Christophe Pignier

Sarcoplasmic reticulum Ca2+ refilling controls recovery from Ca2+-induced Ca2+ release refractoriness in heart muscle

In cardiac muscle Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is initiated by Ca2+ influx via L-type Ca2+ channels. At present, the mechanisms underlying termination of SR Ca2+ release, which are required to ensure stable excitation-contraction coupling cycles, are not precisely known. However, the same mechanism leading to refractoriness of SR Ca2+ release could also be responsible for the termination of CICR. To examine the refractoriness of SR Ca2+ release, we analyzed Na+-Ca2+ exchange currents reflecting cytosolic Ca2+ signals induced by UV-laser flash-photolysis of caged Ca2+. Pairs of UV flashes were applied at various intervals to examine the time course of recovery from CICR refractoriness. In cardiomyocytes isolated from guinea-pigs and mice, &bgr;-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by ≈2-fold. Application of cyclopiazonic acid at moderate concentrations (<10 &mgr;mol/L) slowed down recovery from refractoriness in a dose-dependent manner. Compared with cells from wild-type littermates, those from phospholamban knockout (PLB-KO) mice exhibited almost 5-fold accelerated recovery from refractoriness. Our results suggest that SR Ca2+ refilling mediated by the SR Ca2+-pump corresponds to the rate-limiting step for recovery from CICR refractoriness. Thus, the Ca2+ sensitivity of CICR appears to be regulated by SR Ca2+ content, possibly resulting from a change in the steady-state Ca2+ sensitivity and in the gating kinetics of the SR Ca2+ release channels (ryanodine receptors). During Ca2+ release, the concomitant reduction in Ca2+ sensitivity of the ryanodine receptors might also underlie Ca2+ spark termination by deactivation.

Direct protective effects of poly-unsaturated fatty acids, DHA and EPA, against activation of cardiac late sodium current

Abstract Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic and eicosapentaenoic acids (DHA, EPA) exert ischemic anti-arrhythmic effects. However, their mechanism of action remains unknown. The present study was designed to investigate their potential effect on the regulation of the late sodium current as the basis for their ischemic anti-arrhythmic activity. Human isoforms of wild-type SCN5A and ΔKPQ-mutated cardiac sodium channels were stably transfected in HEK 293 cells and, the resulting currents were recorded using the patch clamp technique in whole cell configuration. In addition to their effect to inhibit peak INa, acute application of DHA and EPA blocked veratridine-induced late sodium current (late INa-Verat) in a concentration — dependent manner with IC50 values of 2.1 ± 0.5 μM and 5.2 ± 0.8 μM,for DHA and EPA, respectively.Channels availability was reduced, resulting in a significant leftward shift of the steadystate inactivation curve by ‒10.0 ± 2.1 mV and ‒8.5 ± 0.2 mV for DHA and EPA, respectively. Similar inhibitory effects of DHA and EPA were also observed on late INa-KPQ. In addition to their role as blocking agents of peak INa, DHA and EPA reduced human late INa. These results could explain the antiarrhythmic properties of DHA and EPA during ischemia or following ischemia-reperfusion.

Sodium late current blockers in ischemia reperfusion: is the bullet magic?

We describe the discovery of the first selective, potent, and voltage-dependent inhibitor of the late current mediated by the cardiac sodium channel Na V1.5. The compound 3,4-dihydro- N-[(2 S)-3-[(2-methoxyphenyl)thio]-2-methylpropyl]-2 H-(3 R)-1,5-benzoxathiepin-3-amine, 2a (F 15845), was identified from a novel family of 3-amino-1,5-benzoxathiepine derivatives. The late sodium current inhibition and antiischemic effects of 2a were studied in various models in vitro and in vivo. In a rabbit model of ischemia-reperfusion, 2a exhibited more potent antiischemic effects than reference compounds KC 12291, ranolazine, and ivabradine. Thus, after a single administration, 2a almost abolished ST segment elevation in response to a transient coronary occlusion. Further, the antiischemic activity of 2a is maintained over a wide range of doses and is not associated with any hemodynamic changes, contrary to conventional antiischemic agents. The unique pharmacological profile of 2a opens new and promising opportunities for the treatment of ischemic heart diseases.

Discovery of novel protease activated receptors 1 antagonists with potent antithrombotic activity in vivo.

Protease activated receptors (PARs) or thrombin receptors constitute a class of G-protein-coupled receptors (GPCRs) implicated in the activation of many physiological mechanisms. Thus, thrombin activates many cell types such as vascular smooth muscle cells, leukocytes, endothelial cells, and platelets via activation of these receptors. In humans, thrombin-induced platelet aggregation is mediated by one subtype of these receptors, termed PAR1. This article describes the discovery of new antagonists of these receptors and more specifically two compounds: 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)penta-1,3-dienyl]benzonitrile 36 (F 16618) and 3-(2-chlorophenyl)-1-[4-(4-fluorobenzyl)piperazin-1-yl]propenone 39 (F 16357), obtained after optimization. Both compounds are able to inhibit SFLLR-induced human platelet aggregation and display antithrombotic activity in an arteriovenous shunt model in the rat after iv or oral administration. Furthermore, these compounds are devoid of bleeding side effects often observed with other types of antiplatelet drugs, which constitutes a promising advantage for this new class of antithrombotic agents.

