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Cardiovascular Research 2004 63(4):653-661; doi:10.1016/j.cardiores.2004.05.010
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Occurrence of a tetrodotoxin-sensitive calcium current in rat ventricular myocytes after long-term myocardial infarction

Julio L Alvareza,b, Eduardo Salinas-Stefanonc, Gerardo Ortac, Tania Ferrerb, Karel Talaverab, Loipa Galánb and Guy Vassort*,a

aINSERM U-390, Physiopathologie Cardiovasculaire, CHU Arnaud de Villeneuve, 371 Avenue du Doyen Gaston Giraud, F-34295 Montpellier Cedex 5, France
bInstituto de Cardiología y Cirugía Cardiovascular, Havana, Cuba
cInstituto de Fisiología, Universidad Autónoma de Puebla, Puebla, Mexico

* Corresponding author. Tel.: +33-4-67-41-52-41; fax: +33-4-67-41-52-42. Email address: vassort{at}montp.inserm.fr

Received 11 December 2003; revised 13 May 2004; accepted 18 May 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: To determine the characteristics of a TTX-sensitive Ca2+ current that occurred only following remodelling after myocardial infarction in Wistar rat. Methods: Using the whole-cell patch-clamp technique, we studied ionic inward current in myocytes isolated from four different ventricular regions of control Wistar rat hearts, or from hearts 4 to 6 months after ligation of the left coronary artery. Inward current characteristics were also analysed in Xenopus laevis oocytes that heterologously expressed the human sodium channel {alpha}-subunit Nav1.5. The effects of oxidative stress by hydrogen peroxide or tert-butyl-hydroxyperoxide as well as those of PKA-dependent phosphorylation, which partly mimic the pathological conditions, were investigated on control cardiomyocytes and Nav1.5-expressing oocytes. Results: In Na-free solution, a low-threshold, tetrodotoxin-sensitive inward current was found in 20 out of 78 cells isolated from 16 post-myocardial infarcted (PMI) cardiomyocytes but not in cardiomyocytes from young and sham rat hearts. This current exhibited kinetics and pharmacological properties similar to the ICa(TTX) current previously reported. ICa(TTX)-like current was critically dependent on extracellular Na+ and was reduced by micromolar Na+ concentrations. Neither in normal rat cardiomyocytes nor in Nav1.5-expressing oocytes could a ICa(TTX)-like current be elicited in Na+-free extracellular solution, even after oxidative stress or PKA-dependent phosphorylation. Conclusions: Our data suggest that ICa(TTX)-like current in PMI myocytes does not arise from classical Na+ channels modified by oxidative stress or PKA phosphorylation and most probably represents a different Na+ channel type re-expressed in some cells after remodelling.

KEYWORDS Arrhythmia; Heart failure; Na-channel; Oxidative phosphorylation; Remodelling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Myocardial remodelling secondary to myocardial infarction implies changes that result in rearrangement of normally existing structures. This process is usually accompanied by arrhythmogenic activities [1,2]. Action potential prolongation is a major contributing factor to the arrhythmia observed in heart failure [1,3]. The cellular and molecular processes that underlie action potential prolongation have been mostly attributed to a down-regulation of K+ currents [1,4]. However, alterations in other ionic channels such as L-type Ca2+ current [4] and fast inward Na+ current [5,6] have also been described.

Ca2+ permeation through Na+ channels and TTX-sensitive Ca2+ currents (ICa(TTX)) have been documented for some time [7]. Detailed characterizations of ICa(TTX) in cardiomyocytes bathed in low or Na-free solutions have appeared [8–10]. These investigators agreed that Ca2+-permeation is critically dependent on the presence of extracellular Na+. This fact, together with the insensitivity of ICa(TTX) to organic and inorganic Ca2+-channel blockers [11] and its sensitivity to TTX, led the investigators to conclude that these currents represent Ca2+ permeation through Na+ channels. However, while Cole et al. [8] essentially proposed that ICa(TTX) represents Ca2+ movement through classical Na+ channels, Aggarwal et al. [9] suggested that it represents a new cardiac Na+ channel. Furthermore, Na+ channels have been suggested to pass Ca2+ ions after PKA-dependent phosphorylation [12]. Although this observation remains controversial, it suggests that channel selectivity could be modulated. Whether ICa(TTX) represent alterations in the behaviour of channel proteins, in their environment, or expression of new isoforms of ion channels is still to be clarified. These factors contribute to the pathogenesis of myocardial remodelling and failure that might also include alterations in ion channel gene expression [13].

