Skeletal muscle VO2 on-kinetics: set by O2 delivery or by O2 utilization?

New insights into an old issue

BRUNO GRASSI Istituto di Tecnologie Biomediche Avanzate, C.N.R., I-20090 Segrate (MI), ITALY; and Department of Medicine, University of California, San Diego; La Jolla, CA 92093-0623

ABSTRACT GRASSI, B. Skeletal muscle VO2 on-kinetics: set by O2 delivery or by O2 utilization? New insights into an old issue. Med. Sci. Sports Exerc., Vol. 32, No. 1, pp. 108 116, 2000. Recent work conducted by our group has expanded knowledge on some basic issues related to pulmonary and skeletal muscle O2 uptake (VO2) on-kinetics. We demonstrated that, in exercising humans during transitions from unloaded pedaling to loaded pedaling below the ventilatory threshold, alveolar VO2 on-kinetics can be taken as a rather close approximation of skeletal muscle VO2 on-kinetics. Experiments conducted on the isolated in situ dog gastrocnemius preparation have shown that, during transitions from rest to contractions corresponding to 70% of the muscle peak VO2, convective O2 delivery to muscle, intramuscular blood flow (Q) versus VO2 maldistribution, and peripheral O2 diffusion are not limiting factors for skeletal muscle VO2 on-kinetics. The latter, therefore, appears to be mainly determined by an intrinsic inertia of skeletal muscle oxidative metabolism, possibly related to acetyl group availability within mitochondria, to regulatory effects on intracellular respiration related to phosphocreatine splitting, and/or to other still not precisely identified control mechanism(s). Evidence from the literature suggests that the limiting factors for skeletal muscle VO2 on-kinetics may vary according to the intensity of muscular contractions or of exercise. Key Words: GAS EXCHANGE KINETICS; SKELETAL MUSCLE, OXIDATIVE METABOLISM

pon a step transition from rest to exercise, or from a lower to a higher workload, O2 uptake (VO2) increase is slower than that of power output (29), following a time course often termed VO2 on-kinetics. Sudden changes in work output are frequently encountered in sports as well as during many activities of everyday life. A faster adjustment of skeletal muscle oxidative metabolism during increases in work rate reduces the need for substrate level phosphorylation, with lesser disturbance for cellular and organ homeostasis (lower degradation of phosphocreatine and glycogen stores, lower accumulation of lactate and H ) and obvious implications for exercise capacity and muscle fatigue. The interest in studies on skeletal muscle VO2 on-kinetics derives also from the fact that such research gives valuable insight into basic mechanisms of metabolic control during muscular contraction. Whereas metabolic steady-state provides a stable scenario to gather data, it is indeed in the transient response where the best insights into system control can be obtained.

0195-9131/00/3201-0108/0 MEDICINE & SCIENCE IN SPORTS & EXERCISE Copyright 2000 by the American College of Sports Medicine Submitted for publication August 1998. Accepted for publication February 1999.

Pulmonary VO2 on-kinetics, as usually determined in a clinical setting or in the exercise physiology laboratory, is known to be: faster in trained than in untrained individuals (14), the effects of training being evident after just a few days (44); slower after bed-rest deconditioning (18); faster during exercises conducted with predominantly slow oxidative than with predominantly fast glycolytic muscles (8,15); faster in children compared with adults (3), whereas it is slower in old subjects compared to young controls (4) during cycle exercise, but not during exercises conducted with muscles commonly utilized during everyday life (17); and slower in patients with respiratory (43) or cardiac (49) diseases, in patients with type II diabetes (47), as well as in heart transplant recipients (27) and in heart and lung transplant recipients (24) compared with healthy untrained controls. General agreement exists for the concept that pulmonary VO2 on-kinetics is an index of the overall conditioning of the integrated pulmonary, cardiovascular, and muscular systems and is the result of a delicate interplay between the various mechanisms regulating O2 delivery to and O2 utili zation by skeletal muscle (13,56,57). Pulmonary VO2 onkinetics, moreover, appears to be more sensitive than max imal aerobic power (VO2max) or the lactate threshold to perturbations such as exercise training (20,44). The 108

