Fundamental importance of Na+–Ca2+ exchange for the pacemaking mechanism in guinea-pig sino-atrial node
1 University Department of Pharmacology, Mansfield Road, Oxford OX1 3QT, UK
Abstract
Na+–Ca2+ exchange (NCX) current has been suggested to play a role in cardiac pacemaking, particularly in association with Ca2+ release from the sarcoplasmic reticulum (SR) that occurs just before the action potential upstroke. The present experiments explore in more detail the contribution of NCX to pacemaking. Na+–Ca2+ exchange current was inhibited by rapid switch to low-Na+ solution (with Li+ replacing Na+) within the time course of a single cardiac cycle to avoid slow secondary effects. Rapid switch to low-Na+ solution caused immediate cessation of spontaneous action potentials. ZD7288 (3 μM), to block If (funny current) channels, slowed but did not stop the spontaneous activity, and tetrodotoxin (10 μM), to block Na+ channels, had little effect, but in the presence of either of these agents, rapid switch to low-Na+ solution again caused immediate cessation of spontaneous action potentials. Spontaneous electrical activity was also stopped following loading of the cells with the Ca2+ chelators BAPTA and EGTA, and by exposure to the NCX inhibitor KB-R7943 (5 μM). When rapid switch to low-Na+ solution caused cessation of spontaneous activity, this was found (using confocal microscopy, with fluo-4 as the Ca2+ probe) to be accompanied by an initial fall in cytosolic [Ca2+], with subsequent appearance of Ca2+ waves. Inhibition of SR Ca2+ uptake with cyclopiazonic acid (CPA, 30 μM) slowed but did not stop spontaneous activity. Rapid switch to low-Na+ solution in the presence of CPA caused abolition of spontaneous Ca2+ transients and a progressive rise in cytosolic [Ca2+]. With ratiometric fluorescence methods (indo-5F as the Ca2+ probe), the minimum level of [Ca2+] between beats was found to be approximately 225 nM, and abolition of beating with nifedipine, acetylcholine or adenosine caused a fall in cytosolic [Ca2+] below this level. These observations support the hypothesis that NCX current is essential for normal pacemaker activity under the conditions of our experiments. A continuous depolarizing influence of current through the NCX protein might result from maintained electrogenic NCX (with 3:1 stoichiometry, supported by a cytosolic [Ca2+] that normally does not fall below 225 nM between beats) and/or from a novel, recently suggested role of the NCX protein to allow a Na+ leak pathway.
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Introduction
A variety of ionic currents are thought to contribute to pacemaker activity in the sino-atrial (SA) node (Brown, 1982). These include L- and T-type Ca2+ currents (Doerr et al. 1989; Huser et al. 2000), hyperpolarization-activated current (Seyama, 1976; DiFrancesco, 1993), delayed rectifier K+ currents (both rapidly and slowly activating components (Shibasaki, 1987; Sanguinetti & Jurkiewicz, 1990), sustained inward current (Guo et al. 1997) and background current (Hagiwara et al. 1992). Na+–Ca2+ exchange (NCX) is recognized to be important in maintaining the Ca2+ balance of the cell, since it is thought to play a major role in Ca2+ extrusion, but has only recently been identified as a potentially important contributor to the pacemaker depolarization. In the steady state, Ca2+ entering during one part of the cardiac cycle (e.g. via L- and T-type Ca2+ channels) must be extruded at another; if most Ca2+ extrusion is through NCX (and assuming a 3:1 stoichiometry, but see later), this extrusion would be associated with NCX-mediated charge entry (over one cardiac cycle) equal to approximately half that contributed by the Ca2+ entry mechanisms. Thus, NCX current may generate a substantial portion of the inward depolarizing current underlying spontaneous pacemaking.
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A recent study by Kang & Hilgemann (2004) has raised the possibility that the contribution of NCX current to pacemaking may occur not only through the conventionally accepted mode of operation, i.e. Na+ entry and Ca2+ extrusion in a 3:1 stoichiometry, but also through an additional transport mode. Although the average stoichiometry of the NCX process was found to be 3.2 Na+ to 1 Ca2+ in this study, it was also reported that, under some conditions, the NCX protein may provide an inward background Na+ current, occurring because of the import of one Na+ with one Ca2+ ion from the extracellular solution, coupled to the export of one Ca2+ ion from the cytosol. Both these modes of transport result in a net inward current, and so both may potentially contribute to pacemaking in the SA node.
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Experimental evidence supporting a role for NCX in pacemaking has come from several recent studies (Ju & Allen, 1998, 1999; Huser et al. 2000; Vinogradova et al. 2002). In two of these, NCX current was reported as being secondary to a rise in subsarcolemmal Ca2+ immediately preceding the upstroke of the action potential, due to Ca2+ release from the SR (Huser et al. 2000; Vinogradova et al. 2002). This was proposed to act as an amplification mechanism at the foot of the SA node action potential, such that Ca2+ entry during the latter part of the pacemaker depolarization triggers Ca2+-induced release of Ca2+ from the SR, generating a significant inward current carried by NCX.
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This proposed contribution of NCX current, secondary to SR Ca2+ release, is becoming more widely accepted, although some debate concerning the importance of the SR in pacemaking remains (Lakatta et al. 2003; Honjo et al. 2003). What is yet to be considered, however, is that NCX may play a fundamental role throughout the pacemaker depolarization. Previous studies in mammalian tissue have focused on the role of NCX current at the foot of the SA node action potential, and have been limited by the use of superfusion techniques to produce a maintained inhibition of NCX with slow onset; this would alter the Ca2+ dynamics of the cell, and so result in secondary changes in other pacemaker currents that are dependent on cytosolic Ca2+ levels. In this study, we circumvent this problem by using a rapid solution-switching system to inhibit NCX within the time course of a single cardiac cycle, allowing us to investigate the effects of NCX inhibition in the absence of secondary changes in other pacemaker currents. We show that inhibition of NCX by rapid switch to a Li+-containing solution caused immediate cessation of beating, demonstrating the fundamental importance of NCX to pacemaking. This is in stark contrast to the effects of inhibition of other established pacemaking currents, such as If(funny current), which result only in a slowing of beating, but not a cessation. The abolition of spontaneous activity on inhibition of NCX was associated with a fall in cytosolic Ca2+ to a level below the minimal level observed between calcium transients in a spontaneously beating cell. Furthermore, a critical contribution of NCX to pacemaking was observed even in the absence of a functional SR, demonstrating that this current is not exclusively dependent on SR Ca2+ release.
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We propose that, in spontaneously beating cells, the cytosolic Ca2+ concentration is always at a level higher than that in resting cells, and that this elevated Ca2+ level supports a level of NCX current throughout the pacemaker potential that is critical to spontaneous beating in these cells. The fundamental importance of NCX for pacemaking may arise from conventional electrogenic NCX (with 3 Na+ ions entering in exchange for 1 Ca2+ ion) and/or from the continuous Na+ leak pathway proposed by Kang & Hilgemann (2004). The precise contribution of each of these mechanisms remains for future study.
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Methods
Isolation of cardiac cells
Male guinea-pigs (weighing 350–500 g) were killed by cervical dislocation following stunning in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 (T.S.O.). Sino-atrial node cells were isolated as previously described (Rigg et al. 2000).
Rapid application of drugs using a local perfusion system
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Solution switches were made using a local perfusion system (Warner Instrument Corp., Hamden, CT, USA) to ensure rapid application of drugs. A triple-barrelled square glass capillary tube was positioned within 100 μm of the cell under study, such that the cell was bathed in solution (36°C) flowing from only one barrel. Lateral movement of the glass capillary tube (driven by a stepper motor), caused the cell to be bathed with solution from a different barrel (changeover < 0.5 s). Solution flow was 100 μl min–1 through each barrel (driven by a syringe pump). Solutions contained (mM): NaCl, 118.5; NaHCO3, 14.5; KCl, 4.2; KH2PO4, 1.18; MgSO4, 1.18; CaCl2, 2.5; and glucose, 11.1; gassed with 95% O2–5% CO2 to maintain a pH of 7.4; NaCl was replaced with LiCl in low-Na+ solutions. Cyclopiazonic acid (CPA), nifedipine (Sigma-Aldrich), BAPTA AM (acetoxy methyl ester form), EGTA AM (Molecular Probes) and ZD7288 (Tocris) were made up as 1–20 mM stock solutions in dimethyl sulphoxide (DMSO) and diluted to the required concentration immediately before use. The maximal concentration of DMSO used in any of these experiments was 0.1%, and was without significant effect on the parameters studied.
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Electrophysiology
Action potentials were recorded using the perforated patch clamp technique (Axopatch 200 amplifier, Axon Instruments). Patch pipettes (3–5 M) contained (mM): KCl, 150; MgCl2, 5; K2ATP, 1; Hepes, 3; pH adjusted to 7.2 with KOH; perforation using amphotericin (Sigma-Aldrich, 250 μg ml–1) occurred 5–10 min after seal formation.
