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Mechanisms by which Epoxyeicosatrienoic Acids (EETs) Elicit Cardioprotection in Rat Hearts
Abstract
Cytochrome P450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce reductions in infarct size in canine myocardium following ischemia-reperfusion injury via opening of either the sarcolemmal K(ATP) (sarcK(ATP)) or mitochondrial K(ATP) (mitoK(ATP)) channel. In the present study, we subjected intact rat hearts to 30 min of left coronary artery occlusion and 2 h of reperfusion followed by tetrazolium staining to determine infarct size as a percent of the area at risk (IS/AAR, %). The results demonstrate that the two major regioisomers of the CYP epoxygenase pathway, 11,12-EET (2.5 mg/kg, iv) and 14,15-EET (2.5 mg/kg, iv) significantly reduced myocardial infarct size (IS/AAR, %) in rats as compared with control (41.9+/-2.3%, 40.9+/-1.2% versus 61.5+/-1.6%, respectively), whereas, a third regioisomer, 8,9-EET (2.5 mg/kg, iv) had no effect (55.2+/-1.4). The protective effect of pretreatment with 11,12- and 14,15-EETs was completely abolished (61.9+/-0.7%, 58.6+/-3.1%, HMR; 63.3+/-1.2%, 63.2+/-2.5%, 5-HD) in the presence of the selective sarcK(ATP) channel antagonist, HMR 1098 (6 mg/kg, iv) or the selective mitoK(ATP) channel antagonist, 5-HD (10 mg/kg, iv) given 10 min after 11,12- or 14,15-EET administration but 5 min prior to index ischemia. Furthermore, concomitant pretreatment with 11,12- or 14,15-EET in combination with the free radical scavenger, 2-mercaptopropionyl glycine (2-MPG), at a dose (20 mg/kg, iv) that had no effect on IS/AAR (57.7+/-1.3%), completely abolished the cardioprotective effect of 11,12- and 14,15-EETs (58.2+/-1.6%, 61.4+/-1.0%), respectively. These data suggest that part of the cardioprotective effects of EETs in rat hearts against infarction is the result of an initial burst of reactive oxygen species (ROS) and subsequent activation of both the sarcK(ATP) and mitoK(ATP) channel.
... The high expression of isoenzymes of the CYP2 family is in line with their known function in the production of EETs from arachidonic acid. This class of fatty acids is well known to be protective in the heart (Lu et al., 2001;Xiao et al., 2004;Cazade et al., 2014) for example, in myocardial ischemia/reperfusion (Gross et al., 2007;Oni-Orisan et al., 2014). Activation of K ATP channels by 11,12-EET and inhibition of T-type calcium channels by 5,6-EET result in membrane 7,14,21,28,35,42,49,56 and 61 days post TAC surgery. ...
... Frontiers in Physiology frontiersin.org repolarization, whereas 11,12-EET and 14,15-EET reduces the infarct size in rat hearts (Gross et al., 2007;Cazade et al., 2014;Oni-Orisan et al., 2014). In a TAC model, cardiac function could be restored by CYP2J2 overexpression (Edin et al., 2011) and changes in expression of CYP450 enzymes and their associated metabolites contribute to the initiation of cardiac hypertrophy (Althurwi et al., 2015). ...
... In a TAC model, cardiac function could be restored by CYP2J2 overexpression (Edin et al., 2011) and changes in expression of CYP450 enzymes and their associated metabolites contribute to the initiation of cardiac hypertrophy (Althurwi et al., 2015). However, most of these studies are based on cardiac myocyte-specific CYP2J2 overexpression, EETs administrated by injection, knockout mouse model of soluble epoxide hydrolase (sEH) or pharmacological inhibition of sEH (Gross et al., 2007;Oni-Orisan et al., 2014). The ecPOR −/− model adds insights into the specific endothelial contribution of metabolites of POR/CYP450 for cardiac function. ...
The cytochrome P450 reductase (POR) transfers electrons to all microsomal cytochrome P450 enzymes (CYP450) thereby driving their activity. In the vascular system, the POR/CYP450 system has been linked to the production of epoxyeicosatrienoic acids (EETs) but also to the generation of reactive oxygen species. In cardiac myocytes (CMs), EETs have been shown to modulate the cardiac function and have cardioprotective effects. The functional importance of the endothelial POR/CYP450 system in the heart is unclear and was studied here using endothelial cell-specific, inducible knockout mice of POR (ecPOR −/− ). RNA sequencing of murine cardiac cells revealed a cell type-specific expression of different CYP450 homologues. Cardiac endothelial cells mainly expressed members of the CYP2 family which produces EETs, and of the CYP4 family that generates omega fatty acids. Tamoxifen-induced endothelial deletion of POR in mice led to cardiac remodelling under basal conditions, as shown by an increase in heart weight to body weight ratio and an increased CM area as compared to control animals. Endothelial deletion of POR was associated with a significant increase in endothelial genes linked to protein synthesis with no changes in genes of the oxidative stress response. CM of ecPOR −/− mice exhibited attenuated expression of genes linked to mitochondrial function and an increase in genes related to cardiac myocyte contractility. In a model of pressure overload (transverse aortic constriction, TAC with O-rings), ecPOR −/− mice exhibited an accelerated reduction in cardiac output (CO) and stroke volume (SV) as compared to control mice. These results suggest that loss of endothelial POR along with a reduction in EETs leads to an increase in vascular stiffness and loss in cardioprotection, resulting in cardiac remodelling.
... These actions were mediated via activation of sarcolemma K ATP (sarcK ATP ) and mitoK ATP channels as well as phosphatidylinositol-3 kinase (PI3K) signaling [31]. Results in canine and rat models of AMI have confirmed the role of K ATP channels in EET-mediated cardioprotection [32][33][34]. ...
... PI3K/Akt signaling [31,36] K ATP channel signaling [26,[31][32][33][34][35]37] STAT3 signaling [40] Opioid signaling [42] eNOS activation [35] Initial ROS formation [34] Inhibition of mPTP opening [35] Reduced chronic left ventricular remodeling a Ischemia-reperfusion (in vivo) Increased eNOS: iNOS ratio [60] Reduced myocardial collagen deposition [58][59][60][61] Reduced cardiac fibroblast activation [59] Reduced inflammatory cytokines [58,61] Reduced macrophage infiltration [59] Reduced oxidative stress [60] Akt, protein kinase B; BNP, B-type natriuretic peptide; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; KATP, ATP-activated K + channel; mPTP, mitochondrial permeability transition pore; NPR-A, natriuretic peptide receptor type-A; OGD, oxygen and glucose deprivation; p42/p44 MAPK, p42/p44 mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; RGR, reoxygenation and glucose repletion; ROS, reactive oxygen species; STAT3, signal transducer and activator of transcription 3. a Chronic ischemia-reperfusion involved reperfusion periods of at least 2 weeks following ischemia. and activator of transcription 3 (STAT3) [40], brain natriuretic peptide (BNP) [41], and opioid receptor [42] signaling in EET-mediated cardioprotection. ...
... PI3K/Akt signaling [31,36] K ATP channel signaling [26,[31][32][33][34][35]37] STAT3 signaling [40] Opioid signaling [42] eNOS activation [35] Initial ROS formation [34] Inhibition of mPTP opening [35] Reduced chronic left ventricular remodeling a Ischemia-reperfusion (in vivo) Increased eNOS: iNOS ratio [60] Reduced myocardial collagen deposition [58][59][60][61] Reduced cardiac fibroblast activation [59] Reduced inflammatory cytokines [58,61] Reduced macrophage infiltration [59] Reduced oxidative stress [60] Akt, protein kinase B; BNP, B-type natriuretic peptide; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; KATP, ATP-activated K + channel; mPTP, mitochondrial permeability transition pore; NPR-A, natriuretic peptide receptor type-A; OGD, oxygen and glucose deprivation; p42/p44 MAPK, p42/p44 mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; RGR, reoxygenation and glucose repletion; ROS, reactive oxygen species; STAT3, signal transducer and activator of transcription 3. a Chronic ischemia-reperfusion involved reperfusion periods of at least 2 weeks following ischemia. and activator of transcription 3 (STAT3) [40], brain natriuretic peptide (BNP) [41], and opioid receptor [42] signaling in EET-mediated cardioprotection. ...
Cardiovascular disease, including acute myocardial infarction (AMI), is the leading cause of morbidity and mortality globally, despite well-established treatments. The discovery and development of novel therapeutics that prevent the progression of devastating consequences following AMI are thus important in reducing the global burden of this devastating disease. Scientific evidence for the protective effects of epoxyeicosatrienoic acids (EETs) in the cardiovascular system are rapidly emerging and suggest that promoting the effects of these cytochrome P450-derived epoxyeicosanoids is a potentially viable clinical therapeutic strategy. Through a translational lens, this review will provide insight into the potential clinical utility of this therapeutic strategy for AMI by 1) outlining the known cardioprotective effects of EETs and underlying mechanisms demonstrated in preclinical models of AMI with a particular focus on myocardial ischemia-reperfusion injury, 2) describing studies in human cohorts that demonstrate a relationship between EETs and associated pathways with coronary artery disease risk, and 3) discussing preclinical and clinical areas that require further investigation in order to increase the probability of successfully translating this rapidly emerging body of evidence into a clinically applicable therapeutic strategy for AMI.
... For instance, 20-HETE, a potent vasoconstrictor, regulates vascular tone, blood flow to specific organs, and sodium and fluid transport to the kidneys [15]. EETs act as an endothelium-derived hyperpolarizing factor and present with potent vasodilatory and antiinflammatory effects [16]. ...
... For instance, 20-HETE, a potent vasoconstrictor, regulates vascular tone, blood flow to specific organs, and sodium and fluid transport to the kidneys [15]. EETs act as an endotheliumderived hyperpolarizing factor and present with potent vasodilatory and anti-inflammatory effects [16]. ...
Through the ACE2, a main enzyme of the renin–angiotensin system (RAS), SARS-CoV-2 gains access into the cell, resulting in different complications which may extend beyond the RAS and impact the Arachidonic Acid (ArA) pathway. The contribution of the RAS through ArA pathways metabolites in the pathogenesis of COVID-19 is unknown. We investigated whether RAS components and ArA metabolites can be considered biomarkers of COVID-19. We measured the plasma levels of RAS and ArA metabolites using an LC-MS/MS. Results indicate that Ang 1–7 levels were significantly lower, whereas Ang II levels were higher in the COVID-19 patients than in healthy control individuals. The ratio of Ang 1–7/Ang II as an indicator of the RAS classical and protective arms balance was dramatically lower in COVID-19 patients. There was no significant increase in inflammatory 19-HETE and 20-HETE levels. The concentration of EETs was significantly increased in COVID-19 patients, whereas the DHETs concentration was repressed. Their plasma levels were correlated with Ang II concentration in COVID-19 patients. In conclusion, evaluating the RAS and ArA pathway biomarkers could provide helpful information for the early detection of high-risk groups, avoid delayed medical attention, facilitate resource allocation, and improve patient clinical outcomes to prevent long COVID incidence.
... EETs derived from CYP2J2 overexpression protect against ischemia-reperfusion in the heart by activation of mito K ATP and p42/p44 mitogen-activated protein kinases (MAPK) [56]. Similarly, 11,12-EET and 14,15-EET significantly reduced myocardial infarct size in ischemic-reperfusion rat model by activation of both the sarc K ATP and mito K ATP channel [57] (Figure 2). cardiomyocytes [49]. ...
... EETs derived from CYP2J2 overexpression protect against ischemia-reperfusion in the heart by activation of mito KATP and p42/p44 mitogen-activated protein kinases (MAPK) [56]. Similarly, 11,12-EET and 14,15-EET significantly reduced myocardial infarct size in ischemicreperfusion rat model by activation of both the sarc KATP and mito KATP channel [57] (Figure 2). Figure 2. The anti-inflammatory effects of EETs in heart disease. EETs protect against inflammatory cardiomyopathy by activating PPARγ, HO-1, and AMPK signaling pathways and inhibiting NF-κB and mTOR signaling pathways. ...
Inflammation plays a crucial role in the initiation and development of a wide range of systemic illnesses. Epoxyeicosatrienoic acids (EETs) are derived from arachidonic acid (AA) metabolized by CYP450 epoxygenase (CYP450) and are subsequently hydrolyzed by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs), which are merely biologically active. EETs possess a wide range of established protective effects on many systems of which anti-inflammatory actions have gained great interest. EETs attenuate vascular inflammation and remodeling by inhibiting activation of endothelial cells and reducing cross-talk between inflammatory cells and blood vessels. EETs also process direct and indirect anti-inflammatory properties in the myocardium and therefore alleviate inflammatory cardiomyopathy and cardiac remodeling. Moreover, emerging studies show the substantial roles of EETs in relieving inflammation under other pathophysiological environments, such as diabetes, sepsis, lung injuries, neurodegenerative disease, hepatic diseases, kidney injury, and arthritis. Furthermore, pharmacological manipulations of the AA-CYP450-EETs-sEH pathway have demonstrated a contribution to the alleviation of numerous inflammatory diseases, which highlight a therapeutic potential of drugs targeting this pathway. This review summarizes the progress of AA-CYP450-EETs-sEH pathway in regulation of inflammation under different pathological conditions and discusses the existing challenges and future direction of this research field.
... NO is a regulator of angiogenesis which enhances vascular permeability, induces extracellular matrix degradation, increases endothelial cell proliferation and migration and stimulates the expression of vascular growth factor (VEGF) [11]. The endothelium is also a major source of critical vasodilators such as epoxyeicosatrienoic acids (EETs) which benefit the endothelium by regulating vascular tone, coagulation, smooth muscle cell proliferation and apoptosis [12]. Epoxidation of arachidonic acid to EETs is catalyzed by a number of cytochrome P450 (CYP) isoforms that demonstrate tissue-specific expression. ...
... Effect of sEH deletion on renal 5,[6][7][8][9][10][11][12][13][14]15-EETs in male and female mice. Values are mean ± S.E., n = 4; significance levels were * p < 0.05 male KO vs male WT; p < 0.05 female KO vs female WT; # p < 0.05 female KO vs male KO. ...
... For instance, 20-HETE, a potent vasoconstrictor, regulates vascular tone, blood flow to specific organs, and sodium and fluid transport to the kidneys [8]. EETs act as an endothelium-derived hyperpolarizing factor and present with potent vasodilatory and antiinflammatory effects [9]. (8,9-EET) (P/N,50351), (±)11,12-epoxyeicosatrienoic acid (11,12-EET)(P/N,50511), (±)14,15-epoxyeicosatrienoic Acid (14,15-EET) (P/N,50651), (±)-5,6-dihydroxyeicosatrienoic acid (5,6-DHET) (P/N, 51211), (±)8,9-dihydroxyeicosatrienoic acid (8,9-DiHT)(P/N,51351), (±)11,12-dihydroxyeicosatrienoic acid (11,12-DHET) (P/N,51511), and (±)14,15-dihydroxyeicosatrienoic acid (14,15-DHET) (P/N,51651). ...
