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1Research Institute of Environmental Medicine, Nagoya University, Nagoya; 2Department of Cardiology, Tokai University School of Medicine, Isehara; and 3Graduate School of Technology, The University of Tokyo, Tokyo, Japan
Submitted 15 June 2006 ; accepted in final form 18 August 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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potassium channel blocker; optical mapping; ventricular tachycardia
In a recent optical mapping study to examine the combined effects of NF and lidocaine in a two-dimensional (2D) subepicardial layer of rabbit ventricular myocardium, we have demonstrated that NF (0.5 µM) alone prolongs VT cycle length and causes its early termination in association with destabilization of the spiral wave dynamics (prolongation of functional block line, frequent local conduction block, and extensive meandering) (1). Preliminary experiments with NF at a lower concentration (0.1 µM) also showed analogous modification of spiral wave reentry (10). On the basis of these observations, we speculated that the NF-induced destabilization of spiral wave reentry may be the result of repolarization delay leading to more frequent wave front-tail interactions, but the issue remains to be substantiated. The mechanism of early VT termination was unclear. The present study was designed to clarity the points in similar 2D rabbit ventricular myocardial tissue preparations. Newly developed software to visualize the wave front and tail of optical action potential signals was employed to analyze their interaction. A phase-mapping method was used to quantify the destabilization and to elucidate the mode of termination of rotors. The results revealed that the wave front during VTs in the presence of NF does frequently encounter its own tail, giving rise to transient breakup of spiral wave or sudden movement of the rotation center, and that early VT termination with NF is the result of either wave front collision with the atrioventricular groove or trapping of the spiral tip in a refractory zone.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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High-resolution optical mapping. The optical mapping system used in this study was described previously (1). After endocardial freezing, the hearts were stained with a voltage-sensitive dye, di-4-ANEPPS. To minimize motion artifacts, we added 15 mM 2,3-butanedione monoxime (BDM) unless otherwise specified. Bipolar electrograms were recorded through widely spaced electrodes, one at the apex of the left ventricle and the other at the lateral wall of the right ventricle, to monitor the whole ventricular excitations. The signals were filtered from 15 to 1,000 Hz and digitized at 1,000 Hz.
The hearts were illuminated with bluish light-emitting diodes; the emitted fluorescence was recorded with a solid-state image-sensing digital video camera (Fastcam-Ultima 40K; Photron) to acquire 8-bit gray scale images from 256 x 256 sites simultaneously at a speed of 750 frames/s. The images covered the anterolateral surface of the left ventricle and part of the anterior surface of the right ventricle. Each acquisition lasted 10.9 s.
To reveal the signal, we subtracted the background fluorescence from each frame and applied low-pass spatial filtering. The fluorescence signals were inverted and then spatially averaged to reduce noise. Spatial resolution after the low-pass filtering was 0.36–0.48 mm. Isochrone maps were generated from the filtered image. Action potential configuration was analyzed after the application of a five-point median filter to the spatially averaged data (13), and then the data were normalized to the range of the maximum and the minimum values in the respective 1,000-frame sample. A time point at 10% depolarization in the upstroke phase and a time point at 90% repolarization in its repolarization phase were identified for each action potential signal, and their interval was measured as APD at 90% repolarization (APD90). The distribution of APD90 values in the recording area was displayed as a color gradient map with 1.33-ms steps, ranging from red (shortest) to blue (longest).
Wave front-tail dynamics during VT were visualized by connecting the 10% depolarization points in the action potential upstrokes as the wave front (red) in the recording area and by connecting the 90% repolarization points as the wave tail (green). We quantified the pattern of wave propagation during VT using the phase-mapping method described by Gray et al. (5). The fluorescence of each pixel at time t, F(t), was plotted against the fluorescence of the same pixel offset by a time interval 
= 25 frames. A cyclic return map of F(t) vs. F(t – ) was constructed, allowing a new parameter, phase 
(t), to be defined as the angle of the coordinate [F(t), F(t – )] around the mean fluorescence for that given pixel, with values between –
and . After the transformation, a new phase (t) movie was produced that included all pixels, in which different phases of the action potential were represented by a continuous color gradient extending from red to purple. Phase singularity (PS) was defined as the point at which all phases converged.
