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Cardiovascular Research 1998 38(3):685-694; doi:10.1016/S0008-6363(98)00048-0
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Preferential inhibition of IKr by MCI-154, a putative cardiotonic Ca2+ sensitizer, in guinea pig atrial cells

Koji Etoa,1,1, Keitaro Hashimotoa and Haruaki Nakayab,*

aDepartment of Pharmacology, Yamanashi Medical University, 1,100 Shimokato, Tamaho-cho, Yamanashi 409-38, Japan
bDepartment of Pharmacology, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan

* Corresponding author. Tel.: +81 (43) 226 2050; Fax: +81 (43) 226 2052; E-mail: nakaya@-med.m.chiba-u.ac.jp

Received 16 October 1997; accepted 22 January 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: To define the electrophysiologic mechanism(s) by which MCI-154, a putative Ca2+ sensitizer, produces a positive inotropic response without a positive chronotropic response, we examined effects of MCI-154 on the action potential of atrial preparations and the membrane currents of atrial myocytes. Methods: The action potentias were recorded from left atrial and sinoatrial node preparations of guinea pigs by the use of standard microelectrode techniques. The whole-cell membrane currents were recorded from enzymatically-dissociated guinea pig atrial myocytes using conventional patch clamp techniques. Results: In isolated left atria, MCI-154 increased the developed tension in a concentration-dependent manner. MCI-154 at concentrations of 10 and 100 µM increased the action potential duration (APD) in left atria stimulated at 0.5 Hz. In sinoatrial node preparations MCI-154 at a concentration of 100 µM produced a negative chronotropic response and prolonged APD. In single right atrial myocytes, MCI-154 at concentrations of 10 and 100 µM failed to increase the inward L-type Ca2+ current, but decreased the delayed rectifier K+ current (IK) in a concentration-dependent manner. MCI-154 decreased IK elicited by short depolarizing pulses more markedly than that induced by long depolarizing pulses. In addition, MCI-154 produced only a little inhibition of IK in the presence of E-4031, a specific blocker of rapidly activating component of IK (IKr). Conclusions: MCI-154 preferentially blocks IKr and the inhibitory action on IKr may be partly involved in the negative chronotropic and positive inotropic responses in atrial preparations.

KEYWORDS MCI-154; Ca2+-sensitizer; Negative chronotropism; The delayed rectifier K+ current; Action potential duration


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
A number of new positive inotropic agents with diverse mechanisms of action have recently emerged as substitute for digitalis glycosides. For instance, many orally active phosphodiesterase (PDE) III inhibitors such as amrinone, milrinone, enoximone, imazodan and vesnarinone have been developed. However, none of them have successfully replaced cardiac glycosides for the treatment of chronic heart failure, because most of this class of agents do not give consistent clinical benefits with long-term therapy [1]. Controlled clinical trials indicated that high doses of amrinone, milrinone, and enoximone failed to improve symptoms or exercise tolerance [2–4]. In addition, these agents exacerbated ventricular arrhythmias, provoked myocardial ischemia, accelerated progression of the heart failure, and increased mortality [5]. Despite the dramatic hemodynamic improvement produced by the PDE III inhibitors, what could lead to the poor long-term clinical results with these agents in terms of survival? Since administration of these drugs increases cyclic AMP and transmembrane Ca2+ influx in myocardial cells, high doses of these drugs could be toxic due to the enhancement of normal and abnormal automaticity [6–8].

