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Cardiovascular Research 2003 59(3):593-602; doi:10.1016/S0008-6363(03)00466-8
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Copyright © 2003, European Society of Cardiology

Characterisation of the Na, K pump current in atrial cells from patients with and without chronic atrial fibrillation

Antony J Workmana,*, Kathleen A Kaneb and Andrew C Rankina

aSection of Cardiology, Division of Cardiovascular & Medical Sciences, University of Glasgow, Royal Infirmary, Glasgow G31 2ER, UK
bDepartment of Physiology and Pharmacology, University of Strathclyde, Glasgow G4 ONR, UK

a.j.workman{at}clinmed.gla.ac.uk

* Corresponding author. Tel.: +44-141-211-1231; fax: +44-141-552-4683.

Received 8 April 2003; revised 16 May 2003; accepted 2 June 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: To assess the contribution of the Na, K pump current (Ip) to the action potential duration (APD) and effective refractory period (ERP) in human atrial cells, and to investigate whether Ip contributes to the changes in APD and ERP associated with chronic atrial fibrillation (AF). Methods: Action potentials and ion currents were recorded by whole-cell patch clamp in atrial myocytes isolated from consenting patients undergoing cardiac surgery, who were in sinus rhythm (SR) or AF (>3 months). Results: In cells from patients in SR, the Ip blocker, ouabain (10 µM) significantly depolarised the membrane potential, Vm, from –80±2 (mean±S.E.) to –73±2 mV, and lengthened both the APD (174±17 vs. 197±23 ms at 90% repolarisation) and ERP (198±22 vs. 266±14 ms; P<0.05 for each, Student’s t-test, n=7 cells, 5 patients). With an elevated pipette [Na+] of 30 mM, Ip was measured by increasing extracellular [K+] ([K+]o) from 0 to 5.4 mM. This produced an outward shift in holding current at –40 mV, abolished by 10 µM ouabain. K+- and ouabain-sensitive current densities were similar, at 0.99±0.13 and 1.12±0.11 pA/pF, respectively (P>0.05; n=9 cells), confirming the K+-induced current as Ip. Ip increased linearly with increasing Vm between –120 and +60 mV (n=25 cells). Stepwise increments in [K+]o (between 0 and 10 mM) increased Ip in a concentration-dependent manner (maximum response, Emax=1.19±0.09 pA/pF; EC50=1.71±0.15 mM; n=27 cells, 9 patients). In cells from patients in AF, the sensitivity of Ip to both Vm and [K+]o (Emax=1.02±0.05 pA/pF, EC50=1.54±0.11 mM; n=44 cells, 9 patients) was not significantly different from that in cells from patients in SR. Within the group of patients in AF, long-term digoxin therapy (n=5 patients) was associated with a small, but significant, reduction in Emax (0.92±0.07 pA/pF) and EC50 (1.35±0.15 mM) compared with non-treatment (Emax=1.13±0.08 pA/pF, EC50=1.76±0.14 mM; P<0.05 for each, n=4 patients). In cells from non-digoxin-treated patients in AF, the voltage- and [K+]o-sensitivity (Emax and EC50) were similar to those in cells from patients in SR. Conclusions: The Na, K pump current contributes to the human atrial cell Vm, action potential shape and ERP. However, the similarity in Ip sensitivity to both [K+]o and Vm between atrial cells from patients with and without chronic AF indicates that Ip is not involved in AF-induced electrophysiological remodelling in patients.

KEYWORDS Na/K-pump; Membrane currents; Atrial function; Arrhythmia (mechanisms); Remodelling

This article is referred to in the Editorial by E. Carmeliet (pages 536–537) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The Na, K pump (Na, K-ATPase) functions to regulate the intracellular Na+ concentration, [Na+]i, by actively extruding 3 Na+ in exchange for 2 K+ [1]. Na, K-ATPase is therefore electrogenic, exerting an outward, hyperpolarising current, Ip, which is known to influence the resting potential, Vm and repolarisation [2]. The Na, K pump current has been measured directly in various cardiac tissues and myocytes isolated from sheep [3], guinea pigs [4], rabbits [5,6] and dogs [7]. However, despite its potential to influence, or be influenced by, cardiac arrhythmias, Ip has not yet been measured directly in cells or tissues from the human heart.

