Omega-3 polyunsaturated fatty acids inhibit transient outward and ultra-rapid delayed rectifier K+currents and Na+current in human atrial myocytes (original) (raw)

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Department of Medicine and Research Centre of Heart

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Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of Medicine, University of Hong Kong

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Pokfulam, Hong Kong, SAR

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China

2

Department of Physiology

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Li Ka Shing Faculty of Medicine, The University of Hong Kong

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Pokfulam, Hong Kong, SAR

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China

*Corresponding author. Tel: +852 2819 9513; fax:

+852 2855 9730

. E-mail address: grli@hkucc.hku.hk

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Department of Medicine and Research Centre of Heart

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Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of Medicine, University of Hong Kong

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Pokfulam, Hong Kong, SAR

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China

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Department of Medicine and Research Centre of Heart

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Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of Medicine, University of Hong Kong

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Pokfulam, Hong Kong, SAR

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China

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Cardiothoracic Unit, Grantham Hospital, Li Ka Shing Faculty of Medicine, Pokfulam, The University of Hong Kong

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Hong Kong, SAR

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China

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Cardiothoracic Unit, Grantham Hospital, Li Ka Shing Faculty of Medicine, Pokfulam, The University of Hong Kong

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Hong Kong, SAR

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China

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Department of Medicine and Research Centre of Heart

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Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of Medicine, University of Hong Kong

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Pokfulam, Hong Kong, SAR

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China

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Department of Medicine and Research Centre of Heart

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Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of Medicine, University of Hong Kong

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14 February 2008

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20 November 2008

Published:

24 November 2008

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Gui-Rong Li, Hai-Ying Sun, Xiao-Hua Zhang, Lik-Cheung Cheng, Shui-Wah Chiu, Hung-Fat Tse, Chu-Pak Lau, Omega-3 polyunsaturated fatty acids inhibit transient outward and ultra-rapid delayed rectifier K+currents and Na+current in human atrial myocytes, Cardiovascular Research, Volume 81, Issue 2, 1 February 2009, Pages 286–293, https://doi.org/10.1093/cvr/cvn322
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Abstract

Aims

The omega-3 (n-3) polyunsaturated fatty acids (omega-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil were recently reported to have an anti-atrial fibrillation effect in humans; however, the ionic mechanisms of this effect are not fully understood. The present study was designed to determine the effects of EPA and DHA on transient outward and ultra-rapid delayed rectifier potassium currents (_I_to and _I_Kur) and the voltage-gated sodium current (_I_Na) in human atrial myocytes.

Methods and results

A whole-cell patch voltage clamp technique was employed to record _I_to and _I_Kur, and _I_Na in human atrial myocytes. It was found that EPA and DHA inhibited _I_to in a concentration-dependent manner (IC50: 6.2 µM for EPA; 4.1 µM for DHA) and positively shifted voltage-dependent activation of the current. In addition, _I_Kur was suppressed by 1–50 µM EPA (IC50: 17.5 µM) and DHA (IC50: 4.3 µM). Moreover, EPA and DHA reduced _I_Na in human atrial myocytes in a concentration-dependent manner (IC50: 10.8 µM for EPA; 41.2 µM for DHA) and negatively shifted the potential of INa availability. The _I_Na block by EPA or DHA was use-independent.

Conclusion

The present study demonstrates for the first time that EPA and DHA inhibit human atrial _I_to, _I_Kur, and _I_Na in a concentration-dependent manner; these effects may contribute, at least in part, to the anti-atrial fibrillation of omega-3 PUFAs in humans.

1. Introduction

Atrial fibrillation (AF) is a common form of cardiac dysrhythmia, and the occurrence of AF increases with age: the prevalence rises from 0.5% of people in their 50s, to 5% of people over the age of 65 years, and to nearly 10% of the population over 80. AF is a major cause of morbidity and mortality as it increases the risk of death, congestive heart failure, and stroke in an ageing population.1,2 It is believed that AF is a lifetime risk in an ageing population, and therefore it is emerging as a major public-health concern.3,4 Anti-arrhythmic drug therapy remains the principal approach for suppressing AF and its recurrence.5

