(Circulation. 1999;100:648-653.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
In Vivo Inhibition of Elevated Myocardial ß-Adrenergic Receptor Kinase Activity in Hybrid Transgenic Mice Restores Normal ß-Adrenergic Signaling and Function
From the Departments of Surgery (S.A.A., A.D.E., K.S., W.J.K.) and Medicine and Biochemistry and the Howard Hughes Medical Institute (R.J.L.), Duke University Medical Center, Durham, NC, and the Department of Medicine/Cardiology, The University of North Carolina at Chapel Hill, Chapel Hill, NC (H.A.R.).
Correspondence to Walter J. Koch, PhD, Laboratory of Molecular Cardiovascular Biology, Box 2606, MSRB Room 471, Duke University Medical Center, Durham, NC 27710. E-mail koch0002{at}mc.duke.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background—The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac ß-adrenergic receptor (ßAR) system, which is critical in the regulation of myocardial function. Expression of the ßAR kinase (ßARK1), which phosphorylates and uncouples ßARs, is elevated in human HF; this likely contributes to the abnormal ßAR responsiveness that occurs with ß-agonist administration. We previously showed that transgenic mice with increased myocardial ßARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous ßARK1 activity in the heart led to enhanced myocardial function.
Methods and Results—We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both ßARK1 and an inhibitor of ßARK1 activity to study the feasibility and functional consequences of the inhibition of elevated ßARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of ßARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial ßARK1 levels remained elevated due to transgene expression, in vitro ßARK1 activity returned to control levels and the percentage of ßARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to ßAR stimulation was restored to normal in the hybrid double-transgenic mice.
Conclusions—Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial ßARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac ßARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.
Key Words: receptors, adrenergic, beta • G proteins • protein kinases • desensitization • heart failure • mice, transgenic • myocardium
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The myocardial ß-adrenergic receptor (ßAR) signaling pathway plays a critical role in the regulation of cardiac contractility. ßARs (ß1 and ß2 subtypes) are the primary myocardial targets of the sympathetic neurotransmitter norepinephrine and the adrenal hormone epinephrine. Activation of ßARs in the heart by these 2 catecholamines leads to positive chronotropic and inotropic action via stimulation of adenylyl cyclase and subsequent increases in cAMP and intracellular Ca2+ release.1 Continued exposure of ßARs to agonists results in a rapid decrease in responsiveness, which is known as desensitization.2 Agonist-dependent desensitization can be initiated by the phosphorylation of activated receptors by members of the family of G protein–coupled receptor kinases (GRK).2 The ßAR kinase-1 (ßARK1; GRK2) is a GRK that specifically phosphorylates activated ß1- and ß2-ARs, leading to desensitization in vitro and in vivo.2 3 4
Heart failure (HF) in humans has been characterized by specific
alterations in the ßAR signaling system. These include selective
down-regulation of ß1ARs by 
50% and
desensitization of the remaining ßARs, which leads to the blunting of
further agonist-mediated stimulation.1 5 6 The enhanced
desensitization of myocardial ßARs is likely due, in part, to the
elevated expression of ßARK1 (3-fold) present in human
HF.7 8 It is generally thought that these changes in the
ßAR system in HF are triggered by increased sympathetic stimulation
of the heart in this disease state.9 The dysfunctional
ßAR signaling, including increased ßARK1 expression and activity,
is a contributing factor to the impaired myocardial
contractility seen in HF.
Our laboratory previously reported that transgenic mice with
cardiac-specific overexpression of ßARK1 to the levels seen in human
HF (3-fold) have significantly depressed agonist-stimulated left
ventricular (LV) function in vivo.4 This study
demonstrated the in vivo action of ßARK1 on
ß1ARs and the importance of this GRK in the
regulation of myocardial function. In the same study, transgenic mice
with cardiac-specific expression of the carboxyl-terminus region of
ßARK1 (ßARKct), which acts as a functional inhibitor of
ßARK1 activity, showed enhanced in vivo basal and agonist-stimulated
LV function.4 ßARKct contains the binding domain
responsible for the specific binding of ßARK1 to the dissociated
ß
subunits of heterotrimeric G proteins
(Gß), a process required for the activation
of this GRK.4 10 11 Therefore, inhibiting normal
endogenous ßARK1 activity can lead to increased cardiac
function in transgenic mice, presumably through the attenuation of
myocardial ßAR desensitization, although other receptor targets for
ßARK1 cannot be ruled out.
