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Am J Physiol Heart Circ Physiol 293: H2710-H2718, 2007. First published July 20, 2007; doi:10.1152/ajpheart.01399.2006
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Aldosterone modulates If current through gene expression in cultured neonatal rat ventricular myocytes

Takao Muto,1,* Norihiro Ueda,6,* Tobias Opthof,2,3 Tomoko Ohkusa,4 Kohzo Nagata,5 Shinsuke Suzuki,4 Yukiomi Tsuji,6 Mitsuru Horiba,6 Jong-Kook Lee,6 Haruo Honjo,6 Kaichiro Kamiya,6 Itsuo Kodama,6 and Kenji Yasui1

1Research Institute of Environmental Medicine, Department of Bio-information Analysis, Nagoya University, Nagoya, Japan; 2Experimental Cardiology Group, Center for Heart Failure Research, Academic Medical Center, Amsterdam, the Netherlands; 3Department of Medical Physiology, University Medical Center Utrecht, Utrecht, the Netherlands; 4Yamaguchi University School of Medicine, Yamaguchi, Japan; 5Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Japan; and 6Research Institute of Environmental Medicine, Department of Cardiovascular Research, Nagoya University, Nagoya, Japan

Submitted 21 December 2006 ; accepted in final form 17 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mineralocorticoid receptor (MR) antagonists decrease the incidence of sudden cardiac death in patients with heart failure, as has been reported in two clinical trials (Randomized Aldactone Evaluation Study and Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study). Aldosterone has been shown to increase the propensity to arrhythmias by changing the expression or function of various ion channels. In this study, we investigate the effect of aldosterone on the expression of hyperpolarization-activated current (If) channels in cultured neonatal rat ventricular myocytes, using the whole cell patch-clamp technique, real-time PCR, and Western blotting. Incubation with 10 nM aldosterone for 17–24 h significantly accelerates the rate of spontaneous beating by increasing diastolic depolarization. If current elicited by hyperpolarization from –50 to –130 mV significantly increases aldosterone by 10 nM (by 1.9-fold). Exposure to aldosterone for 1.5 h increases hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 mRNA by 26.3% and HCN4 mRNA by 47.2%, whereas HCN1 mRNA expression remains unaffected. Aldosterone (24-h incubation) increases the expression of HCN2 protein (by 60.0%) and HCN4 protein (by 84.8%), but not HCN1 protein. MR antagonists (1 µM eplerenone or 0.1 µM spironolactone) abolish the increase of If channel expression (currents, mRNA, and protein levels) by 10 nM aldosterone. In contrast, 1 µM aldosterone downregulated If channel gene expression. Glucocorticoid receptor antagonist (100 nM RU-38486) did not affect the increase of If current by 10 nM aldosterone. These findings suggest that aldosterone in physiological concentrations upregulates If channel gene expression by MR activation in cardiac myocytes and may increase excitability, which may have a potential proarrhythmic bearing under pathophysiological conditions.

eplerenone; spironolactone; automaticity; arrhythmia; HCN gene

ALDOSTERONE (MINERALOCORTICOID hormone) blockade reduces morbidity and mortality in patients with heart failure (28, 29). In particular, mineralocorticoid antagonists decreased the occurrence of sudden cardiac death [29% in the Randomized Aldactone Evaluation Study (RALES), and 21% in the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS)], suggesting that aldosterone may have a pro-arrhythmic effect. Patients with primary aldosteronism are more likely to have left ventricular hypertrophy and stroke than patients with essential hypertension, even when the degree of hypertension is similar in both patient groups (31, 35). Thus aldosterone may have adverse effects on the heart.

