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(Circulation. 1998;98:1783-1789.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Reciprocal In Vivo Regulation of Myocardial G Protein–Coupled Receptor Kinase Expression by ß-Adrenergic Receptor Stimulation and Blockade

Guido Iaccarino, MD; Eric D. Tomhave, BS; Robert J. Lefkowitz, MD; ; Walter J. Koch, PhD

From the Departments of Medicine (G.I., R.J.L.), Surgery (E.D.T, W.J.K.), and Biochemistry (R.J.L.) and Howard Hughes Medical Institute, Duke University Medical Center (R.J.L.), Durham, NC.

Correspondence to Walter J. Koch, PhD, Department of Surgery, Duke University Medical Center, Room 472, MSRB, Research Drive, Durham, NC 27710. E-mail koch0002{at}mc.duke.edu

	Abstract

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Background—Impaired myocardial ß-adrenergic receptor (ßAR) signaling, including desensitization and functional uncoupling, is a characteristic of congestive heart failure. A contributing mechanism for this impairment may involve enhanced myocardial ß-adrenergic receptor kinase (ßARK1) activity because levels of this ßAR-desensitizing G protein–coupled receptor kinase (GRK) are increased in heart failure. An hypothesis has emerged that increased sympathetic nervous system activity associated with heart failure might be the initial stimulus for ßAR signaling alterations, including desensitization. We have chronically treated mice with drugs that either activate or antagonize ßARs to study the dynamic relationship between ßAR activation and myocardial levels of ßARK1.

Methods and Results—Long-term in vivo stimulation of ßARs results in the impairment of cardiac ßAR signaling and increases the level of expression (mRNA and protein) and activity of ßARK1 but not that of GRK5, a second GRK abundantly expressed in the myocardium. Long-term ß-blocker treatment, including the use of carvedilol, improves myocardial ßAR signaling and reduces ßARK1 levels in a specific and dose-dependent manner. Identical results were obtained in vitro in cultured cells, demonstrating that the regulation of GRK expression is directly linked to ßAR signaling.

Conclusions—This report demonstrates, for the first time, that ßAR stimulation can significantly increase the expression of ßARK1, whereas ß-blockade decreases expression. This reciprocal regulation of ßARK1 documents a novel mechanism of ligand-induced ßAR regulation and provides important insights into the potential mechanisms responsible for the effectiveness of ß-blockers, such as carvedilol, in the treatment of heart failure.

Key Words: heart failure • receptors, adrenergic, beta • myocardium • catecholamines

	Introduction

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ß-Adrenergic receptors (ßARs), which couple to the heterotrimeric guanine nucleotide binding (G) protein G_s, are major determinants of cardiac contractility. In the heart, ßARs are targets for catecholamines such as the sympathetic neurotransmitter norepinephrine and the adrenal hormone epinephrine.^{1 2} Catecholamine stimulation of myocardial ßARs triggers a series of transmembrane signaling events through G_s that lead to the increased production of cAMP. In the myocyte, this results in positive inotropy, dromotropy, and chronotropy.^{1 2} Acute agonist (ie, catecholamine) exposure also triggers a series of counterregulatory mechanisms that lead to the functional uncoupling of ßARs, a process known as desensitization.^{3 4} Homologous desensitization of G protein–coupled receptors, such as ßARs, is initiated by the actions of a family of serine/threonine kinases known as the G protein–coupled receptor kinases (GRKs).^{3 4} GRKs normally expressed in the heart, such as the ßAR kinase (ßARK1, or GRK2) and GRK5, are enzymes that are rapidly activated after agonist occupancy of receptors and GRK-mediated receptor phosphorylation and subsequent ß-arrestin binding leads to the loss of G protein coupling.^{3 4}

