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Vol. 292, Issue 1, 381-386, January 2000
The Cardiovascular Research Group, Departments of Medicine and Pathology, The University of Calgary, Calgary, Alberta, Canada
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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This study tested the hypothesis that combination ion channel blockers of the transient outward current (Ito) and the rapid component of the delayed rectifying current (IKr) would produce greater prolongation of the ventricular action potential duration (APD) and increased dispersion of the APD in hypertrophied hearts compared with control hearts. Isolated rabbit hearts were studied 48 ± 5 days postabdominal aortic banding. Left ventricular endocardial and epicardial APDs were significantly greater at baseline in the hypertrophied group than in controls (P < .05). The magnitude of APD prolongation induced by the Ito blocker 4-aminopyridine (4-AP) and combination 4-AP and the IKr blocker dofetilide was greater in the hypertrophied hearts than in the normal hearts (P < .01). Mean APD dispersion was significantly greater in the hypertrophied group than in the control hearts at baseline (P < .05). 4-AP increased APD dispersion by a similar magnitude in the hypertrophied hearts (10 ± 10 ms) and the control hearts (8 ± 8 ms, P = NS), whereas the combination 4-AP and dofetilide increased APD dispersion by a greater magnitude in the hypertrophied hearts (41 ± 28 ms) than the control hearts (21 ± 11 ms, P < .05). Ventricular fibrillation occurred spontaneously in four hypertrophied hearts (40%) during combination drug perfusion and in none of the control hearts (P < .05). Thus, combination Ito and IKr blockers cause greater prolongation APD and increased APD dispersion in left ventricular hypertrophy, and this is associated with the development of ventricular fibrillation.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Ventricular
dilation (Reiter et al., 1988
; Zabel et al., 1996) and some
antiarrhythmic drugs may increase dispersion of ventricular repolarization (Hii et al., 1992; Hohnloser et al., 1995; Zabel et al.,
1997; Gillis et al., 1998c) and predispose to arrhythmia development.
The increased prolongation of the ventricular action potential duration
(APD) and increased dispersion of APD that occur in left ventricular
hypertrophy may be due, in part, to changes in the spatial
heterogeneity of the density of the transient outward current
(Ito) (Hart, 1994; Tomita et al.,
1994; Gillis et al., 1998a), the delayed rectifying current
(IKr) (Kleiman and Houser, 1989;
Furukawa et al., 1994), and the inward rectifying current
(IKl) (Brooksby et al., 1993; Gillis
et al., 1998a). We have previously reported that
BaCl2, in concentrations that block IKl, produces greater prolongation of
the APD in hypertrophied rabbit hearts compared with control hearts
(Gillis et al., 1998a). In contrast, 4-aminopyridine (4-AP) in
concentrations that block Ito and
dofetilide in concentrations that selectively block
IKr did not produce such an effect on
APD in hypertrophied hearts. The effects of combinations of selective
potassium channel blockers on APD and dispersion of APD in hypertrophy
are unknown. We hypothesized that a change in the balance of
repolarizing currents that occur in hypertrophy would result in an
increase in the effects of combination Ito and
IKr blockers on APD and APD dispersion
in hypertrophy compared with controls. Furthermore, we hypothesized
that such an effect might be further exaggerated by increases in
ventricular preload.
Accordingly, the purposes of this study were 1) to compare the effects of modest increases in ventricular preload on APD and dispersion of APD in hypertrophied rabbit hearts compared with control hearts and 2) to compare the effects of the Ito blocker 4-AP, the IKr blocker dofetilide, and combination 4-AP and dofetilide on APD and dispersion of APD in hypertrophied hearts with those in control hearts at baseline and during increased preload.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals and Surgical Procedure.
Male New Zealand White
rabbits weighing 2.5 to 3.0 kg were studied. Left ventricular pressure
overload was induced by partial ligation of the abdominal aorta as
previously described (Gillis et al., 1998a,b). The animals were kept in
the animal care center until the day of study. Animals matched for age
and weight served as controls. The day before study, arterial blood
pressure was measured by inserting a 22-gauge catheter percutaneously
into an ear artery. All procedures performed conformed to the guiding principals of the Canadian Council on Animal Care.