Most effects of aldosterone are mediated by the mineralocorticoid receptor (MR), which belongs to the steroid receptor superfamily. They are mediated by ligand-dependent transcription (23). The main action of aldosterone is acceleration of electrolyte transport in epithelial cells, particularly in the kidney, but also in the salivary gland and colon, where it increases Na+ reabsorption and K+ excretion (4). MRs are also localized in nonepithelial cells, among which are cardiac myocytes (22). Various effects of aldosterone on cardiac ion channels have been reported. Aldosterone increases L-type Ca2+ current and decreases transient outward K+ current (Ito) in adult rat ventricular myocytes (2, 3) and increases the fast Na+ current in adult mouse ventricular myocytes (5). Lalevée et al. (20) reported that aldosterone increases the beating frequency of neonatal rat cardiomyocytes by enhanced T-type and L-type Ca2+ currents following stimulation of gene expression. The expression of Na+-K+-ATPase was also enhanced in aldosterone-treated neonatal rat myocytes (17).

Hyperpolarization-activated current (If) channels are abundantly expressed in sinus node and Purkinje fibers in the adult heart and play an important role in cardiac pacemaking (11, 18). If channels are functionally relevant in the embryonic ventricle (38). Isolated embryonic ventricular myocytes and cultured neonatal myocytes display automaticity. In adult ventricular myocytes, If channels are present, but they do not activate in the physiological potential range (13, 16). In dedifferentiated adult ventricular myocytes in primary culture, If current activates at physiological potentials because of a shift of the activation curve (13). Under pathological conditions such as heart failure and hypertrophy, upregulation of If channels has also been reported in ventricular cells (7, 16).

In the present study, we examined the effects of aldosterone on If channel gene expression in cultured neonatal rat myocytes, using the whole cell patch-clamp technique, real-time PCR, and Western blotting. We also studied the action of MR antagonists (eplerenone and spironolactone) on the effects of aldosterone on If channels and current.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell isolation and culture. Single ventricular myocytes were obtained from the ventricles of 1- to 2-day-old neonatal Wistar rats (Worthington Biochemical). Myocytes were cultured in L-15 with 10% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C. Ventricular myocytes were plated at a density of 105/ml for patch-clamp experiments and at a density of 5 x 105/ml for molecular experiments, respectively. After 48 h cultivation, the medium was replaced by fetal bovine serum-free medium and incubated for 10 h before drug application. All animal procedures were approved by the Animal Care and Use Committee, Research Institute of Environmental Medicine, Nagoya University.

Electrophysiological experiments. Ventricular myocytes were cultured on glass coverslips and were superfused with normal Tyrode solution containing (in mM) 146.9 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.33 NaH2PO4, and 5 HEPES (pH = 7.35). Whole cell patch-clamp was performed using Axopatch 200B (Axon Instruments). Action potentials were recorded in normal Tyrode solution using an internal solution containing (in mM) 60 KOH, 80 KCl, 40 aspartate, 5 HEPES, 10 EGTA, 5 MgATP, 5 Na2-phosphocreatinine, and 0.65 CaCl2 (pH 7.2, pCa 7.96). To isolate If current, 2 BaCl2, 2 NiCl2, and 0.5 4-aminopyridine (in mM) were added to normal Tyrode solution with the same K+-rich internal solution (38). Command generation (voltage and current clamps) and data acquisition were performed with pClamp8 software and a Digidata 1200. The recording pipettes had tip resistances ranging from 3.5 to 4.5 M{Omega} when filled with the internal solution. Cell capacitance was measured by the application of a ramp voltage pulse of 0.5 V/s at a potential ranging between –50 and +70 mV.

The If activation curve was constructed from the amplitude of time-dependent inward current during hyperpolarizing pulses (–140 to –60 mV) from a holding potential of –50 mV at a frequency of 0.1 Hz. The duration of the step was increased from 450 to 2,850 ms, dependent on the applied voltage.

The specific conductance of If was determined for each cell according to the equation:

Formula
where g is the conductance calculated at the membrane potential Vm, I is the current amplitude, and Vrev is the reversal potential. Conductance curves (activation curves) were fitted to the Boltzmann distribution:

Formula
where gmax is maximum conductance, V1/2 is the voltage at half-maximal activation, and k is the slope factor.

