From the * Department of Pharmacology, Department of Biomedical Engineering, and ¶ Department of Medicine, Duke University
Medical Center, Durham, North Carolina 27710; § Department of Biochemistry and Cell Biology, State University of New York, Stony
Brook, Stony Brook, New York 11794; and
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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The biophysical characteristics and subunits underlying calcium-independent transient outward
potassium current (Ito) phenotypes expressed in ferret left ventricular epicardial (LV epi) and endocardial (LV
endo) myocytes were analyzed using patch clamp, fluorescent in situ hybridization (FISH), and immunofluorescent (IF) techniques. Two distinct Ito phenotypes were measured (21-22°C) in the majority of LV epi and LV endo
myocytes studied. The two Ito phenotypes displayed marked differences in peak current densities, activation thresholds, inactivation characteristics, and recovery kinetics. Ito,epi recovered rapidly [
rec,
70 mV = 51 ± 3 ms] with minimal cumulative inactivation, while Ito,endo recovered slowly [
rec,
70 mV = 3,002 ± 447 ms] with marked cumulative inactivation. Heteropoda toxin 2 (150 nM) blocked Ito,epi in a voltage-dependent manner, but had no effect on
Ito,endo. Parallel FISH and IF measurements conducted on isolated LV epi and LV endo myocytes demonstrated that Kv1.4, Kv4.2, and Kv4.3
subunit expression in LV myocyte types was quite heterogenous: (a) Kv4.2 and
Kv4.3 were more predominantly expressed in LV epi than LV endo myocytes, and (b) Kv1.4 was expressed in the
majority of LV endo myocytes but was essentially absent in LV epi myocytes. In combination with previous measurements on recovery kinetics (Kv1.4, slow; Kv4.2/4.3, relatively rapid) and Heteropoda toxin block (Kv1.4, insensitive; Kv4.2, sensitive), our results strongly support the hypothesis that, in ferret heart, Kv4.2/Kv4.3 and Kv1.4
subunits, respectively, are the molecular substrates underlying the Ito,epi and Ito,endo phenotypes. FISH and IF measurements were also conducted on ferret ventricular tissue sections. The three Ito
subunits again showed distinct patterns of distribution: (a) Kv1.4 was localized primarily to the apical portion of the LV septum, LV endocardium, and approximate inner 75% of the LV free wall; (b) Kv4.2 was localized primarily to the right ventricular
free wall, epicardial layers of the LV, and base of the heart; and (c) Kv4.3 was localized primarily to epicardial layers of the LV apex and diffusely distributed in the LV free wall and septum. Therefore, in intact ventricular tissue,
a heterogeneous distribution of candidate Ito
subunits not only exists from LV epicardium to endocardium but
also from apex to base.
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INTRODUCTION |
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Due to their primary importance in both initiating and
modulating repolarization of the cardiac action potential, extensive experimental effort has been devoted to
analysis of potassium channels as potential targets for
antiarrhythmic agents (Rasmusson et al., 1994; Tseng,
1995
; Tamkun et al., 1995
; Barry and Nerbonne, 1996
;
Sanguinetti and Spector, 1997
). In particular, the calcium-independent transient outward potassium current Ito (also referred to as Ito,1; Antzelevitch et al.,
1995
; Campbell et al., 1995
) has recently received
much experimental attention. Ito is present in nearly all
mammalian working cardiac myocyte types, including
both human atrial (Escande et al., 1987
; Shibata et al.,
1988
) and ventricular myocytes (Näbauer et al., 1993
,
1996
; Beuckelmann et al., 1993
; Wettwer et al., 1993
,
1994
; Amos et al., 1996
). Due to its rapid activation kinetics, Ito is the major current responsible for the early
"notch" or phase 1 repolarization characteristic of the
mammalian ventricular action potential. In addition,
due to its slower inactivation kinetics, Ito can also significantly modulate the plateau and early phase 3 repolarization (Campbell et al., 1993a
,b, 1995).
Despite the nearly universal presence of Ito in mammalian working myocardium, it appears to be less
widely appreciated that there may be multiple and
functionally distinct Ito phenotypes. In particular, significant differences in specific gating characteristics of Ito
phenotypes have been reported both among different
species and within different cardiac tissue and myocyte
types from the same species. Three major differences
can be summarized as follows: (a) marked differences
in activation thresholds exist (Campbell et al., 1995),
(b) the kinetics of macroscopic inactivation are either
clearly single exponential (e.g., ferret right ventricle [Campbell et al., 1993a
] and human ventricle [Näbauer
et al., 1996
]) or markedly biexponential (e.g., rabbit
atrium and ventricle [Clark et al., 1988
; Giles and Imaizumi, 1988
]), and (c) the kinetics of recovery from inactivation are either rapid (time constants on the order
of 10-100 ms; e.g., ferret, rat, and human ventricle [Josephson et al., 1984
; Campbell et al., 1993a
; Näbauer et
al., 1996
]) or slow to very slow (time constants on the
order of hundreds of milliseconds to seconds; e.g., rabbit atrium and rabbit and dog ventricle [Liu et al., 1993
;
Antzelevitch et al., 1995
; Clark et al., 1998]). Hence,
there is a 1-2-order-of-magnitude difference in the
time constants of recovery reported for different cardiac Ito phenotypes. Due to these marked differences in
recovery kinetics, very slowly recovering cardiac Ito phenotypes display cumulative inactivation (Aldrich, 1981
)
during rapid and repetitive voltage clamp protocols
(e.g., Clark et al., 1988
), while rapidly recovering phenotypes generally do not (e.g., Campbell et al., 1993a
).
In addition to significant kinetic differences among
Ito phenotypes, within any one given species there may
also exist heterogeneous expression of multiple Ito phenotypes and/or Ito current densities among distinct anatomical regions of the heart (Näbauer et al., 1993,
1996
; Wettwer et al., 1993
, 1994
; Campbell et al., 1995
; Antzelevitch et al., 1995
). For example, recent studies
on human left ventricular epicardial and endocardial
myocytes have indicated a marked difference in both Ito
current density and kinetics of recovery from inactivation between the two myocyte types (epicardial: higher
Ito density, fast recovery; endocardial: lower Ito density, very slow recovery; Näbauer et al., 1993
, 1996
). Thus,
depending on both species and specific anatomical region, there appear to be at least two major Ito phenotypes in mammalian heart capable of exerting functionally distinct frequency-dependent modulatory effects
on repolarization.
The presence of at least two functional Ito phenotypes
suggests the existence of at least two distinct potassium
channel subunits underlying each of the currents. To
date, two main approaches have been used to address
the issue of which
subunit underlies cardiac Ito: (a)
detection of
subunit mRNA levels (generally of whole
cardiac tissue samples), and (b) comparisons of kinetic characteristics of heterologously expressed potassium
channel
subunit clones to native myocyte Ito phenotypes measured in one particular species. Based on
these approaches, Kv1.4, Kv4.2, and Kv4.3 (long and
short isoforms)
subunits have all been recently proposed to underlie "cardiac Ito" (e.g., Po et al., 1993
;
Comer et al., 1994
; Dixon and McKinnon, 1994
; Barry
et al., 1995
; Dixon et al., 1996
; Barry and Nerbonne,
1996
; Xu et al., 1996
; Yeola and Snyders, 1997
; Takimoto et al., 1997
; Ohya et al., 1997
). However, previous
studies have not adequately examined the spatial distribution of both multiple Ito phenotypes and/or heterogeneity of Kv
subunit transcript and protein expression (Dixon and McKinnon, 1994
; Barry et al., 1995
;
Dixon et al., 1996
; Brahmajothi et al., 1996
, 1997
).
