Download Larger Cell Size in Rabbits With Heart Failure Increases Myocardial

Larger Cell Size in Rabbits With Heart Failure Increases
Myocardial Conduction Velocity and QRS Duration
Rob F. Wiegerinck, MSc; Arie O. Verkerk, PhD; Charly N. Belterman, RA;
Toon A.B. van Veen, PhD; Antonius Baartscheer, PhD; Tobias Opthof, PhD; Ronald Wilders, PhD;
Jacques M.T. de Bakker, PhD; Ruben Coronel, MD, PhD
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Background—Patients with heart failure (HF) have an increased QRS duration, usually attributed to decreased conduction
velocity (CV) due to ionic remodeling but which may alternatively result from increased heart size or cellular
uncoupling. We investigated the relationship between QRS width, heart size, intercellular coupling, and CV in a rabbit
model of moderate HF and in computer simulations.
Methods and Results—HF was induced by pressure-volume overload. Heart weight (21.1⫾0.5 versus 10.2⫾0.4 g,
mean⫾SEM; P⬍0.01) and QRS duration (58⫾1 versus 50⫾1 ms; P⬍0.01) were increased in HF versus control.
Longitudinal CV (␪L; 79⫾2 versus 67⫾4 cm/s; P⬍0.01) and transversal subepicardial CV (␪T; 43⫾2 versus 37⫾2 cm/s;
P⬍0.05) were higher in HF than in controls. Transmural CV (␪TM) was unchanged (25⫾2 versus 24⫾1 cm/s; P⫽NS).
Patch-clamp experiments demonstrated that sodium current was unchanged in HF versus control. Immunohistochemical
experiments revealed that connexin43 content was reduced in midmyocardium but unchanged in subepicardium.
Myocyte dimensions were increased in HF by ⬇30%. Simulated strands of mammalian ventricular cells (Luo-Rudy
dynamic model) revealed increased ␪L and ␪T with increased myocyte size; however, increased CV could not
compensate for increased strand size of longitudinally coupled cells, and consequently, total activation time was longer.
Conclusions—Increased myocyte size combined with the observed expression pattern of connexin43 yields increased ␪L
and ␪T and unchanged ␪TM in our nonischemic model of HF. A hypertrophied left ventricle together with insufficiently
increased ␪L and unaltered ␪TM results in a prolonged QRS duration. (Circulation. 2006;113:806-813.)
Key Words: hypertrophy 䡲 heart failure 䡲 electrocardiography 䡲 myocytes 䡲 conduction
P
atients with heart failure (HF) have a poor prognosis.
Mortality is high, and ⬇50% of patients with HF die
suddenly, which suggests an arrhythmogenic origin.1,2
Patients with HF often reveal increased QRS duration3,4
and those with QRS duration ⬎150 ms have a higher
cardiac mortality rate than patients with shorter QRS
duration.4,5 Increased QRS duration is commonly interpreted as slowing of ventricular conduction. Although
slow conduction is one of the prerequisites for the occurrence of reentrant arrhythmias6 and therefore may explain
the increased propensity of patients with HF for lifethreatening arrhythmias,1,2 studies on conduction velocity
(CV) in HF are contradictory.
at day 150 after aortic banding; however, in the same model,
CV was increased after 50 days of aortic banding.11 This
underscores the dynamic nature of HF. In 2 other studies that
included patients with HF, an increased CV was measured12,13 as well. Increased CV was also found in hypertrophied hearts.11–13 In hypertrophy, cell size increases. We
hypothesized that increased cell size underlies both the
increase in CV and the increase in QRS duration. The latter
would increase because it takes more time to activate the
hypertrophied ventricles, despite the increase in CV. The aim
of the present study was to document CV changes and to
correlate CV with QRS duration, connexin43 (Cx43) content,
and cell size in an established nonischemic model of moderate HF,14 in which hypertrophy is the predominant feature and
in which electrical remodeling is negligible. The functional
effects of the experimentally observed changes in cell size
were assessed with computer simulations at various preset
values of intercellular coupling.
