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Cardiovascular Research 2006 70(3):497-508; doi:10.1016/j.cardiores.2006.02.026
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Copyright © 2006, European Society of Cardiology

Effects of a Na+/Ca2+ exchanger inhibitor on pulmonary vein electrical activity and ouabain-induced arrhythmogenicity

Wanwarang Wongcharoenb, Yao-Chang Chenc, Yi-Jen Chena,*, Che-Ming Change, Hung-I Yehf, Cheng-I Lind and Shih-Ann Chenb

aDivision of Cardiovascular Medicine, Taipei Medical University, Wan-Fang Hospital, 111, Hsin-Lung Road, Sec. 3, Taipei, Taiwan
bNational Yang-Ming University, School of Medicine, Division of Cardiology, Veterans General Hospital-Taipei, Taipei, Taiwan
cDepartment of Biomedical Engineering, Taipei, Taiwan
dInstitute of Physiology and, National Defense Medical Center, Taipei, Taiwan
eShin Kong Wu Ho-Su Memorial Hospital and Department of Medicine, Fu Jen Catholic University, Taipei, Taiwan
fDepartment of Internal Medicine, Mackay Memorial Hospital, Nursing and Management College, Taipei, Taiwan

* Corresponding author. Fax: +886 2 29339378; +886 2 28735656. Email address: a9900112{at}ms15.hinet.net

Received 18 October 2005; revised 18 February 2006; accepted 21 February 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objective Pulmonary veins (PVs) are the most important focus for generation of atrial fibrillation. The Na+/Ca2+ exchange (NCX) current is important in PV electrical activity and cardiac glycosides-induced arrhythmias. The purpose of this study was to investigate whether KB-R7943, a NCX current blocker with preferential inhibition of the Ca2+ influx, may alter PV electrophysiological characteristics and reduce glycoside-induced arrhythmogenicity.

Methods Conventional microelectrodes were used to record the effects of KB-R7943 on action potentials and contractility in isolated rabbit PV tissue specimens with and without administration of ouabain. The ionic currents and intracellular calcium were studied in isolated single cardiomyocytes before and after KB-R7943 by the whole-cell patch clamp and indo-1 fluorimetric ratio techniques.

Results KB-R7943 (0, 3, 10, 30µM) concentration-dependently prolonged APD50 and APD90 and decreased the PV firing rates (2.3±1.2Hz, 2.1±1.2Hz, 1.9±0.9Hz, 1.7±1.1Hz, n=7, p<0.05) and incidences of delayed afterdepolarizations (DADs). KB-R7943 (3, 30µM) decreased transient inward currents, Ca2+ transient and sarcoplasmic reticulum Ca2+ content. Ouabain (0, 0.1, 1µM) concentration-dependently increased the PV firing rates and DADs in PVs with spontaneous activity (n=7) and induced nonsustained spontaneous activity (1µM) in the PVs without spontaneous activity (n=14). However, in the presence of KB-R7943 (30µM), ouabain (1µM) did not increase the PV firing rates or induce spontaneous activity in the PVs without spontaneous activity (n=7).

Conclusions KB-R7943 reduces the PV arrhythmogenic activity and prevents the ouabain-induced arrhythmogenicity. Our findings support the role of the NCX current in the PV electrical activity.

KEYWORDS Atrial fibrillation; Glycosides; Na+/Ca2+ exchange current; Pulmonary vein; Triggered activity