We previously reported the existence of a tetrodotoxin-resistant Na+ current (INa(TTX)R) in ventricular myocytes from 4–6-month post-myocardial infarcted (PMI) rats [6]. This chronic infarcted rat heart model of left ventricular dysfunction is clinically relevant and has predicted results in pathophysiologic and pharmacologic studies in man [14]. The INa(TTX)R may provide inward charges in a critical membrane potential range that could be able to alter action potential duration and may trigger ventricular arrhythmias. Additionally, some of these cells also showed an inward, TTX-sensitive Ca2+ current, or ICa(TTX)-like current, in the absence of Na+. Interestingly, we only found this current in PMI cardiomyocytes and not in cardiomyocytes from normal or sham Wistar rat hearts. It was thus our interest to characterize the ICa(TTX)-like current in PMI cardiomyocytes and to test whether its occurrence in this model was related to changes in INa induced by the pathological situation that include oxidative stress [15,16] and chronic catecholamine stimulation [17]. Patch-clamp experiments on cardiomyocytes isolated from control and PMI rats were combined with voltage-clamp recordings of Nav1.5 channel current heterologously expressed in Xenopus laevis oocytes. The ICa(TTX)-like current occurred only in PMI cells. Neither in normal rat cardiomyocytes nor in Nav1.5-expressing oocytes could a ICa(TTX)-like current be elicited in Na+-free solution, even after oxidative stress or PKA-dependent phosphorylation, two situations known to markedly occur during infarction. Our data suggest that ICa(TTX)-like current in PMI myocytes does not arise from classical Na+ channels and most probably represents a different Na+ channel type re-expressed after remodelling.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Isolation of ventricular myocytes
Ventricular myocytes were isolated from the collagenase-perfused heart isolated from urethane-anaesthetized (2 g/kg, IP) young normal (180–230 g), sham or PMI Wistar rats (600–700 g) after it had been cut in four pieces as previously described [4,6]: right ventricle, septum, apex and left ventricle, above the scar in PMI rats. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The yield of well-striated, elongated cells was 60–70%, 50% and 30–40% for young, sham and PMI animals, respectively. Cells were used within 6–8 h.

2.2. Solutions and drugs
The standard extracellular Na+-containing solution was (mM): NaCl 30, NMDG or TEA–Cl 110, CaCl2 5.4, MgCl2 2, glucose 10, HEPES 10; pH 7.4 adjusted with CsOH, or TEA–OH when using TEA–Cl. In some experiments, NaCl was decreased to 10 mM; TEA–Cl or NMDG were then increased to 130 mM. The Na+-free extracellular solution contained no NaCl but NMDG or TEA–Cl 140 mM. The internal solution contained (mM): CsCl 100, TEA 20, EGTA 10, HEPES 10, Na2ATP 5, Na2GTP 0.4; pH 7.3. All salts and compounds were from Sigma but nifedipine (Bayer).

2.3. Voltage clamp recordings
Na+ and TTX-sensitive Ca2+ currents (INa and ICa(TTX)) were recorded at 20–22 °C during 100- or 200-ms depolarizing pulses applied every 4 s from a –100-mV holding potential (HP) using a patch-clamp amplifier (model RK-400; Biologic, France) and filtered at 3 kHz. Currents traces were digitized at a sampling interval of 20–50 µs with a 12-bit analog to digital converter (LabMaster DMA, Scientific Solutions, USA) and the ACQUIS1 software (version 2; CNRS Licence, France). The series resistance (Rs), membrane capacitance (Cm) and time constant of membrane capacitance ({tau}c) were determined on voltage-clamped cells [4,6]. Current data of each cell were normalized to its cell capacitance (current density in pA/pF). Averaged cell capacitances were: 133.3±8.1; 313.5±13.9 and 325.0±12.4 pF in young (n=25), sham (n=38) and PMI cells (n=78), respectively.