Calcium imaging and calcium transients
For confocal microscopy experiments, cells were incubated with fluo-4 AM (1–3 μM) for 10 min at 36°C; > 10 min was allowed for de-esterification of intracellular fluo-4 AM. A Leica TCS NT confocal scanning head was coupled to a DMIRB microscope with a 63x water immersion objective (excitation with 488 nm Ar laser, and emitted fluorescence collected using a 515 nm long-pass filter). Line scan imaging was used to maximize temporal resolution; a single line was repeatedly scanned at an acquisition rate of 385 Hz (2.6 ms per line).
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In some experiments a photomultiplier system (Cairn Instruments, Faversham, UK) was used to record Ca2+ transients in cells incubated with indo-5F AM (Molecular Probes, 4 μM for 10 min), with excitation at 340 nm, and emission collected at 410 and 490 nm. Indo-5F AM was chosen because indo-1 has been shown to slow or stop spontaneous beating in these cells (Rigg et al. 2000). The cytosolic Ca2+ concentration, [Ca2+]i, was calculated from the 410/490 fluorescence signal ratio, r, using the equation:
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where =Fmax/Fmin measured at 490 nm (the ratio of the maximum and minimum fluorescence measured at zero and saturating Ca2+), and KD is the dissociation equilibrium constant between Ca2+ and indo-5F AM (taken as 470 nM; Handbook of Fluorescent Probes and Research Products, ninth edn, Molecular Probes); 410/490 fluorescence ratios for zero Ca2+ and saturating Ca2+ are given by rmin and rmax, while r represents the 410/490 fluorescence ratio measured as a variable during the experiments. An in vivo calibration method was used to determine rmin, rmax, Fmin and Fmax; cells permeabilized with the ionophore ionomycin (in the presence of CPA, to inhibit the SR Ca2+-ATPase, and KB-R7943, to inhibit NCX) were exposed to solutions containing 0 and 5 mM Ca2+, and fluorescence measurements recorded. An adjustment for background fluorescence was also made.
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Statistics
Data are expressed as means ±S.E.M. Statistical significance was evaluated using Student's paired t test. A level of P < 0.05 was considered to be statistically significant.
Results
Inward NCX current was inhibited by reduction of the extracellular concentration of Na+ ions, from 133 to 14.5 mM, by replacement with Li+ (Le Guennec & Noble, 1994; Janvier et al. 1997). Rapid switch to low-Na+ solution caused immediate cessation of spontaneous electrical activity, with at most only one beat occurring after the switch (representative examples in Fig. 1A and B). If the time of exposure to low Na+ was kept short, spontaneous activity could be restarted by rapid switch back to normal Na+ solution. Interestingly, despite the absence of spontaneous action potentials, exposure of cells to low-Na+ solution for more than 3–4 s resulted in the appearance of spontaneous contractions; these did not have the same characteristics as those accompanying spontaneous action potentials (in normal Na+ solution), but were ‘wave like’ in nature. Similar observations were made in five cells, supporting the proposal that NCX makes an essential contribution to pacemaking.
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A and B, rapid switch to low-Na+ solution (during the time indicated by the bar) caused immediate cessation of spontaneous action potentials. Action potentials speedily reappeared following rapid switch back to normal Na+ solution. C, superfusion with the Na+ channel blocker TTX (10 μM) caused a slight slowing in beating rate in this cell. Subsequent rapid switch to low-Na+ solution resulted in an abolition of spontaneous action potentials, with recovery on washout. D, action potentials recorded in the presence of the If blocker ZD7288 (3 μM), which slowed, but did not stop, spontaneous activity. In this cell, ZD7288 reduced the frequency of spontaneous action potential occurrence by 18%, from 201 to 165 beats min–1. Under these conditions, rapid switch to low-Na+ solution again caused cessation of spontaneous activity.
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In a further seven SA node cells, in which spontaneous contractile activity was monitored via a video camera attached to the microscope (but without a patch electrode attached for electrical recording), rapid switch to low-Na+ solution again caused immediate cessation of beating, with at most one beat occurring after the move to low-Na+ solution. Following a period of cessation of beating (2.71 ± 0.64 s, n= 7, P < 0.05) that was considerably greater than the control cycle length (0.60 ± 0.12 s, n= 7, P < 0.05), contractile activity recommenced in low-Na+ solution, but it was apparent that the restored contractions differed from normal ones, resembling wave-like contractions (see below).
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Li+ ions are known to pass through voltage-gated Na+ channels almost as well as Na+ ions (Chandler and Meves, 1965; Hille, 1972), and voltage-gated Na+ channels are thought not to play a major role in pacemaker activity in the majority of mammalian species (but see Lei et al. 2005 for recent observations on mouse SA node). It therefore seems unlikely that the cessation of beating observed on substituting Li+ for Na+ can be ascribed to effects mediated by voltage-gated Na+ channels. Nevertheless, this possibility was tested in experiments in which tetrodotoxin (TTX) was used to block voltage-gated Na+ channels. Figure 1C shows an example trace where application of TTX (10 μM) caused little or no change in the rate of beating, but rapid switch to low-Na+ solution (with Li+ as the replacement, as above) caused immediate cessation of beating that was reversible on restoration of normal Na+ levels. Similar observations were made in a further four cells.
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While switch to low-Na+ solution would be expected to suppress inward NCX current, another possibility to be considered is that If currents activated by hyperpolarization would also be reduced, since Li+ ions are thought to permeate these channels less well than Na+ ions (Ho et al. 1994). To explore whether this could account for the cessation of beating, the effects of the selective If inhibitor ZD7288 (BoSmith et al. 1993) were investigated. In our experiments, ZD7288 (3 μM) was found to reduce If (measured at –80 mV under voltage-clamp conditions) by 73 ± 8% (n= 3); this is comparable to the 78 ± 4% decrease in If observed (at –120 mV) in guinea-pig SA node cells by BoSmith et al. (1993), using 1 μM ZD7288. Despite this substantial inhibition of If, a rapid switch to 3 μM ZD7288 caused only a 14 ± 3% slowing of the beating rate in six further cells studied (P < 0.05) and did not stop the initiation of spontaneous action potentials. However, rapid switch to low-Na+ solution in the presence of 3 μM ZD7288 again caused abolition of spontaneous action potentials in all six cells (Fig. 1D).
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As an alternative method of suppressing NCX current, guinea-pig SA node cells were exposed to KB-R7943, which has been shown to inhibit NCX in guinea-pig cardiac myocytes (Iwamoto et al. 1996; Watano et al. 1996). Rapid switch to KB-R7943 (5 μM) caused cessation of spontaneous beating of SA node cells (n= 8). The time taken for cessation of activity (19.5 ± 3.6 s) was not as rapid as was the case for a switch to low-Na+ solution, but this might be expected if the time taken for this drug to reach the membrane and exert its inhibitory action was slower than was the case in experiments where Li+ was used to replace Na+.
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The effects of low Na+ were explored further in cells loaded with the Ca2+ indicator fluo-4 AM and imaged using confocal microscopy (in the linescan configuration). A series of images from a representative experiment are shown in the upper panel of Fig. 2A. Regular Ca2+ transients, with Ca2+ rising approximately synchronously along the cell, were observed in normal Na+ solution (left panel). A rapid switch to low Na+ occurred just before the next panel, where it can be seen that spontaneous whole cell Ca2+ transients had been completely suppressed. Towards the end of the period of recording shown in the third panel, spontaneous activity resumed, but this was in the form of a Ca2+ wave (starting at a position at the top of the panel and propagating downwards); the next panel shows several more Ca2+ waves (broader and less uniform in magnitude than normal Ca2+ transients). On switching back to normal Na+ solution, activity started to recover, and the final panel shows restoration of spontaneous whole cell Ca2+ transients with synchronous Ca2+ changes along the cell.
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A, the upper panel shows linescan images acquired using confocal microscopy with fluo-4 AM as the Ca2+ probe; distance is represented vertically (bar represents 20 μM), and time horizontally. The lower panel shows Ca2+ transients derived by averaging the fluorescence signal along each scanned line. Successive frames are presented, the first showing Ca2+ transients under control conditions, arising synchronously along the cell. Low-Na+ solution was applied immediately before the second frame, which shows that Ca2+ transients were abolished by the switch to low-Na+ solution; note that cytosolic Ca2+ fell following cessation of activity in low-Na+ solution. The third frame shows a continued fall in cytosolic Ca2+, followed by a Ca2+ wave that starts at the top of the image and propagates downwards. More Ca2+ waves are evident in the fourth frame. Normal Ca2+ transients were quickly restored on return to normal Na+ solution (fifth frame). B, Ca2+ transients were also recorded using indo-5F AM (measured as the ratio of emission at 410 nm to that at 490 nm). Rapid switch to low-Na+ solution again caused cessation of beating and a fall in cytosolic Ca2+, as indicated by the fluorescence ratio. Similar observations were made in a further 7 cells.
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Total fluorescence in these panels was quantified by averaging the signal across the cell and is shown by the traces in the lower panel of Fig. 2A. The red horizontal dashed line indicates the minimum level of fluo-4 AM fluorescence between beats. Note that abolition of spontaneous activity by the switch to low-Na+ solution caused a fall in cytosolic [Ca2+] below the minimum diastolic level observed when the cell was beating (red horizontal dashed line). Similar observations were made in six cells, and when spontaneous activity was halted by a rapid switch to low-Na+ solution, the mean fall in integrated fluo-4 AM fluorescence signal was to 77 ± 5% of the control minimum level between beats (n= 6, P < 0.05).