Clinical Perspective p 813
In animal studies, unchanged CV7,8 and decreased CV9
have been reported in the setting of HF. QRS duration was
increased10 and CV decreased11 in a guinea pig model of HF
Received May 31, 2005; revision received December 7, 2005; accepted December 8, 2005.
From the Departments of Experimental Cardiology (R.F.W., A.O.V., C.N.B., A.B., T.O., J.M.T.d.B., R.C.) and Physiology (A.O.V., R.W.), Academic
Medical Center, University of Amsterdam, Amsterdam, the Netherlands; the Department of Medical Physiology (T.A.B.v.V., T.O., J.M.T.d.B.),
University Medical Center Utrecht, Utrecht, the Netherlands; and the Interuniversity Cardiology Institute of the Netherlands (J.M.T.d.B.), Utrecht, the
Netherlands.
Correspondence to Rob F. Wiegerinck, MSc, Department of Experimental Cardiology, Molecular & Experimental Cardiology Group, Academic
Medical Center, Room M0-109, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail [email protected]
© 2006 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/CIRCULATIONAHA.105.565804
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Wiegerinck et al
Methods
Rabbit Model of HF
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The experimental protocol complied with the “Guide for the Care
and use of Laboratory Animals” published by the US National
Institutes of Health (NIH publication 85-23, revised 1985) and was
approved by the institutional animal experiments committee. HF was
induced by combined volume and pressure overload in 2 sequential
surgical procedures in New Zealand White rabbits as described
previously.14 In that study,14 differences between nonoperated and
sham-operated animals were negligible. Thus, for the present study,
control rabbits were not sham-operated. Before the first operation, a
3-lead ECG was recorded in rabbits premedicated with ketamine
(100 mg/mL, 0.7 mL/kg body weight IM) and xylazine (20 mg/mL,
0.3 mL/kg body weight IM). Control and HF rabbits (at 8 months of
age or when HF with overt signs of dyspnea developed) were
weighed, a 3-lead ECG was recorded, and left ventricular end-diastolic pressure was measured as described previously.14 Rabbits were
heparinized (1000 IU IV) and killed by injection of pentobarbital (60
mg/mL, 5 mL, IV). Hearts were isolated and submerged in Tyrode’s
solution (4°C; for composition, see below). Presence of ascites and
heart and lung weights were documented.
Conduction, QRS Width, and Heart Failure
807
was achieved by using low-resistance pipettes (1.0 to 2.0 M⍀) and
Rs and Cm compensation ⬎80%.
In the cell-attached macropatch experiments, the pipette and bath
solutions contained (in mmol/L) NaCl 140, KCl 5.4, CaCl2 1.8,
MgCl2 1.0, glucose 5.5, and HEPES 5.0 (pH 7.3 [NaOH]). The
pipette command potential was corrected for the resting membrane
potential (Vm) of the myocytes, which was ⫺81.5⫾0.9 and
⫺81.7⫾0.6 mV in control (n⫽37) and failing (n⫽32) myocytes,
respectively, as determined in a separate set of whole-cell patchclamp experiments. INa was defined as the difference between peak
current and the current at the end of the depolarizing voltage step.
Myocyte Dimensions
The size of individual left ventricular myocytes was determined in 6
control and 7 HF animals. Enzymatically isolated myocytes were
studied in a cell chamber on the stage of an inverted microscope
during superfusion with a HEPES solution containing (in mmol/L)
NaCl 148, NaHCO3 1.0, KHCO3 3.3, KH2PO4 1.4, MgCl2 1.9, CaCl2
1.3, glucose 11.0, HEPES 16.8 (37°C, pH 7.3 [NaOH]). Cellular
length and width were measured at a resolution of 3 ␮m in 50
randomly selected viable rod-shaped myocytes per heart.
Immunohistochemistry and Histology
Perfusion of Hearts
After excision of the heart, the aorta was prepared free from adjacent
tissue. In control rabbits, a cannula was inserted into the aorta to
allow retrograde perfusion of the heart. Because retrograde perfusion
is impossible owing to aortic insufficiency in HF, both coronary
arteries were cannulated separately. The hearts were perfused with
Tyrode’s solution containing (in mmol/L) 130 NaCl, 5.6 KCl, 2.2 CaCl2,
0.55 MgCl2, 24.2 NaHCO3, and 11.1 glucose, saturated with 95% O2
and 5% CO2. Myocardial temperature was maintained at 37°C.
