Departments of 1 Pharmacology and 2 Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130
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ABSTRACT |
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Basal contractility
and responses to -adrenoceptor activation are compromised in hearts
from rats with chronic portal vein stenosis. Here we report the effect
of partial ligation of the portal vein on myocardial G protein
expression,
-adrenoceptor-G protein coupling, and
excitation-contraction coupling (ECC). Contractility (dT/dt) was reduced 30-50% in right and left
ventricles, but the rate of relaxation (
dT/dt)
was unaffected. Isoproterenol-induced positive inotropism was
diminished, but there was no difference in ED50. The
concentration-dependent increase in
dT/dt was
unaffected. Gs
and Gi
expression, cholera
toxin- and pertussis toxin-induced ADP-ribosylation, and formation of
the agonist-receptor-Gs complex were unaffected by portal
vein stenosis. Of the components of ECC examined, the
caffeine-sensitive sarcoplasmic reticulum Ca2+ pool was
reduced 35%, although the Ca2+ uptake and release
processes were unchanged; the apparent density of L-type
Ca2+ channels decreased 60% with no change in affinity;
the dihydropyridine Ca2+ channel agonist BAY K 8644 produced relative changes in dT/dt that were similar in
both groups, suggesting normal function in the remaining
Ca2+ channels; and Na+/Ca2+
exchange was reduced 50% in the portal vein stenosis group. These data
suggest that the effect of portal vein stenosis on the myocardium is
the result of alterations to ECC.
isradipine; BAY K 8644; portal hypertension; -adrenoceptor; isoproterenol; G protein; heart; sarcoplasmic reticulum; sodium/calcium
exchange
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INTRODUCTION |
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MARKED HEMODYNAMIC CHANGES occur in humans and experimental animals with cirrhotic liver disease. A hyperdynamic circulation develops that is characterized by increased cardiac output, increased splanchnic blood flow, low total systemic vascular resistance, mild tachycardia, low or normal blood pressure, an increased blood volume, portal hypertension, and reduced responsiveness to vasoconstrictors. These changes can occur whether the disorder is the result of cirrhosis (27, 32, 49), prehepatic portal obstruction (9, 19, 27, 35, 59), or portocaval shunting (18, 33, 58).
Ventricular dilatation, especially of the right heart, and hypertrophy, especially in early cirrhosis, occur in human hearts (43). In animal studies (20), the heart weight in CCl4-induced cirrhotic rats is greater than that in controls, presumably because of chronic ventricular overload. There are no cardiac histological abnormalities in these animals (20). Similarly, Ma and colleagues (45) reported no light microscopic changes in the hearts of bile duct ligation-induced cirrhotic rats. However, these observations do not duplicate what is seen in human cirrhotic patients, most likely because of the long duration of cirrhosis in patients compared with the relatively brief time the experimental animals are cirrhotic. In the portal vein stenosis model, no change in either right or left heart size occurs during the brief interval of 10-12 days postsurgery (Battarbee and Zavecz, unpublished observations), suggesting that either hypertrophy does not occur or impairment is of insufficient duration to result in measurable hypertrophy.
In addition to altered basal hemodynamics, liver disease also disrupts dynamic function. Tilt tests, lower body negative pressure, and other hypotensigenic techniques indicate that both cardiac and peripheral resistance reflex responses are impaired in hepatic disease patients (11, 12, 41, 42) and in experimental portal hypertension (6). Furthermore, cirrhotic patients (1, 8, 25, 34, 38, 46, 54), experimental models of cirrhosis (13, 36), prehepatic portal hypertensive models (6, 7), and cholestatic animals (13-15) exhibit reduced cardiac responses to exogenous and endogenous catecholamines. Studies (26, 43) both in human nonalcoholic cirrhosis and in animal models of nonalcoholic cirrhosis have demonstrated impairment of cardiac contractility in response to various stressors. This development of high-output heart failure with systemic vasodilation has been termed "cirrhotic cardiomyopathy." Although the ventricular dysfunction coexisting with a high cardiac output secondary to reduced peripheral vascular resistance is common in cirrhotic patients (38, 43), the symptoms are usually latent, only appearing under conditions that stress the myocardium, including liver transplantation, surgical portosystemic shunting, transjugular intrahepatic portosystemic stent shunts, mental stress, physical exercise, and pharmacological stimulation (26, 43). Interestingly, portocaval shunting, not hepatocellular disease, is the common factor in the hepatic models used to study the cardiovascular effects of liver disease. Thus it appears that the hyperdynamic circulation results from the shunting of visceral venous blood into the systemic circulation (for an in-depth discussion of cirrhotic cardiomyopathy, see Ref. 43).
