is aldosterone bad for the heart?

Is aldosterone bad for the heart?John W. Funder

Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria, Australia 3168

The recent clinical trials RALES and EPHESUS have

shown that mineralocorticoid receptor (MR) antagon-

ists added to standard of care substantially increase

survival and decrease hospitalization in patients with

heart failure. In both trials aldosterone levels and

sodium status were unremarkable. Most epithelial and

vascular smooth muscle cell MR are normally ‘pro-

tected’ by 11b-hydroxysteroid dehydrogenase, but are

nonetheless normally largely occupied but not acti-

vated by glucocorticoids. When intracellular NADH rises

in response to enzyme blockade or generation of reac-

tive oxygen species MR are activated by glucocorticoids,

mimicking the cardiovascular effects of inappropriate

aldosterone for salt status. This may represent a novel

face of MR activation in vascular inflammation and in

‘non-protected’ tissues, such as cardiomyocytes in

heart failure.

In September 1999, the results of the RALES (RandomizedAldactone Evaluation Study) trial were published [1].In this trial, patients with severe heart failure wererandomized into two groups, one receiving placebo and theother low dose spironolactone (Aldactone), both in additionto current standard of care. The results were impressive, tothe extent that the trial was halted just over half waythrough recruitment: the mineralocorticoid receptor (MR)antagonist spironolactone reduced mortality by 30%, andhospitalization by 35%. No contest: aldosterone is bad forthe heart.

From the vantage point of 2004, the answer has to bequalified. If the question is rephrased ‘Is inappropriate MRactivation bad for the heart?’ the answer is yes. If thequestion is ‘Can inappropriate aldosterone levels forsodium status activate coronary vascular MR?’, againthe answer is yes. Perhaps counterintuitively, what thispaper will argue is that many (most?) instances ofcardiovascular damage follow inappropriate activationof MR by normal levels of glucocorticoids in the context oftissue damage, rather than by aldosterone:salt imbalance.

Aldosterone can be bad for the heart

First, there is no question that inappropriate aldosteronefor salt status can produce massive coronary vascularand cardiac damage. Among the most graphic andilluminating of studies showing this are those ofRicardo Rocha and colleagues [2] (Figure 1). In thesestudies, rats were maintained on 0.9% NaCl solution asdrinking fluid for three weeks and infused withangiotensin II (Ang II). Over the course of the study,

blood pressure rose progressively, whether the ratswere intact, adrenalectomized or given the selectiveMR antagonist eplerenone in their chow, clear evidencefor an angiotensin-driven blood pressure response.

In contrast with the blood pressure response, however,the coronary vascular inflammatory response (Figure 1a)is aldosterone dependent. In eplerenone-treated (Figure 1b)or adrenalectomized animals (Figure 1c), the vascularand perivascular inflammation seen with angiotensininfusion alone is absent; administration of exogenousaldosterone to angiotensin-infused adrenalectomizedrats restores the marked inflammatory response(Figure 1d). These findings, incidentally, provide com-pelling evidence against pathophysiological roles formyocardial synthesis of aldosterone, which is reported tobe increased by both Ang II [3] and (paradoxically) saltadministration [4].

Although aldosterone might play a similar role inConn’s syndrome, and possibly in other forms ofhuman low renin hypertension, in most essential hyper-tensives aldosterone is neither raised nor particularlyinappropriate for salt status. Similarly, in heart failurealdosterone levels are commonly raised only in responseto diuretic therapy. In both circumstances, MR blockadeis clearly beneficial – in essential hypertension byimprovement in surrogate indices, such as bloodpressure, left ventricular hypertrophy and proteinuria[5], and in heart failure by reduction in morbidity andmortality [1,6].

Figure 1. Vascular damage and perivascular inflammatory response in angiotensin

II (Ang II)/salt rats is dependent on mineralocorticoid receptor (MR) activation.

(a) Ang II þ salt alone; (b) Ang II þ salt þ eplerenone; (c) Ang II þ salt þ adrena-

lectomy; (d) Ang II þ salt þ adrenalectomy þ aldosterone. Reproduced from [2].

