lactate formation), and pyruvate dehydrogenase (PDH) (high activity favoring oxidative metabolism), and intracellular PO2 (low PO2 favoring lactate formation). Each of the lactate-favoring pathways is more abundant in type II (fast glycolytic or fast oxidative-glycolytic) muscle fibers, which become increasingly activated as power increases during ramp-incremental exercise. Thus, when the rate of pyruvate formation (via anaerobic glycolysis) exceeds the rate of entry into the mitochondria for use in the tricarboxylic acid (TCA) cycle, the cytosolic redox balance is pushed toward a reduced state (i.e., increased NADH 1 H1), which drives the lactate dehydrogenase reaction to the right and reduces pyruvate to lactate, that is, elevating the cytosolic [lactate]/[pyruvate] ratio. The LT may be modulated by processes that affect myocyte lactate production, which include, for example, endurance exercise training. Mitochondrial expression and capitalization is increased in endurance trained muscle, ̇ 2 at which the LT occurs. Increasing increasing the VO PDH activity using dichloroacetate (DCA) infusion also

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reduces intracellular acidosis and blood lactate accumulation during exercise (Rossiter et al., 2003). Reducing cellular O2 delivery using carbon monoxide inhalation to increase carboxyhemoglobin concentration, reduces the ̇ 2 at which the LT occurs (Koike et al., 1990). The VO empirical observation that blood lactate accumulation increases dramatically and inexorably when muscle venous PO2 reaches B1520 mm Hg (Stringer et al., 1997) reinforces the close association between the matching of O2 delivery to utilization and lactate accumulation (Fig. 10.3). The other component of the process is lactate clearance. In the normal condition, lactate clearance occurs via extracellular cotransport of lactate and a proton (H1) via a monocarboxylate transporter from the muscle cytosolic compartment to the interstitium, and is taken up for oxidation in other tissues. Oxidation may occur in adjacent fibers that have low lactate concentration (typically high-oxidative, type I fibers), cardiac muscle, or other tissues with low [L2] such as liver, kidney, or brain. The metabolic acidosis that occurs as lactate accumulates in the muscle and the blood plays a vital role in the ability to continue to extract O2 from the blood into the myocyte. The acidosis alters the affinity of O2 binding to hemoglobin (the Bohr effect), and facilitates additional O2 unloading into the active muscle, despite capillary PO2 remaining relatively constant (Fig. 10.3). The importance of this is acutely demonstrated in patients with McArdle’s disease, an autosomal recessive genetic condition affecting the enzyme glycogen phosphorylase (Riley et al., 2017). Without the ability to convert breakdown of glycogen to glucose for use in anaerobic glycolysis, lactate does not accumulate during exercise and intolerance occurs at low power outputs with relatively low O2 extraction.

10.2.2.2 The “V-Slope” Relationship The muscular and systemic metabolic effects described earlier that result in an acidosis during ramp-incremental exercise can subsequently be detected at the lung using noninvasive gas exchange and ventilatory measurements. A metabolic acidosis is buffered in the muscle by potassium bicarbonate and in the blood by sodium bicarbonate, to release nonmetabolic CO2. This nonmetabolic CO2 supplements the CO2 produced in the TCA cycle that occurs as oxidative phosphorylation increases. Thus, ̇ 2 and VCO ̇ 2 increase in approximate probelow LT, VO portion (approximately linearly), whereas once LT is ̇ 2 increases in excess of VO ̇ 2 by a rate that exceeded, VCO is very closely associated with the rate of [L2] and [H1] accumulation and [HCO32] diminution, in the blood. This provides the underlying basis of its noninvasive

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FIGURE 10.3 (A) Femoral vein lactate concentration as function of femoral vein PO2 during constant power cycling exercise above LT. Before exercise onset, femoral vein PO2 is high and lactate is low. At the onset of constant power exercise above LT PO2 falls until B1520 mm Hg, after which PO2 approximately stabilizes and femoral vein lactate increases dramatically. (B) Femoral vein constant power exercise above LT plotted against femoral vein hemoglobin saturation and superimposed on the calculated HbO2 dissociation curve for normal pH (7.4) and the curves expected during progressive acidosis (down to pH 7.0). PO2 and HbO2 saturation falls until B20 mm Hg without a change in pH. The onset of a metabolic acidosis is closely coincident with a femoral vein PO2 of B20 mm Hg and, after this, continued HbO2 desaturation occurs via a shift in the dissociation curve to the right (the Bohr effect). Based on Stringer, W., et al., 1994. J. Appl. Physiol. (1985) 76, 14621467 (Stringer et al., 1994).

estimation of LT via the “V-slope method” (Fig. 10.4; Beaver et al., 1986). To understand the V-slope, we will consider the processes influencing to the relative rates of production of CO2 and consumption of O2 in the muscle, and how the results of these processes are expressed at the lung. In newly activated muscle cells in ramp-incremental exercise, the instantaneous ratio of the muscle metabolic CO2 production to O2 consumption (the cellular respiratory quotient, RQ) is likely close to 1.0 during exercise below LT, as activation of glycogenolysis (RQ 5 1.0) is very rapid compared with β-oxidation (RQ 5 0.71). However, the pulmonary equivalent of this ratio, the ̇ 2/VO ̇ 2), is typically respiratory exchange ratio (RER, VCO B0.8 at rest and increases slowly as incremental exercise progresses. In some cases, RER even falls briefly during the first few minutes of exercise (Fig. 10.4). The pulmȯ 2 relative to VO ̇ 2, therefore, at the nary kinetics of VCO onset of ramp-incremental exercise are primarily the result of three events: (1) the instantaneous ratio of the muscle metabolic CO2 production to O2 consumption— termed the cellular RQ; (2) the buffering of CO2 concentration in the muscle and blood; (3) the flow-weighted mixing of blood draining the active muscle with blood draining the tissues from the rest of the body. As discussed, the RQ of individually active muscles cells at exercise onset is likely close to 1.0, meaning that CO2 production equals O2 consumption. However, CO2 concentration is buffered in the muscle cell: increased CO2

production promotes the accumulation of K1HCO32. Another source of intramuscular CO2 buffering occurs in response to PCr breakdown (Wasserman et al., 1997): PCr 1 aH1 -Cr 1 Pi The breakdown of PCr is most rapid during the first 3 min of exercise (Cannon et al., 2013). The overall reaction (termed the Lohmann reaction) results in an acute intracellular metabolic alkalosis, during the time in which hydrogen ions are taken up in the formation of creatine (Cr) and inorganic phosphate (Pi) from PCr (Fig. 10.5). This causes K1 to leave the muscle cell and H1 to enter in response to an intracellular cation shortage. Thus, metabolic CO2, when hydrated, becomes H2CO3 and dissociates to H1 and HCO32, “trapping” CO2 as intracellular bicarbonate. This process effectively and transiently reduces cellular CO2 output relative to O2 consumption, and ultimately contributes to slowing the increase in pul̇ 2 relative to VO ̇ 2. This is one mechanism by monary VCO which the early kinetics of the V-slope appears shallow ̇ 2 in Fig. 10.4). (see the values close to 1 L
min21 VO Another mechanism that contributes to the early kinetics of the V-slope relationship is the flow-weighted mixing of blood draining the active muscles and the rest of the body. It is well known that muscle activation increases as power output increases during rampincremental exercise. We also proposed that activated muscle has an initial RQ close to 1.0, and that for a typical subject with a Western diet, the resting RER is B0.8.

The Coupling of Internal and External Gas Exchange During Exercise Chapter | 10

223

FIGURE 10.5 Intramuscular pH, measured by magnetic resonance spectroscopy, during 15 s of maximal-effort “sprint” exercise and recovery. Note that during the sprint exercise there is a dramatic intracellular alkalosis as H1 is sequestered during PCr breakdown. During recovery the opposite effect is seen, with an acidosis occurring as PCr is resynthesized. Data from Rossiter, H.B., et al., 2002a. Magn. Reson. Mater. Biol. Phys. Med. 14, 175176.

FIGURE 10.4 Noninvasive estimation of the LT (indicated by vertical line) using pulmonary gas exchange and ventilatory variables, as indicated ̇ 2 relative to VO ̇ 2 (i.e., V-slope method; by a disproportionate increase in VCO upper panel) and also to the corroborating indices of the end-tidal fractional concentrations (FET), ventilatory equivalents of CO2 and O2 and RER (lower panels). RER is included to aid in ruling out a specific hyperventilation as ̇ 2. From Rossiter, H.B., 2011. Compr. the cause of increased VCO Physiol. 1, 203244 with permission. Copyright r 2011 American Physiological Society. All rights reserved.

Therefore, blood draining active muscle regions with an RQ close to 1.0 (reduced slightly by transient CO2 buffering) will be mixed in the central circulation with blood draining inactive muscles, and other bodily organs that have an RQ B0.8. The result is a flow-weighted mixed venous blood with an RQ that slowly rises from B0.8 toward 1.0 as the muscles are activated. Therefore, as incremental exercise activates muscle progressively, the pulmonary RER increases toward 1.0 as the mixed venous blood receives a greater and greater contribution from effluent draining active muscles (see the values between ̇ 2 in Fig. 10.4). B1.3 and 2.0 L
min21 VO Overall, therefore, the sub-LT V-slope kinetics are characterized by a kinetic phase during the first B23 min where CO2 accumulation is transiently buffered in the muscle and the blood, followed by an approximately linear increase, which is largely due to a linear increase in muscle activation in response to the incremental power demands of the task. The sub-LT portion of the V-slope kinetics is termed the S1 slope, with the S2 slope being the supra-LT portion. The S1 slope typically averages B0.95 in healthy humans, and is influenced by muscle metabolism. For example, prior glycogen depletion will increase the intramuscular reliance on β-oxidation and reduces the S1 slope (Cooper et al., 1992). A low S1 slope can also result from prior hyperventilation, due to increased rate of CO2 storage early in the ramp (replenishing bodily HCO32 stores): an effect that can undermine the accurate detection of LT (Ozcelik et al., 1999). At LT, the V-slope abruptly increases. The increased CO2 flow from the tissues toward the lung, and resulting

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̇ 2, that occurs at LT is closely matched by an increase VCO ̇ 2 (termed in ventilation (VĖ ), such that the ratio VĖ /VCO the ventilatory equivalent for CO2) does not increase until after B12 min above LT (during a standard rampincremental protocol). Because VĖ is well matched to ̇ 2 during this phase, the mean alveolar PCO2 remains VCO approximately constant (inferred from end-tidal PCO2; PETCO2). This feature, called the “isocapnic buffering” phase, provides the opportunity to use pulmonary measurements to distinguish LT from other potential causes of increased pulmonary CO2 output such as hyperventilation or abruptly reduced work efficiency. During this phase, because VĖ more closely follows the increased CO2 output than O2 uptake, the ventilatory equivalent for ̇ 2) begins to rise, and the PETO2 begins to fall, O2 (VĖ /VO while PETCO2 remains constant (Fig. 10.4). This provides the necessary information to rule out hyperventilation and reduced work efficiency as the causes of the increase in ̇ 2 relative to VO ̇ 2, and discern LT noninvasively. VCO The mechanism underlying the isocapnic buffering period is still unclear, but it may result from the relatively slow adjustment to a new onset acidosis of the carotid bodies, whose neural discharge is stimulated by [H1] (among other variables) (Buckler et al., 1991). The brief delay before the metabolic acidosis stimulates carotid body output, and drives ventilation in excess of that required to maintain arterial PCO2, defines the duration of isocapnic buffering period. It is worth mentioning here that a higher rate of lactate accumulation, that is, in response a ramp-incremental where power is incremented rapidly versus slowly, will shorten the isocapnic buffering period and may make LT more difficult to corroborate. The opposite effect, that is, in response to a slow rampincremental, will lengthen the isocapnic buffering period but will also slow the rate of lactate accumulation, and therefore additional CO2 output is also much lower; this can also make the LT difficult to discern using pulmonary measurements. These are among the reasons why a 10min ramp-incremental exercise test duration is considered optimal. Following isocapnic buffering, VĖ increases out of ̇ 2 and VCO ̇ 2 and causes PETO2 to proportion to both VO rise and PETCO2 to fall (Fig. 10.4). This “compensatory hyperventilation” acts to help buffer the metabolic acidosis by reducing arterial PCO2 (“blowing off” CO2) and promoting the conversion of H1 and HCO32 to H2CO3, which rapidly dissociates to CO2 and H2O. Overall, the S2 slope of the V-slope relationship is .1.0, but the magnitude is particularly sensitive to the rate of [L2] and [H1] accumulation; and therefore particularly sensitive to the ramp rate. A high rate of power increase during ramp-incremental exercise will increase the S2 slope, supporting the notion that it reflects “excess” CO2 derived from bicarbonate buffering of the metabolic

acidosis. Therefore, the S2 slope can range between B1.2 and 2.0 depending on the exercise protocol used (Cooper et al., 1992). The ability to hyperventilate above LT, and elicit a respiratory compensation for the metabolic acidosis, is associated with exercise tolerance during incremental exercise. For example, a metabolic acidosis may increase more rapidly in patients with lung disease where ventilatory limits are attained shortly after LT as compared to those who are able to compensate with a hyperventilation. Also, well-trained athletes, in whom gas exchange limitations are evident at very high cardiac outputs (as measured by an exercise-induced arterial hypoxemia) also have less of a reduction in arterial PCO2 during exercise above LT than those without gas exchange limitation (Dempsey and Wagner, 1999).

