V? O2 ON-KINETICS IN SKELETAL MUSCLE IN NORMOXIA AND HYPOXIA WITH MATCHED CONVECTIVE O2 DELIVERY by James R. McDonald A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama May 6, 2012 Keywords: V? O2 on-kinetics, hypoxia, normoxia, convective O2 delivery Copyright 2012 by James R. McDonald Approved by L.Bruce Gladden, Chair, Professor of Kinesiology David D. Pascoe, Professor of Kinesiology John C. Quindry, Assistant Professor of Kinesiology James L. Sartin, Professor of Anatomy, Physiology & Pharmacology i Abstract The aim of this investigation was to evaluate O2 uptake (V? O2) on-kinetics under hypoxic conditions while holding convective O2 delivery constant in isolated canine muscles in situ. Canine gastrocnemius muscles (n=7) were surgically isolated and then electrically stimulated via the sciatic nerve (8 V, 0.2-ms duration, 50Hz, 200-ms trains) eliciting contractions at a rate of 1 contraction every 2 seconds (about 70% of peak V? O2). The dogs inspired 3 different O2 fractions: 21% (normoxia), 12% (hypoxia), and 8% (severe hypoxia). Convective O2 delivery [blood flow (Qm) x arterial O2 content (CaO2)] to the muscle was held constant by elevating muscle blood flow via a perfusion pump. Blood flow was recorded with an ultrasonic flowmeter while venous O2 saturation was monitored continuously in order to allow measurement of contraction-by-contraction muscle VO2. Relative muscle deoxygenation (?HHbMb) was assessed with continuous near-infrared spectroscopy (NIRS). Arterial blood PO2 (torr) was measured during each stimulation period and the mean per condition was 111 ? 11 for 21% inspired O2, 41 ? 3 for 12% inspired O2, and 30 ? 3 for 8% inspired O2. Venous blood PO2 (torr) measured prior to the end of contractions was significantly different (P< 0.05) among the three conditions (21% = 20 ? 5, 12% = 16 ? 5, 8% = 13 ? 5). The arterial O2 content (CaO2) was significantly lower in the hypoxic conditions and pump perfusion was significantly higher in the hypoxic conditions resulting in matched convective O2 delivery (ml/Kg-1/min-1) that were not different (P > 0.05) among the three different O2 fractions, (21% = 202 ? 57, 12% = 206 ? 57 and 8% = 205 ? 59). There was also no difference in tau, the primary time constant for the VO2 ii on-kinetics among treatments (21% = 10.9 ? 2.4s, 12% = 10.1 ? 1.8s, 8% = 9.2 ? 1.9s ). Additionally, the NIRS data showed no significant time difference in the ? HHb time course, mean response time (MRT) (21% = 15.3 ? 2.7s, 12 % = 14.7 ? 4.4s, 8% = 13.9 ? 3.0s) for each treatment. The primary component of both VO2 and ?HHbMb on-kinetics were characterized by a monoexponential function. Thus in the presence of unchanged (or "matched") convective O2 delivery with arterial hypoxemia, skeletal muscle V? O2 on-kinetics were not significantly impaired indicating there is not a peripheral diffusion limitation due to lower mean capillary PO2. iii Acknowledgments My first and most important I must say thank you to my loving wife, Peggy, for her tolerance, support and willingness to try new things. Without her, this non-traditional student would not have been able to return to Auburn University to pursue this degree. To Dr. L. Bruce Gladden, a teacher, mentor and friend, thank you. Dr. Gladden provided insight, sage advice and encouragement throughout my course of study, allowing me to learn and grow in the field of exercise physiology. Thank you to my committee members, Dr. David Pascoe, Dr. John Quindry and Dr. James Sartin. They provided excellent guidance, feedback, critiques and support throughout this project and my graduate experience. Thank you to Dr. Dean Schwartz who served as outside reader in spite of busy schedule. His critiques and guidance were essential to the finished product. I also need to thank Dr. Mary Rudisill and the Department of Kinesiology for giving me opportunities not usually available to a graduate student. And while there were times I wondered if perhaps I was too quick in accepting the challenges, in the end it made the entire graduate experience much better. Many thanks to fellow graduate students in our laboratory, Dr. Matthew Goodwin and Dr. Andres Hernandez, and Yi Sun. Beyond the Auburn I need to thank Dr. Nicola Lai, Case Western University, Dr. Bruno Grassi, University of Udine and Dr. Michael Hogan, University of California, San Diego for their assistance and guidance during research. Finally, I must return to family, my wife and my three daughters, who are all Auburn grad, so say thank you again for supporting my dream and not letting me forget what?s important in life. iv Table of Contents Abstract .......................................................................................................................................... i Acknowledgments........................................................................................................................ iii List of Tables ................................................................................................................................ v List of Figures .............................................................................................................................. vi I. INTRODUCTION ................................................................................................................ 1 II. LITERATURE REVIEW ...................................................................................................... 4 V? O2 On-Kinetics ............................................................................................................... 4 Metabolic Activation ..................................................................................................... 12 Oxygen Delivery Limitations ........................................................................................ 19 A New Model for V? O2On-Kinetics ................................................................................ 25 Hypoxia Studies .............................................................................................................. 29 Purpose ............................................................................................................................. 35 III. JOURNAL MANUSCRIPT................................................................................................ 36 Abstract ........................................................................................................................... 36 Introduction ..................................................................................................................... 38 Methods and Procedures ................................................................................................. 43 Results ............................................................................................................................. 49 Discussion ....................................................................................................................... 59 Conclusion .................................................................................................................................. 65 References ................................................................................................................................... 67 Cumulative References ............................................................................................................. 72 v List of Tables Table 1, Journal Format: Blood Gas Concentration .................................................................. 50 Table 2 Journal Format: Convective O2 Delivery ..................................................................... 51 Table 3 Journal Format: V? O2 on-kinetics data .......................................................................... 54 Table 4 Journal Format: Near Infrared Spectroscopy (NIRS) data ........................................... 57 Table 5 Journal Format: Force and Fatigue data ...................................................................... 59 vi List of Figures Figure 1: Typical V? O2 Response to change in work rate ............................................................. 7 Figure 2: Pulmonary response to change in work rate ................................................................. 8 Figure 3: VO2 on-kinetics graph ................................................................................................ 11 Figure 4: Oxidative Phosphorylation in the mitochondria ......................................................... 15 Figure 5: Metabolic factors and O2 delivery .............................................................................. 26 Figure 6: New Model of O2 delivery dependence ..................................................................... 27 Figure 1 Journal Format: Mean CaO2 by condition ................................................................... 51 Figure 2 Journal Format: Mean blood flow by condition ......................................................... 52 Figure 3 Journal Format: Mean convective O2 delivery by condition ....................................... 53 Figure 4 Journal Format: Mean contraction-by-contraction V? O2 response by condition??...55 Figure 5 Journal Format: Normalized V? O2 response by condition???????????56 Figure 6 Journal Format: Average NIRS response by condition ............................................... 59 1 I. INTRODUCTION Movement, whether it involves foraging for food, running for your life or scratching an itch, requires muscles to contract. Muscles require some form of energy to power these contractions and this energy comes from the hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and inorganic phosphate (Pi). At rest the levels of ATP are relatively stable, about 5-8 mmol per kilogram (Kg) of wet muscle tissue (15). However, once muscles begin to contract, ATP is used rapidly and must be rapidly replaced for contractions to continue. ATP replenishment comes from three main chemical sources: the phosphogen system, also known as the immediate system, anaerobic glycolysis/glycogenolysis, and oxidative phosphorylation. The latter is the most important source of ATP replacement for mammals (129). Oxidative phosphorylation has the capacity to create substantially more ATP than other metabolic systems; however, there are limitations to the system (34, 46). As early as 1913 Krogh and Lindhard (100) described these ?changes in ventilation, blood flow, pulse rate, respiratory exchange and alveolar CO2 tension,? at the onset of light and heavy exercise. From this starting point, investigations have looked at humans and animals working and exercising in various environments to access V? O2. Studies have examined; maximal O2 consumption (V? O2max for whole body work/exercise or V? O2peak for isolated muscle), the rate of recovery to resting O2 consumption levels after cessation of that work/exercise and the relatively slow O2 uptake kinetics at the beginning of work/exercise. Krogh and Lindhard (100) labeled the relatively slow rise in O2 consumption at the beginning of exercise as an ?oxygen deficit.? They went on to say that the O2 deficit was not compensated for during exercise but 2 was repaid after work ceased, as an ?O2 debt? (99, 101). Some 40 years later, in the 1950s the time course of the oxygen uptake was described by Henry et al. (61, 83, 138). Later, in 1970, di Prampero et al.(61) and Whipp et al. (142) described O2 deficit as having a mono-exponential time curve during moderate work rates. O2 deficit is now often described in terms the rate of change or time it takes to reach steady state V? O2 and thus called oxygen uptake kinetics or V? O2 on-kinetics (46, 79, 85). From this early work, exercise physiologist have continued to attempt to identify the factor or factors that limit the increase in O2 consumption during the transition from rest to work. The investigations have examined two main theories; 1) metabolic activation ? which states that there must be a buildup of metabolic substrates in the muscle before oxidative phosphorylation is turned on - a feedback system (45) and 2) O2 availability - which states that the finite time required to move O2 from the lungs to the muscle and then for the diffusion from the capillary to the mitochondria limits V? O2 - a feed forward system (115). Researchers have conducted experiments with humans and animals using a wide array of technologies to examine whole body, individual muscle and single muscle fiber responses during rest-work transitions. Previous research in our laboratory has used canine skeletal muscle in situ, a model originally used by Stainsby et al. (131) and Piiper et al. (112). This model offers several advantages including; control of blood flow (thus O2 delivery) to the muscle, the ability to measure both arterial and venous O2 saturation at the muscle to derive muscle oxygen consumption (V? O2M) and the ability to stimulate the muscle tetanically, recruiting all muscle fibers during each contraction. The research from our lab has examined V? O2 on-kinetics using convective O2 delivery (44, 49, 52), chemical treatments (50-51, 53, 57) and different exercise intensities (52, 62). The current study will use this same model to examine V? O2 on-kinetics in normoxia and 3 hypoxia with matched convective O2 delivery in an attempt to ascertain if there is an O2 delivery or a diffusion limitation of O2 that is responsible for the a slowing of V? O2 on-kinetics in normally seen under hypoxic conditions. 4 II. REVIEW OF LITERATURE V? O2 ON-KINETICS Humans are rarely in a steady metabolic state. The normal activities of life require movement and movement requires muscle contractions. During the greater part of the day human metabolic energy demand is in transition, i.e. going from sitting to walking or from walking to a more vigourous movement. Traditionally ATP demand during these transitions has been characterized as an immediate square wave increase (Fig. 1). Recent studies (148) has questioned the square wave demand, describing a slower rise in ATP turnover during the first 25 seconds of contractions. This needs to be investigated further, however, whether the increase is square wave or slightly less rapid, there is an immediate increase in ATP demand at the onset of contractions that must be met. In skeletal muscle the supply of ATP is limited; ? 