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(Circulation. 1996;94:1774-1780.)
© 1996 American Heart Association, Inc.

Articles

Antiarrhythmic Effects of Polyunsaturated Fatty Acids

Recent Studies

Jing X. Kang, MD, PhD; Alexander Leaf, MD

the Departments of Medicine, Massachusetts General Hospital, Boston (J.X.K., A.L.), Brockton/West Roxbury Veterans Affairs Medical Center (A.L), and Harvard Medical School, Boston, Mass.

Correspondence to Alexander Leaf, MD, Massachusetts General Hospital, East, Bldg 149, 13th St, Charlestown, MA 02129.

Key Words: fatty acids • fibrillation • death, sudden

	Introduction

Top Introduction References

 
The epidemiological studies of Kromann and Green¹ on the low mortality rate of Greenland Inuits from ischemic heart disease led to the suggestion by Bang et al² that despite the high total fat intake of the Eskimos, this low mortality rate was due to the abundance of n-3 fatty acids from seafood in their diet. This hypothesis initiated research by many investigators into possible antiatherogenic effects of n-3 PUFAs. Much has been learned regarding physiological and biochemical changes induced by this class of essential fatty acids that could have potential antiatherogenic effects; nevertheless, controversy persists in the current literature regarding the clinical evidence for beneficial effects from fish ingestion (the major dietary source of n-3 fatty acids) on the development of coronary heart disease.^{3 4} Studies seeking a resolution to the potential antiatherogenic effects of n-3 PUFA will undoubtedly continue.⁵

Meanwhile, on the basis of earlier sporadic suggestions that n-3 PUFA might possess antiarrhythmic effects,^{6 7} McLennan and coworkers^{8 9} pursued this possibility. They showed in feeding studies in rats that when saturated fats or olive oil is the major dietary fat, a high incidence of fatal, irreversible VF occurs from experimental coronary artery ligation, which was significantly reduced when the dietary fat was vegetable oil but was essentially abolished by fish oil. They have confirmed their basic finding in marmosets.¹⁰

It is not our purpose in this brief review to discuss possible antiatherogenic effects of n-3 PUFA or the role of coronary heart disease in causing malignant ventricular arrhythmias. Clearly, coronary heart disease is the major clinical setting in which malignant ventricular arrhythmias occur today, and myocardial ischemia is the most common trigger eliciting such arrhythmias. Rather, we will focus on our recent studies on the mechanism of the apparent antiarrhythmic actions of PUFA, which to us appears to be on the final common pathway of ventricular malignant arrhythmias, the automaticity or excitability of cardiac tissue, regardless of the settings in which they may occur or of the inciting event or agent. Our efforts have been to confirm the antiarrhythmic action of PUFA in a manner that unequivocally establishes which constituents, if any, of fish or marine oils are responsible for the reported antiarrhythmic effects.

When confirmation was obtained, we would then attempt to identify the mechanism(s) for the antiarrhythmic effect. We did so by identifying an action of PUFA to affect the electric stability of cardiac myocytes, which seemingly can be dissected free from the atherogenic process and results from a direct effect of free PUFA to modulate the excitability or automaticity of cardiac tissue and probably of other excitable tissues through direct interactions with membrane ion channels. Admittedly, we cannot state that some further embellishment initiated by the PUFA on the basic mechanism we describe may not also be contributing to the antiarrhythmic effects, eg, metabolite(s) of these PUFA, regulation of gene expression by these PUFA, and an additional effect on the physical state of the phospholipid bilayer. Further research is needed.

Fig 1 shows the polyunsaturated fatty acids we are discussing. The n-6 class is derived largely from vegetable oils; the n-3 class, primarily from fish oils in today's diets. Both classes of PUFA are necessary for health but must be obtained from our diets. Linoleic acid (C18:2n-6), the parent PUFA of the n-6 class, currently is adequately present in the usual US diet and can be elongated and desaturated to form AA in our bodies. In the chloroplasts of green leaves, algae and phytoplankton linoleic acid can be desaturated to create -linolenic acid (C18:3n-3), the parent of the n-3 class of PUFA. It is largely through ingestion of marine phytoplankton that the n-3 fatty acids enter the food chain and become abundant in marine foods. A few vegetable oils, notably canola oil and flax seed oil, contain -linolenic acid, which our bodies can also elongate and desaturate to form EPA (C20:5n-3) and DHA (C22:6n-3), using and competing in the process with the n-6 fatty acids for the same enzymes. Although the essential status of polyunsaturated fatty acids is generally appreciated, many nutritionists and cardiologists still do not recognize the different and often opposing physiological effects that these two important nutrient classes can exert, so they unfortunately get lumped together under the rubric of "polyunsaturated fats" in dietary recommendations. Both are needed for health.

