© 1995 American Heart Association, Inc.
Articles |
Optical Transmembrane Potential Measurements During Defibrillation-Strength Shocks in Perfused Rabbit Hearts
From the Departments of Medicine and Pathology, Duke University Medical Center, and the Engineering Research Center in Emerging Cardiovascular Technologies, School of Engineering, Duke University, Durham, NC.
Correspondence to Xiaohong Zhou, MD, G82A Volker Hall, Box 201, UAB Station, Birmingham, AL 35294-0019. E-mail xhz@dukebar.crml.uab.edu.
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Abstract |
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Abstract To study the optical transmembrane potential change (
F) induced during shocks, optical recordings were obtained
in 15 isolated perfused rabbit hearts treated with the potentiometric
dye di-4-ANEPPS and diacetyl monoxime. Shock electrodes were sutured on
the right and left ventricles. A laser beam 30 µm in diameter was
used to optically excite di-4-ANEPPS. Fluorescence from a
region 150 µm in diameter was recorded during a shock. In the
macroscopic study (six animals), there were nine recording
spots that were 3 mm apart between the two shock electrodes. In the
microscopic study, there were three recording regions that were
3 mm away from either shock electrode and midway between them, with
nine recording spots that were 30 µm (three animals), 100
µm (three animals), and 300 µm (three animals) apart in each
region. After 20 S1 stimuli, a 10-ms truncated exponential
S2 shock of defibrillation-threshold strength was given
during the plateau of the last S1 action potential. In the
microscopic study, shocks were also given during diastole,
with F recordings at the three recording regions.
Shocks of both polarities were tested. F during the shock was
expressed as a percentage of the fluorescence change during the
S1 upstroke action potential amplitude (the S1
Fapa), ie, F/Fapa%. In the macroscopic
study, the magnitudes of F/Fapa% from recording
spots 1 to 9, numbered from the left to the right
ventricular electrodes, were 77±41%, 46±32%, 32±27%,
28±20%, 37±25%, 24±20%, 33±22%, 37±25%, and 59±29%,
respectively (P<.05 among the nine spots). Depolarization
or hyperpolarization could occur near either shock
electrode with both shock polarities, but the magnitude of
hyperpolarization was 1.8±0.9 times that of
depolarization at the same recording spot when the shock
polarity was reversed (P<.01). In the microscopic study,
the change in F/Fapa% varied significantly over the
microscopic regions examined. The maximum values of
F/Fapa% for hyperpolarizing shocks during
diastole reached only 7±10% of those for shocks during
the plateau (P<.01). During diastole, the time
until a new action potential occurred after the beginning of the shock
was shorter when the membrane was depolarized (1.1±0.5 ms) than when
it was hyperpolarized (12.8±9.1 ms, P<.01). Conclusions
are as follows: (1) A shock can induce either
hyperpolarization or depolarization. (2)
Hyperpolarization or depolarization during a shock
can occur near either the anodal or cathodal shock electrode. (3)
Variation of F/Fapa% exists within a microscopic
region. (4) The effects of a shock during an action potential plateau
are different from those during diastole. (5) The different
responses of the F during a shock affect the excitation latency
during diastole.
Key Words: action potential duration • di-4-ANEPPS • electrical defibrillation • optical transmembrane potential
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Introduction |
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Delivering a strong electrical shock across the heart has long been used to terminate ventricular fibrillation in clinical and experimental practice, yet the mechanism by which the shock affects the heart is still not completely known. Although much work has been performed to study the changes of action potentials following an electrical field stimulus, such as changes of action potential duration and refractory period after a shock,1 2 3 4 5 6 7 only a few studies have used optical recording techniques to study cellular effects during the electrical field stimulus itself. In single isolated cells, these studies showed depolarization at the end of the cell facing the cathode and hyperpolarization at the other end of the cell facing the anode.8 9 In isolated rabbit hearts, depolarization or hyperpolarization of the membrane potential has previously been recorded at a single recording spot3 5 10 and at 64 spots.11
Several mathematical models have been developed to predict the relation
between the shock strength and the responses of the transmembrane
potential, but some of the results of these models are conflicting and
have not yet been verified experimentally.12 13 14 15 For
example, some models predict that the change in transmembrane potential
over space during a shock exhibits a "sawtooth" pattern, with
each sawtooth corresponding to a single cell or group of cells; ie, one
end of a cell or group of cells is depolarized, and the other end is
hyperpolarized.13 14 If the sawtooth pattern exists and
the magnitude of depolarization equals that of
hyperpolarization, the transmembrane potential
changes during a shock should be summed to zero when a single averaged
measurement of transmembrane potential is made from many cells over a
large space. However, only depolarization of the transmembrane
potential was observed during the shock, when a large single optical
recording spot with a diameter of 750 µm and a depth of
several hundred micrometers was recorded by
Dillon,3 5 suggesting that the sawtooth pattern either (1)
is not present, (2) is asymmetrical, or (3) is superimposed on
another pattern of transmembrane potential changes. Recently, Knisley
et al11 and Neunlist and Tung16 have shown
that the optical transmembrane potential, called
m since it represents a weighted
average over the volume of illumination,16 can be either
depolarized or hyperpolarized near an anodal point–stimulating
electrode in myocardium. This differs from the traditional
concept that only depolarization occurs near the cathode and only
hyperpolarization occurs near the
anode.17 18 However, this result is consistent
with another family of models of the myocardium called
bidomain models.12 19
Optical recording techniques have an advantage over
conventional electrical recordings in that they do not
record the shock or stimulus artifacts. Optical recording
has recently been used to investigate the m
changes caused by either point or field electrical stimulation in
isolated cardiac cells9 20 or in the
heart.3 5 10 11 16 One of the disadvantages of optical
recording is the large illumination area in the heart, which
results in recordings from many cells rather than a single
cell. Only a few studies avoided this disadvantage by recording
optical signals in either isolated cardiac cells8 9 or
cultured cardiac cell strands.21 In an attempt to reduce
the influence of optical volume recording, the present
study used an illumination area of 30-µm diameter and recorded
the optical signals in a microscopic region by moving this optical spot
in 30-, 100-, or 300-µm steps between adjacent recording
spots.
