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Journal of Physiology (2001), 537.1, pp. 151-160
© Copyright 2001 The Physiological Society
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ABSTRACT |
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INTRODUCTION |
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In addition to being a Na+-retaining and kalituretic hormone that acts on the kidney, aldosterone is also a cardiovascular hormone. As far back as 1969, a substance with physicochemical properties that were consistent with those of aldosterone was shown to be produced in isolated hearts (Know & Lockett, 1969). More recently, a local cardiac aldosterone synthesis system has been reported in rat (Silvestre et al. 1998) and human hearts (Kayes-Wandover & White, 2000). Cardiac-generated aldosterone might have autocrine or paracrine actions. All components required for specific and selective aldosterone effects are present in the cardiac myocyte. Mineralocorticoid receptors (MRs), which mediate direct regulation patterns of gene expression by this hormone, are expressed in the heart together with 11 
-hydroxysteroid dehydrogenase type II, which ensures the enzymatic protection of MRs against glucocorticoids (Lombes et al. 2000). In addition to the indirect effects of aldosterone on cardiac function during heart failure resulting from Na+ retention, clinical and experimental data suggest that aldosterone may exert direct effects on the heart. In rat, cardiac fibrosis is induced by aldosterone excess secondary to either chronic perfusion or myocardial infarction (see Lijnen & Petrov, 2000, for review). The role of aldosterone during cardiac remodelling has received more attention since the recent randomized aldactone evaluation study of Pitt et al. (1999) and the observation of a stimulation of cardiac aldosterone synthesis during cardiac hypertrophy (Silvestre et al. 1999; Mizuno et al. 2001). Aldosterone is involved in cardiac remodelling (Delcayre & Silvestre, 1999), which is known to be associated with cellular electrophysiological alterations. Notably, genomic regulation of Ca2+ channels and of the transient outward K+ channel has been invoked to account for electrophysiological remodelling (Bénitah et al. 1993; Gómez et al. 1997; Tomaselli & Marbán, 1999). We reported previously that treatment of rat ventricular myocytes with aldosterone for 24 h upregulates L-type Ca2+ current (ICa,L) through a specific genomic pathway (Bénitah & Vassort, 1999). Aldosterone may also play a role in mediating the regulation of other cardiac ion channels that are modulated during heart failure. Alternatively, since Ca2+ plays a key regulatory role in gene transcription (Berridge et al. 1998), ICa,L upregulation by aldosterone might lead to changes in the activity of other channels.
In the present study, we evaluated the effects of long-term aldosterone exposure on the Ca2+-independent 4-aminopyridine (4-AP)-sensitive transient outward K+ current (Ito1), in single rat ventricular myocytes. We demonstrate that aldosterone induces a decrease in Ito1 density, probably via modulation of Ca2+ signalling.
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METHODS |
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This investigation conformed to the European Community guide for the care and use of laboratory animals (French decree no. 87/848 of October 19, 1987).
Cell isolation and incubation
Isolated cardiac ventricular myocytes were prepared using an enzymatic perfusion method. Adult male Wistar rats (250-350 g) were treated with heparin (1000 units kg-1) and anaesthetized with sodium pentobarbital (50 mg kg-1) administered intraperitoneally. The heart was excised rapidly via a thoracotomy and the pericardium was removed. The heart was placed in ice-cold (0 °C) oxygenated Tyrode solution containing (mM): NaCl 130, NaH2PO4 0.4, NaHCO3 5.8, MgCl2 0.5, KCl 5.4, glucose 22, Hepes 25 and insulin 10-3 (titrated to pH 7.4 with NaOH). The aorta was cannulated above the aortic valve and was perfused by gravity (70 cm column height) with warm (37 °C), preoxygenated Tyrode solution supplemented with 0.1 mM EGTA for 2 min. Enzyme solution containing 1 g l-1 collagenase Type II (Worthington) in Tyrode solution supplemented with 0.1 mM CaCl2 was then perfused until the aortic valve was digested (attested by the increased outflow of perfusate). The heart was transferred to a Petri dish containing enzyme solution supplemented with 2 g l-1 bovine serum albumin (BSA). Taking into account the electrical heterogeneity in rat ventricles (Bénitah et al. 1993; Gómez et al. 1997), the apex was selected and gently shaken for 2-3 min at 37 °C to disperse individual myocytes. The resulting cell suspension was filtered through a 250 µm nylon mesh and centrifuged for 3 min at 20 g. The cell pellet was suspended in Tyrode solution supplemented with 0.5 mM CaCl2 and 2 g l-1 BSA and was centrifuged again at the same speed. Finally, the cell pellet was suspended in storage solution comprising Tyrode solution supplemented with 1 mM CaCl2 and 2 g l-1 BSA. This procedure yielded quiescent rod-shaped myocytes that were viable for up to 2 days when incubated at 37 °C in storage solution supplemented with 100 i.u. ml-1 penicillin and 0.1 µg ml-1 streptomycin.
