Growing evidence implicates cardiac energetic metabolism in cardiac arrhythmogenesis. Here, we focused on the role of an alternative pathway of glycolysis, the hexosamines biosynthetic pathway (HBP). This pathway usually represents less than 5% of metabolic pathways, however it has been shown to be increased in some cardiomyopathies. HBP leads to the production of a glycosylation, consisting of the O-linked attachment of a single monosaccharide (N-acetyl-D-glucosamine, O-GlcNAc) to serine and threonine in nuclear and cytosolic proteins. This postranslational protein modification, like phosphorylation/dephosphorylation, may alter many processes, including protein activity & localisation. To determine the role of HBP in cardiac physiopathology. We studied the arrhythmogenesis of acute HBP overactivation by glucosamine administration (30 min) on healthy working rat hearts perfused ex vivo. We performed cellular electrophysiology on left rat ventricular cells using the patch clamp technique. We also measured intracellular calcium transients and contraction using epi-fluorescence (Fura-2) and sarcomere shortening (Ionoptix, USA). Glucosamine administration increases the probability of spontaneous arrhythmia events ex vivo, notably ventricular tachycardia, ventricular fibrillation and atrial fibrillation. Glucosamine perfusion in vitro significantly reduced action potential duration at 90% of repolarization. Glucosamine significantly increases the sustained repolarizing K+ current Isus with no significant modification of Ito or IK1. Furthermore, we observed a calcium transient modification by glucosamine perfusion, notably a significant increase of diastolic calcium and decrease of calcium transient amplitude. Theses observations indicate that HBP participates in the regulation of cardiac electrical properties. To conclude, these data suggest a potential implication of HBP over-activation during arrhythmias.

Superoxide anions have been associated with many aspects of cardiovascular disease. Menadione is a superoxide anion donor that alters the heart’s electrical and mechanical functions. The aim of this study was to demonstrate simultaneous changes in intracellular Ca 2+ ([Ca 2+ ]i) and mechanical activity in intact adult cardiac myocytes, and mechanical activity and electrical activity in isolated whole hearts in order to provide greater insight into the mechanisms associated with the detrimental effects of menadione on the myocardium. Isolated hearts from adult male Wistar rats (n = 11, 200–250 g) were Langendorff perfused at 38°C with a Krebs–Henseleit solution. A saline-filled balloon was placed in the left ventricle (LV) in order to measure diastolic and developed pressure. Monophasic action potentials were simultaneously recorded from the epicardial surface. External stimulation at 5 Hz and intrinsic pacing were used throughout a 10 min control period and 30 min exposure to 50 µM menadione. Single LV myocytes (n = 7 from n = 4 animals) were loaded with the Ca2+-indicator Fura4-AM, stimulated at 1 Hz and exposed to 50 µM menadione. Myocyte length was simultaneously measured with [Ca 2+ ]i using a video edge detection system. In isolated hearts, exposure to menadione significantly decreased contractility and action potential duration (with a simi lar time course); intrinsic heart rate and rhythmicity. Diastolic pressure was significantly increased. In single adult myocytes, menadione caused a significant increase in diastolic [Ca 2+ ]i and a decrease in resting cell length and led to spontaneous release of [Ca 2+ ]i. We conclude that the effects of menadione upon electrical and mechanical activity of the heart are at least in part a consequence of dysregulation of [Ca 2+ ]i handling and the subsequent increase in diastolic [Ca 2+ ] alterations in [Ca 2+ ]i are consistent with the generation of delayed after depolarization arrhythmias.

Monophasic action potentials (MAPs) were recorded from the spongy and compact layers of the yellowfin tuna Thunnus albacares ventricle as stimulation frequency was increased. MAP duration decreased with increase in stimulation frequency in both the spongy and compact myocardial layers, but no significant difference in MAP duration was observed between the layers.

