Introduction

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HF is associated with increased sympathetic activity that maintains blood pressure and oxygen supply. Increased plasma norepinephrine due to the high sympathetic tone activates -adrenoceptors responsible for maintaining inotropy and cardiac output in the compensated stages of HF. This adrenergic overactivation in HF, however, leads to abnormal myocardial -adrenoceptor signaling including -adrenoceptor down-regulation, uncoupling of -adrenoceptor and the stimulatory G protein (G_s), and decreased adenylate cyclase activity (Bristow et al., 1986; Roth et al., 1993; Brodde et al., 1995). An important aspect of this defective myocardial -adrenoceptor signaling is the fact that agonist occupation of -adrenoceptors at high concentrations activates ARK1, which phosphorylates the receptors (Hausdorff et al., 1990). ARK1 is a member of the G protein-coupled receptor kinase family that can phosphorylate agonist-occupied -adrenoceptors, triggering the process of desensitization (Inglese et al., 1993), and the phosphorylated receptors become a target for -arrestin, which uncouples activated receptors from the signaling cascade so that continuous agonist stimulation is prevented (Wilson and Applebury, 1993). It has been shown that ARK1 expression and activity are increased in failing explanted human hearts (Ungerer et al., 1993, 1994). Thus, the increased levels and activity of ARK1 serve as a potential mechanism for the loss of -adrenergic responsiveness seen in HF. Another potential contributing factor to decreased -adrenoceptor signaling in HF is increased levels of the inhibitory G protein (G_i) (Feldman et al., 1988). Increased G_i expression could mitigate cyclic AMP formation and thus contribute to the diminished response to -adrenoceptor stimulation in HF.

In the failing heart, the RAS is also activated, presumably leading to an enhanced local formation of angiotensin II (Hirsch et al., 1991). Because the adrenergic system and RAS are closely interrelated, a therapeutic prevention in HF that inhibits the RAS may also reduce adrenergic activation. In patients with HF, ACE inhibition has been shown to lower cardiac adrenergic drive and partly restore the number of -adrenoceptors (Gilbert et al., 1993). Furthermore, ACE inhibitors can attenuate the reduced -adrenoceptor density and the increased G_i expression in myocardium of rats with HF caused by coronary ligation (Sanbe and Takeo, 1995; Yoshida et al., 2001). It is generally agreed that the beneficial effect of ACE inhibitors on the cardiovascular system is due to prevention of angiotensin II formation. ACE inhibitors not only reduce angiotensin II, however, but also prevent degradation of bradykinin, which may decrease peripheral resistance and improve cardiac function. There is one report to suggest that the beneficial effect of ACE inhibitors on the impaired -adrenergic responsiveness in the failing heart is not primarily caused by modulation of activation of AT₁ receptors, which mediate a major mode of action of angiotensin II, but rather by alternative mechanism (Spinale et al., 1997). Thus, AT₁ receptor blockade, unlike ACE inhibition, failed to normalize cyclic AMP production and myocyte -adrenergic response in experimental canine HF produced by rapid ventricular pacing, although the reduced myocardial -adrenoceptor density was completely reversed (Spinale et al., 1997). These experimental data, however, may stand in contrast to the results of recent clinical trials showing that ACE inhibitors and AT₁ receptor blockers are comparable in improving symptoms and survival in patients with chronic HF (Pitt et al., 2000; Cohn and Tognoni, 2001) if the beneficial effects of ACE inhibition and AT₁ receptor blockade in HF are, in part, due to improvement of altered -adrenergic neuroeffector signal transduction.

The present study was undertaken to address the question of whether AT₁ receptor blockade and ACE inhibition provide potentially differential effects on abnormal -adrenoceptor signaling mechanisms in the failing heart. Rabbits with HF following MI were treated with the ACE inhibitor temocapril (Oizumi et al., 1988) or the AT₁ receptor blocker valsartan (Criscione et al., 1993). The results presented in this study clearly show that AT₁ receptor blockade is equally effective to ACE inhibition in normalizing -adrenoceptor signal transduction derangements, including -adrenoceptor down-regulation, reduced -adrenergic adenylate cyclase activity, increased ARK1 expression, and increased G_i protein content in HF.

