Calcium Cycling Protein Density and Functional Importance to Automaticity of Isolated Sinoatrial Nodal Cells Are Independent of Cell Size
Spontaneous, localized, rhythmic ryanodine receptor (RyRs) Ca2+ releases occur beneath the cell membrane during late diastolic depolarization in cardiac sinoatrial nodal cells (SANCs). These activate the Na+/Ca2+ exchanger (NCX1) to generate inward current and membrane excitation that drives normal spontaneous beating. The morphological background for the proposed functional of RyR and NCX crosstalk, however, has not been demonstrated. Here we show that the average isolated SANC whole cell labeling density of RyRs and SERCA2 is similar to atrial and ventricle myocytes, and is similar among SANCs of all sizes. Labeling of NCX1 is also similar among SANCs of all sizes and exceeds that in atrial and ventricle myocytes. Submembrane colocalization of NCX1 and cardiac RyR (cRyR) in all SANCs exceeds that in the other cell types. Further, the Cx43 negative primary pacemaker area of the intact rabbit sinoatrial node (SAN) exhibits robust positive labeling for cRyR, NCX1, and SERCA2. Functional studies in isolated SANCs show that neither the average action potential (AP) characteristics, nor those of intracellular Ca2+ releases, nor the spontaneous cycle length vary with cell size. Chelation of intracellular [Ca2+], or disabling RyRs or NCX1, markedly attenuates or abolishes spontaneous SANC beating in all SANCs. Thus, there is dense labeling of SERCA2, RyRs, and NCX1 in small-sized SANCs, thought to reside within the SAN center, the site of impulse initiation. Because normal automaticity of these cells requires intact Ca2+ cycling, interactions of SERCA, RyR2 and NCX molecules are implicated in the initiation of the SAN impulse.
We have provided experimental evidence, based on simultaneous confocal images of intracellular Ca2+ and membrane potential,1–4 and numerical modeling,3,5 to support the concept that normal rhythmic pacemaker cell firing is governed by a tight integration of both an intracellular “Ca2+-clock”,6 ie, spontaneous rhythmic sarcoplasmic reticulum (SR) Ca2+ cycling, and a “surface membrane clock”, comprised of the ensemble of sarcolemmal ion channels.5,6 Specifically, we have demonstrated in isolated rabbit SANCs1,2,4 that rhythmic, local Ca2+ releases (LCRs) from SR occur spontaneously during the later part of diastolic depolarization. The LCR period (Ca2+ clock rhythmicity) is tightly controlled by a high degree of basal PKA-dependent protein phosphorylation3; stimulation of β-adrenoreceptors extends the range of LCR periods.2,3 The occurrence of rhythmic LCRs beneath the SANC membrane activate Na+/Ca2+ exchange inward current,1 and this increases the slope of the late part of the diastolic depolarization to ignite an action potential (AP), the shape of which is governed by ion fluxes that are tuned by the membrane clock. The membrane clock, particularly the L-type, channel component, sustains rhythmic operation of the intracellular Ca2+ clock by depleting SR Ca2+ after each AP and by providing Ca2+ influx for SR Ca2+ reloading.3,6
But, although the aforementioned functional evidence provides a strong case for a crucial role of localized submembrane RyR Ca2+ release and subsequent NCX1 activation linked to this localized RyR Ca2+ release in the normal automaticity of SANCs, histological evidence regarding the relative density and localization of RyR-NCX within SANCs have not been demonstrated. In fact, it has been argued that primary pacemaker cells, ie, those small cells near the center of the sinoatrial node that are devoid of Connexin 43 (Cx43),7,8 have a low abundance of SERCA2 and RyRs and are devoid of NCX1; accordingly, the function of these molecules would not be relevant to the normal automaticity of these cells.7,9 In the present study we determined, via immunolabeling and confocal imaging, whether the density and distribution of cRyR, NCX1, SERCA2 differ among SANCs of varying size that had been isolated from rabbit SAN, and compared with the density and location of these proteins in SANCs with those in isolated atrial and ventricle myocytes. We also labeled intact frozen SAN tissue sections to determine whether these Ca2+ cycling proteins are present in the Cx43 negative central nodal region. Finally, we measured AP and Ca2+ release characteristics and spontaneous beating rates in a large number of SANCs of a wide range of sizes to determine the relevance of intracellular Ca2+ cycling to normal automaticity of SANCs that vary in size.
