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Am J Physiol Heart Circ Physiol 294: H58-H65, 2008. First published October 26, 2007; doi:10.1152/ajpheart.00631.2007
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Region of slowed conduction acts as core for spiral wave reentry in cardiac cell monolayers

Joyce W. Lin, Libet Garber, Yue Rosa Qi, Marvin G. Chang, Joshua Cysyk, and Leslie Tung

Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, Maryland

Submitted 1 June 2007 ; accepted in final form 22 October 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pathophysiological heterogeneity in cardiac tissue is related to the occurrence of arrhythmias. Of importance are regions of slowed conduction, which have been implicated in the formation of conduction block and reentry. Experimentally, it has been a challenge to produce local heterogeneity in a manner that is both reversible and well controlled. Consequently, we developed a dual-zone superfusion chamber that can dynamically create a small (5 mm) central island of heterogeneity in cultured cardiac cell monolayers. Three different conditions were studied to explore the effect of regionally slowed conduction on wave propagation and reentry: depolarization by elevated extracellular potassium, sodium channel inhibition with lidocaine, and cell-cell decoupling with palmitoleic acid. Using optical mapping of transmembrane voltage, we found that the central region of slowed conduction always served as the core region around which a spiral wave formed and then revolved following a period of rapid pacing. Because of the localized slowing in the core region, we observed experimentally for the first time an S shape of the spiral wave front near its tip. These results indicate that a small region of slowed conduction can play a crucial role in the formation, anchoring, and modulation of reentrant spiral waves.

arrhythmia; heterogeneity; cell culture; optical mapping

TISSUE HETEROGENEITY CAN DISRUPT regular heart function and facilitate arrhythmia by causing conduction block and acting as functional obstacles that stabilize reentrant waves. Increased heterogeneity is present in diseased cardiac tissue under pathological conditions such as myocardial ischemia or infarction. Drugs that are expected to be antiarrhythmic (based on their cellular effects) in fact can be proarrhythmic (8, 24), possibly because of enhanced tissue heterogeneity.

The importance of heterogeneity in cardiac arrhythmias is well known (26). Heterogeneity has been studied methodically in computational models using regions with prolonged refractoriness (19), elevated extracellular K+ concentration (1, 27), cell-cell decoupling (6), or simulated ischemia (7, 12, 28). Common among these situations is the localized slowing of conduction, which facilitates the formation of conduction block that is a prerequisite for reentry. An experimental model with a comparable level of control does not yet exist, although cultured cardiac cell monolayers hold great promise in this regard. Indeed, the use of patterned cultures of cardiomyocytes has demonstrated how tissue architecture alone may alter local conduction velocity (9, 16) and, in the form of a central zigzag island, can promote the formation of reentry (4).

For this study we developed a dual-zone superfusion chamber for cultured cardiomyocyte monolayers that produces a well-defined central circular zone surrounded by a defined outer zone. In this way, functional heterogeneities that are nonstructural and common in diseased myocardium may be studied. Unlike native tissue, in which the manner and extent of heterogeneity is typically unknown, well-controlled localized heterogeneities can be created and extinguished within a single experiment. Using optical mapping of transmembrane voltage, we investigated the importance of a small central island of slowed conduction, brought about by reduced resting potential, cell excitability or cell-to-cell coupling, on the formation, stability, and modulation of reentrant spiral waves.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Dual-zone chamber. The chamber used in this study consisted of an acrylic base and a polycarbonate lid that were tightly secured during experimentation with screws and a gasket to produce an airtight seal (Fig. 1A). The base contained a 25-mm-diameter well in which to place the coverslip and a glass bottom consisting of a 30-mm-diameter no. 1 coverslip spin-coated with three layers of PSCred (Brewer Science, Rolla, MO), a red photoresist that acted as a fluorescent filter for optical mapping. The chamber lid was 40 mm in diameter and had a 2.5-mm-thick, 25-mm-diameter cylindrical protrusion that fit snugly into the well with a clearance of 1 mm from the bottom. The lid featured one central and four peripheral inlets as well as an outflow annulus (inner diameter 4.5 mm, outer diameter 6 mm) that served to create two distinct zones of flow, a central zone and an outer zone as depicted in Fig. 1, B and D. Flow through the central inlet was actively controlled by a peristaltic pump (Ismatec ISM829; Glattbrugg, Switzerland). Total outflow from the chamber was controlled by a multisyringe pump (KD Scientific KDS230; Holliston, MA) loaded with eight 30-ml plastic syringes connected to the circular annulus. Inflow to the outer zone was passively provided through inlets connected to elevated solution reservoirs, and its rate was equal to the difference between total outflow rate and the central inflow rate. The central inflow and total outflow pump rates used for experiments were 0.59 and 1.6 ml/min, respectively. Also embedded in the chamber lid were 26-gauge platinum wire electrodes located at a height of ~1 mm above the bottom of the chamber for point, line, or field stimulation (Fig. 1C). Further details of the chamber design as well as results of initial testing can be found in Ref. 13.