In the search for other drugs with different mechanisms of action, Ca2+ sensitizing agents such as MCI-154, pimobendan and EMD 53998 have been recently developed although they still inhibit PDE III [7, 9–11]. The PDE III inhibitory action may result in the beneficial hemodynamic effect including vasodilation on both the venous and the arterial side of the peripheral circulation [12]. However, we should still consider the risk of cyclic AMP increase in myocardial cells, which may lead to a marked positive chronotropism and ventricular arrhythmias. We have recently reported that MCI-154, a putative novel Ca2+ sensitizer [9, 10], does not aggravate ventricular arrhythmias elicited by the epinephrine-halothane combination, digitalis, and coronary-ligation in dogs [13]. Several in vivo and in vitro studies have shown that MCI-154 neither increased nor decreased the heart rate in spite of the significant positive ionotropism [13–15]. It is surprising that MCI-154 does not increase the heart rate, although most of new cardiotonic drugs produce positive chronotropic responses. Therefore, it would be interesting to explore why MCI-154 failed to produce a positive chronotropic response in spite of the positive inotropic response. To answer this question, we thought that it would be helpful to examine the effects of MCI-154 on the action potentials of sinoatrial node and atrial preparations. Since the drug prolonged the action potential recorded from these cardiac tissues, we also evaluated the effects of MCI-154 on the membrane current system, with the special reference to the delayed rectifier K+ current (IK), in isolated atrial cells.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Mechanical function study
All experiments were performed in accordance with the regulations of the Animal Care and Use Committee of the Yamanashi Medical University and Chiba University School of Medicine. This investigation conforms with Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23. revised 1985). Guinea pigs weighing 250–300 g were anaesthetized with phenobarbital sodium (30–50 mg/kg i.p.). The heart was dissected following a thoracotomy, and the right and left atrium were isolated. The whole atrial preparations were suspended under a resting tension of 0.5 g in siliconized glass organ baths containing a Krebs–Henseleit solution (20 ml). The composition of the solution was as follows (mM): NaCl, 113; KCl, 4.8; CaCl2, 2.2; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25; D-glucose, 5.5. The medium was maintained at 35±1°C and bubbled with 95% O2–5% CO2. The left atrium was electrically driven by rectangular pulses of 0.5 Hz in frequency, 5 ms in duration, and twice the threshold voltage. The pulses were delivered from an electronic stimulator (DIA Medical DPS 910, Tokyo, Japan). The spontaneous contraction of the right atrium and electrically driven contraction of the left atrium were measured with a force displacement transducer (Nihon Kohden TB-611T, Tokyo, Japan) and an amplifier (Nihon Kohden AP-601G, Tokyo, Japan). The analogue data were put into a microcomputer-aided data acquisition system, Mac Lab 8/e (Analog Digital Instruments, MA, USA), and the heart rate was counted using an on-line data analysis program, Chart 3.3.4 (Analog Digital Instruments). All preparations were allowed to equilibrate for at least 1 h before drug applications. All organ bath experiments were performed in the presence of 1 µM phentolamine, 1 µM propranolol and 1 µM atropine unless otherwise stated. The concentration-response curves for the inotropic and chronotropic effects of drugs were determined in a cumulative manner by increasing its concentration with steps of 0.5 log units.

2.2 Action potential study
The hearts were rapidly removed from guinea pigs weighing 250–300 g and immersed in the oxygenated Krebs–Henseleit solution. Spontaneous beating right atrium and left atrium were dissected from the hearts. The sinoatrial node preparation contained the intercaval area, the crista terminals of the right atrium and the interatrial septum. The preparations were pinned on the bottom of a tissue chamber of about 5 ml volume and continuously superfused at a rate of 10 ml/min with the Krebs–Henseleit solution equilibrated with 95% O2–5% CO2. The composition of the Krebs–Henseleit solution was the same as that used in mechanical function study. The bath temperature was kept constant at 35±1°C.

One end of the left atria was hooked to the lever of a force transducer (Nihon Kohden, TB 651T) connected to an amplifier (Nihon Kohden AP-601G) and the other end was pinned to the bottom of the tissue chamber. In order to obtain a stable impalement, the resting tension of the left atria was adjusted to 200 mg, which was smaller than that in the mechanical function study. Transmembrane potentials were recorded by the standard microelectrode technique, as previously described [16]. In the left atria, the preparation was electrically stimulated at 0.5 Hz with pulses of 1 ms duration at twice the diastolic threshold with bipolar electrodes. Stimuli were delivered from an electronic stimulator (Nihon Kohden S-7272B). Transmembrane action potentials were recorded with glass microelectrodes filled with 3 M KCl, which had a tip resistance of 20–30 M{Omega}. The microelectrode was connected to the input stage of a high-impedance amplifier with capacitance neutralization (Nihon Kohden MZ-4). The amplified signals and developed tension (DT) were displayed on a dual-beam oscilloscope (Nihon Kohden VC-10) and photographed on Polaroid film.

After an equilibration period of 1 h, a stable impalement was obtained and control recordings were made. The preparations were then exposed to solutions containing 10 or 100 µM MCI-154. The recording of the transmembrane potentials was repeated from the same cell during the drug superfusion period. Only the experiments in which a stable impalement was maintained were used for the data analysis and the others were discarded.

