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Circulation. 1995;91:2824-2833

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(Circulation. 1995;91:2824-2833.)
© 1995 American Heart Association, Inc.

Articles

Characteristics of the ß-Adrenergic Receptor Complex in the Epicardial Border Zone of the 5-Day Infarcted Canine Heart

Susan F. Steinberg, MD; HongLu Zhang, MPH; Elena Pak, BS; Geraldine Pagnotta, BS; Penelope A. Boyden, PhD

From the Departments of Medicine (S.F.S.) and Pharmacology (S.F.S., H.-L.Z., E.P., G.P., P.A.B.), Columbia University, New York, NY.

Correspondence to Susan F. Steinberg, MD, Department of Pharmacology, Columbia University, College of Physicians and Surgeons, 630 West 168 St, New York, NY 10032.


*    Abstract
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 *Abstract
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Background The effect of isoproterenol on increasing the peak amplitude of the L-type calcium current is reduced in myocytes dispersed from the epicardial border zone (EBZ) of the 5-day infarcted canine heart when compared with control cells from noninfarcted hearts. This suggests that specific alterations in the ß-adrenergic receptor complex develop in this setting. The present study is an examination of individual components of the ß-adrenergic receptor complex with the aim of elucidating the biochemical defect(s) that might be responsible for the diminished ß-adrenergic receptor responsiveness in the myocytes that survive in the infarcted heart.

Methods and Results We compared components of the ß-adrenergic receptor signaling pathway in membranes prepared from the EBZ of the 5-day infarcted heart and a remote, noninfarcted region (RZ) of the same ventricle as well as the corresponding regions of noninfarcted ventricles. Defects in multiple components of the ß-adrenergic receptor complex were confined to the EBZ of the 5-day infarcted heart. These include a decrease in ß-adrenergic receptor density; diminished basal, guanine nucleotide–, isoproterenol-, forskolin-, and manganese-dependent adenylyl cyclase activities; an increase in the EC50 for isoproterenol-dependent activation of adenylyl cyclase; a diminished level of the {alpha}-subunit of the Gs protein; and an elevated level of the {alpha}-subunit of the Gi protein.

Conclusions Defects in multiple components of the membrane ß-adrenergic receptor complex were identified in the EBZ of the 5-day infarcted canine heart. This constellation of abnormalities would be predicted to impair functional ß-adrenergic responsiveness and contribute to the defect in isoproterenol-dependent stimulation of the L-type calcium current in myocytes isolated from this tissue.


Key Words: receptors, adrenergic, beta • proteins • adenylyl cyclase • myocardial infarction


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Adrenergic receptors are regulated in a complex and dynamic fashion during myocardial ischemia as a result of elevated levels of endogenous catecholamines as well as the consequences of the ischemic event itself. Most studies have focused on an acute ischemic insult, which generally has been reported to lead to an increase in the density of functionally coupled cell surface ß-adrenergic receptors as a result of redistribution from a light vesicular fraction.1 2 3 4 However, the results are inconsistent, with several authors5 6 7 finding no change in ß-adrenergic receptor density using similar experimental paradigms of acute myocardial ischemia. Moreover, although the increase in ß-adrenergic receptor density appears to persist with more prolonged periods of ischemia, catecholamine responsiveness has been reported to decline as a result of inactivation of several of the postreceptor components of this signaling pathway (the stimulatory G protein Gs and the adenylyl cyclase enzyme).2 4 8 9

Whether the changes in the ß-adrenergic receptor complex persist in the subacute phase (5 days) after total coronary artery occlusion in dogs, a time when sustained reentrant ventricular arrhythmias occur,10 11 is not known. Electrophysiological studies designed to determine the effects of sympathetic nerve stimulation on refractoriness and induction of reentrant arrhythmias, in this model, have suggested that sympathetic nerve stimulation preferentially shortens refractoriness in noninfarcted parts of the left ventricle but exerts minimal effects on sites within the epicardial border zone (EBZ).12 These data can be interpreted as evidence for "functional denervation," which could be attributed to the loss of one or more components of the ß-adrenergic receptor complex in the myocytes that survive in the EBZ. However, this concept has not been assessed directly.

In contrast to the limited information regarding adrenergic receptor function in the subacute phase after total coronary occlusion, it is well established that the electrical properties of the epicardial muscle fibers from the healthy heart and from the EBZ 5 days after occlusion differ significantly. For example, fibers from the EBZ display a characteristic triangularization of the action potential, with loss of the rapid phase of repolarization as well as the plateau.13 14 15 Results of initial studies using conventional microelectrode recording techniques on multicellular EBZ muscle fibers suggested that the absence of the plateau phase in the epicardial muscle fibers that survive infarction is related to a decrease in the "slow inward current."16 Subsequent studies using voltage-clamp techniques and myocytes dispersed from the EBZ of the 5-day infarcted heart established that the L-type Ca2+ current is significantly reduced in myocytes from the EBZ.17 18 Moreover, recent studies have demonstrated that the effect of ß-receptor agonists to increase the peak Ca2+ current density is diminished in myocytes from the EBZ.18 19 The diminution in ß-adrenergic receptor responsiveness detected in these myocytes suggests that specific alterations in the ß-adrenergic receptor complex develop in this setting. Accordingly, the goal of the present study was to identify specific defects in the ß-adrenergic receptor complex that might underlie the diminished responsiveness to isoproterenol in the myocytes that survive in the infarcted heart.