Recent experimental and clinical studies have shown that omega-3 polyunsaturated fatty acids (omega-3 PUFAs) from fish oil may be effective in preventing cardiac arrhythmias and sudden death.6–10 The omega-3 PUFAs were found to have a significant anti-arrhythmic action in rat atrial myocytes.11 Human consumption of fish has been associated with a lower incidence of AF in a follow-up study.6,12 Intake of omega-3 PUFAs is found to reduce the incidence of post-operative AF of coronary bypass surgery with a shorter hospital stay.10 A recent study demonstrated that omega-3 PUFAs suppressed congestive heart failure-induced electrical remodelling and AF induction in a dog model.13 Although omega-3 PUFAs have been found to block cardiac voltage-gated sodium current (_I_Na), and L-type Ca2+ current (_I_Ca.L) in cells from animal species,14 the ionic mechanism underlying the anti-AF action is not fully understood in humans, and a study with human atrium is therefore suggested.15 The transient outward and ultra-rapid delayed rectifier K+ currents (_I_to and _I_Kur) are found to play an important role in the repolarization of human atrium;16,17 thus both _I_to and _I_Kur are targets for the anti-AF study.5,18 The aim of this study was to investigate the effects of the omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on _I_to, _I_Kur, and _I_Na in adult human atrial myocytes using a whole-cell patch technique.

2. Methods

2.1 Myocyte preparation

Atrial cells were isolated from specimens of human right atrial appendage obtained from patients undergoing coronary artery bypass grafting. The procedure for obtaining the tissues was approved by the Ethics Committee of the University of Hong Kong, based on the patients' consent. The investigation conforms with the principles outlined in the Declaration of Helsinki (see Cardiovasc Res 1997;**35:**2–4) for use of human tissue. All patients were free from supraventricular tachyarrythmias, and the atria were grossly normal at the time of surgery. Atrial myocytes were enzymatically dissociated as described previously19,20 and/or in Supplementary material online.

2.2 Solutions

The compositions of the Ca2+-free cardioplegic solution, Tyrode solution, and pipette solution used in present study were described previously19,20 and in Supplementary material online.

2.3 Data acquisition and analysis

The whole-cell patch-clamp technique was used for electrophysiological recording at room temperature (22–23°C) as described previously19,20 and in Supplementary material online. Nonlinear curve fitting was performed using Pulsefit (HEKA) and Sigmaplot (SPSS, Chicago, IL, USA). Paired and/or unpaired Student's _t_-test was used as appropriate to evaluate the statistical significance of differences between two group means, and analysis of variance was used for multiple groups. Values of P < 0.05 were considered to indicate statistical significance. Group data are expressed as mean ± SEM.

3. Results

3.1 Effects of polyunsaturated fatty acids on _I_to

Figure 1A shows the time course of peak _I_to recorded in a human atrial myocyte with a 300 ms voltage step to +50 from −50 mV in the absence and presence of EPA. EPA at 10 µM gradually decreased _I_to, and the sustained current (i.e. _I_Kur). The inhibitory effect of the currents only slightly recovered on washout for 10 min. Bovine serum albumin (BSA) was reported to completely reverse the EPA-induced inhibitory effect on Ca2+ current in cardiac myocytes21 or _I_Na in HEK 293 cell line.22 Therefore, a bath solution containing 0.1% BSA was applied to wash EPA. Figure 1B shows that the inhibition of _I_to by EPA was rapidly reversed by 0.1% BSA.

