and Glucocorticoid Receptor-Induced Chronotropic and Hypertrophic ...

RENAL-CARDIAC-VASCULAR

DHEA Prevents Mineralo- and Glucocorticoid Receptor-Induced Chronotropic and Hypertrophic Actions in Isolated Rat Cardiomyocytes Tiphaine Mannic, Mounira Mouffok, Magaly Python, Takehisa Yoshida, Andres D. Maturana, Nicolas Vuilleumier, and Michel F. Rossier Service of Endocrinology, Diabetology, Nutrition, and Hypertension (T.M., M.M., M.P., T.Y., M.F.R.), and Service of Laboratory Medicine (N.V., M.F.R.), Geneva University Hospitals, CH-1211 Geneva 14, Switzerland; Department of Bioengineering (T.Y., A.D.M.), Nagaoka University of Technology, Niigata 940-2188, Japan; and Service of Clinical Chemistry and Toxicology (M.F.R.), Central Institute of the Hospital of Valais, CH-1950 Sion, Switzerland

Corticosteroids have been involved in the genesis of ventricular arrhythmias associated with pathological heart hypertrophy, although molecular mechanisms responsible for these effects have not been completely explained. Because mineralocorticoid receptor (MR) antagonists have been demonstrated to be beneficial on the cardiac function, much attention has been given to the action of aldosterone on the heart. However, we have previously shown that both aldosterone and corticosterone in vitro induce a marked acceleration of the spontaneous contractions, as well as a significant cell hypertrophy in isolated neonate rat ventricular cardiomyocytes. Moreover, a beneficial role of the steroid hormone dehydroepiandrosterone (DHEA) has been also proposed, but the mechanism of its putative cardioprotective function is not known. We found that DHEA reduces both the chronotropic and the hypertrophic responses of cardiomyocytes upon stimulation of MR and glucocorticoid receptor (GR) in vitro. DHEA inhibitory effects were accompanied by a decrease of T-type calcium channel expression and activity, as assessed by quantitative PCR and the patchclamp technique. Prevention of cell hypertrophy by DHEA was also revealed by measuring the expression of A-type natriuretic peptide and BNP. The kinetics of the negative chronotropic effect of DHEA, and its sensitivity to actinomycin D, pointed out the presence of both genomic and nongenomic mechanisms of action. Although the genomic action of DHEA was effective mostly upon MR activation, its rapid, nongenomic response appeared related to DHEA antioxidant properties. On the whole, these results suggest new mechanisms for a putative cardioprotective role of DHEA in corticosteroid-associated heart diseases. (Endocrinology 154: 1271–1281, 2013)

evelopment of many important cardiac dysfunctions like heart hypertrophy, arrhythmias, inflammation, fibrosis, and apoptosis have been linked to an excess of mineralocorticoid production or action. Moreover, spironolactone and more recently eplerenone, 2 antagonists of the mineralocorticoid receptor (MR), have been proved to be clearly beneficial for patients with severe heart failure (1, 2). Two receptors expressed in ventricular cardiomyocytes bind aldosterone with different affinities: the high-affinity MR and the low-affinity glucocorticoid receptor (GR).

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The relative degree of activation of MR and GR can therefore be modulated in vitro by increasing aldosterone concentrations. MR also binds glucocorticoids (cortisol in humans or corticosterone in rodents) with the same high affinity as for aldosterone, with a dissociation constant in nanomolar concentrations (3), whereas GR requires high supraphysiological concentrations of aldosterone to be activated (4). We have recently established in freshly isolated neonatal rat ventricular cardiomyocytes that aldosterone in-

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2013 by The Endocrine Society doi: 10.1210/en.2012-1784 Received July 30, 2012. Accepted January 3, 2013. First Published Online February 8, 2013

Abbreviations: ANP, A-type natriuretic peptide; BNP, B-type natriuretic peptide; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; DTNB, 5,5⬘-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; GR, glucocorticoid receptor; MR, mineralocorticoid receptor.

