Jérôme Roy

Isoprostanes and neuroprostanes: total synthesis, biological activity and biomarkers of oxidative stress in humans.

Isoprostanes (IsoPs) and neuroprostanes (NeuroPs) are formed in vivo by a free radical non-enzymatic mechanism involving peroxidation of arachidonic acid (AA, C20:4 n-6) and docosahexaenoic acid (DHA, C22:6 n-3) respectively. This review summarises our research in the total synthesis of these lipid metabolites, as well as their biological activities and their utility as biomarkers of oxidative stress in humans.

Nonenzymatic lipid mediators, neuroprostanes, exert the antiarrhythmic properties of docosahexaenoic acid

Neuroprostanes are lipid mediators produced by nonenzymatic free radical peroxidation of docosahexaenoic acid (DHA). DHA is associated with a lower atherosclerosis risk, suggesting a beneficial role in cardiovascular diseases. The aim of this study was to investigate the influence of DHA peroxidation on its potentially antiarrhythmic properties (AAP) in isolated ventricular cardiomyocytes and in vivo in post-myocardial infarcted mice. Calcium imaging and biochemical experiments indicate that cardiac arrhythmias induced by isoproterenol are associated with Ca(2+) leak from the sarcoplasmic reticulum after oxidation and phosphorylation of the type 2 ryanodine receptor (RyR2) leading to dissociation of the FKBP12.6/RyR2 complex. Both oxidized DHA and 4(RS)-4-F4t-NeuroP prevented cellular arrhythmias and posttranslational modifications of the RyR2 leading to a stabilized FKBP12.6/RyR2 complex. DHA per se did not have AAP. The AAP of 4(RS)-4-F4t-NeuroP was also observed in vivo. In this study, we challenged the paradigm that spontaneously formed oxygenated metabolites of lipids are undesirable as they are unconditionally toxic. This study reveals that the lipid mediator 4(RS)-4-F4t-neuroprostane derived from nonenzymatic peroxidation of docosahexaenoic acid can counteract such deleterious effects through cardiac antiarrhythmic properties. Our findings demonstrate 4(RS)-4-F4t-NeuroP as a mediator of the cardioprotective AAP of DHA. This discovery opens new perspectives for products of nonenzymatic oxidized ω3 polyunsaturated fatty acids as potent mediators in diseases that involve ryanodine complex destabilization such as ischemic events.

Multifunctional Mitochondrial Epac1 Controls Myocardial Cell Death

Rationale: Although the second messenger cyclic AMP (cAMP) is physiologically beneficial in the heart, it largely contributes to cardiac disease progression when dysregulated. Current evidence suggests that cAMP is produced within mitochondria. However, mitochondrial cAMP signaling and its involvement in cardiac pathophysiology are far from being understood. Objective: To investigate the role of MitEpac1 (mitochondrial exchange protein directly activated by cAMP 1) in ischemia/reperfusion injury. Methods and Results: We show that Epac1 (exchange protein directly activated by cAMP 1) genetic ablation (Epac1−/−) protects against experimental myocardial ischemia/reperfusion injury with reduced infarct size and cardiomyocyte apoptosis. As observed in vivo, Epac1 inhibition prevents hypoxia/reoxygenation–induced adult cardiomyocyte apoptosis. Interestingly, a deleted form of Epac1 in its mitochondrial-targeting sequence protects against hypoxia/reoxygenation–induced cell death. Mechanistically, Epac1 favors Ca2+ exchange between the endoplasmic reticulum and the mitochondrion, by increasing interaction with a macromolecular complex composed of the VDAC1 (voltage-dependent anion channel 1), the GRP75 (chaperone glucose-regulated protein 75), and the IP3R1 (inositol-1,4,5-triphosphate receptor 1), leading to mitochondrial Ca2+ overload and opening of the mitochondrial permeability transition pore. In addition, our findings demonstrate that MitEpac1 inhibits isocitrate dehydrogenase 2 via the mitochondrial recruitment of CaMKII (Ca2+/calmodulin-dependent protein kinase II), which decreases nicotinamide adenine dinucleotide phosphate hydrogen synthesis, thereby, reducing the antioxidant capabilities of the cardiomyocyte. Conclusions: Our results reveal the existence, within mitochondria, of different cAMP–Epac1 microdomains that control myocardial cell death. In addition, our findings suggest Epac1 as a promising target for the treatment of ischemia-induced myocardial damage.