 determination of VO2 on-kinetics offers the additional advantage of not requiring the maximal efforts that could be contraindicated in patients or elderly subjects. Analysis of VO2 on-kinetics could then represent a valuable tool for evaluating the level of aerobic conditioning of patients, subjects, and athletes. To meet this goal, however, a better understanding of the complex mechanisms determining the VO2 on-kinetics would be necessary. These mechanisms have long been a matter of considerable debate, mainly between those who consider the limiting factor to be mainly related to the rate of adjustment of O2 delivery to the exercising muscles (30 32,35) and those who support the concept that the VO2 on-kinetics is mainly set by the inertia of skeletal muscle oxidative metabolism (16,56). According to the latter hypothesis, the rate of adjustment of oxidative phosphorylation during the transition would be mainly determined by levels of cellular metabolic controllers and/or enzyme activation (also see the section entitled What Is Determining Skeletal Muscle VO2 On-Kinetics?). Some recent studies presented in this paper allow more insight into this controversial issue. Another concept that still needed direct confirmation was whether the on-kinetics of pulmo nary or alveolar VO2, as usually determined in the exercise physiology laboratory, can be used to make inference to the VO2 on-kinetics occurring within exercising skeletal muscles. The present paper does not have the ambition of being a review of the many papers that have been published on this topic, also in recent years. Its aim is simply to give an overview of recent work that the author has been conducting, in collaboration with several coworkers (see Acknowledgments) to address some of the issues presented above (25,26,28).

Figure 1Average values ( SD, N 6) of alveolar O2 uptake (VO2alv) and exercising legs O2 uptake (VO2legs) during a transition from unloaded pedaling to loaded pedaling (below the ventilatory threshold) in humans. Vertical hatched line indicates onset of loaded pedaling. Modified from ref. 28, Grassi B., D. C. Poole, R. S. Richardson, D. R. Knight, B. K. Erickson, and P. D. Wagner. Muscle O2 uptake kinetics in humans: implications for metabolic control. J. Appl. Physiol. 80:988 998, 1996. Used with permission of the American Physiological Society.

HUMAN SKELETAL MUSCLE VO2 ON-KINETICS

Until recently, human muscle VO2 on-kinetics had not been determined, mainly as a consequence of the lack of techniques allowing the measurement of limb blood flow (Q) with the time resolution needed for kinetics analysis. In the absence of direct measurements, the kinetics features of human muscle VO2 were inferred by various approaches, such as: 1) extrapolation from the kinetics of pulmonary or alveolar VO2 (13,56,57); 2) mathematical models, again based primarily on pulmonary or alveolar VO2 (9,33); and 3) 31P-nuclear magnetic resonance spectroscopic determination of phosphocreatine (PCr) breakdown at exercise onset (10). The latter approach is supported by the direct proportionality between the products of PCr splitting and muscle or pulmonary VO2, demonstrated in animals (36,40) and, more recently, also in exercising humans (7,39). The indirect or theoretical nature of these approaches should justify the notion that direct measurements of human skel etal muscle VO2 on-kinetics, conducted in conjunction with measurements of pulmonary or alveolar VO2 on-kinetics, were needed.
LIMITING FACTORS FOR VO2 ON-KINETICS

