Cardiovascular disease (CVD)⁴ describes all diseases of the heart and blood vessels including heart disease, stroke and peripheral vascular disease, and is the leading cause of death in western nations. The increased consumption of fats, particularly saturated fats, in the diets of industrialized and developing nations has been linked to the increase in deaths due to coronary heart disease. Epidemiological studies, human intervention trials and animal experiments have shown that dietary fatty acids can modify the risk for cardiovascular disease.

Though there has been a fall in mortality from coronary heart disease in recent years, sudden cardiac deaths associated with fatal arrhythmia remains the cause of most deaths in industrialized societies (Charnock 1991). Mortality statistics from the USA and UK indicate that up to 80% of sudden deaths are due to ventricular fibrillation. Studies such as the cardiac arrhythmia suppression trial (CAST) failed to show any significant decrease in mortality due to coronary artery disease when antiarrhythmic drugs were administered.

Heart rhythms are a result of waves of electrical excitation which spread through the conducting tissues in heart muscle. Individual heart cells known as cardiac myocytes are electrically coupled to each other by membrane structures called gap junctions, which are small pores through which electrical currents can flow from cell to cell. When a region of the heart becomes ischemic, the electrical properties change, leading to arrhythmias. The most common fatal arrhythmia is known as ventricular fibrillation (VF), in which electrical impulses from damaged cardiac muscle cause the normal synchronicity of heart contractions to break down. It is believed that at least half of the deaths due to coronary artery disease in the United States are caused by disturbances in the electrical stability of the heart, terminating in VF (Billman and Leaf 1994). Cardiac arrhythmia occurs during the early and potentially reversible phase of ischemia (Sargent and Riemersma 1990) and after reperfusion. In most cases arrhythmia occurs without previous symptoms and progresses to sudden cardiac death.

Fatty acids are classified into three families: saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids (PUFA). The common saturated fatty acids in our diet are myristic, palmitic and stearic acids derived largely from animal fats, dairy products and manufactured foods. The monounsaturated fatty acid content of our diet is accounted for by oleic acid, the predominant component of canola oil, olive oil and sunola. All mammals can synthesize saturated fatty acids and monounsaturated fatty acids de novo from simple precursors such as glucose or amino acids using fundamental pathways. Linoleic acid [18:2(n-6); LA] and -linolenic acid [18:3(n-3); -LNA], the precursors of the (n-6) and (n-3) family of fatty acids, respectively, are essential fatty acids and have to be supplied by the diet. The main PUFA in the western diet is linoleic acid, found mostly in vegetable oils such as safflower oil, sunflower oil, cottonseed oil, corn oil, soybean oil, etc.

Significant amounts of -LNA are found in green vegetables and in vegetable oils like linseed oil, canola oil and soybean oil. Longer chain PUFA, like arachidonic acid [20:4(n-6); AA], are synthesized in human tissues via chain elongation and desaturation of LA. Arachidonic acid may also originate as such from muscle meat membranes, eggs, organ meats and human milk. Eicosapentaenoic acid [20:5(n-3); EPA] and docosahexaenoic acid [22:6(n-3); DHA] are synthesized via desaturation and chain elongation of -LNA and are also found in high concentrations in fish and fish oils. The metabolism of fatty acids of the (n-3) family (EPA and DHA) and of the (n-6) family (AA) are of particular interest because of the actions of their metabolites (eicosanoids) in vivo. Double bond positions of (n-6) and (n-3) fatty acids are not interconvertible. LA and -LNA compete for desaturation and chain elongation, therefore, a proper balance is essential to optimize AA and DHA in the membranes.

Table 1. Effect of (n-3) polyunsaturated fatty acid supplementation on arrhythmia in experimental animals1 [View Table]

Table 2. Effect of (n-3) polyunsaturated fatty acid supplementation on arrhythmia in vitro1 [View Table]

The antiarrhythmic effects of fish oil in experimental animals have focussed primarily on two models of arrhythmia. They are 1) ischemia induced (arrhythmias occuring after onset of ischemia) and 2) reperfusion arrhythmias.

