There is now a substantial literature demonstrating that there is an active and functioning endocannabinoid (EC) system within cardiovascular tissues. Moreover, there is an equally extensive literature to demonstrate the cardio- and vasculo-protective effects of n-3 long-chain (LC)-PUFA. Since both of these topics have been subject to recent detailed published reviews, this article is not intended to present a comprehensive review of topics that have previously been well covered. However, as is now becoming apparent, there appears to be a close relationship between dietary intervention with n-3 LC-PUFA and changes in tissue levels of EC. The aim of this review is therefore to attempt to draw comparisons between the effects of the EC and n-3 LC-PUFA in the setting of common CVD, and to weigh up the evidence around whether or not EC may play a role in the protective effects of LC-PUFA.
The endocannabinoid system
The EC system constitutes an endogenous signalling system that plays a pivotal role in a variety of centrally and peripherally regulated physiological processes. The system comprises G-protein-coupled cannabinoid (CB) receptors, their endogenous CB (EC) ligands and the associated enzymatic apparatus that controls their synthesis and degradation (as reviewed in( Reference Montecucco and Di Marzo 1 )). The two recognised G-protein coupled receptors are the CB1 and CB2 receptors( Reference Matsuda, Lolait and Brownstein 2 , Reference Munro, Thomas and Abu-Shaar 3 ); CB1 receptors are predominantly expressed in the central nervous system, but are also located in numerous peripheral tissues including cardiac( Reference Batkai, Pacher and Osei-Hyiaman 4 , Reference Pacher, Batkai and Kunos 5 ) vascular( Reference Gebremedhin, Lange and Narayanan 6 , Reference Daly, Ross and Whyte 7 ) and adipose( Reference Nam, Lee and Kim 8 ) tissue. CB2 receptors, while expressed primarily in the periphery, particularly by immune cells( Reference Munro, Thomas and Abu-Shaar 3 ), have also been reported to be present in both the myocardium( Reference Defer, Wan and Souktani 9 ) and endothelial cells( Reference Rajesh, Mukhopadhyay and Batkai 10 ). However, evidence from functional studies also suggests the existence of non-CB receptor targets of the EC, including transient receptor potential vanilloid type-1 channels, PPAR( Reference Pacher, Batkai and Kunos 5 ), the vascular ‘anandamide’ receptor( Reference Zygmunt, Petersson and Andersson 11 ) and the recently de-orphanised receptor GPR55( Reference Pertwee 12 ).
The very presence of these receptors in cardiovascular tissue implies that they are there to mediate responses to endogenous ligands, either as part of cardiovascular homoeostasis, or to participate in pathological processes either as a stress response or as a causative factor. Indeed, endogenous ligands do exist that activate these receptors, the most studied of which are anandamide (AEA) and 2-arachidonyl glycerol (2-AG) and will therefore be the main focus of this review. However, it is worthy of note that various additional endogenous CB receptor ligands have been identified, including docosatetraenyl-ethanolamide, N-arachidonoyl dopamine, virodhamine and noladin (reviewed in( Reference Hiley 13 )).
Endocannabinoid synthesis and sites of action
AEA was first shown to be synthesised from the phospholipid precursor N-arachidonyl-phosphatidyl ethanolamine (NAPE), via hydrolysis of NAPE by a calcium (Ca2+) sensitive, NAPE-selective phospholipase D( Reference Di Marzo, Fontana and Cadas 14 ). However, alternate routes (Fig. 1) of AEA biosynthesis have since been identified, including conversion of NAPE to 2-lyso-NAPE (via phospholipase A2) and subsequently to AEA through a Ca2+ independent mechanism( Reference Sun, Tsuboi and Okamoto 15 ), and also hydrolysis of NAPE by phospholipase C to yield the phospho-AEA, which is further hydrolysed to produce AEA. The synthesis of 2-AG (Fig. 2), which is generated from arachidonic acid-containing phospholipids, can be synthesised via (i) the production of diacylglycerol from phosphatidylinositol via phospholipase Cβ and subsequent hydrolysis by sn1-diacylglycerol lipase to yield 2-AG( Reference Kondo, Kondo and Nakane 16 ) and (ii) through generation (via phospholipase A1) of lyso-phosphatidylinositol from phosphatidylinositol and subsequent hydrolysis by a lyso-phosphatidylinositol selective phospholipase C to yield 2-AG (reviewed in( Reference Muccioli 17 )). In terms of receptor activation by these endogenous ligands (reviewed in( Reference Pertwee and Ross 18 ) ), AEA is a partial agonist at CB receptors, with a marginally higher affinity and markedly higher efficacy for the CB1 receptor compared with the CB2 receptor. 2-AG, on the other hand, has a similar affinity for both CB receptors but has a higher efficacy at the CB2 receptor compared to AEA.