Given these limitations, it is important to correlate levels of expressed Ito channel
subunit proteins in specific anatomical regions of the heart to patterns of
functional Ito current phenotypes measured from individual myocytes isolated from the same anatomical regions.
To begin to address these issues, in this study we first
demonstrate, using whole cell patch clamp, that at least
two kinetically and pharmacologically distinct Ito phenotypes exist in ferret isolated left ventricular epicardial (LV epi)1 and endocardial (LV endo) myocytes. We
designate these two major Ito phenotypes as Ito,epi and
Ito,endo. We then demonstrate, using a combination of
fluorescent in situ hybridization (FISH) and immunofluorescence (IF) techniques, that Kv1.4, Kv4.2, and
Kv4.3 subunit proteins are all expressed in ferret isolated LV myocytes. However, we show that the specific
distribution patterns of these three candidate Ito
subunits are quite heterogeneous among LV epi versus LV
endo myocytes: (a) both Kv4.2 and Kv4.3 are each more predominantly expressed in LV epi than LV endo
myocytes, and (b) Kv1.4 is expressed in the majority of
LV endo myocytes but is essentially absent in LV epi myocytes. These combined patch clamp and FISH/IF results on isolated myocytes therefore strongly suggest
that in ferret heart Kv4.2/Kv4.3 and Kv1.4
subunits, respectively, are the underlying molecular substrates
for the Ito,epi and Ito,endo phenotypes. We then extend
the FISH and IF measurements to sagittal ventricular
tissue sections so as to gain initial insights into overall
distribution patterns of Kv1.4, Kv4.2, and Kv4.3 Ito
subunits in the whole ventricle. We demonstrate that
(a) Kv4.2 is predominantly expressed in LV apical epicardium, (b) Kv1.4 is predominantly expressed in LV
apical endocardium, and (c) Kv4.3 is relatively more
uniformly expressed throughout the LV wall. Hence, in
the intact left ventricle, Kv1.4 and Kv4.2 expression levels vary significantly in the LV wall both transversely (i.e., epicardium-to-endocardium) and sagittally (i.e.,
apex-to-base). In contrast, in right ventricular (RV) tissue, we demonstrate that Kv4.2 is uniformly expressed
across the entire free wall, while Kv1.4 and Kv4.3 are
relatively sparse to absent. This latter result may imply
that Kv4.2
subunits underlie the much more uniformly expressed Ito phenotype in ferret right ventricular myocytes (Campbell et al., 1993a
,b).
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METHODS |
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Myocyte Isolation
Hearts were obtained from 16-30-wk-old male ferrets. Depending on photo period and other environmental factors, ferrets can reach sexual maturity at ~16-32 wk (Bernard et al., 1984);
hence, potential developmental changes in potassium channel
expression occurring during puberty among some of the ferrets
used cannot be definitively ruled out. Myocytes were enzymatically isolated (collagenase, protease, elastase) from the left ventricular free wall via a Langendorff perfusion apparatus exactly
as described (Campbell et al., 1993a
,b, 1996), except for the following slight modification. After 5-10 min of initial enzyme perfusion, the heart was removed from the Langendorff apparatus
and the free wall of the left ventricle was dissected from the adjacent apical, septal, and basal regions, resulting in a tissue sample
corresponding approximately to the middle one- to two-thirds of
the left ventricular wall. Small thin strips (~1 mm thick × 0.5-1 cm
long) were then dissected in a sagittal orientation from both the
partially digested epicardial and endocardial surfaces. These epicardial and endocardial tissue strips were then separately reincubated in fresh enzyme solution, gently shaken at 37°C, and myocytes were harvested at 10-20-min intervals as previously described (Campbell et al., 1993a
,b, 1996). Once isolated, myocytes
were directly stored in normal control Na+- and Ca2+-containing
saline (see below) at room temperature (21-22°C). All voltage
clamp experiments were conducted on myocytes within 6-12 h of isolation.
Electrophysiology, Analysis, and Solutions
All patch clamp recordings were conducted at room temperature
(21-22°C) using the whole cell ruptured patch configuration (Marty and Neher, 1995). Exactly the same equipment, perfusion system, glass tubing for patch pipettes, etc., as previously described in detail (Campbell et al., 1993a
,b, 1996) were used in
the present study. Data were recorded (filtered at 1-2 kHz) on
video tape, and then subsequently digitized (5-10 kHz) off-line
and analyzed using pClamp and FigP software (Campbell et al.,
1993a
,b, 1996). Data are summarized as mean ± SEM.
After attainment of the whole cell configuration in normal
control saline (mM: 144 NaCl, 5.4 KCl, 1 MgSO4, 1.8 CaCl2, 10 HEPES, pH 7.40), Ito was isolated from other overlapping currents by perfusing the myocytes with Na+- and Ca2+-free extracellular "Ito saline" (mM: 144 N-methyl-D-glucamine, 5.4 KCl, 2.3 MgSO4, 0.5 CdCl2, 10 HEPES, pH 7.40). Patch pipettes (3-4.5
M) contained (mM): 140 KCl, 1 MgSO4, 5 Mg-ATP, 5 Tris-creatine phosphate, 0.2 GTP, 5 EGTA, 10 HEPES, pH 7.40. We opted
for 500 µM Cd2+ in the extracellular Ito solution to block the
L-type calcium current, ICa,L, for two reasons: (a) many commonly
used organic ICa,L blockers can exert significant nonspecific
blocking effects on cardiac Ito phenotypes (e.g., Gotoh et al.,
1991
; Lefevre et al., 1991
; Y. Qu and D.L. Campbell, unpublished
observations), thereby potentially complicating analysis (e.g.,
use-dependent effects); and (b) for comparative purposes to previous Ito studies that also used Cd2+ to block ICa,L (e.g., ferret
right ventricular myocytes [Campbell et al., 1993] and human
left ventricular myocytes [Näbauer et al., 1993
, 1996
]). Cd2+ can
exert effects on various potassium currents, including shifts in
both activation and inactivation, and such presently uncharacterized effects may not be identical among the two major Ito phenotypes (Ito,epi, Ito,endo) that we describe. The standard holding potential under all conditions was
70 mV. 5-15 min were routinely allowed to pass after initial attainment of the whole-cell
configuration to allow for stabilization of currents and gating parameters (Campbell et al., 1993a
; Marty and Neher, 1995
). "Leakage correction" was not applied. All chemicals used for making
solutions were obtained from Sigma Chemical Co. Heteropoda
toxin 2 (stored at
20°C; directly added to room temperature Ito
saline at a final concentration of 150 nM immediately before experimental application) was a kind gift of NPS Pharmaceuticals.