_ 2max ) 10.2.2.3 Maximum Oxygen Uptake (VO

_ 2max ) The maximum rate of O2 delivery and utilization (VO is considered a fundamental measure of human endurance ̇ 2 achieved by an individual performance. The greatest VO during a symptom limited ramp-incremental exercise test _ 2peak ) provides a global assessment of car(termed the VO diopulmonary and neuromuscular functioning. The dis_ 2peak and VO _ 2max in this context is not tinction between VO _ 2max requires trivial. The gold standard definition of VO that an increase in power output is accompanied by no ̇ 2 (Hill and Lupton, 1923; Hill and further increases in VO Lupton, 1924). The ramp-incremental is therefore often ̇ 2 prior to the limit of expected to elicit a “plateau” in VO tolerance, where exercise demands continue to increase ̇ 2 reaching its upper limit. However, the incidespite VO ̇ 2-to-power output relationdence of a plateau in the VO ship is actually rare in healthy subjects (estimates suggest as little as one-third; Howley et al., 1995; Day et al., 2003), while the incidence in patients with disease, where limiting pathological symptoms may prevent a plateau in ̇ 2, appears to be even less than in healthy subjects VO (Piepoli et al., 2006; Mezzani et al., 2009). When voluṅ 2, the tarily is terminated without an overt plateau in VO _ _ ̇ 2 term VO2peak is used. VO2peak is the greatest value of VO achieved in the test, and which may or may not represent the “true” maximum rate of O2 delivery and utilization. However, without meeting the traditional criterion (no ̇ 2 with an increase in power), one cannot increase in VO know with any certainty whether or not an individual has _ 2max. attained their “true” VO _ 2max can be confirmed with a verification phase, or VO during additional testing (MacDougall et al., 1991; Day et al., 2003; Midgley et al., 2006; Rossiter et al., 2006). Typically a verification is done using a second maximaleffort test within B5 min of the initial ramp test, using a different power output to the previously achieved peak

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mortality in both health and disease. The increased risk _ 2peak is greater than other comfor mortality of a low VO mon known risk factors such as smoking, obesity, high blood pressure, or preexisting pathologies such as heart failure, diabetes, or chronic obstructive pulmonary disease (COPD) (Kokkinos and Myers, 2010).

10.2.2.4 Determinants of Maximum Oxygen _ 2max ) Uptake (VO The Fick principle provides a quantitative construct to _ 2max. Applying the explore the question of what limits VO Fick principle to gas exchange across the lung and subsequently across the heart yields: ̇ 2 profiles for a single subject during the rampFIGURE 10.6 VO incremental exercise followed by a step change in power to 105% of the peak incremental power. Dashed vertical lines show the start and end of the ramp and constant power components of each protocol. The identical ̇ 2 value elicited at two different power outputs defines the VO _ 2max. VO From Rossiter, H.B., et al., 2006. J. Appl. Physiol. 100, 764770 with permission.

(Fig. 10.6) (Rossiter et al., 2006; Poole and Jones, 2017). _ 2peak is produced by two different power If the same VO _ 2max is confirmed. outputs, then the VO _ 2max in a We believe it is reasonable to use the term VO task-specific context. For example, in the same subject, ̇ 2 achieved during treadmill running is typthe greatest VO ically B5% greater than during cycling; even when subjects are well trained in both activities (e.g., triathletes). The reason for this presumably resides in the ability to engage a greater muscle mass during running than cycling (Calbet et al., 2009). The relative intramuscular pressures and duty cycles generated during each activity also differ, which may contribute to differences in blood flow and distribution of regional muscle O2 delivery between the two activities that favor running. Therefore, while the _ 2max may be closer to that seen during running “true” VO than cycling, is seems reasonable to describe the greatest ̇ 2 achievable during cycling as a task-specific verified VO _ 2max . VO _ 2max represents the most common objective The VO measurement of exercise tolerance in health and disease and is frequently used for an array of clinical assessments, such as providing insight into diagnosis and prognosis, the normalcy of physiological function, symptomatology, the efficacy of drug, device, surgical, or other interventions, and stratification for cardiac transplantation _ 2max may be a little as (Wasserman et al., 2011). VO 10 mL
kg21
min21 in a patient with chronic disease and as great as 85 mL
kg21
min21 in an elite endurance ath_ 2peak is strongly associated with increased lete. A low VO

_ 2 5 Q_ 3 Ca2v O2 V_ A 3 FI2A O2 5 VO where VȦ is the alveolar ventilation, Q ̇ is the cardiac output (all of which is directed to the lung), F is the fractional concentration of O2 in the gas phase, C is the concentration of O2 in the blood phase, and the subscripts I, A, a, and v indicate inspired, alveolar, arterial, and mixed venous, respectively. For simplicity, this equation ignores the effect of different inspired and expired gas volumes that occur when R 6¼ 1. During incremental exercise, FIAO2 undergoes only minimal change in healthy humans (in fact, FIAO2 decreases slightly as FAO2 tends _ 2max). CavO2 on the other to increase approaching VO hand, increases substantially on exercise as CvO2 falls ̇ 2. This approximately hyperbolically in relation to VO ̇ means that VA increases relatively more than Q ̇ during exercise, and that the overall pulmonary VȦ /Q ̇ increases. Teleologically, the apparent discrepancy in this ratio makes sense, as it costs the organism less energy to transport a gas (air) at high rates than a viscous fluid (blood). _ 2max durHowever, this sets the potential for Q ̇ to limit VO ̇ ing exercise as maximal Q is approached. Nevertheless, this pair of equations highlights that any step in the O2 transport pathway, from O2 in the atmosphere (e.g., that determines FIO2) to muscle mitochondrial O2 utilization (a major determinant of CvO2); steps including total and dead space ventilation, convective O2 delivery (including hemoglobin concentration, cardiac output, blood flow distribution), diffusive O2 delivery (including muscle capillarity and muscle diffusing capacity) and oxidative phosphorylation (muscle mitochondrial concentration and _ 2max . enzyme activity) each have the potential to limit VO _ 2max is still widely The source of the limitation to VO debated (Wagner, 2006; Saltin and Calbet, 2006b). Some _ 2max contest that, in health during exercise at sea level, VO is limited by the convective O2 delivery, specifically maximum Q ̇ and/or its distribution (Gonzalez-Alonso and Calbet, 2003; Mortensen et al., 2005; Saltin and Calbet, 2006a). Evidence in support of this notion includes that muscle mitochondrial oxidative capacity generally

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_ 2max (Rowell, 1986). exceeds that of O2 delivery at VO Experimental elevation of O2 delivery, for example under the interventions of hyperoxia (Knight et al., 1993) or blood infusion (Spriet et al., 1986), can result in a greater _ 2max being attained. Also, CavO2 can increase VO _ 2max when leg O2 delivery (Q ̇ 3 CaO2) approaching VO actually begins to fall (Mortensen et al., 2005; Mortensen et al., 2008). Variables downstream of convective O2 delivery, however, such as maximal O2 diffusional conductance in muscle and muscle mitochondrial oxidative capacity also _ 2max. Early evidence supporthave the potential to limit VO _ 2max include the ing the role of the muscle in limiting VO elegant structural and functional studies of Hoppeler et al. (1985), who showed that endurance exercise training results in increased mitochondrial concentration and capillarization, and that these were linearly correlated with the _ 2max: r 5 0.64 with mitochonincrease in whole-body VO drial volume density and r 5 0.76 with capillary-to-fiber ratio (Hoppeler et al., 1985). This suggests muscle variables downstream of convective O2 delivery may also _ 2max. play a pivotal role in determining VO Peter Wagner and colleagues (Roca et al., 1989; Hogan et al., 1991a,b; Roca et al., 1992) added weight to the notion that the periphery plays an important role in _ 2max through investigation of the relative determining VO contribution of each of the primary physiological variables involved in O2 transport and utilization. To achieve _ 2max must lie at the intersecthis Wagner reasoned that VO tion between convective O2 delivery (Fick principle) and diffusive O2 conductance (Fick’s law) (Wagner, 1996). Fick’s law of diffusion states:   _ 2 5 Dm O2 3 PO2cap  PO2mito VO where DmO2 is the muscle diffusion coefficient, multiplied by a PO2 that is determined by the difference between the mean PO2 in the muscle capillary and the _ 2max, in all participants mitochondrion. Approaching VO PO2mito regresses toward a minimum value that is close to (although not identical to) zero. This means that at _ 2max , diffusive O2 conductance can be reasonably charVO acterized by a straight line. This linear function intersects with the curve describing convective O2 delivery to deter_ 2max (Fig. 10.7). By comparing soleus muscle mine the VO and peroneal muscles in rats Behnke et al. (2003) showed _ 2max is achieved by different conthat muscle-specific VO tributions of convective and diffusive O2 transport in each muscle (Fig. 10.7). Computational modeling performed to _ 2max of increases in one variinvestigate the effect on VO able or another involved in this relationship, demonstrated that DmO2 was the most sensitive single variable to _ 2max (Wagner, 2000). In other words, a 40% increase VO increase in DmO2 alone will have a greater influence on

FIGURE 10.7 Relationship between muscle PO2 (PO2m ) and muscle ̇ 2. The interaction of both convective (curve; defined by the Fick prinVO ciple) and diffusive (linear; defined by Fick’s law of diffusion) O2 trans_ 2max (the point of which port are suggested to conflate to determine the VO is where the two lines intersect). Here, this concept is highlighted by modeling data between microvascular PO2 and muscle V_ O2 , as collected during steady state (SS) contractions in rat skeletal muscle during direct stimulations in fibers of contrasting isoforms: slow-twitch type I soleus (sol) versus fast twitch type II peroneal (Per). Diffusive conductance is represented by the slope of the diagonal line, while convective delivery is reflected by the increase in the curved line—both of which can be seen to be higher in the soleus muscle. SS-contractions, Steady-state contracting values. From Behnke, B.J., et al., 2003. J. Physiol. 549, 597605 with permission.

_ 2max than the same 40% increase in Q ̇ alone (or any VO other variable). These authors are quick to note, however, that the starting conditions matter. That is, deconditioned or sedentary individuals will respond to endurance exercise training through large increases in convective and diffusive O2 transport as well as muscle oxidative capacity. However, in these detrained individuals the large training-induced increases in DmO2 and muscle oxidative capacity are likely more important than the accompanying increase in Q.̇ In active healthy subjects however, where peripheral muscles are already well adapted prior to training, increases in convective O2 delivery likely provide the _ 2max primary limitation to the training response in VO (Roca et al., 1992).

