(Circulation. 1999;99:2702-2707.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Enhanced Contractility and Decreased ß-Adrenergic Receptor Kinase-1 in Mice Lacking Endogenous Norepinephrine and Epinephrine
From the Department of Medicine (M.-C.C., H.A.R.), University of North Carolina at Chapel Hill; University of California San Diego School of Medicine (M.R.), LaJolla, Calif; Department of Surgery (W.J.K.), Duke University, Durham, NC; and Department of Biochemistry and Howard Hughes Medical Institute (S.A.T., R.D.P.), University of Washington, Seattle, Wash.
Correspondence to Howard A. Rockman, MD, Department of Medicine, CB#7075, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7075. E-mail hrockman{at}med.unc.edu
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Abstract |
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Background—Elevated circulating norepinephrine (NE) has been implicated in causing the profound ß-adrenergic receptor (ßAR) downregulation and receptor uncoupling that are characteristic of end-stage human dilated cardiomyopathy, a process mediated in part by increased levels of ß-adrenergic receptor kinase (ßARK1). To explore whether chronic sustained NE stimulation is a primary stimulus that promotes deterioration in cardiac signaling, we characterized a gene-targeted mouse in which activation of the sympathetic nervous system cannot lead to an elevation in plasma NE and epinephrine.
Methods and Results—Gene-targeted mice that lack dopamine ß-hydroxylase (dbh-/-), the enzyme needed to convert dopamine to NE, were created by homologous recombination. In vivo contractile response to the ß1AR agonist dobutamine, measured by a high-fidelity left ventricular micromanometer, was enhanced in mice lacking the dbh gene. In unloaded adult myocytes isolated from dbh-/- mice, basal contractility was significantly increased compared with control cells. Furthermore, the increase in ßAR responsiveness and enhanced cellular contractility were associated with a significant reduction in activity and protein level of ßARK1 and increased high-affinity agonist binding without changes in ßAR density or G-protein levels.
Conclusions—Mice that lack the ability to generate NE or epinephrine show increased contractility associated primarily with a decrease in the level of ßARK1 protein and kinase activity. This animal model will be valuable in testing whether NE is required for the pathogenesis of heart failure through mating strategies that cross the dbh-/- mouse into genetically engineered models of heart failure.
Key Words: contractility • catecholamines • heart failure • receptors, adrenergic, beta
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Introduction |
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One of the most important mechanisms for rapidly regulating ß-adrenergic receptor (ßAR) function is agonist-stimulated receptor phosphorylation by G protein–coupled receptor kinases (GRKs), resulting in decreased sensitivity to subsequent catecholamine stimulation.1 ßAR kinase (ßARK1) is a member of this family of GRKs that phosphorylate and regulate a wide variety of receptors that couple to heterotrimeric G proteins.1 2
Activation of the sympathetic nervous system is considered one of the cardinal pathophysiological abnormalities in patients with heart failure3 and frequently precedes the development of overt symptoms.4 Plasma norepinephrine (NE) and renin activity are increased in patients with heart failure and are known prognostic factors for survival.3 Elevated circulating NE and epinephrine have been implicated in contributing to the profound ßAR downregulation and receptor uncoupling that are characteristic of end-stage human dilated cardiomyopathy,5 resulting in subsensitivity to ß-agonist stimulation,6 a process likely mediated by ßARK1.7 Importantly, myocardial ßARK1 mRNA and activity are elevated in human heart failure.8 9 It has been postulated that long-term stimulation of myocardial ßARs may adversely affect cardiomyocyte viability, possibly through cAMP-mediated Ca2+ overload of the cell.10 In this regard, sustained sympathetic activity may both phosphorylate and downregulate phospholamban and act to enhance sarcoplasmic reticulum Ca2+ pump activity in the attempt to maintain contractile and relaxation processes.11 This has led to the hypothesis that chronic sustained sympathetic drive is an important pathological element in the progressive deterioration of the failing heart,12 a concept that is supported by data showing a beneficial outcome in heart failure patients treated with ßAR antagonists13 and a reduction in sympathetic outflow with ACE inhibitors.14
To further explore the role of chronic sustained adrenergic stimulation in the pathogenesis of the failing heart, a model system in which activation of the sympathetic nervous system cannot lead to an elevation in plasma NE would be of value. In this regard, gene-targeted mice that lack dopamine ß-hydroxylase (dbh-/-), the enzyme needed to convert dopamine to NE, were created by homologous recombination.15 16 Adult dbh-/- mice have virtually no endogenous NE or epinephrine.15 16 17 The purpose of the present study was to characterize the cardiovascular phenotype and ßAR signaling pathway in rescued adult dbh-/- mice that are devoid of endogenous NE and epinephrine.
