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      Subtype-specific promoter-driven action potential imaging for precise disease modelling and drug testing in hiPSC-derived cardiomyocytes

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          Translational perspective

          Cardiomyocytes (CMs) generated from human induced pluripotent stem cells are an evolving platform to understand molecular disease mechanism and evaluate cardiovascular drugs. A major limitation of this system is that they represent a heterogeneous mix of ventricular-, atrial-, and nodal-like CMs. By expressing a voltage-sensitive fluorescent protein under the control of lineage-specific promoters, we developed a convenient system allowing high-throughput subtype-specific optical action potential (AP) imaging in these cells. This enables not only quantification of electrical phenotypes in patient-specific CMs but also subtype-specific investigation of drug effects, which may aid both drug development and safety pharmacology in the cardiovascular field.



          Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are increasingly used in disease modelling and drug evaluation. However, they are typically a heterogeneous mix of ventricular-, atrial-, and nodal-like cells based on action potentials (APs) and gene expression. This heterogeneity and the paucity of methods for high-throughput functional phenotyping hinder the full exploitation of their potential. We aimed at developing a method for rapid, sequential, and subtype-specific phenotyping of hiPSC-CMs with respect to AP morphology and single-cell arrhythmias.

          Methods and results

          We used cardiac lineage-specific promoters to drive the expression of a voltage-sensitive fluorescent protein (VSFP-CR) in hiPSC-CMs, enabling subtype-specific optical AP recordings. In a patient-specific hiPSC model of long-QT syndrome type 1, AP prolongation and frequent early afterdepolarizations were evident in mutant ventricular- and atrial like, but not in nodal-like hiPSC-CMs compared with their isogenic controls, consistent with the selective expression of the disease-causing gene. Furthermore, we demonstrate the feasibility of sequentially probing a cell over several days to investigate genetic rescue of the disease phenotype and to discern CM subtype-specific drug effects.


          By combining a genetically encoded membrane voltage sensor with promoters that drive its expression in the major subtypes of hiPSC-CMs, we developed a convenient system for disease modelling and drug evaluation in the relevant cell type, which has the potential to advance the emerging utility of hiPSCs in cardiovascular medicine.

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          Most cited references 23

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          Improving FRET dynamic range with bright green and red fluorescent proteins

          A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of cyan and yellow fluorescent proteins (CFP and YFP), but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light, and complex photokinetic events such as reversible photobleaching and photoconversion. Here, we engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date, and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP in reporters of kinase activity, small GTPase activity, and transmembrane voltage significantly improves photostability, FRET dynamic range, and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action potential firing and RhoA activation in growth cones.
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            Induced pluripotent stem cells: the new patient?

            Worldwide increases in life expectancy have been paralleled by a greater prevalence of chronic and age-associated disorders, particularly of the cardiovascular, neural and metabolic systems. This has not been met by commensurate development of new drugs and therapies, which is in part owing to the difficulty in modelling human diseases in laboratory assays or experimental animals. Patient-specific induced pluripotent stem (iPS) cells are an emerging paradigm that may address this. Reprogrammed somatic cells from patients are already applied in disease modelling, drug testing and drug discovery, thus enabling researchers to undertake studies for treating diseases 'in a dish', which was previously inconceivable.
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              Screening drug-induced arrhythmia [corrected] using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays.

              Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities. Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls. We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.

                Author and article information

                Eur Heart J
                Eur. Heart J
                European Heart Journal
                Oxford University Press
                21 January 2017
                16 June 2016
                16 June 2016
                : 38
                : 4 , Focus Issue on Arrhythmias
                : 292-301
                [1 ]I. Department of Medicine (Cardiology), Klinikum rechts der Isar, Technische Universität München, Munich 81675, Germany
                [2 ]Institute for Molecular Cell Biology, Medical Faculty, University Homburg/Saar, Universität des Saarlandes, Homburg/Saar 66421, Germany
                [3 ]Department of Anatomy and Embryology, Leiden University Medical Center, Leiden 2333, The Netherlands
                [4 ]DZHK (German Centre for Cardiovascular Research)—Partner Site Munich Heart Alliance, Munich 80802, Germany
                [5 ]Institute for Cardiovascular Prevention (IPEK), LMU München, Munich 80336, Germany
                Author notes
                [* ]Corresponding author. I. Department of Medicine, Klinikum rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81675 München, Germany. Tel: +49 89 4140 2350, Fax: +49 89 4140 4900, Email: amoretti@ 123456mytum.de (A.M.); Email: sinnecker@ 123456mytum.de (D.S.); Email: klaugwitz@ 123456mytum.de (K.-L.L.).

                These authors contributed equally to this article.

                See page 302 for the editorial comment on this article (doi: [Related article:]10.1093/eurheartj/ehw380)

                © The Author 2016. Published by Oxford University Press on behalf of the European Society of Cardiology.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

                Page count
                Pages: 10
                Funded by: European Research Council http://dx.doi.org/10.13039/501100000781
                Award ID: MEXT-23208
                Award ID: ERC 261053
                Funded by: German Research Foundation
                Award ID: Mo 2217/1-1
                Award ID: La 1238 3-1/4-1/4-2
                Award ID: Si 1747/1-1
                Funded by: Transregio Research Unit 152
                Funded by: EU Marie Curie FP7-People-2011-IEF Programme
                Award ID: HPSCLQT 29999
                Funded by: Netherlands Institute of Regenerative Medicine
                Basic Science


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