In our previous studies (7, 65) with the portal vein-stenosed rat, we
dissociated hepatocellular dysfunction from the effect of portal
hypertension using the chronic portal vein-stenosed rat, a model of
chronic liver disease in which a hyperdynamic circulation is present
without the hepatocellular damage of cirrhosis. We have
observed depressed contractile function in isolated right and left
ventricular tissue from chronic portal vein-stenosed rats as well as
decreased isoproterenol-induced positive inotropism. -Adrenoceptors
have been shown not to be downregulated in this model, but a greater
fraction of cardiac
-adrenoceptors must be occupied to produce
equivalent absolute increases in dT/dt, the maximum
rate of tension development (65), suggesting that the cause of the
decreased response to isoproterenol is postreceptor. On the other hand,
it is conceivable that the altered responsiveness to
-adrenergic
receptor activation results from a mechanism not directly involving
-adrenergic signal transduction. In the present study, we
have extended our research to include the effects of portal
hypertension with portosystemic shunting on cardiac Gs
and Gi
expression,
-adrenoceptor-Gs
coupling, and excitation-contraction coupling (ECC).
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METHODS |
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Contractile experiments.
Ten to twelve days after portal vein stenosis or sham operation, each
animal was decapitated and the heart was immediately excised and
transferred to a preparative tissue bath containing Krebs-Henseleit
solution (KH) equilibrated with 95% O2-5%
CO2. The buffer contained (in mM) 118 NaCl, 5.8 KCl, 27.2 NaHCO3, 1.0 NaH2PO4, 1.2 MgSO4, 2.5 CaCl2, and 11.1 glucose. The
temperature of the buffer was maintained at 37°C and the pH at 7.4. A small strip of right ventricle was removed, and a left ventricular
papillary muscle was dissected free. The remainder of the heart was
immediately frozen in liquid nitrogen and stored at 70°C for
use in the binding experiments (see below). One end of each muscle was
attached to a rigid support and then placed in an organ bath, where the
other end of the muscle was attached to a Grass FT03C
force-displacement transducer via a length of surgical silk. Muscles
were field stimulated at 2 Hz, with a voltage 50% greater than
threshold and a pulse duration of 4 ms. Resting length was set to the
peak of each muscle's length-tension curve. Isometric tension was
recorded by means of the force-displacement transducer connected to a
Grass model 7D recorder. dT/dt and the maximum rate of
relaxation (
dT/dt) were obtained by
differentiating the output of the channel measuring isometric force. At
the end of each experiment, a graticule was used to measure each
muscle. Tissues were dried and their weights recorded. These
measurements were used to normalize the data for differences in tissue
weight and dimensions.
Experiments in permeabilized muscle fibers. To determine the effect of portal vein stenosis on the Ca2+ sensitivity of the myofilaments and Ca2+ uptake and release from the sarcoplasmic reticulum (SR), left ventricular papillary muscles were chemically skinned with either saponin or Triton X-100 to permeabilize the sarcolemma while leaving the SR intact or to permeabilize both the sarcolemma and the SR membrane, respectively. In this way, the "intracellular" cytosolic concentration of Ca2+ can be controlled and manipulated. The solutions used in these experiments were prepared according to a computer program that takes into account the binding constants of all of the constituents (51). Papillary muscles were removed from left ventricles, and 100- to 150-µm-diameter fiber bundles, 1-2 mm in length, were dissected free in relaxing solution containing 5 mM MgATP, 1 mM Mg2+, 5 mM EGTA, 20 mM imidazole, 15 mM creatine phosphate, and potassium methanesulfonate to yield an ionic strength of 200 mM with pCa > 8.5 adjusted to pH 7.0. One end of each bundle was tied with a human hair to a force transducer (Kent Scientific model TRN001) and the other to a support that positioned the fibers horizontally in a 3-ml tissue bath. The fibers were stretched to a point at which passive tension was just measurable (~0.05 mN). The output of the transducer was digitized and stored on disk for subsequent analysis. All experiments were performed at room temperature. To assess the effect of portal vein stenosis on the sensitivity of the myofilaments to Ca2+, the fiber bundles were permeabilized by superfusion (2 ml/min) with oxygenated relaxing solution containing 0.5% Triton X-100 for 1 h. This treatment permeabilizes both the sarcoplasmic reticular and sarcolemmal membranes (24, 66). Fibers were superfused with relaxing solution for an additional 60 min before exposure to Ca2+. In the experiments that examined the effect of portal vein stenosis on Ca2+ uptake and release from the SR, fibers were chemically permeabilized by superfusion with 50 µg/ml saponin for 30 min to permeabilize the sarcolemma only (24, 66). Saponin was washed out of the tissues, which were then superfused for an additional 30 min in relaxing solution before the start of the experiment.