D-Aldosterone and nifedipine were purchased from Sigma and the cell membrane-permeant Ca2+-specific chelator BAPTA-AM was purchased from Molecular Probes; RU28318 and RU38486 were generously provided by Hoechst Marion Roussel and Exelgyn, respectively.
Electrophysiology
Action potentials (APs) and membrane currents were measured at 23-25 °C using the whole-cell patch-clamp method (Axopatch-1D amplifier, Axon Instruments) with 1-1.8 M
micropipettes. The capacitive current was determined as described previously (Bénitah et al. 1993) and the series resistance was compensated electronically (40-70 %). All electrophysiological recordings elicited at 0.1 Hz were filtered at 2 kHz, digitized and sampled at 10 kHz using a Digidata 1200 series interface and pCLAMP6 software (Axon Instruments). APs were measured in a standard external solution (mM: NaCl 140, MgCl2 1.1, CaCl2 1.8, KCl 4, glucose 10 and Hepes 10, with the pH adjusted to 7.4 with LiOH) while the pipette contained a standard internal solution (mM: KCl 135, MgCl2 4, EGTA 5, glucose 10, Hepes 10, Na2ATP 5 and Na2 creatine phosphate 3, with the pH adjusted to 7.2 with LiOH). APs were elicited by 1.5-fold excitation threshold current pulses of 2.5 ms in duration. After stabilization, 10-20 APs were recorded and averaged for each cell. The 3 mM 4-AP-sensitive outward K+ current (Ito1) was measured as described previously (Bénitah et al. 1993; Gómez et al. 1997) with standard internal solution while the external solution was modified by equimolar replacement of NaCl with 2 mM CdCl2 to block ICa,L and INa, and with 1 mM BaCl2 to block other K+ currents. ICa,L was recorded with standard solutions with KCl replaced by CsCl. Whole-cell currents were evoked in isolated rat cardiomyocytes at 0.1 Hz in 300 ms depolarizing steps to voltages ranging between -50 and +60 mV in +10 mV increments, using a holding potential of -80 mV. For ICa,L, a 500 ms voltage ramp to -40 mV was applied before each pulse to inactivate INa.
For the electrophysiological recordings, 20-40 µl of storage cell suspension was placed into a Petri dish containing 4-5 ml external solution (1:100 dilution). After the establishment of the whole-cell configuration, the bath solution was renewed for 3-5 min until the recording stabilized. This procedure completely abolished any acute effects of the drugs used. Moreover, as for ICa,L (Bénitah & Vassort, 1999), we did not observe any acute effect on Ito1 or APs (data not shown).
Ca2+ imaging
Cells were loaded with the membrane-permeant fluorescent Ca2+-indicator dye fluo-3 acetoxymethyl ester (fluo-3 AM, 5 µM, Molecular Probes) as described previously (Gómez et al. 1996). Imaging was performed with a scanning confocal microscope (LSM 510 coupled to Axioskop 2 FS mot, Zeiss) fitted with a 488 nm argon laser and equipped with a water immersion objective (C-Apochromat 63 
0.9, Zeiss). Emitted fluorescence was measured at wavelengths > 515 nm. Each individual myocyte was imaged 12 times in the line-scan mode at 1.5 ms per line along the longitudinal axis for 1.5 s. After Ca2+ spark measurements, sarcoplasmic reticulum (SR) Ca2+ content was estimated in some cells by rapid caffeine (10 mM) application to release the SR Ca2+ content. During this procedure, the cell was imaged in the line-scan mode along the longitudinal line for 15 s, which allowed us to record both caffeine-evoked Ca2+ release and contraction (estimated by cell shortening). Image processing and analysis were carried out using IDL software (Research Systems).
Statistics
Data are expressed as means ± S.E.M. in the text and figures. Statistical significance was calculated using Student's t test for unpaired samples. Statistical significance was established at P < 0.05.