Antibody Generation
Kv1.4 (monoclonal), Kv4.2 (COOH-terminal polyclonal), and
Kv4.3 (COOH-terminal polyclonal) antibodies were prepared as follows. The anti-Kv1.4 monoclonal antibody K13/31 (Bekele-Arcuri
et al., 1996) was raised against a synthetic peptide (NSHMPYGYAAQARARERERLAHSR; Quality Controlled Biochemical) corresponding to amino acids 13-37 of rat Kv1.4. This sequence is
100% identical to the corresponding sequence of ferret Kv1.4
(Comer et al., 1994
). The anti-Kv4.2 polyclonal antibody Kv4.2C
(Nakahira et al., 1996
) was raised against a synthetic peptide
(CLEKTTNHEFVDEQVFEES; Quality Controlled Biochemical)
corresponding to amino acids 484-502 of rat Kv4.2. The sequence of the corresponding region of ferret Kv4.2 was identical to that of rat Kv4.2 (M.J. Morales, unpublished observations). The anti-Kv4.3 polyclonal antibody Kv4.3C was raised against a synthetic peptide (SPGPNTNIPSITSN; Protein Chemistry Laboratory, Washington University Medical Center, St. Louis, MO)
that matches a unique sequence in the COOH terminus of Kv4.3
that corresponds to amino acids 616-629 of rat Kv4.3 short form
and 635-648 of rat Kv4.3 long form. This amino acid sequence is
down stream from the 19 amino acid insert in the COOH terminus in the long form of Kv4.3 (Takimoto et al., 1997
). This suggests that the Kv4.3 antibody detects both the short and long
forms of the Kv4.3
subunits. A cysteine residue was added to the
NH2 terminus of each peptide to allow coupling to the keyhole
limpet hemocyanin carrier protein, and the coupled peptides
were sent to Caltag Laboratories for injection into rabbits. Sera
were screened using ELISA assays and the antibody was affinity
purified using the Immunopure Antigen/Antibody Immobilization Kit #2 (Pierce Chemical Co.).
Antibody Characterization: Transfected Cells and Immunohistochemistry
The specificity of the Kv1.4 antibody was established in a previous
report (Bekele-Arcuri et al., 1996). The specificity of the Kv4.2
and Kv4.3 antibodies was examined in immunohistochemical experiments on Xenopus oocytes expressing Kv4.2 and Kv4.3 channels. The Kv4.2 and Kv4.3 (short form) cDNAs were obtained
from Lily Jan (University of California, San Francisco, San Francisco, CA) and Jane Dixon and David McKinnon (SUNY, Stony
Brook), respectively. Messenger RNA (Kv4.2, Kv4.3) or distilled
H2O was injected into Xenopus oocytes and incubated for 72 h at
22°C in antibiotic containing Barth's solution as previously described (Comer et al., 1994
). Two electrode voltage-clamp analysis (Comer et al., 1994
) was performed to document the presence of expressed Kv4.2 and Kv4.3 channels. Oocytes expressing
Kv4.2 channels were tested with Kv4.2 and Kv4.3 antibodies, and
cross-reactivity was assessed by preabsorbing the potential
epitopes with Kv4.3 antibody and subsequently incubating with
fluorescently labeled anti-Kv4.2 antibody. Similarly, oocytes expressing Kv4.3 channels were tested with Kv4.3 and Kv4.2 antibodies, and cross-reactivity was assessed by preabsorbing the potential epitopes with Kv4.2 antibody and subsequently incubating
with fluorescently labeled anti-Kv4.3 antibody. Immunofluorescence on oocytes was performed as follows: oocytes were incubated in blocking buffer containing 5% BSA in TBSN (Tris buffered saline with NP40; 155 mM NaCl, 10mM Tris-Cl, pH 7.4, and
0.1% NP40) for 10-16 h at 4°C, and then washed 3 × 5 min in
TBSN at room temperature. Oocytes expressing Kv4.2 and Kv4.3
were incubated with Kv4.2- and Kv4.3-specific primary antibodies
(1:100), respectively, diluted in blocking buffer (indirect IF assay); another set of Kv4.2- and Kv4.3-expressing oocytes were separately incubated with anti-Kv4.2 and -Kv4.3 antibodies (1:100)
diluted in blocking buffer as a preabsorption step. Both of these
incubations were carried out at 4°C for 10-16 h. The oocytes
were washed 5 × 5 min in TBSN. The first set of oocytes incubated with the respective primary antibodies were subsequently
incubated with the secondary antibody, anti-rabbit IgG (1:200)
conjugated with FITC at room temperature for 6 h. A second set
of oocytes preabsorbed with anti-Kv4.2 or -Kv4.3 antibody were
incubated with FITC-labeled anti-Kv4.2 antibody (Kv4.2-expressing oocytes) or FITC-labeled anti-Kv4.3 antibody (Kv4.3-expressing oocytes) for 10-16 h in the dark at 4°C (direct IF assay). The
oocytes were then washed 5 × 10 min in TBSN, dehydrated in
100% methanol, and subsequently treated with BA:BB (one part benzyl alcohol to two parts benzyl benzoate) cleaning solution, mounted, and scanned using a confocal microscope (see below).
Western Blot Analysis
Cardiac membrane proteins were prepared using a slight modification of the protocol previously described by Barry et al. (1995). In brief, protein preparations were obtained from strips of tissue dissected from ferret LV epicardial and endocardial regions (exactly as described above for myocyte isolation). All procedures
were conducted at 4°C and all solutions contained the following
protease inhibitors: iodoacetamide (0.6 mM), 1,10 phenanthroline (0.5 mM), benzamidine (0.5 mM), leupeptin (0.15 µM),
pefebloc (0.5 mM), aprotinin (2 µg/ml), and pepstatin (1 µM).
The tissue strips were homogenized using a polytron (Brinkmann Instruments, Inc.) in 10 vol of 0.25 M sucrose buffer
(whole tissue homogenate), followed by centrifugation at 1,075 g
for 10 min (to remove nuclei and cellular debris). The pellet was
used to prepare cytosolic- and particulate-enriched fractions as
described by Storrie and Madden (1990)
and Dignam (1990)
.
The supernatant was centrifuged at 105,000 g for 1 h at 4°C. The
crude membrane pellet was suspended in Tris-EDTA (TE)
buffer, and then centrifuged at 60,000 g for 30 min. The pellets
were suspended in TE buffer containing 0.6 M KI, incubated on
ice for 15 min, and then centrifuged again at 60,000 g for 30 min,
washed once with TE buffer and centrifuged at 60,000 g. The pellets were resuspended in TE buffer containing 2% deoxycholate and incubated on ice for 1 h. Insoluble materials were removed by centrifugation at 13,175 g for 20 min and the supernatant was aliquoted and used for immunoblot analysis.
Protein concentration was measured using a standard BCA method (Pierce Chemical Co.) and 50 µg of membrane protein with appropriate protein markers were run on SDS PAGE gel (10%), followed by transfer to Hybond-P PVDF membrane (Amersham Corp.). After incubation with PBS-T (0.1% Tween20 in PBS) buffer containing 10% nonfat dry milk powder, the membranes were washed once in PBS-T for 5 min and incubated with appropriate primary antibodies (anti-Kv1.4, 4.2, and 4.3 antibodies). After incubation, the membranes were washed 3 × 5 min and incubated in horseradish peroxidase-conjugated secondary antibody (anti-mouse or -rabbit IgG). Then, the membranes were washed 5 × 10 min, incubated in ECL solution (Amersham Corp.), exposed to film and developed.