In this study, we sought to determine if concomitant myocardial expression of ßARKct could inhibit increased levels of ßARK1 activity (in the elevated range present in human HF) in transgenic mouse hearts. To do this, novel hybrid transgenic mice were created with cardiac-specific overexpression of both ßARK1 and the ßARKct peptide; this effectively turned the heart into a novel "in vivo reaction vessel" that we used to study the interaction between the 2 transgenes. We first examined the functional activity of the ßARK inhibitor peptide at a biochemical level and ultimately studied LV contractility in the intact animal. Our results indicated that the inhibition of elevated myocardial ßARK1 activity by the ßARKct peptide occurs in vivo and that this leads to the reversal of abnormal ßAR responsiveness that accompanies enhanced ßARK1 expression. Thus, ßARK1 is a potential therapeutic target for enhancing myocardial contractility in conditions in which cardiac function is compromised, such as HF.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Experimental Animals
Transgenic mice with cardiac-targeted overexpression of ßARK1 were mated with transgenic mice with cardiac-targeted overexpression of the ßARKct peptide to generate mice that overexpressed both ßARK1 and ßARKct in their hearts.4 In these 2 individual lines of transgenic mice, the ßARK1 and ßARKct transgenes were targeted to the myocardium by using the murine
-myosin heavy chain gene promoter.4 In
the ßARK1 mice, myocardial ßARK1 protein and activity was 3-fold
over endogenous ßARK1 levels; the molar ratio of the
ßARKct peptide to endogenous ßARK1 in the
myocardium of ßARKct animals, as determined by protein
immunoblotting, was approximately 5:1.4
Using 1 parent from each of these 2 individual lines, offspring were
generated; the genotype of these hybrid mice was determined by
polymerase chain reaction on genomic DNA isolated from tail
biopsies.4 Hybrid transgenic mice from these matings
containing both transgenes were used in this study, as were littermates
that were positive for individual transgenes. Importantly, as shown in
Figure 1
, expression of both the ßARK1
and ßARKct transgenes in the hybrid mice did not differ from the
levels expressed in the individual lines. The animals in this study
were handled according to the approved protocols and animal welfare
regulations at Duke University Medical Center and the University of
North Carolina at Chapel Hill.
|
Hemodynamic Evaluation in Intact Anesthetized
Mice
Cardiac catheterization was performed as
described previously.4 12 Mice were anesthetized
with a mixture of ketamine (100 mg/kg IP) and xylazine (2.5
mg/kg IP) and, after endotracheal intubation, were connected to a
rodent ventilator. After bilateral vagotomy, the chest was opened, and
a 1.4 French (0.46 mm) high-fidelity
micromanometer catheter (Millar Instruments)
was inserted into the left atrium, advanced across the mitral valve,
and secured in the left ventricle. The external jugular vein was
cannulated to administer isoproterenol (ISO).
Hemodynamic measurements were recorded at baseline
and 45 to 60 s after the injection of an incremental dose of
ISO.4 12
Protein Immunoblotting
Transgenic mouse hearts were homogenized in ice-cold
buffer (25 mmol/L Tris-HCl [pH 7.5], 5 mmol/L EDTA, 5
mmol/L EGTA, 10 µg/mL leupeptin, 20 µg/mL aprotinin, and 1
mmol/L phenylmethylsulfonyl fluoride). Nuclei and tissue were
separated by centrifugation at 800g for 15
minutes. The crude supernatant was then centrifuged at
20 000g for 15 minutes. Protein concentrations
were determined on the supernatant (cytosolic fraction). Sedimented
proteins (membrane fraction) were resuspended in 50 mmol/L HEPES
(pH 7.3) and 5 mmol/L
MgCl2.4 The immunodetection of
myocardial levels of ßARK1 was performed on an equal amount of
protein from cytosolic extracts from non-transgenic littermate controls
(NLC) and from transgenic mice after immunoprecipitation by using a
monoclonal ßARK1/2 antibody, as described previously.12
The ßARK1 protein (80kDa) was visualized with the monoclonal
antibody raised against an epitope within the carboxyl terminus of
ßARK1 and chemiluminescent detection of anti-mouse IgG conjugated
with horseradish peroxidase (Renaissance, Amersham). ßARKct was
identified with rabbit polyclonal antiserum to the carboxyl terminus of
ßARK14 10 and by chemiluminescent detection of
anti-rabbit IgG.
GRK Activity by Rhodopsin Phosphorylation
The supernatants of the myocardial extracts that contained the
soluble kinases were used to determine GRK activity. Extracts (100 to
150 µg of protein) were incubated with rhodopsin-enriched rod
outer-segment membranes in reaction buffer containing the following
(in mmol/L): MgCl2 10, Tris-Cl 20, EDTA 2,
EGTA 5, and ATP 0.1 (containing [-32P]ATP),
as previously described.4 Reactions were carried out in
the absence and presence of purified Gß (20
pmol) to maximally activate ßARK1.10 11 After
incubating in white light for 15 minutes at room temperature, reactions
were quenched with ice-cold lysis buffer and centrifuged for 15
minutes at 13 000g. Sedimented proteins were
resuspended in 25 µL of protein–gel-loading dye and treated with
12% SDS-PAGE. Phosphorylated rhodopsin was
visualized by autoradiography of dried
polyacrylamide gels and quantified using a Molecular Dynamics
PhosphorImager.