Through the ACE2, a main enzyme of the renin-angiotensin system (RAS), the SARS-CoV2 gains access into the cell, resulting in different complications which may extend beyond the RAS and impact the Arachidonic Acid (ArA) pathway. The contribution of the RAS through ArA pathways metabolites in the pathogenesis of COVID-19 is unknown. We investigated whether RAS components and ArA metabolites can be considered biomarkers of COVID-19. We measured the plasma levels of RAS and ArA metabolites using an LC-MS/MS. Results indicate that Ang1-7 levels were significantly lower, whereas Ang II levels were higher in the COVID-19 patients than healthy control individuals. The ratio of Ang1-7/Ang II as an indicator of the RAS classical and protective arms balance was dramatically lower in COVID-19 patients. There was no significant increase in inflammatory 19-HETE and 20-HETE lev-els. The concentration of EETs was significantly increased in COVID-19 patients, whereas the DHETs concentration was repressed. Their plasma levels were correlated with Ang II concentration in COVID-19 patients. In conclusion, evaluating the RAS and ArA pathway biomarkers could provide helpful information for the early detection of high-risk groups, avoid delayed medical attention, facilitate resource allocation and improve patient clinical outcomes to prevent long COVID incidence.
... A single asterisk represented a statistical difference in comparison with 11,12 -EET-untreated control group (P < 0.05). metastasis [22,24,25]. Therefore, EETs seem to play important roles in many aspects of chronic diseases. ...
The 11,12-epoxy-eicosatrienoic acid (11,12-EET) is formed from arachidonic acid (AA) by cytochrome P450 2J2 (CYP 2J2) epoxygenase and function as an effector in blood vessels. Human endothelial progenitor cells (hEPCs), a preceding cell source for endothelial cells (ECs), involve in the vascular tissue repairing by postnatal neovasculogenesis. However, the effect of 11, 12-EET on hEPCs and neovasculogenesis is not well known. In the current study, we examined the function of 11, 12-EET in hEPCs-mediated neovasculogenesis by using tubular formation analysis, Western Blotting assay, immunofluorescence staining, flow cytometry analysis and zymogram analysis. The results suggest that 11, 12-EET significantly induces neovasculogenesis through the phosphorylation of phosphoinositide 3-kinase (PI3-K)/Akt, endothelial-nitric oxide synthase (e-NOS) and extracellular signal-regulated kinase 1/2 (ERK 1/2) signaling pathways. 11, 12-EET up-regulates the expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and nuclear factor kappa B (NF-κB) proteins. Moreover, 11, 12-EET augments the expression of VE-cadherin and CD31 proteins in hEPCs. 11, 12-EET also augmented Rac1/Rho A signaling cascades, cell migration and an up-regulation of matrix metalloproteinase (MMP) -2 and -9 proteins. These results demonstrate that 11, 12-EET exerts a significant function in the neovasculogenesis of hEPCs.
... The increased level of 14,15-EET benefited certain cardiovascular diseases and protected the cardiovascular system. Moreover, it had been demonstrated that 14,15-EET might reduce infarct size in the rat's heart subjected to regional ischemia and reperfusion [31]. It also induced the phosphorylated eNOS residue (Ser1177 and Thr497) and caused a transient increase of NO production in a cardiomyoblast cell line of rats (H9c2 cells) [20]. ...
The present study aimed to investigate the impacts and underlying mechanisms of 14,15-DHETs on eNOS and vascular endothelial functions. Bovine aortic endothelial cells (BAECs) were treated with various concentrations of 14, 15-DHET. The expressions of eNOS protein and mRNA were observed at different time points. The eNOS expression and phosphorylation were subsequently detected administered with 8,9-DHET, 11,12-DHET, and 14,15-DHET, respectively. Meanwhile, 14,15-DHET action on tube formation was observed in human umbilical vein endothelial cells (HUVECs). Finally, the aorta of male C57BL/6 mice was injected with 14,15-DHET via the tail vein. The impacts of 14,15-DHET (0.4 mg/kg body weight) on the expressions of eNOS protein and mRNA and endothelium-dependent vasodilation (EDV) were detected following 24 h. The expression of eNOS was greatly improved with the 14,15-DHET treatment compared with the BAECs, and eNOS phosphorylation sites at Ser1179, Ser635, and Thr497 were elevated. However, no statistically significant difference was revealed on total eNOS among the 8,9-DHET, 11,12-DHET, and 14,15-DHET treatment groups. Based on the upregulation of eNOS protein, eNOS mRNA levels were increased in BAECs and thoracic aorta of the male C57BL/6 mice treated with 14,15-DHET, suggesting that transcriptional activation was achieved in vascular eNOS. Moreover, 14,15-DHET enhanced tube formation abilities in HUVECs and acetylcholine(ACh)-induced EDV. These findings indicated that 14,15-DHET could improve the vascular endothelial diastolic functions of male C57BL/6 mice, and enhance the ability of tube formation, which might be related to the increase of eNOS expression.
... A crucial aspect is the balance between vasoconstrictive and vasodilatory metabolites of arachidonic acid (116). Vasodilating epoxyeicosatrienoic acids (EETs) have cardioprotective effects (117,118), and increasing EETs via administration of selective soluble epoxide hydrolase inhibitors shows beneficial effects in animal models of ischemia/ reperfusion injury (119)(120)(121)(122). Moreover, lipoxin administration post myocardial infarction improves left ventricular ejection fraction in mice (123). ...
A dysregulated response to systemic inflammation is a common pathophysiological feature of most conditions encountered in the intensive care unit (ICU). Recent evidence indicates that a dysregulated inflammatory response is involved in the pathogenesis of various ICU-related disorders associated with high mortality, including sepsis, acute respiratory distress syndrome, cerebral and myocardial ischemia, and acute kidney injury. Moreover, persistent or non-resolving inflammation may lead to the syndrome of persistent critical illness, characterized by acquired immunosuppression, catabolism and poor long-term functional outcomes. Despite decades of research, management of many disorders in the ICU is mostly supportive, and current therapeutic strategies often do not take into account the heterogeneity of the patient population, underlying chronic conditions, nor the individual state of the immune response. Fatty acid-derived lipid mediators are recognized as key players in the generation and resolution of inflammation, and their signature provides specific information on patients’ inflammatory status and immune response. Lipidomics is increasingly recognized as a powerful tool to assess lipid metabolism and the interaction between metabolic changes and the immune system via profiling lipid mediators in clinical studies. Within the concept of precision medicine, understanding and characterizing the individual immune response may allow for better stratification of critically ill patients as well as identification of diagnostic and prognostic biomarkers. In this review, we provide an overview of the role of fatty acid-derived lipid mediators as endogenous regulators of the inflammatory, anti-inflammatory and pro-resolving response and future directions for use of clinical lipidomics to identify lipid mediators as diagnostic and prognostic markers in critical illness.
... Despite their short lifetime, the polyunsaturated fatty acids' (PUFAs') epoxides, such as the regioisomeric epoxyeicosatrienoic acids (EETs, derived from arachidonic acid, AA, 4) and epoxydocosapentaenoic acids (EDPs, derived from docosohexaenoic acid (DHA, 1) have shown a variety of interesting biological effects (2). Both omega-6 and omega-3 PUFA epoxides are important autocrine/paracrine mediators, and play a critical role in a variety of biological systems, including regulation of inflammation and blood pressure (3,4,5,6,7), pain perception (1,8,9), and angiogenesis (10), and they also have protective effects on various organs such as the heart and brain (9,11,12). In most cases, the epoxide metabolites derived from DHA are more potent than those derived from 4 (structures of DHA and 4 and relevant numbering is shown in Scheme 1). ...
Epoxy PUFAs are endogenous cytochrome P450 metabolites of dietary PUFAs. Although these metabolites exert numerous biological effects, attempts to study their complex biology have been hampered by difficulty in obtaining the epoxides as pure regioisomers and enantiomers. To remedy this, we synthesized 19,20- and 16,17-epoxydocosapentaenoic acids (EDPs, two most abundant EDPs in vivo) by epoxidation of DHA with wild-type (WT) and the mutant (F87V) P450 enzyme BM3 from Bacillus megaterium. WT epoxidation yielded a 4:1 mixture of 19,20:16,17-EDP exclusively as (S,R) enantiomers. Epoxidation with the mutant (F87V) yielded a 1.6:1 mixture of 19,20:16,17-EDP; the 19,20-EDP fraction was approximately 9:1 (S,R):(R,S), but the 16,17-EDP was exclusively the (S,R) enantiomer. To access the (R,S) enantiomers of these EDPs, we used a short (four-step) chemical inversion sequence, which utilizes 2-(phenylthio)ethanol as the epoxide-opening nucleophile, followed by mesylation of the resulting alcohol, oxidation of the thioether moiety, and base-catalyzed elimination. This short synthesis cleanly converts the (S,R)-epoxide to the (R,S)-epoxide without loss of enantiopurity. This method, also applicable to eicosapentaenoic acid and arachidonic acid, provides a simple, cost-effective procedure for accessing larger amounts of these metabolites.
... Epoxyeicosatrienoic acids (EETs) are the cytochrome P-450 monooxidase metabolites of arachidonic acid known to protect mammals against lethal myocardial injury caused by acute ischemia-reperfusion (I/R) insult in various experimental settings (12,33,40). It has been shown that the acute elevation of their levels by exogenous administration of EETs or inhibition of the EET-degrading enzyme soluble epoxide hydrolase (sEH) before ischemia as well as before reperfusion reduces myocardial I/R injury (3,9,13,14,26,29,31,41). Despite strong evidence for cardioprotective effects of EETs, the mechanism(s) by which EETs protect the myocardium against I/R injury is not yet satisfactorily elucidated. ...
... 2 E.g, 5,6-EET was shown to not be effective at promoting the survival of human lung microvascular endothelial cells and 8,9-EET was shown to be the least cardioprotective. 2,5 This is the first study demonstrating that a ligand binding directly at the active site of CYP2J2 alters the site of AA metabolism. 4 As a comparison, we also investigated the effects of the non-cardiotoxic analogues 5-IDN and ZRN on AA metabolism. We found that 5-IDN and ZRN demonstrate competitive inhibition of AA metabolism. ...
Doxorubicin (DOX) is among the oldest and most-used chemotherapeutics. However, its use is limited by a cumulative dose that leads to cardiotoxicity. DOX is an anthracycline quinone that is known to produce reactive oxygen species (ROS). Although ROS play a function in mediating cardiotoxicity, alternative mechanisms that govern its specific cardiotoxicity remain elusive. This is exemplified by the fact that 5-iminodaunorubicin (5-IDN) and zorubicin (ZRN) are relatively noncardiotoxic analogues, even though ZRN forms ROS as efficiently as DOX and 5-IDN produces many fewer ROS.(1) Having a better understanding of the mechanisms underlying DOX cardiotoxicity will be beneficial in the development of efficacious and nontoxic analogues of DOX for cancer chemotherapy.
... Arachidonic acid (AA) is metabolized by cardiac CYP450 enzymes namely CYP2C8, CYP2C9, and CYP2J2 to 4 regioisomeric, cardioprotective epoxyeicosatrienoic acids (EETs) (Figure 2) (Rifkind et al., 1995;Wu et al., 1996). EETs are known to protect the heart against toxic insults mainly by activation of mitochondrial (mitoK ATP ) and sarcolemmal ATP-sensitive potassium channels (Gross et al., 2007;Katragadda et al., 2009;Seubert, 2004). Activation of mitoK ATP reduces intracellular Ca 2þ overload and increases in K þ uptake, moderates the release of stress signals, lowers Dw m dissipation, preserves cellular nucleotides and enhances oxidative phosphorylation (Garlid et al., 2003). ...
Dronedarone and amiodarone are structurally similar antiarrhythmic drugs. Dronedarone worsens cardiac adverse effects with unknown causes while amiodarone has no cardiac adversity. Dronedarone induces preclinical mitochondrial toxicity in rat liver and exhibits clinical hepatotoxicity. Here, we further investigated the relative potential of the antiarrhythmic drugs in causing mitochondrial injury in cardiomyocytes. Differentiated rat H9c2 cardiomyocytes were treated with dronedarone, amiodarone and their respective metabolites namely N-desbutyldronedarone (NDBD) and N-desethylamiodarone (NDEA). Intracellular ATP content, mitochondrial membrane potential (Δψm), and inhibition of carnitine palmitoyltransferase I (CPT1) activity and arachidonic acid (AA) metabolism were measured in H9c2 cells. Inhibition of electron transport chain (ETC) activities and uncoupling of ETC were further studied in isolated rat heart mitochondria. Dronedarone, amiodarone, NDBD and NDEA decreased intracellular ATP content significantly (IC50=0.49, 1.84, 1.07, 0.63 µM respectively) and dissipated Δψm potently (IC50=0.5, 2.94, 12.8, 7.38 µM respectively). Dronedarone, NDBD and NDEA weakly inhibited CPT1 activity while amiodarone (IC50>100 µM) yielded negligible inhibition. Only dronedarone inhibited AA metabolism to its regioisomeric epoxyeicosatrienoic acids (EETs) consistently and potently. NADH-supplemented ETC activity was inhibited by dronedarone, amiodarone, NDBD and NDEA (IC50=3.07, 5.24, 11.94, 16.16 µM respectively). Cytotoxicity, ATP decrease and Δψm disruption were ameliorated via exogenous pre-treatment of H9c2 cells with 11,12-EET and 14,15-EET. Our study confirmed dronedarone causes mitochondrial injury in cardiomyocytes by perturbing Δψm, inhibiting mitochondrial complex I, uncoupling ETC and dysregulating AA-EET metabolism. We postulate cardiac mitochondrial injury is one potential contributing factor to dronedarone-induced cardiac failure exacerbation.
... Due to the rapid switch between oxygen starvation/reperfusion during this phenomenon, the role of ROS was rapidly suspected in IPC ( Bolli et al., 1989;Qiu et al., 1997). An acute increase in ROS was indeed taken in evidence when short periods of ischemia were challenged, resulting in K+ ATP channels activation ( Gross et al., 2007). Furthermore, beneficial effects of IPC are critically blunted when antioxidants are administrated, validating crucial and beneficial impact of ROS in IPC ( Skyschally et al., 2003;Khanna et al., 2008). ...