Fluorescent In Situ Hybridization
Techniques for FISH were performed essentially as previously described by Brahmajothi et al. (1996). Ferret hearts were perfused (Langendorff apparatus) with 4% paraformaldehyde in PBS. Regions of interest were postfixed, incubated in 40% sucrose in
PBS, and subsequently embedded in ornithine carbamyl transferase medium and frozen. Blocks were sectioned using a cryotome (2800E; Leica-Jung Frigocut) at a thickness of 6-7 µm and
laid out on gelatin-coated slides. The sections were then postfixed in paraformaldehyde and incubated in prehybridization
and hybridization buffer with specific sense and antisense oligonucleotide probes labeled with biotin or digoxigenin. The sections were incubated at 42°C for 16-24 h. After hybridization
washes, detection of transcript was carried out using streptavidin-phycoerythrin or antidigoxigenin antibody conjugated with
FITC. Hybridization signals from troponin I cardiac (TnIC) antisense probe was used as positive control and TnIC, Kv1.4, Kv4.2,
and Kv4.3 sense probes were used as negative controls. The fluorescent signals were scanned using an inverted confocal microscope (LSM 410; Carl Zeiss, Inc.) as detailed below.
Immunofluorescence
Techniques for IF were performed essentially as described by
Brahmajothi et al. (1997). Postfixed cryosections were initially blocked with 10% goat serum, washed once with PBS, and incubated with anti-Kv1.4 (1:1,500), anti-Kv4.2 (1:100), and anti-
Kv4.3 (1:50) antibodies for 1 h, and then washed with PBS. For
indirect IF, the sections were incubated with anti-mouse or -rabbit IgG conjugated with fluorescein isothiocyanate, tetramethyl
rhodamine thiocarbamoyl, or sulfonated 7-amino-4-methyl coumarin-3 acetic acid. Colocalization studies were preformed by direct IF; anti-Kv1.4, 4.2, and 4.3 antibodies were directly conjugated with the above fluorochromes. Antimyosin antibody was
used as a positive control. Sections incubated (a) without primary antibodies or (b) preabsorbed primary antibodies (anti-
Kv1.4, 4.2, and 4.3 antibodies previously incubated with myocytes) were used as negative controls. To demonstrate the localization patterns within isolated myocytes, cells were stained with
DiIC18 (dioctadecyl 3,3,3',3' tetramethyl indocarbo cyanine) and
propidium iodide markers that label the sarcolemma and nucleus, respectively. The antibody binding and staining patterns in
isolated myocytes were analyzed by taking 1 µm optical Z sections through the cells. Fluorescence was detected using an inverted confocal microscope equipped with ArKr, HeNe, and HeCd laser
beams under appropriate excitation and emission wavelengths
for each fluorochrome.
Interpretive Limitations of IF Results
Fluorescence intensities.
For numerous reasons (i.e., monoclonal
[Kv1.4] versus polyclonal [Kv4.2, Kv4.3] antibodies employed,
possible differences in number, accessibility, and affinity of targeted epitopes, etc.), a direct quantitative comparison of fluorescence intensities between each of the individual antibodies employed (to estimate and/or compare absolute Kv1.4, Kv4.2, and
Kv4.3 subunit protein expression levels) was precluded. We
therefore wish to emphasize that the specific immunoreactivities/fluorescent intensities that were obtained for any one specific
subunit cannot be quantitatively compared with those obtained for another
subunit type; only qualitative and/or relative comparisons of individual
subunit protein levels are valid.
IF profiles for individual proteins within transverse sections
across the ventricular wall (see Fig. 7) were analyzed by confocal
microscopy, and for comparative purposes the relative fluorescent intensity values of each
subunit protein were normalized
to the maximum fluorescence obtained for that
subunit (i.e.,
maximum relative intensity = 100%).
|
Ito subunit protein localization to specific cardiac cell types.
The IF
results presented on enzymatically isolated LV epi versus LV
endo myocyte preparations (see Fig. 6 and Table II) measure Kv1.4, Kv4.2, and Kv4.3
subunit protein expression levels
within specific myocyte types. As will be described, these measurements indicate marked heterogeneous expression levels of
these three
subunits between LV epi versus LV endo myocytes.
These heterogeneous myocyte
subunit expression levels will
contribute to net IF patterns obtained from intact ventricular tissue sections. However, in ventricular tissue Kv1.4, Kv4.2, and/or
Kv4.3
subunits may be expressed not only in myocytes but also
in numerous nonmyocyte cell types, including fibroblasts, endothelial cells, smooth muscle cells, and neurons. To date, we have
not analyzed the distribution of candidate Ito
subunit expression levels in these various nonmyocyte cell types. We therefore
wish to emphasize that the IF patterns obtained from sagittal sections of ventricular tissue (see Figs. 7 and 8) cannot at present be
attributed exclusively to Kv1.4, Kv4.2, and/or Kv4.3
subunit
protein expression within myocytes.
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RESULTS |
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Basic Observations: Calcium-independent Ito Phenotypes Are Present in both Left Ventricular Epicardial and Endocardial Myocytes
Under our recording conditions (Na+- and Ca2+-free
saline, 500 µM CdCl2; see METHODS) in both ferret LV
epi and LV endo myocytes, calcium-independent transient outward potassium currents could be routinely recorded in response to depolarizing voltage clamp steps.
Representative examples of these transient outward
currents in both LV epi and LV endo myocytes in response to either 500- (LV epi) or 1,000-ms (LV endo)
depolarizing clamp pulses applied from a holding potential (HP) of 70 mV are illustrated in Fig. 1, A and
B. On initial inspection, the transient outward currents in both myocyte types displayed somewhat similar basic
macroscopic gating characteristics: both activated very
rapidly (within milliseconds, with activation of the peak
LV endo current being generally faster, resulting in its
peak being frequently obscured due to overlap with the
capacitive transient), and then displayed a slower phase of macroscopic inactivation to a final steady state level
(i.e., a noninactivated sustained component). Additional measurements (data not shown) wherein the
[K+]o was varied (1-100 mM) indicated that the amplitude of the transient components of these two currents
was indeed a function of [K+]o, verifying that they were
K+ currents. Following the convention established in
previous studies (e.g., Antzelevitch et al., 1995
; Campbell et al., 1995
), we defined the inactivating transient
component (i.e., Ipeak
Isustained) of these currents as
"Ito." We will refer to these two calcium-independent Ito
phenotypes in ferret LV epi and LV endo myocytes as
Ito,epi and Ito,endo, respectively.
|
Electrophysiological Characterization of Left Ventricular Myocyte Ito,epi and Ito,endo Phenotypes
Current density and current-voltage relationships.
There were
no significant differences between LV epi versus LV
endo myocytes in either (a) resting membrane potential (measured in control solution before establishing
voltage clamp; LV epi, Vm = 72.5 ± 0.7 mV, n = 9; LV
endo,
72.6 ± 0.6 mV, n = 10) or (b) density of the inwardly rectifying K+ current, IK1 (measured in response
to 500-ms hyperpolarizing voltage clamp pulses applied
to
80 to
120 mV). However, as illustrated in Fig. 1,
C1 and C2, there were significant differences in both activation thresholds and mean peak current-voltage
relationships between Ito,epi (defined as Ipeak
I500 ms,
n = 7) and Ito,endo (defined as Ipeak
I1000 ms, n = 6).
