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Aldosterone increases voltage-gated sodium current in ventricular myocytes

¹Department of Pharmacology and Toxicology, University of Lausanne, Switzerland; and ²Service of Cardiology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland

	ABSTRACT

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The role of aldosterone in the pathogenesis of heart failure (HF) is still poorly understood. Recently, aldosterone has been shown to modulate the function of cardiac Ca²⁺ and K⁺ channels, thus playing a role in the electrical remodeling process. The goal of this work was to investigate the role of aldosterone on the cardiac Na⁺ current (I_Na). We analyzed the effects of aldosterone on I_Na in isolated adult mouse ventricular myocytes, using the whole cell patch-clamp technique. After 24 h incubation with 1 µM aldosterone, the I_Na density was significantly increased (+55%), without alteration of the biophysical properties and the cell membrane capacitance. Aldosterone (10 nM) increased the I_Na by 23%. In 24-h coincubation experiments, with the use of actinomycin D, cycloheximide, or brefeldin A, the effect of aldosterone on I_Na was abolished. Spironolactone (mineralocorticoid receptor antagonist, 10 µM) prevented the 1 µM aldosterone-dependent I_Na increase, whereas RU-38486 (glucocorticoid receptor antagonist, 10 µM) did not. The action potential duration (APD) was longer in aldosterone-treated (APD₉₀: +53%) than in control myocytes. In addition, the L-type Ca²⁺ current was also upregulated (+48%). We performed quantitative RT-PCR measurements and Western blots to quantify the mRNA and protein levels of Na_v1.5 and Ca_v1.2 (main channels mediating cardiac I_Na and I_Ca), but no significant difference was found. In conclusion, this study shows that aldosterone upregulates the cardiac I_Na and suggest that this phenomenon may contribute to the HF-induced electrical remodeling process that may be reversed by spironolactone.

sodium channels; calcium channels; electrophysiology; spironolactone

The main cardiac voltage-gated Na⁺ channel Na_v1.5 plays a pivotal role in the excitability and conduction of electrical impulses in the heart. Although the structure, function, and pharmacology of Na_v1.5 have been studied in detail, the regulation of its gene expression and trafficking remains poorly understood (18). In recent reports (14, 35, 41), the cell surface density of Na_v1.5 channels has been shown to be regulated by the activity of Nedd4–2, an E3 ubiquitin-protein ligase. The current working model proposes that Na_v1.5 may be ubiquitinated by ubiquitin ligases and that such ubiquitinated channels are recognized by the cellular internalization machinery (35), thus determining the channel density at the cell surface. In kidney distal tubular cells, aldosterone has been shown to indirectly regulate Nedd4–2 activity via stimulation of the expression of the protein kinase Sgk1 that can phosphorylate and inactivate Nedd4–2 by thus far unknown mechanisms (10).

Based on these clinical and molecular findings, the main aim of this work was to investigate whether the cardiac I_Na would also be regulated by pathological concentrations of aldosterone, i.e., ranging from 10 nM to 1 µM. In this study, we report that 24 h treatment of isolated mouse ventricular myocytes with aldosterone increased the I_Na density without affecting its biophysical properties. The effect of aldosterone on I_Na was totally blocked by inhibitors of transcription, protein synthesis, and trafficking, as well as with spironolactone. A similar increase of I_Na was obtained with the glucocorticoid dexamethasone. However, we observed no effect on the mRNA levels of Na_v1.5, the total cellular Na_v1.5 protein pool, and on Nedd4–2 expression and phosphorylation. These results suggest that pathological concentrations of aldosterone increase the density of cardiac Na⁺ channels at the cell surface via the activation of so far unknown genes involved in the regulation of its trafficking and/or membrane stability.

