(Hypertension. 1998;32:831-837.)
© 1998 American Heart Association, Inc.
Scientific Contributions |
Chamber-Specific Alterations of Norepinephrine Uptake Sites in Cardiac Hypertrophy
From the Klinik III für Innere Medizin der Universität zu Köln, Köln (M.B., M.F., C.M., M.M., F.S., O.Z.), and the Institut für Klinische Pharmakologie und Toxikologie der Freien Universität Berlin, Universitätsklinikum Benjamin Franklin, Berlin (M.P.), Germany; and the Scienze Mediche, Universita degli Studi di Brescia, Italy (M.C.).
Correspondence to Michael Böhm, Klinik III für Innere Medizin der Universität zu Köln, Joseph-Stelzmann-Str. 9, 50924 Köln, Germany.
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Abstract—The present study investigated local differences of sympathetic activation and sympathetic neuroeffector defects in nonhypertrophied right and hypertrophied left ventricles in a rat model with renin-induced pressure overload [TG(mREN2)27]. As judged from the depletion of myocardial norepinephrine stores, sympathetic activation was more pronounced in the left than in the right ventricles. In addition, norepinephrine uptake1 carrier sites were reduced in left but unchanged in right ventricles. Gene expression of the carrier was unchanged in stellate ganglia. An increase of Gi
expression and a heterologous adenylyl
cyclase desensitization occurred only in the left but not in the right
ventricles, whereas a reduction of ß-adrenergic receptors was
observed in both chambers. We concluded that general sympathetic
activation can lead to ß-adrenoceptor downregulation but that
pressure overload further increases sympathetic activation involving
norepinephrine uptake mechanisms in the left ventricles,
resulting in heterologous ß-adrenergic desensitization.
Key Words: hypertrophy, cardiac • G proteins • adenylyl cyclase • catecholamines
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Introduction |
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In the failing myocardium, an activation of the tissue renin-angiotensin system (RAS) occurs. In addition, sympathetic stimulation1 produces a decrease of cardiac ß-adrenoceptors2 3 and increased expression of inhibitory G-protein
-subunits
(Gi),3 4 5 resulting in a
desensitization of the adenylyl cyclase and a blunted response to
cAMP-increasing positive inotropic agents.3 In
the failing human heart, reduced catecholamine
stores5 and reduced norepinephrine
uptake1 carrier sites6 have
been observed. It is not resolved whether the general sympathetic
activation or local mechanisms of catecholamine release and
a decreased uptake of norepinephrine within the myocardial chambers
contribute to the extent of postsynaptic alteration of ß-adrenergic
signal transduction. Recently, it became evident that in left
ventricular myocardium in hypertensive cardiac
hypertrophy, adenylyl cyclase desensitization also
occurs.7 8 Therefore, sympathetic activation with
consecutive changes of ß-adrenergic signal transduction could occur
already in the stage of pressure overload–induced cardiac
hypertrophy, thereby contributing to the progression of
compensated cardiac hypertrophy to heart
failure.8 However, it is not resolved whether a
local activation of cardiac RAS or pressure overload could increase
local sympathetic activation. Transgenic rats harboring the mouse renin
gene Ren-2d [TG(mREN2)27] overexpress the
transgene in several tissues and develop fulminant
hypertension.9 A reduction of
ß1-adrenergic receptors and an increase of
Gi proteins have been observed before in
TG(mREN2)27.10 The present study
investigated whether pressure overload imposed on the left ventricles
directly augments sympathetic neuroeffector defects by altering local
concentrations and uptake mechanisms of norepinephrine.