Cellular excitation and alteration in cardiac action potentials that
occur after a defibrillation shock are thought to result from changes
in the transmembrane potential caused by the shock.
Dillon3 5 has used optical recording techniques at
a single cardiac site to show the effects on action potential duration
of electrical shocks delivered during different phases of the action
potential. More information about the spatial distribution of
transmembrane potential changes during a shock is needed. Also,
understanding the mechanism of defibrillation requires knowledge of the
transmembrane potential changes during the shock in addition to action
potential changes following the shock. Therefore, the purpose of the
present study is to determine the m
changes at different recording sites during shocks of
defibrillation-threshold strength in perfused rabbit hearts.
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Materials and Methods |
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Surgical Preparation
Fifteen New Zealand White rabbits weighing 3.5 to 4.5 kg were injected intravenously with sodium nembutal (30 mg/kg body wt) and with 2000 U heparin. The hearts were rapidly excised through a median sternotomy and immersed in cold Tyrode's solution. The hearts were then mounted on a Langendorff perfusion system, and the coronary arteries were continuously perfused via a cannula in the aortic root with Tyrode's solution under a constant pressure of 45 mm Hg. Both ventricles were vented. The heart sat in a silicon cradle, and the posterior surface of the heart from which the recordings were made was exposed to room temperature air. The sinus node was removed to reduce the heart rate so that the ventricles could be regularly paced by S1 stimuli. The Tyrode's solution had the following formula (mmol/L): NaCl 123, CaCl2 1.8, MgCl2 1.1, KCl 4.5, Na2HPO4 1, NaHCO3 20, and glucose 11. The solution was continuously gassed with a mixture of 95% O2/5% CO2, giving a pH of 7.35 to 7.40. Solution temperature was controlled with a thermostatic water bath, and the left ventricular endocardial temperature was monitored by a thermistor and maintained in the range of 36°C to 38°C. The coronary effluent was collected and returned to the perfusion reservoir. A total volume of 4 L Tyrode's solution was continuously recirculated throughout the experiment.
Two round platinum-mesh shock electrodes (10-mm diameter) were sutured
on the right and left ventricles (Fig 1
). The distance
between these two electrodes was 
30 mm. Recordings of
m were made from the posterior portion of
the ventricles between the two patch electrodes. A pair of stainless
steel wires was inserted into the wall of the left ventricle to
record ventricular electrograms and to monitor the
ventricular rhythm. Another pair of stainless steel wires
was inserted into the ventricular apex to pace the
ventricles and to induce ventricular fibrillation. To
measure the surface extracellular potential gradient during the shock,
two tungsten electrodes of 0.1-mm diameter were held 4 mm apart in a
small piece of silicon rubber. These electrodes were placed immediately
adjacent to each laser recording spot as shocks were given. The
two electrodes were positioned so that the imaginary line connecting
the two electrodes was superimposed on the imaginary line connecting
the two shock electrodes. The potential gradient was calculated as the
peak difference between the two potential recordings during the
shock divided by the 4-mm distance between the two recording
electrodes.
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Experimental Protocol
At the beginning of each experiment, the defibrillation
threshold of the heart was determined for a 10-ms truncated exponential
monophasic shock generated by a defibrillator (Ventritex HVS-02)
delivered through two patch-shock electrodes. Episodes of
ventricular fibrillation were induced with 60-Hz
alternating current delivered for 2 seconds to the
ventricular apex via two pacing electrodes.
Ventricular fibrillation was detected by (1) the rapid
activation rate of ventricular electrograms and (2) the
loss of synchronized ventricular contraction.
Defibrillation shocks were given after 15 seconds of fibrillation.
Initial shock intensity was 60 V. The intensity of subsequent shocks
was modified on the basis of the success or failure of the previous
defibrillation shock. The shock intensity was increased by 10 V if the
shock failed to defibrillate or was decreased by 10 V if the shock
succeeded. The lowest voltage resulting in successful defibrillation
was designated as the defibrillation threshold. Unsuccessful
defibrillation shocks were followed by a salvage shock. There was a
4-minute interval between each fibrillation episode. During
ventricular fibrillation, coronary perfusion was
discontinued by interrupting aortic perfusion. Coronary artery
perfusion was resumed immediately after defibrillation. The
defibrillation threshold for both shock polarities was determined. The
higher defibrillation threshold was used for the next test if the
defibrillation thresholds were different.
After measurement of the defibrillation threshold, diacetyl monoxime
was added to the Tyrode's solution at a concentration of 20 mmol/L to
eliminate ventricular contraction. Lack of contraction was
confirmed by a CCD video camera. A voltage-sensitive dye, di-4-ANEPPS,
was dissolved in ethanol,9 added to Tyrode's solution,
and used to record m from spots between
the two patch-shock electrodes. The final concentration of di-4-ANEPPS
in the Tyrode's solution was 0.005 mmol/L. Our pilot experiments
indicated that there was only a small difference in defibrillation
threshold before and after administering di-4-ANEPPS and diacetyl
monoxime (P=NS, authors' unpublished data, 1994).