Materials and Methods
Isolation of SA Node, Atrial and Ventricle Tissue, and Single SA Nodal, Atrial, and Ventricle Cells
We isolated SAN, and single spindle-shaped spontaneously-beating SANCs were isolated from 1.5-kg New Zealand rabbits (Charles River Laboratories, Wilmington, Mass) as described previously.10 We studied rabbit left ventricle tissue, atrial tissue, and single atrial and ventricle myocytes to compare with some characteristics of these to SAN or SANCs.
Immunofluorescence and Immunolabeling of Isolated SANCs
See supplemental materials (available online at http://circres. ahajournals.org) for the standard immunolabeling methods and antibody characteristics, particularly those of NCX antibody, and for detailed method of quantification of antibody labeling in isolated cells. Shortly after washing cells in PBS, we mounted them with Vectashield (H-1000; Vector Laboratories) and visualized their immunofluorescence with a LSM 510 META inverted laser scanning confocal microscope (Carl Zeiss) using a Zeiss 63×1.4 N.A. oil immersion objective or a Zeiss 10×0.45 N.A. water immersion objective.
Studies in the Intact SAN, Atrial, and Ventricle Tissue
Although our primary focus was on isolated SANCs, we also performed RyR, NCX, SERCA2, and Cx43 antibody labeling of the intact SA node. Further, in homogenates of SAN tissue, we compared immunoblottings with these antibodies to those of atrial and ventricular tissue samples. We also ascertained the RyR isoform expression in each of the 3 types of cardiac tissues (see supplemental materials for specific methods of immunolabeling, immunoblotting of RT-PCR of intact tissues).
Functional Measurements in Isolated SANCs
After their isolation, we stored SANCs in KB solution for up to 12 hours at 4°C. We studied isolated SANC functions at 35±0.5°C in a bath solution that had the following composition (in mmol/L): NaCl, 140; KCl, 5.4; MgCl2, 1; HEPES, 5; CaCl2, 1.8; Glucose, 5.5; pH, 7.4. We filled pipettes with a solution that contained (in mmol/L): K-gluconate, 120; NaCl, 10; MgATP, 5; HEPES, 5; KCl, 20; pH, 7.2. We used a perforated patch-clamp technique with 50 μmol/L β-escin11 (Sigma) added to the pipette solution to measure cell capacitance and to record spontaneous APs with an Axopatch-2B patch-clamp amplifier (Axon Instruments). We selected cells for patch clamp experiments based on their regularity of the spontaneous beating, as well as on the relative smoothness and apparent quality of the cellular surface. We made no preferences based on the cell size. In each cell, we monitored the spontaneous beating rate of SANCs (n=333) for at least 40 minutes to insure its stability. We used a standard pClamp program (Molecular Devices Corporation) to store spontaneous SANC AP parameters and an original computer program developed in our laboratory to analyze multiple parameters of the Action Potential (supplemental Figure VIII).
In a random subset of SANCs used for patch clamp experiments we compared cell capacitance to the total projected area of the cell in 2D. To measure SANC area we captured images of cells using an analog video camera Hamamatsu (Hamamatsu Photonics Inc) and calculated the projected area using MetaMorph version 6.1r0 image analysis software (Universal Imaging Corp). We calibrated the system using a standard ocular micrometer.
Pharmacological Perturbations of Continuous Ca2+ Cycling
In a subset of varying size SANCs we simultaneously measured the spontaneous beating rate before and after the interventions that (1) interfere with normal cycling: chelation of Ca2+ (BAPTA-AM, Calbiochem, 25 μmol/L dissolved in DMSO and added to the regular Tyrode buffer); (2) disable RyR function (Ryanodine, Calbiochem, 3 or 30 μmol/L added to Tyrode buffer); or (3) disable NCX function (short period of Li+ substitution for Na+ in the bathing solution).
Confocal Ca2+ Imaging
In addition to recording membrane potential, we performed confocal imaging of Ca2+ releases in a subset of SANCs. We placed cells on the stage of a Zeiss LSM-410 inverted confocal microscope (Carl Zeiss) and loaded them with fluo-3 AM (Molecular Probes), as previously described.4
All results were statistically analyzed with a Student t test, variance analysis, or linear regression analysis when appropriate.