The heart must consume a significant amount of energy to sustain its contractile activity. Although the fuel demands are huge, the stock remains very low. Thus, in order to supply its daily needs, the heart must have amazing adaptive abilities, which are dependent on dioxygen availability. However, in myriad cardiovascular diseases, "fuel" depletion and hypoxia are common features, leading cardiomyocytes to favor low-dioxygen-consuming glycolysis rather than oxidation of fatty acids. This metabolic switch makes it challenging to distinguish causes from consequences in cardiac pathologies. Finally, despite the progress achieved in the past few decades, medical treatments have not improved substantially, either. In such a situation, it seems clear that much remains to be learned about cardiac diseases. Therefore, in this review, we will discuss how reconciling dioxygen availability and cardiac metabolic adaptations may contribute to develop full and innovative strategies from bench to bedside.
... Different pathological conditions such as ischemic cerebrovascular diseases, hypertension, diabetes, kidney diseases, and cancer are linked to changes in 20-HETE production. 11,12 EETs, formed by CYP2C and CYP2J subfamily, are another important group of ArA metabolites with potent vasodilatory and anti-inflammatory effects. 13 They are shown to induce smooth muscle hyperpolarization and relaxation. ...
Inflammatory conditions increase cardiovascular (CV) risk. Some nonsteroidal anti-inflammatory drugs (NSAIDs) that are used to treat pain and inflammation are also associated with CV complications. Inflammation, but not NSAIDs, disrupts the balance of vasodilator and vasoconstrictor components of the renin-angiotensin system (RAS) within the heart. Herein, we report the effect of both inflammation and NSAIDs (rofecoxib, celecoxib and meloxicam) on the physiologically active cytochrome P450 (CYP) metabolites of arachidonic acid (ArA) in the rat with adjuvant arthritis (AA). After oral administration of 7 daily therapeutically equivalent doses of NSAIDs or vehicle, the anti-inflammatory response, as well as the ArA metabolites and drug concentrations in plasma, heart and kidneys were assessed. Inflammation in the form of AA caused a significant tissue-dependent imbalance of ArA metabolites by elevating the ratio of cardiotoxic 20-hydroxyeicosatetraenoic acid over cardioprotective epoxyeicosatrienoic acids in the heart, and reducing the ratio in the kidney. The observed imbalance was augmented by cardiotoxic rofecoxib but not by other examined NSAIDs with known milder cardiotoxicity. The cardio-renal toxicity of NSAIDs with known severe CV side effects may be due to altered CYP-mediated ArA acid metabolism. The ArA metabolism profile may be a marker of NSAIDs safety and toxicity.
... Apart from cyclooxygenases and lipoxygenases, AA is metabolized by an array of cytochrome P450 enzymes to form either epoxy (CYP1A2/2C8/2C9/2J2) or hydroxy metabolites (CYP4A1/4A2/4F12) [2]. Cytochrome P450 metabolites of AA namely epoxyeicosatrienoic acids (EETs) have come into limelight due to their notable vasoprotective, renoprotective and cardioprotective effects [3][4][5][6]. CYP2C8, CYP2C9 and CYP2J2 are the main CYP450 enzymes catalyzing the metabolism of AA to regioisomeric EETs in human heart [7][8][9] (Fig. 1). Apart from CYP2J2, CYP2C8 and CYP2C9 are predominantly expressed in human cardiomyocytes and coronary arteries respectively [10]. ...
Cardiac enzymes such as cytochrome P450 2J2 (CYP2J2) metabolize arachidonic acid (AA) to cardioprotective epoxyeicosatrienoic acids (EETs), which in turn are metabolized by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs). As EETs and less potent DHETs exhibit cardioprotective and vasoprotective functions, optimum levels of cardiac EETs are paramount in cardiac homeostasis. Previously, we demonstrated that dronedarone, amiodarone and their main metabolites, namely N-desbutyldronedarone (NDBD) and N-desethylamiodarone (NDEA), potently inhibit human cardiac CYP2J2-mediated astemizole metabolism in vitro. In this project, we investigated the inhibition of recombinant human CYP450 enzymes (rhCYP2J2, rhCYP2C8, rhCYP2C9)-mediated AA metabolism and human recombinant sEH (rhsEH)-mediated EET metabolism by dronedarone, amiodarone, NDBD and NDEA. A static model describing sequential metabolism was further developed to predict the aggregate effect of dual-inhibition of rhCYP2J2 and rhsEH on the fold-change of 14,15-EET level (CEET(')/CEET). Dronedarone, amiodarone and NDBD inhibit rhCYP2J2-mediated metabolism of AA to 14,15-EET with Ki values of 3.25, 5.48, 1.39 µM respectively. Additionally, dronedarone, amiodarone, NDBD and NDEA inhibit rhsEH-mediated metabolism of 14,15-EET to 14,15-DHET with Ki values of 5.10, 13.08, 2.04, 1.88 µM respectively. Based on static sequential metabolism modelling, dronedarone (CEET(')/CEET =0.85), amiodarone (CEET(')/CEET =0.48) and NDBD (CEET(')/CEET =0.76) were predicted to decrease cardiac 14,15-EET level whereas NDEA (CEET(')/CEET >35.5) was predicted to elevate it. Based on our novel findings, we postulate the differential cardiac exacerbation potential of dronedarone and amiodarone is partly associated with their differential inhibition potencies of cardiac CYP2J2 and sEH.
... Additional regulation of channel activity is also afforded by phosphorylation at the N-and C-termini [181][182][183]. These channels are also known to be activated by stimuli implicated in cardioprotection, such as 11,12-epoxyeicosatrienoic acid (EET) and NO · [184,185]. However, both of these species can elicit protection via pleiotropic mechanisms [186,187], including other K Ca channels [188][189][190]. ...
Mitochondria play an important role in tissue ischemia and reperfusion (IR) injury, with energetic failure and the opening of the mitochondrial permeability transition pore being the major causes of IR-induced cell death.Thus, mitochondria are an appropriate focus for strategies to protect against IR injury.Two widely studied paradigms of IR protection, particularly in the field of cardiac IR, are ischemic preconditioning (IPC) and volatile anesthetic preconditioning (APC).While the molecular mechanisms recruited by these protective paradigms are not fully elucidated, a commonality is the involvement of mitochondrial K+ channel opening.In the case of IPC, research has focused on a mitochondrial ATP-sensitive K+ channel (mitoKATP), but, despite recent progress, the molecular identity of this channel remains a subject of contention.In the case of APC, early research suggested the existence of a mitochondrial large-conductance K+ (BK, big conductance of potassium) channel encoded by the Kcnma1 gene, although more recent work has shown that the channel that underlies APC is in fact encoded by Kcnt2.In this review, we discuss both the pharmacologic and genetic evidence for the existence and identity of mitochondrial K+ channels, and the role of these channels both in IR protection and in regulating normal mitochondrial function.
... Moreover, isolated mouse hearts treated with directly with EETs or a dual-acting compound possessing EET mimetic and sEH inhibitory properties had reduced infarct size and preserved left ventricular developed pressure (LVDP) compared to controls (Batchu et al., 2011). While the underlying protective mechanism of EETs is unknown, results from rat, mouse and canine models have provided consistent evidence suggesting activation of the K ATP channels and phosphatidylinositol-3 kinase (PI3K) signalling are involved in EET-mediated cardioprotection (Gross et al., 2008;Gross, Hsu, Falck, & Nithipatikom, 2007;Seubert et al., 2006). Cardiac sarcolemmal K ATP channels regulate ionic homeostasis under conditions of metabolic stress and have been shown to provide cardioprotective effects toward IR injury (Gumina et al., 2003;Suzuki et al., 2002). ...
The cytochrome P450 monooxygenase system (CYP) is a multigene superfamily of enzymes, which are important in the metabolism foreign and endogenous compounds. CYP isoforms metabolize a number of n-3 and n-6 polyunsaturated fatty acids (PUFA), including linoleic acid (18:2n6, LA), arachidonic acid (20:4n6, AA), ecosapentaenoic acid (20:5n3, EPA) and docosahexaenoic acid (22:6n3, DHA) into bioactive lipid mediators, termed eicosanoids. CYP-derived eicosanoids have numerous effects toward physiological and pathophysiological events within the body, which depends on the type, quantity and timing of metabolites produced. Alterations in fatty acid composition and concentrations have been shown to have a role in cardiovascular disease (CVD). The functional role of CYP isozymes and CYP-derived eicosanoids toward physiological and pathophysiological processes in the heart is a rapidly expanding field of research. Numerous studies have investigated the beneficial and detrimental effects of CYP epoxygenase derived metabolites of AA, epoxyeicosatrienoic acids (EET) and CYP ω-hydroxylase products, hydroxyeicosatetraenoic acids (HETE), toward both cardiac and vascular function and disease. Emerging research is revealing the importance of other lipid mediators generated from CYP isozymes, such as epoxyeicosatetraenoic acids (EEQ) and epoxydocosapentaenoic acids (EDP), formed from the metabolism of EPA and DHA and metabolites of LA. Important determinants such as genetics, gender and age have a role regulating the CYP-derived eicosanoids produced from the metabolism n-3 and n-6 PUFA. Obtaining a better understanding of the complex role CYP-derived eicosanoids have within the heart will provide valuable insight for both basic and clinical researchers investigation cardiovascular disease.
... However, by correcting the RAS imbalance, NSAIDs only fix one piece of a complicated puzzle. Fig. 5 Ratios of relative densities of angiotensin receptors (AT1R, AT2R, Mas) in control, inflamed and NSAIDs treated rats heart and kidneys (a, b, c) represents significant difference between treatments using one-way ANOVA followed by Bonferroni adjustment (p \ 0.05); AA adjuvant arthritis, AA-Control AA not treated, AA-Rof AA treated with rofecoxib, AA-Mel AA treated with meloxicam, AA-CeI AA treated with celecoxib, AA-FIu AA treated with flurbiprofen Alteration in various receptors involved in hemostasis (Mayo et al. 2000;Ling et al. 2009;Sanaee et al. 2011), disease severity (Sanaee et al. 2011), and changes in the biodisposition of physiologically active metabolite of arachidonic acids (Gross et al. 2007) are a few examples of other mechanisms that remain to be studied as potential contributors to the elevation of CV risks. ...
A co-morbidity of inflammatory conditions is increased cardio-renal risks. Additionally, nonsteroidal anti-inflammatory drugs (NSAIDs) which are used to treat pain and inflammation are also associated with increase in such risks. We hypothesized that inflammation and NSAIDs impose the cardio-renal risk through the activation of the renin-angiotensin-system (RAS), a regulating pathway of the renal and cardiovascular homeostasis. We investigated the effect of adjuvant arthritis and NSAIDs on the RAS. Western blotting and ELISA were used to measure the RAS components. Inflammation caused significant imbalances in the cardiac and renal angiotensin converting enzymes, their biologically active angiotensin peptides (AngII and Ang1-7) and the target proteins involved in the peptide-receptor binding (AngII type 1 and type 2, and Ang1-7 receptor, Mas) toward cardio-renal toxicity. However, 7 days treatment of arthritic animals with NSAIDs (rofecoxib, meloxicam, celecoxib and flurbiprofen) restored the constitutive balances, perhaps due to their anti-inflammatory properties. Inflammation exerts its cardio-renal effects by causing imbalance in the RAS. NSAIDs through their anti-inflammatory effect restore this imbalance. Thus, mechanisms other than imbalances in the RAS may be involved in the NSAIDs cardiotoxicity.
... In fact, acidic perfusion in early reperfusion and delaying pH normalization could A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 16 inhibit mPTP during the first minutes of reflow and allow for endogenous protective signaling pathways to be activated by ROS signaling. The delivery of oxygen during acidic perfusion or brief intermittent reperfusions would promote mitochondrial ROS formation, which has been proposed to activate isoforms of PKC through redox signaling [82]. In this regard, different isoforms of PKC appear as critical kinases in the signaling cascade, leading to a reduced probability of mPTP opening after pH normalization. ...
Reperfusion injury is an inherent response to the restoration of blood flow after ischemia. It is a complex process involving numerous mechanisms occurring in the intracellular and extracellular environments, and it is mediated in part by reactive oxygen species (ROS). The imbalance between the cellular formation of free radicals and cells' capacity to defend against them can cause cardiac tissue injuries. In this context, ROS play an essential role in both the organ injury and repair processes. After reperfusion, infiltration into the myocardium of inflammatory leucocytes, such as macrophages and neutrophils, causes further ROS production beyond the initiation of the inflammatory cascade. In this case, ROS overproduction is crucial in cardiac injury, and it can increase the complications related to cardiac reperfusion. In myocardial tissue, ROS can be produced from several sources, such as xanthine oxidase, cytochrome oxidase, cyclooxygenase, mediated unsaturated fatty acid oxidation, oxidation of catecholamines, mitochondrial oxidation, activation of leukocyte nicotinamide adenine dinucleotide phosphate oxidase, iron release, and reduction?oxidation reaction cycling; all of these sources reduce molecular oxygen in the reperfused myocardium. This review discusses about the molecular and therapeutic aspects of cardiac-reperfusion injuries generated by ROS. Experimental and clinical evidence with respect to the use of ischemic preconditioning, Ca2 +, nitric oxide, and conventional antioxidants in cardiac-reperfusion injury are summarized, and causal therapy approaches with various antioxidants are discussed.
... Administration of exogenous 14,15-EET was found to reduce MIRI in rats and dogs (Nithipatikom et al., 2006;Gross et al., 2007). Likewise, exogenous administration of EETs analogues, UA-8, provided significant protection against MIRI (Batchu et al., 2011). ...
Accumulating data suggest that EETs and 20-HETE, cytochrome P450 enzymes (CYPs) metabolites of arachidonic acid (AA), play important roles in cardiovascular diseases. For many years, Cardiotonic Pill (CP), an herbal preparation derived from Salviae Miltiorrhizae Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Borneolum Syntheticum, has been widely used in China for the treatment of coronary artery disease (CAD). However, its pharmacological mechanism has not been well elucidated. The purpose of this study was to investigate the chronic effects of CP on myocardial ischemia-reperfusion injury (MIRI) and AA CYPs metabolism in rats (in vivo) and H9c2 cells (in vitro). The results showed that CP dose-dependently (10, 20 and 40 mg/kg/d, 7 days) mitigated MIRI in rats. The plasma concentrations of EETs in CP-treated (40 mg/kg/d, 7 days) ischemia-reperfusion (I/R) rats were significantly higher (P<0.05) than those in controls. Cardiac Cyp1b1, Cyp2b1, Cyp2e1, Cyp2j3, and Cyp4f6 were significantly induced (P<0.05), CYP2J and CYP2C11 proteins were up-regulated (P<0.05), and AA-epoxygenases activity was significantly increased (P<0.05) after CP (40 mg/kg/d, 7 days) administration in rats. In H9c2 cells, CP also increased (P<0.05) the EETs concentrations and showed protection in hypoxia-reoxygenation (H/R) cells. However, an antagonist of EETs, 14,15-EEZE, displayed a dose-dependent depression of CP's protective effects in H/R cells. In conclusion, up-regulation of cardiac epoxygenases after multiple-dose of CP, leading to elevated concentrations of cardioprotective EETs after myocardial I/R, may be the underlying mechanism, at least in part, for CP's cardioprotective effect in rats.