Fig. 1. Synthetic pathways for anandamide (AEA). NAPE, N-arachidonyl-phosphatidyl ethanolamine; pAEA, phospho-anandamide; NAPE-PLD, NAPE-selective phospholipase D; PLA2, phospholipase A2; PLC, phospholipase C.
Fig. 2. Synthetic pathways for 2-arachidonyl glycerol (2-AG). Lyso-PI, lyso-phosphatidylinositol; DAG, diacylglycerol; PLA1, phospholipase A1; PLCβ, phospholipase C-beta; sn-1-DAGL, sn-1 specific diacylglycerol lipase.
There are now numerous reports that both circulating and tissue EC levels are raised in a variety of cardiovascular-related pathologies, including cerebral( Reference Berger, Schmid and Schabitz 19 ), hepatic( Reference Kurabayashi, Takeyoshi and Yoshinari 20 ) and myocardial( Reference Wagner, Hu and Bauersachs 21 ) ischaemia/reperfusion (I/R) injury, heart failure( Reference Weis, Beiras-Fernandez and Sodian 22 ), diabetic cardiomyopathy( Reference Rajesh, Batkai and Kechrid 23 ), advanced atherosclerosis( Reference Sugamura, Sugiyama and Nozaki 24 ) and obesity-related cardiovascular dysfunction( Reference Quercioli, Pataky and Vincenti 25 ). However, the nature of the specific EC, and its precise role (i.e. whether it is protective or detrimental) varies widely between pathological conditions. For the purposes of this review, the discussion will focus on the potential role of the EC in the setting of three cardiovascular disorders, namely hypertension, atherosclerosis and acute myocardial I/R.
Interactions between long-chain-PUFA and endocannabinoids
There is a significant literature concerning the cardio- and vasculo-protective effects of n-3 LC-PUFA and various underlying mechanisms have been proposed including: anti-inflammatory and anti-oxidant effects; modulation of cardiac ion channels; reduction of TAG; influence on membrane microdomains and downstream cell signalling pathways; improved cardiac mitochondrial function; antithrombotic and antiarrhythmic effects (reviewed in( Reference Adkins and Kelley 26 – Reference Stanley, Khairallah and Dabkowski 29 ) ). What is interesting is that the beneficial effects of n-3 LC-PUFA are observed in very similar settings to those in which changes in levels of EC are observed, which begs the question as to whether or not there are shared or integrated mechanisms through which both the n-3 LC-PUFA and the EC exert their effects. Indeed, there is building evidence of interactions between the n-3 LC-PUFA and activation of the EC system (recently reviewed in( Reference Meijerink, Balvers and Witkamp 30 , Reference Kim, Li and Watkins 31 ), although the findings are variable in terms of whether or not n-3 LC-PUFA raise or lower EC levels. Berger et al ( Reference Berger, Crozier and Bisogno 32 ) were one of the first groups to demonstrate a definitive effect of n-3 LC-PUFA ingestion on raising brain levels of AEA in piglets, and this has since been extended in studies in mice where DHA supplementation significantly altered EC-related metabolites in plasma and brain( Reference Wood, Williams and Pandarinathan 33 ). In contrast, other studies have shown that n-3 LC-PUFA deficiency increases, while supplementation decreases, 2-AG levels in the brains of mice( Reference Watanabe, Doshi and Hamazaki 34 ). Thus the impact of n-3 LC-PUFA on EC levels appears to be dependent upon the EC in question. In terms of the effects of n-3 LC-PUFA on EC levels in peripheral tissues the picture appears to be largely opposite to the effects on central EC levels, as dietary n-3-PUFA supplementation, given as either fish oil( Reference Batetta, Griinari and Carta 35 ) or krill oil( Reference Piscitelli, Carta and Bisogno 36 ) in models of either high-fat feeding or metabolic syndrome, decreases both AEA and 2-AG levels in adipose and heart tissue, effects that are associated with an anti-obesogenic effect and an improvement in glucose tolerance( Reference Rossmeisl, Jilkova and Kuda 37 ). These findings raise the possibility that modulation of the EC system by n-3-PUFA may be an important part of their protective mechanisms of action. The remainder of this review will therefore attempt to synthesise the evidence for the protective mechanisms of ECs and n-3-PUFA to identify where similarities exist and where they do not.
