Effects of diltiazem and nifedipine on transient outward and ultra-rapid delayed rectifier potassium currents in human atrial myocytes

Abstract

1. It is unknown whether the widely used L-type Ca²⁺ channel antagonists diltiazem and nifedipine would block the repolarization K⁺ currents, transient outward current (I_to1) and ultra-rapid delayed rectifier K⁺ current (I_Kur), in human atrium. The present study was to determine the effects of diltiazem and nifedipine on I_to1 and I_Kur in human atrial myocytes with whole-cell patch-clamp technique.

2. It was found that diltiazem substantially inhibited I_to1 in a concentration-dependent manner, with an IC₅₀ of 29.2±2.4 μM, and nifedipine showed a similar effect (IC₅₀=26.8±2.1 μM). The two drugs had no effect on voltage-dependent kinetics of the current; however, they accelerated I_to1 inactivation significantly, suggesting an open channel block.

3. In addition, diltiazem and nifedipine suppressed I_Kur in a concentration-dependent manner (at +50 mV, IC₅₀=11.2±0.9 and 8.2±0.8 μM, respectively). These results indicate that the Ca²⁺ channel blockers diltiazem and nifedipine substantially inhibit I_to1 and I_Kur in human atrial myocytes.

Introduction

The 4-aminopyridine (4-AP)-sensitive transient outward K⁺ current (I_to1) and ultra-rapid delayed rectifier K⁺ current (I_Kur) play important roles in human atrial repolarization (Shibata et al., 1989; Wang et al., 1993; Li et al., 1995; 1996a). Inhibition of I_to1 and/or I_Kur has been found to prolong the action potential duration in human atrium (Courtemanche et al., 1998; 1999; Van Wagoner, 2000). In addition, I_Kur has been reported to be present in the atrium, but not the ventricle of human heart (Li et al., 1996b). Therefore, blockade of I_Kur may be useful in the treatment of patients with atrial fibrillation, but without the risk of ventricular proarrhythmia (Van Wagoner, 2000; Van Wagoner & Nerbonne, 2000; Nattel, 2002).

The benzothiazopine Ca²⁺ channel blocker diltiazem and the dihydropyridine (DHP) Ca²⁺ channel blocker nifedipine are widely used in clinic for the treatment of cardiovascular diseases including hypertension (De Leeuw & Birkenhager, 2002; White, 2003), cardiac angina (Kumar & Hall, 2003), and/or supraventricular arrhythmias (for diltiazem) (Hohnloser et al., 2000). The therapeutic effects are generally believed to be related to the L-type Ca²⁺ channel (I_Ca.L) block (Hohnloser et al., 2000; De Leeuw & Birkenhager, 2001; Kumar & Hall, 2003; White, 2003). Earlier studies demonstrated that nifedipine significantly inhibited I_to1 in rabbit atrial (Gotoh et al., 1991) and rat ventricular (Jahnel et al., 1994) cells. Our recent study found that the phenylalkylamine Ca²⁺ channel blocker verapamil substantially blocked I_Kur, but not I_to1 in human atrial myocytes (Gao et al., 2004). Nevertheless, it is unknown whether diltiazem and nifedipine would affect human atrial repolarization currents. The present study was therefore designed to determine the effects of diltiazem and nifedipine on I_to1 and I_Kur in human atrial myocytes with whole-cell patch-clamp technique.

Methods

Single atrial myocyte preparation

Atrial cells were isolated from specimens of human right atrial appendage obtained from 29 patients (57.7±2.7 years old) undergoing coronary artery bypass grafting. The procedure for obtaining the tissues was approved by the Ethics Committee of the University of Hong Kong based on the patients' consent. All patients were free from supraventricular tachyarrhythmias, and the atria were grossly normal at the time of surgery. After excision, the samples were quickly immersed in oxygenated, nominally Ca²⁺-free cardioplegic solution for transport to the laboratory. Atrial myocytes were enzymatically dissociated as described previously (Du et al., 2003; Gao et al., 2004). Briefly, the myocardial tissue was sliced with a sharp blade, placed in a 15-ml centrifuge tube containing 10 ml of the Ca²⁺-free Tyrode solution (36°C), and gently agitated by continuous bubbling with 100% O₂ for 15 min (5 min at a time in fresh solutions). The chunks were then incubated for 50 min in a similar solution containing 150–200 U ml⁻¹ collagenase (CLS II, Worthington Biochemical, Freehold, NJ, U.S.A.), 0.2 mg ml⁻¹ protease (type XXIV, Sigma Chemical, St Louis, MO, U.S.A.) and 1 mg ml⁻¹ bovine serum albumin (Sigma). Subsequently, the supernatant was discarded. The chunks were re-incubated in a fresh enzyme solution with the same composition but no protease, microscope examination of the medium was performed every 10–15 min to determine the number and the quality of the isolated cells. When the yield appeared to be maximal, the chunks were suspended in a high K⁺ medium containing (mM) 10 KCl, 120 K-glutamate, 10 KH₂PO₄, 1.8 MgSO₄, 10 taurine, 10 HEPES, 0.5 EGTA, 20 glucose, 10 mannitol, pH was adjusted to 7.3 with KOH and gently blown with a pipette. The isolated myocytes were kept at room temperature in the medium at least 1 h before use.