Experimental trials were begun after 10 minutes of exposure of the
heart to the di-4-ANEPPS Tyrode's solution. The heart was paced by a
WPI stimulator with stimulus isolator (WPI Pulsemaster A300). Twenty
S1 stimuli were given at twice diastolic
threshold strength and 2-ms duration with a 300-ms
S1-S1 interval. A 10-ms truncated exponential
monophasic S2 shock of defibrillation-threshold strength
was given from the defibrillator during the plateau of the last
S1 action potential. Since the time required for activation
to propagate from the pacing site to the optical recording site
varied with the location of the different recording sites, the
S1-S2 interval was adjusted during each
experiment in order to deliver the S2 shock during the
plateau, 20 to 30 ms after the upstroke of the last S1
action potential at the recording site. This
S1-S2 interval was chosen because the cells are
refractory at this time. Therefore, the change in
m during the shock can be observed and
measured without being contaminated by the upstroke of a shock-induced
new action potential.9 11
Shocks of each polarity were tested at each recording spot. For the microscopic study described below, after shocks were administered during the plateau of the action potential, shocks with either polarity were also given during the diastolic period at some recording spots. These recordings were made in sequence at each site. First, the last S1-paced action potential was recorded as a control action potential without an S2 shock. Second, the last S1 action potential was recorded with an S2 shock delivered during the plateau of the action potential or during diastole. Third, the last S1 action potential was recorded with an S2 shock with the same timing and strength but of opposite polarity to the previous S2 shock. Shock polarity was tested in random order. The first set of recordings was made from the site closest to the left ventricular shock electrode. After each set of three optical recordings, the recording spot was moved to the next recording spot from the left ventricle toward the right ventricle. The shock potential gradient at each recording spot was calculated by the shock potential recorded by the two tungsten electrodes adjacent to the recording spot and the distance between these two electrodes.
The distance between adjacent spots ranged from macroscopic to
microscopic. In the macroscopic study, which used six animals,
recordings were made from nine spots with 3-mm spacing between
adjacent recording spots (Fig 1
). In the microscopic study,
which involved a total of nine animals, there were three regions from
which nine recordings in a line were made. Two regions were 3
mm away from each shock electrode, and the third was midway between the
two shock electrodes, corresponding to spots 1, 5, and 9 (L, M, and R
in Fig 1). m was optically recorded
sequentially from nine equispaced recording spots in each of
the three regions, with the S2 shock given during the
action potential plateau. There were three subgroups in the microscopic
study, with three perfused hearts studied in each subgroup. Subgroup 1
had 30-µm spacing between adjacent recording spots. Subgroup
2 had 100-µm spacing between adjacent recording spots.
Subgroup 3 had 300-µm spacing between adjacent recording
spots. In all animals in the microscopic study, shocks with both
polarities were given during the action potential plateau at each
recording site and were also given during diastole
at one of nine recording spots at each region, ie, 3 mm apart
from the shock electrodes and midway between two shock electrodes. The
time required for all recordings during each experiment was <2
hours.
Signal Recordings
A 514-nm argon laser beam passed through and was focused by an
objective lens (model 170 10/0.25, Leitz Wetzlar; numerical aperture,
0.25). A region of the epicardium 30 µm in diameter was
illuminated with 0.4 mW or 56.6 W/cm2 of light. The
10-mm diameter of the 30-µm laser beam shown on a monitor by a CCD
video camera was calibrated by a reticle. The CCD video camera was also
used to monitor the heart surface to ensure that there was minimal
motion of the heart during recordings in the microscopic group.
The illumination light power was measured after the experiment by a
digital power meter probe (model 815, Newport) placed in the position
previously occupied by the heart. The laser illumination was turned on
just before and turned off just after each trial to reduce
photobleaching of the dye and the detrimental effect of the laser beam
on the cells. The total illuminating and optical recording
interval during each S1-S2 trial was 600
ms.
Fluorescence light was collected by the objective lens and
passed upward through a dichroic beam splitter with a cutoff wavelength
of 520 nm to separate the reflected illumination from the
fluorescence. The fluorescence light passed through an
eyepiece (Bausch and Lomb; focal length, 1 cm) and then through a
long-pass filter with a cutoff wavelength of 590 nm. An avalanche
photodiode (model TIED69, Texas Instruments) with an active diameter of
1.5 mm was positioned above the eyepiece and filter with a
micromanipulator. The reverse voltage was held constant with an
isolated battery supply and was kept below the breakdown voltage to
ensure linearity of the photodiode response. Magnification at the
photodiode was x10. Therefore, fluorescence light was
collected from a 150-µm-diameter region of heart. This larger
collection area, 150 µm in diameter compared with the 30-µm
illumination area, was used to ensure the collection of all
fluorescence and to reduce noise. The fluorescence
signal was low-pass–filtered at -3 dB and a frequency of 1063 Hz. The
optical system responded in 1 ms to a step increase in
fluorescence. The recordings from the laser system, the
ventricular electrogram, and the shock potential
recordings were monitored and stored on a digitizing
oscilloscope with 12-bit accuracy (model 3001A, Norland) and computer
hard disk (Macintosh IIx, Apple). The sampling rate for the
recordings was 2000 Hz, with which the transmembrane potential
changes during the shock were the same as those when a sampling rate of
8000 was used (authors' unpublished data, 1994).
