Role of epoxyeicosatrienoic acids in protecting the myocardium following ischemia/reperfusion injury

Abstract

Cardiomyocyte injury following ischemia–reperfusion can lead to cell death and result in cardiac dysfunction. A wide range of cardioprotective factors have been studied to date, but only recently has the cardioprotective role of fatty acids, specifically arachidonic acid (AA), been investigated. This fatty acid can be found in the membranes of cells in an inactive state and can be released by phospholipases in response to several stimuli, such as ischemia. The metabolism of AA involves the cycloxygenase (COX) and lipoxygenase (LOX) pathways, as well as the less well characterized cytochrome P450 (CYP) monooxygenase pathway. Current research suggests important differences with respect to the cardiovascular actions of specific CYP mediated arachidonic acid metabolites. For example, CYP mediated hydroxylation of AA produces 20-hydroxyeicosatetraenoic acid (20-HETE) which has detrimental effects in the heart during ischemia, pro-inflammatory effects during reperfusion and potent vasoconstrictor effects in the coronary circulation. Conversely, epoxidation of AA by CYP enzymes generates 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) that have been shown to reduce ischemia–reperfusion injury, have potent anti-inflammatory effects within the vasculature, and are potent vasodilators in the coronary circulation. This review aims to provide an overview of current data on the role of these CYP pathways in the heart with an emphasis on their involvement as mediators of ischemia–reperfusion injury. A better understanding of these relationships will facilitate identification of novel targets for the prevention and/or treatment of ischemic heart disease, a major worldwide public health problem.

1. Introduction

Heart disease and stroke are major causes of illness, disability and death in Western societies, and impose a great burden to national health care systems [1-6]. For example, cardiovascular disease (CVD) accounted for the death of approximately 76,500 Canadians in 2002 [2,4] and over 910,000 individuals in the US [6]. As the population ages and co-morbidities, such as obesity and diabetes become more prevalent, both the human cost and economic burden of CVD will likely increase. Acute myocardial infarction (AMI) continues to be a leading cause of death worldwide [7,8]. Myocardial infarction occurs when ischemia exceeds a critical threshold and overwhelms cellular repair mechanisms that are designed to maintain normal operating function and homeostasis. Ischemia at this critical threshold level results in irreversible myocardial cell damage or death. Such injury contributes to the pathogenesis of heart failure (HF), AMI and sudden death [9]. Advances in early reperfusion therapy, such as thromobolytic drugs, coronary angioplasty or bypass graft surgery, have reduced morbidity, HF and infarct-associated ventricular arrhythmias. Preconditioning (PC) is another powerful cardioprotective strategy that renders the heart resistant to injury [10]. Brief, non-detrimental episodes of ischemia or pharmacological mimetics given prior to a prolonged ischemic event can initiate signaling events that protect the myocardium [10]. Unfortunately, both early reperfusion therapy and cardioprotective drugs given prior to ischemia have limited clinical utility as patients typically present after the onset of ischemia and/or are unable to reach medical facilities [11,12]. In light of the increasing incidence and prevalence of HF after AMI [1,3,4], there is a profound need for a better understanding of the underlying pathophysiology and a need for development of strategies to protect the myocardium from ischemic-reperfusion injury. Thus, novel therapeutic strategies are required in order to prevent the adverse consequences and impact of CVD.

