Cardiovascular Research

Cardiovascular Research 1999 41(1):175-187; doi:10.1016/S0008-6363(98)00202-8
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Characterization of a [Ca²⁺]_i-dependent current in human atrial and ventricular cardiomyocytes in the absence of Na⁺ and K⁺

Olaf F. Köster^*, Gyula P. Szigeti and Dirk J. Beuckelmann

Department of Internal Medicine III, University of Cologne, Joseph-Stelzmann-Strasse 9, 50924 Cologne, Germany

^* Corresponding author. Tel.: +49-(221)-478-4475; fax: +49-(221)-478-6490.

Received 12 January 1998; accepted 6 May 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objectives: In situations of [Ca²⁺]_i-overload, arrhythmias are believed to be triggered by delayed afterdepolarizations, which are generated by a transient inward current I_TI. This study was designed to examine [Ca²⁺]_i-dependent membrane currents in the absence of the Na⁺/Ca²⁺-exchanger as possible contributors to I_TI in human cardiac cells. Methods: The whole cell voltage clamp technique was used for electrophysiological measurements in human atrial and ventricular cardiomyocytes. [Ca²⁺]_i-measurements were performed using the fluorescent Ca²⁺-indicator fura-2. All solutions were Na⁺-free. Voltage-independent [Ca²⁺]_i-transients were elicited by rapid caffeine applications. Results: In atrial myocytes, caffeine induced a transient membrane current in the absence of Na⁺ and K⁺. This current could be suppressed by internal EGTA (10 mM). Cl^– did not contribute to this current. Experiments with different cations suggested non-selectivity for Cs⁺ and Li⁺, whereas N-methyl-D-glucamine appeared to be impermeable. Voltage ramps indicated a linear current–voltage relation in the range of +80 to –80 mV. Fluorescence measurements revealed a dissociation between the time courses of current and bulk [Ca²⁺]_i-signal. In ventricular cardiomyocytes, caffeine failed to induce transient currents in 54 cells from 22 different patients with or without terminal heart failure. Conclusions: In human atrial cardiomyocytes, a [Ca²⁺]_i-dependent nonspecific cation channel is expressed and may contribute to triggered arrhythmias in situations of [Ca²⁺]_i-overload. No evidence could be found for the existence of a [Ca²⁺]_i-dependent chloride current in atrial cells. In ventricular cells, neither a [Ca²⁺]_i-dependent nonspecific cation channel nor a [Ca²⁺]_i-dependent chloride channel seems to be expressed. Possible delayed afterdepolarizations in human ventricular myocardium might therefore be carried by the Na⁺/Ca²⁺-exchanger alone.

KEYWORDS Human; Arrhythmia (mechanisms); Calcium (cellular); Ion channels; Membrane currents; Membrane permeability

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
In situations of [Ca²⁺]_i-overload, cardiac arrhythmias have been attributed to delayed afterdepolarizations, which are carried by a transient inward current I_TI (for review see [1]). In the 1970s this current was extensively studied in cardiac Purkinje fibres [2–4]. It was assumed that I_TI reflects at least two different membrane conductivities: (1) the Na⁺/Ca²⁺-exchanger [4–8]and (2) a [Ca²⁺]_i-dependent nonspecific cation channel [3, 4, 9, 10]. Membrane currents carried by the Na⁺/Ca²⁺-exchanger (I_Na/Ca) have been identified in a large variety of species and tissues, including human atrial myocytes. In the latter, I_Na/Ca contributes to the duration of the action potential and to the generation of delayed afterdepolarizations [11, 12].

The first description of a [Ca²⁺]_i-dependent nonselective cation conductance in cardiac cells was given by Kass et al. in 1978 for calf Purkinje fibres, which exhibited a depolarizing transient inward current under strophanthidin treatment [4]. In 1981, Colquhoun et al. [9]described a [Ca²⁺]_i-dependent nonspecific cation channel, which could be identified on the single-channel level in cultured neonatal rat cardiomyocytes. In the following years similar channels were found in a large variety of species and tissues (for review see [13, 14]. Most of these channels exhibit a voltage-independent behaviour, ohmic conductance, poor selectivity among cations and a single channel conductance around 30 pS. Thus, it was assumed that [Ca²⁺]_i-dependent nonspecific cation channels form a unique class of channels, called CAN-channels ([Ca²⁺]_i-activated nonspecific), although different types seem to exist as well. In cardiac cells, this channel type has been identified in neonatal rat and adult canine and guinea-pig cardiomyocytes [9, 15, 16]. However, the existence of a [Ca²⁺]_i-dependent non-specific cation channel in the human heart is still unknown.

In human atrial myocytes, a [Ca²⁺]_i-dependent and 4-aminopyridine (4-AP) resistant component of the transient outward current I_to has been identified and called brief outward current I_bo [17]. This current exhibited properties, which suggest a dependence on sarcoplasmic reticulum Ca²⁺ release, since it could be inhibited by caffeine and the calcium channel blocking agent Co²⁺. A similar component of I_to could be identified as a [Ca²⁺]_i-dependent chloride current I_Cl(Ca) in atrial, ventricular and Purkinje cells of rabbits [18–22]and in canine ventricular cardiomyocytes [23]. This current generated oscillatory membrane depolarizations in situations of [Ca²⁺]_i-overload or isoproterenol treatment [21–23]. The identification of I_Cl(Ca) as a component of I_to in these species gave rise to the hypothesis, that I_bo in human atrium is believed to be a chloride-current as well. This hypothesis could not be confirmed in a later study by Li et al., who demonstrated that I_to in human atrial cells is completely suppressed when pipette K⁺ is replaced by Cs⁺ and that the remaining currents appear unaltered by [Cl^–]_o-substitution [24]. Furthermore, the results of the latter study do not support a [Ca²⁺]_i-dependence of I_bo.

