From the Department of Cardiology, Cardiovascular Research Institute
Maastricht, Maastricht University, the Netherlands, and the Laboratory of
Experimental Cardiology, University of Leuven, Belgium (K.R.S.).
Correspondence to Marc A. Vos, PhD, Department of Cardiology, Cardiovascular Research Institute Maastricht, University Hospital Maastricht, PO Box 5800, 6202 AZ, Maastricht, Netherlands. E-mail m.vos{at}cardio.azm.nl
Methods and Results—From chronic-AVB dogs with increased heart
weights and TdP, left midmyocardial and right ventricular
myocytes were isolated by enzymatic dispersion. These myocytes were
significantly larger than sinus rhythm (SR) controls. In chronic AVB,
the action potential spike-and-dome configuration was preserved.
However, the action potential duration (APD) at 95% and 50% of
repolarization of the left midmyocardium was significantly
larger in chronic AVB than in SR, with little change in the right
ventricle, causing enhanced interventricular dispersion of
repolarization at slow pacing rates. Treatment with the class III agent
almokalant increased the APD to a much larger extent in chronic-AVB
than in SR myocytes and resulted in a higher incidence of early
afterdepolarizations (EADs). EADs had their takeoff potential between
-35 and 0 mV. There was no evidence that spontaneous sarcoplasmic
reticulum Ca2+ release underlies these EADs.
Conclusions—In the dog, chronic AVB leads to
hypertrophy of both right and left ventricular
myocytes. The repolarization abnormalities predisposing for class
III–dependent TdP in vivo are the results of cellular
electrophysiological remodeling.
Structural adaptations accompany the altered
hemodynamic load in the heart with AVB. Autopsy studies
have demonstrated increased heart weight–to–body weight ratios, with
significant contributions of both the right and left
ventricular mass.4 Morphologically,
the biventricular hypertrophy is characterized
by an eccentric expansion with increased right and left
ventricular diameters, as seen during volume overload.
Therefore, proarrhythmia in dogs with chronic AVB seems to be
based on electrophysiological remodeling in
the presence of bradycardia and cardiac hypertrophy. This
study was designed to investigate the cellular basis of cardiac
electrophysiological and structural changes
in this animal model and to assess their contribution to the
facilitation of TdP induction.
In Vivo Studies
To document the induction of TdP, the class III agent almokalant was
administered intravenously at a concentration of 0.12
mg · kg-1 · 10 minutes infusion
time-1 in 7 of the 9 animals with chronic
AVB.8 Almokalant is a known inhibitor
of the rapidly activating component (IKr)
of the delayed-rectifier K+ current
(IK)9 and increases
regional dispersion of repolarization.8 10
Surface ECG leads and right and left ventricular monophasic
action potential catheters were positioned for simultaneous
on-line recording of the signals. TdP was defined as a
polymorphic ventricular tachycardia
consisting of
Cell Isolation Procedure
Experimental Setup
Myocytes were imaged by a video camera connected to the inverted
microscope (Diaphot 300, Nikon, Inc) of the setup. The length and width
of each cell were measured on a defined area of the monitor with a
x40 microscopic objective. Although the width was not constant along
the length of many cells, we measured this dimension as the estimated
average of the broadest and thinnest part of a cell near its middle.
More than 600 left midmyocardial and >400 right
ventricular cells per group were measured.
For the experiments with microelectrodes, sharp standard glass
electrodes filled with 3.0 mol/L KCl (resistance, 30 to 60 M
For the measurements of intracellular Ca2+
transients and action potentials, the whole-cell variant of the
patch-clamp technique was used.14 Pipettes were
pulled from borosilicate glass and had resistances of 1.0 to 3.0 M
Solutions
Statistical Analysis
Cellular Basis of Biventricular Hypertrophy
Action Potentials in Single Myocytes From Dogs With Chronic
AVB
In SR, the APD at 95% of repolarization (APD95)
increased on slowing of the pacing rate, as expected. Pooled data are
shown in Figure 5
In an additional population of 32 myocytes of the same dogs, we
recorded action potentials during whole-cell patch clamp (chronic
AVB: ncells,LV=7,
ncells,RV=8; SR:
ncells,LV=10, ncells,RV=5).
We confirmed the differences between APD of left versus right ventricle
and chronic AVB versus SR under otherwise similar conditions (data not
shown).
We also examined the peak contraction amplitudes and
[Ca2+]i accompanying the
action potentials. At the pacing CL of 1000 ms, the peak contraction
amplitude measured 4.3±1.9% versus 3.1±3.7% in right
ventricular cells (P=NS) and 7.2±2.2%
versus 5.1±2.1% in left midmyocardial myocytes (P<0.05,
chronic AVB versus SR, respectively). Contraction durations were not
different in these cell groups.