Activation Mapping and CV
Activation times (ATs) were derived from local electrograms recorded with an epicardial multielectrode (247 unipolar electrode
terminals, 1.5⫻1 mm interelectrode distance) and/or with 4 intramural needles (8 electrode terminals/needle, interelectrode distance
0.3 mm) inserted 1 cm apart in a square on the left ventricular free
wall. Electrodes were connected to a data-acquisition system (Biosemi Mark-6). A virtual ground electrode was connected to the aortic
root. Data of 1.8-second episodes (sampling interval 0.5 ms) were
stored on disk. Local AT, defined as the moment of the maximum
negative dV/dt, was determined with a custom interactive analysis
program.15 Pacing from the center of the epicardial multielectrode or
from the most distal located electrode on a needle was performed
with rectangular current pulses at diastolic stimulation threshold
(2-ms duration) at a basic cycle length of 250 ms.
Subepicardial activation maps were constructed, and longitudinal
(␪L) and transversal (␪T) CVs were calculated from the ellipsoid
activation pattern as described previously.16 Transmural CV (␪TM,
perpendicular to the epicardial surface) was calculated from local
ATs measured on 1 needle.
Sodium Current
The fast sodium current (INa) is responsible for the action potential
upstroke and is important for CV.17 Therefore, INa was evaluated by
whole-cell patch-clamp (20°C) and cell-attached macropatch recordings (37°C).18 Myocytes were enzymatically isolated as described
previously.19 Currents were filtered at 5 kHz and digitized at 10 kHz.
Voltage control, data acquisition, and analysis were accomplished
with custom software. The INa current-voltage relationship was
determined by the voltage-clamp protocols diagrammed in Figure 2.
Whole-cell patch-clamp pipettes and bath solutions were as
described previously.20 Current density was calculated by dividing
whole-cell current amplitude by cell capacitance (Cm). Cm was
estimated by dividing the decay time constant of the capacitive
transient in response to 5-mV hyperpolarizing voltage-clamp steps
from ⫺40 mV by the series resistance (Rs). Adequate voltage control
Cryosections (10-␮m thickness) perpendicular or parallel to the
epicardial surface were made from the same area as the CV
measurements. Perpendicular sections incubated with rabbit polyclonal anti-Cx43 antibody (Zymed) and mouse monoclonal anti-Ncadherin antibodies (Sigma) were obtained by the method described
previously.21 Immunolabeled sections were mounted in Vectashield
mounting medium (Vector Laboratories) and examined with a light
microscope with epifluorescence equipment (Nikon Optiphot-2).
Presence of fibrosis was analyzed from parallel sections fixed with
4% paraformaldehyde and stained with picrosirius red. Images from
6 randomly selected fields per section viewed with light microscopy
were digitized, and the amount of fibrosis was analyzed with Image
Pro Plus (version 5.02, Media Cybernetics).
Computer Model
We assessed ␪L and ␪T in linear strands of 100 longitudinally or
transversally coupled subepicardial myocytes. Individual cells of the
strands were described with the Luo-Rudy dynamic model of
mammalian subepicardial ventricular myocytes (LRd model),22 including the transient outward current Ito1.23 As in other studies,24,25
entire cell length (for longitudinal conduction) or width (for transversal conduction) was used as the spatial discretization element in
our computations, with elements connected by the lumped myoplasmic resistance (calculated from the myocyte dimensions and the
myoplasmic resistivity of 150 ⍀/cm) and gap junctional resistance
(calculated from the myocyte dimensions and the gap junctional
resistivity).
Action potential propagation was elicited by injection of an ⬇20%
suprathreshold current pulse (duration 2 ms) into the first cell of the
strand, at a frequency of 4 Hz. The activation wave of the sixth action
potential was analyzed.