Could the endocannabinoid system be a mechanism for the effects of n-3 long-chain-PUFA in hypertension?
Both AEA( Reference Varga, Lake and Huangfu 38 – Reference Pacher, Batkai and Osei-Hyiaman 40 ) and 2-AG( Reference Jarai, Wagner and Varga 41 ) elicit complex vasodilatory( Reference Malinowska, Baranowska-Kuczko and Schlicker 42 ) and cardio-depressive responses in vivo that are sensitive to inhibition by CB1 receptor antagonists( Reference Varga, Lake and Martin 43 ) and are absent in CB1-knockout mice( Reference Jarai, Wagner and Varga 41 ). Together this implies that the CB1 receptor is responsible for mediating the cardiovascular responses to the EC. The simple fact, however, that CB receptor antagonists do not elicit significant blood pressure responses( Reference Batkai, Pacher and Osei-Hyiaman 4 , Reference Hiley 13 ,Reference Lake, Martin and Kunos 39 , Reference Varga, Lake and Martin 43 , Reference Wagner, Varga and Ellis 44 ), and that blood pressure in CB1 knockout mice is comparable with wild-type controls( Reference Jarai, Wagner and Varga 41 ), suggests that EC do not play a tonic role in blood pressure maintenance. However, in hypertensive rats CB1 receptor antagonists increase blood pressure, whereas inhibitors of AEA metabolism normalise blood pressure( Reference Batkai, Pacher and Osei-Hyiaman 4 ), suggesting that under situations of pathophysiological (i.e. hypertensive) stress the EC are produced as a compensatory mechanism. While, as far as the authors are aware, no determination of EC levels has been made in either hypertensive animal models or in clinical samples from hypertensive patients, the increased expression of CB1 receptors in both the heart and endothelium of hypertensive rats( Reference Batkai, Pacher and Osei-Hyiaman 4 ) is suggestive of an up-regulation of the EC system aimed at redressing the balance. The blood pressure lowering effects of EC in hypertension have been attributed to a combination of a tonic suppression of cardiac contractility (and thus cardiac output) along with a direct vasodilator effect.
The effect of n-3 LC-PUFA on blood pressure has long been an issue of controversy. Recent analysis of clinical and epidemiological studies( Reference Cabo, Alonso and Mata 45 ) shows that, while very high (≥3 g/d) doses of n-3 LC-PUFA do produce a small but significant decrease in blood pressure (especially systolic blood pressure), this is evident only in certain groups such as older hypertensive subjects and individuals with hypertriglyceridaemia( Reference Cicero, Derosa and Di Gregori 46 ). Moreover, lower doses of n-3 LC-PUFA as a single treatment are ineffective in lowering blood pressure in mild essential hypertensive patients( Reference Russo, Olivieri and Girelli 47 ) or individuals with metabolic syndrome( Reference Cicero, Derosa and Di Gregori 46 ). Thus, like the EC system, n-3 LC-PUFA only appear to modify blood pressure in the setting of hypertension.