2.4 Heterologous channel expression and voltage-clamp recordings in X. laevis oocytes
Oocytes were removed from female X. laevis frogs anaesthetized by immersion in a solution containing 0.2% 3-aminobenzoic acid ethyl ester (tricaine, Sigma). Oocytes were digested for 90 min with 1.3 mg/ml collagenase (type 1A, Sigma) dissolved in a solution OR2, containing (mM): NaCl 82.5, KCl 2.5, MgCl2 1, HEPES 5; pH 7.6. Oocytes were coinjected into the nucleus with a nanolitre automatic injector (A203XVY; World Precision Instrument, USA) with 2–10 ng of Nav1.5 human {alpha}-subunit and β2/{alpha}1-subunit cDNA clones at a ratio of 1:5 [18,19]. Nav1.5 and β2-clones were kindly provided by Drs. Peter Backx and Eduardo Marbán. Oocytes were then incubated at 18 °C in N-D96 solution containing (mM): NaCl 96, KCl 2, MgCl2 1, HEPES 5 and BaCl2 1; pH 7.6 and supplemented with theophylline 0.5, pyruvate 0.5 and gentamicine 50 µM, for up to 3 days before recording.

Two electrode voltage-clamp recordings were performed at 20–22 °C, using a Warner OC-725C amplifier on oocytes 1 to 3 days after injection. Oocytes were perfused with (mM): NaCl 30, TEA–Cl 66; KCl 2.5, CaCl2 5.4, MgCl2 1, HEPES 5, and BaCl2 1; pH 7.6 [19]. Agarose-plugged electrodes (TW150, World Precision Instruments) were filled with 3 M KCl. Data analysis was performed using pClamp software (Version 7.01, Axon Instruments, USA).

2.5. Statistical evaluation
Results were analysed by the Students' t-test and expressed as means and standard error of means. The criterion for significance was p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Data concerning heart and haemodynamic status of PMI rats used in this study have been previously published [4,6]. Typically in PMI rats, left ventricles were markedly dilated under M-mode echocardiography and heart weight/body weight was increased by 40% despite large infarcted area while end-diastolic pressure demonstrated a significant increase.

In each of the 25 cells isolated from seven young control rat hearts and 38 cells isolated from seven sham 4–6-month-old rats bathed in 10 or 30 mM Na+ solution, a large, partially controlled Na+ current was elicited by –40 mV depolarizations. This fast INa was fully inhibited by 50 µM TTX. After extracellular Na+ removal, ICaL with a threshold potential around –30 mV was the only inward current elicited in these cells.

3.1 Existence of a TTX-sensitive Ca2+ current in PMI rat cardiomyocytes
In a previous study [6], we reported that among the 78 studied PMI cells, 41 of them exhibited a TTX-resistant inward current, INa(TTX)R. After switching to the Na+- and TTX-free solution in 6 out of these 41 cells, an inward current was still present, even at membrane potentials negative to the ICaL activation threshold. Such a low-threshold inward current was also observed in 14 out of the 37 cells that did not show INa(TTX)R implying that 23 out of the 78 PMI cells exhibited only the usual Na+ and L-type Ca2+ currents. The characteristics of this current were further studied in a total of 20 cells that were routinely monitored in Na+- and TTX-free solution (Na+ substituted by TEA (n=12) or NMDG (n=8)). The inward current was reversibly blocked within 10–20 s by the addition of 50 µM TTX (Fig. 1AB). Veratrine (200 µg/ml) significantly and reversibly prolonged its inactivation (n=3; Fig. 1C). The TTX-sensitive inward current was not altered by 100 µM Ni2+ ions (n=8), 3 µM nifedipine (n=5) nor by 2 mM Co2+ ions (n=4), but 100-µM Cd2+ decreased it by 31±5% (n=4; p<0.05; not shown).


Figure 1
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  		Fig. 1 Typical current recordings at indicated voltages in a Na-free, TEA-containing solution on a PMI rat cardiomyocyte. (A) Control conditions (TEA) or in the presence of 50 µM TTX; TTX-sensitive current obtained by numerical subtraction of the individual recordings in the two conditions. (B) The Na+-free, NMDG-containing solution was added with 200-µg/ml veratrine for 40 s (V1) and 160 s (V2). The effects were fully reversible after a 3-min washout (Wash). Note the very slow inactivation and large tail current in the presence of veratrine. A and B, two left ventricular cells.