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Atrial fibrillation (AF) is the most common sustained arrhythmia in clinical medicine. The pulmonary veins (PVs) have been demonstrated to be an important source of the initiation of AF [1,2] and also to have a role in the maintenance of AF [3]. The PVs are known to contain cardiomyocytes with and without pacemaker activity and are suggested to be subsidiary pacemakers [4,5]. Through several mechanisms, PVs have been found to have a high arrhythmogenic potential to induce atrial arrhythmias [6–12]. However, the pathophysiology underlying the arrhythmogenesis of the PVs has not been fully elucidated. Previous studies have indicated that abnormal calcium regulation and triggered activity may underlie PV arrhythmogenic acitivity [13,14]. Honjo et al. [13] demonstrated that Na+–Ca2+ exchanger (NCX) blockers abolished PV electrical activity during rapid pacing in ryanodine treated PVs. Our previous studies have shown that activation of transient inward currents (Iti) with the genesis of delayed afterdepolarizations (DADs) is important for the PV electrical activity [7,11]. It is known that NCX plays a pivotal role in the genesis of Iti and repetitive activity of cardiomyocytes [12]. These findings indicated the importance of the NCX currents in PV arrhythmogenic activity. KB-R7943 is a novel isothiourea derivative that has been reported to preferentially block the Ca2+ influx (reverse) mode of the NCX current [15,16]. Several studies have shown that KB-R7943 may eliminate the ischemia and reperfusion-induced arrhythmias [17,18]. Furthermore, through the prevention of atrial electrical remodeling, KB-R7943 was demonstrated to reduce AF in the canine model and was suggested to be a potentially antiarrhythmic agent [19]. Therefore, it is possible that KB-R7943 may inhibit PV arrhythmogenic activity and reduce the occurrence of AF.

Cardiac glycosides are frequently used in the treatment of heart failure with and without AF. However, the narrow therapeutic range of the cardiac glycosides has resulted in easily inducing cardiac arrhythmias due to intoxication. Cardiac glycosides, such as ouabain may increase the intracellular Ca2+ with the enhancement of the NCX currents through the inhibition of the Na+/K+-ATPase. These effects may shorten the atrial refractory period and aggravate tachycardia-induced atrial electrical remodeling with a predisposition for AF [20,21]. Moreover, it has been reported that cardiac glycosides enhanced PV arrhythmogenic activity and induced atrial tachyarrhythmias in guinea pig [22]. KB-R7943 has been shown to prevent the arrhythmogenic effect of cardiac glycosides [23,24]. It is possible that KB-R7943 may prevent glycoside-induced PV arrhythmogenesis. The purposes of the present study were to investigate the effects of KB-R7943 on the PV electrical activity and evaluate whether KB-R7943 may reduce the ouabain-induced PV arrhythmogenicity.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1 Rabbit PV tissue preparations
The investigation conformed to the institutional Guide for the Care and Use of Laboratory Animals. Rabbits (weight, 1–2kg) were anesthetized with an intraperitoneal injection of sodium pentobarbital (40mg/kg). A mid-line thoracotomy was then performed and the heart with the lungs was removed. For dissection of the PVs, the left atrium was opened by an incision along mitral valve annulus extending from the coronary sinus to the septum in Tyrode's soluton with a composition (in mM) of 137NaCl, 4KCl, 15NaHCO3, 0.5NaH2PO4, 0.5MgCl2, 2.7CaCl2, and 11 dextrose. The PVs were separated from the atrium at the left atrium-PV junction and separated from the lungs at the ending of the PV myocardial sleeves. One end of the preparation, consisting of the PVs and atrium-PV junction, was pinned with needles to the bottom of a tissue bath. The other end was connected to a Grass FT03C force transducer with a silk thread. The adventitia of the PVs faced upwards. The tissue was superfused at a constant rate (3ml/min) with Tyrode's solution which was saturated with a 97% O2–3% CO2 gas mixture. The temperature was maintained constant at 37 °C and the preparations were allowed to equilibrate for 1h before the electrophysiological study.

