Skip Navigation

Cardiovascular Research 2004 64(2):250-259; doi:10.1016/j.cardiores.2004.07.001
© 2004 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Zorn-Pauly, K.
Right arrow Articles by Koidl, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zorn-Pauly, K.
Right arrow Articles by Koidl, B.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

If in left human atrium: a potential contributor to atrial ectopy

Klaus Zorn-Paulya, Peter Schaffera, Brigitte Pelzmanna, Petra Langa, Heinrich Mächlerb, Bruno Riglerb and Bernd Koidla,*

aInstitut für Medizinische Physik und Biophysik, Medizinische Universität Graz, Harrachgasse 21, A-8010 Graz, Austria
bUniversitätsklinik für Chirugie, Medizinische Universität Graz, Auenbruggerplatz 5, A-8036 Graz, Austria

* Corresponding author. Tel.: +43 316 380 4137; fax: +43 316 380 7746. Email address: bernd.koidl{at}meduni-graz.at

Received 11 March 2004; revised 30 June 2004; accepted 1 July 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The left human atrium plays an important role in initiation of atrial fibrillation (AF) and the hyperpolarization activated cation current (If) is a candidate for contributing to abnormal automaticity. However, electrophysiological data concerning If are not available in this cardiac region and we therefore investigated If in human left atrial tissue.

Methods: Human atrial myocytes were isolated from the left atrial appendage (LAA) and the left atrial wall (LAW) obtained from patients undergoing open heart surgery. If was measured with the whole-cell patch-clamp technique.

Results: If densities between –70 and –110 mV were found to be significantly higher in LAA than in LAW cells. Furthermore, in the group of LAA cells the half maximal activation potential (V1/2) was found to be less negative (V1/2 of –84.3±1.9 mV, n=14/9) compared to LAW cells (V1/2 of –97.8±2.1 mV, n=28/9). Beta-adrenergic receptor stimulation with isoproterenol (1 µM) caused an acceleration of current activation and a V1/2 shift to more positive potentials in cells of both regions (LAA: 8.8±2.3 mV, n=6/4 and LAW: 8.9±2.6 mV, n=6/4). Simulations using a mathematical model of the human atrial myocyte demonstrated that If was able to induce spontaneous activity in the model at a regular rhythm due to the interplay of If, Na+/Ca2+ exchange current and Ca2+ release of the sarcoplasmic reticulum (SR).

Conclusions: Our study revealed the presence of If in left atrial myocytes and showed that If parameters depend on atrial region. If current densities were sufficient to convert the mathematical model of a quiescent human atrial cell into a "pacemaker cell". These data support the hypothesis of If as a contributor to abnormal automaticity in human atrial tissue.

KEYWORDS Human left atrial myocytes; Patch clamp; Pacemaker current; Computer modeling; Atrial ectopy

This article is referred to in the Editorial by P. Kohl (pages 195–197) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The cardiac hyperpolarization activated cation current (If) is known to be present not only in regions with primary or secondary pacemaker activity, but also in non-pacemaking regions of atrium and ventricle. In pacemaking regions, If is believed to contribute to spontaneous diastolic depolarization [1,2]. The physiological relevance of If in the working myocardium is not clear, because the activation range is negative to the resting membrane potential and the current density is much smaller compared to sinus node [1,2]. However, it was speculated that If could elicit abnormal automaticity and thus play a role in atrial and ventricular arrhythmias [3–6]. This could be favoured by catecholamine release which is known to shift If activation to less hyperpolarized potentials and/or by a pathological upregulation of If with a concomitant downregulation of the inward rectifier current (IK1), which plays a major role in stabilizing the resting membrane potential [7]. Since atrial IK1 is of a several times smaller amplitude than ventricular IK1 [8,9], the arrhythmogenic potential of a depolarizing current, such as If, should be more pronounced in the atrium [10]. So far, electrophysiological investigations on human atrial If were restricted to the right atrium (right atrial appendage, RAA) [4,11], but the left atrium (LA) is known to play an important role for initiation and maintenance of atrial fibrillation (AF), the most common arrhythmia [12,13]. Furthermore, expression of cardiac ion channels depends on the cardiac region and can differ between right and left atrium as shown for IKr in dogs [14,15]. Therefore, it was the aim of our study to investigate If in two different regions of human LA using tissue from the left atrial appendage (LAA) and the left atrial wall (LAW).