ß-Adrenergic signal transduction alterations in the hypertrophied and
overloaded left ventricles and in the nonhypertrophied right ventricles
of TG(mREN2)27 were analyzed. Gene expression of
uptake1 carrier molecules was studied in
sympathetic cervical ganglia. |
Methods |
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Transgenic Animals
Transgenic animals [TG(mREN2)27] were housed and bred in the animal laboratory of the Max Delbrück Centrum in Berlin, Germany. At the age of 5 weeks, animals were transferred to the animal laboratory of the Klinikum Großhadern, University of Munich (Germany). Sprague-Dawley (SD) control rats were obtained from the Laboratorium für Versuchstierkunde in Hannover (Germany). These were the animals into which the transgene was originally introduced. Animals were housed according to the guidelines of animal care of the University of Munich. Only male rats were used. The animals were maintained on a standard laboratory animal diet (Altromin) and tap water ad libitum. They were exposed to alternating 12-hour dark and light cycles at 20°C to 22°C. The experiments were performed at age 12 to 14 weeks. At this age, hypertension is fully established [ie, TG(mREN2)27 230±8 mm Hg versus SD 115±12 mm Hg]. Animals were killed by a blow on the head, and the hearts were rapidly removed. In TG(mREN2)27 compared with SD, there was a significant increase of absolute and relative heart weights [TG(mREN2)27: 1.82 0.03 g, 4.92 0.11 mg/g; SD: 1.42 0.03 g, 3.78 0.08 mg/g; P<0.001]. Hearts of TG(mREN2)27 exhibited concentric hypertrophy but no dilatation or any signs of heart failure such as excessive scarring. No signs of venous congestion were observed in any other organ. Hydroxyproline content and histological determination of scarring were similar in both groups and have been published recently.11
Preparation of Sympathetic Ganglia
The clavicle, pectoralis major as well as pectoralis minor
muscles, and the ribs were transsected along the ventral axillary line.
The ventral thoracic wall was removed, exposing the cervical
sympathetic trunk and the stellate ganglia, which were located beneath
the first and second ribs. Pre- and postganglionic nerve fibers were
dissected, and the ganglia were removed and immediately frozen in
liquid nitrogen.
Adenylyl Cyclase Determinations
Adenylyl cyclase activity was determined according to Salomon et
al12 with modifications as described
recently.
Membrane Preparation for Receptor and G-Protein
Determinations
Myocardial tissue was chilled in 30 mL ice-cold
homogenization buffer (10 mmol/L Tris-HCl,
1 mmol/L EDTA, 1 mmol/L DTT, pH 7.4). Connective tissue was
trimmed away, myocardial tissue was minced with scissors, and membranes
were prepared with a motor-driven glass-Teflon
homogenizer for 1 minute. Afterward, the membrane
preparation was homogenized by hand for 1 minute with a
glass-glass homogenizer. The homogenate was
spun at 484g (rotor, Beckman JA 20) for 10 minutes. The
supernatant was filtered through 2 layers of cheese cloth, diluted with
an equal volume of ice-cold 1 mol/L KCl, and stored on ice for 10
minutes. This suspension was centrifuged at
100 000g for 30 minutes. For radioligand
binding experiments, the pellet was resuspended in 50 volumes of
incubation buffer (50 mmol/L Tris-HCl, 10 mmol/L
MgCl2, pH 7.4) and homogenized for 1
minute with a glass-glass homogenizer. This suspension
was recentrifuged at 100 000g for 45 minutes. The
final pellet was resuspended in incubation buffer (50 volumes) and was
stored at -70°C. Storage did not alter the results.
Determination of ß-Adrenergic Receptors
Radioligand binding assays were performed in a total
volume of 250 µL incubation buffer (for composition, see above). The
incubation was carried out at 37°C for 60 minutes. All experiments
were performed in triplicate. Myocardial ß-adrenoceptors were studied
using 125I-cyanopindolol as previously
described.3
Determination of Norepinephrine Uptake1
Carrier Sites
Freshly prepared ventricular membranes (200 µg)
were incubated with 3H-nisoxetine (specific
activity, 83 Ci/mmol) in 50 mmol/L Tris-HCl (pH 7.5), 300
mmol/L NaCl, and 5 mmol/L KCl with or without 10 µmol/L
desipramine to determine nonspecific binding, in a total volume of 200
µL. Equilibrium was reached within 20 minutes at 24°C. An
incubation time of 30 minutes was chosen for all subsequent
experiments. Saturation binding assays were carried out at 6
concentrations of 3H-nisoxetine (0.1 to 12
nmol/L). Reactions were terminated by rapid filtration through
GF/C filters and washing with ice-cold buffer (50 mmol/L
Tris-HCl, pH 7.5).
