Polyunsaturated fatty acids come in two classes:

1. The Beneficial Effects of Long-Chain, Polyunsaturated n-3 Fish Oil Fatty Acids on the Cardiovascular SystemAlexander Leaf, M. D.Jackson Professor of Clinical Medicine, EmeritusHarvard Medical School, Boston 02114, USA

2. The structure of this lecture: Back-ground materialTo inform you of the beginning interest in fish and fish oil on heart disease. The current status of the evidence that ingesting fish or fish oils benefit the cardiovascular systems of animals and humans. Author’s humble contributions to our understanding of the mechanisms by which the n-3 fish oil fatty acids prevent fatal ventricular arrhythmias (Sudden Cardiac Death, SCD). Author’s equally humble contribution to the accumulating evidence that n-3 fish oil fatty acids prevent SCD in patients at high risk.

3. BackgroundThe beginning of the interest in fish oil to improve function of the heart occurred early in the 1970’s when two Danish physicians, Bang and Dyerberg, who were aware that the mortality among Greenland Eskimos from heart disease was only about one-tenth that in Denmark and the USA. This was despite the knowledge that the total energy provided from dietary fats was about 38% in all three populations. Dyerberg and Bang then surmised this striking difference in cardiac mortality might be due to the different fats in the diet of the Greenland Inuits and that of Danes and US citizens.

4. Although there were earlier suggestions that polyunsaturated fatty acids might be antiarrhythmic, it was the definitive findings of two Australians McLennan and Charnock, who first demonstrated the antiarrhythmic action of the fish oil fatty acids. Their basic experiment was simple and clear. They fed rats diets for 3- or 4-months in which they could control the major fat component. At the end of the dietary period, they ligated the coronary arteries of the rats and counted the number of animals that died of sustained ventricular fibrillation (VF). In one publication, McLennan reported that slightly more than 40% of animals fed a diet with saturated fat providing 12% of energy calories died of sustained VF.

5. Polyunsaturated fatty acids are essential fatty acids for two reasons. First, they must be obtained from the diet, we cannot synthesize them in our bodies as we can for saturated and monounsaturated fatty acids. Second, they are absolutely essential for optimal development, growth and function of the brain, heart and probably other systems.

6. Polyunsaturated fatty acids come in two classes:

7. The most prevalent in our diet are the n-6 (or also called ω6) class. The parent compound of this class is linoleic acid (LA), an 18-carbon fatty acid with two unsaturated C=C bonds. Since the first of these unsaturated bonds encountered when one counts from the methyl end of the fatty acid chain toward the carboxylic end of the fatty is the 6th carbon atom, hence the appellation n-6 or ω6. This LA can be further desatuated and elongated to form arachidonic acid (C20:4n-6, AA). Arachidonic acid is the physiologically most active member of the n-6 class of fatty acids.

8. The n-3 fatty acids are derived from vertebrate animals in the oceans, the source of n-3 fish oil fatty acids. By contrast the n-6 class of polyunsaturared fatty acids derives from plant seed oils, such as corn oil, sunflower seed, and soy bean oils, the common table and cooking oils, which are much too abundant in our diets. As stated both classes of fatty acids are essential, but for some of their cyclooxegenase, lipoxyngenase and epoxygenase derivatives have actions in our bodies, which oppose each other in important ways. These will be discussed later in this review.

9. The effect of n-3 fish oil fatty acids in preventing arrhythmias in a dog model of sudden cardiac death:To see if we could confirm the surprising findings of McLennan, we turned to a highly reliable dog model of sudden cardiac death. Working with George E. Billman, who prepared the dogs surgically by ligating the left descending coronary artery, producing a large anterior wall infarction and in the same operation placing an hydraulic cuff around the right circumflex artery so that it could be compressed at will. The dogs were then trained to run on a treadmill during the month allowed for them to recover from the surgery and the MI.

11. The Table 1 summarizes our experiments on the dogs:

12. The prevention of fatal arrhythmias by the emulsion of fish oil concentrate (P<0.005), confirms the studies of McLennan and associates. They used feeding experiments, which were criticized because of possible confounding factors occurring in long term feeding studies in animals. We infused the fatty acids just before an ischemic stress in our prepared dogs. We believed that if the n-3 fatty acid infusion were promptly associated with an effect in the protocol we used, we could then feel confident the effect resulted from what had just been infused.

