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(Circulation Research. 2007;100:1605.)
© 2007 American Heart Association, Inc.

Cellular Biology

Store-Operated Ca²⁺ Influx and Expression of TRPC Genes in Mouse Sinoatrial Node

Yue-Kun Ju, Yi Chu, Herve Chaulet, Donna Lai, Othon L. Gervasio, Robert M. Graham, Mark B. Cannell, David G. Allen

From the School of Medical Sciences and Bosch Institute (Y.-K.J., Y.C., D.L., O.L.G., D.G.A.), University of Sydney, and Victor Chang Cardiac Research Institute (H.C., R.M.G.), Australia; and the The Faculty of Medical and Health Sciences (M.B.C.), University of Auckland, New Zealand.

Correspondence to Yue-kun Ju, School of Medical Sciences (F13), University of Sydney, Sydney, NSW 2006, Australia. E-mail ju{at}physiol.usyd.edu.au

	Abstract

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Store-operated Ca²⁺ entry was investigated in isolated mouse sinoatrial nodes (SAN) dissected from right atria and loaded with Ca²⁺ indicators. Incubation of the SAN in Ca²⁺-free solution caused a substantial decrease in resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) and stopped pacemaker activity. Reintroduction of Ca²⁺ in the presence of cyclopiazonic acid (CPA), a sarcoplasmic reticulum Ca²⁺ pump inhibitor, led to sustained elevation of [Ca²⁺]_i, a characteristic of store-operated Ca²⁺ channel (SOCC) activity. Two SOCC antagonists, Gd³⁺ and SKF-96365, inhibited 72±8% and 65±8% of this Ca²⁺ influx, respectively. SKF-96365 also reduced the spontaneous pacemaker rate to 27±4% of control in the presence of CPA. Because members of the transient receptor potential canonical (TRPC) gene family may encode SOCCs, we used RT-PCR to examine mRNA expression of the 7 known mammalian TRPC isoforms. Transcripts for TRPC1, 2, 3, 4, 6, and 7, but not TRPC5, were detected. Immunohistochemistry using anti-TRPC1, 3, 4, and 6 antibodies revealed positive labeling in the SAN region and single pacemaker cells. These results indicate that mouse SAN exhibits store-operated Ca²⁺ activity which may be attributable to TRPC expression, and suggest that SOCCs may be involved in regulating pacemaker firing rate.

Key Words: heart • sinoatrial node • TRPC • store-operated Ca²⁺ channel

	Introduction

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Store-operated Ca²⁺ channels (SOCCs) were first identified in nonexcitable cells based on the observation that depletion of Ca²⁺ stores increase influx of extracellular Ca²⁺.¹ Later a membrane current (I_CRAC) evoked by store depletion was identified and shown to be voltage-insensitive and Ca²⁺-selective.² In nonexcitable cells, store release can be triggered by the activation of phospholipase C (PLC), leading to 1,4,5-inositol-trisphosphate (IP₃) generation, which binds to its receptor (IP₃R) and causes Ca²⁺ release and store depletion (for review see Parekh and Penner³). Ca²⁺ entry through SOCCs may play a role in ensuring that intracellular stores have an adequate Ca²⁺ content to carry out signaling functions. This would be especially important in nonexcitable cells lacking voltage-dependent Ca²⁺-entry pathways.

In excitable cells, Ca²⁺ entry via voltage-sensitive Ca²⁺ channels and other pathways, such as Na⁺/Ca²⁺ exchange, can maintain store Ca²⁺ levels so that the possible role of SOCCs is less clear. Nevertheless, there is increasing evidence that SOCCs exist in various excitable cells, including cardiac ventricular muscle.^4,5 In ventricular myocytes, PLC responses are generally modest with only small amounts of IP₃ being produced.⁶ Thus release of Ca²⁺ through IP₃R is probably too small to modify excitation–contraction coupling. However, atria myocytes express functional IP₃R at 6 to 10 times higher levels than those in ventricules, and Ca²⁺ release from IP₃R may be involved in the generation of arrhythmias.⁷ Furthermore, recent studies have shown that activation of PLC in the heart leads to Ca²⁺ release from perinuclear IP₃R and thereby regulates nuclear-cytoplasmic cycling of transcription factors and alters gene expression.⁸ A similar role may be played by IP₃ and the IP₃R in skeletal muscle and SOCCs have been implicated in IGF-1 induced muscle hypertrophy.^5,9

The identity of the genes that encode SOCCs remains uncertain. Studies of the transient receptor potential (TRP) gene from Drosophila showed that it encodes a PLC-activated Ca²⁺ permeable channel.¹⁰ Subsequently, 7 TRP channel homologues in mammals, termed TRPC1-TRPC7, have been identified and there is considerable evidence to indicate that TRPC1 can encode a SOCC.¹¹ In addition, a recent study showed that overexpression of TRPC3 substantially enhanced SOCC activity in the heart.⁵

The importance of Ca²⁺ release from stores in cardiac pacemaking is now widely accepted.^12–14 In a previous study, we found that activation of the P2Y₁ purinergic receptor by ATP results in modulation of pacemaker firing attirbutable to receptor-coupled PLC activation and depletion of SR Ca²⁺ stores.¹⁵ Because activation of SOCCs also involves PLC, we speculated that SOCCs might be present in pacemaker tissue.

