Effect of intermittent hypoxia on cardiovascular function,

adrenoceptors and muscarinic receptors in Wistar rats

R. Germack *†, F. Leon-Velarde *‡, R. Valdes De La Barra §, J. Farias §,

Experimental Physiology

G. Soto § and J. P. Richalet *

* Laboratoire ‘Réponses cellulaires et fonctionnelles à l’hypoxie’, EA 2363, Université Paris XIII,

93017 Bobigny, France, § Laboratorio de Fisiologia de Altura, Universidad Arturo Prat,
Iquique, Chile and ‡ Laboratorio de Transporte de Oxigeno/IIA, Universidad Cayetano Heredia,
Lima, Peru
(Revised manuscript received 21 February 2002; accepted 9 April 2002)

The usual model of intermittent hypoxia (sleep apnoea) corresponds to repeated episodes of hypoxia from a
few seconds to a few hours interspersed with episodes of normoxia. The aim of this study was to evaluate in rats
the effect of two periods of intermittent exposure for 2 months to hypoxia (IHX1, 24 h in hypoxia (428 Torr),
24 h in normoxia; IHX2, 48 h in hypoxia (428 Torr), 24 h in normoxia) as a new model of hypoxia simulating
intermittent exposure to high altitude experienced by Andean miners. We assessed the haematological
parameters, time course of resting heart rate and systolic blood pressure. We also evaluated the expression of
adrenergic and muscarinic receptors. IHX1 and IHX2 produced an increase in haematocrit, haemoglobin
concentration and mean corpuscular volume as previously seen in most hypoxic models. IHX1 and IHX2
induced a similar sustained elevation of systolic blood pressure (132 ± 2 and 135 ± 3 mmHg, respectively, vs.
the control level of 121 ± 16 mmHg) after 10 days of exposure without change in heart rate. Right ventricular
(RV) hypertrophy (225 ± 13 and 268 ± 15 mg g_1, vs. 178 ± 7 mg g_1) and downregulation of a1-adrenoceptor
(RV: 127 ± 21 and 94 ± 16 fmol mg_1 vs. 157 ± 8 fmol mg_1; left ventricle (LV): 141 ± 5 and 126 ± 9 fmol mg_1
vs. 152 ± 5 fmol mg_1) have been found in both groups, with right ventricular hypertrophy being greater and
a1-adrenoceptor density being lower in IHX2 than in HX1 groups. These data indicate that both parameters are
related to the time of exposure to hypoxia. IHX1 and IHX2 produced the same magnitude of upregulation of
muscarinic receptors (LV, 60 %; RV, 40 %), and no change in b-adrenoceptors. In conclusion, exposure to
intermittent hypoxia led to polycythaemia and RV hypertrophy as observed in other types of hypoxia. A
specific cardiovascular response was seen, that is an increase in blood pressure without change in heart rate,
which was different from the one observed in episodic and chronic hypoxia. Furthermore, this model involved
specific modifications of a1-adrenergic and muscarinic expression. Experimental Physiology (2002) 87.4, 453–460.

Intermittent hypoxia, which is experienced by a great
number of Andean workers (e.g. miners and observatory
workers), is a new model of exposure to hypoxia whereby
periods of stay at higher altitude are interspersed with
periods of stay at sea level. These periods may be as short
as one day to several days (Richalet et al. 2002). This model
is different from acute (sport and tourism) episodic (sleep
apnoea) or chronic (permanent residence) exposure.
Episodic and chronic hypoxia are characterized by marked
cardiovascular effects to offset a global decrease in tissue
oxygen supply, including polycythaemia and an overall
sympathetic stimulation (Moore-Gillon & Cameron, 1985;
Richalet, 1990; Rostrup, 1998; Greenberg et al. 1999;
Neubauer, 2001). In parallel, hypoxic pulmonary vaso-
constriction leads to pulmonary hypertension and a right
ventricular hypertrophy (Rumsey et al. 1999; Neubauer,
2001). Acute and episodic hypoxia have been found to be
associated with an increase in systemic blood pressure and
resting heart rate (Fletcher, 2001). In chronic hypoxia,
these parameters were unchanged or decreased in rats
(Kuwahira et al. 1993; Gonzalez et al. 1998; Favret et al.
2001) or slightly increased in humans (Richalet, 1990;
Antezana et al. 1992). In acute (days) and chronic hypoxia
in vivo, the progressive blunting of the chronotropic
responsiveness has been attributed to a decreased cardiac
sensitivity to adrenergic stimulation (Richalet 1990;
Antezana et al. 1992) and/or an increased cholinergic
activation of the heart (Hartley et al. 1974; Selvamurthy et