Figure 1

Effect of eicosapentaenoic acid on _I_to. (A) Time-dependent effect of 10 µM eicosapentaenoic acid on _I_to elicited by a 300 ms voltage step to +50 from −50 mV (left inset) delivered every 15 s in a typical experiment. _I_to was measured from peak to ‘quasi’-steady-state level. The original current traces at corresponding time points are shown in the right inset. (B ) Eicosapentaenoic acid (10 µM) decreased _I_to in another cell, and the inhibitory effect recovered on washout with 0.1% bovine serum albumin. (C) _I_to traces recorded in a representative cell with the protocol as shown in the inset (0.2 Hz) during control, in the presence of 2 µM diphenyl phosphine oxide-1 for 6 min, and in the co-presence of diphenyl phosphine oxide-1 and 10 µM eicosapentaenoic acid, and washout with 0.1% bovine serum albumin containing 2 µM diphenyl phosphine oxide-1. (D) I–V relationships of _I_to in the presence of 2 µM diphenyl phosphine oxide-1 (control), co-application of diphenyl phosphine oxide-1 and 1, 5, and 10 µM eicosapentaenoic acid (10 min). Eicosapentaenoic acid inhibited _I_to in a concentration-dependent manner (P < 0.05 or P < 0.01 at +10 to +60 mV vs. control). (E). Concentration–response relationship of eicosapentaenoic acid for inhibiting _I_to at +40 mV (n = 5–11 experiments). Symbols are mean data, and solid line is the best-fit Hill equation: E = _E_max/[1+(IC50/C)b], where E is the percentage for inhibiting _I_to at concentration C, _E_max is the maximum effect, IC50 is the concentration for a half of complete inhibition, and b is the Hill coefficient.

Although the _I_to measured was peak to the ‘quasi’-steady-state level, we suspected that the evaluation of the effect of EPA on _I_to might not be accurate. We therefore used the selective Kv1.5 blocker diphenyl phosphine oxide-1 (DPO-1, Sigma-Aldrich)23 to separate _I_to. DPO-1 (2 µM) induced a slight reduction of peak current amplitude and almost a full inhibition of _I_Kur (Figure 1C). _I_to was substantially suppressed by the combination of DPO-1 with 10 µM EPA, and the effect recovered on washout with 0.1% BSA.

Figure 1D illustrates the current–voltage (I_–_V) relationships of _I_to density during control and in the presence of 1, 5, and 10 µM EPA. EPA inhibited _I_to in a concentration-dependent manner. EPA at 1–10 µM suppressed _I_to at test potentials of +10 to +60 mV (n = 7, P < 0.05 or P < 0.01 vs. control), and no significant voltage dependence was observed. The concentration–response relationship for the inhibition of _I_to by EPA from 1 to 50 µM was evaluated at +40 mV (Figure 1D). The IC50 of EPA for inhibiting _I_to was 6.2 µM, with a Hill coefficient of 0.9.

Figure 2A shows the time course of _I_to recorded in the absence or presence of 10 µM DHA in a typical experiment without DPO-1 treatment. DHA slowly decreased _I_to, and the effect reached a steady-state level with over 12 min superfusion. The effect was not reversible by 10 min washout; however, 0.1% BSA bath solution completely reversed DHA's inhibition of _I_to in another representative cell (Figure 2B). DHA, as EPA, inhibited both _I_to and _I_Kur. Original traces are shown in the right of the panels. The inhibition of voltage-dependent _I_to by 5 and 10 µM DHA in the cell pre-treated with 2 µM DPO-1 to suppress _I_Kur was also fully reversed by BSA solution washout (Figure 2C). Figure 2D illustrates the I–V relationships of _I_to during control and in the presence of 1, 5, and 10 µM DHA. DHA at 1–10 µM suppressed _I_to at +10 to +60 mV (n = 6, P < 0.05 or P < 0.01). The IC50 of DHA for inhibiting _I_to was 4.1 µM (Figure 2E), with a Hill coefficient of 0.9.

Figure 2

Docosahexaenoic acid effect on _I_to. (A) Time-dependent effect of 10 µM docosahexaenoic acid on _I_to recorded with the protocol shown in the inset in a typical experiment. Docosahexaenoic acid remarkably suppressed the current, and the inhibition was not reversible on washout. The original current traces at corresponding time points are shown in the right inset. (B) Docosahexaenoic acid decreased _I_to in another cell, and the inhibition fully recovered on washout with 0.1% bovine serum albumin. (C)_I_to traces recorded in a representative cell with the voltage protocol as shown in the inset in the presence of 2 µM diphenyl phosphine oxide-1 (control), co-presence of diphenyl phosphine oxide-1 and 5, 10 µM docosahexaenoic acid, and washout for 10 min with 0.1% bovine serum albumin containing 2 µM diphenyl phosphine oxide-1. (D)I_–_V relationships of _I_to in the presence of 2 µM diphenyl phosphine oxide-1 (control), co-presence of diphenyl phosphine oxide-1 and 1, 5, and 10 µM docosahexaenoic acid (15 min). Docosahexaenoic acid inhibited _I_to in a concentration-dependent manner (P < 0.05 or P < 0.01 at +10 to +60 mV vs. control) (E) Concentration–response relationship of docosahexaenoic acid for inhibiting _I_to at +40 mV (n = 4–9 experiments). Symbols are mean data, and solid line is the best-fit Hill equation.

