© 2000 American Heart Association, Inc.
Integrative Physiology |
Enhanced Dispersion of Repolarization and Refractoriness in Transgenic Mouse Hearts Promotes Reentrant Ventricular Tachycardia
From the Department of Bioengineering (L.C.B.), Cardiovascular Institute (B.L.), and Department of Cell Biology and Physiology (B.-R.C., G.S.), University of Pittsburgh, Pittsburgh, Pa; and Division of Cardiology (G.K.), Brigham and Women’s Hospital, Boston, Mass.
Correspondence to Guy Salama, PhD, Department of Cell Biology and Physiology, University of Pittsburgh, School of Medicine, S314 Biomedical Science Tower, 3500 Terrace St, Pittsburgh, PA 15261. E-mail gsalama+{at}pitt.edu
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Abstract |
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Abstract—The heterogeneous distribution of ion channels in ventricular muscle gives rise to spatial variations in action potential (AP) duration (APD) and contributes to the repolarization sequence in healthy hearts. It has been proposed that enhanced dispersion of repolarization may underlie arrhythmias in diseases with markedly different causes. We engineered dominant negative transgenic mice that have prolonged QT intervals and arrhythmias due to the loss of a slowly inactivating K+ current. Optical techniques are now applied to map APs and investigate the mechanisms underlying these arrhythmias. Hearts from transgenic and control mice were isolated, perfused, stained with di-4-ANEPPS, and paced at multiple sites to optically map APs, activation, and repolarization sequences at baseline and during arrhythmias. Transgenic hearts exhibited a 2-fold prolongation of APD, less shortening (8% versus 40%) of APDs with decreasing cycle length, altered restitution kinetics, and greater gradients of refractoriness from apex to base compared with control hearts. A premature impulse applied at the apex of transgenic hearts produced sustained reentrant ventricular tachycardia (n=14 of 15 hearts) that did not occur with stimulation at the base (n=8) or at any location in control hearts (n=12). In transgenic hearts, premature impulses initiated reentry by encountering functional lines of conduction block caused by enhanced dispersion of refractoriness. Reentrant VT had stable (>30 minutes) alternating long/short APDs associated with long/short cycle lengths and T wave alternans. Thus, optical mapping of genetically engineered mice may help elucidate some electrophysiological mechanisms that underlie arrhythmias and sudden death in human cardiac disorders.
Key Words: arrhythmia • sudden death • mice, transgenic • di-4-ANEPPS • action potential • mapping
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Introduction |
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Cardiac arrhythmias and sudden death can result from rare congenital conditions and damage to the heart and its conduction system induced by ischemia, infarction, or cardiac surgery; drug therapies; and assorted noncardiac diseases.1 Many of the ion channels responsible for the generation of the cardiac action potential (AP) have recently been identified and cloned.2 A link between anomalies of repolarization and arrhythmias is most evident in patients with the congenital long-QT syndrome, in whom the mutations of cardiac ionic channels prolong AP duration (APD), increase electrical heterogeneity, and lead to ventricular tachycardia (VT) and sudden death.3 4 5 6 Abnormalities in ion channel expression and ionic currents have also been reported in ischemia and congestive heart failure, which emphasizes their potential importance in the genesis of arrhythmias.7 8 A unifying hypothesis to account for arrhythmias in a broad range of cardiac conditions is that enhanced spatiotemporal dispersion of repolarization alters the myocardial substrate to initiate and sustain reentrant rhythms.5 6 9 10 11 12 In this setting, a premature beat can encounter a zone of conduction block and propagate unidirectionally to initiate reentry.