Background and purpose:Current strategies to ameliorate cardiac ischaemic and reperfusion damage, including block of the sodium-hydrogen exchanger, are therapeutically ineffective. Here we propose a different approach, block of the persistent sodium current (INaP).Experimental approach:Left ventricular pressure was measured as an index of functional deficit in isolated, Langendorff perfused, hearts from adult rats, subjected to 30 min global ischaemia and reperfusion with vehicle only (control) or riluzole (1-10 mu M) in the perfusate. Cell shortening and intracellular Ca2+ concentrations [Ca2+](i) were measured in adult rat isolated myocytes subjected to hypoxia and re-oxygenation. The block of transient and persistent sodium currents by concentrations of riluzole between 0.01 and 100 mu M were assessed in rat isolated myocytes using patch clamp techniques.Key results:In perfused hearts, riluzole produced a concentration-dependent cardioprotective action, with minor protection from 1 mu M and produced rapid and almost complete recovery upon reperfusion from 3 and 10 mu M. In isolated myocytes, riluzole at 3 and 10 mu M greatly attenuated or prevented the hypoxia- and reperfusion-induced rise in [Ca2+](i) and the contractile deficit. In patch clamp experiments, riluzole blocked the persistent sodium current with an IC50 of 2.7 mu M, whereas the block of the transient sodium current was only apparent at concentrations above 30 mu M.Conclusions and implications:Riluzole preferentially blocked INaP and was protective in cardiac ischaemia and reperfusion. Thus block of the persistent sodium current would be a viable method of ameliorating cardiac ischaemic and reperfusion damage.

L'altération des propriétés contractiles cardiaques observées lors de l'hypertrophie ou l'insuffisance cardiaque (IC) est généralement attribuée à des anomalies de gestion du calcium intracellulaire (1). Si ces anomalies survenant lors de l'hypertrophie ou l'IC gauche ont fait l'objet de nombreux travaux, celles intervenant lors de l'IC droite n'ont été que très peu étudiées. L'objectif de cette étude était de déterminer si le cycle calcique était altéré lors de l'hypertrophie et de l'IC droite. Des rats mâles Wistar (200 g) ont reçu une injection intrapéritonéale de monocrotaline (MCT) à 30 mg/kg pour induire une hypertrophie ou 60 mg/kg pour induire une IC droite. Les animaux furent sacrifiés 3-4 semaines après injection et les cardiomyocytes du ventricule droit isolés enzymatiquement. Le raccourcissement cellulaire et les transitoires calciques ont été enregistrés dans les myocytes préalablement chargés avec l'indicateur calcique fura-4 AM. La caféine (20 mM) a été utilisée pour évaluer le contenu en calcium du réticulum sarcoplasmique. Les sparks calciques on été acquis par microscopie confocale avec l'indicateur calcique fluo-4 AM. Le raccourcissement cellulaire a significativement diminué lors de l'IC (−29 % vs. contrôle). L'amplitude des transitoires calciques a significativement augmenté au stade hypertrophique (+ 43 % vs. contrôle) et lors de l'IC (+ 56 % vs. contrôle). Le contenu en calcium du réticulum sarcoplasmique a également augmenté au stade hypertrophique (+ 20 % vs. contrôle) et au cours de l'IC (+ 61 % vs. contrôle). Les sparks calciques ont vu leur fréquence, leur durée et leur taille augmenter alors que leur intensité a progressivement baissé lors du développement de l'hypertrophie puis de l'IC. L'augmentation du contenu en calcium du réticulum sarcoplasmique au cours de ces stades pathologiques explique l'augmentation observée de l'amplitude des transitoires calciques mais également l'augmentation du nombre de sparks. Cependant, ces anomalies ne semblent pas être directement responsables de la diminution du raccourcissement cellulaire. Nous suggérons qu'une désensibilisation des myofilaments pour le calcium (2) permettrait d'expliquer cette altération de la contraction alors que l'amplitude des transitoires calciques est augmentée. (1) Hasenfuss, G. et Pieske, B. (2002). J. Mol. Cell. Cardiol. 34, 951-969. (2) Lamberts, R.R. et al. (2007). J. Physiol. 582, 695-709.