	MATERIALS AND METHODS

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Cardiomyocytes isolation. Adult mouse ventricular myocytes were isolated from male C57BL/6 mice (25–30 g, Janvier, France). Animals were anesthetized with pentobarbital and, after heparinization, the chest was opened; hearts were excised and subjected to retrograde perfusion (3 ml/min) in a Langendorff apparatus with a solution containing (in mmol/l): 4.75 KCl, 1.2 KH₂PO₄, 35 NaCl, 16 Na₂HPO₄, 25 NaHCO₃, 10 HEPES, 10 glucose, and 134 sucrose; pH 7.4 at 37°C for 3 min. During isolation, 2,3-butanedione oxime (10 mmol/l) was added to the solution to prevent cell damage during the perfusion. Hearts were then perfused with the same solution containing type II collagenase (Worthington Biochemical) for approximately 15 min. Ventricular myocytes were then isolated by several low-speed centrifugations (7 g, 1 min). Myocytes were resuspended in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum (GIBCO), 4% nonessential amino acids (GIBCO), insulin-selenium-transferrin (GIBCO), 100 IU/ml penicillin, and 0.1 mg/ml streptomycin (GIBCO). Cytosine -D-arabino-furanoside (10 µmol/l, Sigma) was added to the medium to inhibit the proliferation of nonmuscle cells. Myocytes were then plated on laminin-coated dishes (Sigma). After an adhesion period of 1 h, myocytes were rinsed twice with serum-free culture medium to eliminate nonadherent cells, dead cells, and debris. The 24-h treatments were conducted in serum-free medium. Animal experiments were performed in accordance with Swiss law. They were submitted and approved by the Veterinary Office of Canton Vaud.

Patch-clamp measurements. The whole cell configuration of the patch-clamp technique was used to record I_Na and action potentials (AP). Experiments were performed at room temperature (22–24°C). Current and AP recordings were performed using VE-2 (Alembic Instruments) and Multiclamp 700A (Axon Instruments) amplifiers, respectively. For I_Na, glass pipettes ( resistance: 1–2 M) were filled with a solution containing (in mmol/l) 60 CsCl, 50 aspartic acid, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 11 EGTA, and 5 Na₂ATP (pH 7.3 with CsOH). For APs, the internal solution was (mmol/l): 10 NaCl, 130 KCl, 2 CaCl₂, 10 glucose, 1 MgCl₂, 10 HEPES, 3 Na₂ATP, and 5 EGTA (pH 7.3 with KOH). Myocytes were bathed with a solution containing (in mmol/l) 137 NaCl, 5 KCl, 2 CaCl₂, 10 glucose, 1 MgCl₂, and 10 HEPES (pH 7.4 with NaOH). For current recordings, external Na⁺ was reduced to 14 mM using N-methyl-glucamine as a Na⁺ substitute, and KCl was replaced by an equal amount of CsCl. The values were not corrected for the measured –16-mV junction potential. The amplitude of I_Na monitored according to a steady-state pulse protocol (a 20-ms depolarizing pulse to –20 mV from a holding potential of –80 mV) was calculated as the difference between the peak inward current and the current measured at the end of the test pulse, and its density (pA/pF) was obtained by dividing I_Na amplitude by the membrane capacitance. Cell capacitance was calculated by using the transient capacitive current caused by a +5-mV pulse from the holding potential. To quantify the voltage dependence of steady-state activation and inactivation, data from individual cells were fitted with the Boltzmann relationship, y·(V_m) = 1/1 + exp[(V_m – V)/K], in which y is the normalized current or conductance, V is the voltage at which half of the available channels are inactivated, K is the slope factor, and V_m is the membrane potential. The voltage dependence of inactivation was determined by measuring current in response to pulses (20 ms) to –20 mV that had been preceded by 500-ms pulses applied in a series of 5-mV incremental voltages. All experiments were started after an equilibration period until peak I_Na was stable. APs were elicited by 40-pA depolarizing current pulses. These stimuli were delivered for a 4-ms test pulse at 0.5 Hz, and the resting potential, the amplitude of the AP, and the AP durations (APDs) from the peak of the AP to the 30, 50, and 90% repolarization level (APD₃₀, APD₅₀, and APD₉₀) were measured. Average of APs (30 per myocyte) recorded from aldosterone-treated myocytes was compared with that of control myocytes.

