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1Laboratoire "Reponses Cellulaires et Fonctionelles à l'Hypoxie," Association pour la Recherche en Physiologie de l'Environnement, Université Paris XIII, 93017 Bobigny, France; and 2Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160
Submitted 12 December 2001 ; accepted in final form 24 June 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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O2 max). Prolonged hypoxia shows reductions in cardiac output (
), maximal heart rate (HR-max), myocardial 
-adrenoceptor (-AR) density, and chronotropic response to isoproterenol. This study tested the hypothesis that exercise training (ET), which attenuates -AR downregulation, would increase HRmax and of acclimatization and result in higher O2 max. After 3 wk of ET, rats lived at an inspired PO2 of 70 Torr for 10 days (acclimatized trained rats) or remained in normoxia, while both groups continued to train in normoxia. Controls were sedentary acclimatized and nonacclimatized rats. All rats exercised maximally in normoxia and hypoxia (inspired PO2 of 70 Torr). Myocardial -AR density and the chronotropic response to isoproterenol were reduced, and myocardial cholinergic receptor density was increased after acclimatization; all of these receptor changes were reversed by ET. Normoxic O2 max (in ml·min-1·kg-1) was 95.8 ± 1.0 in acclimatized trained (n = 6), 87.7 ± 1.7 in nonacclimatized trained (P < 0.05, n = 6), 74.2 ± 1.4 in acclimatized sedentary (n = 6, P < 0.05), and 72.5 ± 1.2 in nonacclimatized sedentary (n = 8; P > 0.05 acclimatized sedentary vs. nonacclimatized sedentary). A similar distribution of O2 max values occurred in hypoxic exercise. was highest in trained acclimatized and nonacclimatized, intermediate in nonacclimatized sedentary, and lowest in acclimatized sedentary groups. ET preserved in acclimatized rats thanks to maintenance of HRmax as well as of maximal stroke volume. preservation, coupled with a higher arterial O2 content, resulted in the acclimatized trained rats having the highest convective O2 transport and O2 max. These results show that ET attenuates -AR downregulation and preserves and O2 max after acclimatization, and support the idea that -AR downregulation partially contributes to the limitation of O2 max after acclimatization in rats.
maximal oxygen consumption; heart rate; stroke volume; tissue oxygen extraction myocardial -adrenoceptors; myocardial cholinergic receptors
O2 max) is not altered substantially after acclimatization (2, 3, 5). Reductions in maximal cardiac output (max) and maximal heart rate (HRmax) are characteristically seen after acclimatization. These features are observed in humans (2, 3, 27, 29) as well as in rats (6, 10-12) acclimatized to hypoxia. The observation that increasing HRmax by atrial pacing increased max and was accompanied by proportionate increases in maximum convective O2 transport (
O2 max) and O2 max of acclimatized rats (10) supports a limiting role of HRmax on O2 max after acclimatization in this species. The low HRmax is accompanied by a decreased chronotropic response to -adrenergic agonists in humans acclimatized to altitude (1, 30) and a reduction in the density of myocardial 
- and -adrenoceptors (AR) of rats exposed to prolonged hypoxia (8, 17, 29, 35). It has been hypothesized that the increased sympathetic activity that characterizes prolonged hypoxia leads to downregulation of myocardial -AR, decreased chronotropic response to -adrenergic agonists, and lower exercise-induced HRmax (1, 30). However, results of recent studies in humans suggest that increased cholinergic drive, rather than reduced response to adrenergic stimulation, is largely responsible for the reduced HRmax of acclimatization (3).
We recently observed that the downregulation of myocardial - and -AR and the upregulation of muscarinic cholinergic (M-Ach) receptors of rats acclimatized to moderate hypoxia [inspired PO2 (PIO2) 110 Torr] were attenuated by hypoxic exercise training (7). However, the mild level of hypoxia resulted in relatively minor changes in the O2 transport system, which made it difficult to assess the role of acclimatization and training on the autonomic control of cardiac function. Most of the studies on the effects of hypoxia on autonomic control of cardiovascular function in humans (1-3, 5, 27, 30) and in rats (6, 10-12) have been performed at low PIO2 levels (
70 Torr or less) where the changes in the O2 transport system are easily detectable. We reasoned that exercise training could provide a tool to influence the autonomic control of cardiac function during acclimatization to PIO2 of <110 Torr. Specifically, this study tested the hypothesis that exercise training would attenuate the downregulation of -AR observed during more severe hypoxia (PIO2 of 70 Torr) and that this would moderate the reduction in HRmax that follows acclimatization. We further hypothesized that preservation of HRmax after acclimatization would result in higher values of max, O2 max and O2 max than those observed in nontrained animals acclimatized to the same PIO2.
