UltraRapid Communication
Structure-Related Initiation of Reentry by Rapid Pacing in
Monolayers of Cardiac Cells
Weining Bian, Leslie Tung
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Abstract—This study examines how a zigzag pattern of conduction, a form of structural heterogeneity frequently found in
old or diseased hearts, affects the vulnerability to reentry during rapid pacing. A central rectangular island (8⫻4 mm)
containing a predefined zigzag pattern was created in cultured isotropic monolayers of neonatal rat ventricular myocytes.
Impulse propagation was optically mapped from 253 sites using voltage-sensitive dye and was anisotropic within the
zigzag island. With increasing interval between neighboring transverse connections (a), relative to the distance between
longitudinal strands (b), transverse conduction velocity (CV) decreased to 66⫾6%, 20⫾2%, and 15⫾2% of CV in the
surrounding isotropic region, whereas longitudinal CV increased to 102⫾8%, 113⫾12%, and 131⫾23% for a:b ratios
of 1:1, 1:5, and 1:9, respectively. During rapid pacing, propagation distal to the island was steered from the side of the
island with more transverse connections (“dominant” side) toward the side with fewer connections (“weak” side).
Increased asymmetry in the pattern accentuated this effect, and resulted in increased rate-dependent differences in CV
on the 2 sides. Consequently, a functional obstacle formed on the weak side, followed by development of single loop
reentry. The reentrant wave revolved around a line of block defined by the border of the island. Reentry chirality was
determined by the weak side location, and the pacing rate needed to initiate reentry decreased with increased asymmetry
in the pattern. In conclusion, reentry is readily induced by rapid pacing in confluent cardiac cell monolayers containing
a central and asymmetric island of zigzag conduction. (Circ Res. 2006;98:e29-e38.)
Key Words: reentry 䡲 arrhythmia 䡲 cardiac electrophysiology 䡲 optical mapping 䡲 cell culture
R
eentry is the circus movement of electrical waves in
cardiac tissue and underlies various arrhythmias, including atrial flutter and fibrillation,1,2 and ventricular arrhythmias that follow myocardial ischemia or infarction.3 To
induce reentry in experimental preparations or computer
models, rapid pacing is typically applied.4 Previous studies
have shown that dynamic instabilities may arise during rapid
pacing as a result of restitution kinetics (for action potential
duration [APD] and conduction velocity [CV]) or calcium
cycling and can produce temporal and spatial alternans, local
conduction block, wave break, and reentry-based
tachycardias or fibrillation.5–10 On the other hand, spatial
heterogeneity of active ionic currents in the cell membrane
has been shown to cause wave break and reentry in the
absence of unstable restitution dynamics.11 However, the
possibility that heterogeneity in tissue structure, in and of
itself, may also serve as a locus for the initiation of reentrant
activity during rapid pacing has not been systematically
investigated. Such was the goal of this study. Our particular
interest lies in a frequent and clinically relevant form of
structural heterogeneity—the zigzag pattern that results from
infiltration of fibrotic tissue into the interstitial spaces between myocardial fibers.
Zigzag conduction was first reported by Spach et al12 in
aged human atria and has also been found in diseased hearts
with dilated cardiomyopathy13 or myocardial infarction.14 It
was first visualized by Koura et al15 in old canine myocardium using high-resolution optical mapping. The presence of
zigzag trajectories leads to immensely slowed transverse
conduction perpendicular to the fiber orientation and promotes the vulnerability to reentrant arrhythmias,12,15,16 but the
detailed mechanism of initiation of reentry remains unclear.
The cell culture approach eliminates much of the complexity found in native tissue preparations (eg, presence of
microvasculature) and the tissue injury from excision for an
ex vivo experimental preparation. Methods of patterned
growth have been used to create linear strands of cultured
cardiac cells for the study of impulse propagation at a
microscopic scale17 and have clarified the functional effects
of tissue expansion and branching geometry.18 –20 Using
advanced cell patterning techniques, we designed and created
simplified 2D tissue models consisting of isotropic monolayers of cardiac cells that contain a central region with a zigzag
structure. Optical mapping was used to monitor the macroscopic electrical activity in the cell monolayers, enabling us
Original received November 4, 2005; revision received January 22, 2006; accepted January 25, 2006.
From the Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, Md. Current address for W.B.: Department of Biomedical
Engineering, Duke University, Durham, NC.
This manuscript was sent to Michael R. Rosen, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to Dr Leslie Tung, Department of Biomedical Engineering, The Johns Hopkins University, 720 Rutland Ave, Baltimore, MD 21205.
E-mail ltung@bme.jhu.edu
© 2006 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000209770.72203.01
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March 3, 2006
to study how this type of structural heterogeneity contributes
to pacing-induced initiation of reentry.