Western blot experiments. Cultured cardiomyocytes were rinsed twice with phosphate-buffered solution, and lysis buffer was added [20 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton X-100, 1 mM PMSF, and complete protease inhibitor cocktail (Roche)]. Lysates were rotated for 60 min at 4°C to allow solubilization of proteins. The soluble fraction of a 10-min centrifugation at 13,000 g was recovered and used for Western blot experiments after dilution in 1x SDS sample buffer. The reagents used were 30% acrylamide/bis-acrylamide (37.5:1 vol/vol) solution from National Diagnostics. N,N,N',N'-Tetramethylethylenediamine, ammonium persulfate, and PMSF were from Acros (Belgium). Nitrocellulose sheets (0.2 µm pore size) were from Schleicher & Schuell. BSA and Bradford reagent solution were purchased from Uptima (France). Horseradish peroxidase-conjugated anti-rabbit secondary antibody and protein-A-Sepharose are from Amersham. Western blots were developed using Super-Signal West-Dura Extended Duration Substrate (Pierce). ECL signals were recorded on Kodak X-OMAT AR film. For the protein gels, 1.5-mm-thick minigels (Bio-Rad system) were cast using a 5–15% acrylamide gradient. Typical runs were carried out at 200 V for 55 min. Proteins were transferred to nitrocellulose using a submarine system: transfer buffer consisted of 18.6 mM Tris, 140 mM glycine, 20% methanol, and 0.1% SDS. Typical transfers were carried out at 100 V for 200 min keeping the transfer apparatus in an ice bath. Membranes were blocked for at least 60 min in blocking solution [5% skim milk solution in Tris-buffered saline 0.1% Tween (TBS-Tween)]. Antibodies and sera were prepared in a 5% BSA solution in TBS-Tween. After 3x 5-min washes in TBS-Tween, membranes were incubated again with blocking solution for 15 min after which secondary antibody was added. After a 1-h incubation at room temperature under agitation, membranes were washed as above and bound immunoglobulins were revealed by ECL.

TaqMan real-time quantitative PCR. RNA was extracted from 2 x 10⁵ myocytes cultured for 24 h using Quiagen RNeasy (Quiagen, Germany). cDNA was synthesized from 500 ng of total RNA using the MU-MLV reverse transcriptase according to the manufacturer protocol (Q-Biogene EMMLV100). Thirty nanograms of cDNA combined with 1x TaqMan Universal Master Mix (Applied Biosystems) and 1 µl of either Na_v1.5, Ca_v1.2 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (Applied Biosystems respectively Mm99999915, Mm00437917 and Mm00451971) were loaded into each well. The 96-well thermal plate was cycled at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, and 60°C for 1 min. Data were collected and analyzed using the threshold cycle (C_t) relative quantification method (24). The GAPDH reference gene was used for data normalization.

Antibodies and reagents. Anti-Na_v1.5 rabbit serum (ASC-005, Alomone) and a monoclonal anti-actin (A2066, Sigma Chemical) antibody were used. Anti-Nedd4–1, anti-Nedd4–2, and anti-phospho-Nedd4–2 antibodies have been described elsewhere (15, 20). A specific Ca_v1.2 (1C) antibody was kindly provided by Dr. J. Hell (University of Iowa). The anti-Sgk1 antibody was purchased at Upstate (07-315). The polyclonal antibody raised against the terminal 14 residues of 1 was kindly provided by Dr. L. Isom (University of Michigan). Actinomycin D, D-aldosterone, and dexamethasone were dissolved in DMSO (100%), and brefeldin A, cycloheximide, RU38486, and spironolactone were prepared in ethanol (100%) until used. The final concentration of DMSO and ethanol alone had no effect on I_Na (data not shown). All drugs were from Sigma (Buchs, Switzerland).

Statistical analyses. Values are expressed as means ± SE. Mann-Whitney test was used when comparing two groups, and Kruskal-Wallis procedure followed by a Dunn’s posttest was used to compare more than two groups. Statistical significance is set at P < 0.05.