Pertussis Toxin–Induced 32P-ADP Ribosylation
32P-ADP ribosylation of
Gi by pertussis toxin was performed for 12
hours at 4°C in a volume of 50 µL containing 100 mmol/L
Tris/HCl, pH 8.0, at 20°C, 25 mmol/L DTT, 2 mmol/L ATP,
1 mmol/L GTP, 50 nmol/L 32P-NAD (800
Ci/mmol), and 20 µg/mL pertussis toxin that had been
activated by incubation with 50 mmol/L DTT for 1 hour at
20°C before the labeling reaction, as described
earlier.3 10
Immunoblotting Techniques
Immunoblotting techniques were performed as
described before.10 The polyclonal antiserum (MB
1) was raised in rabbits against the C-terminal decapeptide of retinal
transducin (KENLKDCGLF) coupled to keyhole limpet hemocyanine. The
antiserum recognized Gi1 and
Gi2 but not Go or
Gi3 (not shown). Blots were stained with an
alkaline phosphatase–labeled goat anti-IgG antiserum.
RNA Preparation
Total RNA from frozen left ventricular tissue
samples was prepared according to the protocol of Chomczynski and
Sacchi.13 Between 50 and 100 µg of total RNA
were obtained from 150 mg tissue. The mean yield did not significantly
differ between myocardium from TG(mREN2)27 and
SD.
Northern Blotting Techniques
Total RNA (10 µg) was separated on a 6% formaldehyde/1.2%
agarose gel, blotted on nylon membranes (Schleicher and Schuell)
by overnight capillary blotting, and fixed by UV irradiation. The
techniques have been recently described by Flesch et
al.11 The membrane was hybridized with a 1.75-kb
cDNA fragment encoding for Gi2. Quantification
of the signals was performed by densitometric analysis using
the Image Quant Densitometric System (Molecular Dynamics).
Reverse Transcription–Polymerase Chain Reaction
Total RNA from stellate ganglia was extracted by a modification
of the method of Chomczynski and Sacchi using the single-step method of
RNA isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction13 using the RNA-Clean Kit (AGS)
according to the manufacturer's instructions. Tissues were
homogenized in RNA-Clean with a glass-Teflon
homogenizer, followed by phenol-chloroform extraction
and 70% ethanol washing of precipitated RNA. cDNA was synthesized from
total RNA (1 µg) by mouse lymphoma virus–derived reverse
transcriptase (BRL) using a random hexamer primer
(Boehringer-Mannheim). After incubations at 42°C for
50 minutes, reverse transcription was terminated by heating at 95°C
for 5 minutes, and 2 µL from the resulting cDNA was removed for
subsequent polymerase chain reaction (PCR). These reactions were
performed in 10 mmol/L Tris-HCl, pH 8.3; 50 mmol/L KCl;
1.5 mmol/L MgCl2; 0.001% (wt/vol) gelatin;
0.2 mmol/L dATP, dCTP, dGTP and dTTP; Taq DNA
polymerase (1.25 U, Boehringer-Mannheim); and 50 pmol of each
primer. Synthetic oligonucleotide primers used were
5'-CAA TGT TTG GCG TTT CCC CTA TC-3' and 5'-CGA CGA CCA TCA GAC AGA
GCA-3' for the uptake1 carrier according to
Ungerer et al14 and 5'-ACC ACA GTC CAT GCC ATC
AC-3' and 5'-TCC ACC ACC CTG TTG CTG TA-3' for GAPDH as an external
control; 34 cycles of 95°C for 45 seconds, 62°C for 45 seconds, and
72°C for 90 seconds followed by 7 minutes at 72°C were performed.
This yielded fragments of 502 bp for the neuronal uptake carrier and
513 bp for GAPDH. Under these conditions, the amplification was linear.
PCR products were separated on 1.5% (wt/vol) agarose gels and
transferred by capillary blotting onto nylon membranes (Hybond N,
Amersham Buchler). Southern blot hybridization was performed with the
hybridization buffer containing 50% formamide, 100 mg/mL salmon sperm
DNA, 6x SSC, and 0.5% SDS. 32P-labeled probes
were prepared by the random primer method using the Prime-It II kit
(Stratagene). The membranes were washed twice with 2x SSC at room
temperature and once with 2x SSC, 0.1% SDS at 50°C for 60 minutes.
Autoradiography was performed, and
autoradiograms were quantified by laser densitometry
(Image Quant). PCR amplification has been shown to be linear within a
range of 28 to 40 cycles (not shown).
