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      Cardiac Subtype-Specific Modeling of K v1.5 Ion Channel Deficiency Using Human Pluripotent Stem Cells

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          Abstract

          The ultrarapid delayed rectifier K + current (I Kur), mediated by K v1.5 channels, constitutes a key component of the atrial action potential. Functional mutations in the underlying KCNA5 gene have been shown to cause hereditary forms of atrial fibrillation (AF). Here, we combine targeted genetic engineering with cardiac subtype-specific differentiation of human induced pluripotent stem cells (hiPSCs) to explore the role of K v1.5 in atrial hiPSC-cardiomyocytes. CRISPR/Cas9-mediated mutagenesis of integration-free hiPSCs was employed to generate a functional KCNA5 knockout. This model as well as isogenic wild-type control hiPSCs could selectively be differentiated into ventricular or atrial cardiomyocytes at high efficiency, based on the specific manipulation of retinoic acid signaling. Investigation of electrophysiological properties in K v1.5-deficient cardiomyocytes compared to isogenic controls revealed a strictly atrial-specific disease phentoype, characterized by cardiac subtype-specific field and action potential prolongation and loss of 4-aminopyridine sensitivity. Atrial K v1.5-deficient cardiomyocytes did not show signs of arrhythmia under adrenergic stress conditions or upon inhibiting additional types of K + current. Exposure of bulk cultures to carbachol lowered beating frequencies and promoted chaotic spontaneous beating in a stochastic manner. Low-frequency, electrical stimulation in single cells caused atrial and mutant-specific early afterdepolarizations, linking the loss of KCNA5 function to a putative trigger mechanism in familial AF. These results clarify for the first time the role of K v1.5 in atrial hiPSC-cardiomyocytes and demonstrate the feasibility of cardiac subtype-specific disease modeling using engineered hiPSCs.

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          Most cited references27

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          2011 ACCF/AHA/HRS focused updates incorporated into the ACC/AHA/ESC 2006 Guidelines for the management of patients with atrial fibrillation: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines developed in partnership with the European Society of Cardiology and in collaboration with the European Heart Rhythm Association and the Heart Rhythm Society.

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            Regional and tissue specific transcript signatures of ion channel genes in the non-diseased human heart.

            The various cardiac regions have specific action potential properties appropriate to their electrical specialization, resulting from a specific pattern of ion-channel functional expression. The present study addressed regionally defined differential ion-channel expression in the non-diseased human heart with a genomic approach. High-throughput real-time RT-PCR was used to quantify the expression patterns of 79 ion-channel subunit transcripts and related genes in atria, ventricular epicardium and endocardium, and Purkinje fibres isolated from 15 non-diseased human donor hearts. Two-way non-directed hierarchical clustering separated atria, Purkinje fibre and ventricular compartments, but did not show specific patterns for epicardium versus endocardium, nor left- versus right-sided chambers. Genes that characterized the atria (versus ventricles) included Cx40, Kv1.5 and Kir3.1 as expected, but also Cav1.3, Cav3.1, Cav alpha2 delta2, Nav beta1, TWIK1, TASK1 and HCN4. Only Kir2.1, RyR2, phospholamban and Kv1.4 showed higher expression in the ventricles. The Purkinje fibre expression-portrait (versus ventricle) included stronger expression of Cx40, Kv4.3, Kir3.1, TWIK1, HCN4, ClC6 and CALM1, along with weaker expression of mRNA encoding Cx43, Kir2.1, KChIP2, the pumps/exchangers Na(+),K(+)-ATPase, NCX1, SERCA2, and the Ca(2+)-handling proteins RYR2 and CASQ2. Transcripts that were more strongly expressed in epicardium (versus endocardium) included Cav1.2, KChIP2, SERCA2, CALM3 and calcineurin-alpha. Nav1.5 and Nav beta1 were more strongly expressed in the endocardium. For selected genes, RT-PCR data were confirmed at the protein level. This is the first report of the global portrait of regional ion-channel subunit-gene expression in the non-diseased human heart. Our data point to significant regionally determined ion-channel expression differences, with potentially important implications for understanding regional electrophysiology, arrhythmia mechanisms, and responses to ion-channel blocking drugs. Concordance with previous functional studies suggests that regional regulation of cardiac ion-current expression may be primarily transcriptional.
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              Efficient generation of human iPSCs by a synthetic self-replicative RNA.

              The generation of human induced pluripotent stem cells (iPSCs) holds great promise for the development of regenerative medicine therapies to treat a wide range of human diseases. However, the generation of iPSCs in the absence of integrative DNA vectors remains problematic. Here, we report a simple, highly reproducible RNA-based iPSC generation approach that utilizes a single, synthetic self-replicating VEE-RF RNA replicon that expresses four reprogramming factors (OCT4, KLF4, and SOX2, with c-MYC or GLIS1) at consistent high levels prior to regulated RNA degradation. A single VEE-RF RNA transfection into newborn or adult human fibroblasts resulted in efficient generation of iPSCs with all the hallmarks of stem cells, including cell surface markers, global gene expression profiles, and in vivo pluripotency, to differentiate into all three germ layers. The VEE-RF RNA-based approach has broad applicability for the generation of iPSCs for ultimate use in human stem cell therapies in regenerative medicine. Copyright © 2013 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Journal
                Front Physiol
                Front Physiol
                Front. Physiol.
                Frontiers in Physiology
                Frontiers Media S.A.
                1664-042X
                06 July 2017
                2017
                : 8
                : 469
                Affiliations
                [1] 1Human Stem Cell Pluripotency Laboratory, Max Planck Institute for Molecular Biomedicine Münster, Germany
                [2] 2Chemical Genomics Centre of the Max Planck Society Dortmund, Germany
                [3] 3Department of Cardiovascular Medicine, Institute of Genetics of Heart Diseases, University of Münster Medical School Münster, Germany
                [4] 4Department of Clinical and Experimental Cardiology, Academic Medical Center, University of Amsterdam Amsterdam, Netherlands
                [5] 5Institute of Human Genetics, University of Münster Münster, Germany
                [6] 6Department of Medical Biology, Academic Medical Center, University of Amsterdam Amsterdam, Netherlands
                Author notes

                Edited by: Zhilin Qu, University of California, Los Angeles, United States

                Reviewed by: Michel Puceat, Institut National de la Santé et de la Recherche Médicale (INSERM), France; Michele Miragoli, University of Parma, Italy

                *Correspondence: Arie O. Verkerk a.o.verkerk@ 123456amc.uva.nl

                This article was submitted to Cardiac Electrophysiology, a section of the journal Frontiers in Physiology

                Article
                10.3389/fphys.2017.00469
                5498524
                28729840
                ca9f2334-f9d4-434f-82e1-44637c674be1
                Copyright © 2017 Marczenke, Piccini, Mengarelli, Fell, Röpke, Seebohm, Verkerk and Greber.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 26 April 2017
                : 19 June 2017
                Page count
                Figures: 5, Tables: 0, Equations: 0, References: 38, Pages: 11, Words: 7798
                Categories
                Physiology
                Original Research

                Anatomy & Physiology
                induced pluripotent stem cells,disease modeling,cardiac differentiation,kv1.5,atrial fibrillation

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