5-8 mmoles per Kg of wet weight and this ATP is only capable of supplying energy for a limited number of contractions. Estimates of ATP demand by muscles per contraction cover a wide range from .15 mM (13) to 3.0 mM (25). To allow contractions to continue ATP must be replenished and within mammalian bodies there are three main sources: phosphocreatine (PCr); anaerobic glycolysis and the comparatively slow, oxidative phosphorylation. ATP is hydrolyzed with each muscle contraction to ADP and organic phosphate (Pi). In the presence of PCr and creatine kinase ADP is recombined with inorganic phosphate from PCr to make ATP in a reversible reaction based on the law of mass action. The equation that describes the process is ADP + PCr ATP + Cr 5 reserves of PCr are higher than ATP in skeletal muscle but still only sufficient for few contractions (144). In all out exercise, such as a 200 meter running event, peak output occurs in ? 1 sec (40) and decreases rapidly over 30 sec(15). This reaction provides a ?temporal buffer? allowing both the glycolytic pathway and the aerobic pathway to begin to supply sufficient ATP to muscle (106). This ?metabolic capacitance? provides the muscle cell with readily available means to meet a wide range of energy demands (134). As PCr is providing ATP extremely quickly the glycolytic pathway is activated. Glycolysis/glycogenolysis, the conversion of glucose or glycogen to ATP produces pyruvate which in the presence of lactate dehydrogenases is quickly converted to lactate (41). Oxidative phosphorylation begins to rise with the first contractions and continues to rise depending on ATP demand. In order that oxidative phosphorylation works there needs to be synchronization between the cardiovascular system, the pulmonary system and the tissues. Oxygen consumption or oxygen uptake is discussed in terms of oxygen uptake per minute or V? O2 and can described for the whole body by the Fick principle: V? O2 = Q x (CaO2 ? CvO2). Cardiac output, Q, represents blood flow to the tissue. Cardiac output is the result of heart rate (HR) times the stroke volume (SV) of the heart minus any peripheral resistance to the blood flow within the arteries. CaO2 represents the arterial blood oxygen content that is being delivered via arteries to arterioles and then to capillaries where O2 can diffuse into tissue cells. Arterial oxygen content depends on alveolar oxygen pressure (PO2), hematocrit, hemoglobin (Hb) and the ability of Hb to bind to available oxygen (47, 85). CvO2 represents mixed venous blood that consists of blood that has off loaded O2 to the cells and blood that has not. The difference between the O2 concentration in arterial blood and mixed venous blood gives the oxygen extraction by the tissue. This equation can be applied to the whole body or to a particular set of 6 tissues such as the liver, heart, or muscles. In previous research in our laboratory we have been investigated V? O2 in isolated muscle in situ, examining muscle blood flow to a muscle, QM, and the arterial and venous difference across that muscle. With an increase to in muscle energy demand such as going from a resting state to some less than exercise intensity below V? O2 max, oxidative phosphorylation normally meets the ATP demand in 2-3 minutes(88) . The rate change for oxidative metabolism can be seen in Figure 1. Note at the start of contractions, time 0, oxidative phosphorylation is providing ATP to meet previous steady state ATP demand and begins to rise with the new demand. In this stylized diagram, there is no time delay or Phase I (this will be discussed shortly) depicted and the rise in O2 consumption follows a curvilinear path that can be described by a monoexponential function. The area above and to the left of the oxidative area on chart the from start of contractions to the steady state is the oxygen deficit.(88) 7 Figure 1. (88) Stylized diagram showing typical VO2 response to a step increase in work rate to a new metabolic steady state. The initial ATP demand at the muscle is shown as a square wave and must be met by the muscle?s immediate energy systems. Under normal conditions oxidative phosphorylation meets the steady state energy demand after ? 2 minutes in healthy individuals. A recent paper has questioned if the ATP demand at the start of exercise is actually a square wave response (148) however whether square wave or a two step process, the ATP demand is sufficiently fast to result in an O2 deficit In humans, measurements of V? O2 are normally based on breath-by-breath gas analysis at the mouth, V? O2 pulmonary (V? O2 P). While this is an excellent way of looking at whole body exercise it makes analysis of on-kinetics slightly more complicated. This can be illustrated in Figure 2. In response to an increase in work-rate there is an initial cardiodynamic phase in which measured V? O2 (measured at the mouth) increases rapidly due to increased cardiac output and an increase venous return to the lungs which does not reflect muscle O2 consumption(85). The Phase I response is not normally modeled for V? O2 on-kinetics. In the Phase II, referred to as the 8 primary response, there is monoexponential increase in V? O2 to meet demand. This rise in oxygen consumption measured via pulmonary V? O2 has been shown to closely reflect muscle V? O2 (56, 123). Phase II is faster in aerobically trained individuals (2, 15, 137, 149) and is slower in sedentary. Also, several diseases such as cardiac failure, chronic obstructive pulmonary disease and peripheral artery disease interfere with normal V? O2 on-kinetics, slowing the response to increased work-rates (1, 7, 17, 45, 88, 110). In Phase III oxidative phosphorylation meets the vast majority of the muscle?s entire ATP demand (78) and at moderate work rates levels off to a new steady state of respiration. Figure 2. (88). Pulmonary O2 adaptation to a step increase in work-rate to a new steady state. Phase I is usually not modeled when examining on-kinetics and Phase II kinetics closely mirror muscle O2 kinetics. 9 At higher intensities, Phase III may exhibit a ?slow component? where VO2 continues to climb. When measuring V? O2 on-kinetics, work rates are usually broken into three categories; 1) work rate below the lactate threshold (LT), (ventilatory threshold (LT) or gas-exchange threshold (GET) are sometimes also used interchangeably in the literature), termed moderate exercise, 2) work rates above LT but below V? O2 max, often termed heavy, and 3) work rates at or above V? O2 max, termed severe or extreme (88, 141). If the work-rate is below the LT, then Phase III should reach a steady state energy production and the V? O2 will stabilize. Above LT, there is a ?slow component? which will be seen as a delay in reaching steady state. The slow component may represent an increasing inefficiency in producing ATP (85) but other mechanisms may play a role and remain one of the key questions in V? O2 on-kinetics (88). At or above V? O2 max, Phase III never reaches steady state and continues to climb until exhaustion (141). As stated above, V? O2 P during Phase II on-kinetics is normally considered to reflect muscle oxygen consumption within ? 10% when working with humans (56, 123). In order to more closely examine muscle on-kinetics other methods have now been developed. These methods include; measuring blood O2 difference across a group of muscles such as the quadriceps in humans (6, 56, 118), or measuring blood O2 difference directly at the muscle in animal models with either muscles in situ, or in isolated muscles or using single muscle fibers in vitro (43, 49, 64, 112), Near-infrared spectroscopy (NIRS) allows investigators to examine the hemoglobin in the muscle to determine the extent of O2 saturation during the initiation of exercise (14, 29, 36, 55). Other methods including phosphorescence quenching techniques and nuclear magnetic resonance (NMR) spectroscopy have been used to examine muscles metabolites (70, 126), myoglobin desaturation (24, 122) and partial pressure of oxygen (PO2) in 10 capillaries and in the cell (117, 120) . These newer methods allow researchers to observe oxygen consumption in the muscle, providing a more precise way to assess on-kinetics. When evaluating on-kinetics, the rate of Phase II change is normally described by a monoexponential mathematical model. The equation to describe this monoexponential rise in O2 consumption follows: V? O2 (t) = V? O2 Bas + ?V? O2 (1 ? e-(t ? TD)/?) In this equation, V? O2 at any time (V? O2 (t)) is equal to the baseline V? O2, (V? O2 Bas) plus the amount of change in V? O2 (?V? O2) from the baseline, also referred to as amplitude, multiplied by 1 minus the exponential function described by the negative result of time (t) minus the time delay before the V? O2 starts to rise (TD) divide by a time constant tau (?). The time constant, ?, is the time required for the V? O2 to rise to 63% of steady-state value (123, 141). A stylized diagram of the on-kinetics with the TD and ? is shown in Figure 3. The time delay can represent the cardiodynamic phase of pulmonary measures but is also found in isolated muscle and has yet to be fully explained (51). Additionally, mean response time, (MRT), the time delay plus the time constant, is sometimes used in the literature to describe on- kinetics. Comparing tau or MRT allows for the comparison the on-kinetics of oxygen consumption under differing experimental conditions including varying work-rates, differing O2 delivery levels, differing substrate levels in the muscles and differing exercise modalities, to name a few methods used to investigate the response in terms of aerobic respiration and energy production by muscle mitochondria to a increased muscle ATP demand. 11 Figure 3. . Idealized from: Grassi B, Hogan MC, Kelley KM, Howlett RA, and Gladden LB. Effects of nitric oxide synthase inhibition by L-NAME on oxygen uptake kinetics in isolated canine muscle in situ. J Physiol 568: 1021-1033, 2005. Onset of contractions (Start); Time delay before a rise in V? O2 (TD). In the search to determine the controller/controllers of O2 consumption during exercise investigators have examined; humans doing whole body and single limb exercise (8, 11-12, 21, 27, 31, 56, 81, 86, 96), animals using whole body exercise (93), isolated animal muscle in situ (49, 63, 66, 112, 131), isolated single muscle fibers in vitro(63, 91, 139) and isolated mitochondria in vitro(145-146). Despite increasingly sophisticated technology, there remains considerable debate over exactly what controls V? O2 on-kinetics (47-48, 77-78, 83, 115, 138). The debate has focused on two main alternatives; 1) the metabolic activation hypothesis which proposes that a build up metabolic substrates within the mitochondria is required for there to be an increase in oxidative phosphorylation and thus O2 consumption - a feedback system (45), and 2) O2 delivery hypothesis which proposes the time required to move O2 from the lungs via blood tau 12 flow to the muscle and/or the time involved in diffusion from the capillary to the mitochondria limits V? O2 - a feed forward system (115). METABOLIC ACTIVATION Before one can discuss the controllers of on-kinetics from a metabolic standpoint it is important to review what is known about overall control of oxidative phosphorylation. Maintaining ATP turnover during muscle contractions requires a multistep process in the mitochondria, described by the chemosmotic model first proposed by Peter Mitchell (109). The details of the reactions and mechanisms involved in this process have been thoroughly described (74, 109), however a brief review follows. The majority of ATP hydrolysis takes place in the cytosol thus ADP and Pi must move from the cytosol to the mitochondria (144). The cytosol also supplies substrates (fats, pyruvate, other small carbon molecules and lactate(15)) to the mitochondria, where they are converted to reducing equivalents by ?-oxidation or the tricarboxylic acid cycle (TCA). The reducing equivalents, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2), donate electrons to the protein complexes in the electron transport chain (ETC) located on the inner membrane of the mitochondria. These electrons are passed between 4 complexes, I-IV. At complex I, III, and IV the energy from the electrons is used to pump H+ ions from the mitochondrial matrix to the intermembrane space between the inner and outer mitochondrial membranes. This creates an electrochemical gradient and establishes a proton motive force. The passage of the H+ ions from the intermembrane space through mitochondrial ATP synthase uses the supplied ADP and Pi to create ATP. The reactions that occur in the ETC can be seen in Fig. 4 and can be summarized by the equation; NADH + H+ +5ADP + 5Pi + O2 ---> 5ATP + NAD+ + 2H2O 13 Figure 4. Oxidative phosphorylation. Stylized depiction of mitochondria with both the inner and outer phospholipid bilayer and the intermembrane space. Within the matrix the citric acid cycle (TCA) supplies reducing substrate to the electron transport chain (ETC). NADH donates two electrons at complex I which leads to a H+ being pumped into the intermembrane space. Succinate donates electrons through FADH2 at complex II. As electrons from complex I and II move down the chain to Complex III and IV more H+ are moved into the intermembrane space. At Complex IV O2 acts as the electron acceptor and forms H2O. The H+ ions create a proton motive force that passively re-enters the matrix through ATP synthase allowing ADP and Pi to join together and form ATP. (109). Based on the reactions involved in mitochondrial repiration any of the following could be involved in regulation of oxidative phosphorylation; NADH, H+, ADP, Pi, ATP and O2. Additionally, because the TCA cycle supplies NADH, the regulatory steps in this cycle also may play a role in turning on oxidative phosphorylation. ADP stimulates cellular respiration and has been proposed as a regulator of oxidative phosphorylation (4, 23). The addition of ADP to isolated mitochondria in vitro maintained in a medium with high concentrations of Pi and substrates rapidly and significantly increases the rate of oxidative phosphorylation (4, 22). 