The Antiarrhythmic Effect of n-3 Polyunsaturated Fatty Acids: Modulation of Cardiac Ion Channels as a Potential Mechanism
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- Xiao, Y., Sigg, D. & Leaf, A. J Membrane Biol (2005) 206: 141. doi:10.1007/s00232-005-0786-z
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Sudden cardiac death remains one of the most serious medical challenges in Western countries. Increasing evidence in recent years has demonstrated that the n-3 polyunsaturated fatty acids (PUFAs) can prevent fatal ventricular arrhythmias in experimental animals and probably in humans. Dietary supplement of fish oils or intravenous infusion of the n-3 PUFAs prevents ventricular fibrillation caused by ischemia/reperfusion. Similar antiarrhythmic effects of these fatty acids are also observed in cultured mammalian cardiomyocytes. Based on clinical observations and experimental studies in vitro and in vivo, several mechanisms have been postulated for the antiarrhythmic effect of the n-3 PUFAs. The data from our laboratory and others have shown that the n-3 PUFAs are able to affect the activities of cardiac ion channels. The modulation of channel activities, especially voltage-gated Na+ and L-type Ca2+ channels, by the n-3 fatty acids may explain, at least partially, the antiarrhythmic action. It is not clear, however, whether one or more than one mechanism involves the beneficial effect of the n-3 PUFAs on the heart. This article summarizes our recent studies on the specific effects of the n-3 PUFAs on cardiac ion channels. In addition, the effect of the n-3 PUFAs on the human hyperpolarization-activated cyclic-nucleotide-modulated channel is presented.
KeywordsCardiac arrhythmiaEicosapentaenoic acidDocosahexaenoic acidHyperpolarization-activated cyclic-nucleotide-modulated channelMutation
Animal data support the clinical observations that a diet high in fish oil, in contrast to saturated fat or monounsaturated olive oil, prevented ventricular fibrillation induced by coronary artery ligation in rats and increased the electrical ventricular fibrillation thresholds in marmosets [37, 38]. The cardioprotective effect of the n-3 PUFAs was further confirmed by Billman and colleagues in a dog model of sudden ventricular fibrillation . In this model, intravenous delivery of an emulsion of fish oil  or of one of the two major n-3 fatty acids EPA and DHA just prior to occluding the left circumflex coronary artery prevented fatal ventricular fibrillation. Dietary flaxseed, which is the richest plant source of one of the n-3 PUFA, α-linolenic acid, was associated with reduced ventricular fibrillation during ischemia-reperfusion in normal and hypercholesterolemic rabbits, possibly via shortening of the action potential duration . To further understand the physiological, biochemical, and biophysical bases of the antiarrhythmic action in vivo by the n-3 PUFAs, Kang and colleagues used cultured neonatal rat cardiac cells as an in vitro model of arrhythmogenesis [24, 25]. Perfusion of a number of agents known to produce lethal arrhythmias in humans, e.g., cardiac glycosides, elevated extracellular Ca2+, isoproterenol, thromboxane, lysophosphatidylcholine, etc., to the cultured myocytes accelerated their beating rates, developed contractures and fibrillations. Addition of EPA or DHA before adding the arrhythmogenic agents prevented fibrillation [24, 25]. If an arrhythmia was induced by one of the toxic agents and EPA or DHA was then added to the perfusate, the arrhythmia stopped and the cells beat regularly again. If the fatty acids were removed from the cardiac cells with delipidated bovine serum albumin in the presence of the arrhythmogenic agents, the arrhythmia resumed [24, 25]. These results suggest that the n-3 PUFAs acted directly on the heart cells without formation of covalent bonds because if they had, the delipidated serum albumin would not have been able to wash away the fatty acids from the myocytes [24, 25].
