Mechanism of beta -Adrenergic Receptor Desensitization in Cardiac Hypertrophy Is Increased beta -Adrenergic Receptor Kinase*

(Received for publication, January 15, 1997, and in revised form, April 3, 1997)

Dong-Ju Choi , Walter J. Koch Dagger , John J. Hunter and Howard A. Rockman §

From the Department of Medicine, University of California, San Diego, School of Medicine, La Jolla, California 92093 and the Dagger  Department of Surgery, Duke University, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

Pressure overload cardiac hypertrophy in the mouse was achieved following 7 days of transverse aortic constriction. This was associated with marked beta -adrenergic receptor (beta -AR) desensitization in vivo, as determined by a blunted inotropic response to dobutamine. Extracts from hypertrophied hearts had approx 3-fold increase in cytosolic and membrane G protein-coupled receptor kinase (GRK) activity. Incubation with specific monoclonal antibodies to inhibit different GRK subtypes showed that the increase in activity could be attributed predominately to the beta -adrenergic receptor kinase (beta ARK). Although overexpression of a beta ARK inhibitor in hearts of transgenic mice did not alter the development of cardiac hypertrophy, the beta -AR desensitization associated with pressure overload hypertrophy was prevented. To determine whether the induction of beta ARK occurred because of a generalized response to cellular hypertrophy, beta ARK activity was measured in transgenic mice homozygous for oncogenic ras overexpression in the heart. Despite marked cardiac hypertrophy, no difference in beta ARK activity was found in these mice overexpressing oncogenic ras compared with controls. Taken together, these data suggest that beta ARK is a central molecule involved in alterations of beta -AR signaling in pressure overload hypertrophy. The mechanism for the increase in beta ARK activity appears not to be related to the induction of cellular hypertrophy but to possibly be related to neurohumoral activation.


INTRODUCTION

The regulation of myocardial beta -adrenergic receptors (beta -ARs)1 involves a process characterized by a rapid loss of receptor responsiveness despite continued presence of agonist. Two classes of kinases regulate receptors through rapid receptor phosphorylation; the second messenger activated protein kinases, such as cAMP-dependent kinase A and protein kinase C (1), and the G protein-coupled receptor kinases (GRK), which phosphorylate only activated receptors leading to a process termed homologous desensitization (2, 3). Of the six known members of the emerging GRK family, GRK2 (commonly known as beta ARK1) and GRK5 appear to be dominantly expressed in the heart (4, 5). Desensitization of agonist-occupied receptors by the primarily cytosolic beta ARK1 requires a membrane-targeting event prior to receptor phosphorylation by a direct physical interaction between residues within the carboxyl terminus of beta ARK and the dissociated, membrane-anchored beta gamma subunits of G proteins (6, 7). Unlike beta ARK1, GRK5 does not undergo agonist-dependent translocation from cytosol to membrane but rather is constitutively membrane-bound (5).

Decreased responsiveness to beta -AR agonists is a characteristic of chronic heart failure. In heart failure, beta -AR desensitization is due to both diminished receptor number (receptor down-regulation) and impaired receptor function (receptor uncoupling) (8), in part related to enhanced beta ARK activity (9, 10). Recent data suggest a step wise increase in plasma norepinephrine levels in individuals from normal cardiac function to asymptomatic left ventricular (LV) dysfunction and symptomatic heart failure (11). Thus, high levels of circulating catecholamines early in the transition from stable cardiac hypertrophy (12) to LV dysfunction may account for the observed beta -AR desensitization in the disease process.

Although cardiac hypertrophy may be regarded as an adaptive response to increased work load (13), ventricular hypertrophy (especially when accompanied by prolonged periods of hypertension) is associated with an increased incidence of heart failure (14). In models of cardiac hypertrophy, heterologous desensitization of adenylyl cyclase associated with down-regulation of beta 1-ARs and increased Gialpha protein are found, which are associated with reduced positive inotropic response to isoproterenol (15, 16).

We have recently shown that beta ARK is a critical modulator of in vivo contractile function (17). Both the beta 1-adrenergic and angiotensin II receptors are targets for beta ARK1-mediated desensitization, whereas selected desensitization of beta 1-ARs occurs with GRK5 (17, 18). Transgenic mice, which overexpress a peptide inhibitor of beta ARK1, show decreased desensitization of beta -ARs supporting the in vivo role for beta ARK1 as a modulator of cardiac function (17). Since abnormalities in beta -adrenergic signal transduction may be one of the earliest changes in the transition from compensated hypertrophy to decompensated heart failure, we wanted to determine whether cardiac hypertrophy is associated with beta -AR desensitization, and we tested the hypothesis that in LV pressure overload, hypertrophy desensitization occurs as a result of enhanced beta ARK1 activity.