Other areas in which the effects of (n-3) PUFA are currently being studied include heart transplants (Grimminger et al. 1996) and restenosis after coronary angioplasty (Leaf et al. 1994). These studies have attempted to elucidate the prophylactic mechanism of fish oil (n-3) PUFA by studying effects on cardiac function, mechanical performance of the heart and ventricular fibrillation threshold. Several mechanisms have been suggested to explain the antiarrhythmic action of (n-3) PUFA, but to date no definite mechanism has been fully validated.

ANIMAL EXPERIMENTS

Abeywardena et al. (1993) examined the effect of long term dietary supplementation of different dietary fatty acids on arrhythmia in rats and marmoset monkeys. The study demonstrated that cardiac eicosanoids were significantly reduced by fish oil feeding, and this subsequently reduced ventricular fibrillation. The association between consumption of different diets and the vulnerability of the myocardium to develop arrhythmias was also studied in adult marmoset monkeys (McLennan et al. 1992). An increase in threshold current required to induce VF was observed after inducing ischemia in animals fed sunflower seed oil diet [(n-6) PUFA] or tuna fish oil diet [(n-3) PUFA] compared to animals fed a saturated fat diet.

In a recent study by Pepe and McLennan (1996), hearts isolated from rats fed fish oil for 16 wk were set up as a working heart model. They found that dietary fish oil prevented and reduced the severity of arrhythmias in ischemia as well as increased the threshold for VF.

Charnock et al. (1992b) studied the effect of (n-3) PUFA on cardiac performance in marmoset monkeys and found the dietary treatment to be beneficial. Both the mechanical performance and the electric stability of animal hearts improved on the (n-3) PUFA diet. Turner et al. (1990) confirmed that the antiarrhythmic effect of dietary (n-3) PUFA is independent of atherosclerosis-induced cardiac vulnerability and is due to their effect on myocardial membrane alone. These studies support the need to further study the mechanism of action of these fatty acids and their influence on the cardiac environment.

The work of Hallaq et al. (1990) using rat cardiac myocytes cultured in a medium supplemented with (n-3)PUFA showed that fish oil prevented VF, and this was attributed to the prevention of increase in cytosolic calcium by (n-3) PUFA. This prophylactic effect of fish oils on VF was studied using ouabain, a potent arrhythmogenic agent, in isolated neonatal cardiac myocytes. Ouabain is a cardiac glycoside and is toxic because it produces excessively high levels of calcium inside the cells. The toxic effects of ouabain were prevented in myocytes incubated with EPA.

Using mongrel dogs Billman et al. (1994) demonstrated that long chain (n-3) PUFA concentrated from fish oil prevented ischemia-induced VF. In that study fish oil fatty acids were infused intravenously before inducing ischemia, and the effect on VF was recorded. In seven of eight animals, the infusion of fish oil emulsion completely prevented acute occurrence of VF. Since the infusion with fish oil emulsion was carried out only 1 h prior to inducing ischemia, the authors suggested that the effects were not mediated via membrane incorporation of (n-3) PUFA but rather via a direct action of (n-3) PUFA on the electrophysiological properties of myocytes.

The effect of adrenergic stimulation on the development of ischemia-induced malignant arrhythmia was studied in isolated multicellular cardiac myocyte preparations using fish oil (n-3) PUFA (Kang and Leaf 1995). In that study unesterified, long-chain PUFA, particularly (n-3) PUFA, but not monounsaturated or saturated fatty acids, were found to effectively prevent and terminate -adrenergic agonist-induced tachyarrhythmias in myocyte preparations. McLennan et al. (1985) found that the number of ventricular ectopic beats and duration of tachycardia or fibrillation was increased in rats fed perirenal sheep fat compared to rats fed sunflower seed oil. In the same rat model, when rats were fed tuna fish oil, VF was prevented during occlusion and reperfusion of the coronary artery, and the severity of arrhythmia was significantly reduced (McLennan et al. 1988).