Materials and Methods
Cell Culture
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All experiments involving animals conformed to the protocols in the
Guide for the Care and Use of Laboratory Animals (NIH Publication
No. 85-23, Revised 1996), and the animal protocol was approved by
the Johns Hopkins Animal Care and Use Committee. Cardiac cells
were dissociated from ventricles of 2-day-old Sprague–Dawley rats
(Harlan, Indianapolis, Ind) using trypsin (Amersham Life Sciences,
Arlington, Ill) and collagenase (Worthington, Lakewood, NJ) and
resuspended in M-199 culture medium (Life Technologies, Rockville, Md) supplemented with 10% FBS (Life Technologies) as
previously described.21 Approximately 1⫻106 cells were plated on
prepared 22-mm diameter circular cover slips. At day 2 following
cell plating, serum was reduced to 2% to reduce proliferation of
noncardiac cells. Such a reduction has been shown to have no
adverse effects on cell size, cellularity index, capture rate,
connexin-43 expression, or conduction velocity.22
Microcontact Printing
Directed cell growth following a predefined zigzag pattern was
achieved using a modified method of microcontact printing of
adhesive proteins.23 Briefly, desired patterns were produced on
3-inch silicon wafers (Ultrasil, Hayward, Calif) using SU-8 photoresist (MicroChem, Newton, Mass) by standard photolithography.
Degassed polydimethyl siloxane (PDMS) prepolymer mixture (10
part of base and 1 part of curing agent by weight) (Sylgard 182, Dow
Corning, Midland, Mich) was poured on the silicon master. After
overnight baking, the solidified PDMS polymer was peeled off the
master, cut into individual stamps and coated with 50 ␮g/mL human
fibronectin (Sigma, St Louis, Mo) for 1 hour. Twenty-two millimeter
diameter glass cover slips (Fisher Scientific, Pittsburgh, Pa) were
spin coated with PDMS prepolymer mixture (Sylgard 184; Dow
Corning), baked in an oven at approximately 70°C for 1 hour and
treated with UV-generated ozone (UVO) for 8 minutes (PSD-UV
system, Novascan Technologies Inc, Ames, Iowa) for sterilization
and increased hydrophilicity. Within 20 minutes after UVO treatment, the fibronectin-coated stamps were washed with deionized and
distilled water and applied to the PDMS surface for 30 to 45 minutes.
The cover slips were then dipped in 0.2% (wt/vol) Pluronic F-127
(Molecular Probes, Eugene, Ore) for 20 minutes to block the regions
uncovered by fibronectin. After rinsing with PBS, the cover slips
were ready for cell plating.
Experimental Setup
Cover slips with cell monolayers were placed in a custom-designed
chamber filled with warm (T⫽36⫾0.5°C) oxygenated Tyrode’s
solution (in mmol/L: 135 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 0.33
NaH2PO4, 5 HEPES, 5 glucose), stained with voltage-sensitive dye
di-4-ANEPPS (10 ␮mol/L) (Molecular Probes) for 5 minutes, and
continually superfused with Tyrode’s solution afterward. A plexiglass cover was placed over the chamber to minimize motion artifact
resulting from solution flow, and a point stimulus was delivered by
a unipolar platinum electrode through one of several holes drilled in
the cover. A field stimulus was delivered by 2 parallel platinum line
electrodes spaced 2.7 cm apart. Transmembrane potentials of cardiac
cells were visualized and monitored by an in-house contact fluorescence imaging (CFI) system that has been previously described24,25
and has now been upgraded as follows. An array of 253 optic fibers
arranged in a 17-mm-diameter hexagon was placed directly under the
experimental chamber to collect the optical fluorescent signals. A red
filter made of a No. 1 glass coverslip spin coated with 3 layers of red
photosensitive resin (PSCred; Brewer Science, Rolla, Mo) was
placed at the bottom of the chamber to block the excitation light and
pass the red emission signal. An LED light source26 consisting of 26
high power green LEDs (Kingbright, Taipei, Taiwan) with an
interference filter (530⫾25 nm) delivered excitation light from
Figure 1. Optical action potential traces recorded during 3-Hz
point pacing at the top edge of the monolayer (viewed from
above). A, Overview of the 253 optical recording sites and the
relative position of the region with zigzag pattern. The gray rectangle (8 mm width, 4 mm height) denotes the region with zigzag
pattern (drawn to scale). Action potential traces recorded over
2-sec interval are shown from sites a, b, and c in the surrounding isotropic region and sites d, e, and f in the zigzag region. B,
Tissue structure of the zigzag region, shown by phase contrast
microscopy. Left image exhibits an overview of the zigzag pattern in the 8⫻4 mm rectangular region. Structural details inside
the black rectangle are enlarged in the right image. The ratio a:b
was defined as the ratio of the interval between neighboring cell
strands (a, measured as center-to-center distance) to the distance between neighboring transverse connections (b). As
exhibited, a⫽300 ␮m, b⫽1500 ␮m, a:b⫽1:5, width of the cell
strand⫽100 ␮m.
above the chamber. Optical signals were low-pass filtered at 320 Hz,
amplified, and sampled at 1 kHz by four 64-channel, 16-bit A/D
acquisition boards (Sheldon Instruments, San Diego, Calif). Data
were displayed and analyzed using software written in Visual C⫹⫹
(Microsoft, Redmond, Wash), LabVIEW (Texas Instrument, Dallas),
and MATLAB (MathWorks, Natick, Mass). A high-resolution
charge-coupled device camera (Hitachi, Tokyo, Japan) with a
50 mm, f/1.4 lens with no. 1 and no. 2 close-up lenses (Nikon,
Tokyo, Japan) was placed above the chamber to capture images of
the cell monolayer and the optical bundle. The images were then
superimposed to verify the position of the optical recording sites
relative to the pattern in the monolayer.