Experimental protocols. Conduction velocity and APD90 were measured during constant (S1) stimulation at the center of the left ventricular free wall at basic cycle lengths (BCLs) of 180–800 ms. A monopolar electrode made of platinum wire was used for stimulation. The longitudinal (LD) and transverse directions (TD) of propagation were determined from the activation maps elicited by S1 stimulation (1). Conduction velocity was measured in a central 18 x 18-mm square around the stimulation site, since measurement in the outer periphery would have been hampered by the sharp curvature of the ventricular surface. Conduction velocities in LD and TD were calculated from the slope of a linear least-squares fit of the activation time plotted against the distance. Data from an area very close to the stimulation site (<2 mm) were excluded to minimize the virtual electrode polarization effects.
We used the dynamic pacing method (11) to characterize the APD restitution property. The center of the left ventricular free wall was initially paced at a BCL of 400 ms, and the BCL was progressively shortened in steps of 5–20 ms. A minimum of 30 stimuli were delivered at a given BCL. APD restitution at a site 5 mm from the stimulation site in TD was analyzed. The APD90 of the final paced action potential was measured at a BCL with no APD alternans. When APD alternans occurred at a shorter BCL, the pacing was interrupted twice to measure the APD90 of both long and short action potential. The BCL was shortened until either a 2:1 block or higher order periodicity occurred. The APD restitution curve was constructed by plotting APD90 against the preceding diastolic interval, and the curve was fitted to a single-exponential function (Origin 7.0; Microcal Software, Northampton, MA).
VTs resulting from spiral-type excitation were induced by modified cross-field stimulation. Eighteen S1 stimuli at a BCL of 400 ms were applied to the apex through a pair of contiguous bipolar electrodes. A 10-ms monophasic rectangular pulse of constant voltage (20 V) was generated by a DC power unit and was delivered from the unit through a 6,700-µF capacitor connected to a power MOS-FET switch. A pair of Ag-AgCl paddle electrodes (7 mm in diameter) were placed on the lateral surface of the left and right ventricles for the DC stimulation (S2) to induce electrical field roughly perpendicular to the S1 excitation from the apex to the base (modified cross-field stimulation). The S1-S2 coupling interval was varied at 10-ms steps to apply S2 during the vulnerable window of the final S1 excitation.
Data were obtained before (baseline) and 20–30 min after application of 0.1 µM NF (Nihon Schering, Osaka, Japan). All the experiments were completed within 120 min of perfusion, during which the conduction velocity, APD90, and APD restitution were unchanged (see Supplemental Table 1; the online version of this article contains supplemental data). We confirmed in pilot experiments of four rabbit hearts that the effects of NF (0.1 µM) on the action potential configuration reached a steady state at 
30 min (see Supplemental Table 2). At the end of experiment, the tissues were fixed in 15% formalin and sectioned parallel to the epicardium at a 8-µm interval. The sections were stained with hematoxylin and eosin to examine the fiber orientation in the mapped area.
Statistical analysis. Group data are expressed as means ± SD. Statistical comparisons were performed using two-way ANOVA with Tukey's test, t-test, or Mann-Whitney U-test when appropriate. Differences were considered significant when the probability value was <0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Figure 1 A shows representative changes in APD in response to NF application. Action potential signals obtained from 16 sites covering an 18 x 18-mm square were superimposed. NF (0.1 µM) caused uniform prolongation of APD at all recording sites. Figure 1B summarizes the changes in APD90 (average of the values at the 16 sites). NF caused significant prolongation of APD90, and the prolongation was greater at longer BCLs. The dispersion of the APD90 values among the 16 recording sites was virtually unaffected (Fig. 1C).