By fitting the inactivation process of _I_to, we found that EPA and DHA (10 µM) had no significant effect on time-dependent inactivation (data not shown). The voltage dependence of activation conductance variable (g) of _I_to was determined from I–V relationships for each cell (Figure 1C) as described previously.24 The normalized activation (g/_g_max) value was fitted to the Boltzmann distribution to obtain half activation (_V_0.5) and slope factor of the current (Figure 3A). The _V_0.5 of _I_to activation positively shifted 6.1 mV with 10 µM EPA (from 18.8 ± 0.8 mV in control to 24.9 ± 0.9 mV in EPA, n = 7, P < 0.01). With 10 µM DHA, the _V_0.5 positively shifted 6.6 mV from 18.1 ± 0.6 mV of control to 24.7 ± 1.1 mV (n = 6, P < 0.01). The slope factor was not significantly changed by EPA or DHA.

The variable (I/_I_max) for the voltage-dependent inactivation (availability) of _I_to was determined with the protocol as shown in the inset of Figure 3A and fitted to the Boltzmann distribution under control conditions and in the presence of 10 µM EPA. The _V_0.5 of _I_to availability was not significantly changed by the application of EPA (−23.1 ± 0.8 mV for control, −26.5 ± 0.8 mV for EPA, n = 6, P = NS). Similarly, DHA at 10 µM had no effect on the _V_0.5 of _I_to availability (−23.5 ± 0.6 mV in control, −26.7 ± 1.1 mV in DHA, n = 6, P = NS). The slope factor was not significantly altered by EPA or DHA.

Figure 3

Effects of eicosapentaenoic acid on voltage dependence and recovery of _I_to from inactivation. (A) Symbols are mean values of voltage-dependent variables for _I_to activation conductance (g/_g_max) and availability (I/_I_max) in the absence or presence of 10 µM eicosapentaenoic acid, and solid lines are fitted to the Boltzmann equation: y = 1/{1 + exp[(_V_m−_V_0.5)/_S_]}, where _V_m is membrane potential, _V_0.5 is the midpoint potential, and S is the slope factor. (B) Mean data for time course of recovery of _I_to from inactivation in the absence and presence of 10 µM eicosapentaenoic acid in seven cells. Data were best fit to mono-exponential function. No change in the recovery time constant of _I_to was observed after the application of eicosapentaenoic acid (n = 7, P = NS).

Time-dependent recovery of _I_to from inactivation was studied with the paired-pulse protocol as shown in the inset of Figure 3B. The recovery curves were fitted to a mono-exponential function in the absence or presence of 10 µM EPA. The recovery time constant of _I_to was slightly slowed by EPA (72.3 ± 15.1 ms in control, 78.1 ± 17.3 ms in EPA, n = 7, P = NS). Similarly, DHA at 10 µM had no significant effect on the recovery time constant (73.6 ± 12.5 ms for control, 77.2 ± 14.4 ms after DHA, n = 6, P = NS) of _I_to from inactivation.

3.2 Effects of eicosapentaenoic acid and docosahexaenoic acid on _I_Kur

Figure 4A displays the time course of _I_Kur in a representative experiment with 10 µM EPA superfusion. EPA remarkably reduced _I_Kur, and the inhibition recovered on washout with 0.1% BSA. Voltage-dependent _I_Kur traces recorded in a typical experiment in the absence or presence of EPA are illustrated in Figure 4B. EPA at 10 µM substantially inhibited both _I_Kur and tail current. EPA inhibited _I_Kur in a concentration-dependent manner (Figure 4C). The IC50 of EPA for inhibiting _I_Kur was 17.5 µM, with a Hill coefficient of 1.4.

