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J Appl Physiol 93: 1265-1274, 2002. First published June 14, 2002; doi:10.1152/japplphysiol.00809.2001
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Vol. 93, Issue 4, 1265-1274, October 2002

Determinants of maximal O2 uptake in rats selectively bred for endurance running capacity

Kyle K. Henderson1, Harrieth Wagner2, Fabrice Favret1, Steven L. Britton3, Lauren G. Koch3, Peter D. Wagner2, and Norberto C. Gonzalez1

1 Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160-7401; 2 Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623; and 3 Functional Genomics Laboratory, Medical College of Ohio, Toledo, Ohio 43614-5804


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

O2 transport during maximal exercise was studied in rats bred for extremes of exercise endurance, to determine whether maximal O2 uptake (VO2 max) was different in high- (HCR) and low-capacity runners (LCR) and, if so, which were the phenotypes responsible for the difference. VO2 max was determined in five HCR and six LCR female rats by use of a progressive treadmill exercise protocol at inspired PO2 of ~145 (normoxia) and ~70 Torr (hypoxia). Normoxic VO2 max (in ml · min-1 · kg-1) was 64.4 ± 0.4 and 57.6 ± 1.5 (P < 0.05), whereas VO2 max in hypoxia was 42.7 ± 0.8 and 35.3 ± 1.5 (P < 0.05) in HCR and LCR, respectively. Lack of significant differences between HCR and LCR in alveolar ventilation, alveolar-to-arterial PO2 difference, or lung O2 diffusing capacity indicated that neither ventilation nor efficacy of gas exchange contributed to the difference in VO2 max between groups. Maximal rate of blood O2 convection (cardiac output times arterial blood O2 content) was also similar in both groups. The major difference observed was in capillary-to-tissue O2 transfer: both the O2 extraction ratio (0.81 ± 0.002 in HCR, 0.74 ± 0.009 in LCR, P < 0.001) and the tissue diffusion capacity (1.18 ± 0.09 in HCR and 0.92 ± 0.05 ml · min-1 · kg-1 · Torr-1 in LCR, P < 0.01) were significantly higher in HCR. The data indicate that selective breeding for exercise endurance resulted in higher VO2 max mostly associated with a higher transfer of O2 at the tissue level.

O2 transport; lung diffusion capacity; muscle diffusion capacity; genetic models


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AEROBIC CAPACITY IS A COMPLEX trait determined by the interplay of genetic and environmental factors. Recent evidence suggests two genetic substrates as contributors to the aerobic phenotype: a complement of genes that determine intrinsic exercise capacity in the untrained state (5) and an additional set of genes that dictate the adaptational response to exercise (4, 6). Although studies in both humans and animals suggest that a genetic component accounts for as much as 70-90% of the total variation in aerobic capacity (25), the individual genes causative of the difference between low and high aerobic capacity remain essentially undefined.

Given such complexity, animal models with minimal genetic as well as environmental variation can be of substantial value for determining the genes causative of variation in aerobic capacity (7). In theory, divergent artificial selection for a complex trait should produce excellent genetic models because contrasting allelic variation is concentrated at the extremes from one generation to the next. A response to selection occurs if sufficient additive genetic variance exists in a population for that trait (12).

Artificial divergent selection of rats was started in 1996 (26) with the purpose of creating low-capacity (LCR) and high-capacity runners (HCR) that could ultimately be developed into contrasting strains for genetic and physiological studies of intrinsic (i.e., untrained) aerobic capacity. Six generations of selection produced LCR and HCR that differed in maximal distance run by 171% (26). The selection process continues; the data presented here were obtained in HCR and LCR rats of generation 7.

Maximal O2 uptake (VO2 max) during exercise is an indication of the capacity of the O2 transport system, i.e., the lungs, cardiovascular system, and musculoskeletal system, to transport and utilize O2 under a given set of conditions, and it is thought to be the result of the interplay between the convective transport of O2 (TO2) to the capillaries of skeletal muscle and the diffusion of O2 from the capillaries to the mitochondria (39). In general, VO2 max correlates with exercise endurance; however, this is not always the case, indicating that different factors determine exercise endurance and VO2 max (9, 10). The objective of these studies was to determine whether the difference in exercise endurance between HCR and LCR is accompanied by differences in VO2 max, and, if so, which components of the O2 transport system differ between groups to explain the differences in performance. To this end, the conductance properties of each major step in the O2 transport chain from the atmosphere to the cells were measured during maximal exercise. The results indicate that the major difference in maximal exercise O2 transport between LCR and HCR centers in the O2 conductance of skeletal muscle, that is, the transport capacity from the muscle microcirculation to the mitochondria.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal model. All procedures were carried out according to the Guide for the Care and Use of Laboratory Animals. The development of the LCR and HCR through generation 6 was described in detail previously (26). Briefly, artificial, divergent, selective breeding was used to create low and high lines for treadmill running capacity. The founder population was 80 male and 88 female genetically heterogeneous rats (N: NIH stock) obtained from a colony maintained at the National Institutes of Health (18). Each rat in the founder population was of different parentage so that selection was not among brothers and sisters, which produced a broader initial genetic variance (19).