Analysis of Results
The changes in fluorescence corresponding to amplitudes
of paced action potentials and m changes
induced during S2 shocks were expressed as a percentage of
the background fluorescence level. The optical signal could not
be interpreted to give a direct indication of the absolute
transmembrane voltage. Fapa was represented by
the change in fluorescence between the maximum depolarization
potential reached by that action potential and the minimum
repolarization potential reached at the end of the previous action
potential. F during an S2 shock was calculated as the
difference between the peak fluorescence deflection (either
depolarization or hyperpolarization) during the
shock and the fluorescence just before the shock. F was
measured as follows: (1) The beginning of a shock was determined from
the signal recorded by the shock potential recording
electrodes. (2) A baseline, determined as the mean value during a 2-ms
interval just before the beginning of the shock, was obtained. (3) Peak
fluorescence change within the interval from the beginning to
10 ms after the beginning of the shock was determined with respect to
the baseline. F during the shock was normalized by dividing it by
the last S1 Fapa and multiplying by 100 to
represent it as a percentage, ie, F/Fapa%.
Depolarization of the membrane potential during a shock was defined as
a more positive membrane potential during the shock than just before
the shock; hyperpolarization was defined as a
membrane potential more negative during than just before the shock. The
maximum dFapa/dt was defined as the maximum time
derivative of the Fapa upstroke from the last
S1 action potential. APD80 was defined as the
interval from the maximum time derivative of the Fapa
upstroke to the time for Fapa to decline to 20% of its
amplitude. The shock APD80 was normalized by dividing it by
the control S1 APD80 and multiplying by 100.
For a shock given during diastole, excitation latency was
measured as the interval between the beginning of the S2
shock and the maximum time derivative during the upstroke of the
S2-induced optical action potential.
F and the action potential duration were expressed as a percentage
to compensate partially for variations (1) in staining by the dye, (2)
in dye washout, and (3) among different animals and different
S1-S2 episodes. A linear subtraction was
performed in which a line passing through the baseline before
S1 and the baseline 450 ms after S1, ie,
after repolarization occurred, was subtracted from the
fluorescence recordings to compensate for decreases in
fluorescence introduced by bleaching of the dye by the laser
illumination. The beat-to-beat variation in fluorescence for
the last S1 Fapa at the same recording
site was also measured to see if the fraction of fluorescence
changes during different trials at the same site was stable. The
variation in fluorescence from beat to beat at the same
recording site was determined by dividing the Fapa
corresponding to the upstroke of the S1 optical action
potential during which the shock was given by the upstroke of the
previous S1 optical action potential and then multiplying
by 100, ie, Fapa%. The maximum variation of the changes
in fluorescence from beat to beat was obtained from each
subgroup of the microscopic group. The repeatability of F was
determined for six recording sites in which shocks with the
same strength and polarity were repeated while recordings were
made at the same site. The F caused by these shocks was determined
and compared to determine the difference in F caused by two
sequential shocks. The data were analyzed by ANOVA
(Student-Newman-Keuls test) and the paired t test. A value
of P<.05 was interpreted as significant. Values are given
as the mean±1 SD.
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Results |
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Magnitude of Fluorescence Change During the Upstroke of the Last S1 Action Potential
Fapa, which indicates the change in the fluorescence corresponding to the last S1 action potential amplitude, was determined as a fraction of the baseline fluorescence before the last S1 pacing stimulus. Fapa in the present study was 6.7±1.3%. The amount of repolarization of the last S1 action potential occurring during the interval between the two measurement times (just before the shock and at the peak fluorescence change during the shock) was 1.5±1.8% of Fapa. The changes in Fapa, ie,
Fapa, of the last S1 action
potential from five consecutive recordings in the microscopic
group were 0.0±0.0%, -0.5±4.7%, -0.8±4.0%, -0.4±5.9%, and
-1.4±6.6% (P>.05 compared with the first
recording), where 0.0±0.0% represents the first
S1 Fapa of the five trials. The changes in
maximum dFapa/dt of the upstroke of the last
S1 action potential from these five consecutive
recordings in the microscopic group were 100±0%, 103±22%,
99±16%, 103±23%, and 102±19% (P>.05 compared with the
first recording), where 100±0% was taken as control from the
first recording of the five trials. Lack of significant changes
in Fapa and maximum dFapa/dt for the
five consecutive recordings indicated that the optical
recordings at the same spot were reproducible and that the dye
and the optical excitation had negligible effects on the
tissue.22 The defibrillation threshold for the anode on
the left ventricle and the cathode on the right ventricle was
3.9±7.4% lower than the defibrillation threshold for the reversed
polarity (P=.055). The lack of significant difference
between the two shock polarities might be due to both shock electrodes
being on the ventricles.
F During Shock in Macroscopic Study
Shocks equal in strength to the defibrillation threshold induced
significant changes in m when given during
the plateau of the action potential. Fig 2 shows the
mean change in F/Fapa% during the S2
shocks. These changes included either depolarization or
hyperpolarization, depending on the shock polarity.
When the shock polarity was positive to the right
ventricular electrode and negative to the left
ventricular electrode, the mean change in
m was depolarization (mean value above the
zero line in the top of Fig 2) across the heart at all nine spots. When
the shock polarity was the opposite, ie, negative in the right
ventricle and positive in the left ventricle, the mean change was
hyperpolarization (mean value below the zero line
in the top of Fig 2) at all spots.
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represents 
, results for the
opposite polarity (L+R-). Normalized APD80 was
significantly longer (P<.05) at spots 1, 2, 3, 8, and 9
(indicated by asterisks) compared with the control action potential
duration, which was the last S1 action potential during the
series of 20 S1 stimuli in which no S2 shock
was given. Brackets represent 1 SD.
