Intracellular calcium modulation of voltage-gated sodium channels in ventricular myocytes

Intracellular calcium modulation of voltage-gatedsodium channels in ventricular myocytes

Simona Casini1, Arie O. Verkerk1, Marcel M.G.J. van Borren1, Antoni C.G. van Ginneken1,Marieke W. Veldkamp1, Jacques M.T. de Bakker1, and Hanno L. Tan1,2*

1Department of Clinical and Experimental Cardiology, Heart Failure Research Center, Academic Medical Center, Universityof Amsterdam, Meibergdreef 9, AZ 1105 Amsterdam, The Netherlands; and 2Department of Cardiology, Academic MedicalCenter, University of Amsterdam, Amsterdam, The Netherlands

Received 28 February 2008; revised 19 September 2008; accepted 22 September 2008; online publish-ahead-of-print 1 October 2008

Time for primary review: 32 days

Aims Cardiac voltage-gated sodium channels control action potential (AP) upstroke and cell excitability.Intracellular calcium (Cai

2þ) regulates AP properties by modulating various ion channels. Whether Cai2þ

modulates sodium channels in ventricular myocytes is unresolved. We studied whether Cai2þ modulates

sodium channels in ventricular myocytes at Cai2þ concentrations ([Cai

2þ]) present during the cardiac AP(0–500 nM), and how this modulation affects sodium channel properties in heart failure (HF), a conditionin which Cai

2þ homeostasis is disturbed.Methods and results Sodium current (INa) and maximal AP upstroke velocity (dV/dtmax), a measure ofINa, were studied at 20 and 378C, respectively, in freshly isolated left ventricular myocytes of controland HF rabbits, using whole-cell patch-clamp methodology. [Cai

2þ] was varied using different pipettesolutions, the Cai

2þ buffer BAPTA, and caffeine administration. Elevated [Cai2þ] reduced INa density

and dV/dtmax, but caused no INa gating changes. Reductions in INa density occurred simultaneouslywith increase in [Cai

2þ], suggesting that these effects were due to permeation block. Accordingly,unitary sodium current amplitudes were reduced at higher [Cai

2þ]. While INa density and gating atfixed [Cai

2þ] were not different between HF and control, reductions in dV/dtmax upon increases in stimu-lation rate were larger in HF than in control; these differences were abolished by BAPTA.Conclusion Cai

2þ exerts acute modulation of INa density in ventricular myocytes, but does not modify INa

gating. These effects, occurring rapidly and in the [Cai2þ] range observed physiologically, may contribute

to beat-to-beat regulation of cardiac excitability in health and disease.

KEYWORDSIon channels;

Na-channel;

Arrhythmia

1. Introduction

Voltage-gated sodium (Naþ) channels control excitability inthe heart and other excitable tissues by generating theaction potential (AP) upstroke. Naþ channel dysfunction invarious conditions may cause life-threatening cardiacarrhythmias by reducing cardiac excitability.1 Intracellularcalcium (Cai

2þ) participates in the regulation of numerouskey physiological processes. Its tight homeostasis supportsstrong and acute changes in Cai

2þ concentrations ([Cai2þ])

through Cai2þ release from Cai

2þ stores [e.g. the sarcoplasmicreticulum (SR)] during systole and reuptake into these storesduring diastole.2 This renders Cai

2þ a potential regulator ofcardiac excitability, as heart rates must vary strongly andacutely (beat-to-beat) to meet metabolic demands. Inmany common diseases associated with impaired excitabilityand life-threatening arrhythmias, Cai

2þ homeostasis is dis-turbed. For instance, in heart failure (HF), diastolic [Cai

2þ]

is increased as Cai2þ reuptake into the SR is delayed and

incomplete. This is particularly prominent at fast heartrates, because diastolic intervals are shorter.3 Yet, whileCai

2þ is known to regulate various ion channels whichcontrol the AP,4–6 it is not fully resolved whether Cai

2þ directlymodulates Naþ channel function and which mechanisms areinvolved. Few studies have investigated a possible relationbetween [Cai

2þ] and Naþ channel function. It was proposedthat Cai

2þ regulates Naþ channel biosynthesis in rat cardiacmyocytes,7,8 while short-term modulation (modulationwithin the time frame of the cardiac AP) was demonstratedin lipid bilayers9,10 and heterologous expression systems.11

In rabbit ventricular myocytes which were cultured for24 h, increase in [Cai

2þ] from 100 to 500 nM caused Naþ

channel gating changes, but left current densityunchanged.12 Still, the effects of changes within the physio-logical range of [Cai

2þ] on Naþ current (INa) of freshly isolatedleft ventricular myocytes have not been reported.

We hypothesized that Cai2þ modulates INa of freshly iso-

lated left ventricular myocytes, and that this modulation* Corresponding author. Tel: þ31 20 5663264; fax: þ31 20 6975458.

E-mail address: [email protected]

Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008.For permissions please email: [email protected].

Cardiovascular Research (2009) 81, 72–81doi:10.1093/cvr/cvn274

by guest on January 29, 2015D

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underlies beat-to-beat variations in INa magnitude, e.g.those that occur upon changes in heart rate. We aimed toestablish how Cai

2þ modulates Naþ channels under physio-logical conditions and during HF.

