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Departments of 1 Biomedical Engineering, 2 Medicine, and 3 Physiology, University of Alabama at Birmingham, Birmingham, Alabama 35294; and 4 Department of Physiology, University of Bern, 3012 Bern, Switzerland
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Organization of cardiac tissue into cell strands and
layers has been implicated in changes of transmembrane potential
(
Vm) during defibrillation. To determine the
shock-induced Vm in such structures, cell
strands of variable width [strand width (SW) = 0.15-2
mm] were grown in culture. Uniform-field shocks with variable
strength [shock strength (SS) = 2-50 V/cm] were
applied across strands during the action potential (AP) plateau, and
Vm were measured optically. Three different
types of Vm were observed. Small
Vm [<40%AP amplitude (APA)] were
linearly dependent on SS and SW and were symmetrically distributed
about a strand centerline with maximal positive and negative
Vm on opposite strand sides being equal.
Intermediate Vm (<200%APA) were strongly
asymmetric with negative Vm > positive
Vm because of a negative time-dependent shift of
Vm at the depolarized side of the strands. For
large Vm (>200%APA), a second time-dependent
shift of Vm to more positive levels was observed in
the hyperpolarized portions of strands, causing reduction of the
Vm asymmetry. We conclude that during application of shocks to cell strands during the AP plateau, passive changes of Vm were followed by two voltage- and
time-dependent shifts of Vm, possibly reflecting
changes of ionic currents or membrane electroporation.
stimulation; optical mapping; voltage-sensitive dyes; cell cultures
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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STRONG ELECTRICAL SHOCKS are used in patients to
terminate atrial and ventricular fibrillation. It is, therefore,
important to understand the mechanisms of interaction between
electrical fields and cardiac tissue. One of the interesting and still
unresolved problems is how an electrical field changes the
transmembrane potential (Vm) of myocytes forming a
cellular network. The classic cable model indicates that
Vm changes (Vm) induced by a
uniform electrical field in structurally continuous tissue are
restricted to a small area near the shock electrodes (12, 31). To
induce Vm far from electrodes, a redistribution
of current between intracellular and extracellular spaces must take
place. This can occur for two reasons: 1) nonuniform
distribution of the extracellular shock field and 2) spatial
variation in the tissue structure. The first mechanism relates
Vm to the spatial derivative of the
extracellular field [the "activating function" (23)].
The second mechanism relates Vm to the formation
of "secondary sources" caused by resistive discontinuities in the
tissue structure (15, 22) or to the rotation of anisotropy axes in
space (29). A combination of structural factors (tissue anisotropy) and
the nonuniform shock field can produce Vm via
the "dog-bone" effect (13, 33).
To investigate the relationship between tissue structure and
Vm during shock application, we employed an
approach in which geometrically defined tissue structures were produced
in culture using the technique of directed cell growth. The effect of
these structures on Vm was measured using
voltage-sensitive dyes. Using this approach, we previously showed (7)
that microscopic intercellular clefts can cause significant
Vm and excite cells during application of
defibrillation type shocks. In the present study, we investigated how
shocks affect Vm in another type of structure, cell
strands. Strands represent a very common and the most simple type of
structural organization of cardiac tissue. Understanding how
Vm changes in strands is a prerequisite for
understanding the mechanisms of Vm changes in other
more complex structures. Previous studies in strand-like structures
such as isolated papillary muscles (35, 36) and cultured cell strands
(8) revealed that application of shocks caused large hyperpolarization
and depolarization on opposite strand borders. It was also shown that
Vm changes were asymmetric during the plateau phase
of the action potential (AP), with hyperpolarization being larger than
depolarization (8, 35). However, it is not known how these results
relate to strands of variable dimensions and to shocks of different
strengths. Therefore, the purpose of the present study was to determine
shock-induced Vm in cell strands as a function
of strand width and shock strength.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Directed Cell Growth
Dissociated cells were obtained from neonatal Wistar rats (2 days old). Cell monolayers with desired growth patterns were produced on glass coverslips according to the previously published procedure (25) with some modifications (S. Rohr and R. Flückiger, unpublished data) that allowed localized coating of glass coverslips with collagen (type IV, Sigma) (5). The growth pattern, schematically shown in Fig. 1, consisted of parallel-oriented cell strands of different widths attached to a rectangular-shaped cell region. This arrangement ensured synchrony of cell contraction in all the strands during cell culturing and, therefore, a similar degree of cell development. The width of the strands was 0.15, 0.3, 0.5, 1, and 2 mm; the strand length was 8-10 mm.