Vrev was calculated from the analysis of tail currents. After a hyperpolarizing pulse to –130 mV of 1-s duration, tail currents were elicited by 10-mV steps to potentials from +10 mV to –50 mV. Vrev values were as follows: control 30.4 ± 2.8 mV, n = 5; aldosterone 33.1 ± 3.0 mV, n = 5; aldosterone and eplerenone 29.5 ± 2.1 mV, n = 5; aldosterone and spironolactone 29.2 ± 1.9 mV, n = 5. These values were not significantly different. We measured voltage dependence of activation and deactivation time constants. Deactivation kinetics were obtained by stepping to potentials from –40 to –80 mV by 10 mV after activating the current by hyperpolarizing pulse to –130 mV. Time constants of activation and deactivation were obtained by fitting the experimental trace to a single exponential curve.

We measured diastolic depolarization rate (DDR) as the slope from the moment of maximum diastolic potential (MDP) during the following 3 mV of depolarization. MDP, DDR, and beating rate were averaged from 10 consecutive action potentials. All experiments were carried out at 35–37°C.

PCR analysis. Total RNA of cultured myocytes was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany). Single-stranded cDNA synthesis was performed with total RNA using oligo(dT) primer and SuperScript II Reverse Transcriptase (Gibco BRL, Life Technologies) after DNase treatment of total RNA. To quantify mRNA expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) 1, HCN2, HCN4, and GAPDH, we applied a real-time fluorogenic 5'-nuclease PCR assay (ABI Prism 7700, Perkin Elmer Biosystems) (26, 38). The primers and probes are listed in Table 1. Respective PCR products were subcloned by TA cloning (pGEM-T Easy, Promega) and were verified by sequencing. Standard curves for the quantification of target molecules were constructed by amplification of successive template quantity of cDNA (from 103 to 107 copies). The threshold cycle was plotted against the logarithm of the initial molecule number of cDNA standards with linear relationships. Slope factors of HCN1, HCN2, HCN4, and GAPDH were –5.516, –3.83, –3.946, and –3.555 (cycles/log decade), respectively. The slope factor of the standard curve for HCN1 was small, but its linearity was excellent (the efficiency of PCR was weak). In a real-time PCR experiment, 150-ng cDNA of each sample were added to each tube.


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  		Table 1. Sequence of PCR primers and sequence-specific probes for HCN1-4 and GAPDH

 
Western blotting for If channel protein. Cultured myocytes were washed twice with PBS and lysed in lysis buffer [10 mM Tris·HCl, 1 mM EDTA (pH 8.0), 2% Triton X, protease inhibitor cocktail (Complete Mini, Roche, no. 11836153001)]. Cell lysates (200 µg of total protein measured by bicinchoninic acid protein assay) were loaded on standard SDS polyacrylamide gels. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane. Membranes were blocked in PBS containing 0.1% Tween 20 and 0.3 or 5% low fat milk for 1 h at room temperature and then incubated for 1 h at room temperature in 1:200 HCN1, 1:500 HCN2, and 1:500 HCN4 primary antibody, respectively (Alomone Laboratories, no. APC-056 for HCN1, no. APC-030 for HCN2, no. APC-052 for HCN4). The immunoblots were developed with horseradish peroxidase-conjugated secondary antibody (Sigma, no. A0545) at 1:15,000 dilution for 1 h at 37°C, followed by enhanced chemiluminescence (SuperSignal West Dura Extended Duration Substrate, Pierce Biotechnology, no. 34075). The intensity of protein bands by chemiluminescence was quantified by a CS Saver and Analyzer (charge-coupled device camera, ATTO & Rise) (36).

Drugs. Aldosterone, spironolactone, and RU-38486 were purchased from Sigma-Aldrich. Eplerenone was provided by Pfizer Japan. Aldosterone and RU-38486 were dissolved in ethanol, and final concentration of ethanol was under 0.1%. Eplerenone and spironolactone were prepared in DMSO. Final concentration of DMSO was 0.1%. 0.1% DMSO or 0.1% ethanol alone did not affect If current (data not shown).