In mammals, the sinoatrial node (SAN) is a heterogeneous tissue. The shape of the action potential and the rate of rise of the pacemaker potential change progressively from the periphery to the center, the latter being the leading pacemaker site.¹⁶ Expression of ionic channels, Ca²⁺ handling proteins, and gap junction proteins in the SAN also vary from center to periphery.¹⁷

In this study, we recorded intracellular Ca²⁺ signals from intact mouse SANs, a preparation in which the structural integrity and activity of the node is preserved. SOCC activity and expression of TRPC gene and proteins were examined in this preparation. These studies indicate that TRPCs might mediate SOCC activity and as a result, regulate pacemaker firing rate.

	Materials and Methods

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For detailed Materials and Methods, please see the supplemental materials (available online at http://circres.ahajournals.org).

SAN Preparation
The right atrium was harvested from anesthetized (pentobarbital sodium 1 mg/kg, i.p.) male BalbC mice (7 to 10 weeks old) and opened under a dissecting microscope to expose the crista terminalis, the intercaval area, and the interatrial septum.¹⁶ A section of right atrial wall containing the SAN region was pinned to a thin Sylgard block and placed in a chamber on an inverted microscope stage. The epicardial surface of this SAN preparation was closest to the objective while the endocardial surface was continuously superfused with modified Tyrode’s solution. All experiments were performed at 33°C.

Ca²⁺ Measurements
The SAN preparation was loaded with the membrane-permeant fluorescent Ca²⁺ indicator, indo-1-AM (10 µmol/L), in Tyrode solution. Fluorescent signals were recorded from a restricted region of the SAN area (about 150x150 µm) by using a rectangular diaphragm (Figure 1A). The analog signals were digitized and the expression (R-R_min)/(R_max-R) calculated, which is linearly related to [Ca²⁺]_i (for details see Kao¹⁸). In some experiments, confocal microscopy was used to collect data from the SAN preparations that were loaded with fluo-4AM (10 µmol/L).

View larger version (36K): [in this window] [in a new window]   Figure 1. A, Transmitted light image of an isolated mouse sinoatrial node (SAN). CT, crista teminalis; RA, right atrium; SVC, superior vena cava. B & C, Spontaneous action potentials and Ca2+ transients recorded from SAN region indicated by rectangular box in A. D & E, 1 µmol/L isoproterenol increased both firing rate and the amplitude of Ca2+ transients recorded from the same region.

mRNA for TRPC Expression
Total RNA was isolated from 20 SANs using a RNAqueous 4PCR isolation kit (Ambion). Specific primers for mTRPC1, mTRPC2, mTRPC3, mTRPC4, mTRPC5, mTRPC6, and mTRPC7 were used for PCR amplification, respectively.¹⁹ To control for potential genomic DNA contamination, a cDNA reaction was performed but in the absence of reverse transcriptase and used as template for PCR reactions.

Immunohistochemistry
Rabbit polyclonal antibodies to TRPC1, TRPC3, TRPC4, and TRPC6 (1:50, 1:100, 1:50, and 1:200 dilution, respectively; Alomone Labs) were used to label the various TRPC isoforms in the SAN whole mount preparations and single pacemaker cells. Antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:100; Alomone Labs), and connexin-43 (3 µg/mL; Chemicon) were also used to identify pacemaker region within the SAN.

Statistics
Data are expressed as mean±SEM, with the number of preparations as n. Statistical test were either Student paired or unpaired t test, and P<0.05 was taken as the level of significance.

	Results

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Spontaneous Ca²⁺ Transients Recorded From the SAN Preparation
The central pacemaker region was identified by anatomic landmarks as shown in Figure 1A.¹⁶ Using conventional glass microelectrodes, we were able to record spontaneous action potentials from this region (Figure 1B, upper panel) that exhibited the low upstroke velocity typical of the primary pacemaker region (Figure 1B, lower panel). The spontaneous firing rate slowed with indo-1AM loading, attributable to the increased [Ca²⁺]_i buffering.¹³ Ca²⁺ transients recorded from the SAN node (Figure 1C) were synchronous with visible contraction of the SAN.

It is well established that ß-adrenergic stimulation increases heart rate via its effect on the pacemaker cells. Therefore, the effects of isoproterenol on pacemaker action potential and Ca²⁺ transient were examined. We found that in the presence of isoproterenol, firing rate increased by 38±7% (n=7, P<0.0001), and the amplitude of Ca²⁺ transients was increased by 69±21% (P<0.01, Figure 1D and 1E). The observations suggest that the Ca²⁺ signals were principally from the pacemaker cells rather than from other cell types in the SAN such as endothelial cells or fibroblasts.²⁰

To confirm this notion, confocal images of the SAN were recorded in the presence of lidocaine (100 µmol/L) to leave only the central SAN cells firing.²¹ Figure 2A, a & b, shows consecutive images recorded from the central SAN, and Figure 2Ac shows a ratio image, demonstrating cyclic [Ca²⁺]_i changes in the central region of the SAN but not surrounding regions. A line scan image across the SAN (Figure 2B) and the time course of [Ca²⁺]_i changes (Figure 2C) show that it is possible to record and characterize spontaneous Ca²⁺ signals arising from the central regions of the SAN.

