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Cardiovascular Research 1998 38(3):788-801; doi:10.1016/S0008-6363(98)00047-9
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Characterization of the hyperpolarization-activated inward current in isolated human atrial myocytes

Uta C Hoppe* and Dirk J Beuckelmann

Department of Medicine III, University of Cologne, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany

* Corresponding author. Tel.: +49 (221) 478-3138; Fax: +49 (221) 478-3163.

Received 17 November 1997; accepted 22 January 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The hyperpolarization-activated inward current (If) has been discussed to contribute to arrhythmias in rat hypertrophied and human failing ventricular myocardium. Cat atrial myocytes were found to exhibit variable size of If. In the present study, we evaluate characteristics of If in human atrial myocardium and investigate if human atria might exhibit any electrophysiological heterogeneity in the diastolic voltage range. Methods and results: The whole-cell patch-clamp technique was used to record If in isolated myocytes from 96 human right atrial appendages. If was observed in 95% (ruptured-patch; 141/146) to 100% (perforated-patch; 18/18) of myocytes showing typical current properties, i.e. time- and voltage-dependence, block by [Cs+]o, permeability for K+, Na+ and Li+, and current increase with raising [K+]o. Using the perforated-patch technique Boltzmann distributions yielded an activation threshold of –60 to –70 mV and half maximal activation at –89.3±0.7 mV (n=18). Isoproterenol (10–6 mol/l) shifted If activation by +7 mV (7/7) using the perforated-patch technique, but only inconsistently shifted If activation using the ruptured-patch method (6/21). Based on the relative current size of If and IK1 three cell types could be distinguished (n=26). In myocytes with a prominent If, If density was found to be larger (in [K+]o 25 mmol/l at –80 mV: –0.78±0.23 pApF–1; n=7) than in cells with predominant IK1 (in [K+]o 25 mmol/l at –80 mV: –0.02±0.01 pApF–1; n=4) (P<0.05). Conclusions: If is present in most human atrial myocytes. Many current properties are similar to those described for If in mammalian pacemaker cells. The relative current size of If and IK1 were found to be variable in different myocytes. Whether If may favor spontaneous diastolic depolarization in individual human atrial myocytes exhibiting predominantly If in vivo remains to be defined, as current size is very small under physiological [K+]o.

KEYWORDS Ion channel; Pacemaker current; Patch clamp; Human atrial myocyte


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Atrial tachyarrhythmias occur most commonly in patients with coronary artery disease or cor pulmonale, but may also occur in patients without any known structural heart disease [1, 2]. Symptoms may reach from discrete palpitations to severe tachycardia-induced cardiomyopathy [3]. In addition to macro- and micro-reentry mechanisms and triggered activity, atrial tachyarrhythmias may be caused by ectopic automaticity and in some patients have been treated effectively by radiofrequency ablation of the abnormal atrial focus [3–7]. However, the underlying ionic nature and membrane currents responsible for such ectopic spontaneous diastolic depolarizations are still unclear.

Escande et al. [8]observed spontaneous pacemaker activity in multicellular preparations of partially depolarized human atrial fibers which were mainly (in 90%) obtained from patients with dilated atria. Since the addition of external cesium prolonged the cycle length of these atrial fibers, it was speculated that the hyperpolarization-activated inward current If might play a role in abnormal diastolic depolarizations of human atrial myocardium. The pacemaker current If is a time- and voltage-dependent non-selective cation inward current, that is typically blocked by external cesium. In sinus node and Purkinje cells, If is considered to be the major current underlying the spontaneous diastolic depolarization phase [9–16].

An If-like current has also been recorded in mammalian (guinea pigs [17], dogs [17, 18], rats [19, 20]and embryonic chicken [21]) and human [22, 23]ventricular myocytes. In spontaneously hypertensive rats, If density was linearly related to the severity of cardiac hypertrophy and was found to be significantly larger than in undiseased control animals [20]. Thus, it was supposed, that an overexpression of If might contribute to the increased propensity of arrhythmias in hypertrophied rat ventricular myocardium [20]. In human ventricular myocytes of patients with end-stage heart failure, we previously observed a trend towards increased If densities compared to non-failing controls [22]. Together with an elevated plasma norepinephrine concentration, [24]diastolic calcium overload [25–27]and a reduction of IK1 in human heart failure [28], If may also favor diastolic depolarizations in individual human myopathic cells.

In atrial tissue of several mammalian species the existence of If has been demonstrated in isolated cells of cats [29–31], rabbits [32], and embryonic chickens [21]and in multicellular preparations of sheeps [33]. In cat atrial myocardium, which included latent pacemaker regions, different cell types were identified based on the relative magnitude of ionic currents elicited during hyperpolarization (If and IK1) [31]. Myocytes exhibiting predominantly an If current were thought to function as subsidiary pacemakers [31].

