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-adrenergic receptor function in fetal sheep
exposed to long-term high-altitude hypoxemia
Center for Perinatal Biology, Departments of Physiology and Obstetrics and Gynecology, Loma Linda University School of Medicine, Loma Linda, California 92350
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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In this study, we hypothesized that a
reduction in 
-adrenergic receptor number or a decrease in functional
coupling of the receptor to the adenylate cyclase system may be
responsible for the blunted inotropic response to isoproterenol
observed in fetal sheep exposed to high altitude (3,820 m) from 30 to
138-142 days gestation. We measured the contractile response to
increasing doses of isoproterenol and forskolin in papillary muscles
from both ventricles, estimated -adrenergic receptor density
(Bmax) and ligand affinity
(Kd) using
[125I]iodocyanopindolol,
and measured adenosine 3',5'-cyclic monophosphate (cAMP)
levels before and after maximally stimulating doses of isoproterenol
and forskolin. Left ventricular wet weight was unchanged, but right
ventricular weight was 20% lower than controls. At the highest
concentration of isoproterenol (10 µM), maximum active tension was 32 and 20% lower than controls in hypoxemic left and right ventricles,
respectively. The contractile response to forskolin was severely
attenuated in both hypoxemic ventricles.
Bmax was unchanged in the left
ventricle, but increased by 55% in the hypoxemic right ventricle.
Kd was not
different from controls in either ventricle. Basal cAMP levels were not
different from controls, but isoproterenol-stimulated and
forskolin-stimulated cAMP levels were 1.4- to 2-fold higher than
controls in both hypoxemic ventricles. The results suggest mechanisms
downstream from cAMP in the -adrenergic receptor pathway are
responsible for the attenuated contractile responses to isoproterenol.
myocardial contractility; isoproterenol; forskolin; iodocyanopindolol
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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DURING CHRONIC HYPOXIA in adults (1, 14, 20, 21),
cardiac output is reduced, although circulating levels of
catecholamines remain elevated. Previous studies have suggested that
increased parasympathetic activity (12), enhanced inactivation of
catecholamines (20), and downregulation of -adrenergic receptors and
adenylate cyclase (2, 15, 32) all play a role in the blunted response to -stimulation. Several studies indicate that left and right ventricular -receptors and adenylate cyclase activity are
differentially regulated by chronic hypoxemia. For example, in adult
rats exposed to high altitude, -adrenergic receptor density
decreased in the left ventricle after 3 wk (15) and remained decreased
after 5 wk (32). In the right ventricle, -receptor density was
unchanged after 3 wk (15), but was decreased after 5 wk (32). Basal and
isoproterenol-stimulated adenylate cyclase activity were significantly reduced in the hypertrophied right ventricle (15), but not the left
ventricle. Similar findings have been reported in newborn sheep made
chronically hypoxemic with right ventricular outflow tract obstruction
and a right-to-left shunt (2, 9, 31). Teitel et al. (31) showed a
dissociation between the chronotropic and inotropic responses to
elevated catecholamine levels. -Adrenergic receptor density and
isoproterenol-stimulated adenylate cyclase activity (2) were decreased
in the left ventricle, but were unchanged in the hypertrophied right
ventricle. The authors suggested that in newborn lambs hypoxemia
downregulates -receptors, whereas pressure overload upregulates
-receptors and causes ventricular hypertrophy. Using the same model,
Doshi et al. (9) showed that the density and affinity of atrial
-adrenergic and muscarinic receptors were unchanged, suggesting that
during chronic hypoxemia atrial and ventricular -receptors are
differentially regulated, resulting in different chronotropic and
inotropic responses.
In fetal sheep exposed to long-term high-altitude hypoxemia (16-18), baseline cardiac output was significantly reduced, owing mainly to a ~35% reduction in right ventricular output. When isoproterenol was infused in utero (18), arterial pressure fell significantly in both normoxic and hypoxemic fetuses, and heart rate increased to a similar extent in both groups. Right ventricular output increased by ~35% in both groups, but left ventricular output increased by only ~15% in hypoxemic fetuses compared with ~40% in normoxic controls, indicating a marked reduction in the left ventricular inotropic response to isoproterenol and a dissociation between the chronotropic and inotropic responses.