A growing body of evidence supports the hypothesis that the actions of GRKs are extremely important in modulating myocardial adrenergic signaling and cardiac function both under normal conditions and in disease states. Several recent studies have shown that GRK levels (eg, ßARK1) and activity are elevated in a variety of cardiovascular disorders. These pathological conditions include human congestive heart failure,⁵ experimental myocardial ischemia,⁶ mild human hypertension,⁷ and pressure overload ventricular hypertrophy.⁸ In the latter study, we have shown that the cardiac hemodynamic dysfunction that accompanies pressure overload ventricular hypertrophy in mice is primarily due to an increase in the expression of ßARK1.⁸ Furthermore, in studies with transgenic mice, we have shown that increased ßARK1 or GRK5 expression and activity in the heart can lead to functional uncoupling and desensitization of myocardial ßARs and subsequent in vivo cardiac dysfunction.^{9 10}

The mechanisms of GRK upregulation associated with these cardiovascular disorders are unknown, but we hypothesize that they may involve enhanced sympathetic nervous activity and high catecholamine levels, triggering enhanced activation and signaling through myocardial ßARs. To explore the possibility that the myocardial expression of GRKs is coupled to the functional state of ßARs, we investigated specific GRK regulation due to long-term activation or antagonism of ßARs. The ß-agonist isoproterenol or the ß-antagonist atenolol was infused into mice through the use of implanted miniosmotic pumps. After long-term treatment with these drugs, we assessed the levels of ßARK1 and GRK5 in the heart through immunoblotting. We also measured myocardial GRK activity. GRK regulation in response to ßAR ligands also was studied in cultured mammalian cells to circumvent the hemodynamic changes associated with the in vivo administration of these drugs. We studied the specific effects on myocardial GRK expression of carvedilol, a novel ß-blocking agent that enhances cardiac performance and survival in human heart failure.¹¹ The mechanisms that account for the effectiveness of ß-blockers in heart failure are not completely understood. In this study, we test the hypothesis that these drugs might be exerting beneficial effects in heart failure through attenuation of ßAR desensitization due to decreased myocardial ßARK1 expression.

	Methods

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Study Design and Miniosmotic Pump Implantation
C57/Bl6 mice (weight, 25 to 30 g) were used in the study. All animal procedures were approved by the Institutional Animal Usage Committee at Duke University. Mice were anesthetized with a mixture of ketamine (10 mg/kg) and xylazine (0.5 mg/kg), and a small incision was made in the skin between the scapulae. A small pocket was created by spreading apart the subcutaneous connective tissue. After insertion of the miniosmotic pump (model 2002; Alzet), the skin incision was closed with a 4.0 catgut suture. Atenolol and isoproterenol were dissolved in 0.002% ascorbic acid, and carvedilol (a generous gift from SmithKline Beecham) was dissolved in 60% DMSO. Pumps were filled to deliver atenolol at the rate of 0.1, 1.0, and 10.0 mg · kg^-1 · d^-1, isoproterenol at the rate of 0.3, 3.0, and 30.0 mg · kg^-1 · d^-1, or carvedilol at the rate of 10.0 mg · kg^-1 · d^-1 over a period of 14 days. As controls, pumps that delivered vehicle (0.002% ascorbic acid or 60% DMSO) were implanted in mice. Heart rates in anesthetized animals were measured by ECG leads after 1 week to ensure drug delivery. At the end of the treatment, the animals were anesthetized and weighed, and their hearts were explanted, rinsed three times in cold PBS, and blotted dry. After weighing, isolated hearts were frozen in liquid nitrogen and stored at -70°C until needed for biochemical studies. The heart weight–to–body weight ratio was then calculated (mg/g).

ßAR Radioligand Binding
Receptor binding on myocardial membranes was performed as previously described using the nonselective ßAR ligand [¹²⁵I]cyanopindolol.^{8 9} Nonspecific binding was determined in the presence of 10 µmol/L alprenolol. Reactions were conducted in 500 µL of binding buffer at 37°C for 1 hour and then terminated by vacuum filtration through glass-fiber filters. All assays were performed in triplicate, and receptor density (in fmol) was normalized to milligrams of membrane protein.