We developed a dominant negative transgenic mouse that overexpresses an
N-terminal fragment of the K+ channel Kv1.1 in
the heart under control of the 
-myosin heavy chain
promoter.13 This truncated channel fragment coassembles
with wild-type Kv1.x subunits, traps heteromeric channels in the
endoplasmic reticulum, and inhibits Kv1.x currents in a dominant
negative manner in heterologous expression systems.14 15
Ventricular myocytes isolated from the dominant negative
transgenic mice exhibited prolonged APDs due to the loss of a slowly
inactivating 4-aminopyridine–sensitive current,
Islow, that is likely encoded, at least in
part, by Kv1.5.13 16 These mice have prolonged QT
intervals, spontaneous nonsustained VT during ambulatory telemetry
monitoring,13 and inducible polymorphic VT during
right ventricular programmed stimulation in
anesthetized, open-chest preparations.17
Here, we applied voltage-sensitive dyes and optical mapping techniques18 19 to demonstrate that the elimination of specific ionic currents involved in the repolarization of the cardiac AP increases the dispersion of repolarization and refractoriness, alters restitution kinetics, and results in an enhanced vulnerability to reentrant VT. A preliminary report of these findings was presented in abstract form.20
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Materials and Methods |
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Perfusion of Mouse Hearts
Animal procedures were approved by the IACUC of the University of Pittsburgh. Mice were anesthetized with fluothane. Hearts were cannulated and perfused (
2.5 mL/min) with standard
oxygenated Tyrode’s solution, producing 60 to 80
mm Hg of aortic pressure. Hearts were stained with 10 µL of
di-4-ANEPPS (2 mmol/L in DMSO) during 5 minutes to monitor
cardiac APs.21 Except for control studies with diacetyl
monoxime (DAM; 15 mmol/L) and cytochalasin D (cyto-D; 5
µmol/L), APs were measured in 1.2 mmol/L free
Ca2+ in the absence of chemicals that block
contraction to avoid adverse effects on AP characteristics and
intercellular coupling. A chamber was used to mechanically stabilize
the heart (Figure 1A
) and to minimize
optical artifacts relative to AP amplitudes.22 23 The
cannula was used to minimize resistance (2 to 5 mm Hg) to flow
(Figure 1B) and to allow a compliance chamber close to the
heart.
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feedback
resistor), AC or DC coupled, amplified (x1, 50, 200, or 1000),
digitized (2.0 kHz per channel, maximum of 3.9 kHz) at 12-bit
resolution (DAP 3200e/214; MicroStar Laboratories), and stored in
computer memory.
Optical Apparatus
The optical apparatus (Figure 1C
) and
computer interface have been previously described.22 24 25
Epifluorescence was used to maximize light collection with a
short working distance lens. An image of the heart was focused on a
12x12-element photodiode array, and 124 diodes were monitored. Image
magnification was x4.5, and 4x4 mm2 tissue
was mapped. Each diode detected APs from an area of 312x312
µm2 (dead space between diodes=0.1 mm) with a
depth of field of 70 µm.26
Analysis of Optical APs
Activation and repolarization patterns were determined from the
124 activation and repolarization time points and displayed as
isochronal maps.22 23 25 Activation times were
calculated from dF/dtmax of AP
upstrokes.22 23 Optical APs and dF/dt were signal
processed as previously described.24 An activation time
was accepted if dF/dtmax was more than the SD of
the background noise. Repolarization times taken at 75% or 90%
recovery to baseline were used to determine APD75
and APD90 (see Figure 2). APs with signal-to-noise ratios of
<10 or excessive movement artifact were eliminated, and the remaining
activation (repolarization) times were triangulated with the use of
Delaunay’s triangulation algorithm to overcome spatially irregular
data sets. Isochronal lines were drawn from these triangles with
linear interpolation and connecting points by lines. Local conduction
velocities were calculated from dF/dtmax at 124
sites as previously described.23 Average velocity was
calculated from the vectorial average of 124 local velocity vectors
oriented between 0 and 
radians. Maximum
(
max) and minimum
(min) conduction velocities are measured
relative to a cartesian coordinate system after pacing of the heart at
the center of the image to elicit elliptical propagation
waves.25 All analysis was automated with in-house
software based on IDL 5.1 (Research Systems).24
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Statistical Analysis
All values are reported as mean±SD. Student’s t
tests were performed on APD and velocities to compare control and
transgenic mice; P<0.05 was considered statistically
significant. APDs (and refractory periods) from the same heart were
averaged from 4 diodes at the base and apex and compared with the use
of paired Student’s t test to determine gradients of
repolarization (and refractoriness). Grouped t tests were
used to determine the statistical significance of gradients of
repolarization and refractoriness between control and transgenic
mice.
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Results |
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A number of control studies were performed to investigate the validity and interpretation of the data. Hearts stained with di-4-ANEPPS exhibited no changes in the intrinsic heart rate, amplitude of electrograms, or excitability measured as the threshold. In 5 hearts, optical APs and electrograms were recorded every 5 minutes for up to 5 hours to determine the stability of the preparations and the incidence of spontaneous arrhythmias. Heart rate and signal amplitudes remained stable for an average of 3.5 hours (maximum 5 hours, minimum 2.9 hours), and none had spontaneous arrhythmias. These control experiments confirm that di-4-ANEPPS has no overt toxic effects on the heart and that the preparation was stable for several hours.