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VTs induced by modified cross-field stimulation. VTs were induced in 15 hearts by DC stimulation before (control) and after NF (0.1 µM) application. In controls, 74 of 93 VTs (79.6%) terminated spontaneously within 30 s (nonsustained), whereas the other 19 (20.4%) persisted >30 s (sustained). Forty-six (49.5%) of the 74 nonsustained VTs terminated within 5 s. Of the 54 VTs in the presence of NF, 52 VTs (96.2%) were nonsustained and the 2 other VTs (3.7%) were sustained. Forty-four (81.5%) of the 52 nonsustained VTs terminated within 5 s. Thus VTs induced in the presence of NF (0.1 µM) terminated earlier than in the control conditions. VT cycle length (VTCL) after NF was significantly longer than under the control conditions (188 ± 31 vs. 154 ± 16 ms, P < 0.05).
Optical images of excitation during VT were analyzed in nine hearts exhibiting visible rotors in the observation area under the control conditions as well as after NF. Figure 2 shows a representative experiment. The record under the control condition (Fig. 2A) was obtained 2 s after VT initiation (see Supplemental Movie 1). Clockwise rotation of wave fronts circulating around a line of functional block (7 mm) with a VTCL of 141 ms can be seen in isochrone maps. The circuit was more or less stable for >10 s and exhibited minimal meandering. A bipolar electrogram obtained during the VT episode showed monomorphic ventricular excitations. Characteristic features of the action potential configuration around the circuit, which were reported previously (1, 10), are recognized; those include slower upstroke at sites close to the pivot points (sites b and d), faster upstroke at sites after passing through the pivot points (sites a and c), clear double potential at the center of functional block line (site e), and no isoelectric segments between action potential signals. The block lines (4–9 mm) run either parallel (70%) or across (30%) the fiber orientation. There was no obvious tissue damage or macroscopic structural discontinuity to anchor the reentrant pathway in a fixed position.
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Qualitatively similar results were obtained in all nine hearts. Thus the characteristic effects of NF on the spiral wave dynamics (prolongation of functional block line, an increase in meandering of circuits, and earlier termination of rotation) (1) have been visualized clearly in experiments at a lower drug concentration (0.1 µM).
Wave front-tail interaction and phase singularity. The mechanisms of NF-induced modification of spiral wave reentry were investigated in terms of the wave front-tail interaction and the PS dynamics. Seven VT episodes with rotors visible in the observation area were analyzed for each before (control) and after application of NF. During VTs under the control conditions, the wave front chased its own tail with a certain distance between them (repolarized zone), and the wave fronts seldom met each other (Fig. 3A, top). The number of PS points in the phase maps was normally (>90%) 1 (a single rotor; Fig. 3A, bottom) and, rarely, increased to 2 for a short period (Fig. 3D). In the presence of NF (0.1 µM), the wave front frequently encountered its own tail, causing transient breakup of the spiral wave (Fig. 3B, top) or sudden movement of the organizational center of rotation to another site (Fig. 3C, top). In the phase maps, the former was recognized as an increase in PS from 1 to 3 (Fig. 3B, bottom), whereas the latter was recognized as a sudden jump in PS site (Fig. 3C, bottom). Figure 3D shows representative changes in PS number per 500 frames (665 ms) after application of NF. Pooled data are summarized in Fig. 3E; the average number of PS points during 500 frames (665 ms) was 1.13 ± 0.14 in the control and 1.63 ± 0.22 after NF (n = 7, P < 0.05).
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In the remaining seven VTs (36.8%) in the presence of NF, the rotors terminated by trapping the spiral tip in a region entirely surrounded by refractory tissue. Figure 5 shows a representative experiment. Isochrone and APD90 maps of three beats before termination are shown in Fig. 5A. In beat 1, activation from the left upper region turned around a short functional block line (yellow) in a clockwise direction. In beat 2, the clockwise rotation was maintained and associated with extension of the functional block line toward the central region. In beat 3, the wave front from the left upper region was blocked in the central region. Because this wave front (red dotted line) was entirely surrounded by a refractory zone, no offspring wavelets emerged, and the VT terminated. Action potential signal tracings from two sites (asterisks) revealed APD alternans during the VT cycles (Fig. 5B), with the longest APD90 of beat 3 (site a) preventing further rotation of the wave front. The APD maps of beat 3 visualized trapping of the entire spiral tip by the long APD90 zone. A similar enhancement of APD alternans preceding the spontaneous termination was observed in all seven VTs of this group. Figure 5C shows phase maps of the final beat (see Supplemental Movie 5). The PS of clockwise rotation shifted from the left upper region to the center and then disappeared. The trajectory of the PS plotted on space and time axes is shown in Fig. 5D.