Statistics. Data are presented as means ± SE. Statistical analysis of data was performed using paired and nonpaired t-test (activation of If currents) or ANOVA and Tukey's post hoc test (other patch-clamp data, quantitative PCR, and Western blotting). Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of aldosterone on action potentials in cultured myocytes. A low concentration (10 nM) of aldosterone was applied to myocytes for 17–24 h. Figure 1 shows representative traces of action potentials. Cultured myocytes showed spontaneous activity with slow diastolic depolarization. Aldosterone accelerated the spontaneous frequency of myocytes. The rate of spontaneous beating, MDP, and DDR are summarized in Table 2. Aldosterone-treated cells had larger DDR than that of control. MR antagonists (1 µM eplerenone or 0.1 µM spironolactone) abolished the increase of beating rate and DDR by aldosterone (Fig. 1 and Table 2). MDP in aldosterone-treated cell tended to be decreased (control: –65.2 ± 2.1 mV, n = 6; aldosterone: –58.0 ± 5.4 mV, n = 7, not significant). The application of only MR antagonist did not affect spontaneous activity (Table 2).


Figure 1
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  		Fig. 1. Effects of aldosterone and mineralocorticoid receptor (MR) antagonists on spontaneous action potentials in cultured neonatal rat myocytes. Myocytes were incubated for 17–24 h without drug, with 10 nM aldosterone, with 10 nM aldosterone + 1 µM eplerenone, or with 10 nM aldosterone + 0.1 µM spironolactone.

 

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  		Table 2. Action potential parameters

 
The increase of If current by aldosterone. We next investigated the effect of aldosterone on If currents in cultured myocytes. Cells were incubated with 10 nM aldosterone for 17–24 h. If current was elicited by hyperpolarizing pulses for 1 s from a holding potential of –50 mV to test potentials ranging from –60 to –130 mV (Fig. 2). Cell capacitance was not affected by aldosterone (control: 27.3 ± 1.7 pF, n = 7; aldosterone-treated cells: 27.8 ± 6.4 pF, n = 7). The inward currents induced by hyperpolarizing steps in aldosterone-treated cells were larger than those in control. Current density of time-dependent current at –130 mV was –1.6 ± 0.2 pA/pF (n = 7) in nontreated cells (control) and –3.1 ± 0.5 pA/pF (n = 7) in aldosterone-treated cells. MR antagonists (1 µM eplerenone or 0.1 µM spironolactone) prevented the increase of If current by 10 nM aldosterone. If current densities in the presence of MR antagonist were significantly smaller than those in control (control: –1.6 ± 0.2 pA/pF, n = 7; eplerenone: –1.2 ± 0.3 pA/pF, n = 7, P < 0.05 vs. control; spironolactone: –0.8 ± 0.1 pA/pF, n = 7, P < 0.05 vs. control).


Figure 2
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  		Fig. 2. The increase of hyperpolarization-activated currents (If) of aldosterone and of aldosterone with MR antagonists (eplerenone and spironolactone, respectively). A: representative If currents. B: current-voltage relationships (I-V curve). Myocytes were incubated for 24 h without drug ({circ}, n = 7), with 10 nM aldosterone (bullet, n = 7), with 10 nM aldosterone + 1 µM eplerenone ({blacktriangleup}, n = 7), and with 10 nM aldosterone + 0.1 µM spironolactone ({blacksquare}, n = 7). P < 0.05: *aldosterone vs. control, {dagger}aldosterone vs. aldosterone + eplerenone, {ddagger}aldosterone vs. aldosterone + spironolactone.

