Journal of Histochemistry and Cytochemistry, Vol. 50, 311-324, March 2002, Copyright © 2002, The Histochemical Society, Inc.


ARTICLE

Heterogeneous Expression of Ca2+ Handling Proteins in Rabbit Sinoatrial Node

Hanny Musa1,a, Ming Lei1,b, Hauro Honjoc, Sandra A. Jonesa, Halina Dobrzynskia, Mathew K. Lancastera, Yoshiko Takagishic, Zaineb Hendersona, Itsuo Kodamac, and Mark R. Boyetta
a School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom
b University Laboratory of Physiology, University of Oxford, Oxford, United Kingdom
c Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan

Correspondence to: Mark R. Boyett, School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK. E-mail: m.r.boyett@leeds.ac.uk


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

We investigated the densities of the L-type Ca2+ current, iCa,L, and various Ca2+ handling proteins in rabbit sinoatrial (SA) node. The density of iCa,L, recorded with the whole-cell patch-clamp technique, varied widely in sinoatrial node cells. The density of iCa,L was significantly (p<0.001) correlated with cell capacitance (measure of cell size) and the density was greater in larger cells (likely to be from the periphery of the SA node) than in smaller cells (likely to be from the center of the SA node). Immunocytochemical labeling of the L-type Ca2+ channel, Na+-Ca2+ exchanger, sarcoplasmic reticulum Ca2+ release channel (RYR2), and sarcoplasmic reticulum Ca2+ pump (SERCA2) also varied widely in SA node cells. In all cases there was significantly (p<0.05) denser labeling of cells from the periphery of the SA node than of cells from the center. In contrast, immunocytochemical labeling of the Na+-K+ pump was similar in peripheral and central cells. We conclude that Ca2+ handling proteins are sparse and poorly organized in the center of the SA node (normally the leading pacemaker site), whereas they are more abundant in the periphery (at the border of the SA node with the surrounding atrial muscle). (J Histochem Cytochem 50:311–324, 2002)

Key Words: L-type Ca2+ channel, Na+-Ca2+ exchanger, ryanodine receptor, Ca2+ pump, sarcoplasmic reticulum


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

THE SINOATRIAL (SA) NODE is a heterogeneous tissue, and the action potential and pacemaker activity change from the periphery (at the border of the SA node with the surrounding atrial muscle) to the center (normally the leading pacemaker site). The heterogeneity is essential for the normal functioning of the SA node. It helps the SA node drive the surrounding atrial muscle and not be suppressed by it, it results in multiple pacemaker mechanisms appropriate for different conditions, and it helps prevent re-entrant arrhythmias involving the SA node (Boyett et al. 2000 ). A breakdown of the normal heterogeneity may be responsible for the functionl deterioration of the SA node with age (Zhang et al. 1998 ). According to the gradient model, the heterogeneity is the result of a gradient in the intrinsic properties of SA node cells from the periphery to the center. Ca2+ is important in the SA node. The L-type Ca2+ current, iCa,L, supports the action potential and pacemaker activity, and there is evidence that Ca2+ release from the sarcoplasmic reticulum (SR) is involved in pacemaking, e.g., by activating inward Na+-Ca2+ exchange current (Hata et al. 1996 ; Rigg and Terrar 1996 ; Li et al. 1997 ; Satoh 1997 ; Ju and Allen 1998 , Ju and Allen 1999 ; Rigg et al. 2000 ; Bogdanov et al. 2001 ). Models of the action potential in the periphery and center of the SA node predict that in the center the density of iCa,L should be ~30% of that in the periphery (Zhang et al. 2000 ). The aims of the study were to (a) determine whether the density of iCa,L and the expression of the L-type Ca2+ channel protein vary as predicted and (b) characterize the expression of other Ca2+ handling proteins (sarcolemmal Na+-Ca2+ exchanger, SR Ca2+ release channel, and SR Ca2+ pump), as well as a protein indirectly involved in Ca2+ handling (Na+-K+ pump). Evidence is presented for decreases in the density of all proteins, apart from the Na+-K+ pump, from the periphery to the center of the SA node.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

SA node cells were isolated as described by Lei and Brown 1996 from 500–1000-g New Zealand White rabbits. For the experiments shown in Fig 1, cells were isolated from throughout the SA node: from a piece of tissue ~1 mm (parallel to the crista terminalis) x 2 mm (perpendicular to the crista terminalis) roughly from the region within the red outline in Fig 4. For experiments shown in the remaining figures, cells were isolated separately from the periphery and centre of the SA node. A piece of tissue (like that above) was divided into three strips [peripheral (closest to the crista terminalis, transitional, and central (most distant from the crista terminalis)] and cells were isolated from the peripheral and central strips after carefully removing subepicardial atrial muscle. Rabbit ventricular and atrial cells were isolated using the Langendorff procedure as previously described by Linz and Meyer 1998 . Dissection of the intact SA node (shown in Fig 4) was carried out as previously described (Boyett et al. 1999 ). The intact SA node was embedded in 10% gelatin (diluted in 0.01 M PBS), frozen in isopentane using liquid N2, and stored at -80C before sectioning.



View larger version (18K):
[in this window]
[in a new window]
 
Figure 1. Correlation of iCa,L density and Cm. (A) Records of iCa,L from large and small cells (Cm of cells shown) elicited by depolarizing clamp pulses (bottom). (B) Current density–voltage relationships for iCa,L in groups of large (n=6) and small (n=6) cells (range of Cm of cells shown). Means ± SEM shown. (C) Plot of density of iCa,L at 0 mV against Cm for different cells. Filled circles, data from present study; open circles, data from rabbit SA node cells from Honjo et al. 1996 ; open squares, data from peripheral (Cm, 65 pF) and central (Cm, 20 pF) rabbit SA node cell models from Zhang et al. 2000 .