	Materials and Methods

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Induction of Myocardial Infarction. All procedures were in accordance with the regulations adopted by the Hokkaido University School of Medicine Animal Care and Use Committee (Sapporo, Japan). Male New Zealand White rabbits (2-2.5 kg) were used in this study. MI was induced by coronary artery ligation, as previously described (Pennock et al., 1997; Maurice et al., 1999), with some modifications. Briefly, rabbits received an intramuscular injection of 0.05 mg/kg atropine and 5.6 mg/kg chlorpromazine 20 min before surgery, followed by anesthetized with an intramuscular injection of 75 mg/kg ketamine. Then, a left thoracotomy was performed through the third or fourth intercostal space, and the large marginal branch of the LCX was identified and ligated in its proximal portion with 5-0 nylon suture. Coronary ligation was considered successful when the free wall of the LV turned pale. For sham-operated animals, the pericardium was incised, but coronary ligation was not performed. Anatomic closure was performed, and residual air was evacuated from thorax using 16-gauge tube attached to a syringe. During the postoperative recovery period, a single dose of ampicillin (250 mg i.m.) was administered. Animals were allowed to recover and then returned to their cages when they were awake and responsive.

Experimental Design and Dosage Rationale. Immediately after surgery, the animals were randomly divided into four groups and treated for 3 weeks. The four groups were 1) sham with no treatment (n = 5), 2) MI with no treatment (n = 5), 3) MI with ACE inhibitor (temocapril; 0.5 mg/kg/day; n = 5), and 4) MI with AT₁ receptor blocker (valsartan; 3 mg/kg/day; n = 5). Because the drugs were administered in drinking water, the concentrations of the drugs in the water were adjusted to the water consumption. Water intake was measured at the beginning of the study to determine whether MI alters drinking activity; however, we found no difference between infarcted and noninfarcted rabbits. The doses of each drug used in this study were previously tested in a pilot study. Thus, myocardial ACE activity was significantly suppressed when rabbits were given temocapril at 0.5 mg/kg/day for 2 weeks, whereas the pressor response to intravenous injection of angiotensin II was significantly reduced when the animals were treated with valsartan at 3 mg/kg/day for 3 weeks. This suggests that the dosage regimen used in this study provides a pharmacological profile consistent with specific effects of ACE inhibition and AT₁ receptor blockade. More importantly, this dosing regimen was confirmed to have no effects on resting mean blood pressure, indicating that the confounding influences of differences in systemic hemodynamics could be minimized.

Hemodynamic Measurements. Three weeks after surgery and treatment, the animals were lightly anesthetized with diethyl ether. Polyethylene catheters (22-gauge; JELCO; Critikon, Tampa, FL) were inserted into the ear artery. Normal saline containing 2 U/ml heparin was continuously infused via the ear arterial catheter to maintain patency of the blood pressure cannula line. Following recovery of anesthesia, the arterial blood pressure and heart rate were continuously monitored using the Surgical Monitoring System (Nihon Kohden, Tokyo, Japan).

Echocardiographic Measurements. The echocardiographic procedure was performed after the animals were sedated with chlorpromazine (5.6 mg/kg, i.m.) and ketamine (50 mg/kg, i.m.). A commercially available echocardiographic system (SONOS 5500; Hewlett Packard, Palo Alto, CA) equipped with an S12 phased-array transducer was used. LVDd and LVDs were measured on the two-dimensional targeted M-mode strip chart recordings. Color flow mapping-guided pulsed-wave Doppler technique was used for the measurements of velocity-time integral (VTI) at the level of the aortic valve. LV ejection fraction (EF) and cardiac index (CI) were calculated by the following equations: EF (%) = [(EDV  ESV)/EDV] × 100; CI (ml/min/kg) = CO/BW; CO = SV × HR; SV = CSA × VTI; CSA = (AoD/2)² × , where EDV and ESV are LV end-diastolic and end-systolic volume, CO is cardiac output, BW is body weight, SV is stroke volume, HR is heart rate, CSA is cross-sectional area and AoD is aortic root dimension.

Measurements of Heart Weight and Infarct Size. After hemodynamic and echocardiographic measurements, the rabbits were anesthetized with an intravenous injection of sodium pentobarbital (50 mg/kg) via the marginal ear vein. The hearts were rapidly excised and rinsed in ice-cold Tris-HCl buffer (75 mM Tris-HCl, 25 mM MgCl₂, 5 mM EDTA, and 1 mM EGTA, pH 7.4, 4°C). Both ventricles were dissected free of connective tissue, fat, major vessels and atria, and weighed. Then, MI size was determined as a percentage of the LV free-wall surface area, as previously described (Maurice et al., 1999). Briefly, areas of the LV free wall that were grossly pale, fibrotic, and thinned were considered to be infarcted. The LV was cut away and opened flat, and a paper tracing of the LV was made with the infarcted area marked. The paper tracing was then cut out and weighed. The tracing of the infarcted area was cut out and weighed separately. The ratio of the weights was used to estimate the percentage of LV infarcted.