2.2 Electrophysiological and pharmacological studies
The transmembrane action potential (AP) of the PVs was recorded by means of machine-pulled glass capillary microelectrodes filled with 3M of KCl and the PV preparation was connected to a WPI model FD223 electrometer under tension with 150mg. The electrical and mechanical events were displayed simultaneously on a Gould 4072 oscilloscope and Gould TA11 recorder. The signals were recorded with DC coupling and a 10-KHz low-pass filter cutoff frequency using a data acquisition system. Signals were recorded digitally with 16-bit accuracy at a rate of 125kHz. An electrical stimuli with a 10-ms duration and suprathreshold strength (30% above the threshold) were provided by a Grass S88 stimulator through a Grass SIU5B stimulus isolation unit. Different concentrations of KB-R7943 (0, 0.3, 3, 10, 30 µM) or ouabain (0.1, 1 µM) were sequentially superfused to test the pharmacological responses. The PV preparations were treated with either or both drugs for at least 20min for each concentration. The 90% and 50% AP durations (APD90, APD50), AP amplitude (APA), potential during the pleateau of the AP and contractile force were measured during 2Hz electrical stimuli before and after the drug administration.

2.3 Electropharmacological study in single PV cardiomyocytes
PV cardiomyocytes from rabbits were enzymatically dissociated through the same procedure as previously described [11]. PV cardiomyocytes with pacemaker activity were identified by the presence of constantly spontaneous beating during perfusion with Tyrode's solution with a composition (in mM) of NaCl 137, KCl 5.4, HEPES 10, MgCl2 0.5, CaCl2 1.8, and glucose 10.

A whole-cell patch-clamp was performed in the PV cardiomyocytes with pacemaker activity before and after the administration of KB-R7943 by an Axopatch 1D amplifier (Axon Instruments, California, USA) at 35±1°C as previously described [25]. The ionic currents were recorded in the voltage-clamp mode [7,8]. Micropipettes were filled with a solution containing (in mM) CsCl 130, MgCl2 1, Mg2ATP 5, HEPES 10, EGTA 10, NaGTP 0.1, and Na2 phosphocreatine 5 (pH of 7.2 with CsOH) for L-type calcium current (ICa–L); containing (in mM) NaCl 20, CsCl 110, MgCl2 0.4, CaCl2 1.75, TEACl 20, BAPTA 5, glucose 5, Mg2ATP 5, and HEPES 10 (pH of 7.25 with CsOH) for NCX current; containing (in mM) CsCl 133, NaCl 5, EGTA 10, Mg2ATP 5, TEACl 20, HEPES 5 (pH 7.3 with CsOH) for Na+ current (INa); and containing (in mM) KCl 20, K aspartate 110, MgCl2 1, Mg2ATP 5, HEPES 10, EGTA 0.5, LiGTP 0.1, and Na2 phosphocreatine 5 (pH of 7.2 with KOH) for Iti and potassium currents.

ICa–L was recorded during depolarization from a holding potential of – 50mV to testing potentials ranging from – 40 to +60 mV in 10-mV steps for 300ms at a frequency of 0.1Hz by means of perforated patch-clamp with amphotericin B. The NaCl and KCl in the external solution were replaced by TEACl and CsCl, respectively.

INa was recorded during depolarization from a holding potential of – 120mV to testing potentials ranging from – 90 to +60 mV in 10-mV steps for 40ms at a frequency of 3Hz at room temperatures (22–24°C) with an external solution containing (in mM): NaCl 5, CsCl 133, MgCl22, CaCl21.8, nifedipine 0.002, HEPES 5 and glucose 5 with a pH of 7.3.

The transient outward current (Ito) was studied with a double-pulse protocol. A 30-ms pre-pulse from – 80 to – 40mV was used to inactivate the sodium channels, followed by a 300-ms test pulse to +60mV in 10-mV steps at a frequency of 0.1 Hz. CdCl2 (200 µM) was added to the bath solution to inhibit ICa–L. Ito was measured as the difference between the peak outward current and steady state current. The delayed rectified outward potassium current (IK) was measured from the peak outward current at the end of 1 s of the depolarization from – 40 to +60mV in 10-mV steps at a frequency of 0.1Hz during the infusion of CdCl2 (200µM) and 4-aminopyridine (2mM) in the bath solution.

Iti was induced during depolarization from a holding potential of – 40mV to +40mV for a duration of 3s and the current was measured on the return to – 40mV. The amplitude of the Iti was measured as the difference between the peak of the transient current and mean of the current just before and after the transient current [7,11].