*    Methods
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 *Methods
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Mongrel dogs of either sex (weight, 12 to 15 kg; age, 1 to 2 years) were used in the present study. Dogs were anesthetized, the pericardium was opened through a left thoracotomy, and the left anterior descending branch of the left coronary artery was totally occluded by the two-stage ligation technique of Harris.20 The chest was then closed, and the animals were allowed to recover. Five days after coronary occlusion, the dogs were reanesthetized, a cardiectomy was performed, and the gross infarct was visualized on the epicardial surface. Tissue was excised from adjacent sites of the EBZ. One section was used to isolate myocytes to investigate the electrophysiological abnormalities that develop in myocytes that survive in the arrhythmic, infarcted heart.19 The other section was frozen for subsequent biochemical studies. Additional tissue samples were obtained from a remote epicardial region (RZ) of the same ventricle. Finally, tissues also were obtained from identical anatomic sites of the left ventricle of noninfarcted hearts so that we would be able to identify any potential regional variations in components of the ß-adrenergic receptor complex that might confound interpretation of the data. In this case, the "EBZ" refers to a thin slice of epicardium of an identical size and from a location corresponding to the EBZ of the infarcted left ventricle. Tissues were frozen rapidly for subsequent membrane preparation and biochemical analyses. The experimental protocol was reviewed and approved by the Columbia University Institutional Animal Care and Use Committee.

Membrane Preparation
Tissues were trimmed of fat and connective tissue, weighed, minced, and homogenized twice for 10 seconds in 4 vol (wt/vol) of ice-cold homogenization buffer (0.25 mol/L sucrose, 0.03 mol/L histidine, and 1 mmol/L EDTA) with a Polytron (Brinkman Instruments, Inc). The crude homogenate was centrifuged at 1500g for 15 minutes to remove large tissue fragments, nuclear debris, and cellular organelles. The supernatant was recentrifuged at 43 000g for 45 minutes, and the pellet was resuspended in homogenization buffer at a protein concentration of 3 to 5 mg/mL. Membranes were stored in aliquots at -70°C. The average membrane protein yield was approximately 0.15% to 0.30% of the initial tissue wet weight and did not differ for EBZ and RZ of infarcted and noninfarcted hearts. Membrane fractions prepared in this manner retained stable receptor binding, adenylyl cyclase activity, and G protein immunoreactivity for at least 2 months but generally were used within 3 weeks of preparation. Membranes from the EBZ and RZ of six infarcted and four noninfarcted dogs were used for receptor binding studies. A separate set of membranes from nine infarcted and nine noninfarcted dogs was used for studies of adenylyl cyclase activity and G proteins. Immunoblot analyses of G protein {alpha}- and ß-subunits were performed on a subset of these membrane preparations as a result of limitations in the amount of epicardial membrane protein available for these analyses.

ß-Adrenergic Receptor Binding Assay
Cyanopindolol (CYP) was radioiodinated to a theoretical specific activity of 2200 Ci/mmol and purified according to methods published previously.21 Binding assays were performed essentially as described previously.22 Briefly, myocardial membranes (35 µg) were incubated for 60 minutes at 37°C with [125I]iodocyanopindolol (ICYP) (4 to 150 pmol/L) in a final volume of 1 mL. The assay buffer contained 0.15 mol/L NaCl, 0.01 mol/L KCl, 0.01 mol/L MgCl2, 0.001 mol/L EDTA, 2 mg/mL dextrose, 1 mg/mL bovine serum albumin, and 0.01 mol/L Tris, pH 7.4. ICYP bound to membrane protein was separated from free, unbound ICYP by rapid vacuum filtration of the entire 1-mL assay volume over glass-fiber filters (Gelman A/E, Gelman Sciences) followed by one wash with 10 mL of 10 mmol/L Tris, pH 7.4. Radioactivity trapped by the filters was detected with a Packard Autogamma Scintillation Spectrophotometer. Specific binding of ICYP, defined as the component of total binding that could be inhibited by excess unlabeled propranolol (1 µmol/L), constituted approximately 85% to 90% of total binding at concentrations of ICYP near the equilibrium dissociation constant. The equilibrium dissociation constant (Kd) and the maximal number of binding sites (Bmax) for ICYP were determined by Scatchard analysis of saturation binding isotherms.

Adenylyl Cyclase Assay
Adenylyl cyclase activity was determined in an assay that monitors the conversion of [{alpha}-32P]ATP to cyclic [32P]AMP as described previously.23 Incubation mixtures contained Tris (0.05 mol/L, pH 7.5), ATP (0.143 mmol/L), an ATP-regenerating system (10 µg creatine phosphate and 14 µg creatine phosphokinase), theophylline (8 mmol/L), MgCl2 (2.5 mmol/L), KCl (10 mmol/L), [{alpha}-32P]ATP (1 to 2x106 cpm per assay tube), and membrane protein (4 µg). MnCl2, forskolin, Gpp(NH)p, and isoproterenol were added at the concentrations indicated in individual experiments. Assays were performed in triplicate for 30 minutes at 37°C in a final volume of 75 µL and were linear with time and protein concentration. Reactions were terminated by the addition of 100 µL of cold stopping solution containing 4.5 mmol/L ATP, 1.4 mmol/L unlabeled cAMP, and 50 000 cpm cyclic [3H]AMP as an internal standard. cAMP was isolated by sequential Dowex and Alumina chromatography. cAMP recovery, as assessed by the recovery of cyclic [3H]AMP, ranged from 70% to 80%.