Adenylyl Cyclase Activity
Crude myocardial membranes (20 to 30 µg of protein) were incubated for 15 minutes at 37°C with [-³²P]ATP under basal conditions or in the presence of either 100 µmol/L isoproterenol or 10 mmol/L NaF, and cAMP was quantified by standard methods as we have described previously.^{8 9}

Protein Immunoblotting
Immunodetection of myocardial levels of ßARK1 was performed on detergent-solubilized extracts after immunoprecipitation, as previously described.⁸ Excised hearts were solubilized in ice-cold RIPA buffer (50 mmol/L Tris-HCl, pH 8.0, 5 mmol/L EDTA, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mmol/L NaF, 5 mmol/L EGTA, 10 mmol/L sodium pyrophosphate, 1 mmol/L phenylmethylsulfonyl fluoride), and ßARK1 was immunoprecipitated from 1 mL of clarified extract (equal protein amounts) using 1:2000 of an anti-ßARK1/2 (GRK2/3) monoclonal antibody^{8 12} and 35 µL of a 50% slurry of Protein A–agarose conjugate agitated for 1 hour at 4°C. After extensive washing,⁸ immune complexes were electrophoresed through 12% polyacrylamide Tris/glycine gels and transferred to nitrocellulose. The 80-kDa ßARK1 protein was visualized using standard enhanced chemiluminescence (ECL kit; Amersham). Immunodetection of GRK5 was performed by Western blotting of myocardial membranes using a polyclonal anti-GRK5 antibody.^{10 13} Quantification of immunoreactive ßARK1 and GRK5 was done by scanning the final autoradiography films and using ImageQuant software (Molecular Dynamics).

Rhodopsin Phosphorylation Assays
Myocardial extracts were prepared through homogenization of excised hearts in 2 mL of ice-cold lysis buffer (25 mmol/L Tris-HCl, pH 7.5, 5 mmol/L EDTA, 5 mmol/L EGTA, 10 µg/mL leupeptin, 20 µg/mL aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride) as described previously.^{8 9 10} Soluble cytosolic fractions and membrane fractions were separated, and GRK activity was assessed in cytosolic fractions (100 to 150 µg of protein) by light-dependent phosphorylation of rhodopsin-enriched rod outer segment membranes in lysis buffer with 10 mmol/L MgCl₂ and 0.1 mmol/L ATP (containing [-³²P]ATP) as described previously.^{8 9 10} Phosphorylated rhodopsin was visualized by autoradiography of dried gels and quantified using a Molecular Dynamics PhosphorImager.

RNA Preparation and Semiquantitative Reverse Transcription–Polymerase Chain Reaction
Total RNA was isolated using RNAzol (Biotech), a one-step guanidinium-based extraction solution.¹⁴ After the treatment of final RNA aliquots with DNase I, 1 µg of total RNA was used for reverse transcription (RT) into cDNA according to standard methods.¹⁵ Equal aliquots of cDNA then was used as templates for the specific amplification of fragments of ßARK1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using Taq DNA polymerase in the presence of [³²P]dCTP. Primer pairs specific for rat ßARK1 and GAPDH sequences have been previously described and were used for amplification of mouse transcripts.¹⁵ These primer pairs amplify the appropriate mRNA in the mouse as revealed by sequencing of amplified products (data not shown). Optimal annealing temperatures for ßARK1 and GAPDH were previously found to be 63°C and 55°C, respectively.¹⁵ The final cycle number used for quantification of the amplified cDNA products was 36 for ßARK1 and 27 for GAPDH, which were previously determined to be in the linear portion of the amplification curve that went to 42 and 35, respectively.¹⁵ Samples were electrophoresed through 1% agarose gel containing ethidium bromide, the polymerase chain reaction (PCR) products were removed from the gel, and ³²P incorporation was measured using liquid scintillation. Relative quantities of ßARK1 were normalized to levels of GAPDH in individual samples as described previously.¹⁵ ßARK1/GAPDH values (in arbitrary units [AU]) from drug-treated hearts are expressed as fold of control (vehicle) mRNA values.