MECHANISMS

It is generally agreed by most authors that the prevention of arrhythmias by fish oil is a result of the interaction of two or more of these mechanisms, but the sequence of action has not been elucidated. The effects of fish oil are most clearly and simply explained by their effects on eicosanoid metabolism, but it is also obvious that other factors interact at one point or other. It has been aptly said that (n-3) PUFA modify so many different factors, it is impossible to attribute all their beneficial effects to one single action (Leaf 1994).

The net result of these actions is vasodilation with less platelet aggregationan antithrombotic effect. By altering the availability of AA in the membrane, a change in the production of eicosanoids and eicosanoid dependent cellular functions may be produced. A reduced ratio of AA/EPA shifts the spectrum of eicosanoid production toward an increase in thromboxane A₃ (TXA₃) and PGI₃ at the expense of TXA₂ and PGI₂, respectively. This shift was found to reduce the risk of VF and sudden cardiac death (SCD) (Coker et al. 1982). The risk of ventricular arrhythmia induced by ischemia was found to be directly proportional to the balance between TXA₂ and PGI₂. Coker and Parrat (1985) found that TXA₂ was released as an early response to occlusion while PGI₂ was released after the onset of ischemia. Drugs that blocked the TXA₂ receptor reduced incidence of ischemia-induced arrhythmia and reperfusion arrhythmia. When local PGI₂ activity was raised, the frequency and severity of both types of arrhythmia was reduced. Parrat et al. (1987) postulated that an increase in PGI₂/TXA₂ balance may be cardioprotective. They also suggest that PGI₂ may be an endogenous anti-arrhythmic agent, which may explain the preventive mechanism of (n-3) PUFA.

The effect of (n-3) PUFA on eicosanoids was also examined in cultured rat ventricular myocytes under normoxic and hypoxic conditions (Oudot et al. 1995). The work showed that (n-3)-rich cardiomyocytes displayed better electromechanical recovery during hypoxia and reoxygenation than (n-6)-rich cardiomyocytes. The authors suggested that dietary (n-3) PUFA contribute to the protection of the heart by modulating eicosanoid synthesis at both the vascular and cardiomyocyte levels.

Another antiarrhythmic mechanism suggested by Abeywardena et al. (1991) describes the existence of different substrate (fatty acid) pools as a result of different dietary lipid supplementation and an inhibitory action of (n-3) PUFA on thromboxane synthetase. The same authors (Abeywardena et al. 1993) examined the effects of various fatty acids on myocardial eicosanoids and on myocardial phospholipids. Fish oil-fed animals had the lowest proportion of LA and AA with a rise in (n-3) fatty acids in the cardiac membrane. These studies have highlighted the importance of the balance of eicosanoids in determining the arrhythmic outcome in ischemia as well as after reperfusion. A recent study by Pepe and McLennan (1996) of isolated perfused hearts from rats fed fish oil concurs with the above studies. Fish oil feeding prevented severe arrhythmias and increased the VF threshold. The authors speculated that the most likely mechanism in this study was the altered fatty acid composition of myocardial membranes or intracellular nonesterified fatty acid pools.

A study of the fatty acid composition of phospholipids of heart sarcolemma after rats were fed one of four different types of oil found that dietary fish oil, unlike the other oils, resulted in a complete change of the sarcolemmal fatty acid pattern (Al Makdessi et al. 1994). The balance of AA that was maintained by the other three oils, namely coconut oil, corn oil and linseed oil, was changed by the presence of the fish oils such that AA was displaced by EPA and DHA. Charnock et al. (1992a) demonstrated in marmoset monkeys that the proportions of AA, EPA and DHA in myocardial phospholipids can be altered by dietary fatty acids, which in turn leads to a shift in the balance between levels of PGI₂ and TXA₂. Fish oil (n-3) PUFA promotes a balance that is beneficial to the prevention of cardiac arrhythmia. These beneficial changes in the marmoset heart were also accompanied by changes in the fatty acid composition of cardiac membrane phospholipids, which links this mechanism with the second mechanism in this review, namely the effect of (n-3) PUFA on membrane phospholipids.

