Cardioprotective effects of CYP-derived epoxy metabolites of docosahexaenoic acid involve limiting NLRP3 inflammasome activation 1.

Impaired mitochondrial function and activation of NLRP3 inflammasome cascade has a significant role in the pathogenesis of myocardial ischemia-reperfusion (IR) injury. The current study investigated whether eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or their corresponding CYP epoxygenase metabolites 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) protect against IR injury. Isolated mouse hearts were perfused in the Langendorff mode with vehicle, DHA, 19,20-EDP, EPA, or 17,18-EEQ and subjected to 30 min of ischemia and followed by 40 min of reperfusion. In contrast with EPA and 17,18-EEQ, DHA and 19,20-EDP exerted cardioprotection, as shown by a significant improvement in postischemic functional recovery associated with significant attenuation of NLRP3 inflammasome complex activation and preserved mitochondrial function. Hearts perfused with DHA or 19,20-EDP displayed a marked reduction in localization of mitochondrial Drp-1 and Mfn-2 as well as maintained Opa-1 levels. DHA and 19,20-EDP preserved the activities of both the cytosolic Trx-1 and mitochondrial Trx-2. DHA cardioprotective effect was attenuated by the CYP epoxygenase inhibitor N-(methysulfonyl)-2-(2-propynyloxy)-benzenehexanamide. In conclusion, our data indicate a differential cardioprotective response between DHA, EPA, and their active metabolites toward IR injury. Interestingly, 19,20-EDP provided the best protection against IR injury via maintaining mitochondrial function and thereby reducing the detrimental NLRP3 inflammasome responses.

Resveratrol improves exercise performance and skeletal muscle oxidative capacity in heart failure.

We investigated whether treatment of mice with established pressure overload-induced heart failure (HF) with the naturally occurring polyphenol resveratrol could improve functional symptoms of clinical HF such as fatigue and exercise intolerance. C57Bl/6N mice were subjected to either sham or transverse aortic constriction surgery to induce HF. Three weeks postsurgery, a cohort of mice with established HF (%ejection fraction <45) was administered resveratrol (~450 mg·kg-1·day-1) or vehicle for 2 wk. Although the percent ejection fraction was similar between both groups of HF mice, those mice treated with resveratrol had increased total physical activity levels and exercise capacity. Resveratrol treatment was associated with altered gut microbiota composition, increased skeletal muscle insulin sensitivity, a switch toward greater whole body glucose utilization, and increased basal metabolic rates. Although muscle mass and strength were not different between groups, mice with HF had significant declines in basal and ADP-stimulated O2 consumption in isolated skeletal muscle fibers compared with sham mice, which was completely normalized by resveratrol treatment. Overall, resveratrol treatment of mice with established HF enhances exercise performance, which is associated with alterations in whole body and skeletal muscle energy metabolism. Thus, our preclinical data suggest that resveratrol supplementation may effectively improve fatigue and exercise intolerance in HF patients.NEW & NOTEWORTHY Resveratrol treatment of mice with heart failure leads to enhanced exercise performance that is associated with altered gut microbiota composition, increased whole body glucose utilization, and enhanced skeletal muscle metabolism and function. Together, these preclinical data suggest that resveratrol supplementation may effectively improve fatigue and exercise intolerance in heart failure via these mechanisms.

Genetic deletion of soluble epoxide hydrolase provides cardioprotective responses following myocardial infarction in aged mice.

