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(Circulation. 2002;106:1410.)
© 2002 American Heart Association, Inc.

Basic Science Reports

KB-R7943 Prevents Acute, Atrial Fibrillation–Induced Shortening of Atrial Refractoriness in Anesthetized Dogs

From the Krannert Institute of Cardiology (A.M., D.P.Z., M.R.) and Department of Medicine, Division of Clinical Pharmacology (S.H.), Wishard Memorial Hospital, Indianapolis, Ind.

Correspondence to Michael Rubart, MD, Wells Center for Pediatric Research, Riley Hospital, 702 Barnhill Dr, Indianapolis, IN 46202. E-mail mrubartv{at}iupui.edu

	Abstract

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Background— To test the hypothesis that Ca²⁺ influx via Na⁺/Ca²⁺ exchange (NCX) underlies atrial fibrillation (AF)–induced shortening of atrial effective refractory period (AERP), we examined the potential of KB-R7943 (KB), a selective inhibitor of Ca²⁺-influx mode NCX, to attenuate this effect.

Methods and Results— Studies were performed in 41 isoflurane-anesthetized dogs. In sinus rhythm dogs, peak AERP changes resulting from intravenous KB infusion ranged from (mean±SEM) 4.4±0.4% (1 mg/kg) to 14.8±2.6% (5 mg/kg; ED₅₀=1.9 mg/kg). AERP was maximally prolonged between 5 and 10 minutes after beginning of KB infusion and returned to baseline values within 30 minutes thereafter. Rapid atrial pacing–induced AF reversibly shortened AERP (P<0.001) in 5 dogs, averaging 14.9±2.1% after 90 minutes of AF. Both the time course and magnitude of mean AERP changes in 5 AF dogs receiving 5 mg/kg KB were indistinguishable from those in 5 sinus rhythm dogs receiving an equivalent KB dose (P>0.05). We measured cardiac tissue and arterial plasma KB concentrations produced by intravenous infusion (1 mg · kg^-1 · min^-1) of 5 mg/kg KB. Plasma drug concentration peaked at the end of KB infusions (30.86±3.26 nmol/L; n=4 dogs) and declined to 0.56±0.19 nmol/L after 100 minutes. The cardiac tissue-to-plasma drug concentration gradient averaged 40 at 100 minutes after start of KB infusion. KB at concentrations achieved in vivo irreversibly blocked NCX-mediated Ca²⁺ influx in isolated canine right atrial myocytes by 60%, but had no significant effect on NCX-dependent Ca²⁺ extrusion.

Conclusion— NCX-mediated Ca²⁺ influx plays an important role in acute, AF-induced AERP shortening.

Key Words: atrium • fibrillation • electrophysiology

	Introduction

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Experimental and clinical studies have demonstrated that atrial fibrillation (AF) shortens the atrial effective refractory period (AERP) and promotes maintenance of AF.^1–3 Verapamil has been shown to attenuate,^3,4 whereas digoxin² and increased extracellular calcium concentration³ have been reported to enhance, these effects of rapid rates, suggesting that AF-induced increases in intracellular calcium load may play a central role in mediating the acute changes in atrial electrophysiological properties.

Increased Ca²⁺ influx via the cardiac Na⁺/Ca²⁺ exchange (NCX) can lead to elevated cellular Ca²⁺ load. Normally, little Ca²⁺ enters via the NCX, but Ca²⁺ entry can increase greatly when intracellular Na⁺ concentration ([Na⁺]_i) rises. Fast rates during sustained tachycardia and fibrillation cause a larger percentage rise in [Na⁺] _i relative to [Ca²⁺]_i^5,6 and increase the fraction of time the myocytes spend at more depolarized potentials. The net effect is an augmented driving force for the Na⁺-Ca²⁺ exchanger in the reverse mode (to bring in Ca²⁺). Because of the stoichiometry of NCX (Na⁺:Ca²⁺=3:1 [or 4:1]⁷), this results in an enhanced net repolarizing current, which shortens action potential duration, and thus refractoriness. Because NCX regulates intracellular concentrations of free Ca²⁺ and Na⁺, it also modulates the cardiac action potential indirectly by way of influencing the activity of other, Ca²⁺- and/or Na⁺-sensitive membrane conductances.

KB-R7943 (KB) is a novel isothiourea derivative that has been reported to preferentially and potently block the Ca²⁺ influx (reverse) mode of the cardiac Na⁺/Ca²⁺ exchanger rather than the extrusion (forward) mode.^8–10 We sought to exploit this property of KB to examine the role of reverse-mode NCX in modulating cardiac electrophysiological properties in dogs in vivo. Furthermore, if increased Ca²⁺ influx via NCX mediates, at least in part, acute, AF-induced shortening of AERP, then one would expect KB to attenuate this effect of AF. Therefore, the second aim of the present study was to assess the effect of KB on changes in atrial refractoriness during rapid pacing–induced acute AF in dogs. Finally, we examined whether KB at concentrations achieved in vivo is capable of selectively inhibiting the Ca²⁺-influx mode of NCX in isolated canine right atrial myocytes.

	Methods

Top Abstract Introduction Methods Results Discussion Conclusions References

 
This study was approved by the Institutional Animal Care and Use Committee of Indiana University in accordance with the Guide for Care and Use of Laboratory Animals (NIH publication No. 86-23, revised in 1985).

