Journal of Cardiac Failure Vol. 12 No. 3 2006
Aldosterone Antagonism Improves Endothelial-Dependent
Vasorelaxation in Heart Failure via Upregulation of
Endothelial Nitric Oxide Synthase Production
HOANG M. THAI, MD, BAO Q. DO, MD, TRUNG D. TRAN, MD, MOHAMED A. GABALLA, PhD,
Background: Altering the renin-angiotensin aldosterone system improve mortality in heart failure (HF)in part through an improvement in nitric oxide (NO)-mediated endothelial function. This study examinedif spironolactone affects endothelial nitric oxide synthase (eNOS) and NO-mediated vasorelaxation in HF.
Methods and Results: Rats with HF after coronary artery ligation were treated with spironolactone for 4weeks. Rats with HF had a decrease (P ! .05) in left ventricular (LV) systolic pressure (130 6 7 versus118 6 6 mm Hg) and LV pressure with respect to time (9122 6 876 versus 4500 6 1971 mm Hg/second)with an increase in LV end-diastolic pressure (4 6 2 versus 23 6 8 mm Hg). Spironolactone did not affecthemodynamics but it improved (P ! .05) endothelial-dependent vasorelaxation at more than 1028 M ace-tylcholine that was abolished with NG-monomethyl-L-arginine. The eNOS levels were decreased (P !.05) in the LV and the aorta; spironolactone restored LV and aortic eNOs levels to normal.
Conclusion: Spironolactone prevents the decrease in eNOS in the LV and aorta and improves NO-dependent vasorelaxation, suggesting that one potential mechanism of spironolactone is an improvementin vasoreactivity mediated though an increase in NO.
Key Words: Endothelial-dependent vasorelaxation, nitric oxide, spironolactone.
Successful therapies aimed at reversing neurohormonal
Aldactone Evaluation Study trial demonstrated a reduction
activation of the renin-angiotensin-aldosterone system
in overall mortality in patients with HF treated with spirono-
(RAAS), such as angiotensin-converting enzyme (ACE) in-
lactone, an aldosterone receptor antagonist, in combination
hibitors and angiotensin receptor blockers (ARB), normalize
therapy with an ACE inhibitorThis is not surprising be-
endothelial dependent vasorelaxation, reduce symptoms, and
cause aldosterone has deleterious effects in patients with
improve survival in heart failure (HF).The Randomized
HF and has been reported to be persistently elevated afterACE inhibitor treatment.Some of these maladaptive effectscaused by aldosterone include the initiation of potassium andmagnesium excretion, which may result in increased arrhyth-
From the Section of Cardiology, Department of Medicine, Southern
mias and coronary vasoconstriction, and the promotion of
Arizona VA Health Care System, Sarver Heart Center, University ofArizona, Tucson, Arizona.
vascular and cardiac fibrosis via fibroblast stimulation.
Manuscript received June 8, 2005; revised manuscript received Decem-
Aldosterone is also been reported to have adverse effects
ber 29, 2005; revised manuscript accepted January 4, 2006.
on vascular endothelial function and has been shown to in-
Reprint requests: Hoang M. Thai, MD, Assistant Professor of Medicine,
Cardiology Section, 1-111C, Southern Arizona VA Health Care System
hibit nitric oxide (NO) in tissue culturA decrease in
Hospital, 3601 S. 6th Avenue, Tucson, AZ 85723.
NO-mediated endothelial vasorelaxation is believed to be re-
Supported in part by grants from the Department of Veterans Affairs, the
sponsible, at least in part, for the increase in vascular tone
American Heart Association, the WARMER Foundation, the HansjorgWyss Foundation, and the Biomedical Research and Education Foundation
seen in HFBased on these potential adverse effects of
aldosterone on endothelial function, this study was designed
to determine if spironolactone alters endothelial nitric oxide
2006 Elsevier Inc. All rights reserved.
(eNOS) production and NO-mediated vasorelaxation in HF.
methods. Phasic aortic pressure was measured, and the electronicmean was determined after withdrawal of the LV catheter into the
Adult male Sprague-Dawley rats (8–10 weeks old) were sub-
jected to myocardial infarction by coronary artery ligation and
Vasorelaxation Response of Thoracic Aortic Segments
randomized to placebo or treatment with spironolactone, givenorally for 4 weeks in the drinking water at a dose of 7 mgkgday.
