This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Steinberg, S. F. Articles by Boyden, P. A. Search for Related Content PubMed PubMed Citation Articles by Steinberg, S. F. Articles by Boyden, P. A.

(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

Top Abstract Introduction Methods Results Discussion References

 
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 -subunit of the G_s protein; and an elevated level of the -subunit of the G_i 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

Top Abstract Introduction Methods Results Discussion References

 
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 authors^{5 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 G_s 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

Top Abstract Introduction Methods Results Discussion References

 
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 - 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 (K_d) and the maximal number of binding sites (B_max) 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 [-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), [-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 -subunit of the heterotrimeric stimulatory G protein G_s (_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 _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 -subunit of the inhibitory G protein G_i (_i1, _i2, and _i3) as well as the -subunit of G_o (_o). Recent studies indicate that _i2 is the major G_i -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-_common, a polyclonal antiserum, previously characterized as antiserum 1398,²⁹ which is strongly reactive against all pertussis toxin–sensitive -subunits; anti-_i1/_i2, an affinity-purified polyclonal antiserum raised against a synthetic peptide corresponding to the shared C-terminal decapeptide sequence of rat _i1 and _i2, which does not cross-react with _i3 or _o; anti-_s, an affinity-purified polyclonal antiserum directed against a synthetic peptide corresponding to the C-terminal decapeptide of rat _s, which specifically recognizes _s; anti-_o, a polyclonal antiserum directed against the amino-terminus of _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-_i1/_i2 and anti-_o) or 1:1000 (anti-_common, anti-_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. [-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 _s-, _i1/_i2-, and ß-subunits were purchased from Upstate Biotechnology Incorporated. Polyclonal anti-_o was purchased from Dupont-New England Nuclear. Polyclonal anti-_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

Top Abstract Introduction Methods Results Discussion References

 
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 1). ß-Adrenergic receptor density was significantly lower in the EBZ of the infarcted heart than in the RZ of the same ventricle (Table 1). The equilibrium dissociation constant (K_d) 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.

View larger version (12K): [in this window] [in a new window]   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.

View this table: [in this window] [in a new window]   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 6).

View larger version (16K): [in this window] [in a new window]   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.

View larger version (21K): [in this window] [in a new window]   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).

View larger version (19K): [in this window] [in a new window]   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).

View larger version (90K): [in this window] [in a new window]   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 -subunits, a membrane preparation from rat brain, which is enriched in o, was included as a control. Results in the two preparations illustrated in this figure are representative of the results for four (for common and i1/i2) or eight (s, o, and ß) separate preparations from infarcted hearts.

View larger version (58K): [in this window] [in a new window]   Figure 6. Immunoblot analysis of G protein -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 o or i1/i2. Both i1/i2 and o immunoreactivities were detectble in membranes from the epicardial layer. In contrast, i1/i2, but not 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 2 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 G_s³¹ ) 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 2). The impaired stimulation by forskolin could result from alterations in G_s, 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 3, and an analysis of the adenylyl cyclase data is presented in Table 2. The V_max 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 2).

View this table: [in this window] [in a new window]   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 4 and Table 2). 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 4, 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 G_s and G_i -subunit expression used cholera toxin– and pertussis toxin–dependent ADP-ribosylation. Table 3 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 _s-subunit and a significantly higher level of _i -subunit in ventricular membranes from the EBZ of the infarcted heart than in the other preparations.

