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Fibroblast-Cardiomyocyte Cross-Talk in Heart Muscle Formation and Function

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Fibroblast-Cardiomyocyte Cross-Talk in Heart Muscle Formation and Function Fibroblast-Cardiomyocyte Cross-Talk in Heart Muscle Formation and Function Doctoral Thesis In partial fulfillment of the requirements for the degree “Doctor rerum naturalium (Dr. rer. nat.)“ in the Molecular Medicine Study Program at the Georg-August University Göttingen submitted by Susanne Franziska Schlick born in Filderstadt, Germany Göttingen, November 2018 Members of the thesis committee Prof. Wolfram Hubertus Institute of Pharmacology and Toxicology Zimmermann (supervisor) University Medical Center Göttingen 1. Referee Robert-Koch-Str. 40 37075 Göttingen [email protected] Prof. Ralf Dressel Department of Cellular and Molecular Immunology 2. Referee Humboldtallee 34 37073 Göttingen, Germany [email protected] Prof. Stefan Luther Biomedical Physics MPI for Dynamics and Self-Organization Am Faßberg 17 37077 Göttingen, Germany Institute of Pharmacology and Toxicology University Medical Center Göttingen/Heart research building (DZHK) Robert-Koch-Str. 40 37075 Göttingen [email protected] II Members of the extended thesis committee Prof. Rüdiger Behr German Primate Center (Deutsches Primatenzentrum -DPZ) Kellnerweg 4 37077 Göttingen [email protected] Prof. Thomas Meyer Molecular Psychocardiology (Molekulare Psychokardiologie) Waldweg 33 37075 Göttingen [email protected] Dr. rer. nat. Antje Ebert Cardiovascular Cell Biology and Systems Medicine University Medical Center Göttingen Robert-Koch-Str. 40 37075 Göttingen [email protected] Date of submission of the Doctoral thesis: 06.11.2018 Date of disputation: 19.12.2018 III Affidavit Here I declare that my doctoral thesis entitled “Fibroblast-Cardiomyocyte Cross-Talk in Heart Muscle Formation and Function” has been written independently with no other sources and aids than quoted. Susanne F. Schlick Göttingen, November, 2018. IV List of publications Title Type of publication Date and location Agonistic and antagonistic roles of fibroblasts and Research article Prog Biophys Mol cardiomyocytes on viscoelastic stiffening of Biol. 2018 Dec 12. engineered human myocardium pii: S0079- 6107(18)30159-7 Susanne F. Schlick*, Florian Spreckelsen*, Malte Tiburcy, Lavanya M. Iyer, Laura Zelarayan, Stefan Luther, Ulrich Parlitz, Wolfram H. Zimmermann, and Florian Rehfeldt *These authors contributed equally to this work X-ray diffraction imaging of cardiac cells and tissue Research article Prog Biophys Mol Biol. 2018 Jun 15.: Jan-David Nicolas, Marten Bernhardt, Susanne F. S0079-6107(18) Schlick, Malte Tiburcy, Wolfram-H. Zimmermann, 30064-6 Amara Khan, Andrea Markus, Frauke Alves, Karl Toischer, Tim Salditt Defined Engineered Human Myocardium With Research article Circulation. 2017 Advanced Maturation for Applications in Heart May Failure Modeling and Repair 9;135(19):1832- Tiburcy M, Hudson J, Balfanz P,Schlick S, Meyer T, 1847. Chang Liao ML, Levent E, Raad F, Zeidler S, Wingender E, Riegler J, Wang M, Gold JD, Kehat I, Wettwer E, Ravens U, Dierickx P, van Laake LW, Goumans MJ, Khadjeh S, Toischer K, Hasenfuss G, Couture LA, Unger A, Linke WA, Araki T, Neel B, Keller G, Gepstein L, Wu JC1, Zimmermann WH. Calcitriol enhances EHM contractile function Poster DGPT 2/2018, Göttingen Effects of Vitamin D- binding protein on engineered heart Talk Meeting foundation muscle development and function Leducq 8/2017 Fibroblast origin determines engineered heart muscle Poster and talk Young DZHK development and function (Poster prize) Retreat 9/2016, Bad Aibling Effects of Vitamin D binding protein on engineered heart Talk Meeting foundation muscle development and function Leducq 6/2016 Tuning hyaluronic acid deposition to enhance engineered Invited talk Dutch German heart muscle function Meeting 3/2016, Leiden, Holland Hyaluronic acid deposition determines engineered heart Invited talk DGPT 3/2016, muscle characteristics and can be pharmacologically Berlin targeted to enhance function Soft matter guided self-assembly of oscillating heart cells Poster and talk SFB937 Retreat into functional macro-myocardium 2015/2016 V Table of contents Table of contents Table of contents ............................................................................................................... I Acknowledgements ........................................................................................................ VI Abstract ............................................................................................................................ IX List of figures .................................................................................................................... X List of tables ................................................................................................................... XII Abbreviations ............................................................................................................... XIII 1 Introduction ........................................................................................................... 