A small aliquot of the solution containing the isolated cells was placed in an open perfusion chamber (1-ml) mounted on the stage of an inverted microscope. Myocytes were allowed to adhere to the bottom of the chamber for 5–10 min and were then superfused at 2–3 ml min⁻¹ with Tyrode solution. Only quiescent rod-shaped cells with clear cross-striations were used. The study was conducted at room temperature (21–22°C).

Solution and drugs

Ca²⁺-free cardioplegic solution for specimen transport contained (in mM) 50 KH₂PO₄, 8 MgSO₄, 5 adenosine, 10 HEPES, 140 glucose, 100 mannitol, 10 taurine, pH was adjusted to 7.3. with KOH. Tyrode solution contained (in mM) 140 NaCl, 5.4 KCl, 1 MgCl₂, 1.0 CaCl₂, 0.33 NaH₂PO₄, 5 HEPES, 10 glucose, pH was adjusted to 7.4 with NaOH. The pipette solution contained (in mM) 20 KCl, 110 K-aspartate, 1.0 MgCl₂, 10 HEPES, 5 EGTA, 0.1 GTP, 5 Na₂-phosphocreatine, and 5 Mg₂-ATP, pH was adjusted to 7.2 with KOH. For I_to1 and I_Kur recording, BaCl₂ (200 μM) and CdCl₂ (200 μM) were added to the superfusion to block I_K1 and I_Ca. Atropine (1.0 μM) was used to minimize possible I_K,ACh contamination during the current recording. Diltiazem was dissolved in distilled water with a stock solution of 100 mM, while a stock (100 mM) of nifedipine was made with DMSO. All the drugs were purchased from Sigma Chemicals Co. (St Louis, MO, U.S.A.).

Data acquisition and analysis

The whole-cell patch-clamp technique was used for electrophysiological recording. Borosilicate glass electrodes (1.2-mm OD) were pulled with a Brown-Flaming puller (model P-97, Sutter Instrument Co., Novato, CA, U.S.A.) and had tip resistances of 2–3 MΩ when filled with pipette solution. The whole-cell membrane currents in voltage-clamp mode were recorded using an EPC-9 amplifier and Pulse software (Heka, Lambrecht, Germany). A 3-M KCl-agar salt bridge was used as reference electrode. Liquid junction potentials were compensated before the pipette touched the cell. After a gigaseal was obtained, the cell membrane was ruptured by gentle suction to establish the whole-cell configuration. The cell membrane capacitance (78.6±4.1 pF, n=46) was directly measured using the lock-in module of the Pulse software, and used for normalizing the current in individual cells. The series resistance (R_s) was 3–8 MΩ and was compensated by 60–80% to minimize voltage errors. Current signals were low-pass filtered at 5 kHz and stored on the hard disk of an IBM compatible computer.

Nonlinear curve fitting was performed using Pulsefit (Heka) and/or Sigmaplot (SPSS Science, Chicago, IL, U.S.A.). Results are presented as mean±s.e. Paired and/or unpaired Student's t-test was used as appropriate to evaluate the statistical significance of differences between two group means, and ANOVA was used for multiple groups. Values of P<0.05 were considered statistical significant.

Results

Effect of diltiazem on I_to1

Figure 1A shows voltage-dependent I_to1 elicited by 300-ms voltage steps to between −40 and +50 from −50 mV (as shown in the inset) at 0. 2 Hz in a representative human atrial myocyte during control, in the presence of diltiazem, and after the drug washout. I_to1 was substantially inhibited by the application of 5 and 50 μM diltiazem, and the effect recovered after washout of the drug for 8 min. Figure 1B illustrates the time-dependent effect of diltiazem on I_to1 activated by the voltage step shown in the left inset. The current measured was peak to ‘quasi'-steady-state level. Diltiazem at 50 μM gradually inhibited I_to1, and the effect reached a steady-state level within 2 min. The current completely recovered upon the drug washout. The original I_to1 traces at the corresponding time points are shown in the right inset of the panel.

Figure 1.

Diltiazem effect on I_to1. (A) Representative voltage-dependent I_to1 (capacitance compensated) recorded in an atrial myocyte with the voltage steps protocol shown in the inset at 0.2 Hz under control conditions (a), in the presence of 5 and 50 μM diltiazem (b, c). I_to1 was substantially inhibited by the application of diltiazem for 6 min, and the effect was recovered by the drug washout for 8 min (d). (B) Time-dependent effect of 50 μM diltiazem on I_to1 elicited by the voltage step shown in the left inset delivered every 10 s in a typical experiment. I_to1 measured was peak to ‘quasi'-steady-state level. The original I_to1 traces at corresponding time points are shown in the right inset of the panel.