Motion artifact was suppressed by placement of the heart in a chamber
to mechanically stabilize the surface of the heart viewed with the
array. One concern is that the isolation, perfusion, and immobilization
of the hearts in the chamber could make them ischemic and
arrhythmic. In mammalian hearts, ischemia produces a marked
decrease in APD, conduction velocity, and force of contraction and
often elicits electrical and mechanical alternans.27
Aortic perfusion pressure, bipolar electrograms (recorded from 1 to
3 sites), APD, and conduction velocity were measured before and after
placement of the hearts in the chamber. In most experiments, aortic
pressure increased by 5 mm Hg when the chamber suppressed
force, whereas electrogram recordings were not altered, and APs
remained stable for several hours. In contrast, low-flow
ischemia (10% to 15% of normal flow) markedly decreased the
conduction velocity and dF/dt of AP upstrokes within 5 minutes. In
addition, changes indicative of ischemia were apparent if the
chamber was tightened. For these reasons, conduction velocity, dF/dt,
and aortic pressure changes were regularly measured to ensure the
chamber did not by itself produce cardiac ischemia or enhance
the vulnerability to arrhythmias.
As an alternative approach to arrest motion artifact, we perfused
several control hearts with agents that interfere with force
development, such as diacetyl monoxime (DAM, 15 mmol/L) and
cytocholasin-D (cyto-D, 5 µmol/L).28 29 30 31 Figures 2A and 2B show APs before (trace 0) and after (traces 1 to 3)
exposure to DAM and cyto-D. DAM (n=5) produced a 2-fold prolongation of
APD within 5 minutes (Figure 2A) and caused a time-dependent
decrease in conduction velocity of 15% in 75 minutes. The
DAM-induced decrease in conduction velocity is consistent with
previous reports that DAM also blocks gap junction
communication.32 Cyto-D (n=4) also prolonged APD 2-fold
within 15 minutes, associated with a prominent plateau phase (Figure 2B). With both agents, APDs remained prolonged but stable for up
to 2 hours. At the concentrations used, both agents arrested
contraction in 5 minutes and caused only minor changes in the voltage
signals immediately after repolarization (<2% on phase IV). This
suggests that the low-amplitude motion artifact that remains after
stabilization of the heart in the chamber is unlikely to significantly
and reproducibly affect the measurement of APD75
or APD90. It also demonstrates that DAM and
cyto-D must be avoided in studies of repolarization and
arrhythmia mechanisms in the mouse.
Figure 2C illustrates the analysis of a single AP trace
with a computer algorithm that automatically identified the activation
and repolarization (at 75% and 90% recovery to baseline) time points.
The automated algorithm annotated each AP on the map to identify these
time points. A manual override made it possible to correct or delete
(typically <5% of the APs) from the analysis errors in the
identification of repolarization time points caused by motion artifact
or high background noise.
Figures 3A and 3B illustrate symbolic
maps of the array; each diode is represented by a square
box with the AP recorded by that diode in its respective location
for a control (Figure 3A) and a transgenic (Figure 3B)
mouse heart. Mouse optical APs have the expected "triangular" shape
(ie, no plateau phase) with rapidly rising upstrokes and brief
durations. When examined on an expanded time base (Figures 3C and 3D), marked prolongation of APD is apparent in transgenic
hearts.
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In sinus rhythm, activation of the ventricles is driven by the Purkinje
fibers of the conduction system of the heart, resulting in a rapid
depolarization of the ventricles in 5 to 6 ms for both control and
transgenic hearts (Figure 4A). When paced
at the center of the left ventricle (300 bpm), activation spread
elliptically, with maximum (
max=0.85±0.13
m/s) and minimum (min=0.33±0.17 m/s)
conduction velocities at 135/315- and 45/225-degree angles,
respectively (n=4 hearts, Figure 4B). The orientation of
max and min was
similar to that of guinea pig left ventricle, where they corresponded
to longitudinal and transverse axes of epicardial fiber
orientations.25 The average anisotropy of conduction
velocities taken as the ratio of max to
min was 2.25 to 1. It should be noted that
pacing produces a "virtual electrode" effect that appears as a zone
of synchronous depolarization around the electrode site (Figure 4B). Local velocity vectors from this zone were automatically
removed from the calculations of conduction velocity.
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When paced at the apex, activation spread from apex to base (Figure 4C). The mean conduction velocity (mostly transverse, with a
component of longitudinal velocity) was similar for control
(M=0.51±0.17 m/s [mean±SD], n=5) and
transgenic (M=0.49±0.13 m/s, n=5) mouse
hearts. In sinus rhythm or when pacing at various sites on the
epicardium, repolarization began near the apex and spread toward the
base of the heart (Figure 4D).
