(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
Department of Surgery, Duke University,
Durham, North Carolina 27710
Pressure overload cardiac hypertrophy in the
mouse was achieved following 7 days of transverse aortic constriction.
This was associated with marked -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.
The regulation of myocardial -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).
Decreased responsiveness to -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.
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 1-ARs and increased
Gi
protein are found, which are associated with reduced
positive inotropic response to isoproterenol (15, 16).
We have recently shown that 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.
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 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.
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 MiceAfter 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 PhosphorylationMyocardial
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. 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).
To confirm 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).
To identify the specific GRK activities in the cytoplasm and membrane
fraction, monoclonal antibodies (10 µg/ml), which specifically bind
to and inhibit 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).
Immunodetection of myocardial levels of
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).
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 [-32P]ATP, and cAMP was quantified (17).
|
Total -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.
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).
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.
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).
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.
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.
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.
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).
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.
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.
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.