Results from Figure 1 indicate that the sustained current (i.e. I_Kur) is also suppressed when I_to1 is inhibited by diltiazem. It would be relatively accurate in evaluating the diltiazem effect on I_to1 if a selective I_Kur inhibitor is available to separate I_to1. It was reported that 4-AP selectively inhibited I_Kur at low concentrations (50 μM, about 50% inhibition) (Wang et al., 1993; Li et al., 1996b), but higher concentrations of 4-AP (>50 μM) also suppressed I_to1 significantly (authors' unpublished observation). Therefore, 4-AP would not be an ideal compound to separate I_to1. We have recently found that verapamil inhibits I_Kur (IC₅₀=3.2 μM) without reducing I_to1 amplitude, while it induces an increase of measured I_to1 in human atrial myocytes (Gao et al., 2004). Therefore, verapamil was used to separate I_to1 as described in the following. We felt that the use of verapamil to limit the possible contamination of I_Kur would not affect drug action on I_to1.

Figure 2A displays representative recordings of I_to1 elicited with the protocol as shown in the inset during control, in the presence of 10 μM verapamil, co-presence of verapamil and 50 μM diltiazem, and washout of diltiazem. After the inhibition of I_Kur by verapamil, inactivation of I_to1 was actually increased, and the measured I_to1 was clearly enhanced as the previous report observed (Gao et al., 2004). Diltiazem at 50 μM substantially suppressed I_to1, and the effect reversed upon dug washout. Figure 2B shows the I–V relationships of I_to1 in seven cells during the pretreatment of 10 μM verapamil to inhibit I_Kur, and after the application of 5, 10, 50, 100, and 200 μM diltiazem, showing that diltiazem inhibits I_to1 in a concentration-dependent manner. The effect was reversed by 90% after washout of diltiazem. Diltiazem at 5–200 μM significantly suppressed I_to1 at voltages from 0 to +60 mV (P<0.05 or 0.01 vs control). Figure 2C illustrates the concentration–response relationship for the inhibition of I_to1 by diltiazem. In six cells completed all the concentrations from 1 to 400 μM, data were fit to the Hill equation: E=E_max/[1+(IC₅₀/C)^b], where E is the effect at concentration C, E_max is the maximal effect, IC₅₀ is the concentration for half-maximal effect, and b is Hill coefficient. IC₅₀ (at +50 mV) was 29.2±2.4 μM with a Hill co-efficient of 0.97±0.06 on the basis of cell-by-cell fits, and E_max was 65.2%.

Figure 2.

Effects of verapamil and diltiazem on I_to1. (A) I_to1 traces recorded with the voltage protocol as shown in the inset of (c) in a representative myocytes during control (a), in the presence of 10 μM verapamil (Verap.) for 6 min (b), co-presence of verapamil and 50 μM diltiazem (Dilt.) for 6 min (c), and washout of diltiazem for 8 min. Verapamil actually induced an increase of measured I_to1 by selectively inhibiting I_Kur. (B) I–V relationships of I_to1 in the presence of 10 μM verapamil (control), co-presence of verapamil and 5, 10, 50, 100, and 200 μM diltiazem (6 min for each concentration), and after the drug washout for 10 min. Diltiazem inhibited I_to1 in a concentration-dependent manner (P<0.05 or 0.01 vs control, and the effect was reversed by 90% after the drug washout. The statistical significance was analyzed by repeated-measures ANOVA. (C) Concentration–response relationship for diltiazem inhibition of I_to1. Symbols are mean data at +50 mV (the error bars are smaller than the size of the data symbol), and solid line is the best-fit Hill equation, IC₅₀=29.2±2.4 μM, Hill co-efficient=0.98±0.08 (n=6), and E_max=65.2%.

Time-dependent kinetics of I_to1 were determined in the presence of 10 μM verapamil. Figure 3a shows current traces (points) upon a 300-ms voltage step to +50 from –50 mV in a representative cell in the presence of 10 μM verapamil (control), and co-presence of verapamil and 50 μM diltiazem. I_to1 was well fitted by a mono-exponential function (solid line) under control conditions with time constant shown. After the application of 50 μM diltiazem, the current trace was no longer fitted by the monoexponential function, but well fitted by a biexponential equation with the time constants (τ₁ and τ₂) shown. A similar finding was obtained in all of the experiments in the presence of 10 and 50 μM diltiazem. Figure 3b illustrates the averaged time constants. The time constant of I_to1 inactivation was reduced from 57.8±2.8 ms of control to 52.1±2.1 and 44.1±1.8 ms respectively (P<0.05 or 0.01 vs control) by the application of 1 and 5 μM diltiazem. At 10 and 50 μM, there was a slower component with a time constant (τ₂) similar to that under control conditions, and a faster component whose time constant (τ₁) decreased to 9.4±0.6 ms from 18.6±2.1 ms as the drug concentration increased (n=7, P<0.01). These results are consistent with high-affinity open-channel block causing rapid current decay. Figure 3c shows the time to peak of I_to1, determined from the onset of depolarization to the current peak. Diltiazem at 50 μM significantly reduced the time to peak of I_to1 at 0 to +60 mV (n=6, P<0.01 vs control), consistent with open-channel block action (Feng et al., 1997).

Figure 3.

