Download Omega-3 polyunsaturated fatty acids inhibit transient outward and

Cardiovascular Research (2009) 81, 286–293
doi:10.1093/cvr/cvn322
Omega-3 polyunsaturated fatty acids inhibit transient
outward and ultra-rapid delayed rectifier K1currents
and Na1current in human atrial myocytes
Gui-Rong Li1,2*, Hai-Ying Sun1, Xiao-Hua Zhang1, Lik-Cheung Cheng3, Shui-Wah Chiu3,
Hung-Fat Tse1, and Chu-Pak Lau1
1
Department of Medicine and Research Centre of Heart, Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of Medicine,
University of Hong Kong, Pokfulam, Hong Kong, SAR, China; 2Department of Physiology, Li Ka Shing Faculty of Medicine, The
University of Hong Kong, Pokfulam, Hong Kong, SAR, China; and 3Cardiothoracic Unit, Grantham Hospital, Li Ka Shing Faculty
of Medicine, Pokfulam, The University of Hong Kong, Hong Kong, SAR, China
Received 14 February 2008; revised 18 November 2008; accepted 20 November 2008; online publish-ahead-of-print 24 November 2008
Time for primary review: 33 days
KEYWORDS
Human;
Atrial myocytes;
Ion channels;
Omega-3 PUFAs
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 (Ito and IKur) and the voltage-gated sodium current (INa) in human atrial myocytes.
Methods and results A whole-cell patch voltage clamp technique was employed to record Ito and IKur,
and INa in human atrial myocytes. It was found that EPA and DHA inhibited Ito in a concentrationdependent manner (IC50: 6.2 mM for EPA; 4.1 mM for DHA) and positively shifted voltage-dependent
activation of the current. In addition, IKur was suppressed by 1–50 mM EPA (IC50: 17.5 mM) and DHA
(IC50: 4.3 mM). Moreover, EPA and DHA reduced INa in human atrial myocytes in a concentrationdependent manner (IC50: 10.8 mM for EPA; 41.2 mM for DHA) and negatively shifted the potential of
INa availability. The INa block by EPA or DHA was use-independent.
Conclusion The present study demonstrates for the first time that EPA and DHA inhibit human atrial Ito,
IKur, and INa 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 Antiarrhythmic 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
* Corresponding author. Tel: þ852 2819 9513; fax: þ852 2855 9730.
E-mail address: [email protected]
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 postoperative 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 (INa), and L-type Ca2þ current (ICa.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 Kþ currents (Ito
and IKur) are found to play an important role in the repolarization of human atrium;16,17 thus both Ito and IKur 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 Ito, IKur, and INa in adult
human atrial myocytes using a whole-cell patch technique.
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008.
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Fish oil on human atrial Ito, IKur, and INa
Figure 1 Effect of eicosapentaenoic acid on Ito. (A) Time-dependent effect of 10 mM eicosapentaenoic acid on Ito elicited by a 300 ms voltage step to þ50 from
250 mV (left inset) delivered every 15 s in a typical experiment. Ito 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 mM) decreased Ito in another cell, and the inhibitory effect recovered on washout
with 0.1% bovine serum albumin. (C) Ito traces recorded in a representative cell with the protocol as shown in the inset (0.2 Hz) during control, in the presence of
2 mM diphenyl phosphine oxide-1 for 6 min, and in the co-presence of diphenyl phosphine oxide-1 and 10 mM eicosapentaenoic acid, and washout with 0.1% bovine
serum albumin containing 2 mM diphenyl phosphine oxide-1. (D) I–V relationships of Ito in the presence of 2 mM diphenyl phosphine oxide-1 (control),
co-application of diphenyl phosphine oxide-1 and 1, 5, and 10 mM eicosapentaenoic acid (10 min). Eicosapentaenoic acid inhibited Ito in a concentrationdependent manner (P , 0.05 or P , 0.01 at þ10 to þ60 mV vs. control). (E). Concentration–response relationship of eicosapentaenoic acid for inhibiting Ito
at þ40 mV (n ¼ 5–11 experiments). Symbols are mean data, and solid line is the best-fit Hill equation: E ¼ Emax/[1þ(IC50/C )b], where E is the percentage
for inhibiting Ito at concentration C, Emax is the maximum effect, IC50 is the concentration for a half of complete inhibition, and b is the Hill coefficient.
2. Methods
2.3 Data acquisition and analysis
2.1 Myocyte preparation
The whole-cell patch-clamp technique was used for electrophysiological recording at room temperature (22–238C) 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.
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.
