Doi:10.1016/j.cardfail.2006.01.002

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 Ó 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.cardfail.2006.01.002 (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.
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