Prostaglandin D2-ethanolamide induces skin cancer apoptosis by suppressing the activity of cellular antioxidants.

The combined incidence of melanoma and non-melanoma skin cancer (NMSC) is greater than the incidence of all other malignancies in the US. Previously, we demonstrated that the endocannabinoid, arachidonoyl-ethanolamide (AEA), was a potent inducer of apoptosis in NMSC. The metabolism of AEA to the prostaglandin, PGD2-EA, was a prerequisite for AEA cytotoxicity. However, the mechanism of PGD2-EA cell death has not been clearly defined. In the present study, we report that PGD2-EA causes apoptosis in melanoma and NMSC cells. Mass spectrometry analysis revealed that PGD2-EA was dehydrated to three J-series prostaglandins; PGJ2-EA, Δ12PGJ2-EA, and 15deoxy,Δ12,14 PGJ2-EA. PGD2-EA inhibited the antioxidant activity of glutathione and thioredoxin which then caused oxidative stress. This increase in oxidative stress was accompanied by the activation of endoplasmic reticulum (ER) stress and apoptosis. The effect of PGD2-EA was independent of DP1, DP2, and PPARγ receptors suggesting that PGD2-EA cytotoxicity was mediated by its metabolic product, 15dPGJ2-EA.

Corrigendum to "Obesity in a model of gpx4 haploinsufficiency uncovers a causal role for lipid-derived aldehydes in human metabolic disease and cardiomyopathy" [Mol Metab 4 (6) (2015) 493-506].

[This corrects the article DOI: 10.1016/j.molmet.2015.04.001.].

Obesity in a model of gpx4 haploinsufficiency uncovers a causal role for lipid-derived aldehydes in human metabolic disease and cardiomyopathy.

OBJECTIVE: Lipid peroxides and their reactive aldehyde derivatives (LPPs) have been linked to obesity-related pathologies, but whether they have a causal role has remained unclear. Glutathione peroxidase 4 (GPx4) is a selenoenzyme that selectively neutralizes lipid hydroperoxides, and human gpx4 gene variants have been associated with obesity and cardiovascular disease in epidemiological studies. This study tested the hypothesis that LPPs underlie cardio-metabolic derangements in obesity using a high fat, high sucrose (HFHS) diet in gpx4 haploinsufficient mice (GPx4(+/-)) and in samples of human myocardium. METHODS: Wild-type (WT) and GPx4(+/-) mice were fed either a standard chow (CNTL) or HFHS diet for 24 weeks, with metabolic and cardiovascular parameters measured throughout. Biochemical and immuno-histological analysis was performed in heart and liver at termination of study, and mitochondrial function was analyzed in heart. Biochemical analysis was also performed on samples of human atrial myocardium from a cohort of 103 patients undergoing elective heart surgery. RESULTS: Following HFHS diet, WT mice displayed moderate increases in 4-hydroxynonenal (HNE)-adducts and carbonyl stress, and a 1.5-fold increase in GPx4 enzyme in both liver and heart, while gpx4 haploinsufficient (GPx4(+/-)) mice had marked carbonyl stress in these organs accompanied by exacerbated glucose intolerance, dyslipidemia, and liver steatosis. Although normotensive, cardiac hypertrophy was evident with obesity, and cardiac fibrosis more pronounced in obese GPx4(+/-) mice. Mitochondrial dysfunction manifesting as decreased fat oxidation capacity and increased reactive oxygen species was also present in obese GPx4(+/-) but not WT hearts, along with up-regulation of pro-inflammatory and pro-fibrotic genes. Patients with diabetes and hyperglycemia exhibited significantly less GPx4 enzyme and greater HNE-adducts in their hearts, compared with age-matched non-diabetic patients. CONCLUSION: These findings suggest LPPs are key factors underlying cardio-metabolic derangements that occur with obesity and that GPx4 serves a critical role as an adaptive countermeasure.

Do fish oil omega-3 fatty acids enhance antioxidant capacity and mitochondrial fatty acid oxidation in human atrial myocardium via PPARγ activation?