View this table: [in this window] [in a new window]   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 -subunit ADP-ribosylation depends on the amount of -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 G_s^{32 33} and ß-subunits modulate ADP-ribosylation of G_i³⁴ ). Therefore, we used quantitative immunoblot analysis to determine whether the changes in G protein -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 5. Using a polyclonal antiserum that is strongly reactive against all pertussis toxin–sensitive -subunits (_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 _i1 and _i2 (but not _i3 or _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 _i2, the predominant pertussis toxin–sensitive -subunit expressed by canine ventricular myocytes.²⁷ Immunoreactivity to _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 _o.^{35 36} In this regard, further experiments established that _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 6). In contrast, _i1/_i2 immunoreactivity was detected in each of these preparations. These results are consistent with the hypothesis that _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 _o and are present in RZ epicardial tissue but are scarce in the EBZ.³⁸ Finally, _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 5). 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 _s and more immunoreactive _i (presumably _i2) than the RZ of the same ventricle or of control, noninfarcted epicardial tissue.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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 G_s protein; and an increase in the G_i 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 al^{43 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 _i, without any change in _s, which contributes importantly to the decrease in ß-adrenergic receptor-dependent cAMP accumulation.^{46 47 48} Results from the present study demonstrate that _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 -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 _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 _o is readily detectable in normal epicardial tissue but _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 -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), _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 _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 _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 _s expression in the EBZ of the 5-day infarcted heart. This change in _s would further contribute to an imbalance in G protein -subunit expression. Taken together, the changes in -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 -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 -subunit expression was not assessed in the present study. There are four known species of ß-subunit and seven different forms of the -subunit.^{49 50} These differences in ß and/or species may be important in overall physiological responsiveness. For example, the ß-subunit can contribute to receptor signaling indirectly by interacting with and deactivating _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-subunits also have been shown to modulate cellular responsiveness by regulating the specificity of ßARK–receptor interactions.⁵⁵ Finally, changes in individual -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 -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 _s-stimulation of adenylyl cyclase, enhanced _i-inhibition of adenylyl cyclase, or enhanced release of ß-subunit dimers on G_i activation that associate with _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 VII^{57 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 _s, and the increase in _i1/_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 EBZ^{18 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

Top Abstract Introduction Methods Results Discussion References

 
1. Mukherjee A, Bush LR, McCoy KE, Duke R, Hagler H, Buja LM, Willerson JT. Relationship between ß-adrenergic receptor numbers and physiological responses during experimental canine myocardial ischemia. Circ Res. 1982;50:735-741. [Abstract/Free Full Text]

2. Vatner DE, Knight DR, Shen YT, Thomas JX, Homcy CJ, Vatner SF. One hour of myocardial ischemia in conscious dogs increases beta-adrenergic receptors but decreases adenylate cyclase activity. J Mol Cell Cardiol. 1988;20:75-82. [Medline] [Order article via Infotrieve]

3. Maisel AS, Motulsky HJ, Insel PA. Externalization of beta-adrenergic receptors promoted by myocardial ischemia. Science. 1985;230:183-186. [Abstract/Free Full Text]

4. Vatner DE, Young MA, Knight DR, Vatner SF. ß-Receptors and adenylate cyclase: comparison of nonischemic, ischemic, and postmortem tissue. Am J Physiol. 1990;258:H140-H144. [Abstract/Free Full Text]

5. Freissmuth M, Schutz W, Weindlmayer-Gottel M, Zimpfer M, Spiss CK. Effects of ischemia on the canine myocardial ß-adrenoceptor-linked adenylate cyclase system. J Cardiovasc Pharmacol. 1987;10:568-574. [Medline] [Order article via Infotrieve]

6. Karliner JS, Stevens M, Woloszyn W, Honbo N, Blaug G, Hoffman JIE. Effects of acute ischemia in the dog on myocardial blood flow, beta receptors and adenylate cyclase activity with and without chronic beta blockade. J Clin Invest. 1989;83:474-481.

7. Heathers GP, Lee PC, Yamada KA, Corr PB. The influence of hypoxia on ß-adrenergic receptors and their intracellular coupling in isolated adult canine myocytes. Coron Artery Dis. 1990;1:97-110.

8. Susanni EE, Manders WT, Knight DR, Vatner DE, Vatner SF, Homcy CJ. One hour of myocardial ischemia decreases the activity of the stimulatory guanine nucleotide regulatory protein G_s. Circ Res. 1989;65:1145-1150. [Abstract/Free Full Text]

9. Devos C, Robberecht P, Nokin P, Waelbroeck M, Clinet M, Camus JC, Beaufort P, Schoenfeld P, Christophe J. Uncoupling between beta-adrenoceptors and adenylate cyclase in dog ischemic myocardium. Naunyn Schmiedebergs Arch Pharmacol. 1985;331:71-75. [Medline] [Order article via Infotrieve]