3. Results
3.1 Effects of polyunsaturated fatty acids on Ito
2.2 Solutions
2þ
The compositions of the Ca -free cardioplegic solution, Tyrode solution, and pipette solution used in present study were described
previously19,20 and in Supplementary material online.
Figure 1A shows the time course of peak Ito recorded in a
human atrial myocyte with a 300 ms voltage step to þ50
from 250 mV in the absence and presence of EPA. EPA at
10 mM gradually decreased Ito, and the sustained current
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G.-R. Li et al.
Figure 2 Docosahexaenoic acid effect on Ito. (A) Time-dependent effect of 10 mM docosahexaenoic acid on Ito 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 Ito in another cell, and the inhibition fully recovered on washout with
0.1% bovine serum albumin. (C) Ito traces recorded in a representative cell with the voltage protocol as shown in the inset in the presence of 2 mM diphenyl
phosphine oxide-1 (control), co-presence of diphenyl phosphine oxide-1 and 5, 10 mM docosahexaenoic acid, and washout for 10 min with 0.1% bovine serum
albumin containing 2 mM diphenyl phosphine oxide-1. (D) I–V relationships of Ito in the presence of 2 mM diphenyl phosphine oxide-1 (control), co-presence of
diphenyl phosphine oxide-1 and 1, 5, and 10 mM docosahexaenoic acid (15 min). Docosahexaenoic acid inhibited Ito in a concentration-dependent manner
(P , 0.05 or P , 0.01 at þ10 to þ60 mV vs. control) (E) Concentration–response relationship of docosahexaenoic acid for inhibiting Ito at þ40 mV (n ¼ 4–9
experiments). Symbols are mean data, and solid line is the best-fit Hill equation.
(i.e. IKur). 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 Ca2þ current in cardiac myocytes21 or
INa 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 Ito by EPA was rapidly reversed by 0.1% BSA.
Although the Ito measured was peak to the ‘quasi’steady-state level, we suspected that the evaluation of
the effect of EPA on Ito might not be accurate. We therefore
used the selective Kv1.5 blocker diphenyl phosphine oxide-1
(DPO-1, Sigma-Aldrich)23 to separate Ito. DPO-1 (2 mM)
induced a slight reduction of peak current amplitude and
almost a full inhibition of IKur (Figure 1C). Ito was substantially suppressed by the combination of DPO-1 with 10 mM
EPA, and the effect recovered on washout with 0.1% BSA.
Figure 1D illustrates the current–voltage (I–V ) relationships of Ito density during control and in the presence of 1,
5, and 10 mM EPA. EPA inhibited Ito in a concentrationdependent manner. EPA at 1–10 mM suppressed Ito at test
potentials of þ10 to þ60 mV (n ¼ 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 Ito by EPA from 1 to 50 mM was evaluated at
þ40 mV (Figure 1D). The IC50 of EPA for inhibiting Ito was
6.2 mM, with a Hill coefficient of 0.9.
Figure 2A shows the time course of Ito recorded in the
absence or presence of 10 mM DHA in a typical experiment
without DPO-1 treatment. DHA slowly decreased Ito, 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 Ito in another representative
cell (Figure 2B). DHA, as EPA, inhibited both Ito and IKur. Original traces are shown in the right of the panels. The inhibition of voltage-dependent Ito by 5 and 10 mM DHA in the
cell pre-treated with 2 mM DPO-1 to suppress IKur was also
fully reversed by BSA solution washout (Figure 2C).
Figure 2D illustrates the I–V relationships of Ito during
control and in the presence of 1, 5, and 10 mM DHA.
DHA at 1–10 mM suppressed Ito at þ10 to þ60 mV (n ¼ 6,
P , 0.05 or P , 0.01). The IC50 of DHA for inhibiting Ito
was 4.1 mM (Figure 2E), with a Hill coefficient of 0.9.
By fitting the inactivation process of Ito, we found that EPA
and DHA (10 mM) had no significant effect on time-dependent
inactivation (data not shown). The voltage dependence of
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Fish oil on human atrial Ito, IKur, and INa
slowed by EPA (72.3 + 15.1 ms in control, 78.1 + 17.3 ms
in EPA, n ¼ 7, P ¼ NS). Similarly, DHA at 10 mM had no significant effect on the recovery time constant (73.6 + 12.5 ms
for control, 77.2 + 14.4 ms after DHA, n ¼ 6, P ¼ NS) of Ito
from inactivation.
