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International audience; Background and Purposes: Several hERG blocking drugs known for their propensity to trigger Torsades de Pointes (TdP) were reported to induce a sympatho-vagal coactivation and to enhance High Frequency heart rate (HFHR) and QT oscillations (HFQT) from telemetric data. The present work aims to characterise the underlying mechanism(s) leading to these autonomic changes.Experimental approach: Effects of 15 torsadogenic hERG blocking drugs (astemizole, chlorpromazine, cisapride, droperidol, ibutilide, dofetilide, haloperidol, moxifloxacin, pimozide, quinidine, risperidone, sotalol, sertindole, terfenadine, thioridazine) were assessed by telemetry in beagle dogs. Hemodynamic effects on diastolic and systolic arterial pressure were analysed from the first doses causing QTc prolongation and/or HFQT oscillations enhancement. Autonomic control changes were analysed with the High Frequency Autonomic Modulation (HFAM) model.Key results: Except moxifloxacin and quinidine, all torsadogenic hERG blockers induced parasympathetic activation or sympatho-vagal coactivation combined with enhancement of HFQT oscillations. These autonomic effects result from reflex compensatory mechanisms in response to mild hemodynamic side effects. These hemodynamic mechanisms were characterised by transient HR acceleration during HF oscillations. A phenomenon of concealed QT prolongation was unmasked for several torsadogenic hERG blockers under β-adrenoceptors blockade by atenolol. Resulting enhancement of HFQT oscillations was shown to contribute directly to triggering of dofetilide induced ventricular arrhythmias.Conclusions and implications: This work supports for the first time a contribution of hemodynamic side properties to ventricular arrhythmias triggering by torsadogenic hERG blocking drugs. These hemodynamic side effects may constitute a second component of their arrhythmic profile acting as a trigger alongside their intrinsic arrhythmogenic electrophysiological properties.

Among two-pore domains K+ channels, twik-related K+ channels (TREK) are characterized by a polymodal activation including membrane stretch and polyunsaturated fatty acids (PUFAS). So far, there is no systematic comparison of PUFAs activation on ITREK-1 and no clear reliable action mechanism.In this study, we performed a thorough comparison of the effects of fatty acids (FA) having between 18 and 22 atoms of carbon and 0 to 6 double bonds and ML402, a well-known direct activator of TREK-1. We observed no relationship between acyl chain length and/or number of double bonds and TREK-1 activation. This suggests that the bilayer-couple hypothesis cannot explain by itself all the activation of TREK-1 by PUFAs. We found that the two strongest activators are linoleic acid and docosahexaenoic acid, respectively the shortest with two double bonds and the longest with six double bonds PUFAs. We also found that the important variability of ITREK-1 activation by PUFAs is related to the variable constitutive activity of TREK-1. Based on activation and washout kinetic analysis using two configurations of patch-clamp, we propose that PUFAs activate TREK-1 mainly following a direct interaction with the channel.

International audience; The transient receptor potential Melastatin 4 (TRPM4) channel is a calcium-activated non-selective cation channel expressed widely. In the heart, using a knock-out mouse model, the TRPM4 channel has been shown to be involved in multiple processes, including β-adrenergic regulation, cardiac conduction, action potential duration and hypertrophic adaptations. This channel was recently shown to be involved in stress-induced cardiac arrhythmias in a mouse model overexpressing TRPM4 in ventricular cardiomyocytes. However, the link between TRPM4 channel expression in ventricular cardiomyocytes, the hypertrophic response to stress and/or cellular arrhythmias has yet to be elucidated. In this present study, we induced pathological hypertrophy in response to myocardial infarction using a mouse model of Trpm4 gene invalidation, and demonstrate that TRPM4 is essential for survival. We also demonstrate that the TRPM4 is required to activate both the Akt and Calcineurin pathways. Finally, using two hypertrophy models, either a physiological response to endurance training or a pathological response to myocardial infarction, we show that TRPM4 plays a role in regulating transient calcium amplitudes and leads to the development of cellular arrhythmias potentially in cooperation with the Sodium-calcium exchange (NCX). Here, we report two functions of the TRPM4 channel: first its role in adaptive hypertrophy, and second its association with NCX could mediate transient calcium amplitudes which trigger cellular arrhythmias.

Up to the middle of the 2000’s, omega 3 polyunsaturated fatty acids were considered has having cardioprotective properties. Patients having a myocardial infarction were supplemented with these fatty acids in secondary prevention of myocardial infarction. Since then, many randomized clinical trials failed to observe the cardioprotective effects previously described. The main hypothesis to explain such change is the systematic prescription of statins to patients following a myocardial infarction, statins interfering with the homeostasis of omega 3 fatty acids. This review discusses the effects of different forms of omega-3 in association with statins on cardiovascular disease and emphasize on the interaction between statins and omega 3 fatty acids leading to the possible need to use higher dose of fatty acids to get a synergistic effect.