	RESULTS

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Aldosterone prolongs the AP duration and upregulates I_Ca in mouse ventricular myocytes. In the present study, we performed short-term (up to 24 h) primary cultures of adult mouse ventricular myocytes that were exposed to high concentrations of aldosterone for different time intervals. First, we carried out a set of experiments aimed at validating this cellular model. The functional consequences of the aldosterone treatment were studied by analyzing the shape of the AP in isolated mouse ventricular myocytes treated or not treated with aldosterone. Figure 1A shows representative traces of AP from mouse ventricular myocytes treated for 24 h with 1 µM aldosterone and control conditions, recorded under current-clamp conditions. Whereas the resting membrane potential was unchanged (control: –73.7 ± 1.1 mV, aldosterone: –73.3 ± 1.2 mV, Fig. 1B), the mean peak AP value (Fig. 1C) tended to be higher in aldosterone conditions (+44.4 ± 2.6 mV) than in control myocytes (+41.3 ± 2.6 mV), but this result did not reach statistical significance. As illustrated in Fig. 1D, repolarization of 90% of the APD was significantly prolonged in myocytes incubated with aldosterone (48.7 ± 3.0 ms) compared with control conditions (31.9 ± 1.2 ms). We then tested whether in mouse ventricular myocytes the L-type Ca²⁺ current (I_Ca) is modulated similarly to what has been reported in rat myocytes (3). Because variations in cell size might account for differences in current amplitudes, we have normalized the measured currents to the membrane capacitance, which was on average 124.1 ± 4.0 pF in control (n = 22) and 132.7 ± 5.8 pF (n = 22) in aldosterone conditions (not significantly different). Figure 2A shows that I_Ca density was increased in mouse myocytes treated with 1 µM aldosterone for 24 h (+48%) compared with control conditions, with no apparent shift in the current-voltage relationship (Fig. 2B). To investigate the mechanisms underlying this current increase, the mRNA levels of the Ca_v1.2 gene (CACNA1C) from myocytes cultured under the two conditions were quantified using specific TaqMan probes (see MATERIALS AND METHODS), but no difference was observed (Fig. 2C). Furthermore, Fig. 2D shows a representative Western blot performed using an anti-Ca_v1.2 antibody (17), illustrating that despite the upregulated I_Ca density, aldosterone did not increase the total amount of cellular Ca_v1.2 protein. Note that to test whether a 1.5-fold increase may have been observed under our experimental conditions, we loaded increasing amounts of myocyte lysate (1x-1.5x–2x) in one gel, and it is apparent that such a difference could have been detected (Fig. 2E).

View larger version (14K): [in this window] [in a new window]   Fig. 1. Aldosterone (Aldo) increases the action potential (AP) duration (APD) in mouse ventricular myocytes. A: superimposition of APs recorded from cardiac cells in absence () or presence () of 1 µM Aldo for 24 h. B–C: resting membrane potential and peak of AP in control (open bars) and Aldo (solid bars) conditions. NS, not significant. D: APD at 30, 50, and 90% repolarization of isolated myocytes in control (open bars) and Aldo (solid bars) conditions. ***P < 0.001, Mann-Whitney; B–D: n = 13–14 cells.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Upregulation of L-type Ca2+ current (ICa) by Aldo. A: representative ICa traces (protocol in inset) obtained from ventricular myocytes in control and after 1 µM Aldo treatment for 24 h. B: current density-voltage relationships of ICa in myocytes after control and Aldo treatment, *P < 0.05, Mann-Whitney. C: quantitative PCR experiments. Bar graph representing amount of Cav1.2 mRNA in control myocytes or treated for 24 h with 1 µM Aldo. Number of mice is indicated in the bars. For each animal, analysis was performed in duplicate; results were obtained as cycle threshold (Ct) and normalized to the reference gene GAPDH. D: representative Western blots showing the levels of expression of Cav1.2 under control and Aldo conditions. Specificity of Cav1.2 antibody was tested using transfected HEK-293 cells. Forty micrograms of protein were loaded for the HEK-293 lysates, and 80 µg of protein were loaded for myocytes lysates. Four of such experiments yielded comparable results. Protein loading was controlled by anti-actin immunoblotting. E: control anti-Cav1.2 Western blot performed with increasing amounts of total protein (40, 60, and 80 µg/lanes).