10. El-Sherif N, Smith RA, Evans K. Canine ventricular arrhythmias in the late myocardial infarction period, 8: epicardial mapping of reentrant circuits. Circ Res. 1981;49:255-265. [Abstract/Free Full Text]

11. Dillon S, Allessie MA, Ursell PC, Wit AL. Influences of anisotropic tissue structure on reentrant circuits in the epicardial border zone of subacute canine infarcts. Circ Res. 1988;63:182-206. [Abstract/Free Full Text]

12. Butrous GS, Gough WB, Restivo M, Yang H, El-Sherif N. Adrenergic effects on reentrant ventricular rhythms in subacute myocardial infarction. Circulation. 1992;86:247-254. [Abstract/Free Full Text]

13. Ursell PC, Gardner PI, Albala A, Fenoglio JJ, Wit AL. Structural and electrophysiologic changes in the epicardial border zone of canine myocardial infarcts during healing. Circ Res. 1985;56:436-451. [Abstract/Free Full Text]

14. Lazzara R, Scherlag BJ. Role of the slow inward current in the generation of arrhythmias in ischemic myocardium. In: Zipes DP, Bailey JC, Elharrar V, eds. Slow Inward Current and Cardiac Arrhythmias. The Hague, Netherlands: Martinus Nijhoff; 1980:339-416.

15. El-Sherif N, Lazzara R. Reentrant ventricular arrhythmias in the late myocardial infarction period, 7: effect of verapamil and D-600 and the role of the `slow channel.' Circulation. 1979;60:605-615. [Abstract/Free Full Text]

16. Boyden PA, Gardner PI, Wit AL. Action potentials of cardiac muscle in healing infarcts: response to norepinephrine and caffeine. J Mol Cell Cardiol. 1988;20:525-537. [Medline] [Order article via Infotrieve]

17. Lue WM, Boyden PA. Abnormal electrical properties of myocytes from chronically infarcted canine heart: alterations in V_max and the transient outward current. Circulation. 1992;85:1175-1188. [Abstract/Free Full Text]

18. Aggarwal R, Boyden PA. Calcium currents in canine myocytes from the epicardial border zone of the infarcted heart. Circulation. 1991;84(suppl II):II-549. Abstract.

19. Aggarwal R, Boyden PA. Effect of isoproterenol and Bay K 8644 on the reduced calcium current of canine myocytes from the epicardial border zone. Biophys J. 1993;64:A399. Abstract.

20. Harris AS. Delayed development of ventricular ectopic rhythms following experimental coronary occlusion. Circulation. 1950;1:1318-1328. [Medline] [Order article via Infotrieve]

21. Engel G, Hoyer D, Berthold R, Wagner H. (±)-[¹²⁵Iodo]Cyanopindolol (ICYP), a new ligand for beta-adrenoceptors: identification and quantitation of subclasses of beta-adrenoceptors in guinea pig. Naunyn Schmiedebergs Arch Pharmacol. 1981;317:277-285. [Medline] [Order article via Infotrieve]

22. Steinberg SF, Jaffe EA, Bilezikian JP. Endothelial cells contain beta adrenoceptors. Naunyn Schmiedebergs Arch Pharmacol. 1984;325:310-313. [Medline] [Order article via Infotrieve]

23. Steinberg SF, Chow YK, Bilezikian JP. Regulation of rat heart membrane adenylate cyclase by magnesium and manganese. J Pharmacol Exp Ther. 1986;237:764-772. [Abstract/Free Full Text]

24. Han HM, Robinson RB, Bilezikian JP, Steinberg SF. Developmental changes in guanine nucleotide binding proteins in the ₁-adrenergic receptor complex in rat myocardium. Circ Res. 1989;65:1763-1773. [Abstract/Free Full Text]

25. Robishaw JD, Smigel MD, Gilman AG. Molecular basis for two forms of the G protein that stimulates adenylate cyclase. J Biol Chem. 1986;261:9587-9590. [Abstract/Free Full Text]

26. Longabaugh JP, Vatner DE, Vatner SF, Homcy CJ. Decreased stimulatory guanosine triphosphate binding protein in dogs with pressure-overload left ventricular failure. J Clin Invest. 1988;81:420-424.