Effects of diltiazem on time-dependent kinetics of I_to1. (a) I_to1 traces recorded from a representative cell upon a 300-ms voltage step to +50 from −50 mV in the presence of 10 μM verapamil (control) and co-presence of verapamil and 50 μM diltiazem. Raw data (points) of I_to1 under control conditions were fitted to a monoexponential function (solid lines, superimposed with raw data) with time constants shown. After the application of 50 μM diltiazem, the data were fitted only by a biexponential equation, with fast and slow time constants (τ₁ and τ₂) shown. (b) Mean values of time constants at +50 mV under control conditions, in the presence of 1, 5, 10, and 50 μM diltiazem. The time constant was reduced by the application of 1 and 5 μM diltiazem (n=7, ^*P<0.05, ^**P<0.01 vs control). Diltiazem at concentrations higher than 10 μM had τ₁ and τ₂. The τ₁ decreased with increasing diltiazem concentration (^##P<0.01 vs 10 μM diltiazem), and the τ₂ did not show significant difference. (c) Time to peak of I_to1 activation at 0 to +60 mV under control conditions and in the presence of 50 μM diltiazem. Diltiazem significantly reduced the time to peak of I_to1 (n=7, ^**P<0.01 vs control). The statistical significance was analyzed by repeated-measures ANOVA.

Voltage dependence of I_to1 activation and inactivation was evaluated in the presence of 10 μM verapamil (control), and co-presence of verapamil and 50 μM diltiazem. The variable (g) of voltage-dependent activation was calculated from I–V relationships for each cell from Figure 2B, based on the formulation g=I/(V_t−V_r) as described previously (Du et al., 2003), where I is the peak current at the test voltage (V_t), and V_r is the measured reversal potential (∼70 mV). The variable (I/I_max) for voltage-dependent inactivation was determined with a protocol as illustrated in Figure 4a, with 1−s conditioning pulses from voltages between −90 and +20 mV, followed by a 300-ms test pulse to +50 mV after a 30-ms interval at −40 mV. Mean data for activation and inactivation, along with best-fit Boltzmann equation to obtain the half activation or inactivation voltage (V_0.5) and the slope (S), under control conditions and in the presence of 50 μM diltiazem are shown in Figure 4b. The voltage dependence of I_to1 activation and inactivation was not affected by the application of diltiazem. V_0.5 and S were 16.9±1.4 and −11.2±0.3 mV for activation, and −29.2±1.1 and 7.5±0.6 mV for inactivation under control conditions. In the presence of 50 μM diltiazem, the corresponding values were 18.4±0.9 and −11.9±0.4 mV for activation (n=7, P=NS), and –30.4±1.4 and 7.6±0.8 mV for inactivation (n=6, P=NS).

Figure 4.

Effects of diltiazem on voltage-dependence and restoration of I_to1. (a) Representative current traces and protocol (inset) used to evaluate voltage-dependent inactivation I_to1 recorded in the presence of 10 μM verapamil. (b) Voltage-dependent variables for I_to1 activation (Act.) and inactivation (Inact.) were fitted to the Boltzmann distribution: y=1/{1+exp[(V_m−V_0.5)/S]}, where V_m is membrane potential, V_0.5 is the midpoint, and S is slope. For activation, V_0.5 and S were 16.9±1.4 and −11.2±0.3 mV for control, and 18.4±0.9 and −11.9±0.4 mV for 50 μM diltiazem (Dilt.) treatment (n=7, P=NS). For inactivation, V_0.5 and S were –29.2±1.1 and 7.5±0.6 mV under control conditions, and −30.4±1.4 and 7.6±0.8 mV in the presence of 50 μM diltiazem (n=6, P=NS). (c) Representative current traces recorded in a typical experiment in the presence of 10 μM verapamil by 300-ms paired pulses to +50 mV after a 30-ms step of −40 mV (to inactivate I_Na) from −80 mV with varying P1 and P2 interval (inset), which are used for assessing time-dependent recovery of I_to1 from inactivation. (d) Mean data for time course of recovery of I_to1 from inactivation in the absence and presence of 50 μM diltiazem in six cells. Data were best fit to monoexponential function. No change in recovery time constant of I_to1 was observed after the application of diltiazem (n=6, P=NS).

Time-dependent recovery of I_to1 from inactivation was studied with a paired-pulse protocol as shown in the inset of Figure 4c. Time course of recovery was well fitted by a monoexponential function with the time constant of 98.6±5.4 ms under control conditions, and 105.8±7.5 ms in the presence of 50 μM diltiazem (n=6, P=NS). The result indicates that diltiazem does not affect recovery of I_to1 from inactivation. In addition, no use-dependent effect of diltiazem (50 μM) on I_to1 (+50 mV) was noted at frequencies from 1 to 3 Hz (n=5, P=NS).