Sinoatrial node cells (SANC) automaticity is generated by functional association between the activity of plasmalemmal ion channels and local diastolic intracellular CaTo investigate the role of L-type CaIn dormant SANC, β-adrenergic stimulation with isoproterenol (ISO) induced spontaneous action potentials (AP) and CaOur study shows a novel role of Ca

Background and Purposes: Several hERG blocking molecules known for their propensity in triggering Torsades de Pointes (TdP) were reported as increasing High Frequency QT oscillations (HFQT). This effect was found as reflecting a sympatho-vagal coactivation. The present work aims to characterise the mechanism(s) leading to this particular state of the autonomic nervous system. Experimental approach: Effects of 20 hERG blockers including 15 torsadogenic molecules were assessed by telemetry in beagle dogs. Electrocardiogram and stroke volume modelled from the pulse contour method were analysed at the first dose level causing either QTc prolongation and/or HFQT increase. Cardiac autonomic control was analysed using the High Frequency Autonomic Modulation (HFAM) model in dogs and in untreated genotyped LQT1 and LQT2 individuals, for comparison. Key results: The sympatho-vagal coactivation induced by torsadogenic molecules is elicited by reflex compensatory mechanisms in response to changes in stroke volume or cardiac output related to hemodynamic off-targets and/or QT prolongation. QTc prolongation was concealed or markedly blunted by the sympathetic component activation in a large proportion of tested torsadogenic drugs. Sympathetic reflex mechanisms in LQT patients similar to that found for dofetilide was also revealed in both patients exhibiting QTc prolongation and concealed QTc prolongation, irrespective to LQT type. Conclusions and implications: QTc prolongation and/or drug-induced hemodynamic side effects enhance beat to beat ventricular repolarisation variability via sympatho-vagal reflex compensatory mechanisms. Considering the sympathetic reflex component via analysis of HFQT oscillations dramatically improves prediction, sensitivity and specificity of drug induced Torsades de pointes risk assessment.

Background and purpose Increase in high-frequency beat-to-beat heart rate oscillations by torsadogenic hERG blockers appears to be associated with signs of parasympathetic and sympathetic co-activation which cannot be assessed directly using classic methods of heart rate variability analysis. The present work aimed to find a translational model that would allow this particular state of the autonomic control of heart rate to be assessed. Experimental approach High-frequency heart rate and heart period oscillations were analysed within discrete 10 s intervals in a cohort of 200 healthy human subjects. Results were compared to data collected in non-human primates and beagle dogs during pharmacological challenges and torsadogenic hERG blockers exposure, in 127 genotyped LQT1 patients on/off β-blocker treatment and in subgroups of smoking and non-smoking subjects. Key results Three states of autonomic modulation, S1 (parasympathetic predominance) to S3 (reciprocal parasympathetic withdrawal/sympathetic activation), were differentiated to build a new model of heart rate variability referred to as high-frequency autonomic modulation. The S2 state corresponded to a specific state during which both parasympathetic and sympathetic systems were coexisting or co-activated. S2 oscillations were proportionally increased by torsadogenic hERG-blocking drugs, whereas smoking caused an increase in S3 oscillations. Conclusions and implications The combined analysis of the magnitude of high-frequency heart rate and high-frequency heart period oscillations allows a refined assessment of heart rate autonomic modulation applicable to long-term ECG recordings and offers new approaches to assessment of the risk of sudden death both in terms of underlying mechanisms and sensitivity.

International audience; 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.

International audience; Non-enzymatic oxygenated metabolites derived from polyunsaturated fatty acids (PUFA) are formed in vivo through free radical reaction under oxidative stress conditions. It has been over twenty-five years since the discovery of cyclic oxygenated metabolites derived from arachidonic acid (20:4 n-6), the isoprostanes, and since then they have become biomarkers of choice for assessing in vivo OS in humans and animals. Chemical synthesis of n-3 PUFA isoprostanoids such as F3-Isoprostanes from eicosapentaenoic acid (20:5 n-3), and F4-Neuroprostanes from docosahexaenoic acid (22:6 n-6) unravelled novel and unexpected biological properties of such omega-3 non-enzymatic cyclic metabolites as highlighted in this review.

International audience; One of the most salient physiological characteristics of cardiac muscle is that a dilated heart pumps more vigorously, a phenomenon known as the Frank-Starling relationship (see Allen and Kentish, 1985). At least two cellular mechanisms participate in this phenomenon: the reduction of the interfilament lattice spacing which favors the formation of cross-bridges (Wang and Fuchs, 1995) and the increased affinity of troponin C (TnC) for calcium (Ca2+) (Babu et al., 1988). In the latter case, it has been established that TnC itself is not the length sensor (Moss et al., 1991). The intracellular structure(s) able to sense changes in cell length has always been challenged and is still not known. We previously observed on intact isolated cardiac cells that active tension is more closely related to passive tension than to sarcomere length per se (Cazorla et al., 1997). This might have some physiological implications in the working heart since we found that sub-epicardial cells are more supple than sub-endocardial cells. In the present work on skinned cells, we studied the relationship between different levels of passive tension (modulated by a mild trypsin digestion) and the shift in pCa50 of tension-pCa relations induced by a stretch of cells from 1.9 to 2.3 microns sarcomere length. A significant correlation was obtained between passive tension and the stretch-induced shift in pCa50, or stretch-sensitivity of the active force. These observations led us to assume that titin might play a role in sensing cell length to modulate the contractile activity. Besides, it is known that myocardial infarcted cells are less sensitive to stretch. We propose that, in such a rat model, alterations of titin might participate in heart failure.