27. Holmer SR, Stevens S, Homcy CJ. Tissue- and species-specific expression of inhibitory guanine nucleotide-binding proteins: cloning of a full-length complementary DNA from canine heart. Circ Res. 1989;65:1136-1140. [Abstract/Free Full Text]

28. Marzo KP, Frey MJ, Wilson JR, Liang BT, Manning DR, Lanoce V, Molinoff PB. ß-Adrenergic receptor–G protein–adenylate cyclase complex in experimental canine congestive heart failure produced by rapid ventricular pacing. Circ Res. 1991;69:1546-1556. [Abstract/Free Full Text]

29. Carlson KE, Brass LF, Manning DR. Thrombin and phorbol esters cause the selective phosphorylation of a G protein other than G_i in human platelets. J Biol Chem. 1989;264:13298-13305. [Abstract/Free Full Text]

30. Neer EJ. Interaction of soluble brain adenylate cyclase with manganese. J Biol Chem. 1978;253:1498-1502. [Abstract/Free Full Text]

31. Darfler FJ, Mahan LC, Koachman AM, Insel PA. Stimulation by forskolin of intact S49 lymphoma cells involves the nucleotide regulatory protein of adenylate cyclase. J Biol Chem. 1982;257:11901-11907. [Free Full Text]

32. Kahn RA, Gilman AG. Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin. J Biol Chem. 1984;259:6228-6234. [Abstract/Free Full Text]

33. Schnabel P, Bohm M, Gierschik P, Jakobs KH, Erdmann E. Improvement of cholera toxin-catalyzed ADP-ribosylation by endogenous ADP-ribosylation factor from bovine brain provides evidence for an unchanged amount of G_s in failing human myocardium. J Mol Cell Cardiol. 1990;22:73-82. [Medline] [Order article via Infotrieve]

34. Neer EJ, Lok JM, Wolf LG. Purification and properties of the inhibitory guanine nucleotide regulatory unit of brain adenylate cyclase. J Biol Chem. 1984;259:14222-14229. [Abstract/Free Full Text]

35. Foster KA, McDermott PJ, Robishaw JD. Expression of G proteins in rat myocytes: effect of KCl depolarization. Am J Physiol. 1990;259:H432-H441. [Abstract/Free Full Text]

36. Luetje CW, Tietje KM, Christian JL, Nathanson NM. Differential tissue expression and development of guanine nucleotide binding regulatory proteins and their messenger RNAs in rat heart. J Biol Chem. 1988;263:13357-13365. [Abstract/Free Full Text]

37. Hewett K, Legato MJ, Danilo P, Robinson RB. Isolated myocytes from adult canine left ventricle: Ca²⁺ tolerance, electrophysiology, and ultrastructure. Am J Physiol. 1983;245:H830-H839.

38. Barber MJ, Mueller TM, Henry DP, Felten SY, Zipes DP. Transmural myocardial infarction in the dog produces sympathectomy in noninfarcted myocardium. Circulation. 1983;67:787-796. [Free Full Text]

39. Bristow MR, Ginsburg R, Umans V, Fowler M, Minobe W, Rasmussen R, Zera P, Menlove R, Shah P, Jamison S, Stinson EB. ß₁- And ß₂-adrenergic receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective ß₁-receptor downregulation in heart failure. Circ Res. 1986;59:297-309. [Abstract/Free Full Text]

40. Feldman AM. Experimental issues in assessment of G protein function in cardiac disease. Circulation. 1991;84:1852-1861. [Free Full Text]

41. Bristow MR, Ginsburg R, Minobe W, Cubicciotti RS, Sageman WS, Lurie K, Billingham ME, Harrison DC, Stinson EB. Decreased catecholamine sensitivity and ß-adrenergic receptor density in failing human hearts. N Engl J Med. 1982;307:205-211. [Abstract]

42. Murphree SS, Saffitz JE. Distribution of ß-adrenergic receptors in failing human myocardium: implications for mechanisms of downregulation. Circ Res. 1989;79:1214-1225.