MATERIALS AND METHODS

Experimental Animals

Adult wild-type (strain C57/B6) and transgenic mice of either sex and 3-6 months of age were used for this study. Two types of transgenic mice were used for this study, 1) mice with cardiac-specific overexpression of a beta ARK1 inhibitor (beta ARKct) shown previously to have diminished desensitization to beta -AR stimulation (17) and 2) transgenic mice homozygous for cardiac-targeted oncogenic ras known to develop severe cardiac hypertrophy in the absence of hemodynamic overload (19). The animals in this study were handled according to the animal welfare regulations of the University of California, San Diego, and the protocol was approved by the Animal Subjects Committee of this institution.

Microsurgical Techniques

Mice were anesthetized with a mixture of ketamine and xylazine (20). After endotracheal intubation, mice were connected to a rodent ventilator. Using microsurgical procedures as described previously (21), the chest cavity was entered in the second intercostal space, and the transverse aorta between the right (proximal) and left (distal) carotid arteries was isolated. Transverse aortic constriction (TAC) was performed by tying a 7-0 nylon suture ligature against a 27-gauge needle, the latter being promptly removed to induce pressure overload cardiac hypertrophy (20, 22). After aortic constriction, the chest was closed, the pneumothorax was evacuated, and the mice were extubated and allowed to recover from the anesthesia. Sham-operated animals underwent the same operation except for aortic constriction.

Hemodynamic Evaluation in Intact Anesthetized Mice

After 7 days of aortic constriction, mice were anesthetized and reweighed, and the left carotid artery (distal to the stenosis) was cannulated with a flame-stretched PE-50 catheter connected to a modified P-50 Statham transducer. Either a 1.4 French (0.46 mm diameter) or 1.8 French (0.61 mm diameter) high fidelity micromanometer catheter (Millar Instruments) was inserted into the right carotid (proximal to the stenosis), and simultaneous aortic pressures were measured. Following bilateral vagotomy, the micromanometer was advanced retrograde into the LV. Hemodynamic measurements were recorded at base line and following 2-min infusions of dobutamine at 0.5, 1.0, 1.5, and 2.0 µg/kg/min. Continuous high fidelity LV and fluid-filled aortic pressure was recorded simultaneously on an 8 channel chart recorder and in digitized form at 2000 Hz for later analysis. Experiments were then terminated, hearts were rapidly excised, and individual chambers were separated, weighed, and frozen in liquid N2 for later biochemical analysis.

GRK Activity by Rhodopsin Phosphorylation

Myocardial extracts were prepared by homogenization of excised hearts in ice-cold lysis buffer (2 ml) (25 mM Tris-Cl (pH 7.5), 5 mM EDTA, 5 mM EGTA, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM PMSF) and centrifuged at 48,000 × g for 30 min. The supernatants, which contain soluble kinases (i.e. beta ARK1), were kept. The pelleted membranes were rehomogenized in lysis buffer containing 250 mM NaCl, put on ice for 30 min to dissociate membrane-bound kinase (i.e. GRK5), and centrifuged at 48,000 × g for 30 min. This supernatant (membrane fraction) was kept. Both the cytosolic and membrane fractions were further purified by adding a slurry of 50% (v/v) diethylaminoethyl Sephacel (pH 7.0) in the presence of 50 mM NaCl for the cytosolic fraction (beta ARK1) and 250 mM NaCl for the membrane fraction (250 mM NaCl was used to dissociate membrane associated GRKs) and incubating at 4 °C for 30 min. To remove NaCl from the suspension, fractions were run over an ion exchange column (18, 23). Final supernatants were eluted with no salt lysis buffer and concentrated using a Centricon (Amicon-30) microconcentrator. Protein concentration was determined by modified Lowry method (Bio-Rad DC Protein Assay kit). Concentrated cytosolic (300-400 µg of protein) and membrane extracts (12-15 µg of protein) from sham and TAC ventricles were incubated with rhodopsin-enriched rod outer segments (ROS) (17) in reaction buffer (25 µl) containing 10 mM MgCl2, 20 mM Tris-Cl, 2 mM EDTA, 5 mM EGTA, and 0.1 mM ATP (containing [gamma -32P]ATP). After incubating in white light for 15 min at room temperature, reactions were quenched with ice-cold lysis buffer (300 µl) and centrifuged for 15 min at 13,000 × g. Sedimented proteins were resuspended in 20 µl of protein-gel loading dye and electrophoresed through 12% SDS-polyacrylamide gels (24). Phosphorylated rhodopsin was visualized by autoradiography of dried polyacylamide gels and quantified using a phoshorimaging system, Molecular Imager GS-250 (Bio-Rad Laboratories).

To confirm beta ARK-dependent phosphorylation of ROS, the protein kinase A inhibitor, PKI (1 µM, Sigma), and heparin (10 µg/ml) were incubated with purified beta ARK, and the capacity to phosphorylate light-activated rhodopsin was determined. Heparin inhibited phosphorylation while PKI did not (data not shown).

To identify the specific GRK activities in the cytoplasm and membrane fraction, monoclonal antibodies (10 µg/ml), which specifically bind to and inhibit beta ARK1/2 (monoclonal C5/1) or GRK4/5/6 (monoclonal A16/17), were incubated with either cytosolic or membrane extracts for 10 min prior to a standard rhodopsin phosphorylation assay (25).

