Species-specific difference in distribution of voltage-gated
L-type Ca2+ channels of cardiac myocytes
Yoshiko
Takagishi1,
Kenji
Yasui2,
Nicholas J.
Severs3, and
Yoshiharu
Murata1
Departments of 1 Teratology and Genetics and
2 Circulation, Research Institute of Environmental Medicine,
Nagoya University, Nagoya 464-8601, Japan; and 3 Department of
Cardiac Medicine, National Heart and Lung Institute, Imperial College
School of Medicine, London SW3 6NP, United Kingdom
 |
ABSTRACT |
Ca2+
influx via sarcolemmal voltage-dependent Ca2+ channels
(L-type Ca2+ channels) is the fundamental step in
excitation-contraction (E-C) coupling in cardiac myocytes.
Physiological and pharmacological studies reveal species-specific
differences in E-C coupling resulting from a difference in the
contribution of Ca2+ influx and intracellular
Ca2+ release to activation of contraction. We investigated
the distribution of L-type Ca2+ channels in isolated
cardiac myocytes from rabbit and rat ventricle by correlative
immunoconfocal and immunogold electron microscopy. Immunofluorescence labeling revealed discrete spots in the surface plasma membrane and transverse (T) tubules in rabbit myocytes. In rat
myocytes, labeling appeared more intense in T tubules than in the
surface sarcolemma. Immunogold electron microscopy extended these
findings, showing that the number of gold particles in the surface
plasma membrane was significantly higher in rabbit than rat myocytes.
In rabbit myocyte plasma membrane, the gold particles were distributed
as clusters in both regions that were associated with junctional
sarcoplasmic reticulum and those that were not. The findings are
consistent with the idea that influx of Ca2+ via surface
sarcolemmal Ca2+ channels contributes to intracellular
Ca2+ to a greater degree in rabbit than in rat myocytes.
rabbit and rat ventricular myocytes; immunoconfocal microscopy; immunoelectron microscopy; excitation-contraction coupling
 |
INTRODUCTION |
EXCITATION-CONTRACTION
COUPLING (E-C coupling) in cardiac muscle involves
Ca2+ entry through sarcolemmal Ca2+ channels
(voltage-dependent L-type Ca2+ channels), followed by a
larger Ca2+ release from sarcoplasmic reticulum (SR) via
ryanodine receptors (RyRs), a process referred to as
Ca2+-induced Ca2+ release (CICR). Though
Ca2+ entry via sarcolemmal Ca2+ channels and
Ca2+ release from the SR both contribute to activation of
contraction in the mammalian heart, the relative importance of these
two events varies with species and region of the heart. In particular,
physiological and pharmacological studies have demonstrated distinct
differences in E-C coupling between rabbits and rats, with a greater
proportion of sarcolemmal Ca2+ influx contributing to
activation of contraction in the rabbit than in the rat (2, 3, 9,
15, 16, 19, 24). A smaller Ca2+ entry elicits a
larger SR Ca2+ release in the rat than in the rabbit,
reflecting a greater dependence on SR Ca2+ in the former
than in the latter, a feature suggested from binding studies to be
linked to a greater L-type Ca2+ channel density in rat T
tubules than in rabbit T tubules (7, 20).
At the ultrastructural level, differences in general
morphological features are consistent with species differences
(13), but detailed comparative information on the spatial
organization of the relevant channels in different species is lacking.
No studies have investigated the distribution of Ca2+
channels in rat cardiac myocytes. Immunofluorescence studies on rabbit
ventricular cardiac myocytes have emphasized the abundance of
Ca2+ channels in the T tubules rather than the surface
plasma membrane (4). Our previous correlative
immunoconfocal microscopy and label-fracture electron microscopy
results revealed that L-type Ca2+ channels are organized in
the form of aggregates in surface plasma membrane and also in T tubules
in guinea pig cardiac myocytes (18), demonstrated by
mathematical analysis to be true clusters (10).
Here we have investigated the distribution of L-type Ca2+
channels in rabbit and rat ventricular myocytes by correlative
immunoconfocal and immunogold electron microscopy to shed further light
on the ultrastructural basis for species-specific differences in
contractile control of cardiac myocytes. Special attention was paid to
localization of the channels in the surface plasma membrane. We
demonstrated a different pattern of immunofluorescently localized
channels in the surface plasma membrane and T tubules between rabbit
and rat myocytes that correlated with the presence of lower levels of
labeling in the surface plasma membrane of rat myocytes than rabbit
myocytes by immunogold electron microscopy.