Experimental Protocol
Before recording, the cover slips were carefully inspected using light
microscopy to exclude any monolayers with broken cell strands,
transverse connections lying out of place in the central patterned area
or underconfluence of cells in the surrounding region. After the
cover slip was placed in the experimental chamber, the central zigzag
pattern was moved to the center of the optical recording area and
aligned horizontally with the rows of recording sites, as shown in
Figure 1A. For measurement of the transverse and longitudinal
conduction velocity (TCV and LCV) in the zigzag region, a 3-Hz
Bian and Tung
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point stimulus was delivered at the top or bottom side (viewed from
above), and left or right side of the monolayer, respectively. To
induce reentry by rapid pacing, the point electrode was placed on the
top or bottom side of the monolayer, so that the excitation wave
propagated transversely across the cell strands in the zigzag pattern.
The excitation threshold was first determined by delivering several
test pulses, and then the stimulus intensity was increased to 1.5⫻
threshold for the remainder of the experiment. A train of 30 pulses
was delivered through the point electrode starting at 3 Hz and
incremented by 1 Hz up to 5 Hz and 0.5 Hz above 5 Hz until reentry
was induced or 1:1 capture (1 stimulus: 1 response) was lost (0.5 Hz
above the maximum capture rate [MCR]). A 2-sec recording was
made at the end of the pulse train that included the last few pulses
and the period immediately following termination of pacing. If
reentry was induced, 2-sec recordings were made at 1 minute, 5
minutes, and 10 minutes after initiation to check the stability of the
reentry. Single or multiple field stimuli were then delivered to
terminate the ongoing reentry. If the reentry was successfully
terminated, the pacing train was reapplied at a rate 0.5 Hz higher than
the previous reentry induction rate (RIR) and then incremented by
0.5 Hz for the determination of MCR. This cycle was repeated until
either loss of capture and identification of MCR was obtained, or
reentry could not be terminated by field stimuli. If MCR was less
than 7 Hz, pinacidil (50 ␮mol/L), a KATP channel opener, was used
to accelerate repolarization, shorten the effective refractory period
and raise MCR (in 8 of 31 monolayers), so that too low an MCR
would not become a confounding factor that could influence the
reentry initiation outcomes (by not permitting high pacing rates to be
applied). Dynamic CV restitution relations were obtained by temporally averaging the last 4 beats of the 30 pulse stimulus trains applied
at the stepwise increasing rates.
Data Analysis
The raw optical signals were detrended by subtracting a fitted
second-order polynomial curve and then low-pass filtered with a
fourth order elliptical filter. Isopotential and isochrone maps were
generated from the processed signals. The activation time was
defined as the instant of maximum positive slope during the
depolarization phase of the action potential. CV was measured along
4 to 5 manually selected paths and averaged spatially and temporally
over 3 to 4 beats. LCV and TCV in the zigzag pattern were
normalized to the CV in the surrounding isotropic tissue (ICV). RIR
for each monolayer was normalized to MCR for that monolayer.
Structure-Related Initiation of Reentry
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monolayers (averaged P⫽0.53⫾0.34). The zigzag structure is
shown in detail in Figure 1B, and the ratio of the separation
between neighboring cell strands (a) to the distance between
neighboring transverse connections (b) was set to 1:1
(a⫽300 ␮m, b⫽300 ␮m), 1:5 (a⫽300 ␮m, b⫽1500 ␮m), or
1:9 (a⫽300 ␮m, b⫽2700 ␮m), with the width of the cell
strand⫽100 ␮m in all cases. These structures have physical
dimensions and patterns intended to mimic the zigzag structures found in infarcted or aged myocardium, albeit detailed
morphological information is limited.14,15 Isochrone maps
derived from the optical signals are shown in Figure 2A and
2B for point stimuli at the top and right sides of the
monolayer. In Figure 2A, it can be seen that the paced wave
front slows maximally in the transverse direction of the
zigzag pattern when it encounters the proximal edge of the
pattern and then accelerates afterward as the result of the
invasion of action currents from the adjacent normal regions
on the left and right sides. In Figure 2B, a small increase in
CV is evident along the longitudinal midline of the zigzag
region, because of less electrotonic loading imposed by the
adjacent normal regions at the upper and lower sides. Figure
2C and 2D summarize LCV, TCV, and the anisotropic ratio
of conduction (AR⫽LCV/TCV) in the zigzag pattern for the
different a:b aspect ratios. TCV and LCV were normalized to
the velocity measured in the surrounding isotropic tissue in
the same monolayer (average ICV⫽17⫾4 cm/sec, n⫽38).
TCV decreased significantly with aspect ratio (0.66⫾0.06,
0.20⫾0.02, and 0.15⫾0.02, for a:b⫽1:1, 1:5, and 1:9, respectively, P⬍0.0001), with significance for all pair-wise combinations. LCV increased slightly but significantly with aspect
ratio (1.02⫾0.08, 1.13⫾0.12, and 1.31⫾0.23 for a:b⫽1:1,
1:5, and 1:9, respectively; P⫽0.0004) with a significant
difference for 1:1 versus 1:9 ratios. Consequently AR increased significantly (1.5⫾0.2, 5.8⫾0.8, and 8.7⫾1.3, for
a:b⫽1:1, 1:5, and 1:9, respectively, P⬍0.0001), with significant differences for all pair-wise comparisons, as shown in
Figure 2D.
Statistical Analysis
Data were expressed as mean⫾SD and analyzed using Student t test
or 1-way ANOVA followed by Tukey post test. Differences were
considered to be significant when P⬍0.05.