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In a further eight cells, Ca2+ transients were measured as indo-5F AM 410/490 nm ratios, allowing quantitative measurements of intracellular Ca2+ concentrations to be obtained following a calibration procedure. As shown in Fig. 2B, rapid switch to low-Na+ solution again caused both cessation of beating and a fall in cytosolic Ca2+ to a level 15 ± 2% (n= 8, P < 0.05) below the minimum diastolic Ca2+ concentration in a beating cell. Minimum diastolic Ca2+ concentration was found to be 225 ± 55 nM in beating cells, and the lowest Ca2+ concentration observed during cessation of beating was 198 ± 72 nM.
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This fall in cytosolic [Ca2+] seems surprising, since low-Na+ solution is expected to inhibit the major pathway of Ca2+ extrusion via NCX; however, Ca2+ might continue to be sequestered by the SR, leading to a fall of Ca2+ in the absence of significant continued Ca2+ entry through voltage-gated channels (since spontaneous electrical activity was abolished). If this were correct, the fall in cytosolic [Ca2+] on switch to low-Na+ solution might not be seen in cells exposed to cyclopiazonic acid (CPA), an inhibitor of SR Ca2+ uptake (Seidler et al. 1989). In experiments using confocal microscopy, application of 30 μM CPA slowed the frequency of, but did not stop, the occurrence of spontaneous Ca2+ transients (frequency reduced by 30 ± 7%; n= 6, P < 0.05). Figure 3A shows that subsequent rapid switch to low-Na+ solution (in the presence of CPA) again caused rapid cessation of spontaneous Ca2+ transients but, in contrast to the observations when CPA was absent, there was a consistent increase in cytosolic [Ca2+] (as indicated by an increase in fluo-4 AM fluorescence; see Fig. 4A). Similar observations were made in all six cells in which fluo-4 AM was used as the Ca2+ indicator, and in a further six cells loaded with indo-5F AM.
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A, Ca2+ transients recorded using confocal microscopy, with fluo-4 AM as the Ca2+ indicator. Application of CPA (30 μM) slowed, but did not stop, the occurrence of spontaneous Ca2+ transients. Note that with CPA present, the Ca2+ transients appeared smaller in magnitude, and slower to both peak and decay, compared with those in control conditions. Rapid switch to low-Na+ solution abolished spontaneous Ca2+ transients, and this was associated with a rise, rather than a fall, in cytosolic Ca2+. B, another cell, showing similar effects of CPA on spontaneous Ca2+ transients. In this cell, rapid switch to KB-R7943 (5 μM), in the continued presence of CPA, resulted in an abolition of Ca2+ transients, with no increase in baseline Ca2+. Subsequent rapid application of low-Na+ solution, in the presence of KB-R7943, did not result in an increase in baseline Ca2+ levels.
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A, spontaneous action potentials recorded from a SA node cell. A rapid switch to 50 mM Na+ solution (bar) caused cessation of spontaneous beating, which returned on washout. Similar observations were made in 3 cells. B, a rapid switch to 75 mM Na+ solution also abolished spontaneous action potentials for approximately the first 3 s of application; two action potentials, of abnormally large amplitude, were subsequently recorded in this cell during application of low-Na+ solution. In two further cells, spontaneous action potentials were completely abolished for the entire period of application of 75 mM Na+ solution. C, spontaneous Ca2+ transients recorded from a representative cell using confocal microscopy (fluo-4 AM). A rapid switch to 50 mM Na+ solution abolished spontaneous Ca2+ transients. In this cell, a localized increase in intracellular Ca2+, accompanied by a gradual global increase in Ca2+, was observed; this may represent Ca2+ release from an overloaded SR. Similar observations were made in 6 cells. D, rapid switch to 75 mM Na+ solution also abolished spontaneous Ca2+ transients. Similar observations were made in 8 cells.
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There are several potential pathways that may underlie the cytosolic elevation of Ca2+ observed on application of low-Na+ solution in the presence of CPA. One possibility is that, on application of low Na+ (at this concentration) NCX reverses, bringing Ca2+ into the cell. To test for this possibility, KB-R7943 was applied in the presence of CPA, since KB-R7943 has been reported to inhibit both forward and reverse modes of NCX (Iwamoto et al. 1996; Watano et al. 1996). As may be seen in Fig. 3B, application of KB-R7943, in the presence of CPA, resulted in a cessation of spontaneous Ca2+ transients, but with little or no rise in baseline Ca2+ levels. A subsequent rapid switch to low-Na+ solution also resulted in little or no change in baseline fluorescence. It may therefore be the case that application of low-Na+ solution in the presence of CPA results in reverse-mode NCX that brings Ca2+ into the cell, causing a gradual elevation of cytosolic [Ca2+].
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If it is the case that NCX reverses on switch to 14.5 mM Na+ solution, this raises the possibility that the cessation of beating on switch to 14.5 mM Na+ solution may not only be due to the removal of an inward current normally contributed by NCX during the pacemaker depolarization, but also due to the imposition of an outward current, due to NCX operating in the Ca2+ influx mode. Indeed, calculation of the reversal potential for NCX (3ENa– 2ECa, where these represent the Nernst equilibrium potentials for Na+ and Ca2+, respectively), assuming intracellular Na+ and Ca2+ concentrations of 7 mM and 225 nM, respectively, yields a value of –190 mV at 14.5 mM extracellular Na+. Hence, following switch to 14.5 mM Na+, NCX would be expected to be operating in reverse-mode at the resting membrane potentials recorded from our SA node cells. Although the aforementioned effects of KB-R7943, which inhibits reverse-mode NCX as well as the conventional mode, do not support the possibility that it is imposition of an outward current that abolishes beating in these cells, this was nonetheless further tested in experiments in which cells were exposed to 50 and 75 mM Na+ solutions. The NCX reversal potentials at 50 and 75 mM Na+ are calculated to be –90 and –59 mV, and so it would be expected that the contribution of any outward current by reverse-mode NCX would be significantly less under these conditions; indeed, a rapid switch to 75 mM Na+ would be expected to result in very little, if any, reversal of NCX. As shown in Fig. 4, rapid switch to either of these concentrations of Na+ resulted in an immediate cessation of both spontaneous action potentials and spontaneous Ca2+ transients. Thus, it is highly likely that the abolition of beating on switch to low-Na+ solution is due to inhibition of an inward current that is normally contributed by NCX during the pacemaker depolarization, and not primarily due to imposition of an outward current.
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If cytosolic [Ca2+] between beats remains at a sufficiently high level that Ca2+ extrusion by NCX contributes inward current that is essential for spontaneous pacemaker activity, an alternative method of suppressing the inward NCX current (and therefore spontaneous activity) would be to apply a Ca2+ chelator, such as BAPTA, to the cytosol to lower [Ca2+]. As illustrated in Fig. 5, application of membrane-permeant BAPTA AM (10 μM), whether by rapid switch (n= 6; data not shown) or by superfusion (n= 6), caused abolition of spontaneous action potentials. A similar cessation of activity was observed in three cells loaded with another Ca2+ chelator, EGTA, by exposure to EGTA AM (10 μM; data not shown).
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Two examples of superfusion with BAPTA AM (10 μM) resulting in abolition of spontaneous action potentials. Note the relatively slow onset of inhibition of beating, presumably reflecting the time taken for diffusion of BAPTA AM into the cell, and subsequent de-esterification.
The data presented above support the possibility that the minimum diastolic level of [Ca2+] between beats may be maintained at a higher level than the resting [Ca2+] in quiescent cells, resulting in a contribution of NCX current throughout the entire pacemaker depolarization. To assess this directly, 5 μM nifedipine was rapidly applied to spontaneously beating SA node cells imaged using confocal microscopy, in order to prevent the generation of action potentials (L-type Ca2+ channels are known to underlie the upstroke of the SA node action potential). As shown in Fig. 6A, abolition of spontaneous beating with nifedipine was associated with a significant decrease in the fluo-4 AM fluorescence level, as compared to minimum levels recorded between spontaneous Ca2+ transients; this was a consistent observation in all six cells studied. Similar falls in Ca2+ levels were observed when either acetylcholine (1 μM, n= 6) or adenosine (1 μM, n= 6) were used to stop spontaneous beating (Fig. 6B and C). This supports the hypothesis that, during beating, cytosolic [Ca2+] concentrations remain higher between beats than those that occur in quiescent cells.
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A, nifedipine, 5 μM (confocal microscopy, fluo-4 AM). B, adenosine, 1 μM (confocal microscopy, fluo-4 AM). C, acetylcholine, 1 μM (conventional fluorescence microscopy, indo-5F AM).