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MATERIALS AND METHODS |
Cell isolation.
Hearts of adult female New Zealand White rabbits and Wistar rats were
excised under deep pentobarbital sodium anesthesia. The method
for cell isolation has been described in detail previously (23). In brief, the heart was rapidly excised and perfused
in a retrograde manner with Ca2+-free Tyrode solution that
contained collagenase (80-100 IU/ml; Yakult Pharmaceutical
Industry) for 10-15 min using a Langendorff apparatus. The
ventricles were separated, minced into small pieces, and infiltrated
through a 200-µm mesh. More than 80% of single ventricular cells
were Ca2+ tolerant and rod shaped. These isolated rat and
rabbit ventricular cells exhibited normal Ca2+ currents
(5, 22), indicating the high yield and maintained viability of the cell.
Fixation.
Freshly isolated cells were fixed with 2% paraformaldehyde in
phosphate-buffered saline (PBS) for 10 min (or up to 1 h).
Antibodies.
The following two antibodies were used for labeling L-type
Ca2+ channels: 1) ccp5, a site-directed antibody
against the sequence 1,691-1,701 of the
1-subunit
of rabbit cardiac L-type Ca2+ channels; this antibody
recognizes the 190-kDa peptide of cardiac Ca2+ channels by
immunoblotting (12); and 2) Manc 1, a
monoclonal antibody raised against rabbit skeletal muscle microsomes
that has been previously characterized (1) and is known to
recognize the
2
-subunit of Ca2+ channels,
an epitope of which is located on the extracellular side of the plasma membrane.
Immunolabeling.
The cells were rinsed thoroughly in PBS and processed with a
centrifugation step between each stage of immunolabeling as described below. The cells were quenched for aldehyde groups in 0.1 M lysine and
blocked using 3% BSA-5% normal goat serum in PBS. If cells were to be
permeabilized, they were incubated in buffered 0.1% Triton X-100 for
10 min before quenching. They were then treated with the primary
antibody overnight (dilution 1:25 for Manc 1 and 1:100 for ccp5 ) at
4°C. After washing in PBS, the myocytes were split into two samples,
one for immunofluorescence labeling and one for immunogold labeling.
Immunofluorescence labeling for confocal microscopy was done by
treatment with biotinylated immunoglobulin and FITC-streptavidin (Amersham Life Sciences, Buckinghamshire, United Kingdom). Each step
was for 1 h at room temperature. After final washing in PBS, the
labeled cells were mounted using Vectashield mounting medium (Vector Labs).
Immunofluorescent-labeled samples were examined by confocal laser
scanning microscopy using a Leica TCS 4D or a Zeiss LSM 510 equipped
with an argon-krypton laser and fitted with the appropriate filter
blocks for the detection of fluorescein. Both single optical sections
and projection views from sets of up to 10-14 consecutive single
optical sections taken at 1.5- to 2.0-µm intervals were examined.
For electron microscopy, the cells labeled with Manc 1 or ccp5 were
treated with anti-mouse or anti-rabbit secondary antibody conjugated to
10-nm-diameter colloidal gold (BioCell International, Cardiff, United
Kingdom) for 1 h at room temperature. Control samples for both
confocal and electron microscopy were treated with normal mouse serum
in place of the primary antibody.
Myocytes were rinsed in PBS and then fixed with 2.5% glutaraldehyde in
PBS for 10 min. They were postfixed with 2% OsO4 for 1 h and, after rinsing, mixed with 18% BSA in PBS. After a few drops of 2.5% glutaraldehyde in PBS were added, the cell suspension became hardened and was cut into small pieces. The blocks were dehydrated and embedded in epoxy resin. Ultrathin sections were prepared and examined with a JOEL electron microscope.