The blood pressure lowering effects of EC are thought to be mediated through an endothelial mechanism involving the release of nitric oxide (NO)( Reference Rossmeisl, Jilkova and Kuda 37 ), via both CB1 ( Reference Malinowska, Kwolek and Gothert 48 ) and non-CB1/CB2 (possibly vanilloid) receptors( Reference Malinowska, Kwolek and Gothert 48 , Reference McCollum, Howlett and Mukhopadhyay 49 ). Conversely, the proposed mechanism(s) by which n-3 LC-PUFA reduce blood pressure are numerous (reviewed in( Reference Cicero, Ertek and Borghi 50 ) ) and include effects on sodium excretion, interference with the renin-angiotensin system and enhancement of endothelial NO production, although with respect to the latter there is a degree of ambiguity between experimental findings. In vitro studies on isolated bovine and ovine blood vessel preparations show that EPA relaxes blood vessels through endothelial NO release( Reference Omura, Kobayashi and Mizukami 51 , Reference Singh, Kathirvel and Choudhury 52 ). However, studies in human subjects are inconsistent with this and have reported no effect of long-term n-3 LC-PUFA supplementation (0·45–3·4 g/d EPA+DHA) on flow-mediated dilatation and/or arterial stiffness (surrogate markers of endothelial function) in either normal subjects( Reference Sanders, Hall and Maniou 53 , Reference Skulas-Ray, Kris-Etherton and Harris 54 ), hypertensives( Reference Woodman, Mori and Burke 55 ) or patients with peripheral vascular disease( Reference Mackay, Ford and Thies 56 ), although microvascular endothelial function is improved in individuals with type II diabetes( Reference Stirban, Nandrean and Gotting 57 ).
At present there is no data around whether or not n-3 LC-PUFA influence EC levels in the vasculature in hypertension. However, the profiles of EC and n-3 LC-PUFA in the setting of hypertension differ substantially: n-3 LC-PUFA are not particularly effective as either anti-hypertensive or NO-releasing agents, while EC are potent endothelium (and presumably NO) dependent vasodilators. On this basis, current evidence suggests that there is no correlation between n-3 LC-PUFA and EC in hypertension.
Could the endocannabinoid system contribute to the anti-atherosclerotic effects of n-3 long-chain-PUFA?
There is a growing evidence base for a role of the EC system in atherosclerotic lesion progression. CB2 activation has been associated with an anti-atherogenic effect on the basis of the following: (i) the phytocannabinoid ∆9-tetrahydrocannabinol reduces atherosclerotic lesion progression and suppresses leucocyte adhesion to the vascular wall in high-fat fed ApoE−/− mice, an effect that is abolished by co-administration with a CB2 selective antagonist (SR144528)( Reference Steffens, Veillard and Arnaud 58 ) ; (ii) the CB2 agonist JWH-133 attenuates smooth muscle cell proliferation and migration( Reference Rajesh, Mukhopadhyay and Hasko 59 ) and (iii) the suppressant effect of the CB1/CB2 receptor agonist WIN 55,212-2 on TNFα and superoxide production in human peripheral blood mononuclear cells is sensitive to CB2 receptor blockade( Reference Germain, Boichot and Advenier 60 ). However, more recent data have shown that neither CB2 receptor activation with a selective agonist (JWH-133) nor CB2 receptor gene deletion modulate atherogenesis in high-fat fed LDLR−/− mice( Reference Willecke, Zeschky and Ortiz Rodriguez 61 ). In contrast to the picture with CB2 receptors, evidence that CB1 activation results in atherogenesis is derived from the findings that patients with advanced coronary artery disease exhibit elevated circulating levels of both AEA and 2-AG along with increased CB1 receptor expression in coronary plaques( Reference Sugamura, Sugiyama and Nozaki 24 ). Taken together with the findings that the CB1 selective antagonist rimonabant (i) attenuates lesion development in a murine model of atherosclerosis via mechanisms involving suppression of proinflammatory gene expression and macrophage recruitment( Reference Dol-Gleizes, Paumelle and Visentin 62 ) and (ii) inhibits vascular smooth muscle cell proliferation and migration( Reference Rajesh, Mukhopadhyay and Hasko 63 ), this has led to the proposal that EC exert a pro-atherogenic effect, signalling through CB1 receptors. However, it should be borne in mind that all of the afore-mentioned studies employed phyto- or synthetic CB ligands, which may behave differently from the endogenous EC, AEA and 2-AG. In fact, AEA attenuates TNFα-induced expression of inter-cellular adhesion molecule 1 and vascular cell adhesion molecule 1 in human coronary artery endothelial cells and attenuates TNFα-stimulated human acute monocytic leukaemia cells monocyte adhesion, both actions being sensitive to CB1 and CB2 receptor blockade( Reference Rajesh, Mukhopadhyay and Batkai 64 ), suggesting that AEA at least exhibits effects that are more in line with an anti-, rather than pro-, atherogenic effect. With regard to 2-AG, there is in fact a dearth of literature about its effects on either atherogenesis or the cellular events involved in the atherogenic process.