In Vivo Experimental Preparation and Electrophysiological Measurements
Forty-one adult mongrel dogs of either sex with a mean weight of 26.7±0.7 kg were initially anesthetized with sodium thiopental (30 mg/kg IV). Anesthesia was maintained by positive-pressure ventilation with pure oxygen supplemented with isoflurane (1.5% to 2.0%). A fluid-filled cannula placed in the left femoral artery was connected to a mechanoelectrical transducer to continuously monitor arterial blood pressure, and a femoral venous cannula was used to administer drugs. For measurements of intracardiac electrograms and myocardial refractoriness, respectively, 7F multipolar catheters were positioned in the right atrium and ventricle, and in the noncoronary cusp of the aorta (His bundle recording). In dogs undergoing rapid atrial pacing, a second 7F bipolar catheter was inserted in the right atrial appendage. To minimize variations in vagal and sympathetic tone, all animals received an initial bolus of atropine and propranolol (0.04 and 0.1 mg/kg, respectively) followed by maintenance infusion for the duration of the experiment (0.007 mg · kg^-1 · h^{- 1} atropine and 0.02 mg · kg^{- 1} · h^-1 propranolol).¹¹ Effective refractory periods (ERPs); AH and HV intervals; QT, QT_c, and QRS durations; and sinus node recovery times were determined as described previously.^{11– 13} To determine the mean atrial fibrillatory cycle length and mean ventricular response rate during AF, the AA and VV intervals, respectively, were measured over a 10-second interval and averaged.

In Vivo Study Protocols
Groups I to V
To study the dose dependence of KB-induced, acute changes in cardiac electrophysiological parameters during sinus rhythm, dogs were assigned to receive KB intravenously at the following doses (in mg/kg): 1 (group I; n=3), 2 (group II; n=3), 3 (group III; n=3), or 5 (group IV; n=5). Group V (n=6) received DMSO diluted in sterile deionized water (Figure 1A). The volume of DMSO equaled that used to dissolve 5 mg/kg KB, the maximum dose administered in this study. Because this amount of DMSO had no significant effect on any of the parameters examined, we did not administer lower vehicle doses. Baseline ERPs were obtained after inhibition of ß-adrenergic and muscarinic receptors three times at each basic drive cycle length (BDCL) and averaged. The solution containing KB or vehicle was then administered intravenously at a rate of 1 mg · kg^-1 · min^-1. Electrophysiological measurements were repeated every 5 to 30 minutes over 120 minutes after completion of intravenous administration.

View larger version (32K): [in this window] [in a new window]   Figure 1. Diagram of experimental protocols. A, Groups I to V; B, Groups VI and VII. SR indicates sinus rhythm; *Time points of ERP measurements; arrows indicate completion of KB or vehicle infusion, respectively. See text for details.

Groups VI and VII
The effect of KB on acute, AF–induced changes in atrial refractoriness was examined in these two groups (Figure 1B). AERPs were measured after blockade of muscarinic and ß-adrenergic receptors three times at a drive cycle length of 400 ms and averaged. Group VI (n=5) then received KB (5 mg/kg IV), and AERP was remeasured 5 minutes after completion of KB infusion. AF was then induced by rapid atrial pacing at a cycle length of 50 ms at an output of 10 mA. Rapid pacing was interrupted every 10 to 15 minutes, and AF was allowed to terminate spontaneously. Immediately on each conversion to sinus rhythm, pacing threshold was redetermined and AERP was remeasured at a cycle length of 400 ms at twice diastolic threshold. S₁S₂ was increased in steps of 2 ms until atrial capture occurred. After the last 15-minute pacing period, AF was not re-induced, and AERP was allowed to return to pre-KB values. In dogs undergoing rapid atrial pacing in the absence of KB (group VII, n=6), AERP was remeasured 10 minutes after beginning of rapid pacing and then every 15 minutes as in group VI (Figure 1B).

In a separate series of 4 experiments, KB was infused intravenously at a dose of 5 mg/kg at a constant rate of 1.0 mg · kg^-1 · min^{- 1}. Arterial blood samples were collected at 1, 2, 3, 5, 7, 10, 20, 25, 40, 55, 70, and 100 minutes after start of KB infusion for subsequent measurements of plasma KB concentrations. Tissue samples were collected from all 4 heart chambers at the end of the observation period for analysis of KB content.

Measurement of Intracellular Free Calcium Concentration in Right Atrial Myocytes
Right atrial myocytes were isolated as described previously.¹⁴ The Tyrode’s solution for cell isolation and experiments contained (in mmol/L) NaCl 136, CaCl₂ 2, KCl 5.6, MgCl₂ 0.8, NaH₂PO₄ 0.33, glucose 10, and HEPES 10 (pH 7.4 adjusted with NaOH). The storage solution contained (in mmol/L) NaCl 136, KCl 5, MgCl₂ 1, NaH₂PO₄ 0.33, glucose 10, HEPES 10, CaCl₂ 0.1, and BSA 0.2% (pH 7.4 adjusted with NaOH). The Na⁺-free solution contained (in mmol/L) tetramethylammonium-chloride 136, KCl 5.6, MgCl₂ 1, HEPES 10, CaCl₂ 2, and glucose 10 (pH 7.4 adjusted with tetramethylammonium-OH).

