A Fast Optical Ion Channel Assay for Assessing Action Potentials in Human Induced Pluripotent Stem Cell Cardiomyocytes

Human induced pluripotent stem cell (iPSC)-based model of cardiac diseases has been proved to be useful and valuable for identifying new therapeutics. However, the use of human iPSC-based model of cardiac diseases for drug screen is hampered by the high-cost and complexity of methods used for reprogramming, in vitro differentiation, and phenotyping. To address the limitations, we first optimized a protocol for reprogramming of human fibroblasts and keratinocytes into pluripotency using single lipofection and the episomal vectors in a 24-well plate format. This method allowed us to generate multiple lines of integration-free and feeder-free iPSCs from seven patients with cardiac diseases and three controls. Second, we differentiated human iPSCs derived from Timothy syndrome patients into cardiomyocytes using a monolayer differentiation method. We found that Timothy syndrome cardiomyocytes showed slower, irregular contractions and abnormal calcium handling compared to controls, which were consistent with previous reports using a retroviral method for reprogramming and using an embryoid body-based method for cardiac differentiation. Third, we developed an efficient approach for recording action potentials and calcium transients simultaneously in control and patient cardiomyocytes using genetically encoded fluorescent indicators, ArcLight and R-GECO1. The dual optical recordings enabled us to observe prolonged action potentials and abnormal calcium handling in Timothy syndrome cardiomyocytes. We confirmed that roscovitine rescued the phenotypes in Timothy syndrome cardiomyocytes and these findings were consistent with previous studies using conventional electrophysiological recordings and calcium imaging with dyes. The approaches using our optimized methods and dual optical recordings will improve iPSC applicability for disease modeling to test potential therapeutics. With those new approaches in hand, next we plan to use the iPSC-based model of Timothy syndrome to investigate novel molecules involved in the pathogenesis of Timothy syndrome and to screen and identify new therapeutic compounds for Timothy syndrome patients.

Download Full-text

Ion Channel Expression and Characterization in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Stem Cells International ◽

10.1155/2018/6067096 ◽

2018 ◽

Vol 2018 ◽

pp. 1-14 ◽

Cited By ~ 15

Author(s):

Zhihan Zhao ◽

Huan Lan ◽

Ibrahim El-Battrawy ◽

Xin Li ◽

Fanis Buljubasic ◽

...

Keyword(s):

Stem Cell ◽

Ion Channels ◽

Ion Channel ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Biological Study ◽

Adrenergic Stimulation ◽

Cell Therapies ◽

Channel Expression ◽

Induced Pluripotent

Background. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are providing new possibilities for the biological study, cell therapies, and drug discovery. However, the ion channel expression and functions as well as regulations in hiPSC-CMs still need to be fully characterized. Methods. Cardiomyocytes were derived from hiPS cells that were generated from two healthy donors. qPCR and patch clamp techniques were used for the study. Results. In addition to the reported ion channels, INa, ICa-L, ICa-T, If, INCX, IK1, Ito, IKr, IKs IKATP, IK-pH, ISK1–3, and ISK4, we detected both the expression and currents of ACh-activated (KACh) and Na+-activated (KNa) K+, volume-regulated and calcium-activated (Cl-Ca) Cl−, and TRPV channels. All the detected ion currents except IK1, IKACh, ISK, IKNa, and TRPV1 currents contribute to AP duration. Isoprenaline increased ICa-L, If, and IKs but reduced INa and INCX, without an effect on Ito, IK1, ISK1–3, IKATP, IKr, ISK4, IKNa, ICl-Ca, and ITRPV1. Carbachol alone showed no effect on the tested ion channel currents. Conclusion. Our data demonstrate that most ion channels, which are present in healthy or diseased cardiomyocytes, exist in hiPSC-CMs. Some of them contribute to action potential performance and are regulated by adrenergic stimulation.

Download Full-text

CSAHi study: Detection of drug-induced ion channel/receptor responses, QT prolongation, and arrhythmia using multi-electrode arrays in combination with human induced pluripotent stem cell-derived cardiomyocytes

Journal of Pharmacological and Toxicological Methods ◽

10.1016/j.vascn.2017.02.001 ◽

2017 ◽

Vol 85 ◽

pp. 73-81 ◽

Cited By ~ 26

Author(s):

Takashi Kitaguchi ◽

Yuta Moriyama ◽

Tomohiko Taniguchi ◽

Sanae Maeda ◽

Hiroyuki Ando ◽

...

Keyword(s):

Stem Cell ◽

Ion Channel ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Qt Prolongation ◽

Electrode Arrays ◽

Drug Induced ◽

Induced Pluripotent

Download Full-text

The Emergence of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) as a Platform to Model Arrhythmogenic Diseases

International Journal of Molecular Sciences ◽

10.3390/ijms21020657 ◽

2020 ◽

Vol 21 (2) ◽

pp. 657 ◽

Cited By ~ 3

Author(s):

Marc Pourrier ◽

David Fedida

Keyword(s):

Stem Cell ◽

Ion Channel ◽

Pluripotent Stem Cell ◽

Molecular Mechanisms ◽

Induced Pluripotent Stem Cell ◽

Transgenic Animal ◽

Cardiac Diseases ◽

Disease Phenotype ◽

Induced Pluripotent

There is a need for improved in vitro models of inherited cardiac diseases to better understand basic cellular and molecular mechanisms and advance drug development. Most of these diseases are associated with arrhythmias, as a result of mutations in ion channel or ion channel-modulatory proteins. Thus far, the electrophysiological phenotype of these mutations has been typically studied using transgenic animal models and heterologous expression systems. Although they have played a major role in advancing the understanding of the pathophysiology of arrhythmogenesis, more physiological and predictive preclinical models are necessary to optimize the treatment strategy for individual patients. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have generated much interest as an alternative tool to model arrhythmogenic diseases. They provide a unique opportunity to recapitulate the native-like environment required for mutated proteins to reproduce the human cellular disease phenotype. However, it is also important to recognize the limitations of this technology, specifically their fetal electrophysiological phenotype, which differentiates them from adult human myocytes. In this review, we provide an overview of the major inherited arrhythmogenic cardiac diseases modeled using hiPSC-CMs and for which the cellular disease phenotype has been somewhat characterized.