Myofilament Ca2+ sensitivity protocol. After permeabilization with Triton X-100, fibers were checked for the presence of functional SR by exposure to 50 mM caffeine. Preparations that responded to caffeine were discarded. pCa-force curves were generated by sequential application of solutions containing 1 mM MgATP, 1 mM Mg2+, 5 mM EGTA, and 20 mM imidazole, potassium methanesulfonate to yield an ionic strength of 200 mM, and Ca2+ (as CaCl2) to achieve the desired pCa. pH was adjusted to 7.0 at room temperature. The data from each group were fit to the Hill equation, and the half-maximum pCa and Hill coefficient (nH) were determined as indexes of myofilament sensitivity and cooperativity, respectively (17).
SR Ca2+ uptake and release. The effect of portal vein stenosis on the uptake and release of Ca2+ by the SR was investigated in permeabilized left ventricular papillary muscle fiber bundles using the ability of high concentrations of caffeine to induce Ca2+ release through the SR Ca2+-release channels. The magnitude of the contracture induced by 30-50 mM caffeine was the index of SR Ca2+ content and release.
In addition to relaxing solution (ionic strength, 150 mM), the following solutions were utilized with the permeabilized fibers: 1) loading solution that contained relaxing solution with the addition of pCa 6.0; and 2) low-EGTA solution made up of relaxing solution with 0.1 mM Mg2+ (lowered from 1 mM to increase myofilament Ca2+ sensitivity and to reduce Mg2+ inhibition of the SR Ca2+ release channels) and 0.05 mM EGTA. After skinning for 20-30 min with 50 µg/ml saponin in relaxing solution to remove the sarcolemma and leave the SR intact, any residual Ca2+ in the SR was released by the application of 50 mM caffeine (in low-EGTA solution). After 1 min, the tissues were washed with relaxing solution for 2 min. The bath solution was changed to the Ca2+ loading solution, and the SR was permitted to take up Ca2+ (pCa 6.0) for 3 min, after which the tissues were washed with low-EGTA solution for 1 min. The Ca2+ content of the SR was then estimated by exposing the fibers to 50 mM caffeine in low-EGTA solution and measuring the consequent contraction. The tissues were washed with low-EGTA solution until force returned to baseline, when the tissue was exposed to caffeine a second time. This second caffeine exposure was used to confirm that all of the caffeine-sensitive Ca2+ had been emptied from the SR (see Fig. 7A). These procedures ensured that loading the SR with Ca2+ always occurred under the same conditions, i.e., the experiments would not be subject to vagaries associated with partially loaded SR before the Ca2+ loading period, and that differences in the response to caffeine between sham-operated and portal vein-stenosed rats were not the result of a defect in uptake rather than release. Loading and subsequent Ca2+ release by caffeine was performed three times in each muscle with identical results each time, indicating that Ca2+ uptake by the SR under these conditions was consistent and reproducible (not shown). Caffeine-induced Ca2+ release from the SR was also examined in intact fibers, i.e., unpermeabilized fibers. Tissues were removed from the animal, placed into tissue baths, and paced as described above with the exception that the stimulation frequency was 0.5 Hz. On stabilization of contractions, electrical pacing was terminated, and 5 min later tissues were exposed to 30 mM caffeine. Once the contracture began to relax, the KH with caffeine was washed out with normal KH, and the tissues were allowed to rest for 30 min with frequent exchanges of KH. The bath solution was then changed to Ca2+-free KH buffer containing 2 mM EGTA (Ca2+-free solution with normal Na+) for 5 min and then exposed to 30 mM caffeine. Ca2+-free solution with normal Na+ stimulates Na+/Ca2+ exchange at rest (4), and in this manner the changes in the response to caffeine in normal KH were examined to determine whether this results from changes in Na+/Ca2+ exchange induced by portal vein stenosis or increased Ca2+ leak from the SR during rest.Determination of dihydropyridine receptor density.
The effect of portal vein stenosis on the density of dihydropyridine
receptors located on L-type Ca2+ channels was assessed
using equilibrium binding of the dihydropyridine antagonist
[3H]isradipine. Rat cardiac membranes were
prepared according to the procedure described by Ebersole et al. (23).
