The immature electrophysiological phenotype of iPSC-CMs still hampers in vitro drug screening: Special focus on I K1

Goversen, Birgit; Heyden, Marcel A G van der; Veen, Toon A.B. van; Boer, Teun P. de

doi:10.1016/j.pharmthera.2017.10.001

Cited by 137 publications

(115 citation statements)

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“…[1][2][3][4] In general, it is challenging to study ion-channels in iPSCs due to the lack or low level of expression but we have used the powerful tool of stem cell technology to analyze the pathophysiology of LCA16. 29 The LCA16 mutation p.Trp53* analyzed in this report is a tryptophan (UGG) to amber (UAG) stop codon variant. 1 An in-frame nonsense mutation accounts for about 12% of inherited genetic disorder.…”

Section: Discussionmentioning

confidence: 84%

Gene augmentation and read-through rescue channelopathy in an iPSC-RPE model of congenital blindness

Shahi

Hermans

Sinha

et al. 2018

Preprint

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PurposeMutations in the KCNJ13 gene are known to cause Leber’s Congenital Amaurosis (LCA16), an inherited pediatric blindness. KCNJ13 gene encodes the Kir7.1 subunit protein which acts as a tetrameric inwardly rectifying potassium ion channel in the retinal pigment epithelium to maintain ionic homeostasis thereby allowing photoreceptors to encode visual information. We sought to determine if genetic approaches might be effective in treating blindness due to mutations in KCNJ13.MethodsWe developed patient-derived hiPSC-RPE carrying an autosomal recessive nonsense mutation in the KCNJ13 gene (c.158G>A, p.Trp53*). We performed biochemical and electrophysiology assays of Kir7.1 function. Both small molecule read-through drug and gene-therapy approaches were tested using this disease-in-a-dish approach.ResultsWe found that the LCA16 hiPSC-RPE had normal morphology but did not express a functional Kir7.1 channel and was unable to demonstrate normal physiology. Following read-through drug treatment, the LCA16 hiPSC cells were hyperpolarized by 30 mV and Kir7.1 current was restored. Similarly, loss-of-function of Kir7.1 channel was circumvented by lentiviral gene delivery to the hiPSC-RPE cells. In either approach, Kir7.1 protein was expressing normally with restoration of membrane potential and Kir7.1 current.ConclusionLoss-of-function mutation in Kir7.1 is a cause of LCA. Using either read-through therapy or gene augmentation, we rescued Kir7.1 channel function in patient-derived iPSC-RPE cells via a precision medicine approach.

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Section: Discussionmentioning

confidence: 84%

Gene augmentation and read-through rescue channelopathy in an iPSC-RPE model of congenital blindness

Shahi

Hermans

Sinha

et al. 2018

Preprint

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“…The third pillar of the CiPA initiative proposes to employ in vitro hiPSC-CMs to confirm the effects of a novel drug predicted by a comprehensive in silico model. hiPSC-CMs and adult ventricular CMs show qualitatively consistent responses to some, but not all drugs (9,20,30). Recently, two computational studies (8,31) quantitatively reported on the main electrophysiological differences between hiPSC-CM and adult ventricular CMs by comparing the Paci 2013 and O'Hara-Rudy models.…”

Section: Toward a Mature Electrophysiological Phenotypementioning

confidence: 99%

Required G1 to Suppress Automaticity of iPSC-CMs Depends Strongly on I1 Model Structure

Fabbri

Goversen

Vos

et al. 2019

Biophysical Journal

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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.

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“…In adult atrial and ventricular CMs, the resting potential is determined primarily by I K1 (Miake, Marbán, & Nuss, ; Silva & Rudy, ), but low I K1 and lack of channel expression are hallmarks of the immaturity of hPSC‐CMs (Goversen, van der Heyden, van Veen, & de, ). K ir 2.1 could be detected in our hESC‐CMs after 25 days in culture, with a significant increase of expression at 75 days.…”

Section: Discussionmentioning

confidence: 99%

Contribution of potassium channels to action potential repolarization of human embryonic stem cell‐derived cardiomyocytes

Wang

Zhu

Tung

2019

British J Pharmacology

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Background and Purpose: The electrophysiological properties of human pluripotent stem cell-derived cardiomyocytes (CMs) have not yet been characterized in a syncytial context. This study systematically characterized the contributions of different repolarizing potassium currents in human embryonic stem cell-derived CMs (hESC-CMs) during long-term culture as cell monolayers. Experimental Approach: The H9 hESC line was differentiated to CMs and plated to form confluent cell monolayers. Optical mapping was used to record the action potentials (APs) and conduction velocity (CV) during electrophysiological and pharmacological experiments. RT-PCR and Western blot were used to detect the presence and expression levels of ion channel subunits. Key Results: Long-term culture of hESC-CMs led to shortened AP duration (APD), faster repolarization rate, and increased CV. Selective block of I Kr , I Ks , I K1 , and I Kur significantly affected AP repolarization and APD in a concentration-and culture time-dependent manner. Baseline variations in APD led to either positive or negative APD dependence of drug response. Chromanol 293B produced greater relative AP prolongation in mid-and late-stage cultures, while DPO-1 had more effect in earlystage cultures. CV in cell monolayers in early-and late-stage cultures was most susceptible to slowing by E-4031 and BaCl 2 respectively. Conclusions and Implications: I Kr , I Ks , I K1 , and I Kur all play an essential role in the regulation of APD and CV in hESC-CMs. During time in culture, increased expressionof I Kr and I K1 helps to accelerate repolarization, shorten APD, and increase CV. We identified a new pro-arrhythmic parameter, positive APD dependence of ion channel block, which can increase APD and repolarization gradients.

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The immature electrophysiological phenotype of iPSC-CMs still hampers in vitro drug screening: Special focus on I K1

Cited by 137 publications

References 107 publications

Gene augmentation and read-through rescue channelopathy in an iPSC-RPE model of congenital blindness

Gene augmentation and read-through rescue channelopathy in an iPSC-RPE model of congenital blindness

Required G1 to Suppress Automaticity of iPSC-CMs Depends Strongly on I1 Model Structure

Contribution of potassium channels to action potential repolarization of human embryonic stem cell‐derived cardiomyocytes

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