(a)

(c)

(b)

(d)

Corresponding author: J.W. Funder ([email protected]).

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www.sciencedirect.com 1043-2760/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tem.2004.03.006

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MR and 11b-HSD2, the specificity-conferring enzyme in

aldosterone target tissues

To support the contention that under such circumstancesMR antagonists are blocking cortisol rather than aldo-sterone from activating MR we need to recapitulate the roleof the specificity-conferring enzyme 11b-hydroxysteroiddehydrogenase (11b-HSD2) in aldosterone action. Over20 years ago, it was found that rat MR had similar affinityfor aldosterone and physiological glucocorticoids, and werepresent at high levels in non-epithelial tissues such ashippocampus, in addition to classic aldosterone targettissues such as the kidney [7]. When the human MR wascloned in 1987 this was shown to be equally true [8], thusposingtwo questions.Thefirst ishow aldosteroneovercomesthe ,100-fold higher plasma free concentrations of gluco-corticoids to occupy and activate MR, and the second thepotential pathophysiology of always-occupied MR in non-epithelial cells, such as neurons and cardiomyocytes.

The first of these questions appeared to be resolved bythe finding that the enzyme 11b-HSD2, which convertscortisol to its receptor-inactive 11-keto congener cortisone,is expressed at high levels in classic aldosterone targettissues (kidneys, colon, sweat glands, salivary glands)[9,10]. Aldosterone has a signature aldehyde group at C18,rather than the inert methyl group in other C21 steroids.This very reactive aldehyde group cyclizes in solution withthe 11b-hydroxyl to form an 11,18 hemiacetal, thus pro-tecting the hydroxyl from enzymatic attack (Figure 2).In addition to classic mineralocorticoid target tissues,11b-HSD2 is also found in blood vessels [11], making thevascular smooth muscle cell a potential physiologicalaldosterone target tissue, just like epithelial cells. Theenzyme is not found in cardiomyocytes or most neurons; insuch tissues MR are essentially always occupied byglucocorticoid, given their high circulating levels andhigh affinity for MR; the physiology of such an always-occupied receptor has rarely been addressed, and to dateremains conjectural.

The limits of cortisol to cortisone conversion

Despite its high concentration (,3.5 £ 106 molecules percell) and its low Km (high affinity) for glucocorticoids, it

seemed inherently unlikely that 11b-HSD2 can totallyinactivate intracellular cortisol, levels of which are,100-fold higher than those of aldosterone [12]. In anexperiment to test this possibility, we found that 11b-HSD2‘debulked’ intracellular glucocorticoids, by converting,90% to receptor-inactive 11-keto congeners, which stillleaves intracellular glucocorticoid levels approximatelytenfold higher than those of aldosterone [13]. Thus,conversion of cortisol to cortisone in aldosterone targettissues adds an order of magnitude in terms of aldosteroneselectivity for MR, just as the tenfold higher extent ofplasma binding of glucocorticoids does. What it also shows,however, is that most MR in such tissues are normallyoccupied but not activated by the tenfold higher concen-trations of intracellular glucocorticoids. How can this be?

We know, for example, that if 11b-HSD2 is deficient, asin the syndrome of apparent mineralocorticoid excess, or isblocked by carbenoxolone administration or licorice abuse,cortisol activates epithelial MR. This is difficult to ascribeto higher intracellular glucocorticoid concentrations,already approximately ten times greater than those ofaldosterone – and so we must ask what else happens whenthe enzyme is deficient or blocked.

NAD, the forgotten cosubstrate for 11b-HSD2

What is often overlooked is that the cosubstrate for theenzyme is NAD (Figure 3). For every molecule of cortisolconverted to cortisone a molecule of NADH is generated.Although 11b-HSD2 is expressed in a minority of kidneycells (perhaps ,5%), its activity is such that renal venouslevels of cortisol are lower, and those of cortisone higher,than in arterial blood. The kidneys account for 20–25% ofcardiac output, so that within aldosterone target cells bothcortisone and NADH are generated at a very high rate.Under resting conditions, levels of intracellular NAD are,700 times those of NADH, which means that generationof NADH by 11b-HSD2 can produce major changes inNADH levels over baseline. The difference in baselineNAD:NADH levels might also account for the essentiallyunidirectional enzyme activity of 11b-HSD2 as a dehydro-genase for physiological glucocorticoids.