10.2.3 Constant Power Exercise ̇ 2 Kinetics and VO _ 2max represents the most common assessment of While VO exercise tolerance in health and disease, it may not be the most applicable assessment to understand the bioenergetic limitations to the activities of daily living (Poole and Jones, 2012). The ability to perform the activities of daily living is dependent on the ability to meet the energy demands of the task in a steady state.

The Coupling of Internal and External Gas Exchange During Exercise Chapter | 10

III

1.25 VO2 (L min–1)

A system in a steady state remains constant over time, but requires continual energy expenditure to maintain it. This condition is also referred to as a “dynamic equilibrium”. Were the energetic processes to cease that acted to maintain the system in steady state, the system would lose energy and regress toward equilibrium. Therefore, a system at equilibrium is stable over time, but no energy is required to maintain that condition. In our context of human exercise, therefore, equilibrium equates to death. A system in a steady state has a higher energetic state than its surroundings. Non-steady-state physiology during constant power exercise therefore reflects progressively increasing energy requirements to maintain system integrity (or “metabolic stability”): this is an unsustainable condition that either must resolve or will continue to challenge stability. The threshold that separates steady-state from non-steady-state physiology during exercise is the CP. CP is commonly measured as the asymptote of the hyperbolic relationship between power and tolerable duration (Fig. 10.2). While this measurement is made in the domain of power output (with units of watts), it actually reflects a metabolic rate that can be expressed in terms of ̇ 2 (Barker et al., 2006). VO It is worth noting here that, were work efficiency and economy to be invariant, exercising under constant conditions, power and metabolic rate would be linearly related. However, work efficiency is reduced above LT by an amount that varies among individuals. Therefore, while the upper limit to a physiological steady state during exercise is an individually determined metabolic rate, this metabolism can be used to perform a range of power outputs depending on the efficiency and economy of the exercise. Thus, strictly, CP should be expressed as a metabolic rate, despite the far-simpler measurement of external power being more typically used (Winter et al., 2016). CP is the greatest metabolic rate for which intramuscular metabolism, pulmonary gas exchange, ventilation, and HR can achieve a steady state (Poole et al., 1988; Jones et al., 2008). The speed with which the body is able to recruit these process to supply intramuscular ATP via ̇ 2 kinetics, is an oxidative phosphorylation, termed VO important determinant of the ability to meet the energy demands of the task in a steady state (Murgatroyd et al., ̇ 2 kinetics are most commonly assessed by mea2011). VO _ 2p on transition to a constant power exercise (a suring VO step increase in power from a resting or low power output to a higher power output). This measurement provides a systemic assessment of the effective functioning and integration of the pulmonary, cardiovascular, and neuromuscular systems (Fig. 10.1). To better understand the mechanisms separating exercise that can be met in a steady state from and nonsteady state, we will discuss the ̇ 2 kinetic responses in the four intensity features of the VO domains: moderate, heavy, very-heavy, and severe.

.

227

II

1.00

τ

I

0.75 0.50 0.25 0 –100 –50

0

50

100

150 200 Time (s)

250

300

350

̇ 2 from breath-by-breath FIGURE 10.8 The three-phase response of VO pulmonary gas exchange measurements on transition from rest to a moderate-intensity square wave exercise bout in a healthy individual. ̇ 2 increases with an expoFollowing an initial abrupt increase (phase I), VO nential time course (phase II) characterized by a time constant (τ) where values of B2030 s are typical of young healthy adults. After B4 time constants have passed, a steady state is attained (phase III). Modified from Rossiter, H.B., et al., 1999. J. Physiol. 518, 921932 with permission.

_ 2p Kinetics 10.2.3.1 Moderate-Intensity VO

_ 2p manifest three distinct Below LT, the kinetics of VO phases (Whipp et al., 1982) (Fig. 10.8). The initial rapid _ 2p (phase I) is followed by a slower, expoincrease in VO nential increase (phase II) before a steady state (phase III) is attained typically in B23 min for healthy humans (Wasserman and Whipp, 1975). Phase I is characterized _ 2p at exercise onset by an initial and rapid increase in VO considered “cardiodynamic” in nature, in the sense that its origin is primarily mediated by an abrupt increase in pulmonary perfusion (Krogh and Lindhard, 1913; Whipp et al., 1982). Once again, consideration of the Fick princi_ 2p 5 Q ̇ 3 CavO2) helps us to understand the ple (VO _ 2p at exercise onset. response of VO Phase I is, therefore, the duration before which large changes in CavO2 are observed at the pulmonary capillary. This delay is due to the intervening venous blood capacitance that resides between the increased O2 extraction at the muscle capillarymyocyte interface at exercise onset, and the pulmonary capillary. The time it takes for this blood to circulate determines the duration of phase I. Therefore, with CavO2 relatively constant during phase I, _ 2p is determined predominantly by the the increase in VO ̇ increase in Q. Q ̇ increases abruptly at exercise onset by rapid parasympathetic withdrawal and also by increased venous return (due to muscle pump), which each contribute to propelling the mixed venous blood through the lungs. Hence, the magnitude of phase I is largely proportional to changes in pulmonary blood flow (Cummin et al., 1986). The effect of this is seen dramatically in patients with pulmonary vascular disease who are unable to abruptly increase pulmonary blood flow, and _ 2p is reduced by B70% comthe increase in phase I VO pared to normal subjects (Sietsema et al., 1986).

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Phase I is followed by the phase II, also termed the _ 2p kinetics (Rossiter “fundamental” component of VO _ et al., 2002c). In phase II, VO2p increases with an approximate mono-exponential time course characterized by a time constant (τ), a parameter that describes the time _ 2p to reach 63% of the final steady-state taken for VO amplitude (Fig. 10.8). This exponential can be characterized as: _ 2 ðtÞ 5 ΔVO _ 2ss 3 ð1  eðtTDÞ= τÞ ΔVO _ 2p at any time, ̇ 2(t) is the increase in VO where ΔVO _ _ ΔVO2ss is the increase in VO2p from the preexercise baseline to the steady state, τ is the time constant of the exponential, and TD is the time delay between the start of exercise and the intersection of the exponential with the _ 2p . baseline VO _ 2p repreThe start of this exponential increase in VO sents the arrival of mixed venous blood at the lung, which has undergone O2 extraction in the active muscle. _ 2p is characterized by the product Therefore, phase II VO of an increasing CavO2 (due to, in healthy subjects, a fall in mixed venous O2 concentration with no change in arterial O2 concentration) and increasing Q.̇ The degree to _ 2p kinetics reflects solely the kinetics which phase II VO ̇ 2, and can therefore be used to proof intramuscular VO vide a noninvasive window to investigate the control mechanisms of oxidative phosphorylation, will be addressed in more detail in the next section of this chap_ 2p kinetics have ter. Suffice it to say, that phase II VO _ 2p kinetics literature been the primary focus of the VO because they are thought to share a close homology with events occurring in the active muscles. Any process that influences the rate of change of pulmonary blood flow and/or CavO2 at exercise onset has the potential to affect _ 2p kinetics. Phase II VO _ 2p kinetics are fast in phase II VO children and endurance trained individuals (where blood flow kinetics are fast and muscle oxidative capacity and capillarity are high) and are progressively slowed with sedentary behavior, aging, and chronic heart, renal, or lung disease (Poole et al., 2005). _ 2p response is followed by phase Phase II of the VO III, which, in moderate-intensity exercise below LT, marks the attainment of a steady state. The amplitude of _ 2p is determined by the the steady state increase in VO functional gain and the power output that, for moderateintensity cycle exercise in healthy subjects, averages B10 mL
W21
min21 (Whipp and Wasserman, 1972; Wasserman and Whipp, 1975). Because the functional gain is invariant among subjects varying in sex, state of training, or state of health (although it has been suggested to be reduced by some B5%20% following nutritional interventions (Larsen et al., 2007; Bailey et al., 2009) and

resistance exercise training (Zoladz et al., 2012)), the _ 2p τ is the primary determinant of the requirephase II VO ment for substrate level phosphorylation and utilization of stored O2 at exercise onset. To put it another way, the _ 2p increase, for any given increase in power kinetics of VO output, determines the magnitude of the O2 deficit (O2D ); that is, the O2 equivalent of the energy transfer of exercise that is not provided from oxidative phosphorylation using O2 acutely transported from the atmosphere: _ 2ss 3 τ O2D 5 ΔVO The sources of the O2D are PCr, O2 associated with hemoglobin, myoglobin or dissolved in muscle, and glycogenolysis accumulating lactate. The capacitance of PCr and stored O2 is limited (Table 10.1); therefore a larger τ will result in more rapid utilization of these finite energy stores and reduced metabolic stability. Similarly, a larger τ will promote a greater reliance on transient lactate accumulation, even during moderate exercise below LT (Cerretelli et al., 1979).

10.2.3.2 Heavy, Very-Heavy, and Severe_ 2p Kinetics Intensity VO

_ 2p During heavy and very-heavy-intensity exercise the VO slow component supplements the moderate-intensity kinet_ 2p slow ics, resulting in a loss of work efficiency. The VO component draws the functional gain for cycling up to as much as B14.5 mL
min21
W21 during very-heavyintensity exercise, compared with B10 mL
min21
W21 in the same individuals below LT (Poole et al., 1994; Whipp, 1994). The difference between heavy and very-heavy_ 2p kinetics is that exercise initiated at a metaintensity VO bolic rate that lies between LT and CP results in a steady state, albeit delayed by up to B15 min, whereas during _ 2max, VO _ 2p continues exercise initiated between CP and VO to increase for as long as the supra-CP exercise task is sustained. _ 2p slow component is still debated The origin of the VO (Poole and Jones, 2012). Early suggestions implicated the energetic cost associated with variables such as increased cardiac and ventilatory work required to maintain supraLT exercise, the additional O2 cost of oxidative lactate clearance, and/or the result of increased body temperature. However, more recent evidence shows that B85% of the _ 2p slow component during cycling or leg exercise VO derives from the active leg muscles (Grassi et al., 1996; Koga et al., 2005; Krustrup et al., 2009; Jones et al., 2011). The causes of the progressive increase energy requirement of constant power exercise above LT effect has been linked to the fatigue process within the active

The Coupling of Internal and External Gas Exchange During Exercise Chapter | 10

muscles themselves (Cannon et al., 2011; Murgatroyd and Wylde, 2011; Grassi et al., 2015; Keir et al., 2016). As previously discussed, high-intensity exercise imposes an intramuscular bioenergetic strain that increases the reliance on substrate level phosphorylation and causes accumulation of metabolites associated with fatigue (Pi, H1, ADP, extracellular K1). This fatigue necessitates increased muscle activation to maintain the constant power output demanded by the task. During exercise initiated at a metabolic rate that lies between LT and CP, the increase in muscle recruitment, and therefore metabolic rate, can stabilize due to, at least, the sufficiency of extracellular lactate and H1 transport and clearance allowing the intramuscular environment to achieve a steady state. By meeting the energy demands of the task through wholly aerobic metabolism, muscle activation, and metabolism stabilize. We define “wholly aerobic” here in a very specific way, as a whole-body event. Although intramuscular ATP requirement is such that lactate is produced and accumulated, its extracellular transport and clearance can match the rate of production (albeit with an elevated muscular, venous, and arterial concentration) and therefore maintain a wholly aerobic state on a whole organism basis, even if individual muscle cells require ongoing supplemental bioenergetics contributions from substrate level phosphorylation. Exercise above CP differs in that muscle activation and metabolism cannot stabilize. The bioenergetic strain of the exercise is sufficiently large such that metabolic stability is lost. This implies that the ATP demands of the task place too great a reliance on substrate level phosphorylation, such that muscle fatigue and progressive muscle recruitment is obligatory. Indeed, as previously discussed, for any given power output the magnitude of substrate level phosphorylation will be proportional to _ 2m. Therefore, τ is proposed to be a strong determiτ VO nant of CP: a large τ (slow kinetics) is related to a low CP. To this end, Murgatroyd et al. (2011) demonstrated a very strong negative association between CP and phase II _ 2p in 14 healthy men, using a cycling task that caused τ VO intolerance in 6 min (Fig. 10.9). This relationship was _ 2p (using maintained during an intervention to alter τ VO priming exercise), suggesting a mechanistic link between _ 2p and CP (Goulding et al., 2017). These findings are τ VO consistent with the hypothesis that accumulation of high rates of substrate level phosphorylation (a large oxygen deficit) lowers the power output that can be maintained in a steady state—the upper limit for heavy-intensity exercise. The precise mechanism(s) by which exercise efficiency is progressively reduced during very-heavyintensity exercise are still debated. The recruitment of poorly efficient muscle fibers (such as poorly oxidative or

