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Am J Physiol Heart Circ Physiol 286: H2393-H2400, 2004; doi:10.1152/ajpheart.01242.2003
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Transmembrane action potential heterogeneity in the canine isolated arterially perfused right atrium: effect of IKr and IKur/Ito block

Alexander Burashnikov, Sandeep Mannava, and Charles Antzelevitch

Masonic Medical Research Laboratory, Utica, New York 13501

Submitted 30 December 2003 ; accepted in final form 4 February 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The role of electrical heterogeneity in development of cardiac arrhythmias is well recognized. The extent to which transmembrane action potential (TAP) heterogeneity contributes to the normal electrophysiology of well-oxygenated atria is not well defined. The principal objective of the present study was to define regional and transmural differences in characteristics of the TAP in isolated superfused and arterially perfused canine right atrial (RA) preparations under baseline, rapidly activating delayed rectifier K+ current (IKr) block, and combined block of ultrarapid delayed rectifier and transient outward K+ current (IKur/Ito block). Superfused preparations that survived generally displayed a triangle-shaped TAP. Exceptions included cells from the crista terminalis, where TAPs with a normal plateau could be recorded. In contrast, most TAPs recorded from throughout the perfused RA displayed a spike-and-dome and/or plateau morphology. The perfused RA displayed a heterogeneous distribution of repolarization, Vmax, and spike-and-dome morphology along the epicardial and endocardial surfaces as well as transmurally, in the region of the upper crista terminalis. IKr block with E-4031 prolonged repolarization homogeneously in the perfused RA, whereas IKur/Ito block using low concentrations of 4-aminopyridine abbreviated action potential duration at 90% repolarization heterogeneously, leading to a reduction in dispersion of repolarization. Our data indicate that the electrical heterogeneities, previously described for the canine ventricle, also exist within the atria and that IKr block does not accentuate and IKur/Ito block reduces RA dispersion of repolarization. Our study also points to major differences in the transmembrane activity recorded using superfused vs. arterially perfused atrial preparations.

atrial electrophysiology; atrial pharmacology; arrhythmias

THE ROLE OF ELECTRICAL HETEROGENEITY in development of cardiac arrhythmias is well recognized (4, 18, 36). Although regional electrophysiological heterogeneities are relatively well defined in the ventricles of the heart (4, 5), the extent to which similar heterogeneities exist in the atria is not well studied.

Although investigations involving superfused atrial preparations have demonstrated diverse action potential morphologies, i.e., plateau (Purkinje-like) and nonplateau (triangular) action potentials (14, 25), there are no comprehensive studies of the three-dimensional distribution of action potential morphologies in well-oxygenated (arterially perfused or in situ) right atria (RA).

Reduction of rapidly activating delayed rectifier K+ current (IKr) has been shown to be associated with an increase of action potential duration (APD) dispersion and ventricular tachyarrhythmias in the clinic and in experimental models (4). Little is known about the effect of IKr reduction on dispersion of repolarization in the atrium. Ventricular proarrhythmia is a major limitation of the use of IKr blockers for the treatment of atrial fibrillation (18, 28). Block of the ultrarapid delayed rectifier K+ current and combined block of IKur and transient outward K+ current (IKur/Ito block) have been suggested to be promising targets for the treatment of atrial fibrillation (20, 28, 35). The effect of IKur/Ito block on atrial electrical heterogeneity has not been reported.

The principal aim of the present study was to examine the three-dimensional distribution of action potential characteristics in isolated arterially perfused canine RA preparations under baseline conditions and after IKr and IKur/Ito block to provide a foundation for our understanding of arrhythmogenic mechanisms that develop in the atria of the heart.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study followed the guidelines of the American Heart Association and was approved by the Masonic Medical Research Laboratory Animal Care and Use Committee.