Discussion
The observations reported here provide an important extension to previous work demonstrating a role for Ca2+, including that released from the SR, in pacemaking mechanisms (Rigg & Terrar, 1996; Ju & Allen, 1998; Huser et al. 2000; Rigg et al. 2000; Vinogradova et al. 2002). Studies in other species have reported that a rise in subsarcolemmal [Ca2+], associated with SR Ca2+ release that precedes the upstroke of the action potential (perhaps triggered by T-type Ca2+ current), leads to depolarizing NCX current that contributes a depolarizing influence at this time (Huser et al. 2000; Bogdanov et al. 2001; Vinogradova et al. 2002). The data presented here, however, demonstrate that the contribution of NCX current to the pacemaker depolarization of guinea-pig SA node cells is not exclusively dependent on a functional SR, since a switch to low-Na+ solution abolished spontaneous beating in the presence of CPA, an inhibitor of SR Ca2+ uptake. Furthermore, if the only contribution of NCX were secondary to SR Ca2+ release, inhibition of SR function might be expected to produce effects on pacemaking that were broadly similar to inhibition of NCX. In guinea-pig SA node cells, however, suppression of SR function is associated with a slowing of rate (Rigg & Terrar, 1996; Rigg et al. 2000; Lakatta et al. 2003), whereas inhibition of NCX abolishes beating. Thus, the essential contribution that NCX makes to pacemaking in this preparation is not limited to current (at the foot of the action potential) that is secondary to localized SR Ca2+ release, but is also due to a significant component that is SR independent. The timing of this SR-independent component need not be limited to the foot of the action potential, but instead may occur throughout the pacemaker depolarization.
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Our experiments using rapid switch to low-Na+ solution to inhibit NCX provide important insights concerning the importance of NCX current and extend the interesting observations of Bogdanov et al. (2001). These authors showed that beating stopped soon after Na+ was replaced by Li+ in the solution bathing rabbit SA node cells, but cytosolic [Ca2+] was not simultaneously monitored in these experiments and, as the authors pointed out, it might be argued that such continuous superfusion with Li+ induces an increase in steady cytosolic [Ca2+] during the diastolic depolarization, which in turn may affect SR loading, refractoriness of the ryanodine receptor, or currents involved in automaticity. In further experiments in which cytosolic [Ca2+] was measured, a rapid ‘spritz’ of low-Na+ solution was found to suppress action potential duration, though a large (approximately 70% of normal) Ca2+ transient (or wave) remained. In our experiments, rapid application of low-Na+ solution consistently suppressed activity within one beat, but a different type of spontaneous activity reappeared after an interval of many seconds, in the form of Ca2+ waves, most likely secondary to spontaneous release of Ca2+ from an overloaded SR (Fig. 2). Our experiments in guinea-pig SA node cells thus differ significantly from those of Bogdanov and co-workers in that rapid application of low-Na+ solution was continued for a sufficiently long period to completely stop spontaneous electrical activity and Ca2+ transients, and (prior to the initiation of Ca2+ waves) this was not accompanied by an elevation of cytosolic [Ca2+].
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Another important observation, shown in Fig. 2, is the fall in cytosolic [Ca2+] that followed cessation of beating in the presence of low-Na+ solution, occurring even though a major mechanism for Ca2+ extrusion (NCX) was suppressed. The mechanism underlying this may involve Ca2+ uptake into the SR, mediated by the Ca2+-ATPase, continuing until the SR becomes overloaded, resulting in spontaneous Ca2+ release and Ca2+ waves. This was supported by the observation that, when SR uptake was inhibited by CPA, a rapid switch to low-Na+ solution again suppressed spontaneous activity, but this was followed by a rise, rather than a fall, in cytosolic [Ca2+]. Hence, when the SR is functioning normally, SR Ca2+ uptake can reduce cytosolic [Ca2+] immediately following cessation of activity due to NCX inhibition.
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Several potential pathways may contribute the Ca2+ that underlies the elevation in cytosolic levels observed on switch to low-Na+ solution in the presence of CPA. The lack of such an elevation of Ca2+ when KB-R7943 is used to inhibit NCX (in the presence of CPA) supports a role for reverse-mode NCX in providing at least some of this Ca2+. This raises the possibility that abolition of spontaneous activity by low-Na+ solution may be due not only to removal of an inward current (i.e. NCX working in the Ca2+ extrusion mode), but also to the imposition of an outward current (reverse-mode NCX) on the pacemaker depolarization. However, since KB-R7943, which inhibits both modes of NCX, completely abolished pacemaker activity, it is highly likely that inhibition of inward NCX current alone is sufficient to cause cessation of spontaneous activity. This is supported further by the observations that beating was abolished by 50 and 75 mM Na+ solutions; for the latter concentration, little or no outward current would be expected, since the NCX reversal potential (–59 mV) is calculated to be close to the minimum diastolic potential of these cells.
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The hypothesis that the cytosolic [Ca2+] remains elevated between beats in spontaneously active SA node cells is supported by our observations that abolition of beating by three differing pharmacological mechanisms (nifedipine, acetylcholine and adenosine) resulted in a fall in [Ca2+] to a level below the minimum recorded between spontaneous Ca2+ transients. The Ca2+ chelators BAPTA and EGTA, which would be expected to greatly reduce the minimum level of cytosolic Ca2+ (regardless of whether the major source was from the SR or Ca2+ entry across the sarcolemma) and hence NCX current, also caused cessation of spontaneous action potentials. It therefore appears that, under the conditions of these experiments, the sequential activation of voltage-dependent ion channels is not by itself sufficient to maintain spontaneous action potentials in the face of an abnormally low cytosolic [Ca2+].
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Another possible effect of low-Na+ solution that must be considered is inhibition of hyperpolarization-activated If currents, since Li+ ions have been reported to permeate less well through these channels (Ho et al. 1994). However, previous experiments have shown that complete block of If slows but does not stop spontaneous beating (Denyer & Brown, 1990). We have also shown that ZD7288 slows but does not prevent initiation of spontaneous action potentials, despite substantial inhibition of If current, but that rapid switch to low-Na+ solution under these conditions again leads to cessation of activity. Furthermore, Bogdanov et al. (2001) have reported that Li+ did not affect If in their experiments. It is therefore unlikely that the effects of low-Na+ solution are due to actions on If. An alternative possibility is that low-Na+ solution might interfere with the depolarizing influence of Ist (sustained inward current; Guo et al. 1995, 1996, 1997), but this seems unlikely, since the channel allows permeation by Na+ and K+ (Guo et al. 1996), and Li+ would also be expected to pass through this channel. It is also unlikely that actions of Li+ on TTX-sensitive voltage-gated Na+ channels account for the abolition of pacemaking, since Li+ passes through these channels almost as easily as Na+ (Chandler & Meves, 1965; Hille, 1972). Furthermore, in our experiments, TTX had little or no effect on pacemaking, while switch to low-Na+ solution in the presence of TTX caused immediate cessation of beating.
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It therefore appears that the major factor suppressing pacemaker activity following switch to low-Na+ solution is inhibition of inward NCX current, either in the form of NCX in its conventional 3 Na+ to 1 Ca2+ ion mode, or in the form of a Na+ leak pathway, as proposed by Kang & Hilgemann (2004), in which import of one Na+ with one Ca2+ ion coupled to export of one Ca2+ ion provides a Na+-conducting pathway. The abolition of spontaneous activity by the Ca2+ chelators BAPTA and EGTA (also seen following excessive loading with Ca2+ indicators, as noted previously by Rigg et al. 2000) is consistent with inhibition of either mode of operation of NCX. The levels of Ca2+ between beats were found to be higher (225 nM) than those in quiescent cells (or in resting atrial and ventricular myocytes), making it likely that there is a maintained component of electrogenic 3:1 NCX throughout the pacemaker depolarization, even if there is an additional Na+ leak through the NCX protein. The mechanisms underlying this maintained elevation of intracellular Ca2+ remain to be determined.
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Although voltage-clamp experiments to measure NCX currents would potentially yield additional information regarding the importance of this current to pacemaking, they are beyond the scope of this study. Simple voltage-clamp experiments would not be helpful, partly because even for conventional 3:1 exchange, the level of currents in a beating cell would vary dynamically with changes in cytosolic Ca2+ during the cardiac cycle, and this would necessarily be modified with conventional voltage-clamp protocols. Furthermore, the contributions from the new modes of operation of NCX require complex experiments that are beyond the scope of this investigation.
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Experiments in whole animal NCX knockout mice (Wakimoto et al. 2000; Cho et al. 2000; Koushik et al. 2001; Reuter et al. 2002) have failed to clarify the role of NCX in pacemaking, since embryos in these models did not survive beyond about 11 days post coitum, and the heartbeat was absent. In the light of the present experiments, it is possible that the lack of heartbeat in these knockout mice may be due to the absence of an essential contribution of NCX during the pacemaker potential. However, other harmful effects of NCX knockout cannot be excluded. It should be noted that the NCX1 knockout described recently by Henderson et al. (2004) is specific to the ventricle, and so provides no insight into the role of NCX in pacemaking.
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Of course, other currents are known to contribute to the pacemaker depolarization, and some of these may be regulated by cytosolic [Ca2+], including If and delayed rectifier K+ currents (Tohse, 1990; Rigg et al. 2003). However, inhibition of many of the ‘established’ pacemaking currents is associated only with a slowing of beating rate in guinea-pig SA node cells, rather than a cessation. The observations reported here are consistent with a fundamental and critical role for NCX in maintaining a depolarizing influence throughout the pacemaker depolarization. We propose that this maintained depolarizing influence of NCX is secondary to a high minimum level of cytosolic [Ca2+] that normally never falls below that which supports Ca2+ extrusion via electrogenic 3:1 NCX, but this mechanism may also be supplemented by a contribution arising from a Na+ leak pathway through the NCX protein.