229

̇ 2 time FIGURE 10.9 Linear regression between CP and phase II VO constant (τ) for supra-CP exercise that causes intolerance in 6 min. From Murgatroyd, S.R., et al., 2011. J. Appl. Physiol. 110, 15981606 with permission.

type II muscle fibers that were thought to require an increased P/W) to sustain the necessary power output (Krustrup et al., 2004a) is commonly implicated. Consistent with this, selective depletion of muscle glycogen or neuromuscular blockade in slow-twitch fibers in humans, exacerbates fast twitch fiber recruitment and _ 2p (Krustrup et al., 2004b, 2008). However, in single VO muscle fibers from the frog, it appears that the highly efficient oxidative fibers are the ones that demonstrate a progressively greater O2 consumption during fatigue, that is, a reduced P/O (Hepple et al., 2010). In humans, magnetic resonance data originally implicated an increased ATP cost of force production (increased P/W) in the active ̇ 2 slow component muscles as the cause of the VO (Rossiter et al., 2002a), although later work added that reduced P/O may also contribute (Cannon et al., 2014). _ 2p slow compoThe finding that, in dog muscle, the VO nent is present even in the absence of the progressive recruitment observed during supra-CP exercise in humans, implies that fatiguing fibers themselves contribute to the inefficiency and it may not only be caused by new recruitment of inefficient fibers (Zoladz et al., 2008). Finally, Grassi et al. (2015) implicated that the SERCA pump system may be more susceptible to the effects of reduced myocyte energetic status than the cross-bridge, meaning that a fall in P/W of the most energetically active cells would be the likely site of the fatigue-associated inefficiency of heavy and very-heavy-intensity exercise. It is worth noting here that although B85% of the _ 2p slow component derives from the large active VO muscles engaged in generating the external locomotor power, the remainder likely comes from the progressive increase in respiratory and cardiac muscle work (among others) required during very-heavy-intensity exercise

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(Dominelli et al., 2015). In addition, because very-heavyintensity exercise is associated with the attainment of very high fractions of maximal cardiac output, increasing _ 2p slow respiratory muscle work may exaggerate the VO component via two mechanisms: (1) direct increase in _ 2p from increased respiratory muscle work; and (2) an VO indirect effect from a reduction in leg muscle blood flow and increased leg muscle fatigue via competition among vascular beds for perfusion—“the respiratory muscle steal effect” (Harms et al., 1997). In support of this latter notion, Cross et al. (2010) showed that reducing respiratory muscle work using heliox breathing (79% helium, 21% O2) during very-heavy-intensity cycling in healthy _ 2p slow component to about half subjects reduced the VO of its normal amplitude. Severe-intensity exercise is distinguished from veryheavy-intensity exercise on the basis that the phase II _ 2max from exercise onset (Whipp, asymptote is above VO _ 2p slow com1996). During severe-intensity exercise a VO _ ponent cannot be observed because VO2max is reached prior to its expression. Likewise, arterial [L2] and [H1] accumulate progressively and rapidly in severe-intensity exercise, which, if continued, is terminated with the _ 2max after 23 min. attainment of VO

10.3 PHYSIOLOGICAL MECHANISMS DISSOCIATING THE LUNG AND MUSCLE GAS EXCHANGE _ 2p ) kinetics are often ̇ 2 (VO Because pulmonary VO _ 2m ), ̇ 2 (VO assumed to represent the kinetics of muscle VO and are therefore often used as a noninvasive window to investigate the control mechanisms of oxidative phosphorylation, it is important to be aware of the situations that ̇ 2 kinetics are likely to result in pulmonary and muscle VO being similar, when they are likely to be different, and the mechanisms that dissociate the muscle gas exchange from the lung. We have already presented mechanisms contributing to dissociating CO2 production in the active muscles ̇ 2 at the lung. In this section we discuss three from VCO mechanisms responsible for dissociating muscle and pul̇ 2 kinetics (O2 stores, transit delays, and flowmonary VO weighted mixing) and the implications of kinetic dissocia_ 2p kinetics as a surrotion for those using pulmonary VO _ 2m kinetics. gate for muscle VO

10.3.1 Oxygen Stores Any change in the volumes of O2 stored in the lung during the exercise transient has the potential to dissociate pulmonary gas exchange measurements made at the mouth from the kinetics of alveolar gas exchange (and _ 2m ). Such changes occur therefore from the kinetics of VO

via changes in the end-expiratory lung volume (depending on the way the particular gas exchange algorithm is programmed) or mixed-expired PO2. End-expiratory lung volume typically falls abruptly at the start of moderate exercise in healthy subjects (Wust et al., 2008). Overall, mixed-expired PO2 is closely related to mixed-expired PCO2, which itself is tightly regulated via chemoreception of arterial PCO2. While gross changes in mixedexpired PO2 generally are not seen in moderate exercise, minor changes may occur among individuals as pulmonary vascular pressure increases causing perfusion of previously poorly perfused (typically apical) lung regions and an improved matching of ventilation to perfusion. In heavy to severe-intensity exercise large changes in endexpiratory lung volume and mixed-expired PO2 are ̇ 2 observed that will contribute to dissociating alveolar VO _ from VO2p if they are not accounted for. While algorithms exist to account for dynamic changes in pulmonary O2 _ 2p stores and calculate alveolar O2 uptake from VO (Beaver et al., 1981; Capelli et al., 2011; Cettolo and Francescato, 2018), the techniques used are far from standardized—as discussed elsewhere by Rossiter (2011). It has been suggested that the dissociation between _ 2p kinetics is likely to be small in healthy alveolar and VO humans (Beaver et al., 1981) because overall changes in pulmonary O2 storage is both minor and has relatively abrupt kinetics (Wust et al., 2008). This mechanism of kinetic dissociation may, however, be enhanced in the elderly (e.g., Taylor and Johnson, 2010) and significant in patients with COPD (e.g., O’Donnell et al. (1998)), where progressive increases in end-expiratory lung volume can _ 2p result in increased pulmonary O2 storage, slowing VO _ kinetics relative to VO2m (Nery et al., 1982; PuenteMaestu et al., 2001). The reduction in O2 stores within the muscle and venous blood during exercise tends to be of a much greater magnitude than changes in lung O2 stores. This _ 2p kinetics must be slowed has led some to suggest that VO _ 2m. This is because utilization by muscle of relative to VO O2 stored within the body, which is manifest as reduced muscle O2 concentration and CvO2, means that not all O2 consumed by the active tissues during the kinetic transient is actually reflected in the measured alveolar gas exchange (Cerretelli and di Prampero, 1987; Lador et al., _ 2p should increase more slowly than VO _ 2m. 2006): thus VO However, under normal conditions in healthy humans the kinetics of Q ̇ act to negate this slowing effect. The effects that Q ̇ kinetics, and its distribution, has on the venous transit delay and the flow-weighted admixture of venous blood draining different vascular beds, means that _ 2p can be slower, the same, or even faster than VO _ 2m VO kinetics (Barstow and Mole, 1987; Barstow et al., 1990; Lai et al., 2009; Rossiter, 2011; Benson et al., 2013). We discuss this in more detail in the following sections.

The Coupling of Internal and External Gas Exchange During Exercise Chapter | 10

10.3.2 Transit Delay _ 2p is temporally dissociated from VO _ 2m in the order of VO some B1020 s (Krustrup et al., 2009), as the venous system and blood flow separating the active muscles from the lungs results in a venous transit delay corresponding to venous effluent leaving the exercising muscles and then arriving at the lungs some time later (Whipp et al., 1982; Rossiter, 2011). This venous transit delay, however, changes during exercise and can dissociate muscle and ̇ 2 kinetics (Barstow et al., 1990; Benson pulmonary VO et al., 2013). It is perhaps simplest to understand the effects of this venous transit delay on kinetic dissociation if one initially considers the hypothetical situation where Q ̇ and venous volume are constant throughout the exercise transient. In this situation the muscle-to-lung transit delay will also remain constant throughout the exercise transient: The venous effluent leaving the muscle at the start of exercise (t 5 0 s) may not reach the lung until 20 s later (i.e., a transit delay of 20 s), while the venous effluent leaving the muscle later in the exercise transient (say at t 5 60 s) will have the same 20 s transit delay (because venous blood flow and volume are constant), reaching the lung at t 5 80 s. This model predicts that the phase I duration of _ 2p, which reflects the initial muscle-to-lung transit VO delay time, will be 20 s, while the kinetics of phase II _ 2p will be identical to those of VO _ 2m . The only differVO _ 2p response will be simply shifted in ence is that the VO _ 2m, and will begin, in this examtime compared with VO ple, 20 s after exercise onset. However, Q,̇ and therefore venous blood flow, does not remain constant throughout the exercise transient, but increases to meet the increased metabolic demands of the exercising muscles (Shoemaker and Hughson, 1999). As _ 2m , this increase in Q ̇ is not instantaneous but with VO occurs in an exponential manner. These dynamics are sensitive to the prior condition of the subject: exercise initiated from rest causes a distinctly biphasic Q ̇ response (an initial abrupt increase followed by an exponential), whereas exercise from unloaded pedaling or a raised work rate results in Q ̇ response that is closer to a monoexponential. As such, in either case, the absolute rate of Q ̇ (in L
min21) is low at the start of the exercise transient, and becomes greater as exercise continues reaching a steady state only in moderate- and heavy-intensity exercise. Thus, because Q ̇ increases throughout the exercise transient, then the muscle-to-lung transit delay becomes progressively shorter as exercise continues (assuming again that venous volume remains constant). Using a similar example as used earlier, while the venous effluent leaving the muscle at the start of exercise (t 5 0 s) may have a time delay of B20 s before it reaches the lung, the venous effluent leaving the muscle later in the exercise

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transient (say at t 5 60 s) may have a much shorter transit delay of B10 s. This is because the increasing Q ̇ speeds blood flow through the venous system before a new steady state is reached. The effect of a constantly decreasing muscle-to-lung transit delay during the exercise transient, _ 2m at the lung (as phase is to distort the expression of VO _ _ 2p is not a simple II VO2p ). In this example therefore, VO _ facsimile of VO2m , shifted by a constant period of time, as it was with the example earlier where blood flow was _ 2p response is instead a constant. Here, the phase II VO time-dependent and nonlinear distortion of the mono_ 2m response, where the time shift exponential muscle VO becomes progressively shorter as the transient continues. The resultant nonlinear time shift causes the phase II _ 2p response to initially increase faster than the correVO _ 2m response, before slowing down as the sponding VO _ VO2p response approaches a steady state. Therefore, the _ 2p kinetics are not purely exponential, meaning that VO _ 2m kinetics by fitting an exponential function inferring VO _ 2p kinetics requires special considerations to phase II VO (Benson et al., 2013, 2017). Because of the nonlinear _ 2p will always have transit time distortion, the phase II VO _ 2m —that is to a different response kinetic to that of VO _ _ say, the kinetics of VO2m and VO2p are, by necessity, dissociated. The degree of this dissociation however, is variable but typically small (see later for more discussion); it _ 2p kinetics to be varies to a degree that allows phase II VO _ a useful surrogate for VO2m, at least in healthy humans (Benson et al., 2013). The overall effect of the transit _ 2m kinetics as they transit delay on its own is to speed VO _ 2p to the lung, counteracting the obligatory slowing of VO kinetics by the reduction in O2 stores as previously discussed. There is, however, a third important influence on the _ 2m to VO _ 2p, which is a result of the flowcoupling of VO weighted admixture of venous blood draining different vascular beds.