Assessment of endurance running capacity. The protocol for estimation of endurance capacity required 2 wk and was started when the rats were 10 wk old (26). The first week consisted of placing the rats on the treadmill (Model Exer-4, Columbus Instruments, Columbus, OH) for increasing duration each day, until the animals were able to run 5 min at 10 m/min on a 15° slope. This exercise duration is insufficient to significantly increase aerobic performance (1, 11). During the second week, each rat underwent a daily endurance trial on 5 consecutive days at a constant slope of 15° and an initial velocity of 10 m/min. Treadmill velocity was increased by 1 m/min every 2 min until the third time a rat could no longer keep pace with the speed of the treadmill. Although this criterion is somewhat arbitrary, it was applied uniformly to all rats of both groups.

For each of the five trials, the total distance run (in m) was used as the estimate of endurance capacity. The single best daily run of five trials for each rat was considered the trial most closely associated with the heritable component of exercise endurance.

Selective breeding. By using the criterion of single best day, the 13 lowest and 13 highest capacity rats of each gender were selected from the founder population and randomly paired for mating. At 10 wk of age, the offspring were tested for running capacity as described above. At each subsequent generation, within-family selection from 13 mating pairs was practiced because it decreases the rate of inbreeding to yield retention of genetic variation and thus increases the overall response to selection (12). Ten LCR and 10 HCR females rats from the extremes of generation 7 rats were selected for the present study. The LCR group was able to run a maximum of 222 ± 17 m over 16.2 ± 0.96 min, whereas the HCR ran 1,590 ± 77 m over 62.8 ± 2.0 min. The average maximal speeds were 17.5 and 40.9 m/min, respectively.

Systemic O2 transport studies. The animals were transported from the Medical College of Ohio to the University of Kansas Medical Center, where the experiments took place. All surgical and experimental procedures were approved by the Animal Care and Use Committee of the University of Kansas Medical Center, an institution accredited by the American Association for the Accreditation of Laboratory Animal Care. The experiments began 2 wk after arrival of the rats at the University of Kansas Medical Center. One day before the exercise protocol, the animals were anesthetized with Nembutal (30 mg/kg ip). A polyethylene catheter (PE-50) was placed in the aortic arch via the left carotid artery, and a PE-10 catheter was advanced into the pulmonary artery via the right jugular vein with the aid of a J-shaped introducer. Adequate placement of the catheters was established by the pressure waveform and was verified at autopsy. The catheters were tunneled subcutaneously, exteriorized at the back of the neck, cut at a length of 4 cm of their emergence from the skin, and flame sealed. The animals were allowed to recover from anesthesia and exercised on the following day. Each animal exercised maximally twice: once in normoxia [inspired PO2 (PIO2) ~145 Torr] and once in hypoxia (PIO2 ~70 Torr). Both runs were carried out on the same day, with the order of the hypoxic and normoxic runs being alternated on successive days. An interval of ~3 h was allowed between runs in each rat. An equal number of HRC and LRC were tested on each day.

Maximal exercise protocol. After measurement of rectal temperature, the animals were placed on a treadmill enclosed in an airtight Lucite chamber adapted for the determination of O2 uptake (VO2) and CO2 production (VCO2) by use of the open-circuit method as described before (20). The catheters were connected, through sampling ports located on the top of the box enclosing the treadmill, to pressure transducers. After 30 min at rest on the treadmill, arterial and mixed venous blood samples were obtained via stopcocks, the blood was replaced with homologous fresh blood, and the treadmill was set at a speed of 10 m/min and an angle of 10°. This work rate was maintained for 2-3 min, after which speed was increased by 4 m/min every 90-120 s, until VO2 max was reached. VO2 max was defined as the VO2 after which an increase in work rate was not associated with a further increase (±5%) in continuously measured VO2. At the highest work rates attained in these experiments, a 5% change in VO2 resulted in a change in effluent %O2 concentration of ~0.006 and 0.004 in normoxic and hypoxic exercise, respectively. These are translated into changes of 6 and 4 mV in the O2 analyzer output, respectively, which is well within the range of detection of the system.