 
To further characterize this current component in our experimental conditions, current-to-voltage relationships and availability curves were established from a holding potential of –100 mV first, in a Na+- and TTX-free solution (all Na+ substituted by TEA or NMDG) and then in the same solution added with 50 µM TTX. Difference current threshold was around –60 mV and maximal peak density (2.23±0.5 pA/pF; n=9) occurred at around –30 mV, a potential just below ICaL threshold. The apparent reversal potential was close to +5 mV indicating a rather poor selectivity (Fig. 2A). Half-inactivation and -activation potentials, determined from the best fits of experimental means to Boltzmann functions, were –67.0 and –34.9 mV, respectively (Fig. 2B). Time for half-reactivation was 21.1±0.7 ms. However, reactivation curve was clearly biphasic showing a delay and could be best fitted by a double exponential (n=4; Fig. 2C).


Figure 2
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  		Fig. 2 Voltage-dependent characteristics of the TTX-sensitive Ca2+ current. (A) Current–voltage relationships established on cells from all regions in a Na+-free (TEA-substituted) solution containing (+) or not (–) TTX. The TTX-sensitive current (7) was estimated by the difference between the current recorded in the absence and presence of TTX (n=5). Inset shows the voltage protocol for obtaining I/V and availability curves. (B) Availability and normalized conductance–voltage ("activation") relations of the TTX sensitive Ca2+ current. Conductance was calculated using the Hodgkin–Huxley equation: gNa=INa/[(Vt–Erev)], assuming a +5 mV reversal potential. The curves are fits of mean values to Boltzmann functions with V1/2=34.4 mV and s=11.2 mV for the activation curve. "Steady-state" inactivation (or availability curves were fitted with V1/2=–67.0 mV and k=8.9 mV (n=5). (C) Time course of recovery from inactivation of ICa(TTX) established with a double 200-ms, 0-mV depolarising pulse protocol. The data were best fitted by a double exponential:

Figure 2

with A0=0, A1=80, A2=20 being the minimal and maximal amplitudes, t1=18.7 and t2=98.4 ms, the half time and d1=6 and d2=20 ms, the time constant of the first and second components, respectively (n=4).

 
These characteristics led us to identify this TTX-sensitive current in PMI cells as the ICa(TTX) previously reported in guinea pig, rat and human cardiomyocytes [8–10].

ICa(TTX) recorded in 20 PMI cells out of 78 investigated showed quite variable amplitude. It appeared to occur with roughly the same frequency and mean amplitude in the four defined regions (septum, apex, left and right ventricles), a result that might however, be attributed to the relatively low number of cells investigated in each region (Table 1). There was no sign of a low-threshold Ca2+ current, ICaT in any of the 78 cells so investigated in 16 chronic PMI rat hearts, nor in control cardiomyocytes.


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  		Table 1 ICa(TTX) distribution and density in PMI rat heart

 
The amplitude of ICa(TTX) in PMI cells was altered by micromolar extracellular Na+ (Fig. 3). In the Na+-free solution, peak density of ICa(TTX) at –40 mV was 2.25±0.4 pA/pF (n=20). Adding 10 µM Na+ to the bathing solution reduced the current while the presence of 30 µM Na+ maximally inhibited ICa(TTX) by 58.3±5.4% (n=4; p<0.05). Further increasing the Na+ concentration enhanced the current elicited at –40 mV. Current kinetics were slow and unaffected by the low Na+ concentrations. In 11 cells, time-to-peak of ICa(TTX) at –40 mV was 3.6±0.4 ms and its inactivation time course could be well described by the sum of two exponentials (Table 2). In the nine other cells, the time-to-peak was larger (5.5±0.1 ms; P<0.05), and the inactivation time course was best fitted by a single exponential. These differences were irrespective of cell origin series resistance and current density.