Confocal Imaging of Antibody Staining of Isolated Cells
Figure 1 shows representative examples of isolated SANCs that vary in size and an atrial cell immunolabeled for Cx43, NCX1, and RyR. Note that only the atrial cell (Figure 1D) shows positive labeling for Connexin 43. Note also that all cells, including the Connexin-negative SANCs, label positively for both NCX1 and RyR. The merged images (lower right segment of each panel) indicate that labeling for NCX1 (in cyan) colocalizes with that of cardiac RyR (in red) beneath the sarcolemma in SANCs (indicated by the color white), regardless of shape or size.
Figure 2 depicts whole cell images and graphed pixel intensities along a line of representative examples of additional SANCs, and of an atrial and ventricle cell, each double labeled by the NCX1 and cRyR antibodies. In left panels, note that whereas NCX1 labeling is present throughout the ventricular cell (attributable to an extensive T-tubular network) in most SANCs and atrial cells, NCX is mostly confined to the sarcolemmal region. Labeling of cRyR is in junctional SR throughout the ventricle cells, and in the atrial cells it is similarly present both at the cell surface and intracellularly; in most smaller and medium-sized SANCs, cRyR is mainly detected in the junctional SR in proximity to the sarcolemma. In large SANCs, and in atrial-like cells isolated from the SAN (Figure 2D), however, cRyR labeling is also present throughout the cytosol. All isolated cells studied (ventricular myocytes, atrial cells, and SANCs, regardless of size) exhibit robust uniform SERCA2 labeling (supplemental Figure I), which strongly suggests presence of an extensive highly-developed SR network throughout all of these cardiac cells.
Figure 2A through 2D (right panels) depicts the individual pixel intensities of NCX1 and cRyR labeling along an arbitrary line positioned across the cell (white lines in left panels). Labeling patterns of cRyR and NCX1 of the pixel-by-pixel plot clearly illustrate that NCX1 and cRyR are highly colocalized in both SANCs and ventricle cells. The density of colocalized NCX1 and cRyR in SANCs, however, is most pronounced beneath the cell membrane; in ventricle cells, the NCX1 and cRyR colocalization is more uniform throughout the cell. The horizontal line in the right panels represents the average pixel values of NCX1 (cyan) and cRyR (red) of the interrogated line. Note that the SANC NCX1 labeling density along the line scan image is greater than that in atrial and ventricle myocytes. Figure 3E illustrates a representative 3D intensity profile of cRyR, and NCX1 labeling and their merged images in the small-sized SANCs (Figure 3C) the submembrane distribution of NCX1 and cRyR proteins is evident. Average cRyR labeling density is equivalent in all 3 cell types.
Figure 3A shows that the distribution of sizes of isolated SANCs were used for confocal imaging of antibody labeling (Figures 1 and 2⇑) extends across a broad range. Figure 3B depicts quantitative profiles, relative to ventricle myocytes, of the average NCX1 and cRyR labeling densities in a randomly selected subset of SANCs of 3 size ranges and in atrial cells. On average, whole cell NCX1 labeling intensity (Figure 3B) appears to be greater in SANCs than in left ventricular cells, whereas whole cell labeling intensity of cRyR does not significantly vary among cell types. Quantitative analysis of labeling intensities within a 0.5 μm subspace beneath the perimeter of the sarcolemma (Figure 3C) confirms the significant increase in both NCX1 and cRyR in these subspaces in SANCs compared with left ventricular or atrial cells, and confirms the degree of their colocalization apparent by visual inspection (Figures 1 and 2⇑).
We also quantified colocalization of cRyR and NCX1 antibody labeling via Pearson correlation coefficient of pixel labeling (Figure 4). Average Pearson correlation coefficient values calculated for 8 to 10 cells of each type ranged from 0.59±0.02 to 0.65±0.06 indicating a significant (and comparable) degree of colocalization between the cardiac RyR and NCX1 in each of these tissues, as expected from visual inspection of the examples. Significantly, these areas of colocalization (shown in white in the cRyR-NCX1 colocalization images, Figure 4 lower right quadrants of Panels A through C) are most prominent along the cell surface membranes in SAN and atrial cells (Panels A and B, respectively), and in addition, are also prominently represented in a distribution compatible with t-tubule membranes in ventricular cells (Panel C). These data also compare favorably with the results of immunoblotting (supplemental Figure IIID). It should be noted, however, that whereas cRyR and NCX1 do appear to be colocalized in the majority of membrane regions examined, there are occasional discrete regions on the membranes of some SANCs, atrial, and ventricular myocytes in which an alternating punctuate pattern of cRyR and NCX1, at or near the limits of optical resolution, is apparently discriminated.