View larger version (23K):
[in this window]
[in a new window]
 
Figure 2. Labeling of L-type Ca2+ channel. (A) Ventricular cell; (B) atrial cell; (C) peripheral SA node cell. (D,E) Central SA node cells. The nucleus is stained red by propidium iodide in some panels in this and other figures.



View larger version (57K):
[in this window]
[in a new window]
 
Figure 3. Labeling of Na+-Ca2+ exchanger. (A) Ventricular cell (B) atrial cell; (C) peripheral SA node cell (D) central SA node cell. (E–G) Sections through crista terminalis (E), periphery (F) and center (G) of the SA node. (H) Section cut perpendicular to the crista terminalis labeled for Cx43. RA, right atrial appendage; CT, crista terminalis; endo, endocardium; epi, epicardium; SEP, interatrial septum.



View larger version (112K):
[in this window]
[in a new window]
 
Figure 4. Photograph of SA node preparation showing positions of the Cx43 (red outline) and Na+-Ca2+ exchange (green outline) negative regions SVC, superior vena cava; SEP, interatrial septum; IVC, inferior vena cava; RA, right atrial appendage; CT, crista terminalis.

The whole-cell patch-clamp technique was used for the recording of iCa,L from single SA node cells with amphotericin-permeabilized patches. An Axopatch-1C amplifier, a Digidata1200A, and pCLAMP software (Axon Instruments; Union City, CA) were used. Pipettes had a tip diameter of ~1–2 µm and a resistance of 3–8 M{Omega}. Pipette solution contained (in mM): 140 KCl, 1.8 MgSO4, 5 HEPES, 1 EGTA, pH 7.4, KOH, amphotericin 200 µg/ml. Cm was obtained from the capacity compensation control of the amplifier. In a previous study (Honjo et al. 1996 ) the accuracy of this method was checked. The series resistance was electronically compensated (>80%) and the current signal was filtered by a low-pass Bessel filter with a cutoff frequency of 5 kHz (-3 dB). Cells were superfused with Tyrode's solution (in mM: 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 glucose, 5 HEPES, pH 7.4, NaOH) at ~1 ml/min at 35C. Three hundred µM lidocaine was added to Tyrode's solution to block the Na+ current, iNa, and 5 µM E-4031 (Eisai Pharmaceuticals; Tokyo, Japan) was added to block the rapid delayed rectifier K+ current, iK,r. It was confirmed that iCa,L could be blocked by 500 nM nisoldipine (Bayer; Newbury, UK). Lidocaine was dissolved in distilled water and ethanol (50:50), E-4031 was dissolved in distilled water, and nisoldipine was dissolved in ethanol to make 10 mM stock solutions.

Immunocytochemistry experiments were carried out using established methods. Single cells were plated on Bunsen burner flame-treated coverslips and allowed to settle for 30 min. From the intact SA node (Fig 4), 18–20 µm tissue sections were cut perpendicular to the crista terminalis through the crista terminalis and intercaval region. Cells and tissue were fixed in 2% paraformaldehyde and washed three times with 0.01 M PBS. Cells and tissue were permeabilized by incubating them in PBS containing 0.1% Triton X-100 for 30 min, washed with PBS, and then blocked in 10% normal donkey serum in PBS for 1 hr. After washing three times with PBS, cells and tissue were incubated with rabbit polyclonal anti-L-type Ca2+ channel {alpha}1C-subunit (anti-CNC1 peptide from Snutch et al. 1991 ; 1:200; provided by Alomone Labs, Jerusalem, Israel), mouse monoclonal anti-Na+-Ca2+ exchanger (clone C2C12; Frank et al. 1992 ; 1:100; provided by Affinity Bioreagents, Golden, CO), mouse monoclonal anti-Na+-K+ pump {alpha}1-subunit (antibody {alpha}6F from Lebovitz et al. 1989 ; 1:20; provided by Developmental Studies Hybridoma Bank, University of Iowa, Ames, IA), mouse monoclonal anti-RYR2 (antibody AE 8.91 from Lewis Carl et al. 1995 ; 1:100; gift from G. Meissner), mouse monoclonal anti-SERCA2 (from Jorgensen et al. 1988 ; cells 1:6000, tissue 1:12000; provided by Affinity Bioreagents), or mouse monoclonal anti-connexin43 antibody (amino acids 252–270; Cx43 1:1000, provided by Chemi-con, Harrow, UK) overnight at 4C. The slides were brought to room temperature, washed three times in PBS, and incubated with secondary antibody, donkey anti-rabbit IgG (1:100) or donkey anti-mouse IgG (1:100) conjugated to FITC (Chemicon) for 1 hr. After washing three times in PBS, coverslips were mounted on microscope slides, sealed with nailpolish, and stored in the dark at 4C for subsequent viewing in a Leica TCS SP laser scanning confocal microscope. All images presented are single optical sections. In the case of single cells, the images were taken approximately midway through the depth of the cell. In the case of tissue sections, neighboring pairs of sections were labeled, one for Cx43 and one for the protein of interest. The presence or absence of Cx43 label was used as a marker, as described in the Results. No labeling above background was obtained when the primary or secondary antibodies were omitted (data not shown). When primary antibody was replaced with normal mouse or rabbit serum (Burry 2000 ) there was also no labeling above background (data not shown). Image analysis was carried out using Scion Image (NIH; Bethesda, MD).

All results are presented as means ± SEM (number of cells). Statistical significance was determined by ANOVA. Linear regression analysis was used for correlations. p<0.05 was considered to indicate a significant difference. All statistical analysis was carried out using SigmaStat software (Jandel Scientific; Chicago, IL).