... The application of mitoK ATP openers mimics IPC whereas K ATP blockers, such as 5-hydroxydecanoate, attenuate cardioprotection, further suggesting the importance of mitoK ATP in IPC protection [47]. Besides the exclusive role of mitochondrial ROS in signaling IPC [7], the initial burst of ROS is correlated with IPC efficacy, and it serves as essential preconditioning stimulus to the activation of mitoK ATP as well as sarcolemmal K ATP (sarcK ATP ) [2,49]. ...
Myocardial ischemia-reperfusion (I/R) injury is experienced by individuals suffering from cardiovascular diseases such as coronary heart diseases and subsequently undergoing reperfusion treatments in order to manage the conditions. The occlusion of blood flow to the tissue, termed ischemia, can be especially detrimental to the heart due to its high energy demand. Several cellular alterations have been observed upon the onset of ischemia. The danger created by cardiac ischemia is somewhat paradoxical in that a return of blood to the tissue can result in further damage. Reactive oxygen species (ROS) have been studied intensively to reveal their role in myocardial I/R injury. Under normal conditions, ROS function as a mediator in many cell signaling pathways. However, stressful environments significantly induce the generation of ROS which causes the level to exceed body's antioxidant defense system. Such altered redox homeostasis is implicated in myocardial I/R injury. Despite the detrimental effects from ROS, low levels of ROS have been shown to exert a protective effect in the ischemic preconditioning. In this review, we will summarize the detrimental role of ROS in myocardial I/R injury, the protective mechanism induced by ROS, and potential treatments for ROS-related myocardial injury.
... AA is often presented as a major player in CHD complications (Hjelte and Nilsson, 2005) and likely responsible for cell damage occurring after ischemia-reperfusion (Oe, 1994). A massive accumulation of AA and lipoxygenase and cytochrome P450 epoxygenase metabolites has been reported in the post-ischemic myocardium (Adamek et al.;, Sexton et al., 2007Gross et al., 2007). However, beside the production of well-characterized detrimental AA-metabolites, production of protective end-products of AA metabolism has also been reported (Gross et al., 2005). ...
Although the Mediterranean diet (MED) is considered the optimal diet to prevent coronary heart disease (CHD), it is still unknown whether adoption of MED may result in improved myocardial resistance to ischemia-reperfusion injury and may potentially prevent ventricular arrhythmias. Accordingly, the first experimental study was carried out to investigate whether a diet low in saturated fats and omega-6 fatty acids (ω6) but rich in plant and marine omega-3 fatty acids (ω3), a typical MED fatty acid profile, may result in smaller infarct size and better left ventricular function (LVF) recovery in a rat model of regional ischemia-reperfusion. Results demonstrate a great accumulation of ω3 and a parallel decrease of arachidonic acid in plasma, cell membranes and cardiac mitochondria. Also, the MED rats developed smaller infarct size compared with the control groups (p<0.01) while LVF recovery was not different in the three groups. The second epidemiologic study was carried out to determine whether ω3 have beneficial antiarrhythmic effects in patients at high risk for fatal ventricular arrhythmias. Two hundred thirty eight patients with implantable cardioverter defibrillators (ICDs) were included at Grenoble University Hospital. The primary end point was time to first ICD event for ventricular tachycardia or fibrillation (VT or VF) or death from cardiac cause. Red blood cells fatty acids was analyzed and the Omega-3 Index was calculated from eicosapentaenoic acid and docosahexaenoic acid. Results did not show significant differences neither in individual omega-3 fatty acids (ALA, EPA and DHA) nor in omega-3 index between quartiles. However, it comes into view that the RBC omega-3 index in these patients (8.6±1.59 to 8,8±1.76) was already at levels that have been previously reported to be cardioprotective.
... In contrast, 12-lipoxygenase and its major product 12-hydroxyeicosatetraenic acid (12-HETE) have been reported to limit myocardial ischemiareperfusion injury [47] and to participate in opioid-induced preconditioning [48]. The cytochrome P450 hydroxylase and its product 20-hydroxyeicosatetraenic acid (20-HETE) exacerbate myocardial injury [49] while cytochrome P450 epoxygenases and their metabolites (epoxyeicosatrienoic acids, EETs) have been proposed to be cardioprotective [50]. However, the observed protective effects of non specific inhibitors of cytochrome P450 against reperfusion injury demonstrate that this enzymatic pathway has an overall detrimental effect on myocardial ischemia-reperfusion injury [24,25]. ...
... Oxo derivatives of DHA and EPA may induce cardioprotection by acting as PPARγ agonists in a mechanism similar to that of nitro fatty acids (71,140). 15d-PGJ 2 and EETs protect against myocardial infarction via ligation of PPARγ and activation of ATP channels, respectively (141,142). EETs contribute to vascular and cardiac physiology through regulating vascular dilation, promoting angiogenesis, and decreasing inflammation and platelet aggregation. In the heart, EETs regulate myocyte contraction and increase coronary blood flow through several mechanisms that involve the activation of TRP ion channels leading to an influx of calcium and through an unidentified receptor that stimulates cAMP production (40). ...
Unsaturated fatty acids are metabolized to reactive products that can act as pro-or anti-inflammatory signaling mediators. Electrophilic fatty acid species, including nitro-and oxo-containing fatty acids, display salutary anti-inflammatory and metabolic actions. Electrophilicity can be conferred by both enzymatic and oxidative reactions, via the homolytic addition of nitrogen dioxide to a double bond or via the formation of α,β-unsaturated carbonyl and epoxide substituents. The endogenous formation of electrophilic fatty acids is significant and influenced by diet, metabolic, and inflammatory reactions. Transcriptional regulatory proteins and enzymes can sense the redox status of the surrounding environment upon electrophilic fatty acid adduction of functionally significant, nucleophilic cysteines. Through this covalent and often reversible posttranslational modification, gene expression and metabolic responses are induced. At low concentrations, the pleiotropic signaling actions that are regulated by these protein targets suggest that some classes of electrophilic lipids may be useful for treating metabolic and inflammatory diseases.
... [5,6] EETs are reported to reduce calcium overload during cardiac ischaemia by activating ATP-sensitive K + channels (KATP). [7][8][9] Opening of the KATP channels allows a K + efflux, resulting in an increased rate of repolarization and a decreased period of Ca 2+ influx during the plateau phase of the action potential. [10,11] In addition, EETs are reported to be promitogenic and attenuate reactive oxygen species level, mitochondrial dysfunction, caspase activation, cell migration and apoptosis. ...
Objectives
To determine the protective role of a soluble epoxide hydrolase(sEH) inhibitor, trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido] cyclohexyloxy} benzoic acid (t-TUCB), in isoproterenol (ISO)-induced myocardial ischaemic injury in vivo.Methods
Cardioprotective activity of t-TUCB was studied against ISO-induced myocardial ischaemic injury in male Wistar rats. Cardioprotection was assessed by measuring elecrocardiographic (EKG), serum lactate dehydrogenase (LDH) and creatine kinase (CK-MB) levels, cardiac calcium and antioxidant levels, and also by measuring infarct size in the cardiac tissue.Key findings Pretreatment with t-TUCB at 3, 10 and 30 mg/kg orally for a period of 14 days significantly prevented the changes in EKG parameters (QTc interval prolongation, ST height depression, pathological Q waves formation and T-wave inversion), serum cardiac biomarkers (CK-MB and LDH), relative heart weight, myocardial calcium levels, infarct size and the oxidative status in the cardiac tissue (lipid peroxidation, catalase and superoxide dismutase levels) when compared with the untreated control animals (P < 0.05).Conclusion
The sEH inhibitor t-TUCB significantly prevents ISO-induced myocardial ischaemic injury in rats. This study provides a preliminary confirmation of the efficacy of t-TUCB by oral administration in rats.
... It has been proposed that the cytoprotective action of EETs is mediated by improving mitochondrial function (31), modulation of the ATP-sensitive K ϩ channels (12), and preventing the loss of mitochondrial membrane potential (23). EETs have also been reported to inhibit ROS production in ischemia-reperfusion injury (25) and prevent oxidant-induced neuronal damage (51), but the effect of EETs in A-induced mitochondrial dysfunction is not known. Therefore, in this study we have addressed the potential effectiveness of 14,15-and 11,12-EET in preventing soluble oligomeric A-induced mitochondrial damage. ...
Amyloid β (Aβ) has long been implicated as a causative protein in Alzheimer's disease (AD). Cellular Aβ accumulation is toxic and causes mitochondrial dysfunction which precedes clinical symptoms of AD pathology. In the present study, we explored the possible use of epoxyeicosatrienoic acids (EETs), epoxide metabolites of arachidonic acid, as therapeutic target against Aβ-induced mitochondrial impairment using cultured neonatal hippocampal astrocytes. Inhibition of endogenous EETs production by a selective epoxygenase inhibitor, MS-PPOH, caused a greater reduction in mitochondrial membrane potential in presence of Aβ (1, 10 µM) exposure versus alone. MS-PPOH pre-incubation also aggravated Aβ-induced mitochondrial fragmentation. Pre-incubation of the cells with either 14,15- or 11,12-EET prevented this mitochondrial depolarization and fragmentation. EETs pre-treatment also further improved the reduction observed in mitochondrial oxygen consumption in presence of Aβ. Pre-incubation of the cells with EETs significantly improved cellular respiration under basal condition and in the presence of the protonophore, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). The uncoupling of ATP synthase from the electron transfer chain that occurred in Aβ-treated cells was also prevented by pre-incubation with EETs. Lastly, cellular reactive oxygen species (ROS) production, a hallmark of Aβ toxicity, also showed significant reduction in presence of EETs. We have previously shown that Aβ reduces EETs synthesis in rat brain homogenates and cultured hippocampal astrocytes and neurons (Sarkar et al, 2011). We conclude that reduction of endogenous EETs may be one of the mechanisms through which Aβ inflicts toxicity and thus supplementing the cells with exogenous EETs improves mitochondrial dynamics and prevents metabolic impairment.
... The latter also possess physiological effects [89]. EETs have been shown to exert cardioprotective effects, for example, reduced infarct size, during ischemia--reperfusion in experimental studies [90][91][92]. 8,9-, 11,12-and 14,15-EETs seem to be particularly important in this context [93]. In addition to the vasodilating effects described earlier, EETs might exert their cardioprotective effects through other mechanisms, for example, sarcolemmal and mitochondrial K + channels, p42/p44 MAPK and PI3K-AKT signaling pathways [88,93]. ...
Introduction:
The P450 enzymes (P450s) mediate the biotransformation of several drugs, steroid hormones, eicosanoids, cholesterol, vitamins, fatty acids and bile acids, many of which affect cardiovascular homeostasis. Experimental studies have demonstrated that several P450s modulate important steps in the pathogenesis of ischemic heart disease (IHD).
Areas covered:
This article discusses the current knowledge on i) the expression of P450s in cardiovascular and renal tissues; ii) the role of P450s in the pathophysiology of IHD, in particular the modulation of blood pressure and cardiac hypertrophy, coronary arterial tone, ischemia-reperfusion injury and the metabolism of cardiovascular drugs; iii) the available evidence from observational studies on the association between P450 gene polymorphisms and risk of myocardial infarction (MI); and iv) suggestions for further research in this area.
Expert opinion:
P450s exert important modulatory effects in experimental models of IHD and MI. However, observational studies have provided conflicting results on the association between P450 genetic polymorphisms and MI. Further, adequately powered studies are required to ascertain the biological and clinical impact of P450s on clinical IHD end-points, that is, fatal and nonfatal MI, revascularization and long-term outcomes post MI. Pharmacogenetic substudies of recently completed cardiovascular clinical trials might represent an alternative strategy in this context.
Myocardial disease, the abnormalities of the cardiac muscle, is the leading cause of death in humans. Eicosanoids represent a large spectrum of lipid mediators with critical roles in physiological and pathophysiological conditions. Arachidonic acid (AA) is the major resource of eicosanoids and is metabolized via cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P450 (CYP) enzymes producing a diverse family of lipid mediators called eicosanoids, including prostanoids, leukotrienes (LTs), epoxyeicosatrienoic acids (EETs), dihydroxyeicosatetraenoic acid (diHETEs), eicosatetraenoic acids (ETEs), and lipoxins (LXs). Beyond the well-established roles of eicosanoids in inflammation and vascular biology, a growing body of evidence showed that eicosanoids, especially CYP450 derived eicosanoids EETs, are preventive and therapeutic targets for many of the myocardial diseases. EETs not only ameliorate the cardiac injury and remodeling in different pathological models, but also attenuate subsequent hemodynamic disturbances and cardiac dysfunction. EETs have direct and indirect protective properties in the myocardium, and thus relieve dietetic cardiomyopathy and inflammatory cardiomyopathy. Moreover, EETs are capable to attenuate the ischemic cardiomyopathy, including the myocardial infarction and cardiac ischemic reperfusion injury. Multiple biological events and signaling networks are targeted during the myocardial protection of EETs, these are including mitochondria hemostasis, angiogenesis, oxidative stress, inflammatory response, metabolic regulation, endoplasmic reticulum (ER) stress and cell death. Additionally, eicosanoids from COX and LOX also have important roles in some of the myocardial diseases, such as cardiac hypertrophy and ischemic heart disease. This chapter summarizes the physiological and pathophysiological significance, and the signal mechanisms of the eicosanoids, especially the EETs, in myocardial diseases.
Arachidonic acid (AA) is closely associated with breast cancer. In addition to the two metabolic pathways regulated by cyclooxygenase and lipoxygenase, AA has a third metabolic pathway through which cytochrome P450 (CYP) enzymes produce hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs). The targeted CYP-mediated pathway of AA can not only kill cancer cells but also inhibit the interstitial microenvironment around a tumor. Therefore, it makes sense to identify potential biomarkers from the AA metabolome for the diagnosis and treatment of breast cancer. This study established a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the analysis of AA and its main metabolites, EETs and HETEs, in MMTV-PyMT mice, a spontaneous breast cancer mouse model. The results showed that there were significant differences in the concentrations of AA, 12-HETE, 19-HETE and 8,9-EET in plasma and tumor tissues between normal and MMTV-PyMT mice. Therefore, the eicosanoids mentioned above may be used as new biomarkers for breast cancer diagnosis. This study provides a new perspective for the recognition and diagnosis of breast cancer.
