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Stiff matrix induces switch to pure β-cardiac myosin heavy chain expression in human ESC-derived cardiomyocytes

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Abstract

Human pluripotent stem cell (hPSC)-derived cardiomyocytes hold great potential for in vitro modeling of diseases like cardiomyopathies. Yet, knowledge about expression and functional impact of sarcomeric protein isoforms like the myosin heavy chain (MyHC) in hPSC-cardiomyocytes is scarce. We hypothesized that ventricular β-MyHC expression alters contraction and calcium kinetics and drives morphological and electrophysiological differentiation towards ventricular-like cardiomyocytes. To address this, we (1) generated human embryonic stem cell-derived cardiomyocytes (hESC-CMs) that switched towards exclusive β-MyHC, and (2) functionally and morphologically characterized these hESC-CMs at the single-cell level. MyHC-isoforms and functional properties were investigated during prolonged in vitro culture of cardiomyocytes in floating cardiac bodies (soft conditions) vs. culture on a stiff matrix. Using a specific anti-β-MyHC and a newly generated anti-α-MyHC-antibody, we found individual cardiomyocytes grown in cardiac bodies to mostly express both α- and β-MyHC-protein isoforms. Yet, 35 and 75 days of cultivation on laminin-coated glass switched 66 and 87 % of all cardiomyocytes to exclusively express β-MyHC, respectively. Twitch contraction and calcium transients were faster for CMs on laminin-glass. Surprisingly, both parameters were only little affected by the MyHC-isoform, although hESC-CMs with only β-MyHC had much lower ATP-turnover and tension cost, just as in human ventricular cardiomyocytes. Spontaneous contractions and no strict coupling of β-MyHC to ventricular-like action potentials suggest that MyHC-isoform expression does not fully determine the hESC-CM differentiation status. Stiff substrate-induced pure β-MyHC-protein expression in hESC-CMs, with several contractile parameters close to ventricular cardiomyocytes, provides a well-defined in vitro system for modeling of cardiomyopathies and drug screening approaches.

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Acknowledgments

The authors thank Alexander Lingk, Torsten Beier and Stefanie Nedel, Molecular and Cell Physiology, Hannover Medical School, for excellent technical assistance. This work was supported by Grants of Deutsche-Forschungsgemeinschaft BR849/31-1, KR1187/21-1, MA2331/16-1, ZW64/4-1, TH903/11-1 and Cluster of Excellence REBIRTH DFG EXC62/3; BMBF-grant 13N12606 and StemBANCC (Innovative Medicines Initiative joint undertaking, Grant agreement no 115439-2, resources of which are composed of financial contribution from the European Union (FP7/2007-2013) and EFPIA companies’ in kind contribution); TECHNOBEAT research project (European Union H2020, GA Number: 668724).

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    Authors and Affiliations

    1. Institute of Molecular and Cell Physiology, Hannover Medical School, Carl Neuberg Str. 1, 30625, Hannover, Germany

      Natalie Weber, Stephan Greten, Meike Wendland, Bogdan Iorga, Cornelia Geers-Knörr, Birgit Piep, Bernhard Brenner & Theresia Kraft

    2. Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany

      Kristin Schwanke, Henning Kempf, Annika Franke, Ulrich Martin & Robert Zweigerdt

    3. Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover Medical School, Hannover, Germany

      Kristin Schwanke, Henning Kempf, Annika Franke, Ulrich Martin & Robert Zweigerdt

    4. HTTG, LEBAO and REBIRTH-Center for Regenerative Medicine, Hannover Medical School, Carl Neuberg Str. 1, 30625, Hannover, Germany

      Kristin Schwanke, Henning Kempf, Annika Franke, Ulrich Martin & Robert Zweigerdt

    5. Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany

      Jan Hegermann & Christoph Wrede

    6. REBIRTH Excellence Cluster, Hannover, Germany

      Jan Hegermann & Christoph Wrede

    7. Institute of Neurophysiology, Hannover Medical School, Hannover, Germany

      Martin Fischer

    8. Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany

      Jan Fiedler, Angelika Pfanne & Thomas Thum

    9. Integrated Research and Treatment Center Transplantation (IFB-Tx) and REBIRTH Excellence Cluster, Hannover Medical School, Hannover, Germany

      Thomas Thum

    10. National Heart and Lung Institute, Imperial College London, London, UK

      Thomas Thum

    11. Department of Physical Chemistry, Faculty of Chemistry, University of Bucharest, Bucharest, Romania

      Bogdan Iorga

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    1. Natalie Weber
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    Natalie Weber and Kristin Schwanke equal first author contribution.

    Theresia Kraft and Robert Zweigerdt equal last author contribution.

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    Weber, N., Schwanke, K., Greten, S. et al. Stiff matrix induces switch to pure β-cardiac myosin heavy chain expression in human ESC-derived cardiomyocytes. Basic Res Cardiol 111, 68 (2016). https://doi.org/10.1007/s00395-016-0587-9

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