[Ca²⁺] _i was recorded by a fluorometric ratio technique. Cardiomyocytes were loaded for 10 to 15 minutes at room temperature with the acetoxymethylester of the Ca²⁺ fluorophores, indo-1 (5 µmol/L + 0.02% [wt/vol] Pluronic F127; Molecular Probes) in 100 µmol/L Ca²⁺ storage solution supplemented with 100 µmol/L ryanodine and 1 µmol/L thapsigargin, to block sarcoplasmic reticulum Ca²⁺ release and uptake, respectively.¹⁵ A small aliquot of the cell suspension was placed in a 40-µL perfusion chamber (Warner Instruments, Hamden, CT) on the stage of a Zeiss inverted microscope (Axioscope) equipped with a x60 Nikon water immersion objective (numeric aperture, 1.2). The microscope was attached to a confocal laser-scanning unit (Zeiss LSM 510). Emitted fluorescence was split by a series of dichroic mirrors and passed through narrow-band-pass (±10 nm) filters centered at 400 and 500 nm whereas indo-1 was excited with the 351-nm line of an argon laser. For measurements of the effect of KB on the rise in [Ca²⁺]_i induced by the removal of extracellular Na²⁺ (0Na⁺), cells were imaged in frame mode at a pixel density of 512x512. At fixed time points (see Figures 7A and 8A), 80 images were taken every 1.9 seconds for each emission wavelength separately. For measurements of the effects of KB on Ca²⁺ decline during twitch Ca²⁺ transients, myocytes were field stimulated at 0.2 Hz via 10-ms square wave pulses with 1.2 times threshold amplitude. The stimuli were delivered by a stimulator (SD9, Grass Instruments) via a pair of platinum electrodes held in position by a micromanipulator. Cells were imaged in line-scan mode.¹⁶ A single line across the entire cell length was repeatedly scanned at a frequency of 500 Hz for 30 seconds, and composite line-scan images were constructed by stacking scan lines vertically. The diameter of the pinhole was set to its maximum for all measurements. Fluorescence signals were digitized at 8-bit resolution and stored on the computer’s hard disk for later analysis.

View larger version (49K): [in this window] [in a new window]   Figure 7. KB (15 nmol/L) reduces Ca2+ influx via NCX in single canine right atrial myocytes. A, Schematic of the experimental protocol. Ca2+ influx via NCX was induced by superfusion of myocytes with Na+-free solution for 1 minute. Sarcoplasmic reticulum function was paralyzed by 1 µmol/L thapsigargin and 10 µmol/L ryanodine. B, Representative recording of the effect of 15 nmol/L KB on Ca2+ influx via NCX, induced by switch to Na+-free solution. C and D, Mean results (±SEM, n=7 cells) for the effect of 15 nmol/L KB on 0Na+-induced rise in [Ca2+]i ( R400/500, C) and resting [Ca2+]i (resting R400/500, D). * P<0.001 vs control. WO indicates washout.

In Vitro Experimental Protocols
Experiments were performed at room temperature. Only quiescent, rod-shaped myocytes with clear cross striations were used. Cells were allowed to adhere to the bottom of the chamber, and then they were superfused at 1 mL/min with normal Tyrode’s solution containing 1 µmol/L thapsigargin and 10 µmol/L ryanodine for 15 minutes to wash out extracellular Ca²⁺ indicator and to allow for intracellular de-esterification of the dye. The experimental protocols for assessment of the effects of KB on NCX-mediated Ca²⁺ flux are outlined in Figures 7A and 8A. Only one cell was studied for each cell aliquot transferred to the bath.

Measurement of Serum and Tissue KB Concentrations
Sample Preparation
Serum and tissue homogenate KB concentrations were determined by high-performance liquid chromatography and mass spectrometry (HPLC)/MS. Briefly, 1 mL of 1N NaOH, 0.5 g NaCl, and 10 ng N-desmethyldiazepam (internal standard) was added to serum or homogenate and extracted with 4 mL of ethyl acetate. The organic layer was dried under a vacuum and reconstituted with 100-µL buffer.

HPLC/MS
HPLC/MS was achieved with a Finnigan Navigator equipped with an atmospheric chemical ionization in positive mode (Atmospheric Pressure Chemical Ionisation) probe and a HP 1100 series chromatography system. Chromatography was performed using a 5-µm Phenomenex C18 Luna column (150x4.6 mm ID) eluted with a buffer consisting of ammonium acetate pH=8/acetonitrile (35/65: vol/vol) at 1 mL/min. KB and N-desmethyldiazepam were detected by selective ion monitoring at 332.3 and 271.5 m/z, respectively. The Atmospheric Pressure Chemical Ionisation probe conditions were cone 20 V, corona pin 3.5 kV, nitrogen 350 L/h, and furnace temperature 550°C. The coefficient of variation at 5 ng/mL KB was 3.5%.

Data Analysis
The ratio of the two fluorescent signals at 400 and 500 nm (R_400/500) was used as an index of [Ca²⁺]_i. Ratios were not converted to [Ca²⁺] _i to avoid uncertainties caused by in vivo calibration of indo-1.¹⁷ Background fluorescence was canceled out by subtracting from each pixel the average fluorescence intensity obtained from a cell-free region. Ratio images were obtained by digitally dividing the background-corrected fluorescent intensities at 400 and 500 nm. For the experiments examining the effect of KB on 0Na⁺-induced rise in [Ca²⁺] _i, the boundaries of the cell being studied were outlined and the average R_400/500 of all pixels inside this region was calculated (MetaMorph software, Universal Imaging Corp) For the measurements of Ca²⁺ decline during twitch calcium transients, the average R_400/500 along each scan line was calculated and plotted as a function of time. The time constant of relaxation of the stimulus-induced Ca²⁺ transient was determined by nonlinear least-squares curve fitting (Levenberg-Marquardt algorithm, Origin Software) beginning from the peak of the calcium transient. Relaxation constants from five consecutive transients were averaged.