Figure 1
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  		Fig. 1. Dual-zone superfusion chamber lid design and photomicrograph of chamber alignment with optical fiber bundle used for mapping. A: chamber configuration. The protrusion of the chamber lid fits into the depression in the base where the coverslip (a) is placed. The floor of the chamber is glass spin-coated with red photoresist (b) and held in place with a rubber gasket (c), metal ring (d), and screws (e). B: 3-dimensional wire schematic of chamber lid design (bottom side up) with view of inflow channels and outflow annulus. C: polycarbonate chamber lid with view of point (f), line (g), and field (h) stimulus electrodes. D: chamber lid placed on fiber bundle. An outline of the transition zone determined by the outflow annulus is shown and separates the central and outer zones of superfusion.

 
Washable blue ink (Quink; Parker Pen, Newhaven, UK) and deionized water were used to visualize the two flow zones as well as any leakage between them. Images were captured at high resolution using a digital camera (Canon Powershot A95; Tokyo, Japan) and analyzed using Photoshop (Adobe, San Jose, CA).

Cell culture. These studies conformed to the protocols of the National Institutes of Heath Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996); the animal protocol used was approved by the Johns Hopkins Animal Care and Use Committee. Neonatal rat ventricular myocytes (NRVMs) were dissociated from 2-day-old Sprague-Dawley rats (Harlan, Indianapolis, IN) and cultured as previously described (4, 5).

Optical mapping. Experiments were performed between days 7 and 9 of culture. Coverslips containing NRVM monolayers were superfused with warmed Tyrode's solution (135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 0.33 mM NaH2PO4, 5 mM HEPES, and 5 mM glucose) containing the voltage-sensitive dye di-4-ANEPPS (10 µM; Molecular Probes, Eugene, OR). After 7 min, the chamber was sealed and the dye was washed out. Action potentials (AP) were optically mapped using a custom-built contact fluorescence imaging system as previously described (18, 25).

Experimental solutions. Tyrode's solution was used to superfuse cells under control conditions. Three agents were applied to the central zone to slow conduction: elevated K+ (cellular depolarization), lidocaine (reduced cellular excitability), and palmitoleic acid (reduced cell-cell coupling). The term "drug" is used to apply to each of them. Palmitoleic acid was prepared with 0.01 or 0.02% DMSO. All chemicals and drugs were obtained from Sigma-Aldrich (St. Louis, MO).