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Correlation Between Density of iCa,L and Cm
Experiments were carried out only on cells showing spontaneous activity. They were spindle- and/or spider-shaped, with no obvious or faint striations. Cells were isolated from the whole of the SA node and distinguished on the basis of size as measured by Cm (large cells tend to be from the periphery and small cells tend to be from the center). iCa,L was elicited by 500-msec depolarizing clamp pulses to various test potentials from a holding potential of -50 mV at 1 Hz (Fig 1A). Fig 1A shows records of iCa,L from large and small cells and Fig 1B shows current–voltage relationships for two groups of cells: large cells with Cm >30 pF and small cells with Cm <30 pF (we have classified cells in a similar manner previously (Honjo et al. 1996 , Honjo et al. 1999 ; Lei et al. 2001 ). The mean density of peak inward current (peak inward current normalized by Cm for each cell) is plotted against the test potential. The density of iCa,L was significantly less in the smaller cells (p<0.01). The filled symbols in Fig 1C show the density of iCa,L in 12 cells at 0 mV plotted against Cm. There is a significant correlation between the density of iCa,L and Cm in the 12 cells (p<0.001; r2 = 0.86). This suggests that in the center of the SA node the density of iCa,L is less than in the periphery. The other symbols in Fig 1C are considered in the Discussion.

Sarcolemmal Ca2+ Handling Proteins
The abundance of proteins in the periphery and center of the SA node was assessed by immunocytochemistry. Fig 2, Fig 3, and Fig 5 Fig 6 Fig 7 show labeling of various cell types (ventricular, atrial, peripheral SA node, central SA node) with various antibodies. Ventricular and atrial cells were labeled as a reference. Rather than isolating cells from the whole of the SA node, cells were separately isolated from tissue taken from the periphery and center of the SA node.



View larger version (68K):
[in this window]
[in a new window]
 
Figure 5. Labeling of Na+-K+ pump. (A) Ventricular cell; (B) atrial cell; (C) peripheral SA node cell; (D) central SA node cell. (E–G) Sections through crista terminalis (E), periphery (F) and center (G) of the SA node. (H) Diagram of section cut perpendicular to the crista terminalis labeled for Cx43 (black, label; white, no label). Abbreviations as in Fig 3.



View larger version (64K):
[in this window]
[in a new window]
 
Figure 6. Labeling of RYR2. (A) Ventricular cell; (B) atrial cell; (C,D) peripheral SA node cells; (E) central SA node cell. (F–I) Sections through right atrial appendage (F), crista terminalis (G), and periphery (H) and center (I) of the SA node. (J) Diagram of section cut perpendicular to the crista terminalis labeled for Cx43 (black, label; white, no label). Abbreviations as in Fig 3.



View larger version (48K):
[in this window]
[in a new window]
 
Figure 7. Labeling of SERCA2. (A) Ventricular cell; (B) atrial cell; (C) peripheral SA node cell; (D) central SA node cell. (E–H) Sections through right atrial appendage (E), crista terminalis (F), and periphery (G) and center (H) of the SA node. Arrow in F highlights an example of labeling of the nuclear envelope. (I) Diagram of section cut perpendicular to the crista terminalis labeled for Cx43 (black, label; white, no label). Abbreviations as in Fig 3.

Fig 2 shows labeling with an antibody to the L-type Ca2+ channel {alpha}1C-subunit. In ventricular cells there was labeling of the outer cell membrane in some cells and internal striated punctate labeling with a periodicity of 1.73 ± 0.04 µm (n=5) (Fig 2A), corresponding to L-type Ca2+ channels in the outer cell membrane and t-tubules, respectively. In atrial cells there was punctate labeling of the outer cell membrane (Fig 2B). In some atrial cells (Fig 2B) there was limited punctate labeling in the cell interior, possibly corresponding to L-type Ca2+ channels in poorly developed t-tubules. Although atrial cells are widely considered not to have t-tubules, some rat atrial cells are reported to have well or poorly developed t-tubules (Forssman and Girardier 1970 ) and guinea pig atrial cells are reported to have short t-tubules (Forbes and Van Niel 1988 ). In peripheral SA node cells there was punctate labeling of the outer cell membrane and little or no internal labeling (Fig 2C), whereas in central cells there was little or no detectable labeling (Fig 2D and Fig 2E).

Fig 3 shows labeling with an antibody to the Na+-Ca2+ exchanger. In ventricular cells there was labeling of the outer cell membrane as well as internal striated (periodicity 1.87 ± 0.02 µm; n=5) punctate labeling (Fig 3A), corresponding to Na+-Ca2+ exchanger in the outer cell membrane and t-tubules, respectively. In atrial cells there was labeling of the outer cell membrane only (Fig 3B). In peripheral SA node cells there was also labeling of the outer cell membrane (Fig 3C), whereas in central cells there was little or no labeling (Fig 3D).