The large standard deviations of the mean values (Fig 2) were caused by
the fact that although depolarization was caused primarily by one shock
polarity and hyperpolarization by the other
polarity, exceptions did occur. When the shock polarity was positive to
the right ventricular electrode and negative to the left
ventricular electrode, depolarization occurred near the
left ventricular shock electrode (spot 1) in five of six
shocks, and hyperpolarization occurred only once
(Table 1). Near the right ventricular shock
electrode (spot 9), depolarization occurred in four of six shocks, and
hyperpolarization occurred in the other two cases
with the same shock polarity. With the opposite polarity, ie, negative
to the right ventricular electrode and positive to the left
ventricular electrode, depolarization was induced near the
left ventricular shock electrode (spot 1) in only one of
six shocks, whereas the other five cases showed
hyperpolarization. Depolarization was induced near
the right ventricular shock electrode (spot 9) in two of
six shocks, whereas the other four cases showed
hyperpolarization. When
hyperpolarization and depolarization at each
recording site were separated into individual groups regardless
of the shock polarity, the amount of
hyperpolarization during the shock was 1.8±0.9
times the amount of depolarization at the same recording spot
when the shock polarity was reversed (P<.01). Reversing
shock polarity usually reversed the polarity of the transmembrane
potential change, ie, from depolarization to
hyperpolarization or from
hyperpolarization to depolarization. Thus, whereas
either shock polarity induced either depolarization or
hyperpolarization, one or the other predominated
for each shock polarity.
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As expected, the extracellular potential gradient field created during
the shock was largest nearest the shock electrodes (spots 1 and 9) and
decreased to a minimum in the region midway between the two shock
electrodes (spots 4, 5, and 6), as shown in the bottom of Fig 2. The
magnitudes of the mean value in F/Fapa% for both
polarities were 77±41%, 46±32%, 32±27%, 28±20%, 37±25%,
24±20%, 33±22%, 37±25%, and 59±29% for spots 1 through 9,
respectively. F near the shock electrodes (spots 1 and 9) was
greater than that farther away from the shock electrodes
(P<.05), consonant with the higher potential gradients at
these two spots. However, F/Fapa was not statistically
different among spots 2 through 8 (P=NS). The correlation
between F during the shock (TP) and the potential gradient (PG) was
weak (r=.45), with a regression equation of
TP=0.3+3.3PG.
Fig 3 shows m
recordings during all of the shocks in one rabbit heart. Shocks
with negative polarity to the left ventricular electrode
and positive polarity to the right ventricular electrode
induced depolarization at all recording spots except spot 3,
where hyperpolarization occurred. Shocks with the
opposite polarity induced hyperpolarization at all
recording spots except spot 3, where a small depolarization
occurred. In the top tracings, depolarization occurred near both anodal
and cathodal shock electrodes (spots 1 and 9), whereas
hyperpolarization occurred near both electrodes in
the bottom tracings, with shock polarity reversed. The responses near
the shock electrodes (Fig 3) were larger than those at the center, and
the degree of hyperpolarization was greater than
the degree of depolarization at the same recording spot. A
large variation in F/Fapa% could occur within a space
of 3 mm, as illustrated by the large changes between spots 2 and 3 and
between spots 3 and 4 (Fig 3).
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Prolongation of Action Potential Duration by Shock
Action potential duration in the macroscopic study was
significantly prolonged by shocks at defibrillation-threshold strength
at spots 1, 2, 3, 8, and 9 (Fig 2) (P<.05). At spots 1 and
9, which were 3 mm away from the shock electrodes, the normalized
action potential duration was 126±9% and 120±13%, respectively. At
the midpoint between the two shock electrodes (spot 5), the normalized
action potential duration was only 101±12% compared with the control
S1 action potential duration of 100% (P=NS).
Thus, those recording spots where the action potential duration
was significantly prolonged were close to the shock electrodes and
received higher potential gradients (Fig 2) than the spots (spots 4, 5,
6, and 7) where action potential duration was not significantly
prolonged. The correlation between normalized APD80 and
F during the shock (TP) was APD80=105+0.17TP
(r=.4, P<.0001). The weak correlation is
indicated by the small r value. For all recording
spots in the macroscopic study, the normalized APD80 for
shocks that induced hyperpolarization was
110±14%, which was not significantly different from that for shocks
that induced depolarization, 113±13%.
F During Shock in Microscopic Study
Since a large variation in m could
occur 3 mm apart (Fig 3), we measured the m
changes during the shock in three microscopic regions with a distance
of 30, 100, or 300 µm between the nine recording spots
at each of the three regions. The potential gradients generated by the
shocks in the three recording regions (L, M, and R in Fig 1)
were 18±2, 7±2, and 19±3 V/cm. m changed
at the nine recording spots in each region for all experiments
(Fig 4). For each of the three distances between
recording spots, the responses predominately observed during
the shock were similar to those seen in the macroscopic study: (1) The
F/Fapa% near the shock electrodes (left and right
ventricular [L and R regions]) was greater than that at
the midpoint (M region). (2) Depolarization or
hyperpolarization during the shock could occur near
either shock electrode with either shock polarity; however,
depolarization was more common when the left ventricular
electrode was the cathode, and hyperpolarization
was more common when it was the anode. (3)
Hyperpolarization was usually greater than
depolarization at the same recording spot when the polarity was
reversed. Hyperpolarization occurred in 78% of the
shock episodes near the anodal shock electrode and 28% near the
cathodal electrode; depolarization occurred in 22% of shock episodes
near the anode and 72% near the cathode.