Experimental protocol. The experimental chamber, solution reservoirs, and flow lines were placed in a metal cage that was internally heated with a hairdryer connected to a variable alternating current power supply to keep the environment at a uniform temperature. Chamber temperature was monitored throughout experiments by using a thermocouple inserted at the base of one of the solution reservoirs and was maintained at 37 ± 1°C. Before mapping, a charge-coupled device camera (Hitachi) was positioned above the chamber to check the alignment of the fiber bundle with the chamber (Fig. 1D) as previously described (4). Cell monolayers were visually inspected for defects (gaps in confluency) under the light microscope. A 2-s recording with 2-Hz point stimulation was made at the start of each experiment to assess overall wave propagation. If necessary, rapid overdrive pacing was used to suppress intrinsic focal activity. Coverslips displaying obvious defects, irregular propagation, or interminable spontaneous activity were excluded from the study.

Drug solutions were introduced through the central inlet to superfuse only the central zone and were allowed to flow for 10 min for the effect to equilibrate before any recordings were taken. Conduction velocity (CV) restitution curves were obtained by increasing stimulation rate from 2 Hz in steps of 1 Hz until 1:1 capture was no longer observed or reentry was initiated. If reentry was not induced through rapid pacing, reentry initiation was attempted using an S1–S2 pacing protocol. A pulse train of point stimuli was applied at the maximum capture rate at which 2:1 block did not occur at the pacing site, followed by a single S2 field pulse. The S1–S2 coupling interval was shortened by 10-ms increments until reentry was observed. Recordings used for analysis were taken starting 3 min after reentry induction to ensure that the spiral wave was sustained and its properties had equilibrated. Washout data were collected by disconnecting the peristaltic pump so that the central inlet became a passive outlet controlled by gravity (with an outflow rate of 1.7 ml/min), allowing the normal Tyrode's solution from the outer zone to immediately flood the central zone.

Data analysis. Data were stored, displayed, and analyzed using software written in LabView (Texas Instruments, Dallas, TX) and Matlab (MathWorks, Natick, MA). Raw optical signals were detrended by subtracting a fitted third-order polynomial curve and then smoothed with a five-point median filter before generation of isopotential and isochrone maps. Activation times were identified at the time of maximum positive slope (dV/dtmax) during action potential depolarization (APD). Cycle length was calculated as the average duration between activation times over a 2-s recording. CV was calculated from isochrone maps by measuring differences in activation times. CV values were averaged along three different paths, each equidistant from the stimulus location, for each AP over a 2-s recording. Separate CV measurements were made for channels comprising the central (drug affected) and the outer (drug free) zones (Fig. 1D). Phase maps were generated by the method of time-delay embedding using a time shift equal to the first zero crossing of the autocorrelation function.

For analysis of spiral waves, reentrant wave fronts under drug and washout conditions were determined at similar phases of rotation and aligned so that the arms of the wave fronts overlaid one another. In the usual cases where the spiral wave detached from the central zone after washout, the wave fronts were shifted so that the tips and arms of both curves were aligned. Area differences between the two curves were calculated in Photoshop and then converted to physical dimensions.

Data are means ± SE and were analyzed using a paired Student's t-test. P values <0.05 were considered to be significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
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Characterization and optimization of the dual zones. Before the monolayer experiments could begin, separation and independence of flow between the two zones had to be validated. Superfusion with ink in either the outer zone (Fig. 2A) or central zone (Fig. 2, B–D) allowed each zone to be visualized. The response of the central and outer zones to different concentrations of ink is shown in Fig. 2E. During whole coverslip superfusion, light absorption at the central and outer zones was comparable, as indicated by the dashed lines, which are nearly horizontal. When ink was superfused in only the central (shaded circles) or outer zone (open circles), light absorption in the region with ink matched that during whole coverslip superfusion with the same ink solution, whereas absorption in the region without ink remained near 0.


