(Circulation. 2000;101:682.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Antisense Inhibition of ß1-Adrenergic Receptor mRNA in a Single Dose Produces a Profound and Prolonged Reduction in High Blood Pressure in Spontaneously Hypertensive Rats
From the Department of Physiology, School of Medicine, University of Florida, Gainesville.
Correspondence to Dr M. Ian Phillips, Department of Physiology, School of Medicine, University of Florida, Box 100274, Gainesville, FL 32610. E-mail mip{at}phys.med.ufl.edu
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Abstract |
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Background—ß-Blockers are the first line of therapy for hypertension. However, they are associated with side effects because of central nervous system (CNS) effects and ß2-adrenergic antagonism. To overcome these problems and provide a long-term ß1-blockade, antisense oligonucleotides against rat ß1-adrenergic receptor (ß1-AR) mRNA (ß1-AS-ODN) were designed and tested for the ability to inhibit cardiac ß1-ARs as well as lower blood pressure in spontaneously hypertensive rats (SHRs).
Methods and Results—Radioligand binding assay showed that a single intravenous injection of ß1-AS-ODN delivered in cationic liposomes significantly decreased cardiac ß1-AR density by 30% to 50% for 18 days (P<0.01), with no effect on ß2-ARs. This was accompanied by marked attenuation of ß1-AR–mediated positive inotropic response in isolated perfused hearts in vitro (P<0.02) and in conscious SHRs monitored by telemetry in vivo (P<0.02). Furthermore, the blood pressure of SHRs was reduced for 20 days, with a 38 mm Hg maximum drop. Heart rate was not significantly decreased. Quantitative autoradiography was performed to assess ß1-AS-ODN effects on the CNS, which demonstrated no changes in ß1-ARs in brain, in contrast to a significant reduction in heart and kidney (P<0.05). For comparison with ß-blockers, the effects of atenolol on cardiovascular hemodynamics were examined, which lowered blood pressure for only 10 hours and elicited appreciable bradycardia in SHRs.
Conclusions—These results indicate that ß1-AS-ODN, a novel approach to specific ß1-blockade, has advantages over currently used ß-blockers in providing a profound and prolonged reduction in blood pressure without affecting heart rate, ß2-ARs, and the CNS. Diminished cardiac contractility resulting from less ß1-AR expression contributes to the antihypertensive effect.
Key Words: gene therapy • receptors, adrenergic, beta • hypertension • contractility
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Introduction |
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The sympathetic nervous system plays a crucial role in the regulation of blood pressure (BP), mainly through activating
- and ß-adrenergic receptors (ß1-ARs) in
the effector organs, including heart, kidney, and blood vessels.
Adrenergic-blocking agents, especially ß-blockers, are commonly used
in the treatment of hypertension, ischemic heart disease, and
arrhythmia (reviewed in Sproat and Lopez1 ).
However, ß-blockers cause several side effects, which are usually
associated with their central nervous system (CNS) reaction (eg, sleep
disturbance, depression, impotence, dizziness, and fatigue) and
ß2-adrenergic antagonistic activity
(eg, increase in peripheral vascular resistance, worsening
of asthma symptoms). In addition, because of their short half-life (3
to 10 hours), ß-blockers must be taken daily to be effective. Because
a cardiovascular disease such as hypertension is a
life-long disorder, longer-lasting treatment without side effects would
be desirable. Although the precise mechanism underlying the antihypertensive effects of ß-blockers remains unclear, it is generally accepted that they antagonize the ß1-AR activity in heart and kidney, decreasing cardiac output and plasma renin activity.1 We have designed a new approach to ß1-blockade that reduces the number of receptors. Antisense oligonucleotides (AS-ODN) or antisense DNA designed specifically against ß1-ARs might represent a new class of ß-blockers. Antisense technique, through a number of mechanisms,2 can effectively downregulate the expression of target proteins. Clinical trials using antisense in targeting AIDS,3 cancer,4 and other genetic and acquired diseases5 indicate their potential clinical usefulness. The antisense approach has several potential advantages over ß-blockers. First, the specificity of AS-ODNs is based on DNA sequence. Second, AS-ODNs do not have direct CNS effects, because of the negligible transport of these highly polar molecules through the blood-brain barrier.6 Third, antisense elements tested in different systems produce long-term effects after single treatment.7 This prolonged effect can be attributed to 2 features of AS-ODN. One is the extended half-life of chemically modified ODN. The half-life of 15- to 20-mer phosphorothioated ODN is 20 to 50 hours in rats and mice after intravenous injection.8 9 The other is associated with the nature of antisense inhibition, which provides a delayed yet prolonged blockade of target proteins distinct from the direct competitive antagonists currently available.
