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Cardiovascular Research Advance Access first published online on October 1, 2008
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Cardiovascular Research, doi:10.1093/cvr/cvn274
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org.

Intracellular calcium modulation of voltage-gated sodium 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,*

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

* Corresponding author. Tel: +31 20 5663264; fax: +31 20 6975458. E-mail address: h.l.tan{at}amc.nl

Received 28 February 2008; revised 19 September 2008; accepted 22 September 2008

Time for primary review: 32 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Cardiac voltage-gated sodium channels control action potential (AP) upstroke and cell excitability. Intracellular calcium (Cai2+) regulates AP properties by modulating various ion channels. Whether Cai2+ modulates sodium channels in ventricular myocytes is unresolved. We studied whether Cai2+ modulates sodium channels in ventricular myocytes at Cai2+ concentrations ([Cai2+]) present during the cardiac AP (0–500 nM), and how this modulation affects sodium channel properties in heart failure (HF), a condition in which Cai2+ homeostasis is disturbed.

Methods and results: Sodium current (INa) and maximal AP upstroke velocity (dV/dtmax), a measure of INa, were studied at 20 and 37°C, respectively, in freshly isolated left ventricular myocytes of control and HF rabbits, using whole-cell patch-clamp methodology. [Cai2+] was varied using different pipette solutions, the Cai2+ buffer BAPTA, and caffeine administration. Elevated [Cai2+] reduced INa density and dV/dtmax, but caused no INa gating changes. Reductions in INa density occurred simultaneously with increase in [Cai2+], suggesting that these effects were due to permeation block. Accordingly, unitary sodium current amplitudes were reduced at higher [Cai2+]. While INa density and gating at fixed [Cai2+] were not different between HF and control, reductions in dV/dtmax upon increases in stimulation rate were larger in HF than in control; these differences were abolished by BAPTA.

Conclusion: Cai2+ exerts acute modulation of INa density in ventricular myocytes, but does not modify INa gating. These effects, occurring rapidly and in the [Cai2+] range observed physiologically, may contribute to beat-to-beat regulation of cardiac excitability in health and disease.

KEYWORDS Ion channels; Na-channel; Arrhythmia


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Voltage-gated sodium (Na+) channels control excitability in the heart and other excitable tissues by generating the action potential (AP) upstroke. Na+ channel dysfunction in various conditions may cause life-threatening cardiac arrhythmias by reducing cardiac excitability.1 Intracellular calcium (Cai2+) participates in the regulation of numerous key physiological processes. Its tight homeostasis supports strong and acute changes in Cai2+ concentrations ([Cai2+]) through Cai2+ release from Cai2+ stores [e.g. the sarcoplasmic reticulum (SR)] during systole and reuptake into these stores during diastole.2 This renders Cai2+ a potential regulator of cardiac excitability, as heart rates must vary strongly and acutely (beat-to-beat) to meet metabolic demands. In many common diseases associated with impaired excitability and life-threatening arrhythmias, Cai2+ homeostasis is disturbed. For instance, in heart failure (HF), diastolic [Cai2+] is increased as Cai2+ reuptake into the SR is delayed and incomplete. This is particularly prominent at fast heart rates, because diastolic intervals are shorter.3 Yet, while Cai2+ is known to regulate various ion channels which control the AP,4–6 it is not fully resolved whether Cai2+ directly modulates Na+ channel function and which mechanisms are involved. Few studies have investigated a possible relation between [Cai2+] and Na+ channel function. It was proposed that Cai2+ regulates Na+ channel biosynthesis in rat cardiac myocytes,7,8 while short-term modulation (modulation within the time frame of the cardiac AP) was demonstrated in lipid bilayers9,10 and heterologous expression systems.11 In rabbit ventricular myocytes which were cultured for 24 h, increase in [Cai2+] from 100 to 500 nM caused Na+ channel gating changes, but left current density unchanged.12 Still, the effects of changes within the physiological range of [Cai2+] on Na+ current (INa) of freshly isolated left ventricular myocytes have not been reported.