2. Methods

2.1 Cell preparation

The investigation conforms with the Guide for the Care and Use ofLaboratory Animals published by the US National Institutes of Health(NIH publication 85–23, revised 1996) and was approved by the insti-tutional animal experiments committee. HF was induced by com-bined volume overload (aortic regurgitation) and pressureoverload (aortic banding) in New Zealand White rabbits.13 Mid-myocardial left ventricular myocytes were isolated from healthy(control) and HF animals.14

cDNA of SCN5A, the gene encoding the a-subunit (Nav1.5) of thehuman cardiac Naþ channel, was prepared in the pCGI vector forbicistronic expression of Nav1.5 and green fluorescent protein.15

Human embryonic kidney (HEK293) cells were transiently trans-fected with 1 mg SCN5A cDNA and 1 mg hb1-subunit cDNA using lipo-fectamine (Gibco BRL, Life Technologies). Transfected HEK293 cellswere cultured in minimum essential medium (Earles salts andL-glutamine) supplemented with non-essential amino acid solution,10% foetal bovine serum, 100 IU/mL penicillin, and 100 mg/mLstreptomycin in a 5% CO2 incubator at 378C for 1–2 days. Onlycells exhibiting green fluorescence were selected for electrophysio-logical experiments.

2.2 Electrophysiology

2.2.1 Data acquisitionMembrane currents and potentials were recorded in the whole-cellconfiguration of the patch-clamp technique using patch pipetteswith a tip resistance of 1.5–2 MV, unless mentioned otherwise. INa

signals were low-pass filtered (cut-off frequency 5 kHz) and digi-tized (20 kHz). Series resistance (Rs) was compensated by �80%.AP measurements were filtered and digitized at 10 and 40–50 kHz,respectively. Voltage control, data acquisition, and analysis of INa

and AP recordings were performed with pClamp8.0/Clampfit (AxonInstruments) and custom-made software, respectively.

2.2.2 Sodium current propertiesIn myocytes, INa was analysed at 208C; at 378C, AP upstroke velocitywas analysed as a measure of INa (see below). At 208C, activation,inactivation, recovery from inactivation, and slow inactivation para-meters were determined using protocols as indicated in Figure 1(cycle time of 2 s for activation, inactivation, and slow inactivation,and 4 s for recovery from inactivation). Data were fitted asdescribed in the Supplementary material online. INa was recordedusing intracellular solutions to which CaCl2 was added to obtain afree [Cai

2þ]¼0, 100, and 500 nM as calculated using WEBMAX Stan-dard software (http://www.stanford.edu/~cpatton/webmaxcS.htm). Pipettes were filled with (mM): NaCl 3, BAPTA 10, Mg-ATP 2,CsOH 140, HEPES 10, pH 7.2 (HCl). The bath solution contained(mM): NaCl 7, CaCl2 1.8, MgCl2 1.2, HEPES 10, glucose 11, CsCl125, nifedipine 0.005, pH 7.3 (CsOH). BAPTA was used as Cai

2þ

buffer, because it provides stringent control over [Cai2þ], thanks to

its fast Cai2þ buffering kinetics.11

INa was defined as the difference between peak current and thecurrent at the end of the depolarizing voltage step. INa density(Table 1) was calculated by dividing maximal INa by cell membranecapacitance (Cm). The mean values for Cm were 161.6+7.8 pF(control, n ¼ 27) vs. 213.0+14.1 pF (HF, n ¼ 28, P ¼ 0.007).Access resistance (Ra) values were 5.1+0.3 MV (control, n ¼ 27)vs. 6.1+0.4 MV (HF, n ¼ 28).

In HEK293 cells, experiments were conducted at 208C and INa waselicited by depolarizing steps from a holding potential of 2140 mV

(cycle time 5 s). Cm and Ra were 11.9+0.9 pF and 5.8+0.7 MV,respectively (n ¼ 10). Acute effects of Cai

2þ on INa were studiedwith a modified dialyzable pipette (Supplementary material onlineand Tang et al.16). The pipette solutions were identical to thoseused for INa measurements in myocytes at 208C (described above,Ca0 and Ca500), while the bath solution (Solution 1) contained(mM): NaCl 10, Na-gluconate 130, HEPES 10, glucose 5.5, CaCl21.8, MgCl2 1.2, pH 7.4 (CsOH). To study possible effects of caffeineon INa, 10 mM caffeine was washed into the bath which contained(mM): NaCl 140, KCl 4.7, CaCl2 1.8, MgCl2 2.0, NaHCO3 4.3,Na2HPO4 1.4, glucose 11.0, HEPES 16.8, pH 7.4 (NaOH). Pipetteswere filled with (mM): CsF 110, CsCl 1.0, EGTA 11, NaF 10, CaCl21.0, MgCl2 1.0, Na2ATP 2.0, HEPES 10, pH 7.3 (CsOH).

