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Cardiovascular Research 1997 33(1):98-109; doi:10.1016/S0008-6363(96)00190-3
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Coronary artery stenosis in rats affects β-adrenergic receptor signaling in myocytes

Leonard G. Meggsa, Har-er Huanga, Baosheng Lia, Peng Lia, Joseph Coupetb, Carl V. Hambya, Masa Akanumac, Yoshihiro Ishikawac and Piero Anversaa,*

aDepartments of Medicine and Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA
bCardiovascular Research Laboratory, Lederle, Pearl River, NY 10965, USA
cDepartment of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA

Received 30 April 1996; accepted 15 August 1996


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The purpose of this study was to determine whether the early chronic ischemic cardiomyopathy produced by non-occlusive coronary artery constriction was characterized by alterations in the regulation of β-adrenoreceptor (β-AR) signaling. Methods: Coronary artery narrowing was surgically induced in rats and the animals sacrified at 7 and 14 days. The changes in the biochemical properties of the multiple components of the β-AR pathway were examined in enzymatically dissociated myocytes. Results: Coronary stenosis, involving an average 55% reduction in luminal diameter, was associated with left ventricular failure and right ventricular dysfunction at both time intervals. A decrease in the quantity of β-AR was detected at 7 days and preceded the loss of high-affinity binding sites. This regulatory modification was characterized by a reduction in β1 and β2 receptors and a shift in the isoproterenol dose response curve indicating a functional correlation between the decrease in β-AR and attenuated inotropic support of the myocardium. The percentage of β-AR binding agonist with high affinity decreased significantly at 14 days along with a further reduction in the density of β1 and β2 receptors. Reconstitution studies with cyc–S49 lymphoma cells did not detect an impairment of Gs{alpha} functional activity, but the quantity of Gi{alpha} was increased at both intervals. Finally, activation of the catalytic unit of adenylyl cyclase by forskolin and GTP was not altered by coronary stenosis, however, basal cyclic AMP in myocytes was depressed at 14 days. Conclusions: Coronary stenosis induces distinct and progressive modifications in the β-AR signaling cascade which may contribute to the impaired ventricular performance in this model of myocardial ischemia.

KEYWORDS Ventricular function; Adrenergic receptors; G-proteins; c-AMP; Rat; Cardiomyopathy; Coronary stenosis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Studies in rodents have indicated that sudden constrictions of the left coronary artery of moderate degree are associated with ECG changes, consistent with transmural myocardial ischemia and marked depression in ventricular performance [1]. The hemodynamic impairment has been shown to persist subacutely and chronically in combination with alterations in contractile protein enzyme activity, myocardial mechanics, loss of myocytes and extensive ventricular remodeling [1, 2]. However, the magnitude of myocyte cell loss is modest, involving at most 10–20% of cells over a period of 1 month [2]. Attenuation in the transmission of adrenergic signals to myocytes via surface β-adrenoreceptor (β-AR) signaling has also been found[3]. However, this defect was observed in the presence of severe ventricular dysfunction, 5 months after coronary artery narrowing (CAN), confirming multiple observations made in the failing heart in humans [4]. Although the changes in the β-AR adenylate cyclase complex in pressure overload hypertrophy [5] and following myocardial infarction [6–8] have been characterized, the role of this system in the early chronic ischemic cardiomyopathy associated with non-occlusive coronary artery constriction is currently unknown. Such a condition leads to an increase in diastolic wall stress only and the development of decompensated eccentric hypertrophy [2]. In particular, it remains to be determined whether short-term diastolic overload secondary to myocardial ischemia is associated with a change in β1-adrenergic receptors on the stressed myocytes of the left ventricular myocardium and whether this proximate event is coupled with corresponding changes in cyclic AMP generation in these cells. Moreover, since β1-postreceptor signaling is modulated by the interaction of the guanine nucleotide binding proteins, Gs{alpha} and Gi{alpha}, with adenylyl cyclase [9], changes in the relative quantities of these regulatory proteins and in the functional activity of Gs{alpha} may have significant implications for the transduction of adrenergic signals in myocytes. It should be recognized that the analysis of these receptors and postreceptor properties is complicated by the heterogeneity of the cell populations in the myocardium which is amplified in ischemic cardiomyopathy [2]. Proliferation of fibroblasts and endothelial cells, and myocytolytic necrosis require that these determinations be performed in isolated myocytes only. These were the objectives of the present study.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animals
Male Sprague-Dawley rats at 2 months of age (Charles River Breeding Laboratories, North Wilmington, MA) were used. CAN was performed in 200 animals; 96 sham-operated rats served as controls. Mortality rate in CAN rats was nearly 50% due to acute ventricular failure and pulmonary edema [2, 10]. Forty-five rats in this group were sacrificed 1 week after CAN whereas 51 were killed at 2 weeks. Forty-seven sham-operated rats were used at 1 week and 49 at 2 weeks. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1985).

2.2. Coronary artery constriction
Under ether anesthesia, thoracotomy was performed, the left coronary artery located and a suture positioned around the vessel 1–2 mm from its origin. An external wire of 275 µm was held in contact with the wall of the exposed coronary artery. The vessel and the wire were ligated and the wire quickly removed to leave a constricted lumen[2, 10]. Control rats were treated similarly except that the ligature was not tied.