[Ca²⁺]_i-dependent currents in the heart may underlie arrhythmogenic mechanisms, such as delayed afterdepolarizations, in conditions of [Ca²⁺]_i-overload (see [1, 25]for review). High levels of [Ca²⁺]_i may lead to spontaneous oscillatory release of Ca²⁺ from the sarcoplasmic reticulum (SR), which may then activate [Ca²⁺]_i-dependent membrane currents. If such [Ca²⁺]_i-oscillations are sufficiently synchronised during diastole these currents may initiate premature action potentials. Therefore, [Ca²⁺]_i-dependent currents may reflect important arrhythmogenic mechanisms in a variety of pathophysiological conditions, which are associated with [Ca²⁺]_i-overload, such as hypertrophy, ischemia and reperfusion, treatment with cardiac glycosides and catecholamines (reviews: [26, 27]).

The aim of this study was to identify [Ca²⁺]_i-dependent membrane currents in the absence of Na⁺/Ca²⁺-exchange current and [Ca²⁺]_i-dependent K⁺-currents in human atrial and ventricular cardiomyocytes.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
All investigations on human tissues conform with the principles outlined in the Declaration of Helsinki and were approved by the ethics committee of the university of Cologne. Patients undergoing therapeutical operations gave informed consent prior to the operation.

2.1 Isolation of human atrial myocytes
Right atrial appendages were obtained from 46 patients (average age 60.4±14.6 S.D. years; 31 males; 15 females) undergoing open heart surgery (33 aorto-coronary bypasses, 5 aortic valve replacements and 8 others). Sinus rhythm was present in 44 cases, atrial fibrillation in 2 cases. Most patients were chronically treated with drugs prior to operation (longacting nitrates or molsidomine: 23, betablockers: 21, diuretics: 19, ACE-inhibitors: 14, calcium-channel-blockers: 8, antilipemic agents: 8, aspirin: 6, cardiac glycosides: 4, others: 18).

Samples were immersed immediately after excision in Bretschneider's cardioplegic solution (15 mM NaCl, 9 mM KCl, 1 mM K-H-2-ketoglutarate, 4 mM MgCl, 18 mM histidine·HCl·H₂O, 180 mM histidine, 2 mM tryptophane, 30 mM mannitol, 0.015 mM CaCl₂), taken to the laboratory, and maintained at 4°C. The cell isolation procedure was similar to that described by Bustamante et al. [53]. Myocardial specimens were chopped with scissors into small chunks of 1 mm³ and placed in flasks, containing 10 ml of a nominally Ca²⁺-free modified Tyrode's solution (135 mM NaCl, 10 mM glucose, 10 mM Hepes–NaOH, 4 mM KCl, 1 mM MgCl₂, 0.33 mM NaH₂PO₄, pH 7.3 with NaOH). All steps were carried out at 37°C and continuous agitation during all steps of the isolation procedure was ensured by oxygen bubbling from the bottom of the flask. This washing procedure was performed for 5 min and repeated twice. Afterwards the tissue was incubated in 10 ml of a similar solution, containing 11 mg Worthington-collagenase type CLS II (347 U/mg; Sigma, St. Louis, USA) and 0.8 mg Protease Type XIV (4.4 U/mg; Sigma). After 30 min the tissue was filled into two separate flasks of 15 ml, each flask was filled with nominally Ca²⁺-free modified Tyrode's solution, and centrifuged (500 g for 1 min). The supernatant was discarded and the remaining tissue was poured into a flask again. The flask was filled with nominally Ca²⁺-free modified Tyrode's solution and the centrifugation procedure was repeated. After removal of the supernatant, the tissue was reincubated in nominally Ca²⁺-free modified Tyrode's solution, containing 11 mg of Worthington-collagenase type CLS II. After 10–20 min (depending on cell yield and morphology), the cell suspension was washed and centrifuged in two steps as described before. Cells were stored in nominally Ca²⁺-free modified Tyrode's solution at room temperature (20°C) and remained viable for hours. Cells were transferred from this medium directly to the examination chamber and superfused with 2 mM Ca²⁺ containing Tyrode's solution. Some cells were contracting spontaneously under these conditions and went over into irreversible contracture. However, only those cells which were not spontaneously contracting in the presence of [Ca²⁺]_o of 2 mM, which were typically shaped and without visible blebs on the surface under these conditions, were used for our experiments.

2.2 Isolation of human ventricular myocytes
Human left ventricular myocardium was obtained from explanted hearts of 22 patients undergoing heart transplantation due to terminal heart failure (17 patients) or from donor hearts without heart failure (5 patients), which could not be transplanted for technical reasons. Heart failure was caused by ischemic (12 patients) or dilated (5 patients) cardiomyopathy. Explanted hearts were immersed in Bretschneider's solution (4°C; see Section 2.1) and taken to the laboratory immediately after explantation. The isolation procedure was previously described in detail [28]. A part of the left ventricular wall (10–20 cm²) was excised with its artery branch. The wall segment was perfused via this arterial branch for 30 min with a nominally Ca²⁺-free modified Tyrode's solution (see Section 2.1) to wash out blood and Ca²⁺. All steps were performed at 37°C and solutions were oxygenated throughout the entire isolation procedure. After the washing phase the segment was perfused in a recirculating manner for 40 min with the same solution containing 25.5 mg/50 ml Worthington-collagenase type CLS II and 3 mg/50 ml protease type XIV. Afterwards, the enzyme was washed out for 15 min with modified Tyrode's solution containing 200 µM Ca²⁺. Myocytes were taken from the midmyocardial region and suspended in modified Tyrode's solution containing 200 µM Ca²⁺. After filtering this suspension through a nylon mesh, [Ca²⁺] was increased in 0.5 mM steps every 15 min up to a final concentration of 2 mM. The suspension was stored at room temperature (20°C) and cells remained viable for up to 5 h.