Mongrel dogs weighing 20–25 kg were anticoagulated with heparin and anesthetized with pentobarbital sodium (30–35 mg/kg iv). The chest was opened via a left thoracotomy, and the heart was excised, placed in a cardioplegic solution consisting of cold (4°C) Tyrode solution containing 8.5 mM extracellular K+, and transported to a dissection tray.

Superfused isolated RA slice preparations. Tissue slice preparations (~1 x 1 cm strips) were isolated from the RA. Pectinate muscle (PM), superior vena cava (SVC), inferior vena cava (IVC), and Bachmann's bundle (BB) slice preparations were pinned epicardial or endocardial surface up. Separated epicardial and endocardial slice preparations (~1 x 0.5 x 0.2 cm strips) were isolated from the crista terminalis (CT). Midmyocardial tissue slices were isolated from the thickest regions of the RA (septal intercaval band and upper CT). The preparations were isolated using a dermatome (Davol Simon Dermatome, Cranston, RI) and then placed in a tissue bath (5 ml volume, 12 ml/min flow rate) and superfused with oxygenated Tyrode solution (37 ± 0.5°C, pH 7.35). The preparations were stimulated at a basic cycle length (BCL) of 700 ms using field or point stimulation (rectangular stimuli of 1- to 3-ms duration, 2–3 times diastolic threshold intensity). The composition of the Tyrode solution was (in mM) 129 NaCl, 4 KCl, 0.9 NaH2PO4, 20 NaHCO3, 1.8 CaCl2, 0.5 MgSO4, and 5.5 D-glucose.

Arterially perfused canine RA. The atria were isolated with a portion of the ventricles attached. The ostium of the right coronary artery was cannulated with polyethylene tubing (1.75 mm ID, 2.1 mm OD), and the preparation was perfused with cold Tyrode solution 120 (8–12°C) containing 8.5 mM extracellular K+. The ventricular branches of the right coronary artery were clamped with metal clips. The entire RA was carefully dissected from the remaining tissues, and then the preparation was unfolded. The ventricular right coronary branches and the atrial branches were ligated using silk ligatures. The preparation was placed in a temperature-controlled bath (8 x 6 x 3 cm) and perfused with Tyrode solution at a rate of 7–8 ml/min and simultaneously superfused with Tyrode solution at a rate of 8–10 ml/min (37 ± 0.5°C) using roller pumps. An air trap was used to avoid bubbles in the perfusion line. The RA preparation consisted of the atrioventricular ring (AVR), sinoatrial node (SAN), PM, CT, BB, SVC, IVC, appendage (APG), septum, fossa ovals, and a rim of right ventricular tissue containing the right coronary artery (Fig. 1). At the end of each experiment, Evans blue dye was perfused to determine nonperfused areas. Five RA were excluded from the study because of the presence of nonperfused regions.



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  		Fig. 1. Regional differences in action potential morphology in arterially perfused canine right atrium (RA) recorded during sinus rhythm (cycle length = 630–800 ms). A: endocardial surface. B: epicardium in regions of Bachmann's bundle (BB) and anterior appendage (APG). C: epicardial surface. LA, left atrium; CT, crista terminalis; PM, pectinate muscle; AVR, atrioventricular ring; IVC, inferior vena cava; SVC, superior vena cava; SAN, sinoatrial node.