Results
Anisotropic Conduction in the Region With Zigzag
Course of Activation
Rectangular islands (8⫻4 mm) containing a predefined zigzag pattern were created in the center of 22-mm diameter
cultured monolayers of neonatal rat ventricular myocytes
(NRVMs). The position of the zigzag pattern relative to the
253 optical recording sites is depicted in Figure 1A and was
verified by superimposing video images of the pattern and
optical recording bundle. Outside the island, cells were
confluent and randomly oriented. The action potentials outside (traces a through c) and inside (traces d through f) the
island were similar, akin to the observations that action
potentials in healed infarcts are like those in normal tissue,
despite deranged cellular connections.14,27 APD at 80% repolarization (APD80) showed no significant difference for the 2
groups of sites inside and outside the zigzag island in 27 of 34
Structure-Related Asymmetric Impulse
Propagation Enhanced by Rapid Pacing
With point stimulation, wave-front propagation around the
central zigzag island is shown in the isopotential maps of
Figure 3 for a zigzag pattern with aspect ratio of 1:9 (see
movie Bian_1.mpg in the online data supplement, available at
http://circres.ahajournals.org, for full animation of Figure 3).
At a 3-Hz pacing rate (Figure 3A), propagation was symmetric around the vertical axis bisecting the island (Map 3). At a
7-Hz pacing rate (Figure 3B), propagation was nearly symmetric, but the wave front on the right side became slightly
more advanced compared with that on the left (Map 3). At a
pacing rate of 9 Hz (Figure 3C), the difference in propagation
times down the 2 sides was even more evident (Map 3). This
difference in propagation was observed only with patterns
with aspect ratios of 1:9, and not with ratios of 1:1 or 1:5.
Closer examination of our patterns revealed a subtle but
significant difference between the left and right sides of the
pattern (Figure 1B). It can be seen that the transverse
connections were not perfectly symmetric with respect to the
central vertical axis of the pattern, and that 1 side (in this case,
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Figure 2. Conduction velocity measurements in zigzag regions with various a:b ratios. A, Isochrone map during 3-Hz point pacing at
the top edge of the monolayer (a:b⫽1:9 in zigzag region). The rectangle indicates the position of the zigzag region. TCV in the region
was measured along the path indicated by the arrow close to the proximal edge where maximum conduction delay occurred. Asterisk
indicates location of stimulus electrode. B, Isochrone map during 3-Hz point pacing at the right edge of the monolayer. LCV in the central region was measured along the path indicated by the arrow passing through the center of the region. C, Changes in LCV and TCV
with varying ratios of a:b. LCVs and TCVs were normalized to ICV in the surrounding region, and values are given in the text. LCVs
increased and TCVs decreased significantly with increases in a:b ratio (P⬍0.0005 for LCVs, P⬍0.0001 for TCVs). Absolute values for
TCVs were 9.8⫾2.3 cm/sec, 3.5⫾1.2 cm/sec, and 2.6⫾0.5 cm/sec for a:b⫽1:1, 1:5, and 1:9, respectively (P⬍0.0001), with pair-wise
significance for 1:1 vs 1:5 and 1:1 vs 1:9 ratios. Absolute values for LCVs were 15⫾3 cm/sec, 20⫾6 cm/sec, and 23⫾4 cm/sec for
a:b⫽1:1, 1:5, and 1:9, respectively (P⫽0.0003), with pair-wise significance for 1:1 vs 1:5 and 1:1 vs 1:9 ratios. D, Increase of anisotropy
ratio (AR) with increase of a:b ratio. AR was calculated as LCV/TCV, and increase was significant (P⬍0.0001). n⫽10 for a:b⫽1:1, 9 for
a:b⫽1:5, and 19 for a:b⫽1:9.
the right) had a greater number of connections compared with
the other side (in this case, the left). We term the 2 sides
to be the “dominant” and “weak” sides, respectively. The
existence of dominant and weak sides has little influence at
low or high pacing rates for aspect ratios of 1:1 or 1:5.
Even with a ratio of 1:9 (Figure 3), propagation was
symmetric at a pacing rate of 3 Hz but became somewhat
asymmetric at 7 Hz and then clearly asymmetric at 9 Hz,
a rate at which CV was greatly slowed and most sensitive
to small differences in source-load conditions. Moreover,
the wave front was always more advanced on the dominant
side of the pattern (in this case, the right side). Also, a
comparison of the time indices for corresponding maps in
Figure 3A through 3C shows an overall slowing of the
wave fronts as pacing frequency increased. Similar results
were observed in 15 other monolayers having the same
pattern. Thus, our results show that asymmetry in microstructure can result in asymmetry in macroscopic propagation that is accentuated at high pacing rates and in
patterns with sparse transverse connections.