3.2 Effects of eicosapentaenoic acid and
docosahexaenoic acid on IKur
Figure 4A displays the time course of IKur in a representative
experiment with 10 mM EPA superfusion. EPA remarkably
reduced IKur, and the inhibition recovered on washout with
0.1% BSA. Voltage-dependent IKur traces recorded in a typical
experiment in the absence or presence of EPA are illustrated
in Figure 4B. EPA at 10 mM substantially inhibited both IKur
and tail current. EPA inhibited IKur in a concentrationdependent manner (Figure 4C). The IC50 of EPA for inhibiting
IKur was 17.5 mM, with a Hill coefficient of 1.4.
Figure 4D shows the time course of IKur in a typical experiment with 10 mM DHA superfusion. DHA, as EPA, remarkably
reduced IKur, and the inhibition recovered on washout with
0.1% BSA solution. Figure 4E illustrates the effect of DHA
on voltage-dependent IKur. DHA substantially decreased IKur
in a concentration-dependent manner (Figure 4F). The
inhibitory effect of DHA on IKur was stronger than that of
EPA. The IC50 of DHA for suppressing IKur was 4.3 mM, with
a Hill coefficient of 1.1.
Figure 3 Effects of eicosapentaenoic acid on voltage dependence and recovery of Ito from inactivation. (A) Symbols are mean values of voltage-dependent
variables for Ito activation conductance (g/gmax) and availability (I/Imax) in the
absence or presence of 10 mM eicosapentaenoic acid, and solid lines are fitted
to the Boltzmann equation: y ¼ 1/{1 þ exp[(Vm 2V0.5)/S]}, where Vm is membrane potential, V0.5 is the midpoint potential, and S is the slope factor. (B)
Mean data for time course of recovery of Ito from inactivation in the absence
and presence of 10 mM eicosapentaenoic acid in seven cells. Data were best fit
to mono-exponential function. No change in the recovery time constant of Ito
was observed after the application of eicosapentaenoic acid (n ¼ 7, P ¼ NS).
activation conductance variable (g) of Ito was determined
from I–V relationships for each cell (Figure 1C) as described
previously.24 The normalized activation (g/gmax) value was
fitted to the Boltzmann distribution to obtain half activation
(V0.5) and slope factor of the current (Figure 3A). The V0.5 of
Ito activation positively shifted 6.1 mV with 10 mM EPA (from
18.8 + 0.8 mV in control to 24.9 + 0.9 mV in EPA, n ¼ 7,
P , 0.01). With 10 mM DHA, the V0.5 positively shifted
6.6 mV from 18.1 + 0.6 mV of control to 24.7 + 1.1 mV (n ¼
6, P , 0.01). The slope factor was not significantly changed
by EPA or DHA.
The variable (I/Imax) for the voltage-dependent inactivation (availability) of Ito 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 mM EPA. The V0.5 of Ito availability was not significantly
changed by the application of EPA (223.1 + 0.8 mV for
control, 226.5 + 0.8 mV for EPA, n ¼ 6, P ¼ NS). Similarly,
DHA at 10 mM had no effect on the V0.5 of Ito availability
(223.5 + 0.6 mV in control, 226.7 + 1.1 mV in DHA, n ¼ 6,
P ¼ NS). The slope factor was not significantly altered by
EPA or DHA.
Time-dependent recovery of Ito 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 mM EPA. The recovery time constant of Ito was slightly
3.3 Influence of eicosapentaenoic acid and
docosahexaenoic acid on human atrial INa
Earlier studies reported that EPA and DHA inhibited cardiac INa
in animal species and in HEK 293 cells expressing cloned Naþ
channels.22,25,26 Here, we evaluated the effect of EPA and
DHA on INa in freshly dissociated human atrial myocytes.
Figure 5A shows the voltage-dependent INa traces recorded in
a human atrial myocyte in the absence or presence of EPA.
EPA at 10 mM remarkably reduced the amplitude of INa, and
the inhibition was fully reversed by washout with 0.1% BSA
bath solution. The I–V relationships of INa density shown in
Figure 5B indicate that EPA significantly inhibits INa at 250 to
25 mV in a concentration-dependent manner. The IC50 of EPA
for inhibiting INa (at 235 mV, Figure 5C) was 10.8 mM, with a
Hill coefficient of 1.2.
DHA also exhibited an inhibitory effect on INa in human
atrial myocytes; nevertheless, the inhibitory action was
weaker than that of EPA (Figure 5D). The IC50 of DHA for
inhibiting INa was 41.2 mM, with a Hill coefficient of 1.4.
Inactivation of INa traces was fitted to mono-exponential
functions before and after 10 mM EPA or 20 mM 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
250 to 240 or 230 mV (n ¼ 7, P , 0.05 vs. control).