Publication of The Physiological Society † Corresponding author: richalet@smbh.univ-paris13.fr

2375
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 454 R. Germak and others
al. 1981; Zhuang et al. 1993). However, Bernardi et al.
(2001) showed that cardiac vagal activity was unchanged
in intermittent hypoxia. These modifications can be
related in part to alterations in the expression of
adrenergic and cholinergic receptors. Indeed, a down-
regulation of b-adrenergic receptors (b-ARs) and
upregulation of muscarinic receptors have been reported
Experimental Physiology
(Kacimi et al. 1993; León-Velarde et al. 1996; Richalet,
1997; Favret et al. 2001). Favret et al. (2001) showed that
a-AR density increased after hypoxic exposure for
1–3 days, but was downregulated after this period in
chronic hypoxia. A similar downregulation of a1-ARs has
been observed in vitro in cardiomyocytes exposed to
chronic hypoxia (Li et al. 1995). Furthermore, a1-ARs are
one of the determinants of the cardiac hypertrophic
response to pulmonary and/or systemic pressure changes
(Benfey, 1990). The hypertrophy induced by a1-AR
stimulation is characterized by increased protein synthesis,
myofibrillar re-organization and the re-expression of fetal
genes (Ikeda et al. 1991; Zimmer et al. 1995). Thus, the a-
and b-adrenergic and cholinergic systems seem to be
involved together in the regulation of the responses of the
heart to hypoxic exposure.
The aim of this study was to establish a new model of
intermittent hypoxia with longer hypoxic exposure than
the usual models, and to determine the cardiovascular
pattern and expression of adrenergic and muscarinic
receptors involved in the control of cardiac function,
which can be different from those observed in episodic and
chronic hypoxia. For this purpose, we evaluated the effects
of two different periods of intermittent hypoxia for
2 months on the haematological parameters, resting heart
rate and systolic blood pressure. The density of adrenergic
and muscarinic cardiac receptors was also determined.
METHODS
Animals and hypoxia procedure
Male Wistar rats (351 ± 6 g, n = 25) were separated into three
groups: NX, normoxia group (control); IHX1, intermittent
hypoxia group 1; IHX2, intermittent hypoxia group 2. For the
hypoxic exposure of IHX1, the hypobaric chamber was brought
to normobaria for 24 h every other day (24 h hypoxia/24 h
normoxia), and for the hypoxic exposure of IHX2, the chamber
was brought to normobaria for 24 h after 2 days in hypoxia (48 h
hypoxia/24 h normoxia). Both groups were exposed to a 4600 m
simulated altitude (428 Torr) for a period of 60 days. The control
group was housed in the same room and in comparable
conditions to the hypoxic groups. In this model we took into
account the metabolic rate of a rat which is 4 times greater
(0.88 ml g_1 h_1) than the metabolic rate of a man (0.23 ml g_1
h_1) (Prosser, 1973). This information was used to calculate the
number of days per rat (1 and 2 days) that corresponded
approximately to the number of days per man (4 or 8 days) in
terms of the effect of exposure to intermittent hypoxia as
experienced by Andean workers. In these groups we measured
the effect of intermittent hypoxia on the haematological
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Exp. Physiol. 87.4
parameters, body weight (BW, g), the time course of changes in
heart rate (HR, beats min_1), systolic blood pressure (SBP,
