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ACC/AHA 2008 Performance Measures for Adults With ST-Elevation and Non-ST-Elevation Myocardial Infarction

ACC/AHA 2008 Guidelines for the Management of Adults With Congenital Heart Disease
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J Am Coll Cardiol, 2006; 47:2074-2085, doi:10.1016/j.jacc.2005.12.064 (Published online 21 April 2006).
© 2006 by the American College of Cardiology Foundation
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PRECLINICAL STUDY

Cellular Basis for Trigger and Maintenance of Ventricular Fibrillation in the Brugada Syndrome Model

High-Resolution Optical Mapping Study

Takeshi Aiba, MD, PhD*, Wataru Shimizu, MD, PhD{dagger},*, Ichiro Hidaka, MS*, Kazunori Uemura, MD*, Takashi Noda, MD, PhD*, Can Zheng, PhD*, Atsunori Kamiya, MD*, Masashi Inagaki, MD*, Masaru Sugimachi, MD, PhD* and Kenji Sunagawa, MD, PhD*

* Department of Cardiovascular Dynamics, Research Institute, National Cardiovascular Center, Suita, Japan
{dagger} Division of Cardiology, Department of Internal Medicine, National Cardiovascular Center, Suita, Japan.

Manuscript received November 10, 2005; revised manuscript received November 25, 2005, accepted December 13, 2005.

* Reprint requests and correspondence: Dr. Wataru Shimizu, Division of Cardiology, Department of Internal Medicine, National Cardiovascular Center, 5-7-1 Fujishiro-dai, Suita, Osaka, 565-8565 Japan. (Email: wshimizu{at}hsp.ncvc.go.jp).

Presented in part at the Scientific Session of the American Heart Association, November 7–10, 2004, and published in abstract form (Circulation 2004;110 Suppl III:III318).


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 Appendix
 References
 
OBJECTIVES: We examined how repolarization and depolarization abnormalities contribute to the development of extrasystoles and subsequent ventricular fibrillation (VF) in a model of the Brugada syndrome.

BACKGROUND: Repolarization and depolarization abnormalities have been considered to be mechanisms of the coved-type ST-segment elevation (Brugada-electrocardiogram [ECG]) and development of VF in the Brugada syndrome.

METHODS: We used high-resolution (256 x 256) optical mapping techniques to study arterially perfused canine right ventricular wedges (n = 20) in baseline and in the Brugada-ECG produced by administration of terfenadine (5 µmol/l), pinacidil (2 µmol/l), and pilsicainide (5 µmol/l). We recorded spontaneous episodes of phase 2 re-entrant (P2R)-extrasystoles and subsequent self-terminating polymorphic ventricular tachycardia (PVT) or VF under the Brugada-ECG condition and analyzed the epicardial conduction velocity and action potential duration (APD) restitutions in each condition.

RESULTS: Forty-one episodes of spontaneous P2R-extrasystoles in the Brugada-ECG were successfully mapped in 9 of 10 preparations, and 33 of them were originated from the maximum gradient of repolarization (GRmax: 176 ± 54 ms/mm) area in the epicardium, leading to PVT (n = 12) or VF (n = 5). The epicardial GRmax was not different between PVT and VF. Wave-break during the first P2R-extrasystole produced multiple wavelets in all VF cases, whereas no wave-break or wave-break followed by wave collision and termination occurred in PVT cases. Moreover, conduction velocity restitution was shifted lower and APD restitution was more variable in VF cases than in PVT cases.

CONCLUSIONS: Steep repolarization gradient in the epicardium but not endocardium develops P2R-extrasystoles in the Brugada-ECG condition, which might degenerate into VF by further depolarization and repolarization abnormalities.

Abbreviations and Acronyms
  AP = action potential
  APD = action potential duration
  APD50 = action potential duration measured at 50% repolarization
  BCL = basic cycle length
  Brugada-ECG = coved-type ST-segment elevation
  Delta-Epi interval = interval from the earliest to the latest epicardial activation
  DR = dispersion of repolarization
  ECG = electrocardiogram/electrocardiography
  GRmax = maximum gradient of repolarization
  ICa = inward calcium current
  IK-ATP = ATP-sensitive potassium current
  INa = sodium current
  Ito = transient outward potassium current
  P2R = phase 2 re-entrant/entry
  RV = right ventricle/ventricular
  Sti-Epi interval = interval from the stimulus to the earliest epicardial activation
  VF = ventricular fibrillation
  VT = ventricular tachycardia