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RESULTS |
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Aldosterone treatment for 48 h decreases Ito1
We recently provided evidence that aldosterone increases the functional expression of ICa,L in rat ventricular myocytes (Bénitah & Vassort, 1999). In the present study, we investigated whether this mineralocorticoid hormone regulates Ito1. Isolated adult rat ventricular myocytes from the apex of the heart were incubated for 48 h at 37 °C in the presence of 100 nM aldosterone. This concentration of aldosterone corresponds to plasma levels observed during hypertension (Brilla et al. 1990). Control recordings were made from cells maintained in the same storage solution, but in the absence of aldosterone, for the same period of time. Aldosterone treatment did not affect the myocyte membrane capacitance (Cm; 124.3 ± 47.2 pF, n = 52 vs. 134.3 ± 63.6 pF, n = 65, for control vs. aldosterone-treated cells, respectively).
Figure 1A shows families of Ito1 traces recorded from cardiomyocytes incubated without or with aldosterone for 48 h. Ito1 tracings were obtained by subtraction of records in the presence of 3 mM 4-AP from those obtained without 4-AP. In both treatment conditions, voltages positive to -10 mV activated time- and voltage-dependent transient outward currents, which rose rapidly to a peak and decayed slowly. After aldosterone treatment, the magnitude of Ito1 was substantially reduced. Since cell-to-cell size variations might account for this difference, the peak amplitude of the current was normalized to Cm to obtain the current density in each cell. The average current densities measured from control and aldosterone-treated cells are plotted as a function of membrane potential in Fig. 1B. Sequential comparisons of individual values show a statistically significant decrease in Ito1 density from potentials positive to 0 mV. For instance, Ito1 density at +60 mV in myocytes treated with aldosterone for 48 h (12.5 ± 3.0 pA pF-1) was significantly reduced compared to controls (19.9 ± 3.4 pA pF-1).
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Figure 1. Aldosterone treatment (48 h) of rat ventricular myocytes decreases Ito1 and lengthens action potential repolarization A, typical pattern of Ito1 generated by myocytes isolated from the apex of the heart and incubated for 48 h at 37 °C in the absence (left) or presence (right) of 100 nM aldosterone. Membrane capacitance (Cm) was 121.1 vs. 112.1 pF for control vs. aldosterone-treated cells, respectively. Whole-cell currents were evoked by 300 ms depolarizing steps to voltages ranging between -50 and +60 mV in +10 mV increments using a holding potential of -80 mV. B, plots of averaged Ito1 densities (maximum current amplitude normalized to Cm) vs. membrane potential obtained from cells that were incubated for 48 h at 37 °C without ( | ||
Variations in Ito1 should be associated with changes in AP duration (APD). APs were elicited by 1.5-fold excitation threshold current pulses of 2.5 ms duration. After stabilization, 10-20 APs were recorded and averaged for each cell. Figure 1C shows typical examples of APs recorded in rat cardiomyocytes incubated for 48 h without or with aldosterone. Analyses were repeated in 11 myocytes for each incubation condition. No difference was observed either in the diastolic potential (ER; -79.1 ± 3.1 vs. -78.8 ± 3.0 mV for control vs. aldosterone-treated cells, respectively) or in the AP amplitude (110.6 ± 12.9 vs. 110.4 ± 12.7 mV for control vs. aldosterone-treated cells, respectively). However, APD was significantly lengthened from 20 % of repolarization after 48 h incubation with aldosterone (4.9 ± 1.3 vs. 8.3 ± 4.8 ms at 20 %; 9.9 ± 3.2 vs. 38.7 ± 17.9 ms at 50 %; and 33.5 ± 12.1 vs. 83.9 ± 15.5 ms at 90 %; control vs. aldosterone-treated cells, respectively). These changes are consistent with the role of Ito1 and ICa,L in shaping APD (Greenstein et al. 2000).
Aldosterone treatment does not modify Ito1 kinetics
Since the aldosterone-induced decrease in Ito1 density could result from modifications of current properties or from an alteration in the number of active channels, kinetic analyses were undertaken. The voltage dependence of inactivation of Ito1 was measured by applying a +40 mV test pulse (300 ms duration) after prepulses of varying amplitude. As shown in Fig. 2A, the voltage dependence of Ito1 inactivation in cells treated with aldosterone for 48 h was nearly identical to that observed in control cells. Each data set was well fitted with a Boltzmann equation to obtain the potential at which the current was half-inactivated (V1/2) and the slope factor (k) (Bénitah et al. 1993; Gómez et al. 1997). Neither of these parameters was altered by aldosterone treatment (5.8 ± 0.2 and 0.8 ± 0.1 mV in the presence of aldosterone vs. 5.4 ± 0.2 and 0.8 ± 0.2 mV in control for V1/2 and k, respectively).