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Measurements were performed between the third and the ninth day in culture. Before each experiment, cell strands were cut off from the common rectangular area with the use of a sharp needle and measurements were performed in the middle portions of the strands. For measurements, monolayers were transferred into a perfusion bath (Fig. 1) that measured 2.5 × 2.5 × 0.5 cm3 and were superfused with a Hanks' balanced salt solution with a composition of (in mM) 137 NaCl, 5.4 KCl, 0.4 KH2PO4, 0.4 NaH2PO4, 0.8 MgSO4, 1.3 CaCl2, 4.2 NaHCO3, 5.0 HEPES, and 5.1 glucose. The pH of the solution was 7.4, and the temperature was kept constant at 35oC.
Optical Recordings of Transmembrane Potential
To measureVm, cells were stained for 3-5
min with 2-3 µM of the fluorescent voltage-sensitive dye RH-237
(Molecular Probes). Two optical systems were used in this study to
record Vm. The first system, described elsewhere
in detail (4, 6), used a 10 × 10 photodiode array (Centronic) for
simultaneous recordings at 96 points. The second system, similar in
overall design, used a larger 16 × 16 photodiode array
(Hamamatsu) and a data acquisition system that allowed recordings at
256 channels. This system was built around an inverted microscope
(Axiovert 135TV, Zeiss). Cells were illuminated at 530-585 nm
using a 100-W mercury lamp, and emitted fluorescence was measured at
>615 nm using the 16 × 16 photodiode array. The array
had diodes with dimensions of 0.95 × 0.95 mm2 and a
center-to-center interdiode distance of 1.1 mm. Microscopic objectives
with magnifications of ×20 and ×40 were used. Additional magnifications of ×1.6 and ×2.5 were provided by a built-in
Bertrand lens. The total optical magnification was in the range between ×20 and ×100, corresponding to an area per diode ranging
from 47 × 47 to 9.5 × 9.5 µm2. The
photocurrents from 252 diodes were converted to voltages; the
background fluorescence was subtracted; and signals were amplified, multiplexed, and digitized at a 12-bit resolution and a sampling rate
of 12 kHz per channel using two data acquisition cards DAP3400 (Microstar Laboratories) installed in a Pentium II personal
computer (Gateway 2000). Software for data acquisition and data
analysis was written using Delphi Pascal (Inprise).
Stimulation and Application of Electrical Shocks
Cells were paced at a cycle length of 500 ms using a bipolar electrode composed of a glass pipette filled with Hanks' solution and a silver wire coiled around the pipette tip. Electrical shocks were applied via two platinum plate electrodes positioned at opposite ends of the perfusing bath (Fig. 1). The electrode dimensions were 1.9 × 0.3 cm2. Monophasic truncated exponential shocks (time constant of 35-38 ms) or rectangular shocks with strengths of 2-50 V/cm and durations of 10-12 ms were used. With both shock waveforms, the distribution of shock-induced changes in transmembrane voltage were similar at equal shock strengths. The voltage gradient (E) produced by the shocks in the bath was measured simultaneously with the optical recordings of Vm by two silver electrodes with a diameter of 0.1 mm and an interelectrode distance of 2 mm. The electrodes were positioned near the mapping area and aligned with the direction of the electrical field. Shocks were delivered 20-25 ms after a stimulation pulse.Typically, up to six measurements were performed at the same location
using shocks of different strengths. In strands of 0.15, 0.3, and 0.5 mm in width, voltage changes were measured simultaneously at different
sites across the strands. Because the imaged area (maximum 872 × 872 µm2) did not cover the width of 1- and 2-mm strands,
the Vm were measured at the strand borders by
sequentially shifting the imaged area from one border to the other and
applying two shocks of the same polarity and strength. In some cases,
measurements were performed at one strand border by changing the shock
polarity. The Vm were not affected by
application of multiple shocks or by multiple exposures to excitation
light: there was no significant difference among 10 consecutive
measurements of Vm induced by 32 V/cm shocks in
0.5-mm strands (n = 3 strands).