The vasorelaxation response of thoracic aortic segments was ex-
All rats were fed with standard rat chow, given water ad libitum,
amined using standard techniques used in our laboratory. Briefly,
and housed in a single room of the animal facility with a 12-hour
a 3.0- to 3.5-mm section of the ascending thoracic aorta was
light/dark cycle and independent ventilation, temperature, and hu-
mounted on a ring apparatus attached to a force transducer. The
midity control. The study was terminated after 10 rats in each of 3
artery segment was attached to stainless steel wire stirrups with
groups (sham rats without treatment, HF rats without treatment,
one wire fixed in place and the other attached to the transducer.
and HF rats treated with spironolactone) were randomized and
The tissue was suspended in 37C bath of Krebs-Henseleit solu-
successfully studied. Physiologic experiments performed in the
tion suffused with 95% oxygen and 5% carbon dioxide. Rings
study animals included measurements of hemodynamic variables
were stretched to a resting tension of 1 gram and allowed to equil-
and quantification of the vasorelaxation response of the thoracic
ibrate for 45 minutes. Rings were precontracted with 60 mM of
aortic cross sectional rings to acetylcholine (ACh). Left ventricu-
KCl for 30 minutes and then returned to Krebs-Henseleit solution
lar and aortic tissue was analyzed for eNOS levels. The experi-
and allowed to equilibrate again for 45 minutes. Rings were con-
stricted with phenylephrine (3 mM) until a steady-state constric-
Accreditation of Laboratory Animal Care accredited facility
tion was obtained. Dose-response studies were performed with
with approval from the animal use committees of the Southern
increasing concentrations of ACh (1029 to 1024 M) and the result-
Arizona Veteran’s Health Care System and the University of
eNOS protein levels are measured using standard immunoblot
Myocardial infarction (MI) was created using techniques stan-
techniques as described previously. Briefly, LV tissue and aortic
dard in our laboratoryBriefly, 3-month-old Sprague-Dawley
tissue were ground up with homogenization buffer (100 mM imid-
rats weighing 250–300 g were anesthetized with inactin and
azole buffer and dH2O) at a concentration 1 mL buffer/0.1 gram
a left thoracotomy was performed. The heart was expressed
tissue using a handheld tissue homogenizer (Polytron, Glen Mills,
from the thorax and a ligature was placed around the proximal
NJ). The homogenized tissue was centrifuged at 10,000g at 4C
left coronary artery. The heart was returned to the chest cavity
for 5 minutes and the supernatant was removed for analysis. In ad-
and the thorax was closed. Rats received acetaminophen (67
dition, 4 ascending thoracic aortic segments from each treatment
mg/mL) in drinking water as postoperative analgesia. One day af-
group were ground using liquid nitrogen using a mortar and pestle.
ter MI, rats were anesthetized with halothane and a 9-lead electro-
The ground tissue was mixed with 100 mL of homogenization
cardiogram performed. Rats with evidence of large MI were
buffer and centrifuged at 5000g at 4C for 5 minutes. The remain-
selected for study and randomized. Briefly, the presence of Q
ing supernatant was concentrated using a Minicon B15 concentra-
waves (O1 mV) in the limb leads (I or aVL) and the sum of
tor (Amicon). Protein concentration was determined using
the R waves in the precordial leads (!10 mV) were used as cri-
standard protein analysis with the Lowry method and linear re-
teria for large MI. Hemodynamically, rats with large MI and HF
gression (Sigma). The supernatant from the left ventricle and
had a LV end-diastolic pressure (EDP) O16 mm Hg. Our labora-
the concentrated supernatant from the thoracic aortic segments
tory has shown that rats selected in this fashion have large MI av-
were fractionated using 7% sodium dodecyl sulfate polyacryl-
eraging 40% of the left ventricle.Rats that had thoracotomy
amide gel electrophoresis. Proteins were then transferred to poly-
but did not have coronary artery ligation were designed as
vinylidene difluoride membranes. Membranes were blocked
sham-operated controls. In our laboratory, this procedure has
overnight at 20C in 5% nonfat dry milk in 1X phosphate buffer
a 40% mortality rate. In this study, there was no mortality in
solution with 0.1% Tween (PBS-T). The membranes were then
rats after randomization and no obvious changes in clinical pre-
washed with PBS-T and incubated with a mouse anti-eNOS immu-
sentations of the rats in the treatment groups.