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A representative experiment is shown in Fig. 6. In the control conditions (Fig. 6A), a PS of counterclockwise rotation was present in the middle of the anterior surface of the left ventricle. A trajectory of the PS revealed that the rotor was more or less stable and exhibited moderate meandering (1,718–2,041 ms). After application of NF (Fig. 6B), a PS of clockwise rotation moved a long distance with a complex trajectory; from the right margin to the upper central region (895–942 ms), going back to the right margin, followed by a traverse in opposite direction toward the posterior surface. The PS reappeared on the anterior surface (left upper region), turned back toward the left upper corner (anatomical boundary), and terminated (1,127–1,250 ms). Action potential signals showed nearly uniform configuration in control (Fig. 6A) compared with marked beat-to-beat variation after NF (Fig. 6B).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Action potential and conduction properties. In this study, we first examined the effects of 0.1 µM NF on the conduction velocity and APD under constant stimulation at a wider range of BCLs (180–800 ms) than in our previous study at 0.5 µM (BCLs 250 and 400 ms) (1). NF (0.1 µM) caused spatially uniform APD prolongation without affecting conduction velocities. The APD prolongation was significant even at a BCL of 180 ms, but it was greater at longer BCLs. The spatial dispersion of APD measured at 16 sites to cover an 18 x 18-mm square was unaffected by NF. NF increased the slope of the APD restitution curve, and the change was associated with an increase in APD alternans. All of these NF-induced alterations in the steady-state and dynamic electrophysiological properties of ventricular muscle can be ascribed to IKr blockade. In action potential clamp experiments, Hua and Gilmour (7) recently demonstrated that IKr contribute importantly to ventricular muscle repolarization during normal and high-frequency stimulation and that APD alternans is regulated substantially by time- and voltage-dependent activation and deactivation of IKr.
VTs of spiral wave reentry. In our 2D ventricular muscle tissue preparations of rabbit heart, spiral wave reentry rotating around functional block lines (single or double loops) can be induced by modified cross-field stimulation in the observation area in more than half (53–61%) of whole VT episodes under the control conditions (1, 10). The VTs induced are polymorphic during the initial several beats (1, 10) but become almost monomorphic during the subsequent beats. Microscopic structural discontinuities in association with anisotropic fiber orientation or epicardial blood vessels may provide a basis for this anchoring behavior.
VTs induced in the presence of NF (0.1 µM) are, consistent with our previous report with NF at 0.5 µM (1), characterized by longer cycle length, polymorphic configuration, and earlier termination than those observed in the controls. The optical images showed that these changes were associated with marked destabilization of rotors, which circulated around much longer and variable functional block lines formed by refractory wake of preceding excitations. We analyzed movies of wave front and wave tail during the rotations and found that a wave front in the presence of NF (0.1 µM) frequently encounters its own tail, resulting in breakup or sudden jump of organizational center of the rotation. The marked destabilization of rotors in the presence of NF cannot be ascribed to a APD restitution slope >1, since the maximum slope after NF, although increased, remained 0.7 on average. Other factors such as short-term cardiac memory, electronic currents, and unstable intracellular Ca2+ cycling also can affect wave stability and may have played a role in enhancing meander of the circuit. The enhanced meandering may have further facilitated the wave front-tail interactions through creation of Doppler-shifted short cycle length in front of moving phase singularities (4, 6).