The living-cell yield was approximately 5–8%. Only cells with clear cross striations without granulation and spontaneous contractions were selected for experiments.

2.3 Recording techniques
Electrophysiological measurements were performed in the whole-cell-mode of the patch-clamp technique [54]using a patch-clamp amplifier (EPC 7, List-Medical, Darmstadt, Germany) with a 100-M feedback resistor. Microelectrodes were fabricated from borosilicate glass capillaries and had resistances of 1.5–3.5 and 3.5–5.0 M for ventricular and atrial cells, respectively. The experimental apparatus was constructed around a Zeiss Axiovert 35 inverted microscope with a photometer attachment (Zeiss, MPM 201) described in detail in [28]. For fluorescence recordings, ultraviolet light emitted from a 75 W xenon arc lamp passed through 10 nm interference filters (340 or 380 nm wavelengths) and was reflected into the objective. Fluorescence emitted from the cell passed through a 510–540 nm bandpass filter to the photomultiplier tube. Fluorescence and current signals were digitised (IDA Interface, Indec Systems, Mountain View, USA) and stored on a personal computer for off-line analysis (sampling rate 100–200 Hz). Membrane capacitance was determined in each cell by integrating the capacitance current upon repolarizing to –80 mV from hyperpolarizing pulses.

2.4 Solutions
Cells were superfused with a modified Tyrode's solution (2 mM CaCl₂, 135 mM NaCl, 10 mM glucose, 10 mM Hepes–NaOH, 4 mM KCl, 1 mM MgCl₂, 0.33 mM NaH₂PO₄, pH 7.3 with NaOH). Extracellular test solutions during the experiments were applied by a fast-solution-exchange system [52]positioned in close proximity (100 µm) to the cell. This system contained four independent microvalves (LFAA 1201718H, Lee, Essex, USA), which were controlled by the stimulation programme. Both, extracellular test solutions and pipette solutions (Tables 1 and 2) were nominally Na⁺-and K⁺-free and contained 20 mM tetraethylammonium (TEA) to inactivate contaminating currents. Na⁺ was replaced by Li⁺, Cs⁺ or N-methyl-D-glucamine (NMDG⁺), when appropriate. In some experiments, [Cl^–] was varied, substituting by glutamate. Caffeine was used to induce Ca²⁺-transients independently from voltage dependent Ca²⁺-currents. In the present study, caffeine (Sigma) was dissolved directly in the extracellular solutions to a final concentration of 1, 2, 5 and 10 mM and was applied in short (250 ms) pulses directly to the cell by the rapid solution-exchange system. Apamin (Sigma) was added to the extracellular solution (1 µM), when indicated. For fluorescence measurements the pipette solution contained 0.05 mM fura-2 pentapotassium salt (Molecular Probes, Eugene, USA). All experiments were carried out at room temperature (20°C).

View this table: [in this window] [in a new window]   Table 1 Extracellular solutions

 

View this table: [in this window] [in a new window]   Table 2 Pipette solutions

 
2.5 Experimental protocol
In experiments where fluorescence recordings were performed, background fluorescence was determined after establishing a gigaseal, but prior to membrane rupture. Pipette solutions were allowed to equilibrate with the cytosol for 2–4 min. In most experiments, test pulses were preceded by five depolarizing prepulses from –60 to +10 mV in order to load the SR with Ca²⁺ to a similar extent. Extracellular solutions were switched to Na⁺-free test solutions prior to the experiments and did not change hereafter, until the experiment was finished. The stimulation protocols for each experiment is given in the figures.

2.6 Statistical analysis
Data included in comparative analysis was corrected for junction potentials, which were calculated by a personal computer, using AXOSCOPE, Version 1.1 (Fa. Axon Instruments, Burlingame, USA) and are presented as means±S.E.M.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Human atrial myocytes
3.1.1 Caffeine activates a transient membrane current in the absence of Na⁺ and K⁺
In all experiments in this paper (if not otherwise stated in the legends to the figures), test pulses were preceded by five depolarizing prepulses from –60 to +10 mV (1 Hz) in order to load the SR with Ca²⁺ to a similar extent and zero currents are indicated in the figures by dotted lines.

To identify contaminating voltage dependent currents, experiments were performed in the absence of caffeine. As shown in Fig. 1A, sustained outward currents could be seen during the test potentials, but no transient currents. L-type-Ca²⁺ currents were small in many cells tested. This feature will be discussed later. Fig. 1B illustrates currents of the same cell, when caffeine (10 mM) was applied for 250 ms, beginning 250 ms after clamping the cell to the test potential. Transient currents were activated 150 ms after onset of the caffeine application with a reversal potential near 0 mV. Digital subtraction of the control-registration from the pulse after addition of caffeine yielded the caffeine-induced currents (Fig. 1C). Similar currents could be observed in a total number of 46 from 81 cells (57%), using either lithium chloride or cesium chloride solutions and caffeine concentrations of 1, 2.5, 5 and 10 mM. The mean reversal potential for 13 cells tested with either Li⁺ (n=6) or Cs⁺ (n=7) as major intra- and extracellular cations was –7.1 (±1.5 S.E.M.) mV and –3.3 (±2.5 S.E.M.) mV, respectively, when using test potentials from –60 to +60 mV (in 30 mV steps) and 10 mM caffeine. The mean current density at –60 mV (–1.13 (±0.26 S.E.M.) pA/pF for Li⁺ and –0.66 (±0.15 S.E.M.) pA/pF for Cs⁺) exhibited a remarkable variability (see Section 4). Currents of this type could never be observed without caffeine application (n=12; only cells in which caffeine previously induced currents). All cells, in which caffeine induced a transient current showed strong and long-lasting contractions upon caffeine application, as judged by visual control in experiments, when no fluorescence measurements were performed.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Effect of caffeine on membrane currents. Pipette- and extracellular solution: LiCl solution. (A) Currents under control conditions (without caffeine). (B) Currents after caffeine application. (C) Caffeine-induced net currents. (D) Current–voltage relation.