noglobulin G antibody (1:500) (Transduction Laboratories) for1 hour. The eNOS was then detected by washing the membrane in
horseradish peroxidase–labeled rabbit anti-mouse immunoglobu-
Four weeks after randomization, rats were anesthetized with thi-
lin G secondary antibody (1:40,000) for 1 hour and exposing it
obutabarbital (100 mg/kg intraperitoneally). A 1- mm microman-
to x-ray film for 30 minutes. The levels of eNOS were measured
ometer-tipped catheter (Millar Instruments, Houston, TX) was
using a photophosphorimaging detection unit, which expressed the
inserted into the right carotid artery. The catheter was advanced
degree of exposure in intensity units.
into the aorta and then into the left ventricle under constant pres-sure monitoring. The zero pressure baselines were obtained by
placing the pressure sensor in 37C saline before measurements.
After a period of stabilization, LV and aortic blood pressure and
Data are expressed as mean 6 standard error (SE). In both the
velocity were recorded and digitized at a rate of 1000 Hz using
physiologic and biochemical measurements, the Student’s t-test
a PC equipped with analog-digital converter and customized soft-
was used to compare sham versus HF and spironolactone treated
ware. From these data, the LV pressure with respect to time (dP/
versus untreated HF rats with a significant P value defined as
dt), and LVEDP was measured according to previously described
Journal of Cardiac Failure Vol. 12 No. 3 April 2006
These eNOS levels in spironolactone-treated HF animalswere similar to noninfarcted sham animals. shows
representative immunoblots of aortic eNOS.
Compared with sham animals, HF animals had a 5-fold
increase in LVEDP, a 9% reduction in systolic blood pres-
sure, a 19% reduction in mean arterial blood pressure, anda 51% reduction in LV dP/dt. There were no changes in he-modynamics or heart weights in HF rats treated with spiro-
Our data showed a moderate decrease in both systolic
nolactone compared with untreated HF rats The
blood pressure and LV dP/dt in HF animals treated with
right ventricular weights increased in the HF rats. This is
spironolactone, but these hemodynamic changes were not
consistent with our previous work showing the increase in
accompanied by reduction in the elevated LVEDP. Despite
right ventricular weight, presumably because of passive
this, heart failure animals treated with spironolactone dem-
pulmonary hypertension in the HF rats.
onstrated a significant restoration of endothelial-dependentvasorelaxation at low to moderate concentrations of ACh.
The NO inhibitor L-NMMA blunted this improvement inendothelial-dependent vasorelaxation. The significant de-
The degree of vasorelaxation in aortic rings of HF ani-
crease of eNOS in the aorta suggests that the impaired en-
mals compared with sham animals in response to ACh
dothelial-dependent vasorelaxation is due to a lack of NO.
was significantly reduced (P ! .05). Treatment of HF ani-
This is confirmed by the restoration of endothelial-dependent
mals with spironolactone improved (P ! .05) endothelial-
vasorelaxation by spironolactone because it increases eNOS
dependent vasorelaxation at ACh concentrations greater
in the left ventricle and aorta. The importance of this process
10 . This effect of spironolactone was abolished
is demonstrated by the attenuation of improved endothelial-
dependent vasorelaxation in MI animals by L-NMMA. The
NMMA), an inhibitor of nitric oxide. To examine the max-
comparison of ACh-mediated vasorelaxation to the response
imal vasodilatory effects in our treated groups we compared
the vasorelaxation response at the highest ACh concentra-
spironolactone is a potent mediator of endothelial-dependent
tion of 1024 with that of an exogenous source of nitric ox-
vasorelaxation, it is not the only mechanism that influences
ide, sodium nitroprusside (1025 M). There was no
the arterial vascular smooth muscle reactivity in HF. This
difference in MI rats treated with spironolactone compared
concept is supported by our phenylephrine data showing
with sham treated with spironolactone (50.3 6 7.0% versus
that, although vasoconstriction is accentuated in MI animals
36.4 6 3.3%, P 5 .2). Interestingly, in MI rats, spironolac-
without treatment, reflecting an activated adrenergic milieu
tone decreased (P ! .05) the maximal vasoconstrictor re-
in heart failure, the vasoconstrictor response to phenyleph-
sponse to phenylephrine (0.1 mM) compared with sham
rine is attenuated in MI animals treated with spironolactone.
