Download Heart Glucocorticoids Activate Cardiac Mineralocorticoid Receptors

Heart
Glucocorticoids Activate Cardiac Mineralocorticoid
Receptors During Experimental Myocardial Infarction
Anastasia S. Mihailidou, Thi Yen Loan Le, Mahidi Mardini, John W. Funder
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Abstract—Myocardial ischemia-reperfusion leads to significant changes in redox state, decreased postischemic functional
recovery, and cardiomyocyte apoptosis, with development and progression of heart failure. Ischemia-reperfusion in the
isolated perfused rat heart has been used as a model of heart failure. Clinically, mineralocorticoid receptor blockade in
heart failure decreases morbidity and mortality versus standard care alone. The effects of corticosteroids on infarct area
and apoptosis were determined in rat hearts subjected to 30 minutes of ischemia and 2.5 hours of reperfusion. Both
aldosterone and cortisol increased infarct area and apoptotic index, an effect half-maximal between 1 and 10 nM and
reversed by spironolactone. Dexamethasone and mifepristone aggravated infarct area and apoptotic index, similarly
reversed by spironolactone. Spironolactone alone reduced infarct area and apoptotic index below ischemia-reperfusion
alone, in hearts from both intact and adrenalectomized rats. The present study shows that cardiac damage is aggravated
by activation of mineralocorticoid receptors by aldosterone or cortisol or of glucocorticoid receptors by dexamethasone.
Mifepristone unexpectedly acted as a glucocorticoid receptor agonist, for which there are several precedents.
Spironolactone protected cardiomyocytes via inverse agonist activity at mineralocorticoid receptors, an effect near maximal
at a relatively low dose (10 nM). Spironolactone acts not merely by excluding corticosteroids from mineralocorticoid receptors
but as a protective inverse agonist at low concentration. Mineralocorticoid receptor antagonists may, thus, provide an
additional therapeutic advantage in unstable angina and acute myocardial infarction. (Hypertension. 2009;54:1306-1312.)
Key Words: myocardial infarction 䡲 cortisol 䡲 spironolactone 䡲 mineralocorticoid receptor 䡲 inverse agonist
C
ardiovascular disease is the leading cause of death
worldwide, with ischemic heart disease a major cause of
morbidity and mortality. Although thrombolytic therapy and
percutaneous coronary interventions reduce mortality from
acute myocardial infarction, additional therapeutic strategies
are needed, given that reperfusion of the ischemic myocardium generates oxygen free radicals1,2 and that impaired myocardial antioxidant defense capacity leads to tissue injury.3
Several clinical trials, the Randomized Aldactone Evaluation
Study4 and the Eplerenone Post-Acute Myocardial Infarction
Heart Failure Efficacy and Survival Study,5 showed decreased mortality with the use of mineralocorticoid receptor
antagonists in addition to standard therapy, evidence for a
role of mineralocorticoid receptor (MR) activation in myocardial infarction and heart failure. In the Eplerenone PostAcute Myocardial Infarction Heart Failure Efficacy and
Survival Study, 32% of the patients were diabetic, and
patients randomized early to treatment derived more benefit
than those randomized later.
Although it is commonly assumed that aldosterone is the
steroid responsible for nonepithelial MR activation in essential hypertension and cardiac failure, initial plasma aldoste-
rone levels in these clinical trials were in the low-normal
range. In contrast, circulating free levels of glucocorticoids
are ⱖ100-fold higher than aldosterone, and MRs have equal
affinity for aldosterone, cortisol, and corticosterone.6,7 Despite higher circulating levels of glucocorticoids, selective
activation of MR by aldosterone in epithelial tissue8,9 and
vascular smooth muscle cells10 is achieved by coexpression
of the enzyme 11␤-hydroxysteroid dehydrogenase type 2.