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Patients
Small pieces (0.1–0.3 g; in case of LAA excision during ablation: ≤0.8 g) of left human atrium (LAA/LAW) were obtained from 18 (9/9) patients undergoing cardiac surgery due to mitral valve disease. They were transported to the laboratory (10–15 min) in saline [16] and immediately used for cell isolation. Most of the patients (16) showed a normal ejection fraction (EF>55), pulmonary artery pressure (PAP<35 mm Hg) and left atrial diameter (LAD<4 cm). The two remaining patients (chronic atrial fibrillation; LAW tissue) had end stage mitral valve disease (EF≤30, PAP≤46, LAD≤4.3). Patient's mean age was 67.1±2.7 years (68.3±4.1/65.8±3.9); 10 patients were female (5/5), 8 patients were male (4/4). Nine patients had chronic atrial fibrillation (4/5), two patients had paroxysmal atrial fibrillation (0/2) and all other patients were in sinus rhythm. The patients were treated with digoxin (4/3), beta-1-blockers (3/1), Ca2+ antagonists (1/0), a class III antiarrhythmic drug (0/1), ACE inhibitors (3/4) and diuretics (4/1). The investigation conforms to the principles outlined in the Declaration of Helsinki [17]. All patients gave informed consent and the use of human tissue was approved by the ethics committee of the Karl-Franzens-University.

2.2 Cell isolation and electrophysiological measurements
Atrial cells were isolated by enzymatic dissociation and mechanical disaggregation using trypsin (Sigma, MO, USA) and collagenase (Type 2, Worthington, NJ, USA) as described in detail previously [11]. The isolated myocytes were stored in cell culture medium (M-199, Sigma), supplemented with 50 µg/ml penicillin and 50 IU/ml streptomycin and were kept in an incubator at 37 °C. Experiments were performed 4–8 h after cell isolation.

Electrophysiological measurements were carried out at 36–37 °C. The external solution contained (in mM): NaCl 137, KCl 25, CaCl2 1.8, MgCl2 1.2, BaCl2 1, MnCl2 2, CdCl2 0.2, 4-aminopyridine 3, glucose 5, HEPES 5, adjusted to pH 7.35 with NaOH. The pipette solution contained (in mM): K+-aspartate 100, KCl 30, Na+-ATP 5, CaCl2 4, EGTA 11, HEPES 10, adjusted to pH 7.2 with KOH. Mn2+, Ba2+, Cd2+ and 4-aminopyridine were added to reduce the interference with potassium and calcium currents. Currents were measured in the whole-cell mode of the ruptured patch-clamp technique using a List L/M EPC-7 amplifier. Electrodes had resistances from 2 to 4 M{Omega} when filled with internal solution.

The amplitude of If was measured as the difference between instantaneous current at the beginning of the hyperpolarizing step and steady-state current at the end of the voltage clamp step and was normalized to cell membrane capacitance. Ionic conductance of If was determined for each cell according to the equation g=If/(Vm–Vrev), where g is the conductance calculated at the membrane potential Vm, If is the current amplitude, and Vrev the reversal potential (–13 mV as described for If in human right atrial myocytes for an external potassium concentration of 25 mM [11]). For calculation of steady-state activation curves, conductances were normalized to the maximal conductance. A Boltzmann equation was fitted to these normalized values.

The voltage-dependency of If activation/deactivation time constants were fitted according to the equation {tau}y=1/({alpha}0exp(–V/V0)+β0exp(V/V0)), whereby the parameters {alpha}0, β0 and V0 had to be determined ({alpha}0, β0 are opening and closing rates at zero voltage V0). Activation and deactivation time constants were calculated by fitting If recordings to a single exponential equation.

Results are presented as means±standard error of the mean (S.E.M.). Statistical significance was tested by Student's t-test and a value of P<0.05 was considered significant.

2.3 Human atrial single cell model
A modeling approach was used to estimate the role of If for eliciting spontaneous activity in the human atrium. Therefore, we used a modified version of a mathematical cell model based on human right atrial myocyte data developed by Nygren et al. [18]. We replaced the Na+ influx by sodium entry due to the stimulus current for the calculation of intracellular ionic concentrations [19]. Since Na+ current assignment produced the same results as the original model (after 20 s and 10 min of pacing) Na+ ions were used as stimulus carrier in all simulations.

If was included in the model according to the equation



Formula

where fNa=0.2677 (fK=1–fNa) and gf is the maximal conductance. The gating variable (y) was described by a differential equation of the form



Formula

where {tau}y is the time constant and y{infty} is the steady-state value of y.

Parameter values of the LAA cell that showed the most positive half-maximal activation voltage (V1/2)=If(LAA)single



Formula



Formula



Formula

and mean parameter values of LAA cells=If(LAA)mean



Formula



Formula



Formula

The atrial cell model was paced for 10 min (1 Hz) to achieve steady state for each scaling of gf and was then simulated for 10 min without stimulation in order to test for the occurrence of spontaneous action potentials (APs) in steady state. The differential equations system of the model was implemented on a personal computer in Simulink 5.0 and a variable step solver (ode 15 s) was used for numerical integration.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
The atrial myocytes used for this study were rod-shaped and showed clear cross striation. Cell membrane capacitance did not differ between the cells isolated from the two left atrial regions (101.9±9.1 for LAA, n=29/9 vs. 116.2±11.5 pF for LAW, n=29/9, P=0.33).

