Cardiac excitation-contraction coupling in the portal hypertensive rat

James H. Zavecz1, Orlando Bueno1, Ronald E. Maloney1, James M. O'Donnell1, Sandra C. Roerig1, and Harold D. Battarbee2

Departments of 1 Pharmacology and 2 Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Basal contractility and responses to beta -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, beta -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. Gsalpha and Gialpha 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; beta -adrenoceptor; isoproterenol; G protein; heart; sarcoplasmic reticulum; sodium/calcium exchange


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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. beta -Adrenoceptors have been shown not to be downregulated in this model, but a greater fraction of cardiac beta -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 beta -adrenergic receptor activation results from a mechanism not directly involving beta -adrenergic signal transduction. In the present study, we have extended our research to include the effects of portal hypertension with portosystemic shunting on cardiac Gsalpha and Gialpha expression, beta -adrenoceptor-Gs coupling, and excitation-contraction coupling (ECC).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

In the experiments in which the effect of portal vein stenosis on the response of the myocardium to beta -adrenoceptor activation with isoproterenol was studied, tissues were equilibrated in KH, and when contractions stabilized, isoproterenol was added to the tissue bath. On attainment of a stable positive inotropic effect, isoproterenol was washed out of the tissue. When contractions returned to the control level, the next concentration of isoproterenol was added. Exposure to the different concentrations of isoproterenol was randomized.

In experiments examining the effect of portal vein stenosis on the relationship between contractile strength and the extracellular Ca2+ concentration, the tissues were equilibrated with KH containing 2.5 mM Ca2+. After force stabilized, the bathing solution was quickly changed to KH with 0.3125 mM Ca2+. When force stabilized, which required 2-3 min, an appropriate amount of Ca2+ was added to produce the next Ca2+ concentration. Concentrations of Ca2+ >7.5 mM were not investigated because of the potential for Ca2+ precipitation with the buffer system and because the maximal response had already been achieved. The brief period of exposure to low Ca2+ did not affect subsequent muscle performance, as indicated by the similarity of force measured at the initial 2.5 mM Ca2+ concentration and that measured at 2.5 mM Ca2+ during the concentration-response experiment (sham: 1,079 ± 117 and 1,147 ± 94 mN/cm2, respectively; stenosed: 634 ± 130 and 660 ± 34 mN/cm2, respectively, in papillary muscle).

Experiments with the dihydropyridine L-type Ca2+ channel activator BAY K 8644 were performed to assess further Ca2+ channel functionality. After contractions stabilized, BAY K 8644 (10-8-3 × 10-6 M) was added cumulatively. Succeeding concentrations were added when the response to the previous concentration stabilized.

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).

Binding assays were performed at 37°C for 30 min in a total volume of 200 µl containing 50 mM Tris · HCl (pH 7.4), 100 µg of membrane protein, 1 mM CaCl2, and 30-600 pM [3H]isradipine (85.8 Ci/mmol; DuPont NEN, Boston, MA). Nonspecific binding, which amounted to 10-20% of total binding, was determined by the addition of 1 µM nitrendipine to appropriate incubation vessels. [3H]isradipine bound was determined by filtration (65). Each assay was performed in duplicate. Receptor density and the apparent equilibrium dissociation constant (Kd) for [3H]isradipine were determined using nonlinear regression (22).

beta -Adrenoceptor-G protein coupling. When Gs binds to the agonist-beta -adrenoceptor complex, forming an agonist-beta -adrenoceptor-Gs complex, the agonist is bound with higher affinity than when it is bound to the receptor only. The fraction of beta -adrenoceptors in this "high-affinity" state is indicative of the coupling of the agonist-receptor complex with Gs. The fraction of beta -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 beta -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 Gsalpha ), AS/7 (directed against carboxy-terminal decapeptide sequences of Galpha i-1 and Galpha i-2), Galpha i-1 (directed against the internal amino acid sequence 159-168 and does not cross react with Galpha i-2), EC/2 (directed against carboxy-terminal decapeptide sequences of Galpha i-3), and GC/2 (directed against amino-terminal decapeptide sequences of Goalpha ) (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 [alpha -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.

