Departments of 1 Physiology, 3 Biochemistry, and 4 Pediatrics; and 2 Institute of Cardiovascular Sciences and Medicine, Faculty of Medicine, University of Hong Kong, Hong Kong, China
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ABSTRACT |
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The effects
of -adrenoceptor stimulation with isoproterenol on electrically
induced contraction and intracellular calcium ([Ca2+]i) transient, and cAMP in
myocytes from both hypertrophied right and nonhypertrophied left
ventricles of rats exposed to 10% oxygen for 4 wk, were significantly
attenuated. The increased [Ca2+]i transient
in response to cholera toxin was abolished, whereas increased cAMP
after NaF significantly attenuated. The biologically active
isoform, Gs
-small (45 kDa), was reduced while the
biologically inactive isoform, Gs
-large (52 kDa),
increased. The increased electrically induced
[Ca2+]i transient and cAMP with 10-100
µM forskolin were significantly attenuated in chronically hypoxic
rats. The content of Gi
2, the predominant
isoform of Gi protein in the heart, was unchanged. Results
indicate that impaired functions of Gs protein and adenylyl cyclase cause
-adrenoceptor desensitization. The impaired function of the Gs protein may be due to reduced
Gs
-small and/or increased Gs
-large, which
does not result from changes in Gi protein. Responses to
all treatments were the same for right and left ventricles, indicating
that the impaired cardiac functions are not secondary to cardiac hypertrophy.
G protein; cardiac hypertrophy
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INTRODUCTION |
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CHRONIC HYPOXIA
leads to cardiac hypertrophy and reduced contractility
(32, 34, 35). It is well established that the responsiveness of cardiac contractility to -adrenoceptor stimulation is reduced in cardiac hypertrophy (3, 44, 47) and heart failure (16).
-adrenoceptor desensitization has been
shown to be due to downregulation of the
-adrenoceptor (9,
19). The postreceptor events, which may also be responsible for
-adrenoceptor desensitization, are not well understood. There is
evidence that the function of the Gs protein, which
mediates the action of
-adrenoceptor stimulation (15, 20, 21,
30), is impaired in the hypertrophied heart (21).
On the other hand, the Gs content was unchanged in the
hypoxic rat heart (18, 21). There was no study on the changes of the two Gs
-subunits (14, 38)
derived from a single gene by alternative splicing of mRNA
(23), a biologically active Gs
-small of an
apparent molecular weight of 45 kDa (14, 31, 49) and a
Gs
-large of an apparent molecular weight of 52 kDa. The
role of adenylyl cyclase (AC), the enzyme that is activated by the
Gs protein and in turn converts ATP into cAMP, is also controversial. Cardiac hypertrophy was associated with reductions in
cellular cAMP (36) and chronic hypoxia with impaired AC
response to forskolin, an activator of AC (30). On the
other hand, it has also been reported that there was no significant
impairment of activity of AC with forskolin stimulation in the right
hypertrophied heart after chronic hypoxia (22, 48). The
Ca2+ influx via the L-type Ca2+
channel, which is enhanced upon
-adrenoceptor stimulation via the
Gs protein/AC/cAMP cascade, is unchanged in the
hypertrophied heart (10, 37, 40).