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Methods |
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Experimental Animals
Details of the gene-targeting strategy used to disrupt the dopamine ß-hydroxylase gene have been published elsewhere.15 Mice used in the present study were 4 to 6 months of age and were of either sex. Mutant offspring were generated from homozygous males mated to heterozygous females. Heterozygous dbh+/- mice have normal levels of NE and epinephrine and were therefore used as controls.16 17 In contrast, homozygous dbh-/- mice have serum and tissue NE levels that are below the level of detection.16 17 Homozygous dbh-/- embryos were rescued from embryonic lethality with the administration of L-threo-3,4-dihydroxyphenylserine (L-DOPS) in the maternal drinking water from day E 9.5 until birth.15 16 L-DOPS is a synthetic amino acid that is converted to NE by aromatic L-amino acid decarboxylase, which is present in all adrenergic cells, bypassing the requirement for DBH.
Experiments were performed in an additional group of genetically altered mice that were heterozygous for ablation of the ßARK1 gene (ßARK1+/-).18 ßARK1+/- heterozygous animals contain a 50% reduction in myocardial levels of ßARK1 protein and GRK activity, which results in increased contractile function compared with wild-type animals.19
Animals in this study were handled according to the animal welfare regulations of the University of North Carolina at Chapel Hill and University of Washington, and the protocol was approved by the animal subjects committees of these institutions.
Physiological Evaluation
Hemodynamic evaluation was performed as
previously described.20 21 22 Mice were anesthetized
with a mixture of ketamine (100 mg/kg) and xylazine (2.5 mg/kg)
given intraperitoneally. After endotracheal
intubation, mice were connected to a rodent ventilator. The left
carotid artery was cannulated with a flame-stretched PE-50 catheter
connected to a modified P-50 Statham transducer. A 1.4F high-fidelity
micromanometer catheter (Millar Instruments) was
inserted into the right carotid and advanced retrogradely into the left
ventricle (LV). Hemodynamic measurements were
recorded before and after bilateral vagotomy and after 2-minute
infusion of incremental doses of dobutamine.
Myocyte Isolation and Contractile Function
Hearts from separate control and
dbh-/- mice were used to isolate
adult myocytes, and contractile function was assessed as previously
described.19 Myocyte cell edges were enhanced and
processed with a video edge motion-detection system (Crescent
Electronics) at a sampling rate of 240 Hz and recorded in digitized
form at 400 Hz.
GRK Activity by Rhodopsin Phosphorylation
Supernatants of cytosolic extracts were prepared as previously
described.19 20 Concentrated (Centricon, Amicon Inc)
cytosolic extract (300 µg of protein) was incubated with
rhodopsin-enriched rod outer segments in 25 µL of lysis buffer with
10 mmol/L MgCl2 and 0.1 mmol/L ATP
containing [
-32P]ATP. The reactions were
incubated in white light for 15 minutes, quenched with 300 µL of
ice-cold lysis buffer, and then centrifuged. Sedimented
proteins were electrophoresed through SDS–12% polyacrylamide
gels, and phosphorylated rhodopsin was visualized by
autoradiography and quantified with a
phosphorimager (Molecular Dynamics).
Immunoblotting
Pelleted membranes (from above) were resuspended in 50
mmol/L HEPES buffer (pH 7.3) containing 5 mmol/L
MgCl2 and electrophoresed on SDS–10%
polyacrylamide gels and transferred to nitrocellulose. The
39-kDa Gi
protein and 2 forms of
stimulatory G (Gs) protein (45 and 53 kDa)
were visualized with 1:1000 dilution of polyclonal antibody (I-20 and
K-20, respectively; Santa Cruz Biotechnology), and detection of
anti-rabbit IgG conjugated with horseradish peroxidase was performed by
enhanced chemiluminescence (ECL; Amersham). Immunoblots
were scanned and quantified with an imaging densitometer
(Bio-Rad).
In separate hearts, immunodetection of myocardial levels of ßARK1 was
performed on cytosolic extracts after immunoprecipitation, as
previously described.20 ßARK1 was immunoprecipitated
from 600 µg of clarified cytosol extract with a 1:1000 (1 µL)
monoclonal anti-ßARK1 (C5/1) antibody and 35 µL of a 50% slurry of
protein A-agarose conjugate agitated for 1 hour at 4°C. Immune
complexes were washed, resuspended in 40 µL of protein-gel loading
buffer, then heated for 3 minutes at 85°C and electrophoresed through
SDS–12% polyacrylamide gels. After transfer to
nitrocellulose, the 80-kDa ßARK1 protein was visualized with the
monoclonal antibody (C5/1) and chemiluminescent detection of anti-mouse
IgG conjugated with horseradish peroxidase (ECL; Amersham).