In our model of chronic hypoxemia, unlike the newborn lamb, plasma
catecholamine levels were not elevated (13), and both left and right
ventricles were exposed to the same levels of hypoxemia and increased
arterial pressure. However, the differences in fetal left and right
ventricular afterload sensitivity and performance (24, 25) led us to
hypothesize that hypoxemia may differentially regulate ventricular
-receptors and adenylate cyclase in fetuses in a manner similar to
that reported for the newborn lamb (2, 9). Such changes would have
important implications for the transition from intrauterine to
extrauterine life. In this study, we extended our previous observations
in utero (16-18) to isolated papillary muscles from fetuses that
were not surgically manipulated before study. We determined whether
1) hypoxemia resulted in left or
right ventricular hypertrophy, 2)
the inotropic response to isoproterenol and forskolin in isometrically
contracting papillary muscle was altered by hypoxemia,
3) the decreased inotropic response to isoproterenol in the left ventricle in utero is related to downregulation of the -adrenergic receptor/adenylate cyclase system,
4) hypoxemia differentially
regulates left and right ventricular -receptors, and
5) changes in -receptor density
and adenosine 3',5'-cyclic monophosphate (cAMP) levels
might explain the decreased cardiac performance.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Seventy-eight time-dated pregnant ewes of a mixed western breed were
obtained from Nebeker Ranch (Lancaster, CA) and randomly separated into
control and long-term hypoxemic groups. The control group
(n = 35) remained at Nebeker Ranch
(altitude ~760 m) until 138-142 days gestation. At 30 days
gestation, the long-term hypoxemic group
(n = 43) was transported to Barcroft
Laboratory, White Mountain Research Station (Bishop, CA; altitude 3,820 m, barometric pressure ~480 Torr) where they remained until
138-142 days gestation. At both locations, the ewes were kept in a
sheltered pen and were provided with alfalfa pellets, mineral
supplements, and clean water ad libitum. On the experimental day, the
ewes were transported to our laboratory at Loma Linda University where
they either underwent immediate study, or, in the case of hypoxemic
ewes awaiting study, a nonocclusive tracheal catheter was surgically
implanted so that N2 gas could be
administered to reestablish hypoxemia immediately after arrival at our
laboratory. In a previous study, maternal blood gases at high altitude
averaged PO2 = 59.1 ± 5.4 Torr,
PCO2 = 30.0 ± 2.5 Torr, and pH = 7.36 ± 0.06 (17). When maternal
PO2 was matched to the high-altitude
value by tracheal nitrogen infusion, fetal arterial
PO2 was 19.3 ± 0.8 Torr compared
with 23.3 ± 0.5 Torr in normoxic fetuses (17). Experiments were
scheduled so that fetuses were 142 days gestation on the experimental
day. The ewes were sedated intravenously with thiamylal (10 mg/kg),
intubated, and kept under deep surgical anesthesia (Halothane, 5% in
oxygen) while we delivered the fetuses through a midline laparotomy.
The fetal hearts were removed via midline thoracotomy and placed
immediately in warm (39°C), heparinized, low-calcium (0.2 mM
Ca2+) Tyrode solution
continuously bubbled with 95%
O2-5%
CO2 for contractile studies or in
ice-cold tris(hydroxymethyl)aminomethane (Tris) buffer for the
-adrenergic receptor assay.
Whole Heart and Ventricular Weights
The fetal heart was dissected free of the great vessels, and wet weights were recorded for the whole heart and the left and right ventricular free walls (Table 1). To determine percent dry weight, small sections of left and right ventricular free wall (1-3 g) were weighed, dried at ~50°C for 5 days, then reweighed. The resulting dry weights were expressed as a percentage of the wet weight.
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Contractile Force
Tissue preparation. The detailed method of muscle preparation and stimulation has been previously described (6). Briefly, four thin papillary muscle strips (0.3-1.1 mm diameter) or trabeculae carnae (0.3-0.6 mm diameter) were excised from the fetal left and right ventricles. The muscles were stretched to the length that produced maximum contractions, suspended in warm (39 ± 0.1°C), oxygenated (95% O2-5% CO2) Tyrode solution, and electrically stimulated at 1 Hz. Each contraction was recorded on an eight-channel polygraph (Gould Electronics, Cleveland, OH). Simultaneously, maximum active tension (Tmax; g) and the maximum rates of rise (+dT/dtmax; g/s) and fall (
dT/dtmax;
g/s) in tension were measured with a microcomputer (6, 8).