We hypothesize that ß1-antisense, through specific inhibition of ß1-AR expression, will decrease the functional sensitivity of ß1-AR–mediated responses in the face of sympathetic activation and thereby achieve an antihypertensive effect. In this study, we designed an AS-ODN complementary to rat ß1-AR mRNA and tested its ability to inhibit ß1-AR density and function in the heart and to reduce BP in spontaneously hypertensive rats.
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Methods |
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Antisense Design and Administration
AS-ODN and inverted ODN control were 15-mer and targeted to the AUG start codon of rat ß1-AR mRNA.10 The sequence of AS-ODN is 5'-CCGCGCCCATGCCGA-3', and the inverted ODN is 5'-AGCCGTACCCGCGCC-3'. This AS-ODN was chosen from 6 AS candidates targeted to different regions of ß1-AR mRNA on the basis of the intensity of cardiac ß1-AR inhibition and reduction of BP in SHRs. These oligonucleotides were modified by backbone phosphorothioation. ODNs delivered with cationic liposomes were injected into the tongue vein.
Preparation of Liposomes and ODN/Liposome Complex
The cationic lipid
1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) was mixed with
helper lipid L--dioleoyl phosphatidylethanolamine (DOPE,
Avanti Polar Lipids) at a 1:1 molar ratio, briefly sonicated, and
stored at 4°C until use. The average diameter of liposomes is 200 to
350 nm.11 ODN/liposome complex was prepared on the day of
use by mixing the desired amounts of ODNs with DOTAP/DOPE to the final
DNA concentration of 300 µg/mL in 5% (wt/vol) dextrose in water and
incubating at room temperature for 60 minutes. Two DNA/lipid molar
ratios, ie, 1:0.5 and 1:2.5, were used in the experiments.
Animal Surgery
Adult male SHRs (250 to 350 g, Harlan, Indianapolis,
Ind) were kept in cages in a room with a 12-hour light-dark
cycle. Animals were fed standard laboratory rat chow and tap water ad
libitum. All care and surgical conditions were approved by the
University of Florida Animal Care Committee.
Telemetric Sensor Implantation
Before implantation, the zero of each radiotransmitter
(TA11PA-C40, Data Sciences) was verified to be 
4 mm Hg. SHRs
were anesthetized with 100 mg/kg ketamine and 15 mg/kg
xylazine, and a midline abdominal incision was made. A fluid-filled
sensor catheter was then inserted into the right femoral artery, and
the tip of the catheter was in the abdominal aorta caudal to the renal
arteries. The rats with implants were allowed to recover for 1
week.
Jugular Vein Cannulation
One week after telemetric implantation, rats were
anesthetized, and a curved catheter made of PE 50 and vinyl
tubing was inserted into the jugular vein. The tubing was led under the
skin of the neck and exposed on the back to allow for drug infusion.
Rats were allowed to recover for 24 hours before experimentation. The
catheters were flushed with 100 U heparin every day to prevent
clogging.