Endocrinology, March 2013, 154(3):1271–1281

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duces a strong chronotropic effect in vitro, markedly increasing the cell beating frequency. This response has been shown to be mediated by a particular isoform of T-type calcium channel, CaV3.2/␣1H, modulated by the redox state of cells and induced upon activation of both receptors, MR and GR (4 –7). Aldosterone is also involved in other cellular specific responses linked to cardiovascular damages. Several lines of evidence demonstrate in vitro that aldosterone induces hypertrophy of cardiomyocytes, including the expression of several hypertrophic markers (A-type natriuretic peptide [ANP], B-type natriuretic peptide [BNP], ANP, BNP, and cardiotrophin-1), changes in cell morphology, and increase of cell capacitance (7, 8). In 10 years, dehydroepiandrosterone (DHEA) putative therapeutic effects have been extensively discussed. DHEA and DHEA sulfate (DHEAS), the main circulating form of DHEA, are steroid hormones principally produced by the adrenal cortex, but they also may be possibly produced by other organs like the heart (9). Since the discovery of this steroid and its metabolites during the first part of the 20th century, many publications have reported a beneficial role for DHEA in many physiological and pathophysiological situations like brain development, aging, osteoporosis, immune rheumatologic diseases, diabetes, obesity, chronic heart failure (particularly linked with oxidative stress), and recently, vascular remodeling diseases, including pulmonary arterial hypertension (10, 11). Other studies have revealed a possible physiological relevance of DHEA production in human heart, demonstrating that DHEA is suppressed in failing heart, preventing its putative cardioprotective action. Further, DHEA could reduce BNP production by cardiomyocytes upon endothelin-1-induced hypertrophy. One group has further suggested that a particular metabolite of DHEA, epiandrosterone, can inhibit L-type Ca2⫹ channels in cardiomyocytes, with a potential role in posthypoxic reoxygenation of isolated perfused hearts (12). Another putative mechanism of DHEA beneficial action has been proposed to be attributed to its free oxygen radical scavenger properties (13). However, despite the fact that DHEA became an important determinant of cardiac pathologies, its exact mechanism of action on cardiomyocytes and its potential antioxidant role have not been totally elucidated yet. In the present study, we intended to test in vitro DHEA effects on isolated neonate rat ventricular cardiomyocytes exposed to high, supraphysiological concentrations of aldosterone able to activate both MR and GR, as well as upon selective stimulation of each receptor separately. The functional role of DHEA was assessed on the chronotropic and hypertrophic responses to MR and GR stimulation and allowed us to highlight a corticosteroid antagonist


role for DHEA mediated in part by its antioxidant properties.

Materials and Methods DMEM was obtained from Invitrogen (Carlsbad, California). All other reagents were from Sigma-Aldrich (St. Louis, Missouri) unless other indication. A pCDNA3 plasmid construct containing the complete sequence coding the human ␣1H-subunit of the CaV3.2 channel flanked by a green fluorescent protein was kindly provided by Pr Edward Perez-Reyes (University of Virginia, Charlottesville, Virginia).

Cell culture Neonatal cardiomyocytes were isolated from 1- to 2-day-old Wistar rat ventricles by digestion with low trypsin-EDTA and type 2 collagenase, and cells were maintained in primary culture as previously described (6). Animals were euthanized in conformity with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (publication no. 85-23) and with the authorization (1012/3134/0-R) of the local County Veterinary Office. Freshly isolated cells were seeded in plastic flasks to allow selective adhesion of cardiac fibroblasts. Thereafter, cardiomyocytes were decanted from the flasks and distributed in laminincoated 35-mm Petri dishes.

Total RNA isolation and mRNA quantification Total RNA from cardiomyocytes was extracted and reverse transcribed using random hexamers as previously described in detail (6). The relative abundance of T-channel and hypertrophic marker mRNAs was assessed respectively by TaqMan or SYBR Green quantitative PCR, and cyclophilin A was used to normalize data. Reactions were performed in LightCycler 480 Probes Master (Roche, Rotkreuz, Switzerland) using a LightCycler 480 real-time thermocycler (Roche). The sequences and concentrations of specific primers and TaqMan probes used have been previously published (5, 7).

Cell contraction frequency Spontaneously contracting cell monolayers were incubated in serum-free DMEM during the indicated times with the appropriate concentration of agents (as mentioned in the figure legends). Cell beating frequency was determined under light microscope. The number of contractions was counted by time unit in 3 or 4 different locations of the dish.

Cell morphology Cardiomyocytes, plated on laminin-coated dishes and exposed to various agents, were placed under an Olympus IX71 inverted microscope, and pictures of the cells were taken with a DP71 CCD camera (Olympus, San Diego, California). The cell surfaces were measured using the ImageJ freeware (available from http://rsbweb.nih.gov/ij/).

Electrophysiological recordings As described previously (6), cardiomyocytes were placed on laminin-coated glass coverslips. HEK-293 cells were cultured on


poly-lysine-coated glass dishes and transfected transiently with the CaV3.2-coding pCDNA3 plasmid using FuGENE 6 (Roche) and according to manufacturer instructions. Calcium currents in HEK-293 cells expressing CaV3.2 were measured 30 hours after transfection. Patch-clamp recordings were performed in the whole-cell configuration. Both types of cells were voltage clamped using an Axopatch 200B amplifier (Axon CNS; Molecular Devices, Sunnyvale, California) at a holding potential of ⫺90 mV. For time course experiments, T-type currents were recorded upon a 200-ms voltage pulse at ⫺30 mV from the ⫺90-mV holding potential. The absence of large contaminations by L-type Ca2⫹ currents at ⫺30 mV was previously demonstrated in cardiomyocytes (6). The currents were filtered at 2 kHz and sampled at 5 kHz using a Digidata 1440A (Axon CNS). The leak was subtracted automatically by a P/4 protocol (pclamp10; Axon CNS). For cardiomyocytes, the bath solution contained 125 mM N-methyl-glucamine, 5 mM 4-aminopyridine, 20 mM tetraethyl-ammonium chloride, 2 mM CaCl2, 2 mM MgCl2, and 10 mM D-glucose and was buffered to pH 7.4 with 10 mM HEPES. The patch pipettes were filled with solution containing 130 mM CsCl, 10 mM EGTA, 3 mM Mg-ATP, 0.4 mM Li-GTP; pH was adjusted to 7.2 with 25 mM HEPES. For HEK-293 expressing the CaV3.2 channels, the bath solution was: 40 mM BaCl2, 80 mM CsCl, and 10 mM HEPES; pH was adjusted to 7.4 with CsOH, and the pipette solution was: 120 mM CsCl, 10 mM EGTA, 10 mM HEPES, and 3 mM Mg-ATP; pH was adjusted to 7.2 with CsOH.