We hypothesized that Cai2+ modulates INa of freshly isolated left ventricular myocytes, and that this modulation underlies beat-to-beat variations in INa magnitude, e.g. those that occur upon changes in heart rate. We aimed to establish how Cai2+ modulates Na+ channels under physiological conditions and during HF.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Cell preparation
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication 85–23, revised 1996) and was approved by the institutional animal experiments committee. HF was induced by combined volume overload (aortic regurgitation) and pressure overload (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 {alpha}-subunit (Nav1.5) of the human cardiac Na+ channel, was prepared in the pCGI vector for bicistronic expression of Nav1.5 and green fluorescent protein.15 Human embryonic kidney (HEK293) cells were transiently transfected with 1 µg SCN5A cDNA and 1 µg hβ1-subunit cDNA using lipofectamine (Gibco BRL, Life Technologies). Transfected HEK293 cells were cultured in minimum essential medium (Earles salts and L-glutamine) supplemented with non-essential amino acid solution, 10% foetal bovine serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin in a 5% CO2 incubator at 37°C for 1–2 days. Only cells exhibiting green fluorescence were selected for electrophysiological experiments.

2.2 Electrophysiology
2.2.1 Data acquisition
Membrane currents and potentials were recorded in the whole-cell configuration of the patch-clamp technique using patch pipettes with a tip resistance of 1.5–2 M{Omega}, unless mentioned otherwise. INa signals were low-pass filtered (cut-off frequency 5 kHz) and digitized (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 (Axon Instruments) and custom-made software, respectively.

2.2.2 Sodium current properties
In myocytes, INa was analysed at 20°C; at 37°C, AP upstroke velocity was analysed as a measure of INa (see below). At 20°C, activation, inactivation, recovery from inactivation, and slow inactivation parameters 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 as described in the Supplementary material online. INa was recorded using intracellular solutions to which CaCl2 was added to obtain a free [Cai2+]=0, 100, and 500 nM as calculated using WEBMAX Standard 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, CsCl 125, nifedipine 0.005, pH 7.3 (CsOH). BAPTA was used as Cai2+ buffer, because it provides stringent control over [Cai2+], thanks to its fast Cai2+ buffering kinetics.11


Figure 1
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  		Figure 1 Sodium current densities and gating properties in control myocytes at various [Cai2+]. (A) Typical examples of whole-cell sodium current (INa) traces recorded at 20°C from control myocytes at [Cai2+]=0 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 voltage dependence of activation. Normalized current was plotted as a function of the membrane potential. On the left part, steady-state inactivation. Peak sodium currents were normalized to maximum values in each cell and plotted as a function of the voltage of the conditioning step. (D) Average fast ({tau}f) and slow ({tau}s) time constants of INa decay plotted as a function of membrane potential. (E) Average time course of recovery from inactivation. Peak sodium currents elicited by P2 were normalized (P2/P1) and plotted as a function of the recovery interval ({Delta}t). (F) Average development of slow inactivation. INa elicited by P2 were normalized (P2/P1) and plotted as a function of the duration of P1. Insets: voltage protocols.

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

In HEK293 cells, experiments were conducted at 20°C and INa was elicited by depolarizing steps from a holding potential of –140 mV (cycle time 5 s). Cm and Ra were 11.9 ± 0.9 pF and 5.8 ± 0.7 M{Omega}, respectively (n = 10). Acute effects of Cai2+ on INa were studied with a modified dialyzable pipette (Supplementary material online and Tang et al.16). The pipette solutions were identical to those used for INa measurements in myocytes at 20°C (described above, Ca0 and Ca500), while the bath solution (Solution 1) contained (mM): NaCl 10, Na-gluconate 130, HEPES 10, glucose 5.5, CaCl2 1.8, MgCl2 1.2, pH 7.4 (CsOH). To study possible effects of caffeine on 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). Pipettes were filled with (mM): CsF 110, CsCl 1.0, EGTA 11, NaF 10, CaCl2 1.0, MgCl2 1.0, Na2ATP 2.0, HEPES 10, pH 7.3 (CsOH).

