fundamental importance of na+–ca2+ exchange for the pacemaking mechanism in guinea-pig sino-atrial...

J Physiol 571.3 (2006) pp 639–649 639

Fundamental importance of Na+–Ca2+ exchange for thepacemaking mechanism in guinea-pig sino-atrial node

Luke Sanders, Stevan Rakovic, Matthew Lowe, Paul A. D. Mattick and Derek A. Terrar

University Department of Pharmacology, Mansfield Road, Oxford OX1 3QT, UK

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 I f (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.

(Resubmitted 18 October 2005; accepted after revision 18 January 2006; first published online 19 January 2006)

Corresponding author D. A. Terrar: University Department of Pharmacology, Mansfield Road, Oxford OX1 3QT, UK.

Email: [email protected]

A variety of ionic currents are thought to contribute topacemaker activity in the sino-atrial (SA) node (Brown,1982). These include L- and T-type Ca2+ currents (Doerret al. 1989; Huser et al. 2000), hyperpolarization-activatedcurrent (Seyama, 1976; DiFrancesco, 1993), delayedrectifier K+ currents (both rapidly and slowly activatingcomponents (Shibasaki, 1987; Sanguinetti & Jurkiewicz,1990), sustained inward current (Guo et al. 1997) andbackground current (Hagiwara et al. 1992). Na+–Ca2+

exchange (NCX) is recognized to be important inmaintaining the Ca2+ balance of the cell, since it is thoughtto play a major role in Ca2+ extrusion, but has only recentlybeen identified as a potentially important contributor tothe 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 assuminga 3:1 stoichiometry, but see later), this extrusion wouldbe associated with NCX-mediated charge entry (over onecardiac cycle) equal to approximately half that contributedby the Ca2+ entry mechanisms. Thus, NCX current maygenerate a substantial portion of the inward depolarizingcurrent underlying spontaneous pacemaking.

A recent study by Kang & Hilgemann (2004) hasraised the possibility that the contribution of NCXcurrent to pacemaking may occur not only throughthe conventionally accepted mode of operation, i.e. Na+

entry and Ca2+ extrusion in a 3:1 stoichiometry, but

C© 2006 The Authors. Journal compilation C© 2006 The Physiological Society DOI: 10.1113/jphysiol.2005.100305

640 L. Sanders and others J Physiol 571.3

also through an additional transport mode. Although theaverage stoichiometry of the NCX process was found to be3.2 Na+ to 1 Ca2+ in this study, it was also reported that,under some conditions, the NCX protein may provide aninward background Na+ current, occurring because of theimport of one Na+ with one Ca2+ ion from the extracellularsolution, coupled to the export of one Ca2+ ion from thecytosol. Both these modes of transport result in a netinward current, and so both may potentially contributeto pacemaking in the SA node.

Experimental evidence supporting a role for NCX inpacemaking 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 beingsecondary to a rise in subsarcolemmal Ca2+ immediatelypreceding the upstroke of the action potential, due toCa2+ release from the SR (Huser et al. 2000; Vinogradovaet al. 2002). This was proposed to act as an amplificationmechanism at the foot of the SA node action potential,such that Ca2+ entry during the latter part of the pacemakerdepolarization triggers Ca2+-induced release of Ca2+ fromthe SR, generating a significant inward current carried byNCX.

This proposed contribution of NCX current, secondaryto SR Ca2+ release, is becoming more widely accepted,although some debate concerning the importance of theSR in pacemaking remains (Lakatta et al. 2003; Honjo et al.2003). What is yet to be considered, however, is that NCXmay play a fundamental role throughout the pacemakerdepolarization. Previous studies in mammalian tissue havefocused on the role of NCX current at the foot of theSA node action potential, and have been limited by theuse of superfusion techniques to produce a maintainedinhibition of NCX with slow onset; this would alter theCa2+ dynamics of the cell, and so result in secondarychanges in other pacemaker currents that are dependenton cytosolic Ca2+ levels. In this study, we circumvent thisproblem by using a rapid solution-switching system toinhibit NCX within the time course of a single cardiac cycle,allowing us to investigate the effects of NCX inhibitionin the absence of secondary changes in other pacemakercurrents. We show that inhibition of NCX by rapid switchto a Li+-containing solution caused immediate cessationof beating, demonstrating the fundamental importance ofNCX to pacemaking. This is in stark contrast to the effectsof inhibition of other established pacemaking currents,such as I f(funny current), which result only in a slowing ofbeating, but not a cessation. The abolition of spontaneousactivity on inhibition of NCX was associated with a fallin cytosolic Ca2+ to a level below the minimal levelobserved between calcium transients in a spontaneouslybeating cell. Furthermore, a critical contribution of NCXto pacemaking was observed even in the absence of afunctional SR, demonstrating that this current is notexclusively dependent on SR Ca2+ release.