BACKGROUND: Pathophysiological responses, including cardiovascular complications, often alter with age. Cardioprotective effects of epoxyeicosatrienoic acids (EETs) toward acute myocardial ischemia-reperfusion injury have been well documented. However, biological relevance of EET-evoked cardioprotection in the ageing myocardium remains unknown. EETs are metabolized to less active metabolites by the enzyme soluble epoxide hydrolase (sEH). This study uses permanent occlusion of the left anterior descending artery (LAD) in young and aged sEH null and WT mice to compare cardiac and mitochondrial function following ischemic injury. METHODS: Age-matched 16 month old (aged) and 3 month old (young) sEH null and littermate wild-type (WT) mice were subjected to permanent occlusion of the left anterior descending coronary artery. Echocardiography was used to assess cardiac structure and function prior-to and 7days post-myocardial infarction with tetrazolium chloride staining to determine infarct size. Mitochondrial ultrastructure was obtained using electron microscopy. Caspase-3, 20S proteasome, aconitase and mitochondrial ETC enzymatic activities were ascertained using established protocols. Mitochondrial respiration was assessed using a Clark electrode in permeabilized cardiac fibers to obtain respiratory control ratios. RESULTS: Markers of cell injury, mitochondrial efficiency and overall cardiac function were preserved in aged sEH null mice, although less robustly than in their young counterparts. While aged animals of both genotypes demonstrated a similar overall age-related decline, sEH deletion consistently demonstrated protection from myocardial ischemic injury regardless of age. CONCLUSION: Our data demonstrates the protection originating from sEH deletion in aged mice was markedly reduced compared to young animals, signifying unavoidable detrimental consequences of biological ageing on cardiac function.

Effect of ischemia reperfusion injury and epoxyeicosatrienoic acids on caveolin expression in mouse myocardium.

BACKGROUND: Caveolins (Cav) are structural proteins that insert into the plasma membrane to form caveolae that can bind molecules important in cardiac signal transduction and function. Cytochrome P450 epoxygenases can metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have known cardioprotective effects. Subsequent metabolism of EETs by soluble epoxide hydrolase reduces the protective effect. AIMS: (1) To assess the effect of ischemia-reperfusion injury on expression and subcellular localization of caveolins. (2) To study the effect of EETs on caveolins. METHODS: Hearts from soluble epoxide hydrolase null (KO) and littermate control (WT) mice were perfused in Langendorff mode and subjected to 20 minutes ischemia followed by 40 minutes reperfusion. Immunohistochemistry, immunoblot, and electron microscopy were performed to study localization of caveolins and changes in ultrastructure. RESULTS: In WT heart, Cav-1 and Cav-3 were present in cardiomyocyte and capillary endothelial cell at baseline. After ischemia, Cav-1 but not Cav-3, disappeared from cardiomyocyte; moreover, caveolae were absent and mitochondrial cristae were damaged. Improved postischemic functional recovery observed in KO or WT hearts treated with 11,12-EET corresponded to higher Cav-1 expression and maintained caveolae structure. In addition, KO mice preserved the Cav-1 signaling after ischemia that lost in WT mice. CONCLUSIONS: Taken together, our data suggest that ischemia-reperfusion injury causes loss of Cav-1 and caveolins, and EETs-mediated cardioprotection involves preservation of Cav-1.

Inhibition of malonyl-CoA decarboxylase reduces the inflammatory response associated with insulin resistance.

We previously showed that genetic inactivation of malonyl-CoA decarboxylase (MCD), which regulates fatty acid oxidation, protects mice against high-fat diet-induced insulin resistance. Development of insulin resistance has been associated with activation of the inflammatory response. Therefore, we hypothesized that the protective effect of MCD inhibition might be caused by a favorable effect on the inflammatory response. We examined if pharmacological inhibition of MCD protects neonatal cardiomyocytes and peritoneal macrophages against inflammatory-induced metabolic perturbations. Cardiomyocytes and macrophages were treated with LPS to induce an inflammatory response, in the presence or absence of an MCD inhibitor (CBM-301106, 10 µM). Inhibition of MCD attenuated the LPS-induced inflammatory response in cardiomyocytes and macrophages. MCD inhibition also prevented LPS impairment of insulin-stimulated glucose uptake in cardiomyocytes and increased phosphorylation of Akt. Additionally, inhibition of MCD strongly diminished LPS-induced activation of palmitate oxidation. We also found that treatment with an MCD inhibitor prevented LPS-induced collapse of total cellular antioxidant capacity. Interestingly, treatment with LPS or an MCD inhibitor did not alter intracellular triacylglycerol content. Furthermore, inhibition of MCD prevented LPS-induced increases in the level of ceramide in cardiomyocytes and macrophages while also ameliorating LPS-initiated decreases in PPAR binding. This suggests that the anti-inflammatory effect of MCD inhibition is mediated via accumulation of long-chain acyl-CoA, which in turn stimulates PPAR binding. Our results also demonstrate that pharmacological inhibition of MCD is a novel and promising approach to treat insulin resistance and its associated metabolic complications.