Cell oxidation measurement Cardiomyocytes in primary culture were incubated as indicated with appropriate agents (5 mM sodium dithionite, 200 ␮M 5,5⬘dithiobis(2-nitrobenzoic acid) (DTNB) or 100 ␮M DHEA) before being detached with a trypsin/EDTA solution, washed by centrifugation in a Krebs buffer, and loaded, by incubation for 30 minutes at 37°C in the dark, with the fluorescent redox probe dichlorofluorescein (dichlorofluorescein, 5 ␮M in dimethylsulfoxide). Probe loading was performed in the continuous presence of the tested agents. Cell fluorescence, proportional to the degree of oxidation, was then analyzed by flux cytometry on a BD FACSCalibur (Becton Dickinson, Allschwil, Switzerland). Only cells displaying a fluorescence level within a predetermined window were considered for the analysis, thus an index of cell oxidation was arbitrarily defined as the median fluorescence times the proportion of total cells that are present in the fluorescence window.

Statistics Results are expressed as the means ⫾ SEM unless stated otherwise. The statistical significance of differences was analyzed by one-way ANOVA followed by post hoc paired or unpaired Student’s t tests between groups. *, **, and *** correspond to P values ⬍.05, ⬍.01, and ⬍.001, respectively.

Results DHEA reduces the corticosteroid-induced chronotropic effect in neonate rat ventricular cardiomyocytes through genomic and nongenomic mechanisms Freshly isolated neonatal rat ventricular cardiomyocytes are able to contract spontaneously in culture. We


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have previously shown that corticosteroids can exert a positive chronotropic action in vitro (5) and that blockade of corticosteroid receptors, either MR or GR, by spironolactone and RU-486, respectively, leads to a proportional inhibition of the aldosterone-induced chronotropic effect (7). To determine whether DHEA can affect this chronotropic action of steroids, the beating frequency of cells has been measured after 24 hours of stimulation of both receptors individually or together (Figure 1A). For this purpose, cells were exposed to 10 nM aldosterone (to activate MR specifically), to 1 ␮M dexamethasone (to activate GR specifically), or to both agents together. Using high, supraphysiological concentrations of aldosterone (1000 nM) also allowed to “quasi pharmacologically” activate both MR and GR together, whereas the addition of spironolactone (10 ␮M) or RU-486 (10 ␮M) under these conditions was expected to antagonize, respectively, MR and GR, as previously published (7). Interestingly, although DHEA did not affect the basal beating frequency, it significantly reduced the steroid-induced chronotropic response after 24 hours, the response being elicited by MR alone, GR alone, or both receptors together. The inhibitory effect of DHEA on the chronotropic response to high aldosterone was concentration dependent, with a significant (P ⬍ .05) inhibition already observed at 0.1 ␮M, and displayed a biphasic response (Figure 1B). Fitting the inhibitory curve to a biphasic exponential decay function revealed time constants at approximately 0.09 ␮M and 30 ␮M, and a maximal effect was observed at 100 ␮M. Interestingly, the sulfated form of DHEA (DHEAS), the major form in vivo of the circulating hormone, behaved like DHEA but with a slightly delayed response (data not shown). Then, we investigated the time course of the DHEA action. After 24 hours of incubation of cells with aldosterone alone (1000 nM), we added 100 ␮M DHEA into the medium and measured the beating frequency after different periods of time. Interestingly, the DHEA effect was very rapid, a significant reduction of the beating frequency being already observed after 1 minute (Figure 1C, left panel, gray bars). After a plateau at 10 minutes, the inhibition continuously increased for a period of at least 6 hours down to the basal levels. Similar results were obtained when DHEA was added after MR activation with 10 nM aldosterone (Figure 1C, middle panel) or after GR activation with 1000 nM dexamethasone (Figure 1C, right panel). This rapid effect of DHEA strongly suggested the presence of a nongenomic mechanism. In order to isolate the putative nongenomic response, the same experiment was performed in the presence of actinomycin D, an efficient inhibitor of mRNA synthesis. The pattern of cell beating

