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(Received for publication, January 15, 1997, and in revised form, April 3, 1997)
From the Department of Medicine, University of California, San
Diego, School of Medicine, La Jolla, California 92093 and the
ABSTRACT Pressure overload cardiac hypertrophy in the
mouse was achieved following 7 days of transverse aortic constriction.
This was associated with marked INTRODUCTION The regulation of myocardial Decreased responsiveness to 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 We have recently shown that MATERIALS AND METHODS 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 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.
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.
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. To confirm To identify the specific GRK activities in the cytoplasm and membrane
fraction, monoclonal antibodies (10 µg/ml), which specifically bind
to and inhibit Immunodetection of myocardial levels of
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 [ Table I.
Adenylyl cyclase activity in sham and hypertrophied hearts
Volume 272, Number 27,
Issue of July 4, 1997
pp. 17223-17229
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
-Adrenergic Receptor Desensitization in Cardiac
Hypertrophy Is Increased -Adrenergic Receptor Kinase*
,
Department of Surgery, Duke University,
Durham, North Carolina 27710
-adrenergic receptor (-AR)
desensitization in vivo, as determined by a blunted
inotropic response to dobutamine. Extracts from hypertrophied hearts
had 
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 -adrenergic
receptor kinase (ARK). Although overexpression of a ARK inhibitor
in hearts of transgenic mice did not alter the development of cardiac
hypertrophy, the -AR desensitization associated with pressure
overload hypertrophy was prevented. To determine whether the induction
of ARK occurred because of a generalized response to cellular
hypertrophy, ARK activity was measured in transgenic mice homozygous
for oncogenic ras overexpression in the heart. Despite
marked cardiac hypertrophy, no difference in ARK activity was found
in these mice overexpressing oncogenic ras compared with
controls. Taken together, these data suggest that ARK is a central
molecule involved in alterations of -AR signaling in pressure
overload hypertrophy. The mechanism for the increase in ARK activity
appears not to be related to the induction of cellular hypertrophy but
to possibly be related to neurohumoral activation.
-adrenergic receptors
(-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 ARK1) and GRK5 appear
to be dominantly expressed in the heart (4, 5). Desensitization of
agonist-occupied receptors by the primarily cytosolic ARK1 requires
a membrane-targeting event prior to receptor phosphorylation by a
direct physical interaction between residues within the carboxyl
terminus of ARK and the dissociated, membrane-anchored 
subunits of G proteins (6, 7). Unlike ARK1, GRK5 does not undergo
agonist-dependent translocation from cytosol to membrane
but rather is constitutively membrane-bound (5).
-AR agonists is a characteristic of
chronic heart failure. In heart failure, -AR desensitization is
due to both diminished receptor number (receptor down-regulation) and
impaired receptor function (receptor uncoupling) (8), in part related
to enhanced 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 -AR
desensitization in the disease process.
1-ARs and increased
Gi
protein are found, which are associated with reduced
positive inotropic response to isoproterenol (15, 16).
ARK is a critical modulator of in
vivo contractile function (17). Both the
1-adrenergic and angiotensin II receptors are targets
for ARK1-mediated desensitization, whereas selected desensitization
of 1-ARs occurs with GRK5 (17, 18). Transgenic mice,
which overexpress a peptide inhibitor of ARK1, show decreased
desensitization of -ARs supporting the in vivo role for
ARK1 as a modulator of cardiac function (17). Since abnormalities in
-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 -AR desensitization, and we tested the hypothesis
that in LV pressure overload, hypertrophy desensitization occurs as a
result of enhanced ARK1 activity.
ARK1 inhibitor (ARKct)
shown previously to have diminished desensitization to -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.
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
(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
[-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).
ARK-dependent phosphorylation of ROS, the
protein kinase A inhibitor, PKI (1 µM, Sigma), and
heparin (10 µg/ml) were incubated with purified ARK, and the
capacity to phosphorylate light-activated rhodopsin was determined.
Heparin inhibited phosphorylation while PKI did not (data not
shown).
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).
