INTRODUCTION

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CHRONIC ENVIRONMENTAL HYPOXIA is characterized by elevated sympathetic activity and decreased myocardial response to -adrenergic agonists (1, 22). A major mechanism responsible for the reduced -adrenergic response is a decrease in the density of myocardial -adrenergic receptors (10, 24), which is thought to develop as a consequence of the prolonged increase in agonist stimulation. An increase in the density of muscarinic M₂ myocardial receptors (11, 25) and downregulation of A₁ adenosinergic receptors (11) may also participate either directly or by modulating the myocardial response to adrenergic stimulation.

A net result of these changes in myocardial autonomic control is a reduction in the maximal heart rate (HR_max) achieved during exercise after acclimatization to chronic hypoxia (4, 9, 18). The reduced HR_max helps explain, in part, the observation that despite a marked increase in blood O₂-carrying capacity, maximal exercise performance, as evidenced by the maximal rate of O₂ consumption (O_2 max), changes little after acclimatization (2, 4). Supporting a limiting role of the reduced HR_max on O_2 max after acclimatization is the observation that increasing HR_max by cardiac pacing results in an increase in maximal cardiac output (_max) and in O_2 max (7).

Although the changes in myocardial -adrenergic and muscarinic receptor function after acclimatization to hypoxia have been well documented, there is little information on the time course of these changes and on the correlation among receptor function, cardiovascular function, and systemic O₂ transport during the course of acclimatization to hypoxia. A study of the correlation among these variables as acclimatization to hypoxia proceeds may provide valuable insight into the role played by changes in the function of myocardial receptors on maximal O₂ transport and exercise capacities.

In contrast to -adrenergic and muscarinic receptors, relatively little is known concerning the effect of hypoxia and of acclimatization to hypoxia on myocardial -adrenergic receptor function. -Adrenergic receptors have been implicated in the development of left ventricular hypertrophy secondary to aortic coarctation (14, 23). Chronic hypoxia is an interesting model because it results in right ventricular hypertrophy (19) with little or no change in left ventricle mass. Comparison of the behavior of right and left ventricular -adrenergic receptors during acclimatization could provide useful information on the possible participation of these receptors in the development of myocardial hypertrophy. Characterization of the time course is important in this respect because early changes in receptor characteristics may signal the onset of responses that are expressed later in the course of hypertrophy development.

The present studies were designed to determine the time course of myocardial ₁- and -adrenergic and cholinergic receptor modification simultaneously with the changes in systemic O₂ transport in maximal exercise during the course of a 21-day period of acclimatization to environmental hypoxia. The rationale for a parallel characterization of myocardial receptors and O₂ transport in maximal exercise was that this approach could provide further insights into the functional significance of the alterations in myocardial receptors during acclimatization to hypoxia. Maximal exercise was chosen because it provides an accurate measure of the capacity of the O₂-transport system to deliver and use O₂. We reasoned that the consequences of the changes in myocardial receptors were more likely to be evidenced during maximal exercise because of the large variations in autonomic nervous system output to the heart that take place in this condition.

	METHODS

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Production of Environmental Hypoxia

Exercise Studies

Surgical procedures. On the day before the exercise test, the animals were removed from the chamber and anesthetized with pentobarbital sodium (35 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 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 immediately after recovery from anesthesia.

Exercise protocol. The protocol has been described earlier (7). After measurement of the rectal temperature, the animals were placed on a treadmill enclosed in a Plexiglas chamber designed for the measurement of O₂ consumption and CO₂ production by the open circuit method. The indwelling catheters were connected, via coiled PE-50 extensions, to sampling ports placed at the top of the box. The animals were maintained at rest in the treadmill for ~30 min, after which arterial and mixed venous (pulmonary artery) blood samples (0.3 ml each) were obtained. The blood withdrawn was replaced with an equal volume of fresh blood obtained from donors, and the exercise test was begun. The treadmill was placed at an angle of 10°, and the speed was set at 10 m/min and maintained at this rate for 2-3 min. The speed was then increased by 4 m/min every 90 s until O_2 max was reached. O_2 max was defined as the O₂ value after which an increase in speed did not result in an increase (±5%) in O₂. Arterial and mixed venous blood samples were obtained during the last 45-60 s of exercise, while O_2 max showed a steady value. The rat was rapidly removed from the treadmill, and its rectal temperature was measured within 30 s of termination of exercise. The results of the animals that did not reach O_2 max as defined were not included in the analysis of the data.

