Original article
Epoxyeicosatrienoic acid prevents postischemic electrocardiogram abnormalities in an isolated heart model

https://doi.org/10.1016/j.yjmcc.2008.09.711Get rights and content

Abstract

Cytochrome P450 epoxygenases metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) which are in turn converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). The main objective of this study was to investigate the protective effects of EETs following ischemic injury using an ex vivo electrocardiogram (EKG) model. Hearts from C57Bl/6, transgenic mice with cardiomyocyte-specific overexpression of CYP2J2 (Tr) and wildtype (WT) littermates were excised and perfused with constant pressure in a Langendorff apparatus. Electrodes were placed superficially at the right atrium and left ventricle to assess EKG waveforms. In ischemic reperfusion experiments hearts were subjected to 20 min of global no-flow ischemia followed by 20 min of reperfusion (R20). The EKG from C57Bl/6 hearts perfused with 1 μM 14,15-EET showed less QT prolongation (QTc) and ST elevation (STE) (QTc = 41 ± 3, STE = 2.3 ± 0.3; R20: QTc = 42 ± 2 ms, STE = 1.2 ± 0.2mv) than control hearts (QTc = 36 ± 2, STE = 2.3 ± 0.2; R20: QTc = 53 ± 3 ms; STE = 3.6 ± 0.4mv). Similar results of reduced QT prolongation and ST elevation were observed in EKG recording from CYP2J2 Tr mice (QTc = 35 ± 1, STE = 1.9 ± 0.1; R20: QTc = 38 ± 4 ms, STE = 1.3 ± 0.2mv) compared to WT hearts. The putative epoxygenase inhibitor MS-PPOH (50 μM) and EET antagonist 14,15-EEZE (10 μM) both abolished the cardioprotective response, implicating EETs in this process. In addition, separate exposure to the KATP channel blockers glibenclamide (1 μM) and HMR1098 (10 μM), or the PKA protein inhibitor H89 (50 nM) during reperfusion abolished the improved repolarization in both the models. Consistent with a role of PKA, CYP2J2 Tr mice had an enhanced activation of the PKAα regulatory II subunit in plasma membrane following IR injury. The present data demonstrate that EETs can enhance the recovery of ventricular repolarization following ischemia, potentially by facilitating activation of K+ channels and PKA-dependent signaling.

Introduction

Heart disease and stroke are major causes of illness, disability and death in Western societies [1]. As populations' age and co-morbidities such as obesity and diabetes become more prevalent, increased costs to heath care systems can be anticipated. Ischemic heart disease (IHD), also known as coronary artery disease (CAD) or coronary heart disease (CHD), results from the damage incurred from the reduction in blood flow and oxygen supply to the heart. Resultant alterations in ionic homeostasis in the ischemic myocardium alterresting membrane potential, the action potential duration (APD) and myocyte excitability resulting in EKG abnormalities [2], [3], [4], [5]. Classical electrocardiographic (EKG) abnormalities associated with ischemic injury are prolonged QT interval and ST-segment elevation (STE) [6], [7], where QT prolongation reflects a decreased repolarization following depolarization of the cardiac ventricles, whereas STE provides a measure of ischemic injury. These EKG changes can manifest as serious arrhythmias, thereby impeding cardiac function, with possible fatal outcomes.

Arachidonic acid (AA), an essential polyunsaturated fatty acid found esterified to membrane phospholipids, may be released by phospholipases following stress stimuli such as ischemia [8]. Free AA can then be metabolized by CYP epoxygenase to four regioisomeric eicosanoid metabolites, epoxyeicosatrienoic acids (5,6-, 8,9-, 11,12-, and 14,15-EET) [9], [10]. Conversion to the corresponding dihydroxyeicosatrienoic acids (5,6-, 8,9-, 11,12-, and 14,15-DHET) by soluble epoxide hydrolase (sEH) reduces their biological activity. EETs are important components of many intracellular signaling pathways in both cardiac and extra cardiac tissues. They activate various stress response systems, such as p42/p44 mitogen activated protein kinases (MAPK); enhance membrane ion channel activity, such as K+ channels; and improve postischemic recovery of left ventricular function [11], [12], [13], [14].

