ARTICLE |
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 50:311324, 2002)
Key Words: L-type Ca2+ channel, Na+-Ca2+ exchanger, ryanodine receptor, Ca2+ pump, sarcoplasmic reticulum
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (30% of that in the periphery (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SA node cells were isolated as described by 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
|
|
|
|
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 12 µm and a resistance of 38 M
. 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 (
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), 1820 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 1C-subunit (anti-CNC1 peptide from
1-subunit (antibody
6F from
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 currentvoltage 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 (
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.
|
|
|
Fig 2 shows labeling with an antibody to the L-type Ca2+ channel 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 (
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.,
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 5E5G 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 5E5G are shown by arrows EG.
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 (JSRSR 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 (
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 7F7H are shown by arrows FH.
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 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
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.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (
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 (
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
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 (30% of that in the peripheral model cell (
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 (
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
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 (
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 (
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 (
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 (
![]() |
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bleeker WK, Mackaay AJC, MassonPévet 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
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
Burry RW (2000) Specificity controls for immunocytochemical methods. J Histochem Cytochem 48:163-166
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
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
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
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
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
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 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 -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
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
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
MassonPévet 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
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
Wang J, Schwinger RHG, Frank K, MüllerEhmsen J, MartinVasallo 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
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
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