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Figure 1. DHEA reduces the corticosteroid-induced chronotropic effects in neonate rat ventricular cardiomyocytes through genomic and nongenomic mechanisms. (A) Cells were exposed to vehicle (basal) or various combinations of aldosterone (10 nM or 1000 nM), dexamethasone (1000 nM), spironolactone (10 ␮M), or RU-486 (10 ␮M) and in the presence or absence of 100 ␮M DHEA, and their beating frequency was determined after 24 hours. Results obtained in control conditions (open bars) were statistically tested for a difference against basal ($), whereas the significance of DHEA effect was assessed by comparison with the corresponding controls (*). Both spironolactone and RU-486, alone or in combination, significantly (P ⬍ .001) reduced the response to 1000 nM aldosterone. Data are the mean values (⫹SEM) from 4 –20 independent experiments. (B) The concentration-dependent inhibitory action of DHEA on 1000 nM aldosterone-induced chronotropic response was determined after 24 hours. DHEA effect was statistically significant (P ⬍ .05) at all concentrations tested. Data were fitted to a biphasic exponential decay function in order to determine the time constant values (corresponding to IC50 values for each step). The dotted line represents the basal beating frequency. (C) The kinetics of the DHEA effect were assessed in cells incubated for 24 hours with 1000 nM aldosterone (left panel), 10 nM aldosterone (middle panel), or 1000 nM dexamethasone (right panel) and in the presence (dark bars) or absence (gray bars) of actinomycin D (2.5 ␮g/ml). The dotted line represents the basal beating frequency. The number of * indicate P values obtained when testing a difference with time 0, and $, P values vs the respective control, in the absence of actinomycin D. Data are the means from 3–5 independent preparations. Aldo, aldosterone; Ctrl, control. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001,$P ⬍ 0.05, $$P ⬍ 0.01, and $$$P ⬍ 0.001.

frequency reduction was not significantly different during the first 30 minutes in the presence (Figure 1C, dark bars) or absence (Figure 1C, gray bars) of actinomycin D (Figure 1C, left panel). In contrast, after 1 hour and later, when the putative genomic action of DHEA was expected to become effective, the reduction of the beating frequency was significantly affected by the presence of actinomycin D that prevented further reduction of the beating frequency. Moreover, in the absence of DHEA, the cell beating frequency remained unchanged in the presence of actinomy-

cin D (data not shown), excluding an action of this drug on the chronotropic response to aldosterone itself. These results allowed us to conclude for the existence of at least 2 distinct mechanisms involved by DHEA, one major, very rapid, nongenomic inhibitory effect, and then a second minor response, developing slowly and due to a genomic mechanism, requiring gene transcription. Interestingly, the ratio between the genomic and nongenomic DHEA effects was different when MR or GR was activated separately (middle and right panels). Indeed, although the


effect of actinomycin D was significant in cells primed with 10 nM aldosterone, it was absent in dexamethasonetreated cells. Both genomic (measured at 24 h) and nongenomic (determined after 15 min) DHEA inhibitory actions were concentration dependent with similar pattern (data not shown). The genomic effect of DHEA involves the modulation of the T-type channel isoform ␣1H To further study the molecular mechanism of the genomic effect of DHEA on cell beating frequency, we investigated the potential modulation of the T-type calcium channels, and particularly of the ␣1H isoform. Indeed, we have previously shown that an increase of the expression and activity of the T-type calcium channel isoform, CaV3.2/␣1H, is required for the aldosterone-induced positive chronotropic action observed in vitro in rat cardiomyocytes (6). Therefore, we analyzed the effect of DHEA on both the expression and activity of ␣1H upon aldosterone stimulation to determine whether it could explain the observed inhibition of the chronotropic response. As shown in Figure 2A, within 24 hours of incubation, ␣1H expression was significantly increased by 80% in response to 10 nM aldosterone and by 250% in response to 1000 nM, and these responses were significantly reduced when cells were incubated from the beginning in the presence of 100 ␮M DHEA. As previously described (6), the 2 antagonists of MR and GR, spironolactone and RU486, respectively, partially reduced ␣1H expression, as compared with aldosterone alone, and the presence of DHEA further reduced ␣1H expression. The concomitant increase of the calcium current amplitude observed after cardiomyocyte treatment with aldosterone (1000 nM) for 24 hours (Figure 2B) was completely prevented in the presence of 100 ␮M DHEA, as shown with the patch-clamp technique. The analysis of the voltage-current relationship (Figure 2C, left panel) revealed that the inhibitory effect of DHEA was proportionally more pronounced at negative voltages (65% inhibition at ⫺20 mV vs 25% at ⫹20 mV), where the contribution of low-threshold T-type calcium channels to the calcium influx is known to be the largest. The effect of DHEA on the T current elicited by MR or GR separately has been also determined at ⫺20 mV (Figure 2C, right panel). Altogether, these results are in agreement with the hypothesis that DHEA affects T-channel activity and expression. DHEA antioxidant properties can explain its nongenomic negative chronotropic effect We have previously shown that the chronotropic action of corticosteroids on rat cardiomyocytes can be modu-