Figure 2
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  		Fig. 2. Profiles of central and outer zones during superfusion with various concentrations of ink. A and C: images with ink in the outer zone only and in the central zone only, respectively, at experimental pump rates (central inflow, 0.59 ml/min; outflow, 1.6 ml/min). B: decrease in size of central zone at low inflow rate (0.44 ml/min). D: increase in size of central zone at high inflow rate (0.78 ml/min). E: independent behavior of the 2 zones at experimental pump rates (central inflow, 0.59 ml/min; outflow, 1.6 ml/min). Light absorption was averaged and normalized from 0 to 1 across regions of interest in central and outer zones of the chamber with concentrations of ink ranging from 0 to 100%. Light measurements from the central and outer zones taken at the same time are connected with a line. Light absorption during whole chamber perfusion is indicated with dashed lines. Ink superfusion in only the central region is indicated by the shaded circles, whereas ink superfusion in only the outer region is indicated by open circles.

 
The images for the central zone (Fig. 2, B–D) show that the size of the central zone varies with the inlet flow rate. The size was measured by calculating average light absorption as a function of radius from the center of the chamber (Fig. 3). As before, ink and water were independently flowed into each zone. In the first set of measurements, total outflow rate was held constant at 1.6 ml/min while central inflow rate was varied from 0.26 to 1.0 ml/min. The two borders of the transition region between zones were measured with ink only in the outer zone (Fig. 3A) or only in the central zone (Fig. 3B). In both cases, decreasing inflow caused both borders to shift toward smaller radii, whereas increasing inflow caused an opposite shift. In a second set of measurements, the central inflow rate was held constant at 0.59 ml/min while the annular outflow rate was varied from 0.8 to 2.4 ml/min. This time, however, an increase in outflow rate caused the outer border of the transition zone to shift toward smaller radii (Fig. 3D) but had no effect on the inner border (Fig. 3C). Thus pump rates of 0.59 ml/min for central inflow and 1.6 ml/min for total outflow (Fig. 3, dashed curves) were judged to be optimal and were used throughout the cell monolayer experiments. At these settings, the center of the transition zone was at 2.5 mm, giving a central zone diameter of 5 mm.


Figure 3
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  		Fig. 3. Transition region characterization for various inflow/outflow rates. Light absorption was calculated as a function of chamber radius (distance from center in mm) for ink superfused in the outer zone only (A and C) or in the central zone only (B and D). Shaded region represents width of annular outflow groove (inner radius, 2.25 mm; outer radius, 3 mm). The pump settings used throughout experimentation are indicated with a dashed curve (central inflow, 0.59 ml/min; outflow, 1.6 ml/min).

 
Central region of slowed conduction. Three drug conditions, 16.2 mM extracellular K+, 200 µM lidocaine, or 10 µM palmitoleic acid, were applied to the central zone to create a localized region of slow conduction. Point stimulation was applied at the edge of each coverslip, and CV and APs were measured in both the central (drug affected) and outer (drug free) zones. Figure 4 shows isopotential maps of wave propagation from point stimulation under control conditions and with drug in the center as well as averaged AP traces in the two zones. All three drug conditions slowed CV in the central zone and created a concavity in the wave front. APD80 increased slightly with elevated K+ and palmitoleic acid but was not altered by lidocaine.


Figure 4
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  		Fig. 4. Effect of drug on wave front propagation and action potential. Isopotential maps during 2 Hz pacing before (control) and after 10 min of drug superfusion with 16.2 mM K+ (A), 200 µM lidocaine (Lido; B), and 10 µM palmitoleic acid (PA; C). Hyperpolarized regions of the coverslip are coded in blue, depolarized regions are in red, and the direction of wave front propagation is indicated by the arrows. Black circle indicates the perimeter of the 5-mm central zone. Action potential (AP) traces averaged from 36 channels lying in a ring in the middle of the outer (control) zone (o; black traces) are superimposed with traces averaged from 19 channels in the central (drug affected) zone (c; red traces).

 
To quantify further the stratification of tissue properties between the central and outer zones, the intrinsic rate-dependency (restitution relations) of CV was measured under the three drug conditions in the central zone and found to be significantly different from those in normal Tyrode's solution in the outer zone (Fig. 5). For elevated K+, CV at 2-Hz pacing decreased to 66 and 46% of control values at 8.1 and 16.2 mM K+, respectively. For 200 µM lidocaine, CV decreased to 61%, and for 10 µM palmitoleic acid, CV decreased to 41%. All decreases in CV were statistically significant. Also shown are control restitution curves for normal Tyrode's solution flowed through the central zone (Fig. 5A). As expected, the curves for the central zone closely matched those of the outer zone and were not statistically different.


