Charlotte Farah

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.

Carnitine deficiency induces a short QT syndrome

BACKGROUNDnShort 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.nnnOBJECTIVEnThe purpose of this study was to confirm a relationship between a short QT syndrome and carnitine deficiency.nnnMETHODSnWe 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.nnnRESULTSnMET88-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.nnnCONCLUSIONnAltogether, these results suggest that long-chain fatty acid metabolism influence the morphology and the electrical function of the heart.

Subendocardial Increase in Reactive Oxygen Species Production Affects Regional Contractile Function in Ischemic Heart Failure

AIMSnHeart failure (HF) is characterized by regionalized contractile alterations resulting in loss of the transmural contractile gradient across the left ventricular free wall. We tested whether a regional alteration in mitochondrial oxidative metabolism during HF could affect myofilament function through protein kinase A (PKA) signaling.nnnRESULTSnTwelve weeks after permanent left coronary artery ligation that induced myocardial infarction (MI), subendocardial (Endo) cardiomyocytes had decreased activity of complex I and IV of the mitochondrial electron transport chain and produced twice more superoxide anions than sham Endo and subepicardial cells. This effect was associated with a reduced antioxidant activity of superoxide dismutase and Catalase only in MI Endo cells. The myofilament contractile properties (Ca(2+) sensitivity and maximal tension), evaluated in skinned cardiomyocytes, were also reduced only in MI Endo myocytes. Conversely, in MI rats treated with the antioxidant N-acetylcysteine (NAC) for 4 weeks, the generation of superoxide anions in Endo cardiomyocytes was normalized and the contractile properties of skinned cardiomyocytes restored. This effect was accompanied by improved in vivo contractility. The beneficial effects of NAC were mediated, at least, in part, through reduction of the PKA activity, which was higher in MI myofilaments, particularly, the PKA-mediated hyperphosphorylation of cardiac Troponin I.nnnINNOVATIONnThe Transmural gradient in the mitochondrial content/activity is lost during HF and mediates reactive oxygen species-dependent contractile dysfunction.nnnCONCLUSIONSnRegionalized alterations in redox signaling affect the contractile machinery of sub-Endo myocytes through a PKA-dependent pathway that contributes to the loss of the transmural contractile gradient and impairs global contractility.

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.

Key role of endothelium in the eNOS-dependent cardioprotection with exercise training

Modulation of endothelial nitric oxide synthase (eNOS) activation is recognized as a main trigger of the cardioprotective effects of exercise training on heart vulnerability to ischemia-reperfusion (IR). However, this enzyme is expressed both in coronary endothelial cells and cardiomyocytes and the contribution of each one to such cardioprotection has never been challenged. The aim of this study was to investigate the role of eNOS from the cardiomyocytes vs. the endothelium in the exercise cardioprotection. Male Wistar rats were assigned to a chronic aerobic training (Ex) (vs. sedentary group; Sed) and we investigated the role of eNOS in the effects of exercise on sensitivity to IR or anoxia-reoxygenation (A/R) at whole heart, isolated cardiomyocytes and left coronary artery (LCA) levels. We observed that exercise increased eNOS activation (Ser1177 phosphorylation) and protein S-nitrosylation in whole heart but not at cardiomyocyte level, suggesting the specific target of endothelial cells by exercise. Consistently, in isolated cardiomyocytes submitted to the A/R procedure, exercise reduced cell death and improved cells contractility, but independently of the eNOS pathway. Next, to evaluate the contribution of endothelial cells in exercise cardioprotection, LCA were isolated before and after an IR procedure performed on Langendorff hearts. Exercise improved basal relaxation sensitivity to acetylcholine and markedly reduced the alteration of endothelium-dependent coronary relaxation induced by IR. Furthermore, inactivation of coronary endothelial cells activity just before IR, obtained with a bolus of Triton X-100, totally suppressed cardioprotective effects of exercise on both left ventricular functional recovery after IR and infarct size, whereas no effect of Triton X-100 was observed in Sed group. In conclusion, these results show that coronary endothelial cells rather than cardiomyocytes play a key role in the eNOS-dependent cardioprotection of exercise.