2.2.3 Upstroke velocity measurements
To investigate INa characteristics at 37°C and physiological Na+ concentrations, we recorded AP upstroke velocities and expressed them as the maximum value of the first derivative of AP upstroke (dV/dtmax). dV/dtmax is a convenient index for INa. Although it slightly overestimates Na+ channel availability, the discrepancies between INa and dV/dtmax are reduced at high temperatures (26–27°C).17 dV/dtmax was measured using an alternate voltage/current-clamp mode with a custom-made amplifier.18 Using dV/dtmax as a measure of Na+ channel availability, steady-state inactivation, recovery from inactivation, and slow inactivation were analysed with the protocols indicated in Figure 2. As a measure of peak current magnitude (Table 2), the maximum dV/dtmax of the Na+ channel availability curve was given. Bath solution (Solution 2) contained (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-ATP 5, and HEPES 10, pH 7.2 (HCl). CaCl2 was added to obtain free [Cai2+]=0 or 500 nM. Data for voltage-dependence of inactivation and recovery from inactivation were fitted as described in the Supplementary material online.


Figure 2
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  		Figure 2 Action potential upstroke velocity as a measure of sodium current magnitude and gating properties in control myocytes at various [Cai2+]. (A) Typical examples of action potential (AP) upstroke velocities recorded at 37°C from control myocytes at [Cai2+]=0 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 of recovery from inactivation. dV/dtmax elicited by P2 were normalized (P2/P1) and plotted as a function of the recovery interval ({Delta}t). (E) Average development of slow inactivation. dV/dtmax elicited by P2 were normalized (P2/P1) and plotted as a function of the duration of P3. Insets: combined voltage/current-clamp protocols. CC, current-clamp.

 


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  		Table 2 Intracellular calcium modulation of sodium channel properties at 37°C

 
To investigate INa availability at various stimulus frequencies and, consequently, at various [Cai2+],3 dV/dtmax were measured in control and HF myocytes at 0.2–4 Hz in the presence or absence of 10 mM intracellular BAPTA. Experiments were conducted at 37°C. The use of BAPTA eliminates calcium (Ca2+) transients and Ca2+ dependent L-type Ca2+ channel inactivation, causing modification of several parameters in the cell, including the normal regulation 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 solution was Solution 2. Pipette solution without BAPTA (Solution 3) contained (mM): K-gluconate 125, KCl 20, NaCl 5, Mg-ATP 5, and HEPES 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).


Figure 6
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  		Figure 6 Relationship between heart rate and action potential upstroke velocity in control and heart failure myocytes. Average relationship between stimulus frequency and maximal action potential upstroke velocity (dV/dtmax) in control and heart failure (HF) myocytes, in the presence or absence of 10 mM BAPTA. Values were normalized to the highest dV/dtmax in each myocyte. Without BAPTA, the reduction in dV/dtmax in response to heart rate increase was larger in HF (n = 13) than in control (n = 11). These differences were abolished by the addition of BAPTA (HF+BAPTA, n = 12; control+BAPTA, n = 13). Asterisk: P < 0.05 between control and HF. Inset: combined voltage/current-clamp protocol. CC, current-clamp.

 
To study acute effects of changing [Cai2+] on Na+ channel availability in the range of [Cai2+] as observed during the cardiac cycle, we created acute increases in Cai2+ by inducing Cai2+ release from the SR with 10 mM caffeine. [Cai2+] and dV/dtmax were simultaneously measured at 37°C using the alternate voltage/current-clamp mode. For each cell, dV/dtmax represents the average of 10 consecutive values recorded at steady-state. Bath solution was Solution 2, pipette solution was Solution 3.