Recently, a modification of the constant infusion thermodilution technique (28) or the utilization of Doppler technology (32) has allowed the determination, with some assumptions, of exercising leg (28) or forearm (32) Q onkinetics in humans. In conjunction with rapid serial collections of arterial and venous blood samples across the exercising limb or muscles, these methods allowed a direct measurement of exercising leg (28) or forearm (32) VO2 on-kinetics. More specifically, a modification of the constant-infusion thermodilution technique, proposed initially by Andersen and Saltin (2) for measurements of steady-state limb Q, significantly improved the time resolution of data collection and allowed the measurement of leg Q (Qleg) in exercising humans during unsteady-state conditions (28). In conjunction with rapid serial collection of arterial (radial) and femoral vein blood samples for the measurement of arteriovenous O2 concentration difference [C(a-v)O2] across the exercising leg, this permitted the determination of the on-kinetics of VO2 across the exercising limb (VO2leg) during the transition from unloaded pedaling to a workload lower than the ventilatory threshold (28). VO2leg was as close as possible to muscle VO2 by utilizing the present techniques. The main results of the study are shown in Figures 1 and 2. Figure 1 shows that the overall response characteristics of VO2 calculated for both legs matched rather closely that of alveolar VO2, despite the interposition of venous blood O2 stores and transit delays from the sites of gas exchange in the muscles to the lungs. Alveolar VO2 on-kinetics, therefore, can be considered a rather close approximation of skeletal muscle VO2 on-kinetics. From Figure 2 it also appears that, for the first 10 15 s of the transition, the kinetics of O2 delivery to muscle (i.e., of Qleg, because CaO2 was constant throughout the transition) was faster than the kinetics of O2 utilization by muscle (i.e., of C(a-v)O2leg). This finding represents indirect evidence in
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 Figure 2Average values of blood flow to the exercising leg (Qleg), arteriovenous concentration difference across the exercising leg [C(a-v)O2 leg] and O2 uptake across the exercising leg (VO2leg) during a transition from unloaded pedaling to loaded pedaling (below the ventilatory threshold) in humans. Vertical hatched line indicates onset of loaded pedaling. Modified from ref. 28, Grassi B., D. C. Poole, R. S. Richardson, D. R. Knight, B. K. Erickson, and P. D. Wagner. Muscle O2 uptake kinetics in humans: implications for metabolic control. J. Appl. Physiol. 80:988 998, 1996. Used with permission of the American Physiological Society.

favor of the hypothesis that, for this type of transition, VO2 on-kinetics was not limited by the rate of adjustment of bulk O2 delivery to muscle. Estimates of transit delays from the sites of gas exchange in the muscle and the site of venous sampling in the femoral vein indicate that they should account only for a relatively minor portion of the observed delay in C(a-v)O2leg increase during the transition (28). A similar study, published by another group shortly after our paper (32) provided data partially different from ours: also these authors reported indeed, for low intensity forearm exercise conducted with the arm below the heart level, a slightly faster forearm Q on-kinetics compared with muscle VO2 on-kinetics; on the other hand, during high intensity exercises, the on-kinetics of the two variables were essentially the same. Exercise intensity could indeed have a significant influence on the O2 delivery versus O2 utilization issue. The results of our study (28) strictly apply only to transitions from unloaded pedaling to loaded pedaling below the ventilatory threshold. Evidence from the literature (23,35) suggests that for transitions involving exercises higher than the ventilatory threshold O2 delivery to muscle may in fact represent one of the limiting factors of VO2 on-kinetics. Transit delays could also in part explain the biphasic nature of VO2leg described in our previously mentioned study (see Fig. 1) (28): an initial rather sluggish VO2leg increase was indeed followed, after 10 15 s of the transition, by a monoexponential increase to the new steady-state. This issue will be discussed in some more detail later in the paper (see the section entitled Is Skeletal Muscle VO2 On-Kinetics Monoexponential?).

