Blebbistatin protects iPSC-CMs from hypercontraction and facilitates automated patch-clamp based electrophysiological study

Li, Wener; Luo, Xiaojing; Ulbricht, Ying; Guan, Kaomei

doi:10.1016/j.scr.2021.102565

Cited by 14 publications

(6 citation statements)

References 33 publications

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“…In fact, sodium current ( I Na ) and I Ca,L are large in any type of CMs and can therefore routinely be measured by APC in hiPSC-CM ( Goversen et al, 2018 ; Li et al, 2019 ). Ion current measurements by APC in hiPSC-CM bearing patient mutations could be of great interest and one recent study used hiPSC-CM derived from patients with Brugada-syndrome AP ( Li et al, 2021 ). In contrast, we are not sure whether ion current measurements in hiPSC-CM from healthy donors will replace “traditional” measurements in expression system ( Lemoine and Christ, 2021 ).…”

Section: Hipsc-cm and Automated Patch Clamp: The Dream Wedding In Exp...mentioning

confidence: 99%

Human induced pluripotent stem cell-derived cardiomyocytes as an electrophysiological model: Opportunities and challenges—The Hamburg perspective

Ismaili

Schulz²,

Horváth³

et al. 2023

Front. Physiol.

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Models based on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are proposed in almost any field of physiology and pharmacology. The development of human induced pluripotent stem cell-derived cardiomyocytes is expected to become a step forward to increase the translational power of cardiovascular research. Importantly they should allow to study genetic effects on an electrophysiological background close to the human situation. However, biological and methodological issues revealed when human induced pluripotent stem cell-derived cardiomyocytes were used in experimental electrophysiology. We will discuss some of the challenges that should be considered when human induced pluripotent stem cell-derived cardiomyocytes will be used as a physiological model.

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Section: Hipsc-cm and Automated Patch Clamp: The Dream Wedding In Exp...mentioning

confidence: 99%

Human induced pluripotent stem cell-derived cardiomyocytes as an electrophysiological model: Opportunities and challenges—The Hamburg perspective

Ismaili

Schulz²,

Horváth³

et al. 2023

Front. Physiol.

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“…Most marketed APC platforms now offer the option to carry out current clamp recordings, but this capability has not been widely used for drug discovery screening apart from experiments to record action potentials from iPSC cardiomyocytes and neurons ( Stoelzle et al, 2011a , b ; Haythornthwaite et al, 2012 ; Goversen et al, 2018 ; Li et al, 2019 , 2021 ; Becker et al, 2020 ). We therefore sought to apply a historical current clamp method used to study ligand-gated ionotropic receptor responses to a modern APC platform, utilizing the same CHO ASIC1a cell line employed in our voltage clamp experiments.…”

Section: Resultsmentioning

confidence: 99%

Development of ASIC1a ligand-gated ion channel drug screening assays across multiple automated patch clamp platforms

Ridley¹,

Manyweathers²,

Tang³

et al. 2022

Front. Mol. Neurosci.

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Human acid-sensing ion channels (ASIC) are ligand-gated ionotropic receptors expressed widely in peripheral tissues as well as sensory and central neurons and implicated in detection of inflammation, tissue injury, and hypoxia-induced acidosis. This makes ASIC channels promising targets for drug discovery in oncology, pain and ischemia, and several modulators have progressed into clinical trials. We describe the use of hASIC1a as a case study for the development and validation of low, medium and high throughput automated patch clamp (APC) assays suitable for the screening and mechanistic profiling of new ligands for this important class of ligand-gated ion channel. Initial efforts to expand on previous manual patch work describing an endogenous hASIC1a response in HEK cells were thwarted by low current expression and unusual pharmacology, so subsequent work utilized stable hASIC1a CHO cell lines. Ligand-gated application protocols and screening assays on the Patchliner, QPatch 48, and SyncroPatch 384 were optimized and validated based on pH activation and nM-μM potency of reference antagonists (e.g., Amiloride, Benzamil, Memantine, Mambalgin-3, A-317567, PcTx1). By optimizing single and stacked pipette tip applications available on each APC platform, stable pH-evoked currents during multiple ligand applications enabled cumulative EC50 and IC50 determinations with minimized receptor desensitization. Finally, we successfully demonstrated for the first time on an APC platform the ability to use current clamp to implement the historical technique of input resistance tracking to measure ligand-gated changes in membrane conductance on the Patchliner platform.

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“…The approach works by injecting simulated currents into a cell in a real-time continuous loop during current clamp experiments (Ortega et al, 2018). I K1 dynamic clamp has been used on iPSC-CMs to attain quiescence at a MP below −70mV so the cells can be paced at a desired frequency (Meijer van Putten et al, 2015; Goversen et al, 2018b; Li et al, 2021; Clark et al, 2022). A dynamically clamped leak-compensation current has also been implemented and used in manual patch-clamp studies with neonatal mouse cardiomyocytes (Ahrens-Nicklas and Christini, 2009), demonstrating the potential of using such an approach with small cardiomyocytes.…”

Section: Discussionmentioning

confidence: 99%

Leak current, even with gigaohm seals, can cause misinterpretation of stem-cell derived cardiomyocyte action potential recordings

Clark

Clerx

Wei

et al. 2022

Preprint

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Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have become an essential tool to study arrhythmia mechanisms. Much of the foundational work on these cells, and the computational models built from the resultant data, has overlooked the contribution of seal-leak current on the immature and heterogeneous phenotype that has come to define these cells. Here, we usein silicoandin vitrostudies to demonstrate how seal-leak current depolarises action potentials (APs), substantially affecting their morphology, even with seal resistances (Rseal) above 1GΩ. We show that compensation of this leak current is difficult due to challenges with recording accurate measures of Rseal during an experiment. Using simulation, we show that Rsealmeasures: 1) change during an experiment, invalidating the use of pre-rupture values, and 2) are polluted by the presence of transmembrane currents at every voltage. Finally, we posit the background sodium current in baseline iPSC-CM models imitates the effects of seal-leak current and is increased to a level that masks the effects of seal-leak current on iPSC-CMs. Based on these findings, we make three recommendations to improve iPSC-CM AP data acquisition, interpretation, and model-building. Taking these recommendations into account will improve our understanding of iPSC-CM physiology and the descriptive ability of models built from such data.

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Blebbistatin protects iPSC-CMs from hypercontraction and facilitates automated patch-clamp based electrophysiological study

Cited by 14 publications

References 33 publications

Human induced pluripotent stem cell-derived cardiomyocytes as an electrophysiological model: Opportunities and challenges—The Hamburg perspective

Human induced pluripotent stem cell-derived cardiomyocytes as an electrophysiological model: Opportunities and challenges—The Hamburg perspective

Development of ASIC1a ligand-gated ion channel drug screening assays across multiple automated patch clamp platforms

Leak current, even with gigaohm seals, can cause misinterpretation of stem-cell derived cardiomyocyte action potential recordings

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