2.3. Functional measurements
Rats were anesthetized with chloral hydrate (300 mg/kg, i.p.), and the external right carotid artery was cannulated with a microtip pressure transducer (model PR 249, Millar Instruments, Houston, TX) which was advanced into the left ventricle for the evaluation of left ventricular pressures and dP/dt. An additional microtip pressure transducer was inserted in the right jugular vein and advanced into the right ventricular chamber for the measurements of right ventricular pressures and dP/dt[2, 10]. Such a characterization of the functional properties of the heart was restricted to 43 sham-operated rats, and 23 and 19 animals at 1 and 2 weeks following CAN.

2.4. Myocyte isolation
Hearts were rapidly excised and myocytes from the left ventricle were enzymatically dissociated [10, 11]. Rectangular, trypan blue excluding cells constituted nearly 80% of all myocytes. The average number of myocytes collected from the left ventricle of sham-operated rats was 17 x 106. The corresponding value in coronary narrowed rats was 12 x 106. The extent of non-myocyte cells present in each preparation in each ventricle was determined by preparing smears of the isolated myocytes which were stained with hematoxylin and eosin. The contribution of interstitial cells was assessed by counting 1000 cells in each preparation and then computing from these counts the respective fractions of myocytes and non-myocytes [10]. Non-myocytes comprised less than 2% of the cell population.

2.5. Myocyte membrane preparation for β-AR
Purified myocytes (2.0 x 106 cells) were homogenized with a Brinkman Polytron (setting 8 x 15 s x2.0) in ice-cold sucrose, 0.25 M; histidine, 0.03 M; EDTA, 1 mM; phenylmethylsulfonyl fluoride (PMSF) 0.1 M buffer. The suspension was centrifuged at 45 000 x g, the supernatant was discarded and the pellet resuspended. Protein concentrations were determined [12] and membranes were stored at –70°C.

2.6. β-AR antagonist binding assay
The radioligand 125I-cyanopindolol (CYP) (specific activity 2200 Ci/mmol: New England Nuclear, Boston, MA), was employed to label β-adrenoreceptors in myocyte membranes. The reaction tubes contained 40–50 µg of membrane protein and increasing concentrations of [125I] CYP in a total volume of 250 µl of reaction buffer (Tris HCl, 50 mM; (pH 7.4); MgCl2, 10 mM; PMSF, 0.1 mM; and EDTA, 1 mM). The reaction was initiated by the addition of membrane protein and continued for 45 min at 37°C. Bound radioactivity was determined by gamma counter. At ligand concentrations equivalent to the KD, specific binding averaged 75–80%. All values in the figures and tables refer to specific binding. To establish whether CAN was associated with a change in the absolute number of β-AR per cell, left ventricular myocytes were washed 5 times with incubation buffer and 1.0 x 105 cells aliquoted into assay tubes. Cell protein was precipitated by the addition of 250 µl of 12% trichloroacetic acid. The suspension was centrifuged at 2000 x g for 15 min. The pellet was resuspended in 0.1N NaOH to determine protein content [12]. Bmax was then converted from fmol per mg protein to binding sites per cell using Avogadro's number: 6.0225 x 1023. At the 1 week interval, 5 CAN and 7 sham-operated rats were included. Corresponding numbers at 2 weeks were 9 controls and 7 CAN.

2.7. β-AR agonist binding studies
Competition curves were performed with the non-selective β-agonist, l-isoproterenol. The reaction tubes contained 40–50 µg of membrane protein, 70 pM [125I]CYP and varying concentrations of l-isoproterenol in a total volume of 250 µl of reaction buffer (Tris-HCl, 50 mM (pH 7.4); MgCl2, 10 mM; PMSF, 0.1 mM; and EDTA, 1 mM). The reaction was initiated by the addition of membrane protein and continued for 45 min at 37°C. The binding assay was terminated by rapid vacuum filtration over glass fiber filters (Gelman Sciences Inc., Ann Arbor, MI). Assays were performed in duplicate with 15 concentrations of l-isoproterenol (10–9 to 10–4 M). Binding data were analyzed by the Ligand program [13]. The F-test was used to determine the best fit, 2 site vs. 1 site model. At 1 week, 10 CAN and 10 control rats were included. At 2 weeks, 10 control and 10 CAN rats were used.

2.8. β1- and β2-receptor subtypes
The relative proportion of β1 and β2 binding sites on myocyte membrane was determined as described by Molinoff[14]. Competition curves were performed with the non-selective radioligand [125I]CYP and the highly selective β1-antagonist, ICI 89406 [15, 16]. The concentration of antagonists varied from 10–9 to 10–4 M. Assays were performed in duplicate with 15 concentrations of ICI 89406. The reaction tubes contained 40–50 µg of membrane protein, 70 pM [125I]CYP and ICI 89406 in a total volume of 250 µl. The reaction was initiated by the addition of membrane protein and continued for 45 min at 37°C. The binding data were analyzed by the Ligand program [13] to determine high- and low-affinity binding constants and the percentage of high- and low-affinity binding sites. The KD value for [125I]CYP was derived by Scatchard analysis and held constant (70 pM), while ICI 89406 was allowed to float to a value for the best fit. At 1 week, 10 CAN and 10 sham-operated rats were included. At 2 weeks, 10 control and 10 CAN rats were utilized.