Experimental Preparation. Animals were studied 48 ± 5 days after abdominal surgery. By this time, significant left ventricular hypertrophy had developed in animals undergoing abdominal aortic constriction compared with age- and weight-matched control animals (P < .05; Table 1). On the day of study, rabbits were pretreated with heparin sulfate (75 U/kg) and then anesthetized with sodium pentobarbital (35 mg/kg). Hearts were rapidly removed through a median sternotomy incision and perfused retrogradely via the aorta with blood buffer-perfusate (10%) hematocrit. The Krebs-Henseleit buffer consisted of 118 mM NaCl, 3.3 mM KCl, 2.0 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 24 mM NaHCO3, 10 mM glucose, 2.0 mM Na pyruvate, and 10 mg/l albumin.
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). Erythrocytes isolated from blood of healthy nonanesthetized sheep were
prepared the morning of study and added to oxygenated Krebs-Henseleit buffer to give a 10% hematocrit (Gillis et al., 1996a, 1998a). The blood-buffer perfusate was warmed to 37°C and equilibrated with
95% O2/5% CO2 in a
custom-made bath containing a Teflon-coated drum. Rotation of the drum
allowed oxygenation of erythrocytes. The perfusate was prefiltered with
a transfusion filter. Coronary sinus and aortic effluent were filtered
and recirculated via Teflon tubing to the oxygenated reservoir.
A latex balloon catheter was inserted into the left ventricle via a
left atriotomy for modulation of left ventricular preload (Reiter et
al., 1988; Zabel et al., 1996). The volume of the intraventricular balloon was changed by hand injections of 1.5 ml of saline. A 4F
open-lumen catheter was attached to the base of the balloon catheter
for monitoring of left ventricular pressure and the derivative of left
ventricular pressure (dP/dt). Hearts were ventricularly paced at a
cycle length of 500 ms (pulse width, 2.0 ms; twice diastolic threshold
intensity) via a bipolar platinum electrode on the lateral wall of the
left ventricle. Platinum electrodes were positioned on the epicardial
surface of the heart for monitoring of the ECG. A plastic ring with
seven evenly spaced electrode holders was mounted around the heart.
Monophasic action potentials were recorded from seven sites on the
epicardial surface of the ventricle via custom-made Ag-AgCl contact
monophasic action potential electrodes: two sites on the right
ventricle and five sites on the left ventricle (Zabel et al., 1996;
Gillis et al., 1998b). One endocardial monophasic action potential was
recorded from the left ventricle via an electrode inserted through the
left ventricular wall. Figure 1
illustrates the location of the monophasic action potential electrodes
and examples of action potential recordings from each site.
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Experimental Protocol.
The perfusion protocol was carried
out in 11 control hearts and 10 hypertrophied hearts. Coronary flow was
maintained at 30 ml/min. Baseline electrophysiologic data were
collected during a 30-min period of perfusion with blood-buffer
containing no drug. Then perfusion with 0.5 mM 4-AP, a concentration
that has been shown to block one component of
Ito (Campbell et al., 1993),
was initiated for 30 min, and electrophysiologic data were collected 15 min after initiation of 4-AP perfusion. Perfusion with the combination
of 0.5 nM 4-AP and 15 nM dofetilide, a concentration that has been
shown to selectively block IKr
(Jurkiewicz and Sanguinetti, 1993), was then initiated for 30 min, and
electrophysiologic data were collected 15 min after initiation of the
drug combination. During each stage, electrophysiologic data were
collected at low preload (no balloon inflation) and during inflation of
the balloon to 1.5 ml. A 2-min recovery interval was allowed before the
next intervention.
Electrophysiologic, Hemodynamic, and Morphologic
Measurements.
The monophasic action potential recordings, left
ventricular pressure, and derivative of left ventricular pressure
(dP/dt) were digitally acquired at 100 Hz/channel on a Compaq 386 system with a data translation (DT 2821) analog and digital
input-output board (Gillis et al., 1998a,b). This board provides a
12-bit resolution, 50-Hz throughput, and software-programmable gains.