[Ca2+]i peaked at
460±171 versus 517±102 nmol/L in the right ventricular
mixture (P=NS, chronic AVB versus SR, respectively) and at
457±146 versus 496±107 nmol/L in the left midmyocardium
(P=NS).
Increased Sensitivity of Chronic-AVB Myocytes to
Almokalant
In the myocytes studied with patch electrodes (n=32), we found
comparable responses to almokalant; likewise, the average increase of
the APD was much larger in the chronic-AVB than the SR cells (Figure 7B
Action potential prolongation was often followed by the occurrence of
EADs in chronic-AVB myocytes but not in SR cells, with the
microelectrode as well as the patch-electrode technique. The
Table
Characteristics of the Almokalant-Induced EADs in Chronic
AVB
We addressed the question of whether spontaneous
Ca2+ release from the sarcoplasmic reticulum
could underlie these EADs, as described earlier for
isoproterenol-induced EADs.19 Figure 9
As to the functional characteristics of chronic-AVB myocytes, we
demonstrated that peak amplitudes of contraction and
[Ca2+]i at 1000-ms pacing
CL were not different from SR cells (or, if anything, were slightly
increased). These data, which are the subject of further
investigations, provide additional support for the notion, based on in
vivo hemodynamic measurements,4
of a compensated form of cardiac hypertrophy.
Cellular Electrophysiological Remodeling in
Chronic AVB
Intrinsic Disturbance of Repolarization
Interventricular Differences of Repolarization
Increased Susceptibility to Almokalant
Our finding that EAD amplitudes were larger in right than in left
ventricular myocytes could be related to the lower takeoff
potentials in the former cells. If indeed
ICaL is the major charge carrier of these
EADs, its triggering at lower takeoff potentials would most likely lead
to larger current amplitudes.
Cellular Basis of In Vivo Proarrhythmia
Another important factor involved in the occurrence of TdP is
ventricular ectopy. In vivo recordings in the dog
with chronic AVB confirm the importance of ventricular
ectopic beats (Figure 1
DAD-triggered action potentials or abnormal automaticity were
also not observed in the chronic-AVB cells. This all may mean that the
substrate for extrasystolic activity involved in the initiation
of TdP is multicellular (eg, phase 2 reentry) or includes the Purkinje
network. In addition, the experimental setup chosen for this study
lacks many of the components affecting arrhythmogenesis in vivo, such
as adrenergic agonists, stretch, and sudden rate changes. For this
reason, the absence of spontaneous action potentials in the single
myocyte does not exclude their generation in vivo.
Limitations of the Study
When we compared the action potential characteristics of our
right ventricular mixture with the data presented
by Sicouri and Antzelevitch,42 we found that the
majority of our cells (93% for SR and 67% for chronic AVB) had a
steep APD/CL relationship, which would identify them as right
ventricular midmyocardial cells.
Conclusions
Received August 19, 1997;
revision received April 22, 1998;
accepted April 22, 1998.
© 1998 American Heart Association, Inc.
Basic Science Reports
Cellular Basis of Biventricular Hypertrophy and Arrhythmogenesis in Dogs With Chronic Complete Atrioventricular Block and Acquired Torsade de Pointes
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—In the dog with chronic
complete atrioventricular block (AVB), torsade de
pointes arrhythmias (TdP) can be induced reproducibly by class
III antiarrhythmic agents. In vivo studies reveal important
electrophysiological alterations of the
heart at 5 weeks of AVB, resulting in increased proarrhythmia.
Autopsy studies indicate the presence of biventricular
hypertrophy. In this study, the cellular basis of
proarrhythmia and hypertrophy in chronic AVB
was investigated.
Key Words: ventricles • arrhythmia • hypertrophy • action potentials • myocytes
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
The dog with
chronic complete atrioventricular block (AVB) has been
described as an animal model of acquired torsade de pointes
arrhythmias (TdP).1 2 Clinically relevant
doses of class III antiarrhythmic agents, either alone (spontaneous
TdP) or in combination with programmed electrical stimulation, can
evoke such a proarrhythmic response in a reproducible manner in the
majority of anesthetized dogs with chronic
AVB.2 3 In vivo studies show that the duration of
AVB is an important determinant of the susceptibility to acquired TdP,
because the latter are rarely inducible at 0 weeks of AVB (acute AVB)
or at sinus rhythm (SR) but are readily inducible at 
5 weeks (chronic
AVB) in most animals.4 The increased
susceptibility to arrhythmias in chronic AVB has been related
to an inhomogeneous prolongation of the monophasic
ventricular action potential (in the left ventricle more
than the right ventricle), leading to enhanced regional dispersion of
repolarization.4 Furthermore, class
III–dependent early afterdepolarizations (EADs) are prominent, which
may explain the more frequent observation of ventricular
ectopic beats5 6 in chronic-AVB
dogs.4
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
The animal experiments were conducted in accordance with the
guidelines of the American Physiological Society
and under the regulations of the Committee for Experiments on Animals
of Maastricht University, Netherlands.