We propose that, in spontaneously beating cells, thecytosolic Ca2+ concentration is always at a level higherthan that in resting cells, and that this elevated Ca2+

level supports a level of NCX current throughout thepacemaker potential that is critical to spontaneous beatingin these cells. The fundamental importance of NCX forpacemaking may arise from conventional electrogenicNCX (with 3 Na+ ions entering in exchange for 1 Ca2+ ion)and/or from the continuous Na+ leak pathway proposedby Kang & Hilgemann (2004). The precise contribution ofeach of these mechanisms remains for future study.

Methods

Isolation of cardiac cells

Male guinea-pigs (weighing 350–500 g) were killed bycervical dislocation following stunning in accordance withthe Home Office Guidance on the Operation of the Animals(Scientific Procedures) Act 1986 (T.S.O.). Sino-atrial nodecells were isolated as previously described (Rigg et al.2000).

Rapid application of drugs using a localperfusion system

Solution switches were made using a local perfusionsystem (Warner Instrument Corp., Hamden, CT, USA)to ensure rapid application of drugs. A triple-barrelledsquare glass capillary tube was positioned within 100 μmof the cell under study, such that the cell was bathedin solution (36◦C) flowing from only one barrel. Lateralmovement of the glass capillary tube (driven by a steppermotor), caused the cell to be bathed with solutionfrom a different barrel (changeover < 0.5 s). Solutionflow was 100 μl min−1 through each barrel (driven by asyringe 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 withLiCl in low-Na+ solutions. Cyclopiazonic acid (CPA),nifedipine (Sigma-Aldrich), BAPTA AM (acetoxy methylester form), EGTA AM (Molecular Probes) and ZD7288(Tocris) were made up as 1–20 mm stock solutions indimethyl sulphoxide (DMSO) and diluted to the requiredconcentration immediately before use. The maximalconcentration of DMSO used in any of these experimentswas 0.1%, and was without significant effect on theparameters studied.

Electrophysiology

Action potentials were recorded using the perforatedpatch clamp technique (Axopatch 200 amplifier, AxonInstruments). Patch pipettes (3–5 M�) contained (mm):KCl, 150; MgCl2, 5; K2ATP, 1; Hepes, 3; pH adjusted

C© 2006 The Authors. Journal compilation C© 2006 The Physiological Society

J Physiol 571.3 Na+–Ca2+ exchange and pacemaking 641

to 7.2 with KOH; perforation using amphotericin(Sigma-Aldrich, 250 μg ml−1) occurred 5–10 min afterseal formation.

Calcium imaging and calcium transients

For confocal microscopy experiments, cells wereincubated with fluo-4 AM (1–3 μm) for 10 min at 36◦C;> 10 min was allowed for de-esterification of intracellularfluo-4 AM. A Leica TCS NT confocal scanning head wascoupled to a DMIRB microscope with a 63× waterimmersion objective (excitation with 488 nm Ar laser, andemitted fluorescence collected using a 515 nm long-passfilter). Line scan imaging was used to maximize temporalresolution; a single line was repeatedly scanned at anacquisition rate of 385 Hz (2.6 ms per line).