International audience; It has been 60 yr since the discovery of reactive oxygen species (ROS) in biology and the beginning of the scientific community's attempt to understand the impact of the unpaired electron of ROS molecules in biological pathways, which was eventually noted to be toxic. Several studies have shown that the presence of ROS is essential in triggering or acting as a secondary factor for numerous pathologies, including metabolic and genetic diseases; however, it was demonstrated that chronic treatment with antioxidants failed to show efficacy and positive effects in the prevention of diseases or health complications that result from oxidative stress. On the contrary, such treatment has been shown to sometimes even worsen the disease. Because of the permanent presence of ROS in organisms, elaborate mechanisms to adapt with these reactive molecules and to use them without necessarily blocking or preventing their actions have been studied. There is now a large body of evidence that shows that living organisms have conformed to the presence of ROS and, in retrospect, have adapted to the bioactive molecules that are generated by ROS on proteins, lipids, and DNA. In addition, ROS have undergone a shift from being molecules that invoked oxidative damage in regulating signaling pathways that impinged on normal physiological and redox responses. Working in this direction, this review unlocks a new conception about the involvement of cellular oxidants in the maintenance of redox homeostasis in redox regulation of normal physiological functions, and an explanation for its essential role in numerous pathophysiological states is noted.-Roy, J., Galano, J.-M., Durand, T., Le Guennec, J.-Y., Lee, J. C.-Y. Physiological role of reactive oxygen species as promoters of natural defenses.

International audience; An imbalance between oxidants and antioxidants that favors the oxidants leads to a disruption of the redox signaling control and/or molecular damage. The action of the oxidants in a non‐enzymatic process generates isoprostanoids from polyunsaturated fatty acids. In this review, we will focus on the effects of ω3‐polyunsaturated fatty acids in two different pathogeneses related to chronic and acute oxidative stress, one in neurodevelopmental, also known as the Rett syndrome, and the second in myocardial infarction and cardiac arrhythmias, respectively. We hypothesize that ω3‐polyunsaturated fatty acid supplementation displays antioxidant properties under a high oxidative stress situation, as in the Rett Syndrome, as well as protective properties of isoprostanoids from polyunsaturated fatty acids in pro‐arrhythmic conditions.

Safety pharmacology aims to predict rare side effects of new drugs. We explored whether rare pro-arrhythmic effects could be linked to the variability of the effects of these drugs on ion currents and whether taking into consideration this variability in computational models could help to better detect and predict cardiac side effects. For this purpose, we evaluated how intra- and inter-individual variability influences the effect of hERG inhibition on both the action potential duration and the occurrence of arrhythmias. Using two computer simulation models of human action potentials (endocardial and Purkinje cells), we analyzed the contribution of two biological parameters on the pro-arrhythmic effects of several hERG channel blockers: (i) spermine concentration, which varies with metabolic status, and (ii) L-type calcium conductance, which varies due to single nucleotide polymorphisms or mutations. By varying these parameters, we were able to induce arrhythmias in 1 out of 16 simulations although conventional modeling methods to detect pro-arrhythmic molecules failed. On the basis of our results, taking into consideration only 2 parameters subjected to intra- and inter-individual variability, we propose that in silico computer modeling may help to better define the risks of new drug candidates at early stages of pre-clinical development.

International audience; 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.

International audience; 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 thecardioprotective polyunsaturated fatty acid, docosahexaenoic acid (DHA). In this study, we investigated whetherintra-cardiac delivery of 4-F4t-NeuroP (0.03 mg/kg) prior to occlusion (ischemia) prevents and protects ratmyocardium from reperfusion damages.Using a rat model of ischemic-reperfusion (I/R), we showed that intra-cardiac infusion of 4-F4t-NeuroPsignificantly decreased infarct size following reperfusion (−27%) and also reduced ventricular arrhythmia scoreconsiderably during reperfusion (−41%). Most notably, 4-F4t-NeuroP decreased ventricular tachycardia andpost-reperfusion lengthening of QT interval. The evaluation of the mitochondrial homeostasis indicates alimitation of mitochondrial swelling in response to Ca2+by decreasing the mitochondrial permeability transitionpore opening and increasing mitochondria membrane potential. On the other hand, mitochondrial respirationmeasured by oxygraphy, and mitochondrial ROS production measured with MitoSox red®were unchanged. Wefound decreased cytochrome c release and caspase 3 activity, indicating that 4-F4t-NeuroP prevented reperfusiondamages and reduced apoptosis. In conclusion, 4-F4t-NeuroP derived from DHA was able to protect I/R cardiacinjuries by regulating the mitochondrial homeostasis

Safety pharmacology aims to predict rare side effects of new drugs. We explored whether rare pro-arrhythmic effects could be linked to the variability of the effects of these drugs on ion currents and whether taking into consideration this variability in computational models could help to better detect and predict cardiac side effects. For this purpose, we evaluated how intra- and inter-individual variability influences the effect of hERG inhibition on both the action potential duration and the occurrence of arrhythmias. Using two computer simulation models of human action potentials (endocardial and Purkinje cells), we analyzed the contribution of two biological parameters on the pro-arrhythmic effects of several hERG channel blockers: (i) spermine concentration, which varies with metabolic status, and (ii) L-type calcium conductance, which varies due to single nucleotide polymorphisms or mutations. By varying these parameters, we were able to induce arrhythmias in 1 out of 16 simulations although conventional modeling methods to detect pro-arrhythmic molecules failed. On the basis of our results, taking into consideration only 2 parameters subjected to intra- and inter-individual variability, we propose that in silico computer modeling may help to better define the risks of new drug candidates at early stages of pre-clinical development.

Acute myocardial infarction leads to an increase in oxidative stress and lipid peroxidation. 4(RS)-4-F

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.

International audience; AbstractBackgroundNanocapsules, 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.