 
To examine whether the increase of If current induced by aldosterone is due to a change in channel kinetics or not, we analyzed the activation curve of If current (Fig. 3). The activation curves fitted to Boltzmann equation gave rise to a V1/2 at –86.5 ± 2.1 mV with a k of 16.8 ± 2.3 mV for control (n = 5), and V1/2 at –88.0 ± 4.9 mV with k of 16.8 ± 4.1 mV for aldosterone-treated myocytes (n = 5) (Fig. 3B). There were no significant differences of V1/2 and k between control and aldosterone-treated myocytes. We analyzed voltage dependence of activation and deactivation time constants of If currents in control and aldosterone-treated cells (Fig. 3, C and D). There were no significant differences for time constant of activation and deactivation between control (n = 5) and aldosterone-treated cells (n = 5). Aldosterone did not alter the If activation.


Figure 3
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  		Fig. 3. Activation and deactivation of If currents. For analysis of activation kinetics, If currents were elicited by hyperpolarization steps (–140 to –60 mV) from a holding potential of –50 mV. The duration of the step was increased from 450 to 2,850 ms. Deactivation kinetics were examined by stepping to potentials from –40 to –80 mV by 10 mV after activating the current by the pulse of –130 mV for 1 s. A: representative traces of If current activation in control. B: averaged activation curves ({circ}, control; bullet, aldosterone-treated cells) were obtained from the time-dependent inward current amplitude during hyperpolarization and fitted by Boltzmann equation. Voltage dependence of time constants of activation (C) and deactivation (D) were compared between control and aldosterone-treated cells. n = 5.

 
We also examined acute (5 min) and short-term (2–3 h) effects of aldosterone on If current in cultured cardiac myocytes. Five minutes after the application of 10 nM aldosterone, If current remained unchanged (–1.2 ± 0.7 and –1.3 ± 0.8 pA/pF at –130 mV, before and after the application of aldosterone, respectively, n = 10). Application of 10 nM aldosterone for 2–3 h elicited a small increase in If current, which failed, however, to reach statistical significance (control: –1.6 ± 0.2 pA/pF, n = 7; aldosterone: –2.1 ± 0.4 pA/pF, n = 5; P = 0.12).

Quantification of HCN mRNA expression. If channels are coded by four distinct genes: HCN1, HCN2, HCN3, and HCN4. Only HCN1, HCN2, and HCN4 are expressed in the hearts (33). We studied the change of mRNA expressions of HCN1, HCN2, and HCN4 by aldosterone. Quantitative PCR revealed the expressions of HCN2, HCN4, and HCN1 genes in neonatal cultured cardiac cells (Fig. 4A). The major HCN gene was HCN2. The application of 10 nM aldosterone for 1.5 h increased HCN2 mRNA by 26.2 ± 4.8% and HCN4 mRNA by 47.2 ± 7.7%, respectively (n = 4) (Fig. 4B). Aldosterone did not affect HCN1 mRNA expression. MR antagonists (1 µM eplerenone or 0.1 µM spironolactone) prevented the increases of HCN2 and HCN4 mRNAs induced by aldosterone. MR antagonists tended to reduce HCN1, HCN2, and HCN4 mRNA expressions to a lower level than in control. There was a significant decrease in HCN2 mRNA expression in spironolactone-treated cells. This may be explained by the fact that 0.1 µM spironolactone binds more strongly to MR than 1 µM eplerenone does (30).


Figure 4
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  		Fig. 4. Changes in mRNA expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) 1, HCN2, and HCN4 genes in cardiac cells by aldosterone and MR antagonists. Cultured myocytes were incubated for 1.5 h without drug, with 10 nM aldosterone, with 10 nM aldosterone + 1 µM eplerenone, or with 10 nM aldosterone + 0.1 µM spironolactone. A: HCN1, HCN2, and HCN4 mRNA levels were determined by a real-time RT-PCR and normalized to GAPDH mRNA levels. HCN2 gene was expressed predominantly (HCN2 > HCN4 > HCN1). B: mRNA expressions were normalized to control in each cell culture. Aldosterone increased HCN2 and HCN4 mRNA expression. *P < 0.05, n = 4.