In some experiments a photomultiplier system (CairnInstruments, Faversham, UK) was used to record Ca2+

transients in cells incubated with indo-5F AM (MolecularProbes, 4 μm for 10 min), with excitation at 340 nm, andemission collected at 410 and 490 nm. Indo-5F AM waschosen because indo-1 has been shown to slow or stopspontaneous beating in these cells (Rigg et al. 2000). Thecytosolic Ca2+ concentration, [Ca2+]i, was calculated fromthe 410/490 fluorescence signal ratio, r, using the equation:

[Ca2+]i = βKD(r − rmin)/(rmax − r)

where β = Fmax/Fmin measured at 490 nm (the ratio ofthe maximum and minimum fluorescence measured atzero and saturating Ca2+), and K D is the dissociationequilibrium constant between Ca2+ and indo-5F AM(taken as 470 nm; Handbook of Fluorescent Probes andResearch Products, ninth edn, Molecular Probes); 410/490fluorescence ratios for zero Ca2+ and saturating Ca2+ aregiven by rmin and rmax, while r represents the 410/490fluorescence ratio measured as a variable during theexperiments. An in vivo calibration method was used todetermine rmin, rmax, Fmin and Fmax; cells permeabilizedwith the ionophore ionomycin (in the presence of CPA,to inhibit the SR Ca2+-ATPase, and KB-R7943, to inhibitNCX) were exposed to solutions containing 0 and5 mm Ca2+, and fluorescence measurements recorded. Anadjustment for background fluorescence was also made.

Statistics

Data are expressed as means ± s.e.m. Statisticalsignificance was evaluated using Student’s pairedt test. A level of P < 0.05 was considered to be statisticallysignificant.

Results

Inward NCX current was inhibited by reduction of theextracellular concentration of Na+ ions, from 133 to14.5 mm, by replacement with Li+ (Le Guennec & Noble,

1994; Janvier et al. 1997). Rapid switch to low-Na+ solutioncaused immediate cessation of spontaneous electricalactivity, with at most only one beat occurring afterthe 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 switchback to normal Na+ solution. Interestingly, despite theabsence of spontaneous action potentials, exposure ofcells to low-Na+ solution for more than 3–4 s resultedin the appearance of spontaneous contractions; these didnot have the same characteristics as those accompanyingspontaneous action potentials (in normal Na+ solution),but were ‘wave like’ in nature. Similar observations weremade in five cells, supporting the proposal that NCX makesan essential contribution to pacemaking.

In a further seven SA node cells, in which spontaneouscontractile activity was monitored via a video cameraattached to the microscope (but without a patch electrodeattached for electrical recording), rapid switch to low-Na+

solution again caused immediate cessation of beating, withat 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 considerablygreater than the control cycle length (0.60 ± 0.12 s,n = 7, P < 0.05), contractile activity recommenced inlow-Na+ solution, but it was apparent that the restoredcontractions differed from normal ones, resemblingwave-like contractions (see below).

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 arethought not to play a major role in pacemaker activity inthe majority of mammalian species (but see Lei et al. 2005for recent observations on mouse SA node). It thereforeseems unlikely that the cessation of beating observed onsubstituting Li+ for Na+ can be ascribed to effectsmediated by voltage-gated Na+ channels. Nevertheless,this possibility was tested in experiments in whichtetrodotoxin (TTX) was used to block voltage-gatedNa+ channels. Figure 1C shows an example trace whereapplication of TTX (10 μm) caused little or no change inthe rate of beating, but rapid switch to low-Na+ solution(with Li+ as the replacement, as above) caused immediatecessation of beating that was reversible on restoration ofnormal Na+ levels. Similar observations were made in afurther four cells.

While switch to low-Na+ solution would be expectedto suppress inward NCX current, another possibility tobe considered is that I f currents activated by hyper-polarization would also be reduced, since Li+ ions arethought to permeate these channels less well than Na+ ions(Ho et al. 1994). To explore whether this could accountfor the cessation of beating, the effects of the selective I f

inhibitor ZD7288 (BoSmith et al. 1993) were investigated.In our experiments, ZD7288 (3 μm) was found to reduce



I f (measured at −80 mV under voltage-clamp conditions)by 73 ± 8% (n = 3); this is comparable to the 78 ± 4%decrease in I f observed (at −120 mV) in guinea-pig SAnode cells by BoSmith et al. (1993), using 1 μm ZD7288.Despite this substantial inhibition of I f, a rapid switchto 3 μm ZD7288 caused only a 14 ± 3% slowing of thebeating rate in six further cells studied (P < 0.05) and didnot stop the initiation of spontaneous action potentials.However, rapid switch to low-Na+ solution in the presenceof 3 μm ZD7288 again caused abolition of spontaneousaction potentials in all six cells (Fig. 1D).

