Human-induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) are a virtually endless source of human cardiomyocytes that may become a great tool for safety pharmacology; however, their electrical phenotype is immature: they show spontaneous action potentials (APs) and an unstable and depolarized resting membrane potential (RMP) because of lack of I K1 . Such immaturity hampers their application in assessing drug safety. The electronic overexpression of I K1 (e.g., through the dynamic clamp (DC) technique) is an option to overcome this deficit. In this computational study, we aim to estimate how much I K1 is needed to bring hiPSC-CMs to a stable and hyperpolarized RMP and which mathematical description of I K1 is most suitable for DC experiments. We compared five mature I K1 formulations (Bett, Dhamoon, Ishihara, O'Hara-Rudy, and ten Tusscher) with the native one (Paci), evaluating the main properties (outward peak, degree of rectification), and we quantified their effects on AP features (RMP, _ V max , APD 50 , APD 90 (AP duration at 50 and 90% of repolarization), and APD 50 /APD 90 ) after including them in the hiPSC-CM mathematical model by Paci. Then, we automatically identified the critical conductance for I K1 ( G K1, critical ), the minimally required amount of I K1 suppressing spontaneous activity. Preconditioning the cell model with depolarizing/hyperpolarizing prepulses allowed us to highlight time dependency of the I K1 formulations. Simulations showed that inclusion of mature I K1 formulations resulted in hyperpolarized RMP and higher _ V max , and observed G K1, critical and the effect on AP duration strongly depended on I K1 formulation. Finally, the Ishihara I K1 led to shorter (À16.3%) and prolonged (þ6.5%) APD 90 in response to hyperpolarizing and depolarizing prepulses, respectively, whereas other models showed negligible effects. Fine-tuning of G K1 is an important step in DC experiments. Our computational work proposes a procedure to automatically identify how much I K1 current is required to inject to stop the spontaneous activity and suggests the use of the Ishihara I K1 model to perform DC experiments in hiPSC-CMs. SIGNIFICANCE In this work, we aim to contribute a method that will facilitate automated dynamic clamp (DC) experiments in which I K1 is injected in induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs). By introducing G K1, critical (minimal I K1 conductance needed to stop automaticity of iPSC-CMs), we are proposing a different approach to setting up DC experiments. These are usually based on the injection of a fixed current density. In contrast, G K1, critical is a parameter that depends on the cell under investigation. Our in silico approach analyzed analogies and differences between I K1 formulations without the confounding factor that can be brought by the variability of iPSC-CMs. It highlighted how much the employed mathematical formulation of I K1 can affect G K1, critical and the action potential waveform in DC experiments.

The establishment of cardiac function in the developing embryo is essential to ensure blood flow and, therefore, growth and survival of the animal. The molecular mechanisms controlling normal cardiac rhythm remain to be fully elucidated. From a forward genetic screen, we identified a unique mutant, grime, that displayed a specific cardiac arrhythmia phenotype. We show that loss-of-function mutations in tmem161b are responsible for the phenotype, identifying Tmem161b as a regulator of cardiac rhythm in zebrafish. To examine the evolutionary conservation of this function, we generated knockout mice for Tmem161b. Tmem161b knockout mice are neonatal lethal and cardiomyocytes exhibit arrhythmic calcium oscillations. Mechanistically, we find that Tmem161b is expressed at the cell membrane of excitable cells and live imaging shows it is required for action potential repolarization in the developing heart. Electrophysiology on isolated cardiomyocytes demonstrates that Tmem161b is essential to inhibit Ca2+ and K+ currents in cardiomyocytes. Importantly, Tmem161b haploinsufficiency leads to cardiac rhythm phenotypes, implicating it as a candidate gene in heritable cardiac arrhythmia. Overall, these data describe Tmem161b as a highly conserved regulator of cardiac rhythm that functions to modulate ion channel activity in zebrafish and mice.

SummaryHuman and mouse embryonic stem (ES) cells have the potential to differentiate to cardiomyocytes in culture. They are therefore of interest for studying early human and mouse heart development, as well as properties of cardiomyocytes from both species, including their responses to cardiac drugs, and, at some point in the future, may represent a source of transplantable cells for cardiac muscle repair. The differentiation protocols that are effective depend in part on the species from which the ES cell lines were derived, and in part on the individual cell lines and the methods used for their propagation prior to differentiation. Here, several methods for generating and characterizing cardiomyocytes from mouse and human ES cells are described, as well as methods for dissociation of cardiomyocytes into single-cell suspensions which are useful both for characterizing cells by antibody staining and electrophysiological measurements, as well as preparing cells for transplantation into (animal) hearts.

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Mutations in GNB5 , encoding the G-protein β5 subunit (Gβ5), have recently been linked to a multisystem disorder that includes severe bradycardia. Here, we investigated the mechanism underlying bradycardia caused by the recessive p.S81L Gβ5 variant. Using CRISPR/Cas9-based targeting, we generated an isogenic series of human induced pluripotent stem cell (hiPSC) lines that were either wild type, heterozygous or homozygous for the GNB5 p.S81L variant. These were differentiated into cardiomyocytes (hiPSC-CMs) that robustly expressed the acetylcholine-activated potassium channel [I(KACh); also known as I K,ACh ]. Baseline electrophysiological properties of the lines did not differ. Upon application of carbachol (CCh), homozygous p.S81L hiPSC-CMs displayed an increased acetylcholine-activated potassium current ( I K,ACh ) density and a more pronounced decrease of spontaneous activity as compared to wild-type and heterozygous p.S81L hiPSC-CMs, explaining the bradycardia in homozygous carriers. Application of the specific I(KACh) blocker XEN-R0703 resulted in near-complete reversal of the phenotype. Our results provide mechanistic insights and proof of principle for potential therapy in patients carrying GNB5 mutations. .