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References
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Abstract
Na+–Ca2+ exchange (NCX) current has been suggested to play a role in cardiac pacemaking, particularly in association with Ca2+ release from the sarcoplasmic reticulum (SR) that occurs just before the action potential upstroke. The present experiments explore in more detail the contribution of NCX to pacemaking. Na+–Ca2+ exchange current was inhibited by rapid switch to low-Na+ solution (with Li+ replacing Na+) within the time course of a single cardiac cycle to avoid slow secondary effects. Rapid switch to low-Na+ solution caused immediate cessation of spontaneous action potentials. ZD7288 (3 μM), to block If (funny current) channels, slowed but did not stop the spontaneous activity, and tetrodotoxin (10 μM), to block Na+ channels, had little effect, but in the presence of either of these agents, rapid switch to low-Na+ solution again caused immediate cessation of spontaneous action potentials. Spontaneous electrical activity was also stopped following loading of the cells with the Ca2+ chelators BAPTA and EGTA, and by exposure to the NCX inhibitor KB-R7943 (5 μM). When rapid switch to low-Na+ solution caused cessation of spontaneous activity, this was found (using confocal microscopy, with fluo-4 as the Ca2+ probe) to be accompanied by an initial fall in cytosolic [Ca2+], with subsequent appearance of Ca2+ waves. Inhibition of SR Ca2+ uptake with cyclopiazonic acid (CPA, 30 μM) slowed but did not stop spontaneous activity. Rapid switch to low-Na+ solution in the presence of CPA caused abolition of spontaneous Ca2+ transients and a progressive rise in cytosolic [Ca2+]. With ratiometric fluorescence methods (indo-5F as the Ca2+ probe), the minimum level of [Ca2+] between beats was found to be approximately 225 nM, and abolition of beating with nifedipine, acetylcholine or adenosine caused a fall in cytosolic [Ca2+] below this level. These observations support the hypothesis that NCX current is essential for normal pacemaker activity under the conditions of our experiments. A continuous depolarizing influence of current through the NCX protein might result from maintained electrogenic NCX (with 3:1 stoichiometry, supported by a cytosolic [Ca2+] that normally does not fall below 225 nM between beats) and/or from a novel, recently suggested role of the NCX protein to allow a Na+ leak pathway.
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Introduction
A variety of ionic currents are thought to contribute to pacemaker activity in the sino-atrial (SA) node (Brown, 1982). These include L- and T-type Ca2+ currents (Doerr et al. 1989; Huser et al. 2000), hyperpolarization-activated current (Seyama, 1976; DiFrancesco, 1993), delayed rectifier K+ currents (both rapidly and slowly activating components (Shibasaki, 1987; Sanguinetti & Jurkiewicz, 1990), sustained inward current (Guo et al. 1997) and background current (Hagiwara et al. 1992). Na+–Ca2+ exchange (NCX) is recognized to be important in maintaining the Ca2+ balance of the cell, since it is thought to play a major role in Ca2+ extrusion, but has only recently been identified as a potentially important contributor to the pacemaker depolarization. In the steady state, Ca2+ entering during one part of the cardiac cycle (e.g. via L- and T-type Ca2+ channels) must be extruded at another; if most Ca2+ extrusion is through NCX (and assuming a 3:1 stoichiometry, but see later), this extrusion would be associated with NCX-mediated charge entry (over one cardiac cycle) equal to approximately half that contributed by the Ca2+ entry mechanisms. Thus, NCX current may generate a substantial portion of the inward depolarizing current underlying spontaneous pacemaking.
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A recent study by Kang & Hilgemann (2004) has raised the possibility that the contribution of NCX current to pacemaking may occur not only through the conventionally accepted mode of operation, i.e. Na+ entry and Ca2+ extrusion in a 3:1 stoichiometry, but also through an additional transport mode. Although the average stoichiometry of the NCX process was found to be 3.2 Na+ to 1 Ca2+ in this study, it was also reported that, under some conditions, the NCX protein may provide an inward background Na+ current, occurring because of the import of one Na+ with one Ca2+ ion from the extracellular solution, coupled to the export of one Ca2+ ion from the cytosol. Both these modes of transport result in a net inward current, and so both may potentially contribute to pacemaking in the SA node.
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Experimental evidence supporting a role for NCX in pacemaking has come from several recent studies (Ju & Allen, 1998, 1999; Huser et al. 2000; Vinogradova et al. 2002). In two of these, NCX current was reported as being secondary to a rise in subsarcolemmal Ca2+ immediately preceding the upstroke of the action potential, due to Ca2+ release from the SR (Huser et al. 2000; Vinogradova et al. 2002). This was proposed to act as an amplification mechanism at the foot of the SA node action potential, such that Ca2+ entry during the latter part of the pacemaker depolarization triggers Ca2+-induced release of Ca2+ from the SR, generating a significant inward current carried by NCX.
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This proposed contribution of NCX current, secondary to SR Ca2+ release, is becoming more widely accepted, although some debate concerning the importance of the SR in pacemaking remains (Lakatta et al. 2003; Honjo et al. 2003). What is yet to be considered, however, is that NCX may play a fundamental role throughout the pacemaker depolarization. Previous studies in mammalian tissue have focused on the role of NCX current at the foot of the SA node action potential, and have been limited by the use of superfusion techniques to produce a maintained inhibition of NCX with slow onset; this would alter the Ca2+ dynamics of the cell, and so result in secondary changes in other pacemaker currents that are dependent on cytosolic Ca2+ levels. In this study, we circumvent this problem by using a rapid solution-switching system to inhibit NCX within the time course of a single cardiac cycle, allowing us to investigate the effects of NCX inhibition in the absence of secondary changes in other pacemaker currents. We show that inhibition of NCX by rapid switch to a Li+-containing solution caused immediate cessation of beating, demonstrating the fundamental importance of NCX to pacemaking. This is in stark contrast to the effects of inhibition of other established pacemaking currents, such as If(funny current), which result only in a slowing of beating, but not a cessation. The abolition of spontaneous activity on inhibition of NCX was associated with a fall in cytosolic Ca2+ to a level below the minimal level observed between calcium transients in a spontaneously beating cell. Furthermore, a critical contribution of NCX to pacemaking was observed even in the absence of a functional SR, demonstrating that this current is not exclusively dependent on SR Ca2+ release.
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We propose that, in spontaneously beating cells, the cytosolic Ca2+ concentration is always at a level higher than that in resting cells, and that this elevated Ca2+ level supports a level of NCX current throughout the pacemaker potential that is critical to spontaneous beating in these cells. The fundamental importance of NCX for pacemaking may arise from conventional electrogenic NCX (with 3 Na+ ions entering in exchange for 1 Ca2+ ion) and/or from the continuous Na+ leak pathway proposed by Kang & Hilgemann (2004). The precise contribution of each of these mechanisms remains for future study.
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Methods
Isolation of cardiac cells
Male guinea-pigs (weighing 350–500 g) were killed by cervical dislocation following stunning in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 (T.S.O.). Sino-atrial node cells were isolated as previously described (Rigg et al. 2000).
Rapid application of drugs using a local perfusion system
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Solution switches were made using a local perfusion system (Warner Instrument Corp., Hamden, CT, USA) to ensure rapid application of drugs. A triple-barrelled square glass capillary tube was positioned within 100 μm of the cell under study, such that the cell was bathed in solution (36°C) flowing from only one barrel. Lateral movement of the glass capillary tube (driven by a stepper motor), caused the cell to be bathed with solution from a different barrel (changeover < 0.5 s). Solution flow was 100 μl min–1 through each barrel (driven by a syringe pump). Solutions contained (mM): NaCl, 118.5; NaHCO3, 14.5; KCl, 4.2; KH2PO4, 1.18; MgSO4, 1.18; CaCl2, 2.5; and glucose, 11.1; gassed with 95% O2–5% CO2 to maintain a pH of 7.4; NaCl was replaced with LiCl in low-Na+ solutions. Cyclopiazonic acid (CPA), nifedipine (Sigma-Aldrich), BAPTA AM (acetoxy methyl ester form), EGTA AM (Molecular Probes) and ZD7288 (Tocris) were made up as 1–20 mM stock solutions in dimethyl sulphoxide (DMSO) and diluted to the required concentration immediately before use. The maximal concentration of DMSO used in any of these experiments was 0.1%, and was without significant effect on the parameters studied.
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Electrophysiology
Action potentials were recorded using the perforated patch clamp technique (Axopatch 200 amplifier, Axon Instruments). Patch pipettes (3–5 M) contained (mM): KCl, 150; MgCl2, 5; K2ATP, 1; Hepes, 3; pH adjusted to 7.2 with KOH; perforation using amphotericin (Sigma-Aldrich, 250 μg ml–1) occurred 5–10 min after seal formation.
Calcium imaging and calcium transients
For confocal microscopy experiments, cells were incubated with fluo-4 AM (1–3 μM) for 10 min at 36°C; > 10 min was allowed for de-esterification of intracellular fluo-4 AM. A Leica TCS NT confocal scanning head was coupled to a DMIRB microscope with a 63x water immersion objective (excitation with 488 nm Ar laser, and emitted fluorescence collected using a 515 nm long-pass filter). Line scan imaging was used to maximize temporal resolution; a single line was repeatedly scanned at an acquisition rate of 385 Hz (2.6 ms per line).