Cardiac hypertrophy in the newborn delays the maturation of fatty acid ß-oxidation and compromises postischemic functional recovery.

During the neonatal period, cardiac energy metabolism progresses from a fetal glycolytic profile towards one more dependent on mitochondrial oxidative metabolism. In this study, we identified the effects of cardiac hypertrophy on neonatal cardiac metabolic maturation and its impact on neonatal postischemic functional recovery. Seven-day-old rabbits were subjected to either a sham or a surgical procedure to induce a left-to-right shunt via an aortocaval fistula to cause RV volume-overload. At 3 wk of age, hearts were isolated from both groups and perfused as isolated, biventricular preparations to assess cardiac energy metabolism. Volume-overload resulted in cardiac hypertrophy (16% increase in cardiac mass, P < 0.05) without evidence of cardiac dysfunction in vivo or in vitro. Fatty acid oxidation rates were 60% lower (P < 0.05) in hypertrophied hearts than controls, whereas glycolysis increased 246% (P < 0.05). In contrast, glucose and lactate oxidation rates were unchanged. Overall ATP production rates were significantly lower in hypertrophied hearts, resulting in increased AMP-to-ATP ratios in both aerobic hearts and ischemia-reperfused hearts. The lowered energy generation of hypertrophied hearts depressed functional recovery from ischemia. Decreased fatty acid oxidation rates were accompanied by increased malonyl-CoA levels due to decreased malonyl-CoA decarboxylase activity/expression. Increased glycolysis in hypertrophied hearts was accompanied by a significant increase in hypoxia-inducible factor-1α expression, a key transcriptional regulator of glycolysis. Cardiac hypertrophy in the neonatal heart results in a reemergence of the fetal metabolic profile, which compromises ATP production in the rapidly maturing heart and impairs recovery of function following ischemia.

Novel soluble epoxide hydrolase inhibitor protects mitochondrial function following stress.

Epoxyeicosatrienoic acids (EETs) are active metabolites of arachidonic acid that are inactivated by soluble epoxide hydrolase enzyme (sEH) to dihydroxyeicosatrienoic acid. EETs are known to render cardioprotection against ischemia reperfusion (IR) injury by maintaining mitochondrial function. We investigated the effect of a novel sEH inhibitor (sEHi) in limiting IR injury. Mouse hearts were perfused in Langendorff mode for 40 min and subjected to 20 min of global no-flow ischemia followed by 40 min of reperfusion. Hearts were perfused with 0.0, 0.1, 1.0 and 10.0 µmol·L(-1) of the sEHi N-(2-chloro-4-methanesulfonyl-benzyl)-6-(2,2,2-trifluoro-ethoxy)-nicotinamide (BI00611953). Inhibition of sEH by BI00611953 significantly improved postischemic left-ventricular-developed pressure and reduced infarct size following IR compared with control hearts, and similar to hearts perfused with 11,12-EETs (1 µmol·L(-1)) and sEH(-/-) mice. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 µmol·L(-1)), or the plasma membrane K(ATP) channels (pmK(ATP)) inhibitor (glibenclamide, 10 µmol·L(-1)) abolished the improved recovery by BI00611953 (1 µmol·L(-1)). Mechanistic studies in H9c2 cells demonstrated that BI0611953 decreased ROS generation, caspase-3 activity, proteasome activity, increased HIF-1∝ DNA binding, and delayed the loss of mitochondrial membrane potential (ΔΨ(m)) caused by anoxia-reoxygenation. Together, our data demonstrate that the novel sEHi BI00611953, a nicotinamide-based compound, provides significant cardioprotection against ischemia reperfusion injury.