Immunoblotting

Immunodetection of myocardial levels of beta ARK1 was performed on cytosolic extracts following immunoprecipitation. Individual control (n = 4) and hypertrophied (n = 4) hearts were homogenized in RIPA (50 mM Tris-Cl (pH 8.0), 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS). beta ARK1 was immunoprecipitated from 1 ml of clarified extract with a 1:1,000 (1 µl) monoclonal anti-beta ARK1 (C5/1) antibody (25) and 35 µl of a 50% slurry of protein A-agarose conjugate agitated for 1 h at 4 °C. Immune complexes were washed 5 times with ice-cold solubilization buffer, and the washed agarose beads were resuspended in 20 µl of protein-gel loading buffer, heated for 3 min at 85 °C, and then electrophoresed through 12% SDS-polyacrylamide gels and transferred to nitrocellulose. The ~80-kDa beta ARK1 protein was visualized with the monoclonal antibody raised against an epitope within the carboxyl terminus of beta ARK1 and chemiluminescent detection of anti-mouse IgG conjugated with horseradish peroxidase (ECL, Amersham Corp.) (17).

Adenylyl Cyclase Activity and beta AR Receptor Density

Myocardial sarcolemmal membranes were prepared by homogenizing whole hearts in ice-cold cyclase buffer A (50 mM HEPES (pH 7.3), 150 mM KCI, 5 mM EDTA). Nuclei and tissue were separated by centrifugation at 800 × g for 10 min, and the crude supernatant was then centrifuged at 20,000 × g for 10 min. Sedimented proteins were resuspended at a concentration of 2-3 mg of protein/ml of assay buffer B (50 mM HEPES (pH 7.3), 5 mM MgCl2). Membranes (30-40 µg of protein) were incubated for 15 min at 37 °C with various agonists (Table I) in 50 µl of assay mixture containing 20 mM Tris-Cl, 0.8 mM MgCl2, 2 mM EDTA, 0.12 mM ATP, 0.05 mM GTP, 0.1 mM cAMP, 2.7 mM phosphoenolpyruvate, 0.05 IU/ml myokinase, 0.01 IU/ml pyruvate kinase, and [alpha -32P]ATP, and cAMP was quantified (17).

Table I. Adenylyl cyclase activity in sham and hypertrophied hearts

Data expressed as pmol/mg protein/min. ISO, isoproterenol; TAC, transverse aortic constriction.

Basal ISO (10-4 M) NaF (10-3 M) Forskolin (10-4 M)

Sham (n = 9) 25.4  ± 2.5 40.6  ± 5.0 292  ± 20 276  ± 28
TAC (n = 9) 18.5  ± 2.2 27.4  ± 2.8a 281  ± 23 227  ± 23

a p, <0.05 compared with sham, one factor ANOVA.

Total beta -AR density was determined by incubating 25 µg of the above membranes with a saturating concentration of 125I-cyanopindolol in 500 µl of binding buffer (17). Nonspecific binding was determined in the presence of 20 µM alprenolol. Binding assays were conducted at 37 °C for 60 min and terminated by rapid vacuum filtration over glass fiber filters, which were subsequently washed and counted in a gamma counter. Specific binding was normalized to membrane protein and reported as picomoles of receptor per milligram of membrane protein.

Statistical Analysis

Data are expressed as mean value ± S.E. To examine the effect of the dobutamine on changes in hemodynamic parameters between the control and TAC groups, a two-way repeated measures analysis of variance (ANOVA) was used. Post hoc analysis with regard to differences in mean values between the groups at a specific dose was conducted with a Newman-Keuls test. To test for statistical difference in adenylyl cyclase activity, a one-factor ANOVA was used. A Student's t test was used to test for statistical difference in the parameters of LV hypertrophy, beta -AR density, and GRK activity. For all analyses, p < 0.05 was considered significant.


RESULTS

Pressure overload cardiac hypertrophy was achieved following 7 days of TAC, which resulted in a significant increase in LV weight to body weight ratio (34%) and LV to tibia length ratio (39%) compared with sham-operated mice (Fig. 1A). The hemodynamic response to chronic aortic constriction was followed by monitoring the pressure gradient between the two carotid arteries (proximal and distal) in anesthetized animals prior to the measurement of ventricular hemodynamics. A significant gradient across the surgically induced stenosis was present at 7 days after TAC (Fig. 1B). Systolic aortic pressure proximal to the stenosis in the TAC group was slightly higher than sham-operated animals but did not reach statistical significance (p = 0.08). This moderate degree of constriction provides an adequate mechanical stimulus for the development of cardiac hypertrophy (Fig. 1A) without the development of cardiac failure (20, 26).