To test this hypothesis, the effects of exercise training on density of myocardial autonomic receptors and on systemic O2 transport during maximal exercise were studied in rats acclimatized to a PIO2 of 70 Torr and in normoxic, nonacclimatized rats.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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380 Torr, which resulted in a PIO2 of 70 Torr. The chamber was opened 5 times/wk for 90 min, during which both sedentary and trained rats were removed from the chamber and exposed to the normoxic environment. The acclimatized and nonacclimatized rats continued to train, under normoxic conditions, at the same work rate as before. The acclimatized sedentary rats were exposed to normoxia for the same time as the acclimatized trained rats but did not exercise. Maximal exercise test. At the end of the training protocol, animals were anesthetized with pentobarbital sodium (30 mg/kg ip). A polyethylene catheter (PE50) was placed in the aortic arch via the left carotid artery, and a PE10 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 verified at autopsy. The catheters were tunneled subcutaneously, exteriorized at the back of the neck, and flame-sealed. The animals were returned to the chamber after recovery from anesthesia.
On the following day, 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 (O2) and CO2 production (CO2) by using the open-circuit method (8). The catheters were connected, through sampling ports located on the top of the box enclosing the treadmill, to pressure transducers. After animals exercised for 30 min on the treadmill, arterial and mixed venous blood samples were obtained via stopcocks, the blood was replaced with homologous fresh blood from the same group, and the treadmill was set at a speed of 10 m/min. This speed was maintained for 2-3 min, after which the treadmill was set at an angle of 10° and the speed increased by 4 m/min every 90-120 s until O2 max was reached. O2 max was defined as the O2 after which an increase in work rate was not associated with a further increase (±5%) in O2. If a plateau in O2 was not observed at the highest two workloads, the animal was discarded. Approximately 80% of all rats achieved O2 max as defined here. Arterial and mixed venous blood samples were obtained during the last 45-60 s of exercise, while O2 and CO2 showed steady values. The box enclosing the treadmill was opened, and the rectal temperature was determined within 30 s of termination of exercise.
Gas exchange and O2 transport determinations. The box enclosing the treadmill was airtight except for the inflow 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 ATPS/min by using 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 using 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. O2 and CO2 were calculated from the inflowing and outflowing O2 concentration difference, the outflowing CO2 concentration, and the outflowing gas flow and were expressed in mililiters STPD per minute per kilogram. Five-second averages of O2 and CO2 values were used to provide data on O2, CO2, and the respiratory exchange ratio throughout the exercise bout. When a O2 plateau was reached, blood sample withdrawal was initiated. Maximal exercise O2 and CO2 values were calculated by using the time-averaged O2 and CO2 data obtained during the 45-60 s of blood sample withdrawal.
Arterial and mixed venous blood samples were analyzed for pH, PO2, and PCO2 by using appropriate electrodes at 38°C, and for Hb concentration and O2 saturation of Hb, and corrected for the rectal temperature by using temperature correction factors for rat blood (13).
Systemic and pulmonary arterial pressures were recorded continuously, with mean pressures obtained by electronic integration. Heart rate (HR) was obtained directly from the systemic arterial pressure tracing.
Arterial O2 content (CaO2) and of mixed venous blood O2 content were calculated from Hb concentration, PO2, and oxyhemoglobin saturation by using an Hb-O2 binding factor of 1.34 ml STPD/g, and an O2 solubility coefficient of 0.003 ml·Torr-1·dl-1. Cardiac output (; in ml·min-1·kg-1) was calculated as the ratio of O2 to arteriovenous O2 content. The rate of convective blood O2 transport (O2) was calculated as the product of x CaO2. The O2 extraction ratio was calculated as arteriovenous O2 content/CaO2. "Ideal" alveolar PO2 (PAO2) was calculated from the alveolar gas equation, assuming PACO2 and arterial PCO2 to be equal. Alveolar ventilation was calculated from the values of CO2 and arterial PCO2. Effective lung diffusing capacity for O2 was calculated from O2 max, arterial PO2, mixed venous PO2, and PAO2 values by using an integration procedure and assuming that all of the difference between PAO2 and arterial PO2 is due to diffusion limitation (13). Lung diffusing capacity for O2 was not calculated in normoxic exercise because of the uncertainty introduced by the nonlinear shape of the oxyhemoglobin dissociation curve at high PO2 values. Mean tissue capillary PO2 was calculated from arterial and mixed venous PO2 values by using a numerical integration procedure. (11, 16). Tissue O2 diffusing capacity (DTO2) was calculated as the ratio of O2 max to tissue capillary PO2.