View larger version (51K): [in this window] [in a new window]   Figure 2. Confocal images of [Ca2+]i in the SAN. The bathing solution contained 100 µmol/L lidocaine and 10 mmol/L 2,3-butanedione monoxime to reduce movement artifacts. A, Consecutive XY images recorded from the central SAN area with a time interval of 0.18 s between a and b. A ratio image of fluorescence intensity is showed in Panel Ac. B, XT ratio image obtained from central SAN pacemaker area. Calibration bar in B indicates the self ratio fluorescence value that is calculated from the pixel values divided by the mean values just before the transient (F/F0). The time scale is the same as in C. C, Normalized fluorescence intensity plotted as a function of time from a region near the lower part of B. (F, the intensity of region plotted; F0, the mean background intensity.) D, Control, XT image (top), and line plot of normalized fluorescence intensity) against time (bottom). E, The effect of 0.5 mmol/L caffeine on [Ca2+]i and firing rate.

Store-Operated Ca²⁺ Influx in Pacemaker Cells
To study Ca²⁺ influx through SOCCs in the SAN, preparations were first incubated in Ca²⁺-free solution resulting in a substantial decline in resting [Ca²⁺]_i and cessation of pacemaker activity (Figure 3A). Reintroduction of Ca²⁺ to the perfusate caused a small rise in [Ca²⁺]_i and a recovery of pacemaker activity (Figure 3B). When SR Ca²⁺ uptake was inhibited with cyclopriazonic acid (CPA, 10 µmol/L), the resting [Ca²⁺]_i increased significantly (Figure 3C). To further empty SR Ca²⁺ stores, SANs were incubated in Ca²⁺-free solution containing CPA for 15 minutes. Reintroduction of Ca²⁺ to the perfusate in the presence of CPA evoked a marked increase in [Ca²⁺]_i (Figure 3D), a characteristic of SOCC activity, as observed in other tissues.²² Ca²⁺ influx in response to reintroduction of Ca²⁺ was 7.1±3.2-fold greater in the presence (Figure 3D) than in the absence of CPA (Figure 3B; P<0.03, n=11), a finding consistent with Ca²⁺ influx being activated in response to SR store depletion. Although it is not feasible to measure the SR store content in this multicellular preparation, the large rise in resting [Ca²⁺]_i in the presence of CPA suggested that SR Ca²⁺ uptake was effectively blocked and, thus, that SR store content was substantially depleted (Figure 3C).

View larger version (19K): [in this window] [in a new window]   Figure 3. Ca2+ influx induced by SR Ca2+ store depletion. A & B, Effect of changing extracellular Ca2+ on [Ca2+]i in the SAN preparation. Extracellular Ca2+ removed (A); extracellular Ca2+ replaced (B). C & D, [Ca2+]i changes in the presence of CPA (10 µmol/L) when in normal extracellular Ca2+ (C) and extracellular Ca2+ replaced after incubating in 0 Ca2+, 0.5 mmol/L EGTA, and CPA for 15 minutes (D).

To examine the possibility that the Ca²⁺ influx was mediated by L-type Ca²⁺ channels or Na⁺/Ca²⁺ exchanger, we tested the effect of nifedipine or the Na⁺/Ca²⁺ exchanger inhibitor, KBR-7943. As shown in Figure 4A, 20µmol/L nifedipine had no effect on SOCC activity. In the presence of 5 µmol/L KBR-7943, Ca²⁺ influx was inhibited by 25±8% of the control (n=6) (Figure 4B). Therefore, even when L-type Ca²⁺ channels or Na⁺/Ca²⁺ exchange are inhibited, store depletion evokes an influx of Ca²⁺as would be expected from the presence of SOCCs.

View larger version (25K): [in this window] [in a new window]   Figure 4. A, The effect of 20 µmol/L nifedipine on Ca2+ influx induced by the SR Ca2+ store depletion. B, The effect of 5 µmol/L KBR-7943 on Ca2+ influx induced by the SR Ca2+ store depletion. C, The effect of 100 µmol/L Gd3+ on Ca2+ influx induced by the SR Ca2+ store depletion. D, The effect of 10 µmol/L SKF-96365 on Ca2+ influx induced by SR store depletion. E, Summary and statistics of the inhibition of Ca2+ entry by pharmacological agents. Bars show the percentage inhibition of peak [Ca2+]i compared with control. Error bars are 1 SE. P<0.01; *P<0.001.

To further confirm that this Ca²⁺ influx was mediated by SOCCs, we applied gadolinium (Gd³⁺), a known SOCC inhibitor. We found that 100 µmol/L Gd³⁺ reduced the maximum [Ca²⁺]_i response by 73±8% (P<0.01, n=4; Figure 4C). It has been reported that SOCCs are also sensitive to SKF-96365 in several cell types.²² We found that 10 µmol/L SKF-96365 inhibited 65±9% of Ca²⁺ influx (P<0.001, n=8; Figure 4D). A summary of these pharmacological evaluations of SOCC activity in SAN is shown in Figure 4E.

Functional Role of SOCCs in the Mouse SAN
Previous studies have demonstrated that in toad and rabbit pacemaker cells, pacemaker activity is modulated by SR Ca²⁺ release.^13,14 To test whether spontaneous pacemaker activity of mouse SAN is also sensitive to SR Ca²⁺ release, the SR Ca²⁺ release antagonist, ryanodine (20 µmol/L) was applied and found to reduced firing rate to 55±11% of control (n=6, P<0.03; Figure 5A, left).

View larger version (29K): [in this window] [in a new window]   Figure 5. A, The effect of 20 µmol/L ryanodine (left) and 10 µmol/L CPA (right) on spontaneous firing rate and [Ca2+]i in intact mouse SAN. B, Summary of SR store modulators on pacemaker firing rate. Bars show the percentage action potential firing rate compared with control. Error bars are 1 SE. P<0.01; *P<0.005 (n=6 in each group). C, The effect of 10 µmol/L SKF-96365+10 µmol/L CPA on spontaneous firing rate and [Ca2+]i. D, Summary of changes in resting Ca2+ and Ca2+ transient. Error bars are 1 SE.* P<0.001 compare to the control. P<0.01 compare to CPA.