Reconstitution of Myocardial Gs Into S49
Cyc- Membranes
Reconstitution assays were performed as described
previously.10
Norepinephrine Determinations
For norepinephrine measurements, tissue
samples were homogenized with a Polytron device in 0.1
mol/L Tris HCl at pH 7.4. After centrifugation
(10 000g, 30 minutes), norepinephrine was
extracted with alumina and determined by high-performance
liquid chromatography with electrochemical detection as
described by Beschi et al.15
Force of Contraction
Experiments were performed on electrically driven (1 Hz) rat
papillary muscles. Papillary muscles of uniform size from the left
ventricles of TG(mREN2)27 and control rats (diameter,
<1.0 mm; length, 3 to 6 mm) were dissected in aerated
bathing solution (for composition, see below) at room temperature. The
preparations were attached to a bipolar platinum stimulating electrode
and suspended individually in 75-mL glass tissue chambers for
recording of isometric contractions. The bathing solution was a
modified Tyrode's solution containing (mmol/L) NaCl 119.8, KCl 5.4,
CaCl2 1.8, MgCl2 1.05,
NaH2PO4 0.42,
NaHCO3 22.6, Na2EDTA 0.05,
ascorbic acid 0.28, and glucose 5.0. It was continuously gassed with
95% O2/5% CO2 and
maintained at 37°C (pH 7.4). Contraction force was measured with an
inductive force transducer (W. Fleck) attached to a Hellige Helco
Scriptor or Gould recorder. Each muscle was stretched to the length
at which contraction force was maximal. The resting force
(approximately 5 mN) was kept constant throughout the experiment.
Preparations were electrically paced at 1 Hz with rectangular pulses of
5 milliseconds in duration (Grass stimulator SD9). The voltage was
approximately 20% greater than threshold. All preparations were
allowed to equilibrate in drug-free bathing solution until complete
mechanical stabilization. Positive inotropic response to
norepinephrine was determined with cumulative
concentration-response curves.
Miscellaneous
Protein was determined according to Lowry et
al16 using bovine serum albumin as the
standard. SDS–polyacrylamide gel electrophoresis was performed
as described by Lämmli.17
Materials
Forskolin was donated by Hoechst AG (Frankfurt, Germany). GTP,
guanylylimidodiphosphate [Gpp(NH)p], ATP, creatine phosphate, and
creatine kinase were purchased from Boehringer-Mannheim;
isobutylmetylxanthine (IBMX) was from EGA-Chemie. The ligands
125I-Cyp and 3H-nisoxetine
were from Amersham Buchler. DTT was from Serva. Pertussis toxin was
from List Biological Laboratories. All other compounds used were of
analytical or best grade commercially available. Only deionized and
double distilled water was used throughout.
Statistics
The data shown are mean±SEM. Statistical significance was
estimated with ANOVA according to Wallenstein et
al.18 A value of P<0.05 was
considered significant. Kd values were
determined graphically in each individual experiment.
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Results |
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Heart Weights and Hemodynamic Changes
In TG(mREN2)27 rats, absolute and relative heart weights were significantly increased. The increase in total heart weight was due to an increase in relative and absolute weights of the left ventricles, whereas no significant differences were observed between the right ventricular weights of the 2 groups.
Adenylyl Cyclase Activity
Basal (-64%)–, Gpp(NH)p (-35%)–, and isoprenaline
(-55%)–stimulated adenylyl cyclase activities were depressed in left
ventricles but were unchanged in right ventricular
membranes from TG(mREN2)27 compared with SD (Figure 1
). The effect of forskolin was slightly
depressed in left ventricular membranes but unchanged in
the right ventricle of TG(mREN2)27.
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ß-Adrenergic Receptors
ß-Adrenoceptors were determined by radioligand
binding experiments using 125I-Cyp.
ß-Adrenergic receptors were reduced in right and left ventricles of
TG(mREN2)27 compared with controls (not shown). The
reduction was similar in both chambers (38±6 versus 19±4 fmol
125I-Cyp/mg protein, right ventricles,
P<0.01; 39±6 versus 24±6 fmol
125I-Cyp/mg protein, n=8 to 10, left ventricles,
P<0.01, not shown). The Kd
values did not differ (not shown).
Stimulatory G-Protein -Subunits
To study whether the function of stimulatory G-protein
-subunits (Gs) is impaired in
TG(mREN2)27, Gs proteins
solubilized from membranes were reconstituted into membranes from S49
cyc- mouse lymphoma cells, which genetically
lack endogenous Gs. After
reconstitution, isoprenaline- and Gpp(NH)p-stimulated adenylyl cyclase
activities were not different whether S49 cyc-
membranes were supplemented with Gs from
TG(mREN2)27 or SD (not shown).