Last, from our data, it appears that spironolactone hasminimal effects as an afterload reducing agent.
There is neurohormonal activation of the RAAS in pa-
Rats with HF had reduced levels of eNOS protein in their
tients with heart failure. Therapies aimed at reversing neu-
LV myocardium (13.9 6 2.8 versus 36.2 6 9.5 intensity
rohormonal activation, such as ACE inhibitor and ARB,
units/mg of protein, P ! .05, ) and thoracic aortas
improve mortality and symptoms. Unfortunately, ACE in-
(25.8 6 3.6 versus 57.3 6 16.6 intensity units/mg, P 5
hibitor and ARB only suppress plasma aldosterone levels
.05, Treatment with spironolactone was associated
transiently. The use of spironolactone in patients with HF
with increased eNOS protein levels compared to untreated
was an attempt to suppress aldosterone more effectively.
HF animals in both LV myocardium (35.6 6 5 versus
This was accompanied by a reduction in overall HF mortal-
13.9 6 2.8 intensity units/mg, P ! .05) and thoracic aortas
ity in patients with refractory HF being treated with ACE
(39.6 6 4.6 versus 25.8 6 3.6 intensity units/mg, P 5 .02).
inhibitorThese improvements in survival outcome
Table 1. Left Ventricular, Aortic Pressures, and Heart Weights in Sham, Heart Failure Rats, and Heart Failure Rats
LV SP (mm Hg) MAP (mm Hg) LVEDP (mm Hg) LV dP/dt (mm Hg/s) LV weight (grams) RV weight (grams)
Values are mean 6 SE; n 5 10 in each group, except for n 5 8 in the LV and RV weights with spironolactone.
HF, heart failure; LV, left ventricular; LVEDP, left ventricular end-diastolic pressure; LV SP, left ventricular systolic blood pressure; MAP, mean arterial
Fig. 2. Changes in endothelial nitric oxide synthase (eNOS) pro-
tein levels. A typical Western blot for eNOS in aortic tissue
with concentrations of eNOS (intensity units/50 mg tissue) in theleft ventricle of rats in heart failure (HF), with and without spiro-
nolactone treatment. First lane, eNOS-positive control; secondlane, HF treated with spironolactone; third and fourth lanes, un-
treated HF; and fifth lane, untreated sham. Note the decrease ineNOS protein levels in untreated HF compared with sham. Spiro-
nolactone treatment restored eNOS level similar to sham control.
of NO is thought to be important. The evidence for
abnormal NO production in HF comes from studies demon-
Fig. 1. Acetylcholine (ACh)-mediated vasorelaxation response of
strating decreased aortic eNOS protein levels in animal
heart failure untreated (heart failure), heart failure treated with
models with HFas well as from data showing
spironolactone alone (heart failure 1 spironolactone), and sham.