Radioligand Binding
Total ßAR density was determined by incubating 25 µg of
cardiac sarcolemmal membranes with a saturating concentration of
[125I]cyanopindolol and 20 µmol/L
alprenolol to define nonspecific binding.4 Competition
binding-isotherms in sarcolemmal membranes were done in triplicate with
80 pmol/L [125I]cyanopindolol and 22 varying
concentrations of ISO (10-14 to
10-4 mol/L) in 250 µL of binding buffer
(50 mmol/L HEPES [pH 7.3], 5 mmol/L
MgCl2, and 0.1 mmol/L ascorbic
acid].4 Assays were done at 37°C for 1 hour and then
filtered over GF/C glass fiber filters (Whatman) that were
washed twice and counted in a counter. Data were analyzed
by nonlinear least-square curve fit (GraphPad Prism).
Adenylyl Cyclase Activity
Cardiac sarcolemmal membranes (20 µg of protein) were
incubated for 15 minutes at 37°C with
[-32P]ATP under basal conditions,
10-4 mol/L ISO to stimulate ßAR, or 10
mmol/L NaF to maximally activate adenylyl cyclase. cAMP
production was quantified by standard methods described
previously.4
Statistical Analysis
Data are expressed as mean±SEM. Unpaired Student's
t tests and 1-way ANOVA were performed for statistical
comparisons except as described otherwise. For all tests,
P<0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Myocardial Expression of ßARK1 and ßARKct
As previously shown, transgenic mice with cardiac-specific expression of ßARK1 had a
3-fold increase in myocardial ßARK1
expression, as assessed by protein immunoblotting,
compared with NLC mice.4 In addition, cardiac-specific
expression of the 30kDa ßARKct peptide was documented by protein
immunoblot in this line of transgenic mice.4
In the hybrid transgenic mice, myocardial expression of both ßARK1
and the ßARKct peptide was identical to that seen in the individual
breeder transgenic lines with cardiac-specific expression of either
protein alone (Figure 1
). Thus, expression of either of these
transgenes, both driven by the same -myosin heavy chain promoter,
does not alter expression of the other transgene, and the hybrid
transgenic mice generated are an intact concomitant model of the 2
independent transgenic lines.
Myocardial ßARK1 Activity
To assess the in vitro functional effect of the ßARKct peptide
on elevated myocardial ßARK1 activity, we assayed heart extracts for
phosphorylation of the G protein-coupled receptor
rhodopsin in the absence and presence of exogenous purified
Gß. We previously showed that
Gß maximally activates ßARK1 by a
membrane-targeting event and that ßARKct peptide action should result
from inhibiting Gß activation of
ßARK1.4 10 11 In addition, our previous studies
demonstrated that myocardial cytosolic GRK activity is primarily due to
ßARK1.12 In this study, adding
Gß maximally stimulated ßARK1 activity,
especially in transgenic extracts overexpressing ßARK1 (Figure 2). As shown in Figure 2, in
transgenic mice expressing ßARKct alone or with ßARK1
overexpression (ßARK1/ßARKct), activation of ßARK1 activity by
exogenous Gß was significantly inhibited.
These data indicate that even in the presence of elevated ßARK1
protein levels, ßARK1 activity can be attenuated in vitro by the
presence of ßARKct.
|
Myocardial ßAR Functional Coupling
To examine the biochemical effects of the 2 transgenes on the
myocardial ßAR system, we assessed receptor-effector coupling in
sarcolemmal membranes from the hearts of NLC and transgenic mice. As
shown in the Table, no difference existed
in total ßAR density between NLC cardiac membranes and those from the
3 transgenic lines, including the hybrid mice. Previously, we showed
that ß1AR in the hearts of ßARK1 mice are
less able to form the high-affinity state of the receptor, which is
coupled to G proteins.4 We confirmed this finding in the
present study and, importantly, showed that concomitant
overexpression of ßARK1 and the ßARKct peptide in ßARK1/ßARKct
mice restores this high-affinity population back to control (NLC)
values (Table).