Chronic atrial fibrillation (AF) shortens the atrial action potential duration (APD) and effective refractory period (ERP), thus contributing to the stabilisation of the arrhythmia [8–13]. The pattern of changes in ionic currents responsible for this is not fully understood. The density of atrial L-type Ca2+ (ICaL) and transient outward K+ (ITO) currents is consistently and markedly reduced in AF or rapid atrial pacing, in dogs [10] and humans [11–13]. However, recent work from our laboratory in human atrial cells [12] suggests that the shortening of APD and ERP by chronic AF cannot be explained by changes in these currents alone, consistent with a mathematical model [14], and data on the main alternatives are presently either unavailable, or in some cases, conflicting [13].

The involvement or otherwise of Ip or Na, K-ATPase in AF-induced atrial electrophysiological remodelling is presently unclear [13]. In sheep, short episodes of rapid atrial pacing increased the expression of atrial Na, K-ATPase [15], although there was no effect on its activity [16]. In the goat model of AF, treatment with the Ip blocker, digoxin, had either no effect on AF-induced changes in atrial ERP and AF inducibility [17], or delayed their reversal [18]. However, Ip was not measured directly in any of these studies, and has not yet, to our knowledge, been recorded in any model of rapid atrial pacing or AF.

It is conceivable that the shortening of atrial APD and ERP by chronic AF in humans might involve an increase in atrial Ip. The aim was to test this hypothesis, by: 1) assessing the contribution of Ip to human atrial cell APD and ERP; 2) measuring Ip directly in atrial cells from patients in sinus rhythm (SR) and AF; 3) comparing the density, voltage- and extracellular [K+] ([K+]o)-sensitivity of Ip between these patient groups.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
The tip of the right atrial appendage was obtained from 25 consenting patients undergoing cardiac surgery. Procedures approved by the institutional research ethics committee were followed. The investigation conforms with the principles outlined in the Declaration of Helsinki [19]. Atrial cells were isolated by enzymatic dissociation and mechanical disaggregation, using protease (Type XXIV, Sigma) and collagenase (Type 1, Worthington), as described in detail previously [12].

Action potentials and ion currents were recorded using the whole cell patch clamp technique. Microelectrodes were pulled (Narishige PP-83) from filamented borosilicate glass tubes (Clark Electromedical) and heat polished to resistances of 3–7 M{Omega}. Cells were superfused at 35–37°C, at 1.5–2 ml/min in a 200 µl perfusion chamber (RC-24E, Warner). An Axopatch-1D amplifier (Axon Instruments) and ‘WinWCP’ software (donated by J Dempster, Strathclyde University) was used to stimulate and record electrical activity. Capacitative transients were compensated electronically prior to recording. Signals were low-pass filtered at 5 kHz and digitised (Digidata 1200 A–D converter, Axon) prior to storage on magnetic and compact discs.

Action potentials were recorded using an extracellular solution containing (mM): NaCl (130), KCl (4), CaCl2 (2), MgCl2 (1), glucose (10) and HEPES (10), and a pipette solution containing: K-aspartate (110), KCl (20), MgCl2 (1), EGTA (0.15), Na2ATP (4), Na2GTP (0.4) and HEPES (5). A liquid junction potential of +7±0.3 mV (n=6) was measured and compensated prior to seal formation. Action potentials were stimulated using 5 ms duration current pulses of 1.2xthreshold strength, with an 8-pulse (S1) conditioning train (75 beats/min). All cells were current-clamped (with hyperpolarising current of <150 pA) initially to a maximum diastolic potential (MDP) of ~–80 mV (measured from the 7th S1 response) and the holding current was kept constant in each cell thereafter. The APD was calculated as the interval between the action potential upstroke and repolarisation to the level of 50, 75 and 90% (APD50, APD75 and APD90, respectively). Action potential restitution was measured by introducing a progressively premature test pulse (S2) after the S1 trains, with S1 and S2 of equal magnitude. From this, the cell’s ERP was measured, as previously [12,20], as the longest S1–S2 interval failing to elicit an S2 response of amplitude >80% [21] of the preceding S1 action potential. The action potential maximum upstroke velocity (Vmax) was measured by automatically scanning phase 0 at high time resolution for the maximum slope between two adjacent voltage samples. Action potentials were recorded before and after superfusion with ouabain (10 µM) for 120 s, and again following its removal. This concentration was chosen to avoid ‘rundown’ of currents during the experimental protocols, since complete Ip block by 1 µM ouabain required 6–10 min superfusion, whilst 10 µM ouabain required only 40–80 s (eg: see Fig. 2).