Isoproterenol dose-response curve. Approximately 3 h after the maximal exercise test was finished, the HR response to increasing doses of isoproterenol, a -AR agonist, was determined in all subgroups under normoxic conditions. After baseline HR measurement, isoproterenol was injected into the pulmonary artery catheter in doses of 0.01, 0.1, 1, and 10 µg/kg, in 0.1 ml of saline.
The next day, animals were euthanized by an overdose of barbiturate (60 mg/kg iv pentobarbital sodium), and the heart and the right gastrocnemius muscle were removed, frozen in liquid nitrogen, and stored at -70°C for determination of myocardial autonomic receptor density and for measurement of skeletal muscle citrate synthase activity.
Myocardial ventricular autonomic receptor measurement. Density of - and -AR and of M-Ach was determined by the radioisotope ligand binding methods by using a procedure described in detail before (7). Myocardial cell membranes were isolated as described (17). Protein content was measured with a dye-binding assay using a commercial kit (BioRad) with bovine serum albumin as standard.
1-AR binding assay. [3H]prazosin, an 1-AR antagonist (Amersham Pharmacia Biotech; specific activity, 81 Ci/mmol) was used to label the receptors. Eight different concentrations of [3H]prazosin, ranging from 0.02 to 1.5 nM, were used in each assay. Unlabeled prazosin (1 µM) was added to determine nonspecific binding. Protein concentration of each sample was adjusted to 40-80 µg/100 µl on the day of the assay. Nonspecific binding averaged 11% of total binding.
-AR binding assay. The procedure used was the same as that described for the 1-AR binding assay, except for the following modification: [3H]CGP 12177 4-[3-[(t-butyl) amino]-2-hydroxypropoxy] benzimidazole-2-one) (Amersham Pharmacia Biotech; specific activity: 51 Ci/mmol), a -AR antagonist, was used to label the receptors. Eight different concentrations of [3H]CGP 12177, ranging from 0.06 to 4 nM, were used. Unlabeled propranolol (10 µM) was added to determine nonspecific binding. The protein concentration was adjusted to 30-60 µg/100 µl on the day of the assay. Nonspecific binding averaged 9% of total binding.
M-Ach-receptor binding assay. The procedure used was the same as the ones described above, except for the following: [3H]QNB (quinuclidinyl benzilate) (Amersham Pharmacia Biotech; specific activity: 55 Ci/mmol), an M-Ach antagonist, was used to label the receptors. Eight different concentrations of [3H]QNB, ranging from 0.01 to 0.8 nM, were used in each assay. Unlabeled atropine (10 µM) was added to determine nonspecific binding. The protein concentration was adjusted to 25-60 µg/100 µl on the day of the assay. Nonspecific binding averaged 7% of total binding.
Radio ligand binding data were analyzed with Ligand, a weighed, nonlinear, least-square curve-fitting computer program (24). For saturation experiments, equilibrium dissociation constants (receptor apparent affinity) and maximum numbers of binding sites were determined by nonlinear regression fitting.
Statistical analysis. Data are presented as means ± SE. One-way ANOVA, followed by a Bonferroni posttest for multiple comparisons, was used to determine the statistical significance between mean values. The effect of acclimatization was evaluated by comparing nonacclimatized sedentary vs. acclimatized sedentary, and nonacclimatized trained vs. acclimatized trained groups. The effect of training was assessed by comparing nonacclimatized sedentary vs. nonacclimatized trained, and acclimatized sedentary vs. acclimatized trained groups. Because the animals exercised in normoxia as well as in hypoxia, the effects of acclimatization and of exercise training were established by making comparisons between groups exercising at the same PIO2. A P value of <0.05 was considered to indicate a significant difference.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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O2 max as defined above; this represents 80% of the total number of rats. The data on myocardial receptor density were obtained in five rats of each group.