2.9. GS{alpha} activity
Membranes of Gs{alpha}-deficient cyc-S49 lymphoma cells[17] were mixed with Gs{alpha} extracted from myocytes [17, 18]. Gs{alpha} was extracted by homogenizing cells in a buffer containing: HEPES, 25 mM (pH 8.0); EGTA, 2 mM (pH 8.0); PMSF, 10 mM; leupeptin, 10 mg/ml; egg white trypsin inhibitor, 5 U/ml; DTT, 1 mM. The crude homogenate was spun 500 x g for 10 min at 4°C. The supernatant was centrifuged 100 000 x g for 40 min at 4°C. The pellet was resuspended in a buffer containing HEPES, 25 mM (pH 8.0); EGTA, 1 mM (pH 8.0); PMSF, 1 mM; leupeptin, 5 mg/ml; egg white trypsin inhibitor, 5 U/ml; DTT, 1 mM; lubrol, 0.1%; and incubated at 4°C for 1 h. The extract was incubated at 30°C for 1 h to inactivate adenylyl cylase, and frozen at –20°C. cyc –S49 lymphoma cells were grown at 37°C in Dulbecco's modified Eagle's medium containing 10% heat-inactivated horse serum and 1% penicillin/streptomycin.

2.10. Reconstitution assay for Gs{alpha}
The method of Salomon was used to measure adenylyl cyclase activity [19]. The reaction mixture was composed of: 20 µl of Gs{alpha} protein (20 µg); 20 µl of cyc –S49 cell membranes (20 µg); HEPES, 20 mM (pH 8.0); MgCl2, 5 mM; EDTA, 0.5 mM; cyclic AMP, 0.1 mM; phosphocreatine, 1 mM; GTP{gamma}S, 100 mM; [32PP]ATP (1.0 x 106 cpm/tube); [3H] cyclic AMP (1.0 x 104 cpm/tube); in a total volume of 100 µl. The reaction was terminated by adding 100 µl of 2% SDS and 800 µl of distilled H2O. [32P] cyclic AMP was then fractionated using Dowex-aluminum sequential column chromatography. The intrinsic adenylyl cyclase activity in Gs{alpha} extracts was negligible. Recovery of [3H] cyclic AMP ranged from 40 to 70%. At 1 week, 4 CAN and 4 sham-operated rats were included. At 2 weeks, 4 control and 4 CAN rats were used.

2.11. Cyclic AMP assay
Agonist-stimulated cyclic AMP generation was measured by radioimmunoassay (Amersham Corp., Arlington Heights, IL). Cells were washed 5 times with an incubation buffer comprised of: NaCl, 117 mM; KCl, 5.7 mM; NaHCO3, 4.4 mM; NaH2SO4, 1.5 mM; MgCl2, 1.7 mM; HEPES, 21.1 mM; glucose, 11.7 mM; amino acids, vitamins, ascorbate 0.05 mM; L-glutamine, 2 mM; CaCl2, 5 µM; insulin, 21 mU/ml; and 0.5% bovine serum albumin. After the final washing, 1.0 x 105 cells were aliquoted into assay tubes. A preincubation period of 30 min preceded initiation of the reaction with 1-isoproterenol. Cyclic AMP formation was determined under basal condition and in response to forskolin (10–4 M), GTP (10–4 M), and l-isoproterenol (10–6 M) and GTP. In a separate set of experiments, dose-response curves were generated by exposing myocytes to increasing concentrations of isoproterenol (10–9-10–4 M) was also determined. The reaction was performed in total volume of 250 µl by incubation at 34°C for 6 min and was terminated by the addition of 250 µl of 12% trichloroacetic acid. Myocytes were sonicated for 15 s at 50% of maximum frequency. The mixture was centrifuged at 2000 x g for 15 min and the pellet saved for protein determination by the method of Lowry. The supernatant was washed 4 times with 2 ml of water saturated ether, ether was evaporated from samples with nitrogen gas and the samples were dried for 3 h in a Speed Vac Concentrator (Savant Instruments, Inc., Hicksville, NY). The dried samples were stored at –20°C until determinations of cyclic AMP by radioimmunoassay were performed. Radioactive counts were determined by gamma counter and expressed as pmol/mg protein/min. At 1 week, 8 CAN and 6 sham-operated rats were included. At 2 weeks, 6 control and 8 CAN rats were used.

2.12. Immunoblot assay
Antisera recognizing rat Gi{alpha}1, Gi{alpha}2 and Gi{alpha}3 subunits were prepared by immunizing rabbits with the C-terminal decapeptide of transducin (KENLKDCGLF) conjugated to keyhole limpet hemocyanin [20]. Immune sera were tested in the immunoblot assay for reactivity with recombinant human Gs{alpha}, Gi{alpha}1, Gi{alpha}3, and mouse Gi{alpha}2 proteins (gift of Dr. Alfred G. Gilman) expressed in lysates of BL21 (DE3) E. coli bacteria. The specificity of antisera reactivity to G proteins was demonstrated by the ability of the immunizing peptide to block antibody binding to G proteins. Moreover, the immune anti-Gi{alpha} sera reacted to immunoblots with recombinant Gi{alpha}1, Gi{alpha}2 and Gi{alpha}3 proteins and did not react with recombinant Gs{alpha} proteins. Myocyte membranes were loaded in an amount of 50 µg/well on 12% acrylamide gels and separated by SDS PAGE [21]. Proteins were transferred to PVDF membranes by semi-dry blotting with transfer buffer (Tris HCl, 50 mM; glycine, 38 mM [pH 8.3], 20% methanol) at 10 V constant voltage for 1 h. Membranes were blocked in Tris-buffered saline (TBS, 20 mM Tris, NaCl, 137 mM; pH 7.6) containing 5% dry milk for 1 h, followed by 1 h incubation with anti-Gi{alpha} immune sera, diluted 1:100, and anti-Gs{alpha} antisera, diluted 1:1000 in 5% dry milk TBS containing 0.05% Tween-20 (TBS-T). Following 4 washes with TBS-T, membranes were incubated 1 h with horseradish-peroxidase-conjugated goat anti-rabbit immunoglobulin affinity-purified antibodies (Jackson ImmunoResearch, West Grove, PA) diluted 1:2000 in 5% dry milk TBS-T. Membranes were washed 5 x with TBS-T, processed with a chemiluminescent kit (Amersham Corporation, Arlington Heights, IL) and exposed to Hyperfilm ECL. At 1 week, 8 CAN and 8 sham-operated rats were included. At 2 weeks, 5 control and 5 CAN rats were used.