3.1 If occurrence
The presence of a hyperpolarization activated inward current (If) was determined in each cell by application of hyperpolarizing voltage steps from –40 to –140 mV in 10 mV steps of 3-s duration (holding potential=–40 mV). Fig. 1 shows current traces obtained in representative cells from LAA (A) and LAW (B). In both atrial regions, the voltage clamp protocol elicited a time-dependent inward current that increased in amplitude and activated more rapidly with progressively more negative test potentials. However, the current in the LAA cell activated at a less negative membrane potential (–70 mV) compared to the LAW cell (–80 mV) and showed a greater density at –140 mV (–3.9 pA/pF in LAA vs. –3.1 pA/pF in LAW). Addition of CsCl (2 mM) reversibly eliminated the time-dependent part of the current in both, the LAA (C) and LAW (D) myocyte. These electrophysiological characteristics and the modulation by Cs+ revealed If to underlie the current in both cells. If could be found in 18 out of 22 LAA myocytes (81%) and in 27 out of 40 LAW myocytes (68%), whereby the pacemaker current was considered to be present if its density was larger than 0.5 pA/pF at –120 mV.


Figure 1
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Original current recordings revealing the existence of the pacemaker current If in a single myocyte from LAA (panel A) and LAW (panel B). Hyperpolarizing steps were applied from a holding potential of –40 to –140 mV (10 mV increment). (C and D) The time dependent component of the recording (i.e. If) could be reversibly blocked in both atrial regions by application of 2 mM CsCl to the bath medium.

 
3.2 Density, kinetics and steady-state activation of If
Fig. 2A illustrates the mean If current–voltage relationships for LAA (closed circles, n=15/9) and LAW cells (open circles, n=28/9). If current densities were significantly higher in LAA than in LAW cells at potentials between –70 and –110 mV (e.g. at –70 mV:–0.47±0.14 pA/pF vs. –0.13±0.04 pA/pF and at –80 mV: –1.22±0.35 pA/pF vs. –0.36±0.01 pA/pF) and the activation threshold in LAA cells was about 10 mV more positive (between –60 and –70 mV) compared to LAW cells (between –70 and –80 mV). At test potentials more negative than –110 mV, If current densities were not significantly different between both groups (e.g. at –120 mV: –3.2±0.7 pA/pF, LAA vs. –1.9±0.2 pA/pF, LAW; P=0.083) which was mainly due to the large current density variability in LAA cells. The inserts in Fig. 2A show current density values of individual myocytes at –80 and –120 mV for both groups. We did not observe a significant influence of the presence or the absence of AF on If densities by either comparing data from the same region (LAA or LAW) or pooling LAA and LAW data into an AF and sinus rhythm (SR) group.


Figure 2
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 (A) Mean current–density to voltage relation in LAA (n=14/9, closed circles) and in LAW (n=28/9, open circles) myocytes. The inserts show current density values at –80 and –120 mV for each individual myocyte of both regions. (B) Mean time constants of If activation in LAA (n=11/7) and LAW (n=17/7) myocytes. The insert illustrates time constants ({tau}) of activation and deactivation of LAA cells (n=6/3), which were used to gain {tau}y (solid line) for If modeling.

 
To estimate {tau}y of the If model, we performed monoexponential fits of current activation and tail current deactivation. Tail currents were elicited in LAA cells (n=6/3) by 10 mV steps from –50 to +20 mV, preceded by a hyperpolarizing step to –120 mV. Current activation was well fitted by a single exponential decay equation at potentials positive to –100 mV. At more negative test potentials in most cases a biexponential fit was more accurate in describing the If time course. However, we decided to describe current activation at all membrane potentials by a monoexponential fit since for the subsequent purpose of modeling only membrane potentials positive to –100 mV were of interest. Fig. 2B shows mean time constants of current activation in LAA (closed circles, n=11/7) and in LAW cells (open circles, n=17/7). If activated significantly faster in cells from LAA than in LAW cells at all membrane potentials (between –80 and –140 mV; e.g. at –80 mV: 1135±87 ms vs. 1661±139 ms and at –140 mV: 60±7 ms vs. 96±13 ms). The insert in Fig. 2B illustrates the mean activation/deactivation values of If and the corresponding {tau}y curve (solid line, {alpha}0=1.2783x10–4, β0=121.609 and V0=9.2404) for LAA cells, which were used to gain the {tau}y equation as described in Methods.

Fig. 3 illustrates the voltage dependence of If activation. A Boltzmann function fitted to the normalized conductances of LAA cells yielded a V1/2 of –84.3±1.9 mV and a slope factor of 10.1±0.6 mV (n=14/9, closed circles). The activation curve of LAW cells (open circles, n=28/9) showed a significant more negative V1/2 value (–97.8±2.1 mV) and a slope factor of 9.6±0.4 mV. The insert in Fig. 3 shows V1/2 values of individual LAA and LAW myocytes.