View larger version (24K): [in this window] [in a new window]   Figure 1. Comparison of the structure of the two dietary classes of essential polyunsaturated fatty acids: n-6 PUFA, found primarily in vegetable oils, and n-3 PUFA, present primarily in fish oils. The nomenclature is based on the number of carbon atoms counting from the methyl end before one reaches the first C=C double bond, hence n-6 and n-3 (also referred to as -6 and -3). The figures of the PUFA are drawn to indicate only the number of carbon and the positions of the C=C unsaturated bonds and carry no implication regarding the configuration of each within cell membranes.

We have reported that an intravenous infusion of n-3 fatty acids just before occlusion of the left circumflex coronary artery of conscious, exercising dogs that had had surgically induced myocardial infarctions slowed the heart rate and prevented the fatal ventricular arrhythmias that would have invariably occurred in these susceptible animals.¹¹ We have now found that 11 of 14 such dogs tested were protected by n-3 PUFA, whereas all 14 developed VF in the control tests done 1 week before and 1 week after the test with the n-3 PUFA infusion (P<.001). Fig 2 is an example from one animal. We purposely used a concentrated, purified fish oil preparation (Pronova, Biocare 6000FA containing 72% n-3 PUFA of which free EPA and DHA made up 33.9% and 25.0%, respectively, with 2 mg/g mixed natural tocopherols and <1.0 IU each of vitamins A and D). Subsequent studies have shown that pure, free EPA (98.4%) and DHA (90.8%) individually are also effective antiarrhythmic agents, whereas their ethyl esters are not effective in these short-term experiments (J.X.K., A.L, and George E. Billman, PhD, unpublished results, 1995). The free fatty acids were administered intravenously just before the ischemic stress was applied, so there could be no doubt that they constituted the active ingredient(s) of fish or fish oils responsible for the antiarrhythmic effects. Dietary studies invariably are confounded by more than a single variable.

View larger version (34K): [in this window] [in a new window]   Figure 2. Prevention of ischemia-induced malignant ventricular arrhythmias in a prepared dog by intravenous administration of n-3 PUFAs. Ventricular electrograms are from the same dog with and without treatment with intravenous n-3 free fatty acids during exercise-plus-ischemia tests.11 The tests were separated by 1 week. During control 2, soy bean oil (Intralipid) lacking n-3 EPA or DHA but containing esterified 7% -linolenic was infused just before the exercise-plus-ischemia test.

We have studied the mechanism of the antiarrhythmic effects of PUFA in vitro with cultured neonatal rat ventricular myocytes, which by the second day in culture grow into aggregates of a few to several hundred cells that beat rhythmically and spontaneously. With an inverted microscope, a video camera, and an edge monitor, the beating rate and amplitude of contraction of a clump of cells can be recorded while the cells are perfused with media of desired composition. Addition of EPA or DHA (2 to 10 µmol/L) invariably causes reversible slowing of the beating rate with no change in the amplitude of contractions.¹² Addition to the perfusing medium of agents known to cause arrhythmias in patients will produce tachyarrhythmias in this preparation. Thus, elevated extracellular Ca²⁺, toxic levels of the cardiac glycoside ouabain (0.1 mmol/L), the ß-adrenergic agent isoproterenol (1 to 5 µmol/L), lysophosphatidyl choline (2 µmol/L), acylcarnitine (2 to 5 µmol/L), and even the calcium ionophore A23187 (30 to 100 µmol/L) will initiate tachyarrhythmias that can be prevented or abolished by low concentrations of n-3 PUFA added to the perfusing medium.^{12 13 14} Fig 3 shows an example of this antiarrhythmic effect of the PUFA on these cultured cardiomyocytes. Noteworthy is the ability of the PUFA (Fig 3C) to abort the violent arrhythmias so that individual cells beating asynchronously return to synchronized, more rhythmic contractions. But adding delipidated BSA (2 mg/mL) to the perfusion medium removes the PUFA from the myocytes and, in the continued presence of the high Ca²⁺ and ouabain, reestablishes the arrhythmia.