14 Chance et al. (23) postulate that changes in [ADP] ?galvanize the function of the respiratory chain in response to energy needs?..? However, Balaban (4) proposed that the regulation of oxidative phosphorylation is more complicated within the cell than a simple feedback system based on [ADP] and [Pi]. He proposed that a more complex model oxidative phosphorylation were three relationships; 1) movement of ATP, ADP and Pi between the cytosol and the mitochondria, 2) the delivery of reducing equivalents, NADH and FADH, to the ETC, and 3) the availability of O2 at Complex IV are all involved in the regulation oxidative phosphorylation (4). Wilson (144) expanded on this view outlining three hypotheses for concerning metabolic mechanisms that may control of respiration rates. The three hypotheses are: 1) The ADP hypothesis, 2) the adenine nucleotide translocase (ANT) hypothesis, and 3) the near equilibrium hypothesis. In the first hypothesis, when [ATP] drops, [ADP] increases and this stimulates respiration. This is a simple feedback model seen in isolated mitochondria as described by Chance et al., (23). Wilson points out, that the ADP hypothesis is limited to situations where mitochondrial respiration is a function of [ADP] alone. Experimental evidence has demonstrated the rate to ATP production can be dependent on [Pi] rather than [ADP] thus may be too simplistic (135). The ANT hypothesis suggests mitochondrial respiration cannot be dependent solely on [ADP] because ADP must move from the cytosol to the mitochondrial matrix to be converted back to ATP. This means that the ratios of [ATP]/[ADP] and the availability of ANT to move ADP into the mitochondria determine the rate of cellular respiration. As with the previous hypothesis, there is no role for [Pi] which has been experimental shown to affect the respiration rate, thus it cannot adequately describe the process. The near equilibrium hypothesis states that the mitochondrial respiration rate is related to the ratio of cellular [ADP]?[Pi]/[ATP], and intra-mitochondrial [NAD+]/[NADH] and that theses substrates are at near equilibrium 15 normally. When exercise begins as stored ATP and PCr reserves are used the [ATP] goes down and [Pi] and [ADP] go up thus stimulating mitochondrial respiration. Combine with this changes in reducing substrate availability [NADH]/[NAD+], ?cellular energy metabolism is well controlled with respect to the rate of ATP synthesis?? (144) The metabolic activation hypothesis for on-kinetics states that there is sufficient O2 available to act as the final electron receptor for the ETC throughout the work-rate transition and that the finite time required for oxidative phosphorylation to reach steady state ATP production is limited by the same factors discussed above (48). Because the supply of NADH directly affected by the TCA cycle, allosteric ( Ca++ ions), enzymatic or acetyl CoA availability could also limit the on-kinetics (47). There are several enzymatic steps in the TCA cycle that could be rate limiting and thus limit the production reducing equivalents, NADH and FADH2. This is one of the first areas of experimentation used to evaluate metabolic activation of on-kinetics. The enzymes that can slow the TCA include; pyruvate dehydrogenase (PDH) that supplies the acetyl-CoA to the cycle, citrate synthase that converts acetyl CoA to citrate, isocitrate dehydrogenase which converts isocitrate to ?-ketoglutarate and ?-ketoglutarate dehydrogenase which converts ?-ketoglutarate to succinyl-CoA. Of these various limiting steps in the TCA cycle, the one that has received the most attention in terms of on-kinetics is PDH. PDH is a prime candidate for a metabolic controller based initially on experiments Timmons et al. (136). The group used dicloroacetate (DCA) infusion to raise pre-exercise PDH levels in humans. The subjects then performed leg kick exercise and O2 deficit was estimated based on the rate of PCr breakdown. The results showed 50% reduction in PCr breakdown during exercise transition with increased PDH levels prior to exercise. The group interpreted this to mean that the oxygen deficit was lower due to the 16 increased PDH providing increased acetyl-CoA to start the TCA cycle. Following this experiment, Howlett et al.(75) treated single frog muscle fibers in vitro with DCA. The treatment significantly increased PDH levels and speeded muscle fiber on-kinetics. These findings indicate that an increase in PDH raised the concentration of the first acetyl-CoA increasing TCA cycle production of NADH and speeding VO2 on-kinetics. Following the single muscle fiber experiment, Grassi et al., (51) used isolated canine muscle in situ treated with DCA to examine on-kinetics. Electrically stimulating the muscle from rest to ? 60% -70% of V? O2peak, the group found that while PDH was elevated the VO2 on-kinetics were not significantly different from a similar trial without DCA. They did find that the ?stockpiling? of acetyl groups in the DCA treated muscle was associated with reduced fatigue(51). Other studies using DCA with humans also showed no speeding of on-kinetics with increased PDH levels or increased aceytl CoA avialability (5, 87, 97, 124). The use of prior exercise has been shown to speed V? O2 on-kinetics in a subsequent bout of moderate exercise (59-60, 62). Gurd et al.(59-60), used moderate ? heavy- moderate cycling protocol to compare the V? O2 on-kinetics for a transition to a moderate work-rate with and without prior heavy (above LT) exercise. Between each exercise bout there was a period of 6 minutes of unloaded cycling. Following the bout of heavy exercise the V? O2 on-kinetics for the 2nd moderate bout of exercise were faster and there was a significant increases PDH levels prior to the 2nd moderate bout. However, the group was unable to determine if the raised level of PDH or another factor such as increased blood flow prior to the moderate bout was responsible for the speeding of the on-kinetics (59-60). The role PDH and acetyl group availability in speeding on-kinetics, remains unclear but elevated acetyl CoA availability at the start of exercise may reduce fatigue and increase efficiency (45). 17 Another part of the oxidative chain which could slow the on-kinetics is found at Complex IV of the ETC where cytochrome c oxidase acts to transfer electrons to oxygen, forming water. Nitric oxide (NO) performs many functions within the body but it is also known to be a competitive inhibitor for the oxygen binding site on cytochrome c oxidase (16, 116). Experiments by Kindig et al. in horses and Jones et al. in humans used an inhibitor of NO, nitro l-arginine methyl ester (L-NAME) and found faster on-kinetics at moderate, heavy , severe and extreme exercise (89-90, 93-94, 143). All of these experiments used pulmonary measures of V? O2 rather than measures of muscle V? O2. There has been only one experiment to date to investigate inhibition of NO in isolated muscle. Grassi et al. using their canine model with L- NAME found inhibition of NO did not speed on-kinetics (53). Further experiments are needed to clarify the effects of NO inhibition on cellular respiration (53, 115). As briefly described above, at the onset of contractions creatine kinase catalyzes the reversible reaction that rephosphorylates ADP to ATP. The enzyme creatine kinase has very high activity and when contractions begin PCr breakdown maintains the [ATP]/[ADP][Pi] ratio close to the resting levels. As discussed earlier [ADP] and [Pi] appear to be powerful stimulators of mitochondrial respiration, so the maintenance of [ATP]/[ADP][Pi] acts as temporal buffer at the start of muscle contractions. As contractions continue the reserves of PCr are rapidly used within a few contractions (144) and [ADP][Pi] rises quickly stimulating oxidative phosphorylation to meet the ATP requirements (58, 105-106, 134, 137, 139-140). The PCr system has been proposed as a controller of oxidative phosphorylation (12, 42, 57). Experimentally, Meyer working with NMR on rat gastrocnemius muscle found a linear relationship between PCr levels and ATP during muscle stimulation and proposed that creatine kinase acted as a chemical capacitor for muscle contractions (105). Later, Rossiter et al., showed 18 that PCr breakdown followed the same kinetics as phase II V? O2P on-kinetics in humans in moderate exercise and as such represented a good estimate of V? O2M (123). His group later went on to show that the ?slow component? of V? O2P on-kinetics, normally seen in heavy exercise above the LT, follows the same kinetics as PCr, again estimating within 10% V? O2M (125). Studies in vitro of isolated mitochondria found a linear relationship between total creatine availability and the time required to reach a steady state in mitochondrial respiration (42). Given this apparent buffering effect of PCr, inhibition of the reaction should result in a speeding of on-kinetics as suggested in the model by Korzeniewski and Zoladz (98). Kindig et al. (92), were the first to look at inhibiting CK in skeletal muscle using iodoacetimide (IA) in isolated frog myocytes. Their results demonstrated a faster drop in intracellular PO2, indicative of increased respiration with CK inhibition. They concluded that CK serves as a ?temporal buffer? to [ATP]/[ADP]?[Pi] ratio at the transition to a higher work rate thus slowing the on- kinetics (92). In 2011, Grassi et al.(57) demonstrated that inhibition of CK speeds V? O2 on- kinetics in an intact muscle in situ. Using isolated canine gastrocnemius complex that this group has used for several other on-kinetic experiments (49-53, 68-69), the group used IA to blocking CK and then stimulated the muscle at a rate to elicit ? 70% of V? O2peak. The results demonstrated faster VO2 on-kinetics with CK inhibition. David Poole, a leader in the field of oxygen uptake kinetics writing in the Journal of Physiology stated that the results ?provide original evidence that muscle creatine kinase constitutes a locus of control for V? O2 on-kinetics in mammalian muscle,? (57, 113). As presented, both pyruvate dehydrogenase and nitric oxide have had equivocal results as possible controllers of oxidative phosphorylation. There is some evidence that increasing acetyl CoA availability can reduces fatigue in a step increase in work-rate but it is unclear if it can 19 speed on-kinetics in either moderate or heavy-severe exercise. Experiments with humans and horses have shown faster on-kinetics with inhibition of NO but isolate muscle preparations have had opposite results. Further study measuring muscle V? O2 is required to determine if indeed NO may be a metabolic controller. The recent work for Grassi et al. (57) gives direct evidence of a metabolic control of mitochondrial respiration with the inhibition of creatine kinase leading to a rapid increase in [ADP]?[Pi]/[ATP] and a speeding of V? O2 on-kinetics. While more work remains to be done, it appears that metabolite ratios are essential to on-kinetics. However, the fact that there is a significant metabolic controller of cellular respiration does not mean that oxygen delivery is not also a regulator of the process. OXYGEN DELIVERY LIMITATIONS The O2 delivery limitation hypothesis postulates that the time required for oxidative phosphorylation to meet ATP demand in a transition from rest to a moderate work rate is the result of the finite time required to move of O2 from the lungs to the muscle cells and/or the diffusion of O2 across the cell to the mitochondria (138). The first line of support for this hypothesis comes from the fact that V? O2 on-kinetics is slower in many disease states that limit cardiac output, limit blood flow or limit O2 saturation. Such diseases include; heart failure (1), heart transplant (20, 54), peripheral artery disease (7), and chronic respiratory disease (110). VO2 on-kinetics is also slowed in sedentary populations and with increasing age (3, 30, 128). In both these cases it appears reduced cardiac output and thus reduced muscle blood flow plays a role. However, in the disease states, and with aging and sedentary individuals there may be metabolic changes in addition to the reduce ability to deliver O2. Another area where one sees slowed on-kinetics is with reduced O2 availability as experienced at altitude or by breathing less than 21% O2 (18-19, 130, 132-133). 20 In order to determine if O2 delivery is the main limiting factor several different methods have been used to reduce O2 delivery to the muscle including; altering cardiac output (Q) by slowing heart rate, changing the position of exercise to reduce stroke volume, chemically reducing the affinity of hemoglobin for O2, and adjusting the O2 content of inspired gases. ?- blockers can be used to reduce Q by inhibiting the sympathetic increase in HR by blocking ?1 adrenergic receptors, restricting HR acceleration and thus cardiac output (15). At the onset of exercise, HR increases initially by the removal of parasympathetic inhibition, vagal tone, to approximately 100 beats per min. The sympathetic nervous system increases the HR above 100 bpm up to the maximum HR (127). Petersen et al.(111) had human subjects transition from unloaded cycling to a work rate of 50% of V? O2max or 67% of V? O2max with propranolol, a ?- blocker, given intravenously and without, the control trial. Propranolol reduced HR and cardiac output and slowed VO2 on-kinetics at both work rates compared to control. The steady state VO2 was not significantly changed between the propranolol trial and the control. The slower HR reduced Q which reduced O2 delivery to the working muscles and slowed the muscle on-kinetics. The following year Hughson (76) used an oral dose of ?-blocker and a single step increase for unloaded pedaling to 100 watts and back to unloaded pedaling to look at both O2 deficit and debt. He found a significant slowing of on-kinetics with the ?