Ito,epi activated at
10 to 0 mV and had a peak density at
+20 mV of +4.03 ± 0.52 pA/pF. In contrast, Ito,endo activated at
30 to
20 mV and had a much lower peak
current density at +20 mV of +0.77 ± 0.16 pA/pF.
Inactivation.
The mean steady state inactivation relationships for Ito,epi (n = 7) and Ito,endo (n = 8) are illustrated in Fig. 2 A (protocols illustrated in inset). Steady
state inactivation relationships for both Ito phenotypes
could be well described as single Boltzmann relationships. However, there were significant differences
among the two inactivation relationships in both V1/2
and k values (Ito,epi: V1/2 = 9 mV, k = 5.45 mV; Ito,endo:
V1/2 =
34 mV, k = 8.05 mV).
|
Kinetics of recovery.
The kinetics of recovery from inactivation of the two LV Ito phenotypes were determined at a HP of 70 mV using a conventional double
pulse protocol (Fig. 3 A2, inset). In the majority of LV epi myocytes (n = 7/9, 78%), recovery was rapid (Fig. 3
A1) and could be well described as a single exponential
with a mean
rec = 51.2 ± 2.8 ms (n = 7; Fig. 3 A2). In
two additional LV epi myocytes, there was a major fast
component of recovery (accounting for 90 and 95% recovery within 200-250 ms), followed by a slower phase (on the order of hundreds of milliseconds), with recovery being complete within 2-3 s (data not illustrated).
In contrast, in the majority of LV endo myocytes (n = 8/11, 73%), recovery was quite slow (Fig. 3 B1) and
could be reasonably described as a single exponential
with a mean
rec = 3,001.8 ± 447.1 ms (Fig. 3 B2). However, in three LV endo myocytes, recovery was rapid (single exponential,
rec = 66.7 ± 3.4 ms; data not
shown); i.e., comparable to Ito,epi. In summary, while in
the majority of LV epi and LV endo myocytes studied
there was a clear distinction between "pure" rapid recovery (Ito,epi) versus "very slow" recovery (Ito,endo), this
kinetic differentiation was not absolute. However, in no
instance was an exclusively "very slow recovery" pattern ever observed in an LV epi myocyte.
|
Effects of Heteropoda toxin 2.
Heteropoda toxins (HPTXs)
are peptides (30-33 amino acids) isolated from the
venom of the spider Heteropoda venatoria. HPTXs block
both native Ito channels in rat ventricular myocytes and Kv4.2 channels expressed in Xenopus oocytes; in contrast, HPTXs have no significant effects on expressed
Kv1.4 channels (Sanguinetti et al., 1997). To determine
whether we could pharmacologically distinguish between ferret LV Ito,epi and Ito,endo, we conducted a preliminary analysis of the effects of HPTX2 (30 amino
acid peptide; sequence given in Fig. 2 of Sanguinetti et
al., 1997
). 150 nM HPTX2 rapidly and reversibly
blocked Ito,epi without any significant effects on the sustained current remaining at 500 ms (Fig. 4 A). However, the degree of block was potential dependent, with
block being relieved with progressive depolarization.
Block decreased from 75.6 ± 5.3% at +10 mV to 21.2 ± 4.8% at +70 mV (n = 3; Fig. 4 B). Assuming a simple
single site binding model, the estimated apparent Kd
for HPTX2 block ranged from 105 nM at +20 mV to
559 nM at +70 mV (Fig. 4 B, inset). In contrast, 150 nM
HPTX2, when perfused over a period of 5-7 min, failed
to produce any significant block of the major slowly recovering Ito,endo phenotype elicited at +50 mV (Fig. 4 C,
n = 2).
|
|
Distribution of Candidate Ito Subunits in Isolated Left
Ventricular Epicardial and Endocardial Myocytes
The rapidly inactivating Kv1.4, Kv4.2, and Kv4.3 clones
have all been recently proposed to form the molecular
basis of the cardiac myocyte calcium-independent Ito
(e.g., Po et al., 1993; Comer et al., 1994
; Barry et al.,
1995
; Barry and Nerbonne, 1996
; Dixon et al., 1996
; Xu
et al., 1996
; Fiset et al., 1997
; Yeola and Snyders, 1997
).
Therefore, the FISH and IF measurements to be described in the next section were designed to determine
whether there was a differential distribution of Kv1.4,
Kv4.2, and/or Kv4.3
subunits between ferret LV epi
and LV endo myocytes, and whether such a differential
distribution correlated with the two major functional Ito
phenotypes described in the preceding sections.
Characterization of Kv1.4, Kv4.2, and Kv4.3 antibodies.
To determine the specificity of the antibodies employed,
IF and immunoblot analyses were first performed. IF
experiments were performed on Xenopus oocytes injected with K+ channel mRNA or sham-injected with
water (Fig. 5, A-I). Oocytes injected with Kv4.2 mRNA
(n = 7) only reacted with the Kv4.2 antibody (Fig. 5,
D-F), while oocytes injected with the short form of Kv4.3 mRNA (n = 10) only reacted with the Kv4.3 antibody
(G-I). Neither antibody stained the sham-injected oocytes (Fig. 5, B and C). Immunoblot analyses for each
antibody were then performed on four different protein preparations obtained from ferret LV epicardial and endocardial regions: (a) sarcolemmal membrane
enriched fractions (Fig. 5, J-L, lanes 2 and 3), (b) cytosolic-enriched fractions (lanes 4 and 5), (c) particulate-enriched fractions (lanes 6 and 7), and (d) whole-tissue
homogenates (lanes 8 and 9). Among the sarcolemmal
membrane-enriched fractions, prominent and specific antibody binding patterns were obtained. The Kv1.4
antibody recognized a single specific band at ~95-100
kD in the LV endo fraction (Fig. 5 J, lane 3); in contrast, there was virtually no reactivity of the Kv1.4 antibody in the LV epi fraction (lane 2). The Kv4.2 antibody specifically recognized a single prominent band
at ~75 kD in the LV epi fraction (Fig. 5 K, lane 2) and a
similar but weaker band in the LV endo fraction (lane
3). Finally, the Kv4.3 antibody recognized a single band
at ~75 kD in both LV epi and LV endo fractions (Fig. 5
L, lanes 2 and 3). In combination with earlier results
(Bekele-Arcuri et al., 1996; Takimoto et al., 1997
), our
IF and immunoblot data strongly suggest that the three antibodies employed detect and specifically bind to
Kv1.4, Kv4.2, and Kv4.3
subunits expressed in ferret
LV epi and LV endo tissues. These results also suggest
that the majority of
subunit proteins detected by the
antibodies are located at cell surfaces.
|
Isolated myocyte analysis.