Hearts were weighed, minced with scissors, and suspended in 2 vol of
50 mM Tris · HCl (pH 7.7 at 25°C) at
4°C. Tissues were homogenized twice with a Polytron homogenizer for
10 s at a setting of 6. The homogenate was centrifuged at 24,000 g for 10 min. The pellet was resuspended in 3 vol of
Tris · HCl and centrifuged at 24,000 g for 10 min. This process was repeated three additional times. The final pellet was suspended in 4 vol of Tris · HCl (1 g original
weight to 4 ml buffer). Membrane preparations were stored at
70°C for no longer than 3 days before use. Membranes were
thawed and diluted 1:9 with Tris · HCl buffer for use
in the binding assays. Protein content was determined as described by
Lowry et al. (39).
-Adrenoceptor-G protein coupling.
When Gs binds to the agonist-
-adrenoceptor complex,
forming an agonist-
-adrenoceptor-Gs complex, the agonist
is bound with higher affinity than when it is bound to the receptor
only. The fraction of
-adrenoceptors in this "high-affinity"
state is indicative of the coupling of the agonist-receptor complex
with Gs. The fraction of
-adrenoceptors in the
high-affinity state and the apparent dissociation constant of an
agonist for the high-affinity state were determined as described
previously (52, 53). Competition binding experiments were
performed using cardiac membranes prepared as previously described
(65). Left ventricles were minced finely and homogenized with a
Polytron PT10 homogenizer (twice at half-speed for 5 s) in 35 ml of
ice-cold buffer containing 5 mM Tris · HCl (pH 7.4),
1 mM MgCl2, 0.25 M sucrose, 1 mM EDTA, and 1 mM
dithiothreitol (DTT). The homogenate was filtered through three layers
of cheesecloth and centrifuged at 12,000 g for 10 min. The
supernatant was centrifuged at 45,500 g for 25 min,
resuspended, and recentrifuged twice. The final pellet was suspended in
2 ml of buffer (50 mM Tris · HCl, pH 7.4, 10 mM
MgCl2, 1 mM EDTA, and 1 mM DTT). A 100-µl aliquot was
removed for protein determination, and the remainder was stored in
liquid nitrogen until used. Approximately 90% of all preparations were
used within 48 h, and the remainder were used within 1 wk of freezing.
Binding assays were performed at 37°C for 30 min in a total volume
of 0.5 ml containing 50 mM Tris · HCl (pH 7.4), 5 mM
MgCl2, 1 mM ascorbic acid, 50 µg of protein, 25-50
pM [125I]iodopindolol (46 Ci/mmol; Amersham
Life Sciences, Arlington Heights, IL), and 15 different concentrations
of isoproterenol (10
11-10
4M)
with or without the addition of 2 mM guanylylimidodiphosphate. Nonspecific binding, which amounted to 10-15% of total binding, was determined by the addition of 100 µM (
)isoproterenol to
appropriate incubation vessels. Binding of
[125I]iodopindolol to
-adrenoceptors was
assessed by filtration (52, 65). Assays were performed in duplicate.
The curves obtained by plotting
[125I]iodopindolol bound vs. the log of the
isoproterenol concentration were analyzed by nonlinear regression (22),
and IC50 values for isoproterenol and the percentage bound
to high- and low-affinity states were determined.
KH and KL, the apparent
Kd for isoproterenol binding to the high- and
low-affinity states, respectively, were calculated by the method of
Cheng and Prusoff (21).
G protein separation and immunoblotting.
G protein expression was assessed using the Western blot technique.
Cardiac membranes containing 50 or 100 µg of protein were mixed with
an equal volume of a buffer containing 0.125 M
Tris · HCl, pH 6.8, 4% SDS, 20% glycerol, and 10%
2-mercaptoethanol (treatment buffer), heated at 95°C for 2 min, and
subjected to SDS-PAGE, 9% acrylamide in 6 M urea (55). After
electrophoresis, proteins were transferred onto Immobilon-P membranes
(Millipore, Bedford, MA) for immunoblotting. After protein transfer,
Immobilon-P membranes were incubated in TBS (20 mM Tris, pH 7.6, and
137 mM NaCl) containing 0.1% Tween plus 5% milk for 2 h at room
temperature with gentle shaking. The membranes were then incubated
overnight with primary antisera (1:1,000 dilution in the same buffer).