In previous studies of human atrial myocardium, If has been recorded in multicellular preparations of atrial appendage fibers [34]and inconsistently in isolated myocytes [35–37], with some investigators not recording any If current at potentials as negative as –120 mV [38]. Until now, only limited data concerning current characteristics of If in human atrial myocardium are available. Since current properties cannot readily be extrapolated from results obtained from animal experiments, the aim of the present study was to investigate the biophysiological characteristics of If in human atrial myocytes and the effects of β-adrenergic stimulation. In addition to a very recently reported study of If in human atria [39], we included current activation kinetics and more detailed investigations of ionic selectivity. To further elucidate a possible functional role of If in human atrium, we investigated, whether there might be any electrophysiological heterogeneity in the diastolic voltage range of human atrial myocardium, that was not obtained from latent pacemaker regions.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Patients
Cells were prepared from small segments of right atrial appendages of 96 patients who underwent coronary bypass surgery (n=80), aortic valve replacement (n=9), mitral valve replacement (n=4) or closure of an interatrial (n=1) or interventricular (n=2) septal defect. Patients' mean age was 60.4±1.2 years; 75 patients were male, 21 were female. 7 patients had atrial fibrillation, the other patients were in sinus rhythm. Digitalis, β-adrenoceptor blocking drugs, Ca2+-antagonists or diuretics were taken by 10, 34, 16 and 22 patients, respectively. Four patients received antiarrhythmic agents (two propafenone, one sotalol, one amiodarone). All patients gave informed consent prior to surgery. The study was approved by the ethical committee of the university. The investigation conforms with the principles outlined in the Declaration of Helsinki [40].

2.2 Cell isolation
Small segments of myocardium sampled from the tip of right atrial appendages were obtained at the time of open heart surgery. Samples were immersed in Bretschneider's cardioplegic solution (mmol/l: NaCl 15, KCl 9, K-H-2-ketoglutarate 1, MgCl 4xH2O 6, histidinexHClxH2O 18, histidine 180, tryptophane 2, mannitol 30, CaCl2 0.015), maintained at 4°C and immediately taken to the laboratory for cell isolation.

Myocytes were isolated using a modification of the procedure described by Bustamante et al. [41]. Specimens were chopped with scissors into cubic chunks (<1 mm3) and were placed in a 25-ml flask containing nominally Ca2+-free modified Tyrode's solution (mmol/l: NaCl 135, KCl 4, MgCl2 1, glucose 10, NaH2PO4 0.33, and HEPES 10; pH 7.3 with addition of NaOH, 37°C). Continuous agitation of the chunks was ensured by oxygenation of the solution and by magnetic stirring at about 10 Hz. Replacement of this solution was performed three times and chunks were incubated for 4 min each time to wash out blood and extracellular Ca2+. Thereafter, the chunks were incubated in 10 ml of a similar solution to which collagenase (type II, 400 IU/ml; Sigma, St. Louis, MO) and protease (type XIV, 0.7 IU/ml; Sigma, St. Louis, MO) were added. The supernatant was removed after 30 min of incubation by centrifugation (100xg for 1–2 min) and discarded. After centrifugation, chunks were incubated in 10 ml of modified Ca2+-free Tyrode's solution supplemented with collagenase alone (type II, 400 IU/ml; Sigma, St. Louis, MO). The second supernatant was again removed by centrifugation after 45 min of incubation and discarded. Tissue and cells were then disaggregated in 15 ml modified Ca2+-free Tyrode's solution. After filtering through a nylon mesh cells were allowed to settle for 30 min. Afterwards Ca2+ was added to a final concentration of 2 mmol/l. Myocytes were stored in this solution at room temperature.

The living-cell yield was approximately 5–10%. Only cells with clear striation without significant granulation were selected for experiments. In the Tyrode's solution, that was used to store myocytes, we infrequently observed myocytes with slow spontaneous contractions. We were not able to patch these spontaneously contracting human atrial myocytes. Therefore, we did not measure any spontaneously contracting cells, although we were investigating the pacemaker current. The resting membrane potential was found to be variable in human atrial myocytes. In atrial myocytes with stable resting membrane potentials, it was –76.1±0.5 mV (n=15). However, action potentials were difficult to record, as many cells did not repolarize after initiation of current clamp. A total of 308 cells yielded results for these experiments; mean cell capacity was 100.5±2.5 pF.

2.3 Solutions
Cells were superfused with a ‘standard’ Tyrode's solution containing (mmol/l) CaCl2 2.0, NaCl 115, KCl 25, MgCl2 1, BaCl2 8 (unless indicated), CdCl2 0.3, 4-aminopyridine 3, HEPES–NaOH 10; pH was adjusted to 7.3 with NaOH. Ba2+ was used to block the inward rectifier potassium current (IK1), which activates in the same voltage range as If, 4-aminopyridine (4-AP) was added to block the transient outward current (Ito1). The slow inward calcium current (ICa), which would interfere with tail current measurements and might activate calcium-dependent currents was blocked by the addition of Cd2+ to the extracellular solution.