Figure 4

Effects of eicosapentaenoic acid and docosahexaenoic acid on _I_Kur. (A) Time course of _I_Kur recorded in a typical experiment with a 100 ms pre-pulse to +40 mV to inactivate _I_to, followed by 200 ms test pulses to +50 mV (inset) every 15 s. Eicosapentaenoic acid gradually reduced _I_Kur, and the inhibitory effect fully recovered on washout with 0.1% bovine serum albumin. (B) Voltage-dependent _I_Kur (capacitance compensated) recorded at 0.2 Hz in a representative cell with a 100 ms pre-pulse to +40 mV to inactivate _I_to, followed by 200 ms test pulses to between −40 and +60 from −50 mV after a 10 ms interval, then to −30 mV (inset) in the absence and presence of 10 µM eicosapentaenoic acid. Eicosapentaenoic acid substantially suppressed _I_Kur. The arrow indicates zero level. (C) Concentration–response relationship of _I_Kur inhibition by eicosapentaenoic acid at +40 mV (n = 7–18 experiments). Symbols are the mean values of inhibitory effect in cells exposed to different concentrations of eicosapentaenoic acid. Solid lines are the best-fit Hill equation. (D) Time-dependent reduction of _I_Kur (recorded with the protocol shown in the inset of A) by 10 µM docosahexaenoic acid, and the effect recovered on washout with 0.1% bovine serum albumin. (E) Voltage-dependent _I_Kur recorded at 0.2 Hz in a representative cell with the protocol shown in the inset of (B) before and after the application of 10 µM docosahexaenoic acid (for 15 min). Docosahexaenoic acid substantially suppressed _I_Kur. (F). Concentration–response relationship of _I_Kur inhibition by docosahexaenoic acid at +40 mV (n = 6–17 experiments). Symbols are the mean values of inhibiting effect in cells exposed to different concentrations of docosahexaenoic acid. Solid lines are the best-fit Hill equation.

Figure 4D shows the time course of _I_Kur in a typical experiment with 10 µM DHA superfusion. DHA, as EPA, remarkably reduced _I_Kur, and the inhibition recovered on washout with 0.1% BSA solution. Figure 4E illustrates the effect of DHA on voltage-dependent _I_Kur. DHA substantially decreased _I_Kur in a concentration-dependent manner (Figure 4F). The inhibitory effect of DHA on _I_Kur was stronger than that of EPA. The IC50 of DHA for suppressing _I_Kur was 4.3 µM, with a Hill coefficient of 1.1.

3.3 Influence of eicosapentaenoic acid and docosahexaenoic acid on human atrial _I_Na

Earlier studies reported that EPA and DHA inhibited cardiac _I_Na in animal species and in HEK 293 cells expressing cloned Na+ channels.22,25,26 Here, we evaluated the effect of EPA and DHA on _I_Na in freshly dissociated human atrial myocytes. Figure 5A shows the voltage-dependent _I_Na traces recorded in a human atrial myocyte in the absence or presence of EPA. EPA at 10 µM remarkably reduced the amplitude of _I_Na, and the inhibition was fully reversed by washout with 0.1% BSA bath solution. The I–V relationships of _I_Na density shown in Figure 5B indicate that EPA significantly inhibits _I_Na at −50 to −5 mV in a concentration-dependent manner. The IC50 of EPA for inhibiting _I_Na (at −35 mV, Figure 5C) was 10.8 µM, with a Hill coefficient of 1.2.