2.2.3 Upstroke velocity measurementsTo investigate INa characteristics at 378C and physiological Naþ con-centrations, we recorded AP upstroke velocities and expressed themas the maximum value of the first derivative of AP upstroke (dV/dtmax). dV/dtmax is a convenient index for INa. Although it slightlyoverestimates Naþ channel availability, the discrepancies betweenINa and dV/dtmax are reduced at high temperatures (26–278C).17

dV/dtmax was measured using an alternate voltage/current-clampmode with a custom-made amplifier.18 Using dV/dtmax as ameasure of Naþ channel availability, steady-state inactivation,recovery from inactivation, and slow inactivation were analysedwith the protocols indicated in Figure 2. As a measure of peakcurrent magnitude (Table 2), the maximum dV/dtmax of the Naþ

channel availability curve was given. Bath solution (Solution 2) con-tained (mM): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.5,and HEPES 5.0, pH 7.4 (NaOH). Pipette solution contained (mM):NMDG-OH 140, BAPTA 10, K-gluconate 125, KCl 20, NaCl 5, Mg-ATP5, and HEPES 10, pH 7.2 (HCl). CaCl2 was added to obtain free[Cai

2þ]¼0 or 500 nM. Data for voltage-dependence of inactivationand recovery from inactivation were fitted as described in theSupplementary material online.

To investigate INa availability at various stimulus frequencies and,consequently, at various [Cai

2þ],3 dV/dtmax were measured incontrol and HF myocytes at 0.2–4 Hz in the presence or absenceof 10 mM intracellular BAPTA. Experiments were conducted at378C. The use of BAPTA eliminates calcium (Ca2þ) transients andCa2þ dependent L-type Ca2þ channel inactivation, causing modifi-cation of several parameters in the cell, including the normal regu-lation of the cardiac AP. Consequently, AP duration is prolonged,especially in HF.19,20 To control the duration of diastolic intervals,we used the combined voltage/current-clamp method. Bath solu-tion was Solution 2. Pipette solution without BAPTA (Solution 3)contained (mM): K-gluconate 125, KCl 20, NaCl 5, Mg-ATP 5, andHEPES 10, pH 7.2 (KOH). Pipette solution with BAPTA contained(mM) BAPTA 10, KOH 40, gluconic acid 20, NaCl 5, Mg-ATP 5,HEPES 10, K-gluconate 85, KCl 20, pH 7.2 (KOH).

To study acute effects of changing [Cai2þ] on Naþ channel avail-

ability in the range of [Cai2þ] as observed during the cardiac cycle,

we created acute increases in Cai2þ by inducing Cai

2þ release fromthe SR with 10 mM caffeine. [Cai

2þ] and dV/dtmax were simul-taneously measured at 378C using the alternate voltage/current-clamp mode. For each cell, dV/dtmax represents theaverage of 10 consecutive values recorded at steady-state. Bathsolution was Solution 2, pipette solution was Solution 3.

2.2.4 Cai21 measurements

[Cai2þ] was measured in Indo1-loaded myocytes (1 Hz stimulation).

Dye-loaded myocytes were excited through a 100� oil-immersionobjective at 340 nm (75W Xenon arc lamp). Intensities of theemitted light at 405 nm (I405) and 505 nm (I505) were recorded, sub-sequently digitized at 1 kHz, and filtered at 100 Hz. A rectangularadjustable slit was used to select a single myocyte and reducebackground fluorescence, which was subtracted offline beforethe ratio values were calculated (I405/I505). Fluorescent ratio was

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translated as [Cai2þ]¼b*Kd(R2Rmin)/(Rmax2R). After calibration of

the setup, b (ratio of maximum to minimum I505) was 2.2,maximal ratio (Rmax) 2.21+0.24, minimal ratio (Rmin) 0.31+0.04, and Kd 250 nM (Data sheet Indo-1, Molecular Probes, EugeneOR, USA).

2.2.5 Single-channel measurementsSingle Naþ channel currents were recorded at 208C from inside-outpatches of ventricular myocytes isolated from control rabbit hearts,in the presence of veratridine (30 mM). Veratridine reduces peak INa,but also induces a non-inactivating current due to long-lasting

channel openings.21 Therefore, the use of veratridine provides amore reliable analysis of unitary current amplitude compared tounmodified Naþ channels.22 Voltage steps (900 ms) were appliedfrom 280 to 240 mV (holding potential 2140 mV, cycle time 5 s).Pipettes were fire polished and had a tip resistance of 2.5–3.5 MV.Signals were filtered and digitized at 1 and 10 kHz, respectively. Bathsolutions were identical to those used as pipette solutions for INa

measurements in myocytes at 208C (Ca0 and Ca500). Pipette solutionwas Solution 1 with the addition of nifedipine (0.005 mM). Unitarycurrent amplitudes were estimated from sweeps exhibiting well-resolved closings as determined by fitting Gaussian distributions.