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 (2.0u2009mg/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.

NO Better Way to Protect the Heart during Ischemia–Reperfusion: To be in the Right Place at the Right Time

Acute myocardial infarction (MI) is one of the leading causes of mortality worldwide. MI is the heart muscle irreversible death secondary to prolonged ischemia. Over the last few decades, medical progress in how and when to restore blood flow to the ischemic area have markedly improved patient survival. Although early heart reperfusion is acknowledged to be the most effective way to limit infarct size, post-ischemic reperfusion is associated with detrimental effects, such as myocardial stunning, ventricular arrhythmias, microvascular dysfunction, and cell death. The molecular mechanisms of these reperfusion injuries remain to be elucidated and their management is very challenging. n nAmong the various therapeutic molecular approaches proposed by experimental studies, nitric oxide (NO) role in protecting heart against MI and reperfusion injuries has been widely assessed and discussed (1–4). NO is a gasotransmitter that is abundantly produced in the cardiovascular system mainly by the NO synthase (NOS) enzymes system. Two isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS), are constitutively expressed in both myocardium and vessels, whereas inducible NOS (iNOS) is detected only in pathological conditions, such as inflammatory and/or oxidative stress. Both eNOS and nNOS are low-NO output Ca2+-dependent enzymes, while iNOS is a high-NO output Ca2+-independent enzyme. In physiological conditions, NOS form homodimers (“coupled” NOS) that catalyze NO production from l-arginine and O2 through electron transfer from NADPH on the reductase domain of one monomer to the oxidase domain of the second monomer. In pathological conditions, such as in the absence of the essential cofactor tetrahydrobiopterin (BH4), eNOS can be “uncoupled” to produce O2− instead of NO. n nIn stress conditions, NO protects tissues through two distinct pathways. In the first one, NO activates the soluble guanylate cyclase (sGC) that initiates cyclic guanosine monophosphate (cGMP) production, leading to the activation of protein kinase G (PKG). As sGC is the major cell receptor for NO and the NO/sGC/cGMP/PKG pathway plays a critical role in both myocardium excitation–contraction coupling and cardiovascular function regulation (5–8), NO cardioprotective role was first attributed to PKG activation (9–11). However, a second pathway in which proteins are directly modified by NO addition to sulfhydryl residues, a process known as S-nitrosylation (SNO), has recently emerged in the scientific literature. Although PKG activation pathway has been largely involved in NO-mediated cardioprotection (11–13), SNO is now taking the front stage and is considered to be a key player in cardioprotection through (i) the transient modification of protein activity and/or (ii) their protection from irreversible oxidation (14–17). Indeed, Sun et al. (18) showed that reduced heart vulnerability to ischemia–reperfusion (IR) following acute ischemic preconditioning is mainly related to SNO signaling and not to PKG activation through the NO–SGC–cGMP pathway. Accordingly, we found that in exercise training-induced cardioprotection against IR injuries, protein SNO level, but not cGMP level, increased during early reperfusion (19). The same year, Methner et al. (20), using a Cre/loxP approach to selectively ablate type I PKG in cardiomyocytes, demonstrated that ischemic post-conditioning reduced infarct size in these mice like in wild type controls. Moreover, they showed that the cardioprotective effect against IR injury of mitochondria-targeted S-nitrosothiol (MitoSNO), which allows NO and S-nitrosothiol accumulation in mitochondria, was comparable in mice that specifically lack PKG in cardiomyocytes and in controls. This indicates that MitoSNO cardioprotective effect is independent of PKG. n nThe current literature strongly supports NO implication in cardioprotection. However, the mechanism is still debated and whether increased NO availability during IR is cytoprotective remains to be demonstrated. Here, we discuss how NO might contribute to protect heart and particularly the importance of NO (i) localization, (ii) concentration, and (iii) time of availability during IR.