 
Transmembrane action potentials (TAPs) were obtained using standard (superfused preparations) or floating (perfused preparations) glass microelectrodes (2.7 M KCl, 10- to 25-M{Omega} direct-current resistance) connected to a high-input impedance amplification system (World Precision Instruments). The signals were displayed on oscilloscopes, amplified, digitized, and analyzed (Cambridge Electronic Design, Cambridge, UK) and stored on computer hard drive or compact disk. Electronic differentiation of the action potential to obtain Vmax was recorded using operational amplifiers or digitally using a sampling rate of 35 kHz. TAPs were obtained from all regions of the perfused RA, except the epicardial AVR (because of epicardial fat) and endocardial APG (because of inaccessibility). Because the SAN of the mature canine heart is an intramural structure, the SAN cells were also not recorded in the present study. TAPs from the SAN could be obtained from the epicardial (but not endocardial) surface of the young dog (15 ± 5 wk old) (29). Whenever possible, we recorded TAPs from endocardial and epicardial surfaces by turning the preparations over without disconnecting the perfusion line. To record midmyocardial TAPs in the perfused RA, we made an oblique transmural cut of the atrial upper CT with a razor blade or scalpel. After >=20 min were allowed for the preparation to heal, TAPs were recorded. The techniques used to record TAPs from the transmural (cut) regions of RA were similar to those routinely used in our laboratory to record TAPs from the transmural (cut) surface of arterially perfused ventricular wedges (9, 32). Because the perfused RA contracted vigorously, a sustained impalement was difficult to maintain. TAPs were often distorted by mechanical displacement of the microelectrodes. TAPs were considered acceptable when at least three consecutive TAPs were identical and phase 0 amplitude was >=90 mV.

Experimental protocols. Superfused tissue slices were allowed to equilibrate for >=2 h before collection of data. Coronary-perfused spontaneously beating RA were equilibrated in the tissue bath until electrically stable, usually 30 min. Five sets of experiments were performed. In the first set of experiments, TAPs were recorded from superfused atrial preparations. In the second set of experiments, we evaluated the electrical (SAN rate and TAPs) and mechanical (contractility) stability of the arterially perfused RA preparation over a period of 6 h. Isometric contractile force was recorded by attaching the intercaval band to a force-displacement transducer (Grass Instruments), as previously described (10). In the third set of experiments, all recordings were obtained at the SAN rhythm from 17 regions of coronary perfused RA (cycle length = 630–800 ms). In the fourth set of experiments, the effect of a specific IKr blocker (E-4031, 1.0 µM) on RA APD heterogeneity was studied in perfused RA. This concentration significantly prolongs APD, induces early afterdepolarization (EAD), and aggravates APD dispersion in canine ventricular isolated tissue slices and perfused wedge preparations (8, 9). Because E-4031-induced APD prolongation is reverse rate dependent, we slowed the spontaneous SAN rhythm by infusion of 1.0 ml of 10% formaldehyde into the SAN region and/or by mechanical crashing of the SAN region. In all these preparations (n = 8), a spontaneous rhythm (regular and/or irregular) persisted. Steady-state pacing at a BCL of <=1,000 ms uninterrupted by spontaneous extrasystole(s) and/or a faster spontaneous rhythm was possible in six of eight RA preparations, which were used for this section of the study. Finally, in the fifth experimental series, the effect of a low concentration of 4-aminopyridine (4-AP, 50 µM) as well as, in separate experiments, the dose-dependent effects of 4-AP (10, 50, and 500 µM) were studied in perfused RA at a pacing BCL of 700 ms. 4-AP at 50 µM is known to completely block canine atrial IKur (IC50 = 5.3 µM) and partially reduce Ito (IC50 = 471 µM) (20). In the fourth and fifth experimental series, seven RA regions were studied, with the exception of the experiments testing dose dependency of 4-AP, where only endocardial CT was used. The TAP recordings were obtained before and 15 min after addition of E-4031 or 4-AP to the coronary perfusate. Vulnerability to atrial arrhythmias was assessed by programmed electrical stimulation applied at a region displaying a short APD region under baseline conditions and in the presence of E-4031 or 4-AP. A pseudo-ECG recording was used to characterize arrhythmic activity, as previously described (10).

Drugs. E-4031 (Eisai) and 4-AP (Sigma-Aldrich) were prepared fresh for each experiment at a stock concentration of 1 and 50 mM, respectively.