Arterial and mixed venous (pulmonary arterial) blood samples were obtained during the last 60-120 s of exercise, while VO2 and VCO2 showed steady values. The box enclosing the treadmill was opened, and the rectal temperature was determined within 30 s of termination of exercise. After the first run, the blood withdrawn in the exercise sample was replaced with homologous fresh blood, and 0.5 ml/100 g of a solution of 0.15 mM NaHCO3 was administered intravenously to correct the metabolic acidosis of maximal exercise. After the last run, the animals were killed with an overdose of pentobarbital sodium, 60 mg/kg iv, and heart and tissue samples were obtained for histological analysis. The results of the histological studies will be reported separately.

Gas exchange and O2 transport determinations. The box enclosing the treadmill is airtight except for the in- and outflow ports, which are independent of one another. PIO2 was adjusted to the desired level by mixing O2 and N2. Flow of the gas mixture entering the treadmill box was maintained constant at ~20 l/min by use of a Cameron Instruments precision gas flow mixer. Inflowing and outflowing O2 concentrations and outflowing CO2 concentration (inflowing gas was CO2 free) were measured continuously and simultaneously by use of an Applied Electrochemistry O2 analyzer and a Columbus Instruments CO2 analyzer, respectively. The output of the O2 and CO2 meters was fed into a computer to provide determination of VO2, VCO2, and respiratory exchange ratio every 5 s. VO2 and VCO2 were calculated from the inflowing and outflowing O2 concentration difference, the outflowing CO2 concentration, and the outflowing gas flow by using standard gas-exchange equations (expressed in ml STPD · min-1 · kg-1). An estimate of the time needed for the gas composition of the box to reach a new steady state after a change in VO2 of the rat was obtained by producing a stepwise change in treadmill gas composition and recording the time necessary for outflow fractional O2 concentration to reach a stable value. At a flow of 19.6 ± 0.5 l/min, a square-wave change in treadmill gas composition was 94% complete in 37.8 ± 1.1 s (n = 5). This time includes the mixing of gas in the box enclosing the treadmill as well as the time delay within the measuring system. These conditions provide ample time to determine whether VO2 has reached a new steady state after work rate is increased, because treadmill speed is changed every 90-120 s.

Arterial and mixed venous blood samples were analyzed for pH, PO2, and PCO2 in a Radiometer ABL 5 blood-gas analyzer at 37°C and for Hb concentration and O2 saturation of Hb by using a Radiometer Osm 3 analyzer. Values measured at 37°C were corrected to the rectal temperature by using temperature correction factors for rat blood (16). Lactate concentration (mmol/l blood) was determined in exercise arterial blood samples by using an enzymatic assay.

Systemic and pulmonary arterial pressures (PAP) were recorded continuously, with mean pressures obtained by electronic integration. Heart rate (HR) was obtained directly from the systemic arterial blood pressure tracing.

O2 contents (ml/dl) of arterial (CaO2) and of mixed venous blood were calculated from measured values of Hb concentration, PO2, and O2 saturation by using an HbO2 binding factor of 1.34 ml STPD/g. This constant was obtained from direct measurement of total blood O2 content (Oxycon, Cameron Instruments, Port Aransas, TX) and of blood Hb concentration by using a spectrophotometric method. In each animal, measured O2 saturation and the corresponding PO2 from all samples were used to estimate standard hemoglobin PO2 for 50% HbO2 saturation at pH 7.4, PCO2 of 40 Torr, and temperature of 37°C (P50). Cardiac output (Q, in ml · min-1 · kg-1) was calculated as the ratio of VO2 to arteriovenous O2 content difference [(a-<A><AC>v</AC><AC>&cjs1171;</AC></A>)CO2]. The rate of TO2 (in ml · min-1 · kg-1) was calculated as the product of Q times CaO2. The O2 extraction ratio was calculated as (a-<A><AC>v</AC><AC>&cjs1171;</AC></A>)CO2/CaO2. Mean tissue capillary PO2 (PcO2, in Torr) and the corresponding value for tissue O2 conductance (DTO2, in ml · min-1 · Torr-1 · kg-1) at maximal exercise were calculated using a numerical integration procedure (34, 40). Effective lung diffusion capacity (DLO2, in ml · min-1 · Torr-1 · kg-1) during maximal hypoxic exercise was calculated by Bohr integration from VO2 max, arterial, mixed venous, and alveolar PO2 values, with the assumption that all of the difference between alveolar and arterial PO2 [(A-a)PO2] is due to diffusion limitation (16). Alveolar ventilation (VA, in ml · min-1 · kg-1) was calculated from VCO2 and arterial PCO2 (PaCO2).