Significant changes of the parameters of cardiac action potentials in the presence of eicosapentaenoic acid
Modulatory Effects of the n-3 PUFAs on Cardiac Ion Channels
Effects of eicosapentaenoic acid on cardiac Na+ currents
Human cardiac Na+ channel
3.9 (INa,peak) 0.9(INa,late)
−19 (INa,peak) ? (INa,late)
To extend understanding of the possible actions of the n-3 PUFAs in human hearts, we investigated their effects on the α-subunit of the human cardiac Na+ channel (hH1α). The hH1α cDNA was transiently transfected into cultured human embryonic kidney (HEK293t) cells. EPA significantly reduced the hH1α Na+ current (INa,α) with an IC50 of 0.51 μM . EPA shifted the V1/2 of the steady-state inactivation by −28 mV (Table 2). In addition, EPA inhibited INa,α with a higher “binding affinity” to hH1α channels in the inactivated state than in the resting state. EPA markedly accelerated the transition from the resting state to the inactivated state and slowed the recovery from the inactivated state to the resting, available for activation state . We are surprised by the result that besides PUFAs, saturated or monounsaturated fatty acids also significantly inhibited INa,α in transfected HEK293t cells . These data indicate that free fatty acids suppress human INa,α with high “binding affinity” to inactivated channels and prolong the duration of inactivation of hH1α channels.
To assess how the PUFAs modulate the activity of cardiac Na+ channels, we introduced specific site-directed point mutations of single amino acids in hH1α to determine whether a single amino-acid mutation would cause a loss or significant reduction of the expected action of the PUFAs on the channel conductance. Three mutated single amino-acid substitutions with lysine were made in the hH1α Na+ channel at domain 4-segment 6 (D4-S6) for F1760, Y1767 and at D1-S6 for N406, sites which are in the putative regions for binding of local anesthetics and batrachotoxin, respectively. EPA or DHA significantly reduced Na+ currents in the HEK293t cells expressing the wildtype, Y1767K, or F1760K of hH1α [60, 61]. The inhibition was voltage- and concentration-dependent with a significant hyperpolarizing shift of the steady-state inactivation (Table 2). In contrast, the mutant N406K was much less sensitive to the inhibitory effect of EPA. Coexpression of N406K with the β1 subunit in HEK293t cells further decreased the inhibitory effect of EPA with a significantly smaller hyperpolarizing shift of the V1/2 of the steady-state inactivation of the Na+ current. These results demonstrate that substitution of asparagine with lysine at site 406 in D1-S6 significantly reduced the inhibitory effect of the PUFAs on the Na+ current, and coexpression with β1 decreased this effect even more. Therefore, asparagine at the 406 site in hH1α appears critical for the inhibition by the PUFAs of cardiac Na+ currents, which may play a significant role in the antiarrhythmic action of the n-3 fatty acids. These findings suggest that the hH1α protein may have a specific binding site for the n-3 PUFAs.
The voltage-gated L-type Ca2+ current (ICa,L) is responsible for the Ca2+-induced Ca2+ release from the sarcoplasmic reticulum into the cytosol of heart cells. Increase in cytoplasmic free calcium concentration is critical for electro-mechanical coupling and contraction of the heart. Intracellular Ca2+ overload, however, can cause cardiac arrhythmias. The inhibitory effects of the n-3 PUFAs on ICa,L may thus partially be responsible for their antiarrhythmic properties. One study using isolated cultured neonatal rat cardiac myocytes showed that the n-3 PUFAs, but not monounsaturated or saturated fatty acids, were antiarrhythmic during Ca2+ overload . The antiarrhythmic effect occurred quickly, but only with the free fatty acid form of the PUFAs, while the ethyl ester or triglyceride forms were not promptly antiarrhythmic . The antiarrhythmic effect is quickly reversed when the free PUFAs are extracted from the cells by adding delipidated bovine serum albumin to the bathing solution. This observation suggests that the PUFAs are neither fully incorporated into membrane phospholipids nor covalently bound to any constituents of the myocyte to produce the antiarrhythmic effect .
Ito, IK, and IK1, are the three major potassium currents across cardiac cell membrane. After analyzing the effects of the n-3 PUFAs on cardiac Na+ and Ca2+ channels, we further examined the effect of these fatty acids on the voltage-activated K+ currents in isolated adult mammalian cardiomyocytes . We found that the two outward K+ currents, the transient outward K+ current (Ito) and the delayed rectifier K+ current (IK), were both inhibited by the n-3 PUFAs, while the inwardly rectifying K+ current (IK1) was unaffected by the n-3 PUFAs. The lack of an effect of the n-3 PUFAs on IK1 may result from differences of channel structures among IK, Ito, and IK1 . DHA produced a concentration-dependent suppression of Ito and IK in adult ferret cardiomyocytes with an IC50 of 7.5 and 20 μM, respectively; but had no effect on IK1. The inhibition of Ito by the PUFAs was also reported by a different laboratory . Honore and colleagues showed that DHA and the n-6 polyunsaturated fatty acid arachidonic acid (C20:4n-6, AA) blocked the major cardiac delayed rectifier K+ channel (Kv1.5) expressed in a Chinese hamster ovarian cell line and IK in cultured mouse and rat cardiac myocytes . Their results demonstrate that the inhibition occurred when DHA was applied extracellularly and not when included in the patch electrode .