CONVECTIVE O2 DELIVERY TO SKELETAL MUSCLE AS A LIMITING FACTOR FOR VO2 ON-KINETICS

As discussed above, we showed (28) that the adjustment of convective O2 delivery to exercising human muscle was 110
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faster during the first 1520 s of the investigated transition than the adjustment of O2 utilization by muscle. This observation does not necessarily demonstrate, however, that convective O2 delivery was not the limiting factor (or one of the limiting factors) for the VO2 on-kinetics. The ideal experimental approach to discriminate the O2 delivery versus O2 utilization issue would be to increase the rate of adjustment of convective O2 delivery to muscle and then determine whether the VO2 on-kinetics becomes faster. Previous studies conducted on humans by following this approach yielded conflicting results (23,27,31). These studies were characterized by some intrinsic limitations. For example, the on-kinetics of convective O2 delivery to muscle could not be directly determined, even though in some of the studies it was inferred from other measurements. Moreover, even assuming they were present, changes in the on-kinetics of convective O2 delivery were presumably rather small. We therefore decided on a more aggressive experimental approach to address the issue and turned to an animal experimental model (isolated in situ dog gastrocnemius preparation) that allowed us: a) to eliminate any delay in convective O2 delivery to the muscle during the rest-to-contraction transition; b) to directly measure such delivery; and c) to measure VO2 directly across the contracting muscle. We hypothesized that, if muscle VO2 on-kinetics was indeed limited by the rate of adjustment of convective O2 delivery, after eliminating any delay in the latter a faster VO2 onkinetics would be observed (25). In the isolated in situ dog gastrocnemius preparation (50), arterial blood perfusing the muscle can come from either the contralateral artery (spontaneous Q) or from a pump controlled by the operator. By utilizing this preparation, it was possible to compare muscle VO2 on-kinetics in a condition of spontaneous adjustment of O2 delivery to muscle (Control) and in a condition in which any delay in the adjustment of such delivery was eliminated by having the muscle pumpperfused, from the last 1530 s of rest and throughout the contraction period, at a constantly elevated Q level, corresponding to the steady-state value during contractions in the presence of spontaneous Q (Fast O2 Delivery). During Fast O2 Delivery, to prevent vasoconstriction and inordinate per fusion pressure increases associated with the high Q, 12 mL min 1 of a 10 2 M adenosine solution were infused intra-arterially by a pump, from 20 to 30 s before contraction onset and throughout the contraction period. Perfusion pressure of the gastrocnemius was monitored via a catheter placed in the artery at the head of the muscle. Isometric tetanic muscle contractions (corresponding to 70% of the muscle peak VO2) were elicited by supramaximal stimulation of the sciatic nerve with trains of stimuli lasting 200 ms, at a rate of 1 contraction 2 s 1 for 3 min. Tetanic contractions were chosen to allow a rapid attainment of a steadystate of force development, as monitored by an isometric myograph attached to the Achilles tendon. Q to muscle was continuously determined in the popliteal vein draining from the gastrocnemius by an ultrasonic flowmeter. Samples of arterial blood entering the muscle and of venous blood from the popliteal vein (as close as possible to the gastrocnemius)
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were drawn at rest and every 57 s of the contraction period for the determination CaO2 and CvO2. The dead-space volume of blood between the point where the vein leaves the gastrocnemius and the site of venous sampling was measured for each dog at the end of the experiment. The timing of each venous sample was then corrected for the time necessary to wash out this dead-space volume. This preparation, therefore, allowed a direct determination of O2 de livery (calculated as Q CaO2) to the working muscle and of VO2 [calculated as Q C(a-v)O2] across the working muscle, at discrete times corresponding to the timing of the blood samples. The main results of the study (25) are shown in Figures 3 and 4. In Figure 3, average values ( SE) of Q CaO2 and VO2 in the two experimental conditions (Control and Fast O2 Delivery) are shown: a complete dissociation of the time courses of the independent variable (Q CaO2) between the two conditions (in Control the variable spontaneously adjusted during the rest-to-contraction transition, whereas in Fast O2 Delivery it was kept constantly elevated throughout the experiment) did not significantly affect the dependent variable, i.e., VO2, whose time courses were remarkably similar in the two conditions. This represents strong evi dence that the Q CaO2 on-kinetics was not a limiting factor for the VO2 on-kinetics. The VO2 on-kinetics was mathematically evaluated and compared in the two conditions by calculating the times necessary for VO2 to reach 50% (t50%, corresponding to the half-time of the response) and 63% (t63%, corresponding to the of a monoexponential response) of the difference between the resting baseline and the steady-state value during contractions. t50% and t63% values in the two experimental conditions are shown in Figure 4: no significant differences were observed. These results demonstrate that, in the present experimental model and for the rest-to-exercise transition studied (rest-to-con tractions corresponding to 70% of VO2 peak), convective O2 delivery to muscle was not a limiting factor for the VO2 on-kinetics. As mentioned above, this conclusion may not

Figure 4 Times necessary for muscle VO2 to reach 50% and 63% of the difference between the resting baseline and the steady-state values during contractions (t50% and t63%, respectively) in the two experimental conditions (Control and Fast O2 Delivery). Data are presented as average values SE, N 6; NS P > 0.05. Modified from ref. 25, Grassi B., L. B. Gladden, M. Samaja, C. M. Stary, and M. C. Hogan. Faster adjustment of O2 delivery does not affect VO2 on-kinetics in isolated in situ canine muscle. J. Appl. Physiol. 85:1394 1403, 1998. Used with permission of the American Physiological Society.