Figure 3
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Voltage dependence of activation in LAA (n=14/9) and LAW cells (n=28/9) calculated by fitting a Boltzmann function to normalized conductances. The insert illustrates individual V1/2 values of both regions.

 
3.3 Beta-adrenergic modulation of If
Since If is a presumptive target for autonomic nervous system regulation (beta-adrenergic stimulation increases If activity), we also investigated the effect of beta-adrenergic stimulation by isoproterenol (1 µM). In both groups of cells, we found an acceleration of If activation (LAA: Fig. 4A, LAW: Fig. 4B) by beta-adrenergic stimulation and a significant shift of V1/2 to more depolarized potentials. This shift amounted for 8.8±2.3 mV in LAA (V1/2 was –87.0±2.3 under control conditions, closed circles and –78.3±2.2 with isoproterenol, closed squares, n=6/4, Fig. 4C) and 8.9±2.6 mV in LAW (V1/2 was –99.2±2.5 under control conditions, open circles and –90.3±2.4 with isoproterenol, open squares, n=6/4, Fig. 4D).


Figure 4
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Beta-adrenergic modulation of If in LAA and LAW myocytes. (A and B) Superimposed original current recordings of a single LAA and LAW cell in "standard" Tyrode solution (control) and after addition of 1 µM isoproterenol (iso). (C and D) Activation curves calculated by fitting a Boltzmann function to normalized conductances showed that iso shifted V1/2 in LAA from –87.0±2.3 to –78.3±2.2 mV (closed circles and squares, respectively, n=6/4) and in LAW from –99.2±2.5 mV to –90.3±2.4mV (open circles and squares, respectively, n=6/4).

 
3.4 Simulation of If
Fig. 5A shows original If recordings from the LAA cell with largest current densities at test potentials between –50 and –80 mV and the simulated current (dashed line=If(LAA)single). Fig. 5B illustrates time constants from this LAA cell (closed circles) used for modeling {tau}y curve (solid line). The external and internal Na+ and K+ concentrations as well as the clamp protocol were identical with the experimental conditions.


Figure 5
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 (A) Original If recordings in response to voltage clamp steps between –50 and –80 mV from an LAA cell that showed the most positive V1/2 value. The dashed lines illustrate the corresponding simulated curves of the If(LAA)single model which were calculated using activation/deactivation data of the cell (panel B, closed circles) for modeling {tau}y (panel B, solid line).

 
3.5 Pacemaking mechanism in the human atrial cell model
If(LAA)single and If(LAA)mean were implemented in the atrial cell model and simulations were performed using different scaling factors for If conductance in order to test for the occurrence of spontaneous action potentials (APs).

Fig. 6 shows selected ionic processes during the first two spontaneous APs (AP1 and AP2) and two spontaneous APs in steady state (AP3 and AP4). The right panel of Fig. 6 (framed box) shows an overlay comparison between a paced steady state AP and corresponding ionic processes of the original Nygren model (1 Hz, dotted line) and AP3 (solid line). First spontaneous APs in steady state (AP3 and AP4) occurred at 35.4% of If(LAA)single conductance (scaling factor 0.354; Fig. 6A). Spontaneous activity was initiated by If inducing additional depolarizing inward current (Figs. 6B and 7AGo) leading to depolarization of the membrane potential (Vm). The depolarized Vm reduced (via decreased driving force) inward current through the Na+/Ca2+ exchanger (INCX; by ~1.4 pA at a time of 0 s in right panel of Fig. 6C), which diminished NCX Ca2+ extrusion leading to a rise of internal Ca2+ concentration ([Ca2+]i; of ~110 nM at a time of 0 s in right panel of Fig. 6D). This small rise in [Ca2+]i was sensed by the release mechanism of the sarcoplasmic reticulum (SR) which responded with a small Ca2+ release further elevating [Ca2+]i. The positive feedback process culminated in an SR Ca2+ release (Ire) of 3.69 nA elevating [Ca2+]i to a maximal value of 0.68 µM. This release was not induced by Ca2+ influx via L-type Ca2+ current (ICa,L). The rise in [Ca2+]i increased inward INCX which reached a maximum of –71.61 pA 1.6 ms after Ire peak. This in turn depolarized Vm to the threshold for activation of sodium current (INa), which peaked to a value of –1.59 nA (Fig. 6E) and brought Vm to an overshoot potential of 13.03 mV. Depolarization of Vm also resulted in activation of ICa,L which induced Ire in the unmodified cell. However, in the If-pacemaker cell this ICaL induced Ire coincided with the trailing edge of the previous SR Ca2+ release and therefore elicited a second, very small second Ire peak (arrow in insert of Fig. 7A). The potassium currents (Fig. 6G, H) repolarized Vm to maximal diastolic potential of –72.57 mV but at this value they could not counterbalance the total inward current due to If, inward INCX and inward background currents. This imbalance caused slow phase-4 depolarization that lead to generation of a subsequent AP (via the mechanism described above) and thus continuous pacemaking.