APD75 and APD90 were longer
in transgenic than in control hearts when measured at 75% or 90%
recovery to baseline (Figure 5A). Similar
findings were reported in isolated ventricular myocytes
from these hearts with the use of patch microelectrodes.13
At any site on the heart, the APD depends on the basic cycle length
(CL) and the local restitution kinetics of the AP after an abrupt
change in CL or a premature impulse. Figure 5B compares the
changes in APD as a function of CL for transgenic and control hearts.
The classic shortening of APD with decreased CL was not observed in
transgenic hearts. The "flat" APD-versus-CL relationship in
transgenic hearts suggests that the "missing" current,
Islow, plays an important role in the
adaptation of APD to changes in heart rate, especially at short
CLs.
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As in guinea pig hearts,25 APDs in mouse hearts were heterogeneous along the epicardium, with APD75 being shorter at the apex (19±3.3 ms) than at the base (29±2.2 ms, n=6, P<0.05). Gradients of APD from apex to base were also found in the presence of the chemical uncouplers 15 mmol/L DAM and 5 µmol/L cyto-D. From apex to base, APD75 increased from 63±4 ms to 69±5 ms (n=3) with DAM and from 45±5 to 50±8 ms (n=3) with cyto-D. The heterogeneity of APDs was more pronounced in transgenic hearts than in control hearts, with APD75 of 32±4.3 ms at the apex and APD75 of 54±5.3 ms at the base (n=7, P<0.005). The spatial dispersion of repolarization from apex to base was also greater in transgenic (10±3 ms, n=7) than control (6±2.7 ms, n=7) hearts.
Dynamic (ie, temporal) heterogeneities of AP amplitude (APA) and
duration are produced by abrupt changes in heart rate or by a premature
impulse.27 Figure 5C demonstrates the response
characteristics of control APs with decreasing S1-S2 interval. Figure 5D plots the restitution kinetics of APA for the apex and base
of control and transgenic mouse hearts. In control hearts, the
restitution curves at the apex and the base decreased gradually with
decreasing S1-S2 interval, until the premature impulse fired during the
refractory period and failed to capture. In contrast, transgenic hearts
had steep restitution curves, particularly at the apex, and failed to
capture at considerably longer S1-S2 intervals
(Table).
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The enhanced dispersion of repolarization and refractoriness resulted
in a highly arrhythmogenic substrate. A single premature stimulus
applied near the apex of transgenic hearts reproducibly elicited
long-lasting (
30 minutes) reentrant VT (n=14/14 hearts). Similar
stimulation at the apex of control hearts failed to elicit reentrant VT
(n=12), except for 1 control heart in which a premature pulse at the
apex elicited a brief run of nonsustained VT (5 beats). Figure 6A shows optical recordings from
the array captured during the initiation of a
representative arrhythmia, and Figure 6B
illustrates traces from 3 locations on an expanded time scale. Each
diode recorded 2 basic beats (CL=200 ms), the premature impulse
(S1-S2=60 ms), and the first 5 beats of the VT. Activation of the basic
paced beat (S1) propagated from apex to base in 17.0 ms (Figure 6C). The premature impulse applied at the apex triggered APs of
lower amplitude (as in Figures 5C and 5D, bottom trace) that
propagated 1 mm toward the base before encountering a line of
refractory myocardium (Figure 6D, black arrow). APs
propagated below the functional line of conduction block from the
anterior to posterior edge of the heart (horizontal white arrow),
reemerged above the line of block 12 ms later (curved arrow), and
spread toward the base of the heart (Figure 6D, diagonal white
arrow). Optical recordings from the region of conduction block
detected 2 distinct depolarizations (seen as a double "hump")
during the premature pulse, corresponding to membrane depolarization
below and above the line of block (Figure 6B, trace 2). The
subsequent APs during VT propagated from the anterior aspect of the
apex toward the posterior aspect base (Figure 6E), demonstrating
that the conduction block was dynamic and not caused by a fixed
anatomical barrier. The wavelength of the reentrant circuit (conduction
velocity of the leading edge (0.27±0.02 m/s, n=4) multiplied by the
mean APD90 (39.8±4.3 ms, n=4) was 10.8
mm, which is consistent with a reentrant circuit around the
perimeter of the mouse heart. Activation patterns during the circus
movement (Figure 6E, white arrow) were noteworthy for the
stability of the AP amplitude, direction, and rate for >45 minutes and
for the reproducibility from heart to heart.