Brugada syndrome is characterized by ST-segment elevation in the right precordial leads (V1 to V3) of electrocardiography (ECG) and a high incidence of ventricular fibrillation (VF) leading to sudden cardiac death (1–4). However, not all of the patients with ST-segment elevation have arrhythmic events (5,6), indicating that additional factors might contribute to development of VF. Previous studies suggest that an accentuation of transient outward potassium current (Ito)-mediated phase 1 notch and loss of action potential (AP) dome in some areas of the right ventricular (RV) epicardium but not endocardium increases transmural dispersion of repolarization (DR), which causes the ST-segment elevation (7–11). The heterogeneous loss of AP dome in the epicardium also increases epicardial DR, and a propagation of AP dome from a site where AP dome is restored to a site where it is lost might develop a local re-excitation called a phase 2 re-entry (P2R), which triggers a circus movement re-entry in the form of VF (8,9,12). It is still unclear, however, to what extent the epicardial DR is required for development of P2R and how phase 2 re-entrant (P2R)-extrasystoles produce VF. Moreover, depolarization abnormality is thought to be one of the potent arrhythmic substrate in the Brugada syndrome (13–17), but it is not fully understood how depolarization and repolarization abnormalities interact and contribute to the development and maintenance of VF in the Brugada syndrome.

To investigate the heterogeneities of cellular repolarization and depolarization and their potential role in the development of re-entrant arrhythmias, we used a technique of high-resolution optical mapping, which allowed us to measure the electrical heterogeneity of APs on the epicardial or endocardial surface (18). We demonstrated that a steep repolarization gradient in the RV epicardium but not in the endocardium plays a key role in initiating P2R. Moreover, further depolarization and repolarization abnormalities degenerate the P2R-induced spiral re-entry into multiple wavelets forming VF in an experimental model of the Brugada syndrome.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 Appendix
 References
 
Canine RV wedge model of the Brugada syndrome.   All animal care procedures were in accordance with the position of the American Heart Association research animal use (November 11, 1984). The methods used for isolation, perfusion, and recording of transmembrane activity from the arterially perfused canine RV (anterior wall) is similar to methods reported with canine wedge preparations (8,9). Briefly, a transmural wedge with dimensions of approximately 2 x 1 x 0.7 cm to 3 x 1.5 x 1 cm was dissected from the free wall of the RV of male dogs (n = 20), cannulated via the branch of right coronary artery, and placed in a small tissue bath. These preparations were arterially perfused between 40 and 60 mm Hg with Tyrode’s solution (35 ± 1°C). The inward calcium current (ICa) and sodium current (INa) block with terfenadine (5 µmol/l), combined with augmentation of ATP-sensitive potassium current (IK-ATP) with pinacidil (2 µmol/l), and INa block with pilsicainide (5 µmol/l) were used to create an experimental model of the Brugada syndrome (8–10,19).

After changing ECG to the coved-type ST-segment elevation mimicking the Brugada syndrome (Brugada-ECG) by administration of these drugs, 1) we recorded the spontaneous occurrence of closely coupled extrasystoles and subsequent non-sustained polymorphic VT (terminated within 5 s) or VF (sustained more than 5 s) during pacing from the endocardium at basic cycle length (BCL) of 2,000 ms (n = 10), and 2) we analyzed restitutions of the epicardial conduction velocity and action potential variable with a single extra stimulus (S2) delivered after every 10th basic beat (S1) paced from the epicardial surface at BCL of 1,000 ms (n = 10).

Transmembrane AP and ECG recording.   A transmural ECG was recorded with Ag-AgCl electrodes, which were placed in the Tyrode’s solution bathing the preparation, 1.0 cm from the epicardial and endocardial surfaces (epicardial, positive pole). The epicardial and endocardial APs were simultaneously recorded from the epicardial and endocardial surfaces with separate intracellular floating microelectrodes (direct current resistance 10 to 20 M{Omega}; 2.7 mmol/l potassium chloride) at positions approximating the transmural axis of the ECG.