11␤-Hydroxysteroid dehydrogenase type 2 reduces the pyridine nicotinamide adenine dinucleotide to reduced
nicotinamide-adenine dinucleotide and converts endogenous
cortisol and corticosterone to their MR-inactive, 11-keto
congeners. The signature aldehyde (CHO) group at carbon 18
of aldosterone cyclizes with the ␤OH group at C11, forming
an enzyme-resistant 11,18-hemiketal. In contrast with vascular smooth muscle cells, 11␤HSD2 is not expressed in
cardiomyocytes,11 and, therefore, cardiomyocyte MRs are
normally occupied (but not activated) by even higher levels
of intracellular glucocorticoids. In “nonprotected” MRs, such
as in cardiomyocytes, endogenous glucocorticoids normally
do not mimic aldosterone but act as MR antagonists.12,13
There is increasing evidence that redox regulation is an
important determinant for the activity of transcription factors.
Received May 26, 2009; first decision June 24, 2009; revision accepted September 15, 2009.
From the Department of Cardiology (A.S.M., T.Y.L.L., M.M.), Royal North Shore Hospital, Sydney, New South Wales, Australia; Sydney Medical
School, University of Sydney (A.S.M., T.Y.L.L., M.M.), Sydney, New South Wales, Australia; Department of Cardiology (M.M.), Westmead Hospital,
Sydney, New South Wales, Australia; Prince Henrys Medical Research Institute (J.W.F.), Clayton, Victoria, Australia.
Correspondence to Anastasia S. Mihailidou, Department of Cardiology, Royal North Shore Hospital, Pacific Highway, St. Leonards, Sydney, NSW
2065, Australia. E-mail [email protected]
© 2009 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.109.136242
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Mihailidou et al
Infarct Size (% IA/AAR)
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Cardiac MR and Myocardial Infarction
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Methods
Male Sprague-Dawley rats (N⫽156; 8 to 12 weeks) were used, and
study protocols were approved by the Joint Royal North Shore
Hospital/University of Technology of Sydney Animal Care and
Ethics Committee. All of the experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of
Animals for Scientific Purposes. Rats were anesthetized with IM
ketamine (60 mg/kg) and xylazine hydrochloride (10 mg/kg) and
then received heparin (250 IU, IP) to prevent formation of microthrombi in the coronary circulation.18 The hearts were rapidly
Infarct Size (% IA/AAR)
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Cellular redox state has been demonstrated to modulate
glucocorticoid hormone action in vivo14 and oxidative stress
and reactive oxygen species generation to regulate expression
of estrogen receptors.15 For every molecule of cortisol converted to cortisone, 1 molecule of nicotinamide adenine
dinucleotide is reduced to reduced nicotinamide-adenine
dinucleotide. Because reduced nicotinamide-adenine dinucleotide has been shown in various systems to activate corepressors for other transcription factors,16,17 changes in redox state
during oxidative stress may drive cardiomyocyte and vascular
smooth muscle cell MR activation by glucocorticoids. We
have used a model of tissue damage with increased levels of
reactive oxygen species, the isolated reperfused rat heart, to
determine the conditions for activation of MRs by physiological glucocorticoids and to explore whether preadministration
of spironolactone may prevent the extent of infarct zone and
apoptosis.
40
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excised and cannulated via the aorta onto a Langendorff apparatus
and retrogradely perfused at a constant flow rate (3 to 4 mL/min)
with Krebs Henseleit-bicarbonate buffer solution, containing (in
mM): 118.0 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3,
2.5 CaCl2, and 5.5 glucose, equilibrated with 95% O2-5% CO2, to
achieve a pH of 7.4 at 35°C. Osmolarity was measured by the
freezing-point depression technique. Regional ischemia/reperfusion
was produced by placing a suture around a prominent branch of the
left coronary artery to occlude flow in the artery (please see the
online Data Supplement at http://hyper.ahajournals.org). Apoptosis
in cardiac tissue was measured in sections cut from paraffinembedded sections by TUNEL (Roche; please see the online
Data Supplement).