Aldosterone upregulates I_Na in mouse ventricular myocytes. After this first set of experiments, we focused on the modulation of the cardiac I_Na by high concentrations of aldosterone, which was the main goal of our study. Figure 3 shows representative examples of voltage-gated inward Na⁺ currents recorded in myocytes treated with (Fig. 3B) or without (Fig. 3A) 1 µM aldosterone for 24 h. As shown in Fig. 3D, the peak I_Na density was significantly higher in aldosterone-treated myocytes (+55%) compared with control conditions. After 24 h incubation with the MR synthetic agonist fludrocortisone (1 µM), the I_Na density was increased by +96% (Fig. 3, C and D). We also treated myocytes with lower concentrations of aldosterone that are in the range of the measured intracardiac concentrations (16 nM) (11) . As shown on the current-voltage curves (Fig. 3E), 10 and 100 nM significantly increased the I_Na peak current by 23% and 35%, respectively. Analysis of the voltage dependence of I_Na activation and inactivation properties at steady state in control and 1 µM aldosterone-treated conditions revealed no difference between the parameters studied (Fig. 4). To study a possible rapid effect of aldosterone, we perfused isolated myocytes that were cultured under control conditions for 4 or 24 h, with 1 µM aldosterone. However, no modification of I_Na was seen under each condition (data not shown). We also tested whether adding 1 µM aldosterone into the patch pipette solution would regulate I_Na. One minute after the rupture of the membrane patch, the control and aldosterone currents were 153 ± 22 pA/pF (n = 5 cells) and 142 ± 21 pA/pF (n = 6), respectively, whereas the I_Na density values after 9 min were 150 ± 20 pA/pF for control currents and 144 ± 17 pA/pF for aldosterone currents. The values at 9 min are not significantly different compared with the 1-min values in both conditions. Finally, in myocytes cultured with 1 µM aldosterone for 4 h, no change in I_Na was observed. These results suggest that a rapid nongenomic effect of aldosterone (26) is not responsible for the increased I_Na.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Aldo stimulates Na+ current (INa) in ventricular myocytes. A–C: representative traces of INa recorded (protocol in inset) in ventricular myocytes after 24 h of incubation in control (A), 1 µM Aldo (B), and 1 µM fludrocortisone (C). D: current densities in control, Aldo, and fludrocortisone conditions. **P < 0.01 and ***P < 0.001, Mann-Whitney. When the holding potential was –120 mV, Aldo increased the INa peak current by 50% (not shown, n = 14–15 cells). E: normalized current density-voltage relationships of INa in control and with increasing Aldo concentrations. *P < 0.05 compared with control.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Steady-state activation and inactivation properties after Aldo treatment. Activation properties were determined from current-voltage (I-V) relationships by normalizing peak INa to driving force and maximal INa and plotting normalized conductance vs. membrane potential (Vm). Protocols used are in insets. Boltzmann curves of individual cells were fitted to steady-state activation data: control V = –35.3 ± 1.6 mV, K = 6.2 ± 0.5; Aldo V = –35.9 ± 1.6 mV; K = 5.9 ± 0.3, where V is the voltage at which half of the available channels are inactivated; and K is the slope factor. Voltage dependence of steady-state inactivation parameters were the following: control V = –75.5 ± 0.7 mV, K = 6.5 ± 0.1, Aldo V = –76.0 ± 0.7 mV; K = 7.0 ± 0.3. Differences between groups were not statistically significant (Mann-Whitney).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Stimulatory effect of Aldo is mediated by mineralocorticoid receptors. A: comparison of INa densities among control, 24 h 1 µM Aldo, and 1 µM Aldo + 10 µM spironolactone (SP). **P < 0.01, Dunn’s posttest. B: current densities of INa in control, 1 µM dexamethasone (DEX), and DEX + 10 µM RU-38486 (glucocorticoid receptor antagonist) conditions. *P < 0.05, Dunn’s posttest. C: current densities of INa recorded from myocytes under control conditions and treated with 1 µM Aldo or 1 µM Aldo + 10 µM RU-38486. *P < 0.05, Dunn’s posttest. D: current densities of INa recorded from HEK-293 cells stably expressing Nav1.5 under control conditions and treated with 1 µM Aldo.