Adult mongrel dogs of either sex and weighing between 22 and 32
kg were used for the experiments. AVB was created according to the
procedure described by Steiner and Kovalik7
(ndogs=9). For a complete description of the
perioperative care, we refer to earlier
publications.2 3 4 5 beats twisting around the baseline in a setting of
prolonged QT(U) duration. These experiments were performed during
anesthesia at the latest 1 week before the dogs were
euthanized for cell isolation. In subsequent cellular experiments,
almokalant was also used. Thus, the response to class III treatment was
tested both in vivo and in vitro in dogs with documented episodes of
TdP and increased heart weights.
Anesthetized chronic-AVB (ndogs=9)
and SR dogs (ndogs=9; similar body weights)
received 10 000 IU heparin IV on thoracotomy. In the former group, AVB
had been present for a duration of 6±1 weeks (range, 5 to 9
weeks). The hearts were excised and washed in a cardioplegic solution
at 5°C to 10°C. Single right and left ventricular
myocytes were isolated simultaneously according to a
procedure adapted from Powell.11 Cannulas were
quickly inserted into and sutured to the left anterior descending and
right coronary arteries under continuous perfusion with
cardioplegic solution. Subsequent perfusion was done at 37°C with (1)
nominally Ca2+-free standard buffer solution for
10 minutes, (2) Ca2+-free solution with
collagenase for 10 minutes, and (3)
Ca2+-free solution with collagenase
plus protease for 20 to 30 minutes. The epicardial surface temperature
and the pH of the cardiac effluent were regularly measured to check for
an adequate perfusion of the tissues. Enzymes were washed out with
0.2 mmol/L [Ca2+] standard buffer solution
for 10 minutes. Finally, the tissue was minced and the cell suspension
filtered and washed. Left ventricular midmyocardial
myocytes were isolated by careful harvesting of the middle third of the
transmural wall of the perfused wedge, as described
earlier.12 Right ventricular myocytes
were harvested as a mixture from the transmural wall. Isolated myocytes
were stored at room temperature in standard buffer solution.
A sample of the cell suspension was transferred to a perfusion
chamber (0.5 mL) and superfused with standard buffer solution at a rate
of 3 mL/min and at 37±0.5°C. Myocytes used for the experiments were
selected on the basis of the following criteria: having sharp
striations, clear contours, transparent cytoplasms without granulations
or blebs, and a resting membrane potential below -75 mV. 
) were
used. Action potentials were recorded with a microelectrode
amplifier (Axoclamp-2B, Axon Instruments, Inc) at cycle lengths (CLs)
of 300, 400, 500, 1000, 2000, 3000, and 4000 ms. The values of action
potential parameters at baseline and during treatment with
almokalant presented throughout the text are the averaged
measurements of 5 beats during steady-state pacing. Myocyte
contractions were recorded with a video edge-motion detector
(Crescent Electronics) at 16-ms temporal
resolution.13 The analog output signals were
digitized and stored on computer hard disk. Cell shortening was
expressed in percent: amplitude in micrometers relative to
cell length.
when filled with 140 mmol/L KCl. Membrane potentials were
recorded in the current-clamp mode (Axopatch-1D, Axon Instruments,
Inc). The data were filtered at 5 kHz, read into a personal computer
through an analog-to-digital converter (2 kHz), and stored for later
analysis. The data acquisition program also controlled the
command potential and various components of the intracellular
Ca2+
([Ca2+]i) measurement
system (Fastlab Software, Indec Systems). The pipette solution
contained Fluo-3 and Fura red (Molecular
Probes).15 The combined use of these
fluorescent indicators was validated in confocal
microscopy.16 Our experimental setup for
[Ca2+]i measurements has
been described elsewhere.15
The standard buffer solution contained (in mmol/L) NaCl
145, KCl 4.0, CaCl2 1.8,
MgCl2 1.0,
Na2HPO4 1.0, glucose 11,
and HEPES 10, pH 7.4 with NaOH at 37°C and bubbled with
O2. In the cold cardioplegic solution, KCl was
set to 8.0 mmol/L. For cell isolation, collagenase
(1.1 mg/mL; type A, Boehringer Mannheim) and protease (0.05
mg/mL; type XIV, Sigma Chemical Co) were used in the presence of BSA
(1.0 mg/mL). The patch-pipette solution contained (in mmol/L)
potassium aspartate 120, KCl 20, MgCl2 0.5, MgATP
5, NaCl 10, HEPES 10, Fluo-3 0.03, and Fura red 0.07, pH 7.2 with KOH.