Isoproterenol-stimulated contractile
response. After the muscles had equilibrated (~1 h),
we recorded baseline control values, then measured the responses to
cumulative doses of isoproterenol (10
10 to
105 M), a nonselective
-adrenergic receptor agonist. The stock solution was taken directly
from 5-ml ampuls of injectable isoproterenol hydrochloride (0.2 mg/ml
Elkins-Sinn, Cherry Hill, NJ). After each addition of isoproterenol, we
allowed the muscles to stabilize (~2 min), and at the plateau of each
new steady state we recorded 20 contractions for later analysis. In
preliminary experiments, increasing the maximum concentration of
isoproterenol from 105 to
104 M did not result in any
additional increase in contractility. Contractile force consistently
reached a new plateau within ~60-90 s after the addition of
isoproterenol and remained stable for up to 15 min in both normoxic
(n = 10) and hypoxemic
(n = 10) fetuses. Some muscle strips
developed rapid (~3 Hz), spontaneous, phasic contractions of reduced
amplitude when isoproterenol concentration was increased above ~0.3
µM. Those muscles were excluded from the study. All experiments were
conducted in the dark to protect isoproterenol from photolytic
degradation.
In preliminary experiments, 10 µM propranolol was added to the bath medium after the muscles had equilibrated to determine whether endogenous catecholamines affected baseline Tmax and +dT/dtmax. After a 20-min incubation there was no significant change in baseline values in both normoxic and hypoxemic fetuses. However, 10 µM propranolol completely blocked the contractile response to 2 µM isoproterenol in both groups.
Forskolin-stimulated contractile
response. In a second set of muscles, we measured the
responses to cumulative doses of forskolin (108 to
105 M), a direct activator
of adenylate cyclase. Forskolin (5 mg vial; Calbiochem) was dissolved
in 180 µl dimethyl sulfoxide (DMSO) and diluted with distilled water
to make a 2.5 mM stock solution in 6% DMSO. After each addition of
forskolin, we allowed the muscles to stabilize (~15 min), and at the
plateau of each new steady-state we recorded 20 contractions for later
analysis. In preliminary experiments, increasing the maximum forskolin
concentration from 10 to 200 µM did not result in any additional
increase in contractile force.
-Adrenergic Receptor Assay
70°C in
sealed cryogenic vials. Samples were stored for no more than 2 mo
before assay.
On the assay day, 5 ml of frozen sample was resuspended in 10 ml
ice-cold 50 mM Tris buffer, then homogenized thoroughly with a glass
mortar and pestle before being passed through two layers of gauze to
remove cellular debris. The homogenate was diluted with an equal volume
of assay buffer (2× stock) to a final protein concentration of
1-2 mg/ml. The resulting membrane suspension contained (in mM) 50 Tris, 4 MgCl2, and 1 ascorbic
acid, pH 7.4, to which was added a cocktail of protease inhibitors in a
final concentration of 76.8 nM aprotinin, 0.83 mM benzamidine, 1 mM iodoacetamide, 1.1 µM leupeptin, 0.7 µM pepstatin-A, and 0.23 mM
phenylmethylsulfonyl fluoride (PMSF).
[125I]iodocyanopindolol
(ICYP) (New England Nuclear) was used to estimate the density of left
and right ventricular -adrenergic receptors. The assay was performed
in triplicate in tubes containing 240 µl of diluted membranes and 10 µl of increasing concentrations of the radioligand (1-1,000 pM).
Nonspecific binding was determined by adding 10 µl of
()isoproterenol (200 µM stock) to one set of assay tubes.
After a 1-h incubation in the dark at 30°C, the membranes were
collected by vacuum filtration on glass fiber filter circles G4 (Fisher
Scientific) and rinsed three times with 5 ml of ice-cold 50 mM Tris and
4 mM MgCl2, pH 7.4, to remove
unbound radiolabel. When the filters were dry, they were placed in 12 × 75 polyethylene tubes, and the radioactivity was measured in a
gamma counter (Packard Autogamma model 5650). The remaining membrane
suspension was analyzed for protein by the Lowry method (19).
cAMP Determination
Isoproterenol-stimulated cAMP production. After a 1-h equilibration in Tyrode solution (see above), actively contracting papillary muscles or trabeculae carnae from normoxic (n = 35) and hypoxemic fetuses (n = 43) were treated with a single bolus of 2 µM isoproterenol hydrochloride. When the contractile response reached its maximum (~45-60 s), the muscles were rapidly frozen by immersion in liquid nitrogen. Untreated left and right ventricular muscle strips from each fetus were also frozen as controls. The frozen muscles were placed in labeled aluminum foil pouches and stored at70°C in sealed cryogenic vials until
assay. In 10 fetuses from each group, we recorded 20 contractions at
baseline and after stimulation with isoproterenol in an attempt to
correlate contractility with cAMP levels.