Inflammation is the body's reaction to pathogenic (biological or chemical) stimuli and covers a burgeoning list of compounds and pathways that act in concert to maintain the health of the organism. Eicosanoids and related fatty acid derivatives can be formed from arachidonic acid and other polyenoic fatty acids via the cyclooxygenase and lipoxygenase pathways generating a variety of pro-and anti-inflammatory mediators, such as prostaglandins, leukotrienes, lipoxins, resolvins and others. The cytochrome P450 pathway leads to the formation of hydroxy fatty acids, such as 20-hydroxyeicosatetraenoic acid, and epoxy eicosanoids. Free radical reactions induced by reactive oxygen and/or nitrogen free radical species lead to oxygenated lipids such as isoprostanes or isolevuglandins which also exhibit pro-inflammatory activities. Eicosanoids and their metabolites play fundamental endocrine, autocrine and paracrine roles in both physiological and pathological signaling in various diseases. These molecules induce various unsaturated fatty acid dependent signaling pathways that influence crosstalk, alter cell-cell interactions, and result in a wide spectrum of cellular dysfunctions including those of the tissue microenvironment. Although the complete role of eicosanoids, including that of the recently elucidated anti-inflammatory specialized pro-resolving lipid mediators (SPMs), e.g. lipoxins, resolvins, protectins and maresins, is not completely understood, the result of unremitting chronic inflammation is fostering early stages of carcinogenesis. Chronic inflammation facilitates the transition from a normal cell to a cancerous one. The disruption of homeostasis across a wide, but identifiable, swath of diverse molecular pathways creates a micromilieu which constitutes an early and necessary step in the 6-step sequence of carcinogenesis for the vast majority of cancers, termed "sporadic cancers".
Background/aims:
Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase (CYP) metabolites of arachidonic acid and have multiple cardiovascular effects. Ophiopogonin D (OP-D) is an important effective monomeric component in Shenmai injection (SM-I). Both have been reported to have a variety of biological functions, including anti-inflammatory, anti-oxidant, and anti-apoptotic effects. We previously demonstrated that OP-D-mediated cardioprotection involves activation of CYP2J2/3 and enhancement of circulating EETs levels in vitro and can be developed as a novel drug for the therapy of myocardial ischemia-reperfusion (MI/R) injury. We therefore hypothesized that the protective effects of OP-D and SM-I against MI/R injury are associated with increased expression of CYP2J3 and enhanced circulating 11,12-EET levels in vivo.
Methods:
A rat model of MI/R injury was generated by ligation of the left anterior descending coronary artery for 40 min, followed by reperfusion for 2 h to determine the protective effects and potential mechanisms of OP-D and SM-I. Electrocardiogram and ultrasonic cardiogram were used to evaluate cardiac function; 2,3,5-triphenyltetrazolium chloride was used to measure myocardial infarct size; hematoxylin and eosin staining and transmission electron microscopy were used to observe the morphology of myocardial tissue; and the expression of related proteins in the mechanistic study was observed by western blot analysis.
Results:
We found that OP-D and SM-I exert protective effects on MI/R injury, including regulation of cardiac function, reduction of lactate dehydrogenase and creatine kinase production, attenuation of myocardial infarct size, and improvement of the recovery of damaged myocardial structures. We found that OP-D and SM-I activate CYP2J3 expression and increase levels of circulating 11,12-EET in MI/R-injured rats.
Conclusion:
We tested the hypothesis that the cardioprotective effects of OP-D and SM-I on MI/R injury are associated with increased expression of CYP2J3 and enhanced circulating 11,12-EET levels in rats. Taken together, our results show that the effects of OP-D and SM-I were also mediated by the activation of the PI3K/Akt/eNOS signaling pathway, while inhibition of the NF-κB signaling pathway and antioxidant and anti-apoptotic effects were involved in the cardioprotective effects of OP-D and SM-I.
Therapeutics for arachidonic acid pathways began with the development of non-steroidal anti-inflammatory drugs that inhibit cyclooxygenase (COX). The enzymatic pathways and arachidonic acid metabolites and respective receptors have been successfully targeted and therapeutics developed for pain, inflammation, pulmonary and cardiovascular diseases. These drugs target the COX and lipoxygenase pathways but not the third branch for arachidonic acid metabolism, the cytochrome P450 (CYP) pathway. Small molecule compounds targeting enzymes and CYP epoxy-fatty acid metabolites have evolved rapidly over the last two decades. These therapeutics have primarily focused on inhibiting soluble epoxide hydrolase (sEH) or agonist mimetics for epoxyeicosatrienoic acids (EET). Based on preclinical animal model studies and human studies, major therapeutic indications for these sEH inhibitors and EET mimics/analogs are renal and cardiovascular diseases. Novel small molecules that inhibit sEH have advanced to human clinical trials and demonstrate promise for cardiovascular diseases. Challenges remain for sEH inhibitor and EET analog drug development; however, there is a high likelihood that a drug that acts on this third branch of arachidonic acid metabolism will be utilized to treat a cardiovascular or kidney disease in the next decade.
Doxorubicin (DOX) is a chemotherapeutic that is used in the treatment of a wide variety of cancers. However, it causes cardiotoxicity partly due to the formation of reactive oxygen species (ROS). CYP2J2 is a human cytochrome P450 that is highly expressed in cardiomyocytes. It converts arachidonic acid (AA) into four different regioisomers of epoxyeicosatrienoic acids (EETs). Using kinetic analyses we show that AA metabolism by CYP2J2 is modulated by DOX. We show that cytochrome P450 reductase (CPR), the redox partner of CYP2J2, metabolizes DOX to 7-deoxydoxorubicin aglycone (7-de-aDOX). This metabolite then binds to CYP2J2, and inhibits and alters the preferred site of metabolism of AA leading to change in the ratio of the EET regioisomers. Furthermore, molecular dynamics (MD) simulations indicate that 7-de-aDOX and AA can concurrently bind to the CYP2J2 active site to produce these changes in the site of AA metabolism. To see if these observations are unique to DOX/7-de-aDOX, we use non-cardiotoxic DOX analogues, ZRN and IDN. ZRN and 5-IDN inhibit CYP2J2-mediated AA metabolism, but does not change the ratio of EET regioisomers. Taken together, we demonstrate that DOX and 7-de-aDOX inhibit CYP2J2-mediated AA metabolism and 7-de-aDOX binds close to the active site to alter the ratio of cardioprotective EETs. These mechanistic studies of CYP2J2 can aid in the design of new alternative DOX derivatives.
Numerous benefits have been attributed to dietary long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFAs), including protection against cardiac arrhythmia, triglyceride-lowering, amelioration of inflammatory, and neurodegenerative disorders. This review covers recent findings indicating that a variety of these beneficial effects are mediated by "omega-3 epoxyeicosanoids", a class of novel n-3 LC-PUFA-derived lipid mediators, which are generated via the cytochrome P450 (CYP) epoxygenase pathway. CYP enzymes, previously identified as arachidonic acid (20:4n-6; AA) epoxygenases, accept eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA), the major fish oil n-3 LC-PUFAs, as efficient alternative substrates. In humans and rodents, dietary EPA/DHA supplementation causes a profound shift of the endogenous CYP-eicosanoid profile from AA- to EPA- and DHA-derived metabolites, increasing, in particular, the plasma and tissue levels of 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP). Based on preclinical studies, these omega-3 epoxyeicosanoids display cardioprotective, vasodilatory, anti-inflammatory, and anti-allergic properties that contribute to the beneficial effects of n-3 LC-PUFAs in diverse disease conditions ranging from cardiac disease, bronchial disorders, and intraocular neovascularization, to allergic intestinal inflammation and inflammatory pain. Increasing evidence also suggests that background nutrition as well as genetic and disease state-related factors could limit the response to EPA/DHA-supplementation by reducing the formation and/or enhancing the degradation of omega-3 epoxyeicosanoids. Recently, metabolically robust synthetic analogs mimicking the biological activities of 17,18-EEQ have been developed. These drug candidates may overcome limitations of dietary EPA/DHA supplementation and provide novel options for the treatment of cardiovascular and inflammatory diseases.
The development of cardiac hypertrophy is a complicated process, which undergoes a transition from compensatory hypertrophy to heart failure, and the identification of new biomarkers and targets for this disease is greatly needed. Here we investigated the development of isoproterenol (ISO)-induced cardiac hypertrophy in an in vitro experimental model. After the induction of hypertrophy with ISO treatment in H9c2 cells, cell surface area, cell viability, cellular reactive oxygen species (ROS), and nitric oxide (NO) levels were tested. Our data showed that the cell viability, mitochondrial membrane potential, and NO/ROS balance varied during the development of cardiac hypertrophy in H9c2 cells. It was also found that the expression of thioredoxin1 (Trx1) and peroxiredoxin2 (Prdx2) was decreased during the cardiac hypertrophy of H9c2 cells. These results suggest a critical role for Trx1 and Prdx2 in the cardiac hypertrophy of H9c2 cells and in the transition from compensated hypertrophy to de-compensated hypertrophy in H9c2 cells, and our findings may have important implications for the management of this disease.
Macrophage polarization determines the timing for the switch from the inflammation phase to the inflammation resolution phase after acute myocardial infarction. The aim of the present study was to investigate whether ω-alkynyl arachidonic acid could mitigate the inflammatory lipid mediators in the regulation of macrophage phenotypes and functions with a special regard to myocardial infarction. We initially discovered that ω-alkynyl arachidonic acid selectively suppressed the up-regulation of inducible nitric oxide synthase (iNOS) over cyclooxygenase-2 (COX-2) in LPS-stimulated macrophages. ω-Alkynyl arachidonic acid also reduced the expression of macrophage M1 biomarkers (e.g., TNF-α, CXCL10, iNOS and IL-6) but increased the expression of macrophage M2 biomarkers (e.g., IL-10 and arginase-1) in LPS-stimulated macrophages. Moreover, ω-alkynyl arachidonic acid markedly enhanced the phagocytotic activity of macrophages against fluorescently-labeled beads or apoptotic H9c2 cardiac cells. We further investigated the in vivo cardioprotective activities of ω-alkynyl arachidonic acid in a mouse model of myocardial infarction. ω-Alkynyl arachidonic acid indeed reduced infarct size, cardiac damage and the leakage of myocardial enzymes CK-MB. Mechanistic studies revealed that ω-alkynyl arachidonic acid suppressed the overexpression and nuclear translocation of glycolytic enzyme PKM2 in LPS-stimulated macrophages. Furthermore, co-immunoprecipitation assay suggested that ω-alkynyl arachidonic acid disrupted the interaction between PKM2 and HIF-1α. Consequently, ω-alkynyl arachidonic acid diminished HIF-1α binding to the HRE sequence in iNOS promoter in response to LPS stimulation. Collectively, ω-alkynyl arachidonic acid may promote the anti-inflammatory M2 polarization of macrophages in acute myocardial infarction via regulating the cross-talk between PKM2, HIF-1α and iNOS.
Biologically active epoxyeicosatrienoic acid (EET) regioisomers are synthesized from arachidonic acid by cytochrome P450 epoxygenases of endothelial, myocardial, and renal tubular cells. EETs relax vascular smooth muscle and decrease inflammatory cell adhesion and cytokine release. Renal EETs promote sodium excretion and vasodilation to decrease hypertension. Cardiac EETs reduce infarct size after ischemia-reperfusion injury and decrease fibrosis and inflammation in heart failure. In diabetes, EETs improve insulin sensitivity, increase glucose tolerance, and reduce the renal injury. These actions of EETs emphasize their therapeutic potential. To minimize metabolic inactivation, 14,15-EET agonist analogs with stable epoxide bioisosteres and carboxyl surrogates were developed. In preclinical rat models, a subset of agonist analogs, termed EET-A, EET-B, and EET-C22, are orally active with good pharmacokinetic properties. These orally active EET agonists lower blood pressure and reduce cardiac and renal injury in spontaneous and angiotensin hypertension. Other beneficial cardiovascular actions include improved endothelial function and cardiac antiremodeling actions. In rats, EET analogs effectively combat acute and chronic kidney disease including drug- and radiation-induced kidney damage, hypertension and cardiorenal syndrome kidney damage, and metabolic syndrome and diabetes nephropathy. The compelling preclinical efficacy supports the prospect of advancing EET analogs to human clinical trials for kidney and cardiovascular diseases.
We have previously reported that epoxyeicosatrienoic acid (EET) has multiple beneficial effects on vascular function; in addition to its antiapoptotic action, it increases insulin sensitivity and inhibits inflammation. To uncover the signaling mechanisms by which EET reduces cardiomyopathy, we hypothesized that EET infusion might ameliorate obesity-induced cardiomyopathy by improving heme oxygenase (HO)-1, Wnt1, thermogenic gene levels, and mitochondrial integrity in cardiac tissues and improved pericardial fat phenotype. EET reduced levels of fasting blood glucose and proinflammatory adipokines, including nephroblastoma overexpressed (NOV) signaling, while increasing echocardiographic fractional shortening and O 2 consumption. Of interest, we also noted a marked improvement in mitochondrial integrity, thermogenic genes, and Wnt 1 and HO-1 signaling mechanisms. Knockout of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in EET-treated mice resulted in a reversal of these beneficial effects including a decrease in myocardial Wnt1 and HO-1 expression and an increase in NOV. To further elucidate the effects of EET on pericardial adipose tissues, we observed EET treatment increases in adiponectin, PGC-1α, phospho-AMP-activated protein kinase, insulin receptor phosphorylation, and thermogenic genes, resulting in a “browning” pericardial adipose phenotype under high-fat diets. Collectively, these experiments demonstrate that an EET agonist increased Wnt1 and HO-1 signaling while decreasing NOV pathways and the progression of cardiomyopathy. Furthermore, this report presents a portal into potential therapeutic approaches for the treatment of heart failure and metabolic syndrome.
NEW & NOTEWORTHY The mechanism by which EET acts on obesity-induced cardiomyopathy is unknown. Here, we describe a previously unrecognized function of EET infusion that inhibits nephroblastoma overexpressed (NOV) levels and activates Wnt1, hence identifying NOV inhibition and enhanced Wnt1 expression as novel pharmacological targets for the prevention and treatment of cardiomyopathy and heart failure.
Listen to this article's corresponding podcast at http://ajpheart.physiology.org/content/early/2017/05/31/ajpheart.00093.2017 .
The opioid receptor family, with associated endogenous ligands, has numerous roles throughout the body. Moreover, the delta opioid receptor (DORs) has various integrated roles within the physiological systems, including the cardiovascular system. While DORs are important modulators of cardiovascular autonomic balance, they are well-established contributors to cardioprotective mechanisms. Both endogenous and exogenous opioids acting upon DORs have roles in myocardial hibernation and protection against ischaemia-reperfusion (I-R) injury. Downstream signalling mechanisms governing protective responses alternate, depending on the timing and duration of DOR activation. The following review describes models and mechanisms of DOR-mediated cardioprotection, the impact of co-morbidities and challenges for clinical translation.