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lated by the redox potential of the cell, oxidants increasing while reducing agents decreasing the beating frequency (4). Furthermore, we have demonstrated that these redox agents directly regulate ␣1H T-type calcium channel activity (6). To determine whether the mechanism of DHEA action on the cardiomyocytes, and particularly its rapid nongenomic effect, was related to its well-described antioxidant properties, we compared its negative chronotropic action elicited within 20 minutes on aldosterone-pretreated cells with that of a strong reducing agent like sodium dithionite. As illustrated in Figure 3A, both DHEA and sodium dithionite significantly reduced the aldosterone-induced chronotropic response in a similar way, without affecting the basal beating frequency. When added together, these agents did not display a cumulative effect. DHEA antioxidant capacity was then evaluated and compared with that of dithionite with the fluorescent cell redox probe, dichlorofluorescein. A cell oxidation index, obtained upon the analysis of the dichlorofluorescein signal by flux cytometry, revealed that cells exposed to DHEA (or dithionite) were much less oxidized than control naïve or aldosterone-pretreated myocytes (Figure 3B). Interestingly, DHEA exerted its antioxidant action even in cells previously exposed to a strong oxidant like DTNB (4). We next tested the acute effects of DHEA on the T-type calcium currents with the patch-clamp technique. Lowthreshold T-type calcium currents were elicited in aldosterone (1000 nM)-pretreated cardiomyocytes by stepping the voltage from a resting potential of ⫺90 to ⫺30 mV, conditions in which putative contaminations by Ltype calcium currents are insignificant (6). Addition of 100 ␮M DHEA rapidly induced a significant decrease of the amplitude of the T-type calcium current within 10 minutes in aldosterone-treated cells (Figure 3C). In contrast, no change in T-type calcium current amplitude was observed upon DHEA addition in control naïve cells (data not shown), a finding in agreement with the lack of effect of DHEA on the basal beating frequency (Figures 1A and 3A). These results suggest that DHEA could directly affect the activity of CaV3.2/␣1H channels whose expression and activity are low in control cells but increased in aldosterone-treated cells (5, 6). To confirm the generality of this hypothesis, we measured the activity of human CaV3.2/␣1H channels transiently expressed in HEK-293 cells (14). The addition of DHEA in this well-characterized cell model significantly reduced the calcium current supported by the CaV3.2/␣1H channels within 10 minutes (Figure 3D). In spite of the fact that the current density was 10 times higher in transfected HEK-293 cells as compared with that recorded in rat cardiomyocytes, the extent

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Figure 2. DHEA reduces ␣1H expression and activity in cardiomyocytes. (A) Cells were incubated for 24 hours in presence of aldosterone alone (10 nM or 1000 nM) or in combination with spironolactone (10 ␮M) or RU-486 (10 ␮M). Incubations were performed in the presence or absence of 100 ␮M DHEA, and ␣1H expression was determined by real-time RT-PCR. Results of controls (open bars) were tested for a difference against basal ($), whereas the significance of DHEA effect was assessed by comparison with the corresponding controls (*). Both spironolactone (P ⬍ .05) and RU-486 (P ⬍ .01) significantly reduced the response to 1000 nM aldosterone. Results are the mean values from 6 –12 independent experiments. (B) Inhibition of the aldosterone-induced increase of calcium currents by DHEA. Examples of representative calcium inward currents elicited by 10-mV voltage step depolarization elicited from a holding potential of ⫺90 mV are shown. Currents were recorded in the whole-cell configuration of the patch-clamp technique as described in Materials and Methods from a naïve cell (Ctrl), from a 24-hour aldosterone (1000 nM)-treated cell (Aldo) and from a 24-hour aldosterone- and DHEA (100 ␮M)-treated cell (Aldo/DHEA). Scale bars correspond to 100 pA (vertical) and 50 ms (horizontal). (C) (Left panel) Current density-voltage curves determined in control cells (n ⫽ 9), aldosterone (1000 nM)-treated cells (n ⫽ 10), and aldosterone plus DHEA (100 ␮M)-treated cells (n ⫽ 16). * indicates current values significantly different from that of Ctrl cells. (Right panel) Current density determined at ⫺20 mV in cells exposed for 24 hours to 10 nM aldosterone, 1000 nM dexamethasone, or 1000 nM aldosterone, in the presence or absence of DHEA (100 ␮M). Results of controls (open bars) were tested for a difference against basal ⫽ dotted line ($), whereas the significance of DHEA effect was assessed by comparison with the corresponding controls (*). Results are the mean values from 5–16 independent cells. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001,$P ⬍ 0.05, $$P ⬍ 0.01, and $$$P ⬍ 0.001.

of inhibition induced by DHEA was similar in the 2 cell types, suggesting the involvement of a same mode of action, possibly related to a direct modulation of the channel activity by the redox state of the cell. After DHEA action, the amplitude of this current was only slightly reduced by the addition of the strong antioxidant dithiothreitol (DTT). Taken altogether, these results strongly suggest that DHEA rapidly and directly inhibits the activity of CaV3.2/

␣1H channels in a nongenomic manner, resulting in a parallel decrease of the frequency of spontaneous beatings in cardiomyocytes overexpressing these channels upon stimulation with aldosterone. DHEA prevents the hypertrophic effect of aldosterone in cardiomyocytes Cardiac hypertrophy is one of the most obvious features of heart failure. It has been shown that aldosterone


