Background and purpose: Ivabradine, a specific and use-dependent I f inhibitor, exerts anti-ischaemic activity purely by reducing heart rate. The aim of this work was to characterize its effect on the predominant HCN channel isoform expressed in human sino-atrial nodes (hSAN), to determine its kinetics in HCN channels from multicellular preparations and ratedependency of its action. Experimental approach: RT-PCR analysis of the four HCN channel isoforms was carried out on RNAs from hSAN. Patch-clamp and intracellular recordings were obtained from CHO cells stably expressing hHCN4 and isolated SAN, respectively. Beating rate of rat isolated atria was followed using a transducer. Key results: hHCN4 mRNAs were predominant in hSAN. Ivabradine induced a time-dependent inhibition of hHCN4 with an IC 50 of 0.5 mM. In rabbit SAN, ivabradine progressively reduced the frequency of action potentials: by 10% after 3 h at 0.1 mM, by 14% after 2 h at 0.3 mM and by 17% after 1.5 h at 1 mM. After 3h, ivabradine reduced the beating rate of rat right atria with an IC 30 of 0.2 mM. The onset of action of ivabradine was use-dependent rather than time-dependent with slower effects than caesium, an extracellular I f blocker. Ivabradine 3 mM decreased the frequency of action potentials in SAN from guinea-pig, rabbit and pig by 33%, 21% and 15% at 40 min, respectively. Conclusions and implications:The use-dependent inhibition of hHCN4 current by ivabradine probably contributes to its slow developing effect in isolated SAN and right atria and to its increased effectiveness in species with rapid SAN activity.

Bedut S, Seminatore-Nole C, Lamamy V, Caignard S, Boutin JA, Nosjean O, Stephan JP, Coge F. High-throughput drug profiling with voltage-and calcium-sensitive fluorescent probes in human iPSC-derived cardiomyocytes. Am J Physiol Heart Circ Physiol 311: H44 -H53, 2016. First published May 3, 2016 doi:10.1152/ajpheart.00793.2015.-Cardiomyocytes derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) are increasingly used for in vitro assays and represent an interesting opportunity to increase the data throughput for drug development. In this work, we describe a 96-well recording of synchronous electrical activities from spontaneously beating hiPSC-derived cardiomyocyte monolayers. The signal was obtained with a fast-imaging plate reader using a submillisecond-responding membrane potential recording assay, FluoVolt, based on a newly derived voltagesensitive fluorescent dye. In our conditions, the toxicity of the dye was moderate and compatible with episodic recordings for Ͼ3 h. We show that the waveforms recorded from a whole well or from a single cell-sized zone are equivalent and make available critical functional parameters that are usually accessible only with gold standard techniques like intracellular microelectrode recording. This approach allows accurate identification of the electrophysiological effects of reference drugs on the different phases of the cardiac action potential as follows: fast depolarization (lidocaine), early repolarization (nifedipine, Bay K8644, and veratridine), late repolarization (dofetilide), and diastolic slow depolarization (ivabradine). Furthermore, the data generated with the FluoVolt dye can be pertinently complemented with a calcium-sensitive dye for deeper characterization of the pharmacological responses. In a semiautomated plate reader, the two probes used simultaneously in 96-well plates provide an easy and powerful multiparametric assay to rapidly and precisely evaluate the cardiotropic profile of compounds for drug discovery or cardiac safety.

1 Exposure to docosahexaenoı¨c acid (DHA), a long-chain polyunsaturated fatty acid, is known to block several ionic currents such as the transient outward current I TO . It has also been reported to activate certain potassium channels. It has been suggested that these effects, observed in single-cell experiments, participate in the antiarrhythmic properties of these compounds in vivo. 2 DHA is highly prone to peroxidation. To investigate the influence peroxidation may have on the effects of DHA on ion channels, we studied I TO and the steady-state outward current I SS in isolated rat ventricular myocytes under ruptured whole-cell patch-clamp conditions. 3 A measure of 10 mm DHA alone reduced I TO , evoked by a pulse to þ 70 mV, by 74.8710.8% (n ¼ 7) and activated a delayed outward current with kinetic properties different from I SS . 4 When an antioxidant, alpha-tocopherol (1 mm), was added together with DHA, the blockade of I TO was reduced to 38.577.7% (n ¼ 8) and the delayed outward current was not activated. a-Tocopherol alone had no effect on these currents. 5 When an oxidant, hydrogen peroxide (1 mm), was applied together with DHA, the blockade of I TO was almost complete (98.471.0%, n ¼ 7) and a large delayed outward current was activated. A measure of 1 mm hydrogen peroxide alone had no effect on these currents. 6 Measurements of nonperoxidized DHA in experimental solutions confirmed the negative relation between DHA concentration and the effects on the currents. 7 We conclude that rather than DHA itself, it is the peroxidation products of DHA that block I TO and activate a delayed outward current in in vitro single-cell experiments. These findings have important implications for the extrapolation of in vitro experimental findings to the antiarrhythmic effects of DHA in vivo because, in vivo, peroxidation of DHA is unlikely to occur.

The development of cell-based assays for high-throughput screening (HTS) approaches often requires the generation of stable transformant cell lines. However, these cell lines are essentially created by random integration of a gene of interest (GOI) with no control over the level and stability of gene expression. The authors developed a targeted integration system in Chinese hamster ovary (CHO) cells, called the cellular genome positioning system (cGPS), based on the stimulation of homologous gene targeting by meganucleases. Five different GOIs were knocked in at the same locus in cGPS CHO-K1 cells. Further characterization revealed that the cGPS CHO-K1 system is more rapid (2-week protocol), efficient (all selected clones expressed the GOI), reproducible (GOI expression level variation of 12%), and stable over time (no change in GOI expression after 23 weeks of culture) than classical random integration. Moreover, in all cGPS CHO-K1 targeted clones, the recombinant protein was biologically active and its properties similar to the endogenous protein. This fast and robust method opens the door for creating large collections of cell lines of better quality and expressing therapeutically relevant GOIs at physiological levels, thereby enhancing the potential scope of HTS. (Journal of Biomolecular Screening 2010:956-967)