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The ability of the premature pulse to trigger a circus movement
arrhythmia depended on its location. Stimuli at the base of
transgenic hearts never elicited VT (n=8 hearts) because when a
premature pulse captures near the base of the heart, the ensuing AP
propagated toward the apex and always encountered excitable
myocardium with shorter refractory periods. Under these
conditions, the activation wave failed to encounter conduction blocks
and to initiate VT. On the other hand, the ability of premature stimuli
at the apex to elicit VT was independent of the basic activation
pattern. For instance, in a heart in sinus rhythm (Figure 6F), a
premature pulse applied on the anteroapical edge of the heart
encountered a functional arc of block and propagated as a clockwise
rotor around the block (Figure 6G). Although the activation
pattern produced by the premature pulse was dependent on its location,
the subsequent reentry patterns typically spread apex to base across
the epicardium (Figure 6H).
A detailed analysis of APs and activation patterns during VT
revealed the presence of long-lasting, stable alternans (n=6 of 7). VT
beats oscillated between large-amplitude, long-duration APs that
propagated at rapid conduction velocities and low-amplitude,
short-duration APs that propagated at slower conduction velocities
(Figure 7A). In 1 heart, alternans
occurred at a 1:2 ratio, with the large-amplitude, rapidly conducting
APs occurring every third beat. Electrogram recordings from the
opposite side of the heart showed that electrical alternations on the
right ventricle had the same oscillations in CL but were
out of phase with those recorded on the left ventricle. The similar
frequencies of electrical alternans on the left and right ventricles
indicated that the alternations were coupled and that the reentrant
circuit involves the perimeter of the mouse heart. Plots of the
fluorescence intensity F(t+
) versus F(t), where =5.6 ms,
produced phase maps oscillating between 2 states (Figure 7B). An
analysis of Poincaré interactivation
intervals33 34 35 (Figure 7C) revealed that the
reentrant circuits oscillate between 2 CLs for >30 minutes. VT beats
with longer CLs probably occur because the leading edge of the previous
wavefront had encountered partially refractory tissue, resulting in a
decrease in conduction velocity and a longer interactivation interval
in the succeeding wavefront. The slower VT beat propagates around the
circuit and allows for greater recovery from refractoriness, thus
ensuring that the tachycardia does not self-extinguish. The
steep slope of the restitution curve in transgenic mice may also play a
role in the maintenance of stable reentrant VT. Variations
in the amplitude of APD alternans from heart to heart may reflect
differences in the length of the reentry pathway, differences in
conduction velocity oscillations, or both.
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The production of mice with specific genetic modifications of cardiac ion channels, cell coupling proteins, and Ca2+ transport proteins has generated a growing interest in methods to investigate the electrophysiology of the intact mouse heart. Optical mapping offers a unique approach to map APs and characterize its electrical properties. In the present study, we refined optical mapping techniques to record high-fidelity APs from multiple sites on the epicardium of control and genetically engineered mouse hearts.
Electrophysiology of the Mouse Ventricle
Optical mapping of APD is particularly challenging in the mouse
ventricle because of the short and triangulated shape of the AP. In
guinea pig hearts, we had shown that the second derivative of the AP
downstroke,
(d2F/dt2)max,
can be relied on to detect the repolarization time point at 97%
recovery to baseline.36 The approach, however, is only
applicable to APs with a stable plateau and rapid downstroke. DAM,
verapamil, and cyto-D have been used to abate contractions
and diminish optical movement artifacts.28 30 31 37 In
hearts from larger mammals, these uncouplers shorten APD, reduce
intercellular coupling, alter restitution kinetics, and act as
antiarrhythmic agents.29 32 38 In mouse ventricle, we
found that DAM and cyto-D blocked contractions, as expected, but
markedly prolonged APD. Hence, in the present study, we relied on
mechanical suppression motion artifact. This, along with the high
signal-to-noise ratio, made it possible to measure
APD50, APD75, and
APD90 with high accuracy and to map
repolarization as well as activation.