As an alternative method of suppressing NCX current,guinea-pig SA node cells were exposed to KB-R7943, whichhas been shown to inhibit NCX in guinea-pig cardiacmyocytes (Iwamoto et al. 1996; Watano et al. 1996).Rapid switch to KB-R7943 (5 μm) caused cessation ofspontaneous beating of SA node cells (n = 8). The timetaken for cessation of activity (19.5 ± 3.6 s) was not asrapid as was the case for a switch to low-Na+ solution,but this might be expected if the time taken for this drug

Figure 1. Spontaneous action potentials recorded from isolated SA node cellsA and B, rapid switch to low-Na+ solution (during the time indicated by the bar) caused immediate cessation ofspontaneous 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 thiscell. Subsequent rapid switch to low-Na+ solution resulted in an abolition of spontaneous action potentials, withrecovery 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 potentialoccurrence by 18%, from 201 to 165 beats min−1. Under these conditions, rapid switch to low-Na+ solution againcaused cessation of spontaneous activity.

to reach the membrane and exert its inhibitory action wasslower than was the case in experiments where Li+ wasused to replace Na+.

The effects of low Na+ were explored further in cellsloaded with the Ca2+ indicator fluo-4 AM and imagedusing confocal microscopy (in the linescan configuration).A series of images from a representative experimentare shown in the upper panel of Fig. 2A. Regular Ca2+

transients, with Ca2+ rising approximately synchronouslyalong the cell, were observed in normal Na+ solution (leftpanel). A rapid switch to low Na+ occurred just beforethe next panel, where it can be seen that spontaneouswhole cell Ca2+ transients had been completely suppressed.Towards the end of the period of recording shown in thethird panel, spontaneous activity resumed, but this was inthe form of a Ca2+ wave (starting at a position at the topof the panel and propagating downwards); the next panelshows several more Ca2+ waves (broader and less uniformin magnitude than normal Ca2+ transients). On switchingback to normal Na+ solution, activity started to recover,



and the final panel shows restoration of spontaneous wholecell Ca2+ transients with synchronous Ca2+ changes alongthe cell.

Total fluorescence in these panels was quantifiedby averaging the signal across the cell and is shownby the traces in the lower panel of Fig. 2A. The redhorizontal dashed line indicates the minimum level offluo-4 AM fluorescence between beats. Note that abolitionof spontaneous activity by the switch to low-Na+ solutioncaused a fall in cytosolic [Ca2+] below the minimumdiastolic level observed when the cell was beating (redhorizontal dashed line). Similar observations were madein six cells, and when spontaneous activity was haltedby a rapid switch to low-Na+ solution, the mean fall inintegrated fluo-4 AM fluorescence signal was to 77 ± 5%

Figure 2. Effects of low-Na+ solution on SA node cell spontaneous Ca2+ transientsA, 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+ solutionwas applied immediately before the second frame, which shows that Ca2+ transients were abolished by the switchto low-Na+ solution; note that cytosolic Ca2+ fell following cessation of activity in low-Na+ solution. The thirdframe shows a continued fall in cytosolic Ca2+, followed by a Ca2+ wave that starts at the top of the image andpropagates downwards. More Ca2+ waves are evident in the fourth frame. Normal Ca2+ transients were quicklyrestored 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 causedcessation of beating and a fall in cytosolic Ca2+, as indicated by the fluorescence ratio. Similar observations weremade in a further 7 cells.

of the control minimum level between beats (n = 6,P < 0.05).

In a further eight cells, Ca2+ transients were measuredas indo-5F AM 410/490 nm ratios, allowing quantitativemeasurements of intracellular Ca2+ concentrations to beobtained following a calibration procedure. As shown inFig. 2B, rapid switch to low-Na+ solution again causedboth cessation of beating and a fall in cytosolic Ca2+ toa level 15 ± 2% (n = 8, P < 0.05) below the minimumdiastolic Ca2+ concentration in a beating cell. Minimumdiastolic Ca2+ concentration was found to be 225 ± 55 nm

in beating cells, and the lowest Ca2+ concentrationobserved during cessation of beating was 198 ± 72 nm.