When [K+]o was varied in the external solution (0, 5, 140 mmol/l), NaCl was adjusted equimolarly. For determination of the extracellular effects of Na+ and Li+, NaCl was replaced with equimolar choline chloride (ChCl) or LiCl; pH was adjusted to 7.3 with the addition of NaOH or KOH where appropriate. In some experiments, atropine 1 µmol/l was added to the ChCl-solution to inhibit any possible potassium currents activated by choline or 4-AP [42, 43].

In some experiments CsCl (concentration as indicated) was added to the external solution to investigate whether Cs+ would block the hyperpolarization-activated inward current. In some experiments, isoproterenol at a concentration of 10–6 mol/l (Sigma, St. Louis, MO) was added to the standard Tyrode's solution to evaluate the effect of β-adrenergic stimulation.

The micropipette electrode solution contained: (mmol/l) KCl 140, MgCl2 1, HEPES–KOH 10, EGTA 5, and Mg–ATP 5; pH was adjusted to 7.2 with KOH. In some experiments, 140 mmol/l KCl was substituted with potassium glutamate 120 mmol/l and KCl 20 mmol/l. In all perforated-patch experiments, the potassium glutamate solution was used and NaCl 8 mmol/l was added to avoid the development of a Donnan potential and the loss of internal Ca2+ through the INaCa, respectively.

2.4 Recording techniques
Experiments were carried out using standard microelectrode ruptured whole-cell patch-clamp techniques [44](unless indicated) with an amplifier PC 501 (Warner Instrument, Hamden USA). Microelectrodes were pulled from borosilicate glass and had tip resistances of 3–5 M{Omega} when filled with the KCl 140 mmol/l solution. Some experiments were performed using the perforated-patch technique (as indicated) developed by Horn and Marty [45]and modified by Rae et al. [46]. The initial 200–500 µm of the pipette tip were filled by suction with the regular pipette solution and the rest was back-filled with pipette solution containing amphotericin B (240 µmol/l), which was prepared just before the experiment. Using this technique, the access resistance stabilized at 6–12 M{Omega} within 6–12 min after forming a G{Omega} seal. Experiments were performed at a temperature of 37±0.5°C. Voltage recordings were corrected for the liquid junction potentials (range –14.8 mV for the solutions containing K+-glutamate in the pipette and [K+]o 5 mmol/l to –1.1 mV for the solutions containing KCl 140 mmol/l in the pipette and [K+]o 140 mmol/l) [47].

Analog filtering of current recordings was done at 3 kHz. Currents were digitized and stored for off-line analysis (pclamp, version 5.5 or 6.0, axon instruments). Cell capacitance was calculated by applying hyperpolarizing 10-mV steps from a holding potential of –80 mV and integrating the current required to charge the membrane when stepping back to –80 mV.

2.5 Data analysis
The size of the hyperpolarization-activated inward current was measured as the difference between the instantaneous current at the beginning of the hyperpolarizing step and the steady-state current at the end of hyperpolarization [19]. Currents were normalized to membrane capacitance to calculate current densities when indicated. Specific conductance of If was determined for each cell according to the equation g=I/(Vm–Vrev), where g is the conductance calculated at the membrane potential Vm, I the current amplitude, and Vrev is calculated from the analysis of tail currents. For the calculation of steady-state activation curves, specific current conductances were normalized to the maximal current conductance to give g/gmax. Boltzmann distributions were fitted to these normalized values: g/gmax=1/{1+exp[(V1/2–Vm)/S]}, where Vm is the membrane voltage, V1/2 is the voltage at half-maximal activation, and S a slope factor at Vm=V1/2. Tail current amplitudes used to evaluate current reversal were measured as the difference between peak current (10 ms after depolarization to omit possible interference of sodium or capacitance currents) and maintained current at the end of the clamp pulse. The relative permeabilities of potassium and sodium (PNa/K) were estimated by fitting the Goldman–Hodgkin–Katz equation [48]to the reversal potential (Vrev): Vrev=58xlog {(PNa/Kx[Na+]o+[K+]o)/(PNa/Kx[Na+]i+[K+]i)}. Kinetics of current activation were calculated using a non-linear least-squares techniques (clampfit, pclamp, version 5.5 or 6.0, axon instruments). Data are presented as mean±standard error of the mean (s.e.m.) when appropriate. The Mann–Whitney non-parametric test and one-way ANOVA for repeated measurements were used for statistical evaluations, and values of P<0.05 were considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Voltage dependence of a hyperpolarization-activated inward current
A family of hyperpolarization steps in 10-mV increments from a holding potential of –40 mV elicited a time-dependent inward current, that increased with more negative potentials. Original current recordings of a single human atrial myocyte (perforated-patch technique) in standard Tyrode's solution containing [K+]o 25 mmol/l are shown in Fig. 1a. Following hyperpolarization cells were depolarized to +20 mV to achieve fast current deactivation. Using the ruptured-patch technique, a hyperpolarization-activated inward current could be recorded in 95% (141 of 148) of the cells investigated. Current activation occurred first at a membrane potential of approximately –80 mV in most of these myocytes. Mean current densities recorded at –130 and –150 mV in these 141 cells were –2.6±0.2 and –4.0±0.2 pApF–1, respectively. Additionally, we investigated 18 atrial myocytes under more physiological intracellular conditions using the perforated-patch method. A hyperpolarization-activated inward current could be elicited in all of these 18 cells. Mean current densities at –80, –130 and –150 mV were –0.7±0.1, –3.8±0.4 and –4.6±0.4 pApF–1, respectively. A Boltzmann distribution, which was fitted to normalized current conductances over a voltage range from –50 to –150 mV, yielded an activation threshold between –60 and –70 mV, a half maximal activation of –89.3±0.7 mV and a slope factor of –12.7±0.7 mV (Fig. 1b). Using the ruptured-patch technique we obtained a variable time-dependent current ‘run-down’ in some cells (–26.6±6.7% at –130 mV after 5 min; n=10), that was not observed using the perforated-patch technique (–2.6±0.9% at –130 mV after 5 min; n=4).