mmHg), and the expression of adrenergic and cholinergic
receptors. All procedures were performed in agreement with the
regulations of the French ‘Ministére de l’Agriculture’ for animal
care.
Heart rate and blood pressure measurement, and
haematological parameters
Resting HR and SBP were measured in conscious rats each day
under normobaric conditions after each hypoxia exposure, using
an inflatable tail-cuff and pressure sensor (RTBP1003-220, Kent
Scientific, Torrington, CT, USA). The signal was sent through a
pre-amplifier to a Workbench data acquisition system. The
average of 7–10 measurements was taken for HR and SBP. Data
were collected and analysed using the RTBP-001-DS worksheet
(Kent Scientific). On the days of measurement, the animals were
acclimated to a movement-limiting Plexiglas chamber for
15–30 min. After vasodilatation of the tail by warming it,
repetitive measurements of systolic pressure were made. The
average of 2 days of measurements before beginning hypoxia was
used as baseline HR and SBP. Body weight (BW) was also
recorded before each BP measurement.
After the last in vivo measurements were performed, the animals
were killed by stunning and cervical dislocation. The blood
sample (200–400 ml) was withdrawn from the cava vein through
a 3 cm incision in the peritoneum immediately after the animal
was killed, when the blood was still under free-flowing
conditions, placed in heparinized microtubules, and spun in a
microcentrifuge for the measurement of the haematocrit (Hct).
The other haematological parameters were measured using a
Coulter Electronics counter (Hialeah, FL, USA). After the blood
samples had been obtained, the hearts were quickly removed and
dissected free of fat and large vessels. The ventricles were
separated from the atria and rapidly put into liquid nitrogen.
They were then immediately placed at _80 °C until use.
Crude membrane preparation
The wet weight of the combined LV plus septum and of the RV
were determined rapidly before membrane preparation. The
ventricles were minced and homogenized in 10 ml hypotonic
buffer (30 mM Tris HCl, 100 mM NaCl, 5 mM MgCl2, 1 mM
EGTA, 10 mg ml_1 leupeptin and 300 mM phenylmethylsulphonyl
fluoride; pH 7.5) with a polytron tissue homogenizer (6 w 5 s
bursts). The suspension was centrifuged at 1000 g for 10 min.
Soon after, the supernatant was centrifuged at 45 000 g for
45 min at 4 °C. The pellet was resuspended in 10 ml binding
buffer (50 mM Tris HCl, 5 mM MgCl2; pH 7.5) and centrifuged at
45 000 g for 45 min at 4 °C. The final pellet was resuspended in
binding buffer to a final concentration of 2–3 mg protein ml_1
and stored at _80 °C for subsequent binding studies. Protein
concentration was determined by dye-binding assay using a
commercial kit (Bio-Rad, München, Germany) and bovine
serum albumin was used as standard. All the products were
purchased from Sigma-Aldrich Chimie (Lyon, France).
Binding studies
Saturation studies were performed with 30–70 mg of crude
membrane protein using [3H]prazosin (a1-antagonist) at
concentrations ranging from 0.025 to 1.5 nM for a1-ARs,
[3H]CGP 12177 (b1/b2-antagonist) at concentrations ranging
from 0.06 to 3 nM for b-ARs or [3H]quinuclidinyl benzilate
(QNB; muscarinic antagonist) at concentrations ranging from
0.01 to 2 nM for muscarinic receptors in a 96-well microplate. To
 Exp. Physiol. 87.4 Effect of intermittent hypoxia on cardiovascular function 455