Figure 2
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  		Fig. 2 Identification of human atrial Na, K pump current, Ip. Whole-cell current changes evoked by K+o and/or ouabain in atrial myocytes from patients in SR. Traces show A) activation of an outward current (I) by increasing [K+]o, current inhibition after decreasing [K+]o, and abolition of K+o-induced current by ouabain; B) non-reversible and relatively slow abolition of Ip by 1 µM ouabain. Bars show concentration and time-course of K+ and ouabain application. C) Histogram comparing mean K+o- and ouabain-sensitive current densities, calculated as peak K+o-induced current minus corresponding current in absence of K+o ({blacksquare}: n=17 cells, 4 patients) and in presence of ouabain at 1 µM (Figure 2: n=9 cells, 3 patients) and 10 µM (Figure 2: n=9 cells, 3 patients). All patients were in SR. Values are means±S.E., NS=P>0.05 vs. K+o-sensitive current.

 
Ip was recorded over a wide voltage range, using solutions selected to minimise contamination from other currents [4,6]. The extracellular solution contained (mM): NaCl (145), CsCl (2), MgCl2 (0.5), glucose (10), HEPES (5), NiCl2 (2), BaCl2 (1). Cs+ was used to block If, IK1 and IKACh; Ni2+ to block INa/Ca, and Ba2+ to block IKP [22]. [Ca2+]o was kept at zero to avoid ICaL. [K+]o was initially kept at zero to record currents in the absence of Ip, prior to its activation. INa was inactivated with a holding potential of –40 mV. The pipette solution contained (mM): Aspartic acid (100), CsCl (20), MgCl2 (2), HEPES (5), MgATP (5), EGTA (10), K-creatine phosphate (5), CsOH (90) and NaOH (30). K+ was replaced by Cs+ to block outward K+ currents. A high degree of intracellular [Ca2+] ([Ca2+]i)-buffering minimised Ca2+-activated currents, and high [Na+]i was used to record Ip at close to full saturation [4,6]. A liquid junction potential (+7±0 mV, n=3) was compensated. The holding current was measured before and after superfusion with 5.4 mM KCl, and following its removal. One or more of the following protocols was then performed. The sensitivity to a cardiac glycoside of the extracellular K+ (K+o)-induced holding current shift was investigated, using ouabain at 1 and 10 µM. The voltage-sensitivity of K+- and glycoside-sensitive currents was determined using voltage ramps (increasing from –120 to +60 mV, at 36 mV/s) or rectangular voltage pulses (300 ms duration, increasing from –120 to +60 mV, at 0.1 Hz). Finally, the sensitivity of the current shift to [K+]o was determined with stepwise-incrementing [K+]o between 0 and 10 mM.