Role of fatty acid uptake and fatty acid beta-oxidation in mediating insulin resistance in heart and skeletal muscle.

Fatty acids are a major fuel source used to sustain contractile function in heart and oxidative skeletal muscle. To meet the energy demands of these muscles, the uptake and beta-oxidation of fatty acids must be coordinately regulated in order to ensure an adequate, but not excessive, supply for mitochondrial beta-oxidation. However, imbalance between fatty acid uptake and beta-oxidation has the potential to contribute to muscle insulin resistance. The action of insulin is initiated by binding to its receptor and activation of the intrinsic protein tyrosine kinase activity of the receptor, resulting in the initiation of an intracellular signaling cascade that eventually leads to insulin-mediated alterations in a number of cellular processes, including an increase in glucose transport. Accumulation of fatty acids and lipid metabolites (such as long chain acyl CoA, diacylglycerol, triacylglycerol, and/or ceramide) can lead to alterations in this insulin signaling pathway. An imbalance between fatty acid uptake and oxidation is believed to be responsible for this lipid accumulation, and is thought to be a major cause of insulin resistance in obesity and diabetes, due to lipid accumulation and inhibition of one or more steps in the insulin-signaling cascade. As a result, decreasing muscle fatty acid uptake can improve insulin sensitivity. However, the potential role of increasing fatty acid beta-oxidation in the heart or skeletal muscle in order to prevent cytoplasmic lipid accumulation and decrease insulin resistance is controversial. While increased fatty acid beta-oxidation may lower cytoplasmic lipid accumulation, increasing fatty acid beta-oxidation can decrease muscle glucose metabolism, and incomplete fatty acid oxidation has the potential to also contribute to insulin resistance. In this review, we discuss the proposed mechanisms by which alterations in fatty acid uptake and oxidation contribute to insulin resistance, and how targeting fatty acid uptake and oxidation is a potential therapeutic approach to treat insulin resistance.

The proinflammatory cytokine tumor necrosis factor-alpha increases the amount of glucose transporter-4 at the surface of muscle cells independently of changes in interleukin-6.

TNFalpha is a proinflammatory cytokine secreted by macrophages in response to bacterial infection. Recently new evidence has emerged suggesting that stressed or injured myocytes produce TNFalpha that then acts as an autocrine and/or paracrine mediator. TNFalpha receptors types 1 and 2 are present in skeletal muscle cells, and muscle cells can secrete, in addition to TNFalpha, other cytokines such as IL-1beta or IL-6. Furthermore, the plasma concentration of TNFalpha is elevated in insulin-resistant states associated with obesity and type 2 diabetes. Here we show that TNFalpha increased the amount of glucose transporter (GLUT)-4 at the plasma membrane and also glucose uptake in the L6 muscle cell line stably expressing GLUT4 tagged with the c-myc epitope. Regardless of the state of differentiation of the L6 cells, TNFalpha did not affect the rate of proliferation or of apoptosis. The stimulatory effects of TNFalpha on cell surface GLUT4 and glucose uptake were blocked by nuclear factor-kappaB and p38MAPK pathway specific inhibitors (Bay 11-7082 and SB220025), and these two pathways were stimulated by TNFalpha. Furthermore, although TNFalpha increased IL-6 mRNA and protein expression, IL-6 did not mediate the effects of TNFalpha on cell surface GLUT4 levels, which also did not require de novo protein synthesis. The results indicate that TNFalpha can stimulate glucose uptake in L6 muscle cells by inducing GLUT4 translocation to the plasma membrane, possibly through activation of the nuclear factor-kappaB and p38MAPK signaling pathways and independently of the production of IL-6.

DHA and 19,20-EDP induce lysosomal-proteolytic-dependent cytotoxicity through de novo ceramide production in H9c2 cells with a glycolytic profile.