The average KB concentration in the arterial plasma between 0 and 10 minutes was obtained by taking the integral of the concentration-versus-time curve between these time limits (Origin Software) and dividing it by the integration interval.

Reagents
KB was a gift from Nippon Organon KK. The compound was dissolved in DMSO (Sigma) to generate a 100 mmol/L stock solution. For intravenous injection, fractions of the stock solution corresponding to doses of 1, 2, 3, and 5 mg/kg body weight were diluted with sterile deionized water to obtain a concentration of 5 mg KB/mL. Propranolol (Ayerst Laboratories) and atropine (American Regent Laboratories) were prepared according to the manufacturer’s specifications. Ryanodine (purity: >99%) and thapsigargin were purchased from Calbiochem and Sigma, respectively.

Statistical Analysis
Data are presented as mean±SEM. ANOVA was used for multiple comparisons. For comparison of three groups, the Tukey honest significant difference test was used to identify where the differences among the groups occurred after the significant ANOVA. In the presence of a significant interaction term by ANOVA, the Neuman-Keuls multiple-comparison procedure was used within each group to identify changes from baseline. Differences were considered significant at P< 0.05.

The dose dependence of changes in AERP (AERP) was analyzed by a nonlinear regression analysis (Origin Software) by applying the function AERP% = AERP_max%/{1+exp[(dose-ED₅₀)/a]}, where AERP_max is the maximal change in AERP, a is a fitting parameter, and ED₅₀ is the dose at which AERP is half maximal.

	Results

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Acute Cardiac Electrophysiological Effects of KB in Dogs With Sinus Rhythm
Figure 2A summarizes the effect of increasing doses of KB on the time course of mean changes in right atrial ERP in dogs with sinus rhythm at a BDCL of 400 ms. Baseline AERPs were 169±5, 174±3, 163±8, and 171±6 ms in dogs receiving 1, 2, 3, and 5 mg/kg KB, respectively. Peak mean changes in AERP occurred 5 to 10 minutes after completion of KB infusion and ranged from 4.4±0.4% after 1 mg/kg to 14.8±2.6% after 5 mg/kg. At all doses, average AERP returned to control values within 30 minutes after intravenous administration. The vehicle alone had no significant effect on mean AERP at any time point after its intravenous infusion (baseline AERP, 170±12 ms). Both the magnitude and time course of KB (5 mg/kg)–induced mean changes in AERP were similar for all three BDCLs (Figure 2B). For example, values for peak AERP averaged 14.5±2.4%, 16.0±2.1%, and 14.8±2.6% at cycle lengths of 250, 300, and 400 ms, respectively (P>0.05). KB (5 mg/kg)–induced peak AERP changes were unaltered in the absence of both propranolol and atropine, averaging 11.5±0.5% (BDCL, 250 ms), 20.5±2.5% (BDCL, 300 ms), and 18±3% (BDCL, 400 ms; n=3; all probability values >0.3 for comparison of AERP at the same BDCLs in the presence and absence of propranolol and atropine). Mean peak AERP increased in a sigmoidal fashion with a rise in KB dose (Figure 2C). On average, a dose of 1.9 mg/kg was needed to obtain half-maximal prolongation of AERP. In contrast to its effect on right atrial refractoriness, 5 mg/kg KB failed to significantly affect right ventricular ERP (baseline ERP, 189±11 ms; P> 0.05; Figure 2D). Similarly, the same dose was found to have no significant effect on average values of spontaneous sinus cycle length (Figure 3A), sinus node recovery time (Figure 3B), AH and HV intervals (Figure 3C), durations of both P wave and QRS complex (Figure 3D), QT and QT_c intervals (Figure 3E), and mean arterial blood pressure (Figure 3F; all probability values >0.1). At all BDCLs, neither atrial nor ventricular pacing thresholds deviated significantly from their respective baseline values throughout the study protocols (data not shown). Over the 1 to 5 mg/kg dose range, no KB-related arrhythmia was observed.

View larger version (28K): [in this window] [in a new window]   Figure 2. KB prolonged atrial, but not ventricular, refractoriness in dogs with sinus rhythm. A, Time course of AERP changes (AERP) in dogs with sinus rhythm receiving KB (1, 2, 3, or 5 mg/kg IV) or vehicle. Ordinate denotes mean change in AERP in percentage from baseline AERP at a BDCL of 400 ms. * P<0.05 vs baseline in groups receiving KB. Vehicle alone at a dose equivalent to 5 mg/kg KB had no significant effect on AERP (P>0.05). SEM bars are omitted for clarity. B, Time course of mean±SEM changes in AERP at BDCLs of 250, 300, and 400 ms after 5 mg/kg KB IV. *P<0.05 vs baseline in the same group. Drive cycle length had no significant effect on time course of KB-induced AERP changes (P=0.86). C, Dose dependence of AERP changes. Peak mean±SEM change in AERP increased progressively with a rise in KB dose (P<0.001). Numbers of experiments per dose are given in parentheses. D, Comparison of time course of changes in right ventricular ERP (VERP) and AERP after intravenous administration of 5 mg/kg KB. Ordinate denotes mean±SEM change in ERP in percentage from baseline ERP at a drive cycle length of 300 ms. *P<0.001 vs baseline in the same group. KB had no significant effect on ventricular ERP (P>0.05). Time 0 in panels A, B, and D marks completion of KB or vehicle infusion, respectively.