Cellular mechanisms responsible for the aldosterone-induced increase in I_Na. Classical aldosterone effects are mediated by the interaction of intracellular MR proteins with promoters of target genes, thus enhancing their transcription (16). Hence, aldosterone modulation of cardiac I_Na may be due to an increase in channel density at the cell surface through stimulation of mRNA transcription and consequent protein expression. To elucidate the cellular mechanisms of aldosterone on I_Na, myocytes were incubated with aldosterone for 24 h in the presence of actinomycin D (inhibitor of transcription, 1 µg/ml), cycloheximide (protein synthesis inhibitor, 10 µg/ml), or brefeldin A [BFA, inhibitor of the protein traffic in the secretory pathway (21), 10 µg/ml]. As illustrated in Fig. 6A, actinomycin D, cycloheximide, or BFA prevented the increase in I_Na induced by aldosterone. Under control conditions, treatment with each compound alone had no significant effect on I_Na (Fig. 6B). Altogether, these results suggest that the aldosterone-induced I_Na increase is dependent on either stimulation of Na_v1.5 expression and/or other genes involved in trafficking of Na_v1.5.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Cellular mechanisms of Aldo -mediated INa stimulation. A: INa densities recorded from myocytes in control or treated with 1 µM Aldo alone or with actinomycin D, cycloheximide, or brefeldin A. **P < 0.01, Dunn’s posttest. B: normalized INa recorded from myocytes in control or treated with actinomycin D, cycloheximide, or brefeldin A alone; groups were statistically not different (Kruskal-Wallis). C: quantitative PCR experiments. Bar graph representing amount of Nav1.5 mRNA in control myocytes or treated with 1 µM Aldo or with 1 µM dexamethasone for 24 h. Number of mice is indicated in bars. For each animal analysis was performed in duplicate; results were obtained as cycle Ct and normalized to the reference gene GAPDH. D: representative Western blots showing levels of expression of Nav1.5, Nedd4–2, phospho-Nedd4–2, Nedd4–1, Sgk1, and 1-Nav subunit under control, Aldo, and dexamethasone conditions. Specificity of Nav1.5 antibody was tested using transfected HEK-293 cells. Forty micrograms of protein were loaded for the HEK-293 lysates, and 80 µg of protein were loaded for myocytes lysates. Three of such experiments yielded comparable results. Protein loading was controlled by anti-actin immunoblotting. E: control anti-Nav1.5 Western blot performed with increasing amounts of total protein (40, 60, and 80 µg/lane).

Recently, we and others (14, 41) obtained evidence that the Na_v1.5 cell membrane density is regulated by the activity of the protein-ubiquitin ligase Nedd4–2. We therefore measured by Western blot analysis the protein levels of Nedd4–2 and phosphorylated (inactive) Nedd4–2 (15) under aldosterone, dexamethasone, and control conditions. The representative Western blots presented in Fig. 6D show no difference between the different tested conditions, again suggesting that other gene products are mediating the aldosterone effect on I_Na. We also tested the expression of Nedd4–1, which is another member of the Nedd4-like family of genes binding to Na_v1.5 (14, 35); Sgk1, a kinase that can phosphorylate Nedd4–2 (10), and the 1-subunit of the Na_v1.5 channel. The expression of all tested proteins was not influenced by either aldosterone or dexamethasone (Fig. 6D).

	DISCUSSION

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About 50% of HF patients die of SCD because of ventricular arrhythmias (8). Two recent large clinical trials (33, 34) provided evidence that MR antagonists, including spironolactone, reduce the mortality due to SCD, supporting the hypothesis that HF-induced hyperaldosteronemia may be proarrhythmogenic. However, thus far the influence of increased levels of aldosterone on cardiac ion channels, which are key molecular players in the genesis of arrhythmias, is still poorly understood.

The main novel finding of this study is that aldosterone also upregulates the I_Na recorded in mouse ventricular myocytes, similarly to what has been previously shown for I_Ca (3). The aldosterone-dependent I_Na increase was abolished by co-incubation with spironolactone, actinomycin D, cycloheximide, or brefeldin A but not with the GR antagonist RU-38486. Interestingly, the levels of expression of Na_v1.5 and Ca_v1.2 mRNAs and proteins were not increased by the steroid hormone treatments.

Mineralocorticoid and glucocorticoid hormones regulate cardiac ion channels. In previous works, Bénitah and coworkers reported that cardiac ion channels can be regulated by aldosterone (2, 3). Using experimental protocols similar to the present study, they showed that aldosterone stimulates I_Ca in rat ventricular myocytes and that I_to was downregulated, both effects leading to a prolongation of the APD (2, 3). In this study, we confirm the effect of aldosterone on APD and I_Ca, using mouse ventricular myocytes (Figs. 1 and 2) but, interestingly, we also show that I_Na is upregulated. Further supporting a role for aldosterone in electrical remodeling processes in heart diseases, a recent study in rat showed that I_Ca and L-type Ca²⁺ channel mRNAs are upregulated 1 wk postinfarction of the left ventricle and that this effect was prevented by using the MR antagonist RU-28318 (30). However, the regulation of I_Na was not investigated in this work. Aldosterone-dependent regulation of I_Ca (2, 3) and I_Na (this study) in ventricular myocytes displays many similarities, suggesting that the mechanisms underlying these effects may be overlapping. However, thus far no study addressed this possibility.