 
Effects of aldosterone on protein expression of HCN genes. We analyzed protein expression of If channel genes (HCN1, HCN2, and HCN4) using Western blotting. The cultured cells were treated with 10 nM aldosterone in the absence and presence of MR antagonists (1 µM eplerenone or 0.1 µM spironolactone) for 24 h. Figure 5A shows representative Western blots of these proteins. Figure 5B shows the relative levels of these proteins to those in control. Aldosterone increased the expression of HCN2 protein (by 60.0 ± 17.1%) and HCN4 protein (by 84.8 ± 9.6%), but not HCN1 protein (n = 5). Both MR antagonists blocked the aldosterone-induced increase of HCN2 and HCN4 proteins.


Figure 5
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  		Fig. 5. Effect of aldosterone and MR antagonists on If channel protein. A: representative Western blots showing HCN1, HCN2, and HCN4 protein expression. Myocytes were incubated for 24 h without drug, with 10 nM aldosterone, with 10 nM aldosterone + 1 µM eplerenone, or with 10 nM aldosterone + 0.1 µM spironolactone. B: summarized data of protein expression from 5 experiments. Data were normalized to control. *P < 0.05, n = 5.

 
Effects of high concentration of aldosterone on If channel. We examined the effects of a high concentration (1 µM) of aldosterone on mRNA expression and current of If channels. One micromolar aldosterone can bind to glucocorticoid receptor (GR) as well as MR. The application of 1 µM aldosterone for 1.5 h (significantly) decreased HCN2 mRNA by 29.4 ± 20.2% and HCN4 mRNA by 50.9 ± 15.3%, respectively (n = 4, vs. control). (Fig. 6, A and B). One micromolar aldosterone did not affect HCN1 mRNA expression.


Figure 6
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  		Fig. 6. Effects of a high concentration of aldosterone on the If channel. Cultured myocytes were incubated for 1.5 h without drug, with 10 nM aldosterone, and with 1 µM aldosterone. A: mRNA expression levels of HCN1, HCN2, and HCN4 were normalized to GAPDH mRNA levels (n = 4). B: mRNA expressions were normalized to control in each cultivation (n = 4). C: If current was activated by hyperpolarization to –130 mV of 1 s from –50 mV. If current was suppressed by 1 µM aldosterone, compared with control. *The difference was significant at P < 0.05.

 
If current was elicited by –130-mV pulses for 1 s from a holding potential of –50 mV. The exposure of 1 µM aldosterone for 17–24 h significantly decreased If current by 43.7% (control: –1.6 ± 0.2 pA/pF, n = 7; 1 µM aldosterone: –0.9 ± 0.2 pA/pF, n = 5, P < 0.05, Fig. 6C).

GR did not mediate the upregulation of If channel by aldosterone. In another series of experiments, we examined the effect of 100 nM RU-38486 (GR antagonist) on the stimulatory action of 10 nM aldosterone on If current. Cells were incubated with 10 nM aldosterone in the presence and the absence of 100 nM RU-38486 for 17–24 h. If current was activated by hyperpolarization to –130 mV of 1 s from –50 mV. RU-38486 did not affect the twofold increase of If current by 10 nM aldosterone (control: –1.0 ± 0.1 pA/pF, n = 6; 10 nM aldosterone + 100 nM RU-38486: –2.0 ± 0.1 pA/pF, n = 5; 100 nM RU38486: –1.1 ± 0.1 pA/pF, n = 4).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we demonstrate 1) that 10 nM aldosterone increases the rate of spontaneous beating of cultured neonatal rat ventricular myocytes, 2) that it increases If currents by upregulation of gene expression of the If channel (mRNAs and proteins for HCN2 and HCN4 genes), and 3) that the latter effect can be abolished by MR antagonists (eplerenone and spironolactone). Furthermore, 4) a GR antagonist does not block the increase in If current, and 5) 10 nM aldosterone do not affect the channel kinetics. Finally, 6) a high concentration (1 µM) of aldosterone inhibits If channel gene expression (If currents and mRNAs for HCN2 and HCN4 genes).