For cholera toxin (CTX)-induced ADP-ribosylation experiments, CTX was activated by preincubation in 50 mM DTT for 10 min at 37°C. Membrane proteins (100 µg) were preincubated in 200 µM guanylylimidodiphosphate for 10 min at 37°C, and then an assay mixture containing 20 mM thymidine, 100 mM NaCl, and 20 mM HEPES (pH 7.7) was added. The activated CTX (5 µg) and 3-5 µCi [alpha -32P]NAD were added to make a final volume of 0.1 ml. The mixture was incubated for 2 h at 30°C, and then 1 ml of ice-cold HEPES (20 mM, pH 7.7) was added and the mixture was centrifuged for 10 min in a Microfuge at 4°C. The pellet was resuspended in 0.5 ml of the same buffer and centrifuged again for 10 min.

The protein pellets were resuspended in treatment buffer and separated by 6 M urea SDS-PAGE as described above for immunoblot analysis. Gels were dried and exposed to X-ray film. Bands on the films were analyzed by densitometry as described above. Five samples each from sham-operated and portal vein-stenosed animals were analyzed. Each sample was analyzed on at least two different gels.

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. Galpha i-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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Effect of portal vein stenosis on DT, dT/dt, and -dT/dt

beta -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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Fig. 1.   Effect of portal vein stenosis on absolute (A) and relative (B) increases in the maximal rate of tension development (dT/dt) induced by isoproterenol in left ventricular papillary muscle. Values are means ± SE of 4-5 experiments. EC50 did not differ between the 2 groups irrespective of the manner in which data were presented (sham-operated rats, 28.8 ± 2.8 nM; portal vein-stenosed rats, 25.0 ± 2.7 nM). Effect of portal vein stenosis on contractile response to isoproterenol was qualitatively similar in right ventricle. * P < 0.05.



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Fig. 2.   Effect of portal vein stenosis on absolute (A) and relative (B) increases in the maximal rate of relaxation (-dT/dt) induced by isoproterenol in right ventricle. Values are means ± SE of 5 experiments. EC50 did not differ between the 2 groups irrespective of the manner in which data were presented (sham-operated rats, 38.5 ± 13.8 nM; portal vein-stenosed rats, 41.4 ± 8.4 nM). Effect of portal vein stenosis on the positive lusitropic action of isoproterenol was qualitatively similar in left ventricle.

Although beta -adrenoceptors are not downregulated after 2 wk of portal vein stenosis, and their affinity for isoproterenol is unaffected (65), a nearly threefold increase in receptor occupancy in hearts from portal vein-stenosed rats is required to produce the same increase in myocardial contractility as in sham-operated rats (65). Therefore, we examined whether a decrease in G protein expression or a defect in beta -adrenoceptor-G protein coupling is responsible for the diminished response to isoproterenol. Figures 3 and 4 illustrate the effect of portal vein stenosis on Gsalpha and Gialpha expression in the left ventricle. Cardiac membranes from both sham-operated and portal vein-stenosed animals were incubated with specific antisera directed against selective peptide sequences in the alpha -subunits of different G protein subtypes. With the antiserum RM/1, which is specific for the alpha -subunit of stimulatory G proteins (Gsalpha ), 3-4 immunopositive bands with a molecular mass of 42-44 kDa were found to be present in cardiac membranes from sham-operated and portal vein-stenosed rats. Representative immunoblots are shown in Fig. 3C. The relative density of the bands from ventricles taken from eight sham-operated and eight portal vein-stenosed rats, all run on the same gel, was determined, and the results are shown in Fig. 4. No difference in Gsalpha expression was detectable between the sham-operated and portal vein-stenosed groups. Expression of the inhibitory G protein subtypes Galpha i-1 and Galpha i-2 was detected using antisera AS/7 (Galpha i-1 and Galpha i-2) and Galpha i-1. EC/2 was used to detect Galpha i-3. Figure 3B shows a representative autoradiograph of immunodetectable Galpha i-1 and Galpha i-2 using AS/7. An intense band with the same mobility as the lower band from spinal cord membranes was detected in the cardiac membranes. This band has been previously identified as Galpha i-2 (64). Two faint immunopositive bands were also observed with AS/7 (Fig. 3B), which could be Galpha i-1. Thus additional tests were performed using antisera selective for Galpha i-1. Results of these studies are shown in Fig. 3A. Spinal cord membranes, which contain Galpha i-1 (64), showed two immunopositive bands with this antisera, but no bands were observed in the cardiac membranes. The autoradiographic intensity of the lower bands (Galpha i-2) seen in Fig. 3B is shown in Fig. 4 for all eight sham-operated and all eight portal vein-stenosed rats, demonstrating that there is no difference in expression of Galpha i-2 in the sham-operated and the portal vein-stenosed groups. No immunopositive bands for Galpha i-3 or Goalpha were detected in the rat heart (data not shown).


