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.

Novel IL1RAPL1 mutations associated with intellectual disability impair synaptogenesis.

Mutations in interleukin-1 receptor accessory protein like 1 (IL1RAPL1) gene have been associated with non-syndromic intellectual disability (ID) and autism spectrum disorder. This protein interacts with synaptic partners like PSD-95 and PTPδ, regulating the formation and function of excitatory synapses. The aim of this work was to characterize the synaptic consequences of three IL1RAPL1 mutations, two novel causing the deletion of exon 6 (Δex6) and one point mutation (C31R), identified in patients with ID. Using immunofluorescence and electrophysiological recordings, we examined the effects of IL1RAPL1 mutant over-expression on synapse formation and function in cultured rodent hippocampal neurons. Δex6 but not C31R mutation leads to IL1RAPL1 protein instability and mislocalization within dendrites. Analysis of different markers of excitatory synapses and sEPSC recording revealed that both mutants fail to induce pre- and post-synaptic differentiation, contrary to WT IL1RAPL1 protein. Cell aggregation and immunoprecipitation assays in HEK293 cells showed a reduction of the interaction between IL1RAPL1 mutants and PTPδ that could explain the observed synaptogenic defect in neurons. However, these mutants do not affect all cellular signaling because their over-expression still activates JNK pathway. We conclude that both mutations described in this study lead to a partial loss of function of the IL1RAPL1 protein through different mechanisms. Our work highlights the important function of the trans-synaptic PTPδ/IL1RAPL1 interaction in synaptogenesis and as such in ID in the patients.