Figure 5
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  		Fig. 5. Conduction velocity (CV) restitution curves for normal Tyrode's solution in central zone (CZ) and normal Tyrode's in outer zone (OZ) (n = 7) (A), 8.1 (n = 4) or 16.2 mM (n = 10) extracellular K+ in CZ and normal Tyrode's (5.4 mM K+) in OZ (n = 14) (B), 200 µM Lido in CZ and normal Tyrode's in OZ (n = 6) (C), and 10 µM PA in CZ and normal Tyrode's in OZ (n = 7) (D). Curves were normalized to following 2-Hz baseline values obtained from OZ for each case: 23.5 (A), 22.2 (B), 24.4 (C), and 21.5 cm/s (D). *P < 0.05, significantly different from OZ value at same cycle length.

 
Role of central zone of slowed conduction on spiral wave induction. Once a region of slow conduction was formed at the center of the monolayer, sustained reentries could be induced simply by rapid pacing in ~65–71% of monolayers, depending on the drug. During stimulation at low pacing rates, propagation with elevated K+ in the central zone occurred symmetrically around the area of slowed conduction. At higher pacing rates, however, intrinsic heterogeneities within the monolayer became unmasked as indicated by the larger error bars at low cycle lengths in the CV restitution curves of Fig. 5. These heterogeneities may have been augmented by the supranormal loading effects on the wave front at the perimeter of the central zone (Fig. 6A), leading to asymmetry in wave propagation around the central zone. Repeated stimulation produced conduction block on one side of the central zone (Fig. 6B), which ultimately resulted in the formation of a spiral wave (Fig. 6C). A similar pattern of spiral wave formation was observed when lidocaine or palmitoleic acid was flowed into the central zone. In most cases, the spiral wave became anchored to the central zone and was sustained.


Figure 6
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  		Fig. 6. Isochrone maps showing initiation of spiral wave reentry around functional heterogeneity in center (black circle) via rapid pacing from point electrodes located at bottom of map. Arrows indicate direction of wave front propagation. Elevated K+ Tyrode's solution is locally superfused in the center, and at 8-Hz rapid pacing, intrinsic heterogeneity in the coverslip causes the wave to veer toward the right (A). After multiple stimulus pulses, block occurs on the right side. The wave propagates up along the left side of the coverslip and then down the right side after it has recovered (B). The wave then reenters the left side of the coverslips and forms a spiral wave anchored to the central zone (C).

 
Influence of central zone of slowed conduction on spiral wave dynamics. The effect of the central region of slowed conduction on reentry dynamics was clearly evident following drug washout. Figure 7 shows representative phase maps of spiral waves under each drug condition before and after washout. With drug, each reentrant wave front was S-shaped near the tip, i.e., concave in the central drug-superfused zone and convex in the outer drug-free zone. Normal propagation in the central zone was restored after washout, whereupon the wave front curvature reverted from concave back to convex. Wave fronts at similar rotational phases before and after drug washout were overlaid (Fig. 7, right) and analyzed as described in METHODS.


Figure 7
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  		Fig. 7. Examples of area analysis. Representative phase maps and corresponding wave front traces with area differences for 16.2 mM K+ (A), 200 µM Lido (B), and 20 µM PA (C). Spiral waves with drug in the center or after washout are shown at left. The activation wave fronts are superimposed in the graphs at right for drug (red trace) and washout (black trace); grid spacing is 5 mm horizontally and 2 mm vertically. Black areas in phase maps represent noisy channels that were excluded from analysis.