|
We assessed functional ßAR coupling by studying the activity of
adenylyl cyclase in myocardial sarcolemmal membranes. Basal and
ISO-stimulated cyclase values normalized to the percentage of
activation achieved with NaF, which was not different between groups
(Figure 3). In cardiac membranes from
ßARK1 animals, basal cyclase activity was significantly depressed
(Figure 3). As in our prior study,4 no difference
existed in basal activity in the ßARKct hearts compared with NLC
hearts (Figure 3) because ßARKct is a cytosolic peptide and is
not present in the membrane fraction. The hybrid transgenic mice
(ßARK1/ßARKct) had basal cyclase activity, which although still
depressed compared with NLC mice, was significantly higher than that
found in ßARK1 cardiac membranes (Figure 3). ISO-stimulated
cyclase activity was also significantly depressed in ßARK1 versus NLC
membranes, whereas the ßARKct membranes had similar ISO-stimulated
adenylyl cyclase activity as NLC membranes (Figure 3). In the
hybrid ßARK1/ßARKct mouse hearts, ISO-stimulated cyclase activity
in myocardial sarcolemmal membranes was significantly increased over
the ßARK1 group but lower than NLC mice (Figure 3). These data
indicate that, in vitro, increased myocardial ßARK1 activity impairs
functional coupling of ßARs, both basally and in response to ISO, and
this impairment can be attenuated by inhibiting membrane targeting of
ßARK1.
|
P<0.05 versus TGßARKct.
In Vivo Cardiac Physiology
To investigate the potential effects of the inhibition of elevated
ßARK1 activity in the hybrid transgenic mice on in vivo myocardial
function, we used cardiac catheterization in
anesthetized intact mice. As shown in Figure 4, ßARK1 overexpression led to a
significantly blunted inotropic response to the highest dose of ISO as
compared with responses in NLC mice. In contrast, ßARKct mice had
enhanced ßAR responsiveness, consistent with the peptide's
effect on reducing ßAR desensitization. Importantly, in the hybrid
ßARK1/ßARKct mice, the response of the maximum first derivative of
LV pressure (LV dP/dtmax) to ISO was
restored to the control values found in NLC mice (Figure 4).
Neither heart rate nor LV systolic pressure were different
between groups. Thus, these data suggest that overexpression of the
ßARKct peptide results in inhibition of the augmented ßAR
desensitization that is induced by elevated ßARK1 activity, which
leads to the normalization of in vivo cardiac
contractility.
|
; n=17), transgenic (TG) ßARK1 (
; n=13),
TGßARKct (•; n=12), and TGßARK1/ßARKct (
; n=9) mice. A, LV
dP/dtmax; B, LV dP/dtmin; C, LV
systolic pressure; and D, heart rate. Data were
analyzed with 4x4 repeated measures ANOVA. If appropriate,
post hoc analysis was performed by Newman-Keuls test.
*P<0.005, 
P<0.0001, §P<0.03 NLC
versus TGßARK1. A significant between-group main effect in response
to ISO was found for LV dP/dtmax (P<0.01)
and LV dP/dtmin (P<0.02). The pattern of
change between groups was statistically different for LV
dP/dtmax (P<0.0001) and LV
dP/dtmin (P<0.01). |
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The results of this study demonstrate the effectiveness of the ßARKct peptide as an inhibitor of increased ßARK1 expression and activity, both in vitro and in vivo. Using novel hybrid transgenic mice with myocardial-targeted concomitant overexpression of ßARK1 and ßARKct, we showed that the presence of a ßARK inhibitor could reverse depressed ßARK1-mediated ßAR coupling, as determined by myocardial ßAR affinity states, adenylyl cyclase activity, and ßAR responsiveness in vivo.
In this study, the hearts of these hybrid transgenic mice were,
effectively, novel in vivo reaction vessels, which allowed us to study
the physiological consequences of the direct action
of 1 transgene on another. This is the first demonstration of 2
competing transgenes being expressed in the hearts of gene-targeted
animals via this methodology. Importantly, expression levels of the
transgene products (driven by the same -myosin heavy chain
promoter) in the hybrid mice were equal to their individual parental
lines, demonstrating that there was no apparent promoter competition,
which could be a problem in hybrid transgenic mice using an
endogenously occurring promoter. The generation of such
hybrid mice by this relatively simple cross-breeding strategy provided
a powerful model for studying in vivo myocardial interactions between
proteins and for dissecting individual phenotypes.
It is becoming increasingly more evident that ßARK1 plays a critical role in myocardial function. As described above, alteration of myocardial ßARK1 activity can have profound effects on in vivo cardiac performance. The importance of ßARK1 in heart function is further supported by the recent findings that increased expression of ßARK1 accompanies attenuated cardiac function in several cardiovascular diseases, including hypertension,13 myocardial ischemia,14 ventricular hypertrophy,15 and HF.7 8 16 It is not clear what triggers increased ßARK1 in these conditions; however, an increased catecholamine level caused by enhanced sympathetic outflow is a likely candidate. In fact, we recently demonstrated that chronic activation of myocardial ßARs led to increased ßARK1 expression in the heart and enhanced myocardial GRK activity.17 Elevated ßARK1 in the failing heart contributes to physiological dysfunction as ßARs become uncoupled from downstream effectors. Moreover, a typical feature of HF is diminished responsiveness to ßAR stimulation.