Cardiovascular disease remains the leading cause of death worldwide. Among many potential targets for pharmacological intervention, a promising strategy involves epoxyeicosatrienoic acid (EET) and soluble epoxide hydroxylase (sEH) inhibition. sEH is the enzyme that converts EET to its less potent metabolite; therefore, EET is upregulated by its inhibitor. EET has pleotropic effects that collectively reduce inflammation, while increasing vasodilation and insulin sensitivity. Recent reports indicate that EET agonists and sEH inhibitors are capable of not only reversing endothelial dysfunction and hypertension, but also of reversing cardiac remodeling, which is a hallmark of cardiomyopathy and the metabolic syndrome. EET agonists and sEH inhibitors are in development as potential therapies, and at least one drug is already in clinical trials. This review examines the activity of EET in biological systems, proposes a series of pathways to explain its mechanism of action, and discusses how these might be exploited for potential therapeutic use.
Soluble epoxide hydrolase (sEH) is a promising therapeutic target for stroke. Both pharmacological inhibition and genetic knockout of sEH have shown protection in experimental models of cerebral ischemia. Additionally, human single-nucleotide polymorphisms in the gene that encodes for sEH, designated ephx2, have been correlated with stroke incidence and outcome. This chapter starts out by introducing sEH in the context of the five other mammalian epoxide hydrolases before delving specifically into sEH biology. Up-to-date research into sEH’s protein structure, role in metabolizing epoxyeicosatrienoic acids, regional localization in brain, as well as subcellular localization are discussed in relation to brain function and disease. The chapter concludes by evaluating the prospect of using sEH inhibitors in clinical trials for the treatment of stroke based on the Stroke Therapy Academic and Industry Roundtable criteria.
Dietary fish oil omega-3 fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), protect against arrhythmia and sudden cardiac death using largely unknown mechanisms. EPA and DHA may serve as efficient alternative substrates of arachidonic acid (AA) metabolizing cytochrome P450 (CYP) enzymes. For many of the CYP isoforms, the n-3 PUFAs are the preferred substrates. Moreover, the CYP enzymes oxygenate EPA and DHA with largely different regioselectivities compared to AA. In particular, the omega-3 double bond that distinguishes EPA and DHA from AA is a preferred site of CYP-catalyzed epoxidation reactions. Given the pivotal role of CYP-dependent AA metabolites in the regulation of vascular, renal and cardiac functions, their replacement by unique sets of epoxy- and hydroxy-metabolites derived from EPA and DHA may have far-reaching physiological implications. The currently available data suggest that some of the vasculo- and cardioprotective effects attributed to dietary n-3 PUFAs may be mediated by CYP-dependent metabolites of EPA and DHA.
There is growing preclinical as well as clinical evidence supporting remote ischemic preconditioning (RIPC), in which short cycles of non-fatal ischemia followed by reperfusion to an organ or tissue distant from the heart elicits cardioprotection. It is the most practical, non-invasive, cost-free, and clinically compatible, secure procedure for reducing ischemia-reperfusion induced injury. The use of a conventional blood pressure cuff on the upper or lower limb in eliciting cardioprotection has expedited its clinical applicability. Endothelium has been documented to respond very quickly to blood flow and hypoxia by releasing different humoral factors such as endothelium derived releasing factor, endothelium derived contracting factor, endothelium derived hyperpolarizing factor. In recent years, there have been studies suggesting the key role of endothelial derived factors in RIPC induced cardioprotection. The signaling cascade involves nitric oxide, gap junctions, epoxyeicosatrienoic (EETs) acids, Ca-activated K(+) channels, angiotensin II, thromboxane A2, superoxide anions and prostacyclin. The present review describes the role of these endothelial derived factors in RIPC induced cardioprotection with possible mechanisms.
This chapter explores the metabolism and disposition of drugs for cardiovascular therapy. The general characteristics of drug metabolizing enzymes, drug transporters, and drug–drug interactions are described in the context of cardiovascular drug disposition, as is the impact of drug metabolites, pharmacogenetics and biomarkers. The body of the chapter is a survey of cardiovascular drug classes with emphasis on primary factors involved in drug metabolism and disposition. The chapter concludes with a brief discussion on the future of cardiovascular drug metabolism.
The cell types comprising brain cellular network include neurons, astrocytes, and capillary microvasculature. Astrocytes extend foot processes and make close contact with neuronal cells and form a continuous layer to ensheath the outer surface of the capillaries and small arterioles to sense neuronal activity and transduce the signal to the cerebral microvessels. The mechanisms through which these three cell types interact to regulate brain function remains obscure. Activated neurons release adenosine that stimulates release of vasoactive factors including reactive oxygen species to induce vasodilatation. The discovery that brain cell types express cytochrome (CYP) ω-hydroxylases and CYP epoxygenases that catalyze carboxylation and epoxidation of the substrate arachidonic acid (AA) to form the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) and the vasodilatory regioisomeric epoxyeicosatrienoic acids (EETs), respectively, advances our knowledge of the mechanisms through which the dynamics of cerebral blood flow are regulated under normal or stress conditions. Both 20-HETE and the EETs target the KCa channel to elicit opposing biological actions. 20-HETE mediates pressure-evoked myogenic cerebral autoregulation, whereas the EETs induce cerebral vasodilatation and couple neuronal activity to hyperemic cerebral blood flow. In addition to their anti-inflammatory, neuroprotection, and vasodilatory actions, the EETs also posses mitogenic and angiogenic properties and promote vessel growth that could be targeted for their anti-tumorigenic and neuroprotective actions. © Springer-Verlag Berlin Heidelberg 2014. All rights are reserved.
Various members of the cytochrome P450 (CYP) superfamily have the capacity of metabolizing omega-6 and omega-3 polyunsaturated fatty acids (n-6 and n-3 PUFAs). In most mammalian tissues, CYP2C and CYP2J enzymes are the major PUFA epoxygenases, whereas CYP4A and CYP4F subfamily members function as PUFA hydroxylases. The individual CYP enzymes differ in their substrate specificities as well as regio- and stereoselectivities and thus produce distinct sets of epoxy and/or hydroxy metabolites, collectively termed CYP eicosanoids. Nutrition has a major impact on the endogenous CYP-eicosanoid profile. "Western diets" rich in n-6 PUFAs result in a predominance of arachidonic acid-derived metabolites, whereas marine foodstuffs rich in n-3 PUFAs shift the profile to eicosapentaenoic and docosahexaenoic acid-derived metabolites. In general, CYP eicosanoids are formed as second messengers of numerous hormones, growth factors and cytokines regulating cardiovascular and renal function, and a variety of other physiological processes. Imbalances in the formation of individual CYP eicosanoids are linked to the development of hypertension, myocardial infarction, maladaptive cardiac hypertrophy, acute kidney injury, stroke and inflammatory disorders. The underlying mechanisms are increasingly understood and may provide novel targets for the prevention and treatment of these disease states. Suitable pharmacological agents are under development and first proofs of concept have been obtained in animal models.
Myocardial infarction (MI) is complicated by ventricular fibrosis and associated diastolic and systolic failure. Emerging studies implicate Wnt1 signaling in the formation of new blood vessels. Epoxyeicosatrienoic acids (EETs)-mediated up- regulation of heme oxygenase-1 (HO-1) protects against the detrimental consequences of MI in several animal models, however, the mechanism(s) by which this occurs remains unclear. The aim of this study was to examine these mechanisms in the LAD ligation animal model of post infarcted heart failure. Specifically, we sought to clarify the mechanistic basis of the interactions of the Wnt1 canonical pathway, HO-1 and associated angiogenesis. Human microvascular endothelial cells (HMECs) were exposed to anoxia and treated with the EET agonist, NUDSA, in the presence and absence of tin mesoporphyrin (SnMP). Increased capillary density, and Wnt1 and HO-1 expression occurred in cells treated with NUDSA. Anoxic HMECs treated with NUDSA and Wnt1 siRNA, exhibited decreased in the expression of β-catenin and the Wnt1 target gene, PPARδ (p<0.05 vs. NUDSA). Furthermore, blocking the Wnt 1 antagonist, Dickkopf 1, by siRNA increased β-catenin and PPARδ expression, and this effect was further enhanced by the concurrent administration of NUDSA. In in vivo experiments, C57B16 mice were divided into 4 groups: sham, mice with MI via LAD ligation and mice with MI treated with NUDSA, with and without SnMP. Increased fractional area change (FAC) and myocardial angiogenesis were observed in mice treated with NUDSA (p<0.05 vs. MI). Increased expression of HO-1, Wnt1, β-catenin, adiponectin, and phospho-endothelial nitric oxide synthetase (p-eNOS), and a decrease in the glycosylated subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, gp91(phox) expression occurred in cardiac tissue of mice treated with NUDSA (p<0.05 vs. MI). SnMP reversed these effects. This novel study demonstrates that increasing the canonical Wnt1 signaling cascade with the subsequent increase in HO-1, adiponectin and angiogenesis ameliorates fibrosis and cardiac dysfunction in a mouse model of MI and support the hypothesis that HO-1 is an integral component of the EETs-adiponectin axis and are central for the control of resistance to fibrosis and vascular dysfunction and in part determine how they influence the cellular/vascular homeostasis and provide insight into the mechanisms involved in vascular dysfunction as well as potential targets for the treatment of CVD.
Copyright © 2015. Published by Elsevier Inc.
Background:
Adipocyte fatty acid-binding protein (FABP4) is a member of a highly conserved family of cytosolic proteins that bind with high affinity to hydrophobic ligands, such as saturated and unsaturated long-chain fatty acids and eicosanoids. Recent evidence has supported a novel role for FABP4 in linking obesity with metabolic and cardiovascular disorders. In this context, we identified FABP4 as a main bioactive factor released from human adipose tissue that directly suppresses heart contraction in vitro. As FABP4 is known to be a transport protein, it cannot be excluded that lipid ligands are involved in the cardiodepressant effect as well, acting in an additional and/or synergistic way.
Objective:
We investigated a possible involvement of lipid ligands in the negative inotropic effect of adipocyte factors in vitro.
Results:
We verified that blocking the CYP epoxygenase pathway in adipocytes attenuates the inhibitory effect of adipocyte-conditioned medium (AM) on isolated adult rat cardiomyocytes, thus suggesting the participation of epoxyeicosatrienoic acids (EETs) in the cardiodepressant activity. Analysis of AM for EETs revealed the presence of 5,6-, 8,9-, 11,12- and 14,15-EET, whereas 5,6-EET represented about 45% of the total EET concentration in AM. Incubation of isolated cardiomyocytes with EETs in similar concentrations as found in AM showed that 5,6-EET directly suppresses cardiomyocyte contractility. Furthermore, after addition of 5,6-EET to FABP4, the negative inotropic effect of FABP4 was strongly potentiated in a concentration-dependent manner.
Conclusions:
These data suggest that adipocytes release 5,6-EET and FABP4 into the extracellular medium and that the interaction of these factors modulates cardiac function. Therefore elevated levels of FABP4 and 5,6-EET in obese patients may contribute to the development of heart dysfunction in these subjects.
To evaluate the effects of Jiashen Prescription (, JSP) on myocardial infarction (MI) size and cardiac function at the early stage of MI in rats.
One hundred male Sprague-Dawley rats were subjected to sham-operation or MI induced by ligating the left anterior descending coronary artery. The rats with MI were treated with vehicle, JSP 3 and 6 g/(kg·d), or losartan 10 mg/(kg·d) for 1 week.
Compared with the vehicle-treated MI rats, 6 g/(kg·d) JSP reduced MI size 3 days after MI (P <0.05), and attenuated the MI-induced increases in left ventricular end-diastolic and end-systolic dimension and decreases in fractional shortening and ejection fraction 1 week after MI (P <0.05). In addition, 6 g/(kg·d) JSP and losartan were equally effective in reducing MI size and enhancing cardiac functional recovery.
JSP reduces MI size and improves cardiac function after MI, suggesting that JSP has potential as a therapy for MI.
1. We examined the effects of epoxyeicosatrienoic acids (EETs), which are cytochrome P450 metabolites of arachidonic acid (AA), on the activities of the ATP-sensitive K+ (KATP) channels of rat cardiac myocytes, using the inside-out patch-clamp technique. 2. In the presence of 100 μM cytoplasmic ATP, the KATP channel open probability (Po) was increased by 240 ± 60% with 0.1 μM 11,12-EET and by 400 ± 54% with 5 μM 11,12-EET (n = 5-10, P < 0.05 vs. control), whereas neither 5 μM 11,12-dihydroxyeicosatrienoic acid (DHET), which is the epoxide hydrolysis product of 11,12-EET, had any effect on Po. 3. The half-maximal activating concentration (EC50) was 18.9 ± 2.6 nM for 11,12-EET (n = 5) and 19.1 ± 4.8 nM for 8,9-EET (n = 5, P = n.s. vs. 11,12-EET). Furthermore, 11,12-EET failed to alter the inhibition of KATP channels by glyburide. 4. Application of 11,12-EET markedly decreased the channel sensitivity to cytoplasmic ATP. The half-maximal inhibitory concentration of ATP (IC50) was increased from 21.2 ± 2.0 μM at baseline to 240 ± 60 μM with 0.1 μM 11,12-EET (n = 5, P < 0.05 vs. control) and to 780 ± 30 μM with 5 μM 11,12-EET (n = 11, P < 0.05 vs. control). 5. Increasing the ATP concentration increased the number of kinetically distinguishable closed states, promoting prolonged closure durations. 11,12-EET antagonized the effects of ATP on the kinetics of the KATP channels in a dose- and voltage-dependent manner. 11,12-EET (1 μM) reduced the apparent association rate constant of ATP to the channel by 135-fold. 6. Application of 5 μM 11,12-EET resulted in hyperpolarization of the resting membrane potential in isolated cardiac myocytes, which could be blocked by glyburide. 7. These results suggest that EETs are potent activators of the cardiac KATP channels, modulating channel behaviour by reducing the channel sensitivity to ATP. Thus, EETs could be important endogenous regulators of cardiac electrical excitability.
While attention has historically focused on mitochondria as the primary source of ROS in myocardial ischemia/reperfusion injury, recent evidence has implicated cytochrome P450 monooxygenases (CYPs) as a significant factor. CYPs represent a large family of enzymes that catalyze the oxidation of endogenous and exogenous compounds. They catalyze arachidonic acid oxidation to a variety of biologically active eicosanoids that regulate ion channels and protein kinases, with effects on vasomotor tone and cardiac inotropy. They also represent a significant source of reactive oxygen species that may target cellular homeostatic mechanisms and mitochondria. In this review, we will consider the contribution of cytochrome P450 enzymes to reperfusion injury and will speculate on whether the mechanism of injury is due to CYP-mediated ROS production or arachidonic acid metabolites.