Forskolin-stimulated cAMP production. After the dose-response curve to forskolin had been constructed (see above), the muscles were rapidly frozen by immersion in liquid nitrogen, then stored at -70°C in sealed cryogenic vials for later determination of cAMP levels. In separate experiments, a second group of muscles was treated with a single bolus of 10 µM forskolin, the maximum concentration used in the dose-response study. When the contractile response reached a new plateau (~10 min), the muscles were rapidly frozen by immersion in liquid nitrogen and stored as described above.
cAMP assay. On the first assay day,
the frozen muscle strips were transferred directly from the
70°C freezer to a Dewar flask filled with liquid nitrogen.
Individual muscle strips were removed from the liquid nitrogen, then
immediately homogenized in 1 ml ice-cold 6% trichloroacetic acid (TCA)
with a glass mortar and pestle. The mortar and pestles were rinsed
twice with 1 ml ice-cold 6% TCA, and the pooled 3-ml volume was
centrifuged at 2,000 g for 15 min at
4°C. The pellet fraction was used for protein determination by the
Lowry method (19). The supernatant was washed four times with 5 ml
water-saturated diethyl ether, then lyophilized (Savant Speedvac
Condenser model SVC-200H) overnight at 60°C. On assay day
2, the dried extract was resuspended
in 1 ml of the 0.05 M acetate buffer provided in Amersham's cAMP
125I-assay system (dual range)
kit. Tissue levels of cAMP were determined using the nonacetylation
assay according to the method described in the Amersham kit. A total of
332 muscle strips from 35 normoxic and 43 hypoxemic animals was
processed.
Data Analysis
Whole heart and ventricular weights. Individual fetal left and right ventricular data were pooled to calculate normoxic and hypoxemic group means.Contractile force. Baseline values recorded at the end of the 1-h equilibration were time averaged and designated the control value (100%). For each fetus, the 20 contractions recorded at each concentration of isoproterenol and forskolin were averaged, expressed as a percentage of the baseline control value, then plotted against log[agonist]. The data were fit to Hill curves using the nonlinear regression analysis algorithms in GraphPAD Prism (GraphPAD Software, San Diego, CA). Pooled data from normoxic and hypoxemic groups were used to fit the curves displayed in Figs. 1 and 3 and to determine the half-maximal effective concentration (EC50) values in Tables 3 and 4.
The 20 contractions recorded after stimulating the muscles with 2 µM isoproterenol were averaged and expressed as a percent of the baseline control value. The resulting individual fetal data were pooled to calculate normoxic and hypoxemic group means.
-Adrenergic receptor and cAMP
assays. The radioactivity measured in triplicate
samples was averaged to produce a single value for each concentration
of the radioligand. Raw counts were converted to femtomoles of
-receptor or cAMP per milligram of protein, and, for
[125I]ICYP, the
resulting data were fit to a rectangular hyperbola using the nonlinear
regression analysis algorithms in Prism. Pooled data from normoxic and
hypoxemic fetuses were used to fit the curves and to calculate
Bmax and
Kd values that
are reported in Fig. 4 and Table 5. Similar results were obtained
whether group data were curve fit or if curve fit parameters from
individual fetuses were averaged.
Statistics
The dose-response curves for isoproterenol and forskolin, and the cAMP data were analyzed in SPSS (SPSS, Chicago, IL) using doubly multivariate repeated-measures analysis of variance for a split-plot design. For all three analyses, ventricle was a within-subjects factor with two levels (left ventricle and right ventricle), and oxygen was the between-subjects factor with two levels (hypoxemia and normoxia). In their respective analyses, isoproterenol and forskolin concentrations were within-subjects factors with 21 and 6 levels, respectively. Tmax and +dT/dt were the measures. In the analysis of the cAMP data, the drug used to stimulate cAMP production was a within-subjects factor with three levels: baseline control (no drug), 2 µM isoproterenol, and 10 µM forskolin. Because the raw cAMP data were not normally distributed, log10-transformed data were analyzed. Eight hypoxemic and three normoxic fetuses were excluded from the repeated-measures analysis because of missing data. Student's t-test was used to compare normoxic and hypoxemic group means for whole heart and ventricular weights, the EC50 values for isoproterenol and forskolin, the bolus of 2 µM isoproterenol, and the Bmax and Kd values for [125I]ICYP. For all comparisons, statistical significance was set at P < 0.05. Results were expressed as means ± SE.| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Whole Heart and Ventricular Wet Weights
Whole heart and left ventricular wet weights were unchanged by long-term hypoxemia; right ventricular wet weight was decreased by ~20% in hypoxemic fetuses (Table 1). Dry weight was slightly increased in the left ventricle but not in the right ventricle of hypoxemic fetuses.Contractile Response to Isoproterenol
At the end of the 1-h equilibration period, Tmax, ±dT/dtmax, and the time course of contraction were similar in normoxic and hypoxemic fetuses as has been found previously (6); for example Tmax in the left ventricle of normoxic and hypoxemic fetuses averaged 0.790 ± 0.175 and 0.812 ± 0.070 g/mm2, respectively, and in the right ventricle it averaged 0.556 ± 0.193 and 0.557 ± 0.071 g/mm2, respectively. Isoproterenol increased contractile force in a dose-dependent manner in both normoxic and hypoxemic fetuses (Fig. 1). In normoxic fetuses, Tmax and +dT/dtmax increased about three- to fourfold in both left and right ventricles, anddT/dtmax
increased about sevenfold in the left ventricle versus about threefold
in the right ventricle (Fig. 1 and Table
2). The hypoxemic left and right ventricles
were less responsive to isoproterenol. In the hypoxemic left ventricle,
the maximum responses were reduced by ~32, ~28, and ~66% for
Tmax,
+dT/dtmax, and
dT/dtmax,
respectively (Fig. 1 and Table 2). In the hypoxemic right ventricle,
all three measures of contractility were decreased by ~20%.