Antisera used were RM/1 (directed against carboxy-terminal decapeptide sequences of Gs), AS/7 (directed against
carboxy-terminal decapeptide sequences of G
i-1 and
G
i-2), G
i-1 (directed against the
internal amino acid sequence 159-168 and does not cross react with
G
i-2), EC/2 (directed against carboxy-terminal
decapeptide sequences of G
i-3), and GC/2 (directed
against amino-terminal decapeptide sequences of Go
) (60,
61). Secondary antibody coupled to horseradish peroxidase was used for
detection of proteins by enhanced chemiluminescence (ECL Western blot
system, Amersham). Immunoreactive bands on X-ray films were digitized
using the gel documentation system (GDS 7500 from UVP) and quantified
using the GelBase software program (UVP) on an IBM computer.
ADP ribosylation of G proteins.
For pertussis toxin (PTX)-catalyzed ADP-ribosylation experiments,
membrane protein (100 µg) was incubated with a mixture containing 50 mM Tris · HCl (pH 8.0), 0.25% Lubrol, 20 mM
thymidine, 1 mM ATP, 5 µM GTP, 20 mM arginine, 50 mM NaCl, 4 µM
MgCl2, 100 mM DTT, 1 µg of PTX, and 3-5 µCi
[-32P]NAD in a final volume of 0.1 ml. The
mixtures were incubated for 2 h at 30°C, after which 1 ml ice-cold
acetone was added and samples were centrifuged for 10 min at 10,000 rpm
in a Microfuge at 4°C.
Chemicals.
Caffeine, isoproterenol, EGTA, creatine phosphate, and creatine
phosphokinase were purchased from Sigma Chemical (St. Louis, MO).
Nitrendipine and BAY K 8644 were purchased from Research Biochemicals
International (Natick, MA). RM/1, AS/7, EC/2, and GC/2 antisera used in
the immunoblots were purchased from DuPont NEN. Gi-1
antiserum was purchased from Calbiochem (San Diego, CA). Other reagents
used for immunoblotting G proteins were the best grade available.
Statistics. Because there were only two groups of animals, the appropriate univariate t-test was used to evaluate the data. P < 0.05 was considered significant.
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RESULTS |
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Basal contractile function.
Table 1 summarizes the effect of 10-12
days of portal vein stenosis on basal developed tension,
dT/dt, and dT/dt in left ventricular papillary muscles and right ventricular strips. Developed tension and dT/dt were decreased 30-46% and
28-50%, respectively. Portal vein stenosis had no significant
effect on the rate of relaxation.
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-Adrenoceptors.
Although the absolute increase in contractility induced by
isoproterenol was markedly decreased by portal vein stenosis (Fig. 1A), there was no difference in
EC50 (sham, 28.8 ± 2.8 nM; stenosed, 25.0 ± 2.7 nM).
Figure 1B demonstrates that the relative change in
dT/dt was not different between the sham-operated and
portal vein-stenosed groups. Similar results were observed in both left and right ventricular tissue (right ventricle not shown). The effect of
portal vein stenosis on the enhancement of
dT/dt
by isoproterenol is shown in Fig. 2. Portal
vein stenosis was without effect in either absolute (Fig. 2A)
or relative terms (Fig. 2B), and the EC50 was not
different (sham, 38.5 ± 13.8 nM; stenosed, 41.4 ± 8.4 nM). A
similar result was observed in the left ventricle (data not shown).
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Extracellular Ca2+.
The effect of portal vein stenosis on the relationship between
extracellular Ca2+ and developed tension was studied in
left ventricular papillary muscle and right ventricular strips.
Developed tension was significantly reduced before any manipulation of
the extracellular Ca2+ concentration in portal
vein-stenosed rats (Table 1) and, as Fig.
5A demonstrates for papillary
muscle, it remained consistently less over the complete range of
Ca2+ concentrations examined. Maximal force development was
observed at the same extracellular Ca2+ concentration in
the sham-operated and portal vein-stenosed groups. Using force
developed in 2.5 mM Ca2+ before the experimental protocol
was performed to normalize the data in Fig. 5A, the two groups
could be compared despite the lower developed tension in the portal
vein stenosis group. This comparison is shown in Fig. 5B and
demonstrates that the relationship between the extracellular
Ca2+ concentration and the relative magnitude of force was
similar in the sham-operated and portal vein-stenosed groups when the data were normalized to account for the lower control tension in the
stenosed group. Similar results were obtained in right ventricle (data
not shown). Furthermore, the time course of the contractile response to
stepped changes in the extracellular Ca2+ concentration was
the same in the sham-operated and portal vein-stenosed groups (data not
shown). There was no difference in the small rise in diastolic tension
between the sham-operated and portal vein-stenosed groups at 5 and 7.5 mM Ca2+.