The Na+-Ca2+ exchanger was also labeled in tissue sections. In the case of tissue sections, pairs of neighboring sections were labeled, one for the gap junction protein connexin43 (Cx43) and one for the protein of interest. Cx43 was used as a marker. Fig 3H shows a tissue section through the thick crista terminalis and thin intercaval region labeled for Cx43. The center of the SA node is known to be located in the intercaval region adjacent to the crista terminalis. In Fig 3H (arrow G) this region lacks green labeling; it is well known that the center of the SA node lacks Cx43 (e.g., Coppen et al. 1999 ). From the intercaval region, SA node tissue rises up the endocardial face of the crista terminalis, where it terminates (Kodama and Boyett 1985 ). The SA node tissue on the crista terminalis is the periphery of the SA node (Fig 3H, arrow F) (Kodama and Boyett 1985 ). The peripheral SA node tissue is labeled green in Fig 3H—the periphery is known to contain Cx43 (Coppen et al. 1999 )—and it is separated by connective tissue from the bulk of the crista terminalis. The bulk of the crista terminalis is atrial muscle and in Fig 3H (arrow E) is also labeled green; atrial muscle also contains Cx43 (e.g. Coppen et al. 1999 ). Fig 3E–3G show labeling of the atrial muscle of the crista terminalis (Fig 3E) and the periphery (Fig 3F) and center (Fig 3G) of the SA node by the antibody to the Na+-Ca2+ exchanger in the neighboring tissue section (Fig 3E–3G approximately correspond to the locations marked by the arrows E to G in Fig 3H). Whereas there was clear labeling of the outer cell membrane of the atrial cells (Fig 3E), there was little labeling of cells in the periphery or center of the SA node (Fig 3F and Fig 3G). In two SA node preparations the extent of the region lacking labeling of the Na+-Ca2+ exchanger was mapped. Fig 4 summarizes data from one preparation. Fig 4 shows a photograph of a preparation. Multiple sections (like that in Fig 3H) were cut perpendicular to the crista terminalis and labeled for either Cx43 or the Na+-Ca2+ exchanger. The red outline in Fig 4 shows the extent of the Cx43-negative region, whereas the green outline shows the extent of the region lacking labeling of the Na+-Ca2+ exchanger. Similar data were obtained from the second preparation.

Fig 5A shows that in ventricular cells there was little labeling by an antibody to the Na+-K+ pump of the outer cell membrane, except at the intercalated discs. In addition, there was internal striated (periodicity 1.91 ± 0.03 µm; n=5) punctate labeling of t-tubules. Fig 5B shows that in atrial cells there was labeling of the outer cell membrane, particularly at intercalated discs. Whether the increase in labeling at the intercalated disc is the result of a high density of the Na+-K+ pump (per unit area of membrane) at this location or the membrane folding at the intercalated disc is not known. In peripheral (Fig 5C) and central (Fig 5D) SA node cells, the pattern of labeling was similar to that of atrial cells. Fig 5E–5G show that in tissue sections there was labeling of the outer cell membrane of atrial cells of the crista terminalis (Fig 5E) and of peripheral (Fig 5F) and central (Fig 5G) SA node cells. The density of labeling was similar in the three regions, in accordance with the data from single cells. Fig 5H summarizes the labeling of Cx43 in the neighboring section. The approximate locations of the images shown in Fig 5E–5G are shown by arrows E–G.

SR Ca2+ Handling Proteins
In ventricular cells, there was internal striated (periodicity 1.83 ± 0.03 µm; n=5) punctate labeling by an antibody to the SR Ca2+ release channel, anti-RYR2 (Fig 6A), corresponding to junctional SR (JSR–SR Ca2+ release site) adjacent to t-tubules. In atrial cells there was labeling adjacent to the outer cell membrane (Fig 6Bi), corresponding to JSR adjacent to the outer cell membrane, as well as internal striated (periodicity 1.94 ± 0.01 µm; n=5) labeling (Fig 6Bii), corresponding to extended JSR (or corbular SR). Extended JSR displays all the anatomic features of JSR but is not associated with the outer cell membrane or t-tubules and is organized at the level of the z-line (Forbes and Van Niel 1988 ; Lewis Carl et al. 1995 ). The internal labeling was separated by a gap from the labeling adjacent to the outer cell membrane. In some peripheral SA node cells there was a similar pattern of labeling as in atrial cells, with both labeling adjacent to the outer membrane (Fig 6Ci) and internal labeling (periodicity 1.86 ± 0.03 µm; n=5; Fig 6Cii). However, in other peripheral SA node cells, punctate labeling adjacent to the outer cell membrane (Fig 6Di) was observed together with random spots of internal labeling (Fig 6Dii). In central SA node cells, there was only punctate labeling adjacent to the outer cell membrane. There was no internal labeling above background intensity (Fig 6E). Fig 6 also shows labeling by anti-RYR2 in tissue sections. In atrial cells in longitudinal section (Fig 6F, from right atrial appendage) there was labeling adjacent to the outer cell membrane and internal striated (periodicity 1.87 ± 0.05 µm; n=5) labeling. In atrial cells in cross-section (Fig 6G, from the crista terminalis) a ring of labeling surrounding (but separated by a gap from) labeling of the cell interior was observed. In the middle of cells there was often no labeling (presumably corresponding to the nucleus). In the center of the SA node there appeared to be labeling adjacent to the outer cell membrane only. There was no evidence of internal labeling (Fig 6I). In the periphery of the SA node two types of labeling were observed: labeling similar to that of atrial cells (labeling adjacent to outer cell membrane and internal labeling; Fig 6Hi) and labeling similar to that of central SA node cells (labeling adjacent to outer cell membrane and no internal labeling; Fig 6Hii). These data are in accordance with the data from single cells. Fig 6J summarizes the labeling of Cx43 in the neighboring section; the approximate locations of the images shown in Fig 6G–6I are shown by arrows G–I.