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The isoproterenol dose-response curves in hypoxemic fetuses were
left-shifted compared with the corresponding curves for normoxic fetuses (Fig. 1). As a result,
EC50 of isoproterenol was
significantly lower in hypoxemic fetuses (Table
3). In the hypoxemic left ventricle, EC50 values were ~18 times lower
for Tmax and
+dT/dtmax and
~55 times lower for
dT/dtmax
than in the normoxic left ventricle. In the hypoxemic right ventricle,
the EC50 values for all three measures of contractility were about three times lower than in the
normoxic right ventricle. The normoxic left ventricle was significantly
less sensitive to isoproterenol than the normoxic right ventricle, as
indicated by the ~6- to 18-fold difference in their
EC50 values (Table 3). In
contrast, the hypoxemic left and right ventricles did not differ in
their sensitivities to isoproterenol.
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When a single bolus of 2 µM isoproterenol was used to stimulate the
muscles (for later cAMP determinations), the contractile response was
significantly reduced in both the hypoxemic left and right ventricles
(Fig. 2).
Tmax and
±dT/dtmax
were reduced by ~30 and ~48%, respectively, in the hypoxemic left
ventricle. Similarly, Tmax and
+dT/dtmax were
reduced by ~30% in the hypoxemic right ventricle but
dT/dtmax
was unchanged.
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Contractile Response to Forskolin
Forskolin increased contractility in a dose-dependent manner in normoxic fetuses (Fig. 3). In both the left and right ventricles, Tmax and ±dT/dtmax increased about two- to fourfold over baseline values. The left ventricle was more sensitive to forskolin than the right ventricle as indicated by the lower EC50 values in Table 4. In hypoxemic fetuses, the contractile response to forskolin was severely blunted, even when the forskolin concentration was increased to 200 µM (data not shown). Because the response was not sigmoidal, meaningful maximum response and EC50 values could not be determined.
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-Adrenergic Receptors
-adrenergic Bmax was ~33% higher in the left ventricle than in the right
ventricle. In hypoxemic fetuses, there was no difference in
-receptor density between left and right ventricles.
Exposure to long-term hypoxemia did not change -receptor
density in the left ventricle. However, there was an ~55% increase
in -receptor density in the hypoxemic right ventricle, indicating
hypoxemic upregulation of right ventricular -receptors. There was no
difference in ligand
Kd between left and right ventricles or between normoxic and hypoxemic fetuses.