Confocal Imaging of Antibody Labeling of Intact SAN
Because all SANCs, regardless of size, show intense SERCA2, NCX1, and cRyR labeling (Figures 1 to 4⇑⇑⇑), and because the cells within center of SAN have been shown on average to be smaller than more peripheral cells,12,9,13 we reasoned that the central Cx43 negative center of SAN must also exhibit labeling for SERCA2, NCX1, and cRyR. Representative, sequential, 10-μm-thick cross-sectional slices of the intact rabbit SAN are illustrated in Figure 5. The crista terminalis and atrial peripheral pacemaker and primary (“central”) pacemaker cell areas are indicated in the bright field image (Figure 5A). Distinct features of the rabbit primary pacemaker cell area are the lack of Cx43 labeling (Figure 5B), as demonstrated previously,7,8 and the presence of the neurofilament protein NF160 labeling (Figure 5C). Figure 5D through 5F illustrates labeling of the intact SAN for NCX1, SERCA2, and cRyR. A striking feature of the figure is that robust labeling, strong NCX1, cRyR, and SERCA2, for Ca2+ regulatory proteins is present not only in the area mostly occupied by atrial and “transitional” or peripheral pacemaker cells, but also within the primary pacemaker area (Cx43-negative, NF160-positive area). Supplement (Figure II) shows antibody staining of the intact node encompassing regions of the atrium and peripheral and central SAN at higher magnification.
Immunoblots and Real Time QPCR
Immunoblots of homogenates of rabbit SAN and atrial and ventricular tissue probed with antibodies for NCX1, cRyR, and SERCA2 confirmed the presence of these proteins in these tissues (supplemental Figure III). RyR3 antibody labeling of Western blots was weaker in SAN than in LV or atrial tissue; and in SAN, RyR3 protein labeling was less than that of the cRyR labeling. Real time qPCR confirmed that the expression of RyR2 was greater than RyR3 in all 3 cardiac tissues (supplemental Figure IV).
Functional Measurements According to Cell Size
The distribution of isolated SANC sizes (measured as a cell area) used for functional studies (Figure 6C) overlaps with that used for antibody labeling (Figure 3A). Figure 6B shows that the distribution of isolated SANC capacitance and their spontaneous beating rates is roughly Gaussian. Figure 6A demonstrates that the beating rate is not related to cell size (indexed by cell capacitance).
Figure VIII shows AP recordings in a representative SANC and indicates how AP parameters were measured. Most of the AP characteristics that we measured in a random subset of cells (supplemental Table I) were comparable to those previously described in the literature.14,15,16 Linear regression analysis failed to detect significant correlations between cell capacitance and any measured AP parameter (supplemental Table II). Representative examples of the scatter plots for some of the AP parameters versus cell size are illustrated in Figure V.
We next determined whether the effects of interfering with normal RyR or NCX1 functioning, both known to affect SANC normal automaticity,1,4 are dependent on cell size. Ryanodine, which selectively interferes with cRyR function, slows or stops the spontaneous beating. Although the magnitude of this ryanodine effect varies between intact SAN and SANCs, and varies among studies, all of studies have demonstrated a negative chronotropic effect of ryanodine.17 Figure 7A (left panel) illustrates the effect of 3 μmol/L ryanodine on the spontaneous beating rate in SANCs that vary in size. A middle trace of Figure 7A shows representative AP recording, and in the right trace intervallogram after application of 3 μmol/L ryanodine. On average, in 24 cells, 3 μmol/L ryanodine produced about 50% decrease in the beating rate, completely independent of cell size. A higher ryanodine concentration (30 μmol/L) abolishes beating in 83% of the cells (5 of 6 cells, see representative example Figure 7C).
Brief Na+ removal from the superfusate (Li+ substitution for Na+) completely suppressed spontaneous beating in all 11 cells studied, regardless of size (Figure 7B, left). Figure 7B (right and middle traces) shows the Li+ effect on spontaneous beating in a representative cell. A main function of NCX1 and cRyR molecular interaction is to cycle Ca2+ into and out of the cytosol. When we superfused single spontaneously beating SANCs with 25 μmol/L BAPTA-AM to buffer intracellular Ca2+ concentration, in all 8 studied cells spontaneous beating stopped. Figure 7D illustrates a representative example.