 
3.1.2 Caffeine-induced current is [Ca²⁺]_i-dependent
Since it is known that caffeine activates a non-specific cation channel in smooth muscle cells of Bufo marinus [29], we investigated whether the caffeine-induced current is activated by caffeine directly or by SR-Ca²⁺ release.

The effect of rapid caffeine application in the presence of 10 mM EGTA in the pipette solution was examined shortly after a whole-cell-patch was established. Under these conditions, caffeine failed to induce a contraction of the cell (judged by visual control) and no transient currents after caffeine application could be observed, as demonstrated in Fig. 2. Similar results were obtained in all cells tested under these conditions (n=7). In these experiments, Ca²⁺-currents were very prominent, compared with those in absence of EGTA (see Section 4).

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Dependence of the caffeine-induced current on [Ca2+]i. Test voltages ranged from –60 to +60 in 30 mV steps. Pipette solution: CsCl solution with 10 mM EGTA. Extracellular solution: NMDG-Cl solution.

 
3.1.3 Do repetitive caffeine applications result in comparable [Ca²⁺]_i-transients and currents?
Caffeine-induced [Ca²⁺]_i-transients may be variable between different cells and preparations due to possible alterations of [Ca²⁺]_i-handling and-sensitivity by the isolation procedure and patient's disease. However, in a given cell, [Ca²⁺]_i-transients and therefore [Ca²⁺]_i-dependent currents should be stable throughout an experiment, as long as experimental conditions remain constant and [Ca²⁺]_i-handling is not fundamentally altered by the experiment. To test this hypothesis, we examined the intra-cell variability of [Ca²⁺]_i-transients and [Ca²⁺]_i-dependent currents, induced by repetitive applications of caffeine. First, variability of the resulting fluorescence in a single cell was tested (Fig. 3A). Fluorescence was defined as the maximum of the traces at 340 nm and the minimum at 380 nm excitation wavelength and is given in relative units (i.e. dimensionless, non-scaled digitised units as received from the photomultiplier). As illustrated in the middle panel of Fig. 3A, the fluorescence signals at both excitation wavelengths did not significantly differ within a single cell under these conditions. Similar results were obtained in seven cells.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 (A) Simultaneous registration of caffeine-induced membrane currents (upper panel) at test potentials of –60 to +60 mV in 30 mV steps and fura-2 fluorescence (middle panel). Pipette- and extracellular solution: CsCl solution. Caffeine application (10 mM, 250 ms) is symbolized by the bold areas in the stimulation protocol (lower panel). (B) Membrane currents under repetitive (5x) caffeine application at +60 mV (upper panel). Pipette- and extracellular solution: LiCl solution.

 
Since fluorescence of non-membrane-associated indicators represents bulk [Ca²⁺]_i rather than subsarcolemmal [Ca²⁺]_i [30, 31], we performed experiments in which caffeine application was repeated five times and [Ca²⁺]_i-dependent peak currents at +60 mV were determined. The amplitudes of the resulting currents may be a more appropriate indicator for subsarcolemmal [Ca²⁺]_i. As illustrated in Fig. 3B, the mean current amplitude variability in a single cell was negligible. Similar results were obtained in four cells.

3.1.4 Ionic nature of the [Ca²⁺]_i-dependent non-specific current
In the absence of Na⁺ and K⁺, [Ca²⁺]_i-dependent currents may represent [Ca²⁺]_i-dependent chloride currents or [Ca²⁺]_i-dependent non-specific cation currents. Since [Ca²⁺]_i-dependent chloride currents have been described in atrial, ventricular and Purkinje cells of rabbits [18–22]and in canine ventricular cardiomyocytes [23], it has been postulated that the 4-AP-resistant component of the transient outward current in human atrial myocytes, called I_bo by Escande et al. [17], may reflect I_Cl(Ca). In the present study, we tested the contribution of chloride to the [Ca²⁺]_i-dependent current by using different intra- and extracellular chloride concentrations. Membrane currents were tested using either CsCl pipette solution with (1) NMDG-Cl (Fig. 4A), (2) NMDG-glutamate extracellular solutions (Fig. 4B), or (3) Cs-glutamate pipette solution with NMDG-Cl extracellular solution (Fig. 4C). According to the Nernst equation, the chloride-reversal-potentials E_Cl^– were calculated as: (1) –1 mV; (2) +41 mV; (3) –46 mV. With respect to the calculated reversal potentials, test potentials ranged from –60 to +60 mV (in 30 mV steps). Fig. 4 illustrates three representative results, showing no clear reversal of the observed currents. A reversal of the current might be expected only negative of –60 mV, since small outward currents can be observed even at –60 mV. Therefore, it must be concluded that the [Ca²⁺]_i-dependent current in human atrial myocytes is not carried by chloride. Since Cs⁺ and Li⁺ appeared to be permeable, as demonstrated in Figs. 1, 3 and 4, a non-specific cation current must be assumed. Furthermore, NMDG⁺ seems to be impermeable (cp. Fig. 4). We tested the permeability for NMDG⁺ by switching the extracellular solution to a 110 mM NMDG⁺ containing solution, after it had been demonstrated that inward currents were observed under experimental conditions described for our Li⁺- and Cs⁺-experiments. In 14 cells, no inward currents could be seen, after the extracellular solutions had been replaced by NMDG-glutamate solution (not shown). Caffeine-induced currents, carried by either Cs⁺ or Li⁺, were identical to those depicted in Fig. 4B. Absence of transient currents in the presence of NMDG⁺ could be demonstrated upon hyperpolarization to –100 mV (not shown), indicating a permeability ratio for NMDG⁺ in comparison with Li⁺ of <0.019, according to the Goldman–Hodgkin–Katz equation. These results suggest complete non-permeability for NMDG⁺, which is in accordance with previous studies on CAN-channels [32]. Since pipette solutions as well as extracellular solutions contained 20 mM TEA⁺, inward currents carried by this cation could be expected at negative potentials, if any permeability is assumed.