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). ARK1 was immunoprecipitated from 1 ml of clarified extract
with a 1:1,000 (1 µl) monoclonal anti-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 ARK1 protein
was visualized with the monoclonal antibody raised against an epitope
within the carboxyl terminus of ARK1 and chemiluminescent detection
of anti-mouse IgG conjugated with horseradish peroxidase (ECL, Amersham
Corp.) (17).
AR Receptor
Density
-32P]ATP, and cAMP was quantified (17).
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.
ABSTRACTTotal -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.
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, -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.
, p < 0.001, t
test with Bonferroni correction for four comparisons.
[View Larger Version of this Image (16K GIF file)]
To determine whether the development of LV hypertrophy is associated
with -AR uncoupling in vivo, cardiac catheterization was
used to measure catecholamine responsiveness in intact anesthetized mice (Fig. 2). Marked blunting of the dobutamine (a
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 -AR desensitization
is associated with the development of cardiac hypertrophy.
Fig. 2. In vivo assessment of LV contractile function in response to
-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 (
, n = 12), and TAC
(
, 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; , 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).
[View Larger Version of this Image (27K GIF file)]
To determine whether alterations in GRK activity could account for the
-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 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
ARK1 in the cytosol of hypertrophied hearts (Fig. 3C).
Consistent with the marked increase in GRK activity, a clear increase
in ARK1 protein levels was observed by Western blotting in hearts
following TAC (Fig. 3C).
Fig. 3. Effect of pressure overload hypertrophy on
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 ARK1, respectively. Shown is a representative
autoradiograph of a dried gel where phosphorylated rhodopsin (Rho) is
visualized. B, the level of 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 ARK1 in cytosolic extracts from individual sham-operated
(lanes 1-4) and individual TAC operated (lanes
5-8) mice. An 80-kDa protein was visualized by Western blotting and chemiluminescence following solubilization of cytosolic extracts and immunoprecipitation. Lane 9, 50 µg of
purified ARK1.
[View Larger Version of this Image (30K GIF file)]
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 -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.
[View Larger Version of this Image (22K GIF file)]
To identify the specific GRK that mediates -AR desensitization in
the hypertrophied heart, monoclonal antibodies that neutralize ARK1
and 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 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 ARK1 or cytosolic extracts with the monoclonal antibody
A16/17 did not inhibit rhodopsin phosphorylation (data not shown). This
indicates that 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
ARK1. Incubation of purified 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 5 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-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.
[View Larger Version of this Image (43K GIF file)]
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 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
ARK1.
-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 -AR density, hypertrophied hearts
had significantly lower isoproterenol-stimulated adenylyl cyclase
activity (Table I). Thus, functional uncoupling of
-ARs as assessed by the physiologic response to -agonist stimulation and adenylyl cyclase activation was hindered with the
development of myocardial hypertrophy. To confirm that the observed
uncoupling of -ARs was not due to the increase in inhibitory G
protein (Gi), we measured the level of Gi
immunoreactivity with antibodies directed against Gi
1-3 (Fig. 6). No difference in the level of
Gi 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 -AR density or to levels of inhibitory G protein.
Fig. 6. Immunodetection of Gi
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
40-kDa Gi 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.
[View Larger Version of this Image (27K GIF file)]
To determine whether the impaired catecholamine responsiveness and
-AR desensitization that accompany the development of cardiac
hypertrophy could be accounted for by the induction of ARK, we used
a strategy of in vivo ARK inhibition achieved with cardiac-specific overexpression of a ARK1 inhibitor in transgenic mice (17). The inhibitor utilized was the carboxyl terminus of ARK1
(ARK1ct), which contains the G-binding domain and competes
with endogenous ARK for binding and subsequent
translocation/activation (6, 7, 17). Transgenic mice overexpressing the
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 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 ARKct inhibitor mice, Fig. 7A). To determine whether this
increase in LV mass was associated with increased ARK activity,
cytosolic extracts from sham- and TAC-operated 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 ARKct mice (Fig.
