Appendix

Table 5: Mean CT-Values of qRT-PCR analyses.

Gene CT-value CamKII 24

cTNT 17.94 CYBA 30.41 GAPDH 18.89 HPRT 27.35

NCX 21.33

NOX2 31.37 NOX4 29.85

PLN 20.42

RAC1 22.77 RAC2 32.85 RYR2 25.32 SERCA 21.9

Figure 35: Verification of primers for amplification of NCF1, NCF2 and NCF4. Expression of NCF1, NCF2 and NCF4 was detected in a heart biopsy of a dilated cardiomyopathy patient but not in iPSC-CMs.

Gene CT-value α-actinin 17.9 α-MHC 23.95 β-MHC 19.22

Figure 36: Original western blots of NADPH oxidase subunits. Bands used for quantification are marked with a red arrowhead. GAPDH was stained additionally on every membrane.

Figure 37: Original western blots of Ca²⁺ handling proteins, α-actinin and reference proteins. Bands used for quantification are marked with an arrowhead.

Figure 38: H2DCF-DA assay trial in iPSC-CMs. H2DCF-loaded iPSC-CM from a healthy donor were observed with confocal laser scanning microscopy in the frames can mode. The mean fluorescence intensity was normalized to the base value. Sample number: 2.

Figure 39: Lucigenin-enhanced chemiluminescence assay trial in CMs. Homogenates of iPSC-CM were treated with respective ·O2- scavengers or enzyme inhibitors and 5 µM lucigenin.

Luminescence intensity was observed 10 min after addition of 300 µM NADPH. Tiron is a ·O2-

scavenger. Diphenyleneiodonium (DPI) is a flavoprotein inhibitor. VAS2870 (VAS) is a NADPH oxidase inhibitor. NG-nitro-L-arginine methyl ester (L-NAME) is a nitric oxide synthase inhibitor. Oxipurinol (OXY) is a xanthine oxidase inhibitor. Rotenone (ROT) is a mitochondrial electron chain inhibitor.

Figure 40: DOX-dependent titin degradation in iPSC-CMs. Western blot of a human heart biopsy sample (hH) and DOX-treated iPSC-CMs from a healthy control donor. Titin was visualized with an antibody that is specific for the C-terminus of titin. N2BA and N2B: Titin isoforms. Data generated by Andrey Fomin.

Acknowledgments

I would like to express my sincere gratitude to everyone who supported me during my doctoral studies over the last years.

First, I especially thank Dr. Katrin Streckfuß-Bömeke for giving me the opportunity to be a doctoral student in her laboratory, for providing me with such an interesting project and for excellent supervision. I also thank Prof. Dr. Gerd Hasenfuß for giving me the chance to work in the Department of Cardiology and Pneumology.

I am very grateful to Prof. Dr. Sigrid Hoyer-Fender for being the first reviewer of my thesis and making it possible for me to participate in the biological doctoral program. I learned scientific thinking during my bachelor’s thesis in her research group and it was very special to me that she supervised me during my doctoral studies.

I thank my second reviewer Prof. Dr. Susanne Lutz for the constructive discussions and her kind advice during our annual committee meetings.

I also extend my thanks to Prof. Dr. Dörthe Katschinski, Prof. Dr. Ernst A. Wimmer and Prof.

Dr. Rüdiger Behr for being part of my examination board despite their busy schedule.

I especially thank the people who donated skin biopsies for this work. They survived cancer and afterwards suffered from heart condition but still donated cells. I admire their courage and kindness.

Thanks go to Dr. Malte Tiburcy and Irina Eckhardt for the generation and analyses of EHMs. I also thank Dr. Andreas Petry for performing EPR experiments and NADPH oxidase western blots.

A big thank goes to the people who took the time to teach and brief me. I thank Dr. Lukas Cyganek for teaching me the measurement of intracellular calcium. I owe particular thanks to Dr. Eriona Heta for the Grx1-roGFP2 construct, the virus generation protocol, which worked like a charm, and her great help with the analyses. I thank Sabrina Becker for her foresightful briefing and teaching of flow cytometry. I also thank Thomas Borchert for writing the helpful makro for the analyses of calcium measurement data.

I thank Malina Seguin, Jérôme Janßen and Dirk Alexander Frick for their hard work during their theses or internships and for giving me the opportunity to learn to teach.

I owe particular thanks to all colleagues from the stem cell lab for their support. Special thanks go to Carmen Klopper and Sandra Georgi for their excellent technical assistance within the ACT project.

thinking and for reviewing my thesis.

I also thank Sabine Rebs, Jessica Spitalieri and Marco Singer for their helpful reviews of my thesis.

Many thanks to my best friend Marco Singer, who always supported me and cheered me up.

My particular thanks go to my mother for her unconditional support and her understanding during busy times in the last years.

I would like to express my deepest gratitude to Jessica for her love, her understanding and her advice.

Curriculum vitae

PhD study program “Biology” at the Georg-August University School of Science (GAUSS), Göttingen

Project: „Modeling anthracycline-induced cardiotoxicity with patient-specific iPSCs“

Institute: Department of Cardiology and Pneumology; University Medical Center Göttingen

Supervisor: Dr. Katrin Streckfuß-Bömeke 10.2011 –

11.2013 Master of Science “Developmental, Neural and Behavioral Biology”, Georg-August-University Göttingen

Master’s Thesis: „Generation and characterization of induced pluripotent stem cells from patients with CPVT”

Graduate assistant at the stem cell laboratory, Department of Cardiology and Pneumology; University Medical Center Göttingen Project: Generation of transgenic ps-hiPSCs containing a

cAMP-biosensor for FRET based analyses Education

09.1999 – 06.2006 Gymnasium Ricarda-Huch in Braunschweig Degree: Abitur diploma (2.7)

 „Generation of a KLF15 homozygous knockout human embryonic stem cell line using paired CRISPR/Cas9n, and human cardiomyocytes derivation”; C. Noack, L. Haupt, W.-H. Zimmermann, K. Streckfuß-Bömeke, L. Zelarayán; August 2017; Stem Cell Research.

 „Assessing the influence of the genetic background on anthracycline-induced cardiotoxicity with human induced pluripotent stem cell-derived cardiomyocytes“; L.

Haupt and K. Streckfuß-Bömeke; October 2016; EMJ Cardiology 4.1.

 „Assessing the influence of the genetic background on anthracycline-induced cardiotoxicity with human induced pluripotent stem cell-derived cardiomyocytes “; L.

Haupt, et al.; poster presentation at the European Society of Cardiology Congress 2016 in Rome.

 „Modeling anthracycline-induced heart failure using hiPSC-derived cardiomyocytes“;

L. Haupt, et al.; poster presentation at the annual meeting of the “Deutschen Gesellschaft für Kardiologie” (German Cardiac Society) 2015 in Mannheim.

Memberships and Grands

 European Society of Cardiology, professional member

 Deutsches Zentrum für Herz-Kreislaufforschung (German Center for Cardiovascular Disease), Young-DZHK member

 Traveling grand of the “Deutschen Gesellschaft für Kardiologie” (German Cardiac Society) for the annual meeting 2015

Affidavit

Here I declare that my doctoral thesis entitled:

“Modeling anthracycline-induced cardiotoxicity with patient-specific iPSCs”

has been written independently with no other sources and aids than quoted.

Luis Peter Haupt

Göttingen, December 2017