APD Measurements
The mouse ventricular AP is triangulated, has no
plateau phase, and is shorter than that of most other mammals. The APD
that we measured with optical techniques (eg,
APD75=23±3 ms) is similar to that reported in
mouse whole-cell voltage-clamp studies.13 16 In addition,
our measurements of APD75 at CL=120 ms in the
presence of 15 mmol/L DAM (47±5 ms) agree with a recent report in
which di-4-ANEPPS and a CCD camera (44±3 ms) were used.39
The gradient of APD from apex to base is also similar to that found in
the guinea pig.25
Activation and Conduction Velocities
The present optical measurements of mean conduction velocity
(M=0.51±0.17 m/s [mean±SD], n=5) in
control mouse hearts are in excellent agreement with those obtained
with electrodes (0.4±0.1 m/s).40 In hearts paced at the
center of the field-of-view of the array, activation spread
elliptically on the left ventricle with maximum
(max=0.85±0.13 m/s) and minimum
(min=0.33±0.17 m/s) velocities. Our results
are similar to those of a recent report in which mouse hearts were
perfused with DAM and optically mapped with a CCD camera
(max=0.63±0.04 m/s,
min=0.38±0.02 m/s) when the 15% to 20%
reduction in conduction velocities caused by 15 mmol/L DAM is
taken into account.39 Conduction velocities in mouse
hearts are fast but within the range reported in guinea
pig,25 rabbit,41 and dog42
hearts. In addition, the orientations of max
and min were closely aligned with the
longitudinal and transverse axes of epicardial fibers, as shown for
guinea pig hearts. This implies that there is a similar fiber
orientation on the mouse and guinea pig epicardium.25
Postrepolarization Refractoriness
Mouse ventricular APs have refractory periods that are
considerably longer than their APDs. At a CL of 200 ms, the refractory
period at the base of the heart was 53.8±12.7 ms compared with an
APD90 of 29±2 ms. This surprisingly long
postrepolarization refractory period was observed in all regions of the
ventricles. Experiments in open-chest anesthetized mice with
programmed stimulation showed even longer refractory periods
(96.7±24.0 ms at 200-ms CL).43 Postrepolarization
refractoriness is likely due to the slow recovery from inactivation of
inward currents (INa and
ICa), which depends in a complex way on the
time and voltage changes of the previous AP downstroke. The refractory
period in the mouse may also be influenced by an increase in
K+ conductance due to the slow inactivation
kinetics of K+ currents that control
repolarization. In contrast with other mammals, normal mouse hearts
have a marked postrepolarization refractoriness, which could be due to
a slower recovery from inactivation of inward currents
(INa and ICa)
or a small increase in resting membrane potential compared with other
mammals. The latter is analogous to the effect of ischemia on
other mammalian hearts, where a depolarization of the resting membrane
potential results in a postrepolarization refractory
period.44
Restitution Kinetics
The most common method of measuring restitution is to plot the APD
of the premature beat as a function of the diastolic
interval. The short duration of the mouse heart AP made it difficult to
measure statistically significant decreases in APDs of the premature
beats, particularly during the steep slope of the restitution curve. We
therefore plotted the restitution of APA, which depends on the
restitution of inward currents (INa and
ICa) and indirectly on
K+ repolarizing currents. Previous studies had
shown that APA decreased with decreasing S1-S2
intervals.45 We found similar results with the
mouse.
Control Versus Transgenic Hearts
We showed that disruption of a component of the repolarizing
K+ current in transgenic mice produces APD
prolongation, increased refractoriness, and enhanced dispersion of
repolarization and refractoriness, resulting in a highly arrhythmogenic
substrate. Alterations in the K+ repolarizing
currents then indirectly affect excitability through changes in
INa (or ICa or
both). Several lines of evidence support the primary role of the
K+ channel alterations as a cause of enhanced
vulnerability to arrhythmia. (1) The rise time of APs,
dF/dtmax, and conduction velocities in control
and transgenic were similar through a broad range of heart rates
(CL=100 to 300 ms), suggesting that no unexpected anomalies in
Na+ channel properties occurred in transgenic
mice. (2) The period of postrepolarization refractoriness (refractory
period minus APD90) was the same in hearts from
control (13.9±4.9 ms) and transgenic (12.24±3.4 ms) mice (n=4 each).
The dispersion of repolarization is not necessarily the same as the
dispersion of refractoriness, particularly in conditions such as
ischemia that promote postrepolarization refractoriness. In
control mice, we found that postrepolarization refractory periods were
uniform at the apex and base of the ventricles and that the dispersions
of repolarization and refractoriness were alike. Moreover,
postrepolarization refractory periods are similar in control and
transgenic mice, suggesting that the recovery of inward currents
(INa and ICa)
was not modified in transgenic mice. Hence, the enhanced dispersion of
refractoriness found in transgenic mice is primarily a consequence of
the enhanced dispersion of repolarization. The highly arrhythmogenic
substrate in transgenic mice may be formed by the steep slope of APA
restitution (ie, a prolonged relative refractory period) at the apex in
combination with the enhanced gradient of refractory periods from apex
to base.