APDs recorded by the monophasic action potential electrodes were
measured from the steepest part of the action potential upstroke to the
level of 90% repolarization. We defined the distance from the
diastolic baseline to the crest of the plateau as the total action
potential amplitude (Gillis et al., 1998b).
; Gillis et al., 1998b). Left ventricular
end-diastolic pressure, peak left ventricular systolic pressure, and
peak positive dP/dt were continuously monitored. Coronary flow was
measured as the coronary sinus effluent ejected via the pulmonary
artery. APDs at 90% (APD90) repolarization, left
ventricular pressures, and dP/dt were measured semiautomatically with a
custom-designed software program. An average of five complexes were
measured at each sampling time. Dispersion of the APD was defined as
the difference between the maximal and minimal recorded APD intervals
(Zabel et al., 1996; Gillis et al., 1998b). Dispersion of ventricular
activation time was defined as the difference between the maximal and
minimal recorded activation times.
Hearts were blotted and weighed at the end of each experiment.
Transverse sections of the heart were cut at the midventricular lead at
the end of each study and fixed in 10% buffered formalin for
light-microscopic study. Sections of paraffin block were stained with
H&E or Gomori trichome (Gillis et al., 1998a). Other sections were
stained by periodic acid Schiff with or without diastase to
outline the cell membranes. Histologic sections were imaged on a
television monitor at ×1000 magnification, and the diameter across the
nucleus of longitudinally oriented myocytes located in the
interventricular septum was measured semiautomatically by marking two
points with a light pen with a Bioquant 4 Image Analysis System
(Rand M Biometrics Inc., Nashville, TN) (Hoshino et al., 1983; Schoen
et al., 1984). The diameters of 80 myocytes were averaged for each experiment.
Statistical Analysis.
Data are presented as means ± S.D. Electrophysiologic and hemodynamic parameters were compared
between the sham and hypertrophied hearts. The effects of increasing
preload, 4-AP, and the combination of 4-AP and dofetilide were compared
within and between groups. Differences between groups were
compared with the paired t test, unpaired t test,
or factorial ANOVA for repeated measures (Montgomery, 1991) where
appropriate. Differences were considered statistically significant at
P < .05.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The characteristics of the control and hypertrophied hearts are shown in Table 1. The hypertrophied animals were slightly heavier than control animals on the day of study (P < .05). The heart mass, left ventricular mass, and ventricular myocyte diameter were significantly (P < .01) greater in the hypertrophied than the control group. The heart-to-body weight ratio was also greater in the hypertrophied group than the control group (P < .05). The mean arterial blood pressure was significantly greater in the hypertrophied animals. Significant histologic abnormalities were not observed in the hypertrophied hearts compared with the control hearts.
Time-Dependent Experiments. APD measured on the epicardial surface of the heart (APDepi) and on the endocardial surface (APDendo) of the left ventricle remained stable over time in the absence of drug perfusion at low and high preload (Table 2). As well, left ventricular systolic pressure and end diastolic pressure remained stable over time at low and high preload (Table 2).
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Changes in Left Ventricular Systolic and Diastolic Pressure. At low preload, the left ventricular systolic pressure was 19 ± 12 mm Hg in the control hearts and 23 ± 16 mm Hg in the hypertrophied hearts (P = NS). Increasing the preload resulted in a significant (P < .05) increase in left ventricular systolic pressure in both the control hearts (35 ± 22 mm Hg) and hypertrophied hearts (42 ± 15 mm Hg). Systolic pressures remained unchanged at a given preload during the perfusion protocol. At low preload, the left ventricular end-diastolic pressure was 1 ± 3 mm Hg in the control hearts and 3± 2 mm Hg in the hypertrophied hearts (P = NS). Increasing preload resulted in a slight (P = NS) increase in the end-diastolic pressure in control hearts (2 ± 3 mm Hg) or hypertrophied hearts (5 ± 2 mm Hg). The left ventricular end-diastolic pressure remained unchanged at low preload during the drug perfusion protocol.
APDs.