7B). To determine whether forced overexpression of the
ARKct would prevent functional -AR desensitization with the
development of cardiac hypertrophy, we performed cardiac
catheterization in sham and TAC mice overexpressing the ARKct
inhibitor. As shown in Fig. 8, the response in LV
dP/dtmax and heart rate to -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
ARK inhibitor (ARKct).
A, left ventricular weight to body weight (LV/BW)
and left ventricular weight to tibia length (LV/T) was
measured in 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 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
ARKct mice; lanes 6-9, individual TAC-operated ARKct
mice. Lanes 2-9 each represents extracts from separate
hearts; lane 10, incubation of ROS with 25 µg of purified
ARK1.
[View Larger Version of this Image (31K GIF file)]
Fig. 8. In vivo contractile function of transgenic mice overexpressing a
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,
LV dP/dtmax) and heart rate
(D, heart rate) with dobutamine infusion in sham-operated
(), n = 10, and TAC-operated () mice,
n = 13. A two-way repeated ANOVA showed no significant difference between sham and TAC ARK inhibitor mice. To determine if
the values for basal dP/dtmax were
different and whether it influenced the response -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).
[View Larger Version of this Image (27K GIF file)]
To address the question of whether the induction of ARK1 in pressure
overload hypertrophy is simply a generalized response to cellular
hypertrophy, we measured the level of 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 ARK
activity in a rhodopsin phosphorylation assay. Despite the development
of severe LV hypertrophy, no difference in the level of ARK activity
was found in mice overexpressing oncogenic ras compared with
wild-type matched controls (Fig. 9). These data suggest
that the increase in 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.
[View Larger Version of this Image (25K GIF file)]
DISCUSSION
The present study demonstrates that -AR desensitization occurs
with the development of pressure overload cardiac hypertrophy, and that
the uncoupling of -ARs with cardiac hypertrophy can be accounted for
by an increase in ARK1. The blunted inotropic response to -AR
stimulation that accompanies LV hypertrophy can be reversed by ARK
inhibition. Furthermore, the mechanism for increased 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, ARK1
is responsible for altered -AR signaling associated with pathologic
states such as pressure overload cardiac hypertrophy.
Several previous experimental studies have demonstrated -AR
desensitization with cardiac hypertrophy (16, 27-29). In two studies,
desensitization was associated with a small decrease in
1-AR subtype and the increase of the inhibitory G
protein Gi, 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 ARK1 subtype.
There also may be a slight increase in GRK5 activity. The impairment in
contractile function with -agonist stimulation following the
development of cardiac hypertrophy (Fig. 2A) is remarkably
similar to that observed with targeted overexpression of ARK1 (17),
providing further evidence that ARK1 is a critical in
vivo modulator of -AR signal transduction.
Although ARK1 is predominantly a cytosolic enzyme, translocation to
the membrane by G is required for desensitization of ARs. In
contrast, GRK5 is always membrane-associated. We used monoclonal
antibodies to discriminate between ARK1 and GRK5 subtypes in the
hypertrophied heart. These experiments demonstrate that in both
cytosolic and membrane fractions, ARK1 was the subtype predominately
responsible for the enhanced GRK activity and suggests that the
majority of membrane activity can be accounted for by ARK1, which is
translocated to the membrane (Fig. 5). Furthermore, the fact that
ARK activity is enhanced in the ARKct mice (in the presence of an
inhibitor of G translocation) without affecting cardiac function,
suggests that just an increased level of ARK will not enhance its
association to the membrane to cause -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
ARK1 and GRK5, can be identified in cardiac tissue; however, the
level of mRNA expression is significantly less than ARK1 and
GRK5. Therefore, it is possible that other GRK subtypes such as ARK2
or GRK6 could account for some of the observed GRK activity.