Figure 1 Sodium current densities and gating properties in control myocytes at various [Cai2þ]. (A) Typical examples of whole-cell sodium current (INa) traces

recorded at 208C from control myocytes at [Cai2þ]¼0 nM (Ca0), 100 nM (Ca100), and 500 nM (Ca500) in response to depolarizing voltage steps (protocol in C, right

part). (B) Average INa densities at Ca0, Ca100, and Ca500 obtained by dividing maximal INa by cell membrane capacitance. (C) On the right part, average voltagedependence of activation. Normalized current was plotted as a function of the membrane potential. On the left part, steady-state inactivation. Peak sodiumcurrents were normalized to maximum values in each cell and plotted as a function of the voltage of the conditioning step. (D) Average fast (tf) and slow(ts) time constants of INa decay plotted as a function of membrane potential. (E) Average time course of recovery from inactivation. Peak sodium currents elicitedby P2 were normalized (P2/P1) and plotted as a function of the recovery interval (Dt). (F) Average development of slow inactivation. INa elicited by P2 werenormalized (P2/P1) and plotted as a function of the duration of P1. Insets: voltage protocols.

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2.2.6 Statistical analysisResults are expressed as mean+ SEM. Unpaired Student’s t-testwas used to study the effects of Cai

2þ on Naþ channel properties,and to compare INa gating, current densities, Cm and Ra betweencontrol and HF. When data were not normally distributed, a

Mann–Whitney test was performed. Paired Student’s t-test wasperformed to compare [Cai

2þ] and dV/dtmax in control myocytesbefore/after addition of caffeine, and INa amplitudes in HEK293cells before/after addition of caffeine or increases in [Cai

2þ]. Toanalyse INa decay, the development of slow inactivation and to

Figure 2 Action potential upstroke velocity as a measure of sodium current magnitude and gating properties in control myocytes at various [Cai2þ]. (A) Typical

examples of action potential (AP) upstroke velocities recorded at 378C from control myocytes at [Cai2þ]¼0 nM (Ca0) and 500 nM (Ca500). (B) Average maximum

AP upstroke velocities (dV/dtmax) of the sodium current (INa) availability curve, as a measure of INa magnitude. (C) Average voltage dependence of inactivation.dV/dtmax values were normalized to the maximum values in each cell and plotted as a function of the voltage of the conditioning step. (D) Average time course ofrecovery from inactivation. dV/dtmax elicited by P2 were normalized (P2/P1) and plotted as a function of the recovery interval (Dt). (E) Average development ofslow inactivation. dV/dtmax elicited by P2 were normalized (P2/P1) and plotted as a function of the duration of P3. Insets: combined voltage/current-clampprotocols. CC, current-clamp.

Table 1 Intracellular calcium modulation of sodium channel properties at 208C

Control myocytes HF myocytes

Ca0 n Ca100 n Ca500 n Ca0 n Ca500 n

Peak currentINa (pA/pF) 250.4+3.8 12 244.5+3.9 5 236.5+3.5$ 10 252.9+5.0 17 237.6+3.5§ 11

ActivationV1/2 (mV) 236.8+1.1 12 236.1+1.2 5 236.5+1.0 10 238.0+0.9 17 238.5+0.8 11k (mV) 4.9+0.2 5.0+0.4 5.2+0.2 4.9+0.2 4.5+0.2#

InactivationV1/2 (mV) 278.0+1.4 10 277.9+1.2 5 278.7+1.2 6 276.6+1.0 11 276.4+0.6 8k (mV) 25.1+0.1 25.2+0.3 25.2+0.2 25.3+0.1 25.6+0.1

Recoverytf (ms) 3.5+0.6 9 3.8+0.9 3 4.1+0.4 8 4.4+0.4 9 4.9+0.5 6ts (ms) 30.3+4.3 31.4+6.5 45.4+8.8 32.6+2.9 31.7+3.7

Slow inactivationA at 1000 ms 0.27+0.02 12 0.26+0.03 5 0.30+0.02 12 0.27+0.02 15 0.26+0.02 14

A at 1000 ms, fraction of channels that enter the slow inactivated state at the end of a 1000 ms step, calculated as 12(P2/P1); Ca0, [Cai2þ]¼0 nM; Ca100,

[Cai2þ]¼100 nM; Ca500, [Cai

2þ]¼500 nM; INa, sodium current density; k, slope factor of voltage-dependence of (in)activation; tf, fast time constant of recov-ery from inactivation; ts, slow time constant of recovery from inactivation; V1/2, voltage of half-maximal (in)activation.

$P , 0.05 vs. control Ca0.§P , 0.05 vs. HF Ca0.#P , 0.05 vs. control Ca500.



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study whether the relationships between dV/dtmax and heart ratewere different in control and HF, we conducted two-way ANOVAwith repetitive measurements followed by a Holm–Sidak test forpost hoc analysis. P , 0.05 indicates statistical significance.

3. Results

3.1 Effects of Cai21 on Na1 channel properties

We first studied the effects of Cai2þ at 208C on INa properties

in myocytes isolated from control rabbits (Table 1 andFigure 1). We used pipette solutions buffered at 0, 100, or500 nM free [Cai

2þ] (Ca0, Ca100, and Ca500) to cover the[Cai

2þ] range found in myocytes of control and HF rabbits3

during the cardiac cycle. Figure 1A shows representativeexamples of INa traces recorded from control myocytes atdifferent [Cai

2þ]. INa density was significantly smaller atCa500 than at Ca0, while values at Ca100 were intermediate(Table 1 and Figure 1B). This reduction was not due tochanges in gating properties. V1/2 and k of activation andinactivation were not different between Ca0, Ca100, andCa500 (Table 1 and Figure 1C), as well the rates of currentdecay (Figure 1D), recovery from inactivation (Figure 1E),and slow inactivation, a conformational state that partlydetermines INa availability at fast heart rates because ofits slow kinetics23 (Table 1 and Figure 1F).