In the present study, we delineated the postreceptor signaling
mechanisms in the right and left hearts of rats exposed to low oxygen
for 4 wk, which has previously been shown to induce right heart
hypertrophy (32, 34, 35) and reduce cardiac contractility
(19, 28). With the use of electrically stimulated twitch
amplitude and intracellular calcium ([Ca2+]i)
transient, and cAMP in isolated ventricular myocytes as parameters, we
determined the changes in ventricular myocytes of chronically hypoxic
and age-matched normoxic rats subjected to manipulations that activated
-adrenoceptor, Gs protein, or AC. The electrically induced [Ca2+]i transient represents the
influx of Ca2+ via the L-type Ca2+ channel upon
electrical stimulation, which then triggers release of Ca2+
from the sarcoplasmic reticulum (SR), leading to a
[Ca2+]i transient and muscle contraction. A
previous study in our laboratory showed that, in a similar experimental
condition, the electrically induced [Ca2+]i
transient is directly related to contractility (51). We
measured the two Gs
isoforms with the low and high
molecular weights in the heart of normoxic and chronically hypoxic
rats. We also measured Gi
2, which is the
predominant isoform of Gi
in the heart (12, 23). In addition, we compared the responses of both the right hypertrophied and left nonhypertrophied ventricles to determine the
relationship between hypertrophy and impaired cardiac function. Results
from this study showed that the functions of both Gs
protein and AC were impaired after chronic hypoxia. The impaired
function of Gs protein was due to a reduction in the
biologically active Gs
-small. The responses in the right
hypertrophied and left nonhypertrophied ventricles were the same,
indicating that impaired cardiac function is not secondary to hypertrophy.
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MATERIALS AND METHODS |
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Chronic hypoxia.
Male Sprague-Dawley rats that weighed 100-150 g at the start of
the experiment were randomly divided into two groups. One group of the
rats (n = 42) was exposed to chronic hypoxia, while the
control (n = 40) was maintained in room air. All the
rats were kept in the same room with the same light-dark cycle. For chronic hypoxia, rats were given inspired oxygen (10% O2)
in a 300-liter acrylic chamber. The hypoxic environment was established with the inflow of a mixture of room air and nitrogen that was regulated by an oxygen analyzer (model 175518A, gold edition, Vacuum
Med) (1, 37a, 39). Carbon dioxide was absorbed by soda lime
granules, and excess humidity was removed by a dessicator. Temperature
was maintained at 19-21°C. The chamber was opened twice a week
for ~1 h to clean the cages and replenish food and water. Rats were
exposed to hypoxia for 4 wk, and experiments were performed immediately
after removal of the rats from the chamber. The rats were weighed and
decapitated, and the hearts were quickly removed. The right
heart of the rats exhibited hypertrophy (Table
1), in agreement with previous
findings (32, 35). The PO2
of arterial blood in the chronically hypoxic rats was reduced by
~35% compared with control.
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Isolation of ventricular myocytes. Ventricular myocytes were isolated from the hearts of male Sprague-Dawley rats using a collagenase perfusion method described previously (11). Immediately after decapitation, the hearts were rapidly removed from the rat and perfused in a retrograde manner at a constant flow rate (10 ml/min) with an oxygenated Joklik modified Eagle's medium supplemented with 1.25 mM CaCl2 and 10 mM HEPES at pH 7.2 at 37°C for 5 min. This was followed by 5-min perfusion with the same medium free of Ca2+. Collagenase was then added to the medium to a concentration of 125 U/ml with 0.1% (wt/vol) bovine serum albumin (BSA). After 35-45 min of perfusion with the medium containing collagenase, the atria were discarded. The right ventricle (RV) and left ventricle (LV) tissues were dissociated separately by shaking in the same oxygenated collagenase-free solution for 5 min at 37°C. Ventricular tissues were cut into small pieces with a pair of scissors followed by stirring with a glass rod for 5 min. The procedure separated the ventricular myocytes from each other. The residue was filtered through 250-µm mesh screens, sedimented by centrifugation at 100 g for 1 min, and resuspended in fresh Joklik solution with 1% BSA. More than 70% of the cells were rod shaped and impermeable to trypan blue. The Ca2+ concentration of the Joklik solution was increased gradually to 1.25 mM within 40 min.
Measurement of twitch amplitude of contraction.