2.13. Quantitation of G protein
The intensity of G protein bands were quantitated by volume integration of Hyperfilm-ECL images (Jandel Scientific, Corte Madera, CA) in which the optical density of each band was averaged over a constant, predetermined area and expressed on a relative densitometric scale ranging from 0 to 100 units. The linear response range of the ECL assay was determined by regression analysis of G protein bands obtained by immunoblotting serial 2-fold dilutions of recombinant G proteins and myocyte membranes (Fig. GR1). The ECL signal for recombinant Gi{alpha} proteins was linear over the range from 12.5 to 50 ng of protein (r = 0.98) and the ECL signal for Gi{alpha} proteins in myocyte membranes was linear between 12.5 and 50 µg of membrane protein (r = 0.97). To correct for differences in loading, a correction factor was calculated for each sample by dividing the intensity of the major peak in the SDS PAGE by the intensity of the peak averaged across all samples (Fig. GR2). The intensity of the G protein ECL signal for each sample was normalized to the amount of protein loaded.


Figure 1
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  		Fig. GR1 Linearity of the ECL immunoblot assay for myocyte membrane protein and recombinant Gi{alpha}2 proteins. Panel A illustrates the ECL signals generated from myocyte membranes in the amounts of 100, 50, 25, 12.5, 6.2 and 3.1 µg in lanes 1–6, respectively, and from recombinant Gi{alpha}2 proteins in the amounts of 100, 50, 25, 12.5, and 6.2 ng in lanes 7–11, respectively. The limits for detection of Gi{alpha} proteins were 12.5 µg of myocyte membranes and 12.5 ng of recombinant protein. Panel B demonstrates that the ECL signal was linear over the range of 12.5–50 µg of myocyte membrane protein ({triangleup}, r = 0.97) and over the range of 12.5–50 ng of recombinant protein ({circ}, r = 0.98). r-Values were calculated by regression analysis.

 

Figure 2
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  		Fig. GR2 Amido black protein staining of individual myocyte samples following SDS PAGE and transfer to PVDF membranes. Left ventricular myocyte membranes from 5 different rats (lanes 1–5) were blotted onto PVDF membranes and stained with 0.1% amido black. A major protein band (arrow) in each profile was quantitated by volume integration with an optical image analysis system to determine the relative amounts of protein loaded on acrylamide gels for the immunoblot assay. Correction factors were calculated for each sample from the densitometric data to adjust for the variations in protein loading among different samples.

 
2.14. Statistical analysis
Values are reported as means ± s.d. Significance between 2 measurements was determined by Student's t-test. Significance in multiple comparisons among independent groups of data, in which analysis of variance and the F-test indicated the presence of significant differences, was evaluated by the Bonferroni method [22]. P-values < 0.05 were considered to be significant. Values of n for each parameter are listed in the text or the legend of each figure.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Ventricular function
The experimental procedure used resulted in a 56 ± 11 and a 54 ± 12% reduction in the luminal diameter of the left coronary artery near its origin at 1 and 2 weeks after surgery, respectively [23]. These changes in linear dimension corresponded to an 81 and 79% decrease in mean cross-sectional area of the coronary artery lumen. These determinations have been obtained in separate groups of animals exposed to an identical protocol since the cell isolation methodology did not allow the assessment of the degree of CAN. The functional characteristics of rats employed for the estimation of coronary narrowing were essentially identical to those described below for the animals utilized in this study. The surgical procedure and coronary stenosis for a period of 7 days did not alter body weight in controls (C = 310 ± 13 g) and experimental rats (E = 290 ± 22 g). Similarly, heart weight was significantly different in the 2 groups of animals (C = 914 ± 57 mg; E = 935 ± 69 mg). The changes in body weight (C = 346 ± 17 g; E = 337 ± 25 g) and heart weight (C = 959 ± 63 mg; E = 987 ± 78 mg) at 14 days after CAN were also not statistically significant.

Table 1 lists only one group of control animals since no difference was found in the hemodynamic profile of sham-operated rats at 1 and 2 weeks after surgery. Body weights of sham-operated rats, 47 at 7 and 49 at 14 days, were 286 ± 15 and 305 ± 24 g, respectively. Corresponding values for 45 CAN rats at 7 days and 51 CAN rats at 14 days were 297 ± 21 and 334 ± 27 g. Table 1 demonstrates that CAN at 7 days was associated with no change in heart rate and a 19% (P < 0.001), 22% (P < 0.001) and 21% (P < 0.001) reduction in systolic, diastolic and mean arterial pressures. In contrast, left ventricular minimal diastolic pressure and end-diastolic pressure were augmented 13.6-fold (P < 0.001) and 2.5-fold (P < 0.001) with coronary stenosis and these elevations were statistically significant. On the other hand, left ventricular systolic pressure was reduced by 18% (P < 0.001). In addition, left ventricular developed pressure, +dP/dt and –dP/dt were found to be decreased 30% (P < 0.001), 22% (P < 0.001) and 23% (P < 0.001), respectively. Similar changes in these physiologic parameters were seen at 2 weeks after narrowing. The effects of CAN on right ventricular function are also presented in Table 1. With the exception of right ventricular end-diastolic and developed pressures, and –dP/dt, the other indices of right side dynamics were not altered. In summary, CAN led to severe impairment of left ventricular performance and right ventricular dysfunction due to reduction in coronary blood flow reserve to the entire left ventricular free wall [1].