[32P]ADP-Ribosylation Assays
Assays of cholera toxin– and pertussis toxin–sensitive G proteins measured the incorporation of [32P]ADP-ribose from [32P]NAD into the appropriate molecular weight membrane proteins as described previously.24 Cholera toxin catalyzes the covalent incorporation of ADP-ribose into the {alpha}-subunit of the heterotrimeric stimulatory G protein Gs ({alpha}s), which couples the ß-adrenergic receptor to stimulation of adenylyl cyclase. Although alternative splicing of a single gene results in two forms of the {alpha}s protein (ie, a 45-kD and a 52-kD form25 ), in agreement with results published by another group,8 26 only the 45-kD substrate for cholera toxin–dependent ADP-ribosylation is identified in canine cardiac membranes. Pertussis toxin catalyzes the covalent incorporation of ADP-ribose into the three related forms of the {alpha}-subunit of the inhibitory G protein Gi ({alpha}i1, {alpha}i2, and {alpha}i3) as well as the {alpha}-subunit of Go ({alpha}o). Recent studies indicate that {alpha}i2 is the major Gi {alpha}-subunit expressed in canine ventricle27 and can be identified on autoradiography as an approximately 40-kD protein band.28

Cholera toxin was preactivated by incubation (at 1 mg/mL) in 25 mmol/L DTT for 20 minutes at 30°C. Labeling with cholera toxin was accomplished by incubating 25 or 50 µg of ventricular membranes in 50 µL of a 300 mmol/L sodium phosphate buffer (pH 7.5) containing 0.4 mmol/L EDTA, 0.8 mmol/L MgCl2, 100 µmol/L NADP, 10 mmol/L thymidine, 5 mmol/L ADP-ribose, 1 mmol/L ATP, 20 mmol/L arginine, 2.5 mmol/L DTT, 0.1 mmol/L Gpp(NH)p, 10 µmol/L [32P]NAD (2 µCi per assay tube), and 100 µg/mL activated cholera toxin for 30 minutes at 30°C. Labeling with pertussis toxin was accomplished by incubating 1 or 2 µg of ventricular membrane in 20 µL of a 50 mmol/L Tris-Cl (pH 8.0) buffer containing 2 mmol/L MgCl2, 1 mmol/L EDTA, 10 mmol/L DTT, 0.1% Lubrol PX, 10 mmol/L thymidine, 10 µmol/L [32P]NAD (1.5 µCi per assay tube), and 20 µg/mL pertussis toxin for 1 hour at 37°C. For each sample, reactions were linear with protein concentration under these assay conditions. Reactions were terminated by the addition of SDS-PAGE sample buffer and boiling for 5 minutes. Electrophoresis was performed on vertical slab gels (resolving gel 12%, stacking gel 4% acrylamide). After proportional counting of the gels using a Betascope Model 603 Blot Analyzer (Betagen Corp), pertussis toxin– and cholera toxin–sensitive G proteins were quantified by relating the number of counts in the band specifically labeled to the specific activity of the [32P]NAD and the protein concentration. All results represent the average of duplicate determinations on each preparation.

Immunoblotting
Samples (150 µg per lane) were electrophoresed on a 12% SDS–polyacrylamide gel and transferred to nitrocellulose. Prestained molecular weight markers were electrophoresed in parallel. Five G protein subunit–specific antisera were used in this study: anti-{alpha}common, a polyclonal antiserum, previously characterized as antiserum 1398,29 which is strongly reactive against all pertussis toxin–sensitive {alpha}-subunits; anti-{alpha}i1/{alpha}i2, an affinity-purified polyclonal antiserum raised against a synthetic peptide corresponding to the shared C-terminal decapeptide sequence of rat {alpha}i1 and {alpha}i2, which does not cross-react with {alpha}i3 or {alpha}o; anti-{alpha}s, an affinity-purified polyclonal antiserum directed against a synthetic peptide corresponding to the C-terminal decapeptide of rat {alpha}s, which specifically recognizes {alpha}s; anti-{alpha}o, a polyclonal antiserum directed against the amino-terminus of {alpha}o; and anti-ß, a polyclonal antiserum raised against an internal decapeptide sequence of the human ß-subunit, which recognizes both the 35- and 36-kD forms of the ß-subunit. These antisera raised against rat and human G protein subunits readily cross-react with canine G proteins, presumably due to the high degree of sequence homology of G proteins in mammalian cells. The nitrocellulose was incubated in 5% dry milk, 50 mmol/L Tris (pH 7.5), 200 mmol/L NaCl, and 0.05% Nonidet P-40 (blocking buffer) for 1 hour at room temperature to block nonspecific binding and then probed with a 1:200 (anti-{alpha}i1/{alpha}i2 and anti-{alpha}o) or 1:1000 (anti-{alpha}common, anti-{alpha}s, and anti-ß) dilution of G protein subunit–specific antiserum in 5% bovine serum albumin, 50 mmol/L Tris (pH 7.5), 200 mmol/L NaCl, 0.05% Nonidet P-40, and 0.02% NaN3 overnight at 4°C. The nitrocellulose was then washed five times, 5 minutes each, with 50 mmol/L Tris (pH 7.5), 200 mmol/L NaCl, and 0.05% Nonidet P-40 and then incubated in blocking buffer for 30 minutes at room temperature. To detect bound primary antibody, we incubated blots for 1 hour at room temperature with 125I-labeled goat anti-rabbit IgG F(ab')2 fragment at a final dilution of 0.67 µCi/mL in blocking buffer. The nitrocellulose was washed seven times as described above, dried, and autoradiographed with Kodak XAR film with intensifying screens at -70°C. In each case, the density of specific immunoreactive bands on the autoradiogram increased linearly with the amount of protein loaded. Accordingly, the relative abundance of individual proteins identified was quantified by scanning densitometry.