Activation of sarcKATP channels can protect the heart against ischemic reperfusion injury by hyperpolarizing the cell, thereby limiting calcium entry, preserving ATP utilization, and maintaining cardiac membrane potential and contractility [12]. Preventing further depolarization of the membrane may avert EKG abnormalities such as QT interval prolongation and STE [12]. Previous studies have shown that EETs are potent activators of vascular and cardiac sarcolemmal ATP-sensitive potassium channels (sarcKATP) [12], [13], [14]. Although the mechanism(s) are unknown, evidence suggests that mouse cardiac sarcKATP channels are activated by EETs directly inhibiting ATP binding, whereas activation of vascular sarcKATP is mediated by a cAMP-Protein Kinase A (PKA) dependent mechanism [14]. There are various reports demonstrating EET-mediated activation of other cardiac ion channels. For example, cell culture models suggest that EETs inhibit cardiac Na+ channels [15], modulate Ca2+ currents [16], [17] and shorten ventricular APD by enhancing Kv4.2 channels in a PKA-dependent manner [18]. Together, these data suggest that EET-mediated action involves modulation of ion channels.

Recently, we reported that transgenic mice with cardiac-specific overexpression of human CYP2J2 or mice with targeted disruption of soluble epoxide hydrolase (sEH null) had increased cardiomyocyte EET biosynthesis, enhanced sarcKATP activity and improved postischemic recovery of left ventricular function [11], [19], [20]. To further examine the cardiac effects of CP-derived eicosanoids towards ischemia–reperfusion induced EKG abnormalities, we evaluated the role of EETs in an ex vivo EKG model. Our initial data shows that elevated levels of EETs attenuate changes in measures of electrocardiogram parameters (QT interval and STE). Moreover, the data suggest that this cardioprotection is mediated by CYP epoxygenase metabolites of AA and involves activation of the PKA and sarcKATP channels and PKA channels.

Section snippets

Animals

Commercially available C57Bl/6 mice were purchased from Charles River Laboratories (Charles River Laboratories, Inc.). Mice with cardiac myocyte-specific over expression of human CYP2J2 (CYP2J2 Tr) [11] were obtained from Dr. Darryl Zeldin (NIEHS, RTP, NC, USA). All studies used mice aged 3–4 months, weighing 25–35 g. Experiments were conducted in strict guidelines provided by the University of Alberta Health Sciences Laboratory Animal Services (HSLAS).

Ex vivo cardiac function and electrocardiogram recording

Hearts were perfused in the Langendorff

Characterization

EKG parameters obtained from the ex vivo model were first characterized by recording changes in C57BL/6 hearts perfused with halofantrine (+/−). Isolated hearts were first perfused for 10 min to obtain stable baseline readings, and then subsequently perfused with different concentrations of halofantrine (+/−) (0, 50 or 100 μM, Fig. 1, Fig. 2). Prolongation of QTc intervals were observed following increasing concentrations of halofantrine (Fig. 2A). These data indicated that our ex vivo model

Discussion

While evidence has begun to identify the significance of cardiac cytochrome P450s to heart function and protection, effects are dependent on the metabolites produced [11], [19], [30], [31], [32], [33], [34]. CYP epoxygenases (EETs) have well established cardioprotective effects within the cardiovascular system [11], [35], whereas metabolites from CYP hydroxylases (20-HETE) can be detrimental. CYP epoxygenase-derived eicosanoids can affect cardiomyocyte function [15], [16], [36] and improve

Acknowledgments

JMS is the recipient of a New Investigator Award from the Heart and Stroke Foundation of Canada and a Health Scholar Award from the Alberta Heritage Foundation for Medical Research.

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    Funding: This work was supported by Canadian Institutes of Health Research Grant (JMS MOP79465), USPHS NIH (JRF GM31278) and the Robert A. Welch Foundation (JRF).

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