Published online before print October 8, 2009, doi: 10.1161/CIRCRESAHA.108.176248
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© 2009 American Heart Association, Inc.
Cellular Biology |
Spiral Waves and Reentry Dynamics in an In Vitro Model of the Healed Infarct Border Zone
From the Division of Cardiology (M.G.C., C.Y.C., B.O., L.X., M.A.Z., E.M., M.R.A.) and the Department of Biomedical Engineering (M.G.C., Y.Z., C.Y.C., L.X., L.T.), The Johns Hopkins University, Baltimore, Md; Medical Scientist Training Program (M.G.C.), University of California at Los Angeles School of Medicine; and Heart Institute (E.M.), Cedars-Sinai Medical Center, Los Angeles, Calif.
Correspondence to M. Roselle Abraham, MD, Department of Cardiology, Johns Hopkins Hospital, 720 Rutland Ave, Ross Building, Room 871, Baltimore, MD 21205. E-mail mabraha3{at}jhmi.edu
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Abstract |
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Rationale: Reentry underlies most ventricular tachycardias (VTs) seen postmyocardial infarction (MI). Mapping studies reveal that the majority of VTs late post-MI arise from the infarct border zone (IBZ).
Objective: To investigate reentry dynamics and the role of individual ion channels on reentry in in vitro models of the "healed" IBZ.
Methods and Results: We designed in vitro models of the healed IBZ by coculturing skeletal myotubes with neonatal rat ventricular myocytes and performed optical mapping at high temporal and spatial resolution.
In culture, neonatal rat ventricular myocytes mature to form striated myocytes and electrically uncoupled skeletal myotubes simulate fibrosis seen in the healed IBZ. High resolution mapping revealed that skeletal myotubes produced localized slowing of conduction velocity (CV), increased dispersion of CV and directional-dependence of activation delay without affecting myocyte excitability. Reentry was easily induced by rapid pacing in cocultures; treatment with lidocaine, a Na+ channel blocker, significantly decreased reentry rate and CV, increased reentry path length and terminated 30% of reentrant arrhythmias (n=18). In contrast, nitrendipine, an L-type Ca2+ channel blocker terminated 100% of reentry episodes while increasing reentry cycle length and path length and decreasing reentry CV (n=16). K+ channel blockers increased reentry action potential duration but infrequently terminated reentry (n=12).
Conclusions: Cocultures reproduce several architectural and electrophysiological features of the healed IBZ. Reentry termination by L-type Ca2+ channel, but not Na+ channel, blockers suggests a greater Ca2+-dependence of propagation. These results may help explain the low efficacy of pure Na+ channel blockers in preventing and terminating clinical VTs late after MI.
Key Words: arrhythmia • Ca2+ channels • cardiac electrophysiology • electrophysiology • mapping • Na+ current • optical mapping
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Introduction |
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In the United States, myocardial infarction (MI) afflicts more than 7.1 million people, with 865 000 new cases each year.1 Late after an MI, surviving myocyte bundles in the infarct, and the infarct border zone (IBZ) have been implicated in the genesis of post-MI reentrant ventricular tachyarrhythmias2 and sudden cardiac death. Currently, the treatment options for post-MI arrhythmias include defibrillator implantation combined with antiarrhythmic drug therapy and/or ablation. Unfortunately, effective drug therapy for post-MI ventricular arrhythmias has been discouraging because they are proarrhythmic,3 lack efficacy,4–7 and cause toxicity.8,9 Understanding the role of ion channels on arrhythmia dynamics is crucial to developing tailored, new drug and gene-based therapies for ventricular tachy-arrhythmias. Currently, technical obstacles preclude 3D mapping in whole heart studies, making 2D and theoretical models attractive options for studying the biophysics of reentrant arrhythmias.