View larger version (27K): [in this window] [in a new window]   Figure 3. Effects of EPA on the arrhythmogenic effect of toxic levels of Ca2+ and ouabain added to the superfusate bathing cultured neonatal rat cardiac myocytes.12 Aa and Ba show the prompt contractures, increased beating rates, and fibrillation caused by these two toxic agents. Ab and Bb show that addition of EPA to the superfusate before the addition of high Ca2+ or ouabain slows the beating rate of the myocytes and prevents the expected arrhythmias. When EPA is added after both the Ca2+ and ouabain, it stops the arrhythmia, but when the EPA is removed by BSA with high Ca2+ and ouabain still present, the arrhythmia recurs.

Albumin has some five to seven binding sites for fatty acids, three of which are of high affinity¹⁵ and can withdraw the free fatty acids from the myocytes, reversing their antiarrhythmic effect. This tells us that it is only the free fatty acid form of the PUFA that by partitioning into the phospholipids of the sarcolemma of the myocytes, exerts the antiarrhythmic effect. If the fatty acids were incorporated into the membrane phospholipids or covalently bound to any membrane constituent, they could not have been removed by the BSA. We also found that adding the ethyl ester or triglyceride of these PUFA had no protective action against induced arrhythmias in these short-term experiments.

The neonatal rat cardiac myocyte preparation was used to assay individual fatty acids for antiarrhythmic activity because slowing of the beating or heart rate correlates well with antiarrhythmic capability.^{11 12} We found that both n-6 and n-3 PUFAs possess this property (Table 1).¹² The anomaly, however, was AA (C:20n-6). In 48 experiments with AA, approximately one third were antiarrhythmic, just like EPA and DHA, but in the other two thirds, AA had no effect or itself provoked violent tachyarrhythmias. The arrhythmic action of AA proved to be due to its potent oxygenated metabolites because AA, together with inhibitors of cyclooxygenase and lipoxygenase, was found to be antiarrhythmic in 28 of 30 additional experiments. Furthermore, the acetylenic analog of AA, eicosatetraynoic acid, which has four triple rather than double C-C bonds and inhibits the enzymes that oxidize AA,^{16 17} also proved antiarrhythmic in all tests. In contrast, the monounsaturated fatty acid oleic acid and the medium- and long-chain saturated fatty acids tested were without antiarrhythmic effects. These observations indicate that the structural requirements for an antiarrhythmic agent that acts in the manner of these PUFAs are a long-chain hydrocarbon with two or more unsaturated bonds and a free carboxylic acid at one end. With this structural guide, we found that all-trans-retinoic acid specifically also is antiarrhythmic.¹⁸

View this table: [in this window] [in a new window]   Table 1. Effects of Various Fatty Acids on the Beating Rate of Isolated Neonatal Rat Cardiac Myocytes12

Because of the arrhythmogenic action of certain metabolites of AA and because linoleic acid (C:18n-6) can be elongated and desaturated in our bodies to form AA, we advise that for possible clinical antiarrhythmic applications, n-3 PUFA, which we have not found to be arrhythmogenic, should be studied.

From the ability of n-3 PUFA to reduce the beating and heart rate and to prevent tachyarrhythmias induced in the cultured myocytes with every agent we have tested, it became evident that n-3 PUFA must be exerting this action on the basic excitability or automaticity of the myocytes. To test this possibility, we determined the electrophysiological effects of these PUFAs on the cultured myocytes in voltage-clamp studies.¹⁹ We observed very definite effects. The resting or diastolic membrane potential of the myocytes was slightly but significantly increased (more negative) and the threshold voltage for the opening of the fast sodium channels was decreased (more positive) in the presence of the PUFA. The result was that a considerably larger electrical stimulus (50%) was required to elicit an action potential in the presence of EPA or DHA (5 to 10 µmol/L) than in their absence. Also of importance was the increased duration of the effective refractory period (150%), causing prolongation of phase 4 of the cardiac electric cycle, despite a small but significant reduction in the action-potential duration. The sum of these two primary electrophysiological effects would account for both the slowing and antiarrhythmic actions of the PUFA observed in both our dog and in vitro cultured myocytes experiments.