Figure 3
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  		Fig. 3 Na+-dependence of the TTX-sensitive Ca2+ current in PMI cardiomyocytes. (A) Superimposed original current tracings elicited by a voltage step to –40- from –90-mV holding potential and recorded in the presence of 10, 30 and 100 µM Na+ added to a TEA-containing solution. (B) Current density-Na+ concentration relation demonstrating an inhibitory effect of the lower Na+ concentrations while 100 µM Na+ restored the amplitude recorded in the Na+-free solution and larger concentrations allowed for much larger currents.

 

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  		Table 2 Inactivation time constants of the inward current elicited at –40 mV and different extracellular Na+ concentrations

 
3.2 Oxidative stress does not induce ICa(TTX)-like currents in normal cardiomyocytes or oocytes expressing Nav1.5 channels
To evaluate the effects of oxidative stress, single cardiomyocytes from young rats and Nav1.5-expressing oocytes were exposed to H2O2 [20] or tert-butyl hydroperoxide, t-BHP [21]. During routine monitoring using PMI cardiomyocytes, INa was evoked at –40-mV depolarisation in a low 10-mM Na+ solution to improve voltage-clamp control and had a density of 10.9±1.9 pA/pF (n=15). Inactivation time course could be well fitted by two exponentials in 9 out of 15 cells and in the 6 other cells by a single exponential (Table 2), irrespective of times to peak current which were 0.60±0.04 and 0.68±0.06 ms. The addition of H2O2 to the low Na+ solution within 1 min decreased INa elicited at –40 mV by 24.6±2.1% (n=3) 54.6±5.1% (n=4) and 56.8±3.2% (n=5), respectively, in 0.1, 1 and 3 mM H2O2, without modifying INa inactivation time course. Then, on applying the Na+-free solution, no inward current could be detected at –40 mV nor in the whole range of potentials negative to –40 mV in any of the seven studied cells in Na+-free solution in the presence of H2O2 (Fig. 4A).


Figure 4
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  		Fig. 4 Oxidative stress by H2O2 did not induce the TTX-sensitive Ca2+ current in control cardiomyocytes or Nav1.5 expressing oocytes. In the presence of 3 mM H2O2, removing Na+ from the perfusing solution fully suppressed the inward current elicited by depolarising pulses to –40 mV in rat control cardiomyocyte initially bathed in 10 mM Na+ (A) or to –30 mV in Nav1.5-expressing oocyte initially bathed in 30 mM Na+ (B). Inset: Current tracings elicited in the indicated conditions.

 
Under control experimental conditions, Nav1.5-expressing oocytes displayed a INa whose activation threshold was around –60 mV, a maximum value at –30 mV and reversal potential at around +30 mV as previously reported [19]. In 10 oocytes, mean INa monitored at –30 mV was 4.76±0.36 µA. Inactivation time course of INa could be well described by two exponentials with time constants of 1.06±0.16 and 6.13±0.74 ms (n=25). No inward current could be detected in the Na+-free solution even when Ca2+ concentration was enhanced to 20 mM. Only 40% of the oocytes displayed an outward current at +30 mV, a fact that could be related to the decrease in open probability of Na+ channels in Na+-free medium [22]. H2O2 applied at concentrations of 3, or even 9 mM, caused no changes in INa amplitude or inactivation kinetics. Under this condition for periods of 5 min or more, removing the extracellular Na+ suppressed the inward current at –30 mV in every oocyte (Fig. 4B). In control or in Na+-free and H2O2-containing solutions, no inward current was seen in the whole range of negative potentials.

Similar experimental series were conducted using 100-µM tert-butyl hydroperoxide, t-BHP. t-BHP decreased INa by 40.5±4.2 % in normal cardiomyocytes (n=4) but not in oocytes (n=8); it did not alter INa inactivation time course. After applying a Na+-free solution with t-BHP, no inward current could be recorded in both cell types (Fig. 5).


Figure 5
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  		Fig. 5 Oxidative stress by t-BHP did not induce the TTX-sensitive Ca2+ current in rat control cardiomyocytes or Nav1.5 expressing oocytes. In the presence of 100 µM t-BHP, removing Na+ from the perfusing solution fully suppressed the inward current elicited by depolarising pulses to –40 mV in rat control cardiomyocyte initially bathed in 10 mM Na+ (A) or to –30 mV in Nav1.5-expressing oocyte initially bathed in 30 mM Na+ (B). Inset: Current tracings elicited in the indicated conditions.