Details of patients’ clinical characteristics and drug treatments were obtained from the case notes and stored in a database (Access, Microsoft). All currents were normalised to cell capacity. [K+]o-response data were fitted iteratively in each cell with variable slope sigmoidal curves (Prism, GraphPad). Values are cell means±S.E. Differences between means were assessed using 2-tailed paired or unpaired Student’s t tests, as appropriate. Differences between incidences of patients receiving specified drugs were compared using a {chi}2 test. P<0.05 was regarded as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Patients’ clinical characteristics
The patients’ characteristics are shown in Table 1. The majority of patients underwent coronary artery bypass graft surgery (76%) and suffered from angina (84%). Of patients in AF at the time of surgery, only those in which AF had persisted for longer than 3 months were included. Half of these patients underwent mitral valve surgery, versus none of those in SR. The medication taken by the patients is detailed in Table 1 and it is noteworthy that none of the patients in SR were taking digoxin, in contrast to 60% of patients in chronic AF. All patients on digoxin had been receiving the drug for longer than 2 months and patients received their routine cardiac drugs on the day of surgery.


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  		Table 1 Patients’ characteristics

 
3.2 Effects of superfusion with ouabain on human atrial cell action potentials and refractoriness
The Ip blocker, ouabain (10 µM) significantly affected the single cell Vm, action potential shape and ERP, as shown in the representative recordings in Fig. 1 and mean data in Table 2. Ouabain depolarised the MDP by ~7 mV (Fig. 1A and Table 2), associated with a marked reduction in the action potential Vmax, overshoot and amplitude and a significant prolongation of early (APD50) and late (APD75 and APD90) repolarisation. Fig. 1B shows the action potential restitution characteristics and ERP measurement in the absence and presence of ouabain, in the same cell as Fig. 1A. The ouabain-induced depolarisation was associated with slowing of the recovery of excitability, flattening of the restitution curve, and marked and significant lengthening of the cell ERP, from 198±22 to 266±14 ms (P<0.05; n=5 cells, 4 patients).


Figure 1
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  		Fig. 1 Effects of ouabain on human atrial cell action potentials and refractoriness. Original action potential recordings from a single atrial cell from a patient in SR, in the absence ({circ}) and presence (bullet) of ouabain (10 µM). A) Superimposed action potentials stimulated by the 7th of a train of conditioning current pulses, S1 (rate: 75 beats/min) showing the effects of ouabain on Vm and action potential configuration. B) Superimposed action potentials elicited, in the same cell, by the 7th and 8th S1 pulses, followed by responses to an increasingly premature test pulse, S2, showing the effects of ouabain on action potential restitution. The cell ERP (solid bars) was calculated as the longest S1–S2 interval failing to elicit an S2 response of amplitude >80% of the preceding S1 action potential. In each case, the S2 response used to measure this interval is labelled ({searrow}).

 

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  		Table 2 Effects of ouabain on human atrial cell action potentials and refractory period

 
3.3 Activation of human atrial Ip by physiological [K+]o was abolished by ouabain
The representative current trace shown in Fig. 2A shows that, using an elevated pipette [Na+] of 30 mM, increasing [K+]o from 0 mM to the physiological value of 5.4 mM produced an outward shift in the holding current at –40 mV, of approximately 1 pA/pF. This response was complete after approximately 60 s of K+ application, and was fully reversed after K+ removal. A second application of K+ produced a current shift of similar magnitude. This was abolished by superfusion with ouabain (10 µM), with a similar time course of effect as that caused by the prior removal of K+. The effect of 1 µM ouabain was also investigated for comparison, in a different cell (Fig. 2B). The K+-induced outward current was again abolished by ouabain, but with a substantially longer time course than at 10 µM. A comparison between the mean current shift produced by the increase in [K+]o and the reverse shift produced by ouabain is shown in Fig. 2C. The K+- and ouabain-sensitive current densities were similar, at 0.99±0.07 and 1.12±0.11 pA/pF (for 10 µM ouabain), respectively (P>0.05; n=9 cells, 3 patients), indicating that the K+-induced current was Ip.