Plasma Norepinephrine Assay. To examine the systemic sympathetic nervous activity, plasma norepinephrine assay was performed. Before the removal of the heart, the blood samples were drawn from the inferior vena cava into tubes containing EDTA-2Na and centrifuged (4°C) to separate the plasma. Plasma norepinephrine levels were measured by high-performance liquid chromatography and normalized to picograms per milliliter of plasma.

Membrane Preparation. After measurements of heart weight and infarct size as above, noninfarcted tissues of the LV free wall were minced with scissors and homogenized in 5 volumes of ice-cold Tris-HCl buffer by the use of a polytron for 15 s. The homogenate was centrifuged at 1000g for 10 min at 4°C. The supernatant was filtered through a single layer of cheesecloth and retained. The pellet was suspended in 5 volumes of cold Tris-HCl buffer and centrifuged again. Membrane fractions in the supernatant were concentrated by centrifugation at 100,000g for 30 min at 4°C. The final pellets were resuspended in cold Tris-HCl buffer and stored at 80°C until used. Protein content was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard.

Radioligand Binding Experiments. The binding of the -adrenoceptor antagonist ()-[¹²⁵I]ICYP (2200 Ci/mmol; PerkinElmer Life Sciences, Boston, MA) was measured by a rapid filtration method similar to that described previously (Hattori et al., 1987). The membrane fractions were diluted further in an incubation medium (50 mM Tris-HCl and 10 mM MgCl₂, pH 7.4) to give a final protein concentration of 0.5 to 1.0 mg/ml. [¹²⁵I]ICYP and all drugs used in this study were prepared in the incubation medium. For saturation experiments, an aliquot of the membrane suspension (100 µl) was incubated with various concentrations of [¹²⁵I]ICYP (12.5-1600 pM) in a final volume of 200 µl. Agonist competition experiments were determined by incubation of 150 pM [¹²⁵I]ICYP with increasing concentrations of isoproterenol (10 pM-1 mM) in the absence or presence of the nonhydrolyzable GTP analog, GppNHp (100 µM). Incubations were carried out for 30 min at 37°C and terminated by adding 5 ml of the ice-cold incubation medium (4°C) to the entire incubation mixture, followed by a rapid filtration over Whatman GF/C glass fiber filters (Clifton, NJ). Each filter was washed three times with an additional 5 ml of the ice-cold medium. The radioactivity of the wet filters was determined in a gamma counter at an efficiency of 75%. All values in binding experiments are the average of triplicates. Nonspecific binding was defined as binding in the presence of 10 µM propranolol. The equilibrium dissociation constant (K_D) and the maximum binding capacity (B_max) were determined by Scatchard analysis. Analysis of the curve for isoproterenol-induced displacement of [¹²⁵I]ICYP was made by the iterative least-squares curve fitting program, as previously described (Hattori et al., 1987). Results were analyzed to identify the presence of one or two classes of binding sites by comparing the sum of squares for each model. The most appropriate model was determined by statistical analysis using an F test of the sum of squares of the residuals.

Adenylate Cyclase Assay. Adenylate cyclase activity was determined in an assay that monitors the conversion of [-³²P]ATP to [³²P]cAMP according to the method of Salomon et al. (1974). The incubation mixture contained 40 mM Tris-HCl (pH 7.5), 0.05 mM cyclic AMP, 0.05 mM ATP, an ATP-regenerating system (5 mM creatine phosphate and 50 U/ml creatine phosphokinase), 0.25 mg/ml bovine serum albumin, 0.5 mM 3-isobutyl-1-methylxanthine, 5 mM MgCl₂, 1 mM dithiothreitol, 1 U/ml adenosine deaminase, [-³²P]ATP (1 µCi per assay; 30 Ci/mmol; PerkinElmer Life Sciences) and membrane protein (50-100 µg per assay). Various agents (isoproterenol, GppNHp, and colforsin dalopate) to stimulate adenylate cyclase activity were included in the incubation mixture. When isoproterenol was used as a stimulant, 0.1 mM GTP was added to the mixture. The final volume of incubation mixture was 100 µl. The mixtures were incubated for 10 min at 37°C, and the reaction was terminated by the addition of a 100 µl aliquot of stopping solution (containing 45 mM ATP, 1.3 mM cyclic AMP, and 2% SDS; adjusted to pH 7.5 by the addition of 2 M Tris base). Assays were performed in triplicate, and the results are expressed as picomoles of cyclic AMP per milligram of protein per 10 min. The assay was linear with regard to time and to protein concentration. Isolation of [³²P]cAMP was accomplished by sequential Dowex and Alumina chromatography using [³H]cAMP (23 Ci/mmol; Amersham Bioscinces UK, Ltd., Little Chalfont, Buckinghamshire, UK) as a recovery marker. The average recovery of cyclic AMP was approximately 60%.