Materials
ICYP was the generous gift of Drs E. Hofferber and W. Hanson, Beiersdorf AG. [{alpha}-32P]ATP, cyclic [3H]AMP, and [32P]NAD were purchased from Dupont-New England Nuclear; Na 125I (as carrier-free Na 125I) was obtained from Amersham; pertussis toxin was purchased from List Biological Company; and cholera toxin, Gpp(NH)p, and forskolin were purchased from Sigma Chemical Company. Polyclonal antibodies against {alpha}s-, {alpha}i1/{alpha}i2-, and ß-subunits were purchased from Upstate Biotechnology Incorporated. Polyclonal anti-{alpha}o was purchased from Dupont-New England Nuclear. Polyclonal anti-{alpha}common was the generous gift of Dr David Manning, University of Pennsylvania. All other chemicals were reagent grade.

Statistical Analysis
Data are presented as mean±SEM. Statistical comparisons were made using Student's t test for paired observations or one-way ANOVA and the Newman-Keuls procedure for comparisons of multiple groups as indicated. Significance was defined at the P<.05 level.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
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ß-Adrenergic Receptor Density
The antagonist radioligand [125I]ICYP was used to compare ß-adrenergic receptor characteristics in membranes from the EBZ and RZ of the infarcted heart. Saturation binding experiments revealed one class of high-affinity ß-adrenergic receptors in each preparation (Fig 1Down). ß-Adrenergic receptor density was significantly lower in the EBZ of the infarcted heart than in the RZ of the same ventricle (Table 1Down). The equilibrium dissociation constant (Kd) for [125I]ICYP binding was not significantly different in the two groups. Furthermore, ß-adrenergic receptor density and antagonist affinity in the RZ of the infarcted ventricle and the two anatomic sites of the left ventricle of noninfarcted hearts were similar.



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  		Figure 1. Plot of representative saturation binding experiment showing that ß-adrenergic receptor density is lower in membranes from the epicardial border zone (EBZ) than in the remote zone (RZ) of the same ventricle. In the experiment shown, the Bmax for the EBZ and RZ was 49.6 and 124.7 fmol/mg, respectively.


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  		Table 1. ß-Adrenergic Receptor Density and Affinity for Antagonists in Epicardial Border Zone and Remote Zone of Infarcted Ventricle and Same Anatomic Regions of Noninfarcted Ventricle

Adenylyl Cyclase Activity
Adenylyl cyclase activity was markedly and consistently diminished in membranes from the EBZ of the 5-day infarcted ventricle compared with membranes from a remote region of the same ventricle or the same anatomic regions of the noninfarcted heart. As will be shown, the defect in adenylyl cyclase activity included basal enzyme activity, enzyme activity stimulated indirectly via the ß-adrenergic receptor (isoproterenol) or G protein [Gpp(NH)p] and more directly with manganese or forskolin (Figs 2 through 6DownDownDownDownDown).



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  		Figure 2. Bar graphs of basal, manganese-, and forskolin-dependent adenylyl cyclase activity (ACA) in the epicardial border zone (EBZ) and remote zone (RZ) of infarcted hearts and the same anatomic regions of noninfarcted hearts. ACA was measured as described in "Methods" in the absence or presence of 10 mmol/L manganese (Mn) or 10 µmol/L forskolin in the assay buffer. Results are the mean±SEM of five separate experiments performed in triplicate.



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  		Figure 3. Plots of Gpp(NH)p-dependent adenylyl cyclase activity (ACA) in the epicardial border zone (EBZ) and remote zone (RZ) of infarcted hearts and the same anatomic regions of noninfarcted hearts. Results are plotted as absolute enzyme activity (left) and are shown as net Gpp(NH)p-dependent stimulation over basal activity, to compensate for differences in nonstimulated enzyme activity between membranes from the EBZ and the RZ of infarcted hearts or the two anatomic areas of the noninfarcted hearts (5.9±1.6, 64.7±12.7, 64.2±5.4, and 73.4±11.5 pmol · mg-1 · min-1, respectively; n=5 for each). Data are plotted as percent of maximal stimulation by Gpp(NH)p (right).



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  		Figure 4. Plots of isoproterenol-dependent adenylyl cyclase activity (ACA) in the epicardial border zone (EBZ) and remote zone (RZ) of infarcted hearts and the same anatomic regions of noninfarcted hearts. Results are plotted as absolute enzyme activity (left). Results are shown as net isoproterenol-dependent stimulation over activity measured in the presence of 0.1 µmol/L Gpp(NH)p, to compensate for differences in enzyme activity between membranes from the EBZ and the RZ of infarcted hearts or the two anatomic areas of the noninfarcted hearts (18.6±3.6, 106.7±18.7, 94.2±8.2, and 105.9±20.0 pmol · mg-1 · min-1, respectively; n=9 for each). Results are plotted as percent of maximal stimulation by isoproterenol (right).