International audience; Neuroprostanes are lipid mediators produced by nonenzymatic free radical peroxidation of docosahex-aenoic 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 þ 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-F 4t-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-F 4t-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-F 4t-neuroprostane derived from nonenzymatic peroxidation of docosahexaenoic acid can counteract such deleterious effects through cardiac antiarrhythmic properties. Our findings demonstrate 4(RS)-4-F 4t-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

International audience; Voltage-gated sodium channels (NaV) are molecular characteristics of excitable cells. Their activation, triggered by membrane depolarization, generates transient sodium currents that initiate action potentials in neurons and muscle cells. Sodium currents were discovered by Hodgkin and Huxley using the voltage clamp technique and reported in their landmark series of papers in 1952. It was only in the 1980's that sodium channel proteins from excitable membranes were molecularly characterized by Catterall and his collaborators. Non-excitable cells can also express NaV channels in physiological conditions as well as in pathological conditions. These NaV channels can sustain biological roles that are not related to the generation of action potentials. Interestingly, it is likely that the abnormal expression of NaV in pathological tissues can reflect the re-expression of a fetal phenotype. This is especially true in epithelial cancer cells for which these channels have been identified and sodium currents recorded, while it was not the case for cells from the cognate normal tissues. In cancers, the functional activity of NaV appeared to be involved in regulating the proliferative, migrative, and invasive properties of cells. This review is aimed at addressing the non-excitable roles of NaV channels with a specific emphasis in the regulation of cancer cell biology.

The cardioprotective effects through prevention of cardiac arrhythmias of long-chain polyunsaturated fatty acids of the n-3 series (PUFAs) have been demonstrated over the last 40 years. The main n-3 PUFAs are eicosapentaenoic acid (C20:5 n-3, EPA) and docosahexaenoic acid (C22:6 n-3, DHA) and both are highly peroxidable due to the presence of skipped dienes. The effects of n-3 PUFAs on cardiac function are controversial, notably due to lack of information on the mechanisms involved. Particularly, it is not well understood which is the active lipid: the PUFA or one of its oxygenated metabolites. Neuroprostanes are lipid mediators produced by non-enzymatic free radical peroxidation of DHA. Plasmatic 4(RS)-4-F4t-NeuroP concentration is negatively correlated with the risk of atherosclerosis suggesting a beneficial active role in some cardiovascular disease. In this study, we show that oxidized DHA and more specifically the isomers 4(RS)-4-F4t-NeuroP possess strong anti-arrhythmic properties (AAP) in isolated ventricular cardiomyocytes and in vivo in post-myocardial infarcted mice. Calcium imaging and biochemical experiments indicate that arrhythmias are associated with Ca2+ leak from the sarcoplasmic reticulum following oxidation and phosphorylation of the type 2 ryanodine receptor (RyR2) leading to dissociation of the FKBP12.6/ RyR2 complex. DHA per se has no AAP. Oxidized DHA as well as 4(RS)-4- F4t-NeuroP prevented posttranslational modifications of the RyR2 and stabilized the complex FKBP12.6/RyR2 to normalize Ca2+. This effect of 4(RS)- 4-F4t-NeuroP was further associated with the suppression in vitro and in vivo of cardiac arrhythmias. Our findings, demonstrates 4(RS)-4-F4t-NeuroP as a mediator of the AAP that exerts cardioprotective characteristics of DHA.

International audience; Long chain fatty acids bind to carnitine and form long chain acyl carnitine (LCAC), to enter into the mitochondria. They are oxidized in the mitochondrial matrix. LCAC accumulates rapidly under metabolic disorders, such as acute cardiac ischemia, chronic heart failure or diabetic cardiomyopathy. LCAC accumulation is associated with severe cardiac arrhythmia including ventricular tachycardia or fibrillation. We thus hypothesized that palmitoyl-carnitine (PC), alters mitochondrial function leading to Ca 2+ dependent-arrhythmia. In isolated cardiac mitochondria from C57Bl/6 mice, application of 10 μM PC decreased adenine nucleotide translocase (ANT) activity without affecting mitochondrial permeability transition pore (mPTP) opening. Mitochondrial reactive oxygen species (ROS) production, measured with MitoSOX Red dye in isolated ventricular cardiomyocytes, increased significantly under PC application. Inhibition of ANT by bongkrekic acid (20 μM) prevented PC-induced mitochondrial ROS production. In addition, PC increased type 2 ryanodine receptor (RyR2) oxidation, S-nitrosylation and dissociation of FKBP12.6 from RyR2, and therefore increased sarcoplasmic reticulum (SR) Ca 2+ leak. ANT inhibition or anti-oxidant strategy (N-acetylcysteine) prevented SR Ca 2+ leak, FKBP12.6 depletion and RyR2 oxidation/S-nitrosylation induced by PC. Finally, both bongkrekic acid and NAC significantly reduced spontaneous Ca 2+ wave occurrences under PC. Altogether, these results suggest that an elevation of PC disturbs ANT activity and alters Ca 2+ handling in a ROS-dependent pathway, demonstrating a new pathway whereby altered FA metabolism may contribute to the development of ventricular arrhythmia in pathophysiological conditions.

Torsade de Pointes (TdP) is a well-described major risk associated with various kinds of drugs. However, prediction of this risk is still uncertain both in preclinical and clinical trials. We tested 45 reference compounds on the model of isolated canine Purkinje fibres. Of them, 22 are clearly associated and/or labelled with a risk of TdP, and 13 others are drugs with published clinical evidence of QT prolongation, with only one or two exceptional cases of TdP. The 10 remaining drugs are without reports of TdP and QT prolongation. The relevance of different indicators such as APD(90) increase, reverse use dependency, action potential triangulation or effect on V(max) was evaluated by comparison with available clinical data. Finally, a complex algorithm called TDPscreen and based on two subalgorithms corresponding to particular electrophysiological patterns was defined. This latter algorithm enabled a clear separation of drugs into three groups: (A) drugs with numerous or several reports (>2 cases) of TdP, (B) drugs causing QT prolongation and/or TdP only, the latter at a very low frequency (< or =2 cases), (C) drugs without reports of TdP or QT prolongation. The use of such an algorithm combined with a database accrued from reference compounds with available clinical data is suggested as a basis for testing new candidate drugs in the early stages of development for proarrhythmic risk prediction.