Mapping studies have revealed that the majority of VTs originate from the IBZ, a substrate that is characterized by nonuniform anisotropic architecture resulting from fibrosis that separates myocyte bundles and gap junction remodeling of surviving myocytes.10 Despite extensive ultrastructural and electrophysiological characterization of the "healed" IBZ, very little information exists regarding reentry dynamics and the role of individual ion channels on reentry. In this study, we adopted a reductionist, tissue engineering approach and created a new 2D in vitro model of the healed IBZ. We used optical mapping to characterize the substrate and study the effects of Na+, Ca2+, and K+ channel blockers on reentrant arrhythmia dynamics. We found that this 2D, coculture model resembled the healed IBZ in several architectural and electrophysiological respects. An increased contribution of the L-type Ca2+ current to impulse propagation was observed in cocultures (consisting of electrically uncoupled myotubes mixed with electrically coupled myocytes11) but not in myocyte-only controls. Low doses of nitrendipine (5 µmol/L), an L-type Ca2+ channel (LTCC) blocker, terminated 100% of reentrant arrhythmias, but high doses of lidocaine (200 µmol/L) only terminated 30% of reentry episodes in cocultures.12 These results may help explain the low efficacy of pure Na+ channel blockers in terminating and preventing ventricular tachycardias that occur late after MI.
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Methods |
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We investigated impulse propagation and arrhythmias by performing optical mapping of novel in vitro models of the healed epicardial10 and lateral13 IBZs generated by coculturing human skeletal myotubes (SkMs) with neonatal rat ventricular myocytes (NRVMs). We used SkMs to simulate fibrosis seen in the healed IBZ because they: (1) lack gap junctions unlike myofibroblasts that express connexin (Cx)43 and Cx4514,15; (2) assume a linear morphology that resembles fibrosis seen in the healed IBZ16; and 3) orient neighboring myocytes into bundles, resulting in a nonuniform anisotropic architecture,10,17 a cardinal feature of the healed IBZ.
The healed "epicardial" IBZ was simulated by plating a mixture of 20% SkMs with 80% NRVMs on 21-mm fibronectin-coated plastic coverslips (Figure 1B and 1C), whereas a model of the healed "lateral" IBZ was created by micropatterning a sector (
=120°, 115 mm2; Figure 5A) composed of a coculture of (20% to 30%) SkMs and (70% to 80%) NRVMs adjacent to an NRVM-only region on fibronectin-coated polydimethylsiloxane-treated glass coverslips. Controls for this study consisted of NRVM-only monolayers. Optical mapping ie, micromapping (20-µm spatial and 125-µs temporal resolution) and macromapping (1 mm spatial and 1-ms temporal resolution) were performed after 9 to 11 days in culture. Reentry was induced by rapid pacing; Na+, Ca2+, or K+ channel blockers were added to stable reentry (5 minutes after reentry initiation), and reentry dynamics were analyzed using custom software written in MATLAB.
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-actinin (white) reveal striations resembling those observed in adult myocytes. E, Immunostaining for Cx43 (white) and nuclear labeling with Hoechst (blue) in a coculture. Arrows point to areas of myotubes that have Hoechst-positive nuclei but lack Cx43.An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
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Results |
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Architectural Characterization
We studied NRVM-only controls (n=50) and cocultures (n=50). Figure 1A shows a representative control, NRVM-only monolayer and Figure 1B and 1C shows a representative transmitted light and corresponding fluorescent microscopy image of a coculture where myotubes are labeled with green fluorescent protein. In culture, NRVMs mature to form striated myocytes (Figure 1D), whereas myoblasts mature to form electrically uncoupled myotubes (100 µm to 2 mm in length) that resemble ingrowths of fibrous tissue in the healed IBZ (Online Figure I).10,17 Furthermore, the myotubes orient bundles of myocytes in several directions (Figure 1B and 1C and Online Figure II), resulting in a nonuniform anisotropic structure, which has been observed in the IBZ because of disarray of the usually parallel-oriented fiber bundles by fibrosis and lateralization of gap junctions.10,17 Figure 1E shows Cx43 immunostaining of a coculture that reveals lateralization of gap junctions in myocytes and lack of gap junctions in myotubes (arrows), both of which are normal features for these cell types. Additionally, impulse conduction in the epicardial border zone is often confined to a 2D plane.18
Electrophysiological Characterization
Electrophysiological studies in transplanted and intact human hearts as well as experimental models of healed MIs reveal that the healed IBZ is characterized by decreased conduction velocity (CV),10,19 increased dispersion of CV,19 and easily inducible, stable reentrant arrhythmias,20–23 all of which are reproduced in this in vitro coculture model.