Hyperpolarization was 78±15%, 28±2%, and
46±23% of the last S1 action potential amplitude at spots
L, M, and R, respectively, which was greater than depolarization at the
same spots (47±18%, 15±10%, and 24±23%) (P<.01). The
change in m varied within a
microscopic region, since the values for each of nine spots did not
form a horizontal straight line (Fig 4). The variation of
F/Fapa% in Table 2 was calculated for
each recording region with the same spacing between
recording spots by subtracting the minimum
F/Fapa% from the maximum for each curve in Fig 4. Table 2 shows the existence of variation of F/Fapa% during
the shock within a microscopic region with a maximum variation of
23±10% to 59±34% of Fapa. Of the 54 curves in Fig 4, 11
crossed zero, indicating that the m change
varied from depolarization to hyperpolarization or
vice versa in the microscopic region. Table 2 also shows the maximum
beat-to-beat variation of Fapa for the last
S1-induced action potential upstrokes at recording
sites in each microscopic group. The mean values for such maximum
variation (Fapa%) was significantly lower than the mean
value of the maximum variation of F/Fapa% caused by the
shocks in each subgroup (P<.01), suggesting that the
variation in F/Fapa% during the shock existed over
space. In addition, in six recording sites where F caused by
a shock was determined repeatedly for the same recording site,
the mean value of difference in F/Fapa% caused by two
sequential shocks was 4.6±2.3%, which was significantly smaller than
the mean value of the maximum variation of F/Fapa%
caused by the shocks over space in each microscopic subgroup
(P<.01). Thus, the variation in F/Fapa% in
each microscopic subgroup was caused mainly by the
m changes over space during a shock, not by
the lack of repeatability of the recordings.
|
). The ordinate gives
|
Fig 5 shows the optically recorded action potentials
from a region 3 mm from the left ventricular shock
electrode in a perfused heart in which the distance between adjacent
recording spots was 100 µm. The potential gradient in this
recording region was 20 V/cm. When the shock polarity was
negative to the left ventricular electrode, the shock
induced depolarization of the optical transmembrane potential. The
amplitude of depolarization became smaller as the recording
spots were moved from left to right in 100-µm steps until the zero
point was crossed and hyperpolarization occurred at
spots 8 and 9. For shocks of the opposite polarity, ie, positive to the
left ventricular electrode,
hyperpolarization became smaller in amplitude as
the recording spots were moved from left to right. Thus, Fig 5
illustrates a large change in the m during
a shock within an 800-µm region.
|
F Caused by Shock During Diastole
Optical recordings were also performed during
diastolic shocks in the nine isolated perfused rabbit
hearts in the microscopic study. The shock was delivered during both
the plateau of the action potential and diastole at three
recording spots (L, M, and R in Fig 1).
Hyperpolarization caused by the shocks during
diastole reached only 3±4%, 3±3%, and 3±5% of the
last S1 action potential amplitude at L, M, and R regions,
respectively, before a new action potential occurred. This degree of
hyperpolarization was only 7±10% of the amplitude
of hyperpolarization when the shock was given
during the action potential plateau (P<.05). For shocks
during diastole, excitation latency, ie, the time after the
beginning of the 10-ms shock when a new action potential occurred, was
shorter when m was depolarized (1.1±0.5
ms) than when it was hyperpolarized (12.8±9.1 ms, P<.01).
Because of the short latency, depolarization caused by the shock could
not be quantified because a new action potential was almost immediately
superimposed on it. Thus, the changes in m
caused by shocks delivered during the plateau of the action potential
differ from those during diastole, and the response of
m varies with shock polarity.
Fig 6 shows optical recordings in which the
shock was delivered during the plateau of action potential and during
diastole. In this example, the recordings were made
at a spot 3 mm away from the left ventricular shock
electrode. The potential gradient at this recording spot was 20
V/cm. For the shocks delivered during the action potential plateau,
depolarization occurred when the shock polarity was negative to the
left ventricular electrode (L-R+ in Fig 6A);
hyperpolarization occurred with the opposite
polarity (L+R- in Fig 6A). During diastole, a much smaller
amount of hyperpolarization was observed (L+R- in
Fig 6B) with the same polarity and strength shock that caused
hyperpolarization during the plateau of the action
potential (L+R- in Fig 6A). The initiation of the action potential
occurred nearly at the beginning of the shock that induced
depolarization (L-R+ in Fig 6B), whereas it occurred 16 ms after the
onset of the shock that induced hyperpolarization
(L+R- in Fig 6B), which is 6 ms after the end of the shock.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study examined the responses of the cardiac transmembrane potential of the perfused heart to shocks of defibrillation strength. To pursue this goal,
m recordings were used to avoid the
shock artifact, which often occurs during conventional microelectrode
recordings and which may obscure the transmembrane potential
during the shock. The main results of the present study are that
(1) a shock can induce either hyperpolarization or
depolarization, (2) hyperpolarization or
depolarization during the shock can occur near either the anodal or
cathodal shock electrode, (3) variation of F during the shock exists
within a microscopic region, (4) the effects of a shock during the
plateau of an action potential are different from those during
diastole, and (5) the different responses of
m during a shock affect the excitation
latency during diastole.
F values in the present study confirm and extend the results of
previous experiments in isolated cells and hearts using optical
recording techniques.3 5 7 8 9 10 11 16 Shock field
stimulation can induce either depolarization or
hyperpolarization, depending on the shock polarity
as reported by others.9 10 The magnitude of F is
greatest near the shock electrode and decreases with increased distance
from the electrode (Figs 2 and 3). Our findings of both depolarization
and hyperpolarization during a shock differ from
the results of Dillon,3 5 who found only depolarization
during a shock. This difference may have arisen because Dillon (1) used
a different shocking electrode configuration than we did, (2) gave
shocks of only a single polarity, and (3) recorded from only a
single spot. This single spot may have been in a region that was always
depolarized by the shock electrode location and polarity used in the
Dillon study.
