Application of redox biosensor mouse models to study redox processes in cardiomyocytes

Reactive oxygen species (ROS) are highly reactive molecules produced in any biological system. When ROS are produced in higher amounts, they are lethal to cells. Therefore cells possess a tight redox regulation through action of various antioxidant defense systems. An imbalance between the ROS produced versus the action of the antioxidants can give rise to a state called oxidative stress. In order to study the consequences of high ROS production in a system, it is essential to develop tools that can measure quantitatively the precise levels of specific ROS or the status of a specific redox couple. Until recently, synthetic probes were used widely to measure ROS in a qualitative manner. However, taking into consideration the limitations of these probes, genetically encoded biosensors have gradually started to replace the relatively non-specific probes. These genetically encoded biosensors can not only visualize the redox nature quantitatively and in real time but also can be targeted to any subcellular compartment of a cell. In line to these necessities, mouse models in which the glutathione redox biosensor Grx1-roGFP2 is expressed in cardiomyocytes and located in two different compartments were applied in the presented thesis. These mouse models allow to study the glutathione redox potential (EGSH) in the cytoplasm versus mitochondrial matrix. The mouse models were used to study the effect of aging on the EGSH of the cytoplasm and mitochondrial matrix in cardiomyocytes. The redox compartmentalization between the two compartments which was observed in young mice seems to disappear in aging animals. Besides applying the mouse models to study the effects of aging on redox regulation, the mito Grx1-roGFP2 mouse model was utilized to study the importance of maintaining the physiological oxygen concentration in order to preserve the reduced EGSH of the mitochondrial matrix as well as the overall mitochondrial functionality. In this part of the thesis, it was observed that upon isolation of mitochondria from cardiac tissue at room air conditions (20% O2), mitochondria seem to almost fully get oxidized However, when the mitochondria are isolated in hypoxia (0.1% O2), the EGSH is preserved demonstrating that the EGSH of the mitochondrial matrix is indeed affected by the change in the pO2 experienced by the mitochondria when isolated from the tissue. Together with the EGSH, other parameters of the mitochondrial electron transport chain like ROS, ATP as well as complex III activity are affected when mitochondria are isolated in 20% O2. In the final part of my thesis, I generated a novel redox biosensor mouse model. In these mice the biosensor consists of the endogenous H2O2 producer D-amino acid oxidase (DAAO) fused with the H2O2 HyPer biosensor. A positive founder line of the DAAO-HyPer, wherein the biosensor is targeted to the nucleus of the cardiomyocytes, was successfully created and characterized. This mouse model is useful to study the development of cardiac dysfunctions in consequence to the generation of the endogenous ROS in the nucleus of the cardiomyocytes