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Effect of variation of intra- and extracellular [Cl–] on the reversal of the caffeine-induced currents (stimulation protocol and scheme of caffeine application was identical to that in Fig. 2). The current–voltage relation of each setting is given in the right column. (A) ECl–=–1 mV. (B) Same cell as in (A), after extracellular solution had been switched to NMDG–glutamate solution. ECl–=+41 mV. (C) ECl–=–46 mV.

 
In conclusion, the ionic permeability of the [Ca²⁺]_i-dependent current indicates that this current is a non-specific cation current and will be referred to as I_CAN in the following sections.

Repetitive caffeine applications at five different voltages during an experiment may lead to variabilities of [Ca²⁺]_i-transients and the resulting currents. Although it has been shown that these variabilities are relatively small, they may lead to misinterpretations of the current–voltage relation concerning rectification. Therefore, ramp protocols were carried out, consisting of four ramps (200 ms) from +80 to –80 mV. With respect to the time course of fura-2 fluorescence and current after caffeine application, it was assumed that once the current has reached its peak, its activation state (i.e. [Ca²⁺]_i) does not change significantly within 200 ms. The first ramp (ramp I) was performed without caffeine application to obtain a control ramp for digital subtraction from the caffeine-induced current. Three more ramps (ramp II, III and IV) were carried out at short intervals after caffeine application. The middle panel of Fig. 5 illustrates two representative registrations from the same cell, one control experiment without caffeine (thin line) and the second one with caffeine application (bold line). All ramps were identical in the control registration, whereas in the test protocol a transient current was activated at the end of ramp II (see the small inward deflection at 900 ms), reached its peak within ramp III and IV and declined thereafter, reaching the zero current line at 3700 ms. After digital subtraction of ramp I from ramp IV, the caffeine-induced net-current was calculated. The current–voltage relations (lower panel) demonstrate a zero-line for the control registration, as expected. The caffeine-induced current reveals an almost linear current–voltage relation without rectification. The reversal potential is –15 mV.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Voltage ramps. The cell was clamped to +80 mV for 200 ms. The first ramp (I) was performed without caffeine application (+80 to –80 mV, 200 ms). Afterwards the cell was clamped back to +80 mV and caffeine application (10 mM, 250 ms) began. Ramps II, III and IV were performed consecutively in short intervals. Between these, the cell was clamped back to +80 mV for 60 ms. Membrane currents are depicted in the middle panel (thin line=control registration without caffeine, bold line=with caffeine application). Notice the different time scale after axis break. Pipette- and extracellular solution: LiCl solution. Lower panel: net current–voltage relation for control registration () and registration with caffeine () are obtained by digital subtraction of ramp I from ramp IV (and hereby capacitive currents are eliminated).

 
3.1.5 Does I_CAN represent a non-selective conductivity through [Ca²⁺]_i-dependent [SK]-K⁺-channels?
[Ca²⁺]_i-dependent K⁺ channels represent an ubiquitous channel type, abundant in a large variety of mammalian and non-mammalian tissues (see [33]for review). In single channel experiments two types of [Ca²⁺]_i-dependent K⁺ channels could be identified: (1) channels with high conductivity of 200–300 pS, called [BK]-channels and (2) channels with low conductivity of 10–14 pS, called [SK]-channels [34]. The former type can be blocked by TEA⁺ from outside in concentrations of 0.1–1 mM and from inside in concentrations of 50–100 mM. The bee venom polypeptide apamin has no effect. The latter type is TEA⁺-resistant, but can be blocked with high selectivity by apamin. Hugues et al. blocked long-lasting afterhyperpolarizations, which are mediated by [Ca²⁺]_i-dependent K⁺ channels, in cultured rat muscle cells and in cultured mouse neuroblastoma cells with apamin in concentrations of 100 nM from the outside of the cell [35, 36].

Since all intra- and extracellular solutions in the present study contained 20 mM TEA⁺, it can be assumed that [Ca²⁺]_i-dependent [BK]-K⁺ channels, if present, were reliably blocked. However, a non-selective conductivity through TEA⁺-resistant [SK]-channels could not be excluded, although it had not been described for this channel type. We tested this hypothesis by using apamin as a tool for a selective blockade. Caffeine was used at a low concentration (1 mM) to allow for longer experiments. Apamin was dissolved in modified Tyrode's solution to a final concentration of 1 µM and was applied by the fast solution-exchange system. Fig. 6 illustrates an experiment in which a gigaseal could be maintained for 30 min. The first registration (upper panel, left) was performed without apamin. Caffeine induced I_CAN with a small amplitude, probably due to low caffeine concentration. The cell was then incubated in the apamin containing solution for 5 min (upper panel, right), 10 min (lower panel, left) and 15 min (lower panel, right) and the experiment repeated. Caffeine induced I_CAN even after 15 min of exposition to apamin and no obvious decrease in current amplitude could be observed. In summary, four cells from three different preparations revealed similar results after 5 min (1 cell), 10 min (2 cells) and 15 min (1 cell) of incubation. These results indicate, that I_CAN is not carried by [Ca²⁺]_i-dependent-[BK]-K⁺ channels.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effect of apamin on ICAN. Caffeine (1 mM, 250 ms) induced ICAN before incubation in apamin (middle panel, left). Hereafter, the cell was incubated for 5, 10 and 15 min in 1 µM apamin containing solution and the experiment was repeated. Pipette- and extracellular solution: CsCl solution.