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cAMP Levels
Figure 5 shows the results from the cAMP assays. Basal unstimulated cAMP levels were not significantly different in normoxic and hypoxemic fetuses or between left and right ventricles. When 2 µM isoproterenol was used to activate-receptor-coupled adenylate cyclase, cAMP concentration increased significantly in both normoxic and hypoxemic fetuses. There were no significant differences between left and right ventricles in either group. However, the increases in
cAMP were ~1.8- and ~1.4-fold higher in hypoxemic left and right
ventricles, respectively, than in the corresponding normoxic ventricle
(Fig. 5). When adenylate cyclase was directly activated with 10 µM
forskolin, a similar pattern emerged. Forskolin-stimulated cAMP levels
were about fivefold higher than basal unstimulated levels in hypoxemic
fetuses, but only about threefold higher than basal unstimulated levels
in normoxic fetuses (Fig. 5). There were no significant differences
between left and right ventricles in either group.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Whole Heart and Ventricular Weights
Several investigators have reported right ventricular hypertrophy after exposure to chronic hypoxemia in adult (15, 32) and newborn (2) mammals, probably mainly due to increased pulmonary artery or intraventricular pressure. We found that right ventricular hypertrophy did not occur in fetal sheep exposed to long-term hypoxemia. Instead, the mass of the right ventricular free wall decreased by ~20% without any compensatory change in the left ventricle. The decrease in right ventricular free wall mass may, in part, help to explain the reduced right ventricular performance previously reported by our laboratory (16-18). Differences in the period of hypoxemic exposure, in pulmonary artery pressure and in the morphology and composition of the fetal myocardium versus the newborn myocardium may account for the differences between our results and those of Bernstein et al. (2). In their study, newborn lambs were exposed to 2 wk of hypoxemia beginning during the first and second weeks of postnatal life; our fetuses were exposed to hypoxemia during days 30-142 of gestation, a period during which myocardial cells are actively dividing (5, 29, 30). During the last week of fetal life and the first week of neonatal life, mitotic activity tapers off and myocyte hypertrophy, especially in the left ventricle, becomes the dominant mechanism for myocardial growth (5, 29, 30). In rats, chronic hypoxemia during fetal life results in marked hyperplasia and delayed transition from hyperplastic to hypertrophic growth in the right ventricle but not in the left ventricle (22). Whether a similar mechanism may be at work in chronically hypoxemic fetal sheep is not known.Contractile Responses to Isoproterenol and Forskolin
In adult and newborn mammals, several investigators have shown that prolonged exposure to elevated catecholamine levels during acute (26) and chronic hypoxemia (1, 2, 12, 15, 18, 21), heart failure (4, 11), and pressure overload (2, 28, 32) results in a marked decrease in myocardial responsiveness to-stimulation. This may, in part, be
explained by downregulation of surface -receptors (2, 9, 15, 32) and
decreased adenylate cyclase activity (2, 15, 32, 33). Recently, we
reported (18) a marked decrease in the left ventricular inotropic response to isoproterenol in fetal sheep exposed to long-term high-altitude hypoxemia. In this study, we hypothesized that the reduced inotropic response to isoproterenol in vivo may be due to
downregulation of the -adrenergic receptor/adenylate cyclase system.
There were several important findings in this study. First,
isoproterenol was a potent positive inotropic agent in both normoxic and hypoxemic fetuses. However, in the hypoxemic left and right ventricles, the inotropic response to isoproterenol was markedly attenuated (Figs. 1 and 2 and Table 2), but sensitivity was greatly increased (Table 3). The changes were more pronounced in the hypoxemic
left ventricle, particularly for
dT/dtmax;
inotropic responsiveness decreased by 66%, but sensitivity increased
55-fold. These results agree with the decreased left ventricular
inotropic response to isoproterenol in vivo (18). However, unlike the results in this study, the right ventricular response in vivo was not
different from normoxic controls. This inconsistency may be the result
of a key difference in methodology. In this study, afterload remained
constant (isometric contraction), but arterial pressure fell
significantly during the isoproterenol infusion in vivo, thereby
reducing afterload and improving stroke volume. Because the right
ventricle is quite sensitive to changes in afterload (17, 24, 25), the
reduction in arterial pressure may have been sufficient to compensate
for an underlying decrease in responsiveness to isoproterenol.
The pattern of the inotropic response to isoproterenol in hypoxemic
fetuses is similar to the inotropic response to extracellular calcium
previously reported (6). In both studies, inotropic responsiveness was
markedly attenuated, but sensitivity was greatly increased. In both
studies, the changes were of a similar magnitude and were more
pronounced in the left ventricle, particularly for dT/dtmax
(see Table 1 and Figs. 1 and 2 in Ref. 6). Because the inotropic
response to isoproterenol depends on calcium delivery, reuptake, and
changes in myofilament sensitivity to calcium, we hypothesize that the
changes in the inotropic response to calcium and isoproterenol in
hypoxemic fetuses are linked by a common, as yet unexplored, mechanism.