After isolation of myocytes for 2 h, measurement of myocyte twitch
amplitude was performed with the use of an optical video system, as
described previously (5, 50). The myocyte was placed in a
perfusion chamber at room temperature under an inverted microscope
(Nikon) and perfused at a rate of 2 ml/min with a Krebs bicarbonate
buffer that contained (in mM) 117 NaCl, 5 KCl, 1.2 MgSO4,
1.2 KH2PO4, 1.25 CaCl2, 25 NaHCO3, and 11 glucose, with 1% dialyzed BSA and a gas
phase of 95% O2-5% CO2, pH 7.4. The myocyte
was field electrically stimulated at a rate of 0.2 Hz with platinum
electrodes connected to a voltage stimulator. The twitch amplitude was
measured with an automatic video analyzer developed in our laboratory
(51). As a prerequisite for the proper operation of this
automatic analyzer, the image of cardiac myocytes projected on the
video camera (SSC-M370CE; Sony) and observed on the video monitor
(PVM-145E; Sony) was first rotated to align horizontally and parallel
to each of the video raster lines. This was achieved by interposing a
-mirror (or a dove prism) between the microscope eyepiece and the
video camera. The video analyzer was interposed between the
video camera and the video monitor, and it generated a positionable
rectangular window that was observed on the video monitor together with
the image of the cell. Light-dark contrast at the edge of the myocyte
provided a marker for measurement of the amplitude of motion. The
amplitude of the marker was directly proportional to the dark image of
contraction, and the action was in real time. The traces of twitch
amplitude were recorded with the use of a two-channel amplifier
recorder system. The amplitude of myocyte motion remained unchanged for at least 10 min, indicating the stability of the preparation.
Measurement of [Ca2+]i. RV and LV myocytes were incubated with 5 µM fura 2-AM in Joklik solution supplemented with 1.25 mM CaCl2 for 30 min. The unincorporated dye was removed by washing the cells twice in fresh incubation solution. The loaded cells were kept at room temperature (25°C) for 30 min before measurement of [Ca2+]i to allow the fura 2-AM in the cytosol to deesterify. A low concentration of fura 2-AM at 5 µM was loaded at a relatively low temperature of 25°C to minimize the effects of compartmentalization of the esters (42).
The ventricular myocytes loaded with fura 2-AM were transferred to the stage of an inverted microscope (Nikon) in a superfusion chamber at room temperature. The inverted microscope was coupled with a dual-wavelength excitation spectrofluorometer (Photo Technical International, South Brunswick, NJ). The myocytes were superfused with a Krebs bicarbonate buffer. The myocytes selected for the study were rod shaped and quiescent with clear striations. They exhibited a synchronous contraction (twitch) in response to suprathreshold 4-ms stimuli at 0.2 Hz delivered by a stimulator (Grass S88) through two platinum field-stimulation electrodes in the bathing fluid. There is a transient rise of [Ca2+]i associated with each contraction of a cardiac muscle cell, and this is widely termed the "[Ca2+]i transient" (2). In many mammalian species, including the rat, the [Ca2+]i transient is mainly due to release of Ca2+ from the SR into the cytoplasm triggered by the Ca2+ influx via the L-type Ca2+ channel on membrane depolarization, and this rise of [Ca2+]i activates the myofilaments to slide and produce cell shortening (4). In the present study, we measured the amplitude of electrically induced [Ca2+]i transient. Fluorescent signals obtained at 340-nm (F340) and 380-nm (F380) excitation wavelengths were stored in a computer for data processing and analysis. The fluorescence ratio (F340/F380) was used to represent [Ca2+]i changes in the myocyte.Assay of cAMP.
The measurement of cAMP was performed according to the method described
previously (6). Samples that contained 3 × 106-6 × 106 freshly isolated
ventricular myocytes after 1.25 mM Ca2+ loading were
incubated in an atmosphere of 5% CO2-95% air for 2 h. Isoproterenol (0.1-10 µM), NaF (1-10 mM), or forskolin
(1-100 µM) was added and incubated for 10 min. At the end of
treatment, the cells were centrifuged for 5 s at 100 g.