Data Analysis
Data analysis was carried out similarly to procedures described previously (7, 8). Shock-induced changes in Vm were measured as a percentage of the change in fluorescence intensity relative to the action potential amplitude (APA). The local activation times were determined at 50% of the APA using linear interpolation between the nearest sampling points. Conduction velocity was calculated using activation times measured at opposite edges of the photodiode array. Activation maps and isopotential maps ofVm distribution were constructed using linear
interpolation and triangulation algorithms. In some of the measurements
carried out with high illumination intensity, photobleaching of the
voltage-sensitive dye caused a decrease in the level of optical signals
during recordings. In these cases, signals were corrected for the
photobleaching by subtracting a linear fit of the signals calculated
during the resting phase of the AP. Data were expressed as means ± SD. Differences were compared using the two-tailed, nonpaired
t-test. They were considered statistically significant if
P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Experiments were carried out in a total of 60 strands from 12 cell
monolayers and 6 cultures. The average conduction velocity was 25.2 ± 4.8 cm/s, and the average maximal rate of rise of the AP upstroke was
108 ± 15 V/s. These data are similar to values reported previously
from experiments in isotropic cell monolayers (5). Shocks were
delivered with a delay of 10.4 ± 3.5 ms after the onset of the AP
upstroke. The magnitude and spatial distribution of the shock-induced
Vm were dependent on both the shock strength and
strand width.
Shock-Induced Vm in Narrow
Strands
Vm were investigated in 14 strands with a width of 0.15 mm. Depending on the shock strength, three
different types of shock-induced Vm were observed.
Symmetric Vm (type I).
Weak shocks induced the simplest type of voltage changes (type I),
which are illustrated in Fig. 2. Figure
2A shows an image of the cell strand and the outline of the
photodiode array. The stimulation electrode was located at the top of
the mapping area, and the shock electrodes were located on the left and
right sides. The stimulation pulse was applied 5 ms after the start of
recording. A rectangular shock with a strength of 1.9 V/cm and a
duration of 12 ms was delivered ~12 ms after the onset of AP. Figure
2B depicts the isopotential map of shock-induced
Vm. The shock caused depolarization of cells in
the left half of the strand and hyperpolarization in the right half.
The map of Vm distribution was rather uniform with isopotential lines running parallel to strand borders, indicating that distribution of Vm across the strand was
essentially one-dimensional.
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Vm
was very similar to the rectangular shape of the shock waveform. Figure
2D shows a Vm profile measured 2 ms
after the shock onset. The maximal levels of depolarization at the left
strand border (site 1) and hyperpolarization at the right
border (site 10) were nearly equal (13.8% and 
15.2%, respectively). Between these locations, there was a linear transition from depolarization to hyperpolarization with no change of
Vm in the middle of the strand. The difference of
Vm across the strand was 29%APA, or 29 mV. This
value is close to the product of the shock strength (1.9 V/cm) and the
distance between the recording spots (0.135 mm), which equaled 26 mV.
Overall, the Vm of this type reflects the
redistribution of shock current according to the predictions of the
passive cable model.
Vm of types II and III.
Figure 3 illustrates two other types of
Vm in a 0.15-mm strand (Fig. 3A) induced
by truncated exponential shocks with a strength of 27 V/cm (Fig.
3B, thin traces) and 39 V/cm (Fig. 3B, thick traces).
The signals are normalized to the corresponding values of APA (not
shown).
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Vm
distribution across the strand was time dependent. During the early
phase of the shock, the Vm distribution was
symmetric, as illustrated by the Vm profile
measured 0.3 ms after the shock onset (Fig. 3C, dashed line).
This indicates that, similar to the effect of weak shocks (Fig. 2), the
initial response of membrane potential to the stronger shock was
passive. Soon after the shock onset, however, membrane potential at all
points within the strand shifted toward more negative levels. As a
result of this shift, the Vm distribution became
asymmetric with hyperpolarization at the left side of the strand being
much greater than depolarization at the right side of the strand. At a
time (t) of 3 ms, the maximal negative and positive
Vm were 180 and 78%APA, respectively. In
the middle portion of the strand (site 6), there was a reversal
of Vm polarity: the initial depolarization was
followed by hyperpolarization. Such shifts of Vm to
more negative levels indicate a net increase of current in the outward
direction, which can be caused by generation of an outward current
within the strand or a decrease of an inward current.