EPA showed effects on Ito, IK, and IK1 similar to DHA. However, AA had a biphasic action on IK: initially inhibition and then activation. The late activation of IK by AA was prevented by pretreatment with indomethacin, a cyclooxygenase inhibitor. This late activation of IK by AA was thus not due to the free AA but to cyclooxygenase metabolites . Monounsaturated and saturated fatty acids, which are not antiarrhythmic, lack any significant effects on the major K+ currents. Our results demonstrate that the n-3 PUFAs inhibited cardiac Ito and IK with higher concentrations (Table 2). Inhibition of the outward K+ currents, Ito and IK, by the n-3 PUFAs would prolong the duration of the action potential rather than shorten it [4, 26]. Therefore, the inhibitory effects of the PUFAs on the ICa,L and INa would have to dominate over the weaker inhibition of the outward K+ currents to explain the observed small, but significant, reduction in the action potential duration of cultured cardiomyocytes  and the electrocardiographic QT interval in dogs (30).
IK1 is essential for maintaining different resting membrane potential in pacemaker or non-pacemaker cardiomyocytes  and in other types of cells . During myocardial ischemia intracellular K+ leaks out due to membrane damage, which can cause membrane depolarization and arrhythmias. A lower resting potential in ischemic (−70 mV) than in nonischemic (−78 mV) human ventricular myocytes has been reported . In addition, intravenous injection of anesthetic thiopental has been shown to increase the incidence of ventricular arrhythmias via depression of IK1 and depolarization of the resting membrane . Our studies have shown that the n-3 PUFAs inhibit IK and Ito, but not IK1 . These effects result in a decrease in the efflux of K+ without a depolarization of cardiac resting membrane potential, which is consistent with our previous finding of slight hyperpolarization of resting cardiomyocytes in the presence of the PUFAs . Therefore, the effects of the n-3 PUFAs on Ca2+ and Na+ channels without inhibition of IK1 may protect the heart from arrhythmias during myocardial ischemia.
OTHER ION CHANNELS
To further examine the effects of the n-3 PUFAs on other cardiac ion channels, we studied the acetylcholine-activated K+ channel (IK,ACh), cardiac Na+-Ca2+ exchanger (NCX1), and cAMP-activated cardiac chloride channel. EPA significantly inhibited cardiac IK,ACh and cAMP-activated Cl− currents . The significance of the inhibition of IK,ACh or Cl− currents is currently not clear. EPA or DHA, but not the saturated fatty acid, stearic acid (C18:0), significantly inhibited cardiac Na+-Ca+ exchanger currents in HEK293t cells transfected with the canine NCX1 cDNA . The suppression was concentration-dependent with an IC50 of 0.82 μM of EPA.
It has been shown that the n-6 fatty acid AA can modulate T-type Ca2+ channels . AA inhibited α1G currents in HEK-293 cells heterologously expressing the T-type Ca2+ channel . The inhibition occurred within a few minutes after perfusion of AA, regardless of preceding exposure to inhibitors of AA metabolism (ETYA and 17-ODYA). Single-channel recordings of cell-free inside-out patches showed that current inhibition was due to a decrease of the open probability without changes in the size of unitary currents. AA shifted the inactivation curve to more negative potentials, increased the speed of macroscopic inactivation, and decreased the extent of recovery from inactivation . During AA perfusion, a tonic current inhibition was observed regardless of whether the channels were held in resting or inactivated states. This result suggests a state-independent interaction with the channel. Compared with the wildtype, the α1G mutants with slow inactivation showed an increased affinity for AA, indicating that the structural determinants of fast inactivation are involved in the AA-channel interaction. Model simulations indicate that AA inhibits T-type currents by switching the channels into a nonavailable conformation and by affecting transitions between inactivated states, which results in the negative shift of the inactivation curve .