3.4 Human atrial Ip was voltage-dependent
The steady-state voltage-dependency of Ip was examined using rectangular voltage pulses, in cells from 9 patients in SR. Fig. 3Ai shows representative currents recorded in the absence and presence of 5.4 mM K+o. All cells displayed steady-state responses between –120 and +20 mV (Fig. 3Ai). A small time-dependent component was observed at +60 mV in 59% of cells, possibly reflecting incomplete K+ current suppression. The currents recorded prior to K+ application were digitally subtracted from those recorded in its presence, as shown in the bottom panel of Fig. 3Ai. The subtraction currents were time-independent between –120 and +60 mV in all cells. The mean current–voltage (I–V) relationship of these currents, shown in Fig. 3Aii, demonstrated that the K+o-sensitive current was voltage-dependent, increasing approximately linearly from 0.47±0.10 pA/pF at –120 mV to 1.36±0.22 pA/pF at +60 mV (P<0.05, n=17 cells). Voltage ramp pulses were also used, to examine and compare the pseudo-steady-state I–V relationships of both K+- and 10 µM ouabain-sensitive currents. Fig. 3Bi shows the current responses to these ramps, prior to and following superfusion with 5.4 mM K+ and after its removal, with the K+-sensitive (subtraction) current in the lower panel. Mean I–V relationship data (Fig. 3Bii), obtained in cells from 12 patients, confirmed that the voltage ramp-evoked K+o-sensitive current displayed a similar voltage-dependence to that of the steady-state current. The voltage-dependent characteristics of the ouabain-sensitive current (Fig. 3C) were similar to those of both the steady-state (Fig. 3A) and pseudo-steady-state (Fig. 3B) currents, indicating that the voltage-dependent characteristics of the K+o-evoked current were those of Ip.


Figure 3
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  		Fig. 3 Voltage-dependency of human atrial Ip. Current–voltage (I–V) relationships for A) steady-state K+o-sensitive currents (n=17 cells, 9 patients); B) pseudo steady-state K+o-sensitive currents (n=25 cells, 12 patients); C) pseudo steady-state ouabain-sensitive currents (n=4 cells, 2 patients) in atrial cells from patients in SR. Upper panels (i) show representative whole-cell current traces and lower panels (ii) mean±S.E. I–V relationships at baseline ({circ}), in the presence of 5.4 mM K+o ({bigtriangleup}) and following either washout of K+ ({square}) or addition of 10 µM ouabain ({diamond}), with digital subtraction of baseline currents from those recorded in the presence of K+ and ouabain indicated by {blacktriangleup} and {diamondsuit}, respectively.

 
3.5 Ip was increased by K+o in a concentration-dependent manner
The amplitude of Ip increased in response to increasing [K+]o, as shown in Fig. 4. Ip was measured from a holding potential of –40 mV, and [K+]o was increased in a stepwise manner between 0 and 10 mM. Ip was near maximal at 5.4 mM K+o. The 10 mM K+o-induced current was abolished by the washout of K+. The concentration–response relationship for the effect of K+o on Ip was examined in cells from 9 patients in SR. In each cell, Ip density was fitted iteratively to logarithmic [K+]o with a variable slope sigmoidal curve, using the Hill equation: Y=Emin+[Emax–Emin]/[1+(x/EC50)P], where Y=Ip density (pA/pF), Emin=Ip at 0 mM (set to 0 pA/pF), Emax=maximum Ip response elicitable by K+o (pA/pF), x=[K+]o (mM), EC50=[K+]o producing 50% of Emax (mM) and P=Hill coefficient. Fig. 5A (open circles) shows the mean Ip density recorded at each [K+]o on a linear scale, with a single hyperbolic curve fitted to these data points using the same procedure as for the individual cells. The mean Emax and EC50, calculated from the individual cells’ curves, were 1.19±0.09 pA/pF and 1.71±0.15 mM, respectively (n=27 cells).


Figure 4
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  		Fig. 4 Extracellular [K+]-sensitivity of human atrial Ip. Original recording of whole cell current at a holding potential of –40 mV in a cell from a patient in SR, showing the effects on Ip amplitude of stepwise increments in [K+]o between 0 and 10 mM.