The inward rectifier potassium current (IK1) was activated from – 40mV to test potentials ranging from – 20 to – 120mV in 10-mV steps for 1s at a frequency of 0.1Hz under the infusion of CdCl2 (200µM) and 4-aminopyridine (2mM) in the bath solution. The amplitudes of the IK1 were measured as 1mM barium-sensitive currents.

The NCX current was elicited by depolarization in 10mV steps from a holding potential of – 40mV to test potentials from – 100 to +100mV for 300ms at a frequency of 0.1Hz (insets in Fig. 3). The amplitudes of the NCX current were measured as 10mM nickel-sensitive currents. The external solution (in mM) consisted of NaCl 140, CaCl2 2, MgCl2 1, HEPES 5 and glucose 10 with a pH of 7.4 and contained strophanthidin (10µM), nitrendipine (10µM) and niflumic acid (100µM) [25].


Figure 3
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  		Fig. 3 The tracings and I–V relationship of NCX currents before and after the administration of KB-R7943 (control, n=34; KB-R7943, 0.3µM, n=10; 3µM, n=8; 10µM, n=8; and 30µM, n=8) in the PV cardiomyocytes. The insets in the current traces show the various clamp protocols.

 
2.4 Measurement of the intracellular calcium
Intracellular calcium ([Ca2+]i) was recorded by a fluorimetric ratio technique. The fluorescent indicator indo-1 was loaded by incubating the myocytes at room temperature for 20–30min with 25µM of indo-1/AM (Sigma Chemical, St. Louis, MO). The PV cardiomyocytes were then perfused with a normal bath solution at 35±1°C for at least 20min to wash out the extracellular indicator and to allow for intracellular deesterification of the indo-1. The background and cell autofluorescence were cancelled out by zeroing the output of the photomultiplier tubes using cells without indo-1 loading. The experiments were performed during superfusion before and after KB-R7943.

Ultraviolet light at a strength of 360nm with a monochromator was used for the excitation of the indo-1 from a xenon arc lamp controlled by a microfluorometry system (OSP100-CA, Olympus, Tokyo, Japan) and the excitation light beam was directed into an inverted microscope (IX-70; Olympus). The emitted fluorescence signals from the indo-1/AM loaded myocytes were digitized at 200Hz. The ratio of flurorescence emissions at 410nm and 485nm was recorded. R410/485 was used as the index of the [Ca2+]i. This approach avoided the uncertainties related to in vivo calibration of the fluorescent Ca2+ indicators. The Ca2+ transient, peak systolic [Ca2+]i, diastolic [Ca2+]i, and decay portion of the Ca2+ transient ({tau}Ca) were measured during a 2Hz field-stimulation with 10-ms twice-threshold strength square-wave pulses. The {tau}Ca was determined by the mono-exponential least-squares fit. The fluorescence ratio data were processed and stored in a computer using software (OSP-SFCA; Olympus). Sarcoplasmic reticulum (SR) Ca2+ content was estimated by adding 20mM caffeine after electric stimulation at 2Hz for at least 30s. The total SR Ca2+ content was measured from the peak amplitude of the caffeine-induced Ca2+ transients.

2.5 Statistical analysis
All quantitative data are expressed as mean±SEM. A paired t-test was used to compare the differences before and after the drug administration in the PV tissue preparations and single cardiomyocytes. Nominal variables were compared by a Chi-square analysis with Yates correction or Fisher's exact test. A p-value lower than 0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1 Effects of KB-R7943 on the PV electrical activity
As the examples show in Fig. 1A, KB-R7943 (3, 10, 30µM) concentration-dependently decreased the firing rates in the PVs with spontaneous activity (Fig. 1B). In the PVs without spontaneous activity, KB-R7943 concentration-dependently prolonged the APD50 and APD90 and KB-R7943 (10, 30 µM) decreased the APA significantly (Fig. 1C, D). Additionally, KB-R7943 (3, 10, 30µM) concentration-dependently shifted the plateau potential to less negative values. KB-R7943 (10, 30µM) decreased the contractile force of the PVs, but did not affect the diastolic tension (Fig. 1C, D). Moreover, KB-R7943 (0, 0.3, 3, 10, 30µM) concentration-dependently decreased the incidence of DADs (n=11; 45%, 45%, 18%, 0%, 0%, p<0.05) significantly.