Figure 1
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  		Fig. 1 Voltage- and time-dependence of the hyperpolarization-activated inward current. (a) Original current recordings show the voltage- and time-dependence of the hyperpolarization-activated inward current in a single human atrial myocyte (perforated-patch technique). Hyperpolarization steps in 10-mV increments were applied from a holding potential of –40 mV. External solution contained (mmol/l) KCl 25, NaCl 115, BaCl2 8, CdCl2 0.3, and 4-aminopyridine 3. (b) The activation curve was calculated by fitting a Boltzmann distribution to normalized current conductances of 18 cells investigated with the permeabilized-patch method over a voltage range of –50 to –150 mV. First current activation occurred between –60 and –70 mV, half-maximal activation and slope factor were –89.3±0.7 mV and –12.7±0.7 mV–1, respectively.

 
We did not obtain any significant influence on If densities (at –130 mV) by patients' age (correlation coefficient r=0.18), gender (male –2.6±1.9 vs. female –2.4±1.5 pA/pF; P=0.97), type of operation (bypass surgery –2.5±1.9 pA/pF vs. valve replacement or septal closure –3.1±1.7 pA/pF; P=0.76) or the presence (–2.5±1.2 pA/pF)/ absence (–2.6±1.8 pA/pF) of atrial fibrillation (P=0.92).

3.2 Effect of extracellular potassium
To determine the effect of extracellular potassium the external K+-concentration was altered and [Na+]o was equimolarly adjusted. In [K+]o-free solution, no time-dependent inward current was observed upon hyperpolarization (n=26). However, similar to rabbit sino-atrial node cells [49]tail currents were present, indicating that current activation occurred (Fig. 2A). Fig. 2 depicts typical current traces from an experiment, where the external K+-concentration was elevated from 0 to 5, 25 and 140 mmol/l (note different scaling) showing that the amplitude of the inward current increased with raising [K+]o. Consistent with observations in rabbit sino-atrial node cells [11, 49]and cat latent pacemaker cells [29]the amplitude of outward If tail currents at our test voltage was nearly unchanged at different [K+]o. Mean current density at –130 mV increased from –0.8±0.9 pApF–1 in 5 mmol/l [K+]o to –2.6±0.8 pApF–1 and –4.9±1.0 pApF–1 in 25 mmol/l [K+]o and 140 mmol/l [K+]o (n=9), respectively. Current density at physiological [K+]o was found to be much smaller than has been reported for mammalian pacemaker cells [11, 49].


Figure 2
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  		Fig. 2 Effect of extracellular potassium on current size. Original current recordings of a single cell superfused with [K+]o 0 mmol/l (A), followed by [K+]o 5 mmol/l (B), [K+]o 25 mmol/l (C) and 140 mmol/l (note different scaling) (D). In K+-free solution no time-dependent inward current was observed. With increasing [K+]o mean current density at –130 mV increased from –0.8±0.9 pApF–1 in 5 mmol/l [K+]o to –2.6±0.8 and –4.9±1.0 pApF–1 in 25 mmol/l [K+]o and 140 mmol/l [K+]o (n=9), respectively.