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  		Figure 5. Immunoblot analyses of G protein subunit expression in epicardial border zone (EBZ) and a remote zone (RZ) of infarcted ventricles. Membranes (150 µg) from the EBZ and the RZ of two infarcted ventricles were subjected to SDS-PAGE and immunoblot analysis with antisera specific for the indicated G protein subunits. In immunoblot analyses with antisera that recognize the pertussis toxin–sensitive {alpha}-subunits, a membrane preparation from rat brain, which is enriched in {alpha}o, was included as a control. Results in the two preparations illustrated in this figure are representative of the results for four (for {alpha}common and {alpha}i1/{alpha}i2) or eight ({alpha}s, {alpha}o, and ß) separate preparations from infarcted hearts.



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  		Figure 6. Immunoblot analysis of G protein {alpha}-subunit expression in membranes from epicardial, midmyocardial, and papillary muscle and myocytes isolated from the dog left ventricle (LV). Membranes (150 µg) were subjected to SDS-PAGE and immunoblot analysis with antisera specific for either {alpha}o or {alpha}i1/{alpha}i2. Both {alpha}i1/{alpha}i2 and {alpha}o immunoreactivities were detectble in membranes from the epicardial layer. In contrast, {alpha}i1/{alpha}i2, but not {alpha}o, immunoreactivity was detectable in myocytes isolated from the LV. Results are from one experiment and are representative of results obtained in three separate sets of membrane preparations.

Fig 2Up illustrates that basal adenylyl cyclase activity in membranes from the EBZ was 10-fold lower than that measured in the remote region of the same ventricle. Manganese (which directly activates the catalytic unit of adenylyl cyclase30 ) and forskolin (which potentiates the activation of the catalytic moiety of adenylyl cyclase by Gs31 ) induced a significant increase in adenylyl cyclase activity in all preparations. However, manganese- and forskolin-dependent adenylyl cyclase activities were markedly reduced in the EBZ of the infarcted heart compared with the RZ of the same ventricle (83% and 87%, respectively) or the same anatomic region of the noninfarcted heart (85% and 91%, respectively; Fig 2Up). The impaired stimulation by forskolin could result from alterations in Gs, changes in its interaction with the catalytic unit, and/or an actual defect in the catalytic moiety of adenylate cyclase. In contrast, the impaired stimulation by manganese argues for a defect in the catalytic unit of adenylyl cyclase.

Stimulation of adenylyl cyclase activity by Gpp(NH)p, a nonhydrolyzable analogue of GTP, also differed in the EBZ compared with the RZ of the same ventricle or either region in the noninfarcted heart. The dose-response curves for Gpp(NH)p-dependent stimulation of adenylyl cyclase activity are illustrated in Fig 3Up, and an analysis of the adenylyl cyclase data is presented in Table 2Down. The Vmax for Gpp(NH)p-dependent stimulation of adenylyl cyclase activity was significantly depressed in membrane preparations derived from the EBZ compared with the RZ of the same ventricle or the same anatomic sites of the noninfarcted ventricle. However, the curves superimposed when the data were normalized to maximal stimulation by Gpp(NH)p since the EC50 values for Gpp(NH)p-dependent stimulation of adenylyl cyclase in the two anatomic regions of the infarcted heart and the corresponding regions of the noninfarcted heart were similar (Table 2Down).


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  		Table 2. Gpp(NH)p- and Isoproterenol-Dependent Adenylyl Cyclase Activity in Epicardial Border Zone and Remote Zone of Infarcted Ventricle and Same Anatomic Regions of Noninfarcted Ventricle

We next measured isoproterenol-dependent adenylyl cyclase activity to probe for ß-adrenergic receptor function. These experiments were performed in the presence of a low concentration of Gpp(NH)p as a cofactor for receptor-dependent stimulation of adenylyl cyclase. Under these conditions, incremental stimulation of adenylyl cyclase by isoproterenol was severely depressed in the EBZ compared with the RZ of the infarcted ventricle or the same anatomic regions of the noninfarcted heart (Fig 4Up and Table 2Up). Moreover, the EC50 for isoproterenol-dependent activation of adenylyl cyclase activity was significantly higher in the EBZ than in the RZ of the infarcted ventricle or the corresponding anatomic sites of the noninfarcted heart. This rightward shift in the curve describing isoproterenol-dependent stimulation of adenylyl cyclase activity in the EBZ of the infarcted heart is best appreciated when the data are normalized to maximal stimulation by isoproterenol (Fig 4Up, top right). The diminished sensitivity to the stimulatory actions of isoproterenol is noteworthy given the significant decrease in ß-adrenergic receptor density in membranes from the EBZ of the infarcted heart.

G Proteins
The first approach to measuring Gs and Gi {alpha}-subunit expression used cholera toxin– and pertussis toxin–dependent ADP-ribosylation. Table 3Down compares the level of substrate for cholera toxin– and pertussis toxin–dependent ADP-ribosylation in membranes from the EBZ and RZ of the infarcted heart and the same anatomic regions of the noninfarcted ventricle. This methodology revealed a significantly lower level of {alpha}s-subunit and a significantly higher level of {alpha}i -subunit in ventricular membranes from the EBZ of the infarcted heart than in the other preparations.