2.3 Whole cell voltage clamp study
Single atrial cells of the guinea pig heart were isolated by an enzymatic dissociation method, as previously described [17]. In brief, the heart was removed from the open-chest guinea pig anaesthetized with pentobarbital sodium, and mounted on a modified Langendorff perfusion system for retrograde perfusion of the coronary circulation with a normal HEPES-Tyrode solution. The perfusion medium was then changed to a nominally Ca2+-free Tyrode solution and then to a solution containing 46 U/ml of collagenase (Yakult, Tokyo, Japan). After digestion, the heart was perfused with Kraft–Brühe (KB) solution. A part of right atrium was dissected and gently shaken up in KB solution to isolate cells. The cell suspension was stored at a low temperature (4°C) for later use. The composition of the normal HEPES-Tyrode solution (in mM) were: NaCl, 135.0; KCl, 5.4; glucose, 5.5; HEPES, 5.0; CaCl2, 1.8; MgCl2, 0.5; NaH2PO4, 0.33. The pH was adjusted to 7.4 with NaOH. Zero-Ca2+ or low Ca2+ HEPES-Tyrode solution was prepared by omitting the CaCl2 or reducing it to 150 µM. The KB medium consisted of (in mM): glutamic acid monopotassium salt, 50.0; KCl, 25.0; taurine, 10.0; KH2PO4, 10.0; EGTA, 0.5; glucose, 10.0; HEPES, 10.0; MgCl2, 3.0. The pH was adjusted to 7.4 with KOH. The solution in the recording pipette contained (in mM): potassium aspartate, 120.0; KCl, 30.0; EGTA, 10.0; HEPES, 5.0; ATP-2 Na, 4.0; MgCl2, 1.0. The pH was adjusted to 7.2 with KOH. In some experiments ICa,L was isolated from other membrane currents by using Cs+-rich external and pipette solutions. The Cs+-rich external solution was prepared by replacing KCl of the normal HEPES-Tyrode solution with equimolar CsCl. The Cs+-rich pipette solution consisted of (in mM): l-aspartate, 110.0; CsOH, 110.0; CsCl, 20.0; EGTA, 10.0; HEPES, 5.0; ATP-K2, 5.0; MgCl2, 1.0. The pH was adjusted to 7.2 with CsOH and HCl. In order to measure IK, ICa,L was blocked by 0.4 µM nisoldipine, a compound which does not affect cardiac K+ currents at this concentration unlike many other Ca2+ channel blockers [18]. All experiments were done at room temperature (26±2°C). The gigaseal patch clamp technique [19]was used in the whole cell clamp configuration with use of patch pipettes having a tip resistance of 3–4 M{Omega}. A patch/whole cell clamp amplifier (EPC-7, List Electronic, Darmstadt, Germany) was used and membrane current signals were displayed on a storage oscilloscope (Nihon Kohden, Model VC-10) and stored in a microcomputer (Compaq, PC-Prolinea) using an analog-to-digital conversion board (Digidata 1200 Interface, Axon Instrument, CA, USA) controlled by pClamp 5.5.7 software (Axon Instrument). Data were digitized at a sampling rate of 4 kHz after filtering at 3 kHz. All experiments were performed using the normal HEPES-Tyrode solution or Cs+-rich Tyrode's solution. All solutions flowed to a recording chamber of 1.0 ml at a speed of about 1.5 ml/min. Stabilization usually occurred within 5–6 min after the patch rupture. The data from the cells in which the membrane currents did not stabilize within 5 min were discarded. The ‘run down’ phenomenon was obvious only in ICa,L, and mean decline of ICa,L for 10 min was 9±3% (n=6) after the stabilization in control conditions. However, IK was relatively stable and the tail current of IK after 10 min was 105±3% (n=6) of the value at the beginning. Mean capacitance of the cells used in this study was 63±3 pF (n=48).

2.4 Drugs
The following drugs were used: MCI-154 (6-[4-(4'-pyridylamino)phenyl]-4,5-dihydro-3(2H)-pyridazinone hydrochloride trihydrate) (Mitsubishi Chemical, Yokohama, Japan), E-4031 (N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide dihydrochloride dihydrate) (Eisai Pharmaceutical, Tokyo, Japan), nisoldipine (Bayer Pharmaceutical, Osaka, Japan), phentolamine hydrochloride, d,l-propranolol hydrochloride and atropine sulfate (Sigma Chemical, St Louise, MO, USA).