All of the chemicals were reagent grade and cell-culture tested
(purchased from Sigma Chemical Co). Almokalant was kindly provided by
Drs L. Carlsson and G. Duker, Astra Hässle, Mölndal,
Sweden.
The data are expressed as mean±SD. Intergroup comparisons were
made by Student's t test for unpaired (chronic AVB versus
SR; right versus left ventricle) and paired (baseline versus treatment)
data groups, respectively, after testing for the normality of
distribution. The 
2 test was used when the
data were presented as a proportion. Differences were
considered significant if P<0.05.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
In Vivo Studies
In our companion study,4 we report on the
electrophysiological consequences of
clinically relevant doses of d-sotalol in dogs with AVB. In
preliminary cellular experiments of the present study, we chose to
use almokalant because this agent exhibits a high specificity of
IKr inhibition at nanomolar to micromolar
concentrations,9 whereas d-sotalol may
also exert other actions.17 18 Accordingly, the
proarrhythmic potential of almokalant was tested in vivo
(ndogs=7). An example of the
electrophysiological consequences of
almokalant treatment and subsequent spontaneous TdP induction is shown
in Figure 1
. At 4±2 weeks of AVB, the
class III agent increased the QT interval from 415±90 to 545±105 ms
(P<0.05) without significant change of the CL of the
idioventricular rhythm (from 1545±300 to 1655±295 ms;
P=NS). Almokalant caused an increase of the monophasic
action potential duration (APD) in the left ventricle from 380±55 to
525±115 ms (P<0.05) and in the right ventricle from
335±65 to 425±110 ms (P<0.05), contributing to an
enhanced interventricular dispersion of repolarization of
45±15 ms at baseline to 100±50 ms during treatment
(P<0.05). In 6 of the 7 dogs, TdP ensued spontaneously; in
the remaining animal, TdP occurred neither spontaneously nor with
programmed electrical stimulation.
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Figure 1. Two runs of spontaneous TdP in a dog with 3
weeks' AVB on administration of almokalant (0.12 mg ·
kg-1 · 10 minutes infusion time-1).
Shown are ECG leads I, II, and aVR and monophasic action potential
(MAP) recordings from the left (LV) and right (RV)
ventricular endocardium. At baseline (1, CONTROL), there is
an idioventricular rhythm with a CL of 1190 ms and a QT
time of 375 ms. At that moment, LV APD is 335 ms, vs 290 ms in RV.
During almokalant treatment (2, ALMOKALANT), both LV and RV action
potentials prolong to 
445 and 350 ms. This inhomogeneous
action potential prolongation contributes to enhancement of
interventricular dispersion of repolarization from 45 ms at
baseline to 95 ms during treatment. Short runs of spontaneous TdP
were present until >20 minutes after end of almokalant infusion.
Characteristically, initiating ventricular complexes occur
before full termination of T wave of ECG and simultaneously
with EADs and triggered action potentials, as recorded by
monophasic action potential catheters in both RV and LV. For estimation
of LV APD during almokalant treatment, descent of repolarization is
drawn artificially.
When weighed directly after excision, the hearts of chronic-AVB
dogs (ndogs=9; 6±1 weeks of AVB) were
significantly heavier than those of SR controls
(ndogs=9): 285±25 versus 222±59 g, respectively
(P<0.05). When corrected for body weight, the difference in
heart weight remained significant: 10.3±1.3 versus 8.5±1.5 g/kg,
respectively (P<0.05). Myocytes were successfully isolated
from the right ventricular free wall and the left
ventricular midmyocardium of all dogs.
Representative photomicrographs of single left
midmyocardial cells are shown in Figure 2. Right ventricular myocytes
showed a similar morphology both in chronic AVB and in SR. All cells
were quiescent during superfusion with the standard buffer solution
containing 1.8 mmol/L [Ca2+]. In the SR
group, right ventricular myocytes were of equal length and
width compared with left midmyocardial myocytes: cell length was
140±10 and 140±16 µm (P=NS), and cell width was
24±2 and 25±2 µm, respectively (P=NS). In the
chronic-AVB group, myocytes from both ventricles were significantly
longer than SR controls, being 172±8 µm (right ventricle) and
158±7 µm (left midmyocardium; both
P<0.05, chronic AVB versus SR). The difference in cell
length of right versus left ventricle in chronic AVB was statistically
significant (P<0.05). By contrast, the width of these cells
was not different from SR controls: 26±1 and 26±2 µm, right
versus left ventricle, respectively (P=NS). Frequency
distributions for cell length and width are shown in Figure 3.