As a further demonstration of the importance of ßARK1 in the cardiovascular system, we previously showed that the complete disruption of the ßARK1 gene in mice leads to a lethal phenotype characterized by cardiac malformations.18 Heterozygous (±) ßARK1-deleted mice have no developmental abnormalities and age normally and, interestingly, these mice were recently found to have a cardiac phenotype of enhanced contractility similar to transgenic mice with ßARKct overexpression.12 Mating the heterozygous ßARK1 (±) knockout mice with the ßARKct mice showed that mice with 50% less ßARK1 expressed in their hearts and expression of ßARKct had further significant enhancement of in vivo cardiac contractility.12 Because the previous study could not delineate a definitive mechanism of ßARKct action on ßARK1 activity, we used the novel hybrid strategy reported here.
In the present study, we directly demonstrated that the ßARKct
peptide can act as an in vivo inhibitor of ßARK1.
Moreover, using transgenic mice showed that ßARKct expression can
inhibit enhanced myocardial ßARK activity that is at the level seen
in human disease. In vitro studies demonstrated that ßARKct could
inhibit enhanced ßARK1 activity in these transgenic hearts by
competing for, and inhibiting, Gß-mediated
membrane translocation. Gß binding to
ßARK1 and subsequent membrane targeting are required steps for
ßARK1 activity directed toward agonist-occupied
receptors.10 11 The myocardial deficits caused by ßARK1
overexpression, including attenuated ISO-stimulated LV
contractility in vivo, decreased adenylyl cyclase
activity, and reduced functional coupling of ßAR,4 were
overcome simply by concomitant overexpression of the ßARKct peptide.
This suggests that inhibiting ßARK1 activity is sufficient to restore
the integrity of myocardial function in vivo.
The critical finding in the present study, that ßARKct can
inhibit enhanced ßARK1 activity in the heart, points to ßARK1 being
a potential target for inhibition in diseases such as HF where ßARK1
is elevated. Interestingly, the level of ßARK1 enhancement seen in
our transgenic mice (3 to 5-fold) was similar to the increased
expression observed in human HF.7 8 Thus, the expression
of ßARKct could be useful in the targeted inhibition of myocardial
ßARK1. Recently, a genetic mouse model of HF was
described19; this model made it possible to test
ßARKct action using hybrid transgenic mice. This murine model of
dilated cardiomyopathy resulted from the ablation
of the gene that encodes the muscle-specific LIM-domain
containing protein (MLP) (-/-).19 We mated cardiac
overexpression of either the ßARKct peptide or
ß2AR into the MLP (-/-)
background.16 Like ßARKct, transgenic mice
overexpressing ß2ARs at >100-fold over
endogenous levels had enhanced in vivo cardiac
contractility.20 Interestingly,
overexpression of ßARKct prevented the development of
cardiomyopathy, whereas
ß2AR overexpression further exacerbated murine
HF. Importantly, MLP (-/-) mice exhibit a 2-fold increase in
cytosolic ßARK1 levels.12 The extraordinary finding that
ßARKct prevents HF in this model, coupled with the present
findings directly demonstrating that ßARKct inhibits enhanced ßARK
activity in vivo, strongly supports the idea that ßARK1 is an
attractive, novel, therapeutic target. Thus, by using hybrid-generating
technology, it will be possible to further exploit the usefulness of
transgenic mouse models to study complex human diseases, such as
hypertension and HF.
Further support for the determination that ßARK1 inhibition results in improved outcomes in HF is the finding that, in mice, long-term treatment with carvedilol, a novel ß-blocking drug that has been used successfully in the treatment of human HF, results in more efficient ßAR coupling associated with a significant decrease in the expression of myocardial ßARK1.17 Thus, several lines of evidence in different models have demonstrated that regardless of how myocardial ßARK1 activity is diminished, ßAR signaling in the heart (and hence cardiac function) is enhanced. In addition to ßARKct expression, heterozygous ßARK1 knockout animals have enhanced cardiac contractility.12 As noted above, carvedilol and other ß-blockers can have a positive effect on the failing heart, which may, in part, be due to the lowering of ßARK1 expression in the heart. It is important to note that inhibition of ßARK1 activity in the heart and enhancement of endogenous ßAR signaling does not seem to produce negative effects on the heart,4 12 16 which is in contrast to the cardiomyopathy seen in transgenic mice with cardiac-specific overexpression of ß1AR.21 This difference in phenotype needs to be further investigated, but it suggests that these 2 mechanisms of increased receptor-effector coupling are intrinsically different. Thus, gene therapy approaches using the ßARKct transgene22 or the development of small-molecule inhibitors of ßARK1 activity could, therefore, be novel therapeutic strategies for the treatment of HF or other cardiovascular diseases that are characterized by desensitized ßARs and enhanced ßARK1 expression and/or activity.