 

Figure 5
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  		Fig. 5 Comparison of atrial Ip characteristics between patients in SR and chronic AF. Ip [K+]o-response curves (A) and characteristics (B) in cells from patients in SR (open symbols; n=27 cells, 9 patients) and chronic AF (filled symbols; n=44 cells, 9 patients). Ip is expressed relative to that at 0 mM K+o. Mean data points were fitted by the Hill equation (see Results). Mean Emax (maximal Ip response to K+o) and EC50 ([K+]o producing 50% of Emax) were calculated from the individual cell curve-fit data. NS=P>0.05 between patient groups. C) Steady-state Ip I–V relationship in cells from patients in SR ({circ}: n=17 cells, 9 patients) and chronic AF (bullet: n=24 cells, 8 patients). All values are means±S.E.

 
3.6 Atrial fibrillation was not associated with a significant alteration of Ip [K+]o- or voltage-sensitivity
The [K+]o-dependency of Ip was examined in 9 patients who were in chronic AF, and compared with that of cells from the 9 patients in SR. Fig. 5A (filled circles) shows the mean Ip density at different [K+]o (0–10 mM) in the cells from patients in AF, with a single hyperbolic curve fitted, using the Hill equation, to the mean data points. The Ip [K+]o-response in cells from the patients in AF was similar to that in cells from the patients in SR (Fig. 5A, open circles). The mean Ip at 5.4 and 10 mM K+o was not significantly different in the cells from patients in SR, at 0.97±0.06 and 1.05±0.08 pA/pF, respectively (n=27 cells) from those in the cells from patients in AF, at 0.85±0.04 and 0.91±0.05 pA/pF, respectively (P>0.05 for each; n=44 cells). Furthermore, the mean EC50 and Emax values were not significantly different in the patients in chronic AF (at 1.54±0.11 mM and 1.02±0.05 pA/pF, respectively) from those measured in the patients in SR, as shown in Fig. 5B. The 5.4 mM K+o-induced Ip steady-state I–V relationship was examined in cells from 8 patients in chronic AF, and compared with that obtained in cells from 9 patients in SR, as shown in Fig. 5C. There was no significant difference in the voltage-dependency of Ip between the two patient groups, with both displaying an approximately linear increase in Ip with increasing voltage.

3.7 Long-term digoxin therapy in patients with AF was associated with a small reduction in Ip
Sixty percent of the patients who were in chronic AF were treated pre-operatively with digoxin, versus none of the patients in SR (Table 1) (P<0.05, {chi}2 test). Since digoxin therapy may be expected to reduce Ip in human atrial tissues [23], the group of patients in chronic AF was sub-divided into those who were treated with digoxin and those who were not. The [K+]o-dependency of the atrial cell Ip was compared between these sub-groups, with the mean Ip values and [K+]o-response curves shown in Fig. 6Ai, and the mean EC50 and Emax in Fig. 6Aii. Digoxin therapy was associated with a small, but significant, reduction in mean Emax, by 19%, from 1.13±0.08 pA/pF (n=23 cells, 5 patients) to 0.92±0.07 pA/pF (P<0.05; n=21 cells, 4 patients). Digoxin therapy was also associated with a small, but significant, reduction in the mean EC50, by 23%, from 1.76±0.14 to 1.35±0.15 mM (P<0.05). The potential influence on Ip of angiotensin converting enzyme (ACE) inhibition was also examined, since Ip was increased in rabbits by captopril treatment [24]. In cells from patients treated with ACE inhibitors, the Ip [K+]o-response Emax and EC50 were 1.29±0.11 pA/pF and 1.98±0.25 mM, respectively (n=11 cells, 4 patients), not significantly different from that in cells from non-treated patients, at 1.12±0.13 pA/pF and 1.51±0.17 mM, respectively (n=16 cells, 5 patients; P>0.05 for each).