 
3.1.6 Time courses of the [Ca²⁺]_i-signal and I_CAN
To compare the time courses of the [Ca²⁺]_i-signal and I_CAN, we performed fluorescence measurements with fura-2. The experimental protocol has been described in Fig. 3A. Background fluorescence was determined for both wavelengths prior to the experiments and digitally subtracted from the experimentally observed fluorescence signals. Hereafter, the fluorescence ratio R_F340/F380 between the wavelengths was calculated. In Fig. 7, [Ca²⁺]_i-signals and currents of a representative cell (same cell as in Fig. 3A) are depicted for each single test voltage. Comparison of the time courses of [Ca²⁺]_i-signals and currents reveal a dissociation between the decreasing phase of the current and the corresponding [Ca²⁺]_i-signal. The upstroke of the current was faster than the upstroke of the [Ca²⁺]_i-signal and inactivation of the current began long before the [Ca²⁺]_i-signal had reached the peak. When the current was completely inactivated, [Ca²⁺]_i was still elevated. Conversely, [Ca²⁺]_i-signals and currents were activated at the same time, without any detectable latency at sampling intervals of 10 ms.

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Time course of [Ca2+]i-transients and ICAN. Same experiment as in Fig. 3A. For each distinct test voltage, two panels are depicted: the upper panels represent the membrane current, the lower panels represent the fura-2 signal. In the inset, time courses of current and fluorescence for +60 mV are shown on an expanded time scale for better comparison. The two lines indicate the time interval between activation and peak amplitude of the current.

 
3.2 Human ventricular cardiomyocytes
For the following experiments, human ventricular cardiomyocytes from the midmyocardial region of the left ventricle were chosen. The stimulation protocol was similar to the protocol we used for atrial cells. Ventricular cells exhibited a much more pronounced contraction upon caffeine than atrial cells, making it difficult to maintain a seal throughout the entire experiment. For this reason, we tested for caffeine concentrations of 1, 2.5, 5 and 10 mM. Caffeine in concentrations as low as 1 mM was sufficient to induce I_CAN in atrial myocytes, as demonstrated in Fig. 6. Only those cells which exhibited a clear contraction upon caffeine-application as an indication for a sufficient Ca²⁺-release from the SR were included in the analysis. This feature was judged by visual control in experiments, where no fluorescence was measured. According to the permeability of Cs⁺ and Li⁺ to the [Ca²⁺]_i-dependent non-specific cation channel in atrial cells, CsCl solution or LiCl solution was used as pipette and extracellular solutions. Fig. 8A shows an example for a cell tested with CsCl solutions. The test solution contained 10 mM caffeine. Prepulses elicited prominent L-type-Ca²⁺ currents. However, no transient currents could be observed after caffeine application. To test whether this feature may be due to the inability of caffeine to induce a sufficient [Ca²⁺]_i-transient in these cells, we performed fluorescence measurements with fura-2 at 340 and 380 nm excitation wavelengths. Fig. 8B illustrates an experiment in which the cell is tested for –60 and +60 mV and 10 mM caffeine. Although caffeine induced a [Ca²⁺]_i-transient, it failed to induce I_CAN at potentials where this current should be large with respect to the expected reversal potential of 0 mV.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Effect of caffeine (10 mM, 250 ms) on ventricular cardiomyocytes. (A) Test voltages ranged from –60 to +60 mV (in 30 mV steps). A prepulse for +10 mV was performed prior to test voltages. (B) Simultaneous fluorescence measurements with fura-2 during caffeine pulses (10 mM, 250 ms) at –60 and +60 mV. Pipette- and extracellular solution: CsCl solution.

 
In summary, caffeine failed to induce I_CAN in 54 cells out of 22 different preparations in test solutions containing caffeine in concentrations of 1 mM (n=15), 2.5 mM (n=10), 5 mM (n=4) and 10 mM (n=25).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
4.1 Human atrial cardiomyocytes
4.1.1 Caffeine induces a transient membrane current in the absence of Na⁺ and K⁺
The results of the present study provide strong evidence for the expression of a [Ca²⁺]_i-dependent ion channel in human atrial myocytes, which can be activated in the absence of Na⁺ and K⁺. We used caffeine as a tool to induce [Ca²⁺]_i-transients independently of contaminating L-type-Ca²⁺ currents. Caffeine is known to have non-specific effects besides its effect on calcium release channel. However, Hansford and Lakatta demonstrated, that after incubation of cardiac cells in nanomolar concentrations of ryanodine, the additional amount of [Ca²⁺]_i released by caffeine is small, suggesting only small non-specific effects in comparison to SR-Ca²⁺-release [37]. Our main reason for selecting caffeine rather than ryanodine as a pharmacological tool to release calcium from intracellular stores, is its fast and reversible effect on [Ca²⁺]_i. Since [Ca²⁺]_i-oscillations are believed to be responsible for the activation of [Ca²⁺]_i-dependent non-specific cation channels (and delayed afterdepolarizations), we felt that caffeine-induced [Ca²⁺]_i-transients may imitate those oscillatory changes in [Ca²⁺]_i better than ryanodine.

In the present study, we were unable to induce a transient membrane current under control conditions, i.e. in absence of caffeine. In these experiments, only a sustained current could be observed, very similar to the depolarization-induced non-specific cation channel in human atrial myocytes, described by Crumb et al. [38]. A direct activation of the transient membrane current by caffeine, as described for a non-specific cation channel in smooth muscle cells of Bufo marinus [29]is unlikely, since in our experiments with 10 mM EGTA in the pipette solution, caffeine failed to induce this current. Therefore it has to be concluded that the caffeine-induced current is activated by [Ca²⁺]_i and that very high [Ca²⁺]_i-concentrations are required. In this study, [Ca²⁺]_i was determined only in a semiquantitative way, but it could be demonstrated that fluorescence at 340 nm was 3–4 times higher during the peak of the caffeine-induced Ca²⁺_i-transients compared with the peak of depolarization- (i.e. calcium channel-) induced transients (not shown).