We assumed that membrane ionic channel density per unit of
membrane surface area did not change with increased cell size. We
ran simulations at normal and reduced intercellular coupling with
control and HF cell dimensions, assuming that cell shape was that of
a rectangular box. CV and total AT per myocyte were calculated
from the difference in AT of cell 20 and cell 80.
Statistical Analysis
Data are presented as mean⫾SEM. A 1-way ANOVA with the
Student-Newman-Keuls test for post hoc analysis was used to test for
a significant differences of ␪L, ␪T, ␪TM, stimulation threshold, QRS
duration, myocyte length and width, resting membrane potential, and
sodium current density (either whole-cell or cell-attached patch
clamp) in HF compared with control hearts. When data were not
normally distributed or variance was not equal, a Kruskal-Wallis
1-way ANOVA on ranks with Dunn’s test for post hoc analysis was
808
Circulation
TABLE 1.
February 14, 2006
Functional Characteristics of Control and HF Rabbits
Control
(n⫽22)
HF
(n⫽44)
BW, kg
4.3⫾0.1
4.2⫾0.1
HW, g
10.2⫾0.4
21.1⫾0.5*
HW/BW, g/kg
2.4⫾0.1
5.0⫾0.1*
LW/BW, g/kg
3.1⫾0.1
4.6⫾0.2*
LVEDP, mm Hg
3.4⫾0.5
20.5⫾1.5*
Ascites
0/22
21/44*
QRS duration, ms
50⫾1
58⫾1*
BW indicates body weight; HW, heart weight; HW/BW, relative heart weight;
LW/BW, relative lung weight; and LVEDP, left ventricular end-diastolic pressure.
Data are mean⫾SEM.
*P⬍0.01 for HF vs control. BW comparisons are by ANOVA; HW, KruskalWallis; HW/BW, Kruskal-Wallis; LW/BW, Kruskal-Wallis; LVEDP, Kruskal-Wallis;
ascites, ␹2; and QRS, ANOVA.
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used. This approach was used to test for significant differences of
heart weight, lung weight, heart weight/body weight ratio, lung
weight/body weight ratio, left ventricular end-diastolic pressure, and
amount of fibrosis in HF compared with control rabbits. The
presence of ascites in HF compared with controls was tested with the
␹2 test for statistical differences. P⬍0.05 indicates statistical
significance.
Results
HF Parameters
Table 1 shows characteristics of HF. There were 3 experimental groups: (1) measurement of CVs (control n⫽8; HF
n⫽15), (2) myocyte dimensions (control n⫽6; HF n⫽7), and
(3) patch-clamp experiments (control n⫽8; HF n⫽22). In
Table 1, data from control (n⫽22) or HF (n⫽44) animals
from each experimental group were lumped together because
there were no significant differences between the parameters
in the 3 subgroups. HF is present as evident from the presence
of ascites and the increase in (relative) heart weight, relative
lung weight, and left ventricular end-diastolic pressure compared with control.
TABLE 2.
CV in Control and HF Rabbits
Control
HF
␪L, cm/s
67⫾4 (n⫽8)
79 ⫾2* (n⫽15)
␪T, cm/s
37⫾2 (n⫽8)
43 ⫾2* (n⫽15)
␪TM, cm/s
24⫾1 (n⫽8)
25 ⫾2 (n⫽10)
Data are in centimeters per second. Data are mean⫾SEM.
*P⬍0.01 for HF vs control. ␪L: longitudinal CV (ANOVA); ␪T: transversal CV
(ANOVA); ␪TM: transmural CV (ANOVA).
control. This is also evident from the 15-ms isochrone (bold
line), which encloses a larger area in the activation map of the
HF than the control rabbit. The stimulation threshold was not
different between HF (165⫾33 ␮A) and control 164⫾25 ␮A
(ANOVA, P⫽NS). Average ␪L and ␪T (Table 2) were higher
in HF than in control animals. ␪TM was not different between
control and HF animals (Table 2).
Sodium Current
Figure 2 shows representative whole-cell patch-clamp (Figure
2A) and cell-attached macropatch (Figure 2B) INa recordings
from control and HF cells. Current density and current-voltage
relationship (Figure 2B) were not different in HF compared with
control in either condition (Figures 2C and 2D).