Inhibition of I_Kur by diltiazem

As described previously (Wang et al., 1993; Li et al., 1996a; Du et al., 2003; Gao et al., 2004), I_Kur was recorded with a 100-ms prepulse to +40 mV to partially inactivate I_to1, followed by 150-ms test pulses to between −40 and +50 from −50 mV after a 10-ms interval, then to −30 mV (as shown in the inset in Figure 5A). Figure 5A displays voltage-dependent I_Kur recorded by the voltage protocol in a typical experiment. Under control conditions the current was rapidly activated by depolarization steps with significant tail current at –30 mV. Diltiazem at 5 and 50 μM substantially inhibited both I_Kur and tail current. The effect was significantly recovered by the drug washout. Figure 5B shows the time-dependent effect of 10 μM diltiazem on I_Kur activated by the voltage protocol shown in the left inset in a representative myocyte. I_Kur measured from zero level to the current at the end of voltage step. I_Kur was gradually inhibited by diltiazem, and recovered upon the drug washout. The original I_Kur traces at corresponding time points are shown in the right inset of the panel.

Figure 5.

Effect of diltiazem on I_Kur. (A) Representative voltage-dependent I_Kur (capacitance compensated) recorded at 0.2 Hz in a typical experiment with a 100-ms prepulse to +40 mV to inactivate I_to1, followed by 150-ms test pulses to between –40 and +50 from –50 mV after a 10-ms interval, then to –30 mV (as shown in the inset of (c) under control conditions (a), in the presence of 5 and 50 μM diltiazem (b, c). I_Kur was substantially suppressed by the application of diltiazem, and the effect was significantly recovered by washout of the drug for 6 min (d). (B) Time-dependent effect of 10 μM diltiazem on I_Kur elicited by 150-ms voltage step to +50 from −50 mV (as shown in the left inset) delivered every 10 s. The original I_Kur races at corresponding time points are shown in the right inset.

Figure 6a displays the I–V relationships of I_Kur (n=8) under control conditions, in the presence of 1, 5, 10, 50, and 100 μM diltiazem, and after washout of the drug, showing that diltiazem blocks I_Kur in a concentration-dependent manner. Figure 6b summarizes the percent reduction of I_Kur by diltiazem at voltages from 0 to +50 mV. Significant inhibitory effect of I_Kur by diltiazem was observed from the low concentration of 1 μM. At concentrations from 10 to 100 μM, diltiazem showed voltage-dependent effect on I_Kur, and the inhibition was stronger at voltages positive to +10 mV (P<0.05 or 0.01 vs 0 mV). Figure 6c shows the concentration–response relationships for suppression of I_Kur at +50 mV by diltiazem in seven cells completed all the concentrations from 0.1 to 200 μM. Mean IC₅₀ was 11.2±0.9 μM with a Hill co-efficient of 0.96±0.07, and E_max was 64%. No use-dependent inhibition of I_Kur (+40 mV) by diltiazem (10 μM) was observed at frequencies from 1 to 3 Hz (n=6, P=NS).

Figure 6.

Concentration-dependent effect of diltiazem on I_Kur. (a) I–V relationships of I_Kur under control conditions, in the presence of 1, 5, 10, 50, and 100 μM diltiazem (6 min for each concentration), and after the drug washout for 10 min. Diltiazem inhibited I_Kur in a concentration-dependent manner, and the effect was reversed by 95% (at +50 mV) after the drug washout. (b) Percent reduction of I_Kur at 0 to +50 mV by diltiazem with multiple concentrations. Diltiazem significantly inhibited I_Kur at concentrations from 1 μM (P<0.05 vs control) to 5, 10, 50, and 100 μM (n=8, P<0.01 vs control). Significant voltage dependence was observed for the drug effect at 10–100 μM, and stronger effect was observed at potentials positive to +10 and +50 mV (P<0.05 or 0.01 vs 0 mV). The statistical significance was analyzed by repeated-measures ANOVA. (c) Concentration–response relationships of I_Kur block by diltiazem at +50 mV. The symbols are mean values of inhibitory effect in cells exposed to different concentrations (0.1–200 μM) of diltiazem. Solid lines were best fit to Hill equation. Mean IC₅₀ was 11.2±0.9 μM, Hill co-efficient was 0.96±0.07, and E_max was 64% (n=7).

Nifedipine effect on I_to1

Figure 7A shows the time-dependent effect of nifedipine on I_to1 activated by a 300-ms voltage to +50 from –50 mV (as shown in the left inset) in a human atrial myocyte. Nifedipine gradually inhibited I_to1, and the effect recovered upon the drug washout. Figure 7B displays voltage-dependent I_to1 recorded with the voltage protocol as shown in the inset in a representative cell under control conditions, and after the application of nifedipine. Nifedipine at 5 and 50 μM substantially suppressed I_to1, and accelerated the inactivation of the current. These effects reversed upon drug washout.

Figure 7.

Inhibition of I_to1 by nifedipine. (A) Time-dependent effect of 30 μM nifedipine on I_to1 elicited by voltage step to +50 from −50 mV (as shown in the left inset) delivered every 10 s. Nifedipine reversibly suppressed I_to1. The original I_to1 traces at corresponding time points are shown in the right inset. (B) Voltage-dependent I_to1 traces recorded with the voltage protocol as shown in the inset in a typical experiment under control conditions (a), in the presence of 5 and 50 μM nifedipine (b, c) for 5 min. I_to1 was significantly inhibited by nifedipine, and the effect recovered upon the drug washout for 6 min.