Key electrophysiological features of the healed IBZ that are reproduced in this model are (1) reduction in CV10,19 at a macroscopic scale (Figure 2A) but normal myocyte excitability10 (Figure 2B and 2C); (2) dispersion in CV at a microscopic scale19 (Figure 2D, 2F, and 2G) but negligible directional differences in impulse propagation at a macroscopic scale10 (Figure 2H); (3) susceptibility to functional block (Figure 2I) and reentrant arrhythmias20–23 (Figure 2J and 2K) following rapid pacing. Remarkably, these features were observed in all cocultures that were mapped (n=50 monolayers) but not in controls (Figure 2E).
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Optical mapping revealed that CV was decreased by 77% (Figure 2A; 5.7±1.1 versus 24.4±4.9 m/sec, P<0.001) and action potential duration (APD) was prolonged by 38% (Figure 2L; 143±29 versus 197±39 ms, P<0.04) in cocultures compared to controls on a macroscopic scale. This decrease in CV was primarily attributable to the underlying architecture and not decreased excitability as demonstrated by similar upstroke velocities measured by micromapping (dF/dtmax=0.33±0.02 versus 0.34±0.02 ms–1; P=0.33, n=7 each, controls and cocultures, respectively; Figure 2B and 2C) and similar resting membrane potential (–77mV in cocultures versus –74 mV in controls) by patch clamp in cocultures and NRVM-only controls.
Myotubes oriented the NRVMs into bundles that resulted in rapid conduction when stimulated along the long axis of the bundles (Online Figure III, A) but slower conduction when stimulated in the transverse direction (Online Figure III, B). Additionally, presence of electrically uncoupled myotubes interposed between myocytes produced slowing of CV but not complete block because of overlap between myotubes and myocytes. Figure 2D shows areas of widely spaced isochrones interspersed with significant slowing, evidenced by crowding of isochrones in a region containing myotubes (labeled with green fluorescent protein), as well as circumvention of wave fronts around myotubes. In contrast, isochrones are uniformly spaced in all areas of control (NRVM-only) monolayers as seen in Figure 2E and Online Figure IV, which show propagation along the transverse and longitudinal axis, respectively. Another interesting feature that has been reported in the healed IBZ is directional differences in activation delay, resulting from variable amounts and architecture of fibrosis that predispose to unidirectional block and reentry. Figure 2F illustrates this point: here, stimulation from the right resulted in a 20-ms activation delay, whereas stimulation from the bottom resulted in an increase in the activation delay to 80 ms attributable to large numbers of myotubes between the stimulation and recording sites. Paradoxically, propagation in cocultures was uniform on a macroscopic scale following point stimulation at the center of the monolayer (Figure 2H), suggesting that, as in the case of the healed IBZ, directional differences in propagation induced by myocyte bundles oriented in several directions on a "microscopic" scale canceled out.
Functional block and reentry can be readily induced in hearts with healed infarcts20–23 and in this in vitro model. In fact, reentrant arrhythmias could be initiated in all cocultures by rapid pacing, and 90% of the reentrant arrhythmias sustained for >5 minutes, making them amenable to pharmacological manipulation. Reentry initiation is illustrated in Figure 2I and 2J; here, uniform impulse propagation was observed at 3-Hz pacing (Figure 2H), but localized conduction block was observed at 4-Hz pacing (Figure 2I), culminating in sustained reentry (Figure 2J); pseudo-ECGs of reentry resembled monomorphic VT (Figure 2K).