13. Effects of n-3 PUFAs on cultured neonatal rat cardiomyocytes:To learn the biochemical or physiologic effects of these n-3 fatty acids, which explain their antiarrhythmic action, the effects of the n-3 PUFAs on cultured neonatal cardiomyocytes were studied. One can quickly remove the hearts from several one to two day old rat pups and separate the individual myocytes enzymatically. The myocytes are then plated on microscope covers-slips and grown in an appropriate culture medium.

16. At this point we had found that the arrhythmias induced in the isolated neonatal rat cardiomyocytes could in every instance be prevented by the prior addition of the EPA or DHA to the superfusate bathing the cells. Adding the EPA or DHA after an arrhythmia was induced, would stop the arrhythmia. It was apparent that the n-3 PUFA were affecting the excitability/automaticity of the cardiomyocytes, so the effects of the n-3 PUFAs on the electrophysiololgy of the myocytes were examined.

17. Heart, brain and muscle are excitable tissues and their function is to generate electrical currents to signal their actions in the body. This they do by activating and then inactivating ion channels in their plasma membranes to allow specific ions to move through their plasma membranes, thus creating ionic currents. In heart cells these ionic currents create action potentials by the sequential opening and closing of fast voltage-dependent sodium currents into the cardiomyocytes – see Fig. 5.

19. Figure 6. Effects of EPA on activation and inactivation of human myocardial Na+ channel (- plus 1-subunits) transiently expressed in HEK293t cells.

20. Our current hypothesis:Our current hypothesis regarding the mechanism of action of the n-3 polyunsaturated fatty acids to prevent fatal arrhythmias is based on their actions to inhibit the fast, voltage-dependent sodium current and the L-type calcium currents. With a myocardial infarction there occurs a gradient of depolarization of cardiomyocytes. In the central core of the ischemic zone cells rapidly depolarize and die. The depolarization results from deficiency of ATP in the ischemic cells causing a dysfunctional Na,K-ATPase and the rise of interstitial K+ concentrations in the ischemic zone.

21. Thus any further small depolarizing stimulus (for example, current of injury) may elicit an action potential, which if it occurs at a vulnerable moment during the cardiac electrical cycle, may initiate an arrhythmia. With non-homogeneous rates of conduction of the action potential in the ischemic tissue reentry arrhythmias are likely. In the presence of the n-3 PUFAs, however, a voltage-dependent shift of the steady state inactivation curve to more hyperpolarized potentials occurs. The consequence of this hyperpolarizing shift is that sodium channel availability is decreased, and the potential necessary to return these Na+ channels in partially depolarized myocytes to a closed but activatable state is physiologically unobtainable.

22. Figure 7. Effects of EPA on resting and inactivated hH1 Na+ channels. Current tracings were evoked by voltage steps from –150 mV to –30 mV (A) and from –70 to –30 mV (B) in the absence and presence of 5 M EPA. Each value represents 6 – 15 cells. Normalized current was calculated as INa(( (EPA)/INa(( (control) from the same corresponding cell.

23. The L-type Ca2+ current,ICa,LNot all fatal cardiac arrhythmias are caused by dysfunction of the Na+ channel. Many serious arrhythmias can be triggered by excessive cytosolic free Ca2+ fluctuations. In clinical practice these may be seen in patients with extensive bone metastases, hyperparathyroidism, immobilization of extremities (which have in common hypercalcemia) and cardiac glycoside toxicity due to the inhibition of the Na-K-ATPase depolarizing the heart cell and allowing increase in cytosolic free calcium concentrations via the Na/Ca exchanger (the high intracellular Na+ moving out of the cell in exchange for Ca2+ increasing in the heart cell).

27. It is of interest to compare the actions of the n-3 fatty acids with that of available phamaceutical drugs both of which inhibit the Na+ and Ca2+ ion channels, such as the Class 1 antiarrhythmic Na+ channel blockers or the L-type Ca2+ channel blockers. There are several striking differences: a) The n-3 fatty acids have been part of the human diet for hundreds of thousands of years, during the time our genes were being adapted to our environment, including the diet of our hunter-gatherer forebears and they are safe. b) By contrast the available antiarrhythmic pharmaceutical drugs are all potentially toxic.

28. Although at present we think that inhibitory effects of the PUFAs on INa and ICaL seem the major effects accounting for their antiarrhythmic actions, we are not unmindful that they affect other sarcolemmal ion currents as well. By whole cell voltage clamp measurements they have been reported to also inhibit K+ currents - the transient outward current, Ito, and the delayed rectifier current, IK, but not the inward rectifying current, IK1. However, these effects on the important repolarizing K+ currents would have the effect to prolong the action potential duration, a potentially proarrhythmic effect whereas the PUFAs, significantly shorten the action potential duration by some 20%.