|
Acknowledgments |
|---|
The authors thank Sandy Duncan and Lan Mao for their excellent technical assistance. R.J.L. is an Investigator of the Howard Hughes Medical Institute. This study was supported, in part, by National Service Research Award HL-09436 (S.A.A.) and grant HL-56687 (H.A.R.) from the National Institutes of Health and a Grant-in Aid from the North Carolina Affiliate of the American Heart Association (W.J.K.).
|
Footnotes |
|---|
Drs Akhter and Eckhart contributed equally to this work.
Received January 29, 1999; revision received April 7, 1999; accepted April 9, 1999.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
-
Brodde O-E. ß-Adrenergic receptors in
failing human myocardium. Basic Res
Cardiol.. 1993;91:35–40.
-
Inglese J, Freedman NJ, Koch WJ, Lefkowitz RJ.
Structure and mechanism of the G protein-coupled receptor kinases.
J Biol Chem.. 1993;268:23735–23738.
[Free Full Text]
-
Freedman NJ, Liggett SB, Drachman DE, Pei G, Caron MG,
Lefkowitz RJ. Phosphorylation and desensitization of
the human ß-adrenergic receptor: involvement of G
protein-coupled receptor kinases and cAMP-dependent protein kinase.
J Biol Chem.. 1995;270:17953–17961.
[Abstract/Free Full Text]
-
Koch WJ, Rockman HA, Samama P, Hamilton RA, Bond RA,
Milano CA, Lefkowitz RJ. Cardiac function in mice overexpressing
the ß-adrenergic receptor kinase or an ßARK
inhibitor. Science.. 1995;268:1350–1353.
[Abstract/Free Full Text]
-
Bristow MR, Ginsburg R, Minobe W, Cubicciotti RS,
Sageman WS, Luni K, Billingham ME, Harrison DC, Stinson EB. Decreased
catecholamine sensitivity and ß-adrenergic-receptor
density in failing human hearts. N Engl J Med. 1982;307:205–211.[Abstract]
-
Feldman AM. Modulation of adrenergic receptors and
G-transduction proteins in failing human ventricular
myocardium. Circulation.. 1993;87:IV27-IV34.
-
Ungerer M, Bohm M, Elce JS, Erdmann E, Lohse MJ.
Expression of ß-arrestins and ß-adrenergic receptor kinases in
the failing human heart. Circulation.. 1993;87:454–463.
[Abstract/Free Full Text]
-
Ungerer M, Parruti G, Bohm M, Puzicha M, DeBlasi A,
Erdmann E, Lohse MJ. Expression of ß-arrestins and
ß-adrenergic receptor kinases in the failing human heart. Circ
Res.. 1994;74:206–213.
[Abstract/Free Full Text]
-
Leimbach WN Jr, Wallin BG, Victor RG, Aylward PE,
Sundlof G, Mark AL. Direct evidence from intraneural recordings
for increased central sympathetic outflow in patients with heart
failure. Circulation.. 1986;73:913–919.
[Abstract/Free Full Text]
-
Koch WJ, Inglese J, Stone WC, Lefkowitz RJ. The binding
site for the beta gamma subunits of heterotrimeric G proteins on
the ß-adrenergic receptor kinase. J Biol Chem.. 1993;268:8256–8260.
[Abstract/Free Full Text]
-
Pitcher JA, Inglese J, Higgins JB, Arriza JL, Casey PJ,
Kim C, Benovic JL, Kwatra MM, Caron MG, Lefkowitz RJ. Role of beta
gamma subunits of G proteins in targeting the ß-adrenergic
receptor kinase to membrane-bound receptors. Science.. 1992;257:1264–1267.
[Abstract/Free Full Text]
-
Rockman HA, Choi D-J, Akhter SA, Jaber M, Giros B,
Lefkowitz RJ, Caron MG, Koch WJ. Control of myocardial contractile
function by the level of ß-adrenergic receptor kinase 1 in
gene-targeted mice. J Biol Chem.. 1998;293:18180–18184.
-
Gros R, Benovic JL, Tan CM, Feldman RD.