In ventricular cells there was labeling by the antibody to the SR Ca2+ pump, anti-SERCA2, adjacent to the outer cell membrane, as well as internal striated (periodicity 1.89 ± 0.02 µm; n=5) labeling and rings of labeling around the nuclei (Fig 7A). This corresponds to network SR (SR Ca2+ uptake site, separate from JSR) adjacent to the outer cell membrane, network SR adjacent to the t-tubules, and the nuclear envelope, respectively. In atrial cells, a similar pattern of labeling was observed (Fig 7B). The internal striated (periodicity 1.93 ± 0.01 µm; n=5) labeling corresponds to network SR adjacent to the z-lines (site of extended JSR) in this case. In peripheral SA node cells there was internal labeling. However, the internal labeling was not striated. Instead, there was random and diffuse labeling of the cell interior (Fig 7C). There was also labeling of the nuclear envelope in peripheral cells. In central SA node cells there was a similar pattern of labeling to that in peripheral cells, but the density of labeling was less (Fig 7D). In tissue sections, in longitudinally or transversely sectioned atrial cells (Fig 7E and Fig 7F), some labeling adjacent to the outer cell membrane, internal labeling (striated in longitudinally sectioned cells; periodicity 2.00 ± 0.03 µm; n=5) and labeling of the nuclear envelope (e.g., Fig 7F, arrow) could be discerned. In peripheral SA node tissue the pattern of labeling was similar to that in atrial tissue (Fig 7G), whereas in central tissue (Fig 7H) less labeling was discernible (apart from clear labeling of the nuclear envelope). Fig 7I summarizes the labeling of Cx43 in the neighboring section. The approximate locations of the images shown in Fig 7F–7H are shown by arrows F–H.

Summary of Single Cell Labeling
With single cells, image analysis was used to measure the total area of labeling within an optical section, approximately midway through the depth of a cell, as well as the total area of the cell in the optical section, from which the density of labeling (percentage of cell area labeled) was calculated. Fig 8A shows that, as expected central SA node cells were significantly smaller in area than peripheral SA node cells. For all cell types, cell area of ventricular cells>atrial cells>peripheral SA node cells>central SA node cells. Fig 8B shows that the density of labeling by the antibody to the L-type Ca2+ channel {alpha}1C-subunit was high in ventricular, atrial, and peripheral SA node cells, but significantly lower in central SA node cells. This finding concerning peripheral and central SA node cells is consistent with the electrophysiological data. Fig 8C shows that the density of labeling by the antibody to the Na+-Ca2+ exchanger of ventricular cells>atrial cells{approx}peripheral SA node cells>central SA node cells. Fig 8D shows that the density of labeling by the antibody to the Na+-K+ pump was significantly higher in ventricular cells than in the other cell types, but not significantly different among atrial and peripheral and central SA node cells. Fig 8E shows that the density of labeling by anti-RYR2 was high in ventricular, atrial, and peripheral SA node cells and significantly less in central SA node cells. Fig 8F shows that the density of labeling by anti-SERCA2 in the central SA node cells was significantly less than in atrial and peripheral SA node cells.



View larger version (29K):
[in this window]
[in a new window]
 
Figure 8. Density of labeling of Ca2+ handling proteins. (A–F) Cell area (A) and density of labeling of L-type Ca2+ channel (B), Na+-Ca2+ exchanger (C), Na+-K+ pump (D), RYR2 (E), and SERCA2 (F) in ventricular, atrial, peripheral SA node, and central SA node cells. Means ± SEM shown. n numbers are shown below bars and statistical differences shown above bars. V, ventricular; A, atrial; P, peripheral SA node; C, central SA node; v, versus.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The present data suggest that the L-type Ca2+ channel, Na+-Ca2+ exchanger, RYR2, and SERCA2 are less abundant in the center of the SA node than in the periphery, whereas the Na+-K+ pump is equally abundant in the two regions. The present data also show that the density of iCa,L is less in smaller cells. In the rabbit and other species, it is known that cells in the center of the SA node are small, whereas cells in the periphery are large (Boyett et al. 2000 ). Taken together, these findings suggest that the density of iCa,L is less in the center of the SA node than in the periphery. This is consistent with the evidence of a lower abundance of the L-type Ca2+ channel in the center than in the periphery.

Validity of Data
In the study of iCa,L, cells were isolated from the whole of the SA node and the cells were subsequently classified according to cell size (Fig 1). Previous studies suggest that the assumption that cell size is an indicator of the site of origin of a cell is reasonable (Honjo et al. 1996 , Honjo et al. 1999 ; Boyett et al. 2000 ; Lei et al. 2000 ; Zhang et al. 2000 ). Classification of cells according to Cm has a number of advantages compared to isolating cells from the periphery and center of the SA node. For example, (a) it is technically straightforward, whereas the isolation of cells from the periphery and center is difficult, and (b) it is effectively continuous (depends only on resolution of measurement of Cm), whereas we have only managed to isolate cells from just two regions of the SA node.

In the present study, ventricular and atrial cells were labeled as a point of reference to check that the labeling pattern seen is consistent with previously published data. In the present study, labeling of the L-type Ca2+ channel and RYR2 in ventricular and atrial cells was similar to that of rabbit ventricle and atrium by Lewis Carl et al. 1995 , and labeling of the Na+-Ca2+ exchanger in ventricular and atrial cells was similar to that of rabbit ventricular cells by Chen et al. 1995 and guinea pig atrial cells by McDonald et al. 2000 . On the basis of Western blotting, the density of the Na+-Ca2+ exchanger has previously been reported in guinea pig right atrium to be 42% of that in right ventricle (McDonald et al. 2000 ), consistent with the data in Fig 8C (see also Wang et al. 1996 ). Lancaster et al. 2000a (also M.K. Lancaster, personal communication) observed similar labeling of the Na+-K+ pump in rabbit ventricle to that observed in ventricular cells in the present study. The Na+-K+ pump in atrial cells has not been labeled previously. Other types of measurement suggest that the density of the Na+-K+ pump is less in the atria than in the ventricles (Wang et al. 1996 ), consistent with Fig 8D. SERCA2 in both ventricular and atrial cells has not been labeled previously. In the present study, single cells and tissues sections were labeled in most cases. The subcellular location of a protein can be better determined with single cells, whereas the position of cells within the SA node is known with greater certainty in the case of tissue sections. The labeling pattern of single cells and tissue sections was similar in the case of labeling of the Na+-K+ pump, RYR2, and SERCA2. In the case of labeling of the Na+-Ca2+ exchanger, the data from single cells and tissue sections agree in showing less labeling of central SA node cells than of atrial cells (Fig 3). However, in tissue sections no labeling of the Na+-Ca2+ exchanger was observed in either the periphery or the center of the SA node whereas, although no labeling was observed in single cells from the center, labeling was observed in single cells from the periphery (Fig 3). This discrepancy could be the result of better access of the antibody to the antigenic site in the case of single cells. In the present study there was little or no labeling of either the L-type Ca2+ channel or the Na+-Ca2+ exchanger in central SA node cells (Fig 2 and Fig 3). This does not mean that the two proteins are absent from the center of the SA node. It is more likely that the density of the proteins in the center is below the detection limit of immunocytochemistry.