2.5 Statistics
All values are expressed as mean±S.E.M. One-way or two-way analysis of variance (ANOVA) with 95% confidence limits followed by a Student's t-test on individual sets of data was performed using analytical software Stat View 4.0 and Super ANOVA (Abacus, CA, USA).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Positive inotropic and negative chronotropic effects of MCI-154 in isolated atrial preparations
Electrically-paced (0.5 Hz) left atrial preparations were exposed to cumulative concentrations of MCI-154 (30 nM-1 mM) in the presence of phentolamine, propranolol and atropine. The changes in the developed tension (DT) after MCI-154 are expressed as a percent increase of the basal DT (1.1±0.1 g, n=5) and are shown in Fig. 1A. The changes in DT reached a new steady state within 5–8 min. The EC50 value for the inotropic effect of MCI-154 was 55±6 µM (n=5).


Figure 1
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  		Fig. 1 Inotropic and chronotropic effects of MCI-154 in isolated guinea pig atria. (A) Positive inotropic effect of MCI-154 in left atria driven at 0.5 Hz. Values are mean±S.E.M. of 5 preparations. Basal developed tension (DT) was 1.1±0.1 g. (B) Negative chronotropic effect of MCI-154 in right atrial preparations. Values are mean±S.E.M. of 5 preparations. Basal heart rate was 172±18 beat/min. * and ** represent a significant change from control value at p<0.05 and p<0.01, respectively.

 
Spontaneously beating right atrial preparations were similarly exposed to cumulative concentrations of MCI-154 (30 nM-1 mM) in the presence of phentolamine, propranolol and atropine. MCI-154 produced no significant changes of beating rate in concentrations less than 30 µM and moderate bradycardia, decreases in beating rate by 10–25 beats/min, in higher concentrations (100 µM-1 mM) (n=5, Fig. 1B). MCI-154 did not induce any arrhythmias in the spontaneously beating right atrial preparations.

3.2 Effects of MCI-154 on action potentials in left and right atrial preparations
Fig. 2A illustrates the representative changes in the action potential configuration and DT after 10 µM MCI-154 in an isolated left atrial preparation stimulated at 0.5 Hz. MCI-154 increased the action potential duration (APD) without affecting the action potential amplitude (APA) and the resting membrane potential (RMP). Changes in APD at 50%, 90% repolarization level (APD50, APD90) and DT after the application of MCI-154 in 10 preparations are summarized in Fig. 2B. After 15 min of superfusion with a solution containing 10 µM MCI-154, DT and APDs reached almost the maximal plateau levels. We also tried to examine the effects of a higher concentration (100 µM) of MCI-154. However, it was difficult to maintain a single impalement for a long time because of the marked positive ionotropism. At 5 min after exposure to 100 µM MCI-154, APD50, APD90 and DT increased from 34±4 ms, 62±6 ms and 51±8 mg to 60±3 ms, 95±8 ms and 253±22 mg, respectively (n=3).


Figure 2
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  		Fig. 2 Effects of MCI-154 on the action potentials (APs) recorded from guinea pig left and right atrial preparations. (A) Superimposed recordings of APs and developed tensions (DTs) obtained before and 15 min after exposure to 10 µM MCI-154 in a left atrial preparation driven at 0.5 Hz. (B) Changes of action potential duration at 50% (APD50) and 90% repolarization level (APD90) and DT after 10 µM MCI-154. Values are mean±S.E.M. of 10 preparations. * and ** represent significant changes from control value at p<0.05 and p<0.01, respectively. (C) Superimposed tracings showing effect of 100 µM MCI-154 on APs of a sinoatrial node preparation. The AP of MCI-154 was recorded at 6 min after drug administration.