Figure 3Average values ( SE, N 6) of O2 delivery to muscle (Q CaO2) and O2 uptake by muscle (VO2), during a rest to submaximal contraction transition, in the two experimental conditions: Control (upper panels) and Fast O2 Delivery (lower panels). Vertical hatched lines indicate contractions onset. Modified from ref. 25, Grassi B., L. B. Gladden, M. Samaja, C. M. Stary, and M. C. Hogan. Faster adjust ment of O2 delivery does not affect VO2 on-kinetics in isolated in situ canine muscle. J. Appl. Physiol. 85:1394 1403, 1998. Used with permission of the American Physiological Society. LIMITING FACTORS FOR VO2 ON-KINETICS

apply to transitions involving exercises of higher metabolic intensity. It may be of interest to observe that, in the Control condition, t50% for Q on- (12.0 2.2 s, mean SE) was significantly lower than t50% for VO2 on- (18.6 1.5 s). Also in this experimental model, therefore, the on-kinetics of O2 delivery to muscle was faster than the on-kinetics of O2 utilization by muscle, confirming previous observations by others (45) with this preparation. Some intrinsic limitations of our study, particularly as far as the extrapolation of the obtained results to exercising humans, are discussed in detail in the paper (25). Our study (25) showed, in strict terms, that bulk convective O2 delivery to muscle was not a significant limiting factor for the VO2 on-kinetics. Apparently, our data did not allow any inference on convective O2 delivery inside the muscle. Indeed, VO2 on-kinetics could be influenced by intramuscular maldistribution of Q/VO2. It is well known that there are both spatial and temporal heterogeneity of Q within active muscle (37,46), and at present it is not known whether this corresponds to VO2 heterogeneity. Q/VO2 maldistribution could in theory influence the VO2 on-kinetics by determining areas of tissue anaerobiosis within the muscle (55). However, in our experimental model, all fibers of the muscle were synchronously activated by electrical stimulation, so that VO2 heterogeneity was presumably absent or significantly reduced compared with the situation of asynchronous and heterogenous fiber activation in physiologically contracting muscle. In Fast O2 Delivery, this re duced or absent VO2 heterogeneity was associated with high Q and with the vasodilation induced by adenosine. Thus, if any Q/VO2 maldistribution was present in Control, it must have been significantly reduced or eliminated in Fast O2 Delivery. The fact that the VO2 on-kinetics was the same in the two experimental conditions suggests that Q/VO2 maldistribution was not a significant issue in determining mus cle VO2 on-kinetics.
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PERIPHERAL O2 DIFFUSION AS A LIMITING FACTOR FOR SKELETAL MUSCLE VO2 ON-KINETICS