View larger version (27K): [in this window] [in a new window]   Figure 3. KB had no significant effect on sinus node automaticity, AV conduction, ventricular de- and repolarization, and arterial blood pressure in dogs with sinus rhythm. Values are mean±SEM from 6 experiments. Time 0 marks completion of intravenous injection of 5 mg/kg KB. SCL, sinus node cycle length; SRTmax, maximal sinus node recovery time; QTc, corrected QT interval; BP, blood pressure. KB had no significant effect on any parameter shown at any time point during the 90-minute period after KB infusion (P>0.05). Data in panels B and C were obtained during atrial pacing at a cycle length of 300 ms. Shortening and lengthening the pacing cycle length to 250 and 400 ms, respectively, similarly failed to reveal significant effects of KB on sinus and AV node function (data not shown). Data in panels A, D, E, and F were obtained during sinus rhythm.

In two separate experiments, intravenous administration of KB at a dose of 7 mg/kg was associated with an irreversible decrease in mean arterial blood pressure by 30 mm Hg. We therefore did not systematically examine KB doses exceeding 5 mg/kg.

Acute, AF-Induced Changes in AERP
Rapid pacing–induced AF in the absence of KB caused reversible shortening of AERP. Baseline AERP in this group was 166±8 ms. Figure 4 (group VII) illustrates the mean change in AERP in percentage from baseline at a BDCL of 400 ms. Mean AERP ranged from -9.0±1.8% at 15 minutes to - 14.9±2.1% at 90 minutes (P< 0.001 versus pre-AF). Within 15 minutes on spontaneous resumption of sinus rhythm, mean AERP returned to a value that did not significantly differ from the pre-AF ERP (P>0.05).

View larger version (36K): [in this window] [in a new window]   Figure 4. KB prevented acute, AF-induced shortening of atrial refractoriness. Shown is a comparison of time courses and magnitudes of changes in AERP in sinus rhythm (SR) dogs receiving KB (group IV) and in dogs with rapid pacing– induced, acute AF in the presence (group VI) and absence (group VII) of KB. Ordinate denotes mean±SEM change in AERP in percentage from baseline AERP at a drive cycle length of 400 ms. *P<0.05 vs baseline in the same group; #P=0.002, group VII vs groups IV and VI. Time 0 indicates completion of KB or vehicle infusion.

Effect of KB on Acute, AF-Induced Shortening of AERP
Figure 4 compares the time course of changes in mean AERP measured at a drive cycle length of 400 ms in dogs with sinus rhythm after intravenous administration of 5 mg/kg KB (group IV) with those in dogs undergoing rapid atrial pacing in the presence (group VI) and absence (group VII) of 5 mg/kg KB, respectively. Mean AERP measured at a BDCL of 400 ms at baseline after pharmacological blockade of ß-adrenergic and muscarinic receptors did not differ significantly among the three groups (group IV, 171±6 ms, n=5; group VI, 162±8 ms, n=5; group VII, 166±8 ms, n=6; P>0.05). At all time points after injection of 5 mg/kg KB, with the sole exception of the 30-minute time point, mean AERP in group VI was not significantly different from that in group IV (P>0.05), but was significantly less than that in group VII between the 15- and 90-minute time points (P=0.002). Within 15 minutes on resumption of sinus rhythm after the final episode of rapid atrial pacing, mean ERP in group VII returned to a value that was not significantly different from pre-AF ERP. No significant change in mean AERP was observed on cessation of rapid pacing in dogs receiving KB.

Neither mean AA nor mean VV intervals were significantly different between rapidly paced dogs in the presence and absence of 5 mg/kg KB (Figure 5A and 5B). Mean arterial blood pressure was significantly higher in group VI than in group VII throughout the duration of the study, including the pre-AF value (P=0.012; Figure 5C). In rapidly paced dogs receiving KB, mean arterial blood pressure at 75 and 105 minutes after drug administration was slightly, yet significantly, lower than pre-AF blood pressure (P=0.024). In contrast, the trend for the mean arterial blood pressure in group VII to decrease over time did not reach statistical significance (P=0.089).

View larger version (35K): [in this window] [in a new window]   Figure 5. Comparison of temporal changes in average AA (A) and VV (B) intervals and mean arterial blood pressure (C) in dogs undergoing rapid atrial pacing in the presence and absence of 5 mg/kg KB. Values are mean±SEM. * P=0.024 vs baseline in the same group; #P=0.012 for group VI vs group VII. Neither mean AA intervals nor mean VV intervals differed significantly between dogs receiving KB and those not receiving KB (P> 0.05).

Plasma and Tissue Concentrations Resulting From KB Infusion In Vivo
Figure 6 shows the time course of mean KB concentration in the arterial plasma produced by intravenous infusions of 5 mg/kg KB at a rate of 1.0 mg · kg^-1 · min^{- 1}. Plasma levels peaked 5 minutes after beginning of KB administration (30.86±3.26 nmol/L) and then declined to subnanomolar concentrations (0.56±0.19 nmol/L) at the end of the observation period. Average right atrial KB concentration at 100 minutes after beginning of KB infusion exceeded the corresponding plasma level by a factor of 41. Similarly high tissue-to-serum concentration gradients were measured for the left atrium (39) and for the right (39) and left (46) ventricle. The average KB plasma concentration during the first 10 minutes after start of KB administration was 15 nmol/L. This concentration was subsequently used to determine the effects of KB on NCX-mediated Ca²⁺ flux in isolated atrial myocytes.