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  		Table 3. G Proteins in Epicardial Border Zone and Remote Zone of Infarcted Ventricle and Same Anatomic Regions of Noninfarcted Ventricle

The extent of G protein {alpha}-subunit ADP-ribosylation depends on the amount of {alpha}-subunit protein substrate but also can be influenced by the degree of endogenous ADP-ribosylation as well as the availability of cofactors required for ADP-ribosylation (ie, endogenous ADP-ribosylation factors can modulate cholera toxin–catalyzed ADP-ribosylation of Gs32 33 and ß{gamma}-subunits modulate ADP-ribosylation of Gi34 ). Therefore, we used quantitative immunoblot analysis to determine whether the changes in G protein {alpha}-subunit levels measured in the [32P]ADP-ribosylation reaction reflect actual changes in G protein subunit expression. A subset of the membrane preparations from control and 5-day infarcted hearts was probed with a panel of polyclonal antisera generated against synthetic peptides derived from the distinct sequences of individual G protein subunits. Results of immunoblot analyses performed on membranes from the EBZ and RZ of two infarcted ventricles, which are representative of the data for the entire group studied, are illustrated in Fig 5Up. Using a polyclonal antiserum that is strongly reactive against all pertussis toxin–sensitive {alpha}-subunits ({alpha}common), we detected 1.8±0.1-fold greater immunoreactive protein in membranes from the EBZ than the RZ of the same ventricle (n=4, P<.05). An antiserum that recognizes both {alpha}i1 and {alpha}i2 (but not {alpha}i3 or {alpha}o) also detected 3.1±0.8-fold more immunoreactive protein in the EBZ compared with the RZ of the same ventricle (n=4, P<.05). The greater immunoreactivity in the EBZ is presumed to represent {alpha}i2, the predominant pertussis toxin–sensitive {alpha}-subunit expressed by canine ventricular myocytes.27 Immunoreactivity to {alpha}o also was detected in epicardial membranes and was 4.9±1.5-fold more abundant in the RZ than in the EBZ (n=8, P<.05). This was somewhat surprising since other groups have provided convincing evidence that ventricular myocytes express only small amounts of {alpha}o.35 36 In this regard, further experiments established that {alpha}o immunoreactivity is readily detected in membranes from the epicardial layer, in lesser amounts in membranes from the midmyocardium and papillary muscle, but not in membranes prepared from myocytes isolated from the midmyocardium of the left ventricle37 (Fig 6Up). In contrast, {alpha}i1/{alpha}i2 immunoreactivity was detected in each of these preparations. These results are consistent with the hypothesis that {alpha}o immunoreactivity in membranes prepared from the epicardial layer derives, in large part, from a contaminating cell population such as nerves, which are enriched in {alpha}o and are present in RZ epicardial tissue but are scarce in the EBZ.38 Finally, {alpha}s immunoreactivity was 2.6±0.3-fold more abundant in the RZ than in the EBZ (n=8, P<.05), whereas ß-subunit expression was equivalent in the RZ and EBZ (ie, immunoreactivity was 1.51±0.4-fold higher in RZ than in EBZ; P=NS; n=8; Fig 5Up). For each antiserum, the amount of immunoreactive protein detected in the RZ of the infarcted ventricle and the two anatomic sites in the noninfarcted heart was similar (data not shown). These data confirm and extend the results of experiments using toxin-catalyzed ADP-ribosylation to measure G protein levels. The studies provide evidence that the EBZ of the 5-day infarcted heart contains less immunoreactive {alpha}s and more immunoreactive {alpha}i (presumably {alpha}i2) than the RZ of the same ventricle or of control, noninfarcted epicardial tissue.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
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*Discussion
down arrowReferences
 
The present study is the first to examine the biochemical determinants of the altered ß-adrenergic response in the thin rim of surviving epicardial tissue that provides the substrate for reentrant tachycardias in the 5-day infarcted canine heart. The data from this study establish that there is no single lesion in the ß-adrenergic receptor signaling pathway in this tissue. Rather, defects in several components of the membrane ß-adrenergic receptor complex are identified, including a decrease in ß-adrenergic receptor density; diminished basal, guanine nucleotide-, isoproterenol-, forskolin-, and manganese-dependent adenylate cyclase activities; a reduced sensitivity to the stimulatory effects of isoproterenol as measured as an increase in the EC50 for isoproterenol-dependent adenylate cyclase activity; a decrease in the Gs protein; and an increase in the Gi protein. This constellation of changes in the ß-adrenergic receptor signaling pathway would be predicted to impair ß-adrenergic receptor responsiveness. Thus, it is reasonable to conclude that abnormalities in several components of the ß-adrenergic receptor complex contribute to the defect in isoproterenol-dependent stimulation of L-type calcium current in myocytes isolated from the EBZ of the 5-day infarcted heart.18