The resulting reduced electric excitability of the myocytes induced by the free PUFA can be simply demonstrated directly on the cultured cardiomyocytes, as shown in Fig 4.¹⁴ By placing platinum electrodes on either side of the coverslip on which the myocytes were cultured, we can stimulate the cells by an imposed external electric field of 15 V to double their spontaneous beating rate. After addition of EPA to the superfusate, the same cells slowed their beating rate and became totally unresponsive to the external imposed electric stimuli. They remained unresponsive when the field strength was increased to 20 V and began to respond only at 25 V, but then only to every other external stimulus. After extraction of the EPA from the myocytes with BSA, the same cells again responded as before to stimuli delivered at 15 V. This enhanced electric stability induced by the PUFA in every individual myocyte may afford protection from VF caused by triggered or reentry aberrant electric depolarizing stimuli resulting from ischemia or other arrhythmic factors.

View larger version (32K): [in this window] [in a new window]   Figure 4. Direct demonstration of the reduced electric excitability of cultured neonatal cardiac myocytes in the presence of n-3 EPA.14 The three tracings were all continuous recordings from the same clump of cells. The top tracing shows that the cells doubled their beating rate in response to stimuli imposed from an external field of 15 V. After addition of EPA to the superfusate, the spontaneous beating rate slowed, and the cells were unresponsive to stimuli imposed by fields of 15 or 20 V, but at 25 V, every other stimulus captured a contraction. Adding BSA and removing the EPA from the superfusate slowed the beating rate, and the cells again responded to the stimuli delivered at 15-V external field strength.

But all electric effects in excitable tissues arise from movement of ions through protein ion channels embedded in the phospholipid membranes of cells. Using the whole-cell patch-clamp technique, Xiao et al²⁰ have shown that these n-3 PUFAs are potent inhibitors of the voltage-gated sodium channels. Inhibition is evident within seconds when 200 nmol/L free EPA is puffed directly onto a myocyte. The IC₅₀ is 4.8 µmol/L, and at 40 µmol/L EPA, the channel is completely inhibited (Fig 5). Fig 6 indicates that this inhibition results from prolongation of the inactivated state of the voltage-gated fast sodium channels by EPA. Only the same PUFAs that are antiarrhythmic inhibit this sodium channel (Table 2). The inhibition is voltage dependent but not use dependent, as expected of a highly lipophilic antiarrhythmic agent.²¹ The PUFAs act to prolong the inactivated state of the voltage-dependent sodium channel. This might account for their ability to prolong the refractory phase of the cardiac cycle,²² to hyperpolarize slightly the resting or diastolic membrane potential,²³ to raise the voltage for the threshold gating of the fast sodium channel, and even to shorten the duration of the action potential.²³

View larger version (13K): [in this window] [in a new window]   Figure 5. Inhibitory response of voltage-gated Na+ channels of neonatal rat cardiac myocytes as a function of the dose of n-3 EPA, determined by the whole-cell patch-clamp method.20

View larger version (15K): [in this window] [in a new window]   Figure 6. EPA modifies the voltage dependence of the steady-state inactivation of the Na+ channels of neonatal cardiac myocytes.20 A, Superimposed original currents recorded in the absence (control) and presence of 10 µmol/L EPA. Currents were elicited by a double-pulse protocol consisting of a 30-ms testing pulse to -30 mV following a 500-ms conditioning prepulse varying from -140 to -30 mV from a holding potential of -80 mV. B, Normalized steady-state inactivation of peak Na+ currents as a function of prepulse voltages with the protocol of A. Extracellular application of 10 µmol/L EPA suppressed INa at prepulses positive to -80 mV. V1/2 for the normalized steady-state inactivation curve of the peak Na+ currents was shifted to the left by EPA.