Prolongation of APD has been proposed as a strategy to protect the heart from triggered activity and arrhythmias through a lengthening of the refractory period.46 More recent data have shown that drugs that prolonged APD and QT interval lead to arrhythmias such as torsade de pointes.47 The precise mechanism by which APD and QT prolongation leads to arrhythmias is not fully understood, but enhanced dispersion of refractoriness has been proposed as a possible explanation.48 In principle, APD prolongation should be antiarrhythmogenic if homogeneous on the ventricle, but when accompanied by an enhanced spatial dispersion of repolarization and refractoriness, it may become proarrhythmic.
Isolated hearts from control or transgenic mice did not exhibit episodes of spontaneous arrhythmias during optical mapping experiments. On the other hand, programmed stimulation with a single premature pulse elicited monomorphic reentrant VT in transgenic (14 of 14) but not control mouse hearts. The location of the premature impulse was critical to its ability to elicit VT. An extra pulse applied near the apex of the heart triggered VT, but pulses applied near the base had no effect. The initiation of VT was characterized by capture of the extra pulse near the apex and its slow propagation due to the relative refractoriness of the tissue. The premature wavefront spread toward the base, encountered a functional line of block, and spread below, then around above the block.
The larger gradient of APD and refractoriness from apex to base
(2-fold) in transgenic mice may result from the direct loss of
nonuniformly distributed K+ channels of the Kv1.x
family or from the increased contribution of other nonuniformly
distributed channels (in the absence of
Islow) to the total current. Of note,
nonuniform distribution of Ito and the
Kv4.x channels has been described,49 50 and
transgenic mice that overexpress a mutated Kv4.2 subunit lack
Ito, fast and have similar prolongation of
APD and QT interval but do not have
arrhythmias.51 Thus, although APD
prolongation may lead to increased early and delayed
afterdepolarizations in mammalian hearts with long APDs, spatial and
temporal dispersions of repolarization and gradients of refractoriness
may be of greater importance in the initiation of reentrant
arrhythmias.
Mechanisms Underlying Stable VT in Transgenic Mice
Spatiotemporal analysis of ventricular
fibrillation (VF) in hearts of larger animals (dog, rabbit, and sheep)
has revealed that VF previously thought to be frenzied and chaotic
actually consists of several reentry circuits or rotors with
frequencies of 8 or 10 Hz.52 53 The reentry circuits in
VF, elicited by premature impulses, appeared to span 2 to 3 mm of
epicardium (unknown depth) and to maintain relatively stable patterns
for 5 to 10 minutes. Thus, reentrant waves are responsible for both VT
and VF, and the elucidation of factors that initiate and stabilize
reentry is fundamental to our understanding of the causes of sudden
cardiac death.
The transgenic mouse described here offers an experimental model of stable VT alternans. The interplay of prolonged APD and enhanced gradient of refractoriness adjusted the wavelength of reentrant circuits (APDxconduction velocity) to match the reentry pathway or the approximate dimensions of the heart. Each rapidly propagating reentry wavefront catches up to its tail and encounters relatively refractory tissue, resulting in a slowing down of conduction. The slow reentrant wavefront propagates through a cycle and then encounters excitable tissue to initiate a fast reentrant beat; hence, the VT alternans are perpetuated. The stable alternans shown in the transgenic mouse hearts in VT is in sharp contrast to reentrant circus movement VT in rings of atrial tissue where CL oscillations preceded termination of VT.54 Simulations of spiral wave breakup predict that electrical alternans leads to spatially disorganized wave activity and a transition from VT to VF.55
The results of the present study emphasize that changes in distribution and abundance of ionic currents alone, in the absence of metabolic injury of anatomical barriers, can increase the vulnerability to reentrant VT. Changes in ion channel abundance and cardiac ionic currents are well documented in pathological conditions such as congestive heart failure.7 8 Clearly, substantial differences exist between humans and mice with respect to the ionic basis of the cardiac AP, the distribution of ion channels, and the dimensions of the heart. Despite this, studies on molecularly engineered mice may help to elucidate common mechanisms that lead to arrhythmias and sudden cardiac death in human disease states.
Study Limitations
A major concern is that motion artifact might limit the validity
and interpretation of APD and repolarization maps in the mouse. All
methods that were used to measure APDs, whether intracellular
electrodes, extracellular electrodes, or voltage-sensitive dyes,
encounter problems caused by muscle contractions, which distort AP
downstrokes and the time course of repolarization. We were unable to
use agents that inhibit contraction such as DAM or cyto-D due to the
changes they caused in APD. We therefore relied on a chamber to inhibit
motion.