The mean APDepi values measured
from one site on the lateral wall of the left ventricle at baseline and
during drug perfusion in the hypertrophied and control hearts are shown
in Fig. 2. APDepi was prolonged in the hypertrophied group compared with the control group at baseline and during perfusion with 4-AP and 4-AP plus dofetilide (P < .05). The drug 4-AP alone and in
combination with dofetilide caused significant (P < .001) prolongation of APDepi in both
hypertrophied and control hearts. However, the magnitude of
APDepi prolongation induced by 4-AP and 4-AP plus
dofetilide was greater in the hypertrophied hearts than in the control
hearts (P < .05 by factorial ANOVA).
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APD Dispersions.
Mean APD dispersions measured at baseline and
during drug perfusion at low and high preload are shown in Fig.
4. APD dispersion was significantly
(P < .05) greater in the hypertrophied group than in
the control group at baseline and during drug perfusion. Drug perfusion
with 4-AP and 4-AP plus dofetilide significantly (P < .001) increased APD dispersion in both groups, and this effect was
greater in the hypertrophied hearts than in the control hearts (P < .005). 4-AP increased APD dispersion by a similar
magnitude in the hypertrophied hearts (10 ± 10 ms) and the
control hearts (8 ± 8 ms; P = NS), whereas 4-AP
plus dofetilide increased APD dispersion by a greater magnitude in the
hypertrophied hearts (41 ± 28 ms) compared with the control
hearts (21 ± 11 ms; P < .05). The effect of 4-AP
plus dofetilide on APD dispersion tended to be further exaggerated in
hypertrophy after an increase in preload (P = .08).
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Spontaneous Ventricular Fibrillation. Spontaneous ventricular fibrillation developed during 4-AP plus dofetilide perfusion in four hypertrophied hearts (40%) and no control hearts (P < .05). Spontaneous ventricular fibrillation did not develop during baseline or 4-AP perfusion in either group.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we have demonstrated that the Ito blocker 4-AP and combination Ito and IKr blockers 4-AP plus dofetilide cause greater prolongation of left ventricular APD and increased dispersion of ventricular repolarization in left ventricular hypertrophy compared with control hearts. Modest increases in preload tended to exaggerate the drug-induced increased dispersion of APD in hypertrophied hearts. Furthermore, spontaneous ventricular fibrillation was more likely to occur in hypertrophied hearts than in control hearts during perfusion with the combination Ito and IKr blockers.
Ventricular hypertrophy induced by pressure or volume overload is
associated with alterations in cardiac electrophysiologic properties
including prolongation of APD (Aronson, 1980; Cerbai et al., 1994;
Hart, 1994; Gillis et al., 1998a) and increased dispersion of
ventricular repolarization (Kowey et al., 1991; Buja et al., 1993;
Davey et al., 1994; Hart, 1994; Gillis et al., 1998b). Changes in ionic
channel current density that are thought to contribute to these
electrophysiologic abnormalities include decreases in
Ito,
IKr, and
IK1 (Brooksby et al., 1993; Gillis et
al., 1998a). We have previously reported that
IK1 and
Ito but not
IKr current densities are
significantly reduced in ventricular myocytes isolated from
hypertrophied rabbit hearts compared with controls (Gillis et al.,
1998a). We also observed that barium in concentrations that selectively
block inward rectifying currents produced greater APD prolongation in
hypertrophied rabbit hearts. In contrast, dofetilide or low
concentrations (0.2 mM) of 4-AP did not significantly prolong APD to a
greater extent in hypertrophied hearts compared with control hearts
(Gillis et al., 1998a). In this study, higher concentrations of 4-AP
did produce greater prolongation of the APD in hypertrophied hearts
compared with control hearts. Thus, the reduction in the two dominant
repolarizing currents in hypertrophied rabbit heart,
Ito and
IK1, is associated with an altered
pharmacodynamic response: exaggeration of the effects of
Ito and
IK1 blockers on prolongation of APD.
Dofetilide in similar concentrations to those used in this study did
not prolong APD to a greater extent in hypertrophied hearts (Gillis et
al., 1998b). However, the combination of
Ito and
IKr blockade further exaggerated the
prolongation of left ventricular endocardial APD in hypertrophy
compared with 4-AP alone. This suggests that a change in the balance of
repolarizing currents in hypertrophy, i.e., a greater dominance of
IKr in repolarization resulting from the decrease in Ito, could result in
an increased pharmacodynamic effect of the combination ion channel blockers.