Theoretical models for a one-dimensional cable predict that during a
stimulation pulse the transmembrane potential is depolarized near the
cathodal electrode and hyperpolarized near the anodal
electrode.17 18 However, the present study
demonstrated that either hyperpolarization or
depolarization during the shock can occur near either anodal or
cathodal shock electrodes (Table 1). These results are
consistent with the study of Knisley et al,11 in
which stimulation produced hyperpolarization in a
region a few millimeters away from a cathodal electrode in the perfused
left ventricle with prefrozen endomyocardium and
midmyocardium, and with the study of Neunlist and
Tung,16 23 which indicated regional depolarization of
cardiac muscle adjacent to an epicardial anode. This phenomenon may be
explained by bidomain models of two-dimensional myocardium,
which predict that although point stimulation with an extracellular
cathodal electrode produces depolarization in the tissue directly
beneath the electrode and in tissue from the electrode in the direction
across fibers, it produces regions of
hyperpolarization a short distance away from the
electrode along fibers because of different anisotropies of the
intracellular and extracellular conductances in the
tissue.11 24 25 The distance from the cathode at which
hyperpolarization first occurs is thought to vary,
depending on fiber orientation with respect to the electrodes. Thus,
small differences in distance to the electrode and in fiber orientation
might explain why depolarization was recorded in five hearts and
hyperpolarization was recorded in one heart at
recording spot 1 (3 mm from the left ventricular
electrode) when it was the cathode and why depolarization was
recorded in two hearts and hyperpolarization
was recorded in four hearts at recording spot 9 (3 mm
from the right ventricular electrode) when it was the
cathode (Table 1).
Our results demonstrate that when a shock is given during the plateau of the action potential, the shock-induced hyperpolarization obtained with one shock polarity is 1.8±0.9 times the shock-induced depolarization obtained with the other shock polarity at the same recording spot. These results are consistent with the results observed in the optical recordings of transmembrane potential for electrical field stimulation given during the action potential plateau in isolated ventricular myocytes9 and in perfused rabbit hearts11 as well as in intracellular recordings for pulses of either polarity given during the action potential plateau in isolated ventricular fibers.26 If the only effect of a shock field on the transmembrane potential was to charge the membrane and the membrane had no rectifying properties, then the magnitude of depolarization during a shock should be the same as the magnitude of hyperpolarization when the shock polarity is reversed.17 This difference in the magnitude of hyperpolarization and depolarization may be caused by the rectifying properties of the ion channels, with a higher impedance to current flow in one direction versus the other across the cell membrane.27 Another possibility is that in addition to charging the membrane, a shock may alter the active processes in the membrane, such as the conductivities of the ionic channels, leading to larger hyperpolarization than depolarization.
The results of the present study show experimentally for the first
time the existence of spatial variation in F caused by a shock
within a microscopic region from 0.24 to 2.4 mm in length (Fig 4). Such
changes varied from 23±10% to 59±34% of the action potential
amplitude (Table 2). F values during the shock could change from
depolarization to hyperpolarization in a region as
small as 0.8 mm (Fig 5).
This phenomenon is difficult to explain by a mathematical model
predicting that the oscillation of the transmembrane
potential changes over space during a shock exhibits a sawtooth
pattern, with each sawtooth corresponding to a single cell or group of
cells.14 According to the single-cell form of this model,
with the exception of cells very near the shock electrodes, the part of
each cell nearest the cathodal electrode is depolarized, and the part
nearest the anodal electrode is hyperpolarized. The optical
recording spot size in the present study may encompass
several cells to hundreds of cells. Thus, we should have recorded a
value near a weighted mean of the transmembrane potential changes over
all of the cells within the recording spot. According to the
sawtooth model, all cells except those close to the shock electrodes
have similar spatial patterns of depolarization and
hyperpolarization with the magnitude of
transmembrane potential directly proportional to the extracellular
potential gradient generated by the shock.13 Since the
potential gradient changed slowly over small distances in the heart
(Fig 2; and authors' unpublished data, 1994), the sawtooth model would
predict only small variation in the transmembrane potential changes
during the shock over a microscopic region instead of the large
variation and occasional changes in polarity that were observed (Fig 4). The sawtooth model predicts a strong linear relation between the
potential gradient and the change in transmembrane potential over
space.13 14 Only a weak correlation (r=.45, Fig 2) was observed.
The changes can be better explained if the sawtooth model is modified
so that each sawtooth occurs over a bundle of cells 2000 µm long
and 200 µm wide instead of over a single cell.13 In this
case, each individual sawtooth is larger than our recording
spot size, so that large changes in transmembrane potential during the
shock could be observed at adjacent recording sites. Other
models can also explain these large variations. The presence of
connective tissue septae or blood vessels may create secondary sources
that cause large variations in transmembrane potential changes near
them.25 Different forms of the bidomain model also predict
such large variations in the transmembrane potential changes caused by
the shock.12 28
One possible reason for the observed result that a shock given during
diastole produced smaller F values than the same
strength shock given during the action potential plateau may be the
higher membrane conductance during diastole than during the
action potential plateau.10 29 A second possible reason
may be a combination of passive and active processes during a shock
delivered during diastole. The large
hyperpolarization that was observed when the shock
was given during the action potential plateau could have been
counteracted by an active process, such as ionic channel activity
inducing inward currents caused by the same strength shock given during
diastole. This explanation is supported by another result
obtained in the present study when the shock was given during
diastole. Excitation latency was shorter when the
transmembrane potential was depolarized (1.1±0.5 ms) than when it was
hyperpolarized (12.8±9.1 ms, P<.01). When shocks, which
induced hyperpolarization during the action
potential plateau, were given during diastole, the upstroke
of the shock-induced action potential could sometimes occur during the
shock interval (<10 ms after the beginning of the shock pulse),
suggesting the opening of sodium channels leading to an inward current
during the shock. This inward current may counteract the
hyperpolarization effect of a shock, resulting in
the smaller transmembrane potential changes (Fig 6). On the other hand,
the hyperpolarization caused by a shock may have
delayed the initiation of a new action potential so that the excitation
latency was longer when the transmembrane potential was hyperpolarized
by this shock.