Figure 5
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  		Figure 5 Relationship between [Cai2+] and action potential upstroke velocity in control myocytes. (A) Typical example of simultaneous recording at 37°C of intracellular calcium ([Cai2+]) 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 [Cai2+] and decrease in dV/dtmax (n = 7–18). (C) Average dV/dtmax at baseline (430.6 ± 22.6 V/s, n = 22) and after addition of 10 mM caffeine (356.1 ± 20.7 V/s, n = 22). (D) Average diastolic [Cai2+] at baseline (86.4 ± 4.0 nM, n = 22) and after addition of 10 mM caffeine (177.1 ± 10.1 nM, n = 22). (E) Average current density of cardiac Na+ channels expressed in HEK293 cells at baseline (–857.4 ± 211.5 pA/pF, n = 6) and after the addition of 10 mM caffeine (–897.1 ± 199.2 pA/pF, n = 6).

 
2.2.4 Cai2+ measurements
[Cai2+] was measured in Indo1-loaded myocytes (1 Hz stimulation). Dye-loaded myocytes were excited through a 100x oil-immersion objective at 340 nm (75W Xenon arc lamp). Intensities of the emitted light at 405 nm (I405) and 505 nm (I505) were recorded, subsequently digitized at 1 kHz, and filtered at 100 Hz. A rectangular adjustable slit was used to select a single myocyte and reduce background fluorescence, which was subtracted offline before the ratio values were calculated (I405/I505). Fluorescent ratio was translated as [Cai2+]=β*Kd(R–Rmin)/(Rmax–R). After calibration of the setup, β (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, Eugene OR, USA).

2.2.5 Single-channel measurements
Single Na+ channel currents were recorded at 20°C from inside-out patches of ventricular myocytes isolated from control rabbit hearts, in the presence of veratridine (30 µM). Veratridine reduces peak INa, but also induces a non-inactivating current due to long-lasting channel openings.21 Therefore, the use of veratridine provides a more reliable analysis of unitary current amplitude compared to unmodified Na+ channels.22 Voltage steps (900 ms) were applied from –80 to –40 mV (holding potential –140 mV, cycle time 5 s). Pipettes were fire polished and had a tip resistance of 2.5–3.5 M{Omega}. Signals were filtered and digitized at 1 and 10 kHz, respectively. Bath solutions were identical to those used as pipette solutions for INa measurements in myocytes at 20°C (Ca0 and Ca500). Pipette solution was Solution 1 with the addition of nifedipine (0.005 mM). Unitary current amplitudes were estimated from sweeps exhibiting well-resolved closings as determined by fitting Gaussian distributions.

2.2.6 Statistical analysis
Results are expressed as mean ± SEM. Unpaired Student's t-test was used to study the effects of Cai2+ on Na+ channel properties, and to compare INa gating, current densities, Cm and Ra between control and HF. When data were not normally distributed, a Mann–Whitney test was performed. Paired Student's t-test was performed to compare [Cai2+] and dV/dtmax in control myocytes before/after addition of caffeine, and INa amplitudes in HEK293 cells before/after addition of caffeine or increases in [Cai2+]. To analyse INa decay, the development of slow inactivation and to study whether the relationships between dV/dtmax and heart rate were different in control and HF, we conducted two-way ANOVA with repetitive measurements followed by a Holm–Sidak test for post hoc analysis. P < 0.05 indicates statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Effects of Cai2+ on Na+ channel properties
We first studied the effects of Cai2+ at 20°C on INa properties in myocytes isolated from control rabbits (Table 1 and Figure 1). We used pipette solutions buffered at 0, 100, or 500 nM free [Cai2+] (Ca0, Ca100, and Ca500) to cover the [Cai2+] range found in myocytes of control and HF rabbits3 during the cardiac cycle. Figure 1A shows representative examples of INa traces recorded from control myocytes at different [Cai2+]. INa density was significantly smaller at Ca500 than at Ca0, while values at Ca100 were intermediate (Table 1 and Figure 1B). This reduction was not due to changes in gating properties. V1/2 and k of activation and inactivation were not different between Ca0, Ca100, and Ca500 (Table 1 and Figure 1C), as well the rates of current decay (Figure 1D), recovery from inactivation (Figure 1E), and slow inactivation, a conformational state that partly determines INa availability at fast heart rates because of its slow kinetics23 (Table 1 and Figure 1F).