In the studies mentioned above, the role of MRs in mediating the effects of aldosterone, i.e., modulation of cardiac currents, is suggested by the fact that the MR antagonist spironolactone blocks its action. However, these findings may also in part reflect the antiglucocorticoid effect of spironolactone when partially binding to GRs, which has been shown to occur with an apparent K_d of 2 µM (9). Moreover, it should be pointed out that the functional role of MRs and their protection from cortisol occupancy by the activity of the 11--hydroxysteroid-dehydrogenase type 2 in cardiac myocytes is still a matter of controversy (13) and, as a result, the specific roles of MRs and/or GRs in these regulatory pathways remain to be clarified. Because the pharmacological approaches used in the mentioned studies (2, 3), as well as our present work, may be biased by the lack of specificity of the MR and GR agonists and inhibitors (12), it may be proposed that this issue should be more adequately addressed using cellular or animal models knocked out for the specific receptors (4).

In the present study, the I_Na was significantly increased by 10 and 100 nM of aldosterone. Such high concentrations may be relevant in the context of HF-induced hyperaldosteronemia because intracardiac aldosterone levels of 16 nM have been reported in rat myocardium (36).

We also obtained evidence that cardiac I_Na is upregulated by the glucocorticoid dexamethasone. Only few studies have addressed the question of regulation of cardiac ion channels by glucocorticoids. Cardiomyocytes from neonatal mice treated for 7 days with dexamethasone displayed increased I_Ca and decreased I_to (42), a finding that is qualitatively similar to the effect of aldosterone on adult rat myocytes (2). It therefore appears that, in the heart, the activation of both gluco- and mineralocorticoid pathways may lead to similar effects on ion channels. However, as mentioned above, the dissection of the two pathways, and their possible overlap, can only be adequately performed using specific knock-out models. In humans, little is known about the role of steroid treatment on cardiac channel function. It should, however, be mentioned that corticosteroid therapy has been reported to be very effective in decreasing the arrhythmic manifestations caused by a loss-of-function mutation of Na_v1.5 in two pediatric patients (38). The findings of the present study may, in part, underlie this beneficial effect.

Cellular mechanisms of aldosterone-dependent regulation of cardiac ion channels. Aldosterone exerts genomic and nongenomic effects on its cellular targets (13). Similarly to the effect on I_Ca in rat ventricular cells (3), we did not observe any short-term (likely nongenomic) effect of aldosterone acute perfusion on mouse myocytes I_Na. In contrast, I_Na was increased after 24 h incubation with aldosterone. Again, as reported for I_Ca (3), inhibition of gene transcription and protein translation inhibited the aldosterone-dependent stimulation of I_Na. In addition, we report here that brefeldin A, known to block the secretory pathway of membrane proteins (21), abolished the I_Na increase caused by aldosterone incubation. These findings suggest that the genes regulated by aldosterone are either encoding the channel itself or proteins involved in channel trafficking or stabilization at the plasma membrane. However, the quantitative RT-PCR and Western blot analysis of the myocytes treated for 24 h with aldosterone and dexamethasone did not reveal any significant increase in mRNA levels nor in the total cellular pool of Na_v1.5 protein, which may have explained the larger I_Na. A similar result was obtained when analyzing the mRNA and total cellular expression of Ca_v1.2 of aldosterone-treated cells. These surprising findings strongly suggest that, in this model, the observed increase in I_Na and I_Ca are due to a redistribution of the channels located in an intracellular pool (48) toward the cell membrane compartment. In fact, such a "shuttling" mechanism is known to be important for the action of many hormones [vasopressin (5) and insulin (43), for instance] on the trafficking of membrane transporters. However, because of a lack of sensitive and reproducible techniques, we were not able to provide direct evidence supporting this hypothesis.