2. Arachidonic acid, CYP epoxygenases and soluble epoxide hydrolase

Arachidonic acid (AA), a polyunsaturated fatty acid normally found esterified to cell membrane glycerophospholipids, can be released by phospholipases in response to several stimuli, such as ischemia [13]. Free AA is then available for metabolism by prostaglandin H2 synthases, lipoxygenases and cytochrome P450 monooxygenases to generate numerous metabolites, collectively termed eicosanoids [14,15]. CYP epoxygenases metabolize AA to four regioisomeric epoxyeicosatrienoic acids (5,6-, 8,9-, 11,12- and 14,15-EETs), all of which are biologically active (Fig. 1) [16,17]. The actions of EETs are terminated by conversion to the corresponding and less biologically active dihydroxyeicosatrienoic acids (DHETs) by epoxide hydrolases [13]. Two major epoxide hydrolases are found in mammalian tissues, the microsomal epoxide hydrolase (mEH) and the soluble epoxide hydrolase (sEH or EPHX2) [14]. Previous work has demonstrated that sEH is the main enzyme involved in the in vivo hydrolysis of the EETs [15,17]. The majority of endogenous EETs (>85%) are esterified to membrane glycerophospholipids, particularly phosphatidylcholine and phosphatidylinositol, where they are generally considered inactive until their release [17]. In this regard, ischemia has been shown to activate cytosolic phospholipase A₂ leading to the release of bioactive eicosanoids from glycerophospholipids [18].

Fig. 1.

CYP epoxygenase and hydroxylase mediated metabolism of arachidonic acid.

EETs are important components of many intracellular signaling pathways in both cardiac and extracardiac tissues. For example, EETs activate Ca²⁺-sensitive K⁺ channels (BK_Ca) in vascular smooth muscle cells resulting in hyperpolarization of the resting membrane potential and vasodilation of the coronary circulation [19-21]. This effect is diminished upon hydrolysis of EETs to DHETs by sEH [22]. Other studies have shown that EETs display anti-inflammatory, thrombolytic and angiogenic properties within the vasculature [23-25]. In endothelial cells, EETs activate mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K)-Akt signaling pathways [25], increase intracellular cAMP levels [24], upregulate expression of nitric oxide synthase [26] and protect against hypoxia-reoxygenation injury [27]. In general, the effects of DHETs on these pathways are less pronounced [23,24]. Within the heart, EETs activate cardiac ATP-sensitive K⁺ (K_ATP) channels [28-31], enhance L-type calcium currents [32,33] and improve postischemic recovery of left ventricular function [31,34-36]. Thus, alteration in the production and/or elimination of EETs may affect steady-state cellular levels of these bioactive eicosanoids in vivo and could potentially influence cardiac function (Fig. 2).

Fig. 2.

Schematic of proposed mechanisms of EET-mediated cardioprotection. CYP2J2-derived eicosanoids, readily removed by sEH, activate K⁺ channels, p42/p44-MAPK, PKA and/or PKC pathways leading to cardioprotection.

Arachidonic acid can be metabolized by CYP epoxygenases and CYP ω-hydroxylases to products that have vastly different physiologic effects. For example, EETs have potent vasodilatory properties [37] and 20-hydroxyeicosatetraenoic acid (20-HETE) has potent vasoconstrictive effects [38]. Therefore, changes in the expression and/or activity of specific CYP epoxygenase and hydroxylase enzymes can alter the delicate balance between EETs and 20-HETE. For instance, recent data has demonstrated that inhibition of CYP ω-hydroxylases results in reduction of infarct size in rats and dogs following ischemic injury, which suggests that 20-HETE has detrimental effects in the heart [39,40]. These investigators originally showed that 20-HETE was released into coronary venous blood in high concentrations at the end of 60 min of ischemia and throughout 3 h of reperfusion. Furthermore, the non-selective CYP inhibitor, miconazole (MIC) and the more selective inhibitors, 17-octadecanoic acid (17-ODYA) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), all reduced 20-HETE release from the ischemic/reperfused heart and produced marked reductions in myocardial infarct size (expressed as a percent of the area at risk) from 19.6 ± 1.7% to 8.4 ± 2.5% (MIC), 5.9 ± 2.2% (17-ODYA) and 10.8 ± 1.8% (DDMS), respectively [39,40]. Conversely, exogenous administration of 20-HETE significantly increased infarct size to 26.9 ± 1.9%. These investigators also showed that at least three isoforms of the ω-hydroxylases were present in canine heart tissue – CYP4A1, CYP4A2 and CYP4F – all of which were markedly inhibited by incubation with 17-ODYA. Reductions in infarct size were also observed by this same group in rat hearts in which MIC,17-ODYA and DDMS were adminstered prior to ischemia or 5 min prior to reperfusion [40] These results suggest that the major effect of inhibiting ω-hydroxylase in rats occurs during reperfusion and is associated with in a decrease in “reperfusion injury” More recently, this same group demonstrated that inhibition of ω-hydroxylases in dog hearts with DDMS reduced infarct size to a degree equal to that of ischemic PC, the “gold standard” in cardioprotection research. These same investigators found that the administration of the 20-HETE antagonist, 20-HEDE, reduced infarct size similar to that observed with ischemic PC and DDMS. Interestingly, the combination of ischemic PC and DDMS produced a significantly greater reduction in infarct size than either intervention alone. Together, the results suggest that these two treatments may be acting via different signaling pathways and may be a potent combination in alleviating the sequelae of ischemia and/or reperfusion injury in a clinical setting, such as coronary artery bypass graft surgery.