Experimental Protocols
After a stabilization period of 10 minutes, we used varying concentrations of steroids in the perfusates. The doses of steroids were
selected to range from physiological levels to supraphysiological
levels used in previous studies.19,20 Although corticosterone is the
physiological steroid in rats, we used cortisol because it has affinity
equal to that of aldosterone for MRs in the rat but substantially lower
affinity for glucocorticoid receptors (GRs) than in cortisol-secreting
species. We used cortisol (10 and 100 nM), aldosterone (1, 10, and
100 nM), dexamethasone (10 and 100 nM), the MR antagonist
spironolactone (10 nM and 1 ␮mol/L), or the GR/progesterone
receptor antagonist mifepristone (RU486; 100 nM and 1 ␮mol/L)
added to the perfusate either alone or in combination before inducing
ischemia and throughout the reperfusion period. The combinations of
the agonist:inhibitor dose ratios were chosen on the basis of the
known kilodalton values for MRs and GRs of these agents. In
separate studies, we added the low molecular weight and highly
(
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+ RU486
Cortisol 10 nM
Figure 1. Myocardial infarct size in hearts
subjected to 30 minutes of ischemia followed
by 2.5 hours during treatment with aldosterone at various concentrations alone and in
combination with other treatments. Top,
Representative cross-sections of the left ventricle stained with 2-3-5-triphenyl tetrazolium
chloride. The effect of the various treatments
on infarct size expressed as percentage of
area at risk (AAR) is shown in the lower
panel. I-R indicates ischemia-reperfusion
alone; Ald, aldosterone; SPIRO, spironolactone; 1 ␮mol/L, Tempol 100 ␮mol/L. Values
are mean⫾SE. *P⬍0.05 vs I-R alone.
10 nM Ald
Ald
60
+ Tempol
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+ SPIRO
+ Tempol
Cortisol 100 nM
Figure 2. Myocardial infarct size expressed as
percent of area at risk (AAR) during treatment
with various concentrations of cortisol and in
combination with other treatments, as indicated in the Methods section. I-R indicates
ischemia-reperfusion alone, RU486 100 nM;
SPIRO, spironolactone 1 ␮mol/L; Tempol,
100 ␮mol/L. Values are mean⫾SE; *P⬍0.05 vs
I-R alone.
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December 2009
Infarct Size (% IA/AAR)
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10 nM
10 nM 100 nM 100 nM
+SPIRO
Dexamethasone
1µM
1µM
+SPIRO
RU486
Figure 3. Effect of various concentrations of dexamethasone
and RU486 alone and in combination with spironolactone on
myocardial infarct size. I-R indicates ischemia-reperfusion alone;
SPIRO, spironolactone 1 ␮mol/L. Values are mean⫾SE;
*P⬍0.05 vs I-R alone; †P⬍0.05 vs RU486.
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permeable superoxide dismutase mimetic Tempol (4-hydroxy2,2,6,6-tetramethylpiperidine-N-oxyl; 100 ␮mol/L) to the perfusate
to scavenge the formation of reactive oxygen species on reperfusion.
To determine the role of endogenous glucocorticoids in our model,
rats were adrenalectomized ⱖ1 day before use and maintained on
standard chow and a 0.9% sodium chloride drinking solution.
Statistical Analysis
The Kolmogorov-Smirnov test confirmed that the data follow a
normal distribution, and, therefore, results are presented as the
mean⫾SEM. Statistical comparisons were performed by 1-way
ANOVA; unpaired Student t test was used to examine differences
between treatments for post hoc analysis, and the Holm-Sidak
correction was adopted to adjust for multiple comparisons. Differences were regarded as statistically significant when P⬍0.05.
Results
The addition of aldosterone to the perfusate increases infarct
size in a dose-dependent manner, with a half-maximal effect
between 1 and 10 nM (Figure 1). This effect is blocked by
spironolactone, interpreted as additional evidence that the
effect is mediated via MRs and also by Tempol, evidence for
the generation of reactive oxygen species in the action of
aldosterone. Figure 2 shows that the effect of aldosterone to
aggravate infarct size is mimicked by cortisol under conditions of tissue damage, again with a maximal effect at 10 nM,
well below the EC50 value for cortisol acting via GRs.