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  		Table 1 Effects of coronary artery narrowing (CAN) on cardiac performance

 
3.2. β-AR antagonist binding
Fig. GR3 illustrates the density of β-AR in myocytes isolated from the left ventricle of sham-operated and CAN rats. The total population of β-AR was reduced after CAN. The KD was not significantly different among the 3 groups: sham-operated, 0.07 ± 0.01 nM; CAN at 7 days, 0.07 ± 0.01 nM; CAN at 14 days, 0.07 ± 0.02 nM. The decrease in β-AR density detected by [125I]CYP reflects labeling of surface and internalized β-receptors. In addition, this variable did not consider the increase in cell protein which occurs with myocyte hypertrophy [1, 2, 10]. CAN was associated with a 31 and 53% increase in the quantity of protein in left ventricular myocytes at 7 and 14 days, respectively (sham-operated, 5.9 ± 1.3 ng/cell; CAN at 7 days 7.7 ± 1.3 ng/cell; CAN at 14 days 9.0 ± 1.5 ng/cell). Thus, the number of β-AR binding sites per cell was derived by determining the number of myocytes per mg protein and dividing this variable by Avogadro's constant[24]. This was then multiplied by the calculated Bmax. In control left ventricular myocytes, this parameter was 37 782 ± 1968 while in myocytes from CAN hearts a value of 39 632 ± 2174 and 39 556 ± 3266 was found at 7 and 14 days after surgery, respectively. The modest increase in β-AR binding sites following CAN was not significant. Thus, the increase in cell size was not accompanied by a corresponding increase in the number of β-AR binding sites per cell leading to a reduction in Bmax. In summary, CAN induced a progressive decrease in β-AR density per unit area of membrane, with practically no change in the total number of β-AR binding sites per cell.


Figure 3
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  		Fig. GR3 Illustrates the effects of coronary artery narrowing (CAN) on the density of β adrenergic receptors (Bmax) on left ventricular myocytes. Results are presented as mean ± s.d. * Indicates a statistically significant difference from control values, P < 0.05. SO = sham-operated controls; CAN7 = coronary artery narrowed rats at 7 days after surgery; CAN14 = coronary artery narrowed rats at 14 days after surgery; (SO, n = 4; CAN7, n = 4; CAN14, n = 4). Statistical comparisons were determined by the analysis of variance and the Bonferroni method.

 
3.3. β-AR agonist binding
1-Isoproterenol competition curves for sham-operated rats were best fit to a 2 site model in which high-affinity (KH) and low-affinity (KL) sites were present. The Ki for the high-affinity site was 27 ± 8 nM and Ki for the low-affinity site was 393 ± 80 nM (n = 4). Analysis of agonist binding curves performed 7 days after CAN also fit best with a 2-site model. The Ki for the high-affinity site was 65 ± 9 nM and Ki for the low-affinity site was 377 ± 188 nM (n = 4). The percentage of high and low-affinity binding sites was 65 ± 11 and 35 ± 11%, respectively, in control animals (n = 4). Corresponding values in CAN rats at 7 days after surgery were 70 ± 7 and 30 ± 7% (n = 4). These small differences were not statistically significant. Computer modeling of agonist binding 14 days after CAN followed an identical pattern. The Ki for the high-affinity site was 42 ± 19 nM and Ki for the low-affinity site was 233 ± 44 nM (n = 4). There was a significant decrease in the percentage of β-AR binding agonist with high-affinity at this time: sham-operated 65 ± 11%, CAN 36 ± 13% (n = 4; P < 0.001). In summary, CAN was not associated with a loss of high-affinity binding sites at 7 days; however, at 14 days the percentage of β-AR binding agonist with high affinity was markedly reduced.

3.4. β1 and β2 receptor subtypes
The proportion of β1 and β2 receptors was determined by computer modeling of competition curves performed with [125I] CYP and the highly selective β1 antagonist, ICI 89406. Binding data indicated that a 2-site model was the preferred fit for ICI 89406. There were no differences in the proportion of β1 and β2 receptor subtypes in sham-operated animals (β1: 78 ± 3.9%; β2: 22 ± 3.9%; n = 6) and CAN rats at 7 days (β1 79 ± 3.0%; β2 21 ± 3.0%; n = 6) and 14 days (β1: 80 ± 2.3%; β2: 20 ± 2.3%; n = 6). The Ki for β1 and β2 was 3.5 ± 0.5 and 334 ± 240 nM for control animals, and 4.9 ± 2.2 and 529 ± 243 nM at 7 days after coronary artery stenosis. Values of Ki for β1 and β2 at 14 days were 5.4 ± 2.2 and 491 ± 131 nM, respectively. None of these differences was statistically significant. The density of β1 receptors was calculated by multiplying Bmax by the fraction of β1 receptors obtained from ICI 89406 [125I] CYP competition curves [25]. A 21% reduction in β1 binding sites (Fig. GR4) was found 7 days after CAN and a 28% decrease at 14 days. Moreover, a 27 and 36% reduction in β2 binding sites was detected at 7 and 14 days after surgery, respectively. In summary, although the proportion of β1 and β2 receptor subtypes remained constant following CAN, β1 and β2 receptor density decreased.