-blocker presumably due to reduce O2 delivery (76). Another method to reduce blood flow to leg muscle in intact humans is to have the individual exercise in a supine position. When comparing upright exercise to supine exercise there is a smaller increase in SV with supine exercise (15). Convertino et al. demonstrated slower on-kinetics with supine cycling compared to upright cycling at a submaximal workload (28). In 1991 Hughson et al. (84) compared supine cycling exercise with upright cycling at 21 three different work rates including maximal effort. In all three cases there was a significant slowing of the O2 on-kinetics in the supine position. With reduced SV in the supine position Q would be lower (Q = HR * SV) and so O2 delivery to the muscle would be lower. To further examine the role of blood flow/O2 delivery on oxygen uptake, Hughson et-al. (82) looked at exercise in the forearm both below and above the heart level. They found blood flow kinetics were faster with the arm below the heart and slower when the arm was above the heart level. Reflecting the changes in blood flow kinetics the V? O2 on-kinetics were significantly slower with the arm above the heart level, pointing to a direct effect of blood flow on oxidative metabolism (82). These experiments demonstrated that reduced cardiac output leading to reduced blood flow does slow on-kinetics. However other experiments have demonstrated that cardiac output and muscle blood flow are usually faster than V? O2 on-kinetics in healthy individuals (33, 138), implying O2 delivery is faster than muscle O2 consumption. One way to examine muscle blood flow is to use thermodilution where cold saline is injected into the femoral vein at one point and then measured further along the same vein, the temperature change and the time distance between the two catheters gives the leg blood flow (LBF) (56). Grassi et al.(56) using this technique examined transitions from unloaded cycling to loaded cycling at a work-rate below LT in humans. During the trials the group also measured pulmonary V? O2 (V? O2p), VO2 across the leg muscles (V? O2leg), LBF, and a-vO2 difference across the leg. The results confirmed that V? O2p during phase II on-kinetics matches closely V? O2leg on- kinetics. The results also found that during pulmonary phase I within the first 10 to 15 seconds of onset bulk delivery of O2 does not appear to limit the leg V? O2 response (56). Bangsbo et al. (6) measured a-vO2 difference and blood transit time across the quadriceps muscle during transition from rest to intense single leg extension exercise with similar results. They found 22 increased LBF and V? O2p within the first 10 -15 sec without a significant increase in VO2leg and a drop in a-vO2 difference after which (in phase II) the on-kinetics at the leg was monoexponential (6). This increase in LBF without an increase in V? O2leg demonstrates that O2 delivery to the leg is faster than O2 demand. In 2003, Grassi et al (55) returned to the cycling model, this time using near infrared spectroscopy (NIRS) to assess oxygen usage in the vastus lateralis muscle during transition from light cycling to a work-rate below LT and above LT. NIRS uses two infrared wavelengths to examine the oxygenation of the heme groups in the blood and muscle. The system is able to track de-oxygenation of hemoglobin and myoglobin (?HHbMb) as well as oxygenated hemoglobin and myoglobin (O2HbMb) and total hemoglobin/myoglobin (THb/Mb). The group found a significant correlation between the mean response time of the ?HHbMb signal from NIRS and the time constant, tau, of V? O2p. During the first 6 ? 10 seconds of transition there was no increase in VO2P neither was there a change in the ?HHbMb signal indicating that more oxygen was available at the muscle than was needed for oxidative phosphorylation (55). This implies that here is a metabolic delay. In an attempt to precisely examine V? O2mus, Grassi et al. (49) used the canine gastrocnemius - superficial digital flexor complex (GS) in situ to examine the relationship between convective O2 delivery and muscle on-kinetics. The experimental protocol consisted of two trials with GS complex electrically stimulated to elicit a step increase from rest to a work- rate at ? 60% - 70% of peak V? O2. In the first trial blood flow was provided to the contacting muscles spontaneously by the animal?s own heart. In the subsequent trial the blood flow was raised by pump perfusion to match the steady state level in the spontaneous trial prior to the start of contractions. This increased O2 delivery to the level needed to sustain ATP production at steady state V? O2 prior to the start of contractions. The a-vO2 difference was measured across the 23 muscle to determined V? O2M. The results showed no significant difference in the on-kinetics between the two treatments and the authors suggested that this supports the hypothesis that metabolic factors are responsible for the on-kinetics timeline (49). Later this same group duplicated these experiments with the muscle contracting at V? O2peak (52). The results from these experiments showed faster on-kinetics and an ? 30% reduction O2 deficit with speeded convective O2 delivery. The group concluded that at V? O2peak oxygen delivery along with metabolic factors contribute to the VO2 on-kinetics.(52) Using isolated rat spinotrapezius muscle and examining the fall in muscle O2 partial pressure (PO2) Behnke et al. (9) found that blood flow and O2 delivery were faster than the on-kinetics of VO2M and the group concluded that oxygen delivery is not limiting factor for on-kinetics in healthy muscle(9). These experimental results support the hypothesis that under O2 delivery is faster than the V? O2 on- kinetics of the muscle and that it is metabolic factors that limit on-kinetics. Another method to test this hypothesis is to raise O2 delivery by having subjects inspire a hyperoxic gas, 50% - 100% O2. Hughson and Kowalchuk (80) had healthy human volunteers perform a series of rest to work transitions breathing different gas mixtures; 14% inspired O2, hypoxia, 21% inspired O2 normoxia and 70% inspired O2, hyperoxia. They found slower on- kinetics in the hypoxia trial but no difference between normoxia and hyperoxia on-kinetics at a moderate level of exercise. Grassi et al. (50) working again with isolated canine gastrocnemius in situ attempted to speed V? O2 on-kinetics by having the animal inspire 100% O2 and in a second trial using RSR-13, a drug shown to right shift the oxyhemoglobin dissociation curve and 100% O2 . This should allow for increased O2 diffusion from the capillary into the working muscle. As with the previous canine experiments the muscle was stimulated to a moderate work-rate, 60% -70% of V? O2peak and the Q was controlled by pump perfusion set to a level equal to a 24 spontaneous steady state blood flow. Comparing normoxia, hyperoxia, and hyperoxia with RSR?13 there was no difference in the V? O2 on-kinetics (50). The authors concluded that enhanced peripheral diffusion does not affect V? O2 on-kinetics at moderate work rates and in normoxia (50). In another experiment examining the role of hyperoxia MacDonald et al. (104) examined both V? O2p and V? O2leg during human leg kicking exercise. They found that hyperoxia did not speed the O2 response at the alveoli or at the leg during the transition to moderate exercise. It appears that an increase in inspired O2 and increased O2 availability does not change V? O2 on-kinetics in moderate exercise again supporting the metabolic activation hypothesis. Another method used to speed V? O2 on-kinetics is the use of ?priming exercises?, a bout of moderate or heavy intensity exercise followed by a short recovery at rest and then a transition to moderate exercise again with the on-kinetics of the second bout of exercise compared to a on- kinetics single transition from rest to a moderate work rate. Gerbino et al. (39) used warm-up exercises below LT and above LT to examine the subsequent exercise bout on-kinetics to a work rate below LT and above LT. The neither priming exercise had an affect on the on-kinetics of the subsequent sub LT exercise. Only supra-LT priming exercise speeded on-kinetics in a subsequent bout of supra LT exercise. The authors suggested that improved muscle perfusion and increased pH were responsible for the faster kinetics (39). MacDonald et al. (103) found similar results where only a prior exercise above LT exercise speeded the on-kinetics of a subsequent exercise bout also above LT. This group also had subject inspire a gas mixture with 70% O2 in addition to priming exercise. They found even faster on-kinetics in the second bout exercise above LT but no difference in a second bout below LT. Their findings support the idea that below the lactate threshold, VO2 on-kinetics are not limited by oxygen delivery however 25 above LT O2 delivery does play a role(103). Other authors (Sheuermann et al. (128), Gurd et al(59-60), Herndandez et al. (62)) have also found speeding of on-kinetics after a set of priming exercises, however the underlying mechanisms are not clear and may be due to increased blood flow prior to the second bout caused by vasodilation, increased muscle pH, or the activation of PDH. Behnke et al. (10) examining muscle PO2 following priming contractions found a shorter time delay (TD) but an unchanged the time constant, ?, for the second bout of exercise. They also found that muscle PO2 was no different between bouts and thus attributed the faster on- kinetics (TD + ?) to metabolic changes in the muscle mitochondria. A NEW MODEL FOR V? O2 ON-KINETICS The above experiments appear to show that oxygen delivery is not a limiting factor in normoxia and that elevated O2 delivery at the onset of contractions does not speed V? O2 on- kinetics at intensities below LT. However, experiments using hypoxia and using hyperoxia in exercise above LT appear to show there are limitations caused by O2 delivery. Thus it appears that VO2 on-kinetics is under the control of both metabolic factors and O2 delivery (24, 79, 88) . Wilson et al. (144, 147) demonstrated that in isolated mitochondrial respiration rate is sensitive to changing intracellular PO2 (PIO2 or PmitoO2) as well as metabolic factors. As discussed earlier in this paper, the metabolic mechanisms for control of respiration rates can be described by three hypotheses: 1) The ADP hypothesis, 2) the adenine nucleotide translocase (ANT) hypothesis, and 3) the near equilibrium hypothesis. The near equilibrium hypothesis appears to give the best explanation of what in occurring in the cell in vivo. The hypothesis proposes that in a steady state, the redox potential of NADH is in equilibrium with NAD+ and that ATP is in equilibrium with ADP and Pi. It goes on to proposes that the rate determining step in respiration occurs at Complex IV in the electron transport chain where O2 is converted to H2O a step that is 26 irreversible. This means that the [ADP]?[Pi]/[ATP] ratio and intramitochondrial [NAD+]/[NADH] along with PO2 interact to control respiration (144). Mitochondrial respiration can occur across a range of PO2 depending on these two ratios. When PO2 is low as might be seen in severe exercise high [ADP]?[Pi]/[ATP] (or intramitocondrial[NAD+]/[NADH]) are required to be elevated to maintain respiration rate. At rest or low work rates with high PO2 and low [ADP]?[Pi]/[ATP] can maintain respiration. A graphic depiction of this can bee seen in Figure 5. Figure 5. Predicted dependence of mitochondrial respiration on partial pressure of O2 and metabolic factors. At rest based on the near equilibrium hypothesis the ration of [ADP]?[Pi]/[ATP] is low near equilibrium. As exercise increase [ATP] drops increasing the ration an allowing respiration to continue with very low PO2. Adapted from Gladden, and Wilson et al. (145) Hogan et al.(64), using the canine GS complex in situ, altered the arterial partial pressure of oxygen (PaO2) during transitions from rest to two different work-rates. The group measured 27 changes in [PCr], [ADP] and [ADP]?[Pi]/[ATP] and found that as PaO2 was reduced, there was greater [PCr] and [ADP] required to meet demand, supporting the Wilsonian model. Applying these findings to VO2 on-kinetics, Poole and Jones (114) have proposed a new model based on a continuum O2 delivery. The continuum includes boht O2 independent on- kinetics and O2 dependent on-kinetics (Fig. 6). In O2 independent zone includes the experimental findings from moderate upright cycling exercise, and various results animal studies; horse exercising in moderate and heavy domain, rat muscle stimulated in the moderate domain and canine GS at moderates rates. The O2 dependent zone includes on-kinetics found in disease, supine cycling, hypoxia and slowed blood flow (114). The authors proposed that at some point on the continuum there is a transition from O2 delivery independent on-kinetics to O2 delivery dependent kinetics and that his constitutes a ?tipping point? beyond which both metabolic and O2 factors are important (114). 28 Figure 6. (88) Representation of limitation of VO2 on-kinetics base on muscle O2 delivery. The O2 independent zone reflects normoxia in healthy humans subjects working at moderate work-rates. The O2 dependent zone represents on-kinetics in individuals with disease, limited oxygen as in hypoxia and supine exercise. Goodwin et al.(44) working in our lab and using the isolated canine GS in situ conducted a series of experiments to attempt to determine the point at which on-kinetics become O2 dependent. Using pump perfusion, convective O2 delivery was slowed by slowing blood flow on kinetics. The experiment used a control blood flow delivery rate based on spontaneous delivery determined prior to the experimental trials. The control was compared with two other treatments, blood flow kinetics slowed by 25 and blood flow kinetics slowed by 50 seconds. The resting blood flow and steady state blood flows where matched so that only the blood flow on- kinetics was different. The slower blood flow kinetics altered the convective O2 delivery. With the slower blood flow kinetics the V? O2 on-kinetics were also slower. Indeed when the mean 29 response time for the three treatments was plotted the results were linear with an R = 0.96. The results appeared to match the predicted O2 dependent model. The authors concluded that spontaneous blood flow to the canine GS operates at ?the nexus of sufficient and insufficient O2 delivery,? in the transition to a moderate work-rate. (44). HYPOXIA STUDIES Reducing the percentage of inspired O2 has been used in both humans and animal models to examine the role of reduce PO2 has on oxygen consumption at rest and during exercise and to examine VO2 on-kinetics. Linnarsson et al., (102) were the first look at the impact of reduced inspired O2 at the start of exercise. They found increased O2 deficit with hypoxia. Several years later Murphy et al.