The impact of n-3 LC-PUFA intervention in atherosclerosis progression has long been a subject of contention, but there is now sufficient evidence from several large-scale randomised trials (DART Trial( Reference Burr, Fehily and Gilbert 65 ), GISSI-Prevenzione Study( 66 ), JELIS Study( Reference Yokoyama, Origasa and Matsuzaki 67 ) ) to demonstrate the effectiveness of n-3 LC-PUFA supplementation in the primary and secondary prevention of CHD. The main effects of n-3 LC-PUFA appear to involve modulation of processes key to atherosclerosis progression and plaque stablisation( Reference von Schacky, Angerer and Kothny 68 , Reference Sekikawa, Curb and Ueshima 69 ) via both direct (i.e. at the level of the plaque) and indirect (i.e. through alterations in lipid metabolism) mechanisms. Since the aim of this article is to identify similarities between the actions of n-3 LC-PUFA and EC, and since EC influences in atherogenesis are related to direct effects on the developing plaque, this discussion will be confined to the direct effects of n-3 LC-PUFA on plaque stability, which are predominantly of an anti-inflammatory and antioxidant nature.
Dietary fish-oil intake has been documented to lower chemoattractant (platelet-derived growth factor and monocyte chemoattractant protein 1) production in mononuclear cell fractions( Reference Baumann, Hessel and Larass 70 ) and decrease surface expression of the adhesion molecules inter-cellular adhesion molecule 1 and vascular cell adhesion molecule 1 in cultured human aortic endothelial cells( Reference Wang, Chen and Lee 71 ), both of which actions would serve to reduce the inflammatory response and subsequent leucocyte infiltration that fuels plaque progression. n-3 LC-PUFA also directly alter plaque morphology by inducing structural changes consistent with increased stability, characterised by an increased fibrous cap thickness and reduced macrophage infiltration( Reference Thies, Garry and Yaqoob 72 ), reduced foam cell infiltration and mRNA expression of matrix metalloproteinase-7, -9 and -12( Reference Cawood, Ding and Napper 73 ), and increased collagen content of the plaques( Reference Matsumoto, Sata and Fukuda 74 ).
These positive effects of n-3 LC-PUFA on atherosclerotic plaque resemble very closely those seen with agents that modulate CB2-mediated effects in the EC system. Although there is evidence of increased CB receptor expression in atherosclerotic plaques, there are no definitive data to show that EC levels (as opposed to the receptors that mediate their effects) are similarly elevated. However, n-3 LC-PUFA do reduce cardiac EC levels in high-fat fed mice( Reference Piscitelli, Carta and Bisogno 36 ) and unpublished results from our own laboratory have shown that both heart and vascular tissue levels of both AEA and 2-AG are markedly elevated in high-fat fed ApoE-/- and that these are normalised by dietary intervention with EPA. At face value it could therefore be argued that the anti-atherogenic effect of n-3 LC-PUFA could be explained by removal of EC, and thus their pro-atherogenic influence mediated by CB1 receptors; however, this would not take account of the possible protective role of the EC exerted via CB2.
Could the endocannabinoid system contribute to the beneficial effects of long-chain-PUFA in IHD?
I/R injury represents the principal cause of tissue damage following acute myocardial infarction (AMI), while the electrical disturbances that occur during an AMI pose a threat to life through sudden arrhythmic death.