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Figure 2. Aldosterone treatment for 48 h does not modify the voltage- and time-dependent properties of Ito1 A, the degree of inactivation of Ito1 was determined by applying a conventional two pulse protocol to cells incubated for 48 h without ( | ||
Comparison of Ito1 recorded in control and aldosterone-treated cells suggested similar rates of activation and inactivation (Fig. 1A). The time to peak, expressed as the time from the onset of voltage step to the peak of the current (tpeak), is plotted against voltage in Fig. 2B. Over the whole range of voltages, tpeak values were not significantly different between myocytes maintained in the absence or presence of aldosterone for 48 h (at +60 mV, 8.7 ± 1.8 vs. 9.4 ± 2.0 ms in 10 control vs. 14 aldosterone-treated cells, respectively). The inactivation kinetics were determined by fitting the decay phase of the current to a monoexponential function to get the time constant (
inac) (Bénitah et al. 1993; Gómez et al. 1997). Pooled values of inac expressed as a function of voltage are shown in Fig. 2C. There were no significant changes in the voltage dependence of the current inactivation kinetics (at +60 mV, 38.3 ± 7.1 vs. 34.8 ± 9.2 ms, in 10 control vs. 14 aldosterone-treated myocytes, respectively).
The inhibitory effect of 48 h of aldosterone treatment on Ito1 density was not associated with significant changes in its voltage- and time-dependent properties. The Ito1 reduction, therefore, is unlikely to be due to modulatory actions on pre-existing channels. Instead, it probably results from a change in either the total number of channels or the fraction of functional channels in the plasma membrane.
MRs mediate Ito1 modulation by aldosterone
Aldosterone exerts its actions on gene expression through activation of cytoplasmic MRs that bind to glucocorticoid response elements (GREs). It has been reported that the glucocorticoid hormone dexamethasone mediates a decrease in the transient outward K+ current in neonatal mouse ventricular myocytes (Wang et al. 1999). To determine whether the glucocorticoid receptors (GRs) and/or MRs are involved in the aldosterone-induced Ito1 decrease, we investigated the effects of incubation for 48 h with aldosterone in the presence of specific MR (RU28318) or GR (RU38486) antagonists (Ratka et al. 1989). As illustrated in Fig. 3, when cardiomyocytes were incubated with aldosterone, Ito1 density at +30 mV was decreased 2-fold. In the presence of 1 µM RU28318, the aldosterone-induced Ito1 decrease was significantly blunted, whereas co-incubation with 1 µM RU38486 did not prevent the decrease of Ito1. These results, observed at all voltages tested, demonstrate that the aldosterone-induced reduction of Ito1 occurs via MRs.
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Figure 3. Co-incubation with the specific MR antagonist RU28318 prevents the down-regulation of Ito1 by aldosterone Bar graphs represent the Ito1 density at +30 mV in myocytes incubated for 48 h in the absence of drugs ( | ||
Ito1 is not altered after 24 h of aldosterone treatment
We have shown previously that aldosterone acts within a day to increase ICa,L (Bénitah & Vassort, 1999). Therefore, in the present study we investigated the effect of 24 h of aldosterone treatment on Ito1. Figure 4A shows the mean current density-voltage relationships for Ito1 from myocytes treated with aldosterone for 24 h and controls. Exposure to 100 nM aldosterone for 24 h did not significantly alter Ito1 density at any voltage compared to controls (at +60 mV, 17.5 ± 6.8 vs. 17.3 ± 7.5 pA pF-1 for control vs. aldosterone-treated cells, respectively). It is worth noting that Ito1 densities were not significantly different in cells incubated for 24 or 48 h without aldosterone. The concentration of aldosterone used might explain the lack of effect of aldosterone exposure for 24 h on Ito1. Therefore, we repeated the experiments with higher (1 µM) and lower (10 nM) concentrations of aldosterone. As summarized in Fig. 4A, aldosterone at both of these concentrations did not significantly alter Ito1 density at any voltage (at +60 mV, 19.3 ± 11.2 and 16.8 ± 9.2 pA pF-1, for 1 µM and 10 nM aldosterone-treated cells, respectively).