(108) compared V? O2 on-kinetics in normoxia and hypoxia by having eight human subjects perform a step transition test on a cycle ergometer while inspiring either a gas with 14 % oxygen and normal air. The steady state V? O2 reached during both treatments was not significantly different however the on-kinetics were significantly slower with the hypoxic treatment. Because blood flow kinetics where the same in the step transitions the authors concluded that the delay was due to the lower arterial O2 concentration (108). Following these findings, Hughson and Kowalchuk (80) examined V? O2 on-kinetics in 6 young men performing cycle ergometer exercise at maximal and sub-maximal levels. The subjects inspired air with O2 percentages of 14%, 21% and 70%. The study found that hypoxia limited maximal V? O2 compared normoxia and hyperoxia. There was no difference in V? O2max between the normoxia and hyperoxia treatments. The hypoxic condition also had significantly slower VO2 on-kinetics compared to the other two conditions (80). Springer et al. (130) compared the changes in ventilation and heart rate response to exercise between adults and children during hypoxia and normoxia. They found that not only are V? O2 on-kinetics slowed but also the heart rate response 30 to exercise (and thus cardiac output) was also slowed in the hypoxic condition. Engelen et al. (35) followed these experiments and examined what happens in heavy exercise with hypoxia. Again using a cycling protocol, subjects inspired air 21%, 15% and 12% oxygen. The cyclist performed work above the LT based on an incremental test to V? O2peak with all three gas mixtures. As with other experiments V? O2 on-kinetics was slowed in the hypoxic conditions however the steady state V? O2 was not different. Additionally, the heart rate was higher at the start but had the same kinetics between treatments indicating that cardiac output could not meet the O2 delivery demand in hypoxia. There was no difference in the steady state V? O2 reached during in any of the treatments and no difference between treatments in the slow component. The group concluded that hypoxia only affects Phase II In 2000, MacDonald et al. (104) used had subjects inspire gas mixtures with 21%, 14%, and 70% oxygen and however rather than using a cycle ergometer they used a step transitions from rest to moderate single leg kick (weighted leg extensions) exercise. Compared to two-leg exercise (cycling), single-leg exercise has been shown to allow significantly higher leg blood flow (LBF)(15) which should increase O2 delivery. The leg kick exercise also allowed the researchers to measure LBF via pulsed and echo Doppler. Additionally they measured the O2 difference at the radial artery and femoral vein. Using the LBF and the radial artery O2 content (CaO2) and femoral vein O2 content (CvO2) to solve the Fick equation they determined V? O2leg as well as measuring VO2P. At rest there were no differences between V? O2p or V? O2leg in hypoxia, normoxia or hyperoxia, despite significantly different CaO2. Once exercise began the V? O2p and the V? O2leg on- kinetics was not significantly different between treatments. The authors postulated that the lack of difference in on-kinetics was due to increased leg blood flow and O2 extraction at the muscle compensating for lower CaO2 in the case of hypoxia (104). Using a similar protocol, Delorey et al. (32), added 31 NIRS to the experiment using the ?HHbMb in the vastus laterialis muscle during exercise as a surrogate for V? O2mus. This group had earlier shown that ?HHbMb accurately reflects O2 delivery and consumption across the working muscle. They stated that when combined VO2P and leg blood flow this (?HHbMb) ?allows for the determination of the time course for local muscle O2 utilization (31). In the hypoxia experiment the subjects initially performed incremental leg kick tests while breathing air with 21% oxygen and then a second test breathing 12% oxygen. These tests established the LT and the moderate work-rate to be used in the trials. On separate days, the subjects returned to perform the step transition from unloaded leg kicks to their pre-established moderate work-rate while breathing either normal air or the hypoxic mixture. Blood flow was measured at the femoral artery and with leg O2 delivery calculated based on LBF and CaO2. The NIRS optical device was placed on the vastus lateralis with the optical sensors set at optimum distance. The results showed VO2P was significantly slower in the hypoxic condition. However, the ?HHbMb response was not significantly different between normoxia and hypoxia. These experimental findings demonstrate that with normal blood flow in hypoxia there is slower on-kinetics. However, the results from MacDonald et al. raise the question of can increased blood flow as seen in single-leg exercise increase O2 delivery sufficiently to compensate for low CaO2. Beyond human studies the impact of low PO2 seen in hypoxia has been studied in muscle in situ and with single muscle fibers in vitro. The findings have helped clarify the results from human studies but the picture is not entirely clear. Hogan and Welch (73) compared hypoxic, normoxic, and hyperoxic conditions in the isolated GS during fatiguing stimulation. They found similar V? O2 and fatigue in all three conditions but there was a significantly different acid-base balance values (73). King et al. (95) used the same muscle preparation and 9% O2 to 32 examine the O2-deficit with various stimulation rates using twitches. They found at using moderate twitch stimulation (2Hz) the O2 deficit was not different between normoxia and hypoxia but at higher stimulation rates , there was a difference. They proposed that at the lower stimulation rates increased blood flow and that along with increased extraction of O2 at the muscle compensated for lower blood O2 content in hypoxia. However at higher stimulation rates these mechanisms could not keep up with demand(95). The picture that emerges from these experiments is that here may be a limitation of not just O2 delivery but also a limitation in diffusion of O2 from the blood to the muscle. Diffusion at the working muscle is described by Fick?s law of diffusion V? O2 = DO2 (PcapO2 ? PmitoO2) where DO2 is the lumped diffusion capacity, PcapO2 is capillary PO2 and PmitoO2 is the PO2 inside the muscle fiber (also called intracellular PO2). Gayeski et al. (37-38) have found intracellular PO2 (PiO2) to be uniform in the muscle fiber suggesting that PmitoO2 would be the same or very close to intracellular levels. Intracellular PO2 measured both in vivo and in situ have been found to be extremely low during exercise, in the range of 1-4 Torr (26, 37, 107, 122). In canine grasilis muscle flash frozen at maximum work rate to be PiO2 was found to be less than 0.5 Torr (38). However even at this low level the PO2 is sufficient to allow full mitochondrial respiration which in situ is estimated to be as low as ? 0.5 Torr (27). Richardson et al. (117, 119- 122) in a series of experiments using H1NMR to examine myoglobin saturation in working human leg muscles during single-leg knee extensions have found that PcapO2 is ? 11 Torr lower in 10% - 12% hypoxia compared to normoxia however the difference in PiO2 was only 1 Torr meaning that the pressure gradient between the capillary and the muscle remains relatively high even in hypoxia. Further they found that while PiO2 drops from rest to exercise it appears to 33 plateau at 50% - 60% exercise intensity and remains at the lower level up to V? O2peak (120, 122). They concluded that the driving pressure from blood to cell is similar in hypoxia and normoxia (117). They went on to propose that diffusion capacity increases with hypoxia to allow for increases in VO2 (117). Hogan et al. performed a series of experiments using the canine GS (64, 67, 71-72) to examine the oxygen diffusion at V? O2peak in hypoxia. The group hypothesized that the lower arterial PO2 found in the capillaries under hypoxic conditions would limit O2 diffusion into the muscle fiber and thus the mitochondria. The first of these experiments used three different levels of hypoxia (12%, 8% and 5% O2). Blood flow was controlled with a pump and the inflow blood pressure was held constant. This resulted in slightly higher muscle blood flow (MBF) in the different hypoxia conditions which the authors attributed to vasodilation caused by the level of hypoxia. They found a linear relationship V? O2peak and O2 delivery at the muscle with the lowest delivery. They also found a similar linear relationship between V? O2peak and PvO2. They did not find a linear relation between PcapO2 and peak V? O2 which was predicted by Fick?s law. The authors speculated that this may have been due to the increased blood flow due to vasodilation. They stated that their results were consistent with the hypothesis that V? O2peak is limited by O2 diffusion in the working muscle (71). In order to examine the PcapO2 and VO2peak relationship and the authors repeated the experiments however this time holding MBF constant (65). Here again they found linear relationship between V? O2peak and both PvO2 and O2 delivery with the lowest inspired O2 content resulting in the lowest V? O2peak. They also found that there was a linear relationship between mean PcapO2 and V? O2peak, with the lowest PcapO2 resulting in the lowest VO2. They proposed that difference between the results from the two experiments was dus to increased diffusion capacity with the higher blood flow in the first 34 experiment. By holding blood flow stable all treatments had the same diffusion capacity and this lead to the linear relationship between PcapO2 and V? O2peak (65). The group went on to examine the relationship between PaO2 levels and metabolic factors that might affect mitochondrial respiration (64). Using to different stimulation rates, one designed to elicit V? O2peak and the another designed to be ? 60%-70% of peak, with normoxia, mild hypoxia (10% O2) and severe hypoxia (7% O2), they examined the oxygen consumption. They found that even at the sub-maximal work rates that VO2 was significantly lower in the two hypoxic conditions. Again in these experiments blood flow was kept at a constant perfusion pressure so that O2 delivery was lower in hypoxia (64). 35 PURPOSE Based on the previous discussion there may be a diffusion limitation at the muscle with low PO2. However, MacDonald et al. (104) demonstrated that with increased LBF in single-leg exercise there is no difference in V? O2 or V? O2 on-kinetics between hypoxia and normoxia which argues against a diffusion limitation. Delorey et al. (32) using the same protocol found slower V? O2 pulmonary on-kinetics however the NIRS data supports the idea that O2 consumption within the muscle was the same in hypoxia and normoxia. On the other hand the canine studies of Hogan et al. support the idea that with low blood PO2 associated with hypoxia leads to a diffusion limitation at least at V? O2peak. However, in these studies MBF remains relatively low and thus O2 delivery is significantly lower in the hypoxic condition. Given that even in hypoxia PiO2 appears to remain above the minimum required to have maximal mitochondrial respiration (117, 120) even working at V? O2max. And that in hypoxia there remains a pressure gradient between the capillary and the muscle fiber that should allow diffusion (117, 121). Then increased O2 delivery should be able to compensate for low CaO2. My hypothesis is that at a moderate work rate muscle V? O2 on-kinetics will not be different between normoxia and hypoxia if convective O2 delivery to the muscle is matched, demonstrating there increased muscle blood flow can overcome any possible diffusion limitation. Therefore the purpose of this experiment is to examine V? O2 on-kinetics under normoxia, hypoxia and severe hypoxia while holding convective O2 delivery constant to evaluate if there are a diffusion limitations between the capillary and the muscle 36 III. Journal Manuscript V? O2 ON-KINETICS IN SKELETAL MUSCLE IN NORMOXIA AND HYPOXIA WITH MATCHED CONVECTIVE O2 DELIVERY ABSTRACT The aim of this investigation was to evaluate O2 uptake (V? O2) on-kinetics under hypoxic conditions while holding convective O2 delivery constant in isolated canine muscles in situ. Canine gastrocnemius muscles (n=7) were surgically isolated and then electrically stimulated via the sciatic nerve (8 V, 0.2-ms duration, 50Hz, 200-ms trains) eliciting contractions at a rate of 1 contraction every 2 seconds (about 70% of peak V? O2). The dogs inspired 3 different O2 fractions: 21% (normoxia), 12% (hypoxia), and 8% (severe hypoxia). Convective O2 delivery [blood flow (Qm) x arterial O2 content (CaO2)] to the muscle was held constant by elevating muscle blood flow via a perfusion pump. Blood flow was recorded with an ultrasonic flowmeter while venous O2 saturation was monitored continuously in order to allow measurement of contraction-by-contraction muscle VO2. Relative muscle deoxygenation (?HHbMb) was assessed with continuous near-infrared spectroscopy (NIRS). Arterial blood PO2 (torr) was measured during each stimulation period and the mean per condition was 111 ? 11 for 21% inspired O2, 41 ? 3 for 12% inspired O2, and 30 ? 3 for 8% inspired O2. Venous blood PO2 (torr) measured prior to the end of contractions was significantly different (P< 0.05) among the three conditions (21% = 20 ? 5, 12% = 16 ? 5, 8% = 13 ? 5). The arterial O2 content (CaO2) was significantly lower in the hypoxic conditions and pump perfusion was significantly higher in the 37 hypoxic conditions resulting in matched convective O2 delivery (ml/Kg-1/min-1) that were not different (P > 0.05) among the three different O2 fractions, (21% = 202 ? 57, 12% = 206 ? 57 and 8% = 205 ? 59). There was also no difference in tau, the primary time constant for the VO2 on-kinetics among treatments (21% = 10.9 ? 2.4s, 12% = 10.1 ? 1.8s, 8% = 9.2 ? 1.9s ). Additionally, the NIRS data showed no significant time difference in the ? HHb time course, mean response time (MRT) (21% = 15.3 ? 2.7s, 12 % = 14.7 ? 4.4s, 8% = 13.9 ? 3.0s) for each treatment. The primary component of both VO2 and ?HHbMb on-kinetics were characterized by a monoexponential function. Thus in the presence of unchanged (or "matched") convective O2 delivery with arterial hypoxemia, skeletal muscle V? O2 on-kinetics were not significantly impaired indicating there is not a peripheral diffusion limitation due to lower mean capillary PO2. 