Membrane Preparation and ß-AR Binding Assay
Four days after intravenous injection of saline
(n=6) or 1 mg/kg inverted ODN (n=6) or 2, 4, 10, or 18 days after
injection of 1 mg/kg ß1-AS-ODN (n=24), animals
were euthanized, and membranes were prepared from heart ventricles as
previously described.12 For saturation experiments, 100
µg membrane protein was incubated in triplicate with 6 concentrations
of [125I](-)-iodocyanopindolol (ICYP, NEN Life
Science, 6.25 to 100 pmol/L) in a total volume of 250 µL containing
50 mmol/L Tris-HCl (pH 7.4), 5 mmol/L
MgCl2 at 36°C for 60 minutes. The nonspecific
and ß2-AR–binding levels were determined in
the presence of 1 µmol/L (±)-alprenolol and 150 nmol/L
CGP20712A (RBI), respectively. Then the reaction mixture was passed
through Whatman GF/B glass fiber filter using a Brandel harvester, and
the bound radioactivity was counted for 1 minute.
Tissue Preparation and Quantitative Autoradiography
Four days after injection of 1 mg/kg
ß1-AS-ODN (n=6) or saline (n=6), rats were
killed, and tissues were removed and frozen in dry ice. Coronal
sections of brain, horizontal sections of heart, and sagittal sections
of kidney (20 µm) were cut on a cryostat (Microm) at -20°C
and mounted on microscope slides. Every seventh slide was stained with
hematoxylin and eosin for histology. Tissue sections were preincubated
in Krebs buffer (mmol/L: NaCl 118.4, KCl 4.7,
MgSO4 1.2, CaCl2 1.27, and
NaH2PO4 10.0, pH 7.1)
containing 0.1 mmol/L GTP, 0.1 mmol/L ascorbic acid, and
10 µmol/L PMSF for 30 minutes at 25°C. Sections were then
incubated in Krebs buffer containing 0.1 mmol/L ascorbic acid and
10 µmol/L PMSF with 100 pmol/L ICYP at 25°C for 150 minutes in
the presence of 1 µmol/L (-)-propranolol, 100
nmol/L ICI 118,551 (ß2-selective
antagonist), or 100 nmol/L CGP20712A
(ß1-selective antagonist) to
distinguish nonspecific, ß1-, and
ß2-bindings. Labeled sections were rinsed in
the same buffer, followed by two 15-minute washes at 37°C in the
buffer, and rinsed in distilled water at 25°C.13 Dried
sections were then exposed to x-ray films. The images were
quantified with a computerized image analysis system
(MCID, Imaging Research) and normalized with 125I
standards. Nonspecific binding was <10% of total binding.
Determination of Effects of ß1-AS and Atenolol on
Cardiovascular Parameters in Response
to ß-Stimulation
Langendorff Heart Perfusion
Forty-eight hours after injection of 1 mg/kg
ß1-AS-ODN (n=9) or inverted ODN (n=6), SHRs
were anesthetized and killed. Hearts were quickly removed and
perfused via the aorta with oxygenated Krebs buffer
(118 mmol/L NaCl, 18.75 mmol/L NaHCO3,
1.2 mmol/L KH2PO4,
4.7 mmol/L KCl, 1.2 mmol/L MgSO4,
1.25 mmol/L CaCl2, 11.1 mmol/L glucose,
and 0.01 mmol/L EDTA) at a constant flow of 7.0 mL/min at 36°C.
Coronary perfusion pressure was measured via a catheter placed
proximal to the aorta and connected to a pressure transducer (Gould
Statham P23ID). A latex balloon filled with water and connected to the
pressure transducer was inserted into the left ventricle through the
left atrium to measure left ventricular
end-diastolic pressure (LVEDP), left
ventricular systolic pressure (LVSP), and developed
left ventricular pressure (dLVP) (dLVP=LVSP-LVEDP). LVEDP
during equilibration was set at 5 to 7 mm Hg. Coronary
perfusion pressure, LVEDP, and LVSP were recorded continuously on a
4-channel recorder (Astro-Med). After baseline values for dLVP and
heart rate (HR) were stable for 5 minutes, isoproterenol (ISO,
nonspecific ß-agonist) was given at 0.01, 0.025, 0.05, and 0.12
µmol/L at 10-minute intervals so as to avoid the effect of
tachyphylaxis.