Statistics. Statistical analysis was performed using a t-test for unpaired data or one-way analysis of variance for unpaired data followed by Bonferroni's test (SigmaStat), as appropriate. Values are means ± SD. P < 0.05 was considered significant. No more than two recordings from the same region of a given atrium were included in the statistical evaluation of regional differences. APD dispersion was determined as the difference between the longest and the shortest APD between RA regions first in each experiment after values from all preparations were averaged.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Superfused vs. arterially perfused atrial preparations. In the initial experimental protocol, we attempted to record TAPs from thin tissue slices isolated from seven RAs. In contrast to thin ventricular tissue slices (including midmyocardial), which survive fairly well under superfused conditions (8, 24), 74% (52 of 70) of superfused atrial tissue slices did not survive. The TAPs of preparations that survived displayed a triangular morphology in all but the endocardial CT region, where plateau-shaped TAPs, along with triangular TAPs, could be recorded (Fig. 2).



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  		Fig. 2. Conversion of plateau-shaped action potentials into triangle-shaped action potentials by switching from perfusion to superfusion mode in endocardial PM and BB, but not CT, regions of RA preparation. Action potentials were recorded before and 20 min after termination of perfusion (with continuous superfusion).

 
In a second experimental series, we recorded TAPs from different sites of isolated arterially perfused RA preparations. In contrast to superfused preparations, the majority of TAPs obtained from throughout the perfused RA displayed a spike-and-dome and/or plateau morphology with no triangulation (Figs. 1 and 2). We hypothesized that triangulation of the TAP in the superfused preparations was due to ischemia. To test this hypothesis, we recorded TAPs before and after stopping coronary perfusion in preparations continuously superfused with oxygenated Tyrode solution (n = 3). Because ischemia slowed sinus rate or caused atrial standstill, the preparations were paced at a BCL of 700 ms. Interruption of coronary flow rapidly (10–20 min) led to triangulation of most plateau-shaped TAPs, with the exception of some endocardial CT TAPs, many of which retained a plateau morphology (Fig. 2). The results suggest that the triangular TAPs recorded from isolated superfused atrial preparations are not normal and point to ischemia as a cause of or major contributor to TAP depression under superfused conditions.

Characteristics of action potentials in arterially perfused RA. Arterially perfused RA preparations were electrically and mechanically stable over a period of 6 h of experiments. At 30 and 330 min after the start of coronary perfusion, SAN spontaneous cycle length, APD at 90% repolarization (APD90), and phasic tension were 704 ± 23 and 715 ± 31 ms, 234 ± 21 and 227 ± 18 ms, and 5.2 ± 2.1 and 4.7 ± 1.8 g, respectively [P = not significant (NS) for all, n = 4–8 in 4 RA]. Our findings are consistent with those of a previous study (22).

The electrophysiological characteristics of TAPs recorded from the endocardial and epicardial surfaces as well as transmural sites (in the upper CT) of the perfused RA are summarized in Table 1. The longest APDs were observed in the endocardial CT and the shortest in the AVR, with all other regions displaying intermediate values. Distribution of APD along the epicardial surface was relatively homogeneous, with the exception of BB, where APD was longest. APD90 differences in RA averaged 37 ± 11 ms between the longest (endocardial upper CT) and shortest (endocardial AVR) TAPs. Dispersion of APD at the epicardial surface was 24 ± 8 ms (between BB and PM). Transmural dispersion of APD averaged 20 ± 7 ms in the upper CT and was much smaller (10 ± 5 ms in low CT) or practically nil in the rest of the RA (Table 1, Fig. 3). TAPs recorded from the midmyocardial region of the upper CT displayed slightly shorter APDs (P = NS) than those recorded at the endocardial surface of the CT but were longer than those recorded in epicardial cells (P = NS; Table 1, Fig. 3). Some cells recorded from the endocardial region of the upper CT displayed a distinct diastolic depolarization and long APD.