Use-dependent block of INa was examined with EPA or DHA
using a train of 15 identical pulses (30-ms) from 2120 to
235 mV. EPA (10 mM) or DHA (20 mM) inhibited INa 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/gmax) of INa 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/Imax) of INa was
determined as illustrated in Figure 6A, and also fitted to
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Figure 4 Effects of eicosapentaenoic acid and docosahexaenoic acid on IKur. (A) Time course of IKur recorded in a typical experiment with a 100 ms pre-pulse to
þ40 mV to inactivate Ito, followed by 200 ms test pulses to þ50 mV (inset) every 15 s. Eicosapentaenoic acid gradually reduced IKur, and the inhibitory effect fully
recovered on washout with 0.1% bovine serum albumin. (B) Voltage-dependent IKur (capacitance compensated) recorded at 0.2 Hz in a representative cell with a
100 ms pre-pulse to þ40 mV to inactivate Ito, followed by 200 ms test pulses to between 240 and þ60 from 250 mV after a 10 ms interval, then to 230 mV
(inset) in the absence and presence of 10 mM eicosapentaenoic acid. Eicosapentaenoic acid substantially suppressed IKur. The arrow indicates zero level. (C) Concentration–response relationship of IKur inhibition by eicosapentaenoic acid at þ40 mV (n ¼ 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 IKur (recorded
with the protocol shown in the inset of A) by 10 mM docosahexaenoic acid, and the effect recovered on washout with 0.1% bovine serum albumin. (E) Voltagedependent IKur 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 mM docosahexaenoic
acid (for 15 min). Docosahexaenoic acid substantially suppressed IKur. (F). Concentration–response relationship of IKur inhibition by docosahexaenoic acid at
þ40 mV (n ¼ 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.
the Boltzmann distribution. Figure 6B shows that 10 mM EPA
shifted the midpoint of INa availability towards negative
potentials. The V0.5 of availability negatively shifted
9.7 mV, from 297.1 + 1.5 mV in control to 2106.9 +
1.7 mV in EPA (n ¼ 7, P , 0.01). There was no shift for the
V0.5 of INa activation conductance with 10 mM EPA
(238.8 + 1.2 mV in control, 239.4 + 1.5 mV in EPA, n ¼ 6,
P ¼ NS). DHA at 20 mM also negatively shifted V0.5 of INa
availability by 6.5 mV (from 296.8 + 1.3 mV in control to
2103.3 + 1.4 mV in DHA, n ¼ 6, P , 0.01) and had no
effect on the V0.5 of INa activation conductance.
Recovery of INa from inactivation was studied with a
paired-pulse protocol. Superimposed currents and the time
course of recovery are illustrated in Figure 6C and D. INa
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 mM EPA (n ¼ 7, P , 0.01). In
another set of experiments, the recovery time constant of
INa was 11.5 + 2.4 ms in control and 16.9 + 1.9 ms with
20 mM DHA (n ¼ 6, P , 0.01).
The development of resting inactivation of INa was determined with a variable duration of conditioning pre-pulse
from 2120 mV to a subthreshold potential (275 mV), followed by a 3 ms step back to 2120 mV prior to a test
pulse to 235 mV.27 Figure 6E shows the voltage protocol
(inset) and superimposed INa traces recorded during the
test pulse in a typical experiment, showing that INa
reduces as the conditioning pre-pulse duration increases.
The normalized INa 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 mM EPA (n ¼ 7,
P , 0.01). In the experiments with 20 mM DHA, the inactivation time constant decreased from 25.3 + 2.5 ms in
control to 16.3 + 3.5 ms in DHA (n ¼ 6, P , 0.01). These
results demonstrate that EPA and DHA significantly increase
the inactivation of INa 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
Fish oil on human atrial Ito, IKur, and INa
291
Figure 6 Influence of INa kinetics by eicosapentaenoic acid. (A) Voltage protocol and superimposed current for determining voltage dependence of availability (I/Imax) of INa. (B) Mean values of voltage dependence of availability
and activation (g/gmax) of INa in the absence and presence of 10 mM eicosapentaenoic acid. Curves were fitted to the Boltzmann distribution. (C)
Voltage protocol and current traces used for determining recovery of INa
from inactivation. (D) Mean values of curves for recovery of INa from inactivation in the absence and presence of 10 mM 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 INa. (F) Inactivation curves of INa at resting states in the absence and presence of 10 mM eicosapentaenoic acid.
Figure 5 Inhibition of INa by eicosapentaenoic acid and docosahexaenoic acid.