Telemetric Monitoring of Live Animals
The effects of ß1-AS-ODN and atenolol on
cardiac dP/dtmax, HR, and systolic blood
pressure (SBP) were compared in the same group of SHRs (n=4). Two days
after catheterization of the jugular vein, control
values were taken and 1 mg/kg ß1-AS-ODN was
injected. Forty-eight hours later, rats were tested for the effect of
ß1-AS-ODN. The rats were allowed to recover
until all the cardiovascular parameters
returned to control values. Then 1 mg/kg atenolol
(ß1-selective antagonist) was
injected, and rats were tested 30 minutes later. For
ß1-stimulation, SHRs were infused with
dobutamine (ß1-selective agonist)
through a jugular vein catheter at 5, 10, 20, and 40 µg ·
kg-1 · min-1. Each
dose was given for 5 minutes continuously and at 1-hour intervals until
all the cardiovascular parameters returned
to baseline values so as to avoid the effect of tachyphylaxis. BP and
HR were sampled every 1 minute. dP/dtmax was
calculated from the slope of the rising pulse-pressure curve and
determined every 1 minute. The difference between values at each dose
and baseline was denoted as 
.
BP Monitoring
Telemetry
Each rat cage was placed on a receiver (RLA1020, Data Sciences)
for measurement of cardiovascular
parameters. Data were collected with a computer-based data
acquisition program (Dataquest LabPRO3.0; Data Sciences). BP and HR
were measured every 10 minutes and averaged every 1 to 24 hours. Before
treatment, SHRs were monitored for a week to get a stable
baseline.
Tail Cuff
Rats were warmed for 20 to 30 minutes in cages on heating pads.
The temperature was controlled at 35°C to 37°C. Then the rats were
placed in a plastic restrainer kept at 37°C. A pneumatic pulse sensor
was attached to the tail. After cuff inflation, SBP was determined as
the first pulsatile oscillation on the descending side of
the pressure curve. HR was determined by manual counting of pulse
numbers per unit time. BP and HR were recorded by a Narco
physiograph. Data values of each rat were taken as an average of at
least 4 stable readings. Baseline was determined by averaging 3 days of
measurements before antisense administration.
Statistical Analysis
Values were expressed as mean±SEM. The difference was
considered statistically significant at P<0.05. An unpaired
t test was used to compare Bmax, dLVP,
and BP in 2 groups. One-way repeated-measures ANOVA and Tukey test were
used to compare dP/dt and HR on dobutamine infusion
in different groups. Pearson product-moment correlation was used to
assess the relationship between ß1-AR
Bmax and dLVP.
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Results |
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Effect on Cardiac ß-AR Density
Figure 1A
shows that a single
intravenous injection of 1 mg/kg
ß1-AS-ODN delivered with cationic liposomes at
a 1:2.5 molar ratio significantly reduced the
ß1-AR density in the SHR hearts for 18 days
(P<0.01). The drop was at its maximum of 47% on day 4,
33% on day 10, and maintained at 29% on day 18. In contrast, there
was no significant change in the Bmax of
ß2-ARs. The KD of
both subtypes remained unaltered
(Table). Consequently, the
ß1/ß2 subtype ratio in
the ventricles was diminished from 
70/30 to 50/50 by
ß1-AS-ODN. Inverted ODN had no effect on either
subtype (Figure 1B).
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). B, Bmax of ß-ARs 4 days after
intravenous injection of saline (solid bar), 1 mg/kg
inverted ODN (open bar), or 1 mg/kg ß1-AS-ODN (shaded
bar). Data represent mean±SEM of each point (n=6).