 
Effects of MCI-154 and E-4031, a class III drug specifically blocking a rapidly activating component of IK (IKr) [20, 21], on the action potential of sinoatrial nodal cells were also evaluated in right atrial preparations. Superimposed recordings of the sinoatrial nodal action potentials after MCI-154 are shown in Fig. 2C. The changes of action potential parameters of sinoatrial nodal preparations are summarized in Table 1. There were no significant differences in any of the baseline values between two groups. MCI-154 at a concentration of 100 µM, which clearly showed a negative chronotropic effect in isolated right atrial preparations, increased the sinus cycle length, concomitantly with an increase in APD and a slight decrease in the maximum diastolic potential (MDP) and APA (Fig. 2C). The increases in APDs and cycle length at 4–6 min after 100 µM MCI-154 were statistically significant although the decreases in APA and MDP did not reach a statistical significance (Table 1). E-4031 at concentrations of 1 and 10 µM similarly increased the sinus cycle length and APDs with slight decreases in MDP and APA (Table 1).


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  		Table 1 Effects of MCI-154 and E-4031 on action potential parameters in sinoatrial node preparations

 
3.3 Effects of MCI-154 on membrane currents in isolated atrial cells
MCI-154 produced positive inotropic and negative chronotropic responses, which were associated with increases in APDs, in guinea pig atrial preparations. In order to examine electrophysiological mechanisms, the effects of MCI-154 on the membrane current system were evaluated in isolated atrial cells. In normal Tyrode's solution, myocytes were depolarized for 2 s from a holding potential of –40 mV to various command voltages at a frequency of 0.25 Hz. To obtain a current-voltage relationship (I–V curve), the command voltage was increased from –40 mV to +60 mV in 10 mV steps. MCI-154 at a concentration of 10 µM did not affect ICa,L, whereas it significantly reduced the time-dependent and tail outward current, as shown in Fig. 3A, B and C. The drug effect at the concentration of 10 µM was partially reversible on washout although the process was very slow (10–15 min).


Figure 3
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  		Fig. 3 Effects of MCI-154 on ICa,L and IK in guinea pig atrial cells. (A) Current traces recorded during 2 s depolarizing pulses to –30, –20, –10, 0, +10, +20, +30, +40, +50, and +60 mV from a holding potential of –40 mV before (left) and after exposure to 10 µM MCI-154 (right, 12 min after drug application) in the normal Tyrode's solution in a right atrial myocyte. Cell capacitance was 54 pF. Arrow head indicates zero current level. (B) Current-voltage relation for the ICa,L peak current and time-dependent current elicited by 2 s depolarizing pulses. The time-dependent current was measured at the end of the depolarizing steps. Data represent mean±S.E.M. of 5 cells before (open circle) and after 10 µM MCI-154 (closed circle). * Significant change from control. (C) Current-voltage relation for tail currents measured on return of membrane voltage to –40 mV from indicated test potentials. Data represent mean±S.E.M. of 5 cells before (open circle) and after 10 µM MCI-154 (closed circle). * Significant change from control. (D) Superimposed tracings showing the effect of 100 µM MCI-154 on ICa,L in the Cs+/Cs+ condition. Test pulses were from –40 to +10 mV for 200 ms. Arrow head indicates zero current level.

 
In order to confirm that MCI-154 does not increase ICa,L, we examined the effect of MCI-154 on ICa,L in the Cs+/Cs+ condition. The membrane potential was depolarized from a holding potential of –40 mV to +10 mV for 200 ms at a frequency of 0.1 Hz (Fig. 3D). After rupture of the cell membrane, peak ICa,L tended to decrease, showing a slight ‘run down’. MCI-154 at concentrations of 10 and 100 µM did not increase ICa,L at 10 min after administration of the drug, nor overcome the ‘run down' (10 µM: from 5.21±0.17 to 4.91±0.20 pA/pF, n=5; 100 µM: from 5.15±0.11 to 4.84±0.15 pA/pF, n=6).