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Fig. 3.   G protein alpha -subunits in membranes from mouse spinal cord (SC) and rat ventricles [sham and portal vein stenosis (pvs)]. Representative immunoblots of Galpha i-1 (A), Galpha i-1 and Galpha i-2 (B), and Gsalpha expression in ventricles from sham-operated and portal vein-stenosed rats. Spinal cord expression was included as an internal control because spinal cord expresses Galpha i-1, Galpha i-2, and Gsalpha . A: antisera selective for Galpha i-1. B: antiserum AS/7 selective for Galpha i-1 and Galpha i-2. C: antiserum RM/1 selective for Gsalpha .



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Fig. 4.   G protein alpha -subunit expression in rat cardiac ventricles. The relative density of bands from immunoblots of ventricular protein from 8 sham and 8 stenosed rats run on the same gel is represented. Protein derived from hearts of the same sham and stenosed rats was used to determine expression of all G proteins examined. Separate gels were run for each different G protein. G protein expression in the portal vein-stenosed (PVS) group is compared with expression in the sham group. Expression of Gsalpha is not compared with expression of Galpha i-2. For Gsalpha , the entire immunopositive area, containing 3-4 relatively distinct bands, was quantified. For Galpha i-2, the band migrating identically to the lower band (Galpha i-2) from spinal cord membranes was quantified. SO, sham operated.

The extent of ADP ribosylation of Gi (with PTX) and Gs (with CTX) was examined to determine whether portal vein stenosis altered G protein function. No differences in ADP ribosylation were observed between the sham-operated and portal vein-stenosed groups (data not shown).

The effect of portal vein stenosis on the coupling of the beta -adrenoceptor agonist-receptor complex to Gs was determined by quantifying the percentage of beta -adrenoceptors in the high-affinity state of the receptor. The results are summarized in Table 2. Although KH was lower in the portal vein stenosis group, suggesting a difference in the stability of the agonist-receptor-G protein complex, the difference between the sham-operated and portal vein-stenosed groups was not significant (P > 0.05). Furthermore, portal vein stenosis-induced portal hypertension and portosystemic shunting did not affect the fraction of receptors in the high-affinity state.

                              
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Table 2.   Effect of portal vein stenosis on affinity state of cardiac beta -adrenoceptors

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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Fig. 5.   A: effect of extracellular Ca2+ concentration on developed tension in left ventricular papillary muscles from sham-operated and portal vein-stenosed rats. Values are means ± SE of 7-8 muscles. Each papillary muscle was equilibrated in normal KH containing 2.5 mM Ca2+ (control data point) before switching to the lowest concentration of Ca2+ (0.3125 mM). Subsequent addition of Ca2+ to attain the next higher Ca2+ concentration was made after force had reached a plateau. B: relative changes in papillary muscle force generation with changes in extracellular Ca2+ concentration. Values represent mean ± SE percentage of the control value in 2.5 mM Ca2+. Similar results were observed in right ventricle (data not shown). * P < 0.05, ** P < 0.01 significantly different from sham.

[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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Table 3.   [3H]isradipine binding to L-type Ca2+ channels in cardiac membranes from left ventricles of sham-operated and portal vein-stenosed rats

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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Fig. 6.   Effect of portal vein stenosis on pCa-force relationship in skinned left ventricular papillary muscle fiber bundles. Values represent means ± SE %maximum response obtained in tissues from sham-operated rats; n = 5. Data from each group were fit to Hill equation and gave a half-maximal pCa of 5.57 and 5.55 for fibers from sham-operated and portal vein-stenosed rats, respectively. Maximal response was 520 ± 100 and 520 ± 180 µN in sham and stenosed rats, respectively. Hill coefficients for sham and stenosed groups were 2.2 ± 0.4 and 2.7 ± 0.4, respectively.

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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Fig. 7.   A: caffeine-induced Ca2+ release from sarcoplasmic reticulum (SR) in permeabilized papillary muscle from a sham-operated rat. After permeabilization and release of SR Ca2+ content with caffeine, muscles were exposed to the following solutions: a, relaxing solution for 2 min; b, Ca2+ loading (pCa 6.0) for 3 min; c, low EGTA for 1 min; d, 50 mM caffeine; e, low EGTA; f, caffeine; and g, relaxing solution. Rapid upward deflection seen in tracing at arrows is an artifact from replacement of bathing solution. B: compilation of the effect of portal vein stenosis on caffeine-induced Ca2+ release determined as shown in A. Values are means ± SE of response to 50 mM caffeine; n = 5-8.