A second finding was that forskolin had a potent positive inotropic effect on the normoxic fetal heart. Overall, the magnitude of the contractile response was similar to that observed for isoproterenol. However, isoproterenol was a potent inotrope when maximal inotropic response and drug sensitivity were compared (see Figs. 1-3 and Tables 2-4). In addition, the response to isoproterenol reached a new plateau within 60-90 s, whereas the response to forskolin required ~10 min to reach a new steady state. The difference in the rate of activation may, in part, be explained by the fact that isoproterenol binds to surface receptors, whereas forskolin has to diffuse across the cell membrane to activate adenylate cyclase. These results agree with previous observations on the relative potencies of isoproterenol, isoprenaline, and forskolin in human (3) and rat (10) hearts.
In hypoxemic fetuses, the inotropic response to forskolin was even more
severely attenuated than the inotropic response to isoproterenol (Fig.
3). In several hypoxemic fetuses, contractility decreased below
baseline values (Fig. 3), suggesting that forskolin may have been
toxic, especially in the right ventricle. Alternatively, forskolin
activates all adenylate cyclases, whereas isoproterenol activates only
the subset of adenylate cyclases that are coupled to the -adrenergic
receptor and to the contractile response (3, 10, 27). Thus the
contractile responses to isoproterenol and forskolin could, in theory,
differ considerably depending on the activity of A-kinase and the
phosphorylation states of membrane and myofilament effector proteins.
In fact, in the isolated perfused rat heart, England and Shahid (10)
showed that isoprenaline was a more potent inotrope than forskolin on a
molar basis and that A-kinase activity, phosphorylase a
content, and the levels of phosphorylated troponin-I and C protein were
much higher after stimulation with isoprenaline than with forskolin,
although forskolin-stimulated cAMP levels were much higher than
isoprenaline-stimulated cAMP levels. They concluded that much of the
cAMP produced by stimulation with forskolin was unavailable to the
A-kinases that are involved in the contractile response because of
compartmentation. It is not clear why these differences may be more
apparent in hypoxemic fetuses than in normoxic fetuses. We speculate
that A-kinase activity and/or the phosphorylation states of
target effector proteins and, hence, their physiological activity may
be reduced after exposure to long-term high-altitude hypoxemia.
-Adrenergic Receptor Density and Agonist Affinity
-adrenergic receptors. Instead, there was a 55% increase in right
ventricular -adrenergic receptor density in hypoxemic fetuses, but
no change in the left ventricle. In addition, there were no changes in
the affinity for
[125I]ICYP in either
ventricle. In normoxic fetuses, the
Bmax for [125I]ICYP in our
study was higher than that previously reported for late-term fetuses
(7, 23) but lower than that reported for normoxic newborn lambs (2). In
our study, normoxic fetal left and right ventricular -receptor
density was ~45 and ~55%, respectively, of that reported for the
2- to 3-wk-old newborn, suggesting that there is an age-related
increase in -receptor density during the early neonatal period.
Hypoxemic fetal left and right ventricular -receptor density was
similar to that reported for chronically hypoxemic newborn lambs (2).
Our results indicate that the attenuated inotropic response to
isoproterenol was not due to downregulation of left and right
ventricular -adrenergic receptors. It is possible that
downregulation did not occur because catecholamine levels were not
chronically elevated in hypoxemic fetuses (13). Alternatively, in
chronically hypoxemic newborn lambs, Bernstein et al. (2) showed that
-receptor density decreased in the left ventricle (exposed to
hypoxemia alone) but did not change in the right ventricle (exposed to
hypoxemia and pressure overload). They suggested that hypoxemia
downregulates -receptors, whereas pressure overload upregulates the
receptor, and concluded that the presence of both factors in the right
ventricle resulted in no change in -adrenergic receptors. In the
fetus, both left and right ventricles were exposed to the combined
effects of hypoxemia and increased arterial pressure. Because the fetal
right ventricle is more sensitive to afterload (16, 17, 24, 25), the
elevated arterial pressure may have had more of an upregulating effect on -receptor density in the right ventricle than in the left ventricle.
cAMP Levels
The fourth major finding in our study was that both isoproterenol-stimulated and forskolin-stimulated cAMP levels were significantly higher in hypoxemic fetuses, but basal unstimulated cAMP levels did not differ from normoxic controls. However, the inotropic responses to isoproterenol and forskolin were significantly higher in normoxic fetuses. Together, these results have several important implications. These data show that the attenuated inotropic responses to isoproterenol and forskolin were not due to attenuated cAMP responses to-adrenergic receptor stimulation. These results strongly suggest that hypoxemia acts downstream of second-messenger production in the signal transduction cascade coupled to -adrenergic receptors. Thus A-kinase, sarcolemmal L-type
Ca2+ channels, the ryanodine
receptor, phospholamban, troponin-I, and C protein are all
possible sites for the effects of hypoxemia. In addition, hypoxemia
could, theoretically, act at the level of the myofilaments, changing
the amount of contractile protein, its calcium affinity, ATPase
activity, and the rate of cross-bridge cycling.
