| dc.contributor.advisor | Streckfuß-Bömeke, Katrin PD Dr. | |
| dc.contributor.author | Maus, Andreas | |
| dc.date.accessioned | 2020-06-24T07:43:07Z | |
| dc.date.available | 2021-02-22T23:50:03Z | |
| dc.date.issued | 2020-06-24 | |
| dc.identifier.uri | http://hdl.handle.net/21.11130/00-1735-0000-0005-13ED-B | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-8029 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-8029 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 610 | |
| dc.title | Human induced pluripotent stem cell models used in the study of doxorubicin-induced cardiomyopathy | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Streckfuß-Bömeke, Katrin PD Dr. | |
| dc.date.examination | 2020-02-24 | |
| dc.description.abstracteng | Doxorubicin (DOX) has been used for decades to treat hematopoietic and solid tumors, although, in a subset of cancer survivors, it causes cardiotoxicity decades after treatment. The mechanisms of anthracycline-induced cardiotoxicity (ACT) are still incompletely understood. Induced pluripotent stem cells (iPSCs)-derived cardiomyocytes have become a valuable tool to study hereditary and structural cardiac conditions in vitro. A former study from our group showed that iPSC-derived cardiomyocytes (iPSC-CMs) from lymphoma patients with ACT were continuously more sensitive to DOX toxicity, demonstrating disorganized myofilament structure and altered calcium handling along with increased cell death and reactive oxygen species (ROS) production compared to control iPSC-CMs.
The aims of this study are to (1) analyze the cellular resorption and distribution of DOX, (2) assess its impact on mRNA translation, (3) address an ACT-associated single-nucleotide polymorphism (SNP) (rs13058338 in the RAC2 gene) in ACT disease progression, (4) test a potential treatment option with TERT overexpression, and finally, (5) distinguish between ACT and unspecific effects of DOX in human cardiomyocytes. To this end, iPSCs from ACT- and dilated cardiomyopathy (DCM)-patients as well as control iPSC lines were differentiated into three-month-old iPSC-CMs and treated with DOX to model the effects of the drug exposure on human cardiomyocytes. In the first part of the study, the cellular resorption and retention of DOX in iPSC-CMs was investigated using high-performance liquid chromatography (HPLC) and positive correlations between DOX treatment time, treatment concentration and intracellular DOX levels were found. Intracellular DOX levels were consistently higher in ACT-iPSC-CMs compared to control iPSC-CMs and DOX remained longer in ACT-iPSC-CMs after treatment compared to control iPSC-CMs. In summary, these experiments suggested little regulation of DOX uptake or efflux in general. In the next chapter, the impact of DOX on mRNA translation was tested using the puromycin-protein incorporation assay. These data showed a significant decrease in translation down to 80% after DOX treatment in all tested iPSC-CMs as compared to untreated controls. In the third part of this work, the SNP rs13058338 was successfully edited in the RAC2 gene in an ACT patient iPSC line and heterozygous and homozygous isogenic gene-corrected ACT-iPSC-lines were established. The established, gene-edited iPSC lines will enable us to unravel the RAC2-SNP-related molecular contributions to ACT predisposition by testing the susceptibility to DOX-induced cardiotoxicity in isogenic ACT-iPSC-CMs. In order to analyze the contribution of cardiomyocyte-specific telomere lengths in ACT, quantitative polymerase chain reaction (qPCR) and quantitative fluorescent in situ hybridization (qFISH) experiments were performed. It was found that telomeres are shorter in iPSC-CMs compared to iPSCs and furthermore, that ACT-iPSC-CMs have shorter telomeres than their control. Telomerase (TERT) activity in cardioprotection was assessed with TERT overexpression in iPSC-CMs using adeno-associated-virus 6 (AAV6) vectors. We did not find measurable TERT-induced effects in the tested ACT-iPSC-CMs regarding apoptosis rates (Annexin V/PI kit for flow cytometry) or extracellular ROS production (Amplex red) after DOX treatment. Finally, DCM-iPSC-CMs were included in the study to identify differences in disease mechanisms that are common in ACT-specific myocardial processes. Therefore, DOX-treated DCM-iPSC-CMs were tested for cell volume and sarcomeric regularity. DOX treatment had differential impacts on sarcomeric integrity of iPSC-CMs from the DCM cohort as compared to the ACT cohort, mainly due to highly deteriorated DCM-iPSC-CMs before DOX treatment. Control iPSC-CMs from the DCM cohort showed a similar decrease in sarcomeric integrity as control iPSC-CMs from the ACT cohort.
In conclusion, this thesis shows that ACT-iPSC-CMs can be used to determine subcellular DOX levels and, in addition, allows assessing the impact of this anthracycline on cellular processes such as mRNA translation. iPSC-CMs from ACT patients are more susceptible to the detrimental effects of DOX treatment than iPSC-CMs from controls or other cardiac disease such as DCM. This tool will allow for identifying the underlying genetic basis and mechanisms of ACT and may enable screening for protective agents. | de |
| dc.contributor.coReferee | Meyer, Thomas Prof. Dr. | |
| dc.contributor.thirdReferee | Shah, Ajay Prof. | |
| dc.contributor.thirdReferee | Walter, Lutz Prof. Dr. | |
| dc.contributor.thirdReferee | Zelarayán, Laura C. PD Dr. | |
| dc.contributor.thirdReferee | Thoms, Sven PD Dr. | |
| dc.contributor.thirdReferee | Behr, Rüdiger Prof. Dr. | |
| dc.subject.eng | induced pluripotent stem cells | de |
| dc.subject.eng | doxorubicin | de |
| dc.subject.eng | ACT | de |
| dc.subject.eng | anthracycline-induced cardiotoxicity | de |
| dc.subject.eng | telomerase | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-21.11130/00-1735-0000-0005-13ED-B-4 | |
| dc.affiliation.institute | Medizinische Fakultät | |
| dc.subject.gokfull | Medizin (PPN619874732) | de |
| dc.subject.gokfull | Kardiologie (PPN619875755) | de |
| dc.description.embargoed | 2021-02-22 | |
| dc.identifier.ppn | 1702118134 | |