Effects of Sodium and Calcium Channel Blockade on Spiral Wave Dynamics
Na+ channel blockers like lidocaine and agents like amiodarone that block Na+, Ca2+, and K+ channels are commonly used for the treatment and/or prevention of sustained ventricular tachycardia in patients with prior MI. We were interested in dissecting out the role of individual ion channels on spiral wave dynamics, so we used specific Na+, Ca2+, and K+ channel blockers, rather than amiodarone. In normal myocardium, lidocaine, exhibits use dependent Na+ channel blockade, slows CV, and flattens CV restitution without affecting APD restitution,24 whereas nitrendipine, which produces use-dependent LTCC blockade, should flatten APD restitution but not significantly affect CV restitution.25
In this study, reentry cycle length (Figure 3A) was significantly increased by 79±38% (263±25 versus 446±29 ms, P<0.001, n=18 monolayers) and 30±14% (242±25 versus 309±35 ms, P=0.03, n=16 monolayers) after perfusion with lidocaine (200 µmol/L) and nitrendipine (5 µmol/L), respectively. This increase in reentry cycle length was attributable to a decrease in reentry CV (–28±23%, 6.2±0.4 versus 4.4±0.5 m/sec, P<0.005, n=11; and –13±11%, 8.0±0.7 versus 6.8±0.6 m/sec, n=8, P=0.01; Figure 3B) and an increase in spiral core size, reflected by increase in the spiral tip path length by 207±160% (16.9±2.7 versus 42.6±5.0 mm/cycle, P<0.001, n=13) and 71±42% (16.5±3.9 versus 27.4±5.5 mm/cycle, P<0.001, n=10) for lidocaine and nitrendipine, respectively (Figure 3C and Online Figure V). Additionally, lidocaine increased reentry APD50 by 28±8% (82±2 versus 106±4 ms, P=0.02, n=15), whereas nitrendipine, produced a small decrease in APD50 (2%±3%, 79±2 versus 77±2 ms, P=0.02, n=12; Figure 3D). The underlying mechanism of the observed changes in reentry APD is probably APD restitution,26,27 where APD prolongs in response to an increase in cycle length. A modest prolongation of APD would be expected under lidocaine superfusion given (1) an insignificant change in the APD restitution relationship and (2) a significant decrease in reentry rate. On the other hand, the small decrease in reentry APD with nitrendipine can be explained by a decrease in Ca2+ influx during the plateau phase of the APD attributable to LTCC blockade that is opposed (to a lesser extent) by the increase in reentry cycle length that tends to prolong APD.
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An unexpected finding was that reentry terminated after nitrendipine superfusion for 4 to 5 minutes, but only 
30% of reentry episodes were terminated by lidocaine despite superfusion for >10 minutes.12 Nitrendipine (Figure 4A), but not lidocaine (Figure 4B) induced wave breaks before reentry termination. These results were confirmed by power spectrum analysis,28 which revealed an additional peak following nitrendipine (Figure 4D and 4E), suggesting wave break, whereas a single dominant frequency was seen during lidocaine superfusion (Figure 4C). Also, optical recordings revealed 2:1 block (resembling graded response29) at the recording site corresponding to the wave break (Figure 4F), whereas recording sites distant to the wave break demonstrated 1:1 capture (Figure 4G).
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Potassium Channel Blockade Does Not Terminate Reentry
Potassium channel blockers like sotalol are often used to prevent ventricular tachy-arrhythmias in conjunction with ICD therapy. We evaluated D-sotalol (300 µmol/L), an IKr blocker, and tetraethylammonium (TEA) (20 mmol/L), a nonspecific K+ channel blocker. These agents prolonged reentry APD (Online Figure VI, A; P<0.005) by 7.6±1.9% during D-sotalol and 3.8±0.8% during TEA superfusion but did not affect reentry CL or induce wave breaks (Online Figure VI, B); D-sotalol did not terminate reentry (n=6), whereas TEA terminated 1/6 reentry, an effect probably stemming from insufficient reentry APD prolongation.
Calcium-Dependent Propagation
Next, we investigated impulse propagation and arrhythmias in the sector model that simulates the lateral IBZ (n=7 monolayers; Figure 5A). The coculture region demonstrated slow propagation (CV was decreased by 54±7% [P<0.01]) relative to the control region during normal tyrode perfusion, and conduction block developed in the coculture region within 5 minutes of nitrendipine superfusion (Figure 5B and 5C).
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Spiral Waves With Transient Concave Wavefront
A novel finding observed in these patterned cultures was the induction of stable, spiral waves with transient concave wave fronts, following rapid pacing. This is illustrated in Figure 6B and the Online Movie, where the spiral wave propagates in the counterclockwise direction. Figure 6A (1 through 5) shows the voltage maps following the 5-point stimuli delivered just before induction of these spiral waves.