Forskolin-stimulated cAMP levels were significantly higher than isoproterenol-stimulated cAMP levels in both normoxic and hypoxemic fetuses. However, the inotropic response to isoproterenol was greater, particularly in hypoxemic fetuses. These results are consistent with the observations of England and Shahid (10), discussed above, and suggest that cAMP may be compartmentalized in the ovine fetal heart. Furthermore, our data suggest that although long-term hypoxemia increased adenylate cyclase activity, proportionately less of the forskolin-stimulated cAMP was available to A-kinases coupled to the contractile response in hypoxemic fetuses than in normoxic fetuses. This suggests that there are other cAMP-dependent pathways whose activity may have been upregulated by exposure to long-term hypoxemia.
Differential Regulation of Left and Right Ventricles
In normoxic fetuses, there was differential sensitivity to-stimulation in the left and right ventricles. The right ventricle was significantly more sensitive to isoproterenol, as indicated by the
lower EC50 values for all three
measures of contractility (Table 3), although -receptor density was
~25% lower than in the left ventricle (Fig. 4 and Table 5). At the
same time, the right ventricle was less sensitive to forskolin, as
indicated by the higher EC50
values for all three measures of contractility (Table 4). Because
isoproterenol-stimulated and forskolin-stimulated cAMP levels and the
maximum inotropic response to forskolin (Fig. 3) were not different
between the left and right ventricles, it is likely that the difference
in sensitivity exists at the level of A-kinase and/or the
effector proteins.
At a maximally stimulating dose of isoproterenol,
dT/dtmax
was ~2.4 times higher in the left ventricle, but
Tmax and
+dT/dtmax were
not significantly different between the two ventricles (Fig. 1 and
Table 2). It is not clear why the maximum rate of relaxation (dT/dtmax)
was higher in the left ventricle. Physiologically, the rate of
relaxation is related to the phosphorylation states of troponin-I and C
protein, phospholamban, and the geometric arrangement of muscle fibers.
It is not clear what mechanism is responsible for the higher rate of
relaxation in the left ventricle.
In hypoxemic fetuses, there were no differences in the inotropic
responsiveness or sensitivity to isoproterenol between the left and
right ventricles. However, the total number of -receptors was higher
in the left ventricular free wall (~85 vs. 73 pmol), although the
density (Fig. 4 and Table 5) was somewhat higher in the right
ventricle. In addition, both isoproterenol-stimulated and
forskolin-stimulated cAMP levels were significantly higher (P < 0.01) in the hypoxemic left
ventricle (Fig. 5), indicating differential regulation of adenylate
cyclase.
In conclusion, there is marked attenuation of the positive inotropic
responses to isoproterenol and forskolin and a significant increase in
sensitivity of the inotropic response to isoproterenol in fetal sheep
exposed to high altitude during days
30-142 of gestation. The decrease
in inotropic responsiveness to -stimulation is not due to
downregulation of myocardial -adrenergic receptors or to
decreased adenylate cyclase activity. Our results strongly suggest that
hypoxemia acts downstream of the second messenger, cAMP, possibly by
decreasing A-kinase activity and/or the functional states of
key effector proteins, including the sarcolemmal L-type Ca2+ channel, the ryanodine
receptor, troponin-I, C protein, and phospholamban. The changes in
inotropy and cAMP levels are consistent with our previous report (6),
which suggested that calcium influx and/or storage and release
from the sarcoplasmic reticulum may be decreased. Alternatively,
phosphodiesterase activity and phosphatase activity may be altered by
long-term hypoxemia. These changes may represent cardioprotective
adaptations that limit metabolic demand by reducing contractility.
Further studies are needed to examine phosphodiesterase, A-kinase and phosphatase activities, and the function of key
effector proteins and to directly assess the calcium current and
intracellular calcium transient.
| |
ACKNOWLEDGEMENTS |
|---|
The authors gratefully acknowledge Al VanVarick, David Trydahl, and the White Mt. Research Station staff for expert technical assistance.
| |
FOOTNOTES |
|---|
This work was supported by National Institute of Child Health and Human Development Grants HD-22190 and HD-03807.
Address reprint requests to R. D. Gilbert.
Received 7 April 1997; accepted in final form 24 August 1997.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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) and chronically hypoxemic (
) fetuses.
Tmax, maximum active tension;
+dT/dtmax,
maximum rate of rise in tension;