Figure 1
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  		Fig. 1 Effects of KB-R7943 on the PV electrical activity. Panel A shows the tracings of the effects of different concentrations of KB-R7943 on the firing rates in the PVs with spontaneous activity. Panel B shows the concentration–response curve of the effects of KB-R7943 on the PV firing rate (n=7). Panel C shows the superimposed tracings of different concentrations of KB-R7943 on the AP configuration and contractile force. The effect of KB-R7943 on the suppression of DADs ({downarrow}) is shown. Panel D shows the concentration–response curve of the effects of KB-R7943 on the APD50, APD90, APA, potential during plateau phase of AP (plateau potential) and contractile force (n=8). *P<0.05, **P<0.01, and ***P<0.001 versus before the KB-R7943 administration.

 
3.2 Effect of KB-R7943 on the membrane currents of PV cardiomyocytes
As the examples show in Fig. 2, KB-R7943 significantly decreased Iti by 59% at 3µM and by 96% at 30µM (Fig. 2B). Fig. 3 shows the tracings and I–V relationship of the effects of KB-R7943 on NCX currents of PV cardiomyocytes. KB-R7943 reduced the reverse mode of NCX currents with an IC50 of 0.36µM and reduced the forward mode of NCX with an IC50 of 5.62µM. Therefore, KB-R7943 preferentially suppressed the reverse mode of NCX currents in PV cardiomyocytes.


Figure 2
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  		Fig. 2 Effects of KB-R7943 on transient inward currents. Panel A shows the tracings of the Iti before and after the administration of KB-R7943 (30µM) in PV cardiomyocytes. Panel B shows the average of Iti before and after KB-R7943 (3µM, n=7; 30µM, n=7). *P<0.05 and **P<0.01 versus before the KB-R7943 administration. The inset in the current traces shows the clamp protocol.

 
Fig. 4 shows the tracings and I–V relationship of the effects of KB-R7943 (3, 30µM) on INa (Fig. 4A) and ICa–L (Fig. 4B) of the PV cardiomyocytes. KB-R7943 (30µM, but not 3µM), decreased ICa–L and INa significantly.


Figure 4
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  		Fig. 4 Effects of KB-R7943 on INa and ICa–L. Panel A shows the tracings and I–V relationship of INa before and after 3µM (n=10) and 30µM (n=9) KB-R7943 in the PV cardiomyocytes. Panel B shows the tracings and I–V relationship of ICa–L before and after 3µM (n=7) and 30µM (n=7) KB-R7943 in the PV cardiomyocytes. *P<0.05 and ***P<0.001 versus before the KB-R7943 administration. The insets in the current traces show the clamp protocol.

 
Fig. 5 shows the tracings and I–V relationship of the effects of KB-R7943 (3, 30µM) on Ito (Fig. 5A), IK (Fig. 5B) and IK1 (Fig. 5C) of the PV cardiomyocytes. KB-R7943 (3, 30µM) reduced the Ito, IK and IK1 significantly.


Figure 5
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  		Fig. 5 Effects of KB-R7943 on Ito, IK and IK1. Panel A shows the tracings and I–V relationship of Ito before and after 3µM (n=7) and 30µM (n=7) KB-R7943 in the PV cardiomyocytes. Panel B shows the tracings and I–V relationship of IK before and after 3µM (n=12) and 30µM (n=10) KB-R7943 in the PV cardiomyocytes. Panel C shows the tracings and I–V relationship of IK1 before and after 3µM (n=7) and 30µM (n=7) KB-R7943 in the PV cardiomyocytes. *P<0.05, **P<0.01 and ***P<0.001 versus before the KB-R7943 administration. The insets in the current traces show the various clamp protocols.