*P<0.01, **P<0.001 vs saline
control.
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Effect on Cardiac Contractility and HR in Response
to ß-Stimulation
Forty-eight hours after injection of 1 mg/kg
ß1-AS-ODN, the cardiac inotropic and
chronotropic responses to ß-stimulation were determined in SHRs in
vitro and in vivo.
First, isolated hearts were perfused with incrementing doses of ISO,
which enhanced HR and contractility via activating
ß-ARs. The dLVP-ISO dose-response curve, which reflected the positive
inotropic effect of ISO, was significantly shifted downward by
ß1-AS-ODN (P<0.02). HR was not
significantly decreased, except at 1 point, ie, 0.01 µmol/L ISO
(P<0.05) (Figure 2).
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An in vivo test was performed in conscious SHRs monitored by
radiotelemetry. ß1-AS-ODN significantly
(P<0.02) dampened the increase in
dP/dtmax in the face of dobutamine
(ß1-selective agonist) (Figure 3A). The change in HR was not
significantly reduced (Figure 3B), which echoed the results in
isolated perfused hearts. Conversely, the response of BP to
dobutamine was biphasic (Figure 3C).
Dobutamine elevated SBP by 5 to 8 mm Hg at low
infusion speed and reduced SBP at higher speed, probably as a result of
the partial ß2-agonistic activity of
dobutamine at high doses and the consequent vasodilatory
effect on BP. Figure 3 also compares the results with
ß1-AS-ODN and atenolol
(ß1-selective antagonist).
Relative to ß1-AS-ODN, 1 mg/kg atenolol
produced a more profound decline in dP/dtmax
and HR during a 5-hour period of time after injection. Moreover, it
reduced basal dP/dtmax (2450±295 mm Hg/s
versus control, 2937±277 mm Hg/s) and caused bradycardia
(295±12 bpm versus control, 365±8 bpm) (P<0.05), whereas
ß1-AS did not change basal
contractility (2922±249 mm Hg/s) or HR (365±12
bpm). But the effects of atenolol on dP/dtmax
and HR were transient. Within 24 hours after atenolol
administration, the inotropic and chronotropic effects of
dobutamine had returned to control levels (data not
shown).
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). Rats were allowed to fully recover between
treatments. During dobutamine infusion, cardiac
dP/dtmax (A), HR (B), and SBP (C) of SHRs were monitored by
telemetry system. Data represent mean±SEM.
*P<0.05, **P<0.01 vs control.
Effect on BP of SHRs
Figure 4 shows the effects of a
single injection of 1 mg/kg ß1-AS-ODN on the BP
of SHRs measured by the tail-cuff method. AS-ODN was delivered with
cationic liposomes at different molar ratios of DNA/lipid, ie, 1:0.5
and 1:2.5. ß1-AS-ODN delivered with liposomes
at a 1:0.5 ratio diminished SBP for 8 days. The maximum drop was
38±5 mm Hg. When the molar ratio was increased to 1:2.5, this
hypotensive effect was drastically prolonged to 20 days. No effect was
seen with inverted ODN.
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) or 1:2.5 (•).
Inverted ODN (1 mg/kg) delivered with liposomes at molar ratio of 1:2.5
(
To compare the effects of ß1-AS-ODN and
atenolol, radiotelemetry was used to monitor BP and HR on a regular
basis. ß1-AS-ODN 1 mg/kg delivered with
liposomes at a 1:0.5 ratio produced a maximum drop of 15 mm Hg in
mean BP (Figure 5A). The antihypertensive
effect lasted for 8 days, which was consistent with the results
measured with the tail cuff. HR was not significantly altered (Figure 5B). In contrast to AS-ODN, although the onset of the
hypotensive effect caused by 1 mg/kg atenolol occurred as early as 20
minutes after injection, it lasted for only 10 hours (Figure 6A). In addition, atenolol caused
considerable bradycardia up to an average of -75 bpm (Figure 6B).