View larger version (13K): [in this window] [in a new window]   Figure 6. Semilogarithmic plot of KB concentration (mean±SEM, n=4 dogs) in arterial plasma over time produced by a 5-minute intravenous infusion of 5 mg/kg KB. Mean±SEM right atrial KB concentration in the same dogs at 100 minutes after start of infusion exceeds the corresponding value in the arterial plasma by a factor of 40. Horizontal bar indicates duration of intravenous KB infusion.

Effect of KB on NCX-Mediated Ca²⁺ Flux
We next evaluated the effects of KB on NCX–mediated Ca²⁺ influx and efflux in single canine right atrial myocytes as previously described.⁹ Figure 7A illustrates the experimental protocol to assess Ca²⁺ influx via NCX. Sarcoplasmic reticulum function was abolished by pretreatment with 1 µmol/L thapsigargin and 100 µmol/L ryanodine. The normal Tyrode’s solution and the Na⁺-free solution contained thapsigargin and ryanodine at concentrations of 1 and 10 µmol/L, respectively. To induce Ca²⁺ influx via NCX, cells were superfused with a Na⁺-free solution for 60 seconds, which changes the electrochemical driving force on NCX in favor of Ca²⁺ entry. Figure 7B (upper panel) demonstrates that transient removal of extracellular Na⁺ caused a reversible increase in [Ca²⁺]_i. Continuous superfusion of the cell for 10 minutes with Tyrode’s solution containing 15 nmol/L KB strongly reduced the 0Na⁺-induced rise in [Ca²⁺]_i and decreased resting R_400/500 levels (Figure 7B, middle panel). Inhibition by KB of Na⁺-free-induced increase in [Ca²⁺]_i was unchanged during a 15-minute washout (Figure 7B, lower panel). Overall, 15 nmol/L KB significantly reduced the mean 0Na⁺-induced increase in [Ca²⁺]_i by 60% (Figure 7C; P=0.001) and the mean resting R_400/500 level by 8% (Figure 7D; P<0.001).

To examine KB effects on Ca²⁺ efflux, we measured the kinetics of [Ca²⁺] _i decline during twitch-activated Ca²⁺ transients. The experimental protocol is shown in Figure 8A. Sarcoplasmic reticulum function was paralyzed as described above. Without sarcoplasmic reticulum function, [Ca²⁺]_i decline is due to forward-mode NCX activity and transport via sarcolemmal Ca²⁺-ATPase. Experiments were carried out in the continued presence of 500 nmol/L (-)Bay-K8644 to enhance Ca²⁺ ingress through L-type Ca²⁺ channels.¹⁸ Figure 8B shows representative calcium transient recordings during control, in the presence of 15 nmol/L KB, and during washout. KB did not significantly alter the time constant of relaxation. Overall, 15 nmol/L KB had no significant effect on [Ca²⁺]_i decline rate (Figure 8C) and peak rise in [Ca²⁺]_i (Figure 8D; P>0.05).

View larger version (20K): [in this window] [in a new window]   Figure 8. KB (15 nmol/L) does not reduce Ca2+ efflux in single right atrial myocytes. A, Schematic of the experimental protocol. Sarcoplasmic reticulum function was abolished by 1 µmol/L thapsigargin and 10 µmol/L ryanodine. Ca2+ influx through L-type Ca2+ channels was increased by 500 nmol/L (-)Bay-K8644. B, KB does not alter [Ca2+]i decline during twitch Ca2+ transients. Shown are representative recordings of changes in [Ca2+]i (R400/500) obtained from a single right atrial myocyte stimulated at 0.2 Hz. Each tracing was obtained by digital averaging of 5 consecutive transients for each experimental condition. Solid lines represent monoexponential fits to the R400/500 values during [Ca2+]i decline. Numbers indicate time constants of [Ca2+]i decay. C, Mean±SEM (n=5 cells) for the effect of 15 nmol/L KB on time constants of [Ca2+]i decline during twitch Ca2+ transients. There were no statistically significant differences. D, Mean±SEM (n=5 cells) for the effect of 15 nmol/L KB on peak rise in [Ca2+]i (R400/500) during field stimulation at 0.2 Hz. There were no significant effects of the drug. Ctl indicates control; WO, washout.

	Discussion

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Major Findings
The results of this study demonstrate that KB at doses 5 mg/kg selectively increased atrial refractoriness in a dose-dependent manner (ED₅₀=1.9 mg/kg). The degree of AERP prolongation was unchanged in the absence of propranolol and atropine, which indicates that it cannot be attributed to KB-induced alterations in muscarinic and/or ß -adrenergic receptor activity. We could not detect an effect of KB on sinus or atrioventricular (AV) node function, or ventricular electrophysiological or hemodynamic effects.

Rapid pacing–induced, acute AF causes reversible shortening of AERP. Both the time course and magnitude of mean changes in AERP in KB-treated dogs with rapid pacing–induced, acute AF did not significantly differ from those in dogs with normal sinus rhythm receiving an equivalent dose of KB. Importantly, average AERP in the presence of KB, but not in its absence, remained largely unchanged on conversion to sinus rhythm after the final AF episode, suggesting that the lack of AERP shortening in treated dogs is unlikely to result from an upward shift of the AERP-versus-time curve. In single canine right atrial myocytes, KB at concentrations achieved in arterial plasma in vivo strongly inhibited reverse-mode, but not forward-mode, NCX activity. These findings support the notion that Ca²⁺ influx via NCX plays an important role in acute, AF-induced shortening of atrial refractoriness.