10.3.3 Flow-Weighted Venous Admixture Although the transit delay time is perhaps the principal ̇ 2 kinetic mechanism underlying muscle-pulmonary VO dissociation, flow-weighted mixing of venous effluent draining different muscle beds also exerts a significant influence. As with the transit delay distortion, this is a dynamic process, in that the mixed venous blood reaching the lungs contains venous effluent from different vascular beds in different ratios, but these ratios change with time during the exercise transient. Computational modeling (Barstow et al., 1990; Benson et al., 2013, 2017) has allowed us to gain insight into this phenomenon that would otherwise prove extremely difficult, if not impossible, to obtain experimentally. Consider the following

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scenario, taken from our modeling studies (Benson et al., 2013) but informed by experimental data (Grassi et al., 1996): At unloaded cycling, that is, immediately before the exercise transient, 57% of the total Q ̇ (derived from blood flow in measured in the femoral vein) flows to the lower limbs to perfuse the muscles involved with turning the cranks. The other 43% flows to all the remaining vascular beds in the body. Therefore, the mixed venous blood flowing back to the lungs contains a mixture of venous blood from “exercising” and “nonexercising” tissues in a 57:43 ratio. As soon as exercise starts (a 100 W, sub-LT constant power protocol in this case), Q ̇ begins to increase in an exponential manner: this increase in blood flow is almost completely directed to the lower limbs. In the study by Grassi et al. (1996) there is also a decrease in blood flow to the nonexercising tissues, with this fractional flow being directed instead to the lower limb along with the exercise-induced increase in Q.̇ This “macro” consideration of kinetic changes in regional blood flow distribution between exercising and nonexercising tissues is a gross simplification of blood flow control, but serves us well for understanding how blood flow distribution and venous admixing effects gas exchange dynamics between muscle and lung. In our example, by 30 s into exercise the fraction of Q ̇ directed to the lower limbs increased from 57% to 75%: 75% of Q ̇ is now directed toward the active limbs, with the remaining 25% going to the nonexercising tissues. By the time a steady state is reached at 3 min into exercise, 80% of Q ̇ goes to the exercising limbs, with the remaining 20% going to the nonexercising tissues. Note that, because Q ̇ increases approximately exponentially, these percentages change with time in a nonlinear manner. The venous effluent draining these two tissue compartments (exercising and nonexercising) is mixed in the venous system in a flow-weighted manner: the relative influence of CvO2 draining the exercising muscle increases as the exercise transient progresses. Thus, the influence of the low O2 concentration in the venous blood flowing out of the exercising muscle becomes greater as exercise progresses. The low CvO2 from the exercising muscle is “diluted” by relatively higher CvO2 from the rest of the body during the early portion of the exercise transient. But because the blood flow to the exercising muscle increases with time, the mixed venous CvO2 becomes progressively closer to the low CvO2 draining the muscle as exercise progresses. We again have a time-dependent and nonlinear distortion of the CvO2 arriving at the lung, relative to the CvO2 that left the exercising limbs. The effect of this flow-weighted mixing in normal _ 2p kinetics tends to speed relative to subjects is that VO _ 2m kinetics. This, together with the transit delay, tends VO _ 2p kinetics relato counteract the obligatory slowing of VO _ 2m due to the reduction in muscle and venous tive to VO

O2 stores. However, these effects interact in a complex manner meaning that the ultimate result is that phase II _ 2p kinetics can be slower, the same or even faster than VO _ 2m depending strongly on the relative ratio of resting VO blood flow distribution (the fraction of Q ̇ directed to the lower limb muscles) and the exercising kinetics Q ̇ and _ 2m. VO

̇ 2 10.4 EVIDENCE THAT PULMONARY VO KINETICS REFLECT INTRAMUSCULAR METABOLISM DURING EXERCISE 10.4.1 Evidence From Computer Simulation It is a commonly held notion in the field that muscle and ̇ 2 kinetics are closely matched, to within pulmonary VO B10% (Grassi et al., 1996; Koga et al., 2005; Krustrup et al., 2009). However, the dissociating effects of O2 stores, transit delays, and flow-weighted mixing of venous blood suggest that simultaneously and directly measured ̇ 2 kinetics are unlikely to be pulmonary and muscle VO closely matched. One approach to address this apparent discrepancy has been to use computational simulations (Benson et al., 2013), based on data from six healthy par_ 2m and ticipants during cycle ergometry where both VO _ VO2p were measured simultaneously (Grassi et al., 1996). Benson et al. (2013) showed that in young healthy _ 2m of 22.0 s, the corresponding humans with normal τ VO _ phase II τ VO2p would be 16.3 s; a kinetic dissociation of _ 2m and phase II 5.7 s or 26% on average. While τ VO _ τ VO2p were strongly correlated in this study (r2 5 0.91 in five of the six participants, see Benson et al., 2013, for further discussion) the finding of a significant dissociation _ 2p as a direct proxy for complicates the use of phase II VO _ VO2m. Although the absolute difference between muscle and ̇ 2 kinetics in young healthy individuals is pulmonary VO relatively small, there are several situations that could cause this kinetic dissociation to increase, to an extent that would undermine using pulmonary gas exchange as a kinetic proxy reflecting intramuscular biogenetics. One such situation is related to Q ̇ kinetics. Although ̇ Q (and therefore muscle blood flow) generally increases in a mono-exponential fashion when exercise is initiated from unloaded pedaling or a raised work rate, there are instances (especially during exercise initiated from rest) where the change in Q ̇ follows a biphasic time course: an early rapid phase of B1030 s and a secondary exponential phase (Fig. 10.10A) (Grassi et al., 1996; Shoemaker and Hughson, 1999; Benson et al., 2013). The early behavior is likely mediated by muscle pump and a rapid-onset vasodilation, with the slower exponential increase determined by local vasodilatory feedback

The Coupling of Internal and External Gas Exchange During Exercise Chapter | 10

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_ 2A and leg VO ̇ 2 (measured as FIGURE 10.11 Phase II alveolar VO twice one leg) using direct Fick measurements. From Grassi, B., et al., 1996. J. Appl. Physiol. 80, 988998 with permission.

FIGURE 10.10 (A) Biphasic exponential fit (solid line) to the experi̇ ) in a young healthy subject (open mental muscle blood flow (Qm ̇ 2 (VO _ 2A ) for squares). (B) Optimized computer simulation of alveolar VO _ 2A measurements the same subject superimposed on the experimental VO (open squares). From Benson, A.P., et al., 2013. J. Appl. Physiol. (1985) 115, 743755 with permission.

mechanisms linked to metabolic demand (Shoemaker and Hughson, 1999). The bi-exponential blood flow kinetics manifests at the lung as a rapidly rising initial portion of _ 2p (Fig. 10.10B) (Benson et al., 2013). Thus, phase II VO _ _ 2m, resulting the VO2p kinetics are speeded relative to VO in a very large kinetic dissociation (33.8 s or 65%). Furthermore, the effect of a rapid early increase in blood flow can be extreme, potentially contributing to pulmo_ 2p overshooting the steady state early in the trannary VO sient as seen in some endurance trained participants (Koppo et al., 2004).

Large kinetic dissociations can also occur in disease states where blood flow is compromised, such as in COPD and chronic heart failure (CHF). A high thoracic pressure during exercise in COPD constrains the amplitude of the resting Q ̇ and its response to exercise (Aliverti et al., 2005). Each of these effects results in greater reductions in muscle and venous O2 concentration during the transient for a given change in power output. The increased contribution of O2 stores to the gas exchange that this necessitates will increase in the dissociation ̇ 2 between the kinetics of muscle and pulmonary VO (Rossiter and Benson, 2010; Rossiter, 2011). _ 2m are each slowed compared to In CHF, Q ̇ and VO health (Poole et al., 2012). The resultant slowed venous blood flow dynamics can exacerbate the influences of the venous transit delay and flow-weighted mixing that dissȯ 2 kinetics (Rossiter and ciate muscle and pulmonary VO Benson, 2010; Rossiter, 2011). In COPD and CHF, the _ 2m may be even more limited than their kinetics of VO _ 2p kinetics suggest. slow VO

10.4.2 Evidence From Direct Measurement ̇ 2 kinetDespite the notion that pulmonary and muscle VO ics match to within 10% on average, computer simulations predict that this kinetic dissociation can be larger; sometimes substantially. What is the evidence from _ 2p and VO _ 2m kinetics are simiexperimental data that VO lar? Sadly, there are very few experiments with combined _ 2p and VO _ 2m to directly kinetic measurements of both VO address this question. The only available data during cycling exercise is from Grassi et al. (1996), who showed that the kinetic relationships between the muscle and the lung were closely coherent (Fig. 10.11). These data are based on six healthy subjects, and confirmed the notion that external respiration closely reflects internal

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respiration of the active muscles, even during the transient phase of exercise. Krustrup et al. (2009) used single-leg knee-extension exercise in seven young healthy individuals during transitions to and from moderate- and heavy-intensity exercise. Like Grassi et al. (1996), they found that the kinetics of ̇ 2 to be not different during the muscle and pulmonary VO on-transient phase of moderate-intensity exercise. During _ 2p the on-transient phase of high-intensity exercise, VO _ kinetics were slightly slower than VO2m , but the two were still strongly correlated. However, during the off-transient (recovery) from both moderate- and heavy-intensity exer_ 2p and VO _ 2m kinetics were significantly different cise, VO from one another and poorly correlated. The reasons for this finding are still not well understood, but the data imply that the replenishment of O2 stores, an increasing transit delay and changes in flow-weighted mixing of venous blood combine in exercise recovery to significantly dissociate muscle and lung gas exchange kinetics. _ 2m Therefore, caution should be used when inferring VO _ kinetics from VO2p measurements during recovery. Larger, meta-analysis, of all available data in cycling and single and dual-legged knee-extension exercise with simultaneous ̇ 2 kinetics on measurement of muscle and pulmonary VO transition during either cycling or knee-extension exercise (n 5 25 measurements on 18 individuals; seven participants completed both moderate- and heavy-intensity exercise) ̇ 2 kinetics when demonstrates a wide dissociation of VO data are considered on an individual basis (Fig. 10.12) (Koga et al., 2014). This may cast into doubt the validity of _ 2p kinetics as a surrogate for using pulmonary phase II VO _ VO2m . However, because the mean bias of these mean data

_ 2p tends to be B2 s are close to identity (phase II τ VO _ greater than τ VO2m ) studies on groups still appear to retain validity that external respiration reflects internal respiration even in the kinetic phase of exercise.