SR Ca²⁺ release can also be affected by reducing SR Ca²⁺ content. Low concentrations of caffeine reduce SR Ca²⁺ content²³ and were found to reduce the both Ca²⁺ transient and firing rate to 87±4% and 75±3% of control respectively (n=4, P<0.01; Figure 2D and 2E). SR Ca²⁺ content can also be reduced by inhibition of SR Ca²⁺ uptake with CPA,²⁴ which has been reported to slow spontaneous activity in single pacemaker cells.²⁵ We found that CPA had a marginal effect on pacemaker firing rate, reducing it to 81±9% control. Both ryanodine and CPA reduced the amplitude of Ca²⁺ transients to 21±5% (n=6, P<0.0004) and 48±4.5% of control (n=7, P<0.0003), respectively. In addition, CPA increased resting [Ca²⁺]_i to 167±37% of control (P<0.0006), whereas ryanodine did not alter resting [Ca²⁺]_i significantly. These effects of SR store modulators on the pacemaker firing rate were confirmed by using intracellular recording of action potentials on intact SANs, which revealed qualitatively similar effects (Figure 5B).

These results suggest that several mechanisms contribute to the role of SR loading in cardiac pacemaking. For instance, whereas depletion of SR stores by CPA and low dose caffeine may activate membrane channels, such as SOCCs, to generate an inward Ca²⁺ current, this would be offset by a decrease of inward Na⁺/Ca²⁺ exchanger current that was caused by reduced SR Ca²⁺ release. Because high concentrations of ryanodine (>10 µmol/L) do not affect store content,²⁶ the reduction the firing rate seen when ryanodine was applied can be explained by its effects on Ca²⁺ release and the Na⁺/Ca²⁺ exchanger current without a direct effect mediated by SOCCs.

To explore the potential implications of SAN SOCC activity for pacemaker function, we examined the effect of SKF-96365 on spontaneous action potential firing rate (Figure 5B). SKF-96365 (10 µmol/L), which blocked SOCCs (Figure 4), slowed firing rate to 64±7% of control (P<0.005, n=5). Moreover, a further reduction in pacemaker firing to 27±4% (P<0.002, n=5) was observed when the SKF-96365 was administered together with the SR Ca²⁺ uptake blocker CPA (Figure 5B and 5C). With this combined treatment, resting [Ca²⁺]_i fell to a level not different from to the control (Figure 5C and 5D). It should be noted that although this combined treatment reduced both the firing rate and Ca²⁺ transients, effects that were fully reversible, the upstroke of action potentials was unchanged (Figure 5C). Thus it seems likely that the effects of SKF on the pacemaker firing were not attributable to blockade of the L-type Ca²⁺ channels.²⁷ A comparison of changes in resting and transient [Ca²⁺]_i is shown in Figure 5D.

Expression of TRPC Gene and Proteins in SAN
It has been suggested that SOCCs are encoded by the TRPC gene family.²⁸ We used RT-PCR to examine mRNA expression of the 7 known mammalian TRPC isoforms in SAN preparations. PCR products were generated using primers specific for TRPC1-TRPC7 isoforms; primers that were initially validated using mouse brain RNA, a tissue known to express all TRPC isoforms. TRPC1, 2, 3, 4, 6, and 7, but not TRPC5, transcripts were detected in SAN (Figure 6Aa). Using HCN4 as a positive control, we showed both mRNA (Figure 6Ab, supplemental Figure I) and protein expression (Figure 6Ba 6Bb) of HCN4 are higher in SAN than right atria.²⁹ HCN4 immunoreactivity was observed in the SAN region but not in the CT and surrounding atrial tissue (Figure 6Ba and 6Bb). In contrast, Cx43 (Figure 6Bc and 6Bd) is absent from the central area of the SAN but is abundant in atria and peripheral SAN as also observed by others.¹⁶ These distinctive protein expression patterns enabled us to identify the central and peripheral SAN.

View larger version (44K): [in this window] [in a new window]   Figure 6. A, TRPC1, 2, 3, 4, 6, and 7 mRNA are expressed in SAN tissue. RT-PCR products of the predicted sizes (between 200 to 500 bp) for the different TRCP species are shown in a. Faint broad bands at <100bp are primer dimers. The presence of the HCN4 RT-PCR product (b) indicates that the tissue used to isolate RNA was from the pacemaker region. In both panels, black bars indicate those the PCR reactions performed in the presence, and open bars (controls) in the absence, of reverse transcriptase. B, Confocal immunofluorescence images of anti-HCN4 and anti-Cx43 in the mouse SAN. Whole mounted preparations of SAN labeled with antibodies to HCN4 (a & b) and Cx43 (c & d) that fluoresce green. DAPI, a nuclear and chromosome counterstain, fluoresces in red.

Polyclonal antibodies raised against human TRPC1, TRPC3, TRPC4, and TRPC6 were used to evaluate the expression of these proteins. We confirmed that there antibodies could detect the TRPC isoforms by Western blot analyses of mouse atrial lysates (supplemental Figure II).

