Journal of Electrocardiology 39 (2006) S2 – S6

www.elsevier.com/locate/jelectrocard

Optical imaging of arrhythmias in tissue cultureB

Leslie Tung, PhD,4 Yibing Zhang, PhD
Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD 21205, USA
Received 28 April 2006; accepted 29 April 2006

Abstract Background: Cardiac cell cultures are becoming an important experimental system of minimal
complexity that captures many of the salient features of myocardial tissue function and are simple
enough that tissue parameters can be controlled systematically. Fundamental mechanisms that
underlie normal and pathological electrophysiology at the tissue level can be studied. Of particular
interest are spiral waves, which underlie many tachyarrhythmias and fibrillation.
Methods: Methods of patterned growth were used to control tissue structure, and contact
fluorescence imaging was used to visualize the spread of electrical waves in confluent monolayers
of neonatal rat ventricular cells stained with voltage-sensitive dye.
Results: Work is summarized regarding anisotropy, multiarmed spirals, cocultures of cardiac cells
and skeletal myoblasts or mesenchymal stem cells, mechanical excitation, attachment of spiral waves
to small anatomical obstacles, perturbation of spiral waves by external electric fields, and structure-
based facilitation of spiral wave formation.
Conclusions: The cultured cell monolayer is a contemporary experimental model encompassing
great versatility for basic studies of wavefront propagation and cardiac arrhythmias.
D 2006 Elsevier Inc. All rights reserved.
Keywords: Optical mapping; Electrophysiology; Reentry; Arrhythmia; Voltage-sensitive dye

Introduction thickness. They have a cellular composition that can be

In recent years, cultured cardiac cell monolayers have
become a contemporary experimental preparation for the
study of fundamental mechanisms that underlie normal and
pathological electrophysiology at the tissue level. Cell
monolayers are 2-dimensional sheets of cells that are
obtained by seeding cell suspensions, obtained by enzymat-
ic dissociation of intact tissue, onto tissue culture surfaces
conducive for cell attachment and spreading. Thus, they
form a synthetic tissue system under the experimenter’s
control that is structurally and functionally intermediate
between the single cell and native tissue. They exhibit the
aggregate behavior of a population of several hundred
thousand cells for centimeter-sized areas and therefore are
useful for studies of functional tissue properties (conduction,
arrhythmia). At the same time, they are a highly simplified
tissue and avoid irregularities in conduction arising from
connective tissue, blood vessels, and variations in tissue
B noncardiac cells such as fibroblasts or stem cells,9,10 which
Financial support: This work was supported by NIH grants
HL66239, RR017073, and an AHA fellowship to Y.Z.
4 Corresponding author. Tel.: +1 410 955 7453; fax: +1 410 502 9814.
E-mail address: ltung@jhu.edu (L. Tung).
0022-0736/$ – see front matter D 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.jelectrocard.2006.04.010
specified, a rapid diffusional access from the bathing
medium, and an absence of injured regions that occurs with
explanted tissue. Methods of oriented or patterned growth1-3
enable explicit control of the tissue architecture of the cell
monolayer, with the power to explore the electrophysiolog-
ical consequences of branched tissue structures4,5 or electric
field stimulation.6-8 With this level of control, the cell
monolayer has attained the status of a biological test bed
for issues dealing with tissue structure, analogous to
2-dimensional computational models of cardiac tissue.
One limitation that should be kept in mind, however, is
that the cell type is presently limited to embryonic (chick) or
neonatal (rat or mouse) heart cells.
An advantage that the tissue culture model has over its in
silico counterpart is its capacity for long-term study,
manipulation, and conditioning (eg, by drugs, gene vectors,
stretch, stimulation) over a period of days to weeks. It is
particularly well suited for studies of the functional
electrophysiological properties of mixtures of cardiac and
would otherwise be difficult if not impossible to predict
given that we still largely do not understand the nature of
the cell-cell interactions. Finally, the electrophysiological
 L. Tung, Y. Zhang / Journal of Electrocardiology 39 (2006) S2 – S6
properties of populations of embryonic stem cells can be
studied in tissue cultured embryoid bodies.11,12
Imaging electrical activity in cell monolayers
Optical mapping with voltage-sensitive dyes is common-
ly used today to record the spread of electrical activity.
Optical recordings provide information regarding action
potential duration, conduction velocity, and diastolic inter-
val. Excellent reviews of approaches used for optical
mapping of cardiac tissue have been published for intact
tissue13 and cell monolayers.14 Cell monolayers are inher-
ently compatible with the focal plane of optical instruments
because they are grown on a flat substrate. Moreover, they
avoid the ambiguities of optical surface recordings of tissue
with regard to the penetration depth of the measurements.
When recorded at a macroscopic scale, cell monolayers have
a minimal motion artifact by virtue of their attachment to a
rigid substrate. No pharmacological intervention (with
excitation-contraction uncouplers) is necessary to reduce
contraction artifacts, as is commonly used with tissue
preparations. The challenge to overcome in spatiotemporal
recordings, however, is the small signal generated by the
single layer of cells. For this reason, calcium is often used as
a surrogate for electrical activity in the cell because of its
close (but not exact) relationship to the action potential and
its much larger signal. However, direct measurements of
transmembrane voltage are preferable, particularly with
respect to parameters related to repolarization (action
potential duration, wavelength). The voltage-sensitive dyes
that are typically used are RH-237, di-4-ANEPPS, or di-8-
ANEPPS (Molecular Probes, Eugene, Ore, USA).
The voltage signals from cell monolayers can be imaged
using a CCD camera and optical lenses.14,15 An alternative
approach that we have adopted is referred to as contact
fluorescence imaging, which is a lensless system. Our
present experimental setup (schematic shown in Fig. 1A)
consists of a bundle of 253 optical fibers 1 mm in diameter,
arranged in a tightly packed 17-mm-diameter hexagonal
Fig. 1. Optical mapping setup for contact fluorescence imaging. A, Schematic. B, Photo of experimental chamber, excitation light source and one end of optical
fiber bundle. C, Other end of bundle connected to eight 32-channel photodetector/preamplifier/filter boards and data acquisition system.
S3
array, above which a cover slip containing a cardiac cell
monolayer is placed in contact. The monolayer is stained
with di-4-ANEPPS. An array of high-power, green light–
emitting diodes delivers excitation light from above
(Fig. 1B). A glass cover slip spin-coated with red photoresist
is placed between the floor of the chamber and the optical
imaging bundle and acts as the emission filter. The other end
of the optical fiber bundle is routed to 8 custom-designed
32-channel printed circuit boards containing photodetectors,
preamplifiers, and filters (Fig. 1C). The output signals are
then stored, displayed, and analyzed on a desktop computer
using custom written software. More details of the system
can be found in Bian and Tung.16 Given a knowledge of
transmembrane potentials through the entire monolayer, it is
possible to calculate a pseudoelectrocardiogram that would
be recorded from a bipolar pair of virtual electrodes
positioned above opposite edges of the monolayer.17
Cardiac arrhythmias in monolayers
The basic principles of impulse propagation and reentry
are discussed in reviews by Rudy,18 Jalife et al,19 and Kleber
and Rudy.20 Optical mapping of cultured strands of cell
monolayers has given insight into mechanisms that can set
the stage for arrhythmia, such as unidirectional conduction
block,21,22 gap junctional coupling and remodeling,23,24
or electroporation resulting from high-strength electrical
pulses.25 Focal and reentrant arrhythmias have been studied
in 2-dimensional monolayers and are summarized in Table 1.
Large sized ( N5 mm) monolayers can support functional
reentry in the form of spiral waves.17,26,41,42
Work in our laboratory on 2-dimesional neonatal rat
ventricular myocyte monolayers
Anisotropic monolayers
Using methods of patterned cell growth via micro-
abrasion and micropatterning techniques, neonatal rat
S4 L. Tung, Y. Zhang / Journal of Electrocardiology 39 (2006) S2–S6