Figure 6
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Selected processes during spontaneous AP initiation (AP1 and AP2) and steady-state oscillations in the modified Nygren human atrial cell model (AP3 and AP4) with If(LAA)single by 0.354 scaled implemented. A paced AP of the unmodified Nygren model (dotted line) and AP3 (solid line) are shown for comparison (framed right box). (A) Membrane voltage, Vm. (B) Hyperpolarization activated cation current, If. (C) Na+/Ca2+ exchange current, INCX. (D) Intracellular calcium concentration, [Ca2+]i. (E) Sodium current, INa. (F) L-type calcium current, ICa,L. (G) Inward rectifier potassium current, IK1 and delayed rectifier potassium current, IK. (H) Sum of transient outward potassium current, It and sustained outward potassium current, Isus.

 

Figure 7
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 (A) Role of NCX Ca2+ efflux as initial trigger for Ire. The solid line shows a simulated AP of the Nygren model without If. Implementing If at simulation time 1 s (arrow) resulted in a spontaneous AP (dashed line). This spontaneous AP could be suppressed by replacing Effluxspon by Effluxstim (beginning at 1 s of simulation time until the end of simulation; dotted line). The inserts show Ire from the stimulated AP (solid line) and the spontaneous and suppressed AP (dashed and dotted lines, respectively, the arrow labels the ICa,L correlated Ire peak). (B) Effect of If scaling using If(LAA)single, If(LAA)mean and If(LAA)mean iso on beat frequency of the single cell model.

 
To confirm that the reduction of Ca2+ efflux by NCX is the main trigger for the SR Ca2+ release, replacement of the NCX Ca2+ efflux in addition to If implementation (see below) should prevent the model cell from pacemaking. Therefore, a stimulated AP without If (Fig. 7A, solid line, NCX Ca2+ efflux=Effluxstim), a spontaneous AP after implementation of If(LAA)single (arrow in Fig. 7A, dashed line, NCX Ca2+ efflux=Effluxspon) and finally the effect of replacing Effluxspon by Effluxstim (dotted line in Fig. 7A) were simulated. Replacement of Effluxspon by Effluxstim synchronous to If implementation resulted in a stable membrane potential without generation of a spontaneous AP. The inserts in Fig. 7A illustrate Ire from the stimulated AP (solid line) and the spontaneous (dashed line) and suppressed AP (dotted line). The arrow labels the Ire peak due to ICa,L.

Furthermore, a spontaneous AP could be suppressed by reduction of INCX immediately before the peak of Ire, indicating that INCX is necessary to bring the membrane potential to activation threshold of INa (data not shown).

3.6 Influence of If parameters on pacemaking in the model
The effects of different If(LAA)single and If(LAA)mean conductances and shifting V1/2 to less negative potentials on eliciting spontaneous activity in the single cell model are shown in Fig. 7B. First spontaneous activity occurred at 35.4% of gf of If(LAA)single (closed circles) at a frequency of 15 beats per minute (bpm). A stepwise increase of the scaling factor up to 1.0 resulted in higher beat frequencies (at a scaling factor of 1.0, which corresponds to the experimentally observed maximal If(LAA)single conductance at [K+]o=25 mM, spontaneous activity was observed at a frequency of 47 bpm). Implementation of If(LAA)mean did not result in spontaneous activity within the parameter values yielded from the experimental data (closed diamonds in Fig. 7B). The If(LAA)mean conductance had to be scaled by a factor of 1.7 to elicit spontaneous activity (at 15 bpm). Further enlargement of If(LAA)mean again initiated beating at higher frequencies. Finally, shifting V1/2 of If(LAA)mean to more positive membrane potentials (by 8 mV as observed under beta-adrenergic stimulation) decreased threshold for evoking spontaneous activity to a scaling factor of 0.9 (14 bpm; If(LAA)mean iso, open diamonds in Fig. 7B).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study provides first evidence of the functional presence of the hyperpolarization activated cation current, If, in the human LA. If was found in both left atrial regions (LAA/82% and LAW/68%) and showed typical properties, i.e. activation at hyperpolarized membrane potentials (with faster activation at more negative potentials) and block by Cs+ [1]. Our data on If occurrence agree with the values reported for human myocardium where If was also shown to be abundant (82% [11] and 95% [4] in the RAA and 75% in the ventricle [6]).