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Figure 4. Exposure to aldosterone for 24 h does not alter Ito1 and lengthens only the late phase of AP repolarization A, current density-voltage relationships for averaged Ito1 recorded in myocytes incubated for 24 h in the absence ( | ||
To confirm the lack of effect of aldosterone on Ito1 after 24 h incubation, we investigated the effect of this treatment on AP characteristics. Figure 4B shows superimposed representative traces of APs recorded in cells incubated for 24 h in the absence or presence of 100 nM aldosterone. In nine control and eight aldosterone-treated myocytes, no differences were observed in ER or AP amplitude (-79.4 ± 4.4 vs. -78.2 ± 3.4 mV for ER and 108.4 ± 13.6 vs. 111.9 ± 8.2 mV for AP amplitude, for control vs. aldosterone-treated cells, respectively). Whereas the APD at 20 % repolarization (APD20) was unaffected (5.6 ± 0.6 vs. 5.7 ± 0.6 ms for control vs. aldosterone-treated cells, respectively), aldosterone significantly lengthened the APD at 50 and 90 % of repolarization (12.8 ± 2.4 vs. 25.8 ± 9.3 ms for APD50 and 34.4 ± 3.7 vs. 77.0 ± 15.2 ms for APD90, for control vs. aldosterone-treated cells, respectively). No significant changes in AP characteristics were noted in control cells incubated without aldosterone for 24 or 48 h.
Aldosterone upregulates ICa,L, which in turn modulates Ito1
The observation that Ito1 was unchanged after 24 h of aldosterone treatment was rather surprising, since we had previously observed an increase in ICa,L in this time frame for myocytes isolated from the whole heart (Bénitah & Vassort, 1999). Therefore, the ICa,L study was repeated on myocytes from the apex of the heart and the aldosterone treatment period was extended to 48 h.
Figure 5A shows families of ICa,L traces elicited in myocytes incubated for either 24 or 48 h in the absence or presence of 100 nM aldosterone. In each cell, depolarizing steps positive to -40 mV evoked an inward current that peaked within ~15 ms of the onset of depolarization and gradually declined, with a maximal current occurring near 0 mV. However, 24 and 48 h of aldosterone treatment substantially increased the magnitude of ICa,L, whereas it was unchanged in control cells. Figure 5B shows the mean current density-voltage relationships of ICa,L from control and aldosterone-treated myocytes. Sequential comparisons show a statistically significant increase in ICa,L density with aldosterone treatment in the -20 to +40 mV voltage range. Peak ICa,L density was 1.4-fold greater when myocytes were treated with aldosterone for 24 h compared to controls (at 0 mV, -18.8 ± 3.4 vs. -13.2 ± 1.7 pA pF-1, respectively). Aldosterone treatment for 48 h led to a further (1.7-fold) increase in peak ICa,L density. In control cells incubated for 48 h, peak ICa,L density at 0 mV (-13.8 ± 2.8 pA pF-1) was not significantly different to that of control cells incubated for 24 h, whereas the peak ICa,L density was significantly larger (-23.0 ± 4.8 pA pF-1, at 0 mV) in cells treated for 48 h with aldosterone. The effects of aldosterone treatment on current density were not associated with significant changes in the voltage- and the time-dependent properties of ICa,L (data not shown).
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Figure 5. Aldosterone-induced increase in ICa,L coordinates Ito1 decrease A, representative ICa,L recorded in myocytes that were maintained at 37 °C for either 24 h (top panels) or 48 h (bottom panels) in a medium without (left column) or with aldosterone (right column). Cm was 112.0 vs. 100.9 pF for 24 h and 122.8 vs. 150.9 pF for 48 h in control vs. aldosterone-treated cells, respectively. Whole-cell current recordings were obtained following the same protocol described in Fig. 1A. However, a voltage ramp (80 mV s-1) to -40 mV was applied before each pulse to inactivate INa. B, plots of averaged ICa,L density vs. membrane potential obtained from cells that were incubated for either 24 h (squares) or 48 h (circles) without (open symbols) or with 100 nM aldosterone (filled symbols). Pooled data from 18 control cells vs. 13 cells treated with aldosterone for 24 h, and 16 control cells vs. 16 cells treated with aldosterone for 48 h. Lines were fitted by eye; *P < 0.05, **P < 0.001. C, bar graph of pooled Ito1 slope conductances (GsIto1) of cardiac cells pretreated without ( | ||