Effects of acclimatization and training on O2 max and convective blood O2 transport. As expected, acclimatization to hypoxia did not result in a significant change in O2 max in the sedentary rats, either during normoxic or hypoxic exercise (Fig. 1; sedentary, nonacclimatized vs. acclimatized). Exercise training, on the other hand, produced the expected increase in O2 max; this occurred in the acclimatized (Fig. 1; acclimatized rats, sedentary vs. trained) as well as in the nonacclimatized rats (Fig. 1; nonacclimatized rats, sedentary vs. trained). The increase in O2 max produced by exercise training, however, was larger in the acclimatized rats. In both hypoxic as well as normoxic exercise, O2 max of acclimatized rats was 30% higher in trained than in sedentary rats, whereas in nonacclimatized rats this difference was 20%. As a result, both in normoxic and hypoxic exercise, O2 max was highest in the acclimatized trained, intermediate in the nonacclimatized trained, and lowest in the acclimatized and nonacclimatized sedentary groups, with no significant difference occurring between the last two groups (sedentary, acclimatized vs. nonacclimatized; normoxic exercise, P = 0.63; hypoxic exercise P = 0.58).
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O2 max increased after acclimatization in both sedentary as well as in trained rats (Fig. 2A), but the increase was larger in the trained rats in which O2 max was 20% higher in the acclimatized rats (Fig. 2A; trained rats, acclimatized vs. non acclimatized), whereas in the sedentary rats, O2 max was 10% higher in the acclimatized group (Fig. 2A; sedentary acclimatized vs. nonacclimatized). Interestingly, exercise training did not increase O2 max in the nonacclimatized rats (Fig. 2A; nonacclimatized, trained vs. sedentary), whereas in the acclimatized rats O2 max was 20% higher in the trained than in the sedentary rats (Fig. 2A; acclimatized, trained vs. sedentary). The effects of acclimatization and exercise training on the two main determinants of O2 max, max and CaO2, are shown in Fig. 2, B and C, respectively. The acclimatized sedentary rats exhibited the characteristic reduction in max (Fig. 2B; sedentary, acclimatized vs. nonacclimatized); despite this decrease, O2 max increased by 10% above the nonacclimatized values (Fig. 2A) owing to the large increase in CaO2 (Fig. 2C). In the trained rats, on the other hand, max remained at essentially identical levels in both acclimatized and nonacclimatized rats during hypoxic exercise and was slightly but not significantly lower in the acclimatized rats during normoxic exercise (Fig. 2B; trained, acclimatized vs. nonacclimatized; P = 0.37). More to the point, max of acclimatized rats reached significantly higher levels in the trained than in the sedentary groups both in hypoxic as well as in normoxic exercise (Fig. 2B; acclimatized, trained vs. sedentary). In other words, exercise training prevented the decrease in max characteristic of acclimatization to severe hypoxia. The preservation of max, combined with the elevated CaO2, led to the significantly higher O2 max of the acclimatized vs. nonacclimatized trained rats (Fig. 2A).
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The higher CaO2 of the acclimatized over the nonacclimatized groups was solely the result of the higher Hb concentration in the former (Table 1). Neither alveolar ventilation nor efficacy of pulmonary gas exchange contributed to maintain a higher PaO2 value in the acclimatized rats (Table 1), which could contribute to the higher CaO2 of acclimatized vs. nonacclimatized rats.
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The lack of effect of exercise training on O2 max of nonacclimatized rats was the result of the lower CaO2 values observed in the trained rats (Fig. 2C; nonacclimatized, trained vs. sedentary). In fact, CaO2 was significantly lower in both the acclimatized and nonacclimatized trained rats relative to their sedentary counterparts (Fig. 2C). This decrease occurred despite PaO2 values that were either higher (normoxic exercise) or not different (hypoxic exercise) in trained vs. sedentary rats (Table 2). Efficacy of pulmonary gas exchange was higher in the trained rats; this was the case in both acclimatized and nonacclimatized rats: A-aPo2 differences were lower in hypoxic and normoxic exercise, and lung diffusing capacity for O2 values in hypoxic exercise were higher in the trained than in the sedentary rats (Table 1). However, despite the improved efficacy of pulmonary gas exchange, oxyhemoglobin saturation was significantly lower in the trained rats (Table 1). This can be attributed to the more severe acidosis developed by the trained rats during maximal exercise (Table 1). It is probable that the more severe acidosis of the trained rats, in turn, was the result of the higher exercise intensity attained by these animals.