Myocardial injury
Elevated levels of tissue and circulating EC have been reported following myocardial I/R injury in both experimental animals( Reference Defer, Wan and Souktani 9 ) and, more recently, in patients with recent AMI( Reference Wang, Jiang and Bu 75 ). The cellular source and exact role of the EC remains a point of contention and has been the subject of a number of comprehensive reviews (e.g.( Reference Pacher and Hasko 76 , Reference Tuma and Steffens 77 ) ). AEA has been shown in some studies to reduce myocardial infarct size in isolated rat hearts( Reference Underdown, Hiley and Ford 78 ), an effect that is sensitive to both CB1 and CB2 receptor antagonists, whereas in others it does not( Reference Lepicier, Bouchard and Lagneux 79 ). Moreover, suppression of AEA metabolism by either pharmacological inhibition or genetic deletion of fatty acid amine hydrolase exacerbates oxidative/nitrative stress-dependent doxorubicin-induced myocardial injury, an effect that is reversed by CB1 antagonism, and increases AEA-induced cardiomyocyte cell death( Reference Mukhopadhyay, Horvath and Rajesh 80 ), further supporting a role for AEA-induced myocardial injury via CB1.
In contrast, reports of a cardioprotective effect of 2-AG are more consistent( Reference Lepicier, Bouchard and Lagneux 79 , Reference Wagner, Abesser and Harvey-White 81 ), although the evidence is similarly conflicting around whether or not selective CB1 and CB2 receptor agonists do( Reference Lepicier, Bouchard and Lagneux 79 ) or do not( Reference Underdown, Hiley and Ford 78 ) mimic 2-AG-induced cardioprotection, suggesting that a site distinct from the classical CB receptors might be involved. 2-AG has also been implicated in myocardial preconditioning (a potent endogenous form of protection against I/R injury( Reference Underdown, Hiley and Ford 78 )) triggered by ischaemic( Reference Bouchard, Lepicier and Lamontagne 82 ), remote( Reference Hajrasouliha, Tavakoli and Ghasemi 83 ) and pharmacological( Reference Wagner, Abesser and Harvey-White 81 ) stimuli, effects largely attributed to activation of CB2, rather than CB1, receptors( Reference Hajrasouliha, Tavakoli and Ghasemi 83 , Reference Montecucco, Lenglet and Braunersreuther 84 ).
The potential of n-3 LC-PUFA to act as direct cardioprotective agents in the setting of AMI (as opposed to preventing cardiac events) has been evident for well over 20 years. Clinically, patients with a high level of fish-oil derived n-3 LC-PUFA consumption have been reported to have smaller infarcts following an AMI and to demonstrate an improved response to coronary thrombolysis( Reference Landmark, Abdelnoor and Urdal 85 ). Experimentally, prolonged (>2 weeks duration) dietary intervention with fish-oil derived n-3 LC-PUFA has been shown to reduce the extent of tissue injury (infarct size) in numerous studies via a broad range of proposed mechanisms including attenuation of platelet function( Reference Zhu, Sievers and Sun 86 ), opening of cardiac K+-activated Ca2+ channels( Reference Ogita, Node and Asanuma 87 ), increased expression of protein kinase Cδ( Reference Hlavackova, Neckar and Jezkova 88 ) and altered fatty acid composition of mitochondrial phospholipids, in particular the mitochondrial antioxidant phospholipid cardiolipin( Reference O'Shea, Khairallah and Sparagna 89 ). Interestingly, n-3 LC-PUFA have also been described to induce cardioprotection similar to ischaemic preconditioning( Reference Abdukeyum, Owen and McLennan 90 ), either when given as a ‘pre-emptive’ infusion into the coronary circulation immediately prior to the onset of I/R( Reference McGuinness, Neilan and Sharkasi 91 ), or following prolonged dietary intervention( Reference Abdukeyum, Owen and McLennan 90 ), possibly through a reduction of reperfusion-induced oxidative stress and induction of heat shock protein 72( Reference Li, Shi and Li 92 ). Moreover, a positive effect on post-infarction ventricular remodelling has also been demonstrated( Reference Fosshaug, Berge and Beitnes 93 ).