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In some experiments a photomultiplier system (Cairn Instruments, Faversham, UK) was used to record Ca2+ transients in cells incubated with indo-5F AM (Molecular Probes, 4 μM for 10 min), with excitation at 340 nm, and emission collected at 410 and 490 nm. Indo-5F AM was chosen because indo-1 has been shown to slow or stop spontaneous beating in these cells (Rigg et al. 2000). The cytosolic Ca2+ concentration, [Ca2+]i, was calculated from the 410/490 fluorescence signal ratio, r, using the equation:
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where =Fmax/Fmin measured at 490 nm (the ratio of the maximum and minimum fluorescence measured at zero and saturating Ca2+), and KD is the dissociation equilibrium constant between Ca2+ and indo-5F AM (taken as 470 nM; Handbook of Fluorescent Probes and Research Products, ninth edn, Molecular Probes); 410/490 fluorescence ratios for zero Ca2+ and saturating Ca2+ are given by rmin and rmax, while r represents the 410/490 fluorescence ratio measured as a variable during the experiments. An in vivo calibration method was used to determine rmin, rmax, Fmin and Fmax; cells permeabilized with the ionophore ionomycin (in the presence of CPA, to inhibit the SR Ca2+-ATPase, and KB-R7943, to inhibit NCX) were exposed to solutions containing 0 and 5 mM Ca2+, and fluorescence measurements recorded. An adjustment for background fluorescence was also made.
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Statistics
Data are expressed as means ±S.E.M. Statistical significance was evaluated using Student's paired t test. A level of P < 0.05 was considered to be statistically significant.
Results
Inward NCX current was inhibited by reduction of the extracellular concentration of Na+ ions, from 133 to 14.5 mM, by replacement with Li+ (Le Guennec & Noble, 1994; Janvier et al. 1997). Rapid switch to low-Na+ solution caused immediate cessation of spontaneous electrical activity, with at most only one beat occurring after the switch (representative examples in Fig. 1A and B). If the time of exposure to low Na+ was kept short, spontaneous activity could be restarted by rapid switch back to normal Na+ solution. Interestingly, despite the absence of spontaneous action potentials, exposure of cells to low-Na+ solution for more than 3–4 s resulted in the appearance of spontaneous contractions; these did not have the same characteristics as those accompanying spontaneous action potentials (in normal Na+ solution), but were ‘wave like’ in nature. Similar observations were made in five cells, supporting the proposal that NCX makes an essential contribution to pacemaking.
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A and B, rapid switch to low-Na+ solution (during the time indicated by the bar) caused immediate cessation of spontaneous action potentials. Action potentials speedily reappeared following rapid switch back to normal Na+ solution. C, superfusion with the Na+ channel blocker TTX (10 μM) caused a slight slowing in beating rate in this cell. Subsequent rapid switch to low-Na+ solution resulted in an abolition of spontaneous action potentials, with recovery on washout. D, action potentials recorded in the presence of the If blocker ZD7288 (3 μM), which slowed, but did not stop, spontaneous activity. In this cell, ZD7288 reduced the frequency of spontaneous action potential occurrence by 18%, from 201 to 165 beats min–1. Under these conditions, rapid switch to low-Na+ solution again caused cessation of spontaneous activity.
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In a further seven SA node cells, in which spontaneous contractile activity was monitored via a video camera attached to the microscope (but without a patch electrode attached for electrical recording), rapid switch to low-Na+ solution again caused immediate cessation of beating, with at most one beat occurring after the move to low-Na+ solution. Following a period of cessation of beating (2.71 ± 0.64 s, n= 7, P < 0.05) that was considerably greater than the control cycle length (0.60 ± 0.12 s, n= 7, P < 0.05), contractile activity recommenced in low-Na+ solution, but it was apparent that the restored contractions differed from normal ones, resembling wave-like contractions (see below).
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Li+ ions are known to pass through voltage-gated Na+ channels almost as well as Na+ ions (Chandler and Meves, 1965; Hille, 1972), and voltage-gated Na+ channels are thought not to play a major role in pacemaker activity in the majority of mammalian species (but see Lei et al. 2005 for recent observations on mouse SA node). It therefore seems unlikely that the cessation of beating observed on substituting Li+ for Na+ can be ascribed to effects mediated by voltage-gated Na+ channels. Nevertheless, this possibility was tested in experiments in which tetrodotoxin (TTX) was used to block voltage-gated Na+ channels. Figure 1C shows an example trace where application of TTX (10 μM) caused little or no change in the rate of beating, but rapid switch to low-Na+ solution (with Li+ as the replacement, as above) caused immediate cessation of beating that was reversible on restoration of normal Na+ levels. Similar observations were made in a further four cells.
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While switch to low-Na+ solution would be expected to suppress inward NCX current, another possibility to be considered is that If currents activated by hyperpolarization would also be reduced, since Li+ ions are thought to permeate these channels less well than Na+ ions (Ho et al. 1994). To explore whether this could account for the cessation of beating, the effects of the selective If inhibitor ZD7288 (BoSmith et al. 1993) were investigated. In our experiments, ZD7288 (3 μM) was found to reduce If (measured at –80 mV under voltage-clamp conditions) by 73 ± 8% (n= 3); this is comparable to the 78 ± 4% decrease in If observed (at –120 mV) in guinea-pig SA node cells by BoSmith et al. (1993), using 1 μM ZD7288. Despite this substantial inhibition of If, a rapid switch to 3 μM ZD7288 caused only a 14 ± 3% slowing of the beating rate in six further cells studied (P < 0.05) and did not stop the initiation of spontaneous action potentials. However, rapid switch to low-Na+ solution in the presence of 3 μM ZD7288 again caused abolition of spontaneous action potentials in all six cells (Fig. 1D).
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As an alternative method of suppressing NCX current, guinea-pig SA node cells were exposed to KB-R7943, which has been shown to inhibit NCX in guinea-pig cardiac myocytes (Iwamoto et al. 1996; Watano et al. 1996). Rapid switch to KB-R7943 (5 μM) caused cessation of spontaneous beating of SA node cells (n= 8). The time taken for cessation of activity (19.5 ± 3.6 s) was not as rapid as was the case for a switch to low-Na+ solution, but this might be expected if the time taken for this drug to reach the membrane and exert its inhibitory action was slower than was the case in experiments where Li+ was used to replace Na+.
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The effects of low Na+ were explored further in cells loaded with the Ca2+ indicator fluo-4 AM and imaged using confocal microscopy (in the linescan configuration). A series of images from a representative experiment are shown in the upper panel of Fig. 2A. Regular Ca2+ transients, with Ca2+ rising approximately synchronously along the cell, were observed in normal Na+ solution (left panel). A rapid switch to low Na+ occurred just before the next panel, where it can be seen that spontaneous whole cell Ca2+ transients had been completely suppressed. Towards the end of the period of recording shown in the third panel, spontaneous activity resumed, but this was in the form of a Ca2+ wave (starting at a position at the top of the panel and propagating downwards); the next panel shows several more Ca2+ waves (broader and less uniform in magnitude than normal Ca2+ transients). On switching back to normal Na+ solution, activity started to recover, and the final panel shows restoration of spontaneous whole cell Ca2+ transients with synchronous Ca2+ changes along the cell.
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A, the upper panel shows linescan images acquired using confocal microscopy with fluo-4 AM as the Ca2+ probe; distance is represented vertically (bar represents 20 μM), and time horizontally. The lower panel shows Ca2+ transients derived by averaging the fluorescence signal along each scanned line. Successive frames are presented, the first showing Ca2+ transients under control conditions, arising synchronously along the cell. Low-Na+ solution was applied immediately before the second frame, which shows that Ca2+ transients were abolished by the switch to low-Na+ solution; note that cytosolic Ca2+ fell following cessation of activity in low-Na+ solution. The third frame shows a continued fall in cytosolic Ca2+, followed by a Ca2+ wave that starts at the top of the image and propagates downwards. More Ca2+ waves are evident in the fourth frame. Normal Ca2+ transients were quickly restored on return to normal Na+ solution (fifth frame). B, Ca2+ transients were also recorded using indo-5F AM (measured as the ratio of emission at 410 nm to that at 490 nm). Rapid switch to low-Na+ solution again caused cessation of beating and a fall in cytosolic Ca2+, as indicated by the fluorescence ratio. Similar observations were made in a further 7 cells.
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Total fluorescence in these panels was quantified by averaging the signal across the cell and is shown by the traces in the lower panel of Fig. 2A. The red horizontal dashed line indicates the minimum level of fluo-4 AM fluorescence between beats. Note that abolition of spontaneous activity by the switch to low-Na+ solution caused a fall in cytosolic [Ca2+] below the minimum diastolic level observed when the cell was beating (red horizontal dashed line). Similar observations were made in six cells, and when spontaneous activity was halted by a rapid switch to low-Na+ solution, the mean fall in integrated fluo-4 AM fluorescence signal was to 77 ± 5% of the control minimum level between beats (n= 6, P < 0.05).