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  		Table 1. Regional action potential characteristics of canine perfused right atria

 


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  		Fig. 3. Transmural distribution of action potentials in canine arterially perfused RA. Action potentials recorded from endocardial (Endo) and epicardial (Epi) surfaces of CT, BB, and PM regions are superimposed. Transmural action potential duration (APD) and spike-and-dome heterogeneities are present in the CT region and absent or insignificant in all other regions of RA. M, midmyocardium.

 
A spike-and-dome morphology of the TAP was identified by a second action potential upstroke (i.e., phase 2 magnitude or notch) with a magnitude of >=1 mV. In a majority of RA (17 of 24), the following pattern of a spike-and-dome morphology distribution was observed. Spike-and-dome was consistently recorded in endocardial CT, BB, SVC, IVC, and septum, as well as epicardial BB, SVC, IVC, and anterior APG. The action potential notch was less consistently observed in epicardial CT, PM, and posterior APG, as well as endocardial AVR. In 3 of 24 preparations, a prominent notch was recorded throughout the RA. In 4 of 24 preparations, no spike-and-dome morphology was observed in any of the RA regions. Transmural spike-and-dome heterogeneities were present in the CT region and were absent or insignificant in all other regions of the RA (Table 1, Fig. 3).

Phase 1 magnitude was remarkably large in some endocardial CT and BB sites, as well as in epicardial BB and anterior APG, reaching up to 40% of the amplitude of phase 0 (Table 1). The endocardial AVR displayed the smallest phase 1 magnitude.

Average Vmax values varied by as much as 122 V/s at different RA sites (Table 1). Endocardial CT, as well as epicardial and endocardial BB and SVC and IVC, showed a similarly large Vmax (Table 1). Vmax values were smaller in epicardial posterior and anterior APG, epicardial and endocardial PM, endocardial AVR, and epicardial CT.

Effect of IKr and IKur/Ito block. IKr reduction (E-4031, 1.0 µM) homogeneously prolonged APD in all RA regions tested, without accentuation of APD dispersions (Fig. 4).



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  		Fig. 4. Effect of rapidly activating delayed rectifier K+ current (IKr) reduction on regional APD and dispersion of repolarization in perfused canine RA. A: typical superimposed transmembrane action potentials (TAPs) recorded from endocardial CT and PM before and after addition of 1.0 µM E-4031 to perfusion solution at a pacing cycle length of 1,000 ms. B and C: effect of IKr block on APD of 7 RA regions (n = 8–12) and APD dispersion (n = 6 each). *P < 0.05. Reduction of IKr produces a homogeneous APD prolongation, without aggravation of APD dispersion. APD90, APD at 90% repolarization.

 
IKur/Ito block (4-AP, 50 µM) significantly reduced phase 1 magnitude and increased the height of phase 1 and 2 amplitude in all RA regions (Fig. 5). In endocardial upper CT, phase 1 magnitude was decreased from 30 ± 6 to 5 ± 3 mV (n = 10, P < 0.001). Although the very early part of repolarization was prolonged, the late phase of repolarization was abbreviated by 4-AP. 4-AP produced a relatively small abbreviation of APD90 in regions initially displaying relatively short APD but a greater APD90 abbreviation in regions displaying a relatively long APD, leading to a reduction in dispersion of repolarization in RA (Fig. 5). No spontaneous or stimulation-induced arrhythmia was recorded before or after E-4031 or 4-AP. EADs were never observed.



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  		Fig. 5. Effect of ultrarapid delayed rectifier K+ current/transient outward K+ current (IKur/Ito) block on RA regional APD and dispersion of repolarization. A: superimposed TAPs obtained from endocardial CT and PM under baseline conditions and in the presence of 50 µM 4-aminopyridine (4-AP). B and C: average data for 4-AP-induced APD90 abbreviation and reduction of APD dispersion. *P < 0.05. Block of IKur/Ito results in inhomogeneous APD shortening, leading to reduction of APD dispersion in perfused canine RA.