Transgenic hearts from mice overexpressing human CYP2J2 have improved postischemic functional recovery as evidenced by studies using Langendorff perfused hearts subjected to ischemia/reperfusion injury [31]. Perfusion with the selective P450 epoxygenase inhibitor N-methylsulphonyl-6-(2-proparglyloxyphenyl)hexanamide (MS-PPOH) for 20 min prior to ischemia resulted in reduction of postischemic LVDP recovery in wild type hearts and abolished the improved postischemic LVDP recovery in CYP2J2 transgenic hearts. These data provided evidence that the cardioprotective effects of CYP2J2 overexpression involved P450 epoxygenase metabolites. Moreover, these data suggest an important role for endogenous P450 epoxygenases in postischemic functional recovery. Mechanistic studies by these investigators [31] suggested that enhanced activation of ATP-sensitive K⁺ channels (K_ATP) were involved in the improved recovery observed in the CYP2J2 transgenic mice. In addition, CYP2J2 overexpressing mice exhibited increased expression of phospho-p42/p44 mitogen-activated protein kinase (MAPK) following ischemia. The addition of the p42/p44 MAPK kinase (MEK) inhibitor PD98059 during reperfusion abolished the cardioprotective effects observed in the CYP2J2 mice. Together, these data suggest that CYP2J2-derived metabolites are cardioprotective following ischemia and the mechanism for this cardioprotection involves activation of K_ATP channels and p42/p44 MAPK.

Many CYP inhibitors lack isoform specificity and also may have effects on other signaling pathways. Reduction in infarct size observed in rat and rabbit hearts following treatment with non-specific CYP inhibitors has been attributed to suppression of CYP-dependent ROS production [41]. CYP2C and CYP2J isozymes are the predominant AA epoxygenases in the cardiovascular system. While CYP2C9 has been shown to generate ROS in coronary arteries [42], CYP2J2 is not a relevant source of ROS [27,42]. These studies highlight the complexity of the CYP enzyme system, emphasize the role of different CYP metabolites in cardioprotection and suggest caution in the interpretation of results when using non-selective CYP inhibitors. Recent epidemiologic data suggests an association between single nucleotide polymorphisms in the genes encoding CYP2J2, CYP2C8, CYP2C9 and EPHX2 and cardiovascular disease risk in humans supporting the functional relevance of the CYP epoxygenase pathway in the heart [43-47].

3. Ischemic injury and cardioprotection

Ischemic heart disease is an underlying cause of most AMIs, congestive HF, arrhythmias and sudden cardiac death. Myocardial ischemia is characterized by inadequate blood flow to the heart resulting in limited glucose, oxygen and delayed metabolic by-product removal. Ultimately, ischemic events result in cellular death and myocyte loss which is the primary pathology behind many CVDs. Myocytes are not easily replaced, although stem cell therapy shows great initial promise in overcoming this problem. Nevertheless, preserving the viability of ischemic myocardium is the major goal for cardioprotection. Cardioprotective mechanisms modulate cell metabolism or signaling pathways directly or via posttranslational modification of key proteins, as well initiate new transcription and translation [10,12]. A diverse spectrum of signals converge onto a few common end effectors that maintain membrane integrity and inhibit cell death [48]. Mitochondria are one of these important convergence points, due to their critical function in cell survival and death; notably, ATP production and apoptosis [10,12,48]. Key mitochondrial proteins, such as several potassium (K⁺) channels [49-53] and the mitochondrial permeability transition pore (mPTP) [48,54-56], act as effectors integrating these upstream signals into cardioprotective responses. The mechanisms by which CYP metabolites alter these important effectors are not well defined and require further experimentation.

