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      Troponin destabilization impairs sarcomere-cytoskeleton interactions in iPSC-derived cardiomyocytes from dilated cardiomyopathy patients

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          Abstract

          The sarcomeric troponin-tropomyosin complex is a critical mediator of excitation-contraction coupling, sarcomeric stability and force generation. We previously reported that induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with a dilated cardiomyopathy (DCM) mutation, troponin T (TnT)-R173W, display sarcomere protein misalignment and impaired contractility. Yet it is not known how TnT mutation causes dysfunction of sarcomere microdomains and how these events contribute to misalignment of sarcomeric proteins in presence of DCM TnT-R173W. Using a human iPSC-CM model combined with CRISPR/Cas9-engineered isogenic controls, we uncovered that TnT-R173W destabilizes molecular interactions of troponin with tropomyosin, and limits binding of PKA to local sarcomere microdomains. This attenuates troponin phosphorylation and dysregulates local sarcomeric microdomains in DCM iPSC-CMs. Disrupted microdomain signaling impairs MYH7-mediated, AMPK-dependent sarcomere-cytoskeleton filament interactions and plasma membrane attachment. Small molecule-based activation of AMPK can restore TnT microdomain interactions, and partially recovers sarcomere protein misalignment as well as impaired contractility in DCM TnT-R173W iPSC-CMs. Our findings suggest a novel therapeutic direction targeting sarcomere- cytoskeleton interactions to induce sarcomere re-organization and contractile recovery in DCM.

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          Most cited references 40

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          Phosphate-binding tag, a new tool to visualize phosphorylated proteins.

          We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn(2+) or Mn(2+)) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn(2+)- and Mn(2+)-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn(2+)-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn(2+)-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn(2+)-Phos-tag SDS-PAGE).
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            Using iPS cells to investigate cardiac phenotypes in patients with Timothy Syndrome

            Individuals with congenital or acquired prolongation of the QT interval, or long QT syndrome (LQTS), are at risk of life threatening ventricular arrhythmia 1, 2. LQTS is commonly genetic in origin but can also be caused or exacerbated by environmental factors1, 3. A missense mutation in the L-type calcium channel CaV1.2 leads to LQTS in patients with Timothy syndrome (TS)4, 5. To explore the effect of the TS mutation on the electrical activity and contraction of human cardiomyocytes (CMs), we reprogrammed human skin cells from TS patients to generate induced pluripotent stem cells (iPSCs), and differentiated these cells into CMs. Electrophysiological recording and calcium (Ca2+) imaging studies of these cells revealed irregular contraction, excess Ca2+ influx, prolonged action potentials, irregular electrical activity and abnormal calcium transients in ventricular-like cells. We found that roscovitine (Ros), a compound that increases the voltage-dependent inactivation (VDI) of CaV1.26–8, restored the electrical and Ca2+ signaling properties of CMs from TS patients. This study opens new avenues for studying the molecular and cellular mechanisms of cardiac arrhythmias in humans, and provides a robust assay for developing new drugs to treat these diseases.
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              Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy.

              Characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure, dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy in patients. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific cellular models, investigating underlying mechanisms, and optimizing therapy. Here, we generated cardiomyocytes from iPSCs derived from patients in a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to control healthy individuals in the same family cohort, cardiomyocytes derived from iPSCs from DCM patients exhibited altered regulation of calcium ion (Ca(2+)), decreased contractility, and abnormal distribution of sarcomeric α-actinin. When stimulated with a β-adrenergic agonist, DCM iPSC-derived cardiomyocytes showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric α-actinin distribution. Treatment with β-adrenergic blockers or overexpression of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (Serca2a) improved the function of iPSC-derived cardiomyocytes from DCM patients. Thus, iPSC-derived cardiomyocytes from DCM patients recapitulate to some extent the morphological and functional phenotypes of DCM and may serve as a useful platform for exploring disease mechanisms and for drug screening.
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                Author and article information

                Contributors
                antje.ebert@med.uni-goettingen.de
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                14 January 2020
                14 January 2020
                2020
                : 10
                Affiliations
                [1 ]Heart Center, Department of Cardiology and Pneumology, Goettingen, Germany
                [2 ]ISNI 0000 0001 2364 4210, GRID grid.7450.6, Institute of Pharmacology, University of Goettingen, ; Robert-Koch-Str. 40, 37075 Goettingen, Germany
                [3 ]ISNI 0000 0004 5937 5237, GRID grid.452396.f, DZHK (German Center for Cardiovascular Research), partner site, ; Goettingen, Germany
                [4 ]ISNI 0000 0001 2172 9288, GRID grid.5949.1, Institute of Physiology II, University of Muenster, ; Muenster, Germany
                [5 ]ISNI 0000 0004 1936 8948, GRID grid.4991.5, Department of Physiology, Anatomy and Genetics, University of Oxford, ; Oxford, OX1 3PT UK
                Article
                56597
                10.1038/s41598-019-56597-3
                6959358
                31937807
                © The Author(s) 2020

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                Funding
                Funded by: FundRef https://doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft (German Research Foundation);
                Award ID: SFB1002 Project A12
                Award ID: SFB1002 Project A12
                Award ID: SFB1002 Project A12
                Award ID: SFB1002 Project A12
                Award ID: SFB 1002 Project C04
                Award ID: SFB1002 Project C04
                Award ID: SFB1002 Project A08
                Award ID: SFB1002 Project D01
                Award ID: SFB 1002 Project A12
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/501100000274, British Heart Foundation (BHF);
                Award ID: RG/17/6/32944
                Award ID: RG/17/6/32944
                Award Recipient :
                Categories
                Article
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                © The Author(s) 2020

                Uncategorized

                cytoskeleton, mechanisms of disease, phosphorylation

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