1. Art and Diseño

Cardiovascular Research 52 (2001) 246–254
www.elsevier.com / locate / cardiores
Spatial alterations of Kv channels expression and K 1 currents in post-MI
remodeled rat heart
B. Huang, D. Qin, N. El-Sherif*
Cardiology Division, Department of Medicine, Box 1199, State University of New York Health Science Center and Veterans Affairs Medical Center,
450 Clarkson Avenue, Brooklyn, NY 11203, USA
Received 21 March 2001; accepted 31 May 2001
Abstract
Keywords: Gap junctions; Gene expression; Infarction; K-channel; Remodeling
1. Introduction
In recent years, the importance of ventricular remodeling after myocardial infarction (MI) on long-term survival
has been better appreciated. Studies from our laboratory
have shown that 3–4 weeks post-MI the noninfarcted
myocardium undergoes significant hypertrophy as part of
the remodeling process [1]. Electrophysiological studies
indicated that the hypertrophic response of the noninfarcted remodeled myocardium engenders an arrhythmogenic
substrate, including prolongation of action potential duration (APD), regional differences in APD, and increased
tendency to early afterdepolarization (EAD)-triggered activity and reentrant tachyarrhythmias [1]. The prolongation
*Corresponding author. Tel.: 11-718-270-4147; fax: 11-718-6303740.
E-mail address: [email protected] (N. El-Sherif).
of APD, 3–4 weeks after MI could be explained, in part,
by downregulation of key K 1 channel genes [2] resulting
in decreased current densities of both components of
transient outward currents; Ito-fast ( f ) and Ito-slow (s) [1]. The
electrophysiological substrates for reentrant tachyarrhythmias are spatial heterogeneity of cardiac repolarization and / or conduction. The hypothesis being tested in this
study is that increased anisotropic properties occurs in the
remodeled post-MI ventricular myocardium. It is proposed
that the changes are caused by regional alterations in the
densities of different Kv channel proteins and currents
resulting in regional variations in APD and hence heterogeneity of cardiac refractoriness. These changes could
provide the electrophysiological substrate for reentrant
excitation. Preliminary results were previously reported
[3].
Time for primary review 22 days.
0008-6363 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 01 )00378-9
Downloaded from by guest on February 6, 2015
Objective: The hypothesis being tested in the present study is that increased anisotropic properties occurs in the remodeled
post-infarction heart due to spatial alterations in Kv channels expression and K 1 currents of the remodeled myocardium. Methods: Three
to 4 weeks post myocardial infarction (MI) in the rat, we measured the two components of the outward K 1 current, Ito-fast ( f ) and Ito-slow(s)
in the epicardium (epi) and endocardium (endo) of noninfarcted remodeled left ventricle (LV) using patch clamp techniques. Alterations in
mRNA and / or protein levels of potassium channel genes Kv1.4, Kv1.5, Kv2.1, Kv4.2 and Kv4.3 were measured in epi, midmyocardium
(mid), and endo regions of LV and in the right ventricle (RV). Results: In sham operated rat heart, the density of Ito-f was 2.3 times
greater in epi compared to endo myocytes. In post-MI heart, the density of Ito-f and Ito-s decreased to a similar degree in LV epi and endo
but the difference in Ito-f density between epi and endo persisted. The mRNA and / or protein levels of Kv1.4, Kv2.1, Kv4.2 and Kv4.3 but
not Kv1.5 decreased to a varying extent in different regions of LV but not in RV of post-MI heart. Conclusions: Our results suggest that
regional downregulation of Kv channels expression and density of K 1 currents can be a significant determinant of increased spatial
electrophysiological heterogeneity and contribute to increased electrical instability of the post-MI heart.  2001 Elsevier Science B.V.
All rights reserved.
B. Huang et al. / Cardiovascular Research 52 (2001) 246 – 254
2. Methods
2.1. Experimental model
2.2. Voltage-clamp recording and current analysis
Two distinct depolarization-activated K 1 currents were
described in adult rat ventricular myocytes [6]. The
characteristics of the fast component is similar to the
4-aminopyridine-sensitive Ito and is termed Ito-f . The slow
component has been termed IK [6] or Ito-s [1,7]. Epicardial
(epi) and endocardial (endo) tissues were obtained from the
free wall of sham-operated rat heart and from the noninfarcted left ventricular (LV) wall of post-MI heart for
recording of Ito-f and Ito-s in epi and endo myocytes.
Details of cell isolation have been previously reported [1].
The composition of external and internal solutions for
recording Ito-f and Ito-s was previously published [1].
Membrane capacitance and series resistance were compensated [1]. The currents were digitally recorded at room
temperature (248C) and analyzed using pCLAMP software
(pCLAMP version 6.02, Axon Instrument Inc., Foster City,
CA, USA). Clampfit software was used to measure amplitudes and time constants of ionic currents. To analyze
the two outward K 1 currents the cell was depolarized from
a holding potential of 2100 mV for 5 s to potentials
ranging from 250 to 160 mV in steps of 10 mV. The
current decay was fitted by two exponentials. The weights
and time constants tf and ts correspond to Ito-f and Ito-s ,
respectively [1,6]. Current density of both Ito-f and Ito-s and
their voltage dependence were analyzed as previously
reported [1].