View this table: [in this window] [in a new window]   Table 2. Comparison of the Blocking Effects of Fatty Acids on Sodium Channels of Cultured Neonatal Cardiac Myocytes20

The PUFAs could conceivably affect conductances of ion channels by altering the physical state ("fluidity") of the membrane phospholipids, indirectly affecting ion channel conductance, or by binding directly to the ion channel proteins. A recent study²⁴ demonstrated that the antiarrhythmic PUFAs allosterically displace dose dependently the specific binding of [³H]batrachotoxinin A 20-benzoate to specific receptor sites on the protein of the -subunit of the sodium channels.

This finding demonstrates that the PUFAs bind to a specific site on the sodium channel protein that prolongs the duration of its inactivated state.^{20 25 26} We hypothesize that the PUFAs may bind to a site on the S4 segment of -subunits of the ion channels that are both positively charged and hydrophobic.²⁷ These segments are thought to serve as voltage sensors and the determinants of change in functional conformation of the channel protein in response to transmembrane voltage signals.²⁷ Thus, it is conceivable that any interaction with these segments may cause major changes in channel conductances. Furthermore, this S4 segment of Na⁺ channels is highly conserved in the voltage-sensitive Ca²⁺ and K⁺ channels.²⁷

Our present working hypothesis²⁴ is that the long, lipophilic, acyl chains of these PUFAs facilitate their partitioning among the acyl chains of the phospholipids comprising the sarcolemma of cardiomyocytes. The C=C unsaturated bonds allow the PUFAs to position themselves by binding to the hydrophobic transmembrane domains of sodium channel proteins so that their free carboxyl group at the lipid-aqueous interface has its negative charge in close proximity to the positively charged amino acids of the sodium voltage sensor,²⁷ modifying the voltage dependence of the sodium channel. The effect of the PUFAs on sodium channels could alone account for most, or all, of the electrophysiological changes observed on the isolated myocytes.¹⁹ Unlike the class I antiarrhythmic drugs that block sodium channels, these PUFAs do not upregulate sodium channels in the myocytes (J.X.K. and A.L., unpublished results, 1996), which has been shown to be the probable mechanism accounting for the toxicity of the class I antiarrhythmic drugs.^{28 29} Furthermore, these PUFAs also modulate K⁺ and Ca²⁺ ion channels (Y.F. Xiao and A.L., unpublished results, 1996), which would also set them apart from class I antiarrhythmic drugs. This is consistent with the conserved amino acid sequences in the voltage sensor segment of the Na⁺, Ca²⁺, and K⁺ channels.²⁷ Recent reports indicate that fatty acids have a variety of effects on different voltage-sensitive ion channels in excitable tissues,^{30 31 32 33 34 35 36 37 38 39 40 41} and some studies have suggested that the effects on ion currents may be mediated through a direct interaction with the ion channel proteins.^{30 35 37 39} Much more work needs to be done to determine fully the mechanism(s) of their antiarrhythmic effects. It will be of interest to learn how they affect other excitable tissues such as nervous and muscular tissues.

It is clear that only the free fatty acid form of these PUFAs can provide immediate prevention against fatal ventricular arrhythmias. Weylandt et al⁴² have shown that loading the membrane phospholipids of neonatal rat myocytes with DHA, which is easily accomplished by culturing the cells in the presence of EPA or DHA in the medium, affords no protection against arrhythmic agents if free EPA or DHA is first removed from the myocytes by washing them with delipidated BSA. However, when EPA or DHA is ingested regularly in the diet, there is ample time for esterases in the intestines and in the plasma or cellular phospholipases to liberate sufficient free PUFA into the nonesterified fraction of the blood lipids to supply the plasma membranes of cardiac myocytes with the free acid form of the PUFAs needed to prevent lethal arrhythmias, as indicated in feeding studies in animals.^{8 9 10 43} It seems probable that the mechanism of the antiarrhythmic effect of the PUFA, whether ingested or administered acutely, is the same, but further evidence to test this assumption is needed.