Ca Releases in Smaller and Larger SANCs
Next we determined characteristics of Ca2+ releases in small and large SANCs. Figure 8A illustrates representative confocal line scan images of Ca2+ in a small and large SANC. Both the AP-induced cytosolic Ca2+ transient ignited by the AP and spontaneous local Ca2+ releases (LCRs) that occur during the later part of diastolic depolarization are similar in both smaller and larger cells. Figure 8B compares the average data for the global cytosolic Ca2+ transient amplitude and LCR characteristics in small and large SANCs.
Our study is the first to describe robust NCX1 labeling of smaller as well as larger SANCs, and to quantify the relative NCX1 labeling of SANCs, regardless of size, relative to that in ventricle and atrial cells. One novel finding is that all isolated SANCs examined, regardless of their size, exhibit robust NCX1 as well as SERCA2 and cRyR cellular labeling densities. Another novel finding of our study is that the entire SA node, including the primary pacemaker area of the intact rabbit SAN, devoid of Connexin 43 antibody labeling, exhibits positive labeling for NCX1, cRyR, and SERCA2. This finding is consistent with that of a previous report showing very marked expression of NCX1 in the SA node or in cells from different parts of the cardiac conduction system.18 This finding is also consistent with our demonstration of robust cRyR, NCX1, and SERCA2 labeling of all isolated SANCs, including small cells which are enriched within the SAN central area. These results on cRyR, and particularly NCX1 labeling in isolated SANCs and within the intact SAN, differ from those of a prior study.7
The local densities of molecules within cells, rather than the average whole cell density, are relevant to functional molecular interactions. Our prior studies of SANC function had demonstrated “crosstalk” between submembrane RyR Ca2+ releases during the later part of the spontaneous diastolic depolarization of SANCs and NCX1 activation that generates inward current at this crucial time in the SANC duty cycle. This cross-talk is a critical factor in the regulation of the normal automaticity of SANCs.1,2,3,4,5 But the morphological basis for this crosstalk had not been demonstrated. Another novel finding of the present study is that the relative distributions of cRyR and NCX1 within SANCs quantitatively differ from those of atrial and ventricle cells: in SANCs, colocalized, high intensity NCX1 and cRyR labeling in the subsarcolemmal region exceeds, by 2-fold, that in atrial or ventricle cells. Quantitative molecular colocalization by confocal imaging of antibody labeling of cRyR and NCX1 largely beneath the sarcolemma (Figure 2), however, does not imply direct physical connection of these molecules, as they, in fact, reside within different organelles, NCX1 within the sarcolemma and cRyR within SR beneath the membrane.
An additional novel finding of the present study, in contrast to a prior report,9 is that the characteristics of intracellular Ca2+ releases of SANCs do not vary with cell size. Further, our functional electrophysiological data clearly indicate that maneuvers that affect Ca2+ cycling in SANCs, ie, chelation of intracellular Ca2+, or inhibition of RyR or NCX1 function, by BAPTA, ryanodine, Li+ substitution for Na+, respectively, markedly slow or abrogate spontaneous SANC beating, irrespective of the SANC size (Figure 7). Thus normal Ca2+ cycling and RyR-NCX “cross-talk” are crucial for the normal automaticity of smaller, as well as for larger, SANCs. Because the SAN impulse is initiated from small SANCs within its central area, our finding implicates potential involvement of Ca2+ cycling and RyR-NCX crosstalk as mechanisms that underlie the initiation of the cardiac impulse.
It is well established that in the intact SAN the initiation of electrical activity of each beat initiates from the central area which contains mostly small SANCs,8,19 and that in the intact SAN the shape of the membrane potential impulses varies between the center and the periphery of the SAN.13,20 However, neither the cycle length, nor the AP characteristics, nor Ca2+ cycling properties, nor ion channel complement measured in isolated SANCs, as in the present study, necessarily report the properties of SANCs within the intact SAN. Indeed, while the impulse is initiated within the SAN center where numerous small SANCs reside, studies of isolated SANCs have failed to demonstrate that smaller isolated SANCs, or isolated balls of small SANCs isolated from the SANC center, beat faster12,21 than larger isolated cells or balls of SANCs isolated from more peripheral areas of the node. Some studies have found, in fact, that smaller central SANCs, or balls of SANCs, beat slower than larger SANCs or balls of SANCs isolated12,21 from the peripheral node. Other studies,15,22 like our study, have failed to detect size-dependent differences in isolated AP characteristics or cycle length in isolated SANCs.