Quantitative immunoblotting of all subtypes would be required to
demonstrate that 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 -AR signaling pathway. We utilized a
genetic model of cellular hypertrophy to demonstrate that the increase
in ARK associated with pressure overload hypertrophy was not simply
a generalized cellular response of increase protein synthesis. ARK
activity in heart extracts from transgenic mice overexpressing
oncogenic ras (which develop massive myocardial hypertrophy)
had no increase in cytosolic 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 ARK activity is required for the development of hypertrophy. Although pressure overload cardiac hypertrophy is associated with enhanced ARK activity, transgenic mice that
overexpress an inhibitor of endogenous ARK activity develop cardiac
hypertrophy to the same extent as wild-type mice (Fig. 7). Since it is
possible that the increase in ARK could have other cellular effects
not inhibited by the ARKct, we cannot definitively prove that ARK is not required for the hypertrophic phenotype. Nonetheless, these data
do suggest that inhibiting endogenous ARK translocation and -AR
desensitization do not prevent the development of the hypertrophy in
response to a mechanical/neurohumoral stimulus.
The mechanism for the increase in 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 ARK activity to enhance -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 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 -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 ARK1 (Fig. 5). Recent evidence suggest
that GRK5, but not 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 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 3.4 mm and LV
end-systolic diameter is 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).
-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 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 -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, ARK activity is increased early in
the disease state and is a potential therapeutic target when
considering reversal of the impaired catecholamine responsiveness.
FOOTNOTES
§ 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:
-AR,
-adrenergic receptor; GRK, G protein-coupled receptor kinase;
ARK, -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 ARK1
and GRK5 and the monoclonal antibodies C5/1 and A16/17.
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 S. V. Naga Prasad, S. A. Laporte, D. Chamberlain, M. G. Caron, L. Barak, and H. A. Rockman Phosphoinositide 3-kinase regulates {beta}2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/{beta}-arrestin complex J. Cell Biol., August 5, 2002; 158(3): 563 - 575. [Abstract] [Full Text] [PDF]
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X. P. Yi, A. M. Gerdes, and F. Li Myocyte Redistribution of GRK2 and GRK5 in Hypertensive, Heart-Failure-Prone Rats Hypertension, June 1, 2002; 39(6): 1058 - 1063. [Abstract] [Full Text] [PDF]
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 S.V. NAGA PRASAD, J. NIENABER, and H.A. ROCKMAN G-Protein-coupled Receptor Function in Heart Failure Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 439 - 444. [Abstract] [PDF]
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 K. Leineweber, I. Heinroth-Hoffmann, K. Ponicke, G. Abraham, B. Osten, and O.-E. Brodde Cardiac {beta}-Adrenoceptor Desensitization Due to Increased {beta}-Adrenoceptor Kinase Activity in Chronic Uremia J. Am. Soc. Nephrol., January 1, 2002; 13(1): 117 - 124. [Abstract] [Full Text] [PDF]
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G. Esposito, A. Rapacciuolo, S. V. Naga Prasad, H. Takaoka, S. A. Thomas, W. J. Koch, and H. A. Rockman Genetic Alterations That Inhibit In Vivo Pressure-Overload Hypertrophy Prevent Cardiac Dysfunction Despite Increased Wall Stress Circulation, January 1, 2002; 105(1): 85 - 92. [Abstract] [Full Text] [PDF]