When the shock strength was increased to 39 V/cm, a third type of
Vm was observed (Fig. 3B, thick traces).
Similar to the case with the 27 V/cm shock, very early
Vm was nearly symmetric, as illustrated by the
Vm profile measured at t = 0.3 ms (Fig. 3D). At t = 3 ms, the Vm
distribution became strongly asymmetric with maximal positive and
negative Vm of 82 and 223%APA,
respectively (ratio of 2.72). Later during the shock, however,
Vm shifted to more positive levels, approaching the
level measured with the weaker, 27 V/cm shock. As a result of this
shift, the degree of Vm asymmetry was reduced:
maximal positive and negative Vm were 66 and
160%APA (ratio of 2.42). These later shifts of Vm
to more positive levels suggest the generation of an inward ionic
current, a decrease of an outward current, or cell electroporation
caused by a large voltage change at the beginning of the shock.
Transitions among three different Vm types at
increasing shock strengths were observed in all 12 strands with a width
of 0.15 mm.
Voltage dependence of Vm.
To determine more precisely the voltage dependence for different types
of Vm, the absolute values of maximal positive
and negative Vm at opposite strand borders were
measured 2 ms after the shock onset as a function of shock strength in
the 0.15-mm strands (n = 14). These data are plotted in Fig.
4. The measuring spots were separated by
0.135 mm (center-to-center interdiode distance). The linear and
symmetric Vm (type I) were observed when shock
strength was less than ~9 V/cm and Vm was less
than ~40%APA. The thin straight line extrapolates this linear
dependence into areas of larger voltages. The slope of this dependence
was 5.3%APA · cm · V
1,
which translates into a length of 0.053 mm (assuming APA = 100 mV).
This is close to one-half the distance between the measuring spots at
the strand borders. The type II asymmetric voltage changes, which
deviated from the passive linear dependence, were observed when
Vm exceeded ~40%APA. The transition to type
III Vm occurred when shock strength was
increased above ~27 V/cm and negative Vm
exceeded ~200%APA.
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Vm in 0.3-mm strands.
Effects of shocks on Vm were investigated in 11 strands with a width of 0.3 mm. Similar to 0.15-mm strands, three types
of Vm were observed in the 0.3-mm strands (not
shown). At the same shock strength, the Vm in
the 0.3-mm strands were larger than in the 0.15-mm strands. Transitions
from one type of Vm to another occurred at a
lower shock strength than in the 0.15-mm strands.
Shock-Induced Vm in Wide Strands
Vm in 0.5-mm strands.
Effects of shocks on Vm were measured in 12 strands
with width of 0.5 mm. Figure 5 shows the
Vm recordings during application of two shocks
with strengths of 10.5 and 24 V/cm, respectively, in one strand. The
weaker shock induced Vm (Fig. 5B, solid
traces) that were similar in shape and spatial distribution to the type II Vm in the 0.15-mm strands with one exception:
there was no symmetric phase in the Vm response at
the beginning of the shock. Contrary to data in Fig. 3B, the
Vm recording in the middle of the strand
(site 5) was negative at all times during the shock. Another
difference was quantitative: compared with the measurements in narrower
strands at a similar shock strength (Fig. 4), Vm in the 0.5-mm strands were much larger and more asymmetric. At t = 3 ms after the shock onset (Fig. 5B, dashed line),
maximal levels of depolarization and hyperpolarization at strand
borders were 62 and 194%APA (ratio of 3.1).
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Vm (Fig. 5B, shaded traces) similar to
the type III in the narrow strands. At t = 3 ms, the
Vm distribution was strongly asymmetric: the maximal positive and negative Vm were 76 and
221%APA (ratio of 2.9). However, subsequent positive shift of
Vm reduced the degree of asymmetry: at t = 8 ms, the maximal positive and negative Vm were
76 and 136%APA (ratio of 1.79). This upward shift of Vm was much more pronounced than in the narrow
strands (Fig. 3). At the edge of the strand (site 9), the
Vm shifted to a level even more positive than
Vm measured at this location with the weaker shock.