Table 1. Effect of intermittent hypoxia on haematological parameters

Hct Hb RBC CHCM VCM
(%) (g dl_1) (w 106 cells ml_1) (g dl_1) (fl)
NX (n = 6) 40 ± 3.4 13.0 ± 1.2 4.6 ± 0.37 32.6 ± 1.1 80 ± 2.5
IHX1 (n = 9) 50 ± 5.8 * 16.2 ± 1.8 * 5.2 ± 0.43 34.6 ± 1.4 * 85 ± 2.7 **
Experimental Physiology

IHX2 (n = 9) 51 ± 5.9 * 16.4 ± 2.3 * 5.1 ± 0.48 34.6 ± 1.2 * 88 ± 2.9 ***
The values are means ± S.D. of the following parameters: haematocrit (Hct), haemoglobin concentration
(Hb), red blood cell count (RBC), mean corpuscular haemoglobin concentration (CHCM) and mean
corpuscular volume (VCM). * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. normoxic control (NX).

the binding buffer, 50 ml radioligand and 50 ml crude membrane

were added to obtain a final volume of 200 ml. The incubation
was carried out in a shaking water bath at 25 °C for 60 min for
a-ARs and muscarinic receptors, and at 37 °C for 60 min for
b-ARs and then stopped by rapid filtration through glass fibre
filters (Filtermats-Receptor binding, Skatron, Lier, Norway)
prewetted in binding buffer. The radioactivity trapped by filters
was measured using a liquid scintillation b-counter (LS6000SC,
Beckman, USA). Non-specific binding was determined in the
presence of 1 mM prazosin, 10 mM propranolol and 10 mM
atropine to a- and b-ARs and muscarinic receptors, respectively.
The radiochemicals were obtained from Amersham Pharmacia
Biotech GmBh (Les Ulis, France).
Data and statistical analysis
The binding parameters (dissociation constant, Kd and receptor
density, Bmax) were determined using a LIGAND non-linear
curve-fitting program (Munson & Rodbard, 1980). The binding
data are expressed as mean ± S.E.M. For the other data, the values
are expressed as mean ± S.D. Differences across all testing
conditions as well as differences between the groups IHX1 and
IHX2 were determined by a repeated measures two-way analysis
of variance for physiological measurements and one-way analysis
of variance ANOVA for receptor expression. Tukey’s post hoc
comparisons were used to identify significant differences among
means. P < 0.05 was considered as statistically significant.

RESULTS
Haematological parameters, heart rate and blood
pressure
Intermittent hypoxia produced polycythaemia, as has been
observed previously in our models and in other models of
hypoxic exposure (Moore-Gillon & Cameron, 1985;
Neubauer, 2001). Table 1 shows all the haematological
parameters for the NX, IHX1 and IHX2 groups. IHX1 and
IHX2 groups showed higher values of Hct, haemoglobin Figure 1
(Hb) and mean corpuscular volume than the NX group. Systolic arterial blood pressure (A) and mean heart
Blood pressure increased after 10 days of hypoxia (IHX1, rate (B) in rats exposed to 60 days of intermittent
132 ± 2 mmHg; IHX2, 135 ± 3 mmHg) when compared hypoxia. NX, normoxia; IHX1, 24 h hypoxia/24 h
with NX (121 ± 16 mmHg; P < 0.05), and when compared normoxia; IHX2, 48 h hypoxia/24 h normoxia.
with the blood pressure values obtained in the same Measurements were made each normobaric day after
animals in normoxia (IHX1, 121 ± 3 mmHg; IHX2, each period of exposure to hypoxia. Heart rate values
were not significantly different at the end of the
121 ± 7 mmHg). Blood pressure remained elevated until
exposure. Blood pressure values from days 15 to 60
the end of the experiment (Fig. 1A). Heart rate was not
in hypoxia were significantly higher than controls for
modified by exposure to hypoxia in either group (IHX1, both IHX1 and IHX2 values; ** P < 0.01.
342 ± 17 beats min_1; IHX2, 345 ± 7 beats min_1) when

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 456 R. Germak and others Exp. Physiol. 87.4

Table 2. Effect of intermittent hypoxia on ventricle weight and weight ratio

Left ventricle Right ventricle LV ratio RV ratio
(mg) (mg) (mg g_1) (mg g_1)
NX (n = 7) 635 ± 22 178 ± 7 1.62 ± 0.07 0.45 ± 0.02
IHX1 (n = 9) 609 ± 12 225 ± 13 a * 1.64 ± 0.03 0.61 ± 0.04 a *
Experimental Physiology