̇ 2 10.4.3 Kinetic Control of Muscle VO _ 2p kinetics have been used substantially to provide a VO window into the intramuscular metabolic control during exercise, whereby the putative mechanisms determining the cellular rate of oxidative phosphorylation in vivo can be noninvasively investigated (Whipp and Mahler, 1980). The overall reaction for oxidative phosphorylation in skeletal muscle can be abbreviated as follows: 5ADP 1 5Pi 1 2NADH 1 2H1 1 O2 -5ATP 1 2NAD1 1 2H2 O Behind this reaction are the major processes contributing to the provision of each substrate, such as delivery of phosphates, reducing equivalents and O2, as well as the activity of the many enzymes feeding into ADP phosphorylation at the mitochondrial inner-membrane-bound ATP synthase. Each these steps identifies a putative site of _ 2m adjustment during exercise. control or limitation of VO The primary focus of research has been to identify the flux control ratio (a concept quantifying the fraction of control exerted by each process in a complex network) of high-energy phosphates to the mitochondrial ATP synthase (ADP, Pi) (Chance and Williams, 1955; Whipp and Mahler, 1980), the supply of reducing equivalents predominantly to complex I of the electron transport chain FIGURE 10.12 A difference plot of the time constant (τ) of muscle O2 consumption determined by direct Fick measurements across the exercising limb and phase II pulmonary O2 uptake from breath-by-breath gas exchange measurements. Measurements were made during moderate-intensity cycle ergometry (filled square) (Grassi et al., 1996), heavy-intensity two-legged knee-extension (open triangle) (Koga et al., 2005), and moderate- (open circle) and heavy- (closed circle) intensity single-legged knee extension (Krustrup et al., 2009). A difference of 6 10% is shown in dashed lines and shaded area and the negative group mean bias is shown as a solid line. From Koga, S., et al., 2014. Med. Sci. Sports Exerc. 46, 860876 with permission.

The Coupling of Internal and External Gas Exchange During Exercise Chapter | 10

(e.g., NADH) (Timmons et al., 1996), the mitochondrial PO2 (Hughson and Morrissey, 1982), and the activity of mitochondrial enzymes that catalyze the overall reaction (Green et al., 1992; Korzeniewski and Zoladz, 2004; Wust et al., 2011).

10.4.3.1 Feedback Control by Intramuscular Phosphates _ 2m kinetics are principally It is generally agreed that VO determined by the rate of ATP hydrolysis and its reactants (Chance et al., 1985), which may act via the following: a feedback control loop dictated by the delivery of intramuscular high-energy phosphates to the mitochondrion (Whipp and Mahler, 1980); thermodynamic control via the phosphorylation potential ([ATP]/[ADP] 1 [Pi]) (Brown, 1992); or a linear dependence on ΔGATP (Meyer and Foley, 1996). Because each is predominantly determined by PCr breakdown, it is often difficult to distinguish among them in exercising humans. Nevertheless, during moderate- to very-heavy-intensity exercise, under normal conditions (e.g., a young healthy human at sea level), evidence suggests that the predominant flux con_ 2m resides in the feedback of ADP to the mitotrol for VO chondrion (Whipp and Mahler, 1980; Meyer, 1988;

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Meyer and Foley, 1996; Rossiter et al., 2002c), which is spatially and temporally buffered by intramuscular PCr. Intramuscular [PCr] provides a useful in vivo proxy of [ADP] (assuming only minimal disturbances in intracellular pH during moderate exercise) (Meyer, 1988), where the breakdown of PCr is intricately associated with the activation of oxidative phosphorylation during exercise. This suggestion is supported by animal experiments _ 2m and PCr were assessed (independently) in where VO both the frog (Mahler, 1985) and rat (Meyer, 1988). It is also supported by studies in humans, which have shown _ 2p kinetics (to within identical PCr and phase II VO B10%) when simultaneously measured at the onset of moderate- and very-heavy-intensity exercise (Rossiter et al., 1999, 2002b) (Fig. 10.13). The homology between _ 2p kinetics support Britton Chance’s PCr and phase II VO original observation in vitro, that respiration is driven once ADP is added to the media bathing isolated mitochondria (Chance and Williams, 1955). That a PCr “slow _ 2p slow component provides component” mirrors the VO _ 2p slow component preadditional evidence that the VO dominantly originates in the active locomotor muscles, and that it is due to an increased phosphate cost of force production (P/W) rather than an increase O2 cost of phosphate production (P/O). ̇ 2 FIGURE 10.13 Simultaneous measurements of pulmonary VO _ 2p ) and phosphocreatine (PCr, from the quadriceps muscle) in a (VO healthy subject on transition to moderate- (A) and very-heavy- (B) _ 2p and intensity bilateral knee-extension exercise. Notice that VO PCr have near-identical dynamics once the transit time delay of ̇ 2 between muscle and lung has being accounted for (vertical VO dashed lines). Of note, the PCr scale is inverted to better demonstrate the close association between the two variables. From Rossiter, H.B., 2011. Compr. Physiol. 1, 203244 with permission. Copyright r 2011 American Physiological Society. All rights reserved.

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The control of oxidative phosphorylation was, therefore, proposed to be a first-order rate reaction, where _ 2m . ADP delivery to the mitochondrial matrix controls VO These kinetics are thought to remain similar over a range of power outputs, and therefore the slope of the inverse _ 2m is dependent on relationship between [PCr] and VO total intramuscular creatine (i.e., PCr 1 Cr; termed the metabolic capacitance), the mitochondrial content or activity (termed the resistance to energy transfer), and the P/O ratio (Meyer, 1988). This model predicts that increasing the total intramuscular Cr pool or increasing mito_ 2m chondrial activity would act to slow or speed VO kinetics, respectively. This inference has been supported by experiments from isolated rat mitochondria in vitro (Glancy et al., 2008), in rat skeletal muscle (Paganini et al., 1997) as well as experiments from humans (Jones ̇ 2 kinetics were linearly et al., 2009b) whereby PCr and VO slowed when total Cr was increased, but linearly speeded when mitochondrial content or activity was increased (although see Jones et al., 2002). However, PCr breakdown occurs in the cytoplasm. So how does the communication occur to link cytoplasmic [PCr] with a process on the inner side of the innermitochondrial membrane bound ATP synthase? A number of elegant studies have provided insight in to identifying the mechanisms of the “creatine shuttle” hypothesis (Fig. 10.14) (Margaria et al., 1965; Whipp and Mahler, 1980; Bessman and Geiger, 1981; Mahler, 1985; Perry et al., 2012). At exercise onset, cytosolic PCr is immediately broken down (proximal to the ATPases at the myofibril and SERCA) to resynthesize ATP via the

extramitochondrial isoform of creatine kinase. This increases cytosolic [Cr], which can enter the mitochondrion intermembrane space via voltage dependent anion channels (VDAC) or porin channels. This results in an increase in [ADP] within the intermembrane space via a reaction catalyzed by the mitochondrial isoform of creatine kinase (mtCK), where ATP is hydrolyzed and PCr is formed. The mtCK is thought to be functionally coupled to the adenine nucleotide translocase (ANT) on the innermitochondrial membrane, which exchanges ADP and ATP between the matrix and intermembrane space. Increased matrix [ADP] provides the feedback signal and substrate necessary to stimulate an increase in oxidative phosphorylation. In the return arm of the shuttle, the ATP that is resynthesized via oxidative phosphorylation is transported back into the intermembrane space by means of the ANT where it reacts with Cr to reform PCr. The PCr then completes the cycle, by diffusing out of the mitochondrion (via the VDAC or porins) to the cytosol where it is now free once again to resynthesize ATP at the myofibril. It is worth noting here that the effect of PCr signaling _ 2m will be strongly influenced by the conto increase VO centrations and activities of mitochondrial enzymes. The greater the mitochondrial protein expression, the more _ 2m for a given change in rapid will be the increase in VO [PCr] (see later). In addition, myocytes with a high expression of glycolytic enzymes may also “short circuit” the creatine shuttle by sequestering ADP and Pi in glycolysis, thereby reducing the potential feedback to the mitochondrion for oxidative phosphorylation (Middlekauff, 2010).

FIGURE 10.14 An illustration of the creatine shuttle hypothesis. In oxidative fibers (A) PCr acts as the key signaling mediator by delivering ADP to the inner-membrane-bound ANT to stimulate oxidative phosphorylation in skeletal muscle. In glycolytic fibers (B), a higher rate of Pi and ADP sequestration in glycolysis is hypothesized to interrupt PCr signaling to the mitochondrion, reducing the contribution of oxidative phosphorylation to ATP provision.

The Coupling of Internal and External Gas Exchange During Exercise Chapter | 10

Perhaps the strongest evidence in support of the role of the creatine kinase shuttle and phosphate feedback in _ 2m comes from data using the isolated frog controlling VO single myocyte preparation (Kindig et al., 2005) and stimulated canine muscle in situ (Grassi et al., 2011). Pharmacological inhibition of creatine kinase, effectively removing the PCr system, and causing rapid .100-fold _ 2m kinetgreater increases in [ADP], markedly speeds VO ics at the onset of contractions. These data support the pivotal of role [PCr] in damping and transducing the signal to increase oxidative phosphorylation at the onset of muscular exercise.

10.4.3.2 Evidence for and Against Control by Reducing Equivalent Provision _ 2m control is the Another suggested mechanism of VO feedforward provision of substrates (in the form of NADH) to the electron transport chain, where a lag in activation of the PDH enzyme complex has been implicated (Timmons et al., 1996, 1998; Howlett et al., 1998, 1999). Evidence from isolated single frog fibers shows that relative mitochondrial NADH concentration, as measured using changes in the autofluorescence of NAD(P)H, show an exponential fall that is preceded by a short time delay (Gandra et al., 2012). This short time delay likely suggests that NADH availability, and the rate of oxidation, is unlikely to limit oxidative phosphorylation. On the contrary, a number of studies (reviewed in Greenhaff et al. 2002) have shown that activation of PDH before exercise via administration of DCA, increases acetyl-CoA entry into the TCA cycle and leads to a reduced O2 deficit. However, in subsequent human studies kinetics of _ 2p (Rossiter et al., 2003), or VO _ 2m (Bangsbo PCr, VO et al., 2002) were not speeded with DCA administration, _ 2p and PCr responses were lower but the amplitudes of VO following DCA administration in humans (Rossiter et al., 2003). The lower amplitude of response may explain why the O2 deficit was reported to be reduced, and suggests that the activities of a large number of regulated mitochondrial enzymes were likely increased following activation of PDH. Similar results were found in the isolated dog gastrocnemius muscle preparation in situ (Grassi et al., 2002), and showed that PDH activation prior to exercise reduced fatigue; again consistent with a reduced O2 deficit for the force developed and a reduced ampli_ 2m slow component. Thus, while rapid tude of the VO delivery of NADH and activation of mitochondrial enzymes early in exercise lessened fatigue and development of work inefficiency, in these studies it did not _ 2m . directly contribute to the control of VO More recently, Gurd et al. (2006, 2008, 2009) pro_ 2m kinetics might be limited by vided evidence that VO NADH availability during the exercise transient,

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particularly in older adults. Muscle biopsies revealed that increase of PDH activity was slow in muscles from older participants and an intervention to elevate PDH activity prior to exercise (using a “priming” exercise bout) was effective in both old (Gurd et al., 2009) and young (Gurd et al., 2006) healthy adults. However, in the older sub_ 2p kinetics were speeded to a greater jects’ phase II VO extent that in younger subjects, suggesting that activation of PDH could provide a stenosis to increasing oxidative phosphorylation in the elderly. Taken together, the current evidence suggests that the provision of substrates to the electron transport chain is adequate at the onset of exercise, and does not control or _ 2p kinetics in young healthy humans. However, limit VO the potential for a reducing equivalent delivery limitation increases in the elderly.