Electrophysiological effects of aldosterone and MR. Upregulation of gene expression of ion channels by aldosterone other than If channels (T-type and L-type Ca2+ channel, Na+-K+-ATPase, Na+ channel) has been reported previously (2, 5, 17, 20). In contrast, Ito channel was downregulated by aldosterone (2).

The activity of the renin-angiotensin-aldosterone system is related to the severity of heart failure, and the concentration of circulating aldosterone is high in decompensated heart failure (12). In addition, Schunkert et al. (32) reported that circulating aldosterone levels correlate positively with left ventricular mass in patients with hypertension. The reduction of morbidity and mortality caused by MR antagonists in patients with heart failure in recent clinical trials (RALES and EPESUS) (28, 29) suggests that excess aldosterone elicits cardiac dysfunction, including arrhythmias. Thus aldosterone production is augmented in cardiac hypertrophy and heart failure and may induce electrical remodeling through modification of the density and/or function of ion channels. In ventricular myocytes of the human failing heart, upregulation of If current has been detected (7, 16). If current was also observed in hypertrophied ventricular myocytes of spontaneously hypertensive rats (6) and aortic stenosis rats (14).

We have demonstrated that 10 nM aldosterone upregulates If channel gene expression in cultured neonatal rat ventricular cells. If current density, mRNA, and protein expression were increased in concert, although If current activation kinetics were not altered. Therefore, we consider that 10 nM aldosterone might augment If current through the increase of the number of If channels. The gene subtypes increased by 10 nM aldosterone were HCN2 (predominantly) and HCN4. In hypertrophied rat hearts, an increase of HCN2 and HCN4 mRNA expression has been reported (17). Our data on activation kinetics (V1/2 of activation curve, time constant of activation and deactivation) compare with those of human HCN2 channel expressed in human embryonic kidney-293 cells (25).

Many studies have shown that V1/2 of the activation curve in ventricular cells is between –88 and –110 mV (5, 7, 17, 19), far more negative than the threshold for activation of the fast Na+ channel (–60 mV). In aldosterone-treated cultured rat ventricular cells in our study, DDR and beating rate of action potential were increased, with the MDP at –58.0 ± 5.4 mV. The resting membrane potential of normal adult ventricular cardiac cells is around –90 mV. Thus an increase in the number of If channels is not expected to cause automaticity. However, in pathological conditions as in heart failure, increased catecholamine concentrations will cause a shift of the If activation curve in the positive direction (1), and this may increase excitability.

MDP in aldosterone-treated cells tended to decrease (control: –65.2 ± 2.1 mV, n = 6; aldosterone: –58.0 ± 5.4 mV, n = 7; not significantly different). Although the mechanism is unknown, De Mello and Motta (10) have reported that the application of 0.83 µM aldosterone for 1 h decreased the resting potential from –80 to –74 mV in rabbit atrial muscle fiber.

Efficacy of MR antagonist on cardiac arrhythmias. The increase of If channel gene expression by aldosterone is completely blocked by MR antagonists, which indicates that this aldosterone effect is mediated by MRs. Transgenic mice overexpressing human MR, where human MR is expressed in all MR-expressing tissues, notably in the kidney and the heart, exhibit cardiac abnormalities without cardiac fibrosis (21). These mice had mild dilated cardiomyopathy with increased heart rate and concomitant cardiac arrhythmias. Recently, Ouvrard-Pascaud et al. (27) reported that mice with cardiac-specific MR overexpression display prolonged ventricular repolarization and lethal arrhythmias. They also demonstrated decreased Ito and increased L-type Ca2+ currents. This model was not associated with cardiac fibrosis. It logically follows that MR antagonists might be capable to prevent such cardiac arrhythmias. Efficacy of MR antagonists (eplerenone and spironolactone) in the prevention of sudden cardiac death was demonstrated in RALES and EPHESUS (28, 29). Spironolactone has been reported to reduce heart rate, normalize heart rate variability, and decrease QT dispersion in patients with heart failure. Moreover, spironolactone decreases ventricular arrhythmias in heart failure (39).