Although there has been considerable recent interest in determining the molecular basis for disease-associated alterations in catecholamine responsiveness, there is a paucity of published data on the integrity of the ß-adrenergic receptor complex in tissues surviving for long periods after total coronary artery occlusion. This is somewhat surprising given that this tissue is believed to provide the substrate for reentrant ventricular tachycardias and is known to differ from normal epicardium in its electrical and pharmacological properties.16 Results of the present study demonstrate that the EBZ of the infarcted heart develops lesions in the ß-adrenergic receptor signaling pathway that, in large part, parallel those identified in certain syndromes of chronic cardiomyopathy in humans. Several laboratories have identified specific derangements in multiple components of the ß-adrenergic receptor signaling pathway (including the ß-adrenergic receptor itself, the G protein, as well as the adenylyl cyclase enzyme) that would be predicted to result in a reduced capacity to generate cAMP in response to stimulation by catecholamines.39 40 For example, diminished cAMP and inotropic responses to ß-receptor agonists have been associated with a decrease in the density of cell surface ß-adrenergic receptors.41 42 Ungerer et al43 44 presented evidence that the decrease in cell surface ß-adrenergic receptor density is associated with enhanced expression of ß-adrenergic receptor kinase (ßARK), which specifically phosphorylates the agonist-occupied form of the ß-adrenergic receptor, thereby playing a pivotal role in the desensitization of ß-receptors. Other investigators find prominent downregulation of the ß1-adrenergic receptor population, whereas the density of ß2-adrenergic receptors remains relatively maintained. This leads to a relative increase in the proportion of ß2-adrenergic receptors in the failing heart, which retain almost full inotropic activity and help to support contractile function in the failing heart.28 39 44 45 Although results establish that the total ß-adrenergic receptor density is decreased in the EBZ of the 5-day infarcted canine heart, future studies will be required to determine whether these changes are accompanied by upregulation of ßARK and/or are associated with alterations in the density and/or proportion of cell surface ß1- and ß2-adrenergic receptors.

The lesion in G proteins in the EBZ of the 5-day infarcted heart also resembles the defect shown to accompany various forms of human heart failure. Here, there is rather consistent evidence for a functionally relevant increase in {alpha}i, without any change in {alpha}s, which contributes importantly to the decrease in ß-adrenergic receptor-dependent cAMP accumulation.46 47 48 Results from the present study demonstrate that {alpha}i expression, measured by pertussis toxin–catalyzed ADP-ribosylation, also is elevated in membranes from the EBZ of the 5-day infarcted canine heart. Insofar as pertussis toxin–dependent ADP-ribosylation is influenced by the amount of {alpha}-subunit protein substrate as well as other factors,34 the immunoblot analyses constitute an important extension of these studies. These experiments establish that the increase in substrate for pertussis toxin–catalyzed ADP-ribosylation in the EBZ of the 5-day infarcted heart reflects increased {alpha}i expression (rather than increased ß-subunit availability) and underscore the importance of combining the techniques of toxin-dependent ADP-ribosylation and immunoblot analysis. The added observation that {alpha}o is readily detectable in normal epicardial tissue but {alpha}o immunoreactivity is reduced in the EBZ of the infarcted heart was unanticipated. It should be emphasized that the immunoblotting methods used in the present study do not incorporate any corrections for potential differences in titer and/or hybridization efficiency between individual antisera. Such corrections would be required to precisely quantify and compare distinct G protein {alpha}-subunits in epicardial tissues from infarcted and noninfarcted hearts. Nevertheless, the results demonstrate that under identical experimental conditions (dilutions of antisera, exposure time for autoradiographs), {alpha}o immunoreactivity in epicardial tissue vastly exceeds that detected in membrane preparations from midmyocardium, papillary muscle, or myocytes isolated from the left ventricle. These results support the tentative conclusion that {alpha}o in epicardial tissue derives from a contaminating cell population, most likely neurons, which are abundant in the epicardial layer and are a rich source of {alpha}o.34 38 Studies using immunodetection techniques on intact tissues would directly test this hypothesis. Alternatively, studies on membranes from individual myocytes isolated from the EBZ and RZ of infarcted hearts also would be revealing. However, the procedure used to isolate epicardial myocytes yields an extremely limiting number of cells17 ; the cell yield is adequate for electrophysiological experiments but does not provide sufficient material for biochemical and/or immunological analyses. Finally, we found diminished {alpha}s expression in the EBZ of the 5-day infarcted heart. This change in {alpha}s would further contribute to an imbalance in G protein {alpha}-subunit expression. Taken together, the changes in {alpha}-subunit expression would be anticipated to result in a range of abnormalities in effector responsiveness. Thus, the diminution in isoproterenol- and Gpp(NH)p-dependent stimulation of adenylyl cyclase presumably represents just one example of the alterations induced by changes in G protein expression. Insofar as G protein {alpha}-subunits play a pivotal role in multiple transmembrane signal transduction processes,49 alterations in signaling through other G protein–dependent pathways, including the modulation of calcium, potassium, and/or sodium channel function, also are likely to occur in the EBZ of the 5-day infarcted heart and could have a significant impact on the electrical and contractile properties of this tissue.

In contrast, the level of G protein ß-subunit expression did not differ between the EBZ and the RZ of the infarcted heart and the same anatomic regions of noninfarcted hearts. However, it should be noted that ß-subunit expression was measured with an antiserum that does not discriminate between the different forms of this protein, and G protein {gamma}-subunit expression was not assessed in the present study. There are four known species of ß-subunit and seven different forms of the {gamma}-subunit.49 50 These differences in ß and/or {gamma} species may be important in overall physiological responsiveness. For example, the ß{gamma}-subunit can contribute to receptor signaling indirectly by interacting with and deactivating {alpha}s. Moreover, this dimer can directly regulate the activity of several types of effector mechanisms, including the adenylyl cyclase enzyme, phospholipases, and ion channels.49 51 52 53 54 G protein ß{gamma}-subunits also have been shown to modulate cellular responsiveness by regulating the specificity of ßARK–receptor interactions.55 Finally, changes in individual {gamma}-subunit subtypes have been reported to lend specificity to the receptor signaling pathway by influencing the ability of an individual G protein to discriminate between individual receptors and effector mechanisms.56 Thus, the results of the present study have not ruled out the possibility that changes in the ß- or {gamma}-subunits of the G protein also contribute to altered specificity of receptor–effector interactions in the EBZ of the 5-day infarcted canine heart. Specific protocols that compare G protein subunit isoform expression in healthy and diseased cardiac tissues and assess the functional importance of disease-induced differences in G protein subunit expression will be required to address this question.