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Figure 3 shows the effects of exercise training and of acclimatization on the determinants of max, i.e., HRmax (top) and maximal stroke volume (SVmax; bottom). Characteristically, HRmax of sedentary rats was significantly lower in the acclimatized animals. Exercise training moderated the decrease in HRmax of acclimatization: HRmax of acclimatized trained rats was significantly higher than that of their sedentary counterparts (acclimatized, sedentary vs. trained) and not different from that of nonacclimatized, trained rats (trained, acclimatized vs. nonacclimatized; normoxic exercise, P = 0.33; hypoxic exercise, P = 0.41) (Fig. 3). HRmax elicited by isoproterenol administration in normoxic conditions was significantly lower in the acclimatized, sedentary rats than in their nonacclimatized counterparts (Fig. 3). Exercise training eliminated this difference; the chronotropic response to isoproterenol after training was essentially the same in acclimatized and nonacclimatized rats (trained, acclimatized vs. nonacclimatized; P = 0.57) (Fig. 3). No significant differences between isoproterenol-induced HRmax and exercise HRmax were observed in any group.
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The lower HRmax of acclimatized rats was accompanied by a significantly lower SVmax (Fig. 3). SVmax and Hrmax contributed in approximately similar proportions to reduce max in the acclimatized vs. nonacclimatized sedentary rats. SVmax increased significantly with exercise training and reached essentially identical values in both acclimatized and nonacclimatized, trained rats (trained, acclimatized vs. nonacclimatized; normoxic exercise, P = 0.58; hypoxic exercise, P = 0.69) (Fig. 3). Because in the sedentary animals SVmax was significantly lower in the acclimatized group (Fig. 3), the exercise training-induced increase in SVmax was relatively larger in the acclimatized than in the nonacclimatized groups.
Effects of acclimatization and training on tissue O2 extraction. Figure 4 is a plot of O2 max as a function of O2 max for all the groups studied. The slope 
O2 max /O2 max represents the average O2 extraction ratio (ER) for each group over the exercise PO2 range investigated. Exercise training significantly increased O2 ER (see also Table 2); however, O2 ER was significantly lower in the acclimatized rats; this was true in both sedentary and trained rats (Fig. 4 and Table 2; acclimatized vs. nonacclimatized).
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Although exercise training resulted in a significant increase in DTO2 (Table 2), there were no differences in DTO2 between acclimatized and nonacclimatized groups, either before or after exercise training, suggesting that acclimatization does not substantially influence tissue O2 diffusive conductance.
Exercise training significantly increased gastrocnemius muscle citrate synthase activity in both nonacclimatized and acclimatized rats (Table 2; sedentary vs. trained). In both sedentary and trained rats, however, muscle citrate synthase activity was significantly lower in the acclimatized rats (Table 2; acclimatized vs. nonacclimatized).
Effect of acclimatization and training on myocardial density of autonomic receptors. As expected, acclimatization to hypoxia induced a decrease in left ventricular -AR density in sedentary rats (Fig. 5; sedentary acclimatized vs. nonacclimatized). Exercise training moderated this reduction: -AR density was lowest in acclimatized sedentary, intermediate in acclimatized trained, and highest in nonacclimatized sedentary and trained with no significant difference observed between these last two groups. Similar effects of acclimatization and training were observed on the density of -AR (Fig. 5). In contrast with the response of -AR, however, exercise training resulted in a small but significant increase in -AR density in the nonacclimatized rats (Fig. 5; nonacclimatized, trained vs. sedentary)
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Left ventricular M-Ach receptor density of the sedentary animals was significantly increased after acclimatization (Fig. 5; sedentary, acclimatized vs. nonacclimatized). This elevation was attenuated by exercise training (Fig. 5; acclimatized, sedentary vs. trained); exercise training, on the other hand, significantly increased M-Ach receptor density of nonacclimatized rats (Fig. 5; nonacclimatized, trained vs. sedentary).
The pattern of right ventricular adrenergic and cholinergic receptor density in response to exercise training and acclimatization was the same as that seen in the left ventricle. No significant effects of hypoxia or training were observed in the affinity of adrenergic or cholinergic receptors to their respective ligands.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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O2 max and was accompanied by reductions in max, HRmax, and SVmax, downregulation of -AR, and decreased chronotropic response to -AR stimulation. Exercise training prevented or attenuated all these changes in the acclimatized rats and resulted in a larger increase in O2 max than that observed after exercise training in nonacclimatized rats.
Experimental design. The design of this study allowed us to assess the effects of acclimatization and exercise training by including groups that underwent acclimatization and training, training without acclimatization, acclimatization alone, and neither training nor acclimatization. Exercise training was initiated 3 wk before acclimatization because preliminary data indicated that this time was sufficient to elicit significant increases in O2 extraction and maximal exercise capacity. Because these studies were directed to test the hypothesis that exercise training prevents or attenuates the decrease in myocardial -AR density that follows acclimatization, exercise training was initiated before the animals were acclimatized to hypoxia. The training protocol was effective in increasing O2 max (Fig. 1), tissue O2 extraction (Fig. 4 and Table 1), and muscle oxidative capacity markers (Table 2).