 
3.3 Ionic selectivity
Measurements of tail currents were used to evaluate the reversal potentials of the hyperpolarization-activated inward current. Tail currents, following a hyperpolarizing step to –120 mV, were elicited by 10-mV depolarization steps (Fig. 3). Tail current amplitudes, normalized to membrane capacitance, obtained from 12 cells superfused with 25 mmol/l [K+]o are plotted as a function of tail step potentials in the I–V relationship shown in Fig. 3b. Best fit through data points gave a linear relationship with a reversal potential of –15.3±0.3 mV. This reversal potential was more positive than would be expected for a pure potassium conductance, indicating a permeability for ions other than potassium. The relative permeability of potassium and sodium (PNa/K) was estimated by fitting the Goldman–Hodgkin–Katz equation [48]to the reversal potential (Vrev). PNa/K ratio calculated for a [Na+]i range of 1–10 mmol/l was 0.45–0.47. Using the perforated-patch method a reversal potential of –14.7±0.6 mV (n=4) was obtained in [K+]o 25 mmol/l, yielding a relative permeability ratio PNa/K of 0.48 for a [Na+]i of 8 mmol/l.


Figure 3
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  		Fig. 3 Current reversal at different extracellular potassium concentrations. (a,b) [K+]o 25 mmol/l and [Na+]o 115 mmol/l. (c,d) [K+]o 140 mmol/l and [Na+]o 0 mmol/l. Following hyperpolarization to –120 mV for 750 ms tail currents were elicited by 10-mV depolarization steps (a,c). Original current recordings (a,c) and I–V relationships (b,d) of normalized tail current amplitudes yielded reversal potentials of –15.3±0.3 and 2.3±0.1 mV in [K+]o 25 mmol/l (n=12) and [K+]o 140 mmol/l (n=11), respectively.

 
To further evaluate the ionic selectivity external NaCl was replaced with equimolar amounts of KCl, ChCl or LiCl. Reversal potentials measured in these [Na+]o-free solutions at various levels of [K+]o are shown in Table 1. Current reversal in the presence of extracellular Li+ indicated a permeability of this cation in addition to potassium. The relative permeability ratio PLi/K calculated from these data was 0.1. The shift of the reversal potential in ChCl for a change of [K+]o from 25 to 50 and to 140 mmol/l (Fig. 3c/d) was close to what had been predicted for a pure potassium conductance under these experimental conditions. Thus, a permeability of any additional extra- or intracellular ions seemed unlikely.


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  		Table 1 Effects of extracellular cations on current reversal in Na+-free solutions

 
Since choline and 4-AP were found to exhibit muscarinic effects activating potassium currents in dog [42]and cat [43]atrial myocytes, respectively, reversal potentials in ChCl (90 mmol/l) solution were also recorded in the presence of external atropine 1 µmol/l (n=4) (Table 1). However, current reversal obtained in the presence and absence of atropine was not significantly different, and atropine application did not result in any shift of the holding current, indicating that no additional potassium conductance was activated under our experimental conditions.

3.4 Effects of extracellular cesium
The extracellular addition of Cs+ 10 mmol/l suppressed the time-dependent inward current in all cells investigated ([K+]o 25 mmol/l, n=19; [K+]o 140 mmol/l, n=11). Since [Cs+]o 10 mmol/l might also inhibit other potassium currents [31], we also evaluated the effect of a lower [Cs+]o concentration (2 mmol/l). Fig. 4 depicts typical current traces of a single atrial myocyte in [K+]o 25 mmol/l before (A) and during (B) the exposure to external Cs+ 2 mmol/l. [Cs+]o 2 mmol/l also suppressed the time-dependent inward current (n=5). Similar to sino-atrial node cells [11, 50], latent pacemaker cells [29]and ventricular myocytes [19]extracellular Cs+ did not abolish outward tail currents. The Cs+-sensitive current was obtained by digital subtraction of currents in the presence from those in the absence of [Cs+]o (Fig. 4C). The current–voltage relationship of the hyperpolarization-activated inward current and of the Cs+-sensitive current were virtually superimposable (Fig. 4D). The Cs+-dependent block was partially reversible upon removal of [Cs+]o. The reversal potential was unchanged before (–14.5±0.5 mV) and after (–14.8±0.6 mV) [Cs+]o application (n=3) (not shown).


Figure 4
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  		Fig. 4 Effects of external Cs+ on the hyperpolarization-activated inward current. Original current traces of a single cell hyperpolarized in [K+]o 25 mmol/l before (A) and during (B) the addition of external Cs+ 2 mmol/l and corresponding I–V relationship (D) demonstrate the Cs+-induced block of the inward current. (C) Superimposed traces of the Cs+-sensitive current were obtained by digital subtraction of current traces recorded in the absence and presence of [Cs+]o.