Fig. 1. The effect of transverse aortic constriction on LV weight and aortic pressure. A, left ventricular weight to body weight (LV/BW) and left ventricular weight to tibia length (LV/T) was measured in sham-operated (n = 24) and 7 days following transverse aortic constriction (TAC) (n = 23). The largest body weight (either preoperative or postoperative) was used to calculate the ratios of (LV) weight to body weight to eliminate potential bias from postoperative weight loss in the aortic-constricted animals. Significant cardiac hypertrophy resulted following TAC. *, p < 0.001 TAC versus sham. B, systolic aortic pressure was measured in the carotid arteries proximal and distal to the region of stenosis created using a suture ligature chronically tied around the transverse aorta. Hemodynamic measurements were performed in 12 sham- and 11 TAC-operated animals. TAC resulted in a significant difference between proximal and distal pressures confirming the presence of a chronic hemodynamic load in vivo in the mice. dagger , p < 0.001, t test with Bonferroni correction for four comparisons.
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To determine whether the development of LV hypertrophy is associated with beta -AR uncoupling in vivo, cardiac catheterization was used to measure catecholamine responsiveness in intact anesthetized mice (Fig. 2). Marked blunting of the dobutamine (a beta 1-agonist) induced inotropic response (Fig. 2A) as well as an attenuated fall in LV dP/dtmin (an index of myocardial relaxation) (Fig. 2B) was observed in mice with cardiac hypertrophy. The LV systolic pressure and heart rate response to dobutamine between the groups were not different (Fig. 2, C and D). These data demonstrate that beta -AR desensitization is associated with the development of cardiac hypertrophy.


Fig. 2. In vivo assessment of LV contractile function in response to beta -agonist stimulation. Cardiac catheterization was performed in the intact anesthetized mice using a 1.8 French high fidelity micromanometer. Parameters measured were heart rate, aortic pressure, LV systolic, and end diastolic pressure, and the maximal and minimal first derivative of LV pressure (LV dP/dtmax and dP/dtmin, respectively). Ten sequential beats were averaged for each measurement. Four measured parameters are shown at base line and after progressive infusion of dobutamine in sham-operated (open circle , n = 12), and TAC (bullet , n = 11) mice. A, LV dP/dtmax; B, LV dP/dtmin; C, LV, systolic pressure; D, heart rate. Data were analyzed with a two-way repeated ANOVA and post hoc analysis with regard to differences in mean values between the groups at a specific dose was conducted with a Newman-Keuls test. *p < 0.001; dagger , p < 0.05; control versus transgenic. The pattern of change between groups was statistically significantly for LV dP/dtmax, p < 0.005 (A) and LV dP/dtmin, p < 0.001 (B).
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To determine whether alterations in GRK activity could account for the beta -AR uncoupling seen in these hypertrophied hearts, extracts were prepared and assayed for phosphorylation of the G protein-coupled receptor rhodopsin. Cytosolic extracts from TAC hearts had approx 3-fold increase in GRK activity compared with extracts from hearts of sham-operated animals (Fig. 3, A and B). Protein immunoblotting was used to detect the level of beta ARK1 in the cytosol of hypertrophied hearts (Fig. 3C). Consistent with the marked increase in GRK activity, a clear increase in beta ARK1 protein levels was observed by Western blotting in hearts following TAC (Fig. 3C).


Fig. 3. Effect of pressure overload hypertrophy on beta ARK activity and protein. A, cytosolic extracts from sham-operated and TAC hearts were measured for their capacity to phosphorylate rhodopsin. 300 µg of cytosolic protein was incubated with 350 pmol of rhodopsin-enriched ROSs in lysis buffer (total volume 25 µl). Phosphorylated rhodopsin was visualized by autoradiography following electrophoresis through 12% SDS-polyacrylamide gels. Lane 1, C, ROS in the absence of heart extract; lanes 2-5, cytosolic extracts from individual sham-operated mice;, lanes 6-9, individual TAC-operated hearts. Each lane (2-9) represents extracts from a separate heart. Lane 10-12, incubation of ROS with 12.5, 25, and 50 µg of purified beta ARK1, respectively. Shown is a representative autoradiograph of a dried gel where phosphorylated rhodopsin (Rho) is visualized. B, the level of beta ARK activity in 12 sham-operated and 11 TAC hearts. Activities were calculated as 32P incorporation (fmol/min/mg of cytosolic protein), *, p < 0.001. C, immunodetection of myocardial level of beta ARK1 in cytosolic extracts from individual sham-operated (lanes 1-4) and individual TAC operated (lanes 5-8) mice. An approx 80-kDa protein was visualized by Western blotting and chemiluminescence following solubilization of cytosolic extracts and immunoprecipitation. Lane 9, 50 µg of purified beta ARK1.
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Since GRK5 is associated with the membrane, the level of GRK activity was measured in cardiac membranes of hypertrophied and non-hypertrophied hearts by assessing their capacity to phosphorylate light-activated rhodopsin (Fig. 4). A 2.5-fold increase in membrane GRK activity was observed. These data suggest that in cardiac hypertrophy, increased GRK levels in both cytosol and membrane fractions may account for the observed desensitization of beta -ARs in vivo.