Western Blot Analysis. Assessment of ARK, G_s, G_i, and G_i-2 contents was conducted using standard SDS-polyacrylamide gel electrophoresis and immunoblotting techniques. Briefly, membrane proteins prepared above were dissolved in an equal volume of 2-fold concentrated sample buffer containing 125 mM Tris-HCl (pH 6.8), 4% SDS, 10% sucrose, 10% 2-mercaptoethanol, and 0.004% bromophenol blue and boiled at 100°C for 3 min. These samples (5 µg) were run on SDS-polyacrylamide gel electrophoresis (8, 10, and 12% polyacrylamide gel for ARK, G_s, and G_i, respectively) and electrotransferred to polyvinylidene diflouride membranes. For ARK and G_i-2, the membranes were blocked for 60 min at room temperature in PBS-Tween buffer (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, and 0.1% Tween 20) containing 1% bovine serum albumin and then incubated overnight at 4°C with mouse monoclonal antibody specifically recognizing ARK1/2 (1:600; Upstate Biotechnology, Lake Placid, NY) and G_i-2 (1:400; NeoMarkers, Fremont, CA), respectively. For G_s and G_i, the membranes were blocked overnight at room temperature in PBS-Tween buffer containing 2% bovine serum albumin and then incubated for 60 min at room temperature with rabbit polyclonal antiserum specifically recognizing G_s (1:8000; Gramsch Laboratories, Schwabhausen, Germany) and G_i (1:300; Oncogene Research Product, Boston, MA), respectively. Then, the membranes were washed for 10 min three times with PBS-Tween buffer and incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (Bio-Rad Laboratories, Hercules, CA) diluted at 1:8000 for ARK, 1:5000 for G_i-2 or anti-rabbit antibody (Bio-Rad Laboratories) diluted at 1:8000 for G_s and G_i for 60 min at room temperature. After being washed for 10 min three times in PBS-Tween buffer, the blots were visualized with the enhanced chemiluminescence detection system (Amersham Bioscinces UK, Ltd.), exposed to X-ray film for 20 s, and analyzed by free software NIH image produced by Wayne Rasband (National Institutes of Health, Bethesda, MD). The intensity of total protein bands per lane was evaluated by densitometry. Negligible loading/transfer variation was observed between samples.

Chemicals. l-Isoproterenol hydrochloride and dl-propranolol hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO), and GppNHp was from Boehringer Mannheim GmbH (Mannheim, Germany). Temocapril hydrochloride was a gift of Sankyo Co. (Tokyo, Japan), valsartan was from Novartis Pharma AG (Basel, Switzerland), and colforsin daropate was from Nihon Kayaku Co. (Tokyo, Japan). Other chemicals used in this study were of the highest purity available from Sigma-Aldrich, Wako Pure Chemical Industries (Osaka, Japan), or Nakalai Tesque, Inc. (Kyoto, Japan).

Statistical Analysis. All data are presented in terms of means ± S.E. Statistical assessment of the data was made by Student's t test for unpaired data or one-way analysis of variance followed by the Tukey-Kramer multiple comparison test to locate differences between groups. A P value <0.05 was considered statistically significant for all analyses.

	Results

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Hemodynamics, Morphology, and Infarct Size. A summary of hemodynamic variables, cardiac chamber weights, and infarct sizes in rabbits 3 weeks after sham surgery or transmural MI without and with temocapril or valsartan treatment is presented in Table 1. The LCX ligation procedure reproducibly resulted in transmural infarction comprising 25 to 35% of the LV wall, but infarct sizes were similar in the MI groups given water, temocapril, or valsartan solution. Heart rate did not differ among groups. In rabbits with MI, systolic blood pressure was significantly reduced compared with sham-operated controls, whereas diastolic blood pressure and mean blood pressure were not changed. Neither temocapril nor valsartan caused further reduction in blood pressure in rabbits with MI. There was no significant difference in body weight among the four groups. The left and right ventricle-to-body weight ratios were significantly increased in untreated MI rabbits compared with controls (26 and 52%, respectively; P < 0.01). In temocapril- and valsartan-treated MI rabbits, however, the development of the cardiac hypertrophy was significantly blunted (P < 0.01 versus untreated MI).