Ischemia and reperfusion both contribute to tissue damage after myocardial infarction. Although many drugs have been shown to reduce infarct size when administered before ischemia, few have been shown to be effective when administered at reperfusion. Moreover, although it is generally accepted that a burst of reactive oxygen species (ROS) occurs at the onset of reperfusion and contributes to tissue damage, the source of ROS and the mechanism of injury is unclear. We now report the finding that chloramphenicol administered at reperfusion reduced infarct size by 60% in a Langendorff isolated perfused rat heart model, and that ROS production was also substantially reduced. Chloramphenicol is an inhibitor of mitochondrial protein synthesis and is also an inhibitor of a subset of cytochrome P450 monooxygenases (CYPs). We could not detect any effect on mitochondrial encoded proteins or mitochondrial respiration in chloramphenicol-perfused hearts, and hypothesized that the effect was caused by inhibition of CYPs. We tested additional CYP inhibitors and found that cimetidine and sulfaphenazole, two CYP inhibitors that have no effect on mitochondrial protein synthesis, were also able to reduce creatine kinase release and infarct size in the Langendorff model. We also showed that chloramphenicol reduced infarct size in an open chest rabbit model of regional ischemia. Taken together, these findings implicate CYPs in myocardial ischemia/reperfusion injury.
A cDNA encoding a P450 monooxygenase was amplified from reverse transcribed rat heart and liver total RNA by polymerase chain reaction using primers based on the 5′- and 3′-end sequences of two rat pseudogenes, CYP2J3P1 andCYP2J3P2. Sequence analysis revealed that this 1,778-base pair cDNA contained an open reading frame and encoded a new 502 amino acid protein designated CYP2J3. Based on the deduced amino acid sequence, CYP2J3 was approximately 70% homologous to both human CYP2J2 and rabbit CYP2J1. Recombinant CYP2J3 protein was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 insect cells using a baculovirus expression system. Microsomal fractions of CYP2J3/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolized arachidonic acid to 14,15-, 11,12-, and 8,9-epoxyeicosatrienoic acids and 19-hydroxyeicosatetraenoic acid as the principal reaction products (catalytic turnover, 0.2 nmol of product/nmol of cytochrome P450/min at 37 °C). Immunoblotting of microsomal fractions prepared from rat tissues using a polyclonal antibody raised against recombinant CYP2J2 that cross-reacted with CYP2J3 but not with other known rat P450s demonstrated abundant expression of CYP2J3 protein in heart and liver. Immunohistochemical staining of formalin-fixed paraffin-embedded rat heart tissue sections using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection localized expression of CYP2J3 primarily to atrial and ventricular myocytes. In an isolated-perfused rat heart model, 20 min of global ischemia followed by 40 min of reflow resulted in recovery of only 44 ± 6% of base-line contractile function. The addition of 5 μm 11,12-epoxyeicosatrienoic acid to the perfusate prior to global ischemia resulted in a significant 1.6-fold improvement in recovery of cardiac contractility (69 ± 5% of base line,p = 0.01 versus vehicle alone). Importantly, neither 14,15-epoxyeicosatrienoic acid nor 19-hydroxyeicosatetraenoic acid significantly improved functional recovery following global ischemia, demonstrating the specificity of the biological effect for the 11,12-epoxyeicosatrienoic acid regioisomer. Based on these data, we conclude that (a) CYP2J3 is one of the predominant enzymes responsible for the oxidation of endogenous arachidonic acid pools in rat heart myocytes and (b) 11,12-epoxyeicosatrienoic acid may play an important functional role in the response of the heart to ischemia.
A cDNA encoding a P450 monooxygenase was amplified from reverse transcribed rat heart and liver total RNA by polymerase chain
reaction using primers based on the 5′- and 3′-end sequences of two rat pseudogenes, CYP2J3P1 andCYP2J3P2. Sequence analysis revealed that this 1,778-base pair cDNA contained an open reading frame and encoded a new 502 amino acid
protein designated CYP2J3. Based on the deduced amino acid sequence, CYP2J3 was approximately 70% homologous to both human
CYP2J2 and rabbit CYP2J1. Recombinant CYP2J3 protein was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 insect
cells using a baculovirus expression system. Microsomal fractions of CYP2J3/NADPH-cytochrome P450 oxidoreductase-transfected
cells metabolized arachidonic acid to 14,15-, 11,12-, and 8,9-epoxyeicosatrienoic acids and 19-hydroxyeicosatetraenoic acid
as the principal reaction products (catalytic turnover, 0.2 nmol of product/nmol of cytochrome P450/min at 37 °C). Immunoblotting
of microsomal fractions prepared from rat tissues using a polyclonal antibody raised against recombinant CYP2J2 that cross-reacted
with CYP2J3 but not with other known rat P450s demonstrated abundant expression of CYP2J3 protein in heart and liver. Immunohistochemical
staining of formalin-fixed paraffin-embedded rat heart tissue sections using the anti-CYP2J2 IgG and avidin-biotin-peroxidase
detection localized expression of CYP2J3 primarily to atrial and ventricular myocytes. In an isolated-perfused rat heart model,
20 min of global ischemia followed by 40 min of reflow resulted in recovery of only 44 ± 6% of base-line contractile function.
The addition of 5 μm 11,12-epoxyeicosatrienoic acid to the perfusate prior to global ischemia resulted in a significant 1.6-fold improvement in
recovery of cardiac contractility (69 ± 5% of base line,p = 0.01 versus vehicle alone). Importantly, neither 14,15-epoxyeicosatrienoic acid nor 19-hydroxyeicosatetraenoic acid significantly improved
functional recovery following global ischemia, demonstrating the specificity of the biological effect for the 11,12-epoxyeicosatrienoic
acid regioisomer. Based on these data, we conclude that (a) CYP2J3 is one of the predominant enzymes responsible for the oxidation of endogenous arachidonic acid pools in rat heart
myocytes and (b) 11,12-epoxyeicosatrienoic acid may play an important functional role in the response of the heart to ischemia.
Arachidonic acid (AA) can be metabolized by cytochrome P450 enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), as well as 19- and 20-hydroxyeicosatetraenoic acids (HETEs). These eicosanoids are potent regulators of vascular tone. However, their role in the ischemic myocardium has not been well investigated. In this study, we used a gas chromatographic–mass spectrometric technique to analyze total EETs, DHETs, and 20-HETE released into coronary venous plasma during coronary artery occlusion and reperfusion in anesthetized dogs. Pentafluorobenzyl esters (PFB-esters) of EETs and PFB-esters/trimethylsilyl ethers (TMS-ethers) of DHETs and 20-HETE were detected in the negative ion chemical ionization (NICI) using methane as a reagent gas. Under the conditions used, all four regioisomers of EET eluted from the capillary gas chromatographic column at similar retention times while four regioisomers of DHETs and 20-HETE eluted separately. The detection limits in plasma samples are 5 pg for total EETs, 40 pg for DHET, and 15 pg for 20-HETE. 14,15-DHET is the major regioisomer detected in the plasma samples while other regioisomers of DHETs are probably present at too low a concentration for detection. During the first 5 to 15 min of coronary occlusion, a slight decrease in the concentration of EETs, 14,15-DHET, and 20-HETE from the control values was observed in coronary venous plasma. At 60 min of occlusion, their concentrations significantly increased and remained elevated during 5 to 60 min of reperfusion. The concentrations decreased at 120 min of reperfusion. The NICI GC–MS was successfully used as a sensitive technique to determine cP450 metabolites of AA in plasma during prolonged occlusion–reperfusion periods. Furthermore, the results indicate that these metabolites may play a role in mediating ischemic–reperfusion injury.
The aims were: (1) to determine if a new ischaemia selective ATP dependent potassium (KATP) channel antagonist, sodium 5-hydroxydecanoate (5-HD), blocks ischaemic preconditioning in dogs; (2) to determine whether a small intracoronary dose of glibenclamide, a classical sulphonylurea KATP channel antagonist, could block ischaemic preconditioning independent of systemic metabolic effects.
Barbitone anaesthetised dogs were subjected to 60 min of left circumflex coronary artery occlusion followed by 5 h of reperfusion. Preconditioning was produced by a single 5 min left circumflex occlusion followed by 10 min of reperfusion prior to the 60 min occlusion period. 5-HD (150 micrograms.kg-1 x min-1) or vehicle was given by intracoronary infusion into the ischaemic region over 20 min, beginning 15 min prior to the 60 min occlusion period in the presence or absence of preconditioning. Glibenclamide (3 micrograms.kg-1 x min-1) was given by intracoronary infusion into the left circumflex artery during the 5 min preconditioning period or during the first 5 min of occlusion in preconditioned or non-preconditioned dogs. Transmural myocardial blood flow was measured by radioactive microspheres and infarct size determined by triphenyltetrazolium staining and expressed as a percent of the area at risk.
There were no differences in haemodynamic variables, myocardial blood flow, area at risk, or blood glucose between groups. Infarct size was markedly reduced in preconditioned dogs compared to control animals, at 7(SEM 2)% v 29(4)%, p < 0.05 The reduction in infarct size by preconditioning was blocked completely by intracoronary 5-HD, or by intracoronary glibenclamide given during preconditioning or during the first 5 min of the prolonged occlusion period. Neither 5-HD nor glibenclamide affected infarct size in the absence of preconditioning at the doses studied.
These results further strengthen the hypothesis that activation of myocardial KATP channels is involved in the mechanism of ischaemic preconditioning in dogs.
Single or multiple brief periods of ischemia (preconditioning) have been shown to protect the myocardium from infarction after a subsequent more prolonged ischemic insult. To test the hypothesis that preconditioning is the result of opening ATP-sensitive potassium (KATP) channels, a selective KATP channel antagonist, glibenclamide, was administered before or immediately after preconditioning in barbital-anesthetized open-chest dogs subjected to 60 minutes of left circumflex coronary artery (LCX) occlusion followed by 5 hours of reperfusion. Preconditioning was elicited by 5 minutes of LCX occlusion followed by 10 minutes of reperfusion before the 60-minute occlusion period. Glibenclamide (0.3 mg/kg i.v.) or vehicle was given 10 minutes before the initial ischemic insult in each of four groups. In a fifth group, glibenclamide was administered immediately after preconditioning. In a final series (group 6), a selective potassium channel opener, RP 52891 (10 micrograms/kg bolus and 0.1 micrograms/mg/min i.v.) was started 10 minutes before occlusion and continued throughout reperfusion. Transmural myocardial blood flow was measured at 30 minutes of occlusion, and infarct size was determined by triphenyltetrazolium staining and expressed as a percent of the area at risk. There were no significant differences in hemodynamics, collateral blood flow, or area at risk between groups. The ratio of infarct size to area at risk in the control group (28 +/- 6%) was not different from the group pretreated with glibenclamide in the absence of preconditioning (31 +/- 6%). Preconditioning produced a marked reduction (p less than 0.002) in infarct size (28 +/- 6% to 6 +/- 2%), whereas glibenclamide administered before or immediately after preconditioning completely abolished the protective effect (28 +/- 6% and 30 +/- 8%, respectively). RP 52891 also produced a significant (p less than 0.03) reduction (28 +/- 6% to 13 +/- 3%) in infarct size. These results suggest that myocardial preconditioning in the canine heart is mediated by activation of KATP channels and that these channels may serve an endogenous myocardial protective role.
Effects of cytochrome P-450 metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETS; 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET), were examined in isolated guinea pig hearts and ventricular myocytes. Addition of 1-16 ng/ml EETs to normal isolated hearts produced no effects on contractility or coronary pressure. In hearts subjected to 60 min of low-flow ischemia, impairment of contractility and declines in heart rate and coronary perfusion pressure were similar in the presence or absence of 1 ng/ml EETs. However, in the presence of either 5,6- or 11,12-EET, recovery was delayed for the first 10 min only. No significant differences were found in any group regarding heart rate, coronary perfusion pressure, or energy metabolite content after 30 min of reperfusion. In myocytes, both 5,6- and 11,12-EET (100 pg/ml, 1.0 ng/ml, and 20 ng/ml) significantly increased cell shortening as well as intracellular calcium concentrations, whereas 8,9- or 14,15-EET was without effect on these parameters. These results describe for the first time the direct effects of various EETs on cardiac cell function as well as their ability to modulate some of the myocardial responses to postischemic reperfusion. The results suggest a potential role for these substances in the response of the heart to pathological insult.
Diazoxide, a selective opener of the mitochondrial ATP-sensitive potassium channel, has been shown to elicit tolerance to ischemia in cardiac myocytes and in perfused heart. However, the mechanism of this cardioprotection is poorly understood. Because reactive oxygen species (ROS) are recognized as important intracellular signaling molecules and have been implicated in ischemic preconditioning, we examined diazoxide-induced ROS production in adult cardiomyocytes. Cells treated with 50 micromol/L diazoxide showed a 173% increase in ROS production relative to baseline. 5-Hydroxydecanoate was found to attenuate the diazoxide-induced increase in ROS generation. The diazoxide-induced increase in ROS also was abrogated by the addition of either the antioxidant N-acetylcysteine (NAC) or N-mercaptopropionylglycine. We also examined the ability of NAC to block the protective effects of diazoxide in the perfused rat heart. After 20 minutes of global ischemia and 20 minutes of reflow, hearts perfused with 100 micromol/L diazoxide before ischemia showed significantly improved postischemic contractile function relative to untreated hearts (84% versus 29% of initial left ventricular developed pressure, respectively). Hearts treated with diazoxide in the presence of 4 mmol/L NAC recovered 53% of initial left ventricular developed pressure, whereas hearts treated with NAC alone recovered 46% of preischemic function. Using (31)P NMR spectroscopy, we found that, similar to preconditioning, diazoxide significantly attenuated ischemia-induced intracellular acidification and enhanced post- ischemic recovery of phosphocreatine levels, both of which were blocked by cotreatment with NAC. These data suggest that the cardioprotective actions of diazoxide are mediated by generation of a pro-oxidant environment.
It has been assumed that all G(i)-coupled receptors trigger the protective action of preconditioning by means of an identical intracellular signaling pathway. To test this assumption, rabbit hearts were isolated and perfused with Krebs buffer. All hearts were subjected to a 30-minute coronary artery occlusion followed by 120 minutes of reperfusion. Risk area was measured with fluorescent particles and infarct size with triphenyltetrazolium chloride staining. Control hearts showed 29.1+/-2.8% infarction of the risk zone. A 5-minute infusion of acetylcholine (0.55 mmol/L) beginning 15 minutes before the 30-minute occlusion resulted in significant protection (9.2+/-2.7% infarction). This protection could be blocked by administration of 300 micromol/L N-2-mercaptopropionyl glycine (MPG), a free radical scavenger, or by 200 micromol/L 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) antagonist, for 15 minutes beginning 5 minutes before the acetylcholine infusion (35.2+/-3.9% and 27.8+/-2.4% infarction, respectively). Similar protection was observed with other known triggers, ie, bradykinin (0.4 micromol/L), morphine (0.3 micromol/L), and phenylephrine (0.1 micromol/L), and in each case protection was completely abrogated by either MPG or 5-HD. In contrast, protection by adenosine or its analog N(6)-(2-phenylisopropyl) adenosine could not be blocked by either MPG or 5-HD. Therefore, whereas most of the tested agonists trigger protection by a pathway that requires opening of mitochondrial K(ATP) channels and production of free radicals, the protective action of adenosine is not dependent on either of these steps. Hence, it cannot be assumed that all G(i)-coupled receptors use the same signal transduction pathways to trigger preconditioning.