3.1. K⁺ channels and cardioprotection

K⁺ channels are membrane proteins involved in many physiological processes, such as regulation of heart rate, muscle contraction and cell volume [57]. Two pharmacologically distinct ATP-sensitive potassium channels (K_ATP) have been identified in cardiomyocytes, sarcolemmal K_ATP (Sarc K_ATP) and mitochondrial K_ATP (mito K_ATP) [58]. sarc K_ATP is activated during cardiac ischemia when cytoplasmic ATP is depleted and this affects membrane excitability. Activation leads to shortening of the cardiac action potential and reduced intracellular calcium overload [59,60]. Although there are no selective sarc K_ATP openers, a number of drugs known to open this channel have been shown to produce beneficial effects in the myocardium in animal models of ischemia/reperfusion injury, and several non-selective inhibitors of sarc K_ATP (such as glibenclamide) block ischemic PC [59,60]. In spite of these findings, the strongest evidence for a role of sarc K_ATP channels in ischemic PC has been demonstrated in the KiR 6.2 knockout mouse where ischemic PC could not be produced [60]. The EETs have been shown to be potent openers of sarc K_ATP by reducing channel sensitivity to ATP in isolated rat myocytes using the inside-out patch clamp technique; however, the exact site on the channel that interacts with EETs remains unknown [30,61]. These authors also showed that membrane hyperpolarization occurred in isolated rat myocytes by 11,12-EET addition, an effect blocked by glibenclamide, the non-selective K_ATP channel inhibitor. A role for the sarc K_ATP channel in mediating the cardioprotective effect of inhibiting ω-hydroxylase at the time of reperfusion in the rat heart was recently described [34]. These investigators showed that the cardioprotective effect resulting from administering MIC and 17-ODYA just prior to reperfusion in rats was completely abolished by HMR 1098, a selective inhibitor of the sarc K_ATP channel. In contrast, the selective mito K_ATP channel inhibitor, 5-hydroxydecanoic acid (5-HD), had no effect. Although it can be assumed that a shift in the 20-HETE/EET balance in favor of the EETs is responsible for the cardioprotective effect of inhibiting 20-HETE synthesis, it remains unknown if the cardioprotective effect of the ω-hydroxylase inhibitors is the result of the action of the EETs on the sarc K_ATP channel in the myocardium.

In contrast to the sarc K_ATP channel, the precise molecular composition of the mito K_ATP channel remains elusive. However, recent studies suggest it is part of a multiprotein complex including the adenine nucleotide transporter (ANT) and succinate dehydrogenase (SDH) [62]. Importantly, pharmacologic data indicate that selective activation of mito K_ATP confers cardioprotection following ischemia and that this channel is the major one mediating ischemic PC [59,60,63,64]. While the precise pathways by which mito K_ATP activation confers cardioprotection remain unknown, potentially beneficial consequences of opening mito K_ATP include partial depolarization of the intramitochondrial membrane, transient swelling of the intramitochondrial space, enhanced respiration via the electron transport chain, reduced mitochondrial calcium overload, and altered production of reactive oxygen species [59,60,64]. Cardioprotective effects of CYP2J2 overexpression involve activation of mito K_ATP channels [31]. Increased flavoprotein fluorescence (a marker of mitochondrial redox status) [63] in CYP2J2 Tr cardiomyocytes is consistent with enhanced mito K_ATP activation in the presence of CYP2J2 overexpression [31]. Moreover, treatment of wild type cardiomyocytes with physiologically relevant concenrations of EETs increased flavoprotein fluorescence [31].