A determinant of VO2 on-kinetics could also be represented by the finite delivery of O2 to mitochondria by diffusion, a process that has been shown to contribute to the limitation of VO2max (54). The hypothesis that peripheral (capillary-to-mitochondria) diffusion of O2 may be a signif icant determinant of VO2 on-kinetics had not been directly tested. Also in this respect previous studies conducted in humans yielded conflicting results and had intrinsic limitations. Linnarsson (34) and MacDonald et al. (35) evaluated pulmonary VO2 on-kinetics in exercising humans breathing hyperoxic mixtures, which should determine an increased capillary PO2 (PcapO2) and thereby increase the driving force for peripheral O2 diffusion. Whereas Linnarsson (34) observed essentially unchanged VO2 on-kinetics in hyperoxia (compared with normoxia), the other group (35) described a faster kinetics for exercise above the ventilatory threshold, but not for exercises below the threshold. An intrinsic limitation of these studies is represented by the fact that the authors could not measure or make any inference to convective O2 delivery to muscle. It is indeed known that O2 delivery to and O2 extraction by skeletal muscle are impeded in hyperoxic animals (12,19), as a consequence of peripheral vasoconstriction, intramuscular blood flow restriction and maldistribution. By the hyperoxic breathing, therefore, Linnarsson (34) and MacDonald et al. (35) were presumably enhancing peripheral O2 diffusion, but at the same time, they could have been impeding convective O2 delivery to muscle. The net result on PcapO2 of the two effects would be difficult to determine. We therefore performed another study (26) using the dog gastrocnemius preparation (see above). Again, in this study, we investigated the transition from rest to electrically stimulated isometric tetanic contractions corresponding to 70% of the muscle peak VO2. The aim of the study was to enhance peripheral O2 diffusion during the rest-to-contraction transition by increasing PcapO2 (but in the presence of a constant convective O2 delivery to muscle) and to determine whether this enhancement was affecting the VO2 on-kinetics. The problem of keeping convective O2 delivery constant was solved by utilizing, as a control condition, the treatment condition (Fast O2 Delivery) of our previous study (25), i.e., a condition characterized by constantly elevated Q and adenosine administration. Because the dogs were breathing ambient air, this condition was named Normoxia. The enhancement of peripheral O2 diffusion was obtained by: a) having the dogs breathe an hyperoxic mixture (FIO2 1.00) (Hyperoxia); b) the administration, in association with hyperoxia, of a drug, 2-(4-{[(3,5-dimethylanilino)-carbonyl]methyl} phenoxy)-2-methylpropionic acid, also known as RSR-13 (Allos Therapeutics, Denver, CO), which, as an allosteric inhibitor of O2-hemoglobin binding, induces a significant rightward shift of the O2hemoglobin dissociation curve (ODC) (1). For the same O2 delivery and VO2, a rightward shift of ODC would of 112
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course increase PcapO2. This condition was termed Hyperoxia RSR13. Also in Hyperoxia and in Hyperoxia RSR13, Q was kept constantly elevated and adenosine was infused. Measured variables were substantially the same as those of our previously mentioned study (25). In addition, PO2 at which 50% Hb was saturated (P50) as well as estimates of mean capillary PO2 (PcapO2, by numerical integration) were calculated for the three experimental conditions. RSR-13 obtained a significant increase of P50 (41 1 Torr in Hyperoxia RSR13 vs 31 1 Torr in Normoxia and Hyperoxia, considered together), indicating a rightward shift of ODC, and a significant increase in PcapO2 (after 15 s of contractions, 197 39 Torr in Hyperoxia RSR13 vs 97 9 Torr in Hyperoxia); the latter was also significantly higher in Hyperoxia than in Normoxia (53 3 Torr). The main results of the study are presented in Figures 5 and

Figure 5Average values ( SE, N 5) of O2 uptake by muscle (VO2) during a rest to submaximal contractions transition, in the three experimental conditions: Normoxia, Hyperoxia, and Hyperoxia RSR13. Vertical hatched lines indicate contraction onset. Modified from ref. 26, Grassi B., L. B. Gladden, C. M. Stary, P. D. Wagner, and M. C. Hogan. Peripheral O2 diffusion does not affect VO2 on-kinetics in isolated in situ canine muscle. J. Appl. Physiol. 85:1404 1412, 1998. Used with permission of the American Physiological Society. http://www.msse.org

 Figure 6 Times necessary for muscle VO2 to reach 50% and 63% of the difference between the resting baseline and the steady-state values during contractions (t50% and t63%, respectively) in the three experimental conditions: Normoxia, Hyperoxia, and Hyperoxia RSR13. Data are presented as average values SE, N 5; NS P > 0.05. Modified from ref. 26, Grassi B., L. B. Gladden, C. M. Stary, P. D. Wagner, and M. C. Hogan. Peripheral O2 diffusion does not affect VO2 on-kinetics in isolated in situ canine muscle. J. Appl. Physiol. 85:1404 1412, 1998. Used with permission of the American Physiological Society.

present paper. The interested reader can be referred to a recent review on the topic (5). In any case, the essentially monoexponential increase in skeletal muscle VO2 during the rest-to-contraction transition, observed in human (28) as well as in canine muscle after convective and diffusive O2 constraints were eliminated or significantly reduced (25,26), appears in agreement with some metabolic models of muscle respiratory control during contraction (10,36,40), according to which a single reaction with first-order kinetics controls muscle VO2. Such a reaction can be identified with ATP resynthesis, the rate of which is directly proportional to creatine concentration, i.e., to one of the products of phosphocreatine splitting. A monoexponential decrease in phosphocreatine concentration during the on-transition has indeed been described by several authors (11,38,42). This hypothesis is supported by the direct proportionality between the products of phosphocreatine splitting and muscle or pulmonary VO2, as demonstrated in animals (36,40) and, more recently, also in exercising humans (7,39).