Figure 6
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  		Fig. 6 Effects of digoxin therapy on atrial Ip, and comparison between non-digoxin-treated patients in SR and AF. A) Comparison of the Ip [K+]o-response between cells from patients in chronic AF treated and not treated with digoxin. B) Comparison of the Ip [K+]o-response between patients in AF not treated with digoxin and those in SR. Concentration-response curves (i) and characteristics (ii) are shown. Values are means±S.E. {blacktriangledown} and {image} =AF, not treated with digoxin (n=21 cells, 4 patients); {blacktriangleup} and {image} =AF, treated with digoxin (n=23 cells, 5 patients); open symbols =SR (n=27 cells, 9 patients). * P<0.05 and NS=P>0.05 between patient sub-groups.

 
3.8 Atrial Ip was similar in the non-digoxin-treated patients in AF to that of the patients in SR
The concentration–response relationship for the effect of K+o on Ip was compared in cells from the patients who were in chronic AF but not treated with digoxin, to that obtained in cells from those patients in SR, none of whom were taking digoxin. Fig. 6B shows that chronic AF, in the absence of the influence of digoxin therapy, was not associated with a significant change in the density or K+o-sensitivity of atrial Ip, with similar mean Ip [K+]o-response curves (Fig. 6Bi) and similar mean EC50 and Emax values (Fig. 6Bii) between the two patient groups.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The Na, K pump current, Ip, was measured directly for the first time to our knowledge in human myocardium, and was shown to have similar characteristics to those recorded in several other species, including the responses to extracellular potassium, voltage and ouabain [3–7]. The half-maximal response to K+o was within the reported range (1.0–2.8 mM), and the Ip-voltage relationship was linear, in line with the majority of studies, although this may have been influenced by incomplete K+ current suppression, since Ip saturation was reported at 0 mV [6]. In addition, evidence has been provided, novel in any species, of a lack of change in atrial Ip associated with chronic AF, and also of a reduction in Ip associated with long-term digoxin therapy.

In cells from patients in chronic AF, Ip displayed a similar density, voltage- and [K+]o-sensitivity to that of cells from patients in SR, indicating that a change in neither the density nor functional properties of atrial Ip is involved in human AF-induced atrial electrophysiological remodelling. This is in line with the reported absence of change in atrial Na, K-ATPase activity following 2 h of AF in sheep [16], and with the lack of effect of digoxin on atrial electrophysiological remodelling produced by 24 h of AF in goats [17], although in the latter model, digoxin delayed the reversal of such remodelling [18]. However, the magnitude of Ip in-vivo may increase in patients during AF, since the rapid rate would increase the time spent in depolarisation. This may increase both [Na+]i and [K+]o, each of which increases Ip [3,4,6,7], as confirmed by the present data, which would contribute to the shortening of atrial APD and ERP during AF. However, Ip cannot be the major factor since atrial electrophysiological remodelling is observed in human atrial isolated cells even when the ionic concentrations are controlled [10–13,25].

Long-term digoxin therapy was associated with a small reduction in atrial Ip. This was consistent with the reported reduction by digoxin therapy in the hyperpolarisation caused by rewarming of human atrial tissue considered due to reactivation of the Na, K pump following [Na+]i-loading [23]. The present magnitude of Ip reduction, of ~20%, corresponded with the reported percentage occupancy of digitalis receptors in chronically digitalised failing human ventricle, measured after protracted washing of tissues in anti-digoxin antibodies [26]. Alternatively, the observed reduction in Ip may represent an adaptive change analogous to the pharmacological remodelling of human atrial ion currents by chronic β-blockade [20]. Such a process would be consistent with the reduction in the number of Na, K-ATPase sites in HeLa cells exposed to ouabain [27]. It is not known whether the Ip reduction was influenced mainly by residual receptor occupancy or by pharmacological remodelling. Since digoxin increased the sensitivity of Ip to K+o, an effect on the Na, K-ATPase {alpha}-2 isoform is a possibility, since its activity is strongly dependent on [K+]o [28]. Long-term ACE inhibition did not affect human atrial Ip, in agreement with the reported lack of effect on human atrial cell action potentials and ERP [20]. There have been no previous reports of effects of ACE inhibitors on human atrial Ip, although ventricular Ip was increased in captopril-treated rabbits, due to a reduction in interstitial and/or intracellular concentrations of angiotensin II, and not to a change in Na, K-ATPase expression [24].