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 S. M. Emani, A. S. Shah, D. C. White, D. D. Glower, and W. J. Koch Right ventricular gene therapy with a {beta}-adrenergic receptor kinase inhibitor improves survival after pulmonary artery banding Ann. Thorac. Surg., November 1, 2001; 72(5): 1657 - 1661. [Abstract] [Full Text] [PDF]
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A. Nakamura, D. G. Rokosh, M. Paccanaro, R. R. Yee, P. C. Simpson, W. Grossman, and E. Foster LV systolic performance improves with development of hypertrophy after transverse aortic constriction in mice Am J Physiol Heart Circ Physiol, September 1, 2001; 281(3): H1104 - H1112. [Abstract] [Full Text] [PDF]
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 G. Iaccarino, J. R. Keys, A. Rapacciuolo, K. F. Shotwell, R. J. Lefkowitz, H. A. Rockman, and W. J. Koch Regulation of myocardial {beta}ARK1 expression in catecholamine-induced cardiac hypertrophy in transgenic mice overexpressing {alpha}1B-adrenergic receptors J. Am. Coll. Cardiol., August 1, 2001; 38(2): 534 - 540. [Abstract] [Full Text] [PDF]
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G. Iaccarino, E. Barbato, E. Cipolleta, A. Esposito, A. Fiorillo, W. J. Koch, and B. Trimarco Cardiac {beta}ARK1 Upregulation Induced by Chronic Salt Deprivation in Rats Hypertension, August 1, 2001; 38(2): 255 - 260. [Abstract] [Full Text] [PDF]
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V. B. Harding, L. R. Jones, R. J. Lefkowitz, W. J. Koch, and H. A. Rockman Cardiac beta ARK1 inhibition prolongs survival and augments beta blocker therapy in a mouse model of severe heart failure PNAS, April 25, 2001; (2001) 91102398. [Abstract] [Full Text]
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 R. P. Erickson and P. E. Graves Genetic Variation in {beta}-Adrenergic Receptors and Their Relationship to Susceptibility for Asthma and Therapeutic Response Drug Metab. Dispos., April 1, 2001; 29(4): 557 - 561. [Abstract] [Full Text]
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M. Iwata, T. Yoshikawa, A. Baba, T. Anzai, I. Nakamura, Y. Wainai, T. Takahashi, and S. Ogawa Autoimmunity Against the Second Extracellular Loop of {beta}1-Adrenergic Receptors Induces {beta}-Adrenergic Receptor Desensitization and Myocardial Hypertrophy In Vivo Circ. Res., March 30, 2001; 88(6): 578 - 586. [Abstract] [Full Text] [PDF]
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A. S. Shah, D. C. White, S. Emani, A. P. Kypson, R. E. Lilly, K. Wilson, D. D. Glower, R. J. Lefkowitz, and W. J. Koch In Vivo Ventricular Gene Delivery of a {beta}-Adrenergic Receptor Kinase Inhibitor to the Failing Heart Reverses Cardiac Dysfunction Circulation, March 6, 2001; 103(9): 1311 - 1316. [Abstract] [Full Text] [PDF]
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 W. E. Schutzer, J. F. Reed, M. Bliziotes, and S. L. Mader Upregulation of G protein-linked receptor kinases with advancing age in rat aorta Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2001; 280(3): R897 - R903. [Abstract] [Full Text] [PDF]
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M. A. Gaballa, A. Eckhart, W. J. Koch, and S. Goldman Vascular {beta}-adrenergic receptor system is dysfunctional after myocardial infarction Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1129 - H1135. [Abstract] [Full Text] [PDF]
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 M. S. Lombardi, A. Kavelaars, P. M. Cobelens, R. E. Schmidt, M. Schedlowski, and C. J. Heijnen Adjuvant Arthritis Induces Down-Regulation of G Protein-Coupled Receptor Kinases in the Immune System J. Immunol., February 1, 2001; 166(3): 1635 - 1640. [Abstract] [Full Text] [PDF]