For quantification of gold labeling, random electron micrographs were
taken (typically ×5,000-20,000) and scanned into a computer. With
the use of NIH Image software 1.61, the length of the plasma membrane
was measured, gold particles were counted (entered manually from the
keyboard), and the density of gold particles per unit length of plasma
membrane was calculated. To compare the distribution of gold labeling
between rabbit and rat, micrographs were collected from 10 rabbit and
10 rat ventricular cells (3 micrographs/cell) from different
experiments. Twenty micrographs that contained at least one peripheral
junctional SR element were collected from 18 rabbit ventricular cells
to examine gold particle density of the plasma membrane in relation to
adjacent junctional SR. The gold particles per unit area
(µm2) were also calculated to estimate background
labeling. Statistical analysis was by paired and unpaired
t-tests.
 |
RESULTS |
Manc 1 and ccp5 antibodies gave similar labeling patterns in each
animal. Use of the Manc 1 antibody does not require detergent treatment
of the specimen, so it is well suited for studying the distribution of
Ca2+ channels in well-preserved plasma membrane at the
ultrastructural level. By contrast, without detergent treatment, ccp5
antibody penetrates less readily to the cell interior.
Immunoconfocal localization.
Confocal microscopic examination consistently revealed distinctive
patterns of L-type Ca2+ channel localization between rabbit
and rat myocytes (Figs. 1 and
2). In rabbit myocytes,
prominent punctate immunofluorescence staining was observed in
both regularly arranged, transversely oriented striations and at the
surface plasma membrane (Figs. 1 and 3).
In rat myocytes, by contrast, the staining was found more intensely in
the regularly spaced transverse striations with little labeling of the
surface plasma membrane (Figs. 2 and 4). The spacing of the fluorescent striations in both rabbit and rat myocytes was 2 µm, corresponding to that of T tubules. The punctate labeling pattern within the striations was much less prominent in rat
than in rabbit myocytes. Higher magnification images clearly revealed
that, whereas the T tubular staining in the rabbit was in the form of
discrete spots, T tubular staining in the rat appeared as intense
continuous linear striations (Fig. 1B and Fig.
2B).

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Fig. 1.
A: immunoconfocal localization of L-type
Ca2+ channels in an isolated rabbit myocyte labeled with
Manc 1 antibody. The labeling is punctate with clearly defined spots at
the peripheral cell surface (arrows) and transverse striations
penetrating into cell. Bar = 10 µm. B: higher
magnification confocal image of a portion of a rabbit myocyte. Note
sharply defined spotlike staining at the cell surface and within
striations. Bar = 10 µm.
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Fig. 2.
A: immunoconfocal localization of L-type
Ca2+ channels in an isolated rat myocyte labeled with Manc
1 antibody. The labeling occurs as intense continuous striations within
the cell. Bar = 10 µm. B: higher magnification
confocal image of a portion of a rat myocyte. The staining is uniformly
distributed in the striations. A hint of labeling is found at the cell
periphery (arrows). Bar = 10 µm.
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Fig. 3.
A-D: selected images from a set of serial
optical sections taken at intervals of 1.7 µm. A rabbit cell labeled
with ccp5 antibody. The punctate fluorescence penetrates into the cell,
demonstrating that L-type Ca2+ channels form series of
discrete foci in the T tubule. The punctate fluorescence is also
irregularly distributed as spots at the cell surface (arrow in
A). Bar = 10 µm.
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Fig. 4.
A-D: selected images from a set of serial
optical sections taken at intervals of 2.0 µm. A rat cell labeled
with ccp5 antibody. The fluorescence is evenly distributed along the
length of the T tubules within the cell. Only faint labeling is
apparent at the cell surface (A). Bar = 10 µm.
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Optical sections taken parallel with the plane of the upper cell
surface allowed en face viewing of the surface staining, visualized as
irregularly distributed, sharply defined spots in rabbit myocytes (Fig.
3A). In contrast, the surface plane showed low-intensity
uniform staining in rat myocytes (Fig. 4A). As the serial
optical sections passed through the cell interior at progressively deeper levels, the punctate fluorescent striations in rabbit myocytes and continuous fluorescent striations in rat myocytes were confirmed to
penetrate into the cell at all planes, in register with the positions
of T tubules (Figs. 3 and 4). All cells in the suspension were
consistently well labeled in the distinctive patterns described. Controls showed no significant fluorescence.