A change in atrial Ip, whether by digoxin therapy as observed here, or by altered voltage and/or ionic conditions during AF in the absence of an electrophysiologically-remodelled Na,K pump would affect the APD and ERP, particularly with the high input resistance of human atrial cells [12,20]. Our results with ouabain suggested the potential for such an effect. It should be noted, however, that although ouabain completely blocked Ip and prolonged the APD and ERP in cells in-vitro, the degree of block in-vivo in patients treated with digoxin would be less (and possibly as low as the observed 19% reduction in Emax), and the consequent effect on the APD and ERP may also, therefore, be predicted to be less than that observed in-vitro. No reports of effects of cardiac glycosides on action potentials in human atrial cells were found, but a small prolongation in atrial ERP was reported in patients administered ouabain [29] or digoxin [30]. However, acute digoxin had no effect on the ERP in goats [17,18] and APD-shortening by digitalis has also been reported [31,32]. In human atrial fibres [31], APD-shortening was secondary to vagal stimulation, but in guinea pig ventricular myocytes [32], it was due to a secondary ionic effect of Ip block, namely attenuation of the normally inward Na+/Ca2+ exchanger current (INa/Ca) by ouabain-induced [Na+]i-loading. The effects of digitalis on the APD and ERP in-vivo therefore result from a complex interaction between direct and secondary effects of Ip blockade and effects of vagal stimulation. It is also the case that the measurement of ERP that is made in single cells is not identical to that made in-vivo, since ERP is conventionally measured in terms of propagation failure, which cannot be measured in single cells. Nevertheless, action potentials of amplitude >80% of normal, as used here to define the cell ERP, have been shown to propagate, with graded responses occurring at lower amplitudes [21]. Additionally, 10 µM ouabain may affect action potentials via currents additional to Ip, such as ICaL [33]. However, since ICaL blockade with nifedipine had no effect on Vm or ERP in human atrial cells [12], this suggests that 10 µM ouabain largely affected these measurements by blocking Ip, supported by a mathematical model [5].

Digoxin is used clinically as an inotropic drug to improve haemodynamic function, and also for ventricular rate control during AF, through central and peripheral augmentation of vagal tone to prolong the AV nodal ERP. Long-term digoxin treatment is positively inotropic by increasing [Ca2+]i, secondary to [Na+]i-loading by Ip blockade, although therapeutic serum concentrations (1–2 nM) are at least 50-fold lower than the minimum 0.1 µM required for acute Ip block [34]. [Na+]i-loading may contribute to APD-shortening in the atrium in-vivo, via a reduced INa/Ca, and [Ca2+]i-loading may also shorten the APD, via electrophysiological remodelling of ICaL and ITO [13]. Either or both of these influences on the APD may explain the reported delay by digoxin of recovery from AF-induced atrial electrophysiological remodelling in goats [18], and may also contribute to the lack of efficacy of digoxin in converting human AF [35]. Of note, in previous studies of human atrial cellular electrophysiological remodelling, the majority of patients in AF were taking digoxin [11,12,25]. However, any influence on the APD of [Na+]i-loading would have been removed by the controlled [Na+]i in each of these studies. Moreover, in the presence of residually-bound or down-regulated digoxin receptors, a reduction in the hyperpolarising and repolarising influences of Ip would contribute a direct APD-lengthening effect, as observed here with ouabain. Thus, the reported APD-shortening associated with chronic AF in human atrial cells [11,12] may have been underestimated.

In conclusion, the present data indicate that chronic AF in humans, in the absence of the influence of digoxin therapy, was not associated with a significant change in the density of atrial Ip, or in its sensitivity to voltage or extracellular K+.

Time for primary review 20 days.


   Acknowledgements
 
We acknowledge the British Heart Foundation for financial support, Julie Russell for isolating the cells and managing the patient database, and the Glasgow Royal Infirmary cardiac surgical operating teams for kindly providing atrial tissue.


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

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