The data are expressed as means ± SE. Statistical analysis was carried out by using a one-way ANOVA. The effect of hypoxia was evaluated by comparing the data of HCR in normoxia vs. HCR in hypoxia and of LCR in normoxia vs. LCR in hypoxia. The effect of selective breeding was evaluated by comparing the data of HCR in hypoxia vs. LCR in hypoxia and that of HCR in normoxia vs. LCR in normoxia. Significance was established with the t-test using the Bonferroni correction for multiple comparisons. A P value <0.05 was considered to indicate a significant difference. The variables for which the one-way ANOVA test indicated a significant difference were further analyzed using a two-way repeated-measures ANOVA for intergroup comparisons at both exercise inspired O2 fraction (FIO2) values in the subset of animals providing complete data at both FIO2 values: 3 HCR and 5 LCR.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Data on five animals of the HCR group and six of the LCR group are presented. These were animals that reached VO2 max as defined above and in which a full set of arterial and venous blood samples could be obtained while the animal maintained a steady work rate for 60-120 s at the highest exercise level.

Body weight (in g) on the day of exercise was significantly lower in HCR than in LCR (215 ± 9 vs. 250 ± 4, P < 0.01). These values were not significantly different from those observed the previous day, immediately before surgery (228 ± 12 and 260 ± 8 for HCR and LCR, respectively). Resting rectal temperature on the day of exercise was 37.8 ± 0.2 and 37.8 ± 0.1°C in HCR and LCR, respectively. The lack of significant change in body weight after surgery, the normal arterial blood acid-base values (see below), and the normal rectal temperature on the day of exercise suggest that the animals recovered well from surgery by the time of exercise.

Table 1 shows the blood acid-base values obtained before the first and second exercise runs, after the animals had been in the treadmill for ~30 min. Because there were no differences in preexercise arterial blood acid-base values between HCR and LCR, the data of both groups were pooled. Although hypoxia resulted in the expected hyperventilation-induced decrease in PaCO2, there was no evidence of residual metabolic acidosis before the second run, probably in some measure because of the administration of NaHCO3 immediately after the first run. In addition to a lack of difference in resting acid-base composition, we observed no consistent difference in O2 transport variables between the first and second run of animals of the same group exercising under the same PIO2, suggesting that prior exercise did not systematically influence the results of the second run.

                              
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  		Table 1.   Resting arterial blood acid-base values obtained before the first and second exercise bout

As expected, hypoxia resulted in a reduction in VO2 max in both groups of rats. In addition, VO2 max in HCR was ~20% higher than LCR in hypoxia and ~12% higher in normoxia (Table 2). Hypoxia had the predicted effect on pulmonary gas exchange in both groups: VA was higher, and alveolar and arterial PO2 were lower, in hypoxia than in normoxia. However, no significant differences in any of these variables were observed between HCR and LCR at either PIO2 level (Table 2). (A-a)PO2 increased significantly (from those in room air) in hypoxia in both groups; although there was a tendency for a higher (A-a)PO2 in the HCR group at both PIO2 levels, this did not reach statistical significance (Table 2). No significant difference in DLO2 was observed during hypoxic exercise between HCR and LCR. DLO2 was not calculated in normoxia because the insensitivity in this calculation when arterial PO2 is in the flat portion of the HbO2 dissociation curve. The data on VA, (A-a)PO2, and DLO2 indicate that efficacy of pulmonary gas exchange was similar in both groups.

                              
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  		Table 2.   Pulmonary gas exchange in maximal exercise

Table 3 shows data on TO2 during maximal exercise for both groups. Although maximal cardiac output (Qmax) was higher in HCR than LCR at both PIO2 levels, the difference reached statistical significance only during hypoxia. CaO2 showed the expected effect of hypoxia in both groups, which was reflected in the values of maximum TO2 (TO2 max), the rate of blood O2 delivery to the tissues. No significant differences were observed between HCR and LCR in either TO2 max, Hb concentration, or CaO2. Standard P50, on the other hand, was slightly but significantly lower in HCR than in LCR (Table 3). It is unlikely, however, that a difference of this magnitude would have a substantial effect on O2 transport.