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Figure 3. DHEA-reducing properties on cardiomyocytes. (A) Cells were exposed for 24 hours to vehicle (basal) or aldosterone (1000 nM). Then, DHEA (100 ␮M), dithionite (5 mM), or both agents together were added for 20 minutes. Beating frequency was measured before and after the addition of DHEA and dithionite. Results of controls (open bars) were tested for a difference against basal ($), whereas the significance of DHEA and dithionite effect was assessed by comparison with the corresponding controls (*). Results are the mean values from 4 independent experiments. (B) Fluorescence of the redox probe dichlorofluorescein was analyzed in cardiomyocytes by flux cytometry, and results were converted into a cell oxidation index as described under Materials and Methods. The significance of DHEA and dithionite effect was assessed by comparison with the corresponding controls (*). Results, expressed as the percentage of the cell oxidation index found in basal conditions, are the mean values from 6 independent experiments. (Inset) Examples of fluorescence distribution in basal and DTNB-treated cells. The cell oxidation index was determined within the M1 fluorescence window. (C) Acute effect of DHEA (100 ␮M) on aldosterone-activated endogenous T-type calcium currents in neonate rat cardiomyocytes. The calcium current density elicited by a single voltage step at ⫺30 mV from a ⫺90-mV holding potential was measured every 30 seconds in a single 24-hour aldosterone-treated neonatal ventricular cardiomyocyte. (Inset) Examples of currents recorded before and after the addition of DHEA, at the indicated times. Scale bars are 25 pA (vertical) and 25 ms (horizontal). (D) Acute effect of DHEA on CaV3.2 calcium channels transiently expressed in HEK-293 cells. Currents were elicited and recorded as described in C. (Inset) Current recorded before and after the addition of DHEA (100 ␮M), and after the addition of DTT (100 ␮M). Scale bares are 100 pA (vertical) and 25 ms (horizontal). Aldo, aldosterone; Ctrl, control; ns, not significant. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001,$P ⬍ 0.05, $$P ⬍ 0.01, and $$$P ⬍ 0.001.

induces hypertrophy of cardiomyocytes via many different pathways (8). Because DHEA is able to decrease cardiomyocytes beating frequency, we wanted to determine whether it can also affect aldosterone-induced cell hypertrophy. To test this possibility, we used 2 different approaches. We first analyzed, by RT-PCR, the expression of hypertrophic markers like ANP and BNP. As shown in Figure 4A, ANP and BNP expression was increased 4 –7 times when cells were stimulated for 24 hours with 1000 nM aldosterone or dexamethasone. Interestingly, low

concentration of aldosterone (10 nM) had no effect, and RU-486 completely abolished the response to high aldosterone, confirming our previous observation that hypertrophy is mostly mediated by GR (7). DHEA completely abolished the ANP response and markedly reduced the BNP elevation. To directly confirm a DHEA action on the cell size, we measured the cell surface as previously described (7). As expected, DHEA was able to completely prevent the high aldosterone-induced increase of the cell surface (Figure 4B


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Figure 4. Hypertrophy induced by GR activation is prevented by DHEA. (A) ANP (left panel) and BNP (right panel) expression was evaluated by real-time RT-PCR in cardiomyocytes exposed for 24 hours to vehicle (basal) or various combinations of aldosterone (10 nM or 1000 nM), dexamethasone (1 ␮M), spironolactone (10 ␮M), or RU-486 (10 ␮M) and in presence or absence of 100 ␮M DHEA. Results of controls (open bars) were tested for a difference against basal ($), whereas the significance of DHEA effect was assessed by comparison with the corresponding controls (*). RU-486 significantly (P ⬍ .01) reduced the response to 1000 nM aldosterone, whereas the inhibition induced by spironolactone was significant (P ⬍ .05) only on BNP. Results are the mean values from 4 –7 independent experiments. (B) DHEA and antioxidant effects on cardiomyocyte morphology changes induced by aldosterone. Freshly isolated myocytes were exposed for 48 hours to aldosterone (1000 nM) alone or in the presence of either DHEA (100 ␮M) or sodium dithionite (5 mM). Control cells (basal) were maintained for 48 hours with vehicle. Cell morphology was then analyzed by microscopy. Representative images obtained under various experimental conditions are shown (scale bars correspond to 20 ␮m). The surface of individual cells (Table 1) was determined with the free software ImageJ (available from http://rsbweb.nih.gov/ij/). Aldo, aldosterone; Ctrl, control; ns, not significant. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001, $P ⬍ 0.05, $$P ⬍ 0.01, and $$$P ⬍ 0.001.

and Table 1). Although stimulation of MR alone by 10 nM aldosterone did not affect the cell size, the dexamethasone/ GR-induced cell hypertrophy was completely prevented by DHEA. Moreover, this significant antihypertrophic action of DHEA was mimicked by sodium dithionite and DTT, suggesting that DHEA effect on hypertrophy was mostly mediated by its antioxidant properties.