Docosahexaenoic acid (DHA) and their CYP-derived metabolites, epoxydocosapentaenoic acids (EDPs), are important fatty acids obtained from dietary sources. While it is known that they have significant biological effects, which can differ between cell type and disease state, our understanding of how they work remains limited. Previously, we demonstrated that DHA and 19,20-EDP triggered pronounced cytotoxicity in H9c2 cells correlating with increased ceramide production. In this study, we examine whether DHA- and 19,20-EDP-induced cell death depends on the type of metabolism (glycolysis or OXPHOS). We cultivated H9c2 cells in distinct conditions that result in either glycolytic or oxidative metabolism. Our major findings suggest that DHA and its epoxy metabolite, 19,20-EDP, trigger cytotoxic effects toward H9c2 cells with a glycolytic metabolic profile. Cell death occurred through a mechanism involving activation of a lysosomal-proteolytic degradation pathway. Importantly, accumulation of ceramide played a critical role in the susceptibility of glycolytic H9c2 cells to cytotoxicity. Furthermore, our data suggest that an alteration in the cellular metabolic profile is a major factor determining the type and magnitude of cellular toxic response. Together, the novelty of this study demonstrates that DHA and 19,20-EDP induce cell death in H9c2 cells with a glycolytic metabolicwct 2 profile through a lysosomal-proteolytic mechanism.

Deficiency of Soluble Epoxide Hydrolase Protects Cardiac Function Impaired by LPS-Induced Acute Inflammation.

Lipopolysaccharide (LPS) is a bacterial wall endotoxin producing many pathophysiological conditions including myocardial inflammation leading to cardiotoxicity. Linoleic acid (18:2n6, LA) is an essential n-6 PUFA which is converted to arachidonic acid (20:4n6, AA) by desaturation and elongation via enzyme systems within the body. Biological transformation of PUFA through CYP-mediated hydroxylation, epoxidation, and allylic oxidation produces lipid mediators, which may be subsequently hydrolyzed to corresponding diol metabolites by soluble epoxide hydrolase (sEH). In the current study, we investigate whether inhibition of sEH, which alters the PUFA metabolite profile, can influence LPS induced cardiotoxicity and mitochondrial function. Our data demonstrate that deletion of soluble epoxide hydrolase provides protective effects against LPS-induced cardiotoxicity by maintaining mitochondrial function. There was a marked alteration in the cardiac metabolite profile with notable increases in sEH-derived vicinal diols, 9,10- and 12,13-dihydroxyoctadecenoic acid (DiHOME) in WT hearts following LPS administration, which was absent in sEH null mice. We found that DiHOMEs triggered pronounced mitochondrial structural abnormalities, which also contributed to the development of extensive mitochondrial dysfunction in cardiac cells. Accumulation of DiHOMEs may represent an intermediate mechanism through which LPS-induced acute inflammation triggers deleterious alterations in the myocardium in vivo and cardiac cells in vitro. This study reveals novel research exploring the contribution of DiHOMEs in the progression of adverse inflammatory responses toward cardiac function in vitro and in vivo.

Cytochrome P450-derived eicosanoids and heart function.

The cytochrome P450 monooxygenase system (CYP) is a multigene superfamily of enzymes, which are important in the metabolism of 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 in 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 CVD.

Lipid oxidation and autophagy in yeast.

Autophagy, a process involved in the degradation and the recycling of long-lived proteins and organelles to survive nitrogen starvation, is generally non-selective. However, recent data suggest that selective forms of autophagy exist, that are able to specifically target several organelles, including mitochondria. Conversely, mitochondrial alterations could trigger autophagy. Such a selective form of autophagy might be involved in the elimination of damaged mitochondria. We reported previously that, mitochondria were early targets of rapamycin-induced autophagy. Here we report that rapamycin-induced autophagy is accompanied by the early production of reactive oxygen species and by the early oxidation of mitochondrial lipid. Inhibition of these oxidative effects by resveratrol largely impaired autophagy of both cytosolic proteins and mitochondria, and delayed subsequent cell death. These results support a role of mitochondrial oxidation events in the activation of autophagy.

SIRT Is Required for EDP-Mediated Protective Responses toward Hypoxia-Reoxygenation Injury in Cardiac Cells.