Taking into account the delay in the Ito1 response, it seemed unlikely that aldosterone regulates Ito1 directly. However, transcriptional gene regulation by Ca2+ has been described in a variety of excitable cell types (Berridge et al. 1998). In cardiomyocytes, the molecular machinery of excitation-contraction coupling also regulates the transcription activity by a process termed excitation- transcription coupling (Atar et al. 1995). Thus, the potential participation of the aldosterone-induced Ca2+ modulation in this regulation should be considered. Consequently, Ito1 was recorded in myocytes that had been pretreated with aldosterone for 24 h and then subjected to an additional 24 h co-incubation with either 100 nM nifedipine or a cell membrane-permeant Ca2+-specific chelator, BAPTA-AM (10 µM). Figure 5C summarizes the results of these pharmacological manipulations. To estimate the slope conductance of Ito1 (GsIto1), the current density-voltage relationships from +10 to +60 mV were fitted with a linear function. On average, a 48 h incubation with aldosterone alone induced an ~30 % decrease in GsIto1 compared with control cells (229.4 ± 54.4 vs. 313.4 ± 70.1 pS pF-1, respectively). Both nifedipine and BAPTA-AM completely blunted the aldosterone-induced decrease in GsIto1 (303.5 ± 64.6 and 297.8 ± 42.6 pS pF-1 for nifedipine- and BAPTA-AM-treated cells in the presence of aldosterone, respectively). Thus, the decrease in cardiac Ito1 after incubation of cells with aldosterone for 48 h might involve ICa,L, because nifedipine treatment prevented this action. This regulation might result from a modulation of [Ca2+]i, since lowering [Ca2+]i with BAPTA blunted the Ito1 decrease in aldosterone-treated cells. Therefore, we examined whether lowering [Ca2+]i by incubating control cells with BAPTA-AM for 24 h, independent of aldosterone treatment, would alter Ito1 density. In eight cells exposed to BAPTA-AM, mean GsIto1 (456.8 ± 91.5 pS pF-1) increased significantly (P < 0.01) compared to 14 control myocytes incubated without any drug for 24 h (306.7 ± 129.3 pS pF-1). These observations further support our hypothesis that long-term regulation of Ito1 might be dependent on [Ca2+]i.
Long-term exposure to aldosterone modulates Ca2+ signalling
Besides the role of ICa,L in cardiac electrogenesis, the subsequent entry of Ca2+ into myocytes regulates Ca2+ loading and triggers Ca2+ release through neighbouring ryanodine receptors (RyRs), leading to cell contraction (Trafford et al. 2001). The Ca2+-dependent opening of RyRs is visualized with a confocal microscope as local [Ca2+]i elevations, termed Ca2+ sparks (Cheng et al. 1993). The upregulation of ICa,L channels after aldosterone treatment might increase cytosolic [Ca2+], which, in turn, activates Ca2+ sparks. Therefore, we measured the occurrence of spontaneous Ca2+ sparks in intact myocytes under our incubation conditions. Figure 6A shows representative line-scan images of myocytes loaded with fluo-3 AM. Ca2+ sparks occurred more frequently in myocytes treated with 100 nM aldosterone. The effects of aldosterone treatment on Ca2+-spark frequency are summarized in Fig. 6B. Although the rate of spontaneous Ca2+ spark occurrence in cells incubated with aldosterone for 24 h showed a tendency to increase, it did not reach statistical significance compared to myocytes maintained for 24 h without aldosterone. However, in myocytes incubated for 48 h with aldosterone, the spontaneous Ca2+-spark frequency increased by 2.6-fold.
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Figure 6. Aldosterone treatment modulates Ca2+ signalling A, sample records from control cells (left panel) and aldosterone-treated cells (right panel) showing a line-scan fluorescence image of spontaneous Ca2+ sparks. B, bar graph showing the spontaneous Ca2+ spark frequencies (Ca2+ spark number normalized to the duration and length of the line-scan). Myocytes were treated for either 24 or 48 h without ( | ||
Two main factors appear to contribute to the probability of spontaneous Ca2+ sparks occurring in resting myocytes: (1) cytosolic [Ca2+] and (2) SR Ca2+ content. Our estimation of SR Ca2+ content, by rapidly applying 10 mM caffeine, did not reveal significant differences. On average, the amplitude of the fluorescence ratio (peak of the fluorescence signal after caffeine application normalized to the signal before caffeine application) was 3.6 ± 0.9 in 14 control myocytes vs. 3.9 ± 0.7 in 14 aldosterone-treated cells. The absence of any change in SR load after 48 h of aldosterone treatment was further confirmed by the analysis of caffeine-induced cell shortening. The percentage cell length at the maximum caffeine-evoked twitch was not significantly different in the absence or presence of aldosterone for 48 h (81.5 ± 5.3 vs. 80.3 ± 5.8 %, respectively).