Taxol is widely used in breast cancer chemotherapy. Its effects are primarily attributed to its anti-mitotic activity. Microtubule perturbators also exert antimetastatic activities which cannot be explained solely by the inhibition of proliferation. Voltage-dependent sodium channels (NaV) are abnormally expressed in the highly metastatic breast cancer cell line MDA-MB-231 and not in MDA-MB-468 cell line. Inhibiting NaV activity with tetrodotoxin is responsible for an approximately 0.4-fold reduction of MDA-MB-231 cell invasiveness. In this study, we focused on the effect of a single, 2-h application of 10nM taxol on the two cell lines MDA-MB-231 and MDA-MB-468. At this concentration, taxol had no effect on proliferation after 7days and on migration in any cell line. However it led to a 40% reduction of transwell invasion of MDA-MB-231 cells. There was no additive effect when taxol and tetrodotoxin were simultaneously applied. NaV activity, as assessed by patch-clamp, indicates that it was changed by taxol pre-treatment. We conclude that taxol can exert anti-tumoral activities, in cells expressing NaV, at low doses that have no effect on cell proliferation. This effect might be due to a modulation of signalling pathways involving sodium channels.

Serotonin (5HT), one of the principal neuromodulators in the mammalian brain, is implicated in a variety of disorders such as pain, depression and schizophrenia. Many aspects of serotonergic transmission remain unknown, necessitating the development of new research tools. Previously, we successfully used viral vectors for cell-specific gene expression in the brain in order to selectively study or modulate the function of targeted neurones (Duale et al. 2007; Wang et al. 2006; Chiti & Teschemacher, 2007). Here we present novel lentiand adenoviral vectors suitable for selective gene expression in raphe 5HT neurones. For targeting we used partial sequences (length 3.6kb, 2kb, and 1kb) of the natural promoter of rat tryptophan hydroxylase 2 (TPH2), the rate limiting enzyme in 5HT synthesis, obtained by PCR from rat brain genomic DNA. Lentiviral vectors for expression of EGFP were prepared using standard protocols (Liu et al. 2008) and stereotaxically microinjected into the rat raphe nuclei (under a mixture of ketamine (60 mg/kg) and medetomidine (250 μg/kg) i.m. anaesthesia). Specificity was then determined by immunofluorescence using anti-GFP and anti-TPH2 antibodies. The 3.6kb and 2kb promoter sequences conferred specific expression (co-localisation >95%), while the specificity of the 1kb promoter was only ~78%. However, native promoters were weak, and expression could only be detected using anti-GFP antibodies. To overcome this limitation, we employed a previously established transcriptional amplification strategy which involves cell-specific coexpression of a potent chimeric transactivator (Liu et al. 2008; Liu et al. 2006). This strategy increased the potency of 3.6kb and 2kb TPH2 promoters, leading to visible EGFP expression, while maintaining 5HT neurone specificity at 99% (n= 700 cells). Adenoviral vectors based on the 3.6kb construct were generated which caused visible EGFP expression in 5HT neurones in organotypic brainstem slice cultures. Moreover, it was possible to visually identify EGFP-positive axons with multiple small varicosities. Using previously established methods (Chiti & Teschemacher, 2007), we made the first microamperometric recordings of quantal 5HT release, and the first patch clamp recordings from EGFP-expressing 5HT neurones of the rat raphe. We believe that these viral vectors have great potential for in vivo and in vitro studies into the function of central 5HT neurones. Chiti Z & Teschemacher AG (2007). FASEB Journal 21, 2540-2550.

The properties of left ventricular cardiac myocytes vary transmurally. This may be related to the gradients of stress and strain experienced in vivo across the ventricular wall. We tested the hypothesis that within the rat left ventricle there are transmural differences in the expression of genes for proteins that are involved in mechanosensitive pathways and in associated physiological responses. Real time reverse transcription polymerase chain reaction was used to measure messenger RNA (mRNA) levels of selected targets in sub-epicardial (EPI) and sub-endocardial (ENDO) myocardium. Carbon fibres were attached to single myocytes to stretch them and to record contractility. We observed that the slow positive inotropic response to stretch was not different between EPI and ENDO myocytes and consistent with this, that the mRNA expression of two proteins implicated in the slow response, non-specific cationic mechanosensitive channels (TRPC-1) and Na/H exchanger, were not different. However, mRNA levels of other targets, e.g. the mechanosensitive K+ channel TREK-1, Brain Natriuretic Peptide and Endothelin-1 receptor B, were significantly greater in ENDO than EPI. No targets had significantly greater mRNA levels in EPI than ENDO. On the basis of these findings, we suggest that the response of the ventricle to stretch will depend upon both the regional differences in stimuli and the relative expression of the mechanosensitive targets and that generally, stretch sensitivity is predicted to be greater in ENDO.