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B. S. Manning, K. Shotwell, L. Mao, H. A. Rockman, and W. J. Koch Physiological Induction of a {beta}-Adrenergic Receptor Kinase Inhibitor Transgene Preserves {beta}-Adrenergic Responsiveness in Pressure-Overload Cardiac Hypertrophy Circulation, November 28, 2000; 102(22): 2751 - 2757. [Abstract] [Full Text] [PDF]
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A. S. Shah, D. C. White, O. Tai, J. A. Hata, K. H. Wilson, A. Pippen, A. P. Kypson, D. D. Glower, R. J. Lefkowitz, and W. J. Koch Adenovirus-mediated genetic manipulation of the myocardial {beta}-adrenergic signaling system in transplanted hearts J. Thorac. Cardiovasc. Surg., September 1, 2000; 120(3): 581 - 588. [Abstract] [Full Text] [PDF]
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A. S. Shah, B. Z. Atkins, J. A. Hata, O. Tai, A. P. Kypson, R. E. Lilly, W. J. Koch, and D. D. Glower Early effects of right ventricular volume overload on ventricular performance and {beta}-adrenergic signaling J. Thorac. Cardiovasc. Surg., August 1, 2000; 120(2): 342 - 349. [Abstract] [Full Text] [PDF]
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R. Ramos-Ruiz, P. Penela, R. B. Penn, and F. Mayor Jr Analysis of the Human G Protein-Coupled Receptor Kinase 2 (GRK2) Gene Promoter : Regulation by Signal Transduction Systems in Aortic Smooth Muscle Cells Circulation, May 2, 2000; 101(17): 2083 - 2089. [Abstract] [Full Text] [PDF]
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S. B. Liggett, N. M. Tepe, J. N. Lorenz, A. M. Canning, T. D. Jantz, S. Mitarai, A. Yatani, and G. W. Dorn II Early and Delayed Consequences of {beta}2-Adrenergic Receptor Overexpression in Mouse Hearts : Critical Role for Expression Level Circulation, April 11, 2000; 101(14): 1707 - 1714. [Abstract] [Full Text] [PDF]
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A. Elorza, S. Sarnago, and F. Mayor Jr. Agonist-Dependent Modulation of G Protein-Coupled Receptor Kinase 2 by Mitogen-Activated Protein Kinases Mol. Pharmacol., April 1, 2000; 57(4): 778 - 783. [Abstract] [Full Text]
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 M. Ungerer, H.-J. Weig, S. Kubert, M. Overbeck, F. Bengel, A. Schomig, and M. Schwaiger Regional pre- and postsynaptic sympathetic system in the failing human heart -- regulation of {beta}ARK-1 Eur J Heart Fail, March 1, 2000; 2(1): 23 - 31. [Abstract] [Full Text] [PDF]
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S. V. Naga Prasad, G. Esposito, L. Mao, W. J. Koch, and H. A. Rockman Gbeta gamma -dependent Phosphoinositide 3-Kinase Activation in Hearts with in Vivo Pressure Overload Hypertrophy J. Biol. Chem., February 18, 2000; 275(7): 4693 - 4698. [Abstract] [Full Text] [PDF]
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C. A. Walker, F. A. Crawford Jr, and F. G. Spinale MYOCYTE CONTRACTILE DYSFUNCTION WITH HYPERTROPHY AND FAILURE: RELEVANCE TO CARDIAC SURGERY J. Thorac. Cardiovasc. Surg., February 1, 2000; 119(2): 388 - 400. [Full Text] [PDF]
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A. D. Eckhart, S. J. Duncan, R. B. Penn, J. L. Benovic, R. J. Lefkowitz, and W. J. Koch Hybrid Transgenic Mice Reveal In Vivo Specificity of G Protein-Coupled Receptor Kinases in the Heart Circ. Res., January 7, 2000; 86(1): 43 - 50. [Abstract] [Full Text] [PDF]
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 L. M. Bohn, R. J. Lefkowitz, R. R. Gainetdinov, K. Peppel, M. G. Caron, and F. Lin Enhanced Morphine Analgesia in Mice Lacking -Arrestin 2 Science, December 24, 1999; 286(5449): 2495 - 2498. [Abstract] [Full Text]
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 O.-E. Brodde and M. C. Michel Adrenergic and Muscarinic Receptors in the Human Heart Pharmacol. Rev., December 1, 1999; 51(4): 651 - 690. [Abstract] [Full Text] [PDF]
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S. Sarnago, A. Elorza, and F. Mayor Jr. Agonist-dependent Phosphorylation of the G Protein-coupled Receptor Kinase 2 (GRK2) by Src Tyrosine Kinase J. Biol. Chem., November 26, 1999; 274(48): 34411 - 34416. [Abstract] [Full Text] [PDF]