Inhibitory G-Protein -Subunits
To study whether differential alterations of
inhibitory G-protein -subunits
(Gi) in the right and in the left ventricles
of TG(mREN2)27 could account for the differences in adenylyl
cyclase activity, the levels of Gi were
determined with pertussis toxin–catalyzed
32P-ADP ribosylation. An increase by 45% of
pertussis toxin substrates was observed in the left ventricles of
TG(mREN2)27, whereas no change was observed in right
ventricular preparations (Figure 2). We then asked whether
the upregulation of Gi occurs at the
pretranslational level. Steady-state mRNA levels were measured
by Northern blot hybridization experiments. Northern blots of total
mRNA with the cDNA fragment encoding for Gi2
hybridized to a single band at 2.4 kb. In left ventricles, the signal
intensity was increased by 40% compared with that of controls (Figure 3). In the right ventricles, no
differences were detected between TG(mREN2)27 and
controls.
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Myocardial Norepinephrine Concentrations
To study whether sympathetic activation is different in right and
left ventricles, myocardial norepinephrine concentrations
were studied in both chambers. Myocardial norepinephrine
levels were reduced by 73% in the left ventricles but by only 28% in
the right ventricles (Figure 4, top).
Taken together, as judged from the myocardial
norepinephrine concentrations, sympathetic activation
appears to be stronger in the pressure-overloaded left ventricles than
in the nonhypertrophied right ventricles. Corresponding to this,
circulating norepinephrine (33.4±5.6 [n=8] versus
25.9±4.7 [n=10] pmol/mL) and epinephrine (110.6±24.1
[n=8] versus 71.2±11.4 [n=10] pmol/mL) serum concentrations were
not significantly changed in TG(mREN2)27 compared with
controls.
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Norepinephrine Uptake1Carrier Sites
To investigate whether an alteration of myocardial
norepinephrine uptake1 carrier sites
could play a role in the differential alterations of
norepinephrine concentrations in the
ventricular chambers, densities of these carrier proteins
were determined by radioligand binding experiments using
the carrier-specific ligand 3H-nisoxetine.
Binding of 3H-nisoxetine was monophasic and
saturable (not shown). Figure 4
, bottom, summarizes the densities in
the right and left ventricles. The decline in
uptake1 carrier sites was 55% in left
ventricles, whereas the density was similar in right ventricles. To
study the mechanisms of the decline in norepinephrine
uptake1 sites, we investigated
uptake1 carrier mRNA by semiquantitative PCR in
the stellate ganglia. Figure 5 shows the
PCR product, which was expected given the primers used. Sequence
analyses revealed a 100% identity of the sequence of the PCR
product with the predicted sequence. No PCR product was
detected in the heart. Analyses showed that there was no
difference between TG(mREN2)27 and control rats (Figure 5).
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At this stage, the question remained whether a 55% decline of
norepinephrine uptake sites in left ventricles is
sufficient to have functional consequences. To address this issue, left
ventricular papillary muscle experiments were performed,
and the sensitivity for the positive inotropic effect of
norepinephrine was determined. The maximal positive
inotropic effect of norepinephrine was reduced, possibly
due to the ß-adrenergic desensitization in TG(mREN2)27
(Figure 6A). However, the effect of
norepinephrine was more potent in TG(mREN2)27,
indicating that the reuptake of norepinephrine from the
synaptic cleft was impaired (Figure 6B).