4.1.2 Ionic nature of the caffeine-induced current
In cardiac cells, [Ca²⁺]_i-dependent membrane currents, which are not attributed to the Na⁺/Ca²⁺-exchange system, have been described as a component of the transient outward current I_to. Escande et al. [17]identified a brief outward current I_bo as a 4-AP-resistant component of I_to in human atrial myocytes. A similar, component of I_to has been identified as a chloride-current in atrial, ventricular and Purkinje cells of the rabbit [18–22]and in canine ventricular cardiomyocytes [23], called I_Cl(Ca). Therefore it was assumed that I_bo may reflect I_Cl(Ca) in human atrium as well. Li et al. were unable to demonstrate a contribution of chloride to I_bo, since I_to was completely suppressed by substitution of pipette K⁺ and the remaining current was unaltered, when [Cl^–]_o was replaced [24]. In the present study, we could not demonstrate a contribution of Cl^– to the caffeine-induced current. Variation of [Cl^–]_i and [Cl^–]_o did not shift the reversal potential in a wide range of test potentials. With respect to these results we conclude that [Ca²⁺]_i-dependent chloride channels are not expressed in human atrial cells. To determine the ionic nature of the caffeine-induced current, we tested the permeability for Cs⁺ and Li⁺, two cations which are generally permeable for CAN channels. The results of the present study demonstrate an almost equal permeability for these cations. In addition, NMDG⁺ as a cation, for which impermeability to CAN channels is generally accepted (see [13]for review), failed to carry inward currents down to –100 mV, when present as the major extracellular cation. Since TEA⁺ was also present in pipette- and extracellular solutions (20 mM), small inward currents at –100 mV would be expected, if permeability for this cation is assumed. Permeability of CAN-channels to the large cations TEA⁺ and Tris has been described in some reports [13]. However, this property was not extensively examined in the present study. A non-selective conductivity through [Ca²⁺]_i-dependent [SK]-K⁺-channels could be ruled out, since apamin in high concentrations (1 µM) was unable to suppress the caffeine-induced current even at low concentrations of caffeine (1 mM).

In conclusion, the results of the present study are in accordance with criteria, which have been formulated for CAN-channels, i.e. [Ca²⁺]_i-dependent activation, non-specific cation-permeability and non-permeability among anions, giving rise to the hypothesis that this current represents a [Ca²⁺]_i-dependent non-specific cation channel in human atrium.

4.1.3 Current–voltage relation of I_CAN
The current densities show a strong variability between different cells and caffeine failed to induce I_CAN in 43% of the cells tested. This may be the consequence of a different Ca²⁺ release capability of the SR due to the isolation procedure, patient's disease and medical treatment. These factors may also influence the caffeine-sensitivity and the function of the SR-ryanodine receptors. Furthermore, L-type-Ca²⁺ currents were very small in many cells (compare Figs. 1, 5 and 6), probably leading to small Ca²⁺-loading of the SR during the prepulses, which may be one reason for the remarkable inter-cell variability of current densities. Suppression of L-type-Ca²⁺ currents in whole cell experiments of human atrial myocytes is a well known feature [39–41, 55], which has been attributed to in vivo pathological processes and isolation procedure [55], patient's age [39], medical treatment [39–41]and atrial dilatation [41]. Ca²⁺-currents with measurable amplitudes were recorded in only 23% of the cells tested, using physiological (2 mM) Ca²⁺-concentrations [39, 41], whereas Escande et al. [55]observed this current in 80% of the cells, when using 2.7 mM [Ca²⁺]_o.

Chronic medical treatment, especially with calcium antagonists, seems to play an important role and is obviously responsible for blocking effects on the channel as well as for a downregulation of the channel receptors [39, 40]. However, in the present study we could demonstrate very large Ca²⁺ currents in all experiments, in which EGTA was present in the pipette solution (compare Fig. 2), which suggests a [Ca²⁺]_i-dependent inactivation of L-type currents. Furthermore, Ca²⁺ currents were more frequently observed, when (in different studies) Na⁺ was present in the extracellular solutions in physiological concentrations. Therefore, we believe that suppression of the L-type-Ca²⁺ currents in the present study is mainly due to high intracellular [Ca²⁺], which reflects altered [Ca²⁺]_i-handling mainly as a result of a decreased Ca²⁺-extrusion capability due to the inhibition of Na/Ca²⁺-exchange in our experiments.

Cardiac CAN-channels reveal an approximately ohmic conduction [9, 16]. In the present study, we performed voltage ramps to minimize the effect of repetitive caffeine applications on the [Ca²⁺]_i-homeostasis and, in consequence, variabilities of the [Ca²⁺]_i-transients. In the range –80 to +80 mV, I_CAN revealed an almost linear current–voltage relation. This behaviour was in accordance with the experiments with distinct voltages. The observed reversal potentials generally matched the calculated reversal potentials for monovalent cations, although in some registrations a small outward current could be observed at 0 mV, shifting the reversal potential slightly in the negative direction. This phenomenon may be due to insufficient equilibration of the cytosol with the pipette solution, although equilibration was allowed at least 2 min prior to each experiment. The limitations of the whole-cell patch-clamp technique in the control of intracellular ionic concentrations have been described in detail in a mathematical model [42]and in experimental studies [43, 44].

However, possible insufficient dialysis of the cell with K⁺-free solution would lead to a contamination of outward currents with K⁺.

4.1.4 Dissociation between [Ca²⁺]_i-signal and current
If we assume that the [Ca²⁺]_i-dependent non-specific cation channel in human atrial cardiomyocytes represents the CAN-channel type, a ligand-dependent operation mode must be expected, i.e. the current is expected to be a linear function of [Ca²⁺]_i. Surprisingly, a dissociation of the time courses of the [Ca²⁺]_i-transient and the current could be observed: the current shows a fast upstroke and reaches a plateau, when [Ca²⁺]_i still continues to increase. The main body of the inactivation of the whole-cell current coincides with the plateau phase of the [Ca²⁺]_i signal. This behaviour suggests that inactivation depends on the change of [Ca²⁺]_i ([Ca²⁺]_i/t) rather than on absolute [Ca²⁺]_i and possibly a faster Ca²⁺-release near the sarcolemma.