The constellation of changes in G protein subunit levels detected in membranes from the EBZ of the infarcted heart would be predicted to compromise the ability of myocytes from the EBZ to generate cAMP (due to either deficient {alpha}s-stimulation of adenylyl cyclase, enhanced {alpha}i-inhibition of adenylyl cyclase, or enhanced release of ß{gamma}-subunit dimers on Gi activation that associate with {alpha}s and thereby interrupt the stimulatory pathway). However, it is unlikely that the defect in cAMP generation arises only as a result of alterations proximal to the adenylyl cyclase enzyme. Rather, the global decrease in adenylyl cyclase activity, including an abnormality when the enzyme is stimulated more directly by forskolin and manganese, suggests that myocytes in the EBZ of the infarcted heart also have a defect in the catalytic moiety of adenylyl cyclase. Of the multiple molecular forms of the adenylyl cyclase enzyme recently identified (types I through VII57 58 59 60 61 ), types V and VI appear to be the most abundant adenylyl cyclase isoforms expressed in ventricular myocardial tissue.57 60 61 62 Recent evidence that the age-dependent decline in adenylyl cyclase enzyme activity in the rat heart correlates with a fall in the steady-state mRNA for the type VI, but not the type V, isoform of adenylyl cyclase62 suggests that isoform-specific changes in the adenylyl cyclase enzyme can contribute to abnormalities in receptor responsiveness. Whether ischemic cardiac insults also lead to functionally important changes in the expression of individual isoforms of the adenylyl cyclase enzyme remains to be determined.

Although normalization of the levels of individual components of the ß-adrenergic receptor complex to membrane protein is a standard approach to investigate membrane receptor-activated signaling pathways in healthy and diseased tissue, this approach may impose certain potential limitations to the present study that must be considered. For example, apart from the presence of lipid droplets, the myocytes in the surviving epicardial tissue are normal at the ultrastructural level (including normal-appearing contractile elements, mitochondria, intercalated discs, and sarcolemma); there is no evidence of cell necrosis.13 Nevertheless, myocytes from the EBZ have been noted to be slightly enlarged compared with myocytes from the noninfarcted heart.17 Therefore, it could be argued that differences in myocardial cell size and protein composition between the EBZ and the RZ could confound interpretation of the data. In addition, an interstitial infiltrate with polymorphonuclear leukocytes and mononuclear cells is present in the EBZ of the 5-day infarcted heart.13 Thus, this tissue may be "diluted" by components from cells that are part of the inflammatory response and are not present in the control tissue. However, the diverse nature of the alterations in components of the receptor signaling mechanism in membranes from the EBZ (eg, the severe defect in basal and forskolin-dependent adenylyl cyclase activity, the moderate decrease in ß-adrenergic receptor density with the shift in the activation constant for isoproterenol-dependent adenylyl cyclase, the decrease in {alpha}s, and the increase in {alpha}i1/{alpha}i2) constitutes strong evidence that we have not merely detected dilutional changes in components of the ß-receptor complex. Nevertheless, it is possible that cytokines, elaborated by the inflammatory cells, act in concert with the ischemic insult to modulate the expression of components of the ß-adrenergic receptor complex in myocytes. Finally, it has been argued that less-refined preparations, which minimize receptor loss during centrifugation, are preferred in studies of ischemia-induced changes in sarcolemmal receptor function.63 However, the finding that a decrease in ß-adrenergic receptor density is associated with a defect in ß-agonist stimulation of the L-type calcium current in myocytes from the EBZ18 19 constitutes compelling evidence that the biochemical derangements in ß-receptor signaling detected in the present study are pathoelectrophysiologically relevant.

In conclusion, the present study identifies specific abnormalities in the ß-adrenergic receptor itself as well as components of the receptor complex distal to the receptor (at the level of the G proteins and catalytic adenylyl cyclase) that develop in the EBZ, the thin rim of surviving epicardial tissue in the 5-day infarcted heart. These findings emphasize the importance of investigating the biochemical determinants and pharmacological properties of the diseased tissues that provide the foci for ventricular arrhythmias during the healing phase of myocardial infarction. Detailed knowledge of the molecular defects in the ß-receptor signaling pathway that contribute to impaired catecholamine responsiveness in myocytes from this region of the heart is predicted to lead to an improved understanding of the pathophysiology of the arrhythmias that arise in the postinfarction period and ultimately should provide more rational strategies for therapeutic interventions.


*    Acknowledgments
 
This work was supported by US Public Health Service–National Heart, Lung, and Blood Institute grants HL-49537 (Dr Steinberg), HL-43731 (Dr Steinberg), and HL-34477 (Dr Boyden) and by an American Heart Association Grant-in-Aid (Dr Boyden).

Received October 24, 1994; revision received December 13, 1994; accepted December 18, 1994.


*    References
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*References
 

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