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Figure 2. Photomicrographs of single myocytes isolated from
left ventricular midmyocardium of a chronic-AVB
(top) and an SR dog (control; bottom). Chronic-AVB myocytes are larger
than their SR controls, with cell lengths of 230 µm (top
right) and 160 µm (top left). Quantitative data for mean myocyte
lengths and widths are presented in Results section.
Bar=50 µm for both panels. Magnification x1000.
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Figure 3. Frequency distributions for lengths and widths of
right ventricular free wall and left midmyocardial myocytes
in chronic-AVB and SR groups. In total, 452 and 403 right and 607 and
676 left ventricular cells are measured in chronic AVB and
SR, respectively. Relatively higher numbers of long cells ( 150
µm) can be observed in chronic AVB.
Action potentials were recorded at various
physiologically relevant pacing CLs with the
microelectrode technique in a total of 51 myocytes (chronic AVB:
ndogs=7, ncells,LV=13,
ncells,RV=9; SR: ndogs=8,
ncells,LV=15,
ncells,RV=14). Representative
action potentials of the 4 different cell groups are shown in Figure 4. In SR, the spike-and-dome
configuration was more pronounced in right ventricular than
left midmyocardial action potentials and more pronounced at longer CLs,
as expected. In chronic AVB, this pattern was preserved or slightly
accentuated (Figure 4).
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Figure 4. Action potentials of single myocytes from dogs
with chronic AVB as a function of pacing CL. In each panel, pacing CLs
are 500, 2000, and 4000 ms from left to right. Action potentials of
left midmyocardial myocytes are more prolonged in chronic AVB than SR.
APD is larger in left than right ventricular cells in both
conditions, especially at long CL. Microelectrode technique. . Chronic AVB
significantly steepened this APD95/CL
relationship in the left midmyocardial myocytes (P<0.05,
chronic AVB versus SR) but not in the right ventricular
cells (P=NS, chronic AVB versus SR). The
interventricular difference of APD95
was therefore significant at all CLs (Figure 5). The same was found for
the APD at 50% of repolarization (APD50). This
parameter increased to the same extent as
APD95 in chronic AVB, again with significant
changes in the left midmyocardial but not the right
ventricular cells. The maximal velocity of repolarization
during phase 3 (measured as the most negative first derivative of the
membrane potential in that phase) was not different between chronic AVB
and SR in both ventricles (data not shown).
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Figure 5. Pooled data of APD95 as a function of
pacing CL using microelectrode technique. In chronic AVB, action
potentials of left midmyocardial myocytes are significantly longer than
of right ventricular myocytes (left panel,
*P<0.05). Also, average APD95 of
chronic-AVB left midmyocardial cells is larger than of SR controls
(left vs right panel; +P<0.05).
We treated both SR and chronic-AVB myocytes with 1 µmol/L
almokalant. A significant prolongation of the action potential was
observed in all cells tested (n=29; microelectrode technique), which
was most pronounced at long pacing CLs. Representative
examples of action potentials of chronic-AVB myocytes during treatment
with almokalant are shown in Figure 6. In
chronic-AVB cells, the relative increase of the
APD95 during almokalant (in the absence of EADs)
was much larger than in SR, independent of the chamber. This is
illustrated by the pooled data of APD95 shown in
Figure 7A. In all cells, the
APD50 increased to the same extent as the
APD95.
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Figure 6. Action potential prolongation by almokalant in a
left midmyocardial and a right ventricular myocyte during
chronic AVB. Thin dashed action potentials are from myocytes under
baseline conditions; thick solid action potentials during treatment
with class III agent. Action potential prolongation is most pronounced
at long pacing CL and in this case, more in left than right ventricle.
Top, Example of an EAD.
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Figure 7. Pooled data of APD95 (in action
potentials without EADs) as a function of pacing CL during treatment
with almokalant. A, Data obtained by microelectrode technique. B,
Patch-electrode technique. Almokalant caused significant increase of
APD95 in all cell groups, regardless of technique used. In
absolute terms, this was much more pronounced and within a broader
range during chronic AVB than in SR. *P<0.05 between
chronic AVB and SR. 
). expresses this increased sensitivity as a larger
proportion of cells in which EADs were observed during treatment with
almokalant. EADs were generated at the longer pacing CL of 2000 ms.
No EADs were observed at CLs of 
500 ms. Whenever EADs appeared, they
caused a significant increase of the APD. In the most sensitive cells,
plateau arrests were often observed, ie, prolonged phases of
depolarization (sometimes lasting several seconds) that were followed
by 1 EADs before a rapid repolarization. Large beat-to-beat
variability of the APD often characterized the class III action, as is
shown in Figure 8.