This fall in cytosolic [Ca2+] seems surprising, sincelow-Na+ solution is expected to inhibit the major



pathway of Ca2+ extrusion via NCX; however, Ca2+ mightcontinue to be sequestered by the SR, leading to a fallof Ca2+ in the absence of significant continued Ca2+

entry through voltage-gated channels (since spontaneouselectrical activity was abolished). If this were correct, thefall in cytosolic [Ca2+] on switch to low-Na+ solutionmight 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 of30 μm CPA slowed the frequency of, but did not stop,the occurrence of spontaneous Ca2+ transients (frequencyreduced by 30 ± 7%; n = 6, P < 0.05). Figure 3A showsthat subsequent rapid switch to low-Na+ solution (in thepresence of CPA) again caused rapid cessation ofspontaneous Ca2+ transients but, in contrast to theobservations when CPA was absent, there was a consistentincrease in cytosolic [Ca2+] (as indicated by an increase influo-4 AM fluorescence; see Fig. 4A). Similar observationswere made in all six cells in which fluo-4 AM was used asthe Ca2+ indicator, and in a further six cells loaded withindo-5F AM.

There are several potential pathways that may underliethe cytosolic elevation of Ca2+ observed on application oflow-Na+ solution in the presence of CPA. One possibilityis that, on application of low Na+ (at this concentration)NCX reverses, bringing Ca2+ into the cell. To test for thispossibility, KB-R7943 was applied in the presence of CPA,

Figure 3. Effects of low-Na+ solution on spontaneous SA node cell Ca2+ transients in the presence ofCPA (to inhibit SR Ca2+ uptake)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, theCa2+ transients appeared smaller in magnitude, and slower to both peak and decay, compared with those in controlconditions. Rapid switch to low-Na+ solution abolished spontaneous Ca2+ transients, and this was associated witha 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 abolitionof Ca2+ transients, with no increase in baseline Ca2+. Subsequent rapid application of low-Na+ solution, in thepresence of KB-R7943, did not result in an increase in baseline Ca2+ levels.

since KB-R7943 has been reported to inhibit both forwardand reverse modes of NCX (Iwamoto et al. 1996; Watanoet al. 1996). As may be seen in Fig. 3B, application ofKB-R7943, in the presence of CPA, resulted in a cessationof spontaneous Ca2+ transients, but with little or no rise inbaseline Ca2+ levels. A subsequent rapid switch to low-Na+

solution also resulted in little or no change in baselinefluorescence. It may therefore be the case that applicationof low-Na+ solution in the presence of CPA results inreverse-mode NCX that brings Ca2+ into the cell, causinga gradual elevation of cytosolic [Ca2+].

If it is the case that NCX reverses on switch to 14.5 mm

Na+ solution, this raises the possibility that the cessationof beating on switch to 14.5 mm Na+ solution may notonly be due to the removal of an inward current normallycontributed 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 forNa+ 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 beexpected to be operating in reverse-mode at the restingmembrane potentials recorded from our SA node cells.Although the aforementioned effects of KB-R7943, which



inhibits reverse-mode NCX as well as the conventionalmode, do not support the possibility that it is impositionof an outward current that abolishes beating in thesecells, this was nonetheless further tested in experiments inwhich cells were exposed to 50 and 75 mm Na+ solutions.The NCX reversal potentials at 50 and 75 mm Na+ arecalculated to be −90 and −59 mV, and so it would beexpected that the contribution of any outward currentby reverse-mode NCX would be significantly less underthese conditions; indeed, a rapid switch to 75 mm Na+

would be expected to result in very little, if any, reversal ofNCX. As shown in Fig. 4, rapid switch to either of theseconcentrations of Na+ resulted in an immediate cessationof both spontaneous action potentials and spontaneousCa2+ transients. Thus, it is highly likely that the abolition ofbeating on switch to low-Na+ solution is due to inhibitionof an inward current that is normally contributed by NCXduring the pacemaker depolarization, and not primarilydue to imposition of an outward current.

If cytosolic [Ca2+] between beats remains ata sufficiently high level that Ca2+ extrusion by

Figure 4. Abolition of spontaneous activity following rapid switch to 50 or 75 mM Na+ solution (withLi+ as the replacement cation)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 thefirst 3 s of application; two action potentials, of abnormally large amplitude, were subsequently recorded in thiscell during application of low-Na+ solution. In two further cells, spontaneous action potentials were completelyabolished for the entire period of application of 75 mM Na+ solution. C, spontaneous Ca2+ transients recordedfrom a representative cell using confocal microscopy (fluo-4 AM). A rapid switch to 50 mM Na+ solution abolishedspontaneous Ca2+ transients. In this cell, a localized increase in intracellular Ca2+, accompanied by a gradualglobal increase in Ca2+, was observed; this may represent Ca2+ release from an overloaded SR. Similar observationswere made in 6 cells. D, rapid switch to 75 mM Na+ solution also abolished spontaneous Ca2+ transients. Similarobservations were made in 8 cells.