10.4.3.3 Limitation by Skeletal Muscle Oxygenation The mitochondrial PO2 needed to sustain high rates of oxidative phosphorylation in skeletal muscle is estimated to be in the range of B3 mm Hg (Wilson et al., 1977; Richardson et al., 1995). Muscle PO2 depends on an adequate rate of O2 diffusion from capillary-to-mitochondria as dictated by Fick’s law (i.e., the product of the difference between capillary PO2 and mitochondrial PO2, and the diffusion coefficient; Wagner, 1996). The O2 diffusion gradient is therefore largely dependent on local muscle ̇ ) matching local metabolic demands blood flow (Qm _ (VO2m ). Assuming arterial O2 concentration remains coṅ /VO _ 2m is stant in normal healthy humans, the ratio of Qm the primary determinant of mean muscle capillary PO2, and therefore CvO2. During exercise spanning the aerobic range, CvO2 has an approximately hyperbolic relationship _ 2m , such that CvO2 falls rapidly at low power outwith VO puts, but further increases in power result in a lesser reduction in CvO2. This is a consequence of the linear ̇ to VO _ 2m relationship and positive intercept relating Qm (Whipp and Ward, 1982; Barstow et al., 1990). However, ̇ /VO _ 2m can result during the transient, the dynamics of Qm in a transient overshoot in CvO2 (i.e., CvO2 falls below the eventual steady state) (Diederich et al., 2002) which may limit the pressure gradient driving O2 diffusion across the capillarymyocyte interface (Rossiter, 2011). The transient profile in CvO2 can therefore provide insight into the dynamic matching of O2 delivery to O2 utilization and thus skeletal muscle oxygenation. There are several approaches used to investigate the dynamics of muscle-PO2-related variables at the onset of exercise, directly at the intracellular level, in the microvasculature, or in combination. The gross features of the adequacy of O2 delivery during the transient phase of exercise are gleaned from measurements by direct Fick

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̇ (and by inferacross the contracting limb. In humans, Qm _ 2m on the ence O2 delivery) adjusts more rapidly than VO transition to moderate exercise (Grassi et al., 1996; Koga et al., 2005; MacPhee et al., 2005; Jones et al., 2012). Such evidence has been used to suggest that O2 delivery _ 2m kinetics (Poole and Jones, 2012). is not limiting to VO However, estimated capillary blood flow kinetics in human skeletal muscle are reported to be similar (or even _ 2m (Ferreira et al., 2005). This slower) than those of VO _ 2m favors O2 delivery as providing a stenosis to VO _ 2p dynamics at the microvascular level. Although VO kinetics are not consistently slowed when O2 delivery is reduced, for example under conditions of hypoxia (Engelen et al., 1996), β blockade (Hughson and Smyth, 1983) or supine exercise (Hughson et al., 1991) _ 2p kinetics were slowed by blood donation (although VO (Burnley et al., 2006) and lower-body positive pressure _ 2p kinet(Williamson et al., 1996)), attempts to speed VO ics by increasing O2 delivery in healthy by hyperoxia (Wilkerson et al., 2006) or elevated [Hb] (Wilkerson et al., 2005) have failed. Perhaps the most powerful evidence of this type against O2 availability limiting kinetics comes from the _ 2m kinetics canine gastrocnemius muscle in situ, where VO can be directly measured while O2 delivery is experimentally manipulated by pump perfusion. In a number of ele_ 2m kinetics were not speeded on transition gant studies, VO to steady-state contractions despite: (1) prior elevation of blood flow to the steady-state level, in combination with adenosine-induced vasodilation (Grassi et al., 1998a); (2) eliminating O2 diffusion limitations by inhalation of a hyperoxic gas mixture and pharmacological infusion reducing the affinity of Hb for O2 (Grassi et al., 1998b). However, using the same model, slowing blood flow _ 2m kinetics (Goodwin et al., 2011). kinetics slowed VO These combined human and animal studies led to the notion of a “tipping point” in microvascular PO2 (Poole and Jones, 2012): above a “critical” capillary PO2, _ 2m kinetics, but increasing O2 delivery does not speed VO below it they are slowed. To investigate these relationships in the intracellular compartment is considerably complex. To this end, Hogan (2001) used phosphorescence quenching to measure PO2 at the onset of stimulated contractions in single frog muscle fibers. The advantages of using this model include assessment of single fiber types in isolation, the fiber is completely bathed in a solution with a known PO2, and the fibers lack myoglobin which simplifies the accurate measurement of intracellular PO2. These studies showed that the fall in PO2 is delayed by B10 s after the onset of contractions and thereafter is well characterized by an exponential with a time constant of B25 s. The noted delay of B10 s after the onset of contractions before PO2 begins to fall provided evidence that O2

supply did not limit appear to limit the kinetics of oxidative phosphorylation at exercise onset. The microvascular PO2 in simulated rat skeletal muscle, also determined using phosphorescence quenching, showed a similar short delay (Behnke et al., 2001). As the microvascular PO2 ̇ /VO _ 2m ratio, the delay in reflects with high fidelity the Qm this in vivo model suggested that tissue O2 delivery does not limit muscle O2 consumption on transition to exercise. However, intramuscular PO2 is difficult to determine in humans. Richardson et al. (2015) used 1H magnetic resonance spectroscopy to measure myoglobin deoxygenation kinetics during moderate- and heavy-intensity calf contractions, and provided possibly the strongest evidence in _ 2m kinethumans that intracellular PO2 does not limit VO ics in these conditions. The findings showed a delay in intramuscular deoxygenation at the onset of exercise and rapid re-oxygenation kinetics upon cessation, implying that intramuscular phosphate feedback and enzyme activities interactions, and not O2 availability, determine the onset kinetics of oxidative metabolism in healthy human skeletal muscles. It should be noted however, that the calf exercise model, required by the limitations of 1H magnetic resonance spectroscopy technique, results in muscle that is more highly perfused during exercise than are the lower limb muscles during large muscle mass, bi-pedal, exercise. It, therefore, remains to be determined whether _ 2m kinetics in intracellular PO2 becomes limiting to VO conditions of whole-body exercise, or in aging or chronic disease where convective and/or diffusive O2 delivery mechanisms are impaired. Another approach commonly employed in humans is near-infrared spectroscopy (NIRS). NIRS provides a noninvasive measure of relative concentrations of skeletal ̇ / muscle microvascular deoxygenation (proportional to Qm _ VO2m ) from the spectroscopic quantification of concentrations in oxyhemoglobin and oxymyoglobin (the combination of which is abbreviated here as HbO2) and deoxyhemoglobin and myoglobin (HHb) (DeLorey et al., 2003; Grassi et al., 2003; Jones et al., 2009a). From these variables, total hemoglobin and myoglobin concentration (tHb) and tissue saturation (StO2 5 HbO2/tHb) can be estimated. Under conditions where tHb is relatively unchanged by exercise, the HHb signal is suggested to provide a close proxy of O2 extraction and therefore the dynamics of CavO2 (where CaO2 is assumed to be constant) (DeLorey et al., 2003; Grassi et al., 2003; Jones et al., 2009a). In health, during the transition to moderate exercise, HHb increases with an exponential-like profile following a delay-like period or even a decrease (the latter taken to reflect O2 delivery is in excess of demands) (DeLorey et al., 2003; Grassi et al., 2003). These features in the microvascular/intramuscular compartment of active skeletal muscle in humans are consistent with the

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direct high-fidelity measurements of microvascular PO2 dynamics from the rat spinotrapezius (Behnke et al., 2001) and direct measures of whole-limb CavO2 in humans (Grassi et al., 1996; Koga et al., 2005; Jones et al., 2012). Technological advances have used time-resolved NIRS, which assesses the diffusion properties of light through tissue to provide an absolute quantification of [HHb] [HbO2], and t[Hb] (Koga et al., 2011; Bowen et al., 2013; Okushima et al., 2015). Positron emission tomography has also been applied to image the coupling of perfusion-to-metabolism in skeletal muscle during exercise (Kalliokoski et al., 2005). These innovative approaches in vivo, which have allowed assessment into the deeper regions of active muscles, suggest that dramatic differences exist across different muscles and different depths in the mechanisms employed by skeletal muscle to maintain O2 delivery during cycling exercise in humans. Okushima et al. (2015) found that the rate of deoxygenation during incremental exercise was greater in the superficial leg muscles (vastus lateralis and vastus medialis), and reached a plateau at high power output ( . 70% peak incremental power). This was in contrast to the deeper muscles (deep rectus femoris and vastus intermedius) where deoxygenation was slower early in ramp exercise, and increased dramatically at high power output (Okushima et al., 2015). It was suggested that this divergent deoxygenation pattern may be due to a greater population of slow-twitch muscle fibers in the deeper muscles, and the differential recruitment profiles and vascular and metabolic control properties of specific fiber populations within superficial and deeper muscle regions. The additional finding that rectus femoris showed a continued increase in t[Hb] unlike the vastus lateralis, suggests that the diffusional surface area (the combined surface area of the muscle capillary endothelium juxtaposed to the erythrocyte) may be better protected in deeper muscles. That is, deeper, more oxidative, muscles may rely more upon O2 diffusion to maintain muscle PO2, while more surface, less oxidative muscle, may rely more upon O2 convection (Okushima et al., 2016). In addition, these control relationships may differ across states of maturation (Chiu et al., 2017) and with aerobic fitness (Kalliokoski et al., 2005).

10.4.3.4 Role of Oxidative Enzyme Activation _ 2m It remains a matter of debate whether the control of VO is related to: (1) the direct effects of ATP usage at the myofibrils and calcium pumps, which provides the necessary feedback to signal increases in oxidative phosphorylation via the products of ATP breakdown (i.e., ADP, Pi); and/or (2) simultaneous activation of pathways of both ATP usage and ATP supply. Given that substrates (ATP) and products (ADP, Pi) of muscle contraction change their concentrations very little (if at all), it was questioned

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whether such homeostasis of key intermediates in pathways of ATP supply and demand during very large swings in ATP turnover could be achieved through a feedback mechanism alone (Hochachka and McClelland, 1997). Such reasoning led to a proposal that ATP supply and demand pathways may be simultaneously activated (in a feedforward mechanism) by a process coined “parallel activation” (Green et al., 1992; Hochachka and Matheson, 1992; Green et al., 1995) or “each step activation” (Korzeniewski and Zoladz, 2004; Zoladz et al., 2014). Parallel activation is hypothesized to occur via a process highly dependent upon intracellular Ca21 flux. _ 2m in the former theory is explained by a Control of VO simple first-order rate reaction (e.g., by an ADP feedback loop) (Chance and Williams, 1955), while the parallel activation theory requires considerably more complex _ 2m . models to explain the kinetic control of VO Parallel activation theory suggests that not only is ATP usage directly involved in the activation of oxidative phosphorylation (ADP feedback), but also direct activation of NADH supply (including glycolysis) and mitochondrial complexes. Evidence supporting parallel activation comes from the demonstration that traininginduced changes in muscle enzyme concentration and ̇ 2 kinetics are too rapid to be explained simply by VO increases in muscle angiogenesis or mitochondrial respiratory protein concentration (Green et al., 1992; McKay et al., 2009; Zoladz et al., 2013, 2014), and therefore the sensitivity of enzyme activity to, for example, cytosolic and mitochondrial Ca21 flux, is also responsive to exercise training. This suggests that mechanisms that precede mitochondrial biogenesis or muscle capillarization, such as Ca21 channel activity, may be responsible for the early ̇ 2 kinetics following exercise training, and speeding of VO therefore that modulation of oxidative enzyme activity by _ 2m control in the Ca21 has the potential to influence VO trained or untrained state. Control of these allosteric processes may be teased out following interventions designed to reduce enzyme inhibition (present under normal rest) prior to exercise and meȧ 2 kinetics. Priming contractions suring the effect on VO (warm up exercise) has been frequently used as a model for this, but priming exercise is complicated by the coincident effects on muscle blood flow and distribution. Animal models where O2 delivery can be controlled may overcome some of this complexity. Priming contractions ̇ 2 kinetic response that in canine muscle produce a VO revert higher-order control characteristics back toward a first-order control characteristic (Hernandez et al., 2010). Similarly prior contractions in single frog fibers demonstrate a faster reduction in intracellular PO2 consistent _ 2m kinetics (Hogan, 2001). These effects with speeded VO are thought likely to be caused by allosteric activation of mitochondrial enzymes that accumulate during contractions and are slow to recover following priming.