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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Fig. 8.   Effect of portal vein stenosis on caffeine-induced Ca2+ release from SR in nonpermeabilized right ventricles. SR Ca2+ concentration was estimated by the magnitude of caffeine-induced contraction. A: representative tracing in a right ventricle from a sham-operated rat. B: effect of portal vein stenosis on caffeine-induced Ca2+ release in intact right ventricle from sham and stenosed rats in normal KH buffer solution (2.5 mM Ca2+) and in Ca2+-free solution with normal Na+ (Ca2+-free). Caffeine was administered in normal buffer after 5 min of no stimulation. Before switching to Ca2+-free solution with normal Na+, muscle was stimulated with several electrical impulses to determine that it was still viable (not shown in tracing). Bars are means ± SE of force developed in response to addition of 30 mM caffeine; n = 5-11.

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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Fig. 9.   Effect of BAY K 8644 on dT/dt of right ventricular strips. Values are means ± SE of %change; n = 4-6. Data were normalized to allow comparison of the effect of BAY K 8644 in tissues with differing muscle stress before drug administration (see Table 1). EC50 of BAY K 8644 was unaffected by portal vein stenosis (sham, 0.16 ± 0.05 µM; stenosed, 0.22 ± 0.09 µM). Similar results were obtained in left ventricle (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -adrenoceptor activation and 2) postreceptor sites that could alter responses to beta -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 beta -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: -379.1 mN/cm2, -62%; stenosed: -155.3 mN/cm2, -39%). The greater decrease in the sham-operated rats (Fig. 8B) suggests that Na+/Ca2+ exchange activity was greater in the sham-operated group than in the portal vein-stenosed group. Because the response to caffeine in the presence of extracellular Ca2+ was greater in sham-operated rats than in portal vein-stenosed rats, a difference in absolute terms might be expected, but not in relative terms if Na+/Ca2+ exchange was not affected. One could speculate that an apparent decrease in Na+/Ca2+ exchange would produce a greater response to caffeine in Ca2+-free solution with normal Na+ in the portal vein-stenosed group because the exchanger is less effective, and therefore, a greater amount of Ca2+ can be taken up by the SR. However, the effect of caffeine in intact fibers suggests that there is less, not more, Ca2+ in the SR of the portal vein-stenosed group. A possible explanation for the reduction in Na+/Ca2+ exchange may lie in the relationship between Ca2+ entry through L-type Ca2+ channels and extrusion by Na+/Ca2+ exchange. It has been suggested that, at equilibrium, the amount of Ca2+ that is removed by Na+/Ca2+ exchange varies directly with the amount of Ca2+ that enters via Ca2+ channel current (ICa) (50, 62) and that the amount of Ca2+ entering via ICa is balanced by an equal amount of Ca2+ leaving the cell at equilibrium. In that case, a chronically reduced Ca2+ entry associated with downregulation of L-type Ca2+ channels could result in decreased Na+/Ca2+ exchange. Although this is circumstantial evidence, it does fit the data. A decrease in Na+/Ca2+ exchange expression would lend credence to this hypothesis.

Alternatively, the reduced response of intact fibers to caffeine in normal KH buffer solution in the portal vein-stenosed group could be the result of a greater leakage of Ca2+ from the SR during rest. In the rat, however, this is complicated by the fact that nearly all Ca2+ leaked from the SR is taken back up into the SR and a decline in the response to caffeine is not observed (5). However, a decrease in the response to caffeine can be observed if Na+/Ca2+ exchange is stimulated during the period of rest (5). The data from permeabilized tissues suggest that the SR from the stenosed group takes up the same amount of Ca2+ as the sham group, at least under controlled loading conditions (Fig. 7B). A larger Ca2+ leak after portal vein stenosis would be expected to produce greater Ca2+ efflux when Na+/Ca2+ exchange is stimulated by Ca2+-free solution with normal Na+, and therefore, a reduced response to caffeine. However, our data showed smaller absolute and relative decrements in the response to caffeine in the portal vein-stenosed group than in the sham-operated group after a 5-min period in Ca2+-free solution with normal Na+ (sham: -379.1 mN/cm2, -62%; stenosed: -155.3 mN/cm2, -39%) (Fig. 8B). This smaller decrement suggests that Na+/Ca2+ exchange was less in the portal vein-stenosed group and is not indicative of increased leakage of Ca2+ from the SR during rest.