The medium was aspirated, the sediment was resuspended in ice-cold
Krebs solution, and the cells were centrifuged again for 5 s at
100 g. The supernatant was aspirated. Ice-cold
ethanol-HCl (0.5 ml) was added, mixed, and left to stand for 5 min at
room temperature. The supernatant was centrifuged at 3,000 g
for 5 min and collected with a pipette. The precipitate was washed with
0.5 ml of ethanol-water (2:1), mixed, and centrifuged at 3,000 g for 5 min. The supernatant was also collected and added
together. Finally, it was evaporated to dryness at 55°C under a
stream of nitrogen. The sediment was stored at 20°C for assay of
cAMP. The pellets were neutralized in 0.1 N NaOH for protein
determination by the method of Lowry et al. (25), using
BSA as a standard.
Western blotting.
In the present study, we measured the levels of Gs and
Gi proteins in washed particulate membrane (WPM), which was
prepared according to the method of Kumar et al. (23). To
prepare WPM from isolated ventricular myocytes, the freshly isolated
myocytes were centrifuged at 100 g, and the pellet was
washed twice with a sucrose- Tris medium (0.25 M sucrose and 25 mM
Tris · HCl, pH 7.5). The washed cell pellet was
suspended and homogenized in a hypotonic membrane buffer
(that contained 20 mM Tris · HCl, pH 7.5, 5 mM
MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitor: 0.4 mM phenylmethylsulfonyl fluoride) with an Ultra Turrax
homogenizer in three bursts (5 s each) at intervals of 30 s. The
homogenate was then centrifuged at 100 g for 5 min to separate the unbroken myocytes, and the supernatant was centrifuged again at 40,000 g for 25 min. The pellet was then
washed once with the membrane buffer (50 mM Tris · HCl, and 150 mM NaCl, pH 7.4), recentrifuged, and finally suspended in the membrane
buffer before storage at 70°C for immunoblotting of specific
Gs and Gi protein isoforms. Protein was
determined by the method of Bradford (7) before using
these fractions.
Drugs and chemicals.
Fura 2-AM, type I collagenase, isoproterenol, propranolol, forskolin,
cholera toxin (CTX), phenylmethylsulfonyl fluoride, Tween 20, dithiothreitol, Tris, EDTA, and alkaline phosphatase-conjugated goat
anti-rabbit IgG were purchased from Sigma Chemical. The
[3H]cAMP assay system was purchased from Amersham
International. Chemicals for SDS-PAGE, electrophoretic transfer of
polypeptide, nonfat dry milk, and nitrocellulose membrane were
purchased from Bio-Rad. Affinity-purified G protein antibodies for
Gs-subunits and Gi were purchased from Calbiochem.
Statistical analysis. Values are expressed as means ± SE. In experiments concerning determination of twitch amplitude and [Ca2+]i, one to three myocytes from a single rat were used. The values obtained from more than one myocyte were averaged, and the mean was used as a single entity for statistical analysis. Paired Student's t-test was used to determine the difference between control and treatment groups. Unpaired Student's t-test was employed to determine the difference among groups. Significance level was set at P < 0.05.
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RESULTS |
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Effects of isoproterenol on the electrically induced contraction in
single ventricular myocytes of normoxic and chronically hypoxic rats.
Electrical stimulation triggered contraction, and the contractile
responses were significantly lower in chronically hypoxic than in normoxic rats (Fig.
1A), in keeping with
the well-established fact that the contractility is reduced after
chronic hypoxia (9, 41). Isoproterenol, a -adrenoceptor
agonist, at the range of 0.01-10 µM dose dependently increased
the electrically induced contraction in the isolated single ventricular
myocyte (Fig. 1B). This response was blocked by 1 µM
propranolol, a
-adrenoceptor antagonist (data not shown). The
response was the same in myocytes from both RV and LV (Fig.
1B).