To determine specific antibody binding patterns to isolated LV epi and LV endo
myocytes, confocal microscopy was employed. Binding
patterns for each of the three antibodies were determined by measuring fluorescence intensity profiles in
successive optical z sections (1-µm thickness) taken
through the entire width of each myocyte type. Fig. 6 illustrates a representative series of such optical z sections (every third section is illustrated) measured from
isolated LV epi (anti-Kv4.2, C1-C6; anti-Kv4.3, D1-
D6) and LV endo (anti-Kv1.4, E1-E6) myocytes. For
controls, standard membrane markers for the sarcolemma (DiIC18; Fig. 6, A1-A6) and nucleus (propidium
iodide; B1-B6) were used. The fluorescence patterns
obtained in the successive optical z sections indicated that binding of all three antibodies was localized to the
outer regions of myocytes, thereby suggesting localization to sarcolemmal proteins. In combination with our
immunoblot data (Fig. 5), these z section results strongly
suggest that sarcolemmally associated Kv1.4, Kv4.2,
and/or Kv4.3 subunits could function as the molecular substrates underlying the two different functional Ito
phenotypes in ferret LV epi and LV endo myocytes.
Distribution of Candidate Ito Subunits in Intact
Ventricular Tissue
In this final section, we extend the parallel FISH and IF
measurements described above for isolated LV myocytes to sagittal sections of whole intact ventricular tissue. These measurements were conducted so as to gain
initial insights into overall ventricular tissue distribution patterns of Kv1.4, Kv4.2, and Kv4.3 mRNA transcript and expressed subunit proteins.
Representative parallel FISH and IF results obtained from such ventricular tissue sections are illustrated in Fig. 7. As controls, sagittal sections taken from the ferret ventricle (orientation in Fig. 7, A) were subjected to hybridization with TnIC antisense or sense probes (Fig. 7, B and C). These data demonstrated that the entire sections were competent for FISH assays, and that these assays produced very low background when using the sense probe for TnIC. Similar results were obtained with sense probes derived from Kv1.4, Kv4.2, and Kv4.3 (data not shown). IF obtained using antimyosin antibody also gave results similar to those illustrated in Fig. 7 B (data not shown), demonstrating that these sections were also competent for IF assays. From these measurements, it was concluded that the observed localization patterns were not artifacts of the tissue preparation. Finally, as controls for the indirect IF results, assays conducted with secondary antibodies (fluorochrome conjugated anti-mouse or -rabbit IgG) alone demonstrated that our indirect fluorescent signals were due to specific binding to the primary antibodies (e.g., Fig. 7, D).
Using FISH, Kv1.4 mRNA was abundantly and uniformly expressed throughout all regions of the ferret
ventricle (Fig. 7, E, E1, and E2). In contrast, Kv4.2
mRNA was heterogeneously expressed, being prominent in apex, RV, LV epi (from both apex to base), and
both basal LV endo and septum, while it was markedly
reduced to absent in both apical LV endo and septum
(Fig. 7, G, G1, and G2). Kv4.3 mRNA was more uniformly expressed, although it was relatively more abundant in the apex and apical LV epi and was reduced in
the RV (Fig. 7, I, I1, and I2). Similar mRNA distribution results for all three subunits were observed in
sagittal sections obtained from a total of seven ferret hearts.
In contrast to the FISH results, indirect IF analysis indicated that Kv1.4 subunit protein was essentially absent from the RV, the LV epi, and the basal region of
the LV, but was uniformly expressed in the apical LV
endo and midmyocardium (free wall and septum) (Fig.
7, F, F1, and F2). Kv4.2
subunit protein was expressed abundantly in the apex, RV, and LV epi (apex and
base) and the basal LV endo and septum, but was markedly reduced to absent in the apical LV endo (Fig. 7, H,
H1, and H2). Finally, Kv4.3
subunit protein was more
uniformly expressed, although it was relatively low to
absent in RV and relatively more abundant in the apex
and LV epi compared with LV endo (Fig. 7, J, J1, and
J2). Therefore, similar to the isolated myocyte studies,
results from the ventricular sections indicate that there
was a general correspondence between differential
Kv4.2 and 4.3 mRNA transcript levels and expressed
subunit proteins, while such a correspondence did not
hold for Kv1.4. A further demonstration of the heterogeneity of the three
subunits across the ventricular
wall is shown by the relative fluorescence intensity profiles measured transversely (Fig. 7, cyan lines 3-5) at
the indicated basal (F-J, 3), mid-ventricular (F-J, 4)
and apical (F-J, 5) regions.
Representative sagittal section colocalization data for
Kv1.4, Kv4.2, and Kv4.3 subunits obtained using direct IF are illustrated in Fig. 8 for Kv1.4 + Kv4.2 (A:
Kv1.4, red; Kv4.2, green), Kv1.4 + Kv4.3 (B: Kv1.4, red;
Kv4.3 green), and Kv4.2 + Kv4.3 (C: Kv4.2, green;
Kv4.3, red; note that in these panels colocalization is represented by a yellow to orange color; refer to legend
for details). Fig. 8 D illustrates the colocalization pattern of all three
subunit proteins (Kv1.4, blue; Kv4.2,
green; Kv4.3, red). From Fig. 8 D it can be seen that
there was a relative abundance of Kv4.2 (green) and
Kv4.3 (red) in the epicardial regions and Kv1.4 (blue)
in the apical endocardial region of the LV, clearly establishing a regional localization. There was also some colocalization of Kv4.2 + Kv4.3 (yellow to orange) in the
epicardial regions and Kv1.4 + Kv4.3 (purple to pink)
in the endocardial region of the LV and the septum.
In summary, subunit protein expression distribution patterns similar to those illustrated in Figs. 7 and 8
were obtained from at least three ferret hearts (Kv4.3,
n = 3; Kv1.4 and Kv4.2, n = 6). The general distribution patterns of the three
subunits were as follows: (a)
Kv1.4 was localized primarily to the apical portion of the
LV septum, LV endo, and approximate inner 75% of the
LV free wall; (b) Kv4.2 was localized primarily to RV
free wall, epicardial layers of the LV, and the base of the
heart; and (c) Kv4.3 was localized primarily to epicardial layers of the LV and base of the heart. Therefore,
in the intact ventricle, there is a substantial heterogeneous distribution of Ito
subunits not only from epicardium to endocardium but also from apex to base.
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DISCUSSION |
---|
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---|
Single LV Epi and LV Endo Myocyte Electrophysiology
Two distinct Ito phenotypes, which we have designated
Ito,epi and Ito,endo, are differentially expressed in ferret
left ventricular epicardial (LV epi) and endocardial
(LV endo) myocytes. Under our recording conditions
(Na+- and Ca2+-free saline, 500 µM Cd2+) Ito,epi and
Ito,endo display significant differences in activation, inactivation, and recovery characteristics and sensitivity to block by HPTX2. In combination, these results strongly
suggest that Ito,epi and Ito,endo are produced by (at least)
two distinct potassium channel subunits that have different molecular mechanisms governing inactivation
and recovery characteristics (e.g., Hoshi et al., 1990
,
1991
; Choi et al., 1991
; Campbell et al., 1995
; Rasmusson et al., 1995a
). In addition to different underlying
subunits, other (presently uncharacterized) factors may
also be contributing to these kinetic differences, including differences in associated
subunits (Rettig et al.,
1994
; Morales et al., 1995
) and homomeric versus heteromeric
subunit assembly (Po et al., 1993
; Sheng et
al., 1993
; Wang et al., 1993
; see colocalization IF data
summarized in Table II).