Hypoxia-reoxygenation (H/R) injury is known to cause extensive injury to cardiac myocardium promoting development of cardiac dysfunction. Despite the vast number of studies dedicated to studying H/R injury, the molecular mechanisms behind it are multiple, complex, and remain very poorly understood, which makes development of novel pharmacological agents challenging. Docosahexaenoic acid (DHA, 22:6n3) is an n - 3 polyunsaturated fatty acid obtained from dietary sources, which produces numerous effects including regulation of cell survival and death mechanisms. The beneficial effects of DHA toward the cardiovascular system are well documented but the relative role of DHA or one of its more potent metabolites is unresolved. Emerging evidence indicates that cytochrome P450 (CYP) epoxygenase metabolites of DHA, epoxydocosapentaenoic acids (EDPs), have more potent biological activity than DHA in cardiac cells. In this study we examined whether EDPs protect HL-1 cardiac cells from H/R injury. Our observations demonstrate that treatment with 19,20-EDP protected HL-1 cardiac cells from H/R damage through a mechanism(s) protecting and enhancing mitochondrial quality. EDP treatment increased the relative rates of mitobiogenesis and mitochondrial respiration in control and H/R exposed cardiac cells. The observed EDP protective response toward H/R injury involved SIRT1-dependent pathways.

A Disintegrin and Metalloprotease-17 Regulates Pressure Overload-Induced Myocardial Hypertrophy and Dysfunction Through Proteolytic Processing of Integrin ß1.

A disintegrin and metalloprotease-17 (ADAM17) belongs to a family of transmembrane enzymes, and it can mediate ectodomain shedding of several membrane-bound molecules. ADAM17 levels are elevated in patients with hypertrophic and dilated cardiomyopathy; however, its direct role in hypertrophic cardiomyopathy is unknown. Cardiomyocyte-specific ADAM17 knockdown mice (ADAM17(flox/flox)/αMHC-Cre; ADAM17(f/f)/Cre) and littermates with intact ADAM17 levels (ADAM17(f/f)) were subjected to cardiac pressure-overload by transverse aortic constriction. Cardiac function/architecture was assessed by echocardiography at 2 and 5 weeks post transverse aortic constriction. ADAM17 knockdown enhanced myocardial hypertrophy, fibrosis, more severe left ventricular dilation, and systolic dysfunction at 5 weeks post transverse aortic constriction. Pressure overload-induced upregulation of integrin ß1 was much greater with ADAM17 knockdown, concomitant with the greater activation of the focal adhesion kinase pathway, suggesting that integrin ß1 could be a substrate for ADAM17. ADAM17 knockdown did not alter other cardiomyocyte integrins, integrin α5 or α7, and HB-EGF (heparin-bound epidermal growth factor), another potential substrate for ADAM17, remained unaltered after pressure overload. ADAM17-mediated cleavage of integrin ß1 was confirmed by an in vitro assay. Intriguingly, ADAM17 knockdown did not affect the myocardial hypertrophy induced by a subpressor dose of angiotensin II, which occurs independent from the integrin ß1-mediated pathway. ADAM17-knockdown enhanced the hypertrophic response to cyclic mechanical stretching in neonatal rat cardiomyocytes. This study reports a novel cardioprotective function for ADAM17 in pressure overload cardiomyopathy, where loss of ADAM17 promotes hypertrophy by reducing the cleavage of cardiac integrin ß1, a novel substrate for ADAM17. This function of ADAM17 is selective for pressure overload-induced myocardial hypertrophy and dysfunction, and not agonist-induced hypertrophy.

Inhibition of Soluble Epoxide Hydrolase Limits Mitochondrial Damage and Preserves Function Following Ischemic Injury.

