Dokument: Role of metabolism and mitochondrial function for stem cell differentiation and stress responses upon genotoxic noxae
| Titel: | Role of metabolism and mitochondrial function for stem cell differentiation and stress responses upon genotoxic noxae | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=71117 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20251028-111121-4 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Westerhoff, Michelle [Autor] | |||||||
| Dateien: |
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| Beitragende: | Reichert, Andreas S. [Gutachter] Gerhard Fritz [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | The anthracycline antibiotic doxorubicin is widely used as chemotherapeutic agent for treatment of solid tumors and hematological malignancies in both adults and children. Despite its clinical efficacy, its application is limited by adverse effects on somatic tissue, particularly in the heart. Doxorubicin treatment can lead to irreversible cardiotoxicity, including arrhythmias, long-term cardiovascular complications or congestive heart failure. Several mechanisms have been implicated in the development of cardiotoxicity, such as induction of DNA damage, involvement in the formation of oxidative stress and damage of organelles, especially reflected in mitochondrial dysfunction. Importantly, mitochondrial malfunction has previously been described under comparatively high and prolonged exposure to doxorubicin, raising the question whether observed effects on mitochondria are downstream events of genotoxic effects on nuclear DNA. In order to decipher the influence of mitochondria in mediating doxorubicin toxicity, a treatment regimen mindful to the clinical situation was applied. An additional challenge in studying these processes was the availability of a human based cardiomyocyte model. For this, a human induced pluripotent stem cells (iPSCs) were established in order to generate cardiac progenitors (CPs) and iPSC derived cardiomyocytes (iCMs) to provide an experimental platform to study cell type- or differentiation-specific responses to doxorubicin treatment in a clinical relevant set-up. The present study aimed to investigate how acute exposure to low concentrations of doxorubicin affects iPSCs and iCMs, with a focus on DNA damage response, mitochondrial function, transcriptional expression and cellular function. With comparison of the response of iPSCs and iCMs upon doxorubicin treatment regimens in iPSCs and iCMs, we sought to gain further insights into stage-dependent susceptibility to doxorubicin and the induction of transcriptional programs that may be involved in the development of cardiotoxic effects. Furthermore, we aimed to understand whether pre-conditioning of iPSCs prior to initiation of cardiac differentiation alters iCM susceptibility towards a second exposure to doxorubicin, thereby testing the hypothesis of a hormesis-like protective effect involving the upregulation of genes involved in stress response pathways.
Several insights into the cellular effects of doxorubicin at clinically relevant concentrations and exposure times were revealed. The results indicate that even short exposures to low concentrations of doxorubicin induced substantial mitochondrial dysfunction and metabolic disturbances, accompanied with transcriptional alterations, which could contribute to long-term cardiotoxicity. Firstly, the present study demonstrated that iPSCs are highly sensitive towards acute exposure to low concentrations of doxorubicin with a significant reduction in cell viability. Furthermore, mitochondria exhibited altered morphology, characterized by increased fragmentation, loss of membrane potential, and reduction in mitochondrial content. It is important to note that these effects occurred independently of DSB formation and doxorubicin-induced DDR activation, suggesting that mitochondrial impairment is a direct and primary target of doxorubicin rather than a secondary consequence of nuclear genotoxicity. The present findings emphasize that the disruption of mitochondrial dynamics and function may play a critical role in the progression of doxorubicin-induced cardiotoxicity, albeit further approaches are needed to validate the observed effects. Secondly, there was evidence of differentiation dependent variations in the susceptibility of iPSCs and iCMs to doxorubicin. In contrast to terminally differentiated cardiomyocytes, iPSCs predominantly activate apoptosis as a protective mechanism against genomic instability. Indeed, iPSCs displayed a higher sensitivity to doxorubicin-induced apoptosis compared to differentiated iCMs, a finding that correlates with the high proliferative capacity of iPSCs and the generally lower threshold of iPSCs to undergo apoptosis. The increased susceptibility of iPSCs may be attributed to mitochondrial stress comprising of mitochondrial membrane potential loss, metabolic disruption, and alterations in the mitochondrial genome. The latter being consistent with the known genotoxic role of Dox via the inhibition of mitochondrial located topoisomerase IIβ. Furthermore, transcriptomic analysis revealed profound effects of doxorubicin treatment on cellular metabolism and cardiac function. In iPSCs, doxorubicin exposure led to downregulation of glycolytic pathway genes, suggesting impaired energy metabolism. Interestingly, we could observe compensatory mechanisms in form of increased mtDNA-encoded OXPHOS genes in iPSCs upon doxorubicin treatment. In contrast, iCMs majorly exhibited a suppression in genes associated with cardiac function (e.g. contractility, calcium homeostasis, and sarcomere organization), highlighting potency of doxorubicin to impair cardiac function even at low concentrations. Furthermore, the activation of extracellular matrix modifying genes in iCMs suggests the promotion of fibrosis under doxorubicin treatment regimens. The present data demonstrates that pre conditioning of cells in pluripotent state is accompanied with transcriptional reprogramming in a way that cardiac differentiation is not adversely affected per se, yet with alterations of cardiac function. Furthermore, we aimed to understand whether a double-treatment approach, where iPSCs were pre conditioned with doxorubicin prior to cardiac differentiation followed by a second encounter with doxorubicin in iCM stage, induces protective adaptations of these cells. Contrary to the expectations, this approach exacerbated mitochondrial dysfunction and increased susceptibility to subsequent doxorubicin exposure. Here, double treated iCMs exhibited a stronger downregulation of genes associated with mitochondrial and cardiac function, along with an increased expression of cell death markers, highlighting has cumulative and persistent adverse effects even with the application of low concentrations, which might explain the increased sensitivity of these cells. Overall, the present results emphasize the potency of doxorubicin, even with acute exposures and low concentrations, resulting in mitochondrial dysfunction, metabolic dysregulation, and transcriptional remodeling possibly mediating cardiotoxic effects. More experimental approaches are needed to understand the relationship of cardiac malfunction and cardiac stem cell pool contribution to the manifestation of cardiotoxicity observed in patients treated with doxorubicin. However, with the iPSC based model, we could reproduce the most common side effects of doxorubicin comprising fibrosis induction and contractile dysfunction of iCMs at comparably low concentrations highlighting the susceptibility of stem cells towards doxorubicin. Thus, future experimental approaches should comprise the application of low concentrations of doxorubicin to examine the underlying mechanisms in doxorubicin induced damage response as well as to determine the possible role of cardiac stem cells in the manifestation of cardiotoxicity. Furthermore, future approaches should implement the modulation of mitochondrial function which subsequently improves cellular resistance to metabolic stress. Moreover, the enhancement of mitochondrial biogenesis, modulation of mitochondrial dynamics, or administration of cardioprotective agents (e.g.: dexrazoxane), could be possible targets to reduce long-term cardiotoxic effects of doxorubicin. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie | |||||||
| Dokument erstellt am: | 28.10.2025 | |||||||
| Dateien geändert am: | 28.10.2025 | |||||||
| Promotionsantrag am: | 01.06.2025 | |||||||
| Datum der Promotion: | 21.08.2025 |