decreased activity of the L-arginine-nitric oxide pathway
Data presented as mean 6 SE; n 5 10 in each group. *P ! .05
in patients with HFIn these studies, NO-mediated
vs heart failure and heart failure 1 spironolactone. sP ! .05 vs
physiologic effects are diminished. An observation that
the decrease in aortic eNOS may have clinical relevancecomes from studies where the serum of patients in HF
were also accompanied by other clinical benefits of aldoste-
has been shown to downregulate eNOS in umbilical vein
rone inhibition such as: an increase in natriuresis,a
decrease in circulating levels of atrial natriuretic peptide, B
The possibility that spironolactone may restore the NO-
type natriuretic peptide, and pro-collagen type III amino-
mediated endothelial dysfunction in HF was first raised in
terminal peptand an improvement in LV ejection
hypertensive patients, where spironolactone increased
fractions while decreasing heart rate variabiand
blood flow and decreased vascular resistThis was
supported by investigators demonstrating that aldosterone
The studies demonstrating spironolactone benefits in HF
antagonism in licorice-induced hypertensive rats restored
raise the possibility that aldosterone antagonism may also
endothelial vasorelaxation and blunted the decrease in vas-
have a significant role in reversing the endothelial dysfunc-
cular eNOS protein levels.This effect on vasorelaxation
tion seen in HF.This is intriguing because there is
appears to be central to spironolactone ability to restore ar-
clinical evidence that the NO-mediated vasorelaxation is
terial elasticity, independent of its diuretic effect.More
severely curtailed in HF.Animal studies using an is-
intriguing, there is evidence that the impaired NO-mediated
chemic animal model of HF corroborated the NO-mediated
endothelial dependent vasorelaxation seen in HF patients
endothelial dysfunction in both large-conduit vessels and
may be related to aldosterone level. Studies have demon-
smaller resistance vessels.Although the mechanisms re-
strated a significant decrease in vascular compliance that
sponsible for the NO-dependent endothelial dysfunction
is inversely proportional to plasma aldosterone levels.
in HF are still under investigation, decreased production
In addition, the aldosterone level in HF has been shownto correlate well to a reduction in NO and eNOS.Finally, in chronic HF patients treated with aldosterone
Table 2. eNOS Protein Levels in the LV and Thoracic Aorta
receptor blockers, forearm blood flow increased in response
in Sham, Heart Failure Rats, and Heart Failure Rats Treated
to ACh; this increase was reversed with L-NMMA, a com-
petitive NO inhibitor,suggesting that aldosterone inhibi-
tion may reverse the NO-mediated endothelial-dependent
vasorelaxation seen in HF. We designed our experimentsto address the control of vasodilation and eNOS regulation
via the direct measurements of ACh-mediated vasorelaxa-
tion of large conduit vessels as well as the quantificationof both myocardial and arterial eNOS levels in HF rats
Concentration of eNOS in the LV and thoracic aorta.
Values are mean 6 SE; n 5 10 in each group.
treated with spironolactone. In our study, we focused on
eNOS, endothelial nitric oxide synthase; HF, heart failure; LV, left
the mechanisms controlling endothelial function in HF.
The importance of the association between endothelial
*P ! .05 HF vs sham.
**P 5 .02 HF vs. HF 1 spironolactone.
dysfunction and mortality risk in HF has been recently
Journal of Cardiac Failure Vol. 12 No. 3 April 2006
emphasized by reports showing that endothelial dysfunction
we have shown that one potential mechanism of action of
can be used to predict mortality risk in patients with HF.
spironolactone appears to be an increase in eNOS in the ves-
In patients treated on high dose ACE-I, there are elevated
sel wall that leads to restoration of the impaired endothelial
aldosterone levels despite inhibition of vascular angiotensin
dependent vasorelaxation seen in HF.
converting Even with this ‘‘aldosterone escape,’’patients treated with ACE-I show an improved outcomesuggesting that while aldosterone level predicts mortality
that it is not the only clinical predictor in patients with heartfailure. In part because of this we did not measure
The authors would like to thank Howard Byrne, Maribeth
aldosterone levels in this study. Furthermore, while
Stansifer, and Nicholle Johnson, BS, for their contributions.
activation of cardiac renin-angiotensin system includingcardiac aldosterone production has been reported in the ratcoronary artery ligation model of heart failure,this
does not result in increases in circulating aldosteronelevels.We have to reconcile this with data showing that
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CURRICULUM VITAE Starkenborglaan 6, 2341 BM, Oegstgeest, The Netherlands Telephone d . [email protected] Birthplace Date of Birth Marital Status Nationality EDUCATION Elementary School High School University University Leiden, The Netherlands, 1978-1983 Specializations Biochemistry, Physical Chemistry, Immunology, During specializations 3 publication
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