LV Function. Echocardiographic studies showed marked differences in LV geometry between rabbits with MI and sham-operated rabbits. Thus, wall thickness and kinetics were decreased, and the LV cavity was prominently dilated in the heart of MI rabbits. As a result, LVDd and LVDs were significantly increased in the untreated MI group compared with sham ligation (Table 2). Furthermore, there was a significant reduction in ejection fraction in untreated MI rabbits. Temocapril or valsartan treatment resulted in a significant improvement of the increases in LVDd and LVDs in rabbits with MI. Ejection fraction showed a trend to increase with temocapril or valsartan treatment. The changes in cardiac index by MI and drug treatment paralleled those in ejection fraction, although the decrease in cardiac index in untreated MI rabbits was not statistically significant.

Plasma Norepinephrine Content. The concentrations of norepinephrine in the plasma of rabbits used for this study are shown in Fig. 1. The plasma norepinephrine level was 3.1-fold higher in untreated MI rabbits than sham-operated controls, reflecting increased systemic sympathetic nervous activation. Temocapril or valsartan treatment significantly reduced the plasma norepinephrine concentration nearly to the level of sham-operated controls.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Plasma norepinephrine concentrations in sham-ligated rabbits, untreated MI rabbits, and MI rabbits treated with temocapril and valsartan. Points are the means ± S.E. of five animals. **, P < 0.01 versus the value obtained with sham-ligated rabbits. #, P < 0.05 versus the value with untreated MI rabbits.

-Adrenoceptor Binding. The specific binding of [¹²⁵I]ICYP to myocardial membranes was saturable and of high affinity in each of the four groups (Fig. 2). Scatchard analyses of the data resulted in a straight line, which indicates a single population of binding sites (Fig. 3A). The number of -adrenoceptors, however, was significantly lower in myocardial membranes from untreated MI rabbits (114 ± 9 fmol/mg of protein; n = 5; P < 0.05) compared with sham-operated controls (165 ± 9 fmol/mg of protein; n = 5). The reduced myocardial -adrenoceptor number in MI rabbits was reversed to the same level as controls by temocapril or valsartan treatment (Fig. 3B). The K_D values for [¹²⁵I]ICYP were comparable among the four groups (sham ligation, 178 ± 14 nM; MI, 144 ± 16 nM; MI + temocapril, 142 ± 10 nM; MI + valsartan, 143 ± 9 nM; n = 5 for each group).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Saturation isotherms for [125I]ICYP binding to myocardial membranes prepared from sham-ligated (A), untreated MI (B), temocapril-treated MI (C), and valsartan-treated MI (D) rabbits. Total binding, specific binding, and nonspecific (in the presence of 10 µM propranolol) binding are plotted. Similar results were obtained with four additional experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   A, Scatchard plots obtained from representative saturation isotherms for [125I]ICYP binding to sham-ligated (open circles) and MI (closed circles) rabbit myocardial membranes. B, bar graph showing the -adrenoceptor densities (Bmax) in myocardial membranes prepared from sham-ligated, untreated MI, temocapril-treated MI, and valsartan-treated MI rabbits. Bars are the means ± S.E. of five experiments. *, P < 0.05 versus the value obtained with sham-ligated rabbits. #, P < 0.05 versus the value with untreated MI rabbits.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Agonist competition curves using 150 pM [125I]ICYP as a competing ligand and isoproterenol as an agonist in the absence (closed circles) and presence (open circles) of 100 µM GppNHp in myocardial membranes prepared from sham-ligated (A) and MI (B) rabbits. The data points represent means of triplicate determinations from a representative experiment. The lines are computer-generated curves fitting the observed data points. KH and KL, dissociation constants for high- and low-affinity competition; %RH and %RL, proportion of receptors adopting the agonist high- and low-affinity state.