Figure 5

Inhibition of _I_Na by eicosapentaenoic acid and docosahexaenoic acid. (A) Voltage-dependent _I_Na traces recorded in a representative myocyte with 30 ms steps to between −70 and −5 from −120 mV at 0.1 Hz during control, in the presence of 10 µM eicosapentaenoic acid, and washout for 10 min with 0.1% bovine serum albumin. (B)I–V relationships of _I_Na during control, in the presence of 5, 10 and 20 µM eicosapentaenoic acid. Eicosapentaenoic acid significantly suppressed _I_Na at potentials of −50 to −10 mV (n = 8, P < 0.05 or P < 0.01 vs. control). (C) Concentration–response relationship of _I_Na inhibition by eicosapentaenoic acid at −30 mV (n = 6–14 experiments). (D) Concentration–response relationship of _I_Na inhibition by docosahexaenoic acid at −30 mV (n = 6–15 experiments). Symbols are the mean values of inhibiting effect in cells exposed to different concentrations of eicosapentaenoic acid or docosahexaenoic acid. Solid lines are the best-fit Hill equation. (E) Voltage dependence of time constant of _I_Na inactivation before and after 10 µM eicosapentaenoic acid (10 min, n = 8, *P < 0.05 vs. control). (F) Voltage dependence of time constant of _I_Na inactivation before and after 20 µM docosahexaenoic acid (15 min, n = 7, *P < 0.05 vs. control). (G) Use dependence of _I_Na before and after 10 µM eicosapentaenoic acid (10 min) at 0.5, 1, 2, and 5 Hz (data values overlapped). _I_Na was normalized by the current elicited by the first pulse at each frequency. (H) Use dependence of _I_Na before and after 20 µM docosahexaenoic acid (15 min) at 0.5, 1, 2, and 5 Hz.

DHA also exhibited an inhibitory effect on _I_Na in human atrial myocytes; nevertheless, the inhibitory action was weaker than that of EPA (Figure 5D). The IC50 of DHA for inhibiting _I_Na was 41.2 µM, with a Hill coefficient of 1.4.

Figure 6

Influence of _I_Na kinetics by eicosapentaenoic acid. (A) Voltage protocol and superimposed current for determining voltage dependence of availability (I/_I_max) of _I_Na. (B) Mean values of voltage dependence of availability and activation (g/_g_max) of _I_Na in the absence and presence of 10 µM eicosapentaenoic acid. Curves were fitted to the Boltzmann distribution. (C) Voltage protocol and current traces used for determining recovery of _I_Na from inactivation. (D) Mean values of curves for recovery of _I_Na from inactivation in the absence and presence of 10 µM eicosapentaenoic acid. Recovery curves were fitted to mono-exponential functions. (E) Voltage protocol and current traces used for determining the development of resting inactivation of _I_Na. (F) Inactivation curves of _I_Na at resting states in the absence and presence of 10 µM eicosapentaenoic acid.

Inactivation of _I_Na traces was fitted to mono-exponential functions before and after 10 µM EPA or 20 µM DHA. The voltage-dependent inactivation time constants are illustrated in Figure 5E and F. EPA or DHA reduced the inactivation time constant; a significant effect was seen only at −50 to −40 or −30 mV (n = 7, P < 0.05 vs. control).

Use-dependent block of _I_Na was examined with EPA or DHA using a train of 15 identical pulses (30-ms) from −120 to −35 mV. EPA (10 µM) or DHA (20 µM) inhibited _I_Na at frequencies of 0.5, 1, 2, 5 Hz; however, the inhibition was use- or rate-independent (Figure 5G and H).

The activation conductance variable (g/_g_max) of _I_Na was determined from I–V relationships for each cell (Figure 5B) and was fitted to the Boltzmann distribution (Figure 6B). The voltage dependence of availability (_I/I_max) of _I_Na was determined as illustrated in Figure 6A, and also fitted to the Boltzmann distribution. Figure 6B shows that 10 µM EPA shifted the midpoint of _I_Na availability towards negative potentials. The _V_0.5 of availability negatively shifted 9.7 mV, from −97.1 ± 1.5 mV in control to −106.9 ± 1.7 mV in EPA (n = 7, P < 0.01). There was no shift for the _V_0.5 of _I_Na activation conductance with 10 µM EPA (−38.8 ± 1.2 mV in control, −39.4 ± 1.5 mV in EPA, n = 6, P = NS). DHA at 20 µM also negatively shifted _V_0.5 of _I_Na availability by 6.5 mV (from −96.8 ± 1.3 mV in control to −103.3 ± 1.4 mV in DHA, n = 6, P < 0.01) and had no effect on the _V_0.5 of _I_Na activation conductance.

Recovery of _I_Na from inactivation was studied with a paired-pulse protocol. Superimposed currents and the time course of recovery are illustrated in Figure 6C and D. _I_Na recovery was complete and well fitted to mono-exponential functions with time constants of 10.6 ± 2.1 ms in control and 18.1 ± 2.5 ms with 10 µM EPA (n = 7, P < 0.01). In another set of experiments, the recovery time constant of _I_Na was 11.5 ± 2.4 ms in control and 16.9 ± 1.9 ms with 20 µM DHA (n = 6, P < 0.01).