Structural Asymmetry and Reentry Induction by
Rapid Pacing
Rapid pacing was applied to monolayers containing central
islands with various zigzag aspect ratios (n⫽10 for a:b⫽1:1,
9 for a:b⫽1:5, and 16 for a:b⫽1:9). No reentry was initiated
when the aspect ratio was 1:1 or 1:5, but single loop reentry
was frequently (13 of 16) formed at a sufficiently high pacing
rate (9.1⫾1.8 Hz, n⫽13) for an aspect ratio of 1:9. Figure 4
depicts 1 such example of reentry induction (see supplemental movie Bian_2.mpg). Sequential isopotential maps in
Figure 4A show that the asymmetric structure not only caused
the wave on the dominant (right) side to propagate around the
zigzag pattern faster than the wave on the weak (left) side, as
seen previously in Figure 3, but also elicited a conduction
block in the weak side of the zigzag region, as indicated by
the pair of white solid lines in Maps 2 and 7. At the same
time, the 2 waves on either side of the zigzag region collided
on the weak side instead of in the center. The collision point
of the 2 waves (denoted by the pairs of opposing white arrows
in Maps 3 and 8) shifted more and more to the weak side with
successive beats. The size and location of the region of
conduction block varied somewhat from monolayer to monolayer but always occurred on the weak side, usually appearing
inside the zigzag region and extending with following beats
outside the region. Occasionally, additional islands of conduction block appeared on the weak side distal to the zigzag
region. Consequently, the wave front on the weak side
became blocked or diverted to the edge of the monolayer,
whereas the wave front from the dominant side was able to
advance through the region of block (which had time to
recover because of its asymmetric location on the weak side)
and collide with the weak-side wave front arising from the
subsequent pacing pulse (Map 9). This resulted in the
annihilation of both waves and left the next wave from
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Figure 3. Isopotential maps during point
pacing. Monolayer containing a central
rectangular region with zigzag pattern
(a:b⫽1:9, a⫽300 ␮m, slightly right dominant, as shown in Figure 5A) was paced
from the top edge (black asterisk
denotes location of the stimulus electrode). A, Monolayer paced at 3 Hz. B,
Monolayer paced at 7 Hz. C, Monolayer
paced at 9 Hz. White dashed line in
maps A3, B3, and C3 denotes the vertical axis of symmetry. Small black arrows
indicate the direction of advancing wave
fronts. Color bar shows relative transmembrane potential normalized from
resting (blue) to overshoot (red) potential.
Trace underneath map is optical recording from a single site denoted by the
small white square. White vertical line in
the trace indicates the recording moment
of the isopotential map, with the timing
shown in upper left corner (time index
has been set to zero in each panel to
coincide with make of stimulus pulse
and appear as 0.252s, 0.432s, and
0.110s in the 3 segments, respectively,
of supplemental movie Bian_1.mpg).
the dominant side free of collision (Map 10) and able to
evolve into a reentrant wave (Maps 11 and 12).
Figure 4B shows the events leading up to the conduction
block in more detail for 6 sites along the conduction pathway
passing through the block site. Responses to the last 17 pulses
of a train of 30 stimuli are shown, as well as reentrant activity
after stimulation was turned off. Conduction proceeded rapidly from site 1 through 6 following each stimulus and
reversed direction during reentry. Successive beats led to a
progressive slowing of conduction from sites 4 to 5, until
conduction block occurred (fourth arrow from left terminating in double line). In the beats leading up to the conduction
block, the amplitudes of the action potentials in sites 4 and 5
decreased with each beat and became increasingly delayed
(decremental conduction). Excitation continued to occur distal to the block in site 6 because of lateral propagation around
the area of block and arrival of the wave from the dominant
side. After 3 more beats, the wave from site 6 (originating
from the 29th beat) penetrated the block at site 5 and collided
with the wave from site 4 (originating from the 30th beat, 5th
arrow from left). The next wave from site 6 (originating from
the 30th beat) then commenced reentry. After pacing was
turned off, reentrant activity persisted, with conduction now
proceeding from site 6 to site 1. The relatively longer delay
from site 6 to site 5 (rightmost arrow) was because of pivoting
of the wave front around the lower left corner of the zigzag
region.
To further investigate the impact of asymmetry in the
zigzag pattern on the initiation of reentry, we designed
additional patterns with various degrees of asymmetry. Pattern Z1 (Figure 5A), referred to as slightly asymmetric, is the
pattern already discussed and contained a zigzag structure
(a:b⫽1:9, a⫽300 ␮m) over the entire rectangular island with
few transverse connections. Pattern Z2 (Figure 5B), referred
to as moderately asymmetric, had a zigzag pattern (a:b⫽1:3,
a⫽300 ␮m) with a moderate number of transverse connections in one half of the island. Pattern Z3 (Figure 5C), referred
to as strongly asymmetric, was similar to Z2 except that the
number of transverse connections was very high in half of the
island (a:b⫽1:1, a⫽300 ␮m) (see supplemental movie
Bian_3.mpg for example of reentry development with this
pattern). Pattern S0 (Figure 5D) was structurally symmetric
(a⫽300 ␮m) and served as the control (lacking any transverse connections). The success rate of initiation of reentry is
summarized in Figure 5E. Reentry induction was 100%
successful with patterns Z2 and Z3, but had 3 failures out of
16 monolayers with pattern Z1 (presumably because of the
weak asymmetry) and totally failed with symmetrical pattern
S0. RIRs, normalized in each monolayer to MCR, were
0.98⫾0.03 (n⫽7), 0.86⫾0.07 (n⫽6), and 0.78⫾0.07 (n⫽6)
for patterns Z1, Z2, and Z3, respectively (Figure 5F) and
decreased significantly with an increase in the degree of
structural asymmetry (P⬍0.0001), with significant differences for Z2 versus Z1 and Z3 versus Z1.