Several studies suggested that potassium channels are involved in the proliferation of cancer cells but the involvement of the large conductance Ca2+-activated K+ channels (BKCa) in the cancerous phenomenon is still controversial. In the present study, we used iberiotoxin, a specific blocker of BKCa, and report the activity of an iberiotoxin-sensitive current in various human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468 and MDA-MB-435s) as well as in normal mammary epithelial cells (HME).Iberiotoxin and NS1619, an activator of BKCa, did not interfere with either cell proliferation or with the invasive properties of the cells, under normal culture conditions. However, extracellular pulses of ATP, which induced transient increases in intracellular Ca2+ concentration, revealed a significant reduction effect of iberiotoxin on cell proliferation.We conclude that the iberiotoxin-sensitive current is not involved in cell proliferation in basal conditions but participates when the intracellular Ca2+ concentration is increased. These experiments also suggest that BKCa channels are not involved in the cancerous transformation and are probably a relic from normal cells.

A voltage-dependent sodium current has been described in the highly invasive breast cancer cell line MDA-MB-231. Its activity is associated with the invasive properties of the cells. The aim of our study was to test whether this current (INa) is sensitive to three representative calcium channel blockers: verapamil, diltiazem and nifedipine. INa was studied in patch-clamp conditions. INa was sensitive to verapamil (IC50=37.6±2.5 μM) and diltiazem (53.2±3.6 μM), while it was weakly sensitive to nifedipine. The tetrodotoxin (TTX) concentration, which fully blocks INa (30 μM), did not affect cell proliferation. Diltiazem and verapamil, at concentrations that do not fully block INa, strongly reduced cell proliferation, suggesting, regarding proliferation, that these molecules act on targets distinct from sodium channels. These targets are probably not other ionic channels, since the current measured at the end of a 500 ms long pulse in the voltage range between −60 and +40 mV was unaffected by verapamil and diltiazem. We conclude that the sodium channel expressed in MDA-MB-231 cells is sensitive to several calcium channel blockers. The present study also underlines the danger of concluding to the possible involvement of membrane channel proteins in any phenomenon on the sole basis of pharmacology, and without an electrophysiological confirmation. Keywords: Breast cancer, invasivity, MDA-MB-231, electrophysiology, calcium channel blockers Introduction Among women, breast cancer is the commonest cancer and the first cause of death. Death occurs primarily after the development of metastasis. One of the problems engendered by chemotherapy is the occurrence of a multidrug resistance phenotype (MDR), which impairs the efficiency of the treatment (Moscow & Cowan, 1988). MDR is mainly associated to the presence of the drug carrier called P-glycoprotein (or P-gp) in the plasmalemma, which extrudes drugs from the cytoplasm (Kartner et al., 1983). Interestingly, this P-gp is blocked by molecules such as verapamil and diltiazem (Cornwell et al., 1987). Thus, in some drug-resistant metastatic cancers, the efficacy of chemotherapy was improved when verapamil was given in association with the usual drugs combination (Timcheva & Todorov, 1996; Belpomme et al., 2000). In breast cancer, the role of ionic channels has been known since the work of Marino et al. (1994). Since this pioneer work, the role of ionic channels in breast cancer cell lines focused mainly on potassium channels (Strobl et al., 1995; Wonderlin et al., 1995; Woodfork et al., 1995; Ouadid-Ahidouch et al., 2000, 2001). The expression and the role of these channels in cell proliferation have been studied, and at least four types of potassium channels exist in the MCF-7 cell line (Wegman et al., 1991; Woodfork et al., 1995; Klimatcheva & Wonderlin, 1999; Ouadid-Ahidouch et al., 2001). The presence of a sodium channel protein has been observed in biopsies of tumours, which developed lymph node metastasis, and in a highly metastatic breast cancer cell line, MDA-MB-231 (Fraser et al., 2002). The current described in the MDA-MB-231 cells is involved in the invasiveness process (Roger et al., 2003). The sodium channel protein found in this cell line is the main cardiac isoform NaV1.5 (Fraser et al., 2002). In cardiac preparations, verapamil and D600 (two phenylalkylamine analogues) have been shown to block INa (McLean et al., 1974; Galper & Catterall, 1978, 1979). In the present study, we investigated if the fast inward sodium current found in MDA-MB-231 cells was sensitive to L-type calcium channel blockers.