When applied to SAN whole mount preparations, immunofluorescence was evident in both the central and peripheral SAN with antibodies against TRPC1, 3, 4, and 6. To further define the cell type expressing TRPCs in the central and peripheral SAN, single pacemaker cells were isolated and labeled with TRPC antibodies. The central or peripheral pacemaker cells were identified by their morphological appearance.¹⁷ Single pacemaker cells isolated from central (Figure 7a) and peripheral (Figure 7b) SAN all showed positive labeling with the TRPC antibodies. As observed in other cell types,^30–32 labeling was predominantly cytoplasmic with the TRPC1 antibody (Figure 7A). The TRPC3 antibody produced the most intense labeling, particularly in central pacemaker cells (Figure 7Ba), and membrane staining was evident in a peripheral pacemaker cell (Figure 7Bb). The TRPC4 antibody exhibited regions of intense labeling near the ends of cells, which may indicate colocalization of TRPC4 with gap junctions³³ (Figure 7Ca and 7Cb). TRPC6 labeling of central pacemaker cell was very weak (Figure 7Da), and in the peripheral pacemaker cells sarcomeric banding patterns were often apparent (Figure 7Db). Negative controls (Figure 7c), in which the antibodies were preincubated with their relevant peptide antigen, confirmed the specificity of TRPC isoform labeling.

View larger version (19K): [in this window] [in a new window]   Figure 7. Confocal immunofluorescence images of anti-TRPCs in single pacemaker cells. Central pacemaker cells (Column a) and peripheral pacemaker cells (Column b) were staining with antibodies to TRPC1 (A), TRPC3 (B), TRPC4 (C), and TRPC6 (D), respectively. Column c, Pacemaker cells stained with antibody preincubated with their peptide antigens. Absence of staining demonstrates specificity of the antibodies. DAPI shown in red. Scale bars=25 µm.

	Discussion

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Advantages of the Intact Mouse SAN Preparation for the Study of Pacemaker Function
Mice are of particular value for studying the mechanisms of cardiac pacemaker function because genetically modified models have been developed and are readily available. The mouse SAN is about 300x150 µm¹⁶ and consists of about 450 pacemaker cells, of which only a few act as leading pacemaker cells at any one time.³⁴ Although studies using isolated mice pacemaker cells have provided valuable electrophysiological data,^21,35 practical difficulties remain, because very few cells show spontaneous activity after enzymatic isolation. Furthermore, it is not always possible to distinguish central from peripheral pacemaker cells, although they differ significantly in their electrophysiological and Ca²⁺ handling properties.^16,21

The small size of the SAN in mouse, which makes single cell isolation more difficult, is an advantage for imaging studies. As the SAN is only 50 µm thick and is transparent, its dimensions are compatible with microscopic fields of view. In the present study we were readily able to record changes in [Ca²⁺]_i in intact SANs. The SAN and surrounding regions were identified by their anatomical landmarks and typical pacemaker action potentials (Figure 1). In addition, we demonstrated the expected sensitivity of spontaneous SAN activity to ß-adrenergic stimulation. The use of a Na⁺ channel blocker, which inactivates peripheral SAN activity but leaves firing from the central area intact, further confirmed the distinction between peripheral and central SAN.³⁶ Moreover, the SAN preparation used in this study is relatively easy to obtain, and the pacemaker function persists for many hours. Thus it offers considerable potential for the study of mouse models including those display abnormal pacemaker function.

Intracellular Ca²⁺ Stores and Store-Operated Ca²⁺ Entry in Pacemaker Cells
Although there is growing evidence that diastolic depolarization can be generated by an inward Na⁺/Ca²⁺ exchange current that is related to Ca²⁺-induced Ca⁺ release from the SR,³⁷ it remains debatable whether SR Ca²⁺ release is essential for cardiac pacemaker function.³⁸ In the present study, we found that ryanodine, CPA, and low concentrations of caffeine all slowed pacemaker firing rate, supporting a role for SR Ca²⁺ release in pacemaker function (Figures 2E, 5A, and 5B).

Substantial Ca²⁺ influx could be induced by SR store depletion, which is a characteristic of SOCC activity (compare Figure 3B and 3D). The increase in [Ca²⁺]_i induced by store depletion was not simply attributable to reduced Ca²⁺ buffering, because it was inhibited by 2 SOCC inhibitors, Gd³⁺ and SKF-96365 (Figure 4C and 4D). We also excluded the possibility that Ca²⁺ influx was attributable to L-type Ca²⁺ channels or reverse mode Na⁺/Ca²⁺ exchanger activity, given that Ca²⁺ entry was still evident after blockade of Ca²⁺ channels with nifedipine or inhibition of the exchanger with KBR-7943 (Figure 4A and 4B). Although the specificity of Gd³⁺ and SKF-96365 remains questionable, similar effects were achieved with both SOCC blockers. This supports the notion that store depletion promotes Ca²⁺ influx mediated via SOCC. In addition, the fact that blocking L-type Ca²⁺ channels had no effect on SOCC activity in SAN strengthens our conclusion that Gd³⁺ and SKF96365 were acting mainly through their effect on SOCCs.