Myocytes with If showing low threshold activation (i.e. activation between –60 and –70 mV) were mainly found in LAA where we also observed significantly higher If current densities (between –70 and –110 mV) and significantly more positive V1/2 values than in LAW cells. The mean V1/2 of LAA (~–84 mV) is similar to that reported for If in the human RAA (–87 mV [11], –89 mV [4]), whereas the mean V1/2 of LAW was found to be more negative (~– 98 mV). The If current densities at hyperpolarized membrane potentials found in LAA myocytes agree to the published data from RAA myocytes (~3.8 pA/pF at –120 mV [11] and at –130 mV [4]).

The nature of the difference in If activation threshold and V1/2 values between LAA and LAW is unclear at present. Molecular cloning revealed that hyperpolarization-activated cyclic nucleotide gated cation channels, from which four subtypes HCN 1–4 are known, encode If-like currents [20]. Data from various studies suggest that HCN4 expression is correlated with spontaneously active cells of the cardiac conduction system, whereas HCN2 expression is found in "non-spontaneously" active myocytes [2]. Both channel types (HCN2 and HCN4) have been detected in the right human atrium [21] and the amount of HCN2 transcripts did not differ between human left (LAA and LAW) and right atrium (RAA and RAW) [5]. Whether differences in the HCN2/HCN4 expression pattern are underlying the observed differences in If activation curves (between LAA and LAW) needs further investigation.

An If with properties as measured in cells of LAA may be of particular interest in contributing to abnormal automaticity in atrium. It is well known that the resting membrane potential stabilizing current IK1 is much smaller in atrium (6–10 fold) than in ventricle and therefore any inward cation current would have a higher arrhythmogenic potential in atrial cells [9,10]. It has also been shown that localized HCN2 overexpression in canine left atrium was able to transform the region near injection in an If-based pacemaker which was sufficient to drive the heart [22] further supporting the arrhythmogenic potential of atrial If.

If was also affected by beta-adrenergic modulation resulting in a shift of V1/2 to more positive potentials (about 9 mV in LAA and LAW), which is similar to right atrial myocytes (6 mV [11] and 7 mV [4]). Although there is no evidence of an elevated sympathetic tone in chronic AF, the sensitivity to beta-adrenergic stimulation may be of interest for the adrenergic type of paroxysmal AF which occurs during states of increased adrenergic activity [23] and in which spontaneous termination is accompanied by a reduction of the elevated sympathetic tone [24].

We used a modeling approach to estimate the arrhythmogenic role of If in the human atrium. This was achieved by implementing two formulations of human atrial If (If(LAA)single and If(LAA)mean) into the established human atrial cell model of Nygren et al. [18]. The If(LAA)single described the LAA cell showing the most positive V1/2 value, the If(LAA)mean was based on the mean If data from LAA. Our simulations demonstrated that If (both formulations) was able to induce spontaneous activity at a regular rhythm. Spontaneous activity was not generated by If solely but due to the interplay of If induced depolarization, subsequent alteration of INCX and SR Ca2+ release. The frequency of activity could be modulated by scaling of gf or shifting V1/2 to more positive potentials as seen under beta-adrenergic stimulation. The If(LAA)single model was able to elicit spontaneous activity at only 35.4% of gf determined experimentally in a [K]o of 25 mM. An elevation of [K]o is known to strongly increase gf (which is utilized in the experiment to amplify If) but also to shift the reversal potential (and also the activation threshold) of the current to more positive potentials [1]. For human left ventricular myocytes, a [K]o of 25 mM was reported to amplify If by 64% compared to [K]o 5.4 mM [3]. Therefore, the reduction of gf by 65.6% in the simulation compensates well for the enlargement of gf in the voltage clamp experiments due to the use of 25 mM [K]o.

It remains open how the If-induced transformation of quiescent atrial myocytes into spontaneously beating cells as shown in our single cell simulations can take place in vivo. Our simulation results suggest the importance of SR Ca2+ release and INCX and confirm experimental work in atrial and sinoatrial pacemaker cells in which diastolic SR Ca2+ release and stimulation of inward NCX was shown to play an essential role in pacemaker activity [25,26]. Furthermore, IK1 downregulation (81%) in a ventricular myocyte model demonstrated a central role of INCX for pacemaking [27].

It is well known that initiation of AF arises from focal activity of locally restricted regions within the atrium (mainly from the pulmonary veins) and therefore AF initiation has to be seen as a local phenomenon [12,13]. This dependence of the focal activity on atrial location is also emphasized by the large regional variability of If density. Therefore, spatial information seems to be a crucial point when estimating the role If as a contributor to atrial ectopy. AF is known to induce ionic current remodeling (for a detailed description see Ref. [28]). Regarding If an increase in mRNA-levels in left and right atrial tissue from chronic AF patients was demonstrated recently [5]. Our data did not indicate a statistically significant difference of If properties between SR and AF. Thus we decided to pool SR and AF data for comparing the two LA regions. In addition, analysis of SR/LAA and SR/LAW data alone showed the same statistically significant differences in If parameters as found for the pooled data. However, due to the large variability of If density even in cells of a given patient (independent of rhythm state) and considering different stages of AF (paroxysmal/persistent/permanent) much larger sample sizes are warranted for a thorough statistical analysis of a correlation between If and AF. Furthermore, we are not entirely able to exclude an influence of clinical parameters and medication on the lack of difference in If properties between SR and AF cells due the limited number of patients (e.g. see Ref. [29] for the influence of digitalis on IK1 in AF patients). However, in the view of focal activity and considering the observed local heterogeneity of If the individual extreme current densities, which are usually lost during averaging and thereby resulting in an underestimation of the arrhythmogenic potential of If, are of particular interest.