Cardioprotective effects of long-chain polyunsaturated fatty acids of the n-3 series (PUFAs) have been demonstrated and represent a novel approach to prevent myocardial infarctions or its consequences. Due to the abundance of double bonds, the main n-3 PUFAs; docosahexaenoic acid (C22: 6 n-3, DHA) are very sensitive to free radical oxidation and can undergo non-enzymatic spontaneous peroxidation under oxidative stress conditions as it occurs in ischemia/reperfusion. In this context, a lot of oxygenated metabolites of PUFAs like Neuroprostanes (NeuroPs) are produced and used as oxidative stress biomarkers but their activities were not determined. We investigated if the pericardial delivery of NeuroPs, protects the myocardium from ischemic damages during and following an ischemia/reperfusion (IR) episode in rats. Cardiac functions, infarct size and arrhythmias were studied and we observed that NeuroPs afford some cardioprotective effect during or after myocardial infarction. Indeed, compared with controls, NeuroPs-treated animals have significantly decreased infarct size (–28%) determined at the end of reperfusion and reduced ventricular arrhythmia score during reperfusion (–38%). Mechanistically, NeuroPs regulates calcium levels by stabilizing RyR2 activity (Roy et al., 2015), which can explain arrhythmias prevention during IR. Also, our results demonstrated an increase of membrane potential (⊗¬m) by the application of NeuroPs. This effect was not due to an augmentation of mitochondrial respiratory chain activity but by the effect leading to the diminution of protons leak. Swelling in response to Ca2+ was prevented by NeuroP, indicating a decrease MPTP opening, which can be explain prevention of cell death during IR. These results suggest a novel pharmacological pathway of n-3 PUFAs and suggest that their well-known cardioprotective effects are mediated by their oxygenated metabolites such as NeuroPs. The author hereby declares no conflict of interest

Voltage-gated ionic channels are known to be involved in oncogenesis. However, only a few studies describe the functional characteristics of these channels or the mechanisms by which they are involved in the proliferation and invasive processes. Breast cancer cells proliferate and migrate under the constant activation of growth factors, hormones, extracellular matrix interactions, etc. It would thus not be surprising if the activity of the ionic channels was modulated by intracellular regulation pathways such as kinases or phosphatases, which in turn can affect oncogenic properties. In the present study, we investigated some of the electrophysiological properties of the fast inward sodium current found in the breast cancer cell line MDA-MB-231 with two configurations of the patch-clamp technique. With perforated patch, a configuration which allows to keep the cytoplam intact, the mean current amplitude was lower, the relative conductance-voltage relationship was shifted to more positive potentials and the recovery from inactivation was accelerated when compared to ruptured patch, where the cytoplasm is dialysed by the intrapipette solution. There was no difference in availability-voltage (pseudo-steady-state inactivation) relationship and in time to peak of the current. These results suggest that regulation mechanisms, possibly involving kinases or phosphatases, are switched off when the cytoplasm is diluted. We propose that such a regulation can modulate the functioning of the channels even in the absence of membrane voltage changes, which in turn can affect oncogenic properties. This finding is of importance when evaluating the physiopathological role of ionic channel in cancer development.

The laboratory exercise described in this article used a simple preparation and a straightforward protocol to illustrate how the neurotransmitter norepinephrine (NE) induces an increase of tension in an artery. This was a practical class designed for undergraduate students of the University of Tours. The students performed several protocols to understand how NE acts to contract aortic ring vessels, which sources of calcium are mobilized, and whether the calcium sensitivity of the contractile regulatory apparatus is involved. The design of this exercise allowed students to participate actively in an exercise demonstrating that many mechanisms are involved and act additively to allow arterial tone to develop. Furthermore, the students were introduced to an isolated organ chamber technique that is used to study cellular mechanisms of many tissues and that is still important for smooth muscle research.

Young tomato plants (Lycopersicon esculentum, 8 days old) were given a heat-wound to a cotyledon. The resulting electrical activity at the hypocotyl level was monitored with intracellular microelectrodes. We observed an original pattern of slow wave potentials (SWPs), consisting of 2-3 slow waves, with associated spikes. The electrophysiological study of the SWPs confirms previous conclusions that the SWPs are due to the inhibition of an active component of the membrane potential. The electrophysiological study of the spikes shows that they fit particularities of putative action potentials (APs). They seem to be triggered by the depolarization accompanying the SWPs and thus can appear late during the SWP. An ionic characterization of the spikes by using different extracellular ionic concentrations and channel blockers suggests that anionic channels might be involved, carrying SO 4 2- ions. The channels activity might be down regulated by the calcium released by the vacuole during the SWPs and APs. A better characterization of the nature of these APs could permit the understanding of the information transmission mechanisms in higher plants.

International audience; Sonic hedgehog (SHH) is a conserved protein involved in embryonic tissue patterning and development. SHH signaling has been reported as a cardio-protective pathway via muscle repair-associated angiogenesis. The goal of this study was to investigate the role of SHH signaling pathway in the adult myocardium in physiological situation and after ischemia-reperfusion. We show in a rat model of ischemia-reperfusion that stimulation of SHH pathway, either by a recombinant peptide or shed membranes microparticles harboring SHH ligand, prior to reperfusion reduces both infarct size and subsequent arrhythmias by preventing ventricular repolarization abnormalities. We further demonstrate in healthy animals a reduction of QTc interval mediated by NO/cGMP pathway leading to the shortening of ventricular cardiomyocytes action potential duration due to the activation of an inward rectifying potassium current sharing pharmacological and electrophysiological properties with ATP-dependent potassium current. Besides its effect on both angiogenesis and endothelial dysfunction we demonstrate here a novel cardio-protective effect of SHH acting directly on the cardiomyocytes. This emphasizes the pleotropic effect of SHH pathway as a potential cardiac therapeutic target.