IS SKELETAL MUSCLE VO2 ON-KINETICS MONOEXPONENTIAL?

Pulmonary VO2 on-kinetics is not exactly monoexponen tial (for a discussion of differences in pulmonary VO2 on-kinetics between sub- and supra-threshold exercises the reader is referred to a recent review by Gaesser and Poole (21). For workloads lower than the lactate or ventilatory threshold, this kinetics is indeed characterized by three distinct phases (see, e.g., 34,57): a first phase (phase I), lasting for the initial 10 15 s of the transition, is followed by a second phase (phase II) of monoexponential increase to the new steady state (phase III). The classic concept is that phase I is cardiodynamic, i.e., is due to the sudden increase of cardiac output when the workload is increased, whereas the phase II response is considered to be mainly the result of a monoexponential VO2 increase occurring at the muscle level, and manifesting its effects at the lungs after a circulatory transit delay. The results of our studies discussed above may not be in complete agreement with this interpretation. A sluggish initial increase in leg or muscle VO2 was indeed described both in our human study (28) and in our canine studies (25,26). Methodological limitations that could in part ex plain this initial sluggish VO2 increase are briefly discussed below and, in more detail, in the mentioned papers. In any case, analysis of residuals for the seven dogs (Control condition) of one of our previously mentioned studies (25; Fig. 7) shows that a monoexponential curve calculated from the onset of contractions (Analysis A, upper panel) provided a poorer fit to the experimental points compared to a curve that leaves out the first 13 VO2 points during the transition (Analysis B, lower panel). In particular, Analysis A underestimated the first VO2 values during the transition, and overestimated most of the second and third VO2 values. Average values of squared residuals were significantly lower for Analysis B than for Analysis A. This was the case for all experimental conditions of both of our canine
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6. In Figure 5, average values of muscle VO2 are shown for the three experimental conditions: the time courses of the variable appear remarkably similar. Individual VO2 onkinetics data were evaluated mathematically as described above for our previous study (25) and the obtained t50% and t63% values are shown in Figure 6: no significant differences were observed among the three conditions. Thus, in this study, after eliminating possible influences of convective O2 delivery, two enhancements of increasing magnitude on peripheral O2 diffusion did not have signifi cant effects on skeletal muscle VO2 on-kinetics. This strongly suggests that, in the present experimental model and for the investigated metabolic transition, muscle VO2 on-kinetics was not influenced by the finite delivery of O2 to mitochondria by diffusion.

WHAT IS DETERMINING SKELETAL MUSCLE VO2 ON-KINETICS?

The observation that, during a rest-to-submaximal contraction transition, both convective and diffusive O2 deliv ery, as well as intramuscular Q/VO2 maldistribution, do not affect skeletal muscle VO2 on-kinetics, supports the hypothesis that the latter is mainly determined by an inertia of muscle oxidative metabolism (16,56). The rate of adjustment of oxidative phosphorylation during the transition would then be mainly determined by levels of cellular metabolic controllers and/or enzyme activation. A possible limiting factor for VO2 on-kinetics could be represented by acetyl group availability within mitochondria, determined by the activity of the pyruvate dehydrogenase complex, as recently suggested both for canine (52) and human (51) muscles. A detailed discussion of the different hypotheses and models dealing with the regulation of skeletal muscle respiration would go well beyond the purposes of the
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 VO2 determined across the heart (53). Moreover, myoglobin O2 stores utilization could have contributed to oxidative metabolism early during the transition (22), and the result ing VO2 could not of course be detected by our measurements, carried out across the muscle. However, the contribution of myoglobin O2 stores, with respect to the measured VO2 across the muscle, should be very small. Assuming a myoglobin concentration of 7 g kg 1 of dog muscle (41), myoglobin O2 stores in 80 g of muscle would be 0.7 mL O2. Even by assuming a 50% myoglobin desaturation, occurring early (1st min) during submaximal contractions (48), this would correspond to a VO2 of only 0.4 mL min 1 100 g 1. In any case, at present it is not clear to which extent transit delays could explain the apparently biphasic skeletal muscle VO2 time course discussed above. If confirmed, however, such biphasic response could bear some influence on the metabolic control models mentioned above (10,36,40).