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Table 1. Proportion of Cells Generating EADs During Almokalant
Treatment
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Figure 8. EADs during treatment with almokalant:
beat-to-beat differences of APD. Continuous recording of 8
action potentials at a pacing CL of 1000 ms. In first beat, a plateau
arrest can be observed that lasts for full CL and is overcome only
after pacing stimulus of next action potential. In consecutive beats,
APD95 varies from 500 to 900 ms. This is an example of
temporal dispersion of repolarization within a single cell.
The takeoff potentials of almokalant-induced EADs in chronic AVB
ranged between -35 and 0 mV. On average, EADs started at significantly
more negative levels in the right ventricle
(nEAD=50) than the left midmyocardium
(nEAD=100): -30±2 versus -19±5 mV,
respectively (P<0.05), whereas the action potentials
analyzed for this purpose had equal resting membrane
potentials. EAD amplitudes were larger in right versus left
ventricular myocytes: 21±6 versus 10±5 mV
(P<0.05). illustrates typical findings for 2 left
midmyocardial myocytes.
[Ca2+]i rose rapidly on
depolarization, reflecting Ca2+ release from the
sarcoplasmic reticulum. This release was followed by a rapid but
incomplete decline of
[Ca2+]i, and
[Ca2+]i typically
remained elevated at 30% above baseline values as long as the
membrane potential did not recover completely (Figure 9A). EADs during
this phase, however, were not associated with distinct new
[Ca2+]i transients. Small
fluctuations of [Ca2+]i
were sometimes observed, in amplitude always <5% of the initial
[Ca2+]i transient. In the
case of cell shortening, the normal twitch contraction was followed by
a relaxation phase during which cell length attained near-resting
levels. However, as with the
[Ca2+]i, full relaxation
awaited full repolarization of the action potential (Figure 9B). In a
few cells and only when large-amplitude EADs were generated, we could
discern small early aftercontractions that followed the EAD upstroke
with a delay of several tens of milliseconds (Figure 9B, arrow).
Similar results on
[Ca2+]i and cell
shortening during EADs were obtained in 30 cells treated with
almokalant.
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Figure 9. Cellular handling of
[Ca2+]i during class III–dependent EADs in
chronic AVB. A, Simultaneous recording of action
potential and [Ca2+]i with current clamping
in whole-cell variant of patch-clamp technique and with
fluorescent indicators Fluo-3 and Fura red in internal pipette
solution. B, Action potential (microelectrode technique) is
recorded simultaneously with cell shortening. A and B,
Left ventricular midmyocardial cells were treated with
almokalant at 1 µmol/L during stimulation at a pacing CL of 4000
ms, leading to generation of EADs. Full relaxation of
[Ca2+]i and contraction, in A and B,
respectively, was delayed until full repolarization of action
potential. Distinct or high-amplitude peaks of
[Ca2+]i (ie, Ca2+
aftertransients) were not observed during EADs, as illustrated in A.
Small aftercontractions following upstroke of EADs were rarely
observed. Example is shown in B (arrow).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
Cellular Hypertrophy in Chronic AVB
The present study confirms the earlier finding of increased
heart weight–to–body weight ratios in adult dogs with chronic
AVB.4 The hearts are enlarged consistent
with eccentric hypertrophy, as described for volume
overload.20 The hypertrophy is
related to increased lengths of both right and left
ventricular myocytes. Myocyte growth has been observed in
most large-animal models of cardiac hypertrophy reported in
the literature (eg, References 21 through 2321 22 23 ). We found that the
biventricular hypertrophy of chronic AVB was
not a homogeneous process: growth responses were larger in
the right than left ventricular myocytes (+23% versus
+13% for cell length, respectively), supporting the autopsy finding of
a larger relative increase of the right than the left
ventricular weight.4 This
differential ventricular growth response resembles the
findings of another animal model of biventricular
hypertrophy related to volume overload (aortocaval
fistula).22 Also, in pigs with heart failure due
to rapid pacing, myocytes were larger in the right than left
ventricle.24 The preponderance of right
ventricular cell growth in chronic AVB may reflect a
greater impact of the altered hemodynamics on that
chamber after the transition from SR to idioventricular
rhythm.
Our data show that myocytes from dogs with chronic AVB have (1) an
intrinsic disturbance of repolarization with prolonged action
potentials at baseline, (2) interventricular differences of
repolarization, and (3) an increased susceptibility to class III
antiarrhythmic agents.