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[3H]isradipine binding.
The density of dihydropyridine binding sites on L-type Ca2+
channels was estimated by measuring the equilibrium binding of
[3H]isradipine, a dihydropyridine antagonist.
Table 3 summarizes the effect of portal
vein stenosis. No change in the affinity of isradipine for its binding
sites was observed, but dihydropyridine receptor density decreased
63%. nH for [3H]isradipine
binding to membranes from both sham-operated and portal vein-stenosed
rats did not differ from 1, indicating that isradipine interacted with
a single site.
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Myofibril Ca2+ sensitivity.
To determine whether the depressed contractile function observed in
cardiac tissue from portal vein-stenosed rats was related to decreased
sensitivity of the myofilaments to Ca2+, the contractile
response of permeabilized left ventricular papillary muscle fiber
bundles to Ca2+ was examined. The effect of portal vein
stenosis is shown in Fig. 6. There was no
difference in myocardial Ca2+ sensitivity (half-maximal
pCa: 5.57 and 5.55 for sham and stenosed groups, respectively), maximum
tension development (520 ± 100 and 520 ± 180 µN for sham and
stenosed groups, respectively), or cooperativity as determined from
nH (2.2 ± 0.4 and 2.7 ± 0.4 for sham and stenosed
groups, respectively) in fibers between sham-operated and portal
vein-stenosed rats.
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Effect of portal vein stenosis on
Ca2+ uptake and release by SR.
The effect of portal vein stenosis on the uptake and release of
Ca2+ by the SR was investigated in left ventricular
papillary muscles in which the sarcolemma was chemically permeabilized.
The contraction produced by high concentrations of caffeine by
promoting Ca2+ release through the SR Ca2+
release channels was used to assess the SR Ca2+ content.
Ca2+ uptake and release were not different in the
sham-operated and portal vein-stenosed groups as evidenced by the
nearly identical responses to caffeine (Fig.
7B). Loading and subsequent release by caffeine was performed three times in each muscle with identical results, indicating that Ca2+ uptake by the SR under these
conditions was consistent and reproducible (not shown). Furthermore,
there was no difference between the sham-operated and portal
vein-stenosed groups in the rate of tension developed in response to
caffeine.
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SR Ca2+ content and
Na+/Ca2+
exchange in intact muscle fibers.
Figure 8 illustrates the effect of portal
vein stenosis on caffeine-induced Ca2+ release in
nonpermeabilized muscle. Figure 8A demonstrates that the
stimulation of Na+/Ca2+ exchange and the
resultant increase in Ca2+ efflux with
Ca2+-free solution with normal Na+ reduces the
SR Ca2+ content. Using this protocol, the effect of portal
vein stenosis on SR Ca2+ content and
Na+/Ca2+ exchange was investigated. Figure
8B presents a comparison between the sham-operated and portal
vein-stenosed groups in normal KH buffer solution (2.5 mM
Ca2+) and in Ca2+-free solution with normal
Na+, which reduces SR reuptake of Ca2+ leaked
during rest by stimulating Na+/Ca2+ exchange.
There was a decrease in the response to caffeine in both left
ventricular papillary muscles (not shown) and right ventricle (Fig.
8B), although the difference was significant only in the right
ventricle. As expected, stimulation of Na+/Ca2+
exchange for 5 min with Ca2+-free solution with normal
Na+ significantly decreased the response to caffeine in the
sham-operated and portal vein-stenosed groups. However, the magnitude
of the decrease was greater in the sham-operated group in absolute as well as relative terms (sham: 379.1 mN/cm2,
62%; stenosed:
155.3 mN/cm2,
39%).
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Effect of BAY K 8644.
BAY K 8644, a dihydropyridine that prolongs the average open time of
L-type Ca2+ channels, increased the development of force as
a function of concentration between 10 nM and 1 µM. As demonstrated
by Fig. 9, BAY K 8644 produced comparable
increments in dT/dt at each concentration in tissues
from sham-operated and portal vein-stenosed animals.