Possibly the most functionally significant difference
between Ito,epi and Ito,endo resides in their kinetics of recovery from inactivation. In the majority of LV epi and
LV endo myocytes studied recovery at a HP of 70 mV
was well-described as a single exponential process; however, the mean
rec of Ito,endo was ~60× slower than Ito,epi. As a result, Ito,endo displays marked cumulative inactivation, while Ito,epi does not. These two major Ito
phenotypes will therefore exert significantly different
frequency-dependent modulatory effects on action potential morphology and repolarization characteristics. For example: (a) in LV epi myocytes, the predicted effects of reducing Ito,epi should only become prominent
at high heart rates; and (b) in LV endo myocytes, the
modulatory effects of Ito,endo should be minimal at normal heart rates (due to cumulative inactivation) but
could become significant at low rates. In addition, if
metabolic factors (e.g., myocyte redox status, second
messengers, phosphorylation) are capable of modulating Ito,endo inactivation/recovery characteristics (e.g.,
Ruppersberg et al., 1991
; Roeper et al., 1997
), then under certain conditions Ito,endo could become a significant repolarizing current at normal heart rates.
In preliminary action potential (AP) measurements,
we have obtained results consistent with these general
functional predictions in some ferret LV epi and LV
endo myocytes. However, as would be predicted by our
IF results, we have also observed significant variability
in both AP morphologies and frequency-dependent
characteristics (0.2-1 Hz), particularly among LV endo
myocytes (D.L. Campbell, unpublished observations).
It is therefore important to note that our previous FISH
and IF measurements (Brahmajothi et al., 1996, 1997
)
demonstrated marked heterogeneous expression patterns of multiple potassium channel
subunit mRNA
transcripts and proteins within different anatomical regions and myocytes of the ferret heart. In combination
with this previous data, our preliminary AP recordings
suggest that, in addition to Ito,epi and Ito,endo, other repolarizing currents are also heterogeneously expressed. In conjunction with future current clamp studies on AP
morphologies, the kinetic properties of each of these
different repolarizing currents need to be quantitatively analyzed before a more realistic understanding of
LV epi versus LV endo myocyte repolarization can be achieved.
Isolated Myocyte Immunolocalization Results: Potential Molecular Substrates of Ito,epi and Ito,endo
Our immunolocalization data on isolated LV epi and
LV endo myocytes (Fig. 6 and Table II) clearly indicate
a correlation between the expression patterns of Kv1.4,
4.2, and 4.3 subunits and the two major functional LV
myocyte Ito phenotypes. Specifically, our data strongly
suggest that Kv4.2/4.3
subunits largely underlie Ito,epi,
and Kv1.4
subunits largely underlie Ito,endo. This hypothesis is further strengthened by the fact that both
Ito,epi and expressed Kv4.2/4.3
subunits (e.g., Tseng
et al., 1996
; Yeola and Snyders, 1997
) display rapid (although not identical) recovery kinetics, while both
Ito,endo and expressed Kv1.4
subunits (e.g., Comer et
al., 1994
; Rasmusson et al., 1995a
) display very slow (although not identical) recovery kinetics with marked cumulative inactivation.
While exclusively rapid versus exclusively slow recovery patterns are the dominant phenotypes in ferret LV
epi versus LV endo myocytes, there was a subpopulation of both (a) LV endo myocytes (27%; n = 3/11)
that displayed rapid single exponential recovery kinetics and (b) LV epi myocytes (22%; n = 2/9) that displayed an additional slower phase of recovery (on the
order of hundreds of milliseconds). The percentage of
LV endo myocytes displaying rapid recovery was therefore similar to the sum of the percentage of isolated LV
endo myocytes displaying colocalization of Kv1.4 + Kv4.2 (10.3 ± 1.7%) and Kv1.4 + Kv4.3 (6.4 ± 2.3%) subunits. In contrast, the percentage of LV epi myocytes displaying a slower component of recovery was
not comparable to the percentage of isolated LV epi
myocytes displaying colocalization of Kv1.4 + Kv 4.2 and Kv1.4 + Kv4.3
subunits. Although caution must
be exercised in directly comparing these two sets of
data, the similarity does allow for speculation that the
coexpression of Kv1.4 and Kv4.2 and/or Kv4.3
subunits within the same LV endo myocyte may underlie
the subpopulation of rapidly recovering Ito,endo phenotypes. At present, our data cannot account for the subpopulation of LV epi myocytes that displayed an additional slow component of recovery. However, in no instance was a "purely slowly recovering" Ito phenotype
observed in an LV epi myocyte. This correlates very well
with the fact that Kv1.4 protein was virtually absent in isolated LV epi myocytes.
Finally, while to date we have not specifically analyzed
Ito subunit expression in ferret isolated RV myocytes,
our IF results on intact ventricular sections (see Interpretive Limitations in METHODS) have implications for our
previous study of the Ito phenotype in ferret isolated RV
myocytes (Campbell et al., 1993a
,b; see Table I). Ferret
RV myocytes almost exclusively express a uniform Ito
phenotype with rapid recovery kinetics. The RV Ito phenotype would therefore appear to correlate with the virtual absence of Kv1.4 and the uniformly abundant expression of Kv4.2
subunits in ferret intact RV tissue.
Furthermore, the RV Ito phenotype displays marked
closed state reverse-use-dependent block by 4-aminopyridine (Campbell et al., 1993b
), a property shared
by expressed Kv4.2 channels (e.g., Tseng et al., 1996
;
Yeola and Snyders, 1997
) but not by Kv1.4 channels
(e.g., Rasmusson et al., 1995b
).
Heteropoda Toxin
Sanguinetti et al. (1997) have reported differential
block of expressed Kv1.4 and Kv4.2 channels by HPTXs
(Kv1.4, insensitive; Kv4.2, sensitive). These investigators
observed that HPTX block of Kv4.2 was relieved with
progressive depolarization, while at a fixed potential
the dose-response curve could be well-described using
a Hill equation formulation with voltage-dependent coefficients ranging from one to two. The effects of
HPTXs on Kv4.3 were not determined in this study.
However, given the extensive sequence homology in
the core region between Kv4.2 and Kv4.3, it is likely that HPTXs would have similar effects on these two
subunits. The results of Sanguinetti et al. (1997)
, in
combination with our HPTX2 results (Ito,epi, sensitive;
Ito,endo, insensitive), further strengthens the hypothesis
that in ferret LV endo and LV epi myocytes Kv1.4 and
Kv4.2/4.3
subunits, respectively, are the major substrates underlying Ito,endo and Ito,epi.
Ferret Ito,epi and Ito,endo: Comparison with Previous Studies of Native Myocyte Ito Phenotypes in Other Species
The characteristics of the two major Ito phenotypes that we
measured in ferret LV epi and LV endo myocytes are very
similar to those recently reported for the two major Ito
phenotypes in human left ventricular epicardial (Amos
et al., 1996; Näbauer et al., 1993
, 1996
) and endocardial
myocytes (Näbauer et al., 1993
, 1996
), although there
are quantitative differences. In both ferret and human
LV, the major Ito,epi phenotype is expressed at relatively
high current densities, activates and inactivates at relatively depolarized potentials, and recovers rapidly (and hence displays little to no cumulative inactivation).