Since we observed an increase in spontaneous Ca2+ spark frequency with aldosterone treatment at a constant SR load, a modification of [Ca2+]i might contribute to this effect. In adult cardiac myocytes, L-type Ca2+ channels are the main pathways for Ca2+ entry. Satoh et al. (1998) reported that the increase in spontaneous Ca2+ spark frequency induced by the dihydropyridine agonist BayK 8644 might result from an increase in ICa,L. Therefore, we examined whether the aldosterone-induced increase in Ca2+ spark frequency observed after 48 h treatment is related to a further increase in ICa,L. We repeated the experiment in which myocytes were incubated with aldosterone for 48 h, except that after the first 24 h incubation period, the incubation solution was supplemented with the L-type Ca2+ channel antagonist nifedipine (0.1 µM). Co-incubation with nifedipine blunted the increase in spontaneous Ca2+-spark frequency in myocytes treated with aldosterone for 48 h to control levels (Fig. 6B). Acute perfusion of cells treated with aldosterone for 48 h with nifedipine (n = 17) also reduced spontaneous Ca2+-spark frequency (by 40 %). On the basis of these experiments, we suggest that the enhancement of Ca2+ sparks after aldosterone treatment might be mediated by an aldosterone-induced increase in ICa,L.
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DISCUSSION |
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The major finding of the present study is that aldosterone induces a decrease in Ito1 in isolated adult cardiac myocytes that seems to be coordinated by a long-term modulation of Ca2+ signalling. An investigation into whether aldosterone has an effect on isolated myocytes was necessary to determine whether this mineralocorticoid hormone has a direct action in heart. The maintenance of electrophysiological characteristics and Ca2+-spark occurrence over the 48 h incubation in control conditions demonstrated that cells were in a healthy state and not dedifferentiated. Our observation thus offers insights into the pathways by which an ion channel may be regulated in heart.
At present, little is known about the effect of aldosterone on cardiac ion channels, in contrast to the relatively wide knowledge about the closely related glucocorticoids. Glucocorticoids up-regulate the transcription of a K+ channel gene, Kv1.5 (Takimoto & Levitan, 1994), and the rat dihydropyridine-sensitive L-type 
1C-subunit gene (Takimoto et al. 1997). It has also been shown that in vivo treatment of mice ventricular myocytes with glucocorticoid agonists causes an approximately 1.6-fold decrease in the amplitude of transient outward K+ current (Wang et al. 1999). Since cardiac MRs bind mineralo- and glucocorticoids with equal affinity (Myles & Funder, 1996), our data are consistent with this notion. Moreover, the specificity of the effects of aldosterone is strengthened by ruling out a role for the GR through the use of a specific GR antagonist.
Theoretically, a change in macroscopic current density could result from changes in single-channel conductance, open probability or channel density. However, due to the lack of alterations in the time- or voltage-dependent properties of Ito1 after aldosterone treatment observed in the present study, we suggest that the Ito1 density decrease is probably due to fewer functional sarcolemmal channels rather than changes in gating kinetics. It is also possible that the maximal channel open probability decreases without any concurrent changes in the potential dependence of opening. In this case, a decrease in Ito1 would appear after brief changes in [Ca2+]i. However, brief changes in [Ca2+]i failed to modify Ito1 in rat ventricular myocytes, even though extracellular Ca2+ had significant effects on Ito1 (Dukes & Morad, 1991). Therefore, the simplest explanation is that an [Ca2+]i increase could initiate the necessary mechanisms that lead either to a decrease in the channel protein or an increase in the rate of channel degradation.