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Sarcoplasmic reticulum release of Ca21 was initially suggested to be the primary signal to activate oxidative phosphorylation via products of ATP hydrolysis (Chance and Williams, 1955), but more recent data have since found Ca21 can also directly activate key enzymes that are involved in the provision of NADH in the TCA cycle (i.e., PDH, isocitrate dehydrogenase) and in the electron transport chain (Glancy et al., 2013). This provides compelling evidence that multiple mechanisms that contribute to controlling the rate of oxidative phosphorylation are likely activated at the onset of contractions, following the initial inward flux of Ca21. This view is supported in sin_ 2m kinetics where, during the onset of gle frog fibers VO contractions, fibers demonstrated a biphasic response profile: and initial activation phase followed by an exponential profile predicted by first-order feedback control (Wust et al., 2013). Direct evidence comes from dog skeletal muscle using direct rapid sampling of intramuscular _ 2m kinetics (Wust phosphates via serial biopsy and VO _ 2m on-transient et al., 2011). These data show that VO kinetics were faster than the accumulation of [ADP] or _ 2m kinetfall in [PCr] alone. Interestingly, the recovery VO ics, measured in the frog single fiber studies, demonstrated a mono-exponential kinetics indicative of firstorder control (Wust et al., 2013). This implies that enzyme activation at exercise onset recovers slowly on cessation. Data to prove these suggestions in humans are complex to obtain, but computational simulations based on combined magnetic resonance spectroscopy measure_ 2p kinetics support ments of muscle phosphates and VO that parallel activation is required for the intramuscular _ 2p ADP response to be translated into the measured VO kinetics (Korzeniewski and Rossiter, 2015). Overall, therefore, while ATP usage supplying ADP and Pi to the mitochondrion via the actions of creatine kinase plays a key role in activating oxidative phosphorylation at exercise onset, the parallel activation of other mechanisms related to mitochondrial and glycolytic enzyme activity likely exert considerable modulation on the rate of oxidative phosphorylation induced by [ADP] at the inner-mitochondrial membrane. These studies provide _ 2m being determined by simple strong evidence against VO first-order control in vivo (for further discussion also see Chapter 17: Muscle Blood Flow and Vascularization in Response to Exercise and Training).

̇ 2 KINETICS IN 10.5 SLOW PULMONARY VO AGING AND CHRONIC DISEASE: WHAT DO THEY TELL US ABOUT EXERCISE LIMITATION? As emphasized throughout this chapter, exercise intolerance is a key symptom in many chronic diseases and one

of the strongest predictors of quality of life and prognosis. This reduction in exercise tolerance is mediated, in large part, by a slower increase in oxidative metabolism to meet energetic demands, which in turn necessitates a greater reliance on substrate level phosphorylation, induction of fatigue and exacerbation of symptoms leading to _ 2p kinetics are reported in intolerance. Slow phase II VO response to aging and in many chronic diseases including CHF, COPD, pulmonary arterial hypertension, peripheral arterial disease, and mitochondrial myopathies, among _ 2p kinetics can offer a powerful others. Assessment of VO effort-independent and noninvasive tool for assessing prognosis (Brunner-La Rocca et al., 1999) and evaluating therapeutic efficacy in clinical practice (Puente-Maestu et al., 2016).

10.5.1 Aging _ 2p kinetics are slowed in older age (Murias and VO _ 2p Paterson, 2015). Longitudinal study suggests that VO kinetics increase by 50% between the ages of 70 and 80 years (Bell et al., 1999). Gurd et al. (2009) used priming _ 2p kinetics in the elderly (warm up) exercise to speed VO by B25%, which occurred coincident with increased muscle oxygenation and PDH activity. This suggests that interventions to increase muscle PO2 and mitochondrial enzyme activity may be targeted in older individuals to help alleviate symptoms of exercise intolerance. Aerobic exercise training in the elderly increased mitochondrial _ 2p kinetics even without enzyme activity and speeded VO a concomitant increase in limb blood flow (Bell et al., _ 2p kinetics, it is 2001). As older age predicts slower VO important that comparison of patients be made with agematched controls to uncover the effects of the disease per _ 2p kinetics. se on VO

10.5.2 Chronic Heart Failure One approach to identify whether a central mechanism _ 2p kinetics in CHF patients has been to measure limits VO ̇ _ 2p kinetics. Q kinetics noninvasively in concert with VO This approach is used in moderately impaired CHF patients, where Q ̇ kinetics (measured via radial artery pulse contour analysis method or impedance cardiography) are either similar (Kemps et al., 2010) or slowed _ 2p (Sperandio et al., 2009) compared with phase II VO kinetics. At first glance, these data seem to suggest that the main target to alleviate exercise limitation in CHF patients would be to increase bulk O2 delivery, because in healthy subjects Q ̇ kinetics are significantly faster than _ 2p (Yoshida and Whipp, 1994). Indeed, increasing bulk VO O2 delivery in CHF patients by cardiac resynchronization _ 2p kinetics and this was underpinned therapy, speeded VO by an increase in stroke volume and left-ventricular

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end-systolic volume (Tomczak et al., 2012). Such data indicate that a limitation to convective O2 delivery is _ 2p kinetics in CHF likely a key mechanism slowing VO patients. However, not all CHF patients are limited by bulk O2 delivery, as a cardiac transplant is unable to _ 2p kinetics despite fasconsistently reverse the slowed VO ̇ ter Q kinetics 2 years postsurgery (Grassi et al., 1997): peripheral maladaptations therefore also likely contribute. Evidence from CHF patients shows clearly that increasing peripheral microvascular O2 delivery may be a better treatment target than simply targeting Q ̇ itself. Slowed Q ̇ kinetics in CHF patients are associated with abnormalities in the microvascular oxygenation profile of the vastus lateralis assessed by NIRS: patients achieving a faster rate of deoxygenation and a deoxygenation “overshoot,” unlike controls (Sperandio et al., 2009). Further evidence for targeting microvascular control in CHF patients is provided using sildenafil, an inhibitor of the cGMP-specific enzyme phosphodiesterase-5 (PDE-5) (Sperandio et al., 2012); however dietary nitrate was _ 2p kinetics unable to influence muscle deoxygenation, VO or exercise tolerance in CHF patients (Hirai et al., 2017). In addition, 12 weeks of high-intensity exercise training in stable CHF patients was associated with increased microvascular oxygenation on transition to exercise without any significant improvements in Q ̇ kinetics (Spee et al., 2016). CHF is a heterogeneous disease, and a “one size fits all” approach has many limitations. Some of the heterogeneity in response to interventions may be due to the relative contribution of the different sites of limitation in these patients. Bowen et al. (2012) used priming exercise to increase muscle oxygenation and activate intramuscular _ 2p and deoxygenenzyme activity prior to measuring VO ation kinetics in CHF patients. This study revealed two distinct groups of CHF patient: moderately impaired _ 2peak was better preserved (Weber patients in whom VO _ 2p kinetics classes A and B; Weber et al., 1982) and VO were more limited by microvascular O2 delivery; and more severe patients (Weber classes C and D) in whom _ 2p increasing muscle oxygenation had less effect on VO kinetics, implicating impairment in mitochondrial oxidative phosphorylation. These noninvasive data confirm direct experimental findings collected in rats with experimentally induced CHF, whereby skeletal muscle microvascular PO2 measurements demonstrated that severe CHF was associated with additional impairments related to mitochondrial function that were not observed in moderate CHF (Diederich et al., 2002). Collectively, these studies suggest that in some CHF patients improving central or microvascular O2 delivery is _ 2p kinetics and reduce a therapeutic target to speed VO exercise limitation, while in more severely impaired

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patients an improvement in O2 delivery would likely have _ 2p kinetics and, by inference, moderate benefit on VO exercise tolerance. The latter group of patients would require interventions to target muscle mitochondrial function to increase exercise tolerance.

10.5.3 Chronic Obstructive Pulmonary Disease Ambulatory patients with COPD have some of the slow_ 2p kinetics measured across different patient popuest VO lations (Rossiter, 2011). They also have slow Q ̇ kinetics and fast microvascular deoxygenation on transition to exercise (Chiappa et al., 2008). Interventions to increase convective O2 delivery by attenuating expiratory flow limitation, such as by breathing a heliox mixture (Chiappa et al., 2009) or using bronchodilators (Berton et al., 2010), speed Q ̇ kinetics, and slow microvascular deoxygenation in COPD patients, and are associated with fas_ 2p kinetics and increased exercise tolerance. ter VO It would be a reasonable assumption that reversing a resting or exercise-induced hypoxemia in COPD patients is the main mechanism of this benefit. However, the benefit can be seen even in patients without hypoxemia or during the early stages of exercise prior to development of hypoxemia (assessed by pulse oximetry). Hyperoxic breathing or noninvasive ventilation interventions in COPD may work by reducing ventilatory work, reducing dynamic hyperinflation and intrathoracic pressure and better distributing blood flow to the locomotor muscles. For example, control subjects with induced expiratory flow _ 2p kinetics on recovery from limitation had slower VO exercise than during normal breathing (Vogiatzis et al., ̇ /VO _ 2m appears to be mini2007). Nevertheless, muscle Qm mally impacted in COPD (Louvaris et al., 2017), and patients terminate exercise with a large muscular power reserve, meaning that peripheral fatigue does not determine their limit of tolerance (Cannon et al., 2016). Other factors may therefore play a key role, such as inspiratory muscle weakness, as this is closely associated with slowed _ 2p kinetics and reduced functional capacity in COPD VO patients (Wolpat et al., 2017). Taken together, these findings are consistent with suggestions that dynamic hyperinflation and symptoms of dyspnea may be the primary drivers of exercise intolerance in this patient population (Guenette et al., 2014; O’Donnell et al., 2014; Abdallah et al., 2017). The finding, however, that dietary nitrate was able to extend time to exhaustion and reduce diastolic and systolic blood pressures in COPD patients (Berry et al., 2015) also suggests targeting vascular impairments may also be a useful therapeutic option in these patients.

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10.5.4 Skeletal Muscle Myopathies Skeletal muscle myopathies, such as McArdle’s disease (myophosphorylase deficiency that impairs glycogen breakdown) or mitochondrial myopathies (electron trans_ 2p port chain dysfunctions), are associated with slow VO kinetics and reduced exercise tolerance. In such conditions, bulk O2 delivery is relatively well preserved such that the locus of limitation has been suggested to be related to mitochondrial impairments alone, with a limitation in the ability to increase muscle O2 extraction and thus increase O2 consumption (Grassi et al., 2007; Grassi _ 2p kinetics, et al., 2009). When combining measures of VO ̇ Q, and microvascular oxygenation, patients with _ 2p kinetics, and McArdle’s disease are able to speed VO increase microvascular deoxygenation by a priming exercise bout (Porcelli et al., 2014). This suggests that prior exercise may act to increase mitochondrial enzyme activ_ 2p kinetics in this patient population. The ity and speed VO mechanism(s) responsible (e.g., increased glucose transport and/or delivery of reducing equivalents, parallel activation of oxidative pathways) in McArdle’s disease are not yet known. It is of interest therefore that the same effect was absent in patients with mitochondrial myopathy (Porcelli et al., 2014). Interventions other than exercise training (Porcelli et al., 2016) to effectively increase oxidative capacity in these patients are yet to be developed. Both groups of patients have a significant increase in exercise tolerance after endurance exercise training (Porcelli et al., 2016). This supports the concept that _ 2p kinetics and better matching of muscle speeding of VO ̇ /VO _ 2m across the exercise transient is a key mechanism Qm associated with increased exercise tolerance (Murias and _ 2p kinetic Paterson, 2015). Overall, the combination of VO measurements with microvascular deoxygenation is effective in dissecting the kinetic relationships internal to exercise respiration and provide insight into therapeutic targets in patients with muscle myopathies.

10.6 CONCLUSIONS The investigations outlined in this chapter demonstrate how an integrative physiological approach, that combines pulmonary gas exchange with measurements of blood flow and/or limb microvascular oxygenation, provides insight into the mechanisms that link internal to external respiration during exercise. The association between muscle and lung gas exchange during dynamic exercise provides us with a noninvasive window of muscle energetics and the processes leading to fatigue and contributing to exercise limitation. Although gas exchange kinetics may be modulated by the intervening circulation and changes

̇ 2 measurements provide in lung gas stores, pulmonary VO valuable insight into normalcy or pathology of exercising muscle.

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