IHX2 (n = 9) 592 ± 9 268 ± 15 a **, b * 1.73 ± 0.02 0.78 ± 0.04 a **, b *

The values are means ± S.E.M. Left ventricle (LV) or right ventricle (RV) ratio: left or right ventricle
weights/body weight. * P < 0.05, ** P < 0.01. a vs. control (NX), b vs. HX1.
Table 3. Effect of intermittent hypoxia on adrenoceptor expression in left and right ventricles
Left ventricle Right ventricle
Kd Bmax Kd Bmax
(nM) (fmol mg_1) (nM) (fmol mg_1)
a1-Adrenoceptor (n = 6) (n = 6)
NX 0.46 ± 0.15 152 ± 5 0.63 ± 0.20 157 ± 8
IHX1 0.47 ± 0.15 141 ± 5 0.63 ± 0.21 127 ± 21
IHX2 0.41 ± 0.13 126 ± 9 * 0.43 ± 0.17 94 ± 16 *
b-Adrenoceptor (n = 6) (n = 4)
NX 0.28 ± 0.03 76 ± 4 0.36 ± 0.06 60 ± 7
IHX1 0.28 ± 0.03 82 ± 7 0.36 ± 0.06 63 ± 7
IHX2 0.31 ± 0.04 84 ± 5 0.38 ± 0.06 60 ± 8
The values are means ± S.E.M. of separate experiments performed in duplicate. The dissociation
constants (Kd) are expressed in nM and the receptor densities (Bmax) in fmol of radioligand bound per mg
of membrane proteins: [3H]prazosin for a1-adrenoceptors and [3H]CGP 12177 for b-adrenoceptors.
* P < 0.05 vs. normoxic control (NX).
Table 4. Effect of intermittent hypoxia on muscarinic receptor expression in left and right ventricles
Left ventricle Right ventricle
Kd Bmax Kd Bmax
(nM) (fmol mg_1) (nM) (fmol mg_1)
(n = 5) (n = 6)
NX 0.18 ± 0.03 735 ± 87 0.26 ± 0.02 796 ± 81
IHX1 0.22 ± 0.03 1175 ± 110 * 0.27 ± 0.04 1123 ± 83 *
IHX2 0.19 ± 0.03 1194 ± 112 * 0.40 ± 0.09 1163 ± 107 *
The values are means ± S.E.M. of separate experiments performed in duplicate. The dissociation
constants (Kd) are expressed in nM and the receptor densities (Bmax) in fmol of [3H]QNB bound per mg
of membrane proteins. * P < 0.05 vs. normoxic control (NX).

compared with NX (335 ± 10 beats min_1), and with their
own heart rate values in normoxia (IHX1, 334 ± 10 beats
min_1; IHX2, 342 ± 23 beats min_1) (Fig. 1B). There was
no statistical difference between the two groups exposed to
intermittent hypoxia, either for blood pressure or heart
rate.
Body and ventricular weights
Intermittent hypoxic exposure for 60 days induced in both
animal groups a significant reduction in body weight gain
compared to NX (IHX1, 378 ± 34 g; IHX2, 348 ± 22 g
versus NX, 398 ± 29 g; P < 0.05). Intermittent hypoxia
resulted in right ventricular hypertrophy as assessed by the
ratio of RV weight to body weight (Table 2). The degree of
hypertrophy was greater in the IHX2 group (22 %). The
wet weight of the LV and the ratio of LV weight to body
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weight of either group were not changed significantly
compared to the control group.
a1- and b-adrenoceptor expression
The a1- and b-adrenoceptor binding characteristics are
shown in Table 3. Figure 2 is an illustration of the binding
experiments. The a1-AR density was lower in hypoxia
without change in affinity whatever the model of hypoxia
as compared to the normoxic control although the difference
between IHX1 group and NX was not significant. The
longest continuous exposure to hypoxia (IHX2) was
associated with a significant decrease in the a1-AR density.
This decrease was greater in RV than in LV when compared
to the control group. The diminution of the a1-AR density
for the IHX1 and IHX2 groups was 7 % and 17 % for the
LV, and 19 % and 40 % for the RV, respectively.
 Exp. Physiol. 87.4 Effect of intermittent hypoxia on cardiovascular function 457

In contrast to the a1-AR expression, intermittent hypoxia
produced no change in the b-AR number in RV as well as
in LV (Table 3). The affinity of b-ARs and a1-ARs was not
modified by intermittent hypoxia.
Muscarinic receptor expression
Muscarinic receptors, implicated in the b-antiadrenergic
Experimental Physiology
measured here, and invasively measured mean arterial
pressure, were shown to be similar in rats exposed to
intermittent hypoxia (Fletcher et al. 1992). Although we
did not measure cardiac output and mean arterial
pressure, the observed 30 % increase in systolic blood
pressure in both groups may reflect systemic hypertension
and an increase in systemic vascular resistance. The