2.3. K 1 channel RNA expression
Details of preparation of RNA from ventricular myocardium, RNase protection assay (RPA) and western blot
analysis have been previously reported [2]. In both sham
and post-MI experimental groups, the left and right atrial
appendages were carefully excised. For the post-MI experimental group the infarct region was carefully separated
from the hypertrophied LV including the septum under a
dissecting microscope. To obtain tissue from epi,
midmyocardium (mid), and endo of LV, the free wall of LV
was dissected free from the rest of the heart and laid out as
a sheet between two glass cover-slips. The two largest
papillary muscles were then dissected from the bulk of the
muscle wall. After being quickly frozen, the inner endo
layer, approximately one third depth of the wall, was first
dissected out with a surgical blade, and then the remaining
sheet was split in two to give the mid and epi regions. The
tissues were rinsed in saline to remove excess blood,
snap-frozen in liquid nitrogen, and stored at 2708C. Total
RNA was extracted from the LV as well as the right
ventricle (RV) using the standard protocol of Chomczynski
and Sacchi [8] of homogenization in acid guanidinium
thiocyanate followed by phenol–chloroform extraction and
ethanol precipitation. The amount of RNA recovered in
each sample was determined spectrophotometrically at a
wavelength of 260 nm and the integrity of each sample
confirmed by analysis on a denaturing agarose gel. For
Western blot analysis, the membranes were incubated with
Kv1.4, Kv1.5, Kv2.1 (Upstate Biotechnology, Lake Placid,
NY, USA) and Kv4.2 / Kv4.3 (that recognizes both Kv4.2
and Kv4.3 and was generously provided by Dixon et al.,
SUNY HSC, Stony Brook, NY).
2.4. RNase protection assay ( RPA)
RPAs were performed through concomitant measurement of cyclophilin genes expression (internal standard)
[2]. RPA was modified from the method described by
Kerig and Melton [9]. Previously described templates [2]
were used to prepare a 32 P-UTP antisense-radiolabeled
cRNA probes (MAXIscriptE, Ambion, Austin, TX, USA).
To differentiate between the specifically protected region
of the probe and any remaining undigested probe, all
probes contain regions of plasmid sequence at one end of
the transcript. Yeast tRNA (10 mg) was used as a negative
control to test for the presence of probe self-complementa-
Downloaded from by guest on February 6, 2015
The experimental protocol was approved by the local
institutional review board and conformed to the principles
outlined in the Declaration of Helsinki (Cardiovascular
Research 1997;35:2–3). Female Sprague–Dawley rats
weighing 200–250 g underwent either left anterior descending coronary artery ligation or sham operation as
previously described [1,4]. Briefly, after anesthesia with 35
mg / kg methohexital, i.p., and local anesthesia with 1%
xylocaine, the trachea was exposed in the midline and the
rats were intubated under direct vision and ventilated with
room air. The chest was opened by anterolateral
thoracotomy, and the pericardium was removed. The heart
was retracted with an apical suture, and the left anterior
descending coronary artery was occluded with a 6-O suture
1–2 mm below the left atrial appendage. Successful
occlusion was confirmed by pallor of the anterior wall of
the left ventricle and ST segment elevation. If neither
changes were observed, the occlusion was re-attempted.
The incision was closed and 100 000 U benzathine penicillin was administered intramuscularly as a prophylaxis
against infection. The rats were extubated and allowed to
recover in individual cages. Sham animals underwent an
identical surgical procedure without coronary ligation. All
rats received standard care, including ad libitum food,
water, and a 12-h day / night cycle.
The rats were studied 3–4 weeks post-MI when compensated hypertrophy has peaked [5]. Post-MI and sham
hearts were collected under deep anesthesia with 50 mg / kg
sodium pentobarbital (i.p.).
247
248
B. Huang et al. / Cardiovascular Research 52 (2001) 246 – 254
2.6. Statistical analysis
For electrophysiological studies, data are presented as
mean6S.E.M. Current amplitude, current density, and time
constants were determined at different test potentials.
When the data could be fit to a physiologically appropriate
model with r 2 .0.95, the parameters for that model were
tested for differences between the sham-operated and postMI groups. In experiments in which the response pattern
could not be fit, repeated measures ANOVA were used to
test for differences between groups. Only linear models fit
the criteria for acceptance, and for these models, the slope
and intercept differences between groups were tested using
multiple regression. In all other cases, localization of
differences between groups was determined using t-tests,
with the Bonferonni correction applied to yield experimental a level of P#0.05.