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  		Table 1 Intracellular calcium modulation of sodium channel properties at 20°C

 
To study whether these findings also apply to physiological temperatures, we repeated these experiments at 37°C, using dV/dtmax as an index of INa magnitude.18 Results were similar to the voltage-clamp studies at 20°C, with dV/dtmax being smaller at Ca500 than at Ca0 and gating properties similar at Ca0 and Ca500 (Table 2 and Figure 2). However, the percentage of dV/dtmax reduction observed at high [Cai2+] was smaller than the INa reduction observed at 20°C. While differences in recording temperatures may cause this discrepancy, future studies must resolve the underlying mechanisms.

As we found no evidence that Cai2+ modulates Na+ channel gating, we hypothesized that its effect to reduce INa magnitude results from impediment of Na+ channel permeation. To test this hypothesis, we conducted paired experiments on human cardiac Na+ channels expressed in HEK293 cells. We measured INa at different [Cai2+] in the same cell by varying [Cai2+] with a dialyzable pipette.16 We observed a similar reduction (~30%) in peak INa amplitude as in myocytes at 20°C when [Cai2+] was increased from 0 to 500 nM (Figure 3B). To verify whether current amplitudes were stable up to the change in intracapillary solution within the same cell, INa amplitudes were plotted against time (Figure 3A, bottom). This analysis indicated that INa amplitudes were stable during recording at [Cai2+]=0 nM, and declined smoothly upon changing the pipette solution to [Cai2+]=500 nM. We took this smooth decrease as being consistent with the (diffusion) time required for the change in intracellular solution.


Figure 3
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  		Figure 3 Sodium current amplitudes in HEK293 cells at various [Cai2+]. (A) Top: typical examples of INa elicited in response to depolarizing voltage clamp steps from –140 to –20 mV at [Cai2+]=0 nM (1) and after dialysis of 500 nM Cai2+ (2). INa were recorded at times indicated by arrows 1 and 2 in the bottom panel. Bottom: current amplitudes were stable in time during the recordings at [Cai2+]=0 nM (Ca0) and during/after wash-in of 500 nM Cai2+ (Ca500). (B) Average peak INa (n = 4) normalized in each cell to the highest INa value obtained with Ca0.

 
3.2 Effects of Cai2+ on unitary Na+ current amplitude
To provide direct evidence for permeation block, we next performed inside-out patch-clamp studies in control myocytes to test the effects of Cai2+ on veratridine-modified single Na+ current amplitudes. The effect of veratridine was first tested on whole-cell INa (Figure 4A). Consistent with previous findings,21 veratridine reduced peak INa amplitudes and induced a non-inactivating current due to long-lasting channel openings (Figure 4A, left panel). Moreover, activation of veratridine-modified Na+ channels occurred already at relatively negative potentials (Figure 4A, right panel). In inside-out patches, resolvability of unitary channel events was only possible in a minority of current traces. This may have been due to long-lasting openings of multiple channels simultaneously, as a consequence of the relatively slow channel inactivation in the presence of veratradine. Nevertheless, two distinct single-channel current amplitudes were observed (Figure 4B), in accordance with a previous study which showed that veratridine induces sub-conductance states of the cardiac Na+ channel.21 Figure 4C, left panel, shows typical examples of single channel currents from two Na+ channel sub-conductance states with 0 and 500 nM Cai2+. When Cai2+ was increased from 0 to 500 nM in paired experiments, the single channel current amplitude decreased to 82.2 ± 2.5% (n = 6 patches). Fitting the pooled data by linear regression yielded 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).