As diltiazem did, nifedipine suppressed both I_to1 and I_Kur. Verapamil at 10 μM was therefore used to inhibit I_Kur to obtain a relatively accurate effect of nifedipine on I_to1. Figure 8A displays representative recordings of I_to1 in the presence of 10 μM verapamil (control), co-presence of verapamil and 5 and 50 μM nifedipine, and washout of nifedipine. Figure 8B illustrates the I–V relationships of I_to1 in seven cells before and after the application of 5, 10, 50, and 100 μM. I_to1 was suppressed by nifedipine in a concentration-dependent manner, and recovered by 80.1% upon washout of nifedipine. Nifedipine significantly inhibited I_to1 at voltages from 0 to +60 mV (P<0.05 or P<0.01 vs control) with 5, 10, 50, and 100 μM nifedipine. The concentration–response relationship for inhibition of I_to1 by nifedipine is illustrated in Figure 8C. On the basis of cell-by-cell fits with the Hill equation in six cells, IC₅₀ was 26.8±2.1 μM with a Hill co-efficient of 0.96±0.05 (n=6), and E_max was 85.1%.

Figure 8.

Effect of nifedipine on I_to1 under conditions of inhibiting I_Kur with 10 μM verapamil. (A) I_to1 traces recorded with the same voltage protocol as shown in the inset in a representative myocyte during control (a, 10 μM verapamil), in the co-presence of verapamil and 5 (b) and 50 (c) μM nifedipine (Nif.) for 6 min, and washout of nifedipine for 10 min (d). (B) I–V relationships of I_to1 under control conditions (10 μM verapamil), in the co-presence of verapamil and 5, 10, 50, 100 μM nifedipine (6 min for each concentration), and after the drug washout for 10 min. Nifedipine (5–100 μM) significantly inhibited I_to1 at voltages from 0 to +60 mV (n=7, P<0.05 or 0.01). The statistical significance was analyzed by repeated-measures ANOVA. (C) Concentration-dependent response of I_to1 to nifedipine. IC₅₀ at +50 mV was 26.8±2.1 μM with a Hill co-efficient of 0.97±0.02 (n=6), and E_max was 85.1%.

Effects of nifedipine on time-dependent inactivation of I_to1 were determined under conditions of I_Kur inhibition by 10 μM verapamil. Figure 9a displays the representative I_to1 traces (points) upon a 300-ms voltage step to +50 from –50 mV, well fitted by a monoexponential function (solid line) under control conditions, but only best fitted by a biexponential function after the application of 50 μM nifedipine with fast and slow time constants (τ₁ and τ₂) shown. Figure 9b summarizes mean values of the time constants studied in seven cells under control conditions, and after the application of 1, 5, 10, and 50 μM nifedipine. In the presence of 1 μM nifedipine, the monoexponential time constant reduced to 39.4±3.7 from 45.2±4.2 ms of control (n=7, P<0.05). At higher concentrations of nifedipine at 5, 10, and 50 μM, there was a slower component with a time constant (τ₂) similar to that under control conditions, and a faster component whose time constant (τ₁) decreased with increasing nifedipine concentration (P<0.01). Figure 9c illustrates mean values of the time to peak of the onset of I_to1 activation before and after the employment of 50 μM nifedipine, showing that nifedipine significantly reduces the time to peak of I_to1 at 0 to +60 mV (n=6, P<0.01 vs control). These results suggest that nifedipine inhibits I_to1 via open channel block.

Figure 9.

Effects of nifedipine on inactivation and time to peak of I_to1. (a) I_to1 traces recorded in a typical experiment upon a 300-ms voltage step to +50 from −50 mV in the presence of 10 μM verapamil (control), and co-presence of verapamil and 50 μM nifedipine. Raw data (points) of I_to1 under control conditions were well fit to a mono-exponential function (solid lines, superimposed with raw data) with time constant shown. The data after application of 50 μM nifedipine were only fit to a biexponential equation with fast and slow time constants (τ₁ and τ₂) shown. (b) Mean values of I_to1 inactivation time constants under control conditions, and in the presence of 1 (n=7, P<0.05 vs control), 5, 10 and 50 μM nifedipine (^#P<0.05, ^##P<0.01 vs 5 μM nifedipine). (c) Time to peak of I_to1 as a function of test potentials under control conditions and in the presence of 50 μM nifedipine. The time to peak of I_to1 was significantly reduced at 0 to +60 mV by the application of diltiazem (n=6, ^**P<0.01 vs control). The statistical significance was analyzed by repeated-measures ANOVA.

No change in voltage-dependent and recovery kinetics of I_to1 was observed with 50 μM nifedipine. V_0.5's of voltage-dependent activation and inactivation of I_to1 were 16.4±2.1 and −28.1±2.4 mV under control conditions, and 18.7±2.8 (n=7) and –31.5±2.9 mV (n=6) after the application of 50 μM nifedipine (P=NS). The time constant (τ) of recovery of I_to1 from inactivation was 97.3±4.9 ms under control conditions, and 89.5±6.2 ms in the presence of 50 μM nifedipine (n=6, P=NS vs control). No use-dependent effect of nifedipine (50 μM) on I_to1 (+50 mV) was observed at frequencies from 1 to 3 Hz (n=6, P=NS).