Table 1
Studies of focal and reentrant arrhythmias in 2-dimensional monolayers of cultured cardiac cells
Study focus Cell type Measurement Reference
Bursting reentrant activity Embryonic chick Ca Bub et al15,26,27
Anatomical reentry Neonatal rat Vm Entcheva et al28
Ischemia-Reperfusion and ectopic beats Neonatal rat Ca Arutunyan et al29-31
Spiral wave generation with decreased cell-cell coupling Embryonic chick Ca Bub et al32,33
Electric shock-induced extrasystole Neonatal rat Vm Fast and Cheek34
Electrically induced and terminated functional reentry Neonatal rat Vm Iravanian et al17
Electromechanical instabilities modulated by microstructure Neonatal rat Ca Yin et al35
Multiarm spiral waves Neonatal rat Vm Bursac et al36,37
Reentry in cocultures of cardiac and skeletal muscle cells Neonatal rat Vm Abraham et al38
Calcium instabilities at rapid pacing rates Neonatal rat Ca Bien et al39
Structure-based initiation of reentry Neonatal rat Vm Bian and Tung16
Reentry in cocultures of cardiac and mesenchymal stem cells Neonatal rat Vm Chang et al40
Listed are the focus of the study, the cell type used, the measurement parameter being imaged by a fluorescent dye (intracellular calcium, Ca, or transmembrane
potential, V m), and reference.