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In a further eight cells, Ca2+ transients were measured as indo-5F AM 410/490 nm ratios, allowing quantitative measurements of intracellular Ca2+ concentrations to be obtained following a calibration procedure. As shown in Fig. 2B, rapid switch to low-Na+ solution again caused both cessation of beating and a fall in cytosolic Ca2+ to a level 15 ± 2% (n= 8, P < 0.05) below the minimum diastolic Ca2+ concentration in a beating cell. Minimum diastolic Ca2+ concentration was found to be 225 ± 55 nM in beating cells, and the lowest Ca2+ concentration observed during cessation of beating was 198 ± 72 nM.
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This fall in cytosolic [Ca2+] seems surprising, since low-Na+ solution is expected to inhibit the major pathway of Ca2+ extrusion via NCX; however, Ca2+ might continue to be sequestered by the SR, leading to a fall of Ca2+ in the absence of significant continued Ca2+ entry through voltage-gated channels (since spontaneous electrical activity was abolished). If this were correct, the fall in cytosolic [Ca2+] on switch to low-Na+ solution might not be seen in cells exposed to cyclopiazonic acid (CPA), an inhibitor of SR Ca2+ uptake (Seidler et al. 1989). In experiments using confocal microscopy, application of 30 μM CPA slowed the frequency of, but did not stop, the occurrence of spontaneous Ca2+ transients (frequency reduced by 30 ± 7%; n= 6, P < 0.05). Figure 3A shows that subsequent rapid switch to low-Na+ solution (in the presence of CPA) again caused rapid cessation of spontaneous Ca2+ transients but, in contrast to the observations when CPA was absent, there was a consistent increase in cytosolic [Ca2+] (as indicated by an increase in fluo-4 AM fluorescence; see Fig. 4A). Similar observations were made in all six cells in which fluo-4 AM was used as the Ca2+ indicator, and in a further six cells loaded with indo-5F AM.
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A, Ca2+ transients recorded using confocal microscopy, with fluo-4 AM as the Ca2+ indicator. Application of CPA (30 μM) slowed, but did not stop, the occurrence of spontaneous Ca2+ transients. Note that with CPA present, the Ca2+ transients appeared smaller in magnitude, and slower to both peak and decay, compared with those in control conditions. Rapid switch to low-Na+ solution abolished spontaneous Ca2+ transients, and this was associated with a rise, rather than a fall, in cytosolic Ca2+. B, another cell, showing similar effects of CPA on spontaneous Ca2+ transients. In this cell, rapid switch to KB-R7943 (5 μM), in the continued presence of CPA, resulted in an abolition of Ca2+ transients, with no increase in baseline Ca2+. Subsequent rapid application of low-Na+ solution, in the presence of KB-R7943, did not result in an increase in baseline Ca2+ levels.
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A, spontaneous action potentials recorded from a SA node cell. A rapid switch to 50 mM Na+ solution (bar) caused cessation of spontaneous beating, which returned on washout. Similar observations were made in 3 cells. B, a rapid switch to 75 mM Na+ solution also abolished spontaneous action potentials for approximately the first 3 s of application; two action potentials, of abnormally large amplitude, were subsequently recorded in this cell during application of low-Na+ solution. In two further cells, spontaneous action potentials were completely abolished for the entire period of application of 75 mM Na+ solution. C, spontaneous Ca2+ transients recorded from a representative cell using confocal microscopy (fluo-4 AM). A rapid switch to 50 mM Na+ solution abolished spontaneous Ca2+ transients. In this cell, a localized increase in intracellular Ca2+, accompanied by a gradual global increase in Ca2+, was observed; this may represent Ca2+ release from an overloaded SR. Similar observations were made in 6 cells. D, rapid switch to 75 mM Na+ solution also abolished spontaneous Ca2+ transients. Similar observations were made in 8 cells.
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There are several potential pathways that may underlie the cytosolic elevation of Ca2+ observed on application of low-Na+ solution in the presence of CPA. One possibility is that, on application of low Na+ (at this concentration) NCX reverses, bringing Ca2+ into the cell. To test for this possibility, KB-R7943 was applied in the presence of CPA, since KB-R7943 has been reported to inhibit both forward and reverse modes of NCX (Iwamoto et al. 1996; Watano et al. 1996). As may be seen in Fig. 3B, application of KB-R7943, in the presence of CPA, resulted in a cessation of spontaneous Ca2+ transients, but with little or no rise in baseline Ca2+ levels. A subsequent rapid switch to low-Na+ solution also resulted in little or no change in baseline fluorescence. It may therefore be the case that application of low-Na+ solution in the presence of CPA results in reverse-mode NCX that brings Ca2+ into the cell, causing a gradual elevation of cytosolic [Ca2+].
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If it is the case that NCX reverses on switch to 14.5 mM Na+ solution, this raises the possibility that the cessation of beating on switch to 14.5 mM Na+ solution may not only be due to the removal of an inward current normally contributed by NCX during the pacemaker depolarization, but also due to the imposition of an outward current, due to NCX operating in the Ca2+ influx mode. Indeed, calculation of the reversal potential for NCX (3ENa– 2ECa, where these represent the Nernst equilibrium potentials for Na+ and Ca2+, respectively), assuming intracellular Na+ and Ca2+ concentrations of 7 mM and 225 nM, respectively, yields a value of –190 mV at 14.5 mM extracellular Na+. Hence, following switch to 14.5 mM Na+, NCX would be expected to be operating in reverse-mode at the resting membrane potentials recorded from our SA node cells. Although the aforementioned effects of KB-R7943, which inhibits reverse-mode NCX as well as the conventional mode, do not support the possibility that it is imposition of an outward current that abolishes beating in these cells, this was nonetheless further tested in experiments in which cells were exposed to 50 and 75 mM Na+ solutions. The NCX reversal potentials at 50 and 75 mM Na+ are calculated to be –90 and –59 mV, and so it would be expected that the contribution of any outward current by reverse-mode NCX would be significantly less under these conditions; indeed, a rapid switch to 75 mM Na+ would be expected to result in very little, if any, reversal of NCX. As shown in Fig. 4, rapid switch to either of these concentrations of Na+ resulted in an immediate cessation of both spontaneous action potentials and spontaneous Ca2+ transients. Thus, it is highly likely that the abolition of beating on switch to low-Na+ solution is due to inhibition of an inward current that is normally contributed by NCX during the pacemaker depolarization, and not primarily due to imposition of an outward current.
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If cytosolic [Ca2+] between beats remains at a sufficiently high level that Ca2+ extrusion by NCX contributes inward current that is essential for spontaneous pacemaker activity, an alternative method of suppressing the inward NCX current (and therefore spontaneous activity) would be to apply a Ca2+ chelator, such as BAPTA, to the cytosol to lower [Ca2+]. As illustrated in Fig. 5, application of membrane-permeant BAPTA AM (10 μM), whether by rapid switch (n= 6; data not shown) or by superfusion (n= 6), caused abolition of spontaneous action potentials. A similar cessation of activity was observed in three cells loaded with another Ca2+ chelator, EGTA, by exposure to EGTA AM (10 μM; data not shown).
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Two examples of superfusion with BAPTA AM (10 μM) resulting in abolition of spontaneous action potentials. Note the relatively slow onset of inhibition of beating, presumably reflecting the time taken for diffusion of BAPTA AM into the cell, and subsequent de-esterification.
The data presented above support the possibility that the minimum diastolic level of [Ca2+] between beats may be maintained at a higher level than the resting [Ca2+] in quiescent cells, resulting in a contribution of NCX current throughout the entire pacemaker depolarization. To assess this directly, 5 μM nifedipine was rapidly applied to spontaneously beating SA node cells imaged using confocal microscopy, in order to prevent the generation of action potentials (L-type Ca2+ channels are known to underlie the upstroke of the SA node action potential). As shown in Fig. 6A, abolition of spontaneous beating with nifedipine was associated with a significant decrease in the fluo-4 AM fluorescence level, as compared to minimum levels recorded between spontaneous Ca2+ transients; this was a consistent observation in all six cells studied. Similar falls in Ca2+ levels were observed when either acetylcholine (1 μM, n= 6) or adenosine (1 μM, n= 6) were used to stop spontaneous beating (Fig. 6B and C). This supports the hypothesis that, during beating, cytosolic [Ca2+] concentrations remain higher between beats than those that occur in quiescent cells.
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A, nifedipine, 5 μM (confocal microscopy, fluo-4 AM). B, adenosine, 1 μM (confocal microscopy, fluo-4 AM). C, acetylcholine, 1 μM (conventional fluorescence microscopy, indo-5F AM).