Figure 4
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  		Fig. GR4 Effect of acute coronary stenosis on β1 (open bars) and β2 (hatched bars) receptor density on left ventricular myocytes. Results are presented as mean ± s.d. * Indicates a statistically significant difference from control values, P < 0.05. SO = sham-operated controls; CAN7 = coronary-artery-narrowed rats at 7 days after surgery; CAN14 = coronary-artery-narrowed rats at 14 days after surgery (SO, n = 6; CAN7, n = 4; CAN14, n = 4). Statistical comparisons were determined by the analysis of variance and the Bonferroni method.

 
3.5. Agonist-stimulated cyclic AMP generation
β-AR-mediated cyclic AMP formation was assessed by incubating myocytes with increasing doses of 1-isoproterenol (Fig. GR5). Cyclic AMP formation increased in a dose-dependent fashion in myocytes from sham-operated rats. Dose-response curves from CAN rats at 7 days were shifted to the right and depressed. A similar analysis at 14 days indicated a marked attenuation of cyclic AMP formation. As shown in Table 2, CAN at 7 days was not accompanied by an alteration in basal or forskolin- stimulated cyclic AMP levels in myocytes. However, at 14 days, basal cyclic AMP generation was reduced (P < 0.02). Similarly, 1-isoproterenol-stimulated cyclic AMP formation was depressed (P < 0.03). In summary, β-agonist-stimulated cyclic AMP generation was attenuated in myocytes 7 and 14 days after CAN, and basal cyclic AMP was reduced at 14 days only.


Figure 5
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  		Fig. GR5 Effects of acute coronary artery narrowing (CAN) on isoproterenol-stimulated cyclic AMP formation in left ventricular myocytes 7 and 14 days after surgery. The values shown represent the increments over basal activity. Dose-response curves were performed with increasing concentrations of the β agonist, 1-isoproterenol, 10–9–10–4 M, and ventricular myocytes. Results are presented as mean ± s.d. * Indicates a statistically significant difference, P < 0.05. Number of determinations at each point equals 3 or 4. Curves were generated by the least-squares non-linear curve fitting method. Statistical comparisons between corresponding points on the curves were determined by the two-tailed unpaired Student's t-test.

 

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  		Table 2 Effects of coronary artery narrowing (CAN) on basal and stimulated cyclic AMP formation in myocytes (fmol cAMP/mg/min)

 
3.6. Gs{alpha} functional activity
The functional activity of Gs{alpha} was assessed using membranes prepared from cyc–S49 lymphoma cells which are deficient in Gs{alpha} [17]. In preliminary studies, cyclic AMP generation was proportional to the amount of cyc–S49 membranes added to the membrane extract and cyclic AMP synthesis remained linear with time for 30 min. Similarly, cyclic AMP generation of cyc–S49 membranes was proportional to the quantity of cardiocyte membrane added (5–20 µg). CAN did not impair the functional activity of Gs{alpha} at 7 or 14 days after surgery (Fig. GR6). In summary, the functional activity of Gs{alpha} was not altered following CAN.


Figure 6
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  		Fig. GR6 Effect of acute coronary artery narrowing (CAN) on the functional activity of Gs{alpha} extracted from left ventricular myocytes 7 and 14 days after surgery. Results are presented as mean ± s.d. Number of determinations in each group equals 6. Statistical comparisons were determined by the analysis of variance and the Bonferroni method.

 
3.7. Immunoblotting with antibodies to Gi{alpha} and Gs{alpha}
Fig. GR7 shows a representative immunoblot of membranes prepared from left ventricular myocytes isolated from control and experimental animals. An increase of the 41 kDa immunoreactive protein was observed in myocytes of coronary-narrowed rats. Densitometric quantitation of the immunoblots showed a 48 and 58% increase of Gi{alpha} at the first and second time interval, respectively (Fig. GR8 A,B). A comparable analysis of immunoblots obtained by employing antibody to Gs{alpha} revealed a decrease of the combined 52 and 45 kDa immunoreactive proteins in myocytes of coronary-narrowed rats at 7 days, whereas no apparent difference was detected at 14 days (Fig. GR9 A). Densitometric quantitation of the immunoblots demonstrated a 41% decrease of Gs{alpha} in left myocytes at 7 days (Fig. GR9 A). In contrast, the change in this parameter at 14 days was not statistically significant (Fig. GR9 B). In summary, acute coronary artery constriction led to a sustained increase in the inhibitory regulatory protein, Gi{alpha}, and to an initial decrease and subsequent normalization in the stimulatory regulatory protein, Gs{alpha}, in left ventricular myocytes.


Figure 7
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  		Fig. GR7 Immunochemical detection of Gi{alpha} subunits in cardiac myocytes isolated from left ventricles of sham-operated and coronary-artery-constricted rats 14 days after surgery. Fifty micrograms of membrane extracts prepared from left ventricular myocytes of 3 different sham-operated rats (lanes 1–3) and 5 different coronary artery constricted rats (lanes 4–8) were separated by SDS PAGE on 12% polyacrylamide gels, transferred to PVDF membranes and processed to detect immunoreactive bands by enhanced chemiluminescence. An increase in the 41 kDa band corresponding to Gi{alpha} was apparent in experimental animals.