 
To assess the contribution of IKur vs. Ito in the development of the phase 1 magnitude, we took advantage of the different reactivation kinetics of the two currents in canine atria (13, 35). Rapidly reactivating IKur, but not relatively slowly reactivating Ito, should contribute importantly to the early phases of a closely coupled premature atrial TAP. Under control conditions, phase 1 of a closely coupled premature beat (S1–S2 = 200–250 ms, S1–S1 = 700 ms) was drastically reduced or eliminated in all cases tested (n = 20) in four RA (Fig. 6). These results point to a much more prominent contribution of Ito than of IKur in formation of phase 1 of the atrial TAP. A similar drastic reduction in the magnitude of phase 1 by 50 µM 4-AP (Fig. 5) suggests that this concentration of 4-AP significantly reduces Ito. The dose-dependent effects of 4-AP on the early phases of the TAP (Fig. 6, B and C) show that an increase of 4-AP concentration from 50 to 500 µM produces only minor additional changes in the TAP notch, indicating again that much of Ito is blocked at 50 µM 4-AP.



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  		Fig. 6. Effects of a premature impulse and concentration-dependent effect of 4-AP on atrial action potential notch. A: a regular TAP (S1–S1 = 700 ms) and the following premature TAP (S1–S2 = 250 ms) recorded in the endocardial upper CT under baseline conditions. B: superimposed TAPs recorded before (control) and after addition of increasing concentrations of 4-AP. C: effect of 4-AP on phase 1 magnitude. All TAPs were obtained from the endocardial surface of upper CT. *P < 0.05 vs. control; n = 6 from 3 preparations.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The principal findings of this study are as follows. 1) Electrophysiological data obtained from superfused atrial tissues, on which a substantial part of our knowledge of atrial function is based, may be unreliable. 2) Important electrical heterogeneities exist in the atria, as in the ventricles, characterized by intraepicardial and intraendocardial, as well as transmural, dispersion of repolarization. 3) In contrast to the response of ventricular myocardium, IKr reduction homogeneously prolongs APD of the RA, without accentuating dispersion of repolarization. 4) IKur/Ito inhibition inhomogeneously abbreviates APD throughout the RA, resulting in reduction of APD dispersion in atria. These findings may aid our understanding of the pathogenesis of reentrant atrial arrhythmias and its pharmacological modulation.

Superfused vs. perfused preparations. Triangular TAPs displaying a depressed plateau, commonly recorded from canine superfused atrial preparations (6, 14, 25), have long been considered to be representative of the typical atrial response. Exceptions to this rule are the longer-duration plateau (Purkinje-like) TAPs recorded from the endocardial surface of the CT of canine isolated superfused RA (14). Vmax is much larger in these Purkinje-like cells than in the cell with a triangle-shaped TAP. Our study compares TAPs recorded from superfused with coronary-perfused canine RA preparations. Whereas a triangular morphology of the TAP, characterized by a depressed plateau, is usually observed in superfused preparations (with the exception of some cells from the endocardial CT), perfused canine atria generally display TAPs with a more prominent plateau phase. A larger Vmax of CT cells in the superfused atrial preparation (14) may be due to their ability to better survive the conditions of superfusion. Ventricular Purkinje fibers are also more resistant to ischemia than the surrounding ventricular myocardium. Also the dramatic difference in cellular electrophysiology between perfused and superfused canine atrial preparations is not observed in the canine ventricle, where TAPs recorded from the isolated ventricular superfused slices are very similar to those recorded from arterially perfused wedge preparations (9, 24, 34). The basis for the difference in TAP morphology and survivability of perfused vs. superfused atrial preparations is not clear but may be related to their ability to resist dissection and ischemia-related injury.