Discussion
The observations reported here provide an important extension to previous work demonstrating a role for Ca2+, including that released from the SR, in pacemaking mechanisms (Rigg & Terrar, 1996; Ju & Allen, 1998; Huser et al. 2000; Rigg et al. 2000; Vinogradova et al. 2002). Studies in other species have reported that a rise in subsarcolemmal [Ca2+], associated with SR Ca2+ release that precedes the upstroke of the action potential (perhaps triggered by T-type Ca2+ current), leads to depolarizing NCX current that contributes a depolarizing influence at this time (Huser et al. 2000; Bogdanov et al. 2001; Vinogradova et al. 2002). The data presented here, however, demonstrate that the contribution of NCX current to the pacemaker depolarization of guinea-pig SA node cells is not exclusively dependent on a functional SR, since a switch to low-Na+ solution abolished spontaneous beating in the presence of CPA, an inhibitor of SR Ca2+ uptake. Furthermore, if the only contribution of NCX were secondary to SR Ca2+ release, inhibition of SR function might be expected to produce effects on pacemaking that were broadly similar to inhibition of NCX. In guinea-pig SA node cells, however, suppression of SR function is associated with a slowing of rate (Rigg & Terrar, 1996; Rigg et al. 2000; Lakatta et al. 2003), whereas inhibition of NCX abolishes beating. Thus, the essential contribution that NCX makes to pacemaking in this preparation is not limited to current (at the foot of the action potential) that is secondary to localized SR Ca2+ release, but is also due to a significant component that is SR independent. The timing of this SR-independent component need not be limited to the foot of the action potential, but instead may occur throughout the pacemaker depolarization.
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Our experiments using rapid switch to low-Na+ solution to inhibit NCX provide important insights concerning the importance of NCX current and extend the interesting observations of Bogdanov et al. (2001). These authors showed that beating stopped soon after Na+ was replaced by Li+ in the solution bathing rabbit SA node cells, but cytosolic [Ca2+] was not simultaneously monitored in these experiments and, as the authors pointed out, it might be argued that such continuous superfusion with Li+ induces an increase in steady cytosolic [Ca2+] during the diastolic depolarization, which in turn may affect SR loading, refractoriness of the ryanodine receptor, or currents involved in automaticity. In further experiments in which cytosolic [Ca2+] was measured, a rapid ‘spritz’ of low-Na+ solution was found to suppress action potential duration, though a large (approximately 70% of normal) Ca2+ transient (or wave) remained. In our experiments, rapid application of low-Na+ solution consistently suppressed activity within one beat, but a different type of spontaneous activity reappeared after an interval of many seconds, in the form of Ca2+ waves, most likely secondary to spontaneous release of Ca2+ from an overloaded SR (Fig. 2). Our experiments in guinea-pig SA node cells thus differ significantly from those of Bogdanov and co-workers in that rapid application of low-Na+ solution was continued for a sufficiently long period to completely stop spontaneous electrical activity and Ca2+ transients, and (prior to the initiation of Ca2+ waves) this was not accompanied by an elevation of cytosolic [Ca2+].
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Another important observation, shown in Fig. 2, is the fall in cytosolic [Ca2+] that followed cessation of beating in the presence of low-Na+ solution, occurring even though a major mechanism for Ca2+ extrusion (NCX) was suppressed. The mechanism underlying this may involve Ca2+ uptake into the SR, mediated by the Ca2+-ATPase, continuing until the SR becomes overloaded, resulting in spontaneous Ca2+ release and Ca2+ waves. This was supported by the observation that, when SR uptake was inhibited by CPA, a rapid switch to low-Na+ solution again suppressed spontaneous activity, but this was followed by a rise, rather than a fall, in cytosolic [Ca2+]. Hence, when the SR is functioning normally, SR Ca2+ uptake can reduce cytosolic [Ca2+] immediately following cessation of activity due to NCX inhibition.
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Several potential pathways may contribute the Ca2+ that underlies the elevation in cytosolic levels observed on switch to low-Na+ solution in the presence of CPA. The lack of such an elevation of Ca2+ when KB-R7943 is used to inhibit NCX (in the presence of CPA) supports a role for reverse-mode NCX in providing at least some of this Ca2+. This raises the possibility that abolition of spontaneous activity by low-Na+ solution may be due not only to removal of an inward current (i.e. NCX working in the Ca2+ extrusion mode), but also to the imposition of an outward current (reverse-mode NCX) on the pacemaker depolarization. However, since KB-R7943, which inhibits both modes of NCX, completely abolished pacemaker activity, it is highly likely that inhibition of inward NCX current alone is sufficient to cause cessation of spontaneous activity. This is supported further by the observations that beating was abolished by 50 and 75 mM Na+ solutions; for the latter concentration, little or no outward current would be expected, since the NCX reversal potential (–59 mV) is calculated to be close to the minimum diastolic potential of these cells.
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The hypothesis that the cytosolic [Ca2+] remains elevated between beats in spontaneously active SA node cells is supported by our observations that abolition of beating by three differing pharmacological mechanisms (nifedipine, acetylcholine and adenosine) resulted in a fall in [Ca2+] to a level below the minimum recorded between spontaneous Ca2+ transients. The Ca2+ chelators BAPTA and EGTA, which would be expected to greatly reduce the minimum level of cytosolic Ca2+ (regardless of whether the major source was from the SR or Ca2+ entry across the sarcolemma) and hence NCX current, also caused cessation of spontaneous action potentials. It therefore appears that, under the conditions of these experiments, the sequential activation of voltage-dependent ion channels is not by itself sufficient to maintain spontaneous action potentials in the face of an abnormally low cytosolic [Ca2+].
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Another possible effect of low-Na+ solution that must be considered is inhibition of hyperpolarization-activated If currents, since Li+ ions have been reported to permeate less well through these channels (Ho et al. 1994). However, previous experiments have shown that complete block of If slows but does not stop spontaneous beating (Denyer & Brown, 1990). We have also shown that ZD7288 slows but does not prevent initiation of spontaneous action potentials, despite substantial inhibition of If current, but that rapid switch to low-Na+ solution under these conditions again leads to cessation of activity. Furthermore, Bogdanov et al. (2001) have reported that Li+ did not affect If in their experiments. It is therefore unlikely that the effects of low-Na+ solution are due to actions on If. An alternative possibility is that low-Na+ solution might interfere with the depolarizing influence of Ist (sustained inward current; Guo et al. 1995, 1996, 1997), but this seems unlikely, since the channel allows permeation by Na+ and K+ (Guo et al. 1996), and Li+ would also be expected to pass through this channel. It is also unlikely that actions of Li+ on TTX-sensitive voltage-gated Na+ channels account for the abolition of pacemaking, since Li+ passes through these channels almost as easily as Na+ (Chandler & Meves, 1965; Hille, 1972). Furthermore, in our experiments, TTX had little or no effect on pacemaking, while switch to low-Na+ solution in the presence of TTX caused immediate cessation of beating.
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It therefore appears that the major factor suppressing pacemaker activity following switch to low-Na+ solution is inhibition of inward NCX current, either in the form of NCX in its conventional 3 Na+ to 1 Ca2+ ion mode, or in the form of a Na+ leak pathway, as proposed by Kang & Hilgemann (2004), in which import of one Na+ with one Ca2+ ion coupled to export of one Ca2+ ion provides a Na+-conducting pathway. The abolition of spontaneous activity by the Ca2+ chelators BAPTA and EGTA (also seen following excessive loading with Ca2+ indicators, as noted previously by Rigg et al. 2000) is consistent with inhibition of either mode of operation of NCX. The levels of Ca2+ between beats were found to be higher (225 nM) than those in quiescent cells (or in resting atrial and ventricular myocytes), making it likely that there is a maintained component of electrogenic 3:1 NCX throughout the pacemaker depolarization, even if there is an additional Na+ leak through the NCX protein. The mechanisms underlying this maintained elevation of intracellular Ca2+ remain to be determined.
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Although voltage-clamp experiments to measure NCX currents would potentially yield additional information regarding the importance of this current to pacemaking, they are beyond the scope of this study. Simple voltage-clamp experiments would not be helpful, partly because even for conventional 3:1 exchange, the level of currents in a beating cell would vary dynamically with changes in cytosolic Ca2+ during the cardiac cycle, and this would necessarily be modified with conventional voltage-clamp protocols. Furthermore, the contributions from the new modes of operation of NCX require complex experiments that are beyond the scope of this investigation.
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Experiments in whole animal NCX knockout mice (Wakimoto et al. 2000; Cho et al. 2000; Koushik et al. 2001; Reuter et al. 2002) have failed to clarify the role of NCX in pacemaking, since embryos in these models did not survive beyond about 11 days post coitum, and the heartbeat was absent. In the light of the present experiments, it is possible that the lack of heartbeat in these knockout mice may be due to the absence of an essential contribution of NCX during the pacemaker potential. However, other harmful effects of NCX knockout cannot be excluded. It should be noted that the NCX1 knockout described recently by Henderson et al. (2004) is specific to the ventricle, and so provides no insight into the role of NCX in pacemaking.
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Of course, other currents are known to contribute to the pacemaker depolarization, and some of these may be regulated by cytosolic [Ca2+], including If and delayed rectifier K+ currents (Tohse, 1990; Rigg et al. 2003). However, inhibition of many of the ‘established’ pacemaking currents is associated only with a slowing of beating rate in guinea-pig SA node cells, rather than a cessation. The observations reported here are consistent with a fundamental and critical role for NCX in maintaining a depolarizing influence throughout the pacemaker depolarization. We propose that this maintained depolarizing influence of NCX is secondary to a high minimum level of cytosolic [Ca2+] that normally never falls below that which supports Ca2+ extrusion via electrogenic 3:1 NCX, but this mechanism may also be supplemented by a contribution arising from a Na+ leak pathway through the NCX protein.
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