Adenylate Cyclase Activity. Basal adenylate cyclase activity, which was measured without using any activator, in myocardial membranes prepared from MI rabbits was significantly lower than in those from sham-operated controls (760 ± 73 versus 997 ± 57 pmol of cAMP/mg of protein/10 min; n = 5 for each group; P < 0.05). Temocapril and valsartan normalized the decreased myocardial adenylate cyclase activity in MI rabbits (951 ± 57 and 874 ± 36 pmol of cAMP/mg of protein/10 min, respectively; n = 5 for each group). In the following text, adenylate cyclase activity stimulated with various agents is therefore expressed as a net increase in stimulation (basal subtracted) to adjust for differences in basal activity. In the presence of 0.1 mM GTP, the adenylate cyclase response to isoproterenol was significantly reduced in untreated MI rabbits compared with sham-operated controls through a wide range of isoproterenol concentrations (Fig. 5). Thus, the overall concentration-response curve for isoproterenol-stimulated adenylate cyclase activity was substantially shifted downward in rabbits with MI. Temocapril and valsartan significantly restored the reduction in adenylate cyclase activity in MI rabbits. Myocardial adenylate cyclase activity stimulated with 100 µM GppNHp, a nonhydrolyzable GTP analog, slightly decreased in MI rabbits, but the difference between sham-operated and MI rabbits was not statistically significant (971 ± 77 versus 740 ± 34 pmol of cAMP/mg of protein/10 min; n = 5 for each). Furthermore, myocardial adenylate cyclase activity stimulated with 10 µM colforsin daropate, a water-soluble forskolin derivative which directly activates adenylate cyclase (Hosono et al., 1990), was substantially the same between sham-operated and MI rabbits (3317 ± 302 versus 2677 ± 178 of pmol cAMP/mg of protein/10 min; n = 5 for each).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effect of isoproterenol on adenylate cyclase activity in myocardial membranes prepared from sham-ligated (open circles), MI (closed circles), temocapril-treated MI (closed triangles), and valsartan-treated MI (closed squares) rabbits. Points (means ± S.E.; n = 5) represent the net increases in cyclic AMP production (basal subtracted). *, P < 0.05 versus the value obtained with sham-ligated rabbits. #, P < 0.05 versus the value with untreated MI rabbits.

Expression of ARK1 Protein. ARK1 immunological detection was performed using C5/1.1. antibody, which recognized the ARK2 carboxyl terminus, sharing homology with ARK1. Although this antibody recognizes ARK1 and ARK2, the single band identified consistently in this study can be considered to represent ARK1 protein because it is the predominant isoform in most tissues, including the heart (Ungerer et al., 1994). Western blot analysis detected a band migrating at 80 kDa in both myocardial membranes from sham-operated and MI rabbits (Fig. 6). The results of densitometric analysis showed a 2.1-fold increase in ARK1 protein expression in MI rabbits compared with sham-operated controls. In MI rabbits treated with temocapril or valsartan, expression of ARK1 protein was reduced nearly to the level of sham-operated controls.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Immunoblot analysis of ARK1 protein in myocardial membranes prepared from sham-ligated, MI, temocapril-treated MI, and valsartan-treated MI rabbits. The upper trace shows representative blots indicating a marked increase in expression of 80-kDa band in MI. The lower trace shows the bar graph comparing the immunostained band for ARK1 in the four groups. Densitometric results are expressed as a percentage of the band obtained with sham ligation in each experiment. Bars are the means ± S.E. of five different experiments. **, P < 0.01 versus the sham ligation value. ##, P < 0.01 versus the MI value.

Expression of G Proteins. The G_s antiserum identified a single band estimated to be 45 kDa in rabbit myocardium, as demonstrated in our previous article (Matsuda et al., 2000). As presented in Fig. 7, quantitative analysis of immunoblots showed no significant difference in the amount of myocardial G_s protein between either untreated or drug-treated MI rabbits and sham-operated controls. Immunoblots obtained using the G_i antiserum showed a single band that migrated with an apparent molecular mass of 40/41 kDa (Fig. 8). Densitometric analysis revealed a 2.3-fold increase in myocardial expression level of G_i in MI rabbits compared with sham-operated controls. This increase in G_i protein in MI rabbits was nearly completely prevented by treatment with temocapril or valsartan (Fig. 8). There are known to be three distinct species of G_i, and regarding the expression in the heart, G_i-2 appears to be the dominant isoform found in ventricular myocardium (Holmer et al., 1989). Thus, the antibody that specifically reacts with G_i-2 was used, which identified one single band with a molecular mass of 40 kDa. The relative amount of immunodetectable G_i-2, as determined by densitometric scanning, was increased in MI rabbits to 244 ± 38% (n = 5; P < 0.01) of sham-operated control. Temocapril and valsartan resulted in a significant improvement in the MI-induced increase in the G_i-2 protein level (134 ± 28 and 129 ± 18%; n = 5 for each; P < 0.05 versus the untreated MI value).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Immunoblot analysis of Gs protein in myocardial membranes prepared from sham-ligated, MI, temocapril-treated MI, and valsartan-treated MI rabbits. The upper trace shows representative blots indicating no difference in expression of 45-kDa band among groups. The lower trace shows the bar graph comparing the immunostained band for Gs in the four groups. Densitometric results are expressed as a percentage of the band obtained with sham ligation in each experiment. Bars are the means ± S.E. of five different experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Immunoblot analysis of Gi protein in myocardial membranes prepared from sham-ligated, MI, temocapril-treated MI, and valsartan-treated MI rabbits. The upper trace shows representative blots indicating a marked increase in expression of the 40/41-kDa band in MI. The lower trace shows the bar graph comparing the immunostained band for Gi in the four groups. Densitometric results are expressed as a percentage of the band obtained with sham ligation in each experiment. Bars are the means ± S.E. of five different experiments. **, P < 0.01 versus the sham ligation value. ##, P < 0.01 versus the MI value.