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Discussion |
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In this study, we designed an in vitro model that reproduces several architectural and electrophysiological features of the healed IBZ (Table). We demonstrated that this model exhibits readily inducible reentrant arrhythmias that are consistently terminated by nitrendipine, an LTCC blocker, but not high doses of lidocaine, a Na+ channel blocker or K+ channel blockers, suggesting increased contribution of the L-type Ca2+ current to impulse propagation attributable to decreased cell–cell coupling. These results may help explain the low efficacy of Na+ channel blockers like lidocaine in terminating sustained ventricular tachycardia in patients with old myocardial infarcts; significantly, these clinical arrhythmias are often terminated by intravenous amiodarone, which when administered acutely, produces use-dependent Na+ and Ca2+ channel blockade, without an appreciable effect on cardiac repolarization.30
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Effects of LTCC Blockade in Cocultures
Impulse propagation in the heart is dependent on active membrane properties determined by the ion channel composition and passive properties determined by architecture, cell size, gap junction function, and distribution.31 In this model, as well as in the healed IBZ, architecture plays an important role in impulse propagation and arrhythmogenesis. Using microelectrode recordings, Ursell et al10 demonstrated that surviving myocytes in the healed IBZ have normal resting membrane potentials and normal action potential morphologies. However, CV is significantly reduced in these regions because of a combination of distortion of myocyte alignment, interstitial fibrosis, and gap junction remodeling. This slow, heterogeneous electric propagation can be located epicardially, endocardially, and/or laterally and produces low voltage, complex, fractionated electrograms32 that are used clinically to identify ablation sites in patients with ventricular arrhythmias.7,33,34
Important insights into arrhythmia mechanisms can be gained by study of impulse propagation and arrhythmia dynamics. However, study of impulse propagation and arrhythmia dynamics in the whole heart is technically challenging, dictating use of reductionist approaches like theoretical and 2D in vitro models. Using a theoretical model, Shaw et al35 demonstrated that as cell–cell coupling decreases, the contribution of the L-type Ca2+ current to action potential propagation increases, whereas that of the Na+ current decreases. In this coculture model, we demonstrate that myocyte excitability is normal but overall cell–cell coupling is reduced because of the presence of electrically uncoupled SkMs interposed between the myocytes. The myotubes also align myocytes into bundles oriented in several directions (Online Figure II) producing a nonuniform anisotropic architecture that contributes to the electrophysiological phenotype. Our micromapping results indicate that on a microscopic scale, impulse propagation is rapid parallel to myocyte fiber direction (Online Figure IV), but slows significantly when it comes across interposed myotubes (Figure 2D and Online Figure VII), producing activation delays and contributing to decrease in macroscopic CV; similar zig-zag activation was observed by Rohr et al36 in cell strands treated with gap junction uncouplers, and in studies on the healed IBZ.19,37 This decreased cell–cell coupling would increase intercellular delays and increase dependence of propagation on the L-type Ca2+ current; this would explain the conduction block observed in the coculture region of the micropatterned sector model following nitrendipine superfusion, and termination of stable reentry with nitrendipine, but not lidocaine.
Reentry Initiation With Rapid Pacing
Clinical and experimental data support reentry as the most common cause of ventricular arrhythmias in healed infarcts. Reentry can be classified into anatomic or functional; mitral isthmus-dependent VT after inferior MI is an example of anatomic reentry38,39: here, the impulse circulates around an anatomic obstacle, the scar.
Functional reentry on the other hand, can be induced even in homogeneous tissues, take the form of spirals and has also been observed in ex vivo whole heart preparations.40 Functional block is essential for reentry initiation; a short wavelength (product of CV and refractory period) favors maintenance of both anatomic and functional reentry by decreasing the size of tissue needed to sustain reentry. Decreased cell–cell coupling combined with nonuniform anisotropic architecture significantly decreases CV,41 thereby decreasing wavelength and thus increasing the likelihood that a reentry circuit or spiral wave would be contained within the IBZ of healed infarcts or a 21 mm coverslip. Rapid pacing or premature stimuli would amplify dispersions in CV and activation times, predisposing to functional block and reentrant arrhythmias. Additionally, heterogeneities would also promote anchoring of spiral waves42 and an ECG pattern of monomorphic VT (Figure 2K). Our experiments reveal that functional block produces wave breaks that precede reentry initiation (Figure 2I and 2J). We hypothesize that electrically uncoupled myotubes or fibrous tissue ingrowths combined with nonuniform anisotropic architecture predispose to areas of source-load mismatch that block at high pacing rates or following premature beats,37,43 resulting in reentry initiation. Slow CV caused by decreased cell–cell coupling also increases the safety factor of propagation,35,44,45 which would promote sustained reentrant arrhythmias.