Inhibition of I_Kur by nifedipine

Figure 10A illustrates the time course of I_Kur during control and after applying 30 μM nifedipine, and washout of the drug in a typical experiment. I_Kur was reversibly decreased by nifedipine. Figure 10B displays the effect of nifedipine on voltage-dependent I_Kur in a representative myocyte. Nifedipine at 5 and 50 μM produced a substantial suppression on I_Kur, and the effect was recovered by the drug washout.

Figure 10.

Effect of nifedipine on I_Kur. (A) Time-dependent effect of 30 μM nifedipine on I_Kur elicited by voltage protocol shown in the left inset delivered every 10 s in a typical experiment. Nifedipine reversibly suppressed I_Kur. (B) Voltage-dependent I_Kur recorded in a representative myocyte by the voltage protocol shown in the inset under control conditions (a), in the presence of 5 and 50 μM nifedipine for 5 min (b, c), and after wash out of the drug for 6 min (d). Nifedipine substantially suppressed I_Kur, and the effect recovered upon the drug washout.

Figure 11a displays the I–V relationships of I_Kur under control conditions, and after the application of 1, 5, 10, 50, and 100 μM nifedipine. I_Kur was inhibited by nifedipine in a concentration-dependent manner, and recovered by 95% of control after washout of the drug. The percent reduction of I_Kur by nifedipine at potentials from 0 to +50 mV is shown in Figure 11b. Significant inhibition of I_Kur was observed from the low concentration of 1 μM. I_Kur at +50 mV was decreased by 7.7±2.6, 25.6±4.2, 48.1±4.8, 69.9±4.3, and 75.9±4.9% at 1, 5, 10, 50, and 100 μM nifedipine, respectively (n=8, P<0.05 or 0.01). Figure 11c shows the concentration-dependent response of I_Kur to nifedipine. On the basis of cell-by-cell fits with Hill equation completed in seven cells, all the concentrations from 0.1 to 200 μM, IC₅₀ was 8.2±0.8 μM with a Hill co-efficient of 1.2±0.1, and E_max was 76%. No use-dependent effect of nifedipine (10 μM) on I_Kur (40 mV) was observed at frequencies from 1 to 3 Hz (n=6, P=NS).

Figure 11.

Nifedipine effect on voltage-dependent I_Kur. (a) I–V relationships of I_Kur under control conditions, in the presence of 1, 5, 10, 50, and 100 μM nifedipine (6 min for each concentration), and after the drug washout for 10 min. Nifedipine inhibited I_Kur in a concentration-dependent manner, and the effect was reversed by 95% (at +50 mV) by washout of the drug for 10 min. (b) Percent inhibition of I_Kur at voltages from 0 to +50 mV by nifedipine with multiple concentrations. Nifedipine significantly inhibited I_Kur at concentrations from 1 to 5, 10, 50, and 100 μM (n=8, P<0.05 or 0.01 vs control), and no voltage-dependent effect was observed for the drug action. The statistical significance was analyzed by the repeated measures ANOVA. (c) Concentration-dependent inhibition of I_Kur by nifedipine at +50 mV. The symbols are mean values of inhibitory effect in seven cells exposed to different concentrations of nifedipine. Solid lines were fit to Hill equation. Mean IC₅₀ was 8.2±0.8 μM, Hill co-efficient was 1.2±0.2, and E_max was 76%.

Discussion

The present study demonstrates that the widely used Ca²⁺ channel blockers diltiazem and nifedipine substantially inhibit the repolarization currents I_to1 and I_Kur in human atrial myocytes. The blockade of human atrial I_to1 and I_Kur by the two Ca²⁺ channel blockers was concentration-dependent.

The benzothiazopine Ca²⁺ channel blocker diltiazem, the dihydropyridine Ca²⁺ channel blocker nifedipine, and the phenylalkylamine Ca²⁺ channel block verapamil were reported to block a variety of cloned K⁺ channels including Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv3.1, Kv4.2, and HERG channel currents expressed in mammalian cell lines and/or Xenopus oocytes (Rampe et al., 1993; Grissmer et al., 1994; Zhang et al., 1997; Rolf et al., 2000; Calmels et al., 2001). Our recent study showed that verapamil inhibited I_Kur, but not I_to1, in human atrium (Gao et al., 2004). The present observation provides the novel information that diltiazem and nifedipine decrease both I_to1 and I_Kur in human atrial myocytes.