For comparisons of mRNA expression between sham
and post-MI myocardium the arbitrary densitometric units
were normalized to the value of the cyclophilin gene and
were statistically compared by one-way ANOVA. The
results were reproducible in two independent determinations, i.e. every sample pair had consistent changes in
level of mRNA expression in the post-MI relative to sham
experimental groups. Differences in level of mRNA expression and immunoreactive protein levels were considered significant at P,0.05 and dispersion from the mean
was noted as mean6S.E.M.
2.5. Western blot analysis
Cardiac cell membrane preparation and Western blot
analysis were performed as described previously by Barry
et al. [10] Sham and post-MI rats cardiac cell membranes
were prepared and protein was fractionated on a 10%
polyacrylamide–SDS gel. Because of anticipated lower
immunoreactive protein level, 200 mg of protein was used
for the Kv1.4 western blot compared to 85 mg for the other
Kv genes. After electrophoretic transfer to polyvinyldifluoride (Bio-Rad Lab., Hercules, CA, USA), the membranes were incubated with antisera against Kv1.4, Kv1.5,
Kv2.1, and Kv4.2 / Kv4.3, at dilutions of 1:1000, 1:500,
and 1:250, respectively. Bound primary antibody was
detected with a 1:10 000 dilution of alkaline phosphataseconjugated goat anti-rabbit IgG and the Western Light
chemiluminescent protein detection kit according to the
manufacturer’s protocol (Tropix Inc., Bedford, MA, USA).
Quantitative immunoreactivity was determined by densitometric analysis of the developed film that was in the linear
ranges with respect to film exposure. Linearity between
amounts of protein and immunoreactive signals were
proved for each Kv channel subunit protein by plotting
different amounts of protein at varying exposure times
against corresponding densitometric units. Quantitative
densitometric analysis was performed (Jandel Scientific,
San Rafael, CA, USA).
3. Results
Electrophysiological data were obtained from eight
sham-operated animals and ten post-MI animals. Cell
membrane capacitance was 13566 pF in epi cells (n536)
and 12767 pF endo cells (n524) and increased in post-MI
group to 228610 pF in epi cells (n574) and 19269 pF in
endo cells (n542) (P,0.001).
Fig. 1 illustrates outward K 1 currents recorded from a
sham myocyte (top) and a 3-week-old post-MI myocyte
(bottom) from an epi (A) and an endo cell (B). The
amplitude of K 1 current is smaller in endo compared to
epi cells both in sham and post-MI myocytes. Although the
post-MI cells have larger membrane capacitance, the K 1
current amplitude was not significantly different between
both cells. Fig. 2 compares the current density of Ito-f and
Ito-s between sham and 3–4 week-old post-MI epi and
endo myocytes, respectively. The densities of Ito-f and Ito-s
of both epi and endo post-MI myocytes were significantly
reduced compared to sham cells. In both sham and post-MI
myocytes the kinetics of inactivation of the outward K 1
current required two exponentials for adequate fit. The
inactivation kinetics of both Ito-f and Ito-s were faster at LV
epi compared to LV endo. Although the density of Ito-f and
Downloaded from by guest on February 6, 2015
tion by intramolecular hybridization, resulting in smaller
than expected protected bands. To account for the relatively greater abundance of internal control mRNA compared to potassium ion channel mRNA in cardiac tissues
and to avoid saturation of autoradiography in hybridizations, the reaction was carried out in the presence of excess
cold UTP (200 mM for cyclophilin), rendering a probe
with less specific activity. To obtain full-length transcripts
and lengthen the shelf life of the cRNA probes for all the
potassium ion channels, transcription was done in the
presence of 25 mM cold UTP. All cRNA probes were
purified prior to use over 5% polyacrylamide / 8 M urea
gel. Concomitant hybridization of the two probes (1310 4
cpm ionic channel cRNA and 1310 4 cpm cyclophilin
cRNA per 10 mg total RNA sample) were carried out at
508C for 18 h followed by digestion with RNase A (250
U / ml) and T1 (10 000 U / ml) [Ambion] at 378C for 30
min. The reaction was terminated by the addition of SDS
and proteinase K followed by phenol–chloroform extraction and ethanol precipitation. The protected fragments
were visualized by autoradiography after electrophoresis
on a 5% polyacrylamide / 8 mol / l urea gel. Quantitative
evaluation was carried out using scanning densitometric
analysis. For comparisons between sham and post-MI
myocardium the arbitary densitometric units were normalized to the value of the cyclophilin gene.
B. Huang et al. / Cardiovascular Research 52 (2001) 246 – 254
249
Ito-s decreased in post-MI myocytes at both LV epi and LV
endo, the inactivation kinetics of both currents were not
significantly changed. The data are summarized in Table 1.
3.1. Regional changes in Kv gene expression and
protein levels in the 3 – 4 week-old post-MI rat ventricle
Fig. 1. Whole cell recording of outward K 1 current from epicardium and
endocardium. Outward K 1 current recorded from sham (top) and a
3-week-old post-MI myocytes (bottom) obtained from epicardium (A) and
endocardium (B), respectively. Although the post-MI cells have larger
membrane capacitance, the current amplitude was not significantly
different between sham and post-MI cells. Note that the amplitude of K 1
current is smaller in endocardial compared to epicardial cells both in
sham and post-MI cells.