Fig. 4. GRK activity in the myocardial membrane fraction. Membrane extracts from individual sham-operated (lanes 1-4), and TAC hearts (lanes 5-8) were assessed for the capacity to phosphorylate rhodopsin (Rho). Lanes 9-11 represent 1:5000, 1:2000, and 1:1000 dilution of purified GRK5, respectively. See Fig. 3 for details.
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To identify the specific GRK that mediates beta -AR desensitization in the hypertrophied heart, monoclonal antibodies that neutralize beta ARK1 and beta ARK2 (C5/1) or GRK4, GRK5, and GRK6 (A16/17) were used to inhibit endogenous GRK activity (25). In a rhodopsin phosphorylation assay using purified beta ARK1 as a control, pre-incubation with the monoclonal antibody C5/1, significantly inhibited phosphorylation. (Fig. 5A). When the monoclonal antibody C5/1 was added to cytosolic extracts from hypertrophied hearts, phosphorylation of rhodopsin was significantly inhibited as shown by the negligible GRK activity (Fig. 5A). Incubation of either purified beta ARK1 or cytosolic extracts with the monoclonal antibody A16/17 did not inhibit rhodopsin phosphorylation (data not shown). This indicates that beta ARK1 accounts for the increase in cytosolic kinase activity with cardiac hypertrophy.


Fig. 5. Determination of specific subtype responsible for increased GRK activity in pressure overload hypertrophy. A, the capacity of the monoclonal antibody C5/1 to inhibit the GRK subtype beta ARK1. Incubation of purified beta ARK1 (50 µg) in the absence (lanes 1 and 2) and presence (lanes 3 and 4) of C5/1. Cytosolic extracts from two individual TAC hearts were incubated in the absence (lanes and 6) and presence (lanes 7 and 8) of C5/1. Lanes 7 and 8 were identical to lanes 5 and 6 except for pre-incubation with the C5/1 monoclonal antibody prior to rhodopsin phosphorylation. B, GRK activity in cardiac membrane. Membrane extracts were incubated in the absence or presence of either monoclonal antibody A16/17 (anti-GRK4/6) or C5/1 (anti-beta ARK1/2). Lanes 1-4, sham-operated hearts; lanes 5-16, TAC-operated hearts. Separate hearts were used for lanes 1-8, whereas extracts from the same hearts were used for lanes 5, 9, and 13; lanes 6, 10, and 14; lanes 7, 12, and 15; and lanes 8, 12, and 16.
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Membrane GRK activity was also studied with monoclonal antibodies since the increase in membrane GRK activity could be due to either enhanced expression of GRK5 or increased membrane translocation of beta ARK1 (a process required for activation) (7). Membrane extracts from sham-operated and TAC mice were incubated in the presence of the monoclonal C5/1 or the monoclonal antibody A16/17 (Fig. 5B). As shown (Fig. 5B, lanes 5-8), a 2.6-fold increase in GRK activity was present in membrane extracts of hypertrophied hearts compared with sham-operated hearts. Incubation with A16/17 (Fig. 5B, lanes 9-12) partially reduced membrane GRK activity (1.6-fold higher than non-hypertrophied controls). In contrast, incubation with the monoclonal antibody C5/1 (Fig. 5B, lanes 13-16) markedly inhibited membrane GRK activity to levels near that of non-hypertrophied control hearts. These data suggest that the predominant GRK responsible for the increase in membrane kinase activity in cardiac hypertrophy is beta ARK1.

beta -AR density was determined in cardiac membranes prepared from sham (n = 12) and TAC (n = 12) operated hearts. No difference was found in the number of receptors between the two groups (21.8 ± 1.3 versus 25.1 ± 2.2, fmol/mg of membrane protein, p = not significant). In contrast to the lack of change in beta -AR density, hypertrophied hearts had significantly lower isoproterenol-stimulated adenylyl cyclase activity (Table I). Thus, functional uncoupling of beta -ARs as assessed by the physiologic response to beta -agonist stimulation and adenylyl cyclase activation was hindered with the development of myocardial hypertrophy. To confirm that the observed uncoupling of beta -ARs was not due to the increase in inhibitory G protein (Gialpha ), we measured the level of Gialpha immunoreactivity with antibodies directed against Gialpha 1-3 (Fig. 6). No difference in the level of Gialpha was observed by immunoblotting in sham and hypertrophied hearts. These data suggest that impaired catecholamine responsiveness with myocardial hypertrophy is not due to alterations in either beta -AR density or to levels of inhibitory G protein.