Cx43 and Fibrosis
Figure 3A shows normal Cx43 expression in all 3 layers of a
control heart, which is accompanied by an almost identical
pattern of N-cadherin expression (mechanical junction). Figure
3B shows a typical pattern of Cx43 in a heart from a rabbit with
HF. Expression in the subendocardium (6/6 hearts) and the
subepicardium (5/6 hearts) is similar to control. Cx43 (but also
N-cadherin) in the midmyocardium is reduced (6/6 hearts). In 1
heart, epicardial Cx43 was reduced, although not to the level
depicted for the midmyocardium. The total amount of fibrosis
was not different between control (21⫾1%, n⫽4) and HF
(20⫾2%, n⫽10; Kruskal-Wallis, P⫽NS).
Myocyte Dimensions
QRS duration (Table 1) was 58⫾1 ms (mean⫾SEM) in HF.
This was longer than in the same animals before the first
operation (49⫾1 ms) and in control animals (age 8 months;
50⫾1 ms; ANOVA, P⬍0.01).
Myocyte length and width were measured in 6 control and 7
HF rabbits (50 myocytes/heart). Figure 4 shows that myocyte
length (191.7⫾3.7 versus 144.9⫾2.4 ␮m, respectively) and
width (32.7⫾1.1 versus 25.2⫾0.5 ␮m, respectively) were
both increased by ⬇30% in HF compared with control
(ANOVA, P⬍0.05).
Conduction Velocity
Simulation Studies
Figures 1A and 1B shows representative subepicardial activation maps. Both ␪L and ␪T were higher in HF than in
We performed computer simulations (Figure 5) to test
whether the experimentally observed increase in myocyte
QRS Duration
Figure 1. Activation maps from a control
(A) and HF (B) rabbit heart. Dots indicate
individual electrodes on the multielectrode, which was placed on the left ventricular free wall. Lines indicate isochrones. Numbers indicate AT (in ms)
relative to the time of stimulation. The
increase in gray scale corresponds with
the 5-ms spaced isochrones. In the
white area, the AT ranges from 0 to 10
ms. Arrows indicate the direction and
magnitude of ␪L and ␪T.
Wiegerinck et al
Conduction, QRS Width, and Heart Failure
809
Figure 2. Fast sodium current (INa) in left
ventricular myocytes isolated from control and HF rabbits. A and B, Typical
whole-cell (A) and cell-attached patchclamp (B) recordings of INa in response to
a voltage clamp step from ⫺120 to ⫺30
mV. C and D, Average INa amplitude
measured with (C) whole cell and (D)
cell-attached patch clamp. Voltageclamp protocol shown as inset. See
Methods for further details. C indicates
control.
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dimensions (see Figure 4) can explain the HF-induced increase in ␪L and ␪T, as well as the increase in total AT as
indicated by the increase in QRS duration. First, we ran
simulations with cell length and width set to 144.9 and 25.2
␮m, respectively, ie, the mean values observed for control
myocytes. We found that total gap junctional conductance
values of 4 and 15 ␮S were required to obtain the experimentally observed ␪L and ␪T, respectively. These values of 4
and 15 ␮S correspond with a gap junctional resistivity (Rj) of
1.59 and 2.43 ⍀/cm2, respectively, and were regarded as
100% of intercellular coupling.
Next, we ran simulations with cell length and width set to
191.7 and 32.7 ␮m, respectively, ie, the mean values observed for failing myocytes. We assumed that the number of
gap junction and membrane ionic channels per unit membrane surface did not change. Figure 6 shows simulated CV
(Figures 6A and 6B) and total AT per myocyte (Figures 6C
and 6D). Longitudinal CV (Figure 6A) increased from 68
cm/s (control) to 81 cm/s (HF) at 100% intercellular coupling; however, the 19% increase in ␪L was not enough to
fully compensate for the 32% increase in myocyte length, and
therefore, total AT/myocyte (Figure 6C) was larger in HF
than control (237 versus 213 ␮s, 11% increase). Transversal
CV (Figure 6B) was 37 cm/s in control and 47 cm/s in HF
animals at 100% intercellular coupling. The 27% increase in
␪T was large enough to almost completely compensate for the
30% increase in cell width. Total AT per myocyte in the
strand of transversally coupled cells (Figure 6D) changed from
68 ␮s in control to 70 ␮s in HF animals (2.5% increase) at 100%
intercellular coupling. To test the dependence of our simulation
results on the degree of intercellular coupling, we repeated our
simulations at 20%, 40%, 60%, and 80% intercellular coupling,
respectively. Reduced coupling resulted in reduced CV, but at
every identical simulated value for intercellular coupling, CV
was larger in HF cells than in control cells. The dashed
horizontal lines in Figures 6A and 6B show that the CV in HF
cells with a coupling of 50% to 60% is similar to that of control
cells with 100% intercellular coupling.