As described earlier (Figure 4), the rate-dependent difference in CV between the weak and dominant sides of the
zigzag region was an important factor that enabled reentry to
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Figure 4. Initiation of reentry during rapid pacing. A, Sequential isopotential maps showing the initiation process during and following
the last 2 pulses in a 30 pulse (10ms duration), 11-Hz pacing train. Same monolayer as in Figure 3. The monolayer was paced at 11 Hz
from the top edge (location of electrode denoted by black asterisk). The direction of wave fronts is indicated by black arrows. Conduction block is indicated by a pair of white solid lines. Collision denoted by 2 pairs of opposing white arrows shifted to the left (weak) side
more and more with successive beats (Maps 3 and 8). The dominant wave from the 2.884s pacing pulse evaded collision and evolved
into a reentrant wave (Maps 10 through 12). Trace at bottom of each map is inverted signal from stimulator, showing timing of each
stimulus. B, Formation of functional block on weak side of zigzag region. The isopotential map is the same as Map 2 in A. Crosses in
isopotential map indicate locations of recording sites. Numbers identify sites corresponding to traces shown on the right, with the stimulus train shown above. Red box indicates time interval during which pacing was turned off, and reentrant activity was present. Black
arrows show that the stimulated waves slowed down successively and finally blocked between sites 4 and 5 (fourth arrow from left).
Vertical black dashed line indicates the instant of time corresponding to the isopotential map.
be initiated. Average CVs were calculated from 4 to 5
manually selected paths oriented along the directions of
propagation just outside the weak and dominant sides of the
zigzag region. The dynamic restitution relation was consistently steeper on the weak side at short cycle lengths
compared with the dominant side, in a manner that became
more pronounced with increasing asymmetry from pattern Z1
to Z3 (Figure 6).
The chirality of the reentrant wave was also directly related
to the asymmetry in geometry, as summarized in Figure 7.
Grouping all reentry episodes induced in patterns Z1, Z2, and
Z3 together, when the dominant side of the pattern was on the
right relative to the position of the pacing source, all of the
reentries (19 of 19) were clockwise. Conversely, when the
dominant side was on the left, all of the reentries (12 of 12)
were counterclockwise. These results also correlated in 100%
of the cases with the location of the collision points and
conduction block being on the weak side (left for dominant
right side and right for dominant left side) during reentry
induction.
Sustained Reentry With a Line of Block
All of the induced reentries were stable and lasted for at least
10 minutes (the time of observation). The reentrant wave
revolved around a line of block that was always located at
either the distal or proximal edge of the rectangular island
containing the zigzag pattern as shown in Figure 8 (also see
supplemental movie Bian_2.mpg). It can be seen that the
length of the line of block was defined by the length of the
edge of the island. The pivot points of the reentrant wave
were exactly at the corners of the island (Maps 2, 3, 6, and 7),
where the wave front could spread rapidly in the transverse
direction. Also note that when the wave front traversed the
inner edge of the island (Maps 1, 2, 7, and 8), the segment
inside the island moved more quickly than that outside the
island, giving a protrusion-like appearance to the wave front.
This was not the case when the wave front traversed the outer
edge of the island (Maps 3, 4, and 5). Similar findings were
obtained in all of our induced reentries (31 monolayers).
Discussion
Rapid activity can occur in vivo from focal activity,28
microreentry,29 or waves emanating from a “mother rotor.’’30
It has been shown that rapid pacing can induce wave breaks
and reentrant activity, primarily from the viewpoint of dynamic instabilities arising from rate-dependent kinetics or
calcium cycling5–9 or spatial gradients in repolarization prop-
Bian and Tung
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Figure 5. The impact of asymmetric structure on reentry induction. Patterns with different degrees of structural asymmetry are
shown in A through D. A, Zigzag pattern Z1 (slightly dominant
right side) with a:b ratio of 1:9 (a⫽300 ␮m) covering the whole
rectangular region. B, Zigzag pattern Z2 (moderately dominant
right side) with a:b⫽1:3 (a⫽300 ␮m) covering only the right half
of the rectangular area, and parallel strands in the left half. C,
Zigzag pattern Z3 (strongly dominant right side) with a:b⫽1:1
(a⫽300 ␮m) covering only the right half of the rectangular area,
and parallel strands in the left half. D, Control pattern S0, containing only parallel cell strands 300 ␮m apart in the rectangular
area with no transverse connections. E, Summary of success
rate of reentry initiation in different patterns. Success rate is
expressed as the number of monolayers in which reentry was
successfully induced divided by the total number of monolayers
in the study containing that pattern. F, Variation of RIR with type
of zigzag pattern. RIR was normalized to MCR for 1:1 response
and decreased significantly as the degree of structural asymmetry increased (P⬍0.0001, 1-way ANOVA). n⫽7 for Z1, n⫽6 for
Z2, and n⫽6 for Z3. Absolute values for RIRs were 9.1⫾1.8 Hz
(n⫽13), 7.6⫾1.9 Hz (n⫽8), and 7.3⫾1.2 Hz (n⫽10) for patterns
Z1, Z2, and Z3 respectively.
erties.11,31,32 On the other hand, the gradients of conduction
and conduction block necessary for the development of
reentry can also arise from other forms of nonuniformity
present in a tissue substrate, including heterogeneity in
cellular coupling and gap junctional proteins29,33,34 or variations in tissue structure at a cellular level.35 Our experimental
results show that (1) structural heterogeneity at the microanatomical level can play an integral role in the development of
reentry; (2) for zigzag structures, slow conduction and asymmetry in the zigzag pathways are key contributing factors;
and 3) the resulting reentry will anchor to the site of structural
discontinuities. Regions of precisely defined microstructure
in the form of a zigzag pattern create a substrate that allows
the reproducible induction of reentry by rapid pacing. Zigzag
activation supports very slow transverse conduction (down to
Structure-Related Initiation of Reentry
e35
Figure 6. CV restitution relations for the dominant and weak
sides just outside the zigzag region. Velocity was measured perpendicular to the isochrone lines. Restitution was obtained
using a dynamic protocol (step increases in pacing rate for 30
beats). n⫽11 for pattern Z1, 8 for Z2, and 9 for Z3. The curves
were fitted by the double exponential function y⫽a 䡠 ebt⫹c 䡠 edt.