Abstract Studies in experimental models suggest that n-3 polyunsaturated fatty acids (PUFAs) improve metabolic and anti-inflammatory/antioxidant capacity of the heart, although the mechanisms are unclear and translational evidence is lacking. In this study, patients ingested a moderately high dose of n-3 PUFAs (3.4 g/day eicosapentaenoic (EPA) and doxosahexaenoic acid (DHA) ethyl-esters) for a period of 2-3 weeks before having elective cardiac surgery. Blood was obtained before treatment and at the time of surgery, and myocardial tissue from the right atrium was also dissected during surgery. Blood EPA levels increased and myocardial tissue EPA and DHA levels were significantly higher in n-3 PUFA-treated patients compared with untreated, standard-of-care control patients. Interestingly, n-3 PUFA patients had greater nuclear transactivation of peroxisome proliferator-activated receptor-γ (PPARγ), fatty acid metabolic gene expression, and enhanced mitochondrial respiration supported by palmitoyl-carnitine in the atrial myocardium, despite no difference in mitochondrial content. Myocardial tissue from n-3 PUFA patients also displayed greater expression and activity of key antioxidant/anti-inflammatory enzymes. These findings lead to our hypothesis that PPARγ activation is a mechanism by which fish oil n-3 PUFAs enhance mitochondrial fatty acid oxidation and antioxidant capacity in human atrial myocardium, and that this preoperative therapeutic regimen may be optimal for mitigating oxidative/inflammatory stress associated with cardiac surgery.

Monoamine oxidase is a major determinant of redox balance in human atrial myocardium and is associated with postoperative atrial fibrillation.

BACKGROUND: Onset of postoperative atrial fibrillation (POAF) is a common and costly complication of heart surgery despite major improvements in surgical technique and quality of patient care. The etiology of POAF, and the ability of clinicians to identify and therapeutically target high-risk patients, remains elusive. METHODS AND RESULTS: Myocardial tissue dissected from right atrial appendage (RAA) was obtained from 244 patients undergoing cardiac surgery. Reactive oxygen species (ROS) generation from multiple sources was assessed in this tissue, along with total glutathione (GSHt) and its related enzymes GSH-peroxidase (GPx) and GSH-reductase (GR). Monoamine oxidase (MAO) and NADPH oxidase were observed to generate ROS at rates 10-fold greater than intact, coupled mitochondria. POAF risk was significantly associated with MAO activity (Quartile 1 [Q1]: adjusted relative risk [ARR]=1.0; Q2: ARR=1.8, 95% confidence interval [CI]=0.84 to 4.0; Q3: ARR=2.1, 95% CI=0.99 to 4.3; Q4: ARR=3.8, 95% CI=1.9 to 7.5; adjusted Ptrend=0.009). In contrast, myocardial GSHt was inversely associated with POAF (Quartile 1 [Q1]: adjusted relative risk [ARR]=1.0; Q2: ARR=0.93, 95% confidence interval [CI]=0.60 to 1.4; Q3: ARR=0.62, 95% CI=0.36 to 1.1; Q4: ARR=0.56, 95% CI=0.34 to 0.93; adjusted Ptrend=0.014). GPx also was significantly associated with POAF; however, a linear trend for risk was not observed across increasing levels of the enzyme. GR was not associated with POAF risk. CONCLUSIONS: Our results show that MAO is an important determinant of redox balance in human atrial myocardium, and that this enzyme, in addition to GSHt and GPx, is associated with an increased risk for POAF. Further investigation is needed to validate MAO as a predictive biomarker for POAF, and to explore this enzyme's potential role in arrhythmogenesis.

Cardioprotection via preserved mitochondrial structure and function in the mPer2-mutant mouse myocardium.

We have previously shown that myocardial infarct size in nonreperfused hearts of mice with a functional deletion of the circadian rhythm gene mPer2 (mPer2-M) was reduced by 43%. We hypothesized that acute ischemia-reperfusion injury (I/R = 30 min I/2 h R) would also be reduced in these mice and that ischemic preconditioning (IPC) (3 × 5 min cycles) before I/R, which enhances protection in wild-type (WT) hearts, would provide further protection in mPer2-M hearts. We observed a 69 and 75% decrease in infarct size in mPer2-M mouse hearts compared with WT following I/R and IPC, respectively. This was coincident with 67% less neutrophil infiltration and 57% less apoptotic cardiomyocytes. IPC in mPer2-M mice before I/R had 48% less neutrophil density and 46% less apoptosis than their WT counterparts. Macrophage density was not different between WT and mPer2-M I/R, but it was 45% higher in mPer2-M IPC mouse hearts compared with WT IPC. There were no baseline differences in cardiac mitochondrial function between WT and mPer2-M mice, but, following I/R, WT exhibited a marked decrease in maximal O2 consumption supported by complex I-mediated substrates, whereas mPer2-M did not, despite no difference in complex I content. Moreover, cardiac mitochondria from WT mice exhibited a very robust increase in ADP-stimulated O2 consumption in response to exogenously added cytochrome c, along with a high rate of reactive oxygen species production, none of which was exhibited by cardiac mitochondria from mPer2-M following I/R. Taken together, these findings suggest that mPer2 deletion preserves mitochondrial membrane structure and functional integrity in heart following I/R injury, the consequence of which is preservation of myocardial viability. Understanding the mechanisms connecting cardiac events, mitochondrial function, and mPer2 could lead to preventative and therapeutic strategies for at risk populations.