G-protein-coupled receptor kinase activity is increased in
hypertension. J Clin Invest.. 1997;99:2087–2093.[Medline]
[Order article via Infotrieve]
-
Ungerer M, Kessebohm K, Kronsbein K, Lohse MJ, Richardt
G. Activation of ß-adrenergic receptor kinase during myocardial
ischemia. Circ Res.. 1996;79:455–460.
[Abstract/Free Full Text]
-
Choi D-J, Koch WJ, Hunter JJ, Rockman HA. Mechanism
of ß-adrenergic receptor desensitization in cardiac
hypertrophy is increased ß-adrenergic receptor kinase.
J Biol Chem.. 1997;272:17223–17229.
[Abstract/Free Full Text]
-
Rockman HA, Chien KR, Choi D-J, Iaccarino G, Hunter JJ,
Ross J Jr, Lefkowitz RJ, Koch WJ. Expression of a ß-adrenergic
receptor kinase 1 inhibitor prevents the development of
myocardial failure in gene-targeted mice. Proc Natl Acad Sci
U S A. 1998;95:7000–7005.
[Abstract/Free Full Text]
-
Iaccarino G, Tomhave ED, Lefkowitz RJ, Koch WJ.
Reciprocal in vivo regulation of myocardial G protein-coupled receptor
kinase expression by ß-adrenergic receptor stimulation and
blockade. Circulation. 1998;98:1783–1789.
[Abstract/Free Full Text]
-
Jaber M, Koch WJ, Rockman HA, Smith B, Bond RA, Sulik
K, Ross J Jr, Lefkowitz RJ, Caron MG, Giros B. Essential role of
ß-adrenergic receptor kinase 1 in cardiac development and function.
Proc Natl Acad Sci U S A. 1996;93:12974–12979.
[Abstract/Free Full Text]
-
Arber S, Hunter JJ, Ross J Jr, Hongo M, Sansig G, Borg
J, Perriard J-C, Chien KR, Caroni P. MLP-deficient mice exhibit a
disruption of cardiac cytoarchitectural organization, dilated
cardiomyopathy, and heart failure. Cell.. 1997;88:393–403.[Medline]
[Order article via Infotrieve]
-
Milano CA, Allen LF, Rockman HA, Dolber PC, McMinn TR,
Chien KR, Johnson TD, Bond RA, Lefkowitz RJ. Enhanced myocardial
function in transgenic mice overexpressing the
ß2-adrenergic receptor. Science.. 1994;264:582–586.
[Abstract/Free Full Text]
-
Port JD, Weinberger HD, Bisognano JD, Knudson OA,
Bohlmeyer TJ, Pende A, Bristow MR. Echocardiographic
and histopathologic characterization of young and old transgenic mice
over-expressing the human ß1-adrenergic receptor. J
Am Coll Cardiol. 1998;31(suppl A):177A. Abstract.
-
Akhter SA, Skaer CS, Kypson AP, McDonald PH,
Peppel KC, Glower DD, Lefkowitz RJ, Koch WJ. Restoration of
ß-adrenergic signaling in failing cardiac ventricular
myocytes via adenoviral-mediated gene transfer. Proc Natl Acad
Sci U S A. 1997;94:12100–12105.
[Abstract/Free Full Text]
This article has been cited by other articles:
 |
|
 |
|
  L. E. Vinge, P. W. Raake, and W. J. Koch Gene Therapy in Heart Failure Circ. Res., June 20, 2008; 102(12): 1458 - 1470. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. K. Pandalai, K. M. McLean, C. F. Bulcao, J. Y. Duffy, K. M. D'Souza, W. H. Merrill, J. M. Pearl, and S. A. Akhter Acute beta-blockade prevents myocardial beta-adrenergic receptor desensitization and preserves early ventricular function after brain death. J. Thorac. Cardiovasc. Surg., April 1, 2008; 135(4): 792 - 798. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. K. Pandalai, C. F. Bulcao, W. H. Merrill, and S. A. Akhter Restoration of myocardial {beta}-adrenergic receptor signaling after left ventricular assist device support J. Thorac. Cardiovasc. Surg., May 1, 2006; 131(5): 975 - 980. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Nordlie, L. E. Wold, B. Z. Simkhovich, C. Sesti, and R. A. Kloner Molecular Aspects of Ischemic Heart Disease: Ischemia/Reperfusion-Induced Genetic Changes and Potential Applications of Gene and RNA Interference Therapy Journal of Cardiovascular Pharmacology and Therapeutics, March 1, 2006; 11(1): 17 - 30. [Abstract] [PDF]
|
|
|
|
 |
|