 
3.3 Adenylate cyclase stimulation does not induce ICa(TTX)-like currents in normal cardiomyocytes or Nav1.5 channel-expressing oocytes
To evaluate the effects of adenylate cyclase stimulation, cardiomyocytes (n=4) from young normal rats and Nav1.5-expressing oocytes (n=7) were exposed to forskolin, FSK. Single cardiomyocytes perfused with a 10-mM Na+ solution showed a non-significant tendency (5.0±2.9%) to increase INa when exposed to 5-µM FSK. After 10 min in this solution, Na+ removal suppressed the inward current observed at –40 mV or lower depolarising potentials (Fig. 6A). Similarly, control cardiomyocytes acutely tested, or preincubated for 4-h, with 1 µM isoproterenol never demonstrated any inward current in the absence of Na+ ions (not shown). FSK treatment of Nav1.5-expressing oocytes decreased INa at –30 mV by 14.3±1.04% and left unaffected its inactivation time course. FSK was unable to allow for any inward current bathed in a Na+-free solution (Fig. 6B).


Figure 6
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  		Fig. 6 β-Adrenergic stimulation did not induce the TTX-sensitive Ca2+ current in rat control cardiomyocytes or Nav1.5 expressing oocytes. In the presence of 5 µM forskolin, FSK, removing Na+ from the perfusing solution fully suppressed the inward current elicited by depolarising pulses to –40 mV in rat control cardiomyocyte initially bathed in 10 mM Na+ (A) or to –30 mV in Nav1.5-expressing oocyte initially bathed in 30 mM Na+ (B). Inset: Current tracings elicited in the indicated conditions.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The most salient feature of the present results, under specific experimental conditions (negative HP, Cs+ instead of K+ ions, low external Na+), is the demonstration of a TTX-sensitive inward current only in PMI ventricular cardiomyocytes from Wistar rats with properties similar to ICa(TTX) previously described in human atrial [10] Sprague–Dawley rat [9] and guinea pig [8] ventricular myocytes. Our results also suggest that this current does not arise from modifications of the Nav1.5-channel selectivity during remodelling.

4.1 Properties of ICa(TTX) recorded in PMI-rat cardiomyocytes
As in previous studies [8–10], ICa(TTX) in PMI cardiomyocytes was fully inhibited by TTX concentrations that abolished INa and exhibited a low reversal potential around +5 mV. It was not blocked by the inhibitors of T- and L-types Ca2+ channels, Ni2+, Co2+ or nifedipine while the well-known Na+ channel "agonist" veratrine markedly prolonged its inactivation. The present results further show that Cd2+ (100 µM) only partially blocked ICa(TTX) in PMI rat cells, in contrast to the full inhibition induced by 20 µM Cd2+ in human cells [10]. These specific experimental conditions allow us to visualize that ICa(TTX) represents Ca2+ permeation through a poorly selective Na+ channel aside of the usual one.

The kinetics of ICa(TTX) in PMI cardiomyocytes are roughly similar to those of the INa we recorded in 10 mM extracellular Na+, i.e. fast kinetics of activation and inactivation and a threshold positive to –60 mV. Half-inactivation of ICa(TTX) occurred at potentials (–67.0 mV) similar to that of the INa (–73 mV) recorded in normal cardiomyocytes superfused with 10 mM extracellular Na+ plus 5.4 mM Ca2+, but less negative than the values previously reported by Lemaire et al. [10] (–96 mV) and Aggarwal et al. [9] (–83.1 mV) using 2 and 3 mM extracellular Ca2+, respectively. Furthermore in our experiments, 45% of the cells showing ICa(TTX) displayed one inactivation constant, while inactivation time course of the other 55% was best fitted by two exponentials. This is similar to our observations in normal cardiomyocytes where INa in 40% of the cells in a 10-mM Na+, 5.4-mM Ca2+ solution inactivates with a single exponential and 60% with two exponentials. However, the values of the time constants are higher, a fact that could be related to changes in gating properties of Na+ channels due to the low-Na+ solution [22]. Our results show many similarities with those of Akaike and Takahashi [7]. They reported the existence of a TTX-sensitive Ca2+ current in neurons from the rat hippocampal CA1 region with half-inactivation and -activation potentials of –72.5 and –42.5 mV, respectively, in 10 mM Ca2+ while in 5.4 mM Ca2+, we found –67.0 and –34.9 mV, respectively, with similar potentials for threshold and peak ICa(TTX). As well, recovery from inactivation was biphasic in both PMI rat cells [5] and CA1 neurons with fast and slow time constants of 18.7 and 98.4 ms and 13 and 120 ms, respectively. The two inactivation time constants of ICa(TTX) in CA1 neurons were faster than those obtained in cardiocytes that showed two inactivation time constants [13].