ßAR Density and Radioligand Binding
Myocardial sarcolemmal membranes were prepared by
homogenization of whole hearts in ice-cold buffer
as previously described.20 21 23 Total ßAR density was
determined by incubation of 25 µg of cardiac sarcolemmal membranes
with a saturating concentration (80 pmol/L) of
[125I]cyanopindolol and 20 µmol/L
alprenolol to define nonspecific binding.23 Typical
nonspecific binding is 40% of the total. Competition-binding
isotherms in sarcolemmal membranes were done in triplicate with 18
varying concentrations of isoproterenol (10-13
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).23 Assays were conducted at 37°C for 60 minutes
and then filtered over GF/C glass fiber filters (Whatman) that were
washed and counted in a gamma counter. Competition isotherms were
analyzed by nonlinear least squares curve fit (GraphPad Prism).
Statistical Analysis
Data are expressed as mean±SEM. Two-way repeated ANOVA was used
to evaluate the hemodynamic measurements under basal
conditions and with dobutamine stimulation. When
appropriate, post hoc analysis was performed with a
Newman-Keuls test. Student's t test with Bonferroni
correction for multiple comparisons was used to assess differences in
isolated myocyte mechanics and for analysis of the biochemical
data. For all analyses, P<0.05 was considered
significant.
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Results |
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Although the dbh-/- mice were smaller than controls, myocardial growth in the adult mouse was proportionate. No significant difference was found between dbh-/- and control mice for LV weight normalized for either body weight or tibia length (Figure 1
). This is consistent with our
previously reported data showing that adult
dbh-/- mice are morphologically
normal and eventually grow to 80% (male) and 88% (female) of
littermate adult weight.15
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Cardiac catheterization was used to measure in vivo
responsiveness to dobutamine in intact anesthetized
dbh-/- and control mice. Before
vagotomy, LV dP/dtmax in the
dbh-/- mice was significantly
greater than in control animals (Figure 2A). As expected, after bilateral
vagotomy, there was a small increase in LV
dP/dtmax that was seen in both genotypes
(Figure 2A). In response to dobutamine, a
significant increase in LV dP/dtmax was seen in
dbh-/- mice compared with control
mice (Figure 2A). The change of LV
dP/dtmax from baseline, 
LV
dP/dtmax, is a measure of ßAR responsiveness.
As shown in Figure 2B, dbh-/-
animals had a marked enhancement of LV dP/dtmax
in response to dobutamine infusion, which suggests
increased ßAR coupling and sensitivity to ß-agonist
stimulation.
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, n=11)
and dbh-/- (
, n=10) mice. A, LV
dP/dtmax; B,
Hemodynamic parameters obtained during
catheterization are shown in Figure 3. Before vagotomy, heart rate in
dbh-/- mice was identical to that
for control animals. After bilateral vagotomy, heart rate increased to
a greater extent in dbh-/- mice,
indicating higher parasympathetic tone in
dbh-/- mice than in control animals
(Figure 3A). With dobutamine infusion, the increase
in heart rate was significantly less in
dbh-/- mice than in control animals,
indicating a reduced chronotropic effect of ß-agonist stimulation in
dbh-/- mice (percent increase from
after vagotomy, control 22.5±2.5% versus
dbh-/- 11.8±1.2%;
P<0.01). LV systolic pressure and
end-diastolic pressures were not statistically different
between groups at baseline or with dobutamine stimulation
(Figure 3B and 3C). These data demonstrate a marked enhancement
of LV contractility and minimal chronotropic effect
with ßAR stimulation in mice chronically lacking NE and
epinephrine. Furthermore, the increased prevagotomy LV
dP/dtmax in
dbh-/- mice with similar heart rate
to controls suggests a state of enhanced contractility.
It is interesting that heart rate before vagotomy was identical to
control in the dbh-/- mice, which
suggests that an elevation in parasympathetic tone may exist in the
dbh-/- mice.
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To test whether dbh-/- mice have
enhanced contractility at a cellular level, cell
mechanics were measured in single adult myocytes. Under conditions of
constant pacing in unloaded, freshly isolated myocytes, the rate of
cell shortening (-dL/dt) and percentage of cell shortening were
significantly higher (33% and 18%, respectively) in
dbh-/- cells than in control cells
(Table). By eliminating the
influence of heart rate and loading conditions on the measurement of
contractile function, we demonstrated that myocytes isolated from
dbh-/- hearts have enhanced cellular
contractility.