 X. Yu, S. Huang, E. Patterson, M. W. Garrett, K. M. Kaufman, J. P. Metcalf, M. Zhu, S. T. Dunn, and D. C. Kem Proteasome degradation of GRK2 during ischemia and ventricular tachyarrhythmias in a canine model of myocardial infarction Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1960 - H1967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. K. Pandalai, J. M. Lyons, J. Y. Duffy, K. M. McLean, C. J. Wagner, W. H. Merrill, J. M. Pearl, and S. A. Akhter Role of the {beta}-adrenergic receptor kinase in myocardial dysfunction after brain death J. Thorac. Cardiovasc. Surg., October 1, 2005; 130(4): 1183 - 1189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Iaccarino, E. Barbato, E. Cipolletta, V. De Amicis, K. B. Margulies, D. Leosco, B. Trimarco, and W. J. Koch Elevated myocardial and lymphocyte GRK2 expression and activity in human heart failure Eur. Heart J., September 1, 2005; 26(17): 1752 - 1758. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Trivedi and M. F. Lokhandwala Rosiglitazone restores renal D1A receptor-Gs protein coupling by reducing receptor hyperphosphorylation in obese rats Am J Physiol Renal Physiol, August 1, 2005; 289(2): F298 - F304. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Williams, J. A. Hata, J. Schroder, E. Rampersaud, J. Petrofski, A. Jakoi, C. A. Milano, and W. J. Koch Targeted {beta}-Adrenergic Receptor Kinase ({beta}ARK1) Inhibition by Gene Transfer in Failing Human Hearts Circulation, April 6, 2004; 109(13): 1590 - 1593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Kirchhefer, L. R Jones, F. Begrow, P. Boknik, L. Hein, M. J Lohse, B. Riemann, W. Schmitz, J. Stypmann, and J. Neumann Transgenic triadin 1 overexpression alters SR Ca2+ handling and leads to a blunted contractile response to {beta}-adrenergic agonists Cardiovasc Res, April 1, 2004; 62(1): 122 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.R. Keys and W.J. Koch The Adrenergic Pathway and Heart Failure Recent Prog. Horm. Res., January 1, 2004; 59(1): 13 - 30. [Abstract] [Full Text]
|
|
|
|
 |
|
 X.-M. Gao, A. Agrotis, D. J. Autelitano, E. Percy, E. A. Woodcock, G. L. Jennings, A. M. Dart, and X.-J. Du Sex Hormones and Cardiomyopathic Phenotype Induced by Cardiac {beta}2-Adrenergic Receptor Overexpression Endocrinology, September 1, 2003; 144(9): 4097 - 4105. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Hata and W. J. Koch Phosphorylation of G Protein-Coupled Receptors: GPCR Kinases in Heart Disease Mol. Interv., August 1, 2003; 3(5): 264 - 272. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. D. Eckhart, T. Ozaki, H. Tevaearai, H. A. Rockman, and W. J. Koch Vascular-Targeted Overexpression of G Protein-Coupled Receptor Kinase-2 in Transgenic Mice Attenuates beta -Adrenergic Receptor Signaling and Increases Resting Blood Pressure Mol. Pharmacol., April 1, 2002; 61(4): 749 - 758. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
H. T. Tevaearai, A. D. Eckhart, K. F. Shotwell, K. Wilson, and W. J. Koch Ventricular Dysfunction After Cardioplegic Arrest Is Improved After Myocardial Gene Transfer of a {beta}-Adrenergic Receptor Kinase Inhibitor Circulation, October 23, 2001; 104(17): 2069 - 2074. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-J. Du Sympathoadrenergic mechanisms in functional regulation and development of cardiac hypertrophy and failure: findings from genetically engineered mice Cardiovasc Res, June 1, 2001; 50(3): 443 - 453. [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Lefkowitz and J. T. Willerson Prospects for Cardiovascular Research JAMA, February 7, 2001; 285(5): 581 - 587. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
X.-J. Du, X.-M. Gao, G. L. Jennings, A. M. Dart, and E. A. Woodcock Preserved ventricular contractility in infarcted mouse heart overexpressing beta 2-adrenergic receptors Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2456 - H2463. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Lefkowitz, H. A. Rockman, and W. J. Koch Catecholamines, Cardiac {beta}-Adrenergic Receptors, and Heart Failure Circulation, April 11, 2000; 101(14): 1634 - 1637. [Full Text] [PDF]
|
|
|
|
|
|
R.-P. Xiao, H. Cheng, Y.-Y. Zhou, M. Kuschel, and E. G. Lakatta Recent Advances in Cardiac {beta}2-Adrenergic Signal Transduction Circ. Res., November 26, 1999; 85(11): 1092 - 1100. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Feldman and C. McTiernan New Insight Into the Role of Enhanced Adrenergic Receptor-Effector Coupling in the Heart Circulation, August 10, 1999; 100(6): 579 - 582. [Full Text] [PDF]
|
|
|
|
 |
|
 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, |