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Effect on ß-AR Distribution in Brain, Heart, and Kidney
Quantitative autoradiography of 6 to 18 tissue
slices in brain, heart, and kidney was analyzed 4 days after
intravenous ß1-AS-ODN
administration (Figure 7). No changes in
the distribution of ß-ARs in the forebrain and brain-stem regions
were detected. This indicated the absence of antisense effect on the
ß-AR expression in CNS. However, ß1-AS-ODN
significantly (P<0.05) reduced
ß1-AR density in cardiac ventricles (from
30.2±2.1 to 20.6±2.5 fmol/mg) and renal cortex (from 26.4±3.1 to
17.4±3.3 fmol/mg). This was consistent with the binding
results. ß2-ARs were not affected in any
tissues (data not shown).
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Discussion |
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This study was designed to compare the effects of a novel ß1-AS-ODN on high BP in a model of hypertension with a currently used ß-blocker. ß1-AS-ODN knocked down ß1-adrenergic activity, resulting in long-term attenuation of BP. The results indicated that ß1-AS-ODN reduced ß1-AR density and cardiac contractility after ß1-stimulation in vitro and in vivo and lowered high BP of SHRs. These findings are consistent with our hypothesis that ß1-AS, through the inhibition of ß1-AR expression, is able to render heart, kidney, and other tissues less sensitive to sympathetic activation, which is a major contributing factor in high BP. A single injection of ß1-AS-ODN effectively decreased the cardiac ß1-AR density, which was accompanied by diminished ventricular contractility and cardiac output in response to ß-stimulation. The antihypertensive effect in SHRs was up to a 38 mm Hg reduction lasting as long as 20 days.
Treatment of ß1-AS-ODN reduced cardiac
ß1-AR density by 50% in 4 days.
Considerable variation is reported in the literature on the half-life
of ß-ARs.12 14 15 From our results with the AS-ODN
inhibition, we deduce that the half-life of cardiac
ß1-ARs is 2 to 4 days to allow for 50%
reduction of ß-AR density within 4 days.
We noted that both tail-cuff and telemetry measures of BP showed a significant drop after antisense treatment. SHRs responded to ß1-AS-ODN to a greater degree of hypotension when subjected to tail cuff versus telemetry. In addition, the baseline measured with the tail cuff was consistently higher than that with telemetry by 20 to 30 mm Hg in the same rats. Bazil et al16 reported a similar phenomenon. They compared the cardiovascular parameters recorded by telemetry, tail cuff, and arterial catheter and observed a more sensitive hypotensive effect of captopril with tail cuff. Tail-cuff measurement of BP involves warming and restraint of rats. It is conceivable that ß1-AS, through the suppression of sympathetic activity, can decrease the BP of animals under stress more effectively.
Cationic liposomes are effective vehicles for gene delivery. It has been shown that liposome entrapment not only improves the cellular uptake of DNA but also protects DNA from degradation and extends its circulation time.17 18 Numerous factors influence the efficiency of cationic liposome-mediated intravenous gene delivery, such as DNA/lipid ratio, selection of lipids, and preparation procedure.18 19 We chose a widely used lipid formula, DOTAP/DOPE, to deliver ß1-AS-ODN at 4 molar ratios (data for ratios 1:1.5 and 1:3.5 are not shown). The optimal ratio of 1:2.5 was determined on the basis of the duration and magnitude of the antihypertensive effects. A profound and prolonged fall in BP of 38 mm Hg up to 20 days was achieved at this ratio, which followed the reduction in ß1-AR binding to a maximum of 47% at day 4, 33% at day 10, and 29% at day 18. This implies that ß1-AS-ODN effectively inhibits the functionally active receptors involved in the BP.