Our findings showed that diltiazem substantially inhibited I_to1 in human atrial myocytes, and significantly accelerated the inactivation of I_to1, and reduced the time to peak of the activation (Figures 1, 2 and 3), suggesting an open-channel block (Feng et al., 1997). No report is available in literature regarding the diltiazem effect on I_to1 to compare the results from the present observation. I_to1 may be encoded by the cloned channel Kv1.4 or Kv4.2/Kv4.3 (Tseng, 1999). A recent report described that Kv1.4 and Kv4.2 currents expressed in Xenopus oocytes were inhibited by 10 and 24% at +50 mV with 100 μM diltiazem (Rolf et al., 2000). The effect is much weaker than that (IC₅₀=29.2 μM, E_max=65%) observed in human atrial native I_to1 (Figure 3).

I_Kur in human atrium is generally believed to be encoded by cloned channel Kv1.5 (Fedida et al., 1993; Wang et al., 1993). It was reported that diltiazem at 100 μM blocked the cloned Kv1.5 channel currents expressed in Xenopus oocytes only by 15% at +50 mV (Rolf et al., 2000), and the compound inhibited the Kv1.5 channel current expressed in mouse fibroblasts with a large K_d of 115 μM (Grissmer et al., 1994). However, diltiazem inhibited I_Kur with IC₅₀ of 11.2 μM in human atrial myocytes (Figures 5 and 6), suggesting that the blocking effect is stronger in human atrial native I_Kur than that in cloned hKv1.5 channel expressed in Xenopus oocytes or mouse fibroblasts (Grissmer et al., 1994; Rolf et al., 2000).

The suppression of native I_to1 by nifedipine was initially reported in rabbit atrial cells (Gotoh et al., 1991), and rat ventricular myocytes (Jahnel et al., 1994). The present observation provides additional evidence that nifedipine also inhibits human atrial I_to1 (Figures 7 and 8). Inactivation of I_to1 was significantly accelerated by nifedipine in human atrial myocytes (Figure 9), suggesting an open-channel block, consistent with the reports from rabbit and rat cardiac myocytes (Gotoh et al., 1991; Jahnel et al., 1994), and cloned Sharker K⁺ channels expressed in Xenopus oocytes (Avdonin et al., 1997).

It has been reported that human atrial I_to1 is mainly encoded by Kv4.3 (Kong et al., 1998). Nifedipine had no significant effect on the kinetics of voltage-dependent activation and inactivation, and the rate of recovery from inactivation of I_to1 in human atrial myocytes. However, nicardipine, another DHP compound, negatively shifted V_0.5 of activation and inactivation, and slowed down the rate of recovery from inactivation of rat Kv4.3L channel currents expressed in HEK 293 cell line (Hatano et al., 2003). These different responses of Kv4.3 to DHPs may be related to the subtle differences of the Kv4.3 sequences (human vs rat), the cell types (native human atrial cells vs HEK 293 cells), or the chemical structures (nifedipine vs nicardipine), etc.

It was reported that nifedipine substantially blocked hKv1.5 current expressed in HEK 293 cells (Zhang et al., 1997) and mouse fibroblast (Grissmer et al., 1994). The present study demonstrated that nifedipine inhibited human atrial I_Kur in a concentration-dependent manner, with an IC₅₀ of 8.2 μM (Figures 10 and 11). The concentration is close to the K_d (6.2 μM) of hKv1.5 current expressed in HEK cells (Zhang et al., 1997), and lower than that of hKv1.5 current expressed in mouse fibroblasts (K_d=92 μM) (Grissmer et al., 1994). No report is available in literature from native cells to compare with the data obtained from the present observation in human atrial myocytes.

I_Kur is found to be functionally expressed in human atrium, but not ventricle (Li et al., 1996b). Therefore, the drugs that specifically inhibit the unique I_Kur may provide a means of preventing atrial fibrillation without the risk of ventricular proarrhythmia (Nattel, 2002). In the present study, we have found that the diltiazem inhibits I_Kur with an IC₅₀ of 11.2 μM. The significant inhibitory effect on I_Kur was observed at low concentration of 1 μM, which is close to the therapeutically relevant plasma (50–300 ng ml⁻¹) concentrations of diltiazem in the treatment of atrial arrhythmias (Singh et al., 1983; Dias et al., 1992; Kelly & O'Malley, 1994), and the I_Ca.L block concentration (1–15 μM) in myocardium (McDonald et al., 1994; Koidl et al., 1997). Nifedipine also significantly inhibited I_Kur at 1 μM, but the concentration is higher than the clinical relevant plasma (10–200 ng ml⁻¹) concentrations (Singh et al., 1983; Kelly & O'Malley, 1994), and the I_Ca.L block concentration (0.05–0.2 μM) in myocardium (McDonald et al., 1994; Koidl et al., 1997). Whether the inhibition of I_Kur and I_to1 by diltiazem or nifedipine would exert a beneficial action on supraventricular arrhythmias remains to be studied.

In summary, the present study has provided the first information that the widely used Ca²⁺ antagonists diltiazem and nifedipine substantially inhibit the repolarization currents I_to1 and I_Kur in human atrial myocytes.

Abbreviations

4-AP

4-aminopyridine

IC₅₀

the concentration for 50% maximum inhibition

I_Ca.L

L-type Ca²⁺ current

I_Kur

ultra-rapid delayed rectifier K⁺ current

I_to1

transient outward K⁺ current