At present, unfortunately, we cannot be definite about the actual circulating concentrations of these PUFAs necessary to provide antiarrhythmic protection. Because some 99.9% of the nonesterified free fatty acids are bound to serum albumin and other circulating proteins,^{15 44} the concentration of the free monomeric fatty acids in plasma water is very low, recently reported to be <20 nmol/L in healthy individuals.^{45 46} But because micellar or laminar aggregations of fatty acids in plasma water may also provide additional sources of free PUFA to partition into the membrane phospholipids, the concentration of the monomeric form alone may not be the pertinent parameter that determines the partitioning of the free PUFA into the phospholipids of the cell membranes. We are certain only that nonesterified free fatty acids can leave their normal albumin carriers rapidly, pass through a low concentration in the aqueous phase of plasma, and partition into the hospitable, lipophilic environment of membrane phospholipids, where they quickly accumulate to much higher concentrations but to similar chemical free energy levels as in the aqueous medium bathing the cells. The similarities observed between the dietary and direct administration of the PUFAs either intravenously to the whole animal or to the medium bathing cultured myocytes make it seem quite probable that both prevent arrhythmia by the same specific mechanisms described.

We currently cannot say whether the evidence will add up to prevention of lethal arrhythmias in patients, but two secondary prevention trials, which unexpectedly showed prevention of ischemia-induced sudden cardiac death, are encouraging. One dietary study⁴⁷ was a prospective, randomized, single-blind, secondary prevention trial that compared the effect of a "Mediterranean -linolenic acid-rich diet to the usual post-infarct prudent diet." The subjects on the more fat-restricted experimental diet receiving the -linolenic acid (C:18n-3) showed a remarkable reduction in mortality and morbidity, including prevention of sudden death, as shown in Fig 7. The other study⁴⁸ was also a randomized, prospective, secondary prevention trial in which subjects who were advised to eat oily fish two or three times weekly were compared with those who were not so advised. This study was done before the possible antiarrhythmic effects on n-3 PUFA were generally appreciated, and sudden deaths were not recorded. The study, however, found no reduction in new events but a 29% reduction in mortality, suggesting a reduction in sudden deaths that make up 50% to 60% of the acute mortality from heart attacks.⁴⁹ In both studies, the survival curves showed a very early beneficial separation of the experimental versus control groups, quite unlike the 2 years required in the cholesterol-lowering trial⁵⁰ before a lower mortality rate was seen. A recent case-control study⁵¹ reported an inverse relationship between fish consumption and sudden cardiac death, suggesting an antiarrhythmic effect from ingestion of fish.

View larger version (25K): [in this window] [in a new window]   Figure 7. Mediterranean -linolenic acid–rich diet in secondary prevention of coronary disease.45 The 605 patients who had just had a myocardial infarction (MI) and therefore were at high risk for a second heart attack were randomized to either a control "prudent diet" or an experimental diet in which saturated fats were reduced and olive oil and a soft margarine made from canola oil (10% -linolenic acid) were added. Increased vegetables and fruits were encouraged in the diet. The study was stopped at a mean follow-up of 27 months because of the striking beneficial outcomes in the experimental group. The risk ratios for the experimental vs control groups show a 70% reduction of all causes of death, which was accounted for by the reduction in cardiovascular deaths. Of interest is the complete suppression of sudden death in the experimental group. These benefits occurred without any changes in serum cholesterol levels. (Prepared from data in Reference 47 with permission of the authors.)

In conclusion, it is evident that an important regulation of cardiac function exists that is affected by these PUFAs, is subject to simple dietary choices, and has largely escaped the attention of investigators and cardiologists. These PUFAs have always been an essential component of human diets⁵² and may potentially provide a simple and safe protection from ischemia-induced and possibly other serious cardiac arrhythmias. With some 250 000 individuals in the United States alone dying annually within an hour of their heart attacks,⁴⁹ these PUFAs may provide significant public health benefit. Carefully planned and executed clinical trials are now needed to determine the antiarrhythmic effectiveness of these fatty acids in humans at high risk of fatal arrhythmias.

	Acknowledgments

 
Studies from our laboratory have been supported in part by research grant DK38165 from the NIDDK, NIH, US Public Health Service.

	Selected Abbreviations and Acronyms

 

AA = arachidonic acid BSA = bovine serum albumin DHA = docosahexaenoic acid EPA = eicosapentaenoic acid PUFA = polyunsaturated fatty acid VF = ventricular fibrillation

Received January 10, 1996; revision received March 21, 1996; accepted April 5, 1996.

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