Optical AP recording.   After staining with the voltage sensitive dye, di-4-ANEPPS (5 µmol/l for 30 min), wedges were stabilized against a flat imaging window. Excitation of the dye’s fluorescence was achieved with 480 ± 15 nm light through a bandpass filter (ANDV8247, Andover, Salem, New Hampshire) from a bluish-green emission diode (E1L51-3B0A4-02, Toyoda Gosei, Aichi, Japan). Fluoresced light from the wedge was split by a dichrotic mirror and narrowed down to the two frequency bands (approximately 540 or 690 nm) through a bandpass filter (ANDV8368 or ANDV7845, respectively, Andover). Then, the dual-wavelength lights were simultaneously focused onto 10-bit 256 x 256 element dual complementary metal oxide semiconductor (C-MOS) censors (Hamamatsu Photonics, Hamamatsu, Japan) with image intensifiers (FASTCAM-Ultima, Photron, Tokyo, Japan) at a 500 frames/s (Fig. 1).


Figure 1
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  		Figure 1 Schematic diagram showing the major components of high-resolution optical mapping of the epicardial (Epi) or endocardial (Endo) surface in an arterially perfused canine right ventricular (RV) wedge preparation. CMOS = complementary metal oxide semiconductor.

 
Both optical signals were digitized at 0.5 kHz, and other amplified signals were digitized at 2 kHz with a 12-bit analog-to-digital converter, stored on the hard disk of a dedicated laboratory computer system, and analyzed with the original software of our laboratory. Therefore, after ratiometry of both signals to subtract a motion artifact, the voltage of the optical signal recorded at each site was automatically displayed in color (lowest, black; greatest, red) and plotted in the 256 x 256 matrix as an isopotential map, and transmembrane APs from 256 sites (16 x 16 units) on the RV epicardial or endocardial surface were displayed in control and in the Brugada-ECG condition with or without arrhythmic events. Moreover, phase analysis was used to display the pattern of wave propagation and wave-break during ventricular tachyarrhythmias (20,21).

Data analysis.   Optical action potential duration (APD) was automatically measured at 50% repolarization (APD50), and the distributions of epicardial and endocardial APD50 were displayed as a repolarization counter map in baseline (control condition) and after changing to the Brugada-ECG with or without P2R-extrasystoles. The epicardial and endocardial DR were calculated from the maximum difference of repolarization times (activation time + APD) in the epicardial and endocardial surfaces, respectively. Transmural DR was calculated from the maximum difference between the epicardial and endocardial repolarization times recorded from the floating microelectrodes. Moreover, the maximum gradient of repolarization (GRmax = maximum Delta-APD50/Delta-distance) in the epicardium and endocardium were calculated in each condition. We also measured depolarization parameters such as the interval from the stimulus to the earliest epicardial activation (Sti-Epi interval) and the interval from the earliest to the latest epicardial activation (Delta-Epi interval) during pacing from the endocardium in control and in the Brugada-ECG condition. Conduction velocity ({theta}) was determined by linear regression of the isochrone distance versus activation time. Lines parallel and perpendicular to the fiber orientation were defined as the direction of longitudinal (L) and transverse (T) propagation, respectively. The optical data at edge of the preparation, with apparent contraction artifact, and noise level more than 20% of AP amplitude were excluded.

Statistical analysis.   Statistical analysis of the data was performed with a Student’s t test for paired data or analysis of variance coupled with Scheffe’s test, as appropriate. Data is expressed as mean ± SD or mean ± SEM. Significance was defined as a value of p < 0.05.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 Appendix
 References
 
Canine wedge model of the Brugada syndrome.   Terfenadine combined with pinacidil and pilsicainide produced the Brugada-ECG in all preparations. There was no arrhythmia in control conditions, whereas combination of the drugs spontaneously developed a P2R-induced short-coupled extrasystole and subsequent polymorphic VT or VF in 9 of 10 preparations (Fig. 2). The QRS interval, QT interval, and J-point level in the ECG were significantly greater in the Brugada-ECG than in the control condition, but those parameters in the Brugada-ECG were not significantly different between beats with and without P2R-extrasystoles (Table 1).


Figure 2
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  		Figure 2 Representative episodes of polymorphic ventricular tachycardia or ventricular fibrillation (VF) in a canine wedge model of the Brugada syndrome. All arrhythmias were spontaneously developed after the electrocardiogram with coved-type ST-segment elevation. Many of the arrhythmias (numbers 1 to 6) terminated within a few seconds, but the others (numbers 7 to 9) with a shorter cycle length (CL) degenerated into VF, which continued more than 5 s.