	Discussion

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In this study, rabbits 3 weeks after the LCX ligation had evidence of chronic compensated HF, including the development of significant right ventricular hypertrophy, as revealed by the increased right ventricle-to-body weight ratio. Echocardiographic studies showed that the LCX ligation in rabbits produced marked LV dilation and global and regional systolic dysfunction. Our echocardiographic assessment showing cardiac dysfunction in MI rabbits is consistent with the results of echocardiographic examinations in previous studies with this animal model (Pennock et al., 1997; Maurice et al., 1999). Furthermore, previous work has stressed that the rabbit LCX-ligation model of MI-induced HF employed in this study appears to provide a recapitulation of the functional abnormalities found in human HF at 3 weeks post-MI, a relatively short time span (Maurice et al., 1999). ACE inhibition with temocapril did not completely improve LV systolic function in MI rabbits but significantly limited LV remodeling and the development of systolic dysfunction. Our results are in good agreement with those from published clinical reports. In patients with HF, long-term treatment with ACE inhibitors can prevent deterioration of LV systolic function without actually restoring it (St. John Sutton et al., 1994). The preventive effect of the AT₁ receptor blocker valsartan on deterioration of LV geometry and systolic performance in MI rabbits was found to be the same as that of ACE inhibition. Furthermore, both treatments similarly improved compensatory hypertrophy in noninfarcted areas including right ventricle. The similar effects of AT₁ receptor blockade and ACE inhibition on cardiac remodeling have been demonstrated previously in a rat model of anterior myocardial infarction (Schieffer et al., 1994).

Both temocapril and valsartan caused a marked inhibition of the increase in circulating plasma norepinephrine in rabbits with MI-induced HF. Consistent with this observation, ACE inhibition or AT₁ receptor blockade has been shown to diminish the elevated plasma norepinephrine levels in canine models of HF caused by coronary embolization (Sabbah et al., 1994) and rapid ventricular pacing (Spinale et al., 1997). HF is accompanied not only by systemic neurohumoral activation but also increased activity of the RAS leading to increased tissue angiotensin II concentrations (Hirsch et al., 1991). Increased tissue concentrations of angiotensin II could represent an important regulatory mechanism to enhance sympathetic nerve activity. Thus, activation of presynaptic AT₁ receptors with angiotensin II facilitates the release of norepinephrine from sympathetic nerve terminals (Clemson et al., 1994). This results in further increase in circulating norepinephrine levels. It should be noted that the inhibitory effects of temocapril and valsartan on plasma norepinephrine levels in our HF model were equivalent. Although bradykinin serves as a potent releaser of norepinephrine from cardiac sympathetic nerve endings, especially when its degradation is prevented by ACE inhibitors (Hatta et al., 1997), it is likely that ACE inhibition with temocapril affected the norepinephrine levels only by attenuating the stimulatory effect of angiotensin II on the sympathetic nerve activity.

It is well known that cardiac -adrenoceptor number is reduced in patients with HF (Bristow et al., 1982). MI-induced HF rabbits exhibited a significant reduction in myocardial -adrenoceptor density. The decreased myocardial -adrenoceptor number in this rabbit LCX-ligation model is primarily due to selective ₁-adrenoceptor down-regulation (Maurice et al., 1999), consistent with what has been seen in the failing human heart (Bristow et al., 1986). In addition to down-regulation of myocardial -adrenoceptors, we showed that the proportion of -adrenoceptors in the high-affinity state, determined by competitive radioligand binding with isoproterenol, was significantly reduced in HF, demonstrating an uncoupling of -adrenoceptors and G_s. Since ARK1 phosphorylates the agonist-occupied -adrenoceptor and thereby uncouples the receptor and G_s, thus attenuating the signal (Hausdorff et al., 1990; Inglese et al., 1993), ARK1 may play a role in down-regulation and functional uncoupling of myocardial -adrenoceptors in the setting of sustained sympathetic activation, as occurs in HF. Exposure to norepinephrine has been shown to evoke a significant up-regulation of ARK1 mRNA in rats (Ungerer et al., 1996). Thus, the increased plasma norepinephrine content could accelerate activation of ARK1. In fact, there was a significant relation between plasma norepinephrine content and cardiac ARK1 expression in the progression of HF in rats produced by coronary ligation (Ishigai et al., 1999). We found that both temocapril and valsartan prevented the increase in ARK1 expression in the rabbit model of MI-induced HF. Furthermore, the total number of -adrenoceptors and the number of the receptors showing high-affinity agonist binding were significantly reversed by these agents. This resulted in normalization of myocardial adenylate cyclase response to isoproterenol, which was depressed in MI rabbits. In light of the view that the increased ARK1 seen in HF can be triggered by catecholamines (Iaccarino et al., 1998), the preventive effects of temocapril and valsartan on myocardial expression of ARK1 may solely result from their abilities to lower plasma norepinephrine levels in rabbits with MI. To our knowledge, the present data represent the first published evidence that ARK1 is a potential target for ACE inhibitors and AT₁ receptor blockers to treat HF.