Ca²⁺-activated K⁺ (K_Ca) channels include large conductance (BK_Ca), voltage-sensitive K⁺ selective proteins expressed in various tissues including heart mitochondria [53]. K_Ca channels can be activated by elevations in intracellular Ca²⁺ and membrane depolarization [65,66]. CYP epoxygenase derived EETs are known activators of BK_Ca channels in vascular smooth muscle [20,67], whereas, CYP ω-hydroxylases derived HETEs are known inhibitors in vascular smooth muscle [38,68,69]. Recent evidence suggests that newly identified K_Ca channels in cardiac mitochondria (mito K_Ca) [53,70] are important mediators of cardioprotection. It is proposed that these mitochondrial K⁺ channels work in concert with mito K_ATP and other mitochondrial proteins in response to ischemia [49]. Activation of K⁺ channels by kinases, such as PKC or PKA, and other unknown signals is predicted to increase mitochondrial K⁺ uptake and in turn reduce Ca²⁺ overload in cardiomyocytes. The cardioprotective mechanisms associated with opening of these channels include a mild uncoupling, depolarization of the intramitochondrial membrane, transient swelling of the intramitochondrial space, enhanced respiration via the electron transport chain and altered production of reactive oxygen species [49,53,59,60,64,70].

3.2. Mitochondrial permeability transition pore

mPTP is a protein complex on the inner mitochondrial membrane which includes ANT, cyclophilin D and the voltage-dependent anion channel (VDAC) [48,49,71]. mPTP remains closed under normal physiological conditions but opens under cellular stress, such as during reperfusion following an ischemic event [48,49,54]. Opening allows free passage of molecules >1.5 kDa which initiate adverse effects, like large osmotic pressure changes and uncoupling of oxidative phosphorylation, that ultimately result in cell death [48,72]. Mitochondrial Ca²⁺ overload, oxidative stress, adenine nucleotide depletion and mitochondrial depolarization contribute to the pore opening. Inhibition of prolonged mPTP opening (high-conductance) during reperfusion with cyclosporine-A (CsA) or sanglifehrin-A (SfA) can reduce cardiomyocyte injury [73-75]. Conversely, evidence suggests that the transient (low-conductance) opening of mPTP during ischemic PC plays a role in cell survival [54]. Cell culture experiments suggest that mito K_ATP openers, such as diazoxide, trigger mPTP opening which can be blocked by CsA or 5HD [54]. Recent evidence demonstrates that multiple cardioprotective kinases, PKA, PKB/Akt and PKC can converge upon glycogen synthase kinase-3β (GSK-3β), which will induce inhibition of the mPTP [76]. While it is very likely that signaling pathways involved in EET-mediated cardioprotection target the mitochondria, it is unknown whether the response involves transient opening of mPTP prior to ischemia or prevents prolonged opening during reperfusion. EETs have been shown to activate sarc K_ATP [30,61] and mito K_ATP channels [31] in the heart, and K_Ca channels [20,67], in vascular smooth muscle cells [38,68,69]. Increased flavoprotein fluorescence in CYP2J2 Tr cardiomyocytes and wild type cardiomyocytes treated with EETs [31], strongly suggest convergence of a cardioprotective signal onto the mitochondria. It is expected that EETs will alter the flavin nucleotide fluorescence and intracellular [Ca²⁺] within the myocytes and most likely alter mPTP opening. It is unknown whether EETs work solely via mito K_ATP channels; however, it is likely they work together with other proteins, such as mito K_Ca channels. Future experiments will help determine whether EETs influence the transient opening or closing of mPTP, as well how they influence intramitochondrial [Ca²⁺] levels.