                              
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  		Table 3.   Convective blood O2 transport in maximal exercise

The major differences between HCR and LCR were seen in the transfer of O2 from blood to tissue (Table 4). Figure 1 shows VO2 max plotted as a function of TO2 max. The straight lines are best fit lines drawn through the origin and are included only to show that the relationships were in fact proportional as PIO2 was changed and also systematically different between groups. The slope of these lines, the average O2 extraction ratio observed over the range of exercise PIO2 investigated, was significantly higher (P < 0.01) in HCR than LCR. This agrees with the O2 extraction ratios calculated for each individual exercise bout, which were also significantly higher in HCR than in LCR rats (Table 4). Mixed venous PO2 and mean tissue PcO2 were lower in HCR, although this difference did not reach statistical significance (Table 4). Because VO2 max was higher in HCR at both PO2 levels, yet PcO2 was similar, a larger O2 flux was obtained with equal PO2 diffusion gradient from capillary to cell in HCR. This is confirmed by the values of calculated DTO2, which were significantly higher in HCR than LCR, both in hypoxia and in normoxia (Table 4). Hypoxia had the predicted effects of significantly lowering (a-<A><AC>v</AC><AC>&cjs1171;</AC></A>)CO2 and mixed venous and PcO2 in both groups. No significant effects of hypoxia on O2 extraction ratio or DTO2 were observed in either HCR or LCR. To strengthen the conclusions that a significant difference exists in capillary-to-cell O2 transfer between HCR and LCR, a two-way repeated-measures ANOVA for intergroup comparisons at both FIO2 values was carried out in the subset of animals that provided complete data at both FIO2: 3 HCR and 5 LCR. This analysis confirmed the presence of significant differences between HCR and LCR in the following variables: VO2 max, Qmax, mixed venous PO2, O2 extraction ratio, and DTO2. On the other hand, no significant difference in mean PcO2 between HCR and LCR was observed by using this approach.

                              
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  		Table 4.   Blood-tissue O2 transfer in maximal exercise



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  		Fig. 1.   Maximal rate of O2 consumption (VO2 max) plotted as a function of the maximal rate of convective blood O2 delivery (TO2 max) calculated as maximal cardiac output (Qmax) times arterial blood O2 content. The slope Delta VO2 max/Delta TO2 max represents the average O2 extraction ratio (a-<A><AC>v</AC><AC>&cjs1171;</AC></A>)CO2/arteriovenous O2 content difference to O2 content of arterial blood. HCR, high-capacity runners; LCR, low-capacity runners. Regression lines were calculated from individual values and drawn through the origin. Bars represent 1 SE on either side of the mean.

Table 5 shows hemodynamic variables in maximal exercise. There was a tendency for HR and mean arterial pressure to decrease with hypoxia in both groups, but this did not reach statistical significance. LCR showed the expected increase in PAP and in the ratio PAP/Q with hypoxia; on the other hand, neither PAP nor PAP/Q increased in hypoxia in HCR, indicating that this group surprisingly did not exhibit hypoxic pulmonary vasoconstriction (HPV).

                              
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  		Table 5.   Hemodynamic variables in maximal exercise

Exercise resulted in the expected metabolic acidosis characterized by reduced plasma bicarbonate concentration and PCO2 in arterial blood, negative base excess, and elevated blood lactate concentration (Table 6). Although hypoxia resulted in lower PaCO2 and higher pH values than normoxia, these features applied equally to HCR and LCR, without significant differences between groups in arterial or venous blood acid-base values either in normoxic or hypoxic exercise. The elevated blood lactate concentration was likely a major cause of the observed metabolic acidosis: however, no differences in blood lactate concentration were observed between HCR and LCR in either normoxic or hypoxic exercise.

                              
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  		Table 6.   Arterial and mixed venous acid-base values in maximal exercise


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The principal findings of this study are 1) VO2 max is higher in rats selectively bred for high exercise endurance capacity, and 2) the major factor related to O2 transport contributing to the higher VO2 max in HCR rats is a higher capacity for O2 transfer at the tissue level. An additional, unexpected, observation is that HCR rats did not develop pulmonary hypertension during acute hypoxia.