Ca2+ is involved in many cellular events as a ubiquitous second messenger. There is also evidence that Ca2+ in the heart is an important mediator of gene expression, leading to the concept of excitation-transcription coupling. Excitation-transcription coupling represents an elementary pathway whereby the electrical activity of a cell feeds back upon and shapes its own genetic programme (Atar et al. 1995). Thus, electrically stimulated contractions activate a hypertrophic gene regulation programme in primary neonatal rat ventricular myocytes (McDermott & Morgan, 1989) and accelerate the rate of protein synthesis in adult feline cardiomyocytes (Ivester et al. 1993). Moreover, Ca2+ influx through L-type Ca2+ channels is critical for early response gene expression (e.g. c-fos, c-myc, c-jun) after stimulation by a variety of stimuli, such as neurotransmitters and growth factors (Morgan & Curran, 1988; McDonough et al. 1997). [Ca2+]i may also affect expression of late-onset genes, including transcription of cardiac ion channels. For example, culturing rat cardiomyocytes in high [Ca2+] medium induces an increase in 1C-subunit gene mRNA abundance (Davidoff et al. 1997). In contrast, mRNA and the number of Na+ channels in neonatal rat cardiomyocytes are reduced by an increase in [Ca2+]i (Chiamvimonvat et al. 1995), while treatment with verapamil, a Ca2+ channel blocker, increases the Na+ channel -subunit mRNA level in rat ventricular myocytes (Duff et al. 1992). Aldosterone produces a specific genomic increase in L-type Ca2+ channel density (Bénitah & Vassort, 1999). After 48 h treatment, this augmentation might be large enough to produce a significant increase in spontaneous Ca2+-spark frequency. In the present study, nifedipine treatment blunted this effect and also the decrease in Ito1. The probability of L-type Ca2+ channel opening is low in myocytes at rest (Cavalié et al. 1986), but remains proportional to channel density. Moreover, the coupling gain between ICa,L and RyRs is very high at negative potentials (Santana et al. 1996). One might be concerned about whether a small increase in [Ca2+]i is sufficient to affect transcription. Cartin et al. (2000) reported a 2-fold increase in Ca2+ spark frequency in intact mouse cerebral arteries, which is sufficient to activate transcription factors, such as cAMP-responsive element binding protein, and to increase c-fos levels. Similarly, we showed here that aldosterone treatment for 48 h increases the occurrence of spontaneous Ca2+ sparks by 2.5-fold. We thus suggest that aldosterone induces Ito1 downregulation secondarily to the increase in ICa,L, which initiates Ca2+ signalling modulation. This interpretation is in line with the observation that L-type Ca2+ channel antagonists inhibited corticosterone-induced gene transcription (Budziszewska et al. 2000). Moreover, in vivo, the Ca2+-dependent regulation of Ito1 might be more pronounced since myocytes are contracting.
Alterations in the abundance of ion channels in cardiac muscle may occur in response to physiological as well as pathophysiological stimuli. During cardiac remodelling, it has long been appreciated that there are important alterations in the number or subtype of ion channels (Bénitah et al. 1993; Tomaselli & Marbán, 1999). In particular, a decrease in Ito1 density is a hallmark of cardiac hypertrophy. However, little is known about the mechanism underlying these phenomena. In this regard, the present work provides a molecular mechanism that might be involved in the regulation of Ito1 density. Among the potential neuromodulators involved in the pathogenesis of left ventricular hypertrophy and heart failure, the renin-angiotensin-aldosterone system (RAAS) might be a major primary stimulus (Holmer & Schunkert, 1996; Hefti et al. 1997). Recently, it was reported that incubation of myocytes with angiotensin II for between 2 and 52 h decreases Ito1 functional expression (Yu et al. 2000; Zhang et al. 2001). Furthermore, the increase in myocardial aldosterone production associated with cardiac remodelling after chronic myocardial infarction is primarily mediated by cardiac angiotensin II (Silvestre et al. 1999). Our data establish that aldosterone treatment reduces Ito1, which suggests a link between Ito1 regulation and the modulation of Ca2+ signalling. Our data also show that, independent of aldosterone treatment, lowering [Ca2+]i by incubating control cells with BAPTA-AM for 24 h increases Ito1 density. These observations further support our hypothesis that long-term regulation of Ito1 might be dependent on [Ca2+]i. In addition, changes in [Ca2+]i have been postulated as a possible trigger for channel remodelling in ischaemia and infarction (Pinto & Boyden, 1999). In the perspective of a more general scheme of cardiac remodelling related to RAAS, one might speculate that angiotensin II-activated aldosterone synthesis alters the regulation of the functional expression of ionic channels during cardiac remodelling.
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Acknowledgements
This study was supported by grants from the European Society of Aldosterone Council and Fondation pour la Recherche Médicale. We thank Dr S. Richard for help with improving the manuscript.
Corresponding author
J.-P. Bénitah: INSERM U390, CHU Arnaud de Villeneuve, 34295 Montpellier, France.
Email: benitah{at}welchlink.welch.jhu.edu
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, n = 10) or with 100 nM aldosterone (
, n = 15). Lines were fitted by eye to the data; *P < 0.05, **P < 0.001. C, representative traces of APs recorded in myocytes incubated for 48 h in control conditions (left) or in the presence of 100 nM aldosterone (right).
) or in the presence of 100 nM aldosterone (
), and with both aldosterone and either 1 µM RU28318 (
) or 1 µM RU38486 (
). n, number of experiments. **P < 0.001.
, n = 30), 100 nM (
, n = 9). B, superimposed representative examples of APs recorded in isolated rat ventricular myocytes incubated for 24 h without (
) or with a membrane-permeant Ca2+ chelator (10 µM BAPTA-AM;