Potential Sites for the Action of the n-3 PUFAs
Membrane phospholipid is one of the possible sites of the action of the n-3 PUFAs on ion channels. Klausner and colleagues showed that these same unsaturated fatty acids, to the extent they were tested, could alter the “fluidity” of membrane phospholipids . This has been thought as a possible means by which addition of free fatty acids to cells might alter the actions of membrane proteins, i.e., channels, carriers and enzymes. However, the concentrations of fatty acids used in their study was some 10-fold or greater than the nM to low μM concentrations of the n-3 PUFAs that inhibited cardiac ion channels in our experiments. At these low concentrations, the molar ratio of the n-3 PUFAs to phospholipds in red cell ghost is less than 1:100 , which is too low to have a general effect of increasing the “fluidity” of the membrane phospholipids.
During the past decade Andersen and colleagues have been rigorously developing and testing a hypothesis that fatty acids and other agents could be affecting ion channels by a primary effect on the cell membrane in the immediate vicinity of the channel protein rather than by a direct action on the channel protein [5, 19, 33, 34]. They have tested their hypothesis on the cationic-permeable short gramicidin channel with two nonionic detergents, Triton X100 and β-octyl glucoside. They reported that these two compounds with no chemical similarity to the PUFAs also affect the Na+ and Ca2+ currents of mammalian channels [5, 33]. They explained their results by alteration of stresses between channel and membrane when the hydrophobic length of the transmembrane channel protein does not match the hydrophobic thickness of the resting membrane phospholipid bilayers (see Fig. 5 in reference 31). This tension exerts locally on the channel protein, which affects the conformational state and conductance of the ion channel. Compounds that can incorporate into the phospholipid membrane close to its junction with a channel protein may change the tension of the channel protein. This would change the conformational state of the transmembrane segment of the channel protein and affect its conductance.
Our recent observations showed that Triton X100 and β-octyl glucoside significantly inhibited Na+ currents of human cardiac Na+ channels transiently expressed in HEK293 cells and shifted the steady-state inactivation to more hyperpolarized potentials . The detergents probably act on the phospholipid cell membrane, which abuts the transmembrane channel protein. Thus, other ion channels, such as Ca2+ channels, were also affected . These effects are similar to those of the n-3 PUFAs. The n-3 PUFAs affect many types of ion channels as we have observed . This low selectivity thus suggests that the modulation of ion channels by the n-3 PUFAs is possible via local action on membrane phospholipids, as the two nonionic detergents do.
The antiarrhythmic action of these fatty acids may result from their significant inhibition of cardiac Na+ and Ca2+ channels. For example, ischemic cardiomyocytes can be partially depolarized (e.g., perhaps −70 mV rather than −90 mV) and more vulnerable. A small depolarizing stimulus (e.g., current of injury) can activate Na+ channels to initiate action potentials and arrhythmias. Fortunately, the n–3 PUFAs can prevent their occurrence by voltage-dependent inhibition and by shifting the steady-state inactivation to more negative potentials [55–65]. Furthermore, in partially depolarized heart cells, the n-3 PUFAs enhance the transition of Na+ channels directly into an inactive state in milliseconds without eliciting an action potential . The result of these events is that the partially depolarized cells are promptly and completely inactivated by the presence of the n-3 fatty acids. The PUFAs have no significant effects on IK1 and HCN channels, which may be important for maintaining normal cardiac function because IK1 channels are critical for maintaining resting membrane potential in most cardiomyocytes and HCN channels are critical for pacemaker cells in the heart.
In summary, our studies and others demonstrate that the n-3 PUFAs can modulate the electrophysiological kinetics of several ion channels. As the effects of these fatty acids on ion channels are fully reversible after extraction with albumin, the PUFAs may act either on channel proteins directly or via non-covalent incorporation into membrane phospholipids. It is also possible that the n-3 PUFAs are able to bind to a specific site on channel proteins and also to non-specifically incorporate into lipid cell membrane. More studies are needed to further dissect the mechanism(s) by which n-3 PUFAs modulate ion channels and protect the heart from arrhythmias.
Our studies have been supported in part by grants DK38165 from NIDDK and by HL62284 from NHLBI of the National Institutes of Health (AL) and American Heart Association (Y-FX). We wish to thank our colleagues who have contributed to the project for several years.