Downloaded from by guest on February 6, 2015
Fig. 2. Comparison of the current density of Ito-f and Ito-s between sham
and 3–4-week-old post-MI left ventricular epicardial and endocardial
myocytes. The density of Ito-f and Ito-s from both epicardial and
endocardial myocytes were significantly reduced in post-MI heart. Note
that the density of Ito-f in epicardial myocytes from sham hearts is
approximately 2.3 times the density of Ito-f in endocardial myocytes. The
same difference persisted in the post-MI heart because the density of Ito-f
was reduced by approximately the same degree in epicardial and
endocardial myocytes.
There are relatively minor differences in the abundance
of the Kv1.5, Kv2.1 and Kv4.3 transcripts across the
ventricular free wall [11,12]. By contrast, Kv4.2 mRNA
was found to be expressed in a steep gradient across the
LV wall [11]. There was no change in the expression of
Kv1.5 mRNA (Fig. 3) or protein level (Fig. 4) across the
LV wall in post MI compared to sham-operated rats. On the
other hand, compared to sham, the mRNA level of Kv2.1
was significantly decreased in post-MI LV by 22, 39 and
38% in epi, mid, and endo zones, respectively (P,0.05,
n56) (Fig. 5). Similarly, the protein levels of Kv2.1 in
post-MI LV were reduced by 21, 26 and 30% in epi, mid
and endo zones, respectively (P,0.05, Fig. 6). In shamoperated rat heart, the mRNA level of Kv4.2 in the epi
region was 2.9 times higher than in the endo region of the
LV free wall (Fig. 7). Significant changes in the mRNA
level between sham-operated and post-MI rats across the
LV free wall were seen in Kv4.2 and Kv4.3 channel
subunits expression. Expression of Kv4.2 channel message
was decreased by 37, 28 and 46% in the epi, mid and endo
zone of post-MI LV, respectively, compared to sham (all
values are significant at P,0.05, n56) (Fig. 7). Similarly,
expression of Kv4.3 channel message was decreased by
31.4, 41 and 21.8% in the epi, mid and endo zones of
post-MI LV, respectively, compared to sham (all values are
significant at P,0.05, n56) (Fig. 8). Fig. 9 shows that the
protein levels of Kv4.2 / 4.3 in post-MI LV wall were
decreased by 49, 53 and 51% in epi, mid and endo zones,
respectively compared to the sham group (P,0.01). There
was no significant change in the protein levels of Kv1.5,
Kv2.1, Kv4.2 / 4.3 between RV of sham and post-MI rats
(Figs 4, 6 and 9).
We have previously reported that the mRNA level of
Kv1.4 is reduced in the 3–4-week post-MI rat LV [2]. In
the previously reported study we did not evaluate changes
in the protein level of Kv1.4 since in a report by Barry et
al. [10] only a trace |97 kDa protein could be detected
using the anti-Kv1.4 antibody. In the present study we
investigated the protein level of Kv1.4 in the RV, LV endo
and LV epi zones in sham and post-MI rat heart as well as
in the rat brain using a commercial anti-Kv1.4 antibody
(Upstate Biotechnology) (Fig. 10). The Kv1.4 protein level
was much higher in the rat brain compared to heart. In the
sham rat heart Kv1.4 protein was detected in RV, LV endo
and LV epi. However, the Kv1.4 protein level was 2.3
times higher in LV endo compared to LV epi and 2.1 times
higher compared to RV. In the post-MI heart the Kv1.4
B. Huang et al. / Cardiovascular Research 52 (2001) 246 – 254
250
Table 1
Characteristics of Ito-f and Ito-s in sham and post-MI cells from epicardial and endocardial myocytes
n
Epicardium
n
Ito-f
Sham
Post-MI
Reduction (%)
35
54
Ito-s
Ito-f
pA / pF
t (ms)
pA / pF
t (ms)
27.262.9
12.561.7**
5262.0
5462.2
7.262.6
3.161.2*
1981673
2012669
54
57
Endocardium
30
46
Ito-s
pA / pF
t (ms)
pA / pF
t (ms)
11.862.8
4.661.6**
113616
111614
7.962.2
3.561.4*
27046144
27866136
61
56
Values are at 160 mV depolarization.* P,0.05, ** P,0.01 vs. sham. t 5decay time constant.
protein level showed no significant change in RV but
significantly decreased by 52 and 48% in LV endo and LV
epi, respectively (P,0.01).
4. Discussion
We have investigated the well-established model of
post-MI remodeling in the rat heart and showed that the
Fig. 4. Western blot analysis of Kv1.5 channel subunit immunoreactive
protein across LV free wall (epi, mid, and end) and in RV in post-MI and
sham hearts (A) Data from 3 to 4-week-old post-MI (n56) and sham
operated (n56) rats. (B) Bar graph showing Kv1.5 immunoreactivity by
measuring the signal for the protein by densitometry. Columns represent
the mean values, with error bars indicating S.E.M. There was no
significant change in the Kv1.5 immunoreactive protein level across LV
free wall, and also no significant change in the Kv1.5 protein level in RV
between the two experimental groups.