Action potential heterogeneity. Although heterogeneities of ventricular electrophysiology are relatively well studied (for review see Refs. 4 and 5), atrial electrical dispersion are less well defined. To our knowledge, no detailed maps of TAP distribution along the epicardial and endocardial surfaces, or transmurally, in well-oxygenated (arterially perfused or in situ) RA are available. In superfused canine RA preparations, the longest APDs are observed in the endocardial caval border of CT, decreasing as a function of distance from the caval border of the CT region (25). APDs in the CT region are reported to be longer than those in the PM region of rabbit RA (31). Regional differences of TAP heterogeneity have been described at the level of single myocytes isolated from the canine RA (13). The longest APDs were observed in myocytes isolated from the CT area. The shortest APDs were observed in AVR regions, and intermediate APDs were recorded from cells isolated from the APG and PM regions of the RA. Our data are largely in agreement with this pattern of regional APD distribution. A prominent distinction is that dispersion of repolarization in perfused atria is relatively small (averaging 37 ms) compared with isolated myocytes and superfused canine RA, in which regional APD differences of 80–120 ms have been reported (13, 25).

The ionic mechanisms underlying the regional difference in APD in canine RA include the presence of the largest L-type Ca2+ current in CT cells, followed by APG, and PM (13). The smallest L-type Ca2+ current is recorded in AVR cells. In contrast, IKr is largest in AVR cells, with smaller values observed in PM, APG, and CT. Ito is smallest in APG. The other RA regions have similar values of Ito. Inwardly rectifying K+ current (IK1), slow delayed rectifier K+ current (IKs), and IKur are evenly distributed in the canine RA (13).

Anyukhovsky and Rosenshtraukh (6) recently reported that endocardial APD is longer than epicardial APD in superfused tissues isolated from the free wall of canine RA. In perfused RA preparations, we found important epicardial-endocardial APD gradients only in the upper CT region. Transmural dispersion of repolarization in the CT region of the RA may have an implication for the generation of atrial arrhythmias. Indeed, amplification of transmural heterogeneities has been shown to underlie the development of ventricular arrhythmia under a variety of conditions, including long-QT and Brugada syndromes, heart failure, ischemia, and reperfusion (1, 2, 11, 23, 30).

Avanzino et al. (7) reported that deep layers of rabbit RA CT display longer APDs and higher TAP with respect to time than TAPs recorded from the surfaces of the atria. These characteristics are similar to those of canine ventricular M cells (24). In our study, isolated canine atrial midmyocardial slices did not survive well, so we probed for the presence of the atrial M cell in the upper CT region of the perfused RA. Distinct M cell characteristics (24) were not detected under these conditions. Vmax and APD of the mid-CT cells were similar to those recorded from endocardial CT. Our data were obtained at a resting SAN cycle length of 630–800 ms. Canine ventricular M cells are not always clearly evident at these rates (24, 34), revealing themselves only at slower rates (cycle length >=1,000 ms) (8, 24, 34). Also the canine left ventricle is much thicker (~1.5–2.5 cm) than the atrial CT (0.4–1.0 cm); therefore, electrotonic forces, known to significantly affect the transmural repolarization gradient (9), are likely to reduce the atrial transmural APD difference.

Regional and transmural distribution of the spike-and-dome morphology of the action potential, well described in canine ventricles (16, 32), has not been delineated in the canine RA. A spike-and-dome configuration of the action potential has been reported in the upper CT and along the ventricular border of the CT region of the superfused canine RA (14, 25). Some of the myocytes isolated from CT and APG regions of canine RA also have a spike-and-dome configuration (13). Tissue slices isolated from adult human APG consistently show a spike-and-dome action potential waveform (12). In our study, an impressive notch was recorded primarily in endocardial BB and CT and epicardial BB and anterior APG, with a substantial transmural difference in the notch magnitude in the upper CT region.

The ventricular distribution of the spike-and-dome morphology is known to contribute to the inscription of the ECG J wave as well as to arrhythmogenicity (32). Heterogeneous loss of action potential dome in the epicardial surface is capable of inducing phase 2 reentry-mediated extrasystoles, which can initiate ventricular arrhythmias (11, 33). This mechanism has been implicated in the arrhythmogenicity attending the Brugada syndrome (3, 11) as well as ischemia-related arrhythmias (17). The presence of a large notch (phase 2 magnitude up to 12 mV) in the perfused canine RA, suggests that a similar mechanism may contribute to arrhythmogenesis in the atrium. This hypothesis remains to be tested.