Aims TRPM2 is a Ca(2+)-permeable cationic channel of the transient receptor potential (TRP) superfamily that is linked to apoptotic signalling. Its involvement in cardiac pathophysiology is unknown. The aim of this study was to determine whether the pro-apoptotic cytokine tumour necrosis factor-α (TNF-α) induces a TRPM2-like current in murine ventricular cardiomyocytes. Methods and results Adult isolated cardiomyocytes from C57BL/6 mice were exposed to TNF-α (10 ng/mL). Western blotting showed TRPM2 expression, which was not changed after TNF-α incubation. Using patch clamp in whole-cell configuration, a non-specific cation current was recorded after exposure to TNF-α (ITNF), which reached maximal steady-state amplitude after 3 h incubation. ITNF was inhibited by the caspase-8 inhibitor z-IETD-fmk, the antioxidant N-acetylcysteine, and the TRPM2 inhibitors clotrimazole, N-(P-amylcinnamoyl) anthranilic acid and flufenamic acid (FFA). TRPM2 has previously been shown to be activated by ADP-ribose, which is produced by poly(ADP-ribose) polymerase 1 (PARP-1). TNF-α exposure resulted in increased poly-ADP-ribosylation of proteins and the PARP-1 inhibitor 3-aminobenzamide inhibited ITNF. TNF-α exposure increased the mitochondrial production of reactive oxygen species (ROS; measured with the fluorescent indicator MitoSOX Red), and this increase was blocked by the caspase-8 inhibitor z-IETD-fmk. Clotrimazole and TRPM2 inhibitory antibody decreased TNF-α-induced cardiomyocyte death. Conclusion These results demonstrate that TNF-α induces a TRPM2 current in adult ventricular cardiomyocytes. TNF-α induces caspase-8 activation leading to ROS production, PARP-1 activation, and ADP-ribose production. TNF-induced TRPM2 activation may contribute to cardiomyocyte cell death.

International audience; Cyclic oxygenated metabolites are formed in vivo through non-enzymatic free radical reaction of n-6 and n-3 polyunsaturated fatty acids (PUFAs) such as arachidonic (ARA C20:4 n-6), adrenic (AdA 22:4 n-6), α-linolenic (ALA 18:3 n-3), eicosapentaenoic (EPA 20:5 n-3) and docosahexaenoic (DHA 22:6 n-3) acids. These cyclic compounds are known as isoprostanes, neuroprostanes, dihomo-isoprostanes and phytoprostanes. Evidence has emerged for their use as biomarkers of oxidative stress and, more recently, the n-3PUFA-derived compounds have been shown to mediate bioactivities as secondary messengers. Accordingly, this review will focus on the cyclic oxygenated metabolites generated from AdA, ALA, EPA and DHA. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance"

Aims Clinical studies failed to prove convincingly efficiency of intravenous infusion of neseritide during heart failure and evidence suggested a pro-adrenergic action of B-type natriuretic peptide (BNP). However, subcutaneous BNP therapy was recently proposed in heart failure, thus raising new perspectives over what was considered as a promising treatment. We tested the efficiency of a combination of oral β1-adrenergic receptor blocker metoprolol and subcutaneous BNP infusion in decompensated heart failure. Methods and results The effects of metoprolol or/and BNP were studied on cardiac remodelling, excitation–contraction coupling and arrhythmias in an experimental mouse model of ischaemic heart failure following postmyocardial infarction. We determined the cellular and molecular mechanisms involved in anti-remodelling and antiarrhythmic actions. As major findings, the combination was more effective than metoprolol alone in reversing cardiac remodelling and preventing ventricular arrhythmia. The association of the two molecules improved cardiac function, reduced hypertrophy and fibrosis, and corrected the heart rate, sympatho-vagal balance (low frequencies/high frequencies) and ECG parameters (P to R wave interval (PR), QRS duration, QTc intervals). It also improved altered Ca 2+ cycling by normalising Ca 2+ -handling protein levels (S100A1, SERCA2a, RyR2), and prevented pro-arrhythmogenic Ca 2+ waves derived from abnormal Ca 2+ sparks in ventricular cardiomyocytes. Altogether these effects accounted for decreased occurrence of ventricular arrhythmias. Conclusions Association of subcutaneous BNP and oral metoprolol appeared to be more effective than metoprolol alone. Breaking the deleterious loop linking BNP and sympathetic overdrive in heart failure could unmask the efficiency of BNP against deleterious damages in heart failure and bring a new potential approach against lethal arrhythmia during heart failure.

The present invention relates to methods and pharmaceutical compositions for cardioprotection of subjects who experienced a myocardial infarction. In particular, the present invention relates to a ligand of the sonic hedgehog signaling pathway for use in the cardioprotection of a subject who experienced a myocardial infarction.

C. Pascarel, F. Brette and J.-Y. Le Guennec. Enhancement of the T-type Calcium Current by Hyposmotic Shock in Isolated Guinea-pig Ventricular Myocytes. Journal of Molecular and Cellular Cardiology (2001) 33 , 0000–0000. It is known that swelling and shrinkage of cardiac cells can modulate their electrical activity. However, the effects of osmotic manipulation on cardiac T-type calcium current (I CaT ) has not been previously reported. In this study, we have examined the effects of cell swelling on I CaT , using the whole cell patch clamp configuration. Isolated guinea-pig ventricular myocytes were swollen by an external hypotonic challenge (0.7 T). We found that I CaT is enhanced during a hypotonic shock. This current has been determined to be the T type calcium current since it is rapidly activated and inactivated, its threshold was at negative potentials and was blocked by 40 μ m Ni 2+ . Disruption of microfilaments by cytochalasin D and of microtubules by colchicine prevented the activation of I CaT during cell swelling. Taxol had no effect. These results indicate that I CaT is increased during cell swelling and this effect needs an intact cytoskeleton.