R values for the dominant and weak sides, respectively, were
0.976 and 0.985 for Z1, 0.992 and 0.977 for Z2, and 0.972 and
0.954 for Z3. Asterisks indicate the frequencies where CV on
the dominant and weak sides differed significantly (P⬍0.05,
paired Student t test). Statistical analysis was not conducted on
the leftmost points which included only 1 sample.
2.6 cm/sec for aspect ratio of 1:9, which is comparable to that
found in periinfarcted tissue,36 old myocardium,15 and infarcted muscle16), which is considered to be important for the
initiation of reentrant arrhythmias.14,36 However, our experiments also suggest that a certain degree of structural asymmetry in the zigzag pattern is necessary for reentry to be
readily initiated. The asymmetry contributes after many beats
Figure 7. Dependence of reentry chirality on asymmetry of the
zigzag structure. The rate of occurrence is displayed as the
number of monolayers in which clockwise or counterclockwise
reentry was induced divided by the number of monolayers with
left or right dominant side. Reentries induced in patterns Z1, Z2,
and Z3 are grouped together. Asterisk indicates position of
stimulus electrode.
e36
Circulation Research
March 3, 2006
Figure 8. Sequential isopotential maps
showing the sustained reentrant activity.
These maps are a continuation of the
same episode shown in Figure 4. The
reentry wave revolved around a line of
block located on the longitudinal interface between the anisotropic central
island (containing the zigzag structure)
and the surrounding isotropic region.
Time index has been reset to 0 at the
instant of time of Map 1.
Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016
to the development of a functional block on the weak side,
typically inside and with subsequent beats, outside the zigzag
region.
Anisotropic Conduction Arising From
Zigzag Activation
Our results support the notion that a zigzag course of
activation underlies the abnormally slow transverse conduction in fibrotic tissue caused by aging, cardiomyopathy, or
myocardial infarction.13,14,33 At the same time, longitudinal
conduction in the zigzag region can be near normal, with
velocity comparable to or higher than that of randomly
oriented cells (isotropic tissue). This results in a very high
degree of anisotropic conduction. The highest aspect ratio
(1:9) in the zigzag pattern resulted in a transverse CV and
anisotropy ratio that respectively were much slower and
higher than that reported by Fast and Kleber37 and Bursac et
al38 in anisotropic monolayer cultures of cardiomyocytes.
Bursac et al found that with increasing AR, LCV increased
and TCV decreased in cultures with small ARs (⬇1.3 to 3.7),
and both LCV and TCV decreased in cultures with large ARs
(⬇3.5 to 5.6). In contrast, we found an increase in LCV and
decrease in TCV over the full and higher range of ARs (1.6
to 8.8) in the zigzag structure. The increased LCV in the
zigzag pattern compared with CV in the surrounding isotropic
tissue was primarily attributable to cell coalignment and
elongation in strands that comprised the zigzag pattern.38 The
trend of a decrease in LCV when the ratio of a:b decreased
can be explained by the conduction slowing effect of a
branching tissue geometry as demonstrated by Kucera et al.20
In their experiments, conduction was significantly slower in
cell strands with multiple branch points compared with those
with just a single branch point, and in strands with interbranch distance of 150 ␮m compared with those with
distance of 300 ␮m. Similarly, longitudinal propagation
along a zigzag pattern with a shorter distance between
neighboring transverse bridges encounters more branch
points per unit length and therefore results in a slower CV.
Structural Asymmetry, CV Restitution, and
Reentry Induction
In our experiments, the slowed transverse conduction of the
zigzag region separated the advancing wave front into two
halves. In control experiments (pattern S0), conduction slowing was symmetric in both halves, so reentry initiation could
not be achieved. However, the introduction of asymmetry
into the zigzag structure led to different slowing of the 2
halves of the advancing wave front, an effect that was
enhanced with an increase in pacing rate and with increased
number of pulses. As demonstrated by Lammers et al.39 rapid
pacing induces a higher degree of inhomogeneity in CV
compared with single or multiple premature beats. Our results
demonstrate that the property of CV restitution (rate dependence of CV) as measured in tissue arises not only from
cellular membrane properties but also from the tissue structure. At low pacing rates, CVs on the dominant and weak
sides of the zigzag pattern are about the same, but at high
pacing rates, CV on the weak side is lower than that on the
dominant side because of fewer transverse connections (Figure 3). Therefore, the weak side has a CV restitution curve
that is steeper than that of the dominant side, with a difference
that increases with increasing asymmetry in the zigzag
pattern (Figure 6). The formation of conduction block on the
weak side of the zigzag pattern (Figure 4B) sets the stage for
the development of reentry having a preset chirality, and
appears to be the result of the steepened slope of CV
restitution that occurs at high pacing rates.