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The enhanced cardiac contractility and ßAR
sensitivity in the dbh-/- mice were
similar to mice with myocardial expression of a peptide
inhibitor of ßARK123 and to
gene-targeted mice with a 50% reduction in ßARK1 levels
(ßARK1+/-).19
Therefore, we investigated ßARK1 expression and activity in these
mice. ßARK1 is a cytosolic enzyme, and our previous studies in mice
demonstrated that GRK activity in those extracts primarily reflects
ßARK1.20 Kinase activity of cytosolic extracts from
dbh-/- hearts was 50% lower than
values obtained from control hearts (Figure 4A and 4B). Consistent with the
reduction in cytosolic kinase activity, the level of ßARK1 protein,
as assessed by immunoblotting, was also significantly
diminished in the hearts of dbh-/-
mice compared with control mice (Figure 4C). These data show
that the enhanced ßAR responsiveness and cellular
contractility in mice lacking NE and
epinephrine are associated with a significant reduction in
ßARK1 activity and protein.
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To determine whether altered levels of G proteins contributed to the
cardiac phenotype, the levels of Gi
and Gs protein in cardiac membranes were
measured by immunoblotting (Figure 5A and 5B). No difference in
Gi protein levels between control (5.0±0.3
arbitrary units [AU]) and dbh-/-
hearts (4.3±0.3 AU) was found by densitometry. Although the higher
molecular weight form of Gs protein was
somewhat lower in the dbh-/- hearts,
this did not reach statistical significance (control versus
dbh-/-: 45-kDa band, 5.6±0.3 versus
5.8±0.4 AU; 53-kDa band, 6.3±0.3 versus 4.6±0.6 AU;
P=NS).
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To investigate the molecular mechanisms for the increased
contractility and enhanced ßAR responsiveness, we
evaluated receptor-effector coupling in sarcolemmal membranes from
hearts of dbh-/- mice and compared
it with that of ßARK1+/- mice. The
total number of ßARs in the dbh-/-
hearts (n=6) (56.1±4.9 fmol/mg membrane protein) was not significantly
different than that in control hearts (n=7) (49.1±6.4 fmol/mg membrane
protein). In contrast, the percentage of ßARs exhibiting
high-affinity binding for isoproterenol was significantly greater in
membranes prepared from dbh-/-
hearts than in those from controls (Figure 6A). Furthermore,
ßAR-Gs coupling in a genetic mouse model in
which myocardial ßARK1 activity was reduced by 50% also showed an
increase in high-affinity agonist binding similar to that observed in
dbh-/- hearts (Figure 6B).
The significantly greater number of high-affinity receptors is
consistent with an increased ability of ßARs to form the
coupled hormone-receptor–G-protein high-affinity state. These changes
occur when receptors are less desensitized.23
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P<0.05
dbh-/- or
ßARK+/- versus respective control,
unpaired t test (n=5 each group). |
Discussion |
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The present study demonstrates that adult dbh-/- mice that lack endogenous NE and epinephrine have the following characteristics: (1) myocardial growth is proportionate to body weight; (2) cellular contractility in isolated myocytes is increased; (3) ßAR responsiveness in vivo is significantly enhanced; and (4) this enhanced contractile state is associated with a significant reduction in ßARK1 and increased high-affinity agonist binding without changes in ßAR density or G-protein levels. Thus, the enhanced inotropy and ßAR responsiveness likely results from decreased ßAR desensitization.