 
3.5 Activation kinetics
Activation kinetics of the original current and of the Cs+-sensitive current were both analyzed. In some cells, If activation exhibited a short delay in onset resulting in a sigmoidal time course, as previously described for sino-atrial node cells [11, 14], Purkinje fibers/cells [12, 51, 52], latent pacemaker cells [29]and ventricular myocytes [18]. Time constants of activation were determined by mono-exponential fits. In cases of sigmoidal time course, the initial ‘delay’ of activation was omitted by extrapolating the exponential function to the onset of the hyperpolarization step as demonstrated in Fig. 5A [12, 18, 51, 52]. In Fig. 5B, {tau}–voltage relationships obtained from 24 cells in [K+]o 25 mmol/l clearly demonstrate that activation got progressively faster with more negative voltages, and that time constants before the addition of [Cs+]o and of the Cs+-sensitive current were similar. Mean {tau}-values varied from approximately 100 ms at –150 mV to several seconds at –80 mV.


Figure 5
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  		Fig. 5 Activation kinetics fitted by mono-exponential functions in [K+]o 25 mmol/l before the addition of external Cs+ and of the Cs+-sensitive current. (A) Since If activation exhibited a sigmoidal time course in some cells, exponential fits were extrapolated to the onset of hyperpolarization omitting the initial ‘delay’ of activation. (B) {tau}–voltage relationship obtained from 24 cells in [K+]o 25 mmol/l clearly demonstrate that activation became progressively faster with more negative voltages, and that time constants before the addition of [Cs+]o and of the Cs+-sensitive current were similar.

 
3.6 Effect of β-adrenergic stimulation
To evaluate the effect of β-adrenergic stimulation cells were first investigated in standard Tyrode's solution. Afterwards the same protocol was applied when myocytes were superfused with the same solution to which isoproterenol 10–6 mol/l had been added. Using the perforated-patch technique, isoproterenol was found to increase current size by shifting current activation to more positive potentials without a change of maximal current amplitude and to accelerate current activation (7/7 cells) (Fig. 6A). Activation curves before and during isoproterenol application calculated by Boltzmann fits of normalized current conductances showed, that isoproterenol 10–6 mol/l shifted the potential of half maximal activation by approximately +7 mV (from –88.2±0.9 to –81.4±0.4 mV; n=7; P<0.05) (Fig. 6B). There was no difference of the current reversal potential under both experimental conditions (n=3) (not shown). Using the ruptured-patch method, a shift of current activation was observed inconsistently (6 of 21 cells).


Figure 6
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  		Fig. 6 Effect of β-adrenergic stimulation. (A) Superimposed original current recordings of a single atrial myocyte (perforated-patch) in ‘standard’ Tyrode's solution (control) and in the same solution to which isoproterenol 10–6 mol/l (Iso) had been added. Isoproterenol led to a shift of current activation to more positive potentials without a change of maximal current amplitude and accelerated current activation. (B) Activation curves calculated by Boltzmann fits of normalized current conductances of 7 myocytes using the permeabilized-patch method showed, that isoproterenol (10–6 mol/l) shifted the potential of half maximal activation by approximately +7 mV (from –88.2±0.9 to –81.4±0.4 mV; n=7; P<0.05).

 
3.7 If versus IK1
To estimate a possible functional significance of If and to investigate, whether human atrial myocytes might exhibit a heterogeneity of ionic currents in the diastolic voltage range, we recorded If and IK1 consecutively in 26 cells. Myocytes were first hyperpolarized in the absence and afterwards in the presence of external Ba2+ 8 mmol/l. IK1 amplitude was measured as the instantaneous inward current of the Ba2+-sensitive current. Relative current densities of If compared to IK1 varied considerably, both between cells from different preparations and between myocytes from individual patients. Some myocytes (n=7) exhibited predominantly an If current (–80 mV, –0.78±0.23 pApF–1; –120 mV, –3.15±0.53 pApF–1) and no obvious or only minor IK1 currents (–80 mV, –0.14±0.06 pApF–1; –120 mV, –0.23±0.09 pApF–1) (‘type 1’) (Fig. 7A). Similar to rabbit sino-atrial node cells [53]the addition of external barium did not shift the mid-activation potential of If significantly in these cells (–90.5±0.6 mV vs. –93.6±0.7 mV in the absence and presence of [Ba2+]o, respectively). In 4 myocytes, we recorded large IK1 currents (–80 mV, –1.31±0.41 pApF–1; –120 mV, –2.18±0.39 pApF–1) compared to If (–80 mV, –0.02±0.01 pApF–1; –120 mV, –0.90±0.22 pApF–1) (‘type 2') (Fig. 7B). If size in cells with minor IK1 was found to be significantly larger than in myocytes with predominant IK1 (P<0.05), although this result has to be judged with the limitation, that experiments were performed using the ruptured patch technique. Cell size was not found to be significantly different between the two cell types (type 1, 82.7±4.3 pF; type 2, 90.6±3.6 pF). In the remaining 15 cells, mean current densities of If at –80 mV were smaller than IK1 densities (–0.05±0.03 pApF–1 vs. –0.48±0.09 pApF–1), whereas at more negative potentials If was larger than IK1 (at –120 mV –1.62±0.25 pApF–1 vs. –1.20±0.21 pApF–1) (‘type 3'). These three categories have no marked transition (Fig. 8) and thus are arbitrary, but were proposed to distinguish mainly between the two extreme forms (types 1 and 2). At face value we were not able to predict which cells would exhibit predominantly If or IK1.