The possibility exists that the conduction block is also the
result of discordant alternans arising from intrinsic repolarization gradients that are enhanced in the presence of rectangular structural barriers, as shown by Pastore and Rosenbaum.40 Although we cannot entirely rule out gradients in
repolarization, our isotropic monolayers of NRVMs possess
Bian and Tung
Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016
macroscopically homogeneous ionic membrane properties
and stable cellular dynamics, as judged by smooth and
circular isochrones during point stimulation,38 moderate
slopes in APD restitution,41 the general absence of wave
breaks during rapid pacing of monolayers lacking central
zigzag islands (data not shown), and electrotonic coupling
that will reduce intrinsic gradients among neighboring cells.42
Furthermore, discordant alternans was not apparent in our
experiments, and the structural barrier was not an anatomical
obstacle but rather a region containing conductive pathways
that would be expected to less effective in augmenting
regional repolarization gradients.
Once formed, the region of conduction block persists and
becomes a functional obstacle. The obstacle tends to grow in
size with time and blocks the wave from the weak side of the
monolayer. This allows the wave from the dominant side
(from the nth pacing pulse) to evade the nth wave from the
weak side and is the key event that permits the dominant side
wave to survive. Advancement through the obstacle is possible because of the longer delay time for the dominant side
wave to reach the obstacle. The unidirectional block is similar
to that which occurs in an anatomical circuit by a premature
stimulus that is applied asymmetrically on 1 side of the
circuit,43 except that here the block evolves over many beats
in the absence of extrastimuli. Subsequently, the nth wave on
the dominant side will either collide with the n⫹1th wave on
the weak side (and repeat with subsequent beats) or, if pacing
has been terminated, evolve into a reentrant wave. As
expected from this scenario, the chirality of the reentrant
wave depends on the asymmetry (right versus left) of the
zigzag pattern (Figure 7). An increase in degree of structural
asymmetry results in a decrease of the pacing rate required for
reentry induction (Figure 5F) and facilitates the onset of
reentry.
Line of Block in Sustained Reentry
The sustained single-loop reentry rotates around a line of
block located along the longitudinal edge of the island
containing the zigzag course of activation. Because this line
of block is fixed at a particular location, it is related to the
anatomical microarchitecture. However, it is different from
conventional inexcitable anatomical obstacles44,45 because
paced wave fronts can pass across the site of block (Figure 3).
It also differs from the functional line of block that occurs in
uniform anisotropic tissue4 or homogeneous46 – 48 isotropic
media because the zigzag line of block remains fixed in
location with a size determined by the width of the zigzag
region. Thus, we see a different type of behavior brought
about by the microanatomical tissue structure. Similarly, lines
of ‘‘apparent block’’ during sustained reentry in the infarct
border zone were described many years ago by Dillon et al
and hypothesized to be the result of nonuniform anisotropy
related to the microanatomical structure.36 Peters and Wit
have speculated that an area of myocardium with enhanced
anisotropy caused by impaired transverse coupling might
form the common central pathway of a reentrant circuit and
define lines of functional block at its longitudinal interface
with the surrounding normal tissue.49 The line of block that
Structure-Related Initiation of Reentry
e37
we observed at the border of the zigzag region is evidence
that supports their hypothesis.
Limitations of the Study
The major limitation of the present study is the simplification
of the 3D tissue architecture in real hearts into a 2D bounded
substrate. Although the central position and minimal size of
the zigzag region lessened the influence of boundaries on
reentry initiation, the initiation mechanism of reentry in 3D
myocardium may not be as straightforward as that shown in
our cultured monolayers of cardiac cells. Moreover, the
portion of the monolayer surrounding the zigzag region
contained randomly oriented cardiomyocytes that yield homogeneously isotropic properties rather than uniformly
anisotropic properties as in real cardiac tissue. This difference
may have enhanced the contrast in functional properties
between the zigzag patterned region and the surrounding area
and facilitated the initiation of reentry even further. Additional experiments, in which the surrounding region of the
monolayer is made to be anisotropic, will be necessary to
resolve this issue.
Conclusion
We have demonstrated the reproducible initiation of reentry
in cultured monolayers of cardiac cells containing a central
region of zigzag structure. We have shown how tissue
microstructure can exert subtle effects on macroscopic behavior, via spatial variation in CV restitution, creation of a
functional obstacle, and formation of a line of block, none of
which is evident during pacing at basal rates. Asymmetry in
the zigzag region, such that 1 side has more transverse
connections than the other side, increases the vulnerability to
reentry, so that rapid pacing can initiate reentrant waves.
Acknowledgments
This project was supported by NIH grant HL66239. We thank
Christopher Chen for his advice on microcontact printing and
method to coat cover slips with PDMS; Felipe Aguel, Zhan Yang
Lim, Yelena Nabutovsky, Barun Maskara, Marvin Chang, Alexander
Jow, and Daniel Loeser for their assistance with development of the
mapping system; Roland Emokpae for supplying the cardiac cells,
and Yibing Zhang for assistance with the movies in the
on-line supplement.
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Structure-Related Initiation of Reentry by Rapid Pacing in Monolayers of Cardiac Cells
Weining Bian and Leslie Tung
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Circ Res. 2006;98:e29-e38; originally published online February 9, 2006;
doi: 10.1161/01.RES.0000209770.72203.01
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