TRPC Expression in the SAN
Having characterized the SAN preparation and provided evidence for the involvement of SOCC activity in pacemaker cell function, we investigated whether SOCC activity could be attributable to TRPCs and, if so, which isoform is involved. We found transcripts for all TRPC isoforms, except TRPC5, in the SAN; absence of TRPC5 expression in the heart has been noted in an earlier report.¹⁹

A recent study of adult ventricular myocytes overexpressing TRPC3 showed abundant SOCC activity that was inhibited with SKF-96365.⁵ We found that TRPC3 expression was particularly prominent in central pacemaker cells and that activity in SAN was also sensitive to SKF-96365, suggesting that TRPC3 might contribute to SOCC activity in mouse pacemaker cells. A quantitative measure of mRNA abundance of TRPC3 in various mouse tissues is shown in the online data supplement (supplemental Figure II). There was no significant difference in TRPC3 expression from different regions of the mouse heart. The predominantly cytoplasmic localization of all the TRPC channels is surprising given that the functional channel should be in the membrane. However, this is not an unusual observation as it has been observed in many cell types^30–32 and with 3 different antibodies to TRPC1.³¹ Given that TRPC3 has recently been shown to undergo constitutive cyclical trafficking between the plasma membrane and intracellular sites,³⁹ it is possible that the failure to identify TRPCs membrane localization may be due to a short membrane retention time.

Possible Physiological Role of SOCC in SAN
Ca²⁺ entry is critical for the cell function, and SOCCs have been suggested to mediate Ca²⁺ entry in both excitable and nonexcitable cells.³ However, whether or not SOCCs contribute to cardiac pacemaking on a beat-to-beat basis remains unclear. We found that the SOCC blocker SKF-96563 slowed firing rate and, when SR Ca²⁺ stores were depleted with CPA, pacemaker firing was further slowed (Figure 5). These observations raise the possibility that the inward current through SOCCs can influence heart rate.

SOCCs have been proposed to provide a pacemaker current in other spontaneously firing cells such as neurons⁴⁰ and interstitial cells of Cajal.⁴¹ In embryonic pacemaker cells, before functional pacemaker ionic channels have developed, IP₃-dependent Ca²⁺ influx through SOCCs has been described and under these circumstances is thought to contribute to pacemaking.⁴² Although SOCCs may generate inward currents² and thereby contribute to diastolic depolarization, store content may also affect Ca²⁺ handling, because release of Ca²⁺ is highly dependent on store Ca²⁺ content.⁴³ In addition, the resting level of [Ca²⁺]_i will also affect calcium extrusion via the Na⁺/Ca²⁺ exchanger, which can generate an inward current. Thus the exact timing of SR calcium release and its influence on pacemaker current(s) is likely to be directly related to SR content which will, in turn, reflect the balance between Ca²⁺ entry and extrusion. Our data suggest that SOCCs provide an additional important Ca²⁺ entry pathway to regulate SAN function.

An alternative or additional role for SOCC/TRPC in pacemaker cells is suggested by a recent study showing that TRPC1 also forms a stretch-activated cation channel.⁴⁴ It has been long known that stretch of the atria increases heart rate,⁴⁵ although the mechanism involved is unknown.⁴⁶ If TRPC1 forms a stretch-activated channel in SAN cells, our finding of SOCCs/TRPCs in these cells raises the possibility that TRPC1 may explain the effect of SAN stretch on heart rate. In this regard, TRPC1 has been suggested to function both as a stretch-sensitive channel and a SOCC in dystrophic skeletal muscle.⁴⁷

In conclusion, we have demonstrated the utility of an intact murine SAN preparation that allows both structural and functional studies of the cardiac pacemaker. Our studies demonstrate SOCC activity in the mouse SAN and raise the possibility that TRPC isoforms are responsible for SOCC activity in SAN cells. This SOCC activity may contribute to the regulation of Ca²⁺ entry and pacemaker firing.

	Acknowledgments

 
Sources of Funding

This work was supported by the National Health and Medical Research Council of Australia (Program grant 354400) and the Health Research Council of New Zealand.

Disclosures

None.

	Footnotes

 
Original received June 28, 2006; resubmission received November 8, 2006; revised resubmission received March 16, 2007; accepted April 24, 2007.