The development of resting inactivation of _I_Na was determined with a variable duration of conditioning pre-pulse from −120 mV to a subthreshold potential (−75 mV), followed by a 3 ms step back to −120 mV prior to a test pulse to −35 mV.27Figure 6E shows the voltage protocol (inset) and superimposed _I_Na traces recorded during the test pulse in a typical experiment, showing that _I_Na reduces as the conditioning pre-pulse duration increases. The normalized _I_Na was plotted against the pre-pulse duration and fitted to mono-exponential functions (Figure 6F). The inactivation time constant reduced from 24.8 ± 3.2 ms in control to 12.3 ± 2.9 ms with 10 µM EPA (n = 7, P < 0.01). In the experiments with 20 µM DHA, the inactivation time constant decreased from 25.3 ± 2.5 ms in control to 16.3 ± 3.5 ms in DHA (n = 6, P < 0.01). These results demonstrate that EPA and DHA significantly increase the inactivation of _I_Na from resting states.

4. Discussion

It is well known that fish oil is rich in the omega-3 PUFAs EPA and DHA.25 A study of the prevention of ventricular fibrillation by dietary fish oil following coronary artery occlusion and reperfusion was reported three decades ago.28 Recent epidemiological studies indicate that fish oil consumption may reduce the risk of ventricular arrhythmias in humans, supporting an anti-arrhythmic effect of omega-3 PUFAs,15,29 although the contradictory result was also reported.30 In addition, Calo et al.10 recently demonstrated that the administration of omega-3 PUFAs (EPA and DHA) induced a reduction of the relative risk of post-operative AF in 54.4% patients who had undergone coronary artery bypass. However, the intake of fish oil supplement has been reported to have no effect or to be pro-arrhythmic in different populations.31,32 Therefore, further clinical observation and cellular investigation are suggested to define the possible anti-AF action of omega-3 PUFAs.15

Earlier cellular studies showed that EPA and DHA inhibited _I_Na in neonatal rat ventricular myocytes26 and in HEK293 cells transfected with the human cardiac Na+ channel hH1,22 L-type Ca2+ current (_I_Ca.L) in rat ventricular cells,33 and the potassium currents _I_K and _I_to in ferret ventricular myocytes.14 It is believed that omega-3 PUFAs modify Na+ channels by directly binding to the channel proteins,34 although an decreased _I_Na was not observed in a recent study in pigs with a fish oil diet for 8 weeks.35 The information obtained from animal species on the effects of omega-3 PUFAs on cardiac ion channels reveals important alterations in the ionic currents that may account for significant cardiac protection against fibrillation. However, we are not aware of PUFAs-mediated alteration of ion channel properties in human cardiac myocytes.

In human heart, the transient outward K+ current _I_to, the ultra-rapid delayed rectifier K+ current _I_Kur, and the rapid and slow components _I_Kr and _I_Ks of delayed rectifier K+ current are important repolarization currents.16,17,36_I_Kur is found to be functionally expressed in human atrium, but not in the ventricle.16 Therefore, the drugs that specifically inhibit the unique _I_Kur may provide a means of preventing AF without the risk of ventricular pro-arrhythmia.5 The inhibition of _I_to and/or _I_Kur was reported to prolong the action potential duration in human atrium.37,38

The present study showed that EPA and DHA significantly inhibited _I_to and _I_Kur in human atrial myocytes (Figures 1–4). The relatively irreversible effects of EPA and DHA on _I_to and _I_Kur are likely related to their directly binding to the channel proteins.34 The suppression of _I_to and _I_Kur is useful for anti-AF.5 Therefore, the inhibitory effects of EPA and DHA on _I_Kur and _I_to would be most likely one of ionic mechanisms of anti-AF observed in humans.6,10,39 However, a reduced _I_to was consistently found in atrial myocytes from patients with AF,40,41 while increased, unchanged, or decreased _I_Kur was reported in different observations.40–42 This may account for the observation of unsuccessful AF prevention.43