Discussion
In a nonischemic rabbit model of moderate HF compared
with control, (1) QRS duration, ␪L, ␪T, and myocyte length
and width are increased; (2) ␪TM, sodium current density,
fibrosis, and subepicardial Cx43 expression are unchanged;
and (3) midmyocardial Cx43 expression is reduced. Computer simulations demonstrate that the increased cell size
results in increased CV at any level of intercellular coupling;
however, ␪L and ␪TM are not large enough to compensate for
the increase in ventricular dimensions in HF and thus result in
an increased QRS width.
Figure 3. Representative immunohistochemical labeling of Cx43 and
N-cadherin in left ventricle in control (A,
n⫽6) and HF (B, n⫽6). Left, Cx43
expression; Middle, N-cadherin (N-cad)
expression; Right, ensemble picture.
Subendocardial (Endo), midmyocardial
(Mid), and subepicardial (Epi) expression
patterns are shown in the upper, middle
and lower rows, respectively. Scale bar:
50 ␮m.
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Circulation
February 14, 2006
Figure 4. Dimensions of isolated myocytes. A,
Typical example of a myocyte from a control (left)
and an HF (right) rabbit. B and C, Mean myocyte
length (B) and width (C) in 6 control (white) and 7
HF (gray) rabbits (50 myocytes/rabbit).
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QRS Duration
QRS prolongation occurs in patients with HF26 and is
associated with increased cardiac mortality.4,5 In patients with
dilated cardiomyopathy, QRS duration increases progressively.3,27 This indicates that even in the presence of complete
bundle-branch block, increased left ventricular mass with
increased CV may cause an increase in QRS duration,
although it cannot be excluded that conduction is slowed in
more severe HF. Indeed, in a study of individuals free of HF
and myocardial infarction, QRS duration was positively
correlated with left ventricular mass and dimensions.28
Sodium Current
In cell-attached patches, INa was unchanged in HF compared
with control animals (Figure 2B). This indicates that the
brain-type sodium channels, located in T-tubules,29 do not
Figure 5. Geometry of simulated strands of (A) longitudinally or
(B) transversally coupled ventricular cells. Individual cells of the
strand were described with the LRd model.22 Cell length (L) and
width (W) were based on experimental data from left ventricular
myocytes isolated from control or failing rabbit hearts (Figure 4).
Rj denotes gap junctional resistivity. See Methods and text for
further details.
change in HF (Figure 2B). Whole-cell INa, carried by both
myocardial-type (located in the intercalated disk30) and braintype sodium channels, is also not changed in HF (Figure 2A).
Any change in CV is therefore attributable to changes in other
parameters. In contrast, INa peak current density was decreased31 in other models. This may imply that patients with
severe HF have a reduced CV that contributes to QRS
prolongation.
Intercellular Coupling and Fibrosis
Reduction of Cx43 may contribute to slowing of conduction.
Cx43 expression is reduced in the left ventricle of patients
with hypertrophic, dilated, or ischemic cardiomyopathies.32–34 These studies did not specifically report on subepicardial Cx43 expression. In the present study, we observed
reduced Cx43 expression in left ventricular midmyocardium
but not in subepicardium. In contrast, in a canine model of
rapid pacing–induced HF, Cx43 expression was reduced,
especially in the subepicardial layer.35 However, in dogs with
left bundle-branch block–related left ventricular dyssynchrony, the lateral wall showed no change in subepicardial
Cx43 expression.36 Lower Cx43 expression levels in subendocardium compared with subepicardium were further exaggerated in dogs with left bundle-branch block associated with
HF compared with controls.36,37 A transmural gradient of
Cx43 expression was present in mice but not in rats.38
Apparently, the transmural gradient of Cx43 expression
differs and changes within the heart, between species, and
with the underlying disease.