F 15845 inhibits persistent sodium current in the heart and prevents angina in animal models

Background and purpose:  Activation of the persistent sodium current in ischaemic myocardium results in calcium overload which is toxic for the cardiomyocyte. Thus, the activity of 3‐(R)‐[3‐(2‐methoxyphenylthio‐2‐(S)‐methylpropyl]amino‐3,4‐dihydro‐2H‐1,5 benzoxathiepine bromhydrate (F 15845), a new selective persistent sodium current blocker, in protecting against the effects of cardiac ischaemia was examined, in both in vitro and in vivo models.

Selective inhibition of persistent sodium current by F 15845 prevents ischaemia-induced arrhythmias

BACKGROUND AND PURPOSE Myocardial ischaemia is associated with perturbations of electrophysiological profile of cardiac myocytes. The persistent sodium current (INap) is one of the major contributors to ischaemic arrhythmias and appears as an attractive therapeutic target. We investigated the effects of F 15845, a new anti‐anginal drug on INap and in integrative models of INap‐induced arrhythmias.

Protease-Activated Receptor-1 Mediates Thrombin-Induced Persistent Sodium Current in Human Cardiomyocytes

After the thrombus formation in cardiac cavities or coronaries, the serine protease thrombin is produced and can therefore reach the myocardial tissue by the active process of extravasation and binds to the G protein-coupled protease-activated receptor-1 (PAR1) expressed in human myocardium. The role of PAR1 was investigated in the thrombin effect on sodium current (INa). INa was recorded in freshly isolated human atrial myocytes by the whole-cell patch-clamp method. Action potentials (AP) were recorded in guinea pig ventricular tissue by the conventional glass microelectrode technique. Thrombin-activated PAR1 induced a tetrodotoxin-blocked persistent sodium current, INaP, in a concentration-dependent manner with an apparent EC50 of 28 U/ml. The PAR1 agonist peptide SFLLR-NH2 (50 μM) was able to mimic PAR1-thrombin action, whereas PAR1 antagonists N3-cyclopropyl-7-((4-(1-methylethyl)-phenyl)methyl)-7H-pyrrolo(3,2-f)quinazoline-1,3-diamine (SCH 203099; 10 μM) and 1-(3,5-di-tert-butyl-4-hydroxy-phenyl)-2-[3-(3-ethyl-3-hydroxy-pentyl)-2-imino-2,3-dihydro-imidazol-1-yl]-ethanone (ER 112787) (1 μM), completely inhibited it. The activated PAR1 involves the calcium-independent phospholipase-A2 signaling pathway because two inhibitors of this cascade, bromoenol lactone (50 μM) and haloenol lactone suicide substrate (50 μM), block PAR1-thrombin-induced INaP.Asa consequence of INaP activation, in guinea pig right ventricle papillary muscle, action potential duration (APD) were significantly increased by 20% and 15% under the respective action of 32 U/ml thrombin and 50 μM SFLLR-NH2, and these increases in APD were prevented by 1 μM tetrodotoxin or markedly reduced by application of 1 μM SCH 203099 or ER 112787. Thrombin, through PAR1 activation, increases persistent component of the Na+ current resulting in an uncontrolled sodium influx into the cardiomyocyte, which can contribute to cellular injuries observed during cardiac ischemia.

Na+ Currents in Cardioprotection: Better to Be Late

We report the discovery of a selective, potent inhibitor of the late current mediated by the cardiac isoform of the sodium channel (Na(V)1.5). The compound, 3,4-dihydro-N-[(2S)-3-[(2-hydroxy-3-methylphenyl)thio]-2-methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2d) (F 15741), blocks the late component of the Na(+) currents and greatly reduces veratridine- or ischemia-induced contracture in isolated tissue and whole heart. The cardioprotective action of 2d was further established in a model of myocardial infarction in the pig in which 2d prevents ischemia-reperfusion damage after 60 min of coronary occlusion and 48 h reperfusion. Under these experimental conditions, only 2d and cariporide reduce infarct size. Remarkably, myocardial protection afforded by 2d occurs in the absence of hemodynamic effects. These data expand the therapeutic potential of late I(Na) blockers and suggest that 2d could be useful in pathologies for which pharmacological treatments are not yet available.

A novel steroid‐like compound F90927 exerting positive‐inotropic effects in cardiac muscle

Here we report a novel steroid‐like compound F90363, exhibiting positive inotropy in vivo and in vitro in various cardiac muscle preparations. F90363 is a racemic mixture composed of the stereoisomers (−)‐F90926 and (+)‐F90927. Only F90927 exerted positive inotropy, while F90926 induced a weak negative inotropy, but only at concentrations 103 times higher than F90927 and most likely resulting from an unspecific interaction. The rapid time course of the action of F90927 suggested a direct interaction with a cellular target rather than a genomic alteration. We could identify the L‐type Ca2+ current ICa(L) as a main target of F90927, while excluding other components of cardiac Ca2+ signalling as potential contributors. In addition, several other signaling pathways known to lead to positive inotropy (e.g. α‐ and β‐adrenergic stimulation, cAMP pathways) could be excluded as targets of F90927. However, vessel contraction and stiffening of the cardiac muscle at high doses (>30 μM, 0.36 mg kg−1, respectively) prevent the use of F90927 as a candidate for drug development. Since the compound may still find valuable applications in research, the aim of the present study was to identify the cellular target and the mechanism of inotropy of F90927.

Reexpression of the Nifedipine‐Resistant Calcium Channel During Dedifferentiation of Adult Rat Ventricular Cardiomyocytes

Nifedipine‐Resistant Calcium Current. Introduction: Using an adult rat ventricular culture model and whole‐cell patch‐clamp technique, we investigated whether the nifedipine‐resistant calcium current observed at the neonatal stage but not at the adult stage could be reobserved under dedifferentiating conditions.