Download Full-text

Application of human induced pluripotent stem cell-derived cardiomyocytes sheets with microelectrode array system to estimate antiarrhythmic properties of multi-ion channel blockers

Journal of Pharmacological Sciences ◽

10.1016/j.jphs.2018.07.011 ◽

2018 ◽

Vol 137 (4) ◽

pp. 372-378 ◽

Cited By ~ 8

Author(s):

Hiroko Izumi-Nakaseko ◽

Mihoko Hagiwara-Nagasawa ◽

Atsuhiko T. Naito ◽

Ai Goto ◽

Koki Chiba ◽

...

Keyword(s):

Stem Cell ◽

Ion Channel ◽

Pluripotent Stem Cell ◽

Microelectrode Array ◽

Induced Pluripotent Stem Cell ◽

Channel Blockers ◽

Ion Channel Blockers ◽

Induced Pluripotent

Download Full-text

Drug-Mediated Shortening of Action Potentials in LQTS2 Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Stem Cells and Development ◽

10.1089/scd.2017.0172 ◽

2017 ◽

Vol 26 (23) ◽

pp. 1695-1705 ◽

Cited By ~ 5

Author(s):

Gary Duncan ◽

Karl Firth ◽

Vinoj George ◽

Minh Duc Hoang ◽

Andrew Staniforth ◽

...

Keyword(s):

Stem Cell ◽

Action Potentials ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Induced Pluripotent

Download Full-text

Dynamic Current Clamp Experiments Define the Functional Roles of IK1 and Ito,f in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

10.1101/135400 ◽

2017 ◽

Cited By ~ 2

Author(s):

Scott B. Marrus ◽

Steven Springer ◽

Eric Johnson ◽

Rita J. Martinez ◽

Edward J. Dranoff ◽

...

Keyword(s):

Stem Cell ◽

Action Potential ◽

Action Potentials ◽

Pluripotent Stem Cell ◽

Phase 1 ◽

Induced Pluripotent Stem Cell ◽

Resting Membrane Potential ◽

Current Clamp ◽

Human Cardiomyocytes ◽

Induced Pluripotent

AbstractThe transient outward potassium current (Ito) plays a key, albeit incompletely defined, role in cardiomyocyte physiology and pathophysiology. In light of the technical challenges of studying adult human cardiomyocytes, this study examines the use of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) as a system which potentially preserves the native cellular milieu of human cardiomyocytes. ISPC-CMs express a robust Ito with slow recovery kinetics and fail to express the rapidly recovering Ito,f which is implicated in human disease. Overexpression of the accessory subunit KChIP2 (which is not expressed in iPSC-CMs) resulted in restoration of a rapid component of recovery. To define the functional role of Ito, dynamic current clamp was used to introduce computationally modeled currents into iPSC-CMs while recording action potentials. However, iPSC-CMs exhibit action potentials with multiple immature physiological properties, including slow upstroke velocity, heterogeneous action potential waveforms, and the absence of a phase 1 notch, thus potentially limiting the utility of these cells as a model of adult cardiomyocytes. Importantly, the introduction of modeled inwardly rectified current (IK1) ameliorated these immature properties by restoring a hyperpolarized resting membrane potential. In this context of normalized action potential morphologies, dynamic current clamp experiments introducing Ito,f demonstrated that there is significant cell-to-cell heterogeneity and that the functional effect of Ito,f is highly sensitive to the action potential plateau voltage in each cell.

Download Full-text

Abstract 026: Optical Recording of Cardiac Action Potentials from Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

Circulation Research ◽

10.1161/res.113.suppl_1.a026 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

B Alexander Yi ◽

Joel M Kralj ◽

Adam E Cohen

Keyword(s):

Stem Cell ◽

High Throughput ◽

Action Potentials ◽

Pluripotent Stem Cell ◽

Gene Mutations ◽

Induced Pluripotent Stem Cell ◽

Patch Clamping ◽

Cardiac Action ◽

Or Gene ◽

Induced Pluripotent

The electrically excitable properties of cardiomyocytes stem from the activity of ion channels that allow the coordinated entry of ions to generate cardiac action potentials. Disruptions in ion channel function either by drugs or gene mutations can lead to cardiac arrhythmias. The ability to screen drugs or gene mutations rapidly for effects on the cardiac action potential would be of interest for both drug discovery as well as for studies of ion channel function; however, the time-consuming and technically challenging nature of conventional patch clamping can limit the ability to perform high throughput screens. Archaerhodopsin3, or Arch, is an Archaebacterial variant of the membrane protein bacteriorhodopsin that binds a retinal fluorophore whose signal is rapidly responsive to changes in membrane potential. Here, we report the use of Arch to optically record action potentials from human induced pluripotent stem cell-derived cardiomyocytes. Human induced pluripotent stem cells that stably express Arch were generated and then differentiated into cardiomyocytes. As compared to patch clamping, Arch faithfully reproduces many of the key features of cardiac action potentials and may be a tool to be used for high throughput electrophysiological screens of cardiomyocytes.

Download Full-text