AIMS: Myocardial ischemia can result in marked mitochondrial damage leading to cardiac dysfunction, as such identifying novel mechanisms to limit mitochondrial injury is important. This study investigated the hypothesis that inhibiting soluble epoxide hydrolase (sEH), responsible for converting epoxyeicosatrienoic acids to dihydroxyeicosatrienoic acids protects mitochondrial from injury caused by myocardial infarction. METHODS: sEH null and WT littermate mice were subjected to surgical occlusion of the left anterior descending (LAD) artery or sham operation. A parallel group of WT mice received an sEH inhibitor, trans-4-[4-(3-adamantan-1-y1-ureido)-cyclohexyloxy]-benzoic acid (tAUCB; 10 mg/L) or vehicle in the drinking water 4 days prior and 7 days post-MI. Cardiac function was assessed by echocardiography prior- and 7-days post-surgery. Heart tissues were dissected into infarct, peri-, and non-infarct regions to assess ultrastructure by electron microscopy. Complexes I, II, IV, citrate synthase, PI3K activities, and mitochondrial respiration were assessed in non-infarct regions. Isolated working hearts were used to measure the rates of glucose and palmitate oxidation. RESULTS: Echocardiography revealed that tAUCB treatment or sEH deficiency significantly improved systolic and diastolic function post-MI compared to controls. Reduced infarct expansion and less adverse cardiac remodeling were observed in tAUCB-treated and sEH null groups. EM data demonstrated mitochondrial ultrastructure damage occurred in infarct and peri-infarct regions but not in non-infarct regions. Inhibition of sEH resulted in significant improvements in mitochondrial respiration, ATP content, mitochondrial enzymatic activities and restored insulin sensitivity and PI3K activity. CONCLUSION: Inhibition or genetic deletion of sEH protects against long-term ischemia by preserving cardiac function and maintaining mitochondrial efficiency.

CYP-epoxygenase metabolites of docosahexaenoic acid protect HL-1 cardiac cells against LPS-induced cytotoxicity Through SIRT1.

Bacterial LPS is an environmental toxin capable of promoting various cardiac complications. Current evidence suggests that LPS-induced myocardial dysfunction emerges as a consequence of compromised quality of cardiac mitochondria. Docosahexaenoic acid (DHA, 22:6n3) is an n-3 polyunsaturated fatty acid (PUFA), which produces a broad spectrum of intrinsic physiological effects including regulation of cell survival and death mechanisms. Although, numerous studies revealed fundamentally beneficial effects of DHA on cardiovascular system, it remains unknown whether these effects were produced by DHA or one of its possibly more potent metabolites. Emerging evidence indicates that cytochrome P450 (CYP) epoxygenase metabolites of DHA, epoxydocosapentaenoic acids (EDPs), produce more potent biological activity compared to its precursor DHA. In this study we investigated whether DHA and its metabolite 19,20-EDP could protect HL-1 cardiac cells against LPS-induced cytotoxicity. We provide evidence that exogenously added or DHA-derived EDPs promote mitochondrial biogenesis and function in HL-1 cardiac cells. Our results illustrate the CYP epoxygenase metabolite of DHA, 19,20-EDP, confers extensive protection to HL-1 cardiac cells against LPS-induced cytotoxicity via activation of SIRT1.

PPARδ signaling mediates the cytotoxicity of DHA in H9c2 cells.