As previously reported in neonatal rat ventricular myocytes and HEK 293 cells expressing human cardiac Na+ sodium channels (hH1),22,26 EPA and DHA inhibited _I_Na in human atrial myocytes in a use-independent way (Figure 5), which is different from that of the anti-arrhythmic drug lidocaine.44 EPA and DHA negatively shifted the potential of _I_Na availability and increased the development of _I_Na inactivation at resting states (Figure 6). These effects would be cardioprotection against cardiac fibrillation.14,25 EPA had a stronger suppression of _I_Na (Figure 5), while DHA showed a stronger inhibition of _I_to and _I_Kur in human atrial myocytes (Figures 1–4). Therefore, fish oil containing both EPA and DHA would be more effective in anti-AF in humans. However, on the other hand, the _I_Na block may be related to the increase of ventricular arrhythmias observed in some people taking fish oil supplement.30,45

The inhibitory effect of EPA (IC50: 6.2 µM) or DHA (IC50: 4.1 µM) on _I_to was slightly stronger in human atrial myocytes than that (IC50: ∼7.5 µM) in ferret ventricular myocytes46 However, the inhibition of _I_Na by EPA (IC50: 10.8 µM), and especially DHA (IC50: 41.2 µM), was weaker in human atrial myocytes than that (IC50: <5 µM) in neonatal rat ventricular cells,26 whereas the inhibitory effect of EPA (IC50: 17.5 µM), and especially DHA (IC50: 4.3 µM), on _I_Kur was stronger in human atrial myocytes than that (IC50: 20–30 µM for DHA) observed in Kv1.5 cell line.47 These differences are likely related to various species and/or types of cells. Nevertheless, the ‘acute’ omega-3 PUFAs effects on cardiac ion channel function may not be completely applicable to the chronic effects of fish oil supplements in the ‘real world’ setting, which is a limitation of an ‘acute’ _in vitro_ study vs. chronic _in vivo_ effect. The information for the concentration levels in cardiac tissue and/or blood plasma remains to be established. It is important to note the superfusion of BSA rapidly reversed the omega-3 PUFAs effect (_Figures 1_, _2_, _4_, and _5_), indicating that omega-3 PUFAs have a high affinity to blood plasma albumin. Therefore, the effects of PUFAs on ion channels observed in the present study would most likely only be reproducible when the cardiac tissue concentration levels and/or free blood plasma concentration levels reached >4–6 µM.

Another limitation of the present study was that the specific _I_Kur blocker DPO-1 used to separate _I_to is a partial open channel blocker and shows less block of Kv1.5 at initial opening of the channel.23 The incomplete inhibition of _I_Kur at initial opening of the channel would underestimate the _I_to inhibition by omege-3 PUFAs.

Although the study with human atrial specimens from the patients with dietary fish oil is indicated,15 an ethical issue arises for such experiments. Here, we only focused the study on the ‘acute’ effects of omega-3 PUFAs on _I_to, _I_Kur, and _I_Na in human atrial cells with the limited human atrial specimens. The effects of omega-3 PUFAs on other ion channel currents (e.g. _I_Ca.L, _I_Kr, and _I_Ks) and action potentials were not verified in human atrial myocytes due to the shortage of human atrial specimens, which occurred because the cardiac coronary artery bypass surgical procedure has been recently improved to avoid cutting atrial tissue. It was reported that DHA inhibited _I_K in ferret ventricular myocytes46 and hERG channel expressed in Chinese hamster ovary cells,48 which may also contribute to anti-arrhythmias.

In summary, the present study demonstrates the novel information that the omega-3 PUFAs EPA and DHA from fish oil inhibited _I_to, _I_Kur, and _I_Na in human atrial myocytes. These effects likely contribute at least in part to the anti-AF action observed in humans.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Funding

The work was supported by Sun Chieh Yeh Heart Foundation of Hong Kong.

Acknowledgement

The authors thank Heather J. Ballard for her critical reading of the manuscript.

Conflict of interest: none declared.

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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Topic:

• atrium
• omega-3 fatty acids
• polyunsaturated fatty acids
• fish oils
• eicosapentaenoic acid
• docosahexaenoic acids
• muscle cells

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