Between t = 8.5 ms and the end of the shock, a new positive
deflection was observed. It appeared near site 4 (Fig. 5B, arrow) at the border between depolarized and
hyperpolarized areas and spread into the hyperpolarized area. The
amplitude of this deflection gradually increased from site 4 to
site 9, reaching 120%APA. This type of activity can be
interpreted as a "diffusion" of Vm from the
depolarized area to the hyperpolarized area with subsequent generation
of a new AP in the area where sodium channels recovered from inactivation.
Figure 5C summarizes measurements of the maximal positive and
negative Vm at opposite strand borders 3 ms
after shock onset in all 12 strands. Similar to the
results in the 0.15-mm strands, the smallest Vm
(~50%APA) were nearly symmetric. The positive Vm was only weakly dependent on shock strength,
quickly reaching a plateau of ~90%APA. The negative
Vm was linearly dependent on shock strength
until Vm of ~200%APA; the rate of
Vm increase then became smaller, and at higher
shock strength it started to decrease. These levels of
Vm for the transitions between different types
of Vm responses were similar to those found in the
0.15-mm strands.
The results obtained in 0.15-, 0.3-, and 0.5-mm-wide strands provide
indications about the involvement of two different currents induced by
large Vm, but it is not clear whether these
currents are generated in the depolarized or hyperpolarized portions of the strands. This is because, in the narrow strands, the depolarized and hyperpolarized areas are close to each other and any charge entering the strand at one side can quickly redistribute across the
strand. Whether these currents are generated at the depolarized or
hyperpolarized regions, however, can be distinguished in very wide
strands [width >> 
(electrotonic space constant)],
where areas of hyperpolarization and depolarization do not interact with each other. According to previously published data (10), the value
of is 360 µm in cultured cell monolayers. Therefore, strands with
a width of 1 or 2 mm should be wide enough to avoid interaction between
polarizations at the opposite strand borders.
Vm in 1- and 2-mm strands.
Effects of shocks on Vm were investigated in 11 strands with a width of 1 mm and 12 strands with a width of 2 mm.
Because these strands were wider than the field of view, measurements of positive and negative Vm were carried out
sequentially; i.e., the field of view was shifted from one border to
the other or, alternatively, measurements were carried out at the same
border with alternating shock polarities. Vm
were similar in the 1- and 2-mm strands. Figure
6 shows Vm
recordings from opposite borders of a 2-mm strand during application of
shocks of variable strength. Positive Vm
measured at site 1 had a shape very similar to the shock
waveform (Fig. 5B). As shocks became stronger, the amplitude of
positive Vm reached a level of ~90%APA and
then did not increase further. Because this area was not influenced by
the hyperpolarization on the opposite side of the strand, it can be
concluded that the reason for the nearly constant
Vm was a net increase of current flowing in the
outward direction in this area (equivalent to reduction of membrane
resistance). On the other side of the strand, the shape of the negative
Vm changed from monotonic for the weakest shock
(Fig. 6, thin trace) to biphasic for stronger shocks (shaded and thick
traces). This indicates an increase of current flow in the inward
direction in the hyperpolarized area of the strand. Similar results
were obtained in other wide strands. Figure 6C summarizes
measurements of maximal positive (site 1) and negative
(site 2) Vm measured 4 ms after shock
onset in 12 strands with a width of 2 mm. The transition
of the negative Vm from monotonic to biphasic
shape occurred when Vm was ~200%APA. This is
similar to the transition from type II to type III
Vm observed in the narrower strands (Figs. 4 and
5).
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Dependence of Vm on Strand Width
Vm measured
2-4 ms after shock onset in strands of variable width. The average
shock strength in these measurements was 8.3 ± 1.5 V/cm (n = 70). To account for the slight variability in the shock strength, the
individual Vm were normalized to a constant shock strength of 8 V/cm. This normalization assumes linear dependence of Vm on shock strength, which is true within
this range of shock strengths. The experimental
Vm are compared with the
Vm predicted by the passive cable model (Fig.