4.2 Origin of ICa(TTX) recorded in PMI-rat cardiomyocytes
Several lines of experimental evidence, notably single-channel recordings, established convincingly that the membrane contains distinct sets of pores, each with its own distinctive selectivity properties. Nevertheless, the prevailing view that ion channel selectivity is preserved during normal electrical activity was recently challenged. In rat ventricular myocytes, Na+ channels were reported to conduct Ca2+ well after cyclic AMP-dependent phosphorylation, the "slip-mode conductance" [12,23]. Besides, in guinea pig and in Sprague–Dawley rat, a Na+-independent, TTX-sensitive current was recorded when the external solution contained no or very little Na+ [8,9,24]. Cole et al. [8] suggested that ICa(TTX) in guinea pig cardiocytes represents Ca2+ flux through the classical Na+ channels, whose selectivity and gating were modified in low-Na+ or Na+-free extracellular solutions. Their main argument was that micromolar extracellular Na+ was able to block ICa(TTX). In contrast, the fact that activation and inactivation kinetics of ICa(TTX) are slower than those of INa recorded in low Na+ solution, as well as that activation and inactivation curves of ICa(TTX) are both shifted to more negative potentials, led Balke's group to propose that ICa(TTX) channels are distinct from classical Na+ channels and are non-interconvertable [8,25]. They recently reinforced this view by showing that an antisense oligonucleotide against H1 inhibits the Nav1.5-generated current but not ICaTTX [26].

The occurrence of a new ionic current, ICa(TTX)-like, only in PMI cells defined by its ionic charge carrier, its pharmacology and its TTX-sensitivity led us to question the nature of the channel protein. Two main possibilities are: alterations in the behaviour of the classical Na+ channels (Nav1.5) induced by environmental changes during remodelling and/or alterations in ion channel gene expression. Our results demonstrate that, as shown by Cole et al. [8], ICa(TTX)-like current in PMI-rat cells is reduced by adding micromolar extracellular Na+ while millimolar concentrations as used by Akaike and Takahashi [7] increase the current (Fig. 3). This observation suggests that Ca2+ and Na+ would compete for a binding site within the Na+ channel pore, similar to the well known Ca2+–Na+ competition at the L-type Ca2+ channel pore [27]. However, after expressing the Nav1.5 channel in Xenopus oocytes, we could not find a ICa(TTX)-like current in Na+-free solution even when Ca2+ concentration was increased from 5.4 to 20 mM. Similarly, no such Ca2+ current could be observed by Nuss and Marban [28] after heterologous expression in CHO while Chen-Izu et al. [25] and Guatimosin et al. [24] (taking into account the surface charge effects, see Chen-Izu et al. [25]) reported that Nav1.5 channels expressed in HEK-293 cells showed a small but noticeable Ca2+ permeation in Na+-free solution. The absence of ICa(TTX) in Xenopus oocytes and also in normal and sham cardiomyocytes suggests that this current is not flowing through classical Nav1.5 channels, thus representing a "new" Na+ channel as indicated by Balke's group [9,25,26]. Nevertheless, since we only found ICa(TTX) in PMI cardiomyocytes, it could be possible that ICa(TTX) in these cells arises from Ca2+ permeation through Nav1.5 channels modified during remodelling in Wistar rats. Also, the fact that the TTX-sensitive Ca2+ permeation in very low or Na-free solutions is seen in guinea pig [8] and Sprague–Dawley rats [9,24,25] but not in control Wistar rats (these results), and in Nav1.5-expressing HEK293 cells [24,25] but not in CHO [28] or Xenopus oocytes (these results) might indicate the existence of an uncontrolled experimental factor which affects Na+ channel selectivity.