Fig. 6. Immunodetection of Gialpha in cardiac membranes from sham and TAC hearts. Excised hearts were homogenized in cold lysis buffer (2 ml) (50 mM HEPES (pH 7.3), 150 mM KCl, 5 mM EDTA, 10 µg/ml leupeptin, 20 µg/ml aprotinin and 1 mM PMSF) and centrifuged at 40,000 × g for 30 min. The pellet membranes were resuspended in 50 mM HEPES buffer (pH 7.3) and electrophoresed on a 10% denaturing gel. After transfer, the approx 40-kDa Gialpha protein was visualized with 1:1,000 dilution of polyclonal antibody (Santa Cruz Biotechnology, I-20) and chemiluminescent detection of anti-rabbit IgG conjugated with horseradish peroxidase. Each lane represents an individual heart. Lanes 1-4, sham-operated; lanes 5-8, TAC. The 42-kDa marker is shown.
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To determine whether the impaired catecholamine responsiveness and beta -AR desensitization that accompany the development of cardiac hypertrophy could be accounted for by the induction of beta ARK, we used a strategy of in vivo beta ARK inhibition achieved with cardiac-specific overexpression of a beta ARK1 inhibitor in transgenic mice (17). The inhibitor utilized was the carboxyl terminus of beta ARK1 (beta ARK1ct), which contains the Gbeta gamma -binding domain and competes with endogenous beta ARK for binding and subsequent translocation/activation (6, 7, 17). Transgenic mice overexpressing the beta ARKct underwent TAC, and the in vivo hemodynamic response to dobutamine was determined 7 days later. Following TAC, hearts from transgenic mice with overexpression of the beta ARKct developed LV hypertrophy to the same degree as wild-type mice (a 38% increase in LV to body weight ratio compared with sham-operated beta ARKct inhibitor mice, Fig. 7A). To determine whether this increase in LV mass was associated with increased beta ARK activity, cytosolic extracts from sham- and TAC-operated beta ARKct mouse hearts were used to test for their capacity to phosphorylate rhodopsin. A 3-fold increase in GRK activity was found in cytosolic extracts from hypertrophied compared with sham-operated beta ARKct mice (Fig. 7B). To determine whether forced overexpression of the beta ARKct would prevent functional beta -AR desensitization with the development of cardiac hypertrophy, we performed cardiac catheterization in sham and TAC mice overexpressing the beta ARKct inhibitor. As shown in Fig. 8, the response in LV dP/dtmax and heart rate to beta -AR stimulation was similar in the hypertrophied transgenic mice compared with sham-operated transgenic liter mates (Fig. 8). Similarly, no statistical difference in the LV dP/dtmin response to dobutamine was found (sham, -9992 ± 786 to -12356 ± 874 mmHg/s; TAC, -9654 ± 952 to -10750 ± 1237, mmHg/s, p = not significant ANOVA).


Fig. 7. The effect of transverse aortic constriction in transgenic mice overexpressing a beta ARK inhibitor (beta ARKct). A, left ventricular weight to body weight (LV/BW) and left ventricular weight to tibia length (LV/T) was measured in beta ARKct animals 7 days following either sham-operation (n = 10) or transverse aortic constriction (TAC) (n = 13). *, p < 0.05 TAC versus SHAM. B, cytosolic extracts from sham-operated and TAC hearts from beta ARKct transgenic mice were measured for their capacity to phosphorylate rhodopsin. Lane 1, C, ROS in the absence of heart extract; lanes 2-5, cytosolic heart extracts from individual sham-operated beta ARKct mice; lanes 6-9, individual TAC-operated beta ARKct mice. Lanes 2-9 each represents extracts from separate hearts; lane 10, incubation of ROS with 25 µg of purified beta ARK1.
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Fig. 8. In vivo contractile function of transgenic mice overexpressing a beta ARK inhibitor following the development of cardiac hypertrophy. Cardiac catheterization was performed in intact anesthetized transgenic animals. The methods used were as in Fig. 2. Shown is LV dP/dtmax (A), heart rate (B), and the difference from base line for LV dP/dtmax (C, Delta LV dP/dtmax) and heart rate (D, Delta heart rate) with dobutamine infusion in sham-operated (open circle ), n = 10, and TAC-operated (bullet ) mice, n = 13. A two-way repeated ANOVA showed no significant difference between sham and TAC beta ARK inhibitor mice. To determine if the values for basal dP/dtmax were different and whether it influenced the response beta -AR stimulation, an analysis of covariance was performed. No significant effect of basal LV dP/dtmax (covariate) was found. Furthermore, just treating basal dP/dtmax as 2 isolated groups using a Student's t test (without correction for multiple comparisons) no significance was reached. No significant difference was found with respect to difference from base line for LV dP/dtmax, and heart rate (C and D).
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To address the question of whether the induction of beta ARK1 in pressure overload hypertrophy is simply a generalized response to cellular hypertrophy, we measured the level of beta ARK activity in transgenic mice homozygous for oncogenic ras overexpression in the heart, which develop cardiac hypertrophy in the absence of hemodynamic overload (19). Compared with wild-type, age-matched controls, overexpression of oncogenic ras resulted in a 62% increase in LV to body weight ratio (5.8 ± 0.35 versus 3.59 ± 0.08 mg/g, p < 0.001). Cytosolic extracts from hearts of these transgenic mice were used to measure beta ARK activity in a rhodopsin phosphorylation assay. Despite the development of severe LV hypertrophy, no difference in the level of beta ARK activity was found in mice overexpressing oncogenic ras compared with wild-type matched controls (Fig. 9). These data suggest that the increase in beta ARK protein and activity in response to pressure overload is not a general phenomenon that accompanies the development of cellular hypertrophy but is specific for pressure overload-induced hypertrophy.