7C, dashed line), which were calculated assuming an electrotonic space
constant () of 300 µm and an APA of 100 mV. In the strands with a
width of 0.15 mm, the positive and negative Vm
were similar (39 ± 9 and 45 ± 7%APA, n = 14, respectively) and close to the linear dependence predicted by the
passive cable model. As strand width became larger, the dependencies
became nonlinear and increasingly asymmetric. The positive
Vm reached a level of 55 ± 8%APA (n = 11) in 0.3-mm strands and did not change significantly in wider
strands. The negative Vm reached a plateau of
152 ± 27%APA (n = 11) in 1-mm strands. The plateau levels
for both positive and negative Vm were
significantly lower than predicted by the passive cable.
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Effect of Potassium Channel Blockers on
Vm Asymmetry
Vm of types II and III, the
asymmetry of the shock-induced Vm response with
larger hyperpolarization than depolarization may be explained by
generation of an outward ionic current. In this case, suppressing this
current with a channel blocker should reduce the degree of the
Vm asymmetry. A potential candidate for such a
current is an outward potassium current, either an inward or a delayed
rectifier. To evaluate the contribution of these currents to the
observed asymmetry, we used a blocker of the inward rectifier current,
BaCl2, and a blocker of the rapid component of the delayed
rectifier current, dofetilide. The effect of BaCl2 in
concentration of 0.1 mM was measured in 13 cell strands with a width of
0.3 mm from 6 cell monolayers. Shocks used had an average strength of
14.9 ± 1.5 V/cm that resulted in a large degree of
Vm asymmetry. Under control conditions, the
average positive and negative Vm measured at the
strand edges, normalized for a shock strength of 15 V/cm, were 96 ± 19 and 244 ± 38%APA (n = 6), respectively. The
average asymmetry ratio was 2.6 ± 0.4. Application of
BaCl2 did not significantly change the degree of Vm asymmetry: the average asymmetry ratio was
2.5 ± 0.5, with positive and negative Vm of 97 ± 17 and 245 ± 35%APA (n = 7), respectively. The
effect of dofetilide in a concentration of 1 µM was measured in five
cell strands with a width of 0.15 mm from two cell monolayers. Shocks
with a strength of 14.3 ± 2.1 V/cm were applied. Similar to the
experiments with BaCl2, dofetilide did not change the
degree of Vm asymmetry. It was 1.93 ± 0.3 (n = 5) under control conditions and 1.90 ± 0.3 in
the presence of dofetilide. The corresponding positive and negative
Vm values were 83 ± 21 and 160 ± 41%APA (control) and 81 ± 22 and 154 ± 48%APA (dofetilide).
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DISCUSSION |
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|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study we applied high-resolution optical mapping to
determine the changes of Vm caused by
defibrillation-type shocks in cell strands of variable width. We
observed three different types of Vm changes during
the plateau phase of the AP. One of the types, characterized by an
asymmetric Vm distribution with negative
Vm at one side of the strand being much larger
than positive Vm on the other side, was observed
previously (8, 35). The new findings regarding this asymmetric
Vm response are the determination of its voltage
dependence and the demonstration that the asymmetric response is caused
by nonlinear Vm changes in the depolarized portions
of the strands. Furthermore, we have found two new types of
Vm responses: 1) small
Vm (<40%APA) were symmetrically distributed
across strands, and 2) very large negative Vm (>200%APA) induced a time-dependent shift
of Vm to more positive levels in the hyperpolarized
portions of the strands with a reduction or complete elimination of
Vm asymmetry.
Symmetric Vm
Vm during the AP plateau within a certain range
of Vm. Changes of Vm with
magnitude <40%APA induced by weak shocks in the narrow strands were
linearly dependent on shock strength, and their spatial distribution
was symmetric, as predicted by the cable model. There was also
quantitative agreement between the experiments and the cable model. The
cable model predicts that the maximal Vm at a
border of a narrow strand (width << ) is equal to one-half the
product of the field strength (E) and the strand width (21). This means that the slope of the dependence
(Vm(E)) is equal to one-half the
strand width or, more precisely, one-half the distance between the
measuring points. From Vm measurements in the
0.15-mm strands (Fig. 4), this slope is 53 µm (assuming APA = 100 mV). This is close to one-half the center-to-center distance between
border diodes in these measurements (67 µm).