To study whether these findings also apply to physiologicaltemperatures, we repeated these experiments at 378C,using dV/dtmax as an index of INa magnitude.18 Resultswere similar to the voltage-clamp studies at 208C, withdV/dtmax being smaller at Ca500 than at Ca0 and gatingproperties similar at Ca0 and Ca500 (Table 2 and Figure 2).However, the percentage of dV/dtmax reduction observedat high [Cai

2þ] was smaller than the INa reduction observedat 208C. While differences in recording temperatures maycause this discrepancy, future studies must resolve theunderlying mechanisms.

As we found no evidence that Cai2þ modulates Naþ channel

gating, we hypothesized that its effect to reduce INa magni-tude results from impediment of Naþ channel permeation.To test this hypothesis, we conducted paired experiments onhuman cardiac Naþ channels expressed in HEK293 cells. Wemeasured INa at different [Cai

2þ] in the same cell by varying[Cai

2þ] with a dialyzable pipette.16 We observed a similar

reduction (�30%) in peak INa amplitude as in myocytesat 208C when [Cai

2þ] was increased from 0 to 500 nM(Figure 3B). To verify whether current amplitudeswere stable up to the change in intracapillary solutionwithin the same cell, INa amplitudes were plotted againsttime (Figure 3A, bottom). This analysis indicated that INa

amplitudes were stable during recording at [Cai2þ]¼0 nM,

and declined smoothly upon changing the pipette solution to[Cai

2þ]¼500 nM. We took this smooth decrease as being con-sistent with the (diffusion) time required for the change inintracellular solution.

3.2 Effects of Cai21 on unitary Na1

current amplitude

To provide direct evidence for permeation block, we next per-formed inside-out patch-clamp studies in control myocytes totest the effects of Cai

2þ on veratridine-modified single Naþ

current amplitudes. The effect of veratridine was first testedon whole-cell INa (Figure 4A). Consistent with previousfindings,21 veratridine reduced peak INa amplitudes and

Figure 3 Sodium current amplitudes in HEK293 cells at various [Cai2þ]. (A)

Top: typical examples of INa elicited in response to depolarizing voltageclamp steps from 2140 to 220 mV at [Cai

2þ]¼0 nM (1) and after dialysis of500 nM Cai

2þ (2). INa were recorded at times indicated by arrows 1 and 2 inthe bottom panel. Bottom: current amplitudes were stable in time duringthe recordings at [Cai

2þ]¼0 nM (Ca0) and during/after wash-in of 500 nMCai

2þ (Ca500). (B) Average peak INa (n ¼ 4) normalized in each cell to thehighest INa value obtained with Ca0.

Table 2 Intracellular calcium modulation of sodium channel properties at 378C

Control myocytes HF myocytes

Ca0 n Ca500 n Ca0 n Ca500 n

Peak currentdV/dtmax (V/s) 435.0+19.5 17 394.1+7.7$ 10 438.1+24.2 12 387.3+15.7§ 13

InactivationV1/2 (mV) 274.9+0.8 17 274.7+1.7 10 274.7+1.5 12 274.1+0.6 13k (mV) 24.4+0.1 24.2+0.1 4.4+0.2 4.1+0.07

Recoverytf (ms) 10.6+2.3 7 8.6+2.1 7 9.7+0.9 8 7.6+1.2 9ts (ms) 36.5+5.4 26.3+5.8 48.0+7.4 46.6+9.2A at 1000 ms 0.11+0.02 14 0.14+0.01 6 0.11+0.02 7 0.12+0.01 8

dV/dtmax, maximal action potential upstroke velocity; other abbreviations, see Table 1.$P , 0.05 vs. control Ca0.§P , 0.05 vs. HF Ca0.

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induced a non-inactivating current due to long-lasting channelopenings (Figure 4A, left panel). Moreover, activation ofveratridine-modified Naþ channels occurred already at rela-tively negative potentials (Figure 4A, right panel). In inside-outpatches, resolvability of unitary channel events was only poss-ible in a minority of current traces. This may have been dueto long-lasting openings of multiple channels simultaneously,as a consequence of the relatively slow channel inactivationin the presence of veratradine. Nevertheless, two distinctsingle-channel current amplitudes were observed (Figure 4B),in accordance with a previous study which showed thatveratridine induces sub-conductance states of the cardiacNaþ channel.21 Figure 4C, left panel, shows typicalexamples of single channel currents from two Naþ channel

sub-conductance states with 0 and 500 nM Cai2þ. When Cai

2þ

was increased from 0 to 500 nM in paired experiments, thesingle channel current amplitude decreased to 82.2+2.5%(n¼ 6 patches). Fitting the pooled data by linear regressionyielded a decrease from 38.5 to 32.2 pS for the ‘larger’channel conductance and from 24.3 to 20.6 pS for the‘smaller’ channel conductance (Figure 4C, right).