Prolongation of the left ventricular action
potential is a consistent observation in myocardial
hypertrophy of different causes in several
species.25 26 27 Cellular
electrophysiological data on right
ventricular hypertrophy are scarce. In
pressure-overload–induced right ventricular
hypertrophy, increases of the APD have been
reported.28 Many of the sarcolemmal ion channels,
exchangers, and pumps as well as intracellular ion transporters that
shape the action potential under normal conditions can show functional
defects leading to delayed repolarization in
hypertrophy.29 30 Other investigators
have shown that this action potential prolongation predisposes to an
increased susceptibility to EADs31 and
ventricular
tachyarrhythmias.32 33 As yet,
the ionic basis of action potential changes in chronic AVB remains to
be elucidated. The APD95 and
APD50 increased to a similar extent, indicating
that the delay in repolarization was most likely due to a
disturbance at the plateau level. From the data in this study,
a prominent role for the transient outward current
(Ito) appears unlikely: the spike-and-dome
configuration of the action potential was preserved or, if anything,
accentuated. In this respect, the dog with chronic AVB differs from the
dog with pacing-induced heart failure.34 In that
model, downregulation of Ito is at least
partly responsible for an attenuation of the notch of the action
potential and an increase of the APD,34 which has
been linked to the generation of lethal ventricular
arrhythmias.35 Additional insights into
the ionic mechanisms of action potential prolongation in chronic AVB
come from the experiments with almokalant.
Carmeliet9 demonstrated that in rabbit
ventricular myocytes, IKr is
fully blocked by 1 µmol/L of the agent, and we have confirmed
this in normal dog ventricular myocytes (data not shown).
After block of IKr during almokalant
treatment, we found that the APD was much larger in chronic-AVB left
and right ventricular myocytes than in SR controls (Figure 7). This implies that ionic currents other than
IKr contribute to the abnormal
repolarization of chronic AVB, even though we cannot exclude that
IKr also plays a role.
To the best of our knowledge, this is the first study to
compare action potential characteristics of single hypertrophied
myocytes isolated from both the right and left ventricles of the same
dog. Interventricular differences of the action potential
are known to exist in the normal myocardium of dogs with
SR,6 36 and this was confirmed by the present
study. In chronic AVB, we found larger APDs in left midmyocardial than
right ventricular myocytes at baseline, in contrast to the
larger degree of hypertrophy in the latter than the former
cells. Yet, the administration of almokalant increased the APD to a
similarly high level in both ventricles. Thus, the presence of
intrinsic repolarization disturbances was unmasked in right
ventricular myocytes. At least 3 explanations could account
for the interventricular differences of repolarization at
baseline: (1) there was a different
electrophysiological remodeling in both
ventricles, (2) the charge carriers involved were the same but their
functional alterations mounted to different amplitudes, or (3) a finer
balance of currents existed during the plateau phase because of a
decrease in outward current and/or an increase in inward current.
Chronic-AVB cells showed an increased susceptibility to
action potential prolongation and EAD generation during class III
treatment. These EADs were observed at long pacing CLs and had takeoff
membrane potentials ranging between -35 and 0 mV. Very small changes
in the balance of inward and outward currents during the
high-resistance plateau can determine the appearance or absence of
EADs. At least 2 possibilities could explain the increased number of
EADs found in this study: (1) they occurred because the APD was
initially prolonged and/or (2) the charge carrier(s) responsible for
their generation were more prominent in myocardial
hypertrophy, eg, increased L-type
Ca2+ current (ICaL)
or more spontaneous sarcoplasmic reticulum Ca2+
release. If the former is true, it follows that any intervention
causing the action potential to shorten would lead to the diminished
appearance of EADs. Preliminary results with the agent levcromakalim,
an IK-ATP activator, support
this hypothesis: we found that in consecutive action potentials with
EADs, levcromakalim initially caused a decrease of the APD, followed by
the disappearance of the EADs and a further shortening of the action
potential. The finding that DADs also remained absent during
this action potential shortening was an argument against the
possibility of spontaneous sarcoplasmic reticulum
Ca2+ release to underlie the class III–dependent
EADs of chronic AVB. Indeed, the characteristics of cytoplasmic
Ca2+ during the EADs confirmed this notion. We
did find that the relaxation of
[Ca2+]i and cell
shortening was slow during action potentials with EADs. The decline of
[Ca2+]i always
paralleled the repolarization regardless of the APD, which suggests
that the voltage dependence of cellular Ca2+
extrusion was maintained as under normal conditions. Our results in
chronic-AVB myocytes resemble those of cesium chloride–induced EADs in
normal ferret ventricular muscle37
and class III–induced EADs in normal rabbit ventricular
myocytes.38 Although our data support an
important role for the initial action potential prolongation, we cannot
exclude the possibility that intrinsic changes of
ICaL may be involved in the increased
number of EADs.