effect, were increased by about 60 % in LV and 40 % in RV polycythaemia alone is not sufficient to explain this
in intermittent hypoxia independent of the time of exposure hypertension since it has not been described in chronic
(Table 4). Unlike the a1-adrenoceptor, the modification of hypoxia. Indeed, the chronically intermittent stimulation
the muscarinic receptor density was greater in LV. The of peripheral chemoreceptors has been shown to be
affinity was not altered by hypoxic exposure in any group. originally implicated in the increase in systemic blood
pressure in hypoxia (Fletcher et al. 1992, Leßke et al. 1997)
DISCUSSION whereas the desensitization of peripheral chemoreceptors
Different types of hypoxic exposure have been described in observed in chronic hypoxia may prevent the development
the literature as alternating periods of hypoxia and of systemic hypertension. It appears also that the increased
reoxygenation as in episodic hypoxia. In these models of
intermittent hypoxia, the cycle length of exposure to
hypoxia ranges from a few seconds to a few hours per day
(Neubauer, 2001). In our model, the period of hypoxia was
longer, from 1–2 days. We estimated the number of days
per rat that would correspond to the number of days per
man in terms of the effect of exposure to intermittent
hypoxia (Prosser, 1973) in order to simulate the inter-
mittent exposure to high altitude experienced by Andean
miners. To establish a new model and characterize the
mechanism of adaptation to this type of intermittent
hypoxia, we evaluated the changes in haematological
parameters, heart rate and systolic blood pressure, as well
as the expression of adrenergic and muscarinic receptors.
Our results showed that adaptation to intermittent
hypoxia led to an increase in systolic blood pressure
without change in heart rate. This pattern is different from
the one observed in episodic and chronic models of hypoxia,
and involved specific modifications in a1-adrenergic and
muscarinic receptor density without changes in b-adreno-
ceptor expression.
Intermittent hypoxia (IHX) in our model produced
polycythaemia as observed in chronic hypoxia and
different models of intermittent hypoxia (Moore-Gillon &
Cameron, 1985; Kacimi et al. 1992). Hb concentration and
Hct increased by around 25 %, which was less than in rats
exposed to chronic hypoxia, with increases of 40 and 70 %,
respectively (Kacimi et al. 1992). No change in Hct level
was found in episodic hypoxia associated with very short
repetitive exposure (30 s of hypoxia; Fletcher et al. 1992).
These data suggest that the degree of changes in blood
parameters depends on the duration of exposure to
hypoxia. Increased Hct, which enhances the vascular
Figure 2
resistance by increasing blood viscosity, contributes to
pulmonary and systemic hypertension. Systemic blood Expression of a1-adrenoceptors. Specific
[3H]prasozin binding to left (A) and right (B)
pressure increases significantly in episodic and progressive
ventricles from rats exposed to 60 days of
hypoxia (Fletcher, 2001) in contrast to chronic exposure intermittent hypoxia. NX, normoxia; IHX1, 24 h
where no changes, or only small variations were observed hypoxia/24 h normoxia; IHX2, 48 h hypoxia/24 h
(Richalet, 1990; Antezana et al. 1992; Kuwahira et al. 1993; normoxia. Each saturation curve is representative of
Gonzalez et al. 1998; Favret et al. 2001). Our results are in an experiment that was performed in duplicate. S.D.
good agreement with those obtained using these models of values not shown are within the size of the symbol.
intermittent hypoxia. Tail-cuff systolic blood pressure, as
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 458 R. Germak and others
activity of adrenergic and renin–angiotensin systems
contributes to the early chronic elevated blood pressure in
intermittent hypoxia (Flecher, 2001). Finally, erythropoietin
may have a direct growth effect on blood vessels and
exacerbate systemic hypertension in already hypertensive
animals (Gogusev et al. 1994). In our model, IHX did not
modify resting heart rate whereas in episodic hypoxia
Experimental Physiology
(sleep apnoea) this variable is increased (Fletcher, 2001)
and it is unchanged or decreased in chronic hypoxia
(Kuwahira et al. 1993; Gonzalez et al. 1998; Favret et al.
2001). This result indicates that a longer exposure time to
hypoxia, even with alternance in normoxia produced a
cardiovascular pattern specific to our model of IHX
switching towards a chronic pattern with respect to resting
heart rate.
In the present model of intermittent hypoxia, hypertrophy
of the right ventricle (RV) was greater in the IHX2 group
(51 %) than in the HX1 group (26 %) indicating that RV
hypertrophy is also related to the duration of exposure to
hypoxia. No significant change was found in the left
ventricle (LV) in either group. Adaptation to hypoxia leads
to a cardiac asymmetry since hypoxia-induced atrophy of
the LV results in reduced oxidative capacity while the RV
is hypertrophied due to pressure overload produced by