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DISCUSSION |
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Studies (7, 10, 13, 16, 29, 37, 40) in both human alcoholic and
nonalcoholic cirrhosis and in nonalcoholic animal models of cirrhosis
have shown that liver disease is associated with impaired basal cardiac
contractile function and diminished responses to -adrenergic
stimulation. In cirrhotic humans, this altered cardiac function has
been termed cirrhotic cardiomyopathy (43). In earlier
studies (7, 65), our laboratory has used the chronic portal
vein-stenosed rat, a hepatic model with extensive portosystemic
shunting without hepatocellular disease, to dissociate the cardiac
effects of portosystemic shunting and hepatocellular disease. The site
of cardiac impairment responsible for the decrease in basal contractile
function is largely unexplored in this model. In the present study, we
have extended our work to include the effect of chronic portal vein
stenosis on 1) sites in myocardial ECC coupling that could
reduce basal contractility as well as the contractile response to
-adrenoceptor activation and 2) postreceptor sites that
could alter responses to
-adrenoceptor activation.
In the current study, basal myocardial contractility was reduced
30-50% in both the right and left ventricle of the chronic portal
vein-stenosed rat (Table 1), and -adrenoceptor responsiveness was
also diminished (Fig. 1), in agreement with previous reports from our
laboratory (7, 65). However, the positive lusitropic action of
isoproterenol was unaltered by portal vein stenosis (Fig. 2).
ECC. The effect of portal vein stenosis on ECC was tested initially by estimating sarcolemmal dihydropyridine receptor density as an indication of L-type Ca2+ channel density. The decrease in dihydropyridine binding sites observed in hearts from portal vein-stenosed animals (Table 3) suggests that the density of L-type Ca2+ channels was reduced. If the remaining channels still gate the inward movement of Ca2+ normally, relative changes in cardiac force in sham-operated and portal vein-stenosed rats would be expected to be similar, and the maximum force developed in both groups should be achieved at the same extracellular Ca2+ concentration. Muscles from the stenosed group, however, would not be expected to generate the same absolute force as the sham group. A between-group difference in the relative change in force development would suggest the possibility of additional effects of portal vein stenosis. This expectation is, of course, founded on the proviso that the sensitivity of the myofilaments to Ca2+ and SR Ca2+ uptake and release are unaffected by portal vein stenosis.
In actively contracting muscle, an increase or decrease in extracellular Ca2+ concentration results in a parallel increase or decrease in Ca2+ entry during the action potential because of the change in its electromotive force (57). This increase or decrease in Ca2+ influx is accompanied by enhancement or attenuation of the intracellular Ca2+ transient (2) and increased or decreased force development (31). In the present study, this relationship between extracellular Ca2+ concentration and force development held for myocardium from both groups. However, at all extracellular Ca2+ concentrations, the force developed by the stenosed group was significantly lower than in the sham-operated group (Fig. 5A). Although this observation could be the result of dysfunction in any of several steps in ECC, it is consistent with decreased Ca2+ influx during muscle contraction. Additional indirect support for the hypothesis that the cardiac effect of portal vein stenosis results from decreased density of L-type Ca2+ channels was obtained by determining the effects of stenosis on the sensitivity of the myofilaments to Ca2+, SR Ca2+ uptake and release, and the positive inotropic effect of the dihydropyridine agonist BAY K 8644. Although a decrease in myofilament Ca2+ sensitivity and/or SR Ca2+ transport would not necessarily negate the hypothesis, the absence of an effect of portal vein stenosis on these aspects of ECC would support the hypothesis. As Fig. 6 illustrates, no difference in myofilament Ca2+ sensitivity was observed (half-maximal pCa was 5.57 and 5.55 in sham-operated and portal vein-stenosed rats, respectively), and maximum tension development occurred at the same Ca2+ concentration in the two groups. With respect to SR Ca2+ uptake and release, a lack of effect of portal vein stenosis on the SR is suggested by the absence of an effect on caffeine-induced Ca2+ release in permeabilized fibers. In permeabilized fibers, where the Ca2+ concentration can be rigidly controlled and uptake into the SR limited to a fixed time interval, there was no difference in the contraction caused by caffeine between the sham-operated and stenosed groups (Fig. 7). Two conclusions about the effect of portal vein stenosis on SR Ca2+ handling are suggested by the absence of a difference between the two experimental groups: 1) there is no direct effect of portal vein stenosis on SR uptake and release; and 2) there is no change in the number of release channels, because the magnitude of a caffeine contraction is dependent not only on the amount of Ca2+ in the SR but also on the number of release channels available