Fig. 9. GRK activity in cytosolic extracts from transgenic mice overexpressing oncogenic ras. The capacity of cytosolic extracts from age-matched, wild-type mice (lanes 1-5) and transgenic mice overexpressing oncogenic ras (lanes 6-10) is tested in a rhodopsin phosphorylation assay. See Fig. 3 for details.
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DISCUSSION

The present study demonstrates that beta -AR desensitization occurs with the development of pressure overload cardiac hypertrophy, and that the uncoupling of beta -ARs with cardiac hypertrophy can be accounted for by an increase in beta ARK1. The blunted inotropic response to beta -AR stimulation that accompanies LV hypertrophy can be reversed by beta ARK inhibition. Furthermore, the mechanism for increased beta ARK levels and activity appear not to be related to the induction of cellular hypertrophy but rather is specific for that which occurs in response to hemodynamic overload. These data demonstrate that in the mouse, beta ARK1 is responsible for altered beta -AR signaling associated with pathologic states such as pressure overload cardiac hypertrophy.

Several previous experimental studies have demonstrated beta -AR desensitization with cardiac hypertrophy (16, 27-29). In two studies, desensitization was associated with a small decrease in beta 1-AR subtype and the increase of the inhibitory G protein Gialpha , which was reversed with angiotensin converting enzyme inhibitor treatment (16, 27). In this study, we show that the marked impairment of catecholamine responsiveness associated with cardiac hypertrophy is related to an increase in GRK activity that predominantly can be accounted for by the increase in beta ARK1 subtype. There also may be a slight increase in GRK5 activity. The impairment in contractile function with beta -agonist stimulation following the development of cardiac hypertrophy (Fig. 2A) is remarkably similar to that observed with targeted overexpression of beta ARK1 (17), providing further evidence that beta ARK1 is a critical in vivo modulator of beta -AR signal transduction.

Although beta ARK1 is predominantly a cytosolic enzyme, translocation to the membrane by Gbeta gamma is required for desensitization of beta ARs. In contrast, GRK5 is always membrane-associated. We used monoclonal antibodies to discriminate between beta ARK1 and GRK5 subtypes in the hypertrophied heart. These experiments demonstrate that in both cytosolic and membrane fractions, beta ARK1 was the subtype predominately responsible for the enhanced GRK activity and suggests that the majority of membrane activity can be accounted for by beta ARK1, which is translocated to the membrane (Fig. 5). Furthermore, the fact that beta ARK activity is enhanced in the beta ARKct mice (in the presence of an inhibitor of Gbeta gamma translocation) without affecting cardiac function, suggests that just an increased level of beta ARK will not enhance its association to the membrane to cause beta -AR desensitization. This is similar to recent findings that suggest that brief cardiac ischemia in an isolated rat heart preparation is of sufficient stimulus to induce membrane GRK activity (30). Our studies here in cardiac hypertrophy go further in that we used monoclonal antibodies to show that a small degree of membrane kinase activity could be accounted for by GRK5.

Tissue distribution of the various members of the GRK family has been assessed by mRNA expression (4). GRK3 and GRK6, in addition to beta ARK1 and GRK5, can be identified in cardiac tissue; however, the level of mRNA expression is significantly less than beta ARK1 and GRK5. Therefore, it is possible that other GRK subtypes such as beta ARK2 or GRK6 could account for some of the observed GRK activity. Quantitative immunoblotting of all subtypes would be required to demonstrate that beta ARK1 and GRK5 are the sole GRKs present in heart tissue.

Induction of myocardial hypertrophy involves activation of several signaling pathways involving key effector molecules such as ras, mitogen-activated protein kinase, and other signaling cascades that are linked to the cell surface through stimulation of G protein-coupled receptors (22, 31, 32). An important issue is whether the increase in GRK activity in response to pressure overload involves specific interactions with the beta -AR signaling pathway. We utilized a genetic model of cellular hypertrophy to demonstrate that the increase in beta ARK associated with pressure overload hypertrophy was not simply a generalized cellular response of increase protein synthesis. beta ARK activity in heart extracts from transgenic mice overexpressing oncogenic ras (which develop massive myocardial hypertrophy) had no increase in cytosolic beta ARK activity (Fig. 9). Clearly, cardiac hypertrophy induced by pressure overload is a very different pathologic state than that induced through forced overexpression of oncogenic ras. Indeed, our previous report of the ras transgenic showed no abnormality in contractility at base line or with isoproterenol stimulation but altered relaxation presumably due to mechanical effects (19). Thus, enhanced GRK activity with LV pressure overload appears not to be directly related to myocyte hypertrophy but perhaps to activation of the sympathetic nervous system and/or the renin angiotensin axis.

A question that we attempted to address in this study is whether increased beta ARK activity is required for the development of hypertrophy. Although pressure overload cardiac hypertrophy is associated with enhanced beta ARK activity, transgenic mice that overexpress an inhibitor of endogenous beta ARK activity develop cardiac hypertrophy to the same extent as wild-type mice (Fig. 7). Since it is possible that the increase in beta ARK could have other cellular effects not inhibited by the beta ARKct, we cannot definitively prove that beta ARK is not required for the hypertrophic phenotype. Nonetheless, these data do suggest that inhibiting endogenous beta ARK translocation and beta -AR desensitization do not prevent the development of the hypertrophy in response to a mechanical/neurohumoral stimulus.