3.3 Effects of Cai21 on AP upstroke velocity

Having established that steady-state increases in [Cai2þ]

reduce INa density in freshly isolated myocytes by impedingion permeation, we next investigated whether this interactionmay be relevant to beat-to-beat modulation of the cardiac AP.

Figure 4 Unitary sodium currents in inside-out patches at various [Cai2þ] in control myocytes. (A) Typical examples of the effect of veratridine on whole-cell

sodium current (INa) recorded at 208C from control myocytes (left) and current–voltage (I–V) relationships of INa (right) in the presence (open circle) and absence(closed circle) of veratridine. (B) Typical examples of single sodium channel currents of different amplitudes (left) and their corresponding Gaussian distributions(right). Due to long-lasting openings of multiple channels simultaneously, the zero current level in the histograms represents the closed state of the sodiumchannel, rather than the real zero current level. (C) Left: typical examples of single sodium channel currents recorded in the presence of 0 and 500 nMcalcium in the bath solution (top: ‘larger’ conductance state, measured at 265 mV; bottom: ‘smaller’ conductance state, measured at 260 mV). Right: I–Vrelationships of pooled data (n ¼ 6) of single channel current amplitudes at [Cai

2þ]¼0 nM (Ca0) and 500 nM (Ca500); circles: ‘smaller’ conductance, squares:‘larger’ conductance. Lines: best fit curves to the equation y¼A�[exp(20.015�Vm)20.1054�exp(0.01�Vm)], assuming Goldman–Hodgkin–Katz behaviourand a reversal potential of þ90 mV.



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To study how changes in [Cai2þ], which occurred acutely

and in the range of [Cai2þ] as observed during the cardiac

cycle in this model,3 impacted on dV/dtmax at 378C, we

conducted simultaneous recordings of dV/dtmax and[Cai

2þ]. Adding 10 mM caffeine to the bath solution causedCai

2þ release from the SR, and resulted in an increase in[Cai

2þ]. This was associated with a reciprocal reductionin dV/dtmax, which occurred instantaneously upon changesin [Cai

2þ] (Figure 5A). Of note, dV/dtmax reduction occurredover the whole range of [Cai

2þ], following depletion of theSR, indicating that [Cai

2þ] acutely reduces INa density in adose-dependent fashion (Figure 5B). Average dV/dtmax

before and after 30 s of caffeine exposure is shown inFigure 5C, and corresponding average [Cai

2þ] in Figure 5D.[Cai

2þ] significantly increased, while dV/dtmax significantlydecreased, with respect to their initial values. To rule outthat these effects were due to a direct action of caffeineon Naþ channels, we studied the effect of the drug on INa

densities of cardiac Naþ channels expressed in HEK293cells. In these cells, caffeine does not induce Ca2þ releasefrom intracellular Ca2þ stores.24 Addition of 10 mM caffeineto the bath solution did not change INa density (Figure 5E).

3.4 Effects of Cai21on Na1 channel properties

in heart failure

To further explore whether this acute modulation of INa

properties by Cai2þ may have pathophysiological relevance,

we studied the effects of Cai2þ on INa properties in a rabbit

model of HF. As reported in earlier studies from our labora-tory,3 Cai

2þ handling is disturbed in this model. In particular,diastolic [Cai

2þ] is significantly increased.

Figure 5 Relationship between [Cai2þ] and action potential upstroke velocity in control myocytes. (A) Typical example of simultaneous recording at 378C of

intracellular calcium ([Cai2þ]) and maximal action potential upstroke velocity (dV/dtmax) in the absence and presence of 10 mM caffeine in control myocytes.

Inset: combined voltage/current-clamp protocol. CC, current-clamp. (B) Average relationship between increase in [Cai2þ] and decrease in dV/dtmax (n ¼ 7–

18). (C) Average dV/dtmax at baseline (430.6+22.6 V/s, n ¼ 22) and after addition of 10 mM caffeine (356.1+20.7 V/s, n ¼ 22). (D) Average diastolic [Cai2þ]

at baseline (86.4+4.0 nM, n ¼ 22) and after addition of 10 mM caffeine (177.1+ 10.1 nM, n ¼ 22). (E) Average current density of cardiac Naþ channelsexpressed in HEK293 cells at baseline (2857.4+211.5 pA/pF, n ¼ 6) and after the addition of 10 mM caffeine (2897.1+199.2 pA/pF, n ¼ 6).

Figure 6 Relationship between heart rate and action potential upstroke vel-ocity in control and heart failure myocytes. Average relationship betweenstimulus frequency and maximal action potential upstroke velocity (dV/dtmax) in control and heart failure (HF) myocytes, in the presence orabsence of 10 mM BAPTA. Values were normalized to the highest dV/dtmax

in each myocyte. Without BAPTA, the reduction in dV/dtmax in response toheart rate increase was larger in HF (n ¼ 13) than in control (n ¼ 11).These differences were abolished by the addition of BAPTA (HFþBAPTA,n ¼ 12; controlþBAPTA, n ¼ 13). Asterisk: P , 0.05 between control andHF. Inset: combined voltage/current-clamp protocol. CC, current-clamp.