The hypothesis proposed in this paper to account for theactivation of ‘glucocorticoid-occupied-but-not-activated’MR, seen when 11b-HSD2 is deficient or blocked (Figure 4),is that the transcriptional activity of a glucocorticoid–MRcomplex is determined by the intracellular levels ofNADH. When NADH levels are relatively high (i.e. when11b-HSD2 is operating normally) NADH is high, and

Figure 2. The enzyme 11b-hydroxysteroid dehydrogenase (11b–HSD2) converts

cortisol to receptor-inactive cortisone; aldosterone is not similarly metabolized

because its C11-OH is protected by cyclization with the signature C18-CHO to give

an 11,18 hemiacetal.

TRENDS in Endocrinology & Metabolism

HOC O

O

OH

Cortisol

HC

HO

C O

O

Aldosterone

O

OC O

O

OH

CortisoneCH2OH CH2OH

CH2OH

11β-HSD2

Mineralocorticoidreceptor

Figure 3. NAD is a cosubstrate for 11b-hydroxysteroid dehydrogenase

(11b–HSD2), which generates relatively high levels of NADH in converting corti-

sol to cortisone.


Cortisol Cortisone

NADHNADHO

C O

O

OH OC O

O

OH

CH2OH CH2OH

11β-HSD2


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somehow inhibits glucocorticoid–MR complexes. A rangeof transcription factors has been shown to be redoxsensitive in other systems, and in particular the corepres-sor activity of CtBP (C-terminal-binding protein) has beenshown to be increased by NADH [14,15]. The hypothesis,yet to be proved, is that similar or parallel effects of redoxstate affect coactivators/corepressors required for gluco-corticoid–MR complex activity; presumably, aldosterone-occupied MR are not similarly constrained. In this context,the non-identical pattern of cofactor recruitment by MRoccupied by aldosterone or cortisol, recently reported byKitagawa et al. [16], may be relevant.

Activation of MR by glucocorticoids: experimental

studies

There are several studies that are best explained by such ahypothesis. In one of these [17], the classic coronaryvascular and perivascular inflammatory response seenwith mineralocorticoid/salt administration (Figure 5) wasreproduced by administration of the 11b-HSD2 blockercarbenoxolone. That this proinflammatory effect was MRmediated is shown by the ability of the selective MRantagonist eplerenone to completely block the carbenox-olone effect. Our interpretation of these data are that when11b-HSD is blocked in vascular smooth muscle cells thenormally inactive glucocorticoid–MR complexes are acti-vated, and set in train the responses seen when MR areactivated by mineralocorticoids. In these studies, theanimals received 0.9% NaCl to drink; whether this isrequired for MR activation by glucocorticoids has yet to be

determined, although it is probably unlikely given thesyndrome of apparent mineralocorticoid excess.

A second, and potentially much more important,mechanism of activating glucocorticoid–MR complexes isby alterations in intracellular redox state as a result ofgeneration of reactive oxygen species. This possibility issupported by the results of another in vivo study on theeffects of MR blockade by eplerenone, in this instance onthe inflammatory and vascular remodeling response toacute coronary angioplasty in pigs [18]. Four weeks aftersuch a procedure, eplerenone preserved the vascularluminal diameter, by blocking the post-traumatic vascularfibrotic response (Figure 6). In this study, however, theanimals were given neither mineralocorticoid nor salt;what the eplerenone would appear to be blocking isglucocorticoid–MR complexes in the vessel wall. In thisinstance, lowered NADH levels in the context of tissuedamage rather than 11b-HSD2 blockade appear to be thetrigger for activation of glucocorticoid–MR complexes.

A role for redox activation of non-epithelial MR–

glucocorticoid complexes?