The proposed cellular mechanisms for EC-induced cardioprotection are plentiful, and some similarities can be drawn to the cardioprotective effects of n-3 LC-PUFA. In particular, as with the n-3 LC-PUFA, inhibition of inflammation and oxidative/nitrosative stress, are believed to be major targets for EC,( Reference Wang, Jiang and Bu 75 , Reference Pacher and Hasko 76 ). However, activation of the myocardial protein kinase C and p38 mitogen-activated protein kinase pro-survival pathways( Reference Abdukeyum, Owen and McLennan 90 ), induction of heat shock protein 72( Reference Lepicier, Bibeau-Poirier and Lagneux 94 ) and generation of NO have all also been implicated in EC-induced cardioprotection. Although there are no reports of n-3 LC-PUFA affecting kinase pathways in the heart, they are known to influence mitogen-activated protein kinase activity in the brain( Reference Rao, Ertley and DeMar 95 ), endothelial cells( Reference Xue, Wan and Song 96 ) and human T-cells( Reference Denys, Hichami and Khan 97 ) and therefore whether or not EC play a role in these effects remains to be explored. In terms of NO production, n-3 LC-PUFA suppress myocardial NO synthase activity in hypertensive hearts( Reference Radosinska, Bacova and Bernatova 98 ) ; whether or not n-3 LC-PUFA influence NO production in the setting of myocardial I/R remains to be explored.
Arrhythmias
Endogenously released EC are implicated as being anti-arrhythmic( Reference Hajrasouliha, Tavakoli and Ghasemi 83 , Reference Krylatov, Ugdyzhekova and Bernatskaya 99 , Reference Krylatov, Uzhachenko and Maslov 100 ) against both ischaemia and reperfusion-induced arrhythmias through an action at CB2 receptors. Likewise, a clear beneficial effect of the n-3 LC-PUFA is seen against I/R-induced arrhythmias; a meta-analysis of twenty-seven experimental studies into the antiarrhythmic effects of n-3 LC-PUFA revealed that they afford significant protection particularly against ventricular tachycardia and ventricular fibrillation( Reference Matthan, Jordan and Chung 101 ). The mechanisms responsible for these antiarrhythmic effects have been thoroughly reviewed( Reference Den Ruijter, Berecki and Opthof 102 ) and are believed to be largely due to direct effects of n-3 LC-PUFA on cardiomyocyte transmembrane currents, resulting in electrophysiological changes such as (i) slowing of the Na+ current (reducing excitability and slowing ventricular conduction), (ii) reduced opening of L-type Ca2+ channels (thus reducing early after-depolarisations), (iii) an effect on the Na+/Ca2+ exchanger (to reduced delayed after-depolarisations) and (iv) reduced spontaneous release of Ca2+ from the sarcoplasmic reticulum (reduced triggered activity).
In terms of the cellular mechanisms underlying the antiarrhythmic effects of EC, there is a paucity of data on this, although studies on the direct electrophysiological effects, at least of AEA, give some insight as to these. Like the n-3 LC-PUFA, AEA suppresses action potential duration and blockade of L-type Ca2+ channels in cardiac myocytes( Reference Li, Ma and Zhang 103 ), but although there are no data concerning the effect of AEA on Ca2+ release from the sarcoplasmic reticulum (a known action of n-3 LC-PUFA), the intriguing observation that AEA inhibits IP3-induced Ca2+ release from the cardiomyocyte nucleus( Reference Currie, Rainbow and Ewart 104 ) suggests that this may be worth pursuing as a common mechanism. However, AEA also exerts electrophysiological effects that are not shared with the n-3 LC-PUFA, such as suppression of the cardiac transient outward potassium current I(to) through a non-CB1/CB2receptor-mediated pathway and augmentation of the ATP-sensitive potassium current I(KATP) through a CB2-dependent mechanism( Reference Li, Ma and Song 105 ), both of which would contribute to an antiarrhythmic effect. Moreover, AEA suppresses noradrenaline release from sympathetic nerves innervating the heart( Reference Kurihara, Nishigaki and Suzuki 106 ), which would potentially reduce catecholamine-related arrhythmias during early ischaemia; this is in contrast with n-3 LC-PUFA, which do not influence cardiac sympathetic tone( Reference Du, Dart and Riemersma 107 ).