Gas exchange measurements. Gas enters and leaves the chamber enclosing the treadmill through separate inflow and outflow tubes; otherwise, the treadmill is airtight. PI_O2 of the gas in the chamber was maintained at the desired value by mixing N₂ and O₂ with the use of a Cameron Instruments Precision Gas Mixer. Incoming airflow rate was maintained constant at ~20 l/min. Inflowing and outflowing O₂ concentrations and outflowing CO₂ concentration (the inflowing gas was CO₂ free) were monitored continuously with the use of an Applied Electrochemistry O₂ analyzer and a Colombus Instruments CO₂ analyzer. The signal from the gas analyzers was fed into a computer to calculate O₂ and CO₂ every 5 s with the use of standard gas exchange equations.

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Experimental protocol. Groups of five to seven littermates exercised in hypoxic conditions (PI_O2 ~70 Torr) after exposure to hypoxia for the following times: 30 min; 24 h; 3, 5, 10, and 21 days. The 30-min group was exposed to an FI_O2 of 0.10 at ambient PB, which resulted in an inspired PO₂ ~70 Torr. In addition to the hypoxic groups, two groups of littermates served as controls and were maintained and exercised in normoxic conditions; one group exercised at the beginning of the 21-day period, whereas the second group exercised at the end of this period.

Studies of Myocardial Autonomic Receptors

Myocardial cell membrane isolation. The procedure used was a slightly modified version of the method of Kacimi et al. (10). The ventricles were weighed and immediately homogenized in 6 ml of buffer (30 mM Tris · HCl, 100 mM NaCl, 5 mM MgCl₂, 1 mM EGTA, 1 mM trypsin inhibitor, 1 mg/ml leupeptin; pH 7.5) with a polytron tissue homogenizer. The suspension was centrifuged at 1,000 g for 10 min at 4°C. The supernatant was transferred to another tube and centrifuged at 50,000 g for 30 min at 4°C. The supernatant was discarded, and the pellet was resuspended with 6 ml of buffer and centrifuged at 50,000 g for 30 min at 4°C. Finally, the pellet was suspended with incubation buffer (50 mM Tris · HCl, 5 mM MgCl₂; pH 7.5) and stored at 80°C. Protein content was measured with a dye-binding assay with the use of a commercial kit [Bio-Rad, (3)] and bovine serum albumin as standard.

₁-Adrenoceptor-binding assay. [³H]prazosin, an ₁-adrenoceptor (AR) antagonist, 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.

-Adrenoceptor-binding assay. The procedure used was the same as that described for the -adrenoceptor-binding assay, except for the following modification: [³H]CGP-12177 [()-4-(3-t-butyl amino-2-hydroxy-propoxy) benzimidazole-2-1], 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 in each assay. 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. Duplicate samples of the membrane preparations were incubated for 1 h at 37°C.

M-Ach receptor-binding assay. The procedure used was the same as the ones described above, except for the following: [³H]QNB [R-()-3-quinuclidinyl benzilate], 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. Duplicate samples of the membrane preparations were incubated for 1 h at 25°C.

Effect of prior surgery and vascular catheterization on myocardial receptor characteristics. An additional group of eight rats underwent surgery and catheter implantation as described in Exercise Studies. After recovery from anesthesia, four rats were placed in hypoxia as described above and the other four remained in normoxia. Twenty-four hours later, the animals were killed by cervical dislocation, and the hearts were removed and prepared for myocardial receptor characterization as described above.

Data Analysis

Statistical Analysis

	RESULTS

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Exercise Studies

As expected, alveolar, arterial, and mixed venous PO₂ values during exercise at 30 min of hypoxia were substantially lower than those observed during normoxic exercise (Table 1). As time of exposure to hypoxia increased, however, there was a tendency for an increase in alveolar PO₂, which was accompanied by an increase in arterial PO₂, and a decrease in the alveolar-arterial PO₂ difference (Table 1). These changes reflect the ventilatory acclimatization and the increased efficacy of pulmonary gas exchange that develop during prolonged hypoxia in this animal model and that tend to minimize the arterial hypoxemia (8). Blood [Hb] increased with time of exposure to hypoxia (Table 1). Although the increase in blood [Hb] late in the hypoxic exposure reflects the increased erythropoiesis, it is likely that the elevation observed at days 1 and 3 was largely the result of the plasma volume contraction known to occur in the first days of hypoxia (20). These changes in [Hb] prevented a larger decrease in arterial blood O₂ content in the early stages of hypoxia (Table 1).