NCX contributes inward current that is essential forspontaneous pacemaker activity, an alternative methodof suppressing the inward NCX current (and thereforespontaneous activity) would be to apply a Ca2+ chelator,such as BAPTA, to the cytosol to lower [Ca2+]. Asillustrated in Fig. 5, application of membrane-permeantBAPTA AM (10 μm), whether by rapid switch (n = 6; datanot shown) or by superfusion (n = 6), caused abolitionof spontaneous action potentials. A similar cessation ofactivity was observed in three cells loaded with anotherCa2+ chelator, EGTA, by exposure to EGTA AM (10 μm;data not shown).

The data presented above support the possibility thatthe minimum diastolic level of [Ca2+] between beats maybe maintained at a higher level than the resting [Ca2+]in quiescent cells, resulting in a contribution of NCXcurrent throughout the entire pacemaker depolarization.To assess this directly, 5 μm nifedipine was rapidly appliedto spontaneously beating SA node cells imaged usingconfocal microscopy, in order to prevent the generationof action potentials (L-type Ca2+ channels are known to



Figure 5. Abolition of spontaneous actionpotentials in the presence of BAPTA AM, to chelateintracellular Ca2+

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 ofBAPTA AM into the cell, and subsequent de-esterification.

underlie the upstroke of the SA node action potential). Asshown in Fig. 6A, abolition of spontaneous beating withnifedipine was associated with a significant decrease in thefluo-4 AM fluorescence level, as compared to minimumlevels recorded between spontaneous Ca2+ transients; thiswas a consistent observation in all six cells studied. Similarfalls in Ca2+ levels were observed when either acetyl-choline (1 μm, n = 6) or adenosine (1 μm, n = 6) wereused to stop spontaneous beating (Fig. 6B and C). Thissupports the hypothesis that, during beating, cytosolic[Ca2+] concentrations remain higher between beats thanthose that occur in quiescent cells.

Figure 6. Abolition of spontaneous Ca2+

transients, by three differing pharmacologicalmechanisms, results in a fall in baselinefluorescence to a level below the minimumobserved between beats (dashed line)A, nifedipine, 5 μM (confocal microscopy,fluo-4 AM). B, adenosine, 1 μM (confocalmicroscopy, fluo-4 AM). C, acetylcholine, 1 μM

(conventional fluorescence microscopy,indo-5F AM).

Discussion

The observations reported here provide an importantextension to previous work demonstrating a role forCa2+, including that released from the SR, in pacemakingmechanisms (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 arise in subsarcolemmal [Ca2+], associated with SR Ca2+

release that precedes the upstroke of the action potential(perhaps triggered by T-type Ca2+ current), leads todepolarizing NCX current that contributes a depolarizinginfluence at this time (Huser et al. 2000; Bogdanov



et al. 2001; Vinogradova et al. 2002). The data presentedhere, however, demonstrate that the contribution of NCXcurrent to the pacemaker depolarization of guinea-pig SAnode cells is not exclusively dependent on a functional SR,since a switch to low-Na+ solution abolished spontaneousbeating in the presence of CPA, an inhibitor of SR Ca2+

uptake. Furthermore, if the only contribution of NCX weresecondary to SR Ca2+ release, inhibition of SR functionmight be expected to produce effects on pacemaking thatwere broadly similar to inhibition of NCX. In guinea-pigSA node cells, however, suppression of SR function isassociated with a slowing of rate (Rigg & Terrar, 1996;Rigg et al. 2000; Lakatta et al. 2003), whereas inhibition ofNCX abolishes beating. Thus, the essential contributionthat NCX makes to pacemaking in this preparation is notlimited to current (at the foot of the action potential) thatis secondary to localized SR Ca2+ release, but is also dueto a significant component that is SR independent. Thetiming of this SR-independent component need not belimited to the foot of the action potential, but instead mayoccur throughout the pacemaker depolarization.