43. Ungerer M, Parruti G, Bohm M, Puzicha M, DeBlasi A, Erdmann E, Lohse MJ. Expression of ß-arrestins and ß-adrenergic receptor kinases in the failing human heart. Circ Res. 1994;74:206-213. [Abstract/Free Full Text]

44. Ungerer M, Bohm M, Elce JS, Erdmann E, Lohse MJ. Altered expression of ß-adrenergic receptor kinase and ß₁-adrenergic receptors in the failing human heart. Circ Res. 1993;87:454-463.

45. Hammond HK, Roth DA, Insel PA, Ford CE, White FC, Maisel AS, Ziegler MG, Bloor CM. Myocardial ß-adrenergic receptor expression and signal transduction after chronic volume-overload hypertrophy and circulatory congestion. Circ Res. 1992;85:269-280.

46. Feldman AM, Cates AE, Veazey WB, Hershberger RE, Bristow MR, Baughman KL, Baumgartner WA, Van Dop C. Increase of the 40,000-mol wt pertussis toxin substrate (G protein) in the failing human heart. J Clin Invest. 1988;82:189-197.

47. Bohm M, Gierschik P, Jakobs KH, Pieske B, Schnabel P, Ungerer M, Erdmann E. Increase of G_i in human hearts with dilated but not ischemic cardiomyopathy. Circulation. 1990;82:1249-1265. [Abstract/Free Full Text]

48. Fu LX, Liang QM, Waagstein F, Hoebeke J, Sylven C, Jansson E, Sotonyi P, Hjalmarson A. Increase in functional activity rather than in amount of Gi- in failing human heart with dilated cardiomyopathy. Cardiovasc Res. 1992;26:950-955. [Abstract/Free Full Text]

49. Simon MI, Strathmann MP, Gautam N. Diversity of G proteins in signal transduction. Science. 1991;252:802-808. [Abstract/Free Full Text]

50. Cali JJ, Balcueva EA, Rybalkin I, Robishaw JD. Selective tissue distribution of G protein gamma subunits, including a new form of the gamma subunits identified by cDNA cloning. J Biol Chem. 1992;267:24023-24227. [Abstract/Free Full Text]

51. Tang WJ, Gilman AG. Type-specific regulation of adenylyl cyclase by G protein ß subunits. Science. 1991;254:1500-1504. [Abstract/Free Full Text]

52. Park D, Jhon D, Lee C, Lee K, Rhee SG. Activation of phospholipase C isozymes by G protein ß subunits. J Biol Chem. 1993;268:4573-4576. [Abstract/Free Full Text]

53. Jelsema CL, Axelrod J. Stimulation of phospholipase A₂ activity in bovine rod outer segments by the beta gamma subunits of transducin and its inhibition by the alpha subunit. Biochemistry. 1987;84:3623-3627.

54. Clapham DE, Neer EJ. New roles for G protein beta-gamma-dimers in transmembrane signalling. Nature. 1993;365:403-406. [Medline] [Order article via Infotrieve]

55. Pitcher JA, Inglese J, Higgins JB, Arriza JL, Casey PJ, Kim C, Benovic JL, Kwatra MM, Caron MG, Lefkowitz RJ. Role of ß subunits of G proteins in targeting the ß-adrenergic receptor kinase to membrane bound receptors. Science. 1992;257:1264-1267. [Abstract/Free Full Text]

56. Kleuss C, Scherubl H, Heschler J, Schultz G, Wittig B. Selectivity in signal transduction determined by gamma subunits of heterotrimeric G proteins. Science. 1993;259.

57. Ishikawa Y, Katsushika S, Chen L, Halnon NJ, Kawabe JI, Homcy CJ. Isolation and characterization of a novel cardiac adenylylcyclase cDNA. J Biol Chem. 1992;267:13553-13557. [Abstract/Free Full Text]

58. Krupinski J, Coussen F, Bakalyar HA, Tang WJ, Feinstein PG, Orth K, Slaughter C, Reed RR, Gilman AG. Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure. Science. 1989;244:1558-1564. [Abstract/Free Full Text]

59. Gao B, Gilman AG. Cloning and expression of a widely distributed (type IV) adenylyl cyclase. Proc Natl Acad Sci U S A. 1991;88:10178-10182. [Abstract/Free Full Text]