Arachidonic acid (AA) can be metabolized by cytochrome P450 (CYP) enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE). These eicosanoids are potent regulators of vascular tone. We developed a liquid chromatography-electrospray ionization-mass spectrometry method to simultaneously determine 5,6-, 8,9-, 11,12-, and 14,15-EETs; 5,6-, 8,9-, 11,12-, and 14,15-DHETs; and 20-HETE. [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE were used as internal standards. These compounds are readily separated on a C18 reverse-phase column using water:acetonitrile with 0.005% acetic acid as a mobile phase. The internal standards, [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE, eluted slightly faster than the natural eicosanoids. The samples were ionized by electrospray with fragmentor voltage of 120 V and detected in a negative mode. The negative ion detection gave a lower background than the positive ion detection for these compounds. These eicosanoids exhibited high abundance of the ions corresponding to [M - 1]-. The m/z = 319, 337, and 319 ions were used for quantitation of EETs, DHETs, and 20-HETE, respectively. The detection limits using selected ion monitoring of these compounds are about 1 pg per injection. The position of functional groups and water content of mobile phase had a significant effect on the sensitivity of detection. Water content of 40% was found to give maximal sensitivity. The method was used to determine EETs, DHETs, and 20-HETE in bovine coronary artery endothelial cells, dog plasma, rat astrocytes, and rat kidney microsome samples.
The heart is richly endowed with K(ATP) channels, which function as biological sensors, regulating membrane potentials and electrical excitability in response to metabolic alterations. We recently reported that the cytochrome P450 metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETs), potently activate cardiac K(ATP) channels by reducing channel sensitivity to ATP. In the present study, we further demonstrate that 11(S),12(R)-EET activated the cardiac K(ATP) channels with an EC(50) of 39.5 nM, whereas 11(R),12(S)-EET was totally inactive. In addition, 11(S),12(R)-EET but not 11(R),12(S)-EET hyperpolarized the resting membrane potentials and shortened the duration of cardiomyocyte action potentials. By studying homologs and analogs of 11,12-EET, we also found that all four EET regioisomers are equipotent activators of the K(ATP) channels, reducing the ATP sensitivity by more than 10-fold; however, neither altered chain length, double bond number, epoxide position, nor methylation of the carboxyl group affected channel inhibitions by ATP. All the fatty epoxides studied are potent K(ATP) channel activators, but the omega-3 homolog was particularly potent, reducing ATP sensitivity 27-fold. Together, the results indicate that the presence of an epoxide group in a particular three-dimensional configuration is a critical determinant for K(ATP) channel activation, and its effect is augmented by a double bond at omega-3 position. The results also suggest that fatty epoxides are important modulators of cardiac electrical excitability.
Human CYP2J2 is abundant in heart and active in the biosynthesis of epoxyeicosatrienoic acids (EETs); however, the functional role of this P450 and its eicosanoid products in the heart remains unknown. Transgenic mice with cardiomyocyte-specific overexpression of CYP2J2 were generated. CYP2J2 transgenic (Tr) mice have normal heart anatomy and basal contractile function. CYP2J2 Tr hearts have improved recovery of left ventricular developed pressure (LVDP) compared with wild-type (WT) hearts after 20 minutes ischemia and 40 minutes reperfusion. Perfusion with the selective P450 epoxygenase inhibitor N-methylsulphonyl-6-(2-proparglyloxyphenyl)hexanamide (MS-PPOH) for 20 minutes before ischemia results in reduced postischemic LVDP recovery in WT hearts and abolishes the improved postischemic LVDP recovery in CYP2J2 Tr hearts. Perfusion with the ATP-sensitive K(+) channel (K(ATP)) inhibitor glibenclamide (GLIB) or the mitochondrial K(ATP) (mitoK(ATP)) inhibitor 5-hydroxydecanoate (5-HD) for 20 minutes before ischemia abolishes the cardioprotective effects of CYP2J2 overexpression. Flavoprotein fluorescence, a marker of mitoK(ATP) activity, is higher in cardiomyocytes from CYP2J2 Tr versus WT mice. Moreover, CYP2J2-derived EETs (1 to 5 micromol/L) increase flavoprotein fluorescence in WT cardiomyocytes. CYP2J2 Tr mice exhibit increased expression of phospho-p42/p44 mitogen-activated protein kinase (MAPK) after ischemia, and addition of the p42/p44 MAPK kinase (MEK) inhibitor PD98059 during reperfusion abolishes the cardioprotective effects of CYP2J2 overexpression. Together, these data suggest that CYP2J2-derived metabolites are cardioprotective after ischemia, and the mechanism for this cardioprotection involves activation of mitoK(ATP) and p42/p44 MAPK.
Cytochrome P450s (CYP) and their arachidonic acid (AA) metabolites have important roles in regulating vascular tone, but their function and specific pathways involved in modulating myocardial ischemia-reperfusion injury have not been clearly established. Thus, we characterized the effects of several selective CYPomega-hydroxylase inhibitors and a CYPomega-hydroxylase metabolite of AA, 20-hydroxyeicosatetraenoic acid (20-HETE), on the extent of ischemia-reperfusion injury in canine hearts. During 60 minutes of ischemia and particularly after 3 hours of reperfusion, 20-HETE was produced at high concentrations. A nonspecific CYP inhibitor, miconazole, and 2 specific CYPomega-hydroxylase inhibitors, 17-octadecanoic acid (17-ODYA) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), markedly inhibited 20-HETE production during ischemia-reperfusion and produced a profound reduction in myocardial infarct size (expressed as a percent of the area at risk) (19.6+/-1.7% [control], 8.4+/-2.5% [0.96 mg/kg miconazole], 5.9+/-2.2% [0.28 mg/kg 17-ODYA], and 10.8+/-1.8% [0.40 mg/kg DDMS], P<0.05, respectively). Conversely, exogenous 20-HETE administration significantly increased infarct size (26.9+/-1.9%, P<0.05). Several CYPomega-hydroxylase isoforms, which are known to produce 20-HETE such as CYP4A1, CYP4A2, and CYP4F, were demonstrated to be present in canine heart tissue and their activity was markedly inhibited by incubation with 17-ODYA. These results indicate an important endogenous role for CYPomega-hydroxylases and in particular their product, 20-HETE, in exacerbating myocardial injury in canine myocardium. The full text of this article is available online at http://circres.ahajournals.org.
Inhibition of 20-hydroxyeicosatrienoic acid (20-HETE), by pretreatment with pharmacological inhibitors of cytochrome P450 (CYP) omega-hydroxylase, has been shown to reduce infarct size in canines when administered prior to ischemia. However, it is unknown whether these agents reduce infarct size when administered just prior to reperfusion and if the sarcolemmal and/or mitochondrial K(ATP) channels (sK(ATP) and mK(ATP)) contribute to cardioprotection. Therefore, we determined whether specific CYP inhibitors for epoxygenases and omega-hydroxylases are cardioprotective when given either prior to ischemia or prior to reperfusion and furthermore, if selective inhibition of the sK(ATP) by HMR-1098 or mK(ATP) by 5-hydroxydecanoic acid (5-HD) could abrogate this effect. Male Sprague-Dawley rats underwent 30 minutes of ischemia followed by 2 hours of reperfusion. Groups received either miconazole (MIC, non-selective CYP inhibitor, 3 mg/kg), 17-octadecynoic acid (17-ODYA, CYP omega-hydroxylase inhibitor, 0,3 or 3 mg/kg), N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS, CYP omega-hydroxylase inhibitor, 0,4 or 4 mg/kg), N-methanesulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH, CYP epoxygenase inhibitor, 3 mg/kg), or vehicle either 10 minutes prior to ischemia or 5 minutes prior to reperfusion. Rats also received either HMR-1098 (6 mg/kg) or 5-HD (10 mg/kg) 10 minutes prior to reperfusion, with subsets of rats also receiving either MIC or 17-ODYA 5 minutes prior to reperfusion. DDMS and 17-ODYA dose dependently reduced infarct size. Rats treated with MIC, 17-ODYA and DDMS, but not MS-PPOH, produced comparable reductions in infarct size when administered prior to ischemia or reperfusion compared to vehicle. HMR-1098, but not 5-HD, also blocked the infarct size reduction afforded by MIC and 17-ODYA. These data suggest a novel cardioprotective pathway involving CYP omega-hydroxylase inhibition and subsequent activation of the sK(ATP) channel during reperfusion.
Preconditioning by one or several brief periods of ischemia activates an endogenous cardioprotective program that increases the resistance of cardiomyocytes to injury by subsequent prolonged periods of ischemia. Ischemic preconditioning can be mimicked by K(+) channel openers and various other substances, a phenomenon termed pharmacological preconditioning. Initially, ischemic preconditioning has been ascribed to the opening of ATP-sensitive K(+) channels at the surface membrane of cardiomyocytes. Since 1997, numerous publications have implicated mitochondrial ATP-sensitive K(+) channels (mK(ATP)) as a major trigger and/or end effector of preconditioning. Diazoxide has been suggested to be a specific activator of mK(ATP) channels, and the substituted fatty acid 5-hydroxydecanoate (5-HD) has been suggested to be a specific inhibitor. However, diazoxide and 5-HD have multiple K(+)-channel-independent actions, and the experimental evidence for an obligatory role of mK(ATP) channels in preconditioning, or even their existence, remains inconclusive. In contrast, surface K(ATP) channels have been well characterized, and we summarize the evidence suggesting that they make a major contribution to preconditioning. We also discuss a number of other factors involved in preconditioning: (1) generation of reactive oxygen species, (2) impairment of fatty acid metabolism, and (3) opening of the mitochondrial permeability transition pore. In the light of these emerging concepts, we critically re-examine the evidence for and against a role of mK(ATP) channels in ischemic and pharmacological preconditioning.
Cytochrome P-450 (CYP) omega-hydroxylases and their arachidonic acid (AA) metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), produce a detrimental effect on ischemia-reperfusion injury in canine hearts, and the inhibition of CYP omega-hydroxylases markedly reduces myocardial infarct size expressed as a percentage of the area at risk (IS/AAR, %). In this study, we demonstrated that a specific CYP omega-hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), markedly reduced 20-HETE production during ischemia-reperfusion and reduced myocardial infarct size compared with control [19.5 +/- 1.0% (control), 9.6 +/- 1.5% (0.40 mg/kg DDMS), 4.0 +/- 2.0% (0.81 mg/kg DDMS), P < 0.01]. In addition, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE, a putative 20-HETE antagonist) significantly reduced myocardial infarct size from control [10.3 +/- 1.3% (0.032 mg/kg 20-HEDE) and 5.9 +/- 1.9% (0.064 mg/kg 20-HEDE), P < 0.05]. We further demonstrated that one 5-min period of ischemic preconditioning (IPC) reduced infarct size to a similar extent as that observed with the high doses of DDMS and 20-HEDE, and the higher dose of DDMS given simultaneously with IPC augmented the infarct size reduction [9.9 +/- 2.8% (IPC) to 2.5 +/- 1.4% (0.81 mg/kg DDMS), P < 0.05] to a greater degree than that observed with either treatment alone. These results suggest an important negative role for endogenous CYP omega-hydroxylases and their product, 20-HETE, to exacerbate myocardial injury in canine myocardium. Furthermore, for the first time, this study demonstrates that the effect of IPC and the inhibition of CYP omega-hydroxylase synthesis (DDMS) or its actions (20-HEDE) may have additive effects in protecting the canine heart from ischemia-reperfusion injury.
Cytochrome P450 (CYP) enzyme 2J2, an epoxygenase predominantly expressed in the heart, metabolizes arachidonic acid to biologically active eicosanoids. One of the CYP2J2 products, 11,12-epoxyeicosatrienoic acid, has several vasoprotective effects. A frequent promoter polymorphism of CYP2J2 decreases gene expression and is associated with coronary artery disease. This association supports the vascular protective role of CYP-derived eicosanoids in cardiovascular disease.
Cytochrome P450 epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which are converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (Ephx2, sEH). To examine the functional role of sEH in the heart, mice with targeted disruption of the Ephx2 gene were studied. Hearts from sEH null mice have undetectable levels of sEH mRNA and protein and cannot convert EETs to DHETs. sEH null mice have normal heart anatomy and basal contractile function, but have higher fatty acid epoxide:diol ratios in plasma and cardiomyocyte cell culture media compared with wild type (WT). sEH null hearts have improved recovery of left ventricular developed pressure (LVDP) and less infarction compared with WT hearts after 20 minutes ischemia. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 to 100 nmol/L) before ischemia abolishes this cardioprotective phenotype. Inhibitor studies demonstrate that perfusion with phosphatidylinositol-3 kinase (PI3K) inhibitors wortmannin (200 nmol/L) or LY294002 (5 micromol/L), the ATP-sensitive K+ channel (K(ATP)) inhibitor glibenclamide (1 micromol/L), the mitochondrial K(ATP) (mitoK(ATP)) inhibitor 5-hydroxydecanoate (100 to 200 micromol/L), or the Ca2+-sensitive K+ channel (K(Ca)) inhibitor paxilline (10 micromol/L) abolishes the cardioprotection in sEH null hearts. Consistent with increased activation of the PI3K cascade, sEH null mice exhibit increased cardiac expression of glycogen synthase kinase-3beta (GSK-3beta) phospho-protein after ischemia. Together, these data suggest that targeted disruption of sEH increases the availability of cardioprotective EETs that work by activating PI3K signaling pathways and K+ channels.
Effect of the free radical scavenger 2-MPG on the reduction of IS/AAR produced by 11,12-and 14,15-EET. 2-MPG was administered 5 min prior to the EETs and 20 min before index ischemia
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Fig. 5. Effect of the free radical scavenger 2-MPG on the reduction of IS/AAR produced by 11,12-and 14,15-EET. 2-MPG was administered 5 min prior to the EETs and 20 min before index ischemia (N = 8/group).
Cytochrome P450 epoxygenase CYP2J2 and the risk of coronary artery disease.
- Spieker M.
- Liao J.K.
Activation of ATP-sensitive K(+) channels by epoxyeicosatrienoic acids in rat cardiac ventricular myocytes
- Lu