International audience; We studied active and passive properties of intact isolated guinea-pig ventricular myocytes in auxotonic conditions. Cells were attached using carbon fibres. The passive properties of the myocytes, in the presence of the stretch-activated channel blocker streptomycin sulphate, could be separated into two groups: stiff cells (stiffness slope=2.88±0.93 nN/m 3 , n=63 cells) and compliant cells (stiffness slope=0.91±0.35 nN/m 3 , n=52 cells). The study and the localization of the different kind of cells indicated that endocardium is mainly constituted of stiff cells (80%) while the epicardium contained more compliant cells (60%). When a longitudinal strain was applied to compliant cells, an increase in resting tension, diastolic sarcomere length and active tension were observed. On the other hand, in stiff cells, it induced an increase in resting tension and active tension with little change of diastolic sarcomere length. In both kinds of cells, strain had no effect on Ca 2+ transient amplitude and shape. Plotting active tension v diastolic sarcomere length also clearly showed two separated populations of cells, corresponding to stiff and compliant cells. The results of the two groups of cells when plotting active tension v resting tension could not be distinguished. We conclude that resting tension is an important factor in the modulation of active tension by stretch in addition to interfilament lattice spacing or sarcomere length.

Ca(2+) signaling plays an important role in the function of dendritic cells (DC), the professional antigen presenting cells. Here, we described the role of Calcium released activated (CRAC) channels in the maturation and cytokine secretion of human DC. Recent works identified STIM1 and Orai1 in human T lymphocytes as essential for CRAC channel activation. We investigated Ca(2+) signaling in human DC maturation by imaging intracellular calcium signaling and pharmalogical inhibitors. The DC response to inflammatory mediators or PAMPs (Pathogen-associated molecular patterns) is due to a depletion of intracellular Ca(2+) stores that results in a store-operated Ca(2+) entry (SOCE). This Ca(2+) influx was inhibited by 2-APB and exhibited a Ca(2+)permeability similar to the CRAC (Calcium-Released Activated Calcium), found in T lymphocytes. Depending on the PAMPs used, SOCE profiles and amplitudes appeared different, suggesting the involvement of different CRAC channels. Using siRNAi, we identified the STIM1 and Orai1 protein complex as one of the main pathways for Ca(2+) entry for LPS- and TNF-α-induced maturation in DC. Cytokine secretions also seemed to be SOCE-dependent with profile differences depending on the maturating agents since IL-12 and IL10 secretions appeared highly sensitive to 2-APB whereas IFN-γ was less affected. Altogether, these results clearly demonstrate that human DC maturation and cytokine secretions depend on SOCE signaling involving STIM1 and Orai1 proteins.

Objectives: The aim of the study was to investigate the mechanisms responsible for provoking and maintaining a large, stretch-induced, increase in the level of resting calcium in single guinea-pig ventricular myocytes. In particular, we wished to test the relative importance of intracellular and extracellular sources of calcium in this phenomenon. Methods: Carbon fibres were used to stretch cells loaded with the fluorescent calcium indicator Indo-1. Sarcomere length and internal calcium activity ([Ca2+]i) were measured. Experimental results from our present and previous studies were compared with those predicted by the OXSOFT HEART (version 4) model of the guinea-pig ventricular myocyte incorporating a stretch-activated channel. Results: The stretch-induced increase in [Ca2+]i was found to be sensitive to removal of [Ca2+] and application of the Ca2+-channel blocker verapamil (1 μM). The phenomenon was not sensitive to disruption of sarcoplasmic reticulum function by ryanodine (1 μM) nor to the Na+ channel blocker TTX (30 μM). Our experimental findings were reproduced in the modelling study. Conclusions: The stretch-induced increase in [Ca2+]i is modulated by extracellular sources of Ca2+ rather than intracellular Ca2+ stores and is not indiscriminately sensitive to blockers of depolarizing current. We propose that the stretch-induced increase in [Ca2+]i may be triggered by activation of stretch-activated channels but that a combination of stretch-activated current and Ca2+-window current maintain the increased levels of resting [Ca2+]i.

International audience

International audience; BACKGROUND AND PURPOSE: In some pathological conditions carnitine concentration is high while in others it is low. In both cases,cardiac arrhythmias can occur and lead to sudden cardiac death. It has been proposed that in ischaemia, acylcarnitine (acyl-CAR), but not carnitine, is involved in arrhythmias through modulation of ionic currents. We studied the effects of acyl-CARs on hERG, K(IR)2.1 and K(v)7.1/minK channels (channels responsible for I(KR), I(K1) and I(KS) respectively). EXPERIMENTAL APPROACH: HEK293 cells stably expressing hERG, K(IR)2.1 or Kv7.1/minK were studied using the patch clamp technique. Free carnitine (CAR) and acyl-CAR derivatives from medium- (C8 and C10) and long-chain (C16 and C18:1) fatty acids were applied intra- and extracellularly at different concentrations. For studies on hERG, C16 and C18:1 free fatty acid were also used. KEY RESULTS: Extracellular long-chain (LCAC), but not medium-chain, acyl-CAR,induced an increase of I(hERG) amplitude associated with a dose-dependent speeding of deactivation kinetics. They had no effect on K(IR)2.1 or Kv7.1/minK currents.Computer simulations of these effects were consistent with changes in action potential profile. CONCLUSIONS AND APPLICATIONS: Extracellular LCAC tonically regulates I(hERG) amplitude and kinetics under physiological conditions. This modulation may contribute to the changes in action potential duration that precede cardiac arrhythmias in ischaemia, diabetes and primary systemic carnitine deficiency.