Another mechanism that has not been explored and which may be investigated in future studies is the role of hydroxylated AA metabolites. AA differs from other fatty acids in that it is a substrate for cyclooxygenase, lipoxygenase and cytochrome P450 pathway enzymes that synthesize bioactive eicosanoids. These metabolites are important modulators of the cell signalling processes that play a major role in heart disease and arrhythmias. Epoxyeicosatetraenoic acid (EET) and hydroxyeicosatetraenoic acid are AA metabolites that are formed by the lipoxygenase and cytochrome P450 pathways. They have proinflammatory action and have been found to be a type of autocrine mediator that acts by altering membrane structure and function (Spector et al. 1994). EET have been found to produce rapid calcium flux in porcine smooth muscle cells into which they are taken up and incorporated into phospholipids. The EET are then inactivated by their conversion into dihydroxyeicosatrienoic acid, which terminates the effect of calcium influx and causes vascular relaxation (Spector et al. 1996). The effect of fish oil supplementation on the different AA pathways and metabolites and their possible role in the antiarrhythmic mechanism of (n-3) PUFA is a new area that needs to be explored.

It has been suggested that diet-induced changes in the fatty acid composition of cardiac muscle cell membranes are associated with development of arrhythmia (Charnock et al. 1985). Hallaq et al. (1992) observed that alterations in the fatty acid composition of the culture medium in which cardiac myocytes were grown also altered myocyte function. Changes in cardiac phospholipids and the NEFA composition brought about by manipulation of dietary fatty acids with (n-3) PUFA has been repeatedly demonstrated as antiarrhythmic (Charnock et al. 1992b, McLennan et al. 1990, McLennan 1993).

The mechanism postulated here is that changes in the lipid composition of cardiac cell membranes induced by dietary modification can influence the availability of Ca²⁺ for excitation-contraction coupling, which in turn could lead to the development of the arrhythmic state. A diet supplemented with fish oil changes the phospholipid composition of the cardiac membrane by reducing the level of (n-6) PUFA and increasing the (n-3) PUFA levels. Membrane phospholipids control the transfer of ions across the membrane, and fatty acid composition of membrane phospholipids may influence the properties of specific ion channels like the calcium and sodium channels (Hallaq et al. 1990).

The activation of phospholipase enzyme is another mechanism that has been proposed to explain degradation of cardiac membranes after long periods of ischemia. Grynberg et al. (1992) found that the activity of phospholipase in the cardiac cell could be influenced by phopholipid fatty acid composition. In their study using cardiomyocytes cultured separately in EPA- or DHA-supplemented media, they found that phospholipase activity was lower in the EPA medium under hypoxic conditions, suggesting that this could explain the decreased membrane degradation during ischemia.

The cardiac sarcoplasmic reticulum (CSR) is an important store of calcium for the activation of myocardial contraction. Relaxation occurs primarily by the energy dependent reuptake of calcium by the CSR. Fish oil supplementation caused a decrease in CSR function, and (n-3) PUFA were found to readily accumulate in CSR phospholipids (Taffet et al. 1993). Dietary (n-3) PUFA also modulate physico-chemical properties of sarcolemma by altering the fatty acid composition of the sarcolemma. The fish oil (n-3) PUFA, EPA and DHA, are taken up by the myocardium and incorporated into membrane phospholipids like phosphatidyl choline and phosphatidyl ethanolamine. This uptake is largely at the expense of arachidonic acid (Swanson and Kinsella 1986).

Several mechanisms have been suggested as to how the increased NEFA levels may trigger arrhythmias. One hypothesis suggests that during ischemia, -oxidation of lipids in mitochondria is inhibited and causes accumulation of intracellular acylcarnitine and acyl-CoA. This acylcarnitine in turn inhibits the Ca²⁺ pump of the sarcoplasmic reticulum and calcium channels, causing an increase in Ca²⁺ levels in the myocardial cells and thus causing arrhythmias (Corr et al. 1984, Huang et al. 1992). Another study lending support to this hypothesis reported the effects of increased NEFA levels on ventricular arrhythmias using ventricular fibrillation threshold (VFT) as an index of arrhythmogenicity (Makiguchi et al. 1991). They observed that the arrhythmogenicity of NEFA was due to a direct effect on the myocardial cells and due to the effect of NEFA esters such as long-chain acylcarnitine and acyl-CoA. They speculated that the effect of NEFA was related to calcium overload in the myocardial cells.