Two main possibilities, not mutually exclusive, were tested in normal rat cardiomyocytes and in Xenopus oocytes expressing Nav1.5 channel for the occurrence of Ca2+ permeation through Nav1.5 channels modified during remodelling: oxidative stress and sustained stimulation of PKA. Evidence exists for a role of oxidative stress [15,16] and of chronic catecholamine stimulation [17]. Both conditions are also known to affect classical INa [21,29–31]. However, neither oxidative stress induced with H2O2 or t-BHP, nor activation of PKA by forskolin, induced ICa(TTX)-like currents in control Wistar rat cardiomyocytes or Nav1.5-expressing oocytes in Na+-free extracellular solution. Here it is to note that oxidative stress (H2O2 or t-BHP) decreased peak INa in normal cardiomyocytes as in frog cardiomyocytes [21] but barely affected Nav1.5 currents in oocytes, as previously shown in both Xenopus oocytes and HEK 293 cells [32].

β-Adrenergic stimulation PKA gives rise to complex effects in Na+ channels in cardiac cells but the prevailing feature is that INa is increased together with a shift in gating properties [29–31]. Although it was not the purpose of this work to further investigate these effects, we found that direct stimulation of adenylate cyclase by FSK induced a modest increase in INa in normal cardiomyocytes but not in Nav1.5-expressing Xenopus oocytes. Yet, the most crucial result was that neither in cardiomyocytes nor in oocytes, PKA activation induced a ICa(TTX)-like current in Na+-free extracellular solution. This result confirms, in part, those of Chandra et al. [33], Nuss and Marban [28] and Hirano and Hiraoka [34] who showed that β-adrenergic stimulation does not change the Na+/Ca2+ selectivity of the Nav1.5 channel.

Aside from experimental artifacts or pathophysiological changes related to remodelling, the ICa(TTX)-like currents recorded in PMI-rat cells could also result from alterations of the genetic program during remodelling. Recent results showed that PMI-rat hearts showed an increased expression of brain type I Na+ channel (Nav 1.1) protein with reversion of the Ia/I isoform ratio to the fetal phenotype and suggested that the increase in the slow component of INa in remodeled-PMI cardiomyocytes is secondary to the increased expression of brain type I Na+ channel [13]. Taking into account that Nav 1.1 are expressed in both heart [35] and hippocampal neuron and the similarities between the properties of ICa(TTX) described by Akaike and Takahashi [7] and in the present results, it could be suggested that the ICa(TTX)-like current found in PMI-rat cardiomyocytes represents the over-expression of Nav 1.1 isoform. Indeed, roles for this channel type were recently demonstrated in ventricular and sinoatrial node mouse cells [36]. That Nav1.1 heterogously expressed channels could carry Ca2+ ions in the absence of Na+ remains to be demonstrated. Moreover, experimental alterations of Na+ channel selectivity and changes in Nav isoforms are not exclusive.

This biophysical approach has allowed us to demonstrate the occurrence of a TTX-sensitive channel with specific characteristics. Although it will hardly carry Ca2+ under control conditions, it will in some part contribute to the depolarising current by letting Na+ to go through, aside of the usual Na+ channel. A last important feature of this study in rat heart is that myocardial infarction after long-term left coronary artery ligation induces similar alterations in all regions including the right ventricle. The increases in both length and width, the decrease in K+ currents [4], and the occurrence of INa(TTX)R [6] and ICa(TTX) in cells isolated from the four selected ventricular regions attest that a large infarcted area has deleterious consequences for the whole heart.


   Acknowledgements
 
The authors wish to thank Dr L. Millán for her help in preparing the cDNA used in this study, and M. Lemallam and P. Bideaux for preparing and controlling the animals. This study was supported in part by grants to J.L. Alvarez from the Ministère de l'Enseignement Supérieur et de la Recherche; Fondation pour la Recherche Médicale, ACC-SV No. 9-1A021A; BIOMED II European grant PL 950287, Conacyt (grant II-77G01 to ES) and by Inserm-Conacyt and Cuba-México exchange programs.


   Notes
 
Time for primary review 25 days


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

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