Figure 7
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  		Fig. 7 Relative current size of If and IK1. Original current recordings of two single human atrial myocytes obtained in ‘standard’ Tyrode's solution in the absence of external barium demonstrate clearly the variability of the relative current size of If and IK1 (note different cell size). The inset demonstrates the ‘instantaneous' current on expanded time scale. (A) A single human atrial myocyte (56.9 pF) exhibiting predominantly If and hardly any IK1. (B) Another human atrial myocyte (149 pF) investigated under the same experimental conditions showed large IK1 compared to If.

 

Figure 8
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  		Fig. 8 Data plots of If density vs. IK1 density measured consecutively in 26 atrial myocytes at –80 mV (A) and –120 mV (B). Some myocytes exhibit predominantly If and hardly any IK1, whereas other cells have a prominent IK1 and only minor If. Transitions between different cell categories are fluent.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
This report describes the presence of a hyperpolarization-activated inward current If in human atrial myocytes and evaluates its biophysiological properties. Upon hyperpolarization an If-like current could be recorded in most human atrial myocytes. Current activation was time-dependent and its amplitude increased with more negative potentials. Similar to rabbit sino-atrial node cells [54]no time-dependent inward current could be recorded upon hyperpolarization in K+-free external solution. With raising extracellular potassium-concentrations the amplitude of the inward current increased, which is consistent with results in mammalian pacemaker cells [11, 12, 14, 15], cat atrial [29]and mammalian [17, 19]and human [22]ventricular cells. Reversal potentials recorded in standard Tyrode's solution indicated a permeability for extracellular Na+ in addition to [K+]o. The permeability ratio PNa/K calculated from the reversal potential in [K+]o 25 mmol/l was 0.48, and, thus, was in the same range as reported for dog (0.6) [17], rat (0.34) [19], and human (0.4) [22]ventricular myocytes at similar external potassium concentrations.

Additionally, we could demonstrate a conductance of extracellular Li+, which has previously been demonstrated for rabbit sino-atrial node cells [15]. Li+-permeability was found to be lower than Na+-permeability. Current reversal potentials in [K+]o 140 mmol/l and in choline-containing solutions were close to the potassium equilibrium potential indicating, that under these experimental conditions there was only a conductance of potassium and not of any additional cation present in our solutions. These results also gave indirect evidence, that there was no permeability of any intracellular anion, like chloride. Since the current reversal and the holding current in choline-solution were unchanged after the addition of atropine (1 µmol/l), it was unlikely that the observed potassium conductance was due to K+-current activation by choline [42]or 4-AP [43].

Extracellular Cs+ at concentrations of 2 and 10 mmol/l both suppressed the time-dependent inward current in human atrial myocytes, but had only minor effects on If outward currents, as has been also observed in rabbit pacemaker cells [11–13, 15, 17, 50], cat atrial cells [29], and rat [19, 23]and human [22]ventricular myocytes.

Current activation got progressively faster at more negative potentials. In some cells, a sigmoidal time course of activation was seen, consistent with recordings of sino-atrial node cells [11, 14, 50], latent pacemaker cells [29]and ventricular myocytes [19]. The {tau}–voltage relationship varied as a function of voltage from approximately 100 ms to several seconds, which has also been described for Purkinje fibers [51]. Time constants before the addition of external Cs+ and of the Cs+-sensitive current were found to be similar. This indicated, that in human atrial myocytes [Cs+]o blocked If effectively, and that no additional time-dependent Cs+-insensitive current was activated upon hyperpolarization.

If is known to be stimulated by β-adrenoceptor agonists [55, 56]through a shift of current activation to more positive potentials (sino-atrial node cells +7 to +15 mV [11, 50], latent pacemaker cells +11 mV [30], rat [20]and human [22]ventricular myocytes +10 mV). Using the perforated-patch technique isoproterenol (10–6 mol/l) was found to shift If activation by +7 mV in human atrial myocytes without a change of maximal current size and to accelerate activation kinetics. Similar to observations in cat latent pacemaker cells [30], β-stimulating effects were obtained only inconsistently using the ruptured-patch method, which might have been caused by a loss of some cytosolic substances due to intracellular dialysis or by current ‘run-down’ [57].