CONCLUSIONS
Recent work conducted by our group has expanded knowledge on some basic issues related to pulmonary and skeletal muscle VO2 on-kinetics. In particular, we have directly confirmed that, in exercising humans, alveolar VO2 on-kinetics can be taken as a rather close approximation of skeletal muscle VO2 on-kinetics (28). Experiments conducted by utilizing the isolated in situ dog gastrocnemius preparation have demonstrated that, during transitions from rest to contractions corresponding to 70% of the muscle peak VO2, convective O2 delivery to muscle (25), intramus cular Q/VO2 maldistribution (25), and peripheral O2 diffusion (26) do not represent limiting factors for skeletal mus cle VO2 on-kinetics. The latter, therefore, appears to be mainly determined by an intrinsic inertia of skeletal muscle oxidative metabolism, possibly related to acetyl group availability within mitochondria (51,52), to regulatory effects on intracellular respiration related to phosphocreatine splitting (10,36,40), and/or to other still not precisely identified control mechanism(s). Other evidence from the literature (see e.g. 23,35) suggests that the limiting factors of skeletal muscle VO2 on-kinetics may vary according to the intensity of muscular contractions or of exercise.
The experiments mentioned in the present paper (refs. 25, 26, and 28) were obtained during two periods spent by the author in Dr. Peter D. Wagners laboratory, at the Division of Physiology, Department of Medicine of the University of California, San Diego. The experiments were conducted in collaboration with Drs. Michael C. Hogan, L. Bruce Gladden, David C. Poole, Russell S. Richardson, Douglas R. Knight, B. Kipp Erickson, Creed M. Stary, Michele Samaja, and Peter D. Wagner. Funding by National Institutes of Health Grants HL-17731, AR-40155, by Allos Therapeutics, and by NATO Collaborative Research Grant n. 972111 is acknowledged. Address for correspondence: Bruno Grassi, M.D., Ph.D., ITBA CNR, Palazzo LITA, Via Fratelli Cervi 93, I - 20090 Segrate (MI), Italy. E-mail: grassi@itba.mi.cnr.it.

Figure 7Analysis of residuals for monoexponential curves fitting muscle VO2 values from the onset of contractions (Analysis A, upper panel) or that leave out the first 13 VO2 values (Analysis B, lower panel). Muscle VO2 values are shown by the points, whereas solid lines indicate fitted curves. Data refer to 6 dogs in Control condition of study cited in ref. 25, Grassi B., L. B. Gladden, M. Samaja, C. M. Stary, and M. C. Hogan. Faster adjustment of O2 delivery does not affect VO2 on-kinetics in isolated in situ canine muscle. J. Appl. Physiol. 85:1394 1403, 1998. Used with permission of the American Physiological Society.

studies (25,26). Thus, as also suggested by our human study (28), during the initial 515 s of contractions muscle VO2 increase appeared to be less pronounced compared with the ensuing phase of monoexponential increase, confirming previous observations by other groups (6,22). On the other hand, a biphasic muscle VO2 response was not described by Mahler (36) and by Piiper et al. (45). The methodological factors that could at least in part explain the initial sluggish VO2 increase relate to transit delays introduced by the fact the VO2 was necessarily determined across the muscle, and not inside of it, where gas exchange occurs. Van Beek and Westerhof (53) applied mathematical methods to their isolated rabbit heart experimental model, in order to account for O2 diffusive and vascular transport delays in the interpretation of the observed venous O2 concentration transients during step changes in heart rate. The resulting mitochon drial VO2 response time was significantly faster than the

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