O_2 max at 30 min of hypoxia decreased to ~60% of the normoxic value and remained at that level for the rest of the hypoxic period (Fig. 1A). Initially, the fall in O_2 max was entirely the result of a decrease in the arteriovenous O₂ difference, with _max remaining unchanged (Fig. 1B). The large decrease in (CaCv)O₂ at this time was largely the result of the reduction in Ca_O2 (Table 1). As hypoxia continued, _max showed a gradual decrease with (CaCv)O₂ following an opposite pattern. The gradual recovery in (CaCv)O₂, in turn, paralleled the increase in Ca_O2 (Table 1 and Fig. 1B). Figure 1C shows that the decrease in _max, in turn, was the combined result of decreases in both HR_max and stroke volume, although their relative contribution varied with time. On the first day of hypoxia, the decrease in _max was entirely due to a decrease in maximal stroke volume (SV_max). As hypoxia continued, HR_max also decreased and increased its contribution to the reduction in _max. At 21 days of hypoxia, HR_max and SV_max had decreased by 13 and 18%, respectively, compared with the normoxic control values.

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Time course of O2-transport variables at maximal exercise during acclimatization. A: maximal rate of O2 transport to the tissues (o2max; ml · min1 · kg1) was calculated as maximal cardiac output (max) × CaO2. Maximal rate of O2 consumption (O2 max) is in ml · min1 · kg1. B: max is in ml · min1 · kg1. Arteriovenous blood O2 content difference [C(av)O2] is in ml/dl. C: maximal stroke volume (SVmax) is in ml/kg. Maximal heart rate (HRmax) is in beats/min. D: mean systemic arterial blood pressure (MABP) is in mmHg. Systemic vascular resistance (SVR) is in mmHg/(ml · min1 · kg1). E: mean pulmonary arterial pressure (PaP) is in mmHg. F: left, right, and total ventricular work (LVW, RVW, and TVW), respectively, are in J · min1 · kg1. *P < 0.05 or better, hypoxia (HX) vs. normoxia (NX).

After an initial decrease at 30 min of hypoxia, mean systemic arterial blood pressure (MABP) and systemic vascular resistance (SVR) values during maximal exercise increased toward the normoxic values (Fig. 1D). At 21 days of hypoxia, MABP was not significantly different from the normoxic control, whereas SVR was significantly higher than in normoxia (Fig. 1D).

Pulmonary arterial pressure in maximal exercise increased markedly and appeared to reach a plateau by 21 days of hypoxia (Fig. 1E). The pulmonary hypertension contributed to a significant increase in the external work performed by the right ventricle (Fig. 1F). This increase, however, was offset by a decrease in the external work performed by the left ventricle due to the lower _max and HR_max. As a result of these opposing changes, the total work performed by the heart during maximal exercise was significantly lower throughout the hypoxic exposure than during normoxia.

Table 2 shows the resting hemodynamic data. It is apparent that, in general, the time course of resting hemodynamics during acclimatization roughly parallels the exercise data, although the changes in resting values are more attenuated than in exercise. Resting heart rate tended to decrease after an initial increase, and this was reflected in a decrease in resting cardiac output, which was accompanied by an increase in SVR without significant changes in MABP. Pulmonary arterial pressure, on the other hand, increased substantially. The resting pressure values are probably a better reflection of the afterload levels faced by the ventricles, and they show that whereas right ventricular afterload increased substantially during acclimatization, left ventricular afterload increased only moderately. Resting right and left ventricular work increased significantly only at 30 min of hypoxia and then decreased toward the control value. This pattern reflects the progressive decreases in heart rate and cardiac output, which offset the blood pressure changes even in the right ventricle.

Body and Ventricular Weights

Density and Affinity of Myocardial Receptors

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Time course of adrenergic and muscarinic receptors during acclimatization. A: -adrenoceptor density (fmol/mg protein) in left and right ventricle (LV and RV). B: -adrenoceptor density (fmol/mg protein) in LV and RV. C: M-Ach-receptor density (fmol/mg protein) in LV and RV. *P < 0.05 or better, HX vs. NX in LV. #P < 0.05 or better, HX vs. NX in RV.

₁-Adrenoceptor density showed a biphasic response in both right and left ventricles (Fig. 2B). In both cases, receptor density increased initially to reach a value significantly higher than the normoxic controls at 3 days of hypoxia. Receptor density then started to decline, and by day 21, it was significantly lower than the normoxic controls in both ventricles.

M-Ach-receptor density increased rapidly in both ventricles in response to hypoxia (Fig. 2C). In the right ventricle, receptor density increased by ~33% above control by day 1 and remained essentially unchanged afterwards. In the left ventricle, after an initial increase of similar magnitude, M-Ach-receptor density continued to increase, reaching a value that was ~80% above control by day 21 of hypoxia.