38 INTRODUCTION The energy demand for contracting muscles during exercise at moderate intensity can normally be met by oxidative phosphorylation. However, at the onset of exercise there is a finite time before O2 consumption rises to a level necessary to support the energy demand. First discussed by Krogh and Lindhard (44), the rate of increase in oxygen consumption by the muscles (measured as V? O2) is characterized by a mono-exponential rise, reaching a steady state after 2-3 minutes (9, 66-67). At exercise intensities above the lactate threshold (LT) the V? O2 may continue to rise in what is described as a ?slow component? (64). The underlying reasons for the relatively slow response of V? O2 on-kinetics continues to be discussed and studied with focus on two hypotheses metabolic activation and O2 delivery (18-19, 34, 38). These two hypotheses are not mutually exclusion giving rise to a view that there is a spectrum responses some that fall in an oxygen dependent zone, while in other circumstances there is sufficient O2 present at the muscle but there is a metabolic substrate limitation (17, 39, 50). Evidence against an oxygen availability limitation is provided by several studies that show muscle blood flow kinetics to be faster than pulmonary V? O2 on-kinetics and faster than V? O2 measured across the muscle (1-2, 24, 28, 37). In a series of classic experiments using isolated canine gastrocnemius (GS) in situ, Grassi et al., demonstrated that elevated blood flow, raised to the required steady state level prior to contractions, did not change muscle V? O2 on- kinetics at moderate (60% of V? O2max) contractions rates(20) The group also demonstrated that even with hyperoxia and use of a drug to right-shift the oxyhemoglobin disassociation curve, which should increase diffusion, there was no speeding of on-kinetics(21). These two experiments suggest that under conditions of normoxia and hyperoxia, there are metabolic factors that limit the speed of the on-kinetics. 39 Experiments that support an oxygen availability limitation to on-kinetics usually restrict O2 availability to the working muscles by slowing heart rate (35, 49) or decreasing blood oxygen content via hypoxia, anemia, or carbon monoxide (10, 30-31, 61). Studies using inspired gas mixtures with less than 21% O2 conducted in humans and animals and appear to demonstrate that low CaO2 slows V? O2 on-kinetics. Linnarsson et al., (46) was the first to investigate the impact of reduced inspired O2 at exercise onset; he found an increase in the O2 deficit. Springer et al. (60) reported slower on- kinetics in both adults and children at the onset of moderate work rate exercise on a cycle ergometer with 15% inspired O2. Similarly, Engelen et al. (11) reported that pulmonary V? O2 (V? O2p) on-kinetics were slowed in hypoxia with heavy exercise, again on a cycle ergometer. Hughson and Kowalchuk (36) also observed a slowing of V? O2 on-kinetics when subjects inspired 14% O2 during the transition to moderate work rate on a cycle ergometer; they speculated that the slower kinetics were due to less O2 delivery to the muscle. These results taken together clearly support an oxygen availability limitation in exercise human muscle. Similar results are seen in experiments on isolated animal muscle. Hogan and Welch (33) compared hypoxic, normoxic, and hyperoxic conditions in the isolated canine GS during fatiguing stimulation. They found V? O2 rose to a similar level and fatigue was not different between the three conditions. However they did find significantly different acid-base balance values between the treatment that implies different levels of anaerobic metabolism in addition to aerobic metabolism(33). King et al. (43) used the same muscle preparation and 9% O2 to examine the O2-deficit with various stimulation rates. They found at using moderate twitch stimulation (2Hz) the O2 deficit was not different between normoxia and hypoxia but at higher stimulation rates, there was a difference. They proposed that at the lower stimulation rates 40 increased blood flow and that along with increased extraction of O2 at the muscle compensated for lower blood O2 content in hypoxia. However at higher stimulation rates these mechanisms could not keep up with demand(43). The picture that emerges from these experiments is that there may low CaO2 may not be only a delivery limitation but also a limitation in diffusion of O2 from the blood to the muscle. However the discovery by Saltin (59) that single leg exercise can significantly raise blood flow to the exercising limb above what is seen in two-legged exercise raised another opportunity to examined hypoxia and delivery versus diffusion limitation. Rowell et al., (58) found, using single leg kick exercise, that cardiac output can rise significantly increasing blood flow to meet the muscle metabolic demand in hypoxia. In 2000, MacDonald et al. (47) used this information to look at on-kinetics in single leg exercise. They had subjects inspire gas mixtures with 21%, 14%, and 70% oxygen and then used a step transitions from rest to moderate single leg kick (weighted leg extensions) exercise. The researchers measured leg blood flow (LBF) via pulsed and echo Doppler. Additionally they measured the O2 difference at the radial artery and femoral vein. Using the LBF and the radial artery O2 content (CaO2) and femoral vein O2 content (CvO2) to solve the Fick equation they determined V? O2leg as well as measuring V? O2p. At rest there were no differences between V? O2p or V? O2leg in hypoxia, normoxia or hyperoxia, this despite significantly different CaO2 between conditions. With the onset of exercise, LBF was significantly higher in the hypoxic condition, however V? O2p and V? O2leg on- kinetics were not significantly different between conditions. The authors postulated that the lack of difference in on-kinetics was due to increased leg blood flow and increased O2 extraction at the muscle compensating for lower CaO2 in the case of hypoxia (47). Using a similar protocol, Delorey et al. (8), examined on-kinetics examining V? O2p, LBF and the change in dexoyhemoglobin 41 (?HHbMb) measured by near infrared spectroscopy (NIRS). NIRS uses infrared light absorption to measure hemoglobin and myoglobin oxygenation, which had previously been shown as an indication of the balance between O2 availability and O2 usage (3, 23). The Delorey group had earlier shown that ?HHbMb accurately reflects O2 delivery and consumption across the working muscle, stating that when combined VO2P, leg blood flow, and ?HHbMb ?allows for the determination of the time course for local muscle O2 utilization? (7). In the hypoxia experiment the subjects initially performed incremental leg kick tests while breathing air with 21% oxygen and then a second test breathing 12% oxygen. Blood flow was measured at the femoral artery and with leg O2 delivery calculated based on LBF and CaO2. .The NIRS optical device was placed on the vastus lateralis with the optical sensors set at optimum distance. The results showed LBF was 35% higher in the hypoxic condition though this was not statistically significant. V? O2p was significantly slower in the hypoxic condition. However, the ?HHbMb response was not significantly different between normoxia and hypoxia, indicating at the muscle O2 delivery and utilization were not different between the two conditions(8). The group concluded that the lower V? O2p in the hypoxic condition despite an increased blood flow and thus increased O2 delivery similar to the normxoic condition was due to limited extraction as the result of a reduced diffusion gradient(8). Diffusion at the working muscle is described by Fick?s law of diffusion V? O2 = DO2 (PcapO2 ? PmitoO2) where DO2 is the lumped diffusion capacity, PcapO2 is capillary PO2 and PmitoO2 is the PO2 inside the muscle fiber (also called intracellular PO2). Gayeski et al. (15-16) have found intracellular PO2 (PiO2) to be uniform in the muscle fiber suggesting that PmitoO2 would be the same or very close to intracellular levels. Intracellular PO2 measured both in vivo and in situ 42 have been found to be extremely low during exercise, in the range of 1-4 Torr (5, 15, 48, 56). In canine grasilis muscle flash frozen at maximum work rate PiO2 was found to be less than 0.5 Torr (16). However even this low level the PO2 is sufficient to allow full mitochondrial respiration which in situ is estimated to be as low as ? 0.5 Torr (6). Richardson et al. (52-56) in a series of experiments using H1NMR to examine myoglobin saturation in working human leg muscles during single-leg knee extensions found that PcapO2 to be approximately 39 Torr in normoxia and as expected lower, approximately 29 Torr in hypoxia(56). Within the muscle fiber, PiO2 was lower in both normoxia and hypoxia down to 3.1 Torr and 2.3 Torr respectively. This difference of less than 1 Torr in PiO2 allowed for a pressure gradient between the capillary and the muscle to provided diffusion even at low PcapO2. Further they found that while PiO2 drops from rest to exercise it appears to plateau at 50% - 60% exercise intensity and remains at the lower level up to V? O2 peak (54, 56). They concluded that the driving pressure for oxygen from blood to cell is similar in hypoxia and normoxia (52). The differing results of MacDonald et al. (47) and Delorey et al. (8) need to be clarified. Can increased O2 delivery compensate for low CaO2 and result V? O2 on-kinetics that are similar to normoxic conditions? Given that even in hypoxia PiO2 is appears to remain above the minimum required for maximal mitochondrial respiration (52, 55), if diffusion can be maintained with the higher blood flow and lower CaO2 then on-kinetics should not change. The hypothesis to be tested in these experiments is that at a moderate work rate muscle V? O2 on-kinetics will not be different between normoxia and hypoxia if convective O2 delivery to the muscle is matched. Therefore the purpose of this experiment is to examine V? O2 on-kinetics under normoxia, hypoxia and severe hypoxia while holding convective O2 delivery constant. 43 METHODS AND PROCEDURES Animals. Eight adult mongrel dogs (canis lupus familiaris) of both sexes were used. The Auburn University Institutional Care and Use Committee approved all procedures. The dogs had ad libitum access to food and water until 24 hours prior to experimental use when both food and water were withheld. Animal Preparation. All animals were housed and maintained at the Auburn University College of Veterinary Medicine prior to experimental use. The animals were anesthetized with an intravenous injection of aqueous pentobarbital sodium (dosage 30 mg per kg-1 body weight). When each animal reached the surgical plane of anesthesia it was intubated, monitored and maintained in a deep, surgical plane of anesthesia throughout the remainder of the experiment with maintenance doses of pentobarbital sodium given as needed. Core temperature was maintained near 37? C. Each animal was ventilated with a respirator (model 613, Harvard Apparatus) at 20 ml per kg and 15- 20 breaths per min. Throughout the experiment arterial blood pH, PCO2, PO2, hemoglobin (Hb) concentration and O2 saturation were monitored. Respirator settings, supplemental sodium bicarbonate and O2, were used as necessary to maintain appropriate physiological levels of arterial blood gases and pH. Surgical preparation. For this study the gastrocnemius plus superficial digital flexor muscle complex (GS) were surgically isolated as described previously (62). Briefly, an incision was made medially in the left hindlimb going from midthigh to the ankle. The overlying 44 muscles (sartorious, gracilis, semiendinosus and semimembranosus) were cut at their insertions and moved aside. Venous outflow from the GS was isolated by ligating all veins draining into the popliteal vein except those from the GS. The popliteal vein was cannulated so that flow (Q) could be measured with a flow-through transit time ultrasonic probe (6NRB440, Transonic Systems, Ithaca, NY). The jugular vein was isolated, and a cannula attached to a reservoir was inserted so that blood from the popliteal vein could be returned to the animal. Once the jugular cannula/reservoir was in place the animal received heparin (3000 units per kg) administered in a bolus (half the total dosage) and then supplemented throughout the experiment. An inline oximeter probe (Opticath model no. U425C, size 4F, Hospira, Lake Forest IL) was inserted into the popliteal vein as close to the muscle as possible to allow continuous measurement of venous O2 saturation. The arterial circulation to the GS was isolated by ligation of all vessels leading from the popliteal artery that did not go to the GS. The right carotid artery was isolated and cannulated. Arterial blood from the right carotid was routed via tubing and a peristaltic pump (Minipuls 3 MP2/HF, Gilson129 Incorporated, Middleton, WI) through another cannula to the isolated popliteal artery supplying the GS. A T-connector in the tubing was connected to a blood pressure transducer (model RP-1500, Narco Bio-systems, Austin, TX) for measuring systemic pressure. The calcaneus, with two tendons attached to the GS was cut from the heel and clamped into a metal bracket for connection to a load cell (Interface SM-250, Scottsdale, AZ) and then attached via a universal coupler to an isometric myograph. The universal coupler allowed the muscle to pull in a direct line with the load cell which prevented any significant torque from being produced. The head of the muscle remained attached to its origin and the femur and the tibia were fixed to the myograph with bone nails to minimize leg movement during contractions. 45 The sciatic nerve was isolated, exposed and the distal stump of the nerve (? 1.5 ? 3.0 cm) was threaded through a small epoxy electrode containing two wire loops which were connected to a stimulator (Grass S48 stimulator, West Warwick, RI). A plastic holder with NIR optodes was placed over the belly of the muscle and held in place by a Velcro strap. The exposed tissue was covered with saline-soaked gauze and a thin, black opaque plastic sheet to reduce possible interference with the NIRS signal (see below). Prior to any experimental runs, optimum length of the muscle was set by progressively stretching the muscle and measuring developed tension elicited by single tetanic train stimuli. Once optimum length was determined, the muscle was allowed to recover for a minimum of 5 min. After all experimental trials were completed the animal was euthanized with an overdose of pentobarbital sodium and saturated potassium chloride. The GS was removed, cleaned of connective tissue and weighed. The muscle wet weight (ww) was used to normalize VO2 to muscle mass. Experimental Design. The experiment consisted of four trials, each separated by a minimum of 35 minutes of rest. For each trial the sciatic nerve was electrically stimulated to elicit tetanic contractions by the GS. Based on earlier experiments (20-21, 26) stimulation at 8 volts, 50 Hz, 0.2 ms pulse duration, 200 ms train duration at a rate of one contraction every 2 s evokes ? 