If glucocorticoid MR activation is redox dependent, theimplications might extend even beyond the role of normallevels of glucocorticoids – rather than raised levels ofaldosterone – in vascular pathology. As noted earlier,11b-HSD2 is not present in tissues such as cardiomyo-cytes, and the much higher affinity of physiological gluco-corticoids for MR than for glucocorticoid receptors meansthat ‘unprotected’ MR are essentially always occupiedby glucocorticoids across the range of diurnal variation(Figure 7). If this is the case, it is difficult to see how suchalways-occupied MR can signal changes in glucocorticoidsignal levels.

What they might signal, however, is changes in cellularredox state. From both in vitro [19] and in vivo [20] studieson cardiomyocytes, glucocorticoid occupancy under normalconditions appears to be tonic inhibitory in nature, block-ing rather than mimicking the effects of aldosterone oncardiomyocyte MR. Very recently, preliminary studies(A.S. Mihailidou et al. Mineralocorticoid receptors incardiovascular disease, 25th Annual Scientific Meeting

Figure 4. Blockade of 11b-hydroxysteroid dehydrogenase (11b–HSD2), or its

congenital deficiency, marginally increases mineralocorticoid receptor (MR)

occupancy by cortisol, but precipitously reduces NADH generation.


Cortisol Cortisone

HOC O

O

OH OC O

O

OH

CH2OH CH2OHNADHNAD

11β-HSD2

Figure 5. Concentrations of the vascular inflammatory markers ED-1, cyclooxygen-

ase-2 (COX-2) and osteopontin (OP) are equally increased by the mineralocorticoid

deoxycorticosterone (DOC) and the 11b-hydroxysteroid dehydrogenase

(11b–HSD2) inhibitor carbenoxolone (CBX), with CBX effect blocked by eplerenone

(EPL). *P , 0.0 versus control (CON) and CBX þ EPL. Reproduced with permission

from [17]. q 2003. The Endocrine Society.

*

0

1

4ED-1

2

*

*

1

COX-2

2 * *

1

3

OP

2

*

CO

N

DO

C

CB

X

CB

X +

EP

L

CO

N

DO

C

CB

X

CB

X +

EP

L

CO

N

DO

C

CB

X

CB

X +

EP

LSco

re fo

r po

sitiv

e st

aini

ng

Figure 6. In the context of tissue damage (without inappropriate aldosterone/salt

or increased glucocorticoid levels), eplerenone reduces constrictive vascular remo-

deling after experimental coronary angioplasty in pigs. On the left, eplerenone

preserves the luminal area compared with placebo (*P , 0.05), with no significant

change in neointimal area (right). Adapted with permission from [18].

*

0

2

4

0

2

4

Lum

inal

are

a(×

106

µm2 )

Intim

al a

rea

(×10

6 µm

2 )

Opinion TRENDS in Endocrinology and Metabolism Vol.15 No.4 May 2004 141



of the High Blood Pressure Research Council of Australia,Melbourne, December 2003) on rapid non-genomic steroideffects have shown cortisol to be a full aldosteroneantagonist in terms of Naþ/Kþ/2Cl- cotransport in rabbitcardiomyocytes; when the intracellular redox state isaltered, however, by addition of oxidized glutathione,cortisol becomes an MR agonist, mimicking rather thanblocking the effect of aldosterone. If glucocorticoid–MRcomplexes in cardiomyocytes can be activated by changesin redox state, such a receptor could not only act as asensor of cellular metabolic state, but also presumably as amediator of a portfolio of responses to such a stressor.Whether or not this is the case and, if so, the range ofgenomic and non-genomic responses to activated gluco-corticoid–MR complexes, remain to be determined.

