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A cell-type and compartment specific analysis of glutathione and hydrogen peroxide

dc.contributor.advisorZimmermann, Wolfram-Hubertus Prof. Dr.
dc.contributor.authorTrautsch, Irina Karoline
dc.titleA cell-type and compartment specific analysis of glutathione and hydrogen peroxidede
dc.contributor.refereeZimmermann, Wolfram-Hubertus Prof. Dr.
dc.description.abstractengReactive oxygen species (ROS) have been recognized to play important roles as messenger molecules. They are involved in the etiology and progression of cardiovascular diseases such as myocardial infarction, cardiac hypertrophy or fibrosis. Investigation of reactive oxygen species and cellular redox alterations has been methodologically challenging. Genetically encoded redox sensors make dynamic measurements of ROS and involved buffering systems in living cells possible. They can be targeted to specific cellular organelles and report compartment specific redox changes. Analysis of sensor response is performed by dual excitation single emission fluorescence microscopy or plate reader based assays. In the present study, we hypothesized that fluorescent redox sensors can be applied in human pluripotent stem cells and stem cell derived cardiomyocytes to investigate cell type and compartment specific differences in redox homeostasis. Genomic integration of two redox sensor systems was performed in human embryonic stem cells. Grx1-roGFP2 reacts and stays in equilibrium with cellular glutathione (GSH) pools, while HyPer can report increases in cellular hydrogen peroxide (H2O2). Grx1-roGFP2 was targeted to cytosol and mitochondria in transgenic stem cells to analyze their GSH balance in the two compartments. Stem cell mitochondria appeared to have a more oxidized GSH pool than the cytosol. Transgenic stem cell lines were also differentiated to cardiomyocytes, but failed to develop spontaneous beating activity. Due to this unexpected phenotype, a lentiviral integration approach was chosen to express Grx1-roGFP2 in cytosol and mitochondria of stem cell derived cardiomyocytes and primary dermal fibroblasts. While fibroblast GSH milieu appeared similar between cytosol and mitochondria, cardiomyocytes had strongly oxidized mitochondria compared to cytosol. As reactive oxygen species can be generated during oxidative phosphorylation in mitochondria, cellular metabolism might play an important role in the regulation of cellular redox homoeostasis. As a model of advanced maturation, stem cell derived cardiomyocytes were used to construct engineered heart muscle. Preliminary proof-of-concept for the analysis of redox sensors in this tissue model is reported in this study. To sense H2O2, we employed an alternative sensor, namely HyPer, which was able to report changes in intracellular H2O2 levels upon extracellular bolus addition of H2O2. Aside from detection of H2O2, also a sensor-producer hybrid construct, HyperDAO, was deployed in a TSA model. Upon stimulation with D-alanine, HyPerDAO produced H2O2 inside transgenic cells. Unfortunately, human embryonic stem cells transgenic for HyPer or HyPerDAO also failed to differentiate into beating cardiomyocytes. In summary, in this thesis tools for the analysis and manipulation of ROS and their buffering systems in a cell- and compartment-specific manner were developed. Further studies are needed to elucidate the mechanisms behind differences in cellular redox balances reported here and will lead to a deeper understanding of redox signaling in cardiac physiology and
dc.contributor.coRefereeUrlaub, Henning Prof. Dr.
dc.subject.enghuman embryonic stem cellsde
dc.subject.engredox sensingde
dc.affiliation.instituteMedizinische Fakultät
dc.subject.gokfullMedizin (PPN619874732)de

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