 
Spiral wave analysis is summarized in Table 1. First, rotationally phase-matched wave fronts during control for each drug (n = 9) were compared between successive rotations to determine the baseline area difference. This value was small, ranging from 0.1 to 0.3 mm2. Average area differences and changes in spiral wave frequency were then calculated over a sample size of n = 9 for each drug. With elevated K+, spiral waves had a longer cycle length and slowed by an average of 32.0 ± 4.8%, with an average area difference at the tip of 10.9 ± 2.1 mm2. With lidocaine, spiral waves slowed by an average of 21.7 ± 5.3% and had an average area difference of 7.3 ± 2.0 mm2. With palmitoleic acid, spiral waves slowed by an average of 14.1 ± 3.5% and displayed an average area difference of 3.5 ± 0.9 mm2. Changes in area and cycle length upon washout were statistically significant for all three drug conditions and mirrored one another. Together, these results suggest that increasingly concave curvature of the wave front at the spiral tip, without a change in curvature of the arms of the spiral wave, is causally related to slowing of rotation of the spiral wave.


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  		Table 1. Effect of washout on spiral waves pinned to drug-superfused center

 
Immediately after washout, cycle length decreased with all three drugs but showed some variability in absolute values, perhaps because of differing degrees of drug reversal, as has been reported previously (22). After 10 min, spiral waves detached in all cases of elevated K+, in 4 of 9 cases of lidocaine, and in 4 of 9 cases of palmitoleic acid (Table 1). Upon detachment, the reentry core would drift and the spiral wave would eventually meander off the coverslip and self-terminate (Fig. 8).


Figure 8
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  		Fig. 8. Voltage maps showing drift and termination of spiral wave when functional heterogeneity in the central zone was eliminated (A–H). Less than 2 min after washout of elevated K+ from the central zone with normal Tyrode's solution, the tip of the spiral wave left the central region and drifted into the edge of the monolayer, thereby terminating the reentry.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
With the use of a novel dual-zone superfusion chamber, a region of local heterogeneity 5 mm in size could be created and washed out in cultured cardiac cell monolayers. Regional slowing of conduction was achieved with elevated extracellular K+, lidocaine, or palmitoleic acid. Optical mapping confirmed that these conditions are sufficient to cause local conduction slowing and facilitate the formation of reentrant spiral waves following a period of rapid pacing. In addition, the central area of slowed conduction provides stability to the spiral wave by forming a stationary core region that acts as an anchor. Because the spiral wave tip passes through the core region, reentry cycle length increases and the rate of rotation slows. The special nature of the core region also affects the shape of the spiral wave by reversing the wave front curvature near the tip and producing an S shape. All of these effects are reversible upon washout: the S-shaped wave front disappears, cycle length decreases, and the spiral wave detaches and drifts off the monolayer edge. These data provide clear experimental evidence that a small (5 mm) localized region of slow conduction can play an important role on cardiac spiral wave dynamics and stability and may explain why some antiarrhythmic drugs may, in fact, be proarrhythmic if their effects are not uniformly expressed within the tissue.

Effect of elevated extracellular K+, lidocaine, and palmitoleic acid on wave propagation. Depressed conduction in cardiac tissue contributes significantly to the formation of reentrant arrhythmias. The agents used in this study slow conduction by three different mechanisms: elevated extracellular K+ depolarizes membrane voltage and reduces excitability (23); lidocaine decreases sodium current and alters sodium channel kinetics (3); and palmitoleic acid decouples cells (10). During point stimulation, local superfusion of these three agents resulted in wave front concavities in the central zone where the drug was applied (Fig. 4), similar to the effect shown in modeling studies with an area of regional ischemia (7, 12, 28) or elevated K+ (1). These concavities reflect a local decrease in CV in the central zone compared with the outer zone (Fig. 5). CV was slowed on average by 54% with 16.2 mM extracellular K+, 39% with 200 µM lidocaine, and 59% with 10 µM palmitoleic acid.