The blood-brain barrier formed by capillary endothelia is permeable only to small lipophilic molecules with a molecular weight of <600 Da (reviewed by Pardridge20 ). Owing to their high hydrophilicity, ODNs undergo negligible transport through blood-brain barrier and have very limited access to CNS.6 The cellular and organ distributions of DNA/liposome complexes with fluorescent labeling were previously studied in mice after intravenous injection, and the results indicated that the complexes were taken up primarily by capillary endothelial cells in most of the peripheral organs, including lung, heart, kidney, and spleen, but were absent in the brain.21 Although brain retention of liposomes after peripheral administration was observed in some cases, it was due to entrapment within the brain microvasculature.22 In our study, autoradiography in brain revealed no detectable changes in the expression and distribution of ß-ARs after intravenous ß1-AS-ODN injection. This provided further evidence that our antisense approach did not have CNS effects.
High specificity based on gene sequence has made antisense an increasingly useful tool in numerous studies and clinical trials. Its success is manifested by the recent approval of the first antisense drug, Vitravene, by the Food and Drug Administration. However, sequence-independent interactions have also been reported with AS-ODNs. High doses are usually responsible for the nonspecific effects,23 but another possible reason is that currently available databases do not cover every gene; thus, homology comparison by BLAST search may not guarantee sequence specificity of AS-ODNs. In our study, ß1-AS-ODN inhibited ß1-AR expression without changing ß2-ARs. Although we hypothesize that this is due to the specificity of the ß1-AS-ODN sequence, we recognize that other possibilities remain, such as indirect effects on regulatory mechanisms.
In patients with chronic heart failure, the severity of the disease closely relates to the decrease in cardiac ß-AR density and functional responsiveness.24 In SHRs treated with ß1-AS-ODN, we observed a marked attenuation of the ß1-AR–mediated positive inotropic response in vitro and in vivo, concurrent with the diminished cardiac ß1-AR level. Therefore, our results indicated a positive correlation between cardiac ß1-AR number and functional sensitivity (correlation coefficient >0.90, P<0.01).
Despite the large decrease in BP, no reflex tachycardia was observed after antisense treatment. However, the suppressive effect of ß1-AS-ODN on HR was less significant than its negative inotropic response. The reason for this is still under investigation. Several possibilities can be considered. First, ß1-AS-ODN did not affect ß2-ARs, which played an important role in the regulation of HR,25 26 although it was not involved in the cardiac contraction. Second, antisense inhibition of ß1-AR expression is gradual and less extensive than with ß-blockers. It is also possible that ß1-ARs may have a larger reserve for controlling HR than contractility. Finally, preliminary evidence in our laboratory suggests that cardiomyocytes preferentially take up AS-ODN, whereas pacemaker cells are less efficient (unpublished data).
The cardiovascular effects of ß1-AS-ODN were compared with those of a hydrophilic ß1-selective antagonist, atenolol. ß1-AS-ODN showed advantages over atenolol in reducing BP and maintaining normal HR. Although the onset of ß1-AS-ODN action was slower than with atenolol, it lasted much longer, 20 days compared with <1 day with atenolol. Furthermore, ß1-AS-ODN did not affect HR, whereas atenolol caused appreciable bradycardia. Bradycardia is a common complaint by patients taking this drug. Atenolol also reduced basal ventricular contractility and HR and thereby reduced resting cardiac output. ß1-AS-ODN is unlikely to alter resting cardiac performance. The results presented here suggest that ß1-AS-ODN may offer a significant improvement over currently used ß-blockers in both prolonged BP reduction and absence of effects on ß2-ARs and CNS.
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Acknowledgments |
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This study was supported by NIH Merit Award HL-27344 and American Heart Association Fellowships 9850002FL and 9704009. The authors thank Debbie Otero for technical assistance in binding assay and Fuxing Tang for liposome preparation.
Received May 28, 1999; revision received August 5, 1999; accepted August 13, 1999.
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