Previous studies have reported that action potential prolongation by a
shock increases when shock strength or the
S1-S2 coupling interval is
increased.1 3 4 5 6 In the present study, at
recording spots that were near the shock electrodes and
therefore exposed to higher potential gradients, APD80 was
significantly prolonged by the defibrillation-threshold shock given
during the action potential plateau,4 30 whereas at spots
midway between the two shock electrodes and exposed to low potential
gradients, APD80 was not obviously
affected.4 30 Action potential prolongation may have
occurred at spots midway between the two shock electrodes if longer
S1-S2 coupling intervals had been used in the
present study, since the action potential is prolonged
significantly more when the shock is given later during the action
potential.1 3 4 5 6 Although prolongation of action potential
duration is thought to be important for both defibrillation and the
initiation of arrhythmias,1 2 3 4 5 7 the mechanism for
prolongation is still not definitely known. It is not clear how the
prolongation of action potential duration is directly related to the
transmembrane potential changes during the shock, since action
potential prolongation occurs in both the depolarized and
hyperpolarized regions (Fig 2) and the correlation between
APD80 and F is not strong (r=.4).
Limitations
Limitations of the Optical Recording System
Optical recording techniques provide advantages over
microelectrode recording techniques for determining
transmembrane potential changes during electric field stimulation.
Optical recordings with voltage-sensitive dye can be
recorded from the whole heart and are not directly affected by the
extracellular shock field. But, optical recordings of
transmembrane potential changes during field stimulation also have
limitations. One limitation is the photobleaching effect, which can
reduce the baseline fluorescence during intense laser
illumination. To reduce the effects of photobleaching, the laser
illumination in the present study was performed only during the
recording for each shock trial, and decreases in baseline
fluorescence during recordings were eliminated by
linear subtractions (see "Materials and Methods"). Also, since
the fractional fluorescence change corresponding to a given
transmembrane voltage change is not constant for all spots, only the
relative changes in the optical transmembrane potentials can be
estimated with respect to the last S1 action potential
upstroke. Therefore, changes in m caused by
a shock were expressed as a percentage of the last S1
optical action potential upstroke. Another limitation of the optical
recording system is the large volume of myocardium
from which fluorescence is collected. The 30-µm area
illuminated by each spot may cover only a few cells. But, the depth of
the optical recording volume may be hundreds of
microns.3 As a result, a single recording is
probably a weighted mean from up to hundreds of cells instead of a
recording from a single cell.16 Thus, the optical
recording volume may cause some information to be missed if
rapid spatial changes in transmembrane potential occur within the
recording volume. The action potential plateau was gradually
repolarizing during the interval when shocks were given; hence, the
hyperpolarization and depolarization measurements
may include this normal repolarization, which was 1.5±1.8% of the
action potential amplitude.
Effects of Diacetyl Monoxime on Transmembrane
Potentials
Diacetyl monoxime (20 mmol/L) was used in the present study to
eliminate the motion artifact during the optical
recording.9 This concentration of diacetyl
monoxime has been reported to reduce the contractile force to zero, but
this effect is reversible.9 31 There are no major effects
of diacetyl monoxime on the membrane electrical properties. For
example, in a study using guinea pig papillary muscles, 20 mmol/L
diacetyl monoxime decreased the action potential amplitude from a
control value of 128 mV in normal Tyrode's solution to 123 mV, whereas
it slightly shortened the action potential duration.31
Thus, there should have been no major influence of diacetyl monoxime on
the amplitude of F during a shock in the present
study.9 Also, action potential prolongation and refractory
period prolongation occurring in the presence of 20 mmol/L diacetyl
monoxime are similar to the prolongation without diacetyl
monoxime.7 Thus, the action potential prolongation
observed in the present study is probably not due to the effects of
diacetyl monoxime.
Conclusion
An optical recording technique was used in the present
study to determine the change in transmembrane potential during shocks
of defibrillation-threshold strength in isolated perfused rabbit
hearts. These shocks induced significant depolarization or
hyperpolarization and significant variation of F
values within a microscopic region. F values for shocks during
diastole were smaller than for shocks during the action
potential plateau. For shocks during diastole, excitation
latency was shorter when the transmembrane potential was depolarized by
the shock than when it was hyperpolarized. Both
hyperpolarization and depolarization caused by
shocks in the plateau of the action potential resulted in prolongation
of action potential duration. The relation between the extracellular
potential gradient caused by the shock, F during a shock, and the
ensuing changes in action potential after the shock are complex,
suggesting that the mechanisms by which a shock defibrillates are also
likely to be complex.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by National Institutes of Health research grants HL-33637, HL-42760, and HL-44066; National Science Foundation Engineering Research Center grant CDR-8622201; American Heart Association, North Caroline Affiliate, Inc, grants NC-93-GS-19 and NC-91-G-14; North Carolina Biotechnology Center grant 9113-ARG-0612; and a grant from the Whitaker Foundation.
Received May 10, 1994; accepted May 8, 1995.
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