Experimental design. The O2 transport system was conceived, for the analysis and interpretation of the data, as composed of four linked conductances: ventilatory convection, alveolar-capillary diffusion, blood convection, and tissue capillary-to-cell diffusion of O2 (37, 38). Maximal exercise was studied because, under these conditions, it is possible to obtain a measure of the capacity of the system to transport and utilize O2. Because the animal preparation allowed us to make a valid appraisal of each of the above four linked O2 conductances, it was possible to determine which ones contributed to differences in overall maximal O2 transport between HCR and LCR and also to determine their relative importance. Each animal ran in normoxia as well as in hypoxia; this was done to determine whether there is a difference between the selectively bred lines in the response to O2 limitation and to provide a reliable estimate of pulmonary and tissue O2 diffusive conductances. O2 conductance values are difficult to interpret in the absence of a clear O2 dependence of peak O2. Given this experimental design, it was possible to obtain an accurate assessment of the various components of the O2 transport system during maximal exercise.

Comparison of the present results with previous data. Table 7 shows values of O2 transport variables obtained during maximal treadmill exercise in untrained rats. It is apparent that there is a relatively large scatter in values. Adequate comparison among studies is hampered by differences in strain, sex, and age and weight of the animals used in the various studies. In addition, differences in the maximal exercise protocol, as well as the presence or absence of vascular catheterization, further complicate the comparison. The VO2 max values observed in the present studies are at the lower end of values reported for animals instrumented with arterial and venous catheters (14, 22), including those obtained by us using the same protocol in male Sprague-Dawley rats (15, 20). Given the differences in sex, strain, and age among the various studies, it is difficult to ascertain which factors contributed to the differences in O2 transport variables among the different studies. The fact remains that, in the present study, VO2 max was significantly higher in HCR than in LCR rats when both groups were studied at the same time under identical experimental conditions.

                              
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  		Table 7.   Comparison of O2 transport values obtained previously in rats with those of this study

Pulmonary ventilation and gas exchange. When normalized for O2 consumption, there were no differences in VA between HCR and LCR, indicating that ventilatory conductance was commensurate with the VO2 in both groups and suggesting that the higher VO2 max of HCR was not the result of a higher ventilatory O2 conductance.

The values for (A-a)PO2 and DLO2 provided measures of efficacy of pulmonary gas exchange. The (A-a)PO2 is influenced by diffusion limitation and by heterogeneity of VA/Q distribution. Hypoxia resulted in a significant increase in (A-a)PO2 in both groups. This is likely to be due principally to diffusion limitation: first, the alveolocapillary PO2 gradient normally decreases in hypoxia; second, the effect of VA/Q heterogeneity on (A-a)PO2 is reduced in hypoxia as a consequence of the nearly linear shape and steepness of the O2 dissociation curve at low PO2 values, whereas the effect of diffusion limitation on (A-a)PO2 is increased. That diffusion limitation is likely to play a role in the (A-a)PO2 differences between groups is suggested by the dependence of (A-a)PO2 on Q (Fig. 2). For any given PIO2, (A-a)PO2 correlated positively with Qmax, with the highest values of Qmax and (A-a)PO2 seen in the HCR group. This suggests that the tendency of HCR to develop higher (A-a)PO2 in maximal exercise is the result of lower pulmonary capillary transit time and not of a lower efficacy of pulmonary gas exchange. This is supported by the observation that DLO2 was not significantly different in HCR and LCR (Table 2). DLO2 takes into account VO2 as well as "ideal" alveolar, arterial, and mixed venous PO2 values. Taken together, the data on (A-a)PO2 and DLO2 indicate that the higher VO2 max of HCR was not the result of differences in ventilatory conductance or in efficacy of pulmonary gas exchange.


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  		Fig. 2.   Alveolar-to-arterial PO2 difference [(A-a)PO2] plotted as a function of Qmax. Bars represent 1 SE on either side of the mean.

Blood O2 convection. TO2 max, the rate of convective transport of O2 from the lungs to the tissue capillaries, is the product of Q and CaO2. Qmax was significantly higher in HCR than in LCR during hypoxic exercise. The lower P50 in the HCR group should enhance VO2 in the lungs, particularly in hypoxia, and thus contribute to increase blood O2 convection. These effects, however, did not materialize in a significantly higher TO2 max. This was due in part to an offsetting effect of a lower Hb and CaO2 in HCR. In conclusion, the data show that the higher VO2 max evidenced by HCR was not the result of a higher rate of convective blood O2 delivery to the tissues.