Previous studies have shown that a tissue subjected to either surgical or chemical sympathectomy elicits an exaggerated response to catecholamine stimulation, a process termed catecholamine supersensitivity.24 25 In the mouse model used in the present study, which is devoid of endogenous NE and epinephrine, we demonstrate that enhanced responsiveness to ßAR stimulation is likely due to a reduction in the level of ßARK1 protein and reduction in ßARK1 activity. Thus, we have further demonstrated that the level of ßARK1 in the normal heart may be modulated by activity of the sympathetic nervous system, an observation supported by previous experimental studies.26 27
Immunoblotting of sarcolemmal membranes with an
antibody to either Gi or
Gs showed no appreciable difference in the
level of these G proteins in dbh-/-
compared with control hearts. In contrast, the changes in ßARK1 are
significant and likely account for the enhancement in ßAR
responsiveness. We have recently shown that mice heterozygous for the
ßARK1 gene deletion, which possess 50% less ßARK1
enzyme than wild-type animals, have enhanced LV
contractility and sensitivity to ßAR
agonists,19 a cardiac phenotype that is
similar to the dbh-/- mice described
herein. In the present study, we also show that both
dbh-/- and
ßARK1+/- hearts have a
significantly greater percentage of receptors in the high-affinity
state (Figure 6
). This enhanced ßAR-effector coupling in
dbh-/- hearts, which is identical to
the findings when myocardial ßARK1 is reduced by 50%, is most
consistent with a mechanism of diminished desensitization
resulting from the reduction in ßARK1 that allows for a greater
number of ßARs to form the coupled hormone-receptor–G-protein
high-affinity state.23
Despite multiple lines of evidence showing enhanced ßAR responsiveness in dbh-/- mice, we were unable to show an increase in membrane adenylyl cyclase activity (basal conditions: control versus dbh-/-, 20.6±5.9 versus 25.4±7.9 pmol · mg protein-1 · min-1, P=NS; isoproterenol [10-4 mol/L]: control versus dbh-/-, 35.7±5.9 versus 42.3±10.2 pmol · mg protein-1 · min-1, P=NS; n=6 for both groups). Although we can only speculate about the apparent lack of enhanced activity, we postulate that the ßARK1 activity present in purified membranes isolated from dbh-/- hearts is sufficient to desensitize ßARs to the same extent as controls in this in vitro assay. Importantly, however, we document that dbh-/- mice have enhanced ßAR responsiveness and reduced ßARK1 activity, which does lead to more ßARs in the high-affinity state.
The dbh-/- mice showed a marked enhancement of LV dP/dtmax in response to ß-agonist stimulation consistent with a state of diminished ßAR desensitization. Desensitization of ßARs requires not only GRK-mediated phosphorylation but also the binding of ß-arrestins, which bind to phosphorylated receptors and interdict further activation of G proteins. The GRKs expressed in the heart are GRK2 (commonly known as ßARK1), GRK3 (ßARK2), GRK5, and GRK6, with ßARK1 being the most abundant. A pivotal role for ßARK1 in the regulation of ßAR signaling is suggested by recent experimental and clinical studies. In a series of studies in gene-targeted mice, we have shown that cardiac-targeted overexpression of ßARK1 results in marked ßAR desensitization,23 whereas overexpression of a peptide inhibitor of ßARK123 or 50% reduction in ßARK119 results in enhanced ßAR sensitivity to ß-agonist stimulation. Furthermore, impaired ßAR responsiveness, which occurs in response to pressure overload hypertrophy, is caused by the increase in myocardial ßARK1 and can be completely reversed on ßARK inhibition.20 Finally, ßARK1 levels are increased in heart extracts from human end-stage heart failure.8 9 In this regard, using several gene-targeted mouse models, we have recently shown that ßARK1 can play a primary role in the pathogenesis of the failing heart.21
A characteristic of chronic heart failure is ßAR desensitization. The trigger for ßAR desensitization in the failing heart is thought be an increase in cardiac neural activity that causes elevation in circulating plasma NE levels.28 It has been generally assumed that activation of the sympathetic nervous system, which precedes the onset of clinically recognized heart failure,4 is detrimental to the failing heart and promotes progressive deterioration in cardiac function.10 12 13 Although the level of plasma NE is a prognostic indicator of survival, it is not known whether the increase in plasma catecholamines is responsible for the long-term deterioration in LV function. A recent study29 showed that selective ventricular denervation in an experimental model of progressive heart failure induced by rapid ventricular pacing could attenuate the decline in cardiac function, which suggests that cardiac nerves play a role in the progression of heart failure. In this regard, the dbh-/- mouse provides a unique opportunity to test the roles the sympathetic nervous system and plasma NE play in the pathogenesis of the failing heart.
In summary, dbh-/- mice that lack the ability to generate NE or epinephrine show enhanced cellular contractility and ßAR responsiveness that are associated with a decrease in the level of ßARK1 protein and kinase activity. The loss of ßARK1-mediated ßAR phosphorylation appears to be the most likely mechanism accounting for this cardiac phenotype. The dbh-/- animal model will prove valuable to test whether NE and epinephrine are required for the pathogenesis of heart failure through mating strategies that cross the dbh-/- mouse into genetically engineered heart failure backgrounds.
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Acknowledgments |
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This work was supported in part by the National Institutes of Health grants HL-56687 (Dr Rockman) and HD-09172 (Dr Palmiter). We gratefully acknowledge Dr Lan Mao for her microsurgical expertise, Kyle Shotwell for technical assistance, and Dr R.J. Lefkowitz for providing purified ßARK1 and the monoclonal antibody C5/1.
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Footnotes |
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Dr Thomas is presently at the Department of Pharmacology, University of Pennsylvania, Philadelphia, Pa.
Received November 10, 1998; revision received February 2, 1999; accepted February 16, 1999.
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