4. Cardioprotective signaling pathways

There is considerable controversy regarding the role of cytochrome P450s in the heart, notably the beneficial versus the detrimental effects of arachidonic acid metabolites [31,34,35,39,40,77,78]. We have demonstrated that EETs play a significant role in the improved postischemic functional recovery in isolated mouse hearts overexpressing CYP2J2 [31,34,35,57,78]. Recent results suggest potential cardioprotective mechanisms and indicate that K_ATP channels, p42/p44-MAPK and PI3K are involved. However, as previously discussed EETs are known to activate a number of signaling elements and ion channels, therefore, further work needs to be done to determine the important protective mediators involved in this novel cardioprotective pathway. Preliminary data suggested that EETs might only partially activate [31] the mito K_ATP channel and could be working synergistically with other cardioprotective sites, such as the sarc K_ATP channel or the mito K_Ca channel. Additional evidence has demonstrated an important role for PKA and PKC in the cardioprotective pathway involving K⁺ channels [79,80,81]. Further experiments will help determine if EET-mediated cardioprotection involves early activation or co-localization of PKA or PKC to the mitochondria.

5. Conclusion

There are numerous reasons for investigating the role of this novel endogenous pathway in myocardial function and ischemic injury. First, CYP-derived metabolites of arachidonic acid play critical roles in modulating fundamental biological processes [68,82]. Second, environmental or genetic factors that alter P450 expression and/or function lead to changes in the production of bioactive eicosanoids [68,82]. Such effects can influence cell and organ function in either an adverse or beneficial manner. Third, CYP isozymes expressed in the heart, notably CYP2J2 [36,83], generate EETs, which have been shown to be cardioprotective [31,36,83]. In contrast, other CYP isozymes (CYP2C and CYP4A) generate products, which are thought to be detrimental to the heart, such as ROS and 20-HETE [39-42]. Traditionally, investigation into the role of CYP isozymes has focused on hepatic and renal drug metabolism and function. There is little known about the importance of this endogenous system within the heart even though the heart contains significant levels of functionally active CYP. Finally, and most importantly, recent human epidemiological evidence has identified associations between CYP2J2 polymorphisms and CAD, and EPHX2 polymorphisms and CHD [43,45,46,84]. These findings provide strong evidence supporting the notion that EETs play an important role in postischemic functional recovery and perhaps infarct size reduction [31,33,36,83]. Recent studies have only begun to address the underlying mechanisms of EET-mediated cardioprotection and highlight the potential for this novel endogenous system as a therapeutic target for CVD. Ultimately, understanding the basic cellular mechanisms of EET-mediated cardioprotection will enhance our knowledge of this important phenomenon and will lead to the development of novel therapeutics for the treatment of cardiovascular diseases. Targeting CYP2J2, ω-hydroxylases or sEH, either through pharmacological or gene therapy methods, represents a novel therapeutic approach to the management of ischemic heart disease in humans.

Abbreviations

AA

arachidonic acid

AMI

acute myocardial infarction

ANT

adenine nucleotide transporter

BK_Ca

large conductance calcium activated potassium channels

CYP

cytochrome P450 monooxygenase

CVD

cardiovascular disease

DHET

dihydroepoxyeicosatrienoic acid

EETs

epoxyeicosatrienoic acid

GSK-3β

glycogen synthase kinase-3β

20-HETE

20-hydroxyeicosatetraenoic acid

HF

heart failure

IPC

ischemic preconditioning

IR

ischemic-reperfusion

K_ATP

ATP-sensitive potassium channel

K_Ca

calcium-activated potassium channel

LVDP

left ventricular developed pressure

MAPK

mitogen activated protein kinase

mEH

microsomal epoxide hydrolase

mito K_ATP

mitochondrial ATP-sensitive potassium channel

mPTP

mitochondrial permeability transition pore

MS-PPOH

N-methylsulphonyl-6-(2-proparglyloxyphenyl) hexanamide

PKA

protein kinase A

PKC

protein kinase C

PI3K

phosphatidylinositol-3 kinase

sarc K_ATP

sarcolemmal ATP-sensitive potassium channel

sEH/Ephx2

soluble epoxide hydrolase

SDH

succinate dehydrogenase

VDAC

voltage-dependent anion channel