 
3.3 Effects of KB-R7943 on the intracellular calcium in PV cardiomyocytes
As the examples show in Fig. 6A, KB-R7943 (3, 30µM) decreased the Ca2+ transient and prolonged the decay time of the Ca2+ transient. However, only KB-R7943 (30µM) significantly decreased the peak systolic [Ca2+]i. Fig. 6B shows the average data of the effect of KB-R7943 (3, 30µM) on the peak systolic [Ca2+]i, diastolic [Ca2+]i, Ca2+ transient, time to peak of Ca2+ transient and decay time of Ca2+ transient. In addition, KB-R7943 decreased SR Ca2+ content by 23% at 3µM and by 38% at 30µM (Fig. 7).


Figure 6
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  		Fig. 6 Effects of KB-R7943 on the Ca2+ transient in the PV cardiomyocytes. Panel A shows the tracings of the Ca2+ transient before and after KB-R7943 (3, 30µM) administration. Panel B shows the average of the peak systolic [Ca2+]i, diastolic [Ca2+]i, Ca2+ transient, time to the peak, and decay portion of the Ca2+ transient ({tau}Ca) before and after 3µM (n=7) and 30µM (n=7) KB-R7943 (3, 30µM). *P<0.05, and **P<0.01 versus before the KB-R7943 administration.

 

Figure 7
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  		Fig. 7 Effects of KB-R7943 on SR Ca2+ content. Panel A shows the tracings of caffeine-induced Ca2+ transient before and after KB-R7943 (3, 30µM) administration. Panel B shows the average of SR Ca2+ content before and after 3µM (n=7) and 30µM (n=7) KB-R7943. **P<0.01 versus before the KB-R7943 administration.

 
3.4 Effects of ouabain on the PV electrical activity
Fig. 8A shows an example of ouabain's effect on the PVs with spontaneous activity. Ouabain (0, 0.1, 1µM) concentration-dependently increased the PV firing rates (Fig. 8B). In the PVs without spontaneous activity, ouabain (1µM) induced PV nonsustained spontaneous activity in 13 (93%) of 14 PVs (Fig. 9A) after the drug administration for 11±3min, and ouabain (0, 0.1, 1µM) increased the incidences of DADs (Fig. 9B, n=14; 43% versus 57% versus 93%, p<0.05). However, ouabain at 0.1µM did not induce spontaneous activity in any PV tissue preparations. Moreover, ouabain (0.1, 1µM) concentration-dependently decreased the APD50 and APD90 (Fig. 9B and C). Ouabain (1µM, but not 0.1µM) increased the contractile force and diastolic tension (tonotropic effect) significantly (Fig. 9B). The effects of ouabain were also investigated in isolated left atrial tissue preparations. Compared to that in the PVs, ouabain (1µM) induced left atrial spontaneous activity with a lower incidence (34%, n=35, p<0.05).


Figure 8
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  		Fig. 8 The effects of ouabain on the PVs with spontaneous activity. (A) The tracings show that ouabain (0.1, 1µM) increased the firing rates in the PVs. (B) The concentration–response curve of the effects of ouabain on the PV firing rate (n=7). **P<0.01 versus before the ouabain administration.

 

Figure 9
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  		Fig. 9 Effects of ouabain on the PVs without spontaneous activity. (A) The tracings show an example of ouabain (1µM) induced PV firing in a PV without spontaneous activity. The development of a phase 4 depolarization was noted. (B) The superimposed tracings show the effects of ouabain (0.1, 1µM) on the AP configuration and contractile force. Note that ouabain (1µM) induced a DAD ({downarrow}) and increased the contractile force and diastolic tension. C, The concentration–response curve of the effects of ouabain on the APD50 and APD90, contractile force and diastolic tension (n=11). *P<0.05, **P<0.01, and ***P<0.001 versus before the ouabain administration.