Increased dispersion of ventricular repolarization has been reported in
left ventricular hypertrophy and is associated with increased risk of
sudden death (Buja et al., 1993; Davey et al., 1994). We previously
reported that increased dispersion of ventricular APD occurs in this
rabbit model of left ventricular hypertrophy and that dofetilide in the
same concentration as used in the current study did not significantly
increase APD dispersion in hypertrophy compared with control hearts
(Gillis et al., 1998b). In this study, 4-AP caused a greater increase
in APD dispersion in hypertrophied hearts than in control hearts.
Furthermore, 4-AP plus dofetilide substantially increased APD
dispersion in hypertrophied hearts compared with control hearts. In
another model of cardiac hypertrophy associated with complete heart
block, the class III drug almokalant was reported to cause greater
prolongation of APD and increased disparities of repolarization
compared with controls (Volders et al., 1998).
Increases in preload have been reported to increase dispersion of
ventricular repolarization in isolated perfused hearts (Zabel et al.,
1996; Calkins et al., 1989) and to increase the propensity to
ventricular arrhythmias (Reiter et al., 1988; Franz et al., 1992). In
this study, modest increases in preload did not substantially increase
APD dispersion at baseline in either control or hypertrophied hearts.
However, the magnitude of APD dispersion tended to be further increased
in hypertrophied hearts compared with control hearts when preload was
increased during perfusion of 4-AP plus dofetilide.
Antiarrhythmic drug-induced dispersion of ventricular repolarization is
associated with increased risk of ventricular proarrhythmia (Hii et
al., 1992) and decreased probability of antiarrhythmic drug efficacy
(Gillis et al., 1998c). In this study, the 4-AP plus dofetilide was
associated with a greater frequency in the development of spontaneous
ventricular fibrillation in hypertrophied hearts compared with control
hearts. In our previous studies, the spontaneous occurrence of
ventricular fibrillation was not observed during the perfusion of 15 nM
dofetilide alone in hypertrophied hearts (Gillis et al., 1998a,b). It
is likely that increased APD dispersion provided the substrate for
ventricular reentry and that ventricular fibrillation may have been
initiated by drug-induced afterdepolarizations. The onset of
ventricular fibrillation was not recorded in this study. Dofetilide has
recently been reported to be safe for use in patients with heart
failure (Pedersen, 1998; Torp-Pedersen et al., 1999). In contrast,
other class I and III antiarrhythmic drugs are proarrhythmic in
patients with ventricular dysfunction or after myocardial infarction
(The Cardiac Arrhythmia Suppression Trial Investigators, 1989; Waldo et
al., 1996). Volders et al. (1998) reported that the class III drug
almokalant causes greater prolongation of APD and a higher incidence of
afterdepolarizations in canine myocytes isolated from hypertrophied
hearts. Thus, this study suggests that combined
Ito and
IKr blockers might be proarrhythmic in
the setting of left ventricular hypertrophy and increased dispersion of
ventricular repolarization.
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Acknowledgments |
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We thank Ronda Ross for manuscript preparation.
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Footnotes |
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Accepted for publication October 5, 1999.
Received for publication July 7, 1999.
1 This study was supported by the Medical Research Council of Canada. Dr. Gillis is a senior scholar of the Alberta Heritage Foundation for Medical Research.
Send reprint requests to: Anne M. Gillis, M.D., FRCPC, Division of Cardiology, The University of Calgary, 3330 Hospital Dr., N.W., Calgary, Alberta, Canada T2N 4N1. E-mail: amgillis{at}ucalgary.ca
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Abbreviations |
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APD, action potential duration; 4-AP, 4-aminopyridine; APD90, action potential duration at 90% repolarization; APDepi, epicardial action potential duration; APDendo, endocardial action potential duration; Ito transient outward current, IKr, rapid component of the delayed rectifying current; IK1, inward rectifying current.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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