	References

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1. Putney JW Jr. A model for receptor-regulated calcium entry. Cell Calcium. 1986; 7: 1–12.[Medline] [Order article via Infotrieve]
2. Hoth M, Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature. 1992; 355: 353–356.[CrossRef][Medline] [Order article via Infotrieve]
3. Parekh AB, Penner R. Store depletion and calcium influx. Physiol Rev. 1997; 77: 901–930.[Abstract/Free Full Text]
4. Seth M, Surnbilla C, Mullen SP, Lewis D, Klein MG, Hussain A, Soboloff J, Gill DL, Inesi G. Sarco(endo)plasmic reticulurn Ca²⁺ ATPase (SERCA) gene silencing and remodeling of the Ca²⁺ signaling mechanism in cardiac myocytes. Proc Natl Acad Sci U S A. 2004; 101: 16683–16688.[Abstract/Free Full Text]
5. Nakayama H, Wilkin BJ, Bodi I, Molkentin JD. Calcineurin-dependent cardiomyopathy is activated by TRPC in the adult mouse heart. FASEB J. 2006; 20: 1660–1670.[Abstract/Free Full Text]
6. Anderson KE, Lambert KA, Woodcock EA. The norepinephrine-stimulated inositol phosphate response in human atria. J Mol Cell Cardiol. 1995; 27: 2415–2419.[CrossRef][Medline] [Order article via Infotrieve]
7. Mackenzie L, Bootman MD, Laine M, Berridge MJ, Thuring J, Holmes A, Li WH, Lipp P. The role of inositol 1,4,5-trisphosphate receptors in Ca²⁺ signalling and the generation of arrhythmias in rat atrial myocytes. J Physiol. 2002; 541: 395–409.[Abstract/Free Full Text]
8. Wu X, Zhang T, Bossuyt J, Li X, McKinsey TA, Dedman JR, Olson EN, Chen J, Brown JH, Bers DM. Local InsP3-dependent perinuclear Ca²⁺ signaling in cardiac myocyte excitation-transcription coupling. J Clin Invest. 2006; 116: 675–682.[CrossRef][Medline] [Order article via Infotrieve]
9. Ju YK, Wu MJ, Chaulet H, Marciniec T, Graham RM, Allen DG. IGF-1 enhances a store-operated Ca²⁺ channel in skeletal muscle myoblasts: involvement of a CD20-like protein. J Cell Physiol. 2003; 197: 53–60.[CrossRef][Medline] [Order article via Infotrieve]
10. Montell C, Rubin GM. Molecular characterization of the Drosophila trp locus: a putative integral membrane protein required for phototransduction. Neuron. 1989; 2: 1313–1323.[CrossRef][Medline] [Order article via Infotrieve]
11. Beech DJ, Xu SZ, McHugh D, Flemming R. TRPC1 store-operated cationic channel subunit. Cell Calcium. 2003; 33: 433–440.[CrossRef][Medline] [Order article via Infotrieve]
12. Rubenstein DS, Lipsius SL. Mechanisms of automaticity in subsidiary pacemakers from cat right atrium. Circ Res. 1989; 64: 648–657.[Abstract/Free Full Text]
13. Ju YK, Allen DG. Intracellular calcium and Na⁺-Ca²⁺ exchange current in isolated toad pacemaker cells. J Physiol. 1998; 508: 153–166.[Abstract/Free Full Text]
14. Bogdanov KY, Vinogradova TM, Lakatta EG. Sinoatrial nodal cell ryanodine receptor and Na⁺-Ca²⁺ exchanger: molecular partners in pacemaker regulation. Circ Res. 2001; 88: 1254–1258.[Abstract/Free Full Text]
15. Ju YK, Huang WB, Jiang L, Barden JA, Allen DG. ATP modulates intracellular Ca²⁺ and firing rate through a P2Y1 purinoceptor in cane toad pacemaker cells. J Physiol. 2003; 552: 777–787.[Abstract/Free Full Text]
16. Verheijck EE, van Kempen MJA, Veereschild M, Lurvink J, Jongsma HJ, Bouman LN. Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution. Cardiovas Res. 2001; 52: 40–50.[Abstract/Free Full Text]
17. Musa H, Lei M, Honjo H, Jones SA, Dobrzynski H, Lancaster MK, Takagishi Y, Henderson Z, Kodama I, Boyett MR. Heterogeneous expression of Ca²⁺ handling proteins in rabbit sinoatrial node. J Histochem Cytochem. 2002; 50: 311–324.[Abstract/Free Full Text]
18. Kao JP. Practical aspects of measuring [Ca²⁺] with fluorescent indicators. In: Methods in Cell Biology(Vol 40): A Practical Guide to the Study of Calcium in Living Cells. Nuccitelli R, ed. 1994. Academic Press, San Diego.
19. Freichel M, Schweig U, Stauffenberger S, Freise D, Schorb W, Flockerzi V. Store-operated cation channels in the heart and cells of the cardiovascular system. Cell Physiol Biochem. 1999; 9: 270–283.[Medline] [Order article via Infotrieve]
20. Camelliti P, Green CR, LeGrice I, Kohl P. Fibroblast network in rabbit sinoatrial node - Structural and functional identification of homogeneous and heterogeneous cell coupling. Circ Res. 2004; 94: 828–835.[Abstract/Free Full Text]
21. Lei M, Jones SA, Liu J, Lancaster MK, Fung SSM, Dobrzynski H, Camelliti P, Maier SKG, Noble D, Boyett MR. Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking. J Physiol. 2004; 559: 835–848.[Abstract/Free Full Text]
22. Flemming R, Xu SZ, Beech DJ. Pharmacological profile of store-operated channels in cerebral arteriolar smooth muscle cells. Brit J Pharmacol. 2003; 139: 955–965.[CrossRef][Medline] [Order article via Infotrieve]
23. Trafford AW, Sibbring GC, Diaz ME, Eisner DA. The effects of low concentrations of caffeine on spontaneous Ca release in isolated rat ventricular myocytes. Cell Calcium. 2000; 28: 269–276.[CrossRef][Medline] [Order article via Infotrieve]
24. Meme W, Leoty C. Cyclopiazonic acid and thapsigargin reduce Ca²⁺ influx in frog skeletal muscle fibres as a result of Ca²⁺ store depletion. Acta Physiol Scand. 2001; 173: 391–399.[CrossRef][Medline] [Order article via Infotrieve]