It was also observed that during acute myocardial infarction, patients with excess saturated fatty acids (sum of 14:0, 16:0 and 18:0) in adipose tissues were most susceptible to ventricular arrhythmias (Abraham et al. 1989). The FFA hypothesis is considered controversial because in one study, despite the elevated plasma FFA levels after ischemia was induced in dogs, no increase in the incidence of arrhythmias was observed nor was VFT altered (Opie et al. 1971)

Another proposed mechanism suggests that altered proportions of AA, EPA or DHA in myocardial NEFA as a result of altered dietary fatty acids could lead to alterations in the NEFA pool that serves as immediate substrates for eicosanoid production. This could then lead to alterations in the production of myocardial TXA₂ and the vulnerability of the heart to develop arrhythmia during partial ischemia (Charnock et al. 1992b). That study demonstrated that after feeding a mixed diet relatively high in saturated fat and containing (n-3) PUFA, the NEFA had a direct effect on the heart by reducing the amount of eicosanoids and was one of the major factors that determined the vulnerability of the heart to develop VF, despite the very low NEFA concentration in the myocardial cell.

A study that looked at the effect of an intravenous infusion of (n-3) PUFA on ischemia-induced VF in exercising dogs (Billman et al. 1994) has once again renewed interest in this mechanism. Intravenous infusion of a fish oil emulsion containing EPA and DHA prior to inducing ischemia successfully prevented ischemia-induced VF. Since infusion was carried out 60 min before inducing ischemia, the possibility that the effect of fish oil was mediated via incorporation of (n-3) PUFA in sarcolemmal membranes was ruled out. The authors attributed the antiarrhythmic effects of fish oil to NEFA and postulated that unesterified PUFA, not saturated or monounsaturated fatty acids, prevented arrhythmias. In a recent study (Kang and Leaf 1996) of isolated myocytes, the authors have demonstrated that PUFA including AA, EPA and DHA can bind to the highly reactive sodium pump and prevent arrhythmias. They also found that since AA is rapidly converted to TXA_2, which is proarrhythmic, only the (n-3) PUFA qualified for possessing antiarrhythmic properties. Saturated and monounsaturated fatty acids did not significantly bind to the Na⁺ channel and were not believed to be antiarrhythmic, although LA was found to be mildly antiarrhythmic. They concluded that (n-3) PUFA did not have to be incorporated into membrane phospholipids because only free acids exhibited antiarrhythmic potential. In another recent study Weylandt et al. (1996) have shown that in cardiac myocytes cultured in NEFA-stripped medium supplemented with EPA and DHA, no antiarrhythmic effects were observed despite the membrane enrichment with EPA and DHA. This further substantiates the claim that (n-3) PUFA exert antiarrhythmic actions as free acids and not in phospholipids. But this in vitro system does not induce the release of NEFA as in an in vivo system where catecholamines and calcium activate phospholipase enzymes, which in turn trigger the release of NEFA following ischemia. The effects of different fatty acids on cardiac arrhythmia are summarized in Figure 2.

In our laboratory, the working hypothesis is that immediately following myocardial infarct NEFA are released from the hydrolysis of membrane phospholipids, and the type of NEFA released determines the arrhythmic response of the myocardium. Our working hypothesis thus compliments that postulated by Leaf and coworkers. However we believe that the incorporation of (n-3) PUFA into myocyte membrane is essential for antiarrhythmic action, so that these fatty acids are readily available for release as free acids to prevent arrhythmias following myocardial ischaemia.

Studies by Anderson et al. (1995) have shown that there is an increase in the release of IP₃ during reperfusion after acute ischemia. In a recent study by Jacobsen et al. (1996), the thrombin stimulated release of IP₃ during myocardial reperfusion was found to be greater than that observed in normoxic tissues. Their study suggests that thrombin is directly proarrhythmic, and the stimulation of IP₃ release initiated reperfusion arrhythmias.