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Discussion |
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Force of contraction is regulated by norepinephrine that is released from sympathetic nerves on stimulation. The inactivation mechanism is mainly a reuptake of norepinephrine into presynaptic stores by uptake1 carrier proteins.19 In situations of neuroendocrine activation, such as in heart failure1 or hypertensive cardiac hypertrophy, the sympathetic drive is increased, producing a desensitization of adenylyl cyclase7 8 20 by a decline of ß1-adrenergic receptors2 8 and an increase of Gi
proteins.3 4 5 These
alterations have also been demonstrated in several models of
hypertensive heart disease.7 8 It is not
completely clear whether circulating catecholamine
concentrations or the increased release of norepinephrine
from the failing heart is responsible for the desensitization. A
decline in uptake1 carrier proteins has been
suggested to be of relevance6 because the
reduction of norepinephrine uptake sites has been reported
to increase the interstitial norepinephrine
concentrations.21 22 Indeed, tracer techniques
have shown that increases in interstitial
norepinephrine concentrations are negatively correlated
with the density of ß-adrenergic
receptors.23
Chamber-Specific Alterations in Myocardial Disease
In patients with dilated cardiomyopathy and
predominant left ventricular failure, ß-adrenergic
receptors are similarly downregulated in both chambers, but an increase
of immunodetectable Gi and a decrease in
guanine nucleotide–stimulated adenylyl cyclase activity
has been observed only in the left but not in the right
ventricles.5 Consistently, myocardial
norepinephrine and neuropeptide Y were more depleted in the
left than in the right ventricles.5 In patients
with primary pulmonary hypertension and isolated right
ventricular failure, ß-adrenoceptor downregulation,
neurotransmitter depletion, and adenylyl cyclase desensitization
occurred only in the right ventricles.5
Therefore, there might be local differences in the regulation of
sympathetic neuroeffector mechanisms. When left ventricular
heart failure is experimentally induced by destruction of the aortic
valve, the reduction of norepinephrine concentrations and
of ß-adrenoceptor downregulation is observed in the left ventricles
only.24 However, when norepinephrine
uptake is impaired by chemical denervation of the heart by treatment of
animals with 6-hydroxydopamine, ß-adrenergic
desensitization is enhanced and the chamber-specific alterations are
abolished.24 These observations emphasize that
norepinephrine-induced uptake mechanisms are critical for
ß-adrenergic desensitization in the postsynaptic membranes.
Furthermore, when heart failure is induced by systemic application of
adriamycin, the reduction of norepinephrine stores
and ß-adrenergic receptors occurs in both ventricles to a similar
extent.25 Thus, mechanical performance of
either ventricle can induce alterations of ß-adrenergic signal
transduction.
The present findings add one pathological condition to those in
which defective norepinephrine uptake plays a role, ie, in
the pressure-loaded ventricle in hypertensive cardiac
hypertrophy. Candidates that produce differential
alterations of presynaptic or postsynaptic sympathetic neuroeffector
mechanisms could be a locally different sympathetic
activation5 or direct stretch and
hemodynamic load–induced alterations of the respective
ventricle. In the present study, we have taken advantage of the
TG(mREN2)27 model, which at the studied age is characterized
by compensated cardiac hypertrophy.15
TG(mREN2)27 develop adenylyl cyclase desensitization due to
a downregulation of ß-adrenergic receptors and an increase of
Gi already in the stage of compensated cardiac
hypertrophy.10 Right
ventricular weights were unchanged, whereas there is a
marked increase in the left ventricular weights. This model
allows the study of the effect of pressure overload on
norepinephrine uptake carriers and adenylyl cyclase
desensitization in comparison to the nonloaded right ventricle.
Norepinephrine Concentrations in
TG(mREN2)27
In left ventricles from TG(mREN2)27, there was a
stronger decline of myocardial norepinephrine stores than
in the right ventricles. Although these measurements cannot
discriminate between the norepinephrine concentrations in
the presynaptic stores and the synaptic cleft, this observation points
toward an impairment of norepinephrine uptake or stronger
sympathetic activation in the pressure-overloaded left compared with
right ventricles. These findings are similar to the data in the right
and left ventricles from patients with predominant left
ventricular failure.5 In
TG(mREN2)27, the transgene is expressed in various tissues
in the presence of a suppressed plasma renin
activity.9 Angiotensin II facilitates
the release of norepinephrine from sympathetic nerve
terminals.19 Therefore, local formation of
angiotensin II by overexpression of the transgene could be
involved in sympathetic activation in the heart. The stronger
activation in the left ventricle can be explained by an additional
sympathetic stimulation due to mechanical overload. Interestingly,
norepinephrine plasma concentrations were not significantly
changed in TG(mREN2)27 compared with controls. This
indicates that the locally released norepinephrine is a