Figure 4
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  		Figure 4 Unitary sodium currents in inside-out patches at various [Cai2+] in control myocytes. (A) Typical examples of the effect of veratridine on whole-cell sodium current (INa) recorded at 20°C 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 sodium channel, rather than the real zero current level. (C) Left: typical examples of single sodium channel currents recorded in the presence of 0 and 500 nM calcium in the bath solution (top: ‘larger’ conductance state, measured at –65 mV; bottom: ‘smaller’ conductance state, measured at –60 mV). Right: I–V relationships of pooled data (n = 6) of single channel current amplitudes at [Cai2+]=0 nM (Ca0) and 500 nM (Ca500); circles: ‘smaller’ conductance, squares: ‘larger’ conductance. Lines: best fit curves to the equation y=Ax[exp(–0.015xVm)–0.1054xexp(0.01xVm)], assuming Goldman–Hodgkin–Katz behaviour and a reversal potential of +90 mV.

 
3.3 Effects of Cai2+ on AP upstroke velocity
Having established that steady-state increases in [Cai2+] reduce INa density in freshly isolated myocytes by impeding ion permeation, we next investigated whether this interaction may be relevant to beat-to-beat modulation of the cardiac AP.

To study how changes in [Cai2+], which occurred acutely and in the range of [Cai2+] as observed during the cardiac cycle in this model,3 impacted on dV/dtmax at 37°C, we conducted simultaneous recordings of dV/dtmax and [Cai2+]. Adding 10 mM caffeine to the bath solution caused Cai2+ release from the SR, and resulted in an increase in [Cai2+]. This was associated with a reciprocal reduction in dV/dtmax, which occurred instantaneously upon changes in [Cai2+] (Figure 5A). Of note, dV/dtmax reduction occurred over the whole range of [Cai2+], following depletion of the SR, indicating that [Cai2+] acutely reduces INa density in a dose-dependent fashion (Figure 5B). Average dV/dtmax before and after 30 s of caffeine exposure is shown in Figure 5C, and corresponding average [Cai2+] in Figure 5D. [Cai2+] significantly increased, while dV/dtmax significantly decreased, with respect to their initial values. To rule out that these effects were due to a direct action of caffeine on Na+ channels, we studied the effect of the drug on INa densities of cardiac Na+ channels expressed in HEK293 cells. In these cells, caffeine does not induce Ca2+ release from intracellular Ca2+ stores.24 Addition of 10 mM caffeine to the bath solution did not change INa density (Figure 5E).

3.4 Effects of Cai2+on Na+ channel properties in heart failure
To further explore whether this acute modulation of INa properties by Cai2+ may have pathophysiological relevance, we studied the effects of Cai2+ on INa properties in a rabbit model of HF. As reported in earlier studies from our laboratory,3 Cai2+ handling is disturbed in this model. In particular, diastolic [Cai2+] is significantly increased.

First, to study whether the nature of INa modulation by Cai2+, as observed in control, is also present in HF, we conducted experiments in myocytes isolated from HF rabbits using pipette solutions with the same steady-state [Cai2+] as in control (0 and 500 nM) at 20°C (INa) and 37°C (dV/dtmax). INa density and gating were similar in control and HF at both temperatures (Tables 1 and 2). Cai2+ exerted similar acute modifications of INa density in HF as in control. Thus, also in HF, INa density and dV/dtmax were significantly smaller at [Cai2+]=500 nM than at [Cai2+]=0 nM, while gating properties were unaltered (Tables 1 and 2).