In the rest of the experiments, IK was isolated from ICa,L using 0.4 µM nisoldipine. The IK of guinea pig atrial myocytes has been reported to consist of two components, IKr (rapidly activating component) and IKs (slowly activating component) [21]. IKr is activated rapidly with moderate depolarizations (between –40 and 0 mV), and fully activated at +30 mV in atrial cell, whereas IKs is activated slowly with a sigmoidal time course at more positive potentials. To test whether MCI-154 specifically blocks one or both components of the IK, the following experiments were conducted. The blocking effect of MCI-154 on IK was prominent with the shorter pulses (200 ms), where IKr predominates, compared to long pulses (5 s) (Fig. 4A and 4B). The MCI-154 (100 µM)-induced decreases in the time-dependent current and the tail current at 0 mV for 200 ms pulses duration were 73±5% and 100% while those at 0 mV for 5 s pulse duration were 45±6% and 75±7% (Fig. 4C, 4D). In addition, MCI-154-induced inhibition of IK was more marked during mild depolarization than during strong depolarization. MCI-154 (100 µM)-induced decreases of the time-dependent current after test pulse to 0 mV, +20 mV, and +60 mV for 200 ms pulse duration were 73±5%, 76±7%, and 22±6%, respectively (Fig. 4C).


Figure 4
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  		Fig. 4 Effects of MCI-154 on IK elicited short and long test pulses. (A) Current traces recorded during 200-ms pulses to –20, 0, +20, +40, and +60 mV from a holding potential of –40 mV before (left) and after exposure to 100 µM MCI-154 (right, 5 min after drug administration) in an atrial cell. The external solution contained 0.4 µM nisoldipine. Cell capacitance was 62 pF. Arrow head indicates zero current level. (B) Current traces recorded during 5-s pulses to –20, 0, +20, +40, and +60 mV from a holding potential of –40 mV before (left) and after exposure to 100 µM MCI-154 (right, 6 min after drug administration) in an atrial cell. The external solution contained 0.4 µM nisoldipine. Cell capacitance was 65 pF. Arrow head indicates zero current level. (C) Graphs showing IK measured at the end of 200-ms test pulses to the indicated test potential (left) and IK,tail measured after repolarizing to –40 mV from the indicated test potential (right). Data represents mean±S.E.M. of 7 cells before (open circle) and after 100 µM MCI-154 (closed circle). * Significant change from control. (D) Graphs showing IK measured at the end of 5 s test pulses to the indicated test potential (left) and IK,tail measured after repolarization to –40 mV from the indicated test potential (right). Data represents mean±S.E.M. of 5 cells before (open circle) and after 100 µM MCI-154 (closed circle). * Significant change from control.

 
In order to test whether MCI-154 inhibits IKs as well as IKr, we examined the effect of MCI-154 on IKs in the presence of E-4031. After the full inhibition of IKr by 5 µM E-4031, MCI-154 only slightly decreased the tail current after long depolarizing pulses (Fig. 5A–5C). In the presence of 5 µM E-4031, MCI-154 at a concentration of 100 µM decreased the tail current amplitude at 0, +40, and +60 mV by 15±6%, 22±6%, and 12±5%, respectively (Fig. 5C). These findings suggest that MCI-154 preferentially blocks IKr.


Figure 5
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  		Fig. 5 Effects of MCI-154 on IKs elicited long test pulses in the presence of E-4031. (A) Current traces recorded during 5 s pulses to –20, 0, +20, +40, and +60 mV from a holding potential of –40 mV in the control condition (left), in the presence of 5 µM E-4031 (middle) and after the addition of 100 µM MCI-154 (right, 6 min after MCI-154). The external solution contained 0.4 µM nisoldipine. Cell capacitance was 72 pF. Arrow head indicates zero current level. (B) (C) Graphs showing IK measured at the end of 5 s test pulses to the indicated test potential (left) and IK,tail measured after repolarization to –40 mV from the indicated test potential (right). Data represents mean±S.E.M. of 8 cells in the absence (open circle) and presence of E-4031 (open square) and E-4031 plus MCI-154 (closed circle). * Significant difference between control and E-4031 alone group. {dagger}Significant difference between E-4031 alone and E-4031 plus MCI-154.

 
The envelope of tails test was also performed to confirm this hypothesis. This test predicts that if IK represents the activation of a single type of channel, then the magnitude of tail current after a given depolarizing pulse of variable duration should increase in parallel with the magnitude of the outward current during the depolarizing pulse [20]. For a given channel type, the ratio of tail currents (IK,tail) to the time-dependent currents activated during the pulses (IK,depo): IK,tail/IK,depo should be constant, regardless of the duration of the pulse. Typical recordings elicited by the depolarizing pulses from a holding potential of –40 to +30 mV for durations ranging from 125 ms to 1.25 s before and after exposure to 100 µM MCI-154 are shown in Fig. 6A, and the ratios of IK,tail/IK,depo obtained from the depolarizing pulses of 125 ms-7 s duration are summarized in Fig. 6B. The control ratio of IK,tail/IK,depo in guinea pig atrial cell was dependent on the duration of the pulse, as described in an earlier report [21]. In the presence of 100 µM MCI-154, there was a significant reduction of IK,tail/IK,depo for depolarizing pulses shorter than 1 s (Fig. 6B), suggesting that MCI-154 might preferentially block IKr.