One question that can be asked is, How do the experiments performed in permeabilized and nonpermeabilized quiescent fibers apply to the observations of depressed contractile function in paced right ventricles and left ventricular papillary muscles (Table 1 and Fig. 1)? The experiments in permeabilized tissues had as their goal the determination of whether the sensitivity of the myofilaments to Ca2+ and SR Ca2+ uptake and release were directly impaired by portal vein stenosis. The results from these experiments suggest that these processes were not directly affected by portal vein stenosis. If these processes are altered in intact contracting muscle, the effect would have to be an indirect one that is removed by permeabilizing the sarcolemma. The data from caffeine-induced contraction in quiescent, intact muscle are probably more indicative of the situation in contracting muscle. It has been shown that the effect of a high concentration of caffeine in intact muscle is similar to the effect of rapid administration of caffeine during a contraction cycle in rat ventricular myocytes, i.e., there is an increase in the Ca2+ transient, a decrease in SR Ca2+ content, and increased extrusion of Ca2+ via Na+/Ca2+ exchange in response to the increase in Ca2+ release (50, 62).

beta -Adrenoceptor responsiveness. Although we had previously demonstrated that hearts from stenosed rats require a threefold greater beta -adrenoceptor occupancy to produce the same absolute increase in force in response to isoproterenol (65), no alteration in myocardial beta -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 beta -adrenoceptors to Gsalpha could explain the effect of portal vein stenosis on beta -adrenoceptor activation. As demonstrated by Figs. 3 and 4, portal vein stenosis did not alter G protein expression.

Failure of the coupling of the beta -adrenoceptor with Gsalpha could cause the effect of portal vein stenosis on beta -adrenoceptor-mediated responses in the heart. It is well known that the beta -adrenoceptor has two affinity states for a bound agonist (28) and that only beta -adrenoceptors coupled to Gsalpha are in the high-affinity state. Fewer beta -adrenoceptors in the high-affinity state would be expected to decrease the efficacy of GTP because there is less agonist-receptor-G protein complex with which GTP can interact. beta -Adrenoceptor-Gsalpha coupling has been shown to be significantly decreased in some models of cardiac hypertrophy and failure, even though beta -adrenoceptor density, determined by antagonist binding, was not reduced (63). This suggests that for any given number of beta -adrenoceptors to which an agonist has bound, there will be a reduced positive inotropic effect (63). Because we (65) had previously found that hearts from portal vein-stenosed rats required a greater beta -adrenoceptor fractional receptor occupancy to produce the same positive inotropic response to isoproterenol as sham-operated rats, the effect of portal vein stenosis on the high- and low-affinity state of the beta -adrenoceptor was investigated. The unchanged fraction of receptors in the high-affinity state (Table 2) clearly shows that portal vein stenosis was without effect on beta -adrenoceptor-G protein coupling. Therefore, beta -adrenoceptor signaling does not appear to be influenced by portal vein stenosis. Indeed, one could argue that the lack of a relative change in the response to beta -adrenoceptor activation in the presence of a decrease in basal cardiac force generation is indicative of no effect on beta -adrenoceptor responsiveness at all. If this is so, then another process must be responsible. Although the process of ECC has not been studied in animals subjected to portal vein stenosis until now, there is evidence from several models of hypertrophy and heart failure in different species, as well as in human congestive heart failure, demonstrating, even in mild pathological states where basal L-type Ca2+ channel peak current density or dihydropyridine binding was unaltered, reduced beta -adrenoceptor responsiveness that coincides with a decrease in the beta -adrenoceptor-mediated enhancement of L-type Ca2+ channel current density (30, 47, 48, 56).

In summary, myocardial contractility was depressed by the induction of portal hypertension by chronic ligation of the prehepatic portal vein. Various aspects of beta -adrenoceptor signaling and ECC were examined in rat right ventricular strips and left ventricular papillary muscles. In permeabilized muscle, myofilament Ca2+ sensitivity and SR Ca2+ uptake and release did not differ between the sham and the stenosed groups. The number of dihydropyridine binding sites was reduced, and in intact muscles, Na+/Ca2+ exchange and the SR Ca2+ content were reduced in the portal vein-stenosed group. No effect of portal vein stenosis on beta -adrenoceptor-G protein coupling and G protein expression or function was observed. The data suggest that in the rat heart, portal vein stenosis-induced myocardial dysfunction is associated with alterations in ECC but not beta -adrenoceptor signaling.


    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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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