 
3.5 Effect of KB-R7943 on the ouabain-induced arrhythmogenicity
In the presence of KB-R7943 (30µM), ouabain (1µM) did not increase the firing rates in the PV with spontaneous activity but decreased it from 1.8±0.4Hz to 1.4±0.3Hz (Fig. 10A, n=7, p<0.05). In the PVs without spontaneous activity, ouabain (1µM) only induced spontaneous activity in 2 (25%) of 8PVs pretreated with KB-R7943 (30µM), which was significantly lower than that without the presence of KB-R7943 (p<0.05). The average time (24±2 versus 11±3min) for ouabain-induced PV spontaneous activity was longer in the PVs pretreated with KB-R7943 than in those without. Moreover, in the presence of KB-R7943 (30µM), the effects of ouabain (1µM) on the APD50 (33±4 to 30±4ms, p>0.05) and APD90 (131±7 to 125±8 ms, p>0.05) were insignificant and the positive tonotropic effect of ouabain was significantly reduced. However, the positive inotropic effect was not affected (Fig. 10B).


Figure 10
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  		Fig. 10 Interactions of KB-R7943 and ouabain on the PV electrical activity. (A) Ouabain did not increase, but decrease the PV firing rates in the presence of 30µM KB-R7943. (B) Effects of ouabain on the AP configuration, contractile force and diastolic tension in the presence of 30µM KB-R7943. The positive tonotropic effect was significantly reduced but the positive inotropic effect was not affected.

 
In the presence of KB-R7943 (30µM), ouabain (1µM) significantly increased the contractile force in a comparable degree as ouabain without KB-R7943 (191±67% versus 239±47%, p>0.05).

This study also investigated the effects of KB-R7943 on ouabain-induced arrhythmias. After ouabain (1µM)-induced PV arrhythmogenic activity (n=7), KB-R7943 (30µM) did not significantly change the firing rates (3.6±0.3 versus 3.7±0.4, p>0.05, Fig. 11).


Figure 11
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  		Fig. 11 Effects of KB-R7943 on ouabain-induced arrhythmias. The tracings show that KB-R7943 (30µM) did not terminate the ouabain (1µM)-induced arrhythmogenic activity.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
4.1 Effects of NCX inhibitors on the PV electrical activity
Abnormalities in intracellular Ca2+ homeostasis are important in the pathophysiology of AF [26]. NCX induces Iti with the genesis of DADs, and an upregulation of NCX has been found in atrial myocytes in AF patients [27]. In this study, we studied the effect of NCX blockers on the PV electrical activity and found that KB-R7943 reduced PV arrhythmogenic activity with the suppression of the Iti, DADs and PV spontaneous activity. Consistent with other studies, the present study showed that KB-R7943 selectively inhibited the NCX currents preferentially on the reverse mode rather than the forward mode [15,17]. The IC50 of the reverse mode in the PVs was similar to the previous study on guinea-pig ventricular cells [15].

Similarly, KB-R7943 was found to have effects on other ionic currents [15,16]. KB-R7943 at 3µM reduced Ito, IK, IK1 and SR Ca2+ content and KB-R7943 at 30µM reduced INa, ICa–L, Ito, IK, IK1 and SR Ca2+ content.

In this study, KB-R7943, both at low and high concentrations, reduced PV arrhythmogenicity through its effects on the triggered activity and automaticity. The effects of KB-R7943 on the decrease of DADs, Iti and PV firing rates could be caused by the reduction in the SR Ca2+ content due to the decrease in the Ca2+ entry during AP, either from inhibition of the reverse mode of the NCX currents (3, 30µM) or the inhibition of ICa–L (30µM). DADs have been proposed to be caused by diastolic Ca2+ release from SR. It has been demonstrated that the SR Ca2+ content determined the spontaneous diastolic Ca2+ release and influenced the pacemaker rates [28]. As the SR Ca2+ content reduced, there would be no longer a diastolic Ca2+ release, which would result in a decrease of DADs. The decrease in spontaneous diastolic Ca2+ release by KB-R7943 also would prolong the diastolic depolarization period and reduce the PV firing rates. In addition, the direct inhibitory effect of high dose of KB-R7943 on the forward mode of NCX currents may also have a role in the decrease of DADs, Iti and PV firing rates. However, it is not clear to what extent the reduction was related to either of these mechanisms. Moreover, the superior antiarrhythmic effect of a higher dose of KB-R7943 may also arise from the nonspecific actions on several ion currents. The reduction in the SR Ca2+ content could also explain the decrease in the Ca2+ transient and a negative inotropic effect caused by KB-R7943. The prolongation of the calcium decay by KB-R7943 (30µM) suggested that the calcium extrusion through the NCX currents was reduced.

