Characterization of the cytosolic and mitochondrial glutathione redox potential using cardiomyocyte specific transgenic Grx1-roGFP2 sensor mice

Abstract

In the field of redox biology, the mainly negative perception of reactive oxygen species has given way to the global concept of an intracellular redox balance. Free radicals like H2O2 not only lead to harmful effects within oxidative stress but are also essential for physiological processes. Redox homeostasis of cardiomyocytes is important for the maintenance of physiological function: disruption of redox balance has been linked to the development of heart failure, cardiac hypertrophy, and other cardiac pathologies. The low weight molecule glutathione, the essential cellular antioxidant, contributes to the maintenance of cellular redox homeostasis. The redox status of the glutathione is reflected by its redox potential (EGSH). The development of transgenic redox sensors based on the green fluorescent protein (roGFP), which are fused to glutaredoxin-1 (Grx1), allows dynamic and specific measurements of EGSH in intact cells and organs. A specific subcellular localization of the redox sensor can be achieved via targeting sequences. Thus, Grx1-roGFP2 redox sensors offer a significant advantage compared to conventional measurements, which often involve destruction of the cell or lack specificity. The aim of this study was to characterize the cytosolic and mitochondrial EGSH in cardiomyocytes using the Grx1-roGFP2 sensor. In two of the four analyzed Grx1-roGFP2 mouse lines, the redox sensor was localized to the mitochondrial matrix, whereas in the other two mouse lines the sensor was non-targeted in the cytoplasm. In all four mouse models, impairment of cardiac performance due to the sensor was excluded by echocardiography. Based on the measurement of the different fluorescence intensities of the Grx1-roGFP2 sensor depending on its oxidation state, isolated single cells in all four mouse lines were analyzed and the Grx1-roGFP2 sensor showed in each mouse line a dynamic response to oxidizing and reducing stimuli. After maximal reduction and oxidation of the Grx1-roGFP2 sensor, the EGSH of the cytosol and the mitochondrial matrix was determined under consideration and exclusion of interfering factors. Here, the cytoplasm showed a significantly more oxidized E_GSH (-278.8 ± 0.7 mV and -280.3 ± 0.7 mV) compared to the mitochondrial matrix (-333.6 ± 0.4 mV and -330.1 ± 0.4 mV). The analysis of whole hearts ex situ of one non-targeted and one mouse line with the sensor targeted to the mitochondrial matrix, showed comparable results as seen in the analysis of isolated cardiomyocytes. The mitochondrial EGSH is consistent with values observed in other studies using roGFP2-based sensors, while the cytosolic EGSH of cardiomyocytes is in contrast more oxidized than in other cell types. The results from this thesis help to shed more light on redox homeostasis in cardiomyocytes to gain a better understanding of redox processes in cardiac physiology and pathology in the future.