Consistent with a cortisol effect via MRs, RU486 100 nM did
not prevent the effect of cortisol 10 nM, and 1 ␮mol/L of
spironolactone blocked the response to 100 nM cortisol; as
for aldosterone, Tempol also blocked the cortisol effect.
To explore whether GR activation might also aggravate
infarct size in response to ischemia and reperfusion, we used
10 and 100 nM dexamethasone in the perfusate, with or
without spironolactone. The results were unanticipated; although dexamethasone increased infarct size at both concentrations, the increase was blunted by spironolactone down to
levels similar to control (Figure 3). As noted above, we
initially interpreted the lack of inhibitory effect of RU486 on
the cortisol response as ruling out an effect via GRs. Figure 3
shows that this interpretation is premature, in that RU486,
commonly considered a GR/progesterone receptor antagonist,
is equipotent with the classic glucocorticoid agonist dexamethasone in terms of increasing infarct size; addition of
spironolactone at equimolar concentrations again reversed
this effect down to control levels.
Because myocardial apoptosis plays a significant role in
postinfarction left ventricular remodeling, we examined the
impact of the various experimental treatments on apoptosis.
Figure 4A and 4B illustrate representative 4⬘,6-diamidino-2phenylindole dihydrochloride (DAPI) and TUNEL staining,
with effects of the various treatments on the apoptotic index
(percentage) shown in Figure 5A and 5B. Consistent with
previous reports, aldosterone (Figure 5A) significantly increased the number of apoptotic cells after infarction. The
proapoptotic effect of aldosterone is mimicked by cortisol
(Figure 5B), with low physiological levels of glucocorticoid
having a notable effect. Both spironolactone and Tempol
prevented the proapoptotic effects of aldosterone and cortisol,
and RU486 did not significantly alter the cortisol effect. The
unexpected effect of dexamethasone and RU486 to aggravate
myocardial infarct size presumably reflects a proapoptotic
effect of both these agents in the heart, as shown in Figure 6,
with the addition of spironolactone reducing the effects of
both to control levels.
In separate studies, we noted another unanticipated result
when spironolactone was perfused alone (Figure 7A). At
concentrations of both 10 nM and 1 ␮mol/L, spironolactone
significantly reduced infarct size below control, in the absence of the added steroid; the protective effect of spironolactone, therefore, is not merely that of blocking MR activation by aldosterone or cortisol in the context of tissue damage.
One possible explanation for this finding may be that, in the
control tissue, a proportion of MRs is reoccupied by endogenous glucocorticoids during the period of ischemia, with
such binding lessened by preincubation with spironolactone.
To explore this possibility, we used rats adrenalectomized 2
to 5 days before study, which again showed a larger infarct
Figure 4. Representative photomicrographs of left
ventricular tissue sections showing TUNEL staining. A, Localization of myocardial nuclei is shown
by DAPI staining bright blue fluorescence. B, Corresponding TUNEL staining (arrows) shown by red
fluorescence of the same section; apoptotic nuclei
(large arrows) correspond with DAPI nuclei present
in A. All of the images are ⫻200 magnification.
A DAPI
B TUNEL
MERGED
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F10 nM F10+RU486
+SPIRO +Tempol
Figure 5. Percentage of apoptotic myocytes during the various
treatments (A and B). Apoptotic index is expressed as the percentage of TUNEL-positive nuclei relative to total DAPI nuclei.
I-R indicates ischemia-reperfusion alone; Ald, aldosterone;
SPIRO, spironolactone 1 ␮mol/L; Tempol⫽100 ␮mol/L; F, cortisol; RU486⫽1 ␮mol/L. *P⬍0.05 vs I-R alone.
area postischemia and an increase in apoptotic index (Figure
7B). Spironolactone perfusion in hearts from adrenalectomized rats significantly reduced both infarct area and apoptosis, indicating that the protective effect of spironolactone
involves mechanism(s) other than competition with endogenous glucocorticoids for the MR.