Downloaded from by guest on February 6, 2015
Fig. 3. Representative comparison of cardiac Kv1.5 gene expression
across LV free wall and in RV in 3–4-week-old post-MI and shamoperated rats by RPA. (A) In these experiments, the samples from LV free
wall and RV contained 10 mg total RNA, and the negative sample
contained 10 mg of yeast RNA. (B) Bar graphs of Kv1.5 mRNA across
LV free wall (epi, mid, and end), and in RV of sham-operated and post-MI
rats. There was no significant change in the mRNA level of Kv1.5 across
LV free wall or in RV of the 3-week post-MI group compared to the
sham-operated group (n56 for each group).
remodeling process is associated with compensatory hypertrophy of the noninfarcted portion of the LV. The
hypertrophic response engenders distinct molecular and
electrophysiological alterations that are potentially deleterious. The changes include downregulation of K 1 channel
genes with consequent decrease of outward K 1 currents
contributing to prolongation of APD of hypertrophied
ventricular myocytes. We have previously shown that the
alterations in duration and configuration of APD is not the
same in epi and endo regions of the LV thus resulting in
increased heterogeneity of repolarization, an important
substrate for reentrant tachyarrhythmias [1]. Spontaneous
and / or induced ventricular tachyarrhythmias have been
reported to occur in the rat model in the late post-MI phase
[1,13,14]. The findings in the present study provide, in
part, the molecular and electrophysiological basis of the
arrhythmogenicity of post-MI heart.
B. Huang et al. / Cardiovascular Research 52 (2001) 246 – 254
251
Fig. 5. Representative comparison of cardiac Kv2.1 gene expression
across LV free wall (epi, mid, and end) and in RV in post-MI and sham
hearts. (A) Data from 3 to 4-week-old post-MI (n56) and sham-operated
rats (n56) by RPA. In these experiments, the samples from LV free wall
and RV contained 10 mg total RNA, and the negative sample contained 10
mg of yeast RNA. (B) Bar graph of Kv2.1 mRNA across LV free wall
(epi, mid, and end), and in RV of sham-operated and post-MI rats. There
was no significant change in the mRNA level of Kv2.1 in RV of the
3–4-week-old post-MI group compared to the sham-operated group.
However, there were significant decreases in the mRNA levels of Kv2.1
across the LV free wall (epi, mid, and end) of the post-MI group
compared to the sham-operated group (P,0.05). Values are
mean6S.E.M.
4.1. Spatial heterogeneity in the expression of Kv
channel subunits and Ito density in the post-MI rat
ventricle
The main consistent electrophysiological abnormality
associated with cardiac hypertrophy is prolongation of
APD [15]. In the remodeled post-MI myocardium, it was
important to ascertain whether the enlargement in myocyte
size is accompanied by proportional or disproportional
changes in key sarcolemmal ion channels that could
contribute to the changes in APD. In this regard, we have
previously reported that the prolongation of APD 3–4
weeks post-MI was not related to alterations in the density
or kinetics of the L-type Ca 21 current which were not
significantly different from control [1]. The electrophysiological observations were consistent with our report of no
change in the mRNA level of the adult isoform of the a 1
subunit of the L-type Ca 21 channel [16]. Several evidences
suggest that changes in repolarizing K 1 currents Ito-f and
Ito-s are major factors in prolongation of APD of post-MI
hypertrophied myocytes [1,15,17]. Dixon and McKinnon
[11] have shown that there is a large gradient of Kv4.2
expression across the rat ventricular wall. Kv4.2 expression in epi muscle was more than eight times higher than
in papillary muscle. However, the difference between
Kv4.2 expression between epi muscle and endo muscle
from the free wall of the LV was approximately 3 to 1.
This is similar to the findings in the present study where
the expression of Kv4.2 in sham animals between epi and
endo myocardium, the later was obtained from the noninfarcted free wall of the LV, was 2.9 to 1. These data also
correlates with our finding that the density of Ito-f in epi
myocytes from sham animals is 2.3 times higher than that
in endo myocytes. In an early study, Clark et al. [18]
showed that peak Ito-f in rat myocytes obtained from epi
and endo free wall was 2.24 versus 0.59 nA, respectively,
at 150 mV depolarization.