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Effects of isoproterenol, CTX, and forskolin on the electrically
induced [Ca2+]i transient
in single ventricular myocytes of normoxic and chronically hypoxic
rats.
To correlate the contractile response to
[Ca2+]i on electrical
stimulation, we determined the electrically induced
[Ca2+]i transient in response to
-adrenoceptor stimulation in myocytes of normoxic and chronically
hypoxic rats. The resting [Ca2+]i was not
different between RV and LV myocytes isolated from hearts of both
chronically hypoxic and age-matched normoxic rats (data not shown). Nor
was the diastolic [Ca2+]i in the RV and LV
myocytes of the two types of rats different from each other (Fig.
2, A and B). The
amplitude of the [Ca2+]i transient was
significantly shorter in myocytes isolated from chronically hypoxic
rats than that from normoxic rats (Fig. 2, A and
B). The difference was 37% in RV and 32% in LV between the two types of rats.
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Effects of isoproterenol, NaF, and forskolin on cAMP accumulation
in ventricular myocytes of normoxic and chronically hypoxic rats.
To further determine whether or not the Gs protein and AC
were impaired in the heart of chronically hypoxic rats, we determined the effect of NaF, a ubiquitous stimulator of AC activity via Gs(43), and forskolin on cAMP accumulation
in the heart of both normoxic and chronically hypoxic rats. We also
determined the effect of isoproterenol. The basal production of cAMP
was not significantly changed in either RV or LV myocytes.
Isoproterenol (0.1-10 µM), NaF (1-10 mM), and forskolin
(1-100 µM) increased the cAMP accumulation in myocytes of both
normoxic and chronically hypoxic rats (Fig.
6, A-C). Like the
[Ca2+]i response, the responses to
isoproterenol, NaF, and forskolin were significantly blunted in
chronically hypoxic rats (Fig. 6, A-C). Again, the
changes were similar in the RV and LV myocytes (Fig. 6,
A-C).
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Gs protein isoforms in ventricular myocytes of
normoxic and chronically hypoxic rats.
Experiments on the [Ca2+]i response in the
present study suggested that Gs protein might be impaired
in the heart of chronically hypoxic rats. To determine whether the
Gs protein was indeed impaired after chronic hypoxia, we
measured the content of the Gs
-small (biologically
active isoform) and Gs
-large (biologically inactive isoform) in the myocytes of chronically hypoxic rats. It was found that
the density of the band of Gs
-small (45 kDa) in the
membranes of both RV and LV myocytes was significantly lower, whereas
that of the band of Gs
-large (52 kDa) was significantly
increased in the chronically hypoxic rats (Fig.
7, A-C). Like the
contractile and [Ca2+]i responses, the
changes were similar in RV and LV. The results indicated that there was
a change in the small and large isoforms of Gs protein
distribution in the hypoxic rat heart.
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Gi protein isoforms in ventricular myocytes of
normoxic and chronically hypoxic rats.
Interaction between Gs and Gi proteins is well
known (13). In this study, we measured the content of the
Gi
2, a predominant isoform in the heart
(12), in the myocytes of normoxic and chronically hypoxic
rats. It was found that the density of the band of
Gi
2 in neither RV nor LV myocyte membranes
was significantly changed in the chronically hypoxic rats (Fig.
8).
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DISCUSSION |
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The most interesting observations in the present study are
1) the stimulatory actions of CTX and NaF, which stimulate
the Gs protein, and of forskolin, which activates AC on the
electrically induced [Ca2+]i transient and
cAMP production in the ventricular myocytes, are significantly
attenuated; and 2) Gs-small, the predominant biologically active Gs
isoform in the heart, is
significantly reduced in the chronically hypoxic rats. The
observations indicate that, in chronically hypoxic rats, the reduced
responsiveness to
-adrenoceptor stimulation is due at least partly
to an impaired function of the Gs protein, which may result
from a reduction in the biologically active Gs
-small,
and AC. So, the reduced responsiveness to
-adrenoceptor stimulation
is due not only to downregulation of the
-adrenoceptor, as shown in
previous studies (19, 27, 48), but is also due to impaired
postreceptor signaling mechanisms, as shown in the present study.