Little is known about the molecular determinants of trafficking and membrane turnover of Na_v1.5. In a recent work (29), interaction of Na_v1.5 with ankyrin-G has been proposed to be necessary for proper targeting of the channel to the plasma membrane. On the other hand, because we have previously shown that, in heterologous expression systems, Na_v1.5 channel density at the plasma membrane may be regulated by E3 ubiquitin ligases of the Nedd4 family (35, 41), we also investigated the expression level of Nedd4–1 and Nedd4–2. However, under these experimental conditions, neither aldosterone nor dexamethasone modified their expression in isolated cardiac myocytes. In Xenopus oocytes, the protein kinase Sgk1, known to be induced by aldosterone in the kidney (7), has been shown to phosphorylate and inactivate Nedd4–2 (10). However, using an anti-phospho-Nedd4–2 antibody (15), we did not see any aldosterone-induced increase in phosphorylation, suggesting that decreased ubiquitination of Na_v1.5 by Nedd4–2 is not involved in the observed phenomenon. These results are in line with the fact that Sgk1 protein was not found to be upregulated (Fig. 6D) and are consistent with the observation that the Sgk1 gene is not increased upon aldosterone treatment in heart (44). The Na_v 1-subunit has been reported to increase the Na_v1.5-mediated currents in expression models (18). However, this protein was not found to be upregulated by aldosterone and is, therefore, not likely to be involved in the observed phenomenon.

A systematic investigation, such as a comprehensive transcriptome analysis of myocytes treated with aldosterone or dexamethasone, would be needed to understand more thoroughly the molecular and cellular mechanisms underlying the observed regulation of cardiac ion channels.

Pathophysiological relevance of aldosterone-dependent I_Na increase. The arrhythmogenic mechanisms underlying SCD in the context of HF are multiple and complex (39). Cardiac Na⁺ current alterations have been implicated in a large number of arrhythmic syndromes caused by mutations of SCN5A, the gene encoding Na_v1.5 (37). However, the role of Na_v1.5 in acquired cardiac disorders such as ischemic heart disease and HF is not well understood. In animal studies, a downregulation of the cardiac Na⁺ channel protein has been reported in dog models of ischemic heart disease (47) and tachyarrhythmia-induced HF (45). In contrast, in guinea pigs in which HF was obtained by banding of the thoracic aorta (chronic pressure overload-induced HF), an I_Na increase of up to about 100% was reported (1). APD prolongation, as observed in this work and in Bénitah’s studies (2, 3, 30), is an important arrhythmogenic factor in the etiology of SCD in the context of HF (39). It can be hypothesized that aldosterone-dependent upregulation of both depolarizing I_Na and I_Ca, as well as downregulation of K⁺ currents, may all participate in a synergistic manner to the AP prolongation. Upregulation of late I_Na, which may contribute to AP prolongation, has been reported in different models of HF (27, 40). Addressing the question whether such late I_Na may also be regulated by steroid hormones represents one of our future tasks. Altogether, these findings illustrate that dysregulation of the cardiac Na⁺ channel expression is very much dependent on the experimental model investigated, and it may be therefore possible that the aldosterone-induced increase in I_Na described here is only present in some specific forms of HF in humans. However, based on the clear beneficial effect of spironolactone and eplerenone on SCD, these aspects deserve to be investigated in future studies.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by a grant of the Swiss National Science Foundation (SNF-Professorship 632-66149.01 to H. Abriel), Fondation Prévot (Geneva), and Fondation Vaudoise de Cardiologie.

	ACKNOWLEDGMENTS

 
We thank Romina Behar for excellent technical and editorial help and Dr. Jean-Daniel Horisberger, Dr. Miguel van Bemmelen, Dr. Jan Loffing, and Dr. Dmitri Firsov for useful suggestions about the manuscript. We also thank Prof. Pavel Kucera for the patch-clamp experiments that were performed in the Department of Physiology (University of Lausanne).

	FOOTNOTES

 

Address for reprint requests and other correspondence: H. Abriel, Dept. of Pharmacology and Toxicology, and Service of Cardiology, Univ. of Lausanne, Bugnon, 27, 1005 Lausanne, Switzerland (e-mail: Hugues.Abriel{at}unil.ch)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^* C. Boixel and B. Gavillet contributed equally to this study.

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