Can the beneficial effects of n-3 long-chain-PUFA in CVD be linked to those of the endocannabinoid system?
From the afore-mentioned discussion, while it is evident that a link between n-3 LC-PUFA and EC is unlikely to exist in the setting of hypertension, there are sufficient similarities regarding the effects of both in atherosclerosis and IHD to consider that a connection exists. Considering the cardinal role that inflammatory cells play in many CVD states, and the similar influence of both n-3 LC-PUFA and EC on inflammatory processes in atherogenesis, this raises the intriguing possibility that EC may be the ‘missing link’ in understanding the mechanisms underlying the vasculo-protective effects of n-3 LC-PUFA. Similarly, common mechanisms exist for both the tissue-sparing and antiarrhythmic effects of both n-3 LC-PUFA and EC, again raising the notion that modulation of the EC system by n-3 LC-PUFA may play a part in the underlying mechanisms. However, counterintuitive to this hypothesis is that while EC levels are consistently seen to be up-regulated in these pathological conditions, n-3 LC-PUFA tend to suppress EC levels. While this may be interpreted as an overall reduction of activity of the EC system by n-3 LC-PUFA, it is probably more probably that these changes are a consequence of a shift in the n-3/n-6 balance of membrane lipids, resulting in compensatory increases in the n-3 LC-PUFA-derived acyl conjugates docosahexaenoyl ethanolamide and eicosapentaenoyl ethanolamine( Reference Wood, Williams and Pandarinathan 33 ). Indeed, it is plausible that these n-3 LC-PUFA–EC conjugates may in part be responsible for some of the beneficial effects of n-3 LC-PUFA, since docosahexaenoyl ethanolamide and eicosapentaenoyl ethanolamine both bind to CB1 and CB2 receptors in human and mouse leucocytes( Reference Yang, Fredman and Krishnamoorthy 108 , Reference Anagnostopoulos, Rakiec and Wood 109 ) and docosahexaenoyl ethanolamide has been shown to exert anti-inflammatory effects in mouse peritoneal and RAW264·7 macrophages( Reference Meijerink, Plastina and Vincken 110 ). Moreover, in contrast to AEA, docosahexaenoyl ethanolamide appears to have a greater affinity for CB2 than for CB1, in human inflammatory cells( Reference Yang, Fredman and Krishnamoorthy 108 ) ; what this likely means at a cellular level is that, in the presence of high n-3 LC-PUFA concentrations, EC are converted to n-3 LC-PUFA–EC conjugates, which then act as ‘surrogate’ CB2 agonists and thus alter the balance between activation of CB1 v. CB2 receptors in favour of CB2 (Fig. 3). However, it must not be overlooked that the EC are known to act at sites other than the classical CB1/CB2 receptors and therefore the n-3 LC-PUFA–EC conjugates may similarly exert actions at sites distinct from CB receptors, although as far as we are aware this has not yet been tested.
Fig. 3. (Colour online) Proposed mechanism of n-3 LC-PUFA–endocannabinoid interaction in atherogenesis and myocardial ischaemia. AEA, anandamide; 2-AG, 2-arachidonyl glycerol; DHEA, docosahexaenoyl ethanolamide; EPEA, eicosapentaenoyl ethanolamine; CB 2 , cannabinoid receptor type 2.
Conclusions
There is no doubt that n-3 LC-PUFA and the EC AEA and 2-AG each demonstrate protective effects in the setting of CVD. Mechanistically speaking, at least in the setting of atherosclerosis and AMI, there are sufficient similarities to suggest that some (but by no means all) relationships exist between the two in these effects. The fact that n-3 LC-PUFA influence endogenous EC levels, possibly resulting in the generation of conjugates that act preferentially on the CB receptors linked to the beneficial effects of the EC, is an attractive explanation for this, but further studies are warranted before this hypothesis can be proven or refuted.
Acknowledgements
None.
Financial support
L. M. was supported by a British Heart Foundation PhD Studentship (FS/07/054/23590). The British Heart Foundation had no role in the writing of this article.
Conflicts of interest
None.
Authorship
Both authors made an equal contribution to gathering the information from the literature for this review. C. W. drafted the final version of the review.