Discussion One novel and important finding of the present study is that DHEA can reduce the corticosteroid-induced chronotropic effects by decreasing the expression and activity of the ␣1H isoform of the T-type calcium channels. We

observed 2 complementary responses; a first, very rapid, nongenomic action of DHEA preceding a second, less rapid, genomic action of DHEA, including changes in mRNA expression, particularly when MR was responsible of the cell beating acceleration. We also present evidence that, like other antioxidant molecules, DHEA can reduce cell oxidation very strongly and therefore, in agreement with our previous studies (4, 6), can also decrease ␣1H activity through this mechanism. The important deleterious consequences of MR activation on the heart function is now well established, considering that MR antagonists can reduce morbidity and mortality of patients with severe heart failure (1). These antagonists are now considered as efficient drugs for reducing arrhythmias in these


Table 1.


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Inhibition of the Hypertrophic Response to Aldosterone by DHEA

Experimental Condition

Mean Surface (␮m2)

95% CI (␮m2)

N

P vs Basal

P vs Ctrl

Basal DHEA Aldo 10 Aldo 10/DHEA Dexa Dexa/DHEA Aldo 1000 Aldo 1000/DHEA Aldo 1000/dithio Aldo 1000/DTT

464 454 391 411 932 449 896 451 443 435

431– 496 424 – 484 316 – 466 360 – 462 763–1100 379 –518 837–955 423– 479 374 –513 374 – 496

310 219 35 38 30 36 295 238 91 46

.339 .076 .138 ⬍.0001 .380 ⬍.0001 .281 .284 .257

.339 .327 ⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001

Freshly isolated cardiomyocytes were incubated for 48 hours under various experimental conditions (DHEA 100 ␮M, aldosterone 10 nM or 1000 nM, dexamethasone 1000 nM, sodium dithionite 5 mM, and DTT 100 ␮M) before analyzing their surface by microscopy with the ImageJ freeware. Abbreviations: Aldo, aldosterone; CI, confidence interval; Dithio, sodium dithionite; N, total number of cells analyzed in each experimental condition. Ctrl are the corresponding conditions without DHEA or antioxidant.

patients (15), and we show here that DHEA mimics some of their effects. Moreover, we demonstrate that DHEA reduces GR-mediated hypertrophy of cardiac cells, possibly also through its antioxidant properties. In the present study, we used systematically high, supraphysiological aldosterone concentration (1000 nM) to stimulate both MR and GR simultaneously and added antagonists for deciphering the role of each receptor. However, because of important concern about the specificity and efficacy of these antagonists under these particular conditions, we also performed the experiments with lower aldosterone concentration (10 nM) or dexamethasone (1000 nM) to selectively activate MR or GR, respectively. DHEA and its sulfate ester form, DHEAS, are the most abundant steroids produced by the adrenals, circulating in serum at supramicromolar concentrations, much higher than those of other steroids, including aldosterone. Furthermore, plasma levels of DHEA(S) decrease with aging and, in some cases, correlate with the severity of heart diseases in males (16, 17). DHEA and aldosterone have been shown to have an inverse synthetic balance in patients with heart failure: DHEA synthesis is reduced in such conditions, whereas aldosterone production is upregulated (9), leading to the suggestion that DHEA could have a very important general cardioprotective role. These considerations led us to study the precise effect of DHEA on aldosterone-stimulated rat ventricular cells. DHEA effect was first tested on the aldosterone-induced positive chronotropic response (Figure 1), which was similar to our previous observations. This allowed us to distinguish clearly between 2 mechanisms of DHEA action on aldosterone-stimulated cardiomyocytes: a first inhibition, rapidly occurring, corresponding to a nongenomic effect of DHEA that could be due to some post-

translational changes of specific proteins, and a second, delayed effect, sensitive to actinomycin D, that can result from expression changes of elements implied in the aldosterone positive chronotropic effect. Interestingly, the genomic effect of DHEA was almost absent when cell beating acceleration was due exclusively to GR stimulation. Numerous electrical mechanisms can lead to arrhythmias, but we have previously demonstrated that the overexpression of T-type calcium channels (and particularly the ␣1H isoform) is essential for the acceleration of the cardiomyocyte contractions induced by aldosterone (5, 6). T-type calcium channels are normally repressed in adult rat cardiomyocytes, but their reexpression can be observed in many contexts of cardiac pathologies (18) and upon aldosterone stimulation (5). In early studies, these channels were proposed to be the a support for a pacemaker current (19). Other studies showed a role for the high thresholdactivated calcium channels, the L-type calcium channels, in epiandrosterone-induced decrease of myocardial contractility. Epiandrosterone is a metabolite of DHEA formed in peripheral tissues. This response was observed in other pathological conditions, such as on myocardial contractile activity during posthypoxic reoxygenation, but, unlike DHEA(S) serum levels, epiandrosterone physiological levels are not known (12). In the present study, L-type Ca2⫹ channels apparently did not play any role in the decrease of the beating frequency induced by DHEA. Indeed, expression of the ␣1C isoform of L-type Ca2⫹ channel was similar in aldosterone-stimulated cells in the presence or absence of DHEA (data not shown). We showed that DHEA is able to significantly decrease ␣1H expression (Figure 2A), but the pathways involved in this mechanism are not clear, nor the steps responsible for