Carnitine deficiency induces a short QT syndrome

BACKGROUND Short QT syndrome is associated with an increased risk of cardiac arrhythmias and unexpected sudden death. Until now, only mutations in genes encoding the cardiac potassium and calcium channels have been implicated in early T-wave repolarization. OBJECTIVE The purpose of this study was to confirm a relationship between a short QT syndrome and carnitine deficiency. METHODS We report 3 patients affected by primary systemic carnitine deficiency and an associated short QT syndrome. Ventricular fibrillation during early adulthood was the initial symptom in 1 case. To confirm the relationship between carnitine, short QT syndrome, and arrhythmias, we used a mouse model of carnitine deficiency induced by long-term subcutaneous perfusion of MET88. RESULTS MET88-treated mice developed cardiac hypertrophy associated with a remodeling of the mitochondrial network. The continuous monitoring of electrocardiograms confirmed a shortening of the QT interval, which was negatively correlated with the plasma carnitine concentration. As in humans, such alterations coincided with the genesis of ventricular premature beats and ventricular tachycardia and fibrillation. CONCLUSION Altogether, these results suggest that long-chain fatty acid metabolism influence the morphology and the electrical function of the heart.

Assessment of Isoprostanes in Human Plasma: Technical Considerations and the Use of Mass Spectrometry

Oxygenated lipid mediators released from non-enzymatic peroxidation of polyunsaturated fatty acids (PUFA) are known to have functional roles in humans. Notably, among these lipid mediators, isoprostanes molecules are robust biomarkers of oxidative stress but those from n-3 PUFA are also bioactive molecules. In order to identify and assess the isoprostanes, the use of mass spectrometry (MS) for analysis is preferable and has been used for over two decades. Gas chromatography (GC) is commonly coupled to the MS to separate the derivatized isoprostanes of interest in biological samples. In order to increase the accuracy of the analytical performance, GC–MS/MS was also applied. Lately, MS or MS/MS has been coupled with high-performance liquid chromatography to assess multiple isoprostane molecules in a single biological sample without derivatization process. However, there are limitations for the use of LC–MS/MS in the measurement of plasma isoprostanes, which will be discussed in this review.

Polymeric nanocapsules prevent oxidation of core-loaded molecules: evidence based on the effects of docosahexaenoic acid and neuroprostane on breast cancer cells proliferation

BackgroundNanocapsules, as a delivery system, are able to target drugs and other biologically sensitive molecules to specific cells or organs. This system has been intensively investigated as a way to protect bioactives drugs from inactivation upon interaction with the body and to ensure the release to the target. However, the mechanism of improved activity of the nanoencapsulated molecules is far from being understood at the cellular and subcellular levels. Epidemiological studies suggest that dietary polyunsaturated fatty acids (PUFA) can reduce the morbidity and mortality from breast cancer. This influence could be modulated by the oxidative status of the diet and it has been suggested that the anti-proliferative properties of docosahexaenoic acid (DHA) are enhanced by pro-oxidant agents.MethodsThe effect of encapsulation of PUFA on breast cancer cell proliferation in different oxidative medium was evaluated in vitro. We compared the proliferation of the human breast cancer cell line MDA-MB-231 and of the non-cancer human mammary epithelial cell line MCF-10A in different experimental conditions.ResultsDHA possessed anti-proliferative properties that were prevented by alpha-tocopherol (an antioxidant) and enhanced by the pro-oxidant hydrogen peroxide that confirms that DHA has to be oxidized to exert its anti-proliferative properties. We also evaluated the anti-proliferative effects of the 4(RS)-4-F4t-neuroprostane, a bioactive, non-enzymatic oxygenated metabolite of DHA known to play a major role in the prevention of cardiovascular diseases. DHA-loaded nanocapsules was less potent than non-encapsulated DHA while co-encapsulation of DHA with H2O2 maintained the inhibition of proliferation. The nanocapsules slightly improves the anti-proliferative effect in the case of 4(RS)-4-F4t-neuroprostane that is more hydrophilic than DHA.ConclusionOverall, our findings suggest that the sensitivity of tumor cell lines to DHA involves oxidized metabolites. They also indicate that neuroprostane is a metabolite participating in the growth reducing effect of DHA, but it is not the sole. These results also suggest that NC seek to enhance the stability against degradation, enhance cellular availability, and control the release of bioactive fatty acids following their lipophilicities.

Non-enzymatic oxidized metabolite of DHA, 4(RS)-4-F4t-neuroprostane protects the heart against reperfusion injury.