Our results implicate that cells from LAA may have a higher arrhythmogenic potential due to larger If than LAW cells. In this context, it is noteworthy that drivers of AF may arise within or at the base of LAA which has also been demonstrated recently in case of focal atrial tachycardia occurring upon AF conversion [30]. Therefore, the LAA which is typically abscised during ablation therapy to eliminate risk of thrombogenesis may also be a source of arrhythmia control. However, to estimate the full arrhythmogenic potential simultaneous information on IK1 (from the same cell where If was measured) is of great importance and a massive IK1 increase as reported for chronic AF [28,29] would abolish spontaneous pacemaker activity. Interestingly, RAA cells with prominent If show a relatively small IK1 [4] which would further emphasize a high arrhythmogenic activity of cells with large If. Ongoing experiments in our laboratory are directed at elucidating this issue for LAA.

In summary, this study is the first electrophysiological characterization of If in left human atrium. We found If densities, activation kinetics and activation range to be dependent on the left atrial region. These results offer a framework for further studies elucidating the correlation between HNC expression and functional If properties. Furthermore, our simulations suggest that human atrial If is able to induce membrane depolarization which is sufficient for the initiation of ectopic activity in the human atrium.


   Acknowledgements
 
This work was supported by the Austrian Science Fund, P-15403-Med.