Docosahexaenoic acid (22:6n3, DHA) is an n-3 polyunsaturated fatty acid (PUFA) known to affect numerous biological functions. While DHA possesses many properties that impact cell survival such as suppressing cell growth and inducing apoptosis, the exact molecular and cellular mechanism(s) remain unknown. Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that regulate many cell pathways including cell death. As DHA acts as a ligand to PPARs the aim of this study was to examine the involvement of PPARδ in DHA-mediated cytotoxicity toward H9c2 cells. Treatment with DHA (100µM) resulted in a significant decline in cell viability, cellular metabolic activity and total antioxidant capacity coinciding with increased total proteasome activities and activity of released lactate dehydrogenase (LDH). No changes in reactive oxygen species (ROS) production or accumulation of lipid peroxidation products were observed but DHA promoted apoptotic cell death as detected by flow cytometry, increased caspase-3 activity and decreased phosphorylation of Akt. Importantly, DHA enhanced PPARδ DNA binding activity in H9c2 cells strongly signifying that the cytotoxic effect of DHA might be mediated via PPARδ signaling. Co-treatment with the selective PPARδ antagonist GSK 3787 (1µM) abolished the cytotoxic effects of DHA in H9c2 cells. Cytotoxic effects of DHA were attenuated by co-treatment with myriocin, a selective inhibitor of serine palmitoyl transferase (SPT), preventing de novo ceramide biosynthesis. LC/MS analysis revealed that treatment with DHA resulted in the accumulation of ceramide, which was blocked by GSK 3787. Interestingly, inhibition of cytochrome P450 (CYP) oxidase with MS-PPOH (50µM) abolished DHA-mediated cytotoxicity suggesting downstream metabolites as the active mediators. We further demonstrate that CYP oxidase metabolites of DHA, methyl epoxy docosapentaenoate (EDP methyl esters, 1µM) (mix 1:1:1:1:1:1; 4,5-, 7,8-, 10,11-, 13,14-, 16,17- and 19,20-EDP methyl esters) and 19,20-EDP cause cytotoxicity via activation of PPARδ signaling leading to increased levels of intracellular ceramide. These results illustrate novel pathways for DHA-induced cytotoxicity that suggest an important role for CYP-derived metabolites, EDPs.

Reserve carbohydrates maintain the viability of Saccharomyces cerevisiae cells during chronological aging.

Glycogen and trehalose are well known to participate in many important cell functions, e.g., protection from stress factors, regulation of cell growth and division, spore formation. Since the aging is a complex process involving many aspects of cell metabolism, it was interesting to study the role of glycogen and trehalose in maintenance of viability of aging cells. We have revealed that cell aging is accompanied by an abrupt fall of glycogen and trehalose contents between the second and third weeks of aging. Simultaneously, we observed a decrease in the activity of glycolytic enzymes, phosphofructokinase and hexokinase. At the same time, the viability of aging cells abruptly declined. Although we found neither glycogen nor trehalose in the cells after the third week of aging, they remained viable for some time, apparently due to development of some compensatory metabolic pathways. In spite of this fact, complete death of the cells occurred by the eighth week of experiment, which confirmed irreplaceability of reserve carbohydrates in yeast cell metabolism. Possible reasons of the inability of aging cells to accumulate glycogen and trehalose are discussed in the work.

PPARγ signaling is required for mediating EETs protective effects in neonatal cardiomyocytes exposed to LPS.

Lipopolysaccharide (LPS) is a bacterial wall endotoxin producing many pathophysiological conditions including myocardial inflammation leading to cardiotoxicity. Epoxyeicosatrienoic acids (EETs) are biologically active metabolites of arachidonic acids capable of activating protective cellular pathways in response to stress stimuli. EETs evoke a plethora of pathways limiting impairments of cellular structures, reducing cell death, and promoting anti-inflammatory reactions in various cell types. Considering EETs are capable of producing various biological protective effects, we hypothesized that EETs would protect rat neonatal cardiomyocytes (NCM) against LPS-induced cytotoxicity. In this study, we used a dual-acting, synthetic analog of EETs, UA-8 [13-(3-propylureido)tridec-8-enoic acid], possessing both EET-mimetic and soluble epoxide hydrolase selective inhibitory properties and 14,15-EET as a model of canonical EET molecules. We found that both UA-8 and 14,15-EET significantly improved cell viability and mitochondrial function of cardiomyocytes exposed to LPS. Furthermore, treatment with UA-8 or 14,15-EET resulted in significant attenuation of LPS-triggered pro-inflammatory response, caspase-3 activation and reduction in the total antioxidant capacity in cardiomyocytes. Importantly, EET-mediated effects were significantly reduced by pharmacological inhibition of peroxisome proliferator-activated receptors γ (PPARγ) suggesting that PPARγ signaling was required for EETs exerted protective effects. Data presented in the current study demonstrate that activation of PPARγ signaling plays a crucial role in EET-mediated protection against LPS-cytotoxicity in cardiomyocytes.