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BackgroundIn this study the effects of a new, highly selective sodium-calcium exchanger (NCX) inhibitor, ORM-10962 were investigated on cardiac NCX current, Ca2+ transients, cell shortening and in experimental arrhythmias. The level of selectivity of the novel inhibitor on several major transmembrane ion currents (L-type Ca2+ current, major repolarizing K+ currents, late Na+ current, Na+/K+ pump current) was also determined.MethodsIon currents in single dog ventricular cells (cardiac myocytes; CM), and action potentials in dog cardiac multicellular preparations were recorded utilizing the whole-cell patch clamp and standard microelectrode techniques, respectively. Ca2+ transients and cell shortening were measured in fluorescent dye loaded isolated dog myocytes. Antiarrhythmic effects of ORM-10962 were studied in anesthetized ouabain (10 μg/kg/min i.v.) pretreated guinea pigs and in ischemia-reperfusion models (I/R) of anesthetized coronary artery occluded rats and Langendorff perfused guinea pigs hearts.ResultsORM-10962 significantly reduced the inward/outward NCX currents with estimated EC50 values of 55/67 nM, respectively. The compound, even at a high concentration of 1 μM, did not modify significantly the magnitude of ICaL in CMs, neither had any apparent influence on the inward rectifier, transient outward, the rapid and slow components of the delayed rectifier potassium currents, the late and peak sodium and Na+/K+ pump currents. NCX inhibition exerted moderate positive inotropic effect under normal condition, negative inotropy when reverse, and further positive inotropic effect when forward mode was facilitated. In dog Purkinje fibres 1 μM ORM-10962 decreased the amplitude of digoxin induced delayed afterdepolarizations (DADs). Pre-treatment with 0.3 mg/kg ORM-10962 (i.v.) 10 min before starting ouabain infusion significantly delayed the development and recurrence of ventricular extrasystoles (by about 50%) or ventricular tachycardia (by about 30%) in anesthetized guinea pigs. On the contrary, ORM-10962 pre-treatment had no apparent influence on the time of onset or the severity of I/R induced arrhythmias in anesthetized rats and in Langendorff perfused guinea-pig hearts.ConclusionsThe present study provides strong evidence for a high efficacy and selectivity of the NCX-inhibitory effect of ORM-10962. Selective NCX inhibition can exert positive as well as negative inotropic effect depending on the actual operation mode of NCX. Selective NCX blockade may contribute to the prevention of DAD based arrhythmogenesis, in vivo, however, its effect on I/R induced arrhythmias is still uncertain.

Ring1 and Yy1 Binding Protein (Rybp) has been implicated in transcriptional regulation, apoptotic signaling and as a member of the polycomb repressive complex 1 has important function in regulating pluripotency and differentiation of embryonic stem cells. Earlier, we have proven that Rybp plays essential role in mouse embryonic and central nervous system development. This work identifies Rybp, as a critical regulator of heart development. Rybp is readily detectable in the developing mouse heart from day 8.5 of embryonic development.Prominent Rybp expression persists during all embryonic stages and Rybp marks differentiated cell types of the heart. By utilizing rybp null embryonic stem cells (ESCs) in an in vitro cardiac differentiation assay we found that rybp null ESCs do not form rhythmically beating cardiomyocytes. Gene expression profiles revealed a down-regulation of terminal cardiac and upregulation of germ line specific markers in the rybp null cardiomyocytes. Furthermore, transcriptome analysis uncovered a number of novel candidate target genes regulated by Rybp.Among these are several important in cardiac development and contractility such as Plagl1, Isl1, This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 2Tnnt2. Importantly, forced expression of rybp in rybp deficient ESCs by a lentiviral vector was able to rescue the mutant phenotype. Our data provides evidence for a previously unrecognized function of Rybp in heart development.and pointing out the importance of germ cell lineage gene silencing during somatic differentiation. IntroductionA complex network of transcription factors governs the temporal and spatial patterns of gene expression in the organs of the developing embryo proper. Transcription factors also have important roles in postnatal and adult life in maintaining the pattern of differentiated gene expression [1][2][3]. Ring1 and Yy1 binding protein (Rybp; or also known as Dedaf (Death Effector Domain Associated Factor), UniGene Mm.321633; MGI:1929059) is a repressor protein that is also a member of the mammalian polycomb repressive complex 1 (PRC1) [4]. Rybp first was described as binding partner for the polycomb group protein (PcG) Ring1A (Ring1; ortholog of Drosophila dRing/Sce) and also was shown to associate with Ring1B (Ring2/Rnf2; ortholog of Drosophila dRing/Sce) and M33 (Pc1; ortholog of Drosophila Pc), components of the PRC1 multiprotein complex [5]. Our previous work showed that Rybp is selectively upregulated in distinct structures and cell types of the developing eye and the central nervous system (CNS) and, may also play a role in the development of more mature neurons [6,7]. We have also shown that rybp is essential for the development of the mouse embryo proper and that homozygous null mouse embryos cannot develop further implantation (embryonic day 5.5 (E5.5)). Notably, in a subset of heterozygous animals and in rybp −/− ↔rybp +/+ chimeras, alt...

BackgroundUse of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects.MethodsTo address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies.ResultsAlthough both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1–50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca2+-cytotoxity ([RTX]>15 µM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes.ConclusionTRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials.

*These two authors contributed equally to this work. †These two senior authors contributed equally to this work. BACKGROUND AND PURPOSEThe reliable assessment of proarrhythmic risk of compounds under development remains an elusive goal. Current safety guidelines focus on the effects of blocking the KCNH2/HERG ion channel-in tissues and animals with intact repolarization. Novel models with better predictive value are needed that more closely reflect the conditions in patients with cardiac remodelling and reduced repolarization reserve. EXPERIMENTAL APPROACHWe have developed a model for the long QT syndrome type-5 in rabbits (LQT5 ) with cardiac-specific overexpression of a mutant (G52R) KCNE1 β-subunit of the channel that carries the slow delayed-rectifier K + -current (I Ks ). ECG parameters, including short-term variability of the QT interval (STV QT ), a biomarker for proarrhythmic risk, and arrhythmia development were recorded. In vivo, arrhythmia susceptibility was evaluated by i.v. administration of the I Kr blocker dofetilide. K + currents were measured with the patch-clamp technique. KEY RESULTSPatch-clamp studies in ventricular myocytes isolated from LQT5 rabbits revealed accelerated I Ks and I Kr deactivation kinetics. At baseline, LQT5 animals exhibited slightly but significantly prolonged heart-rate corrected QT index (QTi) and increased STV QT . Dofetilide provoked Torsade-de-Pointes arrhythmia in a greater proportion of LQT5 rabbits, paralleled by a further increase in STV QT . CONCLUSION AND IMPLICATIONSWe have created a novel transgenic LQT5 rabbit model with increased susceptibility to drug-induced arrhythmias that may represent a useful model for testing proarrhythmic potential and for investigations of the mechanisms underlying arrhythmias and sudden cardiac death due to repolarization disturbances. AbbreviationsHERG, human ether-a-go-go gene; I Ca,L , L-type Ca 2+ current; I K1 , inward rectifier potassium current; I Kr , rapid delayed rectifier potassium current; I Ks , slow delayed rectifier potassium current; I to , transient outward potassium current; KCNE1, potassium voltage-gated channel subfamily E member 1; LQT5, long QT syndrome type 5; minK, minimum sequence required for a potassium current; QTi, heart rate-corrected QT index; STV QT , short-term variability of the QT interval; STV RR , short-term variability of the RR interval; TdP, Torsade-de-Pointes; TG, transgenic; WT, wild type