When strong shocks were applied to the narrow cell strands, the
Vm distribution was also symmetric during the
early phase of the shocks, indicating that this response was passive in
nature. In the wider strands, the initial passive response was not
observed. This was likely because of the dependence of the speed of the passive response on the strand width and on the distance from the
strand edges. The wider the strand and the further the recording spot
from the strand edge, the longer it takes to reach the steady-state Vm. Therefore, in wide strands, the passive
Vm changes can be masked by large nonlinear, active
Vm changes.
Asymmetric Vm
Vm caused by strong shocks was time
dependent. After the early symmetric phase of Vm
changes, an asymmetry in Vm distribution was
established with much larger hyperpolarization on one side of a strand
than depolarization on the other side. Such Vm
asymmetry was previously observed in the strand-like structures when
shocks were applied during the AP plateau (8, 35). Here, we have
demonstrated that this asymmetry was caused by a shift of
Vm to more negative levels in the depolarized
portions of the strands and that the threshold for this shift was ~40
mV above the plateau level. In the narrow strands, this shift was electrotonically transmitted from the depolarized areas to the hyperpolarized areas, causing nonlinear increase of the negative Vm (Fig. 4). In the wide strands, because of the
lack of electrotonic interaction between depolarized and hyperpolarized
areas, such nonlinear increase of negative Vm
was absent (Figs. 5 and 6).
The mechanism underlying the Vm asymmetry is not
known. It has been recently suggested that the
Vm asymmetry is caused by electroporation of the
cell membrane (3). According to this hypothesis,
Vm are symmetric when measured relative to the
zero level of Vm and, because of the
electroporation, they are asymmetric when measured relative to the AP
plateau level. The results of the present study contradict this
hypothesis. First, the degree of Vm asymmetry
was too high to be explained by this mechanism. For example, in the
narrow strands, the negative Vm was
approximately 250%APA and the positive Vm was
~70%APA at a shock strength of 35 V/cm (Fig. 4). With the assumption
that the plateau level was 20 mV (20%APA), the difference between the
negative and positive Vm measured from the zero
level was very large (230 and 90%APA, respectively). Second,
the Vm asymmetry became noticeable when Vm was larger than ~40 mV relative to the AP
plateau level (Fig. 4), which corresponds to an absolute
Vm level of 60 mV. Such a level of
Vm is too low to induce membrane electroporation,
which occurs at Vm of several hundred millivolts
(30). It is more likely that the Vm asymmetry
was caused by changes in the conductance of ionic channels, either by
activation of an outward current caused by depolarization >60 mV or
by inactivation of an inward current. The existence of a mechanism
based on involvement of an ionic current rather than on the membrane
electroporation is also supported by observations that shock-induced
Vm changes were symmetric when shocks were applied
near diastolic potential (8). Possible candidates for such ionic
currents are outward potassium currents (11). However, application of
the channel blockers dofetilide and BaCl2 for these
currents did not change the degree of Vm
asymmetry, indicating that these two currents are not responsible for
the effect of asymmetry. The conclusion that these particular currents
are not involved in the Vm asymmetry is also
supported by the results of simulations (3) in a computer model with Luo-Rudy excitable kinetics (17). This model contains both inward and
delayed rectifier potassium currents. However, either the shock-induced
changes of Vm in a linear cable described by this model were symmetric or the positive Vm was even
slightly larger than the negative Vm. Also, we
observed (unpublished data) no asymmetry of Vm
in linear strands described by an earlier version of Luo-Rudy excitable
kinetics (18). The discrepancy between simulated and experimental
results is not surprising because these models were designed for the
physiological range of Vm. They need to be modified
for large Vm to describe the response of myocardium to strong electrical shocks.
The effect of Vm asymmetry with
hyperpolarization larger than depolarization is in apparent
contradiction with published data on changes in membrane resistance
during the cardiac cycle. The membrane resistance near the plateau
level can be larger than at the resting level (9), which should result
in larger positive than negative Vm, opposite to
the asymmetry observed in both cell cultures and adult tissue (35). The
explanation of this contradiction is that the membrane resistance was
determined in response to small changes of Vm near
the plateau or near the resting level. In our work, however, the
Vm asymmetry was observed for relatively large
Vm changes.