Comparison with Previous Studies
In a previous study of rabbit SA node cells, we failed to observe a correlation between iCa,L density and Cm (Honjo et al. 1996 ). The data from our previous study (with the exception of one point) are shown by the open circles in Fig 1C. However, when we subsequently developed models of action potentials in rabbit peripheral and central SA node cells, with Cm of 65 and 20 pF, respectively, the iCa,L density in the central model cell had to be set at ~30% of that in the peripheral model cell (Zhang et al. 2000 ). The iCa,L densities chosen for the peripheral and central model cells are shown by the open squares in Fig 1C. The discrepancy between experiment and model prompted the present re-examination of the relationship between iCa,L density and Cm. The new data (Fig 1C, filled circles) are consistent with the old data. There is a significant correlation between iCa,L density and Cm, regardless of whether the new data are considered alone (p<0.001) or whether the new and old data are considered together (with or without the point excluded in Fig 1C; p<0.001). The reason why no significant correlation was observed with the original data set is that the range of Cm investigated was not sufficient. The iCa,L densities observed in large and small cells in experiments are comparable to the predicted iCa,L densities from the peripheral and central models (Fig 1C). There are also significant correlations between the densities of iNa, 4-AP-sensitive current, iK,r, slow delayed rectifier K+ current (iK,s), and hyperpolarization-activated current (if) and Cm in rabbit SA node cells (Honjo et al. 1996 , Honjo et al. 1999 ; Lei et al. 2000 , Lei et al. 2001 ).

In various species, including rabbit, in the center of the SA node the cells have been observed by electron microscopy to be "empty," principally because they lack well-ordered myofilaments (Masson-Pevet et al. 1979 ; Boyett et al. 2000 ). In the rabbit at least, there is a progressive decrease in the density of myofilaments from the periphery to the center (Boyett et al. 2000 ). As well as lacking myofilaments, electron microscopy shows that cells in the center of the SA node in the rabbit also contain little SR (about a third of that in atrial cells) (Masson-Pevet et al. 1979 ). The data from the present study are consistent with these findings and provide further information on the organization of the SR in SA node cells. In ventricular cells, SR is known to be abundant and organized in phase with the t-tubules, and this is confirmed by the distribution of the two SR proteins, RYR2 and SERCA2, in this (Fig 6 and Fig 7) and other studies (RYR2; Lewis Carl et al. 1995 ). In atrial cells, SR is also known to be abundant and well organized. Within atrial cells, despite the general lack of well developed t-tubules, SR is organized in a periodic fashion (in phase with the myofilaments) (Forbes and Van Niel 1988 ). RYR2 and SERCA2 labeling, in this (Fig 6 and Fig 7) and other studies (RYR2; Lewis Carl et al. 1995 ), confirms the extended JSR and network SR to be organized in a striated fashion in atrial cells. In atrial cells, in addition to the transversely organized SR, there is SR adjacent to the outer cell membrane, as judged by RYR2 labeling (Fig 6); this is JSR. In contrast to the abundant well-organized SR in ventricular and atrial cells, the SR in SA node cells, as judged by RYR2 and SERCA2 labeling, is sparse and poorly organized (Fig 6 and Fig 7). RYR2 labeling is principally found adjacent to the outer cell membrane (Fig 6), whereas SERCA2 is randomly scattered throughout the cytoplasm (Fig 7).

In contrast to cell size, densities of myofilaments, SR, iNa, 4-AP-sensitive current, iK,r, iK,s, if, iCa,L, L-type Ca2+ channel, Na+-Ca2+ exchanger, RYR2, and SERCA2 (this study and Masson-Pevet et al. 1979 ; Bleeker et al. 1980 ; Honjo et al. 1996 , Honjo et al. 1999 ; Lei et al. 2000 , Lei et al. 2001 ), the density of the Na+-K+ pump does not appear to decrease from the periphery to the center of the rabbit SA node (Fig 5). All of the features that decrease from the periphery to the center are associated with "excitability"—perhaps the Na+-K+ pump does not conform to the general rule because it is also required for vegetative functions of a cell.

Physiological Importance
In the center of the SA node, iCa,L is solely responsible for generation of the action potential, whereas in the periphery iNa is principally responsible (although in the periphery iCa,L is responsible for the action potential plateau as it is in the center), and in this respect it is paradoxical that the density of iCa,L is less in the center than in the periphery. However, in the center various current densities are lower than in the periphery, as explained above, and there is no need for the density of iCa,L to be as high as in the periphery for iCa,L to be able to generate the action potential. In the models of action potentials in the periphery and center of the rabbit SA node (Zhang et al. 2000 ), if the density of iCa,L in the central cell model is set to be the same as in the peripheral cell model, the action potential amplitude and duration become too great and spontaneous activity becomes too slow (as a consequence of the increase in action potential duration).