Figure 6
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  		Fig. 6 IK block by MCI-154 evaluated by the envelope of tails test. Envelopes of tail currents were generated by applying depolarizing pulses of variable duration (125–7000 ms) to +30 mV from holding potentials of –40 mV. Typical superimposed tracings obtained from the same cell are shown in (A). The external solution contained 0.4 µM nisoldipine and the cell capacitance was 58 pF. Arrow head indicates zero current level. The summarized data of the ratios of the tail currents (IK,tail) to the time-dependent currents activated during the depolarizing pulses (IK,depo) from holding potentials of –40 mV is depicted in (B). The ratios are plotted against pulse duration and the inset shows an example of the measurement. Open circle and closed circles indicate the data before (control) and after 100 µM MCI-154. Values are mean±S.E.M. of 9 experiments. * Significant change from the ratio in the control condition.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In the failing heart where intracellular Ca2+ handling is impaired, the ability of sarcoplasmic reticulum for uptake of Ca2+ diminishes and the decay of Ca2+ transient delays beyond the start of relaxation [22]. In these circumstances, exercise or sympathetic stimulation increases the heart rate and hence impairs relaxation (lusitropic dysfunction). Thus, the increase of heart rate not only worsens the hemodynamics but also may aggravate injury of the cardiac muscle with intracellular Ca2+ overload. In fact, previous studies using various inotropic agents with a PDE III inhibitory action have demonstrated that these drugs further aggravate the dysfunction of the failing heart [2–5]. Therefore, new inotropic agents devoid of a positive chronotropic action are being sought for the treatment of the heart failure.

Recently a new classification of positive inotropic actions, based on electrophysiological characteristics, has been proposed by Varró and Papp [23]. According to their classification, class I actions designate positive inotropic mechanisms that enhance the transmembrane Ca2+ influx and class II actions include mechanisms that increase intracellular Na+ activity. Class III and IV actions involve mechanisms that increase Ca2+ sensitivity of the contractile apparatus and those that lengthen the cardiac APD, respectively. Positive inotropic drugs devoid of class I actions are expected to produce little positive chronotropic effect. Indeed, (+)-EMD 60263, a putative Ca2+ sensitizer, has been reported to produce a negative chronotropic response [24]. Even the positive inotropic drugs having class I actions sometimes show a negative or neutral chronotropic effect. For instances, vesnarinone and its derivatives have been shown to produce neutral to negative chronotropic effects [25, 26]. Since these drugs have been shown to inhibit cardiac K+ channel commonly [24, 25, 27, 28], the negative chronotropic effects might be ascribed to the APD-prolonging property, i.e. class IV action. In the present study MCI-154 produced a negative chronotropic response in isolated guinea pig right atrial preparations. Therefore, we thought it would be of interest to examine the effects of MCI-154 on the sinus node action potentials and the delayed rectifier K+ current (IK) of atrial cells.

In isolated atrial cells MCI-154 at concentrations of 10 and 100 µM decreased IK. The inhibitory action of MCI-154 on IK was more prominent when the IK was evoked by short depolarizing pulses than by long pulses. In addition, the MCI-154-induced inhibition of IK was more marked during weakly-depolarizing test pulses than during strongly-depolarizing test pulses (Fig. 4). After the full inhibition of IKr by E-4031, a prototypical IKr channel blocker, MCI-154 at a concentration of 100 µM could only slightly decreased the IK elicited by long depolarizing pulses (Fig. 5). Therefore, the inhibitory effect of MCI-154 on IKs would be small, if any, and MCI-154 preferentially inhibits IKr. In order to confirm this concept, the envelope of tails test was conducted in atrial cells. MCI-154 clearly decreased the IK,tail/IK,depo ratio during short depolarizing pulses (Fig. 6), implying the predominant IKr block by MCI-154.