In this study, we show that the biologically active isoform,
Gs-small, was reduced after chronic hypoxia. This is not
in agreement with the previous finding that there was no change in Gs
-small after hypobaric hypoxia (21). It
should be noted that the rats were subjected to isobaric hypoxia in
this study. We also showed, for the first time, that the biologically
inactive isoform, Gs
-large, was increased. Although
the changes in contents of these two isoforms are believed to be
responsible for the impaired Gs protein function, the roles
of the two isoforms need further study. The mechanism underlying the
alterations in the production of two isoforms in the heart after
chronic hypoxia also needs further study.
As expected, forskolin at 0.1-100 µM increased dose dependently the electrically induced [Ca2+]i and cAMP in the ventricular myocyte of both normoxic and chronically hypoxic rats. It is interesting to note that only to forskolin, at high concentrations (10-100 µM), were the cardiac responses attenuated in the chronically hypoxic rats. This is in agreement with the observation in the Wistar rat subjected to 5 days of hypobaric hypoxia (29) but in disagreement with the observation in the Wistar rat subjected to 21 days of hypobaric hypoxia (22). The attenuated cardiac responses to high concentrations of forskolin in hypoxic rats suggest that only in a more active state is AC function really affected by chronic hypoxia.
It has been shown that, in cardiac hypertrophy, the Gi
protein is increased (8, 17, 29). It has also been shown
that the Gi protein exerts an inhibitory influence on the
Gs protein in the heart (20). In this study,
we showed that there was no significant change in
Gi2 protein, a predominant isoform in the heart. The observation is in agreement with a previous study on rats
subjected to 30 days of hypoxia (21) but not in agreement with a previous study on rats subjected to 5 days of hypoxia
(29).
In another study in our laboratory, we observed that, after chronic
hypoxia, the cardiac responses to -opioid receptor stimulation were
attenuated, and the responses were similar in the right hypertrophied and the left nonhypertrophied ventricles (33). In this
study, we also found that the cardiac responses to
-adrenoceptor
stimulation were similar in both right hypertrophied and left
nonhypertrophied ventricles. The observations suggest that impaired
cardiac responses are most likely due to hypoxia, which induces
hypoxemia, but not necessarily secondary to cardiac hypertrophy.
In conclusion, the present study has provided evidence for the first
time that, after chronic hypoxia, which induces right heart
hypertrophy, the attenuated cardiac responses to -adrenoceptor stimulation are due to impaired functions of Gs protein and
AC. The impaired function of the Gs protein may be due to a
reduction in the biologically active Gs
-small and/or an
increase in the Gs
-large. Similar cardiac responses in
the right hypertrophied and the left nonhypertrophied ventricles are
also unequivocal evidence, suggesting that impaired cardiac responses
are due to hypoxemia, but not necessarily secondary to cardiac
hypertrophy after chronic hypoxia.
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ACKNOWLEDGEMENTS |
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We thank Y. K. Pang for taking care of the rats subjected to chronic hypoxia and C. P. Mok for assistance.
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FOOTNOTES |
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This study was supported by a grant from the Institute of Cardiovascular Sciences and Medicine, Faculty of Medicine, University of Hong Kong.
This study was performed when J. M. Pei and J. J. Zhou were on leave from the Dept. of Physiology, Fourth Military Medical University, Xi'an, China.
Address for reprint requests and other correspondence: T. M. Wong, Dept. of Physiology, Faculty of Medicine, Univ. of Hong Kong, Li Shu Fan Bldg., 5 Sassoon Road, Hong Kong, China (E-mail: wongtakm{at}hkucc.hku.hk).
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.
Received 15 November 1999; accepted in final form 15 May 2000.
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