KB Blocks NCX-Mediated Ca²⁺ Influx but Not Ca²⁺ Efflux
KB has been reported to exert a preferential effect on reverse-mode (Ca²⁺-influx mode) NCX activity,^8–10 although this effect disappeared under certain experimental conditions.¹⁹ It inhibits intracellular Na⁺-dependent Ca²⁺ influx into rat cardiomyocytes or other NCX1-expressing cells with IC₅₀s of 1.2 to 2.4 µmol/L, whereas it blocks extracellular Na⁺-dependent Ca²⁺ efflux from these cells only at higher concentrations (IC₅₀ 30 µmol/L).⁸ KB suppressed 50% of whole-cell outward NCX currents from guinea pig ventricular myocytes or NCX-transfected cells at 0.3 to 0.9 µmol/L,^9,10,20 but much higher concentrations were necessary to inhibit whole-cell inward NCX currents (IC₅₀ 17 µ mol/L).⁹ Although the mechanism underlying the directional selectivity has remained unclear,²¹ Figures 7 and 8 show selective block of NCX-mediated Ca²⁺ influx by 15 nmol/L KB under our experimental conditions.

Our results indicate that intravenous infusion of 5 mg/kg KB results in plasma drug concentrations that are sufficient to strongly inhibit reverse-mode NCX activity in isolated right atrial myocytes. The KB concentration used in our single-cell studies is by a factor of 20 to 160 smaller than previously measured mean 50% inhibitory concentrations (IC₅₀) for reverse-mode NCX activity in isolated cardiac myocytes or NCX1-expressing cells. This discrepancy may be due to differences in KB incubation times. Although the inhibitory action of KB at micromolar concentrations is relatively fast and washable, it was reported recently that the inhibitory potency appears to increase as incubation time is lengthened (>2 minutes), and more time is needed for washout.²¹ KB-induced suppression of NCX-mediated Ca²⁺ influx in our study was unchanged during a 15-minute washout period. Satoh et al⁹ similarly found incomplete recovery from KB-induced block (5 to 10 µmol/L) of reverse-mode NCX in single rat ventricular myocytes at the end of > 30-minute washout periods. Current evidence strongly suggests that KB inhibits NCX activity from the external side in intact cells,^10,20 although it may be capable of inhibiting NCX activity from the cytoplasmic side also.²² The slow recovery or complete lack thereof, respectively, from KB-induced inhibition of NCX-mediated Ca²⁺ influx may therefore reflect slow dissociation of the drug from or irreversible binding to its putative external receptor.²¹

On the basis of electrochemical considerations, NCX is most likely to be operating in the forward mode (Ca²⁺-efflux mode) in resting cardiomyocytes. Because KB selectively inhibits reverse-mode NCX activity under our experimental conditions, the decrease in resting [Ca²⁺]_i in single atrial myocytes does not appear to result from inhibition of NCX-mediated Ca²⁺ influx. Neither an increase in the activity of the sarcolemmal Ca²⁺-ATPase nor a reduction in passive Ca²⁺ influx through open sarcolemmal calcium channels can explain this phenomenon, given that 15 nmol/L KB did not alter the kinetics of [Ca²⁺] _i decline nor did it reduce average peak rise in [Ca²⁺]_i during twitch Ca²⁺ transients. The mechanism underlying KB-induced reduction of resting [Ca²⁺]_i therefore remains obscure.

Possible Mechanism of the Electrophysiological Effects of KB
Prolongation of AERP
Although inhibition of outward NCX by KB may theoretically prolong action potential duration and thus ERP, two observations argue against this explanation. First, KB did not significantly alter peak twitch [Ca²⁺]_i, indicating that under physiological conditions the role of Ca²⁺ entry via NCX is minimal in canine right atrial myocytes, but becomes important during AF with intracellular Na⁺ accumulation. Second, changes in AERP parallel those in plasma drug concentration, whereas protection against AF-induced AERP shortening persists after plasma KB levels have fallen considerably. These findings suggest that KB targets at least two different effectors with different pharmacological properties. Interaction of KB with relatively low-sensitivity, as-yet-undefined effect sites in the atria (such as, eg, K⁺ channels) could be responsible for reversible increases in AERP, whereas lasting protection against AF-induced AERP shortening would result from the relatively higher inhibitory potency to irreversibly block Ca²⁺ influx-mode NCX activity.

KB has been reported to prolong⁹ or shorten action potential duration²¹ and to inhibit Na⁺, L-type Ca²⁺, and K⁺ currents.¹⁰ Importantly, however, the range of KB concentrations used in all previous in vitro studies to examine the effects of the drug on cardiomyocyte electrophysiology exceeds the peak plasma KB concentration in our study by factors of 150 to 500.

The magnitudes of I_Ca.L and I_Na in AV nodal cells and in atrial and ventricular myocytes, respectively, are major determinants of the conduction velocity across the AV junction and in the working myocardium. Because KB at doses 5 mg/kg had no significant effect on AV conduction intervals either during sinus rhythm or during AF, or on QRS duration in our study, it seems unlikely that KB inhibits I_Ca.L or I_Na under our experimental conditions. Also, given that KB did not significantly alter peak twitch [Ca²⁺]_i, and that under our experimental conditions increases in [Ca²⁺]_i associated with the twitch are almost entirely due to inward I_Ca.L,¹⁵ it is unlikely that KB at concentrations seen in vivo exerts an inhibitory effect on I_Ca.L. Finally, reductions in I_Na or I_Ca.L would result in action potential shortening rather than lengthening.