To study whether acute [Cai2+]-mediated changes in INa density may translate into differences in excitability between control and HF under physiological conditions, we studied dV/dtmax at different stimulation rates. We made use of previous findings from our laboratory,3 that, in our model, diastolic [Cai2+] increases in parallel with the stimulation rate, and that this increase is larger in HF than in control. For instance, when pacing rate is increased from 0.2 to 3 Hz, diastolic [Cai2+] increases by 40 nM (from 50 to 90 nM) in control, but by 80 nM (from 145 to 225 nM) in HF. Thus, dV/dtmax was expected to decline at fast heart rates, and this reduction was expected to be more prominent in HF than in control. To better appreciate the changes in dV/dtmax in response to different stimulation rates, values were normalized to the highest dV/dtmax in each myocyte. When stimulation rates were increased in the absence of Ca2+ chelators in the pipette solution, dV/dtmax decreased more in HF than in control. Thus, normalized dV/dtmax was significantly smaller in HF than control at 3 and 4 Hz. Yet, when BAPTA was added to the pipette solution to maintain [Cai2+] at virtually zero levels,11 the differences between control and HF in the dV/dtmax-heart rate relationships were abolished (Figure 6).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
We demonstrated that Cai2+ modulates Na+ channel density without changing its kinetic properties in freshly isolated ventricular myocytes. [Cai2+]-mediated changes in INa density were acute and occurred in the range of [Cai2+] levels found in control and HF. INa reduction was due to decreased Na+ channel conductance. Cai2+-mediated permeation block of the Na+ channel may contribute to beat-to-beat modulation of INa properties as it provides a mechanism by which diastolic [Cai2+] controls INa density. We observed this regulatory mechanism not only in the physiological state, but also in a disease state in which diastolic [Cai2+] is increased, i.e. HF.

4.1 Cai2+-dependent modulation of Na+ channel
Previous single-channel experiments in cardiac myocytes have shown that extracellular Ca2+ reduces Na+ channel unitary current amplitude.21,22,25 Reduction of single-channel conductance was explained by a very fast open channel block, due to a rapid movement of Ca2+ ions into and out of a binding site within the Na+ channel, rather than to changes in the surface charges.25 In contrast, the effects of Cai2+ on Na+ channels were not resolved.

It was proposed that Cai2+ regulates Na+ channel biosynthesis in rat cardiac myocytes.7 Accordingly, the decrease in INa density observed in cultured rat neonatal ventricular myocytes exposed for 24 h to culture medium containing 10 mM Ca2+ and 10 mM K+ (in order to raise [Cai2+]) was attributed to reduced sarcolemmal Na+ channel expression rather than to changes in single-channel conductance or gating.8

While these studies revealed long-term modulation of Na+ channel expression and current density by Cai2+, the findings that single Na+ channel conductance in cells exposed to high [Cai2+] was not different from that in control cells8 contrasted with a study by French et al.,9 in which rat brain-type Na+ channels were inserted into lipid bilayers. In these experiments, either external or internal application of 10 mM Ca2+ reduced single-channel conductance. These experimental findings were reproduced in a computer model of Na+ channels.26 Using the same lipid bilayer system as French et al., Zamponi et al.10 showed that, in the skeletal-muscle isoform of the rat Na+ channel, internally applied millimolar Ca2+ concentrations caused a dose-dependent reduction of single-channel INa. A block of the pore involving multiple sites together with a surface charge effect was suggested as mechanisms.

Wingo et al.11 showed that in tsA201 cells transiently transfected with the cardiac Na+ channel, an increase in [Cai2+] from 0 to 10 µM (with the effect saturating at 1 µM) caused a positive shift of the INa availability curve. A putative EF hand motif located in the C-terminus of the channel was proposed as a Ca2+ binding site and regulator of the Cai2+-dependent effects.11 However, a separate study failed to detect any Ca2+ binding to that region.27

To complement these previous studies, we now provide evidence for a direct modulation of INa by physiological [Cai2+] in freshly isolated cardiomyocytes. We showed that Cai2+ modulates INa density without affecting Na+ channel gating properties. The latter finding contrasts with the report of Wingo et al. This discrepancy may be explained by the differences in the cell types studied. Moreover, it emphasizes that findings obtained in heterologous expression systems may not always be applicable to native myocytes. Further evidence that it is relevant to consider the cell type studied comes from the observation that increases in [Cai2+] from 100 to 500 nM, did cause Na+ channel gating changes in rabbit ventricular myocytes which were cultured for 24 h.12

Our findings that changes in INa magnitude and dV/dtmax in response to changes in [Cai2+] (following Ca2+ dialysis with a perfused pipette, and caffeine administration and its cessation, respectively) occurred instantaneously, argued against a Cai2+ effect on expression and subsequent sarcolemmal insertion of Na+ channels. It is believed that the expression of plasma membrane proteins that must traverse the Golgi apparatus before membrane insertion would require a significantly longer time. However, our single-channel experiments indicated that Cai2+ acts on INa density by modulating Na+ channel permeation. Indeed, Na+ channel conductance was reduced in the presence of Ca500.