Previous studies have shown that NCX currents were similar between PV and left atrium cardiomyocytes [29]. Because PV cardiomyocytes have less negative resting membrane potential and smaller IK1 [7,30], the activation of the reverse mode of NCX with an increase in the SR Ca2+ content will lead to diastolic Ca2+ release and activate the forward mode of NCX with the genesis of DADs and arrhythmias.

It has been shown that KB-R7943 increased the APD in cardiomyocytes [23,24]. Consistently, our results showed that KB-R7943 prolonged APD in the PVs. The reduction in the reverse mode of NCX and potassium currents by KB-R7943 may contribute to the prolongation of the APD. This effect may prevent the genesis of microreentry in the PVs which has been proposed to be one of the mechanisms of PV arrhythmogenicity [31].

4.2 Role of NCX inhibitors in the ouabain-induced arrhythmogenicity
Ouabain-induced arrhythmogenesis is a frequently used model in studying triggered activity-induced cardiac arrhythmias. In this study, we demonstrated that ouabain not only increased the firing rates in the PVs with spontaneous activity but also induced arrhythmogenic activity in the non-spontaneous firing PVs. Ouabain may increase the PV firing rates through the increase of intracellular Ca2+ due to the increase of intracellular Na+ and the decrease of Na+ gradient and Ca2+ extrusion. In contrast to that observed by Honjo et al. [13], this study demonstrated the occurrence of phase 4 depolarization in the PV tissue preparations, which suggested that increased automaticity and triggered activity are the underlying mechanisms of ouabain-induced PV arrhythmogenesis. Moreover, the higher inducible arrhythmogenesis in the PVs than in the atrial tissue suggested a high arrhythmogenic potential of the PVs.

Previous studies have found that KB-R7943 may prevent glycoside-induced atrial and ventricular arrhythmias [23,24]. In this study, we demonstrated that pre-treatment with KB-R7943 prevented the arrhythmogenic activity of ouabain on the PV cardiomyocytes and also prevented the APD shortening by ouabain. The beneficial effects of KB-R7943 may be due to a reduction in the intracellular Ca2+. However, similar to the previous study [32], KB-R7943 did not affect the inotropic effect of cardiac glycosides and did not terminate the ouabain-induced PV arrhythmogenesis. This could be explained by the fact that KB-R7943 did not counteract the inhibition of the Na+/K+-ATPase by ouabain. If ouabain toxicity had progressed sufficiently, the reduction in Ca2+ entry through the reverse mode of NCX may not be able to decrease the Ca2+ overload of the SR.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
This study demonstrated that NCX currents may play a role in the PV electrical activity through a reduction in the SR Ca2+ content. KB-R7943 reduces the PV arrhythmogenenic activity and prevents the ouabain-induced PV arrhythmogenesis. These findings suggest that KB-R7943 may have a potential role in treating AF.


   Acknowledgements
 
The present work was supported by grants NSC 94-2314-B-075-093, NSC 94-2314-B-010-056, NSC 94-2314-B-010-053, VGH 94-204, VGH 94-005, VGH 94-206, VGH 94-009, SKH-TMU-94-01 from Shin Kong Wu Ho-Su Memorial Hospital and MMH E-94003 from Mackay Memorial Hospital.


   Notes
 
Time for primary review 20 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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