pulmonary hypertension (Rumsey et al. 1999) Favret et al.
2001). The lack of hypertrophy of the LV in spite of
systemic hypertension may have been influenced by an
enhanced catabolic or a decreased anabolic state as
demonstrated by a lower weight gain in both hypoxic
groups. We can also postulate that the hypertrophic
stimulus produced by the increase in afterload was not
large enough to produce hypertrophy of the LV. It has
been shown that the activation of a1-ARs stimulates the
hypertrophic response in cardiomyocytes, both in vivo
(Zimmer et al. 1995) and in vitro (Ikeda et al. 1991). The
downregulation of a1-ARs was greater in the hyper-
trophied RV (40 %) than in the LV (17 %). These data
suggest that the downregulation of a1-ARs may be more
related to hypertrophy than to hypoxia. A prolonged
exposure to the agonist results in inactivation and
desensitization of G-protein-coupled receptors, such as
adrenoceptors and muscarinic receptors, by a down-
regulation process (Bünemann et al. 1999). Noradrenaline
(NA, norepinephrine) levels are increased in episodic
hypoxia as a result of sympathetic stimulation (Bao et al.
1997; Greenberg et al. 1999). Thus, an elevated circulating
NA level can explain the downregulation of a1-ARs.
Similar results were obtained in vitro by combining
hypoxia and NA (Li et al. 1995). However, the a1-AR
density was decreased without change in b-AR number, in
contrast to chronic hypoxia where both a1- and b-ARs
were downregulated (Favret et al. 2001). Favret et al. (2001)
showed that the decrease in resting heart rate is consistent
with the downregulation of b-ARs in chronic hypoxia. In
our model, resting heart rate was unchanged, which is in
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Exp. Physiol. 87.4
accordance with the unaltered b-AR density. Furthermore,
it is well known that NA stimulates a-ARs preferentially to
b-ARs (Liggett & Raymond, 1993), which can explain the
downregulation observed only in a1-ARs in IHX. We can
also suggest that the adrenergic stimulation is not sufficient
in IHX (1 or 2 days in hypoxia/1 day in normoxia) to induce
a decrease in b-AR density, particularly when no down-
regulation was reported in chronic hypoxia before 3 days
of exposure (Favret et al. 2001).
Muscarinic receptors are involved in the regulation of
cardiac function by mediating negative chronotropic and
inotropic effects. These receptors have been shown to be
upregulated in chronic hypoxia (Kacimi et al. 1993; León-
Velarde et al. 1996; Favret et al. 2001). Furthermore, this
regulation was rapid and sustained indicating that the
cholinergic system is the first modulated by hypoxia. Our
results are in agreement with these studies. The same
magnitude of upregulation of muscarinic receptors was
found in both ventricles in IHX1 and IHX2 (LV, 60 %; RV,
40 %). It has been shown that chronic heart failure, leading
to an increase in plasma concentrations of catecholamines,
results in a decreased response to isoprenaline and an
increased response to carbachol (Borst et al. 1999).
Furthermore, b-AR stimulation leads to an increase in
muscarinic receptor density. These results are supported
by the downregulation of muscarinic receptors induced by
a chronic b-AR blockade in the rat heart (Marquetant et al.
1992). These findings suggest cross-talk between b-ARs
and muscarinic receptors, which can take place in chronic
hypoxia and partly in intermittent hypoxia since no b-AR
downregulation was observed. However, muscarinic
receptors are downregulated after stimulation by the
agonist like most G-protein-coupled receptors (Haddad &
Roussel, 1998). Thus the upregulation observed in inter-
mittent hypoxia cannot be related to an increase in vagal
activity but rather to a decrease or a lack of change in
cardiac vagal activity, as shown by Bernardi et al. (2001) in
intermittent hypoxia.
In conclusion, intermittent hypoxia led to changes that
were in some ways similar to other models of hypoxia and
also specific to this model: a moderate increase in
haematological constants, a sustained elevation of systolic
blood pressure without modification of resting heart rate,
and the development of right ventricular hypertrophy. The
regulation of cardiac receptors involved an upregulation of
muscarinic receptors and a downregulation of a1-ARs
without change in b-ARs. The density of a1-ARs and the
hypertrophic process were related to the total duration of
exposure to hypoxia, in contrast to muscarinic receptor
expression which was independent of exposure time.
Studies need to be carried out to further our understanding
of the mechanisms of regulation implicated in this new
model of intermittent hypoxia, and especially to elucidate
the physiological responses produced by the changes in
receptor expression.
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Acknowledgements
This study was supported by FONDEF (Chile) D97-11050, and
from INSERM/CONICYT – 1998/1999.

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