Loss of the transcription factor Meis1 prevents sympathetic neurons target-field innervation and increases susceptibility to sudden cardiac death

Although cardio-vascular incidents and sudden cardiac death (SCD) are among the leading causes of premature death in the general population, the origins remain unidentified in many cases. Genome-wide association studies have identified Meis1 as a risk factor for SCD. We report that Meis1 inactivation in the mouse neural crest leads to an altered sympatho-vagal regulation of cardiac rhythmicity in adults characterized by a chronotropic incompetence and cardiac conduction defects, thus increasing the susceptibility to SCD. We demonstrated that Meis1 is a major regulator of sympathetic target-field innervation and that Meis1 deficient sympathetic neurons die by apoptosis from early embryonic stages to perinatal stages. In addition, we showed that Meis1 regulates the transcription of key molecules necessary for the endosomal machinery. Accordingly, the traffic of Rab5+ endosomes is severely altered in Meis1-inactivated sympathetic neurons. These results suggest that Meis1 interacts with various trophic factors signaling pathways during postmitotic neurons differentiation. DOI: http://dx.doi.org/10.7554/eLife.11627.001

MEIS1 variant as a determinant of autonomic imbalance in Restless Legs Syndrome

Restless Legs Syndrome (RLS) is a genetically complex neurological disorder in which overlapping genetic risk factors may contribute to the diversity and heterogeneity of the symptoms. The main goal of the study was to investigate, through analysis of heart rate variability (HRV), whether in RLS patients the MEIS1 polymorphism at risk influences the sympathovagal regulation in different sleep stages. Sixty-four RLS patients with periodic leg movement index above 15 per hour, and 38 controls underwent one night of video-polysomnographic recording. HRV in the frequency- and time- domains was analyzed during nighttime sleep. All RLS patients were genotyped, and homozygotes for rs2300478 in the MEIS1 locus were used for further analysis. Comparison of the sympathovagal pattern of RLS patients to control subjects did not show significant differences after adjustments for confounding factors in frequency-domain analyses, but showed an increased variability during N2 and N3 stages in time-domain analyses in RLS patients. Sorting of RLS patients according to MEIS1 polymorphism reconfirmed the association between MEIS1 and PLMS, and showed a significant increased sympathovagal balance during N3 stage in those homozygotes for the risk allele. RLS patients should be considered differently depending on MEIS1 genotype, some being potentially at risk for cardiovascular disorders.

Antagonism of Nav channels and α1-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine

Ranolazine is a recently developed drug used for the treatment of patients with chronic stable angina. It is a selective inhibitor of the persistent cardiac Na+ current (INa), and is known to reduce the Na+-dependent Ca2+ overload that occurs in cardiomyocytes during ischemia. Vascular effects of ranolazine, such as vasorelaxation,have been reported and may involve multiple pathways. As voltage-gated Na+ channels (Nav) present in arteries play a role in contraction, we hypothesized that ranolazine could target these channels. We studied the effects of ranolazine in vitro on cultured aortic smooth muscle cells (SMC) and ex vivo on rat aortas in conditions known to specifically activate or promote INa. We observed that in the presence of the Nav channel agonist veratridine, ranolazine inhibited INa and intracellular Ca2+ calcium increase in SMC, and arterial vasoconstriction. In arterial SMC, ranolazine inhibited the activity of tetrodotoxin-sensitive voltage-gated Nav channels and thus antagonized contraction promoted by low KCl depolarization. Furthermore, the vasorelaxant effects of ranolazine, also observed in human arteries and independent of the endothelium, involved antagonization of the α1-adrenergic receptor. Combined α1-adrenergic antagonization and inhibition of SMCs Nav channels could be involved in the vascular effects of ranolazine.