Our electrophysiological studies have shown that although Ito-f density in post-MI LV is decreased to a similar
degree across the LV wall significant differences in density
of Ito-f continue to exist between LV epi and endo. The role
of the different K 1 currents and their relative influence in
contributing to action potential profile is different in
different regions of the heart. This can explain the difference in the action potential profile of LV epi and endo
myocytes that was previously reported [1]. Our observa-
Downloaded from by guest on February 6, 2015
Fig. 6. Western blot analysis of Kv2.1 channel subunit immunoreactive
protein across LV free wall (epi, mid, and end) and in RV in post-MI and
sham hearts. (A) Data from 3 to 4-week-old post-MI (n56) and shamoperated (n56) rats. (B) Bar graph showing Kv2.1 immunoreactivity by
measuring the signal for the protein by densitometry. Columns represent
the mean values, with error bars indicating S.E.M. Immunoreactivity of
Kv2.1 protein across LV free wall (epi, mid, and end) was significantly
decreased in post-MI rats compared to sham-operated rats (P,0.05).
There was no significant change in the Kv2.1 immunoreactive protein
level in RV between the two groups.
252
B. Huang et al. / Cardiovascular Research 52 (2001) 246 – 254
tion that there was no change in K 1 gene expression and
Ito density in 3–4 weeks old post-MI rat RV, is further
evidence of the enhanced heterogeneity of post-MI remodeled heart.
4.2. Molecular basis of K 1 channels
Several attempts have been made to correlate channel
subunit genes expression to native K 1 currents. For
example, heterologous expression of Kv4.2 and Kv4.3
mRNA generates channels with kinetics and pharmacological properties similar to Ito-f in cardiac myocytes [12].
Moreover, Kv4.2 mRNA is expressed in a gradient across
LV wall, which correlates well with the gradient of Ito-f
density across the wall [11]. On the other hand, Kv4.3 is
homogeneously expressed across the LV wall [12]. Kv4.2
homomeric and / or Kv4.2–Kv4.3 heteromeric channels are
likely to contribute to the difference of Ito-f in different
parts of the rat ventricle. This can possibly explain the
somewhat slower inactivation kinetics of Ito-f in LV endo in
the present study. Some investigators have shown that
recovery from inactivation was slower for Kv4.3 compared
to Kv4.2 based currents expressed in tsa-201 cells [19]. We
have demonstrated that the protein levels of Kv4.2 / Kv4.3
were significantly decreased in epi and endo of post-MI
remodeled myocardium by 49 and 51%, respectively,
compared to sham. These findings provide the molecular
basis of our electrophysiological observations of decreased
density of Ito-f by 54 and 61% in myocytes obtained from
epi and endo, respectively of post-MI rat heart. The
decreased density of Ito-f is a major contributor to changes
in action potential profile and duration [20].
In contrast to Ito-f , the molecular basis of Ito-s in the rat
has not been well established. In a study by Barry et al.
[10], the authors showed that heterologous expression of
Kv2.1 revealed a slowly activating TEA-sensitive K 1
current similar to Ito-s in adult rat ventricular myocytes and
therefore seemed to be the likely candidate for this current.
Downloaded from by guest on February 6, 2015
Fig. 7. Representative comparison of cardiac Kv4.2 gene expression
across LV free wall (epi, mid, and end) and in RV in post-MI and sham
hearts. (A) Data from 3 to 4-week-old post-MI (n56) and sham-operated
rats (n56) by RPA. In these experiments, the samples from LV free wall
and RV contained 10 mg total RNA, and the negative sample contained 10
mg of yeast RNA. (B) Bar graph of Kv4.2 mRNA across LV free wall
(epi, mid, and end), and in RV of sham-operated and post-MI rats. There
was no significant change in the mRNA level of Kv4.2 in RV of the
post-MI group compared to the sham-operated group. However, there
were significant decreases in the mRNA levels of Kv4.2 across the LV
free wall (epi, mid, and end) of the post-MI group compared to the
sham-operated group (P,0.05). Values are mean6S.E.M.
Fig. 8. Representative comparison of cardiac Kv4.3 gene expression
across LV free wall (epi, mid, and end) and in RV in post-MI and sham
hearts. (A) Data from 3 to 4-week-old post-MI (n56) and sham-operated
rats (n56) by RPA. In these experiments, the samples from LV free wall
and RV contained 10 mg total RNA, and the negative sample contained 10
mg of yeast RNA. (B) Bar graph of Kv4.3 mRNA across LV free wall
(epi, mid, and end), and in RV of sham-operated and post-MI rats. There
was no significant change in the mRNA level of Kv4.3 in RV of the
post-MI group compared to the sham-operated group. However, there
were significant decreases in the mRNA levels of Kv4.3 across the LV
free wall (epi, mid, and end) of the post-MI group compared to the
sham-operated group (P,0.05). Values are mean6S.E.M.
B. Huang et al. / Cardiovascular Research 52 (2001) 246 – 254
Other investigators have suggested that Kv1.4 underlies
the slowly recovering component of Ito , especially in endo
myocytes [19,21,22]. The present study shows that Kv1.4
protein is present in adult rat RV, LV endo and LV epi, and
that the density is at least twice as high in LV endo
compared to RV and LV epi. Furthermore, the inactivation
kinetics of Ito-s in LV endo was slower than that in LV epi,
which may suggest a larger contribution of Kv1.4 to Ito-s in
LV endo. However, the presence of a relatively small
amount of Kv1.4 protein does not constitute enough
evidence for its contribution to Ito-s in the LV.