1 1.1 Cardiovascular diseases .......................................................................................... 1 1.1.1 The failing heart .............................................................................................. 2 1.1.2 Regenerative approaches in cardiovascular medicine .................................... 2 1.2 The cellular components of the myocardium .......................................................... 4 1.2.1 Cardiomyocytes ............................................................................................... 4 1.2.2 Non-myocytes .................................................................................................. 5 1.2.3 Cardiac fibroblasts .......................................................................................... 6 1.3 Crosstalk between cardiac fibroblasts and cardiomyocytes ................................... 8 1.3.1 Direct cell-cell contacts .................................................................................... 9 1.3.2 Paracrine mediators .......................................................................................10 1.3.3 Communication via the extracellular matrix ................................................11 1.4 Viscoelasticity in cardiomyocyte development and function ................................ 12 1.4.1 Viscoelasticity during physiological development of cardiomyocytes ...........12 1.4.2 (Myo)fibroblasts affect myocardial viscoelasticity .........................................13 1.5 Towards better in vitro models of the myocardium .............................................. 15 1.5.1 Limitations of 2D in vitro models for the study of fibroblasts ......................16 1.5.2 Engineered Heart Muscle (EHM) ..................................................................17 I Table of contents 1.6 Hypotheses to be tested......................................................................................... 20 2 Materials and methods ...................................................................................... 21 2.1 Cells ....................................................................................................................... 21 2.1.1 Ventricular cardiac fibroblasts from Lonza ...................................................21 2.1.2 Dilated cardiomyopathy modeling study .......................................................22 2.1.3 Standard cell culture conditions ....................................................................22 2.1.4 Human embryonic kidney SV40 transformed cells .......................................22 2.1.5 Ethics statement ............................................................................................22 2.1.6 Cell culture, maintenance and stock preparation .........................................22 2.1.7 Derivation of human gingiva fibroblasts from biopsies .................................23 2.1.8 Standard cell culture techniques ...................................................................24 2.1.9 Standard culture and passaging of TSA ........................................................25 2.1.10 Human feeder cells .........................................................................................25 2.1.11 Culture of human embryonic stem cells ........................................................25 2.1.12 Derivation of stroma cells from embryonic stem cells ...................................26 2.2 Engineered heart muscle (EHM) .......................................................................... 27 2.2.1 Cell dissociation and casting ..........................................................................27 2.2.2 EHM culture ...................................................................................................28 2.2.3 Generation of engineered connective tissues .................................................28 2.2.4 Collagen I hydrogels for rheology measurements..........................................28 2.2.5 Replacement of B27 ........................................................................................29 2.2.6 EHM dissociation ...........................................................................................30 2.2.7 Irradiation of EHM .........................................................................................30 2.3 Genetic modification of cells ................................................................................. 30 2.3.1 Transfection of TSA cells ................................................................................30
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