In summary, L-type Ca2+ channel immunofluorescence staining
was demonstrated over the surface plasma membrane and in the T tubular membrane in rabbit myocytes, but was more extensive and intense in the
T tubular membrane than in the surface plasma membrane in rat myocytes,
suggesting that L-type Ca2+ channels are concentrated
predominantly in the T tubules in the rat.
Immunogold thin-section electron microscopy.
To determine more precisely the distribution of L-type Ca2+
channels, correlative immunoelectron microscopy was performed in rabbit
and rat myocytes. Gold label was consistently found on the plasma
membrane, located on the extracellular side of the plasma membrane
after labeling with the Manc 1 antibody.
By visual inspection, gold particles were frequently located on the
surface plasma membrane of rabbit ventricular myocytes (Fig.
5). In contrast, they were very sparsely
present on rat ventricular myocytes (Fig.
6). All rabbit cells examined exhibited the gold label; however, rat cells frequently showed no gold label over
the entire cell surface of a given section plane. Quantification of
gold particles on the surface membrane revealed that the number of
L-type Ca2+ channels was significantly greater in
rabbit than in rat myocytes (Table 1).

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Fig. 5.
A thin-sectioned immunolabeled rabbit cell showing
immunogold localization of L-type Ca2+ channels. The gold
particles are distributed in a nonrandom fashion on the surface plasma
membrane with some forming clusters consisting of several particles
(arrows). Bar = 200 nm.
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Fig. 6.
A thin-sectioned immunolabeled rat cell. Only a few gold
particles are found on the surface plasma membrane. Note a gold
particle is found over junctional sarcoplasmic reticulum (jSR) (arrow),
but no particles are present over other jSR elements (arrowhead).
Bar = 200 nm.
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In rabbit myocytes, the gold particles were distributed in a highly
nonrandom fashion over the surface plasma membrane, typically in the
form of clusters consisting of several particles (Fig. 5 and
Fig. 7). On occasion, they were
demonstrated to be located over peripheral junctional SR (Fig. 7),
though some clusters occurred independently of junctional SR (Fig. 5).
Quantification of gold particles over junctional SR and nonjunctional
SR was performed. The number of gold particles per unit membrane length
was significantly higher over junctional SR than over nonjunctional SR
(Table 2), indicating a higher density of
L-type Ca2+ channels over junctional SR than over
nonjunctional SR, especially bearing in mind that only 4.6% area of
the surface plasma membrane was associated with the junctional SR in
the rabbit (14).

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Fig. 7.
A high-magnification view of rabbit surface plasma
membrane. Gold particles (arrow) are located over jSR. Bar = 200 nm.
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In rat myocytes, a corresponding analysis was not feasible, owing to
the dearth of labeling of the plasma membrane (both in junctional SR
and nonjunctional SR regions) (Fig. 6).
Gold labeling was also apparent in T tubules of both rabbit and rat
myocytes (Fig. 8), but the level of
labeling was low and variable compared with the frequency of the
surface membrane labeling of rabbit. This apparent discrepancy between
the levels of labeling observed by immunoconfocal microscopy and
immunogold electron microscopy might be accounted for by poor
penetration of antibody-gold complex into the T tubules. Therefore,
accurate quantification of gold particles in T tubules was not
practicable since it could be expected to give unreliable
results. Nevertheless, gold particles were preferentially and
significantly located in T tubules compared with the rest of the cell
(excluding surface plasma membrane; 2.68 vs. 0 particles/µm2, P < 0.05, paired
t-test).

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Fig. 8.
Gold particles (arrow) are present and associated with T
tubular (T) membranes of rabbit (A) and rat (B)
myocytes. Bar = 200 nm.
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DISCUSSION |
The present findings provide a structural basis for understanding
species' differences in E-C coupling previously revealed in rabbit and
rat cardiac myocytes (2, 3, 9, 15, 16, 19, 24). We
specifically set out to determine whether L-type Ca2+
channels are distributed differently in the surface plasma membrane of
rabbits and rats in a manner that could account for species' differences in physiological and pharmacological properties. For this,
we used isolated cells. The isolated ventricular myocyte preparation
has advantages because viable Ca2+-tolerant cells can be
processed for immunolabeling without prior sectioning and with a
minimum of handling and processing, thereby minimizing artifacts. Also,
these cells exhibited normal Ca2+ currents, indicating the
high yield and maintained viability of the cell (5, 22).