Thus, it would be an oversimplification to assume that the intrinsic spontaneous cycle length, or AP characteristics of individual rabbit SANCs in isolation, are identical to those within intact SAN. The properties of SANCs within the intact SAN depend, not only on the intrinsic properties of SANCs, but also on modulation of intrinsic cell properties by the environment in which SANCs reside.
Extrinsic modulators of intrinsic SANC properties are numerous, and include mechanical and electrotonic forces and autonomic milieu which are heterogeneous throughout the SAN. For example, differences in the AP shape of SANCs within the SAN tissue could be a result of the mutual entrainment between the depolarizing charge, provided by ion channels of single SANC cells, and structural properties of the surrounding inexcitable tissue that imposes the electrical load on a depolarizing cell. It is possible that small cells with less capacitance, and correspondingly with smaller charges, could be more affected by the surrounding electrical load than big cells. SA nodal tissue contains numerous fibroblasts and extensive connective tissue which can occupy as much as 25 to 90% of the SAN.19 In cardiomyocytes, an increase in the fibroblast density decreases the rate of AP upstroke and depolarizes resting potential.23 If this were also to be true for SANCs, then small SANCs within the SAN center may have a less negative maximum diastolic potential and a less steep AP upstroke, because the SAN primary pacemaker area has the highest connective tissue density.19
In addition to bands of fibrous tissue, variable densities and types of gap junction proteins within the node also contribute to local variations in intracellular resistances and contribute to the formation of “local neighborhoods” in which clusters of SANCs reside.24 The myofilament density within SANCs decreases from the peripheral to the central area.25 This results in heterogeneous mechanical stress within the node, after SANC activation. Further, autonomic milieu is heterogeneous throughout the node, with the central area having the greatest density of nerve endings and autonomic receptors on SANCs.26,27
Although the intrinsic properties of isolated SANCs of a given size cannot reflect local properties of clusters of cells of the same size even within the node, or even within its central area, intrinsic properties of SANCs, in addition to extrinsic forces acting on them, must contribute to their function within the intact SAN. Our study, which examined a large number of SANCs in isolation, demonstrates random variation about the average of AP characteristics cycling length (supplemental Figure V, supplemental Tables I and II) and Ca2+ cycling protein density (eg, Figures 2, 3, 4, and 6⇑⇑⇑) of smaller and larger cells. It is not possible from our results to know whether some local “neighborhoods” within the SAN have distributions of SANCs that are skewed from the average. For example, it is possible that small SANCs with the shortest cycle lengths are clustered together within the center of the node.
In the final analysis, a requirement for the supremacy of the central area in initiating the SAN impulse is that the mutually entrained rate of its cells,22 determined by the net balance of intrinsic mechanisms and their modulation by extrinsic factors, exceeds that of other neighborhoods to which the impulse spreads. A phase shift in mutually entrained AP, generated within a SAN neighborhood, must account for the often observed shift in the leading pacemaker site after interventions that act differentially among neighborhoods within the SAN to alter intrinsic SANC properties, eg, vagal or sympathetic stimulation,20,21 or the interaction of SANCs with local extrinsic factors within a neighborhood. The present study establishes that the spontaneous beating of small SANCs, ie, similar to those that are thought to reside within the center of the SAN, as well as larger ones, is profoundly affected by interfering with normal spontaneous cell Ca2+ cycling or with RyR-NCX “crosstalk.” Thus, this crosstalk must be considered among the factors that enable the central SAN area to initiate the spontaneous SAN impulse.
Small isolated SANCs, thought to reside within the SAN center, the site of the initiation of the SAN impulse, as well as larger SANCs, exhibit intense RyR, NCX1, and SERCA2 labeling and dense submembrane NCX/RyR colocalization. The spontaneous beating of small SANCs, as well as larger ones, is profoundly affected by interfering with normal spontaneous cell Ca2+ cycling or with RyR or NCX function and interaction. Ca2+ cycling and NCX-RyR crosstalk within small SANCs within the center of the SAN, therefore, is a potential crucial factor in initiation of the spontaneous SAN impulse.
Sources of Funding
This work was supported, in part, by the Intramural Research Program of National Institute on Aging, National Institute of Health.
↵*These authors contributed equally to this study.
Original received December 12, 2006; revision received April 9, 2007; accepted May 10, 2007.
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