Table 4 shows the values of K_d for all the various receptors investigated in the right and left ventricles. Hypoxia did not influence receptor affinity in any case in either ventricle.

In the animals previously operated, left ventricular myocardial -adrenergic density was 95 ± 8 and 85 ± 2 fmol/mg of protein in normoxia and 24-h hypoxia, respectively. In the right ventricle, the values for normoxia and 24-h hypoxia were 67 ± 4 and 62 ± 1 fmol/mg of protein. These values are not significantly different from the values shown in Fig. 2 for the corresponding time. A similar lack of effect of surgery was observed in the affinity of -adrenergic receptor density that was 0.17 ± 0.01 and 0.18 ± 0.01 nM for normoxia and hypoxia in the left ventricle, respectively, and 0.20 ± 0.02 and 0.18 ± 0.01 nM for the right ventricle. These values are not significantly different from the values shown in Table 4 for the corresponding time. These data indicate that prior surgery and vascular catheterization do not modify adrenergic receptors' characterization.

	DISCUSSION

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These studies represent the first characterization of the time course of myocardial autonomic receptor modification parallel with the changes in maximal exercise capacity during acclimatization to hypoxia in the same animal model. The exercise O₂-transport pattern observed in the present experiments agrees with previous observations in humans and animals that show that acclimatization leads to little or no improvement in O_2 max. The mechanism for this lack of effect of acclimatization on O_2 max appears to be related to the decrease in _max, which offsets the increase in arterial blood O₂ content characteristic of prolonged hypoxia such that the rate at which O₂ is delivered to the exercising muscles does not change substantially. Supporting _max as a factor limiting O_2 max in this case is the observation that increasing HR_max and _max by cardiac pacing increases O_2 max of rats acclimatized to prolonged hypoxia (7). The data presented here show that these offsetting changes match one another rather closely, resulting in relatively uniform values of maximal rate of O₂ transport to the tissues (O_2max) and O_2 max throughout the course of acclimatization (Fig. 1A).

The present studies present a comprehensive picture of the determinants of _max throughout the course of acclimatization and provide clues of the possible role of myocardial receptor modification in this process. Thirty minutes after the onset of hypoxia, _max remained unchanged, after which it decreased gradually as a result of decreases in HR_max and SV_max (Fig. 1B); both of which followed different time courses (Fig. 1C). The initial decrease in SV_max at day 1 of hypoxia may have been the result of the plasma volume contraction that is known to occur at this time (19). The mechanism responsible for the reduction in SV_max later on in the acclimatization process is unclear and may be related to the increased right ventricular afterload (19) or to the conformational changes that occur in the right ventricle as a result of myocardial hypertrophy (20). It is also possible that the decrease in -adrenoceptor density may result in a decreased inotropic response to the elevated sympathetic drive characteristic of heavy exercise and thus contribute to the lower SV_max. A reduction in SV_max is a feature of maximal exercise after acclimatization in humans as well as in this animal model (20).

In contrast with SV_max, HR_max decreased following a gradual course that reached a plateau at 5 days of hypoxia. The changes in -adrenoceptor and M-Ach-receptor density observed in these experiments provide a possible explanation of the mechanisms responsible for the reduction in HR_max. After an initial increase in the right ventricle, hypoxia was associated with a continued and gradual decrease in -adrenoceptor density in both right and left ventricles, which also reached a steady value at 5 days of hypoxia (Fig. 2A). The decrease in -adrenoceptor density observed here agrees with similar findings after 3 wk of hypoxia in this model (10, 24) and shows that receptor density reaches a stable low value relatively early in the acclimatization process. This reduction in density is thought to be due to the prolonged agonist stimulation that results from the increased sympathetic drive of chronic hypoxia (1, 22), but it also could be the consequence of a direct effect of hypoxia on the adrenergic receptor pathway. The time course of resting heart rate is consistent with an increased sympathetic activity; resting heart rate increased significantly at 30 min of hypoxia and continued elevated above the normoxic value until the 3rd day of hypoxia (Table 2). Both resting and maximal heart rate showed a decline that paralleled the decrease in -adrenoceptor density. The decrease in -adrenergic receptor density explains the reduced chronotropic response to -adrenoceptor stimulation characteristic of acclimatization (22). The high positive correlation between ventricular -adrenoceptor density and HR_max is consistent with myocardial -adrenoceptor downregulation as a mechanism for the reduction in HR_max (Fig. 3A).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   A: Boltzmann sigmoidal correlation between HRmax (beats/min) and -adrenoceptor density (fmol/mg protein) during acclimatization. B: Boltzmann sigmoidal correlation between HRmax (beats/min) and M-Ach-receptor density (fmol/mg protein) during acclimatization.