Novel role for thioredoxin reductase-2 in mitochondrial redox adaptations to obesogenic diet and exercise in heart and skeletal muscle.

Increased fatty acid availability and oxidative stress are physiological consequences of exercise (Ex) and a high-fat, high-sugar (HFHS) diet. Despite these similarities, the global effects of Ex are beneficial, whereas HFHS diets are largely deleterious to the cardiovascular system. The reasons for this disparity are multifactorial and incompletely understood. We hypothesized that differences in redox adaptations following HFHS diet in comparison to exercise may underlie this disparity, particularly in mitochondria. Our objective in this study was to determine mechanisms by which heart and skeletal muscle (red gastrocnemius, RG) mitochondria experience differential redox adaptations to 12 weeks of HFHS diet and/or exercise training (Ex) in rats. Surprisingly, both HFHS feeding and Ex led to contrasting effects in heart and RG, in that mitochondrial H2O2 decreased in heart but increased in RG following both HFHS diet and Ex, in comparison to sedentary animals fed a control diet. These differences were determined to be due largely to increased antioxidant/anti-inflammatory enzymes in the heart following the HFHS diet, which did not occur in RG. Specifically, upregulation of mitochondrial thioredoxin reductase-2 occurred with both HFHS and Ex in the heart, but only with Ex in RG, and systematic evaluation of this enzyme revealed that it is critical for suppressing mitochondrial H2O2 during fatty acid oxidation. These findings are novel and important in that they illustrate the unique ability of the heart to adapt to oxidative stress imposed by HFHS diet, in part through upregulation of thioredoxin reductase-2. Furthermore, upregulation of thioredoxin reductase-2 plays a critical role in preserving the mitochondrial redox status in the heart and skeletal muscle with exercise.

Aldehyde stress and up-regulation of Nrf2-mediated antioxidant systems accompany functional adaptations in cardiac mitochondria from mice fed n-3 polyunsaturated fatty acids.

Diets replete with n-3 PUFAs (polyunsaturated fatty acids) are known to have therapeutic potential for the heart, although a specifically defined duration of the n-3 PUFA diet required to achieve these effects remains unknown, as does their mechanism of action. The present study was undertaken to establish whether adaptations in mitochondrial function and stress tolerance in the heart is evident following short- (3 weeks) and long- (14 weeks) term dietary intervention of n-3 PUFAs, and to identify novel mechanisms by which these adaptations occur. Mitochondrial respiration [mO2 (mitochondrial O2)], H2O2 emission [mH2O2 (mitochondrial H2O2)] and Ca2+-retention capacity [mCa2+ (mitochondrial Ca2+)] were assessed in mouse hearts following dietary intervention. Mice fed n-3 PUFAs for 14 weeks showed significantly lower mH2O2 and greater mCa2+ compared with all other groups. However, no significant differences were observed after 3 weeks of the n-3 PUFA diet, or in mice fed on an HFC (high-fat control) diet enriched with vegetable shortening, containing almost no n-3 PUFAs, for 14 weeks. Interestingly, expression and activity of key enzymes involved in antioxidant and phase II detoxification pathways, all mediated by Nrf2 (nuclear factor E2-related factor 2), were elevated in hearts from mice fed the n-3 PUFA diet, but not hearts from mice fed the HFC diet, even at 3 weeks. This increase in antioxidant systems in hearts from mice fed the n-3 PUFA diet was paralleled by increased levels of 4-hydroxyhexenal protein adducts, an aldehyde formed from peroxidation of n-3 PUFAs. The findings of the present study demonstrate distinct time-dependent effects of n-3 PUFAs on mitochondrial function and antioxidant response systems in the heart. In addition, they are the first to provide direct evidence that non-enzymatic oxidation products of n-3 PUFAs may be driving mitochondrial and redox-mediated adaptations, thereby revealing a novel mechanism for n-3 PUFA action in the heart.

Increased propensity for cell death in diabetic human heart is mediated by mitochondrial-dependent pathways.