To avoid detraining effects, the rats of the acclimatized group continued to train during acclimatization. Because exercise capacity is reduced in hypoxia, the animals exercised in normoxia to maintain the same absolute exercise training intensity in both the acclimatized and the nonacclimatized groups. Naturally, normoxic exercise training entailed interruption of hypoxia in the acclimatized rats for 90 min, 5 days/wk. To account for this factor, the sedentary rats of the acclimatized group were also exposed to normoxia for the same time. The assumption was made that the effect of exercise training before dividing the trained group into acclimatized and nonacclimatized subgroups was the same in all rats and that there were no systematic differences in the response to exercise training between the rats that would be eventually assigned to the acclimatized group and those assigned to the nonacclimatized group. Although this assumption was not tested experimentally by measuring O2 max in all animals before the trained group was divided, no consistent differences in performance were observed during the training period among the rats, and subgroup selection was made randomly. The same proportion of animals reached O2 max in the acclimatized and nonacclimatized trained subgroups. Because the acclimatized trained rats lived in hypoxia but trained in normoxia, the protocol approximates the "live high, train low" experimental design introduced by Levine and Stray-Gundersen (21).
Acclimatization was limited to 10 days; previous observations in this animal model showed that oxygen transport variables during maximal exercise, and density of myocardial adrenergic and cholinergic receptors have reached a steady state by this time (8).
Each animal exercised maximally in hypoxia as well as in normoxia. Maximal exercise was chosen because measurement of O2 under these conditions provides an accurate estimate of the capacity of the O2 transport system. Accordingly, it is possible to determine the effect of experimental variables on the individual conductances that compose the O2 transport system. Both normoxic and hypoxic exercise were utilized because this allows a better comparison of the O2 transport system among groups that have been exposed to different PIO2 values. In addition, exercise at different PIO2 values allows determination of the O2 dependence of O2 max.
Effects of acclimatization and exercise training on O2 max. O2 max can be expressed as the product of TO2 max times O2 ER, thus conceptually separating O2 max into two main components: the series of processes involved in transporting O2 from the atmosphere to the tissue capillaries (TO2 max) and those involved in the extraction of O2 from the capillaries by the tissues (O2 ER) (36)
This study was directed to test the hypothesis that the downregulation of myocardial -AR contributes to the reduction in HRmax and max of acclimatization and that the reduction of max, in turn, is one of the factors limiting O2 max after acclimatization. If this hypothesis is correct, it would be expected that interventions that prevent or attenuate myocardial -AR downregulation would 1) prevent or attenuate the decrease in HRmax and max of acclimatization and 2) result in TO2 max and O2 max values higher than those observed in acclimatized animals in which -AR downregulation was not attenuated. The effects of exercise training on HRmax, max and O2 max of acclimatized rats are consistent with the hypothesis proposed. In addition, SVmax also contributed significantly to the preservation of max in the trained acclimatized rats. Maintenance of max through preservation of both HRmax and SVmax in the presence of the increased blood O2-carrying capacity of acclimatization significantly increased TO2 max and resulted in the trained, acclimatized rats achieving the highest O2 max of all groups studied.
Effects of acclimatization and training on autonomic receptor density and HRmax. Acclimatization resulted in the expected decrease in myocardial -AR density (8, 17, 35). This was accompanied by a decrease in the density of -AR and is consistent with the notion that prolonged receptor stimulation due to the high sympathetic drive of hypoxia leads to downregulation of both - and -AR (1, 17, 29). The reduction in the maximal chronotropic response to isoproterenol (Fig. 3) represents the functional correlate of -AR downregulation and illustrates the limited response to -adrenergic agonists, a feature also observed in humans acclimatized to altitude (30). In this study, exercise training attenuated the -AR downregulation of acclimatization and resulted in an increase in the maximal chronotropic response to isoproterenol (Fig. 3) and an increase in exercise HRmax above that of sedentary, acclimatized rats (Fig. 3). The mechanism by which exercise training attenuates the decrease in myocardial density of autonomic receptors that accompany acclimatization cannot be ascertained by this study. Data in rats (19) and humans (4) suggest that chronic exercise training may reduce sympathetic tone. If this is the case, it is possible that the increase in sympathetic activity that occurs in hypoxia may be lessened by previous exercise training and therefore contribute to attenuate the downregulation of -AR.