4.3. Study limitations
We have previously shown regional variations in APD
in the 3–4 week post-MI rat heart [1]. The present study is
an attempt to provide the molecular and ionic basis of
these changes. It should be emphasized, however, that
conclusions regarding the correlation between action potential configuration and current densities remain speculative in the absence of reconstruction of simulated action
potential that incorporates complete data on the density,
time course, and voltage dependence of all currents that
contribute to the repolarization phase [1]. The electrophysiological characteristics of RV myocytes were not
investigated. However, we found no significant changes in
Kv subunits expression in right ventricular myocardium
obtained from 3 to 4-week-old post-MI rat heart. Finally,
the possibility of gender difference in post-MI remodeling
Fig. 10. Western blot analysis of Kv1.4 channel subunit immunoreactive
protein in adult rat brain, and in RV, LV epi and LV end from post-MI and
sham-operated hearts. (A) Data from 3 to 4-week-old post-MI (n56) and
sham-operated (n56) hearts were compared. In the representative gel on
top all samples from rat brain and cardiac tissues were 200 mg. Because
of the higher expression of Kv1.4 in brain sample it was difficult to
resolve the band. On the other hand, cardiac tissue showed a distinct
single band in myocyte protein with molecular mass of |97 kDa. In the
representative gel on bottom the rat brain sample was 50 mg and cardiac
tissue samples were 150 mg. The antibody seems to label two bands in
brain tissue, a faint band with molecular mass of |91 kDa and a distinct
band with molecular mass of |101 kDa. This has been previously
reported by Wickenden et al. [22] and was explained by the presence of
glycosylated and nonglycosylated fractions of the protein. However, in
contrast to Wickenden et al., the Kv1.4 antibody utilized in the present
study (Upstate Biotechnology) recognized a single distinct band in
cardiac tissue with molecular mass of |97 kDa even though some lanes
seem to show a second faint band, e.g. sham LV end sample. (B) The bar
graphs show densitometric measurement of Kv1.4 immunoreactivity in
sham and post-MI tissues. Columns represent mean value, with error bars
indicating S.E.M. Kv1.4 immunoreactivity of LV end was 2.3 times
higher than LV epi and 2.1 times higher than RV. There was no significant
change in Kv1.4 immunoreactivity between sham and post-MI RV. On the
other hand, Kv 1.4 immunoreactivity decreased by 46% in LV epi and by
52% in LV end of post-MI heart compared to sham.
has not been addressed in the present study. Female rats
usually have been utilized as a model for post-MI remodeling [1–5]. The first quantitative analysis of K 1 channel
genes in Sprague–Dawley rats also was done in young
adult female animals [11]. Female sex has long been
associated with a slower rate of cardiac depolarization
based usually on analysis of surface ECG QT interval and
with a higher risk of developing drug-induced torsades de
pointes arrhythmias [23]. Several studies have suggested
that gender difference in the expression and / or modulation
of cardiac K 1 channel genes and currents in some animal
models may underlie these changes [24,25]. There is no
data, however, on gender difference in the downregulation
Downloaded from by guest on February 6, 2015
Fig. 9. Western blot analysis of Kv4.2 / 4.3 channel subunit immunoreactive proteins across LV free wall (epi, mid, and end) and in RV in
post-MI and sham hearts. (A) Data from 3 to 4-week-old post-MI (n56)
and sham operated (n56) rats. (B) Bar graph showing Kv4.2 / 4.3
combined immunoreactivity by measuring the signal for the proteins by
densitometry. Columns represent the mean values with error bars
indicating S.E.M. Immunoreactivity of Kv4.2 / 4.3 across LV free wall
(epi, mid, and end) was significantly decreased in post-MI rats compared
to sham-operated rats (P,0.05). There was no significant change in the
Kv4.2 / 4.3 immunoreactive protein level in RV between the two experimental groups.
253
254
B. Huang et al. / Cardiovascular Research 52 (2001) 246 – 254
of K 1 channel genes and currents and / or the degree of
electrophysiological alteration in the rat post-MI model.
Further investigations are required to address these issues.
5. Conclusion
Regional variations in the downregulation of Kv channels expression and density of K 1 currents can be a
significant determinant of increased spatial electrophysiological heterogeneity and contribute to increased electrical
instability of the post-MI heart.
Acknowledgements
Supported in part by VA MERIT and REAP grants to
NES.
References
Downloaded from by guest on February 6, 2015
[1] Qin D, Zhang Z-H, Boutdjir M, Jain P, El-Sherif N. Cellular and
ionic basic of arrhythmias in post-infarction remodeled ventricular
myocardium. Circ Res 1996;79:461–473.
[2] Gidh-Jain M, Huang B, Jain P, El-Sherif N. Differential expression
of voltage-gated K 1 channel genes in left ventricular remodeled
myocardium after experimental myocardial infarction. Circ Res
1996;79:669–675.