There appears to be general agreement that cardiac G_s is quantitatively unaltered in HF (Brodde et al., 1995). Our Western blotting also revealed no change in G_s expression in myocardium of rabbits with HF. On the other hand, the current study, other animal studies of experimental HF, and studies of human HF demonstrate increases in G_i protein and/or mRNA levels (Feldman et al., 1988; Brodde et al., 1995; Maurice et al., 1999). The mechanisms underlying the increase in G_i expression are not fully understood, but it is speculated to be a consequence of the increased activity of the sympathetic nervous system and hence increased norepinephrine levels. This is based on the findings that chronic treatment of rats with isoproterenol increased myocardial G_i-2 and G_i-3 mRNA (Eschenhagen et al., 1992). Since there is a site on the promoter region of the G_i-2 gene for the cyclic AMP-activated transcriptional factor AP-2 (Weinstein et al., 1988), the increased norepinephrine levels via increasing cyclic AMP and activating the cyclic AMP-response element in the G_i-2 gene may increase its gene expression in HF. This would explain the reason why temocapril and valsartan inhibited the increase in myocardial content of G_i, particularly G_i-2, in rabbits with HF.

Basal adenylate cyclase activity was significantly diminished in myocardial membranes from MI rabbits. This may be related to the increase in G_i. In this connection, it is interesting to note that treatment with pertussis toxin can reverse the decrease in basal adenylate cyclase activity in membranes from failing human heart (Feldman et al., 1988). Thus, temocapril and valsartan improved the diminished basal level of myocardial adenylate cyclase activity in HF, possibly by eliminating the tonic inhibition of G_i. The level of adenylate cyclase activity stimulated with GppNHp should represent a balance of the activities of G_s and G_i. Because of the greater intrinsic activity of G_i compared with G_s for guanine nucleotide analogs (Bokoch et al., 1984), it might be expected that HF could shift the G_s/G_i balance in favor of G_i by the increase in G_i expression, thereby resulting in a decrease in adenylate cyclase activity stimulated with GppNHp. Activation of myocardial adenylate cyclase by GppNHp showed a trend to decrease in untreated MI rabbits compared with sham-operated and temocapril- or valsartan-treated MI animals. As the difference among groups was not statistically significant, however, we interpret these results to indicate that levels and function of G_s are fully operative in our animal model with HF. In addition, the lack of defect in stimulation of adenylate cyclase activity by colforsin daropate would represent no impairment in the levels and activity of the catalytic unit of adenylate cyclase in this HF model.

It is possible that the antiadrenergic properties of temocapril and valsartan are an important component of the therapeutic efficacy of these agents in HF. Thus, their antiadrenergic properties could potentially contribute to some of the beneficial effects of these agents in HF. The improved exercise performance that would be observed with ACE inhibitors and AT₁ receptor blockers may be related to the restoration of the -adrenergic signaling pathway in the heart, just as has been reported with -blocker therapy (Heilbrunn et al., 1989). Furthermore, lowering cardiac adrenergic drive appeared to have a cardioprotective effect (Bristow et al., 1986). This may partly explain the reason why ACE inhibitors and AT₁ receptor blockers, such as -blockers, can improve survival in patients with HF (The CONSENSUS Trial Study Group, 1987; Cohn and Tognoni, 2001).

In summary, the rabbit model of MI-induced HF exhibited diverse derangements in myocardial -adrenoceptor signaling pathway, including -adrenoceptor down-regulation, -adrenoceptor-G_s uncoupling, increased ARK1 expression, and increased G_i expression, that are mostly present in human HF. These derangements in -adrenergic signaling appeared to be associated with elevated activation of the sympathetic nervous system. ACE inhibitor and AT₁ receptor blocker treatment equally led to a complete reversal of the -adrenergic signaling derangements in the heart, which was possibly due to normalization of adrenergic drive. The restoration of -adrenergic balance in the heart may be a relevant mechanism by which ACE inhibition and AT₁ receptor blockade exert beneficial effects in HF.