   Notes
 
Time for primary review 26 days


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

  1. Ludwig A., Zong X., Hofmann F., Biel M. Structure and function of cardiac pacemaker channels. Cell. Physiol. Biochem. (1999) 9:179–186.[CrossRef][Web of Science][Medline]
  2. Biel M., Schneider A., Wahl C. Cardiac HCN channels: structure, function, and modulation. Trends Cardiovasc. Med. (2002) 12:206–212.[CrossRef][Web of Science][Medline]
  3. Cerbai E., Pino R., Porciatti F., et al. Characterization of the hyperpolarization-activated current, If, in ventricular myocytes from human failing heart. Circulation (1997) 95:568–571.[Abstract/Free Full Text]
  4. Hoppe U.C., Beuckelmann D.J. Characterization of the hyperpolarization-activated inward current in isolated human atrial myocytes. Cardiovasc. Res. (1998) 38:788–801.[Abstract/Free Full Text]
  5. Lai L.P., Su M.J., Lin J.L., et al. Measurement of funny current (If) channel mRNA in human atrial tissue: correlation with left atrial filling pressure and atrial fibrillation. J. Cardiovasc. Electrophysiol. (1999) 10:947–953.[Web of Science][Medline]
  6. Cerbai E., Sartiani L., DePaoli P., et al. The properties of the pacemaker current If in human ventricular myocytes are modulated by cardiac disease. J. Mol. Cell. Cardiol. (2001) 33:441–448.[CrossRef][Web of Science][Medline]
  7. Lopatin A.N., Nichols C.G. Inward rectifiers in the heart: an update on IK1. J. Mol. Cell. Cardiol. (2001) 33:625–638.[CrossRef][Web of Science][Medline]
  8. Koumi S., Backer C.L., Arentzen C.E. Characterization of inwardly rectifying K+ channel in human cardiac myocytes. Alterations in channel behavior in myocytes isolated from patients with idiopathic dilated cardiomyopathy. Circulation (1995) 92:164–174.[Abstract/Free Full Text]
  9. Wang Z., Yue L., White M., Pelletier G., Nattel S. Differential distribution of inward rectifier potassium channel transcripts in human atrium versus ventricle. Circulation (1998) 98:2422–2428.[Abstract/Free Full Text]
  10. Opthof T. The membrane current (If) in human atrial cells: implications for atrial arrhythmias. Cardiovasc. Res. (1998) 38:537–540.[Free Full Text]
  11. Porciatti F., Pelzmann B., Cerbai E., et al. The pacemaker current If in single human atrial myocytes and the effect of beta-adrenoceptor and A1-adenosine receptor stimulation. Br. J. Pharmacol. (1997) 122:963–969.[CrossRef][Web of Science][Medline]
  12. Harada A., Sasaki K., Fukushima T., et al. Atrial activation during chronic atrial fibrillation in patients with isolated mitral valve disease. Ann. Thorac. Surg. (1996) 61:104–111.[Abstract/Free Full Text]
  13. Jais P., Weerasooriya R., Shah D.C., et al. Ablation therapy for atrial fibrillation (AF): past, present and future. Cardiovasc. Res. (2002) 54:337–346.[Abstract/Free Full Text]
  14. Li D., Zhang L., Kneller J., Nattel S. Potential ionic mechanism for repolarization differences between canine right and left atrium. Circ. Res. (2001) 88:1168–1175.[Abstract/Free Full Text]
  15. Schram G., Pourrier M., Melnyk P., Nattel S. Differential distribution of cardiac ion channel expression as a basis for regional specialization in electrical function. Circ. Res. (2002) 90:939–950.[Abstract/Free Full Text]
  16. Pelzmann B., Schaffer P., Machler H., Rigler B., Koidl B. Adenosine inhibits the L-type calcium current in human atrial myocytes. Naunyn-Schmiedeberg's Arch. Pharmacol. (1995) 351:293–297.[Web of Science][Medline]
  17. World Medical Association Declaration of Helsinki. Recommendations guiding physicians in biomedical research involving human subjects. Cardiovasc. Res. (1997) 35:2–3.[Free Full Text]
  18. Nygren A., Fiset C., Firek L., et al. Mathematical model of an adult human atrial cell: the role of K+ currents in repolarization. Circ. Res. (1998) 82:63–81.[Abstract/Free Full Text]
  19. Hund T.J., Kucera J.P., Otani N.F., Rudy Y. Ionic charge conservation and long-term steady state in the Luo-Rudy dynamic cell model. Biophys. J. (2001) 81:3324–3331.[Web of Science][Medline]
  20. Accili E.A., Proenza C., Baruscotti M., DiFrancesco D. From funny current to HCN channels: 20 years of excitation. News Physiol. Sci. (2002) 17:32–37.[Abstract/Free Full Text]
  21. Ludwig A., Zong X., Stieber J., Hullin R., Hofmann F., Biel M. Two pacemaker channels from human heart with profoundly different activation kinetics. EMBO J. (1999) 8:2323–2329.
  22. Qu J., Plotnikov A.N., Danilo P. Jr., et al. Expression and function of a biological pacemaker in canine heart. Circulation (2003) 107:1106–1109.[Abstract/Free Full Text]
  23. Coumel P. Paroxysmal atrial fibrillation: a disorder of autonomic tone? Eur. Heart J. (1994) 15:9–16.[Abstract]
  24. Tomita T., Takei M., Saikawa Y., et al. Role of autonomic tone in the initiation and termination of paroxysmal atrial fibrillation in patients without structural heart disease. J. Cardiovasc. Electrophysiol. (2003) 14:559–564.[CrossRef][Web of Science][Medline]
  25. Bogdanov K.Y., Vinogradova T.M., Lakatta E.G. Sinoatrial nodal cell ryanodine receptor and Na+–Ca2+ exchanger: molecular partners in pacemaker regulation. Circ. Res. (2001) 88:1254–1258.[Abstract/Free Full Text]
  26. Lipsius S.L., Bers D.M. Cardiac pacemaking: If vs. Ca2+, is it really that simple? J. Mol. Cell. Cardiol. (2003) 35:891–893.[CrossRef][Web of Science][Medline]
  27. Silva J., Rudy Y. Mechanism of pacemaking in IK1-downregulated myocytes. Circ. Res. (2003) 92:261–263.[Abstract/Free Full Text]
  28. Dobrev D., Ravens U. Remodeling of cardiomyocyte ion channels in human atrial fibrillation. Basic Res. Cardiol. (2003) 98:137–148.[Web of Science][Medline]
  29. Dobrev D., Wettwer E., Kortner A., Knaut M., Schuler S., Ravens U. Human inward rectifier potassium channels in chronic and postoperative atrial fibrillation. Cardiovasc. Res. (2002) 54:397–404.[Abstract/Free Full Text]
  30. Scharf C., Oral H., Chugh A., Hall B., Good E., Cheung P., et al. Acute effects of left atrial radiofrequency ablation on atrial fibrillation. J. Cardiovasc. Electrophysiol. (2004) 15:515–521.[Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Physiol. Rev.Home page
M. Biel, C. Wahl-Schott, S. Michalakis, and X. Zong
Hyperpolarization-Activated Cation Channels: From Genes to Function
Physiol Rev, July 1, 2009; 89(3): 847 - 885.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
E. A. Woodcock, P. M. Kistler, and Y.-K. Ju
Phosphoinositide signalling and cardiac arrhythmias
Cardiovasc Res, May 1, 2009; 82(2): 286 - 295.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
P. Kohl
Cardiac cellular heterogeneity and remodelling
Cardiovasc Res, November 1, 2004; 64(2): 195 - 197.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Zorn-Pauly, K.
Right arrow Articles by Koidl, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zorn-Pauly, K.
Right arrow Articles by Koidl, B.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?