The regional differences in Ca2+ handling proteins within the SA node are expected to result in regional differences in Ca2+ handling in the SA node. Prompted by the results from the present study, we have recently measured intracellular Ca2+ transients in rabbit SA node cells. In cells isolated from the whole of the rabbit SA node, there were significant correlations between the amplitude of the Ca2+ transient, the peak systolic Ca2+ concentration, the diastolic Ca2+ concentration, the duration of the Ca2+ transient, time to peak of the Ca2+ transient, and decay time of the Ca2+ transient with cell size (Lancaster et al. 2000b ). Ca2+ concentrations were less and kinetic parameters were slower in small cells. In addition, ryanodine significantly increased the duration and reduced the amplitude of the Ca2+ transient in large cells but not in small cells (Lancaster et al. 2000b ). These results are consistent with the results from the present study. The paucity of L-type Ca2+ channel, RYR2, and SERCA2 in smaller central cells is expected to result in a smaller Ca2+ transient. The paucity of RYR2 in smaller central cells is expected to result in a prolongation of the time to peak of the Ca2+ transient (because the Ca2+ transient will be the result of Ca2+ entry via the L-type Ca2+ channel). The paucity of SERCA2 and the Na+-Ca2+ exchanger in smaller central cells is expected to result in a prolongation in the relaxation of the Ca2+ transient. The paucity of RYR2 in smaller central cells is expected to result in a decrease in ryanodine sensitivity.

Various studies have shown that Ca2+ release from the SR plays an important role in pacemaking (by modulating iCa,L, Na+-Ca2+ exchange current, delayed rectifier K+ current, and if) in various species, including rabbit (Hata et al. 1996 ; Rigg and Terrar 1996 ; Li et al. 1997 ; Satoh 1997 ; Ju and Allen 1998 , Ju and Allen 1999 ; Bogdanov et al. 2001 ). The present data suggest that intracellular Ca2+ may be handled differently from the periphery to the center and, therefore, the role of intracellular Ca2+ in pacemaking may also vary. In support of this, in the study of Lancaster et al. 2000b , ryanodine significantly slowed pacemaking in large but not in small cells.

Gradient vs Mosaic Models of SA Node
In addition to the gradient model of the SA node, a mosaic model has been proposed in which the regional differences in electrical activity of the SA node are not the result of a gradient in the intrinsic properties of SA node cells from the periphery to the center. Instead, they are the result of variation in the proportions of atrial and SA node cells from the periphery to the center (Verheijck et al. 1998 ). The present data are in accord with the gradient model rather than the mosaic model, in that they show that the density of iCa,L changes with Cm and in terms of cell size, and the densities of the L-type Ca2+ channel, Na+-Ca2+ exchanger, RYR2, and SERCA2 differing between peripheral and central SA node cells. Furthermore, the present data provide no evidence of two cell populations in the periphery or center of the SA node (with the exception of two patterns of RYR2 labeling in peripheral cells; Fig 6). We have recently shown that the gradient model, but not the mosaic model, can explain the electrical properties of SA node tissue (Zhang et al. 2001 ).


  Footnotes

1 These authors made an equal contribution to the study.


  Acknowledgments

Supported by the British Heart Foundation, the Ministry of Education, Science and Culture of Japan, and the Japan Society for the Promotion of Science.

Received for publication May 22, 2001; accepted October 10, 2001.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Bleeker WK, Mackaay AJC, Masson–Pévet M, Bouman LN, Becker AE (1980) Functional and morphological organization of the rabbit sinus node. Circ Res 46:11-22[Abstract]

Bogdanov KY, Vinogradova TM, Lakatta EG (2001) Sinoatrial nodal cell ryanodine receptor and Na+-Ca2+ exchanger: molecular partners in pacemaker regulation. Circ Res 88:1254-1258[Abstract/Free Full Text]

Boyett MR, Honjo H, Kodama I (2000) The sinoatrial node, a heterogeneous pacemaker structure. Cardiovasc Res 47:658-687[Medline]

Boyett MR, Honjo H, Yamamoto M, Nikmaram MR, Niwa R, Kodama I (1999) A downward gradient in action potential duration along the conduction path in and around the sinoatrial node. Am J Physiol 276:H686-698[Abstract/Free Full Text]

Burry RW (2000) Specificity controls for immunocytochemical methods. J Histochem Cytochem 48:163-166[Abstract/Free Full Text]

Chen F, Mottino G, Klitzner TS, Philipson KD, Frank JS (1995) Distribution of the Na+/Ca2+ exchange protein in developing rabbit myocytes. Am J Physiol 268:C1126-1132[Abstract/Free Full Text]

Coppen SR, Kodama I, Boyett MR, Dobrzynski H, Takagishi Y, Honjo H, Yeh H-I, Severs NJ (1999) Connexin45, a major connexin of the rabbit sinoatrial node, is co-expressed with connexin43 in a restricted zone at the nodal-crista terminalis border. J Histochem Cytochem 47:907-918[Abstract/Free Full Text]

Forbes MS, Van Niel EE (1988) Membrane systems of guinea-pig myocardium: ultrastructure and morphometric studies. Anat Rec 222:362-379[Medline]

Forssman WG, Girardier L (1970) A study of the t system in rat heart. J Cell Biol 44:1-19[Abstract/Free Full Text]

Frank JS, Mottino G, Reid D, Molday RS, Philipson KD (1992) Distribution of the Na+-Ca2+ exchange protein in mammalian cardiac myocytes: an immunofluorescence and immunocolloidal gold-labeling study. J Cell Biol 117:337-345[Abstract]

Hata T, Noda T, Nishimura M, Watanabe Y (1996) The role of Ca2+ release from sarcoplasmic reticulum in the regulation of sinoatrial node automaticity. Heart Vessels 11:234-241[Medline]

Honjo H, Boyett MR, Kodama I, Toyama J (1996) Correlation between electrical activity and the size of rabbit sinoatrial node cells. J Physiol 496:795-808[Abstract]