60. Katsushika S, Chen L, Kawabe JI, Nilakantan R, Halnon NJ, Homcy CJ, Ishikawa Y. Cloning and characterization of a sixth adenylyl cyclase isoform: types V and VI constitute a subgroup within the mammalian adenylyl cyclase family. Proc Natl Acad Sci U S A. 1992;89:8774-8778. [Abstract/Free Full Text]

61. Krupinski J, Lehman TC, Frankenfield CD, Zwaagstra JC, Watson PA. Molecular diversity in the adenylylcyclase family: evidence for eight forms of the enzyme and cloning of type VI. J Biol Chem. 1992;267:24858-24862. [Abstract/Free Full Text]

62. Tobise K, Ishikawa Y, Holmer SR, Im MJ, Newell JB, Yoshie H, Fujita M, Susannie EE, Homcy CJ. Changes in type VI adenylyl cyclase isoform expression correlate with a decreased capacity for cAMP generation in the aging ventricle. Circ Res. 1994;74:596-603. [Abstract/Free Full Text]

63. Wolff AA, Hines DK, Karliner JS. Refined membrane preparations mask ischemic fall in myocardial beta-receptor density. Am J Physiol. 1989;257:H1032-H1036.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. Nattel, A. Maguy, S. Le Bouter, and Y.-H. Yeh Arrhythmogenic Ion-Channel Remodeling in the Heart: Heart Failure, Myocardial Infarction, and Atrial Fibrillation Physiol Rev, April 1, 2007; 87(2): 425 - 456. [Abstract] [Full Text] [PDF]

				W. Dun, S. Baba, T. Yagi, and P. A. Boyden Dynamic remodeling of K+ and Ca2+ currents in cells that survived in the epicardial border zone of canine healed infarcted heart Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1046 - H1054. [Abstract] [Full Text] [PDF]

				H. T. Tevaearai, A. D. Eckhart, K. F. Shotwell, K. Wilson, and W. J. Koch Ventricular Dysfunction After Cardioplegic Arrest Is Improved After Myocardial Gene Transfer of a {beta}-Adrenergic Receptor Kinase Inhibitor Circulation, October 23, 2001; 104(17): 2069 - 2074. [Abstract] [Full Text] [PDF]

				X. Yu, M. Zhang, K. Kyker, E. Patterson, J. L. Benovic, and D. C. Kem Ischemic Inactivation of G Protein-Coupled Receptor Kinase and Altered Desensitization of Canine Cardiac {beta}-Adrenergic Receptors Circulation, November 14, 2000; 102(20): 2535 - 2540. [Abstract] [Full Text] [PDF]

				A. Igawa, T. Nozawa, N. Yoshida, N. Fujii, M. Inoue, S. Tazawa, H. Asanoi, and H. Inoue Heterogeneous cardiac sympathetic innervation in heart failure after myocardial infarction of rats Am J Physiol Heart Circ Physiol, April 1, 2000; 278(4): H1134 - H1141. [Abstract] [Full Text] [PDF]

				J. M.B Pinto and P. A Boyden Electrical remodeling in ischemia and infarction Cardiovasc Res, May 1, 1999; 42(2): 284 - 297. [Abstract] [Full Text] [PDF]

				E. A Sosunov, E. P Anyukhovsky, A. Shvilkin, Motoki Hara, S. F Steinberg, P. Danilo Jr., M. R Rosen, N Sydney Moise, J. Merot, V. Probst, et al. Abnormal cardiac repolarization and impulse initiation in German shepherd dogs with inherited ventricular arrhythmias and sudden death Cardiovasc Res, April 1, 1999; 42(1): 65 - 79. [Abstract] [Full Text] [PDF]

				H.-T. Li, N. Y. Honbo, and J. S. Karliner Chronic Hypoxia Increases ß1-Adrenergic Receptor mRNA and Density but Not Signaling in Neonatal Rat Cardiac Myocytes Circulation, December 15, 1996; 94(12): 3303 - 3310. [Abstract] [Full Text]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Steinberg, S. F. Articles by Boyden, P. A. Search for Related Content PubMed PubMed Citation Articles by Steinberg, S. F. Articles by Boyden, P. A.