to interact with caffeine. The experiments performed with the L-type Ca2+ channel agonist BAY K 8644 are also consistent with a decrease in Ca2+ channel density contributing to the reduced contractile function in portal vein-stenosed rats. BAY K 8644 increases the transsarcolemmal Ca2+ influx by lengthening the mean open time of L-type Ca2+ channels. Because the SR Ca2+ and myofilament sensitivity to Ca2+ were unaffected by portal vein stenosis, if the remaining L-type Ca2+ channels function normally, BAY K 8644 should increase force by the same relative increments in tissues from both sham-operated and stenosed animals because the only difference between the two groups is the number of Ca2+ channels. As can be seen in Fig. 9, BAY K 8644 produced similar relative increments in myocardial force in sham-operated and portal vein-stenosed rats. It should be noted that although these experiments on ECC suggest that Ca2+ entry into the myocytes from portal vein-stenosed rats is decreased, actual measurement of the current carried by the L-type Ca2+ channel is necessary to confirm unequivocally the role of the L-type Ca2+ channel in the myocardial action of portal vein stenosis. Aside from the decrease in dihydropyridine binding sites, the only other difference between the portal vein-stenosed and sham-operated groups was a decrease in the response to caffeine in nonpermeabilized muscles in normal KH buffer (Fig. 8B). Although these data do not permit a conclusion to be made concerning the cause of the decrease, possible explanations include a reduced SR Ca2+ content, a change in Na+/Ca2+ exchange, and greater leakage of Ca2+ from the SR during rest (diastole). As mentioned above, the caffeine-induced SR Ca2+-release data shown for skinned fibers (Fig. 7B) indicate that SR Ca2+ uptake and release in the portal vein-stenosed group is unaffected when loading conditions are controlled. These data are inconsistent with a direct effect of portal vein stenosis on SR Ca2+ content. Because no effect of portal vein stenosis on SR Ca2+ content was observed in permeabilized muscle, the decreased response to caffeine in intact fibers would appear to be caused by a mechanism other than SR Ca2+ uptake and release. However, in functioning, nonpermeabilized muscle, a change in the normal relationship between SR uptake of cytosolic Ca2+ and transport of Ca2+ out of the cell by Na+/Ca2+ exchange during rest (diastole) could affect the SR Ca2+ content. In the rat heart, nearly all of the Ca2+ released from the SR is sequestered into the SR by SR Ca2+-ATPase (5). Na+/Ca2+ exchange plays only a minor role in the removal of Ca2+ from the cytoplasm relative to the SR (5). A substantive increase or decrease in Na+/Ca2+ exchange activity in portal vein-stenosed rats, however, might have an inverse effect on the SR Ca2+ content and the amount of Ca2+ available to induce subsequent contractions. To test this hypothesis, nonpermeabilized fibers were exposed to a Ca2+-free solution with normal Na+, which stimulates Na+/Ca2+ exchange, and under these conditions at rest, a decrease in SR Ca2+ content sensitive to caffeine is observed in the rat heart (Ref. 3; compare the response to caffeine with and without Ca2+ in the sham and stenosed groups in Fig. 8B). Therefore, Na+/Ca2+ exchange can be assessed using caffeine to determine the relative amount of Ca2+ remaining in the SR after stimulation of Na+/Ca2+ exchange. If portal vein stenosis was associated with either increased or decreased Na+/Ca2+ exchange, a change in the response to caffeine could be expected. In our study, the decrease in the response to caffeine after Ca2+-free solution with normal Na+ was considerably greater in sham-operated rats in absolute as well as relative terms (sham:-Adrenoceptor responsiveness.
Although we had previously demonstrated that hearts from stenosed rats
require a threefold greater
-adrenoceptor occupancy to produce the
same absolute increase in force in response to isoproterenol (65), no
alteration in myocardial
-adrenoceptor density and affinity occurred
in hearts from portal vein-stenosed rats (44, 65). In the present
study, we investigated whether a change in G protein expression or
coupling of
-adrenoceptors to Gs
could explain the
effect of portal vein stenosis on
-adrenoceptor activation. As
demonstrated by Figs. 3 and 4, portal vein stenosis did not alter G
protein expression.
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ACKNOWLEDGEMENTS |
---|
We thank Stephanie R. Edwards for technical assistance and Dr. Robert E. Godt for providing the program to prepare the solutions for the permeabilized fiber experiments.
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FOOTNOTES |
---|
This study was supported by grants from the American Heart Association, Louisiana Affiliate, the Stiles Foundation, National Institute of Mental Health Grants MH-01231 and MH-40694 (J. M. O'Donnell) and National Institute of Drug Abuse Grant DA-07972 (S. C. Roerig).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. H. Zavecz, Dept. of Pharmacology, Louisiana State Univ. Health Sciences Center, PO Box 33932, Shreveport, LA 71130-3932 (E-mail: jzavec{at}lsumc.edu.
Received 13 October 1998; accepted in final form 4 January 2000.
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