The osmolarity of perfusates containing cortisol, aldosterone, or dexamethasone over a range of concentrations with
or without Tempol (1 ␮mol/L) and spironolactone (10 nM)
did not vary significantly from the control Krebs Henseleitbicarbonate buffer solution (data not shown). In contrast,
osmolarities of perfusates containing spironolactone
(1 ␮mol/L) alone or in combination with aldosterone, corti16
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10 nM 100 nM 100 nM
+SPIRO
Dexamethasone
1µM
1µM
+SPIRO
RU486
Figure 6. Effects of dexamethasone and RU486 on apoptotic
index. I-R indicates ischemia-reperfusion alone; SPIRO, spironolactone 1 ␮mol/L. Values are mean⫾SE; *P⬍0.05 vs I-R alone;
†P⬍0.05 vs RU486 1 ␮mol/L.
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Cardiac MR and Myocardial Infarction
Infarct Size (%) IA/AAR)
Mihailidou et al
Adrx Adrx +SPIRO
0.9% NaCl
Figure 7. Protective effect of spironolactone on infarct size (A)
and apoptotic index (B). Intact animals were untreated or
treated with 10 nM or 1000 nM of spironolactone (SPIRO 10 or
SPIRO 1000); adrenalectomized (Adrx) animals were treated
with SPIRO 1000. I-R indicates ischemia-reperfusion alone.
*P⬍0.05 vs I-R alone; †P⬍0.05 vs Adrx.
sol, or dexamethasone were significantly higher than the
control Krebs Henseleit-bicarbonate buffer (please see the
online Data Supplement). Similarly, perfusates containing
RU486 (1 ␮mol/L) alone or combined with spironolactone
were significantly higher than the control Krebs Henseleitbicarbonate buffer.
Discussion
The present studies confirm and extend previous reports
showing the efficacy of MR blockade in experimental myocardial infarction in vivo21 and in vitro.22,23 In the context of
tissue damage, there are 3 major findings of the present study
of interest. First, the glucocorticoid cortisol was shown at a
low dose to mimic the MR activation produced by the
physiological mineralocorticoid aldosterone. Second, activation of GRs by dexamethasone also produced an increase in
infarct area, an effect counterintuitively mimicked by the
GR/progesterone receptor antagonist RU486. Finally, spironolactone, in addition to blocking the effect of other
perfused steroids, had protective activity in its own right, in
the absence of an added agonist steroid.
There are well-documented adverse cardiovascular effects
of inappropriate levels of aldosterone commonly achieved in
vivo by a combination of salt loading and aldosterone (or
deoxycorticosterone) administration. In the present ex vivo
studies, additional sodium was not required, with inappropriate aldosterone effects presumably reflecting the absence of
glucocorticoid normally limiting MR occupancy by normal
levels of aldosterone. Patients with primary aldosteronism
have much higher rates of atrial fibrillation, stroke, and
myocardial infarction than age-, sex-, and blood pressure–
matched essential hypertensives.24 In the familial syndrome
of glucocorticoid-remediable aldosteronism, affected subjects
who are normotensive exhibit structural and functional car-
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December 2009
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diac changes compared with age-, sex-, and blood pressure–
matched normotensive controls.25 These studies testify to the
direct cardiotoxic effects of elevated aldosterone levels over
and above those of elevated blood pressure, per se. In the
setting of ST-elevation myocardial infarction, a strong association between plasma aldosterone levels (even within the
normal range) and risk of both early and late mortality has
been shown in heart failure,26 in further support of direct
cardiotoxic effects.