 

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  		Table 1. ECG, Repolarization, and Depolarization Parameters in Control and in the Brugada-ECG Condition With or Without Phase 2 Re-Entrant Extrasystoles
 
Epicardial repolarization abnormality develops P2R-extrasystoles.   Figure 3 represents the epicardial and endocardial APD50 contour map and optical APs in the control and in the Brugada-ECG condition with or without P2R-extrasystoles. In the control condition, the epicardial and endocardial APs were almost homogeneous (Figs. 3A and 3D). In contrast, in the Brugada-ECG, the AP morphology in the epicardium but not endocardium changed into heterogeneous, owing to a combination of abbreviated (loss-of-dome) and prolonged (restore-of-dome) APs, resulting in increasing DR in the epicardium rather than in the endocardium (Figs. 3B and 3E). Moreover, further prolonged AP at some areas in the epicardium was closely adjacent to the loss-of-dome APs (arrow), thus producing a repolarization mismatch within a small area and developing a P2R-extrasystole at the loss-of-dome site (Fig. 3C). The APs in the endocardium, however, were less heterogeneous than those in the epicardium even in the Brugada-ECG just before P2R-extrasystoles (Fig. 3F).


Figure 3
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  		Figure 3 Representative action potential duration measured at 50% repolarization (APD50) contour map on the right ventricular epicardium (Epi) and endocardium (Endo) in control condition (A and D, respectively), in the ST-segment elevation (Brugada-ECG) without phase 2 re-entrant (P2R) extrasystoles (B and E, respectively), and in the Brugada-ECG just before P2R extrasystoles (C and F, respectively) and representative optical action potentials at each site (a to c). White arrow = initial site of P2R. DR = dispersion of repolarization; GRmax = maximum gradient of repolarization.

 
The composite data of repolarization and depolarization parameters in the control and in the Brugada-ECG condition with and without P2R-extrasystoles are shown in Table 1. In the Brugada-ECG, the epicardial maximum APD50 was significantly prolonged, whereas the epicardial minimum APD50 was significantly abbreviated compared with those in the control condition, thus significantly increasing the epicardial DR and GRmax. Moreover, the epicardial maximum APD50 was further prolonged in the Brugada-ECG just before P2R-extrasystoles compared with that without P2R-extrasystoles, thus remarkably increasing the epicardial DR and GRmax. The endocardial repolarization parameters, however, were not significantly changed after the Brugada-ECG. Moreover, there was no significant difference in the endocardial repolarization parameters between the Brugada-ECG with and without P2R-extrasystoles. Owing to a different response of APD between the epicardium and endocardium, transmural DR was significantly increased in the Brugada-ECG compared with that in the control condition but was not significantly different between the Brugada-ECG condition with and without P2R-extrasystoles.

Regarding depolarization parameters, the Sti-Epi interval was significantly increased in the Brugada-ECG compared with in the control condition but was not different between the condition with and without P2R-extrasystoles. The Delta-Epi interval was not significantly different among the three conditions.

Threshold to develop P2R-extrasystoles.   A total of 41 episodes of spontaneous P2R-extrasystoles after the Brugada-ECG were successfully mapped in 9 of 10 preparations, and 33 (80%) of them were originated from the GRmax area in the epicardium. As shown in Figure 4, the epicardial GRmax was significantly greater in the Brugada-ECG than in control condition. The GRmax of 99 ms/mm (dashed line) showed that P2R-extrasystoles were spontaneously developed in the Brugada-ECG. In contrast, the endocardial GRmax and transmural DR were greater in the Brugada-ECG condition compared with the control condition but were not different between the Brugada-ECG condition with and without P2R-extrasystoles.


Figure 4
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  		Figure 4 Scatter plots of the maximum gradient of repolarization (GRmax) in the epicardial (Epi) and endocardial (Endo) surfaces (A) and transmural dispersion of repolarization (DR) (B) in control and the ST-segment elevation (Brugada-ECG) condition with (closed circles) or without (open circles) phase 2 re-entrant (P2R) extrasystoles. Values are mean ± SD. *p < 0.05 versus control condition; {dagger}p < 0.05 versus Brugada-ECG condition without P2R-extrasystole by analysis of variance with Scheffe’s test.