(A) Voltage-dependent INa traces recorded in a representative myocyte with
30 ms steps to between 270 and 25 from 2120 mV at 0.1 Hz during control,
in the presence of 10 mM eicosapentaenoic acid, and washout for 10 min with
0.1% bovine serum albumin. (B) I–V relationships of INa during control, in the presence of 5, 10 and 20 mM eicosapentaenoic acid. Eicosapentaenoic acid significantly suppressed INa at potentials of 250 to 210 mV (n ¼ 8, P , 0.05 or P ,
0.01 vs. control). (C) Concentration–response relationship of INa inhibition by
eicosapentaenoic acid at 230 mV (n ¼ 6–14 experiments). (D) Concentration–
response relationship of INa inhibition by docosahexaenoic acid at 230 mV
(n ¼ 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 INa inactivation before and after 10 mM eicosapentaenoic
acid (10 min, n ¼ 8, *P , 0.05 vs. control). (F) Voltage dependence of time constant of INa inactivation before and after 20 mM docosahexaenoic acid (15 min,
n ¼ 7, *P , 0.05 vs. control). (G) Use dependence of INa before and after
10 mM eicosapentaenoic acid (10 min) at 0.5, 1, 2, and 5 Hz (data values
overlapped). INa was normalized by the current elicited by the first pulse at
each frequency. (H) Use dependence of INa before and after 20 mM docosahexaenoic acid (15 min) at 0.5, 1, 2, and 5 Hz.
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
INa in neonatal rat ventricular myocytes26 and in HEK293
cells transfected with the human cardiac Naþ channel
hH1,22 L-type Ca2þ current (ICa.L) in rat ventricular cells,33
and the potassium currents IK and Ito in ferret ventricular
myocytes.14 It is believed that omega-3 PUFAs modify Naþ
channels by directly binding to the channel proteins,34
although an decreased INa 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 Kþ current Ito, the
ultra-rapid delayed rectifier Kþ current IKur, and the rapid
and slow components IKr and IKs of delayed rectifier Kþ
current are important repolarization currents.16,17,36 IKur is
292
found to be functionally expressed in human atrium, but not
in the ventricle.16 Therefore, the drugs that specifically
inhibit the unique IKur may provide a means of preventing
AF without the risk of ventricular pro-arrhythmia.5 The inhibition of Ito and/or IKur was reported to prolong the action
potential duration in human atrium.37,38
The present study showed that EPA and DHA significantly
inhibited Ito and IKur in human atrial myocytes (Figures 1–4).
The relatively irreversible effects of EPA and DHA on Ito and
IKur are likely related to their directly binding to the
channel proteins.34 The suppression of Ito and IKur is useful
for anti-AF.5 Therefore, the inhibitory effects of EPA and
DHA on IKur and Ito would be most likely one of ionic mechanisms of anti-AF observed in humans.6,10,39 However, a reduced
Ito was consistently found in atrial myocytes from patients
with AF,40,41 while increased, unchanged, or decreased IKur
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
Naþ sodium channels (hH1),22,26 EPA and DHA inhibited INa
in human atrial myocytes in a use-independent way
(Figure 5), which is different from that of the antiarrhythmic drug lidocaine.44 EPA and DHA negatively
shifted the potential of INa availability and increased
the development of INa inactivation at resting states
(Figure 6). These effects would be cardioprotection
against cardiac fibrillation.14,25 EPA had a stronger suppression of INa (Figure 5), while DHA showed a stronger inhibition
of Ito and IKur 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 INa 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 mM) or DHA (IC50:
4.1 mM) on Ito was slightly stronger in human atrial myocytes
than that (IC50: 7.5 mM) in ferret ventricular myocytes46
However, the inhibition of INa by EPA (IC50: 10.8 mM), and
especially DHA (IC50: 41.2 mM), was weaker in human atrial
myocytes than that (IC50: ,5 mM) in neonatal rat ventricular
cells,26 whereas the inhibitory effect of EPA (IC50: 17.5 mM),
and especially DHA (IC50: 4.3 mM), on IKur was stronger in
human atrial myocytes than that (IC50: 20–30 mM 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 mM.
Another limitation of the present study was that the
specific IKur blocker DPO-1 used to separate Ito is a partial
open channel blocker and shows less block of Kv1.5 at
G.-R. Li et al.
initial opening of the channel.23 The incomplete inhibition
of IKur at initial opening of the channel would underestimate
the Ito 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 Ito, IKur,
and INa in human atrial cells with the limited human atrial
specimens. The effects of omega-3 PUFAs on other ion
channel currents (e.g. ICa.L, IKr, and IKs) 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 IK 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 Ito, IKur, and INa 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.
Acknowledgement
The authors thank Heather J. Ballard for her critical reading of the
manuscript.
Conflict of interest: none declared.
Funding
The work was supported by Sun Chieh Yeh Heart Foundation of Hong
Kong.
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