 

Figure 8
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  		Fig. GR8 Effects of acute coronary artery stenosis on the quantity of the inhibitory guanine nucleotide binding protein, Gi{alpha}, in left ventricular myocytes at 7 (A) and 14 (B) days after surgery. Results are presented as mean ± s.d. * Indicates a statistically significant difference, P < 0.05. C = sham-operated controls; CAN7 = coronary-artery-narrowed rats at 7 days after surgery; CAN14 = coronary-artery-narrowed rats at 14 days after surgery (7 days: C, n=8; CAN7, n=8; 14 days: C, n=5; CAN14, n=5). Statistical comparisons were determined by the two-tailed unpaired Student's t-test.

 

Figure 9
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  		Fig. GR9 Effects of acute coronary artery stenosis on the relative quantity of the stimulatory guanine nucleotide binding protein, Gs{alpha}, in left ventricular myocytes at 7 (A) and 14 (B) days after surgery. Results are presented as mean±s.d. * Indicates a statistically significant difference, P < 0.05. C = sham-operated controls; CAN7 = coronary artery narrowed rats at 7 days after surgery; CAN14 = coronary artery narrowed rats at 14 days after surgery. Immunoblots of Gs{alpha} proteins in myocyte preparations from 2 sham-operated rats (lanes 1 and 2) and from 2 coronary-artery-constricted rats (lanes 3 and 4) appear above the corresponding bar graphs (7 days: C, n = 8; CAN7, n = 8, 14 days: C, n = 5; CAN14, n = 5). Statistical comparisons were determined by the two-tailed unpaired Student's t-test.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Considerable evidence from in vitro systems supports uncoupling of β-AR from adenylyl cyclase as a proximate mechanism of catecholamine desensitization [9, 26]. Although limited information is available concerning β-AR coupling in heart failure, this adaptation has been documented in pressure [27, 28] and volume overload hypertrophy[29]. Moreover, uncoupling of β-AR was detected 1 day after the initiation of ventricular pacing in dogs, while β1 receptor density remained unchanged [30]. The alteration in agonist binding in this latter model preceded the elevation in plasma catecholamines and left ventricular dysfunction. The depressed activation of adenylyl cyclase was not limited to β agonist but was also observed with G protein and forskolin. In pressure overload, β-AR increases in number and undergoes functional uncoupling from adenylyl cyclase [31]. However, downregulation of β-AR has been a consistent finding of the failing left ventricle in humans [25], suggesting that this modification may reflect chronicity of the disease process [30]. The heightened state of adrenergic activity with ischemia is accompanied by the immediate release of norepinephrine within the myocardium [32, 33] and the rapid onset of ventricular dysfunction [1]. Although direct measurement of synaptic cleft norepinephrine is not feasible, the binding properties of postsynaptic adrenergic receptors reflect this variable [34]. We have previously reported that coronary stenosis of 7 days duration was associated with a downregulation of postsynaptic {alpha}1-adrenoreceptors and a proportionate reduction in norepinephrine-stimulated inositol phosphate generation [10]. The parallel decrease in {alpha}1 and β-AR density in this model supports the premise that neurotransmitter release is enhanced following coronary stenosis. In this regard, coronary artery constriction differs from models of heart failure in which both the onset of ventricular dysfunction and elevation of plasma catecholamines are temporally dissociated from the reduction in β-AR density [30]. However, it must be recognized that the present study does not document when the alterations in β-AR signal transduction actually occurred and that differences may exist between various models of heart failure.

A strength of the present study was the use of a highly purified preparation of adult rat ventricular myocytes to identify and estimate the relative proportion of β-AR subtypes. As previously reported, β1 and β2 receptor subtypes have been identified on cardiac muscle cells[25, 35], with a preponderance of the β1 receptor subtype[35]. Coronary constriction induced a coordinate decrease in the density of β1 and β2 receptor subtypes. A selective downregulation in β1 receptor density has been reported in the failing human myocardium and this modification has recently been shown to be correlated with a reduction in β1 mRNA abundance [25]. Alternatively, β2 receptor density may increase [30], remain unchanged [25] or, as shown here, decrease in the failing heart.

Questions have been raised concerning the significance of changes in the quantity of Gs{alpha} and Gi{alpha} on basal and agonist-stimulated cyclic AMP formation. The level of Gs{alpha} was decreased in myocytes 7 days after coronary artery constriction. However, this alteration was not associated with measurable effect on basal cyclic AMP formation. Conversely, at 14 days, when Gs{alpha} levels had returned to baseline, basal cyclic AMP was depressed. These perturbations in Gs{alpha} were coupled with an increase in Gi{alpha} at both time intervals. These findings raise several possibilities concerning the interaction of inhibitory and stimulatory transducers with the catalytic unit of adenylyl cyclase following CAN. First, a change in Gs{alpha} functional activity cannot account for the depressed basal cyclic AMP formation at 14 days, because reconstitution studies failed to identify such a defect. Secondly, forskolin activation of the catalytic unit was not altered at the 7- and 14-day intervals, implying that the integrity of this membrane component was preserved following coronary artery constriction. Thirdly, the apparent lack of correlation between a depressed Gs{alpha} level and basal cyclic AMP formation at 7 days may suggest that adult rat ventricular myocytes possess spare Gs{alpha}, which serves to offset perturbations in the balance of inhibitory and stimulatory transducers. The finding that basal cyclic AMP was depressed at the 14-day interval, in the absence of a detectable alteration in the quantity or functional activity of Gs{alpha}, casts doubt on the latter contention. The reduced rate of basal cyclic AMP may reflect the marked increase in Gi{alpha} content at 14 days.