Similarly, in both ferret and human LV, the major
Ito,endo phenotype is expressed at relatively low current
densities, activates and inactivates at more hyperpolarized potentials, and recovers slowly (and hence displays
cumulative inactivation). This raises the possibility that
there may be a differential distribution of Kv1.4, 4.2, and/or 4.3
subunits in human LV epi and LV endo
myocytes similar to that which we observe in ferret.
In contrast, Ito phenotypes of both ferret and human
LV myocytes appear to differ from those of rabbit and
canine ventricular myocytes. In rabbit ventricle, only
one major slowly recovering Ito phenotype appears to
be dominant but expressed at different densities in epicardial versus endocardial myocytes (Fedida and Giles,
1991). Hence, in rabbit, Kv1.4 may be the dominant
subunit expressed in both epicardium and endocardium. In canine LV, there also appears to be one major
expressed Ito phenotype displaying relatively slow recovery kinetics but different levels of expression in LV epi,
LV midmyocardial, and LV endo myocytes (e.g., Liu et al.,
1993
; Antzelevitch et al., 1995
). However, it has recently been proposed that Kv4.3
subunits underlie the canine LV Ito (Dixon et al., 1996
). Our results would
therefore suggest, at least with regard to the role(s) of
multiple and distinct Ito phenotypes in modulation of
LV repolarization, that the ferret may provide a better
comparative cellular model for the human heart (e.g.,
Näbauer et al., 1993
, 1996
) than other more commonly studied species.
FISH and Immunolocalization Results: New Insights, Implications, and Limitations
To conclude, we wish to point out that our parallel FISH and IF results on both isolated LV myocytes (Fig. 5 and Table II) and intact ventricular tissue sections (Figs. 7 and 8) yield additional important insights into presently unrecognized but potentially serious limitations of these techniques as they are now commonly employed. Three potentially very important, and interrelated, implications of our results follow.
Transcript mRNA (FISH) versus expressed protein (IF).
For both Kv4.2 and 4.3, there was a general concordance between the distribution of transcript and subunit protein in the ferret ventricle. In contrast, for
Kv1.4, there was a marked disparity between the distribution of transcript and
subunit protein expression, possibly suggesting the presence of some factor that affects translation of the
subunit; e.g., an ancillary
subunit may be needed for protein assembly and insertion into the sarcolemma (Shi et al., 1996
). These very
interesting results suggest differences in transcriptional regulation of the different Ito
subunit proteins
throughout the heart. However, they also clearly indicate that conclusions on the presence of candidate ion
channels and/or
subunits within specific individual
myocyte types cannot always be reliably made based
solely on measurements of transcript mRNA levels obtained from whole tissue and/or whole heart preparations. Our results suggest that application of specific IF
techniques allow for a more accurate analysis of expressed
subunit proteins within specific anatomical
regions, important data that can then be directly correlated with functional patch clamp measurements on
myocytes isolated from the same specific regions.
Heterogeneous subunit expression in different LV myocyte
types and tissue regions.
Our present data corroborates
our previous in situ hybridization results on Kv1.4, 4.2, and 4.3 in isolated myocytes prepared from different
anatomical regions of the ferret heart (Brahmajothi et al.,
1996
). Our new data extend these previous observations by examining, in adjacent sagittal sections, the
distribution of several corresponding expressed
subunit proteins. Our present results indicate that, due to
significant heterogeneous expression of Ito phenotypes
and corresponding
subunits throughout distinct regions of the ventricle (i.e., not only epicardial-to-endocardial but also apical-to-basal; Figs. 7 and 8), very careful attention must be paid to the exact anatomical region from which tissue and/or isolated myocyte
samples are taken for analysis.
Intact ventricular tissue results: myocyte versus nonmyocyte
Ito subunit expression.
While analysis of protein distribution within individual myocyte types is important for
the ultimate identification of the molecular substrates
underlying currents within that particular myocyte
type, analysis of the distribution of Kv
subunit proteins in tissue sections provides important information
concerning the distribution of the different Kv
subunits throughout the ventricular wall. Such information is important since in the intact heart the net process of repolarization results from the overall distribution patterns of multiple potassium channel
subunit
proteins expressed throughout the entire ventricular
myocardium. However, as discussed (see Interpretive
Limitations in METHODS), whole-tissue IF analysis suffers
from the complications of potential
subunit protein expression in various nonmyocyte cell types. For this
reason, we can not at present attribute the fluorescence
signals obtained from the intact ventricular sagittal sections (Figs. 7 and 8) exclusively to Ito
subunit protein
expression in myocytes. Nonetheless, our IF results on
isolated LV epi versus LV endo myocytes (Fig. 6 and Table II) clearly demonstrate heterogeneous expression
gradients of Kv1.4, 4.2, and 4.3
subunits that essentially parallel those observed across the intact LV wall.
These results indicate that heterogeneous myocyte
subunit expression gradients are significantly contributing to the net LV epicardial-to-endocardial fluorescence signals measured in the sagittal tissue sections.
Our sagittal section IF results would therefore suggest
that the distribution of multiple Ito phenotypes in the
mammalian heart, and their potential effects upon net
repolarization characteristics, may be significantly more complicated than presently suspected.
Conclusions
In conclusion, our results demonstrate (a) the presence of (at least) two major functionally distinct Ito phenotypes in ferret LV epi and LV endo myocytes, (b) distinct heterogeneous expression patterns (both epicardial-to-endocardial and apical-to-basal) of Kv1.4, 4.2, and 4.3 subunits in ferret intact LV tissue, and (c) a
parallel
subunit expression gradient at the level of
isolated LV epi and LV endo myocytes, thereby strongly
suggesting that Kv4.2/4.3
subunits largely underlie
Ito,epi, and Kv1.4
subunits largely underlie Ito,endo.
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FOOTNOTES |
---|
Address correspondence to D.L. Campbell, Department of Physiology and Biophysics, School of Medicine and Biomedical Sciences, University at Buffalo, SUNY, 124 Sherman Hall, Buffalo, NY 14214. Fax: 716-829-2344.
Original version received 8 June 1998 and accepted version received 19 February 1999.
R.L. Rasmusson's present address is Department of Medicine, Allegheny University of the Health Sciences, Pittsburgh, PA 15212. D.L. Campbell's, M.V. Brahmajothi's, M. Morales's, and H.C. Strauss's present address is Department of Physiology and Biophysics, School of Medicine and Biomedical Sciences, University at Buffalo, SUNY, Buffalo, NY 14214.The authors thank Janice Del Mar and Michael Sanguinetti for providing us with HPTX2, J.O. McNamara, Sr., for use of equipment, and R.A. Caldwell for evaluating Kv4.2 and Kv4.3 expression in Xenopus oocytes.
This work was supported in part by National Heart, Lung, and Blood Institute grants 54314, 52874, and 19216 and National Institutes of Health equipment grant BIR-9318118.
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Abbreviations used in this paper |
---|
AP, action potential; FISH, fluorescent in situ hybridization; HP, holding potential; HPTX, Heteropoda toxin; IF, immunofluorescent; LV endo, left ventricular endocardial; LV epi, left ventricular epicardial; RV, right ventricular; TnIC, troponin I cardiac.
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