Our experiments using rapid switch to low-Na+

solution to inhibit NCX provide important insightsconcerning the importance of NCX current and extend theinteresting observations of Bogdanov et al. (2001). Theseauthors showed that beating stopped soon after Na+ wasreplaced by Li+ in the solution bathing rabbit SA node cells,but cytosolic [Ca2+] was not simultaneously monitoredin these experiments and, as the authors pointedout, it might be argued that such continuous super-fusion with Li+ induces an increase in steady cytosolic[Ca2+] during the diastolic depolarization, which in turnmay affect SR loading, refractoriness of the ryanodinereceptor, or currents involved in automaticity. In furtherexperiments in which cytosolic [Ca2+] was measured, arapid ‘spritz’ of low-Na+ solution was found to suppressaction potential duration, though a large (approximately70% of normal) Ca2+ transient (or wave) remained. Inour experiments, rapid application of low-Na+ solutionconsistently suppressed activity within one beat, but adifferent type of spontaneous activity reappeared after aninterval of many seconds, in the form of Ca2+ waves, mostlikely secondary to spontaneous release of Ca2+ from anoverloaded SR (Fig. 2). Our experiments in guinea-pig SAnode cells thus differ significantly from those of Bogdanovand co-workers in that rapid application of low-Na+

solution was continued for a sufficiently long period tocompletely stop spontaneous electrical activity and Ca2+

transients, and (prior to the initiation of Ca2+ waves) thiswas not accompanied by an elevation of cytosolic [Ca2+].

Another important observation, shown in Fig. 2, is thefall in cytosolic [Ca2+] that followed cessation of beatingin the presence of low-Na+ solution, occurring eventhough a major mechanism for Ca2+ extrusion (NCX) wassuppressed. The mechanism underlying this may involve

Ca2+ uptake into the SR, mediated by the Ca2+-ATPase,continuing until the SR becomes overloaded, resultingin spontaneous Ca2+ release and Ca2+ waves. This wassupported by the observation that, when SR uptake wasinhibited by CPA, a rapid switch to low-Na+ solution againsuppressed spontaneous activity, but this was followedby a rise, rather than a fall, in cytosolic [Ca2+]. Hence,when the SR is functioning normally, SR Ca2+ uptake canreduce cytosolic [Ca2+] immediately following cessationof activity due to NCX inhibition.

Several potential pathways may contribute the Ca2+

that underlies the elevation in cytosolic levels observedon switch to low-Na+ solution in the presence of CPA.The lack of such an elevation of Ca2+ when KB-R7943 isused to inhibit NCX (in the presence of CPA) supportsa role for reverse-mode NCX in providing at least someof this Ca2+. This raises the possibility that abolitionof spontaneous activity by low-Na+ solution may bedue not only to removal of an inward current (i.e. NCXworking in the Ca2+ extrusion mode), but also to theimposition of an outward current (reverse-mode NCX) onthe pacemaker depolarization. However, since KB-R7943,which inhibits both modes of NCX, completely abolishedpacemaker activity, it is highly likely that inhibition ofinward NCX current alone is sufficient to cause cessationof spontaneous activity. This is supported further bythe observations that beating was abolished by 50 and75 mm Na+ solutions; for the latter concentration, littleor no outward current would be expected, since the NCXreversal potential (−59 mV) is calculated to be close to theminimum diastolic potential of these cells.

The hypothesis that the cytosolic [Ca2+] remainselevated between beats in spontaneously active SA nodecells is supported by our observations that abolition ofbeating by three differing pharmacological mechanisms(nifedipine, acetylcholine and adenosine) resulted ina fall in [Ca2+] to a level below the minimumrecorded between spontaneous Ca2+ transients. The Ca2+

chelators BAPTA and EGTA, which would be expectedto greatly reduce the minimum level of cytosolic Ca2+

(regardless of whether the major source was from theSR or Ca2+ entry across the sarcolemma) and henceNCX current, also caused cessation of spontaneousaction potentials. It therefore appears that, under theconditions of these experiments, the sequential activationof voltage-dependent ion channels is not by itself sufficientto maintain spontaneous action potentials in the face ofan abnormally low cytosolic [Ca2+].