Thus, most current characteristics of If in human atrial myocytes were found to be similar to sino-atrial node cells [11, 13–15, 50], Purkinje cells [12], atrial [29, 30, 32, 33], and ventricular [17–20]myocytes of various mammalian species and to human ventricular cells [22, 23]. These results are also in good agreement with observations of If in human atrial myocardium very recently reported by Porciatti et al. [39]. However, to estimate a possible physiological or pathophysiological significance of If in human atrial myocytes, its activation threshold and relative current size compared to other currents in the diastolic voltage range, especially IK1, have to be considered. Although If can be elicited at physiological potentials in both sinus node and Purkinje cells, it activates at more positive voltages in sino-atrial node preparations [11, 12, 14]. In ventricular myocytes of guinea–pigs, dogs and embryonic chickens If activation thresholds were found to be far more negative than the resting membrane potential [17, 18, 21]. Only in aged rat ventricular cells [19, 20]and human ventricular myocytes [22, 23]an If current could be recorded at potentials overlapping the diastolic voltage range.

In adult mammalian atrial myocardium first activation of If has been observed between –50 and –70 mV [29–31, 33], thus, within the range of the resting membrane potential. However, atrial tissue in these experiments included latent pacemaker regions (internodal pathways). In previous studies of human atrial myocardium obtained from right atrial appendages, activation threshold of If varied between –70 and –140 mV [34–37]. In some studies, If has been recorded only inconsistently [35–37]and Koumi et al. [38]could not find any If current at potentials as negative as –120 mV. These discrepancies might partially have been caused by different experimental conditions. In our experiments using the ruptured-patch method, If activated first at approximately –80 mV. However, under more physiological intracellular conditions using the permeabilized-patch technique, we observed If activation threshold in human atrial myocytes at more positive voltages, between –60 and –70 mV, thus, at potentials close to the resting membrane potential [58]. The difference in activation threshold under both experimental settings might in part have been due to the higher (physiological) internal Ca2+-concentration using the permeabilized-patch method compared to ruptured-patch experiments, that were performed with a pipette solution containing EGTA. Elevation of intracellular Ca2+ from pCa 10 to 7 has been reported to shift the If activation curve by +13 mV in rabbit sino-atrial node cells and to increase maximal current size [50]. Additionally, using the ruptured-patch technique a variable current ‘run-down’ might have led to an underestimation of first current activation [11].

We observed a considerable variability of If size from cell to cell. Since this variability was obtained both using the ruptured- and perforated-patch techniques, it cannot only be attributed to a loss of intracellular calcium [50]due to cell dialysis or to current ‘run-down’ [11]. Wu et al. [31]reported at least four different cell types in cat atrium based on the relative magnitude of If and IK1. Myocytes exhibiting predominantly IK1 or IK1 and If were found to be quiescent, whereas cells with a predominant If current showed spontaneous contractions, and are thought to function as subsidiary pacemakers. However, these authors obtained cells from whole cat atria after the excision of the sino-atrial node, thus, including internodal pathways. Internodal pathways are known to contain morphologically 6 different cell types including cells with ultrastructural characteristics of ‘sinoatrial-nodal' P-cells [59, 60]. In contrast, the tissue in our experiments was obtained from right atrial appendages, which are not contaminated by any specialized conducting pathways. However, similar to Heidbüchel et al. [61]we also observed a heterogeneity of ionic currents in the diastolic voltage range in this human atrial ‘working' myocardium. In myocytes exhibiting an IK1 current, Heidbüchel et al. [61]previously reported about a variable contribution of IK1 and If at voltages negative to –100 mV in different cells consistent with our cell types 2 and 3. Additionally, we observed few myocytes showing no or hardly any IK1 but exhibiting large If currents (type 1) similar to characteristics reported for P-cells. Since we were not able to patch any spontaneously contracting myocytes, we might even have underestimated If size. Basal potassium conductance in human atrial myocardium was found to be mainly due to IK1 channels [61]. Thus, in cells exhibiting no or only minor IK1, small changes in current might easily tip the balance towards depolarization. However, under physiological potassium concentration, especially close to the resting membrane potential, If was found to be very small, which makes it unlikely, that the pacemaker current If plays any physiological role in human atrial myocardium. In human ventricular myocytes from patients with end-stage heart failure, If tended to be larger than in undiseased controls [22]. However, it has yet to be defined, whether under some pathological conditions, such as abnormal automaticity, If is also modulated in atrial myocardium and, thus, with a further increase by β-adrenergic stimulation might favor spontaneous diastolic depolarization in individual human atrial myocytes. Therefore, further studies are necessary to clarify, if cells exhibiting predominantly an If current function as arrhythmogenic sites in human atrium in vivo.

Time for primary review 25 days.


   Acknowledgements
 
Special thanks are due to Prof. Dr. E.R. de Vivie and his colleagues (Department of Cardiac Surgery) for providing the myocardial tissue. The authors thank I. Beckmann for assistance with the cell isolation. Supported by the Deutsche Forschungsgemeinschaft (Be 1113/2-3) and the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (01 KS 9502; ZMMK Projekt 4).


   References
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 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
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