In Vitro Cell Studies
Chinese hamster fibroblast (CHW) cells stably overexpressing ß₁ARs (201±48 fmol/mg) were used.¹⁶ On the day before the experiment, cells were serum starved overnight to induce a state of quiescence, and on the next day, the medium was replaced with fresh medium that contained propranolol (10^-4 M), isoproterenol (10^-4 M), or vehicle (0.002% ascorbic acid). Cells were incubated for 48 hours at 37°C. Cells were washed twice with PBS and then solubilized with ice-cold RIPA buffer. Immunodetection of the specific GRKs were assessed as described.

Statistical Analysis
Data are expressed as mean±SEM. Data for isoproterenol and atenolol were analyzed using ANOVA with posthoc testing performed with Bonferroni's analysis. An unpaired Student's t test was performed to analyze the carvedilol data as well as the RT-PCR data.

	Results

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Heart Weight–to–Body Weight Ratios
Isoproterenol induced a dose-dependent increase in heart size without affecting the body weight (Table 1). This isoproterenol-dependent increase in the heart weight–to–body weight ratio demonstrates the presence of myocardial hypertrophy. Atenolol treatment did not modify body or heart weight. Conversely, carvedilol treatment significantly reduced the cardiac mass, as indicated by the decreased heart weight–to–body weight ratio (Table 1).

View this table: [in this window] [in a new window]   Table 1. Trophic Heart Responses After Drug Treatment

Myocardial ßAR Signaling Properties
Classically, long-term exposure to agonists causes downregulation of ßARs, whereas long-term ß-blockade produces upregulation.² Therefore, ßAR density was measured in the hearts of treated animals. As expected, isoproterenol decreased ßAR density and atenolol treatment induced an increase in the number of ßARs in a dose-dependent manner (Table 2). Carvedilol is an atypical ß-antagonist that has been shown to decrease ßAR density,¹⁷ which was seen after 14 days of treatment (Table 2).

View this table: [in this window] [in a new window]   Table 2. ßAR Density and Membrane Adenylyl Cyclase Activity in Treated Mice

We assessed adenylyl cyclase activity in cardiac membranes to examine the signaling properties of myocardial ßARs after long-term stimulation or blockade. Long-term infusion of isoproterenol resulted in a dampening of adenylyl cyclase activity under basal conditions and after ßAR stimulation, which is consistent with both receptor downregulation and enhanced desensitization (Table 2). In atenolol- and carvedilol-treated animals, there was a dose-dependent increase in adenylyl cyclase activity both under basal conditions and in response to isoproterenol (Table 2). This increase in membrane adenylyl cyclase activity in carvedilol-treated animals occurred despite a significant loss in ßAR density (Table 1).

Myocardial GRK Protein Levels
Long-term stimulation of ßARs with isoproterenol resulted in a significant increase in ßARK1 expression that was related to the dose of the drug (Figure 1A). The analysis of total myocardial ßARK1 levels in atenolol-treated animals demonstrated that ßARK1 regulation is dependent on the degree of ßAR blockade in that the amount of ßARK1 was reduced in a dose-dependent manner (Figure 1B). Interestingly, carvedilol treatment also induced a significant reduction in myocardial ßARK1 expression (Figure 1C). Because other GRKs are expressed in the heart, we investigated whether changes in ßARK1 expression were specific by examining the myocardial levels of GRK5. This GRK is a membrane-bound kinase expressed in the heart that has been shown to desensitize myocardial ßARs in vivo.¹⁰ In contrast to ßARK1, none of the drug treatments affected the expression of GRK5 in cardiac membranes, suggesting that regulation of this enzyme is not dependent on the functional state of ßARs (Figure 2).

View larger version (27K): [in this window] [in a new window]   Figure 1. Myocardial ßARK1 protein levels. Histograms represent mean±SEM in densitometry units of scanned chemiluminescent immunoblots from 4 to 6 hearts at each given dose of (A) isoproterenol (Iso), (B) atenolol (Ate), or (C) carvedilol. Insets show representative immunoblots for each set of mouse hearts. Purified ßARK1 is included as control for protein migration. *P<0.05 versus vehicle.