 
Figure 5A shows the epicardial isopotential map representing the distribution of loss-of-dome and restore-of-dome area in the Brugada-ECG with (beat 2) or without (beat 1) a P2R-extrasystole. Figures 5B and 5C show the depolarization map during the P2R-extrasystole and optical APs at each site on the epicardial surface. At the timing of phase 2 (120 to 190 ms), the restore-of-dome area (orange-red) was larger in the beat 2 than in the beat 1. Moreover, the larger AP dome in the beat 2 moved from a restore-of-dome site (site a and b) to a nearby loss-of-dome site (site d), producing re-excitation at the loss-of-dome site and propagating in a counterclockwise fashion around the refractory region of the epicardium.


Figure 5
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  		Figure 5 Snapshots of a color optical isopotential movie on the epicardial surface for the continuous two beats with (Beat 2) and without (Beat 1) a phase 2 re-entrant extrasystole (P2R-extrasystole) in the Brugada-ECG condition (A). Depolarization map of a P2R-extrasystole (B) and optical action potentials at each site (a to f) and transmural electrocardiogram (ECG) (C). Please see the for accompanying video.

 
P2R-extrasystoles induce polymorphic VT or VF.   The epicardial P2R-extrasystoles produced 12 episodes of self-terminating (<5 s) polymorphic VT and 5 episodes of sustained (≥5 s) VF. The mechanism underlying the difference between the polymorphic VT and VF is shown in representative examples in Figures 6 and 7.Go The epicardial GRmax area (arrow) developed P2R-extrasystole in both cases (Figs. 6A and 7A); however, the epicardial depolarization map paced from the endocardium at BCL of 2,000 ms shows a remarkable conduction delay in the episode of VF (Fig. 7B) compared with that of polymorphic VT (Fig. 6B). We compared the repolarization and depolarization parameters just before the P2R-induced polymorphic VT and VF in Table 2. There was no significant difference in the repolarization parameters between the two groups; however, the depolarization parameters such as QRS, Sti-Epi, and Delta-Epi intervals were significantly longer in the VF group than in the polymorphic VT group.


Figure 6
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  		Figure 6 Representative repolarization and depolarization maps on the epicardial surface in the ST-segment elevation (Brugada-ECG) condition just before non-sustained polymorphic ventricular tachycardia (VT) (A, B), snapshots of phase movie during polymorphic VT originated from the epicardial phase 2 re-entry (C), and optical action potentials at each site (a to f) together with a transmural electrocardiogram (ECG) (D). Open circles = singularity points. APD50 = action potential duration at 50% repolarization. Please see the for accompanying video.

 

Figure 7
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  		Figure 7 Representative repolarization and depolarization maps on the epicardial surface in the ST-segment elevation (Brugada-ECG) condition just before ventricular fibrillation (VF) (A, B), snapshots of phase movie during VF originated from the epicardial phase 2 re-entry (C), and optical action potentials at each site (a to f) together with a transmural electrocardiogram (ECG) (D). Open circles = singularity points. APD50 = action potential duration at 50% repolarization. Please see the for accompanying videos.

 

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  		Table 2. ECG, Optical Repolarization, and Depolarization Parameters Just Before Polymorphic VT or VF in the Brugada-ECG Condition
 
Figures 6C and 6D represent phase map and optical APs, respectively, during the P2R-induced polymorphic VT, showing that re-entry was initiated from the epicardial GRmax area and rotated mainly in the epicardium without wave-break. In contrast, Figures 7C and 7D represent those during the P2R-induced VF, showing that the development of initial P2R was similar to that of polymorphic VT, but the first P2R-wave was broken up into the multiple wavelets, resulting in degenerating VT into VF. The phase singularity points during the first P2R-wave almost coincided with the sites of delayed conduction (Fig. 7B). In all VF cases, the wave was broken up into multiple wandering wavelets during the first P2R-induced extrasystole; however, in the polymorphic VT cases, only 3 of 12 (25%) cases had a wave-break after the second beat, but soon after the wave had been broken, the waves collided and finally terminated.

Conduction and APD restitutions by S1-S2 method.   In another 10 preparations, we analyzed the epicardial conduction velocity and APD restitutions to show the mechanisms underlying the wave-break during the first re-entrant wave in the VF cases. The epicardial longitudinal and transverse conduction velocities ({theta}L and {theta}T) in the VF cases (n = 5) were significantly slower than those in the polymorphic VT cases (n = 5) under the Brugada-ECG condition, and the conduction velocity restitution curve in the VF cases was shifted lower in parallel (Fig. 8).