Dissociation in the time course of [Ca²⁺]_i-dependent whole-cell currents and [Ca²⁺]_i signals have been described in studies of the [Ca²⁺]_i-dependent chloride-channel in rabbit Purkinje cells [20], the [Ca²⁺]_i-dependent K⁺-channel in smooth muscle cells [30]and the Na⁺/Ca²⁺-exchanger in ventricular rat and atrial guinea-pig cardiomyocytes [31, 45]. In these studies, temporal discrepancies between current and [Ca²⁺]_i-signal have been attributed to or at least suggested to be due to subsarcolemmal [Ca²⁺]_i-gradients. Therefore, these currents reflect the subsarcolemmal Ca²⁺-concentration ([Ca²⁺]_sl) rather than bulk Ca²⁺ and are used in some of these studies as an indicator for [Ca²⁺]_sl. It could be demonstrated, that subsarcolemmal Ca²⁺-gradients may also be present in atrial myocytes, which lack a well developed T-tubular system [45]. In human atrial myocytes, a peripheral SR system could be identified, which may form a functional unit with the sarcolemmal dihydropyridine-receptors [46].

Alternative hypotheses for the current/[Ca²⁺]_i-dissociation include a simple blockade of the channel by Ca²⁺-ions, as described for [Ca²⁺]_i-dependent K⁺-channels in skeletal muscle cells [47]or discrepancies between bulk and subsarcolemmal concentrations of caffeine, due to diffusional processes near the sarcolemma. However, these questions require further studies.

4.1.5 Relevance
The physiological role of I_CAN remains unclear, but it can be assumed that it provides an arrhythmogenic mechanism in situations of [Ca²⁺]_i-overload and may be involved in the generation of the transient inward current I_TI and delayed afterdepolarizations. The clinical relevance of such afterdepolarizations could be demonstrated in vivo recently, giving rise to the hypothesis that they are one possible mechanism for the initiation of sustained atrial non-reentry tachycardia in humans [48]. The electrophysiological mechanisms of atrial fibrillation and flutter are very complex and reentry seems to play a major role [49]. Nevertheless, triggered activity caused by delayed afterdepolarizations may be an appropriate substrate for initiation of atrial fibrillation/flutter since it may favour reentry-mechanisms by increasing dispersion of refractoriness in the tissue.

The present concept of the contribution of [Ca²⁺]_i-dependent non-specific cation channels to cardiac arrhythmias was established due to their capability to activate during oscillatory SR-Ca²⁺ release, giving rise to depolarizing membrane currents, such as the transient inward current I_TI. However, this feature has not been described for human cardiomyocytes yet. Bènardeau et al. observed delayed afterdepolarizations in human atrial myocytes, which could be suppressed by substitution of external Na⁺ by Li⁺ [11]. It was concluded that delayed afterdepolarizations in human atrial myocytes are mainly carried by the Na⁺/Ca²⁺ exchanger. Although the present study was not designed to examine the contribution of I_CAN to delayed afterdepolarizations, it must be emphasised that part of the cells developed spontaneous membrane currents in the absence of Na⁺ and K⁺, i.e. in the absence of currents generated by the Na⁺/Ca²⁺ exchanger or [Ca²⁺]_i-dependent K⁺-channels (not shown), suggesting a contribution of this current at least under pathophysiological conditions.

No profound differences concerning the presence or the magnitude of the current were found according to the pathophysiological conditions or drug treatment. The number of patients with atrial fibrillation (2) was too small to expect any statistical significance with respect to the variability of the current.

4.2 Human ventricular cardiomyocytes
We were unable to identify [Ca²⁺]_i-dependent membrane currents in the absence of Na⁺ and K⁺ in human ventricular myocytes. An insufficient load of the SR with Ca²⁺ can be excluded, since all cells showed a strong contraction after caffeine application and currents were not present even in those cells in which SR-Ca²⁺ release could be demonstrated by fura-2-fluorescence. Furthermore, caffeine in concentrations of 10 mM are believed to be sufficient to induce a complete SR-Ca²⁺ release [50]. Therefore we conclude that neither a [Ca²⁺]_i-dependent chloride-, nor a non-specific cation channel is expressed in these cells. Cells were isolated from the midmyocardial region (M-region), according to the suspected region for the generation of delayed afterdepolarizations [51]. However, that the [Ca²⁺]_i-dependent non-specific cation channel may be present in cells from other regions, such as the subepicardial or subendocardial layer, cannot be excluded.

The results of the present study suggest that neither a [Ca²⁺]_i-dependent chloride-, nor a non-specific cation-channel is expressed in human ventricular myocardium. Therefore, we conclude that arrhythmogenic currents in the presence of [Ca²⁺]_i-overload, which are associated with the transient inward current I_TI and delayed afterdepolarizations, might be carried by the Na⁺/Ca²⁺-exchanger alone. However, this hypothesis is based on indirect evidence, i.e. the absence of [Ca²⁺]_i-dependent currents in the absence of Na⁺ and K⁺ and should be confirmed by further studies.

Time for primary review 24 days.

	Acknowledgements

 
This work was supported by the Deutsche Forschungsgemeinschaft (Be 1113/2-3) and the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (01K59502, ZMMK Projekt 4). The visit of Dr. Szigeti was financed by a Hungarian grant (ETT T-06424/93). The expert technical assistance of Iris Beckmann is gratefully acknowledged. Our special thanks are to Prof. E. R. de Vivie (Dept. of Cardiac Surgery, University of Cologne) for providing the myocardial tissue.

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