S. Casini et al.78


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First, to study whether the nature of INa modulation byCai

2þ, as observed in control, is also present in HF, we con-ducted experiments in myocytes isolated from HF rabbitsusing pipette solutions with the same steady-state [Cai

2þ]as in control (0 and 500 nM) at 208C (INa) and 378C (dV/dtmax). INa density and gating were similar in control andHF at both temperatures (Tables 1 and 2). Cai

2þ exertedsimilar acute modifications of INa density in HF as incontrol. Thus, also in HF, INa density and dV/dtmax were sig-nificantly smaller at [Cai

2þ]¼500 nM than at [Cai2þ]¼0 nM,

while gating properties were unaltered (Tables 1 and 2).To study whether acute [Cai

2þ]-mediated changes in INa

density may translate into differences in excitabilitybetween control and HF under physiological conditions, westudied dV/dtmax at different stimulation rates. We madeuse of previous findings from our laboratory,3 that, in ourmodel, diastolic [Cai

2þ] increases in parallel with the stimu-lation rate, and that this increase is larger in HF than incontrol. For instance, when pacing rate is increased from0.2 to 3 Hz, diastolic [Cai

2þ] increases by 40 nM (from 50 to90 nM) in control, but by 80 nM (from 145 to 225 nM) in HF.Thus, dV/dtmax was expected to decline at fast heartrates, and this reduction was expected to be more promi-nent in HF than in control. To better appreciate thechanges in dV/dtmax in response to different stimulationrates, values were normalized to the highest dV/dtmax ineach myocyte. When stimulation rates were increased inthe absence of Ca2þ chelators in the pipette solution, dV/dtmax decreased more in HF than in control. Thus, normal-ized dV/dtmax was significantly smaller in HF than controlat 3 and 4 Hz. Yet, when BAPTA was added to the pipettesolution to maintain [Cai

2þ] at virtually zero levels,11 thedifferences between control and HF in the dV/dtmax-heartrate relationships were abolished (Figure 6).

4. Discussion

We demonstrated that Cai2þ modulates Naþ channel density

without changing its kinetic properties in freshly isolatedventricular myocytes. [Cai

2þ]-mediated changes in INa

density were acute and occurred in the range of [Cai2þ]

levels found in control and HF. INa reduction was due todecreased Naþ channel conductance. Cai

2þ-mediated per-meation block of the Naþ channel may contribute tobeat-to-beat modulation of INa properties as it provides amechanism by which diastolic [Cai

2þ] controls INa density.We observed this regulatory mechanism not only in the phys-iological state, but also in a disease state in which diastolic[Cai

2þ] is increased, i.e. HF.

4.1 Cai21-dependent modulation of Na1 channel

Previous single-channel experiments in cardiac myocyteshave shown that extracellular Ca2þ reduces Naþ channelunitary current amplitude.21,22,25 Reduction of single-channel conductance was explained by a very fast openchannel block, due to a rapid movement of Ca2þ ions intoand out of a binding site within the Naþ channel, ratherthan to changes in the surface charges.25 In contrast, theeffects of Cai

2þ on Naþ channels were not resolved.It was proposed that Cai

2þ regulates Naþ channel biosyn-thesis in rat cardiac myocytes.7 Accordingly, the decreasein INa density observed in cultured rat neonatal ventricular

myocytes exposed for 24 h to culture medium containing10 mM Ca2þ and 10 mM Kþ (in order to raise [Cai

2þ]) wasattributed to reduced sarcolemmal Naþ channel expressionrather than to changes in single-channel conductance orgating.8

While these studies revealed long-term modulation of Naþ

channel expression and current density by Cai2þ, the findings

that single Naþ channel conductance in cells exposed to high[Cai

2þ] was not different from that in control cells8 con-trasted with a study by French et al.,9 in which rat brain-type Naþ channels were inserted into lipid bilayers. Inthese experiments, either external or internal applicationof 10 mM Ca2þ reduced single-channel conductance. Theseexperimental findings were reproduced in a computermodel of Naþ channels.26 Using the same lipid bilayersystem as French et al., Zamponi et al.10 showed that, inthe skeletal-muscle isoform of the rat Naþ channel, intern-ally applied millimolar Ca2þ concentrations caused a dose-dependent reduction of single-channel INa. A block of thepore involving multiple sites together with a surfacecharge effect was suggested as mechanisms.