The mechanism by which KB at doses 5 mg/kg selectively prolongs AERP is unknown. The number of experiments to assess the effects of KB on ventricular refractoriness may have been too small to detect significant changes. However, based on the results in three dogs, it seems unlikely that ventricular and atrial repolarization are equally sensitive to KB. One intriguing explanation is that KB inhibits the ultrarapidly activating delayed rectifier K⁺ current, I_Kur, which is expressed in atrial, but not ventricular, myocytes.

Prevention of AERP Shortening
Calculations based on the Luo-Rudy cell model^6,23 and experimental studies⁵ have previously demonstrated that fast rates during sustained tachycardia and fibrillation cause a larger increase in [Na⁺] _i relative to [Ca²⁺]_i. The reduction in the transmembrane Na⁺ gradient causes a shift of the reversal potential of I_Na/Ca to more hyperpolarized potentials. This shift implies that NCX operates in its reverse mode (bringing in Ca²⁺ and extruding Na⁺ with 3:1 [or 4:1]⁷ stoichiometry) for a longer duration of the action potential, shifting the balance in favor of Ca²⁺ loading. In addition, fast rates increase the fraction of time the myocytes spend at more depolarized potentials, which further augments the driving force for Ca²⁺ entry via NCX. Given that KB at concentrations achieved in vivo induces strong inhibition of reverse-mode (Ca²⁺-influx mode) but not forward-mode (Ca²⁺-efflux mode) NCX activity under our experimental conditions, our results strongly support the notion that increased Ca²⁺ influx via NCX plays an important role in the pathogenesis of acute, AF-induced shortening of atrial refractoriness. An elevation of intracellular free calcium shortens action potential duration, and thus refractoriness, by means of increasing Ca²⁺-dependent inactivation of I_Ca.L, enhancing I_to2, and augmenting I_K.

We think it unlikely that KB induces protection against the ERP shortening effects of acute AF via prolongation of ERP alone for two reasons, as follows. First, clinical studies have shown that treatment with class I, II, and III antiarrhythmic drugs lengthens AERP but fails to prevent short-duration AF-induced AERP shortening.⁴ However, we have not tried ERP-prolonging agents under our experimental conditions to exclude this possibility completely. Second, KB-induced protection against AF-induced ERP shortening is temporally dissociated from its effect on AERP in the present study. Although the average plasma KB level at 100 minutes after beginning of KB infusion was only 2% of its peak at 5 minutes, AERP shortening was not inducible in KB-treated dogs throughout the complete duration of the study. This is consistent with the previous observation that a single 10-minute perfusion period of the Langendorff-perfused whole rabbit heart with Tyrode’s solution containing 3 µmol/L KB followed by a 30-minute period of global ischemia was capable of suppressing ventricular tachyarrhythmias during 45 minutes of reperfusion despite absence of the drug in the reperfusate.²² This is also in accordance with our finding that 10-minute incubation with 15 nmol/L KB is sufficient to exert a lasting and strong inhibition of reverse-mode NCX activity in isolated atrial myocytes. These observations along with the high tissue-to-plasma KB concentration ratio suggest that slow elimination of the drug from the site of action, possibly due to a low dissociation rate from its external receptors, is responsible for the nonparallelism of the plasma pharmacokinetic profile and observed effects on AF-induced AERP shortening.

At no time point during rapid pacing–induced AF was there a significant difference in mean AA intervals in the absence and presence of KB in our study, suggesting that KB does not exert its preventive effect via a reduction in the mean fibrillatory cycle length.

The effects of KB effects could be attributable to inhibition of I_Ca.L by the drug.¹⁰ However, for reasons outlined above, KB at concentrations seen in vivo is unlikely to exert protection via inhibition of I_Ca.L.

Pre-AF mean arterial blood pressure was greater in rapidly paced dogs treated with KB compared with nontreated dogs. Whether differences in blood pressure can account for the different outcome is questionable, because we found no significant relationship between mean blood pressure level and AERP either in the absence (r=0.47, P=0.34) or presence (r=0.41, P=0.42) of KB. Similarly, the slight, albeit statistically significant, decrease in mean arterial blood pressure in KB-treated dogs during AF is unlikely to explain the protective effect of the drug.

Study Limitations
First, ERP measurement was restricted to one atrial site. Thus, the effects of pacing-induced AF and KB on refractoriness in other areas of the atrium or on heterogeneity of refractoriness were not determined. A second limitation is that the findings may be specific to pacing-induced AF in structurally normal atria and may not apply to episodes of AF that occur in structurally abnormal atria. Finally, although KB inhibits outward I_Na/Ca much more potently than other ion channels and transporters,¹⁰ a combined effect of the drug on outward I_Na/Ca and other ion transport mechanisms cannot be excluded.

	Conclusions

Top Abstract Introduction Methods Results Discussion Conclusions References

 
The data from this study strongly support the notion that reverse-mode NCX activity plays an important role in AF-induced AERP shortening. Its inhibition therefore represents a promising target for future pharmacological therapy.

	Acknowledgments

 
This work was supported by the Herman C. Krannert Fund and by the American Heart Association, Midwest Affiliate. We are grateful to the R&D Laboratories of Nippon Organon KK, Osaka, Japan, for generously providing us with KB. We also thank C.J. Arnett, Trevor Tucker, and Emily E. Kerchner for technical assistance and Naomi S. Fineberg for statistical analysis.

Received April 10, 2002; revision received June 5, 2002; accepted June 11, 2002.

	References

Top Abstract Introduction Methods Results Discussion Conclusions References

 
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