Our data show that the interventricular
differences of APD were most pronounced at the longer pacing CLs of
2000 ms, measuring maximally 100 ms at baseline. Because in most
cases the CLs of the idioventricular rhythms of the dogs
were >1500 ms, we consider the cellular findings relevant for the in
vivo situation and find them consistent, at least
qualitatively, with the monophasic action potential findings in the
same dogs before they were euthanized. We do not believe that
interventricular dispersion itself is the direct cause for
the initiation of torsade de pointes, but because it is always
present during arrhythmogenesis, it probably reflects the existence
of dispersion of more closely juxtaposed regions. When using almokalant
in our experiments, we found that this agent promoted temporal
heterogeneity of repolarization with varying APDs and
EADs that appeared in an on-and-off fashion in consecutive beats during
steady-state pacing. Dispersion could be enhanced even more if the
heterogeneity of cellular repolarization occurred out
of phase in various regions of the myocardium. We have
demonstrated such spatial dispersion in
vivo.3 )2 4 ; however, until now
their origin and mechanisms remain obscure. Although others have
demonstrated triggered activity on EADs in Purkinje fibers during in
vitro conditions that mimic proarrhythmia in
vivo,39 40 41 it can be concluded from the
present study that in single cells from the working
myocardium, abnormal impulse generation is limited to EADs
that do not trigger new action potentials in these same cells. This
conclusion is based on our findings that the EADs always (1) arose from
a takeoff membrane potential less negative than -35 mV (above the
threshold for full activation of Na+ current) and
did not show a rapid upstroke as in normal action potentials; (2) had
an amplitude of <30 mV; (3) prolonged the repolarization but did not
take the shape of normal action potentials; (4) were incapable of
inducing normal, if any, contractions; and (5) behaved differently from
the EADs observed in Purkinje fibers or in ventricular
myocytes with takeoff levels closer to the resting membrane potential.
Thus, the characteristics of these membrane responses do not match our
definition of the ventricular action potential.
One of the aspects of this study was to compare the action
potential characteristics of myocytes from the left
midmyocardium with those from the right transmural
ventricular wall. One could argue that this comparison is
not fully adequate, given the findings made in tissue studies that the
shape of the ventricular action potential and the CL
dependence of its duration vary across the free wall of both
chambers.42 Because of the thinness of the right
ventricular wall, we found it difficult to isolate the
cells according to their transmural site of origin in that chamber.
Nevertheless, at long pacing CLs, right ventricular
myocytes consistently had a typical action potential
configuration that was different from that of the left midmyocardial
cells. In addition, the range of APD measured in left and right
ventricular cells, eg, at a CL of 2000 ms, showed an
overlap of only 10% in chronic AVB and of 20% in SR. A "bad pick"
of only epicardial or endocardial cells from the right
ventricular mixture might explain this finding, but this is
unlikely. A more plausible explanation is that the APD differences of
midmyocardial versus epicardial and endocardial myocytes are much less
in the right than the left ventricle. The comparison of right
transmural versus left transmural ventricular myocytes
(instead of right transmural versus left midmyocardial cells, as made
now) would most likely underestimate the interventricular
dispersion of repolarization present in these hearts and was
therefore not applied.
We conclude that AVB of chronic duration leads to
inhomogeneous hypertrophy of right and left
ventricular myocytes. Cellular hypertrophy is
associated with an intrinsic defect of repolarization in the right and
left ventricles. APD differences between left midmyocardial and right
ventricular myocytes cause an enhanced
interventricular dispersion of repolarization, which is
most pronounced at the longer pacing CL. Almokalant increases the APD
to a much larger extent in chronic AVB than in SR, thus further
unmasking the presence of intrinsic repolarization
disturbances. In addition, chronic-AVB cells of both ventricles
show an increased susceptibility to EAD generation. Although the
present study does not elucidate the ionic basis of these action
potential differences or the genesis of ventricular ectopic
beats, it appears that phenotypic alterations of repolarization are
involved in the ventricular arrhythmias and sudden
cardiac death of dogs with chronic AVB.
Acknowledgments
This study was supported by the Wynand M. Pon Foundation
(Leusden, Netherlands). Dr Sipido is a postdoctoral researcher of the
National Fund for Scientific Research Belgium. The European Society of
Cardiology is acknowledged for providing a research
fellowship to Dr Kulcsár. Dr Verduyn is a postdoctoral researcher
of the Netherlands Heart Foundation, project number 94.010. The
authors wish to thank Jet D.M. Leunissen (Department of
Cardiology) and Matthijs J.P. Killian, PhD (Department
of Physiology) for expert technical assistance and Jack P.M. Cleutjens,
PhD, and Mat J.A.P. Daemen, MD, PhD (Department of Pathology) for the
photomicrography. Bela Szabo, MD, PhD (University of Oklahoma Health
Sciences Center) contributed with helpful comments to the
manuscript.
Footnotes
Guest editor for this article was Douglas P. Zipes, MD, Krannert Institute of Cardiology, Indianapolis, Ind.
References
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Abstract
Introduction
Methods
Results
Discussion
References
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