60% VO2peak for the GS in situ. Using this stimulation pattern all animals performed a spontaneous exercise bout, i.e. blood flow to the GS was supplied by the animal?s own cardiac output. The flow and blood pressure were measured (see below). The spontaneous run set the baseline blood flow for the normoxic condition and the calculated O2 delivery from this condition was used to calculate the blood flow the tow hypoxic conditions. The three experimental trials were conducted in random order and included a normoxia trial (animal 46 breathing 21% O2), a hypoxia trial (animal breathing 12% O2) and a severe hypoxia trial (animal breathing 8% O2). In each trial, blood flow to the GS was controlled via the peristaltic pump to match the convective O2 delivery determined in the spontaneous trial. Five minutes prior to each trial, the breathing mixture was changed so subject inspired correct O2 percentage for the trial. Arterial CaO2 was checked via arterial blood sample. Immediately prior to contractions, the blood flow was adjusted to the calculated level to provided matched convective O2 delivery. Measurements. Outputs from the load cell (through a strain gage coupler), ultrasonic flowmeter (T206, Transonic Systems), pressure transducer, the indwelling in-line oximeter probe and NIRS probe were directed to a computerized data acquisition system, the Oxymon MkIII (Artinis Medical systems BV, Zetten, the Netherlands). The sampling rate was 125 Hz. The load cell which was calibrated with known weights before each experiment reaches 90% of full response within 1 ms. The flowmeter was calibrated by graduated cylinder and stopwatch during each experimental trial. The Oximetrix 3 sampled Hb SO2 at a rate of 244 samples per second. The samples were averaged each second resulting and output as a rolling average over 5 seconds. This output has a 90% response time of 5 seconds. The time response is mathematically de- convoluted to account for square wave changes as described by Hernandez et al.(26) Arterial and venous blood samples were drawn anaerobically into 3-ml plastic syringes, prior to commencing stimulation and at the end of stimulation. The venous blood samples were drawn from the catheter draining the muscle. All blood samples were immediately capped and stored in ice water and analyzed within 30 min of the draw on the GEM Premier 3000 (Instrumentation Laboratory, Lexington, MA). Samples were analyzed at 37?C for PO2, PCO2, and pH. Total hemoglobin concentration ([THb]) and SO2 were analyzed by a CO-Oximeter (IL-682, 47 Instrumentation Laboratory, Lexington, MA) set for dog blood. These instruments were calibrated before and during the experiments. V? O2 of the GS was calculated using the Fick principle: V? O2 = Q ? C(a-v)O2, where Q represents blood flow, and C(a-v)O2 is the difference between the arterial and venous blood O2 concentration [O2] measured across the muscle. Contraction-by-contraction V? O2 was determined using methods described by Hernandez et al. (26). Using the data from the Oximeter 3, venous blood O2 concentration was rapidly determined. Arterial O2 concentration was held constant. Blood flow and venous O2 concentration were averaged for each contraction. Using a Microsoft Office Excel macro developed in-house, the data was analyzed for onset of each contraction and then contraction-by-contraction VO2 can be determined by the Fick principle. Kinetics Analysis of V? O2 and ?HHbMb. VO2 and ?HHbMb, relative muscle deoxygenation, kinetics were analyzed via the following mono-exponential function: y(t) = yBas + A(1 ? e-(t ? TD)/?) where YBas is the resting baseline value for V? O2 obtained prior to the contractions (normally 5 sec), A is the amplitude of the V? O2 response between the baseline and the primary component asymptote, TD is the time delay before the mono-exponential rise and ? is the time constant. Mean response time (MRT) was calculated by summing ? and the time delay (TD). The data were fit using a nonlinear least squares program with OrginPro 8.5 (OriginLab, One Roundhouse Plaza, Northhampton, MA). Previously established criteria were used for the line of ?best fit? (57, 65). In this procedure, the variables, A, TD, and ?, are allowed to ?float? with the baseline VO2 held constant at the experimentally measured value. The fitting window was varied to fit Phase II only. The best fit was determined on the basis of minimal values for 48 95% confidence interval for ?, residuals and Chi2. The line of ?best fit? was determined by the best combination of the values above. Near-infrared spectroscopy. Microvascular O2 availability at the muscle was assessed using a near-infrared spectroscopy (NIRS) system (Oxymon Mk III, Artinis Medical Systems BV, Zetten, Netherlands). The system uses two fiber-optic bundles to emit and receive two infrared light wavelengths (760 and 860 nm) which are absorbed by the heme groups in myoglobin and hemoglobin. NIRS does not distinguish between myoglobin and hemoglobin signals, although the myoglobin may account for up to a third of the signal (45). The terms for reduced heme groups (HHbMb), oxygenated heme groups (HbMbO2) and total heme groups (HbMbtot) will be used to represent the three hemoglobin/myoglobin signals. The infrared light used is maximally absorbed at different wavelengths for HHbMb and HbMbO2 and this makes it possible to measure the relative oxygenation of the heme groups. Also absolute changes cannot be calculated because the exact path length of the light cannot be determined (13), and the data are therefore reported in arbitrary units in comparison to a signal that is biased to zero immediately prior to contractions. The two optodes were locked into an epoxy holder attached directly to the belly of the GS. The optodes were held at a distance of 25 mm apart which allows a penetration depth of ?12.5 mm. Signals were biased to zero just prior to first contraction in each experimental trial. Signals were averaged over each contraction using the same procedures outlined above for contraction-by-contraction V? O2. Kinetics for ?HHbMbMb were calculated as discussed above. Statistical Analysis. Data are presented as means ? SD. The data were analyzed using a one-way repeated measures analysis of variance (ANOVA) across the three levels of inspired O2 percentage (21%, 12% and 8%). The level of significance was set at p < 0.05. If a main effects 49 difference was found, a Student-Neuman-Keuls post-hoc test was performed to identify the specific location of any differences. RESULTS The wet weight of the GS muscle was 73.8 g ? 5.4 g and the dry weight was 16.1 g ? 1.1g. The water percentage was 78.1% ? 0.9%. The average water weight was slightly higher than seen in previous studies (17, 27). The higher than normal muscle water percentage was likely the result of higher than normal perfusion pressure resulting in mild edema by the end of the experiments. The additional edema in these muscles may have been the result of follow-on treatments performed for a different study, after the three experimental trials. Resting and steady state values. The resting values and values during the steady state of contractions for pertinent parameters for the study are shown in Table 1. The PaO2 was significantly different among treatments both at rest and during contractions as planned on the basis of the O2 concentration of the inspired gas mixtures. PaCO2 was not significantly different among treatments or between rest and contractions. PvO2 was significantly different among treatments both at rest and during contractions. Also arterial blood pH was not different among treatments nor was it different between resting and steady state levels. Venous pH was significantly different between rest and steady state but not different among treatments 50 Table 1: Blood gases, and pH at rest and during the steady state of contractions. Variable Condition Rest Contractions 21% O2 12% O2 8% O2 21% O2 12% O2 8% O2 PaO2 (Torr) 105 ? 13 41 ? 2? 27 ? 1? 111 ? 11 41 ? 3? 30 ? 3? PaCO2 (Torr) 31 ? 2 30 ? 1 31 ? 3 32 ? 3 30 ? 1 30 ? 4 PvO2 (Torr) 65 ? 6 37 ? 3? 27 ? 1? 20 ? 5* 16 ? 5*? 13 ? 5*? Arterial pH 7.43 ? 0.04 7.46 ? 0.04 7.45 ? 0.06 7.42 ? 0.04 7.45 ? 0.00 7.44 ? 0.01 Venous pH 7.42 ? 0.05 7.44 ? 0.04 7.45 ? 0.06 7.31 ? 0.04* 7.35 ? 0.03* 7.36 ? 0.05* Data are presented as means ? SD. * indicates significance (P< 0.05) from rest to contractions. ? indicates a significant difference between the 21% O2 condition and the two hypoxic conditions. Oxygen Delivery. Values for CaO2, CvO2, C(a-vO2), Q, and convective O2 delivery are presented in Table 2. CaO2 was significantly different among treatments expected, but not different between rest and contractions. CvO2 was not significantly different among treatments at rest; however, during contractions CvO2 was significantly different between the normoxic trial and both the 12% hypoxia and the 8% hypoxia trials. There was no significant difference between the hypoxic treatments. C(a- vO2) difference was significantly different between treatments with the greater absolute difference occurring in normoxia, followed by the 12% hypoxia, and then 8% hypoxia. Blood flow was significantly different among treatments. The convective O2 delivery (Q ? CaO2) was not significantly different among treatments. A graphic depiction of the results can be seen in Figure 1, 2 & 3; where Fig.1 shows the comparison of the average CaO2, Fig. 2 shows the average blood flow for each condition, and Fig. 3 illustrates the convective O2 delivery for each condition. 51 Table 2. Arterial and venous O2 saturation, difference, blood flow and convective O2 delivery. Variable Condition Rest Contractions 21% O2 12% O2 8% O2 21% O2 12% O2 8% O2 CaO2 (mL?dL-1) 21.7 ? 1.7 17.4 ? 2.0 12.5 ? 2.8 21.5 ? 1.8 17.2 ? 2.0 13.3 ? 2.0 CvO2 (mL?dL-1) 20.2 ? 1.9 16.4 ? 1.9 11.7 ? 2.8 6.8 ? 3.6? 4.9 ? 2.9? 3.2 ? 2.3? CaO2-CvO2 (mL?dL-1) 1.4 ? .9 1.4 ? 0.5 1.06 ? 0.7 14.7 ? 2.9? 12.3 ? 1.8? 8.8 ? 2.8? Q (mL?kg- 1?min-1) 903 ? 293? 1213 ? 325? 1552 ? 381? 917 ? 2.6? 1199 ? 308? 1555 ? 342? Q x CaO2 (mL?kg- 1?min-1) 202 ? 58 210 ? 61 193 ? 50 202 ? 58 206 ? 57 205 ? 49 Data are presented as means ? SD. Q: blood flow; Q x CaO2: convective O2 delivery. * indicates significance (P< 0.05) from rest. ? indicates significance (P< 0.05) between treatments. ? indicates significance between rest and contractions and treatments. Per kg indicates per kg wet muscle weight. Figure 1. Mean CaO2 by condition, filled column 21% inspired O2, cross-hatch pattern 12% inspired O2 and squares pattern 8% inspired O2. 0 5 10 15 20 25 52 Figure 2. Mean blood flow (ml?kg-1min-1) by condition; Filled column 21% inspired oxygen, cross-hatched pattern column 12% inspired oxygen, square pattern column 8% inspired oxygen 0 500 100 0 150 0 200 0 53 Figure 3. Mean convective O2 delivery (ml?kg-1?min-1) by condition; Filled column 21% inspired oxygen, cross-hatched pattern column 12% inspired oxygen, square pattern column 8% inspired oxygen V? O2 on-kinetics. Data for the primary time constant (tau), the time delay (TD) and mean response time (MRT), are shown in table 3. There was no significant difference for tau among treatments. There was a significant difference between the TD during normoxia and the two hypoxic trials. TD was not different between the two hypoxic trials. MRT in the normoxic trial was significantly slower than the 8% hypoxic trial; however there was no significant difference between the normoxic trial and the 12% hypoxic trial or between the two hypoxic trials. The amplitude and asymptote for the primary V? O2 for all three trials are presented in Table 3. There were no significant differences in either the amplitude or asymptote of the primary component among trials. There was also no difference in the highest average V? O2 per 0 50 100 150 200 250 54 condition and no significant difference in the slow component in the three conditions. The average contraction-by-contraction V? O2 data are shown in Figure 4 and normalized average V? O2 is shown in Figure 5. The data was normalized by dividing the average VO2 values for each condition by the highest average V? O2 for that condition. Table 3. V? O2 on-kinetics data, primary amplitude, primary asymptote, V? O2peak and slow component Variable 21% O2 12% O2 8% O2 V? O2 TD (s) 7.1 ? 1.5 5.7 ? 1.5* 5.3 ? 1.7* V? O2 Tau (s) 10.9 ? 2.4 10.1 ? 1.8 9.2 ? 1.9 V? O2 MRT (s) 17.9? 3.4 15.7 ? 3.1 14.5 ? 3.3* Primary V? O2 Asymptote (mL?kg ww-1?min-1) 114.2 ? 11.5 116.1 ? 28.0 125.2 ? 21.4 Primary V? O2 Amplitude (mL?kg ww-1 ?min-1) 105.5 ? 13.2 106.0 ? 37.5 101.7 ? 19.8 Maximum V? O2 (mL?kg ww-1?min-1) 137.7 ? 15.1 145.6 ? 32.3 149.0 ? 23.4 Slow Component (mL?kg ww-1? min-1) 23.5 ? 9.4 29.5 ? 15.4 23.8 ? 14.3 Data presented as means ? SD. TD - time delay; tau - time constant; MRT - mean response time: V? O2, mL O2 per kg ww (wet weight) per min. * indicates significant difference (P< 0.05) from 21% oxygen condition treatment. 55 Figure 4. Mean contraction-by-contraction VO2 data for 21% O2 condition (closed square), 12% O2 condition (open circles) and 8% O2 condition (Open diamonds). There was no significant difference in the V? O2 on-kinetic data among conditions. The mean starting V? O2 was higher in the 8% O2 condition but not significantly different from the 12% O2 condition or the 21% O2 condition. 0 50 100 150 0 20 40 60 80 Contractions V? O 2 (ml?k g ww -1 ? min -1 ) 56 Figure 5. Normalized contraction-by-contraction V? O2 data with shown as 21% O2 condition (closed square), 12% O2 condition (open circles) and 8% O2 condition (Open diamonds). To normalize the data each mean contraction-by-contraction V? O2 point was divided by the highest mean V? O2 by condition. There was no significant difference in the normalized V? O2 on-kinetic data among conditions. 0 0 . 2 0 . 4 0 . 