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N. Dzimiri Regulation of beta -Adrenoceptor Signaling in Cardiac Function and Disease Pharmacol. Rev., September 1, 1999; 51(3): 465 - 502. [Abstract] [Full Text] [PDF]
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S. A. Akhter, A. D. Eckhart, H. A. Rockman, K. Shotwell, R. J. Lefkowitz, and W. J. Koch In Vivo Inhibition of Elevated Myocardial {beta}-Adrenergic Receptor Kinase Activity in Hybrid Transgenic Mice Restores Normal {beta}-Adrenergic Signaling and Function Circulation, August 10, 1999; 100(6): 648 - 653. [Abstract] [Full Text] [PDF]
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M.-C. Cho, A. Rapacciuolo, W. J. Koch, Y. Kobayashi, L. R. Jones, and H. A. Rockman Defective beta -Adrenergic Receptor Signaling Precedes the Development of Dilated Cardiomyopathy in Transgenic Mice with Calsequestrin Overexpression J. Biol. Chem., August 6, 1999; 274(32): 22251 - 22256. [Abstract] [Full Text] [PDF]
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J. P. Maurice, A. S. Shah, A. P. Kypson, J. A. Hata, D. C. White, D. D. Glower, and W. J. Koch Molecular beta -adrenergic signaling abnormalities in failing rabbit hearts after infarction Am J Physiol Heart Circ Physiol, June 1, 1999; 276(6): H1853 - H1860. [Abstract] [Full Text] [PDF]
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G. W. Dorn II, N. M. Tepe, J. N. Lorenz, W. J. Koch, and S. B. Liggett Low- and high-level transgenic expression of beta 2-adrenergic receptors differentially affect cardiac hypertrophy and function in Galpha q-overexpressing mice PNAS, May 25, 1999; 96(11): 6400 - 6405. [Abstract] [Full Text] [PDF]
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M.-C. Cho, M. Rao, W. J. Koch, S. A. Thomas, R. D. Palmiter, and H. A. Rockman Enhanced Contractility and Decreased ß-Adrenergic Receptor Kinase-1 in Mice Lacking Endogenous Norepinephrine and Epinephrine Circulation, May 25, 1999; 99(20): 2702 - 2707. [Abstract] [Full Text] [PDF]
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C. V. Carman, M. P. Lisanti, and J. L. Benovic Regulation of G Protein-coupled Receptor Kinases by Caveolin J. Biol. Chem., March 26, 1999; 274(13): 8858 - 8864. [Abstract] [Full Text] [PDF]
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K.-L. Laugwitz, M. Ungerer, T. Schoneberg, H.-J. Weig, K. Kronsbein, A. Moretti, K. Hoffmann, M. Seyfarth, G. Schultz, and A. Schomig Adenoviral Gene Transfer of the Human V2 Vasopressin Receptor Improves Contractile Force of Rat Cardiomyocytes Circulation, February 23, 1999; 99(7): 925 - 933. [Abstract] [Full Text] [PDF]
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G. Iaccarino, P. C. Dolber, R. J. Lefkowitz, and W. J. Koch ß-Adrenergic Receptor Kinase-1 Levels in Catecholamine-Induced Myocardial Hypertrophy : Regulation by ß- but not {alpha}1-Adrenergic Stimulation Hypertension, January 1, 1999; 33(1): 396 - 401. [Abstract] [Full Text] [PDF]
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K. M. Anderson, A. D. Eckhart, R. N. Willette, and W. J. Koch The Myocardial ß-Adrenergic System in Spontaneously Hypertensive Heart Failure (SHHF) Rats Hypertension, January 1, 1999; 33(1): 402 - 407. [Abstract] [Full Text] [PDF]
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G. Iaccarino, E. D. Tomhave, R. J. Lefkowitz, and W. J. Koch Reciprocal In Vivo Regulation of Myocardial G Protein–Coupled Receptor Kinase Expression by ß-Adrenergic Receptor Stimulation and Blockade Circulation, October 27, 1998; 98(17): 1783 - 1789. [Abstract] [Full Text] [PDF]
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G. Iaccarino, H. A. Rockman, K. F. Shotwell, E. D. Tomhave, and W. J. Koch Myocardial overexpression of GRK3 in transgenic mice: evidence for in vivo selectivity of GRKs Am J Physiol Heart Circ Physiol, October 1, 1998; 275(4): H1298 - H1306. [Abstract] [Full Text] [PDF]