Mechanisms Underlying Reentry Termination
Reentry termination can occur because of detachment of the wave from an obstacle, increase in reentry APD that promotes head–tail collision, or interruption of the reentry circuit. The limited length of optical recordings (2 to 4 seconds) did not allow us to capture the termination of reentry. We hypothesize that the mechanism of reentry termination with nitrendipine, an LTCC blocker was decreased safety factor for propagation and conduction block in multiple areas of the monolayer; these conduction blocks would decrease the size of excitable medium available for reentry and consequently terminate reentry. This hypothesis was bolstered by our observation of conduction block in large areas of the monolayer after the termination of reentry. Similar results are illustrated in Figure 5C which demonstrates conduction block only in the coculture region of the sector following nitrendipine superfusion.
We speculate that LTCC-mediated conduction block in reentry circuits may help explain clinical studies where Ca2+ channel blockers have been observed to reduce mortality in patients without significant left ventricular dysfunction,46,47 conditions where the negative inotropic effects of Ca2+ blockers may not be detrimental. Furthermore, our findings may also explain our clinical experience where patients with sustained VT and old infarcts often respond to intravenous amiodarone (which mainly inhibits the depolarization phase of action potentials by use-dependent Ca+2 and Na+ channel blockade) but not lidocaine a relatively pure Na+ channel blocker. In the 3D heart, where an increased number of shunt pathways are available for impulse propagation, pure Ca2+ channel blockers may not be effective,48 and a combination of LTCC and Na+ channel blockade49 may be necessary to terminate or prevent reentrant arrhythmias late after MI.
Mechanism Underlying Spiral Waves With Transient Concave Wavefront
We have demonstrated for the first time the presence of spiral waves with transient concave wavefront in biological tissue. This phenomenon has been observed in experimental and computational studies of chemical reactions,50,51 as well as in the FitzHugh-Nagumo computational model16 by imposing inhomogeneous excitability with specific geometric features.
Limitations
The present study has several limitations. NRVMs used in the study are electrophysiologically different from adult cardiac myocytes.52 Nevertheless, previous studies using patch clamp have demonstrated that the biophysical properties of LTCCs in neonatal and adult rat ventricular myocytes are similar,53 although Na+ channel blockade by lidocaine was more pronounced in NRVMs when compared to adult myocytes.54 Hypertrophy and ion channel remodeling55,56 that occurs in the IBZ is not completely replicated in this in vitro model; despite this drawback, we were still able to reproduce important structural and electrophysiological features of the healed IBZ. Photobleaching of di-4-ANEPPS limited our ability to obtain long-term voltage recordings, resulting in our inability to capture termination of reentry during nitrendipine treatment; hence, we had to rely on the last recording (<2 minutes) before reentry termination for spiral wave analysis. Consequently, the actual changes in specific reentry parameters under maximum Ca2+ channel blockade could be larger than those presented in this study. The macromapping system used in this study is a contact fluorescence imaging system that precludes assessment of architecture concurrently with optical mapping. Lastly, we minimized but were unable to fully eliminate fibroblasts in our cultures by 2 preplating steps. Although fibroblasts may affect the electrophysiological properties of cultures, we assume that they are present to a similar extent in cocultures and controls and hence would not affect the validity of our results.
Conclusion
Our results indicate that a mixture of SkMs and NRVMs forms a substrate that reproduces several architectural and electrophysiological features of the healed IBZ. All reentrant arrhythmias in this model were terminated by LTCC blockade, whereas only 30% were terminated by Na+ channel blockade.12 This study highlights the differential effects of Na+ and Ca+2 channel blockers in an in vitro model of the healed IBZ and may help explain the low efficacy of lidocaine in the treatment of incessant VT episodes in patients with healed infarcts. Furthermore, we have shown for the first time the presence of spiral waves with transient concave wavefront in cardiac tissue, suggesting that this novel arrhythmia phenotype may exist in the in vivo setting.
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Acknowledgments |
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We thank Dr. Rachel R. Smith for graciously providing pathology slides of the in vivo healed IBZ.
Sources of Funding
This study was supported by an American Heart Association Scientist Development Grant (to M.R.A.), the Donald W. Reynolds Foundation, NIH grant HL66239 (to L.T.), and NIH grant MSTP T32GM008042.
Disclosures
None.
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Footnotes |
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This manuscript was sent to Michael Rosen, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
*These authors contributed equally to this work. 
Original received April 5, 2008; revision received September 25, 2009; accepted September 29, 2009.
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