In the present study, aldosterone exacerbated cardiac damage caused by ischemia-reperfusion, in terms both of the
infarct area and the extent of apoptosis, with a half-maximal
effect between 1 and 10 nM, consistent with an effect at
physiological levels of aldosterone (in the rat). Our study is in
contrast to Chai et al,22 who found no significant effect of
aldosterone on extent of infarct area, and in agreement with
several previous studies, which have shown that aldosterone
promotes apoptosis.19,27,28 The effect of aldosterone was
mimicked by cortisol to aggravate myocardial infarct size and
apoptosis, again with an EC50 between 1 and 10 nM. This in
itself is evidence for a cortisol effect via MR, for which
cortisol has considerably higher affinity than for GRs; effects
of physiological steroids via GRs are commonly seen over a
dose range of 10 nM to 1 ␮mol/L. At high cortisol concentrations, cardiomyocyte apoptosis in the ischemia-reperfusion
model may be produced via both MR and GR activation,
given the agonist effects via GR of the more selective
glucocorticoid dexamethasone and of the MR-inactive synthetic ligand RU486. Both cortisol- and aldosterone-induced
cardiac damage were prevented by perfusion of the
membrane-permeable radical scavenger Tempol, indicating
that the steroid-induced cardiac damage depends on oxidative
stress, consistent with previous studies that have shown a
reduction in infarct size with Tempol alone.29
In previous studies, the effect of glucocorticoids during
experimental myocardial infarction has not been uniformly
consistent. In early studies,30,31 protective effects were reported, as well as in more recent studies,32 in which glucocorticoids were shown to attenuate myocardial apoptosis in
neonatal pigs. Similar attenuation of apoptosis by glucocorticoids has been shown in neonatal rat cardiomyocytes.33 In
contrast, Scheuer and Mifflin20 reported detrimental effects
using pharmacological doses of glucocorticoids and no effect
when lower levels were used. Several studies have used the
potent synthetic glucocorticoid dexamethasone in experimental ischemia-reperfusion34 and clinically as dexamethasoneeluting stents to reduce intimal hyperplasia, with mixed
reports for both beneficial outcomes34 –36 and higher restenosis rates in patients with diabetes mellitus.37 In the present
studies, dexamethasone reproducibly increased infarct zone
and apoptotic index at doses of ⱖ10 nM. As for cortisol and
aldosterone, this effect was mitigated by spironolactone,
leading to the initial, puzzling possibility of a dexamethasone
effect via MRs, an unlikely possibility given its GR selectivity and low affinity for MRs.
Our initial spironolactone studies, in which the MR antagonist blocked the effect of both aldosterone and cortisol, we
interpreted as supporting evidence for an action of both
corticosteroids via MRs. In subsequent studies, to be dis-
cussed below, spironolactone had protective effects via MRs
in the absence of corticosteroids. Similarly, in studies designed to exclude high-dose cortisol from GRs by the
addition of the progesterone/GR antagonist RU486, our initial
finding (of no blockade) we interpreted as evidence supporting an MR-mediated effect of cortisol. This interpretation
again had to be tempered by the finding that RU486 alone
exacerbated tissue damage. Therefore, the antagonist studies
proved uninformative in terms of through which receptor
cortisol acts to promote cardiomyocyte apoptosis, and assigning primary proapoptotic action via MRs rests solidly on the
dose-response characteristics of the cortisol effect: at higher
levels, cortisol action presumably aggravates apoptosis via
both MRs and GRs. The spironolactone/RU486 studies
proved illuminating in terms of the unanticipated agonist
effect of RU486 and the inverse agonist effect of spironolactone. We used inverse agonist in the sense of the ability of
spironolactone to activate MRs to produce the effect opposite
or the inverse of that seen with the natural corticosteroid
ligands. RU486 alone aggravated infarct zone and cardiomyocyte apoptosis. Because RU486 has high affinity for GRs
in addition to progesterone receptors and negligible affinity
for MRs, the observed effect might, thus, be mediated by
GRs. Given the demonstrated effects of dexamethasone, we
postulate an agonist effect of RU486 via GRs. Although
commonly RU486 is a GR antagonist, partial agonist activity
has been reported in several studies,38,39 consistent with the
effect found in the present study.