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C. V. Carman, T. Som, C. M. Kim, and J. L. Benovic Binding and Phosphorylation of Tubulin by G Protein-coupled Receptor Kinases J. Biol. Chem., August 7, 1998; 273(32): 20308 - 20316. [Abstract] [Full Text] [PDF]
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 R. B. Penn, R. A. Panettieri Jr., and J. L. Benovic Mechanisms of Acute Desensitization of the beta 2AR-Adenylyl Cyclase Pathway in Human Airway Smooth Muscle Am. J. Respir. Cell Mol. Biol., August 1, 1998; 19(2): 338 - 348. [Abstract] [Full Text]
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H. A. Rockman, D.-J. Choi, S. A. Akhter, M. Jaber, B. Giros, R. J. Lefkowitz, M. G. Caron, and W. J. Koch Control of Myocardial Contractile Function by the Level of beta -Adrenergic Receptor Kinase 1 in Gene-targeted Mice J. Biol. Chem., July 17, 1998; 273(29): 18180 - 18184. [Abstract] [Full Text] [PDF]
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H. A. Rockman, K. R. Chien, D.-J. Choi, G. Iaccarino, J. J. Hunter, J. Ross Jr., R. J. Lefkowitz, and W. J. Koch Expression of a beta -adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice PNAS, June 9, 1998; 95(12): 7000 - 7005. [Abstract] [Full Text] [PDF]
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S. A. Akhter, L. M. Luttrell, H. A. Rockman, G. Iaccarino, R. J. Lefkowitz, and W. J. Koch Targeting the Receptor-Gq Interface to Inhibit in Vivo Pressure Overload Myocardial Hypertrophy Science, April 24, 1998; 280(5363): 574 - 577. [Abstract] [Full Text]
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S. A. Akhter, C. A. Skaer, A. P. Kypson, P. H. McDonald, K. C. Peppel, D. D. Glower, R. J. Lefkowitz, and W. J. Koch Restoration of beta -adrenergic signaling in failing cardiac ventricular myocytes via adenoviral-mediated gene transfer PNAS, October 28, 1997; 94(22): 12100 - 12105. [Abstract] [Full Text] [PDF]
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S. A. Akhter, C. A. Milano, K. F. Shotwell, M.-C. Cho, H. A. Rockman, R. J. Lefkowitz, and W. J. Koch Transgenic Mice with Cardiac Overexpression of alpha 1B-Adrenergic Receptors. IN VIVO alpha 1-ADRENERGIC RECEPTOR-MEDIATED REGULATION OF beta -ADRENERGIC SIGNALING J. Biol. Chem., August 22, 1997; 272(34): 21253 - 21259. [Abstract] [Full Text] [PDF]
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S. V. Naga Prasad, L. S. Barak, A. Rapacciuolo, M. G. Caron, and H. A. Rockman Agonist-dependent Recruitment of Phosphoinositide 3-Kinase to the Membrane by beta -Adrenergic Receptor Kinase 1. A ROLE IN RECEPTOR SEQUESTRATION J. Biol. Chem., May 25, 2001; 276(22): 18953 - 18959. [Abstract] [Full Text] [PDF]
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D. C. White, J. A. Hata, A. S. Shah, D. D. Glower, R. J. Lefkowitz, and W. J. Koch Preservation of myocardial beta -adrenergic receptor signaling delays the development of heart failure after myocardial infarction PNAS, May 9, 2000; 97(10): 5428 - 5433. [Abstract] [Full Text] [PDF]
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V. B. Harding, L. R. Jones, R. J. Lefkowitz, W. J. Koch, and H. A. Rockman Cardiac beta ARK1 inhibition prolongs survival and augments beta blocker therapy in a mouse model of severe heart failure PNAS, May 8, 2001; 98(10): 5809 - 5814. [Abstract] [Full Text] [PDF]
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C. L. Antos, N. Frey, S. O. Marx, S. Reiken, M. Gaburjakova, J. A. Richardson, A. R. Marks, and E. N. Olson Dilated Cardiomyopathy and Sudden Death Resulting From Constitutive Activation of Protein Kinase A Circ. Res., November 23, 2001; 89(11): 997 - 1004. [Abstract] [Full Text] [PDF]
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