M-Ach receptor density was also significantly modified with acclimatization and exercise training, and exercise HRmax showed as high a correlation with MAch as with -AR density (Fig. 6). Accordingly, the myocardial receptor data presented here give equal support to -AR downregulation and to M-Ach receptor upregulation as possible determinants of the changes in HRmax with acclimatization and exercise training. A previous study from our laboratory (6) showed that M-Ach receptor blockade with atropine produced a larger decrease in resting HR in acclimatized than in nonacclimatized rats but had no effect in HRmax in either group. This suggests that, in the rat, cholinergic tone during resting conditions is elevated after acclimatization; however, the reduction in cholinergic drive that accompanies maximal exercise masks this difference. On the other hand, the reduction of HRmax that follows -AR blockade with atenolol was smaller in acclimatized rats (6), a finding consistent with reduced -AR density in this condition. These data suggest that the functional consequences of M-Ach receptor upregulation may have a larger role in controlling resting than exercise HRmax, whereas the consequences -AR downregulation may be principally noticed during maximal exercise.
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Recent studies in humans acclimatized to altitude, however, suggest a different pattern of HR control during maximal exercise (3); in this case, cholinergic blockade abolished the difference in exercise HRmax between acclimatized and nonacclimatized subjects, suggesting that the lower exercise HRmax of acclimatization is entirely due to increased cholinergic tone. These results are in marked contrast with the present observations in rats, since they indicate that cholinergic tone is elevated at rest as well as during maximal exercise in acclimatized humans. Several factors may be responsible for these differences, including species differences in the effects of acclimatization on the autonomic control of HR, differences in the receptor antagonists used, and in the duration of acclimatization and severity of hypoxia.
Effects of acclimatization and exercise training on SVmax. The changes in HRmax secondary to training and acclimatization resulted in parallel changes in max. However, SVmax also contributed significantly to the reduction of max of acclimatization, as well as to the preservation of max by exercise training in acclimatized rats. As seen before in this model (6, 10-12), SVmax decreased after acclimatization in sedentary rats and contributed to the reduction of max in a proportion similar to that of HRmax. SVmax is influenced by HR, preload and afterload levels, and myocardial contractility. Other things being equal, the low HRmax of acclimatization would actually tend to increase SVmax, so it is apparent that other factors participate. A decrease in end-diastolic volume in acclimatized humans (15, 34), perhaps as a result of the reduced plasma volume (31, 32), indicates a low preload. The observation that plasma volume expansion is accompanied by an increase in O2 max of acclimatized subjects (31) is consistent with a limiting role of the reduced preload in maximal O2 transport. Despite the reduced end-diastolic volume, left ventricular ejection fraction, the ratio of peak systolic pressure to enddiastolic volume, and the mean ejection rate are well preserved in acclimatized individuals (34), suggesting that myocardial contractility is normal or even elevated (28). Accordingly, a reduced myocardial contractile function does not appear responsible for the reduced SVmax. Right ventricular afterload is increased in acute hypoxia due to the hypoxic pulmonary vasoconstriction; in chronic hypoxia, vascular remodeling and increased blood viscosity contribute to the pulmonary hypertension (14). Studies in patients lacking a functional subpulmonary ventricle after the Fontan operation showed that normal right ventricular function is necessary to attain max during maximal exercise under hypoxic conditions (9) and suggest that elevated right ventricular afterload may limit SVmax under conditions of high pulmonary vascular resistance. Increased blood viscosity may contribute to reduce SVmax via elevated cardiac afterload, as suggested by the observation that lowering hematocrit at constant blood volume substantially increased SVmax of acclimatized rats (12). The chronically increased after-load may also contribute indirectly to SVmax limitation. Hypertension-induced myocardial hypertrophy leads to altered ventricular diastolic function (33), which could impair ventricular filling. In humans with pulmonary hypertension, the septum tends to be displaced toward the left ventricle, thus compromising left ventricular function (18). Thus the reduction of SVmax after acclimatization may be due to a combination of factors, which include decreased preload and the direct and indirect consequences of the elevated ventricular afterload that results from the combined effects of increased pulmonary vascular resistance and elevated blood viscosity.