References

1 Pitt, B. et al. (1999) The effect of spironolactone on morbidity andmortality in patients with severe heart failure. Randomized aldactoneevaluation study investigators. N. Engl. J. Med. 341, 709–717

2 Rocha, R. et al. (2002) Selective aldosterone blockade preventsangiotensin II/salt-induced vascular inflammation in the rat heart.Endocrinology 143, 4828–4836

3 Silvestre, J.S. et al. (1999) Activation of cardiac aldosterone production

in rat myocardial infarction: effect of angiotensin II receptor blockadeand role in cardiac fibrosis. Circulation 99, 2694–2701

4 Takeda, Y. et al. (2000) Sodium-induced cardiac aldosterone synthesiscauses cardiac hypertrophy. Endocrinology 141, 1901–1904

5 White, W.B. et al. (2003) Effects of the selective aldosterone blockereplerenone versus the calcium antagonist amlodipine in systolichypertension. Hypertension 41, 1021–1026

6 Pitt, B. et al. (2003) Eplerenone, a selective aldosterone blocker, inpatients with left ventricular dysfunction after myocardial infarction.N. Engl. J. Med. 348, 1309–1321

7 Krozowski, Z.S. and Funder, J.W. (1983) Renal mineralocorticoidreceptors and hippocampal corticosterone binding species haveidentical intrinsic steroid specificity. Proc. Natl. Acad. Sci. U. S. A.80, 6056–6060

8 Arriza, J.L. et al. (1987) Cloning of human mineralocorticoid receptorcomplementary DNA: structural and functional kinship with theglucocorticoid receptor. Science 237, 268–275

9 Funder, J.W. et al. (1988) Mineralocorticoid action: target-tissuespecificity is enzyme, not receptor, mediated. Science 242, 583–585

10 Edwards, C.R. et al. (1988) Localisation of 11 b-hydroxysteroiddehydrogenase – tissue specific protector of the mineralocorticoidreceptor. Lancet 2, 986–989

11 Funder, J.W. et al. (1989) Vascular type I aldosterone binding sitesare physiological mineralocorticoid receptors. Endocrinology 125,2224–2226

12 Funder, J.W. (1994) Enzymes and receptors: challenges and futuredirections. Steroids 59, 164–169

13 Funder, J.W. and Myles, K. (1996) Exclusion of corticosterone fromepithelial mineralocorticoid receptors is insufficient for selectivity ofaldosterone action: in vivo binding studies. Endocrinology 137,5264–5268

14 Fjeld, C.C. et al. (2003) Differential binding of NADþ and NADH allowsthe transcriptional corepressor carboxyl-terminal binding protein toserve as a metabolic sensor. Proc. Natl. Acad. Sci. U. S. A. 100,9202–9207

15 Zhang, Q. et al. (2002) Regulation of corepressor function by nuclearNADH. Science 295, 1895–1897

16 Kitagawa, H. et al. (2002) Ligand-selective potentiation of ratmineralocorticoid receptor activation function 1 by a CBP-containinghistone acetyltransferase complex. Mol. Cell. Biol. 22, 3698–3706

17 Young, M.J. et al. (2003) Early inflammatory responses in experi-mental cardiac hypertrophy and fibrosis: effects of 11bhydroxysteroiddehydrogenase inactivation. Endocrinology 144, 1121–1125

18 Ward, M.R. et al. (2001) Eplerenone suppresses constrictive remodel-ing and collagen accumulation after angioplasty in porcine coronaryarteries. Circulation 104, 467–472

19 Sato, A. and Funder, J.W. (1996) High glucose stimulates aldosterone-induced hypertrophy via type I mineralocorticoid receptors in neonatalrat cardiomyocytes. Endocrinology 137, 4145–4153

20 Qin, W. et al. (2003) A transgenic model of aldosterone-driven cardiachypertrophy and heart failure. Circ. Res. 93, 69–76

Figure 7. In tissues such as cardiomyocytes or most neurons, intracellular gluco-

corticoid levels are ,100-fold higher than those of aldosterone, so that mineralo-

corticoid receptors (MR) are essentially always occupied by cortisol. If such

always-occupied receptors are to function as a signal-detecting mechanism, the

variable signal needs to be other than plasma glucocorticoid levels.


Cortisol

Aldosterone

HOC O

O

OH

HC

HO

C O

O

O

Intracellular cortisollevels are~100× higher than those of aldosterone: MRthus always occupied (but notactivated) by cortisol

Mineralocorticoidreceptor

CH2OH

CH2OH

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is aldosterone bad for the heart?

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