Blood-tissue O2 transfer. The main difference in O2 transport between HCR and LCR was observed at the tissue level. This was evidenced in several ways. The O2 extraction ratio was significantly higher in HCR (Table 4, Fig. 1). The O2 extraction ratio is determined by the ratio of tissue diffusive-to-perfusive conductances (30). It is apparent that the higher O2 extraction ratio of HCR was largely the result of the higher tissue diffusive conductance: first, there were no significant differences in TO2 max between HCR and LCR; second, DTO2 was higher in HCR at both levels of PIO2. The differences in diffusive conductance between both groups are better illustrated in Fig. 3, which shows VO2 max plotted as a function of mixed venous (Fig. 3A) and of mean PcO2 (Fig. 3B). In general, the rate of VO2 by the tissues for a given value of effluent blood PO2 reflects the capacity for O2 transfer between the capillary and the cells. A better estimate of this capacity is obtained by relating VO2 max to mean tissue PcO2. There are a number of assumptions involved in this approach. Those related to the calculation of tissue PcO2 have been discussed in detail before (34, 40). In this case, mean PcO2 was calculated from arterial and mixed venous (pulmonary arterial) blood, rather than from skeletal muscle venous PO2. Because most of the O2 utilization in quadrupeds exercising maximally takes place in skeletal muscle (27), the PO2 of mixed venous blood largely reflects the PO2 of the blood draining the exercising muscles. Under these conditions, skeletal muscle cell PO2 is near zero (13, 32), and the mean PcO2 is an adequate representation of the gradient for O2 diffusion from capillary to cell. Within the framework of these assumptions, the slope of the line relating VO2 max and mean PcO2 represents the average tissue O2 transfer capacity (i.e., VO2 max/PO2 gradient = DTO2), a composite parameter determined by all the processes involved in the flow of O2 from the capillary to the mitochondrion. A requirement for the calculation of DTO2 using Fick's law of diffusion is the demonstration of O2 supply dependence of VO2 max, which is clearly shown in Figs. 1 and 3. The slope Delta VO2 max/Delta PcO2 (ml · min-1 · Torr-1 · kg-1) for HCR is 1.18 ± 0.09, and for LCR it is 0.92 ± 0.05 (P < 0.05). This represents the average DTO2 over the PO2 range studied: DTO2 was ~30% higher in HCR than in LCR. These values are in agreement with the DTO2 values calculated for each group in hypoxic and normoxic exercise (Table 4).


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  		Fig. 3.   VO2 max plotted as a function of the mixed venous PO2 (A) and mean tissue capillary PO2 (PcO2; B). The slope Delta VO2 max/Delta PcO2 represents the average tissue diffusing capacity over the inspired PO2 range investigated. Regression lines were calculated from individual values and drawn through the origin. Bars represent 1 SE on either side of the mean.

Our interpretation of these data is that selective breeding resulted in animals with higher diffusive muscle O2 conductance, which enabled a higher VO2 max. The relationship between VO2 max and diffusive muscle conductance is illustrated in Fig. 4. The straight dashed lines with positive slope represent constant values of VO2 max/DTO2 during hypoxic and normoxic exercise. The VO2 max/DTO2 is the capillary-to-cell PO2 gradient, which is represented by the mean PcO2 because mitochondrial PO2 during maximal exercise is very close to zero (see above). The PcO2 values represented in Fig. 4 are the numerical average of the HCR and LCR values. Actual PcO2 was not significantly different in HCR and LCR during hypoxic exercise and was slightly but significantly higher in LCR during normoxic exercise (Table 4). At exercise levels in which the metabolic endpoints characterizing peak exercise (blood lactate, base excess, HR, respiratory exchange ratio) were the same for both groups, VO2 max was significantly higher in HCR than in LCR, despite either similar or lower PO2 diffusion gradient in HCR.


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  		Fig. 4.   VO2 max plotted as a function of diffusive muscle O2 conductance (DTO2). Dashed lines represent constant values of the VO2 max-to-DTO2 ratio. This ratio is the capillary-to-cell PO2 gradient. Because at VO2 max cell PO2 is near zero, the capillary to-cell-PO2 gradient is essentially equal to the PcO2. The values of PcO2 represented in the figure are the numerical averages of the HCR and LCR values for normoxic and hypoxic exercise presented in Table 4. Bars represent 1 SE on either side of mean.

An alternative explanation for the present results is that, during the preliminary tests to determine exercise endurance, HCR ran for longer distances for reasons other than a higher muscle O2 diffusive conductance and the higher exercise intensity had a training effect that resulted in improved muscle DTO2. This possibility seems unlikely given the duration of the "training" period (5 days) as well as the interval between the endurance and the maximal exercise tests (~8 wk).

Although the data suggest that the major factor responsible for the difference in VO2 max resides in the effectiveness of O2 transfer from capillary to mitochondrion, the mechanism responsible for the higher DTO2 in HCR is not clear from this study. Several lines of evidence suggest that a major determinant of skeletal muscle DTO2 is the capillary-to-fiber interface available for O2 diffusion (21, 24, 28). Accordingly, it would be expected that skeletal muscle capillary-to-fiber surface area would be higher in HCR.