Another possible effect of low-Na+ solution that mustbe considered is inhibition of hyperpolarization-activatedI f currents, since Li+ ions have been reported to permeateless well through these channels (Ho et al. 1994). However,previous experiments have shown that complete block ofI f slows but does not stop spontaneous beating (Denyer& Brown, 1990). We have also shown that ZD7288



slows but does not prevent initiation of spontaneousaction potentials, despite substantial inhibition of I f

current, but that rapid switch to low-Na+ solution underthese conditions again leads to cessation of activity.Furthermore, Bogdanov et al. (2001) have reported thatLi+ did not affect I f in their experiments. It is thereforeunlikely that the effects of low-Na+ solution are due toactions on I f. An alternative possibility is that low-Na+

solution might interfere with the depolarizing influenceof I st (sustained inward current; Guo et al. 1995, 1996,1997), but this seems unlikely, since the channel allowspermeation 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-sensitivevoltage-gated Na+ channels account for the abolitionof pacemaking, since Li+ passes through these channelsalmost as easily as Na+ (Chandler & Meves, 1965; Hille,1972). Furthermore, in our experiments, TTX had little orno effect on pacemaking, while switch to low-Na+ solutionin the presence of TTX caused immediate cessation ofbeating.

It therefore appears that the major factor suppressingpacemaker activity following switch to low-Na+ solutionis inhibition of inward NCX current, either in the formof NCX in its conventional 3 Na+ to 1 Ca2+ ion mode,or in the form of a Na+ leak pathway, as proposed byKang & Hilgemann (2004), in which import of one Na+

with one Ca2+ ion coupled to export of one Ca2+ ionprovides a Na+-conducting pathway. The abolition ofspontaneous activity by the Ca2+ chelators BAPTA andEGTA (also seen following excessive loading with Ca2+

indicators, as noted previously by Rigg et al. 2000) isconsistent with inhibition of either mode of operationof NCX. The levels of Ca2+ between beats were found tobe higher (225 nm) than those in quiescent cells (or inresting atrial and ventricular myocytes), making it likelythat there is a maintained component of electrogenic 3:1NCX throughout the pacemaker depolarization, even ifthere is an additional Na+ leak through the NCX protein.The mechanisms underlying this maintained elevation ofintracellular Ca2+ remain to be determined.

Although voltage-clamp experiments to measure NCXcurrents would potentially yield additional informationregarding the importance of this current to pacemaking,they are beyond the scope of this study. Simplevoltage-clamp experiments would not be helpful, partlybecause even for conventional 3:1 exchange, the level ofcurrents in a beating cell would vary dynamically withchanges in cytosolic Ca2+ during the cardiac cycle, andthis would necessarily be modified with conventionalvoltage-clamp protocols. Furthermore, the contributionsfrom the new modes of operation of NCX require complexexperiments that are beyond the scope of this investigation.

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 inpacemaking, since embryos in these models did not survivebeyond about 11 days post coitum, and the heartbeat wasabsent. In the light of the present experiments, it is possiblethat the lack of heartbeat in these knockout mice may bedue to the absence of an essential contribution of NCXduring the pacemaker potential. However, other harmfuleffects of NCX knockout cannot be excluded. It shouldbe noted that the NCX1 knockout described recently byHenderson et al. (2004) is specific to the ventricle, and soprovides no insight into the role of NCX in pacemaking.

Of course, other currents are known to contributeto the pacemaker depolarization, and some of thesemay be regulated by cytosolic [Ca2+], including I f anddelayed rectifier K+ currents (Tohse, 1990; Rigg et al.2003). However, inhibition of many of the ‘established’pacemaking currents is associated only with a slowingof beating rate in guinea-pig SA node cells, ratherthan a cessation. The observations reported here areconsistent with a fundamental and critical role forNCX in maintaining a depolarizing influence throughoutthe pacemaker depolarization. We propose that thismaintained depolarizing influence of NCX is secondaryto a high minimum level of cytosolic [Ca2+] that normallynever falls below that which supports Ca2+ extrusion viaelectrogenic 3:1 NCX, but this mechanism may also besupplemented by a contribution arising from a Na+ leakpathway through the NCX protein.

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Acknowledgements

This work was supported by the Wellcome Trust and the British

Heart Foundation.


fundamental importance of na+–ca2+ exchange for the pacemaking mechanism in guinea-pig sino-atrial...

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