The third major finding of the present study is that
spironolactone not only prevented the deleterious effects of
aldosterone, cortisol, dexamethasone, and RU486 but that,
absent any other added steroid, it consistently reduces infarct
size and apoptosis below control levels. The reduction in
infarct size is comparable with that reported previously both
for spironolactone22 and eplerenone.23 In our studies, the
protective effect of spironolactone persists in adrenalectomized animals, evidence against a blocking effect on retained
or residual endogenous corticosteroids: it is also essentially
equally effective at 10 nM as 1 ␮mol/L in terms of cardioprotection. One formal possibility is that, under such conditions, the damage from ischemia-reperfusion is elevated by
ectopic production of aldosterone by the heart, so that
ischemia-reperfusion alone does not represent a truly basal
state. Although cardiac synthesis of aldosterone has had a
relatively long and disputed40 history, the most recent
reports from vigorously controlled studies on levels of
steroidogenic enzyme mRNA are widely regarded as
providing a definitive negative answer to the question of
cardiac aldosterone synthesis.41,42
This is strong evidence that the protective inverse agonist
effect of spironolactone is via MRs, for which spironolactone
has much higher affinity than for androgen, progesterone, or
GRs. In addition to the report already cited,20 there are cell
culture studies43,44 in which spironolactone alone produced
substantial changes, for example, in integrin expression in
neonatal cardiomyocytes. It also provides additional evidence
that spironolactone commonly acts as an MR antagonist not
merely by excluding aldosterone (or, in the context of tissue
damage, cortisol) from MR, but by occupying MR, and
Mihailidou et al
inducing expression of protective factors. Finally, and equally
important, this effect is seen at concentrations of spironolactone that would occupy only a relatively low percentage of
unprotected cardiomyocyte MRs, normally overwhelmingly
occupied by physiological glucocorticoids.45 This is consistent with, and a probable explanation for, the marked effects
of low-dose spironolactone in the clinical trials (26 m/d
average) of the Randomized Aldactone Evaluation Study4
and eplerenone (43 mg/d) in the Eplerenone Post-Acute
Myocardial Infarction Heart Failure Efficacy and Survival
Study.5
Cardiac MR and Myocardial Infarction
5.
6.
7.
8.
Perspectives
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We have shown that, in the context of tissue damage, normal
circulating levels of cortisol can aggravate tissue damage
during coronary artery occlusion and that maintenance treatment beforehand with spironolactone/acute administration of
spironolactone post hoc might reasonably be expected to
lessen the extent of tissue damage. The effect is (in endocrine
terms) a partial agonist/partial antagonist at cardiomyocyte
MRs in pharmacological terms on an inverse agonist and can
be seen at partial levels of receptor occupancy. These studies
underline the importance of the possible tissue-selective
effects of most “antagonists,” which are commonly partial
agonists/partial antagonists/inverse agonists. In addition, at
the clinical level, they support starting spironolactone or
eplerenone early after myocardial infarction to limit tissue
damage: analysis of the Eplerenone Post-Acute Myocardial
Infarction Heart Failure Efficacy and Survival Study data
showed a 58% reduction in sudden death from cardiac causes
within 30 days of receiving eplerenone. In addition, they
suggest the need for studies on their use in patients referred
for primary percutaneous coronary intervention or STelevation myocardial infarction. A final possibility is the
development of drug-eluting stents in which the agent included is an MR inverse agonist, able even at low dose to
produce marked protective effects after ischemia-reperfusion
injury.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Sources of Funding
This study was supported by a project grant from the National Heart
Foundation of Australia.
Disclosures
J.W.F. is a consultant for Pfizer, Roche, and Schering-Plough; has
received lecture/consulting fees from Merck, Lilly, Daiichi-Sankyo,
and Speedel; and is a member of the board of CBio.
20.
21.
22.
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Glucocorticoids Activate Cardiac Mineralocorticoid Receptors During Experimental
Myocardial Infarction
Anastasia S. Mihailidou, Thi Yen Loan Le, Mahidi Mardini and John W. Funder
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Hypertension. 2009;54:1306-1312; originally published online October 19, 2009;
doi: 10.1161/HYPERTENSIONAHA.109.136242
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MS#: HYPERTENSION/2009/136242
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ONLINE SUPPLEMENT
GLUCOCORTICOIDS ACTIVATE CARDIAC MINERALOCORTICOID RECEPTORS
DURING EXPERIMENTAL MYOCARDIAL INFARCTION
Mihailidou: Cardiac MR and myocardial infarction
Anastasia S. Mihailidou1,2 BSc, PhD, Thi Yen Loan Le1,2 BSc, Mahidi Mardini1,2,3 MBBS,
FRACP, PhD and John W Funder4, MD, PhD, FRACP
1
Department of Cardiology, Royal North Shore Hospital; 2The University of Sydney; 3Department
of Cardiology, Westmead Hospital, Sydney, NSW and 4Prince Henrys Medical Research Institute,
Clayton, Victoria, Australia.