Several lines of evidence indicate that we have sufficiently eliminated
motion artifact to minimize significant errors in repolarization time
points. (1) Optical mapping techniques rely on extensive repetition to
ensure the validity and reproducibility of APDs and are based on
simultaneous measurements from multiple sites. We found
remarkable consistency in our measurements, including
identical findings with APD75 and
APD90. (2) Minimal baseline drift was seen after
repolarization of the AP (<2% estimated error in
APD90), and only minor changes were seen after
the addition of DAM or cyto-D. In the absence of the mechanical
stabilizing chamber, appreciable errors were seen. (3) A reproducible
pattern of repolarization was seen (Figure 4D). This would be
unlikely if major errors of repolarization were present. (4) We
found that the refractory period gradients were similar to those
of APD. The former are not subject to motion artifacts. (5)
Gradients of APD between apex and base remained in the presence of DAM
and cyto-D, despite marked prolongation of APD. Thus, although we
cannot absolutely exclude some component of error due to motion
artifacts, we believe that repolarization maps were quantitatively
accurate and that enhanced dispersion of repolarization underlies the
reentrant arrhythmia in transgenic mice.
To measure restitution kinetics, the stimulating electrodes used to
apply the basic or premature impulses, or both, must have small
dimensions (
250-µm diameter) and the shock strength must be 1.5
times the threshold voltage. Otherwise, the stimuli can readily
depolarize a large percentage of the mouse ventricle and effectively
shock rather than pace these small hearts. We took care to maintain
these conditions.
The optical technique used in the present study imaged the initiation and maintenance of reentrant VT on a 4x4-mm2 area of the epicardial surface of the left ventricle. We did not simultaneously visualize the septum or right ventricular surface, leading to uncertainly regarding the complete path of the reentrant arrhythmia. Bipolar electrograms from the right ventricular surface were monitored, however, and demonstrated continuity with the arrhythmia on the left ventricular epicardial surface. Similarly, optical recordings and programmed stimulation were not performed in the midmyocardium or on the endocardium. Differences in ion channel distribution have been documented across the myocardial layers of larger mammals,50 and we cannot exclude differences in the electrophysiological properties across the myocardium of the mouse heart. We doubt that this will affect the conclusions of the present study, however, given the thinness of the mouse heart wall (<1 mm) and the similar results obtained during epicardial pacing and sinus rhythm with endocardial activation.
An N-terminal fragment of the K+ channel Kv1.1 was used to disrupt repolarizing K+ currents of the Kv1.x family in the hearts of the mice that we studied. Kv1.2, Kv1.4, and Kv1.5 are expressed in the heart2 ; the relative extent and importance of these channels in cardiac repolarization remain uncertain. Thus, the detailed molecular mechanism by which the transgene leads to QT prolongation and arrhythmias remains under investigation. In addition, it is possible that the transgene may affect repolarization in other ways (eg, by binding ß-subunits). Despite these concerns, the results shown here directly link changes in the dispersion of repolarization and refractoriness to an enhanced susceptibility to reentrant VT.
The mouse is not an ideal model for human cardiac electrophysiology or for the human long-QT syndrome. Its small size may limit reentrant circuits, its basal heart rate is >600 bpm, and its APD is short even when corrected for heart rate. However, the mouse model provides the unique advantage of molecular engineering to genetically manipulate ionic currents involved in the repolarization of the AP. In this system, we have shown that manipulations that increase the dispersion of repolarization and refractoriness from apex to base increase susceptibility to reentrant VT. We speculate that in disease states like ischemia, similar changes in repolarization and postrepolarization refractoriness may enhance the propensity to arrhythmias and sudden death in larger mammals, including humans.
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Acknowledgments |
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This study was supported by National Institutes of Health (NIH) grants RO1-HL-57929 and HL-59614 (to Dr Salama); National Heart, Lung, and Blood Institute grant KO8-HL-02843 and a Grant in Aid from the American Heart Association (AHA) (to Dr London); NIH grant RO1-HL-46005 and an Established Investigator Award of the AHA (to Dr Koren); and a predoctoral fellowship from AHA, Western Pennsylvania Affiliate (Dr Baker). The authors thank Scott MacPherson and William Hughes for machining chambers and optical components. The authors are grateful to Gregory Szekeres and James VonHedemann for their help in the design and construction of the computer interface to the array.
Received October 4, 1999; accepted December 1, 1999.
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