The results of this study provide an example of the interplay between convective blood O2 delivery and O2 diffusion at the tissue level as determinants of VO2 max. There is general agreement that interventions that increase blood O2 convection, especially high rates of blood flow, result in improvement in VO2 max (36). However, more recent observations of a dissociation between blood O2 convection and VO2 max in isolated skeletal muscles (23, 33) and intact animals (20, 29) demonstrate the limiting role of O2 diffusion at the tissue level. The present data suggest that an almost exclusive increase in tissue diffusive conductance is a useful strategy to improve VO2 max.

The difference in VO2 max between HCR and LCR was smaller, in relative terms, than the difference in indexes of endurance capacity observed between the lines. The maximal distance run during normoxic exercise in the endurance tests was almost seven times higher, and the maximal run time was approximately four times as long, in HCR than LCR (26). In comparison, normoxic VO2 max was only 12% higher in HCR. This points out the different determinants of endurance and VO2 max (8, 17, 35). Exercise training also results in a relatively small increase in VO2 max compared with the increase observed in endurance capacity (10). Evidence suggesting that the dissociation between endurance and VO2 max reflects the different determinants of these parameters was obtained in the studies of Davies et al. (9), which showed that iron repletion after correction of iron-deficient anemia was accompanied by restoration of VO2 max that closely paralleled the increase in blood Hb concentration. On the other hand, muscle oxidative capacity, muscle mitochondrial content, and endurance capacity remained low. These data suggest that endurance capacity is limited by muscle oxidative capacity but not by muscle O2 delivery (8-10, 17, 35). VO2 max, on the other hand, is determined by the interplay between convective and diffusive O2 conductance (39). The present data suggest that genetic selection for endurance, like exercise training, is not accompanied by proportionate increases in VO2 max. Whether the higher endurance of HCR is accompanied by higher muscle oxidative capacity should be the subject of future research. The data obtained in the present experiments indicate that the strategy followed to increase VO2 max in this case was by enhancing muscle diffusive O2 capacity rather than increasing convective blood O2 delivery.

Pulmonary circulation. An unexpected finding of this study was that PAP did not increase in HCR in response to hypoxia. This was the case both at rest and during exercise and was not the result of a lower Qmax because the PAP/Q under hypoxic conditions was always lower in HCR than LCR, both at rest and during exercise (Fig. 5). The most likely reason for this lack of response is a decreased HPV response in HCR. The mechanism responsible for HPV is not clear, although evidence has accumulated indicating a role of vascular smooth muscle K channels (31, 41). Whether the different response of HCR is due to a difference in K channel behavior or a difference in the balance between pulmonary vasoconstrictors and vasodilators that modulate HPV (2) should be the subject of future research. Whatever the mechanism, a reduced HPV should prove advantageous in hypoxic exercise because it should help maintain a lower right ventricular work for a given Q.


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  		Fig. 5.   Mean pulmonary arterial pressure (PAP) plotted as a function of Q for resting and exercise conditions. The straight dotted lines represent constant PAP-to-Q ratios of 30, 60, and 90 mmHg · l-1 · min-1 · kg-1. Bars represent 1 SE on either side of the mean.

In summary, the data presented here indicate that VO2 max of rats bred selectively for high exercise endurance is significantly greater than that of LCR and that the principal factor responsible for this difference is a higher efficacy of O2 transfer at the tissue level. The structural basis for this remains to be determined. On the other hand, neither ventilatory conductance nor efficacy of pulmonary gas exchange nor blood O2 convection appear to play significant contributory roles in the elevated VO2 max of these animals.


    ACKNOWLEDGEMENTS

The skillful technical assistance of Julie Allen and Mary Nelson is gratefully acknowledged.


    FOOTNOTES

This research was supported by National Heart, Lung, and Blood Institute Grants HL-39443, HL-17731, and HL-64270

Present address of K. K. Henderson: Department of Veterinary Biomedical Sciences, University of Missouri, Columbia, MO 65211.

Address for reprint requests and other correspondence: N. C. Gonzalez, Dept. of Molecular and Integrative Physiology, Univ. of Kansas Medical Center, Kansas City, KS 66160-7401 (E-mail: ngonzale{at}kumc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

June 14, 2002;10.1152/japplphysiol.00809.2001

Received 1 August 2001; accepted in final form 12 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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