Figure 8
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  		Figure 8 Representative epicardial depolarization maps paced from the epicardium by S1-S2 method in the control and ST-segment elevation (Brugada-ECG) condition with polymorphic ventricular tachycardia (PVT) or ventricular fibrillation (VF) (A), and longitudinal (L) and transverse (T) conduction velocity ({theta}) restitution curves in each condition (B). Values are mean ± SEM.

 
In contrast, the epicardial APD was abbreviated and its restitution was flat in the polymorphic VT case under the Brugada-ECG condition, owing to loss of AP dome (Fig. 9B); however, in the VF case, shorter S1-S2 interval (≤300 ms) rather prolonged APD because of restoration of AP dome. Moreover, this restoration was heterogeneous in the epicardial surface, increasing the epicardial DR (Fig. 9C). This "inverse" APD restitution pattern was observed in four of five VF cases but in only one of five polymorphic VT cases under the Brugada-ECG condition.


Figure 9
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  		Figure 9 Representative epicardial repolarization maps paced from the epicardium by S1-S2 method and plot of the restitution of action potential duration at each site (a to e) and superimposed optical action potentials at site b in control condition (A), and the Brugada-ECG condition with polymorphic ventricular tachycardia (PVT) (B) or ventricular fibrillation (VF) (C). APD50 = action potential duration at 50% repolarization; DI = diastolic interval.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
Appendix
 References
 
Repolarization mismatch develops P2R-extrasystoles.   All-or-none repolarization of the ventricular AP and P2R is considered to be one of the potential mechanism of the ST-segment elevation and subsequent VF in the Brugada syndrome (7–9,12); however, because of limitations of conventional electrophysiological recording techniques, it remains unknown to what extent the heterogeneity of APs is required for developing P2R-extrasystoles in the Brugada-ECG. In this study, we conducted a high-resolution optical mapping in canine RV wedge preparation, which allowed a detailed measurement of cellular repolarization and depolarization in the epicardial and endocardial surfaces. First, we photographed the moment that P2R-extrasystoles in the Brugada-ECG occurred and produced re-entrant arrhythmias such as polymorphic VT or VF. A unique topographical distribution of both loss-of-dome and restore-of-dome cells in the epicardium but not endocardium might underlie a key feature of the Brugada phenotype, including coved-type ST-segment elevation and susceptibility to P2R-induced ventricular tachyarrhythmias. It must be essential to develop P2R-extrasystoles that further prolong the epicardial AP results in loss-of-dome at some areas but not at the closely adjacent area, making a steep repolarization mismatch. These data are consistent with some clinical reports that the QT interval is more prolonged in the right precordial leads than in other leads during typical coved-type Brugada-ECG (2,13,22) and that VF in the Brugada syndrome was frequently induced by the specific premature ventricular contractions originated from the free wall of RV outflow tract (23,24).

Ionic backgrounds of Brugada-ECG and P2R-extrasystoles.   Previous experimental studies pharmacologically created the Brugada-ECG by using various drugs and/or conditions capable of causing an outward shift in the current active at the end of phase 1 of RV epicardium (e.g., increase in Ito, IK-ATP, and/or IK-ACh and decrease in ICa and INa) (4,7–10,19). Moreover, a development of P2R on the basis of the all-or-none repolarization phenomenon might depend on a fine balance of Ito, INa, and ICa. We used block of ICa and INa (and other currents) with terfenadine (5 µmol/l), combined with augmentation of IK-ATP with pinacidil (2 µmol/l) and INa block with pilsicainide (5 µmol/l); a combination that is most likely to produce the Brugada-ECG. The reason a loss-of-dome occurred in some areas but not others in the epicardium is expected to be owing to an intrinsic difference in Ito (25). Miyoshi et al. (26) investigated the mechanism of P2R by their mathematical model and suggested that P2R was developed from a boundary area (0.8 cm) between loss-of-dome and restore-of-dome where a fine balance between Ito and ICa,L was required and that ICa,L must play an essential role in the genesis of P2R. This mathematical model supports our data that most of P2R-extrasystoles were developed from a small area (<0.5 cm) of GRmax.

Maintenance of VF.   The only gene linked to the Brugada syndrome is cardiac sodium channel gene, SCN5A (17,27). Moreover, sodium channel blockades often unmask Brugada-phenotype, because a loss of sodium channels function enhances both repolarization and depolarization abnormalities (25,28,29). Our experimental study used a pure sodium channel blocker, pilsicainide, to produce the Brugada-ECG associated with prolonged QRS duration and conduction parameters; however, in the Brugada-ECG condition, the depolarization parameters were not different in beats with and without P2R-extrasystoles. In contrast, slower conduction was closely associated with VF susceptibility. These findings suggest that depolarization disturbance was not directly associated with the development of P2R-extrasystole, a trigger of VF, but might contribute to the maintenance of VF in the Brugada-ECG condition.