View larger version (27K): [in this window] [in a new window]   Figure 2. Myocardial GRK5 protein levels. Histograms represent mean±SEM in densitometry units of scanned chemiluminescent immunoblots from 4 to 6 hearts at each given dose of (A) isoproterenol (Iso), (B) atenolol (Ate), or (C) carvedilol. Insets show representative immunoblots for GRK5 for each drug treatment. P=NS at all doses.

To prove that the regulation of ßARK1 is an intrinsic feature of ßAR stimulation and inhibition and is independent of cellular type and to rule out direct or indirect interference of the drugs with mechanisms other than the functional state of ßARs, such as peripheral hemodynamic changes, we studied cultured mammalian cells (CHW) expressing exclusively the human ß₁AR, thus simulating the predominant ßAR signaling pathway in cardiomyocytes. The analysis of total ßARK1 expression in this model showed that ßAR inhibition using the ß-blocker propranolol induced a significant reduction (25%), whereas ßAR stimulation with isoproterenol induced a similar significant increase in ßARK1 levels (Figure 3). These two opposite-acting drugs did not alter GRK5 expression (data not shown). These findings in a cultured cell system clearly parallel our in vivo findings in the mouse heart.

View larger version (18K): [in this window] [in a new window]   Figure 3. ßARK1 levels in treated CHW-ß1AR cells. Histograms show mean±SEM in densitometry units of scanned immunoblots from 4 experiments performed in duplicate. CTRL indicates control. *P<0.05 versus vehicle.

Myocardial GRK Activity
To assess whether the changes in the protein levels of ßARK1 correspond to an increase in myocardial GRK activity, we examined the soluble GRK activity of cardiac extracts in an in vitro phosphorylation assay using the G protein–coupled receptor rhodopsin as a substrate. We have found that GRK activity in cytosolic fractions is almost entirely due to ßARK1.^{8 9 10} In isoproterenol-treated animals, there was a dose-dependent increase in myocardial GRK activity that was proportional to the increase in ßARK1 protein (Figure 4A). Reciprocally, in atenolol- and carvedilol-treated animals, there was a reduction in GRK activity (Figure 4B and 4C).

View larger version (22K): [in this window] [in a new window]   Figure 4. Myocardial GRK activity. Results shown are mean±SEM from 4 to 6 cytosolic extracts taken from mouse hearts after treatment with doses of (A) isoproterenol (Iso), (B) atenolol (Ate), or (C) carvedilol. Inset in C is representative autoradiograph from dried gel showing reduced rhodopsin (rho) phosphorylation activity in cytosolic extracts from carvedilol-treated hearts. *P<0.05 versus vehicle.

Semiquantitative RT-PCR
To examine the molecular regulation of myocardial ßARK1 expression in response to the modulation of ßAR signaling, we used semiquantitative RT-PCR¹⁵ to analyze mRNA levels in the hearts of mice treated with the highest doses of isoproterenol and atenolol because these hearts have the largest changes in levels of ßARK1 protein. The amplified ßARK1 product was normalized to amplified GAPDH (which was similar in all samples), and values from drug-treated hearts were compared with control (vehicle-treated) mRNA levels. The final cycle lengths used for the quantification of ßARK1 and GAPDH (36 and 27, respectively) were previously found to be in the linear portion of the amplification curve (see Methods). In isoproterenol-treated hearts, ßARK1 mRNA levels were 2-fold higher than those in vehicle-treated control hearts (in fold of control values: 2.10±0.04 for isoproterenol treatment versus 1.00±0.02 for vehicle treatment, n=3 each; P<0.05). Atenolol treatment induced significant lowering of ßARK1 mRNA levels (in fold of control values: 0.63±0.28 for atenolol versus 1.00±0.13 for vehicle, n=5 each; P<0.05). This reciprocal regulation of ßARK1 mRNA expression after isoproterenol and atenolol treatment explains the changes in protein levels already described.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The results of the present study demonstrate that ßARK1 expression in the myocardium is tightly linked to the functional state of ßARs. Using ßAR ligands with opposing actions, we found that ßARK1 expression in the heart was reciprocally regulated after long-term infusion of isoproterenol or the ß-blocker atenolol through the use of implanted miniosmotic pumps. This dynamic relationship between ßAR signaling and the expression of ßARK1 is selective because ßAR inhibition or activation did not affect the expression of GRK5.