The changes in Gi{alpha} content of myocytes reported here are similar to those observed after myocardial infarction during the phase of markedly impaired ventricular pump function [6]. The upregulation in Gi{alpha} levels in the infarcted heart was associated with an increase in Gi{alpha} functional activity, whereas Gs{alpha} content and activity were not altered. The mechanism of this upregulation in Gi{alpha} content has not been identified. However, the gene encoding Gi{alpha} protein has a cyclic AMP response element and it has been suggested that the increase in myocyte cyclic AMP levels early in heart failure may induce Gi{alpha} expression [36]. Of interest, the fold stimulation of cyclic AMP by isoproterenol was comparable in control and experimental myocytes; however, the absolute values were significantly different. A similar relationship has also been found by Kiuchi et al. [30], who reported that basal cyclic AMP levels decline with the duration of cardiac pacing in dogs along with a depression in β-agonist-stimulated cyclic AMP generation. Therefore, the progressive reduction in basal cyclic AMP in myocytes from failing hearts may condition the formation of cyclic AMP in response to β agonist.

The current study which examined the consequences of heart failure in its early stages differs from our previous work that addressed similar issues in the late phases of ventricular decompensation, 5 months after CAN [3]. In the earlier investigation, the reduction in β-AR density was established in crude membrane preparations which have severe limitations because multiple areas of replacement fibrosis are present in the myocardium chronically in this model [1, 2]. In addition, the potential impact of regulatory modifications of β1 and β2-adrenergic receptor subtypes on the contractile properties of the pathologic heart were not examined nor were the affinities of these receptors for 1-isoproterenol measured. This is a significant issue because activation of β1 and β2 receptors elicits different cellular responses at the level of ionic channels, myofilaments and sarcoplasmic reticulum [35]. Adaptations in these receptor subtypes in a state of heightened adrenergic activity may be critical determinants of myocyte performance. Importantly, the interaction of GS{alpha} and Gi{alpha} on the catalytic unit of adenylyl cyclase was not assessed in the previous work as well as the functional activity of GS{alpha}. This is relevant since increases in Gi{alpha} content were demonstrated here at 7 and 14 days after CAN, providing a mechanism for attenuation in the transmission of β-AR signals under this setting.

It must be acknowledged that mechanisms other than the net balance of inhibitory and stimulatory proteins may impose constraints on the catalytic unit of adenylyl cyclase, and impair the efficiency of signal transduction. Isoproterenol-induced cyclic AMP generation is subject to additional regulatory modifications including activation of β-AR, coupling of high-affinity receptors with Gs{alpha}, and stimulation of the catalytic unit. The availability of Gs{alpha} may be limited in hyperadrenergic states in which agonist occupation of β-AR is coupled with translocation of Gs{alpha} to the cytosol [37]. Moreover, increased levels of Gi{alpha} have not been a uniform finding in models of heart failure [37] and purified Gi{alpha} subunits expressed in E. coli do not depress adenylyl cyclase [38]. Additionally, the dissociated β{gamma} dimers of Gi{alpha} may inhibit the catalytic unit by forming a heterotrimer with free Gs{alpha} [39]. Finally, the recent cloning of adenylyl cyclase isoforms [40] has made it possible to examine the expression of mRNA species in the myocardium. A subfamily of cardiac adenylyl cyclases has been identified, composed of type 5 and 6 isoforms[40]. Downregulation of type 5 and 6 mRNAs in pacing-induced heart failure has been coupled with impaired activation of the catalytic unit by β-agonist, G-protein and forskolin [40]. Changes in the rate of transcription or in the stability of the messenger for these isoforms provide yet another potential mechanism by which activity of the catalytic unit may be modified in pathologic states of the myocardium.

In summary, CAN induced distinct and progressive defects in the β-AR signal transduction pathway. Downregulation of β1 and β2 receptor subtypes and perturbations in the quantities of Gi{alpha} and Gs{alpha}, precede the loss of high-affinity binding sites. These alterations are associated with a decrease in the response of myocytes to β agonist and hemodynamic parameters of left ventricular dysfunction. Regulatory modifications in β receptor subtypes and GTP binding proteins may represent proximate events in the depressed myocardial responsiveness to catecholamines following CAN.

4.1. Limitations of the current study
Several limitations have to be acknowledged and considered in the interpretation of the results. (1) The degree of coronary artery constriction induced surgically could not be determined in the same animals employed for the analysis of β-AR density and signaling. (2) The enzymatic dissociation of myocytes may have resulted in the preferential selection of one cell population which may not be representative of the entire heart and may have varied in the different groups of animals. (3) Cell death by ischemic necrosis may have occurred and these damaged myocytes may have been included in the samples used for the different determinations. (4) Since only 80% of the isolated cells retained their cylindrical configuration and were viable, the remaining non-viable component may have influenced the various measurements performed. (5) The protocol employed did not permit an assessment of the functional integrity of β-AR system at the organ level and it is possible that the signaling defects of myocytes may have been reversed by in vivo exposure to pharmacologic interventions. (6) The wide variability in the degree of coronary artery constriction inherent in this model is a factor which has to be taken into account in the evaluation of the data.


   Acknowledgements
 
The expert technical assistance of Maria Feliciano is greatly appreciated. This work was supported by NIH grants HL-38132, HL-39902, HL-40561 and P01-HL-43023.


   Notes
 
* Corresponding author. New York Medical College, Department of Medicine, Vosburgh Pavilion Rm. 302, Valhalla, NY 10595, USA. Tel. +1 914 993-4168; Fax +1 914 993-4406 Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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