Time-Dependent Reduction of the Vm
Asymmetry by Strong Shocks
Vm induced another
time-dependent shift of Vm toward more positive
levels. This shift was generated in the hyperpolarized portions of the
strands and was slower than the negative Vm shift.
As a result of this positive shift, the earlier
Vm asymmetry could be reduced or reversed toward
the end of the shock. Such positive Vm reflects
an increase of current flow in the inward direction that was prominent
with hyperpolarization below ~200 mV from the plateau level (Figs. 4
and 6). As with the outward current, the nature of this inward current
is not known. A possible candidate for this current is the inward
current (If) activated in guinea pig ventricular
myocytes below 120 mV (34). Alternatively, it might be a
nonspecific current caused by membrane electroporation, which can be
produced at transmembrane potentials of several hundred millivolts
(30).
Implications for Cardiac Defibrillation
Presently it is unclear how electrical shocks change Vm of cardiac cells and interrupt fibrillation. The structure of cardiac tissue might play an important role by providing a substrate for Vm changes. The strandlike structures investigated in this study are very common in the heart. They are most prominent in the atria and on the endocardial surface of the ventricles. Therefore, results of this study might be important for understanding the mechanism of atrial defibrillation and the response of the Purkinje system to defibrillation shocks. In addition, the results obtained in cell strands can be applied to intramural cell bundles (28) and cell layers (16) that run across ventricular walls from subendocardium to subepicardium. When the electrical field is oriented across such layers, the shock-induced Vm distribution should be similar to the Vm distribution in cell strands.The efficiency of defibrillation depends on the magnitude of the
shock-induced Vm. In this respect, it is
interesting that increasing shock strength did not increase positive
Vm above ~100%APA (100 mV). Negative
Vm measured several milliseconds after the shock
onset did not increase above ~250%APA (250 mV). Saturation of the
shock-induced Vm changes at increasing shock strength was also observed in intact cardiac tissue (35) and isolated
single cells (14). In experiments with guinea pig papillary muscle, the
maximal levels of depolarization and hyperpolarization were 66 and 99 mV (35), respectively, which are much smaller than
Vm observed in the present study. This
difference is likely because measurements in papillary muscle were done
in the middle muscle sections, thus underestimating the maximal
Vm values achieved at the muscle edges.
According to the results of the present study, saturation of the
positive Vm was likely caused by an increase of
net current flow in the outward direction generated in the depolarized
area, whereas saturation of the negative Vm was
due to inward current flow generated in the hyperpolarized area. In this case, the involvement of ionic currents in shock-induced Vm might provide an opportunity for
pharmacological modulation of Vm and, therefore,
of defibrillation efficacy. Enhancing the Vm
response to electrical shocks might be beneficial because it would
reduce requirements for shock energy.
Limitations
One major limitation of this study is related to differences in structural properties between cell cultures and intact cardiac muscle. Cell cultures lack three-dimensional architecture, anisotropy, and fiber rotation, which are considered to be important factors for defibrillation (26, 29, 32). Another limitation is related to differences in expression of ionic channels and gap junctions. Particularly, the cellular distribution of gap junctions is different in neonatal and adult myocytes (2, 20). This difference might be important for Vm changes at a subcellular scale, but it is not likely to play a significant role in the effects investigated in this study. A more important difference between neonatal and adult myocytes is related to expression of ionic channels. Both inward and outward ionic currents undergo substantial changes during cell development from neonatal to adult phenotype (11, 24), which might affect the nonlinear response of Vm to defibrillation shocks. Therefore, the relation between the tissue structure and shock-inducedVm analyzed in
tissue with defined two-dimensional architecture needs to be verified
in intact tissue.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. André G. Kléber for helpful discussions on the manuscript and Windy Jones and Regula Flückiger Labrada for help with preparation of cell cultures.
| |
FOOTNOTES |
|---|
This work was supported by a grant from The Whitaker Foundation, National Heart, Lung, and Blood Institute Grant HL-42760, and a grant from the Swiss National Science Foundation.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: V. G. Fast, Univ. of Alabama at Birmingham, 1670 University Blvd, VH B149, Birmingham, AL 35294 (E-mail: fast{at}crml.uab.edu).
Received 11 March 1999; accepted in final form 15 September 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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