A growing body of evidence supports a critical role of GRK activity in the determination of cardiac contractility. Studies conducted in transgenic mice have shown that the manipulation of ßARK1 activity in the heart can have profound effects on in vivo cardiac performance. Adding to the importance of ßARK1 in heart function are the recent findings that increased levels of ßARK1 accompany depressed cardiac contractility in several diseases or conditions, such as myocardial ischemia,⁶ ventricular hypertrophy,⁸ hypertension,⁷ and heart failure.⁵ The mechanisms that induce upregulation of ßARK1 in these states are not known. However, because enhanced sympathetic outflow is associated with these conditions, especially heart failure,¹⁸ increased catecholamines may be a triggering mechanism through long-term stimulation of myocardial ßARs.

Long-term isoproterenol administration results in sustained cardiac adrenergic activation, which may mimic the heightened sympathetic nervous system activity observed in cardiovascular disease. Fourteen days of isoproterenol infusion produced cardiac hypertrophy and impairment of ßAR signaling. ßAR density was reduced, and the remaining receptors were desensitized. The increase in ßARK1 expression and activity appears to be responsible for the desensitization because long-term infusion of isoproterenol did not affect GRK5 expression. The mRNA levels for ßARK1 were also increased, supporting the hypothesis of a direct and selective relationship between ßAR signaling and molecular GRK regulation.

Because our results with isoproterenol suggest a biofeedback mechanism linking the functional state of ßARs and myocardial ßARK1 expression, we hypothesized that ß-blockers would reduce ßARK1 levels, leading to improved ßAR signaling. Indeed, long-term treatment with atenolol reduced ßARK1 protein and activity levels in a dose-dependent manner, reaching a maximum reduction of 50%. Lower ßARK1 activity in atenolol-treated animals was associated with enhanced ßAR signaling as measured by adenylyl cyclase activity. The decrease in ßARK1 expression and activity can be attributed to decreased mRNA. Like isoproterenol, atenolol did not affect the expression of GRK5, demonstrating specificity for the regulation of ßARK1. These results demonstrating the specific effects of a ß-blocker on one form of a GRK but not another are in contrast to an earlier study in pigs in which myocardial GRK activity was examined after long-term ß-blockade.¹⁹ Although the authors of this study found an apparent decrease in GRK activity, no specific GRK isoform was examined. Taken together, the present results obtained with atenolol- and isoproterenol-treated mice provide in vivo evidence for the reciprocal regulation of myocardial ßARK1 by the functional state of ßARs. Our data do not rule out minor contributions of other GRKs that are expressed at lower levels in the heart, such as GRK3 and GRK6.

To rule out any possibility that these two opposite-acting drugs regulate ßARK1 through mechanisms independent of myocardial ßAR signaling alterations, such as changes in cardiac hemodynamics, we studied the effects of ßAR antagonism and activation in an in vitro cellular model. We chose CHW cells stably overexpressing the ß₁AR because of the prevalence of this ßAR subtype in the heart. Using isoproterenol or propranolol treatment, we found similar reciprocal regulation of ßARK1. These results demonstrate that regulation of ßARK1 expression is an intrinsic feature of ßAR signaling, apparently independent of cell type. Furthermore, they indicate that regulation of myocardial ßARK1 expression in vivo is due to the direct action of these drugs on myocardial ßARs and not to peripheral effects such as changes in systolic pressure.