1
MS#: HYPERTENSION/2009/136242
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In vitro Ischemia/Reperfusion of the rat heart.
A suture was placed around a prominent branch of the left coronary artery to occlude flow in the
artery. After a stabilisation period of 10 minutes, the suture was passed through a plastic tube and
occlusion effected by placing tension on the suture so that tube compressed the artery for 30
minutes to induce regional myocardial ischemia. The suture was then released to allow reperfusion
for 2.5 hours, and heart rate monitored throughout the protocol. Placing the suture around the
artery without occluding it (sham) had minimal effect on infarct zone. At the end of the
reperfusion phase, the coronary ligature was retightened at the same site and Monastral blue dye
(0.5 %) infused to delineate the normally perfused tissue from the “area-at-risk” (AAR). This
represents the part of heart supplied by the occluded artery, so that the AAR remains unstained.
The heart was cut parallel to the atrioventricular groove through the centre of the at-risk area and
the proximal portion fixed with 10% formalin for paraffin embedding. The distal portion was
frozen at -20oC and 2 hours later was sliced into 2 mm thick sections for morphometry to
determine the ischemic area at risk. These slices were incubated in 1% triphenyl-tetrazolium
chloride (TTC) at 370C for 20 minutes and photographed with a digital camera. TTC reacts with
dehydrogenase enzymes and nicotinamide adenine dinucleotide in viable tissue; ischemic but
viable myocardium was stained red and the infarcted area (IA) remained unstained (pale colour)
by TTC, while the blue area is the perfused (non-ischemic) area, and the non-blue area the AAR.
For examination, the slices were pressed between glass plates to identify the borders between the
at-risk zone and the normal area. Computerized planimetry with the image analysis software
program (National Institute of Health Image J, NIH Image software) was used to assess IA, AAR
and the total area of each heart slice, with slices analysed blind by two independent investigators.
Measurement of apoptosis in cardiac tissue.
Serial cross-sections were cut from paraffin embedded sections for detection of apoptotic
cardiomyocytes by terminal deoxynucleotide transferase-mediated ddUTP nick endlabeling
(TUNEL, Roche, USA). After end-labeling, the sections were counterstained with 4’,6-diamidino2-phenylindole dihydrochloride (DAPI) for total nuclei and examined by fluorescent microscopy
(magnification 200X). An area from the anterior, lateral, septal, posterior and sub-endocardial
regions of each section was randomly selected for evaluation. The apoptotic index was determined
by the number of TUNEL-positive myocyte nuclei expressed as a percentage of the total number
of DAPI-positive nuclei for different regions of the section; again measurements were analysed
blind by two independent observers.
2
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Table S1: Osmolarity of perfusates
Treatment
Osmolarity (Osmol/L)
Control
288 ± 0.3 (21)
Spiro 1µM
305 ± 0.2* (11)
RU486 100nM
291 ± 0.3* (11)
RU486 1 µM
323 ± 0.2* (11)
Aldo + Spiro
305 ± 0.3* (6)
Cort 10 + RU486 100 nM
292 ± 0.5* (6)
Cort 100 + Spiro
307 ± 0.5* (7)
Dex 10 + Spiro
306 ± 0.4* (6)
RU486 1 µM + Spiro
341 ± 0.2† (6)
Table Legends
Table S1. Osmolarity of perfusates for the various treatments: Control; Spironolactone (Spiro, 1
µM); RU486, 100 nM, 1 µM), Aldosterone (Aldo 10 nM) + Spironolactone (Aldo+Spiro) . Values
are mean ± SE, *p<0.05 versus control; †p<0.05 versus spironolactone (ANOVA).
3