Each whole cell can be optically sectioned from the cell surface to the
center of the cell by confocal microscopy. We also used two types of
antibodies for L-type Ca2+ channels. Manc 1 and ccp5
antibodies have different properties, the former being a monoclonal
against an extracellular epitope of the
2
-subunit and
the latter a polyclonal against an intracellular epitope of the
1-subunit of L-type Ca2+ channels. The
2
-subunit is ubiquitously expressed in all types of
high voltage-dependent Ca2+ channels and has extracellular
epitopes (11). The
1-subunit forms the pore
and contains binding sites for toxins, drugs, and the voltage sensors
(11) and is divided into six [more recently, 8 (6)] classes, including
1c (8,
11). The ccp5 antibody recognizes an intracellular domain of the
1-subunit of cardiac Ca2+ channels
(12). It is less advantageous for use in
immunocytochemistry because of the attendant requirement of detergent
treatment of cells to enable the antibody to gain access into the cell
interior; this inevitably results in defects of membrane structure,
especially at the ultrastructural level. The principal results by
immunogold electron microscopy in the present study were thus from
using Manc 1 antibody.
The confocal immunofluorescence studies convincingly show different
distribution patterns of L-type Ca2+ channels between
rabbit and rat ventricular myocytes. In the rabbit, L-type
Ca2+ channels are localized as discrete foci both in the
surface plasma membrane and T tubules, indicating that Ca2+
channels are organized in clusters. This distribution is similar to
that of guinea pig myocytes (18). By contrast, in the rat, Ca2+ channels seem to be located predominantly in T tubules
rather than surface sarcolemma, consistent with pharmacological studies (21) in which [3H]PN200-110 (a ligand
for dihydropyridine receptor) binding sites were reported to
be, on average, threefold more abundant in T tubules than in the
surface plasma membrane in adult rat ventricle. For CICR, the function
of L-type Ca2+ channels is coordinated with that of RyRs.
Thus a close spatial relationship of the two channels is predicted.
Classic ultrastructural studies (14) showed that the T
tubule membrane area associated with the junctional SR was much larger
in rat ventricular myocytes (48%) than in rabbit ventricular myocytes
(21%). The area of the surface plasma membrane associated with the
junctional SR was relatively lower (rat, 7.7%; rabbit, 4.6%). T
tubules were the predominant site of CICR in rat, playing a lesser role
in rabbit ventricular myocytes. We have shown that L-type
Ca2+ channels and RyRs are codistributed along the length
of T tubules in the rat by confocal immunofluorescence microscopy
(17). Also, immunofluorescence study has shown that both
proteins are codistributed in (and associated with) the T tubules and
in a few discrete regions of the surface plasma membrane in rabbit
ventricular cells (4).
Our immunogold electron microscopy showed that the L-type
Ca2+ channel labeling in the rabbit surface plasma membrane
occurs both in regions that overlie junctional SR and those that do
not. Quantitative analysis demonstrates a higher density of L-type Ca2+ channels in the plasma membrane overlying junctional
SR than in that overlying nonjunctional SR. This is consistent with a significant contribution of peripheral coupling to CICR in cardiac E-C
coupling. Even so, our findings imply that a greater area of plasma
membrane than that associated with junctional SR (4.6%) contains
L-type Ca2+ channels. Ca2+ entry via L-type
Ca2+ channels in the surface plasma membrane at
nonjunctional SR regions might contribute directly to myofilament
contraction in the rabbit. Together, the findings provide new
morphological insights into how Ca2+ entry may contribute
to activation of contraction to a greater extent in rabbit than in rat,
how the role of T tubules may predominate in E-C coupling in rat rather
than in rabbit, and why CICR is more prominent in rat than in rabbit
(21), as suggested by a large body of electrophysiological
and pharmacological work (2, 3, 9, 15, 16, 19, 24).
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FOOTNOTES |
Address for reprint requests and other correspondence: Y. Takagishi, Research Institute of Environmental Medicine, Nagoya Univ.,
Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan (E-mail:
taka{at}riem.nagoya-u.ac.jp).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 September 1999; accepted in final form 24 July 2000.
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