The results of the present study demonstrate that long-term ßAR activation triggers mechanisms that lead to the selective increase in ßARK1 mRNA, protein, and activity. Relating this to pathophysiological settings such as in heart failure, the elevated catecholamine levels¹⁸ presumably trigger a series of events, including the upregulation of ßARK1, aimed at compensating for long-term ßAR activation. Importantly, increased ßARK1 leads to both ßAR desensitization and diminished cardiac contractility.^{8 9 10} This explanation supports the "adrenergic hypothesis" of heart failure,²⁰ which proposes that increased cardiac sympathetic drive results in abnormalities of ßAR signaling. We demonstrate here that this includes GRK regulation. Although GRK5 can also desensitize myocardial ßARs in vivo,¹⁰ our findings demonstrate that GRK5 expression is not regulated by ßAR signaling and that this mode of feedback regulation is specific for ßARK1.

If the enhanced ßARK expression and activity in response to ßAR activation in heart failure are maladaptive, then one might predict that treatments that show benefit in the treatment of heart failure through decreased ßAR stimulation would lower levels of ßARK1. We hypothesized that this may play a role in the beneficial effects of certain ß-blockers in the treatment of heart failure. Thus, in addition to atenolol, we examined myocardial GRK expression in mice after long-term treatment with carvedilol, a novel ß-blocker (also possessing -adrenergic receptor antagonism)¹⁷ that has been shown to dramatically increase survival in patients with heart failure.¹¹ Interestingly, 14 days of a carvedilol infusion in the mouse significantly decreased ßARK1 levels in a selective manner. Importantly, the effects of carvedilol on ßARK1 expression are not due to its effects on ßAR density because unlike atenolol, carvedilol decreased ßAR number, suggesting that ßAR density is not a determinant for the improvement in ßAR signaling observed with these drugs. Furthermore, carvedilol and isoproterenol treatments produced similar decreases in ßAR density, yet cAMP production was reciprocally altered, as was ßARK1 expression. The increased adenylyl cyclase activity seen with lower ßARK1 expression by the two ß-blocking agents (Table 2) is consistent with our previous findings in isolated cardiomyocytes in which infection with an adenovirus containing a peptide inhibitor of ßARK1 was found to increase intracellular cAMP accumulation without an alteration in ßAR density.²¹

Our present findings strongly suggest that the increase in ßARK1 levels in heart failure can contribute to attenuated ßAR signaling and cardiac dysfunction. They also specifically demonstrate that antagonism of ßAR signaling leads to a selective reduction in ßARK1, thus raising the hypothesis that reduction in ßARK1 activity participates in the ameliorating effects on heart failure associated with carvedilol treatment. Future studies, perhaps in patients treated with carvedilol, will be required to demonstrate a correlation between lower ßARK1 activity in the heart with salutary effects in heart failure, but our findings support this intriguing possibility. Therefore, inhibition of cardiac ßARK1 activity represents a novel therapeutic target in heart failure. ßARK1 inhibition can be achieved with classic drugs such as ß-blockers or experimentally with more specific and novel therapeutic tools such as gene delivery of DNA encoding a peptide inhibitor of ßARK1, which we have been studying in different model systems.^{21 22} In addition, small molecule pharmaceutical inhibitors of ßARK1 activity can be developed that have the potential to serve as novel therapeutic agents for the treatment of heart failure or other cardiovascular disorders that have a component of ßAR desensitization.

	Acknowledgments

 
This work was supported in part by National Institutes of Health Grant HL-16037 (Dr Lefkowitz); a Fellowship from the American Heart Association, North Carolina Affiliate (Dr Iaccarino); and a Grant-in-Aid from the American Heart Association, North Carolina Affiliate (Dr Koch).

Received March 10, 1998; revision received May 29, 1998; accepted June 3, 1998.

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22. Akhter SA, Skaer CA, Kypson AP, McDonald PH, Peppel KC, Glower DD, Lefkowitz RJ, Koch WJ. Restoration of ß-adrenergic signaling in failing cardiac ventricular myocytes via adenoviral-mediated gene transfer. Proc Natl Acad Sci U S A. 1997;94:12100–12105.[Abstract/Free Full Text]

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