Mechanisms of If channel gene upregulation induced by aldosterone. Ten nanomolar aldosterone upregulates If channel gene expression. The dissociation constant at equilibrium and the half-life of the complex of the binding of aldosterone for MR and GR are 0.52 nM and 140 min, and 14.4 nM and 5 min, respectively (15). Ten nanomolar aldosterone is considered to bind to MR more efficiently than to GR. Furthermore, the GR antagonist 100 nM RU-38486 does not affect the stimulatory action of 10 nM aldosterone on If current. This finding suggests that the increase of If channel gene expression by 10 nM aldosterone mainly depends on MR activation. Plasma aldosterone in patients with severe heart failure has been reported to be 3.25 ± 0.53 nM (n = 5) (12). Rat myocardial aldosterone concentration has been estimated at 16 nM (34). However, in our hands, a high concentration (1 µM) of aldosterone, which will bind to both MR and GR, significantly decreased mRNA expressions of the HCN2 and HCN4 genes, as well as the density of the If current. Overactivation of MR and/or GR by high concentrations of aldosterone may thus suppress If channel gene expression. To clarify the action of high concentration of aldosterone requires further experimental work.

The downstream pathway from the MR to If channel gene expression has not been examined in this study. Sgk1 (serine-threonine kinase), Kirsten Ras GTP-binding protein-2A, and phosphoinositide 3-kinase are involved in the cascade following MR activation (8). Sgk1 has been reported to phosphorylate and inactivate Nedd4-2, which regulates ubiqutination (9). Ubiqutination of cardiac Na+ channels is accelerated by Nedd4-2 (37). Recently, Boixiel et al. (5) reported that the increase of Na+ channel current by aldosterone is mediated by another regulatory mechanism of trafficking of the Na+ channel than Sgk1 and Nedd4-2. In the present study, the increase of If current by 10 nM aldosterone (by 94%) was larger than the increase of mRNA expression (HCN2, by 26%; HCN4, by 47%). Although aldosterone upregulates If channel gene expression, other mechanisms such as trafficking of the channel might underlie the increase of If current by aldosterone.

The production of aldosterone by cardiac myocytes. Aldosterone synthase (CYP11B2) is expressed in the normal rat heart (34). In the isolated perfused heart, angiotensin II or ACTH increase aldosterone production. In cultured neonatal rat cardiomyocytes, CYP11B2 mRNA has been detected, whereas atrial and brain natriuretic peptide reduce its expression (19). In the human failing heart, aldosterone production becomes activated as well (24, 40). In this study, in cultured neonatal rat myocytes, MR antagonists (eplerenone or spironolactone) in the presence of aldosterone decrease If channel density (Fig. 2) and may reduce mRNA expressions of the HCN2 and HCN4 genes to levels even lower than in control (Fig. 4). In addition, in the presence of only MR antagonist (without aldosterone), If channel current was smaller than that in control (If current at –130 mV; control: –1.6 ± 0.2 pA/pF, n = 7; eplerenone: –1.1 ± 0.1 pA/pF, n = 5; spironolactone: –0.9 ± 0.1 pA/pF, n = 6), but the difference was not significant (figure not shown). These findings suggest that cultured myocytes produce aldosterone, which subsequently stimulates If channel gene expression.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (17590720).


   ACKNOWLEDGMENTS
 
We thank Mayumi Hojo and Kyoko Harada for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Yasui, Dept. of Bio-information Analysis, Research Institute of Environmental Medicine, Nagoya Univ., Nagoya 464-8601, Japan (e-mail: kenji{at}riem.nagoya-u.ac.jp)

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.

* T. Muto and N. Ueda contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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