IKr and IKur/Ito block in atria. Dispersion of repolarization is thought to be a major determinant for the development of atrial and ventricular arrhythmias (4, 18, 36). Although reduction of IKr is well known to accentuate dispersion of repolarization in ventricular myocardium (4, 23), our results indicate that such is not the case in canine perfused RA. This observation may help us understand why IKr blockers are largely antiarrhythmic in the atria but often proarrhythmic in the ventricle (28). dl-Sotalol (an IKr blocker) also does not accentuate dispersion of repolarization in canine atria in vivo (19). Although no atrial arrhythmias or EAD were observed under IKr blockade in our study, the K+ channel blocker Cs+ is capable of inducing torsades de pointes-like arrhythmias in canine atria in vivo (21). In contrast to the specific IKr blocker E-4031 used in our study, Cs+ is known to block a number of K+ currents [including IK, IK1, and G protein-gated K+ current (IKACh)]. A recent study shows that patients with inherited long-QT syndrome (including LQT2) may develop "atrial torsades de pointes" (15). However, such atrial arrhythmia may not be very prevalent (26).

Reduction of IKur and IKur/Ito has been suggested to be a useful target in pharmacological treatment of atrial arrhythmias (20, 28, 35). The effect of IKur/Ito reduction on atrial APD heterogeneity was previously unknown. Our data indicate that, in the perfused atrium, IKur/Ito block abbreviates APD and reduces APD dispersion. The former should promote and the latter should suppress reentrant arrhythmias.

It is generally accepted that low concentrations of 4-AP (50 µM) specifically block IKur, with only minor reduction in Ito (~3%) (20). Our results clearly indicate that 50 µM 4-AP drastically reduces or practically eliminates action potential notch (Fig. 5), which is largely due to Ito (Fig. 6). The results suggest that this concentration of 4-AP potently blocks Ito in the perfused RA. The IC50 for 4-AP to block of Ito in the canine atrium is one-third of that reported for the canine ventricles (20).

A prominent example of the pharmacological distinctions encountered in perfused vs. superfused atrial preparations is in response to 50 µM 4-AP. In superfused preparations, 50 µM 4-AP prolongs repolarization of triangle-shaped TAPs (35). In our study, 50 µM 4-AP abbreviated the APD of the plateau-shaped TAPs in the perfused preparations. The APD abbreviation can be explained by a greater recruitment of IKr and IKs due to the 4-AP-induced elevation of the TAP plateau.

Study limitations. The extent to which these observations pertain to other animal species, including humans, is not known. There are interspecies variations in the shape of the atrial action potential and/or their ability to survive under superfused conditions (27). Also triangular TAPs are likely to be normal in the electrically remodeled atria (18). Thus the data obtained in the present study may be applicable for "healthy," but not remodeled, atrium. Our results were obtained in Tyrode solution-perfused isolated atrial preparations that lacked, e.g., autonomic inputs and hormones. These and other blood-related factors are present in vivo and may affect atrial electrophysiology as well as the pharmacological response. Therefore, the electrophysiological characteristics of our preparations may differ in some respects from those encountered in vivo.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grant HL-47678 (C. Antzelevitch) and grants from the American Heart Association (A. Burashnikov and C. Antzelevitch), the Eighth Manhattan Masonic District, and the Masons of New York State and Florida.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the expert technical assistance of Judy Hefferon, Di Hou, and Robert Goodrow.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Burashnikov, Masonic Medical Research Laboratory, 2150 Bleecker St., Utica, NY 13501 (E-mail: sasha{at}mmrl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 DISCUSSION
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 REFERENCES
 

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