Progressive energy deficiency and loss of cardiomyocyte numbers are two prominent factors that lead to heart failure in experimental models. Signals that mediate cardiomyocyte cell death have been suggested to come from both extrinsic (e.g., cytokines) and intrinsic (e.g., mitochondria) sources, but the evidence supporting these mechanisms remains unclear, and virtually nonexistent in humans. In this study, we investigated the sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium (Ca(2+)) using permeabilized myofibers of right atrium obtained from diabetic (n = 9) and nondiabetic (n = 12) patients with coronary artery disease undergoing nonemergent coronary revascularization surgery. Under conditions that mimic the energetic state of the heart in vivo (pyruvate, glutamate, malate, and 100 µM ADP), cardiac mitochondria from diabetic patients show an increased sensitivity to Ca(2+)-induced mPTP opening compared with nondiabetic patients. This increased mPTP Ca(2+) sensitivity in diabetic heart mitochondria is accompanied by a substantially greater rate of mitochondrial H(2)O(2) emission under identical conditions, despite no differences in respiratory capacity under these conditions or mitochondrial enzyme content. Activity of the intrinsic apoptosis pathway mediator caspase-9 was greater in diabetic atrial tissue, whereas activity of the extrinsic pathway mediator caspase-8 was unchanged between groups. Furthermore, caspase-3 activity was not significantly increased in diabetic atrial tissue. These data collectively suggest that the myocardium in diabetic patients has a greater overall propensity for mitochondrial-dependent cell death, possibly as a result of metabolic stress-imposed changes that have occurred within the mitochondria, rendering them more susceptible to insults such as Ca(2+) overload. In addition, they lend further support to the notion that mitochondria represent a viable target for future therapies directed at ameliorating heart failure and other comorbidities that come with diabetes.

Characteristics of tolerance in the guinea pig ileum produced by chronic in vivo exposure to opioid versus cannabinoid agonists.

Few studies have compared the nature of tolerance that develops following chronic opioid treatment with that which develops after chronic cannabinoid exposure in the same tissue and species. The degree and character of tolerance induced by 7 twice daily injections of morphine or 5 daily injections of the cannabinoid receptor agonist, WIN-55,212-2, was examined by comparing the ability of DAMGO, 2-chloroadenosine (CADO) and WIN-55,212-2 to inhibit neurogenic contractions of the longitudinal muscle/myenteric plexus preparation (LM/MP) and the ability of nicotine to elicit contractions in the LM/MP. Chronic morphine treatment resulted in subsensitivity to all inhibitory agonists (rightward shift in IC(50) values of 4-5-fold) and an increased responsiveness to the excitatory effect of nicotine while chronic WIN-55,212-2 exposure resulted in subsensitivity only to WIN-55,212-2 and a reduction in maximum response to both WIN-55,212-2 and DAMGO but no change in responsiveness to CADO. Chronic WIN-55,212-2 treatment significantly reduced CB(1) but not MOR receptor protein abundance while chronic morphine treatment did not change either. Assessment of the distribution of MOR and CB(1) receptors in myenteric neurons revealed distinct individual receptor expression as well as co-localization which was unaffected by either cannabinoid or opioid treatment. Thus, in contrast to the heterologous tolerance that develops after opioid treatment, tolerance in the LM/MP following chronic in vivo WIN-55,212-2 exposure appears to be homologous in character and is accompanied by a selective decrease in CB(1) receptor protein abundance. The data suggest that the cellular basis of tolerance differs between the two systems.

Correlation of the time course of development and decay of tolerance to morphine with alterations in sodium pump protein isoform abundance.

Since the heterologous tolerance that develops after chronic morphine administration has been proposed to be an adaptive process, it follows that the time course of the change in the cellular components should coincide with the time course of the altered responsiveness. This study correlated the time course over which heterologous tolerance develops with changes in the abundance of selected proteins in the guinea-pig longitudinal muscle/myenteric plexus (LM/MP) preparation. Tissues were obtained at various times following a single surgical implantation procedure and heterologous tolerance confirmed by a significant reduction in the sensitivity of the LM/MP to inhibition of neurogenic twitches by morphine, DAMGO, and 2-CADO. Tolerance developed with a delayed onset (significant 2-5-fold reduction in sensitivity by day 4 after pellet implantation) that reached a maximum by 7 days (4-8-fold reduction in responsiveness) that was maintained through 14 days with normal sensitivity spontaneously returning by 21 days post-implantation. Dot blot analysis was used to examine the abundance of the alpha(1) and alpha(3) subunit isoforms of the Na(+)/K(+) ATPase and beta-actin over the same time course. The results showed significant decreases in abundance of the alpha(3) subunit at 4, 7, and 10 days following pellet implantation but no change in beta-actin or the alpha(1) subunit at any time period. These data support the idea that heterologous tolerance following chronic morphine exposure results from a cellular adaptive change that may involve a change in the abundance of the alpha(3) subunit isoform of the Na(+)/K(+) ATPase.