Caloxin-derived peptides for the inhibition of plasma membrane calcium ATPases.

Plasma membrane calcium ATPases (PMCAs) are a family of transmembrane proteins responsible for the extrusion of cytosolic Ca2+ to the extracellular milieu. They are important players of the calcium homeostasis possibly implicated in some important diseases. The reference inhibitors of PMCA extruding activity are on one hand ortho-vanadate (IC50 in the 30 mM range), and on the other a series of 12- to 20-mer peptides named caloxins (IC50 in the 100 µM scale). As for all integral membrane proteins, biochemistry and pharmacology are difficult to study on isolated and/or purified proteins. Using a series of reference blockers, we assessed a pharmacological window with which we could study the functionality of PMCAs in living cells. Using this system, we screened for alternative versions of caloxins, aiming at shortening the peptide backbone, introducing non-natural amino acids, and overall trying to get a glimpse at the structure-activity relationship between those new peptides and the protein in a cellular context. We describe a short series of equipotent 5-residue long analogues with IC50 in the low µM range.

Voltage-sensing optical recording: A method of choice for high-throughput assessment of cardiotropic effects.

INTRODUCTION: Voltage and calcium-sensing optical recording (VSOR and CSOR, respectively) from human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been validated for in vitro evaluation of cardiotropic effects of drugs. When compared to electrophysiological devices like microelectrode array, multi-well optical recordings present a lower sample rate that may limit their capacity to detect fast depolarization or propagation velocity alterations. Additionally, the respective sensitivities of VSOR and CSOR to different cardiac electrophysiological effects have not been compared in the same conditions. METHODS: FluoVolt and Cal520 dyes were used in 96 well format on hPSC-CMs to report sodium channel block by lidocaine and propagation slowing by the junctional uncoupler carbenoxolone at three recording frequencies (60, 120 and 200 Hz) as well as their sensitivity to early and late repolarization delay. RESULTS: Sodium channel block led to a dose-dependent decrease of the VSOR signal rising slope that was improved by an increased sampling frequency. In contrast, the CSOR signal rising slope was only decreased at the highest concentration with no influence from the sampling rate. A similar result was obtained with carbenoxolone. Early repolarization delay by Bay K8644 showed the same effects on VSOR and CSOR signal durations while repolarization slowing by dofetilide had a significantly stronger prolongating effect on the VSOR signal at the lowest concentration. DISCUSSION: VSOR showed a higher capacity to detect sodium channel block, propagation slowing and modest late repolarization delay than CSOR. Increasing the sampling rate improved the detection threshold of VSOR for excitability and conduction velocity alterations.

High-throughput drug profiling with voltage- and calcium-sensitive fluorescent probes in human iPSC-derived cardiomyocytes.

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 voltage-sensitive fluorescent dye. In our conditions, the toxicity of the dye was moderate and compatible with episodic recordings for >3 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.

Meganuclease-driven targeted integration in CHO-K1 cells for the fast generation of HTS-compatible cell-based assays.

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.

Ca2+ current-mediated regulation of action potential by pacing rate in rat ventricular myocytes.

OBJECTIVE: Pacing rate regulates the duration of the cardiac action potential (AP). It also regulates the decay kinetics of the L-type Ca(2+) current (I(Ca-L)) which occurs via modulation of Ca(2+)-dependent inactivation. We investigated whether and how this latter process contributes to frequency-dependent (FD) changes in the AP waveform in rat ventricular cells. METHODS: We recorded APs using a microelectrode technique in rat papillary muscles, and using the whole-cell current patch-clamp technique in single rat ventricular cells. RESULTS: The AP duration (APD) was increased by high rates encompassing the physiological range (0.1-5.7 Hz) in both papillary muscles and single cells. This prolongation was accompanied by concomitant depolarisation (approximately 7 mV at 5.7 Hz) of the membrane potential (MP) in papillary muscles. Equivalent artificial depolarisation of the MP enhanced the FD prolongation in single cells. The FD prolongation was enhanced in presence of the K(+) current blocker 4-aminopyridine (5 mmol/l), and decreased in absence of extracellular Ca(2+). It was antagonised by Ca(2+) channel blockers (Co(2+), nifedipine, nitrendipine) and decreased by use of high EGTA (10 vs. 0.5 mmol/l EGTA) or BAPTA (20 mmol/l) in the patch-pipette. It was prevented by ryanodine or thapsigargin, two drugs that reduce or abolish SR-Ca(2+) function. CONCLUSION: I(Ca-L) contributes to the FD modulation of the AP, which occurs following a sudden change in cardiac frequency in rat ventricular cells. This highly dynamic physiological process is related to SR-Ca(2+) release and occurs through beat-to-beat adaptation of Ca(2+)-dependent inactivation of I(Ca-L).