Abstract Acute myocardial infarction leads to an increase in oxidative stress and lipid peroxidation. 4(RS)‐4‐F4t‐Neuroprostane (4‐F4t‐NeuroP) is a mediator produced by non‐enzymatic free radical peroxidation of the cardioprotective polyunsaturated fatty acid, docosahexaenoic acid (DHA). In this study, we investigated whether intra‐cardiac delivery of 4‐F4t‐NeuroP (0.03 mg/kg) prior to occlusion (ischemia) prevents and protects rat myocardium from reperfusion damages. Using a rat model of ischemic‐reperfusion (I/R), we showed that intra‐cardiac infusion of 4‐F4t‐NeuroP significantly decreased infarct size following reperfusion (−27%) and also reduced ventricular arrhythmia score considerably during reperfusion (−41%). Most notably, 4‐F4t‐NeuroP decreased ventricular tachycardia and post‐reperfusion lengthening of QT interval. The evaluation of the mitochondrial homeostasis indicates a limitation of mitochondrial swelling in response to Ca2+ by decreasing the mitochondrial permeability transition pore opening and increasing mitochondria membrane potential. On the other hand, mitochondrial respiration measured by oxygraphy, and mitochondrial ROS production measured with MitoSox red® were unchanged. We found decreased cytochrome c release and caspase 3 activity, indicating that 4‐F4t‐NeuroP prevented reperfusion damages and reduced apoptosis. In conclusion, 4‐F4t‐NeuroP derived from DHA was able to protect I/R cardiac injuries by regulating the mitochondrial homeostasis. HighlightsThe lipid mediator 4(RS)‐4‐F4t‐neuroprostane derived from non‐enzymatic peroxidation of DHA contributes to cardioprotective properties of this PUFA following an ischemia/reperfusion event.4(RS)‐4‐F4tneuroprostane displays a strong anti‐apoptotic property involving the normalization of calcium homeostasis by the stabilization of ryanodine receptor complex and to a decrease of mPTP opening leading to the reduction of pro‐apoptotic factors.This study suggests that some well‐known effects of n‐3 fatty acids are mediated by their non‐enzymatic cyclic oxygenated metabolites.This discovery opens new perspectives for non‐enzymatic oxidized products of n‐3 polyunsaturated fatty acids as potent preventive therapeutic way in acute myocardial infarction.

Biodegradable Polymeric Nanocapsules Prevent Cardiotoxicity of Anti-Trypanosomal Lychnopholide

Chagas disease is a neglected parasitic disease caused by the protozoan Trypanosoma cruzi. New antitrypanosomal options are desirable to prevent complications, including a high rate of cardiomyopathy. Recently, a natural substance, lychnopholide, has shown therapeutic potential, especially when encapsulated in biodegradable polymeric nanocapsules. However, little is known regarding possible adverse effects of lychnopholide. Here we show that repeated-dose intravenous administration of free lychnopholide (2.0 mg/kg/day) for 20 days caused cardiopathy and mortality in healthy C57BL/6 mice. Echocardiography revealed concentric left ventricular hypertrophy with preserved ejection fraction, diastolic dysfunction and chamber dilatation at end-stage. Single cardiomyocytes presented altered contractility and Ca2+ handling, with spontaneous Ca2+ waves in diastole. Acute in vitro lychnopholide application on cardiomyocytes from healthy mice also induced Ca2+ handling alterations with abnormal RyR2-mediated diastolic Ca2+ release. Strikingly, the encapsulation of lychnopholide prevented the cardiac alterations induced in vivo by the free form repeated doses. Nanocapsules alone had no adverse cardiac effects. Altogether, our data establish lychnopholide presented in nanocapsule form more firmly as a promising new drug candidate to cure Chagas disease with minimal cardiotoxicity. Our study also highlights the potential of nanotechnology not only to improve the efficacy of a drug but also to protect against its adverse effects.

Cardioprotective effects of omega 3 fatty acids: origin of the variability

Since 40 years, it is known that omega-3 poly-unsaturated fatty acids (ω3 PUFAs) have cardioprotective effects. These include antiarrhythmic effects, improvements of autonomic function, endothelial function, platelet anti-aggregation and inflammatory properties, lowering blood pressure, plaque stabilization and reduced atherosclerosis. However, recently, conflicting results regarding the health benefits of ω3 PUFAs from seafood or ω3 PUFAs supplements have emerged. The aim of this review is to examine recent literature regarding health aspects of ω3 PUFAs intake from fish or supplements, and to discuss different arguments/reasons supporting these conflicting findings.

The Complex QT/RR Relationship in Mice

The QT interval reflects the time between the depolarization of ventricles until their repolarization and is usually used as a predictive marker for the occurrence of arrhythmias. This parameter varies with the heart rate, expressed as the RR interval (time between two successive ventricular depolarizations). To calculate the QT independently of the RR, correction formulae are currently used. In mice, the QT-RR relationship as such has never been studied in conscious animals, and correction formulas are mainly empirical. In the present paper we studied how QT varies when the RR changes physiologically (comparison of nocturnal and diurnal periods) or after dosing mice with tachycardic agents (norepinephrine or nitroprusside). Our results show that there is significant variability of QT and RR in a given condition, resulting in the need to average at least 200 consecutive complexes to accurately compare the QT. Even following this method, no obvious shortening of the QT was observed with increased heart rate, regardless of whether or not this change occurs abruptly. In conclusion, the relationship between QT and RR in mice is weak, which renders the use of correction formulae inappropriate and misleading in this species.