Tissue architecture and amount of fibrosis can affect
activation propagation in the setting of HF39; however,
fibrosis was not increased in the present model of HF.
Structural remodeling (except for hypertrophy) was also
absent because no infarction was made.
Conduction Velocity
In guinea pig after 150 days of aortic constriction, heart size,
QRS duration,10 and intracellular resistivity40 were increased
and CV11 was decreased. In a milder stage of HF (50 days of
Wiegerinck et al
Conduction, QRS Width, and Heart Failure
811
Figure 6. Simulation results obtained
with the strands as described in Figure
5. A and B, Effect of myocyte dimensions on CV in strands of longitudinally
(A) and transversally (B) coupled cells.
The dashed line illustrates the conditions
in which there is a balance between the
effects of intercellular coupling and hypertrophy. C and D, Effect of myocyte
dimensions on total AT per myocyte
(TAT/myocyte) in strands of longitudinally
(C) and transversally (D) coupled cells.
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aortic constriction), heart size and CV were increased,11
whereas intracellular resistivity40 and QRS width10 were
unchanged. Other studies reported a decreased subepicardial
CV in the setting of HF9 or no change.7,8 In the present animal
model, we were able to directly relate CV to the increased
cellular dimensions in subepicardium, where Cx43 was unchanged; however, this increased subepicardial CV (combined with unaltered ␪TM) was not enough to compensate for
the increase in myocyte dimensions and hence resulted in an
increased QRS duration. The data obtained in guinea pig
illustrate the dynamic nature of HF. In the present model, HF
is moderate and electrical remodeling is absent, except for
transient outward K⫹ current density (data not shown). This
makes it an ideal model to address the effect of hypertrophy.
In the present study, ␪TM was not changed. In contrast,
Toyoshima et al12 showed that ␪TM, calculated by dividing the
left ventricular wall thickness by the interval between onset
of the QRS complex and the moment of epicardial breakthrough, is increased in hypertrophy, whereas in a canine
model of rapid pacing–induced HF, ␪TM was decreased.35 The
discrepancy between these results might be due to differences
in the amount of Cx43 expression or electrical remodeling.
cell size is at least as important as Cx43 distribution in
transversal propagation.41
The simulations in the present study show that the increase
in ␪T may compensate for the increased dimensions of the
preparation, whereas the increase in ␪L does not compensate
for the increased length of the preparation and results in a
longer total AT. Our simulations with subepicardial cells
indicate that specific combinations of reduced intercellular
coupling and increased myocyte size may yield an unchanged
CV in HF, despite changes in both underlying parameters (cf
the dashed lines in Figures 6A and 6B). This may also explain
the unchanged ␪TM despite reduced midmyocardial Cx43
levels in our rabbit model.
Clinical Implications
QRS prolongation may be associated with increased or
normal CV in patients with HF/hypertrophy; however, the
potential antiarrhythmic effect of increased CV in patients
with HF/hypertrophy may be offset by a disproportional
increase in myocardial mass, which favors the onset of
reentrant arrhythmias.
Study Limitations
Myocyte Dimensions and Simulations
In our computer model, axial resistivity is decreased owing to
pathophysiological growth and results in increased CV.