[3] Huang B, Qin D, Gidh-Jain M, Boutjdir M, El-Sherif N. Spatial
alterations of Kv channel and connexin 43 expression and K 1
current density of post-infarction remodeled left ventricle. Circulation 1998;98(17):I697.
[4] Jain P, Brown Jr EJ, Langenback EG et al. Effects of milrinone on
left ventricular remodeling after acute myocardial infarction. Circulation 1991;84:796–804.
[5] Pfeffer JM, Pfeffer MA, Fletcher PJ, Braunwald E. Progressive
ventricular remodeling in rat with myocardial infarction. Am J
Physiol 1991;260:H1406–H1414.
[6] Apkon M, Nerbonne JM. Characterization of two distinct depolarization-activated K currents in isolated adult rat ventricular
myocytes. J Gen Physiol 1991;97:973–1011.
[7] Dukes ID, Morad M. The transient K current in rat ventricular
myocytes: evaluation of its Ca and Na dependence. J Physiol (Lond)
1991;435:395–420.
[8] Chomczynski P, Sacchi N. Single-step method of RNA isolation by
guanidium thiocynate–phenol–chloroform extraction. Anal Biochem
1987;162:156–159.
[9] Kreig PA, Melton DA. In vitro RNA synthesis with SP6 RNA
polymerase. Methods Enzymol 1987;155:397–415.
[10] Barry DM, Trimmer JS, Merlie JP, Nerbonne JM. Differential
expression of voltage-gated K 1 channel subunits in adult rat heart in
relation to functional K 1 channels. Circ Res 1995;77:361–369.
[11] Dixon JE, McKinnon D. Quantitative analysis of potassium channel
mRNA expression in atrial and ventricular muscle of rats. Circ Res
1994;75:252–260.
[12] Dixon JE, Shi W, Wang H-S et al. Role of the Kv4.3 K 1 channel in
ventricular muscle: a molecular correlate for the transient outward
current. Circ Res 1996;79:659–668.
[13] Opitz CF, Mitchell GF, Pfeffer MA, Pfeffer JM. Arrhythmias and
death after coronary artery occlusion in the rat. Continuous telemetric ECG monitoring in conscious, untethered rats. Circulation
1995;92:253–261.
[14] Belichard P, Savard P, Cardinal R et al. Markedly different effects
on ventricular remodeling result in a decrease in inducibility of
ventricular arrhythmias. J Am Coll Cardiol 1994;23:505–513.
[15] Hart G. Cellular electrophysiology in cardiac hypertrophy and
failure. Cardiovasc Res 1994;28:933–946.
[16] Gidh-Jain M, Huang B, Jain P, Battula V, El-Sherif N. Reemergence
of the fetal pattern of L-type calcium channel gene expression in
non infarcted myocardium during left ventricular remodeling. Biochem Biophys Res Commun 1995;216:892–897.
[17] Rozanski GJ, Xu Z, Zhang K, Patel KP. Altered K 1 current of
ventricular myocytes in rats with chronic myocardial infarction. Am
J Physiol 1998;274(1 Pt 2):H259–H265.
[18] Clark RB, Bouchard RA, Salinas-Stefanon E, Sanchez-Chapula J,
Giles WR. Heterogeneity of action potential waveforms and potassium currents in rat ventricle. Cardiovasc Res 1993;27:1795–1799.
[19] Brahmajothi MV, Camphell DL, Rasmusson RL et al. Distinct
transient outward potassium current (Ito ) phenotypes and distribution
of fast-inactivating potassium channel alpha subunits in ferret left
ventricular myocytes. J Gen Physiol 1999;113:579–600.
¨ ¨ S, Nuss B, Chiamvimonvat N et al. Ionic mechanism of action
[20] Kaab
potential prolongation in ventricular myocytes from dogs with
pacing-induced heart failure. Circ Res 1996;78:262–273.
[21] Nabauer M, Beuckelmann DJ, Uberfuhr P, Steinbeck G. Regional
differences in current density and rate-dependent properties of the
transient outward current in subepicardial and subendcardial
myocytes of human left ventricular. Circulation 1996;93:168–177.
[22] Wickenden AD, Jegla TJ, Kapriellan R, Backx PH. Regional
contributions of Kv1.4, Kv4.2, and Kv 4.3 to transient outward K 1
current in rat ventricle. Am J Physiol 1999;276:H1599–H1607.
[23] Lehmann MH, Hardy S, Archibold D et al. Sex difference in risk of
torsades de pointes with d,l-sotalol. Circulation 1996;94:2535–2541.
[24] Drici MD, Burklow TR, Haridasse V, Glazer RI, Woosley RL. Sex
hormones prolong the QT interval and downregulate potassium
channel expression in the rabbit heart. Circulation 1996;94:1471–
1474.
[25] Liu X-K, Wang W, Ebert SN et al. Female gender is a risk factor for
torsades de pointes in an in vitro animal model. J Cardiovasc Pharm
1999;34:287–294.