Honjo H, Lei M, Boyett MR, Kodama I (1999) Heterogeneity of 4-aminopyridine sensitive current in rabbit sinoatrial node cells. Am J Physiol 276:H1295-1304[Abstract/Free Full Text]

Jorgensen AO, Arnold W, Pepper DR, Kohl SD, Mandel F, Campbell KP (1988) A monoclonal antibody to the Ca2+-ATPase of cardiac sarcoplasmic reticulum cross reacts with slow type I but not fast type II canine skeletal muscle fibres. An immunocytochemical and immunochemical study. Cell Motil Cytoskel 9:164-174[Medline]

Ju Y-K, Allen DG (1998) Intracellular calcium and Na2+-Ca2+ exchange current in isolated toad pacemaker cells. J Physiol 508:153-166[Abstract/Free Full Text]

Ju Y-K, Allen DG (1999) How does ß-adrenergic stimulation increase the heart rate? The role of intracellular Ca 2+(release in amphibian pacemaker cells. J Physiol 516):793-804

Kodama I, Boyett MR (1985) Regional differences in the electrical activity of the rabbit sinus node. Pflugers Arch 404:214-226[Medline]

Lancaster MK, Bennett DL, Cook SJ, O'Neill SC (2000a) Na/K pump {alpha} subunit expression in rabbit ventricle and regional variations of intracellular sodium regulation. Pflugers Arch 440:735-739[Medline]

Lancaster MK, Jones SA, Harrison SM, Boyett MR (2000b) Differences in the intracellular Ca2+ transient within the rabbit sino-atrial node. J Physiol 533P:30

Lebovitz RM, Takeyasu K, Famborough DM (1989) Molecular characterisation and expression of the Na+,K+-ATPase {alpha}-subunit in Drosphila melanogaster. EMBO J 8:193-202[Abstract]

Lei M, Brown HF (1996) Two components of the delayed rectifier potassium current, IK, in rabbit sino-atrial node cells. Exp Physiol 81:725-741[Abstract]

Lei M, Honjo H, Kodama I, Boyett MR (2000) Characterisation of the transient outward K+ current in rabbit sinoatrial node cells. Cardiovasc Res 46:433-441[Medline]

Lei M, Honjo H, Kodama I, Boyett MR (2001) Heterogeneous expression of the delayed rectifier K+ currents, iK,r and iK,s in rabbit sinoatrial node cells. J Physiol 535:703-714[Abstract/Free Full Text]

Lewis Carl S, Felix K, Caswell AH, Brandt NR, Ball WJ, Vaghy PL, Meissner G, Ferguson DG (1995) Immunolocalization of sarcolemmal dihydropryridine receptor and sarcoplasmic reticular triadin and ryanodine receptor in rabbit ventricle and atrium. J Cell Biol 129:673-682

Li J, Qu J, Nathan RD (1997) Ionic basis of ryanodine's negative chronotropic effect on pacemaker cells isolated from the sinoatrial node. Am J Physiol 273:H2481-2489[Abstract/Free Full Text]

Linz KW, Meyer R (1998) Control of L-type calcium current during the action potential of guinea-pig ventricular myocytes. J Physiol 513:425-442[Abstract/Free Full Text]

Masson–Pévet M, Bleeker WK, Mackaay AJC, Bouman LN, Houtkooper JM (1979) Sinus node and atrium cells from the rabbit heart: a quantitative electron microscopic description after electrophysiological localization. J Mol Cell Cardiol 11:555-568[Medline]

McDonald RL, Colyer J, Harrison SM (2000) Quantitative analysis of Na+-Ca2+ exchanger expression in guinea-pig heart. Eur J Biochem 267:5142-5148[Abstract/Free Full Text]

Rigg L, Heath BM, Cui Y, Terrar DA (2000) Localisation and functional significance of ryanodine receptors during ß-adrenoceptor stimulation in the guinea-pig sino-atrial node. Cardiovasc Res 48:254-264[Medline]

Rigg L, Terrar DA (1996) Possible role of calcium release from the sarcoplasmic reticulum in pacemaking in guinea-pig sino-atrial node. Exp Physiol 81:877-880[Abstract]

Satoh H (1997) Electrophysiological actions of ryanodine on single rabbit sinoatrial nodal cells. Gen Pharmacol 28:31-38[Medline]

Snutch TP, Tomlinson WJ, Leonard JP, Gilbert MM (1991) Distinct calcium channels are generated by alternative splicing and are differentially expressed in the mammalian CNS. Neuron 7:45-57[Medline]

Verheijck EE, Wessels A, van Ginneken ACG, Bourier J, Markman MW, Vermeulen JLM, de Bakker JMT, Lamers WH, Opthof T, Bouman LN (1998) Distribution of atrial and nodal cells within rabbit sinoatrial node. Models of sinoatrial transition. Circulation 97:1623-1631[Abstract/Free Full Text]

Wang J, Schwinger RHG, Frank K, Müller–Ehmsen J, Martin–Vasallo P, Pressley TA, Xiang A, Erdmann E, McDonough AA (1996) Regional expression of sodium pump subunit isoforms and Na+-Ca++ exchanger in the human heart. J Clin Invest 98:1650-1658[Abstract/Free Full Text]

Zhang H, Boyett MR, Holden AV, Honjo H, Kodama I (1998) A hypothesis to explain the decline of sinoatrial node function with age. J Physiol 511:76P-77P

Zhang H, Holden AV, Boyett MR (2001) Gradient model versus mosaic model of the sinoatrial node. Circulation 103:584-588[Abstract/Free Full Text]

Zhang H, Holden AV, Kodama I, Honjo H, Lei M, Varghese T, Boyett MR (2000) Mathematical models of action potentials in the periphery and center of the rabbit sinoatrial node. Am J Physiol 279:H397-421[Abstract/Free Full Text]