Spiral wave reentry termination. Information on the effect of IKr block on the dynamics of spiral wave reentry in the ventricular myocardium is still limited. An analysis of computer model of isotropic 2D cardiac tissue showed discrepancies among investigators in the role of the delayed rectifier K+ current (IK) in the regulation of spiral wave dynamics. In their model, Beaumont and Jalife (2) showed that APD is significantly abbreviated (too short for IK activation) near the center of the rotation. Thus IK block prolongs APD only in the periphery and not close to the center. This leads to frequent wave front-tail interaction in the spiral arm without affecting the rotation period. Jalife and colleagues (2, 21) suggested that the inward rectifier K+ current (IK1) may play a much more important role than IK [IKr and IKs (slow component)] in regulation of spiral core dynamics. In their simulation using the phase 1 Luo-Rudy ventricular action potential model, on the other hand, Qu et al. (18) have shown that APD close to the spiral core is longer than in the periphery and that a substantial amount of IK is preserved at the rotation center. They demonstrated that reduction of IK conductance promotes meandering of the spiral core and that quasiperiodic meandering is converted to chaotic meandering that culminates in the breakup of rotors (18).
Our observations in the rabbit heart suggest that IKr plays an essential role in repolarization of the action potential not only in the arm but close to the core of spiral wave reentry. In the absence of NF (control), most spontaneous terminations of spiral-type excitations were the result of mutual annihilation of a pair of rotors with opposite chiralities. NF facilitated the spontaneous termination by two different mechanisms: extinction of rotor(s) after collision with the anatomic boundary, and trapping of the spiral tip in a region entirely surrounded by refractory tissue. The former mode is attributable to considerable meandering of the rotation center, whereas the latter is attributable to APD prolongation of the preceding excitation in association with increased APD alternans near the spiral tip. The latter mode of termination is similar to that reported by Beaumont and Jalife (2) in their 2D cardiac tissue model when sodium current inactivation was slowed in combination with APD prolongation or when the outward component of IK1 was reduced. In a recent theoretical study by Qu and Weiss (19), blockade of time-dependent K+ channel was shown to increase dynamic instability of rotors and to facilitate their self-termination. The present results validate their prediction.
Most of the modification of spiral wave dynamics induced by NF (0.1 µM) may be shared by other IKr blocking drugs, since much higher concentrations (5–100 µM) are required for NF to affect other voltage- and ligand-gated K+ currents (17). However, further experimental studies are required to elucidate the point.
Limitations. In this study using a 2D subepicardial layer of rabbit ventricular myocardium, NF-induced delay and instability of ventricular repolarization were shown to cause frequent collision of spiral wave fronts with their own tails or anatomical boundaries, giving rise to breakup or early extinction of rotors. Extending these results to 3D hearts, especially in larger animals including humans, is not straightforward. If there is sufficient tissue mass, the chance of spontaneous termination of rotors by wave front collision or trapping would be reduced, whereas the enhancement or rotor meander and wave instability may promote breakup in favor of transition from VT to VF. The greater structural discontinuities and functional heterogeneities in diseased hearts would also alter the spatial requirements of spontaneous rotor termination. Thus NF can be not only antiarrhythmic but also proarrhythmic. There are certain clinical reports (in Japanese) showing excessive QT prolongation and torsades de pointes induced by NF (0.1–0.3 mg/kg iv). Plasma concentrations at such therapeutic doses are comparable to the NF concentration (0.1 µM) employed in the present study (17). The proarrhythmia via rotor breakup might be dangerous, since it would promote VF generation and perpetuation. We used BDM as an excitation-contraction uncoupler that is known to alter ionic currents and to reduce the APD restitution slope (13). However, this does not seem to invalidate the present results, because the characteristic modification of the spiral wave dynamics by NF was preserved in the absence of BDM. We cannot completely neglect the potential photodynamic toxicity of the voltage-sensitive dye (di-4-ANEPPS), but this may not have significant effects on the electrophysiological properties of the preparations in our experimental conditions, because there were no time-dependent changes of conduction velocity and action potential configuration up to 120 min of control perfusion (see Supplemental Table 1). There are considerable species differences in the relative contribution of IKr to the repolarization of action potential in ventricular myocytes. Despite these limitations, the present results may provide a new perspective for future development of drugs to prevent sudden arrhythmic death.
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FOOTNOTES |
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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.
* M. Yamazaki and H. Honjo contributed equally to this work. 
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