ventricular myocytes (NRVMs) can be aligned in culture

along a primary axis to produce tissue-like anisotropic
structure, and anisotropic conduction velocity ratios up to
5.6 can be achieved.3 Moreover, the direction of anisotropy
can be varied within a single monolayer, so that the
functional effect of a discontinuity in conduction velocity
direction can be studied. An example of reentrant activity in
a bidirectional, anisotropic monolayer is shown in Fig. 2.
Here, the monolayer has an abrupt change in cellular
orientation as shown in the schematic drawing (Fig. 2B).
The movement of the wavefront is shown at 50-ms time
intervals through one reentry cycle (Fig. 2A). The reentry
pattern is figure-eight reentry, consisting of 2 adjacent and
coupled, counterrotating spiral waves. The movement of
the wavefront can be seen clearly in the isochrone map
(Fig. 2C). The 2 lines of block (thick black lines) lie along
the boundary separating the 2 sections of the monolayer
having different orientations.
Anatomical and functional spiral waves
Self-sustained reentrant activity around anatomical
obstacles can be initiated by rapid pacing and terminated
by a pulsed electric field.28 Functional reentry in the form of
a spiral wave is readily induced in homogeneous isotropic
NRVM monolayers by a double stimulus pulse protocol, in
which an electrical point or area stimulus is applied in the
wake of a planar wave initiated by a line electrode17 and
in anisotropic NRVM monolayers by rapid pacing.36 The
application of rapid pacing to an ongoing spiral wave can
terminate, reset, or accelerate the spiral wave via a process
Fig. 2. Figure-eight reentry in a heterogeneous anisotropic monolayer.
of multiplication into dual-arm or multiarm waves,36 that This example illustrates the capability of growing cells in 2 different
can exhibit complex dynamic behavior such as tip-switching oriented directions in the same monolayer using a microabrasion
and arm-switching.37 technique.3 A, Isopotential maps at 8 different times in a single-reentry
cycle. For each map, the time index is indicated in the upper left corner.
Arrhythmogenic consequences of combinations of cardiac The trace at the bottom indicates the optical potential at a single recording
cells and cells used for cellular therapy site marked by a small white square within the isochrone map, and the
vertical white line indicates the time of the map relative to the recording.
Currently, there is great interest in cellular grafts to The vertical grayscale bar on the left indicates the coding of optical
improve cardiac function, for example, using skeletal potential to intensity, with lowest potential at the bottom and highest
potential at the top. The small white arrows within the map indicate the
muscle myoblasts or mesenchymal stem cells (MSCs). In direction of wave front propagation. B, Schematic showing the direction
collaboration with the Marban/Abraham laboratory at of cell orientation. C, Isochrone map for one cycle of reentry. Numbers on
Hopkins, we have investigated whether mixtures of cardiac isochrones are in milliseconds.
 L. Tung, Y. Zhang / Journal of Electrocardiology 39 (2006) S2 – S6
cells with either cell type might form an arrhythmogenic
substrate.38,40 Reentrant arrhythmias are readily induced by
rapid pacing from a point electrode when the cell density is
10% or greater in skeletal muscle myoblast or in MSC
cocultures. The presence of MSCs also increases action
potential duration of the NRVMs, signifying the importance
of paracrine effects.38
Mechanical excitation
The application of mechanical deformations to cardiac
tissue can produce significant electrophysiological effects
by a process termed mechanoelectric feedback. Mechanical
stimuli in the form of miniature pulsed fluid jets that
impinge upon the surface of confluent NRVM monolayers
can trigger excitation and initiate a propagated wave.43 The
likelihood for mechanical stimulation increases with jet
amplitude and time interval between stimuli and decreases
with the application of stretch-activated channel blockers.
Pinning of spiral waves to small anatomical obstacles and
their control by external electric fields
Experiments conducted on NRVM monolayers contain-
ing small, circular obstacles (0.6-2.6 mm in diameter) show
that spiral waves attach (bpinQ) to the obstacles with a
likelihood that increases with obstacle size.44 Addition of
the sodium channel blocker, lidocaine, destabilizes the
attachment of the spiral wave, with an efficacy that is more
pronounced with smaller obstacle size.44 The application of
electric fields causes strength-dependent slowing of pinned
spiral waves and decreases conduction velocity and wave-
length near the wave tip.45 The make or break of the pulse
can also cause pinned waves to detach.
Structure-based facilitation of the initiation of spiral waves
by rapid pacing
The induction of reentry in cardiac tissue by rapid pacing
has largely been attributed to spatial heterogeneity in ion
channels or cell-cell coupling, to dynamic instabilities
arising from unstable restitution dynamics, or to unstable
calcium dynamics. However, experiments in patterned
NRVM monolayers suggest that heterogeneity in tissue
structure can, in and of itself, serve as a locus for the
initiation of reentry.16
Summary
Fundamental principles governing electrophysiological
function may be best studied in simplified cardiac models.
The cultured cell monolayer is one such experimental
model encompassing great versatility for basic studies of
wave front propagation and cardiac arrhythmias (eg, spiral
waves) when used with optical mapping. With the advent
of methods for patterned growth, cell monolayers have
attained the status of a biological analog to 2-dimensional
computational models of cardiac tissue. Future studies
will focus on quantifying how heterogeneity in tissue
properties contributes toward the genesis and persistence
of cardiac arrhythmias.
S5
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