Increase in cell length in a similar model but one that
compared adult with neonatal myocytes (wild-type and
Cx43⫹/⫺ mice) also resulted in decreased axial resistivity and
increased CV.25
In canine ventricle, normal physiological growth increased
myocyte length and width and ␪L and ␪T in adult dogs
compared with neonatal animals.41 The gap junction distribution also changed with aging. Computer simulations demonstrated that increase in myocyte size and Cx43 distribution
are sufficient to account for the experimental results and that
We directly compared CV of 2D measurements with 1D
simulations. In the measurements, we used point stimulation,
whereas our 1D simulations correspond with plane wave
stimulation in 2D. Therefore, our measurements are subject to
underestimation of planar CV due to wave-front curvature.42
Nevertheless, it is clear that the increase in cell size and CV
are directly related. We used 1D simulations with rectangular
boxlike cells, which does not reflect the mixed contribution of
longitudinal and transverse gap junctions in interdigitating
cell bundles. Length and width but not thickness of the
myocyte could be measured with light microscopy. We used
the width of the myocyte as an estimate for the thickness, but
we cannot exclude an overestimation.
812
Circulation
February 14, 2006
Our model of HF is relatively mild and focused on
hypertrophy, although ascites is a predominant feature. The
model permits study of increases in cell size and decreases in
intercellular coupling. In more severe stages of HF, additional
factors such as fibrosis or electrical remodeling also contribute to pathophysiological changes.
A major cause of HF is ischemia related. We chose a model
of combined volume/pressure overload to exclude infarcted
tissue as a confounding factor. Studies in explanted hearts
involve patients who were in the end stage of HF, whereas
our model reflects a moderate stage of HF. Thus, conclusions
derived from this particular model do not necessarily apply to
other HF models.
Conclusions
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
QRS prolongation was associated with increased heart size
and increased dimensions of left ventricular myocytes in our
nonischemic rabbit model of moderate HF. Also, the animals
with HF had higher left ventricular subepicardial CVs than
control animals, without any change in sodium current
density. Our computer simulations demonstrated that the
increase in myocyte dimensions per se may explain the
increase in subepicardial CV and the unchanged ␪TM despite
the reduction in midmyocardial Cx43 expression. Although
subepicardial CV was increased and ␪TM unchanged, this was
not enough to compensate for increased heart size. This
explains the increased QRS duration, as corroborated by our
simulation study in which total AT was longer in longer
strands with the same number of cells.
Acknowledgments
The authors thank Sara Tasseron, Shirley van Amersfoorth, and
Diana Bakker for their analysis of fibrosis. The authors had full
access to the data and take full responsibility for its integrity. All
authors have read and agree to the manuscript as written.
Disclosures
None.
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CLINICAL PERSPECTIVE
The width of the QRS complex is associated with the duration of complete activation of the ventricles. In hypertrophy and heart
failure, QRS duration is increased. Generally, this increase is interpreted as a decrease in ventricular conduction velocity. We
recorded QRS duration and measured conduction velocity (longitudinal, transversal, and transmural) in the left ventricle in an
animal model of hypertrophy/heart failure. Additionally, we measured the size of left ventricular myocytes and studied the
expression pattern of connexin43 in the left ventricle. QRS duration was increased in animals with heart failure compared with
controls. Myocyte dimensions were increased. Connexin43 expression remained normal in subepicardium but was reduced in
midmyocardium. Transmural conduction velocity was unaltered, and longitudinal and transversal subepicardial conduction
velocities were increased. Computer simulations demonstrated that an increase in myocyte dimensions per se may explain the
increase in subepicardial conduction velocity. Additionally, the increase in myocyte size in combination with the reduction in
midmyocardial connexin43 expression may explain the unchanged transmural conduction velocity. Although subepicardial
conduction velocity is increased and transmural conduction velocity unchanged, this is not enough to compensate for increased
heart size, and hence QRS duration is increased. Therefore, a wide QRS complex, as observed, for example, in patients with heart
failure, does not necessarily indicate slow ventricular conduction but can also be due to hypertrophy alone, which increases the
total time required to activate the ventricles.
Larger Cell Size in Rabbits With Heart Failure Increases Myocardial Conduction Velocity
and QRS Duration
Rob F. Wiegerinck, Arie O. Verkerk, Charly N. Belterman, Toon A.B. van Veen, Antonius
Baartscheer, Tobias Opthof, Ronald Wilders, Jacques M.T. de Bakker and Ruben Coronel
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Circulation. 2006;113:806-813; originally published online February 6, 2006;
doi: 10.1161/CIRCULATIONAHA.105.565804
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