Recently it has been reported that the Ca2+ sensitizer (+)-EMD 60263 inhibits IKr in guinea pig ventricular myocytes [24]. Therefore, the IKr inhibition may not be a special feature of MCI-154. In addition, quinolinone derivatives having PDE-inhibiting properties, such as vesnarinone and OPC-18790, have been also shown to inhibit IK [25, 27–29]. The APD-prolonging effect of these drugs (class IV actions), resulting from the IK inhibition, might contribute to their chronotropic effects as well as inotropic effects.

In this study MCI-154 as well as E-4031 prolonged the APD and the sinus cycle length in isolated sinoatrial node preparations. Therefore, the inhibition of IKr by MCI-154 might play an important role in the negative chronotropic effect. It was reported that a high concentration of E-4031 depolarized the MDP and stopped the spontaneous firing in sinoatrial node cells of the rabbit [30]. Although E-4031 produced a negative chronotropic response concomitantly with an increase in APD and a decrease in MDP, it failed to stop the spontaneous activity. One possible explanation may be that electrotonic influence from surrounding atrial cells might prevent the sinoatrial nodal cells from stopping the spontaneous activity, probably by lessening the decrease in MDP by E-4031. Because of the methodological difficulty in the isolation of guinea pig sinoatrial nodal cells, we could not evaluate the effects of MCI-154 on the nodal IK. Further studies to evaluate the MCI-154 on the membrane current system of the sinoatrial nodal cells may be needed to clarify the precise mechanism of the negative chronotropic action.

MCI-154 has been postulated to be a relatively pure Ca2+ sensitizer with minor PDE III inhibitory activity [9, 10, 31, 32]. In fact, MCI-154 did not increase ICa,L in atrial cells even when ICa,L was isolated from K+ currents using the Cs+-rich condition. Recently we have reported that MCI-154 also failed to increase ICa,L in guinea pig ventricular cells [13]. Therefore, the PDE III inhibitory action of MCI-154, i.e. class I action, may not be important for its positive inotropic effects. However, MCI-154 can prolong APD by inhibiting IKr, as shown in this study. Lengthening of APD may contribute to the Ca2+ load during the plateau of the action potential via the L-type Ca2+ channel, the Na+ channel and/or the Na+-Ca2+ exchanger [23]. Thus, it cannot be concluded that MCI-154 is a pure Ca2+ sensitizer. These electrophysiological properties of MCI-154 contrast well with those of levosimendan, another Ca2+ sensitizer which failed to inhibit IK but increase ICa,L in guinea pig ventricular cells [33].

The lengthening of APD was also observed with (+)-EMD 60263 and vesnarinone [24, 34]. As already discussed, the IK inhibition by the positive inotropic drugs including MCI-154 can lead to neutral to negative chronotropic effects. On the other hand, it should be also kept in mind that excessive IKr inhibition by these drugs can result in marked QT prolongation and torsades de pointes.

In terms of vesnarinone, a positive inotropic drug having class I and IV actions, early clinical trial proved that long term therapy with low dose but not high dose resulted in lower morbidity and mortality and improved quality of life in patients with chronic heart failure [35]. However, a more recent study suggested that the reduction in mortality attributed to vesnarinone cannot be reproduced [36]. One of the differences between vesnarinone and MCI-154 may be the effects on ICa,L:vesnarinone increased ICa,L in ventricular cells [27, 28]while MCI-154 failed to increase the current [13]. It is possible that MCI-154 may produce less intracellular Ca2+ load than vesnarinone in the failing myocardium. On the other hand, the Ca2+-sensitizing (class III) action of MCI-154 may impair the diastolic relaxation [37]. Well-designed clinical trials with Ca2+ sensitizers may be warranted.

In conclusion, the present study has demonstrated that an inhibitory action of MCI-154 on IKr appears to be at least in part involved in the negative chronotropic and positive inotropic effects with its high concentrations in atrial tissues.

Time for primary review 26 days.


   Acknowledgements
 
The authors thank M. Tamagawa at the Department of Pharmacology, Chiba University School of Medicine, for his excellent technical assistance. We also thank Mitsubishi Chemical for the gift of MCI-154 and Eisai Pharmaceutical for the gift of E-4031.


   Notes
 
1 Present address: Department of Medicine (Division of Cardiology), Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku 173, Tokyo, Japan. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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