4.2 Cai2+-dependent modulation of Na+ channel through Cai2+-dependent proteins
While we aimed to establish the direct effect of physiological [Cai2+] on INa properties under control and HF conditions, it must be noted that, besides a direct modulation, Cai2+ can regulate the cardiac Na+ channel through Cai2+-activated proteins. Cai2+-dependent interaction of the Cai2+-binding protein calmodulin (CaM) with an IQ-like motif in the Na+ channel C-terminus may modulate Na+ channel gating in heterologous expression systems27–29 and isolated cardiomyocytes.30 However, the nature of the reported gating changes was not consistent27–30 and binding between CaM and the cardiac Na+ channel was not confirmed in all studies.31 A role for the Ca2+/CaM-dependent protein kinase II (CaMKII) has also emerged. Adenovirus-mediated CaMKII{delta}c overexpression in cultured rabbit myocytes induced a hyperpolarizing shift in voltage-dependent inactivation, increased slow inactivation, and slowed recovery from inactivation in a Cai2+-dependent manner. Moreover, INa decay was slower, and the late component of INa was increased, the latter effect being confirmed in a recent study.30 All these effects were also seen in transgenic mice overexpressing CaMKII{delta}c and they could be reversed with the use of CaMKII inhibitors.12 Of note, none of these studies, which were performed either in heterologous expression systems or in isolated cardiac myocytes, have reported some effects of CaM or CaMKII on INa density. On the other hand, altered Cai2+ homeostasis leading to protein kinase C (PKC) activation was considered the probable cause of reductions in INa and dV/dtmax in transgenic mice overexpressing a constitutively active form of calcineurin.32 These results were in line with previous studies showing a INa decrease following PKC activation.33,34 Although our use of the inside-out configuration of the patch-clamp technique allowed INa recording from patches detached from myocyte cell membranes, it cannot be excluded that INa reduction was (partly) mediated by the activation of the Ca2+-dependent PKC.32–34

4.3 Na+ channel modulation in heart failure
In HF, disturbed Cai2+ handling (in particular, increased diastolic [Cai2+]), ranks among the most consistent changes, found in ventricular myocytes isolated from various animal HF models, and in myocytes from failing human hearts. Previous studies have addressed the chronic effects of HF on INa density, but these results were not consistent. Downregulation of INa was found in different canine HF models35–37 and in human ventricular cells.36 Decrease in INa density may reduce excitability, thereby slowing myocardial conduction and contributing to re-entrant arrhythmias. However, Wiegerinck et al.38 provided no evidence for reduced INa in the volume/pressure overload HF rabbit model as used by us, in line with the present study and other findings which did not reveal changes in INa in dogs with pacing-induced HF.39 The contrasting results might be due to species/model differences.

To complement these studies, we demonstrated that acute reduction in INa density following increases in Cai2+, as observed at fast heart rates, is more prominent in HF. This finding agrees with observations that HF patients are particularly susceptible to reductions in cardiac excitability at fast heart rates.40 Several studies have shown that increased electrocardiographic QRS width, a clinically useful marker of cardiac excitability, is an independent predictor of sudden (arrhythmia-induced) death in HF patients.41,42 Future studies aimed at preventing arrhythmias and sudden death in HF may target the regulation mechanism revealed here.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
H.L.T was supported by the Royal Netherlands Academy of Arts and Sciences, the Netherlands Heart Foundation NHS 2002B191, and the Bekales Foundation.


   Acknowledgements
 
The authors thank Charly N.W. Belterman, Diane Bakker, and Cees A. Schumacher for technical support, and Jan W.T. Fiolet for discussions.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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