stronger determinant of postsynaptic signal transduction defects than
circulating plasma catechols. In support of this notion, a report
indicated a significant correlation of norepinephrine
spillover from the heart but not of plasma concentrations of
norepinephrine to the prognosis of patients with heart
failure.26
ß-Adrenergic Desensitization
If sympathetic activation is different in right and left
ventricles from TG(mREN2)27, one would suggest that
postsynaptic alterations of the ß-adrenergic receptor/adenylyl
cyclase system would also be different. The reduction of ß-adrenergic
receptors was similar in both right and left ventricular
tissue, even though the norepinephrine concentrations were
more strongly reduced in the left ventricles. The stimulation of
adenylyl cyclase by saturating concentrations of isoprenaline was
unchanged in the right but reduced in the left ventricles. The
preserved maximal effect in the right ventricle can be explained by the
well-documented presence of a receptor reserve in the rat
myocardium,2 which maintains the
maximal effect despite a certain degree of receptor downregulation. The
depression of the isoprenaline effect in left ventricular
membranes is due to the slightly more pronounced relation of
ß-adrenergic receptors and the identified postreceptor event, ie, an
increase of Gi in the left ventricle. However,
Gi was only increased on mRNA and protein
level in the left but not the right ventricles. After treatment of rats
with isoprenaline, the amount of Gi protein is
increased.27 In neonatal rat
cardiomyocytes, it has been shown that the increase of
Gi only occurs at high
norepinephrine concentrations and after prolonged exposure
to norepinephrine, whereas the decline of ß-adrenoceptors
is more sensitive to
norepinephrine.28 Therefore, the
increase of Gi mRNA, protein levels, and
Gi-related pertussis toxin substrates is most
likely mediated by the stronger increase of ß-adrenergic drive in the
left ventricles than in the right ventricles of
TG(mREN2)27.
Norepinephrine Uptake1Carrier Sites
Norepinephrine uptake is inversely related to the
number of ß-adrenergic receptors in a model of experimental heart
failure.23 Thus, alterations of uptake carrier
sites could contribute to the different degree of
norepinephrine depletion and ß-adrenergic desensitization
in right and left ventricles. There was a 55% decline of
norepinephrine uptake sites. The decline in uptake carriers
apparently is due to an increase in protein degradation, reduced
translation, or altered posttranslational processing rather than to a
reduced gene expression of the protein because gene expression was not
altered in stellate ganglia. The observed reduction of
uptake1 carrier sites is functionally relevant,
as demonstrated by the increased sensitivity of the hypertrophied
ventricles to the positive inotropic effect of
norepinephrine. The decline in
uptake1 sites and the left shift of the
concentration-response curve of norepinephrine corresponds
to the observations made previously in failing human
myocardium.6 Infusion of
norepinephrine into nondiseased dogs with a presumably
intact norepinephrine uptake did not reduce ß-adrenergic
receptor number even though norepinephrine plasma
concentrations were increased by a factor of
15.29 These findings emphasize a role for an
intact norepinephrine uptake mechanism to protect the heart
from ß-adrenergic desensitization. Evidence for a chamber-specific
regulation has been provided by data in rats on right heart failure due
to monocrotaline treatment. In these rats, uptake carrier sites were
reduced in the right but not left ventricles. In support of these data
are recent observations in rats with left ventricular
myocardial infarction. In the noninfarcted regions of the left
ventricles, but not in the right ventricles, isoprenaline- and
Gpp(NH)p-stimulated adenylyl cyclase activity was
reduced.30 This was associated with an increased
turnover of norepinephrine in left but not right
ventricles.31
Conclusions
Degree and mechanisms of ß-adrenergic desensitization in cardiac
hypertrophy due to pressure overload and in heart failure
can be different in both chambers, indicating that not only systemic
but also local control of cardiac contractility is
altered in these pathological conditions. The reduction of
uptake1 carrier sites appears to be
pathophysiologically relevant in altering
recapture of norepinephrine at sympathetic nerve endings,
thus potentiating or prolonging receptor activation and promoting
ß-adrenergic desensitization. It most likely is induced by pressure
overload of the left ventricles. Because the presynaptic alterations
are chamber-specific and occur already before the occurrence of overt
heart failure, they could contribute to the progression of left or
right ventricular dysfunction in various pathological
conditions such as heart failure, cor pulmonale, or
ventricular dysfunction in various valve diseases.
Presynaptic alterations of the sympathetic nerve terminals could
provide additional targets for the pharmacological treatment of these
disabling conditions.
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft. We thank Bodo Cremers for expert assistance.
Received December 19, 1997; first decision January 12, 1998; accepted July 7, 1998.
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References |
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