Electrophysiologic mechanism of VF in the Brugada syndrome has been considered to be re-entry because of high inducibility and reproducibility of VT/VF by programmed electrophysiologic stimulation (3,6,14,30), although it is still unclear how VF re-entry is maintained in the Brugada syndrome. In this study, most of the polymorphic VT was single or figure-of-eight type re-entry with no wave-break and terminated within a few seconds (Fig. 6C). In contrast, wave-break in VF group occurred during the first re-entrant wave and took place at sites of the delayed epicardial conduction (Fig. 7B). Wu et al. (31) demonstrated that Ca2+ and fast Na+ current inhibition turned fast VF into slow VF by flattering APD restitution and increasing conduction time. In this Brugada model, however, VF was characterized as the shorter cycle length and multiple wandering wavelets (Fig. 7C) in spite of the slower conduction (Fig. 8), because APD restitution was not flat but rather an "inverse" pattern (Fig. 9), thus increasing dispersion of repolarization during tachycardia. Krishnan and Antzelevitch (25) had demonstrated the incremental arrhythmogenesis of Na+ channel dysfunction in the RV epicardium during tachycardia. Flecainide also rate-dependently slowed down the conduction velocity. Thus, fast Na+ current inhibition strongly enhances both heterogeneity of repolarization and conduction slowing during tachycardia in the Brugada-ECG model, which can easily break up the spiral re-entry, thus degenerating polymorphic VT into VF with multiple wavelets.

Clinical implication.   Previous clinical study suggested that induction of VF by programmed ventricular stimulation depended on the severity of depolarization abnormalities such as a longer QRS duration or His-ventricular interval but did not predict the recurrence of cardiac events in symptomatic Brugada syndrome (14,15). Moreover, depolarization and repolarization abnormalities in this syndrome are now considered to be closely correlated (16,29,32,33), supporting our data that both repolarization and depolarization abnormalities were important in the development of VF. Our results, for the first time, revealed how repolarization and depolarization abnormalities interact in developing a trigger of premature ventricular complexes and in maintaining VF in the Brugada-ECG condition. A steep repolarization gradient in the epicardium introduced P2R-extrasystoles and subsequent non-sustained polymorphic VT, and further increased depolarization and repolarization abnormalities maintained VF, thus increasing risk of sudden cardiac death.

Study limitations.   First, we mapped the epicardial or endocardial surface separately in each condition. Therefore, the two-dimensional mapping technique used in this study provides only limited insights into the number of spiral waves and these re-entrant patterns and could not directly evaluate the relationship between the transmural gradient of repolarization and arrhythmogenesis in the Brugada-ECG condition. A second limitation is the size of wedge preparation. It is unclear whether a polymorphic VT or VF in the wedges can result in those with larger hearts. Third, we pharmacologically created, similarly to the methods of previous studies, the Brugada-phenotype, which could not be a complete surrogate for the Brugada syndrome. Finally, with optical mapping, there is a major concern about motion artifacts that can greatly distort the AP recorded, but our ratio-metric methods can reduce motion artifacts without using an uncoupler.


   Appendix
Top
 Abstract
 Methods
 Results
 Discussion
 Appendix
References
 
For accompanying videos to Figures 5, 6, and 7, please see the online version of this article.


   Footnotes
 
This work was supported by grants from Japan Cardiovascular Research Foundation (Dr. Aiba) and the Japan Foundation of Cardiovascular Research (Dr. Aiba), Health Sciences Research grants from the Ministry of Health, Labour, and Welfare (Dr. Shimizu), Research grants for Cardiovascular Diseases (15C-6) from the Ministry of Health, Labour, and Welfare (Dr. Shimizu), Japan Science and Technology Agency (Dr. Sunagawa), and a Health and Labour Sciences Research grant for research on medical devices for analyzing, supporting, and substituting the function of the human body from the Ministry of Health Labour, and Welfare of Japan (Dr. Sunagawa).


   References
Top
 Abstract
 Methods
 Results
 Discussion
 Appendix
 References
 

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