25. Sanders L, Rakovic S, Lowe M, Mattick PA, Terrar DA. Fundamental importance of Na⁺-Ca²⁺ exchange for the pacemaking mechanism in guinea-pig sino-atrial node. J Physiol. 2006; 571: 639–649.[Abstract/Free Full Text]
26. Ju YK, Allen DG. Does Ca²⁺ release from the sarcoplasmic reticulum influence heart rate? Clin Exp Pharmacol Physiol. 2001; 28: 703–708.[CrossRef][Medline] [Order article via Infotrieve]
27. Merritt JE, Armstrong WP, Benham CD, Hallam TJ, Jacob R, Jaxa-Chamiec A, Leigh BK, McCarthy SA, Moores KE, Rink TJ. SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem J. 1990; 271: 515–522.[Medline] [Order article via Infotrieve]
28. Vazquez G, Wedel BJ, Aziz O, Trebak M, Putney JW Jr. The mammalian TRPC cation channels. Biochim Biophys Acta. 2004; 1742: 21–36.[Medline] [Order article via Infotrieve]
29. Zicha S, Fernandez-Velasco M, Lonardo G, L’Heureux N, Nattel S. Sinus node dysfunction and hyperpolarization-activated (HCN) channel subunit remodeling in a canine heart failure model. Cardiovas Res. 2005; 66: 472–481.[Abstract/Free Full Text]
30. Bergdahl A, Gomez MF, Wihlborg AK, Erlinge D, Eyjolfson A, Xu SZ, Beech DJ, Dreja K, Hellstrand P. Plasticity of TRPC expression in arterial smooth muscle: correlation with store-operated Ca2+ entry. Am J Physiol Cell Physiol. 2005; 288: C872–C880.[Abstract/Free Full Text]
31. Wang X, Pluznick JL, Wei P, Padanilam BJ, Sansom SC. TRPC4 forms store-operated Ca²⁺ channels in mouse mesangial cells. Am J Physiol Cell Physiol. 2004; 287: C357–C364.[Abstract/Free Full Text]
32. Wang W, O’Connell B, Dykeman R, Sakai T, Delporte C, Swaim W, Zhu X, Birnbaumer L, Ambudkar IS. Cloning of Trp1beta isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein. Am J Physiol. 1999; 276: C969–C979.[Medline] [Order article via Infotrieve]
33. Song X, Zhao Y, Narcisse L, Duffy H, Kress Y, Lee S, Brosnan CF. Canonical transient receptor potential channel 4 (TRPC4) co-localizes with the scaffolding protein ZO-1 in human fetal astrocytes in culture. Glia. 2005; 49: 418–429.[CrossRef][Medline] [Order article via Infotrieve]
34. Boyett MR, Dobrzynski H, Lancaster MK, Jones SA, Honjo H, Kodama I. Sophisticated architecture is required for the sinoatrial node to perform its normal pacemaker function. J Cardiovas Electrophysiol. 2003; 14: 104–106.[CrossRef][Medline] [Order article via Infotrieve]
35. Cho HS, Takano M, Noma A. The electrophysiological properties of spontaneously beating pacemaker cells isolated from mouse sinoatrial node. J Physiol. 2003; 550: 169–180.[Abstract/Free Full Text]
36. Yamamoto M, Honjo H, Niwa R, Kodama I. Low-frequency extracellular potentials recorded from the sinoatrial node. Cardiovasc Res. 1998; 39: 360–372.[Abstract/Free Full Text]
37. Vinogradova TM, Maltsev VA, Bogdanov KY, Lyashkov AE, Lakatta EG. Rhythmic Ca²⁺ oscillations drive sinoatrial nodal cell pacemaker function to make the heart tick. Ann N Y Acad Sci. 2005; 1047: 138–156.[Abstract/Free Full Text]
38. Lipsius SL, Bers DM. Cardiac pacemaking: If vs. Ca²⁺, is it really that simple? J Mol Cell Cardiol. 2003; 35: 891–893.[CrossRef][Medline] [Order article via Infotrieve]
39. Smyth JT, Lemonnier L, Vazquez G, Bird GS, Putney JW Jr. Dissociation of regulated trafficking of TRPC3 channels to the plasma membrane from their activation by phospholipase C. J Biol Chem. 2006; 281: 11712–11720.[Abstract/Free Full Text]
40. LeBeau AP, Van Goor F, Stojilkovic SS, Sherman A. Modeling of membrane excitability in gonadotropin-releasing hormone-secreting hypothalamic neurons regulated by Ca²⁺-mobilizing and adenylyl cyclase-coupled receptors. J Neurosci. 2000; 20: 9290–9297.[Abstract/Free Full Text]
41. Walker RL, Koh SD, Sergeant GP, Sanders KM, Horowitz B. TRPC4 currents have properties similar to the pacemaker current in interstitial cells of Cajal. Am J Physiol Cell Physiol. 2002; 283: C1637–C1645.[Abstract/Free Full Text]
42. Mery A, Aimond F, Menard C, Mikoshiba K, Michalak M, Puceat M. Initiation of embryonic cardiac pacemaker activity by inositol 1,4,5-trisphosphate-dependent calcium signaling. Mol Biol Cell. 2005; 16: 2414–2423.[Abstract/Free Full Text]
43. Diaz ME, Graham HK, O’Neill SC, Trafford AW, Eisner DA. The control of sarcoplasmic reticulum Ca content in cardiac muscle. Cell Calcium. 2005; 38: 391–396.[CrossRef][Medline] [Order article via Infotrieve]
44. Maroto R, Raso A, Wood TG, Kurosky A, Martinac B, Hamill OP. TRPC1 forms the stretch-activated cation channel in vertebrate cells. Nat Cell Biol. 2005; 7: 179–185.[CrossRef][Medline] [Order article via Infotrieve]
45. Bainbridge FA. The influence of venous filling upon the rate of the heart. J Physiol. 1915; 50: 65–84.[Free Full Text]
46. Kohl P, Hunter P, Noble D. Stretch-induced changes in heart rate and rhythm: clinical observations, experiments and mathematical models. Prog Biophys Mol Biol. 1999; 71: 91–138.[CrossRef][Medline] [Order article via Infotrieve]
47. Vandebrouck C, Martin D, Colson-Van Schoor M, Debaix H, Gailly P. Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers. J Cell Biol. 2002; 158: 1089–1096.[Abstract/Free Full Text]

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