Influence of central region of slowed conduction on initiation and stability of spiral wave reentry. After 10 min of local superfusion with drug in the center, rapid pacing was used to initiate spiral wave reentry. The central heterogeneity produces differences in the restitution properties of the central and outer zones (Fig. 5) that result in a localized zone of slow conduction that can assist in spiral wave generation (4). Our data also indicate that the central heterogeneity provides an anchor to which the spiral wave can attach. Spiral waves rotating about a region with depressed conduction remain stably attached to the region and turn more slowly compared with their rate after restoration of conduction in the region (Table 1).

Drift and termination of the spiral wave were observed upon washout for all cases of central superfusion with elevated K+ as well as in approximately one-half of all cases with lidocaine or palmitoleic acid (Table 1). Loss of anchoring likely reflects the effectiveness of drug washout. Spiral waves that were anchored with elevated K+ terminated within 1–2 min of washout, whereas the lidocaine and palmitoleic acid reentries took 2–10 min to terminate.

In addition to aiding in the formation and anchoring of spiral waves, heterogeneity at the core of the reentry can also affect spiral wave characteristics such as the shape of the wave front. A novel experimental finding of this study is the appearance of a concave curvature near the spiral tip for all three drug conditions. Concavity at the tip allows a higher source-to-load ratio and faster local CV (11), which permits the wave front to maintain its rotational speed and carry on despite being in a region of depressed conduction. In the only report of which we are aware (28), an S-shaped wave front can be seen in a computational simulation in which a spiral wave partially invades an area of moderate ischemia to which it is anchored. In our experiments, the S-shape reverted to a purely convex shape upon washout and was quantified by area differences (Fig. 7). The area differences were negligible in the arms of the spiral wave lying in the outer zone and were maximal in the region of altered curvature near the spiral tip.

The area differences were not the same for all three drugs. One may expect that the magnitude of the area difference varies with the dosage of the drug that was used and with the degree of washout. Differences in the mechanisms of conduction slowing and safety factors for propagation (15) among the drugs also may have been contributing factors.

Performance of the dual-zone superfusion chamber. Although it is relatively straightforward to incorporate functional heterogeneity into a computer model, developing such a model experimentally is nontrivial. Geometrically well-defined regions of heterogeneity can be achieved in cell cultures by restricting cell access to the bath by a glass coverslip (20) but are not easily reversed. Several groups have developed flow chambers that can selectively and reversibly superfuse a given area of a cell monolayer with drug (2, 14, 17, 21). However, none of the designs resulted in the creation of a sharply defined, scalable island with uniform properties distinct from the remainder of the monolayer that would be valuable in the study of the initiation or maintenance of reentry.

The dual-zone superfusion chamber presented in this work is capable of producing such an island of heterogeneity that also can be switched on or off. The 5-mm diameter of the central zone is adequate to allow its macroscopic effects on wave propagation or spiral wave reentry to be expressed. The ability to reversibly create a central zone of superfusion allows for the comparison of propagation and reentry under drug and washout conditions in the same preparation. Finally, the size of the central zone can be varied by either changing the diameter of the outflow annulus or varying the relative values of inflow and outflow rates (Figs. 2, A–D, and 3).


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Funding for this work was provided by National Institutes of Health Grants R01 HL66239, R21 RR017073, and R21 EB006171.


   ACKNOWLEDGMENTS
 
We thank Rajesh Babu Sekar and Brett Eaton for providing the cell cultures used in this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Tung, Dept. of Biomedical Engineering, The Johns Hopkins Univ., 720 Rutland Ave., Baltimore, MD 21205 (e-mail: ltung{at}jhu.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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 RESULTS
 DISCUSSION
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E. M. Aliot, W. G. Stevenson, J. M. Almendral-Garrote, F. Bogun, C. H. Calkins, E. Delacretaz, P. D. Bella, G. Hindricks, P. Jais, M. E. Josephson, et al.
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