Pioneering work by Hallaq et al. (1990, 1992) attempted to explain the effect of (n-3)PUFA on calcium channels. They demonstrated that DHA prevents toxic arrhythmias more effectively than EPA by modulating L-type calcium channels in the sarcolemma of cardiac myocytes, which prevents cytosolic free calcium levels from increasing to toxic levels. They also believe that the (n-3) PUFA are not merely acting as calcium channel blockers but as modulators, or valves, that control the influx and efflux of calcium to maintain normal contractility of the myocytes (Leaf 1988).

Hallaq et al. (1990) showed that the protective effect of (n-3) PUFA may not be due to the change in membrane phospholipids, but instead may be due to specific actions of (n-3) PUFA and their metabolites. Ouabain inhibits the Na⁺,K⁺-ATPase pump and changes the electrophysiology of the myocardial cell causing sodium ions to accumulate. The increasing concentration of sodium ions in turn causes accumulation of calcium ions resulting in increased contraction and spontaneous beating rate in the myocytes. In myocytes incubated with AA there was no change after exposure to ouabain, while in myocytes incubated with EPA, ouabain toxicity was prevented. The authors suggest that EPA incorporated in membrane phospholipids, unlike AA, blocked ouabain binding which prevented the Ca²⁺ levels from increasing and thus preserved normal physiological levels of calcium. To further understand the role of calcium channels in the protective effect of (n-3)PUFA, the same group (Hallaq et al. 1992) studied isolated rat cardiac myocytes using nitrendipine, an inhibitor of the L-type calcium channel. This time they found that EPA protected the cardiac myocytes from ouabain toxicity by preventing the increase in free calcium to toxic levels via direct action on the calcium channels, not due to the incorporation of the (n-3)PUFA in membrane phospholipids. This study demonstrated that fish oil fatty acids appeared to exert a dual effect; they prevented excessive calcium influx but at the same time increased calcium influx when it was insufficient. Figure 3 presents the link between the inositol lipid cycle and calcium levels and the possible effects in initiating arrhythmia within myocardial cells.

Fish oil has been found to affect phospholipase enzyme activity. Activation of phospholipase A₂ causes elevation of intracellular calcium. Phospholipase D activity is associated with the sarcolemma and is believed to be involved in second messenger signalling systems. Dai et al. (1995) reported that sarcolemmal phospholipase D is activated by unsaturated fatty acids like arachidonic and oleic acids.

TXA₂ and prostacyclin, both derived from arachidonic acid, have potent but opposite actions on both vascular smooth muscles and platelets. TXA₂ is a potent vasoconstrictor of arteries and promotes platelet aggregation, whereas prostacyclin is a vasodilator and inhibits platelet aggregation. Inhibitors and receptors have also been used to study the effect of thromboxane on arrhythmias. Studies in which UK38485, a specific inhibitor of thromboxane, was administered to greyhound dogs prior to occlusion of the left anterior descending artery (LAD) demonstrated that thromboxane is a major factor in the genesis of arrhythmias induced by acute myocardial infarction and reperfusion (Coker 1982). In another study by the same authors (Coker and Parrat 1985), intravenous injection of a thromboxane antagonist, AH23848, before and during acute myocardial ischemia was found to effectively block thromboxane receptors and to produce an antiarrhythmic effect. Blocking TXA₂-endoperoxide receptors has also been shown to reduce ischemic injury to the myocardium (Hock et al. 1986).

Thrombin is the final enzyme in the procoagulant pathway and is an important regulator of various cellular events. One important role of thrombin is the activation of phospholipase C, which is involved in the generation of second messengers and the mobilization of calcium stores. A study of the effects of thrombin on the heart suggested that it might prolong repolarization and cause electrical abnormalities which could lead to ischemia (Steinberg et al. 1991). The effect of diet or fish oil supplementation on thrombin has not been studied, but this could be a possible mechanism and warrants further investigation.

CONCLUSION