The mechanism for the increase in beta ARK is not certain. Recent evidence suggest that both phosphatidylinositol 4,5-bisphosphate (33, 34) (although concentration-dependent (35)) and protein kinase C (36) can directly regulate beta ARK activity to enhance beta -AR phosphorylation and initiate desensitization. G protein-coupled signaling cascades that involve protein kinase C are generally considered to be importantly involved in the regulation of cell growth, which occurs in response to mechanical stimuli (37, 38) and receptor stimulation (31). Whether this may account for the small but significant greater responsiveness in the hypertrophied transgenic mice overexpressing beta ARKct is not certain.

GRK5 is expressed mostly in the heart compared with other tissues (5). It does not undergo agonist-dependent translocation from cytosol to membrane but rather is constitutively membrane bound (5) and will effectively phosphorylate and desensitize beta -ARs both in vitro and in vivo (18, 25). Our results in membrane fractions demonstrate that the predominant GRK activity in the membrane of hypertrophied hearts is beta ARK1 (Fig. 5). Recent evidence suggest that GRK5, but not beta ARK1, can be inhibited by Ca2+/calmodulin (39). Calmodulin appears to be an important molecule in cardiomyocyte growth as shown by the proliferative and hypertrophic response of atrial myocytes when overexpressed in transgenic mice (40). Although it is possible that in hypertrophied hearts GRK5 activity was partially inhibited by calmodulin, it is not known whether intracellular calmodulin levels were increased in these hearts with pressure overload cardiac hypertrophy. Nonetheless, our data suggest that the increased membrane GRK activity is due to enhanced beta ARK translocation.

The high fidelity catheters used for these experiments were either a 1.8 French or a 1.4 French micromanometer, which have diameters of 0.6 and 0.46 mm, respectively. The length of the sensor is 2.5 mm. We have previously assessed LV end-diastolic and end-systolic diameter by echocardiography and show that, for normal mice of approximate weight and age used in this study, the average diameter of the ventricle is 3.73 mm (end-diastole) and 2.2 mm (end-systole), whereas for mice with moderate hypertrophy, LV end-diastolic diameter is approx 3.4 mm and LV end-systolic diameter is approx 2 mm (26). Thus, the diameter of the catheter is considerably smaller than the diameter of the ventricle in either control or hypertrophied hearts and suggests that catheter size is unlikely to influence the measurement of hemodynamic variables.

Cardiac hypertrophy that develops following acute pressure overload is clearly different from that which occurs secondary to hypertension or valvular disease in the clinical setting, and thus, these data must be interpreted as such. In this regard, we have previously investigated the degree of myocardial injury in this overload model of mechanical-induced hypertrophy. In developing this mouse model of hypertrophy, we have chosen to constrict the transverse aorta that, in contrast to ascending aortic constriction, avoids excessive overload on the left ventricle because of the position of the innominate artery proximal to the constriction, allowing for a low resistance outlet (20, 21). Furthermore, we have performed histological examinations on hypertrophied ventricles and find that only the occasional heart will have small localized foci of cardiac damage accounting for less than 5% of the ventricle (41).

beta -AR desensitization has been shown to occur in end-stage human heart failure and is considered to be an important alteration leading to contractile dysfunction and impaired exercise tolerance (42). As we observed in hypertrophy, this desensitization in part, may be secondary to elevated beta ARK protein and activity (9, 10). Chronic pressure overload resulting from hypertension is a leading cause of chronic heart failure (14), and the onset of hypertrophy is a well accepted prognostic indicator for subsequent cardiac dysfunction and morbid events (43). Whether beta -AR desensitization, which occurs in hypertensive cardiac hypertrophy (44), is a factor in the pathological process for the transition from compensatory hypertrophy to overt heart failure is unknown. This study demonstrates that in an animal model of pressure overload hypertrophy, beta ARK activity is increased early in the disease state and is a potential therapeutic target when considering reversal of the impaired catecholamine responsiveness.


FOOTNOTES

*   This work was supported in part by the National Institutes of Health Grant HL56687 (to H. A. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Dept. of Medicine, University of North Carolina at Chapel Hill, CB 7075, Chapel Hill, NC 27599-7075. Tel.: 919-966-5201; FAX: 919-966-1743.
1   The abbreviations used are: beta -AR, beta -adrenergic receptor; GRK, G protein-coupled receptor kinase; beta ARK, beta -adrenergic receptor kinase; LV, left ventricular; TAC, transverse aortic constriction; PMSF, phenylmethylsulfonyl fluoride; ROS, rod outer segment; ANOVA, analysis of variance.

ACKNOWLEDGEMENT

We gratefully acknowledge Dr. R. J. Lefkowitz for careful review of the manuscript and for providing purified beta ARK1 and GRK5 and the monoclonal antibodies C5/1 and A16/17.


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