Exercise training, on the other hand, increased SVmax of the acclimatized rats to levels that were essentially identical to those of nonacclimatized rats. Exercise training did not modify either right ventricular afterload or the polycythemia of acclimatization (Table 2); accordingly, other factors must have contributed to the increase in SVmax. Exercise training is accompanied by an increase in plasma volume, which could offset the effects of acclimatization and tend to restore normal ventricular preload levels (32). Exercise-trained rats show an increase in ventricular compliance and improved diastolic relaxation (22, 39, 40). Increased ventricular diastolic compliance is also observed in endurance athletes (20); this may facilitate ventricular filling and contribute to increase stroke volume. Exercise training is also associated with increased contractile function of isolated rat cardiomyocytes (23, 38). Thus a combination of exercise-induced increased preload, coupled with improved diastolic compliance and enhanced contractility, could participate in the increased SVmax of acclimatized trained rats. The possible role of these factors in the changes in SVmax observed in this study cannot be ascertained and should be the subject of further study.
The combined data of this and previous studies from our laboratory (6) show that HRmax is one of the factors that contribute to the reduction in max of acclimatization in the rat and supports the notion that HRmax reduction is linked to -AR downregulation rather than to the upregulation of M-Ach receptors. The beneficial effect of exercise training on O2 max is due, in part, to preservation of HRmax after acclimatization. However, this study also shows that a reduction in SVmax is another important mediator of the low max of acclimatized rats and that the increase in SVmax brought about by exercise training plays a critical role in the preservation of O2 max in acclimatized, trained rats.
Effects of acclimatization and exercise training on tissue O2 extraction. The increase in O2 max induced by exercise training was partially offset by the reduction in O2 ER that accompanied acclimatization. The reduction in O2 ER was relatively smaller (7-9%) than that of max (12-14%), but, in contrast to the reduction in max, the effect of acclimatization on O2 ER persisted after exercise training. This suggests that acclimatization reduces O2 ER by mechanisms that are different from those by which exercise training increases O2 ER.
O2 ER is determined by the ratio of diffusive-toperfusive tissue O2 conductances, represented by the ratio -DTO2/, where is the slope of the blood O2 absorption curve (25). DTO2 was higher in the trained than in the sedentary rats; this is consistent with the known effects of exercise training on the determinants of DTO2 (26). On the other hand, the lack of difference in DTO2 between acclimatized and nonacclimatized rats suggests that the lower O2 ER is not due to a lower DTO2 in the acclimatized rats.
Tissue perfusive O2 conductance, the product , tends to increase as a result of the increase in , owing to the high Hb concentration of acclimatization. of the sedentary rats, calculated from the ratio of arteriovenous O2 content to arteriovenous PO2, was 35% higher in the acclimatized than in the nonacclimatized rats. max, on the other hand, was only 13% lower in the acclimatized rats. Accordingly, the product max was nearly 20% higher in the acclimatized sedentary rats. The difference was even larger in the trained rats, in which max values were essentially the same in both groups.
The lower O2 ER contributed, albeit in a smaller proportion than the reduction in max, to the limitation of O2 max in the acclimatized sedentary animals, thus offsetting the effect of the increase in O2 max observed in the acclimatized trained animals. It is interesting to point out that, although the combination of exercise training and acclimatization tends to increase O2 max by increasing both determinants of blood O2 convection, i.e., max and CaO2, the increase in these variables tends to reduce tissue O2 extraction by increasing O2 perfusive conductance. This phenomenon illustrates the self-limiting effects of increased blood flow and blood O2 content on tissue O2 extraction (37).
A decrease in muscle oxidative capacity may have also contributed to the decrease in tissue O2 extraction, as suggested by the lower values of gastrocnemius muscle citrate synthase activity in the acclimatized rats.
In summary, the results of the present studies confirm previous data form our laboratory regarding the effects of acclimatization to hypoxia on systemic O2 transport in maximal exercise. The data also suggest that the effect of acclimatization on autonomic control of cardiac function may differ in rats and in humans. Exercise training prevents the decreases in HRmax, SVmax, and max of acclimatization and results in a higher O2 max than in trained, nonacclimatized rats, despite a lower tissue O2 extraction in acclimatized rats. The attenuation by exercise training of the acclimatization-induced decreases in myocardial -AR density and on the chronotropic response to isoproterenol suggests that the low HRmax is due in part to reduced myocardial -AR density. These data, together with previous observations, support the notion that O2 max of acclimatization in the rat is limited in part by a reduction in max.
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Present address of K. K. Henderson: Department of Veterinary Biomedical Sciences, University of Missouri-Columbia, 1600 E. Rollins, Columbia, MO 65222-5120.
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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.
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O2 max at sea level and altitude. Respir Physiol 106: 329-343, 1996.