Wingo et al.11 showed that in tsA201 cells transientlytransfected with the cardiac Naþ channel, an increase in[Cai

2þ] from 0 to 10 mM (with the effect saturating at1 mM) caused a positive shift of the INa availability curve.A putative EF hand motif located in the C-terminus of thechannel was proposed as a Ca2þ binding site and regulatorof the Cai

2þ-dependent effects.11 However, a separatestudy failed to detect any Ca2þ binding to that region.27

To complement these previous studies, we now provideevidence for a direct modulation of INa by physiological[Cai

2þ] in freshly isolated cardiomyocytes. We showed thatCai

2þ modulates INa density without affecting Naþ channelgating properties. The latter finding contrasts with thereport of Wingo et al. This discrepancy may be explainedby the differences in the cell types studied. Moreover, itemphasizes that findings obtained in heterologousexpression systems may not always be applicable to nativemyocytes. Further evidence that it is relevant to considerthe cell type studied comes from the observation thatincreases in [Cai

2þ] from 100 to 500 nM, did cause Naþ

channel gating changes in rabbit ventricular myocyteswhich were cultured for 24 h.12

Our findings that changes in INa magnitude and dV/dtmax inresponse to changes in [Cai

2þ] (following Ca2þ dialysis with aperfused pipette, and caffeine administration and its cessa-tion, respectively) occurred instantaneously, argued againsta Cai

2þ effect on expression and subsequent sarcolemmalinsertion of Naþ channels. It is believed that the expressionof plasma membrane proteins that must traverse the Golgiapparatus before membrane insertion would require a sig-nificantly longer time. However, our single-channel experi-ments indicated that Cai

2þ acts on INa density bymodulating Naþ channel permeation. Indeed, Naþ channelconductance was reduced in the presence of Ca500.

4.2 Cai21-dependent modulation of Na1 channel

through Cai21-dependent proteins

While we aimed to establish the direct effect of physiologi-cal [Cai

2þ] on INa properties under control and HF conditions,it must be noted that, besides a direct modulation, Cai

2þ canregulate the cardiac Naþ channel through Cai

2þ-activated



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proteins. Cai2þ-dependent interaction of the Cai

2þ-bindingprotein calmodulin (CaM) with an IQ-like motif in the Naþ

channel C-terminus may modulate Naþ channel gating inheterologous expression systems27–29 and isolated cardio-myocytes.30 However, the nature of the reported gatingchanges was not consistent27–30 and binding between CaMand the cardiac Naþ channel was not confirmed in allstudies.31 A role for the Ca2þ/CaM-dependent proteinkinase II (CaMKII) has also emerged. Adenovirus-mediatedCaMKIIdc overexpression in cultured rabbit myocytesinduced a hyperpolarizing shift in voltage-dependent inacti-vation, increased slow inactivation, and slowed recoveryfrom inactivation in a Cai

2þ-dependent manner. Moreover,INa decay was slower, and the late component of INa wasincreased, the latter effect being confirmed in a recentstudy.30 All these effects were also seen in transgenic miceoverexpressing CaMKIIdc and they could be reversed withthe use of CaMKII inhibitors.12 Of note, none of thesestudies, which were performed either in heterologousexpression systems or in isolated cardiac myocytes, havereported some effects of CaM or CaMKII on INa density. Onthe other hand, altered Cai

2þ homeostasis leading toprotein kinase C (PKC) activation was considered the prob-able cause of reductions in INa and dV/dtmax in transgenicmice overexpressing a constitutively active form of calci-neurin.32 These results were in line with previous studiesshowing a INa decrease following PKC activation.33,34

Although our use of the inside-out configuration of thepatch-clamp technique allowed INa recording from patchesdetached from myocyte cell membranes, it cannot beexcluded that INa reduction was (partly) mediated by theactivation of the Ca2þ-dependent PKC.32–34

4.3 Na1 channel modulation in heart failure

In HF, disturbed Cai2þ handling (in particular, increased dia-

stolic [Cai2þ]), ranks among the most consistent changes,

found in ventricular myocytes isolated from various animalHF models, and in myocytes from failing human hearts. Pre-vious studies have addressed the chronic effects of HF on INa

density, but these results were not consistent. Downregula-tion of INa was found in different canine HF models35–37 andin human ventricular cells.36 Decrease in INa density mayreduce excitability, thereby slowing myocardial conductionand contributing to re-entrant arrhythmias. However,Wiegerinck et al.38 provided no evidence for reduced INa inthe volume/pressure overload HF rabbit model as used byus, in line with the present study and other findings whichdid not reveal changes in INa in dogs with pacing-inducedHF.39 The contrasting results might be due to species/model differences.

To complement these studies, we demonstrated thatacute reduction in INa density following increases in Cai

2þ,as observed at fast heart rates, is more prominent in HF.This finding agrees with observations that HF patients areparticularly susceptible to reductions in cardiac excitabilityat fast heart rates.40 Several studies have shown thatincreased electrocardiographic QRS width, a clinicallyuseful marker of cardiac excitability, is an independentpredictor of sudden (arrhythmia-induced) death in HFpatients.41,42 Future studies aimed at preventing arrhyth-mias and sudden death in HF may target the regulationmechanism revealed here.

Supplementary material

Supplementary material is available at CardiovascularResearch online.

Acknowledgements

The authors thank Charly N.W. Belterman, Diane Bakker, and CeesA. Schumacher for technical support, and Jan W.T. Fiolet fordiscussions.

Conflict of interest: none declared.

Funding

H.L.T was supported by the Royal Netherlands Academy of Arts andSciences, the Netherlands Heart Foundation NHS 2002B191, and theBekales Foundation.

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Intracellular calcium modulation of voltage-gated sodium channels in ventricular myocytes