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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 alpha 2delta -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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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Table 1.   Comparison of gold labeling between rabbit and rat surface sarcolemma

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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Table 2.   Quantification of gold labeling in the rabbit surface sarcolemma

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 2delta -subunit and the latter a polyclonal against an intracellular epitope of the alpha 1-subunit of L-type Ca2+ channels. The alpha 2delta -subunit is ubiquitously expressed in all types of high voltage-dependent Ca2+ channels and has extracellular epitopes (11). The alpha 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 alpha 1c (8, 11). The ccp5 antibody recognizes an intracellular domain of the alpha 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).


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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