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the aldosterone-induced positive regulation of the expression of this channel. DHEA has been proposed to act through its own putative receptor in cardiomyocytes, and ␴-1 receptor is in this context a relevant candidate. This receptor is largely expressed in heart, its gene contains a steroid-binding component, and the protein has been shown to interact with DHEA (20). Some studies have described ␴-1 receptor as linked to several ionic channels and to calcium influx in the heart (21, 22) and, therefore, to regulate cardiac contractility and rhythm (20, 23), but there is no evidence at the moment for a specific action on the T-type calcium channels. The physiological relevance of this receptor in the human heart and its role in DHEA action remain, however, to be clearly demonstrated. DHEAS appeared to have the same negative chronotropic effect on cardiomyocytes as DHEA (data not shown), but the response was slightly delayed, possibly because of the slower entry of the sulfated steroid into the cells. It is, however, not known whether both DHEA and DHEAS act through the same signaling pathways. The early nongenomic negative chronotropic response of DHEA upon aldosterone treatment is extremely efficient and robust. Because recently published data indicated a protective role for DHEA against oxidative stress-induced endothelial cell dysfunctions (24), the nongenomic response of DHEA on rat cardiomyocyte beating frequency was compared with that of well-characterized reducing agents. Tamagno et al (13) have shown that DHEA can affect superoxide radicals and acts as a metal chelator. We showed here that DHEA can modulate the redox state of cardiac cells (Figure 3), and its effect on beating frequency was mimicked by antioxidant agents. Concomitantly, DHEA acts directly on the ␣1H channel, reducing its activity, in a manner similar to what we previously showed for antioxidant agents (6). Recently, it has been shown that DHEA inhibits the 3 recombinant Tchannel isoforms expressed in NG108-15 cell line, as well as native T-channels in pulmonary artery smooth muscle cells with kinetics and affinities similar to those we have observed in cardiac and HEK cells (25). In that study, the effect of DHEA was independent of the androgen or estrogen receptors and implicated a pertussis toxin-sensitive G protein, putative mechanisms that remain to be determined in cardiomyocytes. Aldosterone is also considered hypertrophic, acting on cardiac cells via genomic and nongenomic signaling events. Processes implied in aldosterone-induced hypertrophy can be mediated by protein kinase C- and MAPKdependent mechanisms (8). Besides, the negative regulation of hypertrophy in cardiac cells by DHEA is also effective on endothelin-1-stimulated cardiomyocytes, in-


ducing a decrease in cell size and BNP mRNA expression (9). The fact that DHEA is able to counteract the hypertrophic response to both corticosteroids and endothelin-1 suggests that its action is through a common general mechanism, not specifically linked to a given receptor. This assumption is somehow supported by the ability of antioxidant agents to mimic DHEA action in a nonadditive manner. It is, however, difficult to assess whether this antihypertrophic action of DHEA is genomic or nongenomic in nature, because the establishment of hypertrophy (by aldosterone or endothelin-1) requires gene expression, and hypertrophy is not easily reversible. The distinction between a direct antioxidant effect of the DHEA molecule itself and an action of DHEA on a redox-sensitive genomic pathway remains to be made. In conclusion, we found that DHEA reduces the positive chronotropic and hypertrophic responses of isolated cardiomyocytes to MR and GR stimulation. Concerning the chronotropic effect, we have distinguished 2 different pathways induced by DHEA. The first, nongenomic, very rapid response to DHEA can be explained in part by its ability to affect the redox potential of the cells and, consequently, the activity of the T-type calcium channel isoform, ␣1H. The second response, requiring gene expression, involves pathways that remain to be fully elucidated. The antihypertrophic effect of DHEA could be explained in part by its reducing power, but some cellular pathways involving MAPK could also be responsible for a part of the response, a mechanism that we did not test in the present study. Hopefully, these results should motivate further investigations of cardiac signaling pathways involved in DHEA cardioprotective action with the aim of targeting pharmacologically these pathways in order to help preventing, or at least reducing, the burden represented by cardiac diseases in our society.

Acknowledgments We tank Dr M. Frias for his help in obtaining neonate rat cardiomyocytes and to Ms A. Diemand for linguistic corrections of the manuscript. Address all correspondence and requests for reprints to: Dr Tiphaine Mannic, Service of Endocrinology, Diabetology, and Nutrition, University Hospital of Geneva, 4 Rue Gabrielle-Perret-Gentil, CH-1211 Geneva 14, Switzerland. E-mail: [email protected]. This work was supported by the Swiss National Science Foundation Grant 310030-130545, the Foundation Endocrinologie (Geneva), and Novartis Foundation (M.F.R.) and by Telemaque, Gustave and Simone Prevost, and De Reuter Foundations (N.V.). Disclosure Summary: The authors have nothing to disclose.


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