The density of M-Ach receptors increased almost immediately after the onset of hypoxia and remained elevated in both ventricles (Fig. 2B). An increase in left ventricular M-Ach-receptor density was observed previously in rats exposed to hypoxia for 30 days (11, 25). The present results extend these observations to the right ventricle and show that the response to hypoxia is rapid and sustained. The mechanism responsible for the elevated M-Ach activity is not known. M-Ach-receptor stimulation mediates the negative chronotropic and inotropic effects of acetylcholine. It is not known if acclimatization to hypoxia is associated with changes in vagal output to the heart, which could help explain the increased myocardial M-Ach-receptor density. The larger effect of maximal doses of atropine in the resting heart rate of acclimatized than in nonacclimatized rats can be explained by the present findings of elevated myocardial M-Ach-receptor density (5). Regardless of the mechanism, however, there is a negative correlation between myocardial M-Ach-receptor density and HR_max (Fig. 3B). The picture that emerges from these results is that acclimatization to hypoxia is characterized by gradual changes in -adrenergic and M-Ach receptors, both of which are highly correlated with the changes in exercise HR_max observed simultaneously (Fig. 3). The direction of these changes is consistent with the idea that the reduction in HR_max, and by extension the limitation of maximal exercise capacity, characteristic of acclimatization, is the result of intrinsic changes in myocardial receptor function that are ultimately translated into a decreased response to adrenergic stimulation and an increased response to cholinergic stimulation. The resulting decrease in HR_max tends to lower the maximal rate of cardiac work and therefore reduce myocardial O₂ consumption. The strong correlation between changes in myocardial receptor characteristics and chronotropic responses to maximal exercise could represent the presence of a mechanism that tends to preserve the balance between myocardial O₂ delivery and utilization during maximal exercise in severe hypoxia (21).

The present experiments have also shown that hypoxia influences myocardial -adrenergic receptor density. Stimulation of -adrenoceptors mediates increases in myocardial contractility, and these effects are thought to be complementary to those of -adrenoceptor stimulation (6, 12). In addition, it has been suggested that stimulation of -adrenoceptors is involved in myocardial hypertrophy, although evidence on this subject is controversial, with some data supporting (23) and others opposing (13, 17) this possible effect. The present studies showed a similar pattern of response to hypoxia in both ventricles; -adrenoceptor density increased early after hypoxic exposure, after which it decreased to reach levels below the normoxic controls (Fig. 2B). In contrast, hypoxia resulted in right ventricular hypertrophy (Table 3) and pulmonary hypertension (Fig. 1E), transient left ventricular weight loss (Table 3), and, initially, systemic hypotension (Fig. 1D). The different patterns of weight gain between the right and left ventricle, in the presence of a similar time course of -adrenoceptor-density change in both ventricles, do not appear to support a role of these receptors as mediators of myocardial hypertrophy. In addition to the possible functional role of the changes in -adrenoceptor density, the mechanism of their change is also difficult to explain. Although the progressive decrease in -adrenoceptor density could be explained, at least in part, by the elevated agonist stimulation, the biphasic response of -adrenoceptors is not easily explained by this factor. The increase in SVR does suggest an increased -adrenergic vasoconstrictor tone, a feature that has been associated with this model of hypoxia (15). If this is the case, it is not clear why -adrenoceptor density continued to increase until the 3rd day of hypoxia before it started to decline. It is apparent that additional research is necessary to elucidate the role of myocardial -adrenoceptors in the acclimatization process.

In summary, the present studies correlate the changes in O₂ transport and maximal exercise capacity with the alterations in myocardial receptor characteristics before and during acclimatization to hypoxia. The results show that the lack of effect of acclimatization on O_2 max is correlated with a parallel lack of change in the rate of delivery of O₂ to the exercising muscles and that this is due to offsetting changes in blood O₂ content and cardiac output. A major factor in the decrease in cardiac output is a gradual decrease in HR_max, which correlates highly with the changes in myocardial -adrenergic and M-Ach-receptor density, suggesting that the reduction in HR_max is linked to intrinsic myocardial receptor modification during the acclimatization process. This could represent a mechanism that helps preserve the balance between myocardial O₂ delivery and utilization during exercise in severe hypoxia. Hypoxia also results in marked changes in the density of -adrenergic receptor density, the mechanism and functional relevance of which remain unclear.