6 0 . 8 1 0 20 40 60 80 No rma lized VO 2 ( ml? kg ww -1 ? min -1 ) Contractions 57 NIRS. The mean response of the NIRS ?[HHbMb] signal in the different protocols is shown in Figure 4 and the tau, TD and MRT are given in Table 3. There were no significant differences between tau, TD or MRT among trials can be seen in Figure 4. Table 3. NIRS Deoxygenated Heme Groups ?[HHbMb] TD (sec) 6.7 ? 0.7 5.7 ? 1.5 5.8 ? 0.6 HHb Tau (sec) 7.9 ? 2.4 8.8 ? 3.9 8.1 ? 2.9 HHb MRT (sec) 14.9 ? 2.6 14.5 ? 4.6 13.9 ? 3.0 Data are presented as means ? SD. There was no significant difference between conditions for time delay, TD, primary time constant, tau or mean response time, MRT 58 Figure 6. Mean NIRS data for ?HHbMb for the three conditions; 21% O2 (closed squares), 12% O2 (open circles) and 8% O2 (open diamonds). There was no significant difference between mean HHbMb on-kinetics. 0 10 20 30 40 50 0 20 40 60 80 Contractions ? HH bM b ( ?M ) 59 Fatigue. Table 4 contains the force and fatigue results for the three trials. There were no significant differences among treatments in peak force or final force. There was a significant difference in the fatigue index between normoxia and severe hypoxia (8% iO2) but no difference between 21% and 12%, or 12% and 8% inspired O2. There was also no order effect in relation to fatigue index. Table 4. Force and Fatigue index Variable Contractions 21% O2 12% O2 8% O2 Peak Force (kN?kg-1) 6.2 ? 1.0 6.3 ? 1.1 6.4 ? 1.0 Final Force (kN?kg-1) 6.0 ? 0.8 6.0 ? 0.9 6.0 ? 1.0 Fatigue Index % 1.9% 3.4% 5.3%* Data presented as means ? SD. There was no significant difference in peak force or final force among treatments. * indicates a significant difference from the 21% O2 treatment DISCUSSION The major finding of this is study is that under conditions moderate hypoxia, 12% inspired O2 and severe hypoxia, 8% inspired O2, normal V? O2 on-kinetics can be maintained by increasing blood flow so that O2 delivery to the muscle matches O2 delivery with normoxia, 21% inspired O2. The fact that tau, the V? O2 primary amplitude and the steady state V? O2 were not different among treatments supports the idea that with higher blood flow the diffusion driving pressure was sufficient to meet the muscle O2 demand despite lower capillary PO2. In this experiment this appears to be due to an increase in the diffusion capacitance in hypoxia. 60 V? O2 on-kinetics. In previous work from our laboratory we have studied VO2 on-kinetics by modifying blood flow via pump perfusion to change convective O2 delivery to an isolated muscle. The findings include; 1) under normoxic condition at a work rate less than VO2peak on- kinetics are not speeded by either increased O2 delivery prior to commencement of contractions or increased arterial O2 saturation combined with increased delivery prior to contraction (20-21), 2) With the isolated muscle working at or near V? O2peak, increased blood flow and thus O2 delivery prior to contractions does speed on-kinetics (22), and 3) By reducing convective O2 delivery by slowing blood flow to the muscle in normoxia one can slow on-kinetics in a manner consistent with oxygen dependent zone described by Poole and Jones (17, 50). These studies led to this current research into the role of O2 delivery in hypoxic conditions. Earlier work with humans and in animal models has found that under conditions of hypoxia O2 deficit is increased and V? O2 on kinetics are slowed (11, 36, 43, 60). Many of the human studies used upright cycling and hypoxia at various work rates less than V? O2max. During upright cycling in hypoxia, O2 delivery to the leg muscles is reduced compared to the normoxic condition and this results in slower pulmonary on-kinetics (11, 36, 60). However, Rowell et al., (58) found that leg blood flow is increased in hypoxia in single leg exercise when compared to upright cycling. They went on to find that in single-leg knee extension exercise under conditions of hypoxia (10% -11% O2) that increased blood flow to the leg maintained V? O2peak compared to normoxia. They postulated that the heart could meet O2 demand by increasing blood flow along with increased muscle O2 extraction. Using a similar approach Richardson et al., (53) examined a range of single leg work rates up to a peak level with inspired O2 levels of 21%, normoxia and 12%, hypoxia. They found a significantly lower VO2peak between normoxia and hypoxia but only a trend toward lower V? O2 with work rates from 50% to 90%. Cardiac output and LBF 61 significantly increased in hypoxia compared to what was seen in normoxia until exercise reached ? 80% of V? O2peak. They concluded that at 50% to 90% of V? O2peak increased O2 extraction allowed V? O2 rise at a rate not significantly different from that of the normoxic condition. Later MacDonald et al., (47) used moderate intensity single leg kicking to examine V? O2 on-kinetics in normoxia and hypoxia. They found that LBF was significantly higher in hypoxia and that muscle V? O2 on-kinetics was the same between normoxia and hypoxia. They concluded that the increased blood flow and thus increased oxygen delivery along with increased extraction ameliorating the effects of low CaO2 (47). DeLorey et al., (8) used a similar protocol to examine muscle de-oxygenation via NIRS while also looking at LBF and V? O2p. They found that V? O2p was slower in hypoxia despite an approximate 35% increase in LBF. Their NIRS results, the de-oxy hemoglobin (?HHbMb) showed not significant difference between conditions indicating that at the muscle the oxygen delivery and utilization were similar. They did find a tendency toward a lower ?HHbMb in hypoxia which they suggested this was due to decrease extraction because of reduced diffusion pressure gradient. The results of the current experiment supports the idea that increased blood flow and O2 delivery can compensate for low arterial O2 levels and can maintain the diffusion driving pressure. We saw no difference in the V? O2 muscle on-kinetics between treatments with higher blood flow and thus increased convective O2 delivery. Additionally, there were no significant differences in the steady state V? O2 achieved between three conditions. As can be seen in figure 4, average V? O2 profiles were the similar for the three treatments and as can be seen in table 3, the is no difference in the primary time constant. There was significant difference in TD between the normoxia and the two hypoxic conditions which is probably due to the significantly higher blood flow rate prior to contractions due to increasing the pump output prior to the start of contractions. Also, the 8% hypoxia treatment had an 62 observably higher resting V? O2 prior to contraction however; this was not significantly higher than the 21% and 12% conditions. The O2 consumption for the moderate work rate was not statistically different as seen in V? O2 amplitude. Taken together these findings support the results in MacDonald et al. (47) and supported the hypothesis that matched convective O2 delivery can compensate for low CaO2. Hogan et al., (29), using the same isolated muscle preparation and similar levels of hypoxia without increased muscle blood flow, reported significantly different V? O2 between the treatments both at moderate work rates and at a work rate designed to elicit V? O2PEAK. However blood flow was held constant in these experiments and thus O2 delivery was significantly different even at a moderate work rate.. Hogan et.al, postulated that in hypoxia the lower V? O2 was due to a diffusion limitation as a result of low capillary PO2 The findings in this experiment at 60% -70% VO2 peak found no difference in V? O2 with increased O2 delivery and that diffusion was not limited with the higher flow despite the low PcapO2. As Richardson et al. point out in the human leg kick studies ?a major cause of increased V? O2 is an increase in DO2 not a change in PO2? (52). Interestingly there was lower absolute O2 extraction, C(a-vO2) difference between normoxia, hypoxia 12% and hypoxia 8% with greater extraction occurring in normoxia and lower in both hypoxia treatments. This is similar to what has been seen in human single leg kick preparations (8). The fact that there was no significant difference in tau for either hypoxic treatment though there was different extraction appears to support the idea of increased diffusion capacity in hypoxia. NIRS. As mentioned in the introduction the NIRS ?HHbMb signal is a composite of both myoglobin and hemoglobin de-oxygenation and estimates of the role of myoglobin in the signal during exercise continue to be debated (14, 45) however there is general agreement that 63 the ?HHbMb reflects the balance between O2 delivery and O2 extraction (7, 12, 23). In this study ?HHbMb and muscle V? O2 on- kinetics were essentially the same confirming that in our model NIRS data is an excellent substitute for muscle oxygen consumption. As can be seen in Table 4 there was no significant difference in the TD between ?HHbMb and V? O2m. The time delay for V? O2m is consistent with previous experiments using the canine GS (20, 27) and with the delay in the fall of PO2 at the onset of contraction in single muscle fibers in vitro (40, 63). This supports the hypothesis that there is a matching of O2 delivery and utilization during the first few seconds of an increase in work.(25). There was no difference in the tau ?HHbMb between the normoxic and either hypoxic condition again demonstrating that O2 availability and use were similar. Diffusion limitation. Gayeski et al. (15-16) have found intracellular PO2 to be uniform in the muscle fiber suggesting that PmitoO2 would be the same or very close to PiO2. Intracellular PO2 measured both in vivo and in situ have been found to be extremely low during contractions, in the range of 1-4 Torr (5, 15, 48, 56) and in canine grasilis muscle flash frozen at maximum work rate to be as low as 0.5 Torr (16). However even at this low level the PO2 is sufficient to allow full mitochondrial respiration which in situ is estimated to be > 0.5 Torr (6). Richardson et al., (52-56) in a series of experiments using H1NMR to examined myoglobin saturation in working human leg muscles during single-leg knee extensions has found that PcapO2 is ? 11 Torr, from 39 Torr in normoxia to 29 Torr in 12% hypoxia. While the drop in PiO2 difference in the muscle and thus PmitoO2 was only 1 Torr, 3.1 Torr in normoxia to 2.3 Torr in hypoxia. Further they found that while PmitoO2 drops from rest to exercise it appears to plateau at 50% - 60% exercise intensity and remains at the lower level (? 3 Torr) up to VO2 peak (54, 56). They concluded that the driving pressure from capillary to muscle cell is similar in hypoxia and normoxia and thus diffusion capacity must increases with hypoxia to allow for 64 similar V? O2 despite lower PcapO2 (52). In this experiment we did not measure PcapO2, however using PvO2 as a surrogate, we saw a significant lower PvO2 in both hypoxic conditions. However, the results, similar V? O2, and similar V? O2 on-kinetics indicate that diffusion driving pressure was maintained in all conditions. Using Fick?s law as mentioned above for V? O2 to remain the same between conditions despite apparently significantly lower PvO2 (? PcapO2) in the hypoxic conditions, the diffusing capacity would need to increase. This is similar to what has been seen in human single leg kick preparations (8). Force and Fatigue: There was no difference between peak force and final force between treatments. The fatigue index for the 8% hypoxia was significantly greater than for normoxia but not different from 12% hypoxia and the 12% hypoxia did not have a significantly greater fatigue index compared to normoxia. There was a significant difference between the venous pH at rest and at the end of exercise in all three treatments (normoxia - 7.42 ? 0.05 rest, 7.31 ? 0.04 end, 12% hypoxia - 7.44 ? 0.04 rest, 7.35 ? 0.03 end, 8% hypoxia - 7.45 ? 0.06 rest, 7.36 ? 0.05 end) but final pH was not different between treatments. Lactate was not significantly different between treatments. These data taken together demonstrate that in each treatment mitochondrial respiration was able to meet the work demand with the majority of energy coming from oxidative sources. Limitations. The major limitation of this study was that we did not explore VO2peak along with the moderate stimulation protocol. In earlier study Hogan et al. (32) found a dissociation of VO2peak with matched convective O2 delivery in normoxia and hypoxia. It would have been interesting to compare data but in the Hogan study the blood flow and thus O2 delivery was at slightly lower than spontaneous rate in normoxia resulting in a slightly lower O2 delivery to the muscle and the hypoxic treatment used a blood flow to match the lower normoxic 65 delivery rate. In this model O2 delivery was calculated to match spontaneous delivery in normoxia in two hypoxic condtions. A further study at VO2 peak using a similar protocol would help clarify the question of O2 diffusion verses delivery as the limiting factor. The canine GS model offers many advantages in the study of on-kinetics but it also has limitations. The muscle is highly oxidative and homogeneous so any conclusions need to be interpreted with caution particularly if being applied to a human leg model. The muscle is performing isometric contractions with all motor units firing synchronously unlike exercise where recruitment of fibers is required and work is done. Additionally, while we were able to control bulk blood flow via the perfusion pump we were not able to measure what was happening with capillary blood flow in the three different conditions. While I do not suspect that there was additional capillary recruitment at the higher blood flow based on the work of Kindig et al.,(41-42), the increased blood flow may have increased the red blood cell to capillary surface contact as suggested by Poole et al., (51). This would account for increased diffusion from capillary to muscle fiber and might alleviate the need for a change in the diffusion capacity at the interface between capillary and muscle. In either case there would be increased O2 available to meet demand. Conclusions. For the first time this study has demonstrated that increase O2 delivery can maintain VO2 on-kinetics under conditions of moderate and severe hypoxia. 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